Endothelial Progenitor Cells and Vascular Biology

Current Diabetes Reviews, 2005, 1, 41-58

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Endothelial Progenitor Cells and Vascular Biology in Diabetes Mellitus: Current Knowledge and Future Perspectives Gian P. Fadini*, Carlo Agostini** and Angelo Avogaro* Department of Clinical and Experimental Medicine, * Division of Metabolic Diseases, ** Clinical Immunology and Hematology, University of Padova, School of Medicine Abstract: A growing amount of evidence demonstrates that Endothelial Progenitor Cells (EPCs) are involved in adult neovasculogenesis and maintenance of vascular integrity. EPC decrease and dysfunction are related to atherosclerosis and cardiovascular disease (CVD), and it has been proposed that the level of circulating EPCs may be used as a surrogate index of cumulative cardiovascular risk. Moreover, many experimental approaches reveal that exogenous EPC injection stimulates blood flow recovery in critical limb and myocardial ischemia, providing a new therapeutic tool for CVD. Diabetes Mellitus is a clinical condition characterized by a high incidence of CVD and is indeed associated with alterations in EPC physiology. In this review we focus on the relationships between EPCs and vascular biology, with particular regard to Diabetes Mellitus and future therapeutical implications.

Keywords: Endothelium, Diabetes, Angiogenesis. INTRODUCTION Studies on endothelial progenitor cells (EPCs) began in 1997, when Asahara and colleagues isolated putative progenitors implicated in adult neovasculogenesis, challenging the paradigm that de novo vessel formation exclusively occurs during embryonic development [1,2]. Subsequent studies addressed EPC pathophysiology in various clinical conditions associated with cardiovascular risk (CVD), and revealed that circulating progenitor cells are likely to be involved in many aspects of atherosclerotic disease [3], while a role for EPCs in tumoral angiogenesis and wound repair have also been postulated [4]. The discovery that a subtype of peripheral blood mononuclear cells (PBMCs) can differentiate into endothelial cells [6] has led to a new field of cardiovascular science, recently including some clinical trials of cellular therapy for limb and myocardial ischemia [7]. Endothelial Progenitor Cells Circulating EPCs are thought to be a subset of bone marrow-derived PBMCs, expressing immature surface markers common to hematopoietic stem cells, such as CD34 and CD133, and endothelial lineage markers [8]. EPCs can be isolated from peripheral, umbilical cord, and bone marrow blood [8]. CD34 represents a marker of immature stem cells that is often used to characterize EPCs together with other surface antigens. However, as CD34 is also

*Address correspondence to this author at the Department of Clinical and Experimental Medicine, Division of Metabolic Diseases, University of Padova, School of Medicine, Via Giustiniani 2, 35128 Padova, Italy; Tel: 0039-049-8212178; Fax: 0039-049-8754179; E-mail: angelo.avogaro@ unipd.it 1573-3998/05 $50.00+.00

expressed at lower levels on mature endothelial cells, most recent studies used CD133, a marker of more immature hematopoietic stem cells that is now considered the best surface marker to define, identify and isolate circulating EPCs. CD133 (also known as AC133 or prominin) is a highly conserved antigen with unknown biological activity, which is expressed on hematopoietic stem cells, but not on mature endothelial cells and monocytes [9-12]. Even if the exact phenotype of EPCs has not been definitively established yet, there is general agreement for the use of at least one additional marker reflecting endothelial commitment: the most used is Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2 or KDR), while others are Platelet-Endothelial Cells Adhesion Molecule-1 (PECAM-1 or CD31), Vascular Endothelial-cadherin, von Willebrand Factor, c-kit, Tie-2 and VEGFR-1 [13]. In most published studies, the amounts of circulating EPCs are determined by a culture method [14-18]: EPCs are defined as fibronectin adherent peripheral blood-derived cells uptaking acetilated LDL and binding Ulex-selectin in culture, and then further characterized by the expression of surface markers. At present there is no general agreement on the methods to define EPCs, and different studies used different way of identification and isolation, sometimes making results not easily concordant and comparable. However, it should be noted that the culture method is time-dependent and that results vary largely depending on culture conditions. In fact endothelial cells cultured from peripheral blood do not correspond to the actual population of circulating EPCs, but include mature circulating endothelial cells, shed off the vessel wall, and monocyte/macrophage-derived cells assuming endothelial phenotype in culture [19]. Moreover EPC count in vitro does not provide information on the absolute number of © 2005 Bentham Science Publishers Ltd.

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circulating EPCs [20], because they depend not only on the initial number of progenitors, but also on adhesion, proliferation and survival of plated EPCs, resulting from complex cellular interactions in the culture environment. Notably, the phenotype of these cells rapidly changes when they are expanded and cultured ex vivo: for example continuous cultivation was shown to increase endothelial marker protein expression and decrease markers of immaturity, supporting the hypothesis that cultivation induces differentiation and, possibly, senescence of EPCs [13]. These considerations suggest that it may be difficult to define the “true” population of EPCs. Some authors showed that prolonged culture of PBMCs in supplemented endothelial basal medium positively select a population of outgrowing endothelial-like cells with functional characteristics of self-renewing cells, which may well fulfill the definition of EPCs [21]. Recently, Peichev et al. showed that circulating CD34+CD133+KDR+ cells give rise to endothelial cells in vitro and thus functionally correspond to the definition of EPCs: therefore, three-fluorescence analysis of this cell subset may be another simple and elegant way to

unambiguously identify and quantify circulating EPCs without culturing them [22]. EPCs in Cardiovascular Diseases It is commonly believed that EPCs are mobilized from bone marrow upon various stimuli (Table 1A) [23]. Acute tissue ischemia is considered the strongest stimulus for EPC mobilization from the bone marrow to the peripheral circulation [24,25]. Then, EPCs are believed to localize specifically at site of ischemia, where they should increase blood vessel growth [26,27]: EPC ability to improve blood supply in peripheral and myocardial critical ischemia has been demonstrated in several experimental animal models [28-30] and recently confirmed in preliminary data from small clinical trials in humans [31,32]. A growing amount of data show that there are many other stimuli for EPC increase, including physiological events, such as physical exercise [33], pathophysiological conditions, like vascular trauma [34], growth factors and cytokines (VEGF, bFGF, SDF-1, GM-CSF, PlGF, EPO) [3, 35-38], and drugs such as statins, angiotensin converting

Table 1. Conditions and stimuli associated with EPC increase and decrease. Table 1A EPC Increase

References

Clinical Conditions Tissue Ischemia

Takahashi et al. Nat Med 1999 [23]

Acute myocardial infarction

Shintani et al. Circulation 2001 [107]

Unstable Angina

George et al. Eur Heart J 2004 [108]

Vascular Trauma

Gill et al. Circ Res 2001 [34]

Physical Exercise

Adams et al. Arterioscler Throm Vasc Biol 2004 [25] Laufs et al. Circulation 2004 [33]

Growth Factors, Cytokines and Chemokines VEGF

Gill et al. Circulation 2003 [34]

bFGF

Wang et al. Am J Physiol Heart Circ Physiol 2004 [177]

SDF-1

Yamaguchi et al. Circulation 2003 [178]

GM-CSF

Cho et al. Circulation 2003 [61]

Pl-GF

Tamarat et al. Am J Pathol 2004 [60]

IL-1β

Amano et al. J Mol Cell Cardiol 2004 [180]

EPO

Bahlmann et al. Kidney Int 2003 [35] Bahlmann et al. Blood 2003 [36]

Angiopoietin-1

Yamaguchi et al. Circulation 2003 [178]

Statins

Dimmeler et al. J Clin Invest 2001 [74] Walter et al. Circulation 2002 [64]

ACEI

Min et al. Cardiovasc Drugs Ther 2004 [127]

PPAR-γ agonists

Wang et al. Circulation 2004 [142]

Drugs

Hormones Estrogens

Strehlow et al. Circulation 2003 [68]

Endothelial Progenitor Cells and Vascular Biology in Diabetes Mellitus

Current Diabetes Reviews, 2005, Vol. 1, No. 1 43 Table 1. (Contd.….)

Table 1B EPC Decrease

References

Type 2 Diabetes Mellitus

Tepper et al. Circulation 2002 [17]

Type 1 Diabetes Mellitus

Loomans et al. Diabetes 2004 [18]

Chronic Renal Failure

Choi et al. Arterioscler Thromb Vasc Biol 2004 [16)

Hypercholesterolemia

Chen et al. Clin Sci (Lond) 2004 [39]

Risk Factors

Vasa et al. Circ Res 2001 [14]

Sigarette Smoking

Kondo et al. Arterioscler Thromb Vasc Biol 2004 [40]

Stable Coronary Artery Disease

Heeschen et al. Circulation 2004, Vasa et al. Circ Res 2001 [38] Eizawa et al. Heart 2004 [110]

Aging

Rauscher et al. Circulation 2003 [41] Edelberg et al. Circ Res 2002 [179]

Table 2. Clinical trials of cell therapy for myocardial or peripheral artery disease. Right external column shows how many diabetic patients received bone marrow- or peripheral blood-derived progenitor cells for therapeutic angiogenesis. BMC = Bone Marrow Cells. PBMCs = Peripheral Blood Mononucleas Cells. Clinical Setting Acute Myocardial Infarction

Reference

Cell type

Diabetic Patients/Total Patients

24h cultured BMCs

0/10

BMC vs EPCs

4/20

Fresh BMCs

3/30

Peripheral blood CD34+ cells

3/10

Bone marrow CD133+ cells

?/12 (not specified)

CD133+ BMC

0/6

Tse et al. Lancet 2003 [166]

Fresh BMCs

6/8

Fuchs et al. J Am Coll Cardiol 2003 [167]

Fresh BMCs

5/10

Perin et al. Circulation 2003 [168]

Fresh BMCs

4/14

BMCs

0/6

BMCs vs PBMCs

18/25

Peripheral blood CD34+ cells

0/2

Higashi et al. Circulation. 2004 [169]

Fresh BMCs

0/7

Esato et al. Cell Transplant. 2002 [171]

Fresh BMCs

0/8

Strauer et al. Circulation 2002 [163] Assmus et al. Circulation 2002: TOPCARE-AMI study [31] Wollert et al. Lancet 2004: BOOST study [164] Kang et al. Lancet 2004 [181] Stamm et al. Thorac Cardiovasc Surg 2004 [165]

Chronic myocardial ischemia

Stamm et al. Lancet 2003 [162]

Li et al. J Card Surg. 2003 [170] Peripheral Artery Disease

Tatehishi-Yuyama et al. Lancet 2002: TACT study [154] Kudo et al. Int Angiol 2003 [155]

enzyme inhibitors, estrogens and peroxisomal proliferator activating receptor-γ. Currently, it is suggested that increase in EPCs may be one of the mechanisms by which these stimuli would ameliorate vascular function: high EPC levels could help to maintain endothelial integrity and stimulate angiogenesis in ischemic organs. On the contrary, reduction in circulating EPCs has been demonstrated in the presence of risk factors for atherosclerosis, endothelial dysfunction, hypercholesterolemia, aging, smoking, chronic renal failure, coronary artery

disease (CAD), cerebral vascular disease and diabetes mellitus (Table 1B) [14, 16, 40-44]. Progenitor cell impairment in these clinical conditions is thought to account, at least in part, for the associated high cardiovascular risk or the development and progression of CVD. This growing amount of data has become the rational basis of cell-therapy for peripheral or myocardial critical ischemia [45]. At present, besides some clinical trials that used different types of bone marrow- or peripheral bloodderived cells (Table 2), only one study has been published on

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Fig. (1). A normal endothelium and the mechanisms responsible for endothelial dysfunction in diabetes mellitus.

cell therapy with EPCs for heart repair after acute myocardial infarction [31]. It has confirmed the promising results on animals, providing evidence for a new helpful therapeutic tool for CVD. However, many questions are unsolved, including, among the most relevant, mechanisms by which EPCs stimulate vasculogenesis, and possible harmful effects of progenitor cell infusion in patients with established CVD. EPCS IN DIABETES MELLITUS Diabetes mellitus is characterized by a three-to-four fold increase in cardiovascular risk. Vasculopathies associated with diabetes include excessive blood vessel formation (retinopathy, glomerular nephropathy) and accelerated atherosclerosis leading to CAD, peripheral artery disease (PAD), and cerebrovascular disease. Diabetic vasculopathy is an important source of morbidity and mortality: it is characterized by high prevalence, early development, rapid progression, bilaterality, and involvement of multiple distal arterial segments. The severity of macrovascular complications in diabetes seems to be due to profound impaired collateralization of vascular ischemic beds [46], but mechanisms that hinder ischemia-induced neovascularization in diabetes remain elusive. In diabetic patients collateralization is insufficient to overcome the loss of blood flow through occluded arteries, leading to ischemia, and often non-traumatic limb amputation. However, a few studies focused on factors that may impair ischemia-induced neovascularization in diabetes: mechanisms such as alterations in VEGF expression and signalling or in inflammation-related pathways have been proposed [47]. Moreover, diabetes mellitus is typically considered a clinical condition characterized by early and widespread endothelial dysfunction [48]. By means of soluble factors that can alternatively mediate vasoconstriction or vaso-

dilation, the endothelium is crucially involved in maintenance of adequate vascular tone and function. The term endothelial dysfunction refers to a condition in which endothelium loses its physiological ability to promote vasodilation, fibrinolysis and antiaggregation. Mechanisms by which diabetes induces endothelial dysfunction include reduced nitric oxide (NO) bioavailability, resistance to non-metabolic effects of insulin in type 2 diabetes, hyperglycemia and oxidative stress, common to type 1 and type 2 diabetic patients [49-52]. Recently, we have shown that non-complicated type 2 diabetic patients have a decreased conversion of L-arginine to NO, and that elevated free fatty acids associated with insulin resistance may impair vascular function by inhibiting inwardly rectifying potassium channels, a candidate component of the Endothelium-Derived Hyperpolarizing Factor [53-55]. From a pathophysiological point of view, the endothelial dysfunction in diabetes appears to be determined by a widespread condition of oxidative stress: this condition activates many intracellular pathways that converge with hyperglycemia and insulin resistance-stimulated pathways [56-58] (Fig. 1). Emerging evidence indicating that bone marrow-derived EPCs participate in postnatal neovascularization led to the hypothesis that alterations in EPC number or function could be involved in the pathogenesis of vascular complications in diabetes. Moreover, Lambiase et al. have shown that poor coronary collateral development, which is typical of diabetes, may be related to low levels of circulating EPCs [59]. A possible role for EPCs in diabetic vascular disease was first investigated in diabetic mice. Infusion of human CD34positive leukocytes, as an EPC-enriched population, was able to accelerate blood flow restoration in diabetic nude mice with experimental hindlimb ischemia, but did not in non-


Current Diabetes Reviews, 2005, Vol. 1, No. 1 45

Fig. (2). EPC origin and fate in the peripheral blood: EPCs may derive either directly from bone marrow or indirectly from peripheral differentiation of HSCs. The vascular and bloodstream environment may determine whether EPCs take part in endothelial repair and neoangiogenesis or undergo apoptosis. HSCs = Hematopoietic Stem Cells; VSMCs = Vascular Smooth Muscle Cells.

diabetic animals [5]. The reason for different response to EPC administration between diabetic and non-diabetic mice was not clear, but it could be due to the fact that blood flow restoration in non-diabetic animals was largely provided by physiological ischemia-induced neovascularization, which is hampered in diabetic animals. Therefore, it is possible that exogenous cells are of little value for animals that have a fully functional pool of progenitor cells, and can help only in animals with EPC reduction or compromised function. Indeed, reduced angiogenic potential of EPCs has been more recently demonstrated in diabetic animals [60]. In nondiabetic mice, transplantation of bone marrow-derived progenitors isolated from non-diabetic animals induced neovascularization in a model of hindlimb ischemia. Administration of diabetic progenitors in ischemic hindlimbs of non-diabetic mice improved to a lesser extent the angiogenic response, while, in diabetic mice, injection of non-diabetic progenitors was still more efficient than that of diabetic progenitors, suggesting that the diabetic vascular environment was more likely to affect endogenous EPC proangiogenic potential rather than the response to exogenous progenitor cell administration. Indeed, the authors reported that diabetes reduced the ability of bone marrow-derived mononuclear cells to differentiate into EPCs and that diabetic EPCs were unable to participate in vascular-like structure formation, in vitro . However, these studies did not determine whether poor angiogenic potential of progenitor cells in diabetes is specifically related to dysfunction of EPCs, other cellular populations or both.

conditions, including diabetes. In this light, EPC alterations were demonstrated in patients with CAD and were shown to correlate negatively with common risk factors for atherosclerotic disease [7].

EPCs were first defined as putative progenitors for neovasculogenesis in the adult human organism. It appeared soon after that alterations in the number and function of EPCs could be related to the impaired new vessel growth in ischemic organs, which is typical of several clinical

The concept that EPCs are involved both in early and advanced events of the development of cardiovascular diseases is of crucial importance for diabetology since diabetes mellitus is widely associated with endothelial dysfunction as well as with poor new vessel generation.

Subsequently, Hill et al. demonstrated that levels of circulating EPCs are inversely related to vascular function and global cardiovascular risk, also in healthy subjects [15]: they reported a strong negative correlation between the number of circulating EPCs and the patients’ combined Framingham risk score, while the measurement of flowmediated brachial reactivity also revealed a significant negative correlation between endothelial function and EPC levels. These observations confirmed the hypothesis that circulating EPCs may contribute to ongoing endothelial repair by means of providing a circulating pool of cells that could form a cellular patch at sites of denuding injury or a cellular reservoir to replace dysfunctional endothelium. It is well known that extension of neighboring mature endothelial cells is responsible for repair of denuded vessel walls, but growing evidence supports the idea that also circulating EPCs take part in reendotheliziation at sites of endothelial injury [61-65] (Fig. 2). Given this new possible role of EPCs it becomes clear that EPC reduction and dysfunction may be involved both in endothelial dysfunction, as an earlier event in the atherogenetic process [66], and in impaired collateralization in the presence of vascular obstruction by a plaque, as an advanced event leading to clinical manifestation of the atherosclerotic disease [67].

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Reduction in circulating EPCs and functional impairment of cultured EPCs has been reported both in type 1 and type 2 diabetic patients. Tepper et al. showed that PBMC-derived EPCs isolated from type 2 diabetics displayed a proliferation rate in culture decreased by 48% compared to control subjects, a weaker adherence to activated human umbilical vein endothelial cells (HUVEC) and a 2,5 fold reduced incorporation into vascular structures in vitro. Loomans et al. reported almost identical results in type 1 Diabetic Patients [17,18]. These data suggest that EPC dysfunction may represent a mechanism by which diabetic patients exhibit impaired ability to form collaterals. The rate of EPC proliferation from plated PBMCs in diabetic patients was inversely correlated with the levels of glycated hemoglobin, suggesting a possible relation between glucose control and EPC function. Poor adhesion of EPCs to HUVECs demonstrated altered cell-to-cell interactions which could indicate that EPCs are recruited less avidly in vivo at sites of ischemia, as well that reendothelization by means of bone-marrow derived cells is less likely to take place in the presence of EPC dysfunction. EPC pathophysiology in diabetes, and that in other clinical conditions associated with alterations in progenitor cells, have two aspects closely related one to another. From a merely quantitative point of view, low levels of circulating EPCs in basal and stimulated conditions may be insufficient to restore blood flow in ischemic tissues: in this light, augmentation of circulating EPCs by means of transplantation of expanded autologous progenitors would represent a promising therapeutic tool for critical ischemia syndromes. From a qualitative point of view, functional impairment would not allow residue EPCs to take part in ischemia-induced neovascularization, and would limit beneficial effects of autologous EPC administration. Putative Mechanisms Leading To Decreased Circulating EPCs Mechanisms underlying EPC reduction in diabetes are largely unknown: weak bone marrow mobilization, impaired peripheral differentiation, and short survival in peripheral blood are all candidates. Bone marrow origin of EPCs have been clearly demonstrated in mice transplanted with bone marrow from transgenic donors expressing beta-galactosidase transcriptionally regulated by the endothelial cell-specific Tie-2 promoter, and in mice transplanted with green fluorescent protein-positive bone marrow [68]. Altered ischemia-induced neovascularization in diabetic patients may be due to an insufficient EPC release from bone-marrow: stem cells mobilization from the bone marrow depends on the complex interactions in the local microenvironment, which consist of stromal mesenchimal cells, osteoblasts, fibroblasts and mature endothelial cells. Mobilizing cytokines allow stem cells to undergo transendothelial migration and to pass into the peripheral blood by means of attenuating stromal cell-stem cell interactions and by rearranging extracellular matrix.

Avogaro et al.

Ischemia is considered the strongest stimulus for EPC mobilization from bone marrow, through the upregulation of VEGF and SDF-1 (Stromal Derived Factor-1). Once in the blood stream, it seems that progenitor cell recruitment in regenerating tissues is mediated by hypoxic gradients via HIF-1 (Hypoxic Inducible Factor-1)-induced expression of SDF-1 [69]. Gill et al. have reported that vascular trauma, in the form of burn or coronary artery bypass grafting, is followed by a marked increase in circulating EPCs that peaks at 6-12 hours, resembling very closely VEGF increase and kinetics [34]. It has been recently shown that the expression of angiogenic factors VEGF and HIF-1 are reduced in the hearts of diabetic patients during acute coronary syndromes in respect to control subjects, and that myocardial infarct size in the rat is increased in hyperglycemic conditions and is associated with a reduced expression of the HIF-1 gene [70,71]. Therefore, poor collateral formation in response to myocardial ischemia may be due to a weaker stimulus from the ischemic heart to bone marrow. Accordingly, we noted (not published) that vascular trauma in the form of coronary or peripheral artery angioplasty was not able to increase EPC levels in diabetic patients. This observation needs further study because of the limited number of cases, but it could shed light on a new aspect of diabetes-related EPC dysfunction. Moreover, impaired cell-to-cell interactions of EPCs cultured from diabetic subjects could reflect alterations in the so-called “stem cell niche” that hampers cytokines-induced mobilization of stem cells. Indeed, dysfunction of mesenchymal cells in diabetes has been confirmed by studies that showed diabetic fibroblasts with impaired migration, reduced production of metalloproteinases and VEGF in response to ischemia, and correlated those alterations with the degree of hyperglycemia [47]. Molecular mechanisms that regulate EPC release in peripheral blood are not fully understood, but some data suggest a role for the phosphatidylinositol-3 kinase (PI3K)/Protein kinase-B (Akt/PKB) pathways. Statins, VEGF, EPO, estrogen and exercise are able to increase circulating EPCs and are all well-known to induce PI3-K/Akt activation [72,73]. Accordingly, Dimmeler et al. reported that pharmacological inhibition of PI3-K or the overexpression of a dominant negative Akt construct abolished statin- and VEGF-induced EPC proliferation, differentiation and increase both in vitro and in vivo, in animals [74]. Because eNOS seems essential for bone marrow mobilization of progenitor cells [75], stimuli that increase circulating EPCs could act by a PI3-K/Akt-dependent activation of NO synthase within both bone marrow stromal and stem cells [76]. Some data suggest that an adequate NO regulation is also needed for the angiogenic properties of EPCs: eNOS inhibition by the compound L-NAME significantly reduced microtubule formation by EPCs in a culture model of angiogenesis [77]. Dysfunction of all these subcellular pathways may be involved in the defective mobilization of EPCs in diabetic patients: indeed, diabetes mellitus is characterized by altered activation of PI3-K/Akt pathways and by reduced NO bioavailability: using a stable isotope approach, we have reported that the overall systemic fraction of L-arginine


converted to NO is lower in type 2 diabetic patients than in normal subjects [53,55] (Fig. 3). EPCs reside in the bone marrow environment and are mobilized upon appropriate stimulation [28]. However another reasonable speculative hypothesis is that general uncommitted precursors are continuously released from bone marrow to peripheral blood at a basal rate. Thus, progenitors may be subjected to all the influences, humoral factors or cell-to-cell interactions, taking place in the blood stream and in the vascular wall, which would determine whether they survive, differentiate into endothelial-committed progenitors, or undergo apoptosis (Fig. 2). The concept of peripheral differentiation of bone marrow-derived progenitor cells into EPCs has not been deeply investigated, however some authors report that EPC variations are not always concordant with the levels of all hematopoietic progenitor cells. This should lead to hypothesize that mobilizing stimuli are specifically targeted to endothelial-committed progenitors in the bone marrow, but it is also plausible that they act in the peripheral blood by inducing differentiation of generic precursors into EPCs. Accordingly, we noted that the percentage of EPCs among the CD34+ pool vary widely from patient to patient and, in the same patient, under different pathophysiological conditions, indicating possible peripheral differentiation rather than bone-marrow


mobilization. Endothelial commitment of generic hematopoietic precursors in peripheral blood may involve surface receptors, stimulated in states of ischemia [78]. As the amount of circulating leukocytes is determined by the balance between proliferation and apoptosis, and as diabetes is typically considered a clinical condition characterized by increased oxidative stress, apoptosis may well be related to EPC reduction in diabetic patients. In their study Loomans et al. could not find any difference in the mean levels of Annexin V binding to cultured EPCs from diabetic patients compared with controls [18]. Annexin V binding to externalized phosphatidilserine on apoptotic cells is a simple assay to reveal an early stage of the apoptotic process. Then, authors should not be surprised in finding that, after 4 or 7 days of culture under normoglycemic conditions, EPCs derived from diabetic patients contained the same amount of early apoptotic cells as from healthy subjects. In that case, levels of Annexin V binding would depend more on culture conditions than on cell origin. Indeed there is much data supporting that EPCs might decrease because of increased apoptosis. Bruhl et al. revealed a dose-dependent relation between levels of p21Cip1, that controls cell cycle progression and apoptosis in mature endothelial cells, and levels of circulating EPCs in double and single p12Cip1 knockout mice [79]. According to

Fig. (3). EPC mobilization from bone marrow to peripheral blood in response to tissue ischemia involves many interactions with bone marrow stromal cells in the so-called “stem-cell niche”. Activation of the PI3-K/Akt pathway and release of nitric oxide seem necessary for this function. EPCs then localise at sites of ischemia, where VEGF and HIF-1a genes are upregulated.

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Strehlow et al. estrogen increased EPC numbers through a decreased apoptosis rate, via a caspase-8-dependent pathway [68]. A TUNEL staining study demonstrated that incubation of EPCs with C-reactive protein significantly increased apoptosis and that this effect was prevented by pretreatment with a PPAR-γ ligand [77]. Also exercise training was able to increase EPCs by means of reduced apoptosis, quantified by an immunoenzymatic assay of cytoplasmic histoneassociated DNA fragments [33]. Therefore, it is possible that apoptosis is involved in EPC reduction in diabetic patients, but further studies in vivo are needed to confirm this hypothesis. It is worthy to note that despite culturing in a normal glucose environment, EPCs retained functional differences between diabetics and controls, suggesting that diabetesinduced EPC dysfunction is present at the level of the gene expression profile, making EPCs “remember” their metabolic descent in culture. This observation resembles closely the phenomenon of the so-called hyperglycemic memory. This refers to the persistence or progression of hyperglycemia-induced microvascular alterations during subsequent periods of normal glucose homeostasis. The most striking example is the development of diabetic retinopathy in dogs during a post-hyperglycemic period of euglycemia. Results from the Epidemiology of Diabetes Interventions and Complications study indicate that hyperglycemic memory also occurs in human patients [80]. Hyperglycemia-induced mitochondrial superoxide production may provide an explanation for the development of complications during post-hyperglycemic periods of normal glycemia. Hyperglycemia-mediated increase in superoxide might induce mutations in mitochondrial DNA, leading to defective subunits of the electron-transport complexes that would cause increased superoxide production even at normal glucose concentrations [81]. Preliminary results of DNA microarrays reported by Loomans et al. indicate that differential patterns of gene expression in diabetic EPCs resemble many alterations already seen in diabetes and high oxidative stress in general, such as upregulation of plasminogen activator inhibitor-1 and osteopontin. However, recent works demonstrated that EPCs display a gene expression profile that confers resistance to oxidative stress similar to that of stem cells [82,83]. The so-called “stemness” genes, that include catalase, manganese superoxide dismutase and gluthathione peroxidase encoding genes, are oxidative stress-associated genes providing protection against pro-oxidant conditions, with upregulated DNA repair and detoxifier systems. Compared to HUVECs, outgrowing cultured EPCs exhibited lower basal concentration of Reactive Oxygen Species (ROS) and milder ROS increase after incubation with high hydrogen peroxide or a redox cycler. Accordingly, only combined inhibition of all antioxidant enzymes led to ROS elevation and EPC dysfunction. The resistance to oxidative stress has been related to the ability of EPCs to preserve mitochondrial integrity under conditions of oxidative stress. These data show that, consistent with their progenitor cell character, EPCs are more protected against oxidative stress than mature endothelial cells, and therefore it seems unlikely that decrease and dysfunction of EPCs is mediated by increased oxidative stress.

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Oxidative stress plays a crucial role in the pathogenesis of late diabetic complications [81] as well as in the entire atherogenic process, including endothelial dysfunction [52]. ROS, normally produced at low amounts, can act as a second messenger in many cellular responses, while in pathological conditions higher amounts of ROS can cause DNA damage, cellular toxicity, or even apoptosis. In the entire organism, of all cells, vascular wall cells are the most directly exposed to systemic oxidative stress. Thus, even if EPCs are relatively resistant to oxidative stress in culture, in vivo pro-oxidant conditions may affect other cells involved in the complex cellular network in the blood stream and in the vascular wall that interact with EPCs to determine their function and fate. For example Imanishi et al. demonstrated that oxidized lowdensity lipoprotein induces EPC senescence and cellular dysfunction [84]. Moreover, in endothelium exposed to vasculature-damaging agents several enzymes that can produce ROS are upregulated: we have recently shown that gene expression of NAD(P)H oxidase, a major vascular sources of ROS [85-87], is increased in circulating PBMCs from type 2 diabetic patients depending upon metabolic control [88]. Remembering that EPCs are a subset of PBMCs and partly derive from PBMCs, it is easily to imagine how oxidative stress could reduce EPCs in clinical conditions with high oxidative stress, such as diabetes. Furthermore, EPCs have been found to be sentitive to the DNA-damaging agent cisplatin that activates proapoptotic pathways leading to cytochrome C release from the mitochondrial inner membrane, suggesting that EPCs may not be resistant to all death-inducing signaling. In diabetes oxidative stress actually arises from several intracellular and extracellular pathways: mitochondrial dysfunction, activation of stress-related signaling pathways including nuclear factorkB (NF-kB), p38 MAPK, NH2-terminal Jun kinases/stressactivated protein kinases (JNK/SAPK), generation of advanced glycosylation end-products (AGE), and activation of protein kinase C (PKC) [81,89]. Thus, it appears that such a hostile vascular environment like that of diabetic patients may well negatively influence EPC proliferation, differentiation and function. In summary, this brief discussion highlights the fact that reduction in circulating EPCs in diabetic patients may find its explanation in at least three pathophysiological models: the first one we can call “central” and is at present the most credited, proposing that weak bone marrow mobilization account for low levels of EPCs in peripheral blood. The other two we can call “peripheral” and hypothesize that decreased circulating EPCs may derive from impaired endothelial commitment of circulating hemotopoietic stem cells, or that EPCs subjected to the unfavorable vascular environment of diabetic subjects undergo senescence or apoptosis. Putative Metabolic Dysfunction

Alterations

Leading

to

EPC

What the metabolic alterations are that account for EPC dysfunction in diabetes remain largely unknown. Given that impaired function has been demonstrated in EPCs cultured from both type 1 and type 2 diabetics [17,18], it has been proposed that hyperglycemia is the common feature that


affects EPC proliferation, differentiation and function. However, there are very few data available in the literature on this topic. Schatteman et al. [30] reported that supplementation of culture medium with high glucose (30 mM) had no significant effect on EPC proliferation and differentiation from PBMCs of healthy subjects. They also noted that there were no differences in the numbers of EPCs that could be obtained in culture from type 1 and type 2 diabetic patients, but that CD34+ cells from type 1 diabetic patients gave rise to less mature endothelial cells than from type 2 diabetic patients: since type 1 and type 2 diabetic patients had comparable glycemic control, this alteration could not be attributed to glucose toxicity. Given that one major difference between type 1 and type 2 diabetes is that the former are hypoinsulinemic and the latter are hyperinsulinemic, the different behavior of EPCs in culture may be related to insulin levels. Accordingly, the authors showed that supplemental insulin (3 µΜ) induced generation of more spindle-shaped cells from PBMCs of healthy subjects, suggesting that high insulin shifted progenitor cells from proliferation to differentiation. Indeed, the endothelial fraction of CD34+ cells cultured in hyperinsulinemic conditions was initially increased, but decreased as cells remained in culture long term. In this light, high insulin levels would reduce circulating EPCs by means of inducing their differentiation into mature endothelial cells or even other cell types. Changing the phenotype of immature progenitor cells may disclose potential harmful effects [90]. While some kind of bone marrow cells can contribute to atherosclerosis and vascular remodeling [91-97], EPCs exhibit the property of preventing neointimal formation in animal models, suggesting that only this specific cell type may have antiatherogenic properties. However, upon pathological stimulations, such those associated with hyperinsulinemia, EPCs may transdifferentiate to display other phenotypes derived from CD34+ population, such as smooth muscle cells, fibroblasts or monocytes, which could in turn take part in atherosclerosis. Studies both in vitro and in vivo demonstrate that hyperinsulinemia and insulin resistance are associated with endothelial dysfunction [98]. It is well known that insulin acts as a weak vasodilatator through signaling pathways involving PI3-K and activation of eNOS via phosphorylation of a critical serine residue by the Akt/PKB [99-101]. Thus, a defective activation of the PI3-K/Akt pathways, which has been proposed as a biochemical mechanism of EPC dysfunction and has been confirmed in ex vivo experiments from type 2 diabetic patients, may be involved in the alterations of EPC proliferation and differentiation induced by high insulin in culture. Although high glucose had no direct effects on EPCs in this in vitro model, it may play a role in vivo anyhow. In vivo, hyperglycemia alters many molecules that have an impact on leukocyte and endothelial cell function; it leads to mitochondrial dysfunction and induces oxidative stress by increasing the production of ROS, directly via glucose metabolism and auto-oxidation, and indirectly though the activation of PKC [89], mithocondrial oxidases, and NAD(P)H oxidase. In cultured rabbit aortic endothelial cells,


high glucose increases mitochondrial respiratory chain flux as a result of overproduction of electron donors by the Kreb’s cycle, and then raises the proton gradient above a threshold value which causes a marked increase in superoxide production. In turn, ROS stimulate signaling pathways similar to that of hyperglycemia, such as upregulation of transforming growth factor-beta 1, plasminogen activator inhibitor-1, and activation of protein kinase C, mitogen-activated protein kinases and transcription factors. Indeed, many of these cellular effects of hyperglycemia are blocked by antioxidants [102]. Thus, high glucose may have effects on EPCs in vivo that are not observable in the culture systems. Accordingly, we have previously reported that hyperglycemia acutely increases activity of both MAP kinases and PKC in human circulating PBMCs in vivo [103-105]: the same effects are likely to intervene also in circulating EPCs, as a subgroup of PBMCs, even if future studies will be needed to confirm this hypothesis. Clinical studies clearly confirm that hyperglycemia impairs endothelium-dependent vasodilation [106], and that the correction of hyperglycemia improves endothelial function, in humans. Moreover, in both type 1 and type 2 diabetic patients, alterations in EPC number and function have been negatively correlated with levels of glycated hemoglobin, reflecting a possible link between glycemic control and progenitor cell impairment. In apparent contrast with these data which have been reported in the literature, we feel that a stronger inverse correlation is present between the numbers of circulating EPCs and blood glucose at time of blood collection in type 2 diabetic patients. This would indicate that rapid variations in glucose control could influence the EPC count: in that case it is more likely that low circulating EPCs reflect a shortened peripheral survival rather than a weak bone-marrow mobilization. According to this hypothesis, preliminary data from our patients confirm that rapid metabolic recompensation is followed by an increase in circulating EPCs. In conclusion, we discussed briefly that even if some studies do not show clear effects of high glucose on cultured EPCs, in vivo hyperglycemia may well influence EPC function. Then we discussed the putative role of hyperinsulinemia, as the typical metabolic alteration of type 2 diabetic patients. Despite experimental data suggesting a possible role for both hyperglycemia and hyperinsulinemia, more studies will be needed to determine which metabolic alteration of diabetes mellitus is causally related to EPC reduction and dysfunction. We believe that alterations in EPC biology relate to the pathophysiology of diabetes in general somehow regardless of the different pathogenesis of type 1 and type 2 diabetes. Finally, since both type 1 and type 2 diabetes are characterized by increased systemic oxidative stress, we would propose a unifying hypothesis in which oxidative stress may be the leading cause of EPC alteration in both type 1 and type 2 diabetes, regardless of the very different pathogenesis. Pathophysiological Models Regardless of the mechanisms, it is believed that diabetes mellitus induces EPC reduction and dysfunction. Thus,

50


according to the classical theory, poor endothelial repair capacity would lead to endothelial dysfunction and damage, while poor ischemia-induced neoangiogenesis would lead to the clinical manifestations of vascular occlusive atherosclerotic diseases (Fig. 4A). EPC dysfunction has been hailed as a novel concept in the pathogenesis of vascular complications of diabetes, but currently there are no data in the literature on the relationships between EPCs and vascular disease in diabetic patients. EPCs are found to be increased in all acute coronary syndromes [107,108], but data on stable CAD are unclear and inconsistent [109], while there are no data on EPCs and PAD. Vasa et al. reported a 40% reduction in EPCs from a cohort of patients with CAD, compared with age-matched healthy volunteers: however the CAD group included not only patients with stable disease, but also patients with acute coronary syndromes or myocardial infarction, clinical events known to be followed by EPC increase [14]. Heeschen et al. found no differences in CD34+CD133+ cells between patients with chronic myocardial ischemia and healthy controls, but only reported poorer angiogenic properties of progenitor cells from patients than from controls [38]. Eizawa et al. showed a 40% reduction in CD34+ cells in patients with stable CAD, but they did not evaluate endothelial progenitors [110]. Thus, the literature provides no definitive data on EPCs in stable vascular diseases and further studies are needed to confirm that EPC alterations are involved in the pathogenesis of vascular complications of diabetes. For example, it might be of interest to know whether EPC reduction directly correlates with severity of the atherosclerotic disease both from a pathological and clinical point of view. Indeed, if dysfunction of progenitor cells has a causative role in vascular disease, then one would expect that, being equal

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other risk factors, patients with more severe disease might have a more profound EPC reduction or dysfunction. Another fascinating hypothetic model is that EPC alterations might precede, and possibly, cause the development of type 2 diabetes, exactly as endothelial dysfunction can precede diagnosis of diabetes [111]. CVD and diabetes are so closely connected that the so-called “common soil” hypothesis has been proposed [112]. This hypothesis implies that CVD might not be simply a consequence of diabetes but that these two conditions are a single entity sharing common pathophysiological mechanisms, the clue of which is endothelial dysfunction: in large arteries endothelial dysfunction leads to clinical CVD, whereas in the microcirculation intimately coupled to metabolically active, insulin-sensitive tissues, endothelial dysfunction leads to type 2 diabetes [113]. In this setting, classical risk factors, such as age and smoking, may negatively affect EPC physiology, thus favoring endothelial dysfunction, which, in turn, would predispose to type 2 diabetes, hypertension and CVD (Fig. 4C). Future studies will eventually shed light on this new scenario. One possible approach could be to evaluate function and number of circulating EPCs in subjects with impaired glucose tolerance before the diagnosis of diabetes. In this case causal factors other than long lasting-hyperglycemia should be taken into consideration. Obviously these considerations regard only type 2 diabetes, but it should be noted that a decrease in a subset of stem/progenitor cells might also be involved in the exhaustion of pancreatic islet beta cells during the development of type 1 diabetes [114-116]. Mathews et al. demonstrated that bone marrow-derived cells are recruited to

Fig. (4). Possible pathophysiological models for EPC reduction in Diabetes Mellitus and its vascular complications. A) Classic model in which EPC impairment contributes to endothelial dysfunction and cardiovascular disease (CVD) development and progression. B) The bystander hypothesis: EPC reduction is the effect and not the cause of CVD. C) The “common-soil” theory with respect to EPCs: EPC alterations may precede and determine the development of both CVD and diabetes.


sites of pancreatic β-cell injury, using an animal model of type 1 diabetes transplanted with green fluorescent-bone marrow from congenic mice. The majority of donor-derived cells were hematopoietic and were located in the pancreatic interstitium, while some cells developed vascular endothelial phenotype and were located into the recipient’s pancreas. Though the authors were not able to show significant transdifferentiation of donor-derived cells into pancreatic βcells, EPC homing to site of pancreatic injury may be a relevant adaptive response during the development of type 1 diabetes. In the clinical setting, EPC administration may represent an adjuvant therapy to favor engraftment and survival of transplanted pancreatic islet by means of improving blood supply and preventing islet ischemia [114]. Another possible pathophysiological model is that EPCs are merely bystanders of what happens in the vascular environment of diabetic patients: altered vascular biology may also be the cause and not the consequence of EPC dysfunction (Fig. 4B). In general pathology one of the ways to study an unclear cause-effect relationship is to interrupt the chain of events at some points: indeed treatment of EPC reduction with exogenous EPC administration seemed to prevent vascular damage in animal models, but the mechanisms by which EPCs exert this effect are not completely known. Many Authors reported that EPCs actively take part in new vessel generation, and even can undertake transdifferentiation into cardiomyocytes [117119]. However the rate of incorporation of EPCs into new vessels and regenerating tissues dramatically varies between 0% to 90% in the different studies [120], and some reported that implanted EPCs are not integrated in the vessels, but localize adjacent to them. For these reasons other authors believe that exogenous cultured progenitor cells contribute to angiogenesis only in a paracrine way, by means of providing an optimal cocktail of growth factors and cytokines [121]. Hence, the bystander hypothesis cannot be ruled out a priori. Therapeutical Implications There is evidence that some drugs that positively affect vascular function in diabetic patients, also improve function and number of circulating EPCs. Thus, it appears that the vasculoprotective effect of these compounds may be partly due to their action on EPCs. Conversely, the existence of molecules acting on EPCs can be used to positively condition cultured EPCs before therapeutic transplantation. Angiotensin Converting Enzyme Inhibitor (ACEI) Angiotensin II (At-II) plays an important role in the development of many cardiovascular diseases, inducing vascular remodeling and myocardial hypertrophy. Many of these effects seem to be mediated by increased oxidative stress through the induction of NAD(P)H oxidase, subsequent activation of NF-KB pathways and reduced NO bioavailability [122]. ACE inhibitors have been used extensively in the management of patients with hypertension and heart failure. Over the past decade, a large body of evidence has emerged indicating that ACE inhibition also favorably affects the vasculature. Experimental studies demonstrated that ACE inhibitors improve endothelial function and have a host of


other beneficial effects on the arterial wall, while clinical trials demonstrated that treatment with these agents reduces the risk for acute ischemic events, improves the function of the arterial endothelium and can retard the anatomic progression of atherosclerosis [123,124]. The TREND study has documented a significant improvement in the endothelium-dependent vasodilation in the epicardial coronary arteries in patients with establishes CAD by quinapril [125]. Enalapril has also been shown to improve endothelial function in the peripheral arteries of patients with CAD by increasing the peripheral bioavailability of NO [126]. Min et al. showed that 4-week ramipril treatment (5 mg daily) of patients with stable CAD was associated with an increase in the number of circulating EPCs, similar to those obtained with statins, and in the functional activity of EPCs, as assessed by their proliferation, migration, adhesion and in vitro vasculogenesis capacity [127]. Moreover, Imanishi et al. demonstrated that in vitro At-II improves VEGF-induced human EPC differentiation and network formation through the upregulation of VEGF-R2, and that this effect is inhibited by the angiotensin type 1 receptor antagonist valsartan or a PKC inhibitor [128]. According to these data in vitro and in vivo, At-II might reduce EPCs in vivo by means of shifting EPC state from proliferation and self-renewal to differentiation into mature endothelial cells or even into smooth muscle cells. Thus, ACEI may maintain the pool of circulating progenitors and reduce accumulation of cells with harmful phenotype, involved in vascular and cardiac remodeling. Results from the MICRO-HOPE trial on 3577 diabetic patients revealed that ramipril was beneficial for cardiovascular events in people with diabetes independently of the decrease in blood pressure, providing large clinical evidence for pleiotropic effects of ACEI in diabetics [129]. A putative positive action on progenitor cells may be involved in these vasculoprotective effects in diabetic patients, who are thought to have a more profound EPC reduction and dysfunction. However, at present no data are available on chronic effects of ACEI on EPCs and thus more data are necessary to confirm that blocking At-II has positive effects on EPCs and that this will contribute to the clinical benefit of ACEI therapy. Hy dr oxy - M e thy l - Glutar y l - C o - E nz y me A -I nhibitor s (Statins) Large clinical trials such as the 4S, WOSCOP, CARE, and HPS suggest that the clinical benefits of statins are not limited to cholesterol reduction and are not associated with base-line cholesterol levels [130]. It is a common belief that statins improve endothelial function, independently of their lipid-lowering capacities, by preventing the synthesis of isoprenoid intermediates of the cholesterol biosynthetic pathway that serve as important lipid attachments for the posttranslational modification of a variety of cell signalling proteins such as members of the Ras and Rho GTPase family [131]. The inhibition of these pathways by statins increases endothelial NO synthase (eNOS) expression and provides an

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antiproliferative effect. Indeed, pleiotropic effects of statins include increase in NO bioavailability and plaque stability, reduced inflammation [132] and platelet aggregability [133]. Dimmeler and Llevadot have reported that 4-week treatment with simvastin (20 mg/kg/die) increased circulating EPCs in mice, and that statin supplemetation in culture medium increased proliferation, differentiation and function, while it decreased senescence of EPCs, by means of modulating various cell cycle regulatory proteins via the PI3-K pathway [74,134-135]. The authors have confirmed that 4-week treatment with atorvastatin (40 mg) increase and stimulate EPCs in patients with stable CAD, and improves the differentiation into cardiomyogenic cells [136,137]. In the study by Tepper et al. the authors could not report higher levels of EPCs in diabetic patients receiving statins compared with patients not receiving statin medication at time of blood collection. This result apparently contradicts other data published in the literature, but may find its explanation in the different therapy duration in various studies: this cross-observational study enrolled patients that were taking statins from a period of month-to-years, while other studies focused on short-term effects of statin therapy on EPCs. Therefore, at least three hypotheses can be made: 1) that beneficial effects of statin therapy on EPC biology are transient; 2) that diabetes prevents the beneficial effects of statin on EPCs; 3) that in treated patients EPC levels reflect the balance between hyperlipidemia-associated EPC reduction and statin-induced EPC increase: thus, EPC reduction and dysfunction would be underestimated when diabetic patients on long-term statin therapy are considered. Currently, there are no data available in literature on the long-term effects of statins on EPC number and function, and more data are needed to establish whether the action on EPCs can be properly included among the pleiotropic effect of statins on humans. Peroxisome Proliferator-Activated Agonists (Thiazolidinediones)

Receptor-Gamma

Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) agonists, such as the glitazone drugs, are a new class of insulin sensitizers used clinically to treat diabetes: they are able to lower glucose and lipid levels in patients with type 2 diabetes and also have antiatherosclerotic and antihypertensive effects [138]. PPAR-γ agonists appear to improve endothelial function independently of their insulin sensitization effects and inhibit intimal hyperplasia after balloon injury in both diabetic and non-diabetic animal models and after coronary stent implantation in patients with type 2 diabetes mellitus [139,140]. They also favorably limit vascular inflammation and decrease circulating levels of Creactive protein (CRP) [141], which serves not only as a marker but also as a mediator of atherosclerosis and has been reported to induce EPC dysfunction. Wang et al. reported that rosiglitazone caused an increase in colony formation by EPCs isolated from human circulating PBMCs, and attenuated neointimal formation after femoral angioplasty, by means of modulating angiogenic progenitor cells behavior in the vascular wall [142]. In vitro PPAR-γ ligands inhibit endothelial proliferation and promote endothelial apoptosis, but these effects may be concentration specific and may be attributed to pharmacological activity independent of PPAR-

γ: hence in vitro effects of thiazolidinediones on EPCs should be cautiously interpreted. In the work by Verma et al. CRP promoted apoptosis of EPCs and reduced their survival, differentiation and angiogenic properties [80]. All these effects were prevented by supplementation of the culture medium with rosiglitazone, which hampered CRP-induced NO reduction in EPCs [143]. Despite these promising data, the positive effects on number and function of circulating EPCs have not been confirmed in vivo, in humans, and much more data are needed to elucidate this new aspect of glitazone pleiotropy. Estrogens Despite discordant results from large epidemiological studies and clinical trials, estrogens are thought to have cardioprotective effects and to modulate the inflammatory response in atherosclerosis. Accumulating data provide convincing evidence that some metabolites of estradiol, particularly catecholestradiols and methoxyestradiols, are biologically active and mediate multiple effects on the cardiovascular and renal systems that are largely independent of estrogen receptors [144]. These protective effects are mediated in part by the inhibition of the ability of vascular smooth muscle cells, cardiac fibroblasts, and glomerular mesangial cells to migrate, proliferate, and secrete extracellular matrix proteins, as well as by an improvement in lipid profile and in vascular endothelial cell function [145]. The work by Strehlow et al. demonstrated that, in mice subjected to ovariectomy, estrogen deficiency significantly decreased EPCs circulating in the peripheral blood and residing in the bone marrow, while these effects were completely prevented by estrogen replacement [68]. They also reported that women with increased estrogen plasma concentrations displayed profoundly increased levels of circulating EPCs. Further insights into estrogen effects on EPCs have been provided by Asahara et al. which showed that bone marrow-derived EPCs significantly take part in estrogen-stimulated reendothelization after carotid injury in ovariectomized wild-type mice [146]. These in vitro and in vivo data support the hypothesis that beneficial effects of estrogens on cardiovascular risk may be partly attributed to the positive effects they exert on the levels of circulating EPCs. Concluding, growing data suggest that EPC biology might be considered a therapeutical target in cardiovascular medicine. Some of the drugs currently used for their vasculoprotective effects seem to positively affect EPC behavior, thus it is possible that effects on EPCs are part of the pleiotropic effects of these drugs. Given the high prevalence of CVD in diabetes, it appears that more convincing proof supports the use of these vasculoprotective agents in diabetic patients. Physical Exercise Regular physical activity is associated with a decrease in the incidence of cardiovascular events. Physical training


improves coronary endothelial function in non-diabetic healthy subjects [147,148], and in patients with CAD, PAD or heart failure. Epidemiological data confirm the benefit of physical training for improving blood glucose control and for preventing the development of diabetes mellitus: exercise may improve endothelial function in type 2 diabetic patients too, but no studies have yet been done. However, despite clear epidemiological data, the underlying mechanisms are incompletely understood. Some data suggest that physical training reduces vascular oxidative stress by upregulation of extracellular superoxide dismutase and by increase in eNOS activity [149]. In a study on mice, exercise reduced the apoptosis of spleen-derived EPCs in a NO-dependent manner [33], while Adams et al. showed that a maximal stress test in patients with CAD increases the number of circulating EPCs, and that the extent is comparable to a 4-week statin therapy [25]. Thereafter, exercise-related beneficial effects on CVD may rely in part on modulation of EPCs. While exercise above the ischemic threshold in CAD patients may increase cardiovascular events, it is safe and feasible in patients with PAD: long-term physical training may lead to a solid increase in the pool of circulating EPCs, thereby enhancing neovascularization and alleviation of symptoms. The “Diabetic Paradox” Interest linking diabetes mellitus and EPC pathophysiology is also part of the so-called “diabetic paradox”, by which diabetic patients, who have poor blood vessel growth in ischemic conditions, also have increased retinal neovascularization leading to diabetic retinopathy [150]. It has been demonstrated that bone marrow-derived cells with functional hemangioblast activity are involved in retinal neovascularization [151,152]. Moreover VEGF, known to have a potent stimulatory effect on EPC proliferation and on angiogenesis, is significantly elevated in the ocular fluid of diabetic patients, while it is decreased in ischemic nonretinal tissues [58]. It has been proposed that the retina responds differently to ischemia in comparison with tissue outside the central nervous system. A role for EPCs in the pathogenesis and progression of diabetic retinopathy has not been established yet. Data from our patients do not show any difference in the number of circulating EPCs between diabetic patients with and without retinopathy. Nontheless investigators should note that bone marrow-cell transplantation might have adverse effects on diabetic retinopathy. However, among more than 900 patients enrolled in clinical trials of therapeutic neoangiogenesis with progenitor cells infusion or cytokines for critical ischemia there have been no reports of increased incidence or progression of retinopathy [32] (Table 2). Cell Therapy Clinical trials of therapeutic angiogenesis with cytokines have failed to extrapolate the hopeful results from animal studies into the clinical setting. The growing amount of experimental data indicating a role for EPCs in neovasculogenesis, together with the observations of reduced levels of EPCs in patients at risk for and with established


CAD, has led to the concept of cell therapy for critical ischemia syndromes [32,153]. Indeed, it was proposed that diabetes mellitus induces a poor response to angiogenic stimulations with humoral factors: supplying the software (cytokines) would not be sufficient in the absence of the hardware (vascular progenitor cells) [121]. The promising results from experimental studies promoted the initiation of clinical trials, with the first result published in 2002. Investigators of the TACT study randomized patients with PAD to receive standard therapy or intramuscular injection of bone marrow-derived mononuclear cells: they reported an increase in transcutaneous oxygen pressure and pain-free walking, and improvement in rest pain [154]. While no other clinical trials in PAD have been published [155], many other authors reported results of cell therapy studies in patients with chronic ischemic heart failure. However, only in the TOPCARE-AMI (Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction) trial, specifically ex vivo expanded EPCs were used to stimulate repair after reperfused acute myocardial infarction, and induced global left ventricular function at 4 month follow-up in 20 patients, compared to a nonrandomized matched reference group [31]. The TACT study showed that symptoms of PAD ameliorate after progenitor cell-mediated increase in oxygen supply to the tissues. However it remains unclear whether, in the heart, EPCs act preferentially by improving tissue perfusion or by regenerating cardiac myocytes: a recent study could not demonstrate hematopoietic stem cells transdifferentiation into cardiomyocytes in myocardial infarct, using reporter transgenes [156,157]. Most of the preclinical studies were performed with young and healthy animals, but, in the clinical setting, patients often have an established CAD, are of advantage age, and have multiple risk factors that may limit the effects of cell-therapy. The notion that patient groups that would gain more benefit from new cell-based clinical strategies, such as diabetic patients, are the same in which EPC dysfunction has been demonstrated, suggests that EPCs isolated from these patients for autologous transplantation may retain their dysfunction in vivo thus limiting therapeutic neovascularization. Therefore, it is desirable that more efforts will be carried out to understand the mechanisms of EPC dysfunction and to design strategies to improve EPC function ex vivo before therapeutic transplantation. However, it should be noted that, to achieve a functional improvement, EPCs would need to be ex vivo cultured and expanded before transplantation [158]. Given the possible dangers arising from these procedures, laboratories with Good Manufacturing Practice accreditation are required, but they are at present not widespread, at least in some countries [159,160]. Alternative strategies for research centers unsupplied with credited laboratories would be to use large amounts of freshly isolated bone marrow-derived CD133+ cells or peripheral blood-derived CD133+ cells mobilized after GM-

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CSF administration, that could be transplanted directly without culture.

eNOS

=

Endothelial Nitric Oxide Synthase

ROS

=

Reactive Oxygen Species

Currently, there are no specific studies in the literature on therapeutic revascularization with cell therapy for diabetic patients, however many studies also involved diabetics and there have been no reports that, among a total of almost 150 patients treated, diabetics (almost fifty) displayed a poorer response to cell therapy than non-diabetic patients [161-171] (Table 2). This observation may cautiously lead to hope that the limit imposed to cell-therapy outcomes by the demonstrated EPC dysfunction in diabetic patients is more speculative than factual.

At-II

=

Angiotensin-II

ACEI

=

Angiotensin Converting Enzyme Inhibitor

As heart disease in diabetes mellitus is due not only to accelerated coronary atherosclerosis, but also to a wide range of biochemical, metabolic and structural microvascular alterations, unified in the definition of diabetic cardiomyopathy [172-174], it is likely that, in diabetic patients, PAD would gain more benefit from EPC therapy than ischemic heart disease. PAD is a striking source of morbidity and disability in diabetic subjects and is the leading cause of non-traumatic amputations in western countries [175,176]. Moreover, because of the severe and widespread vascular involvement, patients are often not candidates for standard revascularization. For these reasons we would encourage the development of a new EPC-based therapeutic tool for diabetic patients with disabling “no-option” critical lower extremities ischemia: it could make the difference between saving and losing a limb.

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CONCLUSIONS The recent discovery of circulating vascular progenitor cells has disclosed very wide experimental and clinical perspectives. Given the striking medical, social and economic impact of cardiovascular complications of diabetes, researchers involved in diabetic medicine should not remain indifferent to these novel opportunities. In this review we discussed some of the aspects linking endothelial progenitor cells to the altered vascular biology of diabetes mellitus. The literature supports the notion that EPC alterations are probably involved in the pathogenesis of vascular diseases in diabetics, but at present many questions remain unanswered. Future studies will be needed to consolidate our current knowledge and lay the foundations for innovative biological therapies.

[12] [13] [14]

[15] [16] [17]

ABBREVIATIONS EPCs

=

Endothelial Progenitor Cells

CVD

=

Cardiovascular Diseases

CAD

=

Coronary Artery Disease

PAD

=

Peripheral Artery Disease

PBMCs

=

Peripheral Blood Mononuclear Cells

VEGF

=

Vascular Endothelial Growth Factor

NO

=

Nitric Oxide

[18] [19] [20] [21]

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Endothelial Progenitor Cells and Vascular Biology in Diabetes Mellitus [22]

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[32] [33] [34] [35] [36] [37] [38]

[39]

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[41]

[42]

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