CXC Chemokine Receptor4’s Role In Pathogenesis Of Oxygen-Induced Retinopathy And Its Mechanism

The retinal neovascularization is a main cause of blindnesses worldwide. Retinal neovascular diseases include proliferative diabetic retinopathy (PDR), retinopathy of prematurity (ROP), central retinal vein occlusion (CRVO) and age related macular degeneration (AMD). They feature in pathological retinal neovascularization induced by relative hypoxia.Formation of retinal vessels involves in vasculogenesis and angiogenesis/neovascularization. Vasculogenesis refers to primary vascular network formed by endothelial progenitor cells, which generated from bone marrow hematopoietic stem cells, and differentiated into mature endothelial cells to form vessels. This is how blood vessels are developed originally. Angiogenesis are always considered as pathological processes. As in retina, when it subjected to hypoxia insults, vascular basement membrane degradated, endothelial cells sprout out from previous existed blood vessels, proliferate and form vascular lumens. In vivo and in vitro studies showed, retinas suffered from hypoxia or ischemia insults can release numerous cellular factors such as HIF-1alpha, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), nsulin-like growth factor (IGF-1), transformation growth factor books (TGF-beta), etc. These factors effect on vascular endothelial cells, accelerate mitosis of endothelial cell, which lead to angiogenesis.SDF-1belongs to the CXC chemokine sub-family, possessing cellular chemotactic unctions. SDF-1and receptor CXCR4constitute the SDF-1/CXCR4axis. When SDF-1and CXCR4combine together the downstream signals were activated, it could mediate nflammation, HIV infection and homing of hematopoietic stem cells. The SDF-1/CXCR4axis also takes important roles in tumor metastasis and (?)eovascularization, including ocular neovascularization. It was found that SDF-1can nduce endothelial progenitor cells migrate into retina ischemia region and participate in (?)eovascularization. AMD3100(commercial name Plerixafor(?)) is a specific antagonists of CXCR4. AMD3100can effectively blocking the combining of SDF-1to CXCR4and silence down cellular signal transductions of receptor CXCR4. It has been proved, by blocking SDF-1/CXCR4signal, AMD3100could inhibit tumor angiogenesis and HIV infection.However, whether CXCR4plays role in formations of hypoxia induced retinal neovascularization is unknown yet. Therefore, in this study, mice models of oxygen-induced retinopathy and hypoxic cell model of human umbilical vein endothelial cells (HUVECs) will be established. Blocking the SDF-1/CXCR4signaling with its specific antagonist AMD3100, the formations of RNVs in OIR model, proliferations and expressions of neovascular factors in HUVECs are evaluated. Discuss the role of CXCR4in the pathogenesis and progression of hypoxia induced retinal neovascularization and the possible mechanism.Part OneEstablishment of Mice Model of Oxygen-Induced Retinopathy and Cultivation of Human Umbilical Vein Endothelial CellsPurpose:Establish oxygen induction retinopathy (OIR) mice model; harvest human umbilical vein endothelial cells (HUVECs) by collagenase I digestion, identify and build hypoxic cell model.Methods:Obtain20C57BL/6mice at7th postnatal days (P7), dividing into normal group (raised in normoxia until P17) and OIR model group (place a glass container with oxygen concentration at75℃up/below2℃from P7to P12, then were reared in normoxia until P17) randomly. At P17, all mice puppies were anesthetized subjected to retinal FITC-Dextran angiography or made into retinal section with HE staining. Quantify retinal NVs by counting endothelial cells (ECs) nucleus breakthrough the internal limiting membrane (ILM) of retina. Qualify retinal neovascular vessels under fluorescence microscope. Bring a segment of fresh umbilical cord (20cm-25cm) to the aseptic condition; wash the umbilical vein with D-hank’s solution. Filled umbilical vein with20ml collagenase Ⅰ, digested for20min in37℃, collected and cultured cells with endothelial cell medium (ECM), observe, pass the cells and indentify them with vWF immuno-fluorescence staining. Draw cell growing curve and evaluate CoCl2(0μg/L,20μg/L,50μg/L,100μg/L,200μg/L and400μg/L) impact on HUVECs as hypoxia model by MTT assay.Results:OIR mice possess massive ECs nucleus protruding into the ILM than normal mice number, some of them formed neovascular clusters (49sections in50sections are positive in RNV, the rate is98%). The number of neovascular ECs protruded into the ILM is (0.20±0.447) in normal group and (31.60±2.07) in OIR model, the difference is statistically significant. Retinal FITC-Dextran angiography showed irregular structures of retinal vessels expanded, with no perfusion area at the posterior pole, neovascular plexus in periphery of retina with fluorescence leakages, proving OIR modeling method to be successful. Primary harvested cells grew at the bottom in single layer. Cells were rounded, after passsges they stretched in spindle-look, rich in cytoplasm, with clear oval nucleus. Immunofluorescence stains showed numerous positive particles located in cytoplasm, while the nucleus area was negative. Cells cultivations were showed to be stable. Growing speed were exaggerated in the first2to3days after one passage; the mimic hypoxia agent CoCl2could suppress HUVEC survives as dosage increased.Conclusion:OIR mice model can be set up as Smith described; harvest human umbilical vein endothelial cells (HUVECs) by collagenase Ⅰ digestion, and establish chemical hypoxia cell model by administrating appropriate dose of CoCl2.Part TwoCXCR4’s Role in the Pathogenesis of Oxgen-Induced Retinopathy Objective:Explore CXCR4’s roles in the pathogenesis and progress of oxygen-induced retinopathy and its possible mechanism.Methods:1hundred P7C57BL/6mice were divided into5groups randomly (eight mice each group):normal group, OIR models group, AMD3100large-dosage and small-dosage group, model control group. Except for normal group mice, the rests were put in the hyperoxic incubator for5days (P7to P12) and returned to normal environment till P17to establish OIR models. OIR model group received no other treatment, AMD3100large-dosage group received1μL of100μg/μL AMD3100injected into vitreous cavity once at P12when taken out from oxygen incubator; AMD3100small dosage mice received1μl of50μg/μL AMD3100injected into vitreous cavity once at P12, and model control mice received1μL of BSS at the same time. Observe HE stained-paraffin section of retina (five mice each group) and retinal FITC-Dextran angiography (five mice each group), quantify the number of endothelial cells nucleus protruding into ILM. Four mice and six mice each group are used for detection of retinal HIF-la and VEGF mRNA and protein by RT-PCR and western bolt asssy, respectively.Results:AMD3100(large doses and small doses) reduced neovascular ECs breakthrough ILM by retinal sections. Normal group average number of ECs nucleus is for each section (0.01+/-0.12), to oxygen model group average for each section (30.33±1.51), there is a significant difference between statistical significance (t=-49.35, P＜0.01). Theaverage number of endothelial nuclei breakthrough ILM in large dose AMD3100treated group was is (13.50±1.87), compared to oxygen model there is a significant difference between statistical significance (t=17.17, P＜0.01). FITC-Dextran angiography showed retinal structures are normal in large doses of AMD3100treatment (did not see the significant new blood vessels, restrain effect of new blood vessels is dose dependent); expression level of HIF-la protein and VEGF protein were significantly lower than that in OIR model mice in a dose-dependent manner.Conclusion:Vitreous cavity injection of CXCR4antagonist AMD3100reduced RNVs in OIR mice, and suppressed the upregulated mRNA and protein levels of HIF-1alpha and VEGF, suggesting the SDF-1/CXCR4signaling’s role in the pathogenosis of hypoxia induced retinal neovascularization, this may largely related to the expression of HIF-la and VEGF. Part ThreeCXCR4’s Role in Hypoxic Change of Human Umbilical Vein Endothelial Cells and its MechanismObjective:Discuss the effect of CXCR4signaling on hypoxic changes of cultured HUVECs and the possible mechanism.Methods:Detect proliferations rate of HUVEC under different concentrations of AMD3100(50,100,200,400,800μmol/L), and AMD3100(50or100μmol/L) combined CoCl2(50or100μ g/L) by MTT assay. Expressions of HIF-1α and VEGF mRNA were quantified at0h,1h,2h,4h,6h,12h,24h after100μg/L CoCl2treatment in HUVECs by RT-PCR; protein levels of HIF-1alpha and VEGF in HUVECs at0h,6h,12h and24h after100μg/L CoCl2addition were detected by western bolting. AMD3100(50nM and100nM) were added to cell medium1hour ahead of100μg/L CoCl2treatment,24h later HIF-1alpha and VEGF mRNA and protein levels were evaluated.Results:AMD3100inhibited HUVECs proliferation as its doses increased; the proliferation rate of HUVECs under800μmol/L AMD3100is80%of that in normal group. After100μg/L CoCl2were added, mRNA expression of HIF-1α and VEGF reached to the peak in6h-12h, and then dropped at24h; protein expression of HIF-1α and VEGF increased continuously from0h to24h;1h of AMD3100pretreatment suppressed100μg/L CoCl2-induced increasing of HIF-1α and VEGF both in mRNA and protein level.Conclusion:Blocking the CXCR4signaling in hypoxic HUVECs possess an obvious anti-neovascular effect. SummaryInhibition of SDF-1/CXCR4signal pathway reduced hypoxia-induced retinal neovascularization in mice model; downregulated cell proliferation rates, expressions of HIF-1α and VEGF in hypoxic HUVECs. Hypoxia irritates sensitities of CXCR4receptors, the downstream signals are activated, and cytokines related to NVs are synthesized and released into retina microenviroment, inducing RNVs in the end.