Ang II-treated Mice Research Articles

Changes in endogenous sulfur dioxide pathway in angiotensin II- induced myocardial hypertrophy in mice

Objective To explore the changes in the endogenous sulfur dioxide (SO2) pathway in the myocardial hypertrophy induced by the angiotensin Ⅱ(Ang Ⅱ) in mice. Methods Fourteen healthy C57BL mice, 9 weeks old, were randomly divided into control group(n=7) and Ang Ⅱ group(n=7), and capsule osmotic pump with pre-loaded 9 g/L saline and Ang Ⅱ was implanted into the back of each mouse subcutaneously.After 2 weeks, the mice were executed.The heart weight/body weight (HW/BW) and the left heart weight/full heart weight (LVW/HW) of the mice were measured.The microstructure of the cardiac myocyte was observed by hematoxylin-eosin (HE) staining under the microscope.The expression of myocardial alpha myosin heavy chain (α-MHC) was detected by immunohistochemistry and Western blot methods.SO2 enzymes aspartate aminotransferase 1 (AAT1) and AAT2 protein expression were detected by Western blot method.Myocardial SO2 content and AAT activity were measured by high performance liquid chromatography with fluorimetric detection and colometric method. Results Compared with control group, the HW/BW and LVW/HW in mice of Ang Ⅱ group were significantly increased(all P<0.01), the cardiac myocytes were hypertrophy, and α-MHC positive staining in the cytoplasm of myocardium was weakened.Moreover, Western blot data showed that α-MHC protein expression in heart tissue of Ang Ⅱ-treated mice was decreased significantly (all P<0.05). Simultaneously, the data showed that AAT2 protein expression, SO2 content and AAT activity in heart tissue of Ang Ⅱ-treated mice were also decreased markedly[(1.093±0.131) μmol/g protein vs.(0.737±0.233) μmol/g protein, P<0.05; (7.979±1.317) U/mg protein vs.(6.470±0.516) U/mg protein, P<0.01]. Furthermore, there was a negative correlation between LVW/HW and cardiac SO2 content in heart tissue (r=-0.56, P<0.05). Conclusions Myocardial endogenous SO2/AAT2 pathway is down-regulated in the development of myocardial hypertrophy induced by Ang Ⅱ in mice. Key words: Myocardial hypertrophy; Sulfur dioxide; Angiotensin Ⅱ

Cardiac STAT3 Deficiency Impairs Contractility and Metabolic Homeostasis in Hypertension.

Signal transducer and activator of transcription 3 (STAT3) protects the heart from acute ischemic stress. However, the importance of STAT3 to the heart in chronic stress, such as hypertension, is not known. To study this, we used cardiomyocyte-targeted STAT3 knockout (KO) mice and Angiotensin II (ANG II) infusion by osmotic minipumps. After 4 weeks, ANG II induced similar cardiac hypertrophy in wild type (WT) and cardiac Cre-expressing control (CTRL) mice with no impairment of cardiac function. In contrast, STAT3 KO mice exhibited reduced contractile function but similar hypertrophy to CTRL mice. Ejection fraction and fractional shortening decreased by 22.5 and 27.3%, respectively. Since STAT3 has direct protective effects on mitochondrial function, we examined rates of glucose and oleate oxidation by isolated perfused hearts using a Langendorff system. Hearts of ANG II-treated STAT3 KO and CTRL mice had similar rates of oleate oxidation as saline-infused WT mice. Rates of glucose oxidation were similar between hearts of WT plus saline and CTRL plus ANG II mice; however, glucose oxidation was increased by 66% in hearts of ANG II-treated STAT3 KO mice. The ratio of maximal ATP yield from glucose to fatty acid oxidation was 21.1 ± 3.1 in hearts of ANG II-treated STAT3 KO mice vs. 12.6 ± 2.2 in hearts of ANG II-treated CTRL mice. Lactate production was also elevated in hearts of ANG II-treated STAT3 KO mice by 162% compared to ANG II-treated CTRL mice. Our findings indicate that STAT3 is important for maintaining contractile function and metabolic homeostasis in the hypertensive heart, and STAT3 deficiency promotes a switch toward glucose utilization.

Abstract P604: Astrocyte-dependent Regulation of Vascular Tone: Role in Hypertension

Clinical studies support that untreated hypertension (HT) accelerates the development of vascular cognitive impairment (VCI). Yet, the underlying mechanisms for VCI are not known. In a recent study we demonstrated the role of astrocytes in the regulation of parenchymal arteriole (PA) steady-state vascular tone. Here we hypothesized hypertension results in structural and functional changes to the neurovascular unit resulting in enhanced astrocytic TRPV4 channel-dependent Ca 2+ increases contributing to augmented pressure-induced PA constriction . Functional studies were conducted in brain slices from angiotensin II (AngII) treated mice (600 ng/Kg/min, 28 days). PA arterioles within brain slices were perfused and pressurized and myogenic-evoked diameter changes measured using video microscopy. In addition, using the GLAST-CreERT2 ; R26-lsl-GCaMP3 mice we measure myogenic-evoked Ca 2+ changes in perivascular astrocytes. We demonstrate that HT increases pressure-induced PA tone by 11.14% at 30 mmHg and 12.97% at 60 mmHg (10.88 to 22.02 and 15.46 to 28.43% of tone, P&lt;0.05 and P&lt;0.01, respectively). In ANG II-treated mice, PA myogenic-evoked responses significantly increased astrocytic Ca 2+ oscillations frequency (119.4%, 0.0366 to 0.0803 Hz, P&lt;0.0001). A significant increase in astrocytic Ca 2+ oscillation frequency was also observed after 2 min of AngII (500 nM) bath application (44.8%, 0.0366 to 0.053 Hz, P&lt;0.01) in brain slices from AngII treated mice. Furthermore, using the model of spontaneous hypertensive rat (SHR) we observed that HT differentially increases vascular density and the number of vascular pericytes in cortical layers with highest neuronal densities (L III-V). Finally, while aquaporin 4 (AQP4) expression pattern was not different in the gray matter of SHR compared with WKY rats, a significant increase in unpolarized AQP4 expression was observed in the white matter of SHR. Taken together, this evidence indicates that HT induces functional and structural changes to the neurovascular unit favoring the development of regional brain hypoperfusion likely contributing to the development of VCI.

Abstract 001: Hypertension Induces Heightened Activity of the Adaptive Immune System

Adaptive immunity plays a key role in the pathogenesis of hypertension, but how does hypertension affect immunity? To study this, splenic dendritic cells (DC) and peritoneal macrophages were isolated from normotensive and hypertensive angiotensin II (AngII)-infused C57BL6/J mice (490 ng/kg/min, 2 wk). These antigen presenting cells (APCs) were loaded with ovalbumin (OVA) or the OVA MHC class I epitope SIINFEKL (SKL) for 3h. Then, splenocytes from OT-I mice, containing OVA-specific, CD8 + T cells (OT-I cells), were added for 4h. Flow cytometry showed significantly more activated T cells expressing CD69 when stimulated with APCs from AngII-treated mice than equivalent cells from naïve mice (As example, DC+OVA: 2.9 ± 0.4% vs. 1.8 ± 0.3% ,P&lt;0.05; DC+SKL: 13.9 ± 0.9 % vs. 7.4 ± 1.1%, P&lt;0.01;). To study antigen presentation in vivo, we immunized AngII and sham treated mice with OVA and adjuvant. Consistently more OVA-specific CD8 + T cells were induced in the blood (2.7 ± 0.2% vs. 1.2 ± 0.4%, P&lt;0.05) and the spleen (2.6 ± 0.3% vs. 1.5 ± 0.2%, P&lt;0.05) of hypertensive mice vs. sham when measured by flow using a H-2k b -SKL tetramer. RIP-mOVA mice were also used to study if hypertension affects APC cross-presentation of self-antigens. This transgenic mouse line expresses membrane-bound OVA in pancreatic islet β cells and kidney proximal tubular cells. When OT-I cells (5x10 6 ) are injected into RIP-mOVA mice, they activate by cross-presentation of OVA and cause insulitis and diabetes. RIP-mOVA mice were made hypertensive with either AngII or L-NAME. When OT-I cells were infused into hypertensive RIP-mOVA mice, they rapidly developed much higher blood glucose levels as compared to equivalently treated normotensive RIP-mOVA mice. For example, comparing mice 3 weeks after AngII and 1 wk after OT-I cells to normotensive mice 1 wk after OT-I cells, blood glucose was 331 ± 47 mg/dl in the hypertensive mice vs. 168 ± 39 mg/dl in the normotensive controls. Also, far more pancreatic islets were infiltrated by T cells in the hypertensive mice (AngII 78%, 31 of 40; L-NAME 72%, 21 of 29; control 13%, 10 of 75). In conclusion, hypertension itself is associated with higher activity of APC presentation of foreign and self-antigens which may explain why hypertension induces inflammation.

Abstract 085: The Role of CD70 in Vascular Function

We have recently shown that CD70 is important in formation of effector memory T cells and that these cells seem to enhance the hypertensive response and end organ damage caused by repeated hypertensive stimuli. CD70 has traditionally been identified on antigen presenting cells and is thought to interact with CD27 on T cells, promoting proliferation and memory cell formation. The precise cell types that express CD70 and the role of CD70 on non-immune cells has not previously been investigated. In our prior study, we showed that hypertension is associated with a marked increase in CD70 surface expression on dendritic cells (DCs) and macrophages. To examine the specific effect of DC CD70, we performed adoptive transfer of DCs from hypertensive WT mice to normotensive CD70 -/- mice and from hypertensive CD70 -/- mice to normotensive WT mice. The recipient mice were treated with a normally subpressor dose of ang II (140 ng/kg/min). Interestingly the CD70 -/- recipients were protected from the development of hypertension, despite receiving WT DCs from hypertensive donors. In contrast, the WT recipeints that received CD70 -/- DCs developed modest hypertension, indicating that non-DC sources of CD70 likely contribute to the hypertensive phenotype. Studies of mesenteric vascular reactivity showed that CD70 -/- mice have markedly impaired endothelium-dependent vasodilatation to acetylcholine compared to WT mice (43±10 vs 25 ± 3 %) at baseline. In contrast, there were no differences in relaxation responses to sodium nitroprusside. In additional experiments, we showed that human umbillical vein endothelial cells express CD70 mRNA and that this is increased by &gt; 30 fold by laminar shear stress (15 dynes/cm2) compared to oscillatory shear. Finally, using immunohistochemistry, we identified CD70 protein localized to resistance vessels of the kidney of ang II-treated mice. These data identify a new role of CD70 in modulating vascular function independent of its role on antigen presenting cells in memory T cell formation.

Angiotensin II increases coronary fibrosis, cardiac hypertrophy and the incidence of myocardial infarctions in ApoE-/-Fbn1C1039G+/- mice

Objective Apolipoprotein E deficient mice (ApoE-/-) with a heterozygous mutation in the fibrillin-1 gene (Fbn1C1039G+/-) develop increased arterial stiffness, accelerated plaque progression, spontaneous plaque ruptures, myocardial infarction (MI) and sudden death. In the present study we used angiotensin II (Ang II) infusion to assess the responsiveness of the ApoE-/-Fbn1C1039G+/-mouse model to risk factors of atherosclerosis, such as high blood pressure and inflammation.Methods and results Osmotic minipumps filled with phosphate buffered saline or Ang II (1,000 ng/kg/day) were implanted in ApoE-/- Fbn1C1039G+/-mice at week 14 of Western diet (WD). After 4 weeks of therapy, we were not able to detect an increase in blood pressure, but we observed severe aneurysms in both the thoracic and abdominal aorta of Ang II treated mice. Plaque size and composition in the proximal ascending aorta was not altered. Nevertheless, coronary fibrosis, cardiac hypertrophy and a 6-fold increase in the occurrence of MI in Ang II-treated mice was observed, which did not result in a higher mortality.Conclusion The study showed that Ang II treatment in ApoE-/-Fbn1C1039G+/-mice increased cardiac hypertrophy, coronary fibrosis and the incidence of MI, without affecting blood pressure, plaque vulnerability and survival.

Abstract 434: Sirt4 Accelerates Ang II-induced Pathological Cardiac Hypertrophy by Inhibiting Mnsod Activity

Aims: Oxidative stress contributes to the development of cardiac hypertrophy and heart failure. One of the mitochondrial sirtuins, Sirt4, is highly expressed in the heart, but its function remains unknown. The aim of the present study was to investigate the role of Sirt4 in the pathogenesis of pathological cardiac hypertrophy and the molecular mechanism by which Sirt4 regulates mitochondrial oxidative stress. Methods and results: Male C57BL/6 Sirt4 knockout mice, transgenic mice exhibiting cardiac-specific overexpression of Sirt4 (Sirt4-Tg) and their respective controls were treated with angiotensin II (Ang II). At 4 weeks, hypertrophic growth of cardiomyocytes, fibrosis and cardiac function were analyzed. Sirt4 deficiency conferred resistance to Ang II infusion by significantly suppressing hypertrophic growth, and the deposition of fibrosis. In Sirt4-Tg mice, aggravated hypertrophy and reduced cardiac function were observed compared with non-transgenic mice following Ang II treatment. Mechanistically, Sirt4 inhibited the binding of manganese superoxide dismutase (MnSOD) to Sirt3, another member of the mitochondrial sirtuins, and increased MnSOD acetylation levels to reduce its activity, resulting in elevated reactive oxygen species (ROS) accumulation upon Ang II stimulation. Furthermore, inhibition of ROS with MnTBAP, a mimetic of SOD, blocked the Sirt4-mediated aggravation of the hypertrophic response in Ang II-treated Sirt4-Tg mice. Conclusions: Sirt4 promotes hypertrophic growth and cardiac dysfunction by increasing ROS levels upon pathological stimulation. These findings reveal a role of Sirt4 in pathological cardiac hypertrophy, providing a new potential therapeutic strategy for this disease.

Chronic p38 mitogen-activated protein kinase inhibition improves vascular function and remodeling in angiotensin II-dependent hypertension.

Introduction:An excess of angiotensin II (Ang II) causes hypertension and vascular injury. Activation of mitogen-activated protein kinase p38 (p38-MAPK) plays a substantial role in Ang II-dependent organ damage. Recently, we showed that p38-MAPK activation regulates the pressor response to Ang II. This study evaluates the effect of chronic p38-MAPK inhibition in Ang II-dependent hypertension.Materials and methods:C57Bl/6J mice were infused with Ang II for 14 days and either treated with the p38-MAPK inhibitor BIRB796 (50 mg/kg/day) or the vehicle as the control. We assessed vascular function in the aorta and isolated perfused kidneys.Results:Chronic p38-MAPK inhibition did not alter blood pressure at the baseline, but attenuated Ang II-induced hypertension significantly (baseline: 122 ± 2 versus 119 ± 4 mmHg; Ang II: 173 ± 3 versus 155 ± 3 mmHg; p < 0.001). In addition, BIRB796 treatment improved vascular remodeling by reducing the aortic media-to-lumen ratio and decreasing the expression of the membrane metalloproteinases (MMP) MMP-1 and MMP-9. Moreover, renal vascular dysfunction induced by chronic Ang II infusion was significantly ameliorated in the BIRP796-treated mice. Acute p38-MAPK inhibition also improved vascular function in the aorta and kidneys of Ang II-treated mice, highlighting the important role of p38-MAPK activation in the pathogenesis of vascular dysfunction.Conclusions:Our findings indicated there is an important role for p38-MAPK in regulating blood pressure and vascular injury, and highlighted its potential as a pharmaceutical target.

SIRT4 accelerates Ang II-induced pathological cardiac hypertrophy by inhibiting manganese superoxide dismutase activity.

Oxidative stress contributes to the development of cardiac hypertrophy and heart failure. One of the mitochondrial sirtuins, Sirt4, is highly expressed in the heart, but its function remains unknown. The aim of the present study was to investigate the role of Sirt4 in the pathogenesis of pathological cardiac hypertrophy and the molecular mechanism by which Sirt4 regulates mitochondrial oxidative stress. Male C57BL/6 Sirt4 knockout mice, transgenic (Tg) mice exhibiting cardiac-specific overexpression of Sirt4 (Sirt4-Tg) and their respective controls were treated with angiotensin II (Ang II, 1.1 mg/kg/day). At 4 weeks, hypertrophic growth of cardiomyocytes, fibrosis and cardiac function were analysed. Sirt4 deficiency conferred resistance to Ang II infusion by significantly suppressing hypertrophic growth, and the deposition of fibrosis. In Sirt4-Tg mice, aggravated hypertrophy and reduced cardiac function were observed compared with non-Tg mice following Ang II treatment. Mechanistically, Sirt4 inhibited the binding of manganese superoxide dismutase (MnSOD) to Sirt3, another member of the mitochondrial sirtuins, and increased MnSOD acetylation levels to reduce its activity, resulting in elevated reactive oxygen species (ROS) accumulation upon Ang II stimulation. Furthermore, inhibition of ROS with manganese 5, 10, 15, 20-tetrakis-(4-benzoic acid) porphyrin, a mimetic of SOD, blocked the Sirt4-mediated aggravation of the hypertrophic response in Ang II-treated Sirt4-Tg mice. Sirt4 promotes hypertrophic growth, the generation of fibrosis and cardiac dysfunction by increasing ROS levels upon pathological stimulation. These findings reveal a role of Sirt4 in pathological cardiac hypertrophy, providing a new potential therapeutic strategy for this disease.

Combination of angiotensin II and l-NG-nitroarginine methyl ester exacerbates mitochondrial dysfunction and oxidative stress to cause heart failure.

Mitochondrial dysfunction has been implicated as a cause of energy deprivation in heart failure (HF). Herein, we tested individual and combined effects of two pathogenic factors of nonischemic HF, inhibition of nitric oxide synthesis [with l-N(G)-nitroarginine methyl ester (l-NAME)] and hypertension [with angiotensin II (AngII)], on myocardial mitochondrial function, oxidative stress, and metabolic gene expression. l-NAME and AngII were administered individually and in combination to mice for 5 wk. Although all treatments increased blood pressure and reduced cardiac contractile function, the l-NAME + AngII group was associated with the most severe HF, as characterized by edema, hypertrophy, oxidative stress, increased expression of Nppa and Nppb, and decreased expression of Atp2a2 and Camk2b. l-NAME + AngII-treated mice exhibited robust deterioration of cardiac mitochondrial function, as observed by reduced respiratory control ratios in subsarcolemmal mitochondria and reduced state 3 levels in interfibrillar mitochondria for complex I but not for complex II substrates. Cardiac myofibrils showed reduced ADP-supported and oligomycin-inhibited oxygen consumption. Mitochondrial functional impairment was accompanied by reduced mitochondrial DNA content and activities of pyruvate dehydrogenase and complex I but increased H2O2 production and tissue protein carbonyls in hearts from AngII and l-NAME + AngII groups. Microarray analyses revealed the majority of the gene changes attributed to the l-NAME + AngII group. Pathway analyses indicated significant changes in metabolic pathways, such as oxidative phosphorylation, mitochondrial function, cardiac hypertrophy, and fatty acid metabolism in l-NAME + AngII hearts. We conclude that l-NAME + AngII is associated with impaired mitochondrial respiratory function and increased oxidative stress compared with either l-NAME or AngII alone, resulting in nonischemic HF.

Thrombospondin-4 knockout in hypertension protects small-artery endothelial function but induces aortic aneurysms.

Thrombospondin-4 (TSP-4) is a multidomain calcium-binding protein that has both intracellular and extracellular functions. As an extracellular matrix protein, it is involved in remodeling processes. Previous work showed that, in the cardiovascular system, TSP-4 expression is induced in the heart in response to experimental pressure overload and infarction injury. Intracellularly, it mediates the endoplasmic reticulum stress response in the heart. In this study, we explored the role of TSP-4 in hypertension. For this purpose, wild-type and TSP-4 knockout (Thbs4(-/-)) mice were treated with angiotensin II (ANG II). Hearts from ANG II-treated Thbs4(-/-) mice showed an exaggerated hypertrophic response. Interestingly, aortas from Thbs4(-/-) mice treated with ANG II showed a high incidence of aneurysms. In resistance arteries, ANG II-treated wild-type mice showed impaired endothelial-dependent relaxation. This was not observed in ANG II-treated Thbs4(-/-) mice or in untreated controls. No differences were found in the passive pressure-diameter curves or stress-strain relationships, although ANG II-treated Thbs4(-/-) mice showed a tendency to be less stiff, associated with thicker diameters of the collagen fibers as revealed by electron microscopy. We conclude that TSP-4 plays a role in hypertension, affecting cardiac hypertrophy, aortic aneurysm formation, as well as endothelial-dependent relaxation in resistance arteries.

Angiotensin 1-7 Protects against Angiotensin II-Induced Endoplasmic Reticulum Stress and Endothelial Dysfunction via Mas Receptor.

Angiotensin 1–7 (Ang 1–7) counter-regulates the cardiovascular actions of angiotensin II (Ang II). The present study investigated the protective effect of Ang 1–7 against Ang II-induced endoplasmic reticulum (ER) stress and endothelial dysfunction. Ex vivo treatment with Ang II (0.5 μM, 24 hours) impaired endothelium-dependent relaxation in mouse aortas; this harmful effect of Ang II was reversed by co-treatment with ER stress inhibitors, l4-phenylbutyric acid (PBA) and tauroursodeoxycholic acid (TUDCA) as well as Ang 1–7. The Mas receptor antagonist, A779, antagonized the effect of Ang 1–7. The elevated mRNA expression of CHOP, Grp78 and ATF4 or protein expression of p-eIF2α and ATF6 (ER stress markers) in Ang II-treated human umbilical vein endothelial cells (HUVECs) and mouse aortas were blunted by co-treatment with Ang 1–7 and the latter effect was reversed by A779. Furthermore, Ang II-induced reduction in both eNOS phosphorylation and NO production was inhibited by Ang 1–7. In addition, Ang 1–7 decreased the levels of ER stress markers and augmented NO production in HUVECs treated with ER stress inducer, tunicamycin. The present study provides new evidence for functional antagonism between the two arms of the renin-angiotensin system in endothelial cells by demonstrating that Ang 1–7 ameliorates Ang II-stimulated ER stress to raise NO bioavailability, and subsequently preserves endothelial function.

P-165: Protective role of nucleotidases against the development of hypertension

Background Hypertension is associated with a hypertrophic remodeling of big arteries, oxidative stress and inflammation. Extracellular nucleotides, released under stress condition, play a role in various physiopathological conditions through P2 receptors activation. Hydrolysis of nucleotides is achieved by nucleoside triphosphate diphosphohydrolases, in particular NTPDase1 (CD39) which is highly expressed in the arterial wall. Together with ecto-5’nucleotidase (CD73), these ectoenzymes generate vasoprotective adenosine. The contribution of the purinergic signaling in cardiovascular diseases, hypertension in particular, remains to be established. Here, we investigate the potential benefit of extracellular nucleotidase in experimental hypertension by treating hypertensive mice with the potato ATPDase, Apyrase. Methods Experimental hypertension was induced by Angiotensin II (AngII, subcutaneous 1mg/kg/day, osmotic pumps). Endogenous CD39 arterial expression and serum activity of hypertensive mice was evaluated by quantitative RT-PCR and by HPLC respectively (incubated with fluorescent etheno-ADP as a substrate). To replete mice with exogenous nucleotidase activity mice were treated or not with Apyrase (APY 90U/kg/day and 500U/kg ip every 3 days). Blood pressure was recorded daily by tail cuff plethys-mography until day 12 the day of the sacrifice. Then, media thickness of thoracic aorta was measured by histomorphometry, oxidative stress was evaluated with dihydroethidium staining and Immune cells infiltration was assessed by Immunofluorescence using pan immune cells marker (CD45) or macrophages marker (F4/80). Data were corroborated by quantitative RT-PCR. Results We found a decrease in both CD39 arterial expression and serum activity in AngII-treated mice, suggesting a link of the endogenous ectonucleotidase CD39 to hypertension. In accordance with a protective role of nucleotidase activity, blood pressure increase and hypertrophic arterial remodeling were significantly reduced in AngII/APY-treated mice compared to AngII alone. Moreover, RT-qPCR and immunofluorescence experiments evidenced decreased perivascular immune cells infiltration and oxidative stress in AngII/APY-treated mice compared to AngII-treated mice. Conclusions Nucleotidase activities protect against vascular alterations associated to hypertension while the expression of CD39 decreases with the pathology. Nucleotidase may represent new therapeutic treatment for vascular diseases.

Cardiac acetylcholine inhibits ventricular remodeling and dysfunction under pathologic conditions

Autonomic dysfunction is a characteristic of cardiac disease and decreased vagal activity is observed in heart failure. Rodent cardiomyocytes produce de novo ACh, which is critical in maintaining cardiac homeostasis. We report that this nonneuronal cholinergic system is also found in human cardiomyocytes, which expressed choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). Furthermore, VAChT expression was increased 3- and 1.5-fold at the mRNA and protein level, respectively, in ventricular tissue from patients with heart failure, suggesting increased ACh secretion in disease. We used mice with genetic deletion of cardiomyocyte-specific VAChT or ChAT and mice overexpressing VAChT to test the functional significance of cholinergic signaling. Mice deficient for VAChT displayed an 8% decrease in fractional shortening and 13% decrease in ejection fraction compared with angiotensin II (Ang II)-treated control animals, suggesting enhanced ventricular dysfunction and pathologic remodeling in response to Ang II. Similar results were observed in ChAT-deficient mice. Conversely, no decline in ventricular function was observed in Ang II-treated VAChT overexpressors. Furthermore, the fibrotic area was significantly greater (P < 0.05) in Ang II-treated VAChT-deficient mice (3.61 ± 0.64%) compared with wild-type animals (2.24 ± 0.11%). In contrast, VAChT overexpressing mice did not display an increase in collagen deposition. Our results provide new insight into cholinergic regulation of cardiac function, suggesting that a compensatory increase in cardiomyocyte VAChT levels may help offset cardiac remodeling in heart failure.

Abstract 075: Proinflammatory T Helper 17 Cells as an Indicator of Hypertension

Objectives: T cells and interleukin 17 (IL17) have been shown to be critical in the development of hypertension (HTN). This implicates a significant role of IL17 producing T helper 17 (Th17) cells in HTN. Thus, the objective of our study was to investigate if Th17 cell levels in the peripheral blood (PB) and the bone marrow (BM) are correlated with blood pressure. Methods: Saline or Ang II was chronically infused into C57BL6 mice (1000ng Ang II/kg/min using osmotic pumps) for 3 weeks. This resulted in an increase in MAP of 45±10 mmHg. BM and PB from saline and Ang II-treated mice were analyzed using FACS. A parallel human study was conducted using blood samples obtained from hypertensive patients (n = 8, systolic BP ≥125 mmHg) and normotensive subjects (n = 10, systolic BP &lt;125 mmHg). FACS analysis was used to examine changes in the inflammatory cells levels in these patients. Results: We observed 87% and 36% increases in both Sca-1 + c-Kit + Lin - hematopoietic stem cell (HSC; 0.23±0.04% vs. 0.43±0.05%) and Sca-1 + c-Kit - Lin - lymphoid progenitors (10.8±1.1% vs. 14.7±2.9%) in the BM of Ang II infused mice. These are upstream stem/progenitor cells for T cells. This was associated with 30% and 190% increases in CD4 + IL17 + Th17 cells in the BM and PB (1.20±0.09% vs. 1.61±0.19 % and 4.8±2.0 % vs. 14.7±3.8 % respectively) in the Ang II infused mice. Importantly, there were 58% and 206% increases of angiotensin II type 1 receptor (AT1R) expressing CD4 + T cells in the BM and PB of Ang II HTN mice (0.40±0.04 % vs. 0.63±0.14 % and 1.1±0.2 % vs 3.4±1.1 % respectively) and ~ 85% of these cells were also positive for IL-17. Consistent with mouse data, analysis of PB showed a 470% increase of Th17 cells in HTN patients (0.48±0.18 % vs 2.72±1.2%). Conclusions: We observed (i) Increased hematopoietic stem/progenitor cells in Ang II HTN mice; (ii) increased Th17 cells in both HTN mice and humans and (iii) the majority of AT1R expressing CD4 + T cells was Th17 cells. Taken together, these observations indicate that Th17 cells may be an important indicator of those destined to develop HTN and suggest that these cells may represent a novel therapeutic target.

Abstract 074: An Obligatory Role for B Cells in the Development of Angiotensin II-dependent Hypertension

Background: Clinical hypertension is associated with raised serum antibody levels. However, no studies have examined whether B cells and antibodies play causative roles in the pathogenesis of hypertension. We investigated whether experimental hypertension is similarly associated with elevated IgG production, and if B cell/antibody deficiency affords protection against hypertension and associated vascular remodeling. Methods and Results: Ang II-dependent hypertension in mice was associated with B cell activation/maturation as revealed by a: (1) a 25% increase in the proportion of splenic B cells that expressed the activation marker CD86; (2) an 80% increase in numbers of splenic plasma cells, (3) a 500% increase in serum IgG levels; and (4) marked accumulation of IgG deposits in the aortic adventitia. Indicative of a causative role for B cells, B cell activating factor-receptor-deficient (BAFF-R-/-) mice, which lack mature B cells, displayed blunted hypertensive responses to chronic angiotensin II infusion (Δ30±4 mmHg) compared to wild-type (WT) mice (Δ41±5 mmHg). Importantly, adoptive transfer of B cells into BAFF-R-/- mice restored the hypertensive response. BAFF-R-/- mice showed no evidence of Ang II-induced increases in aortic IgG accumulation. They also had 80% fewer F4/80+ macrophages, 60% fewer CD4+ T cells, and 70% reduction in TGF-β expression and collagen in their aortas compared to Ang II-treated wild-type mice. Ang II doubled aortic pulse-wave velocity, a measure of vessel stiffening, whereas BAFF-R-/- mice were largely protected (70% reduced) from this effect. Finally, pharmacological depletion of B cells with an anti-CD20 antibody blunted Ang II-induced hypertension (Δ37±9 mmHg vs Δ57±6 mmHg). Conclusions: We provide the first evidence that B cells are activated during development of hypertension and contribute to vessel remodeling and elevated blood pressure. These findings suggest B cells and/or the IgG antibodies they produce could be targeted for anti-hypertensive therapy.

Abstract P106: The Role of Inflammasomes and Angiotensin II Type 1 Receptor in the Pressor Responses of Aged Mice to Angiotensin II

Introduction: The prevalence of hypertension increases with age. Chronic low-grade inflammation commonly occurs with ageing, and inflammasomes are important initiators of inflammatory responses. We tested whether aged mice have enhanced pressor responses to angiotensin II (Ang II) and whether this is associated with exaggerated pro-inflammatory and vascular contractile responses. We also tested whether MCC950, a NLRP3 inflammasome inhibitor, reduces blood pressure (BP) in Ang II-treated aged mice. Methods: Young (8-12 weeks) and aged (24-30 months) male C57Bl6 mice were left untreated, or either treated with vehicle or a slow-pressor dose of Ang II (0.28 mg/kg/day) for 28 days. Another group of aged mice were treated with either Ang II + saline or Ang II + MCC950 (10 mg/kg/day) for 10 days. We measured systolic BP, mRNA expression of inflammatory markers and components of the renin-angiotensin system, and vascular contractile responses. Results: In young mice, Ang II caused a gradual increase in BP (final BP: 141.6 ± 8.3 mmHg), whereas BP increased by ~20 mmHg from baseline in aged mice, and continued to increase for 28 days (final BP: 155 ± 12.4 mmHg) (n=8-9, P&lt;0.05). Ageing alone increased renal expression of AT1a receptors, NLRP3, Caspase-1, IL-1β, IL-33, CCR2, CCL7 and CCL8 by at least 1.5-fold (n=7-8, all P&lt;0.05). Maximum contractile responses of mesenteric artery were 1.8-fold greater to Ang II and 1.2-fold greater to phenylephrine in aged vs young mice (n=4, both P&lt;0.05). BP was not different between Ang II + saline-treated aged mice (BP: 138.8 ± 6.8 mmHg) and Ang II + MCC950-treated aged mice (BP: 144.7 ± 9.6 mmHg) (n=6-7, P&gt;0.05). Conclusions: Aged mice have enhanced pressor responses to Ang II and this is associated with exacerbated pro-inflammatory and vascular contractile responses. The NLRP3 inflammasome does not appear to contribute to Ang II-induced hypertension in aged mice.

M2 macrophage accumulation in the aortic wall during angiotensin II infusion in mice is associated with fibrosis, elastin loss, and elevated blood pressure.

Macrophages accumulate in blood vessels during hypertension. However, their contribution to vessel remodeling is unknown. In the present study, we examined the polarization state of macrophages (M1/M2) in aortas of mice during hypertension and investigated whether antagonism of chemokine receptors involved in macrophage accumulation reduces vessel remodeling and blood pressure (BP). Mice treated with ANG II (0.7 mg·kg(-1)·day(-1), 14 days) had elevated systolic BP (158 ± 3 mmHg) compared with saline-treated animals (122 ± 3 mmHg). Flow cytometry revealed that ANG II infusion increased numbers of CD45(+)CD11b(+)Ly6C(hi) monocytes and CD45(+)CD11b(+)F4/80(+) macrophages by 10- and 2-fold, respectively. The majority of macrophages were positive for the M2 marker CD206 but negative for the M1 marker inducible nitric oxide synthase. Expression of other M2 genes (arginase-1, Fc receptor-like S scavenger receptor, and receptor-1) was elevated in aortas from ANG II-treated mice, whereas M1 genes [TNF and chemokine (C-X-C motif) ligand 2] were unaltered. A PCR array to identify chemokine receptor targets for intervention revealed chemokine (C-C motif) receptor 2 (CCR2) to be upregulated in aortas from ANG II-treated mice, while flow cytometry identified Ly6C(hi) monocytes as the main CCR2-expressing cell type. Intervention with a CCR2 antagonist (INCB3344; 30 mg·kg(-1)·day(-1)), 7 days after the commencement of ANG II infusion, reduced aortic macrophage numbers. INCB334 also reduced aortic collagen deposition, elastin loss, and BP in ANG II-treated mice. Thus, ANG II-dependent hypertension in mice is associated with Ly6C(hi) monocyte and M2 macrophage accumulation in the aorta. Inhibition of macrophage accumulation with a CCR2 antagonist prevents ANG II-induced vessel fibrosis and elevated BP, highlighting this as a promising approach for the future treatment of vessel remodeling/stiffening in hypertension.

Src Family Kinases (SFK) Mediate Angiotensin II-Induced Myosin Light Chain Phosphorylation and Hypertension.

Angiotensin (Ang) II is the major bioactive peptide of the renin–angiotensin system (RAS); it contributes to the pathogenesis of hypertension by inducing vascular contraction and adverse remodeling, thus elevated peripheral resistance. Ang II also activates Src family kinases (SFK) in the vascular system, which has been implicated in cell proliferation and migration. However, the role of SFK in Ang II-induced hypertension is largely unknown. In this study, we found that administration of a SFK inhibitor SU6656 markedly lowered the level of systemic BP in Ang II-treated mice, which was associated with an attenuated phosphorylation of the smooth-muscle myosin-light-chain (MLC) in the mesenteric resistant arteries. In the cultured human coronary artery smooth muscle cells (SMCs), pretreatment with SU6656 blocked Ang II-induced MLC phosphorylation and contraction. These results for the first time demonstrate that SFK directly regulate vascular contractile machinery to influence BP. Thus our study provides an additional mechanistic link between Ang II and vasoconstriction via SFK-enhanced MLC phosphorylation in SMCs, and suggests that targeted inhibition of Src may provide a new therapeutic opportunity in the treatment of hypertension.

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.