Kelly J. Peyton

Nitric Oxide Induces Heme Oxygenase-1 Gene Expression and Carbon Monoxide Production in Vascular Smooth Muscle Cells

Since recent studies demonstrate that vascular smooth muscle cells synthesize two distinct guanylate cyclase-stimulatory gases, NO and CO, we examined possible regulatory interactions between these two signaling molecules. Treatment of rat aortic smooth muscle cells with the NO donors, sodium nitroprusside, S-nitroso-N-acetyl-penicillamine, or 3-morpholinosydnonimine, increased heme oxygenase-I (HO-1) mRNA and protein levels in a concentration and time-dependent manner. Both actinomycin D and cycloheximide blocked NO-stimulated HO-1 mRNA and protein expression. Nuclear run-on experiments demonstrated that NO donors increased HO-1 gene transcription between 3- and 6-fold. In contrast, NO donors had no effect on the stability of HO-1 mRNA. Incubation of vascular smooth muscle cells with the membrane-permeable cGMP analogues, dibutyryl cGMP and 8-bromo-cGMP, failed to induce HO-1 gene expression. Treatment of vascular smooth muscle cells with NO donors also stimulated the production and release of CO, as demonstrated by the CO-dependent increase in intracellular cGMP levels in coincubated platelets. Finally, incubating vascular smooth muscle cells with interleukin-1 beta and tumor necrosis factor-alpha induced NO synthesis and also significantly increased the level of HO-1 protein. The cytokine-stimulated production of both NO and HO-1 protein in smooth muscle cells was blocked by the NO synthase inhibitor methyl-L-arginine. These results demonstrate that exogenously administered or endogenously released NO stimulates HO-1 gene expression and CO production in vascular smooth muscle cells. The ability of NO to induce HO-catalyzed CO release from vascular smooth muscle cells provides a novel mechanism by which NO might modulate soluble guanylate cyclase and, thereby, vascular smooth muscle cell and platelet function.

Carbon monoxide inhibits apoptosis in vascular smooth muscle cells

OBJECTIVE Carbon monoxide (CO) is generated from vascular smooth muscle cells via the degradation of heme by the enzyme heme oxygenase-1. Since smooth muscle cell apoptosis is associated with numerous vascular disorders, we investigated whether CO regulates apoptosis in vascular smooth muscle. METHODS AND RESULTS Treatment of cultured rat aortic smooth muscle cells with a combination of cytokines (interleukin-1beta, 5 ng/ml; tumor necrosis factor-alpha, 20 ng/ml; interferon-gamma, 200 U/ml) for 48 h stimulated apoptosis, as demonstrated by DNA laddering, annexin V binding, and caspase-3 activation. However, the exogenous administration of CO inhibited cytokine-mediated apoptosis. The antiapoptotic action of CO was partially dependent on the activation of soluble guanylate cyclase and was associated with the inhibition of mitochondrial cytochrome c release and with the suppression of p53 expression. Incubation of smooth muscle cells with the cytokines also resulted in a pronounced increase in heme oxygenase-1 protein after 24 h of stimulation. The addition of the heme oxygenase inhibitor, zinc protoporphyrin-IX, or the CO scavenger, hemoglobin, stimulated apoptosis following 24 h of cytokine exposure. CONCLUSIONS These results demonstrate that CO, either administered exogenously or endogenously derived from heme oxygenase-1 activity, inhibits vascular smooth muscle cell apoptosis. The ability of CO to block smooth muscle cell apoptosis may play an important role in blocking lesion formation at sites of vascular injury.

Heme oxygenase-1 attenuates vascular remodeling following balloon injury in rat carotid arteries

The heme oxygenase-1 (HO-1) system of heme catabolism has been proposed to exert protective actions upon the cardiovascular system. This investigation examined the influence of HO-1 induction on vascular remodeling following arterial injury. Rats were subjected to left carotid artery (LCA) balloon injury following pre-treatment with either vehicle, the HO-1 inducer hemin (50 mg/kg, SC), or concomitant treatment with hemin and the HO-1 inhibitor tin-protoporphyrin IX (SnPP-IX; 50 micromol/kg, IP). Animals were injected daily for 14 days post-injury, after which animals were sacrificed and tissues obtained. Western blot analyses revealed vascular HO-1 induction after 2 and 16 days of hemin treatment. Positive immunostaining for HO-1 was detected in the endothelial and adventitial layers following 48 h of hemin treatment and positive medial staining for HO-1 after 16 days of hemin treatment. The injured LCA of hemin-treated animals demonstrated significantly attenuated neointimal (NI) area (-57%), NI thickness (-58%), and NI area/medial wall area ratio (-40%) compared to the injured LCA of vehicle controls. The cross-sectional medial wall areas of both LCA and uninjured RCA were also significantly reduced in the hemin-treated animals. SnPP-IX treatment, however, completely restored the NI area, NI thickness, NI area/medial wall area ratio, and partially restored the medial wall area towards control levels. These results directly implicate HO-1 and the products of heme catabolism in attenuating the arterial response to injury and ensuing vascular wall remodeling.

Transforming Growth Factor-β1 Stimulates l-Arginine Transport and Metabolism in Vascular Smooth Muscle Cells : Role in Polyamine and Collagen Synthesis

Background—Transforming growth factor-β1 (TGF-β1) contributes to arterial remodeling by stimulating vascular smooth muscle cell (VSMC) growth and collagen synthesis at sites of vascular injury. Because l-arginine is metabolized to growth-stimulatory polyamines and to the essential collagen precursor l-proline, we examined whether TGF-β1 regulates the transcellular transport and metabolism of l-arginine by VSMCs. Methods and Results—TGF-β1 increased l-arginine uptake, and this was associated with a selective increase in cationic amino acid transporter-1 (CAT-1) mRNA. In addition, TGF-β1 stimulated l-arginine metabolism by inducing arginase I mRNA and arginase activity. TGF-β1 also stimulated l-ornithine catabolism by elevating ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT) activity. TGF-β1 markedly increased the capacity of VSMCs to generate the polyamine putrescine and l-proline from extracellular l-arginine. The TGF-β1–mediated increase in putrescine and l-proline production was rever...

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.

Physiological cyclic stretch directs L-arginine transport and metabolism to collagen synthesis in vascular smooth muscle

Application of cyclic stretch (10% at 1 hertz) to vascular smooth muscle cells (SMC) increased L‐arginine uptake and this was associated with a specific increase in cationic amino acid transporters (CAT‐2) mRNA. In addition, cyclic stretch stimulated L‐arginine metabolism by inducing arginase I mRNA and arginase activity. In contrast, cyclic stretch inhibited the catabolism of L‐arginine to nitric oxide (NO) by blocking inducible NO synthase expression. Exposure of SMC to cyclic stretch markedly increased the capacity of SMC to generate L‐proline from L‐arginine while inhibiting the formation of polyamines. The stretch‐mediated increase in L‐proline production was reversed by methyl‐L‐arginine, a competitive inhibitor of L‐arginine transport, by hydroxy‐L‐arginine, an arginase inhibitor, or by the ornithine aminotransferase inhibitor L‐canaline. Finally, cyclic stretch stimulated collagen synthesis and the accumulation of type I collagen, which was inhibited by L‐canaline. These results demonstrate that cyclic stretch coordinately stimulates L‐proline synthesis by regulating the genes that modulate the transport and metabolism of L‐arginine. In addition, they show that stretch‐stimulated collagen production is dependent on L‐proline formation. The ability of hemodynamic forces to up‐regulate L‐arginine transport and direct its metabolism to L‐proline may play an important role in stabilizing vascular lesions by promoting SMC collagen synthesis.—Durante, W., Liao, L., Reyna, S. V., Peyton, K. J., Schafer, A. I. Physiological cyclic stretch directs L‐arginine transport and metabolism to collagen synthesis in vascular smooth muscle. FASEB J. 14, 1775–1783 (2000)

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.

Platelet-Derived Growth Factor Stimulates Heme Oxygenase-1 Gene Expression and Carbon Monoxide Production in Vascular Smooth Muscle Cells

Recent studies indicate that vascular smooth muscle cells (VSMCs) generate CO from the degradation of heme by the enzyme heme oxygenase-1 (HO-1). Because platelet-derived growth factor (PDGF) modulates various responses of VSMCs, we examined whether this peptide regulates the expression of HO-1 and the production of CO by rat aortic SMCs. Treatment of SMCs with PDGF resulted in a time- and concentration-dependent increase in the levels of HO-1 mRNA and protein. Both actinomycin D and cycloheximide blocked PDGF-stimulated HO-1 mRNA and protein. In addition, PDGF stimulated the production of reactive oxygen species by SMCs. Both the PDGF-mediated generation of reactive oxygen species and the induction of HO-1 protein was inhibited by the antioxidant N-acetyl-L-cysteine. Incubation of platelets with PDGF-treated SMCs resulted in a significant increase in platelet cGMP concentration that was reversed by treatment of SMCs with the HO-1 inhibitor tin protoporphyrin-IX or by addition of the CO scavenger hemoglobin to platelets. In contrast, the nitric oxide inhibitor methyl-L-arginine did not block the stimulatory effect of PDGF-treated SMCs on platelet cGMP. Finally, incubation of SMCs with the releasate from collagen-activated platelets induced HO-1 protein expression that was blocked by a neutralizing antibody to PDGF. These results demonstrate that either administered exogenously or released by platelets, PDGF stimulates HO-1 gene expression and CO synthesis in vascular smooth muscle. The ability of PDGF to induce HO-1-catalyzed CO release by VSMCs may represent a novel mechanism by which this growth factor regulates vascular cell and platelet function.

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.

Lysophosphatidylcholine Regulates Cationic Amino Acid Transport and Metabolism in Vascular Smooth Muscle Cells ROLE IN POLYAMINE BIOSYNTHESIS

Lysophosphatidylcholine (lyso-PC) is a major component of atherogenic lipids that stimulate vascular smooth muscle cell (SMC) proliferation. Because cationic amino acids are metabolized to growth-stimulatory polyamines, we examined whether lyso-PC regulates the transcellular transport and metabolism of cationic amino acids by vascular SMC. Treatment of SMC with lyso-PC initially (0–2 h) decreased cationic amino acid uptake, whereas longer exposures (6–24 h) progressively increased transport. Kinetic studies indicated that lyso-PC-induced inhibition was associated with a decrease in affinity for cationic amino acids, but the stimulation was mediated by an increase in transport capacity. Lyso-PC strongly induced the expression of cationic amino acid transporter-2 mRNA while modestly elevating the level of cationic amino acid transporter-1 mRNA. In addition, lyso-PC stimulated intracellular cationic amino acid metabolism by inducing ornithine decarboxylase activity and mRNA expression and also by inducing arginase activity in vascular SMC. In contrast, lyso-PC inhibited the catabolism ofl-arginine to nitric oxide by blocking inducible nitric oxide synthase expression. Lyso-PC increased markedly the capacity of SMC to generate putrescine, a polyamine, from extracellularl-ornithine and l-arginine. The lyso-PC-mediated increase in the production of putrescine was reversed by N G-methyl-l-arginine, a competitive inhibitor of cationic amino acid transport, or by α-difluoromethylornithine, an ornithine decarboxylase inhibitor. The formation of putrescine from l-arginine was also prevented by arginase inhibitorN G-hydroxy-l-arginine. These results demonstrate that lyso-PC stimulates polyamine synthesis in vascular SMC by inducing the expression of the genes that regulate both the transport and metabolism of cationic amino acids. The actions of lyso-PC in stimulating cationic amino acid uptake and directing their metabolism to growth-stimulatory polyamines while simultaneously inhibiting the synthesis of antiproliferative NO, may contribute to lyso-PC-induced SMC proliferation and atherosclerotic lesion formation.