Inhibition of glutaminolysis alleviates myocardial fibrosis induced by angiotensin II / 生理学报

The present study was aimed to investigate the role and mechanism of glutaminolysis of cardiac fibroblasts (CFs) in hypertension-induced myocardial fibrosis. C57BL/6J mice were administered with a chronic infusion of angiotensin II (Ang II, 1.6 mg/kg per d) with a micro-osmotic pump to induce myocardial fibrosis. Masson staining was used to evaluate myocardial fibrosis. The mice were intraperitoneally injected with BPTES (12.5 mg/kg), a glutaminase 1 (GLS1)-specific inhibitor, to inhibit glutaminolysis simultaneously. Immunohistochemistry and Western blot were used to detect protein expression levels of GLS1, Collagen I and Collagen III in cardiac tissue. Neonatal Sprague-Dawley (SD) rat CFs were treated with 4 mmol/L glutamine (Gln) or BPTES (5 μmol/L) with or without Ang II (0.4 μmol/L) stimulation. The CFs were also treated with 2 mmol/L α-ketoglutarate (α-KG) under the stimulation of Ang II and BPTES. Wound healing test and CCK-8 were used to detect CFs migration and proliferation respectively. RT-qPCR and Western blot were used to detect mRNA and protein expression levels of GLS1, Collagen I and Collagen III. The results showed that blood pressure, heart weight and myocardial fibrosis were increased in Ang II-treated mice, and GLS1 expression in cardiac tissue was also significantly up-regulated. Gln significantly promoted the proliferation, migration, mRNA and protein expression of GLS1, Collagen I and Collagen III in the CFs with or without Ang II stimulation, whereas BPTES significantly decreased the above indices in the CFs. α-KG supplementation reversed the inhibitory effect of BPTES on the CFs under Ang II stimulation. Furthermore, in vivo intraperitoneal injection of BPTES alleviated cardiac fibrosis of Ang II-treated mice. In conclusion, glutaminolysis plays an important role in the process of cardiac fibrosis induced by Ang II. Targeted inhibition of glutaminolysis may be a new strategy for the treatment of myocardial fibrosis.

Inhibitor of growth protein-2 silencing alleviates angiotensin Ⅱ-induced cardiac remodeling in mice by reducing p53 acetylation / 南方医科大学学报

OBJECTIVE@#To investigate the effect of inhibitor of growth protein-2 (Ing2) silencing on angiotensin Ⅱ (AngⅡ)-induced cardiac remodeling in mice and explore the underlying mechanism.@*METHODS@#An adenoviral vector carrying Ing2 shRNA or empty adenoviral vector was injected into the tail vein of mice, followed 48 h later by infusion of 1000 ng · kg-1 · min-1 Ang Ⅱ or saline using a mini-osmotic pump for 42 consecutive days. Transthoracic echocardiography was used to assess cardiac geometry and function and the level of cardiac hypertrophy in the mice. Masson and WGA staining were used to detect myocardial fibrosis and cross-sectional area of cardiomyocytes, and myocardial cell apoptosis was detected with TUNEL assay. Western blotting was performed to detect myocardial expressions of cleaved caspase 3, ING2, collagen Ⅰ, Ac-p53(Lys382) and p-p53 (Ser15); Ing2 mRNA expression was detected using real-time PCR. Mitochondrial biogenesis, as measured by mitochondrial ROS content, ATP content, citrate synthase activity and calcium storage, was determined using commercial assay kits.@*RESULTS@#The expression levels of Ing2 mRNA and protein were significantly higher in the mice with chronic Ang Ⅱ infusion than in saline-infused mice. Chronic infusion of AngⅡ significantly increased the left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) and reduced left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) in the mice. Ing2 silencing obviously alleviated AngⅡ-induced cardiac function decline, as shown by decreased LVEDD and LVESD and increased LVEF and LVFS, improved myocardial mitochondrial damage and myocardial hypertrophy and fibrosis, and inhibited cardiomyocyte apoptosis. Chronic AngⅡ infusion significantly increased myocardial expression levels of Ac-p53(Lys382) and p-p53(Ser15) in the mice, and Ing2 silencing prior to AngⅡ infusion lessened AngⅡ- induced increase of Ac-p53(Lys382) without affecting p53 (ser15) expression.@*CONCLUSION@#Ing2 silencing can inhibit AngⅡ-induced cardiac remodeling and dysfunction in mice by reducing p53 acetylation.

Expression of LRG-1 in mice with hypertensive renal damage and its significance / 中南大学学报(医学版)

OBJECTIVES@#Long-term elevated blood pressure may lead to kidney damage, yet the pathogenesis of hypertensive kidney damage is still unclear. This study aims to explore the role and significance of leucine-rich alpha-2-glycoprotein-1 (LRG-1) in hypertensive renal damage through detecting the levels of LRG-1 in the serum and kidney of mice with hypertensive renal damage and its relationship with related indexes.@*METHODS@#C57BL/6 mice were used in this study and randomly divided into a control group, an angiotensin II (Ang II) group, and an Ang II+irbesartan group. The control group was gavaged with physiological saline. The Ang II group was pumped subcutaneously at a rate of 1.5 mg/(kg·d) for 28 days to establish the hypertensive renal damage model in mice, and then gavaged with equivalent physiological saline. The Ang II+irbesartan group used the same method to establish the hypertensive renal damage model, and then was gavaged with irbesartan. Immunohistochemistry and Western blotting were used to detect the expression of LRG-1 and fibrosis-related indicators (collagen I and fibronectin) in renal tissues. ELISA was used to evaluate the level of serum LRG-1 and inflammatory cytokines in mice. The urinary protein-creatinine ratio and renal function were determined, and correlation analysis was conducted.@*RESULTS@#Compared with the control group, the levels of serum LRG-1, the expression of LRG-1 protein, collagen I, and fibronectin in kidney in the Ang II group were increased (all P<0.01). After treating with irbesartan, renal damage of hypertensive mice was alleviated, while the levels of LRG-1 in serum and kidney were decreased, and the expression of collagen I and fibronectin was down-regulated (all P<0.01). Correlation analysis showed that the level of serum LRG-1 was positively correlated with urinary protein-creatinine ratio, blood urea nitrogen, and blood creatinine level in hypertensive kidney damage mice. Serum level of LRG-1 was also positively correlated with serum inflammatory factors including TNF-α, IL-1β, and IL-6.@*CONCLUSIONS@#Hypertensive renal damage mice display elevated expression of LRG-1 in serum and kidney, and irbesartan can reduce the expression of LRG-1 while alleviating renal damage. The level of serum LRG-1 is positively correlated with the degree of hypertensive renal damage, suggesting that it may participate in the occurrence and development of hypertensive renal damage.

Protective Effect and Mechanism of Kuntai Capsule on Angiotensin II -Induced Hypertension in Ovariectomized Rats / 中国结合医学杂志

OBJECTIVE@#To explore the protective effect and mechanism of Kuntai (KT) Capsule on angiotensin II (Ang II)-induced hypertension in ovariectomized (OVX) rats.@*METHODS@#Fifty-four rats were randomly divided into 6 groups according to a random number table, 9 in each group: control, OVX sham+Ang II, OVX, OVX+Ang II, OVX+Ang II +E2, and OVX+Ang II +KT. OVX rats model was constructed by retroperitoneal bilateral ovariectomy. After 4 weeks of pretreatment with KT Capsule [0.8 g/(kg·d) and 17- β -estradiol (E2, 1.2 mg/(kg·d)] respectively, Ang II was injected into a micro-osmotic pump with a syringe to establish a hypertensive rat model. Blood pressure of rat tail artery was measured in a wake state of rats using a non-invasive sphygmomanometer. Blood pressure changes were compared between the intervention groups (OVX+Ang II +KT, OVX+Ang II +E2) and the negative control group (OVX+Ang II). Serum malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were detected respectively. The expressions of oxidative stress-related protein superoxide dismutase2 (SOD2) and anti-thioredoxin (TRX), autophagy marker protein [beclin1, light chain (LC) 3 II/I ratio and autophagy canonical pathway protein phosphatidylinositol 3-kinase (PI3K)/serine/threonine kinase (AKT)-mammalian target of rapamycin (mTOR)] were evaluated by Western blotting.@*RESULTS@#Compared with the OVX+Ang II group, the systolic blood pressure of OVX+Ang II +KT group was significantly lowered (P<0.05) but not the diastolic blood pressure. Besides, SOD2 and TRX protein levels in mycardial tissues were significantly reduced in the OVX+Ang II +KT group compared with the OVX+Ang II group (P<0.05). Oxidative stress serum markers MDA and SOD were down- and up-regulated in the OVX+Ang II +KT group, respectively (P<0.05). Compared with OVX+Ang II group, the levels of cardiac proteins beclin-1 and LC3II/LC3 I in OVX+Ang II +KT group were also up-regulated (P<0.05), and the expression levels of p-PI3K, p-AKT and mTOR protein were down-regulated (P<0.05).@*CONCLUSION@#KT could protect blood pressure of Ang II-induced OVX rats by inhibiting oxidative stress and up-regulating protective autophagy.

Role of Brain Angiotensin-II in Development of Experimental Diabetic Nephropathy in Wistar Rats

Abstract The renin-angiotensin-aldosterone system (RAAS) plays a key role in diabetic nephropathy (DN). Angiotensin-II secreted during the RAAS pathway increases nephropathy. It stimulates oxidative stress which can quench nitric oxide. Reduced nitric oxide level aggravates Ang-II-induced vasoconstriction. Ang-II has also emerged as a central mediator of the glomerular hemodynamic changes that are associated with renal injury. Deletion of ACE2 is also noted due to increased Ang-II level which leads to the development of DN. We hypothesize that nephropathy caused by Ang-II in the periphery may be controlled by brain RAAS. ACE inhibitors and ARBs may show the renoprotective effect when administered through ICV without crossing the blood-brain barrier. DN was observed after 8 weeks of diabetes induction through alloxan. Administration of captopril and valsartan once and in combined therapy for 2 weeks, significantly reduced urine output, blood urea nitrogen, total protein in the urine, serum cholesterol, serum creatinine, serum triglycerides, and kidney/body weight ratio as compared to diabetic control rats. Further, combination therapy significantly increased the body weight and serum nitrate level as compared to diabetic control animals. However, increased ACE2 levels in the brain may reduce the sympathetic outflow and might have decreased the peripheral activity of Ang-II which shows beneficial effects in DN.

Uso de anticorpos monoclonais por pacientes com covid-19 interfere na concentração sérica de angiotensina II? / Does the use of monoclonal drugs by patients with COVID-19 interfere with the serum concentration of angiotensin II? / ¿El uso de fármacos monoclonales por pacientes con COVID-19 interfiere con la concentración sérica de angiotensina II?

Introdução:uma vez conhecidos os mecanismos de patogênese do SARS-CoV-2, vários métodos de tratamento para a COVID-19 foram desenvolvidos, dentre eles destaca-se o uso dos anticorpos monoclonais para o contexto de pacientes em estágios graves da doença. Objetivo: compreender se o uso dos anticorpos monoclonais para tratamento da COVID-19 grave interfere nos níveis séricos da angiotensina II. Metodologia:Para a realização dessa pesquisa foram selecionados através do DeCS e MeSH os descritores "COVID-19", "Angiotensin II" e "Antibodies, Monoclonal" e seus respectivos "entry terms" sugeridos pela base MeSH. Posteriormente,utilizando-se os operadores booleanos OR e AND, foi montada uma estratégia de busca, a qual foi utilizada nas bases de dados PUBMED, EMBASE, Web of Science, Cochrane Library e Scopus, sem restrição dedata de publicação ou idioma. Resultados:ao final do processo de seleção dos artigos, 29 foram selecionados para a leitura e análise completa. Nesta revisão, foram abordados diferentes tipos de anticorpos monoclonais, os quais foram oportunamente agrupados de acordo com o seu mecanismo de ação. Conclusão: foi possível concluir que das cinco classes de anticorpos monoclonais tratadas neste trabalho, três potencialmente podem causar alterações nos níveis séricos de angiotensina II (AU).

Introduction:once the mechanisms of pathogenesis of SARS-CoV-2 are known, several methods of treatment for COVID-19 have been developed, among them the use of monoclonal antibodies for the context of patients in severe stages of the disease. Purpose:to understand whether the use of monoclonal antibodies for the treatment of severe COVID-19 interferes with serum angiotensin II levels. Methodology:For this research were selected through DeCS and MeSH the descriptors "COVID-19", "Angiotensin II" and "Antibodies, Monoclonal" and their respective entry "Terms" suggested by the MeSH database. Subsequently, using the boolean operators OR and AND, a search strategy was set up, which was used in the databases PUBMED, EMBASE, Web of Science, Cochrane Library and Scopus, without restriction of publication date or language. Results:at the end of the article selection process, 29 were selected for reading and full analysis. In this review, different types of monoclonal antibodies were addressed, which were opportunely grouped according to their mechanism of action. Conclusion:it was possible to conclude that of the five classes of monoclonal antibodies treated in this study, three potentially can cause changes in serum levels of angiotensin II (AU).

Introducción:Una vez conocidos los mecanismos de patogénesis del SARSCoV-2, se desarrollaron variosmétodos de tratamiento para el COVID-19, entre ellos, el uso de anticuerpos monoclonales para el contexto de pacientes en fases graves de la enfermedad. Objetivo:Comprender si el uso de anticuerpos monoclonales para el tratamiento de la COVID-19 grave interfiere en los niveles séricos de angiotensina II. Metodología:Los descriptores "COVID-19", "Angiotensina II", "Anticuerpos, Monoclonales" y sus respectivos "entry terms" (términos de entrada) sugeridos por el MeSH fueron seleccionados a través de DeCS yMeSH. Posteriormente, utilizando los operadores booleanos OR y AND, se estableció una estrategia de búsqueda que se utilizó en las bases de datos PUBMED, EMBASE, Web of Science, Cochrane Library y Scopus, sin restricción de fecha de publicación ni de idioma. Resultados:Al final del proceso de selección de artículos, se seleccionaron 29 artículos para su lectura y análisis completos. En esta revisión se han abordado diferentes tipos de anticuerpos monoclonales, que se han agrupado oportunamente según su mecanismo de acción. Conclusión:Se pudo concluir que de las cinco clases de anticuerpos monoclonales tratados en este trabajo, tres pueden potencialmente causar alteraciones en los niveles séricos de angiotensina II (AU).

Effect and mechanism of leonurine on pressure overload-induced cardiac hypertrophy in rats / 中国中药杂志

To investigate the effects of leonurine(Leo) on abdominal aortic constriction(AAC)-induced cardiac hypertrophy in rats and its mechanism. A rat model of pressure overload-induced cardiac hypertrophy was established by AAC method. After 27-d intervention with high-dose(30 mg·kg~(-1)) and low-dose(15 mg·kg~(-1)) Leo or positive control drug losartan(5 mg·kg~(-1)), the cardiac function was evaluated by hemodynamic method, followed by the recording of left ventricular systolic pressure(LVSP), left ventricular end-diastolic pressure(LVESP), as well as the maximum rate of increase and decrease in left ventricular pressure(±dp/dt_(max)). The degree of left ventricular hypertrophy was assessed based on heart weight index(HWI) and left ventricular mass index(LVWI). Myocardial tissue changes and the myocardial cell diameter(MD) were measured after hematoxylin-eosin(HE) staining. The contents of angiotensin Ⅱ(AngⅡ) and angiotensin Ⅱ type 1 receptor(AT1 R) in myocardial tissue were detected by ELISA. The level of Ca~(2+) in myocardial tissue was determined by colorimetry. The protein expression levels of phospholipase C(PLC), inositol triphosphate(IP3), AngⅡ, and AT1 R were assayed by Western blot. Real-time quantitative PCR(qRT-PCR) was employed to determine the mRNA expression levels of β-myosin heavy chain(β-MHC), atrial natriuretic factor(ANF), AngⅡ, and AT1 R. Compared with the model group, Leo decreased the LVSP, LVEDP, HWI, LVWI and MD values, but increased ±dp/dt_(max) of the left ventricle. Meanwhile, it improved the pathological morphology of myocardial tissue, reduced cardiac hypertrophy, edema, and inflammatory cell infiltration, decreased the protein expression levels of PLC, IP3, AngⅡ, AT1 R, as well as the mRNA expression levels of β-MHC, ANF, AngⅡ, AT1 R, c-fos, and c-Myc in myocardial tissue. Leo inhibited AAC-induced cardiac hypertrophy possibly by influencing the RAS system.

Captopril related kidney damage: renal afferent arteriolar responses to angiotensin II and inflammatory signaling / 生理学报

Captopril can have nephrotoxic effects, which are largely attributed to accumulated renin and "escaped" angiotensin II (Ang II). Here we test whether angiotensin converting enzyme-1 (ACE1) inhibition damages kidneys via alteration of renal afferent arteriolar responses to Ang II and inflammatory signaling. C57Bl/6 mice were given vehicle or captopril (60 mg/kg per day) for four weeks. Hypertension was obtained by minipump supplying Ang II (400 ng/kg per min) during the second 2 weeks. We assessed kidney histology by periodic acid-Schiff (PAS) and Masson staining, glomerular filtration rate (GFR) by FITC-labeled inulin clearance, and responses to Ang II assessed in afferent arterioles in vitro. Moreover, arteriolar H2O2 and catalase, plasma renin were assayed by commercial kits, and mRNAs of renin receptor, transforming growth factor-β (TGF-β) and cyclooxygenase-2 (COX-2) in the renal cortex, mRNAs of angiotensin receptor-1 (AT1R) and AT2R in the preglomerular arterioles were detected by RT-qPCR. The results showed that, compared to vehicle, mice given captopril showed lowered blood pressure, reduced GFR, increased plasma renin, renal interstitial fibrosis and tubular epithelial vacuolar degeneration, increased expression of mRNAs of renal TGF-β and COX-2, decreased production of H2O2 and increased catalase activity in preglomerular arterioles and enhanced afferent arteriolar Ang II contractions. The latter were blunted by incubation with H2O2. The mRNAs of renal microvascular AT1R and AT2R remained unaffected by captopril. Ang II-infused mice showed increased blood pressure and reduced afferent arteriolar Ang II responses. Administration of captopril to the Ang II-infused mice normalized blood pressure, but not arteriolar Ang II responses. We conclude that inhibition of ACE1 enhances renal microvascular reactivity to Ang II and may enhance important inflammatory pathways.

Zofenopril antitumor activity in mice bearing Ehrlich solid carcinoma: Modulation of PI3K/AKT signaling pathway

Angiotensin-II (AgII) is thought to be crucial for tumor growth and progression. Moreover, hydrogen sulfide (H2S) performs a controversial action in cancer pathology. Zofenopril (ZF) is an angiotensin-converting enzyme (ACE) inhibitor with H2S donating properties. Hence, this study aims at investigating the tumor suppressor activity of ZF and elucidating the involved trajectories in Ehrlich's solid tumor (EST)-bearing mice. EST was induced by the intradermal injection of Ehrlich's ascites carcinoma cells into femoral region. All parameters were assessed after 28 days post-inoculation or one-week thereafter. ZF treatment resulted in significant reduction of tumor weights with marked decrease in IL-6 and VEGF levels in serum, and tumor Ag II and CEA contents. Additionally, the administration of ZF downregulated the tumor gene expression of cyclin-D, ACE-1, and Bcl2 and upregulated the proapoptotic gene, BAX. Moreover, ZF increased CBS gene expression, which is a major contributor to cellular H2S production. In addition, ZF was able to reduce the protein expression of PI3K, pAKT, pGSK-3ß, and NFκB. Our study has provided novel insights into the possible mechanisms by which ZF may produce its tumor defeating properties. These intersecting trajectories involve the interference between PI3K/Akt and CBS signaling pathways

Inibição combinada do receptor AT1 e produtos finais de glicação avançada na nefropatia diabética / Combined inhibition of AT1 receptor and advanced glycation end products in diabetic nephropathy

O diabetes mellitus (DM) é um grande problema de saúde pública, que afeta cerca de 463 milhões de pessoas no mundo e pode alcançar 700 milhões de pessoas até 2045. A nefropatia é uma das complicações microvasculares do diabetes e a maior causa de insuficiência renal. Uma vez que a ativação do receptor AT1 pela angiotensina II causa vasoconstrição da artéria aferente, e que o acúmulo de produtos finais de glicação avançada (AGEs) causam glicotoxicidade intracelular de células mesangiais, podocitárias e tubulares, foi avaliado se o tratamento combinado de olmersatana (OLM) e piridoxamina (PYR) é capaz de melhorar a nefropatia diabética quando comparado aos tratamentos isolados. Para isto, o diabetes mellitus experimental foi induzido em 50 camundongos C57BL/6 pela administração de estreptozotocina (50 mg/kg/dia via intraperitoneal por 5 dias). Os animais foram divididos em cinco grupos: controle, diabéticos, diabéticos tratados com OLM (20 mg/Kg/dia), diabéticos tratados com PYR (400 mg/Kg/dia) e diabéticos tratados com OLM e PYR (20 mg/Kg/dia e 400 mg/Kg/dia, respectivamente) Os tratamento durou 16 semanas. Como resultado, os camundongos que receberam STZ desenvolveram hiperglicemia e doença renal, diminuição de peso, poliúria, polidipsia, polifagia, e albuminúria, além de aumento de frutosamina, ferro, uréia e fosfatase alcalina na urina, diminuição da atividade de catalase no rim e aumento de excreção dos AGEs na urina. Histologicamente, houve aumento de área glomerular e do espaço de BowmanO tratamento com OLM atenuou a albuminúria e atenuou o declínio da função renal, uma vez que o OLM melhorou a polidipsia, polifagia, poliúria, marcadores bioquímicos de disfunção renal e parâmetros histológicos, apesar de não apresentar ação sobre a fosfatase alcalina, uréia, ferro, frutosamina e ácido úrico. O tratamento isolado com PYR melhorou a maioria dos parâmetros alterados pela doença, entre eles a ingestão alimentar, ingestão hídrica, volume urinário, creatinina sérica, excreção de albumina na urina, ACR, AGEs no tecido renal e urina, ureia na urina e todos os parâmetros morfológicos analisados, contudo sem efeito sobre a fosfatase alcalina, ferro, frutosamina e ácido úrico. O tratamento combinado (PYR e OLM) melhorou a polifagia, polidipsia e poliúria comparado aos animais diabéticos sem tratamento, entretanto não houve diferença comparado a cada tratamento isolado. Os parâmetros histológicos e bioquímicos (creatinina no soro, albumina na urina, ACR e uréia) também apresentaram melhorara em relação aos animais diabéticos sem tratamento, mas não em relação aos animais em tratamento isolado. Tanto PYR quanto OLM não influenciaram a excreção de AGEs na urina. Esses dados nos levam a concluir que o tratamento combinado não ofereceu efeitos benéficos adicionais quando comparado aos tratamentos isolados, e que o tratamento que mais ofereceu benefícios foi o tratamento isolado com PYR nos parâmetros metabólicos, morfológicos e de função renal. (AU)

Inhibition of CXCR2 alleviates the development of abdominal aortic aneurysm in Apo E-/- mice

ABSTRACT Purpose To investigate the relationship between atherosclerotic abdominal aortic aneurysm (AAA) and CXC chemokine receptor type 2 (CXCR2). Methods Mouse AAA model was established by embedding angiotensin-II pump (1000 ng/kg/min) in ApoE-/- mice. Mice were received SB225002, a selective CXCR2 antagonist, for treatment. Blood pressure was recorded, and CXCR2+ macrophages were examined by flow cytometry analysis. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining was performed to detect cell apoptosis of abdominal aortic aneurysms. Macrophages were isolated from ApoE-/- mice and treated with Ang II and/or SB225002. Dihydroethidium staining was carried out to determine reactive oxygen species (ROS) activity. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the production of IL-1β and TNF-α. The corresponding gene expressions were measured using real-time polymerase chain reaction (PCR), western blot, and immunohistochemistry staining. Results We found that Ang II activated the expression of CXCR2 in monocytes during the formation of AAA. Inhibition of CXCR2 significantly reduced the size of AAA, attenuated inflammation and phenotypic changes in blood vessels. Ang II-induced macrophages exhibited elevated ROS activity, and elevated levels of 1β and TNF-α, which were then partly abolished by SB225002. Conclusions CXCR2 plays an important role in AAA, suggesting that inhibiting CXCR2 may be a new treatment for AAA.

Effects of SET7 on angiotensin II-mediated proliferation and collagen synthesis of myocardial fibroblasts and its mechanisms / 中南大学学报(医学版)

OBJECTIVES@#Silence of SET domain containing lysine methyltransferase 7 (SET7) alleviates myocardial tissue injury caused by ischemia-reperfusion. But the effects of SET7 on angiotensin II (Ang II)-induced myocardial fibroblast proliferation and the collagen synthesis are not clear. The purpose of this study was to explore the effect of SET7 on the proliferation and collagen synthesis of myocardial fibroblasts and its mechanisms.@*METHODS@#Myocardial fibroblasts were isolated and identified by immunofluorescence. Myocardial fibroblasts were randomly divided into 4 groups: a control group (cells were normally cultured), an Ang II group (cells were treated with 100 nmol/L Ang II for 24 h), a siCtrl group (cells were transfected with siRNA control and were then treated with 100 nmol/L Ang II for 24 h), and a siSET7 group (cells were transfected with siRNA SET7 and were then treated with 100 nmol/L Ang II for 24 h). Cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assay were used to evaluate cell proliferation. Real-time PCR was used to detect the mRNA levels of SET7, collagen I, collagen III, and α-smooth muscle actin (α-SMA). Western blotting was used to detect the protein expression of SET7, collagen I, collagen III, α-SMA, sonic hedgehog (Shh), ptched1 (Ptch1), and glioma-associated oncogene homolog 1 (Gli1).@*RESULTS@#Fluorescence microscopy showed positive vimentin staining, and myocardial fibroblasts were in good condition. As compared to the control group, the mRNA and protein levels of SET7 in the Ang II group were significantly upregulated; cell proliferation rate and EdU fluorescence intensity in the Ang II group were significantly increased; the mRNA and protein levels of collagen I, collagen III, and α-SMA were significantly upregulated (all @*CONCLUSIONS@#Silence of SET7 gene inhibits Ang II-induced proliferation and collagen synthesis of myocardial fibroblasts. Shh signaling pathway may be involved in this process.

Macrophage exosomes transfer angiotensin II type 1 receptor to lung fibroblasts mediating bleomycin-induced pulmonary fibrosis / 中华医学杂志(英文版)

BACKGROUND@#Macrophages are involved in the pathogenesis of idiopathic pulmonary fibrosis, partially by activating lung fibroblasts. However, how macrophages communicate with lung fibroblasts is largely unexplored. Exosomes can mediate intercellular communication, whereas its role in lung fibrogenesis is unclear. Here we aim to investigate whether exosomes can mediate the crosstalk between macrophages and lung fibroblasts and subsequently induce fibrosis.@*METHODS@#In vivo, bleomycin (BLM)-induced lung fibrosis model was established and macrophages infiltration was examined. The effects of GW4869, an exosomes inhibitor, on lung fibrosis were assessed. Moreover, macrophage exosomes were injected into mice to observe its pro-fibrotic effects. In vitro, exosomes derived from angiotensin II (Ang II)-stimulated macrophages were collected. Then, lung fibroblasts were treated with the exosomes. Twenty-four hours later, protein levels of α-collagen I, angiotensin II type 1 receptor (AT1R), transforming growth factor-β (TGF-β), and phospho-Smad2/3 (p-Smad2/3) in lung fibroblasts were examined. The Student's t test or analysis of variance were used for statistical analysis.@*RESULTS@#In vivo, BLM-treated mice showed enhanced infiltration of macrophages, increased fibrotic alterations, and higher levels of Ang II and AT1R. GW4869 attenuated BLM-induced pulmonary fibrosis. Mice with exosomes injection showed fibrotic features with higher levels of Ang II and AT1R, which was reversed by irbesartan. In vitro, we found that macrophages secreted a great number of exosomes. The exosomes were taken by fibroblasts and resulted in higher levels of AT1R (0.22 ± 0.02 vs. 0.07 ± 0.02, t = 8.66, P = 0.001), TGF-β (0.54 ± 0.05 vs. 0.09 ± 0.06, t = 10.00, P < 0.001), p-Smad2/3 (0.58 ± 0.06 vs. 0.07 ± 0.03, t = 12.86, P < 0.001) and α-collagen I (0.27 ± 0.02 vs. 0.16 ± 0.01, t = 7.01, P = 0.002), and increased Ang II secretion (62.27 ± 7.32 vs. 9.56 ± 1.68, t = 12.16, P < 0.001). Interestingly, Ang II increased the number of macrophage exosomes, and the protein levels of Alix (1.45 ± 0.15 vs. 1.00 ± 0.10, t = 4.32, P = 0.012), AT1R (4.05 ± 0.64 vs. 1.00 ± 0.09, t = 8.17, P = 0.001), and glyceraldehyde-3-phosphate dehydrogenase (2.13 ± 0.36 vs. 1.00 ± 0.10, t = 5.28, P = 0.006) were increased in exosomes secreted by the same number of macrophages, indicating a positive loop between Ang II and exosomes production.@*CONCLUSIONS@#Exosomes mediate intercellular communication between macrophages and fibroblasts plays an important role in BLM-induced pulmonary fibrosis.

Aqueous humor renin, angiotensin I, and angiotensin II activity in primary open-angle glaucoma / Atividade no humor aquoso da renina, angiotensina I e angiotensina II no glaucoma primário de ângulo aberto

ABSTRACT Purpose: The renin-angiotensin system is involved in the pathogenesis of retinal ischemic conditions and glaucoma. Our objective was to evaluate the renin, angiotensinconverting enzyme 1, and angiotensin-converting enzyme 2 activities in aqueous humor and blood samples of patients with and without primary open-angle glaucoma. Methods: We analyzed samples from 56 participants who underwent ocular surgeries. The patients were divided into two groups: patients with cataract alone (n=28) and patients with cataract and primary open-angle glaucoma (n=28). Venous blood (2 ml) and aqueous humor (150 µl, via paracentesis) samples were collected during phacoemulsification (cataract only) or glaucoma surgery (cataract and primary open-angle glaucoma). The serum and aqueous humor renin, angiotensin-converting enzyme 1, and angiotensin-converting enzyme 2 activities of all patients were evaluated by fluorimetric assays, and results were analyzed by using multivariate regression analysis. Results: Both the aqueous humor renin activity and renin activity aqueous humor/serum ratio were significantly lower in patients with cataract and primary open-angle glaucoma than in patients with cataract only [(mean ± SE): 0.018 ± 0.006 ng/ml/h vs 0.045 ± 0.009 ng/ml/h, p<0.001; 0.05 ± 0.02 vs 0.13 ± 0.05, p=0.025]. Multivariate analyses showed a significant relationship between lower aqueous humor renin activity and primary open-angle glaucoma [coefficient (±SE): -0.029 ± 0.013, p=0.026]. Conclusions: Our results showed that patients with primary open-angle glaucoma had lower aqueous humor renin activity. As timolol eye drops were used by most of the primary open-angle glaucoma patients, we propose that a large sample of washed-out patients should be studied in the future to discriminate the involvement of b-blocker treatment in the aqueous humor renin activity.

RESUMO Objetivo: O sistema renina-angiotensina está envolvido na patogênese das condições isquêmicas retinianas e no glaucoma. Nosso objetivo foi avaliar a atividade da renina, enzima conversora de angiotensina 1 e 2 no humor aquoso, e amostras de sangue de pacientes com e sem glaucoma primário de ângulo aberto. Métodos: Foram analisadas amostras de 56 participantes submetidos à cirurgia ocular. Os pacientes foram divididos em dois grupos: pacientes com catarata apenas (n=28), e pacientes com catarata e glaucoma primário de ângulo aberto (n=28). Amostras de sangue venoso (2ml) e humor aquoso (150 µl, via paracentese) foram coletadas durante a facoemulsificação (apenas catarata) ou cirurgia de glaucoma (catarata e glaucoma primário de ângulo aberto). As atividades sérica do humor aquoso de renina, enzima conversora de angiotensina 1 e enzima conversora de angiotensina 2 de todos os pacientes foram avaliadas por ensaios fluorimétricos, e os resultados foram analisados por regressão multivariada. Resultados: Tanto a atividade da renina no humor aquoso quanto à razão humor aquoso/soro da atividade da renina foram significativamente menores nos pacientes com catarata e glaucoma primário de ângulo aberto do que em pacientes com catarata apenas [(média ± DP): 0,018 ± 0,006 ng/ml/h vs 0,045 ± 0,009 ng/ml/h; p<0,001 e 0,05 ± 0,02 vs 0,13 ± 0,05; p=0,025]. Análises multivariadas mostraram uma releção significativa entre menor atividade de renina no humor aquoso e glaucoma primário de ângulo aberto [coeficiente (±erro padrão): -0,029 ± 0,013; p=0,026]. Conclusões: Como a maioria dos pacientes com glaucoma primário de ângulo aberto usavam o colírio de timolol, estudos futuros envolvendo um maior número de pacientes e retirada prévia do tratamento são necessários para se discriminar o envolvimento do uso de betabloqueadores na atividade da renina no humor aquoso.

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

Aliskiren attenuates cardiac dysfunction by modulation of the mTOR and apoptosis pathways

Aliskiren (ALS) is well known for its antihypertensive properties. However, the potential underlying the molecular mechanism and the anti-hypertrophic effect of ALS have not yet been fully elucidated. The aim of the present study was to investigate the role of ALS in mammalian target of rapamycin (mTOR) and apoptosis signaling using in vivo and in vitro models of cardiac hypertrophy. A rat model of cardiac hypertrophy was induced by isoproterenol treatment (5 mg·kg-1·day-1) for 4 weeks, with or without ALS treatment at 20 mg·kg-1·day-1. The expression of hypertrophic, fibrotic, and apoptotic markers was determined by RT-qPCR. The protein expression of apoptotic markers mTOR and p-mTOR was assessed by western blot analysis. The proliferation of H9C2 cells was monitored using the MTS assay. Cell apoptosis was analyzed using flow cytometry. In vivo, isoproterenol-treated rats exhibited worse cardiac function, whereas ALS treatment reversed these dysfunctions, which were associated with changes in p-mTOR, Bcl-2, Bax, and cleaved caspase-3 expression, as well as the number of apoptotic cells. In vitro, H9C2 cardiomyocyte viability was significantly inhibited and cardiac hypertrophy was induced by Ang II administration, but ALS reversed Ang II-induced H9C2 cardiomyocyte hypertrophy and death. Furthermore, Ang II triggered the activation of the mTOR and apoptosis pathways in hypertrophic cardiomyocytes that were inhibited by ALS treatment. These results indicated that ALS alleviated cardiac hypertrophy through inhibition of the mTOR and apoptosis pathways in cardiomyocytes.

MicroRNA-23a knockdown attenuates angiotensin Ⅱ induced hypertrophy in rat H9c2 cells via activating PTEN and AMPK pathway / 中华心血管病杂志

Objective: To investigate if microRNA (miR) -23a knockdown could attenuate angiotensin Ⅱ(AngⅡ) induced cardiac hypertrophy by activating phosphatase and tensin homolog deleted on chromosome ten(PTEN) and AMP-activated protein kinase(AMPK) pathway. Methods: Rat H9c2 cells were cultured in DMEM high glucose medium and put in 5% CO(2) incubator at 37 ℃(normal group). After 48 hours of culture, H9c2 cells were stimulated with 10 nmol/L AngⅡ to establish cell hypertrophy model (AngⅡgroup). The H9c2 cells were inoculated in a 6-well cell culture plate and cultured in an incubator at 37 ℃. When the confluence degree of cell growth was about 70%, the cells were transfected with different reagents, and 24 hours after transfection, 10 nmol/L AngⅡ was used to interfere with the cells. The H9c2 cells were divided into different groups according to the reagents, namely AngⅡ+anti-miR group(transfected with miR-23a inhibitor), Ang Ⅱ+NC group(transfected with miR-23a inhibitor negative control), Ang Ⅱ+anti-miR+si-PTEN group(cotransfected with miR-23a inhibitor and PTEN small interference RNA(siRNA)), and AngⅡ+anti-miR+si-NC group(cotransfected with miR-23a inhibitor and PTEN siRNA negative control). The surface area of single cell was measured by Image J software.The mRNA expression levels of α-actin 1 (ACTA1) and β-myosin heavy chain (β-MHC) and miR-23a were detected by quantitative real-time PCR(qRT-PCR). The expression levels of PTEN and AMPK signal pathway related proteins were detected by Western blot. In order to verify whether miR-23a targets PTEN gene, double luciferase reporter gene experiment was performed. The luciferase reporter gene vector recombinant plasmids of wild type pGL-WT-PTEN and mutant pGL-MUT-PTEN were constructed and prepared after normal sequencing. H9c2 cells was inoculated into 24-well cell culture plate and cultured overnight in 37 ℃ incubator. The cells were co-transfected with miR-23a mimic or miR-23a mimic negative control and wild type or mutant reporter gene recombinant plasmid. Forty-eight hours after transfection, firefly luciferase activity and sea kidney luciferase activity were measured, and the ratio of them was recorded as relative luciferase activity. Results: Compared with the normal group, the cell surface area, the mRNA expression levels of ACTA1, β-MHC and miR-23a were significantly higher, while the protein expression levels of PTEN and p-AMPK were significantly lower in the Ang Ⅱ group(all P<0.05). The results of double luciferase reporter gene assay showed that the relative luciferase activity of cells co-transfected with miR-23a mimic and wild-type reporter gene recombinant plasmid was lower than that of miR-23a mimic negative control (P<0.05), and PTEN served as the target gene of miR-23a. In AngⅡ+anti-miR group the mRNA expression levels of miR-23a, ACTA1 and β-MHC were lower, and the cell surface area was smaller, while the protein expression levels of PTEN and p-AMPK were higher than that in AngⅡ group and AngⅡ+NC group(all P<0.05). Compared with AngⅡ+anti-miR group, the cell surface area was bigger, the expression of ACTA1 and β-MHC mRNA was up-regulated, and the protein expression levels of PTEN and p-AMPK were down-regulated in Ang Ⅱ+anti-miR+si-PTEN group(all P<0.05). Conclusion: Inhibition of miR-23a can attenuate Ang Ⅱ-induced hypertrophy in H9c2 cells through targeting PTEN and activating AMPK signaling pathway.

Gαq-RGS2 loop activator modulates the activity of vario us agonists on isolated heart and aorta of normal rats

The Gαq-RGS2 loop activator, 1-(5-chloro-2-hydroxyphenyl)-3-(4-(trifluoromethyl)-phenyl)-1H-1,2,4-triazol-5(4H)-one has demonstrated Gαq signaling inhibitor activity. Therefore, we aimed to study the effect of Gαq-RGS2 loop activator on isolated heart and aorta of normal rats. Heart and aorta were isolated from the sacrificed rats (n=6) and mounted on the langendroff's and organ bath assembly, respectively. The effect of various receptor-dependent (acetylcholine, angiotensin II and adrenaline) and independent (calcium chloride and sodium nitroprusside) agonists in absence and presence of Gαq-RGS2 loop activator on left ventricular systolic pressure (LVSP) and the contractile responseswere evaluated in isolated heart and aorta, respectively. Gαq-RGS2 loop activator (100 µM) significantly attenuated the adrenaline (p<0.001,) and angiotensin II (p<0.001) induced increase in LVSP in isolated heart and contractile response of adrenaline (p<0.01) and angiotensin II (p<0.01) in the aorta. However, effect calcium chloride did not significantly alter by Gαq-RGS2 loop activator. The effect of acetylcholinewas significantly (p<0.01, p<0.05) increased by Gαq-RGS2 loop activator in isolated heart and aorta. The effect of sodium nitroprusside significantly (p<0.01) potentiated by Gαq-RGS2 loop activator (100 µM) in isolated heart while it did not significantly alters in the aorta. Ultimately, the Gαq-RGS2 loop activator modulated the action of receptor-dependent agonists in isolated heart and aorta

Injuria renal fetal y neonatal: una complicación de la terapia antihipertensiva materna. Reporte de caso / Fetal and neonatal renal injury: a complication of maternal hypertensive therapy: case presentation

Los Inhibidores de la enzima convertidora de angiotensina II (IECAs) y antagonistas de los receptores de angiotensina II (ARA II) son drogas usadas comúnmente en el manejo de hipertensión arterial, sin embargo, su uso en el embarazo está asociado con toxicidad fetal.1, 2 La acción de la angiotensina II requiere su unión a dos receptores; AT1, involucrado en el control de la tensión arterial y AT2, probablemente encargado del crecimiento fetal. 2 La angiotensina II es esencial en la hemodinamia sistémica y la filtración glomerular fetal y neonatal. La disminución de la perfusión placentaria por efecto hipotensor en el bloqueo del sistema renina angiotensina aldosterona materno puede determinar hipotensión fetal sistémica, disminución de la filtración glomerular, oligoamnios e insuficiencia renal, anormalidades tubulares, hipoplasia craneal y alto riesgo de muerte perinatal. 2 Reportamos el caso de prematuro de 30 semanas con oligohidramnios severo y exposición materna a olmesartan. Al nacimiento presentó dificultad respiratoria; imposibilidad de mantener una adecuada tensión arterial a pesar de los esfuerzos para conseguir mejorar su tono vascular; anuria sin respuesta a diuréticos; alteraciones craneales; alteraciones metabólicas severas con consecuencias fatales. El tratamiento de hipertensión materna con inhibidores de la enzima convertidora de angiotensina II y los antagonistas de los receptores de angiotensina II está asociada con toxicidad fetal por lo que su uso debe ser evitado durante el embarazo.

Angiotensin II converting enzyme (ACE) inhibitors and angiotensin II receptor antagonists (ARBs) are drugs for general use in the management of arterial hypertension, however their use in gestational hypertension is related to the Fetal toxicity. 1, 2 The action of angiotensin II requires its binding to two receptors; AT1, involved in the control of blood pressure and AT2, probably responsible for fetal growth.2 Angiotensin II is essential in systemic hemodynamics and fetal and neonatal glomerular filtration. The decrease in placental perfusion due to hypotensive effect in the blockade of the maternal rennin angiotensin aldosterone system can determine systemic fetal hypotension, decreased glomerular filtration, oligohydramnios and renal insufficiency, tubular abnormalities, cranial hypoplasia and high risk of perinatal death. 2 We report the case of prematurity of 30 weeks with a history of severe oligohydramnios and maternal exposure to olmesartan. At birth the patient presented in particular respiratory distress; inability to maintain adequate blood pressure despite efforts to improve your vascular tone; anuria without response to diuretics; cranial alterations; metabolic alterations with fatal consequences. The treatment of maternal hypertension with inhibitors of the angiotensin II convective enzyme and angiotensin II receptor antagonists is associated with fetal toxicity and should therefore be avoided during pregnancy