Transfection of inducible nitric oxide synthase gene causes apoptosis in vascular smooth muscle cells

BACKGROUND: Excess production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in a variety of physiological processes including vascular remodeling. To elucidate whether endogenous NO generated by iNOS is involved in the programmed cell death (apoptosis) of the vasculature, iNOS cDNA- expressing construct was transfected into rat and human vascular smooth muscle cells (VSMCs) by lipofection. METHODS AND RESULTS: VSMCs transiently transfected with iNOS cDNA functionally expressed 130 kd iNOS protein with full catalytic activity to generate massive NO in proportion to the doses of cDNA used; its enzymatic activity as well as NO production was completely blocked by an NOS inhibitor, NG-monomethyl-L-arginine (LNMMA). Overexpression of iNOS led to a marked inhibition of DNA synthesis as well as induction of apoptosis in VSMCs. Evidence for apoptotic cell death was provided by internucleosomal DNA fragmentation by agarose gel electrophoresis, positive staining for TdT-mediated dUTP biotin nick end-labeling, and appearance of hypodiploid cells by flow cytometry analysis. Apoptosis after transfection with iNOS cDNA was abrogated by LNMMA. Transfection of iNOS cDNA caused accumulation of the tumor suppressor gene p53 but not of bcl-2, which was also blocked by LNMMA. CONCLUSIONS: These results demonstrate that massive generation of endogenous NO derived from iNOS overexpression leads to a marked apoptosis in VSMCs, thus suggesting an important role of NO as a proapoptotic factor for VSMCs in the process of vascular remodeling.     

Is smooth muscle growth in primate arteries regulated by endothelial nitric oxide synthase?

PURPOSE: We investigated whether control of constitutive endothelial cell nitric oxide synthase (cNOS) and nitric oxide (NO) by changes in shear stress might be important for the regulation of smooth muscle cell (SMC) growth and vascular diameter. METHODS: Bilateral femoral arteriovenous fistulas were placed in baboons to increase the blood flow in the external iliac arteries. At 2 months, the fistula was ligated on one side to restore normal flow (flow switch). RESULTS: In response to flow switch and a decrease in shear stress, iliac artery lumenal area decreased and SMC proliferation was induced. A decline in NO production, cNOS messenger RNA (mRNA), and protein were associated with these biological effects. In a subset of animals with iliac arteries under high flow, infusion of N(omega)-nitro-L-arginine, an inhibitor of cNOS, did not induce proliferation. CONCLUSION: Shear stress can regulate cNOS, vasoconstriction, and SMC proliferation. A decrease in nitric oxide may be necessary, but is not sufficient to induce SMC proliferation in response to a decrease in blood flow.     

Platelets inhibit the induction of nitric oxide synthesis by interleukin-1 beta in vascular smooth muscle cells

We have investigated the role of platelets in regulating the hemostatic and vasomotor properties of vascular smooth muscle. Experiments were performed to examine the effect of the releasate from activated platelets on the production of nitric oxide from interleukin-1 beta (IL-1 beta)-treated cultured rat aortic smooth muscle cells. Treatment of vascular smooth muscle cells with IL-1 beta resulted in significant accumulation of nitrite in the culture media and in marked elevation of intracellular cyclic guanosine monophosphate (GMP) levels. The releasate from collagen-aggregated platelets blocked the IL-1 beta-mediated production of nitrite and the accumulation of cyclic GMP in smooth muscle cells in a platelet number-dependent manner. In functional assays, the perfusates from columns containing IL-1 beta-treated smooth muscle cells relaxed detector blood vessels without endothelium and the addition of IL-1 beta-treated smooth muscle cells to suspensions of platelets inhibited their thrombin-induced aggregation. The simultaneous treatment of smooth muscle cells with IL-1 beta and the platelet releasate abolished both the vasorelaxing activities of the perfusates and the inhibition of platelet aggregation. Platelet releasates treated with a neutralizing antibody to platelet-derived growth factor (PDGF) failed to block IL-1 beta-induced nitric oxide production by the smooth muscle cells, as measured by both biochemical and functional assays. The platelet releasate from a patient with gray platelet syndrome likewise failed to block IL-1 beta-induced nitrite release by smooth muscle cells. These results demonstrate that platelets downregulate the production of nitric oxide by IL-1 beta-treated vascular smooth muscle cells through the release of PDGF. This effect may represent a novel mechanism by which platelets regulate vasomotor tone and thrombus formation at sites of vascular injury.     

Inducible nitric oxide synthase expression in smooth muscle cells and macrophages of human transplant coronary artery disease

BACKGROUND: The inducible isoform of the nitric oxide synthase (iNOS) produces large amounts of nitric oxide in response to cytokine stimulation. Previous investigations have demonstrated iNOS expression in the setting of acute and chronic rejection in experimental cardiac transplant models. The goal of this study was to investigate whether iNOS is upregulated in human transplant coronary artery disease (TCAD), a major cause of late mortality after cardiac transplantation. METHODS AND RESULTS: We studied 15 patients with TCAD and 10 with normal coronary arteries. In situ hybridization and immunohistochemistry were used in tissue sections to localize iNOS mRNA and protein, respectively. The presence of peroxynitrite was indirectly assessed by immunostaining with an anti-nitrotyrosine antibody. Normal coronary arteries had no evidence of iNOS expression. In contrast, 30 of 36 coronary artery segments with TCAD (83%) were immunostained by the iNOS antibody. The presence of iNOS mRNA was demonstrated in these vessels by in situ hybridization. Specific cell markers identified iNOS-positive cells as neointimal macrophages and smooth muscle cells. Nitrotyrosine immunoreactivity colocalized with iNOS expression in arteries with TCAD, distributed in macrophages and smooth muscle cells. CONCLUSIONS: iNOS mRNA and protein are expressed in human arteries with TCAD, where they are associated with extensive nitration of protein tyrosines. These findings indicate that the high-output nitric oxide pathway and possibly the oxidant peroxynitrite might be involved in the process leading to the development of TCAD.     

Aspirin and salicylate enhances the induction of inducible nitric oxide synthase in cultured rat smooth muscle cells

Aspirin and sodium salicylate enhance to a similar extent the production of nitric oxide (NO) in cultured smooth muscle cells following stimulation by interleukin-1beta (IL-1beta). The similar potencies of aspirin and sodium salicylate indicate that acetylation of cellular macromolecules is not essential for the enhancement of NO production. The failure of added prostaglandin E2 (PGE2) or Thromboxane A2 (TXA2) to overcome the effects of aspirin or sodium salicylate indicates that these effects are not simply the result of inhibition of prostaglandin synthesis. The enhancement of NO production occurs dependent of the effects of these agents on induction of inducible nitric oxide synthase (iNOS) expression by IL-1beta. Aspirin and sodium salicylate enhance the induction of iNOS expression by IL-1beta. We previously reported that pretreatment of vascular smooth muscle cells (VSMCs) with high glucose decreased the response of the cells by IL-1beta, that is, the induction of iNOS expression and NO production. We investigated the effect of aspirin and sodium salicylate on the response by IL-1beta of VSMCs pretreated with high glucose (25 mM). Aspirin and sodium salicylate ameliorate the down-regulation of iNOS expression and the decrease of NO production caused by pretreatment with high glucose (25 mM). These results suggest a possible therapeutic role in atherosclerotic disease and diabetes mellitus for aspirin and sodium salicylate by enhancing the level of iNOS expression and NO production.     

Serotonin inhibits nitric oxide synthesis in rat vascular smooth muscle cells stimulated with interleukin-1

The elimination half-life of fluoride is significantly increased in patients with chronic renal failure. This led us to conduct a study of variations of its plasma levels in 35 patients receiving dialysis treatment. In this population, there is a gaussian distribution of the values before and after the hemodialysis session, with a significant decrease in the averages. Furthermore, there is a highly significant correlation between fluoride levels before and after the dialysis session (P < 0.00001), and also between the amount of time in hemodialysis (in months) and the average fluoride level before dialysis (r = 0.624; P = 0.008). The presence of a group of patients consuming fluoride waters such as Vichy St-Yorre Water was easily identified by their excessive fluoride levels (above 100 micrograms/l), which could have a tendency to increase the risks of this group.     

Vascular smooth muscle nitric oxide synthase anomalies in Dahl/Rapp salt-sensitive rats

Salt-sensitive hypertension in the Dahl/Rapp rat (S strain) is prevented by L-arginine. Based on the observations that dexamethasone prevented the antihypertensive effect of L-arginine in these animals and the suggestion that a locus in or near an inducible nitric oxide synthase (NOS) gene on chromosome 10 cosegregated with hypertension in some F2 crosses that utilized the S rat, the present study explored the hypothesis that the vascular smooth muscle isoform of inducible NOS (NOS2) was abnormal in S rats. Primary cultures of aortic smooth muscle cells from S rats demonstrated impaired inducible NO production, which improved with increased L-arginine in the medium. Sequence analysis identified a single T-->C transversion that produced an amino acid substitution (S714P) between the FAD and FMN binding sites and a restriction fragment length polymorphism. This restriction fragment length polymorphism was present only in S rats. The mutation of NOS2 and the role of this enzyme in the pathogenesis of salt-sensitive hypertension in the Dahl/Rapp rat require further investigation.     

Failure of L-NAME to cause inhibition of nitric oxide synthesis: role of inducible nitric oxide synthase

We addressed the hypothesis that administration of nitric oxide synthase inhibitor, NG -nitro-L-arginine methyl ester (L-NAME) does not result in a sustained suppression of nitric oxide (NO) synthesis, because of a compensatory expression of inducible nitric oxide synthase (iNOS). L-NAME was administered in the drinking water (0.1-1.0 mg/ml) for 7 days to guinea pigs and rats. Nitric oxide synthesis was assessed by [1] ex vivo formation of nitrite in blood vessels and intestine [2] tissue levels of cGMP [3] iNOS gene expression by RT-PCR [4] NADPH diaphorase staining [5] direct assessment of NO release in tissue explants using a microelectrode/electrochemical detection system. Chronic L-NAME administration elevated intestinal cGMP and nitrite levels in guinea pigs (p < 0.05). In rats, intestinal nitrite levels were comparable in control and L-NAME treatment groups, whereas direct assessment of NO release defined a marked increase in the L-NAME group. Chronic L-NAME resulted in an induction of iNOS gene expression in rats and guinea pigs and novel sites of NADPH diaphorase staining in the intestine. We conclude that iNOS expression is responsible for a compensatory increase or normalization of NO synthesis during sustained administration of L-NAME.     

Inhibition by the JAK/STAT pathway of IFNgamma- and LPS-stimulated nitric oxide synthase induction in vascular smooth muscle cells

Inducible nitric oxide synthase (iNOS) is induced in many cell types by cytokines and lipopolysaccharide (LPS). Cytokine signal transduction is believed to be mediated primarily through the JAK/STAT pathway. We therefore examined the effects of a JAK2-specific inhibitor, an antisense oligonucleotide to JAK2, and electroporation of neutralizing anti-STAT1 and anti-STAT3 antibodies on IFNgamma- and LPS-stimulated induction of iNOS in vascular smooth muscle cells. Unexpectedly, we found that the JAK/STAT pathway suppresses IFNgamma- and LPS-stimulated iNOS induction in these cells. In contrast, the JAK/STAT pathway appears to have a positive role in iNOS induction in RAW 264.7 macrophages.     

Dietary beta-adrenoceptor agonists have a persistent effect on nitric oxide synthesis in rat cultured smooth muscle cells

Several compounds including lipopolysaccharide and sympathomimetics stimulate the expression of the inducible nitric oxide synthase in vascular smooth muscle cells. We evaluated the effect of clenbuterol on nitric oxide (NO) production by vascular smooth muscle cells of the rat aorta in culture. Wistar rats were divided into three diet groups (control, clenbuterol and washout). Aortic vascular smooth muscle cells from rats from these 3 diet groups were cultured in the presence and absence of lipopolysaccharide and/or beta-adrenoceptor agonists. NO release was measured by Griess reagent. Clenbuterol or salbutamol added to cells from control rats potentiated lipopolysaccharide-induced NO release. Cells from rats fed on clenbuterol, in a medium without beta-adrenoceptor agonists, showed a similar potentiation, even after a 10-day washout period. The addition of beta-adrenoceptor agonists to the latter cells did not increase NO production. NG-Nitro-L-arginine decreased nitrite production in lipopolysaccharide-stimulated cells. Our results demonstrate that dietary clenbuterol has a persistent 'ex vivo' effect on lipopolysaccharide-induced NO production by cultured vascular smooth muscle cells.     

Selective anesthetic inhibition of brain nitric oxide synthase

BACKGROUND: It has been postulated that nitric oxide (NO) is a neurotransmitter involved in consciousness, analgesia, and anesthesia. Halothane has been shown to attenuate NO-mediated cyclic guanosine monophosphate accumulation in neurons, and a variety of anesthetic agents attenuate endothelium-mediated vasodilation, suggesting an interaction of anesthetic agents and the NO-cyclic guanosine monophosphate pathway. However, the exact site of anesthetic inhibitory action in this multistep pathway is unclear. The current study examines effects of volatile and intravenous anesthetic agents on the enzyme nitric oxide synthase (NOS) in brain. METHODS: NOS activity was determined by in vitro conversion of [14C]arginine to [14C]citrulline. Wistar rats were decapitated and cerebellum quickly harvested and homogenized. Brain extracts were then examined for NOS activity in the absence and presence of the volatile anesthetics halothane and isoflurane, and the intravenous agents fentanyl, midazolam, ketamine, and pentobarbital. Dose-response curves of NOS activity versus anesthetic concentration were constructed. Effects of anesthetics on NOS activity were evaluated by analysis of variance. RESULTS: Control activities were 57.5 +/- 4.5 pmol.mg protein-1.min-1 in the volatile anesthetic experiments and 51.5 +/- 6.5 pmol.mg protein-1.min-1 in the intravenous anesthetic experiments. NOS activity was not affected by ketamine (< or = 1 x 10(-4) M), pentobarbital (< or = 5 x 10(-5) M), fentanyl (< or = 1 x 10(-5) M), and midazolam (< or = 1 x 10(-5) M). Halothane decreased NOS activity to 36.7 +/- 2.5 (64% of control, P < 0.01 from control), 23.8 +/- 4.3 (41%, P < 0.01 from control and < 0.05 from 0.5% halothane), 25.2 +/- 3.8 (44%, P < 0.01 from control and < 0.05 from 0.5% halothane), and 19.7 +/- 2.8 (34%, P < 0.01 from control and < 0.05 from 0.5% halothane) pmol.mg protein-1.min-1 at 0.5, 1.0, 2.0, and 3.0% vapor. Isoflurane decreased NOS activity to 48.9 +/- 6.1 (85% of control), 46.0 +/- 3.2 (80%, P < 0.05 from control), 40.3 +/- 5.1 (70%, P < 0.05 from control), and 34.2 +/- 4.0 (60%, P < 0.05 from control and 0.5% and 1.0% isoflurane) pmol.mg protein-1.min-1 at 0.5, 1.0, 1.5, 2.0% vapor, respectively. CONCLUSIONS: Volatile anesthetics inhibit brain NOS activity in an in vitro system, but the intravenous agents examined have no effect at clinically relevant concentrations. This inhibition suggests a protein-anesthetic interaction between halothane, isoflurane, and NOS. In contrast, intravenous agents appear to have no direct effect on NOS activity. Whether intravenous agents alter signal transduction or regulatory pathways that activate NOS is unknown.     

Chronic inhibition of nitric oxide synthase in Heymann nephritis

Effects of nitric oxide (NO) synthase inhibition on blood pressure and on the course of Heymann nephritis was examined in rats. L-NG-nitroarginine-methylester (L-NAME, 10 mg/100 ml in the drinking water for 12 weeks) was used as an inhibitor of NO synthase. Urinary excretion of guanosine 3',5'-cyclic monophosphate (cGMP), a second messenger of NO, was used as an indirect estimate of NO activity. Rats were divided into the following groups: control, nephritis, L-NAME, and nephritis-L-NAME. Urinary cGMP excretion was lower in the nephritis group (p < 0.05) and in the nephritis-L-NAME group (p < 0.005) compared with controls. Plasma atrial natriuretic peptide (ANP) levels were elevated in the nephritis (p < 0.001) and in the nephritis-L-NAME groups (p < 0.05. L-NAME treatment alone did not have any effect on plasma ANP levels. Blood pressure rose progressively in all L-NAME-treated rats. Most marked albuminuria developed in the nephritis-L-NAME group. No differences in the immunohistological findings were observed between the nephritis and the nephritis-L-NAME groups. NO synthase inhibition causes hypertension and aggravates albuminuria in chronic nephritis. Moreover, nephritis itself may decrease then production of cGMP either as a consequence of blunted NO activity or, in addition, because of ANP resistance. It appears that NO synthase inhibition does not change the immunological course of Heymann nephritis but rather the increased hemodynamic load makes the course of nephritis worse.     

Role of nuclear factor-kappaB activation in cytokine- and sphingomyelinase-stimulated inducible nitric oxide synthase gene expression in vascular smooth muscle cells

Inflammatory cytokines, such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF alpha), are known to activate sphingomyelinase (SMase) and nuclear factor-kappaB (NF-kappaB) in certain cell types, which also stimulate inducible nitric oxide synthase (iNOS) gene in vascular smooth muscle cells (VSMCs). However, it remains unknown whether the SMase pathway is involved in iNOS gene expression in VSMCs. Therefore, the present study was designed to examine whether SMase induces iNOS gene expression via the NF-kappaB activation pathway similar to that of IL-1beta and TNF alpha in cultured rat VSMCs. Neutral SMase, although less potently than IL-1beta and TNF alpha, stimulated nitrite/nitrate (NOx) production, and iNOS messenger RNA and protein expression, as assessed by Northern and Western blot analyses, respectively. Neutral SMase, IL-1beta, and TNF alpha activated NF-kappaB, as revealed by electrophoretic mobility shift assay, and its nuclear translocation, as demonstrated by immunocytochemical study. Neutral SMase potentiated NOx production, iNOS expression, and NF-kappaB activation stimulated by TNF alpha, but not by IL-1beta. Aldehyde peptide proteasome inhibitors completely blocked NOx production, iNOS expression, NF-kappaB activation, and its nuclear translocation induced by cytokines and neutral SMase. IL-1beta and TNF alpha, but not neutral SMase, caused a transient decrease in IkappaB-alpha protein levels, whereas IkappaB-beta protein expression was not affected by either agent. Proteasome inhibitors prevented cytokine-mediated IkappaB-alpha degradation. Several cell-permeable ceramide analogs (C2, C6, and C8), hydrolysis products of sphingomyelin, activated NF-kappaB less potently than neutral SMase, but had no effect on NOx production. These results demonstrate an essential role of NF-kappaB activation in mediation of neutral SMase-induced iNOS expression, but distinct from the proteasome-mediated IkappaB-alpha degradation by cytokines, suggesting the possible involvement of an additional signaling pathway(s).