[Specification for genetic diagnosis of congenital heart disease].

Congenital heart disease (CHD) is one of the most common congenital malformations and a major cause of mortality among neonates and children. Conventional methods for the diagnosis of CHD have relied on clinical features and imaging findings. With the rapid development of genetic techniques, to identify the cause of CHD through genetic diagnosis has gained great significance for the early diagnosis, treatment, and prevention of CHD. However, currently there is still a lack of norms and standards for the genetic diagnosis of CHD. In view of this, experts from the relevant fields have formulated the present norm by integrating the latest research advances on CHD-related genes with the current clinical practice on the diagnosis and treatment of CHD and status quo of genetic diagnosis in China. The norm has been recommended by the Cardiology Section of the Chinese Medical Education Association, the Medical Genetics Branch and the Heart Group of Pediatric Surgery Branch of the Chinese Medical Association, which has formulated the procedures and norms of genetic testing, prenatal diagnosis, and genetic counseling for CHD, with an aim to provide reference for clinicians as the standards for the integrated diagnosis, early treatment, and prevention of CHD.

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 (L­DOPA) is an amino acid, converted by the enzyme aromatic L­amino acid decarboxylase to dopamine. The uptake of L­DOPA by cells of the proximal tubular epithelium of the kidney is controlled by the L­type 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 second­order mesenteric arteries and kidneys of spontaneously hypertensive rats. The present study aimed to investigate the physiological role of the SLC7A8 gene in L­DOPA handling by kidney cells. Selective upregulation of SLC7A8 mRNA and protein levels was achieved by adenoviral transduction of NRK­52E cells, which retain several properties of proximal tubular epithelial cells. In addition, L­DOPA uptake was determined using high performance liquid chromatography; NRK­52E cells expressing SLC7A8 exhibited increased uptake of L­DOPA. The results of the present study suggested that SLC7A8 may serve a critical role in blood pressure control through regulating L­DOPA 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.

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)-ß, 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.

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.

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

OBJECTIVES: Retinal blood vessels may develop vasculopathy and apoptosis in response to hypertension. The present study was aimed at testing the role of losartan, a specific antagonist of angiotensin II receptor type 1 receptor in regulation of vascular apoptosis in retinal vasculature with 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. RESULTS: Losartan treatment for 4-8 weeks reduced blood pressure of SHR to the normal levels seen in WKY. The losartan-treated SHR showed marked improvement of retinal vascular morphology compared with untreated SHR. The retinal blood networks of the treated SHR developed lower degrees of vasculopathy and apoptosis. TUNEL and transmission electron microscopy also revealed that losartan exerted its protective effects not only on endothelial cells but on pericytes as well. The blood vessels of losartan-treated animals also showed decreased expression of bax with elevation of B-cell CLL/lymphoma 2. CONCLUSION: Treatment with losartan, a medicine that lowers blood pressure by blocking angiotensin II receptor type 1 receptor, can protect the retinal vasculature against hypertensive vascular injury by inhibiting apoptosis of vascular cells and by preventing hypertensive retinopathy.

[Isolation, culture and identification of two types of endothelial progenitor cells from peripheral blood in rabbits].

AIM: To investigate how to isolate, culture and identify two types of endothelial progenitor cells from peripheral blood in rabbits. METHODS: Mononuclear cells(MNCs) were isolated from rabbit peripheral blood. Endothelial progenitor cells (EPCs) and endothelial outgrowth cells (EOCs) were obtained from MNCs through different ways of isolation and culture. Two types of cells were assessed by DiI-ac-LDL uptake and lectin binding, and then they were identified by immunofluorescence of flk-1, immunocytochemistry of CD34 and VIII factor related antigen and vasculogenesis activity in vitro. RESULTS: Two types of endothelial progenitor cells were obtained from rabbit peripheral blood through different ways of isolation and culture. EPCs on the seventh day and EOCs on the sixteenth day were positive for ac-LDL uptake and lectin binding, and both of them expressed CD34, flk-1 and VIII factor related antigen. EOCs were assembled into primitive vascular tube-like structures when plated in matrigel. CONCLUSION: EPCs and EOCs could be obtained from rabbit peripheral blood when different ways of isolation and culture were performed. The system of cell culture can be applied to subsequent experiments in cell transplantation.

[Effects of endothelial progenitor cells and endothelial outgrowth cells in repair of injured carotid vessel: a comparative study with rabbits].

OBJECTIVE: To compare the effects of endothelial progenitor cells (EPCs) and endothelial outgrowth cells (EOCs) on the repair of injured vessels. METHODS: Mononuclear cells (MNCs) were isolated from rabbit peripheral blood by density-gradient centrifugation. EPCs and EOCs were obtained from the culture of MNCs and labeled with the cell dye CM-DiI for cells tracking. Eighteen rabbits were made into models of balloon-injured common carotid artery and then divided into 2 equal groups to undergo injection of the suspensions of EPCs or EOCs. Nine rabbits underwent injection of normal saline as control group. Four weeks after transplantation, the rabbits underwent venous injection of Evans blue, and then were killed with the injured vessels taken out. Fluorescence-labeled both types of cells, endothelial regeneration rate and IA/MA ratio were detected. RESULTS: Four weeks after transplantation, fluorescence-labeled EPCs and EOCs were detected within the tunica intima, mostly in the neointima and on the luminal surface of injured vessel. The endothelialization area of denuded vessel of the EPC and EOC groups were 91.6% +/- 3.6% and 89.1% +/- 6.3% respectively, both significantly larger than that of the control group (62.1% +/- 7.5%, both P < 0.01), however, without significant difference between the 2 former groups (P = 0.50). The intima area/media area ratio of the EPC and EOC groups were 0.48 +/- 0.11 and 0.44 +/- 0.06, both significantly lower than that of the control group (0.88 +/- 0.14, both P < 0.01), however, without significant difference between the 2 former groups (P = 0.59). CONCLUSION: Transplantation of both EPCs and EPCs accelerate the reendothelialization and reduce the neointimal formation with similar effects.