[Comparison and analysis of lincRNAs expression profile in the hypothalamic-pituitary-ovarian axis of anestrous and estrous primiparous sows].

The normal estrus in weaned primiparous sows has a great impact on pig production and abnormal estrus is the main reason for the elimination of primiparous sows. In this study, we studied the long intergenic noncoding RNAs (lincRNAs) in the hypothalamic-pituitary-ovarian axis of anestrous and estrous primiparous sows. These long intergenic noncoding RNAs (lincRNAs) were screened and compared through RNA-seq analysis. The expression profiles of lincRNAs were obtained and their characteristics and functions were preliminarily analyzed. There are 3519 novel lincRNAs identified in the hypothalamic-pituitary-ovarian axis of anestrous and estrous primiparous sows. Compared with estrous primiparous sows, 17 differentially expressed lincRNAs were indentified, including 12 up-regulated lincRNAs and 5 down-regulated lincRNAs (FC≥2, P<0.05). The four lincRNA transcripts obtained through selection were verified by qRT-PCR, which are consistent with the RNA-seq results. The GO, KEGG pathway, and lincRNA-mRNA co-expression network analysis of these 17 lincRNAs revealed that these lincRNAs were mainly involved in reproductive activities, such as oocyte meiosis mature, ovarian cells differentiation and granulosa cells apoptosis. The results enriched the data resources of pig lincRNAs and provided useful information for further research about the reproductive performance of primiparous sows.

MicroRNA expression profile and functional analysis reveal their roles in contact inhibition and its disruption switch of rat vascular smooth muscle cells.

Contact inhibition and its disruption of vascular smooth muscle cells (VSMCs) are important cellular events in vascular diseases. But the underlying molecular mechanisms are unclear. In this study we investigated the roles of microRNAs (miRNAs) in the contact inhibition and its disruption of VSMCs and the molecular mechanisms involved. Rat VSMCs were seeded at 30% or 90% confluence. MiRNA expression profiles in contact-inhibited confluent VSMCs (90% confluence) and non-contact-inhibited low-density VSMCs (30% confluence) were determined. We found that multiple miRNAs were differentially expressed between the two groups. Among them, miR-145 was significantly increased in contact-inhibited VSMCs. Serum could disrupt the contact inhibition as shown by the elicited proliferation of confluent VSMCs. The contact inhibition disruption accompanied with a down-regulation of miR-145. Serum-induced contact inhibition disruption of VSMCs was blocked by overexpression of miR-145. Moreover, downregulation of miR-145 was sufficient to disrupt the contact inhibition of VSMCs. The downregulation of miR-145 in serum-induced contact inhibition disruption was related to the activation PI3-kinase/Akt pathway, which was blocked by the PI3-kinase inhibitor LY294002. KLF5, a target gene of miR-145, was identified to be involved in miR-145-mediated effect on VSMC contact inhibition disruption, as it could be inhibited by knockdown of KLF5. In summary, our results show that multiple miRNAs are differentially expressed in contact-inhibited VSMCs and in non-contact-inhibited VSMCs. Among them, miR-145 is a critical gene in contact inhibition and its disruption of VSMCs. PI3-kinase/Akt/miR-145/KLF5 is a critical signaling pathway in serum-induced contact inhibition disruption. Targeting of miRNAs related to the contact inhibition of VSMCs may represent a novel therapeutic approach for vascular diseases.

Knockdown of the HDAC1 promotes the directed differentiation of bone mesenchymal stem cells into cardiomyocytes.

Failure of the directed differentiation of the transplanted stem cells into cardiomyocytes is still a major challenge of cardiac regeneration therapy. Our recent study has demonstrated that the expression of histone deacetylase 1 (HDAC1) is decreased in bone mesenchymal stem cells (BMSCs) during their differentiation into cardiomyocytes. However, the potential roles of HDAC1 in cardiac cell differentiation of BMSCs, as well as the mechanisms involved are still unclear. In current study, the expression of HDAC1 in cultured rat BMSCs is knocked down by lentiviral vectors expressing HDAC1-RNAi. The directed differentiation of BMSCs into cardiomyocytes is evaluated by the expression levels of cardiomyocyte-related genes such as GATA-binding protein 4 (GATA-4), Nirenberg, Kim gene 2 homeobox 5 (Nkx2.5), cardiac troponin T (CTnT), myosin heavy chain (MHC), and connexin-43. Compared with that in control BMSCs, the expression of these cardiomyocyte-related genes is significantly increased in these HDAC1 deficient stem cells. The results suggest that HDAC1 is involved in the cardiomyocyte differentiation of BMSCs. Knockdown of the HDAC1 may promote the directed differentiation of BMSCs into cardiomyocytes.

Downregulation of HDAC1 is involved in the cardiomyocyte differentiation from mesenchymal stem cells in a myocardial microenvironment.

Under myocardial microenvironment, bone marrow-derived mesenchymal stem cells (MSCs) can transdifferentiate into cardiomyocytes (CMs). However, the role of histone deacetylase 1 (HDAC1) in this directed differentiation process remains unclear. The current study is to determine the acetylation regulatory mechanisms that may be involved in the directed CM differentiation from MSCs. MSCs isolated from male Sprague-Dawley (SD) rats were marked with Ad-EGFP and co-cultured with CMs. Flow cytometry was used to sort EGFP-positive (EGFP+) MSCs from the co-culture system. Then, the expression of cardiac troponin T (cTnT) in these MSCs was detected by immunofluorescence assay. In addition, HDAC1 levels at different co-culture times were measured by quantitative real-time polymerase chain reaction (QT-PCR) and Western blotting. At 4 days after co-culture with CMs, the MSCs began to expression detectable levels of cTnT. The expression of HDAC1 in CMs was much lower than that in MSCs. After co-culture with CMs, the expression of HDAC1 in MSCs was significantly decreased in a time dependent manner. In addition, our recent study has also identified that knockdown of the HDAC1 could promote the directed differentiation of MSCs into CMs. The results suggest that HDAC1 has a negative correlation with cardiac cell differentiation from MSCs under a myocardial microenvironment. HDAC1 might play an important role in the directed differentiation of MSCs into CMs in heart.

The effects of simvastatin on left ventricular hypertrophy and left ventricular function in patients with essential hypertension.

This study is to evaluate the effects of Simvastatin on left ventricular hypertrophy and left ventricular function in patients with essential hypertension. Untreated or noncompliance with drug treatment patients with simple essential hypertension were treated with a therapy on the basis of using Telmisartan to decrease blood pressure (BP). There were 237 patients who had essential hypertension combined with left ventricular hypertrophy as diagnosed by echocardiography, taken after their BPs were decreased to meet the values of the standard normal. Among them, there were only 41 out of the original 237 patients, 17.3%, who had simple essential hypertension combined with left ventricular hypertrophy without any other co-existing disease. They were the patients selected for this study. All patients were randomly, indiscriminately divided into two groups: one was the control group (Group T), treated with the Telmisartan-based monotherapy; the other was the target group (Group TS), treated with the Telmisartan-based plus simvastatin therapy. The changes of left ventricular hypertrophy and left ventricular function were rediagnosed by echocardiography after 1 year. The results we obtained from this study were as follows: (i) The average BPs at the beginning of the study, of simple essential hypertension combined with left ventricular hypertrophy, were high levels (systolic blood pressure (SBP) 189.21 ± 19.91 mm Hg, diastolic blood pressure 101.40 ± 16.92 mm Hg). (ii) The Telmisartan-based plus simvastatin therapy was significantly effective in lowering the SBP (128.26 ± 9.33 mm Hg vs. 139.22 ± 16.34 mm Hg). (iii) After the 1-year treatment, the parameters of left ventricular hypertrophy in both groups were improved. Compared to group T, there were no differences in the characteristics of the subjects, including interventricular septum, left ventricular mass, left ventricular mass index, ejection fraction, left atrium inner diameter at baseline. The patients' interventricular septum (Group TS 10.30 ± 1.80 mm vs. Group T 10.99 ± 1.68 mm, P < .05), LVM (Group TS 177.43 ± 65.40 g vs. Group T 181.28 ± 65.09 g, P < .05), and LVMI (Group TS 100.97 ± 37.33 g/m(2) vs. Group T 106.54 ± 27.95 g/m(2), P < .05), all dropped more prominently (P < .05) in group TS; the ejection fraction rose more remarkably in group TS (Group TS: 57.50 ± 16.41% to 65.43 ± 11.60%, P < .01 while showing no change in Group T); the left ventricular hypertrophy reversed more significantly and the left ventricular systolic function improved more. (iv) The left atrium inner diameter of Group TS decreased (P < .01), the ratio of E/A, which indicates the left ventricular diastolic function, continued to drop further, showing no change to the trend of left ventricular diastolic function declination. Patients who have hypertension with left ventricular hypertrophy usually suffer other accompanying diseases at the same time. Telmisartan-based plus Simvastatin treatment can significantly reduce SBP, reverse left ventricular hypertrophy, improve the left ventricular systolic function, but it has no effect on reversing the left ventricular diastolic function. This experiment indicated that Simvastatin can reverse left ventricular hypertrophy and improve left systolic function.

[Effects of simvastatin on vasa vasorum and aortic endothelial function in rats].

OBJECTIVE: To investigate the effect of hyperlipidemia on vasa vasorum and vascular endothelial growth factor (VEGF) and study the role of vasa vasorum in arteriosclerosis. METHODS: Thirty SD rats were randomized into normal control, hyperlipidemic and simvastatin treatment groups (n=10). In simvastatin group, hyperlipidemia was induced by a 4-week administration of atherogenic diet followed by a 16-week treatment with simvastatin at the daily dose of 10 mg/kg, and the rats in hyperlipidemic rats received no treatment. The changes in the aorta and vasa vasorum were examined, and serum lipid concentration and VEGF and NO levels were measured. RESULTS: Compared with the control group, the hyperlipidemic rats showed significantly thickened intima and media aorta and increased vasa vasorum density with lowered NO level, but VEGF underwent no significant changes. Simvastatin treatment significantly reduced the thickness of the intima and media aorta and increased vasa vasorum density in comparison with those in hyperlipidemic group. Simvastatin treatment also significantly increased VEGF and NO levels and a positive correlation was noted between their levels. CONCLUSION: Hyperlipidemia can impair the vasa vasorum and aortic endothelial function. Simvastatin increases VEGF and NO and promotes neogenesis of the vasa vasorum for the benefit of the aortic function.

[Leptin promotes neointimal formation by stimulating vascular smooth muscle cell proliferation through leptin receptor].

OBJECTIVE: To evaluate the role of leptin in neointimal formation and related mechanisms. METHODS: Femoral arterial injury was induced in wild-type (Wt, n = 10), leptin-deficient (Lep(-)/-, n = 12), and leptin receptor-deficient (LepR(-)/-, n = 10) mice. Leptin treatment studies (tail vein injection of adenovirus expressing murine leptin on the RSV promoter, ad-leptin) were performed on Lep(-)/- (n = 5) and LepR(-)/- (n = 4) mice. Intimal (I) and medial (M) areas were measured and the ratio of I/M was calculated. Smooth muscle cells were detected by smooth muscle alpha-actin staining using an alpha-actin monoclonal antibody. Cellular proliferation was analyzed with BrdU Staining Kit and the number of BrdU-positive cells was counted manually. Plasma leptin level was measured by ELISA. RESULTS: The I/M ratio of Lep(-)/- and LepR(-)/- mice was significantly lower than that in Wt separately (Lep(-)/- vs. Wt = 0.80 +/- 0.14 vs. 1.50 +/- 0.22, P < 0.01; LepR(-)/- vs. Wt = 0.55 +/- 0.20 vs. 1.50 +/- 0.22, P < 0.05). Plasma leptin level was significantly increased in Lep(-)/- and LepR(-)/- mice post leptin treatment. I/M was significantly increased in Lep(-)/- mice receiving ad-leptin compared with untreated Lep(-)/- mice (P < 0.05), while I/M was similar between LepR(-)/- mice with and without ad-leptin treatment (P > 0.05). The changes on number of positive alpha-actin and BrdU stained smooth muscle cells were consistent with the neointimal formation findings in various groups. CONCLUSIONS: Mice lacking leptin or the leptin receptor were protected from neointimal formation following vascular injury. Leptin treatment increased neointimal formation in Lep(-)/- but not in LepR(-)/- mice, suggesting leptin receptor activation and vascular smooth muscle cell proliferation played a pivotal role on neointimal formation post-injury in this model, giving an evidence that high plasma leptin level is a risk factor for neointimal formation.

[Factor V Leiden mutation leads to enhanced atherosclerosis in apolipoprotein E deficient mice].

OBJECTIVE: Factor V Leiden (FvL) causing activated protein C resistance is a genetic risk factor for venous thrombosis in humans, and it's effect on atherosclerosis is controversial. We evaluated the effect of FvL mutation on atherosclerosis in apolipoprotein E deficient mice fed with normal diet. METHODS: Degree of atherosclerosis and tissue fibrin deposition were determined in Fv+/+ApoE-/-, FvQ/+ApoE-/- and FvQ/QApoE-/- mice. RESULTS: In the presence of ApoE deficiency, homozygous FvL significantly increased atherosclerosis coverage in ApoE-/- mice (FvQ/QApoE-/- vs. Fv+/+ApoE-/-=5.0%+/-1.1% vs. 2.2%+/-0.4%, P<0.005) and tissue fibrin deposition in atherosclerotic lesion (FvQ/QApoE-/- vs. Fv+/+ApoE-/-=3.4% +/- 0.5% vs. 1.8%+/-0.4%, P<0.05). The atherosclerotic lesion of FvQ/+ApoE-/- mice was intermediate between FvQ/Q ApoE-/- and Fv+/+ApoE-/-, and there was no significant difference comparing with any of them. CONCLUSIONS: These observations demonstrate that homozygous FvL could promote atherosclerosis and fibrin deposition in apolipoprotein E deficient mice suggesting that Factor V mutation could be an important genetic risk factor for the enhanced atherosclerosis in human.

[Triptolide-eluting stent prevents porcine coronary artery in-stent restenosis by affecting PCNA and P27(kip1) expression].

OBJECTIVE: To investigate the effectiveness and safety of triptolide-eluting stents implanted in porcine coronary arteries for restenosis prevention, and its effect on the expression of proliferating cell nuclear antigen (PCNA) and P27(kip1). METHODS: Ten triptolide-eluting stents and 10 stainless steel stents (control) were implanted in 20 porcine coronary arteries at random. Four weeks later, angiography of the arteries was performed along with also histopathological and immunochemical examinations. RESULTS: The in-stent minimal lumen diameter of triptolide group was significantly greater, and the neointimal area significantly smaller, than those of the control group (P<0.05). PCNA expression was significantly lower while P27(kip1) protein significantly higher in triptolide group than in the control group (P<0.05). CONCLUSION: Triptolide-eluting stent can effectively inhibit neointimal formation to prevent restenosis in porcine coronary artery 4 weeks after implantation, probably by inhibiting P27(kip1) expression and consequently vascular smooth muscle cell proliferation.

[Isolation, in vitro culture and identification of cardiac stem cells from neonatal SD rats].

OBJECTIVE: To isolate and culture cardiac stem cells (CSCs) in vitro and evaluate their potential of differentiation into functional cardiac myocytes. METHODS: Myocardial tissues obtained from neonatal SD rats were cut into pieces of 0.5-1.0 mm(3), and digested twice for 5 min at 37 degrees C; with 0.2% trypsin and 0.1% collagenase II. The remaining tissues were cultured in complete explant culture medium (CEM) at 37 degrees C; in the presence of 5% CO(2). About a week later, a layer of fibroblast-like cells was generated from the adherent explants. These cells were passaged and seeded at about 1x10(6) cells/ml in poly-D-lysine-coated multi-well plates in cardiosphere-growing medium. When beating of the cultured cells was observed (at week 2), flow cytometry and immunohistochemistry were performed for identification of the primary and passaged cells. RESULTS: The primary cells were successfully cultured from the digested myocardial tissue, and flow cytometry demonstrated the phenotype of c-kit(+)CD31(+)CD34(-)CD45(-)CTnT(-). After cell passage for about two weeks, single beating cells and cell clusters with synchronized contraction were seen microscopically, and their phenotype was converted to c-kit(+)CD31(-)CD34(-)CD45(-) CTnT(+). Immunohistochemistry staining identified CTnT expression in the passaged cells but not in the primary cells. CONCLUSIONS: A cell population with the phenotype c-kit(+)CD31(+)CD34(-)CD45(-)CTnT(-) has been obtained from neonatal SD rat heart, which possesses the potential to differentiate in vitro into beating cardiac myocytes and express CTnT protein.