Masafumi Kuzuya

Taxol inhibits neointimal smooth muscle cell accumulation after angioplasty in the rat.

Despite significant improvements in the primary success rate of the medical and surgical treatments for atherosclerotic disease, including angioplasty, bypass grafting, and endarterectomy, secondary failure due to late restenosis continues to occur in 30-50% of individuals. Restenosis and the later stages in atherosclerotic lesions are due to a complex series of fibroproliferative responses to vascular injury involving potent growth-regulatory molecules (such as platelet-derived growth factor and basic fibroblast growth factor) and resulting in vascular smooth muscle cell (VSMC) proliferation, migration, and neointimal accumulation. We show here, based on experiments with both taxol and deuterium oxide, that microtubules are necessary for VSMCs to undergo the multiple transformations contributing to the development of the neointimal fibroproliferative lesion. Taxol was found to interfere both with platelet-derived growth factor-stimulated VSMC migration and with VSMC migration and with VSMC proliferation, at nanomolar levels in vitro. In vivo, taxol prevented medial VSMC proliferation and the neointimal VSMC accumulation in the rat carotid artery after balloon dilatation and endothelial denudation injury. This effect occurred at plasma levels approximately two orders of magnitude lower than that used clinically to treat human malignancy (peak levels achieved in this model were approximately 50-60 nM). Taxol may therefore be of therapeutic value in preventing human restenosis with minimal toxicity.

Deficiency of the Cysteine Protease Cathepsin S Impairs Microvessel Growth

Abstract— During angiogenesis, microvascular endothelial cells (ECs) secrete proteinases that permit penetration of the vascular basement membrane as well as the interstitial extracellular matrix. This study tested the hypothesis that cathepsin S (Cat S) contributes to angiogenesis. Treatment of cultured ECs with inflammatory cytokines or angiogenic factors stimulated the expression of Cat S, whereas inhibition of Cat S activity reduced microtubule formation by impairing cell invasion. ECs from Cat S–deficient mice showed reduced collagenolytic activity and impaired invasion of collagens type I and IV. Cat S–deficient mice displayed defective microvessel development during wound repair. This abnormal angiogenesis occurred despite normal vascular endothelial growth factor and basic fibroblast growth factor levels, implying an essential role for extracellular matrix degradation by Cat S during microvessel formation. These results demonstrate a novel function of endothelium-derived Cat S in angiogenesis.

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.

Neointima formation in a restenosis model is suppressed in midkine-deficient mice

Neointima formation is a common feature of atherosclerosis and restenosis after balloon angioplasty. To find a new target to suppress neointima formation, we investigated the possible role of midkine (MK), a heparin-binding growth factor with neurotrophic and chemotactic activities, in neointima formation. MK expression increased during neointima formation caused by intraluminal balloon injury of the rat carotid artery. Neointima formation in a restenosis model was strongly suppressed in MK-deficient mice. Continuous administration of MK protein to MK-deficient mice restored neointima formation. Leukocyte recruitment to the vascular walls after injury was markedly decreased in MK-deficient mice. Soluble MK as well as that bound to the substratum induced migration of macrophages in vitro. These results indicate that MK plays a critical role in neointima formation at least in part owing to its ability to mediate leukocyte recruitment.

Effect of MMP-2 Deficiency on Atherosclerotic Lesion Formation in ApoE-Deficient Mice

Objective—Although it has been reported that matrix metalloproteinase (MMP)-2 is a major proteinase in atherosclerotic plaque lesions, there is no direct evidence of the role of MMP-2 in atherosclerotic lesion formation. In the present study we determined the role of MMP-2 in atherosclerosis plaque development using apolipoprotein E-deficient (apoE−/−) mice. Methods and Results—To generate MMP-2–deficient, apoE-deficient mice (MMP-2−/−:apoE−/−), MMP-2−/− mice were crossed with apoE−/− mice. After 8 weeks of feeding with a lipid-rich diet, morphological and biochemical studies of the aortic sinus and arch were conducted. A significant reduction of the atherosclerotic plaque in the aortic sinus and arch with the decrease in smooth muscle cell-positive area was observed in MMP-2−/−:apoE−/− mice compared with that of MMP-2+/+:apoE−/− mice. Macrophage- and collagen-positive areas were less in aortic sinus but not in aortic arch in MMP-2−/−:apoE−/− mice. There was no difference of MMP-9 mRNA expression in the plaque lesion between the 2 genotypes. A much lower level of mRNA expression of TIMP-1 and TIMP-2 was detected in the atherosclerotic plaque lesions of MMP-2−/−:apoE−/− mice than in those of MMP-2+/+:apoE−/− mice. Conclusions—MMP-2 contributes to the development of atherosclerosis in apoE−/− mice.

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.

Role of lipoprotein-copper complex in copper catalyzed-peroxidation of low-density lipoprotein

The oxidative modification of low-density lipoprotein (LDL) is suggested to play an important role in the pathogenesis of atherosclerosis. The present study examined the role of the formation of LDL-copper (Cu) complex in the peroxidation of LDL. The amount of copper bound to LDL increased during incubation performed with increasing concentrations of CuSO4. More than 80% of the copper bound to the LDL particle was observed in the protein phase of LDL, suggesting that most of the copper ions formed complexes with the ligand-binding sites of apoprotein. The addition of histidine (1 mM), known to form a high affinity complex with copper, and EDTA (1 mM), a metal chelator, during the incubation of LDL with CuSO4 prevented the formation of both thiobarbituric acid-reactive substances (TBARS) and LDL-Cu complexes. EDTA inhibited the copper-catalyzed ascorbate oxidation whereas histidine had no effect, suggesting that the copper within the complex with histidine is available to catalyze the reaction, in contrast to EDTA. These observations indicate that the preventive effect of histidine on the copper-catalyzed peroxidation of LDL is not simply mediated by chelating free copper ions in aqueous phase. Evidence that copper bound to LDL particle still has a redox potential was provided by the observed increase in TBARS content during incubation of LDL-Cu complexes in the absence of free copper ions. The addition of either histidine or EDTA to LDL-Cu complexes inhibited the formation of TBARS by removing copper ions from the LDL forming the corresponding complexes. However, there still remained small amounts of copper in the LDL particles following the treatment of LDL-Cu complexes with histidine or EDTA. The copper ions remaining in the LDL particle lacked the ability to catalyze LDL peroxidation, suggesting that there may be two types of copper binding sites in LDL: tight-binding sites, from which the copper ions are not removed by chelation, and weak-binding sites, from which copper ions are easily removed by chelators. The formation of TBARS in the LDL preparation during incubation with CuSO4 was comparable to the incubation with FeSO4. In contrast, the formation of TBARS in the LDL-lipid micelles by CuSO4 was nearly eliminated even in the presence of ascorbate to promote metal-catalyzed lipid peroxidation, although a marked increase in TBARS content was observed in the LDL-lipid micelles with FeSO4, and with FeCl3 in the presence of ascorbate.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of inducible nitric oxide synthase in T lymphocytes and macrophages of cholesterol-fed rabbits

While endothelial nitric oxide synthase has been reported to be expressed in the endothelial cells of normal and atherosclerotic vessels, there are few reports about inducible nitric oxide synthase (iNOS). We investigated the expression of iNOS and its relation to inflammatory cells in atheroma. New Zealand White rabbits were fed 1 of 4 diets: (i) normal diet for 9 weeks; (ii) normal plus 1% cholesterol diet (atherogenic diet) for 9 weeks; (iii) atherogenic diet for 9 weeks, then normal diet for 9 weeks; (iv) atherogenic diet for 9 weeks, then the normal diet for 36 weeks. The aortas were examined by immunohistochemical staining for anti-iNOS antibody, as well as antibodies for macrophages, T lymphocytes, and muscle actin. No iNOS was detected in normal aortas, intimal thickenings, or fatty streaks. Although iNOS was detected in necrotic cores of advanced plaque, it was not seen in smooth muscle-derived cells or endothelial cells but was found in some macrophage-derived cells and in T lymphocytes. In regressive atherosclerotic aortas, iNOS was detected only in necrotic cores, not in macrophage-derived cells but in T lymphocytes. These findings suggest that T lymphocytes and some macrophages induce iNOS through cytokine production in atheroma. This is the first report of iNOS expression in atheromatous plaque.

Mechanisms Underlying the Impairment of Ischemia-Induced Neovascularization in Matrix Metalloproteinase 2–Deficient Mice

Matrix metalloproteinases (MMPs) have been implicated in the process of neovascularization. However, the exact roles of individual MMPs in vessel formation are poorly understood. To study the putative role of MMP-2 in ischemia-induced neovascularization, a hindlimb ischemia model was applied to MMP-2+/+ and MMP-2−/− mice. Serial laser Doppler blood-flow analysis revealed that the recovery of the ischemic/normal blood-flow ratio in MMP-2−/− young and old mice remained impaired throughout the follow-up period. At day 35, microangiography and anti–l-lectin immunohistochemical staining revealed lesser developed collateral vessels and capillary formation in both old and young MMP-2−/− mice compared with the age-matched MMP-2+/+ mice. An aortic-ring culture assay showed a markedly impaired angiogenic response in MMP-2−/− mice, which was partially recovered by supplementation of the culture medium with recombinant MMP-2. Aorta-derived endothelial cells or bone marrow–derived endothelial progenitor cell (EPC)-like c-Kit+ cells from MMP-2−/− showed marked impairment of invasive or/and proliferative abilities. At day 7, plasma and ischemic tissues of vascular endothelial growth factor protein were reduced in MMP-2−/−. Flow cytometry showed that the numbers of EPC-like CD31+c-Kit+ cells in peripheral blood markedly decreased in MMP-2–deficient mice. Transplantation of bone marrow–derived mononuclear cells from MMP-2+/+ mice restored neovascularization in MMP-2−/− young mice. These data suggest that MMP-2 deficiency impairs ischemia-induced neovascularization through a reduction of endothelial cell and EPC invasive and/or proliferative activities and EPC mobilization.

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.