Small interfering RNA targeting PGC-1α inhibits VEGF expression and tube formation in human retinal vascular endothelial cells

AIMTo determine whether small interfering RNA (siRNA) of PGC-1α could inhibit vascular endothelial growth factor (VEGF) expression and tube formation in human retinal vascular endothelial cells (hRVECs).METHODShRVECs transfected with peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) siRNA were incubated for 24h and then placed into a normoxic (20%, O2) or hypoxic (1%, O2) environment for another 16h. PGC-1α mRNA and protein levels were detected by real-time PCR and Western blot. VEGF mRNA and protein levels were detected by real-time PCR and ELISA. Cell proliferation was evaluated by BrdU incorporation assay. Forty-eight hours after siRNA transfection, hRVECs were planted into Matrigel-coated plates and cultured under normoxic (20%, O2) or hypoxic (1%, O2) conditions for another 48h. The tube formation of hRVECs was observed under an optical microscope and quantified by counting the number of branch points and calculating the total tube length.RESULTSPGC-1α mRNA and protein levels were significantly reduced by PGC-1α siRNA, and VEGF mRNA and protein levels also decreased significantly. The percentage of BrdU-labeled cells in siPGC-1α groups were significantly decreased compared with control siRNA groups under normoxia and hypoxia in cell proliferation assay. In the tube formation assay, PGC-1α siRNA treated cells formed significantly fewer tubes.CONCLUSIONBlocking PGC-1α expression can inhibit VEGF expression in hRVECs and inhibit their ability to form tubes under both normoxic and hypoxic conditions.

Effects and mechanism of miRNA-24 on the proliferation,migration and tube formation of vascular endothelial cells

Objective:To investigate the effect and mechanism of miRNA-24 on the proliferation,migration and tube formation of vascular endothelial cells.Methods:Human umbilical vein endothelial cells(HUVECs)were assigned into control,microRNA(miR)-24 overexpression and anti-miR-24 groups.The proliferation and migration abilities of HUVECs were detected by methyl thiazolyl tetrazolium(MTT)assay and scratch wound healing assay,respectively.The ability of HUVECs to form tubular structures was evaluated by a tube formation assay.The mRNA and protein expressions of vascular endothelial growth factor(VEGF)and transcription factor Sp1 were determined by RT-PCR,immunocytochemistry and western blotting,respectively.Results:The miR-24 overexpression group exhibited decreased cell proliferation and migration,and expressions of VEGF and Sp1 compared with the control group(P <0.01).No tube-like network structure was formed in the miR-24 overexpression group.However,inhibition of miR-24 in HUVECs markedly increased cell proliferation and migration,enhanced tube formation and expressions of VEGF and Sp1(P<0.05 or P<0.01).Conclusion:MiR-24 suppressed the proliferation,migration and tube formation of HUVECs,and the mechanism might be related to the down-regulation of VEGF expression.Sp1 might participate in this regulation process.

The Effect of Xuefu Zhuyu Decoction(血府逐瘀汤) on in vitro Endothelial Progenitor Cell Tube Formation

Objective： To observe the effect of Xuefu Zhuyu Decoction （血府逐瘀汤）-containing serum （XFZYD-CS） on endothelial progenitor cell （EPC） tube formation in vitro. Methods： Mononuclear cells from rat bone marrow were prepared in a Ficoll density gradient centrifuge. EPCs were separated by the differential attachment method, and observed with inverted microscope for the effect of XFZYD-CS on EPC tube formation. Results： After one day, EPCs exposed to the serum containing 5%, 10% and 15% XFZYD-CS formed typical tubes or vessel networks. The tube formation time was two days ahead of the control group and the size of most tubes in the serum groups was smaller than in the control group. Conclusion： XFZYD-CS could induce EPC angiogenesis and hasten tube formation, especially in capillary vessels. The study provides experimental evidence for the plausibility of Xuefu Zhuyu Decoction in the treatment of ischemic diseases.

VISUAL OBSERBATION OF HCFC141b GAS HYDRATE FORMATION/DECOMPOSITION PROCESS OUTSIDE OF A TUBE

In order to design a kind of heat exchanger suitable to the indirect-touched gas hydrate cool storage vessel, a visual observation of HCFC141b gas hydrate formation/decomposition process was presented through a self-designed small-scale visualization apparatus of gas hydrate cool storage. Based on the shooted photos and recorded temperatures, the formation/decomposition process of HCFC141b are described, some characteristics are concluded, and some suggestions of designing heat exchanger are indicated according to the specific characteristics of HCFC141b gas hydrate formation/decomposition process.

Leptin activates the JAK/STAT pathway to promote angiogenesis in RF/6A cells in vitro

AIM: To investigate the effect of leptin on the angiogenesis of RF/6 A cells(monkey retinal choroidal endothelial cells) in vitro and test the cellular signaling in the mechanism.METHODS: RF/6 A cells were cultured in vitro and randomly divided into four groups: normal control, with leptin at 50, 100, 200 ng/m L for cell counting kit-8(CCK8). RF/6 A cell proliferation and migration were examined by Transwell assays, while RF/6 A cell tube formation by Matrigel assay. JAK2, p-JAK2, STAT3, and p-STAT3 protein expression was measured by Western blotting. Cells were then divided into the following treatment groups: control, 100 ng/m L leptin and AG-490(100 ng/m L leptin+10 μmol/L AG-490) for examinations of RF/6 A cellular behaviour again. Analysis of differences was carried out using one-way ANOVA and least significant difference(LSD).RESULTS: RF/6 A cell proliferation, migration and cell tube formation were promoted significantly by leptin in a dose-dependent manner(P<0.05). Western blotting showed that leptin up-regulated p-JAK2 and p-STAT3 expression levels. Treatment with the JAK/STAT pathway inhibitor, AG-490, decreased leptin-induced p-JAK2 and p-STAT3 expression, and inhibited cell proliferation, migration and cell tube formation induced by leptin(P<0.05). CONCLUSION: Leptin can promote RF/6 A cell angiogenesis in vitro via activation of the JAK2/STAT3 signaling pathway.

Intravitreal injection of resveratrol inhibits laser-induced murine choroidal neovascularization

AIM:To determine the effects of intravitreal resveratrol(RSV)on murine laser-induced choroidal neovascularization(CNV).METHODS:The toxicity of RSV to choroidal endothelial cell(CEC)was measured using thiazolyl blue tetrazolium bromide(M一)assay.Effects of RSV on choroidal endothelial cell(CEC)migration were evaluated with a modified Boyden chamber assay,while tube formation was evaluated in a 2-D gel assay.CNV was induced by laser photocoagulation in mice.The effects of intravitreal injection of RSV on CNV development were evaluated by fluorescein angiography(FA),confocal analysis of isolectin B4 labeled choroidal flat mounts,and histologic examination of CNV membranes.Immunostaining was used to analyze the expression and phosphorylation of vascular endothelial growth factor receptor 2(VEGFR2).RESULTS:No significant cell toxicity was observed in CEC if the concentration of RSV was less than 200 pmol/L(P>0.05).RSV inhibited vascular endothelial growth factor(VEGF)-induced CEC migration(P<0.05)and tube formation(P<0.05)invitro.Furthermore,intravitrealinjectionof RSV significantly inhibited laser induced CNV formation in mice.The FA leakage,CNV volume and CNV area analysis revealed that there were 41%,45%,and 58%reduction in RSV-treated eyes(1.691±0.1032,178163±78623μm^3 and 6508±619.0μm^2,respectively)compared with those in control(2.724±0.08447,379676±98382μm3and16576±2646μm^2,respectively;P<0.05).Phospho-VEGFR2expression was much weaker in the sections of CNV lesions in RSV injected mice compared with that in control(P<0.05).CONCLUSION:Intravitreal injection of RSV exerts an inhibitory effect on CNV,which may through suppressing endothelial cell migration,tube formation and VEGFR2 phosphorylation.

Heparanase-1 is downregulated in chemoradiotherapy orbital rhabdomyosarcoma and relates with tumor growth as well as angiogenesis

AIM:To determine the role of heparanase-1(HPSE-1)in orbital rhabdomyosarcoma(RMS),and to investigate the feasibility of HPSE-1 targeted therapy for RMS.METHODS:Immunohistochemistry was performed to analyze HPSE-1 expression in 51 cases of orbital RMS patients(including 28 cases of embryonal RMS and 23 cases of alveolar RMS),among whom there were 27 treated and 24 untreated with preoperative chemoradiotherapy.In vitro,studies were conducted to examine the effect of HPSE-1 silencing on RMS cell proliferation and tube formation of human umbilical vein endothelial cells(HUVECs).RD cells(an RMS cell line)and HUVECs were infected with HPSE-1 sh RNA lentivirus at a multiplicity of infection(MOI)of 10 and 30 separately.Real-time PCR and Western blot were applied to detect the m RNA and protein expression levels of HPSE-1.Cell viability of treated or control RD cells was evaluated by cell counting kit-8(CCK-8)assay.Matrigel tube formation assay was used to evaluate the effect of HPSE-1 RNAi on the tube formation of HUVECs.RESULTS:Immunohistochemistry showed that the expression rate of HPSE-1 protein was 92.9%in orbital embr yonal RMS and 91.3%in orbital alveolar RMS.Tissue from alveolar orbital RMS did not show relatively stronger staining than that from the embryonal orbital RMS.However,despite the types of RMS,comparing the cases treated chemoradiotherapy with those untreated,we have observed that chemoradiotherapy resulted in weaker staining in patients’tissues.The expression levels of HPSE-1 declined significantly in both the m RNA and protein levels in HPSE-1 sh RNA transfected RD cells.The CCK-8 assay showed that lentivirus-mediated HPSE-1 silencing resulted in significantly reduced RD cells viability in vitro.Silencing HPSE-1 expression also inhibited VEGF-induced tube formation of HUVECs in Matrigel.CONCLUSION:HPSE-1 silencing may be a promising therapy for the inhibition of orbital RMS progression.

Primary culture and identification about brain microvascular endothelial cells of rabbits

Objective:To establish a simple and efficient culture method of primary rabbit brain microvascular endothelial cells,provide important carriers and tool cells for the research of related cerebrovascular diseases.Methods:The cerebral cortexes of rabbits were collected aseptic and inoculated after cutting,passing through cell sieve,bovine serum albumin density gradient centrifugation,typeⅡcollagenase digestion,finally inoculated and cultured.The cultured cells were identified by cell morphological observation and angiogenesis experiment.Results:Under the inverted microscope,the cells were short fusiform or polygonal,and grew in clusters and adhere to the wall.After the cells were densely fused,they would be in a typical monolayer flat,“pebbled"mosaic arrangement.Tube formation test had the ability to form tubes structure.Conclusion:This method can successfully separate and cultivate primary rabbit brain microvascular endothelial cells.