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Aortic aneurysms (AAs) are a major public health challenge, featured by a progressive impairs in aortic wall integrity that drives to aortic dilation and, in end stage, to its rupture. Despite important advances in the surgical treatment of aortic aneurysms, there is currently no pharmacological intervention that prevents their development, reduces their expansion, or avoids their rupture. In addition to classic risk factors such age or gender, several heritable connective tissue disorders have been associated with AA developing, highlighting the role of extracellular matrix (ECM) genes alterations in the developing of AA. In this sense, we have recently demonstrated that global deletion of the cellular communicating network factor 2 (CCN2), previously known as connective tissue growth factor (CTGF) due to its role in the extracellular matrix formation, predisposes to early and lethal AAs development after Angiotensin II (Ang II) infusion in mice. Here, we detail the protocol to induce and detect AAs generation in inducible global CCN2 knockout mice after Ang II infusion which allow the characterization of CCN role in AA development and may help to the development of pharmacological target for AA treatment., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Renin-angiotensin system (RAS) plays an indispensable role in regulating blood pressure through its effects on fluid and electrolyte balance. As an aside, cumulative evidence from experimental to clinical studies supports the notion that dysregulation of RAS contributes to the pro-inflammatory, pro-oxidative, and pro-fibrotic processes that occur in pulmonary diseases like asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and acute lung injury (ALI). Pharmacological intervention of the various RAS components can be a novel therapeutic strategy for the treatment of these respiratory diseases. In this chapter, we first give a recent update on the RAS, and then compile, review, and analyse recent reports on targeting RAS components as treatments for respiratory diseases. Inhibition of the pro-inflammatory renin, angiotensin-converting enzyme (ACE), angiotensin (Ang) II, and Ang II type 1 receptor (AT1R) axis, and activation of the protective ACE2, AT2R, Ang (1-7), and Mas receptor axis have demonstrated varying degrees of efficacies in experimental respiratory disease models or in human trials. The newly identified alamandine/Mas-related G-protein-coupled receptor member D pathway has shown some therapeutic promise as well. However, our understanding of the RAS ligand-and-receptor interactions is still inconclusive, and the modes of action and signaling cascade mediating the newly identified RAS receptors remain to be better characterized. Clinical data are obviously lacking behind the promising pre-clinical findings of certain well-established molecules targeting at different pathways of the RAS in respiratory diseases. Translational human studies should be the focus for RAS drug development in lung diseases in the next decade., (Copyright © 2023. Published by Elsevier Inc.)

Angiotensin II (AngII) induces disruption of mitochondrial homeostasis and oxidative stress. Nuclear receptor NR4A1 (Nur77) plays an important role in vascular smooth muscle cells (VSMCs) function. However, the role of Nur77 in AngII-induced mitochondrial dynamics and oxidative stress in VSMCs remains unknown. In an in vitro model of AngII-treated cells, we discovered that Nur77 knockout aggravated AngII-induced oxidative stress in VSMCs, whereas activation of Nur77 by celastrol diminished them. Concomitantly, disturbance of mitochondrial dynamics induced by AngII was further exacerbated in Nur77 deficient VSMCs compared to wild-type (WT) VSMCs. Interestingly, Nur77 deletion increased mitochondrial fission but not fusion as evidenced by upregulated fission related genes (Fis1 and Drp1) but not fusion (Opa1 and Mfn2) under AngII stimulation in VSMCs. Mechanically, Nur77 could directly bind to the promoter regions of Fis1 and Drp1 and repress their transcription. Furthermore, we observed that Nur77 additionally promoted mitochondrial homeostasis by increasing mitophagic flux in a transcription-independent manner upon AngII challenge. By using an in vivo model of AngII-induced abdominal aortic aneurysm (AAA), we finally validated the protective role of Nur77 involved in the mitochondrial fission process and mitophagic flux in aortas, which was correlated with the occurrence and development of AAA in AngII-infused mice. Our data defines an essential role of Nur77 in regulating oxidative stress by maintaining mitochondrial homeostasis in VSMCs via both transcription-dependent and transcription-independent manner, supporting the therapeutic potential of Nur77 targeting in vascular diseases., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Effects of hypertrophic challenge on small-conductance, Ca 2+ -activated K + (SK2) channel expression were explored in intact murine hearts, isolated ventricular myocytes and neonatal rat cardiomyocytes (NRCMs). An established experimental platform applied angiotensin II (Ang II) challenge in the presence and absence of reduced p21-activated kinase (PAK1) (PAK1 cko vs. PAK1 f/f , or shRNA-PAK1 interference) expression. SK2 current contributions were detected through their sensitivity to apamin block. Ang II treatment increased such SK2 contributions to optically mapped action potential durations (APD 80 ) and their heterogeneity, and to patch-clamp currents. Such changes were accentuated in PAK1 cko compared to PAK1 f/f , intact hearts and isolated cardiomyocytes. They paralleled increased histological and echocardiographic hypertrophic indices, reduced cardiac contractility, and increased SK2 protein expression, changes similarly greater with PAK1 cko than PAK1 f/f . In NRCMs, Ang II challenge replicated such increases in apamin-sensitive SK patch clamp currents as well as in real-time PCR and western blot measures of SK2 mRNA and protein expression and cell hypertrophy. Furthermore, the latter were enhanced by shRNA-PAK1 interference and mitigated by the PAK1 agonist FTY720. Increased CaMKII and CREB phosphorylation accompanied these effects. These were rescued by both FTY720 as well as the CaMKII inhibitor KN93, but not its inactive analogue KN92. Such CREB then specifically bound to the KCNN2 promoter sequence in luciferase assays. These findings associate Ang II induced hypertrophy with increased SK2 expression brought about by a CaMKII/CREB signaling convergent with the PAK1 pathway thence upregulating the KCNN2 promoter activity. SK2 may then influence cardiac electrophysiology under conditions of cardiac hypertrophy and failure., (Copyright © 2021. Published by Elsevier Ltd.)

It is well known that lectin-like oxidized low-density lipoprotein (ox-LDL) and its receptor LOX-1, angiotensin II (AngII) and its type 1 receptor (AT 1 -R) play an important role in the development of cardiac hypertrophy. However, the molecular mechanism is not clear. In this study, we found that ox-LDL-induced cardiac hypertrophy was suppressed by inhibition of LOX-1 or AT1-R but not by AngII inhibition. These results suggest that the receptors LOX-1 and AT1-R, rather than AngII, play a key role in the role of ox-LDL. The same results were obtained in mice lacking endogenous AngII and their isolated cardiomyocytes. Ox-LDL but not AngII could induce the binding of LOX-1 and AT1-R; inhibition of LOX-1 or AT1-R but not AngII could abolish the binding of these two receptors. Overexpression of wild type LOX-1 with AT1-R enhanced ox-LDL-induced binding of two receptors and phosphorylation of ERKs, however, transfection of LOX-1 dominant negative mutant (lys266ala / lys267ala) or an AT1-R mutant (glu257ala) not only reduced the binding of two receptors but also inhibited the ERKs phosphorylation. Phosphorylation of ERKs induced by ox-LDL in LOX-1 and AT 1 -R-overexpression cells was abrogated by an inhibitor of Gq protein rather than Jak2, Rac1 or RhoA. Genetically, an AT1-R mutant lacking Gq protein coupling ability inhibited ox-LDL induced ERKs phosphorylation. Furthermore, through bimolecular fluorescence complementation analysis, we confirmed that ox-LDL rather than AngII stimulation induced the direct binding of LOX-1 and AT 1 -R. We conclude that direct binding of LOX-1 and AT1-R and the activation of downstream Gq protein are important mechanisms of ox-LDL-induced cardiomyocyte hypertrophy., (Copyright © 2021. Published by Elsevier Ltd.)

Under stress, the heart undergoes extensive remodeling resulting in cardiac fibrosis and hypertrophy, ultimately contributing to chronic heart failure (CHF). Alterations in microRNA levels are associated with dysfunctional gene expression profiles involved in the pathogenesis of heart failure. We previously showed that myocardial infarction-induced microRNA-enriched extracellular vesicles (EVs) contribute to the reduction in antioxidant enzymes by targeting Nrf2 signaling in CHF. MicroRNA-27a (miRNA-27a) is the predominant microRNA contained in cardiac fibroblast-derived EVs contributing to oxidative stress along with hypertrophic gene expression in cardiomyocytes. In the present study, we observed that miRNA-27a passenger strand (miRNA-27a*) was markedly upregulated in the non-infarcted area of the left ventricle of rats with CHF and encapsulated into EVs and secreted into the circulation. Bioinformatic analysis revealed that PDZ and LIM domain 5 (PDLIM5) is one of the major targets of miRNA-27a*, playing a major role in cardiac structure and function, and potentially contributing to the progression of cardiac hypertrophy. Our in vivo data demonstrate that PDLIM5 is down-regulated in the progression of heart failure, accompanied with the upregulation of hypertrophic genes and consistent with alterations in miRNA-27a*. Moreover, exogenous administration of miRNA27a* mimics inhibit PDLIM5 translation in cardiomyocytes whereas a miRNA27a* inhibitor enhanced PDLIM5 expression. Importantly, we confirmed that infarcted hearts have higher abundance of miRNA-27a* in EVs compared to normal hearts and further demonstrated that cultured cardiac fibroblasts secrete miRNA27a*-enriched EVs into the extracellular space in response to Angiotensin II stimulation, which inhibited PDLIM5 translation, leading to cardiomyocyte hypertrophic gene expression. In vivo studies suggest that the administration of a miRNA-27a* inhibitor in CHF rats partially blocks endogenous miR-27a* expression, prevents hypertrophic gene expression and improves myocardial contractility. These findings suggest that cardiac fibroblast-secretion of miRNA27a*-enriched EVs may act as a paracrine signaling mediator of cardiac hypertrophy that has potential as a novel therapeutic target., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: It has been noted that dysregulation of microRNAs (miRNAs) contributes to the formation of abdominal aortic aneurysm (AAA), a vascular disease associated with progressive aortic dilatation and degradation, and pathological infiltration and activation of inflammatory cells, such as macrophages. Our microarray data revealing that miR-144-5p was the top 1 downregulated miRNA in mouse AAA tissues as compared to normal aortas motivated us to explore its role in AAA development., Methods: We profiled miRNA and mRNA expression in Angiotensin II (Ang II)- (n = 3) and saline-infused abdominal aortas (n = 4) via Agilent microarrays, and further validated the data with real-time QPCR. In vivo, miR-144-5p or control agomirs were given to Apoe -/- mice with Ang II infusion-induced AAA. In vitro, mouse RAW 264.7 macrophages and human THP-1 macrophage-like cells were transfected with miR-144-5p or control agomirs/antagomirs, and oxidized Low Density Lipoprotein (ox-LDL) was used to stimulate M1 macrophage polarization., Results: Based on the microarray and real-time QPCR validation data, we identified miR-144-5p as a novel downregulated miRNA in AAA tissues. Overexpression of miR-144-5p by utilizing its specific agomirs in vivo significantly attenuated Ang II-induced aortic dilatation and elastic degradation in Apoe -/- mice and improved their survival. AAA incidence was reduced by miR-144-5p as well. MiR-144-5p polarized macrophages to M2 type in Ang II-infused aortas. Further, the expression levels of two predictive targets for miR-144-5p, Toll Like Receptor 2 (TLR2) and ox-LDL Receptor 1 (OLR1), were higher in AAA specimens, and negatively correlated to miR-144-5p (Pearson correlation coefficient r < -0.9, P < .01). These two molecules were then confirmed as novel miR-144-5p targets via dual-luciferase assay. MiR-144-5p agomirs suppressed ox-LDL-induced upregulation of M1 macrophage markers, including interleukin 1β (IL1β), tumor necrosis factor α (TNFα), prostaglandin-endoperoxide synthase 2 (PTGS2) and nitric oxide synthase 2 (NOS2), in macrophages probably by targeting TLR2. MiR-144-5p also inhibited the signaling transduction of pathways downstream to TLR2 and OLR1, including NF-κB and ERK1/2 pathways, whose abnormal activation contributed AAA formation., Conclusion: Our work suggests miR-144-5p as a novel regulator for AAA pathology. Management of miR-144-5p and its targets TLR2 and OLR1 provides therapeutic potential for limiting AAA formation., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Hypertension is an independent risk factor for atrial fibrillation (AF), although its specific mechanisms remain unclear. Previous research has been focused on cyclic stretch, ignoring the role of high hydrostatic pressure. The present study aimed to explore the effect of high hydrostatic pressure stimulation on electrical remodeling in atrial myocytes and its potential signaling pathways. Experiments were performed on left atrial appendages from patients with chronic AF or sinus rhythm, spontaneously hypertensive rats (SHRs) treated with or without valsartan (10 mg/kg/day) and HL-1 cells were exposed to high hydrostatic pressure using a self-developed device. Whole-cell patch-clamp recordings and western blots demonstrated that the amplitudes of I Ca,L , I to , and I Kur were reduced in AF patients with corresponding changes in protein expression. Angiotensin protein levels increased and Ang1-7 decreased, while focal adhesion kinase (FAK) and Src kinase were enhanced in atrial tissue from AF patients and SHRs. After rapid atrial pacing, AF inducibility in SHR was significantly higher, accompanied by a decrease in I Ca,L , upregulation of I to and I Kur , and a shortened action potential duration. Angiotensin upregulation and FAK/Src activation in SHR were inhibited by angiotensin type 1 receptor inhibitor valsartan, thus, preventing electrical remodeling and reducing AF susceptibility. These results were verified in HL-1 cells treated with high hydrostatic pressure, and demonstrated that electrical remodeling regulated by the FAK-Src pathway could be modulated by valsartan. The present study indicated that high hydrostatic pressure stimulation increases AF susceptibility by activating the renin-angiotensin system and FAK-Src pathway in atrial myocytes., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: In the present study, we used a two-kidney-two-clip (2k2c) stroke-prone renovascular hypertension rat model (RHRSP) to investigate the protective effects of ligustrazine (TMP) on cerebral arteries and to examine PI3K/Akt pathway behavior under this protection., Methods: The cerebral artery remodeling was induced by 2k2c-induced renovascular hypertension. Brain basilar artery tissues were isolated and their histological changes were detected through H&E and EVG staining, α-SMA IHC staining, and transmission electron microscopy at four, eight, and twelve weeks after 2k2c surgery, both with and without TMP treatment. Meanwhile, the ET-1, Ang II, and NO levels in basilar arteries and plasma were determined. Furthermore, the PTEN expression and the activation of PI3K/Akt in basilar artery tissues were detected through IHC and Western Blot. In addition, the primary basilar artery smooth muscle cells (BASMCs) were cultured and TMP protection of BASMCs stimulated with ET-1/Ang II in the presence or absence of insulin-like growth factor 1 (IGF-1) was determined., Results: TMP attenuated basilar artery remodeling, decreased ET-1 and Ang II levels and increased NO level in basilar arteries and plasma of RHRSP rats. Moreover, TMP reduced BASMCs proliferation upon ET-1/Ang II stimulation. We also found that TMP could effectively suppress the activation of PI3K/Akt in 2k2c-RHRSP rat basilar artery and ET-1/Ang II stimulated BASMCs. Most importantly, IGF-1, as an activator of PI3K/Akt, could damage the protective effect of TMP., Conclusions: TMP exerts its protective effects and prevents basilar artery remodeling in RHRSP rats at least partly through the inhibition of PI3K/Akt pathway., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Acute lung injury is one of the most popular consequences of hepatic ischemia/reperfusion (I/R) injury. Recently it was documented that renin-angiotensin system plays a key role in tissue inflammation, generation of reactive oxygen species (ROS) and tumor necrosis factor-alpha (TNF-α) (the principal liver injury mediators) during I/R., Material and Methods: We investigated the effect of acute versus chronic usage of angiotensin converting enzyme inhibitor (captopril) on liver inflammation and lung injury caused by hepatic ischemia for 1h followed by 24h reperfusion. Forty adult Wistar male rats were divided into sham, I/R, I/R-acute captopril (100 mg/kg, 24 and 1.5 h before surgery) and I/R-chronic captopril (10 mg/kg/day for 28 days before surgery) groups., Results: We found captopril pretreatment significantly decreased liver damage indices, adhesion molecules, and TNF-α level in hepatic and tracheal tissues. Histologically, acute captopril pretreatment significantly decreased hepatic Kupffer cells number and lung α-smooth muscle actin expression more than chronic pretreatment. Increased tracheal tone, in response to acetylcholine, was suppressed by acute and chronic captopril pretreatment., Conclusion: Angiotensin II plays a key role in tissue inflammation and airway hyperresponsiveness (AHR) via enhancing production of TNF-α. With more protection observed in lung, acute captopril could attenuate liver-induced lung injury via lowering TNF-α; a suggested possible mediator of airway hyperreactivity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Atrial fibrillation (AF) is prevalent in hypertension and elevated angiotensin II (Ang II); however, the mechanisms by which Ang II leads to AF are poorly understood. Here, we investigated the basis for this in mice treated with Ang II or saline for 3 weeks. Ang II treatment increased susceptibility to AF compared to saline controls in association with increases in P wave duration and atrial effective refractory period, as well as reductions in right and left atrial conduction velocity. Patch-clamp studies demonstrate that action potential (AP) duration was prolonged in right atrial myocytes from Ang II treated mice in association with a reduction in repolarizing K + currents. In contrast, APs in left atrial myocytes from Ang II treated mice showed reductions in upstroke velocity and overshoot, as well as greater prolongations in AP duration. Ang II reduced Na + current (I Na ) in the left, but not the right atrium. This reduction in I Na was reversible following inhibition of protein kinase C (PKC) and PKCα expression was increased selectively in the left atrium in Ang II treated mice. The transient outward K + current (I to ) showed larger reductions in the left atrium in association with a shift in the voltage dependence of activation. Finally, Ang II caused fibrosis throughout the atria in association with changes in collagen expression and regulators of the extracellular matrix. This study demonstrates that hypertension and elevated Ang II cause distinct patterns of electrical and structural remodeling in the right and left atria that collectively create a substrate for AF., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cardiac tyhrotropin-releasing hormone (TRH) is overexpressed in the hypertrophied left ventricle (LV) of spontaneously hypertensive rats (SHR) and its inhibition prevents both hypertrophy and fibrosis. In a normal heart, the TRH increase induces fibrosis and hypertrophy opening the question of whether TRH could be a common mediator of left ventricular hypertrophy (LVH). We used angiotensin II (AngII) as an inductor of LVH to evaluate if the blockade of LV-TRH prevents hypertrophy and fibrosis in mice. We challenged C57BL/6 adult male mice with an infusion of AngII (osmotic pumps; 2 mg/kg.day) to induce LVH. Groups of mice were injected with an intracardiac siRNA-TRH or scrambled siRNA (siRNA-Con). Body weight, water intake and systolic arterial blood pressure (SABP) were measured daily. AngII significantly increased water intake and SABP (p < .05). Cardiac hypertrophy (heart weight/body weight) was evident in the group with the normal cardiac TRH system. In fact, it was found an AngII-induced increase of TRH precursor mRNA (p < .05) in conjunction with elevated TRH levels measured by immunohistochemistry and western blot. These changes were not observed in the AngII + siRNA-TRH group. Furthermore, AngII increased significantly (p < .05) BNP (hypertrophic marker), collagens I and III and TGF-β (fibrosis markers) expression in the group with the native cardiac TRH system. These increases were attenuated in the groups with the TRH system blocked despite the high blood pressure. Similar and stronger results were observed "in vitro" with NIH3T3 and H9C2 cell culture models, where, when the TRH system is blocked, AngII stimulus was not able to induce the markers of its fibrotic and hypertrophic effects, so we believe that these effects are independent of any other physiological modifications. Our results point out that cardiac TRH is required for AngII-induced hypertrophic and fibrotic effects., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Myocardial fibroblast activation coupled with extracellular matrix production is a pathological signature of myocardial fibrosis and is governed mainly by transforming growth factor TGFβ-Smad2/3 signaling. Targeting the ubiquitous TGFβ leads to cellular homeostasis deregulation with adverse consequences. We previously showed the anti-fibrotic effects upon downregulation of 90-kDa heat shock protein (Hsp90), a chaperone that associates to the TGFβ signaling cascade. In the present study, we use a fluorescent-labeled Hsp90 protein inhibitor (CTPR390-488) with specific Hsp90 binding properties to reduce myocardial pro-fibrotic events in vitro and in vivo. The mechanism of action involves the disruption of TGFβRI-Hsp90 complex, resulting in a decrease in TGFβ signaling and reduction in extracellular matrix collagen. In vivo, decreased myocardial collagen deposition was observed upon CTPR390-488 treatment in a pro-fibrotic mouse model. This is the first study demonstrating the ability of an engineered Hsp90 protein inhibitor to block collagen expression, reduce the motility of myocardial TGFβ-activated fibroblasts and ameliorate angiotensin-II induced cardiac myocardial fibrosis in vivo., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cilostazol (Ciloz) a potent Type III phosphodiesterase inhibitor is effective against inflammation, insulin resistance and cardiomyopathy. However, the effect of Ciloz on obesity-associated left ventricular diastolic dysfunction has not been explored yet. Hence, we examined the effect of Ciloz on cardiac remodelling and dysfunction in non-obese and obese-insulin resistant mice infused with AngiotensinII (AngII). Male C57BL/6 J mice were initially subjected to 19 weeks of chow or high fat diet (HFD) regimen and thereafter animals were randomised for AngII (1500 ng/kg/min, s.c) infusion or saline and Ciloz (50 mg/kg, p.o) for another 1 week. Obese and non-obese mice infused with AngII exhibited significant diastolic dysfunction and features of heart failure with preserved ejection fraction (HFpEF) since a decrease in fractional shortening and no change in ejection fraction were observed when compared with respective controls. Administration of AngII and Ciloz in HFD fed mice significantly improved the left ventricular function compared with AngII infused HFD mice as evinced from the echocardiographic data. Further, Ciloz treatment significantly reduced cardiomyocyte area, interstitial and perivascular fibrosis; and collagen deposition. Moreover, Ciloz reduced the inflammatory milieu in the heart as evinced by decreased F4/80 + and CD68 + cells; IL-1β and IL-6 gene transcripts. Quantitative assessment of the expression levels revealed substantial upregulation of MMP-9 (pro- and mature-forms) and α-SMA in the left ventricle of AngII infused HFD-fed mice, which was considerably suppressed by Ciloz regimen. The beneficial effect of Ciloz was associated with the normalization in gene expression of hypertrophic and fibrotic markers. Likewise, Ciloz administration markedly reduced the AngII and HFD induced TGF-β1/SMAD3 and Akt/mTOR signalling. Additionally, AngII administered and HFD-fed mice showed increased glycolytic flux, which was considerably diminished by Ciloz treatment as indicated from suppressed PKM2, HK-2, PDK-2, HIF-1α mRNA and GLUT-1 protein expression. Taken together, Ciloz might be therapeutically exploited against AngII and obesity-associated diastolic dysfunction thereby preventing overt heart failure., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cardiac hypertrophy is a common response of cardiac myocytes to stress and a predictor of heart failure. While in vitro cell culture studies have identified numerous molecular mechanisms driving hypertrophy, it is unclear to what extent these mechanisms can be integrated into a consistent framework predictive of in vivo phenotypes. To address this question, we investigate the degree to which an in vitro-based, manually curated computational model of the hypertrophy signaling network is able to predict in vivo hypertrophy of 52 cardiac-specific transgenic mice. After minor revisions motivated by in vivo literature, the model concordantly predicts the qualitative responses of 78% of output species and 69% of signaling intermediates within the network model. Analysis of four double-transgenic mouse models reveals that the computational model robustly predicts hypertrophic responses in mice subjected to multiple, simultaneous perturbations. Thus the model provides a framework with which to mechanistically integrate data from multiple laboratories and experimental systems to predict molecular regulation of cardiac hypertrophy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cardiac fibrosis is a common pathologic consequence of stress insult to the heart and is characterized by abnormal deposition of fibrotic extracellular matrix that compromises cardiac function. Cardiac fibroblasts are key mediators of fibrotic remodeling and are regulated by secreted stress-response proteins. The matricellular protein connective tissue growth factor (CTGF), or CCN2, is strongly produced by injured cardiomyocytes and although it is considered a pro-fibrotic factor in many organ systems, its role in cardiac fibrosis is controversial. Here we adopted a cell-specific genetic approach to conditionally delete CCN2 in either cardiomyocytes or activated fibroblasts. Fibrosis was induced by angiotensin II-based neurohumoral stimulation, an insult that strongly induces CCN2 expression from cardiomyocytes and to a lesser extent in fibroblasts. Remarkably, only CCN2 deletion from activated fibroblasts inhibited the fibrotic remodeling while deletion from cardiomyocytes (the main source of CCN2 in the heart) had no effects. In vitro experiments revealed that although efficiently secreted by both fibroblasts and cardiomyocytes, only fibroblast-derived CCN2 is proficient in its ability to fully activate fibroblasts. These results overall indicate that although secreted into the extracellular matrix, CCN2 acts in an autocrine fashion. Secretion of CCN2 by cardiomyocytes is not pro-fibrotic, while fibroblast-derived CCN2 can modulate fibrosis in the heart. In conclusion we found that cardiomyocyte-derived CCN2 is dispensable for cardiac fibrosis, while inhibiting CCN2 induction in activated fibroblasts is sufficient to abrogate the cardiac fibrotic response to angiotensin II. Hence, CCN2 is an autocrine factor in the heart., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Hypertension is associated with hypertrophy and hyperplasia of smooth muscle cells (SMCs). Disintegrin and metalloproteinase 17 (ADAM17) is a membrane-bound enzyme reported to mediate SMC hypertrophy through activation of epidermal growth factor receptor (EGFR). We investigated the role of ADAM17 in Ang II-induced hypertension and end-organ damage. VSMC was isolated from mice with intact ADAM17 expression (Adam17 f/f ) or lacking ADAM17 in the SMC (Adam17 f/f /Cre Sm22 ). Human VSMCs were isolated from the aorta of donors, and ADAM17 deletion was achieved by siRNA transfection. Ang II suppressed proliferation and migration of Adam17-deficient SMCs, but did not affect apoptosis (mouse and human), this was associated with reduced activation of EGFR and Erk1/2 signaling. Adam17 f/f /Cre Sm22 and littermate Adam17 f/f mice received saline or Ang II (Alzet pumps, 1.5mg/kg/d; 2 or 4weeks). Daily blood pressure measurement in conscious mice (telemetry) showed suppressed hypertension in Adam17 f/f /Cre Sm22 mice during the first week of Ang II infusion, but by the second week, it become comparable to that in Adam17 f/f mice. EGFR activation remained suppressed in Adam17 f/f /Cre Sm22 -Ang II arteries. Ex vivo vascular function and compliance assessed in mesenteric arteries were comparable between genotypes. Consistent with the transient protection against Ang II-induced hypertension, Adam17 f/f /Cre Sm22 mice exhibited significantly lower cardiac hypertrophy and fibrosis, and renal fibrosis at 2weeks post-Ang II, however this protection was abolished by the fourth week of Ang II infusion. In conclusion, while Adam17-deficiency suppresses Ang II-induced SMC remodeling in vitro, in vivo Adam17-deficiency provides only a transient protective effect against Ang II-mediated hypertension and end-organ damage., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The renin-angiotensin system (RAS) is a key regulator of blood pressure and blood volume homeostasis. The RAS is primarily comprised of the precursor protein angiotensinogen and the two proteases, renin and angiotensin-converting enzyme (ACE). Angiotensin I (Ang I) is derived from angiotensinogen by renin, but appears to have no biological activity. In contrast, angiotensin II (Ang II) that has a variety of biological functions in the cells is converted from Ang I through removal of two-C-terminal residues by ACE. The physiological effects of Ang II are due to Ang II signaling through specific receptor binding, resulting in muscle contraction leading to increased blood pressure and volume. To modulate RAS, three classes of drugs have been developed: (1) renin inhibitors to prevent angiotensinogen conversion to Ang I, (2) ACE inhibitors, to prevent Ang I processing to Ang II and (3) angiotensin receptor blockers, to inhibit Ang II signaling through its receptor. Studies using the RAS inhibitors and Ang II demonstrated that RAS signaling mediates actions of Ang II in the regulation of proliferation and differentiation of specific hematopoietic cell types, especially in the red blood cell lineage. Accumulating evidence indicates that RAS regulates EPO, an essential mediator of red cell production, for human anemia and erythropoiesis in vivo and in vitro. The regulation of EPO expression by Ang II may be responsible for maintaining red blood cell homeostasis. This review highlights the biological roles of RAS for blood cell and EPO homeostasis through Ang II signaling. The molecular mechanism for Ang II-induced EPO production of the cell or tissue type-specific expression is discussed., (2017 Published by Elsevier Inc.)

Background: To address the impact of antidiabetic drugs on cardiovascular safety is a matter of clinical concern. Preclinical studies revealed that various protective effects of dipeptidyl peptidase-4 inhibitor (DPP4i) on cardiovascular disease; however, its impact of on hypertension remains controversial., Methods and Results: Teneligliptin (TEN; 10mg/kg/day/p.o.) ameliorates hypertension and cardiac remodeling by normalizing a rise of angiotensin-II (AngII) that specifically observed in spontaneously hypertensive rats (SHR). TEN had no effects on vasculature and concentrations of the DPP4-related vasoactive peptides (bradykinin, neuropeptide Y, and atrial natriuretic peptide). The primary action of TEN on BP lowering was due to restoring the AngII-induced manifestation of congestive heart failure observed in SHR. Sodium-proton pump exchanger type 1 (NHE-1) is a regulator of intracellular acidity (pHi) and implicated pathophysiological role in cardiac remodeling occurred in diseased myocardium. Cardiac NHE-1 expression level was increased in SHR and this was restored in TEN-treated SHR. AngII directly augmented cardiac NHE-1 expression and its activity that contributed to hypertrophic response. TEN attenuated the AngII-induced cardiac hypertrophy with decline in pHi via suppression of NHE-1. Loss of NHE-1 activity by specific inhibitor or RNA silencing promoted intracellular acidification and consistently attenuated the AngII-mediated cardiac hypertrophy., Conclusion: The present study revealed the protective actions of TEN on hypertension and comorbid cardiac remodeling via AngII/NHE-1 axis and the novel pathophysiological roles of intracellular acidification via NHE-1 in cardiac hypertrophy., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cellular senescence has emerged as an important player in both physiology and pathology. Excessive reactive oxygen species (ROS) is known to mediate cellular senescence. NADPH oxidases are major sources for ROS production in the vascular wall; the roles of different NADPH oxidase isoforms in cellular senescence remain unclear, however. We investigated the roles of two NADPH oxidase isoforms in mitochondrial dysfunction during angiotensin II (Ang II)-induced cellular senescence of human aortic vascular smooth muscle cells (VSMCs). Ang II (10(-7)M) stimulated ROS generation, exhibiting early increases between 30 and 60min and sustained increases between 24h and 72h, and induced VSMCs senescence after 48h or 72h treatment as assessed with senescence-associated β-galactosidase activity and the expression of two cell cycle inhibitors, p21 and p16. ROS scavengers and membrane-permeable catalase (catalase-PEG) reduced Ang II-stimulated cellular senescence. Furthermore, small interfering RNA (siRNA) of NADPH oxidase catalytic subunit Nox1, but not that of another isoform Nox4, inhibited Ang II-induced cellular senescence. Nox1 siRNA inhibited both early and sustained ROS increases induced by Ang II. In addition, a mitochondrial-specific antioxidant, mitoQ10, effectively inhibited Ang II-induced ROS increases and cellular senescence. Ang II decreased ATP synthesis and induced mitochondrial membrane depolarization, which were attenuated by pre-treating cells with Nox1 siRNA, mitoQ10 or catalase-PEG. The effect of Ang II on the mitochondrial regulator peroxisome-proliferator-activated receptor gamma coactivator-1α (PGC-1α) and its downstream genes was examined. Ang II stimulated S570 phosphorylation of PGC-1α with concomitant decreases in catalase and uncoupling protein-2 (UCP-2) levels between 12h and 72h, which were inhibited by Nox1 siRNA. Knockdown of both catalase and UCP-2 mimicked Ang II-induced VSMC senescence. These results suggested that Ang II-stimulated Nox1 activation mediates mitochondrial dysfunction, probably by decreasing PGC-1α activity and increasing mitochondrial oxidative stress, and leads to cellular senescence of VSMCs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Angiotensin II (AngII) type 1 receptor blockers (ARBs) have been effectively used in hypertension and cardiac remodeling. However, the differences among them are still unclear. We designed this study to examine and compare the effects of several ARBs widely used in clinics, including Olmesartan, Candesartan, Telmisartan, Losartan, Valsartan and Irbesartan, on the ACE-AngII-AT1 axis and the ACE2-Ang(1-7)-Mas axis during the development of cardiac remodeling after pressure overload. Although all of the six ARBs, attenuated the development of cardiac hypertrophy and heart failure induced by transverse aortic constriction (TAC) for 2 or 4weeks in the wild-type mice evaluated by echocardiography and hemodynamic measurements, the degree of attenuation by Olmesartan, Candesartan and Losartan tended to be larger than that of the other three drugs tested. Additionally, the degree of downregulation of the ACE-AngII-AT1 axis and upregulation of the ACE2-Ang(1-7)-Mas axis was higher in response to Olmesartan, Candesartan and Losartan administration in vivo and in vitro. Moreover, in angiotensinogen-knockdown mice, TAC-induced cardiac hypertrophy and heart failure were inhibited by Olmesartan, Candesartan and Losartan but not by Telmisartan, Valsartan and Irbesartan administration. Furthermore, only Olmesartan and Candesartan could downregulate the ACE-AngII-AT1 axis and upregulate the ACE2-Ang(1-7)-Mas axis in vitro. Our data suggest that Olmesartan, Candesartan and Losartan could effectively inhibit pressure overload-induced cardiac remodeling even when with knockdown of Ang II, possibly through upregulation of the expression of the ACE2-Ang(1-7)-Mas axis and downregulation of the expression of the ACE-AngII-AT1 axis. In contrast, Telmisartan, Valsartan and Irbesartan only played a role in the presence of AngII, and Losartan had no effect in the presence of AngII in vitro., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Nucleic acid aptamers can be served as drugs, carriers and diagnostic probes in living systems. Before recruiting aptamers, their pharmacological characteristics should be determined. Here we intended to investigate four important properties of isolated ssDNA anti-angiotensin II aptamers (FLC112 and FLC125) including hemolytic activity, cytotoxicity, immunogenicity and serum stability through in vitro and in vivo models. The hemolytic effect and cytotoxicity potential of aptamers were measured through hemolysis test and MTT assay respectively. In the following test, the humoral immune responses to aptamers in BALB/c mice were assessed. The human serum stability of aptamers was also determined using real-time PCR (qPCR). The results of this study revealed that the FLC112 aptamer with its unique structure had slightly higher cytotoxicity and hemolysis effect (9.14% and 0.1 ± 0.037% respectively) relative to FLC125 (8.07% and 0.08 ± 0.045% respectively) at the highest concentration (5 μM). FLC112 showed ignorable immune response in mice and barely higher than FLC125. Serum stability test confirmed that FLC112 with 12 h had more nuclease stability than FLC125 with 8 h. Aptamer molecule analysis revealed that the structure, sequense composition and motifs are the determinative parameters in aptamer pharmacological properties., (Copyright © 2016. Published by Elsevier Ltd.)

The AT1 receptor (AT1R) mediates the manifold actions of angiotensin II in the cardiovascular system. This study probed the molecular mechanisms that link altered redox status to AT1R expression in cardiac fibroblasts. Real-time PCR and western blot analysis showed that H2O2 enhances AT1R mRNA and protein expression via NADPH oxidase-dependent reactive oxygen species induction. Activation of NF-κB and AP-1, demonstrated by electrophoretic mobility shift assay, abolition of AT1R expression by their inhibitors, Bay-11-7085 and SR11302, respectively, and luciferase and chromatin immunoprecipitation assays confirmed transcriptional control of AT1R by NF-κB and AP-1 in H2O2-treated cells. Further, inhibition of ERK1/2, p38 MAPK and c-Jun N-terminal kinase (JNK) using chemical inhibitors or by RNA interference attenuated AT1R expression. Inhibition of the MAPKs showed that while ERK1/2 and p38 MAPK suffice for NF-κB activation, all three kinases are required for AP-1 activation. H2O2 also increased collagen type I mRNA and protein expression. Interestingly, the AT1R antagonist, candesartan, attenuated H2O2-stimulated AT1R and collagen mRNA and protein expression, suggesting that H2O2 up-regulates AT1R and collagen expression via local Angiotensin II generation, which was confirmed by real-time PCR and ELISA. To conclude, oxidative stress enhances AT1R gene expression in cardiac fibroblasts by a complex mechanism involving the redox-sensitive transcription factors NF-κB and AP-1 that are activated by the co-ordinated action of ERK1/2, p38 MAPK and JNK. Importantly, by causally linking oxidative stress to Angiotensin II and AT1R up-regulation in cardiac fibroblasts, this study offers a novel perspective on the pathogenesis of cardiovascular diseases associated with oxidative stress., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cardiac fibrosis is one of the major components of the healing mechanism following any injury of the heart and as such may contribute to both systolic and diastolic dysfunction in a range of pathophysiologic conditions. Canonically, it can occur as part of the remodeling process that occurs following myocardial infarction or that follows as a response to pressure overload. Integrins are cell surface receptors which act in both cellular adhesion and signaling. Most importantly, in the context of the continuously contracting myocardium, they are recognized as mechanotransducers. They have been implicated in the development of fibrosis in several organs, including the heart. This review will focus on the involvement of integrins and integrin-related proteins, in cardiac fibrosis, outlining the roles of these proteins in the fibrotic responses in specific cardiac pathologies, discuss some of the common end effectors (angiotensin II, transforming growth factor beta 1 and mechanical stress) through which integrins function and finally discuss how manipulation of this set of proteins may lead to new treatments which could prove useful to alter the deleterious effects of cardiac fibrosis., (Published by Elsevier Ltd.)

Activating transcription factor 3 (ATF3) is an adaptive-response protein induced by various environmental stresses and is implicated in the pathogenesis of many disease states. However, the role of ATF3 SUMOylation in hypertension-induced vascular injury remains poorly understood. Here we investigated the function of ATF3 SUMOylation in vascular endothelial cells (ECs). The expression of ATF3 and small ubiquitin-like modifier 1 (SUMO1) was increased in angiotensin II (Ang II)-induced human umbilical vein endothelial cells (HUVECs). Microscopic analyses further revealed that the expression of ATF3 and SUMO1 is upregulated and colocalized in the endothelium of thoracic aortas from Ang II-induced hypertensive mice. However, Ang II-induced upregulation of ATF3 and SUMO1 in vitro and in vivo was blocked by Ang II type I receptor antagonist olmesartan. Moreover, Ang II induced ATF3 SUMOylation at lysine 42, which is SUMO1 dependent. ATF3 SUMOylation attenuated ATF3 ubiquitination and in turn promoted ATF3 protein stability. ATF3 or SUMO1 knockdown inhibited Ang II-induced expression of inflammatory molecules such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8. Wild type ATF3 but not ATF3-K42R (SUMOylation defective mutant) reduced the production of nitric oxide (NO), a key indicator of EC function. Consistently, ginkgolic acid, an inhibitor of SUMOylation, increased NO production in HUVECs and significantly improved vasodilatation of aorta from Ang II-induced hypertensive mice. Our findings demonstrated that ATF3 SUMOylation is involved in Ang II-induced EC inflammation and dysfunction in vitro and in vivo through inhibiting ATF3 ubiquitination and increasing ATF3 protein stability., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Ischemia-reperfusion (I/R) injury is one of the most important pathologic processes causing acute kidney injury. Human atrial natriuretic peptide (hANP) has various effects, including renal protection. The purpose of the present work was to study the effects of intrarenal angiotensin II (Ang II) and investigate the potential of hANP to prevent kidney injury., Materials and Methods: Male Sprague-Dawley rats were divided into three groups as follows: (1) sham; (2) I/R (30 min of bilateral renal ischemia followed by 6 h reperfusion); and (3) I/R + hANP (I/R injury + continuous intravenous infusion of hANP at 0.025 μg/kg/min). After 6 h of reperfusion, both renal and plasma Ang II concentrations were measured. Urinary angiotensinogen and neutrophil gelatinase-associated lipocalin were measured before ischemia and 2, 4, and 6 h after reperfusion. To evaluate the renal-protective effects of hANP, serum creatinine was determined 6 and 24 h after reperfusion. In addition, mitochondrial oxygen consumption in kidney cortex was measured in the presence of Ang II and hANP., Results: Renal Ang II concentrations were 24.5 ± 3.9 and 14.2 ± 3.4 pg/mg renal weight in the I/R and I/R + hANP groups, respectively. Urinary angiotensinogen and neutrophil gelatinase-associated lipocalin excretions were elevated after I/R injury. Treatment with hANP significantly attenuated this effect after 4 and 6 h. Oxygen consumption in renal mitochondria increased with the addition of Ang II, which was also attenuated by hANP., Conclusions: Production of intrarenal Ang II was attenuated by hANP, indicating a potential to diminish renal I/R injury., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Delineation of mechanisms underlying the regulation of fibrosis-related genes in the heart is an important clinical goal as cardiac fibrosis is a major cause of myocardial dysfunction. This study probed the regulation of Discoidin Domain Receptor 2 (DDR2) gene expression and the regulatory links between Angiotensin II, DDR2 and collagen in Angiotensin II-stimulated cardiac fibroblasts. Real-time PCR and western blot analyses showed that Angiotensin II enhances DDR2 mRNA and protein expression in rat cardiac fibroblasts via NADPH oxidase-dependent reactive oxygen species induction. NF-κB activation, demonstrated by gel shift assay, abolition of DDR2 expression upon NF-κB inhibition, and luciferase and chromatin immunoprecipitation assays confirmed transcriptional control of DDR2 by NF-κB in Angiotensin II-treated cells. Inhibitors of Phospholipase C and Protein kinase C prevented Angiotensin II-dependent p38 MAPK phosphorylation that in turn blocked NF-κB activation. Angiotensin II also enhanced collagen gene expression. Importantly, the stimulatory effects of Angiotensin II on DDR2 and collagen were inter-dependent as siRNA-mediated silencing of one abolished the other. Angiotensin II promoted ERK1/2 phosphorylation whose inhibition attenuated Angiotensin II-stimulation of collagen but not DDR2. Furthermore, DDR2 knockdown prevented Angiotensin II-induced ERK1/2 phosphorylation, indicating that DDR2-dependent ERK1/2 activation enhances collagen expression in cells exposed to Angiotensin II. DDR2 knockdown was also associated with compromised wound healing response to Angiotensin II. To conclude, Angiotensin II promotes NF-κB activation that up-regulates DDR2 transcription. A reciprocal regulatory relationship between DDR2 and collagen, involving cross-talk between the GPCR and RTK pathways, is central to Angiotensin II-induced increase in collagen expression in cardiac fibroblasts., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In addition to cell surface membranes, numerous G protein-coupled receptors (GPCRs) are located on intracellular membranes including the nuclear envelope. Although the role of numerous GPCRs at the cell surface has been well characterized, the physiological function of these same receptors located on intracellular membranes remains to be determined. Here, we employ a novel caged Ang-II analog, cAng-II, to compare the effects of the activation of cell surface versus intracellular angiotensin receptors in intact cardiomyocytes. When added extracellularly to HEK 293 cells, Ang-II and photolysed cAng-II increased ERK1/2 phosphorylation (via AT1R) and cGMP production (AT2R). In contrast unphotolysed cAng-II did not. Cellular uptake of cAng-II was 6-fold greater than that of Ang-II and comparable to the HIV TAT(48-60) peptide. Intracellular photolysis of cAng-II induced an increase in nucleoplasmic Ca(2+) ([Ca(2+)]n) that was greater than that induced by extracellular application of Ang-II. We conclude that cell-permeable ligands that can access intracellular GPCRs may evoke responses distinct from those with access restricted to the same receptor located on the cell surface., (Copyright © 2015 Elsevier Inc. All rights reserved.)

There has been a growing appreciation that G protein-coupled receptor (GPCR) functional selectivity (viz. biased signaling), in particular between G protein- and β-arrestin-dependent signaling, can be achieved with specific ligands, and that such directed signaling represents a promising avenue for improving drug efficacy and therapy. Thus, for any given GPCRs it is important to define means to pharmacologically characterize and classify drugs for their propensity to bias signaling. Here we describe an experimental protocol and step-by-step approach to assess functional selectivity between Gαq and β-arrestin-dependent responses using the prototypical angiotensin II (AngII) type 1 receptor (AT1R) expressed in HEK 293 cells. The protocol describes the expression of Bioluminescence Resonance Energy Transfer (BRET) sensors for either Gαq or β-arrestin with AT1R, and the use of the operational model of pharmacological agonism to quantify ligand bias. Such methods are equally applicable to other GPCRs and their downstream signaling effectors., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Cardiac remodeling, a hallmark of heart disease, is associated with intense auto- and paracrine signaling leading to cardiac fibrosis. We hypothesized that the specific mediator of Gq/11-dependent RhoA activation p63RhoGEF, which is expressed in cardiac fibroblasts, plays a role in the underlying processes. We could show that p63RhoGEF is up-regulated in mouse hearts subjected to transverse aortic constriction (TAC). In an engineered heart muscle model (EHM), p63RhoGEF expression in cardiac fibroblasts increased resting and twitch tensions, and the dominant negative p63ΔN decreased both. In an engineered connective tissue model (ECT), p63RhoGEF increased tissue stiffness and its knockdown as well as p63ΔN reduced stiffness. In 2D cultures of neonatal rat cardiac fibroblasts, p63RhoGEF regulated the angiotensin II (Ang II)-dependent RhoA activation, the activation of the serum response factor, and the expression and secretion of the connective tissue growth factor (CTGF). All these processes were inhibited by the knockdown of p63RhoGEF or by p63ΔN likely based on their negative influence on the actin cytoskeleton. Moreover, we show that p63RhoGEF also regulates CTGF in engineered tissues and correlates with it in the TAC model. Finally, confocal studies revealed a closely related localization of p63RhoGEF and CTGF in the trans-Golgi network., (Copyright © 2015. Published by Elsevier Ltd.)

Angiotensin II (Ang II) induces cardiac hypertrophy and fibrosis in part by stimulating endothelin (ET-1) transcription. The involvement of the epigenetic machinery in this process is largely undefined. In the present study, we examined the epigenetic maneuvering underlying cardiac hypertrophy and fibrosis following ET-1 transactivation by Ang II. In response to Ang II stimulation, core components of the mammalian chromatin remodeling complex (Brahma-related gene 1, or Brg1, and Brahma or Brm) and histone H3K4 methylation complex (Ash2, absent, small, or homeotic discs 2, or Ash2 and WD domain repeat 5, or Wdr5) were recruited to the ET-1 promoter region in endothelial cells. Over-expression of Brg1/Brm or Ash2/Wdr5 enhanced while depletion of Brg1/Brm or Ash2/Wdr5 attenuated Ang II-induced ET-1 transactivation. Endothelial-specific knockdown of Brg1/Brm or Ash2/Wdr5 ameliorated cardiac hypertrophy both in vitro and in vivo. More important, Brg1/Brm interacted with Ash2/Wdr5 on the ET-1 promoter to catalyze H3K4 methylation. The crosstalk between Brg11/Brm and Ash2/Wdr5 was mediated by myocardin-related transcription factor A (MRTF-A). In conclusion, our data have unveiled an epigenetic complex that links ET-1 transactivation in endothelial cells to Ang II-induced cardiac hypertrophy and fibrosis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Chronic hypertension induces vascular remodeling. The most important factor for hypertension treatment is reducing the risk of cardiovascular disease. OW1 is a novel imperatorin derivative that exhibits vasodilative activity and antihypertensive effects in two-kidney one-clip (2K1C) renovascular hypertensive rats. It also inhibited vascular remodeling of the thoracic aorta in a previous study. Here, the inhibitory effects and mechanisms of OW1 on arterial vascular remodeling were investigated in vitro and in 2K1C hypertensive rats in vivo. OW1 (20μM, 10μM, 5μM) inhibited Ang II-induced vascular smooth muscle cells (VSMCs) proliferation and ROS generation in vitro. OW1 also reversed the Ang II-mediated inhibition of α-SMA levels and stimulation of OPN levels. Histology results showed that treatment of 2K1C hypertensive rats with OW1 (20, 40, and 80mg/kg per day, respectively for 5weeks) in vivo significantly decreased the number of VSMCs, the aortic cross-sectional area (CSA), the media to lumen (M/L) ratio, and the content of collagen I and III in the mesenteric artery. Western blot results also revealed that OW1 stimulated the expression of α-SMA and inhibited the expression of collagen I and III on the thoracic aorta of 2K1C hypertensive rats. In mechanistic studies, OW1 acted as an ACE inhibitor and affected calcium channels. The suppression of MMP expression and the MAPK pathway may account for the effects of OW1 on vascular remodeling. OW1 attenuated vascular remodeling in vitro and in vivo. It could be a novel candidate for hypertension intervention., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Neuroinflammation plays a role in the pathology of epilepsy and in cognitive impairment. Angiotensin II (AII) and the angiotensin receptor type 1 (AT1) have been shown to regulate seizure susceptibility in different models of epilepsy. Inhibition of AT1 attenuates neuroinflammatory responses in different neurological diseases. In the present study, we showed that the protein expression of AII and AT1 was increased in activated microglia following lithium pilocarpine-induced status epilepticus (SE) in rats. Furthermore, the AT1 receptor antagonist, losartan, significantly inhibited SE-induced cognitive impairment and microglia-mediated inflammation. Losartan also prevented SE induced neuronal loss in the hippocampus and exerted neuroprotection. These data suggest that losartan improves SE-induced cognitive impairment by suppressing microglia mediated inflammatory responses and attenuating hippocampal neuronal loss. Overall, our findings provide a possible therapeutic strategy for the treatment of cognitive impairment in epilepsy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Angiotensin II (Ang II) stimulates endothelin (ET-1) transcription, which contributes to cardiac hypertrophy and fibrosis. We have previously reported that myocardin related transcription factor A (MRTF-A) is indispensable for ET-1 transcription in vascular endothelial cells under hypoxic conditions, indicating that MRTF-A might mediate Ang II-induced pathological hypertrophy. Here we report that Ang II augmented the expression of MRTF-A in cultured endothelial cells and in the lungs of mice with cardiac hypertrophy. Over-expression of MRTF-A enhanced, whereas depletion of MRTF-A attenuated, transcriptional activation of ET-1 gene by Ang II. MRTF-A deficiency ameliorated Ang II induced cardiac hypertrophy and fibrosis in mice paralleling diminished synthesis and release of ET-1. Mechanistically, MRTF-A was recruited to the ET-1 promoter by c-Jun/c-Fos (AP-1) in response to Ang II treatment. Once bound, MRTF-A altered the chromatin structure by modulating histone acetylation and H3K4 methylation on the ET-1 promoter. More importantly, mice with endothelial-specific MRTF-A silencing by lentiviral particles phenocopied mice with systemic MRTF-A deletion in terms of Ang II-induced pathological hypertrophy. In conclusion, we data have unveiled a MRTF-A-containing complex that links ET-1 transactivation in endothelial cells to cardiac hypertrophy and fibrosis by Ang II., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Cardiac hypertrophy is controlled by a highly connected signaling network with many effectors of cardiac myocyte size. Quantification of the contribution of individual pathways to specific changes in shape and transcript abundance is needed to better understand hypertrophy signaling and to improve heart failure therapies. We stimulated cardiac myocytes with 15 hypertrophic agonists and quantitatively characterized differential regulation of 5 shape features using high-throughput microscopy and transcript levels of 12 genes using qPCR. Transcripts measured were associated with phenotypes including fibrosis, cell death, contractility, proliferation, angiogenesis, inflammation, and the fetal cardiac gene program. While hypertrophy pathways are highly connected, the agonist screen revealed distinct hypertrophy phenotypic signatures for the 15 receptor agonists. We then used k-means clustering of inputs and outputs to identify a network map linking input modules to output modules. Five modules were identified within inputs and outputs with many maladaptive outputs grouping together in one module: Bax, C/EBPβ, Serca2a, TNFα, and CTGF. Subsequently, we identified mechanisms underlying two correlations revealed in the agonist screen: correlation between regulators of fibrosis and cell death signaling (CTGF and Bax mRNA) caused by AngII; and myocyte proliferation (CITED4 mRNA) and elongation caused by Nrg1. Follow-up experiments revealed positive regulation of Bax mRNA level by CTGF and an incoherent feedforward loop linking Nrg1, CITED4 and elongation. With this agonist screen, we identified the most influential inputs in the cardiac hypertrophy signaling network for a variety of features related to pathological and protective hypertrophy signaling and shared regulation among cardiac myocyte phenotypes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Cardiac fibrosis is a substantial problem in managing multiple forms of heart disease. Fibrosis results from an unrestrained tissue repair process orchestrated predominantly by the myofibroblast. These are highly specialized cells characterized by their ability to secrete extracellular matrix (ECM) components and remodel tissue due to their contractile properties. This contractile activity of the myofibroblast is ascribed, in part, to the expression of smooth muscle α-actin (αSMA) and other tension-associated structural genes. Myofibroblasts are a newly generated cell type derived largely from residing mesenchymal cells in response to both mechanical and neurohumoral stimuli. Several cytokines, chemokines, and growth factors are induced in the injured heart, and in conjunction with elevated wall tension, specific signaling pathways and downstream effectors are mobilized to initiate myofibroblast differentiation. Here we will review the cell fates that contribute to the myofibroblast as well as nodal molecular signaling effectors that promote their differentiation and activity. We will discuss canonical versus non-canonical transforming growth factor-β (TGFβ), angiotensin II (AngII), endothelin-1 (ET-1), serum response factor (SRF), transient receptor potential (TRP) channels, mitogen-activated protein kinases (MAPKs) and mechanical signaling pathways that are required for myofibroblast transformation and fibrotic disease. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium "., (© 2013.)

Fibrosis, which is defined as excessive accumulation of fibrous connective tissue, contributes to the pathogenesis of numerous diseases involving diverse organ systems. Cardiac fibrosis predisposes individuals to myocardial ischemia, arrhythmias and sudden death, and is commonly associated with diastolic dysfunction. Histone deacetylase (HDAC) inhibitors block cardiac fibrosis in pre-clinical models of heart failure. However, which HDAC isoforms govern cardiac fibrosis, and the mechanisms by which they do so, remains unclear. Here, we show that selective inhibition of class I HDACs potently suppresses angiotensin II (Ang II)-mediated cardiac fibrosis by targeting two key effector cell populations, cardiac fibroblasts and bone marrow-derived fibrocytes. Class I HDAC inhibition blocks cardiac fibroblast cell cycle progression through derepression of the genes encoding the cyclin-dependent kinase (CDK) inhibitors, p15 and p57. In contrast, class I HDAC inhibitors block agonist-dependent differentiation of fibrocytes through a mechanism involving repression of ERK1/2 signaling. These findings define novel roles for class I HDACs in the control of pathological cardiac fibrosis. Furthermore, since fibrocytes have been implicated in the pathogenesis of a variety of human diseases, including heart, lung and kidney failure, our results suggest broad utility for isoform-selective HDAC inhibitors as anti-fibrotic agents that function, in part, by targeting these circulating mesenchymal cells., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Fat1 is an atypical cadherin that controls vascular smooth muscle cell (VSMC) proliferation and migration. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (Nox1) is an important source of reactive oxygen species (ROS) in VSMCs. Angiotensin II (Ang II) induces the expression and/or activation of both Fat1 and Nox1 proteins. This study tested the hypothesis that Ang II-induced Fat1 activation and VSMC migration are mediated by Nox1-dependent ROS generation and redox signaling. Studies were performed in cultured VSMCs from Sprague–Dawley rats. Cells were treated with Ang II (1 μmol/L) for short (5 to 30 min) or long term stimulations (3 to 12 h) in the absence or presence of the antioxidant apocynin (10 μmol/L), extracellular-signal-regulated kinases 1/2 (Erk1/2) inhibitor PD98059 (1 μmol/L), or Ang II type 1 receptor (AT1R) valsartan (1 μmol/L). siRNA was used to knockdown Nox1 or Fat1. Cell migration was determined by Boyden chamber assay. Ang II increased Fat1 mRNA and protein levels and promoted Fat1 translocation to the cell membrane, responses that were inhibited by AT1R antagonist and antioxidant treatment. Downregulation of Nox1 inhibited the effects of Ang II on Fat1 protein expression. Nox1 protein induction, ROS generation, and p44/p42 MAPK phosphorylation in response to Ang II were prevented by valsartan and apocynin, and Nox1 siRNA inhibited Ang II-induced ROS generation. Knockdown of Fat1 did not affect Ang II-mediated increases in Nox1 expression or ROS. Inhibition of p44/p42 MAPK phosphorylation by PD98059 abrogated the Ang II-induced increase in Fat1 expression and membrane translocation. Knockdown of Fat1 inhibited Ang II-induced VSMC migration, which was also prevented by valsartan, apocynin, PD98059, and Nox1 siRNA. Our findings indicate that Ang II regulates Fat1 expression and activity and induces Fat1-dependent VSMC migration via activation of AT1R, ERK1/2, and Nox1-derived ROS, suggesting a role for Fat1 downstream of Ang II signaling that leads to vascular remodeling.

Background: Pulmonary fibrosis occurs in approximately 60% of patients with acute respiratory distress syndrome and has been significantly correlated with a poor outcome. The overexpression of angiotensin (Ang) II can induce lung inflammation and fibrosis. This observation, coupled with the knowledge that Ang-(1-7) is considered to be an endogenous antagonist of Ang II, led us to hypothesize that Ang-(1-7) would prevent lung remodeling in patients with acute respiratory distress syndrome., Materials and Methods: The protocol involved five groups: (1) control, (2) lipopolysaccharide (LPS), (3) losartan as a positive control group, (4) Ang-(1-7), and (5) [D-Ala7]-Ang-(1-7) (A779), an antagonist of the Ang-(1-7) receptor. Acute lung injury was induced by an intratracheal injection of LPS 5 mg/kg in C57BL/6 mice. Losartan (10 mg/kg) was administered by gavage daily, starting from 1 d before LPS stimulation. Ang-(1-7) or A779 in saline (100 ng/kg/min) was infused subcutaneously 1 h before acute lung injury induction for 3 or 7 d. The lung tissues were harvested for analysis at day 3 or 7 after injection of LPS., Results: LPS stimulation resulted in significantly increased inflammation, edema, and lung collagen production. With Ang-(1-7) treatment, the lung fibrosis score and hydroxyproline level were significantly reduced, and the expression of transforming growth factor-β and Smad2/3 were decreased on days 3 and 7. Losartan attenuated lung fibrosis similarly to Ang-(1-7) after LPS exposure. In the A779 group, a tendency was seen to aggravate collagen deposition and lung remodeling., Conclusions: These findings indicate an antiremodeling role for Ang-(1-7) in acute lung injury, similar to the blocker of Ang II receptor, that might be at least partially mediated through an Ang-(1-7) receptor., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Dysfunction of iron homeostasis has been shown to be involved in ageing, Parkinson's disease and other neurodegenerative diseases. Increased levels of labile iron result in increased reactive oxygen species and oxidative stress. Angiotensin II, via type-1 receptors, exacerbates oxidative stress, the microglial inflammatory response and progression of dopaminergic degeneration. Angiotensin activates the NADPH-oxidase complex, which produces superoxide. However, it is not known whether angiotensin affects iron homeostasis. In the present study, administration of angiotensin to primary mesencephalic cultures, the dopaminergic cell line MES23.5 and to young adult rats, significantly increased levels of transferrin receptors, divalent metal transporter-1 and ferroportin, which suggests an increase in iron uptake and export. In primary neuron-glia cultures and young rats, angiotensin did not induce significant changes in levels of ferritin or labile iron, both of which increased in neurons in the absence of glia (neuron-enriched cultures, dopaminergic cell line) and in the N9 microglial cell line. In aged rats, which are known to display high levels of angiotensin activity, ferritin levels and iron deposits in microglial cells were enhanced. Angiotensin-induced changes were inhibited by angiotensin type-1 receptor antagonists, NADPH-oxidase inhibitors, antioxidants and NF-kB inhibitors. The results demonstrate that angiotensin, via type-1 receptors, modulates iron homeostasis in dopaminergic neurons and microglial cells, and that glial cells play a major role in efficient regulation of iron homeostasis in dopaminergic neurons., (© 2013.)

Prolactin (PRL) cells of the Mozambique tilapia, Oreochromis mossambicus, are osmoreceptors by virtue of their intrinsic osmosensitivity coupled with their ability to directly regulate hydromineral homeostasis through the actions of PRL. Layered upon this fundamental osmotic reflex is an array of endocrine control of PRL synthesis and secretion. Consistent with its role in fresh water (FW) osmoregulation, PRL release in tilapia increases as extracellular osmolality decreases. The hyposmotically-induced release of PRL can be enhanced or attenuated by a variety of hormones. Prolactin release has been shown to be stimulated by gonadotropin-releasing hormone (GnRH), 17-β-estradiol (E2), testosterone (T), thyrotropin-releasing hormone (TRH), atrial natriuretic peptide (ANP), brain-natriuretic peptide (BNP), C-type natriuretic peptide (CNP), ventricular natriuretic peptide (VNP), PRL-releasing peptide (PrRP), angiotensin II (ANG II), leptin, insulin-like growth factors (IGFs), ghrelin, and inhibited by somatostatin (SS), urotensin-II (U-II), dopamine, cortisol, ouabain and vasoactive intestinal peptide (VIP). This review is aimed at providing an overview of the hypothalamic and extra-hypothalamic hormones that regulate PRL release in euryhaline Mozambique tilapia, particularly in the context on how they may modulate osmoreception, and mediate the multifunctional actions of PRL. Also considered are the signal transduction pathways through which these secretagogues regulate PRL cell function., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Marine teleost fishes drink environmental seawater to compensate for osmotic water loss, and the amount of water intake is precisely regulated to prevent dehydration or hypernatremia. Unlike terrestrial animals in which thirst motivates a series of drinking behaviors, aquatic fishes can drink environmental water by reflex swallowing without searching for water. Hormones are key effectors for the regulation of drinking. In particular, angiotensin II and atrial natriuretic peptide are likely candidates for physiological regulators because of their potent dipsogenic and antidipsogenic activities, respectively. In the eel, these hormones act on the area postrema in the medulla oblongata, a circumventricular structure without blood-brain barrier, which then regulates the activity of the glossopharyngeal-vagal motor complex. These motor neurons in the hindbrain innervate the upper esophageal sphincter muscle and other swallowing-related muscles in the pharynx and esophagus for regulation of drinking. Thus, the neural circuitry for drinking in fishes appears to be confined within the hindbrain. This simple mechanism is much different from that of terrestrial animals in which thirst sensation is induced through hormonal actions on the subfornical organ and organum vasculosum of the lamina terminalis that are located in the forebrain. It seems that the neural and hormonal mechanism that regulates drinking behavior has evolved from fishes depending on the availability of water in their natural habitats., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The renin-angiotensin system (RAS) plays a key pathogenic role in heart failure. The adverse effects of angiotensin II (Ang II), a major player of the RAS, contributes to the development of heart failure. Heart failure is accompanied by significant perturbations in cardiac energy metabolism that can both decrease cardiac energy supply and decrease cardiac efficiency. Recent evidence suggests that Ang II might be involved in these perturbations in cardiac energy metabolism. Furthermore, new components of the RAS, such as angiotensin converting enzyme 2 and Ang1-7, have been reported to exert beneficial effects on cardiac energy metabolism. As a result, a further understanding of the relationship between the RAS and cardiac energy metabolism has the potential to improve the control of heart failure, and may lead to the development of new therapies to treat heart failure. This review summarizes what effects the RAS has on cardiac energy metabolism, highlighting how Ang II can induce cardiac insulin resistance and mitochondrial damage, and what role reactive oxygen species and sirtuins have on these processes., (© 2013.)

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Patients with AF have up to seven-fold higher risk of suffering from ischemic stroke. Better understanding of etiologies of AF and its thromboembolic complications are required for improved patient care, as current anti-arrhythmic therapies have limited efficacy and off target effects. Accumulating evidence has implicated a potential role of oxidative stress in the pathogenesis of AF. Excessive production of reactive oxygen species (ROS) is likely involved in the structural and electrical remodeling of the heart, contributing to fibrosis and thrombosis. In particular, NADPH oxidase (NOX) has emerged as a potential enzymatic source for ROS production in AF based on growing evidence from clinical and animal studies. Indeed, NOX can be activated by known upstream triggers of AF such as angiotensin II and atrial stretch. In addition, treatments such as statins, antioxidants, ACEI or AT1RB have been shown to prevent post-operative AF; among which ACEI/AT1RB and statins can attenuate NOX activity. On the other hand, detailed molecular mechanisms by which specific NOX isoform(s) are involved in the pathogenesis of AF and the extent to which activation of NOX plays a causal role in AF development remains to be determined. The current review discusses causes and consequences of oxidative stress in AF with a special focus on the emerging role of NOX pathways., (Copyright © 2013 Elsevier Ltd. All rights reserved.)