Angiotensin II Treatment Research Articles

Prostaglandin E2 Inhibition Promotes Human Aortic Smooth Muscle Cell Differentiation

INTRODUCTIONIn a mouse model of abdominal aortic aneurysms (AAAs) induced by chronic angiotensin II (AngII) infusion, the effectiveness of cyclooxygenase‐2 (COX‐2) inhibition in attenuating AAA progression is associated with maintenance of a differentiated smooth muscle cell (SMC) phenotype. However, the mechanisms responsible for COX‐2‐dependent aortic SMC phenotypic modulation are unknown. Therefore, we assessed the hypothesis that COX‐2 reduces human aortic SMC (hASMC) differentiation by the action of prostaglandin E2 (PGE2).METHODSCultured hASMCs were treated with varying concentrations of AngII, PGE2, or inhibitors of COX‐2 and PGE2 synthetic pathway. The effects of these treatments on the expression of SMC differentiation markers and components of these pathways were examined.RESULTSCOX‐2 inhibition with celecoxib significantly reduced PGE2 levels and increased expression of alpha‐actin (marker of differentiation), whereas exogenous PGE2 treatment reduced hASMC differentiation. AngII treatment resulted in increased protein expression of the inducible PGE2 synthase ‐ microsomal PGE2 synthase (mPGES‐1) and decrease in alpha‐actin. Inhibition of mPGES‐1 and PGE2 by 15‐deoxyPGJ2 resulted in increased alpha‐actin whereas mPGES‐1 knockdown reduced PGE2 synthesis, and promoted hASMC differentiation.CONCLUSIONCOX‐2 inhibition by celecoxib may increase hASMC differentiation by inhibiting production of mPGES‐1‐derived PGE2. Specific inhibition of mPGES‐1 may serve as a potential target for attenuating AAA progression by maintaining a differentiated SMC phenotype.FUNDINGNIH HL083122

High‐Mobility Group Box 1 (HMGB1) in Angiotensin II induced hypertension

Hypertension is the most critical predisposing factor for the development of cardiovascular diseases. While effective pharmacological control of hypertension using angiotensin receptor blockers has been implemented, the clinical course of the disease remains progressive. Consequently, new and innovative approaches for treating hypertension are needed. A growing body of evidence suggests that inflammatory cytokines play important roles in the pathophysiology of cardiovascular disease. Our lab has shown that blocking inflammatory molecules and their transcription factor, nuclear transcription factor kappa B (NF‐kB), within the paraventricular nucleus (PVN) attenuates sympathetic activity and hypertension. However, it remains unknown how inflammation is activated during the development of hypertension. We proposed that the interaction between the inflammatory cytokine protein high‐mobility group box 1 (HMGB1) and its toll‐like receptor, TLR4, results in the up‐regulation of NF‐kB, thereby contributing to the development of hypertension. We performed in vitro and in vivo studies to understand the role played by HMGB1 in the development of hypertension. We treated mouse Neuro‐2a cells with control medium or medium containing Angiotensin II (AngII). Immunofluorescence and immunoblot analysis of AngII‐treated N2a cells correlated increases in HMGB1 levels with up‐regulation of inflammatory markers. In addition, we examined the levels of HMGB1 in the PVN from AngII and saline‐treated rats using RT‐PCR and western blotting. AngII treatment resulted in an increase in blood pressure and this was accompanied by an increase in HMGB1 in the PVN of rats. These findings indicate a potential for HMGB1 in the PVN as a possible mechanism of AngII‐induced hypertension.

Evidence for time‐dependent and adaptive mechanisms in the mitral valve following prolonged Angiotensin II infusion

Activation of angiotensin II (AngII) signaling has been implicated in the pathogenesis of human myxomatous mitral valve prolapse (MVP). To determine whether chronic AngII infusion can elicit molecular and phenotypic manifestations of MVP, we implanted C57BL/6J mice with osmotic minipumps delivering AngII (1000ng/kg/min) or saline for 2 or 4 weeks. We probed changes in mRNA (qRT-PCR) and protein (immunohistochemistry) levels in mitral valves, and assessed valve function by echocardiography. In line with previous findings from human MVP, 2-week AngII infusion was associated with increased pro-fibrotic (e.g. TGFβ2 and CTGF) and “classical” pro-calcific (Wnt/β-catenin) gene expression in mouse mitral valves. Increased immunofluorescence of phospho-SMAD2 and nuclear β-catenin suggested canonical activation of these pathways. Increased gene expression of the transcription factor Runx2 (1.8 fold-change [FC]) and the proliferation marker KI67 (3.9 FC) were also recaptured by AngII. Surprisingly, we found that increases in Runx2 and KI67 mRNA were attenuated (both ~1.3FC) following 4 weeks of AngII, and transcriptional changes in TGFβ and Wnt/β-catenin pathway target genes were no longer evident. The incidence of mitral regurgitation after 2 weeks of AngII (38%) did not increase when mice were treated for 4 weeks (40%). Collectively, these data suggest that there may be adaptive molecular changes occurring following prolonged AngII treatment, which oppose fibrogenic and proliferative signaling pathways. Future studies aimed at identifying key protective and deleterious molecular changes following prolonged AngII infusion will be critical to understanding mechanisms contributing to evolution of MVP.

Stimulus-selective induction of the orphan nuclear receptor NGFIB underlies different influences of angiotensin II and potassium on the human adrenal gland zona glomerulosa-specific 3β-HSD isoform gene expression in adrenocortical H295R cells.

In the adrenal, the type I 3β-hydroxysteroid dehydrogenase (HSD3B1) is expressed exclusively in the zona glomerulosa (ZG), where aldosterone is produced. Angiotensin II (AngII) and potassium (K(+)) are the major physiological regulators of aldosterone synthesis. However, their respective roles in regulation of aldosterone synthesis are not fully defined, particularly in terms of transcriptional regulation of steroidogenic enzyme genes. We previously showed that AngII can stimulate expression of HSD3B1. But, K(+) responsiveness of this gene has remained unexplored. Here, we report that K(+) stimulation lacks the ability to induce HSD3B1 expression in human adrenocortical H295R cells. Both AngII and K(+) were able to enhance transcription of the aldosterone synthase gene (CYP11B2). Promoter analysis revealed that although both AngII and K(+) activate transcription from the Ca(2+)/cAMP-responsive element (CRE) located in the CYP11B2 promoter, the orphan nuclear receptor NGFIB-responsive element (NBRE) located in the HSD3B1 promoter fails to respond to K(+), being only able to enhance transcription after AngII treatment. We found that induction of de novo protein synthesis of NGFIB occurs only after AngII treatment. This sharply contrasts with the phosphorylation that occurs in response to both AngII and K(+) on the CREB/ATF family transcription factor ATF2. Chromatin immunoprecipitation assay confirmed that the NGFIB protein occupies the HSD3B1 promoter only after AngII, while ATF2 binds to the CYP11B2 promoter in response to both AngII and K(+). These data provide evidence that downstream signals from AngII and K(+) can be uncoupled in the regulation of HSD3B1 in the human adrenocortical H295R cells.

Angiotensin II mediates angiotensin converting enzyme type 2 internalization and degradation through an angiotensin II type I receptor-dependent mechanism.

Angiotensin-converting enzyme type 2 (ACE2) is a pivotal component of the renin-angiotensin system, promoting the conversion of angiotensin II (Ang-II) to Ang-(1-7). We previously reported that decreased ACE2 expression and activity contributes to the development of Ang-II-mediated hypertension in mice. The present study aimed to investigate the mechanisms involved in ACE2 downregulation during neurogenic hypertension. In ACE2-transfected Neuro-2A cells, Ang-II treatment resulted in a significant attenuation of ACE2 enzymatic activity. Examination of the subcellular localization of ACE2 revealed that Ang-II treatment leads to ACE2 internalization and degradation into lysosomes. These effects were prevented by both the Ang-II type 1 receptor (AT1R) blocker losartan and the lysosomal inhibitor leupeptin. In contrast, in HEK293T cells, which lack endogenous AT1R, Ang-II failed to promote ACE2 internalization. Moreover, this effect could be induced after AT1R transfection. Furthermore, coimmunoprecipitation experiments demonstrated that AT1R and ACE2 form complexes, and these interactions were decreased by Ang-II treatment, which also enhanced ACE2 ubiquitination. In contrast, ACE2 activity was not changed by transfection of AT2 or Mas receptors. In vivo, Ang-II-mediated hypertension was blunted by chronic infusion of leupeptin in wildtype C57Bl/6, but not in ACE2 knockout mice. Overall, this is the first demonstration that elevated Ang-II levels reduce ACE2 expression and activity by stimulation of lysosomal degradation through an AT1R-dependent mechanism.

Long Noncoding RNA–MicroRNA–mRNA

A large fraction of mammalian genomes is transcribed into noncoding RNAs.1 Despite the prevalence in the genome, up until recently, noncoding RNAs were regarded as junk RNAs that do not code for protein and, thereby, have no biological purpose. However, over the past decade, it has become increasingly evident that noncoding RNAs can and do serve important regulatory functions in pathophysiological processes.1 Short noncoding RNAs called microRNAs, typically 20 to 24 nucleotides in length, have been widely reported to post-transcriptionally regulate gene expression by binding to partially complementary sequences in target mRNAs or promoting mRNA degradation and translational repression.2 Another class of noncoding RNAs, typically >200 nucleotides in length, called long noncoding RNAs (lncRNAs) have been recently discovered to regulate gene expression not only at the post-transcriptional level, but also at transcriptional and epigenetic levels.3 Notably, lncRNAs have also been reported to function as microRNA sponges, altering microRNA expression levels by binding to and sequestering microRNAs.4,5 In the present study, Wang et al6 present evidence that a lncRNA modulates the ability of a microRNA to target an mRNA, which plays a role in the development of cardiac hypertrophy. To identify microRNAs whose expression is altered by hypertrophic stimulation, the authors6 performed microRNA microarray analysis and quantitative reverse transcription–polymerase chain reaction (qRT-PCR) on neonatal mouse cardiomyocytes treated with or without angiotensin II (Ang-II). In particular, microRNA-489 (miR-489) was found to be most downregulated microRNA in response to Ang-II treatment. The authors used several methods to study the role of miR-489 in cardiac hypertrophy. First, they infected cardiomyocytes with adenoviruses encoding miR-489 to overexpress miR-489 and subsequently treated the cardiomyocytes with Ang-II. Second, they transfected cardiomyocytes with miR-489 antagomirs (anta-489) to knockdown the expression of miR-489, followed by treatment of the cardiomyocytes with …

Angiotensin II upregulates endothelial lipase expression via the NF-kappa B and MAPK signaling pathways.

BackgroundAngiotensin II (AngII) participates in endothelial damage and inflammation, and accelerates atherosclerosis. Endothelial lipase (EL) is involved in the metabolism and clearance of high density lipoproteins (HDL), the serum levels of which correlate negatively with the onset of cardiovascular diseases including atherosclerosis. However, the relationship between AngII and EL is not yet fully understood. In this study, we investigated the effects of AngII on the expression of EL and the signaling pathways that mediate its effects in human umbilical vein endothelial cells (HUVECs).Methods and FindingsHUVECs were cultured in vitro with different treatments as follows: 1) The control group without any treatment; 2) AngII treatment for 0 h, 4 h, 8 h, 12 h and 24 h; 3) NF-κB activation inhibitor pyrrolidine dithiocarbamate (PDTC) pretreatment for 1 h before AngII treatment; and 4) mitogen-activated protein kinase (MAPK) p38 inhibitor (SB203580) pretreatment for 1 h before AngII treatment. EL levels in each group were detected by immunocytochemical staining and western blotting. HUVECs proliferation was detected by MTT and proliferating cell nuclear antigen (PCNA) immunofluorescence staining. NF-kappa B (NF-κB) p65, MAPK p38, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and phosphorylated extracellular signal-regulated kinase (p-ERK) expression levels were assayed by western blotting. The results showed that the protein levels of EL, NF-κB p65, MAPK p38, JNK, and p-ERK protein levels, in addition to the proliferation of HUVECs, were increased by AngII. Both the NF-kB inhibitor (PDTC) and the MAPK p38 inhibitor (SB203580) partially inhibited the effects of AngII on EL expression.ConclusionAngII may upregulate EL protein expression via the NF-κB and MAPK signaling pathways.

Abstract 231: Hydrogen Sulfide Promotes Macrophage Polarization to Reduce Renal Inflammation in Angiotensin-II Induced Hypertension

Ageing is associated with structural and functional changes in the kidney and the presence of hypertension in this population further contributes to renal complications leading to renovascular sclerosis. Emerging evidence suggests that hydrogen sulfide (H 2 S) pathway participates in the pathogenesis of hypertension. Furthermore, chronic inflammation is known to play a critical role in ageing and development of hypertension. In this study, we hypothesized that angiotensin-II (Ang-II)-induced hypertension causes renal fibrosis by sustained activation of pro-inflammatory M1 macrophage promoting the release of inflammatory cytokines and treatment with H 2 S attenuates renal injury and fibrosis by promoting alternate activation of anti-inflammatory M2 macrophage. C57BL/6J (wild type, WT) mice, aged 18 months, were infused with Ang-II (1000ng/Kg/min) or saline using osmotic pumps for 4 weeks and treated without or with H 2 S in drinking water (30μM/L). Animal groups were 1) Saline, 2) Saline + H 2 S, 3) Ang-II, 4) Ang-II + H 2 S. Blood pressure was measured weekly and renal ultrasound was done to measure hemodynamic parameters. Systolic and diastolic blood pressure was increased in mice receiving Ang-II (160.73 ± 4.5 and 129.10 ± 4.9 respectively) and treatment with H 2 S showed significant reduction (134.5 ± 4.8 and 101.1 ± 4.5 respectively). Ultrasound revealed increased renal artery and intra-renal cortical artery resistive index in Ang-II mice (0.73 ± 0.03) which was significantly decreased following H2S treatment (0.61 ± 0.2). Ang-II increased the protein expression and mRNA levels of pro-inflammatory M1 macrophage marker, CD40, and TNF-α and MCP-1, whereas, H2S treatment increased the expression of anti-inflammatory M2 macrophage marker, CD206, and IL-10. Fluorescent probe H2DCF-DA showed increased intracellular reactive oxygen species with Ang-II treatment which was abrogated with H2S treatment. Our data suggests that H 2 S treatment in Ang-II-induced hypertension attenuates inflammation and hypertension by reducing classical activation of inflammatory M1 macrophage and promotes alternate activation of anti-inflammatory M2 macrophage.

Role of Two Types of Angiotensin II Receptors in Colorectal Carcinoma Progression

Angiotensin II (Ang-II) is a bioactive peptide associated closely with the progression and metastasis of colorectal cancer (CRC). We examined the expression and role of 2 Ang-II receptor types in 20 cases of CRC. Ang-II type 1 receptor (AT1R) protein was localized to the plasma membrane, whereas Ang-II type 2 receptor (AT2R) protein was localized to the nuclei. AT1R expression showed a direct correlation with tumor stage and liver metastasis, whereas AT2R expression showed an inverse correlation. A knockdown study of the AT1R or AT2R with Ang-II treatment was performed to reveal their individual roles in a mouse rectal cell line CMT93, which expresses both Ang-II receptor types. AT2R knockdown showed that the AT1R was associated with tumor growth, survival, invasion and VEGF-A secretion in CMT93 cells in a dose-dependent manner. In contrast, AT1R knockdown showed that the AT2R was associated with increased VEGF-A secretion at low Ang-II concentrations, whereas high concentrations of Ang-II inhibited tumor growth, survival, invasion and VEGF-A secretion. Thus, the AT1R showed a monophasic protumoral effect, while the AT2R showed a biphasic amphitumoral effect. Our findings suggest that a high angiotensinogen condition in the liver might evoke the antitumoral role of the AT2R in CRC cells.

YIA5 RGS-1 Regulates Leukocyte Trafficking in Atherosclerosis and Aortic Aneurysm Formation through Chemokine Receptor Desensitisation

The regulation of macrophage recruitment and retention into the vascular wall is critical in the progression of atherosclerosis and aortic aneurysm formation. This can be mediated by chemokine activation of...

Abstract 106: Prevention of Abdominal Aortic Aneurysm by Anti-MiRNA-712 or Anti-miR-205 in Angiotensin II--Infused Mice

Abdominal aortic aneurysm (AAA) is characterized by weakening of the vessel wall, followed by progressive expansion of the diseased aortic segment. MicroRNAs (miRNAs) have emerged as key regulator of gene expression in the cardiovascular diseases and may play a key role in therapeutically targeting AAA development. Although,vascular wall degradation by matrix metalloproteases (MMPs) is the key mechanism in AAA development, their targeting through miRNAs have never been studied. We identified microRNA-712 (miR-712) as a novel Angiotensin II(AngII)-sensitive miRNA which is upregulated in the abdominal aortic endothelium of AngII-infused mice. Mechanistically, we identified that miR-712 directly regulates two key endogenous inhibitors of MMP: tissue inhibitor of metalloproteinase 3 (TIMP3) and reversion inducing cysteine-rich protein with kazal motifs (RECK). Furthermore, inhibition of miR-712 by subcutaneous injection of anti-miR-712 significantly decreased MMP activity in the AngII-infused abdominal aorta wall, prevented the dilatation of aortae and significantly reduced AAA incidence from 80% (8/10) to 20% (2/10), compared to its mismatched control in ApoE -/- mice. Interestingly, based on the seeding sequence, we identified miR-205 as the human homolog of miR-712. miR-205 was also upregulated by AngII treatment and like miR-712 regulated MMPs activity via TIMP3 and RECK. Moreover, inhibition of miR-205 dramatically inhibits AngII-induced AAA development. We also found that miR-205 was significantly upregulated in the aortic sections of AAA patients in comparison to the healthy controls. Our findings demonstrate that AngII-sensitive miRNAs, miR-712 and miR-205, regulate MMP activity through TIMP3 and RECK and play important role in the pathogenesis of AAA. These results suggest that targeting these miRNAs using their inhibitors may hold promise as a therapeutic strategy to prevent the development of AAA.

Abstract 149: Regulator of G-Protein Signaling-1 Modulates Leukocyte Trafficking in Atherosclerosis and Aortic Aneurysm Formation Through Chemokine Receptor Desensitization

The regulation of macrophage recruitment and retention into the vascular wall is critical in the progression of atherosclerosis and aortic aneurysm formation. This can be mediated by chemokine activation of multiple G-protein coupled receptors. The Regulator of G-Protein Signaling-1 (RGS1) acts to deactivate the intracellular response to sustained chemokine stimulation. We have found that RGS1 is upregulated with atherosclerotic plaque progression and with monocyte-macrophage activation but its role is unknown. Rgs1-/- macrophages have significantly enhanced migratory responses to atherogenic chemokines and have impaired desensitization to chemokine re-stimulation (p<0.001). In vivo, RGS1 has a role in the accumulation of macrophages in atherosclerotic lesions and during Angiotensin II (AngII) aortic aneurysm rupture. In the absence of RGS1, atherosclerosis and macrophage accumulation is attenuated in early lesions in the aortic root and aortas of ApoE-/- mice (p<0.001). Rgs1-/- mice are protected from AngII induced aneurysm rupture compared to ApoE-/- mice with 94% survival vs. 56%. Rgs1-/- mice have significantly fewer CD11b+ myeloid cells and CD14+ macrophages in aortas than ApoE-/- mice (p<0.05) after 5 days of AngII infusion. Following bone marrow transplantation, recipient mice receiving ApoE-/- bone marrow were more susceptible to aortic aneurysm rupture (p=0.0124), indicating bone marrow-derived RGS1 is required for aneurysm rupture. Furthermore, AngII treatment increased systolic blood pressure to a greater extent in Rgs1-/- mice than ApoE-/- mice suggesting aneurysm formation in these mice is independent of AngII induced hypertension and this is mediated by vascular-derived RGS1. To gain insight into the mechanism by which RGS1 regulates trafficking, we selectively labelled inflammatory monocytes in vivo to track their movement into aortas following AngII infusion. We found an accumulation of labelled CD45+ cells in the aortas of ApoE-/- mice from day 3 to day 5 but not in Rgs1-/- mice indicating RGS1 as a regulator of macrophage retention in aortic aneurysms. These findings identify a novel role for RGS1 in leukocyte retention in vascular inflammation, highlighting RGS1 as a potential target in cardiovascular disease.

Abstract 377: Angiotensin II Drives Murine Ly-6chi Monocyte Trafficking from the Spleen to the Aneuvrismal Aorta Through B Cells

Abdominal aortic aneurysm (AAA) is widespread among elderly people and results in progressive expansion and rupture of the aorta with high mortality. AAA is histologically characterized by medial degeneration and various degrees of chronic adventitial inflammation. In particular, macrophages initially accumulate within the site of aneurysm. Early depletion of circulating monocytes protects against the disease. Two monocyte subsets with different migratory behaviours and functions have been described in mice, ‘inflammatory’ Ly6C+ monocytes (classical) and ‘patrolling’ Ly6C- monocytes (nonclassical). In this study, we aim to understand and clarify the role of monocyte-derived cells in AAA development. We monitored monocyte mobilisation up to 28 days in a mouse model of inflammatory AAA, which consists of implanting subcutaneous osmotic pumps delivering Angiotensin II (AngII) at a rate of 1000 ng/kg/min into C57BL6 ApoE-/- male mice between 8 and 12 weeks old. We found that AAA initiation is translated by a rapid and temporary mobilisation of classical monocytes at day 3-6 after AngII followed by non classical monocytes at days 7-9. We showed that classical monocytes are mobilized from the spleen, then recruited in the abdominal adventitia of ApoE-/- in response to AngII treatment as monitored through a pulse labelling method, along with an increased proteasic activity at the abdominal vessel wall as seen by fluorescence tomography. Importantly, we found that classical monocyte number positively correlated to stages of AAA. Splenectomy performed prior to AAA initiation prevented classical monocyte mobilization at day 3-6 after AngII and significantly protected against AAA. We found similar results in ApoE-/- Rag2-/- mice reconstituted with B cell-depleted splenocytes and treated with AngII, suggesting that splenic B cells are responsible for early deleterious mobilization of classical monocytes. Taken together, the data show that splenic classical monocytes directly participate to AAA pathology and could be exploited to improve the prognosis of patients harbouring AAA.

Abstract 122: STAT3 Mediates Toll-Like Receptor 4--Dependent Abdominal Aortic Aneurysm Formation in Angiotensin II--Treated ApoE-/- Mice

Abdominal aortic aneurysm (AAAs) is a life threatening disease and currently the only treatment is surgical or endovascular repair. Using an animal model for AngiotensinII (AngII) mediated AAA formation in the ApoE-/- mouse, we demonstrate that AAA formation is dependent on the activation of the JAK/STAT pathway. STAT3 activity was increased in the abdominal aortas of Ang II treated ApoE-/- mice. Treatment of ApoE-/- mice with AngII and the p-STAT3 inhibitor S3I-201 decreased the incidence of AAA formation and aortic diameter by greater than 50% compared to placebo. While AngII treatment increased activity of MMP2 and MMP9 in aortas of ApoE-/- mice, S3I-201 treatment inhibited p-STAT3 stimulation and decreased MMP2 and MMP9 activity to levels similar to those in control aortas. The increase in p-STAT3 levels and the secretion of MMP2 and MMP9 in AngII treated bone marrow derived macrophages (BDMs) from ApoE-/- mice was inhibited by pretreatment with S3I-201. Interestingly, AngII stimulated TLR4 expression in AAAs and in BDMs from ApoE-/- mice. Based on these findings we determined the role of TLR4 on AAA formation and p-STAT3 stimulation in ApoE-/-TLR4-/- mice. The incidence of AAA formation, increase in aortic diameter and MMP2 and MMP9 activity were markedly decreased in AngII treated ApoE-/-TLR4-/- mice compared to ApoE-/- controls. Importantly, AngII stimulation of p-STAT3 was also decreased in ApoE-/-TLR4-/- mice compared to control. Similar effects on AngII stimulated p-STAT3 and activity of MMP2 and MMP9 were demonstrated in BMDs from ApoE-/-TLR4-/- mice. Treatment of ApoE-/- mice with the TLR4 inhibitor Eritoran (Eisai, Inc) also decreased the incidence and severity of AAAs, MMPs secretion and p-STAT3 activity. Finally, we determined that S3I-201 decreased AngII stimulated TLR4 expression in AngII treated ApoE-/- mice and in BDM from ApoE-/- mice compared to placebo. These data supported the conclusion that AngII stimulation of AAAs is mediated via a TLR4 dependent activation of p-STAT3 and that both p-STAT3 and TLR4 might be potential therapeutic targets for the treatment of AAAs.

Opposite Effects of Gene Deficiency and Pharmacological Inhibition of Soluble Epoxide Hydrolase on Cardiac Fibrosis

Arachidonic acid-derived epoxyeicosatrienoic acids (EETs) are important regulators of cardiac remodeling; manipulation of their levels is a potentially useful pharmacological strategy. EETs are hydrolyzed by soluble epoxide hydrolase (sEH) to form the corresponding diols, thus altering and reducing the activity of these oxylipins. To better understand the phenotypic impact of sEH disruption, we compared the effect of EPHX2 gene knockout (EPHX2 −/−) and sEH inhibition in mouse models. Measurement of plasma oxylipin profiles confirmed that the ratio of EETs/DHETs was increased in EPHX2 −/− and sEH-inhibited mice. However, plasma concentrations of 9, 11, 15, 19-HETE were elevated in EPHX2 −/− but not sEH-inhibited mice. Next, we investigated the role of this difference in cardiac dysfunction induced by Angiotensin II (AngII). Both EPHX2 gene deletion and inhibition protected against AngII-induced cardiac hypertrophy. Interestingly, cardiac dysfunction was attenuated by sEH inhibition rather than gene deletion. Histochemical staining revealed that compared with pharmacological inhibition, EPHX2 deletion aggravated AngII-induced myocardial fibrosis; the mRNA levels of fibrotic-related genes were increased. Furthermore, cardiac inflammatory response was greater in EPHX2 −/− than sEH-inhibited mice with AngII treatment, as evidenced by increased macrophage infiltration and expression of MCP-1 and IL-6. In vitro, AngII-upregulated MCP-1 and IL-6 expression was significantly attenuated by sEH inhibition but promoted by EPHX2 deletion in cardiofibroblasts. Thus, compared with pharmacological inhibition of sEH, EPHX2 deletion caused the shift in arachidonic acid metabolism, which may led to pathological cardiac remodeling, especially cardiac fibrosis.

Cardiac hypertrophy is negatively regulated by miR-541.

Heart failure is a leading cause of death in aging population. Cardiac hypertrophy is an adaptive reaction of the heart against cardiac overloading, but continuous cardiac hypertrophy is able to induce heart failure. We found that the level of miR-541 was decreased in angiotensin II (Ang-II) treated cardiomyocytes. Enforced expression of miR-541 resulted in a reduced hypertrophic phenotype upon Ang-II treatment in cellular models. In addition, we generated miR-541 transgenic mice that exhibited a reduced hypertrophic response upon Ang-II treatment. Furthermore, we found miR-541 is the target of microphthalmia-associated transcription factor (MITF) in the hypertrophic pathway and MITF can negatively regulate the expression of miR-541 at the transcriptional levels. MITFce/ce mice exhibited a reduced hypertrophic phenotype upon Ang-II treatment. Knockdown of MITF also results in a reduction of hypertrophic responses after Ang-II treatment. Knockdown of miR-541 can block the antihypertrophic effect of MITF knockdown in cardiomyocytes upon Ang-II treatment. This indicates that the effect of MITF on cardiac hypertrophy relies on the regulation of miR-541. Our present study reveals a novel cardiac hypertrophy regulating pathway that was composed of miR-541 and MITF. Modulation of their levels may provide a new approach for tackling cardiac hypertrophy.

Renal responses to intra‐arterial infusion of a peroxynitrite scavenging agent with or without angiotensin II in anesthetized rats (1134.4)

Peroxynitrite (OONO‐) is continually formed in the biological tissue by the spontaneous reaction between nitric oxide and superoxide. However, its potential role in the regulation of renal hemodynamics and excretory function in control condition as well as in the condition of elevated angiotensin II (AngII) level is not yet clearly defined. In the present study, we examined the responses to a OONO‐ scavenging agent, mercaptoethyl guanidine (MEG; 5 µg/kg/min for 75 min) infused directly into the left renal artery in anesthetized (Inactin; 100 mg/kg; i.p.) rats pretreated with (n=5) or without (n=5) AngII (1 ng/kg/min). The MEG dose was effective in blocking the increased renal blood flow (RBF) response to a bolus dose infusion of OONO‐ (40 µg/kg). RBF was recorded by the Transonic flow probe placed on the left renal artery and glomerular filtration rate (GFR) was determined by inulin clearance. In control rats (n=5), MEG infusion did not alter renal vascular resistance (RVR), RBF or GFR but caused significant decreases in urine flow (V, 9.5±1.4 to 7.8±1.5 μL/min/g; ‐20.2±6.7%), sodium excretion (UNaV, 1.69±0.38 to 1.34±0.43 μmol/min/g; ‐29.3±14.2%) and in fractional excretion of sodium (FENa, 1.04±0.33 to 0.87±0.36%; ‐29.3±15%). In a separate group (n=5), AngII treatment caused an increase of 18.0±4.1% in RVR (baseline value, b; 14.8±1.9 mmHg/mL/min/g) and the decreases of 14.4±3.6% in RBF (b, 7.2 mL/min/g), 17.1±6.2% in V (b, 6.7±2.3 μL/min/g), 35.6±14.4% in UNaV (b, 1.17±0.61 μmol/min/g) and 37.4±12.5% in FENa (b, 0.9±0.6%) without appreciable change in GFR. However, MEG infusion in these AngII treated rats caused similar decreases in renal excretory parameters (‐19.7±10.9% in V, ‐31.8±13.4% in UNaV and ‐33.3±8.4% in FENa) without significant changes in RVR, RBF or GFR. These data indicate that the endogenous OONO‐ exerts minimal hemodynamic effect but contributes to the maintenance of renal excretory function both in control condition as well as in the condition of elevated AngII level.

Angiotensin II activates the proteasome and stimulates vascular smooth muscle cell proliferation (866.10)

Growing evidence implicates an important role of the proteasome in cardiovascular disease. However, the role of the proteasome in hypertension is understudied. We showed previously that proteasome inhibition attenuated angiotensin hypertension‐induced aortic hypertrophy in vivo. This study tested the hypothesis that proteasome inhibition attenuates angiotensin‐induced vascular smooth muscle proliferation.Experiments were performed on a rat embryonic aortic cell line, A7r5 (ATCC) and human umbilical artery vascular smooth muscle primary cells (Cell Applications) (HUAVSM). The cells were treated for 48 hours with vehicle (0.01% DMSO), angiotensin II (AngII) (100 nM), a proteasome inhibitor, bortezomib (Bort) (5 nM) or both (AngII+Bort). Cell number and viability were assessed by flow cytometry. Cells were also stained for Ki67, a marker of proliferating cells, and DAPI to stain DNA. Adenoviral infection was used to co‐express a green fluorescent protein fused with a ubiquitination signal sequence (GFPu) and a red fluorescent protein (RFP) in the cell. The GFPu to RFP ratio was taken as an inverse index of ubiquitin‐proteasome system (UPS) activity.Compared to vehicle, Ang II treatment increased UPS activity. Cell number was increased by AngII in both A7r5 and HUAVSM cells. AngII treatment increased Ki67 staining compared to vehicle in both A7r5 and HUAVSM cells. Treatment with Bort alone did not affect cell viability or cell number in either A7r5 or HUAVSM cells. On the other hand, Bort treatment abolished the AngII‐induced increase in UPS activity, cell number and Ki67 staining.Collectively, these data suggest that AngII‐induced vascular smooth muscle cell proliferation is mediated, at least in part, via activation of the UPS. This effect may contribute to AngII hypertension induced vascular remodeling.Grant Funding Source: Supported by HL072166, HL085629 (X.J.W.), HL109964 (Y.L.), NIGMS: P20GM103443 SD BRIN (D.M., H.F.)

Endothelial NADPH Oxidase-2 Promotes Interstitial Cardiac Fibrosis and Diastolic Dysfunction Through Proinflammatory Effects and Endothelial-Mesenchymal Transition

This study sought to investigate the effect of endothelial dysfunction on the development of cardiac hypertrophy and fibrosis. Endothelial dysfunction accompanies cardiac hypertrophy and fibrosis, but its contribution to these conditions is unclear. Increased nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX2) activation causes endothelial dysfunction. Transgenic mice with endothelial-specific NOX2 overexpression (TG mice) and wild-type littermates received long-term angiotensin II (AngII) infusion (1.1 mg/kg/day, 2 weeks) to induce hypertrophy and fibrosis. TG mice had systolic hypertension and hypertrophy similar to those seen in wild-type mice but developed greater cardiac fibrosis and evidence of isolated left ventricular diastolic dysfunction (p < 0.05). TG myocardium had more inflammatory cells and VCAM-1-positive vessels than did wild-type myocardium after AngII treatment (both p < 0.05). TG microvascular endothelial cells (ECs) treated with AngII recruited 2-fold more leukocytes than did wild-type ECs in an in vitro adhesion assay (p < 0.05). However, inflammatory cell NOX2 per se was not essential for the profibrotic effects of AngII. TG showed a higher level of endothelial-mesenchymal transition (EMT) than did wild-type mice after AngII infusion. In cultured ECs treated with AngII, NOX2 enhanced EMT as assessed by the relative expression of fibroblast versus endothelial-specific markers. AngII-induced endothelial NOX2 activation has profound profibrotic effects in the heart in vivo that lead to a diastolic dysfunction phenotype. Endothelial NOX2 enhances EMT and has proinflammatory effects. This may be an important mechanism underlying cardiac fibrosis and diastolic dysfunction during increased renin-angiotensin activation.

Angiotensin II Removes Kidney Resistance Conferred by Ischemic Preconditioning

Ischemic preconditioning (IPC) by ischemia/reperfusion (I/R) renders resistance to the kidney. Strong IPC triggers kidney fibrosis, which is involved in angiotensin II (AngII) and its type 1 receptor (AT1R) signaling. Here, we investigated the role of AngII/AT1R signal pathway in the resistance of IPC kidneys to subsequent I/R injury. IPC of kidneys was generated by 30 minutes of bilateral renal ischemia and 8 days of reperfusion. Sham-operation was performed to generate control (non-IPC) mice. To examine the roles of AngII and AT1R in IPC kidneys to subsequent I/R, IPC kidneys were subjected to either 30 minutes of bilateral kidney ischemia or sham-operation following treatment with AngII, losartan (AT1R blocker), or AngII plus losartan. IPC kidneys showed fibrotic changes, decreased AngII, and increased AT1R expression. I/R dramatically increased plasma creatinine concentrations in non-IPC mice, but not in IPC mice. AngII treatment in IPC mice resulted in enhanced morphological damage, oxidative stress, and inflammatory responses, with functional impairment, whereas losartan treatment reversed these effects. However, AngII treatment in non-IPC mice did not change I/R-induced injury. AngII abolished the resistance of IPC kidneys to subsequent I/R via the enhancement of oxidative stress and inflammatory responses, suggesting that the AngII/AT1R signaling pathway is associated with outcome in injury-experienced kidney.