Ang II-induced Increase Research Articles

AT2R agonist NP‐6A4 mitigates aortic stiffness and proteolytic activity in mouse model of aneurysm

Clinical and experimental studies show that angiotensin II (AngII) promotes vascular pathology via activation of AngII type 1 receptors (AT1Rs). We recently reported that NP‐6A4, a selective peptide agonist for AngII type 2 receptor (AT2R), exerts protective effects on human vascular cells subjected to serum starvation or doxorubicin exposure. In this study, we investigated whether NP‐6A4–induced AT2R activation could mitigate AngII‐induced abdominal aortic aneurism (AAA) using AngII‐treated Apoe−/− mice. Male Apoe−/− mice were infused with AngII (1 µg/kg/min) by implanting osmotic pumps subcutaneously for 28 days. A subset of mice was pre‐treated subcutaneously with NP‐6A4 (2.5 mg/kg/day) or vehicle for 14 days prior to AngII, and treatments were continued for 28 days. NP‐6A4 significantly reduced aortic stiffness of the abdominal aorta induced by AngII as determined by ultrasound functional analyses and histochemical analyses. NP‐6A4 also increased nitric oxide bioavailability in aortic tissues and suppressed AngII‐induced increases in monocyte chemotactic protein‐1, osteopontin and proteolytic activity of the aorta. However, NP‐6A4 did not affect maximal intraluminal aortic diameter or AAA incidences significantly. These data suggest that the effects of AT2R agonist on vascular pathologies are selective, affecting the aortic stiffness and proteolytic activity without affecting the size of AAA.

Dietary fructose enhances angiotensin II-stimulated Na+ transport via activation of PKC-α in renal proximal tubules.

Angiotensin II (ANG II) stimulates proximal nephron transport via activation of classical protein kinase C (PKC) isoforms. Acute fructose treatment stimulates PKC and dietary fructose enhances ANG II's ability to stimulate Na+ transport, but the mechanisms are unclear. We hypothesized that dietary fructose enhances ANG II's ability to stimulate renal proximal tubule Na+ reabsorption by augmenting PKC-α activation and increases in intracellular Ca2+. We measured total and isoform-specific PKC activity, basal and ANG II-stimulated oxygen consumption, a surrogate of Na+ reabsorption, and intracellular Ca2+ in proximal tubules from rats given either 20% fructose in their drinking water (fructose group) or tap water (control group). Total PKC activity was measured by ELISA. PKC-α, PKC-β, and PKC-γ activities were assessed by measuring particulate-to-soluble ratios. Intracelluar Ca2+ was measured using fura 2. ANG II stimulated total PKC activity by 53 ± 15% in the fructose group but not in the control group (-15 ± 11%, P < 0.002). ANG II stimulated PKC-α by 0.134 ± 0.026 but not in the control group (-0.002 ± 0.020, P < 0.002). ANG II increased PKC-γ activity by 0.008 ± 0.003 in the fructose group but not in the control group (P < 0.046). ANG II did not stimulate PKC-β in either group. ANG II increased Na+ transport by 454 ± 87 nmol·min-1·mg protein-1 in fructose group, and the PKC-α/β inhibitor Gö6976 blocked this increase (-96 ± 205 nmol·min-1·mg protein-1, P < 0.045). ANG II increased intracellular Ca2+ by 148 ± 53 nM in the fructose group but only by 43 ± 10 nM in the control group (P < 0.035). The intracellular Ca2+ chelator BAPTA blocked the ANG II-induced increase in Na+ transport in the fructose group. We concluded that dietary fructose enhances ANG II's ability to stimulate renal proximal tubule Na+ reabsorption by augmenting PKC-α activation via elevated increases in intacellular Ca2+.

Apelin-13 alleviated cardiac fibrosis via inhibiting the PI3K/Akt pathway to attenuate oxidative stress in rats with myocardial infarction-induced heart failure.

The present study aimed to determine whether apelin-13 could attenuate cardiac fibrosis via inhibiting the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway to inhibit reactive oxygen species in heart failure (HF) rats. HF models were established by inducing ischemia myocardial infarction (MI) through ligation of the left anterior descending artery in Sprague–Dawley (SD) rats. MI-induced changes in hemodynamics and cardiac function were reversed by apelin-13 administration. The increases in the levels of collagen I, collagen III, α-smooth muscle actin (SMA), and transforming growth factor-β (TGF-β) in the heart of MI rats and cardiac fibroblasts (CFs) treated with angiotensin (Ang) II were inhibited by apelin-13. The levels of PI3K and p-Akt increased in Ang II-treated CFs, and these increases were blocked by apelin-13. The PI3K overexpression reversed the effects of apelin-13 on Ang II-induced increases in collagen I, collagen III, α-SMA, and TGF-β, NADPH oxidase activity and superoxide anions in CFs. Apelin-13 reduced the increases in the levels of NADPH oxidase activity and superoxide anions in the heart of MI rats and CFs with Ang II treatment. The results demonstrated that apelin-13 improved cardiac dysfunction, impaired cardiac hemodynamics, and attenuated fibrosis of CFs induced by Ang II via inhibiting the PI3K/Akt signaling pathway to inhibit oxidative stress.

Study on protective effect of vanillic acid from Astragalus membranaceus on hypertensive cardiac remodeling based on network pharmacology screen

To identify and verify the active ingredients from Astragalus membranaceus on hypertensive cardiac remodeling based on network pharmacology and heart RNA-sequencing data. The monomers of A. membranaceus and their intervention target database were established by using network pharmacology. The genes associated to cardiac remodeling were then screened by analyzing cardiac RNA-sequencing data. An overlap between genes related to cardiac remodeling and targets of ingredients form A. membranaceus was collected to obtain monomers with protective effect on hypertensive cardiac remodeling. Angiotensin Ⅱ(AngⅡ)-induced mouse cardiac remodeling model was used to validate the protective effect of active ingredients from A. membranaceus on hypertensive cardiac remodeling. Finally, a total of 81 monomers and 1 197 targets were enrolled in our database. Mouse RNA-sequencing data showed that 983 genes were significantly up-regulated and 465 genes were down-regulation in myocardial tissues of the cardiac remodeling mice as compared with blank group mice, respectively. Ninety-two genes were found via overlapping between genes related to cardiac remodeling and targets, involving 59 monomers from A. membranaceus. Further research found that vanillic acid(VA) could intervene 27 genes associated with hypertensive cardiac remodeling, ranking top 1. Meanwhile, VA could significantly inhibit AngⅡ-induced increase in ratio of heart weight to body weight and heart weight to tibial length, ANP and BNP mRNA levels in myocardial tissues, myocardial tissue damage, cardiac fibrosis level and cardiac hypertrophy level in vivo. Those results showed that network pharmacology screen-based VA has protective effect on AngⅡ-induced cardiac remodeling.

AT1 and AT2 receptors modulate renal tubular cell necroptosis in angiotensin II-infused renal injury mice

Abnormal renin-angiotensin system (RAS) activation plays a critical role in the initiation and progression of chronic kidney disease (CKD) by directly mediating renal tubular cell apoptosis. Our previous study showed that necroptosis may play a more important role than apoptosis in mediating renal tubular cell loss in chronic renal injury rats, but the mechanism involved remains unknown. Here, we investigate whether blocking the angiotensin II type 1 receptor (AT1R) and/or angiotensin II type 2 receptor (AT2R) beneficially alleviates renal tubular cell necroptosis and chronic kidney injury. In an angiotensin II (Ang II)-induced renal injury mouse model, we found that blocking AT1R and AT2R effectively mitigates Ang II-induced increases in necroptotic tubular epithelial cell percentages, necroptosis-related RIP3 and MLKL protein expression, serum creatinine and blood urea nitrogen levels, and tubular damage scores. Furthermore, inhibition of AT1R and AT2R diminishes Ang II-induced necroptosis in HK-2 cells and the AT2 agonist CGP42112A increases the percentage of necroptotic HK-2 cells. In addition, the current study also demonstrates that Losartan and PD123319 effectively mitigated the Ang II-induced increases in Fas and FasL signaling molecule expression. Importantly, disruption of FasL significantly suppressed Ang II-induced increases in necroptotic HK-2 cell percentages, and necroptosis-related proteins. These results suggest that Fas and FasL, as subsequent signaling molecules of AT1R and AT2R, might involve in Ang II-induced necroptosis. Taken together, our results suggest that Ang II-induced necroptosis of renal tubular cell might be involved both AT1R and AT2R and the subsequent expression of Fas, FasL signaling. Thus, AT1R and AT2R might function as critical mediators.

LncRNA FAF inhibits fibrosis induced by angiotensinogen II via the TGFβ1-P-Smad2/3 signalling by targeting FGF9 in cardiac fibroblasts

The dysregulation of Long noncoding RNAs (lncRNAs) has been implicated in many cardiovascular diseases, including cardiac fibrosis. However, the functions and mechanisms of lncRNAs in cardiac fibroblasts (CFs) have not been fully elucidated. First, we observed a correlation between cardiac remodeling (CR) and lncRNA FAF (FGF9-associated factor, termed FAF) expression in the heart. In vitro, we found that the expression of lncRNA FAF was altered in CFs, whereas it behaved inconsistently in cardiomyocytes (CMs). Next, we investigated the effects of lncRNA FAF on angiotensinogen II (Ang II)-induced cardiac fibrosis in neonatal rat CFs and explored the mechanism underlying these effects. In this study, lncRNA FAF was enriched in CFs and was associated with cardiac fibrosis. Upregulation of lncRNA FAF significantly restrained Ang II-induced increases in cell proliferation, differentiation and collagen accumulation of CFs. Moreover, we found that the function of lncRNA FAF was mainly realized through Transforming growth factor β1 (TGFβ1) secretion and then downregulated phosphorylation of Smad2/3. Additional analysis revealed that Fibroblast growth factor 9 (FGF9) is a direct target of lncRNA FAF, as the overexpression of lncRNA FAF could increase the expression of FGF9 and knockdown of the FGF9 expression could attenuate the down-regulation of lncRNA FAF on TGFβ1-P-Smad2/3 pathway. Furthermore, knockdown of the FGF9 expression also abolished the inhibitory effect of FAF on fibrosis. In summary, we demonstrated that the overexpression of lncRNA FAF could inhibit fibrosis induced by Ang II via the TGFβ1-P-Smad2/3 signalling by targeting FGF9 in CFs.

Centaurium Erythraea Extracts Exert Vascular Effects through Endothelium- and Fibroblast-dependent Pathways.

Centaurium erythraea is a plant used in traditional medicine for several cardiovascular disorders, namely hypertension, but there is no scientific evidence able to provide a molecular basis for its claimed antihypertensive effects. After a preliminary screen of extracts obtained from sequential extraction of C. erythraea aerial parts, effects of the methanolic fraction (MFCE) on changes in perfusion pressure of isolated rat mesenteric vascular bed (MVB) and in rat cardiac fibroblasts proliferation were investigated, gathering information on the mechanisms involved in endothelium-dependent effects and their dependence on a pro-proliferative stimulus. The HPLC-DAD determination of the phenolics content of MFCE revealed the presence of 22 phenolic compounds. MFCE reduced (63.3 ± 3.9%; n = 4) perfusion pressure in MVB and almost completely abrogated the Ang II-induced increase in cardiac fibroblasts proliferation. Reduction of the perfusion pressure caused by MFCE was endothelium-dependent and occurred in parallel with an increase in NO release. These effects were inhibited by muscarinic receptor antagonists, by L-NAME (a NO synthase inhibitor), and by ODQ (a soluble guanylate cyclase inhibitor). Experiments revealed that effects required the involvement of K+ channels, being inhibited by tetraethylamonium (TEA; a Ca2+ activated K+ channels inhibitor) and by glibenclamide (an ATP-sensitive K+ channels inhibitor). In conclusion, extracts from C. erythraea, particularly the compounds present in the MFCE, induce endothelium-dependent vasodilation and prevent fibroblast proliferation induced by angiotensin II, which can account for the claimed antihypertensive effects of C. erythraea in traditional medicine.

Sodium butyrate attenuates angiotensin II-induced cardiac hypertrophy by inhibiting COX2/PGE2 pathway via a HDAC5/HDAC6-dependent mechanism.

Sodium butyrate (NaBu) is reported to play important roles in a number of chronic diseases. The present work is aimed to investigate the effect of NaBu on angiotensin II (Ang II)‐induced cardiac hypertrophy and the underlying mechanism in in vivo and in vitro models. Sprague Dawley rats were infused with vehicle or Ang II (200 ng/kg/min) and orally administrated with or without NaBu (1 g/kg/d) for two weeks. Cardiac hypertrophy parameters and COX2/PGE2 pathway were analysed by real‐time PCR, ELISA, immunostaining and Western blot. The cardiomyocytes H9C2 cells were used as in vitro model to investigate the role of NaBu (2 mmol/L) in inhibition of Ang II‐induced cardiac hypertrophy. NaBu significantly attenuated Ang II‐induced increase in the mean arterial pressure. Ang II treatment remarkably increased cardiac hypertrophy as indicated by increased ratio of heart weight/body weight and enlarged cardiomyocyte size, extensive fibrosis and inflammation, as well as enhanced expression of hypertrophic markers, whereas hearts from NaBu‐treated rats exhibited a significant reduction in these hypertrophic responses. Mechanistically, NaBu inhibited the expression of COX2/PGE2 along with production of ANP and phosphorylated ERK (pERK) stimulated by Ang II in in vivo and in vitro, which was accompanied by the suppression of HDAC5 and HDAC6 activities. Additionally, knocking down the expression of HDAC5 and HDAC6 via gene‐editing strategy dramatically blocked Ang II‐induced hypertrophic responses through COX2/PGE2 pathway. These results provide solid evidence that NaBu attenuates Ang II‐induced cardiac hypertrophy by inhibiting the activation of COX2/PGE2 pathway in a HDAC5/HDAC6‐dependent manner.

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

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

Abstract P3052: A Muscadine Grape Extract Improves Hypertension-Induced Aortic Damage in Conjunction With a Reduction in Fibrosis, Oxidative Stress, and Inflammation

Hypertension-induced arterial stiffness contributes to both reduced vascular and cardiac function. While the pathophysiology of aortic stiffness is not completely understood, significant vascular remodeling occurs in response to stimuli such as vascular stress and Ang II. The goal of this study was to determine whether a polyphenol-enriched muscadine grape skin and seed extract (MGE) improves Ang II-induced aortic remodeling. Sprague-Dawley rats (male, 8 weeks old) received normal drinking water (Control), 0.2 mg total phenolics/mL in their drinking water (MGE), 24 μg/kg/h of Ang II via osmotic minipump (Ang II), or both Ang II and MGE (Ang II/MGE, pre-treated with MGE for 1 week prior to Ang II treatment). MGE had no effect on systolic blood pressure, measured by tail cuff plethysmography, in normotensive or Ang II-treated rats. Aortic stiffness, determined by echocardiographic pulse wave velocity, was increased 2.5-fold in Ang II-treated animals but was significantly improved by co-administration of MGE. MGE prevented the Ang II-induced increase in aortic media thickness (Control: 95.8 ± 2.1 μm, Ang II: 135.1 ± 3.7 μm, and Ang II/MGE: 118.2 ± 2.9 μm) and lumen diameter (Control: 2.7 ± 0.2, Ang II: 2.1 ± 0.1 mm, and Ang II/MGE: 2.6 ± 0.1 mm). Nuclear phosphorylated ERK (pERK) in aortic medial cells was increased by 131% in Ang II-treated rats compared to Controls (p &lt; 0.05); MGE co-administration prevented this by 83% (p &lt; 0.001). Ang II-induced aortic fibrosis, measured by total collagen and collagen III, was significantly attenuated in hypertensive animals treated with MGE, in association with reduced pSMAD2 and CTGF. 4-HNE, a marker of lipid peroxidation, and subunits of NADPH oxidases--Nox1, Nox2, p-p47phox--were significantly upregulated by Ang II and normalized with MGE. The inflammatory markers IL-6 and NF-κB were also increased in Ang II-treated aortas (8.9- and 12.7-fold, respectively) and attenuated by MGE. This study is the first to demonstrate that Ang II-induced vascular remodeling is prevented by co-administration of MGE. MGE reduced aortic stiffness, in association with a decrease in fibrotic, oxidative stress, and inflammatory signaling, suggesting that MGE supplementation may improve vascular function in hypertensive patients.

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

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

PKCδ Mediates Mineralocorticoid Receptor Activation by Angiotensin II to Modulate Smooth Muscle Cell Function

Angiotensin II (AngII) and the mineralocorticoid receptor (MR) ligand aldosterone both contribute to cardiovascular disorders, including hypertension and adverse vascular remodeling. We previously demonstrated that AngII activates MR-mediated gene transcription in human vascular smooth muscle cells (SMCs), yet the mechanism and the impact on SMC function are unknown. Using an MR-responsive element-driven transcriptional reporter assay, we confirm that AngII induces MR transcriptional activity in vascular SMCs and endothelial cells, but not in Cos1 or human embryonic kidney-293 cells. AngII activation of MR was blocked by the MR antagonist spironolactone or eplerenone and the protein kinase C-δ (PKCδ) inhibitor rottlerin, implicating both in the mechanism. Similarly, small interfering RNA knockdown of PKCδ in SMCs prevented AngII-mediated MR activation, whereas knocking down of MR blocked both aldosterone- and AngII-induced MR function. Coimmunoprecipitation studies reveal that endogenous MR and PKCδ form a complex in SMCs that is enhanced by AngII treatment in association with increased serine phosphorylation of the MR N terminus. AngII increased mRNA expression of the SMC-MR target gene, FKBP51, via an MR-responsive element in intron 5 of the FKBP51 gene. The impact of AngII on FKBP51 reporter activity and gene expression in SMCs was inhibited by spironolactone and rottlerin. Finally, the AngII-induced increase in SMC number was also blocked by the MR antagonist spironolactone and the PKCδ inhibitor rottlerin. These data demonstrate that AngII activates MR transcriptional regulatory activity, target gene regulation, and SMC proliferation in a PKCδ-dependent manner. This new mechanism may contribute to synergy between MR and AngII in driving SMC dysfunction and to the cardiovascular benefits of MR and AngII receptor blockade in humans.

Blackberry and Raspberry Attenuate the Increase in Blood Pressure Elicited by Angiotensin II in Mice (P06-054-19)

ObjectivesTo examine whether diet supplementation with blackberry or raspberry attenuate angiotensin (Ang) II-induced increase in blood pressure (BP) and decrease oxidative stress in the kidneys of mice. MethodsTwelve-week male C57BL/6 J mice were fed AIN-93 M diets alone (control and Ang II groups) or supplemented with 10% w/w freeze-dried blackberry (BL + Ang II) or raspberry (RB + Ang II) powder for eight weeks. At week 4, mice were implanted with subcutaneous osmotic minipumps that delivered 0.9% saline (Control) or Ang II (1 μg/kg body weight/day) for another four weeks. BP was measured weekly by tail-cuff plethysmography and validated by direct measurement at endpoint using a BP catheter. Kidney expression of pro-oxidant enzymes such as NADPH oxidases (Nox) and antioxidant enzymes such as superoxide dismutates (SOD) were also measured. Results were analyzed using ANOVA followed by Tukey-Kramer post-hoc test. ResultsAs expected, Ang II significantly increased systolic BP (SBP; 185 ± 4, n = 6, vs 132 ± 2 mmHg, n = 5, P < 0.001) and diastolic BP (124 ± 4, n = 6, vs 96 ± 2 mmHg, n = 4, P < 0.001) compared to control. Supplementation with BL and RB significantly attenuated this increase in SBP (143 ± 7 mmHg, n = 5, P < 0.0001 and 137 ± 4 mmHg, n = 5, P < 0.0001, respectively) and DBP (100 ± 6 mmHg, n = 5, P < 0.003 and 90 ± 3 mmHg, n = 5, P < 0.0001, respectively). Regarding the expression of NADPH oxidases in the kidney, Nox2 was significantly decreased by supplementation with BL (0.68 ± 0.07-fold, n = 5, P = 0.0005) and RB (0.70 ± 0.03-fold, n = 5, P = 0.0008) compared to Ang II (1.15 ± 0.12-fold, n = 5). Nox1 expression was up-regulated by Ang II compared to control (2.03 ± 0.16 vs 1.00 ± 0.00-fold, n = 5, P = 0.0001), but this was not prevented by supplementation with either berry. Nox4 expression was not affected by any of the treatments. Lastly, Ang II did not change the expression of SOD1 and SOD2 in the kidney, however, supplementation with RB significantly increased SOD1 (P = 0.02) and SOD2 (P = 0.005) expression compared to the other groups. ConclusionsOur findings indicate that supplementation with BL and RB decrease BP in mice which may be related to a decrease in oxidative stress in the kidney. Studies are underway to further elucidate the mechanism by which BL and RB exert their antihypertensive effects. Funding SourcesArkansas Biosciences Institute and Lewis Foundation Grant Program.

Pharmacological inhibition of TRPV4 channels protects against ischemia-reperfusion-induced renal insufficiency in neonatal pigs.

Renal vasoconstriction, an early manifestation of ischemic acute kidney injury (AKI), results in renal hypoperfusion and a rapid decline in kidney function. The pathophysiological mechanisms that underlie ischemia-reperfusion (IR)-induced renal insufficiency are poorly understood, but possibilities include alterations in ion channel-dependent renal vasoregulation. In the present study, we show that pharmacological activation of TRPV4 channels constricted preglomerular microvessels and elicited renal hypoperfusion in neonatal pigs. Bilateral renal ischemia followed by short-term reperfusion increased TRPV4 protein expression in resistance size renal vessels and TRPV4-dependent cation currents in renal vascular smooth muscle cells (SMCs). Selective TRPV4 channel blockers attenuated IR-induced reduction in total renal blood flow (RBF), cortical perfusion, and glomerular filtration rate (GFR). TRPV4 inhibition also diminished renal IR-induced increase in AKI biomarkers. Furthermore, the level of angiotensin II (Ang II) was higher in the urine of IR- compared with sham-operated neonatal pigs. IR did not alter renal vascular expression of Ang II type 1 (AT1) receptors. However, losartan, a selective AT1 receptor antagonist, ameliorated IR-induced renal insufficiency in the pigs. Blockade of TRPV4 channels attenuated Ang II-evoked receptor-operated Ca2+ entry and constriction in preglomerular microvessels. TRPV4 inhibition also blunted Ang II-induced increase in renal vascular resistance (RVR) and hypoperfusion in the pigs. Together, our data suggest that SMC TRPV4-mediated renal vasoconstriction and the ensuing increase in RVR contribute to early hypoperfusion and renal insufficiency elicited by renal IR in neonatal pigs. We propose that multimodal signaling by renal vascular SMC TRPV4 channels controls neonatal renal microcirculation in health and disease.

Role of PLD-PKCζ signaling axis in p47phox phosphorylation for activation of NADPH oxidase by angiotensin II in pulmonary artery smooth muscle cells.

We sought to determine the mechanism by which angiotensin II (ANGII) stimulates NADPH oxidase-mediated superoxide (O2 .- ) production in bovine pulmonary artery smooth muscle cells (BPASMCs). ANGII-induced increase in phospholipase D (PLD) and NADPH oxidase activities were inhibited upon pretreatment of the cells with chemical and genetic inhibitors of PLD2, but not PLD1. Immunoblot study revealed that ANGII treatment of the cells markedly increases protein kinase C-α (PKC-α), -δ, -ε, and -ζ levels in the cell membrane. Pretreatment of the cells with chemical and genetic inhibitors of PKC-ζ, but not PKC-α, -δ, and -ε, attenuated ANGII-induced increase in NADPH oxidase activity without a discernible change in PLD activity. Transfection of the cells with p47phox small interfering RNA inhibited ANGII-induced increase in NADPH oxidase activity without a significant change in PLD activity. Pretreatment of the cells with the chemical and genetic inhibitors of PLD2 and PKC-ζ inhibited ANGII-induced p47phox phosphorylation and subsequently translocation from cytosol to the cell membrane, and also inhibited its association with p22phox (a component of membrane-associated NADPH oxidase). Overall, PLD-PKCζ-p47phox signaling axis plays a crucial role in ANGII-induced increase in NADPH oxidase-mediated O2 .- production in the cells.

Mitofusin 2 Participates in Mitophagy and Mitochondrial Fusion Against Angiotensin II-Induced Cardiomyocyte Injury.

BackgroundMitochondrial dynamics play a critical role in mitochondrial function. The mitofusin 2 (MFN2) gene encodes a mitochondrial membrane protein that participates in mitochondrial fusion to maintain and operate the mitochondrial network. Moreover, MFN2 is essential for mitophagy. In Ang II-induced cardiac remodeling, the combined effects of MFN2-mediated mitochondrial fusion and mitophagy are unclear. This study was designed to explore a novel strategy for preventing cardiomyocyte injury via modulation of mitochondrial dynamics.MethodsWe studied the function of MFN2 in mitochondrial fusion and mitophagy in Ang II-stimulated cardiomyocyte injury. Cardiomyocyte injury experiments, including reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), and apoptosis rate of cardiomyocytes were performed. The mitochondrial morphology in cardiomyocytes was examined via transmission electron microscopy (TEM) and confocal microscopy. Autophagic levels in response to Ang II were examined by immunoblotting of autophagy-related proteins. Moreover, PINK1/MFN2/Parkin pathway-related proteins were examined.ResultsWith stimulation by Ang II, MFN2 expression was progressively reduced. MFN2 deficiency impaired mitochondrial quality, resulting in exacerbated mitochondrial damage induced by Ang II. The Ang II-induced increases in ROS production and apoptosis rate were alleviated by MFN2 overexpression. Moreover, MFN2 alleviated the Ang II-induced reduction in MMP. MFN2 promoted mitochondrial fusion, and MFN2 promoted Parkin translocation and phosphorylation, leading to mitochondrial autophagy. The effects of MFN2 overexpression were reversed by autophagy inhibitors.ConclusionMitofusin 2 promotes Parkin translocation and phosphorylation, leading to mitophagy to clear damaged mitochondria. However, the beneficial effects of MFN2 were reversed by autophagy inhibitors. Additionally, MFN2 participates in mitochondrial fusion to maintain mitochondrial quality. Thus, MFN2 participated in mitophagy and mitochondrial fusion against Ang II-induced cardiomyocyte injury.

Novel molecular mechanisms in the inhibition of adrenal aldosterone synthesis: Action of tolvaptan via vasopressin V2 receptor‐independent pathway

We investigated the inhibitory effect and associated molecular mechanisms of tolvaptan on angiotensin II (AngII)-induced aldosterone production in vitro and in vivo. In vitro, H295R human adrenocarcinoma cells were incubated with 1μmol·L-1 arginine vasopressin (AVP) or dDAVP, or tolvaptan (0.1, 1, and 3μmol·L-1 ) in the presence and absence of 100nmol·L-1 of AngII. In vivo, Sprague-Dawley rats were treated with tolvaptan 0.05% in the diet for 6days in the presence and absence of 200pmol·min-1 AngII. Tolvaptan suppressed AngII-induced aldosterone production in a dose-dependent manner in H295R cells, whereas neither AVP nor dDAVP in the presence or absence of AngII altered aldosterone production, suggesting the vasopressin V2 receptor was not involved in the inhibitory effect of tolvaptan on aldosterone synthesis. In addition, tolvaptan inhibited the AngII-induced increase in aldosterone synthase (CYP11B2) protein levels without suppressing CYP11B2 mRNA expression. Notably, tolvaptan increased the levels of unfolded protein response (UPR) marker DDIT3 and eIF2α phosphorylation (a UPR-induced event), which could block the translation of CYP11B2 mRNA into protein and thereby inhibit aldosterone production. In vivo, tolvaptan significantly inhibited AngII-induced increases in serum and adrenal aldosterone levels and CYP11B2 protein levels. This anti-aldosterone effect was associated with a reduction in the elevated systolic and diastolic BP. Tolvaptan inhibited AngII-stimulated aldosterone production via a V2 receptor-independent pathway, which can counteract or even surpass its potential activating effect of diuresis-induced aldosterone secretion in certain aldosterone-mediated pathological conditions.

Evidence that Regulator of G‐Protein Signaling 5 Protein Modulates Emotional Behaviors in Adult Mice

Many actions of angiotensin II (Ang II), such as oxidative stress and inflammation in the cerebral vasculature and brain, are mediated by the Ang II type 1 receptor, a G‐protein coupled receptor (GPCR). Ang II‐induced brain inflammation may contribute to emotional abnormalities, such as anxiety and depression. Regulator of G‐protein signaling (RGS) proteins modulate responses to extracellular signals such as Ang II acting through GPCRs by terminating G‐protein activity. Among the many types of RGS proteins, RGS5 is expressed in the brain and vasculature and modulates Ang II‐induced pressor responses and vascular contractility. We hypothesized that RGS5 deficiency would worsen Ang II‐induced anxiety‐ and depression‐like behavior, cerebral vascular oxidative stress, and brain inflammation. Adult male wild‐type (RGS5+/+) and RGS5‐deficient (RGS5−/−) mice were treated with Ang II (1 mg/kg/d) or vehicle (saline) for 21 days using osmotic minipumps. Systolic blood pressure (SBP, measured using tail cuff), anxiety (measured using elevated plus maze) and depression (measured using tail suspension test)‐like behaviors, and spontaneous locomotor activity were assessed in conscious mice; superoxide levels (measured using chemiluminesence) were examined in isolated cerebral arteries, and protein expression of VCAM‐1, NOX4 and eNOS were examined in left brain hemispheres. SBP was lower in RGS5−/− compared to RGS5+/+ mice (SBP in RGS5+/+=153±3 mmHg, n=22; RGS5−/− =140±5 mmHg, n=23, P&lt;0.05), suggesting that RGS5 contributes to blood pressure maintenance. Ang II treatment caused a greater increase in SBP in RGS5−/− vs RGS5+/+ mice (eg. ΔSBP at Day 17 in Ang II–treated RGS5+/+= 16±7mmHg, n=10; Ang II‐treated RGS5−/−= 37±5 mmHg, n=10, P&lt;0.05), confirming that RGS5 modulates Ang II‐induced pressor responses. In vehicle‐treated RGS5−/− mice, anxiety‐like behavior was increased compared to vehicle‐treated RGS5+/+ mice (time spent in open arms [secs] in veh‐treated RGS5+/+=79±12, n=7; veh‐treated RGS5−/−=36±5, n=8, P&lt;0.05), suggesting that RGS5 deficiency causes anxiety‐like behavior. There was no effect of Ang II treatment on anxiety‐like behavior in RGS5−/− mice when compared with Ang II‐treated RGS5+/+. RGS5 deficiency alone had no effect on depression‐like behavior, however combined RGS5 deficiency and Ang II treatment resulted in increased depression‐like behavior compared to Ang II treatment in RGS5+/+ mice (time immobilized [secs] in Ang II‐treated RGS5+/+=170±10, n=10; Ang II‐treated RGS5−/−=202±10, n=11, P&lt;0.05). Ang II treatment or RGS5 deficiency had no effect on spontaneous locomotor activity, cerebrovascular superoxide levels, and protein expression of VCAM‐1, NOX4 and eNOS. Overall, these data suggest that RGS5 deficiency increases vulnerability to Ang II‐induced increases in blood pressure, and that RGS5 deficiency differentially influences Ang II‐induced anxiety‐ and depression‐like behaviors.Support or Funding InformationThis work was supported by a Bower, Bennet and Bennet Endowed Chair Research Award from the College of PharmacyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Microglial Cells Impact Gut Microbiota and Gut Pathology in Angiotensin II-Induced Hypertension.

Increased microglial activation and neuroinflammation within autonomic brain regions have been implicated in sustained hypertension, and their inhibition by minocycline-an anti-inflammatory antibiotic-produces beneficial effects. These observations led us to propose a dysfunctional brain-gut communication hypothesis for hypertension. However, it has been difficult to reconcile whether an anti-inflammatory or antimicrobial action is the primary beneficial effect of minocycline in hypertension. Accordingly, we utilized chemically modified tetracycline-3 (CMT-3)-a derivative of tetracycline that has potent anti-inflammatory activity-to address this question. Test the hypothesis that central administration of CMT-3 would inhibit microglial activation, attenuate neuroinflammation, alter selective gut microbial communities, protect the gut wall from developing hypertension-associated pathology, and attenuate hypertension. Rats were implanted with radiotelemetry devices for recording mean arterial pressure. Ang II (angiotensin II) was infused subcutaneously using osmotic mini-pumps to induce hypertension. Another osmotic mini-pump was surgically implanted to infuse CMT-3 intracerebroventricularly. Intracerebroventricular CMT- 3 infusion was also investigated in SHR (spontaneously hypertensive rats). Physiological, pathological, immunohistological parameters, and fecal microbiota were analyzed. Intracerebroventricular CMT-3 significantly inhibited Ang II-induced increases in number of microglia, their activation, and proinflammatory cytokines in the paraventricular nucleus of hypothalamus. Further, intracerebroventricular CMT-3 attenuated increased mean arterial pressure, normalized sympathetic activity, and left ventricular hypertrophy in Ang II rats, as well as in the SHR. Finally, CMT-3 beneficially restored certain gut microbial communities altered by Ang II and attenuated pathological alterations in gut wall. These observations demonstrate that inhibition of microglial activation alone was sufficient to induce significant antihypertensive effects. This was associated with unique changes in gut microbial communities and profound attenuation of gut pathology. They suggest, for the first time, a link between microglia and certain microbial communities that may have implications for treatment of hypertension.

Assessing the role of extracellular vesicles in renin-angiotensin system signalling in cardiomyocyte hypertrophy

Assessing the role of extracellular vesicles in renin-angiotensin system signalling in cardiomyocyte hypertrophy