Fernanda R.C. Giachini

Toll-like receptor 4 contributes to blood pressure regulation and vascular contraction in spontaneously hypertensive rats

Activation of Toll-like receptors (TLR) induces gene expression of proteins involved in the immune system response. TLR4 has been implicated in the development and progression of cardiovascular diseases. Innate and adaptive immunity contribute to hypertension-associated end-organ damage, although the mechanism by which this occurs remains unclear. In the present study we hypothesize that inhibition of TLR4 decreases blood pressure and improves vascular contractility in resistance arteries from spontaneously hypertensive rats (SHR). TLR4 protein expression in mesenteric resistance arteries was higher in 15 weeks-old SHR than in same age Wistar controls or in 5 weeks-old SHR. In order to decrease activation of TLR4, 15 weeks-old SHR and Wistar rats were treated with anti-TLR4 antibody or non-specific IgG control antibody for 15 days (1µg per day, i.p.). Treatment with anti-TLR4 decreased mean arterial pressure as well as TLR4 protein expression in mesenteric resistance arteries and interleukin-6 (IL-6) serum levels from SHR when compared to SHR treated with IgG. No changes in these parameters were found in Wistar treated rats. Mesenteric resistance arteries from anti-TLR4-treated SHR exhibited decreased maximal contractile response to noradrenaline compared to IgG-treated-SHR. Inhibition of cyclooxygenase-1 (Cox) and Cox-2, enzymes related to inflammatory pathways, decreased noradrenaline responses only in mesenteric resistance arteries of SHR treated with IgG. Cox-2 expression and thromboxane A2 release were decreased in SHR treated with anti-TLR4 compared with IgG-treated-SHR. Our results suggest that TLR4 activation contributes to increased blood pressure, low grade inflammation and plays a role in the augmented vascular contractility displayed by SHR.

Cigarette Smoking and Erectile Dysfunction: Focus on NO Bioavailability and ROS Generation

INTRODUCTION Thirty million men in the United States suffer from erectile dysfunction (ED) and this number is expected to double by 2025. Considered a major public health problem, which seriously affects the quality of life of patients and their partners, ED becomes increasingly prevalent with age and chronic smoking is a major risk factor in the development of ED. AIM To review available evidence concerning the effects of cigarette smoking on vascular changes associated with decreased nitric oxide (NO) bioavailability and increased reactive oxygen species (ROS) generation. METHODS We examined epidemiological and clinical data linking cigarette smoking and ED, and the effects of smoking on vascular NO bioavailability and ROS generation. MAIN OUTCOME MEASURES There are strong parallels between smoking and ED and considerable evidence supporting the concept that smoking-related ED is associated with reduced bioavailability of NO because of increased ROS. RESULTS Cigarette smoking-induced ED in human and animal models is associated with impaired arterial flow to the penis or acute vasospasm of the penile arteries. Long-term smoking produces detrimental effects on the vascular endothelium and peripheral nerves and also causes ultrastructural damage to the corporal tissue, all considered to play a role in chronic smoking-induced ED. Clinical and basic science studies provide strong indirect evidence that smoking may affect penile erection by the impairment of endothelium-dependent smooth muscle relaxation or more specifically by affecting NO production via increased ROS generation. Whether nicotine or other products of cigarette smoke mediate all effects related to vascular damage is still unknown. CONCLUSIONS Smoking prevention represents an important approach for reducing the risk of ED. The characterization of the components of cigarette smoke leading to ED and the mechanisms by which these components alter signaling pathways activated in erectile responses are necessary for a complete comprehension of cigarette smoking-associated ED.

Increased activation of stromal interaction molecule-1/Orai-1 in aorta from hypertensive rats: a novel insight into vascular dysfunction.

Disturbances in the regulation of cytosolic calcium (Ca2+) concentration play a key role in the vascular dysfunction associated with arterial hypertension. Stromal interaction molecules (STIMs) and Orai proteins represent a novel mechanism to control store-operated Ca2+ entry. Although STIMs act as Ca2+ sensors for the intracellular Ca2+ stores, Orai is the putative pore-forming component of Ca2+ release–activated Ca2+ channels at the plasma membrane. We hypothesized that augmented activation of Ca2+ release–activated Ca2+/Orai-1, through enhanced activity of STIM-1, plays a role in increased basal tonus and vascular reactivity in hypertensive animals. Endothelium-denuded aortic rings from Wistar-Kyoto and stroke-prone spontaneously hypertensive rats were used to evaluate contractions because of Ca2+ influx. Depletion of intracellular Ca2+ stores, which induces Ca2+ release–activated Ca2+ activation, was performed by placing arteries in Ca2+ free-EGTA buffer. The addition of the Ca2+ regular buffer produced greater contractions in aortas from stroke-prone spontaneously hypertensive rats versus Wistar-Kyoto rats. Thapsigargin (10 &mgr;mol/L), an inhibitor of the sarcoplasmic reticulum Ca2+ ATPase, further increased these contractions, especially in stroke-prone spontaneously hypertensive rat aorta. Addition of the Ca2+ release–activated Ca2+ channel inhibitors 2-aminoethoxydiphenyl borate (100 &mgr;mol/L) or gadolinium (100 &mgr;mol/L), as well as neutralizing antibodies to STIM-1 or Orai-1, abolished thapsigargin-increased contraction and the differences in spontaneous tone between the groups. Expression of Orai-1 and STIM-1 proteins was increased in aorta from stroke-prone spontaneously hypertensive rats when compared with Wistar-Kyoto rats. These results support the hypothesis that both Orai-1 and STIM-1 contribute to abnormal vascular function in hypertension. Augmented activation of STIM-1/Orai-1 may represent the mechanism that leads to impaired control of intracellular Ca2+ levels in hypertension.

PYK2/PDZ-RhoGEF Links Ca2+ Signaling to RhoA

Objective—Ras homolog gene family member A (RhoA)/Rho-kinase-mediated Ca2+ sensitization is a critical component of constrictor responses. The present study investigates how angiotensin II activates RhoA. Methods and Results—Adenoviral vectors were used to manipulate the expression of regulator of G protein signaling (RGS) domain containing Rho-specific guanine exchange factors (RhoGEFs) and proline-rich tyrosine kinase 2 (PYK2), a nonreceptor tyrosine kinase, in primary rat vascular smooth muscle cells. As an evidence of RhoA activation, RhoA translocation and MYPT1 (the regulatory subunit of myosin light chain phosphatase) phosphorylation were analyzed by Western blot. Results showed that overexpression of PDZ-RhoGEF, but not p115-RhoGEF or leukemia-associated RhoGEF (LARG), enhanced RhoA activation by angiotensin II. Knockdown of PDZ-RhoGEF decreased RhoA activation by angiotensin II. PDZ-RhoGEF was phosphorylated and activated by PYK2 in vitro, and knockdown of PDZ-RhoGEF reduced RhoA activation by constitutively active PYK2, indicating that PDZ-RhoGEF links PYK2 to RhoA. Knockdown of PYK2 or PDZ-RhoGEF markedly decreased RhoA activation by A23187, a Ca2+ ionophore, demonstrating that PYK2/PDZ-RhoGEF couples RhoA activation to Ca2+. Conclusions—PYK2 and PDZ-RhoGEF are necessary for angiotensin II–induced RhoA activation and for Ca2+ signaling to RhoA.

Impaired Vasodilator Activity in Deoxycorticosterone Acetate-Salt Hypertension Is Associated With Increased Protein O-GlcNAcylation

Hyperglycemia, which increases O-linked &bgr;-N-acetylglucosamine (O-GlcNAc) proteins, leads to changes in vascular reactivity. Because vascular dysfunction is a key feature of arterial hypertension, we hypothesized that vessels from deoxycorticosterone acetate and salt (DOCA-salt) rats exhibit increased O-GlcNAc proteins, which is associated with increased reactivity to constrictor stimuli. Aortas from DOCA rats exhibited increased contraction to phenylephrine (Emax [mN]=17.6±4 versus 10.7±2 control; n=6) and decreased relaxation to acetylcholine (47.6±6% versus 73.2±10% control; n=8) versus arteries from uninephrectomized rats. O-GlcNAc protein content was increased in aortas from DOCA rats (arbitrary units=3.8±0.3 versus 2.3±0.3 control; n=5). PugNAc (O-GlcNAcase inhibitor; 100 &mgr;mol/L; 24 hours) increased vascular O-GlcNAc proteins, augmented phenylephrine vascular reactivity (18.2±2 versus 10.7±3 vehicle; n=6), and decreased acetylcholine dilation in uninephrectomized (41.4±6 versus 73.2±3 vehicle; n=6) but not in DOCA rats (phenylephrine, 16.5±3 versus 18.6±3 vehicle, n=6; acetylcholine, 44.7±8 versus 47.6±7 vehicle, n=6). PugNAc did not change total vascular endothelial nitric oxide synthase levels, but reduced endothelial nitric oxide synthaseSer-1177 and AktSer-473 phosphorylation (P<0.05). Aortas from DOCA rats also exhibited decreased levels of endothelial nitric oxide synthaseSer-1177 and AktSer-473 (P<0.05) but no changes in total endothelial nitric oxide synthase or Akt. Vascular O-GlcNAc–modified endothelial nitric oxide synthase was increased in DOCA rats. Blood glucose was similar in DOCA and uninephrectomized rats. Expression of O-GlcNAc transferase, glutamine:fructose-6-phosphate amidotransferase, and O-GlcNAcase, enzymes that directly modulate O-GlcNAcylation, was decreased in arteries from DOCA rats (P<0.05). This is the first study showing that O-GlcNAcylation modulates vascular reactivity in normoglycemic conditions and that vascular O-GlcNAc proteins are increased in DOCA-salt hypertension. Modulation of increased vascular O-GlcNAcylation may represent a novel therapeutic approach in mineralocorticoid hypertension.

Activation of the ET-1/ETA pathway contributes to erectile dysfunction associated with mineralocorticoid hypertension.

INTRODUCTION The cavernosal tissue is highly responsive to endothelin-1 (ET-1), and penile smooth muscle cells not only respond to but also synthesize ET-1. AIM Considering that ET-1 is directly involved in end-organ damage in salt-sensitive forms of hypertension, we hypothesized that activation of the ET-1/ET(A) receptor pathway contributes to erectile dysfunction (ED) associated with mineralocorticoid hypertension. METHODS Wistar rats were uninephrectomized and submitted to deoxycorticosterone acetate (DOCA)-salt treatment for 5 weeks. Control (Uni [uninephrectomized control]) animals were uninephrectomized and given tap water. Uni and DOCA-salt rats were simultaneously treated with vehicle or atrasentan (ET(A) receptor antagonist, 5 mg/Kg/day). Cavernosal reactivity to ET-1, phenylephrine (PE), ET(B) receptor agonist (IRL-1620) and electric field stimulation (EFS) were evaluated in vitro. Expression of ROCKalpha, ROCKbeta, myosin phosphatase target subunit 1 (MYPT-1), and extracellular signal-regulated kinase 1/2 (ERK 1/2) were evaluated by western blot analysis. ET-1 and ET(A) receptor mRNA expression was evaluated by real-time reverse-transcriptase polymerase chain reaction. Voltage-dependent increase in intracavernosal pressure/mean arterial pressure (ICP/MAP) was used to evaluate erectile function in vivo. MAIN OUTCOME MEASURE ET(A) receptor blockade prevents DOCA-salt-associated ED. RESULTS Cavernosal strips from DOCA-salt rats displayed augmented preproET-1 expression, increased contractile responses to ET-1 and decreased relaxation to IRL-1620. Contractile responses induced by EFS and PE were enhanced in cavernosal tissues from DOCA-salt hypertensive rats. These functional changes were associated with increased activation of the RhoA/Rho-kinase and ERK 1/2 pathways. Treatment of rats with atrasentan completely prevented changes in cavernosal reactivity in DOCA-salt rats and restored the decreased ICP/MAP, completely preventing ED in DOCA-salt rats. CONCLUSION Activation of the ET-1/ET(A) pathway contributes to mineralocorticoid hypertension-associated ED. ET(A) receptor blockade may represent an alternative therapeutic approach for ED associated with salt-sensitive hypertension and in pathological conditions where increased levels of ET-1 are present.

Upregulation of intermediate calcium-activated potassium channels counterbalance the impaired endothelium-dependent vasodilation in stroke-prone spontaneously hypertensive rats

Endothelial dysfunction has been linked to a decrease in nitric oxide (NO) bioavailability and attenuated endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation. The small (SK(Ca)) and intermediate (IK(Ca)) calcium-activated potassium channels play a key role in endothelium-dependent relaxation. Because the repressor element 1-silencing transcription factor (REST) negatively regulates IK(Ca) expression, we hypothesized that augmented REST and decreased IK(Ca) expression contributes to impaired endothelium-dependent vasodilation associated with hypertension. Acetylcholine (ACh) responses were slightly decreased in small mesenteric arteries from male stroke-prone spontaneously hypertensive rats (SHRSPs) versus arteries from Wistar Kyoto (WKY) rats. Incubation with N-nitro-L-arginine methyl ester (L-NAME; 100mumol/L) and indomethacin (100mumol/L) greatly impaired ACh responses in vessels from SHRSP. Iberiotoxin (0.1mumol/L), which is a selective inhibitor of large-conductance K(Ca) (BK(Ca)) channels, did not modify EDHF-mediated vasodilation in SHRSP or WKY. UCL-1684 (0.1mumol/L), which is a selective inhibitor of SKCa channels, almost abolished EDHF-mediated vasodilation in WKY and decreased relaxation in SHRSP. 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; 10mumol/L) and charybdotoxin (0.1mumol/L), which are both IKCa inhibitors, produced a small decrease of EDHF relaxation in WKY but completely abrogated EDHF vasodilation in SHRSP. EDHF-mediated relaxant responses were completely abolished in both groups by simultaneous treatment with UCL-1684 and TRAM-34 or charybdotoxin. Relaxation to SK(Ca)/IK(Ca) channels agonist NS-309 was decreased in SHRSP arteries. The expression of SK(Ca) was decreased, whereas IK(Ca) was increased in SHRSP mesenteric arteries. REST expression was reduced in arteries from SHRSP. Vessels incubated with TRAM-34 (10mumol/L) for 24h displayed reduced REST expression and demonstrated no differences in IK(Ca). In conclusion, IK(Ca) channel upregulation, via decreased REST, seems to compensate deficient activity of SK(Ca) channels in the vasculature of spontaneously hypertensive rats.

O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway

AIMS Glycosylation with β-N-acetylglucosamine (O-GlcNAcylation) is one of the most complex post-translational modifications. The cycling of O-GlcNAc is controlled by two enzymes: UDP-NAc transferase (OGT) and O-GlcNAcase (OGA). We recently reported that endothelin-1 (ET-1) augments vascular levels of O-GlcNAcylated proteins. Here we tested the hypothesis that O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of the RhoA/Rho-kinase pathway. METHODS AND RESULTS Incubation of vascular smooth muscle cells (VSMCs) with ET-1 (0.1 μM) produces a time-dependent increase in O-GlcNAc levels. ET-1-induced O-GlcNAcylation is not observed when VSMCs are previously transfected with OGT siRNA, treated with ST045849 (OGT inhibitor) or atrasentan (ET(A) antagonist). ET-1 as well as PugNAc (OGA inhibitor) augmented contractions to phenylephrine in endothelium-denuded rat aortas, an effect that was abolished by the Rho kinase inhibitor Y-27632. Incubation of VSMCs with ET-1 increased expression of the phosphorylated forms of myosin phosphatase target subunit 1 (MYPT-1), protein kinase C-potentiated protein phosphatase 1 inhibitor protein (protein kinase C-potentiated phosphatase inhibitor-17), and myosin light chain (MLC) and RhoA expression and activity, and this effect was abolished by both OGT siRNA transfection or OGT inhibition and atrasentan. ET-1 also augmented expression of PDZ-Rho GEF (guanine nucleotide exchange factor) and p115-Rho GEF in VSMCs and this was prevented by OGT siRNA, ST045849, and atrasentan. CONCLUSION We suggest that ET-1 augments O-GlcNAcylation and this modification contributes to increased vascular contractile responses via activation of the RhoA/Rho-kinase pathway.

Determination of Adenosine Effects and Adenosine Receptors in Murine Corpus Cavernosum

This study tested the hypothesis that adenosine, in murine corpora cavernosa, produces direct relaxation of smooth muscle cells and inhibition of contractile responses mediated by sympathetic nerve stimulation. Penes were excised from anesthetized male C57BL/6 mice, dissected, and cavernosal strips were mounted to record isometric force. Adenosine, 2-chloroadenosine (stable analog of adenosine), and 2-phenylaminoadenosine (CV1808) (A2A/A2B agonist) produced concentration-dependent relaxations of phenylephrine-contracted tissues. Relaxation to 2-chloroadenosine was inhibited, in a concentration-dependent manner, by 2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH58261; A2A antagonist; 10–9–10–6 M) and N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamida (MRS1706; A2B antagonist; 10–8–10–6 M). The combination of both antagonists abrogated 2-chloroadenosine-induced relaxation. Electrical field stimulation (EFS; 1–32 Hz) of adrenergic nerves produced frequency-dependent contractions that were inhibited by compounds that increase adenosine levels, such as 5′-iodotubercidin (adenosine kinase inhibitor), erythro-9-(2-hydroxy-3-nonyl)adenine (adenosine deaminase inhibitor), and dipyridamole (inhibitor of adenosine transport). The adenosine A1 receptor agonist N6-cyclopentyladenosine (C8031) right-shifted contractile responses to EFS, with a significant inhibitory effect at 10–6 M. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine (C101) (10–7 M) enhanced contractile responses to EFS and eliminated the inhibitory effects of 5′-iodotubercidin. Dipyridamole and 5′-iodotubercidin had no effect on adenosine-mediated relaxation. In summary, adenosine directly relaxes cavernosal smooth muscle cells, by the activation of A2A/A2B receptor subtypes. In addition, adenosine negatively modulates sympathetic neurotransmission, by A1 receptor subtype activation, in murine corpora cavernosa. Adenosine may subserve dual roles in modulating the physiological mechanisms of erection in mice.

Extracellular Signal–Regulated Kinase 1/2 Activation, via Downregulation of Mitogen-Activated Protein Kinase Phosphatase 1, Mediates Sex Differences in Desoxycorticosterone Acetate-Salt Hypertension Vascular Reactivity

Extracellular signal–regulated kinase (ERK)1/2 has been reported to play a role in vascular dysfunction associated with mineralocorticoid hypertension. We hypothesized that, compared with female rats, an upregulation of ERK1/2 signaling in the vasculature of male rats contributes to augmented contractile responses in mineralocorticoid hypertension. Uninephrectomized male and female Sprague-Dawley rats received desoxycorticosterone acetate (DOCA) pellets (200 mg per animal) and saline to drink for 3 weeks. Control uninephrectomized rats received tap water to drink. Blood pressure, measured by telemetry, was significantly higher in male DOCA rats (191±3 mm Hg) compared with female DOCA rats (172±7 mm Hg; n=5). DOCA treatment resulted in augmented contractile responses to phenylephrine in aorta (22±3 mN; n=6) and small mesenteric arteries (13±2 mN; n=6) from male DOCA rats versus uninephrectomized male rats (16±3 and 10±2 mN, respectively; P<0.05) and female DOCA rats (15±1 and 11±1 mN, respectively). ERK1/2 inhibition with PD-98059 (10 &mgr;mol/L) abrogated increased contraction to phenylephrine in aorta (14±2 mN) and small mesenteric arteries (10±2 mN) from male DOCA rats, without any effects in arteries from male uninephrectomized or female animals. Compared with the other groups, phosphorylated ERK1/2 levels were increased in the aorta from male DOCA rats, whereas mitogen-activated protein kinase phosphatase 1 expression was decreased. Interleukin-10 plasma levels, which positively regulate mitogen-activated protein kinase phosphatase 1 activity, were reduced in male DOCA-salt rats. We speculate that augmented vascular reactivity in male hypertensive rats is mediated via activation of the ERK1/2 pathway. In addition, mitogen-activated protein kinase phosphatase 1 and interleukin 10 play regulatory roles in this process.