Angiotensin II induces Fat1 expression/activation and vascular smooth muscle cell migration via Nox1-dependent reactive oxygen species generation.

Fat1 is an atypical cadherin that controls vascular smooth muscle cell (VSMC) proliferation and migration. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (Nox1) is an important source of reactive oxygen species (ROS) in VSMCs. Angiotensin II (Ang II) induces the expression and/or activation of both Fat1 and Nox1 proteins. This study tested the hypothesis that Ang II-induced Fat1 activation and VSMC migration are mediated by Nox1-dependent ROS generation and redox signaling. Studies were performed in cultured VSMCs from Sprague­Dawley rats. Cells were treated with Ang II (1 µmol/L) for short (5 to 30 min) or long term stimulations (3 to 12 h) in the absence or presence of the antioxidant apocynin (10 µmol/L), extracellular-signal-regulated kinases 1/2 (Erk1/2) inhibitor PD98059 (1 µmol/L), or Ang II type 1 receptor (AT1R) valsartan (1 µmol/L). siRNA was used to knockdown Nox1 or Fat1. Cell migration was determined by Boyden chamber assay. Ang II increased Fat1 mRNA and protein levels and promoted Fat1 translocation to the cell membrane, responses that were inhibited by AT1R antagonist and antioxidant treatment. Downregulation of Nox1 inhibited the effects of Ang II on Fat1 protein expression. Nox1 protein induction, ROS generation, and p44/p42 MAPK phosphorylation in response to Ang II were prevented by valsartan and apocynin, and Nox1 siRNA inhibited Ang II-induced ROS generation. Knockdown of Fat1 did not affect Ang II-mediated increases in Nox1 expression or ROS. Inhibition of p44/p42 MAPK phosphorylation by PD98059 abrogated the Ang II-induced increase in Fat1 expression and membrane translocation. Knockdown of Fat1 inhibited Ang II-induced VSMC migration, which was also prevented by valsartan, apocynin, PD98059, and Nox1 siRNA. Our findings indicate that Ang II regulates Fat1 expression and activity and induces Fat1-dependent VSMC migration via activation of AT1R, ERK1/2, and Nox1-derived ROS, suggesting a role for Fat1 downstream of Ang II signaling that leads to vascular remodeling.

Obesity induced by neonatal treatment with monosodium glutamate impairs microvascular reactivity in adult rats: role of NO and prostanoids.

BACKGROUND AND AIM: given that obesity is an independent risk factor for the development of cardiovascular diseases we decided to investigate the mechanisms involved in microvascular dysfunction using a monosodium glutamate (MSG)-induced model of obesity, which allows us to work on both normotensive and normoglycemic conditions. METHODS AND RESULTS: Male offspring of Wistar rats received MSG from the second to the sixth day after birth. Sixteen-week-old MSG rats displayed higher Lee index, fat accumulation, dyslipidemia and insulin resistance, with no alteration in glycemia and blood pressure. The effect of norepinephrine (NE), which was increased in MSG rats, was potentiated by L-nitro arginine methyl ester (L-NAME) or tetraethylammonium (TEA) and was reversed by indomethacin and NS-398. Sensitivity to acetylcholine (ACh), which was reduced in MSG rats, was further impaired by L-NAME or TEA, and was corrected by indomethacin, NS-398 and tetrahydrobiopterin (BH4). MSG rats displayed increased endothelium-independent relaxation to sodium nitroprusside. A reduced prostacyclin/tromboxane ratio was found in the mesenteric beds of MSG rats. Mesenteric arterioles of MSG rats also displayed reduced nitric oxide (NO) production along with increased reactive oxygen species (ROS) generation; these were corrected by BH4 and either L-NAME or superoxide dismutase, respectively. The protein expression of eNOS and cyclooxygenase (COX)-2 was increased in mesenteric arterioles from MSG rats. CONCLUSION: Obesity/insulin resistance has a detrimental impact on vascular function. Reduced NO bioavailability and increased ROS generation from uncoupled eNOS and imbalanced release of COX products from COX-2 play a critical role in the development of these vascular alterations.

Aldosterone and angiotensin II synergistically stimulate migration in vascular smooth muscle cells through c-Src-regulated redox-sensitive RhoA pathways.

OBJECTIVE: Synergistic interactions between aldosterone (Aldo) and angiotensin II (Ang II) have been implicated in vascular inflammation, fibrosis, and remodeling. Molecular mechanisms underlying this are unclear. We tested the hypothesis that c-Src activation, through receptor tyrosine kinase transactivation, is critically involved in synergistic interactions between Aldo and Ang II and that it is upstream of promigratory signaling pathways in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: VSMCs from WKY rats were studied. At low concentrations (10(-10) mol/L) Aldo and Ang II alone did not influence c-Src activation, whereas in combination they rapidly increased phosphorylation (P<0.01), an effect blocked by eplerenone (Aldo receptor antagonist) and irbesartan (AT1R blocker). This synergism was attenuated by AG1478 and AG1296 (inhibitors of EGFR and PDGFR, respectively), but not by AG1024 (IGFR inhibitor). Aldo and Ang II costimulation induced c-Src-dependent activation of NAD(P)H oxidase and c-Src-independent activation of ERK1/2 (P<0.05), without effect on ERK5, p38MAPK, or JNK. Aldo/Ang II synergistically activated RhoA/Rho kinase and VSMC migration, effects blocked by PP2, apocynin, and fasudil, inhibitors of c-Src, NADPH oxidase, and Rho kinase, respectively. CONCLUSIONS: Aldo/Ang II synergistically activate c-Src, an immediate signaling response, through EGFR and PDGFR, but not IGFR transactivation. This is associated with activation of redox-regulated RhoA/Rho kinase, which controls VSMC migration. Although Aldo and Ang II interact to stimulate ERK1/2, such effects are c-Src-independent. These findings indicate differential signaling in Aldo-Ang II crosstalk and highlight the importance of c-Src in redox-sensitive RhoA, but not ERK1/2 signaling. Blockade of Aldo/Ang II may be therapeutically useful in vascular remodeling associated with abnormal VSMC migration.

Gender-specific vascular effects elicited by chronic ethanol consumption in rats: a role for inducible nitric oxide synthase.

BACKGROUND AND PURPOSE: Epidemiological data suggest that the risk of ethanol-associated cardiovascular disease is greater in men than in women. This study investigates the mechanisms underlying gender-specific vascular effects elicited by chronic ethanol consumption in rats. EXPERIMENTAL APPROACH: Vascular reactivity experiments using standard muscle bath procedures were performed on isolated thoracic aortae from rats. mRNA and protein for inducible NO synthase (iNOS) and for endothelial NOS (eNOS) was assessed by RT-PCR or western blotting, respectively. KEY RESULTS: In male rats, chronic ethanol consumption enhanced phenylephrine-induced contraction in both endothelium-intact and denuded aortic rings. However, in female rats, chronic ethanol consumption enhanced phenylephrine-induced contraction only in endothelium denuded aortic rings. After pre-incubation of endothelium-intact rings with L-NAME, both male and female ethanol-treated rats showed larger phenylephrine-induced contractions in aortic rings, compared to the control group. Acetylcholine-induced relaxation was not affected by ethanol consumption. The effects of ethanol on responses to phenylephrine were similar in ovariectomized (OVX) and intact (non-OVX) female rats. In the presence of aminoguanidine, but not 7-nitroindazole, the contractions to phenylephrine in rings from ethanol-treated female rats were greater than that found in control tissues in the presence of the inhibitors. mRNA levels for eNOS and iNOS were not altered by ethanol consumption. Ethanol intake reduced eNOS protein levels and increased iNOS protein levels in aorta from female rats. CONCLUSIONS AND IMPLICATIONS: Gender differences in the vascular effects elicited by chronic ethanol consumption were not related to ovarian hormones but seemed to involve the upregulation of iNOS.

Maternal diabetes affects specific extracellular matrix components during placentation.

During embryo implantation, invasive trophoblast cells mediate embryo invasion into the decidualized stroma, forming a rich network of lacunae that connect the embryonic tissues to the maternal blood vessels. Placentation is probably guided by the composition and organization of the endometrial extracellular matrix. Certain pathological conditions that occur during pregnancy, including diabetes, have been linked to abnormal placental morphology and consequent fetal morbidity. We used immunoperoxidase techniques to identify members of the collagen, proteoglycan and glycoprotein families in the various compartments of the rat placenta and to determine whether experimentally induced diabetes affects placental morphology and alters the distribution of these molecules during pregnancy. Single injections of alloxan (40 mg kg(-1) i.v.) were used to induce diabetes on day 2 of pregnancy in Wistar rats. Placentas were collected on days 14, 17, and 20. Type I and III collagen, as well as the proteoglycans decorin and biglycan, were found to be distributed throughout the placentas of control and diabetic rats. In both groups, laminin expression decreased at the end of pregnancy. In contrast, fibronectin was detected in the labyrinth region of diabetic rats at all gestational stages studied, whereas it was detected only at term pregnancy in the placentas of control rats. These results show for the first time that some extracellular matrix molecules are modulated during placental development. However, as diabetic rats presented increased fibronectin deposition exclusively in the labyrinth region, we speculate that diabetes alters the microenvironment at the maternal-fetal interface, leading to developmental abnormalities in the offspring.

Endothelin-1, but not Ang II, activates MAP kinases through c-Src independent Ras-Raf dependent pathways in vascular smooth muscle cells.

OBJECTIVE: Endothelin-1 (ET-1) and angiotensin II (Ang II) activate common signaling pathways to promote changes in vascular reactivity, remodeling, inflammation, and oxidative stress. Here we sought to determine whether upstream regulators of mitogen-activated protein kinases (MAPKs) are differentially regulated by ET-1 and Ang II focusing on the role of c-Src and the small GTPase Ras. METHODS AND RESULTS: Mesenteric vascular smooth muscle cells (VSMCs) from mice with different disruption levels in the c-Src gene (c-Src(+/-) and c-Src(-/-)) and wild-type (c-Src(+/+)) were used. ET-1 and Ang II induced extracellular signal-regulated kinase (ERK) 1/2, SAPK/JNK, and p38MAPK phosphorylation in c-Src(+/+) VSMCs. In VSMCs from c-Src(+/-) and c-Src(-/-), Ang II effects were blunted, whereas c-Src deficiency had no effect in ET-1-induced MAPK activation. Ang II but not ET-1 induced c-Src phosphorylation in c-Src(+/+) VSMCs. Activation of c-Raf, an effector of Ras, was significantly increased by ET-1 and Ang II in c-Src(+/+) VSMCs. Ang II but not ET-1-mediated c-Raf phosphorylation was inhibited by c-Src deficiency. Knockdown of Ras by siRNA inhibited both ET-1 and Ang II-induced MAPK phosphorylation. CONCLUSIONS: Our data indicate differential regulation of MAPKs by distinct G protein-coupled receptors. Whereas Ang II has an obligatory need for c-Src, ET-1 mediates its actions through a c-Src-independent Ras-Raf-dependent pathway for MAPK activation. These findings suggest that Ang II and ET-1 can activate similar signaling pathways through unrelated mechanisms. MAP kinases are an important point of convergence for Ang II and ET-1.

The influence of improved glycaemic control with chlorpropamide on microvascular reactivity and nitric oxide synthase activity in diabetic rats.

Hyperglycaemia is a primary cause of vascular complications in diabetes. A hallmark of these vascular complications is endothelial cell dysfunction, which is partly due to reduced production of nitric oxide. The aim of this study was to verify the influence of improved glycaemic control with chlorpropamide on microvascular reactivity, endothelial nitric oxide synthase (e-NOS) expression, and NOS activity in neonatal streptozotocin-induced diabetic rats (n-STZ). Diabetes was induced by STZ injection into neonates Wistar rats. n-STZ diabetic rats were treated with chlorpropamide (200 mg kg(-1), 15 days, by gavage). The changes in mesenteric arteriolar and venular diameters were determined in anaesthetized control and n-STZ diabetic rats, before and after topical application of acetylcholine, bradykinin and sodium nitroprusside (SNP). We also assessed e-NOS expression (using polymerase chain reaction after reverse transcription of mRNAs into cDNAs) and NOS activity (conversion of L-arginine to citrulline) in the mesenteric vascular bed of chlorpropamide-treated n-STZ, vehicle-treated n-STZ, and control rats. In n-STZ, chlorpropamide treatment reduced high glycaemic levels, improved glucose tolerance and homoeostatic model assessment (HOMA-beta), and restored NOS activity. Impaired vasodilator responses of arterioles and venules to acetylcholine, bradykinin and SNP were partially corrected by chlorpropamide treatment in n-STZ. We concluded that improved metabolic control and restored NOS activity might be collaborating with improved microvascular reactivity found in chlorpropamide-treated n-STZ.

Endothelin-1 contributes to the sexual differences in renal damage in DOCA-salt rats.

We investigated whether gender differences in renal damage in DOCA-salt hypertension are associated with effects of ovarian hormones and/or endothelin-1 (ET-1). Renal injuries and renal pre-pro-ET-1 mRNA expression were enhanced in male and female ovariectomized (OVX) DOCA rats versus female DOCA rats. Treatment with estrogen plus progesterone or progesterone, but not estrogen alone, attenuated renal damage and pre-pro-ET-1 mRNA expression in OVX DOCA rats. The ETA antagonist BMS182874 greatly ameliorated renal damage in male and OVX DOCA rats. In conclusion, the ovarian hormones have a protective role on the renal structural alterations in female DOCA rats by modulating effects of ET-1, via ETA receptors.

Toll-like receptor 4 inhibition reduces vascular inflammation in spontaneously hypertensive rats.

AIMS: Hypertension is associated with increased levels of circulating cytokines and recent studies have shown that innate immunity contributes to hypertension. The mechanisms which hypertension stimulates immune response remain unclear, but may involve formation of neo-antigens that activate the immune system. Toll like receptor 4 (TLR4) is an innate immune receptor that binds a wide spectrum of exogenous (lipopolysaccharide) and endogenous ligands. TLR4 signaling leads to activation of nuclear factor kappa B (NFκB) and transcription of genes involved in inflammatory response. We previously demonstrated that TLR4 blockade reduces blood pressure and the augmented vascular contractility in spontaneously hypertensive rats (SHR). Here we hypothesized that inhibition of TLR4 ameliorates the vascular inflammatory process by a NFκB signaling pathway. MAIN METHODS: SHR and Wistar rats were treated with anti-TLR4 antibody (1µg/day) or unspecific IgG for 15days (i.p.). KEY FINDINGS: Anti-TLR4 treatment decreased production of reactive oxygen species and expression of IL-6 cytokine in mesenteric resistance arteries from SHR, when compared with IgG-treated SHR. Anti-TLR4 treatment also abolished the increased vascular reactivity to noradrenaline observed in IgG-treated SHR, as described before, and inhibition of NFκB decreased noradrenaline responses only in IgG-treated SHR. Mesenteric arteries from SHR treated with anti-TLR4 displayed decreased expression of MyD88, but not TRIF, key molecules in TLR4 signaling. Phosphorylation of p38 and NF-κB p65 were decreased in arteries from anti-TLR4-treated SHR versus IgG-treated SHR. SIGNIFICANCE: Together, these results suggest that TLR4 is a key player in hypertension and vascular inflammatory process by a NFκB signaling pathway.

Intracellular calcium stores and oscillatory contractions in arteries from genetically hypertensive rats.

Strips of tail artery from stroke-prone spontaneously hypertensive rats (SHRSP), but not from normotensive Wistar Kyoto (WKY) rats, exhibit oscillatory activity after stimulation with norepinephrine. In addition, oscillatory activity is observed in response to tetraethylammonium (TEA) in vessels from both SHRSP and WKY rats. Mechanistically, the oscillatory contractions are associated with calcium (Ca2+)-driven action potentials. We have tested the hypothesis that intracellular Ca2+ stores participate in the generation of norepinephrine-induced oscillatory contractions in tail arteries from SHRSP. Additionally, the role of intracellular Ca2+ stores on TEA-induced contractions were evaluated. Contractile force in strips of tail artery from SHRSP and WKY rats was measured, using standard muscle bath procedures, and the effect of interventions that affect the storage of intracellular Ca2+ on the oscillatory contractions was evaluated. Depletion of intracellular Ca2+ stores, with ryanodine, or inhibition of Ca2+ uptake into the sarcoplasmic reticulum (SR), with thapsigargin and cyclopiazonic acid (CPA), did not inhibit oscillatory contractions induced by norepinephrine in SHRSP vessels. However, these agents inhibited the amplitude of TEA-induced contractions in WKY strips. Bay K 8644 and A23187 inhibited TEA-induced oscillatory contractions in WKY vessels. In SHRSP tail artery Bay K 8644 inhibited both norepinephrine and TEA-induced contractions, while A23187 did not have any effect. The phospholipase C inhibitor, NCDC (3X 10(-5) M), blocked oscillatory activity induced by norepinephrine in SHRSP tail artery and TEA-induced oscillations both in SHRSP and WKY vessels. These observations suggest that Ca2+ release and Ca2+ uptake into intracellular Ca2+ stores are not involved in the contraction-relaxation cycles that characterize norepinephrine-induced oscillatory activity in SHRSP tail artery. Similarly, SR Ca2+ stores may modulate but are not essential for TEA-induced oscillatory contractions.

Cyclosporin A attenuates skeletal muscle damage induced by crotoxin in rats.

This work was undertaken to determine the role of the calcineurin pathway on the necrosis of skeletal muscle induced by crotoxin, the major component of the venom of Crotalus durissus terrificus. Rats were treated with cyclosporin A (CsA), a calcineurin inhibitor, for 5 days and, in the 6th day, received an intramuscular injection of crotoxin into the tibialis anterior muscle. Rats were also treated with diclofenac, a non-steroidal anti-inflammatory drug, for 5 days and, on the 6th day, injected with crotoxin. All treated groups were sacrificed 24 h after injection of crotoxin. Tibialis anterior and soleus muscles were removed, frozen and stored in liquid nitrogen. Histological sections were stained with Toluidine Blue and assayed for acid phosphatase. The results show that CsA, but not diclofenac, is able to significantly minimize myonecrosis promoted by crotoxin. In conclusion, CsA attenuates skeletal muscle necrosis induced by crotoxin, indicating that the calcineurin pathway is essential for crotoxin myotoxic activity. The myoprotective effect of CsA is not related to its anti-inflammatory effect since diclofenac, a cyclo-oxygenase inhibitor, was not able to produce myoprotection.

Role of nitric oxide in myotoxic activity induced by crotoxin in vivo.

This study was aimed to determine the role of nitric oxide on the skeletal myotoxic activity induced by crotoxin, the major component of the venom of Crotalus durissus terrificus. Rats were treated with N(G)-nitro-L-arginine methyl ester (L-NAME), a non-selective inhibitor of nitric oxide synthase or vehicle for 4 days, and on the 5th day received an intramuscular injection of crotoxin into the tibialis anterior muscle. Rats were also treated with aminoguanidine bicarbonate salt or 7-nitroindazole, inhibitors of the inducible and neuronal isoforms of nitric oxide synthase, respectively, for 4 days and on the 5th day injected with crotoxin. All treated groups were sacrificed 24 h after injection of crotoxin. Tibialis anterior and soleus muscles were removed, frozen and stored in liquid nitrogen. Histological sections were stained with toluidine blue and assayed for acid phosphatase. The results show that L-NAME significantly minimizes myonecrosis induced by crotoxin and both aminoguanidine and 7-nitroindazole partially prevented myonecrosis induced by crotoxin. Based on the present results we conclude that nitric oxide is a very important intracellular signaling molecule that mediates crotoxin myotoxic activity.

Effects of estrogen on the vascular system.

The cardiovascular protective actions of estrogen are partially mediated by a direct effect on the vessel wall. Estrogen is active both on vascular smooth muscle and endothelial cells where functionally competent estrogen receptors have been identified. Estrogen administration promotes vasodilation in humans and in experimental animals, in part by stimulating prostacyclin and nitric oxide synthesis, as well as by decreasing the production of vasoconstrictor agents such as cyclooxygenase-derived products, reactive oxygen species, angiotensin II, and endothelin-1. In vitro, estrogen exerts a direct inhibitory effect on smooth muscle by activating potassium efflux and by inhibiting calcium influx. In addition, estrogen inhibits vascular smooth muscle cell proliferation. In vivo, 17beta-estradiol prevents neointimal thickening after balloon injury and also ameliorates the lesions occurring in atherosclerotic conditions. As is the case for other steroids, the effect of estrogen on the vessel wall has a rapid non-genomic component involving membrane phenomena, such as alteration of membrane ionic permeability and activation of membrane-bound enzymes, as well as the classical genomic effect involving estrogen receptor activation and gene expression.

The effects of cyclopiazonic acid on intracellular Ca2+ in aortic smooth muscle cells from DOCA-hypertensive rats.

We tested the hypothesis that cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, increases intracellular Ca2+ concentration ([Ca2+]) in aortic myocytes and that the increase in [Ca2+]i is higher in aortic cells from deoxycorticosterone acetate (DOCA)-hypertensive rats. Male Sprague-Dawley rats, 250-300 g, underwent uninephrectomy, received a silastic implant containing DOCA (200 mg/kg) and had free access to water supplemented with 1.0% NaCl and 0.2% KCl. Control rats were also uninephrectomized, received normal tap water, but no implant. Intracellular Ca2+ measurements were performed in aortic myocytes isolated from normotensive (Systolic blood pressure = 120 +/- 3 mmHg; body weight = 478 +/- 7 g, N = 7) and DOCA-hypertensive rats (195 +/- 10 mmHg; 358 +/- 16 g, N = 7). The effects of CPA on resting [Ca2+]i and on caffeine-induced increase in [Ca2+]i after [Ca2+]i depletion and reloading were compared in aortic cells from DOCA and normotensive rats. The phasic increase in [Ca2+]i induced by 20 mM caffeine in Ca(2+)-free buffer was significantly higher in DOCA aortic cells (329 +/- 36 nM, N = 5) compared to that in normotensive cells (249 +/- 16 nM, N = 7, P < 0.05). CPA (3 microM) inhibited caffeine-induced increases in [Ca2+]i in both groups. When the cells were placed in normal buffer (1.6 mM Ca2+, loading period), after treatment with Ca(2+)-free buffer (depletion period), an increase in [Ca2+]i was observed in DOCA aortic cells (45 +/- 11 nM, N = 5) while no changes were observed in normotensive cells. CPA (3 microM) potentiated the increase in [Ca2+]i (122 +/- 30 nM, N = 5) observed in DOCA cells during the loading period while only a modest increase in [Ca2+]i (23 +/- 10 nM, N = 5) was observed in normotensive cells. CPA-induced increase in [Ca2+]i did not occur in the absence of extracellular Ca2+ or in the presence of nifedipine. These data show that CPA induces Ca2+ influx in aorta from both normotensive and DOCA-hypertensive rats. However, the increase in [Ca2+]i is higher in DOCA aortic cells possibly due to an impairment in the mechanisms that control [Ca2+]i. The large increase in [Ca2+]i in response to caffeine in DOCA cells probably reflects a greater storage of Ca2+ in the SR.

Calcium handling by vascular myocytes in hypertension.

Calcium ions (Ca2+) trigger the contraction of vascular myocytes and the level of free intracellular Ca2+ within the myocyte is precisely regulated by sequestration and extrusion mechanisms. Extensive evidence indicates that a defect in the regulation of intracellular Ca2+ plays a role in the augmented vascular reactivity characteristic of clinical and experimental hypertension. For example, arteries from spontaneously hypertensive rats (SHR) have an increased contractile sensitivity to extracellular Ca2+ and intracellular Ca2+ levels are elevated in aortic smooth muscle cells of SHR. We hypothesize that these changes are due to an increase in membrane Ca2+ channel density and possibly function in vascular myocytes from hypertensive animals. Several observations using various experimental approaches support this hypothesis: 1) the contractile activity in response to depolarizing stimuli is increased in arteries from hypertensive animals demonstrating increased voltage-dependent Ca2+ channel activity in hypertension; 2) Ca2+ channel agonists such as Bay K 8644 produce contractions in isolated arterial segments from hypertensive rats and minimal contraction in those from normotensive rats; 3) intracellular Ca2+ concentration is abnormally increased in vascular myocytes from hypertensive animals following treatment with Ca2+ channel agonists and depolarizing interventions, and 4) using the voltage-clamp technique, the inward Ca2+ current in arterial myocytes from hypertensive rats is nearly twice as large as that from myocytes of normotensive rats. We suggest that an alteration in Ca2+ channel function and/or an increase in Ca2+ channel density, resulting from increased channel synthesis or reduced turnover, underlies the increased vascular reactivity characteristic of hypertension.

Gender differences in vascular expression of endothelin and ET(A)/ET(B) receptors, but not in calcium handling mechanisms, in deoxycorticosterone acetate-salt hypertension.

We determined if the increased vascular responsiveness to endothelin-1 (ET-1) observed in male, but not in female, DOCA-salt rats is associated with differential vascular mRNA expression of ET-1 and/or ET A/ET B receptors or with functional differences in Ca2+ handling mechanisms by vascular myocytes. Uninephrectomized male and female Wistar rats received DOCA and drinking water containing NaCl/KCl. Control rats received vehicle and tap water. Blood pressure and contractile responses of endothelium-denuded aortic rings to agents which induce Ca2+ influx and/or its release from internal stores were measured using standard procedures. Expression of mRNA for ET-1 and ET A/ET B receptors was evaluated by RT-PCR after isolation of total cell RNA from both aorta and mesenteric arteries. Systolic blood pressure was higher in male than in female DOCA rats. Contractions induced by Bay K8644 (which activates Ca2+ influx through voltage-operated L-type channels), and by caffeine, serotonin or ET-1 in Ca2+-free buffer (which reflect Ca2+ release from internal stores) were significantly increased in aortas from male and female DOCA-salt compared to control aortas. DOCA-salt treatment of male, but not female, rats statistically increased vascular mRNA expression of ET-1 and ET B receptors, but decreased the expression of ET A receptors. Molecular up-regulation of vascular ET B receptors, rather than differential changes in smooth muscle Ca2+ handling mechanisms, seems to account for the increased vascular reactivity to ET-1/ET B receptor agonists and higher blood pressure levels observed in male DOCA-salt rats.

Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain.

O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2 ± 2 vs 7.9 ± 1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4 ± 2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3 ± 2 vs 7.5 ± 2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1 ± 2 vs 7.4 ± 2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca(2+)/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

Mechanisms of endothelial dysfunction in obesity-associated hypertension.

Obesity is strongly associated with high blood pressure, dyslipidemia, and type 2 diabetes. These conditions synergistically increase the risk of cardiovascular events. A number of central and peripheral abnormalities can explain the development or maintenance of high blood pressure in obesity. Of great interest is endothelial dysfunction, considered to be a primary risk factor in the development of hypertension. Additional mechanisms also related to endothelial dysfunction have been proposed to mediate the development of hypertension in obese individuals. These include: increase in both peripheral vasoconstriction and renal tubular sodium reabsorption, increased sympathetic activity and overactivation of both the renin-angiotensin system and the endocannabinoid system and insulin resistance. The discovery of new mechanisms regulating metabolic and vascular function and a better understanding of how vascular function can be influenced by these systems would facilitate the development of new therapies for treatment of obesity-associated hypertension.

Improvement of metabolic parameters and vascular function by metformin in obese non-diabetic rats.

AIMS: Metformin is an insulin sensitizing agent with beneficial effects in diabetic patients on glycemic levels and in the cardiovascular system. We examined whether the metabolic changes and the vascular dysfunction in monosodium glutamate-induced obese non-diabetic (MSG) rats might be improved by metformin. MAIN METHODS: 16 week-old MSG rats were treated with metformin for 15 days and compared with age-matched untreated MSG and non-obese non-diabetic rats (control). Blood pressure, insulin sensitivity, vascular reactivity and prostanoid release in the perfused mesenteric arteriolar bed as well as nitric oxide production and reactive oxygen species generation in isolated mesenteric arteries were analyzed. KEY FINDINGS: 18-week-old MSG rats displayed higher Lee index, fat accumulation, dyslipidemia, insulin resistance and hyperinsulinemia. Metformin treatment improved these alterations. The norepinephrine-induced response, increased in the mesenteric arteriolar bed from MSG rats, was corrected by metformin. Indomethacin corrected the enhanced contractile response in MSG rats but did not affect metformin effects. The sensitivity to acetylcholine, reduced in MSG rats, was also corrected by metformin. Indomethacin corrected the reduced sensitivity to acetylcholine in MSG rats but did not affect metformin effects. The sensitivity to sodium nitroprusside was increased in preparations from metformin-treated rats. Metformin treatment restored both the reduced PGI2/TXA2 ratio and the increased reactive oxygen species generation in preparations from MSG rats. SIGNIFICANCE: Metformin improved the vascular function in MSG rats through reduction in reactive oxygen species generation, modulation of membrane hyperpolarization, correction of the unbalanced prostanoids release and increase in the sensitivity of the smooth muscle to nitric oxide.

Endogenous testosterone increases leukocyte-endothelial cell interaction in spontaneously hypertensive rats.

AIMS: Inflammation may have an important role in the beginning and in the progress of cardiovascular diseases. Testosterone exerts important effects on vascular function, which is altered in arterial hypertension. Thus, the aim of this study was to evaluate the influence of endogenous testosterone on leukocyte behavior in post-capillary venules of the mesenteric bed of spontaneously hypertensive rats (SHR). MAIN METHODS: 18 week-old intact SHR, castrated SHR and normotensive rats (intact Wistar) were used. Blood pressure was measured by tail plethysmography and serum testosterone levels by ELISA. Leukocyte rolling, adhesion and migration were evaluated in vivo in situ by intravital microscopy. KEY FINDINGS: Castration significantly reduced blood pressure and reversed the increased leukocyte rolling and adhesion observed in SHRs. Leukocyte counts and other hemodynamic parameters did not differ among groups. SHRs displayed increased protein expression of P-selectin and ICAM-1 in mesenteric venules when compared to intact Wistar. Castration of SHRs restored the protein expression of the cell adhesion molecules. SIGNIFICANCE: The findings of the present study demonstrate the critical role of endogenous testosterone mediating the effects of hypertension increasing leukocyte-endothelial cell interaction. Increased expression of cell adhesion molecules contribute to the effects of endogenous testosterone promoting increased leukocyte rolling and adhesion in SHRs.