2K-1C Rat Aortas Research Articles

Sarcoplasmic reticulum calcium ATPase (SERCA) proteolysis by matrix metalloproteinase-2 contributes to vascular dysfunction in early hypertension

AimsHypertension is associated with an increased activity of matrix metalloproteinase (MMP)-2 in the vasculature, which, in turn, proteolyzes extra- and intracellular proteins that lead to vascular dysfunction. The activity of sarcoplasmic reticulum calcium ATPase (SERCA) is decreased in the aortas of hypertensive rats. Increased activity of MMP-2 proteolyzed SERCA in rat heart during ischemia and reperfusion injury, thus impairing cardiac function. Therefore, we examined whether increased activity of MMP-2 in early hypertension contributes to proteolyze SERCA in the aortas, thus leading to maladaptive vascular remodeling and dysfunction. Main methodsMale Sprague-Dawley rats were submitted to two kidney-one clip (2K-1C) or Sham surgery and treated with doxycycline. Systolic blood pressure (SBP) was assessed by tail-cuff plethysmography. After 7 days, aortas were collected for zymography assays, Western blot to SERCA, ATPase activity assay, vascular reactivity, Ki-67 immunofluorescence and hematoxylin/eosin stain. Key findingsSBP was increased in 2K-1C rats and doxycycline did not reduce it, but decreased MMP-2 activity and prevented SERCA proteolysis in aortas. Cross sectional area, media to lumen ratio and Ki-67 were all increased in the aortas of hypertensive rats and doxycycline decreased Ki-67. In 2K-1C rats, arterial relaxation to acetylcholine was impaired and doxycycline ameliorated it. Significancedoxycycline reduced MMP-2 activity in aortas of 2K-1C rats and prevented proteolysis of SERCA and its dysfunction, thus ameliorating hypertension-induced vascular dysfunction.

O65 SARCOPLASMIC CALCIUM ATPASE (SERCA) PROTEOLYSIS BY MATRIX METALLOPROTEINASE (MMP)-2 CONTRIBUTES TO VASCULAR DYSFUNCTION IN EARLY HYPERTENSION

Background and objective: Hypertension-induced increased matrix metalloproteinase (MMP)-2 activity proteolyzes extra- and intracellular proteins in aortas and contributes to vascular dysfunction. MMP-2 proteolyzes sarcoplasmic reticulum calcium ATPase (SERCA) in the heart during ischemia and reperfusion injury, thus impairing cardiac function. SERCA activity is also impaired in the arteries of hypertensive animals. We investigated whether hypertension-induced increased MMP-2 activity in aorta contributes to SERCA proteolysis, vascular remodeling and dysfunction. Methods: Aortas from male Sprague-Dawley (SD) rats, incubated or not with angiotensin II (AngII) and MMP inhibitors, ONO-4817 or doxycycline (Doxy), were used for zymography and co-immunoprecipitation of SERCA with MMP-2. Male SD rats were also submitted to two kidney-one clip (2K-1C) or Sham surgery and treated with Doxy from third to seventh day post-surgery. Systolic blood pressure (SBP) was assessed basally and daily by tail-cuff plethysmography. After 7 days, aortas were collected for gel and in situ zymography, western blot to SERCA, ATPase activity assay, vascular reactivity to phenylephrine and acetylcholine, Ki-67 immunofluorescence and hematoxylin/eosin stain. A7r5 cells were incubated or not with AngII, in the presence or absence of SERCA inhibitor, tapsigargin, to assess intracellular calcium concentrations. Results: AngII increased MMP-2 activity in aortas and MMP-2 co-immunoprecipitated with SERCA. SBP was increased in 2K-1C rats and Doxy did not reduce it, but decreased MMP-2 activity and prevented SERCA proteolysis in aortas. Intracellular calcium concentrations were increased in A7r5 cells incubated with AngII and/or SERCA inhibitor, tapsigargin. Cross sectional area, media to lumen ratio and Ki-67 were all increased in the aortas of hypertensive rats and Doxy decreased Ki-67 cell proliferation. In 2K-1C rats, arterial contraction to phenylephrine was accentuated but Doxy ameliorated only the impaired relaxation to acetylcholine. Conclusion: Doxy reduced MMP-2 activity in the aortas of 2K-1C rats and prevented the ATPase activity impairment and MMP-2-induced SERCA proteolysis and vascular dysfunction.

Ang II-induced contraction is impaired in the aortas of renovascular hypertensive animal model.

Renin-angiotensin system plays a critical role in blood pressure control, and the abnormal activation of the AT1 receptor contributes to the development of renovascular hypertension. This study aimed to evaluate the underlying cellular signaling for AT1 receptor activation by Ang II and to compare this mechanism between aortas from 2K-1C and 2K rats. Effects of antagonists and inhibitors were investigated on Ang II-induced contractions in denuded or intact-endothelium aortas. The AT1 receptor antagonist abolished Ang II-induced contraction in 2K-1C and 2K rat aortas, while AT2 and Mas receptors antagonists had no effect. Endothelial nitric oxide synthase inhibition increased the maximal effect (Emax) of Ang II in 2K, which was not changed in 2K-1C aortas. It was associated with lower eNOS mRNA levels in 2K-1C. Endothelium removal increased the Emax of Ang II in 2K-1C and mainly in 2K rat aortas. Nox and COX inhibition did not alter Ang II-induced contraction in 2K and 2K-1C rat aortas. However, AT1 expression was higher in 2K-1C compared to 2K rat aortic rings, whereas expression of phosphorylated (active) IP3 receptors was lower in 2K-1C than in 2K rats. These results demonstrate that endothelium removal impairs Ang II-stimulated contraction in the aorta of 2K-1C rats, which is associated with the reduction of IP3 receptor phosphorylation and activation. In addition, eNOS plays a critical role in Ang II-induced contraction in 2K rat aortas. It is possible that the high Ang II plasma levels could desensitize AT1 receptor in 2K-1C rats, leading to impaired IP3 receptors activation.

Type I collagen proteolysis by matrix metalloproteinase-2 contributes to focal adhesion kinase activation and vascular smooth muscle cell proliferation in the aorta in early hypertension

IntroductionIncreased matrix metalloproteinase (MMP)-2 activity contributes to increase vascular smooth muscle cell (VSMC) proliferation in the aorta in early hypertension by cleaving many proteins of the extracellular matrix. Cleaved products from type I collagen may activate focal adhesion kinases (FAK) that trigger migration and proliferation signals in VSMC. We therefore hypothesized that increased activity of MMP-2 proteolyzes type I collagen in aortas of hypertensive rats, and thereby, induces FAK activation, thus leading to increased VSMC proliferation and hypertrophic remodeling in early hypertension. MethodsMale Sprague-Dawley rats were submitted to renovascular hypertension by the two kidney-one clip (2K1C) model and treated with doxycycline (30 mg/kg/day) by gavage from the third to seventh-day post-surgery. Controls were submitted to sham surgery. Systolic blood pressure (SBP) was measured daily by tail-cuff plethysmography and the aortas were processed for zymography and Western blot for MMP-2, pFAK/FAK, integrins and type I collagen. Mass spectrometry, morphological analysis and Ki67 immunofluorescence were also done to identify collagen changes and VSMC proliferation. A7r5 cells were stimulated with collagen and treated with the MMP inhibitors (doxycycline or ARP-100), and with the FAK inhibitor PND1186 for 24 h. Cells were lysed and evaluated by Western blot for pFAK/FAK. Results2K1C rats developed elevated SBP in the first week as well as increased expression and activity of MMP-2 in the aorta (p < 0.05 vs. Sham). Treatment with doxycycline reduced both MMP activity and type I collagen proteolysis in aortas of 2K1C rats (p < 0.05). Increased pFAK/FAK and increased VSMC proliferation (p < 0.05 vs. Sham groups) were also seen in the aortas of 2K1C and doxycycline decreased both parameters (p < 0.05). Higher proliferation of VSMC contributed to hypertrophic remodeling as seen by increased media/lumen ratio and cross sectional area (p < 0.05 vs Sham groups). In cell culture, MMP-2 cleaves collagen, an effect reversed by MMP inhibitors (p < 0.05). Increased levels of pFAK/FAK were observed when collagen was added in the culture medium (p < 0.05 vs control) and MMP and FAK inhibitors reduced this effect. ConclusionsIncrease in MMP-2 activity proteolyzes type I collagen in the aortas of 2K1C rats and contributes to activate FAK and induces VSMC proliferation during the initial phase of hypertension.

THE ROLE OF MATRIX METALLOPROTEINASE (MMP)-2 TO TYPE I COLLAGEN TURNOVER CONTRIBUTING TO HYPERTROPHIC ARTERIAL REMODELING IN RENOVASCULAR HYPERTENSION

Objective: Type I collagen proteolytic products contribute to VSMC proliferation via integrin receptor and focal adhesion kinase (FAK) activation. We then evaluated whether the early activation of MMP-2 in hypertension induces VSMC proliferation, due to type I collagen proteolysis, which may activate integrin and FAK to develop hypertrophic arterial remodeling. Design and method: Sprague Dawley rats were submitted to 2K1C surgery to induce hypertension and they were orally treated with doxycycline or vehicle (five days at first week (acute) and eight weeks at ten weeks of hypertension (chronic)); (CEUA-USP 165/2019). In the aorta, we evaluated the expression of MMP-2 and type I collagen by Western blotting; MMP-2 activity by gelatin and in situ zymography, arterial morphology by HE, and collagen deposition by picrosirius red. All statistical analyzes used two-way ANOVA followed by Tukey post hoc test with p &lt; 0.05. Results: Our results showed that 2K1C rats developed high systolic blood pressure at the first week (156 ± 13 mmHg vs. Sham (p &lt; 0,001)), which was progressively higher at ten weeks (219 ± 6 mmHg, p &lt; 0,0001). Moreover, there was a significant increase in the MMP-2 expression and activity in the 2K1C rats at the first and tenth weeks of hypertension (p &lt; 0.05). Treatment with doxycycline reduced MMP-2 activity only in the acute situation (p &lt; 0.05). The 2K1C showed increased levels of type I collagen as well as its potential degradation products (p &lt; 0.05) in the aorta during the first week of hypertension. However, the deposition of collagen was notably resynthesized in the aortas of chronic 2K1C rats (p &lt; 0.05). Conclusions: Treatment with doxycycline reduced such effects in hypertensive rats. At this moment, we concluded that increased activity of MMP-2 may contribute to type I collagen proteolysis in aortas at early stages of hypertension, with its potential re-synthesis at late stages. This proteolytic effect of MMP-2 on collagen may contribute to stimulating VSMCs proliferation through integrins and FAK, mechanisms that we are currently investigating.

C-type natriuretic peptide-induced relaxation through cGMP-dependent protein kinase and SERCA activation is impaired in two kidney-one clip rat aorta

AimsHypertension underlies endothelial dysfunction, and activation of vasorelaxation signaling with low dependence on nitric oxide (NO) represents a good alternative for vascular modulation. C-type natriuretic peptide (CNP) causes relaxation by increasing cyclic guanosine 3′,5′-monophosphate (cGMP) or Gi-protein activation through its natriuretic peptide receptor-B or -C, respectively. We have hypothesized that CNP could exerts its effects and could overcome endothelial dysfunction in two kidney-one clip (2K-1C) hypertensive rat aorta. Here, we investigate the intracellular signaling involved in CNP effects in hypertension. Materials and methodsThe 2K-1C hypertension was induced in male Wistar rats (200 g). CNP-induced vascular relaxation and cGMP production were investigated in rat thoracic aortas. The natriuretic peptide receptor-B and -C localization was evaluated by immunofluorescence. Calcium mobilization was assessed in endothelial cells from rat aortas. Key findingsCNP induced similar relaxation in normotensive and 2K-1C hypertensive rat aortas, which increased after endothelium removal. CNP-induced relaxation involved natriuretic peptide receptor-B and -C activation in 2K-1C rats. Nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) counter-regulated CNP-particulate GC (pGC) activation in aortas. CNP reduced endothelial calcium and increased cGMP production, which was lower in 2K-1C. CNP-induced cGMP-dependent protein kinase (PKG) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) activation was impaired in 2K-1C rat aorta. SignificanceOur results indicated CNP triggered relaxation through its natriuretic peptide receptor-B and -C in 2K-1C rat aortas, and that CNP-induced relaxation overcomes endothelial dysfunction in hypertension. In addition, NOS and sGC activities counter-regulate CNP-pGC activation to induce vascular relaxation.

Chronic intermittent hypobaric hypoxia provides vascular protection in the aorta of the 2-kidney, 1-clip rat model of hypertension.

Many studies have demonstrated that chronic intermittent hypobaric hypoxia (CIHH) can reduce blood pressure in spontaneously hypertensive rats and renovascular hypertensive (RVH) rats in which endothelial dysfunction is determined as a critical factor. However, whether CIHH can regulate vasodilation of the aorta in RVH rats remains unknown. The purpose of this study was to investigate the effect of CIHH on impaired relaxation of the aorta in the 2-kidney, 1-clip (2K1C) RVH rat model. The results showed CIHH improved the impaired endothelium-dependent relaxation in the 2K1C rat aorta. The endothelial dysfunction was prevented by the p38 antagonist SB203580, but not by the ERK1/2 antagonist PD98059 or JNK antagonist SP600125. Furthermore, the expression of p-eNOS, HIF-1α, and HIF-2α increased while that of p-p38 and BMP-4 decreased in CIHH-treated aortas from 2K1C rats. Finally, the p-eNOS expression was upregulated and the p-p38 expression was downregulated by pre-incubation of SB203580 or the BMP-4 antagonist Noggin with the aorta. CIHH ameliorated the impairment of endothelium-dependent relaxation through upregulating the expression of p-eNOS, which may be mediated by the inhibition of BMP-4/p-p38 MAPK, and upregulating the expression of HIFs in the 2K1C rat aorta.

Quercetin decreases the activity of matrix metalloproteinase-2 and ameliorates vascular remodeling in renovascular hypertension

Background and aimsIncreased activity of matrix metalloproteinase (MMP)-2 is observed in aortas of different models of hypertension, and its activation is directly mediated by oxidative stress. As quercetin is an important flavonoid with significant antioxidant effects, the hypothesis here is that quercetin will reduce increased MMP-2 activity by decreasing oxidative stress in aortas of hypertensive rats and then ameliorate hypertension-induced vascular remodeling. MethodsMale two-kidney one-clip (2K1C) hypertensive Wistar rats and controls were treated with quercetin (10 mg/kg/day) or its vehicle for three weeks by gavage. Rats were then analyzed at five weeks of hypertension. Systolic blood pressure (SBP) was determined by tail-cuff plethysmography. Aortas were used to determine MMP activity by in situ zymography and reactive oxygen species (ROS) levels by dihydroethidium. Western blot was performed to detect focal adhesion kinase (FAK) and phosphorylated-FAK levels. ResultsSBP was increased in 2K1C rats and only a borderline reduction in SBP was observed after treating 2K1C rats with quercetin. Cross-sectional area and the number of vascular smooth muscle cells were significantly increased in aortas of hypertensive rats, and quercetin reduced them. Quercetin reduced ROS levels in aortas of 2K1C rats and the increased activity of gelatinases in situ. However, quercetin did not affect the levels of tissue inhibitor of MMP (TIMP)-2 and did not interfere with FAK and p-FAK levels in aortas of hypertensive rats. Furthermore, different concentrations of quercetin did not directly reduce the activity of human recombinant MMP-2 in vitro. ConclusionsQuercetin reduces hypertension-induced vascular remodeling, oxidative stress and MMP-2 activity in aortas.

Endothelial nitric oxide synthase and cyclooxygenase are activated by hydrogen peroxide in renal hypertensive rat aorta

In this work, we hypothesized that cyclooxygenase (COX) activity can be regulated by nitric oxide (NO) and hydrogen peroxide (H2O2). In the renal hypertension (2K-1C), phenylephrine (PE)-induced contraction was lower than in normotensive (2K) rat aortas. This impaired contraction is due to NO/H2O2- induced vasodilation. We evaluated the effects of H2O2 on the activity of COX and endothelial NO-Synthase (eNOS) in 2K-1C rat aortas stimulated with PE. Responses for PE or H2O2 were evaluated in 2K-1C and 2K rat aortas, without or with inhibitors for COX (Indomethacin) or eNOS (L-NAME). COX isoforms expression was evaluated by Western blotting. eNOS inhibition was tested on thromboxane A2 (TXA2) and prostacyclin (PGI2) production. PE-induced contraction was lower in 2K-1C than in 2K. Indomethacin reduced PE-induced contraction in 2K, but it had no effect in 2K-1C. L-NAME reversed indomethacin-induced effect in 2K and it normalized PE-induced contraction in 2K-1C to the normotensive levels. COX-1 and COX-2 expression, TXA2 and PGI2 production were higher in 2K-1C than in 2K. eNOS inhibition did no modify TXA2/PGI2 production. In low concentrations, H2O2 induced relaxation only in 2K that was abolished by L-NAME while the contractions induced by high concentrations were abolished by indomethacin in both 2K and 2K-1C. The activity/expression of COX, and TXA2/PGI2 production were increased in 2K-1C, which were not modified by eNOS. High levels of H2O2 increased the endothelial COX activity, which induced contraction. Therefore, an high increase in H2O2 production may increase COX-induced vasoconstriction rather than eNOS-induced relaxation, which might contribute to aggravate hypertension.

Matrix Metalloproteinase-2 Activity is Associated with Divergent Regulation of Calponin-1 in Conductance and Resistance Arteries in Hypertension-induced Early Vascular Dysfunction and Remodelling.

Matrix metalloproteinase (MMP)-2 participates in hypertension-induced maladaptive vascular remodelling by degrading extra- and intracellular proteins. The consequent extracellular matrix rearrangement and phenotype switch of vascular smooth muscle cells (VSMCs) lead to increased cellular migration and proliferation. As calponin-1 degradation by MMP-2 may lead to VSMC proliferation during hypertension, the hypothesis of this study is that increased MMP-2 activity contributes to early hypertension-induced maladaptive remodelling in conductance and resistance arteries via regulation of calponin-1. The main objective was to analyse whether MMP-2 exerts similar effects on the structure and function of the resistance and conductance arteries during early hypertension. Two-kidney, one-clip (2K-1C) hypertensive male rats and corresponding controls were treated with doxycycline (30 mg/kg/day) or water until reaching one week of hypertension. Systolic blood pressure was increased in 2K-1C rats, and doxycycline did not reduce it. Aortas and mesenteric arteries were analysed. MMP-2 activity and expression were increased in both arteries, and doxycycline reduced it. Significant hypertrophic remodelling and VSMC proliferation were observed in aortas but not in mesenteric arteries of 2K-1C rats. The contractility of mesenteric arteries to phenylephrine was increased in 2K-1C rats, and doxycycline prevented this alteration. The potency of phenylephrine to contract aortas of 2K-1C rats was increased, and doxycycline decreased it. Whereas calponin-1 expression was increased in 2K-1C mesenteric arteries, calponin-1 was reduced in aortas. Doxycycline treatment reverted changes in calponin-1 expression. MMP-2 contributes to hypertrophic remodelling in aortas by decreasing calponin-1 levels, which may result in VSMC proliferation. On the other hand, MMP-2-dependent increased calponin-1 in mesenteric arteries may contribute to vascular hypercontractility in 2K-1C rats. Divergent regulation of calponin-1 by MMP-2 may be an important mechanism that leads to maladaptive vascular effects in hypertension.

Relaxation induced by the nitric oxide donor and cyclooxygenase inhibitor NCX2121 in renal hypertensive rat aortas

There is an imbalance between contractile and relaxing endothelial factors in hypertension which induces vascular dysfunction. It involves cyclooxygenase (COX) due to production of vasoconstrictor agonists such as thromboxane A2 (TXA2). In this work we used the NO-NSAID vasodilator compound NCX2121 that has nitric oxide (NO) in its structure and the non-selective COX inhibitor indomethacin. This class of pro-drug has been demonstrated hypotensive effect in animal models of hypertension without alteration in the arterial pressure of normotensive rats. It has been pointed a direct vasodilator effect of NO-NSAIDS which mechanism remains unclear. This study aimed to evaluate the cellular mechanisms involved in the NCX2121-induced relaxation in renal hypertensive two kidney-one clip (2K-1C) rat aorta with intact endothelium and denuded aortas. Our data demonstrated that NO was directly released from the compound NCX2121 in the cytosol of the vascular smooth muscle cells. The relaxation induced by NCX2121 was similar in 2K-1C and normotensive two kidney (2K). It was impaired by the endothelium removal and NO-Synthase (NOS) inhibition in both rat aorta groups. The inhibition of the main NO target, soluble guanylyl-cyclase (sGC) with ODQ, abolished NCX2121-induced relaxation in denuded aorta. The expression of COX-1 and COX-2 as well the produced stable analog of Thromboxane A2 (TXA2) were higher in 2K-1C than in 2K. NCX2121 decreased the augmented production of TXA2 in 2K-1C rat aortas. In intact-endothelium aorta, ODQ impaired the relaxation induced by NCX2121 in 2K but it had no effect in 2K-1C rat aorta. Taken together, our results show that NCX2121-induced relaxation is due to the direct NO release inside the vascular smooth muscle cells and sGC activation in denuded aortas, whereas in intact endothelium aortas, NCX2121-induced relaxation could be due to eNOS activation and inhibition of prostanoids production. In addition, relaxation-induced by NCX2121 is not greater in normotensive than in hypertensive rat aortas because COX activity is higher in 2K-1C than in 2K rat aortas.

Enhanced vasorelaxation effect of endogenous anandamide on thoracic aorta in renal vascular hypertension rats.

Emerging evidence has indicated that anandamide (AEA) is able to stimulate vasorelaxation in both spontaneously hypertensive rats (SHRs) and L-NAME-induced hypertensive rats. Yet it remains unknown whether AEA modulates vasomotion of the aorta in renovascular hypertensive (RVH) rats. The aim of present study is to explore the effect of AEA on the relaxation of thoracic aortas in two-kidney one-clip (2K1C)-induced RVH rats. It is demonstrated that AEA stimulates a pronounced relaxation in the aortas of 2K1C rats compared with sham rats. The enhanced relaxation caused by AEA in aortas from 2K1C rats was diminished in the presence of the cannabinoid receptor-1 (CB1 ) antagonist AM251 and the CB2 receptor antagonist AM630. Likewise, the vasodilation action of AEA was blocked in L-NAME-treated or endothelium-denuded aortas. The Western blot results revealed that the expression of CB1 and CB2 receptors was increased in the 2K1C rat aortas compared with sham rats. The phosphorylation of endothelial nitric oxide synthase (p-eNOS) at the activation site Ser1177 was enhanced in AEA-treated rings from 2K1C rats in both time-dependent anddose-dependentmanners. The augmented p-eNOS expression was inhibited by the co-treatment with AM251 or AM630. Taken together, the present study demonstrated that AEA enhanced endothelium-dependent aortic relaxation through activation of both CB1 and CB2 receptors and P-eNOS/NO pathway in 2K1C rats.

Imbalance between matrix metalloproteinase (MMP)‐2 and calponin‐1 in early hypertensive vascular remodeling

Increased matrix metalloproteinase (MMP)‐2 contributes to hypertension‐induced chronic vascular remodeling. Calponin is a contractile protein, which is cleaved by MMP‐2, and a differentiation marker of vascular smooth muscle cells (VMSCs). Reduced calponin levels are associated with phenotype switch which leads to migration and remodeling. We examined whether there is a relationship between MMP‐2 and calponin‐1 with arterial remodeling in the early and chronic phases of two‐kidney, one‐clip (2K1C) hypertension. Systolic blood pressure (SBP) was assessed in sham and 2K1C rats at 1, 2 and 10 weeks by tail‐cuff plethysmography. Aortic MMP‐2 and calponin‐1 were determined by Western blot. Quantitative morphometry was performed in aortic sections to analyze structural changes. SBP increased from 152 ± 8 mmHg in 1 week of hypertension to 180 ± 8 mmHg and 194 ± 3 mmHg in 2 and 10 weeks (p&lt;0.05 vs. Sham). We observed increased MMP‐2 and reduced calponin‐1 in aortas of 2K1C rats at 1 week of hypertension (P&lt;0.05 r=‐0.74), followed by early, non‐significant changes in arterial structure. Increased MMP‐2 and calponin were observed at 2 and 10 weeks of hypertension (P&lt;0.05), which were accompanied by increased aortic media thickness and media/lumen (P&lt;0.05). These results indicate that an imbalance between MMP‐2 and calponin‐1 occurs only in early hypertension and may be a starting point of chronic arterial remodeling. Fundação de Amparo a Pesquisa do Estado de São Paulo, Conselho Nacional de Desenvolvimento Científico e Tecnológico.

Phenylephrine activates eNOS Ser1177 phosphorylation and nitric oxide signaling in renal hypertensive rat aorta

The endothelial nitric oxide synthase (eNOS) plays an important role in the control of the vascular tone. This work aimed to evaluate the role of an α1-adrenoceptor agonist phenylephrine (PE) on eNOS activity and downstream signaling pathway activation in normotensive (2K) and renal hypertensive (2K-1C) intact-endothelium rat aortas. Concentration-effect curves were performed for PE in intact-endothelium aortas from 2K and 2K-1C rats, in the absence of or in the presence of NOS or soluble guanylyl cyclase (sGC) inhibitor. Intact endothelium aortas were stimulated with PE in organ chambers and eNOS Ser1177/Thr495 phosphorylation expression was evaluated by western blot. Nitric Oxide (NO) production was evaluated in isolated endothelial cells from 2K and 2K-1C rat aortas by flow-cytometry using NO selective fluorescent probe, DAF-2DA. The sGC activity/expression was also evaluated. PE-induced contractile response is lower in 2K-1C than in 2K intact-endothelium rat aorta. This is due to higher eNOS Ser1177 phosphorylation in 2K-1C, which induces the eNOS overactivation. It was abolished by NOS or sGC inhibition. Phenylephrine reduces NO production in 2K as compared to the basal level, but it is not modified in 2K-1C. In PE-stimulated endothelial cells, the NO production is higher in 2K-1C than in 2K. Phenylephrine induces higher cGMP production in 2K-1C than in 2K, despite the lower expression of sGC in 2K-1C. Our results suggest that alpha1-adrenoceptor activation contributes to the increased activity of the enzyme eNOS by Ser1177 phosphorylation in 2K-1C intact-endothelium aorta, which consequently decreases PE-induced contractile response.

Propranolol does not increase the contractile response to epinephrine in endothelium-intact aorta from renal hypertensive rat

Renal hypertension (2K-1C) is characterized by the increased sympathetic activity and oxodative stress induced by angiotensin II. Therefore, the epinephrine plasma levels can potentiate the contractile response in 2K-1C rat aorta. This study aimed to evaluate the effect of inhibition of β-adrenoceptor (β-AR) activation on the contractile response induced by epinephrine in intact endothelium (E+) and in denuded aorta (E−) of 2K-1C rats compared to normotensive sham-operated rats (2K). All the procedures were performed in accordance with the standards and policies of Animal Care and Use Committee of the University of São Paulo. Male rats (180–200 g) were anesthetized with tribromoethanol and after a midline laparotomy a silver clip was placed around the left renal artery. Normotensive two-kidney rats (2K) were only submitted to laparotomy. The systolic blood pressure (SBP) was measured by an indirect tail-cuff method 6 weeks after surgery. Rats were considered to be hypertensive when systolic arterial pressure was higher than 160 mm Hg. Rats were killed and the aortic rings were isolated. Concentration–effect curves were constructed for epinephrine in E+ and E− aortas in the absence (Control) or after incubation with 10 μM propranolol (P+) for 20 min. The maximal effect (ME, in grams of tension) and the potency (pD2) were evaluated. Propranolol did not change the contractile response induced by epinephrine in aortas E− from 2K rats (Control, ME: 2.9 ± 0.2 g; pD2: 7.74 ± 0.09; n = 9) and P+ (ME: 3.7 ± 0.3 g; pD2: 7.48 ± 0.19; n = 4) and 2K-1C (Control, ME: 2.1 ± 0.1 g; pD2: 7.92 ± 0.10; n = 7) and P+ (ME: 2.2 ± 0.2 g; pD2: 8.44 ± 0.78; n = 5). In E+ aorta, propranolol increased the ME to epinephrine in 2K rat aortas from 2.6 ± 01 g; n = 11 to 3.1 ± 0.2 g; n = 6) (P <0.01) and it reduced the pD2 values in 2K-1C rat aortas from 7.37 ± 0.16; n = 9 to 6.70 ± 0.22; n = 4) (P <0.05). Our results indicate that the contractile response induced by epinephrine involves the activation of β-AR in the endothelial cells of 2K rat aortas that leads to a lower contractile responses of 2K rat aorta but not in 2K-1C hypertensive rat aorta. Supported by FAPESP and CNPq .

Hydrogen peroxide modulates phenylephrine-induced contractile response in renal hypertensive rat aorta

Endothelium-derived factors play an important role in vascular tone control. This study aimed to evaluate how endothelium and reactive oxygen species (ROS) contribute to phenylephrine (PE)-induced contraction in renovascular hypertensive (2K–1C) and normotensive (2K) rats aortas. The effects of the superoxide scavenger Tiron (0.1mM and 1mM) or catalase (30U/ml, 90U/ml, 150U/ml and 300U/ml) on the PE-induced contraction were evaluated in both intact endothelium (E+) and denuded (E−) aortas. Endothelium removal increased the PE-induced contractions. The maximum contractile response decreased only in 2K–1C rat E+ aorta, and catalase (30U/ml, 90U/ml, 150U/ml) partially reversed this effect. Endothelium increased the basal hydrogen peroxide (H2O2) production in 2K and 2K–1C rats aortas. PE-stimulated H2O2 production was higher in 2K–1C (E+/E−) than in 2K (E+/E−). Inhibition of the enzymes cyclooxygenase, NADPH-oxidase, xanthine-oxidase, and superoxide dismutase reduced the PE-stimulated H2O2 production in 2K–1C rat aorta. The decreased contraction to PE in 2K–1C rat aorta is partially due to endothelial H2O2 production; however, in denuded aorta, it contributes to maintaining the contractile response. Superoxide plays an important role on the PE-induced contraction in 2K rat denuded aorta, whereas in 2K–1C rat aorta, it is H2O2 that plays an important role in this effect.

Decreased number of caveolae in endothelial cells impairs the relaxation induced by acetylcholine in hypertensive rat aortas

The present study was designed to investigate the contribution of endothelial cell caveolae to vascular relaxation in aortas from a normotensive (2K) and renal hypertensive (2K-1C) rat. For that purpose, concentration–effect curves to acetylcholine were constructed in 2K and 2K-1C intact endothelium aortic rings, in the absence or in the presence of the caveolae disassembler methyl-β-ciclodextrin. The potency (pD 2) and the maximum relaxant effect to acetylcholine were greater in 2K than in 2K-1C aortas. Methyl-β-ciclodextrin reduced the pD 2 in 2K and the maximum relaxant effect in both 2K and 2K-1C. The quantification of the caveolae number by electronic microscopy has shown a larger number of caveolae in 2K than in 2K-1C endothelial cells, which was reduced by methyl-β-ciclodextrin in both 2K and 2K-1C. The production of NO stimulated with acetylcholine was greater in 2K than in 2K-1C endothelial cells, and this effect was impaired by methyl-β-ciclodextrin in both 2K and 2K-1C. The cytosolic Ca 2+ concentration ([Ca 2+]c) was simultaneously measured in endothelial and smooth muscle cells stimulated with acetylcholine by confocal image of aortic slices. Acetylcholine produced a greater [Ca 2+]c increase in 2K than in 2K-1C endothelial cells, which response was inhibited by methyl-β-ciclodextrin only in 2K cells. In smooth muscle cells the reduction of [Ca 2+]c was higher in 2K than in 2K-1C. This effect was inhibited by methyl-β-ciclodextrin only in 2K cells. Taken together, our results suggest that the decreased number of caveolae in the endothelial cells from 2K-1C rat aortas is involved in the impaired effect of acetylcholine on [Ca 2+]c and NO.

URETHRAL DIVERTICULAE IN WOMEN: DISCREPANCIES BETWEEN MRI AND SURGICAL FINDINGS

Endothelium-derived factors play an important role in vascular tone control. This study aimed to evaluate how endothelium and reactive oxygen species (ROS) contribute to phenylephrine (PE)-induced contraction in renovascular hypertensive (2K–1C) and normotensive (2K) rats aortas. The effects of the superoxide scavenger Tiron (0.1 mM and 1 mM) or catalase (30 U/ml, 90 U/ml, 150 U/ml and 300 U/ml) on the PE-induced contraction were evaluated in both intact endothelium (E+) and denuded (E−) aortas. Endothelium removal increased the PE-induced contractions. The maximum contractile response decreased only in 2K–1C rat E+ aorta, and catalase (30 U/ml, 90 U/ml, 150 U/ml) partially reversed this effect. Endothelium increased the basal hydrogen peroxide (H2O2) production in 2K and 2K–1C rats aortas. PE-stimulated H2O2 production was higher in 2K–1C (E+/E−) than in 2K (E+/E−). Inhibition of the enzymes cyclooxygenase, NADPH-oxidase, xanthine-oxidase, and superoxide dismutase reduced the PE-stimulated H2O2 production in 2K–1C rat aorta. The decreased contraction to PE in 2K–1C rat aorta is partially due to endothelial H2O2 production; however, in denuded aorta, it contributes to maintaining the contractile response. Superoxide plays an important role on the PE-induced contraction in 2K rat denuded aorta, whereas in 2K–1C rat aorta, it is H2O2 that plays an important role in this effect.

Lercanidipine reduces matrix metalloproteinase-2 activity and reverses vascular dysfunction in renovascular hypertensive rats

Increased expression/activity of matrix metalloproteinases (MMPs), especially MMP-2, plays a role in the vascular alterations induced by hypertension, and increased oxidative stress is a major factor activating MMPs. Here, we hypothesized that lercanidipine, a calcium channel blocker, could attenuate the increases in oxidative stress and MMP-2 expression/activity in the two-kidney, one-clip (2K-1C) hypertensive rats. Sham-operated or 2K-1C hypertension rats were treated with lercanidipine 2.5 mg/kg/day (or vehicle) starting three weeks after hypertension was induced. Systolic blood pressure was monitored weekly. After five weeks of treatment, aortic rings were isolated to assess endothelium-dependent and independent relaxations. Quantitative morphometry of structural changes in the aortic wall were studied in hematoxylin/eosin sections. Aortic MMP-2 levels were determined by gelatin zymography. Aortic MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by quantitative real-time RT-PCR. Plasma thiobarbituric acid reactive substances concentrations were determined using a fluorometric method. Lercanidipine attenuated 2K-1C hypertension (224 ± 12 versus 183 ± 11 mm Hg in 2K-1C rats and 2K-1C + Lercandipine rats, respectively; P < 0.01) and prevented the reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Increased MMP-2 and Pro-MMP-2 levels were found in the aortas of 2K-1C rats (all P < 0.05). Lercandipine attenuated 2K-1C-induced increases in MMP-2 by more than 60% and blunted 2K-1C-induced increases in oxidative stress (both P < 0.001). While hypertension-induced significant aortic wall hypertrophy and approximately 9-fold increases in the ratio of MMP-2/TIMP-2 mRNA expression (both P < 0.05), lercandipine did not affect these changes. These results suggest that lercanidipine produces antihypertensive effects and reverses the endothelial dysfunction associated with 2K-1C hypertension, probably through mechanisms involving antioxidant effects leading to lower MMP-2 activation.

Vitamin C improves the effect of a new nitric oxide donor on the vascular smooth muscle from renal hypertensive rats

Impaired relaxation induced by the new nitric oxide (NO) donor [Ru(NH.NHq)(terpy)NO +] 3+ (TERPY) has been observed in the aortic rings from renal hypertensive rats (2K-1C). An increased production of reactive oxygen species (ROS) in the aortas from 2K-1C rats are capable of reducing NO bioavailability. Therefore, this study aimed at investigating the effects of an antioxidant (vitamin C) on the relaxant effect of NO released from TERPY on the 2K-1C rat aorta. As for vascular reactivity, the potency of TERPY is greater in the control rats (2K) than in 2K-1C whereas the maximum relaxation (ME) is not significantly different between the 2K and 2K-1C rat aortas. The relaxation of TERPY is potentiated only in the 2K-1C aortic ring treated with vitamin C. TERPY has a lower effect in decreasing cytosolic Ca 2+ concentration ([Ca 2+]c) in vascular smooth muscle cells (VSMCs) from 2K-1C rats. This effect is also potentiated in 2K-1C aortic cells treated with vitamin C, but it is not altered in 2K cells. The basal cytosolic NO concentration ([NO]c) is lower in 2K-1C than in 2K cells, and the bioavailability of the NO released from TERPY is larger in 2K than in 2K-1C VSMCs. The superoxide radical concentration ([ O 2 - ]) is higher in the 2K-1C aorta, and vitamin C reduces the [ O 2 - ] in the 2K-1C aorta. Taken together, these results show that in the aortas of renal hypertensive 2K-1C rats, released NO from the new NO donor is not available to produce a similar effect in 2K aorta due to increased [ O 2 - ].