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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^*,2

^* I. Medizinische Klinik und Deutsches Herzzentrum,
Institut für Pathologie der Technischen Universität München;
II. Medizinische Klinik und
Urologische Klinik der Universität Ulm;
^|| Urologische Abteilung der Klinik Biberach;
^¶ Urologische Abteilung der Klinik Heidenheim; and
^# Micromet AG, Munich, Germany

² Correspondence: E-mail: d_zohlnhoefer{at}yahoo.com

SPECIFIC AIMS

Rapamycin combines antiproliferative and anti-inflammatory properties and reduces restenosis after angioplasty. We investigated transcriptional programs governing neointima formation in a human organ culture model with respect to 1) whether there are time-dependent changes in gene expression after angioplasty; 2) how the pathophysiological mechanisms of neointima formation relate over time; 3) whether there is evidence for involvement of hematopoietic progenitor cells (HPC) in human neointima formation; and 4) how rapamycin acts on transcriptional changes after angioplasty.

PRINCIPAL FINDINGS

1. Changes in gene expression occur in a time-dependent manner with a maximal alteration 21 days after injury
Angioplasty was performed on segments of renal arteries extracted during routine nephrectomies with an inflation pressure of 9 bar for 60 s. Untreated arterial segments served as controls (n=5). After ballooning, segments were stored within 15 min (n=6) or cultured for 21 (n=4) and 56 days (n=5) in a 1:1 mixture of Waymouth’s MB 752/1 and Ham F12 nutrient mixture supplemented with 15% FCS. Rapamycin was added at a concentration of 100 ng/mL for 21 (n=3) and 56 days (n=4).

Of 2312 genes analyzed with cDNA array technology, 264 were regarded as differentially expressed over the time course studied. Criteria for differential expression were a descriptive P<0.05, a minimum ratio of 2.5 and a minimal difference of 0.1 within group medians. Two-dimensional hierarchical clustering of differentially expressed genes revealed up-regulation of the majority of genes (n=212), whereas 44 genes showed consistent down-regulation over the time course of neointima formation. Eight genes showed inconsistent but differential regulation.

Balloon angioplasty led to significant up-regulation of 65 genes immediately after angioplasty. 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. Therefore, vascular injury had the strongest effect on gene expression during the first three weeks.

2. Early activation of inflammation-associated genes is followed by profound up-regulation of proliferation- and apoptosis-associated genes
We performed functional clustering of all 264 differentially expressed genes. Of these, 45 genes were related to inflammation, 52 to extracellular matrix production and adhesion, and 95 to proliferation and apoptosis—a total of 192 genes representing 72% of all differentially expressed genes (Fig. 1 ).

View larger version (14K): [in this window] [in a new window]   Figure 1. Functional cluster analysis of differentially expressed genes. Time course dependent up- or down-regulation of genes associated with inflammation (left columns), proliferation/apoptosis (center columns) and adhesion/cytoskeleton/ECM (right columns). The value on the vertical axis indicates the number of regulated genes, the number of genes that have been described to be expressed in HPC, or other immature bone marrow cells and is indicated by the shaded areas within the columns.

Nearly equal activation in all three functional groups was observed immediately after balloon injury (Fig. 1 , left), when 53 genes showed altered expression. 114 genes (Fig. 1 , center) were regulated after 21 days, indicating major changes in gene expression took place at this point. The majority of differentially expressed genes after 21 days was associated with proliferation such as CDK4, PCNA, or VEGF, suggesting a predominant role of proliferation and apoptosis in the later time points. The relative impact of inflammation was maximal shortly after angioplasty and decreased over time.

3. Balloon angioplasty leads to a gene expression pattern promoting recruitment and activation of inflammatory and hematopoietic progenitor cells
Inflammation-associated genes regulated in our study included genes such as IL-1ß, IL-8, or prostaglandin G/H synthase 1 (COX-1), and less studied genes such as granulocyte chemotactic protein 2 (GCP-2), endothelial monocyte-activating peptide II (EMAP-II), or ß-thromboglobulin (ß-TG).

Fifty-seven of the 192 genes belonging to the three groups have been described as being expressed in hematopoietic progenitor cells or other immature bone marrow cells (Fig. 1 , shaded part of columns). Likewise, we found an up-regulation of SDF-1, MMP-9, CD157, JAK1, and oncostatin M receptor ß. This finding supports the notion that recruitment, proliferation, and differentiation of progenitor cells may play an important role in the pathogenesis of human neointima formation.

4. Rapamycin prevents induction of a proadhesive and proinflammatory gene expression pattern as well as induction of HPC promotors
Statistical analysis yielded 117 genes significantly down-regulated by rapamycin in the human organ culture model. Most down-regulated genes were related to proliferation/apoptosis such as VEGF-C, caspase-8, or HIF1- . Rapamycin treatment also reduced expression of many genes related to inflammation such as IL-8, EMAP-II, GCP-2, or COX-1. A lower ß-thromboglobulin (NAP-2) expression was shown on the protein level (Fig. 2 B, D).

View larger version (48K): [in this window] [in a new window]   Figure 2. Validation of array data by PCR and immunohistochemistry. A) Verification of altered mRNA levels after angioplasty by gene-specific PCR for ß-thromboglobulin, COX-1, and GADD45. Rapamycin-dependent down-regulation of mRNA-levels is shown for ß-thromboglobulin and COX-1. B) Densitometric analysis of mRNA expression ±SEM for ß-thromboglobulin, COX-1, GADD45, OSMBR, and JAK1. *P < 0.05 over time course, °P< 0.05 between untreated and rapamycin-treated samples. C) Densitometric analysis of mRNA expression ±SEM for thrombospondin-1, MMP-2, MMP-9, SDF-1, thrombin receptor, and PGlF1+2. *P < 0.05 over time. D) Immunohistochemical staining with an antibody against ß-thromboglobulin. ß-thromboglobulin protein expression after angioplasty in the organ culture model (left) is reduced by rapamycin treatment (right). Scale bars indicate 100 µm.

Rapamycin additionally reduced expression of genes related to HPC such as CD157, JAK1, or the oncostatin M receptor (Fig. 2B ), hence modulating this mechanism of HPC recruitment contributing to neointima formation.

CONCLUSIONS AND SIGNIFICANCE

Rapamycin reduces risk of in-stent restenosis in humans. We show that after angioplasty: 1) changes in gene expression occur in a time-dependent manner with a maximal alteration 21 days after injury; 2) inflammation has a higher early effect while proliferation and apoptosis dominate later alterations in gene expression; 3) balloon angioplasty leads to a gene expression pattern indicative of facilitated recruitment and activation of inflammatory as well as hematopoietic progenitor cells; and 4) rapamycin prevents induction of a proadhesive and proinflammatory gene expression pattern as well as induction of HPC promoters.

We analyzed gene expression using cDNA array technology immediately at 21 days, and 56 days after balloon dilation. It became evident that changes in gene expression are time dependent, with peak alteration after 21 days, and that only a few genes are regulated exclusively after 56 days. We conclude that 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. Since they release 80% of the total drug dose within in the first 30 days after placement, restenosis is unlikely to depend on late effects.

The coordinated induction of a proinflammatory gene expression pattern in our model provides a rationale for profound leukocyte recruitment after balloon dilation. Cytokines such as IL-8, EMAP-II, or GCP-2 were up-regulated after angioplasty and enhanced the migration of granulocytes. We observed persistent up-regulation of ß-thromboglobulin. Cleavage of this cytokine results in active NAP-2, which promotes adhesion and transendothelial migration of neutrophil granulocytes.

In addition, we found an up-regulation of genes related to HPC. Likewise, IL-8 induces mobilization of hematopoietic progenitor cells. Enhanced release of stem and progenitor cells from bone marrow has been shown to be caused by MMP-9, a matrix metalloproteinase known to be up-regulated by IL-8 as well as by SDF-1. SDF-1 itself induces chemotaxis of HPC. SDF-1 was recently identified as playing an important role in murine neointima formation. Thus, it is conceivable that hematopoietic progenitor cells contribute to some extent to the cell content of murine neointima. Because 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.

The macrolide antibiotic rapamycin has been shown to dramatically decrease human in-stent restenosis in several clinical studies. As rapamycin slows graft vasculopathy in humans, its beneficial effect on that type of occlusive vascular disease may have implications for its effectiveness in reducing postangioplasty restenosis. In a cardiac allograft model, rapamycin reduced IL-8 expression and strongly diminished neutrophil infiltration. In our study, induction of IL-8 after angioplasty was completely inhibited by rapamycin treatment. Rapamycin treatment led to coordinated suppression of the CXC chemokines 6-8 (GCP2, ß-thromboglobulin, IL-8) or EMAP-II, which play an important role in adhesion, migration, and activation of circulating neutrophils and monocytes. Reduced neutrophil recruitment and activation following rapamycin treatment may lead to reduced recruitment of HPC to the vessel wall and reduction of neointima formation.

Rapamycin profoundly inhibits proinflammatory gene expression pattern and promoters of HPC after vascular injury. We provide new data demonstrating how rapamycin may reduce recruitment of HPC to vessel wall in humans; this may explain the high effectiveness of rapamycin in reducing restenosis.

Our model represents an excellent tool for scrutinizing new drugs developed to prevent restenosis. Pleiotropic properties will be indispensable for evaluation of any drug that may come into competition with rapamycin, the most effective drug for reducing restenosis in humans.

View larger version (39K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2431fje;

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