Shigeru Kanda

Deficiency of Gelatinase A Suppresses Smooth Muscle Cell Invasion and Development of Experimental Intimal Hyperplasia

Background—Although it has been demonstrated that matrix metalloproteinases (MMPs) play an important role in the arterial remodeling in atherosclerosis and restenosis, it is not clear which MMP is involved in which process. To define the role of MMP-2 in arterial remodeling, we evaluated the influence of the targeted deletion of the MMP-2 gene on vascular remodeling after flow cessation in the murine carotid arteries. Methods and Results—The left common carotid arteries of wild-type and MMP-2–deficient mice were ligated just proximal to their bifurcations, and the animals were then processed for morphological and biochemical studies at specific time points. MMP-2 activity and mRNA levels increased in ligated carotid arteries of wild-type mice on the basis of observation by gelatin zymography and quantitative real-time RT-PCR. There was significantly less intimal hyperplasia in MMP-2–deficient mice at 2 and 4 weeks after ligation than there in wild-type mice. Arterial explants from the aorta of MMP-2–deficient mice showed that smooth muscle cell (SMC) migration was inhibited in comparison with wild-type mice. The chemoattractant-directed invasion through a reconstituted basement membrane barrier was significantly reduced in cultured SMCs derived from MMP-2–deficient mice, although no difference was observed in SMC migration across the filter or in proliferative response between the control and MMP-2–deficient mice. Conclusions—In a mouse carotid artery blood flow cessation model, MMP-2 contributes to intimal hyperplasia mainly through the SMC migration from the media into the intima by degrading and breaching the extracellular matrix proteins surrounding each cell and the internal elastic lamina.

Induction of Macrophage VEGF in Response to Oxidized LDL and VEGF Accumulation in Human Atherosclerotic Lesions

The interaction between macrophages and oxidatively modified low density lipoprotein (Ox-LDL) appears to play a central role in the development of atherosclerosis, not only through foam cell formation but also via the induction of numerous cytokines and growth factors. The current study demonstrated that Ox-LDL upregulated vascular endothelial growth factor (VEGF) mRNA expression in RAW 264 cells, a monocytic cell line, in a time- and concentration-dependent manner and that Ox-LDL stimulated VEGF protein secretion from the cells. Lysophosphatidylcholine, a component of Ox-LDL, also enhanced VEGF mRNA expression in RAW 264 cells and VEGF secretion from RAW 264 cells, with a maximal effect at a concentration of 10 micromol/L lysophosphatidylcholine. Immunohistochemical studies showed that human early atherosclerotic lesions exhibited intense VEGF immunoreactivity in subendothelial macrophage-rich regions of the thickened intima. In atherosclerotic plaques, VEGF staining was also observed in foam cell-rich regions adjacent to the lipid core or the neovascularized basal regions of plaque consisting predominantly of smooth muscle cells. High-power-field observation revealed that VEGF was localized in the extracellular space as well as at the macrophage cell surface. These observations suggest the possible involvement of Ox-LDL in the development of human atherosclerosis through VEGF induction in macrophages.

Green tea catechins inhibit the cultured smooth muscle cell invasion through the basement barrier

Epidemiological studies suggest that green tea consumption is associated with a reduced risk of cardiovascular disease. Antioxidative properties of green tea flavonoids, catechins, have been believed to be involved in the antiatherogenic effect of green tea, since catechins inhibit low density lipoprotein oxidation. The migration of vascular smooth muscle cells (SMCs) from the tunica media to the subendothelial region is a key event in the development and progression of atherosclerosis and post-angioplasty vascular remodeling. Matrix metalloproteinases (MMPs) play a key role in these processes of SMC migration. In the present study, we investigated the effect of catechins on the gelatinolytic activity of MMP-2 that was derived from cultured bovine aortic SMCs. We also investigated the effect of catechins on the SMC invasion through the reconstituted basement membrane barrier. A major constituent of green tea catechins, (-)-epigallocatechin gallate (EGCG), inhibited the gelatinolytic activity of MMP-2 and concanavalin A (ConA)-induced pro-MMP-2 activation without the influence of membrane-type MMP expression in SMCs. EGCG also inhibited the SMC invasion through the basement membrane barrier in a concentration-dependent manner without any influence of SMC migration across the basement membrane protein thin-coated filter. The antagonistic effects of other catechins, namely (-)-epigallocatechin (EGC) and (-)-epicatechin gallate (ECG), on gelatinolytic activity of MMP-2, ConA-induced pro-MMP-2 activation, or PDGF-BB-directed SMC invasion were much less pronounced than those of EGCG. Also, (+)-catechin and (-)-epicatechin failed to show any effect. These findings may suggest that the anti-invasive and anti-metalloproteinase activities involve at least part of the anti-atherogenic action of catechin in accordance with the antioxidant properties of catechin.

Rho-Rho kinase is involved in smooth muscle cell migration through myosin light chain phosphorylation-dependent and independent pathways

Although Rho, a small GTPase, has been demonstrated to play an important role in the smooth muscle contraction and relaxation, little is known about the involvement of Rho protein in smooth muscle cell (SMC) migration. In this study the role of Rho-Rho kinase pathway was examined in SMC migration induced by platelet-derived growth factor (PDGF) and lysophosphatidic acid (LPA). C3 transferase, a specific inhibitor of Rho, blocked SMC migration induced by PDGF and LPA. Y-27632, a specific inhibitor of Rho kinase, a direct target molecule of Rho, inhibited PDGF and LPA-induced SMC migration in a concentration dependent manner. Although rapid increase in myosin light chain (MLC) phosphorylation in SMC treated with LPA was observed, no enhanced MLC phosphorylation was detected in response to PDGF. Y-27632 suppressed LPA-induced as well as basal level of MLC phosphorylation. ML-9, a specific inhibitor of myosin light chain kinase (MLCK), inhibited PDGF and LPA-induced SMC migration without the suppression of MLC phosphorylation at 5 min incubation, suggesting that MLCK may contribute to SMC migration via mechanism other than MLC phosphorylation. These results suggest that Rho-Rho kinase pathway is implicated in SMC migration and that different signaling pathways downstream of Rho-Rho kinase may be involved in LPA and PDGF-induced SMC migration. MLC phosphorylation via Rho-Rho kinase pathway appears to be implicated in LPA-dependent SMC migration. Whereas PDGF-mediated SMC migration is independent of increased MLC phosphorylation and other target molecules downstream of Rho-Rho kinase seem to be involved.

Increased Expression of Elastolytic Cysteine Proteases, Cathepsins S and K, in the Neointima of Balloon-Injured Rat Carotid Arteries

The matrix-degrading activity of several proteases are involved in the accelerated breakdown of extracellular matrix associated with vascular remodeling during the development of atherosclerosis and vascular injury-induced neointimal formation. Previous studies have shown that the potent elastolytic cysteine proteases, cathepsins S and K, are overexpressed in atherosclerotic lesions in human and animal models. However, the role of these cathepsins in vascular remodeling remains unclear. In the present study, the expressions of cathepsin S and K and their inhibitor cystatin C were examined during arterial remodeling using a rat carotid artery balloon-injury model. The increase in both cathepsin S and K mRNA levels was observed from day 1 and day 3 through day 14 following the induction of balloon injury, respectively. Western blotting analysis revealed that both cathepsin S and K protein levels also increased in the carotid arteries during neointima formation, coinciding with an increase elastolytic activity assayed using Elastin-Congo red, whereas, no significant change in the expressions of cystatin C mRNA and protein was observed during follow-up periods after injury. Immunohistochemistry, Western blot, and in situ hybridization showed that the increase of cathepins S and K and the decrease of cystatin C occurred preferentially in the developing neointima. These findings suggest that cathepsin S and K may participate in the pathological arterial remodeling associated with restenosis.

Glycation cross-links inhibit matrix metalloproteinase-2 activation in vascular smooth muscle cells cultured on collagen lattice

Aims/hypothesis. Extracellular matrix glycation has been proposed to contribute to the arterial stiffness observed in aging and diabetes. We examined whether matrix protein glycation regulates the proleolytic process through the manipulation of matrix metalloproteinases (MMPs) activation, using collagen fibrils model. Methods. Vascular smooth muscle cells were cultured on control or glycated collagen fibrils. Matrix metalloproteinase-2 activation and the production of tissue inhibitors of metalloproteinase (TIMPs) were measured in the conditioned medium by using gelatin zymography and immunoblotting. Membrane type 1 matrix metalloproteinase (MT1-MMP) expression was also measured in cell lysates. Results. When smooth muscle cells were cultured on collagen fibrils, pro-MMP-2 processing to active form was observed in the conditioned medium in coincidence with the increased MT1-MMP expression and the suppressed TIMP-2 production. Culturing smooth muscle cells on glycated collagen fibrils inhibited MMP-2 activation and attenuated MT1-MMP expression without the alteration of TIMP-2 production compared with control fibrils, indicating the possible mechanism of the suppression of MT1-MMP expression for the inhibition of MMP-2 activation on glycated collagen fibrils. Inclusion of aminoguanidine, an inhibitor of cross-linking formation, during collagen glycation restored the MMP-2 activation, suggesting the role of cross-links on the inhibition of MMP-2 activation. Conclusion/interpretation. These observations suggest that glycation-induced cross-linking formation in interstitial collagen contributes to arterial stiffness in aging and diabetes through the manipulation of matrix metalloproteinase activation along with the reduction of the susceptibility to proteolytic enzymes. [Diabetologia (2001) 44: 433–436]

Inhibition of angiogenesis on glycated collagen lattices

Summary Advanced glycation endproduct (AGE) accumulation in extracellular matrix proteins has been demonstrated in diabetic patients with a significant correlation with the severity of diabetic complications. AGE accumulation induces matrix protein cross-link formation, resulting in an increased stiffness of matrix fibres and the reduction of the susceptibility of matrix proteins to proteolytic degradation. We examined whether glycation-induced collagen cross-linking may affect vascular endothelial cell behaviours such as invasion, proliferation and differentiation, using the in vitro angiogenesis model of capillary-like structure formation in three-dimensional matrices of collagen type I. Endothelial cells cultured on collagen gel with angiogenic factors (the combination of fibroblast growth factor-2 and vascular endothelial growth factor) invaded the underlying collagen matrix, and organized capillary-like cord structures in the gel. We found that endothelial cell invasion into glycated collagen gel was significantly attenuated without any effect on proteinase activity including cell-associated plasminogen activator and matrix metalloproteinase in the conditioned medium. In addition, subsequent capillary-like cord formation was also inhibited in glycated collagen gel. In contrast, endothelial cell proliferation was enhanced on glycated collagen gel with or without angiogenic factors compared with control collagen gel. These results suggest that the structural alterations of extracellular matrix proteins through the glycation-induced cross-link formation affect the interaction between endothelial cell and extracellular matrix, resulting in the impairment of an adequate neovascularization in diabetic patients. [Diabetologia (1998) 41: 491–499]

Epigallocatechin-3-gallate binding to MMP-2 inhibits gelatinolytic activity without influencing the attachment to extracellular matrix proteins but enhances MMP-2 binding to TIMP-2.

Although epigallocatechin-3-gallate (EGCg), a dominant component of green tea catechins, has been demonstrated to have anti-gelatinase properties, the molecular mechanisms by which EGCg blocks gelatinolytic activities remain unknown. We investigated whether EGCg may affect matrix metalloproteinase-2 (MMP-2) binding to native and denatured-type I collagen, and binding to the tissue inhibitor of metalloproteinase-2 (TIMP-2). Here, we report that EGCg forms a reversible complex with MMP-2, resulting in the inhibition of gelatinolytic activity of MMP-2. EGCg had no effect on the MMP-2 binding to immobilized native and denatured-type I collagen, but significantly enhanced pro- and activated MMP-2 binding to TIMP-2, as assessed by immunoprecipitation. These findings provide a new understanding of the molecular mechanisms underlying the inhibitory effect of EGCg on the gelatinolytic activity of MMP-2.

VEGF Protects Against Oxidized LDL Toxicity to Endothelial Cells by an Intracellular Glutathione-Dependent Mechanism Through the KDR Receptor

Abstract—Although the accumulation of vascular endothelial growth factor (VEGF) has been observed in human atherosclerotic lesions, the exact role of this growth factor in atherogenesis remains unknown. We hypothesized that VEGF in the vascular wall might have a preventive effect on endothelial cell damage during atherosclerosis. To test our hypothesis, we examined whether VEGF protects against the toxicity of oxidized low density lipoprotein (Ox-LDL) in cultured endothelial cells derived from bovine aortas (BAECs). Preincubation of BAECs with VEGF prevented Ox-LDL-induced toxicity in a preincubation time- and VEGF concentration-dependent manner. Addition of N&ohgr;-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor, did not reverse the protective effect of VEGF on Ox-LDL toxicity. Incubation of BAECs with VEGF increased intracellular glutathione (GSH) content in a time-dependent manner. Combined addition of VEGF and l-buthionine sulfoximine, a GSH synthesis inhibitor, reversed both GSH levels and the protective effect of VEGF on Ox-LDL-induced cytotoxicity. Placenta growth factor, which ligates to the VEGF Flt-1 receptor but not KDR/Flk-1, failed to prevent Ox-LDL toxicity and had no effect on intracellular GSH levels. An anti-KDR antibody completely blocked these beneficial activities of VEGF. These results suggest that VEGF prevents Ox-LDL-induced endothelial cell damage via an intracellular GSH-dependent mechanism through the KDR/Flk-1 receptor.

A novel model of occlusive thrombus formation in mice

A novel model to induce occlusive thrombus formation was developed in mice in vivo. Mice were simultaneously treated with ligation and cuff placement at the left carotid artery. At 7 days after the treatment, occlusive thrombus was observed at the intracuff region, but not in the distal and proximal regions of the cuff, and not induced by a single treatment of ligation or cuff placement. The plasma levels of von Willebrand factor (vWF), which represent the endothelial status, were significantly increased in combined treatment of ligation and cuff placement 1 day after the operation. Whereas no significant changes in plasma vWF were observed in either single treatment of ligation or cuff placement. The expression of vWF, considered to be the endothelial marker, was detected on the luminal surface distal and proximal to the cuff and the carotid artery in the single treatment groups treated with either ligation or cuff placement, but was not detected in the intracuff region. Furthermore, the binding of Griffolia Simplicifolia Lectin-I (GSL-I) and endothelial nitric oxide synthase (eNOS) expression indicating the endothelial integrity was not detected in the intracuff region. Intermittent injections of ancrod, which decreases the plasma fibrinogen, inhibited occlusive thrombus formation in the intracuff region. The expression of eNOS was detected at the distal and proximal but not the intracuff region of the carotid artery treated with ancrod. Daily administration of aspirin significantly suppressed the thrombus formation in this model. These results indicate that occlusive thrombus formation accompanied by endothelial damage or dysfunction is induced by the combined application of ligation and cuff placement at the carotid artery, and suggest that this endothelial damage or dysfunction may be one pathogenesis of thrombogenesis in this model.