Rapamycin attenuates vascular wall inflammation and ... - CiteSeerX

©2004 FASEB

The FASEB Journal express article 10.1096/fj.04-2431fje. Published online November 16, 2004.

Rapamycin attenuates vascular wall inflammation and progenitor cell promoters after angioplasty Thomas G. Nührenberg,*,1 Rainer Voisard,‡,1 Felicitas Fahlisch,‡ Martina Rudelius,† Jürgen Braun,║ Jürgen Gschwend,§ Margaratis Kountides,¶ Tina Herter,‡ Regine Baur,‡ Vinzenz Hombach,‡ Patrick A. Baeuerle,# and Dietlind Zohlnhöfer* *I. Medizinische Klinik und Deutsches Herzzentrum, †Institut für Pathologie der Technischen Universität München, Munich, Germany; ‡II. Medizinische Klinik und §Urologische Klinik der Universität Ulm, Ulm, Germany; ║Urologische Abteilung der Klinik Biberach, Germany; ¶ Urologische Abteilung der Klinik Heidenheim, Germany; and #Micromet AG, Munich, Germany 1

T. G. Nührenberg and R. Voisard contributed equally to this work.

Corresponding author: Dietlind Zohlnhöfer, Deutsches Herzzentrum München, Lazarettstrasse 36, München 80636, Germany. E-mail: [email protected] ABSTRACT Rapamycin combines antiproliferative and antiinflammatory properties and reduces neointima formation after angioplasty in patients. Its effect on transcriptional programs governing neointima formation has not yet been investigated. Here, we systematically analyzed the effect of rapamycin on gene expression during neointima formation in a human organ culture model. After angioplasty, renal artery segments were cultured for 21 or 56 days in absence or presence of 100 ng/ml rapamycin. Gene expression analysis of 2312 genes revealed 264 regulated genes with a peak alteration after 21 days. Many of those were associated with recruitment of blood cells and inflammatory reactions of the vessel wall. Likewise, chemokines and cytokines such as M-CSF, IL-1β, IL-8, β-thromboglobulin, and EMAP-II were found up-regulated in response to vessel injury. Markers indicative for a facilitated recruitment and stimulation of hematopoetic progenitor cells (HPC), including BST-1 and SDF-1, were also induced. In this setting, rapamycin suppressed the coordinated proadhesive and proinflammatory gene expression pattern next to down-regulation of genes related to metabolism, proliferation, and apoptosis. Our study shows that mechanical injury leads to induction of a proinflammatory, proadhesive gene expression pattern in the vessel wall even in absence of leukocytes. These molecular events could provide a basis for the recruitment of leukocytes and HPC. By inhibiting the expression of such genes, rapamycin may lead to a reduced recruitment of leukocytes and HPC after vascular injury, an effect that may play a decisive role for its effectiveness in reducing restenosis. Key words: hematopoetic progenitor cells ● smooth muscle cells

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estenosis is the most significant limitation of percutaneous angioplasty procedures. After stent implantation, it is mainly caused by neointima formation. This process was formerly considered to be primarily directed by dedifferentiation of medial smooth Page 1 of 21 (page number not for citation purposes)

muscle cells (SMC) and their migration to the intima, followed by proliferation and production of abundant extracellular matrix (1). Lately, it has been shown that recruitment of inflammatory cells is an essential step in the pathogenesis of neointima formation in humans (2). In various animal models, reduction of leukocyte recruitment by selective blockade of adhesion molecules significantly reduced neointima formation and restenosis (3–5). More recently, it has been shown that a distinct part of neointimal SMC develops in mice from bone-marrow-derived cells (6). Likewise, an unexpectedly high rate of restenosis in humans treated with G-CSF and thereby having mobilized peripheral progenitor cells underlines the impact of HPC recruitment to the vessel wall in a clinical setting (7). With the use of drug-eluting stents, neointima formation can be dramatically decreased by local delivery of rapamycin, an anti-proliferative as well as anti-inflammatory drug (8–10). However, it is poorly understood how rapamycin acts on several aspects of neointima formation, such as increased arterial inflammation (2) and the involvement of invading HPC (6). In addition, our understanding of early transcriptional mechanisms in humans remains scarce, as vascular tissue is not available shortly after angioplasty. We therefore sought to identify early molecular mechanisms of human neointima formation using a human ex vivo organ culture model. In this study, we performed differential gene expression analysis of atherosclerotic renal arteries at three different time points after angioplasty and investigated the impact of rapamycin on gene expression after vascular injury. METHODS Preparation of organ cultures During routine nephrectomies, parts of the renal arteries of 15 patients (11 male/4 female; mean age ± SD: 66.5±10) were extracted. All patients gave informed consent, and the study was approved by the Institutional Ethics Committee. Sections were made at 4-mm intervals perpendicular to the vessel wall axis. During transport to the laboratory and preparation, the segments were stored in sterile HEPES-buffered (15 mM) culture medium (DMEM) without serum supplement. Balloon injury in vitro Angioplasty was performed as reported before (11) with an inflation pressure of 9 bar for 60 s. Untreated arterial segments served as controls (n=5). Sample cultivation and fixation After ballooning, the segments were either stored within 15 min (n=6) or transferred to six-well plates (Tecnomara, Fernwald, Germany) and cultured for 21 (n=4) and 56 days (n=5) in a 1:1mixture (vol/vol) of Waymouth’s MB 752/1 and Ham’s F-12 nutrient mixture (Cambrex, Vervier, Belgium) supplemented with 15% fetal calf serum (Cambrex, Vervier) at 37°C in 5% carbon dioxide. Culture medium was exchanged every second or third day. Rapamycin was added at a concentration of 100 ng/ml for 21 (n=3) and 56 days (n=4). Samples used for gene expression analysis (n=22) were snap-frozen and kept in liquid nitrogen until

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mRNA isolation. The remaining samples were fixed in 4% buffered paraformaldehyde (Merck, Darmstadt, Germany) and embedded in paraffin. Isolation of mRNA, global RT-PCR, labeling of cDNA probes, and hybridization to cDNA arrays mRNA isolation, cDNA synthesis, and PCR amplification were performed as described previously (12, 13). Aliquots of 25 ng of each cDNA were labeled with digoxigenin-11-dUTP (Roche Diagnostics, Mannheim, Germany) during PCR, and each probe was hybridized to three arrays (Clontech Atlas human cancer 1.2, human 1.2, and cardiovascular arrays, BD Biosciences, Heidelberg, Germany) as described. Detection of filter bound probes was performed by the digoxigenin detection system (Roche Diagnostics). Developed films were scanned and analyzed using the Array Vision™ software (Imaging Research Inc., St. Catharines, Canada). Background was subtracted, and signals were normalized to nine housekeeping genes present on each filter, whereby the average signal of the housekeeping genes was set to one and the background to zero. Confirmation of array data by gene-specific PCR A selection of differential hybridization signals was confirmed by gene-specific PCR. Amplification was performed using 2.5 ng of each cDNA in a 25 µl reaction containing PCR buffer (Sigma-Aldrich, Munich, Germany). PCR products were subjected to electrophoresis on a 2% agarose gel containing ethidium bromide (0.5 µg/ml). Images of the gels with a resolution of 28 pixels/cm were analyzed using the Array Vision software. Background was set to zero, and the mean signal intensity of β-actin (GeneBank accession: X00351) was set to one. Morphometry of human organ cultures The effect of rapamycin on neointimal thickening was studied at 21 and 56 days after ballooning (for detailed information, see ref 11) Immunohistochemical analysis For histology and immunohistochemistry, specimens were fixed in 4% formaldehyde (pH 7.0) and embedded in paraffin. Serial paraffin sections (4 µm) were deparaffinized, dehydrated, and, for antigen retrieval, pressure-cooked for 4 min in citrate buffer (10 mM, pH 6.0), followed by blocking of endogenous peroxidase (1% H2O2/methanol; 15 min) and preincubation with 4% dried skim milk in Antibody Diluent (DakoCytomation, Hamburg, Germany). Immunostaining employed the streptavidin-horseradish-peroxidase technique (Dako ChemMate Detection Kit). A primary antibody against β-thromboglobulin (a kind gift from Dr.E.Brandt, Forschungszentrum Borstel, Germany) was used at a dilution of 1:200. Statistical analysis Gene expression values are reported as median expression values of each group. All other values are means ± SE. Significance of differential expression over the time course (Control, 15 min, 21 and 56 days) was analyzed by the Kruskas-Wallis-test. Rapamycin treatment was compared with


control using the Mann-Whitney’s U test. A descriptive P < 0.05 was regarded as significant. Subsequently genes with a minimum ratio of 2.5 and a minimal difference of 0.1 within their group medians were considered as differentially expressed. Hierarchical clustering of average linkage clustering with the centered correlation metric was used (Gene Cluster/Treeview; ref 14). The same statistical tests were used for the data obtained by densitometric PCR analysis. RESULTS Morphometric analysis of human organ culture As shown previously (11), neointima formation in the cultured specimens could be analyzed by morphometric analysis. Twenty-one days after angioplasty, the neointimal thickness was 1.44 ± 2.27% of the vessel lumen with a further increase up to 6.11 ± 3.81% of the vessel lumen after 56 days. Treatment with rapamycin could completely inhibit neointima formation after 21 days and showed a reduction of neointima by 31 to 4.20 ± 4.25% of the vessel lumen after 56 days of treatment. Time-dependent changes in gene expression after angioplasty Of the 2312 genes analyzed, 264 genes were regarded as differentially expressed over the time course studied. Criteria for differential expression have been applied in previous studies from our group (12) and others (15). Two-dimensional hierarchical clustering of differentially expressed genes revealed the up-regulation of the vast majority (n=212) of genes (Fig. 1, groups A–D), whereas 44 genes showed consistent down-regulation over the time course of neointima formation (Fig. 1, group E). Eight genes showed inconsistent but differential regulation over the time course. As shown in Fig. 1, balloon angioplasty led to significant up-regulation of 65 genes immediately after angioplasty. Thereof, a small number of genes (n=16) showed rapid but only transient activation (Fig. 1, group E). After 21 days, 133 additional genes exhibited increased expression. After 56 days of culture, only a few more genes (n=14) were found to be up-regulated for the first time (Fig. 1). Therefore, vascular injury had the strongest impact on gene expression after 21 days. Similarity to gene expression in human neointima from in-stent restenosis As we had already analyzed the gene expression of native human neointima from in-stent restenosis retrieved by helix cutter atherectomy in a previous study (12), we were here interested in genes that have been regulated in a similar manner. In fact, we could identify 36 genes (14%) that showed congruent alteration in both studies. Thereof, 30 were up-regulated such as MMP-9, MRP 8&14, CD13, COX-1, or β-thromboglobulin (Table 1, upper part). Additionally, six genes showed reduced expression after angioplasty in vivo and ex vivo like desmin and frizzled-related FrzB (Table 1, lower part).


Functional clustering reveals major changes in transcription of genes associated with proliferation, apoptosis, extracellular matrix production, adhesion, and inflammation To gain a more detailed insight into the molecular mechanisms governing neointima formation, we performed functional clustering of all 264 differentially expressed genes (Figs. 2 and 3). To this end, we assigned the genes to six functional groups, consisting of genes involved in transcription, inflammation, proliferation/apoptosis, signal transduction, extracellular matrix/adhesion/cytoskeleton and cell metabolism. In detail, 45 genes were related to inflammation (Fig. 3A), 52 to extracellular matrix production and adhesion of leukocytes (Fig. 3B) and 95 to proliferation and apoptosis (Fig. 3C); adding together to a number of 192 genes and representing 72% of all differentially expressed genes. Portraying the time course of their regulation (Fig. 2), almost equal activation in all three groups was observed immediately after balloon injury (Fig. 2, left group of bars) when 53 genes showed altered expression; 114 genes (Fig. 2, middle group of bars) were additionally regulated after 21 days, indicating that major changes in gene expression took place at this time point of culture. Indeed, the majority of differentially expressed genes after 21 days was associated with proliferation such as CDK4, PCNA, or VEGF (Fig. 3C). This suggests a predominant role of proliferation and apoptosis in the later time points of the organ culture model. The timedependent gene regulation further underlines the known impact of smooth muscle cell proliferation in neointima formation. Interestingly, they also demonstrate that early inflammation is a key pathogenic step after balloon angioplasty. Inflammation-associated genes regulated in our study were described previously genes like IL1β, IL-8, or prostaglandin G/H synthase 1 (COX-1) but also less studied genes like βthromboglobulin (β-TG) or EMAP-II (Fig. 3A). Changes in the transcription of genes related to extracellular matrix production consisted in a higher expression of several collagen subtypes as well as in the down-regulation of cytoskeletal components like desmin or genes involved in the regulation of smooth muscle cell contractility like cardiac phospholamban or myosin light chain kinase (MLCK; Fig. 3B). To our surprise, 57 of the 192 genes belonging to the three groups have previously been described to be expressed in hematopoetic progenitor cells or other immature bone marrow cells (shaded parts of clomuns in Fig. 2, marked with # in Fig. 3). Likewise, we found an upregulation of SDF-1, MMP-9, CD157, and the oncostatin M receptor-β. Effects of treatment with rapamycin for 21 and 56 days on gene expression As rapamycin-coated stents dramatically reduce restenosis in humans (8–10), we were interested in delineating mechanisms by which rapamycin affects neointima formation. Therefore, we compared gene expression after angioplasty with respect to rapamycin treatment. The statistical analysis yielded 117 genes that were significantly down-regulated by rapamycin in the human organ culture model (Fig. 4B and C). According to the antiproliferative properties of rapamycin, most of the down-regulated genes were related to proliferation/apoptosis, such as JAK1, VEGF-C, caspase-8, or HIF1-α (Fig. 4A). Furthermore, rapamycin treatment also reduced the expression of many genes related to


inflammation and adhesion/extracellular matrix formation (Fig. 4A), for example βthromboglobulin (NAP-2), IL-8 (NAP-1), or the collagen subtypes COL1A2 and COL2A1. Based on the differentially regulated genes during neointima formation in our model, 39 showed simultaneously down-regulation by rapamycin such as the inflammation-associated genes βthromboglobulin, IL-8, EMAP-II, or COX-1 (Fig. 4B). In high accordance with a previous study (16), no genes were observed to be up-regulated by treatment with rapamycin. As the majority of genes altered after angioplasty in the organ culture model belonged to the above mentioned three functional groups, rapamycin acts thus on the major pathogenic steps of neointima formation. Moreover, rapamycin also reduced the expression of genes related to HPC such as CD157, JAK1, or the oncostatin M receptor-β. Validation of cDNA array data by gene-specific PCR Gene expression data obtained by cDNA array analysis were confirmed by gene-specific PCR for 11 genes that may play a role in the recruitment of leukocytes such as β-thromboglobulin (NAP-2) or IL-8 (NAP-1). Genes related to HPC such as the oncostatin M receptor β, MMP-9 and SDF-1 could also be verified. Representative PCR gels of three genes are shown in Fig. 5A, and the results of the densitometrical and statistical analysis in Fig. 5B and C. Verification of up-regulated β-thromboglobulin protein expression by immunohistochemistry To further validate the involvement of β-thromboglobulin, an important chemokine for activation and chemotaxis of neutrophils, in neointima formation, we performed an immunohistochemical analysis. β-Thromboglobulin protein expression could be verified in the organ culture model after angioplasty (Fig. 5D, left panel), whereas rapamycin treatment reduced the intensity of βthromboglobulin staining (Fig. 5D, right panel). DISCUSSION Rapamycin reduces the risk of in-stent restenosis in humans (8–10). However, the underlying mechanisms of how rapamycin governs neointima formation are so far not understood in detail. Here, we systematically investigated gene expression of renal arteries after angioplasty in absence of leukocytes and the effect of rapamycin treatment. We show that 1) changes in gene expression occur in a time-dependent manner with a maximal alteration 21 days after injury; 2) major changes take place in genes related to proliferation and apoptosis, extracellular matrix and adhesion, and inflammation; 3) balloon angioplasty leads to a gene expression pattern promoting recruitment and activation of inflammatory and hematopoetic progenitor cells; 4) rapamycin treatment reduced neointima formation by its anti-proliferative effects; and 5) rapamycin prevents the induction of a proadhesive and proinflammatory gene expression pattern in our model. Time-dependent regulation of gene expression after balloon angioplasty A time-dependent gene expression analysis of arteries after angioplasty has not been reported so far. Here, we systematically analyzed gene expression using cDNA array technology immediately, 21 days, and 56 days after balloon dilation. As expected, far more genes were up-


regulated as compared with the 44 consistently down-regulated genes. In both groups, we found genes known to be regulated in neointima formation like MMP-2, MMP-9, CD13, COX-1, VEGF, or desmin. Furthermore, we show that the mechanical injury alone induces profound changes in gene expression. A number of those genes have been reported to be regulated by mechanical strain like IL-8, COX-1, VEGF, FGF-2, AKT1, or thrombomodulin (17–21). Regarding the horizontal cluster axis, it is evident that changes in gene expression are a timedependent event. A large group of genes was only up-regulated after 21 days (Fig. 1A and B), whereas other genes showed sustained up-regulation (Fig. 1B and D). Among the latter, many genes were related to cell adhesion as well as extracellular matrix formation like MMP-14, MMP-11, and several collagen subtypes. As only a few genes were regulated exclusively after 56 days, we conclude that the later restenosis is a sequel of early, misguided wound healing. This concept is supported by the impressive reduction of in-stent restenosis by rapamycin-coated stents (8–10). Since they release ~80% of the total drug dose within in the first 30 days after placement, restenosis is unlikely to be dependent on late effects. Balloon angioplasty leads to enhanced inflammation in absence of leukocytes Whereas the involvement of proliferation in neointima formation is well known, the impact of inflammatory processes leading to leukocyte recruitment after vascular injury has been underlined more recently (2, 22). To get a more detailed understanding of inflammatory mechanisms affected in this model, we clustered all genes into functional groups according to information existing in the literature. As 192 or 72% of all differentially expressed genes were linked either to inflammation (Fig. 3A), to extracellular matrix production and adhesion (Fig. 3B), or to proliferation and apoptosis (Fig. 3C), we focused on these groups. The proinflammatory gene expression pattern shown in Fig. 3A gives an impressive rationale for profound leukocyte recruitment after balloon dilation. Interestingly, many of these cytokines act on cells with myeloid origin. Likewise, the activation and induced migration of monocytes respectively macrophages has been reported for EMAP-II (23), MST1 (24), and MCSF (25). Other cytokines like IL-8, β-thromboglobulin, or GCP-2 were also up-regulated after angioplasty and enhance all the migration of granulocytes (26–28). The macrophage-related proteins 8 and 14, also induced in neointima from human in-stent restenosis, are closely linked to inflammatory processes, enhancing neutrophil chemotaxis and adhesion (29). In addition, we observed the persistent up-regulation of CD13 and CD32, both markers of granulocytes as well as of βthromboglobulin. Cleavage of this cytokine through monocyte proteases results in active NAP-2 (30), which promotes adhesion and transendothelial migration of neutrophil granulocytes (27). The mechanically induced activation of this gene expression pattern will favor leukocyte recruitment and thereby restenosis, as inflammatory cell density in neointima correlates significantly with the extent of restenosis (2).


A subset of genes related to the recruitment and expansion of bone marrow-derived progenitor cells is induced by angioplasty Moreover, we found an up-regulation of genes related to HPC. Likewise, IL-8 induces the mobilization of hematopoetic progenitor cells (31). Enhanced release of stem and progenitor cells from the bone marrow has also been shown to be caused by MMP-9, a matrix metalloproteinase known to be up-regulated by IL-8 (31) as well as by SDF-1 (32). SDF-1 itself induces chemotaxis of HPC (33). Lately, SDF-1 was identified to play an important role in murine neointima formation (34). Thus, it is conceivable that hematopoetic progenitor cells contribute to some extent to the cell content of murine neointima (6). However, little is known about the transferability of those observations to humans. As IL-8, MMP-9, and SDF-1 were all up-regulated in our model, we provide evidence that those mechanisms may also apply to humans. Strikingly, we observed changes in other genes involved in the recruitment of hematopoetic progenitor cells like CXCR4 (33), ITGB7 (35,) or PlGF (36), which have so far not been related to early and chronic stages of neointima formation. Taken together our findings support the notion that recruitment, proliferation, and differentiation of progenitor cells play an important role in the pathogenesis of human neointima formation. Treatment with rapamycin reduces the expression of genes associated with inflammation and hematopoetic progenitor cells The macrolide antibiotic rapamycin has recently shown to dramatically decrease human in-stent restenosis in several clinical studies (8–10). However, its exact mode of action after vascular injury remains unclear as it exerts pleiotropic effects on the different cell types of the vascular wall. It has been thoroughly shown that rapamycin inhibits SMC migration and proliferation by its effect on mTOR (37, 38). On the molecular level, vascular injury induces the sustained activation of Akt, an upstream regulator of mTOR (20). This is conforming to a subsequent activation of mTOR in human neointima (16). Rapamycin representing an mTOR inhibitor may therefore block the activation of the mTOR-pathway, which is important for SMC replication after arterial injury (39). Interestingly, treatment of SMC with rapamycin leads to profound changes in gene expression and reduces adhesiveness of SMC for monocytic cells (16). In consequence, it will be important to identify further basic mechanisms by which rapamycin inhibits restenosis after angioplasty. According to previous data, it reduced the expression of genes associated with proliferation and apoptosis as well as of extracellular matrix production (16). Of note, all collagen subtypes showed profound down-regulation by rapamycin while the matrix metalloproteinases were not regulated. Besides, postangioplasty restenosis, graft vasculopathy, and hyperlipidemia-induced atherosclerosis share several pathophysiological mechanisms. Thus, bone marrow derived cells may give rise to many of the SMCs that contribute to arterial remodeling (6). As rapamycin also slows graft vasculopathy in humans (40) and the progression of atherosclerosis (41), its mode of action in those diseases may have implications for its striking effectiveness in reducing postangioplasty restenosis.


In a cardiac allograft model, rapamycin reduced IL-8 expression and thereby strongly diminished neutrophril infiltration (42). Likewise, the induction of IL-8 after angioplasty in our study was completely inhibited by rapamycin treatment. More impressive, rapamycin treatment greatly reduced the expression of the CXC chemokines 6-8 (GCP2, β-thromboglobulin, IL-8) as well as CD73 or EMAP-II, which all play an important role in adhesion and migration of circulating granulocytes and monocytes. Moreover, rapamycin has been shown to induce differentiation in a myogenic cell line (37). In our model, rapamycin treatment reduced the expression of several genes that could foster immature, still differentiating cells in the vessel wall. Thus, CD157, also known as bone marrow stromal antigen 1, is essential for generation of cyclic ADP-ribose, which itself stimulates the proliferation of human hematopoetic progenitors (43). Furthermore, rapamycin reduced the expression of the oncostatin M receptor, which is also involved in the stimulation of hematopoetic progenitors (44). Interestingly, coexpression of JAK1 enhances oncostatin M receptor expression (45). Conversely, rapamycin completely suppressed JAK1 induction after vascular injury and may thus prevent oncostatin M-induced stimulation of hematopoetic progenitors and stromal cells. Taken together, rapamycin has profound inhibitory effects on the proinflammatory gene expression pattern and on promoters of HPC after vascular injury. Since there is little data elucidating a possible recruitment of HPC in human vasculature, we provide here new and interesting data how rapamycin may reduce the recruitment of HPC to the vessel wall in humans. In consequence, this may explain the high effectiveness of rapamycin in reducing restenosis, seeing that the extent of recruited leukocytes correlates with neointima formation (2). Limitations of the study Although we report here for the first time a detailed description of the gene expression changes induced by angioplasty in humans ex vivo, we cannot exclude that there may be differences between renal arteries, as used in our study, and coronary arteries. However, the rather low number of genes that are congruently regulated in native neointima and in our organ culture model may be due to several other reasons: the samples represent different time points during the process of neointima formation. While the early mechanisms of neointima formation were analyzed in the organ culture model, the tissue of native neointima was retrieved 3-8 months after angioplasty and reflects the late and chronic phase of the pathophysiological process. In addition, the influence of infiltrating blood cells was not monitored in the organ culture model, whereas the samples from in-stent-restenosis contain a higher diversity of cells. The absence of leukocytes and hematopoietic progenitor cells in the organ culture model may also explain the relative low amount of neointimal tissue. On the one hand, this is further underlining the importance of infiltrating blood cells and HPC for enhanced neointima formation. On the other hand, the organ culture model demonstrates that a pro-adhesive environment is induced selectively in the vessel wall itself even in absence of leukocytes facilitating the recruitment of leukocytes and HPC that is attenuated by rapamycin.


Future prospects Our study provides impressive data concerning the alteration in gene expression after angioplasty. However, the significance of the individual changes for the later development of restenosis remains to be elucidated by further in vivo and in vitro studies. On the other hand, our model represents an excellent tool to scrutinize new drugs developed to prevent restenosis. According to our data, pleiotropic properties will be indispensable for any drug founding a keen competition with rapamycin, which is so far the most effective drug reducing restenosis in humans. ACKNOWLEDGMENTS This study was supported by a postdoctoral fellowship awarded to D. Zohlnhöfer by the Deutsche Forschungsgemeinschaft (DFG, Zo 104/1-1, Zo 104/2-1) and by grants awarded to R. Brandl by the Deutsche Forschungsgemeinschaft (DFG Br1583/1-2). We wish to thank Renate Hegenloh for perfect technical assistance. REFERENCES 1.

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Table 1 Genes congruently regulated in human neointima retrieved of in-stent-restenosis and in the human organ culture model after angioplasty

Native Control

Native Neoint ima

OCM Control

OCM 21 days

OCM 56 days

Fold change in native tissue

0.007

0.150

0.138

0.289

0.000

+

20.1

+

2.1

M54995 U85611 X06234 X06233 M31932 M34570 X59932 AF019562 U14188 U35735 Z36715

BCL-2 binding athanogene-1 (BAG-1) beta thromboglobulin; neutrophil activating peptide 2 (NAP2) calcium & integrin-binding protein (CIB) calgranulin A; (MRP8) calgranulin B; (MRP14) CD32 antigen; FC-gamma RII-A collagen 6 alpha 2 subunit (COL6A2) c-src kinase (CSK); protein-tyrosine kinase cyl DNAX activation protein 12 ephrin A4 precursor (EFNA4) erythrocyte urea transporter (UTE) ets domain protein elk-3

0.085 0.141 0.055 0.313 0.000 0.010 0.098 0.054 0.014 0.202 0.000

1.147 0.505 1.054 1.137 0.457 0.363 0.634 0.695 0.608 0.548 0.262

0.000 0.265 0.000 0.065 0.071 0.000 0.127 0.089 0.000 0.029 0.041

0.817 0.837 0.133 0.904 0.586 0.136 0.474 0.697 0.428 0.705 0.326

0.331 0.554 0.348 0.613 0.742 0.000 0.398 0.350 0.108 0.488 0.441

+ + + + + + + + + + +

13.4 3.6 18.7 3.6 457.9 33.5 6.4 12.6 41.0 2.7 263.1

+ + + + + + + + + + +

818.5 3.1 349.0 13.7 10.3 137.0 3.7 7.8 428.5 23.8 10.6

M76673 J03909 M62880 U05875 J05070 M22324

FMLP-related receptor I (FMLPRII) gamma-interferon-inducible protein; IP-30 integrin beta 7 subunit precursor (ITGB7) interferon-gamma (IFN-gamma) receptor beta matrix metalloproteinase 9 (MMP9) microsomal aminopeptidase N; glycoprotein CD13

0.009 0.033 0.067 0.037 0.012 0.009

0.182 0.224 0.297 0.290 0.191 0.367

0.000 0.000 0.000 0.079 0.020 0.000

0.116 0.176 0.175 0.387 0.368 0.239

0.107 0.251 0.004 0.276 0.057 0.402

+ + + + + +

18.4 6.6 4.4 7.6 15.0 36.8

+ + + + + +

117.0 252.0 175.6 4.9 17.3 403.0

M25665 U63717 M86400 X54936 M63193 M59979 M63488 U85625

neutrophil NADPH oxidase factor 1; p47-PHOX osteoclast stimulating factor phospholipase A2 placenta growth factors 1 + 2 (PLGF1 + PLGF2 ) platelet- derived endothelial cell growth factor prostaglandin G/H synthase 1, COX-1 replication protein A 70-kDa subunit (RPA70) ribonuclease 6 precursor

0.059 0.032 0.064 0.032 0.106 0.028 0.021 0.002

0.764 0.519 0.665 0.177 0.906 0.549 0.386 0.274

0.000 0.404 0.208 0.117 0.139 0.000 0.000 0.000

0.187 0.721 0.647 0.642 0.583 0.450 0.169 0.100

0.017 0.825 0.472 0.233 0.514 0.632 0.074 0.000

+ + + + + + + +

12.7 16.0 10.3 5.4 8.5 18.8 17.4 93.8

+ + + + + + + +

187.5 2.0 3.1 5.5 4.2 633.0 170.0 100.5

X85106 J03040

ribosomal protein S6 kinase II alpha 2 secreted protein acidic and rich in cysteine (SPARC)

0.009 0.374

0.306 0.999

0.000 0.284

0.040 0.783

0.143 0.666

+ +

31.6 2.7

Genbank accession no.

Name

Max. fold change in OCM

A S83171

+ 41.0 + 2.7 Page 15 of 21 (page number not for citation purposes)

M32313 X14787

steroid 5-alpha reductase 1 (SRD5A1) thrombospondin 1 precursor (THBS1; TSP1)

0.013 0.012

0.149 0.126

0.070 0.405

0.202 0.477

0.126 1.088

+ +

10.4 9.8

+ +

2.9 2.7

U59167 X97335 U24163 V00568

desmin (DES) dual-specificty A-kinase anchoring protein 1 frizzled-related FrzB (FRITZ) c-myc oncogene GTP-binding protein ras associated with diabetes (RAD1) serum response factor (SRF)

1.179 0.119 0.199 0.121

0.100 0.000 0.000 0.000

1.078 0.137 0.243 0.635

0.653 0.099 0.373 0.132

0.454 0.000 0.000 0.131

-

0.1 0.0 0.0 0.0

-

0.4 0.0 0.0 0.2

0.308 0.325

0.083 0.095

0.866 0.335

0.386 0.188

0.060 0.000

-

0.3 0.3

-

0.1 0.0

B

L24564 J03161


Fig. 1

Figure 1. Hierarchical cluster analysis of data from time course after balloon angioplasty. Genes were selected for this analysis if their expression level deviated by at least a factor of 2.5 among groups and reached a descriptive P < 0.05. Each gene is represented by a single row and each time point by a single column. For each expression value, median mRNA level in each group normalized to mRNA expression level of housekeeping genes is represented by a gray value; intensity according to expression level as indicated in scale at bottom. A cluster comprising all 264 differentially expressed genes is shown on left. Groups A-D indicate upregulated genes. Group A: upregulation at 21 days, no effect by rapamycin treatment. Group B: upregulation at 21 days, inhibited by rapamycin treatment. Group C: sustained upregulation, no effect by rapamycin treatment. Group D: sustained upregulation, inhibited by rapamycin treatment. Group E: downregulated and transiently expressed genes. On right, amplified gene clusters of each group. Page 17 of 21 (page number not for citation purposes)

Fig. 2

Figure 2. Time course dependent up- or down-regulation of functionally clustered, differentially expressed genes associated with inflammation (left columns), proliferation/apoptosis (central columns), and adhesion/cytoskeleton/ECM (right columns). Value on vertical axis is number of regulated genes; number of genes that have previously been described to be expressed in HPC or other immature bone marrow cells is indicated by shaded area within columns.


Fig. 3

Figure 3. Functional cluster analysis of differentially expressed genes associated with inflammation (A), adhesion/cytoskeleton/ECM (B), and proliferation/apoptosis (C). Expression values are visualized as described in Fig. 1.


Fig. 4

Figure 4. Influence of rapamycin on gene expression. A) Percentual influence of rapamycin on gene expression in different functional groups. A deviation by at least a factor of 2.5 between the single groups was regarded as relevant influence. B) Functional cluster image showing genes differentially regulated both in organ culture model after angioplasty as well as by rapamycin treatment. C) Functional cluster image of genes regulated by rapamycin only that showed no significant alteration after angioplasty. Expression pattern of each gene is displayed as a horizontal strip. Each column is a single time point. For each expression value, median of mRNA level of single experiments normalized to mRNA expression level of housekeeping genes is represented by gray value according to signal intensity scale at bottom. Page 20 of 21 (page number not for citation purposes)

Fig. 5

Figure 5. Validation of array data by PCR and immunohistochemistry. A) Verification of altered mRNA levels after angioplasty by gene-specific PCR for β-thromboglobulin, COX-1, and GADD45. Rapamycin-dependent downregulation of mRNA-levels is shown for β-thromboglobulin and COX-1. B) Densitometric analysis of mRNA expression ±SE for βthromboglobulin, COX-1, GADD45, OSMBR and JAK1. *P < 0.05 over the time course, °P < 0.05 between untreated and rapamycin-treated samples. C) Densitometric analysis of mRNA expression ±SE for thrombospondin-1, MMP-2, MMP-9, SDF-1, thrombin receptor, and PGlF1+2. *P < 0.05 over time course. D) Immunohistochemical staining with an antibody against β-thromboglobulin: β-thromboglobulin protein expression after angioplasty in organ culture model (left) is reduced by rapamycin treatment (right). Page 21 of 21 (page number not for citation purposes)