Effect of angiotensin-(1-7) on angiotensin II-induced rat renal interstitial fibroblast activation

AIM: To explore the influence of angiotensin-(1-7) on angiotension II (Ang II)-induced activation and extracellular matrix secretion in rat renal interstitial fibroblasts (NRK-49F cells). METHODS: The NRK-49F cells were maintained and sub-cultured, then the cells were divided into control group, Ang II group, Ang-(1-7) group and Ang II+Ang-(1-7) group. The expression of α-smooth muscle actin(α-SMA),transforming growth factor β₁ (TGF-β₁) and insulin-like growth factor I(IGF-I) was detected by the method of immunocytochemistry when the cells were cultured for 72 h. The content of TGF-β₁, IGF-I and collagen type I(Col I) in the cultured supernatants were measured by ELISA. RESULTS: In control group and Ang-(1-7) group, only basic expression of α-SMA and almost no expression of TGF-β₁, IGF-I and Col I were observed. Compared with control group, the expression of α-SMA, TGF-β₁, IGF-I and Col I was increased in Ang II group. Compared with Ang II group, the expression of α-SMA, TGF-β₁, IGF-I and Col I was significantly decreased in Ang II+Ang-(1-7) group.CONCLUSION: Ang-(1-7) inhibits the activation of renal interstitial fibroblasts and decreases the Ang II induced secretion of Col I by suppressing TGF-β₁ and IGF-I expression.     

Angiotensin-(1-7) increases expression of ABCA1 and decreases content of cholesterol in THP-1-derived foam cells via AC-cAMP pathway

AIM: To establish the THP-1-derived foam cell formation and to evaluate the effects of angiotensin-(1-7) and MDL (an inhibitor of adenylate cyclase) on the expression of ATP-binding cassete transporter A1(ABCA1) and the content of cholesterol. METHODS: THP-1-derived macrophages were treated with oxidized low-density lipoprotein(ox-LDL) to develop into foam cells. The foam cells were divided into 4 groups: control group, MDL group, Ang-(1-7) group and MDL+Ang-(1-7) group. At 24 h after treatment, the content of cAMP was measured by ELISA. The mRNA and protein levels of ABCA1 were determined by real-time RT-PCR and Western blotting, respectively. The content of cholesterol was detected by high performance liquid chromatography. RESULTS: The cAMP, the mRNA and protein levels of ABCA1 in Ang-(1-7) group were significantly higher, and the content of cholesterol was significantly lower than those in control group (P<0.05). On the contrary, the cAMP, the mRNA and protein levels of ABCA1 in MDL group were significantly lower and the content of cholesterol was significantly higher than those in control group (P<0.05). The results in MDL+Ang-(1-7) group were between Ang-(1-7) group and control group. CONCLUSION: Ang-(1-7) inhibits the formation of foam cells by promoting the expression of ABCA1 and decreasing the content of cholesterol. MDL partly antagonizes the effect of Ang-(1-7) by inhibiting the adenylate cyclase and decreasing the content of cAMP.     

Effects of angiotensin-(1-7) on calcification in vascular smooth muscle cells and its signal transduction pathway

AIM: To clarify the effects of angiotensin-(1-7) on the calcification in rat vascular smooth muscle cells(VSMCs) and its signal transduction.METHODS: Calcification of cultured rat VSMCs was prepared by incubation of VSMCs with β-glycerophosphate.Calcification was confirmed by Von Kossa staining.The cells were treated with angiotensin-(1-7).The calcium content,alkaline phosphatases activity,osteocalcin and Cbfa1 mRNA expression were also measured.RESULTS: Angiotensin-(1-7) inhibited the increases of calcium content,alkaline phosphatases activity（P>0.05）,osteocalcin concentration and Cbfa1 mRNA expression in calcified VSMCs（P<0.05）,and the effects of angiotensin II on calcium content,alkaline phosphatases activity,osteocalcin concentration and Cbfa1 mRNA expression in calcified VSMCs were also inhibited （P<0.05）.Angiotensin-(1-7) increased cAMP concentration in calcified VSMCs（P<0.05）and selective PKA inhibitor blocked the effects of angiotensin-(1-7) on calcium content,alkaline phosphatases activity,osteocalcin concentration and Cbfa1 mRNA expression（P<0.05）.CONCLUSION: Angiotensin-(1-7) can inhibit beta-glycerophosphate-induced calcification in VSMCs through cAMP-PKA-Cbfa1 pathway and antagonize the effect of angiotensin II on calcification in VSMCs.     

Effects of perindopril at different doses on cardiac function and ACE2/Ang-(1-9)/Ang-(1-7) axis of ischemic cardiac dysfunction rabbits

AIM: To investigate the different dose of perindopril on cardiac function in the rabbits with ischemic cardiac dysfunction. METHODS: Male rabbits weighing 2.5~3.0 kg(n=30) were randomly divided into 3 groups(n=10):high dose perindopril group(HD group), low dose perindopril group(LD group) and cardiac dysfunction group(CD group). The Left anterior descending coronary artery of the rabbits was ligatured for model preparation. In HD group, the rabbits were treated with perindopril split normal saline solution(1 g/L)2 mL·kg^-1·d^-1. In LD group, the rabbits were treated with perindopril split normal saline solution(0.33 g/L)2 mL·kg^-1·d^-1. In CD group, the rabbits were treated with normal saline solution 2 mL·kg^-1·d^-1. Four weeks after treatment, the cardiac function was measured via echocardiography, the mRNA expression of angiotensin-converting enzyme 2(ACE2) and angiotensin type 2 receptor(AT2R) was analyzed by real-time PCR, serum angiotensin(Ang)-(1-9) and Ang-(1-7) levels were detected by ELISA. RESULTS: Compared with CD group, the cardiac function of the 2 groups treated with perindopril was significantly improved(P<0.01), and more improvement in HD group was observed than LD group(P<0.05). The serum angiotensin(Ang)-(1-9) and Ang-(1-7) level and the mRNA expression of ACE2 and AT2R in the 2 groups treated with perindopril were significantly improved(P<0.01). Compared with LD group, the mRNA expression of ACE2 and AT2R and the serum levels of Ang-(1-9) in HD group were significant improved(P<0.05), while no difference of serum Ang-(1-7) level was observed. Correlation analysis revealed that the improvement of the cardiac function was associated with serum Ang-(1-9) level, mRNA expression of ACE2 and AT2R(P<0.01), but has no significant correlation with serum Ang-(1-7) level. CONCLUSION: High dose of perindopril may improve more cardiac function in ischemic cardiac dysfunction model in rabbits. The mechanism may relate to increasing serum Ang-(1-7) level to activate AT2R.     

Ang(1-7) attenuates palmitic acid-induced injury of Min6 cells

AIM: To investigate the effect of angiotensin (1-7)[Ang (1-7)] on palmitic acid (PA)-induced injury of Min6 cells and the potential protective mechanisms of autophagy. METHODS: Cultured Min6 cells were divided into 7 groups:control group, PA group, PA+Ang(1-7) group, PA+Ang(1-7)+A779 group, PA+Ang(1-7)+rapamycin group, Ang(1-7) group and A779 group. The function of Min6 cells was detected by glucose-stimulated insulin release test. Intracellular reactive oxygen species (ROS) production was measured by ROS assay kit. Apoptosis was analyzed by flow cytometry with Annexin V/PI staining. Autophagy-related proteins were determined by Western blot. RESULTS: Compared with control group, the secretion of insulin from Min6 cells in PA group was significantly decreased (P<0.05), and the apoptotic rate was increased (P<0.05). The ratio of LC3-Ⅱ/LC3-I was significantly increased (P<0.05). Compared with PA group, the insulin secretion in PA+Ang(1-7) group was increased (P<0.05). The intracellular ROS level and the A779 and LC3-Ⅱ/LC3-I were significantly decreased (P<0.05). This protective effect of Ang(1-7) was partially blocked by A779 and rapamycin. CONCLUSION: Ang(1-7) attenuates PA-induced Min6 cell injury, and its protective mechanism may be related to inhibiting the activity of autophagy.     

Angiotensin-(1-7) protects H9c2 cardiac cells against high glucose-induced injury and inflammation by inhibiting the interaction between TLR4 activation and necroptosis

AIM: To investigate whether angiotensin-(1-7)[Ang-(1-7)] protects H9c2 cardiac cells against high glucose (HG)-induced injury and inflammation by inhibiting the interaction between Toll-like receptor 4 (TLR4) activation and necroptosis. METHODS: The expression levels of receptor-interacting protein 3 (RIP3; an indicator of necroptosis) and TLR4 were determined by Western blot. Cell viability was measured by CCK-8 assay. The activity of lactate dehydrogenase (LDH) in the culture medium was measured with a commercial kit. The releases of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were measured by ELISA. The intracellular level of reactive oxygen species (ROS) was analyzed by 2', 7'-dichlorfluorescein-diacetate (DCFH-DA) stating followed by photofluorography. Mitochondrial membrane potential (MMP) was examined by rhodamine 123 staining followed by photofluorography. RESULTS: After the H9c2 cardiac cells were treated with HG (35 mmol/L glucose) for 24 h, the expression of RIP3 was obviously increased. Co-treatment of the cells with 30 μmol/L TAK-242 (an inhibitor of TLR4) attenuated the up-regulation of RIP3 induced by HG. Furthermore, the expression of TLR4 was significantly increased after the cells were exposed to HG for 24 h, and co-treatment of the cells with 100 μmol/L necrostatin-1 (Nec-1; a specific inhibitor of necroptosis) and HG for 24 h attenuated the up-regulation of TLR4 expression induced by HG. Moreover, 1 μmol/L Ang-(1-7) simultaneously blocked the up-regulation of the RIP3 and TLR4 induced by HG. On the other hand, co-treatment of the cells with 1 μmol/L Ang-(1-7), 30 μmol/L TAK-242 or 100 μmol/L Nec-1 and HG for 24 h attenuated HG-induced injuries and inflammatory response, leading to the increase in the cell viability, and the decreases in the activity of LDH, ROS generation, MMP loss as well as the releases of IL-1β and TNF-α. CONCLUSION: Ang-(1-7) protects H9c2 cardiac cells against HG-induced injury and inflammation by inhibiting the interaction between TLR4 activation and necroptosis.     

Role of apolipoprotein E in cholesterol efflux mediated by ABCA1 and ABCG1

AIM:To investigate the role of apolipoprotein E(ApoE) in cholesterol efflux mediated by ATP-binding cassette transporter A1(ABCA1) and ATP-binding cassette transporter G1(ABCG1). METHODS:RAW 264.7 cells were seeded in either 6-well or 24-well plates, and then incubated with 20 mg/L low-density lipoprotein receptor gene knockout(LDLr^-/-) mouse lipoprotein 20 mg/L ApoE gene knockout(ApoE^-/-) mouse lipoprotein or culture medium alone. The changes of intracellular lipid content were measured by transmission electron microscopy and enzymatic colorimetric method. The cholesterol efflux was determined by liquid scintillation. The mRNA and protein levels of ABCA1 and ABCG1 were detected by real-time PCR and Western blotting, respectively. RESULTS:The ApoE^-/- mouse lipoprotein increased the content of intracellular cholesterol ester by 60% compared with the control cells. In addition, ApoE^-/- mouse lipoprotein treatment decreased the cholesterol efflux to apolipoprotein A-I(ApoA-I) and high-density lipoprotein(HDL) compared with LDLr^-/- mouse lipoprotein treatment. ApoE^-/- mouse lipoprotein treatment inhibited the mRNA and protein levels of ABCA1 and ABCG1 compared with LDLr^-/- mouse lipoprotein treatment. CONCLUSION:Apolipoprotein E plays an important role in the cholesterol efflux of macrophages, which is associated with its regulatory effect on the expression of ABCA1 and ABCG1.     

Angiotensin-(1-7)/Mas receptor axis protects cardiomyocytes against high glucose-induced injury by modulating nuclear factor-κB pathway

AIM: To study whether the angiotensin-(1-7)[Ang-(1-7)]/Mas receptor axis protects cardiomyocytes against high glucose(HG)-induced injury by inhibiting nuclear factor-κB(NF-κB) pathway. METHODS: The cell viability was measured by CCK-8 assay. The intracellular levels of reactive oxygen species(ROS) were detected by DCFH-DA staining. The number of apoptotic cells was tested by Hoechst 33258 nuclear staining. Mitochondrial membrane potential(MMP) was examined by JC-1 staining. The levels of NF-κB p65 subunit and cleaved caspase-3 protein were determined by Western blotting. RESULTS: Treatment of H9c2 cardiac cells with 35 mmol/L glucose(HG) for 30, 60, 90, 120 and 150 min significantly enhanced the levels of phosphorated(p) NF-κB p65, peaking at 60 min. Co-treatment of the cells with 1 μmol/L Ang-(1-7) and HG for 60 min attenuated the up-regulation of p-NF-κB p65 induced by HG. Co-treatment of the cells with Ang-(1-7) at concentrations of 0.1~30 μmol/L and HG for 24 h inhibited HG-induced cytotoxicity, evidenced by an increase in cell viability. On the other hand, 1 μmol/L Ang-(1-7) ameliorated HG-induced apoptosis, oxidative stress and mitochondrial damage, indicated by decreases in the number of apoptotic cells, cleaved caspase-3 level, ROS generation and MMP loss. However, the above cardioprotective effects of Ang-(1-7) were markedly blocked by A-779, an antagonist of Ang-(1-7) receptor(Mas receptor). Similarly, co-treatment of H9c2 cardiac cells with 100 μmol/L PDTC(an inhibitor of NF-κB) and HG for 24 h also obviously reduced the above injuries induced by HG. CONCLUSION: Ang-(1-7)/Mas receptor axis prevents the cardiomyocytes from the HG-induced injury by inhibiting NF-κB pathway.     

Effects of angiotensin Ⅱ on ATP binding cassette transporter A1 in THP-1 derived foam cells

AIM:To study the influence of angiotensin Ⅱ (AngⅡ) on ATP-binding cassette transporter A1 in THP-1 derived foam cells. The variance of the expression of ABCA1, the content and the effluent rate of cholesterol were also investigated. METHODS:The regulatory effect of AngⅡ on the expression of ABCA1 mRNA and protein in THP-1 derived form cells were measured by RT-PCR and Western blotting. The effect of variance of cholesterol content was measured by zymochemistry via-fluorospectrophotometer, cholesterol effluent was measured by liquid scintillator. RESULTS:A positive facilitative effect of Ang Ⅱon form cells was observed. Total cholesterol content were increased significantly by Ang Ⅱ treatment (P<0.05). The mRNA and protein of ABCA1 were down-regulated significantly by Ang Ⅱ stimulation (P<0.05). Irbesartan reduced the total cholesterol content significantly (P<0.05). Meanwhile, the increase in the effluent rate of cholesterol and the expression of ABCA1 were observed (P<0.05). CONCLUSION:The effects of Ang Ⅱ on the formation of foam cells and atherosclerosis may be correlated to the activation of AT1 receptor and down-regulation of ABCA1.     

Effect of homocysteine on expression of interleukin-8 mRNA and protein in THP-1 macrophages

AIM: To investigate the effect of homocysteine (Hcy) on expression of interleukin-8 (IL-8) mRNA and protein in THP-1-derived macrophages (THP-1 macrophages). METHODS: Cultured THP-1 monocytes were induced to macrophages by 0.1 μmol/L PMA treatment for 72 hours, then the differentiated THP-1 macrophages were incubated with homocysteine (0.01 mmol/L-0.20 mmol/L) for 24 hours, or with 0.10 mmol/L Hcy for various time up to 48 hours. IL-8 protein in THP-1 supernatants was measured by ELISA, and IL-8 mRNA expression was detected by semiquantitive RT-PCR. RESULTS: Compared with control, Hcy significantly increased the expression of IL-8 protein in a concentration-dependent manner. 0.05 mmol/L, 0.10 mmol/L and 0.20 mmol/L Hcy increased IL-8 production by 1.28 fold, 1.32 fold and 1.55 fold, respectively (P＜0.01). IL-8 production were elevated significantly 3 h after treatment with 0.10 mmol/L Hcy. In addition, Hcy also increased IL-8 mRNA expression in a concentration-and time-dependent manner. CONCLUSION: Hcy may contribute to atherogenesis by inducing IL-8 expression and secretion in THP-1 macrophages.     

Roles of NPC1 and NPC2 in intracellular lipid homeostasis

Niemann-pick protein C1 (NPC1) is a large integral membrane glycoprotein that resides in late endosomes, whereas niemann-pick protein C2 (NPC2) is a small soluble protein found in the lumen of lysosomes. NPC1 protein is believed to facilitate the transport of lipids, particularly cholesterol, from late endosomes/lysosomes to the Golgi apparatus, endoplasmic reticulum and plasma membrane. NPC2 primarily plays a role in the egress of cholesterol and glycolipids from lysosomes. Mutations in either NPC1 or NPC2 result in aberrant lipid transport from endocytic compartments, which results in lysosomal storage of a complex mixture of lipids, primarily cholesterol and glycosphingolipids. The NPC proteins regulate sterol homeostasis through production of LDL cholesterol-derived oxysterols. Oxysterols are endogenous ligands for the liver X receptors(LXRs), which can upregulate ATP binding cassette transporter A1(ABCA1) expression. ABCA1 may have antiatherogenic effects through the efflux of it-mediated cholesterol. Meanwhile, NPC1 heterozygote mutation confers substantial resistance to lesional necrosis and lesional macrophage apoptosis. Study of the NPC proteins will help us for further understanding of the mechanisms involved in atherogenesis.     

Effects of B7-1 gene expression on the biological characteristics of hepatocarcinoma cells

AIM:Modification of tumor cells with B7-1 (CD80) costimulatory adhesion molecules has been proposed as a means to develop therapeutic cancer vaccines for use in human immunotherapy.METHODS:Glycosyl-phosphatidylinositol (GPI)-anchored B7-1 was transfered into QK10341 cell membranes with the plasmid of pcDNA3.1(+)/GPI-B7-1 by lipofectamine transfection. Then the GPI-B7-1 protein was isolated and purified from QK10341 cells. The SKOV3 cells incubated with GPI-B7-1 protein resulted in stable incorporation of B7-1 on SKOV3 cell membranes. Expression of B7-1 in tumors by protein transfer created an immunogenic tumor cell that induced antitumor immunity. The growth curve of T cells, the change of Fas (CD95) expression on cell membranes, and level of cytokines secreted from CTL were determined by MTT, FCM, and ELISA, respectively. The cytotoxicity alteration of the CTL was also studied.RESULTS:Compared with SKOV3 cells, B7-1-SKOV3 cells more effectively induced the proliferation of effector lymphocytes and the generation of specific lytic activity (P<0.01). The level of cytokine secretion also increased.CONCLUSION:The costimulation signal of B7-1 is required for the activation and proliferation of T lymphocytes. The B7-1 expression in SKOV3 tumor cells can increase their immunogenicity and induce more effective T lymphocytes activation.     

Effects of angiotensin II and losartan on collagen synthesis in rat hepatic stellate cells

AIM: To investigate the effects of angiotensin II (Ang II) and AT_1a receptor antagonist (losartan) collagen synthesis in rat hepatic stellate cells (HSCs). METHODS: ① Rat HSCs were isolated, cultured and identified. ② Rat HSCs were incubated in the medium with different concentrations of AngII or losartan, then the quantity of collagen was examined by -proline release assay. RESULTS: ① The yield of HSCs was 2×10⁷-3×10⁷/per rat, their viability and purity was more than 95% and 90%, respectively. ② The yield of collagen in HSCs significantly got a rise in a concentration-dependent manner when HSCs were incubated with AngII (10^－6mol/L-10^－10 mol/L) (P<0.05). While HSCs were influenced by the antagonist of AT_1a (10^－6 mol/L-10^－9 mol/L), the quantity of collagen dropped greatly (P<0.05). CONCLUSIONS: Ang II stimulates HSCs to produce more collagen. Losartan inhibits the cell to synthesize collagen via AT_1a receptor (P<0.05). The results indicate that Ang II may play an important role in the development of hepatic fibrosis, and using AT_1a antagonist may offer a new strategy to prevent hepatic fibrosis.     

Exercise training attenuates hypertension progression via activation of central ACE2-Ang(1-7)-Mas axis in prehypertensive rats

AIM: To investigate the effects of exercise training on the progression from prehypertension to hypertension, blood pressure regulation and the angiotensin-converting enzyme 2 (ACE2)-angiotensin (Ang) (1-7)-MAS axis activation in cardiovascular centers, and to elucidate the central mechanisms of exercise training postponing hypertension progression. METHODS: The male spontaneously hypertensive rats (SHR; n=20, 5 weeks old) and normotensive Wistar Kyoto (WKY) rats (n=20) were randomly assigned to sedentary (Sed) group and exercise training (ExT) group. The trained rats run on a treadmill in moderate-intensity for 20 weeks. Systolic blood pressure (SBP) was measured by tail-cuff method. The baroreflex sensitivity (BRS) was assessed by intravenous injection of phenylephrine. The expression of ACE2 and MAS receptor at mRNA and protein levels in baroreflex centers were determined by real-time PCR and Western blot, respectively. Alterations of BRS were evaluated before and after intracerebroventricular injection of MAS receptor agonist Ang (1-7) and its antagonist A779, respectively. RESULTS: Compared with SHR+Sed group, exercise training since prehypertension significantly postponed the development of hypertension, delayed the hypertension progression, and decreased SBP in both SHR and WKY rats (P<0.05). Exercise training enhanced blood pressure regulation and improved the BRS in SHR (P<0.01). The expression of ACE2 and MAS receptor at mRNA and protein levels in the baroreflex centers (rostral ventrolateral medulla, nucleus tract solitarius and paraventricular nucleus) were up-regulated in SHR+ExT group (P<0.05). Central administration of A779 abolished the benefits of exercise-induced improvement of BRS in SHR+ExT group (P<0.01). In contrast, Ang(1-7) improved the BRS in both SHR+Sed group and SHR+ExT group (P<0.05). CONCLUSION: Exercise training postpones hypertension progression and improves blood pressure regulation, which may be associated with the activation of central ACE2-Ang(1-7)-Mas axis.