ABSTRACT
Objective To investigate the effect of over-expression of ROBO4 on permeability of human renal glomerular endothelial cells (HRGECs) in high glucose medium.Methods HRGECs infected with recombinant lentiviral vector ROBO4 were cultured in high glucose or low glucose medium in vitro.The protein levels of ROBO4 and ARF6 in each group were detected by Western blotting.The endothelial permeability was measured by the effiux of fluorescein isothiocyanate-dextran (FITC-Dextran)permeated through the monolayer endothelial cells using Transwell cell model system.The cell viability after lentivirus transfection was measured by CCK8 assay.Results The transfection rate of lentiviruses in HRGECs reached 80％ 72h after,and obvious overexpression of ROBO4 protein was in transformed cells compared with the empty vector group (P＜0.05).The lentivirus-mediated ROBO4 transfection did not affect cell viability of HRGECs.Compared with the low glucose group,the expression of ROBO4 increased obviously after 12h,but declined after 24h (P＜0.05),and reached to minimun after 72h (P＜0.05).On the contrary,the expression of ARF6 increased after 12h,and the increase reached to the maximum after 72h (P＜0.05).Furthermore,the vascular permeability increased gradually after 24h,and reached to the maximum after 72h (P＜0.05) in high glucose group.Compared with the empty vector group,the over-expression of ROBO4 inhibited the expression of ARF6 significantly,and the FITC-Dextran permeability reduced obviously.Conclusion Over-expression of ROBO4 may significantly enhance the barrier functions of HRGEC in high glucose medium,and ROBO4 activation may be a potential therapeutic approach in diabetic nephropathy.
ABSTRACT
Objective To investigate the expression of ROBO4/ARF6 and its correlation to glomerular endothelial cells (MGECs) permeability at each pathological stage of diabetic kidney disease (DKD). Methods Thirty patients with DKD were enrolled (patient group), and their renal tissues obtained by biopsy were divided into incipient, manifest and advanced stages (10 each) with Tervaert pathological staging, and confirmed normal renal tissues adjacent to the renal angiomyolipoma were assigned as control group. Immunohistochemistry method of MaxVision was used to detect the expression of ROBO4/ARF6 in the specimens, and then the correlation of the expression with the clinicopathological parameters (patients' HbA1c, albuminuria, serum creatinine, eGFR) were determined using statistical software SPSS 21.0. Results ROBO4 was abundantly but ARF6 rarely expressed in normal MGECs. In DKD patients, ROBO4 was mainly expressed in MGECs, while ARF6 was expressed in both MGECs and renal tubular epithelial cells. Compared with the control group, the positive intensity of ROBO4 in patient group decreased significantly (P0.05), but was positively correlated with 24h-urinary albumin (r=0.603, P<0.01) and HbA1c (r=0.582, P<0.01). Conclusion The low expression of ROBO4 and high expression of ARF6 in MGECs of DKD patients suggest that ROBO4/ARF6 signaling pathway may participate in the DKD glomerular endothelial and vascular pathological injury and urinary protein increase.
ABSTRACT
Objective To observe the effects of Lipopolysaccharide (LPS) in different concentrations on expression of Robo4 in human umbilical vein endothelial cells (HUVECs). Methods HUVECs were cultured in vitro and randomly divided into 3 groups: control group, low dose LPS group (10 μg/mL) and high dose LPS group (100 μg/mL). Robo4 protein level was detected by immunofluorescence staining and western blot , and Robo4 mRNA level was measured by real-time PCR. Results Robo4 protein and Robo4 mRNA in high dose LPS group were 0.49 ± 0.08 and 0.23 ± 0.08 respectively , which were significantly decreased than those (1.35 ± 0.15 and 0.97 ± 0.17) in control group(P 0.05). Conclusion High dose LPS (100 μg/mL) could down-regulate expression of Robo4 in HUVECs.
ABSTRACT
AlM: To explore the roles of neuronal axon-guidance molecules Slit3 and Robo4 receptor in corneal neovascularization ( CNV ) by study their expression in neovascularized cornea of rats. METHODS: CNV models were established by implantation pellets containing basic fibroblast growth factor ( bFGF ) into corneal stroma. CNV models were measured by biomicroscopy photography. lmmunohistochemical staining and imaging analysis system were used to detect the expression of Slit3 and Robo4 in the models after 1, 4, 7, 10 and 14d. RESULTS:The area of CNV and the expression of Slit3, Robo4 were increased in CNV models compared to that in normal cornea and reached highest level on 7d. And the expression level of Slit3 and Robo4 were significantly correlated with the size of CNV on every time point except 1d (r=0. 84-0. 91, all P CONCLUSlON: The expression of Slit3 and Robo4 may be related to the CNV development. They are potential therapeutical target for CNV.
ABSTRACT
Objective To observe the expression of axon guidance cues Slit2 and Robo4 in lung tissue of rat with acute lung injury (ALI) and explore the function of Slit2 and Robo4 in ALI.Methods Forty-eight Sprague-Dawley rats were randomly (random number) divided into control group (n =24) and ALl group (n =24).ALI model was reproduced by cecum ligation and puncture (CLP).The control group only experienced a simulated operation without CLP.Both groups were further divided into 3 subgroups with 8 rats in each subgroup:12 h,24 h,and 48 h subgroups.artery blood gas analysis,lung tissue wet/dry weight (W/D) ratio,lung histopathologic changes,pulmonary microvascular permeability were observed.The serum tumor nocrosis factor-α (TNF-α) was measured with enzyme linked immunosorbent assay (ELISA).The expression of Slit2 and Robo4 mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR).The expression of Slit2 and Robo4 protein in lung tissues was assessed by immunohistochemistry.Date were analyzed by one-way ANOVA with SPSS version 13.0 software.Statistical significance was established at a P value of less than 0.05.Results Compared with the control group,in ALI rats at different time points,partial pressure of oxygen in arterial blood (PaO2) decreased significantly,lung W/D weight ratio and pulmonary microvascular permeability,the serum TNF-α increased significantly (all P ＜ 0.05),histopathology of lung revealed signs of injury.The expression of Slit2 mRNA in lung tissues was decreased markedly after CLP compared with control group [(0.56±0.13) vs.(0.87±0.05),F=41.39,P＜0.05,(0.42±0.10) vs.(0.85±0.07),F=93.54,P＜0.05,(0.26±0.08) vs.(0.89 ±0.09),F=227.05,P＜0.05].but there were no significant difference in expression of Robo4 mRNA in lung tissue between ALI group and control group [(0.86±0.07) vs.(0.83±0.05),F=0.695,P＞0.05,(0.82±0.05) vs.(0.89±0.08),F=2.061,P ＞ 0.05,(0.86 ± 0.08) vs.(0.86 ± 0.05),F =0.035,P ＞ 0.05].Immunohistochemistry study showed Slit2 protein was mainly expressed on the extracellular surface of vascular endothelial cells,while lung epithelial cell nuclei and endochylema.Robo4 protein was only expressed on the extracellular surface of vascular endothelial cells.Compared with the control group,expression of Slit2 protein in lung tissue in ALI group decreased markedly [(0.37 ± 0.05) vs.(0.45 ± 0.07),F =6.82,P ＜ 0.05,(0.32±0.06) vs.(0.47±0.09),F=23.54,P＜0.05,(0.28±0.07) vs.(0.46±0.06),F=28.01,P ＜ 0.05].As good as RT-PCR,there were no significant difference in expression of Robo4 protein in lung tissue between two groups [(0.53±0.04) vs.(0.52±0.05),F=0.155,P＞0.05,(0.53± 0.09) vs.(0.50±0.05),F=0.498,P＞0.05,(0.55±0.06) vs.(0.56±0.07),F=0.073,P ＞ 0.05].Conclusions Lung tissues of control group rats express Slit2 and Robo4.The decreased Slit2 mRNA and protein expressions in the lung tissue of rat with ALI caused by CLP may be associated with the occurrence of ALI.