Ang II-induced Contraction Research Articles

I-mfa, Mesangial Cell TRPC1 Channel, and Regulation of Glomerular Filtration Rate.

Inhibitor of MyoD family A (I-mfa) is a cytosolic protein. Its function in kidney is unknown. The aim of the present study was to examine the regulatory role of I-mfa on glomerular filtration rate (GFR). GFR was measured by transdermal measurement of FITC-sinitrin clearance in conscious wild type (WT) and I-mfa knockout (KO) mice. Cell contractility was assessed in a single human or mouse mesangial cell. Single cell RNA sequence (scRNA-seq), Western blot, and Ca2+ imaging were used to evaluate the effects of I-mfa on TRPCs at messenger, protein and functional levels in MCs. In KO mice, GFR was significantly lower than that in WT mice. In WT mice, knocking down I-mfa selectively in mesangial cells using targeted nanoparticle/siRNA delivery system significantly decreased GFR. In human mesangial cells, overexpression of I-mfa significantly blunted the Ang II-stimulated contraction, and knockdown of I-mfa significantly enhanced the contractile response. Consistently, the Ang II-induced contraction was significantly augmented in primary mesangial cells isolated from KO mice. The exaggerated response was restored by re-introducing I-mfa. Furthermore, scRNA-seq showed an increase in trpc1 messenger and Western blot showed an increase in TRPC1 protein abundance in I-mfa KO mouse mesangial cells. TRPC1 protein abundance was decreased in HEK cells overexpressing I-mfa. Ca2+ imaging experiments showed that downregulation of I-mfa significantly enhanced Ang II-stimulated Ca2+ entry in human mesangial cells. Finally, TRPC1 inhibitor, Pico145 significantly blunted Ang II-induced mesangial cell contraction. I-mfa positively regulated GFR by decreasing mesangial cell contractile function through inhibition of TRPC1-mediated Ca2+ signaling.

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.

Reactivity of renal and mesenteric resistance vessels to angiotensin II is mediated by NOXA1/NOX1 and superoxide signaling.

Activation of NADPH oxidase (NOX) enzymes and the generation of reactive oxygen species and oxidative stress regulate vascular and renal function and contribute to the pathogenesis of hypertension. The present study examined the role of NOXA1/NOX1 function in vascular reactivity of renal and mesenteric resistance arteries/arterioles of wild-type and Noxa1-/- mice. A major finding was that renal blood flow is less sensitive to acute stimulation by angiotensin II (ANG II) in Noxa1-/- mice compared with wild-type mice, with a direct action on resistance arterioles independent of nitric oxide (NO) bioavailability. These functional results were reinforced by immunofluorescence evidence of NOXA1/NOX1 protein presence in renal arteries, afferent arterioles, and glomeruli as well as their upregulation by ANG II. In contrast, the renal vascular response to the thromboxane mimetic U46619 was effectively blunted by NO and was similar in both mouse genotypes and thus independent of NOXA1/NOX1 signaling. However, phenylephrine- and ANG II-induced contraction of isolated mesenteric arteries was less pronounced and buffering of vasoconstriction after acetylcholine and nitroprusside stimulation was reduced in Noxa1-/- mice, suggesting endothelial NO-dependent mechanisms. An involvement of NOXA1/NOX1/O2•- signaling in response to ANG II was demonstrated with the specific NOXA1/NOX1 assembly inhibitor C25 and the nonspecific NOX inhibitor diphenyleneiodonium chloride in cultured vascular smooth muscle cells and isolated mesenteric resistance arteries. Collectively, our data indicate that the NOX1/NOXA1/O2•- pathway contributes to acute vasoconstriction induced by ANG II in renal and mesenteric vascular beds and may contribute to ANG II-induced hypertension.NEW & NOTEWORTHY Renal reactivity to angiotensin II (ANG II) is mediated by superoxide signaling produced by NADPH oxidase (NOX)A1/NOX1. Acute vasoconstriction of renal arteries by ANG was blunted in Noxa1-/- compared with wild-type mice. NOXA1/NOX1/O2•- signaling was also observed in ANG II stimulation of vascular smooth muscle cells and isolated mesenteric resistance arteries, indicating that it contributes to ANG II-induced hypertension. A NOXA1/NOX1 assembly inhibitor (C25) has been characterized that inhibits superoxide production and ameliorates the effects of ANG II.

Function of connexin 43 and RhoA/LIMK2/Cofilin signaling pathway in transient changes of contraction and dilation of human umbilical arterial smooth muscle cells

Post-induction hypotension, a common complication after propofol-based induction regimen, is a life-threatening challenge for anesthesiologists especially when unexpected pre-induction hypertension characterized by angiotensin release and increased vascular tone was presented by the same patient. Gap junctions (GJs) composed of connexin 43 (Cx43) have been considered a key factor in regulating vascular contraction and dilation. We aimed to explore the role of Cx43-GJs during peri-induction blood pressure fluctuation and elucidate the underlying mechanisms. Human umbilical arterial smooth muscle cells (HUASMCs) were pretreated by short-term Angiotensin Ⅱ (Ang Ⅱ) with or without subsequent propofol treatment to simulate transient contraction and dilation of vascular smooth muscle cells during anesthesia induction. F-actin polymerization, a classic indicator of HUASMCs constriction, was determined by F-actin staining assay. Both the function and expression of Cx43-GJs during transient contraction and dilation of HUASMCs, and their potential regulation of downstream Ca2+/RhoA/LIMK2/Cofilin signaling pathway were explored via different targeting inhibitors and siRNAs. Ang Ⅱ pretreatment significantly induced F-actin polymerization that indicate cell contraction, accompanied by enhanced GJs function on HUASMCs. With the inhibition of Cx43 GJs by the specific inhibitor, Gap26, and Cx43-siRNA, Ang Ⅱ-induced F-actin polymerization was reversed accompanied with the decrease of intracellular Ca2+ mobility and the RhoA/LIMK2/Cofilin signaling pathway activity. We also noticed that propofol application could inhibit GJs function, the same as Gap26. Simultaneously, intracellular Ca2+ mobility and RhoA/LIMK2/Cofilin signaling pathway activity on HUASMCs were both downregulated, finally resulting in downstream reduction of F-actin polymerization. The function of Cx43-GJs lies in the center of Ang Ⅱ-induced contraction of HUASMCs, which potentially regulates intracellular Ca2+ mobility as well as RhoA/LIMK2/Cofilin signaling pathway activity. Propofol can reverse this effect induced by Ang Ⅱ through suppressing the function of Cx43-GJs.

Estrogen normalizes maternal HFD-induced vascular dysfunction in offspring by regulating ATR.

Previous studies have shown that female offspring are resistant to fetal high-fat diet (HFD)-induced programming of heightened vascular contraction; however, the underlying mechanisms remain unclear. The present study tested the hypothesis that estrogen plays a key role in protecting females from fetal programming of increased vascular contraction induced by maternal HFD exposure. Pregnant rats were fed a normal diet (ND) or HFD (60% kcal from fat). Ovariectomy (OVX) and 17β-estradiol (E2) replacement were performed on 8-week-old female offspring. Aortas were isolated from adult female offspring. Maternal HFD exposure increased angiotensin II (Ang II)-induced contractions of the aorta in adult OVX offspring, which was abrogated by E2 replacement. The AT1 receptor (AT1R) antagonist losartan (10 μM), but not the AT2 receptor (AT2R) antagonist PD123319 (10 μM), completely blocked Ang II-induced contractions in both ND and HFD offspring. In addition, HFD exposure caused a decrease in endothelium-dependent relaxations induced by acetylcholine (ACh) in adult OVX but not OVX-E2 offspring. However, it had no effect on sodium nitroprusside (SNP)-induced endothelium-independent aorta relaxation in any of the six groups. Maternal HFD feeding increased AT1R, but not AT2R, leading to an increased AT1R/AT2R ratio in HFD-exposed OVX offspring, associated with selective decreases in DNA methylation at the AT1aR promoter, which was ameliorated by E2 replacement. Our results indicated that estrogen play a key role in sex differences of maternal HFD-induced vascular dysfunction and development of hypertensive phenotype in adulthood by differently regulating vascular AT1R and AT2R gene expression through a DNA methylation mechanism.

Phosphodiesterase 4D promotes angiotensin II-induced hypertension in mice via smooth muscle cell contraction

Hypertension is a common chronic disease, which leads to cardio-cerebrovascular diseases, and its prevalence is increasing. The cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway participates in multiple cardiovascular diseases. Phosphodiesterase (PDE) 4 has been shown to regulate PKA activity via cAMP specific hydrolysis. However, whether PDE4-cAMP-PKA pathway influences hypertension remains unknown. Herein, we reveal that PDE4D (one of PDE4 isoforms) expression is upregulated in the aortas of experimental hypertension induced by angiotensin II (Ang II). Furthermore, knockout of Pde4d in mouse smooth muscle cells (SMCs) attenuates Ang II-induced hypertension, arterial wall media thickening, vascular fibrosis and vasocontraction. Additionally, we find that PDE4D deficiency activates PKA-AMP-activated protein kinase (AMPK) signaling pathway to inhibit myosin phosphatase targeting subunit 1 (MYPT1)-myosin light chain (MLC) phosphorylation, relieving Ang II-induced SMC contraction in vitro and in vivo. Our results also indicate that rolipram, a PDE4 inhibitor, may be a potential drug for hypertension therapy.

Angiotensin II-Induced Vasoconstriction via Rho Kinase Activation in Pressure-Overloaded Rat Thoracic Aortas.

Angiotensin II (Ang II) induces vasoconstriction through myosin light chain (MLC) kinase activation and MLC phosphatase inactivation via phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) by Rho kinase. However, the detailed mechanism underlying Rho kinase activation by Ang II is still unknown. We investigated the mechanism of Ang II-induced vasoconstriction mediated by Rho kinase in pressure-overloaded rat thoracic aortas. Pressure-overloaded rats were produced by coarctation of the suprarenal abdominal aorta in four-week-old male Wistar rats. The contractile response to Ang II was significantly enhanced in the pressure-overloaded rats. Ang II-induced vasoconstriction was attenuated by inhibitors of Rho kinase, extracellular signal-regulated kinase 1 and 2 (Erk1/2), and epidermal growth factor receptor (EGFR) in both the sham-operated and pressure-overloaded rats. The Ang II-induced vasoconstriction was attenuated by a Janus kinase 2 (JAK2) inhibitor in only the pressure-overloaded rats. The protein levels of MYPT1 and JAK2 increased only in the pressure-overloaded rat thoracic aortas. These results suggested that Ang II-induced contraction is mediated by Rho kinase activation via EGFR, Erk1/2, and JAK2 in pressure-overloaded rat thoracic aortas. Moreover, Ang II-induced contraction was enhanced in pressure-overloaded rats probably because the protein levels of MYPT1 and JAK2 increased in the thoracic aortas.

C21 preserves endothelial function in the thoracic aorta from DIO mice: role for AT2, Mas and B2 receptors.

Compound 21 (C21), a selective agonist of angiotensin II type 2 receptor (AT2R), induces vasodilation through NO release. Since AT2R seems to be overexpressed in obesity, we hypothesize that C21 prevents the development of obesity-related vascular alterations. The main goal of the present study was to assess the effect of C21 on thoracic aorta endothelial function in a model of diet-induced obesity (DIO) and to elucidate the potential cross-talk among AT2R, Mas receptor (MasR) and/or bradykinin type 2 receptor (B2R) in this response. Five-week-old male C57BL6J mice were fed a standard (CHOW) or a high-fat diet (HF) for 6 weeks and treated daily with C21 (1 mg/kg p.o) or vehicle, generating four groups: CHOW-C, CHOW-C21, HF-C, HF-C21. Vascular reactivity experiments were performed in thoracic aorta rings. Human endothelial cells (HECs; EA.hy926) were used to elucidate the signaling pathways, both at receptor and intracellular levels. Arteries from HF mice exhibited increased contractions to Ang II than CHOW mice, effect that was prevented by C21. PD123177, A779 and HOE-140 (AT2R, Mas and B2R antagonists) significantly enhanced Ang II-induced contractions in CHOW but not in HF-C rings, suggesting a lack of functionality of those receptors in obesity. C21 prevented those alterations and favored the formation of AT2R/MasR and MasR/B2R heterodimers. HF mice also exhibited impaired relaxations to acetylcholine (ACh) due to a reduced NO availability. C21 preserved NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways. In conclusion, C21 favors the interaction among AT2R, MasR and B2R and prevents the development of obesity-induced endothelial dysfunction by stimulating NO release through PKA/p-eNOS and AKT/p-eNOS signaling pathways.

COMPOUND 21 IMPROVES VASCULAR FUNTION IN THE THORACIC AORTA FROM OBESE MICE: IMPLICATION OF AT2, MAS AND B2 RECEPTORS

Objective: The angiotensin AT2 Receptor (AT2R) is known to form heterodimers with either the Mas (MasR) or the bradykinin B2 receptor (B2R). Compound 21 (C21), a highly selective AT2R agonist, induces vascular relaxation through stimulation of nitric oxide (NO). Since AT2R is overexpressed in obese models, we hypothesize that C21 might prevent the development of vascular alterations associated with obesity. The aim of this study was to assess the effect of C21 on endothelial function in a model of diet-induced obesity and the potential crosstalk between AT2R, MasR and/or B2R in this response. Design and method: Eleven-week-old male C57BL6J mice (n = 20/group) were fed a standard (Chow) or a very high-fat diet (HF) for 6 weeks. Half of mice per group were simultaneously treated with C21 (1 mg/kg/day in drinking water, Chow-C21, HF-C21). Vascular reactivity experiments were performed in rings from thoracic aorta. NO production was determined in human endothelial cells (EA.hy926) using DAF-2DA (10-5 M). Results: Thoracic aortas from HF mice exhibited impaired relaxations to acetylcholine (Ach) compared to the Chow group. This alteration was prevented by treatment with C21 [Ach, ChowEmax = 85,6 ± 1,7%; HFEmax = 63,8 ± 3,0% vs HF-C21Emax = 86,0 ± 1,8%, P < 0.001]. In addition, NO availability calculated from the area under the Ach curve in presence and absence of L-NAME (NO synthase inhibitor) was significantly lower in HF mice and increased by C21 treatment [Chow = 237 arbitrary units (AU), HF = 168 AU vs HF-C21 = 239 AU; P < 0.001]. Aortic rings from HF mice exhibited stronger contractions to angiotensin II (Ang II, 10-9-10-6 M). PD123177 (AT2R antagonist, 10-7 M), A779 (MasR antagonist, 10-6 M) or HOE-140 (B2R antagonist, 10-7 M) significantly enhanced Ang II-induced contractions in Chow- but not in HF-rings. HF-C21 aortas exhibited similar responses to Chow rings. In EA.hy926 cells, C21 (10-6 M) significantly increased levels of phosphorylated eNOS and NO production. Both effects were abolished by PD123177, A779 or HOE-140, respectively. Conclusions: In conclusion, our data suggest that C21 improves endothelial dysfunction induced by HF-feeding due to an increase in endothelial NO availability via AT2R activation. In addition, both MasR and B2R are implicated in this response and seem necessary for mediating C21 effects.

Maternal high-fat diet increases vascular contractility in adult offspring in a sex-dependent manner.

A maternal high-fat diet (HFD) is a risk factor for cardiovascular diseases in offspring. The aim of the study was to determine whether maternal HFD causes the epigenetic programming of vascular angiotensin II receptors (ATRs) and leads to heightened vascular contraction in adult male offspring in a sex-dependent manner. Pregnant rats were treated with HFD (60% kcal fat). Aortas were isolated from adult male and female offspring. Maternal HFD increased phenylephrine (PE)-and angiotensin II (Ang II)-induced contractions of the aorta in male but not female offspring. NG-nitro-L-arginine (ʟ-NNA; 100 μM) abrogated the maternal HFD-induced increase in PE-mediated contraction. HFD caused a decrease in endothelium-dependent relaxations induced by acetylcholine in male but not female offspring. However, it had no effect on sodium nitroprusside-induced endothelium-independent relaxations of aortas regardless of sex. The AT1 receptor (AT1R) antagonist losartan (10 μM), but not the AT2 receptor (AT2R) antagonist PD123319 (10 μM), blocked Ang II-induced contractions in both control and HFD offspring in both sexes. Maternal HFD increased AT1R but decreased AT2R, leading to an increased ratio of AT1R/AT2R in HFD male offspring, which was associated with selective decreases in DNA methylation at the AT1aR promoter and increases in DNA methylation at the AT2R promoter. The vascular ratio of AT1R/AT2R was not significantly different in HFD female offspring compared with the control group. Our results indicated that maternal HFD caused a differential regulation of vascular AT1R and AT2R gene expression through a DNA methylation mechanism, which may be involved in HFD-induced vascular dysfunction and the development of a hypertensive phenotype in adulthood in a sex-dependent manner.

Autophagy inhibition by chloroquine prevents increase in blood pressure and preserves endothelial functions

Purpose: To determine the effects of lysosomal inhibition of autophagy by chloroquine (CHQ) onhypertension-associated changes in the endothelial functions.
 Method: Angiotensin II (Ang II)-treated human endothelial cell line EA.hy926 and renovascularhypertensive rats were subjected to CHQ treatment (in vitro: 0.5, 1, and 2.5 μM; in vivo: 50 mg/kg/dayfor three weeks). Changes in the protein expressions of LC3b II (autophagosome formation marker) andp62 (autophagy flux marker) were assessed using immunoblotting. Cell migration assay, tubuleformation assay (in vitro), and organ bath studies (in vivo) were performed to evaluate the endothelialfunctions. Hemodynamic parameters were measured as well.
 Results: A higher expression of LC3b II and a reduced expression of p62 observed in the Ang II-treatedendothelial cells, as well as in the aorta of the hypertensive rats, indicated enhanced autophagy.Treatment with CHQ resulted in reduced autophagy flux (in vitro as well as in vivo) and suppressed AngII-induced endothelial cell migration and angiogenesis (in vitro). The treatment with CHQ was alsoobserved to prevent increase in blood pressure in hypertensive rats and preserved acetylcholineinducedrelaxation in phenylephrine-contracted aorta from the hypertensive rats. In addition, chloroquineattenuated Ang II-induced contractions in the aorta of normotensive as well as hypertensive rats.
 Conclusion: These observations indicated that CHQ lowers the blood pressure and preserves thevascular endothelial function during hypertension.
 Keywords: Angiotensin II, Autophagy, Chloroquine, Endothelial function, Hypertension, Vasculardysfunction

In utero hypoxia altered Ang II-induced contraction via PKCβ in fetal cerebral arteries.

Cerebral circulation is important in fetal brain development, and angiotensin II (Ang II) plays vital roles in regulation of adult cerebral circulation. However, functions of Ang II in fetal cerebral vasculature and influences of in utero hypoxia on Ang II-mediated fetal cerebral vascular responses are largely unknown. This study investigated the effects and mechanisms of in utero hypoxia on fetal middle cerebral arteries (MCA) via Ang II. Near-term ovine fetuses were exposed to in utero hypoxia, and fetal MCA responses to Ang II were tested for vascular tension, calcium transient, and molecular analysis. Ang II caused significant dose-dependent contraction in control fetal MCA. Ang II-induced MCA constriction was decreased significantly in hypoxic fetuses. Neither losartan (AT1R antagonist, 10-5 mol/L) nor PD123,319 (AT2R antagonist, 10-5 mol/L) altered Ang II-mediated contraction in fetal MCA. Phenylephrine-mediated constriction was also significantly weaker in hypoxic fetuses. Bay K8644 caused similar contractions between the two groups. Protein expression of L-type voltage-dependent calcium channels was unchanged. There were no differences in caffeine-mediated vascular tension or calcium transients. Contraction induced by PDBu (PKC agonist) was obviously weaker in hypoxic MCA. Protein expression of PKCβ was reduced in the hypoxic compared with the control, along with no differences in phosphorylation levels. The results showed that fetal MCA was functionally responsive to Ang II near term. Intrauterine hypoxia reduced the vascular agonist-mediated contraction in fetal MCA, probably via decreasing PKCβ and its phosphorylation, which might play protective effects on fetal cerebral circulation against transient hypoxia.

Analysis of angiotensin II-Induced rat urinary bladder contractions in the presence of angiotensin II receptors blockers

Objective An application of a specific analysis on recordings obtained from urinary bladder (UB) preparations influenced with Angiotensin II (AngII) and AngII receptor (ATR) blockers was performed. Methods UB preparations were divided as follows: group 1 stimulated with AngII only; group 2:PD123319 (ATR type-2 blocker)+AngII; group 3:Losartan (ATR type-1 blocker)+AngII. The averaged time and force parameters of the contractions were processed by a spline interpolation and graphic images of the different patterns of the contractile activity were obtained. Results The speed of AngII-induced UB contraction, when PD123319 was administered, was significantly higher than those, registered by the application of AngII alone and Losartan + AngII. The presence of Losartan markedly delayed the speed of the overall AngII-induced contraction. Conclusion The study indicates the contribution of both ATR subtypes for the development of AngII-induced UB contraction. Our results showed that probably ATR mediate a reciprocal dynamic response to AngII in the bladder.

Renal transplantation increases angiotensin II receptor-mediated vascular contractility associated with changes of epigenetic mechanisms.

Hypertension is one of the most common complications following renal transplantation, and it increases the risk of graft loss and other cardiovascular diseases. Previous studies have revealed that the use of angiotensinII (AngII) blockers for preventing and treating hypertension is closely associated with higher survival following renal transplantation. However, the cellular and molecular mechanisms by which the vascular contractility of the recipient is altered in response to AngII following renal transplantation have not been fully elucidated. In the present study, using the Fisher‑Lewis rat kidney transplantation model, the blood pressure(BP) of the conscious transplant recipient was measured following the intravenous administration of AngII. In addition, the mechanisms underlying the AngII-mediated vascular contractility via the type1 and type2AngII receptors (AT1R and AT2R, respectively) in large and small-resistance blood vessels were determined in the recipient after renal transplantation. The results showed that renal transplantation significantly increased the AngII-stimulated BP of the rats. Additionally, exvivo contractility experiments using aorta and mesenteric arteries revealed that the contractions induced by AngII were significantly strengthened in the recipient following renal transplantation, and were associated with an increased intracellular Ca2+ concentration. Losartan almost eradicated the AngII-induced contractions whereas PD-123319 had no apparent effects on the AngII-induced contractions in the aorta and mesenteric arteries of the recipient. Furthermore, the expression levels of AT1R but not AT2R were significantly increased in the vasculature of the recipient following renal transplantation, which exhibited a close association with selective DNA demethylation detected in the promoter region of the vascular AT1aR gene. These results indicate that changes of recipient vascular AT1R gene expression, occurring through a mechanism involving DNA methylation, increase the vascular contractility in response to AngII. This may lead to the increased risk of hypertension following renal transplantation.

Rho-kinase and the nitric oxide pathway modulate basilar arterial reactivity to acetylcholine and angiotensin II in streptozotocin-induced diabetic mice.

Diabetes mellitus comprises a heterogeneous group of metabolic disorders with underlying hyperglycemia and secondary cardiovascular complications. Growing evidence suggests that vascular dysfunction is among the most important causes of diabetic cardiovascular disease. Therefore, we determined whether streptozotocin (STZ)-induced diabetes in mice affects blood pressure and cerebral arterial responsiveness to angiotensin (Ang) II and acetylcholine (ACh), which are important modulators of cerebrovascular autoregulation. Diabetes was induced using a single intraperitoneal injection of STZ (50mg/kg). Blood pressure was measured in conscious mice using the indirect tail-cuff method. Functional studies of the isolated arteries' response to vasoactive substances were performed using a micro-organ-bath system at 60days after STZ injection. Systolic, diastolic, and mean blood pressures significantly increased at days 45 and 60 in the STZ-induced diabetic mice. In the isolated basilar arteries, ACh-induced relaxation, which is dependent on nitric oxide (NO) production from endothelial cells, decreased. In contrast, Ang II-induced contraction, mediated via rho-kinase activation in the smooth muscle, increased in the diabetic mice. There was significantly greater relaxation in the precontracted isolated basilar arteries of diabetic mice that had been treated with Y27632, a rho-kinase inhibitor, than in the control mice arteries. Pretreatment with Nω-nitro-L-arginine (L-NAME), an NO synthase inhibitor, significantly enhanced Ang II-induced contraction and Y27632-induced relaxation in the control basilar arteries but not in the STZ-induced diabetic mice arteries. These results suggest that decreased NO bioavailability and enhanced rho-kinase activity in basilar arteries contribute to altered reactivity to ACh and Ang II, respectively, in STZ-induced diabetic mice.

The Role of Cyclooxygenase Enzymes in the Effects of Losartan and Lisinopril on the Contractions of Rat Thoracic Aorta.

It was suggested that prostaglandins which are synthesized by cyclooxygenase (COX) enzymes contribute to the actions of angiotensin-converting enzyme (ACE) inhibition and angiotensin AT1 receptor antagonism and there is an interaction between ACE signaling pathway and COX enzymes. We aim to investigate the role of COX enzymes in the effects of losartan, an angiotensin II (Ang II) receptor antagonist or lisinopril, an ACE inhibitor, on the contractions of rat thoracic aorta in isolated tissue bath. Responses of losartan (10-6, 10-5, 10-4 M), lisinopril (10-6, 10-5, 10-4 M), and non-selective COX inhibitor dipyrone (10-4, 7 × 10-4, 2 × 10-3 M) alone to the contractions induced by phenylephrine (Phe) (10-7 M), potassium chloride (KCl) (6 × 10-2 M), Ang II (10-8 M) and responses of losartan or lisinopril in combination with dipyrone to the contractions induced by Phe or KCl were recorded. When used alone, dipyrone and losartan inhibited Phe, KCl, and Ang II-induced contractions, whereas lisinopril inhibited only Phe and Ang II-induced contractions. Inhibition of COX enzymes (COX-3, COX-3 + COX-1, COX-1+ COX-2 + COX-3 by dipyrone 10-4, 7 × 10-4, 2 × 10-3 M, respectively) augmented the relaxant effects of losartan or lisinopril. Also, dipyrone potentiated the effect of lisinopril on KCl-induced contractions. We suggest that dipyrone increases the smooth-muscle relaxing effects of losartan or lisinopril and that COX enzyme inhibition may have a role in the enhancement of this relaxation.

Chronic restraint stress increases angiotensin II potency in the rat carotid: role of cyclooxygenases and reactive oxygen species.

To investigate the mechanisms underlying the effects of chronic restraint stress on the vascular contractile response induced by angiotensin (Ang) II in rat carotid. Concentration-response curves for AngII were obtained in endothelium-intact or endothelium-denuded carotid rings, in the absence or presence of SC-560 (COX-1 inhibitor), SC-236 (COX-2 inhibitor), wortmannin (PI3 K-Akt inhibitor), ML171 (NOX-1 inhibitor), VAS2870 (NOX-4 inhibitor), tiron (O2- scavenger) or PEG-catalase (H2 O2 scavenger). 6-ketoPGF1α , TXB2 , O2- or H2 O2 levels and superoxide dismutase and catalase activity or expression were also measured in rat carotid. Stress increased AngII potency in rat carotid. Muscular COX-1 or COX-2-derived metabolites negatively modulated AngII-induced contraction in control rat carotid. Endothelial COX-1 or COX-2-derived metabolites positively modulated AngII-induced contraction in stressed rat carotid. PI3 K-Akt, NOX-1, NOX-4, O2- and H2 O2 positively modulated AngII-induced contraction in stressed rat carotid. Stress increased 6-ketoPGF1α or H2 O2 generation and reduced catalase activity in rat carotid. Protein expression of COX-1, NOX-4 or p-Akt was increased in stressed rat carotid. Stress increases AngII potency in rat carotid by a mechanism that involves the increased generation of PGI2 and H2 O2 and the activation of Akt pathway. Such mechanism could play a pathophysiological role in cardiovascular diseases correlated with stress.

Abstract P213: Vascular Dysfunction and Aberrant Vascular NOTCH3 Signalling in Hypertension and Cerebral Autosomal Dominant Subcortical Infarcts and Leukoencephalopathy (CADASIL)

Hypertension (HT) and CADASIL are clinical conditions of small vessel disease. Vascular dementia is a major feature in CADASIL, and a serious consequence of HT. CADASIL is a monogenic condition due to mutations in NOTCH3 , which is expressed almost exclusively in VSMCs. We hypothesised that altered NOTCH3 signalling in CADASIL and HT are associated with small vessel disease. Small arteries from gluteal biopsies from CADASIL patients (n=14), HT patients (n=3) and healthy controls (n=10) were investigated. Vascular function was assessed by myography. Cultured VSMCs were used to assess signaling through NOTCH3, NO, ER stress (gene array) and Rho kinase (ELISA). CADASIL and HT patients exhibited endothelial dysfunction (Max response: CADASIL 41.7±3%, HT 54.1±2% vs Control 98.2±4%). Pre-incubation with N-acetyl-cysteine ameliorated vasorelaxation. Only CADASIL displayed impaired endothelium-independent relaxation (Max response: CADASIL 53±1.9% vs Control 93±8.9%) and contraction (Max response: CADASIL 78±1.3% vs control 102±5%) (p<0.05). AngII-induced contraction was elevated in HT (98%), yet reduced in CADASIL (28%) (vs control 64% max contraction: p<0.05), despite VSMCs from both conditions displaying increased AT 1 R mRNA expression (HT: 5.1; CADASIL: 3.8; fold vs control; p<0.05). VSMCs from CADASIL and HT have decreased expression of CAMK1, SIRT2 and VEGFA; important in NO signalling (0.5 fold; p<0.05 vs control). VSMC levels of NOTCH3 and NOTCH ligand, JAG1, were increased in CADASIL (3.5, 2.5 fold) and HT (3.0, 2.6 fold, p<0.05). Downstream targets, HEY1 and HEYL, were elevated in CADASIL (3.8, 4.2 fold) and HT (1.9, 2.6 fold) (p<0.05). CADASIL but not HT VSMCs exhibited increased expression of ER stress markers. Rho kinase activity was increased in VSMCs from CADASIL (2.5 fold) and HT (2 fold) vs control (p<0.05). These data demonstrate that in CADASIL and HT, vascular dysfunction, is associated with aberrant NOTCH3 and Rho kinase signalling. In CADASIL, but not HT, endothelium-independent relaxation and ER stress were increased. Our results demonstrate a putative role for NOTCH3 -Rho kinase in vascular dysfunction in conditions of small vessel disease and suggest that ER stress and oxidative stress may be important in vascular injury in CADASIL.

The bioflavonoid quercetin synergises with PPAR-γ agonist pioglitazone in reducing angiotensin-II contractile effect in fructose-streptozotocin induced diabetic rats.

This study investigated the effects of combined minimal concentrations of quercetin and pioglitazone on angiotensin II-induced contraction of the aorta from fructose-streptozotocin (F-STZ)-induced type 2 diabetic rats and the possible role of superoxide anions (O2(-)) and nitric oxide (NO) in their potential therapeutic interaction. Contractile responses to Ang II of aortic rings from Sprague-Dawley (SD) and F-STZ rats were tested following pre-incubation of the tissues in the vehicle (DMSO; 0.05%), quercetin (Q, 0.1μM), pioglitazone (P, 0.1μM) or their combination (P+Q; 0.1μM each). The amount of superoxide anion was evaluated by lucigenin-enhanced chemiluminescence and dihydroethidium fluorescence, and NO by assay of total nitrate/nitrite, and 4-Amino-5-Methylamino-2',7'-Difluorofluorescein (DAF-FM) diacetate. The synergistic reduction of Ang II-induced contraction of diabetic but not normal aorta with minimally effective concentrations of P+Q occurs through inhibiting O2(-) and increasing NO bioavailability. This finding opens the possibility of maximal vascular protective/antidiabetic effects with low dose pioglitazone combined with quercetin, thus minimizing the risk of adverse effects.

Relaxin deficiency attenuates pregnancy-induced adaptation of the mesenteric artery to angiotensin II in mice.

Pregnancy is associated with reduced peripheral vascular resistance, underpinned by changes in endothelial and smooth muscle function. Failure of the maternal vasculature to adapt correctly leads to serious pregnancy complications, such as preeclampsia. The peptide hormone relaxin regulates the maternal renal vasculature during pregnancy; however, little is known about its effects in other vascular beds. This study tested the hypothesis that functional adaptation of the mesenteric and uterine arteries during pregnancy will be compromised in relaxin-deficient (Rln(-/-)) mice. Smooth muscle and endothelial reactivity were examined in small mesenteric and uterine arteries of nonpregnant (estrus) and late-pregnant (day 17.5) wild-type (Rln(+/+)) and Rln(-/-) mice using wire myography. Pregnancy per se was associated with significant reductions in contraction to phenylephrine, endothelin-1, and ANG II in small mesenteric arteries, while sensitivity to endothelin-1 was reduced in uterine arteries of Rln(+/+) mice. The normal pregnancy-associated attenuation of ANG II-mediated vasoconstriction in mesenteric arteries did not occur in Rln(-/-) mice. This adaptive failure was endothelium-independent and did not result from altered expression of ANG II receptors or regulator of G protein signaling 5 (Rgs5) or increases in reactive oxygen species generation. Inhibition of nitric oxide synthase with l-NAME enhanced ANG II-mediated contraction in mesenteric arteries of both genotypes, whereas blockade of prostanoid production with indomethacin only increased ANG II-induced contraction in arteries of pregnant Rln(+/+) mice. In conclusion, relaxin deficiency prevents the normal pregnancy-induced attenuation of ANG II-mediated vasoconstriction in small mesenteric arteries. This is associated with reduced smooth muscle-derived vasodilator prostanoids.