Arteries Of Hypertensive Rats Research Articles

Carvacrol inhibits TRPM7‐like current in native myocytes from mesenteric artery of Hypertensive Rats

Carvacrol, a phenolic monoterpene, is found in several plants of Origanum genus and has been described to cause vasodilatation in mesenteric artery from normotensive rats by reducing Ca2+ influx in vascular smooth muscle cells (VSMC). Then, the aim of this study was to investigate the effect of carvacrol on TRPM6/TRPM7 channels in VSMC from normotensive and hypertensive rats. Under whole‐cell voltage‐clamp, with voltage‐dependent Ca2+ and K+ channels blocked, currents generated by TRPM6/TRPM7 channels were measured by symmetrical voltage ramp commands to potentials between −100 to +100 mV, from a holding potential of 0 mV, displaying outward rectification in physiological extracellular medium. In VSMC isolated from hypertensive rats, carvacrol (100 and 300 μM) inhibit significantly both inward (−80 mV) and outward (+80 mV) currents [(current values at − 80 mV: (−3.42 ± 0.47 pA/pF; N = 5, control); (−1.07 ± 0.07 pA/pF, N = 5, p<0.05, carvacrol 100 μM); (−1.86 ± 0.34 pA/pF, N = 5, p<0.05, carvacrol 300 μM); (current values at + 80 mV: −6.21 ± 0.57 pA/pF, N = 5, control); (−3.53 ± 0.28 pA/pF, N = 5, p<0.01, carvacrol 100 μM); (−2.29 ± 0.27 pA/pF, N = 5, p<0.001, carvacrol 300 μM)]. This effect was similar to that induced by elevating extracellular Mg2+ (2.5 mM, N=5). In the presence of 2‐APB (100 μM, N=5), a TRPM7 blocker, no additional effect in TRPM7/TRPM6 whole‐cell current was observed after perfusion with carvacrol (300 μM; N=5), suggesting that probably, the effect of carvacrol is through inhibition of TRPM7‐like currents (ITRPM7) in VSMC. Additionally, since the sensitivity of TRPM7 channels to Mg2+ is an essential condition, we examined whether the above described current is inhibited by high concentrations of intracellular Mg2+. In this condition, the magnitude of TRPM7‐like current was significantly decreased inward and outward currents, in both myocytes, from hypertensive and normotensive rats, confirming that ITRPM were ITRPM6/TRPM7. In conclusion, these data demonstrate that carvacrol inhibited ITRPM6/TRPM7, in myocytes isolated from mesenteric artery of hypertensive and normotensive rats. This study provides the first electrophysiological evidence of TRPM7‐like current in native VSMC from mesenteric artery.Support or Funding InformationFundação de Amparo à pesquisa do estado da Bahia (FAPESB) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

TRPC3 channel confers cerebrovascular remodelling during hypertension via transactivation of EGF receptor signalling.

Ionic perturbation in vascular smooth muscle cells contributes to cerebrovascular remodelling in the setting of hypertension, but the role of transient receptor potential (TRP) channel superfamily remains unknown. The present study was conducted to define the contribution of TRP channels to cerebrovascular remodelling. By integrating quantitative PCR, western blotting, patch clamping, and Ca(2+) imaging, we identified TRP channel, subfamily canonical, member 3 (TRPC3) as the channel subtype most considerably elevated in basilar arteries of two-kidney, two-clip stroke-prone hypertensive rats. Importantly, administration of pyrazole 3 (Pyr3), a TRPC3 channel blocker, attenuated cerebrovascular remodelling. During hypertension, epidermal growth factor receptor (EGFR) was transactivated, as evidenced by marked EGFR phosphorylation, increased pro-HB-EGF shedding, and elevated activity of ADAM17 (HB-EGF sheddase). ADAM17 activity was increased owing to enhanced activation rather than elevated expression. Remarkably, Pyr3 treatment suppressed EGFR transactivation in hypertension. In proliferating basilar artery smooth muscle cells or basilar arteries of hypertensive rats, co-immunoprecipitation assay revealed an interaction between TRPC3 and ADAM17 upon Ang II stimulation. Collectively, we demonstrated that enhanced EGFR transactivation, due to increased TRPC3 expression and functional coupling of TRPC3/ADAM17, resulted in cerebrovascular remodelling. Therefore, TRPC3-induced EGFR transactivation may be therapeutically exploited to prevent hypertension-induced cerebrovascular remodelling.

Effect of magnesium supplementation on blood pressure and vascular reactivity in nitric oxide synthase inhibition-induced hypertension model

The aim of this study was to assess the effect of oral magnesium supplementation (Mg-supp) on blood pressure (BP) and possible mechanism in nitric oxide synthase (NOS) inhibition-induced hypertension model. Hypertension and/or Mg-supp were created by N-nitro-l-arginine methyl ester (25 mg/kg/day by drinking water) and magnesium-oxide (0.8% by diet) for 6 weeks. Systolic BP was measured weekly by tail-cuff method. The effects of hypertension and/or Mg-supp in thoracic aorta and third branch of mesenteric artery constriction and relaxation responses were evaluated. NOS-inhibition produced a gradually developing hypertension and the magnitude of the BP was significantly attenuated after five weeks of Mg-supp. The increased phenylephrine-induced contractile and decreased acetylcholine (ACh)-induced dilation responses were found in both artery segments of hypertensive groups. Mg-supp was restored ACh-relaxation response in both arterial segments and also Phe-constriction response in thoracic aorta but not in mesenteric arteries. The contributions of NO, prostaglandins and K+ channels to the dilator response of ACh were similar in the aorta of all the groups. The contribution of the NO to the ACh-mediated relaxation response of mesenteric arteries was suppressed in hypertensive rats, whereas this was corrected by Mg-supp. The flow-mediated dilation response of mesenteric arteries in hypertensive rats failed and could not be corrected by Mg-supp. Whereas, vascular eNOS protein and magnesium levels were not changed and plasma nitrite levels were reduced in hypertensive rats. The results of this study showed that Mg-supp lowered the arterial BP in NOS-inhibition induced hypertension model by restoring the agonist-induced relaxation response of the arteries.

0416 : Vascular smooth muscle cells are responsible for a prothrombotic phenotype of spontaneously hypertensive rat arteries

The hypothesis that hypertension confers a hypercoagulable state arises from the complications associated with hypertension, stroke and myocardial infarction. Our objective was to determine whether spontaneous hypertension causes changes in the thrombin generating capacity of the vascular wall. We used spontaneously hypertensive rats (SHR) compared with Wistar rats. The addition of thoracic aorta rings of SHR to a Wistar or SHR plasma pool resulted in a greater increase in thrombin generation compared to the addition of equivalent rings from Wistar. Comparison of 5 week-old and 12 week-old rats indicate that established hypertension is required to induce increased thrombin generation within the vessel wall. Whereas no difference was observed for endothelial cells, thrombin formation was higher at the surface of cultured aortic smooth muscle cells (SMCs) from SHR than from Wistar. Exposure of negatively-charged phospholipids was higher on SHR than on Wistar aortic rings as well as on SMCs. Tissue factor activity was higher in SHR SMCs. Twelve week-old SHR exhibited accelerated FeCl3-induced thrombus formation in carotid arteries and the resulting occlusive thrombi are disaggregated by blockade of glycoprotein Ibα-von Willebrand factor interactions. SHR SMCs were more sensitive to thrombininduced proliferation than Wistar SMCs. This cellular effect was totally abolished by a protease-activated receptor 1 inhibitor. The prothrombotic phenotype of the SHR vessel wall was due to the ability of SMCs to support greater thrombin generation and resulted in accelerated occlusive thrombus formation after arterial injury, which is sensitive to glycoprotein Ibα-von Willebrand factor inhibitors.

Tanshinone IIA Prevents Rat Basilar Artery Smooth Muscle Cells Proliferation by Inactivation of PDK1 During the Development of Hypertension.

Basilar vascular smooth muscle cells (BASMCs) hyperplasia is a prominent feature of cerebrovascular remodeling and stroke during the development of hypertension. Tanshinone IIA (Tan) has been reported to exhibit a protective effect against the pathological features of hypertension. Previous studies have shown that phosphoinostitide-3 kinase (PI3K)/3'-phosphoinostitide dependent kinase (PDK1)/AKT pathway is involved in the regulation of proliferation of various cell types. Therefore, there may be a crosstalk between Tan antihypertension processes and PI3K/PDK1/AKT proliferative effect in BASMCs. To test this hypothesis, we used a 2-kidney, 2-clip hypertension model to examine the effect of Tan on PI3K/PDK1/AKT pathway by cellular, molecular, and biochemical approaches. Our results revealed that the abundance of PDK1 in plasma was paralleled with an increase in blood pressure and the cross-sectional area of basilar artery in hypertensive rats. Tan decreased blood pressure and hypertension-induced PDK1 phosphorylation but produced no effect on the phosphorylation of PI3K. Moreover, Tan attenuated endothelin 1 induced the activation of PDK1/AKT pathway in rat BASMCs. Tan could inhibit cell cycle transition by regulating the expression of cyclin D1 and p27, in turn, prevent proliferation of BASMCs. Our study provides a novel mechanism by which Tan prevents cerebrovascular cell proliferation during hypertension, and thus Tan may be a potential therapeutic agent for cerebrovascular remodeling and stroke.

Vascular smooth muscle cells are responsible for a prothrombotic phenotype of spontaneously hypertensive rat arteries.

The hypothesis that hypertension induces a hypercoagulable state arises from the complications associated with hypertension: stroke and myocardial infarction. Here, we determine whether hypertension causes changes in the thrombin-generating capacity of the vascular wall. We used spontaneously hypertensive rats (SHR) compared with Wistar rats. The addition of thoracic aortic rings of SHR to a Wistar or SHR plasma pool resulted in a greater increase in thrombin generation compared with equivalent rings from Wistar. This increase occurred in 12- but not 5-week-old rats and was prevented by an angiotensin II-converting enzyme inhibitor, indicating that established hypertension is required to induce increased thrombin generation within the vessel wall. Whereas no difference was observed for endothelial cells, thrombin formation was higher on aortic smooth muscle cells (SMCs) from SHR than on those from Wistar. Exposure of negatively charged phospholipids was higher on SHR than on Wistar rings, as well as on cultured SMCs. Tissue factor activity was higher in SHR SMCs. Twelve-week-old SHR exhibited accelerated FeCl3-induced thrombus formation in carotid arteries, and the resulting occlusive thrombi were disaggregated by blockade of glycoprotein Ibα-von Willebrand factor interactions. SHR SMCs were more sensitive to thrombin-induced proliferation than Wistar SMCs. This effect was totally abolished by a protease-activated receptor 1 inhibitor. The prothrombotic phenotype of the SHR vessel wall was due to the ability of SMCs to support greater thrombin generation and resulted in accelerated occlusive thrombus formation after arterial injury, which was sensitive to glycoprotein Ibα-von Willebrand factor inhibitors.

Tumor necrosis factor-α inhibition attenuates middle cerebral artery remodeling but increases cerebral ischemic damage in hypertensive rats.

Hypertension causes vascular inflammation evidenced by an increase in perivascular macrophages and proinflammatory cytokines in the arterial wall. Perivascular macrophage depletion reduced tumor necrosis factor (TNF)-α expression in cerebral arteries of hypertensive rats and attenuated inward remodeling, suggesting that TNF-α might play a role in the remodeling process. We hypothesized that TNF-α inhibition would improve middle cerebral artery (MCA) structure and reduce damage after cerebral ischemia in hypertensive rats. Six-week-old male stroke-prone spontaneously hypertensive rats (SHRSP) were treated with the TNF-α inhibitor etanercept (ETN; 1.25 mg·kg(-1)·day(-1) ip daily) or PBS (equivolume) for 6 wk. The myogenic tone generation, postischemic dilation, and passive structure of MCAs were assessed by pressure myography. Cerebral ischemia was induced by MCA occlusion (MCAO). Myogenic tone was unchanged, but MCAs from SHRSP + ETN had larger passive lumen diameter and reduced wall thickness and wall-to-lumen ratio. Cerebral infarct size was increased in SHRSP + ETN after transient MCAO, despite an improvement in dilation of nonischemic MCA. The increase in infarct size was linked to a reduction in the number of microglia in the infarct core and upregulation of markers of classical macrophage/microglia polarization. There was no difference in infarct size after permanent MCAO or when untreated SHRSP subjected to transient MCAO were given ETN at reperfusion. Our data suggests that TNF-α inhibition attenuates hypertensive MCA remodeling but exacerbates cerebral damage following ischemia/reperfusion injury likely due to inhibition of the innate immune response of the brain.

Sequential effects of aerobic training upon muscular and renal arteries in hypertensive rats (706.8)

This study evaluated if aerobic training determine an arteriolar remodeling in locomotor and non‐locomotor skeletal muscle, heart and kidney of hypertensive (SHR) and normotensive (WKY) rats.Were used SHR and WKY male rats 10 weeks‐age submitted or not to aerobic training (AT, 1hr, 5 times/week, in treadmill). Rats were trained for 1, 2, 4, 8 and 12 weeks (55% of maximal capacity) or kept sedentary for 0 and 12 weeks (n=8/group). At the end of each time, mean arterial pressure (MAP) and heart rate (HR) were measured. Heart and locomotor (soleus, gastrocnemius) and non‐locomotor (temporalis) skeletal muscles (SM), heart and kidneys were harvested for arteriolar wall/lumen ratio measurements. Results were compared by ANOVA, using Bonferroni as post‐test. p蠄0.05 was considered as significant.Both SHR and WKY improved physical performance after 6 and 12 weeks of AT (S0:1.02±0.3 vs 1.62±0.4, T6:1.32±0.2 vs 2.09±0.3, T12:1.92±0.4 vs 2.40±0.4 km/h, WKY vs SHR). AT induced a rest bradycardia in WKY (326±4 to 294±3bpm), and in SHR (385±7 to 357±2bpm). Only SHR reduced MAP after AT (174±2 to 164±1mmHg). SHR showed increased arteriolar wall/lumen ratio compared to WKY (soleus: 0.19±0.02 vs 0.10±0.01µm; gastrocnemius red fibers (GRF): 0.20±0.02 vs 0.10±0.01µm; gastrocnemius white fibers (GWF): 0.21±0.01 vs 0.11±0.01µm; and temporalis: 0.19±0.01 vs 0.13±0.01µm; heart: 0.22±0.02 vs 0.12±0.02µm; kidney: 0.26±0.03 vs 0.15±0.01µm). On the other hands, AT decreased arteriolar wall/lumen ratio in SHR in locomotor SM (soleus: ‐47%; GRF: ‐50%; GWF: ‐52%), non‐locomotor SM (temporalis: ‐32%), and in heart ‐45%), with no changes in Kidney arterioles.The data indicates that AT induced beneficial vascular remodeling after hemodynamic adjusts, improving arteriolar wall/lumen ratio of SHR, and these modifications seems to be depending also of metabolic and systemic (hemodynamic and sympathetic) mechanisms.Grant Funding Source: Supported by FAPESP

Pathogenesis of focal cytoplasmic necrosis of the smooth muscle cells in hypertensive rat arterial media.

Hypertensive rat arteries exhibited severe medial smooth muscle cell injury and necrosis. Electron microscopic observations showed the smooth muscle cells of these arteries exhibited characteristics of focal cytoplasmic necrosis forming new cytodemarcating membrane between the healthy cytoplasm and necrotic cytoplasm. When the focal necrotic cytoplasm disappeared from the injured smooth muscle cells, it left it with a moth-eaten leaf-like appearance (moth-eaten necrosis). At an advanced stage of injury, smooth muscle cells changed to islet-like cell bodies with newly formed basement membranes around them, and further islet-like cell bodies and cell debris disappeared leaving lamellar and reticular basement membranes.In hypertensive rats injected with nitroblue tetrazolium (NBT), formazan deposits were observed in the medial cells and nitrotyrosine, a biomarker of peroxynitrite, were immunohistochemically observed in the arterial media. Nick-end positive extranuclear small granular bodies, which might have derived from focal necrotic cytoplasm and nucleus, were detected in the arterial media using DNA nick-end labeling method. Based on electron microscopical and histochemical findings, we conjectured that the focal cytoplasmic necrosis of the smooth muscle cells in the arterial media depended on injury arising from mitochondria-derived oxidants.

Perivascular macrophages mediate endothelium dysfunction in the middle cerebral artery of hypertensive rats

Hypertension increases macrophages (MΦ) around the cerebral arteries. These MΦ may release mediators that could modulate endothelial function. We hypothesized that MΦ depletion would improve middle cerebral artery (MCA) endothelial function in stroke‐prone spontaneously hypertensive rats (SHRSP). Liposome‐encapsulated clodronate (CLOD) was used to deplete MΦ, control rats received PBS containing liposomes; rats were treated from 6–12 weeks of age. Data are mean±SEM, PBS vs CLOD. Dilation data is shown for the highest concentration of the dilator. SHRSP+CLOD had less perivascular MΦ around the cerebral arteries (0.7±0.1 vs 0.3±0.1, n=5, p<0.01). Pressurized MCAs from SHRSP+CLOD showed improved dilation to ADP (change from baseline: 32.8±3.4 vs 50.8±9.7μm, n=6, p<0.05). Endothelial function was further assessed with acetylcholine (Ach)±N‐nitro‐L‐arginine methyl ester (L‐NAME) by wire myography (n=7). MCAs from SHRSP+CLOD had improved dilation to Ach (%KCl constriction: 64.8±5.0 vs 51.8±4.4, p<0.05), which was blunted by L‐NAME (%KCl constriction: 80.8±7.6 vs 79.8±5.0). Endothelium‐independent dilation to sodium nitroprusside was unchanged (%KCl constriction: 39.4±3.2 vs 43.1±2.1). These data suggest that perivascular MΦ might reduce endothelial nitric oxide generation in MCAs from SHRSP.

Involvement of Rab28 in NF-κB Nuclear Transport in Endothelial Cells

Our previous proteomic analysis revealed the expression of Rab28 in arteries of rats. However, the function of Rab28 in mammalian cells, and its role in vessels are still unknown. Coarctation of abdominal aorta above left kidney artery in rat was used as hypertensive animal model. FX-4000 cyclic strain loading system was used to mimic the mechanical condition on vascular cells during hypertension in vitro. Immunofluorescence and co-immunoprecipitation (Co-IP) were used to identify distribution and interaction of Rab28 and nuclear factor kappa B (NF-κB). Rab28 expression was significantly increased in carotid arteries of hypertensive rats. High cyclic strain induced Rab28 expression of endothelial cells (ECs) through a paracrine control of vascular smooth muscles cells (VSMCs), which at least partly via angiotensin II (Ang II). Rab28 knockdown decreased proliferation of ECs, while increased apoptosis and migration. Immunofluorescence revealed that Ang II stimulated the co-translocation of Rab28 and NF-κB from cytoplasm into nucleus. Knockdown of Rab28 attenuated NF-κB activation. Co-IP of NF-κB p65 and Rab28 indicated their interaction. Our results revealed that Rab28, as a novel regulator of NF-κB nuclear transport, might participate in the disturbance of EC homeostasis.

Transcriptional Upregulation of α 2 δ-1 Elevates Arterial Smooth Muscle Cell Voltage-Dependent Ca 2+ Channel Surface Expression and Cerebrovascular Constriction in Genetic Hypertension

A hallmark of hypertension is an increase in arterial myocyte voltage-dependent Ca 2+ (Ca V 1.2) currents that induces pathological vasoconstriction. Ca V 1.2 channels are heteromeric complexes composed of a pore-forming Ca V 1.2α 1 with auxiliary α 2 δ and β subunits. Molecular mechanisms that elevate Ca V 1.2 currents during hypertension and the potential contribution of Ca V 1.2 auxiliary subunits are unclear. Here, we investigated the pathological significance of α 2 δ subunits in vasoconstriction associated with hypertension. Age-dependent development of hypertension in spontaneously hypertensive rats was associated with an unequal elevation in α 2 δ-1 and Ca V 1.2α 1 mRNA and protein in cerebral artery myocytes, with α 2 δ-1 increasing more than Ca V 1.2α 1 . Other α 2 δ isoforms did not emerge in hypertension. Myocytes and arteries of hypertensive spontaneously hypertensive rats displayed higher surface-localized α 2 δ-1 and Ca V 1.2α 1 proteins, surface α 2 δ-1:Ca V 1.2α 1 ratio, Ca V 1.2 current density and noninactivating current, and pressure- and depolarization-induced vasoconstriction than those of Wistar-Kyoto controls. Pregabalin, an α 2 δ-1 ligand, did not alter α 2 δ-1 or Ca V 1.2α 1 total protein but normalized α 2 δ-1 and Ca V 1.2α 1 surface expression, surface α 2 δ-1:Ca V 1.2α 1 , Ca V 1.2 current density and inactivation, and vasoconstriction in myocytes and arteries of hypertensive rats to control levels. Genetic hypertension is associated with an elevation in α 2 δ-1 expression that promotes surface trafficking of Ca V 1.2 channels in cerebral artery myocytes. This leads to an increase in Ca V 1.2 current-density and a reduction in current inactivation that induces vasoconstriction. Data also suggest that α 2 δ-1 targeting is a novel strategy that may be used to reverse pathological Ca V 1.2 channel trafficking to induce cerebrovascular dilation in hypertension.

Impaired flow-induced arterial remodeling in DOCA-salt hypertensive rats

Arteries from young healthy animals respond to chronic changes in blood flow and blood pressure by structural remodeling. We tested whether the ability to respond to decreased (-90%) or increased (+100%) blood flow is impaired during the development of deoxycorticosterone acetate (DOCA)-salt hypertension in rats, a model for an upregulated endothelin-1 system. Mesenteric small arteries (MrA) were exposed to low blood flow (LF) or high blood flow (HF) for 4 or 7 weeks. The bioavailability of vasoactive peptides was modified by chronic treatment of the rats with the dual neutral endopeptidase (NEP)/endothelin-converting enzyme (ECE) inhibitor SOL1. After 3 or 6 weeks of hypertension, the MrA showed hypertrophic arterial remodeling (3 weeks: media cross-sectional area (mCSA): 10±1 × 10(3) to 17±2 × 10(3) μm(2); 6 weeks: 13±2 × 10(3) to 24±3 × 10(3) μm(2)). After 3, but not 6, weeks of hypertension, the arterial diameter was increased (Ø: 385±13 to 463±14 μm). SOL1 reduced hypertrophy after 3 weeks of hypertension (mCSA: 6 × 10(3)±1 × 10(3) μm(2)). The diameter of the HF arteries of normotensive rats increased (Ø: 463±22 μm) but no expansion occurred in the HF arteries of hypertensive rats (Ø: 471±16 μm). MrA from SOL1-treated hypertensive rats did show a significant diameter increase (Ø: 419±13 to 475±16 μm). Arteries exposed to LF showed inward remodeling in normotensive and hypertensive rats (mean Ø between 235 and 290 μm), and infiltration of monocyte/macrophages. SOL1 treatment did not affect the arterial diameter of LF arteries but reduced the infiltration of monocyte/macrophages. We show for the first time that flow-induced remodeling is impaired during the development of DOCA-salt hypertension and that this can be prevented by chronic NEP/ECE inhibition.

AMP-activated protein kinase activator AICAR acutely lowers blood pressure and relaxes isolated resistance arteries of hypertensive rats

AMP-activated protein kinase (AMPK) activity may alter blood pressure by directly influencing vascular tone. The purpose of this study is to examine if these effects occur acutely in a model of hypertension. Using distinct groups of Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) we compare baseline aortic and mesenteric artery AMPK activation (by immunoblotting), hemodynamic (blood pressure and heart rate via carotid catheter) and biochemical responses to an acute injection of AMPK activator 5-aminoimidazole-4-carboxyamide-1-β-D-ribofuranoside (AICAR) in vivo and vasomotor responses of isolated mesenteric vessels to AICAR exposure in vitro using myography. Mean arterial pressure (MAP) decreased from 196 ± 3 to 122 ± 15 mmHg (P < 0.001) during the 30 min following AICAR injection in SHR (an effect partially prevented by NOS inhibitor L-NAME), but in WKY MAP was unaffected by AICAR. Basal AMPK activation (phosphorylation of AMPK activation site threonine 172) was reduced by approximately 50% in aorta of SHR vs. WKY (0.49 ± 0.1 vs. 1.0 ± 0.1 arbitrary units, P < 0.001), and was improved approximately 1.6-fold in SHR but not in WKY aorta 30 min following AICAR injection. In isolated vessel experiments, dose-dependent vasorelaxation to AICAR was similar in mesenteric arteries of SHR and WKY, although responses were more reliant on nitric oxide in SHR vs. WKY. The ability of AICAR to improve vascular AMPK activation, and to generate parallel reductions in blood pressure and relaxation of SHR resistance vasculature, highlights the potential importance of AMPK in the regulation of blood pressure and vascular tone.

RhoA guanine exchange factor expression profile in arteries: evidence for a Rho kinase-dependent negative feedback in angiotensin II-dependent hypertension

Sustained overactivation of RhoA is a common component for the pathogenesis of several cardiovascular disorders, including hypertension. Although activity of Rho proteins depends on Rho exchange factors (Rho-GEFs), the identity of Rho-GEFs expressed in vascular smooth muscle cells (VSMC) and participating in the control of Rho protein activity and Rho-dependent functions remains unknown. To address this question, we analyzed by quantitative RT-PCR the expression profile of 28 RhoA-GEFs in arteries of normotensive (saline-treated) and hypertensive (ANG II-treated) rats. Sixteen RhoA-GEFs were downregulated in mesenteric arteries of hypertensive rats, among which nine are also downregulated in cultured VSMC stimulated by ANG II (100 nM, 48 h), suggesting a direct effect of ANG II. Inhibition of type 1 ANG II receptors (losartan, 1 μM) or Rho kinase (fasudil, 10 μM) prevented ANG II-induced RhoA-GEF downregulation. Functionally, ANG II-induced downregulation of RhoA-GEFs is associated with decreased Rho kinase activation in response to endothelin-1, norepinephrine, and U-46619. This work thus identifies a group of RhoA-GEFs that controls RhoA and RhoA-dependent functions in VSMC, and a negative feedback of RhoA/Rho kinase activity on the expression of these RhoA-GEFs that may play an adaptative role to limit RhoA/Rho kinase activation.

L-Arginine restores endothelial nitric oxide synthase-coupled activity and attenuates monocrotaline-induced pulmonary artery hypertension in rats

L-arginine can attenuate pulmonary hypertension (PH) by a mechanism that are not fully understood. This study investigated the molecule mechanism of L-arginine attenuating PH. Sprague Dawley rats were treated with monocrotaline (MCT) with or without L-arginine for 3 or 5 wk. Right ventricular systolic pressure (RVSP), right heart hypertrophy, survival rate, pulmonary artery wall thickness, nitric oxide (NO) concentration, and superoxide anion (O(2)(*-)) generation in the lung were measured. Expressions of endothelial nitric oxide synthase (eNOS) and heat shock protein 90 (HSP90), phosphorylation of eNOS at Ser(1177), and the association of eNOS and HSP90 in the lung were determined by Western blot and immunoprecipitation experiments. MCT increased RVSP, right heart hypertrophy, mortality, pulmonary artery wall thickness, and O(2)(*-) generation and decreased eNOS and HSP90 expression and association, phosphorylation of eNOS at Ser(1177), and NO production. L-arginine decreased RVSP, right heart hypertrophy, mortality, O(2)(*-) generation, and pulmonary artery wall thickness and increased NO production. L-arginine increased eNOS expression, phosphorylation of eNOS at Ser(1177), and association of eNOS and HSP90 without significantly altering HSP90 expression. L-arginine may act through three pathways, providing a substrate for NO generation, preserving eNOS expression/phosphorylation, and maintaining the association of eNOS and HSP90, which allows restoration of eNOS activity and coupling activity, to maintain the balance between NO and O(2)(*-) and delay the development of PH.

Gene Delivery of Cytochrome P450 Epoxygenase Ameliorates Monocrotaline-Induced Pulmonary Artery Hypertension in Rats

Pulmonary arterial hypertension (PAH) is a life-threatening disease that leads to progressive pulmonary hypertension, right heart failure, and death. Endothelial dysfunction and inflammation were implicated in the pathogenesis of PAH. Epoxyeicosatrienoic acids (EETs), products of the cytochrome P450 epoxygenase metabolism of arachidonic acid, are potent vasodilators that possess anti-inflammatory and other protective properties in endothelial cells. We investigated whether gene delivery with the human cytochrome P450 epoxygenase 2J2 (CYP2J2) ameliorates monocrotaline (MCT)-induced pulmonary hypertension in rats. Significant pulmonary hypertension developed 3 weeks after the administration of MCT, but gene therapy with CYP2J2 significantly attenuated the development of pulmonary hypertension and pulmonary vascular remodeling, without causing changes in systemic arterial pressure or heart rate. These effects were associated with increased pulmonary endothelial NO synthase (eNOS) expression and its activity, inhibition of inflammation in the lungs, and transforming growth factor (TGF)-β/type II bone morphogenetic protein receptor (BMPRII)-drosophila mothers against decapentaplegic proteins (Smads) signaling. Collectively, these data suggest that gene therapy with CYP2J2 may have potential as a novel therapeutic approach to this progressive and oftentimes lethal disorder.

Loss of cerebrovascular Shaker-type K+ channels: a shared vasodilator defect of genetic and renal hypertensive rats

The cerebral arteries of hypertensive rats are depolarized and highly myogenic, suggesting a loss of K(+) channels in the vascular smooth muscle cells (VSMCs). The present study evaluated whether the dilator function of the prominent Shaker-type voltage-gated K(+) (K(V)1) channels is attenuated in middle cerebral arteries from two rat models of hypertension. Block of K(V)1 channels by correolide (1 micromol/l) or psora-4 (100 nmol/l) reduced the resting diameter of pressurized (80 mmHg) cerebral arteries from normotensive rats by an average of 28 +/- 3% or 26 +/- 3%, respectively. In contrast, arteries from spontaneously hypertensive rats (SHR) and aortic-banded (Ao-B) rats with chronic hypertension showed enhanced Ca(2+)-dependent tone and failed to significantly constrict to correolide or psora-4, implying a loss of K(V)1 channel-mediated vasodilation. Patch-clamp studies in the VSMCs of SHR confirmed that the peak K(+) current density attributed to K(V)1 channels averaged only 5.47 +/- 1.03 pA/pF, compared with 9.58 +/- 0.82 pA/pF in VSMCs of control Wistar-Kyoto rats. Subsequently, Western blots revealed a 49 +/- 7% to 66 +/- 7% loss of the pore-forming alpha(1.2)- and alpha(1.5)-subunits that compose K(V)1 channels in cerebral arteries of SHR and Ao-B rats compared with control animals. In each case, the deficiency of K(V)1 channels was associated with reduced mRNA levels encoding either or both alpha-subunits. Collectively, these findings demonstrate that a deficit of alpha(1.2)- and alpha(1.5)-subunits results in a reduced contribution of K(V)1 channels to the resting diameters of cerebral arteries from two rat models of hypertension that originate from different etiologies.

Liver growth factor antifibrotic activity in vivo is associated with a decrease in activation of hepatic stellate cells.

The antifibrotic activity of Liver Growth Factor (LGF), a liver mitogen, was previously demonstrated in several models of rat liver fibrosis and even in extrahepatic sites, such as carotid artery in hypertensive rats and rat CdCl2-induced lung fibrosis. In the present study, we have attempted to examine in depth its mechanism of antifibrotic action in bile duct-ligated (BDL) rats, with special emphasis on its activity in fibrogenic liver cells. BDL rats received either LGF 9 microg/week for 2 or 3 weeks (BDL+LGF, n=20/group) or saline (BDL+S, n=20/group), at times 0, week 2, or week 5 after operation. Groups were compared in terms of liver alpha-smooth muscle actin (SMA) content (western blotting and immunohistochemistry), hepatic apoptosis, liver desmin content (western blotting), and serum endothelin-1 (ELISA). LGF produced a marked decrease in liver alpha-SMA content compared with saline-injected rats, especially evident at longer times (5w and 8w; p<0.05). Moreover, LGF did not seem to influence HSC proliferation, as shown by measuring liver desmin content. The antifibrotic activity exerted by LGF seems to be closely related to a modulation of the activation state of fibrogenic liver cells (activated HSC and myofibroblasts) in BDL rats.

INVOLVEMENT OF PROLYLCARBOXYPEPTIDASE IN THE EFFECT OF RUTAECARPINE ON THE REGRESSION OF MESENTERIC ARTERY HYPERTROPHY IN RENOVASCULAR HYPERTENSIVE RATS

1. Previous studies indicate that rutaecarpine blocks increases in blood pressure and inhibits vascular hypertrophy in experimentally hypertensive rats. The aim of the present study was to determine whether the effects of rutaecarpine are related to activation of prolylcarboxypeptidase (PRCP). 2. Renovascular hypertensive rats (Goldblatt two-kidney, one-clip (2K1C)) were developed using male Sprague-Dawley rats. Chronic treatment with rutaecarpine (10 or 40 mg/kg per day) or losartan (20 mg/kg per day) for 4 weeks to the hypertensive rats caused a sustained dose-dependent attenuation of increases in blood pressure, increased lumen diameter and decreased media thickness, which was accompanied by a similar reduction in the media cross-sectional area : lumen area ratio in mesenteric arteries compared with untreated hypertensive rats. 3. Angiotensin (Ang) II expression was significantly increased in mesenteric arteries of hypertensive rats compared with sham-operated rats. No significant differences in plasma AngII levels were observed between untreated hypertensive and sham-operated rats. Hypertensive rats treated with high-dose rutaecarpine had significantly decreased Ang II levels in both the plasma and mesenteric arteries. 4. Expression of PRCP protein or kallikrein mRNA was significantly inhibited in the right kidneys and mesenteric arteries of hypertensive rats. However, expression of PRCP protein and kallikrein mRNA was significantly increased after treatment with rutaecarpine or losartan (20 mg/kg per day). 5. The data suggest that the repression of increases in systolic blood pressure and reversal of mesenteric artery remodelling by rutaecarpine may be related to increased expression of PRCP in the circulation and small arteries in 2K1C hypertensive rats.