William Durante

ARGINASE: A CRITICAL REGULATOR OF NITRIC OXIDE SYNTHESIS AND VASCULAR FUNCTION

1 Arginase is the focal enzyme of the urea cycle hydrolysing l‐arginine to urea and l‐ornithine. Emerging studies have identified arginase in the vasculature and have implicated this enzyme in the regulation of nitric oxide (NO) synthesis and the development of vascular disease. 2 Arginase inhibits the production of NO via several potential mechanisms, including competition with NO synthase (NOS) for the substrate l‐arginine, uncoupling of NOS resulting in the generation of the NO scavenger, superoxide and peroxynitrite, repression of the translation and stability of inducible NOS protein, inhibition of inducible NOS activity via the generation of urea and by sensitization of NOS to its endogenous inhibitor asymmetric dimethyl‐l‐arginine. 3 Upregulation of arginase inhibits endothelial NOS‐mediated NO synthesis and may contribute to endothelial dysfunction in hypertension, ageing, ischaemia–reperfusion and diabetes. 4 Arginase also redirects the metabolism of l‐arginine to l‐ornithine and the formation of polyamines and l‐proline, which are essential for smooth muscle cell growth and collagen synthesis. Therefore, the induction of arginase may also promote aberrant vessel wall remodelling and neointima formation. 5 Arginase represents a promising novel therapeutic target that may reverse endothelial and smooth muscle cell dysfunction and prevent vascular disease.

Hyperhomocysteinemia Decreases Circulating High-Density Lipoprotein by Inhibiting Apolipoprotein A-I Protein Synthesis and Enhancing HDL Cholesterol Clearance

We previously reported that hyperhomocysteinemia (HHcy), an independent risk factor of coronary artery disease (CAD), is associated with increased atherosclerosis and decreased plasma high-density lipoprotein cholesterol (HDL-C) in cystathionine &bgr;-synthase–/apolipoprotein E–deficient (CBS−/−/apoE−/−) mice. We observed that plasma homocysteine (Hcy) concentrations are negatively correlated with HDL-C and apolipoprotein A1 (apoA-I) in patients with CAD. We found the loss of large HDL particles, increased HDL-free cholesterol, and decreased HDL protein in CBS−/−/apoE−/− mice, and attenuated cholesterol efflux from cholesterol-loaded macrophages to plasma in CBS−/−/apoE−/− mice. ApoA-I protein was reduced in the plasma and liver, but hepatic apoA-I mRNA was unchanged in CBS−/−/apoE−/− mice. Moreover, Hcy (0.5 to 2 mmol/L) reduced the levels of apoA-I protein but not mRNA and inhibited apoA-1 protein synthesis in mouse primary hepatocytes. Further, plasma lecithin:cholesterol acyltransferase (LCAT) substrate reactivity was decreased, LCAT specific activity increased, and plasma LCAT protein levels unchanged in apoE−/−/CBS−/− mice. Finally, the clearance of plasma HDL cholesteryl ester, but not HDL protein, was faster in CBS−/−/apoE−/− mice, correlated with increased scavenger receptor B1, and unchanged ATP-binding cassette transporter A1 protein expression in the liver. These findings indicate that HHcy inhibits reverse cholesterol transport by reducing circulating HDL via inhibiting apoA-I protein synthesis and enhancing HDL-C clearance.

Hyperhomocystinemia Impairs Endothelial Function and eNOS Activity via PKC Activation

Objective—A risk factor for cardiovascular disease, hyperhomocystinemia (HHcy), is associated with endothelial dysfunction. In this study, we examined the mechanistic role of HHcy in endothelial dysfunction. Methods and Results—Through the use of 2 functional models, aortic rings and intravital video microscopy of the cremaster, we found that arterial relaxation in response to the endothelium-dependent vessel relaxant, acetylcholine or the nitric oxide synthase (NOS) activator (A23187), was significantly impaired in cystathionine β-synthase null (CBS−/−) mice. However, the vascular smooth muscle cell (VSMC) response to the nitric oxide (NO) donor (SNAP) was preserved in CBS−/− mice. In addition, superoxide dismutase and catalase failed to restore endothelium-dependent vasodilatation. Endothelial nitric oxide synthase (eNOS) activity was significantly reduced in mouse aortic endothelial cells (MAECs) of CBS−/− mice, as well as in Hcy-treated mouse and human aortic endothelial cells (HAECs). Hcy-mediated eNOS inhibition—which was not rescued by adenoviral transduction of superoxide dismutase and glutathione peroxidase, or by tetrahydrobiopterin, sepiapterin, and arginine supplementations in MAEC—was associated with decreased protein expression and increased threonine 495 phosphorylation of eNOS in HAECs. Ultimately, a protein kinase C (PKC) inhibitor, GF109203X (GFX), reversed Hcy-mediated eNOS inactivation and threonine 495 phosphorylation in HAECs. Conclusions—These data suggest that HHcy impairs endothelial function and eNOS activity, primarily through PKC activation.

Role of carbon monoxide in cardiovascular function.

Carbon monoxide (CO) is an endogenously derived gas formed from the breakdown of heme by the enzyme heme oxygenase. Although long considered an insignificant and potentially toxic waste product of heme catabolism, CO is now recognized as a key signaling molecule that regulates numerous cardiovascular functions. Interestingly, alterations in CO synthesis are associated with many cardiovascular disorders, including atherosclerosis, septic shock, hypertension, metabolic syndrome, and ischemia‐reperfusion injury. Significantly, restoration of physiologic CO levels exerts a beneficial effect in many of these settings, suggesting a crucial role for CO in maintaining cardiovascular homeostasis. In this review, we outline the actions of CO in the cardiovascular system and highlight this gas as a potential therapeutic target in treating a multitude of cardiovascular disorders.

Regulation of homocysteine metabolism and methylation in human and mouse tissues

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine (Hcy) metabolism involves multiple enzymes; however, tissue Hcy metabolism and its relevance to methylation remain unknown. Here, we established gene expression profiles of 8 Hcy metabolic and 12 methylation enzymes in 20 human and 19 mouse tissues through bioinformatic analysis using expression sequence tag clone counts in tissue cDNA libraries. We analyzed correlations between gene expression, Hcy, S‐adenosylhomocysteine (SAH), and S‐adenosylmethionine (SAM) levels, and SAM/SAH ratios in mouse tissues. Hcy metabolic and methylation enzymes were classified into two types. The expression of Type 1 enzymes positively correlated with tissue Hcy and SAH levels. These include cystathionine β‐synthase, cystathionine‐γ‐lyase, paraxonase 1, 5,10‐methylenetetrahydrofolate reductase, betaine:homocysteine methyltransferase, methionine adenosyl‐transferase, phosphatidylethanolamine N‐methyltransferases and glycine N‐methyltransferase. Type 2 enzyme expressions correlate with neither tissue Hcy nor SAH levels. These include SAH hydrolase, methionyl‐tRNA synthase, 5‐methyltetrahydrofolate:Hcy methyltransferase, S‐adenosylmethionine decarboxylase, DNA methyltransferase 1/3a, isoprenylcysteine carboxyl methyltransferases, and histone‐lysine N‐methyltransferase. SAH is the only Hcy metabolite significantly correlated with Hcy levels and methylation enzyme expression. We established equations expressing combined effects of methylation enzymes on tissue SAH, SAM, and SAM/SAH ratios. Our study is the first to provide panoramic tissue gene expression profiles and mathematical models of tissue methylation regulation.—Chen, N. C., Yang, F., Capecci, L. M., Gu, Z., Schafer, A. I., Durante, W., Yang, X.‐F., Wang, H. Regulation of homocysteine metabolism and methylation in human and mouse tissues. FASEB J. 24, 2804–2817 (2010). www.fasebj.org

Hyperhomocysteinemia Promotes Inflammatory Monocyte Generation and Accelerates Atherosclerosis in Transgenic Cystathionine β-Synthase–Deficient Mice

Background— Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease. Monocytes display inflammatory and resident subsets and commit to specific functions in atherogenesis. In this study, we examined the hypothesis that HHcy modulates monocyte heterogeneity and leads to atherosclerosis. Methods and Results— We established a novel atherosclerosis-susceptible mouse model with both severe HHcy and hypercholesterolemia in which the mouse cystathionine &bgr;-synthase (CBS) and apolipoprotein E (apoE) genes are deficient and an inducible human CBS transgene is introduced to circumvent the neonatal lethality of the CBS deficiency (Tg-hCBS apoE−/− Cbs−/− mice). Severe HHcy accelerated atherosclerosis and inflammatory monocyte/macrophage accumulation in lesions and increased plasma tumor necrosis factor-&agr; and monocyte chemoattractant protein-1 levels in Tg-hCBS apoE−/− Cbs−/− mice fed a high-fat diet. Furthermore, we characterized monocyte heterogeneity in Tg-hCBS apoE−/− Cbs−/− mice and another severe HHcy mouse model (Tg-S466L Cbs−/−) with a disease-relevant mutation (Tg-S466L) that lacks hyperlipidemia. HHcy increased monocyte population and selective expansion of inflammatory Ly-6Chi and Ly-6Cmid monocyte subsets in blood, spleen, and bone marrow of Tg-S466L Cbs−/− and Tg-hCBS apoE−/− Cbs−/− mice. These changes were exacerbated in Tg-S466L Cbs−/− mice with aging. Addition of l-homocysteine (100 to 500 &mgr;mol/L), but not l-cysteine, maintained the Ly-6Chi subset and induced the Ly-6Cmid subset in cultured mouse primary splenocytes. Homocysteine-induced differentiation of the Ly-6Cmid subset was prevented by catalase plus superoxide dismutase and the NAD(P)H oxidase inhibitor apocynin. Conclusion— HHcy promotes differentiation of inflammatory monocyte subsets and their accumulation in atherosclerotic lesions via NAD(P)H oxidase–mediated oxidant stress.

Far Infrared Therapy Inhibits Vascular Endothelial Inflammation via the Induction of Heme Oxygenase-1

Objective—Survival of arteriovenous fistulas (AVFs) in hemodialysis patients is associated with both far infrared (FIR) therapy and length polymorphisms of the heme oxygenase-1 (HO-1) promoter. In this study, we evaluated whether there is an interaction between FIR radiation and HO-1 in regulating vascular inflammation. Methods and Results—Treatment of cultured human umbilical vein endothelial cells (ECs) with FIR radiation stimulated HO-1 protein, mRNA, and promoter activity. HO-1 induction was dependent on the activation of the antioxidant responsive element/NF-E2-related factor-2 complex, and was likely a consequence of heat stress. FIR radiation also inhibited tumor necrosis factor (TNF)-&agr;–mediated expression of E-selectin, vascular cell adhesion molecule-1, intercellular cell adhesion molecule-1, monocyte chemoattractant protein-1, interleukin-8, and the cytokine-mediated adhesion of monocytes to ECs. The antiinflammatory action of FIR was mimicked by bilirubin, and was reversed by the HO inhibitor, tin protoporphyrin-IX, or by the selective knockdown of HO-1. Finally, the antiinflammatory effect of FIR was also observed in patients undergoing hemodialysis. Conclusions—These results demonstrate that FIR therapy exerts a potent antiinflammatory effect via the induction of HO-1. The ability of FIR therapy to inhibit inflammation may play a critical role in preserving blood flow and patency of AVFs in hemodialysis patients.

Platelet-derived growth factor stimulates LAT1 gene expression in vascular smooth muscle: role in cell growth

Platelet‐derived growth factor (PDGF) contributes to vascular disease by stimulating the growth of vascular smooth muscle cells (SMCs). Since amino acids are required for cell growth, the present study examined the effect of PDGF on system L amino acid transport, which is the predominant cellular pathway for the uptake of essential amino acids. System L amino acid transport was monitored by measuring the uptake of L‐leucine. Treatment of SMCs with PDGF stimulated L‐leucine transport in a concentration‐ and time‐dependent manner, and this was associated with a selective increase in LAT1 mRNA and protein. PDGF failed to induce the expression of the other system L transport proteins, LAT2 and the heavy chain of the 4F2 cell surface antigen. The induction of LAT1 by PDGF was dependent on de novo RNA and protein synthesis and on mTOR activity. Serum, thrombin, and angiotensin II likewise stimulated L‐leucine transport by inducing LAT1 expression. Inhibition of system L amino acid transport by the model substrate 2‐aminobicyclo‐(2,2,1)‐heptane‐2‐carboxylic acid blocked growth factor‐mediated SMC proliferation and induced SMC apoptosis, whereas it had no effect on quiescent cells. These results demonstrate that growth factors stimulate system L amino acid transport by inducing LAT1 gene expression and that system L amino acid transport is essential for SMC proliferation and survival. The capacity of vascular mitogens to induce LAT1 expression may represent a basic mechanism by which these agents promote cell growth and provide a novel therapeutic target for treatment of vasculoproliferative disorders.

Heme Oxygenase Inhibitor Restores Arteriolar Nitric Oxide Function in Dahl Rats

Abstract—Vascular tissues express heme oxygenase (HO), which metabolizes heme to form carbon monoxide (CO). CO relaxes vascular smooth muscle but inhibits nitric oxide (NO) formation. Decreased NO synthesis may contribute to salt-induced hypertension in Dahl salt-sensitive (DS) rats. The current study examines the hypothesis that elevated levels of endogenous CO contribute to NO dysfunction in salt-induced hypertensive DS rats. Male DS rats were placed on high-salt (8% NaCl, HS) or low-salt (0.3% NaCl, LS) diets for 4 weeks. With respect to the LS group, the HS group’s blood pressure and carboxyhemoglobin levels were elevated, and abdominal aortas showed 6-fold higher HO-1 protein levels. Experiments used isolated pressurized first-order gracilis muscle arterioles superfused with oxygenated modified Krebs buffer. An inhibitor of NO synthase, N &ohgr;-nitro-l-arginine methyl ester (L-NAME), caused concentration-dependent vasoconstriction in both groups, with attenuated responses in HS arterioles. HS arterioles also showed attenuated vasodilatory responses to an endothelium-dependent vasodilator, acetylcholine. Acute pretreatment with an inhibitor of HO, chromium mesoporphyrin, enhanced vascular responses to L-NAME and acetylcholine in both groups but abolished the differences between HS and LS arterioles. These data show that HO-1 protein levels and CO production are increased in HS rats. Arteriolar responses to L-NAME and acetylcholine are impaired in HS rats compared with LS animals, and this difference can be abolished by an inhibitor of endogenous CO production. These results suggest that elevated levels of endogenous CO contribute to arteriolar NO dysfunction in DS rats with salt-induced hypertension.

Hyperhomocysteinemia impairs endothelium-derived hyperpolarizing factor–mediated vasorelaxation in transgenic cystathionine beta synthase–deficient mice

Hyperhomocysteinemia (HHcy) is associated with endothelial dysfunction (ED), but the mechanism is largely unknown. In this study, we investigated the role and mechanism of HHcy-induced ED in microvasculature in our newly established mouse model of severe HHcy (plasma total homocysteine, 169.5 μM). We found that severe HHcy impaired nitric oxide (NO)- and endothelium-derived hyperpolarizing factor (EDHF)-mediated, endothelium-dependent relaxations of small mesenteric arteries (SMAs). Endothelium-independent and prostacyclin-mediated endothelium-dependent relaxations were not changed. A nonselective Ca(2+)-activated potassium channel (K(Ca)) inhibitor completely blocked EDHF-mediated relaxation. Selective blockers for small-conductance K(Ca) (SK) or intermediate-conductance K(Ca) (IK) failed to inhibit EDHF-mediated relaxation in HHcy mice. HHcy increased the levels of SK3 and IK1 protein, superoxide (O(2)(-)), and 3-nitrotyrosine in the endothelium of SMAs. Preincubation with antioxidants and peroxynitrite (ONOO(-)) inhibitors improved endothelium-dependent and EDHF-mediated relaxations and decreased O(2)(-) production in SMAs from HHcy mice. Further, EDHF-mediated relaxation was inhibited by ONOO(-) and prevented by catalase in the control mice. Finally, L-homocysteine stimulated O(2)(-) production, which was reversed by antioxidants, and increased SK/IK protein levels and tyrosine nitration in cultured human cardiac microvascular endothelial cells. Our results suggest that HHcy impairs EDHF relaxation in SMAs by inhibiting SK/IK activities via oxidation- and tyrosine nitration-related mechanisms.