Ang II-induced Hypertension Research Articles

PROXIMAL TUBULE-SPECIFIC DELETION OF AT1A RECEPTORS ATTENUATES CIRCULATING AND INTRATUBULAR ANGIOTENSIN II-INDUCED HYPERTENSION IN MICE

Objective: The present study used a novel mouse model with proximal tubule-specific knockout of AT1a receptors in the kidney, PT-Agtr1a-/-, to test the hypothesis that intratubular angiotensin II (Ang II) and AT1a receptors in the proximal tubules are required for maintaining normal blood pressure and the development of Ang II-induced hypertension. Design and method: To test our hypothesis, eight groups (n = 7–15 per group) of adult male wild-type, global Agtr1a-/-, and PT-Agtr1a-/-, and PT-Nhe3-/- mice were infused with Ang II (1.5 mg/kg/day, i.p.), or overexpressed an intracellular Ang II fusion protein (Ad-sglt2-ECFP/Ang II) in the proximal tubules of the kidney for 2 weeks. Basal blood pressure and the pressor responses, glomerular filtration rate (GFR) and 24 h urinary natriuretic response, and the pressure-natriuresis response were determined. Results: Basal telemetry blood pressure were ∼15 ± 3 mmHg lower in PT-Agtr1a-/- than wild-type mice and ∼13 ± 3 mmHg higher than Agtr1a-/- mice (P < 0.01). Basal glomerular filtration was ∼23.9% higher (P < 0.01), whereas fractional proximal tubule Na+ reabsorption was lower in PT-Agtr1a-/- mice (P < 0.01). Deletion of AT1a receptors in the proximal tubules augmented the pressure-natriuresis response (P < 0.01), and natriuretic responses to salt loading or Ang III infusion (P < 0.01). Ang II induced hypertension in wild-type, PT-Agtr1a-/- and PT-Nhe3-/- mice, but the pressor response was ∼16 ± 2 mmHg lower in PT-Agtr1a-/- and PT-Nhe3-/-mice (P < 0.01). Deletion of AT1a receptors or NHE3 in the proximal tubules attenuated ∼50% of Ang II-induced hypertension in wild-type mice (P < 0.01), but blocked intracellular Ang II fusion protein-induced hypertension in PT-Agtr1a-/- mice (P < 0.01). Conclusions: Taken together, the results of the present study provide new insights into the critical role of intratubular Ang II/AT1 (AT1a)/NHE3 pathways in the proximal tubules in normal blood pressure control and the development of Ang II-induced hypertension.

ROLE OF VASCULAR NOX5 IN ANG II-MEDIATED PRO-FIBROTIC AND PRO-INFLAMMATORY SIGNALLING IN THE KIDNEY

Objective: Mice expressing human Nox5 (hNox5) in vascular smooth muscle cells (VSMC) exhibit increased contraction, ROS generation, cardiac fibrosis and increased pulse pressure. In kidneys, hNox5 in podocytes is associated with albuminuria, hypertension and inflammation. Here we questioned whether VSMC Nox5 influences kidney function and damage in Ang II-induced hypertension. Design and method: Wildtype (WT) and mice expressing hNOX5 in SMC (Nox5+SM22+) were studied. Mice were infused with Ang II (600 ng/Kg/day) for 28 days. Blood pressure was assessed by tail-cuff, kidney damage by histology, and mRNA and protein expression by qPCR and western-blotting, respectively. Inflammatory infiltrate was assessed by FACS. Results: Ang II increased blood pressure (mmHg) in WT (162.7 ± 9) and Nox5+SM22+ (162.3 ± 10). Albuminuria was increased in Nox5+SM22+ versus WT mice (10.2 ± 6 vs 1.27 ± 0.3 mg/mmol, p < 0.05) and by Ang II in both groups (Nox5: 44.8 ± 18; WT: 58.7 ± 12 mg/mmol). Kidneys from Ang II-infused Nox5+SM22+ mice exhibited significant perivascular fibrosis and inflammatory cell infiltration compared to WT. Kidney expression of vimentin (AU: 1.01 ± 0.05 vs WT 0.85 ± 0.03) and aSMA (AU: 0.44 ± 0.03 vs WT 0.33 ± 0.01), markers of myofibroblast differentiation, was increased in Nox5 mice (p < 0.05), an effect that was augmented by Ang II. In kidneys from Nox5+SM22+ mice, levels of macrophage F4/80+ cells were increased (%: 24 ± 2 vs WT 18 ± 1, p < 0.05). Ang II increased macrophage homing in kidneys from WT mice (34 ± 3%, p < 0.05 vs non-treated), but did not further increase it in Nox5 mice (35 ± 5%). Levels of cytotoxic CD8+ T cells and expression of IL-1b and IRAK were increased by Ang II in Nox5 mice. Conclusions: Our study suggests that Nox5 in VSMCs induces renal fibrosis and pro-inflammatory signalling by Ang II, likely through increased ROS generation.

GAMMA-DELTA T CELLS MEDIATE ALPHA-BETA T CELL ACTIVATION IN ANGIOTENSIN II-INDUCED HYPERTENSION

Objective: 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. Design and method: Fourteen to 15-week-old aged C57BL/6J male mice were infused or not with Ang II (490 ng/kg/min, SC) for 7 and 14 days (n = 5–7). γδ and αβ T cells were isolated from peripheral lymph nodes and spleen and αβ T cells were cultured alone or with γδ T cells (5:1) in presence of anti-CD3 antibodies plus or minus Ang II. 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 (r2 = 0.74, P < 0.01) and CD8 + CD69 + T cells (r2 = 0.64, 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.

PROXIMAL TUBULE-SPECIFIC DELETION OF AT1A RECEPTORS ATTENUATES ANGIOTENSIN II-INDUCED HYPERTENSION BY INCREASING GLOMERULAR FILTRATION AND THE PRESSURE-NATRIURESIS RESPONSE

Objective: The present study tested the hypothesis that Ang II and AT1a receptors in the proximal tubules are required for normal blood pressure homeostasis and full development of Ang II-induced hypertension. Design and method: Six groups (n = 7–12 per group) of adult male wild-type (WT) and PT-Agtr1a-/- mice were infused with or without Ang II for 2 weeks (1.5 mg/kg/day, i.p.). Glomerular filtration rate (GFR) was measued using fluorescein-labeled sinistrin, and natriuretic responses were determined under basal conditions and after Ang II infusion. Results: Basal systolic blood pressure were ∼13 ± 3 mmHg lower in PT-Agtr1a-/- than WT mice (P < 0.01). However, basal GFR was higher in PT-Agtr1a-/- mice (WT: 160.4 ± 7.0 μl/min vs. PT-Agtr1a-/-: 186.0 ± 10 μl/min, P < 0.01). Basal 24 h urinary Na+ excretion (UNaV) was higher in PT-Agtr1a-/- than WT mice (P < 0.01). In response to Ang II infusion, both WT and PT-Agtr1a-/- mice developed hypertension with similar pressor response to Ang II in WT (43 ± 3 mmHg, P < 0.01) and PT-Agtr1a-/- mice (39 ± 5 mmHg, P < 0.01). However, systolic blood pressure was still 16 ± 3 mmHg lower in PT-Agtr1a-/- mice (P < 0.01). Ang II decreased GFR to 132.2 ± 7.0 μl/min in WT mice (P < 0.01), and to 129.4 ± 18.6 μl/min in PT-Agtr1a-/- mice (P < 0.01), respectively. In WT mice, UNaV increased from 139.3 ± 22.3 μmol/24 h in the control group to 196.4 ± 29.6 μmol/24 h in the Ang II-infused group (P < 0.01). In PT-Agtr1a-/- mice, UNaV increased from 172.0 ± 10.2 μmol/24 h in the control group to 264.7 ± 35.4 μmol/24 h in the Ang II-infused group (P < 0.01). The pressor response to Ang II was attenuated, while the natriuretic response was augmented by losartan in WT and PT-Agtr1a-/- mice (P < 0.01). Finally, proximal tubule-specific deletion of AT1a receptors significantly augmented the pressure-natriuresis response and natriuretic responses to acute saline infusion (P < 0.01) or a 2% high salt diet for 2 weeks (P < 0.01). Conclusions: The present study supports the hypothesis that Ang II and AT1a receptors in the proximal tubules are required for normal blood pressure homeostasis and the development of Ang II-induced hypertension.

GENETIC BACKGROUND DOMINATES FIBROTIC AORTIC REMODELING DURING ANGIOTENSIN-INDUCED HYPERTENSION IN MICE

Objective: Many induced genetic mutations affect aortic structure and function in mice, but less is known about the influence of background strain. This study quantifies and compares the aortic phenotype in angiotensin II (AngII)-induced hypertension across different strains of wild-type mice. Design and method: Adult C57BL/6J and 129SvEv mice were studied before and after induction of hypertension via subcutaneous infusion of AngII (1000 ng/kg/min) for two weeks, which elevated blood pressure similarly in both strains (+34% vs. +32%, systolic). Excised descending thoracic aortas were subjected to a series of isobaric vasocontraction and vasorelaxation tests as well as novel biaxial stiffness testing. Immuno-histological studies quantified medial and adventitial composition as well as CD45+ cellular infiltration. Results: Baseline aortic geometry and biomechanical properties were similar across strains, consistent with the existence of general homeostatic mechanical targets. Nevertheless, aortic remodeling due to AngII-induced hypertension differed dramatically between strains, with gross over-adaptive remodeling (exuberant thickening of the media and adventitia) in C57BL/6J (+89% thickness increase) but under-adaptive remodeling in 129SvEv mice (+12% thickness increase). Importantly, vasoconstrictive strength was lower in C57BL/6J than 129SvEv mice, both before (-4% vs. -11% contractile change in outer diameter (δOD), p = 0.020; -8 vs. -24% contractile change in circumferential stress (δσ), p = 0.023) and after (-10% vs. -22% contractile δOD, p < 0.001; -24 vs. -46% contractile δσ, p < 0.001) hypertension. CD45+ cell content was markedly higher in C57BL/6J than 129SvEv mice following hypertension (p < 0.001). When considering individual vessels, wall thickness correlated with both CD45+ content (p = 0.034) and total contractility (p = 0.001), with contractility the strongest predictor statistically. Finally, layer-specific biomechanical analyses revealed increased adventitial load bearing in the hypertensive 129SvEv aorta under passive conditions, which, however, it is able to normalize by its increased contractility. Conclusions: Genetic modifiers play key roles in the different hypertensive aortic remodeling between C57BL/6J and 129SvEv mice (Figure) as well as mixed C57BL/6;129SvEv mice. The lower fibrotic response in 129SvEv mice results from their lower inflammatory state but also their greater aortic vasoresponsiveness to AngII, which lowers the wall-stress associated mechano-stimulus for remodeling. Remodeling in mixed-background mice was dominated by the 129SvEv strain.

Activation of the Cholinergic Anti-inflammatory Pathway Attenuated Angiotension II-Dependent Hypertension and Renal Injury.

Background: Angiotensin II (AngII) induces renal fibrosis, characterized by fibroblast proliferation, inflammatory cell infiltration and excessive extracellular matrix deposition, all of which was relevant closely to hypertension. The vagus nerve-related cholinergic anti-inflammatory pathway (CAP) modulates local and systemic inflammatory responses. The aim of present study was to determine the effect of CAP on renal inflammation and fibrosis. Methods and Results: AngII-induced hypertension was induced in vivo by 14-days low-dose AngII infusion from osmotic minipumps. We used GTS-21 dihydrochloride, a selective nicotinic acetylcholine receptor agonist. Daily intraperitoneal GTS-21 injection and/or vagotomy started after hypertension was confirmed and continued for 4 weeks. The elevated blood pressure caused by AngII was significantly attenuated by GTS-21. Improved baroreflex sensitivity was observed after GTS-21 administration. Masson stain and immunoblotting revealed that deposition of excessive fibrosis and overexpression of inflammatory cytokines induced by AngII was reduced by GTS-21. To determine the role of autonomic control in CAP, unilateral vagotomy was performed. Vagotomy weakened the effect of CAP on AngII-induced hypertension. In vitro, GTS-21 suppressed NF-κB activation, attenuated AngII-induced epithelial-mesenchymal transition and reduced inflammation and fibrosis in NRK-52E cells; α-bungarotoxin (α-Bgt, an α7-nAChR selective antagonist) partly inhibited these effects. Conclusion: CAP protected against AngII-induced hypertension via improvement in autonomic control, suppression of NF-κB activation, and reduction of renal fibrosis and inflammatory response.

Therapeutic Assessment of Combination Therapy with a Neprilysin Inhibitor and Angiotensin Type 1 Receptor Antagonist on Angiotensin II-Induced Atherosclerosis, Abdominal Aortic Aneurysms, and Hypertension.

Combined neprilysin (NEP) inhibition (sacubitril) and angiotensin type 1 receptor (AT1R) antagonism (valsartan) is used in the treatment of congestive heart failure and is gaining interest for other angiotensin II (AngII)-related cardiovascular diseases. In addition to heart failure, AngII promotes hypertension, atherosclerosis, and abdominal aortic aneurysms (AAAs). Similarly, NEP substrates or products have broad effects on the cardiovascular system. In this study, we examined NEP inhibition (with sacubitril) and AT1R antagonism (with valsartan) alone or in combination on AngII-induced hypertension, atherosclerosis, or AAAs in male low-density lipoprotein receptor–deficient mice. Preliminary studies assessed drug delivery via osmotic minipumps for simultaneous release of sacubitril and/or valsartan with AngII over 28 days. Mice were infused with AngII (1000 ng/kg per minute) in the absence (vehicle) or presence of sacubitril (1, 6, or 9 mg/kg per day), valsartan (0.3, 0.5, 1, 6, or 20 mg/kg per day), or the combination thereof (1 and 0.3, or 9 or 0.5 mg/kg per day of sacubitril and valsartan, respectively). Plasma AngII and renin concentrations increased 4-fold at higher valsartan doses, indicative of removal of AngII negative feedback on renin. Sacubitril doubled plasma AngII concentrations at lower doses (1 mg/kg per day). Valsartan dose-dependently decreased systolic blood pressure, aortic atherosclerosis, and AAAs of AngII-infused mice, whereas sacubitril had no effect on atherosclerosis or AAAs but reduced blood pressure of AngII-infused mice. Combination therapy with sacubitril and valsartan did not provide additive benefits. These results suggest limited effects of combination therapy with NEP inhibition and AT1R antagonism against AngII-induced hypertension, atherosclerosis, or AAAs.SIGNIFICANCE STATEMENTThe combination of valsartan (angiotensin type 1 receptor antagonist) and sacubitril (neprilysin inhibitor) did not provide benefit above valsartan alone on AngII-induced hypertension, atherosclerosis, or abdominal aortic aneurysms in low-density lipoprotein receptor–deficient male mice. These results do not support this drug combination in therapy of these AngII-induced cardiovascular diseases.

Mitochondrial activity regulates the differentiation of skin-derived mesenchymal stem cells into brown adipocytes to contribute to hypertension

BackgroundBrown adipocytes (BAs) are major components of brown adipose tissue (BAT), which is involved in blood pressure regulation. BAs are derived from multiple progenitors, including PDGFRα+ adipose-derived stem cells (ASCs). Skin-derived mesenchymal stem cells (S-MSCs) have the capacity to differentiate into adipocytes; however, their ability to differentiate into BAs remains unexplored. We aim to study the ability and regulatory mechanism of the differentiation of S-MSCs into BAs and the direct role of BAT in blood pressure regulation.MethodsProtein expression was measured by flow cytometry or Western blotting, and gene mRNA levels were quantified by real-time quantitative PCR (RT-PCR). To induce the differentiation of S-MSCs into BAs, S-MSCs were stimulated with a brown adipogenic cocktail comprising insulin, IBMX, dexamethasone, triiodothyronine (T3), and rosiglitazone for the indicated periods. The oxygen consumption rate (OCR) was measured with an XF24 Extracellular Flux Analyzer. Mitochondrial mass was determined by flow cytometry and fluorescence staining. Hypertension was induced in WT mice by infusion of angiotensin II (Ang II), and systolic blood pressure (SBP) was measured using a tail cuff. Interscapular brown adipose tissue (iBAT)-deficient mice were generated by surgical removal of the iBAT depot, after which the animals were allowed to recover for 6 days. Aortic, iBAT, and heart tissue sections were analyzed by hematoxylin and eosin (HE) staining.ResultsWe found that in vitro, S-MSCs isolated from the mouse dermis expressed the stem cell markers CD90/105 and PDGFRα and readily differentiated into BAs. Mitochondrial biogenesis and oxygen consumption were markedly increased during differentiation of S-MSCs into BAs. In vivo, iBAT was converted to white adipose tissue (WAT) in Ang II-induced hypertensive mice. We assessed the direct role of BAT in blood pressure (BP) regulation by using iBAT-deficient mice (generated by surgical removal of iBAT) and C57BL/6 (wild-type (WT)) mice and found that Ang II-induced BP elevation and vascular damage were markedly aggravated in iBAT-deficient mice compared with WT mice.ConclusionsThis study demonstrates that PDGFRα+ S-MSCs are able to differentiate into BAs and that this differentiation is regulated by mitochondrial activity. We also show that BAT plays a direct role in ameliorating Ang II-induced hypertension. The therapeutic potential of BAT for the prevention of hypertension-induced organ remodeling thus warrants further investigation.Graphical abstract. Schematic of the in vitro differentiation of PDGFRα+ S-MSCs into BAs via a process regulated by mitochondrial activity. BAT plays a direct role in Ang II-induced hypertension and target organ remodeling

MiR-634 inhibits human vascular smooth muscle cell proliferation and migration in hypertension through Wnt4/β-catenin pathway.

MicroRNAs (miRNAs) have been regarded as modulators in vascular pathologies, including hypertension. Dysregulated proliferation and migration of VSMCs (vascular smooth muscle cells) contributes to vascular remodeling during hypertension. miR-634 was reported to be dysregulated in hypertensive patients. The involvement of miR-634 in hypertension and the role of miR-634 on VSMCs proliferation and migration were then evaluated. Firstly, HASMCs (human aortic smooth muscle cells) were incubated with 2 μM angiotensin (Ang) II for 12 hours to establish the cell model of Ang II-induced hypertension. Results showed that Ang II treatment promoted proliferation and migration of HASMCs. Secondly, miR-634 was down-regulated in the hypertensive patients, and reduced in Ang II-induced HASMCs in a time dependent manner. Functional assays revealed that Ang II promoted proliferation and migration of HASMCs were suppressed by miR-634 mimic. Lastly, miR-634 targeted 3' untranslated region (UTR) of Wnt4, and reduced Wnt4 expression in HASMCs. miR-634 inhibited β-catenin nuclear translocation. Over-expression of Wnt4 counteracted the suppressive effects of miR-634 on Ang II-induced proliferation and migration of HASMCs. In conclusion, miR-634 inhibited HASMCs proliferation and migration through inactivation of Wnt4/β-catenin pathway.

HDAC5 inhibition reduces angiotensin II-induced vascular contraction, hypertrophy, and oxidative stress in a mouse model

Non-specific histone deacetylase (HDAC) inhibition reduces high blood pressure in essential hypertensive animal models. However, the exact HDAC isoforms that play a critical role in controlling hypertension are not known. Here, we investigated the role of HDAC5 in vascular contraction, hypertrophy, and oxidative stress in the context of angiotensin II (Ang II)-induced hypertension.Genetic deletion of HDAC5 and treatment with class IIa HDAC inhibitors (TMP269 and TMP195) prevented Ang II-induced increases in blood pressure and arterial wall thickness. Hdac5-knockout mice were also resistant to the thromboxane A2 agonist (U46619)-induced vascular contractile response. Furthermore, the expression of Rho-associated protein kinase (ROCK) 2 was downregulated in the aortas of Ang II-treated Hdac5-knockout mice. Knockdown of HDAC5, RhoA, or ROCK2 reduced collagen gel contraction, whereas silencing of ROCK1 increased it. VSMC hypertrophy reduced on knocking down HDAC5, ROCK1, and ROCK2. Here we showed that genetic deletion of HDAC5 and pharmacological inhibition of class IIa HDACs ameliorated Ang II-induced ROS generation. Moreover, ROCK1 and ROCK2, the downstream targets of HDAC5, influenced ROS generation. The relative protein levels of HDAC5, ROCK1, and ROCK2 were increased both in the cytoplasm and nuclear fraction in response to Ang II stimulation in vascular smooth muscle cells. Inhibition of HDAC5 expression or activity reduced vascular hypertrophy, vasoconstriction, and oxidative stress in the Ang II-induced hypertension model. These findings indicate that HDAC5 may serve as a potential target in the treatment of hypertension.

Site-1 Protease-Derived Soluble (Pro)Renin Receptor Contributes to Angiotensin II-Induced Hypertension in Mice.

Activation of PRR ([pro]renin receptor) contributes to enhancement of intrarenal RAS and renal medullary α-ENaC and thus elevated blood pressure during Ang II (angiotensin II) infusion. The goal of the present study was to test whether such action of PRR was mediated by sPRR (soluble PRR), generated by S1P (site-1 protease), a newly identified PRR cleavage protease. F1 B6129SF1/J mice were infused for 6 days with control or Ang II at 300 ng/kg per day alone or in combination with S1P inhibitor PF-429242 (PF), and blood pressure was monitored by radiotelemetry. S1P inhibition significantly attenuated Ang II-induced hypertension accompanied with suppressed urinary and renal medullary renin levels and expression of renal medullary but not renal cortical α-ENaC expression. The effects of S1P inhibition were all reversed by supplement with histidine-tagged sPRR termed as sPRR-His. Ussing chamber technique was performed to determine amiloride-sensitive short-circuit current, an index of ENaC activity in confluent mouse cortical collecting duct cell line cells exposed for 24 hours to Ang II, Ang II + PF, or Ang II + PF + sPRR-His. Ang II-induced ENaC activity was blocked by PF, which was reversed by sPRR-His. Together, these results support that S1P-derived sPRR mediates Ang II-induced hypertension through enhancement of intrarenal renin level and activation of ENaC.

Qingda granule attenuates angiotensin II-induced cardiac hypertrophy and apoptosis and modulates the PI3K/AKT pathway

Qingda granule (QDG), simplified from Qingxuan Jiangya Decoction, is a well-known traditional Chinese medicine formula that has been used for decades to treat hypertension. However, the cardioprotective effects of QDG on Ang II-induced hypertension remain unknown. This study aimed to investigate the effects of QDG on hypertension-induced cardiac hypertrophy and apoptosis, as well as explore its underlying mechanisms. Mice were infused with Ang II (500 ng/kg/min) or saline solution as control, then administered oral QDG (1.145 g/kg/day) or saline for two weeks. QDG treatment attenuated the elevation in blood pressure caused by Ang II, as well as the decreased left ventricle ejection fractions and fractional shortening. Moreover, QDG treatment significantly alleviated the Ang II-induced elevation of the ratio of heart weight to tibia length, as well as cardiac injury, hypertrophy, and apoptosis. In cultured H9C2 cells stimulated with Ang II, QDG partially reversed the increase in cell surface area and number of apoptotic cells, up-regulation of hypertrophy markers ANP and BNP, and activation of caspases-9 and -3. QDG also partially reversed Ang II-induced accumulation of reactive oxygen species (ROS), depolarization of the mitochondrial membrane, release of cytochrome C, up-regulation of Bax, and decrease in levels of p-PI3K, p-AKT, and Bcl-2. These results suggest that QDG can significantly attenuate Ang II-induced hypertension, cardiac hypertrophy and apoptosis, and it may exert these effects in part by suppressing ROS production and activating the PI3K/AKT signaling pathway.

Blocking of interleukin-1 suppresses both angiotensin II-induced renal inflammation and hypertension

Abstract Background Clinical hypertension is associated with renal inflammation and elevated circulating levels of proinflammatory cytokines. IL-1 receptor antagonist (IL-1Ra) is one of the most important anti-inflammatory cytokines and plays a crucial role in inflammation. Inhibition of IL-1 may contribute to modulation of the Angiotensin II (AngII)-induced hypertension response. This study aimed to elucidate the effects of IL-1Ra and anti-IL-1beta antibody (01BSUR) on AngII-induced hypertension and renal inflammation. Methods and results To determine the contribution of IL-1Ra to AngII-induced renal inflammation, male wild-type (WT) and IL-1Ra-deficient (IL-1Ra−/−) mice were infused with AngII (1000ng/kg/min) using subcutaneous osmotic pumps for 14 days. We checked blood pressure, histological change, and several mRNA expressions 14 days after infusion. Fourteen days after infusion, systolic blood pressure (197±5 vs 169±9 mmHg, p<0.05) in IL-1Ra−/− mice significantly increased compared with WT mice. Furthermore, on day 14 of AngII infusion, plasma IL-6 was 5.9-fold higher in IL-1Ra−/− versus WT mice (p<0.001); renal preproendothelin-1 mRNA expression was also significantly higher in IL-1Ra−/− mice (p<0.05). To examine renal function, we analyzed 24-hour urinary protein excretion and serum levels of blood urea nitrogen, creatinine, and uric acid in IL-1Ra−/− and WT mice. On day 14 of Ang II infusion, all levels increased significantly in IL-1Ra−/− mice compared with WT mice, suggesting that IL-1Ra deficiency reduced renal function following Ang II infusion. In addition, renal histology revealed that glomerular injury (Figure upper panels: PAS staining) and tubulointerstitial fibrosis (Figure lower panels: Elastica Masson staining) increased significantly in Ang II-infused IL-1Ra−/− versus Ang II-infused WT mice. Finally, we administrated 01BSUR to both IL-1Ra−/− and WT mice, and 01BSUR treatment decreased AngII-induced hypertension (162±17 vs 204±6 mmHg, p<0.05) and renal damage (glomerular injury and fibrosis of the tubulointerstitial area) in both IL-1Ra−/− and WT mice compared with IgG2a treatment. These findings suggest that 01BSUR suppresses Ang II-induced inflammation and renal injury. Conclusions Inhibition of interleukin-1 by both endogenous IL-1Ra and exogenous 01BSUR decreased AngII-induced hypertension and renal damage in mice, suggesting suppression of IL-1 may provide an additional strategy to protect against renal damage in hypertensive patients. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): JSPS KAKENHI

Fibroblast Nox2 (NADPH Oxidase-2) Regulates ANG II (Angiotensin II)-Induced Vascular Remodeling and Hypertension via Paracrine Signaling to Vascular Smooth Muscle Cells.

The superoxide-generating Nox2 (NADPH oxidase-2) is expressed in multiple cell types. Previous studies demonstrated distinct roles for cardiomyocyte, endothelial cell, and leukocyte cell Nox2 in ANG II (angiotensin II)-induced cardiovascular remodeling. However, the in vivo role of fibroblast Nox2 remains unclear. Approach and Results: We developed a novel mouse model with inducible fibroblast-specific deficiency of Nox2 (fibroblast-specific Nox2 knockout or Fibro-Nox2KO mice) and investigated the responses to chronic ANG II stimulation. Fibro-Nox2KO mice showed no differences in basal blood pressure or vessel wall morphology, but the hypertensive response to ANG II infusion (1.1 mg/[kg·day] for 14 days) was substantially reduced as compared to control Nox2-Flox littermates. This was accompanied by a significant attenuation of aortic and resistance vessel remodeling. The conditioned medium of ANG II-stimulated primary fibroblasts induced a significant increase in vascular smooth muscle cell growth, which was inhibited by the short hairpin RNA (shRNA)-mediated knockdown of fibroblast Nox2. Mass spectrometric analysis of the secretome of ANG II-treated primary fibroblasts identified GDF6 (growth differentiation factor 6) as a potential growth factor that may be involved in these effects. Recombinant GDF6 induced a concentration-dependent increase in vascular smooth muscle cell growth while chronic ANG II infusion in vivo significantly increased aortic GDF6 protein levels in control mice but not Fibro-Nox2KO animals. Finally, silencing GDF6 in fibroblasts prevented the induction of vascular smooth muscle cell growth by fibroblast-conditioned media in vitro. These results indicate that fibroblast Nox2 plays a crucial role in the development of ANG II-induced vascular remodeling and hypertension in vivo. Mechanistically, fibroblast Nox2 may regulate paracrine signaling to medial vascular smooth muscle cells via factors, such as GDF6.

A new mechanism for the sex differences in angiotensin II-induced hypertension: the role of macula densa NOS1β-mediated tubuloglomerular feedback.

Females are protected against the development of angiotensin II (ANG II)-induced hypertension compared with males, but the mechanisms have not been completely elucidated. In the present study, we hypothesized that the effect of ANG II on the macula densa nitric oxide (NO) synthase 1β (NOS1β)-mediated tubuloglomerular feedback (TGF) mechanism is different between males and females, thereby contributing to the sexual dimorphism of ANG II-induced hypertension. We used microperfusion, micropuncture, clearance of FITC-inulin, and radio telemetry to examine the sex differences in the changes of macula densa NOS1β expression and activity, TGF response, natriuresis, and blood pressure (BP) after a 2-wk ANG II infusion in wild-type and macula densa-specific NOS1 knockout mice. In wild-type mice, ANG II induced higher expression of macula densa NOS1β, greater NO generation by the macula densa, and a lower TGF response in vitro and in vivo in females than in males; the increases of glomerular filtration rate, urine flow rate, and Na+ excretion in response to an acute volume expansion were significantly greater and the BP responses to ANG II were significantly less in females than in males. In contrast, these sex differences in the effects of ANG II on TGF, natriuretic response, and BP were largely diminished in knockout mice. In addition, tissue culture of human kidney biopsies (renal cortex) with ANG II resulted in a greater increase in NOS1β expression in females than in males. In conclusion, macula densa NOS1β-mediated TGF is a novel and important mechanism for the sex differences in ANG II-induced hypertension.

T cell-derived extracellular vesicles are elevated in essential HTN.

Extracellular vesicles (EVs) are novel mediators of cell-to-cell communication and appear to mediate the pathogenesis of hypertension (HTN). However, the mechanisms underlying the involvement of EVs in HTN remain unclear. The adaptive and innate immune systems play an important role affecting the kidney and vasculature in animal models of HTN. Evolving evidence shows that immune cell-derived EVs can modulate the immune system in a paracrine fashion and therefore may mediate the effects of inflammation in the pathogenesis of HTN. Therefore, we aimed to understand if specific subtypes of leukocyte/immune cell-derived EVs are altered in essential HTN using an in vivo model of angiotensin II (ANG II)-induced HTN. After 4 wk of ANG II treatment, EVs were isolated from the blood and kidney. EV origin and counts were characterized with Imaging Flow Cytometry, antibody panels targeting platelets, endothelial cells, and leukocytes including B and T cells, monocytes, and neutrophils. Leukocyte-derived EVs (CD45+) were elevated in the circulation and kidney tissue in ANG II-induced HTN. Subgroup analysis depicted T cell-derived EVs (CD3+) to be significantly elevated in ANG II-induced HTN (3.50e+5 particles/mL) compared with control groups (9.16e+4 particles/mL, P = 0.0106). T cell-derived EVs also significantly correlated with systolic blood pressure levels (r2 = 0.898, P = 0.0012). In summary, leukocyte-derived EVs, and more specifically T cell-derived EVs (CD3+), are elevated in ANG II-induced HTN in the circulation and kidney tissue and correlate well with blood pressure severity. EVs from the circulation and kidney may be sensitive biomarkers for HTN and end-organ damage and may lead to new mechanistic insights in this silent disease.

Dual deficiency of angiotensin-converting enzyme-2 and Mas receptor enhances angiotensin II-induced hypertension and hypertensive nephropathy.

Angiotensin‐converting enzyme‐2 (ACE2) and Mas receptor are the major components of the ACE2/Ang 1‐7/Mas axis and have been shown to play a protective role in hypertension and hypertensive nephropathy individually. However, the effects of dual deficiency of ACE2 and Mas (ACE2/Mas) on Ang II‐induced hypertensive nephropathy remain unexplored, which was investigated in this study in a mouse model of hypertension induced in either ACE2 knockout (KO) or Mas KO mice and in double ACE2/Mas KO mice by subcutaneously chronic infusion of Ang II. Compared with wild‐type (WT) animals, mice lacking either ACE2 or Mas significantly increased blood pressure over 7‐28 days following a chronic Ang II infusion (P < .001), which was further exacerbated in double ACE2/Mas KO mice (P < .001). Furthermore, compared to a single ACE2 or Mas KO mice, mice lacking ACE2/Mas developed more severe renal injury including higher levels of serum creatinine and a further reduction in creatinine clearance, and progressive renal inflammation and fibrosis. Mechanistically, worsen hypertensive nephropathy in double ACE2/Mas KO mice was associated with markedly enhanced AT1‐ERK1/2‐Smad3 and NF‐κB signalling, thereby promoting renal fibrosis and renal inflammation in the hypertensive kidney. In conclusion, ACE2 and Mas play an additive protective role in Ang II‐induced hypertension and hypertensive nephropathy. Thus, restoring the ACE2/Ang1‐7/Mas axis may represent a novel therapy for hypertension and hypertensive nephropathy.

Angiotensin II Stimulates the Proliferation and Migration of Lymphatic Endothelial Cells Through Angiotensin Type 1 Receptors.

Background/AimThe proliferation and migration of lymphatic endothelial cells (LECs) is essential for lymphatic vessel growth (also known as lymphangiogenesis), which plays a crucial role in regulating the tissue fluid balance and immune cell trafficking under physiological and pathological conditions. Several growth factors, such as VEGF-C, can stimulate lymphangiogenesis. However, the effects of angiotensin II (Ang II) on the proliferation and migration of mouse LECs and the underlying potential mechanisms remain unknown.MethodsWild-type mice were infused with Ang II (1,000 ng/kg/min) for 1–2 weeks. Murine LECs were stimulated with Ang II (500 nM) or saline for 12–48 h. Cell proliferation was determined with 5-bromo-2-deoxyuridine (BrdU) incorporation assays, while cell migration was assessed by scratch wound healing and transwell chamber assays. The gene expression profiles were obtained by time series microarray and real-time PCR analyses.ResultsAng II treatment significantly induced lymphangiogenesis in the hearts of mice and the proliferation and migration of cultured LECs in a time-dependent manner. This effect was completely blocked by losartan, an angiotensin II type 1 receptor (AT1R) antagonist. The microarray results identified 1,385 differentially expressed genes (DEGs) at one or more time points in the Ang II-treated cells compared with the control saline-treated cells. These DEGs were primarily involved in biological processes and pathways, including sensory perception of smell, the G protein coupled receptor signaling pathway, cell adhesion, olfactory transduction, Jak-STAT, alcoholism, RIG-I-like receptor and ECM-receptor interaction. Furthermore, these DEGs were classified into 16 clusters, 7 of which (Nos. 13, 2, 8, 15, 7, 3, and 12, containing 586 genes) were statistically significant. Importantly, the Ang II-induced alterations the expression of lymphangiogenesis-related genes were reversed by losartan.ConclusionThe results of the present indicate that Ang II can directly regulate the proliferation and migration of LECs through AT1R in vivo and in vitro, which may provide new potential treatments for Ang II-induced hypertension and cardiac remodeling.

Abstract P012: Cytochrome P450 1B1 Generated 17β-Estradiol Metabolite 2-Methoxyestradiol Inhibits 12/15-Lipoxygenase To Protect Against Angiotensin II-induced Hypertension, Autonomic Dysfunction And Renal Injury In Female Mice

17β-estradiol (E2) via its cytochrome P450 (CYP) 1B1 generated metabolite 2-methoxyestradiol (2ME) protects from angiotensin (Ang) II-induced hypertension in female mice. Gene disruption of 12/15 lipoxygenase (LOX) that metabolizes arachidonate (A) into 12(S)- and 15(S)-HETE minimizes Ang II-induced hypertension in male mice. We performed this study to determine if 2-ME generated by CYP1B1 from E2 mediates its protective effect against Ang II-induced hypertension via LOX inhibition in female mice. Systolic blood pressure (SBP, mmHg, tail-cuff) in response to Ang II (700 ng/Kg/min, sc, miniosmotic pump, 2 weeks) was greater ( P &lt;0.05, n=4-5) in ovariectomized (OVX) than intact Cyp1b1 +/+ / Alox15 +/+ (174±2 vs 135±5), and intact or OVX- Cyp1b1 +/+ / Alox15 –/– (120±4 and 125±5) mice. Moreover, SBP in response to Ang II was greater in trans-2,3',4,5'-tetramethoxystilbene (CYP1B1 inhibitor, 300 μg/Kg, every 3rd day, ip)-treated intact Cyp1b1 +/+ / Alox15 +/+ than Cyp1b1 +/+ / Alox15 –/– (164±3 vs 132±3, n=6-8, P &lt;0.05) mice. Also, mean arterial pressure (MAP, mmHg, radiotelemetry) and low frequency to high-frequency oscillation ratio (LF/HF ratio, power spectral analysis), the index of baroreflex sensitivity impairment, in response to Ang II was greater ( P &lt;0.05, n=4) in OVX- Cyp1b1 +/+ / Alox 15 +/+ (160±3; 2.9±0.1) than in OVX- Cyp1b1 +/+ / Alox15 –/– (119±7; 2±0.1) mice. However, MAP and LF/HF ratio in response to Ang II was lower in 2ME (1.5 mg/g, every 3rd day, ip)-treated OVX- Cyp1b1 +/+ / Alox15 +/+ (108±3; 1.6±0.1) and OVX- Cyp1b1 +/+ / Alox 15 –/– (107±2; 1.6±0.1) mice (n=4). Plasma12(S)-HETE level (ELISA), and renal injury assessed by urine albumin/creatinine ratio in response to Ang II was lower in 2ME than its vehicle (dimethyl sulfoxide)-treated OVX- Cyp1b1 +/+ / Alox 15 +/+ mice (1616±30 vs 2162±55, pg/mL, and 2.7±0.3 vs 1.9±0.1; n=4, P &lt;0.05). Ang II did not increase 12(S)-HETE or albumin/creatinine ratio in Cyp1b1 +/+ / Alo x 15 –/– mice. These data suggest that that E2-CYP1B1 generated metabolite 2ME by inhibiting A-LOX signaling protects against Ang II-induced hypertension, impaired baroreflex, and renal injury in female mice. Therefore, LOX inhibitors could be useful in the treatment of Ang II-induced hypertension and associated renal injury in females.

Abstract P140: Myeloid Activation in Hypertension and Effects on Bone: A New End Organ?

Hypertension affects approximately one-half of the adult population. Another common disease is osteoporosis, which affects 53 million people in the United States. Clinical studies have shown an association between hypertension and bone loss. We hypothesized that experimental hypertension would be associated with osteoporosis. In Angiotensin (Ang) II-induced hypertension, there is an increase in hematopoietic stem cells (HSCs) expressing the master myeloid transcription factor PU.1 (2.99 ± 0.92) vs. sham (0.17 ± 0.04,p=0.007). Here we reported an increase an osteoclasts number per mm 2 by Tartrate-resistant acid phosphatase staining in DOCA-salt hypertension (27.4 ± 2.3) vs. sham (12.09 ± 1.7, p=0.0002). Using microCT analysis of femoral bone, we found that Ang II-induced hypertension caused striking bone loss as reflected by decreases in cortical bone area, cortical thickness and trabecular number (see Table). We also examined bone strength using a three-point biomechanical testing. In keeping with the micro-CT data, both forms of hypertension were associated with increased bone fragility, reflected by several parameters including maximum force to failure and rigidity (see Table). Recent studies have implicated T cells in the genesis of osteoporosis, and in keeping with this, we found almost two-fold increase in both CD4 + and CD8 + T cells in the marrow of mice with DOCA-salt hypertension. Thus, in two models of experimental hypertension there is marked bone loss and the development of bone fragility, potentially related to T cell accumulation and cytokine release. These findings might explain the association between osteoporosis and hypertension.