Mechanism of eNOS gene transfer inhibition of vascular smooth muscle cell proliferation

Endothelial nitric oxide synthase (eNOS) is responsible for the production of nitric oxide (NO) in blood vessels. NO has been shown to be involved in the inhibition of vascular smooth muscle cell (VSMC) proliferation. In the present study, the eNOS gene was transferred into rat aortic smooth muscle cells by using an adenoviral vector, and the effect of endogenously produced NO on VSMC proliferation was investigated. The presence of eNOS in eNOS-transfected cells was confirmed by immunocytochemistry and Western blot analysis. eNOS transfection resulted in inhibition of VSMC proliferation. This effect was accompanied by increased levels of p53 and p21. This effect was abrogated in the presence of the protein kinase A (PKA) inhibitor Rp-8-bromoadenosine 3',5'-cyclic monophosphothioate. The increased levels of p53 and p21 observed in eNOS-transfected cells were reduced in the presence of the PKA inhibitor. These data suggest that p21 and p53 play a role in the inhibition of proliferation in eNOS-transfected cells and that levels of these two proteins are regulated by PKA.     

IGF-1 induces iNOS expression via the p38 MAPK signal pathway in the anti-apoptotic process in pulmonary artery smooth muscle cells during PAH

Abstract

Apoptosis and cell proliferation are two important cellular processes that determine the accumulation of pulmonary artery smooth muscle cells (PASMC) during pulmonary arterial hypertension (PAH). Insulin-like growth factor 1 (IGF-1) is an endocrine and autocrine/paracrine growth factor that circulates at high levels in the plasma and is expressed in most cell types. IGF-1 has major effects on development, cell growth and differentiation, also tissue repair. Inducible nitric oxide synthase (iNOS) has been shown to serve many vasoprotective roles in vascular smooth muscle cells (VSMCs) including inhibition of VSMC proliferation and migration and stimulation of endothelial cell growth. In this study, we investigated the involvement of iNOS in the process of IGF-1-induced inhibition of PASMC apoptosis. We also examined the role of p38 mitogen-activated protein kinase (MAPK) in the IGF-1-induced iNOS activation. Our results show that exogenous IGF-1 induced the up-regulation of iNOS in PASMC. Immunofluorescence of IGF-1 and iNOS showed a decreased immunostaining of both IGF-1 and iNOS in the cytoplasm and the perinucleus under serum deprivation condition. iNOS inhibition in PASMC in vitro markedly induced IGF-1-mediated anti-apoptosis as assessed by the cell viability measurement, Western blot, mitochondrial potential analysis and nuclear morphology determination. A p38 MAPK inhibitor blocked all the effects of IGF-1 on iNOS. Our findings suggest that IGF-1 inhibits cells apoptosis in PASMC by activating the p38 MAPK–iNOS transduction pathway. This mechanism may contribute to the accumulation of PASMC in early human PAH.     

Gene expression of cyclin-dependent kinase inhibitors and effect of heparin on their expression in mice with hypoxia-induced pulmonary hypertension

The balance between cell proliferation and cell quiescence is regulated delicately by a variety of mediators, in which cyclin-dependent kinases (CDK) and CDK inhibitors (CDKI) play a very important role. Heparin which inhibits pulmonary artery smooth muscle cell (PASMC) proliferation increases the levels of two CDKIs, p21 and p27, although only p27 is important in inhibition of PASMC growth in vitro and in vivo. In the present study we investigated the expression profile of all the cell cycle regulating genes, including all seven CDKIs (p21, p27, p57, p15, p16, p18, and p19), in the lungs of mice with hypoxia-induced pulmonary hypertension. A cell cycle pathway specific gene microarray was used to profile the 96 genes involved in cell cycle regulation. We also observed the effect of heparin on gene expression. We found that (a) hypoxic exposure for two weeks significantly inhibited p27 expression and stimulated p18 activity, showing a 98% decrease in p27 and 81% increase in p18; (b) other CDKIs, p21, p57, p15, p16, and p19 were not affected significantly in response to hypoxia; (c) heparin treatment restored p27 expression, but did not influence p18; (d) ERK1/2 and p38 were mediators in heparin upregulation of p27. This study provides an expression profile of cell cycle regulating genes under hypoxia in mice with hypoxia-induced pulmonary hypertension and strengthens the previous finding that p27 is the only CDKI involved in heparin regulation of PASMC proliferation and hypoxia-induced pulmonary hypertension.     

Pulmonary artery smooth muscle cells from normal subjects and IPAH patients show divergent cAMP-mediated effects on TRPC expression and capacitative Ca2+ entry

Pulmonary vascular remodeling due to overgrowth of pulmonary artery smooth muscle cells (PASMC) is a major cause for the elevated vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Increased cytosolic Ca(2+) concentration, resulting from enhanced capacitative Ca(2+) entry (CCE) and upregulated transient receptor potential (TRP) channel expression, is involved in stimulating PASMC proliferation. The current study was designed to determine the impact of cAMP, a second messenger that we hypothesized would blunt aspects of PASMC activity, as a possible contributor to IPAH pathophysiology. Short-term (30 min) pretreatment with forskolin (FSK; 10 muM), a direct activator of adenylyl cyclase, in combination with the cyclic nucleotide phosphodiesterase inhibitor isobutylmethylxanthine (IBMX; 200 muM), attenuated CCE in PASMC from normal subjects, patients without pulmonary hypertension (NPH), and patients with IPAH. The FSK-mediated CCE inhibition was independent of protein kinase A (PKA), because the PKA inhibitor H89 negligibly affected the decrease in CCE produced by cAMP. By contrast, longer (4 h) treatment with FSK (with IBMX) attenuated CCE in normal and NPH PASMC but enhanced CCE in IPAH PASMC. This enhancement of CCE was abolished by PKA inhibition and associated with an upregulation of TRPC3. In addition, cAMP increased TRPC1 mRNA expression in IPAH (but not in normal or NPH) PASMC, an effect blunted by H89. Furthermore, iloprost, a prostacyclin analog that increases cAMP, downregulated TRPC3 expression in IPAH PASMC and FSK-mediated cAMP increase inhibited IPAH PASMC proliferation. Although a rapid rise in cellular cAMP decreases CCE by a PKA-independent mechanism, sustained cAMP increase inhibits CCE in normal and NPH PASMC but increases CCE via a PKA-dependent pathway in IPAH PASMC. The divergent effect of cAMP on CCE parallels effects on TRPC expression. The results suggest that the combined use of a PKA inhibitor and cAMP-elevating drugs may provide a novel approach for treatment of IPAH.     

Activation of cGMP-dependent protein kinase Ibeta inhibits interleukin 2 release and proliferation of T cell receptor-stimulated human peripheral T cells

Several major functions of type I cGMP-dependent protein kinase (cGK I) have been established in smooth muscle cells, platelets, endothelial cells, and cardiac myocytes. Here we demonstrate that cGK Ibeta is endogenously expressed in freshly purified human peripheral blood T lymphocytes and inhibits their proliferation and interleukin 2 release. Incubation of human T cells with the NO donor, sodium nitroprusside, or the membrane-permeant cGMP analogs PET-cGMP and 8-pCPT-cGMP, activated cGK I and produced (i) a distinct pattern of phosphorylation of vasodilator-stimulated phosphoprotein, (ii) stimulation of the mitogen-activated protein kinases ERK1/2 and p38 kinase, and, upon anti-CD3 stimulation, (iii) inhibition of interleukin 2 release and (iv) inhibition of cell proliferation. cGK I was lost during in vitro culturing of primary T cells and was not detectable in transformed T cell lines. The proliferation of these cGK I-deficient cells was not inhibited by even high cGMP concentrations indicating that cGK I, but not cGMP-regulated phosphodiesterases or channels, cAMP-dependent protein kinase, or other potential cGMP mediators, was responsible for inhibition of T cell proliferation. Consistent with this, overexpression of cGK Ibeta, but not an inactive cGK Ibeta mutant, restored cGMP-dependent inhibition of cell proliferation of Jurkat cells. Thus, the NO/cGMP/cGK signaling system is a negative regulator of T cell activation and proliferation and of potential significance for counteracting inflammatory or lymphoproliferative processes.     

LncRNA NRON alleviates atrial fibrosis via promoting NFATc3 phosphorylation

Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) plays a dominant role in the development of pulmonary arterial hypertension (PAH). Some studies and our previous work found that disturbance of fatty acid metabolism existed in PAH. However, the mechanistic link between fatty acid catabolism and cell proliferation remains elusive. Here, we identified an essential role and signal pathway for the key rate-limiting enzyme of mitochondrial fatty acid β-oxidation, carnitine palmitoyltransferase (CPT) 1, in regulating PASMC proliferation in PAH. We found that CPT1 was highly expressed in rat lungs and pulmonary arteries in monocrotaline-induced PAH, accompanied by decreased adenosine triphosphate (ATP) production and downregulation of the AMPK-p53-p21 pathway. Platelet-derived growth factor (PDGF)-BB upregulated the expression of CPT1 in a dose- and time-dependent manner. PASMC proliferation and ATP production induced by PDGF-BB were partly reversed by the CPT1 inhibitor etomoxir (ETO). The overexpression of CPT1 in PASMCs also promoted proliferation and ATP production and subsequently inhibited the phosphorylation of AMPK, p53, as well as p21 in PASMCs. Furthermore, AMPK was activated by ETO, which increased the expression of p53 and p21, and the proportion of cells in the cell cycle G₂/M phase in response to PDGF-BB stimulation in PASMCs. Our work reveals a novel mechanism of CPT1 regulating PASMC proliferation in PAH, and regulation of CPT1 may be a potential target for therapeutic intervention in PAH.

     

Nitric oxide-induced inhibition of smooth muscle cell proliferation involves S-nitrosation and inactivation of RhoA

Nitric oxide (NO) acts as a vasoregulatory molecule that inhibits vascular smooth muscle cell (SMC) proliferation. Studies have illustrated that NO inhibits SMC proliferation via the extracellular signal-regulated kinase (ERK) pathway, leading to increased protein levels of the cyclin-dependent kinase inhibitor p21^Waf1/Cip1. The ERK pathway can be pro- or antiproliferative, and it has been demonstrated that the activation status of the small GTPase RhoA determines the proliferative fate of ERK signaling, whereby inactivation of RhoA influences ERK signaling to increase p21^Waf1/Cip1 and inhibit proliferation. The purpose of these investigations was to examine the effect of NO on RhoA activation/S-nitrosation and to test the hypothesis that inhibition of SMC proliferation by NO is dependent on inactivation of RhoA. NO decreases activation of RhoA, as demonstrated by RhoA GTP-binding assays, affinity precipitation, and phalloidin staining of the actin cytoskeleton. Additionally, these effects are independent of cGMP. NO decreases SMC proliferation, and gene transfer of constitutively active RhoA (RhoA^63L) diminished the antiproliferative effects of NO, as determined by thymidine incorporation. Western blots of p21^Waf1/Cip1 correlated with changes in proliferation. S-nitrosation of recombinant RhoA protein and immunoprecipitated RhoA was demonstrated by Western blotting for nitrosocysteine and by measurement of NO release. Furthermore, NO decreases GTP loading of recombinant RhoA protein. These findings indicate that inactivation of RhoA plays a role in NO-mediated SMC antiproliferation and that S-nitrosation is associated with decreased GTP binding of RhoA. Nitrosation of RhoA and other proteins likely contributes to cGMP-independent effects of NO. cell signaling; posttranslational modification; vascular disease     

Activation of peroxisome proliferation–activated receptor-γ inhibits transforming growth factor-β1-induced airway smooth muscle cell proliferation by suppressing Smad–miR-21 signaling

The aims of the current study were to examine the signaling mechanisms for transforming growth factor-β1 (TGF-β1)-induced rat airway smooth muscle cell (ASMC) proliferation and to determine the effect of activation of peroxisome proliferation–activated receptor-γ (PPAR-γ) on TGF-β1-induced rat ASMC proliferation and its underlying mechanisms. TGF-β1 upregulated microRNA 21 (miR-21) expression by activating Smad2/3, and this in turn downregulated forkhead box O1 (FOXO1) mRNA expression. In addition, TGF-β1–Smad–miR-21 signaling also downregulated phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression and thus de-repressed the PI3K–Akt pathway. Depletion of PTEN reduced the nuclear FOXO1 protein level without affecting its mRNA level. Inhibition of the PI3K–Akt pathway or proteasome function reversed PTEN knockdown-induced nuclear FOXO1 protein reduction. Our study further showed that loss of FOXO1 increased cyclin D1 expression, leading to rat ASMC proliferation. Preincubation of rat ASMCs with pioglitazone, a PPAR-γ activator, blocked TGF-β1-induced activation of Smad2/3 and its downstream targets changes of miR-21, PTEN, Akt, FOXO1, and cyclin D1, resulting in the inhibition of rat ASMC proliferation. Our study suggests that the activation of PPAR-γ inhibits rat ASMC proliferation by suppressing Smad–miR-21 signaling and therefore has a potential value in the prevention and treatment of asthma by negatively modulating airway remodeling.     

Molecular mechanisms of nitric oxide-induced growth arrest and apoptosis in fetal pulmonary arterial smooth muscle cells

Superoxide plays an important role in pulmonary arterial smooth muscle cell (SMC) proliferation and survival. The rapid reaction between superoxide and nitric oxide (NO) to form peroxynitrite suggests that endothelium-derived NO may influence adjacent SMC growth. To investigate this possibility, we determined the dose-dependent effects of NO on the proliferation and viability of pulmonary arterial SMC isolated from fetal lambs (FPASMC). Using fluorescence microscopy we found a dose-dependent decrease in superoxide levels in FPASMC treated with the NO donor spermine NONOate. This was associated with an increase in peroxynitrite-mediated protein nitration. At doses between 50 and 250 microM, spermine NONOate attenuated serum-induced FPASMC proliferation resulting in a G(0)/G(1) cell cycle arrest. This process involved a decrease in levels of cyclin A and an increase in the nuclear localization of p21 and p27. Furthermore, 500 microM spermine NONOate decreased viable cell number by inducing programmed cell death: FPASMC treated with 500 microM spermine NONOate displayed a loss of mitochondrial membrane potential, followed by caspase activation and DNA fragmentation. These data suggest that NO inhibits superoxide-induced proliferation of FPASMC and at higher doses induces apoptosis. NO donors may therefore prove to be useful therapeutic tools to treat diseases resulting from excessive proliferation of vascular smooth muscle.     

DMF inhibits PDGF-BB induced airway smooth muscle cell proliferation through induction of heme-oxygenase-1

Background

Airway wall remodelling is an important pathology of asthma. Growth factor induced airway smooth muscle cell (ASMC) proliferation is thought to be the major cause of airway wall thickening in asthma. Earlier we reported that Dimethylfumarate (DMF) inhibits platelet-derived growth factor (PDGF)-BB induced mitogen and stress activated kinase (MSK)-1 and CREB activity as well as IL-6 secretion by ASMC. In addition, DMF altered intracellular glutathione levels and thereby reduced proliferation of other cell types.

Methods

We investigated the effect of DMF on PDGF-BB induced ASMC proliferation, on mitogen activated protein kinase (MAPK) activation; and on heme oxygenase (HO)-1 expression. ASMC were pre-incubated for 1 hour with DMF and/or glutathione ethylester (GSH-OEt), SB203580, hemin, cobalt-protoporphyrin (CoPP), or siRNA specific to HO-1 before stimulation with PDGF-BB (10 ng/ml).

Results

PDGF-BB induced ASMC proliferation was inhibited in a dose-dependant manner by DMF. PDGF-BB induced the phosphorylation of ERK1/2 and p38 MAPK, but not of JNK. DMF enhanced the PDGF-BB induced phosphorylation of p38 MAPK and there by up-regulated the expression of HO-1. HO-1 induction inhibited the proliferative effect of PDGF-BB. HO-1 expression was reversed by GSH-OEt, or p38 MAPK inhibition, or HO-1 siRNA, which all reversed the anti-proliferative effect of DMF.

Conclusion

Our data indicate that DMF inhibits ASMC proliferation by reducing the intracellular GSH level with subsequent activation of p38 MAPK and induction of HO-1. Thus, DMF might reduce ASMC and airway remodelling processes in asthma.     

Apolipoprotein E binding to low density lipoprotein receptor-related protein-1 inhibits cell migration via activation of cAMP-dependent protein kinase A

Smooth muscle cell migration and proliferation contribute to neointimal hyperplasia and vascular stenosis after endothelial denudation. Previous studies revealed that apolipoprotein E (apoE) is an effective inhibitor of platelet-derived growth factor-directed smooth muscle cell migration and proliferation and that the anti-migratory function is mediated via apoE binding to low density lipoprotein receptor-related protein-1 (LRP-1). This study was undertaken to identify the intracellular pathway by which apoE binding to LRP-1 results in inhibition of smooth muscle cell migration. The results showed that apoE increased intracellular cAMP levels 3-fold after 5 min, and the increase was sustained for more than 1 h. As a consequence, apoE also increased protein kinase A (PKA) activity in smooth muscle cells. Importantly, suppression of PKA activity with a cell-permeable peptide inhibitor of PKA abolished the inhibitory effect of apoE on smooth muscle cell migration. These results indicated that apoE inhibition of smooth muscle cell migration is mediated via the activation of cAMP-dependent PKA. Additional experiments revealed that apoE also inhibited fibroblasts migration toward platelet-derived growth factor by a similar mechanism of cAMP-dependent PKA activation. It is noteworthy that apoE failed to increase cAMP levels or inhibit migration of LRP-1-negative mouse embryonic fibroblasts and LRP-1-deficient smooth muscle cells. Taken together, these findings established the mechanism by which apoE inhibits cell migration, i.e. via cAMP-dependent protein kinase A activation as a consequence of its binding to LRP-1.     

cAMP activates BKCa channels in pulmonary arterial smooth muscle via cGMP-dependent protein kinase

The signal transduction mechanisms defining the role of cyclic nucleotides in the regulation of pulmonary vascular tone is currently an area of great interest. Normally, signaling mechanisms that elevate cAMP and guanosine-3',5'-cyclic monophosphate (cGMP) maintain the pulmonary vasculature in a relaxed state. Modulation of the large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channel is important in the regulation of pulmonary arterial pressure, and inhibition (closing) of the BK(Ca) channel has been implicated in the development of pulmonary hypertension. Accordingly, studies were done to determine the effect of cAMP-elevating agents on BK(Ca) channel activity using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMC) of the fawn-hooded rat (FHR), a recognized animal model of pulmonary hypertension. Forskolin (10 micro M), a stimulator of adenylate cyclase and an activator of cAMP-dependent protein kinase (PKA), and 8-4-chlorophenylthio (CPT)-cAMP (100 micro M), a membrane-permeable derivative of cAMP, opened BK(Ca) channels in single FHR PASMC. Treatment of FHR PASMC with 300 nM KT5823, a selective inhibitor of cGMP-dependent protein kinase (PKG) activity inhibited the effect of both forskolin and CPT-cAMP. In contrast, blocking PKA activation with 300 nM KT5720 had no effect on forskolin or CPT-cAMP-stimulated BK(Ca) channel activity. These results indicate that cAMP-dependent vasodilators activate BK(Ca) channels in PASMC of FHR via PKG-dependent and PKA-independent signaling pathways, which suggests cross-activation between cyclic nucleotide-dependent protein kinases in pulmonary arterial smooth muscle and therefore, a unique signaling pathway for cAMP-induced pulmonary vasodilation.     

S-nitrosothiols inhibit uterine smooth muscle cell proliferation independent of metabolism to NO and cGMP formation

S-nitrosothiols (RSNOs) are important mediators of nitric oxide (NO) biology. The two mechanisms that appear to dominate in their biological effects are metabolism leading to the formation of NO and S-nitrosation of protein thiols. In this study we demonstrate that RSNOs inhibit uterine smooth muscle cell proliferation independent of NO. The antiproliferative effects of NO on vascular smooth muscle are well defined, with the classic NO-dependent production of cGMP being demonstrated as the active pathway. However, less is known on the role of NO in mediating uterine smooth muscle cell function, a process that is important during menstruation and pregnancy. The RSNOs S-nitrosoglutathione and S-nitroso-N-acetyl pencillamine inhibited growth factor-dependent proliferation of human and rat uterine smooth muscle cells (ELT-3). Interestingly, these cells reduced RSNOs to generate NO. However, use of NO donors and other activators of the cGMP pathway failed to inhibit proliferation. These findings demonstrate the tissue-specific nature of responses to NO and demonstrate the presence of a RSNO-dependent but NO-independent pathway of inhibiting DNA synthesis in uterine smooth muscle cells.     

Cross talk between NO and cyclic nucleotide phosphodiesterases in the modulation of signal transduction in blood vessel.

An increase in cAMP and/or cGMP induces vasodilation which could be potentiated by endothelium or NO-donors. Cyclic nucleotide phosphodiesterases (PDE) are differently distributed in vascular tissues. cAMP hydrolyzing PDE isozymes in endothelial cells are represented by PDE2 (cGMP stimulated-PDE) and PDE4 (cGMP insensitive-PDE), whereas in smooth muscle cells PDE3 (cGMP inhibited-PDE) and PDE4 are present. To investigate the role of NO in vasodilation induced by PDE inhibitors, we studied the effects of PDE3- or PDE4-inhibitor alone and their combination on cyclic nucleotide levels, on relaxation of precontracted aorta and on protein kinase implication. Furthermore, the direct effect of dinitrosyl iron complex (DNIC) was studied on purified recombinant PDE4B. The results show that: 1) in endothelial cells PDE4 inhibition may up-regulate basal production of NO, this effect being potentiated by PDE2 inhibition; 2) in smooth muscle cGMP produced by NO inhibits PDE3 and increases cAMP level allowing PDE4 to participate in vascular contraction; 3) protein kinase G mediates the relaxing effects of PDE3 or PDE4 inhibition. 4) DNIC inhibits non competitively PDE4B indicating a direct effect of NO on PDE4 which could explain an additive vasodilatory effect of NO. A direct and a cGMP related cross-talk between NO and cAMP-PDEs, may participate into the vasomodulation mediated by cAMP activation of protein kinase G.     

Calcitonin gene-related peptide activates different signaling pathways in mesenteric lymphatics of guinea pigs

The effects of calcitonin gene-related peptide (CGRP) on constriction frequency, smooth muscle membrane potential (V(m)), and endothelial V(m) of guinea pig mesenteric lymphatics were examined in vitro. CGRP (1-100 nM) caused an endothelium-dependent decrease in the constriction frequency of perfused lymphatic vessels. The endothelium-dependent CGRP response was abolished by the CGRP-1 receptor antagonist CGRP-(8-37) (1 microM) and pertussis toxin (100 ng/ml). This action of CGRP was also blocked by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine (L-NNA; 10 microM), an action that was reversed by the addition of L-arginine (100 microM). cGMP, adenylate cyclase, cAMP-dependent protein kinase (PKA), and ATP-sensitive K+ (K+(ATP)) channels were all implicated in the endothelium-dependent CGRP response because it was abolished by methylene blue (20 microM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 microM), dideoxyadenosine (10 microM), N-[2-(p-bromociannamylamino)-ethyl]-5-isoquinolinesulfonamide-dichloride (H89; 1 microM) and glibenclamide (10 microM). CGRP (100 nM), unlike acetylcholine, did not alter endothelial intracellular Ca2+ concentration or V(m). CGRP (100 nM) hyperpolarized the smooth muscle V(m), an effect inhibited by L-NNA, H89, or glibenclamide. CGRP (500 nM) also caused a decrease in constriction frequency. However, this was no longer blocked by CGRP-(8-37). CGRP (500 nM) also caused smooth muscle hyperpolarization, an action that was now not blocked by L-NNA (100 microM). It was most likely mediated by the activation of the cAMP/PKA pathway and the opening of K+(ATP) channels because it was abolished by H89 or glibenclamide. We conclude that CGRP, at low to moderate concentrations (i.e., 1-100 nM), decreases lymphatic constriction frequency primarily by the stimulation of CGRP-1 receptors coupled to pertussis toxin-sensitive G proteins and the release of NO from the endothelium or enhancement of the actions of endogenous NO. At high concentrations (i.e., 500 nM), CGRP also directly activates the smooth muscle independent of NO. Both mechanisms of activation ultimately cause the PKA-mediated opening of K+(ATP) channels and resultant hyperpolarization.     

Inhibition of endogenous TRP1 decreases capacitative Ca2+ entry and attenuates pulmonary artery smooth muscle cell proliferation

Pulmonary vascular medial hypertrophy due to proliferation of pulmonary artery smooth muscle cells (PASMC) greatly contributes to the increased pulmonary vascular resistance in pulmonary hypertension patients. A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) is an important stimulus for cell growth in PASMC. Resting [Ca2+]cyt, intracellularly stored [Ca2+], capacitative Ca2+ entry (CCE), and store-operated Ca2+ currents (I(SOC)) are greater in proliferating human PASMC than in growth-arrested cells. Expression of TRP1, a transient receptor potential gene proposed to encode the channels responsible for CCE and I(SOC), was also upregulated in proliferating PASMC. Our aim was to determine if inhibition of endogenous TRP1 gene expression affects I(SOC) and CCE and regulates cell proliferation in human PASMC. Cells were treated with an antisense oligonucleotide (AS, for 24 h) specifically designed to cleave TRP1 mRNA and then returned to normal growth medium for 40 h before the experiments. Then, mRNA and protein expression of TRP1 was downregulated, and amplitudes of I(SOC) and CCE elicited by passive depletion of Ca2+ from the sarcoplasmic reticulum using cyclopiazonic acid were significantly reduced in the AS-treated PASMC compared with control. Furthermore, the rate of cell growth was decreased by 50% in AS-treated PASMC. These results indicate that TRP1 may encode a store-operated Ca2+ channel that plays a critical role in PASMC proliferation by regulating CCE and intracellular [Ca2+](cyt).     

Cinaciguat, a soluble guanylate cyclase activator, augments cGMP after oxidative stress and causes pulmonary vasodilation in neonatal pulmonary hypertension

Although inhaled NO (iNO) therapy is often effective in treating infants with persistent pulmonary hypertension of the newborn (PPHN), up to 40% of patients fail to respond, which may be partly due to abnormal expression and function of soluble guanylate cyclase (sGC). To determine whether altered sGC expression or activity due to oxidized sGC contributes to high pulmonary vascular resistance (PVR) and poor NO responsiveness, we studied the effects of cinaciguat (BAY 58-2667), an sGC activator, on pulmonary artery smooth muscle cells (PASMC) from normal fetal sheep and sheep exposed to chronic intrauterine pulmonary hypertension (i.e., PPHN). We found increased sGC α(1)- and β(1)-subunit protein expression but lower basal cGMP levels in PPHN PASMC compared with normal PASMC. To determine the effects of cinaciguat and NO after sGC oxidation in vitro, we measured cGMP production by normal and PPHN PASMC treated with cinaciguat and the NO donor, sodium nitroprusside (SNP), before and after exposure to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an sGC oxidizer), hyperoxia (fraction of inspired oxygen 0.50), or hydrogen peroxide (H(2)O(2)). After treatment with ODQ, SNP-induced cGMP generation was markedly reduced but the effects of cinaciguat were increased by 14- and 64-fold in PPHN fetal PASMC, respectively (P < 0.01 vs. controls). Hyperoxia or H(2)O(2) enhanced cGMP production by cinaciguat but not SNP in PASMC. To determine the hemodynamic effects of cinaciguat in vivo, we compared serial responses to cinaciguat and ACh in fetal lambs after ductus arteriosus ligation. In contrast with the impaired vasodilator response to ACh, cinaciguat-induced pulmonary vasodilation was significantly increased. After birth, cinaciguat caused a significantly greater fall in PVR than either 100% oxygen, iNO, or ACh. We conclude that cinaciguat causes more potent pulmonary vasodilation than iNO in experimental PPHN. We speculate that increased NO-insensitive sGC may contribute to the pathogenesis of PPHN, and cinaciguat may provide a novel treatment of severe pulmonary hypertension.     

Autoinhibition of endothelial nitric oxide synthase (eNOS) in gut smooth muscle by nitric oxide

Nitric oxide in the gut is produced by nNOS in enteric neurons and by eNOS in smooth muscle cells. The eNOS in smooth muscle is activated by vasoactive intestinal peptide (VIP) released from enteric neurons. In the present study, we examined the effect of nitric oxide on VIP-induced eNOS activation in smooth muscle cells isolated from human intestine and rabbit stomach. NOS activity was measured as formation of the 1:1 co-product, l-citrulline from l-arginine. VIP caused an increase in l-citrulline production that was inhibited by NO in a concentration dependent manner (IC(50)~25 microM; maximal inhibition 72% at 100 microM NO). Basal l-citrulline production, however, was unaffected by NO. The effect was not mediated by cGMP/PKG since the PKG inhibitor KT5823 had no effect on eNOS autoinhibition. The autoinhibition was selective for NO since the co-product l-citrulline had no effect on VIP-induced NOS activation. Similar effects were obtained in rabbit gastric and human intestinal smooth muscle cells. The results suggest that NO produced in smooth muscle cells as a result of the activation of eNOS by VIP exerts an autoinhibitory restraint on eNOS thereby regulating the balance of the VIP/cAMP/PKA and NO/cGMP/PKG pathways that regulate the relaxation of gut smooth muscle.     

BMP-2 inhibits proliferation of human aortic smooth muscle cells via p21Cip1/Waf1

Bone-morphogenetic proteins (BMP)-2 and -7, multifunctional members of the transforming growth factor (TGF)-beta superfamily with powerful osteoinductive effects, cause cell cycle arrest in a variety of transformed cell lines by activating signaling cascades that involve several cyclin-dependent kinase inhibitors (CDKIs). CDKIs in the cip/kip family, p21(Cip1/Waf1) and p27(Kip1), have been shown to negatively regulate the G1 cyclins and their partner cyclin-dependent kinase proteins, resulting in BMP-mediated growth arrest. Bone morphogens have also been associated with antiproliferative effects in vascular tissue by unknown mechanisms. We now show that BMP-2-mediated inhibition of platelet-derived growth factor (PDGF)-stimulated human aortic smooth muscle cell (HASMC) proliferation is accompanied by increased levels of p21 protein. Antisense oligodeoxynucleotides specific for p21 attenuate BMP-2-induced inhibition of proliferation when transfected into HASMCs, demonstrating that BMP-2 inhibits PDGF-stimulated proliferation of HASMCs through induction of p21. Whether p21-mediated induction of cell cycle arrest by BMP-2 sets the stage for osteogenic differentiation of vascular smooth muscle cells, ultimately leading to vascular mineralization, remains to be investigated.