Angiotensin II-induced Hypertensive Mice Research Articles

Sirtuin 7 ameliorates cuproptosis, myocardial remodeling and heart dysfunction in hypertension through the modulation of YAP/ATP7A signaling.

Myocardial fibrosis is a typical pathological manifestation of hypertension. However, the exact role of sirtuin 7 (SIRT7) in myocardial remodeling remains largely unclear. Here, spontaneously hypertensive rats (SHRs) and angiotensin (Ang) II-induced hypertensive mice were pretreated with recombinant adeno-associated virus (rAAV)-SIRT7, copper chelator tetrathiomolybdate (TTM) or copper ionophore elesclomol, respectively. Compared with normotensive controls, reduced SIRT7 expression and augmented cuproptosis were observed in hearts of hypertensive rats and mice with decreased FDX1 levels and increased HSP70 levels. Notably, intervention with rAAV-SIRT7 and TTM strikingly prevented DLAT oligomers aggregation, and elevated ATP7A and TOM20 expressions, contributing to the alleviation of cuproptosis, mitochondrial injury, myocardial remodeling and heart dysfunction in spontaneously hypertensive rats and Ang II-induced hypertensive mice. In cultured rat primary cardiac fibroblasts (CFs), rhSIRT7 alleviated CuCl2, Ang II or elesclomol-induced cuproptosis and fibroblast activation by blunting DLAT oligomers accumulation and downregulating α-SMA expression. Additionally, conditioned medium from rhSIRT7-pretreated CFs remarkably mitigated cellular hypertrophy and mitochondrial impairments of neonatal rat cardiomyocytes, as well as cell migration and polarization of RAW 264.7 macrophages. Importantly, verteporfin reduced CuCl2-induced cuproptosis, mitochondrial injury and fibrotic activation in CFs. Knockdown of ATP7A with si-ATP7A blocked cellular protective effects of rhSIRT7 and verteporfin in CFs. In conclusion, SIRT7 attenuates cuproptosis, myocardial fibrosis and heart dysfunction in hypertension through the modulation of YAP/ATP7A signaling. Targeting SIRT7 is of vital importance for developing therapeutic strategies in hypertension and hypertensive heart disorders.

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.

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.

Galantamine Reduces Blood Pressure and Renal Injury in Female Angiotensin II-induced Hypertensive Mice

Inflammation is a known culprit in the development and progression of hypertension. The autoimmune disease, systemic lupus erythematosus (SLE), is an important model of hypertension in the setting of chronic kidney inflammation. We have shown that administration of an acetylcholinesterase inhibitor, galantamine, attenuates the hypertension, kidney inflammation, and kidney injury in female SLE mice by activating the vagally-mediated cholinergic anti-inflammatory pathway. Although SLE hypertension represents a specific population of patients, we hypothesized that galantamine would have similar anti-inflammatory effects in the angiotensin II (AngII)-induced hypertensive mouse, which more commonly models essential hypertension. Female C57BL/6 mice were implanted with minipumps at 9 weeks of age for subcutaneous delivery of either AngII (1000 ng/kg/min) or saline (controls), and galantamine hydrobromide (4 mg/kg/day) or vehicle, for 21 days. At 12 weeks, carotid catheters were implanted and mean arterial pressure (MAP) was measured in conscious mice for two consecutive days. Mice were then euthanized, and kidneys collected. Markers of kidney inflammation and injury, including interleukin (IL)-6 and kidney injury molecule (KIM)-1, were measured by ELISA and normalized to total kidney protein. MAP (mmHg) was higher in AngII-treated mice compared to controls (174±4 vs. 133±3; n=4-5, p=0.0023). Galantamine lowered MAP in AngII mice (157±8; n=7, p=0.2567) without altering MAP in controls (133±4; n=5, p=0.9999). Renal IL-6 (pg/μg) was not different between AngII and control mice (0.004±0.0001 vs. 0.005±0.0006; p=0.6685). Although galantamine lowered IL-6 in both groups, it was only significant in controls (0.003±0.0002; p=0.0134) and not AngII mice (0.003±0.0003; p=0.4677). Renal cortical KIM-1 (pg/μg) trended higher in AngII mice compared to controls (2.53±0.50 vs 0.63±0.16; p=0.0741). Galantamine lowered KIM-1 in AngII mice (1.18±0.39, p=0.2064), but not controls (1.45 ± 0.59; p=0.6285). In summary, galantamine protected female mice from hypertension and renal injury in a model of AngII hypertension. This suggests that acetylcholinesterase inhibitor therapy may be effective in patients with hypertension. Studies funded by NIH R01HL153703 and K01HL139859 for KWM. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Resolvin E1/ChemR23 Protects Against Hypertension and Vascular Remodeling in Angiotensin II-Induced Hypertensive Mice.

Inflammation plays a critical role in the development of hypertension and vascular remodeling. Resolvin E1 (RvE1), as one of the specialized proresolving lipid mediators, promotes inflammation resolution by binding with a G protein-coupled receptor, ChemR23 (chemerin receptor 23). However, whether RvE1/ChemR23 regulates hypertension and vascular remodeling is unknown. Hypertension in mice was induced by Ang II (angiotensin II) infusion (750 ng/kg per minute), and RvE1 (2 µg/kg per day) was administered through intraperitoneal injection. Loss of ChemR23 was achieved by mice receiving intravenous injection of adeno-associated virus 9-encoding shRNA against ChemR23. Aortic ChemR23 expression was increased in Ang II-induced hypertensive mice and that ChemR23 was mainly expressed on vascular smooth muscle cells (VSMCs). RvE1 lowered blood pressure, reduced aortic media thickness, attenuated aortic fibrosis, and mitigated VSMC phenotypic transformation and proliferation in hypertensive mice, which were all reversed by the knockdown of ChemR23. Moreover, RvE1 reduced the aortic infiltration of macrophages and T cells, which was also reversed by ChemR23 knockdown. RvE1 inhibited Ccl5 expression in VSMCs via the AMPKα (AMP-activated protein kinase α)/Nrf2 (nuclear factor E2-related factor 2)/canonical NF-κB (nuclear factor κB) pathway, thereby reducing the infiltration of macrophages and T cells. The AMPKα/Nrf2 pathway also mediated the effects of RvE1 on VSMC phenotypic transformation and proliferation. In patients with hypertension, the serum levels of RvE1 and other eicosapentaenoic acid-derived metabolites were significantly decreased. RvE1/ChemR23 ameliorated hypertension and vascular remodeling by activating AMPKα/Nrf2 signaling, which mediated immune cell infiltration by inhibiting the canonical NF-κB/Ccl5 pathway, and regulated VSMC proliferation and phenotypic transformation. RvE1/ChemR23 may be a potential therapeutic target for hypertension.

Browning of perivascular adipose tissue prevents vascular dysfunction and reduces hypertension in angiotensin II-infused mice.

Hypertension is a world-leading cause of cardiovascular disease and premature deaths. Vascular tone is in part regulated by perivascular adipose tissue (PVAT) that releases pro and anticontractile factors. In hypertension, dysfunctional PVAT is observed and studies have indicated a causal relationship between dysfunctional PVAT and vascular damage in hypertension. The phenotype of PVAT on resistance vessels is primarily white adipose tissue. The present study investigates the impact of a changed phenotype, i.e., browning of PVAT, on vascular function and the development of hypertension. Browning was induced by β3-adrenergic agonist in control and angiotensin II-induced hypertensive mice. Studied parameters included blood pressure by tail-cuff plethysmography and vascular function by wire myography. Browning was confirmed through an immunohistochemical and gene analysis approach. The anticontractile effect of PVAT is lost in untreated hypertensive mice and vascular tone and blood pressure are increased. Browning of PVAT resulted in a maintained anticontractile effect, improved endothelial function, and reduced development of hypertension. Phenotype of PVAT is a major determinant of PVAT health during hypertensive conditions. Our data clearly demonstrates that browning of PVAT, i.e. changing the phenotype of PVAT, protects the vascular function and counteract the development of hypertension. This study provides novel insights into how PVAT can be protected in pathologies and thus limit the development of hypertension.

Cytosolic full-length PTEN-induced putative kinase 1 enhances mitophagic potential and mtDNA integrity in angiotensin II-induced hypertensive mice

PTEN-induced putative kinase 1 (PINK1) is an essential molecule in mitophagy process in mammalian cells. The primary purpose of this study was to determine the expression and subcellular localization of PINK1 under physiological fluidic conditions in endothelial cells (ECs) and elucidate the implication these phenomena in the endothelial homeostasis and disease prevention. Unidirectional laminar shear stress (LSS) significantly elevated both mRNA and protein expression levels of the full-length PINK1 (FL-PINK1) in ECs. Extensive mitochondrial fractionation assays and confocal microscopic analyses demonstrated significantly reduced FL-PINK1 accumulation in the mitochondrial compartment yet increased in the cytosolic pool of FL-PINK1 after exposed to prolonged high LSS (15 dyne/cm2 for 48h). We also observed that mitophagy flux was significantly decreased without changes in mitochondrial membrane structures and membrane potential under the LSS condition. Moreover, increased cytosolic PINK1 increased mitophagic sensitivity to the cumulative fragmented mitochondria in the ECs. When treated with angiotensin II (AngII), LSS-preconditioning significantly decreased mtDNA lesions and displayed rapid Parkin recruitment followed by mitophagy induction in response to a mitochondrial uncoupler, carbonyl cyanide m-chlorophenyl hydrazone, -mediated oxidative stress condition. In addition, voluntary running exercise-preconditioning showed elevated PINK1 expression and enhanced mitophagy induction and reduced mtDNA lesions in the AngII-induced hypertensive mice. Taken together, LSS specifically increases a cytosolic pool of FL-PINK1, which elevates mitophagic sensitivity to dysfunctional mitochondria in endothelial cells. Our data suggest that enhanced unidirectional laminar shear stress promotes mitochondrial quality control and cell protection mechanisms via a PINK1-dependent pathway. This work was supported by funding from the National Institute of Heart, Lung, and Blood Institute at the National Institutes of Health (R01 HL126952 to J.Y.P.); and The American Heart Association/Beatrice F. Nicoletti research grant (19POST34450157 to J.C.S). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Andrographolide improves the dysfunction of endothelial progenitor cells from angiotensin II-induced hypertensive mice through SIRT1 signaling

Endothelial progenitor cells (EPCs) are crucial for the maintenance of vascular homeostasis. The dysfunction of EPCs contributes to the endothelial damage in hypertension. Andrographolide (AGP) is a traditional Chinese patent medicine that has been reported to have protective effects on cardiovascular system. However, the effect of AGP on the function of EPCs in hypertension remains unknown. In this study, we aimed to elucidate the effect of AGP on EPCs and the underlying mechanisms. In vivo, the blood pressure and endothelial function (indicated by endothelial dependent vasodilation) of AGP-fed angiotensin II (Ang II)-infused hypertensive mice were examined. In vitro, the function of EPCs isolated from bone marrow were evaluated by tube formation, migration, and adhesion assay. Additionally, a silent information regulator 1 (SIRT1) inhibitor/agonist and a small interfering RNA (si-RNA) targeting SIRT1 were used to determine the pathway involved. The results showed that AGP not only reduced blood pressure, improved endothelial function in hypertensive mice but also restored the dysfunction of EPCs of hypertension in vitro. Mechanistically, AGP up-regulated SIRT1 expression, decreased the Bax/Bcl-2 ratio and the expression level of Cleaved caspase-3, thus inhibiting the apoptosis of Ang II induced EPCs. However, the beneficial effects of AGP on EPCs disappeared after the inhibition or the knockdown of SIRT1. To summarize, this study demonstrates for the first time that AGP improves the dysfunction of EPCs through SIRT1-mediated anti-apoptotic effects. Our findings might provide a novel therapeutic strategy for treating vascular damage in 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

Swimming Exercise Alleviates Endothelial Mitochondrial Fragmentation via Inhibiting Dynamin-Related Protein-1 to Improve Vascular Function in Hypertension.

Regular exercise has been recommended clinically for all individuals to protect against hypertension but the underlying mechanisms are not fully elucidated. We recently found a significant mitochondrial fragmentation in the vascular endothelium of hypertensive human subjects. In this study, we investigated whether exercise could restore endothelial mitochondrial dynamics and thus improve vascular function in hypertension. Vascular endothelial mitochondrial morphological alterations were examined in patients with hypertension and hypertensive animal models. Furthermore, swimming exercise-induced endothelial mitochondrial dynamics and vascular function changes were investigated in spontaneously hypertensive rats (SHRs). Mitochondrial fragmentation with an elevated mitochondrial fission mediator Drp1 (dynamin-related protein-1) was observed in the mesenteric artery endothelium from hypertensive patients. A similar mitochondrial fragmentation with increased Drp1 expression were exhibited in the aortic endothelium of angiotensin II-induced hypertensive mice and SHRs. Interestingly, swimming exercise significantly reduced vascular Drp1 expression and alleviated endothelial mitochondrial fragmentation, thus improving blood pressure in SHRs. In cultured endothelial cells, angiotensin II exposure induced Drp1 upregulation, mitochondrial fragmentation and dysfunction, and reduced nitric oxide production, which was blunted by Drp1 genetic reduction or its inhibitor Mdivi-1. Mdivi-1 administration also ameliorated endothelial mitochondrial fragmentation, vascular dysfunction and blood pressure elevation in SHRs while swimming exercise plus Mdivi-1 treatment provided no additional benefits, suggesting that Drp1 inhibition may partially contribute to swimming exercise-conferred anti-hypertensive effects. These findings suggest that swimming exercise alleviates endothelial mitochondrial fragmentation via inhibiting Drp1, which may contribute to exercise-induced improvement of vascular function and blood pressure in hypertension.

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.

Effects of 3-methyladenine, an autophagy inhibitor, on the elevated blood pressure and arterial dysfunction of angiotensin II-induced hypertensive mice

Autophagy is an intracellular degradation system that disassembles cytoplasmic components through autophagosomes fused with lysosomes. Recently, it has been reported that autophagy is associated with cardiovascular diseases, including pulmonary hypertension, atherosclerosis, and myocardial ischemia. However, the involvement of autophagy in hypertension is not well understood. In the present study, we hypothesized that excessive autophagy contributes to the dysfunction of mesenteric arteries in angiotensin II (Ang II)-induced hypertensive mice. Treatment of an autophagy inhibitor, 3-methyladenine (3-MA), reduced the elevated blood pressure and wall thickness, and improved endothelium-dependent relaxation in mesenteric arteries of Ang II-treated mice. The expression levels of autophagy markers, beclin1 and LC3 II, were significantly increased by Ang II infusion, which was reduced by treatment of 3-MA. Furthermore, treatment of 3-MA induced vasodilation in the mesenteric resistance arteries pre-contracted with U46619 or phenylephrine, which was dependent on endothelium. Interestingly, nitric oxide production and phosphorylated endothelial nitric oxide synthase (p-eNOS) at S1177 in the mesenteric arteries of Ang II-treated mice were increased by treatment with 3-MA. In HUVECs, p-eNOS was reduced by Ang II, which was increased by treatment of 3-MA. 3-MA had direct vasodilatory effect on the pre-contracted mesenteric arteries. In cultured vascular smooth muscle cells (VSMCs), Ang II induced increase in beclin1 and LC3 II and decrease in p62, which was reversed by treatment of 3-MA. These results suggest that autophagy inhibition exerts beneficial effects on the dysfunction of mesenteric arteries in hypertension.

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.