Karni S. Moshal

H2S protects against methionine-induced oxidative stress in brain endothelial cells.

Homocysteine (Hcy) causes cerebrovascular dysfunction by inducing oxidative stress. However, to date, there are no strategies to prevent Hcy-induced oxidative damage. Hcy is an H2S precursor formed from methionine (Met) metabolism. We aimed to investigate whether H2S ameliorated Met-induced oxidative stress in mouse brain endothelial cells (bEnd3). The bEnd3 cells were exposed to Met treatment in the presence or absence of NaHS (donor of H2S). Met-induced cell toxicity increased the levels of free radicals in a concentration-dependent manner. Met increased NADPH-oxidase-4 (NOX-4) expression and mitigated thioredxion-1(Trx-1) expression. Pretreatment of bEnd3 with NaHS (0.05 mM) attenuated the production of free radicals in the presence of Met and protected the cells from oxidative damage. Furthermore, NaHS enhanced inhibitory effects of apocynin, N-acetyl-l-cysteine (NAC), reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), Nomega-nitro-l-arginine methyl ester (L-NAME) on ROS production and redox enzymes levels induced by Met. In conclusion, the administration of H2S protected the cells from oxidative stress induced by hyperhomocysteinemia (HHcy), which suggested that NaHS/H2S may have therapeutic potential against Met-induced oxidative stress.

Mitochondrial matrix metalloproteinase activation decreases myocyte contractility in hyperhomocysteinemia

Cardiomyocyte N-methyl-d-aspartate receptor-1 (NMDA-R1) activation induces mitochondrial dysfunction. Matrix metalloproteinase protease (MMP) induction is a negative regulator of mitochondrial function. Elevated levels of homocysteine [hyperhomocysteinemia (HHCY)] activate latent MMPs and causes myocardial contractile abnormalities. HHCY is associated with mitochondrial dysfunction. We tested the hypothesis that HHCY activates myocyte mitochondrial MMP (mtMMP), induces mitochondrial permeability transition (MPT), and causes contractile dysfunction by agonizing NMDA-R1. The C57BL/6J mice were administered homocystinemia (1.8 g/l) in drinking water to induce HHCY. NMDA-R1 expression was detected by Western blot and confocal microscopy. Localization of MMP-9 in the mitochondria was determined using confocal microscopy. Ultrastructural analysis of the isolated myocyte was determined by electron microscopy. Mitochondrial permeability was measured by a decrease in light absorbance at 540 nm using the spectrophotometer. The effect of MK-801 (NMDA-R1 inhibitor), GM-6001 (MMP inhibitor), and cyclosporine A (MPT inhibitor) on myocyte contractility and calcium transients was evaluated using the IonOptix video edge track detection system and fura 2-AM. Our results demonstrate that HHCY activated the mtMMP-9 and caused MPT by agonizing NMDA-R1. A significant decrease in percent cell shortening, maximal rate of contraction (-dL/dt), and maximal rate of relaxation (+dL/dt) was observed in HHCY. The decay of calcium transient amplitude was faster in the wild type compared with HHCY. Furthermore, the HHCY-induced decrease in percent cell shortening, -dL/dt, and +dL/dt was attenuated in the mice treated with MK-801, GM-6001, and cyclosporin A. We conclude that HHCY activates mtMMP-9 and induces MPT, leading to myocyte mechanical dysfunction by agonizing NMDA-R1.

Mitochondrial Mechanism of Microvascular Endothelial Cells Apoptosis in Hyperhomocysteinemia

An elevated level of homocysteine (Hcy) limits the growth and induces apoptosis. However, the mechanism of Hcy‐induced programmed cell death in endothelial cells is largely unknown. We hypothesize that Hcy induces intracellular reactive oxygen species (ROS) production that leads to the loss of transmembrane mitochondrial potential (Δψm) accompanied by the release of cytochrome‐c from mitochondria. Cytochrome‐c release contributes to caspase activation, such as caspase‐9, caspase‐6, and caspase‐3, which results in the degradation of numerous nuclear proteins including poly (ADP‐ribose) polymerase (PARP), which subsequently leads to the internucleosomal cleavage of DNA, resulting cell death. In this study, rat heart microvascular endothelial cells (MVEC) were treated with different doses of Hcy at different time intervals. Apoptosis was measured by DNA laddering and transferase‐mediated dUTP nick‐end labeling (TUNEL) assay. ROS production and MP were determined using florescent probes (2,7‐dichlorofluorescein (DCFH‐DA) and 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐benzamidazolocarbocyanin iodide (JC‐1), respectively, by confocal microscopy. Differential gene expression for apoptosis was analyzed by cDNA array. The results showed that Hcy‐mediated ROS production preceded the loss of MP, the release of cytochrome‐c, and the activation of caspase‐9 and ‐3. Moreover the Hcy treatment resulted in a decrease in Bcl2/Bax ratio, evaluated by mRNA levels. Caspase‐9 and ‐3 were activated, causing cleavage of PARP, a hallmark of apoptosis and internucleosomal DNA fragmentation. The cytotoxic effect of Hcy was blocked by using small interfering RNA (siRNA)‐mediated suppression of caspase‐9 in MVEC. Suppressing the activation of caspase‐9 inhibited the activation of caspase ‐3 and enhanced the cell viability and MP. Our data suggested that Hcy‐mediated ROS production promotes endothelial cell death in part by disturbing MP, which results in subsequent release of cytochrome‐c and activation of caspase‐9 and 3, leading to cell death. J. Cell. Biochem. 98: 1150–1162, 2006.

Cardioprotective Role of Sodium Thiosulfate on Chronic Heart Failure by Modulating Endogenous H2S Generation

Background/Aims: Sodium thiosulfate (STS) has been shown to be an antioxidant and calcium solubilizer, but the possible role of STS in dysfunctional ventricles remains unknown. Here, we assessed the effects of STS in the failing heart. Methods: Heart failure was created by an arteriovenous fistula (AVF). Mice were divided into 4 groups: sham, AVF, sham + STS, and AVF + STS. STS (3 mg/ml) was supplemented with drinking water for 6 weeks in the appropriate surgery groups after surgery. Results: M-mode echocardiograms showed ventricular contractile dysfunction with reduced aortic blood flow in AVF mice, whereas STS treatment prevented the decline in cardiac function. Ventricular collagen, MMP-2 and -9, and TIMP-1 were robustly increased with a decreasing trend in adenylate cyclase VI expression; however, STS supplementation reversed these effects in AVF mice. Among 2 enzymes that produce endogenous hydrogen sulfide (H2S), cystathionine-γ-lyase (CSE) expression was attenuated in AVF mice with no changes in cystathionine-β-synthase (CBS) expression. In addition, reduced production of H2S in AVF ventricular tissue was normalized with STS supplementation. Moreover, cardiac tissues were more responsive to H2S when AVF mice were supplemented with STS compared to AVF alone. Conclusions: These results suggested that STS modulated cardiac dysfunction and the extracellular matrix, in part, by increasing ventricular H2S generation.

Early induction of matrix metalloproteinase-9 transduces signaling in human heart end stage failure

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.

Mitochondrial mechanism of oxidative stress and systemic hypertension in hyperhomocysteinemia

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. J. Cell. Biochem.

Cytochrome P450 (CYP) 2J2 gene transfection attenuates MMP-9 via inhibition of NF-κβ in hyperhomocysteinemia

Hyperhomocysteinemia (HHcy) is associated with atherosclerotic events involving the modulation of arachidonic acid (AA) metabolism and the activation of matrix metalloproteinase‐9 (MMP‐9). Cytochrome P450 (CYP) epoxygenase‐2J2 (CYP2J2) is abundant in the heart endothelium, and its AA metabolites epoxyeicosatrienoic acids (EETs) mitigates inflammation through NF‐κβ. However, the underlying molecular mechanisms for MMP‐9 regulation by CYP2J2 in HHcy remain obscure. We sought to determine the molecular mechanisms by which P450 epoxygenase gene transfection or EETs supplementation attenuate homocysteine (Hcy)‐induced MMP‐9 activation. CYP2J2 was over‐expressed in mouse aortic endothelial cells (MAECs) by transfection with the pcDNA3.1/CYP2J2 vector. The effects of P450 epoxygenase transfection or exogenous supplementation of EETs on NF‐κβ‐mediated MMP‐9 regulation were evaluated using Western blot, in‐gel gelatin zymography, electromobility shift assay, immunocytochemistry. The result suggested that Hcy downregulated CYP2J2 protein expression and dephosphorylated PI3K‐dependent AKT signal. Hcy induced the nuclear translocation of NF‐κβ via downregulation of IKβα (endogenous cytoplasmic inhibitor of NF‐κβ). Hcy induced MMP‐9 activation by increasing NF‐κβ–DNA binding. Moreover, P450 epoxygenase transfection or exogenous addition of 8,9‐EET phosphorylated the AKT and attenuated Hcy‐induced MMP‐9 activation. This occurred, in part, by the inhibition of NF‐κβ nuclear translocation, NF‐κβ–DNA binding and activation of IKβα. The study unequivocally suggested the pivotal role of EETs in the modulation of Hcy/MMP‐9 signal. J. Cell. Physiol. 215: 771–781, 2008.

Homocysteine decreases blood flow to the brain due to vascular resistance in carotid artery.

An elevated level of Homocysteine (Hcy) is a risk factor for vascular dementia and stroke. Cysthathionine beta Synthase (CBS) gene is involved in the clearance of Hcy. Homozygous individuals for (CBS-/-) die early, but heterozygous for (CBS-/+) survive with high levels of Hcy. The gamma-Amino Butyric Acid (GABA) presents in the central nervous system (CNS) and functions as an inhibitory neurotransmitter. Hcy competes with GABA at the GABA(A) receptor and affects the CNS function. We hypothesize that Hcy causes a decrease in blood flow to the brain due to increase in vascular resistance (VR) because of arterial remodeling in the carotid artery (CA). Blood pressure and blood flow in CA of wild type (WT), CBS-/+, CBS-/+ GABA(A)-/- double knockout, and GABA(A)-/- were measured. CA was stained with trichrome, and the brain permeability was measured. Matrix Metalloproteinases (MMP-2 and MMP-9), tissue inhibitor of metalloproteinase (TIMP-3, TIMP-4), elastin, and collagen-III expression were measured by real-time polymerase chain reaction (RT-PCR). Results showed an increase in VR in CBS-/+/GABA(A)-/-double knockout>CBS-/+/>GABA(A)-/- compared to WT mice. Increased MMP-2, MMP-9, collagen-III and TIMP-3 mRNA levels were found in GABA(A)-/-, CBS-/+, CBS-/+/GABA(A) double knockout compared to WT. The levels of TIMP-4 and elastin were decreased, whereas the levels of MMP-2, MMP-9 and TIMP-3 increased, which indirectly reflected the arterial resistance. These results suggested that Hcy caused arterial remodeling in part, by increase in collagen/elastin ratio thereby increasing VR leading to the decrease in CA blood flow.

Mitochondrial MMP activation, dysfunction and arrhythmogenesis in hyperhomocysteinemia.

Chronic volume/pressure overload-induced heart failure augments oxidative stress and activates matrix metalloproteinase which causes endocardial endothelial-myocyte (EM) uncoupling eventually leading to decline in myocardial systolic and diastolic function. The elevated levels of homocysteine (Hcy), hyperhomocysteinemia (HHcy), are associated with decline in cardiac performance. Hcy impairs the EM functions associated with the induction of ventricular hypertrophy leading to cardiac stiffness and diastolic heart failure. Hcy-induced neurological defects are mediated by the NMDA-R (N-methyl-D-aspartate (NMDA) receptor) activation. NMDA-R is expressed in the heart. However, the role of NMDA-R on cardiac function during HHcy is still in its infancy. The blockade of NMDA-R attenuates NMDA-agonist-induced increase in the heart rate. Hcy increases intracellular calcium and activates calpain and calpain-associated mitochondrial (mt) abnormalities have been identified in HHcy. Mitochondrial permeabilization and uncoupling in the pathological setting is fueled by redox stress and calcium mishandling. Recently the role of cyclophilin D, a component of the mitochondrial membrane permeability transition pore, has been identified in cardiac-ischemia. Mechanisms underlying the potentiation between NMDA-R activation and mitochondrial defects leading to cardiac dysfunction during HHcy remain to be elucidated. This review addresses the mitochondrial mechanism by which Hcy contributes to the decline in mechano-electrical function and arrhythmogenesis via agonizing NMDA-R. The putative role of mitochondrial MMP activation, protease stress and mitochondrial permeability transition in cardiac conduction during HHcy is discussed. The review suggests that Hcy increases calcium overload and oxidative stress in the mitochondria and amplifies the activation of mtMMP, causing the opening of mitochondrial permeability transition pore leading to mechano-electrical dysfunction.

Homocysteine‐induced myofibroblast differentiation in mouse aortic endothelial cells

Differentiation of myofibroblast, as evidenced by α‐smooth muscle actin (α‐SMA) expression, is largely mediated by transforming growth factor‐β1 (TGF‐β1). This mechanism often follows inflammatory events such as endothelial damage due to oxidative stress, which can further leads to vascular thickening, stiffness, and fibrosis. We hypothesized that hyperhomocysteinemia (HHcy)‐induced oxidative stress lead to vascular stiffness, in part due to endothelial–myofibroblast differentiation and alteration of collagen homeostasis in the extracellular matrix (ECM). We tested our hypothesis in vitro using mouse aortic endothelial cells (MAEC). Our result shows that Hcy induces α‐SMA and collagen type‐1 expression in MAEC as evidenced by immunoblot and confocal imaging. RT‐PCR shows robust increase of α‐SMA and collagen type‐1 mRNA level in Hcy‐induced condition. We demonstrated that Hcy induces autophosphorylation of focal adhesion kinase (FAK) (a member of the protein tyrosine kinase (PTK) family) at Tyr‐397. PP2 (general PTK inhibitor) as well as FAK siRNA abrogates Hcy‐mediated α‐SMA formation. In addition to that, Hcy‐mediated TGF‐β1 induction was inhibited by TGF‐β R1 kinase inhibitor II (ALK5 inhibitor II) and attenuated FAK phosphorylation and α‐SMA expression. Furthermore, we showed that Hcy activates ERK‐44/42 (extracellular signal‐regulated kinase) pathway and augments collagen type‐1 deposition. Studies with pharmacological ERK blocker, PD98059 and ERK siRNA attenuated ERK‐44/42 phosphorylation and collagen type‐1 synthesis. Taken together our results demonstrate that Hcy‐mediated TGF‐β1 upregulation triggers endothelial–myofibroblast differentiation secondary to FAK phosphorylation and that Hcy‐induced ERK activation is involved in ECM remodeling by altering collagen type‐1 homeostasis. J. Cell. Physiol. 209: 767–774, 2006.