Molecular Pathways of Endothelial Cell Activation for - Semantic Scholar

Current Vascular Pharmacology, 2005, 3, 11-39

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Molecular Pathways of Endothelial Cell Activation for (Targeted) Pharmacological Intervention of Chronic Inflammatory Diseases Joanna M. Kuldo1, Ken Ichi Ogawara2, Naomi Werner1, Sigridur A. Ásgeirsdóttir1, Jan A.A.M. Kamps1, Robbert J. Kok3 and Grietje Molema1,* 1

Dept. Pathology and Laboratory Medicine, Medical Biology section, and 3Dept. Pharmacokinetics and Drug Delivery, Groningen University Institute for Drug Exploration (GUIDE), Groningen, Netherlands; 2Graduate School of Natural Sciences and Technology, Okayama University, Okayama, Japan Abstract: In chronic inflammatory conditions, endothelial cells actively recruit immune cells from the circulation into the underlying tissue and participate in angiogenesis to support the continuous demand for oxygen and nutrients. They do so in response to activation by cytokines and growth factors such as tumour necrosis factor α (TNFα), interleukin-1 (IL-1), vascular endothelial growth factor (VEGF), and fibroblast growth factors (FGFs). Receptor triggering initiates intracellular signal transduction leading to activation of nuclear factor κB (NFκB), mitogen activated protein kinase (MAPK) activity, and nitric oxide and reactive oxygen species production, among others. As a result, adhesion molecules, cytokines and chemokines, and a variety of other genes are being expressed that mediate and control the inflammatory process. In recent years, different classes of drugs have been developed that interfere with selected enzymes involved in the intracellular signalling cascades. In endothelial cell cultures, they exert potent inhibitory effects on the expression of genes, while several studies also report on in vivo effectiveness to confine the inflammatory responses. To prevent undesired toxicity and to improve drug behaviour and efficacy, drug carrier systems have been developed that selectively deliver the therapeutics into the activated endothelial cells. The above subjects are recapitulated to give an overview on the status of development of endothelial cell directed therapeutic strategies to pharmacologically interfere with chronic inflammatory diseases.

Keywords: Endothelial cells, chronic inflammation, angiogenesis, signal transduction, drugs, pharmacology, vascular drug targeting. INTRODUCTION In recent years, knowledge regarding endothelial cell (dys)function in physiological processes and pathophysiological conditions has greatly increased. Extensive effort in this area of research is dedicated to the development of drugs that can selectively inhibit endothelial cell activation for the treatment of chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease and atherosclerosis. An increased understanding of intracellular signalling pathways during cell activation has led to a shift from development of antibodies and peptides that block specific molecules controlling leukocyte – endothelial cell interaction to small chemical entities that affect endothelial cell activation. A new advance is furthermore the development of so called vascular drug targeting strategies that aim to increase specificity of the newly developed drugs. Incorporation of drugs in carrier systems can facilitate selective targeting, resulting in reduced side effects of the highly potent and often intrinsically toxic drugs.

*Address correspondence to this author at the Dept. Pathology and Laboratory Medicine, Medical Biology section, Endothelial Cell Research & Vascular Drug Targeting Group, Hanzeplein 1, 9713 GZ Groningen, Netherlands; Tel: +31-50-3610115; Fax: +31-50-3619911; Email: [email protected]; www.rug.nl/med/faculteit/disciplinegroepen/plg/

1570-1611/05 $50.00+.00

In this review, we summarise current knowledge on molecular signalling pathways involved in endothelial cell activation during inflammatory processes and inflammation associated angiogenesis. Drugs identified to interfere with gene expression driven by these pathways and developments in vascular drug targeting strategies for the treatment of chronic inflammation are discussed. Although many of the drugs discussed have been studied for their effects on other cell types as well, cell type specific regulatory mechanisms do not allow simple extrapolation from one cell type to the other. Therefore, this review will focus on observations in primary endothelial cells and endothelial cell lines in vitro, and on endothelial cell related effects observed in vivo. ENDOTHELIAL CELL FUNCTION IN HEALTH AND DISEASE The blood vessels in our body are highly heterogeneous with regard to architecture and function. The larger blood vessels that consist of endothelium, smooth muscle cells, connective tissue and elastic elements, transport blood through the body. The endothelium is actively involved in blood pressure control via, among others, release of the vasodilator prostacyclin and the vasorelaxant gas nitric oxide as well as the production of vasoconstrictors including endothelin-1 and the arachidonic acid metabolites thromboxane A2 and prostaglandin H2 [1].

© 2005 Bentham Science Publishers Ltd.

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Current Vascular Pharmacology, 2005, Vol. 3, No. 1

In the microva scular bed of organs, the endothelial cells in general reside on a basal lamina and are only supported by sparsely distributed pericytes that cover the endothelium with finger-like protrusions. Pericyte presence is a prerequisite for vessel maturation and proper vascular function, since loss of pericytes correlates with endothelial hyperplasia and increased endothelial permeability, among others [2,3]. Endothelial cells exert four main functions while in addition organ-specific activities can be attributed to endothelial cells in their respective microvascular beds. First of all, they function as a (semipermeable) barrier for transport of soluble molecules. Furthermore, the endothelium maintains haemostatic balances via the production of procoagulants (von Willebrand factor, Tissue Factor) and the fibrinolytic plasminogen activator inhibitor-1, coagulation inhibitors thrombomodulin and Tissue Factor pathway inhibitor, and tissue type and urokinase-type plasminogen activator. Other factors including thrombin, antithrombin and heparan sulphate proteoglycans, platelet activating factor and platelet factor-4 also play a prominent role in haemostatic

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control by the vessel wall in conditions of physical damage and cellular stress [1]. Coordinated recruitment of leukocytes, including lymphocytes, monocytes and neutrophils, into underlying tissue, and active participation in neovascularisation processes during inflammatory processes and malignant growth are other essential tasks of endothelial cells (Fig. 1) [4]. In most microvascular beds except those of spleen, lungs and liver, the postcapillary venule endothelium interacts with leukocytes to facilitate migration into tissues [5]. Atherosclerosis is an exception as this disease is characterised by monocyte transmigration over the macrovascular aortic wall following initial lipoprotein entrapment in the subendothelial matrix [6]. In inflammation, activation of endothelial cells results in intracellular signalling pathways as described below. Consequently, the endothelial cells produce, in a strictly regulated fashion, a series of cell adhesion molecules, chemokines and cytokines, which guide leukocytes into underlying tissue and co-stimulate them to become fully activated. Ongoing leukocyte recruitment, cellular activation

Fig. (1). The role of endothelial cells in leukocyte recruitment and neovascularisation during chronic inflammatory diseases. A: Top: schematic representation of the interaction between leukocytes and endothelium mediated by cell adhesion molecules (transiently) expressed on both cell types. Bottom: microscopic detail of a Crohn’s disease lesion in the human colon, where excessive leukocyte infiltration into the tissue parenchyma resulted in tissue destruction. B: Top: schematic representation of the behaviour of endothelial cell under the influence of an angiogenic stimulus. After activation, the basal lamina is degraded by the endothelial cells that subsequently migrate into the underlying tissue, proliferate and maturate into a new vessel. For clarity, other cells types involved in these processes, including pericytes, fibroblasts, and immune cells, are not depicted. Bottom: microscopic detail of a Crohn’s disease lesion in the human colon where the inflammatory response is accompanied by excessive neovascularisation. (Microscopic pictures: courtesy Dr. G. Dijkstra, Dept. Gastroenterology, University Hospital Groningen) Abbreviations: EC: endothelial cell; IgSF: immunoglobulin super family; sLex : sialyl Lewis X.

Molecular Pathways of Endothelial Cell Activation

and cell death induction all put a heavy demand on the local oxygen supply, leading to new blood vessel formation. This active role in maintaining a chronic inflammatory status and their easy accessibility for blood borne drugs make the endothelium an attractive target cell for therapeutic intervention. SIGNAL TRANSDUCTION PATHWAYS AND GENE EXPRESSION IN ENDOTHELIUM The focus of this review is on intracellular signalling cascades in endothelial cells that are active during chronic inflammatory diseases and that can be pharmacologically interfered with. TNFα and IL-1 represent the two proinflammatory cytokines that have been most extensively studied in this respect. Occasionally referral will be made to the effects of lipopolysaccharide (LPS, or endotoxin). Besides inducing TNFα production when administered in vivo, LPS can directly activate endothelial cell signalling via intracellular signal transduction pathways comparable to those reported for TNFα and IL-1. As the pro-angiogenic factors VEGF and FGF-2 are also prominently involved in endothelial cell activation during inflammation [7], signal transduction pathways relayed via their respective receptors will be discussed as well. Furthermore, interactions with blood borne cells can influence the status of the endothelial cells by affecting gene expression and consequent cellular function. Understanding the pathways involved in cellular responses to these stimuli during inflammatory disease initation and progression forms the basis for the rational development of (targeted) therapeutic strategies. Intracellular Signalling Mediated by Pro-Inflammatory Cytokines Cytokines and growth factors are produced as soluble extracellular (glyco)proteins or are released from storage in the extracellular matrix (ECM) in response to different stimuli. They affect the activation status of endothelial cells per se by receptor-mediated activation of intracellular signalling cascades resulting in changes in gene expression. One of the first steps in the immune response in inflammation is cytokine-mediated activation of endothelial cells. Within minutes, the endothelial cells secrete P-selectin that is stored in Weibel-Palade bodies, and start to produce E-selectin, the counter ligand for sialyl LewisX on the leukocytes. Also, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecules ICAM-1, 2, and -3, members of the immunoglobulin superfamily and receptors for leukocyte expressed integrins Very Late Antigen (VLA)-4 and Leukocyte Function Antigen (LFA)-1, respectively, are upregulated upon activation by proinflammatory cytokines. Together with cytokines and chemokines such as IL-6, IL-8 and Fraktalkine, cell adhesion molecules actively initiate leukocyte – endothelial cell contact and facilitate subsequent rolling, activation and transendothelial migration of the leukocytes. Different leukocyte subsets employ different combinations of adhesion molecules, cytokines, and chemokines for rolling and transendothelial migration, while also heterogeneity in endothelial behaviour affects the combinations required [8]. In vitro studies demonstrated a role for both LFA-1 and


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VLA-4 in T lymphocyte transendothelial migration, while neutrophil transmigration is mainly controlled by LFA-1. Whereas chemokine activated T-lymphocytes could interact with resting human umbilical vein endothelial cells (HUVEC) via LFA-1 / ICAM-1 interaction, VLA-4 involvement required additional endothelial cell activation [9]. Monocytes, on the other hand, can use an ICAM-1 / ICAM-2 independent pathway employing β2 integrin Mac-1 for transendothelial migration [10]. In vivo, P- and Eselectin, and VLA-4 were shown to be essential for memory T lymphocyte rolling onto activated skin endothelium, while E-selectin and VLA-4, but not P-selectin were important for lymphocyte infiltration into the skin tissue [11]. These differential and overlapping functions of cytokines, chemokines, and adhesion molecules on inflammatory leukocyte recruitment demonstrate the complexity of control of leukocyte - endothelial cell interactions in disease. Pro-inflammatory cytokines also affect endothelial haemostatic properties. For example, they induce the expression of one of the main initiators of the extrinsic pathway of blood coagulation, Tissue Factor, thereby inducing a pro-coagulant state [1]. In addition, Ludwig and colleagues recently put forward a role for activated platelets in facilitating leukocyte – endothelial cell interactions as a potential mechanism in leukocyte recruitment [12]. In a model of chronic skin inflammation, platelets form aggregates with leukocytes via platelet P-selectin – leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) interaction. In the skin microvasculature, the aggregates subsequently interact with the endothelium via platelet GPIIb/IIIa integrin – endothelial ICAM-1 and / or platelet PSGL-1 – endothelial P-selectin. This may enhance overall leukocyte rolling and facilitate the inflammatory process. TNFα Induced Signalling in Endothelial Cells TNFα is mainly produced by cells of haematopoietic origin. It is formed as type II transmembrane protein in which the receptor-binding moiety is located in the Cterminal part of the extracellular domain. TNFα is released after disintegrin and metalloproteinase cleavage of the “spacer” region that links the receptor-binding and transmembrane parts [13]. The soluble and receptor-binding form of TNFα exists as a trimer, and as such it reacts with TNF receptor-1 (TNFR1) and TNFR2. In endothelial cells, as in most other cells in the body, TNFα-induced effects are mediated by TNFR1 [14]. After binding to TNFR1 on the endothelial cells, TNFα activates multiple pathways that lead to an inflammatory status of the endothelial cell (Fig. 2) [15]. TNFR1 activation provides a docking site for accessory proteins TNFR associated factor 2 (TRAF2) and TNFR1-associated death domain TRADD that form the branching point for the pro-inflammatory and proapoptotic signalling pathways [13]. NFκB inducing kinase NIK activates inhibitor of κB (IκB) kinases IKKα and IKKβ to guide the signal transduction relay system towards activation of the transcription factor Nuclear Factor κ B (NFκB) [16]. IKKs form a catalytic subunit responsible for phosphorylation, site-specific ubiquitinylation and subsequent proteasomal degradation of IκB. Moreover, IKKα itself can activate the expression of NFκB responsive genes upon cytokine stimulation of cells [17]. Whether this

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Fig. (2). Signalling pathways that become active in endothelial cells upon interleukin-1 (IL-1) and tumour necrosis factor α (TNFα) activation. The activation leads to the expression of pro-inflammatory genes, including cell adhesion molecules, cytokines and chemokines, and cell survival genes. Abbreviations: IKK: IκB kinase; IκB: inhibitor κB; IRAK: IL-1 receptor-1 associated kinase; NEMO: NFκB essential modulator; NF κB: nuclear factor κB; NIK; NF κB inducing kinase; PI3K: phosphoinositol-3 kinase; PKC: protein kinase C; ROS: reactive oxygen species.

direct IKKα driven gene expression also takes place in endothelium needs to be established. The IKK complex furthermore contains a regulatory subunit called NFκB essential modulator (NEMO) or IKKγ that has also been studied as a target molecule for therapeutic purposes [18]. Upon degradation of IκB, the nuclear localisation sequence of NFκB, a dimer usually consisting of p65 (RelA) and p50 (NFκB1), targets the protein into the nucleus. Here NFκB binds to DNA and upon interaction with co-activators and other components of the gene transcription machinery, it activates transcription of a range of inflammatory genes. Besides being controlled by IKK activity, reactive oxygen species prominently affect NFκB activity [19]. Following TNFα induced NFκB activation, endothelial cells express functionally related genes, including the pro-inflammatory adhesion molecules E-selectin, ICAM-1, and VCAM-1, cytokines IL-6 and IL-8, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), among others [14,20]. In parallel, genes involved in cell signalling and apoptosis/cell proliferation control are downregulated [21]. The induction of IκB gene transcription and translation by NFκB, and IKKβ autophosphorylation in its inhibitory loop both represent feedback mechanisms that ensure that NFκB dependent transcription is only transiently activated upon stimulation [22]. Enhanced nuclear localisation of NFκB in endothelium in rheumatoid arthritis and Crohn’s disease has been reported [23,24]. Besides inducing a pro-inflammatory status, NFκB signalling also leads to the expression of so called protective

genes. It is assumed that for the purpose of maintaining integrity, these protective genes prevent the endothelium from going into apoptosis upon activation. At the same time, they down-regulate the endothelial pro-inflammatory response. Protective genes that are expressed by endothelium under stress conditions include the zinc finger protein A20 and the bcl-related gene A1 [25]. Furthermore, inducible hemeoxygenase-1 (HO-1), involved in heme catabolism, was shown to antagonise inflammation by inhibiting NFκB and activated protein-1 (AP-1) transcription factor driven gene expression in human pulmonary artery endothelium [26]. Several reports have also implicated control of COX-2 enzyme activity by HO-1 in endothelial cells, the exact consequences of which on cell function are by now unknown [27,28]. One of the downstream targets of TNFα induced phosphoinositol-3 (PI3) kinase is the serine/threonine protein kinase Akt, which also plays an important role in endothelial cell anti-apoptosis signalling [29]. In bovine carotid artery endothelial cells, for example, PI3 kinase/Akt signalling attenuated caspase-8 activity, whereas MAPK signalling controlled by MAPK-kinase MEK-1 impaired caspase-9 activity [30]. MAPK also becomes activated in endothelium upon TNFα activation. The 3 major MAPK signalling pathways at present known are the extracellular-regulated protein kinase (ERK) pathway, the c-Jun NH2 – terminal kinase (JNK) pathway, and the p38 MAPK pathway. They are involved in a network of signalling routes leading to a variety of cellular responses, including cell proliferation, transformation and


differentiation (ERK regulated), and apoptosis, stress responses and inflammation (JNK and p38 MAPK regulated) [31,32]. Of the MAPK-family, p38 MAPK is involved in TNFα-driven IKK activation, as shown e.g., in HUVEC and in the endothelial cell derived cell line ECV304 [33]. Furthermore, p38 MAPK can control mRNA stability of inducible cytokines TNFα, IL-1 and IL-8 that are short lived and contain an AU-rich element in their 3’-untranslated region that is responsible for their high turnover rate. Upon proper stimulation, p38 MAPK substrate MAPKAP K2 phosphorylates AU-binding proteins, as a result of which the mRNA becomes stabilised [34,35]. In rheumatoid arthritis patients, Schett and colleagues reported the presence of phosphorylated (i.e., activated) p38 MAPK in the endothelial cells in diseased lesions [36]. TNFα induced protein kinase C (PKC) activation is responsible for downstream activation of the two MAPK family members p42 MAPK (ERK2) and p44 MAPK (ERK1) [37]. JNK activation in HUVEC was shown to be controlled by TRAF2 - apoptosis signal regulated kinase1 (ASK1) - stress-activated protein kinase ERK kinase 1 (SEK1) complex, leading to ICAM-1 and VCAM-1 expression [38]. TNFα stimulation of human pulmonary artery endothelial cells can also induce the atypical protein kinase isoenzyme PKCζ, leading to a quick association with ICAM1. As a result, phosphorylation and clustering of cell surface ICAM-1 and activation of endothelial adhesiveness occurred [39]. Other data also implied a critical role of PKCζ in regulating TNFα-induced oxidant generation and in signalling the activation of NFκB and ICAM-1 gene transcription [40]. Consequently, this PKC isoform may also be considered of interest as a target for pharmacological intervention of endothelial cell activation. IL-1 Induced Signalling in Endothelial Cells IL-1 includes two agonists, IL-1α and IL-1β, with IL-1α remaining mostly in the cytosol and associated with the plasma membrane and IL-1β being the cytokine matured by proteolytic enzyme cleavage. Based on sequence homology and the presence of key structural patterns, new members of the IL-1 family were more recently identified [41]. The presence of more than one type of IL-1 molecule implies the existence of more than one type of IL-1 receptor. Studies indeed have shown the presence of two types of receptors (IL-1R), of which IL-1RI binds IL-1α with higher affinity than IL-1β. In contrast, IL-1RII binds IL-1β more avidly than IL-1α. Human endothelial cells exclusively express IL1RI, indicating that IL-1RII is dispensable for IL-1 signalling in this cell type [42]. The cell activation pathways mediated by IL-1 signal transduction overlap to a large extent with the TNFα activation pathway (Fig. 2) [16,37]. When IL-1β, hereafter referred to as IL-1, binds IL-1RI, intracellularly located accessory protein AcP increases the binding affinity of interleukin to the receptor. Through activation of IL-1RIassociated kinases or IRAKs, various adaptor proteins are subsequently recruited to activate NFκB inducing kinase NIK. From this point downward the NFκB pathway proceeds as described for TNFα activation [43]. IL-1 induced


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hydrolysis of sphingomyelin to ceramide also facilitates signalling via NFκB [44]. Furthermore, in human pulmonary vascular endothelium, IL-1 induced IL-8 mRNA and protein expression could be attributed to p38 MAPK activity [45]. IL-1 induced IL-6, COX-2, collagenase-1, and stromelysin-1 expression were also shown to be p38 MAPK controlled in HUVEC [46]. One of the similarities between TNFα and IL-1 induced gene expression comprises the upregulation of the protective gene A20. This gene encodes a zinc finger protein, which in the case of IL-1 induced expression was able to interfere with signal transduction upstream of NFκB [47]. Differences between TNFα- and IL-1 controlled signalling pathways and downstream effects in endothelial cells have however also been reported. For example, in rat pulmonary artery endothelial VA cells, the IL-1 induced expression of Manganese superoxide dismutase (MnSOD), an anti-oxidant enzyme acting in the first line of defence to detoxify oxygen radicals, was NFκB dependent. In contrast, TNFα mediated MnSOD expression could be most efficiently blocked by mitochondrial electron transport chain inhibitors and Nacetylcysteine, linking TNFα induced MnSOD expression to reactive oxygen species [48]. In human pulmonary microvascular endothelial cells, IL1 but not TNFα treatment significantly induced both COX-2 mRNA levels and protein activity. This observed differential effect could be explained by the fact that TNFα alone was not able to modify p38 MAPK activity, in the specific cell type studied a prerequisite for COX-2 gene expression induction [49]. These cell type dependent control mechanisms in signal transduction possibly reflect differences in scaffolding protein composition. These scaffolding proteins can be considered as protein kinase ‘insulators’ or docking sites that compartmentalise the various molecules involved in intracellular signalling [50,51]. As a result, duration, intensity and outcome of the signalling process are highly controlled by the scaffolding proteins. Different cell types as well as related cell types located in different sites in the body likely express different scaffolding proteins and hence control their signal transduction relay system in their own way. The observation that a combination of TNFα and IL-1 led to potentiation of COX-2 mRNA and activity induction is of interest considering the fact that during chronic inflammatory diseases multiple cytokines will be locally produced [49]. Also, synergistic effects of TNFα plus IL-1 and other inflammation associated cytokine combinations on HUVEC cell adhesion molecule expression were reported [52]. Especially for the development of new chemical entities (NCEs) aiming to interfere with endothelial cell activation, this complexity of effects mediated by cytokine combinations should be carefully considered in choosing in vitro and in vivo screening tests to study effectiveness of the new compounds. With the advent of advanced techniques like cDNA microarrays, real time RT-PCR, and protein arrays, more detailed information on the complexity of changes in endothelial gene expression patterns has become available. Genes initially not primarily associated with endothelial cell function and responses to inflammatory stimuli have been

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identified. For example, Bandman et al. showed in cultures of coronary artery endothelium that in response to IL-1 and TNFα treatment over 200 genes were regulated. Ninety-nine of these genes were previously not associated with endothelial cell function, but rather associated with leukocyte function or related to antiviral and antibacterial defence [53]. At present, most of these data on complex gene and protein expression patterns have been generated in in vitro systems, and data on their in vivo relevance are awaited. Yet, by choosing experimental strategies that encompass these new techniques, adaptation of our attitude towards looking beyond the well-recognised endothelial gene families is required. Intracellular Signalling Angiogenesis

in

Inflammation-Associated

Although being studied mostly for its role in tumour growth, angiogenesis is also a hallmark of chronic inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease [54,55]. Moreover, in atherosclerosis a relation exists between levels of the main angiogenesis promoting factor VEGF and disease progression [56]. Recombinant as well as endogenous human VEGF could induce atherosclerotic plaque formation and progression in animals [57]. Furthermore, the Angiotensin II regulated angiogenic factor CYR61, a heparin binding, secreted cystein rich protein, colocalised with AngII in small vessels in human atherosclerotic tissues [58]. Prostaglandin PGE2, produced under both normal and various inflammatory conditions, affected endothelial cell adhesion and survival signalling that depends on the angiogenesis associated integrin αvβ3 [59]. These observations, in combination with the finding that NFκB activation can be regulated via αvβ3 [60,61] emphasise the definite association between inflammatory and angiogenic signalling pathways in endothelial cells. Three different cellular processes, i.e., angiogenic sprouting, intussusceptive growth and seeding of endothelial progenitor cells in angiogenic blood vessels, are considered to contribute to neovascularisation [62]. The exact features of the angiogenic sprouting process vary depending on the organ and the location within the organ, and the factors controlling the angiogenic process [63-65]. In general, the first step in this process is the activation of the endothelial cells in response to hypoxic conditions. Production of especially matrix metalloproteinase (MMP)-2, or gelatinase A, by the stimulated endothelial cells subsequently leads to degradation of the surrounding basement membrane [66,67]. Angiopoietins and their receptors Tie-1 and Tie-2 control endothelial sensitivity to other factors by affecting vessel stabilization [68]. Moreover, Angiopoietins were recently shown to control MMP-9 expression by endothelial cells [69]. Degradation of the ECM allows the endothelial cells to migrate into the tissue, where they proliferate and differentiate to form a new vessel. To mature, vessels need to be surrounded by basement membrane and supporting cells including pericytes and/or smooth muscle cells. A complex interplay between growth factors like platelet-derived growth factor (PDGF) and transforming growth factor (TGF-β) guide the attraction of the supporting cells. Finally, a new basement membrane is produced that contains among others

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collagen IV, laminin 8/10, perlecan, nidogen, and fibronectin [70-73]. Besides VEGF and FGF that are discussed below, the Angiopoietins are considered essential factors regulating the angiogenic response. Angiopoietin-1 signals via Angiopoietin receptor Tie-2 to PI3 kinase dependent Akt activation and cell survival [74]. Tie-2 binding of antagonistic Angiopoietin-2, which is primarily expressed in sites of vascular remodelling, on the other hand, destabilises the blood vessels to become prone to the effects of other angiogenic factors. If these factors are absent under these conditions, the endothelial cells go into apoptosis as a consequence of which vessel regression takes place [75,76]. Tie-2 activation may furthermore be the cause of dysfunctional endothelial responses to inflammatory stimuli, as it decreases TNFα driven leukocyte-endothelial interaction through inhibition of NFκB activation via A20 binding inhibitor of NFκB activation-2 [77]. Disease activity in early psoriatic arthritis was recently shown to be associated with a significant increase in Angiopoietin-2 expression, which coincided with increased VEGF and TGFβ levels [78]. VEGF Induced Signalling The five members of the VEGF protein family VEGF-A, -B, -C, -D, -E, and the two placenta growth factors PlGF1 and PlGF2 play a crucial role in the well-orchestrated process leading to new blood vessel formation. The VEGF family members bind in a distinct pattern to three structurally related receptor tyrosine kinases (RTKs), i.e., VEGF receptor-1 (VEGFR-1, also known as Flt-1), VEGFR2 (KDR/Flk-1), and VEGFR-3 (Flt-4), whereas PlGF-1 and 2 can interact with VEGFR-1 [79]. PlGF-2 can furthermore bind to neuropilin-1, which is a non-tyrosine kinase receptor. VEGF and PlGF synergise in inducing angiogenesis [80], and more recently a function for PlGF-1 as vascular survival factor has been proposed [81]. VEGFR-1 and -2 are expressed, at different levels, by endothelial cells, monocytes and smooth muscle cells. VEGFR-3, in the adult, is found mostly in the lymphatic vessels [82,83]. An increase in expression of both VEGF and its receptors has been reported in inflammatory disease [54,55]. VEGF is a multifunctional cytokine that is necessary for normal vasculature development. VEGF variant production can be strongly upregulated via hypoxia inducible factor HIF-1. HIF-1 is a transcription factor that becomes activated under hypoxic conditions as well as under the influence of growth factors such as insulin like growth factor and epidermal growth factor (EGF) [84]. Various other cytokines and growth factors, including PDGF BB, TNFα, TGFβ, and IL-1 stimulate the expression of VEGF, in some cases by increasing VEGF mRNA stability instead of through increased gene transcription [85]. Binding of VEGF to its receptor leads to receptor dimerisation and subsequent kinase activation (Fig. 3). The biological activities of VEGF, including the induction of blood vessel permeability, endothelial cell migration, proliferation, survival and differentiation, are transduced mainly by VEGFR-2 [86]. VEGF activates the MAPK family members ERK1/2 via PKC, thereby stimulating



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Fig. (3). Main signalling relay systems active in endothelial cells upon vascular endothelial cell growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) induced activation. The activation of the cells lead to cellular processes that facilitate blood vessel permeability, endothelial cell proliferation, migration and survival. Abbreviations: NO: nitric oxide; PGI2: prostaglandin I2; PI3K: phosphoinositol-3 kinase; PKC: protein kinase C.

endothelial cell proliferation, whereas p38 MAPK signalling leads to actin reorganisation and cell migration signalling, possibly through nitric oxide (NO) and prostacyclin production [70,86]. Furthermore, increased eNOS protein and NO production in HUVEC stimulated for 24h with VEGF could be efficiently blocked by calcium chelators and PI3 kinase inhibitors [87,88]. While VEGF induced increase in NO production was shown to be (partly) transcriptionally controlled, VEGF induced synthesis of the vascular permeability inducer platelet activating factor (PAF) in bovine aortic endothelial cells (BAEC) was directed via p38 MAPK, controlling the conversion of lysoPAF to PAF. In addition, ERK1/2, PLCγ and PKC were essential in this response, while PI3 kinase acted as a negative regulator of PAF synthesis [89]. The PI3 kinase pathway is furthermore engaged in VEGF signalling leading to vascular hyperpermeability and cell migration via cytoskeletal regulatory protein focal adhesion kinase activation [90,91]. Protection from apoptosis in endothelial cells by VEGF occurs via the induction of expression of anti-apoptotic genes Bcl-2, A1, and survivin, among others [92-94]. Another endothelial cytoprotective phenomenon consists of VEGF driven expression of decay-accelerating factor under the control of PKC mediated p38 MAPK activity. As a result, complement deposition and activation becomes significantly downregulated. This may represent a mechanism by which endothelial cells are protected from complement-mediated lysis during inflammation associated

angiogenesis [95]. VEGF induced cell survival signalling is furthermore activated via the PI3 kinase/Akt route, leading to downregulation of both p38 MAPK activity [96] and caspase-3 activity [97]. The complexity of VEGFR mediated signal transduction in relation to the (patho)physiological effects of VEGF was recently shown in an elegant study by Issbrucker et al. In mice exposed to normobaric hypoxia for 24 hrs, VEGF induced brain vessel hyperpermeability could be counteracted by the treatment with the p38 MAPK inhibitor SB203580. In the chorioallantoic membrane (CAM) assay, however, inhibition of VEGFR signalling controlled p38 MAPK activation led to enhanced angiogenesis, an effect accompanied by prolonged ERK1/2 activity, endothelial cell survival, and activation of plasminogen [98]. Considering the importance of both vascular hyperpermeability and endothelial cell survival in angiogenesis, a concerted action between VEGF and other cytokines likely controls the spatiotemporal functionality of molecular switches like p38 MAPK. VEGFR-2 induced signalling in endothelial cells regulates the expression of an array of genes. Besides the ones involved in endothelial cell migration, proliferation, and survival, they include adhesion molecule CD34, Tie-2 precursor, Angiopoietin-2, heparin binding epidermal growth factor-like growth factor, Mif1, COX-2, and early growth response EGR-3 [99,100]. The in vitro kinetics of induction

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of mRNA levels differed significantly between genes, ranging from as early as 0.5 hrs after start of VEGFR-2 signalling in the case of EGR-3 to peak levels at 24 hrs for Angiopoietin-2 [100]. FGF Induced Signalling The fibroblast growth factor (FGF) family consists of over 20 structurally related, heparin binding polypeptides, of which FGF-1 (acidic FGF) and FGF-2 (basic FGF) are the most extensively studied. In contrast to FGF-1, FGF-2 is ubiquitously present, although its transcription occasionally is regulated, e.g., by mildly oxidised LDL species produced in hypercholesterolemic conditions [101]. Data from cancer research imply a role for FGF-2 and its binding protein FGFBP in the control of the angiogenic switch that occurs during tumour growth progression [102]. The direct role of FGFs on endothelial cell behaviour includes effects on cell proliferation, migration and differentiation, although knowledge on their molecular signal transduction pathways is less detailed compared to those induced by VEGF. Similar to VEGF and Angiopoietin signalling, the cellular effects of FGFs are mediated via receptors containing tyrosine kinase activity. Endothelial cells in vitro express FGFR1 more abundantly than FGFR2 [103]. To form an active ternary complex of FGF and its receptor, heparan sulphate proteins are a prerequisite [104]. Studies with corneal endothelial cells showed that FGF-2induced endothelial cell migration required activation of a PI3 kinase-like enzyme and/or lipoxygenase products [105]. In bovine aortic endothelial cells, arachidonic acid metabolites induced calcium influx and gave rise to cyclooxygenase products, both controlling cell proliferation and motility [106]. The mitogenic effects of FGF-2 were also primarily controlled by PI3 kinase activity via which cell cycle regulating cyclin dependent kinase Cdk4 expression was upregulated [107]. Furthermore, in chorocapillary endothelial cells, FGF-2 induced cell proliferation via ERK1/2 activation [108]. In bovine capillary endothelial cells, p38 MAPK negatively regulated endothelial cell differentiation by decreasing the expression of differentiation-specific proteins such as the Notch ligand Jagged1. Inhibition of p38 MAPK function led to increased vessel formation, although most of them appeared nonfunctional [109]. More recently, a role of Raf kinase in FGF2 based endothelial cell survival signalling has been reported. In HUVEC, FGF-2 activated Raf-1 via p21activated protein kinase-1 phosphorylation, leading to Raf-1 mitochondrial translocation and protection from stress induced apoptosis. In contrast, VEGF activated Raf-1 protected the cells from the extrinsic receptor signalling based pathway of apoptosis via Src kinase and MEK-1. Both pro-angiogenic factors possibly cooperate to alter the threshold of endothelial susceptibility to apoptosis and thereby control resistance of angiogenic endothelium against cytotoxic agents [110]. In HUVEC, FGF-2 induced a complex gene expression pattern that partly overlapped the gene expression pattern induced by VEGF. Genes modulated included extracellular matrix proteins, proteases and protease inhibitors, growth and migration regulators, transcription factors, oxidative stress responders and DNA-repair proteins [99].

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Although the various signalling pathways induced by isolated angiogenic factors are now being unravelled at a fast pace, the in vivo pro-angiogenic microenvironment is complicated in that it contains many cytokines. These cytokines can all affect each other’s action, depending on their concentration and the composition of the cytokine mixture present. Synergistic interaction of FGF-2 with VEGF on the activation of the angiogenesis associated plasminogen system was observed in vitro in bovine aortic and lymphatic endothelial cells [111]. Similarly, VEGF and Angiopoietin-1 individually and in combination affected critical signalling pathways such as p44/42 and p38 MAPK, and Akt. In a study using freshly cut rat aorta rings, time dependent differences in generating distinct signalling patterns of p44/42 MAPK and Akt phosphorylation by VEGF and Ang-1 were observed. Maximal stimulatory effects were obtained by treating the cultures with a VEGF/Ang-1 combination [112]. Control of angiogenesis via non-cytokine proteins has also been established. Recently, Qi et al reported on the ability of tissue inhibitor of metalloproteinases type 3 to inhibit VEGF induced angiogenesis by blocking VEGF binding to VEGFR-2 and consequent inhibition of downstream signalling [113]. Similarly, blockade of FGF-2 binding to its receptor by ganglioside species shed by tumour cells negatively influenced FGF-2 induced endothelial cell proliferation, thereby actively modulating the activity of the angiogenic factor [114]. Furthermore, integrins α5β1 and αvβ3, transmembrane molecules essential for maintaining endothelial cell viability and integrity during FGF- and VEGF-induced angiogenesis [115], represent key-regulators of angiogenesis that can interfere with the molecular processes involved in growth factor induced signalling. Signalling Events Induced by Cell-Cell Interactions Much attention on mechanisms of endothelial cell activation is directed towards cytokine, chemokine and growth factor mediated pathways. Cell-cell interaction based activation, however, is also a relevant route by which endothelial cell activation can be initiated, extended or even brought to a fully advanced state. In one of the early reports on this contact dependent endothelial cell activation, Lou and colleagues showed that phorbol ester activated T lymphocytes could activate human brain microvascular endothelium to express E-selectin, VCAM-1 and ICAM-1. The thus activated endothelium also showed an increase in production of the pro-inflammatory cytokine IL-6 and the chemoattractant IL-8 [116]. Similarly, we and others showed that anti-CD3 / cytokine activated T lymphocytes were capable of activating HUVEC to express E-selectin, VCAM-1 [117], IL-6, IL-8, MCP-1, and Tissue Factor [118] in a contact dependent way. Activation may be partly CD40/CD40L dependent, indicating that in diseases in which the endothelium (over) expresses CD40, ligation with CD40L expressing T cells or activated platelets may be the underlying cause of endothelial activation [119-121]. CD40 ligation furthermore can induce endothelial COX-2 expression [122,123]. Induction of angiogenesis upon crosslinking of endothelial CD40 was observed in human skins engrafted on SCID mice. This cellular reaction coincided


with the expression of the pro-angiogenic factors VEGF, FGF-2, Angiopoietin, endoglin, and Tie-2 [124]. Besides activating endothelium via CD40 ligation, platelets can also induce endothelial COX-2 expression via platelet produced thromboxane A2. Upon TXA2 receptor activation, HUVEC showed increased expression of COX-2 with concomitant production of PGI2, a response controlled by p42/44 MAPK activation [125]. LPS mediated endothelial cell activation can be controlled in a paracrine manner via polymorphomononuclear cell (PMN) or neutrophil NADPHoxidase derived oxidant signalling. Absence of PMNNADPH oxidase either in neutropenic mice or in neutropenic mice replenished with PMN from NADPH-oxidase knockout mice, significantly reduced LPS induced cell activation [126]. Leukocyte-endothelial cell contact based activation is by no means a unidirectional process. Endothelial cells can promote full activation of T lymphocytes via transcostimulatory interactions [127,128]. Although being studied in a transplantation setting, these cell activation and costimulation pathways that involve both transmembrane and soluble factors are likely to prevail in inflammatory processes as well. Endothelial chemokines juxtaposed to VCAM-1 were furthermore shown to be capable of augmenting VLA-4 dependent lymphocyte tethering and adhesion through rapid activation of lymphocyte Gi protein coupled receptor signalling. The subsequent clustering of VLA-4 at the lymphocyte-substrate contact zone, a classical example of insideout signalling via integrins, resulted in enhanced avidity of VLA-4 to VCAM-1 [129]. Additionally, endothelial cell adhesion molecules by themselves can actively participate in cellular signalling. Through cross-linking during adhesive interactions with leukocytes, the cytoplasmic domain of E-selectin is phosphorylated, leading to ERK1/2 activation [130]. Similarly, cross-linking of ICAM-1 and VCAM-1 on HUVEC led to ERK1/2 activation and subsequent IL-8, RANTES and VCAM-1 production [131,132], and small GTPase p21Rho mediated Rac activation facilitating monocyte transmigration, respectively [133]. These data imply that E-selectin, ICAM-1 and VCAM-1 can control, but possibly also initiate endothelial intracellular signalling pathways during inflammation when being cross-linked by their natural counter ligands on the leukocytes. Other Molecules that Induce Signalling in Endothelial Cells Besides TNFα and IL-1, various others molecules can affect endothelial cell function and as such influence inflammatory disease activity. Among them is LPS, a key mediator of sepsis and consequent acute respiratory distress syndrome and multiple organ dysfunction syndrome. Toll like receptor 4 (TLR4) is the transducing receptor for LPS that is constitutively expressed by the endothelium [134]. TLR4 signalling relays via p38 and p42/44 MAPK, NFκB and AP-1, leading to the expression of cell adhesion molecules, chemokines and cytokines, and enzymes including COX-2 [135-137]. Furthermore, IL-6 and its structurally related cytokine family member oncostatin M (OSM) [138], IL-18 [139,140], and monocyte chemoattractant protein-1 (MCP-1) can strongly affect endothelial


19

cell activation status in inflammatory diseases. HUVEC exposure to MCP-1 resulted in rapid phosphorylation of p38 MAPK as well as ERK1/2 and c-Jun N-terminal kinase [141]. The serine protease thrombin also induced p38 MAPK signalling in HUVEC, leading to IL-8 and MCP-1 production via a functional link between the thrombin G protein coupled receptor and p38 MAPK [142]. The acute phase protein C-reactive protein caused cell adhesion molecule and MCP-1 production in HUVEC, with the production of the latter chemokine being controlled by peroxisome proliferator-activated receptor-α or PPAR-α [143]. Cyclophilin A is an intracellular protein that is secreted by smooth muscle cells and macrophages during oxidative stress and LPS activation. In patients with sepsis and rheumatoid arthritis, Cyclophilin A plasma levels were shown to be significantly increased. Jin and colleagues recently demonstrated that this protein could effectively induce p38, ERK1/2, JNK, and NFκB activation and concurrent E-selectin and VCAM-1 expression in HUVEC at nM concentration [144]. Oxidised low-density lipoprotein (ox-LDL) induced abnormalities in endothelial cell function, which may be relevant for the progression of atherosclerosis lesions. In BAEC, 50-200 µg/ml ox-LDL significantly downregulated NO production under basal and thrombin activated conditions which was paralleled by an increase in superoxide (O2·-) radicals [145]. Moreover, within 5 minutes after start of BAEC incubation with ox-LDL, activation of NFκB became evident, which could be blocked by a monoclonal antibody against the endothelial lectin-like ox-LDL receptor1 [146]. Also implicated in cardiovascular diseases is Angiotensin-II (AngII), the dominant effector of the reninangiotensin system. In HUVEC AngII activated NFκB, which was mediated by reactive oxygen species as well as p38 MAPK, leading to cell adhesion molecule expression, among others [147]. Furthermore, AngII regulated the expression of the limiting NAD(P)H oxidase subunit (gp91phox), which is responsible for proatherosclerotic oxidative stress and corresponding superoxide anion formation in endothelial cells [148]. Complement activation product C5a stimulated HUVEC to express a complex gene expression pattern similar to that observed by activation with either TNFα or LPS. Besides the expected upregulation of cell adhesion molecules, cytokines, chemokines and related receptors, downregulation of genes involved in angiogenesis and drug metabolism were observed [149]. Advanced glycosylation end products (AGE) are produced in diabetes, various neurodegenerative diseases, and other chronic diseases including atherosclerosis [150]. The products of non-enzymatic glycation and oxidation of proteins and lipids can activate endothelium via Receptor for AGE (RAGE) to express inflammatory cell adhesion molecules [151]. Both AGE and RAGE were shown to be present in endothelial cells in inflamed rheumatoid synovia [151]. The recently identified high mobility group protein-1 was also capable of activating RAGE to induce proinflammatory signalling, as demonstrated in HMEC-1 microvascular cells in vitro. This latter signal transduction pathway was mediated by MAPK and transcription factors

20


Kuldo et al.

NFκB and Sp1, and was partly due to TNFα autocrine stimulation [152]. Basic knowledge on cytokine, growth factor and cell-cell contact induced cellular responses is an essential starting point for understanding the molecular processes taking place in endothelial cells during an inflammatory response. The challenge for the future is to map the complexity of the signalling cascades in the in vivo situation in the different stages of inflammation. Only by this means, (rational combinations of) relevant molecular targets and (rational combinations of) relevant inhibitors of these targets can be proposed for effective pharmacological intervention strategies. DRUGS AFFECTING ENDOTHELIAL SIGNALLING IN INFLAMMATION

CELL

Several strategies have been explored to interfere with inflammatory processes. These include the development of neutralising antibodies and small chemicals that block adhesion molecules on leukocytes and endothelial cells, and antibodies neutralising pro-inflammatory and pro-angiogenic cytokines and growth factors [13, 153-157]. New in this respect is the development of so called high affinity blockers of cytokines consisting of fusion proteins containing a constant region of IgG and extracellular domains of distinct cytokine receptor components involved in binding and neutralisation of the cytokine [158]. Although many of these strategies were successful in preclinical studies, the outcome of clinical trails has been inconsistent, with the majority of studies showing limited effects. Likely, the redundancy in and overlapping functions of adhesion molecules, cytokines and chemokines for efficient leukocyte recruitment allows for alternative molecules to be used upon blocking a specific one. In addition, since different leukocyte subsets employ different combinations of adhesion molecules and cytokines/chemokines, blockade of recruitment of one subset at a specific stage of disease progression may not significantly alter disease outcome [156]. In the following section, focus will be on studies addressing pharmacological effects of chemical compounds that can interfere with intracellular signalling events associated with cytokine and angiogenic growth factor mediated endothelial cell activation. One important advantage of these compounds is their potential to inhibit the expression of multiple proteins involved in leukocyte recruitment. However, since their target molecules are ubiquitously expressed throughout the body and likely functional in homeostatic control, their lack of cell specificity is considered disadvantageous. Incorporation in cell selective drug targeting constructs may overcome this drawback. For many of the drugs and NCEs discussed, no detailed information on their intracellular availability in vitro and their in vivo pharmacokinetic profile is available, limiting apt appreciation of their potential for future application in the clinic as well as in drug targeting strategies. NFκB Inhibitors The observation that the NFκB cascade is not only regulated by kinases but also by reactive oxygen species

initially led to the study of the effects of anti-oxidants on NFκB driven cell activation. Numerous natural and synthetic antioxidants, including vitamins E and C, curcumin, caffeic acid, capsaicin, dihydropyridines, and pyrollidine dithiocarbamate (PDTC), were shown to inhibit NFκB dependent cell adhesion molecule and/or cytokine expression in endothelial cells in vitro [159]. Vitamin C furthermore could also block a redox-insensitive part of the NFκB activation pathway. This effect, brought about by high millimolar concentrations of Vitamin C, was mediated by activation of the p38 MAPK enzyme, which in turn interfered with IκB phosphorylation [33]. The propenenitrile derivatives BAY 11-7821 and Bay 117083 could also block E-selectin, VCAM-1 and ICAM-1 expression by HUVEC. They irreversibly inhibited TNFα induced IκB phosphorylation, leaving constitutive IκB phosphorylation unaffected. Although they exerted (undesired) activation of different kinases, in vivo reduction of rat carrageenan paw oedema and rat adjuvant arthritis was observed without the occurrence of concurrent toxicity. Whether the pharmacologic activities were associated with interference with reactive oxygen species was not investigated [160]. Cysteine containing derivatives can also inhibit NFκB signalling. Homocysteine has been intensively studied after the discovery that homocysteinuria is associated with an increased incidence of vascular disease. Homocysteine almost completely inhibited the TNFα induced expression of adhesion molecules by HUVEC at millimolar concentration. Data suggest that homocysteine selectively inhibits the NFκB pathway in endothelium without affecting IκB-α phosphorylation and degradation, in contrast to other thiol agents such as PDTC and N-acetylcysteine [161,162]. There is evidence that homocysteine can directly modify proteins, leading to protein damage in the homocysteinyl-ation process. N-acetylcysteine, on the other hand, inhibited activation of c-Jun N-terminal kinase, p38 MAPK, redoxsensitive AP-1 and NFκB transcription factor activities, thereby regulating the expression of numerous genes involved in inflammatory processes [162]. Other drugs effecting NFκB mediated signalling are those inhibiting degradation of phosphorylated IκB by the proteasomes [163], the natural products gliotoxin and parthenolide [164,165], peptides that interact with the NEMO protein in the IKK signalsome [18], and nonsteroidal anti-inflammatory drugs including aspirin that independent from COX inhibition interfere with NFκB activation [147,166]. MOL294, a non-peptide inhibitor of NFκB, acts by inhibiting the cellular redox protein thioredoxin. As a result, TNFα mediated expression of cell adhesion molecules VCAM-1, ICAM-1 and E-selectin by HUVEC was inhibited [167]. Using a cell based reporter assay for NFκB dependent gene activation in HUVEC, Takehana and colleagues identified from a chemical library the pyridine analogue APC0576 that inhibited at low µM concentrations IL-1 induced expression of IL-8 and MCP-1. The molecular basis for this inhibition is of interest, in that it did not interfere with NFκB DNA binding, IκB degradation, or NFκB RelA protein phosphorylation. Possibly it interacts with crucial


transcription co-activators, a mechanism of action that implies possible inhibition of gene expression driven by other transcription factor as well [168]. Some caution should be exerted when applying NFκB inhibitors, since NFκB is not only the driving force for inflammation, but is also a molecular switch controlling the balance between cell death and cell survival [169]. Whereas the phenomenon of activation induced cell death is essential for containment of an immunological response, endothelial cells should refrain from this controlled cell death mechanism. Not only would the endothelial cells become pro-coagulant at an early stage of apoptosis, they can also start to express adhesion molecules under these conditions, thereby possibly augmenting the inflammatory response [170,171]. Furthermore, loss of endothelial cell integrity could eventually lead to loss of whole body integrity. In this respect, targeted delivery of NFκB inhibitors into inflammation associated activated endothelium in which activated NFκB controls cellular activation can be considered a valuable approach in circumventing the undesired side effects. p38 MAPK Inhibitors Most of the kinase inhibitors are ATP-competitive and targeted towards the phosphorylated high activity form of the kinase [172]. Taking into consideration that almost 500 protein kinases are encoded by the human genome, the development of inhibitors specific for a certain (class of) disease associated protein kinase as a treatment modality is an essential prerequisite.


21

administered p.o. in an LPS model at 50 mg/kg in mice, and at 25 mg/kg in rats [180]. The question whether (part of) the anti-inflammatory effects could be attributed to inhibition of endothelial cell activation was not addressed in animal studies reported. Data on a clinical study with RWJ67657, however, shed some light on this issue. In this study, the drug was administered orally to healthy volunteers in doses ranging from 350 to 1400 mg, 30 minutes before a one-minute infusion of LPS. Besides inhibition of neutrophil activation, all doses led to a decrease in circulating soluble ICAM and soluble E-selectin levels [181]. Especially this latter result indicates that inhibition of endothelial cell activation had occurred, as Eselectin production is specific for activated endothelial cells. Whether this endothelial cell inhibitory effect was a direct effect of the drug on endothelial cell intracellular signalling or an indirect effect resulting from inhibition of leukocyte activation is unknown. Similar to NFκB, the role of p38 MAPK in cellular and organ homeostasis is complex, and hence the consequences of interfering with its function on whole body homeostasis unclear. Based on the positive outcome of pre-clinical pharmacological tests, a number of p38 MAPK inhibitors have now advanced to clinical trials [35]. Whether in humans p38 MAPK inhibitor treatment will lead to an increase in macrophage cytokine production and concurrent reduced capacity of bacterial clearance as observed in mice [182] remains to be established. Glucocorticosteroids

One of the most extensively studied groups of drugs that inhibit p38 MAPK is the group of the pyridinylimidazole compounds [173]. SB203580, one of the first analogues described, blocked TNFα induced MCP-1 [174], and VCAM-1 expression by HUVEC, with ICAM-1 levels being unaltered [175]. The effect on VCAM-1 was mRNA independent, i.e., the regulation took place at the posttranscriptional level. IL-8 production by LPS stimulated human pulmonary arterial endothelial cells was blocked in a concentration dependent manner, with 10 µM SB203580 completely inhibiting the production of this cytokine [176]. Similarly, p38 MAPK inhibitor SB202190 could block LPS induced IL-8 production in HUVEC, ranging from 20% inhibitory effect at 1 µM to over 80% inhibition at 100 µM SB202190 [177].

Well-known and extensively used anti-inflammatory therapeutics are the glucocorticosteroids. General features of glucocorticoid action have been divided into non-genomic and genomic effects that occur after binding of the corticosteroid to the glucocorticoid receptor (GR) in the cytoplasm [183]. The thus created monomeric or dimeric complex is released from chaperone proteins, enabling translocation into the nucleus where it can bind to glucocorticoid responsive elements found in promoters of anti-inflammatory genes, among others. The non-genomic effects are relayed by the monomeric glucocorticoid/GR complex that can affect the activity of NFκB and AP-1 components Fos/Jun. This leads to inhibition of expression of genes encoding inflammatory cytokines, receptors, and adhesion molecules [184,185].

P38 MAPK inhibitors have been extensively studied in animal models of arthritis, inflammatory angiogenesis, ischaemia/reperfusion and septic shock (for a concise review on this subject see [35]). In a rat model of arthritis, SB203580 administered p.o. at 60 mg/kg 5 times a week significantly inhibited IL-6 production. In a mouse air pouch granuloma model where angiogenesis occurs alongside the inflammatory process, SB220025 administered at 30 mg/kg twice daily strongly inhibited granuloma associated angiogenesis with parallel attenuation of TNFα and IL-1β production [178]. When administered to mice 30 min before LPS/D-galactosamine challenge as a model of septic shock, 100 mg/kg SB203580 could inhibit TNFα production to 13% of initial levels [179]. P38 MAPK inhibitor RWJ67657 also inhibited TNFα production to a significant extent when

Although glucocorticoids have been mostly studied for their effects on immune cells, some data on their effects on endothelial cell activation are available. Dexamethasone and budesonide inhibited TNFα induced IL-8 synthesis by HUVEC in a dose dependent manner [186]. In porcine aortic endothelial cells, methylprednisolone at a concentration of 200 ng/ml could inhibit LPS and TNFα induced E-selectin and IL-8 mRNA levels. Of note is the observation that in combination with cyclosporin A and Rapamycin, drugs that are well known for their ability to inhibit IL-2 production and IL-2 driven lymphocyte division, this inhibitory effect was significantly enhanced. Also in vivo, pre-treatment of rats with 1 mg/kg methylprednisolone alone or in combination with cyclosporin A at 10 mg/kg, immediately before TNFα infusion was able to downregulate E-selectin

22


Kuldo et al.

mRNA levels in the heart. Possibly, the in vivo effect was mediated by an induction of IκB mRNA, as was shown to occur in vitro [187]. Moreover, evidence of glucocorticoid therapy associated inhibition of NFκB activity and increase in vascular endothelium associated IκB protein in colon in patients with active inflammatory bowel disease has been provided [23;188]. When applied to endothelial cells in vitro, dexamethasone stimulated eNOS activity via direct activation of PI3 kinase and Akt phosphorylation with a maximal 2.7 fold increase at 100 nM drug concentration [189]. In vivo, administration of 40 mg/kg of dexamethasone 1 hr before ischaemia/reperfusion in cremaster muscle prevented local leukocyte adhesion. In addition, in a myocardial infarct model, dexamethasone pre-treatment resulted in a decreased infarct size and tissue oedema, the effects being GR mediated and NO-dependent [189]. A direct effect of the treatment on endothelial cells may be the underlying cause, as in inflammatory bowel disease associated endothelium also an in vivo relation was found between NO production capacity and leukocyte recruitment capacity [190]. In our laboratory, we exploited gene expression array analysis to study the effects of dexamethasone on TNFα induced gene expression in HUVEC (Table 1) [191]. Of the 33 genes that were upregulated more than 5 fold in HUVEC after 6 h incubation with TNFα, 24 were downregulated by at least 50% upon incubation with 100 nM dexamethasone. Responsive genes included Interleukin family members, and growth factor FGF3, PDGF-B and TGFβ2, among others (Table 1). Not only was the gene expression profile dependent on the activation period (50 genes were upregulated after 6h of TNFα activation, whereas only 28 were still elevated after 24h of activation), also the effects of the drug were dependent on the time interval of incubation. Dexamethasone inhibitory action on gene expression was quite pronounced at 6 hrs of incubation, yet only limited effects were observed after 24h incubation, emphasising the sometimes neglected dynamics of drug induced modulation of gene expression. Non-Steroidal Inhibitors

Anti-Inflammatory

Drugs:

COX-2

Whereas cyclooxygenase isoenzyme COX-1 is constitutively expressed in various tissues, COX-2 expression is induced in cells exposed to inflammatory conditions, among others in endothelial cells in angiogenesis and atherosclerosis [192,193]. As such, COX-2 is an attractive target for therapeutic intervention [194]. Over one hundred years old, aspirin is one of the inhibitors of the COX enzyme system therapeutically most extensively applied. In patients at risk of cardiovascular events, aspirin was shown to exert anti-platelet activity. Furthermore, inflammation-induced endothelial dysfunction could be counteracted by aspirin, possibly through modulation of the cytokine cascade [195]. In vitro, aspirin inhibited at 10 µg/ml IL-1 induced Thromboxane A2 production by HUVEC over 80%, while being devoid of effects on PGF1α and PGE2 [196]. In recent years, a new mode of anti-inflammatory action of aspirin has been

unravelled that is dependent on lipid mediators and COX-2 [185]. In the presence of ω-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA), IL-1 activated HUVEC that were co-treated with aspirin converted EPA to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. For this conversion, COX-2 enzyme activity was pivotal. In neutrophils exposed to these HEPE derivatives at low nanomolar concentrations, trihydroxy-containing antiinflammatory lipoxins 15R-LX5 and tri-HEPE derivatives were produced, inhibiting in vitro endothelial transmigration to approximately 50%. In a murine dorsal air-pouch model, 100 ng i.v. injected 18R-tri HEPE derivative inhibited leukocyte trafficking to 50%, an inhibition similar to that observed upon treatment of mice with the aspirin/COX-2 induced anti-inflammatory lipid aspirin-triggered lipoxin (ATL)a [197]. Stable ATL analogues could furthermore inhibit VEGF induced endothelial proliferation at low nanomolar concentration in vitro [198]. Inhibitors of COX-2 activity have drawn special attention within the angiogenesis research field as they potently block endothelial cell pro-angiogenic behaviour in various in vitro and in vivo models, even though they affect enzyme systems downstream of NFκB and MAPK control. Upregulation of COX-2 in endothelial cells is followed by production of PGI2 and PGE2. These prostaglandins promote the expression of pro-angiogenic factors and support cellular processes leading to endothelial cell proliferation, migration and maturation [199]. Selective inhibition of COX-2 by NS-398 or celecoxib significantly downregulated the synthesis of these prostaglandins with a resulting increase in endothelial cell apoptosis index in FGF-2 induced corneal angiogenesis [192,196]. Various COX-2 specific and COX-1/COX-2 nonspecific inhibitors furthermore suppressed integrin αvβ3 dependent activation of endothelial cells, leading to inhibition of endothelial cell spreading and migration in vitro, and inhibition of angiogenesis in vivo [200]. Production of VEGF and cell proliferation mediated by endothelial αvβ3 engagement could be inhibited with a combination of COX-1 and COX-2 specific inhibitors (SC560 and NS398, respectively) at 1 µM concentration. Both inhibitors could also strongly block αvβ3 dependent microtubule formation in vitro. Of note was the complete absence of microtubules upon blockade of COX-1, whereas COX-2 inhibition still allowed some tubes to form [201]. In inflammation associated cardiovascular diseases, COX-2 inhibitors have been studied in clinical trials. In coronary artery disease patients on background therapy with aspirin and statins, treatment with celecoxib significantly improved endothelium dependent vasodilation with concomitant reduction of markers of inflammation and oxidative stress [202]. In hypertensive patients, COX-2 inhibitor treatment reversed endothelial cell dysfunction as reflected by improved brachial artery flow-mediated dilation [203]. In inflammatory animal models, COX inhibitors demonstrated differential effects on inflammation associated COX activity. In a mouse air pouch granuloma model aspirin treatment in high (200 mg/kg) and low (10 mg/kg) dose regimens inhibited PGE 2 levels, whereas the COX-2 specific inhibitor nimesulide at 5 mg/kg increased PGE2 levels with a concurrent increase in granuloma vascularity and dry weight [204]. In contrast, in a rat model of endotoxin shock, the


Table 1.


23

Inhibition of TNFα Induced Gene Expression in HUVEC by 100 nM Dexamethasone [191], as Determined by cDNA Expression Array Analysis Using Atlas Human Cytokine/Cytokine Receptor Array (http://www.clontech.com/atlas/ genelists/index.shtml) TNFα

TNFα + 100 nM Dexamethasone

Gene Adj.Intensitya

Adj.Intensitya

Inhibition

SHH

11

0

100%

FGF3

9

0

100%

CD40-L

14

0

100%

GMF-beta

20

0

100%

CD30L

15

0

100%

RYK

16

0

100%

IL-12B

15

0

100%

IFN-beta

20

0

100%

IL-7

18

1

94%

PDGFB

21

2

90%

TGF-beta2

19

2

89%

NT-4

9

1

89%

CC CKR1

11

2

82%

ERBB-3

11

2

82%

VEGFR3

15

3

80%

IL-2

10

2

80%

IL-1

22

5

77%

L2RA

12

3

75%

Notch4

7

2

71%

IFN-alpha-R

6

2

67%

IL-8

338

140

59%

Lunatic fringe

7

3

57%

GDNPF

7

3

57%

HJ1

62

30

52%

Cells were stimulated for 6 h with 100 ng/ml TNFα. Where appropriate the stimulation was carried out in the presence of 100 nM Dexamethasone. Listed genes showed at least 5 fold increased expression after TNFα stimulation and 50% reduction with the drugs tested. a Adjusted intensity is hybridisation-signal value after subtraction of the background signal.

COX-2 specific inhibitor NS-398 significantly inhibited the increase in PG production [205]. Some of the at first sight conflicting data observed in these studies may find their basis in the fact that COX-2 can also drive the synthesis of anti-inflammatory cyclo-pentenone prostaglandins involved in resolution of inflammation [206]. Possibly, disease model specific kinetics of production of these regulatory PGs hamper interpretation of results presented. In cancer therapy, COX-2 inhibitors sensitise tumour cells to apoptotic signals. Using a systematic chemical approach to synthesise a series of celecoxib and rofecoxib

derivatives, Zhu and colleagues defined a new class of compounds that could induce apoptosis via interference with Akt and ERK2 signalling. This activity was independent of COX-2 inhibitory effects [207]. If induction of endothelial cell apoptosis proofs to be advantageous for resolving chronic inflammatory processes, possibly in the future this new class of compounds may be developed for inflammatory diseases as well. Lack of cell specificity of these, and other drugs discussed, may be circumvented by targeted delivery of the drugs to the inflamed endothelium, as described below.

24


Inhibitors of Kinase Activity to Interfere with Angiogenesis Pharmacologic interference at the level of angiogenesis associated processes has been mostly studied for the treatment of cancer. Therapeutic intervention includes inhibition of matrix metalloproteinase activity, neutralisation of pro-angiogenic cytokines by specific monoclonal antibodies or high affinity decoy receptors, and interference with endothelial cell – extracellular matrix interaction and cell survival signalling. Furthermore, drugs that directly induce endothelial apoptosis, necrosis, or expression of antiangiogenic proteins are effective in inhibiting neovascularisation [157,208,209]. Another rational therapeutic approach for the treatment of angiogenesis is blockade of the intracellular signalling pathways in the pro-angiogenic endothelium. This should lead to inhibition of endothelial cell proliferation, migration and maturation without induction of concomitant apoptotic characteristics and consequent prothrombotic and proadhesive features. Most of the kinase inhibitors have been developed for interfering with tumour cell associated signal transduction, among others those affecting PI3 kinase and protein kinase C [210,211]. For interfering with endothelial cell angiogenic signalling, these inhibitors may prove useful as well. Yet, they have been less extensively studied than the class of RTK inhibitors. This latter group of drugs includes inhibitors of VEGF-, PDGF-, FGF-2 and Tie-2 receptor signalling that act immediately downstream of receptor dimerisation. Although specificity for angiogenesis associated receptor signalling theoretically means that they are devoid of overt toxicity in the body, clinical studies for cancer treatment suggest that drug associated toxicity may be a factor limiting their application. Studies on the exact localisation of the toxicity are awaited, yet heterogeneity in basal endothelial cell signalling status in different microvascular beds is a likely basis for these observations. Almost all reported receptor tyrosine kinase inhibitors interfere with phosphorylation of the kinase activation loop tyrosines. Inhibitors of the inactivated form of the enzyme are considered more potent than inhibitors of the activated kinase [212]. From high-throughput screening of indolinone libraries, SU-5416 and SU-6668 were identified and developed into drugs that are now in clinical testing. SU5416 inhibited VEGF induced HUVEC proliferation in vitro in nM concentrations [213], while SU-6668 exhibited an inhibitory effect on signalling induced by multiple growth factors, including VEGF, FGF-2 and PDGF [214]. The more recently developed indolinone SU-10944 inhibited VEGF induced Tissue Factor production in HUVEC with an IC50 of ~ 100 nM and VEGF induced neovascular growth in rat corneas. Both VEGFR-1 and VEGFR-2 inhibitory activity could be demonstrated [215]. A certain degree of specificity in VEGFR kinase inhibitory activity is exhibited by ZD6474, although more recently an inhibitory effect on EGFR signalling in tumour cells was reported as well [216]. ZD6474 is an anilinoquinazoline with structural overlap with the anilinophthalazine receptor tyrosine kinase inhibitor PTK787 [217,218]. ZD6474 could potently inhibit VEGF induced HUVEC proliferation (IC50 ~ 60 nM) and counteract VEGF

Kuldo et al.

induced hypotension in rats [219]. PTK787 and other anilino-phtalazines inhibited VEGF and FGF-2 induced angiogenesis in vitro in bovina aortic and bovine adrenal cortex-derived microvascular endothelium [220]. Intraperitoneal administration of 50 mg/kg PTK787 twice a day inhibited intimal thickening of coronary arteries in rat allograft arteriosclerosis, which was accompanied by a diminished number of arteries afflicted [221]. Another recently reported drug with proven inhibitory effects on growth factor induced signalling in endothelial cells in vitro and in vivo is CEP-7055, a dimethyl glycine prodrug [222]. The observation that hepatocyte growth factor (HGF) driven angiogenesis is PI3 kinase and MAPK controlled led Fan and colleagues to investigate the effects of PI3 kinase inhibitors wortmannin and LY294002, and MEK inhibitor PD98059 on in vivo HGF induced angiogenesis. Mice implanted with a polyether polyurethane scaffold impregnated with HGF showed a strong reduction of neovessel ingrowth into the scaffold upon local treatment with the drugs [223]. Considering its pronounced expression in lesions of inflammatory disease in endothelial cells that are actively involved in vascular remodelling [224], the development of inhibitors with specificity for Tie-2 is awaited. Possibly, the recently reported nakijiquinone library of RTK inhibitors will provide candidate compounds for this receptor [225]. In the treatment of cancer, inhibitors of kinases also actively influence tumour cell signal transduction. Pharmacological effects reported in tumour models therefore may represent an effect on tumour cells alone, on tumour endothelium alone, or a combined effect on both tumour cells and tumour endothelium. Depending on the expression pattern of the receptors in the various cell types involved in the pathology of inflammatory disorders, either a single or multiple target cell based effect of these drugs can be expected to occur. Anti-angiogenic therapy not only inhibits neovascularisation, but also reverses the anergic status of the angiogenic endothelium towards facilitating leukocyte recruitment. For example, SU-6668 treatment of HUVEC rendered the endothelium more prone to TNFα stimulation to express cell adhesion molecules and to facilitate monocyte transendothelial migration [226]. A potential downside of treating chronic inflammatory conditions with angiogenesis inhibitors may therefore be an increase in leukocyte recruitment due to enhanced responsiveness of the endothelium to pro-inflammatory cytokines. Detailed in vivo studies are needed to further characterise the effects of antiangiogenic drug treatment on these processes. Other Drugs Affecting Angiogenesis Besides the above-described receptor tyrosine kinase inhibitors, a broad array of therapeutics with varying chemical compositions and mechanisms of action has been shown to inhibit pathologic angiogenesis. Without trying to be complete, a few examples of pre-clinical studies on treatment of chronic inflammatory conditions with drugs that exert anti-angiogenic effects are briefly discussed below. Storgard and colleagues reported on the anti-arthritic effects of RGD peptides in rabbits. These peptides interfere



at the level of endothelial cell – ECM interaction through αvβ3 integrin [227]. As such they induce anoikis, i.e., apoptosis induction by loss of interaction with the ECM. Based on the in vitro observation that apoptotic endothelial cells exert pro-coagulant behaviour and start to express adhesion molecules, deterioration of the inflammatory process might have taken place during treatment. Would this have occurred, it clearly did not exert a detrimental effect on the final outcome of the treatment. Angiostatin, the plasminogen cleavage product that exerts anti-angiogenic activity through interference with endothelial cell proliferation and apoptosis, was recently shown to inhibit plaque angiogenesis and atherosclerosis, an effect, which was paralleled by a reduced localisation of macrophages [228]. Both IL-10 and Rapamycin are known for their inhibitory effects on the immune system. They can, however, also influence endothelial cell behaviour in an indirect or direct manner. In mouse hind limb ischaemia, a model of hypoxia and inflammation controlled angiogenesis, the antiinflammatory cytokine IL-10 strongly controlled the angiogenic process and concomitant VEGF production [229]. Concentration as low as 10 ng/ml of Rapamycin could control HUVEC responsiveness to VEGF stimulation [230]. Furthermore, in human pulmonary artery endothelial rapamycin induced the expression of HO-1 [26], which may subsequently counteract pro-inflammatory signal transduction, gene expression and protein functions as described. As is the case with other inhibitors of pro-inflammatory signal transduction, in the majority of in vivo studies no experimental evidence was generated to allow the conclusion that the effects observed were a result of direct interference of the drugs with the inflammatory endothelium. The outcome of the treatment on disease activity is, however, such that drugs with anti-angiogenic effects, being either directly or indirectly inflicted, likely obtain a position as a therapeutic modality for chronic inflammation in the future. A summary of the classes of drugs interfering with endothelial cell behaviour in inflammatory conditions as discussed above is given in Table 2. UNDERSTANDING ENDOTHELIAL PONSES TO DRUGS IN VIVO

CELL

RES-

New technological developments in molecular biology, immunology, pharmacology and other disciplines of (bio) medical research have enabled us to decipher the pathways along which vascular endothelium performs its functions in health and disease. In the majority of studies, however, isolated endothelial cells in static culture systems have been utilised. In such an experimental set up, endothelial cell lines are often the cell type of choice because of ease of handling [245-248]. Yet, they may be less useful for pharmacological studies investigating e.g., complex gene expression profiles, as immortalised cells often have lost specific regulatory functions. Primary endothelial cells freshly isolated from human umbilical cord veins or arteries, human (fore)skin, or other human and non-human organs [4] do not have this drawback. Yet, primary endothelium derived from different

25

blood vessels, and microvascular and macrovascular beds have distinct and characteristic gene expression profiles [249], rationalising careful consideration in choosing the appropriate endothelial cell type for in vitro studies. All in vitro systems, however, have intrinsic disadvantages that can easily lead to misinterpretation of data. One trait of microvascular endothelium in organs and tissues that is difficult to mimic is the precise content of the extracellular matrix and the microenvironment that the endothelium experiences within the organ. These components are of great importance as they regulate endothelial cell behaviour. Similarly, shear stress brought about by laminar or turbulent blood flow, the latter being one of the main factors in initiation of atherosclerosis [6], significantly affects gene expression regulation in endothelial cells [250-252]. The question therefore is justified whether the in vitro observations regarding endothelial cell behaviour and the effects of pharmacological interference in endothelial cell regulatory pathways have a predictive value for in vivo effects. To improve understanding of endothelial cell biology, in our laboratory we developed experimental protocols to study in vivo endothelial cell gene expression profiles in health and disease, and upon pharmacological intervention. From cryostat cut sections of organs and diseased tissues endothelial cells were dissected using laser dissection microscopy. By quantitative RT-PCR, RNA levels of multiple pre-determined genes were established [253,254]. The application of linear amplification protocols will enable us in the future to perform quantitative RT-PCR array and/or microarray analysis on the endothelial cell specific RNAs (Fig. 4). Using this technique, a map of the complex molecular mechanisms underlying endothelial cell (dys)function in vivo can be generated, forming the basis for the identification of new targets for therapy. Besides animal tissues, human tissues can serve to study endothelial cell behaviour in vivo. By this means, interspecies differences in intracellular regulatory pathways as described for isolated endothelial cells [255] may be disclosed. In the field of drug development and target finding this will be an important step forward to bridge the gap between pre-clinical and clinical research. Furthermore, these techniques are critical to study in vivo endothelial cell responses to (targeted) drugs and the relation between endothelial gene expression profiles and disease outcome. VASCULATURE DIRECTED DRUG TARGETING FOR SELECTIVE INTERFERENCE WITH ENDOTHELIAL CELL ACTIVATION IN INFLAMMATION Drug delivery aims at targeting and subsequent delivery of drugs (in)to selected target cell populations to increase drug efficacy while concomitantly reducing toxicity. The word ‘drug’ in this respect encompasses pharmacologically active agents in its broadest sense, including chemicals, toxins, plasmids encoding therapeutic genes and small interfering RNAs, and immune effector cells, among others. Pharmaceutical companies have become strongly interested in drug delivery technologies, as “the synthesis of the medicine is only one part of the drug. Without delivery you just won’t have successful treatment” [256].

26


Table 2.

Kuldo et al.

Drugs Exerting Pharmacological Effects on Endothelial Cell Activation Induced by Pro-Inflammatory or Pro-Angiogenic Stimuli. A Selection of the Drugs Summarised is Discussed in More Detail in the Text

Molecular target

Drug class

Drugs

References

NFκBa

antioxidants

Vitamins C, E Curcumin Caffeic acid phenethyl ester PDTC

[33] [231] [232;233] [139;233;234]

propenenitrile derivatives

BAY 11-7082 BAY 11-7083,BAY 11-7821

[235] [160]

cysteine derivatives

Homocysteine N-acetyl cysteine

[161] [139]

thioredoxin inhibitor

MOL294

[167;236]

various

NSAIDs gliotoxin parthenolide NEMO inhibitory peptide APC0576

[147;166] [237;238] [239] [18] [168]

P38 MAPK

pyridinylimidazole

SB203580 SB202190 SB220025 RWJ67657

[98;174;176] [174;240] [178] [241;242]

Various inflammation associated targets

Glucocorticoids

(Methyl)prednisolone Dexamethasone Budesonide

[187;188] [186;189] [186]

COX-2

NSAIDs

Aspirin NS-398 Celecoxib Nimesulide

[197;243] [196;200;244] [192;203;244] [204]

Receptor Tyrosine Kinase (RTK)

indolinone

SU-5416 SU-6668 SU-10944

[213] [226] [215]

anilino derivatives

ZD6474 PTK787

[216;218;219] [217]

various

CEP-7055 Nakijiquinones (Tie-2 selective inhibitors ?)

[222] [225]

various

PD98059 (MEK inhibitor) RGD-peptides Angiostatin IL-10 Rapamycin

[110;223;244] [227] [228] [229] [26;187;230]

Various angiogenesis inhibitors

a

Abbreviations: COX: cyclooxygenase; IL: interleukin; MAPK: mitogen activated protein kinase; NEMO: NFκB essential modulator; NSAIDs: non-steroidal anti-inflammatory drugs; PDTC: pyrollidine dithiocarbamate

In the following section, we will briefly introduce the basic principles of drug targeting, and subsequently focus on new developments in this area of research for the targeted pharmacological interference of endothelial cell activation in inflammation. For an overview of organ specific drug

targeting strategies employed so far, and for in-depth reviews on specific issues, the reader is referred to [257] and special issues of Advanced Drug Delivery Reviews, the Journal of Controlled Release, and Pharmaceutical Research.



27

Fig. (4). Studying in vivo endothelial cell responses by combining microdissection of endothelium from tissue biopsies with gene expression measurement. To analyse the expression of endothelial genes, total RNA can be isolated from a whole tissue biopsy and applied for quantitative (real time) RT-PCR using endothelial cell specific primers (upper panel). Microarray analysis with RNA isolated from a whole tissue will predominantly demonstrate gene expression patterns in non-endothelial, more abundantly present cell types, with the risk of masking gene expression of the endothelial cells. Alternatively, RNA can be isolated from endothelial cells purified by Laser Dissection Microscopy (LDM) (lower panel). The relatively small amount of RNA isolated from dissected cells can be increased using linear amplification protocols prior to microarray analysis.

The Drug Targeting Concept In the case of passive drug targeting / drug delivery, the drug loaded carrier is taken up by cells not specifically targeted to, including cells of the monocyte-phagocytic system (MPS). This is followed by release of the drug into the cells’ interior and occasionally, the drug is released from the cells into the tissue environment or into the systemic circulation. An example of passive drug targeting is the direct administration of liposomes complexed with a plasmid encoding eNOS into rabbit coronary circulation. As a result of local transfection followed by eNOS expression, ischaemia / reperfusion induced NFκB activity, and VCAM1 and ICAM-1 expression in the microvascular endothelium were reduced. In parallel, intramyocardial neutrophil and T lymphocyte populations were halved in the eNOS transfected hearts [258]. Another example of untargeted, passive drug delivery is the induction of remission of experimental arthritis in rats by single i.v. treatment with prednisolon-phosphate loaded, long circulating PEGylated liposomes. This effect was likely brought about by inhibition of joint associated macrophage activation and availability of the corticosteroid for other inflammatory cells in the inflamed sites upon local release [259].

Active drug targeting / drug delivery aims at the selective delivery of drugs using targeting moieties that specifically bind to epitopes (over)expressed on the target cells only. By this means, only in the target cells intracellular concentrations of the drugs are achieved that are sufficient for a pharmacological effect while unwanted side effects elsewhere in the body are circumvented. For active drug targeting, a pharmacologically active molecule is incorporated in a (macromolecular) construct consisting of a carrier molecule, a homing ligand (this can be a separate entity or an intrinsic part of the carrier), which specifically binds to the target cells, and a drug (Fig. 5). By including drugs in, or attaching drugs to the carrier molecule, the pharmacokinetic characteristics of the drugs are overruled by the pharmacokinetic behaviour of the carrier system. Carrier systems used for this purpose include liposomes, (recombinant) monoclonal antibodies and fragments thereof, soluble polymers, modified plasma proteins, microspheres and nanoparticles, lipoproteins, polymeric micelles, cellular systems, and viral vectors. Targeting moieties encompass peptides, antibodies and antibody fragments, and saccharide based structures, among others that specifically recognise the target epitopes on the cells aimed at [260-265].

28


Kuldo et al.

Fig. (5). Selective delivery of drugs into inflammatory endothelial cells by drug targeting. The drug targeting conjugate consists of a carrier (e.g., a liposome, a nanoparticle, or an adenovirus), a homing ligand that specifically binds to the target epitopes (e.g., E-selectin, ICAM-1) on the endothelium, and a drug (e.g., an inhibitor of intracellular signal transduction or a plasmid encoding a therapeutic gene). After binding to the target epitope, the construct is internalised via the endocytotic pathway, with subsequent release of the drug from the conjugate in the lysosomal compartment of the cell. Availability of the drug in the desired cell compartment (e.g., cytoplasm, nucleus) is a prerequisite for its subsequent pharmacologic effects.

Target Epitopes for Endothelial Drug Targeting in Inflammation For endothelial cell specific drug targeting strategies, accessibility of the target epitopes is not a problem. Therefore, in theory, all carriers mentioned above could be exploited for this purpose. The longer the circulation time of the drug-carrier conjugate, the longer the exposure of the target endothelium to the conjugate will be. This may be advantageous from a point of view that the kinetics of disease induced target epitope expression may spatiotemporally differ between endothelial cell subsets in the diseased site. The choice of the target epitope forms the basis for the selectivity of the drug targeting strategy. Ideally, target epitope expression is restricted to the endothelial cells in the inflamed lesion, thereby circumventing accumulation of the targeted drugs into non-diseased endothelium [266]. At present, one of the few molecules that have been identified to meet this selectivity requirement is E-selectin. E-selectin is absent on normal, resting endothelial cells in the (micro)vascular beds throughout the body but it becomes upregulated during an inflammatory insult. 99Tc-labelled anti-E-selectin Fab’ fragment enabled discrimination between actively inflamed and silent joints in rheumatoid arthritis patients [267]. Similarly, active lesions in patients with inflammatory bowel disease could be imaged with 111 In-labelled anti-E-selectin F(ab’)2 antibodies [268]. Concern exists regarding the presence of soluble E-selectin

in the systemic circulation that can interfere with the homing of E-selectin targeted carriers. Theoretically, soluble Eselectin could function as a link leading to clearance of the E-selectin specific antibody / antibody-drug conjugate and thereby distract the drug delivery construct from its target. The data from these two studies, however, imply that the levels of soluble E-selectin in these patients are too low to affect the homing potential of the antibody molecules. Under pro-inflammatory conditions, endothelial cells also exhibit an increased expression of the cell adhesion molecules VCAM-1 and ICAM-1. Both have been shown to be internalised [269,270]. These molecules, however, do not meet the requirements of unique expression by activated endothelial cells. VCAM-1 is constitutively expressed by a subset of microvascular endothelium in the kidney, whereas a variety of (endothelial) cells in the body express ICAM-1. Yet, VCAM-1 and ICAM-1 may still be exploited as target epitopes for drug targeting purposes, as long as the therapeutic advantages outweigh the undesired effects of delivery of the drugs in non-target cells. αvβ3 integrin is a macrophage associated molecule that mediates virus clearance, among others. During the angiogenic phase of a chronic inflammatory reaction αvβ3 and αvβ5 integrin are upregulated by the endothelium. αv integrin based vascular targeting has most extensively been studied in animal tumour models and cancer patients [271]. The observation that many chronic inflammatory diseases are accompanied by profound neovascularisation intensified


research efforts investigating the potentials of αv integrinbased targeting for the treatment of inflammatory conditions. EDB-Fn is a 91 amino acid domain of Fibronectin, which is inserted in the Fibronectin molecule by alternative splicing. It accumulates around neovascular structures in tissues undergoing angiogenesis. A 123I-labelled dimeric antiEDB-Fn single chain Fv (ScFv) antibody fragment selectively localised in aggressive tumour sites with active angiogenesis [272]. One of the advantages of this target epitope is that the molecule is identical in mouse, rabbit, and human [273]. Consequently, carrier-drug conjugates aimed at this target that are tested in animal models of disease can subsequently be applied for testing in clinical phase. In contrast, antibodies against cell adhesion molecules in one species are rarely recognising adhesion molecules in another species. By applying peptide based homing ligands conjugated to a carrier system, this drawback of antibodybased constructs aimed at adhesion molecules may in theory be circumvented. Using advanced technologies including serial analysis of gene expression and in vivo phage display, new target epitopes and targeting ligands have been put forward. Although these technologies have been most extensively applied to identify tumour growth associated angiogenic endothelium [274,275], also peptides recognising rheumatoid arthritis associated, synovium specific microvascular endothelium and endothelial cells residing in atherosclerotic tissue have been selected [276,277]. Application of these ligands for vascular drug targeting is awaited. Cellular handling of the target epitope determines the fate of the drug-carrier conjugate and of the drug after binding. Effector molecules that need to be locally active when delivered on the outside of the cell include blood coagulation inducing factors [278], and immune effector cells that can lyse the target endothelium [279,280]. These strategies have been exploited for therapy of solid tumours, but are likely not the appropriate effector mechanism for treatment of chronic inflammatory diseases, as they will compromise the blood supply of the inflamed tissue too extensively. More appropriate approaches encompass the delivery of drugs that interfere with intracellular signalling pathways that lead to endothelial cell activation. Intracellularly active drugs include inhibitors of the signal transduction pathways as discussed above, plasmids encoding genes or small interfering RNAs, and RNA aptamers that inhibit target molecules involved in the cell activation processes. During trafficking in the various cell compartments, the active compounds should be released from the endosomal or lysosomal vesicles and find their way to their target (i.e., kinases involved in signal transduction, cytoplasmic receptors, RNAs and / or binding proteins in the nucleus). The significance of proper intracellular trafficking emphasises that the drugs / plasmids have to possess certain characteristics to meet trafficking requirements [281]. Endothelial Drug Targeting Strategies for Treatment of Inflammation It is known for several years that E-selectin is an internalising ligand that routes to the lysosomes [282]. Consequently, it can be exploited for the intracellular


29

delivery of drugs that exert their effects inside the cells, as in the lysosomes break down of the carrier and subsequent release of the drug can take place. One of the first applications of anti-E-selectin antibodies as a homing ligand for the delivery of drugs into endothelial cells was reported by Spragg et al. They conjugated anti-E-selectin antibody to different types of liposomes and demonstrated selective delivery of the cytotoxic agent doxorubicin in activated endothelium, with resultant endothelial cell death [283]. We were the first ones to show that drugs interfering with intracellular signal transduction can be delivered in a pharmacologically active form in endothelial cells without effecting cell viability [284]. We chemically conjugated the glucocorticoid dexamethasone to an anti-E-selectin antibody, resulting in the so-called AbEsel-dexa conjugate. Using an in house prepared antibody against protein-conjugated dexamethasone, we could follow the trafficking of the conjugate inside the HUVEC after binding using ImmunoElectron Microscopy. Both the carrier protein and the AbEseldexa conjugate were routed into multivesicular bodies (Fig. 6). By exploiting 125I-labelled AbEsel-dexa conjugate we could furthermore demonstrate that in time, the conjugate was degraded in the lysosomes of the endothelial cells (Fig. 6) [285]. Pharmacological effects of thus selectively delivered dexamethasone in activated endothelial cells were subsequently identified using cDNA gene expression array analyses combined with gene verification by (quantitative) RT-PCR [191]. We however, also showed using radiolabelled Ab Esel-dexa immunoconjugate and AbEsel-PEGImmunoliposomes that the latter drug targeting construct could theoretically deliver over 1,000 fold more drug into the activated endothelium due to its superior drug loading capacity [286]. AbEsel-dexa immunoconjugate and AbEselPEG-Immunoliposomes homed to endothelium in inflamed tissue upon i.v. injection into mice with a skin delayed type hypersensitivity inflammatory reaction [286]. Both construct are now being studied for their pharmacological effects and effects on disease outcome in mouse models of chronic inflammation. For gene therapy purposes, selective targeting to the endothelium has the advantage of good accessibility for the cargo-vehicles, in contrast to the limited accessibility of e.g., tumour cells or immune effector cells infiltrated into the tissue. Anti-E-selectin antibody - immunoliposomes were shown to be able deliver plasmid DNA into activated endothelium [287]. Also tropism of adenoviruses could be modified using a bispecific antibody combining adenovirus and E-selectin specificity [288]. We recently showed that direct conjugation of the anti-E-selectin antibody to the adenovirus is also possible, resulting in a gene delivery construct with specificity for activated endothelium in vitro and in vivo [289]. Therapeutic genes of interest for delivery into inflammatory endothelium include eNOS to reduce leukocyte infiltration by inhibiting cell adhesion molecule expression, HO-1 and dominant negative IκB to inhibit proinflammatory signalling pathways as discussed. In contrast to the quick lysosomal routing of E-selectin and αvβ3 binding carriers, anti-ICAM-1 antibody coated nanoparticles were retained in TNFα activated HUVEC in early endosomes for a prolonged period of time before trafficking to the lysosomes. When these anti-ICAM-1

30


Kuldo et al.

Fig. (6). Anti-E-selectin antibody – Dexamethasone immunoconjugate is internalised and degraded in activated endothelial cells in vitro. Using Immuno-Electron Microscopy, the immunoconjugate could be found in multivesicular bodies 1 hour after incubation with TNFα activated HUVEC. Both the anti-E-selectin antibody carrier (A) and the Dexamethasone attached to the carrier (B) were found in multivesicular structures representative of endosomal/lysosomal routing of the conjugate [284]. Degradation of the immunoconjugate could be demonstrated using radiolabeled immunoconjugate (C). After loading of TNFα activated HUVEC with 125I-immunoconjugate, in time low molecular weight conjugate degradation products were retrieved from the culture supernatant (closed symbols). Up to 80% of the immunoconjugate was degraded in 16 hrs, while the remainder (open symbols) stayed intact [285].

nanoparticles were loaded with the antioxidant catalase, they exerted effective protection of the HUVEC against oxidative stress [269]. For the delivery of drugs into endothelial cells aimed at angiogenesis related target epitopes, the cyclic RGD peptide specifically binding to αvβ3/αvβ5 integrin has been extensively exploited. Systemic injection of the RGD peptide conjugated to the apoptosis inducing heptapeptide dimer D(KLAKLAK)2 decreased signs of collagen induced arthritis in mice, with a concurrent increase in apoptosis of the synovial blood vessels [290]. The observed effects may also be brought about by a combination of inducing endothelial and macrophage apoptosis, since the low molecular weight RGD-D(KLAKLAK)2 is likely to be taken up by this latter cell type as well. To improve blood circulation time and affinity of binding for its target receptor, RGD peptide and other peptides specific for disease associated target molecules can be conjugated to high molecular weight proteins [291,292]. The resulting drug carriers have the advantage of higher drug loading capacity compared to peptide carriers. Alternatively, conjugation to PEGylated liposomes or artificial virus like particles or viruses can lead to a multivalent construct that binds to its target with high affinity [293]. Many drugs under development are poorly water-soluble, and as such difficult to include in a drug-targeting construct. Immunomicelles may in this respect provide a solution to this problem [294]. Although they have not been tested in a

vascular drug targeting set up, they may be suitable for this purpose, taking into consideration that they can contain a significant amount of drug and do not encounter cellular barriers to find their endothelial target. Furthermore, in the case of poorly internalising carrier molecules selective for endothelial cells in the diseased sites, conjugation with streptavidin may help to facilitate internalisation properties [295]. Alternatively, conjugation onto a protein or polymer backbone may facilitate the desired cross-linking of target molecules to trigger the internalisation process. CONSIDERATIONS FOR THE DEVELOPMENT OF INFLAMMATORY ENDOTHELIAL DRUG TARGETING STRATEGIES Although many drug targeting concepts aimed at inflammatory endothelium have been validated with respect to their targeting specificity, limited data has been provided so far on pharmacological effectiveness of the strategies under study. Not only are data on pharmacokinetic behaviour, essential for establishing proper treatment schedules, scarce [296], immunogenicity of the drug targeting constructs should be carefully considered, as repeated administration protocols for the treatment of chronic inflammatory diseases are possibly elemental for success. Furthermore, in contrast to other cell types studied for drug targeting purposes, endothelial cells do not per se excessively internalise proteins via receptor-mediated endocytosis. Investigations describing internalisation


capacities of endothelial target molecules complexed with drug-carrier conjugates have not been published extensively, and data on in vivo drug-carrier conjugate internalisation capacity of endothelium are lacking. It is however likely that intracellular delivery of sufficient drug molecules can only be achieved by employing drug carriers with a high drug loading capacity. For protein based carrier molecules, high drug loading often changes the pharmacokinetic behaviour and whole body distribution of the carrier due to alterations in physicochemical features. Only if the drug is highly potent, requiring only a few drug molecules to be delivered in the endothelium to induce an effect, or if new conjugation technologies are being employed, protein carriers should be considered for vascular drug targeting. In all other cases, carriers with higher drug loading capacity, among others antibody or peptide targeted liposomes, nanoparticles and polymers, are more likely to lead to a therapeutic success. As with regular drugs, elimination of drug targeting constructs from the body is an essential process to maintain body homeostasis. Drug targeting constructs are often cleared by macrophages in liver and spleen via scavenger receptor systems. If accessible, also other resident macrophages and MPS associated cells throughout the body may scavenge the drug targeting constructs in a non-specific way. If the drug is released in these cells in a pharmacologically active form, the drug may influence the function of these cells. This may bring about undesired or at first sight inexplicable side effects, both locally as well as systemically since resident macophages can be a source of a variety of cytokines.


31

effects in vivo. Using double-fluorescence immunostaining, Solorzano and colleagues demonstrated that phosphorylated Erk and Akt were constitutively expressed in endothelial cells in liver tumour metastases, and that SU-6668 treatment blocked activation of these signalling intermediates in the endothelium [298]. Ideally, for all proteins involved in intracellular signalling these kinds of detection methods should become available for use on both animal and human tissue biopsies. The rapid unravelling of the molecular processes taking place during endothelial cell activation in chronic inflammatory diseases forms the basis for the development of new chemical entities that can selectively interfere with these processes. An improved understanding of the challenges ahead combined with the over four decades of experience in the drug delivery field opens the road to the development of endothelial cell directed therapeutics for clinical use. ABBREVIATIONS AP-1

=

Activated protein-1

ATL

=

Aspirin triggered lipoxin

AGE

=

Advanced glycosylation end products

BAEC

=

Bovine aortic endothelial cells

COX

=

Cyclooxygenase

ECM

=

Extracellular matrix

EGF

=

Epidermal growth factor

CONCLUDING REMARKS

FGF

=

Fibroblast growth factor

Endothelial cells are attractive target cells for pharmacological intervention of chronic inflammatory diseases. They are key players in the disease, easy accessible for (vascular targeted) drugs, and their number is limited compared to other cell types involved in maintenance of disease activity. Some key issues need to be addressed in future research, as they are vital for development of drugs and drug targeting constructs to become successful treatment modalities. The first issue in this respect is endothelial cell heterogeneity. Not only have different endothelial cell subsets in an organ different functions related to organ physiology, they also behave differently under pathophysiological stress [8,63,297]. The microscopically observed phenotypic differences between microvascular beds throughout the body point to differences in function that are poorly understood at present. It is likely that endothelial cell responses to drug treatment are also microvascular bed dependent.

GR

=

Glucocorticoid receptor

HEPE

=

Hydroxy-eicosapentaenoic acid

HO-1

=

Hemeoxygenase-1

HUVEC

=

Human umbilical vein endothelial cells

ICAM-1

=

Intercellular adhesion molecule-1

IκB

=

Inhibitor of κB

IKK

=

IκB kinase

IL (R)

=

Interleukin (receptor)

IRAK

=

IL-1R-associated kinase

LDL

=

Low density lipoprotein

LFA

=

Leukocyte Function Antigen

LPS

=

Lipopolysaccharide

LX

=

Lipoxin

Another issue is species-specificity in endothelial cell responses [255], implying that extrapolation of data obtained in one species to situations in another species is hazardous. Mapping endothelial cell gene and protein expression patterns using technologies applicable to animal and human tissue biopsies is therefore fundamental for the development of new drugs.

MAPK

=

Mitogen activated protein kinase

MCP-1

=

Monocyte chemoattractant protein-1

MPS

=

Monocyte-phagocytic system

NCE

=

New chemical entity

NEMO

=

NFκB essential modulator

For studying the effects of drugs on signalling pathways in endothelium it will be of importance to develop techniques that enable visualisation of the localisation of the

NFκB

=

Nuclear factor κB

NO

=

Nitric oxide

32


Kuldo et al.

(e/i)NOS

=

(Endothelial / inducible) NO synthase

[14]

PDGF

=

Platelet derived growth factor

[15]

PDTC

=

Pyrollidine dithiocarbamate

PI3 kinase =

Phosphoinositol-3 kinase

[16]

PKC

=

Protein kinase C

[17]

PlGF

=

Placental growth factor

RAGE

=

Receptor of advanced glycosylation end products

RTK

=

Receptor tyrosine kinase

[18]

[19]

(Mn)SOD =

(Manganese) Superoxide Dismutase

TGFβ

=

Transforming growth factor β

TLR4

=

Toll like receptor-4

TNFα

=

Tumour necrosis factor α

TNFR

=

Tumour necrosis factor α receptor

VCAM-1

=

Vascular cell adhesion molecule-1

[22]

VLA

=

Very late antigen

[23]

VEGF (R) =

Vascular endothelial growth factor (receptor)

[20] [21]

[24]

REFERENCES [1] [2]

[3] [4]

[5] [6] [7]

[8]

[9] [10]

[11] [12]

[13]

The endothelial cell in health and disease. Vane JR, Born GVR and Welzel D (eds), Schattauer Verlagsgesellschaft mbH, Stuttgart, 1995. Ball HJ, McParland B, Driussi C and Hunt NH. Isolating vessels from the mouse brain for gene expression analysis using laser capture microdissection. Brain Res Brain Res Protoc 2002; 9: 20613. Hellstrom M, Gerhardt H, Kalen M, Li X, Eriksson U, Wolburg H, et al. Lack of pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis. J Cell Biol 2001; 153: 543-53. Griffioen AW and Molema G. Angiogenesis: potentials for pharmacological intervention in the treatment of cancer, cardiovascular diseases and chronic inflammation. Pharmacol Rev 2000; 52: 237-68. Von Andrian UH and Mackay CR. T-cell function and migration. Two sides of the same coin. N Engl J Med 2000; 343: 1020-34. Lusis AJ. Atherosclerosis. Nature 2000; 407: 233-41. Salven P, Hattori K, Heissig B and Rafii S. Interleukin-1alpha promotes angiogenesis in vivo via VEGFR-2 pathway by inducing inflammatory cell VEGF synthesis and secretion. FASEB J 2002; 16: 1471-3. Lim YC, Garcia-Cardena G, Allport JR, Zervoglos M, Connolly AJ, Gimbrone MA, Jr., et al. Heterogeneity of endothelial cells from different organ sites in T-cell subset recruitment. Am J Pathol 2003; 162: 1591-601. Ding Z, Xiong K and Issekutz TB. Chemokines stimulate human T lymphocyte transendothelial migration to utilize VLA-4 in addition to LFA-1. J Leukoc Biol 2001; 69: 458-66. Shang XZ and Issekutz AC. Contribution of CD11a/CD18, CD11b/CD18, ICAM-1 (CD54) and -2 (CD102) to human monocyte migration through endothelium and connective tissue fibroblast barriers. Eur J Immunol 1998; 28: 1970-9. Issekutz AC and Issekutz TB. The role of E-selectin, P-selectin and very late activation antigen-4 in T lymphocyte migration to dermal inflammation. J Immunol 2002; 168: 1934-9. Ludwig RJ, Schultz JE, Boehncke WH, Podda M, Tandi C, Krombach F, et al. Activated, not resting, platelets increase leukocyte rolling in murine skin utilizing a distinct set of adhesion molecules. J Invest Dermatol 2004; 122: 830-6. Palladino MA, Bahjat FR, Theodorakis EA and Moldawer LL. Anti-TNF-alpha therapies: the next generation. Nat Rev Drug Discov 2003; 2: 736-46.

[25] [26]

[27]

[28]

[29] [30]

[31] [32] [33] [34] [35] [36]

[37]

Hallenbeck JM. The many faces of tumour necrosis factor in stroke. Nat Med 2002; 8: 1363-8. Itoh T and Arai KI. Mechanisms of signal transduction. In: The Cytokine Network and Immune Function,(Ed. Theze Jacques), Oxford University Press, Oxford; 1999 pp. 169-90. May MJ and Ghosh S. Signal transduction through NF-kappa B. Immunol Today 1998; 19: 80-8. Yamamoto Y, Verma UN, Prajapati S, Kwak YT and Gaynor RB. Histone H3 phosphorylation by IKK-alpha is critical for cytokineinduced gene expression. Nature 2003; 423: 655-9. May MJ, D'Acquisto F, Madge LA, Glockner J, Pober JS and Ghosh S. Selective inhibition of NF-kappaB activation by a peptide that blocks the interaction of NEMO with the IkappaB kinase complex. Science 2000; 289: 1550-4. Chen XL, Zhang Q, Zhao R, Ding X, Tummala PE and Medford RM. Rac1 and superoxide are required for the expression of cell adhesion molecules induced by tumour necrosis factor-alpha in endothelial cells. J Pharmacol Exp Ther 2003; 305: 573-80. Pahl HL. Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene 1999; 18: 6853-66. Viemann D, Goebeler M, Schmid S, Klimmek K, Sorg C, Ludwig S, et al. Transcriptional profiling of IKK2/NF-kappa B- and p38 MAP kinase-dependent gene expression in TNF-alpha-stimulated primary human endothelial cells. Blood 2004; 103: 3365-73. Makarov SS. NF-kappaB as a therapeutic target in chronic inflammation: recent advances. Mol Med Today 2000; 6: 441-8. Thiele K, Bierhaus A, Autschbach F, Hofmann M, Stremmel W, Thiele H, et al. Cell specific effects of glucocorticoid treatment on the NF- kappaBp65/IkappaBalpha system in patients with Crohn's disease. Gut 1999; 45: 693-704. Marok R, Winyard PG, Coumbe A, Kus ML, Gaffney K, Blades S, et al. Activation of the transcription factor nuclear factor-kappaB in human inflamed synovial tissue. Arthritis Rheum 1996; 39: 583-91. Stroka DM, Badrichani AZ, Bach FH and Ferran C. Overexpression of A1, an NF-kappaB-inducible anti-apoptotic bcl gene, inhibits endothelial cell activation. Blood 1999; 93: 3803-10. Visner GA, Lu F, Zhou H, Liu J, Kazemfar K and Agarwal A. Rapamycin induces heme oxygenase-1 in human pulmonary vascular cells: implications in the antiproliferative response to rapamycin. Circulation 2003; 107: 911-6. Li Volti G, Seta F, Schwartzman ML, Nasjletti A and Abraham NG. Heme oxygenase attenuates angiotensin II-mediated increase in cyclooxygenase-2 activity in human femoral endothelial cells. Hypertension 2003; 41: 715-9. Kushida T, Li VG, Quan S, Goodman A and Abraham NG. Role of human heme oxygenase-1 in attenuating TNF-alpha-mediated inflammation injury in endothelial cells. J Cell Biochem 2002; 87: 377-85. Gabay C. Cytokine inhibitors in the treatment of rheumatoid arthritis. Expert Opin Biol Ther 2002; 2: 135-49. Zhang L, Himi T, Morita I and Murota S. Inhibition of phosphatidylinositol-3 kinase/Akt or mitogen-activated protein kinase signaling sensitizes endothelial cells to TNF-alpha cytotoxicity. Cell Death Differ 2001; 8: 528-36. Herlaar E and Brown Z. p38 MAPK signalling cascades in inflammatory disease. Mol Med Today 1999; 5: 439-47. English JM and Cobb MH. Pharmacological inhibitors of MAPK pathways. Trends Pharmacol Sci 2002; 23: 40-5. Bowie AG and O'Neill LA. Vitamin C inhibits NF-kappa B activation by TNF via the activation of p38 mitogen-activated protein kinase. J Immunol 2000; 165: 7180-8. Kotlyarov A, Neininger A, Schubert C, Eckert R, Birchmeier C, Volk HD, et al. MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis. Nat Cell Biol 1999; 1: 94-7. Kumar S, Boehm J and Lee JC. p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases. Nat Rev Drug Discov 2003; 2: 717-26. Schett G, Tohidast-Akrad M, Smolen JS, Schmid BJ, Steiner CW, Bitzan P, et al. Activation, differential localization and regulation of the stress- activated protein kinases, extracellular signalregulated kinase, c-JUN N-terminal kinase and p38 mitogenactivated protein kinase, in synovial tissue and cells in rheumatoid arthritis. Arthritis Rheum 2000; 43: 2501-12. May MJ, Wheeler-Jones CP, Houliston RA and Pearson JD. Activation of p42mapk in human umbilical vein endothelial cells


[38]

[39]

[40]

[41]

[42] [43] [44]

[45]

[46]

[47] [48]

[49]

[50] [51] [52]

[53]

[54] [55]

[56] [57]

by interleukin-1 alpha and tumour necrosis factor-alpha. Am J Physiol 1998; 274: C789-C798. Yoshizumi M, Fujita Y, Izawa Y, Suzaki Y, Kyaw M, Ali N, et al. Ebselen inhibits tumour necrosis factor-alpha-induced c-Jun Nterminal kinase activation and adhesion molecule expression in endothelial cells. Exp Cell Res 2004; 292: 1-10. Javaid K, Rahman A, Anwar KN, Frey RS, Minshall RD and Malik AB. Tumour Necrosis Factor-{alpha} Induces Early-Onset Endothelial Adhesivity by Protein Kinase C{zeta}-Dependent Activation of Intercellular Adhesion Molecule-1. Circ Res 2003; 92: 1089-97. Rahman A, Anwar KN and Malik AB. Protein kinase C-zeta mediates TNF-alpha-induced ICAM-1 gene transcription in endothelial cells. Am J Physiol Cell Physiol 2000; 279: C906C914. Debets R, Timans JC, Homey B, Zurawski S, Sana TR, Lo S, et al. Two novel IL-1 family members, IL-1 delta and IL-1 epsilon, function as an antagonist and agonist of NF-kappa B activation through the orphan IL-1 receptor-related protein 2. J Immunol 2001; 167: 1440-6. Mantovani A, The interleukin-1 system. In: The Cytokine Network and Immune Functions,(Ed. Theze Jacques), Oxford University Press, Oxford; 1999 pp. 85-96.. O'Neill LA and Dinarello CA. The IL-1 receptor/toll-like receptor superfamily: crucial receptors for inflammation and host defense. Immunol Today 2000; 21: 206-9. Masamune A, Igarashi Y and Hakomori S. Regulatory role of ceramide in interleukin (IL)-1 beta-induced E-selectin expression in human umbilical vein endothelial cells. Ceramide enhances IL-1 beta action, but is not sufficient for E-selectin expression. J Biol Chem 1996; 271: 9368-75. Hashimoto S, Matsumoto K, Gon Y, Maruoka S, Takeshita I, Hayashi S, et al. p38 Mitogen-activated protein kinase regulates IL-8 expression in human pulmonary vascular endothelial cells. Eur Respir J 1999; 13: 1357-64. Ridley SH, Sarsfield SJ, Lee JC, Bigg HF, Cawston TE, Taylor DJ, et al. Actions of IL-1 are selectively controlled by p38 mitogenactivated protein kinase: regulation of prostaglandin H synthase-2, metalloproteinases and IL-6 at different levels. J Immunol 1997; 158: 3165-73. Heyninck K and Beyaert R. The cytokine-inducible zinc finger protein A20 inhibits IL-1-induced NF- kappaB activation at the level of TRAF6. FEBS Lett 1999; 442: 147-50. Rogers RJ, Monnier JM and Nick HS. Tumour necrosis factoralpha selectively induces MnSOD expression via mitochondria-tonucleus signaling, whereas interleukin-1beta utilizes an alternative pathway. J Biol Chem 2001; 276: 20419-27. Said FA, Werts C, Elalamy I, Couetil JP, Jacquemin C and Hatmi M. TNF-alpha, inefficient by itself, potentiates IL-1beta-induced PGHS-2 expression in human pulmonary microvascular endothelial cells: requirement of NF-kappaB and p38 MAPK pathways. Br J Pharmacol 2002; 136: 1005-14. Pouyssegur J, Volmat V and Lenormand P. Fidelity and spatiotemporal control in MAP kinase (ERKs) signalling. Biochem Pharmacol 2002; 64: 755-63. Burack WR and Shaw AS. Signal transduction: hanging on a scaffold. Curr Opin Cell Biol 2000; 12: 211-6. Raab M, Daxecker H, Markovic S, Karimi A, Griesmacher A and Mueller MM. Variation of adhesion molecule expression on human umbilical vein endothelial cells upon multiple cytokine application. Clin Chim Acta 2002; 321: 11-6. Bandman O, Coleman RT, Loring JF, Seilhamer JJ and Cocks BG. Complexity of inflammatory responses in endothelial cells and vascular smooth muscle cells determined by microarray analysis. Ann N Y Acad Sci 2002; 975: 77-90. Paleolog EM. Angiogenesis in rheumatoid arthritis. Arthritis Res 2002; 4 Suppl 3: S81-S90. Kanazawa S, Tsunoda T, Onuma E, Majima T, Kagiyama M and Kikuchi K. VEGF, basic-FGF and TGF-beta in Crohn's disease and ulcerative colitis: a novel mechanism of chronic intestinal inflammation. Am J Gastroenterol 2001; 96: 822-8. Celletti FL, Waugh JM, Amabile PG, Brendolan A, Hilfiker PR and Dake MD. Vascular endothelial growth factor enhances atherosclerotic plaque progression. Nat Med 2001; 7: 425-9. Celletti FL, Hilfiker PR, Ghafouri P and Dake MD. Effect of human recombinant vascular endothelial growth factor165 on


[58]

[59]

[60]

[61]

[62] [63]

[64] [65]

[66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76]

[77] [78] [79] [80]

33

progression of atherosclerotic plaque. J Am Coll Cardiol 2001; 37: 2126-30. Hilfiker A, Hilfiker-Kleiner D, Fuchs M, Kaminski K, Lichtenberg A, Rothkotter HJ, et al. Expression of CYR61, an angiogenic immediate early gene, in arteriosclerosis and its regulation by angiotensin II. Circulation 2002; 106: 254-60. Dormond O, Bezzi M, Mariotii A and Ruegg C. Prostaglandin E2 promotes integrin avb3-dependent endothelial cell adhesion, Racactivation and spreading through cAMP/PKA dependent signalling. J Biol Chem 2002; 277: 45838-46. Scatena M and Giachelli C. The alpha(v)beta3 integrin, NFkappaB, osteoprotegerin endothelial cell survival pathway. Potential role in angiogenesis. Trends Cardiovasc Med 2002; 12: 83-8. Klein S, de Fougerolles AR, Blaikie P, Khan L, Pepe A, Green CD, et al. Alpha 5 beta 1 integrin activates an NF-kappa B-dependent program of gene expression important for angiogenesis and inflammation. Mol Cell Biol 2002; 22: 5912-22. Carmeliet P and Jain RK. Angiogenesis in cancer and other diseases. Nature 2000; 407: 249-57. Pettersson A, Nagy JA, Brown LF, Sundberg C, Morgan E, Jungles S, et al. Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular endothelial growth factor. Lab Invest 2000; 80: 99115. Romano M, Sironi M, Toniatti C, Polentarutti N, Fruscella P, Ghezzi P, et al. Role of IL-6 and its soluble receptor in induction of chemokines and leukocyte recruitment. Immunity 1997; 6: 315-25. Guo P, Xu L, Pan S, Brekken RA, Yang ST, Whitaker GB, et al. Vascular endothelial growth factor isoforms display distinct activities in promoting tumour angiogenesis at different anatomic sites. Cancer Res 2001; 61: 8569-77. Stetler Stevenson WG. Matrix metalloproteinases in angiogenesis: a moving target for therapeutic intervention. J Clin Invest 1999; 103: 1237-41. McCawley LJ and Matrisian LM. Matrix metalloproteinases: multifunctional contributors to tumour progression. Mol Med Today 2000; 6: 149-56. Rafii S, Lyden D, Benezra R, Hattori K and Heissig B. Vascular and haematopoietic stem cells: novel targets for anti-angiogenesis therapy? Nat Rev Cancer 2002; 2: 826-35. Das A, Fanslow W, Cerretti D, Warren E, Talarico N and McGuire P. Angiopoietin/Tek interactions regulate mmp-9 expression and retinal neovascularization. Lab Invest 2003; 83: 1637-45. Matsumoto T and Claesson-Welsh L. VEGF receptor signal transduction. Sci STKE 2001; 2001: RE21. Jones N, Iljin K, Dumont DJ and Alitalo K. Tie receptors: new modulators of angiogenic and lymphangiogenic responses. Nat Rev Mol Cell Biol 2001; 2: 257-67. Carmeliet P. Angiogenesis in health and disease. Nat Med 2003; 9: 653-60. Kalluri R. Basement membranes: structure assembly and role in tumour angiogenesis. Nat Rev Cancer 2003; 3: 422-33. Babaei S, Teichert-Kuliszewska K, Zhang Q, Jones N, Dumont DJ and Stewart DJ. Angiogenic actions of angiopoietin-1 require endothelium-derived nitric oxide. Am J Pathol 2003; 162: 1927-36. Holash J, Maisonpierre PC, Compton D, Boland P, Alexander CR, Zagzag D, et al. Vessel cooption, regression and growth in tumours mediated by angiopoietins and VEGF. Science 1999; 284: 1994-8. Niu Q, Perruzzi C, Voskas D, Lawler J, Dumont DJ and Benjamin LE. Inhibition of Tie-2 Signaling Induces Endothelial Cell Apoptosis, Decreases Akt Signaling and Induces Endothelial Cell Expression of the Endogenous Anti-Angiogenic Molecule, Thrombospondin-1. Cancer Biol Ther 2004; 3. Hughes DP, Marron MB and Brindle NP. The antiinflammatory endothelial tyrosine kinase Tie2 interacts with a novel nuclear factor-kappaB inhibitor ABIN-2. Circ Res 2003; 92: 630-6. Fearon U, Griosios K, Fraser A, Reece R, Emery P, Jones PF, et al. Angiopoietins, growth factors and vascular morphology in early arthritis. J Rheumatol 2003; 30: 260-8. Nagy JA, Dvorak AM and Dvorak HF. VEGF-A(164/165) and PlGF: roles in angiogenesis and arteriogenesis. Trends Cardiovasc Med 2003; 13: 169-75. Carmeliet P, Moons L, Luttun A, Vincenti V, Compernolle V, De Mol M, et al. Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and

34


[81] [82]

[83]

[84] [85] [86] [87]

[88] [89]

[90] [91] [92] [93]

[94] [95]

[96]

[97]

[98]

[99] [100] [101]

[102]

plasma extravasation in pathological conditions. Nat Med 2001; 7: 575-83. Shih SC, Ju M, Liu N and Smith LE. Selective stimulation of VEGFR-1 prevents oxygen-induced retinal vascular degeneration in retinopathy of prematurity. J Clin Invest 2003; 112: 50-7. Barleon B, Reusch P, Totzke F, Herzog C, Keck C, Martiny-Baron G, et al. Soluble VEGFR-1 secreted by endothelial cells and monocytes is present in human serum and plasma from healthy donors. Angiogenesis 2001; 4: 143-54. Parenti A, Brogelli L, Filippi S, Donnini S and Ledda F. Effect of hypoxia and endothelial loss on vascular smooth muscle cell responsiveness to VEGF-A: role of flt-1/VEGF-receptor-1. Cardiovasc Res 2002; 55: 201-12. Pugh CW and Ratcliffe PJ. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med 2003; 9: 677-84. Ferrara N. Molecular and biological properties of vascular endothelial growth factor. J Mol Med 1999; 77: 527-43. Kliche S and Waltenberger J. VEGF receptor signaling and endothelial function. IUBMB Life 2001; 52: 61-6. Papapetropoulos A, Garcia Cardena G, Madri JA and Sessa WC. Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells. J Clin Invest 1997; 100: 3131-9. Hood JD, Meininger CJ, Ziche M and Granger HJ. VEGF upregulates ecNOS message, protein and NO production in human endothelial cells. Am J Physiol 1998; 274: H1054-8. Bernatchez PN, Allen BG, Gelinas DS, Guillemette G and Sirois MG. Regulation of VEGF-induced endothelial cell PAF synthesis: role of p42/44 MAPK, p38 MAPK and PI3K pathways. Br J Pharmacol 2001; 134: 1253-62. Wu MH, Guo M, Yuan SY and Granger HJ. Focal adhesion kinase mediates porcine venular hyperpermeability elicited by vascular endothelial growth factor. J Physiol 2003; 552.3: 691-9. Qi JH and Claesson-Welsh L. VEGF-induced activation of phosphoinositide 3 kinase is dependent on focal adhesion kinase. Exp Cell Res 2001; 263: 173-82. Gerber HP, Dixit V and Ferrara N. Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells. J Biol Chem 1998; 273: 13313-6. Nor JE, Christensen J, Mooney DJ and Polverini PJ. Vascular endothelial growth factor (VEGF)-mediated angiogenesis is associated with enhanced endothelial cell survival and induction of Bcl- 2 expression. Am J Pathol 1999; 154: 375-84. O'Connor DS, Schechner JS, Adida C, Mesri M, Rothermel AL, Li F, et al. Control of apoptosis during angiogenesis by survivin expression in endothelial cells. Am J Pathol 2000; 156: 393-8. Mason JC, Lidington EA, Yarwood H, Lublin DM and Haskard DO. Induction of endothelial cell decay-accelerating factor by vascular endothelial growth factor: a mechanism for cytoprotection against complement-mediated injury during inflammatory angiogenesis. Arthritis Rheum 2001; 44: 138-50. Gratton JP, Morales-Ruiz M, Kureishi Y, Fulton D, Walsh K and Sessa WC. Akt down-regulation of p38 signaling provides a novel mechanism of vascular endothelial growth factor-mediated cytoprotection in endothelial cells. J Biol Chem 2001; 276: 3035965. Yilmaz A, Kliche S, Mayr-Beyrle U, Fellbrich G and Waltenberger J. p38 MAPK inhibition is critically involved in VEGFR-2mediated endothelial cell survival. Biochem Biophys Res Commun 2003; 306: 730-6. Issbrucker K, Marti HH, Hippenstiel S, Springmann G, Voswinckel R, Gaumann A, et al. p38 MAP kinase-a molecular switch between VEGF-induced angiogenesis and vascular hyperpermeability. FASEB J 2003; 17: 262-4. Jih YJ, Lien WH, Tsai WC, Yang GW, Li C and Wu LW. Distinct regulation of genes by bFGF and VEGF-A in endothelial cells. Angiogenesis 2001; 4: 313-21. Abe M and Sato Y. cDNA microarray analysis of the gene expression profile of VEGF- activated human umbilical vein endothelial cells. Angiogenesis 2001; 4: 289-98. Chen CH, Jiang T, Yang JH, Jiang W, Lu J, Marathe GK, et al. Low-density lipoprotein in hypercholesterolemic human plasma induces vascular endothelial cell apoptosis by inhibiting fibroblast growth factor 2 transcription. Circulation 2003; 107: 2102-8. Czubayko F, Liaudet Coopman ED, Aigner A, Tuveson AT, Berchem GJ and Wellstein A. A secreted FGF-binding protein can

Kuldo et al.

[103] [104]

[105] [106]

[107]

[108]

[109]

[110] [111]

[112] [113]

[114]

[115] [116]

[117]

[118]

[119] [120]

[121]

[122]

serve as the angiogenic switch in human cancer. Nat Med 1997; 3: 1137-40. Javerzat S, Auguste P and Bikfalvi A. The role of fibroblast growth factors in vascular development. Trends Mol Med 2002; 8: 483-9. Miao HQ, Ishai Michaeli R, Atzmon R, Peretz T and Vlodavsky I. Sulphate moieties in the subendothelial extracellular matrix are involved in basic fibroblast growth factor sequestration, dimerization and stimulation of cell proliferation. J Biol Chem 1996; 271: 4879-86. Rieck PW, Cholidis S and Hartmann C. Intracellular signaling pathway of FGF-2-modulated corneal endothelial cell migration during wound healing in vitro. Exp Eye Res 2001; 73: 639-50. Antoniotti S, Fiorio PA, Pregnolato S, Mottola A, Lovisolo D and Munaron L. Control of endothelial cell proliferation by calcium influx and arachidonic acid metabolism: a pharmacological approach. J Cell Physiol 2003; 197: 370-8. Lee HT and Kay EP. Regulatory role of PI 3-kinase on expression of Cdk4 and p27, nuclear localization of Cdk4 and phosphorylation of p27 in corneal endothelial cells. Invest Ophthalmol Vis Sci 2003; 44: 1521-8. Zubilewicz A, Hecquet C, Jeanny JC, Soubrane G, Courtois Y and Mascarelli F. Two distinct signalling pathways are involved in FGF2-stimulated proliferation of choriocapillary endothelial cells: a comparative study with VEGF. Oncogene 2001; 20: 1403-13. Matsumoto T, Turesson I, Book M, Gerwins P and Claesson-Welsh L. p38 MAP kinase negatively regulates endothelial cell survival, proliferation and differentiation in FGF-2-stimulated angiogenesis. J Cell Biol 2002; 156: 149-60. Alavi A, Hood JD, Frausto R, Stupack DG and Cheresh DA. Role of Raf in vascular protection from distinct apoptotic stimuli. Science 2003; 301: 94-6. Pepper MS, Mandriota SJ, Jeltsch M, Kumar V and Alitalo K. Vascular endothelial growth factor (VEGF)-C synergizes with basic fibroblast growth factor and VEGF in the induction of angiogenesis in vitro and alters endothelial cell extracellular proteolytic activity. J Cell Physiol 1998; 177: 439-52. Bergers G and Benjamin LE. Tumourigenesis and the angiogenic switch. Nat Rev Cancer 2003; 3: 401-10. Qi JH, Ebrahem Q, Moore N, Murphy G, Claesson-Welsh L, Bond M, et al. A novel function for tissue inhibitor of metalloproteinases-3 (TIMP3): inhibition of angiogenesis by blockage of VEGF binding to VEGF receptor- 2. Nat Med 2003; 9: 407-15. Slevin M, Kumar S, He X and Gaffney J. Physiological concentrations of gangliosides GM1, GM2 and GM3 differentially modify basic-fibroblast-growth-factor-induced mitogenesis and the associated signalling pathway in endothelial cells. Int J Cancer 1999; 82: 412-23. Friedlander M, Brooks PC, Shaffer RW, Kincaid CM, Varner JA and Cheresh DA. Definition of two angiogenic pathways by distinct alpha v integrins. Science 1995; 270: 1500-2. Lou J, Dayer J-M, Grau G-E and Burger D. Direct cell/cell contact with stimulated T lymphocytes induces the expression of cell adhesion molecules and cytokines by human brain microvascular endothelial cells. Eur J Immunol 1996; 26: 3107-13. Molema G, Cohen Tervaert JW, Kroesen BJ, Helfrich W, Meijer DKF and de Leij LFMH. CD3 directed bispecific antibodies induce an increased lymphocyte - endothelial cell interaction in vitro. Br J Cancer 2000; 82: 472-9. Monaco C andreakos E, Young S, Feldmann M and Paleolog E. T cell-mediated signaling to vascular endothelium: induction of cytokines, chemokines and tissue factor. J Leukoc Biol 2002; 71: 659-68. Mach F, Schonbeck U and Libby P. CD40 signaling in vascular cells: a key role in atherosclerosis? Atherosclerosis 1998; 137 Suppl: S89-S95. Zhou L, Stordeur P, de Lavareille A, Thielemans K, Capel P, Goldman M, et al. CD40 engagement on endothelial cells promotes tissue factor-dependent procoagulant activity. Thromb Haemost 1998; 79: 1025-8. Henn V, Slupsky JR, Grafe M, Anagnostopoulos I, Forster R, Muller-Berghaus G, et al. CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature 1998; 391: 591-4. Dongari-Bagtzoglou AI, Thienel U and Yellin MJ. CD40 ligation triggers COX-2 expression in endothelial cells: evidence that


[123]

[124] [125]

[126] [127]

[128]

[129]

[130]

[131]

[132]

[133]

[134]

[135]

[136] [137]

[138]

[139]

[140]

CD40-mediated IL-6 synthesis is COX-2-dependent. Inflamm Res 2003; 52: 18-25. Garlichs CD, Geis T, Goppelt-Struebe M, Eskafi S, Schmidt A, Schulze-Koops H, et al. Induction of cyclooxygenase-2 and enhanced release of prostaglandin E(2) and I(2) in human endothelial cells by engagement of CD40. Atherosclerosis 2002; 163: 9-16. Reinders ME, Sho M, Robertson SW, Geehan CS and Briscoe DM. Proangiogenic function of CD40 ligand-CD40 interactions. J Immunol 2003; 171: 1534-41. Caughey GE, Cleland LG, Gamble JR and James MJ. Upregulation of endothelial cyclooxygenase-2 and prostanoid synthesis by platelets. Role of thromboxane A2. J Biol Chem 2001; 276: 37839-45. Fan J, Frey RS and Malik AB. TLR4 signaling induces TLR2 expression in endothelial cells via neutrophil NADPH oxidase. J Clin Invest 2003; 112: 1234-43. Denton MD, Geehan CS, Alexander SI, Sayegh MH and Briscoe DM. Endothelial cells modify the costimulatory capacity of transmigrating leukocytes and promote CD28-mediated CD4(+) T cell alloactivation. J Exp Med 1999; 190: 555-66. Khayyamian S, Hutloff A, Buchner K, Grafe M, Henn V, Kroczek RA, et al. ICOS-ligand, expressed on human endothelial cells, costimulates Th1 and Th2 cytokine secretion by memory CD4+ T cells. Proc Natl Acad Sci U S A 2002; 99: 6198-203. Grabovsky V, Feigelson S, Chen C, Bleijs DA, Peled A, Cinamon G, et al. Subsecond induction of alpha4 integrin clustering by immobilized chemokines stimulates leukocyte tethering and rolling on endothelial vascular cell adhesion molecule 1 under flow conditions. J Exp Med 2000; 192: 495-506. Hu Y, Szente B, Kiely JM and Gimbrone MA, Jr. Molecular events in transmembrane signaling via E-selectin. SHP2 association, adaptor protein complex formation and ERK1/2 activation. J Biol Chem 2001; 276: 48549-53. Sano H, Nakagawa N, Chiba R, Kurasawa K, Saito Y and Iwamoto I. Cross-linking of intercellular adhesion molecule-1 induces interleukin-8 and RANTES production through the activation of MAP kinases in human vascular endothelial cells. Biochem Biophys Res Commun 1998; 250: 694-8. Lawson C, Ainsworth M, Yacoub M and Rose M. Ligation of ICAM-1 on endothelial cells leads to expression of VCAM-1 via a nuclear factor-kappaB-independent mechanism. J Immunol 1999; 162: 2990-6. van Wetering S, van den BN, van Buul JD, Mul FP, Lommerse I, Mous R, et al. VCAM-1-mediated Rac signaling controls endothelial cell-cell contacts and leukocyte transmigration. Am J Physiol Cell Physiol 2003; 285: C343-C352. Faure E, Thomas L, Xu H, Medvedev A, Equils O and Arditi M. Bacterial lipopolysaccharide and IFN-gamma induce Toll-like receptor 2 and Toll-like receptor 4 expression in human endothelial cells: role of NF-kappa B activation. J Immunol 2001; 166: 201824. Cuschieri J, Gourlay D, Garcia I, Jelacic S and Maier RV. Modulation of endotoxin-induced endothelial function by calcium/calmodulin-dependent protein kinase. Shock 2003; 20: 176-82. Chen JX, Berry LC, Christman BW and Meyrick B. Glutathione mediates LPS-stimulated COX-2 expression via early transient p42/44 MAPK activation. J Cell Physiol 2003; 197: 86-93. Li X, Tupper JC, Bannerman DD, Winn RK, Rhodes CJ and Harlan JM. Phosphoinositide 3 kinase mediates Toll-like receptor 4-induced activation of NF-kappa B in endothelial cells. Infect Immun 2003; 71: 4414-20. Taupin JL, Minvielle S, Theze J, Jacques Y and Moreau JF. The interleukin-6 family of cytokines and their receptors. In: The Cytokine Network and Immune Functions. (Ed. Theze Jacques), Oxford University Press, Oxford; 1999 pp. 31-44. Morel JC, Park CC, Woods JM and Koch AE. A novel role for interleukin-18 in adhesion molecule induction through NF kappa B and phosphatidylinositol (PI) 3-kinase-dependent signal transduction pathways. J Biol Chem 2001; 276: 37069-75. Gerdes N, Sukhova GK, Libby P, Reynolds RS, Young JL and Schonbeck U. Expression of interleukin (IL)-18 and functional IL18 receptor on human vascular endothelial cells, smooth muscle cells and macrophages: implications for atherogenesis. J Exp Med 2002; 195: 245-57.

Current Vascular Pharmacology, 2005, Vol. 3, No. 1 [141] [142]

[143]

[144]

[145]

[146]

[147]

[148]

[149]

[150] [151]

[152]

[153]

[154]

[155] [156] [157]

[158]

35

Werle M, Schmal U, Hanna K and Kreuzer J. MCP-1 induces activation of MAP-kinases ERK, JNK and p38 MAPK in human endothelial cells. Cardiovasc Res 2002; 56: 284-92. Marin V, Farnarier C, Gres S, Kaplanski S, Su MS, Dinarello CA, et al. The p38 mitogen-activated protein kinase pathway plays a critical role in thrombin-induced endothelial chemokine production and leukocyte recruitment. Blood 2001; 98: 667-73. Pasceri V, Cheng JS, Willerson JT, Yeh ET and Chang J. Modulation of C-reactive protein-mediated monocyte chemoattractant protein-1 induction in human endothelial cells by anti-atherosclerosis drugs. Circulation 2001; 103: 2531-4. Jin ZG, Lungu AO, Xie L, Wang M, Wong C and Berk BC. Cyclophilin A Is a Proinflammatory Cytokine that Activates Endothelial Cells. Arterioscler Thromb Vasc Biol 2004; 24: 118191. Cominacini L, Rigoni A, Pasini AF, Garbin U, Davoli A, Campagnola M, et al. The binding of oxidized low density lipoprotein (ox-LDL) to ox-LDL receptor-1 reduces the intracellular concentration of nitric oxide in endothelial cells through an increased production of superoxide. J Biol Chem 2001; 276: 13750-5. Cominacini L, Pasini AF, Garbin U, Davoli A, Tosetti ML, Campagnola M, et al. Oxidized low density lipoprotein (ox-LDL) binding to ox-LDL receptor-1 in endothelial cells induces the activation of NF-kappaB through an increased production of intracellular reactive oxygen species. J Biol Chem 2000; 275: 12633-8. Costanzo A, Moretti F, Burgio VL, Bravi C, Guido F, Levrero M, et al. Endothelial activation by angiotensin II through NFkappaB and p38 pathways: Involvement of NFkappaB-inducible kinase (NIK), free oxygen radicals and selective inhibition by aspirin. J Cell Physiol 2003; 195: 402-10. Rueckschloss U, Quinn MT, Holtz J and Morawietz H. Dosedependent regulation of NAD(P)H oxidase expression by angiotensin II in human endothelial cells: protective effect of angiotensin II type 1 receptor blockade in patients with coronary artery disease. Arterioscler Thromb Vasc Biol 2002; 22: 1845-51. Albrecht EA, Chinnaiyan AM, Varambally S, Kumar-Sinha C, Barrette TR, Sarma JV, et al. C5a-induced gene expression in human umbilical vein endothelial cells. Am J Pathol 2004; 164: 849-59. Yan SF, Ramasamy R, Naka Y and Schmidt AM. Glycation, inflammation and RAGE: a scaffold for the macrovascular complications of diabetes and beyond. Circ Res 2003; 93: 1159-69. Basta G, Lazzerini G, Massaro M, Simoncini T, Tanganelli P, Fu C, et al. Advanced glycation end products activate endothelium through signal-transduction receptor RAGE: a mechanism for amplification of inflammatory responses. Circulation 2002; 105: 816-22. Fiuza C, Bustin M, Talwar S, Tropea M, Gerstenberger E, Shelhamer JH, et al. Inflammation-promoting activity of HMGB1 on human microvascular endothelial cells. Blood 2003; 101: 265260. Haug CE, Colvin RB, Delmonico FL, Auchincloss H Jr. TolkoffRubin N, Preffer FI, et al. A phase I trial of immunosuppression with anti-ICAM-1 (CD54) mAb in renal allograft recipients. Transplantation 1993; 55: 766-72. Kawasaki K, Yaoita E, Yamamoto T, Tamatani T, Miyasaka M and Kihara I. Antibodies against intercellular adhesion molecule-1 and lymphocyte function-associated antigen-1 prevent glomerular injury in rat experimental crescentic glomerulonephritis. J Immunol 1993; 150: 1074-83. Brekke OH and Sandlie I. Therapeutic antibodies for human diseases at the dawn of the twenty- first century. Nat Rev Drug Discov 2003; 2: 52-62. Ulbrich H, Eriksson EE and Lindbom L. Leukocyte and endothelial cell adhesion molecules as targets for therapeutic interventions in inflammatory disease. Trends Pharmacol Sci 2003; 24: 640-7. Ferrara N, Hillan KJ, Gerber HP and Novotny W. Case history: Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov 2004; 3: 391400. Economides AN, Carpenter LR, Rudge JS, Wong V, Koehler-Stec EM, Hartnett C, et al. Cytokine traps: multi-component, highaffinity blockers of cytokine action. Nat Med 2003; 9: 47-52.

36


[159]

[160]

[161]

[162] [163] [164]

[165]

[166] [167]

[168]

[169]

[170] [171]

[172] [173] [174]

[175]

[176]

[177]

[178]

Cominacini L, Pasini AF, Pastorino AM, Garbin U, Davoli A, Rigoni A, et al. Comparative effects of different dihydropyridines on the expression of adhesion molecules induced by TNF-alpha on endothelial cells. J Hypertens 1999; 17: 1837-41. Pierce JW, Schoenleber R, Jesmok G, Best J, Moore SA, Collins T, et al. Novel inhibitors of cytokine-induced IkappaBalpha phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo. J Biol Chem 1997; 272: 21096-103. Roth J, Goebeler M, Ludwig S, Wagner L, Kilian K, Sorg C, et al. Homocysteine inhibits tumour necrosis factor-induced activation of endothelium via modulation of nuclear factor-kappa b activity. Biochim Biophys Acta 2001; 1540: 154-65. Zafarullah M, Li WQ, Sylvester J and Ahmad M. Molecular mechanisms of N-acetylcysteine actions. Cell Mol Life Sci 2003; 60: 6-20. Smolen JS and Steiner G. Therapeutic strategies for rheumatoid arthritis. Nat Rev Drug Discov 2003; 2: 473-88. Kwok BH, Koh B, Ndubuisi MI, Elofsson M and Crews CM. The anti-inflammatory natural product parthenolide from the medicinal herb Feverfew directly binds to and inhibits IkappaB kinase. Chem Biol 2001; 8: 759-66. Lopez-Franco O, Suzuki Y, Sanjuan G, Blanco J, HernandezVargas P, Yo Y, et al. Nuclear factor-kappa B inhibitors as potential novel anti-inflammatory agents for the treatment of immune glomerulonephritis. Am J Pathol 2002; 161: 1497-505. Karin M, Yamamoto Y and Wang QM. The IKK NF-kappa B system: a treasure trove for drug development. Nat Rev Drug Discov 2004; 3: 17-26. Misra-Press A, McMillan M, Cudaback E, Qabar M, Ruan F, Nguyen M, et al. Identification of a novel inhibitor of the NF-kB pathway. Current Medicinal Chemistry-Anti-inflammatory & Antiallergy Agents 2002; 1: 29-39. Takehana K, Konishi A, Oonuki A, Noguchi M, Fujita K, Iino Y, et al. APC0576, a novel inhibitor of NF-kappaB-dependent gene activation, prevents pro-inflammatory cytokine-induced chemokine production in human endothelial cells. Biochem Biophys Res Commun 2002; 293: 945-52. Chen LW, Egan L, Li ZW, Greten FR, Kagnoff MF and Karin M. The two faces of IKK and NF-kappaB inhibition: prevention of systemic inflammation but increased local injury following intestinal ischemia- reperfusion. Nat Med 2003; 9: 575-81. Bombeli T, Schwartz BR and Harlan JM. Endothelial cells undergoing apoptosis become proadhesive for nonactivated platelets. Blood 1999; 93: 3831-8. Hebert MJ, Gullans SR, Mackenzie HS and Brady HR. Apoptosis of endothelial cells is associated with paracrine induction of adhesion molecules: evidence for an interleukin-1beta-dependent paracrine loop. Am J Pathol 1998; 152: 523-32. Alton G, Schwamborn K, Satoh Y and Westwick JK. Therapeutic modulation of inflammatory gene transcription by kinase inhibitors. Expert Opin Biol Ther 2002; 2: 621-32. Salituro FG, Germann UA, Wilson KP, Bemis GW, Fox T and Su MS. Inhibitors of p38 MAP kinase: therapeutic intervention in cytokine- mediated diseases. Curr Med Chem 1999; 6: 807-23. Goebeler M, Kilian K, Gillitzer R, Kunz M, Yoshimura T, Brocker EB, et al. The MKK6/p38 stress kinase cascade is critical for tumour necrosis factor-alpha-induced expression of monocytechemoattractant protein-1 in endothelial cells. Blood 1999; 93: 857-65. Pietersma A, Tilly BC, Gaestel M, de Jong N, Lee JC, Koster JF, et al. p38 mitogen activated protein kinase regulates endothelial VCAM-1 expression at the post-transcriptional level. Biochem Biophys Res Commun 1997; 230: 44-8. Hashimoto S, Gon Y, Matsumoto K, Maruoka S, Takeshita I, Hayashi S, et al. Selective inhibitor of p38 Mitogen-activated protein kinase inhibits lipopolysaccharide-induced Interleukin-8 expression in human pulmonary vascular endothelial cells. J Pharmacol Exp Ther 2000; 293: 370-5. Hippenstiel S, Soeth S, Kellas B, Fuhrmann O, Seybold J, Krull M, et al. Rho proteins and the p38-MAPK pathway are important mediators for LPS-induced interleukin-8 expression in human endothelial cells. Blood 2000; 95: 3044-51. Jackson JR, Bolognese B, Hillegass L, Kassis S, Adams J, Griswold DE, et al. Pharmacological effects of SB 220025, a selective inhibitor of P38 mitogen-activated protein kinase, in

Kuldo et al.

[179]

[180]

[181]

[182]

[183] [184] [185] [186] [187]

[188]

[189]

[190]

[191]

[192]

[193] [194] [195]

[196]

[197]

angiogenesis and chronic inflammatory disease models. J Pharmacol Exp Ther 1998; 284: 687-92. Badger AM, Bradbeer JN, Votta B, Lee JC, Adams JL and Griswold DE. Pharmacological profile of SB 203580, a selective inhibitor of cytokine suppressive binding protein/p38 kinase, in animal models of arthritis, bone resorption, endotoxin shock and immune function. J Pharmacol Exp Ther 1996; 279: 1453-61. Wadsworth SA, Cavender DE, Beers SA, Lalan P, Schafer PH, Malloy EA, et al. RWJ 67657, a potent, orally active inhibitor of p38 mitogen-activated protein kinase. J Pharmacol Exp Ther 1999; 291: 680-7. Fijen JW, Tulleken JE, Kobold AC, De Boer P, Van Der Werf TS, Ligtenberg JJ, et al. Inhibition of p38 mitogen-activated protein kinase: dose-dependent suppression of leukocyte and endothelial response after endotoxin challenge in humans. Crit Care Med 2002; 30: 841-5. Blink B.van den, Juffermans NP, ten Hove T, Schultz MJ, van Deventer SJ, van der PT, et al. p38 mitogen-activated protein kinase inhibition increases cytokine release by macrophages in vitro and during infection in vivo. J Immunol 2001; 166: 582-7. Losel R and Wehling M. Nongenomic actions of steroid hormones. Nat Rev Mol Cell Biol 2003; 4: 46-56. Barnes PJ and Karin M. Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 1997; 336: 1066-71. Gilroy DW, Lawrence T, Perretti M and Rossi AG. Inflammatory Resolution: new opportunities for drug discovery. Nat Rev Drug Discov 2004; 3: 401-16. Nyhlen K, Linden M andersson R and Uppugunduri S. Corticosteroids and interferons inhibit cytokine-induced production of IL-8 by human endothelial cells. Cytokine 2000; 12: 355-60. Charreau B, Coupel S, Goret F, Pourcel C and Soulillou JP. Association of glucocorticoids and cyclosporin A or rapamycin prevents E-selectin and IL-8 expression during LPS- and TNFalpha-mediated endothelial cell activation. Transplantation 2000; 69: 945-53. Ardite E, Panes J, Miranda M, Salas A, Elizalde JI, Sans M, et al. Effects of steroid treatment on activation of nuclear factor kappaB in patients with inflammatory bowel disease. Br J Pharmacol 1998; 124: 431-3. Hafezi-Moghadam A, Simoncini T, Yang E, Limbourg FP, Plumier JC, Rebsamen MC, et al. Acute cardiovascular protective effects of corticosteroids are mediated by non-transcriptional activation of endothelial nitric oxide synthase. Nat Med 2002; 8: 473-9. Binion DG, Rafiee P, Ramanujam KS, Fu S, Fisher PJ, Rivera MT, et al. Deficient iNOS in inflammatory bowel disease intestinal microvascular endothelial cells results in increased leukocyte adhesion. Free Radic Biol Med 2000; 29: 881-8. Asgeirsdottir SA, Kok RJ, Everts M, Meijer DKF and Molema G. Delivery of pharmacologically active dexamethasone into activated endothelial cells by dexamethasone-anti-E-selectin immunoconjugate. Biochem Pharmacol 2003; 65: 1729-39. Leahy KM, Ornberg RL, Wang Y, Zweifel BS, Koki AT and Masferrer JL. Cyclooxygenase-2 inhibition by celecoxib reduces proliferation and induces apoptosis in angiogenic endothelial cells in vivo. Cancer Res 2002; 62: 625-31. Stemme V, Swedenborg J, Claesson H and Hansson GK. Expression of cyclo-oxygenase-2 in human atherosclerotic carotid arteries. Eur J Vasc Endovasc Surg 2000; 20: 146-52. Woods JM, Mogollon A, Amin MA, Martinez RJ and Koch AE. The role of COX-2 in angiogenesis and rheumatoid arthritis. Experimental and Molecular Pathology 2003; 74: 282-90. Kharbanda RK, Walton B, Allen M, Klein N, Hingorani AD, MacAllister RJ, et al. Prevention of inflammation-induced endothelial dysfunction: a novel vasculo-protective action of aspirin. Circulation 2002; 105: 2600-4. Caughey GE, Cleland LG, Penglis PS, Gamble JR and James MJ. Roles of cyclooxygenase (COX)-1 and COX-2 in prostanoid production by human endothelial cells: selective up-regulation of prostacyclin synthesis by COX-2. J Immunol 2001; 167: 2831-8. Serhan CN, Clish CB, Brannon J, Colgan SP, Chiang N and Gronert K. Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and transcellular processing. J Exp Med 2000; 192: 1197-204.

Molecular Pathways of Endothelial Cell Activation [198]

[199] [200]

[201] [202]

[203]

[204] [205] [206] [207]

[208]

[209] [210] [211] [212] [213]

[214]

[215]

[216]

[217]

[218]

Fierro IM, Kutok JL and Serhan CN. Novel lipid mediator regulators of endothelial cell proliferation and migration: aspirintriggered-15R-lipoxin A(4) and lipoxin A(4). J Pharmacol Exp Ther 2002; 300: 385-92. Iniguez MA, Rodriguez A, Volpert OV, Fresno M and Redondo JM. Cyclooxygenase-2: a therapeutic target in angiogenesis. Trends Mol Med 2003; 9: 73-8. Dormond O, Foletti A, Paroz C and Ruegg C. NSAIDs inhibit alpha V beta 3 integrin-mediated and Cdc42/Rac-dependent endothelial-cell spreading, migration and angiogenesis. Nat Med 2001; 7: 1041-7. Murphy JF, Steele C, Belton O and Fitzgerald DJ. Induction of cyclooxygenase-1 and -2 modulates angiogenic responses to engagement of alphavbeta3. Br J Haematol 2003; 121: 157-64. Chenevard R, Hurlimann D, Bechir M, Enseleit F, Spieker L, Hermann M, et al. Selective COX-2 inhibition improves endothelial function in coronary artery disease. Circulation 2003; 107: 405-9. Widlansky ME, Price DT, Gokce N, Eberhardt RT, Duffy SJ, Holbrook M, et al. Short- and long-term COX-2 inhibition reverses endothelial dysfunction in patients with hypertension. Hypertension 2003; 42: 310-5. Gilroy DW, Tomlinson A and Willoughby DA. Differential effects of inhibition of isoforms of cyclooxygenase (COX- 1, COX-2) in chronic inflammation. Inflamm Res 1998; 47: 79-85. Futaki N, Takahashi S, Kitagawa T, Yamakawa Y, Tanaka M and Higuchi S. Selective inhibition of cyclooxygenase-2 by NS-398 in endotoxin shock rats in vivo. Inflamm Res 1997; 46: 496-502. Gilroy DW, Colville-Nash PR, Willis D, Chivers J, Paul-Clark MJ and Willoughby DA. Inducible cyclooxygenase may have antiinflammatory properties. Nat Med 1999; 5: 698-701. Zhu J, Song X, Lin HP, Young DC, Yan S, Marquez VE, et al. Using cyclooxygenase-2 inhibitors as molecular platforms to develop a new class of apoptosis-inducing agents. J Natl Cancer Inst 2002; 94: 1745-57. Huang J, Frischer JS, Serur A, Kadenhe A, Yokoi A, McCrudden KW, et al. Regression of established tumours and metastases by potent vascular endothelial growth factor blockade. Proc Natl Acad Sci U S A 2003; 100: 7785-90. Cristofanilli M, Charnsangavej C and Hortobagyi GN. Angiogenesis modulation in cancer research: novel clinical approaches. Nat Rev Drug Discov 2002; 1: 415-26. Berrie CP. Phosphoinositide 3-kinase inhibition in cancer treatment. Expert Opin Investig Drugs 2001; 10: 1085-98. Goekjian PG and Jirousek MR. Protein kinase C inhibitors as novel anticancer drugs. Expert Opin Investig Drugs 2001; 10: 2117-40. Bilodeau MT, Fraley ME and Hartman GD. Kinase insert domaincontaining receptor kinase inhibitors as anti-angiogenic agents. Expert Opin Investig Drugs 2002; 11: 737-45. Fong TA, Shawver LK, Sun L, Tang C, App H, Powell TJ, et al. SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumour vascularization and growth of multiple tumour types. Cancer Res 1999; 59: 99-106. Laird AD, Vajkoczy P, Shawver LK, Thurnher A, Liang C, Mohammadi M, et al. SU6668 is a potent antiangiogenic and antitumour agent that induces regression of established tumours. Cancer Res 2000; 60: 4152-60. Patel N, Sun L, Moshinsky D, Chen H, Leahy KM, Le P, et al. A selective and oral small molecule inhibitor of vascular epithelial growth factor receptor (VEGFR)-2 and VEGFR-1 inhibits neovascularization and vascular permeability. J Pharmacol Exp Ther 2003; 306: 838-45. Ciardiello F, Caputo R, Damiano V, Caputo R, Troiani T, Vitagliano D, et al. Antitumour effects of ZD6474, a small molecule vascular endothelial growth factor receptor tyrosine kinase inhibitor, with additional activity against epidermal growth factor receptor tyrosine kinase. Clin Cancer Res 2003; 9: 1546-56. Bold G, Altmann KH, Frei J, Lang M, Manley PW, Traxler P, et al. New anilinophthalazines as potent and orally well absorbed inhibitors of the VEGF receptor tyrosine kinases useful as antagonists of tumour-driven angiogenesis. J Med Chem 2000; 43: 2310-23. Hennequin LF, Stokes ES, Thomas AP, Johnstone C, Ple PA, Ogilvie DJ, et al. Novel 4-anilinoquinazolines with C-7 basic side chains: design and structure activity relationship of a series of


[219]

[220] [221] [222]

[223]

[224]

[225]

[226]

[227] [228]

[229]

[230]

[231]

[232]

[233]

[234]

[235] [236]

37

potent, orally active, VEGF receptor tyrosine kinase inhibitors. J Med Chem 2002; 45: 1300-12. Wedge SR, Ogilvie DJ, Dukes M, Kendrew J, Chester R, Jackson JA, et al. ZD6474 inhibits vascular endothelial growth factor signaling, angiogenesis and tumour growth following oral administration. Cancer Res 2002; 62: 4645-55. Tille JC, Wood J, Mandriota SJ, Schnell C, Ferrari S, Mestan J, et al. Vascular endothelial growth factor (VEGF) receptor-2 antagonists inhibit. J Pharmacol Exp Ther 2001; 299: 1073-85. Lemstrom KB, Krebs R, Nykanen AI, Tikkanen JM, Sihvola RK, Aaltola EM, et al. Vascular endothelial growth factor enhances cardiac allograft arteriosclerosis. Circulation 2002; 105: 2524-30. Ruggeri B, Singh J, Gingrich D, Angeles T, Albom M, Chang H, et al. CEP-7055: a novel, orally active pan inhibitor of vascular endothelial growth factor receptor tyrosine kinases with potent antiangiogenic activity and antitumour efficacy in preclinical models. Cancer Res 2003; 63: 5978-91. Sengupta S, Sellers LA, Li RC, Gherardi E, Zhao G, Watson N, et al. Targeting of mitogen-activated protein kinases and phosphatidylinositol 3 kinase inhibits hepatocyte growth factor/scatter factor-induced angiogenesis. Circulation 2003; 107: 2955-61. Uchida T, Nakashima M, Hirota Y, Miyazaki Y, Tsukazaki T and Shindo H. Immunohistochemical localisation of protein tyrosine kinase receptors Tie-1 and Tie-2 in synovial tissue of rheumatoid arthritis: correlation with angiogenesis and synovial proliferation. Ann Rheum Dis 2000; 59: 607-14. Kissau L, Stahl P, Mazitschek R, Giannis A and Waldmann H. Development of natural product-derived receptor tyrosine kinase inhibitors based on conservation of protein domain fold. J Med Chem 2003; 46: 2917-31. Zhang H and Issekutz AC. Down-modulation of monocyte transendothelial migration and endothelial adhesion molecule expression by fibroblast growth factor: reversal by the antiangiogenic agent SU6668. Am J Pathol 2002; 160: 2219-30. Storgard CM, Stupack DG, Jonczyk A, Goodman SL, Fox RI and Cheresh DA. Decreased angiogenesis and arthritic diseasein rabbits treated with an αvβ3 antagonist. J Clin Invest 1999; 103: 47-54. Moulton KS, Vakili K, Zurakowski D, Soliman M, Butterfield C, Sylvin E, et al. Inhibition of plaque neovascularization reduces macrophage accumulation and progression of advanced atherosclerosis. Proc Natl Acad Sci U S A 2003; 100: 4736-41. Silvestre JS, Mallat Z, Duriez M, Tamarat R, Bureau MF, Scherman D, et al. Antiangiogenic effect of interleukin-10 in ischemia-induced angiogenesis in mice hindlimb. Circ Res 2000; 87: 448-52. Guba M, von Breitenbuch P, Steinbauer M, Koehl G, Flegel S, Hornung M, et al. Rapamycin inhibits primary and metastatic tumour growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med 2002; 8: 128-35. Motterlini R, Foresti R, Bassi R and Green CJ. Curcumin, an antioxidant and anti-inflammatory agent, induces heme oxygenase1 and protects endothelial cells against oxidative stress. Free Radic Biol Med 2000; 28: 1303-12. Li D, Saldeen T, Romeo F and Mehta JL. Oxidized LDL upregulates angiotensin II type 1 receptor expression in cultured human coronary artery endothelial cells: the potential role of transcription factor NF-kappaB. Circulation 2000; 102: 1970-6. Rose F, Zeller SA, Chakraborty T, Domann E, Machleidt T, Kronke M, et al. Human endothelial cell activation and mediator release in response to Listeria monocytogenes virulence factors. Infect Immun 2001; 69: 897-905. Aoki M, Nata T, Morishita R, Matsushita H, Nakagami H, Yamamoto K, et al. Endothelial apoptosis induced by oxidative stress through activation of NF-kappaB: antiapoptotic effect of antioxidant agents on endothelial cells. Hypertension 2001; 38: 4855. Hamuro M, Polan J, Natarajan M and Mohan S. High glucose induced nuclear factor kappa B mediated inhibition of endothelial cell migration. Atherosclerosis 2002; 162: 277-87. Henderson WR Jr. Chi EY, Teo JL, Nguyen C and Kahn M. A small molecule inhibitor of redox-regulated NF-kappa B and activator protein-1 transcription blocks allergic airway inflammation in a mouse asthma model. J Immunol 2002; 169: 5294-9.

38


[237]

[238]

[239]

[240]

[241]

[242]

[243]

[244]

[245] [246]

[247] [248]

[249]

[250] [251]

[252]

[253]

[254]

[255]

Wilson SJ, Leone BA anderson D, Manning A and Holgate ST. Immunohistochemical analysis of the activation of NF-kappaB and expression of associated cytokines and adhesion molecules in human models of allergic inflammation. J Pathol 1999; 189: 26572. Farkas S, Herfarth H, Rossle M, Schroeder J, Steinbauer M, Guba M, et al. Quantification of mucosal leucocyte endothelial cell interaction by in vivo fluorescence microscopy in experimental colitis in mice. Clin Exp Immunol 2001; 126: 250-8. Molestina RE, Miller RD, Lentsch AB, Ramirez JA and Summersgill JT. Requirement for NF-kappaB in transcriptional activation of monocyte chemotactic protein 1 by Chlamydia pneumoniae in human endothelial cells. Infect Immun 2000; 68: 4282-8. Nwariaku FE, Chang J, Zhu X, Liu Z, Duffy SL, Halaihel NH, et al. The role of p38 map kinase in tumour necrosis factor-induced redistribution of vascular endothelial cadherin and increased endothelial permeability. Shock 2002; 18: 82-5. Westra J, Kuldo JM, De Boer P, van Rijswijk MH, Molema G and Limburg PC. E-selectin expression and chemokine production in activated endothelial cells are inhibited by p38 MAPK (mitogen activated protein kinase) inhibitor RWJ67657. (Submitted) Kuldo JM, Westra J, Asgeirsdottir SA, Kok RJ, Oosterhuis K, Rots MG, et al. TNFα versus IL-1β induced inflammatory gene expression and the differential effects of NFκB and p38 MAP kinase inhibitors in endothelial cells in vitro. (Submitted) Perretti M, Chiang N, La M, Fierro IM, Marullo S, Getting SJ, et al. Endogenous lipid- and peptide-derived anti-inflammatory pathways generated with glucocorticoid and aspirin treatment activate the lipoxin A4 receptor. Nat Med 2002; 8: 1296-302. Jones MK, Wang H, Peskar BM, Levin E, Itani RM, Sarfeh IJ, et al. Inhibition of angiogenesis by nonsteroidal anti-inflammatory drugs: Insight into mechanisms and implications for cancer growth and ulcer healing. Nat Med 1999; 5: 1418-23. Bouis D, Hospers G, Meijer C, Molema G and Mulder N. Endothelium in vitro: a review of human vascular endothelial cell lines for blood vessel-related research. Angiogenesis 2001; 4: 102. Unger RE, Krump-Konvalinkova V, Peters K and Kirkpatrick CJ. In vitro expression of the endothelial phenotype: comparative study of primary isolated cells and cell lines, including the novel cell line HPMEC-ST1.6R. Microvasc Res 2002; 64: 384-97. Mutin M, Dignat-George F and Sampol J. Immunologic phenotype of cultured endothelial cells: quantitative analysis of cell surface molecules. Tissue Antigens 1997; 50: 449-58. van Leeuwen EBM, Wisman GBA, Cohen Tervaert JW, Palmans LL, van Wijk RT, Veenstra R, et al. An SV40 large T-antigen immortalized human umbilical vein endothelial cell line for antiendothelial cell antibody detection. Clinical Experimental Rheumatology 2001; 19: 283-90. Chi JT, Chang HY, Haraldsen G, Jahnsen FL, Troyanskaya OG, Chang DS, et al. Endothelial cell diversity revealed by global expression profiling. Proc Natl Acad Sci U S A 2003; 100: 106238. Topper JN and Gimbrone MA, Jr. Blood flow and vascular gene expression: fluid shear stress as a modulator of endothelial phenotype. Mol Med Today 1999; 5: 40-6. Bartling B, Tostlebe H, Darmer D, Holtz J, Silber RE and Morawietz H. Shear stress-dependent expression of apoptosisregulating genes in endothelial cells. Biochem Biophys Res Commun 2000; 278: 740-6. Bongrazio M, Baumann C, Zakrzewicz A, Pries AR and Gaehtgens P. Evidence for modulation of genes involved in vascular adaptation by prolonged exposure of endothelial cells to shear stress. Cardiovasc Res 2000; 47: 384-93. Asgeirsdottir SA, Werner N, Harms G, van den Berg A and Molema G. Analysis of in vivo endothelial cell activation applying RT-PCR following endothelial cell isolation by laser dissection microscopy. Ann NY Acad Sci 2002; 973: 586-9. Asgeirsdottir SA, Werner N, Berg Avd, Schraa AJ, Moorlag HE and Molema G. In vivo endothelial cell activation in acute inflammation: expression levels of inflammation-related gene in microdissected endothelial cells. (Submitted) Yao L, Setiadi H, Xia L, Laszik Z, Taylor FB and McEver RP. Divergent inducible expression of P-selectin and E-selectin in mice and primates. Blood 1999; 94: 3820-8.

Kuldo et al. [256] [257] [258]

[259]

[260] [261] [262] [263] [264] [265] [266]

[267]

[268] [269]

[270]

[271] [272]

[273]

[274] [275] [276] [277] [278]

[279]

Basu P. News Feature: Technologies that deliver. Nat Med 2003; 9: 1100-1. Molema G and Meijer DKF, Drug Targeting: Organ-Specific Strategies. Wiley-VCH, Weinheim, 2001. Iwata A, Sai S, Nitta Y, Chen M, Fries-Hallstrand R, Dalesandro J, et al. Liposome-mediated gene transfection of endothelial nitric oxide synthase reduces endothelial activation and leukocyte infiltration in transplanted hearts. Circulation 2001; 103: 2753-9. Metselaar JM, Wauben MH, Wagenaar-Hilbers JP, Boerman OC and Storm G. Complete remission of experimental arthritis by joint targeting of glucocorticoids with long-circulating liposomes. Arthritis Rheum 2003; 48: 2059-66. Maurer N, Fenske DB and Cullis PR. Developments in liposomal drug delivery systems. Expert Opin Biol Ther 2001; 1: 923-47. Molema G. Drug Targeting: basic concepts and novel advances. In: Drug targeting - organ specific strategies, (Eds. Molema G and Meijer DKF), Wiley-VCH, Weinheim, New York; 2001 pp. 1-22. Duncan R. The dawning era of polymer therapeutics. Nat Rev Drug Discov 2003; 2: 347-60. Hudson PJ and Souriau C. Engineered antibodies. Nat Med 2003; 9: 129-34. O'Riordan CR, Song A and Lanciotti J. Strategies to adapt adenoviral vectors for targeted delivery. Methods Mol Med 2003; 76: 89-112. Ehrhardt C, Kneuer C and Bakowsky U. Selectins-an emerging target for drug delivery. Adv Drug Deliv Rev 2004; 56: 527-49. Everts M, Schraa AJ, de Leij LFMH, Meijer DKF and Molema G, Vascular endothelium in inflamed tissue as a target for site selective delivery of drugs. In: Drug Targeting - Organ Specific Strategies, (Eds. Molema G and Meijer DKF), Wiley-VCH, Weinheim, New York; 2001 pp. 171-97. Jamar F, Houssiau FA, Devogelaer JP, Chapman PT, Haskard DO, Beaujean V, et al. Scintigraphy using a technetium 99m-labelled anti-E-selectin Fab fragment in rheumatoid arthritis. Rheumatology (Oxford) 2002; 41: 53-61. Bhatti M, Chapman P, Peters M, Haskard D and Hodgson HJF. Visualizing E-selectin in the detection and evaluation of inflammatory bowel disease. Gut 1998; 43: 40-7. Muro S, Cui X, Gajewski C, Murciano JC, Muzykantov VR and Koval M. Slow intracellular trafficking of catalase nanoparticles targeted to ICAM-1 protects endothelial cells from oxidative stress. Am J Physiol Cell Physiol 2003; 285: C1339-C1347. Ricard I, Payet MD and Dupuis G. VCAM-1 is internalized by a clathrin-related pathways in human endothelial cells but its alpha4beta1 integrin counter-receptor remains associated with the plasma membrane in human T lymphocytes. Eur J Immunol 1998; 28: 1708-18. Sipkins DA, Cheresh DA, Kazemi MR, Nevin LM, Bednarski MD and Li KCP. Detection of tumour angiogenesis in vivo by αvβ3targeted magnetic resonance imaging. Nat Med 1998; 4: 623-6. Santimaria M, Moscatelli G, Viale GL, Giovannoni L, Neri G, Viti F, et al. Immunoscintigraphic detection of the ED-B domain of fibronectin, a marker of angiogenesis, in patients with cancer. Clin Cancer Res 2003; 9: 571-9. Nilsson F, Kosmehl H, Zardi L and Neri D. Targeted delivery of tissue factor to the ED-B domain of fibronectin, a marker of angiogenesis, mediates the infarction of solid tumours in mice. Cancer Res 2001; 61: 711-6. Carson-Walter EB, Watkins DN, Nanda A, Vogelstein B, Kinzler KW and St Croix B. Cell surface tumour endothelial markers are conserved in mice and humans. Cancer Res 2001; 61: 6649-55. Pasqualini R, Arap W and McDonald DM. Probing the structural and molecular diversity of tumour vasculature. Trends Mol Med 2002; 8: 563-71. Lee L, Buckley C, Blades MC, Panayi G, George AJ and Pitzalis C. Identification of synovium-specific homing peptides by in vivo phage display selection. Arthritis Rheum 2002; 46: 2109-20. Houston P, Goodman J, Lewis A, Campbell CJ and Braddock M. Homing markers for atherosclerosis: applications for drug delivery, gene delivery and vascular imaging. FEBS Lett 2001; 492: 73-7. Huang X, Molema G, King S, Watkins L, Edgington TS and Thorpe PE. Tumour infarction in mice by antibody-directed targeting of tissue factor to tumour vasculature. Science 1997; 275: 547-50. Niederman TM, Ghogawala Z, Carter BS, Tompkins HS, Russell MM and Mulligan RC. Antitumour activity of cytotoxic T


[280]

[281]

[282]

[283]

[284]

[285]

[286]

[287] [288]

lymphocytes engineered to target vascular endothelial growth factor receptors. Proc Natl Acad Sci U S A 2002; 99: 7009-14. Schraa AJ, Kok RJ, Botter SM, Withoff S, Meijer DKF, de Leij LFMH, et al. RGD-modified anti-CD3 antibodies redirect cytolytic capacity of cytotoxic T lymphocytes towards alphav-beta3 expressing endothelial cells. Int J Cancer 2004; 112: 279-85. Kok RJ, Ásgeirsdóttir SA and Verweij WR, Development of proteinaceous drug targeting constructs using chemical and recombinant DNA approaches. In: Drug Targeting - Organ specific strategies, (Eds. Molema G and Meijer DKF), Wiley-VCH, Weinheim, New York; 2001 pp. 275-308. von Asmuth EJ, Smeets EF, Ginsel LA, Onderwater JJ, Leeuwenberg JF and Buurman WA. Evidence for endocytosis of Eselectin in human endothelial cells. Eur J Immunol 1992; 22: 251926. Spragg DD, Alford DR, Greferath R, Larsen CE, Lee KD, Gurtner GC, et al. Immunotargeting of liposomes to activated vascular endothelial cells: a strategy for site-selective delivery in the cardiovascular system. Proc Natl Acad Sci U S A 1997; 94: 8795800. Everts M, Kok RJ, Asgeirsdottir SA, Melgert BN, Moolenaar TJM, Koning GA, et al. Selective delivery of dexamethasone to activated endothelial cells using an E-selectin directed immunoconjugate. J Immunol 2002; 168: 883-9. Kok RJ, Everts M, Asgeirsdottir SA, Meijer DKF and Molema G. Cellular handling of a dexamethasone-anti-E-selectin immunoconjugate by activated endothelial cells: comparison with free dexamethasone. Pharm Res 2002; 19: 1730-5. Everts M, Koning GA, Kok RJ, Asgeirsdottir SA, Vestweber D, Meijer DKF, et al. In vitro cellular handling and in vivo targeting of E-selectin directed immunoconjugates and immunoliposomes used for drug delivery to inflamed endothelium. Pharm Res 2003; 20: 64-72. Tan PH, Manunta M, Ardjomand N, Xue SA, Larkin DF, Haskard DO, et al. Antibody targeted gene transfer to endothelium. J Gene Med 2003; 5: 311-23. Harari OA, Wickham TJ, Stocker CJ, Kovesdi I, Segal DM, Huehns TY, et al. Targeting an adenoviral gene vector to cytokineactivated vascular endothelium via E-selectin. Gene Ther 1999; 6: 801-7.

Current Vascular Pharmacology, 2005, Vol. 3, No. 1 [289]

[290]

[291]

[292]

[293] [294]

[295]

[296]

[297]

[298]

39

Ogawara KI, Rots MG, Kok RJ, Moorlag HE, van Loenen A, Meijer DKF, et al. A novel strategy to modify adenovirus tropism and enhance transgene delivery to activated endothelial cells in vitro and in vivo. Hum Gene Ther 2004; 15: 433-43. Gerlag DM, Borges E, Tak PP, Ellerby HM, Bredesen DE, Pasqualini R, et al. Suppression of murine collagen-induced arthritis by targeted apoptosis of synovial neovasculature. Arthritis Res 2001; 3: 357-61. Kok RJ, Schraa AJ, Bos EJ, Moorlag HE, Asgeirsdottir SA, Everts M, et al. Preparation and functional evaluation of RGD-modified proteins as alpha(v)beta(3) integrin directed therapeutics. Bioconjug Chem 2002; 13: 128-35. Schraa AJ, Kok RJ, Moorlag HE, Bos EJ, Proost JH, Meijer DKF, et al. Targeting of RGD-modified proteins to tumour vasculature: a pharmacokinetic and cellular distribution study. Int J Cancer 2002; 102: 469-75. Muller K, Nahde T, Fahr A, Muller R and Brusselbach S. Highly efficient transduction of endothelial cells by targeted artificial virus-like particles. Cancer Gene Ther 2001; 8: 107-17. Torchilin VP, Lukyanov AN, Gao Z and PapahadjopoulosSternberg B. Immunomicelles: Targeted pharmaceutical carriers for poorly soluble drugs. Proc Natl Acad Sci U S A 2003; 100: 603944. Wiewrodt R, Thomas AP, Cipelletti L, Christofidou-Solomidou M, Weitz DA, Feinstein SI, et al. Size-dependent intracellular immunotargeting of therapeutic cargoes into endothelial cells. Blood 2002; 99: 912-22. Proost JH. Pharmacokinetic/Pharmacodynamic modelling in drug targeting. In: Drug Targeting - Organ Specific Strategies, (Eds. Molema G and Meijer DKF), Wiley-VCH, Weinheim, New York; 2001 pp. 333-70. Hickey MJ, Kanwar S, McCafferty DM, Granger DN, Eppihimer MJ and Kubes P. Varying roles of E-selectin and P-selectin in different microvascular beds in response to antigen. J Immunol 1999; 162: 1137-43. Solorzano CC, Jung YD, Bucana CD, McConkey DJ, Gallick GE, McMahon G, et al. In vivo intracellular signaling as a marker of antiangiogenic activity. Cancer Res 2001; 61: 7048-51.