Angiotensin II-induced Vascular Dysfunction Research Articles

Abstract P125: Long-term Exposure Of Angiotensin II Enhanced Vascular Contraction Via Activation Of Protein Kinase C Beta

Protein kinase C (PKC) β is linked to lymphoma and diabetes complications including diabetic retinopathy, nephropathy and neuropathy. Also, recent studies revealed that PKCβ activation also accelerates atherosclerotic plaque formation in diabetes. These findings strongly indicate that PKCβ is a potential target for the treatment of diabetic vascular complications. Although PKCβ is associated with diabetic vascular dysfunction, role of PKCβ in hypertension remains unclear. Since hypertension is one of most important risk factor for vascular diseases including atherosclerosis, we here examined whether PKCβ is involved in angiotensin II-induced vascular dysfunction. Treatment with angiotensin II (100 nM) for 30 min strongly increased phosphorylation at the PKCβ activation site at S660 in aorta from Wistar rat (n=4). Treatment of rat aorta with angiotensin II (100 nM) for 24 hr increased contractile responses to serotonin (5-HT, Maximum contraction, control vs. Angiotensin II, 92.2±20.0 vs 142.1±20.5%, P<0.05, n=6) or endothelin-1 (ET-1, 157.4±30.6 vs 214.1±24.3%, P<0.05, n=8). In contrast, angiotensin II did not affect vascular contractile response to noradrenaline (n=4). The enhanced contraction induced by angiotensin II was blocked by CGP53353 (1 μM), a PKCβ inhibitor (5-HT, Angiotensin II vs. CGP53353+Angiotensin II,197.8±27.8 vs. 167.4±17.6%, P<0.05, n=4-5). Pretreatment with LY333531 (1 μM), another PKCβ inhibitor, also prevented angiotensin II-induced vascular dysfunction (P<0.05, n=3-4). Next, we generated PKCβ knockout rat using rGONAD. Consistent with the results using PKCβ inhibitors, angiotensin II-induced enhanced contractile responses to 5-HT (122.6±15.6 vs 111.0±16.3%, n=4) and ET-1 (137.9±39.9 vs 139.2±30.1%, n=4) were dismissed in PKCβ knockout rat. We also confirmed that phorbol12-myristate13-acetate (0.05-5 μg/ml), a PKC activator, enhanced vascular contractile responses to 5-HT and ET-1 in dose-dependent manner (P<0.05, n=4). We conclude that long exposure of angiotensin II (24 hr) causes increase in vascular contractile responses to 5-HT and ET-1 and the augmented contraction are due to activation of PKCβ. Our result suggests that PKCβ might be associated with angiotensin II-related vascular dysfunction.

Mineralocorticoid Receptor in Myeloid Cells Mediates Angiotensin II-Induced Vascular Dysfunction in Female Mice

Enhanced mineralocorticoid receptor (MR) signaling is critical to the development of endothelial dysfunction and arterial stiffening. However, there is a lack of knowledge about the role of MR-induced adipose tissue inflammation in the genesis of vascular dysfunction in women. In this study, we hypothesize that MR activation in myeloid cells contributes to angiotensin II (Ang II)-induced aortic stiffening and endothelial dysfunction in females via increased pro-inflammatory (M1) macrophage polarization. Female mice lacking MR in myeloid cells (MyMRKO) were infused with Ang II (500 ng/kg/min) for 4 weeks. This was followed by determinations of aortic stiffness and vasomotor responses, as well as measurements of markers of inflammation and macrophage infiltration/polarization in different adipose tissue compartments. MyMRKO mice were protected against Ang II-induced aortic endothelial stiffening, as assessed via atomic force microscopy in aortic explants, and vasorelaxation dysfunction, as measured by aortic wire myography. In alignment, MyMRKO mice were protected against Ang II-induced macrophage infiltration and M1 polarization in visceral adipose tissue (VAT) and thoracic perivascular adipose tissue (tPVAT). Collectively, this study demonstrates a critical role of MR activation in myeloid cells in the pathogenesis of vascular dysfunction in females associated with pro-inflammatory macrophage polarization in VAT and tPVAT. Our data have potential clinical implications for the prevention and management of cardiovascular disease in women, who are disproportionally at higher risk for poor outcomes.

P2Y11 Agonism Prevents Hypoxia/Reoxygenation- and Angiotensin II-Induced Vascular Dysfunction and Intimal Hyperplasia Development.

Vascular dysfunction in cardiovascular diseases includes vasomotor response impairments, endothelial cells (ECs) activation, and smooth muscle cells (SMCs) proliferation and migration to the intima. This results in intimal hyperplasia and vessel failure. We previously reported that activation of the P2Y11 receptor (P2Y11R) in human dendritic cells, cardiofibroblasts and cardiomyocytes was protective against hypoxia/reoxygenation (HR) lesions. In this study, we investigated the role of P2Y11R signaling in vascular dysfunction. P2Y11R activity was modulated using its pharmacological agonist NF546 and antagonist NF340. Rat aortic rings were exposed to angiotensin II (AngII) and evaluated for their vasomotor response. The P2Y11R agonist NF546 reduced AngII-induced vascular dysfunction by promoting EC-dependent vasorelaxation, through an increased nitric oxide (NO) bioavailability and reduced AngII-induced H2O2 release; these effects were prevented by the use of the P2Y11R antagonist NF340. Human vascular SMCs and ECs were subjected to AngII or H/R simulation in vitro. P2Y11R agonist modulated vasoactive factors in human ECs, that is, endothelial nitric oxide synthase (eNOS) and endothelin-1, reduced SMC proliferation and prevented the switch towards a synthetic phenotype. H/R and AngII increased ECs secretome-induced SMC proliferation, an effect prevented by P2Y11R activation. Thus, our data suggest that P2Y11R activation may protect blood vessels from HR-/AngII-induced injury and reduce vascular dysfunctions. These results open the way for new vasculoprotective interventions.

α1AMPK deletion in myelomonocytic cells induces a pro-inflammatory phenotype and enhances angiotensin II-induced vascular dysfunction.

Immune cell function involves energy-dependent processes including growth, proliferation, and cytokine production. Since the AMP-activated protein kinase (AMPK) is a crucial regulator of intracellular energy homeostasis, its expression and activity may also affect innate and adaptive immune cell responses. Therefore, we aimed to investigate the consequences of α1AMPK deletion in myelomonocytic cells on vascular function, inflammation, and hypertension during chronic angiotensin II (ATII) treatment. We generated a mouse strain with α1AMPK deletion in lysozyme M+ myelomonocytic cells. Compared to controls, chronic ATII infusion (1 mg/kg/day for 7 days) lead to increased vascular oxidative stress and aggravated endothelial dysfunction in LysM-Cre+ x α1AMPKfl/fl mice. This was accompanied by an increased aortic infiltration of CD11b+F4/80+ macrophages and enhanced pro-inflammatory cytokine release (tumour necrosis factor-alpha, interferon-gamma, and interleukin-6). Mechanistically, we found that increased expression of C-C chemokine receptor 2 (CCR2) in α1AMPK deficient myelomonocytic cells facilitated their recruitment to the vascular wall. In addition, expression of the ATII receptor type 1a and the oxidative burst was increased in these cells, indicating an increased susceptibility towards pro-oxidant stimuli. In summary, α1AMPK deletion in myelomonocytic cells aggravates vascular oxidative stress and dysfunction by enhancing their recruitment to the vascular wall and increasing their susceptibility towards pro-oxidant stimuli. Our observations suggest that metabolic control in myelomonocytic cells has profound implications for their inflammatory phenotype and may trigger the development of vascular disease.

FGF21 Prevents Angiotensin II-Induced Hypertension and Vascular Dysfunction by Activation of ACE2/Angiotensin-(1–7) Axis in Mice

Fibroblast growth factor 21 (FGF21) is a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity. However, the role of FGF21 in hypertension remains elusive. Here we show that FGF21 deficiency significantly exacerbates angiotensin II-induced hypertension and vascular dysfunction, whereas such negative effects are reversed by replenishment of FGF21. Mechanistically, FGF21 acts on adipocytes and renal cells to promote induction of angiotensin-converting enzyme 2 (ACE2), which in turn converts angiotensin II to angiotensin-(1-7), then inhibits hypertension and reverses vascular damage. In addition, ACE2 deficiency strikingly abrogates these beneficial effects of FGF21 in mice, including alleviation of angiotensin II-associated hypertension and vascular damage. Otherwise, pharmaceutical inhibition of angiotensin-(1-7) attenuates the protective effect of FGF21 on angiotensin II-induced vascular dysfunction, but not on hypertension. Thus, FGF21 protects against angiotensin II-induced hypertension and vascular impairment by activation of the ACE2/angiotensin-(1-7) axis via fine-tuning the multi-organ crosstalk between liver, adipose tissue, kidney, and blood vessels.

A heptameric peptide purified from Spirulinasp. gastrointestinal hydrolysate inhibits angiotensin I-converting enzyme- and angiotensinII-induced vascular dysfunction in human endothelial cells.

In this study, a marine microalga Spirulina sp.-derived protein was hydrolyzed using gastrointestinal enzymes to produce an angiotensin I (Ang I)-converting enzyme (ACE) inhibitory peptide. Following consecutive purification, the potent ACE inhibitory peptide was composed of 7 amino acids, Thr-Met-Glu-Pro-Gly-Lys-Pro (molecular weight, 759 Da). Analysis using the Lineweaver-Burk plot and molecular modeling suggested that the purified peptide acted as a mixed non-competitive inhibitor of ACE. The inhibitory effects of the peptide against the cellular production of vascular dysfunction-related factors induced by Ang II were also investigated. In human endothelial cells, the Ang II-induced production of nitric oxide and reactive oxygen species was inhibited, and the expression of inducible nitric oxide synthase (iNOS) and endothelin-1 (ET-1) was downregulated when the cells were cultured with the purified peptide. Moreover, the peptide blocked the activation of p38 mitogen-activated protein kinase. These results indicated that this Spirulina sp.-derived peptide warrants further investigation as a potential pharmacological inhibitor of ACE and vascular dysfunction.

Assessment of Vascular Dysfunction and Inflammation Induced by Angiotensin II in Mice.

Vascular inflammation in cardiovascular diseases is recognized to be linked with immune cell activation. Recruitment of immune cells into the vessel wall is an early step in angiotensin II-induced vascular dysfunction and arterial hypertension. Exploring the role of monocytes and macrophages in angiotensin II-induced hypertension and vascular inflammation in mouse models highlights the importance of these pathophysiological processes. Here we describe our routinely used protocols concerning angiotensin II-induced hypertension, assessment of blood pressure, vascular function, and immune cell infiltration.

Genetic and Pharmacologic Inhibition of the Chemokine Receptor CXCR2 Prevents Experimental Hypertension and Vascular Dysfunction.

The recruitment of leukocytes to the vascular wall is a key step in hypertension development. Chemokine receptor CXCR2 mediates inflammatory cell chemotaxis in several diseases. However, the role of CXCR2 in hypertension development and the underlying mechanisms remain unknown. Angiotensin II (490 ng·kg-1·min-1) or deoxycorticosterone acetate (DOCA) salt-induced mouse hypertensive models in genetic ablation, pharmacologic inhibition of CXCR2, and adoptive bone marrow transfer mice were used to determine the role of CXCR2 in hypertension (measured by radiotelemetry and tail-cuff system), inflammation (verified by flow cytometry and quantitative real-time polymerase chain reaction [PCR] analysis), vascular remodeling (studied by haematoxylin and eosin and Masson's trichrome staining), vascular dysfunction (assessed by aortic ring), and oxidative stress (indicated by nicotinamide adenine dinucleotide phosphate [NADPH] oxidase activity, dihydroethidium staining, and quantitative real-time PCR analysis). Moreover, the blood CXCR2+ cells in normotensive controls and hypertension patients were analyzed by flow cytometry. Angiotensin II significantly upregulated the expression of CXCR2 mRNA and protein and increased the number of CD45+ CXCR2+ cells in mouse aorta (n=8 per group). Selective CXCR2 knockout (CXCR2-/-) or pharmacological inhibition of CXCR2 markedly reduced angiotensin II- or DOCA-salt-induced blood pressure elevation, aortic thickness and collagen deposition, accumulation of proinflammatory cells into the vascular wall, and expression of cytokines (n=8 per group). CXCR2 inhibition also ameliorated angiotensin II-induced vascular dysfunction and reduced vascular superoxide formation, NADPH activity, and expression of NADPH oxidase subunits (n=6 per group). Bone marrow reconstitution of wild-type mice with CXCR2-/- bone marrow cells also significantly abolished angiotensin II-induced responses (n=6 per group). It is important to note that CXCR2 blockade reversed established hypertension induced by angiotensin II or DOCA-salt challenge (n=10 per group). Furthermore, we demonstrated that CXCR2+ proinflammatory cells were higher in hypertensive patients (n=30) compared with normotensive individuals (n=20). Infiltration of CXCR2+ cells plays a pathogenic role in arterial hypertension and vascular dysfunction. Inhibition of CXCR2 pathway may represent a novel therapeutic approach to treat hypertension.

Context-dependent effects of SOCS3 in angiotensin II-induced vascular dysfunction and hypertension in mice: mechanisms and role of bone marrow-derived cells.

Carotid artery disease is a major contributor to stroke and cognitive deficits. Angiotensin II (Ang II) promotes vascular dysfunction and disease through mechanisms that include the IL-6/STAT3 pathway. Here, we investigated the importance of suppressor of cytokine signaling 3 (SOCS3) in models of Ang II-induced vascular dysfunction. We examined direct effects of Ang II on carotid arteries from SOCS3-deficient (SOCS3(+/-)) mice and wild-type (WT) littermates using organ culture and then tested endothelial function with acetylcholine (ACh). A low concentration of Ang II (1 nmol/l) did not affect ACh-induced vasodilation in WT but reduced that of SOCS3(+/-) mice by ∼50% (P < 0.05). In relation to mechanisms, effects of Ang II in SOCS3(+/-) mice were prevented by inhibitors of STAT3, IL-6, NF-κB, or superoxide. Systemic Ang II (1.4 mg/kg per day for 14 days) also reduced vasodilation to ACh in WT. Surprisingly, SOCS3 deficiency prevented most of the endothelial dysfunction. To examine potential underlying mechanisms, we performed bone marrow transplantation. WT mice reconstituted with SOCS3(+/-) bone marrow were protected from Ang II-induced endothelial dysfunction, whereas reconstitution of SOCS3(+/-) mice with WT bone marrow exacerbated Ang II-induced effects. The SOCS3 genotype of bone marrow-derived cells did not influence direct effects of Ang II on vascular function. These data provide new mechanistic insight into the influence of SOCS3 on the vasculature, including divergent effects depending on the source of Ang II. Bone marrow-derived cells deficient in SOCS3 protect against systemic Ang II-induced vascular dysfunction.

Abstract 32: Coagulation Factor XI and Thrombin Mediate Angiotensin II-induced Vascular Dysfunction

Background: Multicellular interactions of platelets, coagulation factors, leukocytes and the vessel wall play pivotal roles in activating coagulation and precipitating arterial and venous thrombosis. High levels of angiotensin II (ATII) cause arterial hypertension by a complex inflammatory pathway requiring inflammatory leukocyte recruitment and reactive oxygen species (ROS) production within the vessel wall coupled to vascular dysfunction. How platelets, coagulation factors, leukocytes and the vessel wall cooperate to promote vascular inflammation is unclear. Methods and results: Here we delineate a novel non-thrombotic, FXI-dependent, pro-inflammatory coagulation pathway that substantially regulates vascular tone and endothelial function. We can demonstrate that pharmacological inhibition of the synthesis of FXI by an antisense-oligonucleotide (ASO) attenuates ATII-induced vascular dysfunction in acetylcholine-induced vascular relaxation. ATII-induced arterial hypertension, vascular inflammation and leukocyte infiltration are equally diminished by FXI ASO treatment. In addition, vascular ROS formation and fibrotic remodeling is blocked by FXI ASO treatment in ATII-infused mice. Accordingly, intravital microscopy video imaging (IVM) shows that ATII-induced leukocyte rolling as well as adhesion in the carotid artery is reduced by inhibition of the synthesis of FXI, whereas acute infusion of recombinant FXI is not able to restore ATII-driven leukocyte adhesion. Hence FXI is required continuously for the ATII-induced vascular pathology. We show that this pathway does not depend on FXII-dependent activation and that FXII-deficiency does not prevent from ATII-induced vascular dysfunction and inflammation. Conclusion: Our results provide novel insight into coagulation-inflammation circuits and point to broader utility of specific FXI-targeted anticoagulants beyond indications as antithrombitic agents in cardiovascular diseases.

Abstract 11888: MyD88 Expressed by Myeloid Cells is Essential for Angiotensin II-Induced Vascular Dysfunction, Inflammation and Arterial Hypertension

Background: Angiotensin II (ATII) causes hypertension and promotes infiltration of inflammatory cells into the vessel wall. Vascular superoxide formation and shear stress mediated inflammatory remodeling of conduit arteries was shown to be myeloid differentiation factor 88 (MyD88) dependent, but the exact mechanism are unknown. Objective: The goal of this study was to determine the mechanism, how MyD88 contributes to the development of ATII-induced vascular dysfunction and arterial hypertension. Methods and Results: MyD88 deficiency profoundly attenuated ATII-induced (1 mg/kg/d for 7 days) blood pressure increase (measured by radio telemetry) and vascular dysfunction (assessed by aortic ring relaxation studies). Additionally vascular superoxide levels as well as mRNA expression levels of VCAM-1, iNOS, Nox2 were decreased in ATII-infused MyD88 -/- mice compared to WT controls. Aortic flow cytometric analysis revealed that ATII-induced infiltration with CD11b + Ly6C high inflammatory monocytes was significantly dampened in MyD88 -/- mice. ATII led to an increased expression of inflammatory monocyte markers in blood and aorta, which was blocked in MyD88 -/- mice, indicating a role of MyD88 for myeloid cell differentiation. Additionally less IFN-gamma + NK cells were detected in the vessel wall of ATII-treated MyD88 -/- mice and aortic lysates showed reduced mRNA levels of several proinflammatory cytokines like IL-12 and IL-1beta. Bone marrow transfer experiments further revealed a protective effect of MyD88 deficiency in inflammatory cells represented by a reduction of vascular dysfunction and inflammation. Conclusion: We provide first evidence that MyD88 expressed by bone marrow-derived cells plays an essential role in ATII-induced vascular dysfunction and arterial hypertension. Our data indicate that MyD88 is important for cytokine production and might be required for ATII-induced differentiation of monocytes into an inflammatory phenotype.

Abstract 610: Context-Dependent Effects of Suppressor of Cytokine Signaling 3 (SOCS3) in Angiotensin II-Induced Vascular Dysfunction: Mechanisms and Role of Bone Marrow-Derived Cells

Angiotensin II (Ang II) promotes vascular disease and hypertension, in part, by activating the interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway. Although SOCS3 regulates this pathway in the immune system, its role in vascular disease and hypertension is unknown. In this study, we investigated the role of SOCS3 in a model of Ang II-induced vascular disease. To exam direct effects, carotid arteries from wild-type (WT) and SOCS3 haplodeficient (SOCS3 +/- ) mice were incubated with Ang II for 22 hrs, followed by examination of endothelial function using acetylcholine (Ach). Relaxation to Ach was similar in all arteries incubated with vehicle. A low concentration of Ang II (1 nmol/L) did not affect Ach-induced vasodilation in WT mice, but reduced that of SOCS3 +/- mice by ~50% (P&lt;0.05). Ang II-induced impairment was prevented by inhibitors of STAT3, IL-6, NF-κB, or a scavenger of superoxide. Responses to nitroprusside were similar in all groups. We also tested the impact of SOCS3 in vivo by systemically infusing Ang II (1.4 mg/kg per day) for 14 days via osmotic mini-pumps. Ach-induced vasodilation in carotid and resistance arteries in brain from WT mice was reduced by ~60% (P&lt;0.05). Surprisingly, deficiency in SOCS3 prevented the majority of Ang II-induced endothelial dysfunction without affecting the pressor response to Ang II. Lethally irradiated WT mice reconstituted with SOCS3 +/- bone marrow were protected from Ang II-induced endothelial dysfunction (P&lt;0.05), while reconstitution of irradiated SOCS3 +/- mice with WT bone marrow exacerbated Ang II-induced vascular dysfunction (P&lt;0.05). WT into WT and SOCS3 +/- into SOCS3 +/- bone marrow chimeras exhibited vascular function consistent with non-irradiated controls. The pressor response to Ang II was reduced by ~50% in WT mice reconstituted with bone marrow from SOCS3 +/- mice (P&lt;0.05). These data suggest SOCS3 exerts divergent local versus systemic effects on Ang II-induced vascular dysfunction. In the face of SOCS3 deficiency, bone marrow-derived cells protect against Ang II-induced vascular dysfunction and hypertension.

Oxidative inhibition of the vascular Na+-K+ pump via NADPH oxidase-dependent β1-subunit glutathionylation: Implications for angiotensin II-induced vascular dysfunction

Glutathionylation of the Na+-K+ pump’s β1-subunit is a key molecular mechanism of physiological and pathophysiological pump inhibition in cardiac myocytes. Its contribution to Na+-K+ pump regulation in other tissues is unknown, and cannot be assumed given the dependence on specific β-subunit isoform expression and receptor-coupled pathways. As Na+-K+ pump activity is an important determinant of vascular tone through effects on [Ca2+]i, we have examined the role of oxidative regulation of the Na+-K+ pump in mediating angiotensin II (Ang II)-induced increases in vascular reactivity. β1-subunit glutathione adducts were present at baseline and increased by exposure to Ang II in rabbit aortic rings, primary rabbit aortic vascular smooth muscle cells (VSMCs), and human arterial segments. In VSMCs, Ang II-induced glutathionylation was associated with marked reduction in Na+-K+ATPase activity, an effect that was abolished by the NADPH oxidase inhibitory peptide, tat-gp91ds. In aortic segments, Ang II-induced glutathionylation was associated with decreased K+-induced vasorelaxation, a validated index of pump activity. Ang II-induced oxidative inhibition of Na+-K+ ATPase and decrease in K+-induced relaxation were reversed by preincubation of VSMCs and rings with recombinant FXYD3 protein that is known to facilitate deglutathionylation of β1-subunit. Knock-out of FXYD1 dramatically decreased K+-induced relaxation in a mouse model. Attenuation of Ang II signaling in vivo by captopril (8mg/kg/day for 7 days) decreased superoxide-sensitive DHE levels in the media of rabbit aorta, decreased β1-subunit glutathionylation, and enhanced K+-induced vasorelaxation. Ang II inhibits the Na+-K+ pump in VSMCs via NADPH oxidase-dependent glutathionylation of the pump’s β1-subunit, and this newly identified signaling pathway may contribute to altered vascular tone. FXYD proteins reduce oxidative inhibition of the Na+-K+ pump and may have an important protective role in the vasculature under conditions of oxidative stress.

Abstract 192: Depletion of Inflammatory Monocytes Reduces Endothelial Nitric Oxide Synthase Uncoupling and iNOS Derived Nitrosative Stress in Angiotensin II-induced Vascular Dysfunction

Background Endothelial nitric oxide synthase (eNOS) uncoupling occurs in disease states of vascular dysfunction and amplifies vascular oxidative stress and loss of NO bioactivity. Inflammatory myelomonocytic cells as a major source of superoxide are critical for angiotensin-II induced vascular dysfunction; however their role in mediating eNOS uncopuling has not been defined yet. Methods and results Angiotensin II (1mg/kg/d, 7d) increased the number of CD11b + Gr-1 low iNOS + monocytes and macrophages in mouse aorta (verified by flow cytometry) in paralell to increasing nox4 and heme oxygenase 1 expression (assessed by Western blot analysis) and inducing eNOS uncoupling, measured by differential NOS dependent superoxide formation in the endothelial layer and glutathionylation of eNOS. Toxin mediated ablation of macrophages in LysM iDTR transgenic mice restored all of these parameters. Conversely, ablation of LysM + cells reduced iNOS expression and normalized vascular levels of tetrahydrobiopterin (measured by high-performance liquid chromatography) and expression of the GTP cyclohydrolase-1, essential for tetrahydrobiopterin synthesis, all of which were increased by angiotensin II in control mice. Conclusion Depletion of LysM + monocytes reduces angiotensin II induced eNOS uncopuling in vascular dysfunction, and simultaneously protects from iNOS mediated nitrosative stress: These findings highlight the role of vascular inflammatory cells in disturbed NO-bioactivity and vascular function

Protection of protease-activated receptor 2 mediated vasodilatation against angiotensin II-induced vascular dysfunction in mice

BackgroundUnder conditions of cardiovascular dysfunction, protease-activated receptor 2 (PAR2) agonists maintain vasodilatation activity, which has been attributed to increased cyclooxygenase-2, nitric oxide synthase and calcium-activated potassium channel (SK3.1) activities. Protease-activated receptor 2 agonist mediated vasodilatation is unknown under conditions of dysfunction caused by angiotensin II. The main purpose of our study was to determine whether PAR2-induced vasodilatation of resistance arteries was attenuated by prolonged angiotensin II treatment in mice. We compared the vasodilatation of resistance-type arteries (mesenteric) from angiotensin II-treated PAR2 wild-type mice (WT) induced by PAR2 agonist 2-furoyl-LIGRLO-amide (2fly) to the responses obtained in controls (saline treatment). We also investigated arterial vasodilatation in angiotensin II-treated PAR2 deficient (PAR2-/-) mice.Results2fly-induced relaxations of untreated arteries from angiotensin II-treated WT were not different than saline-treated WT. Treatment of arteries with nitric oxide synthase inhibitor and SK3.1 inhibitor (L-NAME + TRAM-34) blocked 2fly in angiotensin II-treated WT. Protein and mRNA expression of cyclooxygenase-1 and -2 were increased, and cyclooxygenase activity increased the sensitivity of arteries to 2fly in only angiotensin II-treated WT. These protective vasodilatation mechanisms were selective for 2fly compared with acetylcholine- and nitroprusside-induced relaxations which were attenuated by angiotensin II; PAR2-/- were protected against this attenuation of nitroprusside.ConclusionsPAR2-mediated vasodilatation of resistance type arteries is protected against the negative effects of angiotensin II-induced vascular dysfunction in mice. In conditions of endothelial dysfunction, angiotensin II induction of cyclooxygenases increases sensitivity to PAR2 agonist and the preserved vasodilatation mechanism involves activation of SK3.1.

Regarding Article “Receptor Activity-Modifying Protein-1 Augments Cerebrovascular Responses to Calcitonin Gene-Related Peptide and Inhibits Angiotensin II-Induced Vascular Dysfunction”

HomeStrokeVol. 42, No. 2Regarding Article “Receptor Activity-Modifying Protein-1 Augments Cerebrovascular Responses to Calcitonin Gene-Related Peptide and Inhibits Angiotensin II-Induced Vascular Dysfunction” Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBRegarding Article “Receptor Activity-Modifying Protein-1 Augments Cerebrovascular Responses to Calcitonin Gene-Related Peptide and Inhibits Angiotensin II-Induced Vascular Dysfunction” Kazushi Tsuda, MD, FAHA Kazushi TsudaKazushi Tsuda Cardiovascular and Metabolic Research Center Kansai University of Health Sciences Osaka, Japan (Tsuda) Search for more papers by this author Originally published30 Dec 2010https://doi.org/10.1161/STROKEAHA.110.604272Stroke. 2011;42:e24Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 1, 2010: Previous Version 1 To the Editor:We read with great interest the recent article by Dr Chrissobolis and colleagues1 dealing with the cerebrovascular responses in the transgenic mice with overexpression of the receptor activity-modifying protein-1 (RAMP1) for calcitonin gene-related peptide (CGRP) receptors. The results of their study demonstrated that the responses to CGRP in carotid and basilar arteries in vitro as well as cerebral arterioles in vivo were selectively enhanced in human RAMP1 transgenic mice compared with controls. In addition, the authors presented that angiotensin II-induced oxidative stress and endothelial dysfunction was prevented after expression of RAMP1. The authors propose the hypothesis that RAMP1 may be an important mediator of vascular protection and a new therapeutic target in vascular disease.Several studies have reported that enhanced activity of the sympathetic nervous system might actively participate in the pathogenesis of vascular dysfunction. In a study we presented previously, the changes in norepinephrine release induced by CGRP was investigated in the rat central nervous system.2 In an in vitro study, we showed that CGRP inhibited the stimulation-evoked norepinephrine release in a dose-dependent manner. It was also demonstrated that a dihydropyridine-sensitive calcium channel agonist, Bay K 8644, significantly reversed the inhibitory effect of CGRP on norepinephrine release, indicating that CGRP might partially interact with dihydropyridine-sensitive calcium channels and modulate intracellular calcium mobilization. Furthermore, we showed that the inhibitory action on CGRP on norepinephrine release was significantly attenuated in spontaneously hypertensive rats compared with normotensive rats.3 In the peripheral tissues, Ohhashi and Jacobowitz4 observed that CGRP might have reduced the electric stimulation-induced contraction of rat vas deferens, suggesting that CGRP might inhibit norepinephrine release during adrenergic nerve stimulation. It can be speculated that the sympatholytic action of CGRP might be a defense against vascular dysfunction. Because angiotensin II may stimulate the sympathetic neurotransmission in the central nervous system,5 we would like to know whether changes in sympathetic nervous activity might be associated with the magnitude of the RAMP1 overexpression or whether CGRP might suppress the angiotensin II-induced sympathetic hyperactivity in the study of Dr Chrissobolis and colleagues. Further studies should be performed to assess more thoroughly the interactions between CGRP and the sympathetic nervous system and their role in the protective effect against the angiotensin II-induced vascular dysfunction in the RAMP1 transgenic mice.Kazushi Tsuda, MD, FAHA Cardiovascular and Metabolic Research Center Kansai University of Health Sciences Osaka, JapanDisclosuresNone.FootnotesStroke welcomes Letters to the Editor and will publish them, if suitable, as space permits. Letters must reference a Stroke published-ahead-of-print article or an article printed within the past 3 weeks. The maximum length is 750 words including no more than 5 references and 3 authors. Please submit letters typed double-spaced. Letters may be shortened or edited. Include a completed copyright transfer agreement form (available online at http://stroke.ahajournals.org and http://submit-stroke.ahajournals.org).

Resurrecting Hope for Antioxidant Treatment of Cardiovascular Disease

Oxidative stress has been associated with cardiovascular disease and the development of hypertension in both humans and in animal models. Antioxidant therapy has been successful in ameliorating disease in animals, but the results from human studies have been largely disappointing. Despite these negative results, a wealth of evidence suggests that oxidative stress is involved in the development of cardiovascular disease both in humans and animal models. Because global attenuation of cellular reactive oxygen species (ROS) may not be advantageous, more recent therapeutic strategies target specific subcellular sources of oxidative stress. Most studies implicate plasmalemmal membrane-bound NADPH oxidase as a key mediator of ROS generation in cardiovascular disease. However, in the current issue of Circulation Research , Dikalova et al1 demonstrate that specific attenuation of mitochondrial derived oxidants prevents the development of angiotensin II–induced hypertension in the mouse. Perhaps more interesting is the mechanism: inhibition of a mitochondrial ROS-triggered activation of NADPH oxidase, interrupting a vicious cycle of ROS production. The novel approach of targeting the inciting source of ROS (mitochondria) to prevent accelerated oxidant production may hold more promise for humans by eliminating pathological but not homeostatic sources of ROS. Seminal observations by Harrison et al2 showed evidence of increased oxidative stress within the systemic vasculature in an Angiotensin II–induced rat model of hypertension, piquing interest in a possible etiologic role for ROS in this condition. In animal models, stimulating ROS production can induce hypertension,2,3 whereas treatment with a variety of antioxidants including vitamin C and E, or targeted inhibition of enzymatic oxidant production, can reduce blood pressure.3–6 Hypertensive patients demonstrate increased NAD(P)H oxidase activity7 with elevated levels of hydrogen peroxide in plasma.8 Antioxidant treatment leads to enhanced vasodilation in both forearm and coronary arteries of human hypertensive subjects,9,10 indicating a functional vasomotor …

Angiotensin II-induced vascular dysfunction is mediated by the AT1A receptor in mice.

Many of the actions of angiotensin II (Ang II) are mediated by angiotensin type 1 receptors (AT1), of which there are 2 pharmacologically indistinguishable subtypes (AT1A and AT1B). The purpose of this study was to evaluate the effect of an AT1A homozygous deletion (AT1A-/-) on vascular reactivity. AT1A-/- mice and control littermates (AT1A+/+) were infused with vehicle (saline) or Ang II (1000 ng x kg(-1) x min(-1)) for 7 days by osmotic pumps. Systolic pressure was increased in AT1A+/+ mice (Delta45+/-8 mm Hg, P<0.0001) but unchanged in AT1A-/- mice (Delta5+/-3 mm Hg, P>0.13) on day 7. The carotid artery response to the vasodilators acetylcholine (ACh), nitroprusside, and papaverine and to the vasoconstrictors phenylephrine, U46619, 5-hydroxytryptamine (5-HT), and KCl were not different between vehicle-infused AT1A+/+ and AT1A-/- animals. Carotid relaxation to ACh was impaired and contraction to 5-HT was increased in Ang II-infused AT1A+/+ mice. Ang II did not affect carotid responses in AT1A-/- mice. Superoxide, measured by lucigenin (5 micromol/L), and hydroethidine staining were not different between AT1A+/+ and AT1A-/- mice after vehicle or Ang II infusion, suggesting that it was not contributing to the altered ACh and 5-HT responses. The Rho-kinase inhibitor Y-27632 (1 micromol/L) attenuated the 5-HT response in both vehicle- and Ang II-infused AT1A+/+ mice. Moreover, concentration-dependent relaxation to Y-27632 and RhoA protein expression were not different in vehicle- or Ang II-infused AT1A+/+. These data demonstrate that the AT1A receptor is required for Ang II-induced changes in carotid artery function.