Exogenous Angiotensin II Research Articles

When the liver is in poor condition, so is the heart - cardiac remodelling in MASH mouse models.

Metabolic dysfunction-associated steatohepatitis (MASH) confers a risk for cardiovascular diseases in patients. Animal models may help exploring the mechanisms linking liver and heart diseases. Hence, we explored the cardiac phenotype in two MASH mouse models: foz/foz mice fed a high-fat diet (HFD) for 24 or 60 weeks and C57BL/6J mice fed a high-fat-, high-cholesterol-, and high-fructose diet for 60 weeks. Angiotensin II (AngII) was used as an additional cardiovascular stressor for 4 weeks in 10 weeks HFD-fed foz/foz mice. Foz/foz mice with fibrosing MASH developed cardiac hypertrophy with adverse cardiac remodelling not seen in WT similarly fed the HFD. AngII caused hypertension and up-regulated the expression of genes contributing to pathological cardiac hypertrophy (Nppa, Myh7) more severely so in foz/foz mice than in controls. After 60 weeks of HFD, while liver disease had progressed to burn-out non steatotic MASH with hepatocellular carcinoma in 50% of the animals, the cardiomyopathy did not. In an independent model (C57BL/6J mice fed a fat-, cholesterol- and fructose-rich diet), moderate fibrosing MASH is associated with cardiac fibrosis and dysregulation of genes involved in pathological remodelling (Col1a1, Col3a1, Vim, Myh6, Slc2a1). Thus, animals with MASH present consistent adverse structural changes in the heart with no patent alteration of cardiac function even when stressed with exogenous AngII. Liver disease, and likely not overfeeding or aging alone, is associated with this cardiac phenotype. Our findings support foz/foz mice as suitable for studying links between MASH and heart structural changes ahead of heart failure.

TLR4 and AT1R mediate blood-brain barrier disruption, neuroinflammation, and autonomic dysfunction in spontaneously hypertensive rats

Angiotensin II (AngII) is implicated in neuroinflammation, blood-brain barrier (BBB) disruption, and autonomic dysfunction in hypertension. We have previously shown that exogenous AngII stimulates Toll-like receptor 4 (TLR4) via AngII type 1 receptor (AT1R), inducing activation of hypothalamic microglia ex vivo, and that AngII-AT1R signaling is necessary for the loss of BBB integrity in spontaneously hypertensive rats (SHRs). Herein, we hypothesized that microglial TLR4 and AT1R signaling interactions represent a crucial mechanistic link between AngII-mediated neuroinflammation and BBB disruption, thereby contributing to sympathoexcitation in SHRs. Male SHRs were treated with TAK-242 (TLR4 inhibitor; 2 weeks), Losartan (AT1R inhibitor; 4 weeks), or vehicle, and age-matched to control Wistar Kyoto rats (WKYs). TLR4 and AT1R inhibitions normalized increased TLR4, interleukin-6, and tumor necrosis factor-α protein densities in SHR cardioregulatory nuclei (hypothalamic paraventricular nucleus [PVN], rostral ventrolateral medulla [RVLM], and nucleus tractus solitarius [NTS]), and abolished enhanced microglial activation. PVN, RVLM, and NTS BBB permeability analyses revealed complete restoration after TAK-242 treatment, whereas SHRs presented with elevated dye leakage. Mean arterial pressure was normalized in Losartan-treated SHRs, and attenuated with TLR4 inhibition. In conscious assessments, TLR4 blockade rescued SHR baroreflex sensitivity to vasoactive drugs, and reduced the SHR pressor response to ganglionic blockade to normal levels. These data suggest that TLR4 activation plays a substantial role in mediating a feed-forward pro-hypertensive cycle involving BBB disruption, neuroinflammation, and autonomic dysfunction, and that TLR4-specific therapeutic interventions may represent viable alternatives in the treatment of hypertension.

OR04-5 Stimulation of Protein Disulfide Isomerase Activity by Activation of The Renin-Angiotensin System

Understanding the molecular and cellular mechanisms that are affected by activation of the renin-angiotensin system (RAS) are important for development of novel therapeutic strategies to more specifically treat hypertension and reduce cardiovascular risk. RAS regulates blood pressure (BP) in part via its effector molecule, angiotensin II (AngII), a potent vasopressor that stimulates the production of reactive oxygen species (ROS). Protein disulfide isomerase (PDI) is a multifunctional oxidoreductase that catalyzes thiol/disulfide interchange reactions within the endoplasmic reticulum and regulates ROS production. Also, PDI is present in circulation and at the cell surface of platelets, lymphocytes, erythrocytes and endothelial cells and plays a critical role in initiating thrombus formation. However, the interaction of RAS and PDI remain unclear. We hypothesized that RAS activation would lead to increased PDI levels thus contributing to oxidative stress. First, we studied the in vitro effects of AngII on EA.hy926, a human endothelial cell line, and measured PDI activity. Our results show that AngII dose and time dependently increased extracellular PDI activity (p<0.05, n=6); an event that was reduced to baseline levels by preincubation with 0.5 uM losartan, an AngII type 1 receptor antagonist (ARB). Consistent with these results, AngII stimulated: 1) a 3-fold rise of extracellular PDI levels as determined by ELISA (p<0.05, n=4); 2) a 2.5-fold increase in PDI mRNA levels by RT-PCR (p<0.05, n=4); and 3) increased PDI protein levels by western blot analyses that were blocked by losartan. We also studied the effect of AngII on extracellular PDI activity in differentiated HL-60 cells, a human polymorphonuclear neutrophil cell line, and observed significantly increased extracellular PDI activity following incubation with AngII (p<0.05, n=4). We then characterized the in vivo effects of exogenous AngII infusion on PDI in Sprague-Dawley rats, a model of increased BP, inflammation and target organ damage. We studied three conditions: (1) control; (2) AngII infused (80 ng/min x 28 days); (3) AngII + ARB (10 mg losartan/kg/day x 21 days). AngII infusion led to significant increases in plasma PDI levels that were reduced by 20% following ARB treatment (p<0.05, n=5/group). We then studied a model of naturally increased AngII and hypertension, the Otsuka Long Evans Tokushima Fatty rats (OLETF) and their strain controls that were randomly assigned to the following groups: (1) untreated strain controls; (2) untreated OLETF; (3) OLETF + ARB. OLETF rats had increased BP and significantly greater circulating PDI activity than control rats that was likewise blocked by ARB treatment (p<0.05; n=6/group). Thus, RAS activation represents a novel mechanism for increased PDI levels and activity that may contribute to the deleterious effects of disordered RAS in cardiovascular disease.

Arcuate Nucleus Angiotensin II Increases Arterial Pressure and Sympathetic Nerve Activity in Part via Inhibition of Neuropeptide Y Projections to the Hypothalamic Paraventricular Nucleus

Angiotensin II (AngII) acts centrally to increase arterial pressure (AP) and sympathetic nerve activity (SNA) mainly via AngII type 1 receptors (AT1R). Multiple brain areas have been identified as targets of AngII, including the arcuate nucleus (ArcN). However, the neural pathway that mediates AngII actions in the ArcN remains unknown. Recent studies suggest that AT1R expression in the ArcN is restricted to Neuropeptide Y (NPY) neurons, which project to the hypothalamic paraventricular nucleus (PVN). Therefore, we hypothesized that, in the ArcN, AngII increases AP and SNA in part by suppressing the activity of tonically sympathoinhibitory NPY neurons that innervate the PVN. To test this hypothesis, in α‐chloralose anesthetized male SD rats, we determined if blockade of PVN NPY Y1 receptors (Y1R) with bilateral nanoinjections of BIBO3304 (60 pmol/60nL) elicits smaller increases in AP and SNA 60 min after nanoinjection of exogenous AngII into the ArcN, compared to 60 min after ArcN injections of artificial cerebrospinal fluid (aCSF). As we reported before, after ArcN aCSF, blockade of PVN Y1R increased (n=3; all P&lt;0.05) mean AP (MAP, 99±4 to 111± 4 mmHg), lumbar SNA (LSNA, 99±9 to 130±12 % control) and splanchnic SNA (SSNA, 105±9 to 142±10 % control). Bilateral nanoinjection of AngII into the ArcN slowly increased (n=4; all P&lt;0.05) MAP (101±4 to 116±3 mmHg), LSNA (100 to 153±10 % control) and SSNA (100 to 151±6 % control). However, 60 min after ArcN AngII, blockade of PVN Y1R did not further increase MAP (to 122±2 mmHg), LSNA (to 174±17 % control), and SSNA (to 176±22 % control), compared to the changes observed after PVN aCSF injections (ArcN AngII+PVN aCSF: n=5; MAP, 120±4 to 123±3 mmHg; LSNA, 160±20 to 174±22 % control; SSNA, 172±21 to 193±20 % control). In summary, ArcN injections of AngII markedly attenuated the pressor and sympathoexcitatory effects of blockade of PVN NPY Y1R. Therefore, we conclude that ArcN AngII increases MAP, LSNA, and SSNA, in part, via inhibition of NPY neuronal projections to the PVN.Support or Funding InformationSupported in part by NIH HL128181.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 257: Activation of the Renin-Angiotensin II-Aldosterone-System Leads to Increase in Protein Disulfide Isomerase

Activation of the Renin-Angiotensin-Aldosterone-System (RAAS) is proposed to play a role in the development of insulin resistance and type II diabetes. Angiotensin II (AngII) is a principal effector molecule that binds AT1 receptors (AT1R) in various tissues. Recent data shows that activated endothelial cells secrete protein disulfide isomerase (PDI), a multifunctional protein that has been shown to be critical to the initiation and regulation of thrombus formation. However, the effects of RAAS on PDI regulation are unknown. We hypothesized that RAAS activation would lead to increased PDI levels thus contributing to inflammation. First, we studied the in vitro effects of AngII on EA.hy926 human endothelial cells and measured PDI activity. Our results show that AngII increased PDI activity; an event that was blocked by preincubation with 0.5 M losartan, an AT1R antagonist (ARB) (P&lt;0.05, n=6). We then studied the in vivo effects of exogenous AngII infusion in Sprague–Dawley rats under the following conditions: (1) control; (2) AngII infused; (3) AngII + ARB. In these rats, AngII infusion led to significant increases in plasma PDI levels that were partially prevented by ARB treatment (P&lt;0.05, n&gt;5). We then studied the obese Otsuka Long Evans Tokushima Fatty (OLETF) (n = 6/group) rats, a model of naturally increased AngII and RAAS mediated insulin resistance and hypertension. OLETF and their lean strain controls were randomly assigned to the following groups: (1) untreated Long Evans Tokushima Otsuka (lean, control); (2) untreated OLETF; (3) OLETF + ARB. Our results show that OLETF rats had increased insulin resistance and significantly greater circulating PDI activity than control rats (P&lt;0.05) that was likewise blocked by ARB treatment (P&lt;0.05). To assess the relevance to humans of these findings we measured circulating PDI levels in patients with type 2 diabetes. In our cohort, PDI activity was significantly greater in patients with type 2 diabetes than non diabetics (P&lt;0.001, n=134). Our data suggest that RAAS activation represents a novel mechanism for PDI secretion. Thus we posit that PDI may contribute to the deleterious effects of RAAS-mediated vascular disease.

Angiotensin receptor 1 blockade reduces secretion of inflammation associated cytokines from cultured human carotid atheroma and vascular cells in association with reduced extracellular signal regulated kinase expression and activation

BackgroundA number of studies have suggested that angiotensin II (AII) receptor type 1 (ATR1) blocking drugs (ARBs) have anti-inflammatory effects however the mechanisms responsible are poorly investigated. ObjectiveTo determine the role of extracellular signal regulated kinase (ERK)1/2 in ARB induced anti-inflammatory effects within human carotid atherosclerosis. MethodsAtheroma samples obtained from patients undergoing carotid endarterectomy were cultured with and without ATR1 (irbesartan), ERK1/2 (PD98059), AII ([Sar1, Ile8]-AII) and angiotensin converting enzyme (ACE)2 (DX600) blockade. The in vitro effects of ATR1 and ERK1/2 blockade and exogenous AII on serum stimulated healthy, primary vascular cells were also investigated. Outcome was assessed by measuring cytokine, (interleukin (IL)-6, IL-8, C–C motif chemokine (CCL)2, C-X-C motif chemokine (CXCL)5, osteoprotegerin (OPG), osteopontin (OPN), CXCL16), concentrations in supernatants and phosphorylated ERK1/2 in the tissue lysates using ELISA. ERK1/2 expression in the tissue was assessed using Western blotting. ResultsIrbesartan reduced concentrations of IL-6, IL-8, CCL2, CXCL5, OPG, OPN and CXCL16 in both atheroma and primary vascular cell culture supernatants. The reduction in cytokine levels in the atheroma supernatant was correlated to a reduction in ERK1/2 expression in the tissue. Inhibition of ERK1/2 downregulated IL-6, IL-8 and CXCL5 in both atheroma and cell culture supernatants. AII and ACE2 blockade had no impact on cytokine or active ERK1/2 levels in the atheroma culture. ConclusionOur findings suggest that ATR1 blockade downregulates atheroma tissue ERK1/2 expression leading to a reduction in cytokine production and that a non-AII agonist ATR1 signalling response may induce expression of these inflammation associated cytokines in the atheroma.

Angiotensin II (AngII) is rapidly metabolized in neuronal cell culture media as determined by liquid chromatography‐tandem mass spectrometry (LC MS/MS)

Previous reports using AngII‐sensitive neuronal cell culture models suggest that AngII induces chronic changes in various cell signaling proteins (e.g. kinases &amp; transcription factors). Many of these studies stimulated cultured neurons with a single administration of exogenous AngII and harvested neurons 1–3 days later to investigate changes in protein levels. One limitation of such “chronic” AngII stimulation studies is the lack of evidence indicating the length of time exogenous AngII remains in neuronal cell culture media. Herein, using LC MS/MS, we tested the hypothesis that exogenous AngII is quickly metabolized to other angiotensin peptides in neuronal cell culture media. AngII (104.62pg/μl) was added to cultured CATH.a neurons and media was collected 15 min – 24 hr later for LC MS/MS analysis. Media levels of AngII rapidly and significantly decreased over time (52.8pg/μl AngII at 15 min; 4.2pg/ul at 3 hr; 0.37pg/μl at 24 hr, p&lt;0.05, n = 3–4). At early time points (15 min – 3 hr), levels of AngIII and Ang1–7, two peptides generated from AngII, increased with a subsequent decrease at later times. Levels of AngI and AngIV did not significantly change. These studies provide new insight into the half‐life and metabolism of AngII in neuronal cell culture media and suggest previous studies using a single administration of AngII to study “chronic” AngII intra‐neuronal signaling pathways be reevaluated. NIH R01HL103942

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.

Mesangial cells are responsible for orchestrating the renal podocytes injury in the context of malignant hypertension

Two populations of renal cells fully possess functional contractile cell apparatus: mesangial cells and podocytes. Previous studies demonstrated that in the context of malignant hypertension overproduction of Angiotensin-II by the contracting mesangial cells aggravated hypercellularity and apoptosis of adjacent cell populations. The role of podocytes in pathogenesis of malignant hypertension is unclear. We investigated responsiveness of normal vs. hyperglycaemic podocytes to pressure in a model of malignant hypertension. Rat renal podocytes and mesangial cells were subjected to high hydrostatic pressure, using an in vitro model of malignant hypertension. Part of them was pre-exposed to hyperglycaemic medium. Alternatively, the cells were cultured in conditioned medium collected from mesangial cells pre-exposed to pressure. Angiotensin-II was significantly increased in normoglycaemic mesangial cells subjected to pressure, triggering enhanced proliferation and apoptosis. No augmented Angiotensin-II, proliferation or apoptosis were evident in pressure-exposed normoglycaemic podocytes. In hyperglycaemic mesangial cells, but not podocytes, basal Angiotensin-II and apoptosis were augmented, along with abrogated proliferation. Challenge with exogenous Angiotensin-II or Angiotensin-II-containing conditioned medium, induced apoptosis both in podocytes and mesangial cells. 1. Unlike mesangial cells, podocytes do not respond to high pressure or hyperglycaemia per se. Resultantly, neither high pressure nor hyperglycaemia, trigger apoptosis of podocytes in vitro. However, surplus of Angiotensin-II, amply produced in vivo by the adjacent mesangial cells, would seem to be sufficient for initiating apoptosis of both mesangial cells and podocytes. 2. Hyperglycaemia abrogates cell replication. Resultantly, in diabetic patients regeneration of renal tissue damaged by the incidence of malignant hypertension may become compromised or completely lost.

Important role of the angiotensin II pathway in producing matrix metalloproteinase-9 in human thoracic aortic aneurysms

BackgroundThe precise pathologic mechanisms underlying human thoracic aortic aneurysms (TAAs) remain uncertain, except that matrix metalloproteinase-9 (MMP-9) is considered a key enzyme for the degradation of extracellular matrix in aneurysm walls. The aim of this study was to elucidate the significance of the angiotensin II (AngII) pathway to MMP-9 production in human TAA walls. Methods and resultsWe examined the activation of Smad2, a common downstream molecule of AngII and transforming growth factor β (TGF-β) pathways, and the expression of MMP-9 in human nonsyndromic TAA walls. We observed significant increases in Smad2 activation and MMP-9 expression, associated with disruption of elastic lamellae. Using human TAA walls in ex vivo culture, we investigated whether AngII and/or TGF-β pathways are essential for MMP-9 production. Unexpectedly, TGF-β receptor inhibitor had no effect on MMP-9 production. We used PD98059, an inhibitor of extracellular signal–regulated kinase (ERK) activation, and demonstrated that PD98059 dramatically reduced MMP-9 production with attenuation of Smad2 activation. Moreover, exogenous AngII resulted in increases in Smad2 activation and MMP-9 production, in an ERK-dependent manner. ConclusionOur findings indicate that the AngII/ERK pathway has an important role in the production of MMP-9 in human nonsyndromic TAA walls.

Effect of angiotensin II on expression of alveolar epithelial sodium channel in rat

To research the effect of exogenous angiotensin II (AngII) on alveolar fluid clearance (AFC) and alveolar epithelial sodium channel (ENaC) expression in rats. Fifteen healthy Sprague-Dawley (SD) rats were randomly divided into control group, AngII group and AngII type 1 (AT1) receptor blocker ZD7155 pretreatment group, with 5 rats in each group. Exogenous AngII 10 μg× kg(-1)×min(-1)was administered by micro pump via catheter in left jugular vein in AngII group and ZD7155 pretreatment group, whereas control group rats received only normal saline. ZD 7155 10 mg/kg was injected intraperitoneal 30 minutes before administration of exogenous AngII in ZD7155 pretreatment group. The pathological changes in lung were observed after 6 hours. AFC was estimated by Evans-blue labeled 5% albumin. The mRNA and protein expression of ENaC were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. AFC in AngII group was significantly lower than that of control group [(6.16 ± 3.01)% vs. (16.10 ± 3.46)%, P<0.01], and AFC in ZD7155 pretreatment group was significantly higher than that of AngII group [(10.60 ± 2.05)% vs. (6.16 ± 3.01)%, P<0.05]. α-ENaC mRNA expression was significantly increased in AngII group compared with control group (0.663 ± 0.068 vs. 0.236 ± 0.030, P<0.01), but significantly decreased in ZD7155 pretreatment group when compared with AngII group (0.386 ± 0.061 vs. 0.663 ± 0.068, P<0.01). There was no significant difference in β-ENaC and γ-ENaC mRNA expression among three groups. Compared with control group, α-ENaC protein was significantly increased in AngII group (0.343 ± 0.053 vs. 0.145 ± 0.030, P<0.01), but β-ENaC and γ-ENaC proteins were significantly decreased (β-ENaC: 0.286 ± 0.038 vs. 0.512 ± 0.055, γ-ENaC : 0.144 ± 0.040 vs. 0.460 ± 0.066, both P<0.01). Compared with AngII group, α-ENaC protein was significantly lower(0.228 ± 0.045 vs.0.343 ± 0.053, P<0.01), whereas β-ENaC and γ-ENaC proteins were significantly higher (β-ENaC: 0.358 ± 0.043 vs. 0.286 ± 0.038, γ-ENaC: 0.220 ± 0.033 vs. 0.144 ± 0.040, both P<0.05) in ZD7155 pretreatment group. Exogenous AngII modulates ENaC expression of gene and protein by AT1 receptor pathway, attenuates AFC, and aggravates lung edema.

Contribution of Bradykinin B2 Receptors to the Inhibition by Valsartan of Systemic and Renal Effects of Exogenous Angiotensin II in Salt-Repleted Humans

To investigate whether bradykinin (BK) participates in the inhibition of renal effects of exogenous angiotensin II (AngII) by AngII type 1 receptor (AT1R) blockade, eight salt-repleted volunteers underwent four p-aminohippurate- and inulin-based renal studies of AngII infusion at increasing rates of 0.625, 1.25, and 2.5 ng.kg.min(-1) for 30 min. Studies 1 and 2 were preceded by 3 days of placebo, whereas studies 3 and 4 used 240 to 320 mg.day(-1) valsartan. Bradykinin B2-type receptor (BKB2R) antagonist icatibant (50 mug.kg(-1)) was coinfused in studies 2 and 4. Mean blood pressure (MBP), glomerular filtration rate (GFR), renal blood flow (RBF), and renal sodium excretion (UNaV) were measured. In study 1, MBP rose by 12.8%, UNaV decreased by 68%, and GFR and RBF also fell (p < 0.001 for all). In study 2, GFR and RBF fell as in study 1, but the rise in MBP and the fall in UNaV were accentuated [+20.0%, analysis of variance (ANOVA), p < 0.02 versus study 1 and -80.0%, p < 0.05, respectively]. In study 3, AngII had no effects, and in study 4, renal hemodynamics remained unaffected, but MBP still rose and UNaV fell (ANOVA, p < 0.02 and 0.005 versus study 3, respectively). Icatibant accentuated AngII-induced changes in MBP and UNaV. Previous AT1R blockade prevented any systemic and renal effects of AngII, but significant changes in MBP and UNaV still followed AngII plus icatibant even after AT1R blockade. BK, through BKB2Rs, participates in the inhibitory action of AT1R blockers toward actions of exogenous AngII on MBP and UNaV in healthy humans.

Regulation of resistin by cyclic mechanical stretch in cultured rat vascular smooth muscle cells

Resistin has a potential role in atherosclerosis; however, the molecular mechanism underlying the increase in resistin expression in atherosclerosis remains unclear. As mechanical stretch plays an important role in atherosclerosis, in the present study we sought to investigate the cellular and molecular mechanisms underlying the regulation of resistin by cyclic mechanical stretch in VSMCs (vascular smooth muscle cells). VSMCs from thoracic aorta of adult Wistar rats were cultured and subjected to cyclic stretch. Cyclic mechanical stretch significantly increased resistin protein and mRNA expression as compared with control cells without stretch. The specific p38 MAPK (mitogen-activated protein kinase) inhibitor SB203580, the antioxidant N-acetylcysteine and p38 MAPK siRNA (small interfering RNA) attenuated the induction of resistin protein by cyclic stretch. Cyclic stretch significantly increased the phosphorylation of p38 MAPK, whereas pre-treatment with SB203580 and N-acetylcysteine significantly inhibited this effect. Cyclic stretch significantly increased ROS (reactive oxygen species) production, and pre-treatment with N-acetylcysteine significantly inhibited stretch-induced ROS production. Cyclic stretch also increased STAT3 (signal transducer and activator of transcription 3)-binding activity and resistin promoter activity, and resistin promoter activity was abolished when STAT3 in the promoter area was mutated. Pre-treatment with SB203580 and N-acetylcysteine significantly attenuated resistin promoter activity induced by cyclic stretch. Cyclic stretch increased the secretion of AngII (angiotensin II) and resistin from cultured VSMCs. Exogenous AngII increased resistin expression, and AngII receptor inhibition attenuated this effect. In conclusion, cyclic mechanical stretch increases resistin expression in cultured rat VSMCs. Stretch-induced resistin expression is mediated through ROS, and the p38 MAPK and STAT3 pathways. Therefore resistin induced by cyclic stretch may contribute to the pathogenesis of atherosclerosis under haemodynamic overload.Key

Effects of ANP upon ion pump activity and gene expression in aortic smooth muscle cells from spontaneously hypertensive rats

To explore the effects of atrial natriuretic peptide (ANP) upon the activities of Na(+), K(+)-ATPase, Ca(2+)-ATPase and mRNA expression levels of Na(+), K(+)-ATPase alpha(1)-subunit and plasma membrane Ca(2+)-ATPase isoform 1 (PMCA1) in cultured thoracic aortic vascular smooth muscle cells (ASMCs) isolated from spontaneously hypertensive rats (SHR). ASMCs isolated from 14-week-old male SHR and Wistar-Kyoto (WKY) rats were interference-cultured in different doses of ANP and Angiotensin II (AngII). The contents of ANP and AngII in supernatant from ASMCs were measured by radioimmunoassay. The activities of the above two ATPases were measured by biochemistry and enzymology. RT-PCR assay was employed to determine the relative levels of Na(+), K(+)-ATPase alpha(1)-subunit and PMCA1 mRNA in ASMCs. The ANP level of supernatant in SHR ASMCs was significantly lower than those from WKY control [(7.3 +/- 2.4) pg x 10(-6) cells vs (19.3 +/- 3.3) pg x 10(-6) cells, P < 0.01] while the content of AngII in SHR ASMCs was significantly higher than those from WKY control [(57 +/- 4) pg x 10(-6) cells vs (44 +/- 4) pg x 10(-6) cells, P < 0.01]. The activity of Na(+), K(+)-ATPase [(4.3 +/- 0.8) micromol x h(-1) x mg(-1) vs (5.3 +/- 1.0) micromol x h(-1) x mg(-1)], Ca(2+)-ATPase [(3.2 +/- 0.7) micromol x h(-1) x mg(-1) vs (4.5 +/- 0.7) micromol x h(-1) x mg(-1)] in ASMCs from SHR were significantly lower than those from WKY control (both P < 0.01). The mRNA expression of Na(+), K(+)-ATPase alpha(1)-subunit (0.524 +/- 0.025 vs 0.704 +/- 0.116), PMCA1 (0.193 +/- 0.030 vs 0.547 +/- 0.045) significantly decreased in ASMCs from SHR versus the WKY control (both P < 0.01). As compared with SHR control, exogenous ANP improved obviously the activities of Na(+), K(+)-ATPase, Ca(2+)-ATPase and expression of alpha(1)-subunit, PMCA1 mRNA in a does-dependent manner (P < 0.05-P < 0.01). Exogenous AngII (1 x 10(-9), 1 x 10(-8), 1 x 10(-7) mol/L) significantly repressed activities of Ca(2+)-ATPase and attenuated the expression of PMCA1 mRNA (P < 0.05-P < 0.01). Only AngII (1 x 10(-7) mol/L) significantly inhibited the activity of Na(+), K(+)-ATPase and attenuated the expression of Na(+), K(+)-ATPase alpha(1)-subunit mRNA (both P < 0.05). ANP antagonized the effects of AngII (1 x 10(-7) mol/L) upon the activities of two ATPases and the expression of Na(+), K(+)-ATPase alpha(1)-subunit PMCA1 mRNA (P < 0.05-P < 0.01). AngII (1 x 10(-7) mol/L) increased the Na(+), K(+)-ATPase activity and the expression of Na(+), K(+)-ATPase alpha(1)-subunit mRNA, repressed the Ca(2+)-ATPase activity and the expression of PMCA1 mRNA in ASMCs from WKY rat (P < 0.05-P < 0.01). ANP antagonized the effects of AngII (1 x 10(-7) mol/L) upon the activity of Ca(2+)-ATPase and the expression of PMCA1 mRNA (P < 0.05-P < 0.01), but did not antagonize the effects of AngII (1 x 10(-7) mol/L) upon the activity of Na(+), K(+)-ATPase and the expression of alpha(1)-subunit mRNA in ASMCs from WKY rats (P > 0.05). The decreased activities of Na(+), K(+)-ATPase and Ca(2+)-ATPase may be related to the abnormal autocrine of ANP and AngII in ASMC of SHR. ANP can antagonize the effects of AngII upon the activities of two ATPases and the expression of Na(+), K(+)-ATPase alpha(1)-subunit PMCA1 mRNA.

Sympathoexcitatory Response to Exogenous Angiotensin II in the Rostral Ventrolateral Medulla Depends on Local Iontotropic Glutamate Receptors

Glutamate and angiotensin II (AngII) regulate the excitability of sympathoexcitatory neurons in the rostral ventrolateral medulla (RVLM). However, limited evidence is available regarding whether these two neurotransmitters interact within the RVLM to regulate sympathetic nerve activity (SNA) and arterial blood pressure (ABP). Therefore, the present study examined whether blockade of either iontotropic glutamate or AngII‐type 1 receptors modulated the sympathoexcitatory response to exogenous AngII or glutamate, respectively. In urethane‐chloralose anesthetized rats, unilateral injection (60nL) of the AT1R antagonist losartan (1nmol, n=3) into the RVLM did not affect the increase in renal SNA (114±12 vs 97±14%) and mean ABP (27±2 vs 24±1 mmHg) to 1nmol glutamate. Losartan did eliminate the increase in renal SNA (50±6 vs 0±0%) and ABP (20±2 vs 0±0 mmHg) evoked by 60pmol AngII. In marked contrast, unilateral injection of the iontotropic glutamate receptor antagonist kynurenic acid (3nmol, n=6) into the RVLM eliminated the AngII‐evoked increase in renal SNA (50±6 vs 1±1%) and mean ABP (24±1 vs 0±1mmHg) but not the sympathoexictatory response to 1nmol carbachol (renal SNA: 45±2%; mean ABP: 32±2mmHg, n=3). These findings suggest that iontotropic glutamate receptors mediate the sympathoexcitatory response to exogenously applied AngII in the RVLM. Supported by NIH HL090826, AHA 0630202N, AHA0815372D

Renal Response to Angiotensin II is Blunted in Sodium-sensitive Normotensive Men

In hypertension, sodium sensitivity (SS) of blood pressure is associated with renal hemodynamic abnormalities related to increased activity of the renal renin-angiotensin aldosterone system (RAAS). The renal mechanisms of SS in normotensives are unknown. Therefore, we studied whether SS is related to renal hemodynamics and renal responsiveness to angiotensin II (AngII) in young healthy adults. Blood pressure (mean arterial pressure (MAP)) and renal function were measured in 34 healthy men after 1-week low-sodium diet (LS; 50 mmol Na(+)/24 h), 1-week high-sodium diet (HS; 200 mmol Na(+)/24h), and 1-week HS-ACEi (enalapril 20 mg/day). The responses of effective renal plasma flow (ERPF; (131)I-Hippuran clearance) to graded infusion of AngII were assessed during each condition. The sodium-induced change in MAP ranged from -7 to +14 mm Hg. SS (a sodium-induced increase in MAP >3 mm Hg) was present in 13 subjects. ERPF was lower in SS subjects during LS and during HS-ACEi. The AngII-induced decrease in ERPF was blunted in SS on LS (-25 +/- 6 vs. -29 +/- 7% in sodium-resistant (SR) subjects, P < 0.05) and on HS (-30 +/- 5 vs. -35 +/- 6%, P < 0.05). The blunting was corrected by angiotensin-converting enzyme inhibitors (ACEi) (-36 +/- 6 vs. -37 +/- 7%). SS normotensive subjects have a blunted renal response to exogenous AngII. This is ameliorated by ACEi, supporting a role for inappropriately high intrarenal RAAS activity. As these findings cannot be attributed to subclinical renal hypertensive damage, high intrarenal RAAS activity and altered renal hemodynamics may be primary phenomena underlying SS.

Angiotensin II Type 2 Receptor Provides an Endogenous Brake During Inflammation-Induced Microvascular Fluid Leak

The dual actions of angiotensin II (AngII) on microvascular fluid leak remain enigmatic. Our hypothesis was that the AngII type 2 (AT2) receptor decreases microvascular fluid leak during inflammation. The purposes of this study were to determine the activity of the AT2 receptor during stimulation by endogenous AngII, during stimulation by exogenous AngII, and during inflammation. Hydraulic permeability (L(p)) of rat mesenteric venules was measured using a microcannulation technique. L(p) was measured during perfusion with the AT1 receptor antagonist, ZD7155, and also with exogenous AngII during AngII type 1 receptor (AT1) blockade. Inflammation was induced with platelet activating factor (PAF), and L(p) was measured during perfusion of AngII with AT1 blockade and also with an AT2 receptor agonist, CGP42112. AT2 receptor activation by endogenous AngII slightly decreased L(p) over that of the control (p=0.02). Exogenous AngII increased L(p) fivefold (L(p)=4.83+/-1.32; p < 0.001). Addition of AT1 receptor blockade decreased L(p) by 74% (to 1.24+/-0.03; p < 0.01). PAF activation increased L(p) fourfold (L(p)=4.49+/-0.74; p < 0.0001). After PAF activation, exogenous AngII then decreased L(p) by 39% (to 2.74+/-0.12; p < 0.01). Exogenous AngII during AT1 receptor blockade after PAF activation decreased L(p) by 61% (from 4.49+/-0.74 to 1.77+/-0.22; p < 0.0001), and selective AT2 receptor stimulation after PAF activation decreased L(p) by 69% (from 4.49+/-0.74 to 1.40+/-0.04; p < 0.001). This study further supports a dual role for AngII. AngII increases microvascular fluid leak during basal conditions but appears to decrease it during inflammation. Alterations in AT2 receptor activity may be responsible for these different effects.

6A3-5/Osa2 is an Early Activated Gene Implicated in the Control of Vascular Smooth Muscle Cell Functions

Vascular smooth muscle cells (VSMC) growth plays a key role in the pathophysiology of vascular diseases. However, the molecular mechanisms controlling gene transcription in VSMC remain poorly understood. We previously identified, by differential display, a new gene (6A3-5) overexpressed in proliferating rat VSMC. In this study, we have cloned the full-length cDNA by screening a rat foetal brain cDNA library and investigated its functions. The 6A3-5 protein shows 4 putative conserved functional motifs: a DNA binding domain called ARID (AT-rich interaction domain), two recently described motifs (Osa Homology Domain), and a nuclear localization signal. The deduced protein sequence was observed to be 85% identical to the recently described human Osa2 gene. Immunolabelling, using an anti-6A3-5/Osa2 monoclonal antibody, showed a nuclear localization of the 6A3-5/Osa2 protein. In addition, PDGF upregulated 6A3-5/Osa2 expression at both the transcript and protein levels in a dose and time-dependent fashion. The pattern of upregulation by PDGF was reminiscent of the early responsive gene c-fos. The PDGF-induced upregulation of 6A3-5/Osa2 and proliferation of VSMC were significantly inhibited in a dose and sequence-dependent fashion by an antisense, but not by sense, scrambled or mismatched oligonucleotides directed against 6A3-5/Osa2. In VSMC of aortas derived from hypertensive (LH) rats, 6A3-5/Osa2 is overexpressed as compared to that in normotensive (LL) rats. The 6A3-5/Osa2-gene expression is downregulated by an ACE inhibitor and upregulated by exogenous AngiotensinII in LH rats. In summary, these results indicate that 6A3-5/Osa2 is an early activated gene that belongs to a new family of proteins involved in the control of VSMC growth.

Angiotensin II (AT1) receptors and NADPH oxidase regulate Cl- current elicited by beta1 integrin stretch in rabbit ventricular myocytes.

Direct stretch of β1 integrin activates an outwardly rectifying, tamoxifen-sensitive Cl− current (Cl− SAC) via focal adhesion kinase (FAK) and/or Src. The characteristics of Cl− SAC resemble those of the volume-sensitive Cl− current, ICl,swell. Because myocyte stretch releases angiotensin II (AngII), which binds AT1 receptors (AT1R) and stimulates FAK and Src in an autocrine-paracrine loop, we tested whether AT1R and their downstream signaling cascade participate in mechanotransduction. Paramagnetic beads coated with mAb for β1-integrin were applied to myocytes and pulled upward with an electromagnet while recording whole-cell anion current. Losartan (5 μM), an AT1R competitive antagonist, blocked Cl− SAC but did not significantly alter the background Cl− current in the absence of integrin stretch. AT1R signaling is mediated largely by H2O2 produced from superoxide generated by sarcolemmal NADPH oxidase. Diphenyleneiodonium (DPI, 60 μM), a potent NADPH oxidase inhibitor, rapidly and completely blocked both Cl− SAC elicited by stretch and the background Cl− current. A structurally unrelated NADPH oxidase inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF, 0.5 and 2 mM), also rapidly and completely blocked Cl− SAC as well as a large fraction of the background Cl− current. With continuing integrin stretch, Cl− SAC recovered upon washout of AEBSF (2 mM). In the absence of stretch, exogenous AngII (5 nM) activated an outwardly rectifying Cl− current that was rapidly and completely blocked by DPI (60 μM). Moreover, exogenous H2O2 (10, 100, and 500 μM), the eventual product of NADPH oxidase activity, also activated Cl− SAC in the absence of stretch, whereas catalase (1,000 U/ml), an H2O2 scavenger, attenuated the response to stretch. Application of H2O2 during NADPH oxidase inhibition by either DPI (60 μM) or AEBSF (0.5 mM) did not fully reactivate Cl− SAC, however. These results suggest that stretch of β1-integrin in cardiac myocytes elicits Cl− SAC by activating AT1R and NADPH oxidase and, thereby, producing reactive oxygen species. In addition, NADPH oxidase may be intimately coupled to the channel responsible for Cl− SAC, providing a second regulatory pathway.

A Forskolin Derivative, Colforsin Daropate Hydrochloride, Inhibits the Decrease in Cortical Renal Blood Flow Induced by Noradrenaline or Angiotensin II in Anesthetized Rats

A forskolin derivative, colforsin daropate hydrochloride (CDH), acts directly on adenylate cyclase to increase the intracellular cyclic adenosine monophosphate levels which produce a positive inotropic effect and a lower blood pressure. However, little is known about the effects of CDH on the renal function. We used laser Doppler flowmetry to measure the cortical renal blood flow (RBF) in male Wistar rats given a continuous intravenous infusion of CDH and evaluated the effects of CDH on the noradrenaline (NA) and angiotensin II (AngII) induced increases in blood pressure and reductions in RBF. Continuous intravenous administration of CDH at 0.25 µg/kg/min did not affect the mean arterial pressure (MAP), but increased heart rate and RBF. Continuous intravenous administration of CDH at high doses (0.5–0.75 µg/kg/min) decreased the MAP, with little effect on the RBF. The administration of exogenous NA (1.7 µg/kg) increased the MAP and decreased the RBF. However, a bolus injection of NA did not decrease the RBF during continuous intravenous administration of CDH, and CDH did not affect the NA-induced increase in MAP. The administration of exogenous AngII (100 ng/kg) increased MAP and decreased RBF and heart rate, but a bolus injection of AngII did not decrease RBF during continuous intravenous administration of CDH. These results suggest that CDH plays a protective role against the pressor effects and the decrease in RBF induced by NA or AngII.