Induction of vascular endothelial growth factor receptor expression in human umbilical vein endothelial cells after repeated bevacizumab treatment in vitro

AIM： To investigate the mechanism underlying the loss of responsiveness to anti-vascular endothelial growth factor（VEGF） treatment after repeated injections for choroidal neovascularization, VEGF and VEGF receptor（VEGFR） expressions were evaluated following repeated bevacizumab treatments in hypoxic human umbilical vein endothelial cells（HUVECs） in vitro.METHODS： HUVECs were incubated under hypoxic conditions in two media of different bevacizumab concentrations（1.0 or 2.5 mg/m L） for 17 h, and then in a new medium without bevacizumab for 7h. This procedure was repeated twice more. A culture with an identical volume of excipients served as the control. Cytotoxicity and cell proliferation were assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and Ki-67 assays, respectively. Levels of VEGF and VEGFR were assessed using enzyme-linked immunosorbent assay and Western blot respectively.RESULTS： Cytotoxic effects were not reported for either bevacizumab concentration. Cell proliferation was not reduced after anti-VEGF treatments. VEGF level after single treatment was significantly higher than that of the control and after repeated treatments. Phosphorylated VEGFR-2 expression increased significantly after singleand repeated bevacizumab treatments compared with the control. The 1.0 mg/m L bevacizumab induced significantly higher expressions of VEGFR-2 than the 2.5 mg/m L in single and repeated treatment groups.CONCLUSION： Bevacizumab treatment of HUVECs elevated VEGFR expression in both single and repeated treatments, indicating a mechanism for the reduced efficacy of anti-VEGF therapy in ocular neovascular disorders.

Mesenchymal stem cells-derived exosomes ameliorate blue light stimulation in retinal pigment epithelium cells and retinal laser injury by VEGF-dependent mechanism

AIM： To observe the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells（h UCMSCs） on the expression of vascular endothelial growth factor-A（VEGF-A） in blue light injured human retinal pigment epithelial（RPE） cells and laser-induced choroidal neovascularization（CNV） in rats.METHODS： Exosomes were isolated from h UCMSCs and characterized by transmission electron microscope and Western blot. MSCs-derived exosomes were cultured with RPE cells exposed to blue light. The m RNA and protein expression of VEGF-A were determined by real time-polymerase chain reaction（PCR） and Western blot, respectively. Immunofluorescence assay was used for the detection of the expression level of VEGF-A. We injected different doses of MSCs-derived exosomes intravitreally to observe and compare their effects in a mouse model of laserinduced retinal injury. The histological structure of CNV in rats was inspected by hematoxylin-eosin（HE） staining and fundus fluorescein angiography. The expression of VEGF-A was detected by immunohistochemistry.RESULTS： Exosomes exhibited the typical characteristic morphology（cup-shaped） and size（diameter between 50 and 150 nm）. The exosomes marker, CD63, and h UCMSCs marker, CD90, showed a robust presence. In vitro, MSCsderived exosomes downregulated the m RNA（Exo-L： t=6.485, 7.959, 9.286; Exo-M： t=7.517, 10.170, 13.413; Exo-H： t=10.317, 12.234, 14.592, P〈0.05） and protein（Exo-L： t=2.945, 4.477, 6.657; Exo-M： t=4.713, 6.421, 8.836; Exo-H：t=6.539, 12.194, 12.783; P〈0.05） expression of VEGF-A in RPE cells after blue light stimulation. In vivo, we found that the MSCs-derived exosomes reduced damage, distinctly downregulated VEGF-A（Exo-H： t=0.957, 1.382; P〈0.05）, and gradually improved the histological structures of CNV for a better visual function（Exo-L： 0.346, Exo-M： 3.382, Exo-H： 8.571; P〈0.05）. CONCLUSION： MSCs-derived exosomes ameliorate blue light stimulation in RPE cells and laser-induced retinal injury via downregulation of VEGF-A.

Inhibition of vascular endothelial growth factor gene expression by T7-siRNAs in cultured human retinal pigment epithelial cells

Background Retinal pigment epithelial (RPE) cells play an important role in the occurrence of choroidal neovascularization (CNV). Vascular endothelial growth factor (VEGF) as a positive regulatory growth factor is produced by the RPE in an autocrine or paracrine manner, promoting CNV development. Duplexes of 21 nt RNAs, known as short interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference when introduced into mammalian cells. We searched for an efficient siRNA to interfere with VEGF expression in RPE cells and shed light on the treatment of CNV.Methods Human primary RPE (hRPE) cells were cultured and identified. Three pairs of siRNAs were designed according to the sequence of VEGF 1-5 extrons and synthesized by T7 RNA polymerase transcription in vitro. To evaluate the inhibitory activity of T7-siRNAs, hRPE cells were transfected via siPORT Amine. The interfering effect of T7-siRNAs in hRPE cells was examined by semiquantitative reverse transcription-polymerase chain reaction and immunofluorescence. Results Three pairs of T7-siRNAs synthesized by in vitro transcription with T7 RNA polymerase suppressed VEGF gene expression with efficiency from 65% to 90%. T7-siRNA (B), targeted region at 207 nt to 228 nt and double stranded for 21 nt with 2 nt UU 3’ overhangs, was the most effective sequence tested for inhibition of VEGF expression in hRPE cells. Compared with nontransfected cells, the mean fluorescence in hRPE cells transfected with T7-sRNAs was significantly less (P<0.01). siRNA with a single-base mismatch and ssRNA(+) did not show suppressing effect. Furthermore, it was found that siRNAs had a dose dependent inhibitory effect (5 to 10 pmol).Conclusion T7-siRNA can effectively and specifically suppress VEGF expression in hRPE cells and may be a new way to treat CNV.

Immunohistochemical study in dural arteriovenous fistula and possible role of ephrin-B2 for development of dural arteriovenous fistula

Background Although there were several clinical and experimental studies discussing the pathogenesis of dural arteriovenous fistula (DAVF), the pathological process leading to intracranial DAVF so far remains unknown In this study, we investigated the expression of vascular growth factors in order to elucidate the possible role of these factors for the development of DAVF and to study the biological activity of this uncommon lesion Methods We examined the histological features, proliferative and angiogenic capacities of the tissue specimens obtained from 6 patients who underwent surgery at our institution Immunohistochemical staining for vascular endothelial growth factor (VEGF), its receptors Flk 1 and Flt 1, ephrin B2, MIB 1 and proliferating cell nuclear antigen (PCNA) was performed using standard immunohistochemical techniques Results A positive immunostaining was found for all antibodies studied except MIB 1, whereas nuclear endothelial expression of PCNA was observed in only 3/6 cases VEGF stained positive in all of the available specimens (6/6) Flk 1 showed a positive immunoreaction in only 2/6 cases and Flt 1 in 4/6 cases Ephrin B2 was expressed in the majority (5/6) of the cases Conclusions These results support the hypothesis that DAVFs might be acquired dynamic vascular malformations with low biological activity Vascular growth factors like VEGF and ephrin B2 might play a pivotal role in the formation of DAVF