Xian Wu Cheng

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.

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.

A Simple Method of Plaque Rupture Induction in Apolipoprotein E–Deficient Mice

Objective—The development of a murine model of atherosclerotic plaque rupture. Methods and Results—The left common carotid arteries of male apolipoprotein E (apoE)–deficient mice (9 weeks old) were ligated just proximal to their bifurcations. After 4 weeks on a standard diet, the mice received polyethylene cuff placement just proximal to the ligated site, and the animals were then processed for morphological studies at specific time points. Ligation of the carotid artery in apoE-deficient mice for 4 weeks induced marked intimal hyperplasia, which is a lipid- and collagen-rich lesion that contains a number of macrophages, T lymphocytes, and smooth muscle cells. Subsequently, the cuff placement evoked intraplaque hemorrhage and plaque rupture with fibrin(ogen)-positive luminal thrombus in this region accompanying a decrease in collagen content as well as an increase in apoptotic cells in the intima within a few days after cuff placement. Conclusions—We demonstrated the murine model of human plaque rupture, which is simple, fast, and highly efficient. This model would help us not only to understand the mechanism of human plaque rupture but also to assess various already-known and as-yet-unknown agents in the future.

Role for Cysteine Protease Cathepsins in Heart Disease Focus on Biology and Mechanisms With Clinical Implication

The extracellular matrix (ECM) of the heart is composed largely of elastin and collagen and plays many roles in cardiac wall and valve homeostasis. Maintenance of a healthy cardiac system relies on controlled biosynthesis, maturation, function, and breakdown of ECM proteins. Dysregulation of proteolytic enzymes may disrupt these normal biological processes in myocardium-coronary-valve disease (CCVD). Substantial evidence supports the involvement of matrix metalloproteinase (MMP) and serine protease families in this process (reviewed elsewhere1,2). Cysteinyl proteases have received much less consideration in this regard, even though cardiovascular cells and macrophages with greatly expanded lysosomal compartments figure prominently in the pathogenesis of CCVD. Lysosomal cysteine proteases, generally known as cathepsins, were discovered in the second half of the 20th century. In the initial years after their discovery, cysteinyl cathepsins were shown to localize in lysosomes and endosomes and to function there to degrade unwanted intracellular or endocytosed proteins.3–5 However, the recent recognition of the inducible cathepsins F, K, S, B, and L led to the unraveling of their molecular functions in inflammatory and/or autoimmune diseases such as atherosclerosis,6–11 obesity,12–14 rheumatoid arthritis,15,16 cardiac repair,17 cardiomyopathy,18–20 and cancer.21 Most strikingly, we have now discovered that these cathepsins can be secreted into and function within the extracellular spaces. The observations of cathepsin expression and activity in failing cardiac tissues22–24 and valve tissues25–27 from humans and animals and in cultured media of cardiomyocytes,22,24 cardiac fibroblasts,22 vascular smooth muscle cells,28 endothelial cells,12 and macrophages6,10 have significantly broadened our understanding of their potential roles in cardiovascular pathogenesis. Furthermore, recent studies have shown that pharmacological cathepsin inhibition exhibits cardiovascular protective actions in animal models. …

Cysteine Protease Cathepsins in Atherosclerosis-Based Vascular Disease and Its Complications

Atherosclerosis-based vascular disease is an inflammatory disease characterized by extensive remodeling of the extracellular matrix architecture of the arterial wall. Although matrix metalloproteinases and serine proteases participate in these pathological events, the discovery of cysteine protease cathepsins, such as cathepsins K, S, L, and B, and cystatin C, and their tissue distribution has suggested that at least some of them participate in cardiovascular disease. Studies on vascular cells have shown that atherosclerosis-associated inflammatory cytokines augment cysteinyl cathepsin expression and activity. Novel insight into cathepsin functions has been made possible by the generation and in-depth analysis of knockout and transgenic mice. These studies have provided direct evidence implicating cathepsins in atherosclerosis-based vascular disease through the activation, liberation, and modification of angiogenic growth factors, cytokines, and proteases associated with lipid metabolism, cell events (migration, invasion, proliferation, and apoptosis), angiogenesis, and matrix protein remodeling. Furthermore, evaluation of the feasibility of cathepsins as a diagnostic tool has revealed that the serum cathepsins S and L and the endogenous inhibitor cystatin C hold promise as biomarkers of coronary artery disease and aneurysm formation. The goal of this review is to summarize the available information regarding the mechanistic contributions of cathepsins in atherosclerosis-based vascular disease.

Elastolytic Cathepsin Induction/Activation System Exists in Myocardium and Is Upregulated in Hypertensive Heart Failure

Cathepsins are cysteine proteases that participate in various types of tissue remodeling. However, their expressions during myocardial remodeling have not been examined. In this study, we investigated their expressions in the left ventricular (LV) myocardium of rats and humans with hypertension-induced LV hypertrophy or heart failure (HF). Real-time PCR and immunoblot analysis revealed that the abundance of cathepsin S mRNA or protein in the LV tissues was greater in rats or humans with HF than in those with hypertrophy or in control subjects. Immunostaining showed that cathepsin S was localized predominantly to cardiac myocytes and coronary vascular smooth muscle cells, but also overlapped in part with macrophages. Elastic lamina fragmentations significantly increased in the LV intramyocardial coronary arteries of HF rats. The amount of elastolytic activity in the extract of the LV myocardium was markedly increased for HF rats compared with controls, and this activity was mostly because of cathepsin S. Although the amount of elastin mRNA was increased in the LV myocardium of HF rats, the area of interstitial elastin was not. The expression of interleukin 1β was increased in the LV myocardium of HF rats, and this cytokine was found to increase the expression and activity of cathepsin S in cultured neonatal cardiomyocytes. These results suggest that cathepsin S participates in pathological LV remodeling associated with hypertension-induced HF. This protease is, thus, a potential target for therapeutics aimed at preventing or reversing cardiac remodeling.

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.

Exercise Training Stimulates Ischemia-Induced Neovascularization via Phosphatidylinositol 3-Kinase/Akt-Dependent Hypoxia-Induced Factor-1α Reactivation in Mice of Advanced Age

Background— Exercise stimulates the vascular response in pathological conditions, including ischemia; however, the molecular mechanisms by which exercise improves the impaired hypoxia-induced factor (HIF)-1&agr;–mediated response to hypoxia associated with aging are poorly understood. Here, we report that swimming training (ST) modulates the vascular response to ischemia in aged (24-month-old) mice. Methods and Results— Aged wild-type mice (MMP-2+/+) that maintained ST (swimming 1 h/d) from day 1 after surgery were randomly assigned to 4 groups that were treated with either vehicle, LY294002, or deferoxamine for 14 days. Mice that were maintained in a sedentary condition served as controls. ST increased blood flow, capillary density, and levels of p-Akt, HIF-1&agr;, vascular endothelial growth factor, Fit-1, and matrix metalloproteinase-2 (MMP-2) in MMP-2+/+ mice. ST also increased the numbers of circulating endothelial progenitor cells and their function associated with activation of HIF-1&agr;. All of these effects were diminished by LY294002, an inhibitor of phosphatidylinositol 3-kinase; enhanced by deferoxamine, an HIF-1&agr; stabilizer; and impaired by knockout of MMP-2. Finally, bone marrow transplantation confirmed that ST enhanced endothelial progenitor cell homing to ischemic sites in aged mice. Conclusions— ST can improve neovascularization in response to hypoxia via a phosphatidylinositol 3-kinase–dependent mechanism that is mediated by the HIF-1&agr;/vascular endothelial growth factor/MMP-2 pathway in advanced age.

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]