Angiotensin II Treatment Research Articles

Deoxyelephantopin—a novel PPARγ agonist regresses pressure overload-induced cardiac fibrosis via IL-6/STAT-3 pathway in crosstalk with PKCδ

Pathological cardiac hypertrophy is associated with ventricular fibrosis leading to heart failure. The use of thiazolidinediones as Peroxisome Proliferator-Activated Receptor-gamma (PPARγ)-modulating anti-hypertrophic therapeutics has been restricted due to major side-effects. The present study aims to evaluate the anti-fibrotic potential of a novel PPARγ agonist, deoxyelephantopin (DEP) in cardiac hypertrophy. AngiotensinII treatment in vitro and renal artery ligation in vivo were performed to mimic pressure overload-induced cardiac hypertrophy. Myocardial fibrosis was evaluated by Masson's trichrome staining and hydroxyproline assay. Our results showed that DEP treatment significantly improves the echocardiographic parameters by ameliorating ventricular fibrosis without any bystander damage to other major organs. Following molecular docking, all-atomistic molecular dynamics simulation, reverse transcription-polymerase chain reaction and immunoblot analyses, we established DEP as a PPARγ agonist stably interacting with the ligand-binding domain of PPARγ. DEP specifically downregulated the Signal Transducer and Activator of Transcription (STAT)-3-mediated collagen gene expression in a PPARγ-dependent manner, as confirmed by PPARγ silencing and site-directed mutagenesis of DEP-interacting PPARγ residues. Although DEP impaired STAT-3 activation, it did not have any effect on the upstream Interleukin (IL)-6 level implying possible crosstalk of the IL-6/STAT-3 axis with other signaling mediators. Mechanistically, DEP increased the binding of PPARγ with Protein Kinase C-delta (PKCδ) which impeded the membrane translocation and activation of PKCδ, downregulating STAT-3 phosphorylation and resultant fibrosis. This study, therefore, for the first time demonstrates DEP as a novel cardioprotective PPARγ agonist. The therapeutic potential of DEP as an anti-fibrotic remedy can be exploited against hypertrophic heart failure in the future.

#3293 ESTRADIOL REPLACEMENT MITIGATED BLOOD PRESSURE ELEVATION VIA SUPPRESSION OF NCC IN ANGIOTENSIN II-INFUSED OVARIECTOMIZED FEMALE RATS

Abstract Background and Aims Female rats excrete more urine sodium than male rats with similar blood pressure (BP). However, it is not clear whether the sex difference in pressure natriuresis is conserved in menopausal rats. We assessed whether the natriuretic response and renal sodium transporter activity in ovariectomized female rats infused with angiotensin II (ANGII) could be affected by estrogen supplementation. Method Six-week-old female Sprague-Dawley rats (n=30) were ovariectomized (OVX, n =20) or sham-operated (n=10) (−4 weeks, −4W). After 2 weeks, subcutaneous estradiol was administered to half of the OVX rats (n=10) for 4 weeks (-2W∼2W). ANGII was infused via the osmotic minipump in 60% of the rats for 2 weeks (0W∼2W). Six groups (female saline: FS, female ANGII: FA, OVX saline: OS, OVX ANGII: OA, OVX estradiol saline: OES, OVX estradiol ANGII: OEA) were sacrificed after 2 weeks of the ANGII infusion (2W). We also analyzed human data from the Korean Genome and Epidemiology Study (KoGES). Results ANGII-induced BP elevation was highest in the OA group, followed by the FA and OEA groups. Urine sodium tended to increase after ANGII infusion in FA and OEA but decreased in OA. Western blot results showed that cortical phosphorylated sodium chloride cotransporter (pNCC) tended to be augmented by ANGII treatment in FA and OA, but mitigated in OEA. Cortical ANGII type 2 receptor (AT2R) expression was significantly higher in estradiol-treated OVX rats than in OVX rats, irrespective of ANGII infusion. Similarly, the enhanced pNCC in ANGII-treated distal convoluted tubular cells was reversed by estradiol treatment. In KoGES data, urine sodium was significantly lower in postmenopausal women than in premenopausal women, despite the higher BP. Conclusion Estradiol reversed BP elevation and augmented the natriuretic response in ANGII-infused OVX rats and distal tubular cells via suppression of pNCC and increase of AT2R.

ROLE OF METALLOPROTEINASES AND ROS GENERATION IN THE AORTA OF ELASTASE-2 KNOCKOUT MICE IN ABDOMINAL AORTIC ANEURYSM MODEL

Objective: Elastase-2 (ELA-2) enzyme generates angiotensin-II (AngII) in arteries and is implicated in abnormal vascular remodeling in animal models. Our group demonstrated previously that ELA-2 knockout (ELA-2KO) mice were less susceptible to developing an aorta dilation in response to AngII infusion. This study hypothesizes that metalloproteinases and ROS generation in ELA-2KO mice mediate less susceptibility to aorta dilation and abdominal aortic aneurysm (AAA). Design and method: Male wild type (C57bl/6, WT) and ELA-2KO (CELA-2aTm1Bdr) mice were treated with either saline or AngII (2.28g/g/min) for twenty-eight days by subcutaneously mini-pumps infusion. Mice were divided into four groups: WT treated with saline (WT+SAL, n = 5), ELA-2KO treated with saline (ELA-2KO+SAL, n = 5), WT treated with AngII (WT+AngII,n = 5), and ELA-2KO treated with AngII (ELA-2KO+AngII, n = 5). The vascular ultrasound (US) was done before treatment and once a week during 28 days of treatment. The MMP- 2 and MMP-9 analyzes were done by zymography, ROS generation by DHE analyses, and alfa-actin expression by immunofluorescence in all aorta mice groups. ANOVA and Kruskal Wallis tests were used to compare the average between groups. This study is approved by CEUA (n.131/2019). Results: AngII treatment was associated with increased MMPs expression in the WT group aorta (WT+AngII vs. WT+SAL p<0.05). The aorta ELA-2KO group showed no difference in MMPs in response to AngII infusion compared to ELA-2KO+SAL group (p = 0.81). The ROS generation had upregulated in aorta ELA-2KO+Ang II mice compared to ELA-2KO+SAL (p<0.001) and aorta WT+AngII compared with WT+SAL (p<0.0001). AngII treatment was associated with decreased ROS generation in the aorta ELA-2KO+AngII group compared with WT+AngII (p = 0.0051). The alfa-actin expression was similar in all groups. Our data demonstrate that AngII treatment did not increase the expression of MMPs in aorta ELA-2KO but was associated with an upregulation for ROS generation. Conclusions: Taken together, this suggests that ELA-2KO mice's resistance to developing an aorta dilation/AAA is related to MMPs and ROX generation. Our results also highlight the importance of the local/tissue response in the pathophysiology of AAA.

Scleraxis Alters Arterial Smooth Muscle Phenotypic Switching and Exacerbates Arterial Stiffness

Arterial stiffness (AS) is an impairment in arterial relaxation and function in blood pressure regulation and is associated with the development of multiple cardiovascular diseases including hypertension, atherosclerosis, and adverse outcomes such as stroke, heart failure, and death. AS comprises an increase in vascular wall components (extracellular matrix proteins or vascular smooth muscle cell (VSMC) composition) or cellular malfunction, or a combination of these factors. Preliminary data from our lab showed that using the Transverse Aortic Constriction (TAC) model, the transcription factor scleraxis is upregulated in the aortic wall on the pre-TAC/high pressure side versus post-TAC/low pressure side. Scleraxis also induces cellular phenotype activation of fibroblasts to myofibroblasts in cardiac fibrosis and mediates epithelial to mesenchymal transition in development. Angiotensin II (AngII) induces hypertension and increases stiffness in arteries, and we found that it also up-regulates scleraxis expression, suggesting the possibility that scleraxis may alter arterial wall composition. In this study, we investigated the role of scleraxis expression in VSMC, and its effect when over-expressed along with AngII treatment in small resistance arteries and aorta. In vivo, we upregulated scleraxis in VSMC using a Cre/LoxP approach and the tamoxifen-inducible Myh11cre mouse, with or without AngII delivery by micro-osmotic pump, then isolated 3rd order mesenteric arteries and assessed arterial function and stiffness by pressure myography. We harvested aortas for immunohistology staining and gene expression analysis. In vitro, we upregulated scleraxis in VSMC with or without AngII treatment and measured their proliferation by flow cytometry. Our findings show that scleraxis induces stiffness in mesenteric arteries, but not aorta by induction of VSMC proliferation. Scleraxis upregulation with AngII infusion exacerbates vascular stiffness in mesenteric arteries due to significant changes in arterial wall components, reducing internal lumen diameter and smooth muscle relaxation. At the molecular level, extracellular matrix protein expression is reduced in aortas. In in vitro studies, scleraxis-AngII treated VSMC exhibit lower proliferative capacity, distinct morphological changes and higher contractile gene expression, indicative of a hypertrophic contractile phenotype altering overall arterial stiffness. Our study shows that scleraxis alone can induce AS by regulating VSMC phenotype toward proliferation. In contrast, arterial stiffness is exacerbated in scleraxis overexpression and AngII-infused vessels by inducing VSMC towards a hypertrophic contractile phenotype. Therefore, scleraxis alters vascular smooth muscle phenotypic switching and arterial stiffness through different mechanisms, depending on the presence of AngII, which may have different pathological impacts depending on the specific underlying conditions. This work is supported by funding from Heart & Stroke Foundation and BMO Studentship. 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.

Protective role of short chain fatty acids against Angiotensin II-induced mitochondrial dysfunction in brain endothelial cells: Potential role of Heme Oxygenase 2

Objectives: Short chain fatty acids (SCFAs), the main metabolites released from the gut microbiota, are reduced during hypertension. SCFAs play a beneficial role on the cardiovascular system. However, the effect of SCFAs on cerebrovascular endothelial cells is yet to be uncovered. In this study, we used brain endothelial cells to investigate the in vitro effect of SCFAs on heme oxygenase 2 (HO-2) and mitochondrial function following Angiotensin-II treatment. Methods: Brain human microvascular endothelial cells were treated with Angiotensin II (Ang-II, 500nM for 24hours) in the presence and absence of SCFAs cocktail (1mM; acetate, propionate, and butyrate) and/or HO-2 inhibitor (SnPP 5 μM). At the end of the treatment, HO-2, endothelial markers (p-eNOS, NO production), inflammatory markers (TNFα, NFkB-p50 and -p65), calcium homeostasis, mitochondrial membrane potential, mitochondrial ROS and hydrogen peroxide (H 2 O 2 ), and mitochondrial respiration were determined in all groups of treated cells. Results: Our data showed that SCFAs rescued HO-2 after Angiotensin II treatment. Additionally, SCFAs rescued Angiotensin II-induced eNOS reduction, mitochondrial membrane potential impairment and mitochondrial respiration damage. On the other hand, SCFAs reduced Angiotensin II-induced inflammation, calcium dysregulation, mitochondrial ROS, and H 2 O 2 . All these beneficial effects of SCFAs on endothelial cells and mitochondrial function occurred through HO-2. Conclusions: SCFAs treatment restored endothelial cells and mitochondrial function following Angiotensin II-induced oxidative stress. SCFAs exert these beneficial effects by acting on HO-2. 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.

Abstract 525: Angiotensin Ii Type 1a Receptor Deletion In Vascular Smooth Muscle Cells Prevent Abdominal Aortic Aneurysm Formation Induced By Angiotensin Ii Plus β-aminopropionitrile

Abdominal aortic aneurysm (AAA) is multifactorial disease resulting from changes in the aortic wall structure, such as medial degradation and inflammation, causing life-threatening AAA rupture. Angiotensin II (AngII) type 1A receptor (AT 1A R) upon AngII binding mediates blood pressure elevation and several chronic cardiovascular diseases including AAA. Treatment with AngII plus β-aminopropionitrile (BAPN), a lysyl oxidase inhibitor, in normolipidemic C57BL6 mice similarly induces AAA. However, we have limited understanding whether smooth muscle AT 1A R affects AAA formation induced by AngII plus BAPN. Thus, the subject of this study is to test the contribution of smooth muscle AT 1A R to AngII-dependent AAA development in normocholesterolemic mice. At 8 to 12 week of age, smooth muscle AT 1A R silenced mice (knock-in Tagln-Cre +/- crossed with AT 1A R flox/flox mice) and control mice were infused with AngII (1 μg/kg/min) via osmotic mini-pump for 4 weeks and treated with 1 g/L BAPN containing water for the first 2 weeks or sham-operated for mini-pump implantation. After 4 weeks, significant increases in maximum external aortic diameter and structural alterations were observed in the control mice, which was attenuated in smooth muscle AT 1A R silenced mice (1.67 mm vs. 1.22 mm, p<0.05). AngII plus BAPN treatment increased perivascular collagen III positive cell ratio, which was normalized in smooth muscle AT 1A R silenced mice (p<0.01). Evaluation of blood pressure at week 4 in conscious mice showed significant reduction in AngII-induced hypertension in smooth muscle AT 1A R silenced mice (p<0.01). Furthermore, in contrast to control mice, no death duet to aortic rupture was observed in smooth muscle AT 1A R silenced mice. In conclusion, our study has demonstrated the significant contribution of smooth muscle expressing AT 1A R in mediating AAA development and rupture in response to AngII plus BAPN treatment. It is possible that attenuation of hypertension in the AT 1A R silenced mice may provide additional protection against AAA rupture.

Angiotensin II Increases Oxidative Stress and Inflammation in Female, But Not Male, Endothelial Cells.

Women are at elevated risk for certain cardiovascular diseases, including pulmonary arterial hypertension, Alzheimer's disease, and vascular complications of diabetes. Angiotensin II (AngII), a circulating stress hormone, is elevated in cardiovascular disease; however, our knowledge of sex differences in the vascular effects of AngII are limited. We therefore analyzed sex differences in human endothelial cell response to AngII treatment. Male and female endothelial cells were treated with AngII for 24h and analyzed by RNA sequencing. We then used endothelial and mesenchymal markers, inflammation assays, and oxidative stress indicators to measure female and male endothelial cell functional changes in response to AngII. Our data show that female and male endothelial cells are transcriptomically distinct. Female endothelial cells treated with AngII had widespread gene expression changes related to inflammatory and oxidative stress pathways, while male endothelial cells had few gene expression changes. While both female and male endothelial cells maintained their endothelial phenotype with AngII treatment, female endothelial cells showed increased release of the inflammatory cytokine interleukin-6 and increased white blood cell adhesion following AngII treatment concurrent with a second inflammatory cytokine. Additionally, female endothelial cells had elevated reactive oxygen species production compared to male endothelial cells after AngII treatment, which may be partially due to nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX2) escape from X-chromosome inactivation. These data suggest that endothelial cells have sexually dimorphic responses to AngII, which could contribute to increased prevalence of some cardiovascular diseases in women. The online version contains supplementary material available at 10.1007/s12195-023-00762-2.

Levocarnitine regulates the growth of angiotensin II-induced myocardial fibrosis cells via TIMP-1.

This study aimed to explore the effects of tissue inhibitor of metalloproteinases-1 (TIMP-1) on levocarnitine (LC)-mediated regulation of angiotensin II (AngII)-induced myocardial fibrosis (MF) and its underlying mechanisms. H9C2 cells were treated with AngII for 24 h to induce fibrosis. The cells were then treated with LC or transfected with TIMP-1-OE plasmid/si‑TIMP-1. Cell apoptosis, viability, migration, and related gene expression were analyzed. AngII treatment significantly upregulated Axl, α-SMA, and MMP3 expression (P < 0.05) and downregulated STAT4 and TIMP1 expression (P < 0.05) relative to the control levels. After transfection, cells with TIMP-1 overexpression/knockdown were successfully established. Compared with that of the control, AngII significantly inhibited cell viability and cell migration while promoting cell apoptosis (P < 0.05). LC and TIMP-1-OE transfection further suppressed cell viability and migration induced by Ang II and upregulated apoptosis, whereas si-TIMP-1 had the opposite effect. Furthermore, LC and TIMP-1-OE transfection downregulated Axl, AT1R, α-SMA, collagen III, Bcl-2, and MMP3 expression caused by AngII and upregulated caspase 3, p53, and STAT4 expression, whereas si-TIMP-1 had the opposite effect. TIMP-1 is therefore a potential therapeutic target for delaying MF progression.

Pkd1 is necessary for full cardiac AngII-induced hypertrophy but not for full acute AngII-induced calcium handling changes.

The renin-angiotensin system (RAS) regulates blood pressure, extracellular fluid and electrolyte balance, cardiac inotropy, and vascular resistance. In the heart, angiotensin II (AngII) can induce hypertrophy directly by activating Protein Kinase D (PKD), a signaling kinase known to contribute to cardiac remodeling. Little is known, however, about the acute and chronic functional effects of AngII-induced PKD activation on calcium (Ca) handling. To investigate this, we used hearts from cardiac-specific PKD1 knockout (cKO) mice and their WT littermates. We measured Ca transients (CaT) and Ca spark (CaSp) frequencies in cardiomyocytes at 0.5 and 1 Hz pacing under acute and chronic AngII stimulation (100 nM in vitro and 3 mg/kg/day in osmotic minipump, respectively). Sarcoplasmic reticulum (SR) Ca load was assessed by rapid caffeine-induced Ca release. At baseline and after acute AngII treatment, CaT amplitude and tau decay, CaSp frequency and SR Ca load were similar in WT vs. PKD1 cKO. This was also the case for chronic exposure to AngII except that the tau decay was significantly slower in PKD1 cKO myocytes. In agreement with previous reports, echocardiographic measurements revealed that PKD1 cKO mice were also more resistant to chronic AngII-induced hypertrophy (LVPW increase 24.69 ± 2.74% vs. 54.73 ± 7.73 %) and ejection fraction reduction (4.08 ± 1.5% vs. 12.67 ± 0.52%). We conclude that despite PKD being necessary for the remodeling changes induced by chronic AngII, it does not alter the amplitude and kinetics of CaT and spontaneous Ca release, except for the slowed decay kinetics in the PKD1 cKO after chronic AngII treatment and decreased acute SR Ca load.

PS-BPB04-2: P2RX7 DEFICIENCY OR P2RX7 ANTAGONISM BLUNTS ANGIOTENSIN II-INDUCED HYPERTENSION, VASCULAR INJURY AND CD8+ T CELL ACTIVATION

Objectives: Innate and adaptive immune cells contribute to hypertension and end-organ damage. High blood pressure (BP) causes cardiovascular injury and the release of damage-associated molecule patterns such as adenosine triphosphate (ATP). ATP can bind to the purinergic receptor P2X7 (P2RX7) on innate immune cells triggering interleukin-1β; release, which drives further immune activation. Elevated plasma ATP levels was observed in hypertensive patients. We hypothesized that P2rx7 knockout or P2RX7 antagonism would blunt angiotensin II (AngII)-induced BP elevation and cardiovascular injury through decreased immune activation. Design and method: Ten-to-12-week-old male C57BL/6J wild-type (WT) and P2rx7-/- mice were infused or not with AngII (1000 ng/kg/min) for 14 days. A second group of AngII-infused WT mice was also infused with the P2RX7 antagonist AZ10606120 (694 ng/kg/min) or vehicle. BP was determined by telemetry, plasma ATP using a bioluminescence assay, mesenteric artery function using pressurized myography, cardiac left ventricle (LV) function and mass by ultrasound and activated immune T cell infiltration in aortic perivascular adipose tissue (PVAT) by flow cytometry. Results: Plasma ATP was 2.2 fold higher in AngII-infused compared to sham-treated mice (P &lt; 0.05). AngII-induced systolic BP elevation was reduced by P2rx7 deficiency (12 mm Hg, P &lt; 0.05) or P2RX7 antagonism (27 mm Hg, P &lt; 0.01). AngII induced a 26% decrease in LV fractional shortening (FS, P &lt; 0.05) and a 1.5 fold increase in LV mass/body weight (BW) in WT mice (P &lt; 0.001), which were exaggerated in P2rx7-/- mice (FS: 38% lower and LVmass/BW: 1.14 fold higher, P &lt; 0.05), but not in mice receiving AZ10606120. AngII treatment caused a 26% reduction in the dilatation response of mesenteric arteries to acetylcholine in WT mice (P &lt; 0.05), but not in P2rx7-/- or AZ10606120-treated mice. AngII promoted a 3.8 fold increase in CD69+CD8+ T cell infiltration in aortic PVAT of WT mice (P &lt; 0.001), but not in P2rx7-/- or AZ10606120-treated mice. Conclusion: P2rx7 knockout or antagonism attenuates AngII-induced BP elevation, vascular injury, and infiltration of activated CD8+ T cells into aortic PVAT. P2rx7 knockout exacerbated AngII-induced cardiac dysfunction and hypertrophy, whereas P2RX7 antagonism did not.

Neurons of the median preoptic nucleus contribute to chronic angiotensin II-salt induced hypertension in the rat.

Experiments were designed to test the hypothesis that median preoptic (MnPO) neurons are necessary for the full hypertensive response to chronic angiotensin II (AngII) in rats consuming a high salt diet. The MnPO is implicated in many of the physiologic actions of AngII, primarily acting as a downstream nucleus to AngII binding at circumventricular organs such as the organum vasculosum of the lamina terminalis (OVLT). We have previously shown a prominent effect of lesion of the OVLT on the chronic hypertensive effects of AngII in rats consuming high salt. Additionally, we have shown that lesion of the MnPO attenuated the hypertensive response to chronic intravenous infusion of AngII in rats. However, whether MnPO neurons or fibers of passage contribute to this response is not clear. Male Sprague Dawley rats were randomly assigned to either sham (SHAM; n=8) or ibotenic acid lesion of the MnPO (MnPOx; n=6). In the MnPOx group, 200 nl of ibotenic acid in phosphate buffer saline (5 μg/μl) was injected into each of 3 predetermined coordinates targeted at the entire MnPO. After a week of recovery, rats were instrumented with radiotelemetric pressure transducers, provided 2.0% NaCl diet and distilled water ad libitum and given another week to recover. After 3 days of baseline measurements, osmotic minipumps were implanted subcutaneously in all rats for administration of AngII at a rate of 150 ng/kg/min. Blood pressure measurements were made for 14 days after minipump implantation. By day 7 of AngII treatment, blood pressure responses appeared to plateau in both groups while the hypertensive response was markedly attenuated in MnPOx rats (MnPOx, 122 ± 6 mmHg; SHAM, 143 ± 8 mmHg). These results support the hypothesis that neurons of the MnPO are involved in the central pathway mediating the chronic hypertensive effects of AngII in rats consuming a high salt diet.

Abstract 10706: Transnitrosation From the NO-Receptor Soluble Guanylyl Cyclase to Oxidized Thioredoxin-1 Has a Potential Cardiovascular Protective Effect Against Oxidative Stress

Soluble guanylyl cyclase (GC1) is an α/β heterodimer producing cGMP when stimulated by nitric oxide (NO). The NO-GC1-cGMP pathway is essential for cardiovascular homeostasis. Oxidative stress is known to disrupt the NO-GC1-cGMP pathway via oxidation of GC1 cysteines, which makes it unresponsive to NO stimulation. Under these conditions, GC1-α subunit “moonlights” by increasing cellular S-nitrosation via specific transfer of its nitrosothiols to cysteines of other proteins (transnitrosation). One of the SNO-targets identified by Mass Spectrometry (MS) was the oxidized form of Thioredoxin1 (oTrx1), which is unidirectionally transnitrosated by GC1 with αC610 as a SNO-donor and C73 of Trx1 as the SNO-recipient. Because oTrx1 itself drives transnitrosation, we sought and MS-identified SNO-proteins targeted by both GC1 and Trx1, suggesting a SNO-GC1→oTrx1→targets cascade. We investigated the (patho)physiological relevance of GC1-dependent transnitrosation activity by creating a knock-in (KI) mice with replacement of αC610 to a serine (αC610S). Biotin switch assays confirm that, under oxidative stress (Angiotensin II (AngII) treatment), global and Trx1 levels of S-nitrosation (SNO) were drastically reduced in lungs and hearts of KI mice compared to WT but the NO-stimulated GC1 activity was similar between WT and KI. Importantly, telemetry recordings indicated that 2 weeks of AngII treatment led to a more severe increase in BP and a more prolonged QTc in KI mice. The AngII-induced rise in MBP was 27.6 ± 2.5 mmHg in KI vs 21.6 ± 1.2mmHg in WT, while AngII-increase in QTc, reflecting delayed repolarization, was 12.53 ± 2.30ms in KI vs. 8.713 ± 2.72ms for the WT. The elevated BP with AngII treatment correlated with higher increase in cardiac hypertrophy, oxidation (measured by DHE staining) and fibrosis (picric acid staining) in KI mice. The AngII-induced increase in a) hypertrophy in KI was 0.63 ± 0.07 vs. 0.24 ± 0.08 mg/g in WT, b) cardiac oxidation was 0.19 ± 0.07 in KI and negligible in WT, and c) fibrosis was 1.75 (A.U) in KI vs. 1.31 in WT. These more severe cardiovascular dysfunctions in KI mice in response to AngII suggest that transnitrosation by GC1, potentially using oTrx1 as a nitrosothiol relay, is a mechanism of protection against oxidative stress.

Cardiomyocyte p38 MAPKα suppresses a heart–adipose tissue–neutrophil crosstalk in heart failure development

Although p38 MAP Kinase α (p38 MAPKα) is generally accepted to play a central role in the cardiac stress response, to date its function in maladaptive cardiac hypertrophy is still not unambiguously defined. To induce a pathological type of cardiac hypertrophy we infused angiotensin II (AngII) for 2 days via osmotic mini pumps in control and tamoxifen-inducible, cardiomyocyte (CM)-specific p38 MAPKα KO mice (iCMp38αKO) and assessed cardiac function by echocardiography, complemented by transcriptomic, histological, and immune cell analysis. AngII treatment after inactivation of p38 MAPKα in CM results in left ventricular (LV) dilatation within 48 h (EDV: BL: 83.8 ± 22.5 µl, 48 h AngII: 109.7 ± 14.6 µl) and an ectopic lipid deposition in cardiomyocytes, reflecting a metabolic dysfunction in pressure overload (PO). This was accompanied by a concerted downregulation of transcripts for oxidative phosphorylation, TCA cycle, and fatty acid metabolism. Cardiac inflammation involving neutrophils, macrophages, B- and T-cells was significantly enhanced. Inhibition of adipose tissue lipolysis by the small molecule inhibitor of adipocytetriglyceride lipase (ATGL) Atglistatin reduced cardiac lipid accumulation by 70% and neutrophil infiltration by 30% and went along with an improved cardiac function. Direct targeting of neutrophils by means of anti Ly6G-antibody administration in vivo led to a reduced LV dilation in iCMp38αKO mice and an improved systolic function (EF: 39.27 ± 14%). Thus, adipose tissue lipolysis and CM lipid accumulation augmented cardiac inflammation in iCMp38αKO mice. Neutrophils, in particular, triggered the rapid left ventricular dilatation. We provide the first evidence that p38 MAPKα acts as an essential switch in cardiac adaptation to PO by mitigating metabolic dysfunction and inflammation. Moreover, we identified a heart–adipose tissue–immune cell crosstalk, which might serve as new therapeutic target in cardiac pathologies.

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&amp;lt;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

Abstract P114: Differential Expression Of Angiotensin Ii Receptors In Aorta Of Ela2ko Mice With Abdominal Aortic Aneurysm.

Elastase-2 (ELA-2) enzyme generates angiotensin-II (AngII) in arteries and is implicated in abnormal vascular remodeling in animal models.Our group demonstrated previously that ELA-2 knockout (ELA-2KO) mice were less susceptible to develop an aorta dilation in a response to AngII infusion. In this study, we hypothesize that susceptibility to an aorta dilation and abdominal aortic aneurysm (AAA) is mediated by AngII receptors (AT1 and AT2). Methods: Male wild type (C57bl/6, WT) and ELA-2KO (CELA-2aTm1Bdr) mice were treated with either saline or AngII (2.28g/g/min) for twenty-eight days by subcutaneously mini-pumps infusion. Mice were divided into four groups: WT treated with saline (WT+SAL, n=5), ELA-2KO treated with saline (ELA-2KO+SAL, n=5), WT treated with AngII (WT+AngII,n=5), and ELA-2KO treated with AngII (ELA-2KO+AngII, n=5). Protein expression of AT1 and AT2 receptor, CD68, and α-actin was measured by immunofluorescence in formalin-fixed, paraffin-embedded aorta tissue. ANOVA and Kruskal Wallis tests were used to compare the average fluorescent intensity between groups. Results: AngII treatment was associated with increased AT1 receptor expression in a WT group, but not with AT2 receptor expression (vs. WT+SAL,p=0.06 and p=0.31, respectively). The ELA-2KO group showed no difference in AT1 receptor expression in a response to AngII infusion compared to a ELA-2KO+SAL group (p=0.81). While ELA-2KO+Ang II mice had lower AT2 expression compared to ELA-2KO+SAL (p=0.05). The α-actin expression was similar in all groups. WT+AngII group expressed more CD68 expression in a response to an AngII treatment that was increased in a WT group (p=0.03), but not in a ELA-2KO group. Conclusion: Our data demonstrate that Ang II treatment did not increase an expression of AT1 receptor in ELA-2KO, but was associated with a downregulation of AT2 receptors. Taken together, this suggests That ELA-2KO mice resistance to developing an aorta dilation/AAA is related to downregulation of Ang II receptors. Our results also highlight the importance of the local/tissue response in the pathophysiology of AAA.

Contrasting effects of sleep fragmentation and angiotensin-II treatment upon pro-inflammatory responses of mice

Disordered sleep promotes inflammation in brain and peripheral tissues, but the mechanisms that regulate these responses are poorly understood. One hypothesis is that activation of the sympathetic nervous system (SNS) from sleep loss elevates blood pressure to promote vascular sheer stress leading to inflammation. As catecholamines produced from SNS activation can directly regulate inflammation, we pharmacologically altered blood pressure using an alternative approach-manipulation of the renin-angiotensin system (RAS). Male C57BL6/J mice were treated with angiotensin or captopril to elevate and reduce blood pressure, respectively and then exposed to 24-h of sleep fragmentation (SF) or allowed to sleep (control). Pro- and anti-inflammatory cytokine gene expression and as endothelial adhesion gene expression as well as serum glucocorticoids (corticosterone) were measured. RAS manipulation elevated cytokines and endothelial adhesion expression in heart and aorta while SF increased cytokine expression in peripheral tissues, but not brain. However, there were interactive effects of angiotensin-II and SF upon cytokine gene expression in hippocampus and hypothalamus, but not prefrontal cortex. SF, but not RAS manipulation, elevated serum corticosterone concentration. These findings highlight the contrasting effects of RAS manipulation and SF, implying that inflammation from SF is acting on different pathways that are largely independent of RAS manipulation.

Angiotensin II enhances bacterial clearance via myeloid signaling in a murine sepsis model

Sepsis, defined as organ dysfunction caused by a dysregulated host-response to infection, is characterized by immunosuppression. The vasopressor norepinephrine is widely used to treat low blood pressure in sepsis but exacerbates immunosuppression. An alternative vasopressor is angiotensin-II, a peptide hormone of the renin-angiotensin system (RAS), which displays complex immunomodulatory properties that remain unexplored in severe infection. In a murine cecal ligation and puncture (CLP) model of sepsis, we found alterations in the surface levels of RAS proteins on innate leukocytes in peritoneum and spleen. Angiotensin-II treatment induced biphasic, angiotensin-II type 1 receptor (AT1R)-dependent modulation of the systemic inflammatory response and decreased bacterial counts in both the blood and peritoneal compartments, which did not occur with norepinephrine treatment. The effect of angiotensin-II was preserved when treatment was delivered remote from the primary site of infection. At an independent laboratory, angiotensin-II treatment was compared in LysM-Cre AT1aR-/- (Myeloid-AT1a-) mice, which selectively do not express AT1R on myeloid-derived leukocytes, and littermate controls (Myeloid-AT1a+). Angiotensin-II treatment significantly reduced post-CLP bacteremia in Myeloid-AT1a+ mice but not in Myeloid-AT1a- mice, indicating that the AT1R-dependent effect of angiotensin-II on bacterial clearance was mediated through myeloid-lineage cells. Ex vivo, angiotensin-II increased post-CLP monocyte phagocytosis and ROS production after lipopolysaccharide stimulation. These data identify a mechanism by which angiotensin-II enhances the myeloid innate immune response during severe systemic infection and highlight a potential role for angiotensin-II to augment immune responses in sepsis.

DECREASED URINARY LEVELS OF SIRT1 AS NON-INVASIVE BIOMARKER OF EARLY RENAL DAMAGE IN HYPERTENSION

Objective: Hypertension and diabetes mellitus (DM) produce renal injury. Sirtuins have become important players in renal damage, but their value as biomarkers is poorly assessed. The aims of the study were to evaluate the sirtuin1 levels (SIRT1), and miRNAs miR-34a and miR-200, that regulate SIRT1 expression in urinary cell pellet from hypertensive patients with incipient renal damage with and without diabetes. In addition, to mimic disease conditions and deepen into the implications of SIRT1 in podocyte injury, employed cultured podocytes subjected to high glucose (HG) and angiotensin II (Ang II) concentrations. Design and method: We quantified urinary SIRT1 and claudin 1 (CLDN1) mRNA and miR-34a and miR-200a levels by quantitative real-time polymerase chain reaction (RT-qPCR) from urinary cell free pellet from patients and in cultured human immortalized podocytes treated with HG and ANG II. Protein levels and subcellular localization were analyzed by Western blot and fluorescence analyses by confocal microscopy. Results: We found decreased SIRT1 levels in patients with increased urinary albumin excretion (UAE), the lowest with diabetes presence, and a strong association with UAE, discriminating incipient renal damage. In vitro experiments also showed SIRT1 overall decreases in podocyte cultures under treatment conditions. In urine samples, miR-34a was reduced and miR-200a increased, both related to UAE levels. However, both miRNAs were generally increased in podocyte cultures under high glucose and angiotensin-II treatment. Conclusions: Results evidenced reduction of urinary SIRT1 from hypertensives with or without DM. The decrease of SIRT1 and increase of CLDN1 in patients and podocytes subjected to stress support the implication of both in renal injury and evidence a reflection in urinary levels. The alterations found in miR-34a and miR-200a both in patients’ urine and podocyte cultures reveal novel points to further address their implication in renal injury through SIRT1 regulation. Interestingly, SIRT1 mRNA levels and miR-200a are related to increases in UAE and discriminate the presence of UAE in patients. In summary, SIRT1 mRNA measurements is an easily accessible and non-invasive method to early characterize renal damage in patients.

Immune cells play a crucial role in the development of a cardiac dysfunction in cardiac p38 MAPKa KO mice after pressure overload

QuestionCardiac metabolic remodeling is one of the early changes driving the progression of heart failure and is present even before overt cardiac dysfunction. Previously we showed that the tamoxifen inducible deletion of cardiomyocyte specific p38 MAPKa (KO) leads to a pronounced left ventricular dilation with a strongly impaired heart function just within 2 days of angiotensin II (ANGII) treatment. Additionally, a strong infiltration of immune cells in the cardiac tissue can be observed.Methods and ResultsUnder baseline conditions no impairment of the heart function in KO mice can be observed. ANGII treatment for 2 days induces a strong left ventricular dilation and poor heart function, indicated by a reduced ejection fraction and an increase in end‐systolic and end‐diastolic volume. KO mice do not get used to constant ANGII treatment even after 7 days of ANGII treatment and cardiac function continues to deteriorate. KO mice transiently loose weight as a consequence of ANGII treatment. On the fourth to fifth day of ANGII treatment, KO animals gain weight again, although the heart function continues to deteriorate. FACS analysis of cardiac tissue revealed a strong infiltration of neutrophils in KO hearts after 48h of ANGII treatment (4.5‐fold increase compared to control hearts). A strong correlation between reduced ejection fraction and increasing amount of neutrophils in the cardiac tissue suggests a particularly important role of neutrophils in the development of cardiac dysfunction. Also an increase of macrophages (1.6‐fold increase), CD4+ T‐helper cells (2.5‐fold increase) and CD8+ cytotoxic T‐cells (2.5‐fold increase) as well as B‐cells (2‐fold increase) could be shown in KO hearts compared to control hearts. After long term ANGII treatment for 7 days, however, the number of neutrophils in the hearts decreased (10‐fold decrease in control hearts and 73‐fold decrease in KO hearts compared to 48h of ANGII treatment) and a strong increase in M2‐like macrophage infiltration, mainly in control hearts (1.6‐fold increase compared to 48h ANGII treatment), could be observed. The infiltrated macrophages were also positive for CCR2, which indicates they originated from monocytes. B‐ and T‐cells were still elevated in KO hearts after 7 days of ANGII treatment. Also in control hearts an increase of B‐ and T‐cells could be observed. In addition, fibrosis developed in the cardiac tissue, which could be shown by histological staining of collagen I and FACS analysis targeting αSMA+ cardiac fibroblasts.ConclusionImmune cells and in particular neutrophils play an important role in the development of cardiac dysfunction after ANGII induced pressure overload in p38 MAPKa KO mice. Over time, however, the number of immune cells in control mice also increases, which underlines the peculiarity of the first neutrophil influx after 48h ANGII treatment for the impairment of cardiac function.

Abstract 463: The Smooth-Muscle-Cell-Angiotensin II-Sensitive LncRNA Controls Cell Division Fidelity And Mitochondrial Organization

Reactivation of the cell cycle and increase in proliferation rate (hyperplasia) is a common response of vascular smooth muscle cells (SMC) to modifications of their environment during remodeling. Although SMC hyperplasia is a predominant feature of many vascular diseases, SMC can also increase their mass within the remodeled vessel wall by enlarging their size and becoming hypertrophic. Hypertrophy is usually accompanied by cell cycle defects, cell polyploidy and binucleation, and senescence. However, the molecular mechanisms favoring SMC hypertrophy and their repercussion on SMC phenotype are not fully understood. Long-non-coding-RNAs (LncRNAs) are epigenetic regulators of gene expression, and they have been identified as modulators of cell division. We recently discovered a novel lncRNA, SAS (SMC-Angiotensin II-Sensitive), whose expression was markedly decreased in multiple models of SMC dedifferentiation, suggesting that SAS could regulate SMC phenotype and function. Publicly available transcriptional datasets revealed that SAS is preferentially expressed in SMC-rich tissues, including the aorta, in humans and mice. Yet, the functional relevance of SAS in SMC has never been investigated. Knockdown of SAS reduces proliferation, cell arrest and migration in aortic and renal artery-derived SMC treated with Platelet Derived Growth Factor. SAS knockdown was also associated with distinct SMC morphological changes including increase in cell size and binucleation demonstrating hypertrophy. Together, these observations suggest that decrease in SAS causes SMC hypertrophy due to defects in cell cycle completion and cytokinesis. Interestingly, SAS expression is decreased in response to Angiotensin-II in cultured VSMC and in the aorta of hypertensive mice (2 Kidney-1 Clip model), suggesting a role in mediating hypertension induced SMC hypertrophy. Similarly, to Angiotensin-II treatment, SAS knockdown promoted senescence. Furthermore, SAS deficient cells present mitochondria hyperfusion and increased oxygen consumption that correlates with the observed exacerbated senescence. Altogether, our results indicate that SAS is a potent regulator of VSMC morphology and is required for proper cell division and mitochondria organization.