Different migratory and proliferative properties of smooth muscle cells of coronary and femoral artery

In the human coronary arteries, the intima begins to thicken from early adolescence and shows progressive thickening with age. We compared the response to vascular injury of the coronary and femoral arteries using a canine model. Both incorporation of 5-bromo-2'-deoxyuridine (BrdU) and neointimal formation after balloon injury were significantly greater in the coronary artery than in the femoral artery. Also, the proliferative and migratory activities of coronary smooth muscle cells (SMCs) were significantly greater than those of femoral SMCs in vitro. The level of phosphorylated myosin light chain (phospho-MLC) was higher in coronary SMCs than in femoral SMCs. Y-27632, a specific inhibitor of Rho-kinase, significantly inhibited the PDGF-induced migration of both coronary and femoral SMCs. In contrast, the migration of coronary SMCs, but not femoral SMCs, was inhibited by ML-9, a specific inhibitor of myosin light chain kinase (MLCK). These findings suggest that the contribution of Rho-kinase and MLCK differs between the different arteries. They also suggest that a neointima develops more easily in the coronary artery than in the femoral artery because of the greater proliferative and migratory activity of coronary SMCs. Differential activation of MLC might partly explain the increased proliferation and migration of coronary SMCs.     

Matrix metalloproteinase-9 is necessary for the regulation of smooth muscle cell replication and migration after arterial injury

Matrix metalloproteinases (MMPs) and, in particular, MMP-9 are important for smooth muscle cell (SMC) migration into the intima. In this study, we sought to determine whether MMP-9 is critical for SMC migration and for the formation of a neointima by using mice in which the gene was deleted (MMP-9(-/-) mice). A denuding injury to the arteries of wild-type mice promoted the migration of medial SMCs into the neointima at 6 days, and a large neointimal lesion was observed after 28 days. In wild-type arteries, medial SMC replication was approximately 8% at day 4, 6% at day 6, and 4% at day 8 and had further decreased to 1% at day 14. Intimal cell replication was 65% at 8 days and had decreased to approximately 10% at 14 days after injury. In MMP-9(-/-) arteries, SMC replication was significantly lower at day 8. In addition, SMC migration and arterial lesion growth were significantly impaired in MMP-9(-/-) arteries. SMCs, isolated from MMP-9(-/-) mouse arteries, showed an impairment of migration and replication in vitro. Thus, our present data indicate that MMP-9 is critical for the development of arterial lesions by regulating both SMC migration and proliferation.     

Estrogen attenuates the adventitial contribution to neointima formation in injured rat carotid arteries

OBJECTIVE: This study tested, in ovariectomized rats, whether (1) adventitial activation plays a role in the vascular injury response, and (2) inhibition of adventitial activation and the subsequent wave of cell proliferation moving from adventitia to neointima contributes to the estrogen-induced attenuation of neointima formation in balloon injured carotid arteries. METHODS: Ovariectomized Sprague-Dawley rats were treated with either 17 beta-estradiol or vehicle beginning 72 h prior to balloon injury of the right common carotid artery and were sacrificed at 0, 3, 7, 14 and 28 days after injury. BrdU was administered 18 h and 12 h prior to sacrifice in order to quantitate mitotic activity in adventitia, media and neointima of the damaged vessel at specified times post injury. RESULTS: Adventitial activation, evidenced by positive BrdU staining, was evident on the day of injury, peaked on day 3 and was resolved by day 7, thus preceding neointima formation. Numbers of BrdU labeled cells in adventitia on day 3 were significantly reduced in estrogen treated rats compared to controls. BrdU labeled cells were undetectable in media on the day of injury, appeared at day 3 and disappeared by day 14. Neointima appeared at day 7 and increased in area throughout the period of observation. Neointimal area and numbers of BrdU labeled cells in neointima were significantly reduced in estrogen treated rats compared to controls. These findings suggest that there is a wave of cell proliferation moving in an adventitia-to-lumen direction following endoluminal injury of the rat carotid artery and that estrogen modulates this proliferative response to injury. CONCLUSION: These results support the hypothesis that adventitial activation contributes to the vascular injury response and that estrogen reduces this contribution.     

大鼠胸主动脉球囊损伤对细胞凋亡及凋亡相关基因Fas、Bcl-2表达的影响

目的探讨大鼠胸主动脉球囊损伤后细胞凋亡和凋亡相关基因表达的变化规律。方法将30只400~500 g的雄性SD大鼠随机分为2组,手术组(n=24)行球囊扩张损伤大鼠胸主动脉术;对照组(n=6)不行球囊损伤,作为正常对照。分别于术后2、7、14、28 d取胸主动脉应用HE染色、TUNEL法、免疫组化和计算机图像分析仪进行形态学、细胞凋亡、增殖细胞核抗原(PCNA)、凋亡基因Fas;抗凋亡基因Bcl-2表达水平检测。结果对照组管壁处于非增殖状态;手术组球囊损伤后7 d形成新生内膜,血管平滑肌细胞(VSMC)增殖活跃;14 d内膜明显增厚,但VSMC增殖已减弱;28 d内膜继续缓慢增厚,管腔明显狭窄。动脉损伤后Fas表达和TUNEL法测定的凋亡规律一致,两周内凋亡较明显,但细胞凋亡高峰时间(中膜7 d、内膜14 d)迟于增殖高峰(中膜2 d、内膜7 d),两周后凋亡与增殖均明显下降。动脉损伤后抗凋亡基因Bcl-2表达下调,在中膜和内膜分别在7 d1、4 d达最低水平,后回升,与凋亡基因Fas表达呈明显负相关(r=-0.878,P<0.001)。结论动脉球囊损伤后,平滑肌细胞的凋亡呈现规律性变化,可能在管腔狭窄的病理过程中具有重要作用。     

Early remodeling of saphenous vein grafts: proliferation, migration and apoptosis of adventitial and medial cells occur simultaneously with changes in graft diameter and blood flow

BACKGROUND: This study was designed to determine how migration and proliferation of adventitial and medial cells correlate temporally with hemodynamic characteristics including changes in diameter and blood flow in vein grafts. METHODS: Male mongrel dogs underwent end-to-side reversed saphenous vein bypass grafting across a ligated femoral artery. Hemodynamic parameters were assessed by duplex ultrasound. Proliferating cells were labeled 24-48 h after grafting with 5-bromo-2'-deoxyuridine (BrdU). Grafts were removed on postoperative days 2, 5, 7 and 14. Immunohistochemistry was performed on graft sections with antibodies to nuclear antigen Ki-67 (MIB-1), BrdU and smooth muscle cell markers. Apoptosis was identified by modified TUNEL staining. Proliferating/apoptotic cells were quantified digitally. RESULTS: Mean luminal cross-sectional area, blood flow and velocity increased from day 2 to 7. Cell proliferation was evident in adventitia and media on day 2, maximum on day 5 and decreased significantly by day 14. Apoptosis was maximum on day 5. Early proliferating cells (BrdU labeled) localized in the adventitia and media on day 2 were more prevalent in the neointima on days 5-14 suggesting inward migration. On day 2, proliferating cells stained positive only for vimentin. By days 5-14, proliferating cells stained for alpha-smooth-muscle actin, a phenotype characteristic of myofibroblasts. CONCLUSION: These results indicate that cell proliferation and apoptosis occur simultaneously within the adventitia and media of the vein during the first week following grafting, when changes in diameter and blood flow are greatest. In addition, proliferating adventitial cells subsequently migrate inwards to contribute to the formation of neointima.     

平滑肌细胞与基质在静脉桥内膜增生的时空变化

目的 :研究移植静脉桥内膜增厚过程中平滑肌细胞 （SMC）增殖、表型转化和细胞外基质积聚的时间变化与空间分布。方法 :将兔自体颈浅静脉桥移植到颈动脉 ,在术后 3,8,15 d和 2 ,6个月取血管桥行免疫组化染色。结果 :静脉桥中层 SMC在术后 3d表现受损并开始增殖 ,8d迁移到内层并形成新内膜层 ,SMC表型从正常成熟型向胚胎型转化。术后 15 d,SMC增殖率达高峰 ,2月后迅速下降 ,且绝大部分细胞从胚胎型转变为成熟表型 ,但在术后6个月 ,仍有少量胚胎型 SMC存在。新内膜层自形成后在整个观察期呈进行性增厚。内膜层细胞外基质主要成分 : 型胶原、硫酸肝素、硫酸皮肤素。术后 2月基质大多堆积在内膜深层细胞稀疏区。结论 :静脉桥新内膜的形成起源于表型转化胚胎型 SMC的迁移 ,细胞增殖和基质积聚使新内膜增厚。而胚胎型 SMC在新生内膜的持续存在 ,可能促使基质不断堆积和新内膜层进行性增厚 ,易使移植静脉桥造成狭窄     

血管去内皮损伤后平滑肌细胞凋亡的实验观察

目的 了解血管去内皮损伤后新内膜形成及平滑肌细胞(SMCs)凋亡的时相变化。方法用氮气干燥剥脱大鼠颈动脉内皮复制血管损伤模型,HE染色光镜形态计量内膜/中膜比(I/M),末端脱氧核苷酸转移酶(TdT)介导的荧光素d-uTP缺口末端标记(TRNEL)方法观察不同时间新内膜形成(I/M)及VSMC凋亡。结果 血管去内皮后四天,在SMCs增生形成的新内膜中有TUNEL法证实的细胞凋亡,I/M加比,凋亡指数(TUNELI)分别为0.12±0.06,2.4±1.98。在七天,内膜明显增厚(I/M为0.6±0.15,与四天比,P<0.05),TUNELI达到最大值(为9.3±3.8,与四天比,P<0.05)。到第14天,内膜明显增厚约为中膜的1.25倍(I/M比1.25±0.14,与七天比,P<0.05),TUNELI逐渐减小(为8.75±4.01;与七天比,P>0.05)。损伤后21天,内膜开始变薄(I/M比为0.98±0.41,与14天比,P<0.05),凋亡水平进一步下降(TUNELI为6.58±3.97,但差异无显著性,P=NS)。结论 血管壁对损伤刺激诱发增生反应的同时激活凋亡机制。凋亡调节血管壁细胞数和内膜增厚演变。细胞凋亡的平衡失调可能是动脉粥样硬化(AS)、再狭窄(RS)等血管疾病发生的机制之一。     

Tranilast inhibits vascular smooth muscle cell growth and intimal hyperplasia by induction of p21(waf1/cip1/sdi1) and p53

Tranilast, which is an antiallergic drug, has a potent effect on preventing postangioplasty restenosis. To elucidate this mechanism, we studied the effect of tranilast on the proliferation of vascular smooth muscle cells (SMCs) in vitro and in vivo. Tranilast decreased the growth rate of SMCs stimulated by either 10% FBS or platelet-derived growth factor. The IC50 value, evaluated as cell number, was 100 micromol/L. These inhibitory effects were associated with inhibition of the retinoblastoma gene product (pRb) phosphorylation. Because pRb phosphorylation is regulated by cyclin-dependent kinases (CDK), we investigated CDK2 and CDK4 activities and the expression of CDK inhibitor p21(waf1/cip1/sdi1) (p21). When SMCs were stimulated by 10% FBS or platelet-derived growth factor, CDK2 and CDK4 activities reached a maximum near the G1/S transition. Tranilast suppressed their activities by >80% without reduction of CDK2/cyclin E and CDK4/cyclin D1 protein levels. These inhibitory effects were associated with enhanced expression of p21 and elevated complexing of p21 with CDK2/CDK4. Next, rat balloon-injured carotid artery was analyzed for intimal thickening and p21 expression. Tranilast-treated rats had a 70% (P<0.001) smaller neointima/media area ratio at 14 days after balloon injury compared with the controls. Immunohistochemical staining demonstrated that, in tranilast-treated rats, p21 was already present in the neointima at day 7 and strongly expressed throughout the neointima at day 14. In control rats, p21 was not observed in the neointima at day 7 but was sparsely expressed at day 14. These data demonstrate that inhibition of CDK2/CDK4 activities by the increased expression of p21 may be one mechanism by which tranilast inhibits SMC proliferation and prevents postangioplasty restenosis.     

Fish oil supplementation prevents neointima formation in nonhypercholesterolemic balloon-injured rabbit carotid artery by reducing medial and adventitial cell activation

We asked whether balloon-injured neointima formation in the presence of high/low serum cholesterol (CT) levels might be affected by dietary supplementation with fish oil (FO). To test this hypothesis, we examined the differentiation, proliferation, or apoptosis profile of smooth muscle cell (SMC) and adventitial cell response to a mild injury induced via a Fogarty catheter in the carotid artery of adult rabbits that had been fed a standard chow or 0.5% CT-enriched diet starting 4 weeks before the lesion. One week before surgery, animals received FO supplementation. This regimen was continued for the following 3 weeks. The effect of FO on the early proliferative/migratory response of carotid SMCs was also examined in 2- and 7-day-injured normocholesterolemic rabbits. As controls, animals subjected to 3-week endothelial injury and animals kept on a 7-week CT diet were used. Carotid cryosections from the various animal groups were evaluated for morphometry (image analysis), differentiation (immunofluorescence with monoclonal antibodies specific for smooth muscle markers, ie, myosin isoforms, SM22, and fibronectin), proliferation (bromodeoxyuridine incorporation), and apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling). FO treatment significantly reduced the development of intimal thickening in normocholesterolemic rabbits but had no efficacy in the presence of relatively higher serum CT levels. At day 2 (adventitia) and day 7 (neointima, media, and adventitia), the proliferation index of SMCs in FO-treated injured rabbits was markedly lower than in untreated injured controls. Concomitantly with the antiproliferative effect, FO was able to decrease the size of 2 cell types involved in the cell growth response to endothelial injury, namely, the "fetal-type" medial SMC subpopulation and the fibroblast-derived adventitial myofibroblasts. Thus, in our experimental conditions, a low CT level is a permissive condition for FO to prevent neointima formation to a considerable extent. This event is attributable to the early postinjury effect of FO on SMC/adventitial cell proliferation/differentiation patterns.     

舒心益脉煎对气滞血瘀型大鼠动脉内膜损伤后细胞凋亡及基因蛋白产物的影响

探讨气滞血瘀型大鼠胸主动脉内膜剥脱后内膜增生中，舒心益脉煎对血管平滑肌细胞凋亡及相关基因Fas抗原和Fas配体的影响。将58只大鼠随机分为假且10只，舒心益脉煎组和对照组各24只。后两组大鼠行胸主动脉内膜剥脱术，舒心益脉煎组动物于术前7d至术后14d每日接受舒心益脉煎治疗。术后14d处死动物，用免疫组织化学法测定Fas抗原与Fas配体在血管中的表达，原位末端标记法测定凋亡细胞。术后14d血管新生内膜中有平滑肌细胞凋亡；舒心益脉煎显著增加新生内膜中Fas抗原与Fas配体的表达，增加血管平滑肌细胞凋亡，减少新生内膜面积。说明舒心益脉煎可能通过Fas系统调节动脉血管内皮损伤修复过程中血管平滑肌细胞凋亡，从而抑制新生内膜形成。     

Directed migration of smooth muscle cells to engineer plaque-resistant vein grafts.

PURPOSE: To test the hypothesis that controlled perivascular release of tissue plasminogen activator (tPA) can generate cleaved extracellular matrix (ECM) chemotactic gradients to guide the migration of vascular smooth muscle cells (SMCs) away from the lumen, thereby limiting neointima formation. METHODS: This hypothesis was tested in rabbit models in which the perivascular surface of vein bypass grafts was treated with microspheres releasing tPA (MS-tPA), microspheres containing no drug (MS-blank), or phosphate buffered saline (PBS). Vein graft segments harvested after 7 days were then evaluated for elastin content, proliferating SMCs, intima-to-media (I/M) ratio, and inflammation; late impact on neointima formation was also examined. RESULTS: The 7-day results demonstrated cleaved elastin gradients and proliferating SMCs that assumed a more peripheral distribution in the MS-tPA group than MS-blank and PBS controls (p<0.05). At 28 days, vein grafts treated with MS-tPA showed a mean I/M ratio (0.35+/-0.04) that was 63.5% lower than PBS controls (0.96+/-0.07, p<0.005) and 43.5% lower than MS-blank specimens (0.62+/-0.08, p<0.05). CONCLUSIONS: Perivascular release of tPA modifies ECM gradients, directionally guides SMC migration away from the lumen, and limits neointima formation.     

SDF-1alpha/CXCR4 axis is instrumental in neointimal hyperplasia and recruitment of smooth muscle progenitor cells

Recent evidence infers a contribution of smooth muscle cell (SMC) progenitors and stromal cell-derived factor (SDF)-1alpha to neointima formation after arterial injury. Inhibition of plaque area and SMC content in apolipoprotein E-deficient mice repopulated with LacZ+ or CXCR4-/- BM or lentiviral transfer of an antagonist reveals a crucial involvement of local SDF-1alpha and its receptor CXCR4 in neointimal hyperplasia via recruitment of BM-derived SMC progenitors. After arterial injury, SDF-1alpha expression in medial SMCs is preceded by apoptosis and inhibited by blocking caspase-dependent apoptosis. SDF-1alpha binds to platelets at the site of injury, triggers CXCR4- and P-selectin-dependent arrest of progenitor cells on injured arteries or matrix-adherent platelets, preferentially mobilizes and recruits c-kit-/platelet-derived growth factor receptor (PDGFR)-beta+/lineage-/sca-1+ progenitors for neointimal SMCs without being required for their differentiation. Hence, the SDF-1alpha/CXCR4 axis is pivotal for vascular remodeling by recruiting a subset of SMC progenitors in response to apoptosis and in concert with platelets, epitomizing its importance for tissue repair and identifying a prime target to limit lesion development.     

Endoglin is overexpressed after arterial injury and is required for transforming growth factor-beta-induced inhibition of smooth muscle cell migration

Endoglin is a homodimeric membrane glycoprotein primarily expressed on endothelial cells. In association with transforming growth factor (TGF)-ss receptors I and II, it can bind TGF-beta1 and -beta3 and form a functional receptor complex. There is increasing evidence that endoglin can modulate the cellular response to TGF-beta, a factor implicated in vascular lesion formation in human and experimental models. The purpose of this study was to analyze the expression of endoglin in normal and balloon-injured porcine coronary arteries and in normal and atherosclerotic human coronary arteries and to determine its ability to mediate the effects of TGF-beta on the migration of vascular smooth muscle cells (SMCs). In normal porcine coronary arteries, endoglin was of low abundance and was found primarily on endothelial cells and adventitial fibroblasts, as well as on a minority of medial SMCs. On days 3, 7, and 14 after angioplasty, endoglin was present not only on endothelial cells but also on adventitial myofibroblasts and medial SMCs of porcine coronary arteries. By day 28, few or no cells expressed endoglin. In situ hybridization revealed that endoglin mRNA expression appeared to be highest in endothelial cells on days 3, 7, and 14 days after injury and absent thereafter. With a second balloon injury, a similar pattern of endoglin protein and mRNA expression was observed. In human vascular tissue, endoglin immunolabeling was higher in endarterectomy specimens removed from diseased coronary arteries than in normal internal mammary arteries. In vitro, antisense oligonucleotides to endoglin decreased its expression and antagonized the TGF-beta-mediated inhibition of human and porcine SMC migration. In summary, upregulation of endoglin occurs during arterial repair and in established atherosclerotic plaques and may be required for modulation of SMC migration by TGF-beta.     

Circulating smooth muscle progenitor cells in arterial remodeling

The proliferation and migration of vascular smooth muscle cells (SMCs) from the media toward the intimal layer are key components in vascular proliferative diseases. In addition, the differentiation of circulating bone marrow-derived mononuclear cells (BMMCs) into SMCs has been described to contribute to lesion progression in experimental models of atherosclerosis, transplant arteriosclerosis, and neointima formation. In vitro, CD14⁺ BMMCs from peripheral blood acquire a spindle-shaped phenotype and express specific SMC markers in response to platelet-derived growth factor-BB. However, the ‘trans-differentiation’ capacity of BMMCs into definitive SMCs in vivo remains a highly controversial issue. Whereas SMCs within atherosclerotic plaques have been demonstrated to be exclusively of local origin, more severe injury models have shown a wide diversity of SMCs or smooth muscle-like cells derived from BMMCs. In hindsight, these discrepancies may be attributed to methodological differences, e.g., the use of high-resolution microscopy or the specificity of the SMC marker proteins. In fact, the analysis of mouse strains that express marker genes under the control of a highly specific smooth muscle-myosin heavy chain (SM-MHC) promoter and a time-course analysis on the dynamic process of neointima formation have recently shown that BMMCs temporarily express α-smooth muscle actin, not SM-MHC. Additionally, BM-derived cells disappear from the neointimal lesion after the inflammatory response to the injury has subsided. Although CD14⁺/CD68⁺ have important paracrine effects on arterial lesion progression, BMMCs account for more of the ‘SMC-like macrophages’ than the highly ‘trans-differentiated’ and definitive SMCs in vivo. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".     

Stem cell factor deficiency is vasculoprotective: unraveling a new therapeutic potential of imatinib mesylate

Evidence suggests that bone marrow (BM) cells may give rise to a significant proportion of smooth muscle cells (SMCs) that contribute to intimal hyperplasia after vascular injury; however, the molecular pathways involved and the timeline of these events remain poorly characterized. We hypothesized that the stem cell factor (SCF)/c-Kit tyrosine kinase signaling pathway is critical to neointimal formation by BM-derived progenitors. Wire-induced femoral artery injury in mice reconstituted with wild-type BM cells expressing yellow fluorescent protein was performed, which revealed that 66+/-12% of the SMCs (alpha-smooth muscle actin-positive [alphaSMA(+)] cells) in the neointima were from BM. To characterize the role of the SCF/c-Kit pathway, we used c-Kit deficient W/W(v) and SCF-deficient Steel-Dickie mice. Strikingly, vascular injury in these mice resulted in almost a complete inhibition of neointimal formation, whereas wild-type BM reconstitution of c-Kit mutant mice led to neointimal formation in a similar fashion as wild-type animals, as did chronic administration of SCF in matrix metalloproteinase-9-deficient mice, a model of soluble SCF deficiency. Pharmacological antagonism of the SCF/c-Kit pathway with imatinib mesylate (Gleevec) or ACK2 (c-Kit antibody) also resulted in a marked reduction in intimal hyperplasia. Vascular injury resulted in the local upregulation of SCF expression. c-Kit(+) progenitor cells (PCs) homed to the injured vascular wall and differentiated into alphaSMA(+) cells. Vascular injury also caused an increase in circulating SCF levels which promoted CD34(+) PC mobilization, a response that was blunted in mutant and imatinib mesylate-treated mice. In vitro, SCF promoted adhesion of BM PCs to fibronectin. Additionally, anti-SCF antibodies inhibited adhesion of BM PCs to activated SMCs and diminished SMC differentiation. These data indicate that SCF/c-Kit signaling plays a pivotal role in the development of neointima by BM-derived PCs and that the inhibition of this pathway may serve as a novel therapeutic target to limit aberrant vascular remodeling.     

Vascular remodeling in mice lacking the cytoplasmic domain of tissue factor

Tissue factor (TF), the cell surface receptor for the serine protease FVIIa supports cell migration by interaction with the cytoskeleton. Intracellular signaling pathways dependent on the cytoplasmic domain of TF modify cell migration and may alter vascular remodeling. Vascular remodeling was analyzed in a femoral artery injury and a blood flow cessation model in mice with a targeted deletion of the 18 carboxy-terminal intracellular amino acids of TF (TF(Deltact/Deltact)) and compared with TF wild-type mice (TF(wt/wt)). Morphometric analysis revealed a decrease in the intima/media ratio after vascular injury in arteries from TF(Deltact/Deltact) compared with TF(wt/wt) mice (femoral artery injury: 2.4+/-0.3 TF(wt/wt) versus 0.6+/-0.3 TF(Deltact/Deltact), n=9 to 10, P=0.002; carotis ligation: 0.45+0.11 TF(wt/wt) versus 0.22+0.03 TF(Deltact/Deltact), n=12 to 14, P=0.09). This was caused by an increase in the media by 54% (P=0.04) in the femoral artery model and by 32% (P=0.03) after carotis ligation and was associated with an increased number of proliferating cells. Isolated aortic smooth muscle cells (SMCs) of TF(wt/wt) mice showed an increased migratory response toward the TF ligand active site-inhibited FVIIa that was abolished in TF(Deltact/Deltact) SMC. In contrast, the unstimulated proliferation rate was increased in TF(Deltact/Deltact) SMC compared with TF(wt/wt) SMCs. Thus, retention of SMCs attributable to a migratory defect and increased proliferation results in thickening of the media and in decrease in neointima formation after arterial injury. TF cytoplasmic domain signaling alters vascular remodeling and, thereby, may play a role in the development of restenosis, atherosclerotic disease, and neovascularization.     

Increased secretion of basement membrane-degrading metalloproteinases in pig saphenous vein into carotid artery interposition grafts.

Late saphenous vein bypass graft failure in humans involves medial and neointimal thickening as the result of migration and proliferation of vascular smooth muscle cells (SMCs). Recent work on angioplasty indicates that basement membrane-degrading metalloproteinases (MMPs) cooperate with growth factors to mediate SMC migration and proliferation. We sought evidence here for a similar role in experimental vein grafts in pigs. Tissue levels and secretion of MMP-2 and MMP-9 were compared by quantitative zymography in veins and grafts removed 2 to 168 days after implantation. Pro and active forms of MMP-2 were present in veins, but levels were increased in vein grafts after 7 days (4- and 6-fold, respectively) and 28 days (3-fold for both), returning to values in veins after 168 days. MMP-9 was not detected in veins, was increased in grafts after 2 days, further increased after 7 days (6-fold) and 28 days (15-fold), and declined to undetectable levels by 168 days. Immunocytochemistry detected increased expression of MMP-2 and MMP-9 with the same time course. MMP-2 was widely distributed, whereas MMP-9 was concentrated in highly proliferative SMCs at the superficial layers of the thickened neointima. We conclude that increased production of the basement membrane-degrading MMP-2 and MMP-9 spanned the period of neointima formation and SMC proliferation in experimental vein grafts. MMPs therefore constitute new therapeutic targets for reducing late vein graft failure.     

Matrix metalloproteinase-9 overexpression enhances vascular smooth muscle cell migration and alters remodeling in the injured rat carotid artery

Matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis of atherosclerosis as well as intimal hyperplasia after vascular injury. We used Fischer rat smooth muscle cells (SMCs) overexpressing MMP-9 to determine the role of MMP-9 in migration and proliferation as well as in vessel remodeling after balloon denudation. Fischer rat SMCs were stably transfected with a cDNA for rat MMP-9 under the control of a tetracycline-regulatable promoter. In this system, MMP-9 was overexpressed in the absence, but not in the presence, of tetracycline. In vitro SMC migration was determined using a collagen invasion assay as well as a Boyden chamber assay. In vivo migration was determined by measuring the invasion into the medial and intimal layers of transduced SMCs seeded on the outside of the artery. Transduced SMCs were also seeded on the luminal surface, and the effect of local MMP-9 overexpression on vascular structure was measured morphometrically at intervals up to 28 days. MMP-9 overexpression enhanced SMC migration in both the collagen invasion assay and Boyden chamber in vitro, increased SMC migration into an arterial matrix in vivo, and altered vessel remodeling by increasing the vessel circumference, thinning the vessel wall and decreasing intimal matrix content. These results demonstrate that MMP-9 enhances vascular SMC migration in vitro and in vivo and alters postinjury vascular remodeling.