Role of redox signaling and poly (adenosine diphosphate-ribose) polymerase activation in vascular smooth muscle cell growth inhibition by nitric oxide and peroxynitrite
Article Outline
- Abstract
- Abstract
- Materials and methods
- Results
- Inhibition of smooth muscle cell growth and DNA synthesis by nitric oxide and peroxynitrite
- Cytotoxic and apoptotic effects of nitric oxide and peroxynitrite
- Role of redox-sensitive mechanisms in mediating the effects of nitric oxide and peroxynitrite
- Role of PARP in mediating the effects of peroxynitrite
- Role of nitric oxide–signaling pathways and superoxide in the antiproliferative effects of SNAP
- Peroxynitrite-mediated protein nitration and cytotoxicity
- Discussion
- Conclusion
- Author contributions
- Acknowledgment
- References
- Copyright
Purpose
The vascular mediator, nitric oxide regulates vascular smooth muscle cell proliferation and can react with superoxide to form peroxynitrite, a highly reactive free radical. The intracellular mechanisms by which nitric oxide and peroxynitrite inhibit smooth muscle cell growth remain undefined, as is the potential role of peroxynitrite formation in the antiproliferative effects of nitric oxide. We sought to define the intracellular effects and signaling mechanisms of nitric oxide and peroxynitrite in smooth muscle cells.
Methods
Cultured rat aortic smooth muscle cells were treated with exogenous nitric oxide or peroxynitrite and inhibitors of nitric oxide and redox signaling pathways. Cell growth, DNA synthesis, apoptosis, cyclic guanosine 3′-5′ monophosphate (cGMP) levels, poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) activity, and cytotoxicity were assayed. Peroxynitrite formation was determined by nitrotyrosine immunoblotting. Vasoreactivity was assessed in isolated rat aortic rings after treatment with nitric oxide/peroxynitrite and redox agents.
Results
Both exogenous nitric oxide and peroxynitrite decreased cell growth and DNA synthesis of cultured rat aortic smooth muscle cells, but peroxynitrite-induced growth arrest was irreversible and associated with apoptosis and cytotoxicity. Inhibition of guanylate cyclase, PARP activity, mitogen-activated protein kinase, or bypass of ornithine decarboxylase did not reverse growth arrest by nitric oxide. The antioxidants N-acetylcysteine, ascorbate, and glutathione selectively reversed growth inhibition by nitric oxide but not by peroxynitrite. Antioxidants did not impair nitric oxide–induced cGMP generation in smooth muscle cells or nitric oxide–induced vasodilatation of isolated aortic rings. Nitric oxide treatment did not result in peroxynitrite formation and augmentation of superoxide levels did not induce peroxynitrite-like effects. Peroxynitrite-induced cytotoxicity and apoptosis were not reversed by antioxidants or PARP inhibition, because peroxynitrite activated PARP in J774 macrophages but failed to activate PARP in smooth muscle cells.
Conclusions
Exogenous nitric oxide induces reversible cytostasis in smooth muscle cells by a redox-sensitive mechanism independent of peroxynitrite formation and distinct from the nitric oxide vasodilating mechanism. Peroxynitrite does not activate PARP selectively in smooth muscle cells and induces redox-independent smooth muscle cell cytotoxicity and apoptosis. Thus, the antiproliferative effects of nitric oxide and peroxynitrite on smooth muscle cells use divergent intracellular pathways with distinct redox sensitivities. These findings are relevant to the pathogenesis of vascular disease and the potential application of nitric oxide–based therapy for vascular disease.
Clinical Relevance
Vascular smooth muscle cell proliferation is an important component of atherosclerosis, vein graft failure, and arterial restenosis, and is known to be regulated by the vascular signaling molecule nitric oxide. Nitric oxide can combine with the free radical superoxide to form the unstable metabolite peroxynitrite, which has been detected in human vascular lesions. This study examines the role of peroxynitrite in mediating the antiproliferative effects of nitric oxide. We identify important differences in the effects and intracellular mechanisms of nitric oxide and peroxynitrite in regulating vascular smooth muscle cell proliferation and programmed cell death. Defining the differential effects of these free radicals in vascular cells is important to our understanding of the pathogenesis of vascular disease and the development of novel therapy aimed at treating proliferative vascular lesions.
Proliferation of vascular smooth muscle cells (SMCs) is important in the pathogenesis of atherosclerosis, in-stent restenosis, vein graft failure, and fibromuscular dysplasia. One of the most important factors regulating SMC proliferation is the endogenous vascular signaling molecule nitric oxide (NO), which is normally produced by endothelium and is generated during vascular inflammation in hypertension, arterial injury. and atherosclerosis. Nitric oxide, surprisingly, has both antiproliferative and proatherogenic effects in the vessel wall. Nitric oxide was initially considered vasoprotective because augmentation of NO activity inhibits neointimal hyperplasia and SMC proliferation after arterial injury.1 However, cholesterol-induced atherosclerosis is increased by endothelial NO synthesis,2 which indicated that NO can be proatherogenic. Nitric oxide is critical in angiogenesis and arteriogenesis, and is decreased in clinical conditions predisposing to proliferative vascular disease, including smoking, diabetes mellitus, hypertension, and hypercholesterolemia.3
Despite the importance of NO in regulating SMC proliferation and the development of vascular disease, the intracellular mechanisms that mediate the antiproliferative effects of NO remain unclear. Nitric oxide induces expression of the cell cycle arrest molecules p53 and p21; however, SMCs lacking p53 or p21 genes are still sensitive to NO growth arrest.4 Nitric oxide inhibits the expression or activity of a number of growth-related genes and enzymes, including ornithine decarboxylase,5 ribonucleotide reductase,6 cyclins,7 and mitogen-activated protein kinase (MAPK).5 Nitric oxide-induced growth arrest in cancer cells is sensitive to the antioxidant glutathione,8 and endogenous free radicals are known to modulate SMC growth,9 suggesting that free radical interactions may be involved in growth inhibition of SMCs by NO.
Nitric oxide readily combines with other endogenous free radicals, and an important reaction of NO is with superoxide (O2−) to form peroxynitrite (ONOO−). Peroxynitrite was initially described as a highly reactive and potentially toxic radical causing DNA damage, lipid peroxidation, and apoptosis.10, 11 Subsequent studies demonstrated noncytotoxic effects, including activation of matrix metalloproteinases12 and poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) to cause energy depletion and cell death.11 Peroxynitrite can form in SMCs from exogenous NO and superoxide generated by nicotinamide adenine dinucleotide hydrogen (NADH)/nicotinamide adenine dinucleotide phosphate hydrogen (NADPH).9 Formation of peroxynitrite has been detected in human atherosclerotic plaques13 and in cultured SMCs after cytokine stimulation.14
There are conflicting data on the mechanisms by which peroxynitrite inhibits cell growth. Peroxynitrite induces lipid peroxidation10 and activates caspases involved in apoptosis,15 but it can also be protective against oxidant-induced apoptosis.16 The role of peroxynitrite in mediating the antiproliferative effects of NO in SMCs remains undefined. The purpose of this study was to test the hypothesis that peroxynitrite formation is involved in the inhibition of SMC proliferation by NO and to define the intracellular mechanisms responsible for growth inhibition by NO and peroxynitrite.
Materials and methods
Cell culture and materials
Rat aortic SMCs were derived, characterized, and maintained as previously described6 in Dulbecco Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and used in passages 5 to 20 for all studies. The mouse macrophage cell line J774 was obtained from the American Type Culture Collection (ATCC, Manassas, Va) and maintained in DMEM with 10% FBS. Cells were routinely tested for mycoplasma by both polymerase chain reaction–based and fluorescence assays. All chemicals were from Sigma-Aldrich (St Louis, Mo) except for peroxynitrite, which was from Calbiochem (San Diego, Calif). Peroxynitrite concentration was determined before each experiment using the extinction coefficient of 1670 M/cm at 302 nm.
Proliferation and DNA synthesis assay
The effect of exogenous agents on assays of SMC proliferation and DNA synthesis were done as previously described.6, 17 Proliferation studies were performed for 48 hours, and cell counts were analyzed by Coulter counter (Coulter Electronics, Hileah, Fla), with experimental agents replenished every 24 hours. Smooth muscle cells were growth-arrested by treatment with serum-free media for 24 hours, and DNA synthesis was determined by 5-bromo-2-deoxyuridine incorporation into DNA 18 hours after restimulation with serum-contained media and experimental agents as described.
Detection of protein nitration
Cytosolic proteins were extracted (Tris-HCl, 250 mM; sodium dodecylsulfate [SDS], 2.5%), sonicated, centrifuged, separated (30 μg) on 4% to 20% gradient Tris gels, and transferred to nitrocellulose membranes. Membranes were blocked with 1% nonfat milk, hybridized with a mouse monoclonal antibody (1 μg/mL) to nitrotyrosine (Cayman Chemical, Ann Arbor, Mich), and visualized with a sheep antimouse secondary antibody and chemiluminescent technique (ECL-Plus, Amersham, Piscataway, NJ). Nitrated bovine serum albumin (NT-BSA) was used as a positive control.
Cytotoxicity, live/dead, and apoptosis assays
Growth-arrested SMCs in 96-well plates were treated with experimental agents and cytotoxicity was assessed by lactate dehydrogenase (LDH) release using a colorimetric enzyme-immunoassay (Roche, Indianapolis, Ind). Data are expressed as a percentage of maximal LDH release (determined by lysis with Triton-X100 detergent). Cell membrane integrity and metabolic function were determined by fluorescent exclusion of ethidium (4 μM) and the energy-dependent enzymatic conversion of calcein (2 μM, L-3224, Molecular Probes, Eugene, Ore), respectively. Apoptosis was determined by two methods using photometric enzyme-immunoassay (Roche) for cytoplasmic histone-associated DNA fragments or propidium iodide flow cytometry for apoptotic nuclei, as we have previously described.6
Cyclic guanosine 3′-5′ monophosphate and superoxide assay
Cellular cyclic guanosine 3′-5′ monophosphate (cGMP) was measured after treatment with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) and experimental agents. The cGMP was extracted in 95% ethanol and quantified with a competitive colorimetric immunoassay (Assay Designs, Ann Arbor, Mich). Superoxide levels were determined every 30 seconds for 2 minutes using a chemiluminescent assay (Calbiochem) in a luminometer (Turner Designs TD-2020, Sunnyvale, Calif).
Poly(ADP-ribose) polymerase assay
Cells in 60-mm plates were treated for 15 minutes with peroxynitrite, harvested, pelleted, and resuspended in 0.5 mL of assay buffer (56 mM Hepes, pH 7.5; 28 mM KCl, 28 mM NaCl, 2 mM MgCl2, 0.01% digitonin, 0.125 μM 3H-NAD+) for 20 minutes. Cells were pelleted, resuspended in 200 μL ice-cold 50% trichloroacetic acid (TCA) for 4 hours at 4°C; re-pelleted, washed with ice-cold 5% TCA, and then solubilized in 2% SDS/0.1N NaOH before determination of incorporation of NAD+ into cellular proteins by scintillation counting.
Isolated tissue-bath protocol
Isometric contractile force was determined in de-endothelialized rat thoracic aorta helical strips (0.25 × 1 cm) in isolated perfused organ baths, as previously described.18 After an initial phenylephrine challenge (PE, 10 μmol/L), tissues were washed and incubated for 1 hour with vehicle, N-acetylcysteine (0.1 mM), glutathione (0.1 mM), or ascorbate (0.1 mM) and a concentration response curve to PE (1× 10−9−10−5 M) was performed. Tissues were washed again, contracted with a half-maximal concentration of PE (% max: control, 48.3 ± 7.2; NAC, 46.8 ± 10.3; ascorbate, 54.5 ± 7.4; glutathione, 47.3 ± 5.9; P > .05 by analysis of variance), and cumulative S-nitroso-N-acetylpenicillamine (SNAP) concentration response curves (10−9 −1 × 10−5 M) determined after 1-hour incubation with experimental agents. Data are reported as a percentage of the half-maximal contraction to PE.
Statistical analysis
All experiments were performed in triplicate or quadruplicate and repeated with similar results at least three times. Results shown are mean ± standard deviation. Data were analyzed using analysis of variance or unpaired t test as appropriate, and differences were considered significant at P < .05.
Results
Inhibition of smooth muscle cell growth and DNA synthesis by nitric oxide and peroxynitrite
Both peroxynitrite and the NO donor SNAP inhibited SMC proliferation and DNA synthesis (Fig 1, A and B), as we have previously reported for SNAP.6 However, recovery from treatment with peroxynitrite and NO demonstrated substantial differences. Cells initially treated with NO resumed cell proliferation by day 7, but cells treated with two concentrations of peroxynitrite demonstrated similar failure of subsequent proliferation (Fig 1, C).
Fig 1.
Nitric oxide and peroxynitrite and smooth muscle cell growth and apoptosis. Smooth muscle cells were exposed to peroxynitrite (ONOO) and S-nitroso-N-acetylpenicillamine (SNAP) as indicated and either (A) cell counts were determined after 48 hours or (B) DNA synthesis was assayed after 18 hours (*P < .05 vs control). C, Cells were treated for 24 hours with the indicated agents, after which the media was replaced with fresh growth media daily and cell counts were determined after 2 and 7 days. (*P < .05 vs day 2 control, **vs day 7 control). Smooth muscle cells were exposed to peroxynitrite and SNAP as indicated for 18 hours and (D) lactate dehydrogenase (LDH) release and (E) DNA fragmentation was determined (*P < .05 vs respective controls). F, Detection of sub-G1 apoptotic nuclei (black arrows) by flow cytometry after peroxynitrite treatment as indicated for 18 hours. All data are mean ± SD of triplicate wells from one of four to eight similar experiments.
Cytotoxic and apoptotic effects of nitric oxide and peroxynitrite
The failure of SMCs to recover from growth inhibition by peroxynitrite suggests potentially irreversible cytotoxicity or apoptosis. Exogenous peroxynitrite, but not the NO donor SNAP, caused a concentration-dependent increase in cell death (Fig 1, D) and SMC apoptosis (Fig 1, E and F) that was not seen with NO treatment. SNAP induced statistically significant but small increases in apoptosis and LDH release at very high concentrations (1-2 mM); however, these were not seen at the concentrations required for inhibition of proliferation and DNA synthesis (0.2-0.5 mM; Fig 1, A and B).
Role of redox-sensitive mechanisms in mediating the effects of nitric oxide and peroxynitrite
The antioxidants ascorbate (0.1-0.5 mM), glutathione (0.1 mM), and N-acetylcysteine (0.1 mM) all significantly reversed inhibition of DNA synthesis and cell growth by SNAP (Fig 2, A and B). The reversal by antioxidants of the NO growth inhibition may be due to destruction of the biologic activity or free radical activity of NO. To evaluate this, we determined the effect of antioxidants on two other NO effects, the accumulation of intracellular cGMP and vasorelaxation. In the presence of antioxidants, SNAP activated guanylate cyclase, which increased cGMP, and the antioxidants themselves did not activate guanylate cyclase (Fig 2, C). Ascorbate and N-acetylcysteine did not significantly alter the concentration-response curve for SNAP vasorelaxation, and glutathione actually potentiated its activity (Fig 2, D) and correspondingly potentiated SNAP-induced cGMP accumulation (Fig 2, C). In contrast to their effect with NO, the antioxidants did not reverse the effects of peroxynitrite on DNA synthesis, cell growth, or cytotoxicity (Fig 3). The antioxidants themselves had no significant effect on DNA synthesis, LDH release, apoptosis, or cell growth (Fig 2, Fig 3).
Fig 2.
Effect of antioxidants on S-nitroso-N-acetylpenillamine (SNAP)-induced growth arrest, cyclic guanosine 3′-5′ monophosphate (cGMP) levels, and vasodilation. Smooth muscle cells were treated with the indicated combinations of SNAP (0.2 mM), ascorbate (0.5 mM), N-acetyl-cysteine (NAC; 0.1 mM), and glutathione (0.1 mM), and assays performed for (A) DNA synthesis after 18 hours, (B) cell proliferation after 48 hours, and (C) cGMP levels after 30 minutes. (*P < .05 vs SNAP alone). Data are mean ± SD of triplicate wells from one of four to eight similar experiments. D, Vasorelaxation in rat aorta treated with increasing concentrations of SNAP in the presence of the antioxidants ascorbate, NAC, and glutathione (all 0.1 mM). Data are mean ± SD of six to nine rings per experimental group. (*P < .05 vs SNAP alone).
Fig 3.
Effect of antioxidants on the antiproliferative effect of peroxynitrite. A, Cells were treated with peroxynitrite (ONOO; 270 μM) plus either ascorbate (0.5 mM), N-acetyl-cysteine (NAC; 0.1 mM), or glutathione (0.1 mM), and DNA synthesis was measured. B, Cells were treated with peroxynitrite (180uM) plus either ascorbate (0.5 mM), NAC (0.5 mM) or glutathione (0.5 mM) for 48 hours and cell counts were determined. C, Cells were treated with peroxynitrite (360uM) plus either ascorbate (0.5 mM), NAC (0.5 mM), or glutathione (0.5 mM) for 18 hours, and lactate dehydrogenase (LDH) release was measured as described (*P < .05 vs control). Data are mean ± SD of triplicate samples from one of four to eight similar experiments.
Role of PARP in mediating the effects of peroxynitrite
The PARP inhibitor 3-aminobenzamide (3-AB, 1 mM) had no significant effect in reversing peroxynitrite-induced inhibition of DNA synthesis (Fig 4, A), cytotoxicity (Fig 4, B), and apoptosis (Fig 4, C). To investigate why inhibition of PARP did not reverse these effects of peroxynitrite on SMC, we measured PARP activation by peroxynitrite in SMCs and the macrophage cell line J774 (Fig 4, D). Peroxynitrite (360 μM) induced PARP activity in macrophages that was completely inhibited by this concentration of 3-AB but failed to induce PARP activity in SMCs. This concentration of peroxynitrite abolished SMC proliferation (Fig 1), indicating that PARP activation did not play a role in growth inhibition by peroxynitrite.
Fig 4.
Effect of poly (adenosine diphosphate-ribose) polymerase (PARP) inhibition on growth inhibition by peroxynitrite and S-nitroso-N-acetylpenillamine (SNAP). A, Cells were treated with peroxynitrite (270 μM), SNAP (0.2 mM), and 3-aminobenzamide (3-AB, 1 mM), and DNA synthesis was determined. B, Smooth muscle cells were treated with peroxynitrite (360 μM) with or without 3-AB (1 mM) for 18 hours and lactate dehydrogenase (LDH) release was measured. C, Apoptosis was determined using DNA fragmentation assay after exposure of smooth muscle cells to peroxynitrite (135 μM) with or without 3-AB (1 mM). D, Effect of peroxynitrite on PARP enzyme activity in smooth muscle cells and macrophages (J774) after a 15-minute exposure to the indicated agents. *P < .05 vs control. Data are mean ± SD.
Role of nitric oxide–signaling pathways and superoxide in the antiproliferative effects of SNAP
Signaling pathways known to be activated by NO in SMCs were individually inhibited to determine their role in the antiproliferative effect of NO. The MAPK inhibitor PD98059, the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, (ODQ) or bypass of ornithine decarboxylase with putrescine (0.1 and 0.5 mM) all failed to reverse NO inhibition of DNA synthesis, indicating these pathways are not critical to NO-induced growth arrest (Fig 5, A). Putrescine (0.1 mM) completely reversed inhibition of SMC proliferation by α-difluoromethylornithine (DMFO), a specific inhibitor of ornithine decarboxylase, but did not reverse the antiproliferative effect of NO (Fig 5, B). Putrescine alone significantly increased the number of SMCs (159% ± 11% of control).
Fig 5.
Role of nitric oxide signaling pathways and superoxide in the antiproliferative effect of S-nitroso-N-acetylpenillamine (SNAP). A, Growth arrested smooth muscle cells were treated with growth media containing the experimental agents indicated for 18 hours before analysis of DNA synthesis. SNAP (0.2 mM), PD 98059 (mitogen-activated protein kinase inhibitor, 0.1 mM), and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, guanylate cyclase inhibitor, 1 μM). B, Smooth muscle cells in growth media were treated with SNAP (0.1 mM), putrescine (1 mM) and α-difluoromethylornithine (DMFO, 0.3 mM) in the combinations indicated for 48 hours and cells counts determined. C, Cells were treated with experimental agents as indicated for 18 hours and DNA synthesis measured (*P < .05 vs .05 SNAP, **P < .05 vs 0.2 SNAP). Xanthine (100 μM) and xanthine oxidase (XO, 1 mU/mL), superoxide dismutase (SOD, 50 U/mL). (D) Cells were treated with SNAP (0.2 mM) ± xanthine and XO (100 μM, 1 mU/mL) for 18 hours and lactate dehydrogenase (LDH) release was measured. All data are mean ± SD of triplicate determinations from one of three to six similar experiments.
Administration of xanthine/xanthine oxidase increased superoxide concentrations in both the media and cellular lysates approximately 10-fold (data not shown). The addition of xanthine/xanthine oxidase to SNAP potentiated the antiproliferative effects at SNAP concentrations of 0.05 mM and 0.2 mM (Fig 5, B), but the addition of superoxide dismutase (50 U/mL) failed to reverse the potentiation by xanthine/xanthine oxidase. The addition of superoxide (xanthine/xanthine oxidase) to SNAP did not mimic peroxynitrite-like cytotoxicity (Fig 5, D), and superoxide alone had no significant effects on DNA synthesis or LDH release (Fig 5, C and D).
Peroxynitrite-mediated protein nitration and cytotoxicity
Peroxynitrite exposure caused increased protein nitration (Fig 6) at concentrations relevant to inhibition of SMC growth (<300 μM). This was not seen with the NO donor SNAP, even at concentrations (2 mM) far exceeding those required for growth arrest of SMCs (ie, 0.2 mM; Fig 1, A). Because peroxynitrite induced both apoptosis and cellular release of LDH (Fig 1), we assayed energy-dependent transport and membrane integrity after short, 90-minute peroxynitrite exposures. Smooth muscle cells treated with peroxynitrite for 90 minutes demonstrated immediate cytotoxicity, as evidenced by loss of membrane transport and integrity, whereas SNAP-treated SMCs did not differ in appearance from control cells (Fig 6).
Fig 6.
Detection of nitrotyrosined proteins and cytotoxicity. Cells were treated with experimental agents as indicated and harvested after 18 hours for detection of nitrated proteins. Nitrotyrosine bovine serum albumin (NT-BSA) was used as positive control. A, Blot shown is representative of five independent experiments. Smooth muscle cells were treated with the indicated agents for 90 minutes and membrane function and permeability was assessed as described. SNAP, S-nitroso-N-acetylpenillamine; ONOO, peroxynitrite. A, Control; (B), SNAP, 1.0 mM; (C), peroxynitrite, 280 μM; (D), peroxynitrite, 980 μM; (E), methanol (positive control). Photomicrographs are from one of four similar experiments at ×100 original magnification.
Discussion
The major findings in this study are:
Peroxynitrite activation of PARP mediates its cytotoxicity in macrophages11 and cardiac myocytes,19 but data in vascular SMCs are conflicting. Initial studies indicated that PARP inhibitors prevented peroxynitrite-induced impairment of mitochondrial respiration in rat aortic SMCs11; however, peroxynitrite-induced PARP enzyme activity was not directly measured. In subsequent studies, PARP inhibition did not prevent peroxynitrite-induced apoptosis in SMCs.20 We find that PARP activation does not mediate peroxynitrite-induced apoptosis or cytotoxicity in SMCs, and we identify the responsible mechanism as an SMC-specific lack of peroxynitrite-induced PARP activation (Fig 4, D). This finding resolves the conflicting previous reports.11, 20
Lipid peroxidation10 by peroxynitrite may mediate the loss of plasma membrane integrity and cell death ≤90 minutes (Fig 6). In preliminary studies, there was a corresponding loss of mitochondrial membrane potential (determined by JC-1 fluorescence) with peroxynitrite application to SMCs (data not shown). This immediate cytotoxicity of peroxynitrite was not reproduced by its free radical components superoxide (Fig 5, C) or NO (Fig 1). This immediate cytotoxicity may predominate over other delayed effects of peroxynitrite such as DNA damage, changes in mitochondrial gene expression,21 or apoptosis, which each take hours to complete.
To our knowledge, the specific role that intracellular peroxynitrite formation plays in NO-induced growth arrest has not been previously studied, and we could not demonstrate peroxynitrite-like effects of NO even with augmented levels of superoxide generation. Augmentation of superoxide levels did potentiate NO-induced inhibition of DNA synthesis (Fig 5, C); however, this was not reversible by superoxide dismutase, suggesting that the effect was due to oxidants other than superoxide. The identity of these non-superoxide oxidants remains to be elucidated, because attempts to decrease endogenous production of oxidants with diphenyliodonium (inhibitor of NADPH oxidase) or catalase resulted in substantial cytotoxicity (data not shown). Despite the widespread use of xanthine/xanthine oxidase as an experimental source of superoxide, a similar undefined superoxide dismutase–insensitive oxidant was noted in activation of matrix metalloproteinases by xanthine/xanthine oxidase.12
Our finding that exogenous NO does not induce significant intracellular peroxynitrite formation also raises the question of where peroxynitrite does arise from in vascular lesions.13, 22 We studied exogenous NO as would be potentially derived from the endothelium or therapeutic administration. Peroxynitrite activity (nitrotyrosine immunoreactivity) has been shown to co-localize with inducible NO synthase expression13 and may arise in SMCs from simultaneous NADPH oxidase and inducible NO synthase activity within the cell. We specifically did not use cytokine stimulation to simultaneously generate superoxide and NO because of the known confounding nonoxidant effect of inflammatory cytokines on SMC growth.23
Numerous antiproliferative mechanisms have been described for NO in SMCs,4, 5, 6, 7 and thus it is not surprising that inhibition of individual signaling pathways did not reverse NO growth arrest. We identify the mechanism of NO growth arrest as being redox-sensitive, cGMP-independent, and distinct from the vasodilatory effect of NO. This redox-sensitive mechanism could involve S-nitrosylation of intracellular proteins. The divergence of NO signaling between the antiproliferative and vasoreactive effects was confirmed by bioassay of the effects of antioxidants on the vasorelaxing capability of NO (Fig 2, D), which demonstrated that these effects of NO have opposing sensitivities to antioxidants. Thus, distinct signal-transduction mechanisms mediate the antiproliferative and vasorelaxing capabilities of NO with opposing sensitivities to redox-modulating agents such as glutathione. This distinction may be significant if therapeutic agents are designed to inhibit or activate these downstream signaling pathways as oxidant stress becomes a therapeutic target in the vasculature.
Conclusion
The role that PARP activation plays in mediating the cytotoxic effects of peroxynitrite in SMC has clinical significance because glucose-induced activation of PARP mediates the endothelial dysfunction of diabetes mellitus.24 Thus, our finding that PARP activation does not mediate peroxynitrite-induced or NO-induced suppression of SMC growth is relevant to PARP becoming a potential therapeutic target in cardiovascular and inflammatory disease.
Nitric oxide is a well-described regulator of SMC growth3 and has a distinct profile from peroxynitrite in terms of cytostasis, cytotoxicity, and apoptosis in SMCs. Peroxynitrite formation occurs in vascular lesions and may regulate SMC accumulation by cytotoxic mechanisms. These findings help define the intracellular mechanisms of these important nitrogen and oxygen radicals in the vasculature and may allow development of antiproliferative therapy targeting their distinct signaling pathways.
Author contributions
We thank Minakshi Sarkar and Judy Tweedie-Hardman for valuable technical assistance and Louis Messina for critical discussion.
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Competition of interest: none.
Supported by a University of California, San Francisco (UCSF) Faculty Development Award and a Mentored Clinician Scientist Development Award (HL04435) from the National Institutes of Health (NIH) National Heart, Lung, and Blood Institute (NHLBI), the Lifeline Foundation, and the William J. von Leibig Foundation to Dr Sarkar. Dr Huang is the recipient of a Medical Fellowship Award from the Giannini Family Foundation. Dr Lin was supported by Nafzinger Surgical Society fellowship. Dr Watts is an Established Investigator of the American Heart Association. Additional support to Dr Sarkar was received from the Pacific Vascular Research Foundation, the UCSF Academic Senate and Committee on Research and NIH (PO1 HL70687) to Dr Watts.
PII: S0741-5214(07)01768-5
doi:10.1016/j.jvs.2007.11.006
© 2008 The Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.
