Angiotensin II-induced Hypertension Research Articles

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

Single cell atlas of cd45+ cells in angiotensin II-induced hypertension

Abstract Hypertension has been recently identified as an inflammatory disease. Immune cell infiltration is a characteristic feature in the vasculature and the kidneys in experimental hypertension, but the unique nature of such inflammatory infiltrates has not yet been comprehensively characterised. Accordingly, we aimed to provide in-depth characteristics of immune cells in the vasculature and the kidneys in experimental hypertension. To achieve this, we used single-cell RNA-sequencing of leukocytes (CD45+ cells were sorted) using the 10x Genomics platform in the aortas and the kidneys of male 12-week-old C57BL/6J mice (n=16–17/group) upon AngII (490 ng/min/kg) or sham buffer 14-day infusion, using osmotic minipumps. Samples were pooled to analyse three independent replicates. Bioinformatics analysis used Seurat/R to identify immune cell subpopulations and characterise differentially expressed genes (DEGs), pathways, and interactions signatures. Ang II infusion increases the total number of CD45+ leukocytes in the aorta (346.7±89.1 vs. 1210±214.3; p=0,048), while in the kidneys, this was much less pronounced (1.1±0.5 fold vs. sham). Fifteen leukocyte populations/clusters were identified in the aorta and kidney based on their unique markers. In the aorta, shifts in numerous populations were evident, with the most significant differences in tissue-resident macrophages and activated tissue-resident macrophages, monocyte-derived dendritic cells and NK cells (Figure 1). Kidneys did not display such profound changes. The transcriptomic profile analysis showed 767 significant DEGs in the aorta and only 35 in the kidney. CellChat analysis also indicated more robust interactions between the immune cells in the aorta than in kidneys. These included Ifitm1, Apoe, Il1b, and C1q a/b/c, which were shared between aorta and kidney and may play an immunoregulatory role, affecting smooth muscle cell proliferation and arterial vascular remodelling. Top up-regulated leukocyte genes in the aorta included Ccl8, Ccl3, Cxcl2, Lyz2, and Spp1, while in the kidney, Cd74, Cst3, Fos, Fcer1g, Tyrobp, and Ccl4. GO pathway signatures of aortic leukocyte DEGs revealed pathways related to leukocyte migration, cytokine production, T cell activation, and leukocyte activation and adhesion. Cell-specific analysis showed that macrophage subpopulations most strongly increased in Ang II-induced hypertension displayed the most pronounced changes in the transcriptome profiles and cell-cell interactions. Comprehensive single-cell RNA sequencing identifies tissue-resident macrophages, monocyte-derived dendritic cells, and NK cells as most affected leukocyte subpopulations in hypertensive vasculature. Differentially expressed genes support the role of these cells in vascular remodelling and propagation of inflammation, further supporting the identification of these cells as potential future targets for therapeutic interventions in hypertension. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Research Council

Inhibition of coagulation factor XI attenuates inflammation in myocardial ischemia/reperfusion injury

Abstract Background Increasing evidence suggests that FXI is an attractive target for antithrombotic therapy because it reduces thrombosis without increasing bleeding risk. Patients with decreased levels of FXI are at reduced risk of cardiovascular diseases and thromboembolic events. We have already revealed that depletion of FXI inhibits the vascular coagulation-inflammatory circuit in angiotensin II-induced arterial hypertension. However, the effect of FXI depletion on cardiac inflammation and vascular function in ischemia/reperfusion (I/R) injury is unknown. Purpose Using FXI-depleted mice to investigate the role of FXI in inflammation response post cardiac I/R injury. Methods 8–12 weeks old C57BL/6J male mice were injected intraperitoneally with FXI antisense oligonucleotide (FXI ASO) or scrambled controls for a period of 2 weeks. Then we temporarily ligated the left anterior descending (LAD) for 45 minutes to induce myocardial ischemia, followed by the reperfusion for 72 hours (ischemia/reperfusion injury, IRI). Myocardial inflammation and vascular function were analyzed by flow cytometry, real-time PCR, vascular relaxation studies from isolated aortic segment in organ chamber and chemiluminescence photon counting of oxidative burst in whole blood. Results FXI ASO treatment reduced hepatic FXI mRNA levels and prolonged activated partial thromboplastin time. Compared to scrambled ASO injected mice, oxidative burst from whole blood and endothelial dysfunction were decreased in FXI ASO injected mice with IRI. Compared to control groups, depletion of FXI attenuated cardiac infiltration of CD45+CD11b+ cells, especially LyG-LyChigh monocytes and LyG+ neutrophils at day 3 in I/R injury. Furthermore, the expression levels of adhesion molecules and expression of pro-inflammatory cytokines, such as Vcam-1, CCL2, IL-6 and IL-1b, in ischemic myocardium were significantly decreased in the FXI depleted mice of I/R injury. Conclusion Our results suggest that Inhibition of FXI leads to reduction of vascular dysfunction and ROS production in I/R injury, as well as attenuation of the inflammatory response and the influx of inflammatory cells in ischemic myocardium. This indicates that FXI could be a potential target for further treatment in cardiac IRI. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): TICARDIO project has received funding from the European Union's Horizon 2020 research and Innovation programme under the Marie Skłodowska-Curie grant agreement No 813409.

Activation of the Mas receptors by AVE0991 and MrgD receptor using alamandine to limit the deleterious effects of Ang II-induced hypertension.

The MrgD receptor agonist, alamandine (ALA) and Mas receptor agonist, AVE0991 have recently been identified as protective components of the renin-angiotensin system. We evaluated the effects of ALA and AVE0991 on cardiovascular function and remodeling in angiotensin (Ang) II-induced hypertension in rats. Sprague Dawley rats were subject to 4-week subcutaneous infusions of Ang II (80 ng/kg/min) or saline after which they were treated with ALA (50 μg/kg), AVE0991 (576 μg/kg), or ALA+AVE0991 during the last 2 weeks. Systolic blood pressure (SBP) and heart rate (HR) values were recorded with tail-cuff plethysmography at 1, 15, and 29 days post-treatment. After euthanization, the heart and thoracic aorta were removed for further analysis and vascular responses. SBP significantly increased in the Ang II group when compared to the control group. Furthermore, Ang II also caused an increase in cardiac and aortic cyclophilin-A (CYP-A), monocyte chemoattractant protein-1 (MCP-1), and cardiomyocyte degeneration but produced a decrease in vascular relaxation. HR, matrix metalloproteinase-2 and -9, NADPH oxidase-4, and lysyl oxidase levels were comparable among groups. ALA, AVE0991, and the drug combination produced antihypertensive effects and alleviated vascular responses. The inflammatory and oxidative stress related to cardiac MCP-1 and CYP-A levels decreased in the Ang II+ALA+AVE0991 group. Vascular but not cardiac angiotensin-converting enzyme-2 levels decreased with Ang II administration but were similar to the Ang II+ALA+AVE0991 group. Our experimental data showed the combination of ALA and AVE0991 was found beneficial in Ang II-induced hypertension in rats by reducing SBP, oxidative stress, inflammation, and improving vascular responses.

Abstract P345: Inhibition Of Nuclear Receptor Retinoid-related Orphan Receptor γt Attenuates Interleukin-17A Production And Ameliorates Angiotensin II-induced Neuroinflammation And Hypertension In Rats

Interleukin (IL)-17A, a key inflammatory mediator produced primarily by T helper (Th) 17 cells, has been implicated in the pathogenesis of cardiovascular diseases including hypertension and heart failure. IL-17A can access the brain by disrupting blood-brain barrier (BBB) integrity to induce neuroinflammation. We previously reported that IL-17A contributes to angiotensin (ANG) II-induced hypertension via promoting neuroinflammation and sympathetic excitation. The nuclear receptor retinoid-related orphan receptor γt (RORγt) is a master transcription factor regulating Th17 cell differentiation. Here, we sought to determine whether systemic inhibition of RORγt attenuates IL-17A production and diminishes ANG II-induced neuroinflammation and hypertension. Sprague-Dawley rats received a 2-week subcutaneous (sc) infusion of ANG II (150 ng/kg/min) combined with daily sc injection of a RORγt inhibitor digoxin or vehicle (VEH). Compared with the control animals, blood pressure (162±5* vs 118±3 mmHg, *P<0.05) and sympathetic tone as indicated by blood pressure change in response to ganglionic blockade (55±5* vs 23±3 mmHg) were increased in ANG II+VEH rats, which were reduced (38-41%*) in ANG II+digoxin rats. ANG II+VEH rats also had elevated RORγt mRNA in peripheral blood mononuclear cells (PBMCs: 3.38±0.70* vs 1.12±0.22) and higher IL-17A levels in plasma (23.22±5.85 * vs 4.30±0.66 pg/mL), along with increased IL-17A levels in cerebrospinal fluid (CSF: 11.89±1.91* vs 2.01±0.55 pg/mL), mRNA of IL-17A (3.02±0.48* vs 1.07±0.17), cytokines tumor necrosis factor (TNF)-α (2.78±0.45* vs 1.04±0.12) and IL-1β (2.57±0.53* vs 1.03±0.14) in the hypothalamic paraventricular nucleus (PVN), a key cardiovascular-related center in the brain. Although RORγt mRNA was unchanged in PBMCs, levels of IL-17A in plasma and CSF, mRNA of IL-17A, TNF-α and IL-1β in the PVN was reduced (41-62 %*) in ANG II+digoxin rats. Additionally, mRNA of caveolin-1 (2.26±0.39* vs 1.06±0.17), a marker of the BBB permeability, was increased in the PVN in ANG II+VEH rats and decreased (43%*) in ANG II+digoxin rats. These data suggest that RORγt inhibition improves ANG II-induced hypertension and sympathetic activation probably by reducing IL-17A production and neuroinflammation.

Abstract P230: Hypertensive Rats Show Increased Expression Of Flavin Monooxygenases And Diminished Hypertensive Response To Trimethylamine, A Gut Bacterial Product And An Industry Air Pollutant

Flavin-dependent monooxygenases (FMOs) are involved in a wide range of biological processes. Those enzymes catalyze oxygenation of a wide range of compounds like amines. Increasing evidence suggests a contribution of trimethylamine (TMA), a gut-bacteria product and industry pollutant, to cardiovascular disease. TMA is oxidized by FMOs to trimethylamine oxide (TMAO), an emerging marker of cardiovascular risk. We hypothesized that blood pressure phenotype may be associated with altered expression of FMOs.Gene expression and proteins of FMO1, FMO3 and FMO5, were evaluated in the kidneys, liver, lungs, small and large intestines harvested from normotensive, male, Wistar-Kyoto (WKY), spontaneously hypertensive (SHRs) and WKY with angiotensin II-induced hypertension (WKY-ANG) rats. Plasma concentrations of TMA and TMAO were measured at baseline and after the intravenous administration of TMA at dose of 45, 135 or 405μmol/kg using LC-MS. mRNA expression level of FMO1, FMO3 and FMO5 was examined. The obtained results showed that WKY, SHR and WKY-ANG rats revealed expression of FMOs in the following order of magnitude FMO3>FMO1>>FMO5.The highest expression of FMOs was found in the liver and lungs, the lowest in small intestine. In the liver and lungs, SHRs showed significantly higher expression of FMO3 than WKY (0.44 ± 0.04 vs. 0.24 ± 0.02 for liver and 0.73±0.03 vs. 0.35±0.03 a.u. for lungs respectively). It was found that, at the baseline, SHR showed significantly higher TMAO plasma concentration than WKY and WKY-ANG (10.52 ± 0.97, 3.31 ± 0.57 and 6.11 ± 0.55 μmol/L, respectively). TMA plasma level was not significantly higher in SHR than in WKY and WKY-ANG 0.15 μmol/L ± 0.01, 0.14 μmol/L ± 0.02 and 0.09 ± 0.02 μmol/L, respectively. 20 min after the infusion, SHR showed significantly higher plasma TMAO/TMA ratio than WKY and WKY-ANG, for all examined TMA doses. In conclusion, hypertensive rats show increased expression and activity of FMO3. Increased activity of FMOs may contribute to blood pressure phenotype due to altered metabolism of endogenous and exogenous amines such as TMA.

Abstract P062: Interaction Of Cytochrome P450 1B1 And 12/15 Lipoxygenase In Paraventricular Nucleus In The Regulation Of Angiotensin II-induced Hypertension In Female Mice

Recently, we showed that 17β-estradiol (E2)-cytochrome P450 (CYP) 1B1-generated metabolite 2-methoxyestradiol (2-ME) in the paraventricular nucleus (PVN) protects from angiotensin (Ang) II-induced hypertension. Moreover, we showed that 2-ME produces this action by inhibiting the cytosolic phospholipase (cPLA) 2 α/arachidonic acid (AA)-cyclooxygenase (COX)-generated metabolites prostaglandin (PG)E 2 and thromboxane (TX)A 2 exerting prohypertensive effects via prostanoid receptors EP1, EP3, and TP, respectively, in female mice. 12/15 lipoxygenase (ALOX15), which also metabolizes AA, is present in the brain in neural tissue, endothelial cells, microglia, and astrocytes. This study was conducted to determine the contribution of central 12/15 lipoxygenase (ALOX15) and its relationship to CYP1B1 in Ang II-induced hypertension in the female mice. ALOX15 knockdown in the PVN by transduction with adenovirus (Ad)-ALOX15-short hairpin (sh)RNA (200 nL, bilaterally 7.04х10 11 pfu/mL) but not its control Ad-scrambled (Scr)-shRNA (6.4х10 11 pfu/mL) produced a greater reduction in systolic blood pressure (SBP, mmHg) measured by tail-cuff in response to Ang II (700 ng/kg/min, subcutaneous, osmotic pump, two weeks) in intact Cyp1b1 knockout (KO, E2 present but lacking 2-ME) than wild-type (WT, endogenous E2 and 2-ME present) female mice (Day 12: 177±4 vs 143±1 and 133±3 vs 124±1, respectively, P<0.05, n=4/group). Moreover, administration of ALOX15-generated AA metabolite 12(S)-hydroxyeicosatetraenoic acid (HETE) in a dose (50 ng/ 2 μL/ every 3rd day, intracerebroventricularly) that did not alter the mean arterial pressure (MAP) alone produced a greater increase in MAP in response to Ang II in ovariectomized (both E2 and 2-ME are lacking) than intact Alox15 KO female mice (Day 0 vs Day 12: 103±4 vs 167±2 and 103±2 vs 128±1, respectively, P<0.05, n=4/group). These results suggest that E2, via its CYP1B1-derived metabolite 2-ME, inhibits the generation of ALOX15/AA metabolite 12(S)-HETE in the brain to protect against Ang II-induced hypertension in female mice. Thus, 2-ME and/or ALOX15/12(S)-HETE inhibitors could be useful for treating hypertension and its pathogenesis in females.

Abstract P316: ANGIOTENSIN II-INDUCED LEFT VENTRICLE HYPERTROPHY AND DYSFUNCTION WERE EXAGGERATED BY P2RX7 KNOCKOUT BUT UNAFFECTED BY P2RX7 PHARMACOLOGIC BLOCKADE

Introduction: We have demonstrated that the P2X7 receptor (P2RX7), which activates immune cells via binding of extracellular adenosine triphosphate, plays a role in hypertension and vascular injury. Angiotensin (Ang) II-induced hypertension, vascular injury and perivascular infiltration of activated CD8 + T cells were attenuated by P2rx7 knockout or P2RX7 pharmacologic blockade. However, whether P2RX7 is involved in Ang II-induced cardiac dysfunction and hypertrophy remains unknown. Hypothesis: We hypothesized that Ang II-induced left ventricular (LV) dysfunction and hypertrophy will be blunted equally by P2rx7 knockout and P2RX7 antagonism. Methods: Ten-to-12-week-old male C57BL/6J wild-type (WT) and P2rx7 -/- mice were infused or not with Ang II (1000 ng/kg/min) for 14 days. A second group of WT mice infused with Ang II was infused with the P2RX7 antagonist AZ10606120 (694 ng/kg/min) or vehicle for 14 days. Cardiac LV function and remodeling were determined by ultrasound, and hypertrophic markers in cardiac ventricles by reverse transcription-quantitative PCR (RT-qPCR). Results: Ang II-induced increase in LV mass/body weight (6.3±0.2 vs 4.2±0.2 mg/g, P <0.001) and decrease in fractional shortening in WT mice (32.5±3.1% vs 43.8±2.4%, P <0.05) were exaggerated in P2rx7 -/- mice (7.3±0.5 mg/g and 20.2±3.1%, P <0.05), but not in mice receiving AZ10606120 (6.2±0.4 mg/g and 28.9±3.3%). Ang II-induced atrial natriuretic peptide ( Nppa /ribosomal protein S16 [ Rps16 ], 6.8±2.3 vs 1±0.1, P <0.001) and α-skeletal actin 1 expression ( Acta1 / Rps16 , 6.3±1.3 vs 1±0.2, P <0.001) tended to be further increased in P2rx7 -/- mice ( Nppa / Rps16 : 13.5±2.4 and Acta1 / Rps16 : 11.9±2.4), but not in AZ10606120-treated mice ( Nppa / Rps16 : 5.5±0.7 and Acta1 / Rps16 : 5.6±0.8). Conclusion: Ang II-induced LV hypertrophy and dysfunction were exaggerated by P2rx7 knockout but were not affected by P2RX7 pharmacologic blockade.

Abstract 046: Induction Of Endothelial Sirt3 After Onset Of Hypertension Rescues Endothelial Function

Endothelial dysfunction plays a key role in the pathogenesis of hypertension and represents a major risk factor for cardiovascular disease. Despite treatment with multiple drugs, only 1 in 4 patients has blood pressure under control. We discovered a new synergistic control point with the potential to address this problem. We have shown that Sirt3 level is reduced in essential hypertension and endothelial Sirt3 overexpression attenuates endothelial dysfunction and hypertension. We proposed that induction endothelial Sirt3 after onset of hypertension rescues endothelial function and reduces hypertension. To test this hypothesis, we used genetic- and pharmacological Sirt3 induction. To genetically induce the expression of endothelial Sirt3 we used Sirt3 flox/flox mice crossed with VeCad-Cre mice treated with low dose of 4-hydroxytamoxifen (4HOT, i.p. 0.3 mg/20 g daily, 5 days) which does not have off-target cardiovascular effects. Mice underwent telemetry placement and ten days later received 4-week osmotic pumps containing vehicle (saline) or angiotensin II (0.7 mg/kg/day). 14-days later half of mice received 4HOT to induce endothelial Sirt3 overexpression. Treatment of mice with 4HOT after onset of hypertension slightly reduced blood pressure but most importantly completely rescued endothelial-dependent relaxation, normalized mitochondrial O 2 . - and restored endothelial nitric oxide. Second, we tested if pharmacological induction of Sirt3 by hexafluoro (i.p. 8 mg/kg, 4 days) after onset of angiotensin II-induced hypertension improves endothelial function and reduces hypertension. It was found that hexafluoro substantially reduced systolic blood pressure, significantly diminished vascular mitochondrial O 2 . - and improved endothelial nitric oxide. To test the role of endothelial Sirt3 in pharmacological effect of hexafluoro we used endothelial specific Sirt3 knockout mice and angiotensin II model of hypertension. Interestingly, hexafluoro was not effective in endothelial Sirt3 deficient mice supporting critical role of endothelial Sirt3. These in vivo studies demonstrate therapeutic potential of genetic and pharmacological endothelial Sirt3 induction after onset of hypertension for treatment of endothelial dysfunction.

Broad-acting therapeutic effects of miR-29b-chitosan on hypertension and diabetic complications

MicroRNA miR-29 promotes endothelial function in human arterioles in part by targeting LYPLA1 and increasing nitric oxide production. In addition, miR-29 is a master inhibitor of extracellular matrix gene expression, which may attenuate fibrosis but could also weaken tissue structure. The goal of this study was to test whether miR-29 could be developed as an effective, broad-acting, and safe therapeutic. Substantial accumulation of miR-29b and effective knockdown of Lypla1 in several mouse tissues were achieved using a chitosan-packaged, chemically modified miR-29b mimic (miR-29b-CH-NP) injected systemically at 200μg/kg body weight. miR-29b-CH-NP, injected once every 3days, significantly attenuated angiotensin II-induced hypertension. In db/db mice, miR-29b-CH-NP treatment for 12weeks decreased cardiac and renal fibrosis and urinary albuminuria. In uninephrectomized db/db mice, miR-29b-CH-NP treatment for 20weeks significantly improved myocardial performance index and attenuated proteinuria. miR-29b-CH-NP did not worsen abdominal aortic aneurysm in ApoE knockout mice treated with angiotensin II. miR-29b-CH-NP caused aortic root fibrotic cap thinning in ApoE knockout mice fed a high-cholesterol and high-fat diet but did not worsen the necrotic zone or mortality. In conclusion, systemic delivery of low-dose miR-29b-CH-NP is an effective therapeutic for several forms of cardiovascular and renal disease in mice.

Empagliflozin prevents angiotensin II-induced hypertension related micro and macrovascular endothelial cell activation and diastolic dysfunction in rats despite persistent hypertension: Role of endothelial SGLT1 and 2

SGLT2 inhibitors (SGLT2i) showed pronounced beneficial effects in patients with heart failure but the underlying mechanisms remain unclear. We evaluated the effect of empagliflozin, selective SGLT2i, on hypertension-induced cardiac and vascular dysfunction.Male Wistar rats received diet with or without empagliflozin (30 mg/kg/day). After 1 week, a hypertensive dose of Ang II (0.4 mg/kg/day) was administered using osmotic mini-pumps for 4 weeks. Systolic blood pressure was determined by sphygmomanometry, the cardiac function by echocardiography and ex vivo (coronary microvascular endothelial cell activation, LV remodeling and fibrosis responses), and the systemic micro and macrovascular endothelial cell activation ex vivo. Empagliflozin treatment did not affect the Ang II-induced hypertensive response. Ang II treatment increased LV mass and induced LV diastolic dysfunction, fibrosis, collagen I and ANP expression, and infiltration of macrophages. In the vasculature, it caused eNOS upregulation in the aorta and down-regulation in mesenteric microvessels associated with increased oxidative stress, ACE, AT1R, VCAM-1, MCP-1, MMP-2, and MMP-9 and collagen I expression, increased endothelial SGLT1 staining in the aorta, mesenteric and coronary microvessels, increased SGLT1 and 2 protein levels in the aorta. All Ang II-induced cardiac and vascular responses were reduced by the empagliflozin treatment.Thus, the SGLT2i effectively attenuated the deleterious impact of Ang II-induced hypertension on target organs including cardiac diastolic dysfunction and remodeling, and endothelial cell activation and pro-atherosclerotic, pro-fibrotic and pro-remodeling responses in macro and microvessels despite persistent hypertension.

Age and Blood Pressure Contribute to Aortic Cell and Tissue Stiffness Through Distinct Mechanisms.

Aortic stiffening is strongly associated with both aging and hypertension, but the underlying mechanisms remain unclear. We hypothesized that aging-induced aortic stiffness is mediated by a mechanism differing from hypertension. We conducted comprehensive in vivo and in vitro experiments using multiple rat models to dissect the different mechanisms of aortic stiffening mediated by aging and hypertension. A time-course study in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) normotensive rats showed more pronounced aging-associated aortic stiffening in SHR versus WKY. Angiotensin II-induced hypertension was associated with more significant aortic stiffening in older versus young WKY rats. Hypertension aggravated aging effects on aortic wall thickness and extracellular matrix content, indicating combinational effects of aging and hypertension on aortic stiffening. Intrinsic stiffness of isolated aortic vascular smooth muscle cells (VSMCs) increased with age in WKY rats, although no significant difference between older SHR and older WKY VSMCs was observed in 2-dimensional culture, reconstituted 3-dimensional tissues were stiffer for older SHR versus older WKY. A selective inhibitor that reduced hypertension-mediated aortic stiffening did not decrease age-related stiffening in aortic VSMCs and aortic wall. Integrin β1 and SM22 (smooth muscle-specific SM22 protein) expression were negligibly changed in WKY VSMCs during aging but were markedly increased by hypertension in older versus young WKY VSMCs. A notable shift of filamin isoforms from B to A was detected in older WKY VSMCs. Our results indicate distinct mechanisms mediating aging-associated aortic VSMC and vessel stiffness, providing new insights into aortic stiffening and the pathogenesis of hypertension in the elderly.

Hinokitiol produces vasodilation in aortae from normal and angiotensin II- induced hypertensive rats via endothelial-dependent and independent pathways

Hinokitiol is a natural bioactive compound with numerous pharmacological properties. Here, we aimed to examine hinokitiol's effects on vascular relaxation. Cumulative relaxation responses to hinokitiol were assessed in isolated aortae from normotensive and angiotensin II-induced hypertensive rats in the presence and absence of selective inhibitors. Hinokitiol produced vasodilation of phenylephrine preconstricted aortae using both normotensive and hypertensive rats. In normotensive rats, hinokitiol's vasodilation was reduced by endothelial denudation and nitric oxide synthase (NOS), guanylate cyclase, and cyclooxygenase inhibition. Also, hinokitiol vasodilation was attenuated by β-receptors, adenylate cyclase, Ca2+-activated K+ channels and hyperpolarization inhibition. Moreover, hinokitiol exhibited a blocking activity on Ca2+ mobilization through voltage dependent Ca2+ channels (VDCC). However, its effect was not changed by muscarinic receptor and Sarc-K+ ATP channels blocking but was enhanced by blocking voltage-dependent K+ channels. However, in angiotensin II-induced hypertension, hinokitiol vasodilating activity was attenuated by NOS inhibition and it blocked Ca2+ mobilization through VDCC, while its vasodilation was partially attenuated by Sarc-K+ ATP channels blocking. However, the vasodilating effect of hinokitiol was not attenuated by either cyclooxygenase, β-receptor, Ca2+-activated K+ channels, or voltage-dependent potassium channels inhibition, but was enhanced by blocking hyperpolarization. Hinokitiol's vasodilating effect in normotensive and hypertensive vessels is mediated through both endothelium-dependent and endothelium-independent mechanisms.

Dahl salt-resistant rats are protected against angiotensin II-induced hypertension

Angiotensin II is a potent endogenous vasoconstrictor that induces oxidative stress in hypertensive rodent models. Dahl salt-resistant (SR) rats are protected against hypertension after high salt or high fructose intake. However, whether these rats are also protected against angiotensin II-mediated hypertension has not been investigated. Dahl salt-sensitive (SS) and SR rats were infused with angiotensin II (10 or 50 ng/kg/min) or vehicle via a mini-osmotic pump for 2 weeks. Blood pressure was measured using the tail-cuff method. Paraffin sections of the thoracic aortas and kidneys were stained using hematoxylin/eosin or Masson trichrome. Renal gene expression was measured using reverse transcription-quantitative polymerase chain reaction. Angiotensin II (50 ng/kg/min) induced hypertension in SS rats, but not in SR rats, although low doses of angiotensin II (10 ng/kg/min) transiently increased blood pressure in SS rats. Angiotensin II (50 ng/kg/min) did not induce morphological changes in the aortic walls or kidneys. Angiotensin II (50 ng/kg/min) induced the expression of At1rb, Nox2, Il-17ra, Il-23r, Tgf-β, Il-1β and Il-6 in SS rats, but not in SR rats. In conclusion, SR rats were protected against angiotensin II-induced hypertension. This result implies that the genetic trait that determines salt sensitivity may also determine susceptibility to hypertension in response to vasoconstrictors.

Resolvin D2 prevents cardiovascular damage in angiotensin II-induced hypertension

Introduction: Vascular functional and structural alterations induced by hypertension are greatly influenced by low- grade chronic inflammation. Resolution of inflammation is orchestrated by specialized lipid pro-resolving mediators (SPMs), which derive from n3 fatty acids (PUFAs). Previous evidence suggest that SPM prevent vascular damage in several pathological situations including atherosclerosis or vascular restenosis. Among SPMs, resolvins (Rv) seem to have beneficial effects in some cardiovascular pathologies, but little is known about their effect on cardiovascular damage in hypertension. Objective: The aim of this study was to evaluate the effects of resolvin D2 (RvD2) in blood pressure and cardiovascular damage associated with hypertension. Material and methods: aorta, mesenteric resistance arteries (MRA), heart and peritoneal macrophages were taken from C57BL/6J mice, infused or not with angiotensin II (AngII; 1,44mg/kg/day; for 14 days) in presence or absence of RvD2 (100ng/mice, every second day, started before AngII infusion). Blood pressure was measured by tail-cuff plethysmography. Cardiac and vascular function and structure were studied with wire and pressure myographs, confocal microscopy, histological staining, and echocardiography. Circulating leucocyte were analyzed by flow cytometry and macrophages behavior by electrophysiology. Gene expression was analyzed with RT-PCR. Results: Aorta or heart from AngII-infused mice showed altered expression of enzymes and receptors involved in SPMs biosynthesis and signaling. We also observed a downregulation of SPMs in heart tissues from these mice including 17R-RvD1 and RvE3. Treatment with RvD2 partially prevented the increase in blood pressure and in the content of circulating immune cells induced by AngII. RvD2 treatment also improved cardiac hypertrophy, fibrosis and dysfunction. Moreover, RvD2 treatment reduced vascular hypercontractility and endothelial dysfunction induced by AngII likely because of enhanced NO and PGI2 availability. RvD2 normalized AngII-induced vascular remodeling by decreasing media thickness and number of vascular smooth muscle cell, while it did not affect vascular stiffness. Finally, RvD2 reduced aortic and cardiac leukocyte infiltration and shifted macrophage phenotype towards a pro-resolving phenotype. Conclusion: Our data shows that RvD2 treatment limits the cardiovascular alterations induced by hypertension. These findings highlight that activating resolution mechanisms by treatment with RvD2 may represent a novel therapeutic strategy for the treatment of cardiovascular alterations associated to 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.

Type 1 Angiotensin Receptors on CD11c-Expressing Cells Protect Against Hypertension by Regulating Dendritic Cell-Mediated T Cell Activation.

Type 1 angiotensin (AT1) receptors are expressed on immune cells, and we previously found that bone marrow-derived AT1 receptors protect against Ang (angiotensin) II-induced hypertension. CD11c is expressed on myeloid cells derived from the bone marrow, including dendritic cells (DCs) that activate T lymphocytes. Here, we examined the role of AT1 receptors on CD11c+ cells in hypertension pathogenesis. Mice lacking the dominant murine AT1 receptor isoform, AT1a, on CD11c+ cells (dendritic cell [DC] AT1aR knockout [KO]) and wild-type (WT) littermates were subjected to Ang II-induced hypertension. Blood pressures were measured by radiotelemetry. DC AT1aR KO mice had exaggerated hypertensive responses to chronic Ang II infusion with enhanced renal accumulation of effector memory T cells and CD40+ DCs. CCL5 (C-C motif chemokine ligand 5) recruits T cells into injured tissues, and CCR7 (C-C motif chemokine receptor 7) facilitates DC and T cell interactions in the kidney lymph node to allow T cell activation. DCs from the hypertensive DC AT1aR KO kidneys expressed higher levels of CCL5 and CCR7. mRNA expressions for CCR7 and tumor necrosis factor-α were increased in CD4+ T cells from the renal lymph nodes of DC AT1aR KO mice. During the second week of Ang II infusion when blood pressures between groups diverged, DC AT1aR KO mice excreted less sodium than WTs. Expressions for epithelial sodium channel subunits were increased in DC AT1aR KO kidneys. Following activation of the renin angiotensin system, AT1aR stimulation on DCs suppresses renal DC maturation and T cell activation with consequent protection from sodium retention and blood pressure elevation.

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.

Mediterranean G6PD variant rats are protected from Angiotensin II-induced hypertension and kidney damage, but not from inflammation and arterial stiffness

RationaleEpidemiological studies suggest that individuals in the Mediterranean region with deficiency of glucose-6-phosphate dehydrogenase (G6PD) are less susceptible to cardiovascular diseases. However, our knowledge regarding the effects of G6PD deficiency on pathogenesis of vascular diseases caused by factors, like angiotensin II (Ang-II), which stimulate synthesis of inflammatory cytokines and vascular inflammation, is lacking. Furthermore, to-date the effect of G6PD deficiency on vascular health has been controversial and difficult to experimentally prove due to a lack of good animal model. ObjectiveTo determine the effect of Ang-II-induced hypertension (HTN) and stiffness in a rat model of the Mediterranean G6PD (G6PDS188F) variant and in wild-type (WT) rats. Methods and resultsOur findings revealed that infusion of Ang-II (490 ng/kg/min) elicited less HTN and medial hypertrophy of carotid artery in G6PDS188F than in WT rats. Additionally, Ang-II induced less glomerular and tubular damage in the kidneys – a consequence of elevated pressure – in G6PDS188F than WT rats. However, Ang-II-induced arterial stiffness increased in G6PDS188F and WT rats, and there were no differences between the groups. Mechanistically, we found aorta of G6PDS188F as compared to WT rats produced less sustained contraction and less inositol-1,2,3-phosphate (IP3) and superoxide in response to Ang-II. Furthermore, aorta of G6PDS188F as compared to WT rats expressed lower levels of phosphorylated extracellular-signal regulated kinase (ERK). Interestingly, the aorta of G6PDS188F as compared to WT rats infused with Ang-II transcribed more (50-fold) myosin heavy chain-11 (MYH11) gene, which encodes contractile protein of smooth muscle cell (SMC), and less (2.3-fold) actin-binding Rho-activating gene, which encodes a protein that enhances SMC proliferation. A corresponding increase in MYH11 and Leiomodin-1 (LMOD1) staining was observed in arteries of Ang-II treated G6PDS188F rats. However, G6PD deficiency did not affect the accumulation of CD45+ cells and transcription of genes encoding interleukin-6 and collagen-1a1 by Ang-II. ConclusionsThe G6PDS188F loss-of-function variant found in humans protected rats from Ang-II-induced HTN and kidney damage, but not from vascular inflammation and arterial stiffness.

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.