Continuous angiotensin-II Infusion Research Articles

Abstract Tu018: Integrin alpha 1 plays a critical role in angiotensin II-induced abdominal aortic aneurysm rupture

Background: Abdominal aortic aneurysm (AAA) is a life-threatening condition without an established specific therapy. This study aims to identify a new therapeutic target for preventing AAA rupture. Methods and Results: Angiotensin II (AngII)-infused ApoE-/- mice develop AAA, an established model of AAA. To identify molecular pathways contributing to AAA development, we conducted single-cell RNA sequence analysis using a dataset of aortic tissues harvested from mice infused with AngII for 4 weeks (GSE186865). Gene set enrichment analysis revealed upregulation of integrin- and TGFB-related pathways in AngII-stimulated smooth muscle cells (SMCs). Integrin α1 (Itga1) constitutes half of the α1β1 integrin duplex and serves as a major collagen receptor in SMCs. Male wild-type (n=37) and Itga1 knockout (n=36) mice with an ApoE-/- background received a continuous infusion of AngII for 4 weeks to develop AAA. Kaplan-Meier analysis indicated that ablating Itga1 significantly suppressed AAA rupture (p=0.009), without affecting the maximum diameter of the abdominal aorta. Considering extracellular matrix degradation as a critical hallmark of AAA rupture, we examined the abundance of major matrix metalloproteinases (MMPs) in the aortic wall, specifically MMP2 and MMP9, two major MMPs in the aorta. Gelatin zymography revealed that ablation of Itga1 suppressed AngII-induced MMP9 increase, without affecting MMP2 abundance. The expression of MMP9 is controlled by inflammation and proliferation signals; hence, we focused on these signals. qPCR analysis (n=8 per group) demonstrated that AngII-induced upregulation of Tnfa (p<0.001) and Tgfb1 (p=0.026) was mitigated by Itga1 ablation in aortic tissues. Conclusion: The integrin pathway is activated in the tissue of AAA tissue and deletion of Itga1 suppresses AAA rupture. Here we showed that the Itga1-Tgfb1-MMP9 axis would be a novel molecular target to inhibit AAA rupture.

Przewaquinone A inhibits Angiotensin II-induced endothelial diastolic dysfunction activation of AMPK

BackgroundEndothelial dysfunction (ED), characterized by markedly reduced nitric oxide (NO) bioavailability, vasoconstriction, and a shift toward a proinflammatory and prothrombotic state, is an important contributor to hypertension, atherosclerosis, and other cardiovascular diseases. Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) is widely involved in cardiovascular development. Przewaquinone A (PA), a lipophilic diterpene quinone extracted from Salvia przewalskii Maxim, inhibits vascular contraction. PurposeHerein, the goal was to explore the protective effect of PA on ED in vivo and in vitro, as well as the underlying mechanisms. MethodsA human umbilical vein endothelial cell (HUVEC) model of ED induced by angiotensin II (AngII) was used for in vitro observations. Levels of AMPK, endothelial nitric oxide synthase (eNOS), vascular cell adhesion molecule-1 (VCAM-1), nitric oxide (NO), and endothelin-1 (ET-1) were detected by western blotting and ELISA. A mouse model of hypertension was established by continuous infusion of AngII (1000 ng/kg/min) for 4 weeks using osmotic pumps. Following PA and/or valsartan administration, NO and ET-1 levels were measured. The levels of AMPK signaling-related proteins in the thoracic aorta were evaluated by immunohistochemistry. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) were measured using the tail cuff method. Isolated aortic vascular tone measurements were used to evaluate the vasodilatory function in mice. Molecular docking, molecular dynamics, and surface plasmon resonance imaging (SPRi) were used to confirm AMPK and PA interactions. ResultsPA inhibited AngII-induced vasoconstriction and vascular adhesion as well as activated AMPK signaling in a dose-dependent manner. Moreover, PA markedly suppressed blood pressure, activated vasodilation in mice following AngII stimulation, and promoted the activation of AMPK signaling. Furthermore, molecular simulations and SPRi revealed that PA directly targeted AMPK. AMPK inhibition partly abolished the protective effects of PA against endothelial dysfunction. ConclusionPA activates AMPK and ameliorates endothelial dysfunction during hypertension.

Role of atrial lymphatics in atrial myocardial fibrosis and atrial fibrillation: a human and mouse study

Abstract Background/Introduction Cardiac lymphatics maintain myocardial fluid and immune cell homeostasis, but its effects on atrial fibrosis and atrial fibrillation (AF) have not been clarified. Purpose To address this issue, we first evaluated the left atrial appendage (LAA) in humans histologically and biologically. We then studied mice to elucidate the mechanisms. Methods Human LAA samples were collected from 34 consecutive AF patients during cardiovascular surgery. They were assigned to the paroxysmal AF (PAF) group (n=17, 70.9±11.6 years) and the persistent AF (PeAF) group (n=17, 72.1±7.2 years). In the mouse experiments, 8-week-old male C57BL/6 mice were administrated subcutaneously with vehicle or Angiotensin II (AngII) (2.0 mg/kg/day) for 4 weeks. In some mice, VEGFC, a pro-lymphangiogenesis inducer, was administrated subcutaneously at a dose of 50 μg/kg/day simultaneously with AngII. AF was induced by transesophageal burst pacing in vivo, and by burst pacing in isolated perfused hearts using a Langendorff apparatus. Results In human LAA samples, mRNA expression of lymphangiogenesis-related genes (LYVE1, PROX1, and VEGFR3) was significantly lower in the PeAF group compared to the PAF group (p=0.037, 0.029, and 0.041). The number of LYVE1-positive atrial lymphatic endothelial cells (LECs) was also significantly lower in the PeAF group compared to the PAF group (p=0.026). There was a negative correlation between the number of LYVE1-positive atrial LECs and the area percentage of atrial myocardial fibrosis (r=-0.609, p<0.001). In mouse experiments, continuous infusion of AngII suppressed the mRNA expression of lymphangiogenesis-related genes, including Lyve1, Prox1, and Vegfr3 (n=6 in each group, p<0.001, p=0.003, and p<0.001, respectively). AngII infusion reduced the number of LYVE1-positive atrial LECs (p<0.001) and increased the mRNA expression of fibrosis-related genes (Tgfb, Col1a1, and Col3a1). The area percentage of fibrosis in atrial myocardium was also increased by AngII. Treatment with VEGFC for 4 weeks effectively reversed the effects of AngII, i.e., suppression of lymphangiogenesis-related genes expression, reduction of LYVE1-positive atrial LECs, increase in fibrosis-related genes expression, and increase in atrial fibrosis. Additionally, VEGFC treatment attenuated AngII-induced enhancement of vulnerability to AF in in vivo experiments (n=8 in each group, p=0.007) and in isolated perfused hearts (n=8 in each group, p=0.007). Conclusions Our study with human LAA demonstrated a strong association between atrial lymphatics and atrial myocardial fibrosis/progression of AF. Our mouse study showed that VEGFC reversed AngII -induced increase in atrial myocardial fibrosis and vulnerability to AF, possibly via promotion of lymphangiogenesis. Defects of lymphangiogenesis may be involved in atrial myocardial fibrosis and AF, and may be a therapeutic target for atrial cardiomyopathy.

Synthetic growth hormone-releasing hormone agonist ameliorates the myocardial pathophysiology characteristic of heart failure with preserved ejection fraction.

To test the hypothesis that the activation of the growth hormone-releasing hormone (GHRH) receptor signalling pathway within the myocardium both prevents and reverses diastolic dysfunction and pathophysiologic features consistent with heart failure with preserved ejection fraction (HFpEF). Impaired myocardial relaxation, fibrosis, and ventricular stiffness, among other multi-organ morbidities, characterize the phenotype underlying the HFpEF syndrome. Despite the rapidly increasing prevalence of HFpEF, few effective therapies have emerged. Synthetic agonists of the GHRH receptors reduce myocardial fibrosis, cardiomyocyte hypertrophy, and improve performance in animal models of ischaemic cardiomyopathy, independently of the growth hormone axis. CD1 mice received 4- or 8-week continuous infusion of angiotensin-II (Ang-II) to generate a phenotype with several features consistent with HFpEF. Mice were administered either vehicle or a potent synthetic agonist of GHRH, MR-356 for 4-weeks beginning concurrently or 4-weeks following the initiation of Ang-II infusion. Ang-II-treated animals exhibited diastolic dysfunction, ventricular hypertrophy, interstitial fibrosis, and normal ejection fraction. Cardiomyocytes isolated from these animals exhibited incomplete relaxation, depressed contractile responses, altered myofibrillar protein phosphorylation, and disturbed calcium handling mechanisms (ex vivo). MR-356 both prevented and reversed the development of the pathological phenotype in vivo and ex vivo. Activation of the GHRH receptors increased cAMP and cGMP in cardiomyocytes isolated from control animals but only cAMP in cardiac fibroblasts, suggesting that GHRH-A exert differential effects on cardiomyocytes and fibroblasts. These findings indicate that the GHRH receptor signalling pathway(s) represents a new molecular target to counteract dysfunctional cardiomyocyte relaxation by targeting myofilament phosphorylation and fibrosis. Accordingly, activation of GHRH receptors with potent, synthetic GHRH agonists may provide a novel therapeutic approach to management of the myocardial alterations associated with the HFpEF syndrome.

ELASTASE-2 KNOCKOUT IS ASSOCIATED WITH LESS SUSCEPTIBLE AORTA DILATION IN RESPONSE TO ANGIOTENSIN II STIMULATION IN THE MICE MODEL

Objective: Elastase-2 (ELA-2), a chymotrypsyn-serine protease elastase family member 2A, is expressed in several tissues such as the lungs, heart, liver, kidneys, brain, and vessels. ELA-2 was found to generate angiotensin II (AngII) in arteries and contribute to resistance arteries and vascular remodeling in animal models. It has also been shown that continuous infusion of AngII induces abdominal aortic aneurysm (AAA) in mice. Therefore, we sought to investigate the role of ELA-2 in an aortic dilation, which is a risk factor to AAA development, in a mouse model. Design and method: Male wild type (C57bl/6, WT) and ELA-2 knockout (CELA-2aTm1Bdr; ELA-2 KO) mice were treated with saline or angiotensin II (AngII, 2.28 g/g/min) for twenty-eight days by subcutaneously mini-pumps infusion. To evaluate the role of elastase-2 in a AAA development, mice were divided into four groups: WT treated with saline (WT+SAL), ELA-2KO treated with saline (ELA-2KO+SAL), WT treated with AngII (WT+AngII), and ELA-2KO treated with Ang II (ELA-2KO+AngII). The vascular ultrasound (US) was done before treatment and once a week during 28 days of treatment. The abdominal aorta measurements such as maximum aortic diameter, aortic para-renal diameter, aortic infra-renal diameter, and aortic supra-renal diameter were collected. A dilated aorta is a risk factor aortic rupture/aneurysm. Statistical analyses were performed by the Shapiro Wilk Normality test, two-way ANOVA with Sidak's multiple comparisons test. This study is approved by the Ethics Committee (CEUA-131/2019). Results: The most dilated aorta as measured by the maximum aorta diameter was detected in WT+AngII group and was 60% higher than in the WT+Saline (p = 0.01). There was no significant difference in the maximum aorta diameter between ELA-KO+SAL vs. ELA-2KO+AngII (p = 0.8). The measurement of dilation in aortic para-renal and aortic infra-renal showed that AngII induced a dilation in WT+AngII (p < 0.05), while not modified these parameters in ELA-2KO+AngII group. The aortic supra-renal diameter parameters and weight showed no differences between groups. Conclusions: These results suggest that ELA-2KO mice might be less susceptible to develop an aorta dilation. Therefore, Elastase-2 is a candidate factor that contributes to the formation and/or development of abdominal aortic aneurysm.

Longitudinal Testing of Retinal Blood Flow in a Mouse Model of Hypertension by Laser Speckle Flowgraphy.

PurposeThe purpose of this study was to investigate the applicability of laser speckle flowgraphy (LSFG) for a longitudinal study of blood flow parameters in mice before, during, and after continuous infusion of angiotensin-II.MethodsNormotensive C57BL/6J mice were imaged by LSFG at one (n = 22) or three sessions (n = 10). Two additional cohorts were imaged by LSFG before, during, and after continuous infusion of angiotensin-II by minipump for 2 or 4 weeks (n = 6 and 8, respectively). Retinal blood flow, vascular resistance, and total area of retinal vascular flow, a surrogate of vascular remodeling and vasoconstriction, were determined at each time point.ResultsDuring infusion of angiotensin-II for 2 weeks, decreased retinal blood flow and area of vascular flow, as well as increased vascular resistance, were observed. These changes were reversed 1 week after the end of angiotensin-II infusion. In mice infused with angiotensin-II for 4 weeks, decreased retinal blood flow and increased vascular resistance persisted at 6 weeks postinfusion, despite a decrease in blood pressure.ConclusionsArterial hypertension, induced by continuous angiotensin-II infusion, results in reduced retinal blood flow, increased vascular resistance, and decrease in area of intravascular blood flow within retinal arterioles and venules. Sustained vasoconstriction 6 weeks after the end of a 4-week period of angiotensin-II infusion may indicate vascular remodeling after a period of chronic hypertension.Translational RelevanceRetinal LSFG is useful for serial investigation of blood flow in mouse models and provides a novel approach for translational studies on the microvascular effects of hypertension in vivo.

Effects of Isorhamnetin in Human Amniotic Epithelial Stem Cells in vitro and Its Cardioprotective Effects in vivo.

Cardiac hypertrophy and fibrosis are major pathophysiologic disorders that lead to serious cardiovascular diseases (CVDs), such as heart failure and arrhythmia. It is well known that transforming growth factor β (TGFβ) signaling pathways play a major role in the proliferation of cardiac hypertrophy and fibrosis, which is mainly stimulated by angiotensin II (AgII). This study aimed to investigate the cardioprotective potential of isorhamnetin (ISO) in human amniotic epithelial stem cells (hAESCs) through global gene expression analysis and to confirm its beneficial effects on cardiac hypertrophy and fibrosis in the AgII-induced in vivo model. In vitro, biological processes including TGFβ, collagen-related functions, and inflammatory processes were significantly suppressed in ISO pretreated hAESCs. In vivo, continuous AgII infusion using an osmotic pump induced significant pathological fibrosis and myocardial hypertrophy, which were remarkably suppressed by ISO pretreatment. ISO was found to reverse the enhanced TGFβ and Collagen type I alpha 1 mRNA expression induced by AgII exposure, which causes cardiovascular remodeling in ventricular tissue. These findings indicate that ISO could be a potential agent against cardiac hypertrophy and fibrosis.

A traditional herbal medicine rikkunshito prevents angiotensin II-Induced atrial fibrosis and fibrillation

BackgroundRikkunshito (RKT), a traditional herbal medicine, has been demonstrated to exert anti-inflammatory, anti-apoptotic, and anti-fibrotic effects in several organs. This study tested the hypothesis that RKT can suppress angiotensin II (AngII)-induced inflammatory atrial fibrosis and ameliorate enhanced vulnerability to atrial fibrillation (AF). MethodsEight-week-old male C57BL/6 mice were subcutaneously infused with either vehicle or AngII (2.0 mg/kg/day) for 2 weeks. Water or RKT at a dose of 1000 mg/kg/day were orally administered once daily for 2 weeks. Morphological, histological, and biochemical analyses were performed. AF was induced either by transesophageal burst pacing in vivo or by burst/extrastimuli in isolated perfused hearts using a Langendorff apparatus. ResultsRKT at a dose of 1000 mg/kg/day for 2 weeks attenuated atrial interstitial fibrosis and profibrotic and proinflammatory signals induced by continuous infusion of AngII. RKT attenuated AngII-induced enhanced vulnerability to AF in in vivo experiments and in isolated perfused hearts. Atractylodin, an active component of RKT, exhibited antifibrotic activity comparable to that of RKT. RKT reversed AngII-induced suppression of sirtuin 1 (Sirt1) translocation to the nuclei. RKT suppressed AngII-induced phosphorylation of IκB, overexpression of p53, and cellular apoptotic signals and apoptosis. All of the antagonizing effects of RKT against AngII were attenuated by a concomitant treatment with a growth hormone secretagogue receptor (GHSR)-inhibitor. ConclusionOur results demonstrated that RKT prevented atrial fibrosis and attenuated enhanced vulnerability to AF induced by AngII. The results also suggested that potentiating the GHSR-Sirt1 pathway is involved in these processes.

Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration

Vascular smooth muscle cells (VSMCs) proliferation and migration play a fundamental role during the process of hypertensive angiopathy. Angiotensin-II (Ang-II) is one of the robust phenotype-modulating agents, which changes VSMCs to efficiently proliferate and migrate. The mechanism of the proliferation and migration is not well understood yet. Septin4, as a member of GTP binding protein family, is widely expressed in the eukaryotic cells and considered to be an essential component of the cytoskeleton which is involved in many important physiological processes. We approved that Septin4 expression was upregulated in mouse aorta by continuous infusion of Ang-II and in cultured VSMCs treated with Ang-II. Overexpression of Septin4 led to lower level of autophagy and decreased capacity of proliferation and migration. In order to identify the mechanism by which Septin4 interacts with these processes, we blocked autophagy by chloroquine (CQ). After inhibiting the autophagy, the ability of proliferation and migration was further restrained in the Septin4 overexpression VSMCs. In conclusion, our results indicated that during the process of VSMCs proliferation and migration induced by Ang-II, Septin4 modulated autophagy and thus regulated the activity of proliferation and migration.

1440Anti-inflammatory adipokine, omentin, attenuates angiotensin II-induced abdominal aortic aneurysm formation in apolipoprotein-E knockout mice

Abstract Background Abdominal aortic aneurysm (AAA) is an increasing and life-threatening disease. Obesity is associated with an increased risk of AAA. Omentin is a circulating adipokine, which is downregulated by obesity. Recently we have demonstrated that omentin is an anti-inflammatory adipokine that prevents the development of atherosclerosis in apolipoprotein-E knockout (apoE-KO) mice. Here we examined whether omentin could modulate angiotensin II-induced AAA formation in apoE-KO mice. Methods and results To overexpress human omentin in apoE-KO mice, apoE-KO mice were crossed with transgenic mice expressing the human omentin gene in fat tissue under the control of AP2 promoter (OMT-Tg mice). Circulating levels of human omentin in apoE-KO/OMT-Tg mice were approximately threefold higher than those in healthy human subjects, whereas human omentin was undetectable in apoE-KO mice. There were no differences in body weight, blood pressure and heart rate between apoE-KO/OMT-Tg and apoE-KO mice. We also subjected apoE-KO/OMT-Tg and apoE-KO mice at 24 weeks of age to continuous angiotensin II-infusion by using osmotic mini pumps for 4 weeks, which is a widely-accepted model of experimental AAA. ApoE-KO/OMT-Tg mice exhibited a lower incidence of AAA formation and a reduced maximal diameter of AAA determined by direct measurement and ultrasound imaging as compared with apo-E KO mice. In histological analyses with van Gieson staining, apoE-KO/OMT-Tg mice showed attenuated disruption of medial elastic fibers in response to angiotensin II compared with apo-E KO mice. ApoE-KO/OMT-Tg mice also displayed reduced mRNA levels of matrix metalloproteinase (MMP) 2 and MMP9 as well as pro-inflammation genes including interleukin (IL)-6 in aortic walls compared with apo-E KO mice. Treatment of human monocyte-derived macrophages with human omentin protein attenuated LPS-stimulated expression of MMP9, TNF-α and IL-6. Omentin treatment also reduced LPS-induced activation of MMP9 in cultured media of human macrophages as evaluated by gelatinolytic zymography. Omentin treatment increased phosphorylation levels of Akt in human macrophages. The suppressive effects of omentin on inflammatory response in macrophages were reversed by treatment with LY294002, which is an inhibitor of PI3 kinase/Akt signaling. Conclusion These data suggest that omentin acts as an adipokine that can attenuate angiotensin II-induced development of AAA through suppression of MMP activation and inflammatory response in the vascular wall.

Abstract 16934: Targeted Cardiac Delivery of miR-29b Mimic via Ultrasound-Mediated Microbubble Cavitation Attenuates Myocardial Fibrosis

Introduction: Cardiac fibrosis contributes to adverse ventricular remodeling in ischemic or hypertensive heart disease, increases heart failure risk, and is associated with loss of microRNA-29b (miR-29b). Plasmid or i.v. injection of miR-29b mimic has proven the concept that restoring miR-29b blunts cardiac fibrosis, but these are inefficient, non-targeted delivery strategies with low clinical translational potential. We hypothesized that ultrasound (US) mediated cavitation of microbubbles (MBs) loaded with miR-29b mimic would inhibit cardiac fibrosis. We first tested effects of US-targeted cavitation of miR29b-MBs (UTMC) on cardiac fibroblasts in vitro , then performed in vivo studies in a mouse model of angiotensin II (ANGII)-induced hypertension/fibrosis. Methods: Cationic lipid MBs were loaded with miR-29b mimic or negative control (NC) miRNA, placed with cultured neonatal cardiac fibroblasts, then treated with 10 sec pulsed US ( f =1.3 MHz, MI 1.6, 4 frames/burst, 4 bursts/second). Cells were harvested at 24 hrs to measure downstream fibrotic mediators. Mice received continuous ANGII infusion via osmotic pump and UTMC with miRNA-loaded MBs at days 0, 3, 7. Serial echo was performed; the heart was harvested on day 10. Results: In cardiac fibroblasts, UTMC treatment with MBs loaded with miR-29b mimic vs NC miRNA caused a 543±51% increase in miR-29b ( p <0.01) and knockdown in transcripts for collagen 1A1 (26±6%, p <0.01), collagen 1A2 (53±8%, p <0.01), collagen III (31±5%, p <0.01), and fibrillin (43±10%, p <0.05) ( n =4/group). ANGII mice treated with UTMC + miR-29b mimic vs. NC miRNA ( n =8-9/group) had downregulated cardiac fibrillin (37±8%, p <0.05); trends of lower collagen IA1, 1A2, and III by RT-PCR; decreased cardiac α-SMA protein by immunoblot ( p <0.05); and preservation of ejection fraction and fractional shortening ( p <0.01). Conclusion: UTMC-mediated delivery of miR-29b mimic blunts pathologic expression of fibrosis markers and is associated with preservation of left ventricular function in ANGII-induced murine cardiac fibrosis. This is the first demonstration of UTMC-delivery of a miRNA mimic for attenuating cardiac fibrosis, and has promising implications for targeted RNA-based approaches for treating cardiac disease.

Angiotensin‐II‐Induced Increase in Blood Pressure in Female and Male Mice

The kidney regulates blood pressure by controlling urinary salt and water excretion. Angiotensin II (AngII), a key player in controlling blood pressure, constricts blood vessels, stimulates vasopressin and aldosterone release, stimulates thirst, and increases sodium absorption from the proximal tubule. All effects cause increased blood pressure. Sex differences in the regulation of arterial blood pressure involve events surrounding the renin‐angiotensin‐aldosterone system. Evidence generally supports a protective mechanism for females with AngII‐induced hypertension; although, conflicting evidence exists. The purpose of this study was to investigate sex differences in CD‐1 mice under AngII administration for a period of 10 days. We hypothesized that female mice would be protected from AngII‐induced increase in mean arterial pressure (MAP) compared to male mice and that the renal handling of salt and water contributes to this protection. Four‐week old CD‐1 mice were obtained from Envigo, Inc. (Indianapolis, IN). Two studies, one with females and one with males, were conducted. Mice were placed in metabolic cages and consumed normal diet and water, ad libitum, for 15 days. After the first 5 days, an Alzet™ minipump containing either AngII (n=5) or vehicle (n=5) was implanted subcutaneously in each mouse to ensure continuous AngII infusion (1 mg/kg/min). Daily measurements included MAP via the tail‐cuff method (Kent Scientific, CODA System), urinary output, and urinary sodium excretion. MAP (mmHg) was not different between females and males in the 5‐day control period (85.1 ± 3.7 vs 81.7 ± 4.0, respectively). AngII‐treatment increased MAP in both females and males (110.3 ± 4.4 and 105.8 ± 3.0, p<0.001, compared to respective control period). Interestingly, AngII‐treated females showed higher urine output than AngII‐treated males (9.7 ± 0.8 vs 7.4 ± 0.4 ml/day, p<0.05) and also had higher sodium excretion (242 mEq/day vs 166 mEq/day, p<0.01). Absolute food intake in females and males was not different, however, females consumed more food per body weight. We conclude that female mice are not protected from AngII‐induced increase in MAP compared to male mice for a 10‐day AngII infusion period. However, based on the sodium excretion data, protection may be observed later from increased urine and sodium output in females. Results suggest that the renal handling of salt and water could be an important determinant of sex differences in blood pressure regulation.Support or Funding InformationNational Institute of General Medical Sciences of National Institutes of Health through Grant Number 8P20GM103447This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tumor necrosis factor: a mechanistic link between angiotensin-II-induced cardiac inflammation and fibrosis.

Continuous angiotensin-II infusion induced the uptake of monocytic fibroblast precursors that initiated the development of cardiac fibrosis; these cells and concurrent fibrosis were absent in mice lacking tumor necrosis factor receptor 1 (TNFR1). We now investigated their cellular origin and temporal uptake and the involvement of TNFR1 in monocyte-to-fibroblast differentiation. Within a day, angiotensin-II induced a proinflammatory environment characterized by production of inflammatory chemokines, cytokines, and TH1-interleukins and uptake of bone marrow-derived M1 cells. After a week, the cardiac environment changed to profibrotic with growth factor and TH2-interleukin synthesis, uptake of bone marrow-derived M2 cells, and the presence of M2-related fibroblasts. TNFR1 signaling was not necessary for early M1 uptake, but its absence diminished the amount of M2 cells. TNFR1-knockout hearts also showed reduced levels of cytokine expression, but not of TH-related lymphokines. Reconstitution of wild-type bone marrow into TNFR1-knockout mice was sufficient to restore M2 uptake, upregulation of proinflammatory and profibrotic genes, and development of fibrosis in response to angiotensin-II. We also developed an in vitro mouse monocyte-to-fibroblast maturation assay that confirmed the essential role of TNFR1 in the sequential progression of monocyte activation and fibroblast formation. Development of cardiac fibrosis in response to angiotensin-II was mediated by myeloid precursors and consisted of 2 stages. A primary M1 inflammatory response was followed by a subsequent M2 fibrotic response. Although the first phase seemed to be independent of TNFR1 signaling, the later phase (and development of fibrosis) was abrogated by deletion of TNFR1.

Abstract 75: TNF Receptor 1 Signaling: a Mechanistic Link between Cardiac Inflammation and Fibrosis

Background: We previously showed in several models of cardiac hypertrophy and failure that, in the absence of cell death, cardiac interstitial fibrosis was mediated by the uptake of monocyte-derived fibroblast precursor cells; fibrosis and concurrent cardiac remodeling were blocked by deletion of monocyte chemoattractant protein-1 (MCP-1), as well as of tumor necrosis factor receptor-1 (TNFR1). We now investigated the cellular origin, kinetics of uptake, and subpopulation of fibroblast precursors in the angiotensin-II (Ang-II)-challenged heart. Methods: Mice with genetic deletion of TNFR1 (TNFR1-KO mice) were irradiated and rescued with bone marrow from wild-type (WT) mice, and were subjected to continuous infusion of Ang-II for 7 days. Flow cytometry was performed on isolated cells, immunostaining on perfusion-fixed tissue, quantitative PCR on whole heart mRNA isolations. Results: In WT mice, Ang-II induced the early uptake of monocytes which became M1 macrophages (CD86+CD45+) in an M1/Th1 environment. Monocytes entering the heart after 2-3 days polarized to M2 macrophages (CD301+CD45+) in an M2/Th2 milieu with concurrent appearance of collagen-producing CD301+ fibroblasts. M1 cells produced TNF, whereas M2 cells did not; both expressed TNFR1. Ang-II-exposed TNFR1-KO hearts showed similar cardiac infiltration of M1 cells, but the amount of M2 cells was significantly lower. They also had reduced expression of M1 and M2 cytokines, but not of Th1 and Th2 interleukins. Transplantation of WT bone marrow to TNFR1-KO mice before Ang-II exposure restored cardiac fibrosis, uptake of M2 cells, and expression of cytokines to levels observed in Ang-II-exposed WT animals. Many TNFR1+ cells in chimeric TNFR1-KO/WT were committed to the fibroblast lineage. Conclusion: Our data show that monocytic fibroblast precursor cells originated in the bone marrow, and that signaling through TNFR1 was required for their uptake and maturation into collagen-producing M2/fibroblasts that mediated the development of cardiac fibrosis in response to Ang-II. They also suggest a mechanistic link between inflammation and fibrosis, i.e. pro-inflammatory, M1-produced TNF initiates pro-fibrotic M2/fibroblast maturation via TNFR1 signaling.

Angiotensin-II type 1 receptor-mediated Janus kinase 2 activation induces liver fibrosis

Activation of the renin angiotensin system resulting in stimulation of angiotensin-II (AngII) type I receptor (AT1R) is an important factor in the development of liver fibrosis. Here, we investigated the role of Janus kinase 2 (JAK2) as a newly described intracellular effector of AT1R in mediating liver fibrosis. Fibrotic liver samples from rodents and humans were compared to respective controls. Transcription, protein expression, activation, and localization of JAK2 and downstream effectors were analyzed by real-time polymerase chain reaction, western blotting, immunohistochemistry, and confocal microscopy. Experimental fibrosis was induced by bile duct ligation (BDL), CCl4 intoxication, thioacetamide intoxication or continuous AngII infusion. JAK2 was inhibited by AG490. In vitro experiments were performed with primary rodent hepatic stellate cells (HSCs), Kupffer cells (KCs), and hepatocytes as well as primary human and human-derived LX2 cells. JAK2 expression and activity were increased in experimental rodent and human liver fibrosis, specifically in myofibroblastic HSCs. AT1R stimulation in wild-type animals led to activation of HSCs and fibrosis in vivo through phosphorylation of JAK2 and subsequent RhoA/Rho-kinase activation. These effects were prevented in AT1R(-/-) mice. Pharmacological inhibition of JAK2 attenuated liver fibrosis in rodent fibrosis models. In vitro, JAK2 and downstream effectors showed increased expression and activation in activated HSCs, when compared to quiescent HSCs, KCs, and hepatocytes isolated from rodents. In primary human and LX2 cells, AG490 blocked AngII-induced profibrotic gene expression. Overexpression of JAK2 led to increased profibrotic gene expression in LX2 cells, which was blocked by AG490. Our study substantiates the important cell-intrinsic role of JAK2 in HSCs for development of liver fibrosis. Inhibition of JAK2 might therefore offer a promising therapy for liver fibrosis.

TNF receptor 1 signaling is critically involved in mediating angiotensin-II-induced cardiac fibrosis

Angiotensin-II (Ang-II) is associated with many conditions involving heart failure and pathologic hypertrophy. Ang-II induces the synthesis of monocyte chemoattractant protein-1 that mediates the uptake of CD34+CD45+ monocytic cells into the heart. These precursor cells differentiate into collagen-producing fibroblasts and are responsible for the Ang-II-induced development of non-adaptive cardiac fibrosis. In this study, we demonstrate that in vitro, using a human monocyte-to-fibroblast differentiation model, Ang-II required the presence of tumor necrosis factor-alpha (TNF) to induce fibroblast maturation from monocytes. In vivo, mice deficient in both TNF receptors did not develop cardiac fibrosis in response to 1week Ang-II infusion. We then subjected mice deficient in either TNF receptor 1 (TNFR1-KO) or TNF receptor 2 (TNFR2-KO) to continuous Ang-II infusion. Compared to wild-type, in TNFR1-KO, but not in TNFR2-KO hearts, collagen deposition was greatly attenuated, and markedly fewer CD34+CD45+ cells were present. Quantitative RT-PCR demonstrated a striking reduction of key fibrosis-related, as well as inflammation-related mRNA expression in Ang-II-treated TNFR1-KO hearts. TNFR1-KO animals also developed less cardiac remodeling, cardiac hypertrophy, and hypertension compared to wild-type and TNFR2-KO in response to Ang-II. Our data suggest that TNF induced Ang-II-dependent cardiac fibrosis by signaling through TNFR1, which enhances the generation of monocytic fibroblast precursors in the heart.

Abstract 8742: Deletion of CIKS (Act1 or traf3ip2) Abrogates Angiotensin-II-induced Cardiac Hypertrophy in vitro and in vivo

Chronic elevation of Angiotensin-II (Ang-II) can lead to cardiac hypertrophy and heart failure. Ang-II activates NF-κB and AP-1, and induces pro-inflammatory cytokines and pro-growth factors. The adapter molecule CIKS (connection to IKK and SAPK/JNK; also known as Act1 or traf3ip2) lies upstream of IKK and JNK, and mediates both autoimmune and inflammatory diseases. We hypothesized that CIKS plays a causal role in Ang-II-induced cardiac hypertrophy. The heart and body weights were similar in both wild type (WT; C57Bl/6) and CIKS null mice (C57Bl6 background). However, continuous infusion of Ang-II (1.5 μg/kg/min for 7 days; n=8/group) led to cardiac hypertrophy (heart wt/tibial length, 38% increase vs saline, P <0.01) and upregulation of CIKS mRNA (2-fold, P <0.05) and protein (3.1-fold, P <0.01) in WT, but not CIKS null mice ( P <0.001 vs WT+Ang-II). Ang-II increased heart rate and systolic blood pressure in both, but levels trended higher in the CIKS null (not significant). Ang-II increased NF-κB and AP-1 DNA binding activities, and induced ANP, cytokine (TNF-α, IL-18), growth factor (WISP1), and MMP (MMP-2, -9, and -14) expression/activity in WT, but not CIKS null mice. Ang-II treatment of adult cardiomyocytes from WT resulted in significant hypertrophy (increased cell surface area and protein without DNA synthesis; both at least P <0.01), PI3K, Akt, p70 S6K and ribosomal S6 protein activation, and ANP, TNF-α, IL-18, WISP1, and MMPs expression. Moreover, Ang-II induced CIKS mRNA (at 1 h, 5.1-fold, P <0.001) and protein (at 2 h, 3.78-fold, P <0.01) in a time- and dose-dependent manner. Importantly, CIKS knockdown (lentiviral shRNA) blunted Ang-II induced IKKβ and JNK activities, p65 and c-Jun phosphorylation, and NF-κB and AP-1 DNA binding and reporter gene activities, cytokine, WISP1, and MMPs in WT-cardiomyocytes. These results were recapitulated in CIKS null cardiomyocytes. Our results demonstrate that CIKS plays a critical role in Ang-II-mediated cardiac hypertrophy in vivo and cardiomyocyte hypertrophy in vitro thorough induction of IKK/NF-κB and JNK/AP-1-dependent cytokine, growth factor, and MMP expression. CIKS is a potential therapeutic target in cardiac hypertrophy and congestive heart failure.

Prolonged Infusion of Angiotensin II in apoE−/− Mice Promotes Macrophage Recruitment with Continued Expansion of Abdominal Aortic Aneurysm

Angiotensin II (AngII) infusion initiates abdominal aortic aneurysm (AAA) development due to medial disruption and results in luminal dilation and thrombus formation. The objective of this study was to determine whether AAA progressed during protracted AngII infusion. Male apoE(-/-) mice were infused with AngII using miniosmotic pumps. On day 27, suprarenal aortic luminal diameters were ultrasonically measured to identify mice exhibiting AAAs. Mice were designated to three groups with similar mean luminal dilation. Group 1 mice were sacrificed on day 28. Group 2 and 3 mice were subsequently infused with saline or AngII, respectively, for an additional 56 days. In Group 2, saline infusion-after the initial 28 days of AngII infusion-led to an immediate decrease in systolic blood pressure. Over the subsequent 56 days of saline infusion, there were no aneurysm-related deaths or significant changes in luminal diameter. In contrast, continuous AngII infusion in Group 3 maintained persistently increased systolic blood pressure, with aneurysmal rupture-associated deaths, increased luminal diameters, and tissue remodeling. Aortic aneurysmal segments that expanded during continuous AngII infusion exhibited macrophage accumulation in regions of medial disruption, predominantly on the adventitial aspect. Macrophages immunostained for CD206 more than for iNOS, consistent with an M2 phenotype. In conclusion, prolonged AngII infusion promotes AAA expansion, and is associated with enhanced rupture rates and increased macrophage infiltration.

Angiotensin-II type 1 receptor and NOX2 mediate TCF/LEF and CREB dependent WISP1 induction and cardiomyocyte hypertrophy

Angiotensin-II (Ang-II) plays a key role in myocardial hypertrophy, remodeling and failure. We investigated whether Ang-II-induced cardiomyocyte hypertrophy is dependent on WNT1 inducible signaling pathway protein 1 (WISP1), a pro growth factor. Ang-II induced hypertrophy and WISP1 expression in neonatal rat cardiomyocytes (NRCM), effects that were significantly inhibited by pre-treatment with the AT1 antagonist losartan and by WISP1 knockdown. Further, Ang-II induced WISP1 was superoxide-dependent, and inhibited by DPI, an inhibitor of NADPH oxidases, and by knockdown of NOX2. AT1 was physically associated with NOX2 both in vitro and in vivo, and Ang-II increased this interaction in vivo. Ang-II induced WISP1 expression via superoxide/Akt/GSK3β/β-catenin/TCF/LEF and by Akt-dependent CREB activation. Further, Ang-II also activated CREB via superoxide-mediated p38 MAPK and ERK activation. Continuous infusion of Ang-II for 7days induced myocardial hypertrophy in rats, and was associated with increased Akt, p-Akt, p-p38 MAPK, p-ERK1/2, and WISP1 expression. These results demonstrate that Ang-II induced cardiomyocyte hypertrophy is mediated through AT1, NOX2 and the induction of WISP1, and may involve the direct interaction of AT1 with NOX2. Thus targeting both WISP1 and NOX2 may have a therapeutic potential in improving cardiomyocyte survival and growth following myocardial injury and remodeling. This article is part of a Special Issue entitled ‘Possible Editorial’.

Monocytic fibroblast precursors mediate fibrosis in angiotensin-II-induced cardiac hypertrophy

Angiotensin-II (Ang-II) is an autacoid generated as part of the pathophysiology of cardiac hypertrophy and failure. In addition to its role in cardiac and smooth muscle contraction and salt retention, it was shown to play a major role in the cardiac interstitial inflammatory response and fibrosis accompanying cardiac failure. In this study, we examined a model of Ang-II infusion to clarify the early cellular mechanisms linking interstitial fibrosis with the onset of the tissue inflammatory response. Continuous infusion of Ang-II resulted in increased deposition of collagen in the heart. Ang-II infusion also resulted in the appearance of distinctive small, spindle-shaped, bone marrow-derived CD34 +/CD45 + fibroblasts that expressed collagen type I and the cardiac fibroblast marker DDR2 while structural fibroblasts were CD34 −/CD45 −. Genetic deletion of monocyte chemoattractant protein (MCP)-1 (MCP-1-KO mice) prevented the Ang-II-induced cardiac fibrosis and the appearance of CD34 +/CD45 + fibroblasts. Real-time PCR in Ang-II-treated hearts revealed a striking induction of types I and III collagen, TGF-β1, and TNF mRNA expression; this was obviated in Ang-II-infused MCP-1-KO hearts. In both wild-type and MCP-1-KO mice, Ang-II infusion resulted in cardiac hypertrophy, increased systolic function and hypertension which were not significantly different between the WT and MCP-1-KO mice over the 6-week course of infusion. In conclusion, the development of Ang-II-induced non-adaptive fibrosis in the heart required induction of MCP-1, which modulated the uptake and differentiation of a CD34 +/CD45 + fibroblast precursor population. In contrast to the inflammatory and fibrotic response, the hemodynamic response to Ang-II was not affected by MCP-1 in the first 6 weeks.