AT1 Expression Research Articles

Abstract 659: Curcumin Ameliorates Cardiac Dysfunction in the Ovariectomized Diabetic mRen2.Lewis Rat by Inhibiting Renin Angiotensin System

Diabetes is a well-recognized independent risk factor for the development of cardiovascular disease in women. Moreover, hypertension and loss of estrogen are additional complicating factors to diabetic heart disease. Curcumin, an active compound in the widely used spice turmeric, exerts therapeutic potential in diabetes mellitus; however, the underlying mechanisms are not fully understood. The present study was designed to investigate the potential of curcumin to ameliorate early cardiac dysfunction in insulin- and estrogen-depleted hypertensive mRen2.Lewis rats by altering the renin angiotensin system as one of the major contributors to the development of diabetic cardiomyopathy. Ovariectomy (OVX) was performed at 10 weeks of age and diabetes (D) subsequently induced at 11 weeks with streptozotocin (65 mg/kg). Only animals that showed hyperglycemia greater than 200 mg/dl were considered diabetic. OVX-D rats were given curcumin at a dose of 200 mg/kg/day (OVX-D & CUR; n=7) or vehicle (OVX-D & VEH; n=8) by gastric gavage starting two days after streptozotocin injection. Intact non-diabetic mRen2.Lewis rats served as controls (C; n=9). After four weeks, concomitant insulin and estrogen depletion induced systolic and diastolic dysfunction, indicated by decreased fractional shortening (FS; C: 60.0 ± 1.5 vs OVX-D & VEH: 43.7 ± 2.2 %; p<0.05) and Tissue Doppler velocity at the level of mitral annulus (e’; C: 75.0 ± 3.4 vs OVX-D & VEH: 58.6 ± 5.6 mm/s; p<0.05) that was associated with increased blood pressure (BP; C: 131 ± 3 vs 179 ± 2 mmHg; p<0.05), elevated plasma angiotensin II (Ang II; C: 20 ± 4 vs 32 ± 2 pg/ml; p<0.05), and cardiac expression of Ang II type 1 receptor (AT1; C: 100 ± 12 vs 206 ± 33 %; p<0.05). Curcumin treatment did not affect BP (164 ± 6 mmHg; p>0.05) but attenuated cardiac systolic (FS: 53 ± 3%, p<0.05) and diastolic dysfunction (e’: 74 ± 3 mm/s; p<0.05) while reducing AT1 expression (120 ± 12 %; p<0.05) in the diabetic heart. It also decreased plasma Ang II to the levels not different from the controls (21.6 ± 3 pg/ml; p>0.05). In conclusion, the present study suggests that curcumin treatment, in a blood pressure independent manner, may ameliorate early cardiac dysfunction in estrogen- and insulin-depleted mRen2.Lewis rats by reducing Ang II signaling pathway.

Cardiotonic steroids induce anti-angiogenic and anti-proliferative profiles in first trimester extravillous cytotrophoblast cells

ObjectivePreeclampsia (preE), is characterized by abnormal placental invasion and function. Marinobufagenin (MBG), a cardiotonic steroid (CTS), inhibits cytotrophoblast (CTB) cell functions that are critical for normal placental development. This study tests the hypothesis that CTSs induce anti-angiogenic and anti-proliferative effects in CTB cells. MethodsHuman extravillous CTB cells of the line Sw-71, derived from first trimester chorionic villus tissue, were incubated with 0, 0.1, 1, 10, and 100 nM of each of three CTSs (MBG, cinobufatalin (CINO) and ouabain (OUB)) for 48 h. Thereafter, levels of pro-angiogenic (vascular endothelial growth factor (VEGF165), placental growth factor (PlGF)) and anti-angiogenic (soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng)) factors were measured in culture media using ELISA kits. Expression of three receptors (VEGF receptor 1 (VEGFR1), angiogenic angiotensin type 1 receptor (AT1) and anti-angiogenic angiotensin type 2 receptor (AT2)) were assayed using immunoblotting (western blots) in cell lysates. ResultssFlt-1 and sEng secretion were increased while VEGF165 and PIGF were decreased in the culture media of CTB cells treated with 1 nM or more of each CTSs (p < 0.01 for each). The AT2 receptor expression was up-regulated (p < 0.05) in CTB cells treated with 1 nM or more of MBG and CINO and with 100 nM OUB, while AT1 and VEGFR1 expressions decreased (p < 0.05) with 1 nM or more of MBG and 10 nM or more of CINO and OUB. ConclusionsCTSs influence extravillous CTB cells to induce an anti-angiogenic and anti-proliferative profile.

Identification and validation of suitable reference genes for RT-qPCR analysis in mouse testis development.

RT-qPCR is a commonly used method for evaluating gene expression; however, its accuracy and reliability are dependent upon the choice of appropriate reference gene(s), and there is limited information available on suitable reference gene(s) that can be used in mouse testis at different stages. In this study, using the RT-qPCR method, we investigated the expression variations of six reference genes representing different functional classes (Actb, Gapdh, Ppia, Tbp, Rps29, Hprt1) in mice testis during embryonic and postnatal development. The expression stabilities of putative reference genes were evaluated using five algorithms: geNorm, NormFinder, Bestkeeper, the comparative delta C(t) method and integrated tool RefFinder. Analysis of the results showed that Ppia, Gapdh and Actb were identified as the most stable genes and the geometric mean of Ppia, Gapdh and Actb constitutes an appropriate normalization factor for gene expression studies. The mRNA expression of AT1 as a test gene of interest varied depending upon which of the reference gene(s) was used as an internal control(s). This study suggested that Ppia, Gapdh and Actb are suitable reference genes among the six genes used for RT-qPCR normalization and provide crucial information for transcriptional analyses in future studies of gene expression in the developing mouse testis.

Effects of estrogen on ACE-Ang II-AT1 axis in ovariectomy and hypoxic pulmonary hypertension rats

To explore the effects of estrogen (E2) on angiotensin converting enzyme-angiotensin II-angiotensin type 1 receptor (ACE-Ang II-AT1) axis in hypoxic pulmonary hypertension rats. A total of 60 healthy female Sprague-Dawdley (SD) rats were divided randomly into 6 groups (n = 10 each) of sham operation, pure ovariectomy (OVX), pure hypoxia,OVX+hypoxia,OVX+E2 and OVX+hypoxia+E2. Abdominal cavity was opened for sham operation group and bilateral ovaries were left intact without any other procedure. The pure OVX group underwent oophorectomy. The pure hypoxia group were placed into a low-oxygen environment (24 hour, 8 weeks). The OVX+hypoxia group were placed into a low-oxygen environment after bilateral oophorectomy. The OVX+E2 group received a subcutaneous injection of E2 (20 µg×kg(-1)×d(-1)) after bilateral oophorectomy. The OVX+hypoxia+E2 group had an injection of E2 and was placed into a low-oxygen environment after bilateral oophorectomy. The rats were feed continuously for 8 weeks to establish hypoxic pulmonary hypertension model. The mean pulmonary artery pressure (mPAP) was measured after bloodletting. Then right ventricle hypertrophy index (RVHI) and hematoxylin-eosin pulmonary artery remodeling (HPSR) were observed. And electron microscope was employed to observe pulmonary arteriolar ultrastructure. The methods of radio-immunity assay, ultraviolet spectroscopy, Western blot and reverse transcription PCR were used to measure the levels of CE,Ang II and AT1 in sera, lung and pulmonary artery. The vascular walls of pure hypoxia and OVX+hypoxia groups became thickened and lumen narrowed.mPAP and RVHI were (32.4 ± 2.2) mmHg (1 mmHg = 0.133 kPa),0.331 ± 0.032 and (37.9 ± 1.6) mmHg,0.433 ± 0.033. Both were significantly higher than those of Sham operation group ((12.6 ± 1.8) mmHg,0.233 ± 0.029) (both P < 0.05); the above parameters of OVX+Hypoxia+E2 group changed little. And mPAP ((26.1 ± 1.4) mmHg) was significantly higher than that in sham operation group (P < 0.05) while the difference in RVHI between OVX+ Hypoxia+ E2 (0.267 ± 0.040) and sham operation groups had no statistical significance (P > 0.05). Compared with Sham operation group, the expression levels of ACE,Ang II and AT1 in pure OVX, pure hypoxia and OVX+ hypoxia groups rose markedly (all P < 0.05).However, the OVX+E2 and OVX+ hypoxia+E2 groups had no obvious change (all P > 0.05). The intervention effect of E2 for hypoxic pulmonary hypertension may be partly mediated by the down-regulated expressions of ACE and AT1 in lung tissue resulting in the reduced activity of ACE-AngII-AT1 axis.

Increased proinflammatory markers and lipoperoxidation in obese individuals: Inicial inflammatory events?

BackgroundChronic inflammation associated to obesity increases the risk for developing insulin resistance (IR), hyperglycemia, hypertension and hyperlipidemia. The initial factors involved in generating the inflammatory events in the obesity remain unclear. Therefore, this study was aimed to determine inflammatory and oxidative markers in the blood of obese individuals with normal clinical and biochemical parameters and with or without IR. MethodsNineteen obese non-diabetic and nine lean subjects were studied for serum levels of TNF-α, IL-1β, adiponectin, angiotensin II, insulin, malondialdehyde (MDA) and the expressions of RAGE (advanced glycation end product receptor), AT1 (Ang II receptor), s100A12 protein (RAGE ligand) and nuclear factor-κB (NF-κB) in circulating mononuclear cells (CMC) by available antibodies and commercial kits. CMC were also cultured to determine pro-inflammatory mediators. ResultsInsulin was increased in obese subjects with IR. Decreased serum adiponectin in obese individuals and increased TNF-α, IL-1β and CMC bearing RAGE, AT1 and s100A12 in obese individuals without IR were found. High values of serum MDA in obese subjects were observed. Similar TNF-content in cultures from obese and controls, increased cellular IL-1β content in cultures from obese individuals without IR and high content of MDA in supernatants from obese individual cultures were observed. ConclusionsThe inflammatory events were mainly observed in obese individuals without IR. The absent of inflammatory events and high levels of insulin in obese subjects with IR, suggest a protector role of insulin for developing inflammatory events. These data can represent initial aspects of the chronic inflammation observed in the obesity.

Effects of exercise training on circulating and skeletal muscle renin-angiotensin system in chronic heart failure rats.

BackgroundAccumulated evidence shows that the ACE-AngII-AT1 axis of the renin-angiotensin system (RAS) is markedly activated in chronic heart failure (CHF). Recent studies provide information that Angiotensin (Ang)-(1–7), a metabolite of AngII, counteracts the effects of AngII. However, this balance between AngII and Ang-(1–7) is still little understood in CHF. We investigated the effects of exercise training on circulating and skeletal muscle RAS in the ischemic model of CHF.Methods/Main ResultsMale Wistar rats underwent left coronary artery ligation or a Sham operation. They were divided into four groups: 1) Sedentary Sham (Sham-S), 2) exercise-trained Sham (Sham-Ex), sedentary CHF (CHF-S), and exercise-trained CHF (CHF-Ex). Angiotensin concentrations and ACE and ACE2 activity in the circulation and skeletal muscle (soleus and plantaris) were quantified. Skeletal muscle ACE and ACE2 protein expression, and AT1, AT2, and Mas receptor gene expression were also evaluated. CHF reduced ACE2 serum activity. Exercise training restored ACE2 and reduced ACE activity in CHF. Exercise training reduced plasma AngII concentration in both Sham and CHF rats and increased the Ang-(1–7)/AngII ratio in CHF rats. CHF and exercise training did not change skeletal muscle ACE and ACE2 activity and protein expression. CHF increased AngII levels in both soleus and plantaris muscle, and exercise training normalized them. Exercise training increased Ang-(1–7) in the plantaris muscle of CHF rats. The AT1 receptor was only increased in the soleus muscle of CHF rats, and exercise training normalized it. Exercise training increased the expression of the Mas receptor in the soleus muscle of both exercise-trained groups, and normalized it in plantaris muscle.ConclusionsExercise training causes a shift in RAS towards the Ang-(1–7)-Mas axis in skeletal muscle, which can be influenced by skeletal muscle metabolic characteristics. The changes in RAS circulation do not necessarily reflect the changes occurring in the RAS of skeletal muscle.

Exercise Training suppresses vascular fibrosis in aging obesity induced rats.

[Purpose]The aim of this study was to investigate the effects of exercise training (ET) on vascular fibrosis in aging model rats with diet-induced obesity.[Methods]Twenty-four male Sprague-Dawley rats were divided into 3 groups: Aging control (A-C), A-C with high fat diet (AHF), AHF with ET (AHF + ET). Aging was induced by D-galactose (D-gal) and obesity was induced by HFD (60% fat) for 9 weeks. The experimental rats performed swimming (60 min/day, 5 days/week) for 8 weeks. All rat aorta samples were harvested for RT-PCR and morphologic analyses.[Results]The exercise training significantly decreased levels of AT-1, TGF-ß and Coll-1 gene expression compared to AHF group. The AHF + ET group showed a reduced collagen accumulation in the aorta media compared to AHF group.[Conclusion]These results suggest that ET could protect the aging obesity aorta against down-regulation of fibrotic factors (AT-1, TGF-ß and Coll-1 gene) and fibrosis by inhibition of collagen accumulation in the aorta media.

Angiotensin II and 1-7 during aging in Metabolic Syndrome rats. Expression of AT1, AT2 and Mas receptors in abdominal white adipose tissue

Renin-Angiotensin System (RAS) plays an important role in the development of Metabolic Syndrome (MS) and in aging. Angiotensin 1-7 (Ang 1-7) has opposite effects to Ang II. All of the components of RAS are expressed locally in adipose tissue and there is over-activation of adipose RAS in obesity and hypertension. We determined serum and abdominal adipose tissue Ang II and Ang 1-7 in control and MS rats during aging and the expression of AT1, AT2 and Mas in white adipose tissue. MS was induced by sucrose ingestion during 6, 12 and 18 months. During aging, an increase in body weight, abdominal fat and dyslipidemia were found but increases in aging MS rats were higher. Control and MS concentrations of serum Ang II from 6-month old rats were similar. Aging did not modify Ang II seric concentration in control rats but decreased it in MS rats. Ang II levels increased in WAT from both groups of rats. Serum and adipose tissue Ang 1-7 increased during aging in MS rats. Western blot analysis revealed that AT1 expression increased in the control group during aging while AT2 and Mas remained unchanged. In MS rats, AT1 and AT2 expression decreased significantly in aged rats. The high concentration of Ang 1-7 and adiponectin in old MS rats might be associated to an increased expression of PPAR-γ. PPAR-γ was increased in adipose tissue from MS rats. It decreased with aging in control rats and showed no changes during aging in MS rats. Ang 1-7/Mas axis was the predominant pathway in WAT from old MS animals and could represent a potential target for therapeutical strategies in the treatment of MS during aging.

NADPH oxidase mediates altered vascular responses in allergic mice (1065.10)

Asthma is associated with adverse cardiovascular outcomes, though the cause is unclear. We have shown vascular inflammation and altered vascular responses in allergic mice (Ponnoth et al., AJP 2008, 2010). In this study, we investigated the contribution of NADPH oxidase to angiotensin II (Ang II)‐mediated aortic responses in a mouse model of allergic asthma developed in this laboratory. Mice were divided into control (CON) and allergen sensitized‐challenged (SEN) groups. Mice were sensitized (i.p.) on days 1, 6 with 0.2μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11‐13. Ang II type 1 receptor (AT1) antagonist losartan was given i.p. as a single bolus dose (20mg/kg) on day 14 to SEN mice. Organ bath studies were done on day 14 and Western Blot analyses were done to determine aortic expression of AT1, NOX 1, 2 and 4 as well as p22phox and p67phox, the subunits of NOX. In the SEN group, Ang II (10μM) produced higher aortic contraction (33.48±3.26% vs. 18.06±3.07% in CON; p&lt;0.05) that was lowered with the use of apocynin (10μM; 33.48±3.26% vs.13.11±6.89), while no effect was observed in CON. SEN mice had lower endothelial response to acetylcholine (30.95±5.73% vs. 57.6±4.3% in CON; p&lt;0.05), that was improved with i.p. losartan (56.1±4.39%). AT1 expression was unchanged in SEN mice. However, the expression of NOX4 (1.27±0.06 vs. 0.99±0.09) and p22phox (3.45±0.35 vs. 1.35±0.62) were significantly higher in SEN mice compared to CON. Treatment with losartan significantly attenuated the increases in NOX4 (from1.27±0.06 to 0.67±0.11) and p22phox (from3.45±0.35 to 0.26±0.11) in SEN mice. These data suggest that most likely NOX4, through p22phox association, may contribute to altered Ang ll and endothelial responses in allergic mice, and that losartan can improve these outcomes.Grant Funding Source: Supported by HL027339, HL094447

Aldosterone-induced inflammatory response of mesangial cells via angiotension II receptors

In this study, we investigated whether AngII receptors (AT1a and AT2) contributed to the development of the aldosterone-induced inflammatory response of rat mesangial cells (RMCs). RMCs were isolated from the glomeruli of normal or diabetic rats which were produced by injection of streptozotocin, and cultured in high-glucose media. In order to evaluate the effects of aldosterone, the expression of AT1a, AT2, NF-κB and MCP-1 was detected. In addition, in order to evaluate the role of Ang II receptors, AT1a and AT2 genes were blocked and the expression of NF-κB and MCP-1 was detected. Moreover, for assessing the relationship between NF-κB and MCP-1, the NF-κB gene was blocked and MCP-1 expression was detected. Aldosterone significantly increased AT1a, AT2, NF-κB and MCP-1 levels in RMCs in a dose-dependent manner, whereas eplerenone (EPI), a selective aldosterone antagonist, partly inhibited the effects of aldosterone. When AT1a and AT2 genes were blocked, the expression of NF-κB and MCP-1 was greatly inhibited. Moreover, when the NF-κB gene was silenced, the expression of MCP-1 was reduced. We demonstrated that aldosterone induced an inflammatory response in RMCs cultured in high-glucose media via the AT1a and AT2 pathways.

Impact of repeated intravenous bone marrow mesenchymal stem cells infusion on myocardial collagen network remodeling in a rat model of doxorubicin-induced dilated cardiomyopathy

Bone marrow mesenchymal stem cells (MSCs) transplantation improved cardiac function and reduced myocardial fibrosis in both ischemic and non-ischemic cardiomyopathies. We evaluated the effects of repeated peripheral vein injection of MSCs on collagen network remodeling and myocardial TGF-β1, AT1, CYP11B2 (aldosterone synthase) gene expressions in a rat model of doxorubicin (DOX)-induced dilated cardiomyopathy (DCM). Thirty-eight out of 53 SD rats survived at 10 weeks post-DOX injection (2.5 mg/kg/week for 6 weeks, i.p.) were divided into DCM blank (without treatment, n = 12), DCM placebo (intravenous tail injection of 0.5 mL serum-free culture medium every other day for ten times, n = 13), and DCM plus MSCs group (intravenous tail injection of 5 × 10(6) MSCs dissolved in 0.5 mL serum-free culture medium every other day for 10 times, n = 13). Ten untreated rats served as normal controls. At 20 weeks after DOX injection, echocardiography, myocardial collagen content, myocardial expressions of types I and III collagen, TGF-β1, AT1, and CYP11B2 were compared among groups. At 20 weeks post-DOX injection, 8 rats (67%) survived in DCM blank group, 9 rats (69%) survived in DCM placebo group while 13 rats (100 %) survived in DCM plus MSCs group. Left ventricular end-diastolic diameter was significantly higher and ejection fraction was significantly lower in DCM blank and DCM placebo groups compared to normal control rats, which were significantly improved in DCM plus MSCs group (all p < 0.05 vs. DCM blank and DCM placebo groups). Moreover, myocardial collagen volume fraction, types I and III collagen, myocardial mRNA expressions of TGF-β1, AT1, CYP11B2, and collagen I/III ratio were all significantly lower in DCM plus MSCs group compared to DCM blank and DCM placebo groups (all p < 0.05). Repeated intravenous MSCs transplantation could improve cardiac function by attenuating myocardial collagen network remodeling possibly through downregulating renin-angiotensin-aldosterone system in DOX-induced DCM rats.

Vitamin D receptor-modulated Hsp70/AT1 expression may protect the kidneys of SHRs at the structural and functional levels

Previous hypertension studies have shown that low levels of vitamin D are linked to elevated renin-angiotensin system. The heat shock protein 70 regulates signaling pathways for cellular oxidative stress responses. Hsp70 has been shown to protect against angiotensin II-induced hypertension and exert a cytoprotective effect. Here, we wanted to evaluate whether the vitamin D receptor (VDR) associated with Hsp70/AT1 expression may be involved in the mechanism by which paricalcitol provides renal protection in spontaneously hypertensive rats (SHRs). One-month-old female SHRs were treated for 4 months with vehicle, paricalcitol, enalapril, or a combination of both paricalcitol and enalapril. The following were determined: blood pressure; biochemical parameters; fibrosis; apoptosis; mitochondrial morphology; and VDR, AT1 receptor, and Hsp70 expression in the renal cortex. Blood pressure was markedly reduced by enalapril or the combination but not by paricalcitol alone. However, VDR activation, enalapril or combination, prevented fibrosis, the number of TUNEL-positive apoptotic cells, mitochondrial damage, and NADPH oxidase activity in SHRs. Additionally, high AT1 receptor expression, like low Hsp70 expression (immunohistochemical/immunofluorescence studies), was reversed in the renal cortices of paricalcitol- and/or enalapril-treated animals (SHRs), and these changes were most marked in the combination therapy group. Finally, all of the recovery parameters were consistent with an improvement in VDR expression. Data suggest that Hsp70/AT1 modulated by VDR is involved in the mechanism by which paricalcitol provides renal protection in SHRs. We propose that low AT1 expression through VDR induction could be a consequence of the heat shock response Hsp70-mediated cell protection.

A Mechanism Underlying Hypertensive Occurrence in the Metabolic Syndrome: Cooperative Effect of Oxidative Stress and Calcium Accumulation in Vascular Smooth Muscle Cells

Several lines of evidence indicate that reactive oxygen species (ROS) overproduction under the metabolic syndrome condition is the leading cause of cardiovascular events. Calcium is an important stimulus for vasoconstriction and plays a pivotal role in the development of hypertension. Here, we investigate whether a relationship exists between metabolic syndrome-induced mitochondrial ROS overproduction and Ang II-mediated Ca2+ release in vascular smooth muscle cells (VSMC). The effect of mitochondrial ROS on AT1 expression, and Ca2+ and IP3 generation was studied in 2 VSMC models of metabolic syndrome using fura-2/AM probes and ELISA-based assay. Ang II-mediated aortic ring contraction in SD rats fed with high-fat diet (HFD) was measured using a force transducer connected to chart recorder. In the metabolic syndrome, almost 2-fold increased mitochondrial O2 - significantly upregulated AT1 expressions by ~60%, companied by elevated Ca2+ and IP3 levels in VSMC and enhanced aortic rings contraction. All these increments were blocked by rotenone (inhibitor of mitochondrial respiratory chain complex I), ruthenium red (inhibitor of calcium uniporter), cyclosporin A (inhibitor of mitochondrial permeability pore), and N-acetylcysteine. Therefore, in the states of metabolic syndrome, ROS overproduction in mitochondrial complex I enhances Ang II-mediated vascular contraction via an AT1-dependent pathway. In addition, the import of Ca2+ from endoplasmic reticulum to mitochondria via calcium uniporter and mitochondrial permeability pore seems to serve as a mechanism to further aggravate mitochondrial damage and vascular dysfunction that may contribute to the occurrence of hypertension.

Cross-talk between mineralocorticoid receptor/angiotensin II type 1 receptor and mitogen-activated protein kinase pathways underlies aldosterone-induced atrial fibrotic responses in HL-1 cardiomyocytes

BackgroundAldosterone is increasingly recognized for its involvement in atrial structural remodeling. However, the precise molecular mechanisms and signal pathways underlying aldosterone-induced atrial fibrosis are unknown. MethodsWestern blotting was used to investigate the effects of aldosterone on the expression of mineralocorticoid receptor (MR), angiotensin II type I receptor (AT1), mitogen-activated protein kinases (MAPKs), and fibrotic marker proteins in cultured HL-1 cardiomyocytes. ResultsAldosterone upregulated MR and AT1 expressions in a concentration-dependent and time-dependent manner. Aldosterone (10−6M) significantly and time-dependently increased activation of the extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p38MAPK pathways, and the protein expression of collagen 1A and 3A (COL1A and COL3A), transforming growth factor (TGF)-β1, and α-smooth muscle actin (SMA). Pre-treatment with eplerenone (10−10M) prevented the increased expression of MR, MAPK signals and the above profibrotic molecules, but amplified the increase in AT1 level stimulated by aldosterone (10−6M). Pre-treatment with losartan (10−10M) or MAPK pathway inhibitors (U0126 or SP600125) abolished aldosterone-induced MR upregulation and significantly inhibited the expression of the above fibrotic marker proteins, indicating the critical role of MR and the requirement for active AT1 in the development of aldosterone-induced atrial fibrosis. ConclusionsElevated MR activity plays a central role in aldosterone-mediated activation of the MAPK signaling pathway and subsequent profibrotic effects in HL-1 atrial cells. MR/AT1 and the MAPK signaling pathway interact to trigger the molecular mechanism underlying the aldosterone-induced atrial fibrotic response. Our results support the view that MR blockade in conjunction with AT1 blockade can prevent the occurrence of atrial fibrillation.

The renin–angiotensin system plays a major role in voiding dysfunction of ovariectomized rats

AimsThe renin–angiotensin system (RAS) plays a major role in cardiovascular diseases in postmenopausal women, but little is known about its importance to lower urinary tract symptoms. In this study we have used the model of ovariectomized (OVX) estrogen-deficient rats to investigate the role of RAS in functional and molecular alterations in the urethra and bladder. Main methodsResponses to contractile and relaxant agents in isolated urethra and bladder, as well as cystometry were evaluated in 4-month OVX Sprague–Dawley rats. Angiotensin-converting enzyme activity and Western blotting for AT1/AT2 receptors were examined. Key findingsCystometric evaluations in OVX rats showed increases in basal pressure, capacity and micturition frequency, as well as decreased voiding pressure. Angiotensin II and phenylephrine produced greater urethral contractions in OVX compared with Sham group. Carbachol-induced bladder contractions were significantly reduced in OVX group. Relaxations of urethra and bladder to sodium nitroprusside and BAY 41-2272 were unaffected by OVX. Angiotensin-converting enzyme activity was 2.6-fold greater (p<0.05) in urethral tissue of OVX group, whereas enzyme activity in plasma and bladder remained unchanged. Expressions of AT1 and AT2 receptors in the urethra were markedly higher in OVX group. In bladder, AT1 receptors were not detected, whereas AT2 receptor expression was unchanged between groups. 17β-Estradiol replacement (0.1mg/kg, weekly) or losartan (30mg/kg/day) largely attenuated most of the alterations seen in OVX group. SignificanceProlonged estrogen deprivation leads to voiding dysfunction and urethral hypercontractility that are associated with increased ACE activity and up-regulation of angiotensin AT1/AT2 receptor in the urethral tissue.

Abstract 406: Improved Angiotensin II Type 2 Receptor Expression and Function in Transglutaminase 2 Knock-Out Mice Treated with Angiotensin II

Objective: Transglutaminase 2 (TG2) induces transamidation and cross-linking of proteins that can modulate receptor interaction. Angiotensin II (AngII) may regulate TG2 and AngII type 2 receptor (AT2) expression and function. We hypothesized that AngII may impair AT2 expression and function through TG2. Methods: TG2-knockout mice (TG2-K/O, 8 weeks old, 6 for each group) and age-matched wild type (WT) mice were treated or not with AngII (400 ng/kg/min) for 14 days. Vascular reactivity was assessed in response to sodium nitroprusside (SNP, 10 nM to 1 mM), in mesenteric arteries pre-contracted with norepinephrine (10 μM). AT2 function was assessed by concentration-response curve to the selective AT2 agonist Compound 21 (C21, 1nM to 1 μM) in mesenteric arteries pre-contracted with norepinephrine. AT2 expression in aorta was evaluated by immunoblotting. Results: C21-induced relaxation was similar in untreated WT, and in untreated TG2-K/O. C21-induced relaxation was improved only in AngII-treated TG2-K/O (2-fold increase vs untreated TG2-K/O, P&lt;0.001). SNP dependent relaxation was similar in all groups. AT2 receptor expression was similar in untreated WT and untreated TG2-K/O. AT2 was reduced by AngII in WT (-36±6% vs untreated WT, P&lt;0.01), and significantly increased in TG2-K/O (+53±4% vs untreated TG2-K/O, P&lt;0.001). Conclusions: AngII fails to reduce AT2 expression and function in TG2-K/O mice. Therefore, AngII may negatively modulate AT2 receptor expression and function through TG2.

Dietary arachidonic acid and docosahexaenoic acid regulate liver fatty acid desaturase (FADS) alternative transcript expression in suckling piglets

Molecular regulation of fatty acid desaturase (Fads) gene expression by dietary arachidonic acid (ARA) and docosahexaenoic acid (DHA) during early post-natal period, when the demand for long chain polyunsaturated fatty acids (LC-PUFA) is very high, has not been well defined. The objective of the current study was to determine regulation of liver Fads1, Fads2 and Fads3 classical (CS) and alternative transcripts (AT) expression by dietary ARA and DHA, within the physiological range present in human breast milk, in suckling piglets. Piglets were fed one of six milk replacer formula diets (formula-reared groups, FR) with varying ARA and DHA content from days 3–28 of age. The ARA/DHA levels of the six formula diets were as follows (% total fatty acid, FA/FA): (A1) 0.1/1.0; (A2) 0.53/1.0; (A3-D3) 0.69/1.0; (A4) 1.1/1.0; (D2) 0.67/0.62; and (D1) 0.66/0.33. The control maternal-reared (MR) group remained with the dam. Fads1 expression was not significantly different between FR and MR groups. Fads2 expression was down-regulated significantly in diets with 1:1 ratio of ARA:DHA, compared to MR. Fads2 AT1 expression was highly correlated to Fads2 expression. Fads3 AT7 was the only Fads3 transcript sensitive to dietary LC-PUFA intake and was up-regulated in the formula diets with lowest ARA and DHA contents compared to MR. Thus, the present study provides evidence that the proportion of dietary ARA:DHA is a significant determinant of Fads2 expression and LC-PUFA metabolism during the early postnatal period. Further, the data suggest that Fads3 AT7 may have functional significance when dietary supply of ARA and DHA are low during early development.

GW24-e1171 Effect of different ARB-eluting stents on coronary intimal hyperplasia in vivo

ObjectivesInvestigate the difference of inhibiting intima proliferation between telmisartan, valsartan and sirolimus eluting stent.MethodsTelmisartan eluting stent (TES), valsartan eluting stent (VES), sirolimus eluting stent (SES) and polymer eluting stent (PES)...

PTH-102 Preliminary Evidence for a Role of the Renin Angiotensin System in Intestinal Fibrosis in Crohn’S Disease Using Angiotensin Receptor Immunohistochemistry

IntroductionFibrosis is a major limitation to successful non-surgical treatment of Crohn’s disease (CD) and is a cause of significant morbidity. The pathophysiological determinants of intestinal fibrosis in CD remain uncertain....

Effects of rosuvastatin on expression of angiotensin-converting enzyme 2 after vascular balloon injury in rats.

ObjectiveTo investigate the effects and mechanisms of rosuvastatin on angiotensin -converting enzyme 2 (ACE2) in the process of neointimal formation after vascular balloon injury in rats, and to explore the effects of ACE2 and rosuvastatin in restenosis.MethodsThirty-six Wistar rats were randomly allocated into three groups: control group (n = 12), surgery group (n = 12), and statin group (n = 12). Aortic endothelial denudation of rats was performed using 2F balloon catheters. At days 14 and 28 after injury, aortic arteries were harvested to examine the following. Intimal thickening was examined by hematoxylin and eosin staining. We measured angiotensin II (Ang II) and angiotensin 1-7 (Ang-[1–7]) levels by a radioimmunological method or enzyme-linked immunosorbent assay. Protein and mRNA expression of ACE2 and Ang II type 1 receptor (AT1) were investigated by immunohistochemistry, Western blots, and Reverse transcriptase-polymerase chain reaction (RT-PCR). We measured changes in proliferating cell nuclear antigen (PCNA) by immunohistochemistry. The level of phosphorylated extracellular signal regulated kinase 1/2 (P-ERK1/2) was evaluated by Western blotting.ResultsProliferation of vascular smooth muscle cells (VSMC) and intimal thickening were higher at day 14 after vascular balloon injury in the surgery group compared with the control group. Proliferation of VSMC was decreased by day 28 after injury, while intimal thickening continued. With rosuvastatin treatment, the extent of VSMC proliferation and intimal thickening was reduced at day 14 and 28 after injury. Ang II and P-ERK levels were significantly increased, Ang-(1–7) levels were significantly decreased, mRNA and protein expressions of ACE2 were significantly decreased, and AT1 expression was significantly increased at days 14 and 28 after vascular balloon injury in the surgery group compared with the control group. PCNA expression was higher in the surgery group than in the control group, and it was significantly decreased after being given rosuvastatin. Expression of ACE2 mRNA and protein, and Ang-(1–7) levels were significantly increased, while AT1 expression and levels of Ang II and P-ERK were significantly decreased in the statin group compared with the surgery group.ConclusionsExpression of ACE2 mRNA and protein is decreased in the process of intimal thickening after balloon injury. The inhibitory effect of rosuvastatin on intimal thickening is related to upregulation of ACE2, an increase in Ang-(1–7), downregulation of AT1, and activation of the P-ERK pathway.