Localization and effects of hepatocyte growth factor on smooth muscle cells during neointimal formation after balloon denudation

The migration and proliferation of smooth muscle cells (SMCs) may play a key role in tissue remodeling after arterial wall injury. We investigated the localization and effects of hepatocyte growth factor (HGF) in rabbit carotid arteries after balloon denudation. Immunoreactivity for HGF and the c-Met receptor was clearly observed in neointimal SMCs. The immunoreactivity was not restricted to proliferating cells but was seen even in non-dividing cells in the basal layer of the neointima 4 and 6 weeks after balloon denudation. The distribution of platelet-derived growth factor (PDGF)-positive cells paralleled that of proliferating SMCs. The SMCs in the basal layer of the neointima at 4 and 6 weeks were positive for matrix metalloproteinase (MMP)-2 and membrane type 1-MMP which can activate the proform of MMP-2. HGF significantly stimulated the migration but not proliferation of cultured SMCs. Our results suggest that HGF and PDGF act in coordination to promote the proliferation and migration of SMCs in the earlier phases of neointimal formation and that HGF as well as MMP-2 contribute to the later stages by facilitating the migration but not replication of SMCs. Accepted: 19 March 1999     

Expression of T-cadherin in the rat carotid artery wall after balloon injury and in different rat organs

T-cadherin is an unusual glycosilphosphatidylinositol (GPI)-anchored member of the cadherin family of cell adhesion proteins. In contrast to classical cadherins, tissue distribution of T-cadherin so far remained unknown. We examined tissue distribution of T-cadherin in rats using Western blotting and immunohistochemical method. Our results show that T-cadherin is expressed in all types of muscles (cardiac, striated, and smooth muscles), in brain neurons, and spinal cord, in the vessel endothelium, at the apical pole of intestinal villar epithelium, in the basal layer of skin, and eosophagal epithelium. Blood-derived and lymphoid cells as well as connective tissue were T-cadherin-negative. The highest level of T-cadherin expression was revealed in the cardiovascular system. Although T-cadherin was detected in smooth muscle cells, its role in the intimal thickening and restenosis is not known. We examined T-cadherin expression within 1-28 days after balloon injury of rat left carotid arteries. T-cadherin expression was valued immunohistochemically with semiquantitative method. In uninjured arteries, T-cadherin was expressed in endothelial (vWF-positive) cells, and smooth muscle (alpha-actin-positive) cells (SMCs). After denudation of arterial wall, T-cadherin was present both in the media and neointima. We revealed dynamics of T-cadherin expression in the media of injured artery: an essential increase being registered at the stage of cell migration and proliferation in the media and neointima (1-7 days), followed by its decrease to the baseline level (10-28 days). The high upregulation of T-cadherin expression in the media and neointima during migration and proliferation of vascular cells after vessel injury enables us to suggest the involvement of T-cadherin in vessel remodeling after balloon catheter injury.     

Changes in osteopontin mRNA expression during phenotypic transition of rabbit arterial smooth muscle cells

 Transition from a contractile to a synthetic phenotype appears to be an early key event during the development of intimal thickening after arterial wall injury. We examined the expression of osteopontin mRNA, proliferation, and phenotypic properties of smooth muscle cells (SMCs) in rabbit neointima after balloon denudation and in primary culture. A strong osteopontin mRNA signal was detected in the thickened intima 1 week after balloon denudation and in the surface layer of the intima 2 weeks after balloon denudation. Ki-67 immunohistochemistry showed that osteopontin mRNA expression increased when SMCs entered the proliferating phase in the intima. Rabbit arterial SMCs on type I collagen after 1 day of primary culture with growth factors, as well as freshly isolated cells, were in the G₀ phase (contractile phenotype) and did not express osteopontin mRNA. After 3 days of culture, most cells entered the G_1B phase (synthetic phenotype) and expressed osteopontin mRNA. In the absence of growth factors, most cells transferred to the G_1A phase (intermediate phenotype) after 3 and 7 days, but did not express osteopontin mRNA. Our findings indicate that the osteopontin gene provides a marker that can be used to distinguish the phenotypic properties of vascular SMCs. Accepted: 22 November 1996     

Medial and adventitial macrophages are associated with expansive atherosclerotic remodeling in rabbit femoral artery

Expansive vascular remodeling is considered a feature of vulnerable plaques. Although inflammation is upregulated in the media and adventitia of atherosclerotic lesions, its contribution to expansive remodeling is unclear. We investigated this issue in injured femoral arteries of normo- and hyperlipidemic rabbits fed with a conventional (CD group; n=20) or a 0.5% cholesterol (ChD group; n=20) diet. Four weeks after balloon injury of the femoral arteries, we examined vascular wall alterations, localization of macrophages and matrix metalloproteases (MMP)-1, -2, -9, and extracellular matrix. Neointimal formation with luminal stenosis was evident in both groups, while expansive remodeling was observed only in the ChD group. Areas immunopositive for macrophages, MMP-1, -2 and -9 were larger not only in the neointima, but also in the media and/or adventitia in the injured arterial walls of the ChD, than in the CD group. Areas containing smooth muscle cells (SMCs), elastin and collagen were smaller in the injured arterial walls of the ChD group. MMP-1, -2 and -9 were mainly localized in infiltrating macrophages. MMP-2 was also found in SMCs and adventitial fibroblasts. Vasa vasorum density was significantly increased in injured arteries of ChD group than in those of CD group. These results suggest that macrophages in the media and adventitia play an important role in expansive atherosclerotic remodeling via extracellular matrix degradation and SMC reduction.     

Expression of adhesion molecule T-cadherin is increased during neointima formation in experimental restenosis

Phenotypic modulation, migration and proliferation of vascular smooth muscle cells (SMCs) are major events in restenosis after percutaneous transluminal angioplasty. Surface cell adhesion molecules, essential to morphogenesis and maintenance of adult tissue architecture, are likely to be involved, but little is known about cell adhesion molecules expressed on SMCs. T-cadherin is a glycosyl phosphatidylinositol-anchored member of the cadherin superfamily of adhesion molecules. Although highly expressed in vascular and cardiac tissues, its function in these tissues is unknown. We previously reported increased expression of T-cadherin in intimal SMCs in atherosclerotic lesions and proposed a role for T-cadherin in phenotype control. Here we performed immunohistochemical analysis of spatial and temporal changes in vascular T-cadherin expression following balloon catheterisation of the rat carotid artery. T-cadherin expression in SMCs markedly increases in the media early (1-4 days) after injury, and later (day 7-28) in forming neointima, especially in its preluminal area. Staining for monocyte/macrophage antigen ED-1, proliferating cell nuclear antigen and smooth muscle alpha-actin revealed that spatial and temporal changes in T-cadherin level coincided with the peak in cell migration and proliferation activity during neointima formation. In colchicine-treated cultures of rat aortic SMCs T-cadherin expression is increased in dividing M-phase cells but decreased in non-dividing cells. Together the data support an association between T-cadherin expression and SMC phenotype.     

Specific Matrix Metalloproteinases Play Different Roles in Intraplaque Angiogenesis and Plaque Instability in Rabbits

Background

Ectopic angiogenesis within the intima and media is considered to be a hallmark of advanced vulnerable atherosclerotic lesions. Some studies have shown that specific matrix metalloproteinases (MMPs) might play different roles in angiogenesis. Therefore, we investigated the predominant effects of specific MMPs in intraplaque angiogenesis and plaque instability in a rabbit model of atherosclerosis.

Methods and Results

New Zealand rabbits underwent balloon injury of the abdominal artery and ingestion of a high-cholesterol (1%) diet to establish an atherosclerotic animal model. At weeks 4, 6, 8, 10, and 12 after balloon injury, five rabbits were euthanized and the abdominal aorta was harvested. Blood lipid analysis, intravascular ultrasound imaging, pathologic and immunohistochemical expression studies, and western blotting were performed. From weeks 4 to 12, the expression of MMP-1, -2, -3, and -9 and vascular endothelial growth factor A (VEGF-A) increased with atherosclerotic plaque development in the abdominal aorta, while the expression of MMP-14 substantially decreased. The vulnerability index (VI) gradually increased over time. Intraplaque neovessels appeared at week 8. The microvessel density (MVD) was greater at week 12 than at week 8. The VI, MVD, and VEGF-A level were positively correlated with the MMP-1, -2,-3, and -9 levels within plaques. Negative correlations were noted between the MMP-14 level and the VI, MVD, and VEGF-A level.

Conclusion

Upregulation of MMP-1, -2, -3, and -9 and downregulation of MMP-14 may contribute to intraplaque angiogenesis and plaque instability at the advanced stage of atherosclerosis in rabbits.     

Vanin-1 pantetheinase drives smooth muscle cell activation in post-arterial injury neointimal hyperplasia

The pantetheinase vanin-1 generates cysteamine, which inhibits reduced glutathione (GSH) synthesis. Vanin-1 promotes inflammation and tissue injury partly by inducing oxidative stress, and partly by peroxisome proliferator-activated receptor gamma (PPARγ) expression. Vascular smooth muscle cells (SMCs) contribute to neointimal hyperplasia in response to injury, by multiple mechanisms including modulation of oxidative stress and PPARγ. Therefore, we tested the hypothesis that vanin-1 drives SMC activation and neointimal hyperplasia. We studied reactive oxygen species (ROS) generation and functional responses to platelet-derived growth factor (PDGF) and the pro-oxidant diamide in cultured mouse aortic SMCs, and also assessed neointima formation after carotid artery ligation in vanin-1 deficiency. Vnn1(-/-) SMCs demonstrated decreased oxidative stress, proliferation, migration, and matrix metalloproteinase 9 (MMP-9) activity in response to PDGF and/or diamide, with the effects on proliferation linked, in these studies, to both increased GSH levels and PPARγ expression. Vnn1(-/-) mice displayed markedly decreased neointima formation in response to carotid artery ligation, including decreased intima:media ratio and cross-sectional area of the neointima. We conclude that vanin-1, via dual modulation of GSH and PPARγ, critically regulates the activation of cultured SMCs and development of neointimal hyperplasia in response to carotid artery ligation. Vanin-1 is a novel potential therapeutic target for neointimal hyperplasia following revascularization.     

Matrix metalloproteinase 2 (gelatinase A) is related to migration of keratinocytes.

The role of matrix metalloproteinases (MMPs) in cell migration was studied by measuring cell growth, migration, and production of MMP-2 and -9 in oral mucosal and skin keratinocytes cultured in the presence of synthetic MMP inhibitors. MMP-2 was the major gelatinolytic MMP produced by these cells while MMP-9 was produced at a low basal level. Inhibitor effects on MMP-9 production were therefore studied in keratinocytes stimulated by tumor necrosis factor alpha (TNFalpha). Tetracycline analogues at concentrations that inhibited the production of MMP-2 but not MMP-9 were able to drastically inhibit migration of both mucosal and skin keratinocytes. Tetracycline analogues also inhibited keratinocyte growth, an effect not found for the other inhibitors tested. Heterocyclic carbonate-derived compounds (LWs) that inhibited MMP-9 but not MMP-2 production had no effect on cell migration. Batimastat, a potent MMP inhibitor, did not have any effect on MMP production or cell growth but did inhibit keratinocyte migration. Tumor growth factor beta (TGFbeta) increased keratinocyte migration as well as both cell-associated and secreted MMP-2 production in wounded cell cultures. The secreted enzyme was partially converted into an active form. In this model batimastat totally blocked TGFbeta-promoted keratinocyte migration. Immunostaining of keratinocytes advancing into the wound revealed that MMP-2 was localized in extracellular matrix contactlike structures against the endogenously produced laminin-5-rich matrix. MMP-9 was localized diffusely along the cell membranes. Using in situ hybridization we observed that in chronically inflamed human gingiva MMP-2 is expressed in epithelium extending into subepithelial connective tissue. These results suggest that MMP-2 plays a specific role in epithelial migration, possibly by detaching the advancing cells from the pericellular matrix or by activating other MMPs.     

Gastric gelatinase B/matrix metalloproteinase-9 is rapidly increased in Helicobacter felis-induced gastritis

It has previously been shown that matrix metalloproteinase-9 (MMP-9) levels, originating from macrophages, are considerably increased in human Helicobacter pylori-associated gastritis. Here, the early kinetics of the MMP-9 response resulting from Helicobacter infection in C57BL/6 and MMP-9 knock-out mice using the murine Helicobacter felis model were examined. H. felis infection induced severe gastritis in the murine stomach at just 2 weeks after infection. Before gastritis, an increase was observed in MMP-9-positive cells detected by immunohistochemistry in the basal lamina propria. This finding was corroborated by gelatin zymography of stomach samples. As the gastritis increased so did the concentration of MMP-9 and the incidence of gastric MMP-9-positive cells, their location corresponding to that of macrophages. In contrast, systemic levels of MMP-9 remained unchanged. When MMP-9-deficient mice were infected with H. felis, no significant difference in gastritis development was detected compared with disease development in wild-type animals. We conclude that MMP-9 production is an early event in the response to gastric Helicobacter infection, a feature that may favor the recruitment of immune cells early during infection. At later stages, however, the increased levels of MMP-9 may damage the integrity of the stomach mucosa.     

Role of the hemopexin domain of matrix metalloproteinases in cell migration

The biological functions of matrix metalloproteinases (MMPs) extend beyond extracellular matrix degradation. Non-proteolytic activities of MMPs are just beginning to be understood. Herein, we evaluated the role of proMMPs in cell migration. Employing a Transwell chamber migration assay, we demonstrated that transfection of COS-1 cells with various proMMP cDNAs resulted in enhancement of cell migration. Latent MMP-2 and MMP-9 enhanced cell migration to a greater extent than latent MMP-1, -3, -11 and -28. To examine if proteolytic activity is required for MMP-enhanced cell migration, three experimental approaches, including fluorogenic substrate degradation assay, transfection of cells with catalytically inactive mutant MMP cDNAs, and addition of hydroxamic acid-derived MMP inhibitors, were employed. We demonstrated that the proteolytic activities of MMPs are not required for MMP-induced cell migration. To explore the mechanism underlying MMP-enhanced cell migration, structure-function relationship of MMP-9 on cell migration was evaluated. By using a domain swapping approach, we demonstrated that the hemopexin domain of proMMP-9 plays an important role in cell migration when examined by a transwell chamber assay and by a phagokinetic migration assay. TIMP-1, which interacts with the hemopexin domain of proMMP-9, inhibited cell migration, whereas TIMP-2 had no effect. Employing small molecular inhibitors, MAPK and PI3K pathways were found to be involved in MMP-9-mediated cell migration. In conclusion, we demonstrated that MMPs utilize a non-proteolytic mechanism to enhance epithelial cell migration. We propose that hemopexin homodimer formation is required for the full cell migratory function of proMMP-9.     

Systemic tissue inhibitor of metalloproteinase-1 gene delivery reduces neointimal hyperplasia in balloon-injured rat carotid artery

Metalloproteinases (MMP)-2 and MMP-9 play a role in smooth muscle cell (SMC) migration from the media to the intima following arterial injury. Intravenous administration of adenovirus encoding tissue inhibitor of metalloproteinase-1 (TIMP-1) into balloon-injured rat arteries (3 x 10(11) viral particles/rat; n=7) resulted in a transient expression of TIMP-1 and a significant inhibition of neointima thickening within 16 days ( approximately 40% vs. control; P=0.012). Three days after injury, the number of intimal SMCs was decreased by approximately 98% in TIMP-1-treated rats. However, no alteration was seen in intimal SMC proliferation after 13 days of injury. Therefore, our results show that systemic gene transfer of TIMP-1 is a promising approach in early restenosis treatment.     

Possible involvement of Cyclic-GMP-dependent protein kinase on matrix metalloproteinase-2 expression in rat aortic smooth muscle cells

Degradation and resynthesis of the extracellular matrix (ECM) are essential during tissue remodeling. Expansion of the vascular intima in atherosclerosis and restenosis following injury is dependent upon smooth muscle cell (SMC) proliferation and migration. The migration of SMC from media to intima critically depends on degradation of ECM protein by matrix metalloproteinases (MMPs). MMP inhibitors and eNOS gene transfer have been shown to inhibit SMC migration in vitro and neointima formation in vivo. Nitric oxide (NO) and cyclic-GMP have been implicated in the inhibition of VSMC migration. But, there are few studies addressing the role of NO signaling pathways on the expression of MMPs. Here we reported the involvement of cyclic-GMP-dependent protein kinase (PKG) (an important mediator of NO and cGMP signaling pathway in VSMC) on MMP-2 expression in rat aortic SMC. The goal of the present study was to gain insight into the possible involvement of PKG on MMP-2 in rat aortic SMC. MMP-2 protein and mRNA level and activity were downregulated in PKG-expressing cells as compared to PKG-deficient cells. In addition, the secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2) was increased in PKG-expressing cells as compared to PKG-deficient cells. PKG-specific membrane permeable peptide inhibitor (DT-2) reverses the process. Interestingly, little or no changes of MMP-9 were observed throughout the study. Taken together our data suggest the possible role of PKG in the suppression of MMP-2.     

Acceleration of matrix metalloproteinase-1 production and activation of platelet-derived growth factor receptor beta in human coronary smooth muscle cells by oxidized LDL and 4-hydroxynonenal

Increases in matrix metalloproteinases (MMPs) at atherosclerotic lesions are involved in the migration of smooth muscle cells (SMCs) into the intima and to the rupture of plaques, being implicated in the progression of atherosclerosis. The present study examined the mechanisms underlying the production of MMP-1, interstitial collagenase-1, induced by oxidized low-density lipoprotein (oxLDL) and 4-hydroxynonenal (4-HNE), factors proposed to play a pivotal role in atherogenesis, in human coronary SMCs. oxLDL promoted the production of MMP-1 with the preceding phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Immunoprecipitation of platelet-derived growth factor receptor beta (PDGFR-beta) revealed that oxLDL induced tyrosine phosphorylation of the receptor. Inhibition of the activation of PDGFR-beta and ERK1/2 resulted in a suppression of the production of MMP-1. Consistently, 4-HNE also elicited the production of MMP-1 with the preceding phosphorylation of PDGFR-beta and ERK1/2. The 4-HNE-induced production of MMP-1 was prevented when the activation of PDGFR-beta and ERK1/2 was inhibited. The present results suggest that the activation of PDGFR-beta and ERK1/2 is involved in the production of MMP-1 in oxLDL- and 4-HNE-stimulated human coronary SMCs.     

Mechanism of inhibition of matrix metalloproteinase-9 induction by NO in vascular smooth muscle cells.

Vascular smooth muscle (VSM) cell migration is a critical step in the development of a neointima after angioplasty. Matrix metalloproteinases (MMPs) degrade the basement membrane and extracellular matrix, facilitating VSM cell migration. Recently, we demonstrated that nitric oxide (NO) inhibits interleukin-1 beta (IL-1 beta)-stimulated MMP-9 induction in rat aortic VSM cells. In this study, we examined the hypothesis that NO inhibits MMP-9 induction by attenuating superoxide generation and extracellular signal-regulated kinase (ERK) activation. Stimulation of VSM cells with IL-1 beta significantly (P < 0.05) increased superoxide production, ERK activation, and MMP-9 induction. Pretreatment of VSM cells with the NO donor DETA NONOate significantly (P < 0.05) decreased IL-1 beta-stimulated superoxide generation. In addition, pretreatment of VSM cells with a specific ERK pathway inhibitor, PD-98059, or DETA NONOate inhibited IL-1 beta-stimulated ERK activation and MMP-9 induction. Direct exposure of VSM cells to increased superoxide levels by treatment with xanthine/xanthine oxidase increased ERK activation and MMP-9 induction, whereas pretreatment of cells with PD-98059 significantly (P < 0.05) inhibited xanthine/xanthine oxidase-stimulated ERK activation and MMP-9 induction. We conclude that NO inhibits IL-1 beta-stimulated MMP-9 induction by inhibiting superoxide generation and subsequent ERK activation.     

The role of matrix metalloproteinases 2 and 9 during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

Matrix metalloproteinases (MMPs) are implicated in a wide range of physiological and pathological processes, including morphogenesis, wound healing, angiogenesis, inflammation, and cancer. The purpose of this study was to characterize the role of MMPs as depicted by the expression of MMP-2 and MMP-9 during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis. Male Wistar rats were distributed into three groups of 10 animals each and treated with 4-nitroquinoline 1-oxide solution at 50 ppm through their drinking water for 4, 12, and 20 weeks. Ten animals were used as control group. No histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure; however, immunoexpression of MMP-2 was noticed. The same picture occurred to MMP-9, in which positive expression was detected for this immunomarker. MMP-2 and MMP-9 showed positive expression either in pre-neoplastic lesions at 12 weeks following carcinogen exposure or in well-differentiated squamous cell carcinoma induced after 20 weeks of treatment with 4NQO. Taken together, our results support the belief that MMP-2 and MMP-9 play important role during malignant transformation and conversion of oral mucosa as assessed by immunohistochemistry.     

Matrix metalloproteinases 9 and 2 are necessary for the migration of Langerhans cells and dermal dendritic cells from human and murine skin

Dendritic cells migrate from the skin to the draining lymph nodes. They transport immunogenic MHC-peptide complexes, present them to Ag-specific T cells in the T areas, and thus generate immunity. Migrating dendritic cells encounter physical obstacles, such as basement membranes and collagen meshwork. Prior work has revealed that matrix metalloproteinase-9 (MMP-9) contributes to mouse Langerhans cell migration. In this study, we use mouse and human skin explant culture models to further study the role of MMPs in the migration and maturation of skin dendritic cells. We found that MMP-2 and MMP-9 are expressed on the surface of dendritic cells from the skin, but not from other sources. They are also expressed in migrating Langerhans cells in situ. The migration of both Langerhans cells and dermal dendritic cells is inhibited by a broad spectrum inhibitor of MMPs (BB-3103), by Abs to MMP-9 and -2, and by the natural tissue inhibitors of metalloproteinases (TIMP), TIMP-1 and TIMP-2. Inhibition by anti-MMP-2 and TIMP-2 define a functional role for MMP-2 in addition to the previously described function of MMP-9. The importance of MMP-9 was emphasized using MMP-9-deficient mice in which Langerhans cell migration from skin explants was strikingly reduced. However, MMP-9 was only required for Langerhans cell migration and not maturation, since nonmigrating Langerhans cells isolated from the epidermis matured normally with regard to morphology, phenotype, and T cell stimulatory function. These data underscore the importance of MMPs, and they may be of relevance for therapeutically regulating dendritic cell migration in clinical vaccination approaches.     

Salvianolic acid B attenuates MMP-2 and MMP-9 expression in vivo in apolipoprotein-E-deficient mouse aorta and in vitro in LPS-treated human aortic smooth muscle cells

Salvianolic acid B (Sal B), a water-soluble antioxidant derived from a Chinese medicinal herb, is believed to have multiple therapeutic and preventive against human vascular diseases, including atherosclerosis and restenosis. To elucidate the underlying cellular mechanisms, we produced hypercholesterolemia by feeding apo-E-deficient mice a 0.15% cholesterol diet and inflammation in human aortic smooth muscle cells (HASMCs) with the endotoxin lipopolysaccharide (LPS), focusing on the metallopreteinases MMP-2 and MMP-9, the relevant signal transduction pathways and the effects of Sal B. Immunohistochemical analyses indicated apo-E-deficient mice fed a 0.15% cholesterol diet for 12 weeks exhibited thickened intima and elevated levels of MMP-2 and MMP-9 in aortic sections, both of which were attenuated by 0.3% Sal B dietary supplement. Western blotting and zymography analyses indicated that unstimulated HASMCs exhibited basal levels of protein and activity levels for MMP-2 and barely detectable levels for MMP-9, both of which were markedly upregulated by LPS, which also induced cell migration. Sal B significantly attenuated upregulations of both MMPs as well as the LPS-induced cell migration through the inactivation of MMP-2 and MMP-9 protein synthesis as well as the downregulation of the extracellular-signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH(2)-terminal kinase (JNK). These results demonstrate that Sal B has anti-migration properties on smooth muscle cells and may explain its anti-atherosclerotic properties. This novel mechanism of action of Sal B, in addition to its previously reported inhibition of LDL oxidation, may help explain its efficacy in the treatment of atherosclerosis.