Ang II-induced Endothelial Dysfunction Research Articles

Ticagrelor protects against AngII-induced endothelial dysfunction by alleviating endoplasmic reticulum stress

Ticagrelor has been reported to decrease cardiovascular mortality compared with clopidogrel. This benefit cannot be fully explained by the more efficient platelet inhibition. Many studies demonstrated that ticagrelor improved endothelial function, leaving the mechanism elusive though. The present study aims to investigate whether ticagrelor protects against endothelial dysfunction induced by angiotensinII (AngII) through alleviating endoplasmic reticulum (ER) stress. Male Sprague Dawley rats were infused with AngII or vehicle and administrated with ticagrelor or vehicle for 14 days. Reactive oxygen species (ROS) was detected. Aortas from normal mice were incubated with endoplasmic reticulum stress inducer tunicamycin with or without ticagrelor. Vasorecactivity was measured on wire myography. Rat aortic endothelial cells (RAECs) were pretreated with ticagrelor followed by AngII or tunicamycin. Endothelial nitric oxide synthase (eNOS) phosphorylation and ER stress markers were determined by western blotting. Impaired endothelial function, induction of ER stress, reduced eNOS phosphorylation and elevated ROS generation was restored by ticagrelor treatment in vivo. In addition, tunicamycin induced endothelial dysfunction was improved by ticagrelor. In vitro, the induction of ER stress and inhibited eNOS phosphorylation in REACs exposed to AngII as well as tunicamycin was reversed by co-culturing with ticagrelor. In conclusion, ticagrelor protects against AngII-induced endothelial dysfunction via alleviating ER stress.

Matrix metalloproteinase-2 knockout prevents angiotensin II-induced vascular injury.

Matrix metalloproteinases (MMPs) have been implicated in the development of hypertension in animal models and humans. Mmp2 deletion did not change Ang II-induced blood pressure (BP) rise. However, whether Mmp2 knockout affects angiotensin (Ang) II-induced vascular injury has not been tested. We sought to determine whether Mmp2 knockout will prevent Ang II-induced vascular injury. A fourteen-day Ang II infusion (1000 ng/kg/min, SC) increased systolic BP, decreased vasodilatory responses to acetylcholine, induced mesenteric artery (MA) hypertrophic remodelling, and enhanced MA stiffness in wild-type (WT) mice. Ang II enhanced aortic media and perivascular reactive oxygen species generation, aortic vascular cell adhesion molecule-1 and monocyte chemotactic protein-1 expression, perivascular monocyte/macrophage and T cell infiltration, and the fraction of spleen activated CD4+CD69+ and CD8+CD69+ T cells, and Ly-6Chi monocytes. Study of intracellular signalling showed that Ang II increased phosphorylation of epidermal growth factor receptor and extracellular-signal-regulated kinase 1/2 in vascular smooth muscle cells isolated from WT mice. All these effects were reduced or prevented by Mmp2 knockout, except for systolic BP elevation. Ang II increased Mmp2 expression in immune cells infiltrating the aorta and perivascular fat. Bone marrow (BM) transplantation experiments revealed that in absence of MMP2 in immune cells, Ang II-induced BP elevation was decreased, and that when MMP2 was deficient in either immune or vascular cells, Ang II-induced endothelial dysfunction was blunted. Mmp2 knockout impaired Ang II-induced vascular injury but not BP elevation. BM transplantation revealed a role for immune cells in Ang II-induced BP elevation, and for both vascular and immune cell MMP2 in Ang II-induced endothelial dysfunction.

A salt-sensing kinase in T lymphocytes, SGK1, drives hypertension and hypertensive end-organ damage.

We previously showed that angiotensin II (Ang II) increases T cell production of IL-17A, and that mice deficient in IL-17A have blunted hypertension and attenuated renal and vascular dysfunction. It was recently shown that salt enhances IL-17A production from CD4+ T cells via a serum- and glucocorticoid-regulated kinase 1-dependent (SGK1-dependent) pathway. Thus, we tested the hypothesis that SGK1 signaling in T cells promotes hypertension and contributes to end-organ damage. We show that loss of T cell SGK1 results in a blunted hypertensive response to Ang II infusion by 25 mmHg. Importantly, renal and vascular inflammation is abrogated in these mice compared with control mice. Furthermore, mice lacking T cell SGK1 are protected from Ang II-induced endothelial dysfunction and renal injury. Loss of T cell SGK1 also blunts blood pressure and vascular inflammation in response to deoxycorticosterone acetate-salt (DOCA-salt) hypertension. Finally, we demonstrate that the Na+-K+-2Cl- cotransporter 1 (NKCC1) is upregulated in Th17 cells and is necessary for the salt-induced increase in SGK1 and the IL-23 receptor. These studies demonstrate that T cell SGK1 and NKCC1 may be novel therapeutic targets for the treatment of hypertension and identify a potentially new mechanism by which salt contributes to hypertension.

Abstract P220: Matrix Metalloproteinase 2 Knockout Protects from Angiotensin II-induced Vascular Injury

Objective: Matrix metalloproteinase-2 (MMP2) participates in the mechanisms of vascular injury in atherosclerosis. Whether MMP2 plays a role in angiotensin (Ang) II-induced hypertension and vascular remodeling is unknown. We hypothesized that Mmp2 knockout ( Mmp2 -/- ) would prevent Ang II-induced hypertension and vascular injury. Methods and Results: Fourteen days of Ang II infusion (1000 ng/kg/min, SC) increased systolic blood pressure (SBP, 161±9 vs 122±3 mm Hg, P <0.05) and decreased vasodilatory responses to acetylcholine (33±5 vs 83±3%, P <0.001), increased the media/lumen (4.8±0.4 vs 3.1±0.1%, P <0.001) and media cross-sectional area (7223±467 vs 5346±336 μm 2 , P <0.05) and enhanced stiffness ( P <0.05), as shown by a leftward shift of the stress/strain relationship of mesenteric arteries in wild-type mice. Ang II enhanced aortic (73±6 vs 6±1 relative fluorescence units [RFU]/μm 2 , P <0.001) and perivascular adipose tissue (PVAT) reactive oxygen species generation (76±11 vs 12±1 RFU/μm 2 , P <0.001), aortic VCAM-1 (17±3 vs 5±1 RFU/μm 2 , P <0.001) and MCP-1 expression (71±14 vs 11±3 RFU/μm 2 , P <0.001), PVAT monocyte/macrophage (1.8±0.3 vs 0.1±0.1 % of PVAT, P <0.001) and T cell infiltration (56±14 vs 16±9 cells/μm 2 , P <0.05) and the fraction of spleen activated CD4 + CD69 + (17±2 vs 10±1 % of CD4+ T cells, P <0.001), CD8 + CD69 + T cells (11±1 vs 5±1 % of CD4+ T cells, P <0.001) and Ly-6C hi monocytes (53±6 vs 25±2 % event, P <0.001). Ang II increased phosphorylation of epidermal growth factor receptor 1.9±0.2-fold and extracellular-signal-regulated kinase 1/2 1.4±0.1-fold in vascular smooth muscle cells isolated from mesenteric arteries of wild-type mice ( P <0.05). Mmp2 knockout prevented or reduced all of the above ( P <0.05) except SBP elevation. Bone marrow transplantation experiments revealed that Ang II-induced hypertension was impaired in absence of immune cell MMP2 and endothelial dysfunction was blunted or reduced in absence of immune or vascular cell MMP2 ( P <0.05). Conclusions: Mmp2 knockout prevented Ang II-induced vascular injury but not hypertension. Bone marrow transplantation experiments revealed a complex relationship of immune and vascular cell MMP2 in the development of Ang II-induced hypertension and endothelial dysfunction.

Gene silencing of endothelial von Willebrand Factor attenuates angiotensin II-induced endothelin-1 expression in porcine aortic endothelial cells.

Expression of endothelin (ET)-1 is increased in endothelial cells exposed to angiotensin II (Ang II), leading to endothelial dysfunction and cardiovascular disorders. Since von Willebrand Factor (vWF) blockade improves endothelial function in coronary patients, we hypothesized that targeting endothelial vWF with short interference RNA (siRNA) prevents Ang II-induced ET-1 upregulation. Nearly 65 ± 2% silencing of vWF in porcine aortic endothelial cells (PAOECs) was achieved with vWF-specific siRNA without affecting cell viability and growth. While showing ET-1 similar to wild type cells at rest, vWF-silenced cells did not present ET-1 upregulation during exposure to Ang II (100 nM/24 h), preserving levels of endothelial nitric oxide synthase activity similar to wild type. vWF silencing prevented AngII-induced increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity and superoxide anion (O2−) levels, known triggers of ET-1 expression. Moreover, no increase in O2− or ET-1 levels was found in silenced cells treated with AngII or NOX-agonist phorbol ester (PMA 5 nM/48 h). Finally, vWF was required for overexpression of NOX4 and NOX2 in response to AngII and PMA. In conclusion, endothelial vWF knockdown prevented Ang II-induced ET-1 upregulation through attenuation of NOX-mediated O2− production. Our findings reveal a new role of vWF in preventing of Ang II-induced endothelial dysfunction.

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.

Deficiency of T-regulatory cells exaggerates angiotensin II-induced microvascular injury by enhancing immune responses.

T-regulatory lymphocyte (Treg) adoptive transfer prevented angiotensin (Ang) II-induced hypertension and microvascular injury. Scurfy mice are deficient in Treg because of a mutation in the transcription factor forkhead box P3 (Foxp3) gene. Enhanced Ang II effects in the absence of Treg would unambiguously demonstrate their vascular protective role. We hypothesized that adoptive transfer of Scurfy vs. wild-type T cells will exacerbate Ang II-induced microvascular damage in T and B-cell-deficient recombination-activating gene 1 (Rag1) knockout mice. Rag1 knockout mice were injected with vehicle, 10(7) T cells from wild-type or Scurfy mice or 10 (6)wild-type Treg alone or in combination with Scurfy T cells, and then infused or not with Ang II (490 ng/kg per min, subcutaneous) for 14 days. Ang II increased SBP in all the groups, but DBP only in wild-type and Scurfy T-cell groups. Ang II-induced endothelial dysfunction and oxidative stress in perivascular adipose tissue (PVAT) of mesenteric arteries of the wild-type T-cell group, whereas these were exaggerated in the Scurfy T-cell group. Ang II enhanced microvascular remodeling and stiffness in vehicle and Scurfy T-cell groups. Ang II increased monocyte chemotactic protein-1 expression in the vascular wall and PVAT, monocyte/macrophage infiltration and proinflammatory polarization in PVAT and the renal cortex, and T-cell infiltration in the renal cortex only in the Scurfy T-cell group. Treg coinjection in the vehicle and Scurfy T-cell groups prevented or reduced the effects of Ang II. FOXP3+ Treg deficiency exaggerates Ang II-induced microvascular injury by modulating innate and adaptive immune responses.

Interference with PPARγ in endothelium accelerates angiotensin II-induced endothelial dysfunction.

The ligand activated nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) in the endothelium regulates vascular function and blood pressure (BP). We previously reported that transgenic mice (E-V290M) with selectively targeted endothelial-specific expression of dominant negative PPARγ exhibited endothelial dysfunction when treated with a high-fat diet, and exhibited an augmented pressor response to angiotensin II (ANG II). We hypothesize that interference with endothelial PPARγ would exacerbate ANG II-induced endothelial dysfunction. Endothelial function was examined in E-V290M mice infused with a subpressor dose of ANG II (120 ng·kg(-1)·min(-1)) or saline for 2 wk. ANG II infusion significantly impaired the responses to the endothelium-dependent agonist acetylcholine both in basilar and carotid arteries from E-V290M but not NT mice. This impairment was not due to increased BP, which was not significantly different in ANG II-infused E-V290M compared with NT mice. Superoxide levels, and expression of the pro-oxidant Nox2 gene was elevated, whereas expression of the anti-oxidant genes Catalase and SOD3 decreased in carotid arteries from ANG II-infused E-V290M mice. Increased p65 and decreased Iκ-Bα suggesting increased NF-κB activity was also observed in aorta from ANG II-infused E-V290M mice. The responses to acetylcholine were significantly improved both in basilar and carotid arteries after treatment with Tempol (1 mmol/l), a scavenger of superoxide. These findings provide evidence that interference with endothelial PPARγ accelerates ANG II-mediated endothelial dysfunction both in cerebral and conduit arteries through an oxidative stress-dependent mechanism, suggesting a role for endothelial PPARγ in protecting against ANG II-induced endothelial dysfunction.

Abstract P006: Liraglutide Attenuates Cardiac Fibrosis and Vascular Dysfunction in a Non-diabetic Angiotensin II-infusion Model via Anti-inflammatory and Anti-oxidant Mechanisms

Glucagon-like peptide-1 (GLP-1) based therapies are used to treat type II diabetes via increasing insulin secretion and inhibiting glucagon production. Recent evidence suggests that activating the GLP-1 receptor may also mediate direct vaso-protective effects. Therefore the objective of the study was to determine whether GLP-1R stimulation conferred cardio- and vaso-protection in a non-diabetic setting using the angiotensin (Ang) II infusion model of hypertension and cardiovascular dysfunction. Male C57Bl/6J mice (4-6 months) were assigned to one of the following 4 week treatment protocols: 1) vehicle (saline), 2) Ang II (800ng/kg/day), 3) Ang II + liraglutide (30μg/kg/day), 4) Ang II + liraglutide (300μg/kg/day). All treatments were administered via osmotic mini-pumps (s.c). After 4 weeks the effect of liraglutide treatment on blood pressure, vascular function and cardiac remodelling was examined. Liraglutide (both doses) attenuated Ang II-induced increase in systolic blood pressure (Ang II: 175.3 ± 8.6mmHg vs Ang II+Lirag (30) 150.2 ± 6.4 mmHg or Ang II+Lirag (300): 145.4 ± 6.9 mmHg) without affecting blood glucose levels. Liraglutide (both doses) completely prevented Ang II-induced endothelial dysfunction (% maximum relaxation: Ang II=50.7 ± 7.8%; Ang II+Lirag (30)=82.7 ± 5.8; Ang II+Lirag (300)=81.5 ± 6.1%). In the heart, liraglutide prevented Ang II-induced cardiomyocyte hypertrophy (n=7-10; p&lt;0.05) and reduced collagen deposition (% collagen expression: Ang II=4.4 ± 0.5 vs Ang II+Lirag(300)=2.9 ± 0.3; n=7-9; p&lt;0.01). This anti-fibrotic effect was attributed to reduced fibroblast/myofibroblast expression as well as decreased inflammation with reduced NFκB and MCP-1 expression and decreased oxidative stress with a significant reduction in superoxide production using high dose of liraglutide. Overall, stimulation of GLP-1R in a non-diabetic setting protected against Ang II-mediated cardiac hypertrophy, cardiac fibrosis and vascular dysfunction, indicating potential for use of GLP-1 based therapies in treatment of cardiovascular disease independent of diabetes.

Abstract P123: Retinol-Binding Protein 7 (RBP7) Regulates Endothelial Function via an Adiponectin-Dependent Mechanism in Angiotensin II-Infused Mice

The transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) regulates vascular function and protects against endothelial dysfunction. We reported that mice expressing dominant-negative PPARγ in endothelium exhibit an enhanced pressor response to angiotensin II (Ang II). Given that RBP7 is a PPARγ target expressed in endothelium, we hypothesized that loss of RBP7 would augment endothelial dysfunction caused by Ang II. Using osmotic minipumps, RBP7 knockout (KO) mice and wild type (WT) littermates were infused with subpressor Ang II (120 ng/kg/min, 2 weeks) or saline. Systolic blood pressure (by tail cuff) was not different between KO and WT mice and was not altered by infusion of saline or Ang II. Acetylcholine (ACh, 30 μM) mediated relaxation of carotid arteries was not different between groups during saline infusion, but was selectively impaired in KO mice during Ang II (44±5% in KO vs 71±3% in WT, p&lt;0.05). Aortic superoxide, assessed by hydroethidine staining, was higher after Ang II in KO compared with WT mice. Pre-incubation of carotid arteries with a superoxide scavenger Tempol (1 mM, 30 min) restored ACh-induced relaxation in Ang II-infused KO mice (74±6% in Tempol-KO vs 46±8% in KO). To identify molecular mechanisms, RNA Sequencing was performed using carotid arteries from WT and KO mice fed high fat diet (HFD) or normal chow for 8 weeks. Adiponectin (Adipo), a known PPARγ target gene, was increased ~6-fold in HFD-fed WT mice, a response that was markedly blunted in KO mice. Immunohistochemistry revealed that Adipo was expressed specifically in endothelial cells (co-immunostaining with endothelial specific CD31). Rosiglitazone, a PPARγ ligand, increased carotid artery Adipo protein in WT, but not KO mice. To test the importance of Adipo, carotid arteries from Ang II-infused WT and KO mice were incubated with vehicle or full-length Adipo (5 μg/mL) for 12 hrs. Adipo incubation ameliorated Ang II-induced endothelial dysfunction in KO mice (ACh, 30 μM: 86±3% Adipo-KO vs 58±8% KO) while having no effect in WT mice (ACh, 30 μM: 83±1% Adipo-WT vs 87±3% WT). In conclusion, RBP7 protects the endothelium from oxidative stress-induced dysfunction caused by Ang II. Our data suggest an important role for Adipo in mediating this protective response.

Abstract 002: Dynamic T Cell-Antigen Presenting Cell Interactions and Direct T Cell Activation Within the Vascular Wall During Hypertension

T cells are now known to be vital to the development of experimental hypertension. Hypertension is associated with significant accumulation of T cells into the perivascular fat surrounding the aorta and renal vasculature. While a hypertension-specific neoantigen has been implicated in T cell activation, it is not known whether vascular-infiltrating T cells recognize and are locally activated by an antigen within the vessel wall. We developed live-cell imaging of explanted aortas to identify whether cognate antigens are presented to T cells within the vessel wall of hypertensive mice, evidenced by slower T cell velocities and a greater number of T cells interacting with antigen presenting cells (APCs). Splenic T cells were isolated from normotensive vehicle-treated (nT cells) and hypertensive angiotensin II (Ang II)-infused (0.7mg/kg/day; 14 days; hT cells) C57BL6/J mice. Following anti-CD3/CD28 stimulation (48 hours), cells were fluorescently labelled and co-incubated simultaneously (16 hours) with explanted aorta from normotensive or hypertensive CD11c-YFP mice, where APCs are fluorescently labelled. In CD11c-YFP mouse aorta alone, we detected a ~2-fold increase in CCR5 ligand (CCL3, CCL4 and CCL5) secretion from hypertensive mouse aorta compared to vehicle-treated mouse aorta (*P&lt;0.05; n = 4). Using 2-photon microscopy, we observed a greater number (~2-fold) of hT cells compared to nT cells within Ang II-infused mouse aorta (390 ± 113 Vs 198 ± 49). Importantly, time-lapse recordings of hypertensive mouse aorta revealed hT cells exhibited significantly slower velocity (hT cells 2.0 μm/min Vs nT cells 4.8 μm/min; **P&lt;0.01, n=8-12), and a greater proportion of interactions with APCs (hT cells 4.4 ± 1.1 Vs nT cells 0.8 ± 0.4%). Moreover, activation of local vascular T cells by incubating hypertensive aorta with anti-CD3/CD28 antibodies (16 hours) augmented Ang II-induced endothelial dysfunction (67.5 ± 2.0 Vs Ang II alone 54.5 ± 3.7% maximal relaxation). We have the first evidence that vascular-infiltrating T cells are presented with cognate antigens by APCs within the vessel wall during hypertension; direct activation of these T cell infiltrates further impairs endothelial function, which may promote the development of hypertension.

Baicalin attenuates angiotensin II-induced endothelial dysfunction

Angiotensin II (Ang II) has been shown to activate multiple downstream pathways resulting in endothelial dysfunction and oxidative stress. Baicalin, a natural flavone, exerts anti-oxidant and anti-apoptotic effects in cardiovascular diseases. In the present study, we hypothesized that baicalin has beneficial effects in Ang II-induced endothelial cells injury. Here, we shown that baicalin improved endothelial fuction impaired by Ang II through promoting endothelial-dependent vasodilation and suppressing the apoptosis of HUVECs in which baicalin decreased the expression of bax and cleaved caspase-3, and increased bcl-2 expression. Additionally, baicalin significantly conversed Ang II to angiotensin-1-7 [Ang-(1–7)] by activating angiotensin-converting enzyme 2 (ACE2) and Mas receptor mRNA expression and protein expression. Moreover, treatment with baicalin significantly reduced cell oxidative damage induced by Ang II through MDA/ROS decrease and NO/T-AOC increase. This antioxidant capacity was related to the increases of PI3K, phosphor-AKT (Ser-473) and phosphor-eNOS (Ser-1177). In conclusion, our results implicate that baicalin could protect endothelial cells from Ang II-induced endothelial dysfunction and oxidative stress via modulating the expression of bax, bcl-2 and cleaved caspase-3, activating ACE2/Ang-(1-7)/Mas axis and up-regulating PI3K/AKT/eNOS pathway.

Abstract 237: Role of Runx2 in Vascular Responses to Angiotensin II-Induced Hypertension

Increases in local angiotensin II (ANGII) signaling have been implicated in a number of diseases, including atherosclerosis, hypertension, and aneurysm. Endothelial dysfunction is strongly associated with increased ANGII signaling and the pathogenesis of these diseases. While increases in Runx2 have been implicated in cardiovascular calcification and vascular stiffening in diabetes, little is known regarding the role of Runx2 in the regulation of endothelial function. In the present study, we hypothesized that Runx2 is a key mediator of impaired vascular function and increased vascular wall stiffness provoked by ANGII. Wild-type mice (WT) and littermates that were deficient in one copy of Runx2 (HET) were implanted with osmotic minipumps containing saline or a pressor dose of ANGII (1000 ng/kg/min). Blood pressure was measured non-invasively using tail-cuff methods (CODA) and mice were sacrificed following 14 days of treatment. Changes in endothelium-dependent relaxation (relaxation to acetylcholine, R ACH ) and passive pressure-diameter relationships were measured in vitro (Living Systems arteriograph). In WT mice, treatment with ANGII for 14 days resulted in profound impairment in R ACH (85 ± 4% vs 47 ± 14% p&lt;0.05) and slight reductions in vascular diameter at high distending pressures (~10% reduction in maximal diameter). While Runx2 deficiency did not alter R ACH or vascular distensibility/compliance in saline-treated mice, loss of one copy of Runx2 in ANGII-treated mice dramatically improved R ACH compared to their wild-type, ANGII-treated littermates. Interestingly, impairments in the pressure-diameter relationship with ANGII infusion were not affected by loss of one copy of Runx2. Critically, reductions in Runx2 did not significantly alter the pressor response to ANGII. In conclusion, our data suggest that Runx2 may play an integral role in the pathogenesis of ANGII-induced endothelial dysfunction, with these changes occurring independently of alterations in extracellular matrix remodeling and vascular stiffness. These data suggest that Runx2 may play a key, context-dependent role mediating vascular responses to ANGII.

Genetic Deletion of Retinol‐Binding Protein 7 (RBP7), a PPARγ Target Gene, Accelerates Angiotensin II‐mediated Endothelial Dysfunction

Mice expressing dominant-negative peroxisome proliferator-activated receptor γ (PPARγ) in endothelium exhibit an augmented pressor response to angiotensin II (Ang II). The mechanisms responsible for protective actions of PPARγ are unclear. RBP7 is an intracellular RBP and a PPARγ target gene highly expressed in endothelium. We hypothesized that RBP7 may normally mediate protective effects and that deletion of RBP7 may augment Ang II-induced endothelial dysfunction. RBP7 knockout (KO) and wild type (WT) mice were infused with subpressor or pressor doses of Ang II (120 or 1000 ng/kg/min, osmotic minipumps) or saline for 2 weeks. Systolic blood pressure (SBP) was measured using tail-cuff and carotid artery function was assessed using a wire myograph. SBP and the carotid artery responses to acetylcholine (ACh) and nitroprusside (SNP) were not different between saline-infused KO and WT mice suggesting that KO of RBP7 per se does not affect baseline vascular function. Subpressor Ang II did not alter SBP in either group, but diminished relaxation in response to ACh, but not SNP, in KO mice (at 30 µM: ACh 41±3% in KO vs 72±6% in WT, p<0.05). Pressor Ang II increased SBP (WT: 121±2 to 152±6 mmHg, KO: 128±4 to 159±6 mmHg) and impaired responses to ACh and SNP to a similar extent in both WT and KO mice. Superoxide, measured by hydroethidine staining, was significantly higher in aorta after subpressor Ang II in KO compared with WT mice, suggesting that superoxide may contribute to impaired ACh responses in KO mice. We conclude that loss of RBP7 augments Ang II-mediated endothelial dysfunction, possibly through an oxidative stress-dependent mechanism.

Role of Arginases in the Angiotensin II-Induced Hypertension-Associated Endothelial Dysfunction in the Rat Aorta

Increases in arginase activity have been reported in a variety of diseases associated with vascular dysfunction. As arginases compete with NO synthase for their common substrate L-arginine, an increased arginase activity might decrease the bioavailability of nitric oxide (NO), a major vasoprotective factor. Moreover, red wine polyphenols (RWPs) have been shown to prevent the endothelial dysfunction in several rat models, at least in part, through protection of NO bioavailability. The aim of the present study was to determine whether an increased arginase activity contributes to the Ang II-induced hypertension-associated endothelial dysfunction in the rat aorta, and, if so, to determine the protective effect of RWPs. Rats received either RWPs (150 mg/kg/day) or apocynin, an antioxidant and an inhibitor of NADPH oxidase (100 mg/kg/day), in the drinking water for 7 days before the infusion of Ang II (0.4 mg/kg/day) for 14 days. Arterial pressure was measured in conscious rats. After euthanasia, vascular reactivity was assessed in organ chambers, arginases activity by a colorimetric assay, and expression level of arginases by immunofluorescence. Ang II-induced hypertension is associated with an endothelial dysfunction characterized by a reduced relaxation and an increased contraction in response to acetylcholine. Moreover, Ang II infusion increased both expression and activity of arginases. Treatment with RWPs and apocynin prevented the Ang II-induced hypertension and endothelial dysfunction, and the associated increased arginase expression and activity. Altogether, these findings suggest that Ang II-induced hypertension and endothelial dysfunction are associated with an increased expression and activity of vascular arginases, which can be prevented by RWPs and apocynin. These results constitute a real contribution in the understanding of the physiopathology of endothelial dysfunction arterial high blood pressure induced by the angioyensine II. Besides, they offer real perspectives in prevention treatment of these cardiovascular disorders.

LACK OF ANGIOPOIETIN LIKE-2 PROTECTS AGAINST ANGIOTENSIN II-INDUCED CEREBRAL ENDOTHELIAL DYSFUNCTION IN MICE

Angiopoietin like-2 (angptl2) is a recently identified pro-inflammatory and pro-oxidative protein that is elevated in patients with chronic inflammatory and cardiovascular diseases. Its role in regulating endothelial function is largely unknown, and there is no report of the impact of angptl2 in the cerebrovasculature. We hypothesized that the lack of angptl2 improves endothelial cell stress resistance and prevents cerebral endothelial dysfunction induced by a sub-pressor dose of angiotensin II (angII). Subcutaneous infusion of angII (200 ng/kg/min, n=15; osmotic mini-pump), or saline as control (n=15), was performed in 20- to 22-week-old angptl2 knock-down (KD) mice and wild-type (WT) littermates for 14 days. In isolated cerebral arteries of saline-treated KD and WT mice, global acetylcholine (ACh)-induced vasodilations were similar. However, the contribution of endothelium-derived relaxing factors differed: while eNOS-derived O2-/H2O2 contributed to dilation in WT, eNOS-derived NO (P<0.001) was involved in KD mice. Furthermore, angII induced endothelial dysfunction only in WT mice (P<0.01). This was reversed by acute addition of either N-acetylcysteine (10 μM), an anti-oxidant, apocynin (10 μM), a non-selective NADPH oxidase (Nox) inhibitor, or indomethacin (10 μM), a non-selective cyclooxygenase (COX) inhibitor, suggesting that angII-induced endothelial dysfunction in WT mice was related to oxidative stress, Nox activity and/or COX-derived constrictor-derivatives. In KD mice treated with angII, on the other hand, apocynin worsened endothelium-dependent dilation, while N-acetylcysteine and indomethacin were ineffective, suggesting remodeling of the Nox systems, low levels of oxidative stress and COX-derived constrictor derivatives. The demonstration that the lack of angptl2 expression is associated with NO-dependent dilation, prevented angII-induced endothelial dysfunction, and led to a deleterious effect of apocynin, suggests that lowering angptl2 protects cerebral endothelial function and leads to a potential compensatory Nox dilatory pathway, ultimately reinforcing cerebral endothelial cell stress resistance.

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.

Abstract 456: Role of Signal Transducer and Activator of Transcription protein (STAT)-1 in the Angiotensin II-induced Cardiovascular Damage

Background: Inflammation participates importantly in hypertensive organ damage. Angiotensin II (Ang II) plays a crucial role in hypertension and induces inflammation. An essential mediator of inflammation is the transcription factor STAT1 which is activated by interferon but also by Ang II. We hypothesized that activation of STAT1 during Ang II infusion upregulates chemokines, enhances chemotaxis and consequently results in heart fibrosis and vessel dysfunction independent on blood pressure and hypertrophy Methods: C57BL/6, C57BL/6 receiving candesartan (5mg/kg) and STAT1-/- mice received infusion of Ang II 1.5 μg/g/day or placebo for 4 weeks (n=9/group). Blood pressure was measured using tail cuff pletysmography. Expression of chemokines Cxcl10, Cxcl9 and MCP-1 as well as Nos2 were investigated. Small mesenteric arteries (SMA) were mounted in a wire myograph to assess their function. Cardiac hypertrophy and inflammation (CD45 staining) and fibrosis (hydroxyproline assay) were determined. Results: Ang II caused expression of IFNg in C57BL/6 and STAT1-/- in vitro and in vivo. Blood pressure and cardiac hypertrophy did not differ between angiotensin treated C57BL/6 and STAT1-/- mice. Ang II increased in C57BL/6 expression of STAT1 dependent genes of chemokines and Nos2 (Cxcl10: 4.8-fold, Cxcl9: 3.4-fold, MCP-1: 6.6-fold, Nos2: 2.6-fold; all P&lt;0.05) whereas this remained abolished in STAT1-/- mice. Ang II lead in STAT1-/- mice as compared to C57BL/6 to decreased cardiac CD45 number/view (C57BL/6-AngII: 27±4 STAT1-/--AngII: 11±5; P&lt;0.05) and reduced cardiac fibrosis (hydroxyproline assay: C57BL/6-AngII: 80.6±11.8μmol/l versus STAT1-/- -AngII: 55.0±8.4μmol/l; P&lt;0.05). Mesenteric arteries of STAT1-/- mice were fully protected against angiotensin induced endothelial dysfunction. Candesartan (AT1 receptor antagonist) inhibited action of Ang II in C57BL/6 and reduced but not reversed expression of chemokines in the heart. Conclusions: Lack of STAT1 revealed protection against Ang II-induced cardiac fibrosis and endothelial dysfunction. We suggest that STAT1-induced chemokine activation induces chemotaxis into hypertensive target organs and modifies cardiac fibrosis and vascular dysfunction in a blood pressure-independent manner.

Characterization of blood pressure and endothelial function in TRPV4-deficient mice with l -NAME- and angiotensin II-induced hypertension

Transient receptor potential vanilloid type 4 (TRPV4) is an endothelial Ca2+ entry channel contributing to endothelium‐mediated dilation in conduit and resistance arteries. We investigated the role of TRPV4 in the regulation of blood pressure and endothelial function under hypertensive conditions. TRPV4‐deficient (TRPV4−/−) and wild‐type (WT) control mice were given l‐NAME (0.5 g/L) in drinking water for 7 days or subcutaneously infused with angiotensin (Ang) II (600 ng/kg per minute) for 14 days, and blood pressure measured by radiotelemetry. TRPV4−/− mice had a lower baseline mean arterial pressure (MAP) (12‐h daytime MAP, 94 ± 2 vs. 99 ± 2 mmHg in WT controls). l‐NAME treatment induced a slightly greater increase in MAP in TRPV4−/− mice (day 7, 13 ± 4%) compared to WT controls (6 ± 2%), but Ang II‐induced increases in MAP were similar in TRPV4−/− and WT mice (day 14, 53 ± 6% and 37 ± 11%, respectively, P < 0.05). Chronic infusion of WT mice with Ang II reduced both acetylcholine (ACh)‐induced dilation (dilation to 10−5 mol/L ACh, 71 ± 5% vs. 92 ± 2% of controls) and the TRPV4 agonist GSK1016790A‐induced dilation of small mesenteric arteries (10−8 mol/L GSK1016790A, 14 ± 5% vs. 77 ± 7% of controls). However, Ang II treatment did not affect ACh dilation in TRPV4−/− mice. Mechanistically, Ang II did not significantly alter either TRPV4 total protein expression in mesenteric arteries or TRPV4 agonist‐induced Ca2+ response in mesenteric endothelial cells in situ. These results suggest that TRPV4 channels play a minor role in blood pressure regulation in l‐NAME‐ but not Ang II‐induced hypertension, but may be importantly involved in Ang II‐induced endothelial dysfunction.

Abstract 300: Targeted Deletion of Matrix Metalloproteinase 2 Prevents Angiotensin II-Induced Endothelial Dysfunction and Vascular Remodeling, Oxidative Stress and Inflammation

Background: Matrix metalloproteinase 2 (MMP2) is involved in vascular remodeling in atherosclerosis. Whether MMP2 plays a role in angiotensin (Ang) II-induced hypertension, vascular remodeling, oxidative stress and inflammation is unknown. We hypothesized that Mmp2 knockout will prevent Ang II-induced vascular injury. Methods: Ten to 12-week-old male Mmp2 knockout ( Mmp2 -/- ) and wild type (WT) mice were infused with Ang II (1000 ng/kg/min, sc) for 14 days. Systolic blood pressure (SBP) was measured by telemetry. Mesenteric arteries (MA) were studied by pressurized myography. NADPH oxidase activity was evaluated by lucigenin chemiluminescence, and aortic reactive oxygen species (ROS) generation using dihydroethidium staining. Aortic expression of vascular cell adhesion protein 1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1), and monocyte/macrophage infiltration were assessed by immunofluorescence. Spleen T cells and monocytes were assessed by flow cytometry. Results: Ang II increased SBP by 50 mmHg ( P &lt;0.01), decreased vasodilatory responses to acetylcholine by 70 % ( P &lt;0.01), induced MA hypertrophic remodeling, indicated by a 1.5-fold increase ( P &lt;0.01) in media-to-lumen ratio and 1.3-fold increase ( P &lt;0.05) in media cross-sectional area, and enhanced MA stiffness ( P &lt;0.01), as shown by a leftward shift of the stress/strain relationship, in WT mice. Ang II increased NADPH oxidase activity 1.4-fold in aorta ( P &lt;0.05), 2-fold in the heart ( P &lt;0.01) and 2.6-fold in the renal cortex ( P &lt;0.01) and aortic ROS generation 25-fold in WT mice ( P &lt;0.01). Ang II increased aortic VCAM-1 and MCP-1 expression 3- and 6-fold, respectively, and monocyte/macrophage infiltration 8-fold in WT ( P &lt;0.05). Ang II increased ≥1.8-fold spleen activated CD4 + CD69 + and CD8 + CD69 + T cells and pro-inflammatory Ly-6C hi monocytes ( P &lt;0.001) in WT mice. Mmp knockout prevented or reduced all of the above except SBP elevation ( P &lt;0.05). Conclusion: MMP2 plays a role in Ang II-induced endothelial dysfunction, vascular remodeling, oxidative stress and inflammation but not in blood pressure elevation.