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     

eNOS gene transfer inhibits smooth muscle cell migration and MMP-2 and MMP-9 activity

Vascular smooth muscle cell (SMC) migration is a critical step in the development of neointima after angioplasty. Matrix metalloproteinases (MMPs) degrade the basement membrane and the extracellular matrix, facilitating SMC migration. Transfer of the endothelial nitric oxide synthase (eNOS) gene to the injury site inhibits neointima formation. Neither the signaling pathways leading to NO-mediated inhibition of SMC migration and proliferation nor the alterations in these pathways have been characterized. We hypothesize that NO inhibits SMC migration in part by regulating MMP activity. To test this hypothesis, we transfected cultured rat aortic SMCs with replication-deficient adenovirus containing bovine eNOS gene and analyzed the conditioned medium for MMP activity. We observed that eNOS gene transfer significantly (P<0.05) inhibited SMC migration and significantly (P<0.05) decreased MMP-2 and MMP-9 activities in the conditioned medium. Similarly, addition of the NO donor DETA NONOate and 8-bromo-cGMP to the culture medium significantly decreased MMP-2 and MMP-9 activities in the conditioned medium collected 24 hours after treatment. Furthermore, Western blot analysis of the conditioned medium collected from eNOS gene-transfected SMCs showed a significant increase in tissue inhibitor of metalloproteinases-2 (TIMP-2) levels. Our data suggest that NO decreases MMP-2 and MMP-9 activities and increases TIMP-2 secretion, and this shifts the balance of MMP activity, which may favor the inhibition of cell migration because of inhibition of extracellular matrix degradation.     

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.     

Enhanced superoxide production in experimental venous bypass graft intimal hyperplasia: role of NAD(P)H oxidase

-Vein graft intimal hyperplasia, due to smooth muscle cell (SMC) proliferation, remains a limiting factor in long-term vein graft patency. Increased superoxide production regulates SMC mitogenesis and contributes to reduced NO bioactivity in systemic models of vascular disease. We compared superoxide production in experimental venous bypass grafts with ungrafted veins and determined its enzymatic sources and cellular localization. Vascular superoxide production was measured in vein grafts and control jugular veins obtained from normocholesterolemic rabbits undergoing jugular vein-carotid artery interposition bypass grafting. Surgical isolation of the contralateral jugular vein, without bypass grafting, provided an additional control for the effects of surgical manipulation. Superoxide production was increased 3-fold in vein grafts compared with control veins. Systematic stimulation and inhibition of specific oxidases revealed that the major source of increased vein graft superoxide production was a membrane-associated NAD(P)H-dependent oxidase. Western blotting of vascular homogenates demonstrated corresponding increases in NAD(P)H oxidase p22phox (membrane-associated) and p67phox (cytosolic) subunits in vein grafts compared with jugular veins. There was marked intimal hyperplasia in vein grafts, and immunohistochemical staining of vessel cryosections revealed increased p22phox-expressing cells in vein grafts that were predominantly intimal SMCs. Superoxide generation is increased in experimental vein grafts compared with ungrafted veins. The principal source of increased superoxide generation in vein grafts is an NAD(P)H oxidase, expressed by intimal SMCs. These findings suggest a role for NAD(P)H oxidase-mediated superoxide production in the proliferative response to vascular injury in vein grafts.     

Reduced neointima in vein grafts following a blockage of cell recruitment from the vein and the surrounding tissue

OBJECTIVE: Accumulation of intimal smooth muscle cells (SMC) is an important event in vein graft-stenosis. Different SMC sources have been reported, but their interrelations have been poorly studied. In a mouse vein graft model we investigated whether recipient-derived intimal SMCs are recruited from the surrounding tissue and whether blockage of SMC recruitment from the surrounding tissue and/or the donor vein will reduce neointimal formation. METHODS: To detect recipient-derived cells, wild-type veins were implanted into ROSA26 transgenic mice. To block cell recruitment from the surrounding tissue, implanted veins were isolated with a tube-shaped plastic film. To exclude vein-derived cells in the neointimal formation, acellular veins were implanted. RESULTS: In vein grafts isolated from the surrounding tissue the recipient contribution became minimal, but the total number of SMCs was not decreased. Acellular grafts contained an equal number of intimal SMCs as cellular controls after 4 weeks. Isolation of acellular grafts from the surrounding tissue decreased the number of intimal SMCs by 90%. CONCLUSIONS: Recipient-derived SMCs are mainly recruited from the surrounding tissue. Cell recruitment from either the vein or the surrounding tissue is enough to form a neointima. Therefore, a simultaneous inhibition of both these sources is needed to reduce accumulation of intimal SMCs.     

Early effects of arterial hemodynamic conditions on human saphenous veins perfused ex vivo

Exposure to the arterial hemodynamic environment is thought to be a potential trigger for the pathological remodeling of saphenous vein grafts. Using matched pairs of freshly isolated human saphenous vein, we analyzed the early effects of ex vivo hemodynamic conditions mimicking the venous (native) compared with arterial (graft) environment on the key components of vascular remodeling, ie, matrix metalloproteinase (MMP)-9 and MMP-2 and cell proliferation. Interestingly, we found that arterial conditions halved latent MMP-9 (50+/-11%, P=0.01) and MMP-2 (44+/-6%, P=0.005) levels relative to matched vein pairs maintained ex vivo under venous perfusion for up to 3 days. Immunostaining supported decreased MMP levels in the innermost area of arterially perfused veins. Either decreased synthesis or increased posttranslational processing may decrease MMP zymogen levels. Biosynthetic radiolabeling showed that arterial perfusion actually increased MMP-9 and MMP-2 production. When we then examined potential pathways for MMP zymogen processing, we found that arterial conditions did not affect the expression of MT-MMP-1, a cell-associated MMP activator, but that they significantly increased the levels of superoxide, another MMP activator, suggesting redox-dependent MMP processing. Additional experiments indicated that increased superoxide under arterial conditions was due to diminished scavenging by decreased extracellular superoxide dismutase. Arterial perfusion also stimulated cell proliferation (by 220% to 750%) in the majority of vein segments investigated. Our observations support the hypothesis that arterial hemodynamic conditions stimulate early vein graft remodeling. Furthermore, physiological arterial flow may work to prevent pathological remodeling, particularly the formation of intimal hyperplasia, through rapid inactivation of secreted MMPs and, possibly, through preferential stimulation of cell proliferation in the outer layers of the vein wall.     

Relationship between type IV collagen degradation,metalloproteinase activity and smooth muscle cell migration and proliferation in cultured human saphenous vein

The relationship between degradation of basement membranes, metalloproteinase (MMP) activity and smooth muscle cell (SMC) migration and proliferation has not been previously investigated in any intervention study. We used adenoviral overexpression of tissue inhibitors of metalloproteinases (TIMPs) in cultured human saphenous veins. By immunocytochemistry, the percentage of medial SMC surrounded by basement membrane type IV collagen (Coll-IV) decreased from 93+/-1 to 77+/-4% and 82+/-1% (n=18, both P<0.001) after 7 and 14 days of culture, respectively, while all SMC that migrated to the neointima lacked Coll-IV. Overexpression of TIMP-1 or TIMP-3 significantly increased the percentage of medial SMC surrounded by Coll-IV (94+/-2 or 98+/-2%, respectively, both P<0.01 vs. no treatment) and decreased the number of neointimal SMC. Some 44+/-18 and 30+/-6%, respectively, of BrdU or PCNA labeled medial SMC remained surrounded by type IV collagen and this was not affected by overexpression of TIMP-1 or TIMP-3. We conclude that MMPs mediate loss of basement membrane and this is closely related to SMC migration. SMC proliferation does not require complete basement membrane degradation, which itself does not require MMPs in proliferating SMC.     

Accelerated arteriosclerosis of vein grafts in inducible NO synthase(-/-) mice is related to decreased endothelial progenitor cell repair

Inducible NO synthase (iNOS) is expressed by macrophages and smooth muscle cells in atherosclerotic lesions. Previously, we have established a mouse model for vein graft arteriosclerosis by grafting autologous jugular veins or vena cava to carotid arteries. Using this model, we studied the role of iNOS in the development of vein graft arteriosclerosis in iNOS(-/-) mice. Four weeks after grafting, neointimal hyperplasia of vein grafts in iNOS(-/-) mice was increased 2-fold compared with that of wild-type controls. Neointimal lesions contained mainly MAC-1+ macrophages and alpha-actin+ smooth muscle cells (SMCs) in both vein grafts of iNOS(-/-) and iNOS(+/+) mice. Immunofluorescence analysis revealed that increased iNOS expression in neointimal macrophages and SMCs of wild-type, but not iNOS(-/-), mice coincided with increased vascular endothelial growth factor (VEGF) expression in vein grafts. When vein grafts were performed in iNOS(-/-)/TIE2-LacZ transgenic mice expressing LacZ gene only in endothelial cells, the number of beta-galactosidase+ cells in iNOS(-/-) vein grafts were significantly decreased. Furthermore, treatment with the NOS inhibitor NG-nitro-L-arginine methyl ester resulted in delayed endothelial progenitor cell attachment, whereas L-arginine intake through drinking water enhanced endothelial repair. Interestingly, local application of VEGF to iNOS(-/-) vein grafts restored endothelial progenitor homing and reduced neointimal lesions, whereas the VEGF receptor inhibitor SU1498 increased the lesion formation. Additionally, iNOS-deficient SMCs showed a low level of VEGF production in response to interleukin 1beta stimulation. Thus, iNOS deficiency accelerates neointima formation by abrogating VEGF production and endothelial progenitor cell attachment and differentiation.     

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.     

In vitro differences between venous and arterial-derived smooth muscle cells: potential modulatory role of decorin

OBJECTIVE: We analyzed the phenotypic and functional differences between venous and arterial smooth muscle cells (SMC) and the role of decorin in modulating these differences. METHODS AND RESULTS: SMC were isolated from the jugular veins and carotid arteries of male white New Zealand rabbits. Venous SMC demonstrated increased proliferation (2-fold, p<0.001), migration (1.7-fold, p<0.001), and collagen synthesis (4-fold, p<0.001), with decreased adhesion to collagen and fibronectin (1.2-fold, p<0.01) compared to arterial SMC. Higher levels of gelatinase activity (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinase (TIMP) were also observed in venous SMC. Venous SMC demonstrated increased expression of SMemb and decreased expression of SM1--markers of a dedifferentiated and differentiated phenotype, respectively. Arterial SMC produced increased levels of the inhibitory proteoglycan, decorin, compared to venous SMC. Conditioned medium from arterial SMC (ASMC-CM) significantly decreased DNA synthesis, collagen synthesis, and gelatinase activity in venous SMC. Removal of decorin from ASMC-CM by immunoprecipitation significantly reversed the inhibitory effects of ASMC-CM on venous SMC proliferation and collagen synthesis but did not affect gelatinase activities. CONCLUSION: Venous SMC are more dedifferentiated and demonstrate increased proliferative and synthetic capacity than arterial SMC. Differential decorin expression between arterial and venous SMC contributes to these differences in biologic behavior. Venous SMC properties may contribute to accelerated atherosclerosis in venous bypass grafts.     

Expression of intercellular adhesion molecules in human saphenous veins: effects of inflammatory cytokines and neointima formation in culture

Atherosclerosis causes occlusions in as many as 50% of human saphenous vein coronary artery bypass grafts. Monocyte infiltration is an early step in saphenous vein-graft atherosclerosis, however, comparatively little is known of its underlying mechanisms. As a first approach, we sought to define the occurrence, location and regulation of leukocyte adhesion molecules in human saphenous vein before and after surgical preparation for grafting, during neointima formation in culture and on stimulation with inflammatory cytokines. We compared the distribution of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1) and platelet endothelial cell adhesion molecule (PECAM-1 or CD-31) in endothelial cells and smooth muscle cells (SMCs), using immunocytochemistry. ICAM-1 was expressed on endothelial cells before culture and on both endothelial cells and medial or neointimal SMCs after culturing vein for 14 days in 30% foetal bovine serum or after culturing for 24 h with TNF-alpha. Relative tissue levels of ICAM-1 measured by Western blotting were significantly elevated by culturing freshly-isolated (0.02+/-0.01 to 0.18+/-0.03) and surgically-prepared (0.02+/-0.01 to 0.14+/-0.03; n=6) veins or following TNF-alpha treatment of surgically-prepared veins (0.04+/-0.01 to 0.32+/-0.11, n=7). VCAM-1 was undetectable before or after culturing but was strongly upregulated on endothelial cells by incubation with the cytokines TNF-alpha, IL-1alpha or interferon-gamma. PECAM-1 was expressed constitutively on endothelial cells. We conclude that human saphenous vein expresses several adhesion molecules capable of mediating monocyte migration. The increased expression of ICAM-1 in SMC after culturing or cytokine treatment and of VCAM-1 in endothelial cells suggests that interactions with beta1 and beta2 integrins are important pathways for stimulated monocyte ingress into human saphenous vein grafts.     

Regression of hypertrophied rat pulmonary arteries in organ culture is associated with suppression of proteolytic activity, inhibition of tenascin-C, and smooth muscle cell apoptosis.

Increased elastase activity and deposition of the matrix glycoprotein tenascin-C (TN), codistributing with proliferating smooth muscle cells (SMCs), are features of pulmonary vascular disease. In pulmonary artery (PA) SMC cultures, TN is regulated by matrix metalloproteinases (MMPs) and mechanical stress. On attached collagen gels, MMPs upregulate TN, leading to SMC proliferation, whereas on floating collagen, reduced MMPs suppress TN and induce SMC apoptosis. We now investigate the response of SMCs in the whole vessel by comparing attached and floating conditions using either normal PAs derived from juvenile pigs or normal or hypertrophied rat PAs that were embedded in collagen gels for 8 days. Normal porcine PAs in attached collagen gels were characterized by increasing activity of MMP-2 and MMP-9 assessed by zymography and TN deposition detected by Western immunoblotting and densitometric analysis of immunoreactivity. PAs on floating collagen showed reduced activity of both MMPs and deposition of TN. Tenascin-rich foci were associated with proliferating cell nuclear antigen immunoreactivity, and TN-poor areas with apoptosis, by terminal deoxynucleotidyl transferase-mediated nick end labeling assay, but no difference in wall thickness was observed. Although normal rat PAs were similar to piglet vessels, hypertrophied rat PAs showed an amplified response. Increased elastase, MMP-2, TN, and elastin deposition, as well as SMC proliferating cell nuclear antigen positivity, correlated with progressive medial thickening on attached collagen, whereas reduced MMP-2, elastase, TN, and induction of SMC apoptosis accompanied regression of the thickened media on floating collagen. In showing that hypertrophied SMCs in the intact vessel can be made to apoptose and that resorption of extracellular matrix can be achieved by inhibition of elastase and MMPs, our study suggests novel strategies to reverse vascular disease.     

Allogenic immune response promotes the accumulation of host-derived smooth muscle cells in transplant arteriosclerosis

OBJECTIVE: Smooth muscle cells (SMCs) involved in intimal hyperplasia during transplant vasculopathy are derived both from the graft and the host. Here, the role of an allogenic immune response in the accumulation of host-derived SMCs in the neointima was explored. METHODS: Infrarenal aorta was transplanted from female F344 to male Lewis rats with or without immunosuppression by cyclosporine A (CsA). Accumulation of host-derived SMCs and inflammatory cells in the grafts, SMC proliferation, and apoptosis were analyzed by immunohistochemistry and real-time polymerase chain reaction (PCR) for the SRY gene. Finally, SMCs were seeded in an allogenic or isogenic manner after balloon injury to carotid arteries and SMC survival was estimated. RESULTS: Proliferating graft SMCs and infiltrating leukocytes were observed in the intima early after transplantation. In parallel, inflammatory cells and immunoglobulins infiltrated the media and apoptosis of medial SMCs occurred, leading to destruction of this layer. CsA decreased the number of SRY+ SMCs in the lesions, restricted medial destruction, and improved survival of allogenic SMCs after seeding in injured arteries. CONCLUSIONS: Development of intimal thickenings during transplant vasculopathy involves an allogenic immune response, which promotes accumulation of host-derived SMCs and apoptosis of resident graft SMCs.     

Focal expression of angiotensin II type 1 receptor and smooth muscle cell proliferation in the neointima of experimental vein grafts: relation to eddy blood flow

Eddy flow has been shown to promote focal smooth muscle cell (SMC) proliferation and neointimal formation in experimental vein grafts. This study focuses on whether the angiotensin II type 1 (AT(1)) receptor mediates these events. Experimental vein grafts with and without eddy flow were created in the rat. Losartan was used to assess the influence of the AT(1) receptor on SMC proliferation. In vein grafts with eddy flow, apparent focal expression of AT(1) mRNA and protein was found in the leading region of the proximal focal neointima, where eddy flow occurred, but not in the trailing region, where eddy flow diminished, at days 5, 10, 20, and 30. The rate of SMC proliferation in the leading region (10.9+/-1.4%, 19.5+/-2.2%, 12.2+/-2.0%, and 6.9+/-1.3% at these times, respectively) was significantly higher than that in the trailing region (9.5+/-1.8%, 15.3+/-2.0%, 8.2+/-1.9%, and 3.2+/-0.7%) in these vein grafts. When eddy flow was prevented in engineered vein grafts, no apparent location difference was found in the distribution of AT(1) receptor mRNA and protein in the neointima, and the rate of SMC proliferation (5.3+/-1.0%, 5.8+/-0.9%, 3.4+/-1.0%, and 3.7+/-0.9% at days 5, 10, 20, and 30, respectively) was reduced significantly. In vein grafts with losartan, the rate of SMC proliferation in the leading region of the neointima (9.4+/-1.8%, 10.1+/-1.3%, 8.3+/-0.9%, and 4.2+/-0. 5% at days 5, 10, 20, and 30, respectively) was significantly lower than that in vein grafts without losartan. These results suggested that eddy flow upregulated the AT(1) receptor, which in turn mediated focal SMC proliferation in the neointima of experimental vein grafts.     

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.     

Role of Rho-associated kinase in neointima formation after vascular injury

BACKGROUND: The Rho/Rho-associated kinase (Rho-kinase) system is implicated in various cellular functions, including migration, proliferation, and apoptosis. Because a possible role of the system is suggested in neointima formation after vascular injury, we sought to examine whether a new specific Rho-kinase inhibitor, Y27632, prevents neointima formation of the balloon-injured rat carotid artery, and if so, to investigate the effects of Y27632 on migration, proliferation, and apoptosis of smooth muscle cells (SMCs) in the injured artery. METHODS AND RESULTS: Y27632 was administered intraperitoneally from 1 day before to 14 days after vascular injury. Treatment with Y27632 inhibited phenylephrine-induced Rho-kinase activation in the carotid artery on the basis of immunoblotting against the phosphorylated myosin-binding subunit of myosin phosphatase. Y27632 markedly prevented neointima formation at days 7 and 14. In controls, BrdU(+) proliferating and TUNEL(+) apoptotic SMCs were transiently and coincidentally increased in the neointima, with a peak at day 7. Y27632 significantly increased the neointimal TUNEL(+) SMCs at days 7 and 14, but not BrdU(+) SMCs. Y27642 significantly decreased the number of intimal SMCs at day 4, while not affecting the number of BrdU(+) or TUNEL(+) SMCs. Reendothelialization after balloon injury was not significantly affected by Y27632 at days 7 and 14. CONCLUSIONS: Y27632 inhibited neointima formation by enhancing SMC apoptosis and probably by suppressing early SMC migration. Therefore, a role of Rho-kinase is suggested in neointima formation after vascular injury.     

Smooth muscle cell matrix metalloproteinase production is stimulated via alpha(v)beta(3) integrin

This study tests the hypothesis that alpha(v)beta(3) integrin receptors play a critical role in smooth muscle cell (SMC) migration after arterial injury and facilitate migration through the upregulation of matrix metalloproteinase (MMP) activity. We showed that beta(3) integrin mRNA was upregulated by SMCs in the balloon-injured rat carotid artery in coincidence with MMP-1 expression and early SMC migration. Treatment with the beta(3) integrin-blocking antibody F11 significantly decreased SMC migration into the intima at 4 days after injury, from 110.8+/-30.8 cells/mm(2) in control rats to 10.29+/-7.03 cells/mm(2) in F11-treated rats (P=0.008). By contrast, there was no effect on medial SMC proliferation or on medial SMC number in the carotid artery at 4 days. In vitro, we found that human newborn SMCs produced MMP-1 but that adult SMCs did not. This was possibly due to the fact that newborn SMCs expressed alpha(v)beta(3) integrin receptors, whereas adult SMCs did not. Stimulation of newborn (alpha(v)beta(3)+) SMCs with osteopontin, a matrix ligand for alpha(v)beta(3), increased MMP-1 production from 114.4+/-35.8 ng/mL at 0 nmol/L osteopontin to 232.5+/-57.5 ng/mL at 100 nmol/L osteopontin. Finally, we showed that stimulation of newborn SMCs with platelet-derived growth factor-BB and osteopontin together increased the SMC production of MMP-9. Thus, our results support the hypothesis that SMC alpha(v)beta(3) integrin receptors play an important role in regulating migration by stimulating SMC MMP production.     

Expression of matrix metalloproteinases in cardiac allograft vasculopathy and its attenuation by anti MMP-2 ribozyme gene transfection

OBJECTIVE: Proliferation and migration of vascular smooth muscle cells (SMCs) causes intimal thickening during cardiac allograft vasculopathy (CAV). This process requires the degradation or remodeling of extracellular matrix (ECM) surrounding the cells. Imbalance between degradation and accumulation of ECM also contributes to the development of CAV. In this study, we investigated the contribution of matrix metalloprotenases (MMPs), enzymes regulating ECM turnover, to the development of CAV. METHODS: Donor hearts from male DBA mice were heterotopically transplanted to male B10.D2 recipient mice, and harvested at days 15 and 30 post transplantation. We examined expression MMP-2, -3, -9 and -13 of graft vessels using immunohistochemistry. To clarify the role of MMP-2 in CAV, anti MMP-2 ribozyme was delivered into donor hearts just before transplantation, mediated by a hemagglutinating virus of Japan-liposome complex to specifically suppress MMP-2 activity. RESULTS: All MMPs were immunopositive in SMCs from the slightly thickened neointima at day 15. In the advanced stage of intimal thickening at day 30, in addition to increased number of SMCs, accumulation of collagenous fibers was observed; expression of MMP-3, -9 and -13 was decreased. In contrast, MMP-2 expression remained distinctly positive throughout the progression of the vascular remodeling. After the gene transfer of MMP-2 ribozyme, luminal occlusion was significantly decreased compared to non-treated allografts [25.0+/-6.5 vs. 55.1+/-7.0% (P<0.05)] at day 30 post transplantation. CONCLUSION: MMP-2 is a principle MMP throughout the progression of the vascular remodeling in CAV. Anti MMP-2 therapy could therefore be one of the candidates for a supplemental therapy for CAV.     

Selective gene silencing of either MMP-2 or MMP-9 inhibits invasion of human saphenous vein smooth muscle cells

Activation of matrix metalloproteinases (MMPs) facilitates smooth muscle cell (SMC) invasion, an important event in the development of intimal hyperplasia in saphenous vein (SV) bypass grafts. In this study, we performed selective gene silencing using small inhibitory RNA (siRNA) oligonucleotides to examine the relative contributions of MMP-2 and MMP-9 to the invasiveness of cultured human SV-SMCs. Cultures were established from human SV obtained from patients undergoing coronary artery bypass surgery. Transfection of SV-SMCs with MMP-2 siRNA selectively reduced MMP-2 secretion and inhibited invasion through a Matrigel barrier. Supplementation of medium with recombinant MMP-2 overcame these effects. Similarly, transfection of SV-SMCs with MMP-9 siRNA selectively reduced MMP-9 secretion and subsequent invasion, effects reversed by recombinant MMP-9 supplementation. Neither MMP-2 nor MMP-9 siRNA inhibited SV-SMC migration in the absence of a Matrigel barrier. Our data demonstrate that selective gene-silencing of either MMP-2 or MMP-9 markedly reduces the invasive capacity of cultured human SV-SMCs, indicating that these MMPs play distinct non-overlapping roles in SV-SMC invasion in vitro. Specific manipulation of either MMP-2 or MMP-9 may therefore provide a valuable strategy for prevention of SV graft stenosis in man.