William Durante

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.

Vascular Smooth Muscle Cell Heme Oxygenases Generate Guanylyl Cyclase–Stimulatory Carbon Monoxide

BACKGROUND Carbon monoxide (CO), like nitric oxide (NO), stimulates soluble guanylyl cyclase and thereby raises intracellular levels of cGMP. We examined the endogenous capacity of vascular smooth muscle cells (SMCs) to produce CO from heme through the activity of heme oxygenases. METHODS AND RESULTS Cultured SMCs from rat aorta (RASMCs) expressed immunoreactive inducible heme oxygenase-1 (HO-1) and constitutive HO-2. Treatment of RASMCs with hemin and sodium arsenite, which are inducers of HO-1, stimulated RASMC cGMP without stimulating nitrite release or inducible NO synthase expression, and the induced elevations of cGMP were not inhibited by the NO synthase inhibitor NG-methyl-L-arginine. Induced CO from RASMCs likewise caused elevation of cGMP levels in platelets coincubated with the vascular cells. Zinc protoporphyrin IX, an inhibitor of HO, reversed the inducible increases in platelet cGMP. CONCLUSIONS These results indicate that vascular SMCs have both constitutive and inducible HO activity, and they respond to specific stimuli to generate guanylyl cyclase-stimulatory CO in the same SMCs and in coincubated platelets.

Heme oxygenase-1 in growth control and its clinical application to vascular disease

Heme oxygenase‐1 (HO‐1) catalyzes the degradation of heme to carbon monoxide (CO), iron, and biliverdin. Biliverdin is subsequently metabolized to bilirubin by the enzyme biliverdin reductase. Although interest in HO‐1 originally centered on its heme‐degrading function, recent findings indicate that HO‐1 exerts other biologically important actions. Emerging evidence suggests that HO‐1 plays a critical role in growth regulation. Deletion of the HO‐1 gene or inhibition of HO‐1 activity results in growth retardation and impaired fetal development, whereas HO‐1 overexpression increases body size. Although the mechanisms responsible for the growth promoting properties of HO‐1 are not well established, HO‐1 can indirectly influence growth by regulating the synthesis of growth factors and by modulating the delivery of oxygen or nutrients to specific target tissues. In addition, HO‐1 exerts important effects on critical determinants of tissue size, including cell proliferation, apoptosis, and hypertrophy. However, the actions of HO‐1 are highly variable and may reflect a role for HO‐1 in maintaining tissue homeostasis. Considerable evidence supports a crucial role for HO‐1 in blocking the growth of vascular smooth muscle cells (SMCs). This antiproliferative effect of HO‐1 is mediated primarily via the release of CO, which inhibits vascular SMC growth via multiple pathways. Pharmacologic or genetic approaches targeting HO‐1 or CO to the blood vessel wall may represent a promising, novel therapeutic approach in treating vascular proliferative disorders.

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...

The induction of nitric oxide synthase activity is inhibited by TGF-β1, PDGFAB and PDGFBB in vascular smooth muscle cells

Abstract The effect of transforming growth factor- β 1 (TGF- β 1 ) and platelet-derived growth factor (PDGF) was investigated on the induction of nitric oxide synthase activity caused by interleukin-1β in cultured smooth muscle cells from rat aorta. TGF- β 1 , PDGF AB and PDGF BB but not PDGF AA inhibited in a concentration-dependent manner the production of nitrite, an oxidation product of nitric oxide, evoked by interleukin-1β. The growth factors alone did not stimulate the release of nitrite. The addition of interleukin-1β-treated smooth muscle cells to suspensions of indomethacin-treated human washed platelets inhibited the aggregation evoked by thrombin whereas no effect was observed with untreated cells. Platelet aggregation was not inhibited by smooth muscle cells that had been pretreated with interleukin-1β in combination with either TGF- β 1 , PDGF AB or PDGF BB but not with PDGF AA . These observations demonstrate that platelet-derived products such as TGF-β and PDGFs inhibit the induction of nitric oxide synthase activity in vascular smooth muscle cells.

Platelet-derived Growth Factor Regulates Vascular Smooth Muscle Cell Proliferation by Inducing Cationic Amino Acid Transporter Gene Expression

Since recent studies demonstrated that platelet-derived growth factor (PDGF) induces vascular smooth muscle cell (SMC) proliferation by stimulating polyamine synthesis, we examined whether the transcellular transport of L-ornithine, the cationic amino acid precursor of polyamines, could regulate the mitogenic response of PDGF. Treatment of SMC with PDGF stimulated DNA and putrescine synthesis, and this was enhanced further by increasing the extracellular concentration of L-ornithine. The potentiating effect of L-ornithine was reversed by the competitive inhibitor of cationic amino acid transport, methyl-L-arginine, or by preventing putrescine formation with α-difluoromethylornithine. Cationic amino acid uptake by SMC was Na-independent and was mediated by both a high and low affinity carrier system. Treatment of SMC with PDGF initially (0-2 h) decreased basic amino acid transport, while longer exposures (6-24 h) progressively increased uptake. Kinetic studies indicated that PDGF-induced inhibition was associated with a decrease in affinity for cationic amino acids, while the stimulation was mediated by an increase in transport capacity. Endogenous PDGF released by collagen-activated platelets likewise up-regulated cationic amino acid transport in SMC. Reverse transcriptase-polymerase chain reaction detected the presence of mRNA encoding two distinct cationic amino acid transporter (CAT) proteins, CAT-1 and CAT-2B. Treatment of SMC with PDGF strongly induced the expression CAT-2B mRNA and modestly elevated the level of CAT-1 mRNA. These results demonstrate that PDGF-induced polyamine synthesis and SMC mitogenesis are dependent on the transcellular transport of L-ornithine. The capacity of PDGF to up-regulate the transport of L-ornithine by inducing the expression of the genes for CAT-1 and CAT-2B may modulate its mitogenic effect by providing SMC with the necessary intracellular precursor for polyamine biosynthesis.

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 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.