Abstract-Diabetes mellitus (DM) is a primary risk factor for cardiovascular disease. Although recent studies have demonstrated an important role for extracellular matrix metalloproteinases (MMPs) in atherosclerosis, little is known about the effects of hyperglycemia on MMP regulation in vascular cells. Gelatin zymography and Western blot analysis revealed that the activity and expression of 92-kDa (MMP-9) gelatinase, but not of 72 kDa (MMP-2) gelatinase, were significantly increased in vascular tissue and plasma of two distinct rodent models of DM. Bovine aortic endothelial cells (BAECs) grown in culture did not express MMP-9 constitutively; however, chronic (2-week) incubation with high glucose medium induced MMP-9 promoter activity, mRNA and protein expression, and gelatinase activity in BAECs.On the other hand, high glucose culture did not change MMP-9 activity from vascular smooth muscle cells or macrophages. Electron paramagnetic resonance studies indicate that BAECs chronically grown in high glucose conditions produce 70% more ROS than do control cells. Enhanced MMP-9 activity was significantly reduced by treatment with the antioxidants polyethylene glycol-superoxide dismutase and N-acetyl-L-cysteine but not by inhibitors of protein kinase C. In conclusion, vascular MMP-9 activity is increased in DM, in part because of enhanced elaboration from vascular endothelial cells, and oxidative stress plays an important role. This novel mechanism of redox-sensitive MMP-9 expression by hyperglycemia may provide a rationale for antioxidant therapy to modulate diabetic vascular complications. (Circ Res. 2001;88:1291-1298.)Key Words: endothelium Ⅲ atherosclerosis Ⅲ gelatinase Ⅲ oxidative stress Ⅲ remodeling C ardiovascular complications are the leading cause of morbidity and mortality in patients with diabetes mellitus (DM). 1,2 Because the onset and progression of complications are delayed in patients with good glycemic control, 3 hyperglycemia is thought to be an important regulator of vascular lesion development. Recent studies indicate that elevated glucose concentrations can induce dysfunction of several intracellular signal transduction cascades, including modulation of protein kinase C (PKC), activation of mitogenactivated protein kinase, generation of reactive oxygen species (ROS), and accumulation of advanced glycation end products (AGEs). 4,5 However, the underlying mechanisms between hyperglycemia and vascular disease remain unclear.Matrix metalloproteinases (MMPs) are members of a family of Zn 2ϩ -and Ca 2ϩ -dependent endopeptidases, which are essential for cellular migration and tissue remodeling in both physiological and pathological conditions. 6 MMPs are secreted by many types of cells as proenzymes. On activation by proteolytic cleavage, activated enzymes are capable of degrading many extracellular matrix components. Because MMPs appear to be involved in monocyte invasion and vascular smooth muscle cell migration, derangement of MMP regulation is considered to be a critical factor in the development of...

Abstract-Advanced age is associated with endothelial dysfunction and increased risk for atherosclerosis. However, the mechanisms for these observed effects are not clear. To clarify the association between aging and loss of endothelial function, young human aortic endothelial cells (HAECs), senescent HAECs transfected with control vector, and immortalized HAECs containing human telomerase reverse transcriptase (hTERT) were compared for expression of endothelial nitric oxide synthase (eNOS) and production of NO. To investigate a specific function modulated by endothelial NO, adhesion of monocytes under basal conditions as well as after exposure to TNF-␣ was assessed. A decrease in eNOS mRNA, protein, and activity was observed in endothelial cells at senescence as compared with young HAEC; this effect was blunted in hTERT cells. In all cells, shear stress induced a greater increase in the expression of eNOS protein with the final result being higher levels in hTERT compared with senescent cells. Basal monocyte binding was significantly elevated on aged endothelial cells compared with parental and hTERT cells. Exposure of TNF-␣ resulted in a 2-fold increase in monocyte adhesion in senescent cells, whereas this effect was reduced in cells transfected with hTERT. Prior exposure to fluid flow significantly reduced subsequent monocyte adhesion in all groups. These studies demonstrate that replicative aging results in decreased endothelial expression of eNOS accompanied by enhanced monocyte binding. Key Words: aging Ⅲ telomerase Ⅲ atherosclerosis Ⅲ nitric oxide Ⅲ shear stress V ascular endothelial cells play an important role in maintaining normal vascular homeostasis by producing a balance of paracrine factors such as nitric oxide (NO) and angiotensin II. Many of these factors are known not only to play an important role in the regulation of vessel tone, but also have dramatic effects on vascular structure. Nitric oxide, for example, is not only a potent vasodilator, but it also inhibits platelet adhesion and reactivity, smooth muscle proliferation, and leukocyte adhesion. 1 Because each of these processes is thought to be important in the development of atherosclerosis, nitric oxide is hypothesized to be an important endogenous antiatherogenic molecule.Nitric oxide is produced from the conversion of L-arginine to L-citrulline by the enzyme nitric oxide synthase (NOS). 2 In endothelial cells, the constitutive isoform, eNOS (NOSIII) is responsible for endothelium-derived NO production. In vivo, the predominant physiological stimulus for NO is wall shear stress. 3 NO is produced in response to not only the specific level of shear but also to acute changes in blood flow.Interestingly, eNOS expression is also shear responsive with increasing levels of laminar shear stress resulting in enhanced eNOS mRNA and protein. 4 Because NO has important regulatory function in atherogenesis, increased eNOS expression by laminar shear stress has been hypothesized to be a factor in the resistance of specific vascular segments to atheros...

Quertermous T. In vivo genetic profiling and cellular localization of apelin reveals a hypoxia-sensitive, endothelial-centered pathway activated in ischemic heart failure. Am J Physiol Heart Circ Physiol 294: H88-H98, 2008. First published September 28, 2007 doi:10.1152/ajpheart.00935.2007.-Signaling by the peptide ligand apelin and its cognate G protein-coupled receptor APJ has a potent inotropic effect on cardiac contractility and modulates systemic vascular resistance through nitric oxide-dependent signaling. In addition, there is evidence for counterregulation of the angiotensin and vasopressin pathways. Regulatory stimuli of the apelin-APJ pathway are of obvious importance but remain to be elucidated. To better understand the physiological response of apelin-APJ to disease states such as heart failure and to elucidate the mechanism by which such a response might occur, we have used the murine model of left anterior descending coronary artery ligation-induced ischemic cardiac failure. To identify the key cells responsible for modulation and production of apelin in vivo, we have created a novel apelin-lacZ reporter mouse.Data from these studies demonstrate that apelin and APJ are upregulated in the heart and skeletal muscle following myocardial injury and suggest that apelin expression remains restricted to the endothelium. In cardiac failure, endothelial apelin expression correlates with other hypoxia-responsive genes, and in healthy animals both apelin and APJ are markedly upregulated in various tissues following systemic hypoxic exposure. Experiments with cultured endothelial cells in vitro show apelin mRNA and protein levels to be increased by hypoxia, through a hypoxia-inducible factor-mediated pathway. These studies suggest that apelin-expressing endothelial cells respond to conditions associated with heart failure, possibly including local tissue hypoxia, and modulate apelin-APJ expression to regulate cardiovascular homeostasis. The apelin-APJ pathway may thus provide a mechanism for systemic endothelial monitoring of tissue perfusion and adaptive regulation of cardiovascular function.congestive heart failure; endothelium; gene expression APJ IS A SEVEN TRANSMEMBERANE domain G protein-coupled receptor for which apelin remains the only known ligand (24). Apelin is a highly conserved 77 amino-acid prepropeptide, cleaved to shorter peptides in various tissues (33). Given the cell and developmental specific pattern of expression of apelin and APJ in vascular and cardiac structures and initial studies in developmental model organisms, it is likely that this pathway has a fundamental role in embryogenesis of the cardiovascular system (9,12,15,19,31,38). In the adult cardiovascular system, both APJ and apelin are expressed in the endothelium of heart, kidney, and lung, and APJ is expressed by myocardial cells and some vascular smooth muscle cells (6,20,21).A growing body of literature suggests that the apelin-APJ pathway has direct effects on both cardiac and vascular functions. Data from experimental models...

Glassford AJ, Yue P, Sheikh AY, Chun HJ, Zarafshar S, Chan DA, Reaven GM, Quertermous T, Tsao PS. HIF-1 regulates hypoxia-and insulin-induced expression of apelin in adipocytes. Am J Physiol Endocrinol Metab 293: E1590-E1596, 2007. First published September 18, 2007 doi:10.1152/ajpendo.00490.2007.-Apelin, a novel peptide with significant cardioactive properties, is upregulated by insulin in adipocytes. However, the mechanism by which insulin promotes apelin production is unknown. Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor involved in the angiogenic and metabolic responses to tissue hypoxia, has been shown to be activated by insulin in various settings. We therefore hypothesized that HIF-1 regulates insulin-mediated apelin expression in adipocytes. 3T3-L1 cells were differentiated into adipocytes in culture. For experiments, serum-starved 3T3-L1 cells were exposed to insulin and/or a 1% O 2 environment. Apelin expression was assessed using quantitative real-time PCR and ELISA. To directly assess the role of HIF-1 in apelin production, we differentiated mouse embryonic fibroblasts (MEFs) containing a targeted deletion of the HIF-1␣ gene into adipocytes and measured their response to insulin and hypoxia. Apelin expression in mature 3T3-L1 adipocytes was increased significantly by insulin and was attenuated by pharmacological inhibition of insulin signaling. Exposure of cells to either hypoxia or the chemical HIF activators cobalt chloride (CoCl2) and dimethyloxaloylglycine (DMOG) resulted in significant upregulation of apelin, consistent with a role for HIF in apelin induction. Moreover, hypoxia-, CoCl2-, DMOG-, and insulin-induced apelin expression were all attenuated in differentiated HIF-1␣-deficient MEFs. In summary, in cultured 3T3-L1 adipocytes and differentiated MEFs, HIF-1 appears to be involved in hypoxiaand insulin-induced apelin expression.