Hyperhomocysteinemia decreases vascular reactivity and is associated with cardiovascular morbidity and mortality. However, pathogenic mechanisms that increase oxidative stress by homocysteine (Hcy) are unsubstantiated. The aim of this study was to examine the molecular mechanism by which Hcy triggers oxidative stress and reduces bioavailability of nitric oxide (NO) in cardiac microvascular endothelial cells (MVEC). MVEC were cultured for 0-24 h with 0-100 microM Hcy. Differential expression of protease-activated receptors (PARs), thioredoxin, NADPH oxidase, endothelial NO synthase, inducible NO synthase, neuronal NO synthase, and dimethylarginine-dimethylaminohydrolase (DDAH) were measured by real-time quantitative RT-PCR. Reactive oxygen species were measured by using a fluorescent probe, 2',7'-dichlorofluorescein diacetate. Levels of asymmetric dimethylarginine (ADMA) were measured by ELISA and NO levels by the Griess method in the cultured MVEC. There were no alterations in the basal NO levels with 0-100 microM Hcy and 0-24 h of treatment. However, Hcy significantly induced inducible NO synthase and decreased endothelial NO synthase without altering neuronal NO synthase levels. There was significant accumulation of ADMA, in part because of reduced DDAH expression by Hcy in MVEC. Nitrotyrosine expression was increased significantly by Hcy. The results suggest that Hcy activates PAR-4, which induces production of reactive oxygen species by increasing NADPH oxidase and decreasing thioredoxin expression and reduces NO bioavailability in cultured MVEC by 1) increasing NO2-tyrosine formation and 2) accumulating ADMA by decreasing DDAH expression.

In hypertension, an increase in arterial wall thickness and loss of elasticity over time result in an increase in pulse wave velocity, a direct measure of arterial stiffness. This change is reflected in gradual fragmentation and loss of elastin fibers and accumulation of stiffer collagen fibers in the media that occurs independently of atherosclerosis. Similar results are seen with an elevated level of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), which increases vascular thickness, elastin fragmentation, and arterial blood pressure. Studies from our laboratory have demonstrated a decrease in elasticity and an increase in pulse wave velocity in HHcy cystathionine β synthase heterozygote knockout (CBS(-/+)) mice. Nitric oxide (NO) is a potential regulator of matrix metalloproteinase (MMP) activity in MMP-NO-TIMP (tissue inhibitor of metalloproteinase) inhibitory tertiary complex. We have demonstrated the contribution of the NO synthase (NOS) isoforms, endothelial NOS and inducible NOS, in the activation of latent MMP. The differential production of NO contributes to oxidative stress and increased oxidative/nitrative activation of MMP resulting in vascular remodeling in response to HHcy. The contribution of the NOS isoforms, endothelial and inducible in the collagen/elastin switch, has been demonstrated. We have showed that an increase in inducible NOS activity is a key contributor to HHcy-mediated collagen/elastin switch and resulting decline in aortic compliance. In addition, increased levels of Hcy compete and suppress the γ-amino butyric acid-receptor, N-methyl-d-aspartate-receptor, and peroxisome proliferator-activated receptor. The HHcy causes oxidative stress by generating nitrotyrosine, activating the latent MMPs and decreasing the endothelial NO concentration. The HHcy causes elastinolysis and decrease elastic complicance of the vessel wall. The treatment with γ-amino butyric acid-receptor agonist (muscimol), N-methyl-d-aspartate-receptor antagonist (MK-801), and peroxisome proliferator-activated receptor agonists (ciprofibrate and ciglitazone) mitigates the cardiovascular dysfunction in HHcy [corrected].

Homocysteine (Hcy) induces matrix metalloproteinase (MMP)-9 in microvascular endothelial cells (MVECs). We hypothesized that the ERK1/2 signaling pathway is involved in Hcy-mediated MMP-9 expression. In cultured MVECs, Hcy induced activation of ERK, which was blocked by PD-98059 and U0126 (MEK inhibitors). Pretreatment with BAPTA-AM, staurosporine (PKC inhibitor), or Gö6976 (specific inhibitor for Ca(2+)-dependent PKC) abrogated ERK phosphorylation, suggesting the role of Ca(2+) and Ca(2+)-dependent PKC in Hcy-induced ERK activation. ERK phosphorylation was suppressed by pertussis toxin (PTX), suggesting the involvement of G protein-coupled receptors (GPCRs) in initiating signal transduction by Hcy and leading to ERK activation. Pretreatment of MVECs with genistein, BAPTA-AM, or thapsigargin abrogated Hcy-induced ERK activation, suggesting the involvement of the PTK pathway in Hcy-induced ERK activation, which was mediated by intracellular Ca(2+) pool depletion. ERK activation was attenuated by preincubation with N-acetylcysteine (NAC) and SOD, suggesting the role of oxidation in Hcy-induced ERK activation. Pretreatment with an ERK1/2 blocker (PD-98059), staurosporine, folate, or NAC modulated Hcy-induced MMP-9 activation as measured using zymography. Our results provide evidence that Hcy triggers the PTX-sensitive ERK1/2 signaling pathway, which is involved in the regulation of MMP-9 in MVECs.

Chronic hyperhomocysteinemia (HHcy) is an important factor in development of arterial hypertension. HHcy is associated with activation of matrix metalloproteinases (MMPs); however, it is unclear whether HHcy-dependent extracellular matrix (ECM) accumulation plays a role in arterial hypertrophy and hypertension. We tested the hypothesis that in HHcy the mechanism of arterial hypertension involves arterial dysfunction in response to ECM accumulation between endothelial and arterial smooth muscle cells and subsequent endothelium-myocyte (E-M) uncoupling. To decrease plasma Hcy, dietary supplementation with 3-deazaadenosine (DZA), the S-adenosylhomocysteine hydrolase inhibitor, was administered to cystathionine ␤-synthase (CBS) knockout (KO) mice. Mice were grouped as follows: wild type (WT; control), WTϩDZA, CBSKO, and CBSKOϩDZA (n ϭ 4/group). Mean aortic blood pressure and heart rate were monitored in real time with a telemetric system before, during, and after DZA treatment (6 wk total). In vivo aorta function and morphology were analyzed by M-mode and Doppler echocardiography in anesthetized mice. Aorta MMP activity in unfixed cryostat sections was measured with DQ gelatin. Aorta MMP-2, MMP-9, and connexin 43 expression were measured by RT-PCR and Western blot analyses, respectively. HHcy caused increased aortic blood pressure and resistance, tachycardia, and increased wall thickness and ECM accumulation in aortic wall vs. control groups. There was a linear correlation between aortic wall thickness and plasma Hcy levels. MMP-2, MMP-9, and connexin 43 expression were increased in HHcy. In the CBSKOϩDZA group, aortic blood pressure and levels of MMP and connexin 43 were close to those found in control groups. However, removal of DZA reversed the aortic lumen-to-wall thickness ratio in CBSKO mice, suggesting, in part, a role of vascular remodeling in the increase in blood pressure in HHcy. The results show that arterial hypertension in HHcy mice is, in part, associated with arterial remodeling and E-M uncoupling in response to MMP activation. cardiovascular dysfunction; aorta; echocardiography; extracellular matrix remodeling; in situ matrix metalloproteinase activity; cystathionine ␤-synthase; connexin; DQ gelatin; telemetry EXPERIMENTAL AND CLINICAL studies have shown that chronic elevation of homocysteine (Hcy) levels, known as hyperhomocysteinemia (HHcy), is a significant independent risk factor for cardiovascular disease (31), particularly in the development of arterial hypertension and atherosclerosis (3,24,26). The mechanisms by which HHcy affects arterial wall and induces arterial hypertension have remained unclear. It has been shown that HHcy leads to arterial damage (4) and increased arterial wall stiffness (2,22).Hcy is a product of methionine metabolism that under normal conditions is converted to cystathionine by cystathionine ␤-synthase (CBS). It has been established that mice carrying a disrupted CBS gene are adequate models for HHcy. Systemic vascular cells lacking CBS (7,8) are the prime target for...

Hyperinsulinemia accompanying insulin resistance (IR) is an independent risk factor for stroke. The objective is to examine the cerebrovascular actions of insulin in Zucker obese (ZO) rats with IR and Zucker lean (ZL) control rats. Diameter measurements of cerebral arteries showed diminished insulin-induced vasodilation in ZO compared with ZL. Endothelial denudation revealed vasoconstriction to insulin that was greater in ZO compared with ZL. Nonspecific inhibition of nitric oxide synthase (NOS) paradoxically improved vasodilation in ZO. Scavenging of reactive oxygen species (ROS), supplementation of tetrahydrobiopterin (BH 4 ) precursor, and inhibition of neuronal NOS or NADPH oxidase or cyclooxygenase (COX) improved insulin-induced vasodilation in ZO. Immunoblot experiments revealed that insulin-induced phosphorylation of Akt, endothelial NOS, and expression of GTP cyclohydrolase-I (GTP-CH) were diminished, but phosphorylation of PKC and ERK was enhanced in ZO arteries. Fluorescence studies showed increased ROS in ZO arteries in response to insulin that was sensitive to NOS inhibition and BH 4 supplementation. Thus, a vicious cycle of abnormal insulin-induced ROS generation instigating NOS uncoupling leading to further ROS production underlies the cerebrovascular IR in ZO rats. In addition, decreased bioavailability and impaired synthesis of BH 4 by GTP-CH induced by insulin promoted NOS uncoupling.

Formation of homocysteine (Hcy) is the constitutive process of gene methylation. Hcy is primarily synthesized by de-methylation of methionine, in which s-adenosyl-methionine (SAM) is converted to s-adenosyl-homocysteine (SAH) by methyltransferase (MT). SAH is then hydrolyzed to Hcy and adenosine by SAH-hydrolase (SAHH). The accumulation of Hcy leads to increased cellular oxidative stress in which mitochondrial thioredoxin, and peroxiredoxin are decreased and NADH oxidase activity is increased. In this process, Ca2+-dependent mitochondrial nitric oxide synthase (mtNOS) and calpain are induced which lead to cytoskeletal de-arrangement and cellular remodeling. This process generates peroxinitrite and nitrotyrosine in contractile proteins which causes vascular dysfunction. Chronic exposure to Hcy instigates endothelial and vascular dysfunction and increases vascular resistance causing systemic hypertension. To compensate, the heart increases its load which creates adverse cardiac remodeling in which the elastin/collagen ratio is reduced, causing cardiac stiffness and diastolic heart failure in hyperhomocysteinemia.

A decrease in vascular elasticity and an increase in pulse wave velocity in hyperhomocysteinemic (HHcy) cystathionine-beta-synthase heterozygote knockout (CBS(-/+)) mice has been observed. Nitric oxide (NO) is a potential regulator of matrix metalloproteinase (MMP) activity in MMP-NO-tissue inhibitor of metalloproteinase (TIMP) inhibitory tertiary complex. However, the contribution of the nitric oxide synthase (NOS) isoforms eNOS and iNOS in the activation of latent MMP is unclear. We hypothesize that the differential production of NO contributes to oxidative stress and increased oxidative/nitrative activation of MMP, resulting in vascular remodeling in response to HHcy. The overall goal is to elucidate the contribution of the NOS isoforms, endothelial and inducible, in the collagen/elastin switch. Experiments were performed on six groups of animals [wild-type (WT), eNOS(-/-), and iNOS(-/-) with and without homocysteine (Hcy) treatment (0.67 g/l) for 8-12 wk]. In vivo echograph was performed to assess aortic timed flow velocity for indirect compliance measurement. Histological determination of collagen and elastin with trichrome and van Gieson stains, respectively, was performed. In situ measurement of superoxide generation using dihydroethidium was used. Differential expression of eNOS, iNOS, nitrotyrosine, MMP-2 and -9, and elastin were measured by quantitative PCR and Western blot analyses. The 2% gelatin zymography was used to assess MMP activity. The increase in O(2)(-) and robust activity of MMP-9 in eNOS(-/-), WT+Hcy, and eNOS(-/-)+Hcy was accompanied by the gross disorganization and thickening of the ECM along with extensive collagen deposition and elastin degradation (collagen/elastin switch) resulting in a decrease in aortic timed flow velocity. Results show that an increase in iNOS activity is a key contributor to HHcy-mediated collagen/elastin switch and resulting decline in aortic compliance.

Extracellular matrix (ECM) turnover is regulated by matrix metalloproteinases (MMPs) and plays an important role in cardiac remodeling. Previous studies from our lab demonstrated an increase in gelatinolytic‐MMP‐2 and ‐9 activities in endocardial tissue from ischemic cardiomyopathic (ICM) and idiopathic dilated cardiomyopathic (DCM) hearts. The signaling mechanism responsible for the left ventricular (LV) remodeling, however, is unclear. Administration of cardiac specific inhibitor of metalloproteinase (CIMP) prevented the activation of MMP‐2 and ‐9 in ailing to failing myocardium. Activation of MMP‐2 and ‐9 leads to induction of proteinase activated receptor‐1 (PAR‐1). We hypothesize that the early induction of MMP‐9 is a key regulator for modulating intracellular signaling through activation of PAR and various downstream events which are implicated in development of cardiac fibrosis in an extracellular receptor mediated kinase‐1 (ERK‐1) and focal adhesion kinase (FAK) dependent manner. To test this hypothesis, explanted human heart tissues from ICM and DCM patients were obtained at the time of orthotopic cardiac transplants. Quantitative analysis of MMP‐2 and ‐9 gelatinolytic activities was made by real‐time quantitative zymography. Gel phosphorylation staining for PAR‐1 showed a significant increase in ICM hearts. Western blot and RT‐PCR analysis and in‐situ labeling, showed significant increased expression of PAR‐1, ERK‐1and FAK in ICM and DCM. These observations suggest that the enhanced expression and potentially increased activity of LV myocardial MMP‐9 triggers the signal cascade instigating cardiac remodeling. This early mechanism for the initiation of LV remodeling appears to have a role in end‐stage human heart failure.