Overexpression of UHRF1 promoted the proliferation of vascular smooth cells via the regulation of Geminin protein levels.

Geminin is an inhibitor of DNA replication licensing and cell cycle. Our previous study demonstrates that Geminin plays an important role in regulating phenotypic diversity and growth of vascular smooth cells (VSMCs). Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1) is an epigenetic coordinator, whose RING domain confers intrinsic E3 ligase activity, mediating the ubiquitination of several proteins and the protein-protein interaction. Aberrant expression of UHRF1 was related to aggressiveness of multiple human malignancies, where knockdown of UHRF1 led to decreased proliferation of cancer cells. However, it is unclear whether proper UHRF1 function is involved in aberrant proliferation and phenotypic switching of VSMCs via altering Geminin protein levels. In present study, in UHRF1-overexpressing A10 cells, 3H-thymidine and 5-ethynyl-20-deoxyuridine (EdU) and CCK8 were used to examine the proliferation of VSMCs. RT-PCR and Western blot analyses were performed to investigate whether UHRF1-mediated effects were achieved by altering Geminin expression in VSMCs. RNA-seq analysis was performed to dissect related mechanisms or signaling pathways of these effects. The results of in vitro experiments suggested that UHRF1 prompted proliferation and cell cycle of VSMCs via the down-regulation of Geminin protein levels with no change in Geminin mRNA expression. Besides, PI3K-Akt signaling pathway was increased upon UHRF1 up-regulation. Our study demonstrated that overexpressing UHRF1 was involved in VSMCs proliferation through reducing inhibitory Geminin protein levels to promote cell cycle as well as activating PI3K-Akt signaling. This may provide key knowledge for the development of better strategies to prevent diseases related to VSMCs abnormal proliferation.

Different effects of neuropeptide Y on proliferation of vascular smooth muscle cells via regulation of Geminin.

The proliferation-promoting effect of neuropeptide Y (NPY) always functions in low-serum-cultured vascular smooth muscle cells (VSMCs), and the phenotypic switch of VSMCs is regulated by concentrations of serum. Whether the property of the NPY proliferative effect in VSMCs relies on phenotype of VSMCs is unclear. We aimed to explore the role of NPY on proliferation of different VSMC phenotypes in the pathogenesis of atherosclerosis. By stimulating A10 cells with 200 nM NPY in 0.5 or 10% serum, 3H-thymidine and 5-ethynyl-2'-deoxyuridine (EdU) and CCK8 measurements were used to detect VSMC proliferation. RT-PCR and Flow cytometry were performed to detect the factors involved in different properties of the NPY proliferative effect in VSMCs. Instead of facilitating proliferation, NPY had no significant effect on the growth of VSMCs when cultured in 10% serum (VSMCs stayed at synthetic states). The underlying mechanism may be involved in down-regulation of Y1 receptor (P < 0.05 vs. Vehicle) and up-regulation of Geminin (P < 0.05 vs. Vehicle) in 10% serum-cultured VSMCs co-incubated with 200 nM NPY. Besides, modulation of Geminin was effectively blocked by the Y1 receptor antagonist. The stimulation of NPY on proliferation of VSMCs could be a double-edged sword in the development of atherosclerosis and thus provides new knowledge for therapy of atherosclerosis.

Clinical efficiency and safety analysis of transcatheter closure of multiple atrial septal defects in adults.

BACKGROUND: Transcatheter closure of atrial septal defects (ASDs) is currently a reliable alternative to surgery, even though challenging in patients with multiple ASDs. HYPOTHESIS: The aim of this study was to evaluate the clinical efficiency and safety of transcatheter closure in multiple ASDs. METHODS: Multiple ASDs were diagnosed by transthoracic echocardiography (TTE) or transesophageal echocardiography (TEE). The occlusive condition and distance between 2 adjacent ASDs were measured by TTE examination. Then, the number and size of the occluder(s) was determined. TTE examinations were performed after transcatheter closure as follow-up. RESULTS: The transcatheter procedure was successful in 15 patients with multiple ASDs, using a single occluder in 9 patients and 2 occluders in the remaining 6 patients. Overall, 21 ASD occluders were implanted. During a follow-up period of 6 mo to 5 y, a slight residual shunt was found in 1 patient without any symptoms; a moderate residual shunt was identified at the inferior vena cava and the occluder was removed by surgery 1 mo after procedure. Other complications, including endocarditis, arrhythmia, thromboembolism, and atrioventricular valve damage were not recorded in any of the 15 patients during the follow-up period. CONCLUSION: Transcatheter closure of multiple ASDs is safe and efficient. Two occluders are necessary for the distance of 2 ASDs more than 7 mm, and a single occluder is sufficient for those 7 mm or less.

RNA interference targeting ORC1 gene suppresses the proliferation of vascular smooth muscle cells in rats.

BACKGROUND: The proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the pathogenesis of vascular diseases such as atherosclerosis and postangioplasty restenosis. The largest subunit of the origin recognition complex (ORC), ORC1, plays a critical role during the initiation of DNA replication in eukaryotes. However, the involvement of ORC1 in the initiation of DNA replication in VSMCs has not been studied yet. OBJECTIVE: The aim of this study was to silence ORC1 gene selectively by using RNA interference and analyze the effects of ORC1 gene on the proliferation and apoptosis of rat VSMCs. METHODS: Freshly isolated rat VSMCs were transfected with siRNA targeting ORC1 gene capsulated in liposome. ORC1 protein expression was determined by Western blotting and ORC1 mRNA level by RT-PCR. DNA synthesis was analyzed by (3)H thymidine ((3)H-TdR) incorporation and cell proliferative activity and cell cycle distribution by flow cytometry. Two apoptosis-related proteins, Bax and Bcl-2, were examined immunohistochemically. RESULTS: Down-regulation of ORC1 mRNA and protein expression was observed in rat VSMCs at 24 h after transfection with the three pairs of siRNA targeting ORC1 gene and this reduction persisted at least 7 days post-transfection. Down-regulation of ORC1 mRNA (60%) and protein (80%) expression was observed at 72 h post-transfection in the cells transfected with B-ORC1 siRNA. A significant decrease in (3)H thymidine incorporation was observed in rat VSMCs with ORC1 gene silencing after serum challenge, but not in the non-silenced control. A significant increase in the proliferation index and a significant decrease in the percentage of cells at G(0)/G(1) phase after serum challenge were observed in the non-silenced control, but not in ORC1 gene silenced cells. A significant increase in the ratio of Bcl-2/Bax was observed after serum challenge in the non-silenced control, but only a slight increase was found in the ORC1 gene silenced cells. ORC1 gene silencing disappeared 7 days after transfection. Continuous serum challenge stimulated VSMCs to synchronously reenter the cell cycle as evidenced by increases in [(3)H] thymidine incorporation, the proliferation index, and the ratio of Bcl-2/Bax, as non-silenced cells were induced to resume cell cycle progression by the addition of 15% fetal bovine serum to the culture medium. CONCLUSION: ORC1 gene silencing causes rat VSMCs to enter a reversible G(0) quiescent, growth arrested state; thus, ORC1 gene may be an important new target for suppressing VSMCs proliferation.

Clinical efficiency and safety analysis of transcatheter interventional therapy for compound congenital cardiovascular abnormalities.

OBJECTIVE: To investigate the efficiency and safety of transcatheter interventional therapy for compound congenital cardiovascular abnormalities. METHODS: From Nov 2001 to Jun 2006, a total of 36 patients (17 male, 19 female), aged 17.20 +/- 10.52, with compound congenital cardiovascular abnormalities underwent transcatheter interventional procedure. These patients included 11 with perimembranous ventricular septal defect (PVSD) and patent ductus arteriosus (PDA), 8 patients with PVSD and atrial septal defect (ASD), 8 patients with ASD and PDA, 7 patients with ASD and pulmonary stenosis (PS), 1 patient with ASD and mitral stenosis(MS), 1 patient with coarctation of aorta (COA) and PDA. According to the principle of "easy first, hard second," balloon valvuloplasties of PS or MS were performed before the closure of PVSD, and of PDA and ASD. Electrocardiogram and transthoracic echocardiogram were examined at 4 days, 1, 2, 6 and 12 months, respectively, after each procedure. RESULTS: Transcatheter interventional therapy for compound congenital cardiovascular abnormalities was successful in all patients. Among these, 2 occluders were planted in each of 27 patients, 7 patients with ASD combined with PS and 1 patient with ASD combined with MS underwent successfully performed balloon valvuloplasty and ASD closure, 1 patient with COA combined with PDA underwent successfully performed balloon valvuloplasty and subsequent covered stent implantation. No patient encountered serious adverse events during the (30.5 +/- 14.6) months of follow-up. CONCLUSIONS: Transcatheter interventional therapy for compound congenital cardiovascular abnormalities could obtain satisfactory results with technical feasibility.

[Expression of origin recognition complex 1 gene in DNA replication of vascularsmooth muscle cells].

OBJECTIVE: To explore the expression of origin recognition complex 1 (ORC1) during the DNA replication of vascular muscle cells (VSMC). METHODS: VSMC of thoracic aorta in rats were obtained by the adherence method of tissue culture. The cell synchrony was obtained by the method of double-thymidine block, colchicine treatment and serum starvation. The expression of ORC1 mRNA at different cell cycles of VSMC was determined by RT-PCR and the protein expression of ORC1 was analyzed by Western blot. RESULTS: Cultured VSMC were identified by light microscope and immunocytochemistry. Significant expression of ORC1 mRNA and protein in a quiescent stage of VSMC were not observed. Upon synchronization, the expression of ORC1 mRNA was significantly higher at G(1)/S phase of VSMC than that at S and G(2)/M phases. The expression of ORC1 protein followed same changes as the ORC1 mRNA expression at different stages of cell cycles. CONCLUSION: ORC1 may be an important regulatory factor at the initiation of proliferative process of VSMC.