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Idiopathic pulmonary fibrosis (IPF) is a severe disorder leading to progressive and irreversible loss of pulmonary function. In this study we investigated the anti-fibrotic effect of vitamin D using a mouse model of IPF. Lung fibrosis was induced with bleomycin in vitamin D-sufficient and vitamin D-deficient C57BL/6 mice. We found that treatment with active vitamin D analog paricalcitol prevented mouse body weight loss and alleviated lung fibrosis, whereas vitamin D deficiency severely aggravated lung injury. At the molecular level, paricalcitol treatment suppressed the induction of fibrotic inducer TGF-β and extracellular matrix proteins α-SMA, collagen type I and fibronectin in the lung, whereas vitamin D deficiency exacerbated the induction of these proteins. Interestingly, bleomycin treatment activated the local renin–angiotensin system (RAS) in the lung, manifested by the induction of renin, angiotensinogen, angiotensin II and angiotensin receptor type 1 (AT1R). Paricalcitol treatment suppressed the induction of these RAS components, whereas vitamin D deficiency enhanced the activation of the lung RAS. We also showed that treatment of bleomycin-induced vitamin D-deficient mice with AT1R antagonist losartan relieved weight loss, substantially ameliorated lung fibrosis and markedly blocked TGF-β induction in the lung. Moreover, we demonstrated that in lung fibroblast cultures, TGF-β and angiotensin II synergistically induced TGF-β, AT1R, α-SMA, collagen type I and fibronectin, whereas 1,25-dihydroxyvitamin D markedly suppressed the induction of these fibrotic markers. Collectively, these observations strongly suggest that vitamin D mitigates lung fibrosis by blocking the activation of the lung RAS in this mouse model of IPF.

Disordered sleep promotes inflammation in brain and peripheral tissues, but the mechanisms that regulate these responses are poorly understood. One hypothesis is that activation of the sympathetic nervous system (SNS) from sleep loss elevates blood pressure to promote vascular sheer stress leading to inflammation. As catecholamines produced from SNS activation can directly regulate inflammation, we pharmacologically altered blood pressure using an alternative approach-manipulation of the renin-angiotensin system (RAS). Male C57BL6/J mice were treated with angiotensin or captopril to elevate and reduce blood pressure, respectively and then exposed to 24-h of sleep fragmentation (SF) or allowed to sleep (control). Pro- and anti-inflammatory cytokine gene expression and as endothelial adhesion gene expression as well as serum glucocorticoids (corticosterone) were measured. RAS manipulation elevated cytokines and endothelial adhesion expression in heart and aorta while SF increased cytokine expression in peripheral tissues, but not brain. However, there were interactive effects of angiotensin-II and SF upon cytokine gene expression in hippocampus and hypothalamus, but not prefrontal cortex. SF, but not RAS manipulation, elevated serum corticosterone concentration. These findings highlight the contrasting effects of RAS manipulation and SF, implying that inflammation from SF is acting on different pathways that are largely independent of RAS manipulation.

Cardiac fibrosis is an underlying cause of diastolic dysfunction, contributing to heart failure. Substance P (SP) activation of the neurokinin-1 receptor (NK-1R) contributes to cardiac fibrosis in hypertension. However, based on in vitro experiments, this does not appear to be via direct activation of cardiac fibroblasts. While numerous cells could mediate the fibrotic effects of SP, herein, we investigate mast cells (MC) as a mechanism mediating the fibrotic actions of SP, since MCs are known to play a role in cardiac fibrosis and respond to SP. Spontaneously hypertensive rats (SHR) were treated with the NK-1R antagonist L732138 (5 mg/kg/d) from 8 to 12 weeks of age. L732138 prevented increased MC maturation of resident immature MCs. NK-1R blockade also prevented increased cardiac MC maturation in angiotensin II-infused mice. MC-deficient mice were used to test the importance of MC NK-1Rs to MC activation. MC-deficient mice administered angiotensin II did not develop fibrosis; MC-deficient mice reconstituted with MCs did develop fibrosis. MC-deficient mice reconstituted with MCs lacking the NK-1R also developed fibrosis, indicating that NK-1Rs are not required for MC activation in this setting. In conclusion, the NK-1R causes MC maturation, however, other stimuli are required to activate MCs to cause fibrosis.

Oxidative stress has been suggested to play a role in brain damage during carbon monoxide (CO) poisoning. Severe poisoning induced by CO at 3000 ppm, but not 1000 ppm, enhances hydroxyl radical (˙OH) production in the rat striatum, which might be mediated by NADPH oxidase (NOX) activation associated with Ras-related C3 botulinum toxin substrate (Rac) via cAMP signaling pathway activation. CO-induced ˙OH production was suppressed by antagonists of angiotensin II (AngII) type 1 receptor (AT1R) and type 2 receptor (AT2R) but not an antagonist of the Mas receptor. Suppression by an AT1R antagonist was unrelated to peroxisome proliferator-activated receptor γ. Angiotensin-converting enzyme inhibitors also suppressed CO-induced ˙OH production. Intrastriatal AngII at high concentrations enhanced ˙OH production. However, the enhancement of ˙OH production was resistant to inhibitors selective for NOX and Rac and to AT1R and AT2R antagonists. This indicates a different mechanism for ˙OH production induced by AngII than for that induced by CO poisoning. AT1R and AT2R antagonists had no significant effects on CO-induced cAMP production or ˙OH production induced by forskolin, which stimulates cAMP production. These findings suggest that the renin-angiotensin system might be involved in CO-induced ˙OH production in a manner independent of cAMP signaling pathways.

The proximal tubule is a particularly important site for ageing-related kidney damage. Sirtuin 1 (SIRT1), an NAD+ (nicotinamide adenine dinucleotide)-dependent deacetylase in the proximal tubule, may be involved in renal injury associated with ageing. However, the mechanisms of SIRT1 regulation remain to be elucidated. We recently reported that angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP)-deficient mice displayed age-associated renal function decline and tubulointerstitial fibrosis. Our data showed that SIRT1 protein expression was reduced in ATRAP-deficient mice, although the relationship between ATRAP deficiency and age-associated renal fibrosis is still not fully understood. It is, therefore, necessary to investigate how ATRAP affects SIRT1 protein expression to resolve ageing-associated kidney dysfunction. Here, since ageing studies are inherently lengthy, we used an ex vivo model of the proximal tubule to determine the role of ATRAP in SIRT1 protein expression. We first generated a clonal immortalised human renal proximal tubule epithelial cell line (ciRPTEC) expressing AT1R and ATRAP. Using this cell line, we demonstrated that ATRAP knockdown reduced SIRT1 protein expression in the ciRPTEC but did not alter SIRT1 mRNA expression. Thus, ATRAP likely mediates SIRT1 protein abundance in ciRPTEC.

Activation of the renin-angiotensin system (RAS) is associated with hypertension and heart disease. However, how RAS activation causes cardiac lesions remains elusive. Here we report the involvement of Wnt/β-catenin signaling in this process. In rats with chronic infusion of angiotensin II (Ang II), eight Wnt ligands were induced and β-catenin activated in both cardiomyocytes and cardiac fibroblasts. Blockade of Wnt/β-catenin signaling by small molecule inhibitor ICG-001 restrained Ang II-induced cardiac hypertrophy by normalizing heart size and inhibiting hypertrophic marker genes. ICG-001 also attenuated myocardial fibrosis and inhibited α-smooth muscle actin, fibronectin and collagen I expression. These changes were accompanied by a reduced expression of atrial natriuretic peptide and B-type natriuretic peptide. Interestingly, ICG-001 also lowered blood pressure induced by Ang II. In vitro, Ang II induced multiple Wnt ligands and activated β-catenin in rat primary cardiomyocytes and fibroblasts. ICG-001 inhibited myocyte hypertrophy and Snail1, c-Myc and atrial natriuretic peptide expression, and abolished the fibrogenic effect of Ang II in cardiac fibroblasts. Finally, recombinant Wnt3a was sufficient to induce cardiomyocyte injury and fibroblast activation in vitro. Taken together, these results illustrate an essential role for Wnt/β-catenin in mediating hypertension, cardiac hypertrophy and myocardial fibrosis. Therefore, blockade of this pathway may be a novel strategy for ameliorating hypertensive heart disease.

This study aimed to study the impact of a combination of maternal and post-weaning high-fat diets and whether resveratrol was beneficial. Sprague-Dawley dams were fed either chow or a high-fat diet, before mating, during pregnancy, and into lactation. At weaning, their offspring were randomly fed chow or a high-fat diet. Four experimental groups were generated: CC (maternal/postnatal chow diet), HC (maternal high-fat/postnatal chow diet), CH (maternal chow/postnatal high-fat diet), and HH (maternal/postnatal high-fat diet). A fifth group consisted of HH plus resveratrol. The 4 month-old offspring of HH group had higher body weight, higher levels of plasma triglycerides, leptin, angiotensin I and angiotensin II and abnormal intraperitoneal glucose tolerance test results, which fulfilled the features of metabolic syndrome. The dysregulation of the renin-angiotensin system was seen in multiple organs. Sirtuin 1 expression/abundance was reduced by a maternal/postnatal high-fat diet, in all the organs examined. Resveratrol ameliorated most of the features of metabolic syndrome and molecular alterations. The administration of a high-fat diet in both periods showed interactive metabolic effects in the plasma and many organs. Our results suggest that a maternal high-fat diet sensitizes offspring to the adverse effects of subsequent high-fat intake on multiple organs.

Vascular T-type Ca2+ channels (CaV3.1 and CaV3.2) play a key role in arterial tone development. This study investigated whether this conductance is a regulatory target of angiotensin II (Ang II), a vasoactive peptide that circulates and which is locally produced within the arterial wall. Patch clamp electrophysiology performed on rat cerebral arterial smooth muscle cells reveals that Ang II (100 nM) inhibited T-type currents through AT1 receptor activation. Blocking protein kinase C failed to eliminate channel suppression, a finding consistent with unique signaling proteins enabling this response. In this regard, inhibiting NADPH oxidase (Nox) with apocynin or ML171 (Nox1 selective) abolished channel suppression highlighting a role for reactive oxygen species (ROS). In the presence of Ni2+ (50 µM), Ang II failed to modulate the residual T-type current, an observation consistent with this peptide targeting CaV3.2. Selective channel suppression by Ang II impaired the ability of CaV3.2 to alter spontaneous transient outward currents or vessel diameter. Proximity ligation assay confirmed Nox1 colocalization with CaV3.2. In closing, Ang II targets CaV3.2 channels via a signaling pathway involving Nox1 and the generation of ROS. This unique regulatory mechanism alters BKCa mediated feedback giving rise to a “constrictive” phenotype often observed with cerebrovascular disease.

Angiotensin-converting enzyme inhibitors (ACEis) are beneficial in patients with chronic kidney disease (CKD). Yet, their clinical effects after kidney transplantation (KTx) remain ambiguous and local renin-angiotensin system (RAS) regulation including the ‘classical’ and ‘alternative’ RAS has not been studied so far. Here, we investigated both systemic and kidney allograft-specific intrarenal RAS using tandem mass-spectrometry in KTx recipients with or without established ACEi therapy (n = 48). Transplant patients were grouped into early (12 years). Patients on ACEi displayed lower angiotensin (Ang) II plasma levels (P P P P P

Neuropeptide release in the brain has traditionally been difficult to observe. Existing methods lack temporal and spatial resolution that is consistent with the function and size of neurons. We use cultured “sniffer cells” to improve the temporal and spatial resolution of observing neuropeptide release. Sniffer cells were created by stably transfecting Chinese Hamster Ovary (CHO) cells with plasmids encoding the rat angiotensin type 1a receptor and a genetically encoded Ca2+ sensor. Isolated, cultured sniffer cells showed dose-dependent increases in fluorescence in response to exogenously applied angiotensin II and III, but not other common neurotransmitters. Sniffer cells placed on the median preoptic nucleus (a presumptive site of angiotensin release) displayed spontaneous activity and evoked responses to either electrical or optogenetic stimulation of the subfornical organ. Stable sniffer cell lines could be a viable method for detecting neuropeptide release in vitro, while still being able to distinguish differences in neuropeptide concentration.

Oxidative stress in renal mesangial cell causes diabetic glomerular changes. High glucose levels and angiotensin II (Ang II) are known to stimulate superoxide production in renal mesangial cells. However, it has been unclear whether Ang II stimulation and pre-conditioning with high glucose affects the same pathway of superoxide production in renal mesangial cells or not. In this study, we examined the levels of oxidative stress under Ang II stimulation in renal mesangial cells preincubated for six hours at various glucose levels. Intracellular levels of reactive oxidative species (ROS) were measured using dihydroethidium or 5′,6′-chloromethyl- 2′,7′ dichlorodihydro-fluorescein diacetate, which facilitates the detection of intracellular ROS under real-time fluorescent microscope. Ang II-induced elevated intracellular ROS levels were detected only when the cells were pre-incubated with high levels of glucose (13.5 mM, 27.8 mM), but was not detected under normal glucose condition (5.5 mM). Production of Ang II-induced intracellular ROS was higher under pre-treatment with 27.8 mM glucose compared to pretreatment with 13.5 mM glucose level. This ROS production in mesangial cells was induced within several minutes of the initiation of Ang II stimulation under high glucose levels. The production of intracellular ROS was significantly reduced in the presence of angiotensin II type1-receptor (AT1R) antagonist, whereas it was augmented in the presence of angiotensin II type2-receptor antagonist. In conclusion, Ang II-induced oxidative stress was augmented by high glucose levels and ROS levels were further alleviated in the presence of AT1R antagonists.

Abnormal renin-angiotensin system (RAS) activation plays a critical role in the initiation and progression of chronic kidney disease (CKD) by directly mediating renal tubular cell apoptosis. Our previous study showed that necroptosis may play a more important role than apoptosis in mediating renal tubular cell loss in chronic renal injury rats, but the mechanism involved remains unknown. Here, we investigate whether blocking the angiotensin II type 1 receptor (AT1R) and/or angiotensin II type 2 receptor (AT2R) beneficially alleviates renal tubular cell necroptosis and chronic kidney injury. In an angiotensin II (Ang II)-induced renal injury mouse model, we found that blocking AT1R and AT2R effectively mitigates Ang II-induced increases in necroptotic tubular epithelial cell percentages, necroptosis-related RIP3 and MLKL protein expression, serum creatinine and blood urea nitrogen levels, and tubular damage scores. Furthermore, inhibition of AT1R and AT2R diminishes Ang II-induced necroptosis in HK-2 cells and the AT2 agonist CGP42112A increases the percentage of necroptotic HK-2 cells. In addition, the current study also demonstrates that Losartan and PD123319 effectively mitigated the Ang II-induced increases in Fas and FasL signaling molecule expression. Importantly, disruption of FasL significantly suppressed Ang II-induced increases in necroptotic HK-2 cell percentages, and necroptosis-related proteins. These results suggest that Fas and FasL, as subsequent signaling molecules of AT1R and AT2R, might involve in Ang II-induced necroptosis. Taken together, our results suggest that Ang II-induced necroptosis of renal tubular cell might be involved both AT1R and AT2R and the subsequent expression of Fas, FasL signaling. Thus, AT1R and AT2R might function as critical mediators.

Activation of the renin-angiotensin system may initiate chronic kidney disease. We hypothesised that renal hypoxia is a consequence of hemodynamic changes induced by angiotensin II and occurs prior to development of severe renal damage. Male Sprague-Dawley rats were infused continuously with angiotensin II (350 ng/kg/min) for 8 days. Mean arterial pressure (n = 5), cortical (n = 6) and medullary (n = 7) oxygenation (pO2) were continuously recorded by telemetry and renal tissue injury was scored. Angiotensin II increased arterial pressure gradually to 150 ± 18 mmHg. This was associated with transient reduction of oxygen levels in renal cortex (by 18 ± 2%) and medulla (by 17 ± 6%) at 10 ± 2 and 6 ± 1 hours, respectively after starting infusion. Thereafter oxygen levels normalised to pre-infusion levels and were maintained during the remainder of the infusion period. In rats receiving angiotensin II, adding losartan to drinking water (300 mg/L) only induced transient increase in renal oxygenation, despite normalisation of arterial pressure. In rats, renal hypoxia is only a transient phenomenon during initiation of angiotensin II-induced hypertension.

The Renin Angiotensin System (RAS), a key regulator of blood pressure has been linked to metabolic disorders. We have previously reported that adipose overexpression of angiotensinogen in mice (Agt-Tg) induces obesity, in part mediated by adipose tissue inflammation, through yet unidentified mechanisms. Hence, we hypothesize that adipose tissue enrichment of angiotensinogen leads to activation of inflammatory cascades and endoplasmic reticulum (ER) stress, thereby, contributing to obesity. We used wild type (Wt), Agt-Tg and Agt-knockout (KO) mice along with 3T3-L1 and human adipocytes treated with RAS, ER stress and inflammation inhibitors. ER stress and pro-inflammation markers were significantly higher in Agt-Tg compared to Wt mice and captopril significantly reduced their expression. Furthermore, in vitro treatment with Ang II significantly induced ER stress and inflammation, whereas angiotensin II receptor inhibitor, telmisartan reduced RAS effects. Moreover, miR-30 family had significantly lower expression in Agt-Tg group. MiR-708-5p and -143-3p were upregulated when RAS was overexpressed, and RAS antagonists reduced miR-143-3p and -708-5p in both mouse adipose tissue and adipocytes. Activation of RAS by Ang II treatment, increased inflammation and ER stress in adipocytes mainly via AT1 receptor, possibly mediated by miR-30 family, -708-5p and/or -143-3p. Hence, RAS and mediating microRNAs could be used as potential targets to reduce RAS induced obesity and related comorbid diseases.

Both the activation of the renin angiotensin aldosterone system (RAAS) and elevations of circulating Fibroblast Growth Factor-23 (FGF-23) have been implicated in the pathogenesis of left ventricular hypertrophy (LVH) in chronic kidney disease. To investigate potential cross-talk between RAAS and FGF-23, we administered angiotensin II (Ang II) to wild-type rodents and the Hyp mouse model of excess FGF-23. Ang II administration for four weeks to wild-type rodents resulted in significant increases in systolic blood pressure and LVH. Unexpectedly, FGF-23 circulating levels were increased by 1.5–1.7 fold in Ang II treated animals. In addition, Ang II treatment increased expression of FGF-23 message levels in bone, the predominant tissue for FGF-23 production, and induced expression of FGF-23 and its co-receptor α-Klotho in the heart, which normally does not express FGF-23 or α-Klotho in physiologically relevant levels. Hyp mice with elevated FGF-23 exhibited increased blood pressure and LVH at baseline. Ang II administration to Hyp mice resulted further increments in blood pressure and left ventricular hypertrophy, consistent with additive cardiovascular effects. These findings suggest that FGF-23 may participate in unexpected systemic and paracrine networks regulating hemodynamic and myocardial responses.

MicroRNAs participate in a variety of physiological and pathophysiological processes in various organs including the heart. Our previous work revealed that the level of miR-199a-5p was significantly higher in failing hearts than in control hearts. However, whether it is associated with the progression of heart failure (HF) and mediates cardiomyocyte apoptosis remained unclear. In the present study, we used various biochemical and molecular biological approaches to investigate the changes in miR-199a-5p levels in failing hearts in a rat model induced by acute myocardial infarction. We found that miR-199a-5p levels in the heart increased with the progression of HF, and overexpression of miR-199a-5p significantly increased apoptosis in untreated H9C2 cells and potentiated angiotensin II-induced apoptosis. Thus, our results indicate that miR-199a-5p is involved in the progression of HF and mediates cardiomyocyte apoptosis. We also confirmed that JunB, a member of the activator protein-1 transcription factor family, is one of direct targets of miR-199a-5p via a dual-luciferase reporter assay and mutagenesis on the 3′ untranslated region of the JunB gene. Consistent with the above findings, overexpression of JunB in H9c2 cells suppressed cell apoptosis. Based on our findings, miR-199a-5p induces apoptosis by targeting JunB.

Mesangial cells stimulated with high glucose (HG) exhibit increased intracellular angiotensin II (AngII) synthesis that is correlated with the upregulation of AngII target genes, such as profibrotic cytokines. The intracrine effects of AngII can be mediated by several molecules transferred to other cells via exosomes (Exos), which play a key role in cellular communication under many physiological and pathological conditions. The aim of this study was to investigate the effects of exosomes derived from HG-stimulated human mesangial cells (HG-HMCs) on normal unstimulated HMCs. Exosomes from HMCs (C-Exos) and HG-HMCs (HG-Exos) were obtained from cell culture supernatants. HMCs were incubated with C-Exos or HG-Exos. HG stimulus induced a change in the amount but not the size of Exos. Both C-Exos and HG-Exos contained angiotensinogen and renin, but no angiotensin converting enzyme was detected. Compared with HMCs treated with C-Exos, HMCs treated with HG-Exos presented higher levels of fibronectin, angiotensinogen, renin, AT1 and AT2 receptors, indicating that HG-Exos modified the function of normal HMCs. These results suggest that the intercellular communication through Exos may have pathophysiological implications in the diabetic kidney.

Increased activation of the renin-angiotensin system is involved in the onset and progression of cardiometabolic diseases, while natriuretic peptides (NP) may exert protective effects. We have recently demonstrated that sacubitril/valsartan (LCZ696), a first-in-class angiotensin receptor neprilysin inhibitor, which blocks the angiotensin II type-1 receptor and augments natriuretic peptide levels, improved peripheral insulin sensitivity in obese hypertensive patients. Here, we investigated the effects of sacubitril/valsartan (400 mg QD) treatment for 8 weeks on the abdominal subcutaneous adipose tissue (AT) phenotype compared to the metabolically neutral comparator amlodipine (10 mg QD) in 70 obese hypertensive patients. Abdominal subcutaneous AT biopsies were collected before and after intervention to determine the AT transcriptome and expression of proteins involved in lipolysis, NP signaling and mitochondrial oxidative metabolism. Both sacubitril/valsartan and amlodipine treatment did not significantly induce AT transcriptional changes in pathways related to lipolysis, NP signaling and oxidative metabolism. Furthermore, protein expression of adipose triglyceride lipase (ATGL) (Ptime*group = 0.195), hormone-sensitive lipase (HSL) (Ptime*group = 0.458), HSL-ser660 phosphorylation (Ptime*group = 0.340), NP receptor-A (NPRA) (Ptime*group = 0.829) and OXPHOS complexes (Ptime*group = 0.964) remained unchanged. In conclusion, sacubitril/valsartan treatment for 8 weeks did not alter the abdominal subcutaneous AT transcriptome and expression of proteins involved in lipolysis, NP signaling and oxidative metabolism in obese hypertensive patients.

MicroRNA-21 (mir-21) induced by angiotensin II (AngII) plays a vital role in the development of pulmonary fibrosis, and the NLRP3 inflammasome is known to be involved in fibrogenesis. However, whether there is a link between mir-21 and the NLRP3 inflammasome in pulmonary fibrosis is unknown. Angiotensin-converting enzyme 2/angiotensin(1–7) [ACE2/Ang(1–7)] has been shown to attenuate AngII-induced pulmonary fibrosis, but it is not clear whether ACE2/Ang(1–7) protects against pulmonary fibrosis by inhibiting AngII-induced mir-21 expression. This study’s aim was to investigate whether mir-21 activates the NLRP3 inflammasome and mediates the different effects of AngII and ACE2/Ang(1–7) on lung fibroblast apoptosis and collagen synthesis. In vivo, AngII exacerbated bleomycin (BLM)-induced lung fibrosis in rats, and elevated mir-21 and the NLRP3 inflammasome. In contrast, ACE2/Ang(1–7) attenuated BLM-induced lung fibrosis, and decreased mir-21 and the NLRP3 inflammasome. In vitro, AngII activated the NLRP3 inflammasome by up-regulating mir-21, and ACE2/Ang(1–7) inhibited NLRP3 inflammasome activation by down-regulating AngII-induced mir-21. Over-expression of mir-21 activated the NLRP3 inflammasome via the ERK/NF-κB pathway by targeting Spry1, resulting in apoptosis resistance and collagen synthesis in lung fibroblasts. These results indicate that mir-21 mediates the inhibitory effect of ACE2/Ang(1–7) on AngII-induced activation of the NLRP3 inflammasome by targeting Spry1 in lung fibroblasts.

Preeclampsia (PE) is a common pregnancy-related complication, and polymorphisms in angiotensinogen (AGT), angiotensin-converting enzyme (ACE), and angiotensin II type 1 receptor (AT1R) are believed to contribute to PE development. We implemented a hybrid study to investigate the influence of maternal and fetal ACE I/D, ACE G2350A, AGT M235T, AGT T174M, and AT1R A1166C polymorphisms on PE in Han Chinese women. Polymorphisms were genotyped in 1,488 subjects (256 patients experiencing PE, along with their fetuses and partners, and 360 normotensive controls with their fetuses). Transmission disequilibrium tests revealed that ACE I/D (P = 0.041), ACE G2350A (P = 0.035), and AT1R A1166C (P = 0.018) were associated with maternal PE. The log-linear analyses revealed that mothers whose offspring carried the MM genotype of AGT M235T had a higher risk of PE (OR = 1.54, P = 0.010), whereas mothers whose offspring carried the II genotype of ACE I/D or the GG genotype of ACE G2350A had a reduced risk (OR = 0.58, P = 0.039; OR = 0.47, P = 0.045, respectively). Our findings demonstrate that fetal ACE I/D, ACE G2350A, AGT M235T, and AT1R A1166C polymorphisms may play significant roles in PE development among pregnant Han Chinese women.

Rivaroxaban is an oral direct factor Xa inhibitor approved for the treatment of stroke and systemic thromboembolism in patients with non-valvular atrial fibrillation. Despite its efficacy, rivaroxaban therapy results in adverse effects and complications, such as bleeding. Angiotensin II (AngII) is implicated in many cardiovascular conditions, such as hypertension and heart failure. In this study, we investigate whether AngII influences anticoagulant effects of rivaroxaban by using an experimental mouse model with type 2 diabetes mellitus and advanced glycation end product (AGE)-exposed human umbilical vein endothelial cells (HUVECs). We found that AngII promoted the anticoagulant effects of rivaroxaban in KKAy mice. The combination of rivaroxaban and AngII enhanced in vivo tissue factor pathway inhibitor (TFPI) activity and induced TFPI expression and activity in AGE-exposed HUVECs. Angiotensin type 2 receptor (AT2R) and Mas antagonists attenuated the AngII-enhanced anticoagulant action of rivaroxaban in vivo, and abolished the increased endothelial TFPI expression and activity. However, angiotensin type 1 receptor (AT1R) antagonist exerted no effects. Additionally, combination of rivaroxaban and AngII induced aortic AT2R and Mas expression. Our data suggest that the anticoagulant effects of rivaroxaban are promoted by AngII via AT2R and Mas signaling. These findings are significant for the clinical administration of rivaroxaban.

The renin-angiotensin system (RAS) is a principal determinant of arterial blood pressure and fluid and electrolyte balance. RAS component dysregulation was recently found in some malignancies and correlated with poor patient outcomes. However, the exact mechanism of local RAS activation in tumors is still unclear. Here, we find that the local angiotensin II predominantly exists in the hypoxic regions of tumor formed by nasopharyngeal carcinoma CNE2 cells and breast cancer MDA-MB-231 cells, where these tumor cells autocrinely produce angiotensin II by a chymase-dependent rather than an angiotensin converting enzyme-dependent mechanism. We further demonstrate in nasopharyngeal carcinoma CNE2 and 5–8F cells that this chymase-dependent effect is mediated by increased levels of lactate, a by-product of glycolytic metabolism. Finally, we show that the enhanced angiotensin II plays an important role in the intracellular accumulation of HIF-1α of hypoxic nasopharyngeal carcinoma cells and mediates the radiation-resistant phenotype of these nasopharyngeal carcinoma cells. Thus, our findings reveal the critical role of hypoxia in producing local angiotensin II by a lactate-chymase-dependent mechanism and highlight the importance of local angiotensin II in regulating radioresistance of hypoxic tumor cells.

Myeloid differentiation 1 (MD-1), a secreted protein interacting with radioprotective 105 (RP105), plays an important role in Toll-like receptor 4 (TLR4) signalling pathway. Previous studies showed that MD-1 may be restricted in the immune system. In this study, we demonstrated for the first time that MD-1 was highly expressed in both human and animal hearts. We also discovered that cardiac-specific overexpression of MD-1 significantly attenuated pressure overload-induced cardiac hypertrophy, fibrosis, and dysfunction, whereas loss of MD-1 had the opposite effects. Similar results were observed for in vitro angiotensin II-induced neonatal rat cardiomyocyte hypertrophy. The antihypertrophic effects of MD-1 under hypertrophic stimuli were associated with the blockage of MEK-ERK 1/2 and NF-κB signalling. Blocking MEK-ERK 1/2 signalling with a pharmacological inhibitor (U0126) greatly attenuated the detrimental effects observed in MD-1 knockout cardiomyocytes exposed to angiotensin II stimuli. Similar results were observed by blocking NF-κB signalling with a pharmacological inhibitor (BAY11–7082). Our data indicate that MD-1 inhibits cardiac hypertrophy and suppresses cardiac dysfunction during the remodelling process, which is dependent on its modulation of the MEK-ERK 1/2 and NF-κB signalling pathways. Thus, MD-1 might be a novel target for the treatment of pathological cardiac hypertrophy.

Pirfenidone (PFD), an anti-fibrotic small molecule drug, is used to treat fibrotic diseases, but its effects on myocardial infarction (MI)-induced cardiac fibrosis are unknown. The aim of this study was to determine the effects of PFD on MI-induced cardiac fibrosis and the possible underlying mechanisms in rats. After establishment of the model, animals were administered PFD by gavage for 4 weeks. During the development of MI-induced cardiac fibrosis, we found activation of a positive feedback loop between the angiotensin II type 1 receptor (AT1R)/phospho-p38 mitogen-activated protein kinase (p38 MAPK) pathway and renin-angiotensin system (RAS), which was accompanied by down-regulation of liver X receptor-α (LXR-α) expression. PFD attenuated body weight, heart weight, left ventricular weight, left ventricular systolic pressure, and ±dp/dtmax changes induced by MI, which were associated with a reduction in cardiac fibrosis, infarct size, and hydroxyproline concentration. Moreover, PFD inhibited the AT1R/p38 MAPK pathway, corrected the RAS imbalance [decreased angiotensin-converting enzyme (ACE), angiotensin II, and angiotensin II type 1 receptor expression, but increased ACE2 and angiotensin (1-7) activity and Mas expression] and strongly enhanced heart LXR-α expression. These results indicate that the cardioprotective effects of PFD may be due, in large part, to controlling the feedback loop of the AT1R/p38 MAPK/RAS axis by activation of LXR-α.

Exercise training (ExT) has been reported to benefit hypertension; however, the exact mechanisms involved are unclear. We hypothesized that ExT attenuates hypertension, in part, through the renin-angiotensin system (RAS), reactive oxygen species (ROS), and glutamate in the paraventricular nucleus (PVN). Two-kidney, one-clip (2K1C) renovascular hypertensive rats were assigned to sedentary (Sed) or treadmill running groups for eight weeks. Dizocilpine (MK801), a glutamate receptor blocker, or losartan (Los), an angiotensin II type1 receptor (AT1-R) blocker, were microinjected into the PVN at the end of the experiment. We found that 2K1C rats had higher mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA). These rats also had excessive oxidative stress and overactivated RAS in PVN. Eight weeks of ExT significantly decreased MAP and RSNA in 2K1C hypertensive rats. ExT inhibited angiotensin-converting enzyme (ACE), AT1-R, and glutamate in the PVN, and angiotensin II (ANG II) in the plasma. Moreover, ExT attenuated ROS by augmenting copper/zinc superoxide dismutase (Cu/Zn-SOD) and decreasing p47phox and gp91phox in the PVN. MK801or Los significantly decreased blood pressure in rats. Together, these findings suggest that the beneficial effects of ExT on renovascular hypertension may be, in part, through the RAS-ROS-glutamate pathway in the PVN.

Angiogenesis and inflammation are implicated in aortic aneurysm and atherosclerosis and regulated by angiopoietin-2 (Angpt2). The effect of Angpt2 administration on experimental aortic aneurysm and atherosclerosis was examined. Six-month-old male apolipoprotein E deficient (ApoE−/−) mice were infused with angiotensin II (AngII) and administered subcutaneous human Fc-protein (control) or recombinant Angpt2 (rAngpt2) over 14 days. Administration of rAngpt2 significantly inhibited AngII-induced aortic dilatation and rupture of the suprarenal aorta (SRA), and development of atherosclerosis within the aortic arch. These effects were blood pressure and plasma lipoprotein independent and associated with Tie2 activation and down-regulation of monocyte chemotactic protein-1 (MCP-1) within the SRA. Plasma concentrations of MCP-1 and interleukin-6 were significantly lower in mice receiving rAngpt2. Immunostaining for the monocyte/macrophage marker MOMA-2 and the angiogenesis marker CD31 within the SRA were less in mice receiving rAngpt2 than controls. The percentage of inflammatory (Ly6Chi) monocytes within the bone marrow was increased while that in peripheral blood was decreased by rAngpt2 administration. In conclusion, administration of rAngpt2 attenuated angiotensin II-induced aortic aneurysm and atherosclerosis in ApoE−/− mice associated with reduced aortic inflammation and angiogenesis. Up-regulation of Angpt2 may have potential therapeutic value in patients with aortic aneurysm and atherosclerosis.

Cardiovascular and renal pathologies are frequently associated with an activated renin-angiotensin-system (RAS) and increased levels of its main effector and vasoconstrictor hormone angiotensin II (Ang II). Angiotensin-converting-enzyme-2 (ACE2) has been described as a crucial enzymatic player in shifting the RAS towards its so-called alternative vasodilative and reno-protective axis by enzymatically converting Ang II to angiotensin-(1-7) (Ang-(1-7)). Yet, the relative contribution of ACE2 to Ang-(1-7) formation in vivo has not been elucidated. Mass spectrometry based quantification of angiotensin metabolites in the kidney and plasma of ACE2 KO mice surprisingly revealed an increase in Ang-(1-7), suggesting additional pathways to be responsible for alternative RAS activation in vivo. Following assessment of angiotensin metabolism in kidney homogenates, we identified neprilysin (NEP) to be a major source of renal Ang-(1-7) in mice and humans. These findings were supported by MALDI imaging, showing NEP mediated Ang-(1-7) formation in whole kidney cryo-sections in mice. Finally, pharmacologic inhibition of NEP resulted in strongly decreased Ang-(1-7) levels in murine kidneys. This unexpected new role of NEP may have implications for the combination therapy with NEP-inhibitors and angiotensin-receptor-blockade, which has been shown being a promising therapeutic approach for heart failure therapy.

Curcumin exerts beneficial effects on cardiovascular diseases, including hypertension. However, its mechanisms are unknown. We propose that curcumin prevents the development of hypertension by regulating AT1 receptor (AT1R) expression in arteries. The present study examined how curcumin regulates AT1R expression in vascular smooth muscle cells and investigated the physiological significance of this regulation in angiotensin (Ang) II-induced hypertension. The results showed that curcumin decreased AT1R expression in a concentration- and time-dependent manner in vascular smooth muscle cells. Using luciferase reporters with an entire AT1 or a mutant AT1R in A10 cells, the AT1R promoter activity was inhibited by 10−6M curcumin, and the proximal element (from −61 to +25 bp) of the AT1R promoter was crucial for curcumin-induced AT1R down-regulation. An electrophoretic mobility shift assay showed that curcumin decreased specificity protein 1 (SP1) binding with the AT1R promoter in A10 cells. Curcumin treatment reduced Ang II-induced hypertension in C57Bl/6J mice, which was accompanied by lower AT1R expression in the arteries and decreased Ang II-mediated vasoconstriction in the mesenteric artery. These findings indicate that curcumin down-regulates AT1R expression in A10 cells by affecting SP1/AT1R DNA binding, thus reducing AT1R-mediated vasoconstriction and subsequently prevents the development of hypertension in an Ang II-induced hypertensive model.

Renin angiotensin system (RAS) is a key hormonal system which regulates the cardiovascular function and is implicated in several autoimmune diseases. With the discovery of the angiotensin-converting enzyme 2 (ACE2), a protective axis of RAS namely ACE2/Ang-(1–7)/Mas that counteracts the deleterious ACE/AngII/AT1R axis has been established. This axis is emerging as a novel target to attenuate ocular inflammation. However, the underlying molecular mechanisms remain unclear. We investigated the hypothesis that enhancing the activity of the protective axis of RAS by subretinal delivery of an AAV8 (Y733F)-ACE2 vector would protect against the ocular inflammation in experimental autoimmune uveitis (EAU) mice through regulating the local immune responses. Our studies demonstrated that increased ACE2 expression exerts protective effects on inflammation in EAU mouse by modulating ocular immune responses, including the differentiation of Th1/Th17 cells and the polarization of M1/M2 macrophages; whereas the systemic immune responses appeared not affected. These effects were mediated by activating the Ang-(1–7)/Mas and inhibiting the MAPK, NF-κB and STAT3 signaling pathways. This proof-of-concept study suggests that activation of ocular ACE2/Ang-(1–7)/Mas axis with AAV gene transfer modulates local immune responses and may be a promising, long-lasting therapeutic strategy for refractory and recurrent uveitis, as well as other inflammatory eye diseases.

It has been shown that angiotensin I-converting enzyme inhibitors (ACEIs) and angiotensin II type-1 receptor blockers (ARBs) can decrease tumor growth and tumor-associated angiogenesis and inhibit metastasis. Epidermal growth factor receptor (EGFR) mutations are found in approximately 30% of patients with advanced non-small cell lung cancer (NSCLC) in East Asia and in 10–15% of such patients in Western countries. We retrospectively identified 228 patients with histologically confirmed advanced NSCLC and 73 patients with early stage disease; 103 of these patients took antihypertensive drugs and 112 received treatment with EGFR tyrosine kinase inhibitors (TKIs). There was a significant difference in progression-free survival after first-line therapy (PFS1) between the ACEI/ARB group and the non-ACEI/ARB group. For the patients treated with TKIs, there was a significant difference in PFS but not in overall survival (OS) between the ACEI/ARB group and the non-ACEI/ARB group. For the patients with advanced NSCLC, there was a significant difference in PFS1 between the ACEI/ARB group and the non-ACEI/ARB group. ACEI/ARB in combination with standard chemotherapy or TKIs had a positive effect on PFS1 or OS, regardless of whether the lung cancer was in the early or advanced stage.

The renin-angiotensin system (RAS) plays pathogenic roles in renal and cardiovascular disorders, but whether it is involved in colitis is unclear. Here we show that RenTgMK mice that overexpress active renin from the liver developed more severe colitis than wild-type controls. More than 50% RenTgMK mice died whereas all wild-type mice recovered. RenTgMK mice exhibited more robust mucosal TH17 and TH1/TH17 responses and more profound colonic epithelial cell apoptosis compared to wild-type controls. Treatment with aliskiren (a renin inhibitor), but not hydralazine (a smooth muscle relaxant), ameliorated colitis in RenTgMK mice, although both drugs normalized blood pressure. Chronic infusion of angiotensin II into wild-type mice mimicked the severe colitic phenotype of RenTgMK mice and treatment with losartan [an angiotensin type 1 receptor blocker (ARB)] ameliorated colitis in wild-type mice, confirming a colitogenic role for the endogenous RAS. In human biopsies, pro-inflammatory cytokines were suppressed in patients with inflammatory bowel disease who were on ARB therapy compared to patients not receiving ARB therapy. These observations demonstrate that activation of the RAS promotes colitis in a blood pressure independent manner. Angiotensin II appears to drive colonic mucosal inflammation by promoting intestinal epithelial cell apoptosis and mucosal TH17 responses in colitis development.

Respiratory syncytial virus (RSV) infection is a major cause of severe lower respiratory illness in infants and young children, but the underlying mechanisms responsible for viral pathogenesis have not been fully elucidated. To date, no drugs or vaccines have been employed to improve clinical outcomes for RSV-infected patients. In this paper, we report that angiotensin-converting enzyme-2 (ACE2) protected against severe lung injury induced by RSV infection in an experimental mouse model and in pediatric patients. Moreover, ACE2 deficiency aggravated RSV-associated disease pathogenesis, mainly by its action on the angiotensin II type 1 receptor (AT1R). Furthermore, administration of a recombinant ACE2 protein alleviated the severity of RSV-induced lung injury. These findings demonstrate that ACE2 plays a critical role in preventing RSV-induced lung injury and suggest that ACE2 is a promising potential therapeutic target in the management of RSV-induced lung disease.

The mechanism by which chronic metabolic acidosis (CMA) regulates sodium (Na+)-chloride (Cl−) cotransporter (NCC) in the renal distal convoluted tubules remains unexplored. We examined the role of STE20/SPS1-related proline/alanine-rich kinase (SPAK) and with-no-lysine kinase 4 (WNK4) on expression of NCC in mouse models of CMA. CMA was induced by NH4Cl in wild type mice (WTA mice), SPAK and WNK4 knockout mice. The quantities of Ncc mRNA, expression of total NCC, phosphorylated (p)-NCC, SPAK and WNK4 in the kidneys as well as NCC inhibition with hydrochlorothiazide and Na+ balance were evaluated. Relative to WT mice, WTA mice had similar levels of Ncc mRNA, but increased expression of total and p-NCC, SPAK and WNK4 and an exaggerated response to hydrochlorothiazide which could not be observed in SPAK or WNK4 knockout mice with CMA. In WTA mice, increased plasma renin activity, aldosterone and angiotensin II concentrations accompanied by a significantly negative Na+ balance. High Na+ diet abolished the enhanced NCC expression in WTA mice. Furthermore, an angiotensin II type 1 receptor blocker rather than a mineralocorticoid receptor antagonist exerted a marked inhibition on Na+ reabsorption and NCC phosphorylation in WTA mice. CMA increases WNK4-SPAK-dependent NCC phosphorylation and appears to be secondary to previous natriuresis with volume-dependent angiotensin II activation.

Cardio-renal fibrosis plays key roles in heart failure and chronic kidney disease. We sought to determine the effects of renal denervation (RDN) on cardiac and renal fibrosis in rats with isoproterenol induced cardiomyopathy. Sixty male Sprague Dawley rats were randomly assigned to Control (n = 10) and isoproterenol (ISO)-induced cardiomyopathy group (n = 50). At week 5, 31 survival ISO-induced cardiomyopathy rats were randomized to RDN (n = 15) and Sham group (n = 16). Compared with Control group, ejection fraction was decreased, diastolic interventricular septal thickness and left atrial dimension were increased in ISO-induced cardiomyopathy group at 5 week. After 10 weeks, cardio-renal pathophysiologic results demonstrated that the collagen volume fraction of left atrio-ventricular and kidney tissues reduced significantly in RDN group compared with Sham group. Moreover the pro-fibrosis factors (TGF-β1, MMP2 and Collagen I), inflammatory cytokines (CRP and TNF-α) and collagen synthesis biomarkers (PICP, PINP and PIIINP) concentration significantly decreased in RDN group. Compared with Sham group, RDN group showed that release of noradrenaline and aldosterone were reduced, angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/angiotensin II type-1 receptor (AT1R) axis was downregulated. Meanwhile, angiotensin-converting enzyme 2 (ACE2)/angiotensin-1-7 (Ang-(1-7))/mas receptor (Mas-R) axis was upregulated. RDN inhibits cardio-renal fibrogenesis through multiple pathways, including reducing SNS over-activity, rebalancing RAAS axis.

Angiotensin (Ang) II plays an important role in the brain as a neurotransmitter and is involved in psychological stress reactions, for example through activation of the sympatho-adrenomedullary system. We investigated the effects of centrally administered Ang II on the micturition reflex, which is potentially affected by the sympatho-adrenomedullary system and brain Ang II receptors in urethane-anesthetized (1.0 g/kg, intraperitoneally) male rats. Central administration of Ang II (0.01, 0.02 and 0.07 nmol per rat, intracerebroventricularly, icv) but not vehicle rapidly and dose-dependently decreased the urinary bladder intercontraction interval, without altering the bladder detrusor pressure. Central administration of antagonists of Ang II type 1 but not type 2 receptors inhibited the Ang II-induced shortening of intercontraction intervals. Administration of the highest dose of Ang II (0.07 nmol per rat, icv) but not lower doses (0.01 and 0.02 nmol per rat, icv) elevated the plasma concentration of adrenaline. Bilateral adrenalectomy reduced Ang II-induced elevation in adrenaline, but had no effect on the Ang II-induced shortening of the intercontraction interval. These data suggest that central administration of Ang II increases urinary frequency by acting on brain Ang II type 1 receptors, independent of activation of the sympatho-adrenomedullary system.

Alzheimer’s disease is characterized by neuronal loss and cerebral accumulation of amyloid-β protein (Aβ) and lowering the generation of Aβ is a pivotal approach in the strategy of Alzheimer’s disease treatment. Midlife hypertension is a major risk factor for the future onset of sporadic Alzheimer’s disease and the use of some antihypertensive drugs may decrease the incidence of Alzheimer’s disease. However, it is largely unknown how the blood pressure regulation system is associated with the pathogenesis of Alzheimer’s disease. Here we found that the deficiency of angiotensin type 1a receptor (AT1a), a key receptor for regulating blood pressure, significantly decreased Aβ generation and amyloid plaque formation in a mouse model of Alzheimer’s disease. The lack of AT1a inhibited the endocleavage of presenilin-1 (PS1), which is essential for γ-secretase complex formation and Aβ generation. Notably, the ligand of AT1a, angiotensin II, enhanced Aβ generation, PS1 endocleavage and γ-secretase complex formation. Our results suggest that AT1a activation is closely associated with Aβ generation and brain amyloid accumulation by regulating γ-secretase complex formation. Thus, removal of life style factors or stresses that stimulate AT1a to elevate blood pressure may decrease Aβ generation and brain amyloid accumulation, thereby preventing the pathogenesis of Alzheimer’s disease.

Angiotensin II is one of the key regulatory peptides implicated in the pathogenesis of liver disease. The mechanisms underlying the salubrious role of α-tocopherol and β-carotene on liver pathology have not been comprehensively assessed. Here, we investigated the mechanisms underlying the role of Angiotensin II on hepatic damage and if α-tocopherol and β-carotene supplementation attenuates hepatic damage. Hepatic damage was induced in Apoe−/−mice by infusion of Angiotensin II followed by oral administration with α-tocopherol and β-carotene-enriched diet for 60 days. Investigations showed fibrosis, kupffer cell hyperplasia, hepatocyte degeneration and hepatic cell apoptosis; sinusoidal dilatation along with haemorrhages; evidence of fluid accumulation; increased ROS level and increased AST and ALT activities. In addition, tPA and uPA were down-regulated due to 42-fold up-regulation of PAI-1. MMP-2, MMP-9, MMP-12, and M-CSF were down-regulated in Angiotensin II-treated animals. Notably, α-tocopherol and β-carotene treatment controlled ROS, fibrosis, hepatocyte degeneration, kupffer cell hyperplasia, hepatocyte apoptosis, sinusoidal dilatation and fluid accumulation in the liver sinusoids and liver enzyme levels. In addition, PAI-1, tPA and uPA expressions were markedly controlled by β-carotene treatment. Thus, Angiotensin II markedly influenced hepatic damage possibly by restraining fibrinolytic system. We concluded that α-tocopherol and β-carotene treatment has salubrious role in repairing hepatic pathology.

Dietary fats and sodium are both palatable and are hypothesized to synergistically contribute to ingestive behavior and thereby obesity. Contrary to this hypothesis, C57BL/6J mice fed a 45% high fat diet exhibited weight gain that was inhibited by increased dietary sodium content. This suppressive effect of dietary sodium upon weight gain was mediated specifically through a reduction in digestive efficiency, with no effects on food intake behavior, physical activity, or resting metabolism. Replacement of circulating angiotensin II levels reversed the effects of high dietary sodium to suppress digestive efficiency. While the AT1 receptor antagonist losartan had no effect in mice fed low sodium, the AT2 receptor antagonist PD-123,319 suppressed digestive efficiency. Correspondingly, genetic deletion of the AT2 receptor in FVB/NCrl mice resulted in suppressed digestive efficiency even on a standard chow diet. Together these data underscore the importance of digestive efficiency in the pathogenesis of obesity and implicate dietary sodium, the renin-angiotensin system and the AT2 receptor in the control of digestive efficiency regardless of mouse strain or macronutrient composition of the diet. These findings highlight the need for greater understanding of nutrient absorption control physiology and prompt more uniform assessment of digestive efficiency in animal studies of energy balance.

The known angiotensin II (AngII) physiological effect of aldosterone synthesis and secretion is mediated by either Gq/11 proteins or βarrestin1 (βarr1), both of which can couple to its type 1 receptors (AT1Rs), present in adrenocortical zona glomerulosa (AZG) cell membranes. In the present study, we examined the relative potencies of all the currently used in the clinic AT1R antagonist drugs (angiotensin receptor blockers, ARBs, or sartans) at preventing activation of these two signaling mediators (G proteins and βarrs) at the AngII-bound AT1R and, consequently, at suppression of aldosterone in vitro. All ARBs were found to be potent inhibitors of G protein activation at the AT1R. However, candesartan and valsartan were the most potent at blocking AngII-induced βarr activation at this receptor, among the tetrazolo-biphenyl-methyl derivatives, translating into excellent efficacies at aldosterone suppression in H295R cells. Conversely, irbesartan and losartan were largely G protein-selective inhibitors at the AT1R, with very low potency towards βarr inhibition. As a result, they were very weak suppressors of βarr1-dependent aldosterone production in H295R cells. These findings provide important pharmacological insights into the drug class of ARBs and medicinal chemistry insights for future drug development in the field of AngII antagonism.

Pulmonary fibrosis is a serious lung disorder that can lead to respiratory failure. Here we show that transgenic mice expressing active renin from the liver (RenTgMK) developed progressive pulmonary fibrosis leading to impaired pulmonary function. Histological analyses revealed a marked increase in extracellular matrix (ECM) deposition and decrease in alveolar size in the lungs of RenTgMK mice compared to wild-type (WT) littermates, accompanied with increased expression of ECM proteins and fibrogenic factors. The increase in lung fibrosis led to a substantial decrease in respiratory system compliance. Two-week treatment with aliskiren (renin inhibitor) or losartan (AT1 antagonist) ameliorated pulmonary ECM deposition, blocked the induction of ECM proteins and fibrogenic factors and improved respiratory compliance in RenTgMK mice, confirming a critical role of the renin-Ang II-AT1 cascade in promoting pulmonary fibrogenesis. However, when RenTgMK mice were treated with hydralazine (a smooth muscle relaxant), the blood pressure was normalized but the lung fibrotic abnormalities, fibrogenic gene induction and pulmonary elasticity were not corrected. Moreover, intratracheal instillation of lipopolysaccharide induced more severe lung injury in RenTgMK mice compared to WT littermates. These observations demonstrate that the renin-angiotensin system is a key mediator of lung fibrosis and its pro-fibrotic effect is independent of blood pressure.