Angiotensin II Exposure Research Articles

Abstract 3133: Selective Vulnerability Of Coronary Arteries To Hypercholesterolemia And Angiotensin II-induced Hypertension

Hypercholesterolemia and hypertension are major risk factors for cardiovascular disease. We aimed to determine their concomitant effects on coronary atherogenesis. An SR-BI (an HDL receptor) suppression and doxycycline-inducible angiotensin II (AngII) cassette was inserted downstream of the ApoE promoter to ablate ApoE expression and overproduce AngII. The resulting mutants (ApoE SA/SA ) exhibited mild coronary atherosclerosis after Western diet feeding. Doxycycline induced AngII-induced hypertension and drastically accelerated coronary atherosclerosis, with lesions exhibiting endothelial erosion, myeloid cell infiltration, and plaque rupture, resulting in myocardial infarction, heart failure, and male-propensity of sudden death. AngII, but not norepinephrine-induced hypertension, led to severe coronary atherogenesis under hypercholesterolemia, which was abolished by losartan. Notably, norepinephrine constricted femoral arteries, but it dilated coronary arteries. In contrast to coronary arteries, femoral arteries did not develop atherosclerosis. Proteomic analyses revealed distinctive differences between the two vascular beds. Coronary vasodilation to acetylcholine was highly susceptible to the risk factors, compared to femoral artery, and was markedly attenuated by AngII exposure and prostaglandin inhibition. Interestingly, coronary prostaglandin synthesis was suppressed in atherogenesis, and elevated coronary capacity to produce prostaglandins following methotrexate administration was associated with ameliorated early coronary endothelial dysfunction and improved cardiovascular survival, featuring markedly reduced lesional myeloid cells. Coronary arteries from patients with hypertension and hypercholesterolemia showed substantially impaired endothelium-dependent vasodilation, compared to internal mammary arteries of similar risk exposure, whereas their endothelium-independent vasodilation was intact. Thus, the combination of hypercholesterolemia and AngII-induced hypertension drastically promoted coronary atherosclerosis with spontaneous lesion rupture, which confers a selective vulnerability of coronary vasodilation to prostaglandin inhibition and AngII exposure.

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.

Involvement of Senescence and Mitochondrial Fission in Endothelial Cell Pro-Inflammatory Phenotype Induced by Angiotensin II.

Angiotensin II (AngII) has a crucial role in cardiovascular pathologies, including endothelial inflammation and premature vascular aging. However, the precise molecular mechanism underlying aging-related endothelial inflammation induced by AngII remains elusive. Here, we have tested a hypothesis in cultured rat aortic endothelial cells (ECs) that the removal of AngII-induced senescent cells, preservation of proteostasis, or inhibition of mitochondrial fission attenuates the pro-inflammatory EC phenotype. AngII stimulation in ECs resulted in cellular senescence assessed by senescence-associated β galactosidase activity. The number of β galactosidase-positive ECs induced by AngII was attenuated by treatment with a senolytic drug ABT737 or the chemical chaperone 4-phenylbutyrate. Monocyte adhesion assay revealed that the pro-inflammatory phenotype in ECs induced by AngII was alleviated by these treatments. AngII stimulation also increased mitochondrial fission in ECs, which was mitigated by mitochondrial division inhibitor-1. Pretreatment with mitochondrial division inhibitor-1 attenuated AngII-induced senescence and monocyte adhesion in ECs. These findings suggest that mitochondrial fission and endoplasmic reticulum stress have causative roles in endothelial senescence-associated inflammatory phenotype induced by AngII exposure, thus providing potential therapeutic targets in age-related cardiovascular diseases.

Angiotensin II triggers RIPK3-MLKL-mediated necroptosis by activating the Fas/FasL signaling pathway in renal tubular cells.

Our earlier studies proved that RIPK3-mediated necroptosis might be an important mode of renal tubular cell death in rats with chronic renal injury and the necroptotic cell death can be triggered by tumor necrosis factor-α (TNF-α) in vitro, but the triggering role of angiotensin II (AngII), which exerts notable effects on renal cells for the initiation and progression of renal tubulointerstitial fibrosis, is largely unknown. Here, we identified the presence of necroptotic cell death in the tubular cells of AngII-induced chronic renal injury and fibrosis mice and assessed the percentage of necroptotic renal tubular cell death with the disruption of this necroptosis by the addition of necrostatin-1 (Nec-1). Furthermore, the observation was further confirmed in HK-2 cells treated with AngII and RIPK1/3 or MLKL inhibitors. The detection of Fas and FasL proteins led us to investigate the contribution of the Fas/FasL signaling pathway to AngII-induced necroptosis. Disruption of FasL decreased the percentage of necroptotic cells, suggesting that Fas and FasL are likely key signal molecules in the necroptosis of HK-2 cells induced by AngII. Our data suggest that AngII exposure might trigger RIPK3-MLKL-mediated necroptosis in renal tubular epithelial cells by activating the Fas/FasL signaling pathway in vivo and in vitro.

Assessment of Cardiac Function of Rbm 20−/− Rats Using Pressure‐Volume Loop Analysis After 28 Days of Angiotensin II Treatment

RNA binding motif 20 (Rbm20), a muscle specific splicing factor for the sarcomeric protein titin, affects diastolic and systolic properties of the heart per the Frank‐Starling mechanism. Failure to alter titin expression from the predominant fetal isoform N2BA to the shorter, adult N2B isoform in Rbm20−/− (KO) rats, results in impaired cardiomyocyte relaxation, increased compliance of the ventricular walls, and dilated cardiomyopathy. These pathologies are similar to those seen in humans with this mutation, which often results in early, sudden death in both species. Angiotensin II (AII) acutely causes vasoconstriction, and chronically results in cardiac hypertrophy, characterized by an increase in cardiac wall stiffness. We hypothesized that 28 days of treatment with AII would increase the stiffness of the myocardium, resulting in improved cardiac function in KO rats. KO rats and age‐matched wild type (WT) controls were fitted with subdermal osmotic pumps, delivering AII continuously (0.25 μL·hr −1) for 28 days. After AII exposure, cardiac function was assessed in vivo using pressure‐volume loop analysis. Under isoflurane anesthesia, loops were recorded under baseline, brief inferior vena cava occlusion, and hypertonic saline infusion (20–40 μL) conditions. The rat was then heparinized, and blood collected for cuvette calibration. No group differences (p<0.05) were found in cardiac output (23.3±0.8 KO; 26.9±2.5 mL·min−1 WT), heart rate (295±21 KO; 341±16 b·min−1 WT), and stroke volume (80.8±9.1 KO; 79.5±7.6 μL WT). However, significant reductions were found in contractility (preload recruitable stroke work; 41.6±10.8 KO; 86.9±8.2 mmHg WT), stroke work (5.13±0.73; 8.13±0.86 mmHg·min WT), and ejection fraction (49.8±4.2 KO; 74.7±7.4 % WT) in KO rats. KO rats were able to maintain stroke volume, despite impaired contractility, due to a significant increase in preload (169±16 KO; 113±7 μL WT) and decrease in afterload (84.4±8.7 KO; 113±9 mmHg WT). We conclude that KO rats failed to improve contractility with 28 days of AII treatment, but maintained normal cardiac output through compensatory changes in preload and afterload.Support or Funding InformationThis project is supported in part by a grant from the National Institute of General Medical Sciences (2P20GM103432) from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Connective tissue growth factor expression after angiotensin II exposure is dependent on transforming growth factor-β signaling via the canonical Smad-dependent pathway in hypertensive induced myocardial fibrosis.

Introduction:Transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) are often described as the initial pro-fibrotic mediators upregulated early in fibrosis models dependent on angiotensin II (Ang-II). In the present study, we explore the mechanistic link between TGF-β and CTGF expression by using a novel TGF-β trap.Materials and methods:NIH/3T3 fibroblasts were subjected to TGF-β with or without TGF-β trap or 1D11 antibody, CTGF or CTGF plus TGF-β for six or 24 hours, and then used for quantitative real-time polymerase chain reaction (qRT-PCR) or immunocytochemistry. Male C57BL/6 mice were infused with Ang-II and randomly assigned TGF-β trap for six or 24 hours. Hearts were harvested for histological analyses, qRT-PCR and western blotting.Results:Exogenous TGF-β-induced fibroblasts resulted in significant upregulation of CTGF, TGF-β and type I collagen transcript levels in vitro. Additionally, TGF-β promoted the differentiation of fibroblasts into α-SMA+ myofibroblasts. CTGF expression was reduced by the addition of TGF-β trap or neutralizing antibody, confirming that its expression is dependent on TGF-β signaling. In contrast, exogenous CTGF did not appear to have an effect on fibroblast production of pro-fibrotic transcripts or fibroblast differentiation. Ang-II infusion in vivo led to a significant increase in TGF-β and CTGF mRNA expression at six and 24 hours with corresponding changes in Smad2 phosphorylation (pSmad2), indicative of increased TGF-β signaling. Ang-II animals that received the TGF-β trap demonstrated reduced CTGF mRNA levels and pSmad2 at six hours, suggesting that early CTGF expression is dependent on TGF-β signaling.Conclusions:We demonstrated that CTGF expression is dependent on TGF-β signaling both in vitro and in vivo in a model of myocardial fibrosis. This also suggests that early myocardial CTGF mRNA expression (six hours) after Ang-II exposure is likely dependent on latent TGF-β activation via the canonical Smad-dependent pathway in resident cardiac cells.

O22-7 - Calmodulin, Which Dissociated from Cardiac Ryanodine Receptor, Plays a Pivotal Role in Driving Pathological Cardiac Hypertrophy

Calmodulin (CaM) binding to cardiac ryanodine receptor (RyR2) (CaM-RyR2) is a critical regulator of SR Ca release. Our recent studies indicated that CaM-RyR2 binding affinity is reduced in pathological conditions. However, the roles of CaM released from RyR2 are unknown. Here, we tested whether RyR2-dissociated CaM play a pivotal role in pathological cardiac hypertrophy. Results: First, we tested whether treatment with angiotensin II (AngII), which stimulates pathologic cardiac hypertrophy, reduced CaM-RyR2 binding and whether CaM moves to nucleus using immunofluorescence. After AngII exposure, there are significant shifts of CaM from RyR2 to nucleus. To further test whether this nuclear CaM accumulation comes from CaM-RyR2, we used dantrolene, which is known to restore the CaM-RyR2 binding affinity, or suramin, which completely deplete CaM from RyR2. Treatment with Dantrolene and AngII dramatically reduced nuclear CaM accumulation vs. AngII alone. On the other hands, suramin significantly increased the nuclear CaM accumulation, suggesting that nuclear CaM accumulation comes from RyR2-CaM. To gain further information about this relation, we measured the kinetics of CaM movement after treatment with AngII. After exposure of AngII, CaM-RyR2 dissociation was faster than nuclear CaM association rate, indicating that CaM-RyR2 dissociation was the trigger for nuclear CaM translocation. Conclusions: Nuclear accumulation of CaM, which came from RyR2-dissociated CaM, may be an important step in driving pathological cardiac hypertrophy.

Protective effect of hydrogen sulphide against myocardial hypertrophy in mice.

Cardiac hypertrophy is a critical component of phenotype in the failing heart. Recently, increasing evidence has demonstrated that oxidative stress plays an important role in the pathogenesis of myocardial hypertrophy. In the present study, we generated a mouse model of transverse aortic constriction (TAC) to investigate whether hydrogen sulfide (H2S) has protective effects against cardiac hypertrophy. Left ventricular structure was analyzed by two-dimensional echocardiography. Oxidative stress was evaluated by measuring malondialdehyde, superoxide dismutase, glutathione peroxidase and reactive oxygen specie in the myocardium. Angiotensin II (Ang-II) was used to induce cardiomyocyte hypertrophy. Neonatal rat cardiomyocytes pretreated with H2S donor sodium hydrosulfide prior to Ang-II exposure were used to determine the involvement of Nrf2 and PI3K/Akt pathway in the antioxidant effects of H2S. Our findings showed that H2S could protect against cardiac hypertrophy by attenuating oxidative stress. The antioxidant roles of H2S in myocardial hypertrophy probably depend on the activation of PI3K/Akt signaling, which consequently increases Nrf2 activity and HO-1 and GCLM expression. In summary, H2S may exert antioxidant effect on cardiac hypertrophy via PI3K/Akt-dependent activation of Nrf2 pathway.

Hydrogen sulfide suppresses angiotensin II-stimulated endothelin-1 generation and subsequent cytotoxicity-induced endoplasmic reticulum stress in endothelial cells via NF-κB.

The effects of hydrogen sulfide (H2S) and the nuclear factor κB (NF-κB) signaling pathway in angiotensinII (AngII)-induced endothelin-1 (ET-1) expression and subsequent cytotoxicity remain unclear. The present study aimed to investigate the hypothesis that H2S protects human umbilical vein endothelial cells (HUVECs) against AngII‑stimulated ET‑1 generation and subsequent cytotoxicity‑induced endoplasmic reticulum stress via the NF‑κB signaling pathway. The results of the present study demonstrated that AngII significantly upregulated the expression levels of ET‑1, glucose‑regulated protein 78, CCAAT‑enhancer‑binding protein homologous protein, phosphorylated (p)‑p65 and inducible nitric oxide synthase; stimulated nitric oxide production; suppressed the expression and activity of cystathionine-γ-lyase (CSE), a H2S synthetase; and decreased cell viability. Conversely, BQ788 (an ET‑1 receptor antagonist) exhibited an inhibitory effect on the AngII‑mediated suppression of CSE expression and activity in HUVECs. The effects of AngII were abrogated by sodium hydrosulfide (NaHS, an H2S donor), BQ788 or pyrrolidinedithiocarbamic acid (PDTC, an inhibitor of NF‑κB). Furthermore, pretreatment with NaHS or PDTC attenuated AngII‑induced apoptosis and cleaved caspase-12 generation. The pretreatment of HUVECs with BQ788 prior to AngII exposure mimicked the inhibitory effect of NaHS on the expression of p‑p65 induced by AngII. In conclusion, the present study provides evidence that exogenous H-2S attenuates AngII‑induced inflammation and cytotoxicity via inhibition of the ET‑1/NF‑κB signaling pathway in HUVECs.

CaV1.2 Interaction with AT1R Reduces Receptor Internalization

The cardiac L type calcium channel is a multi-subunit complex that includes CaV1.2 as the pore-forming subunit that co-assembles with the auxiliary CaVa2d1 subunit and with the CaVb subunit. The main role of these auxiliary subunits is to modify the amount of channels inserted in the plasma membrane. Similarly, trafficking of these channels is controlled by direct interaction with other proteins such as some G-protein coupled receptors (GPCR). Angiotensin II (AngII) type I receptor (AT1R) is a GPCR well known to be involved in the intracellular calcium regulation at the heart, modulating cardiomyocytes contractibility. In this work we show, by co-immunoprecipitation, that AT1R forms a macro-complex with CaV1.2 in primary culture of newborn rat cardiomyocytes and in an heterologous system. Standard biochemical approaches were used to demonstrate that these proteins colocalize at the plasma membrane and to determine the region of CaV1.2 responsible for this interaction. The functional impact of this complex was studied in terms of L-type currents inhibition and in terms of internalization of AT1R. Although inhibition of endogenous L-type current in cardiomyocytes was found to be dependent on the CaVb2 transcriptional start site (TSS) expressed, the presence of the macro-complex was found to be independent on the variant expressed. The effect of this interaction over AT1R internalization was studied in cells stimulated with AngII at different times. As expected, in cell expressing only the AT1R and stimulated with AngII, the receptor is readily internalized after 15 min, however, in cells expressing also CaV1.2, AT1R internalization (and CaV1.2) is observed only after 30 minutes of AngII stimulation. Overall, our results demonstrate that AT1R interaction with CaV1.2 increases the residence time at the plasma membrane of the receptor and induce L-type channel internalization after prolonged AngII exposure.

Abstract 18216: Essential Role of Platelet Glycoprotein Ibα Dependent Thrombin-FXI Feedback Loop in Arterial Hypertension, Vascular Dysfunction and Inflammation

Interactions of platelets, leukocytes and the vessel wall play pivotal roles in activating coagulation and precipitating thrombosis. High levels of angiotensin II (ATII) cause arterial hypertension by a complex inflammatory pathway requiring leukocyte recruitment and reactive oxygen species production within the vessel wall coupled to vascular dysfunction. Here we report an upregulation of tissue factor, thrombin-dependent endothelial cell VCAM-1 expression and integrin α4- and platelet-dependent leukocyte adhesion to arterial conductance vessels by ATII in vivo (1mg/kg/d for 7d). ATII-induced vascular dysfunction and Ccl2, VCAM-1 and Ly6C mRNA expression were ameliorated by thrombin inhibition, platelet-depletion as well as in hIL-4R/Ibα mice missing the extracellular ligand binding domains of GPIbα. Blockade of TF during ATII administration also attenuated vascular dysfunction and reduced vascular oxidative stress. Platelet rich plasma (PRP) of ATII-treated mice showed an increased thrombin evoked endogenous thrombin potential (ETP), whereas PRP from mice with pharmacological inhibition of FXI production or of hIL-4R/Ibα mice failed to amplify ETP following chronic ATII exposure. These data show that a FXI-dependent thrombin generation feedback loop requires GPIbα on platelets and suggest that TF-initiated coagulation promotes additional thrombin formation on platelets to cause vascular inflammation. The therapeutic potential of blocking FXI synthesis via antisense oligonucleotides was generalized by experiments with 5/6 nephrectomized (5/6 Nx) rats. 5/6 Nx rats showed after 4 wk of surgery an endothelial dysfunction, increased blood pressure, vascular inflammation and oxidative stress, as well as a elevated ETP compared to sham whereas pharmacological inhibition of FXI synthesis attenuated these effects. Our results reveal a critical role of platelet GPIbα to promote localized thrombin amplification and supporting a FXI-thrombin feedback loop in ATII-induced vascular inflammation. By using a FXI targeted approach, we propose a novel therapeutic possibility to interrupt this heterotypic cellular coagulation-inflammatory circuit.

ARC Knockout Does Not Alter Apoptotic Enzyme Activities in Skeletal Muscle in Response to Angiotensin II Exposure

Although apoptotic signalling does not cause complete skeletal muscle fiber elimination, apoptotic protease activation contributes to contractile protein degradation. The anti‐apoptotic protein apoptosis repressor with caspase recruitment domain (ARC) inhibits both intrinsic and extrinsic apoptotic pathways. Interestingly, although ARC expression levels are relatively high in mature skeletal muscle, its importance in mediating apoptotic signalling is underexplored. Therefore, the purpose of this study was to examine if ARC knockout (KO) would alter apoptotic enzyme activities in response to angiotensin II (AngII) exposure. Wild‐type (WT) and ARC KO mice were implanted with a subcutaneous placebo or AngII‐releasing pellet. No differences in caspase‐3, ‐8, or ‐9 activity were detected between groups in red gastrocnemius; however, a trend (p=0.07) towards an interaction effect was observed for calpain activity. In white gastrocnemius, there was a significant increase in caspase‐3 activity with AngII treatment; however, no differences were seen between genotypes. Although a trend (p=0.06) towards decreased caspase‐8 activity with AngII treatment was observed, no significant differences were detected. A significant decrease in caspase‐9 activity with AngII treatment was also observed. Finally, an interaction effect was detected for calpain activity. In conclusion, apoptotic enzyme activities are not increased in ARC KO compared to WT mice in response to AngII exposure. This may be due to adaptations occurring during development which may counteract the absence of ARC.

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.

Antibody therapy can enhance AngiotensinII-induced myocardial fibrosis

BackgroundMyocardial fibrosis is a pathological process that is characterized by disrupted regulation of extracellular matrix proteins resulting in permanent scarring of the heart tissue and eventual diastolic heart failure. Pro-fibrotic molecules including transforming growth factor-β and connective tissue growth factor are expressed early in the AngiotensinII (AngII)-induced and other models of myocardial fibrosis. As such, antibody-based therapies against these and other targets are currently under development.ResultsIn the present study, C57Bl/6 mice were subcutaneously implanted with a mini-osmotic pump containing either AngII (2.0 μg/kg/min) or saline control for 3 days in combination with mIgG (1 mg/kg/d) injected through the tail vein. Fibrosis was assessed after picosirius red staining of myocardial cross-sections and was significantly increased after AngII exposure compared to saline control (11.37 ± 1.41%, 4.94 ± 1.15%; P <0.05). Non-specific mIgG treatment (1 mg/kg/d) significantly increased the amount of fibrosis (26.34 ± 3.03%; P <0.01). However, when AngII exposed animals were treated with a Fab fragment of the mIgG or mIgM, this exacerbation of fibrosis was no longer observed (14.49 ± 2.23%; not significantly different from AngII alone).ConclusionsThese data suggest that myocardial fibrosis was increased by the addition of exogenous non-specific antibodies in an Fc-mediated manner. These findings could have substantial impact on the future experimental design of antibody-based therapeutics.

Angiotensin II stimulates sympathetic neurotransmission to adipose tissue

Angiotensin II (AngII) facilitates sympathetic neurotransmission by regulating norepinephrine (NE) synthesis, release, and uptake. These effects of AngII contribute to cardiovascular control. Previous studies in our laboratory demonstrated that chronic AngII infusion decreased body weight of rats. We hypothesized that AngII facilitates sympathetic neurotransmission to adipose tissue and may thereby decrease body weight. The effect of chronic AngII infusion on the NE uptake transporter and NE turnover was examined in metabolic (interscapular brown adipose tissue, ISBAT; epididymal fat, EF) and cardiovascular tissues (left ventricle, LV; kidney) of rats. To examine the uptake transporter saturation isotherms were performed using [3H]nisoxetine (NIS). At doses that lowered body weight, AngII significantly increased ISBAT [3H]NIS binding density. To quantify NE turnover, alpha-methyl-para-tyrosine (AMPT) was injected in saline-infused, AngII-infused, or saline-infused rats that were pair-fed to food intake of AngII-infused rats. AngII significantly increased the rate of NE decline in all tissues compared to saline. The rate of NE decline in EF was increased to a similar extent by AngII and by pair feeding. In rats administered AngII and propranolol, reductions in food and water intake and body weight were eliminated. These data support the hypothesis that AngII facilitates sympathetic neurotransmission to adipose tissue. Increased sympathetic neurotransmission to adipose tissue following AngII exposure is suggested to contribute to reductions in body weight.

289 UPREGULATION OF CAVEOLIN PROTEINS BY ANGIOTENSIN II ENHANCES DETRUSOR CONTRACTION

INTRODUCTION AND OBJECTIVES: Bladder smooth muscle (BSM) cells release Angiotensin II (AngII) in response to mechanical stress and the angiotensin system is upregulated with bladder obstruction. AngII-mediated bladder contractions require intact caveolae, membrane microdomains involved in signal transduction regulation. These microdomains were found to be altered in hypertrophic detrusor muscle from obstructed bladders. This study investigated the effect of AngII on caveolin expression and whether changes in caveolins induced by AngII are associated with functional alterations. METHODS: Rat bladder tissue without mucosa was challenged for 8 hours with exogenous AngII, or with continuous electrical field stimulation (EFS) in the presence or absence of the AngII receptor-1 (AT1R) antagonist losartan (los). Changes in amplitude of contractions in response to treatments were recorded during the 8 hours. The effect of EFS on the release of endogenous AngII was determined by enzyme immunoassay. After stimulation, caveolin gene and protein expression was determined by real time rt-PCR and western blot. The interaction between AT1R and caveolin proteins was investigated by immunoprecipitation. RESULTS: Exposure of BSM tissue to AngII upregulated Cav-1, Cav-2 and Cav-3 gene and protein expression compared to non-stimulated tissue. Caveolin gene expression was similarly upregulated after 8 hours of EFS, but remained unchanged if EFS was delivered in the presence of los. Release of endogenous AngII was increased in EFS stimulated tissue compared to control. Continuous AngII exposure increased the amplitude of spontaneous activity compared to non-stimulated tissue. In addition, the amplitude of EFSinduced contractions was augmented after 8 hours. However, the increase in EFS-induced contractions was prevented by los. AT1R co-precipitated with Cav-1, Cav-2 and Cav-3. CONCLUSIONS: The caveolin protein-AT1R interaction is consistent with the localization of AT1R in caveolae and its regulation by caveolin proteins. The effect of AT1R inhibition on caveolin expression during mechanical perturbations indicates that autocrine/paracrine AngII positively regulates caveolin expression. These findings suggest that endogenous AngII released by BSM initiates a positive feedback response in which AT1R-activated detrusor contractions that are facilitated by caveolae become augmented by an enhanced caveolin-AT1R interaction. These findings may have important implications for pathophysiologic processes associated with altered AngII secretion such as the hypertrophic response to bladder obstruction.

Remodelling of the Cardiac NCX1-TRPC3 Signaling Complex Promotes Angiotensin Ii-Induced Arrhythmogenesis

TRPC3 has been demonstrated as a player in structural remodeling of the heart, while its proarrhythmogenic potential is incompletely understood. using a TRPC3 transgenic overexpression mouse model, we examined the impact of TRPC3 expression on cardiac actions of Angiotensin II (AngII) in whole hearts (Langendorff perfusion) and in isolated single ventricular myocytes. At low TRPC3 expression (WT mice) AngII elicited a previously described 3-phasic inotropic response without generating significant arrhythmias. By contrast, at high TRPC3 expression (TRPC3 transgenic mice; TRPC3-TG), AngII induced substantial impairment of cardiac contractile function associated with prominent bigeminal beats and ventricular tachycardia. Calcium imaging experiments revealed a dramatic increase in diastolic Ca2+ levels associated with spontaneous sarcomere shortenings during AngII exposure of TRPC3-TG myocytes. More detailed electrophysiological characterization demonstrated AngII-induced depolarization, distortion of action potential morphology and enhanced frequency of delayed afterdepolarizations (DADs) in TRPC3-TG. This phenotype was not observed in WT mice. Analysis of NCX-tail currents suggested that AngII-induced arrhythmias were based on profound NCX-1 forward mode-associated depolarization. On the contrary, our analysis of Ca2+ handling in TRPC3-TG myocytes at basal conditions clearly indicated suppressed forward-mode NCX-1 action as expected from enhanced Na+ entry at TRPC3- NCX-1 signalplexes. Immunocytochemistry demonstrated a partial disruption of the tight colocalization of TRPC3 and NCX-1 confined to the T-tubuli system during AngII stimulation of TRPC3-TG myocytes. Our results confirm the concept of a close physical and functional coupling between TRPC3 and NCX-1, which results in a digitalis-like modulation of cardiac function by TRPC3 overexression in non-stimulated myocardium. AngII is suggested to disrupt the organization of TRPC3- NCX-1 signalplexes, thereby enabling NCX-1-mediated distortion of AP morphology, DADs and arrhytmogenesis.

Regulation and Role of Connective Tissue Growth Factor in AngII-Induced Myocardial Fibrosis

Exposure of rodents to angiotensin II (AngII) is a common model of fibrosis. We have previously shown that cellular infiltration of bone marrow-derived progenitor cells (fibrocytes) occurs before deposition of extracellular matrix and is associated with the production of connective tissue growth factor (CTGF). In the present study, we characterized the role of CTGF in promoting fibrocyte accumulation and regulation after AngII exposure. In animals exposed to AngII using osmotic minipumps (2.0 μg/kg per min), myocardial CTGF mRNA peaked at 6 hours (21-fold; P < 0.01), whereas transforming growth factor-β (TGF-β) peaked at 3 days (fivefold; P < 0.05) compared with saline control. Early CTGF expression occurred before fibrocyte migration (1 day) into the myocardium or ECM deposition (3 days). CTGF protein expression was evident by day 3 of AngII exposure and seemed to be localized to resident cells. Isolated cardiomyocytes and microvascular endothelial cells responded to AngII with increased CTGF production (2.1-fold and 2.8-fold, respectively; P < 0.05), which was abolished with the addition of anti-TGF-β neutralizing antibody. The effect of CTGF on isolated fibrocytes suggested a role in fibrocyte proliferation (twofold; P < 0.05) and collagen production (2.3-fold; P < 0.05). In summary, we provide strong evidence that AngII exposure first resulted in Smad2-dependent production of CTGF by resident cells (6hours), well before the accumulation of fibrocytes or TGF-β mRNA up-regulation. In addition, CTGF contributes to fibrocyte proliferation in the myocardium and enhances fibrocyte differentiation into a myofibroblast phenotype responsible for ECM deposition.

369 The Effects of Aging on Myocardial Fibrosis Development Secondary to Angiotensinii Exposure

Myocardial fibrosis is a pathological process that is characterized by excess deposition and lack of clearance of extracellular matrix (ECM) proteins resulting in permanent scarring of the heart tissue and eventual organ dysfunction. There is a direct correlation seen between the progression of heart failure and aging, which is not well understood. Our objective is to characterize the underlying mechanisms of fibrosis development in young and aged mice in order to investigate the effects of age. We exposed young (8wk) and old (40wk) mice to angiotensinII (AngII) or saline for 3d, an established model of hypertension. We assessed mRNA expression and protein production of growth factors in myocardium. We also assessed the degree of cellular infiltrate and fibrosis. At baseline, the myocardium of old mice was more fibrotic when compared to young mice as assessed by % collagen (11.1±1.3%; 4.9±1.2%). As well, old mice had significantly higher baseline mRNA expression of fibrotic factors (CTGF, TGFbeta, PDGF, FGF and Col1a1). After AngII exposure for 3d there was an equal increase in fibrosis observed in old and young myocardium (absolute increase of 5.5±1.3%; 5.5±1.9%), however significant cellular infiltration that occurred in young mice by 1d was not significant in old mice until 3d of AngII exposure. While all fibrotic factors assessed increased in young mice in response to AngII exposure, only CTGF (7.2±2.6fold) increased significantly in old mice after AngII. AngII exposure resulted in a similar increase in myocardial fibrosis in old and young animals. It was therefore surprising that old myocardium had significantly less cellular infiltration and reduced fibrotic factor upregulation after exposure to AngII, compared to that observed in young myocardium. This suggests that older mice have; 1- an altered pathway of ECM regulation, or 2 - different cellular response to stimuli. This study provides novel insights towards understanding the signaling mechanisms involved in myocardial fibrosis development in aged animals.

Abstract P224: Connective Tissue Growth Factor Plays a Key Role in Myocardial Fibrosis After Angiotensin II Exposure

Background: AngiotensinII (AngII) exposure to rodents is a common model of fibrosis, characterized by hypertension, hypertrophy and eventual deposition of excess extracellular matrix (ECM) proteins resulting in organ dysfunction. We have previously shown that cellular infiltration of bone marrow derived progenitor cells (fibrocytes) occurs prior to ECM deposition and is associated with production of the connective tissue growth factor (CTGF) and transforming growth factor beta (TGF-β). The objective of this project was to characterize the role of CTGF in promoting fibrocyte recruitment and subsequent fibrosis after AngII exposure. Methods: Mice were treated with AngII or saline using an osmotic mini-pump at 2.8mg/kg/day. After 6hr to 7d hearts were excised and embedded in paraffin or prepared for mRNA isolation. Immunohistochemistry was used to determine extent and localization of CTGF protein. Quantitative RT-PCR was used to determine relative CTGF and TGF-β mRNA levels. Primary cardiomyocyte and fibrocyte cultures were isolated from neonatal or 3d AngII exposed animals respectively. Primary cultures were stimulated to determine the source (qRT-PCR) and function (proliferation, migration and differentiation) of CTGF. Results: In animals exposed to AngII, CTGF mRNA peaked the earliest at 6hr (21-fold; p&lt;0.01) when compared to TGFbeta, which peaked at 3d (5-fold; p&lt;0.05). Concurrent CTGF protein expression was evident by 3d of AngII exposure and appeared localized to the cardiomyocytes. Findings were confirmed using isolated cardiomyocytes, which significantly increased expression of CTGF in response to AngII (2-fold; p&lt;0.05). While CTGF did not promote fibrocyte migration in transwell chamber assay it promoted significant fibrocyte proliferation in vitro (2-fold; p&lt;0.05). Conclusion: We provide strong evidence that AngII exposure first results in the production of CTGF by cardiomyocytes. Furthermore, we have shown that CTGF does not promote migration as a chemokine but instead contributes to proliferation of fibrocytes once recruited from the bone marrow into the myocardium.