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. Author manuscript; available in PMC: 2012 Jul 1.
Published in final edited form as: J Vasc Surg. 2011 Apr 14;54(1):182–191.e24. doi: 10.1016/j.jvs.2010.12.070

A Link between Smooth Muscle Cell Death and Extracellular Matrix Degradation during Vascular Atrophy

Richard D Kenagy a,*, Seung-Kee Min b,*, Eileen Mulvihill a, Alexander W Clowes a
PMCID: PMC3129478  NIHMSID: NIHMS271534  PMID: 21493032

Abstract

Objective

We have previously reported that high blood flow induces neo-intimal atrophy in polytetrafluoroethylene (PTFE) aorto-iliac grafts and that a tight external PTFE wrap of the iliac artery induces medial atrophy. In both non-human primate models, atrophy with loss of smooth muscle cells and extracellular matrix (ECM) begins within 4 days. We hypothesize that matrix loss is linked to cell death, but the factors and mechanisms involved are not known. The purpose of this study was to determine commonly regulated genes in these two models, which we hypothesized would be a small set of genes that might be key regulators of vascular atrophy.

Methods

DNA microarray analysis (Illumina Sentrix Human Ref-8; ~23,000 genes) was performed on arterial tissue from the wrap model (n=9) and graft neointima from the graft model (n=5) one day after wrapping or the switch to high flow, respectively. Quantitative polymerase chain reaction (qRT-PCR) was also performed. Expression of this vascular atrophy gene set was also studied in two in vitro models of Fas ligand-induced cell death (cultured smooth muscle cells and organ cultured arteries).

Results

By microarray analysis fifteen genes were found to be regulated in the same direction in both atrophy models − 9 up-regulated and 6 down-regulated. Of these genes, 7 of 9 up-regulated genes were confirmed by RT-qPCR in both models. Upregulated genes included ECM degrading enzymes (ADAMTS4, tissue plasminogen activator, and hyaluronidase 2), possible growth regulatory factors (chromosome 8 open reading frame 4 [TC1] and leucine-rich repeat family containing 8), a differentiation regulatory factor (musculoskeletal embryonic nuclear protein 1), a dead cell removal factor (ficolin 3), and a prostaglandin transporter (solute carrier organic anion transporter family member 2A1). Five down-regulated genes were confirmed but only in one or the other model. Of the 7 up-regulated genes, ADAMTS4, tissue plasminogen activator, hyaluronidase 2, solute carrier organic anion transporter family member 2A1, leucine-rich repeat family containing 8, and chromosome 8 open reading frame 4 (TC1) were also up-regulated in vitro in cultured smooth muscle cells or cultured iliac artery by treatment with FasL, which causes cell death. However, blockade of caspase activity with ZVAD inhibited FasL-mediated cell death, but not gene induction.

Conclusion

A total of 7 gene products were up-regulated in two distinctly different in vivo non-human primate vascular atrophy models. In addition, induction of cell death by FasL in vitro induced 6 of these genes, including the ECM degrading factors ADAMTS4, hyaluronidase 2, and tissue plasminogen activator, suggesting a mechanism by which the program of tissue atrophy coordinately removes extracellular matrix as cells die. These genes may be key regulators of vascular atrophy.

A novel approach for the treatment of restenotic stented arteries, vein grafts, and arterio-venous fistulas would be to induce atrophy of the established intimal lesion. This would enable treatment of only affected patients rather than all patients as required when prevention of neointimal hyperplasia is the strategy1. Intimal atrophy occurs naturally at late times in stented arteries in rats, pigs and the majority of humans2–5, but little is known about its regulation. Therefore, we have established two different models of vascular atrophy in baboons. In the first model, neointima forms over 2 months in aorto-iliac polytetrafluoroethylene (PTFE) grafts and is then induced to regress in response to a marked increase in blood flow following construction of a femoral arterio-venous fistula6–7. In the second model, the media of a normal iliac artery regresses in response to a tight PTFE wrap8. In both models, loss of smooth muscle cells (SMCs) and matrix degradation are apparent by four days 7–8, and SMC death (TUNEL labeling) is observed in the PTFE graft model by one day9. We hypothesize that matrix loss is linked to cell death, but the factors and mechanisms involved are not known. For example, we previously tested the hypothesis that nitric oxide is required for graft neointimal atrophy based on the observations that endothelial nitric oxide synthase is increased in the regressing graft endothelium10 and that nitric oxide can inhibit SMC proliferation11. However, we found that pharmacological blockade of NOS did not affect the neointima7. To further test the hypothesis that ECM loss is linked to cell death, we have determined gene expression after one day in both models of vascular atrophy to define a subset of genes common to both models and have determined the effect of FasL-induced cell death on induction of these genes in vitro.

METHODS

Baboon Vascular Atrophy Models

PTFE graft

10 male baboons received bilateral aorto-iliac, 60 μm internodal distance, 4mm un-reinforced, polytetrafluoroethylene (PTFE; GoreTex) bypass grafts as described previously7. Two months after the bypass surgery, a distal 1 cm arteriovenous fistula was created distal to the patent graft between the superficial femoral artery and the femoral vein. Femoral artery flow was measured by duplex ultrasound just before and 1 day after fistula formation.

PTFE wrap

The common iliac arteries of 10 male baboons were tightly wrapped with a piece of 60 μm internodal distance, un-reinforced PTFE graft material by wrapping the collapsed artery and a 2.5 mm diameter rod together and removing the rod after sewing the graft material closed 8. The contralateral artery was dissected free from the surrounding tissue as a control.

Animal care and procedures were conducted at the University of Washington Regional Primate Research Center in accordance with state and federal laws and under protocols approved by the University of Washington Institutional Animal Care and Use Committee and the Regional Primate Research Center. Animal care and handling complied with the “Guide for the Care and Use of Laboratory Animals” issued by the Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, Washington DC, National Academies Press, 1996 (http://stills.nap.edu/readingroom/books/labrats/)

RNA isolation and quantification

PTFE graft intimal tissue or wrapped and unwrapped arteries were harvested 1 day after fistula surgery or wrapping, respectively, and snap-frozen in RNAlater. Total RNA was prepared from graft neointimas and iliac arteries using the RNeasy fibrous tissue midi kit as described by the manufacturer (Qiagen). Briefly, frozen tissue was pulverized under liquid nitrogen, homogenized in extraction buffer, and treated with proteinase K. RNA from cultured cells was obtained using the RNeasy mini kit (Qiagen) with the syringe and needle method of shearing. RNA preparations were treated with DNase1 prior to purification on the kit columns. RNA quality was assessed using the RNA 6000 Nano assay on the 2100 Bioanalyzer (Agilent Palo Alto, Calif); A260/280 ratios and yield were determined using the Nanodrop ND1000 spectrophotometer (Thermo Scientific).

Microarray analysis

The Illumina Sentrix Human Ref-8 system (Illumina Inc., San Diego, CA) was used according to the manufacturer’s instructions. In brief, total RNA was used for cDNA synthesis, followed by amplification and labeling to create biotin-cRNA probes (Ambion MessageAmp kit). Hybridization, washing, blocking, development, and scanning were performed as directed by the manufacturer. Summarized bead intensities for non-control probes were quantile normalized. Several quality metrics were applied and indicated RNA degradation in two graft model samples and one sample of the wrap model. These three a rrays plus their paired arrays were removed from the analysis. Statistical significance of gene expression differences was computed using Significance Analysis of Microarrays (SAM) software (http://www-stat.stanford.edu/~tibs/SAM/)12 without a threshold for fold change.

Five collections of gene sets (GO Biological Process, BP; GO Molecular Function, MF; GO Cellular Component, CC; and the C1 and C2 sets from Subramanian et al.13) were utilized for gene set analysis. Information about C1 and C2 sets can be found at: http://www.broad.mit.edu/gsea/msigdb/index.jsp. The software used has been described13–14. Analysis was restricted to gene sets with a minimum of 10 and a maximum of 500 genes. If a particular gene set is enriched in the wrapped or high flow group, the online Results files of log2 expression contrasts (e.g. wrapped to unwrapped) are presented online and have the word “positive” in the file name if a gene set is enriched in the wrapped or high flow group and “negative” in the file name if it is enriched in the unwrapped or normal flow groups.

Quantitative RT-PCR

RT-qPCR was performed using the 7500 Fast real-time PCR system (Applied Bio Foster systems, City, CA) according to the manufacturer’s instructions using Taqman primers and probes purchased from Applied Biosystems and SensiMix master mix from Quantace Ltd. Human probes for Gas6 and PLAT did not work in baboon tissues; therefore, new primers were chosen using Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and obtained from Integrated DNA Technologies (Coralville, Ia): Gas6 (human): 5′-CGGAATCTGGTCATCAAGGT-3′ and 5′-TCTTCTCCGTTCAGCCAGTT-3′; PLAT (human): 5′-CCCAGATCGAGACTCAAAGC-3′ and 5′-TGGGGTTCTGTGCTGTGTAA-3′: 18S (mammalian): 5′-AAACGGCTACCACATCCAAG-3′ and 5′-CCTCCAATGGATCCTCGTTA-3′. RT-qPCR for Gas6 and PLAT was performed using SYBR Green SensiMix DNA kit (Quantace) according to the manufacture’s instruction. Melting curves and size of product were tested to determine specificity. 18S was used as an endogenous control.

In Vitro Studies

Baboon aortic SMCs were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% calf serum. SMCs were seeded at 20,000/cm2 in 6-well plates with 10% calf serum. The next day the cell layer was washed with phosphate-buffered saline and medium was changed to DMEM without serum plus recombinant human soluble FasL/FLAG fusion protein (50ng/ml), anti-FLAG (2 μg/ml) and 1.5 μg/ml cycloheximide. After 6 and 24 hours RNA was extracted using RNeasy kits as described above. For experiments with ZVAD, ZVAD (50 μM) was added (or equivalent DMSO to controls and FasL treated cultures) 30 minutes prior to the addition of FasL, anti-FLAG, and cycloheximide as above. After 24 hours medium was removed and spun at 1000g to remove floating cells for counting. Cell layers were harvested for RNA as above.

For organ culture experiments, baboon iliac arteries were obtained from the Washington National Primate Research Center tissue program. Periadventitial tissue was removed and the arteries were opened longitudinally. Arterial tissue (1.5 cm length) was incubated in a 12-well plate containing 2 ml of 10 mM HEPES/DMEM, pH 7.4, with or without FasL/FLAG fusion protein (100ng/ml), anti-FLAG (2 μg/ml), and 1.5 ug/ml cycloheximide. Arteries were snap-frozen 6 hours later and stored at −80°C until extraction of RNA using fibrous RNeasy kits as described above.

Statistical analysis

Values reported are the mean ± SEM. Statistical differences (other than microarray data) were tested using a one-tailed Wilcoxon signed rank test.

RESULTS

PTFE grafts in three animals were found occluded just before the fistula formation, so fistulas were made in 7 animals. Unfortunately RNA from one wrapped artery and two graft neointimas was degraded and was excluded from further analysis. A total of 9 pairs of wrapped arteries and 5 pairs of graft intima were analyzed by microarray.

Genes regulated during vascular atrophy in vivo

Paired comparisons of gene expression by microarray were made between wrapped and unwrapped arteries and between high flow and normal flow PTFE graft intimas. In the wrapped arteries, we found 404 upregulated genes and 407 down regulated genes among the 24,357 genes examined using an estimated false discovery rate of <1% (online Table 1). In the graft neointimas, we found 58 upregulated genes and 26 down regulated genes (online Table 2). In this case, we used a 10% false discovery rate for the analysis of the graft intimas, because of the small number of samples. Comparison of the genes that were significantly changed in the two models demonstrates the degree of difference between these two models (Fig. 1). However, we did identify 15 genes regulated in the same direction in both models (Table I). The correlation of expression between the two models for these 15 genes was significant (Spearman’s r= 0.76, P<.001).

Fig. 1.

Fig. 1

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Venn diagram of significantly regulated genes during flow-mediated graft neointimal atrophy and wrap-mediated medial arterial atrophy. Fifteen genes were increased or decreased in both baboon models.

Table I.

Genes regulated in both models of vascular atrophy

Gene RefSeq # Name Wrap Graft
ADAMTS4 NM_005099 A Disintegrin and Metalloproteinase with Thrombospondin motifs 4 2.94 (0) 1.58 (0)
FCN3 NM_173452 ficollin 3 2.00 (0) 2.26 (0)
MUSTN1 NM_205853 musculoskeletal embryonic nuclear protein 1 (Mustang 1) 1.98 (0) 3.00 (0)
HIST2H2AC NM_003517 histone 2H2ac 1.74 (0) 1.47 (0)
PLAT NM_000931 tissue plasminogen activator 1.66 (0) 1.47 (8.15)
LRRC8 XM_026998 leucine-rich repeat family 8 containing 1.62 (0) 1.37 (8.15)
C8orf4 NM_020130 TC (thyroid cancer) 1 1.49 (0) 1.81 (0)
SLCO2A1 NM_005630 Solute carrier organic anion transporter family, member 2A1 (PGT) 1.39 (0.17) 2.15 (0)
HYAL2 NM_003773 hyaluronidase 2 1.34 (0) 1.47 (8.22)
FABP3 NM_004102 fatty acid binding protein 3 0.74 (0.84) 0.64 (6.30)
FMOD NM_002023 fibromodulin 0.77 (0.71) 0.60 (6.30)
OGN NM_014057 osteoglycin 0.77 (0.58) 0.57 (6.30)
MATN2 NM_030583 matrilin 2 0.77 (0.32) 0.70 (6.30)
TNNC1 NM_003280 troponin C type 1 0.56 (0) 0.73 (8.18)
GAS6 NM_000820 growth arrest specific protein 6 0.66 (0) 0.67 (6.30)
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Values are the fold of unwrapped or normal flow, respectively. Values in parentheses are q values from SAM analysis.

To validate the microarray data, RT-qPCR of the 15 genes was performed. All but one of the 9 genes upregulated in wrapped arteries were verified (Fig. 2A-I). These included 3 genes involved in ECM regulation – ADAMTS4, PLAT (tissue plasminogen activator), and HYAL2 (hyaluronidase 2). The other five genes were MUSTN1 (Mustang 1), FCN3 (ficolin 3), SLCO2A1 (solute carrier organic anion transporter 2A1, also called the prostaglandin transporter or PGT), C8orf4 (thyroid cancer 1 or TC1), and LRRC8 (leucine rich repeat containing 8). In the graft intimas PLAT, HYAL2, MUSTN1, FCN3, SLCO2A1, and C8orf4 showed the same trend (P=.063; only 4 paired samples were available for analysis), and ADAMTS4 was previously verified at the protein and mRNA levels15. Among the 6 downregulated genes, MATN2 (matrilin 2), TNNC1 (troponin C), and FABP3 (fatty acid binding protein 3) were verified in the wrap model (Fig. 2J-O), while GAS6 and OGN (osteoglycin) demonstrated a trend to be decreased in the graft (P=.063).

Fig. 2.

Fig. 2

Fig. 2

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RT-qPCR of genes found by microarray analysis to be regulated in the PTFE wrap and PTFE graft models of vascular atrophy. (A) ADAMTS4, see Kenagy et al{Kenagy, 2009 #12545} for graft data, (B) Ficolin 3 (FCN3), (C) Mustang 1 (MUSTN1), (D) Histone 2H2A (HIST2H2A), (E) tissue plasminogen activator (PLAT), (F) LRRC8, (G) C8orf4 or TC1, (H) SCLO2A1 or the prostaglandin transporter, PGT, (I) hyaluronidase 2 (HYAL2). Each point represents a triplicate determination of paired samples of RNA from an individual animal’s iliac arteries or PTFE graft intimas. Values are expressed as the ratio of wrapped/unwrapped or high blood flow/normal blood flow. The dotted line represents a ratio of 1 and the continuous line the mean value. *P<.02; #P=.063

Gene Set Analysis of atrophy in vivo

The results of gene set analysis of the microarray data were consistent with the results from analysis of individual genes in that a comparison of the two atrophy models yielded a small number of gene sets regulated in common. The two models shared only 4 gene sets of the Biological Process collection. Three were significantly higher (GO:0045892 negative regulation of transcription, DNA-dependent; GO:0006974 response to DNA damage stimulus; and GO:0042127 regulation of cell proliferation; gene set lengths of 42, 23, and 40, respectively) and one was significantly lower (GO:0050819 negative regulation of coagulation; gene set length of 11) comparing wrapped to unwrapped and high flow to normal flow (see online Figs. I–IV).

Gene regulation during apoptosis of SMCs in vitro

We have found a small number of genes that are regulated in the same manner in two models of vascular atrophy. Since cell death is a significant aspect of both of these models, it is possible that these genes are regulated as part of a cell death pathway. To explore this possibility, we studied the effect of Fas ligand (FasL) on gene expression; we have previously shown that FasL at this dose kills ~30% of baboon SMCs by 24 hours15. Of the 7 up-regulated genes, we found that tissue plasminogen activator (PLAT), the prostaglandin transporter (SLCO2A1), C8orf4, and LRRC8, were increased by FasL in vitro (Fig. 3A-H), but hyaluronidase 2 (HYAL2), Mustang 1 (MUSTN1), and ficolin 3 (FCN3) were not changed. We have previously reported that ADAMTS4 mRNA is increased 5 fold after FasL treatment15. As a positive control we showed that MCP-1, which is induced by FasL16, was also increased by FasL (27 ± 18 and 12 ± 6 fold of control at 6 and 24 hours, respectively; mean ± SD of two experiments).

Fig. 3.

Fig. 3

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The effect of FasL on expression of (A) tissue plasminogen activator (PLAT), (B) hyaluronidase2 (HYAL2), (C) LRRC8, (D) prostaglandin transporter (SLCO2A1), (E) C8orf4 (TC-1), (F) ficolin 3 (FCN3), and (G) Mustang 1 (MUSTN1) in cultured SMCs expressed as fold of 6 or 24 hour control. * P<.05 vs control; mean ± SEM of 3 experiments.

The observation that only 5 of 7 genes that were up-regulated in vivo were also up-regulated in the cultured SMC death model raised the possibility that these differences may result from the lack of endothelial cells or the lack of a physiological ECM in the in vitro model of cell death. Therefore, we also studied the effect of a 6 hour treatment with FasL on the expression of the up-regulated genes in cultured baboon arteries. In contrast to the cultured SMCs, FasL induced hyaluronidase 2 in the artery (Fig 4B). Tissue plasminogen activator (PLAT), the prostaglandin transporter (SLCO2A1), C8orf4, and ADAMTS4 were induced in the artery to a similar degree as observed in SMCs (Fig. 4A, D, E, H). Ficolin 3 and MUSTN1 were not induced by FasL, and the modest stimulation of LRRC8 was not statistically significant.

Fig. 4.

Fig. 4

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The effect of FasL on expression of (A) tissue plasminogen activator (PLAT), (B) hyaluronidase2 (HYAL2), (C) LRRC8, (D) prostaglandin transporter (SLCO2A1), (E) C8orf4 (TC-1), (F) ficolin 3 (FCN3), and (G) Mustang 1 (MUSTN1) in arterial organ culture expressed as fold of control. * P<.05 vs control; † P=.06; mean ± SEM of 3–4 experiments.

To determine whether blocking apoptosis would prevent induction of the atrophy related genes by FasL, we used the pan-caspase inhibitor, ZVAD. This series of experiments was performed with a different baboon SMC line than the one used for data presented in figure 3. FasL induced the prostaglandin transporter (SLCO2A1), C8orf4, and ADAMTS4, but not PLAT and LRRC8. This is not surprising given the variability of gene induction also observed in vivo (Fig. 2). We found that addition of 50 μM ZVAD completely blocked the production of floating SMCs by FasL at 24 hours, but ZVAD did not inhibit FasL-mediated increases in ADAMTS4, C8orf4, and SLCO2A1 (Table II).

Table II.

Effect of ZVAD on FasL-induced gene expression and number of floating SMCs

FasL FasL + ZVAD
ADAMTS4 2.19 ± 0.54 2.09 ± 0.53
C8orf4 3.28 ± 0.67 3.58 ± 0.51
LRRC8 1.23 ± 0.25 1.44 ± 0.17
PLAT 0.66 ± 0.14 0.73 ± 0.15
SLCO2A1 2.50 ± 0.46 4.52 ± 1.02
Floating SMCs 12.0 ± 4.3 0.2 ± 0.1
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Values are expressed as the fold of control (mean ± SEM of 3 experiments).

DISCUSSION

We have demonstrated that only a small number of genes are up-regulated in both non-human primate models of vascular atrophy, namely medial atrophy in the tightly- wrapped iliac artery and neointimal atrophy in a PTFE graft subjected to a switch from normal to high blood flow. The comparison of two distinctly different models of atrophy, one caused by changes in shear stress and the other caused by changes in wall stress, was purposely used to narrow the results obtained from microarray analysis. We chose the one day time point, at which time there is increased cell death and decreased cell proliferation in the PTFE graft model, but before significant changes occur in wall mass9. Since there is essentially no proliferation in the artery model, we expected that there might be changes in both models in genes related to cell death. Our results support the conclusion that these commonly regulated genes may play a fundamental role in vascular atrophy. In addition, the fact that 6 of 7 atrophy-associated genes are also induced by the ligand for the death receptor Fas, further suggests that these genes are an important part of the vascular cell death program. These ideas are supported by the observation that one of the genes, ficolin 3, binds to and mediates the uptake of apoptotic cells by macrophages17. While there are few macrophages in the regressing tissue in the baboon atrophy models7–8, SMCs also can phagocytose dead cells18. Also, a third of the up-regulated genes degrade components of the ECM, loss of which is a major feature of vascular atrophy. For example, the ECM constitutes ~80% of neo-intimal volume6,19 and loss of cross-sectional area at 4 days in both models occurs with no change in nuclear density consistent with a loss of ECM along with cells. While there is a preferential loss of versican in the PTFE graft ECM, components of the ECM appear to be lost proportionately in the wrapped arteries8,20. Tissue plasminogen activator activates plasminogen to plasmin, and plasmin degrades numerous ECM components and can also activate some matrix degrading matrix metalloproteinase family members 21–22. Hyaluronidase 2 degrades the pro-migratory and pro-proliferative ECM that hyaluronan forms with versican23, and it also generates ~20 kD fragments of hyaluronan that may induce MCP1 and other inflammatory factors24–25. ADAMTS4 can degrade a number of ECM proteins including versican, aggrecan, biglycan, matrilins, and brevican26–31.

Cell death and ECM degradation

Our data add to the literature linking cell death to enzymatic degradation of ECM. Several classes of proteases are increased in dying cells32–35 and may be responsible for maintaining the cell-ECM balance by removing the ECM associated with dying cells. They can also cause cell death via reduction of matrix/cell attachment factors required for viability. For example, Kovanen and colleagues reported that chymase causes SMC death by degrading the integrin-binding matrix factor, fibronectin36. Of particular relevance to our study, plasmin generated from plasminogen by tissue plasminogen activator can cause SMC death37. Since tissue plasminogen activator is induced by FasL, it may be involved in a positive feedback loop to facilitate cell death. In addition, it is intriguing that tissue plasminogen activator inhibits the accumulation of intimal SMCs and causes positive arterial remodeling after carotid artery injury38. With regards to ADAMTS4, both SMC death and ADAMTS4 are increased in the PTFE graft neointima one day after the switch to high blood flow7, 15 and dying intimal SMCs express ADAMTS415. Furthermore, we have shown that ADAMTS4 cleaves versican, a major constituent of graft neointima15, 26. The cleavage fragments of versican may play an important role during neo-intimal atrophy39. This has been shown recently for regression of cardiac outflow tract and interdigital tissue in the mouse40–41. ADAMTS5, 9, and 20 cooperatively clip versican at the Glu-441-Ala-442 bond (the same bond cleaved by ADAMTS442) and generate an N-terminal fragment that increases apoptosis in interdigital web cells. Threshold levels of the versican N-terminal fragment may be required for tissue regression.

Potential functions of other atrophy-associated genes

The function of the other up-regulated gene products associated with atrophy is less clear. SLCO2A1 (PGT) transports prostaglandins into cells where they can be degraded43–44. Prostaglandins of the D, E, and F series are transported at a high rate, thromboxane at an intermediate rate, and prostacyclin analogs at a low rate45–46. Since SMCs have been reported to synthesize PGE2, PGI2, and PGD247–50, increased SLCO2A1 might be expected to have the greatest impact on reducing local levels of PGE2 and PGD2, which utilize the receptors, EP1 through EP4 and DP1 and 2, respectively. While prostaglandin receptor expression has not been measured in these atrophy models and these receptors can mediate opposite effects (e.g. PGE2 receptors EP1 and EP3 induce vasoconstriction, whereas EP2 and EP4 induce vasodilatation51), it is of interest that EP4 appears to mediate intimal hyperplasia. Activation of EP4 increases ductus intimal cushion formation52 and PGE2 may also activate the thromboxane receptor53–54, knockout of which leads to decreased neointima formation after arterial injury55. These data suggest the possibility that SLCO2A1 mediated loss of local PGE2 might remove a pro-proliferative signal. C8orf4, which is overexpressed in thyroid and breast cancers56–57, has been shown to cause anchorage-independent growth when overexpressed in immortalized epithelial cells58, to mediate IL-1β-induced proliferation of a dendritic-like cell line59, and to inhibit Chibby60, a negative regulator of β catenin signaling. β catenin stimulates SMC proliferation and inhibits cell death31,61. Musculoskeletal embryonic nuclear protein 1 (MUSTN1) is required for differentiation of chondrocytes and myoblasts62–63. Whether MUSTN1 is affecting SMC differentiation is uncertain. Message levels of SMC differentiation genes such as SM22α (TAGLN), smoothelin (SMTN), desmin (DES), and calponin (CNN1) are decreased in the wrap model, but not in the graft neointima. Other smooth muscle differentiation genes such as smooth muscle myosin heavy chain, smooth muscle alpha actin (ACTA2), and caldesmon are not altered in either model (online Tables 1 and 2). Finally, LRRC8, a putative four-pass transmembrane protein with many leucine-rich repeats in the extracellular domain, is required for B-cell development64. Thus, tissue plasminogen activator, SLOC2A1, and MUSTN1 may provide anti-proliferative and pro-differentiation functions, and C8orf4 may be a counter-regulatory factor to moderate cell death.

Potential role of FasL in vivo

How cell death and gene expression are regulated during vascular atrophy is not evident. Nevertheless, it is known that FasL/Fas interactions mediate SMC apoptosis during spiral artery remodeling, after arterial injury, and after increased shear stress in vitro65–67. This pathway may be operative in the baboon models, since shear stress may be increased in the tightly wrapped artery, as well as in the PTFE graft. Further, FasL induces 6 of the 7 atrophy-associated up-regulated genes in cultured SMCs and arteries (Figs. 3 and 4). The observation that activation of Fas in apoptosis-resistant cells increases MMP2 and MMP9 in a caspase-independent manner68 also links Fas activation to the baboon atrophy models, since baboon SMCs are apoptosis resistent (i.e. cycloheximide is required with FasL for killing) and we have previously reported that levels of MMP2 and MMP9 are increased after 7 days of high blood flow in the PTFE graft model69. The in vitro data indicate that FasL-mediated atrophy gene induction is independent of caspase-induced cell death (Table II), suggesting the possibility that a signaling cascade observed in glioblastoma cells that consists of a src kinase, PI3K, and PKB may be involved68. FasL also induces MCP-1 in SMCs in vitro (our results and 16), and increased blood flow increases MCP-1 in the PTFE graft model after 4 days (online table 8C in Hsieh et al9), but not after 1 day (online Tables I and II). In addition, induction of MCP-1 is partially mediated by IL-1α (Schaub et al. 2000), which also induces ADAMTS4 and hyaluronidase 270–71. These data suggest that a Fas/IL-1α pathway may be operative in vivo during atrophy and account for the up-regulation of some of the atrophy-associated genes.

Vascular cells expressing atrophy-associated genes

While the cells expressing atrophy-associated genes in vivo has not been determined, we have demonstrated that cultured SMCs regulate the expression of tissue plasminogen activator, LRRC8, SLCO2A1, C8orf4, and ADAMTS4. However, endothelial cells also express tissue plasminogen activator, SLCO2A1, ADAMTS4, hyaluronidase, and ficolin 372–73. The endothelium is clearly the cell exposed to the greatest shear stress and many genes have been reported to be regulated by shear stress, including tissue plasminogen activator. Our observation that hyaluronidase 2 was not increased in cultured SMCs, but was increased in the cultured artery, is consistent with endothelial cell expression of this gene.

In conclusion, we have found 7 genes up-regulated in two distinctly different models of non-human primate vascular atrophy. The majority of these genes are also up-regulated during induction of cell death in vitro, including the ECM degrading factors tissue plasminogen activator, ADAMTS4, and hyaluronidase-2. Further studies are required to determine the specific roles of these and the other atrophy-associated gene products and their suitability as therapeutic targets to induce vascular atrophy in established restenotic disease.

Supplementary Material

Acknowledgments

Supported by National Institutes of Health HL30946 and RR00166

We thank the staff of the Washington National Primate Research Center for their assistance with animal surgery and care. We thank Lynn Amon, PhD, for normalization and quality analysis of microarray data; Richard Beyer, PhD, for the gene set analysis; Steve Schwartz, MD PhD, for helpful discussion; and Lihua Chen, PhD, for technical help with microarrays. We also thank Olivier Defawe and Suzanne Justice for assistance with the baboon operations and tissue processing.

Footnotes

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References

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