Protective Effects of MicroRNA-126 on Human Cardiac Microvascular Endothelial Cells Against Hypoxia/Reoxygenation-Induced Injury and Inflammatory Response by Activating PI3K/Akt/eNOS Signaling Pathway.

OBJECTIVE: This study explored the protective effects of the microRNA-126 (miR-126)-mediated PI3K/Akt/eNOS signaling pathway on human cardiac microvascular endothelial cells (HCMECs) against hypoxia/reoxygenation (H/R)-induced injury and the inflammatory response. METHODS: Untreated HCMECs were selected for the control group. After H/R treatment and cell transfection, the HCMECs were assigned to the H/R, miR-126 mimic, mimic-negative control (NC), miR-126 inhibitor, inhibitor-NC, wortmannin (an inhibitor of PI3K) and miR-126 mimic + wortmannin groups. Super oxide dismutase (SOD), nitric oxide (NO), vascular endothelial growth factor (VEGF) and reactive oxygen species (ROS) were measured utilizing commercial kits. Quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were performed to detect miR-126 expression and the mRNA and protein expression of inflammatory factors. Western blotting was used to determine the expression of key members in the PI3K/Akt/eNOS signaling pathway. ACCK-8 assay and flow cytometry were employed to examine cell proliferation and apoptosis, respectively. The angiogenic ability in each group was detected by the lumen formation test. RESULTS: Compared to the control group, p/t-PI3K, p/t-Akt and p/t-eNOS expression, NO, VEGF and SOD levels, cell proliferation and in vitro lumen formation ability were decreased, while the ROS content, interleukin (IL)-6, IL-10 and tumor necrosis factor (TNF)-α expression and cell apoptosis were significantly increased in the H/R, mimic-NC, miR-126 inhibitor, inhibitor-NC, wortmannin and miR-126 mimic + wortmannin groups. Additionally, in comparison with the H/R group, the miR-126 mimic group had elevated p/t-PI3K, p/t-Akt and p/t-eNOS expression, increased NO, VEGF and SOD contents, and strengthened cell proliferation and lumen formation abilities but also exhibited decreased ROS content, reduced IL-6, IL-10 and TNF-α expressions, and weakened cell apoptosis, while the miR-126 inhibitor and wortmannin group exhibited the opposite results. Furthermore, decreased p/t-PI3K, p/t-Akt and p/t-eNOS expressions, decreased NO, VEGF and SOD contents, cell proliferation and lumen formation abilities, as well as increased ROS content, increased IL-6, IL-10 and TNF-α expression, and increased cell apoptosis were observed in the miR-126 mimic + wortmannin group compared to themiR-126 mimic group. CONCLUSIONS: These findings indicated that miR-126 protects HCMECs from H/R-induced injury and inflammatory response by activating the PI3K/Akt/ eNOS signaling pathway.

Antagonistic effects of endostatin-vascular endothelial growth inhibitor chimeric recombinant adenovirus on homocysteine-induced vascular endothelial cells injury in vitro and in vivo.

BACKGROUND: The study is inclined to investigate the antagonistic effects of endostatin-vascular endothelial growth inhibitor chimeric recombinant adenovirus (Ad-hENDO-VEGI) on homocysteine (Hcy)-induced vascular endothelial cells (VECs) injury in vitro and in vivo. METHODS: Human VECs cell line ECV304 was selected and infected with Ad-hENDO-VEGI. The LDH leakage, SOD activity, and MDA levels were measured by the automatic biochemical analyzer. Cell survival rate was counted by Trypanblau dying. The TNF-α and MCP-1 protein expressions were detected by ELISA assay. The protein expressions of fusion protein of Ad-hENDO-VEGI, nuclear factor kappa B p65 (NF-kappa B p65), and NF-kappa B inhibitor alpha (I-kappa B-α) were detected by Western blotting. A rat model of hyper-homocysteinemia was constructed. Thirty-six Wistar rats were randomly divided into 3 groups: the control group, the model group, and the Ad-hENDO-VEGI group. Serum Hcy levels in rats were measured with enzymatic cycling method. Endothelial vasodilation function was evaluated with the treatment of sodium nitroprusside and acetylcholine. RESULTS: After Ad-hENDO-VEGI infection, high expressions (41 kD) of fusion proteins in ECV304 cells were observed. The injury severity of Hcy on ECV304 cells had a dose-dependent manner, and the injury reached a steady stage at 1.0 mmol/L. Thus, 1.0 mmol/L Hcy was selected for further experiments. With an increase of Ad-hENDO-VEGI in ECV304 cells after Hcy treatment, LDH leakage, MDA, TNF-α, MCP-1, and nuclear NF-kappa B p65 protein expression were gradually decreased, and cell survival rate, SOD activity, and I-kappa B-α protein expression were gradually increased. Compared with the control group, the model group had a higher Hcy level and attenuated vasodilator response. The Ad-hENDO-VEGI group exhibited a lower Hcy level and enhanced vasodilator response than the model group. CONCLUSION: These results indicated that Ad-hENDO-VEGI could down-regulate NF-kappa B p65 expression and suppress inflammatory response, thereby alleviating Hcy-induced VECs injury.

The effects of tanshinone IIA on hypoxia/reoxygenation-induced myocardial microvascular endothelial cell apoptosis in rats via the JAK2/STAT3 signaling pathway.

OBJECTIVE: This study aims to investigate the effects of tanshinone IIA on hypoxia/reoxygenation (H/R)-induced myocardial microvascular endothelial cell (MMEC) apoptosis in rats. METHODS: MMECs from 10-days aged rats were isolated, cultured and identified, which were then divided into following groups: control group, control+tanshinone IIA (50µM) group, H/R model group, H/R+tanshinone IIA (5µM) group, H/R+tanshinone IIA (50µM) pre-treatment group, H/R+AG490 (50µM) pre-treatment group and H/R+AG490 (50µM)+tanshinone IIA (50µM) pre-treatment group. MTT assay, TUNEL staining and flow cytometry were used to measure the cellular viability and apoptosis. Western-blot were performed to detect protein expressions in JAK2/STAT3 signaling pathway. RESULTS: Compared with control group, H/R group showed decreased cell viability, increased apoptosis rate, increased proportions of cells into G0/G1 phase, decreased proportions of cells in S phase and G2/M phase, as well as up-regulated expressions of JAK2, STAT3, p53, Bax, Caspase-3, pJAK2 and pSTAT3, and down-regulated Bcl-2 expression (all P<0.05). Compared with H/R group, H/R+tanshinone IIA (5µM) group, H/R+tanshinone IIA (50µM) group H/R+AG490 (50µM) group and H/R+AG490 (50µM)+tanshinone IIA (50µM) group had increased cell viability, decreased apoptosis rate, reduced proportions of cells into G0/G1 phase, elevated proportions of cells in S phase and G2/M phase, as well as down-regulated expressions of JAK2, STAT3, p53, Bax, Caspase-3, pJAK2 and pSTAT3, elevated expression of Bcl-2 (all P<0.05). The most remarkable changes were observed in H/R+AG490 (50µM)+tanshinone IIA (50µM) group. CONCLUSION: Tanshinone IIA may attenuate H/R-induced MMEC apoptosis in rats by inhibiting the JAK2/STAT3 signaling pathway and regulating the expressions of p53, Bax, Caspase-3 and Bcl-2, which may provide a protective effect of tanshinone IIA for MMECs.

[Effect of pretreatment with qishen yiqi dropping pills on right cardiac function of patients undergoing valve replacement].

In this study, 120 patients with rheumatic heart disease undergoing valve replacement were randomly divided into the control group and the Qishen group, with 60 cases in each group. Before the operation, the control group was given routine heart and diuretic treatments and placebo of Qishen Yiqi dropping pills for seven days (0.5 g each time, three times a day); While the Qishen group was given Qishen Yiqi dropping pills for seven days (0.5 g each time after meal, three times a day) on the basis of the routine treatments. The right ventricular end-diastolic volume (RVEDV), end-systolic volume (RVESV), stroke volume (SV) and right ventricular ejection fraction (RVEF) were detected after the operation. The results showed that patients in the two groups showed significantly lower right ventricular end diastolic volume (RVEDV), right ventricular end systolic volume (RVESV) and stroke volume (SV) decreased than that before the operation, but with significantly higher Ejection fraction (RVEF) significantly than that before the operation. However, the Qishen group showed a significantly lower right heart function reduction than the control group, with the statistical significance in the differences (P < 0.05). This indicated that the pretreatment with Qishenyiqi Drop Pills showed a remarkable efficacy in the improvement of right ventricular function after valve replacement.

[Surgical treatment of congenital heart diseases in pediatric patients with body weight less than 10 kg].

OBJECTIVE: To report the experience of the surgical treatment of congenital heart diseases (CHD) in pediatric patients with body weight less than 10 kg. METHODS: Between January 2000 and December 2007, 105 children with CHD, aged 2 months to 3 years and weighing between 3.5 to 10 kg, underwent surgical treatment. Of the 105 patients, 56 were concomitant with moderate to severe pulmonary hypertension or repeated pulmonary infections, and 35 with complex cardiac abnormalities. Operations were performed through median sternotomy with moderate hypothermic cardiopulmonary bypass in 88 cases, with normothermic extracorporeal circulation on beating heart in 14 cases, and with deep hypothermic circulatory arrest in 3 cases. RESULTS: There were 5 early deaths (4.8%). During a follow-up of 2 month to 3 year, 97 survivors with corrective procedure had no late mortality or complications with NYHA class I of cardiac function, excepting 2 cases with little residual shunt. Three survivors with palliative procedure enjoyed higher quality of life. CONCLUSIONS: The surgical treatment of CHD in pediatric patients with body weight less than 10 kg seems to be feasible and safe, with satisfactory early and long-term results.