| The majority of eye diseases that lead to vision loss result from retinal neovascularization, often in response to retinal ischemia, such as diabetic retinopathy, retinopathy of prematurity and central retinal vein occlusion. When ischemia takes place in organs such as the heart and brain, growth of collateral vessels may be beneficial. However, neovascularization secondary to ischemia in the eye is always harmful and causes bleeding, retinal edema, fibrovascular proliferation, and even retinal detachment. Currently, there is no satisfactory treatment to block the retinal neovascularization and vascular leakage. Laser photocoagulation, vascular endothelial growth factor (VEGF) inhibitors, photodynamic therapy, angiostatic steroids and vitrectomy have been reported to regress neovascularization to different degrees, but often fail to completely inhibit it, associating with recidivation or normal retinal injury, or other complications, and may ultimately aggravate the ischemia and hypoxia. Studies showed that the function of retinal vascular endothelium decreased in patients with retinal ischemic diseases. Therefore, the key to control the retinal neovascularization is to repair the damaged vascular endothelium and alleviate ischemia and hypoxia in the retina. Endothelial progenitor cells (EPCs) have the capacity to differentiate into mature endothelial cells, facilitate endothelial repair and angiogenesis in vivo. If EPCs can effectively migrate to the injured retina and maintain normal function, it is possible to repair the damaged vascular endothelium, give rise to the formation of functional retinal vessels, and ultimately alleviate ischemia and hypoxia. However, there is still no stable and efficient method for tracking transplanted EPCs in retina.Objective:The current study sought to establish a simple, reliable, fluorescent labeling method for tracking EPCs with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), 1,1'-dilinoleyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-AcLDL) and green fluorescent protein (GFP)in laser-injured mouse retina, and observed the role of EPCs in retinal vascular repair.Methods:(1) EPCs were isolated by hydroxyethyl starch sedimentation and density gradient centrifugation from human umbilical cord blood mononuclear cells, and cultivated. Then EPCs were identified by cytomorphology, specific markers detected by flow cytometry, immunofluorescence observed by confocal microscope and electron microscope. (2) EPCs were labeled with CFSE, Dil-AcLDL and GFP. The morphology of labeled EPCs were observed by inverted optical microscope. The survival capability and the adhesion ability of labeled EPCs were measured by Trypan blue staining and adherent cells count respectively. Fluorescence microscopy was used to observe the label stability and the fluorescence intensity during the extended culture period. The proportion of labeled cells was detected by flow cytometry. (3) The mouse retinal vascular injured model was estabalished by injured the retinal veins and capillarity network around the optic disc with retinal laser photocoagulation. Labeled EPCs were transplanted into the vitreous cavity of pigmented mouse model. Fundus photography, paraffin section, retinal frozen section, angiography and flat mounted were used to obeserved the distribution, fluorescence duration and intensity change of labeled cells, and the repair of retinal vascular injury after the transplantation of EPCs.Results:(1) EPCs were isolated from human umbilical cord blood and the cells presented numberous cell clusters and cobblestone morphology. Most adherent cells were double stained by Dil-AcLDL and FITC-UEA-I, and the flow cytometry showed that the cells were positive for CD34, CD 133 and VEGFR-2. W-P body was observed by electron microscope. (2) Our data indicated that EPCs labeled with CFSE presented intense green fluorescence and the positive rate was above 95%; while EPCs labeled with Dil-AcLDL presented red fluorescence and the positive rate was about 80%. The fluorescence intensity gradually decreased in the cells at the end of 4 weeks. GFP transduced EPCs showed efficiency more than 30% at the end of 4 weeks, and fluorescence intensity gradually strengthened. EPCs labeled with the three methods maintained normal morphology. The survival capability and the adhesion ability were not statistically different between labeled and unlabeled EPCs. (3) The mouse retinal vascular injured model was estabalished by retinal laser photocoagulation. The paraffin section and HE staining of retinal displayed the injury of laser photocoagulation and the repair by EPCs transplantation for 4 weeks. The retinal frozen section displayed distribution of labeled cells in the retinal layers in 4 weeks. Evans blue angiography of the retina displayed the retinal capillarity network clearly and fluorescence leakage was observed around photocoagulated spots in the laser-injured mouse model. One week after transplantation of CFSE labeled EPCs, the fluorescent cells were identified around the photocoagulated lesions. Four weeks after transplantation, fluorescent tube-like structures were observed in the retinal vascular networks. The results indicated that EPCs could participate to the repair of injured retinal vessels.Conclusions:(1) EPCs could be labeled by CFSE and Dil-AcLDL, be monitored in vivo for at least 4 weeks. CFSE has more advantages for short time tracing. Combining CFSE with retinal frozen section and Evans blue angiography contributite a multi-angle observation to tracking EPCs. GFP could transduce EPCs by lentivirus and has the potential for long time tracing. (2) The mouse retinal vascular injured model could be estabalished by retinal laser photocoagulation. EPCs could be transplanted into eyes by intravitreal injection. EPCs could localize directly to retinal injured sites and participate to the repair of injured retinal vessels.
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