II-induced Hypertensive Mice Research Articles

Abstract MP18: Evidence for An Important Physiological Role of Bradykinin B 2 Receptors in The Renomedullary Interstitial cells of The Kidney in Angiotensin II-induced Hypertension in Mice

Bradykinin is a potent vasoactive peptide of the kallikrein-kinin system and activates two different classes of G protein-coupled receptors, B 1 and B 2 , in the kidney. In the present study, we tested the hypothesis that deletion of B 2 receptors selectively in renomemdullary interstitial cells (RMICs) of the kidney medulla lowers basal blood pressure and attenuates angiotensin II (Ang II)-induced hypertension. To test the hypothesis, we generated a novel mouse model with deletion of B 2 receptors selectively in RMICs of the medulla (RMIC- Bdkrb2 -/- ) using inducible Tenascin-C-CreER2/Bdkrb2 fl/fl recombination approach. Mice with deletion of B 2 receptors selectively in RMICs developed and grew normally without structural abnormalities in the kidney medulla. Basal telemetry systolic (SBP), diastolic, and mean arterial blood pressure were significantly lower in RMIC- Bdkrb2 -/- than WT mice (WT: 123 ± 3 mmHg vs. RMIC- Bdkrb2 -/- : 106 ± 3 mmHg, P <0.01) (n=12-15). The hypotensive phenotype in RMIC- Bdkrb2 -/- mice was associated with significant decreases in 24 h urine output (WT: 1.86 ± 0.15 mL/24 h vs. RMIC- Bdkrb2 -/- : 1.09 ± 0.15 mL/24 h, P <0.05) and urine osmolality (WT: 1893 ± 441 mOsm/Kg H 2 O vs. RMIC- Bdkrb2 -/- : 1311 ± 78 mOsm/Kg H 2 O, P <0.01) (n=12=15). Deletion of Bdkrb2 receptors in RMICs significantly decreases COX-2, eNOS, ENaC, and AQP2 mRNA expression ( P <0.01 vs. WT), but not PGE 2 or prorenin receptor mRNA expression in the kidney medulla (n.s.) (n=6-10). In response to Ang II, SBP increased to 166 ± 6 mmHg in WT mice ( P <0.01 vs. Basal), but it increased only to 139 ± 5 mmHg in RMIC- Bdkrb2 -/- mice ( P <0.01 vs. WT+Ang II) (n=10-12). Ang II-induced hypertension was completely blocked by oral administration of AT 1 receptor blocker losartan (20 mg/kg/day, p.o.) in WT and RMIC- Bdkrb2 -/- mice (n=10). By comparison, infusion of DDAVP (1-deamino-8-D-arginine vasopressin, ~0.5 mg/kg/day, i.p., for 2 weeks) had no effect on blood pressure in WT and RMIC- Bdkrb2 -/- mice (n.s.) (n=8). Similarly, infusion of Ang II or DDAVP for 2 weeks significantly increased urine osmolality in WT mice ( P <0.01), and restored urine osmolality in RMIC- Bdkrb2 -/- mice to the WT basal level (1995 ± 315 mOsm/Kg H 2 O, P <0.01) (n=8-12). In conclusion, the present study demonstrates for the 1 st time that bradykinin B 2 receptors in RMICs of the kidney medulla plays an important role in maintaining basal blood pressure and urine osmolality homeostasis and the development of Ang II-induced hypertension.

Sex differences and role of lysyl oxidase-like 2 in angiotensin II-induced hypertension in mice.

Hypertension, a disease with known sexual dimorphism, accelerates aging-associated arterial stiffening, partly because of the activation of matrix remodeling caused by increased biomechanical load. In this study, we tested the effect of biological sex and the role of the matrix remodeling enzyme lysyl oxidase-like 2 (LOXL2) in hypertension-induced arterial stiffening. Hypertension was induced by angiotensin II (ANG II) infusion via osmotic minipumps in 12- to 14-wk-old male and female mice. Blood pressure and pulse wave velocity (PWV) were measured noninvasively. Wire myography and uniaxial tensile testing were used to test aortic vasoreactivity and mechanical properties. Aortic wall composition was examined by histology and Western blotting. Uniaxial stretch of cultured cells was used to evaluate the effect of biomechanical strain. LOXL2's catalytic function was examined using knockout and inhibition. ANG II infusion-induced hypertension in both genotypes and sexes. Wild-type (WT) males exhibited arterial stiffening in vivo and ex vivo. Aortic remodeling with increased wall thickness, intralamellar distance, higher LOXL2, and collagen I and IV content was noted in WT males. Female mice did not exhibit increased PWV despite the onset of hypertension. LOXL2 depletion improved vascular reactivity and mechanics in hypertensive males. LOXL2 depletion improved aortic mechanics but worsened hypercontractility in females. Hypertensive cyclic strain contributed to LOXL2 upregulation in the cell-derived matrix in vascular smooth muscle cells (VSMCs) but not endothelial cells. LOXL2's catalytic function facilitated VSMC alignment in response to biomechanical strain. In conclusion, in males, arterial stiffening in hypertension is driven both by VSMC response and matrix remodeling. Females are protected from PWV elevation in hypertension. LOXL2 depletion is protective in males with improved mechanical and functional aortic properties. VSMCs are the primary source of LOXL2 in the aorta, and hypertension increases LOXL2 processing and shifts to collagen I accumulation. Overall, LOXL2 depletion offers protection in young hypertensive males and females.NEW & NOTEWORTHY We examined the effect of sex on the evolution of angiotensin II (ANG II)-induced hypertension and the role of lysyl oxidase-like 2 (LOXL2), an enzyme that catalyzes matrix cross linking. While ANG II led to hypertension and worsening vascular reactivity in both sexes, aortic remodeling and stiffening occurred only in males. LOXL2 depletion improved outcomes in males but not females. Thus males and females exhibit a distinct etiology of hypertension and LOXL2 is an effective target in males.

MiPEP31 alleviates Ang II-induced hypertension in mice by occupying Cebpα binding sites in the pri-miR-31 promoter

BackgroundPrevious studies have shown that peptides encoded by noncoding RNAs (ncRNAs) can be used as peptide drugs to alleviate diseases. We found that microRNA-31 (miR-31) is involved in the regulation of hypertension and that the peptide miPEP31, which is encoded by the primary transcript of miR-31 (pri-miR-31), can inhibit miR-31 expression. However, the role and mechanism of miPEP31 in hypertension have not been elucidated.MethodsmiPEP31 expression was determined by western blot analysis. miPEP31-deficient mice (miPEP31−/−) were used, and synthetic miPEP31 was injected into Ang II-induced hypertensive mice. Blood pressure was monitored through the tail-cuff method. Histological staining was used to evaluate renal damage. Regulatory T (Treg) cells were assessed by flow cytometry. Differentially expressed genes were analysed through RNA sequencing. The transcription factors were predicted by JASPAR. Luciferase reporter and electrophoretic mobility shift assays (EMSAs) were used to determine the effect of pri-miR-31 on the promoter activity of miPEP31. Images were taken to track the entry of miPEP31 into the cell.ResultsmiPEP31 is endogenously expressed in target organs and cells related to hypertension. miPEP31 deficiency exacerbated but exogenous miPEP31 administration mitigated the Ang II-induced systolic blood pressure (SBP) elevation, renal impairment and Treg cell decreases in the kidney. Moreover, miPEP31 deletion increased the expression of genes related to Ang II-induced renal fibrosis. miPEP31 inhibited the transcription of miR-31 and promoted Treg differentiation by occupying the Cebpα binding site. The minimal functional domain of miPEP31 was identified and shown to regulate miR-31.ConclusionmiPEP31 was identified as a potential therapeutic peptide for treating hypertension by promoting Treg cell differentiation in vivo. Mechanistically, we found that miPEP31 acted as a transcriptional repressor to specifically inhibit miR-31 transcription by competitively occupying the Cebpα binding site in the pri-miR-31 promoter. Our study highlights the significant therapeutic effect of miPEP31 on hypertension and provides novel insight into the role and mechanism of miPEPs.

Sex Differences and Role of Lysyl Oxidase Like 2 (LOXL2) in Angiotensin II-Induced Hypertension in Mice.

Hypertension, a disease with known sexual dimorphism, accelerates aging associated arterial stiffening, in part due to the activation of matrix remodeling caused by increased biomechanical load. In this study, we tested the effect of biological sex and the role of the matrix remodeling enzyme lysyl oxidase like 2 (LOXL2) in hypertension induced arterial stiffening. Angiotensin II (Ang II) was delivered using osmotic pumps in Loxl2+/- and WT male and female mice. Blood pressure and pulse wave velocity (PWV) were measured noninvasively to assess hypertension and aortic stiffness. Wire myography and uniaxial tensile testing were used to test aortic vasoreactivity and mechanical properties. Aortic wall composition was examined by histology and Western blotting. The effect of biomechanical strain on LOXL2 expression and secretion by vascular smooth muscle (VSMC) and endothelial cells (EC) was evaluated by uniaxial cyclic stretching of cultured cells. The role of LOXL2 catalytic function on VSMC alignment in response to mechanical loading was determined with LOXL2 inhibition and knockout. Ang II infusion induced hypertension in WT and Loxl2+/- mice of both sexes and increased PWV in WT males but not in Loxl2+/- males, WT females, or Loxl2+/- females. LOXL2 depletion protected males from Ang II mediated potentiation of vasoconstriction but worsened in females and improved aortic mechanical properties in both sexes. Histological analysis showed increased aortic wall thickness in hypertensive WT males but not females and increased intralamellar distance in both sexes, that was ameliorated in Loxl2+/- mice. Western blotting revealed increased collagen I, decreased collagen IV, and increased LOXL2 accumulation and processing in hypertensive mice. Hypertensive cyclic strain contributed to LOXL2 upregulation in the cell-derived matrix in VSMCs but not ECs. LOXL2 catalytic function facilitated VSMC alignment in response to biomechanical strain. In males, arterial stiffening in hypertension is driven both by VSMC response and matrix remodeling. Females exhibit a delayed onset of Ang II-induced hypertension with minimal ECM remodeling but with VSMC dysfunction. LOXL2 depletion ameliorates arterial stiffening and preserves functional contractility and aortic structure in male hypertensive mice. LOXL2 depletion improves aortic mechanics but worsens aortic contractility in hypertensive females. VSMCs are the primary source of LOXL2 in the aorta and hypertension increases LOXL2 processing and shifts to collagen I accumulation. Overall, LOXL2 depletion offers protection in young hypertensive males and females.

DELETION OF BRADYKININ TYPE 2 RECEPTORS SELECTIVELY IN THE RENOMEDULLARY INTERSTITIAL CELLS OF THE KIDNEY MEDULLA ATTENUATES ANGIOTENSIN II-INDUCED HYPERTENSION IN MICE

Objective: The present study tested the hypothesis that deletion of vasoactive peptide bradykinin B2 receptors selectively in the renomemdullary interstitial cells (RMICs) of the kidney medulla attenuates angiotensin II-induced hypertension in mice. Design and method: To test the hypothesis, we employed inducible Tenascin-C-CreER2 and bradykinin B2 receptor-floxed recombination approach to generate a novel mouse model with deletion of B2 receptors selectively in the RMICs of the kidney medulla (RMIC-B2R-/-). Basal blood pressure and urine osmolality and their responses to angiotensin II (Ang II, ∼500 μg/kg/day, i.p., for 2 weeks) were determined in adult male wildtype (WT) and RMIC-B2R-/- mice (n=10 per group), respectively. Results: Mice with deletion of B2 receptors selectively in RMICs of the kidney medulla developed normally without structural abnormalities in the kidney medulla. However, basal telemetry systolic, diastolic, and mean arterial blood pressure were significantly lower in RMIC-B2R-/- than WT mice (WT: 123 ± 3 mmHg vs. RMIC-B2R-/-: 106 ± 3 mmHg, P<0.01). The hypotensive phenotype in RMIC-B2R-/- mice was associated with significant decreases in 24 h urine output (WT: 1.86 ± 0.15 mL/24 h vs. RMIC-B2R-/-: 1.09 ± 0.15 mL/24 h, P<0.05) and urine osmolality (WT: 1893 ± 441 mOsm/Kg H2O vs. RMIC-B2R-/-: 1311 ± 78 mOsm/Kg H2O, P<0.01). In response to Ang II infusion (∼0.5 mg/kg/day, i.p., for 2 weeks), systolic blood pressure increased to 166 ± 6 mmHg in WT mice (P<0.01 vs. Basal), but it increased only to 139 ± 5 mmHg in RMIC-B2R-/- mice (P<0.01 vs. WT). By comparison, infusion of DDAVP (1-deamino-8-D-arginine vasopressin, ∼0.5 mg/kg/day, i.p., for 2 weeks) did not significantly alter blood pressure in WT and RMIC-B2R-/- mice (n.s.). However, infusion of Ang II for 2 weeks significantly increased urine osmolality in WT mice (P<0.01), and restored urine osmolality in RMIC-B2R-/- mice to the WT basal level (1995 ± 315 mOsm/Kg H2O, P<0.01). Conclusions: In conclusion, the present study demonstrates for the 1st time that bradykinin B2 receptors in the RMICs of the kidney medulla plays an important role in maintaining basal blood pressure and urine osmolality homeostasis and the development of Ang II-induced hypertension.

Histamine H2-receptor antagonism improves conduit artery endothelial function and reduces plasma aldosterone level without lowering arterial blood pressure in angiotensin II–hypertensive mice

Aldosterone through the mineralocorticoid receptor MR has detrimental effects on cardiovascular disease. It reduces the bioavailability of nitric oxide and impairs endothelium-dependent vasodilatation. In resistance arteries, aldosterone impairs the sensitivity of vascular smooth muscle cells to nitric oxide by promoting the local secretion of histamine which activates H2 receptors. The present experiments tested in vivo and ex vivo the hypothesis that systemic H2-receptor antagonism reduces arterial blood pressure and improves vasodilatation in angiotensin II–induced chronic hypertension. Hypertension was induced by intravenous infusion of angiotensin II (60 ng kg−1 min−1) in conscious, unrestrained mice infused concomitantly with the H2-receptor antagonist ranitidine (27.8 µg kg−1 min−1) or vehicle for 24 days. Heart rate and arterial blood pressure were recorded by indwelling arterial catheter. Resistance (mesenteric) and conductance (aortae) arteries were harvested for perfusion myography and isometric tension recordings by wire myography, respectively. Plasma was analyzed for aldosterone concentration. ANGII infusion resulted in elevated arterial blood pressure and while in vivo treatment with ranitidine reduced plasma aldosterone concentration, it did not reduce blood pressure. Ranitidine improved ex vivo endothelial function (acetylcholine 10−9 to 10−6 mol L−1) in mesenteric resistance arteries. This was abolished by ex vivo treatment with aldosterone (10−9 mol L−1, 1 h). In aortic segments, in vivo ranitidine treatment impaired relaxation. Activation of histamine H2 receptors promotes aldosterone secretion, does not affect arterial blood pressure, and protects endothelial function in conduit arteries but promotes endothelial dysfunction in resistance arteries during angiotensin II–mediated hypertension. Aldosterone contributes little to angiotensin II–induced hypertension in mice.

Depletion of follicular B cell-derived antibody secreting cells does not attenuate angiotensin II-induced hypertension or vascular compliance.

Marginal zone and follicular B cells are known to contribute to the development of angiotensin II-induced hypertension in mice, but the effector function(s) mediating this effect (e.g., antigen presentation, antibody secretion and/or cytokine production) are unknown. B cell differentiation into antibody secreting cells (ASCs) requires the transcription factor Blimp-1. Here, we studied mice with a Blimp-1 deficiency in follicular B cells to evaluate whether antibody secretion underlies the pro-hypertensive action of B cells. 10- to 14-week-old male follicular B cell Blimp-1 knockout (FoB-Blimp-1-KO) and floxed control mice were subcutaneously infused with angiotensin II (0.7 mg/kg/d) or vehicle (0.1% acetic acid in saline) for 28 days. BP was measured by tail-cuff plethysmography or radiotelemetry. Pulse wave velocity was measured by ultrasound. Aortic collagen was quantified by Masson's trichrome staining. Cell types and serum antibodies were quantified by flow cytometry and a bead-based multiplex assay, respectively. In control mice, angiotensin II modestly increased serum IgG3 levels and markedly increased BP, cardiac hypertrophy, aortic stiffening and fibrosis. FoB-Blimp-1-KO mice exhibited impaired IgG1, IgG2a and IgG3 production despite having comparable numbers of B cells and ASCs to control mice. Nevertheless, FoB-Blimp-1-KO mice still developed hypertension, cardiac hypertrophy, aortic stiffening and fibrosis following angiotensin II infusion. Inhibition of follicular B cell differentiation into ASCs did not protect against angiotensin II-induced hypertension or vascular compliance. Follicular B cell functions independent of their differentiation into ASCs and ability to produce high-affinity antibodies, or other B cell subtypes, are likely to be involved in angiotensin II-induced hypertension.

CD4+ T-Cell Legumain Deficiency Attenuates Hypertensive Damage via Preservation of TRAF6.

T cells are central to the immune responses contributing to hypertension. LGMN (legumain) is highly expressed in T cells; however, its role in the pathogenesis of hypertension remains unclear. Peripheral blood samples were collected from patients with hypertension, and cluster of differentiation (CD)4+ T cells were sorted for gene expression and Western blotting analysis. TLGMNKO (T cell-specific LGMN-knockout) mice (Lgmnf/f/CD4Cre), regulatory T cell (Treg)-specific LGMN-knockout mice (Lgmnf/f/Foxp3YFP Cre), and RR-11a (LGMN inhibitor)-treated C57BL/6 mice were infused with Ang II (angiotensin II) or deoxycorticosterone acetate/salt to establish hypertensive animal models. Flow cytometry, 4-dimensional label-free proteomics, coimmunoprecipitation, Treg suppression, and in vivo Treg depletion or adoptive transfer were used to delineate the functional importance of T-cell LGMN in hypertension development. LGMN mRNA expression was increased in CD4+ T cells isolated from hypertensive patients and mice, was positively correlated with both systolic and diastolic blood pressure, and was negatively correlated with serum IL (interleukin)-10 levels. TLGMNKO mice exhibited reduced Ang II-induced or deoxycorticosterone acetate/salt-induced hypertension and target organ damage relative to wild-type (WT) mice. Genetic and pharmacological inhibition of LGMN blocked Ang II-induced or deoxycorticosterone acetate/salt-induced immunoinhibitory Treg reduction in the kidneys and blood. Anti-CD25 antibody depletion of Tregs abolished the protective effects against Ang II-induced hypertension in TLGMNKO mice, and LGMN deletion in Tregs prevented Ang II-induced hypertension in mice. Mechanistically, endogenous LGMN impaired Treg differentiation and function by directly interacting with and facilitating the degradation of TRAF6 (tumor necrosis factor receptor-associated factor 6) via chaperone-mediated autophagy, thereby inhibiting NF-κB (nuclear factor kappa B) activation. Adoptive transfer of LGMN-deficient Tregs reversed Ang II-induced hypertension, whereas depletion of TRAF6 in LGMN-deficient Tregs blocked the protective effects. LGMN deficiency in T cells prevents hypertension and its complications by promoting Treg differentiation and function. Specifically targeting LGMN in Tregs may be an innovative approach for hypertension treatment.

Resolvin D1 attenuates Ang II-induced hypertension in mice by inhibiting the proliferation, migration and phenotypic transformation of vascular smooth muscle cells by blocking the RhoA/mitogen-activated protein kinase pathway.

The proliferation, migration and phenotypic transformation of vascular smooth muscle cells contribute to vascular remodeling and hypertension. Resolvin D1 (RvD1) is a specialized pro-resolving lipid mediator that has been shown to have anti-inflammatory effects and can protect against different cardiovascular diseases. However, the role and mechanism of RvD1 in hypertension are not clear. The current study investigated the role of RvD1 in Ang II-induced hypertensive mice and Ang II-stimulated rat vascular smooth muscle cells. The results showed that RvD1 treatment significantly attenuated hypertension and vascular remodeling, as indicated by decreases in blood pressure, aortic media thickness and collagen deposition. In addition, RvD1 inhibited the proliferation, migration and phenotypic transformation of vascular smooth muscle cells (VSMCs) in vivo and in vitro . Notably, the protective effects of RvD1 were mediated by the Ras homolog gene family member A (RhoA)/mitogen-activated protein kinase (MAPK) signaling pathway. In conclusion, our findings demonstrated the potential benefits of RvD1 as a promising therapeutic agent in the treatment of vascular remodeling and hypertension.

GENETIC DELETION OF AT1A RECEPTORS SELECTIVELY IN THE RENOMEDULLARY INTERSTITIAL CELLS OF THE KIDNEY MEDULLA ATTENUATES ANGIOTENSIN II-INDUCED HYPERTENSION

Objective: The present study tested the hypothesis that genetic deletion of angiotensin II (Ang II) AT1 (AT1a) receptors selectively in the renomemdullary interstitial cells (RMICs) of the kidney medulla attenuates the development of Ang II-induced hypertension in mice. Design and method: To test the hypothesis, we used inducible Tenascin-C-CreER2 and Agtr1a-floxed recombination approach to generate a novel mouse model with deletion of AT1a receptors selectively in the RMICs of the kidney (RMIC-Agtr1a-/-). Adult male wildtype (WT) and RMIC-Agtr1a-/- mice (n = 10 per group) were implanted with a telemetry probe and infused with or without Ang II via an osmotic minipump (∼500 μg/kg/day, i.p.) for 2 weeks. Results: Mice with deletion of AT1a receptors selectively in RMICs of the kidney medulla were fertile and grew normally without apparent structural or histological abnormality in the kidney medulla. However, telemetry systolic, diastolic, and mean blood pressure were significantly lower in adult male RMIC-Agtr1a-/- than WT mice (WT: 122 ± 4 mmHg vs. RMIC-Agtr1a-/-: 102 ± 3 mmHg, P < 0.01). Interestingly, this hypotensive phenotype in RMIC-Agtr1a-/- mice was associated with significant decreases in 24 h urine output (WT: 1.56 ± 0.15 mL/24 h vs. RMIC-Agtr1a-/-: 0.98 ± 0.13 mL/24 h, P < 0.01), urinary sodium (WT: 207.9 ± 7.3 μmol/24 h vs. RMIC-Agtr1a-/-: 145.2 ± 5.4 μmol/24 h, P < 0.01), and potassium excretion (WT: 221.0 ± 6.7 μmol/24 h vs. RMIC-Agtr1a-/-: 160.8 ± 10.6 μmol/24 h, P < 0.01), respectively. Moreover, urine osmolality was markedly decreased in RMIC-Agtr1a-/-mice (WT: 1724 ± 335 mOsm/Kg H2O vs. RMIC-Agtr1a-/-: 1128 ± 102 mOsm/Kg H2O, P < 0.01). Ang II infusion significantly increased telemetry systolic blood pressure to 148 ± 5 mmHg in WT mice, but it increased systolic blood pressure only to 129 ± 8 mmHg in RMIC-Agtr1a-/- mice (P < 0.01). Furthermore, infusion of Ang II for 2 weeks failed to restore urine osmolality in RMIC-Agtr1a-/- mice (1226 ± 110 mOsm/Kg H2O, n.s.). Conclusions: In conclusion, the results of the present study support the hypothesis that AT1a receptors in the RMICs of the kidney medulla plays a critical role in maintaining basal blood pressure homeostasis and the development of Ang II-induced hypertension.

YAP-galectin-3 signaling mediates endothelial dysfunction in angiotensin II-induced hypertension in mice.

Vascular endothelial dysfunction is regarded as an early event of hypertension. Galectin-3 (Gal-3) is known to participate in various pathological processes. Whilst previous studies showed that inhibition of Gal-3 effectively ameliorates angiotensin II (Ang II)-induced atherosclerosis or hypertension, it remains unclear whether Ang II regulates Gal-3 expression and actions in vascular endothelium. Using techniques of molecular biology and myograph, we investigated Ang II-mediated changes in Gal-3 expression and activity in thoracic aortas and mesenteric arteries from wild-type and Gal-3 gene deleted (Gal-3-/-) mice and cultured endothelial cells. The serum level of Gal-3 was significantly higher in hypertensive patients or in mice with chronic Ang II-infusion. Ang II infusion to wild-type mice enhanced Gal-3 expression in the aortic and mesenteric arteries, elevated systolic blood pressure and impaired endothelium-dependent relaxation of the thoracic aortas and mesenteric arteries, changes that were abolished in Gal-3-/- mice. In human umbilical vein endothelial cells, Ang II significantly upregulated Gal-3 expression by promoting nuclear localization of Yes-associated protein (YAP) and its interaction with transcription factor Tead1 with enhanced YAP/Tead1 binding to Gal-3 gene promoter region. Furthermore, Gal-3 deletion augmented the bioavailability of nitric oxide, suppressed oxidative stress, and alleviated inflammation in the thoracic aorta of Ang II-infused mice or endothelial cells exposed to Ang II. Our results demonstrate for the first time that Ang II upregulates Gal-3 expression via increment in YAP nuclear localization in vascular endothelium, and that Gal-3 mediates endothelial dysfunction contributing to the development of hypertension.

Protection against angiotensin II-induced hypertension in mice lacking sphingosine kinase 1 in vascular smooth muscle cells

Abstract Background Sphingosine-1-phosphate (S1P), a lipid mediator produced by sphingosine kinases (Sphks), significantly contributes to the maintenance of cardiovascular homeostasis. Mice lacking Sphk1 globally are protected against angiotensin II-induced hypertension, yet, due to pleiotropic action of S1P, it has been challenging to identify a specific cell type responsible for this protection. Purpose The aim of this study was to investigate the role of Sphk1 expressed in vascular smooth muscle cells (VSMCs) in the development of angiotensin II (AngII)-induced hypertension in mice. Methods Male, 12-week-old C57BL/6J wild type (WT) or VSMC-specific, Cre-mediated, Sphk1 knockout (Sphk1VSMC−/−) mice underwent either Sham or AngII (490 ng/min/kg s.c.) treatment for 2 weeks, using a surgically implanted osmotic minipump. Blood pressure was measured by the tail-cuff method. Vascular structure and function were examined ex vivo using wire (vasoconstrictor/vasorelaxant responses) and pressure myography (myogenic tone, structural and elasticity parameters). Gene set enrichment analysis (GSEA) was performed using transcriptomic profiles of the isolated mesenteric arteries (MA, RNASeq). Expression of proteins was analyzed by Western blot. Results Deletion of Sphk1 in VSMCs resulted in 5x and 40x lower expression of Sphk1 mRNA in MA and aorta compared to WT mice respectively. A 2-week infusion of AngII resulted in a development of higher systolic blood pressure (SBP) in WT mice compared to Sphk1VSMC−/− mice (mean SBP ± SEM: 168±4.3 mmHg vs. 132±4.4 mmHg respectively, p<0.05). Studies of ex vivo mesenteric artery (MA) function revealed diminished both, depolarization-induced (in response to KCl), as well as G protein-coupled receptor-elicited (in response to phenylephrine and thromboxane A2 analogue) contraction of MA derived from hypertensive Sphk1VSMC−/− mice compared to WT mice. In the same time, significantly impaired endothelial vasorelaxation was observed in MA of hypertensive Sphk1VSMC−/− compared to hypertensive WT mice. Moreover, MA from Sphk1VSMC−/− mice were characterized by the significantly reduced myogenic response evoked by intravascular pressure elevation (maximal myogenic response ± SEM: 19.7±1.8% for WT normotensive, 26.2±1.8% for WT AngII vs. 19.0±2.5% for Sphk1VSMC−/− AngII mice), and an increased stiffness compared to WT mice. GSEA found that the lack of Sphk1 gene in VSMCs significantly downregulated molecular pathways related to smooth muscle contraction. In line with transcriptomic studies, significantly reduced level of the Rho-associated protein kinase (Rock) has been observed in MA of hypertensive Sphk1VSMC−/− compared to hypertensive WT mice using Western blot. Conclusions This study revealed that mice lacking Sphk1 gene in VSMCs are protected against the development of AngII-induced hypertension likely due to the lowered myogenic tone in resistance arteries, which may be mediated by autocrine effects of Sphk1 on Rock proteins. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The National Science Centre, Poland

Targeting the blood–brain barrier disruption in hypertension by ALK5/TGF-Β type I receptor inhibitor SB-431542 and dynamin inhibitor dynasore

IntroductionIn this study, we aimed to target two molecules, transforming growth factor-beta (TGF-β) and dynamin to explore their roles in blood–brain barrier (BBB) disruption in hypertension. MethodsFor this purpose, angiotensin (ANG) II-induced hypertensive mice were treated with SB-431542, an inhibitor of the ALK5/TGF-β type I receptor, and dynasore, an inhibitor of dynamin. Albumin-Alexa fluor 594 was used to assess BBB permeability. The alterations in the expression of claudin-5, caveolin (Cav)-1, glucose transporter (Glut)-1, and SMAD4 in the cerebral cortex and the hippocampus were evaluated by quantification of immunofluorescence staining intensity. ResultsANG II infusion increased BBB permeability to albumin-Alexa fluor 594 which was reduced by SB-431542 (P < 0.01), but not by dynasore. In hypertensive animals treated with dynasore, claudin-5 immunofluorescence intensity increased in the cerebral cortex and hippocampus while it decreased in the cerebral cortex of SB-431542 treated hypertensive mice (P < 0.01). Both dynasore and SB-431542 prevented the increased Cav-1 immunofluorescence intensity in the cerebral cortex and hippocampus of hypertensive animals (P < 0.01). SB-431542 and dynasore decreased Glut-1 immunofluorescence intensity in the cerebral cortex and hippocampus of mice receiving ANG II (P < 0.01). SB-431542 increased SMAD4 immunofluorescence intensity in the cerebral cortex of hypertensive animals, while in the hippocampus a significant decrease was noted by both SB-431542 and dynasore (P < 0.01). ConclusionOur data suggest that inhibition of the TGFβ type I receptor prevents BBB disruption under hypertensive conditions. These results emphasize the therapeutic potential of targeting TGFβ signaling as a novel treatment modality to protect the brain of hypertensive patients.

DP1 (Prostaglandin D2 Receptor 1) Activation Protects Against Vascular Remodeling and Vascular Smooth Muscle Cell Transition to Myofibroblasts in Angiotensin II-Induced Hypertension in Mice.

Vascular smooth muscle cell (VSMC) phenotype transition plays an essential role in vascular remodeling. PGD2 (Prostaglandin D2) is involved in cardiovascular inflammation. In this study, we aimed to investigates the role of DP1 (PGD2 receptor 1) on VSMC phenotype transition in vascular remodeling after Ang II (angiotensin II) infusion in mice. VSMC-specific DP1 knockout mice and DP1flox/flox mice were infused with Ang II for 28 days and systolic blood pressure was measured by noninvasive tail-cuff system. The arterial samples were applied to an unbiased proteome analysis. DP1f/f Myh11 (myosin heavy chain 11) CREERT2 R26mTmG/+ mice were generated for VSMC lineage tracing. Multiple genetic and pharmacological approaches were used to investigate DP1-mediated signaling in phenotypic transition of VSMCs in response to Ang II administration. DP1 knockout promoted vascular media thickness and increased systolic blood pressure after Ang II infusion by impairing Epac (exchange protein directly activated by cAMP)-1-mediated Rap-1 (Ras-related protein 1) activation. The DP1 agonist facilitated the interaction of myocardin-related transcription factor A and G-actin, which subsequently inhibited the VSMC transition to myofibroblasts through the suppression of RhoA (Ras homolog family member A)/ROCK-1 (Rho associated coiled-coil containing protein kinase 1) activity. Moreover, Epac-1 overexpression by lentivirus blocked the progression of vascular fibrosis in DP1 deficient mice in response to Ang II infusion. Our finding revealed a protective role of DP1 in VSMC switch to myofibroblasts by impairing the phosphorylation of MRTF (myocardin-related transcription factor)-A by ROCK-1 through Epac-1/Rap-1/RhoA pathway and thus inhibited the expression of collagen I, fibronectin, ED-A (extra domain A) fibronectin, and vinculin. Thus, DP1 activation has therapeutic potential for vascular fibrosis in hypertension.

NLRP3 inflammasome contributes to endothelial dysfunction in angiotensin II-induced hypertension in mice

AimsInflammation is a key feature of endothelial dysfunction induced by angiotensin (Ang) II. The purpose of this study was to explore the role of Nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome in endothelial dysfunction in Ang II-induced hypertension. Materials and methodsWe analyzed blood pressure and vascular function of wild-type (WT) and Nlrp3 knockout (Nlrp3−/−) mice, treated with Ang II. In vitro, we mainly tested the endothelial nitric oxide synthase (eNOS) phosphorylation expression of human umbilical vein endothelial cells (HUVECs). Key findingsHere we showed that 14-day Ang II infusion into mice resulted in the elevation of blood pressure, NLRP3 expression, serum interleukin (IL)-1β level, and the decline of endothelium-dependent relaxation function, p-eNOS-Ser1177 expression in aortas. Nlrp3 deficiency reduced Ang II-induced blood pressure elevation and endothelial dysfunction. In vitro, NLRP3 was involved in the effect of Ang II on reducing p-eNOS-Ser1177 expression. Moreover, the direct effect of IL-1β on vascular endothelial injury could be observed in both vivo and vitro. SignificanceOur result demonstrates that the NLRP3 inflammasome is critically involved in the detrimental effects of Ang II on vascular endothelium in hypertension via the activation of IL-1β, placing NLRP3 as a potential target for therapeutic interventions in conditions with endothelial dysfunction in hypertension.

Neohesperidin Protects Angiotensin II-Induced Hypertension and Vascular Remodeling.

Vascular remodeling due to hypertension is one of the major health challenges facing countries around the world. Neohesperidin, a flavonoid glycoside found in citrus fruits, is an antioxidant. Neohesperidin has been studied for a variety of diseases in addition to hypertension. In this study, angiotensin II was used to induce hypertension in mice (490 ng/kg/min, 14 days). We used H&E, Masson, immunofluorescence, dihydroethidine and qPCR to evaluate the effect of Nehesperidin (50 mg/kg/day, 16 days) on pathological hypertension in mice. Estimating the effect of Nehesperidin on human umbilical vein endothelial cells and vascular smooth muscle cells stimulated by angiotensin II. We found that neohesperidin inhibited angiotensin II-induced hypertension in mice. Neohesperidin reduced angiotensin II-induced vascular hypertrophy, fibrosis, inflammation and oxidative stress in vivo. Neohesperidin inhibited angiotensin II-induced ROS and DNA damage in human umbilical vein endothelial cells. Neohesperidin inhibited angiotensin II-induced migration of vascular smooth muscle cells. The results showed that Nehesperidin acts as an antioxidant and could significantly inhibit angiotensin II induced hypertension and vascular remodeling in vitro and in vivo.

Phosphodiesterase 4D promotes angiotensin II-induced hypertension in mice via smooth muscle cell contraction

Hypertension is a common chronic disease, which leads to cardio-cerebrovascular diseases, and its prevalence is increasing. The cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway participates in multiple cardiovascular diseases. Phosphodiesterase (PDE) 4 has been shown to regulate PKA activity via cAMP specific hydrolysis. However, whether PDE4-cAMP-PKA pathway influences hypertension remains unknown. Herein, we reveal that PDE4D (one of PDE4 isoforms) expression is upregulated in the aortas of experimental hypertension induced by angiotensin II (Ang II). Furthermore, knockout of Pde4d in mouse smooth muscle cells (SMCs) attenuates Ang II-induced hypertension, arterial wall media thickening, vascular fibrosis and vasocontraction. Additionally, we find that PDE4D deficiency activates PKA-AMP-activated protein kinase (AMPK) signaling pathway to inhibit myosin phosphatase targeting subunit 1 (MYPT1)-myosin light chain (MLC) phosphorylation, relieving Ang II-induced SMC contraction in vitro and in vivo. Our results also indicate that rolipram, a PDE4 inhibitor, may be a potential drug for hypertension therapy.

Thrombin Inhibition Prevents Endothelial Dysfunction and Reverses 20-HETE Overproduction without Affecting Blood Pressure in Angiotensin II-Induced Hypertension in Mice.

Angiotensin II (Ang II) induces hypertension and endothelial dysfunction, but the involvement of thrombin in these responses is not clear. Here, we assessed the effects of the inhibition of thrombin activity by dabigatran on Ang II-induced hypertension and endothelial dysfunction in mice with a particular focus on NO- and 20-HETE-dependent pathways. As expected, dabigatran administration significantly delayed thrombin generation (CAT assay) in Ang II-treated hypertensive mice, and interestingly, it prevented endothelial dysfunction development, but it did not affect elevated blood pressure nor excessive aortic wall thickening. Dabigatran’s effects on endothelial function in Ang II-treated mice were evidenced by improved NO-dependent relaxation in the aorta in response to acetylcholine in vivo (MRI measurements) and increased systemic NO bioavailability (NO2− quantification) with a concomitant increased ex vivo production of endothelium-derived NO (EPR analysis). Dabigatran treatment also contributed to the reduction in the endothelial expression of pro-inflammatory vWF and ICAM-1. Interestingly, the fall in systemic NO bioavailability in Ang II-treated mice was associated with increased 20-HETE concentration in plasma (UPLC-MS/MS analysis), which was normalised by dabigatran treatment. Taking together, the inhibition of thrombin activity in Ang II-induced hypertension in mice improves the NO-dependent function of vascular endothelium and normalises the 20-HETE-depedent pathway without affecting the blood pressure and vascular remodelling.

New Insights into the Critical Importance of Intratubular Na+/H+ Exchanger 3 and Its Potential Therapeutic Implications in Hypertension.

The sodium (Na+) and hydrogen (H+) exchanger 3 (NHE3), known as solute carrier family 9 member 3 (SLC9A3), mediates active transcellular Na+ and bicarbonate reabsorption in the small intestine of the gut and proximal tubules of the kidney. The purpose of this article is to review and discuss recent findings on the critical roles of intestinal and proximal tubule NHE3 in maintaining basal blood pressure (BP) homeostasis and their potential therapeutic implications in the development of angiotensin II (Ang II)-dependent hypertension. Recently, our and other laboratories have generated or used novel genetically modified mouse models with whole-body, kidney-specific, or proximal tubule-specific deletion of NHE3 to determine the critical roles and underlying mechanisms of NHE3 in maintaining basal BP homeostasis and the development of Ang II-induced hypertension at the whole-body, kidney, or proximal tubule levels. The new findings demonstrate that NHE3 contributes to about 10 to 15 mmHg to basal blood pressure levels, and that deletion of NHE3 at the whole-kidney or proximal tubule level, or pharmacological inhibition of NHE3 at the kidney level with an orally absorbable NHE3 inhibitor AVE-0657, attenuates ~ 50% of Ang II-induced hypertension in mice. The results support the proof-of-concept hypothesis that NHE3 plays critical roles in physiologically maintaining normal BP and in the development of Ang II-dependent hypertension. Our results also strongly suggest that NHE3 in the proximal tubules of the kidney may be therapeutically targeted to treat poorly controlled hypertension in humans.

Mutagenesis of the Cleavage Site of Pro Renin Receptor Abrogates Angiotensin II-Induced Hypertension in Mice.

[Figure: see text].