Role of oxidative stress in angiotensin II-induced enhanced expression of Gi(alpha) proteins and adenylyl cyclase signaling in A10 vascular smooth muscle cells

We have previously reported that angiotensin II (ANG II) treatment of A10 vascular smooth muscle cells (VSMCs) increased inhibitory G proteins (G(i) protein) expression and associated adenylyl cyclase signaling which was attributed to the enhanced MAP kinase activity. Since ANG II has been shown to increase oxidative stress, we investigated the role of oxidative stress in ANG II-induced enhanced expression of G(i)alpha proteins and examined the effects of antioxidants on ANG II-induced enhanced expression of G(i)alpha proteins and associated adenylyl cyclase signaling in A10 VSMCs. ANG II treatment of A10 VSMCs enhanced the production of O(2)(-) and the expression of Nox4 and P47(phox), different subunits of NADPH oxidase, which were attenuated toward control levels by diphenyleneiodonium (DPI). In addition, ANG II augmented the expression of G(i)alpha-2 and G(i)alpha-3 proteins in a concentration- and time-dependent manner; the maximal increase in the expression of G(i)alpha was observed at 1 to 2 h and at 0.1-1.0 microM. The enhanced expression of G(i)alpha-2 and G(i)alpha-3 proteins was restored to control levels by antioxidants such as N-acetyl-L-cysteine, alpha-tocopherol, DPI, and apocynin. In addition, ANG II also enhanced the ERK1/2 phosphorylation that was restored to control levels by DPI. Furthermore, the inhibition of forskolin-stimulated adenylyl cyclase activity by low concentrations of 5'-O-(3-triotriphosphate) (receptor-independent G(i) functions) and ANG II-, des(Glu(18),Ser(19),Glu(20),Leu(21),Gly(22))atrial natriuretic peptide(4-23)-NH(2) (natriuretic peptide receptor-C agonist), and oxotremorine-mediated inhibitions of adenylyl cyclase (receptor-dependent functions) that were augmented in ANG II-treated VSMCs was also restored to control levels by antioxidant treatments. In addition, G(s)alpha-mediated diminished stimulation of adenylyl cyclase by stimulatory hormones in ANG II-treated cells was also restored to control levels by DPI. These results suggest that ANG II-induced enhanced levels of G(i)alpha proteins and associated functions in VSMCs may be attributed to the ANG II-induced enhanced oxidative stress, which exerts its effects through mitogen-activated protein kinase signaling pathway.     

Nitric oxide modulates Gi-protein expression and adenylyl cyclase signaling in vascular smooth muscle cells

We have previously shown that treatment of rats with the nitric oxide (NO) synthase inhibitor N6-nitro-L-arginine methyl ester for 4 weeks resulted in the augmentation of blood pressure and enhanced levels of Gialpha proteins. The present studies were undertaken to investigate if NO can modulate the expression of Gi proteins and associated adenylyl cyclase signaling. A10 vascular smooth muscle cells (VSMC) and primary cultured cells from aorta of Sprague-Dawley rats were used for these studies. The cells were treated with S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) for 24 h and the expression of Gialpha proteins was determined by immunobloting techniques. Adenylyl cyclase activity was determined by measuring [32P]cAMP formation for [alpha-32P]ATP. Treatment of cells with SNAP (100 microM) or SNP (0.5 mM) decreased the expression of Gialpha-2 and Gialpha-3 by about 25-40% without affecting the levels of Gsalpha proteins. The decreased expression of Gialpha proteins was reflected in decreased Gi functions (receptor-independent and -dependent) as demonstrated by decreased or attenuated forskolin-stimulated adenylyl cyclase activity by GTPgammaS and inhibition of adenylyl cyclase activity by angiotensin II and C-ANP4-23, a ring-deleted analog of atrial natriuretic peptide (ANP) that specifically interacts with natriuretic peptide receptor-C (NPR-C) in SNAP-treated cells. The SNAP-induced decreased expression of Gialpha-2 and Gialpha-3 proteins was not blocked by 1H[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase, or KT5823, an inhibitor of protein kinase G, but was restored toward control levels by uric acid, a scavenger of peroxynitrite and Mn(111)tetralis (benzoic acid porphyrin) MnTBAP, a peroxynitrite scavenger and a superoxide dismutase mimetic agent that inhibits the production of peroxynitrite, suggesting that NO-mediated decreased expression of Gialpha protein was cGMP-independent and may be attributed to increased levels of peroxynitrite. In addition, Gsalpha-mediated stimulation of adenylyl cyclase by GTPgammaS, isoproterenol, and forskolin was significantly augmented in SNAP-treated cells. These results indicate that NO decreased the expression of Gialpha protein and associated functions in VSMC by cGMP-independent mechanisms. From these studies, it can be suggested that NO-induced decreased levels of Gi proteins and resultant increased levels of cAMP may be an additional mechanism through which NO regulates blood pressure.     

Cyclic GMP modulates the expression of Gi protein and adenylyl cyclase signaling in vascular smooth muscle cells

We have recently shown that the nitric oxide (NO) donor, SNAP, decreased the expression of Giα proteins and associated functions in vascular smooth muscle cells. Because NO stimulates soluble guanylyl cyclase and increases the levels of guanosine 3′,5′-cyclic monophosphate (cGMP), the present studies were undertaken to investigate whether cGMP can also modulate the expression of Gi proteins and associated adenylyl cyclase signaling. A10 vascular smooth muscle cells (VSMCs) and primary cultured cells from aorta of Sprague Dawley rats were used for these studies. The cells were treated with 8-bromoguanosine 3′,5′-cyclic monophosphate (8Br-cGMP) for 24 h and the expression of Giα proteins was determined by immunobloting techniques. Adenylyl cyclase activity was determined by measuring [³²P]cAMP formation for [α-³²P]ATP. Treatment of cells with 8-Br-cGMP (0.5 mM) decreased the expression of Giα-2 and Giα-3 by about 30–45%, which was restored towards control levels by KT5823, an inhibitor of protein kinase G. On the other and hand, the levels of Gsα protein were not altered by this treatment. The decreased expression of Giα proteins by 8Br-cGMP treatment was reflected in decreased Gi functions. For example, the inhibition of forskolin (FSK)-stimulated adenylyl cyclase activity by low concentrations of GTPγS (receptor-independent Gi functions) was significantly decreased by 8Br-cGMP treatment. In addition, exposure of the cells to 8Br-cGMP also resulted in the attenuation of angiotensin (Ang) II- and C-ANP_4–23 (a ring-deleted analog of atrial natriuretic peptide [ANP]-mediated inhibition of adenylyl cyclase activity (receptor-dependent functions of Gi). On the other hand, Gsα-mediated stimulations of adenylyl cyclase by GTPγS, isoproterenol and FSK were significantly augmented in 8Br-cGMP-treated cells. These results indicated the 8Br-cGMP decreased the expression of Giα proteins and associated functions in VSMCs. From these studies, it can be suggested that 8Br-cGMP-induced decreased levels of Gi proteins and resultant increased levels of cAMP may be an additional mechanism through which cGMP regulates vascular tone and thereby blood pressure.     

Peroxynitrite inhibits the expression of G(i)alpha protein and adenylyl cyclase signaling in vascular smooth muscle cells

We previously showed that S-nitroso-N-acetylpenicillamine, a nitric oxide donor, decreased the levels and functions of G(i)alpha proteins by formation of peroxynitrite (ONOO(-)) in vascular smooth muscle cells (VSMC). The present studies were undertaken to investigate whether ONOO(-) can modulate the expression of G(i)alpha protein and associated adenylyl cyclase signaling in VSMC. Treatment of A-10 and aortic VSMC with ONOO(-) for 24 h decreased the expression of G(i)alpha-2 and G(i)alpha-3, but not G(s)alpha, protein in a concentration-dependent manner; expression was restored toward control levels by (111)Mn-tetralis(benzoic acid porphyrin) and uric acid, but not by 1H[1,2,4]oxadiazole[4,3-a]quinoxaline-1-one (ODQ) and KT-5823. cGMP levels were increased by approximately 50% and 150% by 0.1 and 0.5 mM ONOO(-), respectively, and attenuated toward control levels by ODQ. In addition, 0.5 mM ONOO(-) attenuated the inhibition of adenylyl cyclase by ANG II and C-type atrial natriuretic peptide (C-ANP(4-23)), as well as the inhibition of forskolin-stimulated adenylyl cyclase activity by GTPgammaS, whereas, the G(s)-mediated stimulations were augmented. In addition, 0.5 mM ONOO(-) decreased phosphorylation of ERK1/2 and p38 MAP kinase and enhanced JNK phosphorylation but did not affect AKT1/3 phosphorylation. These results suggest that ONOO(-) decreased the expression of G(i) proteins and associated functions in VSMC through a cGMP-independent mechanism and may involve the MAP kinase signaling pathway.     

Angiotensin II enhances the expression of Gialpha in A10 cells (smooth muscle): relationship with adenylyl cyclase activity

In the present studies, we have investigated the effect of angiotensin II (AII) on guanine nucleotide regulatory protein (G protein) expression and functions in A10 smooth muscle cells. AII treatment of A10 cells enhanced the levels of inhibitory guanine nucleotide regulatory protein (Gi) as well as Gi mRNA and not of stimulatory guanine nucleotide regulatory protein (Gs) in a concentration-dependent manner as determined by immunoblot and Northern blot analysis, respectively. AII-evoked increased expression of Gialpha-2 and Gialpha-3 was inhibited by actinomycin D treatment (RNA synthesis inhibitor). The increased expression of Gialpha-2 and Gialpha-3 by AII was not reflected in functions, because the GTPgammaS-mediated inhibition of forskolin-stimulated adenylyl cyclase and the receptor-mediated inhibition of adenylyl cyclase by AII and C-ANP4-23 [des(Gln18, Ser19, Gln20, Leu21, Gly22) ANP4-23-NH2] were not augmented but attenuated in AII-treated A10 cells. The attenuation was prevented by staurosporine (a protein kinase C inhibitor) treatment. On the other hand, AII treatment did not affect the expression and functions of stimulatory guanine nucleotide regulatory protein (Gs), however, the stimulatory effects of 5'-O-(3-thiotriphosphate), isoproterenol, and N-ethylcarboxamide adenosine (NECA) on adenylyl cyclase activity were inhibited to various degrees by AII treatment. Staurosporine reversed the AII-evoked attenuation of isoproterenol- and NECA-stimulated enzyme activity. From these results, it can be suggested that AII, whose levels are increased in hypertension, may be one of the possible contributing factors responsible for exhibiting an enhanced expression of Gi protein in hypertension.     

Suppression of soluble adenylyl cyclase protects smooth muscle cells against oxidative stress-induced apoptosis

Apoptosis of vascular smooth muscle cells (VSMC) significantly contributes to the instability of advanced atherosclerotic plaques. Oxygen radicals are an important cause for VSMC death. However, the precise mechanism of oxidative stress-induced VSMC apoptosis is still poorly understood. Here, we aimed to analyse the role of soluble adenylyl cylclase (sAC). VSMC derived from rat aorta were treated with either H₂O₂ (300 µmol/L) or DMNQ (30 µmol/L) for 6 h. Oxidative stress-induced apoptosis was prevented either by treatment with 30 µmol/L KH7 (a specific inhibitor of sAC) or by stable sAC-knockdown (shRNA-transfection). A similar effect was found after inhibition of protein kinase A (PKA). Suppression of the sAC/PKA-axis led to a significant increase in phosphorylation of the p38 mitogen-activated protein kinase under oxidative stress accompanied by a p38-dependent phosphorylation/inactivation of the pro-apoptotic Bcl-2-family protein Bad. Pharmacological inhibition of p38 reversed these effects of sAC knockdown on apoptosis and Bad phosphorylation, suggesting p38 as a link between sAC and apoptosis. Analysis of the protein phosphatases 1 and 2A activities revealed an activation of phosphatase 1, but not phosphatase 2A, under oxidative stress in a sAC/PKA-dependent manner and its role in controlling the p38 phosphorylation. Inhibition of protein phosphatase 1, but not 2A, prevented the pro-apoptotic effect of oxidative stress. In conclusion, sAC/PKA-signaling plays a key role in the oxidative stress-induced apoptosis of VSMC. The cellular mechanism consists of the sAC-promoted and protein phosphatase 1-mediated suppression of p38 phosphorylation resulting to activation of the mitochondrial pathway of apoptosis.     

Enhanced expression of Gialpha protein and adenylyl cyclase signaling in aortas from 1 kidney 1 clip hypertensive rats

We have previously shown the augmented levels of Gialpha-2 and Gialpha-3 proteins (isoforms of inhibitory guanine nucleotide regulatory protein (G-protein)), and not of Gsalpha, in the hearts and aortas of spontaneously and experimentally induced hypertensive rats. The increased expression of Gialpha and blood pressure was restored toward WKY levels by captopril treatment, suggesting a role for angiotensin (Ang) II in the enhanced expression of Gialpha protein and blood pressure. This study was undertaken to investigate whether 1 kidney 1 clip (1K-1C) hypertensive rats that exhibit enhanced levels of Ang II also express enhanced levels of Gialpha proteins. Aortas from 1K-1C hypertensive rats were used. The expression of G-proteins was determined at protein levels with immunoblotting techniques, using specific antibodies for different isoforms of G-proteins. The levels of Gialpha-2 and Gialpha-3 proteins were significantly higher in aortas from 1K-1C hypertensive rats than in control rats; Gsalpha levels were unchanged. The inhibitory effect of low concentrations of guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) on forskolin (FSK)-stimulated adenylyl cyclase (AC) activity was significantly enhanced in aortas from 1K-1C hypertensive rats; the inhibitory effect of C-ANP(4-23), which specifically interacts with the atrial natriuretic peptide (ANP)-C receptor, and Ang II on AC was attenuated. GTPgammaS, isoproterenol, glucagon, NaF, and FSK stimulated the AC activity in aortas from control and hypertensive rats to varying degrees; however, the stimulations were significantly lower in hypertensive rats than in control rats. These data suggest that aortas from 1K-1C hypertensive rats exhibit enhanced expression of Gialpha proteins and associated functions.     

NAD(P)H oxidase participates in the signaling events in high glucose-induced proliferation of vascular smooth muscle cells

Reactive oxygen species (ROS) have been implicated in the pathogenesis of vascular dysfunction in diabetes mellitus, and NAD(P)H oxidase is known as the most important source of ROS in the vasculatures. To determine whether NAD(P)H oxidase is a major participant in the critical intermediary signaling events in high glucose (HG, 25 mM)-induced proliferation of vascular smooth muscle cells (VSMC), we investigated in explanted aortic VSMC from rats the role of NAD(P)H oxidase on the HG-related cellular proliferation and superoxide production. VSMC under HG condition had increased proliferative capacity that was inhibited by tiron (1 mM), a cell membrane permeable superoxide scavenger, but not by SOD, which is not permeable to cell membrane. The nitroblue tetrazolium staining in the HG-exposed VSMC was more prominent than that of VSMC under normal glucose (5.5 mM) condition, which was significantly inhibited by DPI (10 microM), an NAD(P)H oxidase inhibitor, but not by inhibitors for other oxidases such as NADH dehydrogenase, xanthine oxidase, and nitric oxide synthase. In the VSMC under HG condition, the enhanced NAD(P)H oxidase activity with increased membrane translocation of Rac1 was observed, but the protein expression of p22phox and gp91phox was not increased. These data suggest that HG-induced changes in VSMC proliferation are related to the intracellular production of superoxide through enhanced activity of NAD(P)H oxidase.     

Adenosine stimulates guanylate cyclase activity in vascular smooth muscle cells

Good evidence exists to indicate that the vasodilating effect of adenosine is mediated by cell surface receptors on vascular smooth muscle cells. The mechanism of transmembrane signal transduction for adenosine, however, is not fully understood. Since cGMP is a second messenger known to mediate vasodilation, I have examined the effect of adenosine on the intracellular concentration of cGMP in vascular smooth muscle cells from rat aorta. I found that adenosine at 10(-9) to 10(-5) M led to an increase in intracellular cGMP levels in a dose-dependent fashion. The effect of adenosine on cyclic guanosine inorganic monophosphate (cGMP) could be mimicked by the A-type receptor agonists N6-cyclohexyladenosine and 5'-N-ethylcarboxamidoadenosine and was attenuated by the A-receptor antagonist theophylline. The order of potency of the adenosine analogues was N6-cyclohexyladenosine greater than 5'-N-ethylcarboxamidoadenosine greater than adenosine. These findings suggest that the effect of adenosine on cGMPi is mediated by A1-type cell surface receptors. Concerning the mechanism by which adenosine could elevate cGMPi, I found that the effect of adenosine on cGMPi was potentiated by the cGMP phosphodiesterase-specific inhibitor M & B 22948. Moreover, I found that N6-cyclohexyladenosine, 5'-N-ethylcarboxamidoadenosine, and adenosine stimulated a guanylate cyclase in homogenates of the cultured smooth muscle cells in a dose-dependent fashion with the same order of potency as their effects on cGMPi. Further evidence was obtained to indicate that adenosine and its analogues stimulated a particulate guanylate cyclase activity, whereas they did not alter soluble guanylate cyclase activity. Since cGMP is known as a second messenger mediating relaxation of vascular smooth muscle cells, the results obtained in this study could suggest that adenosine exerts its vasorelaxing effect by activating an Ai-receptor-linked guanylate cyclase.     

Regulation of cGMP-dependent protein kinase expression by soluble guanylyl cyclase in vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) undergo many phenotypic changes when placed in culture. Several studies have shown that the levels of expression of soluble guanylyl cyclase (sGC) or cGMP-dependent protein kinase (PKG) are altered in cultured VSMC. In this study the mechanisms involved in the coordinated expression of sGC and PKG were examined. Pro-inflammatory cytokines that increase the expression of type II NO synthase (inducible NO synthase, or iNOS) decreased PKG expression in freshly isolated, non-passaged bovine aortic SMC. However, in several passaged VSMC lines (i.e. bovine aortic SMC, human aortic SMC, and A7r5 cells), PKG protein expression was not suppressed by cytokines or NO. sGC was highly expressed in non-passaged bovine aortic SMC but not in passaged cell lines. Restoration of expression of sGC to passaged bovine SMC using adenovirus encoding the alpha1 and beta1 subunits of sGC restored the capacity of the cells to increase cGMP in response to NO. Furthermore, treatment of these sGC-transduced cells with NO donors for 48 h resulted in decreased PKG protein expression. In contrast, passaged rat aortic SMC expressed high levels of NO-responsive sGC but demonstrated reduced expression of PKG. Adenovirus-mediated expression of the PKG catalytically active domain in rat aortic SMC caused a reduction in the expression of sGC in these cells. These results suggest that there is a mechanism for the coordinated expression of sGC and PKG in VSMC and that prolonged activation of sGC down-regulates PKG expression. Likewise, the loss of PKG expression appears to increase sGC expression. These effects may be an adaptive mechanism allowing growth and survival of VSMC in vitro.     

Modulation of Nrf2 expression alters high glucose-induced oxidative stress and antioxidant gene expression in mouse mesangial cells

Reactive oxygen species (ROS) play an important role in the pathogenesis of diabetic nephropathy. Nuclear factor erythroid 2-related factor 2 (Nrf2) can up-regulate the expression of antioxidant genes and protect cells from oxidative damage. The current study is aimed at examining the effect of modulation of Nrf2 expression on high glucose-induced oxidative stress and Nrf2-targeting antioxidant expression in mouse mesangial cells. In this study, mouse mesangial cells were transiently transfected with Nrf2-plasmid or the Nrf2-specific siRNA. The high glucose-induced intracellular ROS, malondialdehyde, cell proliferation, and TGF-β1 secretion were measured. The levels of Nrf2, heme oxygenase-1 (HO-1), γ-glutamylcysteine synthethase (γ-GCS) expression, and nuclear expression of Nrf2 in mouse mesangial cells were determined. We found that high glucose induced ROS and malondialdehyde generation in mouse mesangial cells. Induction of Nrf2 over-expression reduced the high glucose-induced ROS and malondialdehyde production, inhibited cell proliferation and TGF-β1 secretion, accompanied by up-regulating the expressions of HO-1 and γ-GCS in mouse mesangial cells. However, knockdown of Nrf2 expression displayed reverse effects in mouse mesangial cells. All these results indicated that Nrf2 and its downstream antioxidants, HO-1 and γ-GCS, are negative regulators of high glucose-induced ROS-related mouse mesangial cell dysfunction.     

Role of PKC and TGF-beta receptor in glucose-induced proliferation of smooth muscle cells

The role of protein kinase C (PKC) and transforming growth factor (TGF)-beta in the proliferation of vascular smooth muscle cells (SMCs) under a high glucose condition was investigated. [3H]-thymidine incorporation under 20 mM glucose was significantly accelerated compared with that under 5.5 mM glucose, and this increase was inhibited by an anti-TGF-beta antibody or a PKC-beta specific inhibitor, LY333531. The amount of active and total TGF-beta1 in the conditioned media did not differ between 5.5 and 20 mM glucose. However, the expression of TGF-beta receptor type II under 20 mM glucose was significantly increased, but that of the TGF-beta receptor type I was not. This increased expression of the TGF-beta receptor type II was prevented by LY333531. These observations suggest that the increased expression of the TGF-beta receptor type II via PKC-beta plays an important role in the accelerated proliferation of SMCs under a high glucose condition, leading to the development of diabetic macroangiopathy.     

Eriodictyol inhibits high glucose-induced oxidative stress and inflammation in retinal ganglial cells

Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes mellitus and is considered as a leading cause of blindness. Oxidative stress and inflammation are significant drivers for the development of DR. Eriodictyol, a flavonoid compound, was proved to possess anti-inflammatory, antioxidative, and antidiabetic activities. However, the role of eriodictyol in DR has not been unveiled. In the current study, we explored the protective effects of eriodictyol on high glucose (HG)-induced rat retinal ganglial cells (RGCs). The results suggested that eriodictyol improved cell viability of HG-induced rat RGC-5 cells in a dose-dependent manner. Eriodictyol reduced the reactive oxygen species production and increased the activities of superoxide dismutase, glutathione peroxidase and catalase in rat RGC-5 cells in response to HG stimulation. The production of proinflammatory cytokines including tumor necrosis factor alpha and interleukin-8 was diminished after eriodictyol treatment. Eriodictyol also suppressed cell apoptosis induced HG in rat RGC-5 cells. Furthermore, eriodictyol enhanced the nuclear translocation of nuclear factor erythroid-2 (E2)-related factor 2 (Nrf2) and elevated the expression of antioxidant enzyme heme-oxygenase-1 (HO-1). These findings suggested that eriodictyol protects the RGC-5 cells from HG-induced oxidative stress, inflammation, and cell apoptosis through regulating the activation of Nrf2/HO-1 pathway.     

Role of the actin cytoskeleton in angiotensin II signaling in human vascular smooth muscle cells

Angiotensin II (Ang II) regulates vascular smooth muscle cell (VSMC) function by activating signaling cascades that promote vasoconstriction, growth, and inflammation. Subcellular mechanisms coordinating these processes are unclear. In the present study, we questioned the role of the actin cytoskeleton in Ang II mediated signaling through mitogen-activated protein (MAP) kinases and reactive oxygen species (ROS) in VSMCs. Human VSMCs were studied. Cells were exposed to Ang II (10-7 mol/L) in the absence and presence of cytochalasin B (10-6 mol/L, 60 min), which disrupts the actin cytoskeleton. Phosphorylation of p38MAP kinase, JNK, and ERK1/2 was assessed by immuno blotting. ROS generation was measured using the fluoroprobe chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (4 micromol/L). Interaction between the cytoskeleton and NADPH oxidase was determined by evaluating the presence of p47phox in the Triton X-100 insoluble membrane fraction. Ang II significantly increased phosphorylation of p38MAP kinase, JNK, and ERK1/2 (two- to threefold above control, p < 0.05). Cytochalasin B pretreatment attenuated p38MAP kinase and JNK effects (p < 0.05) without altering ERK1/2 phosphorylation. ROS formation, which was increased in Ang II stimulated cells, was significantly reduced by cytochalasin B (p < 0.01). p47phox, critically involved in NADPH oxidase activation, colocalized with the actin cytoskeleton in Ang II stimulated cells. Our data demonstrate that Ang II mediated ROS formation and activation of p38MAP kinase and JNK, but not ERK1/2, involves the actin cytoskeleton in VSMCs. In addition, Ang II promotes interaction between actin and p47phox. These data indicate that the cytoskeleton is involved in differential MAP kinase signaling and ROS generation by Ang II in VSMCs. Together, these studies suggest that the cytoskeleton may be a central point of crosstalk in growth- and redox-signaling pathways by Ang II, which may be important in the regulation of VSMC function.     

Interferon regulatory factor-1 as a positive regulator for high glucose-induced proliferation of vascular smooth muscle cells

High glucose-induced proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development of diabetic vascular diseases. However, molecular mediators responding for the proliferation of VSMCs remain to be determined. In this study, VSMCs were isolated from the rat thoracic aorta, and two cell models with Irf-1 knockdown and overexpression were established by transfecting cells with pGCsi-FU-Irf-1 and pGC-FU-Irf-1, respectively. Subsequently, high glucose was added to cells to induce proliferation. Proliferation assays were performed to see whether Irf-1 was involved in high glucose-induced proliferation of VSMCs. In addition, the expression of Irf-1 was detected in VSMCs stimulated with high glucose and the thoracic aorta of diabetic rats to confirm the relationship between Irf-1 expression and the proliferation of hyperglycemia-dependent VSMCs. The results showed that Irf-1 expression was significantly higher in the thoracic aorta of diabetic rats and VSMCs stimulated with high glucose than that in nondiabetic rats and untreated cells. Overexpression of Irf-1 accelerated the proliferation of VSMCs, and down-regulation of Irf-1 expression significantly depressed the proliferative ability of VSMCs under high-glucose conditions, indicating that Irf-1 was a positive regulator for high glucose-induced proliferation of VSMCs. It could be presumed that Irf-1 is associated with the accelerated proliferation of VSMCs in diabetic vascular diseases and may prove to be a potential target gene for disease treatment.     

Rho expression and activation in vascular smooth muscle cells

Phenotypic modulation of smooth muscle cells (SMC) involves dramatic changes in expression and organization of contractile and cytoskeletal proteins, but little is known of how this process is regulated. The present study used a cell culture model to investigate the possible involvement of RhoA, a known regulator of the actin cytoskeleton. In rabbit aortic SMC seeded into primary culture at moderate density, Rho activation was high at two functionally distinct time-points, first as cells modulated to the "synthetic" phenotype, and again upon confluence and return to the "contractile" phenotype. Rho expression increased with time, such that maximal expression occurred upon return to the contractile state. Transient transfection of synthetic state cells with constitutively active RhoA (Val14RhoA) caused a reduction in cell size and reorganization of cytoskeletal proteins to resemble that of the contractile phenotype. Actin and myosin filaments were tightly packed and highly organised while vimentin localised to the perinuclear region; focal adhesions were enlarged and concentrated at the cell periphery. Conversely, inhibition of endogenous Rho by C3 exoenzyme resulted in complete loss of contractile filaments without affecting vimentin distribution; focal adhesions were reduced in size and number. Treatment of synthetic state SMC with known regulators of SMC phenotype, heparin and thrombin, caused a modest increase in Rho activation. Long-term confluence and serum deprivation induced cells to return to a more contractile phenotype and this was augmented by heparin and thrombin. The results implicate RhoA for a role in regulating SMC phenotype and further show that activation of Rho by heparin and thrombin correlates with the ability of these factors to promote the contractile phenotype.     

High glucose increases the expression of Gq/11alpha and PLC-beta proteins and associated signaling in vascular smooth muscle cells

The levels and activity of protein kinase C and diacylglycerol were shown to be upregulated in diabetes/hyperglycemia; however, studies on the expression of upstream signaling molecules of phosphatidylinositol turnover were lacking. The present study was therefore undertaken to examine whether hyperglycemia/diabetes could also modulate the expression of Gqalpha and phospholipase C-beta (PLC-beta) proteins and associated phosphatidylinositol turnover signaling in aortic vascular smooth muscle cells (VSMCs) and A10 VSMCs exposed to high glucose. Aortic VSMCs from streptozotocin-diabetic rats exhibited an increased expression of Gqalpha and PLC-beta1 proteins (60% and 30%, respectively) compared with control cells as determined by Western blot analysis. The pretreatment of A10 VSMCs with high glucose (26 mM) for 3 days also augmented the levels of Gqalpha, G11alpha, PLC-beta1 and -beta2 proteins by about 50, 35, 30, and 30%, respectively, compared with control cells that were restored to control levels by endothelin-1 (ET-1), ET types A and B (ET(A) and ET(B)) receptors, and angiotensin II type 1 (AT1) receptor antagonists. In addition, ET-1-stimulated inositol triphosphate formation was also significantly higher in VSMCs exposed to high glucose, whereas the basal levels of inositol triphosphate were not different between the two groups. Furthermore, the treatment of A10 VSMCs with angiotensin II and ET-1 also significantly increased the levels of Gq/11alpha and PLC-beta proteins that were restored toward control levels by ET(A)/ET(B) and AT1 receptor antagonists. These results suggest that high glucose augments the expression of Gq/11alpha, PLC-beta, and mediated signaling in VSMCs, which may be attributed to AT1, ET(A), and ET(B) receptors.     

Raf-1 kinase mediates adenylyl cyclase sensitization by TNF-alpha in human airway smooth muscle cells

Tumor necrosis factor (TNF)-alpha is a potent inflammatory cytokine implicated in the exacerbation of asthma. Chronic exposure to TNF-alpha has been reported to induce G protein-coupled receptor desensitization, but adenylyl cyclase sensitization, in airway smooth muscle cells by an unknown mechanism. Cyclic AMP, which is synthesized by adenylyl cyclases in response to G protein-coupled receptor signals, is an important second messenger involved in the regulation of the airway muscle proliferation, migration, and tone. In other cell types, TNF-alpha receptors transactivate the EGF receptor, which activates raf-1 kinase. Further studies in transfected cells show that raf-1 kinase can phosphorylate and activate some isoforms of adenylyl cyclase. Cultured human airway smooth muscle cells were treated with TNF-alpha in the presence or absence of inhibitors of prostaglandin signaling, protein kinases, or G(i) proteins. TNF-alpha caused a significant dose- (1-10 ng/ml) and time-dependent (24 and 48 h) increase in forskolin-stimulated adenylyl cyclase activity, which was abrogated by pretreatment with GW5074 (a raf-1 kinase inhibitor), was partially inhibited by an EGF receptor inhibitor, but was unaffected by pertussis toxin. TNF-alpha also increased phosphorylation of Ser(338) on raf-1 kinase, indicative of activation. IL-1beta and EGF sensitization of adenylyl cyclase activity was also sensitive to raf-1 kinase inhibition by GW5074. Taken together, these studies link two signaling pathways not previously characterized in human airway smooth muscle cells: TNF-alpha transactivation of the EGF receptor, with subsequent raf-1 kinase-mediated activation of adenylyl cyclase.