Ang II-induced Hypertension Research Articles

Downregulation of Brain Gα12 Attenuates Angiotensin II-Dependent Hypertension.

Angiotensin II (Ang II) activates central Angiotensin II type 1 receptors to increase blood pressure via multiple pathways. However, whether central Gα proteins contribute to Ang II-induced hypertension remains unknown. We hypothesized that Angiotensin II type 1 receptors couple with Gα12 and/or Gαq to produce sympatho-excitation and increase blood pressure and downregulation of these Gα-subunit proteins will attenuate Ang II-dependent hypertension. After chronic infusion of Ang II (s.c. 350 ng/kg/min) or vehicle for 2 weeks, Ang II evoked an increase in Gα12 expression, but not Gαq in the rostral ventrolateral medulla of Sprague-Dawley rats. In other studies, rats that received Ang II or vehicle infusion s.c. were simultaneously infused i.c.v. with a scrambled (SCR) or Gα12 oligodeoxynucleotide (ODN; 50 µg/day). Central Gα12 ODN infusion lowered mean blood pressure in Ang II infused rats compared with SCR ODN infusion (14-day peak; 133 ± 12 vs. 176 ± 11 mm Hg). Compared to the SCR ODN group, Ang II infused rats that received i.c.v. Gα12 ODN showed a greater increase in heart rate to atropine, an attenuated reduction in blood pressure to chlorisondamine, and an improved baroreflex sensitivity. In addition, central Gα12 and Gαq ODN pretreatment blunted the pressor response to an acute i.c.v. injection of Ang II (i.c.v., 200 ng). These findings suggest that central Gα12 protein signaling pathways play an important role in the development of chronic Ang II-dependent hypertension in rats.

Chronic central miR-29b antagonism alleviates angiotensin II-induced hypertension and vascular endothelial dysfunction

Dysregulation of miR-29 has been revealed in multiple diseases, but its role in the development of hypertension and vascular endothelial dysfunction has not been defined. Here, we found that, compared with the wild-type (WT) Wistar rats, miR-29b was robustly upregulated in spontaneously hypertensive rats (SHRs), while CTRP6 was distinctly downregulated. There were two miRNA-responding-elements (MREs) for miR-29 in the 3′-UTR of CTRP6 mRNA, and the luciferase activity assay revealed that miR-29b directly targeted CTRP6 mRNA. Intraventricular injection was applied to deliver the miR-29b mimic or miR-29b inhibitor (4 mg/kg) into SHRs once two weeks from 10th week. Downregulation of miR-29b could increase serum CTRP6 content in SHRs, decrease the arterial systolic pressure, reduce serum concentrations of Ang II and ET-1, and enhance serum NO content. Meanwhile, we demonstrated that inhibition of miR-29b increased the phosphorylation of ERK1/2 to activate PPARγ, an inducer of Ang II. Finally, miR-29b expression was manipulated in, and CTRP6 recombinant protein was applied to incubate with the primary aortic endothelial cells. Inhibition of miR-29b increased CTRP6 expression, improved cell proliferation and migration, suppressed secretion of Ang II and ET-1, and decreased ROS accumulation and LDH release, displaying a similar effect to the CTRP6 recombinant protein. Moreover, the CTRP6 recombinant protein could antagonize the suppressive effect of miR-29b on activation of the ERK/PPARγ axis and function of aortic endothelial cells. In conclusion, miR-29b antagonism can alleviate Ang II-induced hypertension and vascular endothelial dysfunction through activating the CTRP6/ERK/PPARγ axis.

Abstract P2029: Cytochrome P450 (CYP) 1B1-Estradiol Metabolite 2-Methoxyestradiol Protects Against Angiotensin II-induced Hypertension By Inhibiting CYP4A1 Expression <And> 20-Hydroxyeicosatetraenoic Acid (20-HETE) Production In The Female Mice

Previously we reported that angiotensin (Ang) II-induced hypertension is mediated by group IV cytosolic phospholipase A 2 α (cPLA 2 α) activation resulting in arachidonic acid (AA) release, and generation predominantly of eicosanoids with prohypertensive effects. Androgen dihydrotestosterone increases the expression of AA metabolizing enzyme cytochrome P450 (CYP) 4A1 and production of 20-HETE in the male rats and mice and an increase in blood pressure (BP). However, female mice that are resistant to Ang II-induced hypertension produce lower renal levels of 20-HETE, compared with males. Cyp4a12a, which has been identified as the predominant 20-HETE producing synthase in the mouse kidney, is expressed in very low levels in the female compared to male mice. Previously, we showed that the protection against Ang II-induced hypertension and associated pathogenesis in the female mice are mediated by CYP1B1-estradiol (E2) generated metabolite 2-methoxyestradiol (2-ME). These observations raised the possibility that CYP1B1-E2 metabolite 2-ME might protect against Ang II-induced hypertension by inhibiting expression of CYP4A1 and 20-HETE production. To test this hypothesis, we examined the effect of 20-HETE receptor antagonist 20-SOLA on Ang II (700 ng/kg/min, s.c. for two weeks)-induced hypertension in 8 weeks old wild type intact, ovariectomized (OVX), and intact Cyp1b1 -/- female mice. The systolic blood pressure (SBP, mmHg) was measured by tail-cuff. At the end of 2 weeks, Ang II caused greater increase in SBP in OVX- Cyp1b1 +/+ (161±6) and Cyp1b1 -/- intact female mice (165±2) compared to the intact Cyp1b1 +/+ mice (128±7) (p<0.05, n=4-5). 20-SOLA (10 mg/kg/day, s.c.), the 20-HETE receptor antagonist minimized Ang II-induced increase in SBP in OVX- Cyp1b1 +/+ (116±4, Δ45) and Cyp1b1 -/- female mice (135±9, Δ30) (P<0.05, n= 4-5). The expression of CYP4A1 was increased in the kidney of OVX- Cyp1b1 +/+ and intact Cyp1b1 -/- compared to intact Cyp1b1 +/+ mice, which was inhibited by E2 in the former but not the later mice; however, 2-ME inhibited CYP4A1 expression in the intact Cyp1b1 -/- mice. These data suggest that CYP1B1-E generated metabolite 2-ME protects against Ang II-induced hypertension by inhibiting the expression of CYP4A1 and production of 20-HETE.

Abstract 077: Proximal Tubule-Specific Deletion of the NHE3 (na + /h + Exchanger 3) in the Kidney Attenuates Angiotensin II-Induced Hypertension in Mice

The Na + /H + exchanger 3 (NHE3), a ~85 kDa protein encoded by the SLC9A3 gene, is the most important Na + transporter in the proximal tubules of the kidney. Angiotensin II (ANG II) is well-recognized to increase proximal tubule Na + reabsorption by stimulating NHE3 expression and activity in the proximal tubules. However, a direct cause and effect relationship between ANG II and NHE3 in the proximal tubules in ANG II-induced hypertension has not been determined previously. The present study directly tested the hypothesis that NHE3 in the proximal tubules of the kidney is required for the development of ANG II-induced hypertension using proximal tubule-specific NHE3 knockout mice (PT- Nhe3 -/- ). Specifically, PT- Nhe3 -/- mice were generated using the SGLT2-Cre / Nhe3 loxlox approach, whereas ANG II-induced hypertension was studied in 12 groups (n=5-12 per group) of adult male and female wild-type (WT) and PT- Nhe3 -/- mice. Under basal conditions, systolic (SBP), diastolic (DBP), and mean arterial blood pressure (MAP) were significantly lower in male and female PT- Nhe3 -/- than WT mice ( P <0.01). A high pressor, 1.5 mg/kg/day, i.p., or a slow pressor dose of ANG II, 0.5 mg/kg/day, i.p., for 2 weeks significantly increased SBP, DBP, and MAP in male and female WT mice ( P <0.01), but the hypertensive response to ANG II was markedly attenuated in male and female PT- Nhe3 -/- mice ( P <0.01). ANG II impaired the pressure-natriuresis response in WT mice, whereas proximal tubule-specific deletion of NHE3 improved the pressure-natriuresis response in ANG II-infused PT- Nhe3 -/- mice ( P <0.01). AT 1 receptor blocker losartan completely blocked ANG II-induced hypertension in both WT and PT- Nhe3 -/- mice ( P <0.01). However, inhibition of nitric oxide synthase with L-NAME had no effect on ANG II-induced hypertension in WT or PT- Nhe3 -/- mice (n.s.). Furthermore, ANG II-induced hypertension was significantly attenuated by an orally absorbable NHE3 inhibitor AVE0657. In conclusion, NHE3 in the proximal tubules of the kidney is required for the full development of ANG II-induced hypertension. Our results suggest that NHE3 in the proximal tubules may be therapeutically targeted to treat hypertension induced by ANG II or associated with increased NHE3 expression in the proximal tubules.

Abstract P1113: P2x7 Receptor Plays a Role in Angiotensin II-Induced Hypertension and Vascular Remodeling

Introduction: Inflammasome activation by binding of ATP released from damaged cells to the purinergic receptor P2X7 (P2RX7) could play a role in hypertension and vascular injury through release of interleukin (IL)-1β and immune cell activation. Elevated ATP levels were observed in the renal interstitial fluid of angiotensin (Ang) II-infused rats, and treatment of these rats with a non-selective P2 receptor blocker prevented Ang II-induced inflammation and renal damage. P2RX7 knockout ( P2rx7 –/– ) prevented deoxycorticosterone acetate-salt-induced blood pressure (BP) elevation and renal damage. However, it is unknown whether P2RX7 plays a role in Ang II-induced BP elevation and vascular damage. We hypothesize that Ang II-induced hypertension, vascular injury, and inflammation will be blunted in P2rx7 -/- mice. Methods: Ten to 12-week-old male C57BL/6J male wild-type (WT) and P2rx7 –/– mice were sham-treated or infused with Ang II (490ng/kg/min) for 14 days. BP was determined by telemetry. Mesenteric artery function and remodeling was assessed using pressurized myography. P2RX7 expression in spleen immune cells was determined by flow cytometry. IL-1β secretion from bone marrow-derived macrophages (BMDM) isolated from WT and P2rx7 –/– mice was assessed by ELISA. Results: Stimulation with lipopolysaccharides and ATP caused IL-1β release in WT (339±162 pg/mL) but not P2rx7 –/– BMDMs. In WT mice, Ang II increased P2RX7 expression in dendritic cells (mean fluorescence intensity: 1927±120 vs 3983±983) and macrophages (2541±265 vs 3314±273 respectively). P2rx7 knockout reduced Ang II-induced systolic (159±3 vs 165±7 mm Hg) and diastolic BP elevation (110±4 vs 126 ±7 mm Hg) compared to WT mice. Acetylcholine-induced vascular relaxation was unaffected by Ang II or P2rx7 knockout. Ang II increased the media-to-lumen ratio in WT (4.71±0.18% vs 3.89±0.21%, P <0.01) but not in P2rx7 –/– mice. Conclusion: This study demonstrated that P2RX7 plays a role in Ang II-induced hypertension and vascular remodeling.

Abstract P120: Leukocyte and Lymphocyte-Derived Extracellular Vesicles are Elevated in Angiotensin-Induced Hypertension

Infiltration of macrophages and T cells in kidneys and vasculature may play a role in the development of hypertension (HTN) and extracellular vesicles (EVs) have emerged as novel cell-to-cell communicators in the pathogenesis HTN. Immune cell-derived EVs can reflect the pathophysiological activity of the parental cell and mediate immune stimulation or inhibition in paracrine fashion. We investigated subgroups of circulating leukocyte-derived EVs (LEVs) in angiotensin II (Ang II)-induced HTN with or without candesartan (C) or hydralazine, hydrochlorothiazide, and reserpine (HHR). Ang II was delivered via osmotic mini pumps (500 ng/kg/min; n=15) and with C (10 mg/kg/day, n=5) or HHR (30 mg/kg/day, n=5) in drinking water for 4 weeks to 129S6 mice. Systolic blood pressure (SBP) was measured daily by tail-cuff manometry. After 4 weeks, mice were euthanized and citrated blood collected. EV isolation was performed by differential centrifugation, followed by EV phenotyping with imaging flow cytometry. We used EV surface markers for leukocyte (CD45), T- and B-cells (CD3,CD19), neutrophils (Ly6g+/CD11b-) and monocytes/macrophages (Ly6g-/CD11b+). Ang II infused mice had significantly higher SBP compared to normotensive controls (NC) (n=5) (173.79 ± 4.9mmHg vs 129.8 ± 6.2mmHg, p <0.0001) and C/HHR treated mice (134.42 ± 7.5mmHg (C) and 138.21 ± 5.2mmHg (HHR), p = NS). Leukocyte-derived EV counts (CD45+ EVs for Ang II: 2.16E+06 particles/ml; NC: 6.15E+05 particles/mL; p=0.0001) and T cell-derived EV counts (CD3+) (Ang II: 2.14E+06 particles/ml, NC: 4.46E+05 particles/mL; p=0.0001] increased after 4 weeks in Ang II-infused mice compared to NC. In C and HHR treated groups, leukocyte- and T cell-derived EV concentrations were similar to NC, suggesting a BP effect rather than drug class effect. CD45+ and CD3+ EV concentrations correlated significantly with SBP [(r2=0.9343; p= 0.0004) and (r2= 0.8982; p= 0.0012), respectively]. In conclusion, leukocyte- and T cell-derived EVs were increased in Ang II-induced HTN and correlated significantly with SBP. Therefore, leukocyte- and T cell-derived EVs are potential bio-markers in HTN. Further studies are required to understand the functional role of leukocyte- and T cell-derived EVs in the pathogenesis of HTN.

Abstract P1108: Serum Immunoglobulins are Not Necessary for Experimental Hypertension

The role of B cells and serum immunoglobulins (Ig) in hypertension is unclear. Elevated serum IgG has been observed in some hypertensive patients and in animal models of hypertension. Mice with a severe B cell deficiency due to lack of B cell activating factor receptor (BAFF-R) and mice lacking the cytokine interleukin 21 (IL21) - which is a potent inducer of B cell Ig class switching and high affinity Ig production - are protected from Ang II-induced hypertension and exhibit reduced serum IgG levels. Thus, we sought to determine whether hypertensive IgGs play a causative role in hypertension and end-organ dysfunction. First, we purified serum IgG from normotensive and hypertensive wild type (WT) mice and adoptively transferred equal amounts of IgG by intraperitoneal injection into normotensive recipient mice followed by infusion of a sub-pressor dose of angiotensin II (Ang II) for 2 weeks. Flow cytometric analysis of aortas revealed a modest increase in F4/80+ macrophages (p=0.04) and CD8+ T cells (p=0.02) in mice that received hypertensive IgG. However, there was no difference in endothelial dependent relaxation, renal inflammation, albuminuria, and blood pressure (BP) between the two groups. As an alternative method to investigate the role of class-switched high affinity Igs, we studied mice with genetic deletion of activation-induced deaminase (AID) which is an enzyme necessary for somatic hypermutation (to generate high affinity Igs) and isotype class switching. AID -/- mice exhibit undetectable IgG and higher levels of IgM in serum compared to WT mice. Ang II infusion for 4 weeks revealed no difference in BP, aortic inflammation, renal inflammation, and albuminuria. We also studied mice with deficiency of the IgM heavy chain (uMT). These mice exhibit undetectable levels of all immunoglobulins and are severely deficient in mature B cells. uMT-/- mice are also not protected from hypertension induced by Ang II infusion or deoxycorticosterone acetate (DOCA-salt) treatment. Together, these results demonstrate that while elevated serum immunoglobulins seen in hypertensive animals and humans may represent a biomarker of aberrant immune activation, they are likely not playing a causal role in hypertension pathophysiology.

Abstract 102: Genetic Deletion of Sirtuin 3 (SIRT3) in the Mitochondria of the Proximal Tubules Aggravates Angiotensin II-Induced Hypertension by Impairing the Pressure-Natriuresis Response in Proximal Tubule-Specific SIRT3 Knockout Mice

Sirtuin 3 (SIRT3), a key mitochondrial protein in the sirtuin family, plays a key role in maintaining normal mitochondrial function via its anti-oxidative, anti-aging, and anti-inflammatory effects. Conversely, angiotensin II (ANG II), a key peptide of the renin-angiotensin system, plays a key role in the development of cardiovascular and hypertensive injury through its vasoconstrictive, pro-oxidative, pro-growth, and proinflammatory actions, in part by suppressing mitochondrial SIRT3 expression and inducing mitochondrial dysfunction. The present study tested the hypothesis that genetic deletion of SIRT3 selectively in the proximal tubules of the kidney aggravates ANG II-induced hypertension in proximal tubule (PT)-specific SIRT3 knockout mice (PT-SIRT3-KO). PT-SIRT3-KO mice were generated using the SGLT2-Cre/SIRT3-loxP approach. ANG II-dependent hypertension was induced by infusing a slow pressor dose of ANG II, 0.5 mg/kg/day, i.p., and a 2% Na + diet for 2 weeks in adult male wildtype (WT) and PT-SIRT3-KO mice (n=7-12 per group). PT-SIRT3-KO mice developed normally, and showed no abnormal cardiac and kidney structures. Under basal conditions, systolic, diastolic and mean arterial pressure were significantly lower (WT: 119 ± 3 mmHg vs. PT-SIRT3-KO: 111 ± 3 mmHg, P <0.05), whereas urinary Na + excretion was significantly higher in PT-SIRT3-KO than WT mice (WT: 167.9 ± 5.8 μmol/24 h vs. PT-SIRT3-KO: 217.9 ± 7.9 μmol/24 h, P <0.05), without altering urinary K + excretion. ANG II infusion significantly increased systolic blood pressure by 24 ± 3 mmHg in WT mice ( P <0.01), whereas it increased systolic blood pressure by 38 ± 3 mmHg in PT-SIRT3-KO mice ( P <0.01). Conversely, ANG II-induced hypertension was associated with a marked natriuretic response in WT mice (547.7 ± 19.9 μmol/24 h), which was attenuated in PT-SIRT3-KO mice (312.1 ± 11.1 μmol/24 h, P <0.01). Proximal tubule-specific SIRT3 deletion slightly increased the kidney to body wt. ratio (n.s.), but significantly attenuated the heart to body wt. ratio in PT-SIRT3-KO mice ( P <0.01). Our results suggest that genetic deletion of mitochondrial SIRT3 in the proximal tubules of the kidney aggravates ANG II-induced hypertension by impairing the pressure-natriuretic response in PT-SIRT3-KO mice.

Abstract 023: γδ T Cells Mediate αβ T Cell Activation in Angiotensin II-Induced Hypertension

Objectives: Both innate and adaptive immune cells have been shown to play a role in hypertension and vascular injury. Recently, we demonstrated that a small subset of “innate-like” T lymphocytes, expressing the γδ T cell receptor (TCR) rather than the αβ TCR, plays a key role in hypertension and vascular injury. We demonstrated an increased number and activation (CD69 + ) of αβ and γδ T cells during the development of hypertension caused by angiotensin (Ang) II infusion, and that deficiency in γδ T cells prevented Ang II-induced hypertension, resistance artery endothelial dysfunction and spleen T-cell activation in mice. We hypothesized that γδ T cells mediate activation of other T cells in hypertension. Methods: C57BL/6 male mice were infused with Ang II (490 ng/kg/min, SC) for 7 and 14 days (n=5-7). All mice were 14-15 week-old at the end of the study. Spleen T cell profile was determined by flow cytometry. γδ and αβ T cells were isolated using magnetic beads from peripheral lymph nodes and spleen from C57BL/6 male mice treated or not with Ang II for 14 days. αβ T cells were cultured alone or with γδ T cells (5:1) in presence of anti-CD3 antibodies plus or minus Ang II, and αβ T cell phenotype was evaluated by flow cytometry. Results: Close correlations were demonstrated between the number (#) of activated CD69 + γδ T cells and CD4 + CD69 + T cells (r 2 =0.74, P <0.01) and CD8 + CD69 + T cells (r 2 =0.64, P <0.01) after 7-day Ang II infusion. Correlations were also shown between the # of CD27 + CD69 + γδ T cells and CD4 + CD69 + T cells (r 2 =0.76, P <0.001) and CD8 + CD69 + T cells (r 2 =0.65, P <0.01) after 7-day Ang II infusion. In vitro , Ang II increased the fraction of CD69 + αβ T cells when αβ T cells were co-cultured with γδ T cells isolated from control mice (% of αβ T cells: 19.9±2.9 vs. 16.4±2.6, P <0.001) but not when cultured alone. The fractions of CD69 + (% of CD69 + αβ T cells: 35.7±2.7 vs. 18.9±1.5, P <0.001) and CCR6 + αβ T cells (% of CCR6 + αβ T cells: 27.6±2.4 vs. 19.2±3.5, P <0.05) were increased when αβ T cells were co-cultured with γδ T cells isolated from Ang II-infused compared to control mice. Conclusions: These results suggest that γδ T cells mediate activation of αβ T cells in Ang II-induced hypertension. Targeting γδ T cells may contribute to reduce the low-grade inflammation found in hypertension.

Abstract P2014: Prolyl Endopeptidase (PEP) is the Main Angiotensin (1-7) Forming Enzyme From Angiotensin II (1-8) in the Circulation: Implications for Hypertension

The AngII(1-8)-Ang(1-7) axis of the Renin Angiotensin System (RAS) encompasses three enzymes that form Ang(1-7) directly from AngII: Angiotensin Converting Enzyme 2 (ACE2), Prolyl Carboxypeptidase (PRCP) and Prolyl Endopeptidase (PEP). We studied their relative contribution to Ang(1-7) formation in-vivo and also ex-vivo in serum, lungs and kidneys using models of genetic ablation coupled with pharmacological inhibitors. In WT mice infusion of AngII resulted in a rapid increase of plasma Ang(1-7). In a model of ACE2 and PRCP double genetic ablation AngII infusion resulted in a similar increase in Ang(1-7) as in WT (505 ± 46 vs 482 ± 63 pg/ml, respectively), showing that the bulk of Ang(1-7) formation in circulation is essentially independent of ACE2 and PRCP. By contrast, a PEP inhibitor, ZPP, obliterated the rise in plasma Ang(1-7) after infusing AngII(1-8) to control WT mice. In a genetic mouse model of PEP deficiency (PEP -/- ) the increase in Ang(1-7) was also blunted as compared to WT mice (277±41 and 423±25 pg/ml, respectively p=0.01). The rate of recovery from acute AngII(1-8)-induced hypertension, in PEP -/- mice moreover, was delayed as compared to WT mice (Figure). In ex vivo studies PEP inhibition with ZZP reduced Ang(1-7) formation from Ang(1-8) in serum and in lung lysates. By contrast, in kidney lysates the absence of ACE2, but not PEP, obliterated Ang1-7 formation from added AngII. We conclude that PEP is the main enzyme responsible for AngII conversion to Ang(1-7) in the circulation and in the lungs. PEP, not ACE2, is responsible for protecting against AngII-induced hypertension.

Abstract P148: Transcriptomic and Proteomic Analysis of CD4 + T Cells to Identify Sex Differences in Angiotensin II Signaling Pathways

T cells are involved in hypertension pathogenesis in both males and postmenopausal females while premenopausal females are resistant to T cell-mediated Ang II-induced hypertension. The goals of this study were (1) to identify T cell specific proteomic pathways associated with postmenopausal susceptibility to hypertension (2) to identify T cell specific transcriptomic changes associated with premenopausal protection from hypertension. Proteomic analysis was performed on splenic CD4 + T cells isolated from premenopausal and postmenopausal females (VCD, 160 mg/kg/day i.p. 20d) following Ang II infusion (800 ng/kg/min 14d). 384 proteins from CD4 + T cells were identified as differentially expressed following Ang II infusion in premenopausal females. 285 proteins from CD4 + T cells were identified as differentially expressed between premenopausal and postmenopausal females following Ang II infusion. Gene ontology (GO) analysis of pre vs. postmenopausal proteins identified enriched pathways associated with RNA binding, chaperone activity and cellular stress responses. Transcriptomic changes were analyzed, via RNAseq, on isolated splenic CD4 + T cells from premenopausal females, with and without Ang II infusion. Thirty-four genes were identified as differentially expressed in CD4 + T cells following Ang II infusion. GO analysis of Ang II upregulated genes revealed an enrichment of five distinct molecular functions, including antioxidant activity. In a subsequent study to validate the RNAseq, we confirmed that Ang II increased CD4 + T cell mRNA expression of calprotectin (S100a8/S100a9), a calcium and zinc binding protein complex that contributes to antioxidant defense (S100a8: Con 1.0 ± 0.4 vs Ang II 5.0 ± 0.8*; S100a9: Con 1.0 ± 0.4 vs Ang II 6.0 ± 0.8*, *P<0.05 vs Con). Furthermore, we determined that Ang II did not increase calprotectin expression in mice lacking estrogen receptor α (ERKO) (S100a8: ERKO 0.1 ± 0.4 vs ERKO/Ang II 0.3 ± 0.7; S100a9: ERKO 0.2 ± 0.5 vs ERKO/Ang II 0.3 ± 0.6). The current studies demonstrate a role for estrogen in Ang II-induced T cell gene expression and signal transduction, and begin to elucidate the molecular mechanisms of female protection from T cell-mediated hypertension.

G Protein-Coupled Estrogen Receptor Protects From Angiotensin II-Induced Increases in Pulse Pressure and Oxidative Stress.

Our previous work showed that the G protein-coupled estrogen receptor (GPER) is protective in the vasculature and kidneys during angiotensin (Ang) II-dependent hypertension by inhibiting oxidative stress. The goal of the current study was to assess the impact of GPER deletion on sex differences in Ang II-induced hypertension and oxidative stress. Male and female wildtype and GPER knockout mice were implanted with radiotelemetry probes for measurement of baseline blood pressure before infusion of Ang II (700 ng/kg/min) for 2 weeks. Mean arterial pressure was increased to the same extent in all groups, but female wildtype mice were protected from Ang II-induced increases in pulse pressure, aortic wall thickness, and Nox4 mRNA. In vitro studies using vascular smooth muscle cells found that pre-treatment with the GPER agonist G-1 inhibited Ang II-induced ROS and NADP/NADPH. Ang II increased while G-1 decreased Nox4 mRNA and protein. The effects of Ang II were blocked by losartan and Nox4 siRNA, while the effects of G-1 were inhibited by adenylyl cyclase inhibition and mimicked by phosphodiesterase inhibition. We conclude that during conditions of elevated Ang II, GPER via the cAMP pathway suppresses Nox4 transcription to limit ROS production and prevent arterial stiffening. Taken together with our previous work, this study provides insight into how acute estrogen signaling via GPER provides cardiovascular protection during Ang II hypertension and potentially other diseases characterized by increased oxidative stress.

Reduction of glyoxalase 1 (GLO1) aggravates cerebrovascular remodeling via promoting the proliferation of basilar smooth muscle cells in hypertension

Uncontrollable vascular smooth cell proliferation is responsible for vascular remodeling during hypertension development. Glyoxalase 1 (GLO1), the major enzyme detoxifying methylglyoxal, has a critical role in regulating proliferation of several cell types. However, little is known whether GLO1 is involved in cerebrovascular remodeling and basilar smooth muscle cell (BASMC) proliferation during hypertension. Here we explored the role of GLO1 in angiotensin II (Ang II)-induced cerebrovascular remodeling and proliferation of BASMCs and the underlying mechanisms. The protein expression of GLO1 in basilar arteries from hypertensive mice was decreased, and GLO1 expression was negatively correlated with medial cross-sectional area and blood pressure in basilar arteries during hypertension. Knockdown of GLO1 promoted while overexpression of GLO1 prevented Ang II-induced cell proliferation and cell cycle transition in BASMCs. These results were related to the inhibitory effects of GLO1 on PI3K/AKT/CDK2 cascade activation upon Ang II treatment. In addition, in vivo study, GLO1 overexpression with adeno-associated virus harboring GLO1 cDNA improved cerebrovascular remodeling in basilar artery tissue during Ang II-induced hypertension development. These data indicate that GLO1 reduction mediates cerebrovascular modeling via PI3K/AKT/CDK2 cascade-dependent BASMC proliferation. GLO1 acts as a negative regulator of hypertension-induced cerebrovascular remodeling and targeting GLO1 may be a novel therapeutic strategy to prevent hypertension-associated cardiovascular complications such as stroke.

LncRNA AK094457 promotes AngII-mediated hypertension and endothelial dysfunction through suppressing of activation of PPARγ

Hypertension is one of the major risk factors for cardiovascular disease worldwide and is striking more young people, which is characterized by impaired vascular endothelial function. To find the functional lncRNAs associated with hypertension, high throughput lncRNA microarray were used to analyze expression profile of the lncRNAs in the aortic vascular endothelial cells (VECs) of spontaneously hypertensive rats (SHRs). The tail vein injection of siRNA was used to study the influence of lncRNA AK094457 inhibition on endothelial function in vivo. In vitro, endothelial function was studied in endothelial cells transfected with lncRNA AK094457-overexpressed vectors and siRNAs. pPPARγ and iNOS protein levels were detected with Western blot. Elisa assay was used to analyze the secretion of AngII, ET-1, ROS and LDH level. The nitrite/nitrate (NO2−/NO3−) concentration was measured using a colorimetric assay. LncRNA AK094457 was a most upregulated lncRNA in SHRs. It is showed that downregulation of AK094457 significantly reduced rat arterial pressure, increased activation of endothelial PPARγ, and suppressed serum contents of AngII and NO in vivo. Furthermore, results from gain-and-loss of function in primary aortic endothelial cells indicated that AK094457 negatively regulated activation of PPARγ and promoted AngII-mediated endothelial dysfunction, manifested by decreased capacities of cell proliferation and migration, and increased levels of ROS production and LDH release. In conclusion, lncRNA AK094457 is identified as a key regulator in blood pressure and endothelial function, which can increase AngII-induced hypertension and endothelial dysfunction via suppression of PPARγ.

Proximal Tubule-Specific Deletion of the NHE3 (Na + /H + Exchanger 3) in the Kidney Attenuates Ang II (Angiotensin II)-Induced Hypertension in Mice

The present study directly tested the hypothesis that the NHE3 (Na+/H+ exchanger 3) in the proximal tubules of the kidney is required for the development of Ang II (angiotensin II)-induced hypertension using PT-Nhe3-/- (proximal tubule-specific NHE3 knockout) mice. Specifically, PT-Nhe3-/- mice were generated using the SGLT2-Cre/Nhe3loxlox approach, whereas Ang II-induced hypertension was studied in 12 groups (n=5-12 per group) of adult male and female wild-type (WT) and PT-Nhe3-/- mice. Under basal conditions, systolic blood pressure, diastolic blood pressure, and mean arterial blood pressure were significantly lower in male and female PT-Nhe3-/- than WT mice (P<0.01). A high pressor, 1.5 mg/kg per day, intraperitoneal or a slow pressor dose of Ang II, 0.5 mg/kg per day, intraperitoneal for 2 weeks significantly increased systolic blood pressure, diastolic blood pressure, and mean arterial blood pressure in male and female WT mice (P<0.01), but the hypertensive response to Ang II was markedly attenuated in male and female PT-Nhe3-/- mice (P<0.01). Ang II impaired the pressure-natriuresis response in WT mice, whereas proximal tubule-specific deletion of NHE3 improved the pressure-natriuresis response in Ang II-infused PT-Nhe3-/- mice (P<0.01). AT1 receptor blocker losartan completely blocked Ang II-induced hypertension in both WT and PT-Nhe3-/- mice (P<0.01). However, inhibition of nitric oxide synthase with L-NG-Nitroarginine methyl ester had no effect on Ang II-induced hypertension in WT or PT-Nhe3-/- mice (not significant). Furthermore, Ang II-induced hypertension was significantly attenuated by an orally absorbable NHE3 inhibitor AVE0657. In conclusion, NHE3 in the proximal tubules of the kidney may be a therapeutical target in hypertension induced by Ang II or with increased NHE3 expression in the proximal tubules.

Hypoxia inducible factor 1\u03b1 in vascular smooth muscle cells promotes angiotensin II-induced vascular remodeling via activation of CCL7-mediated macrophage recruitment

The process of vascular remodeling is associated with increased hypoxia. However, the contribution of hypoxia-inducible factor 1α (HIF1α), the key transcription factor mediating cellular hypoxic responses, to vascular remodeling is established, but not completely understood. In the angiotensin II (Ang II)-induced vascular remodeling model, HIF1α was increased and activated in vascular smooth muscle cells (VSMCs). Selective genetic disruption of Hif1a in VSMCs markedly ameliorated Ang II-induced vascular remodeling, as revealed by decreased blood pressure, aortic thickness, collagen deposition, inflammation, and aortic stiffness. VSMC Hif1a deficiency also specifically suppressed Ang II-induced infiltration of CD45+CD11b+F4/80+CD206− M1 macrophages into the vessel. Mechanistically, HIF1α deficiency in VSMCs dramatically suppressed the expression of CCL7, a chemokine critical for macrophage recruitment. Bioinformatic analysis and chromatin immunoprecipitation assays revealed three functional hypoxia-response elements in the Ccl7 promoter, indicating that Ccl7 is a direct HIF1α target gene. Blocking CCL7 with antibody in vivo alleviated Ang II-induced hypertension and vascular remodeling, coincident with decreased macrophage infiltration. This study provides direct evidence that HIF1α activation in VSMCs exacerbates Ang II-induced macrophage infiltration and resultant vascular remodeling via its target gene Ccl7, and thus may serve as a potential therapeutic target for remodeling-related vascular disease.

Angiopoietin-Like 7 Contributes to Angiotensin II-Induced Proliferation, Inflammation and Apoptosis in Vascular Smooth Muscle Cells

Introduction: Angiotensin II (AngII) induces hypertension and pathophysiological vascular thickening and atherosclerosis. This study aims to validate the effects of Angiopoietin-like 7 (ANGPTL7) in AngII-induced hypertension. Methods: ANGPTL7 in blood samples were determined by quantitative real-time polymerase chain reaction. AngII-induced cell growth were detected by CCK-8. Cell cycle arrest and cell apoptosis by downregulation of ANGPTL7 were detected by flow cytometric assay. AngII-induced inflammation was evaluated by Western blotting and ELISA. Results: ANGPTL7 was highly expressed in patients with hypertension. AngII promoted cell viability and the expression level of ANGPTL7 in vascular smooth muscle cells (VSMC). Downregulation of ANGPTL7 inhibited AngII-induced cell proliferation and cell inflammation. Moreover, ANGPTL7 expression decreases also promoted cell apoptosis. Conclusions: Downregulation of ANGPTL7 reversed AngII-induced cell proliferation and cell inflammation and promoted apoptosis in AngII-induced VSMC cells. Therefore, ANGPTL7 can be a potential target in AngII-induced hypertension.

Smooth muscle-specific TMEM16A expression protects against angiotensin II-induced cerebrovascular remodeling via suppressing extracellular matrix deposition

Cerebrovascular remodeling is the leading factor for stroke and characterized by increased extracellular matrix deposition, migration and proliferation of vascular smooth muscle cells, and inhibition of their apoptosis. TMEM16A is an important component of Ca2+-activated Cl− channels. Previously, we showed that downregulation of TMEM16A in the basilar artery was negatively correlated with cerebrovascular remodeling during hypertension. However, it is unclear whether TMEM16A participates in angiotensin II (Ang II)-induced vascular remodeling in mice that have TMEM16A gene modification. In this study, we generated a transgenic mouse that overexpresses TMEM16A specifically in vascular smooth muscle cells. We observed that vascular remodeling in the basilar artery during Ang II-induced hypertension was significantly suppressed upon vascular smooth muscle-specific overexpression of TMEM16A relative to control mice. Specifically, we observed a large reduction in the deposition of fibronectin and collagen I. The expression of matrix metalloproteinases (MMP-2, MMP-9, and MMP-14), and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were upregulated in the basilar artery during Ang II-induced hypertension, but this was suppressed upon overexpression of TMEM16A in blood vessels. Furthermore, TMEM16A overexpression alleviated the overactivity of the canonical TGF-β1/Smad3, and non-canonical TGF-β1/ERK and JNK pathways in the basilar artery during Ang II-induced hypertension. These in vivo results were similar to the results derived in vitro with basilar artery smooth muscle cells stimulated by Ang II. Moreover, we observed that the inhibitory effect of TMEM16A on MMPs was mediated by decreasing the activation of WNK1, which is a Cl−-sensitive serine/threonine kinase. In conclusion, this study demonstrates that TMEM16A protects against cerebrovascular remodeling during hypertension by suppressing extracellular matrix deposition. We also showed that TMEM16A exerts this effect by reducing the expression of MMPs via inhibiting WNK1, and decreasing the subsequent activities of TGF-β1/Smad3, ERK, and JNK. Accordingly, our results suggest that TMEM16A may serve as a novel therapeutic target for vascular remodeling.

Rosmarinic acid improves hypertension and skeletal muscle glucose transport in angiotensin II-treated rats

BackgroundRosmarinic acid (RA) is a natural pure compound from herbs belonging to the Lamiaceae family, such as rosemary, sage, basil, and mint. The antioxidant, angiotensin-converting enzyme inhibitory, and vasodilatory effects of RA have been revealed. Angiotensin II (ANG II) is a potent agent that generates hypertension and oxidative stress. Hypertension and skeletal muscle insulin resistance are strongly related. The aim of this study was to evaluate the effects of acute and chronic RA treatment on blood pressure and skeletal muscle glucose transport in ANG II-induced hypertensive rats.MethodsEight-week-old male Sprague Dawley rats were separated into SHAM and ANG II-infused (250 ng/kg/min) groups. ANG II rats were treated with or without acute or chronic RA at 10, 20, or 40 mg/kg. At the end of the experiment, body weight, liver and heart weights, oral glucose tolerance, skeletal muscle glucose transport activity, and signaling proteins were evaluated.ResultsBoth acute and chronic RA treatment decreased systolic, diastolic, and mean arterial blood pressure. Only acute RA at 40 mg/kg resulted in a reduction of fasting plasma glucose levels and an induction of skeletal muscle glucose transport activity. These effects might involve increased ERK activity in skeletal muscle. Meanwhile, chronic RA treatment with 10, 20, and 40 mg/kg prevented ANG II-induced hyperglycemia.ConclusionsBoth acute and chronic RA treatment attenuated ANG II-induced cardiometabolic abnormalities in rats. Therefore, RA would be an alternative strategy for improving skeletal muscle glucose transport and protecting against ANG II-induced hypertension and hyperglycemia.

SAT-109 NEW INSIGHTS INTO THE NOVEL ROLES OF ANGIOTENSIN AT1A RECEPTORS IN THE PROXIMAL TUBULES OF THE KIDNEY IN ANGIOTENSIN II-INDUCED HYPERTENSION AND KIDNEY INJURY

It is well-recognized that the renin-angiotensin system plays powerful endocrine, paracrine, and intracellular roles in the physiological regulation of blood pressure, cardiovascular and kidney function, as well as in the development of hypertension and kidney diseases. The present study tested the hypothesis that genetic deletion of AT1a receptors selectively in the proximal tubules of the kidney attenuates angiotensin II (ANG II)-induced hypertension and renal ischemia-reperfusion injury using global and proximal tubule-specific AT1a receptor knockout mouse models. Global and proximal tubule-specific AT1a receptor-knockout mice (PT-AT1a-KO) were bred, or generated in this laboratory using the standard Cre/LoxP recombination approach. The hypothesis was tested in the following experimental protocols. Three groups (n=12 per group) of age- and body wt.-matched adult male C57BL/6J (WT), global AT1a-KO (Agtr1a-/-), and PT-AT1a-KO mice were first used to measure basal systolic, diastolic, and mean arterial blood pressure using the telemetry and tail-cuff techniques. Another two groups of WT, AT1a-KO, and PT-AT1a-KO mice (n=8-12 per group) were infused with saline (control) or a pressor dose of ANG II at 1.5 mg/kg/day, i.p., for 2 weeks. Additional three groups of WT, AT1a-KO and PT-AT1a-KO mice (n=8-12 per group) were treated with sham surgery (control) or 45 min bilateral renal ischemia, followed by reperfusion for 24 h or 7 days. Blood pressure and renal functional responses, and glomerular, tubuointerstitial, and perivascular injury were determined accordingly. Under basal conditions, systolic, diastolic and mean arterial blood pressure was 23 ± 3 mmHg lower in global AT1a-KO (p<0.01 vs. WT), and 12 ± 3 mmHg lower in PT-AT1a-KO mice (p<0.01 vs. WT). 24 h urine output and urinary sodium excretion were significantly higher in AT1a-KO and PT-AT1a-KO than in WT mice (p<0.01). Urine osmolality was significantly lower in AT1a-KO mice (p<0.01), but to a much less extent in PT-AT1a-KO mice (p<0.05). The kidney wt. to body wt. ratio was smaller in AT1a-KO mice (p<0.01), but was not different between WT and PT-AT1a-KO mice. In response to ANG II infusion, systolic, diastolic and mean arterial blood pressure were significantly increased by 45 ± 5 mmHg (p<0.01 vs. baseline) in WT mice, but only by 25 ± 3 mmHg in PT-AT1a-KO mice (p<0.01 vs. baseline). The ANG II-induced hypertension was completely prevented in global AT1a-KO mice (n.s. vs. baseline). In response to induction of renal ischemia-reperfusion injury, WT mice developed significant glomerular, cortical tubulointerstitial and perivascular injury 24 h or 7 days after renal ischemia-reperfusion was induced (p<0.01), as expected. However, both acute and chronic ischemia-reperfusion renal injury was not attenuated in global AT1a-KO mice (n.s. vs. WT). Surprisingly, however, acute and chronic ischemia-reperfusion renal injury was significantly attenuated in PT-AT1a-KO mice 24 h or 7 days after renal ischemia-reperfusion was induced (p<0.01 vs. WT or AT1a-KO). The results of the present study provide the direct evidence for an important role of ANG II and AT1a receptors in the proximal tubules of the kidney in maintaining physiological blood pressure homeostasis, and in the full development of ANG II-dependent Hypertension and renal ischemia-reperfusion injury in mice.