[Angiotensin II increases ROS production in cardiac fibroblasts by inducing p22phox over-expression].

OBJECTIVE: To investigate the changes in the reactive oxygen species (ROS) in rat cardiac fibroblasts exposed to angiotensin II (Ang II) treatment and explore the possible pathways that mediate ROS production. METHODS: In vitro cultured fetal rat cardiac fibroblasts treated with apocynin (APO, 100 micromol/L), Ang II (10(-7) mol/L), or APO+Ang II (10(-7) mol/L Ang II was added 1 h after 100 micromol/L APO), and the ROS levels and p22phox expression in the cells were detected using fluorescent microscope and immunohistochemistry, respectively. RESULTS: Compared with the normal control cells, Ang II treatment of the cardiac fibroblasts resulted in significantly increased ROS production, the effect of which was inhibited by the application of APO. p22phox expression was hardly detected by immunohistochemistry in the control cells, but over-expressed in AngII-treated cells. APO substantially decreased the over-expression of p22phox induced by Ang II. CONCLUSION: Ang II increases ROS production in fetal rat cardiac fibroblasts probably by inducing p22phox over-expression.

[Angiotensin II type 2 receptors participate in the regulation of inflammatory cytokine secretion in adult rat hypertrophied cardiomyocytes].

OBJECTIVE: To investigate the effect of angiotensin II (AngII) type 2 (AT2) receptors on pressure overload-induced inflammatory cytokine secretion in adult rat hypertrophied cadiomyocytes. METHODS: Rat models of left ventricular hypertrophy induced by pressure overload was established by placing a band around the abdominal aortic of the rats, from which the hypertrophied cadiomyocytes were isolated and purified 8 weeks later. The isolated cardiomyocytes were treated with AngII plus losartan or AngII plus PD123319, and 36 h after the treatments, the expression levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and IL-6 in the supernatant were detected using radioimmunoassay. RESULTS: AngII induced TNF-alpha and IL-1beta secretion from the hypertrophied cardiomyocyets, and pretreatment of the cells with PD123319, but not losartan, decreased their secretion. IL-6 level was not detected in the supernatant. CONCLUSION: AngII-induced the expression of inflammatory cytokines in adult rat hypertrophied cardiomyocytes is mediated mainly by AT2, not by AT1 receptors.

[The influence of angiotensin II receptor blockade on the expressions of Col I and TIMP-1 mRNA in cardiac fibroblast of adult rat].

OBJECTIVE: To investigate the role of angiotensin II receptor type 2 (AT2) on collagen metabolism in cardiac fibroblast, and the difference between AT2 and angiotensin II receptors type 1 (AT1). METHODS: Adult rat cardiac fibroblasts were isolated and cultured. The cells were divided into 4 groups: Angiotensin II (Ang II), Ang II + Losartan, Ang II + PD123319, Ang II + Losartan + PD123319. Semiquantitative reverse transcription PCR was used to determine the mRNA levels of collagen I (Col I ) and tissue inhibitor of metalloproteinase 1 (TIMP-1). RESULTS: Losartan-treatment making AT1 blockade decreased Col I mRNA to 71.8% in cardiac fibroblasts, AT2 blockade from PD123319-treatment decreased Col I mRNA to 81.5%, co-treatment of both AT1 and AT2 blockade decreased Col I mRNA to 50.9% ; the AT1 blockade with Losartan-treatment decreased TIMP-1 mRNA to 88.1% in cardiac fibroblasts, AT2 blockade by PD123319-treatment decreased TIMP-1 mRNA to 75.4%, Both AT1 and AT2 blockade by co-treatment decreased TIMP-1 mRNA to 44.1%. CONCLUSION: AT2 receptor is involved in collagen metabolism in cardiac fibroblast by regulating the levels of Col I and TIMP-1 mRNA.

The signalling of AT2 and the influence on the collagen metabolism of AT2 receptor in adult rat cardiac fibroblasts.

OBJECTIVES: Angiotensin (Ang) II exerts its roles on cardiac fibroblasts by two receptors: type I (AT1) and type 2 (AT2). The role of AT1 has been well known, but less is known about AT2. The present study was designed to explore signal pathways of AT2 and observe whether AT2 is involved in the collagen metabolism of cardiac fibroblasts. METHODS AND RESULTS: Adult rat cardiac fibroblasts were extracted, cultured and treated with Ang-II alone, Ang-II plus losartan or PD123319. G protein-coupled receptors signalling pathway finder gene arrays were used to analyse expression changes of 96 genes associated with 11 signal pathways. With a 10-fold change in threshold, 7 genes were differentially expressed specific to AT1 blockade associated to 5 signal pathways including PKC, PLC, MAPK, NO/cGMP and NFkappaB; while 24 genes were specific to AT2 blockade related to 10 signal pathways including cAMP/PKA, Ca2+, PKC, PTK, MAPK, PI-3K, NO/cGMP, Rho, NFkappaB and JAK-STAT. RT-PCR were used to confirm the results of arrays and measure collagen (Col) I and tissue inhibitor of metalloproteinase (TIMP)-1 mRNA levels. AT2 blockade decreased Col I and TIMP-1 mRNA levels compared to the Ang II-treated group, which was similar with AT1 blockade. CONCLUSION: In adult rat cardiac fibroblasts, AT2 was obviously distinct from AT1 in signalling responses. AT2 appeared to spread more widely than AT1. AT2 might be involved in the collagen metabolism of rat cardiac fibroblasts by regulating Col I and TIMP1 mRNA levels.

[Role of AT2 receptors on angiotensin II-induced tumor necrosis factor alpha and interleukin 1 beta secretion in adult rat cardiac fibroblasts].

OBJECTIVE: To investigate the role of AT2 receptors in the secretion of tumor necrosis factor alpha (alpha-TNF) and interleukin 1 beta (IL1 beta) in adult rat cardiac fibroblasts. METHODS: Adult rat cardiac fibroblasts in in vitro culture were divided into control, Ang II, AngII + Losartan, and AngII + PD123319 groups with corresponding treatments. Radioimmunoassay was used to determine alpha-TNF and IL1 beta levels in the supernatant of the treated cardiac fibroblasts. RESULTS: Ang II treatment resulted in significantly increased alpha-TNF and IL1 beta levels. Compared with AngII group, IL1 beta level was decreased by 69.1% and 78.7% and alpha-TNF by 58.7% and 65.9% after blocking AT1 and AT2 receptors, respectively. CONCLUSION: AT2 receptors are involved in alpha-TNF and IL1 beta secretions in cardiac fibroblasts.

Angiotensin II receptors subtypes mediate diverse gene expression profile in adult hypertrophic cardiomyocytes.

1. Although the systemic and cardiac renin-angiotensin systems are known to be activated in the setting of pressure overload, the actions and signaling mechanisms of angiotensin (Ang) II via AT(1) and AT(2) receptors in hypertrophic cardiomyocytes (CM) remain largely unclear. 2. Hypertrophic CM were prepared from rats with aortic banding for 8 weeks, cultured and then treated as follows: (i) 1 micromol/L AngII for 24 h; (ii) 10 micromol/L losartan (an AT(1) receptor antagonist) for 1 h followed by 1 micromol/L AngII for 24 h; and (iii) 10 micromol/L PD123319 (an AT(2) receptor antagonist) for 1 h followed by 1 micromol/L AngII for 24 h. Changes in the expression of genes following stimulation of AT(1) and AT(2) receptors specific to G-protein-coupled receptor (GPCR) signaling pathways were tested using GEArray (Superarray, Bethesda, MD, USA). The effects of AngII, acting via AT(1) and AT(2) receptors, on the expression of tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 were confirmed by reverse transcription-polymerase chain reaction and radioimmunoassay. 3. The genes regulated via stimulation of AT(1) receptors were mainly restricted to the signaling pathways including cAMP/protein kinase (PK) A, Ca(2+), PKC, protein tyrosine kinase, mitogen-activated protein kinases, phosphatidylinositol 3-kinase and nuclear factor-kappaB. In addition to these pathways related to activation of AT(1) receptors, four additional signaling pathways were found to be associated with stimulation of AT(2) receptors, including phospholipase C, nitric oxide/cGMP, Rho and Janus kinase/signal transducer and activator of transcription. Blockade of AT(2) receptors decreased the mRNA and protein expression of TNF-alpha and IL-1beta, whereas blockade of AT(1) receptors had no such effect. 4. In conclusion, in hypertrophic CM, AngII leads to distinct signaling responses mediated by AT(1) and AT(2) receptors. Stimulation of AT(2) receptors appears to have a greater influence on GPCR-signaling than stimulation of AT(1) receptors. Angiotensin II enhances the synthesis and secretion of TNF-alpha and IL-1beta in hypertrophic CM, which is mediated by AT(2), but not AT(1), receptors.

[Alteration of signal transduction-associated gene expression in rat cardiac fibroblasts induced by blocking angiotensin II receptors].

To investigate the molecular mechanism of angiotensin II (Ang II) receptor activation in adult rat cardiac fibroblasts, the expressions of cell signal transduction-associated genes were studied by using cDNA microarray. Cardiac fibroblasts of adult Sprague-Dawley rats (230~250 g) were isolated and cultured. The cells were divided into 4 groups: Ang II, Ang II + losartan, Ang II + PD123319, Ang II + losartan + PD123319. The expressions of Ang II receptors were studied by immunohistochemical staining. Total RNA was extracted and purified. After cDNA synthesis and biotin-16-dUTP labeling, the probes were denatured and hybridized with GEArray Q Series mouse G Protein-coupled Receptors Signaling Pathway Finder Gene Array (MM-025) containing 96 genes associated with 11 pathways. The arrays were scanned with a Uniscand1000 scanner and further analyzed with GEArray Analyzer software. RT-PCR was used to further confirm the results of gene microarray. The results of immunohistochemical staining showed that the expression of Ang II type 2 (AT2) receptor was evidently induced by Ang II stimulation when Ang II type 1 (AT1) receptor was blocked. The results of gene array indicated that blocking AT1 receptor changed 34 genes (more than 2 folds), 30 were down-regulated and 4 were up-regulated. The maximum change was not beyond 20 folds. The following 9 pathways were activated: cAMP/PKA, Ca2+, PKC, PLC, MAPK, PI-3 kinase, NO-cGMP, Rho, NF-kappaB pathways. Blockade of AT2 receptor caused 64 genes changing more than 2 folds (48 were down-regulated and 16 were up-regulated). Eleven pathways were basically activated. The change of the following 7 genes was over 30 folds: Cyp19a1 (37 folds), Il1r2 (42 folds), Cflar (53 folds), Bcl21 (31 folds), Pik3cg (278 folds), Cdkn1a (90 folds), Agt (162 folds). According to the activated extent, the signal transduction pathways in turn were PI-3 kinase, NF-kappaB and JAK-STAT pathways. Blocking both AT1 and AT2 receptors changed 46 genes more than 2 folds (36 were down-regulated and 10 were up-regulated). Eleven pathways were basically activated. The results of RT-PCR of IL-1beta and TNF-alpha confirmed the observations in gene microarray. Our results show that Ang II can induce a high expression of AT2 receptor in adult rat cardiac fibroblasts when AT1 receptor is blocked, and the signal mechanism of AT2 receptor is clearly different from that of AT1 receptor.

[Construction and identification of tetracycline-inducible human hepatocyte growth factor eukaryotic expression vector].

OBJECTIVE: To construct a tetracycline-inducible eukaryotic expression vector containing human hepatocyte growth factor (HGF) cDNA. METHODS: Human HGF cDNA fragment was obtained by PCR from pUC-SRalpha/HGF plasmid and inserted into the restriction site between Mlu I and Sal I of the tetracycline-inducible eukaryotic expression vector pBI-L. pBI-L-HGF was constructed by DNA recombination in vitro, and was identified by restriction endonucleases digestion and sequencing. RESULTS: The fragment of pBI-L-HGF digested with restriction endonucleases well corresponded to expectation, and the sequence of inserted HGF cDNA was correct according to the GenBank. CONCLUSION: The tetracycline-inducible eukaryotic expression vector of human HGF pBI-L-HGF has been constructed successfully, which allows further study of HGF gene therapy with much safety and easy manipulation.

[Construction and identification of tetracycline-inducible rat Smad7 eukaryotic expression vector].

OBJECTIVE: To construct a tetracycline-inducible eukaryotic expression vector of rat Smad7. METHODS: The total RNA was extracted from normal rat kidney with Trizol agent. Rat Smad7 cDNA fragment was cloned by RT-PCR, and was inserted into the restriction site between Nhe I and Hind III of the inducible eukaryotic expression vector pBI-L by tetracycline. pBI-L-Smad7 was constructed by digestion and ligation, and detected by restriction endonuclease digestion and sequencing. RESULTS: The recombinant eukaryotic expression vector pBI-L-Smad7 was constructed correctly as confirmed by restriction endonuclease digestion and sequencing. The fragment of pBI-L-Smad7 digested with restriction endonucleases and the sequence of inserted Smad7 cDNA were consistent with the results of theoretical analysis. CONCLUSION: The tetracycline- inducible eukaryotic expression vector of rat Smad7, pBI-L-Smad7, is constructed successfully, which may facilitate further clinical study of Smad7 gene therapy for tissue and organ fibrosis.

Neuroprotection of insulin against oxidative stress-induced apoptosis in cultured retinal neurons: involvement of phosphoinositide 3-kinase/Akt signal pathway.

In order to investigate the neuroprotection of insulin in retinal neurons, we used retinal neuronal culture as a model system to study the protective effects of insulin against H2O2-induced cytotoxicity and apoptotic death. Primary retinal neuronal cultures were grown from retinas of 0-2-day old Sprague-Dawley rats. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Apoptotic cell death was evaluated by the TdT-mediated digoxigenin-dUTP nick-end labeling assay, and by DNA laddering analysis. Phosphoinositide 3-kinase (PI3K) activity was measured using phosphoinositide 4,5-bisphophate and [gamma-32P]ATP as substrate. Western blot analysis with anti-phospho-Akt (pS473) antibody was performed to examine the level of phosphorylated Akt. We observed that treatment with 100 microM H2O2 for 24 h significantly decreased cell viability and induced apoptotic death of retinal neurons, and that pretreatment with 10 nM insulin significantly inhibited or attenuated H2O2-induced cytotoxicity and apoptosis. Pretreatment with LY294002, a specific PI3K inhibitor, abolished the cytoprotective effect of insulin. Insulin also strongly activated both PI3K and the downstream effector Akt. These results suggest that insulin protects retinal neurons from oxidative stress-induced apoptosis and that the PI3K/Akt signal pathway is involved in insulin-mediated retinal neuroprotection.

[Identification of up-regulated genes induced by angiotensin II in cardiac fibroblasts].

To identify up-regulated genes in adult rat cardiac fibroblasts (CF) induced by angiotensin II (Ang II), suppression subtractive hybridization (SSH) was performed between the CF stimulated by Ang II (tester) and unstimulated CF (driver) to generate subtractive cDNA library. The library was screened with dot blots hybridization to further verify the differentially expressed cDNA clones. Partial positive clones (19 up-regulated genes) were sequenced and BLAST analyzed. Twelve up-regulated genes related to extracellular matrix, cell cycle, intracellular signal transduction, cell cytoskeleton, cell metabolism and 7 new expressed sequence tags (EST) were acquired (GenBank accession number: CN382808, CN382809, CN382810, CN382811, CN382812, CN382813, CN382814). Our data reveal that SSH is a powerful technique of high sensitivity for the detection and cloning of up-regulated genes expressed in CF induced by Ang II, which may be helpful to clarify the mechanism of cardiac remodeling.

[Changes of endogenous ouabain in rat serum and certain tissues after exposure to simulated weightlessness].

OBJECTIVE: To investigate the changes of endogenous ouabain (EO) in rat serum and some tissues after exposure to simulated weightlessness and to investigate its possible pathophysiology. METHOD: Male Wistar rats were randomly divided into control group (Con) and 1 week tail-suspension group (TS). Enzyme-linked immunosorbent assay (ELISA) was used to detect the content of EO in serum, hypothalamus, pituitary, adrenal gland, kidney, heart and liver. RESULT: Compared with Con, EO increased significantly in serum, hypothalamus, adrenal gland and kidney after tail suspension (P<0.05). CONCLUSION: Simulated weightlessness induced changes of EO in serum and some tissues, which may have some effects on the regulation of hydro-electrolyte metabolism and cardiovascular functions.