Deye Yang

Expression of microRNA-122 contributes to apoptosis in H9C2 myocytes.

The microRNAs (miRNAs) can post‐transcriptionally regulate gene expression and heart development. The Pax‐8 gene knockout mice have apparent heart abnormalities. This study investigated the role of miRNAs in regulation of cardiac apoptosis and development in the knockout mice. MicroRNA microarrays demonstrated differential expression of microRNAs between Pax‐8−/− and Pax‐8+/− mice, confirmed by real‐time PCR. The miR‐122 was up‐regulated by 1.92 folds in Pax‐8−/− mice. There were ventricular septum defects in Pax‐8−/− mice, and increased numbers of apoptotic cells in the left ventricular wall and interventricular septum in Pax‐8−/− mice. In H9C2 myocytes, treatment with miR‐122 mimics or miR‐122 inhibitor affects the expression of CCK‐8 and activity of Caspase‐3. The miR‐122 is up‐regulated in the myocytes of Pax‐8−/− mice and may participate in the apoptotic gene expression and pathogenesis of heart development defect.

Role of microRNA-29b in angiotensin II-induced epithelial-mesenchymal transition in renal tubular epithelial cells

Angiotensin II (Ang II) has been proven to induce epithelial-mesenchymal transition (EMT). The aim of the present study was to determine the role of microRNA-29b (miR-29b) during Ang II-induced EMT. For this purpose, we used spontaneously hypertensive rats (SHRs) and age-matched Wistar-Kyoto (WKY) rats. The levels of Ang II and its receptor in the kidneys of the SHRs are significantly higher than those in the age-matched WKY rats. As shown by RT-qPCR, the expression of miR-29b in the renal cortex was lower in the SHRs than in the WKY rats. For in vitro experiments, NRK-52E renal tubular epithelial cells were treated with 10(-7) M Ang II; we found that the expression of miR-29b was decreased in the cells treated with Ang II. In addition, transfection of the NRK-52E cells with miR-29b inhibitor led to the downregulation of miR-29b in these cells, and increased the expression of transforming growth factor (TGF)-β, α-smooth muscle actin (α-SMA) and collagen I (Col I). Similar results were observed with the induction of Ang II expression in the NRK-52E cells. By contrast, the upregulation of miR-29b by transfection with miR-29b mimics inhibited the overexpression of these genes induced by Ang II. These results suggest that miR-29b plays an important role in Ang II-induced EMT.

The defects in development and apoptosis of cardiomyocytes in mice lacking the transcriptional factor Pax-8

BACKGROUND Cardiac-specific deletion of ALK3 is lethal in mid-gestation with ventricular septum malformations (VSM). This study was designed to define the Pax-8s role in heart development and cardiomyocyte apoptosis. METHODS Pathologic changes in the hearts of Pax-8 or ALK3 knockout and wild type control mice were determined by light and electron microscopy. Analysis of cardiomyocyte apoptosis was performed by TUNEL. The effect of Pax-8 gene deficiency on caspase-3 activity was examined after transfecting Pax-8 siRNA into cultured myoblast cell line. RESULTS Mice with ALK3 or Pax-8 gene knockout but not wild type control animals showed the development of VSM. Increased cardiomyocyte apoptosis was found in homozygotes. Echocardiography showed that Pax-8 homozygote mice developed malfunction of the heart. Furthermore, the caspase-3 activity was significantly higher in the cells treated with Pax-8 siRNA as compared to those treated with negative control siRNA in H9C2 (2-1) cell line. CONCLUSIONS The Pax-8 gene may play a crucial role in heart development and regulating cardiocyte apoptosis. Knockout of Pax-8 may exert a similar effect on myocardial morphology and apoptosis as those seen in ALK3 knockouts. Furthermore, the ventricular septum malformations could be partially attributed to accelerated cardiomyocyte apoptosis.

BMPR IA downstream genes related to VSD.

Cardiac-specific deletion of the receptor IA of bone morphogenetic protein (BMP) (ALK3) by Cre recombinase driven under the α-MHC promoter is lethal in mid-gestation with defects in the interventricular septum [ventricular septum defect (VSD)]. Analysis of expression of the ALK3 downstream genes is important to identify the signaling pathway for interventricular septum development. The mRNA expression level of a control group was compared with that of a test group. ALK3 downstream genes were screened using polymerase chain reaction (PCR)-select cDNA subtraction and microarray. It was found that the mice with an ALK3 knockout gene produced a VSD. The expression of some genes such as platelet-activating factor acetylhydrolase (PAF) and Pax-8 was down-regulated in the test group. Pax-8 gene expression was down-regulated by 7.1 times in the test group and expressed specifically in the 11.5-d embryonic (E11.5) heart. Furthermore, the expression of the protein-tyrosine kinase of the focal adhesion kinase subfamily (PTK) and β subtype protein 14-3-3 was up-regulated in the test group. PTK gene expression was up-regulated by 3.7 times in the test group. These data provided support that the ALK3 gene plays an important role during heart development. The PAF and Pax-8 genes could be important ALK3 downstream genes in the BMP signaling pathway during interventricular septum development. PTK and β subtype protein 14-3-3 might be regulatory factors in this pathway.

Reduced expression of FXYD domain containing ion transport regulator 5 in association with hypertension

Experimental evidence indicates that hypertension is a multifactorial disorder and that the products of several genes may contribute to its development. The aim of this study was to investigate the expression of hypertension-related genes in spontaneous hypertensive rats (SHRs). A microarray screening for hypertension-related genes was conducted in SHRs and Wistar-Kyoto (WKY) rats using total-RNA extracted from second-order mesenteric arteries and kidneys. The FXYD5 mRNA expression in vascular smooth muscle cells (VSMCs) was silenced by RNA interference (RNAi). Meanwhile, the FXYD5 mRNA overexpression in renal tubular epithelial cells (RTECs) was induced by the recombinant plasmid pcDNA3.1(+)-FXYD5. The expression of FXYD5 mRNA was found to be 14.8-fold lower in SHR rats compared to that in WKY rats (P<0.01). The levels of FXYD5 mRNA expression were the highest in kidneys of SHR 13-week-old rats when the blood pressure reached the highest levels. The down-regulated FXYD5 mRNA expression inhibited the migration of smooth muscle cells (P<0.01) and cell membrane Na⁺-K⁺-ATPase activity (P<0.01). Up-regulated FXYD5 mRNA expression enhanced the renal tubular epithelial cell membrane Na⁺-K⁺-ATPase activity (P<0.05) and cell proliferation (P<0.05). FXYD5 is related to the migration of smooth muscle cells and cell membrane Na⁺-K⁺-ATPase activity in rodents. The results of the present study suggest that FXYD5 may have profound impact on the regulation of blood pressure, and that this gene may be a potential target for anti-hypertensive therapy.

Increased SLC7A8 expression mediates L-DOPA uptake by renal tubular epithelial cells

The kidney serves a central role in the control of blood pressure through the release of vasoactive substances and the urinary excretion of Na+. Patients with essential hypertension usually exhibit persistent high blood pressure accompanied by Na+ retention. L-dihydroxyphenylalanine (L‑DOPA) is an amino acid, converted by the enzyme aromatic L‑amino acid decarboxylase to dopamine. The uptake of L‑DOPA by cells of the proximal tubular epithelium of the kidney is controlled by the L‑type amino acid transporter 2 (LAT2). LAT2 belongs to the solute carrier family 7 (SLC7) of amino acid transporters and is coded by the SLC7A8 gene. SLC7A8 expression is increased in the second‑order mesenteric arteries and kidneys of spontaneously hypertensive rats. The present study aimed to investigate the physiological role of the SLC7A8 gene in L‑DOPA handling by kidney cells. Selective upregulation of SLC7A8 mRNA and protein levels was achieved by adenoviral transduction of NRK‑52E cells, which retain several properties of proximal tubular epithelial cells. In addition, L‑DOPA uptake was determined using high performance liquid chromatography; NRK‑52E cells expressing SLC7A8 exhibited increased uptake of L‑DOPA. The results of the present study suggested that SLC7A8 may serve a critical role in blood pressure control through regulating L‑DOPA uptake in renal epithelial cells of the proximal tubule.

Pax8 plays a pivotal role in regulation of cardiomyocyte growth and senescence

Congenital heart disease (CHD) is a worldwide health problem, particularly in young populations. In spite of the advancement and progress in medical research and technology, the underlying causative factors and mechanisms of CHD still remain unclear. Bone morphogenetic protein receptor IA (ALK3) mediates the development of ventricular septal defect (VSD). We have recently found that paired box gene 8 (Pax8) may be the downstream molecule of ALK3. Paired box gene 8 plays an essential role in VSD, and apoptosis and proliferation imbalance leads to septal dysplasia. Recent studies have also disclosed that cellular senescence also participates in embryonic development. Whether programmed senescence exists in cardiac organogenesis has not ever been reported. We hypothesized that together with various biological processes, such as apoptosis, enhanced cellular senescence may occur actively in the development of Pax8 null mice murine hearts. In H9C2 myogenic cells, Pax8 overexpression can rescue caspase‐dependent apoptosis induced by ALK3 silencing. Senescent cells and senescence‐associated mediators in Pax8 knockout hearts increased compared with the wild‐type ones in an age‐dependent manner. These results suggest that Pax8 maybe the downstream molecule of ALK3, it mediates the murine heart development perhaps via cellular senescence, which may serve as a mechanism that compensates for the cell loss via apoptosis in heart development.

GW24-e0815 The role of microRNA-29b in renal fibrosis induced by angiotensin II

Objectives Primary study have demonstrated that the Angiotensin II (Ang II) contents and the its receptor density in the kidney of young spontaneously hypertensive rats (SHR) were significantly higher than age-matched Wistar-Kyoto rats (WKY). Ang II has been shown to induce renal fibrosis, including Epithelial-Mesenchymal Transition (EMT) and tubulointerstitial fibrosis. This study investigated the role of microRNAs (miRNAs) in renal fibrosis induced by Ang II. Methods Differential expression of miRNAs in renal cortex between young SHR and age-matched WKY was demonstrated by miRNA microarray, and confirmed by real-time PCR. Results The miR-29b was down-regulated by 48% in SHR. In NRK-52E renal tubular epithelial cells and NRK-49F renal interstitial fibroblasts, treatment withAng II reduced the expression of miR-29b. Down-regulation of miR-29b by transfection of miR-29b inhibitor significantly increased the expression of transforming growth factor (TGF-β), α-smooth muscle actin (α-SMA), Collagen (Col ) of NRK-52E cells and the expression of TGF-β, Col and matrix metalloproteinase-2 (MMP-2) of NRK-49F cells. Treatment of 10-7M Ang II caused similar result, while up-regulation of miR-29b by transfection of miR-29b mimics 24 hrs before Treatment of Ang II resulted in obvious down-regulation of these genes. Conclusions We conclude that miR-29b is down-regulated in renal cortex of young SHR and may participate in renal fibrosis induced by Ang II.

Reduced expression of FXYD domain containing ion transport regulator 5 (FXYD5) in hypertension

l-nitroarginine methyl ester (L-NAME, 40 mg/kg, i.p.) significantly inhibited post exercise hypotension (25±11 and 5±3mm Hg, respectively; P<0.05). In addition, the superoxide anion generation was decreased, while the plasma nitrite production and serine phosphorylation of endothelial nitric oxide synthase were significantly elevated in spontaneously hypertensive rats at 30 min after the termination of exercise. Taken together, these data demonstrate that the increased phosphorylation of endothelial nitric oxide synthase plays a crucial role in the reduction of arterial pressure following a single bout of dynamic exercise in spontaneously hypertensive rats.

Protection of retinal vasculature by losartan against apoptosis and vasculopathy in rats with spontaneous hypertension

Methods Losartan potassium was administered to spontaneously hypertensive rats (SHR). Blood pressure was measured in SHR as well as normotensive Wistar– Kyoto rats (WKY). Eye fundus was examined in living animals and then tissue specimens were collected for histochemistry by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’triphosphate nick end labeling (TUNEL), immunohistochemistry and transmission electron microscopy.