Protective Role of Short-term Aerobic Exercise Against Zinc Oxide Nanoparticles-Induced Cardiac Oxidative Stress Via Possible Changes of Apelin, Angiotensin II/Angiotensin II Type I Signalling Pathway.

This study examined the protective role of short-term aerobic exercise on ZnO NPs-induced cardiac oxidative stress and possible changes of apelin, angiotensin II (AngII) and angiotensin II type I receptor (AT1R) signalling pathway. Thirty-five male Wistar rats were randomized into five groups of seven rats, including control, saline, ZnO NPs, exercise and exercise + ZnO NPs groups. The animal in ZnO NPs and exercise + ZnO NPs groups received 1 mg/kg of ZnO NPs. Rats underwent the treadmill exercise program. Treatments lasted four weeks, 5 days/week. After 4 weeks of treatment, superoxide dismutase (SOD) activity, malondialdehyde (MDA), apelin, Ang II and AT1R concentration were measured in heart tissue.Cardiac MDA, Ang II and AT1R levels significantly increased while SOD activity and apelin levels significantly decreased following ZnO NPs administration. The aerobic exercise induced a significant increase in the SOD activity and apelin levels and a significant decrease in the enhanced MDA, Ang II and AT1R levels in the heart of ZnO NPs-exposed rats. These results suggest that the exercise-induced attenuation of the Ang II-AT1R signalling pathway is mediated by reduced lipid peroxidation, augmented antioxidant defence and enhanced apelin synthesis that may be a protective mechanism to prevent and/or treatment ZnO NPs-induced cardiac oxidative stress.

ACE2 internalization induced by a SARS-CoV-2 recombinant protein is modulated by angiotensin II type 1 and bradykinin 2 receptors.

AIMS: Angiotensin-converting enzyme 2 (ACE2) is a key regulator of the renin-angiotensin system (RAS) recently identified as the membrane receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we aim to study whether two receptors from RAS, the angiotensin receptor type 1 (AT1R) and the bradykinin 2 receptor (B2R) modulate ACE2 internalization induced by a recombinant receptor binding domain (RBD) of SARS-CoV-2 spike protein. Also, we investigated the impact of ACE2 coexpression on AT1R and B2R functionality. MATERIALS AND METHODS: To study ACE2 internalization, we assessed the distribution of green fluorescent protein (GFP) signal in HEK293T cells coexpressing GFP-tagged ACE2 and AT1R, or B2R, or AT1R plus B2R in presence of RBD alone or in combination with AT1R or B2R ligands. To estimate ACE2 internalization, we classified GFP signal distribution as plasma membrane uniform GFP (PMU-GFP), plasma membrane clustered GFP (PMC-GFP) or internalized GFP and calculated its relative frequency. Additionally, we investigated the effect of ACE2 coexpression on AT1R and B2R inhibitory action on voltage-gated calcium channels (CaV2.2) currents by patch-clamp technique. KEY FINDINGS: RBD induced ACE2-GFP internalization in a time-dependent manner. RBD-induced ACE2-GFP internalization was increased by angiotensin II and reduced by telmisartan in cells coexpressing AT1R. RBD-induced ACE2-GFP internalization was strongly inhibited by B2R co-expression. This effect was mildly modified by bradykinin and rescued by angiotensin II in presence of AT1R. ACE2 coexpression impacted on B2R- and AT1R-mediated inhibition of CaV2.2 currents. SIGNIFICANCE: Our work contributes to understand the role of RAS modulators in the susceptibility to SARS-CoV-2 infection and severity of COVID-19.

Endothelial dysfunction after androgen deprivation therapy and the possible underlying mechanisms.

INTRODUCTION: Androgen deprivation therapy (ADT) is a key treatment modality in the management of prostate cancer (PCa), especially for patients with metastatic disease. Increasing evidences suggest that patients who received ADT have increased incidence of diabetes, myocardial infarction, stroke, and even mortality. It is important to understand the pathophysiological mechanisms on how ADT increases cardiovascular risk and induces cardiovascular events, which would provide important information for potential implementation of preventive measures. METHODS: Twenty-six 12-week-old male SD rats were divided into four groups for different types of ADTs including: the bilateral orchidectomy group (Orx), LHRH agonist group (leuprolide), LHRH antagonist group (degarelix), and control group. After treated with drug or adjuvant injection every 3 weeks for 24 weeks, all rats were sacrificed and total blood were collected. Aorta, renal arteries, and kidney were preserved for functional assay, immunohistochemistry, western blot, and quantitative reverse-transcription polymerase chain reaction. RESULTS: In vascular reactivity assays, aorta, intrarenal, and coronary arteries of all three ADT groups showed endothelial dysfunction. AT1R and related molecules at protein and messenger RNA (mRNA) level were tested, and AT1R pathway was shown to be activated and played a role in endothelial dysfunction. Both ACE and AT1R mRNA levels were doubled in the aorta in the leuprolide group while Orx and degarelix groups showed upregulation of AT1R in the kidney tissues. By immunohistochemistry, our result showed higher expression of AT1R in the intrarenal arteries of leuprolide and degarelix groups. The role of reactive oxygen species in endothelial dysfunction was confirmed by DHE fluorescence, nitrotyrosine overexpression, and upregulation of NOX2 in the different ADT treatment groups. CONCLUSION: ADT causes endothelial dysfunction in male rats. GnRH receptor agonist compared to GnRH receptor antagonist, showed more impairment of endothelial function in the aorta and intrarenal arteries. Such change might be associated with upregulation and activation of AngII-AT1R-NOX2 induced oxidative stress in the vasculature. These results help to explain the different cardiovascular risks and outcomes related to different modalities of ADT treatment.

Angiotensin II enhances the proliferation of Natural Killer/T-cell lymphoma cells via activating PI3K/Akt signaling pathway.

The present study was to determine the roles of Angiotensin (Ang) II in the growth of lymphoma in nude mice and the proliferation and viability of the human Natural Killer/T (NK/T)-cell lymphoma cell line SNK-6, and the activation of downstream signaling pathway. Lymphoma samples and corresponding normal tissues were obtained from lymphoma patients. Proliferation of SNK-6 cells was detected by CCK8 or MTT assay. The levels of Ang II and its receptor Ang II type 1 receptor (AT1R) were higher in lymphoma tissues than those in control tissues. Ang II increased the lymphoma volume and size in nude mice, the proliferation and viability and the proliferating cell nuclear antigen (PCNA) and Ki67 levels of SNK-6 cells. Losartan, an antagonist of AT1R, reduced lymphoma volume and size in nude mice, and the proliferation and viability and the PCNA and Ki67 levels of SNK-6 cells. The levels of phosphorylated phosphatidylinositol 3-kinase (p-PI3K) and phosphorylated protein kinase B (p-Akt) were increased by Ang II and then reduced by losartan in SNK-6 cells. The proliferation and viability of SNK-6 cells were increased by Ang II, but these increases were inhibited by PI3K inhibitor wortmannin and Akt inhibitor MK2206. The increases of PCNA and Ki67 induced by Ang II were inhibited by wortmannin or MK2206 in SNK-6 cells. These results indicate that Ang II/AT1R is activated in lymphoma, and Ang II promotes the progression of lymphoma in nude mice and the proliferation and viability of SNK-6 cells via activating PI3K/Akt signaling pathway.

Cutaneous inflammation differentially regulates the expression and function of Angiotensin-II types 1 and 2 receptors in rat primary sensory neurons.

Neuropathic and inflammatory pain results from cellular and molecular changes in dorsal root ganglion (DRG) neurons. The type-2 receptor for Angiotensin-II (AT2R) has been involved in this type of pain. However, the underlying mechanisms are poorly understood, including the role of the type-1 receptor for Angiotensin-II (AT1R). Here, we used a combination of immunohistochemistry and immunocytochemistry, RT-PCR and in vitro and in vivo pharmacological manipulation to examine how cutaneous inflammation affected the expression of AT1R and AT2R in subpopulations of rat DRG neurons and studied their impact on inflammation-induced neuritogenesis. We demonstrated that AT2R-neurons express C- or A-neuron markers, primarily IB4, trkA, and substance-P. AT1R expression was highest in small neurons and co-localized significantly with AT2R. In vitro, an inflammatory soup caused significant elevation of AT2R mRNA, whereas AT1R mRNA levels remained unchanged. In vivo, we found a unique pattern of change in the expression of AT1R and AT2R after cutaneous inflammation. AT2R increased in small neurons at 1 day and in medium size neurons at 4 days. Interestingly, cutaneous inflammation increased AT1R levels only in large neurons at 4 days. We found that in vitro and in vivo AT1R and AT2R acted co-operatively to regulate DRG neurite outgrowth. In vivo, AT2R inhibition impacted more on non-peptidergic C-neurons neuritogenesis, whereas AT1R blockade affected primarily peptidergic nerve terminals. Thus, cutaneous-induced inflammation regulated AT1R and AT2R expression and function in different DRG neuronal subpopulations at different times. These findings must be considered when targeting AT1R and AT2R to treat chronic inflammatory pain. Cover Image for this issue: doi: 10.1111/jnc.14737.

Planejamento e desenvolvimento de ligantes não peptídicos com atividade agonista enviesada no receptor de angiotensina II tipo 1 empregando-se técnicas de modelagem molecular e ensaios in vitro / Drug design of non-peptidic biased agonists for angiotensin II type 1 receptor using molecular modeling techniques and in vitro tests

A insuficiência cardíaca (IC) é uma síndrome de elevada morbimortalidade, correspondendo a um grave problema de saúde pública. Uma das abordagens terapêuticas para IC consiste no uso de antagonistas do receptor de angiotensina II do tipo 1 (AT1R), conhecidos como sartanas. Estudos apontam que uma nova classe de compostos, os agonistas enviesados, é capaz de induzir a sinalização da via da ß-arrestina sem ativação da via da proteína G. Essa seletividade funcional é particularmente interessante, pois a via dependente da proteína G é responsável pelo aumento da pressão arterial, morte celular e fibrose tecidual, levando a hipertrofia cardíaca e progressão da IC. No entanto, a via da ß-arrestina está associada com renovação celular e aumento do inotropismo. Além disso, estudos in vivo sugerem que agonistas enviesados poderiam corresponder a uma terapia superior à dos antagonistas convencionais, que bloqueiam ambas as vias. Apesar do potencial terapêutico, esses compostos possuem estrutura peptídica e, por isso, tem sua administração restrita à via intravenosa. A resolução da estrutura cristalográfica do AT1R permitiu estudos de modelagem molecular mais acurados. Tendo isso em mente, nesse trabalho foram propostos agonistas enviesados de natureza não peptídica para o AT1R por meio de técnicas de modelagem molecular e validação das hipóteses levantadas por ensaios in vitro. Foram realizados estudos de dinâmica molecular com o AT1R (PDB ID: 4YAY) em uma bicamada lipídica e ensaios de ancoramento molecular da angiotensina II (AngII) e do ligante enviesado TRV027. As poses de ancoramento molecular selecionadas foram utilizadas em dinâmicas de complexo, que revelaram diferenças entre os sistemas apo (sem nenhum ligante) e holo (com o ligante no sitio de ligação). Nossos resultados sugerem que o TRV027 induz um padrão exclusivo de ligações de hidrogênio e de estrutura secundária, enquanto que a AngII afeta os resíduos do bolso hidrofóbico do sitio de ligação, principalmente a conformação do Trp2536.48. Com base nas simulações, três farmacóforos foram criados e utilizados de maneira complementar em triagens virtuais na base de dados ZINC15, resultando na seleção de cinco compostos. Um desses compostos apresentou afinidade pelo receptor AT1R e, ainda que estudos complementares de ativação de vias especificas sejam necessários para que o composto possa ser classificado como agonista enviesado, já se constitui em molécula potencialmente promissora. Além disso, esses estudos permitiram a proposição de estruturas inéditas que podem vir a ser hits no processo de desenvolvimento de agonistas enviesados para AT1R. Portanto, como continuidade desse trabalho, essas moléculas serão sintetizadas e investigadas quanto à possível interação com o receptor.

Heart Failure (HF) is a common syndrome with high morbimortality, being considered a serious public health problem. One of the therapeutic approaches for HF consists in the use of the sartan class, which are angiotensin II type 1 receptor (AT1R) antagonists. Recent studies have shown that a new class of compounds, known as biased agonists, is able to induce signaling via ß-arrestin without G-protein activation. This functional selectivity is particularly interesting since G-protein dependent signaling is responsible for cell death and cardiac tissue fibrosis, which leads to cardiac muscle hypertophy and HF progression. On the other hand, ß-arrestin signaling is associated with cellular renewal and increased inotropism. In vivo studies suggests that biased agonists could correspond to a superior therapy over conventional angiotensin II type 1 receptor antagonists, which blocks cell signaling as a whole, however their peptidic structure restricts their use to intravenous administration. Moreover, the AT1R crystal structure determination holds great promise for more accurate molecular modeling studies. With that being said, the aim of this work was to plan and develop new non-peptidic biased agonists for ATR1 employing molecular modeling techniques and in vitro tests for hypothesis validation. Molecular dynamics (MD) simulations of the refined AT1R crystal (PDB ID: 4YAY) embedded in a lipid bilayer and molecular docking studies with angiotensin II (AngII) and TRV027 (biased agonist) were conducted. Selected docking poses from both ligands underwent complex MD simulations revealing differences between apo (ligand free) and holo (ligand in the binding site) systems. Our results suggest that TRV027 induces an exclusive hydrogen bond and secondary structure pattern, while AngII affects the hydrophobic pocket conformation, mainly Trp253. Based on the simulations, three pharmacophore models were created and used in virtual screenings in the ZINC15 database, resulting in the selection of five compounds that were tested in vitro. One of the compounds displayed affinity for AT1R and is a promising molecule. Nonetheless, it needs further pathway activation characterization in order to be a classified as a biased agonist. Furthermore, these results have contributed significantly for the proposition of new structures that could be hits with biased agonist activity for AT1R. Thus, for future works, we point out the necessity for synthesis and characterization of this new compounds

Vasoactive intestinal peptide infusion reverses existing myocardial fibrosis in the rat.

Congestive cardiac failure has become one of the major health challenges of the 21st century and new therapies are needed to address this problem. The concentration of vasoactive intestinal peptide (VIP) in the heart has been shown to decrease as fibrosis (the pathology leading to heart failure) increases and to become undetectable in end stage cardiomyopathy. We sought to determine whether replenishment of myocardial VIP might treat myocardial fibrosis and therefore represent a new therapeutic target. Wistar Kyoto rats on a high (4.4%) salt diet were randomised to zero time control, 4 week infusion of VIP (5 pmol/kg/min) or vehicle control infusion. Myocardial VIP concentration was measured by radioimmunoassay, fibrosis was quantitated by computerised histomorphometry and changes in pro-fibrotic mediators were measured by quantitative rt-PCR. Myocardial VIP increased significantly in VIP treated rats compared with vehicle treated controls (P < 0.01) while fibrosis in the VIP treated rats was significantly lower than in both the zero time control (P < 0.05) and the vehicle infused control (P < 0.0005). Although all six profibrotic mediators which were measured increased over the 4 week experimental period VIP infusion only affected angiotensinogen (Agt) and angiotensin receptor type 1a (AT1a) expression. In both instances VIP caused a significant decrease in messenger rna expression (Agt P < 0.01 and At1a P < 0.01) compared with vehicle infused controls. We conclude that VIP infusion increased myocardial VIP concentration and was able to reverse existing myocardial fibrosis suggesting a possible therapeutic role for a VIP based therapy in cardiac failure.

Candesartan inhibits inflammation through an angiotensin II type 1 receptor independent way in human embryonic kidney epithelial cells.

Besides stimulating vasoconstriction, Angiotensin II is also well known in inducing reactive oxygen species and promoting inflammatory phenotype switch via its type 1 receptor. In clinic, Angiotensin II type 1 (AT1) receptor blocker like candesartan has been widely applied as an antihypertensive medication. We previous have demonstrated that a higher dose of candesartan plays a protective role after kidney injury. However, whether candesartan could exhibit anti-inflammatory effects remains unclear. Here, by stimulating isolated human embryonic kidney epithelial cells with tumor necrosis factor-α (TNF-α), we observed the anti-inflammation capacity of candesartan ex vivo. It was found that pre-treat with candesartan significantly suppressed transforming growth factor-ß (TGF-ß) and interleukin-6 (IL-6) expression after incubation with TNF-α. Surprisingly, silence of angiotensin II type 1 receptor has little effects on reducing TGF-ß or IL-6 products. Furthermore, candesartan inhibited TNF-α-induced oxidative stress in the primary cultured tubular epithelial cells. Overall, our data indicates that candesartan suppresses TNF-α-induced inflammatory cytokine production by inhibiting oxidative stress, rather than block AT1 receptor activity.

Whole body sodium depletion modifies AT1 mRNA expression and serotonin content in the dorsal raphe nucleus.

Angiotensin II (Ang II) acts on Ang II type 1 (AT1) receptors located in the organum vasculosum and subfornical organ (SFO) of the lamina terminalis as a main facilitatory mechanism of sodium appetite. The brain serotonin (5-HT) system with soma located in the dorsal raphe nucleus (DRN) provides a main inhibitory mechanism. In the present study, we first investigated the existence of Ang II AT1 receptors in serotonergic DRN neurones. Then, we examined whether whole body sodium depletion affects the gene expression of the AT1a receptor subtype and the presumed functional significance of AT1 receptors. Using confocal microscopy, we found that tryptophan hydroxylase-2 and serotonin neurones express AT1 receptors in the DRN. Immunofluorescence quantification showed a significant reduction in 5-HT content but no change in AT1 receptor expression or AT1/5-HT colocalisation in the DRN after sodium depletion. Whole body sodium depletion also significantly increased Agtr1a mRNA expression in the SFO and DRN. Oral treatment with the AT1 receptor antagonist losartan reversed the changes in Agtr1a expression in the SFO but not the DRN. Losartan injection into either the DRN or the mesencephalic aqueduct had no influence on sodium depletion-induced 0.3 mol L-1 NaCl intake. The results indicate the expression of Agtr1a mRNA in the DRN and SFO as a marker of sodium depletion. They also suggest that serotonergic DRN neurones are targets for Ang II. However, the function of their AT1 receptors remains elusive.

BRET-based assay to monitor EGFR transactivation by the AT1R reveals Gq/11 protein-independent activation and AT1R-EGFR complexes.

The type 1 angiotensin II (AngII) receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR), which leads to pathological remodeling of heart, blood vessels and kidney. End-point assays are used as surrogates of EGFR activation, however these downstream readouts are not applicable to live cells, in real-time. Herein, we report the use of a bioluminescence resonance energy transfer (BRET)-based assay to assess recruitment of the EGFR adaptor protein, growth factor receptor-bound protein 2 (Grb2), to the EGFR. In a variety of cell lines, both epidermal growth factor (EGF) and AngII stimulated Grb2 recruitment to EGFR. The BRET assay was used to screen a panel of 9 G protein-coupled receptors (GPCRs) and further developed for other EGFR family members (HER2 and HER3); the AT1R was able to transactivate HER2, but not HER3. Mechanistically, AT1R-mediated ERK1/2 activation was dependent on Gq/11 and EGFR tyrosine kinase activity, whereas the recruitment of Grb2 to the EGFR was independent of Gq/11 and only partially dependent on EGFR tyrosine kinase activity. This Gq/11 independence of EGFR transactivation was confirmed using AT1R mutants and in CRISPR cell lines lacking Gq/11. EGFR transactivation was also apparently independent of ß-arrestins. Finally, we used additional BRET-based assays and confocal microscopy to provide evidence that both AngII- and EGF-stimulation promoted AT1R-EGFR heteromerization. In summary, we report an alternative approach to monitoring AT1R-EGFR transactivation in live cells, which provides a more direct and proximal view of this process, including the potential for complexes between the AT1R and EGFR.

Angiotensin II system in the nucleus tractus solitarii contributes to autonomic dysreflexia in rats with spinal cord injury.

BACKGROUND: Autonomic dysreflexia (AD) is a potentially life-threating complication after spinal cord injury (SCI), characterized by episodic hypertension induced by colon or bladder distension. The objective of this study was to determine the role of impaired baroreflex regulation by the nucleus tractus solitarii(NTS) in the occurrence of AD in a rat model. METHODS: T4 spinal cord transection animal model was used in this study, which included 40 Male rats Colorectal distension (CD) was performed to assess AD and compare the changes of BP, HR, and BRS, six weeks after operation. After that, SCI rats with successfully induced AD were selected. Losartan was microinjected into NTS in SCI rats, then 10, 30, 60 minutes later, CD was performed to calculate the changes of BP, HR, and BRS in order to explicit whether Ang II system was involved in the AD occurrence. Ang II was then Intra-cerebroventricular infused in sham operation rats with CD to mimic the activation of Ang II system in AD. Finally, the level of Ang II in NTS and colocalization of AT1R and NMDA receptor within the NTS neurons were also detected in SCI rats. RESULTS: Compared with sham operation, SCI significantly aggravated the elevation of blood pressure (BP) and impaired baroreflex sensitivity (BRS) induced by colorectal distension; both of which were significantly improved by microinjection of the angiotensin receptor type I (AT1R) antagonist losartan into the NTS. Level of angiotensin II (Ang II) in the NTS was significantly increased in the SCI rats than sham. Intracerebroventricular infusion of Ang II also mimicked changes in BP and BRS induced by colorectal distension. Blockade of baroreflex by sinoaortic denervation prevented beneficial effect of losartan on AD. CONCLUSION: We concluded that the activation of Ang II system in NTS may impair blood pressure baroreflex, and contribute to AD after SCI.

Effects of moderate­intensity endurance exercise on angiotensin II and angiotensin II type I receptors in the rat heart.

The current study was aimed at examining the effects of moderate­intensity endurance exercise on the expression of angiotensin II (AngII) and AngII type 1 receptor (AT1R) in the rat heart. Male Sprague­Dawley rats were randomly divided into the control group (n=20) and moderate­intensity endurance exercise group (n=20). Cardiac hypertrophy was induced by treadmill endurance training for 8 weeks. The mRNA expression of AngII and AT1R were assessed by reverse transcription­quantitative polymerase chain reaction. The immune response positive area and optical density of AngII and AT1R was measured by immunohistochemistry. AngII was primarily expressed in the cytoplasm and membrane, however infrequently in coronary vascular wall smooth muscle cells. AT1R was primarily expressed in the coronary vessel wall smooth muscle, rarely in cardiac cells. The mRNA expression of cardiac AngII was significantly increased after the 8­week exercise period, while AT1R was significantly decreased. Immunohistochemistry indicated a significant increase in the AngII immune­positive area and optical density after the 8­week exercise. The AT1R immune­positive area and optical density was significantly reduced following the 8­week exercise. In conclusion, subsequent to 8­weeks endurance training, the AngII expression was increased and the AT1R expression was decreased. AT1R may expand the coronary artery, thereby increasing coronary blood flow and ensuring the energy supply of heart during exercise. The expression change in AngII does not reflect the character of cardiac hypertrophy. The exercise­induced change in the expression of AngII and AT1R may be a protective mechanism to avoid cardiac pathological hypertrophy.

Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling.

G-protein-coupled receptors (GPCRs) play critical roles in regulating physiological processes ranging from neurotransmission to cardiovascular function. Current methods for tracking GPCR signaling suffer from low throughput, modification or overexpression of effector proteins, and low temporal resolution. Here, we show that peroxidase-catalyzed proximity labeling can be combined with isobaric tagging and mass spectrometry to enable quantitative, time-resolved measurement of GPCR agonist response in living cells. Using this technique, termed "GPCR-APEX," we track activation and internalization of the angiotensin II type 1 receptor and the ß2 adrenoceptor. These receptors co-localize with a variety of G proteins even before receptor activation, and activated receptors are largely sequestered from G proteins upon internalization. Additionally, the two receptors show differing internalization kinetics, and we identify the membrane protein LMBRD2 as a potential regulator of ß2 adrenoceptor signaling, underscoring the value of a dynamic view of receptor function.

Expression of Angiotensin II Types 1 and 2 Receptors in Endometriotic Lesions.

BACKGROUND/AIM: The aim of this study was to evaluate the gene and protein expression of angiotensin type (AT) 1, AT2 receptors in endometriotic lesions and its relation to prostaglandin (PG) synthases. MATERIALS AND METHODS: Endometriosis samples were obtained from 32 patients with endometriotic cysts. Endometrial tissues were obtained during operations for benign gynecological conditions. The expression of the AT1 and AT2 receptor mRNA and that of PG-endoperoxide synthase 2 and microsomal PGE2 synthase-1 (mPGES-1) was examined by quantitative RT-PCR. Immunohistochemical staining was performed for these receptors. RESULTS: AT1 and AT2 receptor proteins were mostly located in endometrial glandular epithelium and some stromal cells. Immunoreactivity of the receptor proteins was observed in both the eutopic endometrium and endometriotic lesions. The AT1/AT2 ratio in endometriotic cysts (median 7.29, range 1.88-187.60) was significantly increased compared with that in the eutopic endometrium in the proliferative-phase in controls (median 1.01, range 0.37-2.09, p < 0.001). There was a relationship between the AT1 mRNA expression and that of mPGES-1 mRNA in the endometriotic cysts (r = 0.394089, p < 0.05). There was a significant relationship between the mRNA expression of the AT2 receptor and that of mPGES-1 in eutopic endometrium of non-endometriotic control (r = 0.610714, p < 0.05). CONCLUSION: Renin-angiotensin system may play an important role in the pathophysiology of endometriosis.

Free Fatty Acids Activate Renin-Angiotensin System in 3T3-L1 Adipocytes through Nuclear Factor-kappa B Pathway.

The activity of a local renin-angiotensin system (RAS) in the adipose tissue is closely associated with obesity-related diseases. However, the mechanism of RAS activation in adipose tissue is still unknown. In the current study, we found that palmitic acid (PA), one kind of free fatty acid, induced the activity of RAS in 3T3-L1 adipocytes. In the presence of fetuin A (Fet A), PA upregulated the expression of angiotensinogen (AGT) and angiotensin type 1 receptor (AT1R) and stimulated the secretion of angiotensin II (ANG II) in 3T3-L1 adipocytes. Moreover, the activation of RAS in 3T3-L1 adipocytes was blocked when we blocked Toll-like receptor 4 (TLR4) signaling pathway using TAK242 or NF-κB signaling pathway using BAY117082. Together, our results have identified critical molecular mechanisms linking PA/TLR4/NF-κB signaling pathway to the activity of the local renin-angiotensin system in adipose tissue.

Effects of Angiotensin Inhibitor Valsartan on the Expression of the Angiotensin II 1 Receptor, Matrix Metalloproteinases -2 and -9 in Human Bladder Cancer Cell Lines.

BACKGROUND: In order to investigate how valsartan-the angiotensin II 1 receptor (AT1R) antagonist-affects the expressions of AT1R antigen, matrix metalloproteinases (MMPs) -2 and -9 in carcinoma of urinary bladder (CUB) cell lines with different invasive abilities. METHODS: Three cell lines, EJ-M3, EJ, and BIU-87, with different invasive abilities were cultured and treated with valsartan. Cell proliferation states were determined by the methyl thiazolyl tetrazolium (MTT) method. The expressions at protein level and gene level were determined by Western blot and real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR), respectively. The invasive abilities and migratory abilities of the three cell lines were determined by Transwell in vitro cell invasion assay and wound healing assay, respectively. RESULTS: MTT results show that valsartan can inhibit the proliferation of CUB cells, and the inhibition effect is enhanced with the increase of concentration. CONCLUSIONS: AngII promotes the MMP2 and MMP9 expressions (both protein and gene levels) in CUB cells through AT1R, but their expressions can be effectively inhibited by valsartan, the AngII inhibitor. AngII inhibitor may become a novel drug that can inhibit CUB metastasis and prolong the survival of CUB patients.

Quantifying biased signaling in GPCRs using BRET-based biosensors.

There has been a growing appreciation that G protein-coupled receptor (GPCR) functional selectivity (viz. biased signaling), in particular between G protein- and ß-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 ß-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 ß-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.

Avaliação dos eixos ECA / ANG II / AT1 e ECA-2 / ANG (1-7) / MAS em fibroblastos de gengiva e ligamento periodontal humanos estimulados por IL-1ß e sua contribuição na produção e regulação de citocinas e quimiocinas / Evaluation ACE /ANG II /AT1 and ACE-2 /ANG (1-7) /MAS axis in gingival and periodontal ligament human fibroblasts stimulated with IL-1ß and its contribution in the production of cytokines and chemokines

O Sistema renina-angiotensina (SRA) tem sido relatado como um importante modulador de processos inflamatórios e imunológicos, incluindo a doença periodontal (DP). Estudos sugerem neste sistema um eixo alternativo (ECA-2 /ANG(1-7) /MAS) que atuaria como um contra-regulador de efeitos mediados pelo clássico eixo (ECA /ANGII /AT1). Sabe-se que bactérias periodontopatogênicas, como a Porphyromonas gingivalis (Pg), possuem componentes bioativos de membrana (ex. lipopolissacarídeos-LPS) capazes de induzir uma forte resposta imune no hospedeiro devido à liberação de citocinas nas células, entre elas Interleucina (IL)- 1ß. Neste contexto, fibroblastos são as células mais abundantes nos tecidos periodontais e possuem em sua superfície celular receptores necessários para o reconhecimento da invasão bacteriana, ativando cascatas intracelulares, que levam à produção de citocinas. O objetivo deste estudo foi verificar se os eixos ECA/ ANGII/ AT1 e ECA-2/ ANG(1-7)/ MAS contribuem para a produção e/ ou regulação de citocinas inflamatórias (CI) por fibroblastos de gengiva humana (HGF) e ligamento periodontal humano (HPLF) estimulados por IL-1ß. Após o pré-tratamento com Losartan e Ang (1-7) ou silenciamento mediado por RNA de interferência (RNAi) de AT1, HGF e HPLF foram estimulados por IL-1ß por 3 horas (RNAm) ou 24 horas (proteína). Expressão de RNAm para AT1, MAS, ECA, ECA-2, IL-1ß, TNF-α, IL-6, IL-8, IL-10, TGF-ß, CXCL12, RANK-L e OPG foram avaliados por RT-qPCR e das proteínas IL-6, IL-8, ECA e ECA-2 por ELISA. Foi realizado também Western Blot para detecção de AT1 e ECA nos extratos celulares e dosagem de nitrito no sobrenadante das culturas. Ambos os subtipos de fibroblastos mostraram aumento da expressão de RNAm para AT1, IL-1ß, IL-6, IL-8, TNF-α e OPG, quando estimulados por IL-1ß. No entanto, apenas em HPLF foi observado aumento para MAS, ECA e TGF-ß. Losartan e Ang (1-7) não modularam o transcrito, a secreção de CI e nem a produção de nitrito no sobrenadante das culturas, tanto em HGF como em HPLF. O silenciamento do receptor AT1 reduziu a secreção de IL-6 e IL-8 induzida por IL-1ß em cultura de HGF e HPLF e aumentou a expressão gênica de OPG somente em HGF. Estes resultados sugerem que o silenciamento de AT1, mas não o bloqueio farmacológico deste receptor pelo antagonista Losartan, em HGF e HPLF, pode controlar a produção de IL-6 e IL-8, que por sua vez contribuem para a patogênese periodontal.(AU)

The renin-angiotensin system (RAS) has been reported as an important modulator of inflammatory and immune responses, including periodontal disease (PD). Studies suggest an alternative axis as part of this system (ACE-2 / ANG (1-7) / MAS) that would act as counter-regulatory to the classical axis (ECA / ANGII / AT1). It is known that periodontal bacteria such as Porphyromonas gingivalis (Pg) have bioactive components in their membrane (such as lipopolysaccharide-LPS) capable of inducing a strong immune response in the host due to the release of cytokines in cells, including interleukin (IL) - 1ß. In this regard, fibroblasts are the most abundant cells in periodontal tissues and receptors needed for the recognition of bacterial invasion by activating intracellular cascades that lead to cytokine production. The aim of this study was to determine whether the axes ACE / ANGII / AT1 and ACE-2 / ANG (1-7) / MAS contribute to the production and / or regulation of inflammatory cytokines (IC) by fibroblasts of human gingiva (HGF) and human periodontal ligament (HPLF) stimulated IL-1ß. After pre-treatment with Losartan, Ang (1-7) or silencing mediated by RNA interference (RNAi) of AT1, HGF and HPLF were stimulated by IL-1ß for 3 hours (RNAm) or 24 hours (protein). Expression mRNA for AT1, MAS, ACE, ACE-2, IL-1ß, TNF-α, IL-6, IL-8, IL-10, TGF-ß, CXCL12, RANK-L and OPG was assessed by RT- qPCR and proteins IL-6, IL-8, ACE and ACE-2 by ELISA. Western Blot for the detection of AT1 and ECA and dosage of nitrite was also performed. Experiments stimulated by IL-1ß showed a positive control for gene expression AT1, IL-1ß, IL-6, IL-8, TNF-α and OPG in HGF and HPLF and MAS, ACE and TGF-ß only HPLF. Losartan and Ang (1-7) did not modulate the transcription and secretion of IC and no nitrite production in the culture supernatant of HGF and HPLF. The silencing AT1 reduced IL-6 secretion and IL-8 induced by IL- ß in cultured HGF and HPLF and increased OPG gene expression only HGF. These results suggest that silencing AT1, but not pharmacological blockade of this receptor by Losartan in HPLF and HGF, can control the production of IL-6 and IL-8, which in turn contribute to the pathogenesis of periodontal disease.(AU)

Endogenous hydrogen sulfide is associated with angiotensin II type 1 receptor in a rat model of carbon tetrachloride-induced hepatic fibrosis.

The present study aimed to investigate the effects of endogenous hydrogen sulfide (H2S) on the expression levels of angiotensin II type 1 receptor (AGTR1) in a rat model of carbon tetrachloride (CCl4)­induced hepatic fibrosis. A total of 56 Wistar rats were randomly divided into four groups: Normal control group, model group, sodium hydrosulfide (NaHS) group, and DL­propargylglycine (PAG) group. Hepatic fibrosis was induced by CCl4. The rats in the PAG group were intraperitoneally injected with PAG, an inhibitor of cystathionine­Î³­lyase (CSE). The rats in the NaHS group were intraperitoneally injected with NaHS. An equal volume of saline solution was intraperitoneally injected into both the control and model groups. All rats were sacrificed at week three or four following treatment. The serum levels of hyaluronidase (HA), laminin protein (LN), procollagen III (PcIII), and collagen IV (cIV) were detected using ELISA. The serum levels of alanine transaminase (ALT), aspartate transaminase (AST), and albumin (ALB) were detected using an automatic biochemical analyzer. The liver mRNA expression levels of CSE were detected by reverse transcription­quantitative polymerase chain reaction. The liver expression levels of AGTR1 and the plasma expression levels of H2S were detected using western blot analyses. The results indicated that the severity of hepatic fibrosis, the serum expression levels of HA, LN, PcIII, cIV, ALT, and AST, the liver expression levels of CSE and AGTR1, and the plasma expression levels of H2S were significantly higher in the PAG group, as compared with the model group (P<0.05). Conversely, the expression levels of ALB were significantly lower in the PAG group, as compared with the model group. In addition, the severity of hepatic fibrosis, the serum expression levels of HA, LN, PcIII, cIV, ALT, and AST, the liver expression levels of CSE and AGTR1, and the plasma expression levels of H2S were significantly lower in the NaHS group, as compared with the model group (P<0.05). These results suggest that endogenous H2S is associated with CCl4­induced hepatic fibrosis in rats, and may exhibit anti­fibrotic effects. Furthermore, H2S reduced the liver expression levels of AGTR1, which may be associated with the delayed progression of hepatic fibrosis.

Chronic treatment with qiliqiangxin ameliorates aortic endothelial cell dysfunction in diabetic rats.

Qiliqiangxin (QL), a traditional Chinese medicine, has been shown to be beneficial for chronic heart failure. However, whether QL can also improve endothelial cell function in diabetic rats remains unknown. Here, we investigated the effect of QL treatment on endothelial dysfunction by comparing the effect of QL to that of benazepril (Ben) in diabetic Sprague-Dawley rats for 8 weeks. Cardiac function was evaluated by echocardiography and catheterization. Assays for acetylcholine-induced, endothelium-dependent relaxation (EDR), sodium nitroprusside-induced endothelium-independent relaxation, serum nitric oxide (NO), and nitric oxide synthase (NOS) as well as histological analyses were performed to assess endothelial function. Diabetic rats showed significantly inhibited cardiac function and EDR, decreased expression of serum NO and phosphorylation at Ser(1177) on endothelial NOS (eNOS), and impaired endothelial integrity after 8 weeks. Chronic treatment for 8 weeks with either QL or Ben prevented the inhibition of cardiac function and EDR and the decrease in serum NO and eNOS phosphorylation caused by diabetes. Moreover, either QL or Ben suppressed inducible NOS (iNOS) protein levels as well as endothelial necrosis compared with the diabetic rats. Additionally, QL prevented the increase in angiotensin-converting enzyme 1 and angiotensin II receptor type 1 in diabetes. Thus, chronic administration of QL improved serum NO production, EDR, and endothelial integrity in diabetic rat aortas, possibly through balancing eNOS and iNOS activity and decreasing renin-angiotensin system expression.