| Backgrounds Choroidal neovascularization (CNV) secondary to agerelated macular degeneration (AMD) causes severe visual loss in older adults.As life expectancy increases and the global population ages, CNV, especiallythe one secondary to AMD, is increasing as a socio-medical problem. There isgrowing evidence that CNV involves both angiogenesis (the development ofnew blood vessels from the local vasculature) and vasculogenesis (defined as therecruitment and in situ differentiation of vascular endothelial cells fromcirculating bone marrow-derived cells, BMCs). Bone marrow-derivedmsenchymal stem cells (BMSCs), a subset of BMCs, possess multilineagedifferentiation potential and probably can serve as a carrier for cell-basedtherapies for CNV.A number of genetic and environmental factors have been identified risk for neovascular AMD. Diabetes mellitus (DM), a common chronic disease, hasgradually elevated in incidence and prevalence year by year and beenemphasized as a global public health problem. Recent epidemiologicalinvestigations showed that diabetes might have an association with neovascularAMD, however, this concept is still in debate now. The reduction anddysfunction of BMCs and BMSCs in diabetic patients has been extensivelyreported, but there is no report on the influence of hyperglycemia oncontribution of BMCs and BMSCs to CNV.Aims To investigate the influence of hyperglycemia on contribution ofBMCs and BMSCs to CNV and try to detect underlying molecular mechanisms.Methods The wild-type C57BL/6J mice were randomly divided into threegroups: the non-diabetic control group, the streptozotocin induced diabetic (DM)group and diabetes post-treated with insulin (DM+INS) group. In diabetes groupand insulin post-treated group, mice were injected intraperitoneally withstreptozotocin (60mg/kg/d) for5days to induce diabetes.7days after the fifthinjection, mice with glucose levels>16.7mol/l were included in the study. Aftersuccessfully induced diabetes, mice in DM+INS group were subcutaneousinjected with10U/kg/d of insulin until the end of the experiment, and the bloodglucose levels were monitored. CNV was induced by laser photocoagulation.The CNV severity was evaluated by fundus fluorescence angiography (FFA),choroidal flatmount examination and hematoxylin&eosin staining (HE) on day14after photocoagulation. The expression of vascular endothelial growth factor(VEGF) and stromal cell derived factor-1(SDF-1) were examined by enzymelinked immunosorbent assay (ELISA) and quantitative real-time polymerasechain reaction (qRT-PCR) on day3after photocoagulation.Green fluorescent protein (GFP) chimeric mice were developed by transplanting bone marrow cells from GFP transgenic mice to C57BL/6J mice.The degree of chimerism was detected by flow cytometry. The GFP chimericmice were randomly divided into two groups: the control group and the DMgroup. Choroidal flatmount and immunofluorescence were performed tomeasure GFP positive cells (GFP cells) recruitment and the expression of VEGFand SDF-1on day3after photocoagulation. BMCs expression of vascularendothelial cell marker CD31, vascular smooth muscle cell marker α-SMA andmacrophage cell marker F4/80in CNV were also examined byimmunofluorescence to see the differentiation status on day14afterphotocoagulation.Isolation and characterization of C57BL/6J mice BMSCs was carried outwith standard protocols. Oil red O staining and Alizarin Red S staining wereused to reveal the lipid accumulation and calcium deposition respectively. Thethird-passage BMSCs were transduced with adenoviral vectors carrying GFPreporter gene (Ad-GFP) and the transduction efficiency was observed at24hourafter infection. The Ad-GFP transduced BMSCs (2×106per mouse) wereinjected into mice via tail veins several hours after lasering and choroidalflatmount was performed to measure BMSCs recruitment in CNV lesions on day3after lasering. Human BMSCs was also cultured and identified. BMSCs-RPEcells co-culturing system was established. Then the migration of BMSCs wasobserved under three kinds of culture condition in which low glucose media (D-5.5mmol/l glucose), high glucose media (D+,25mmol/l glucose) or osmoticpressure equal media (M+,5.5mmol/l glucose plus19.5mmol/l mannitol) wasadded. The levels of VEGF and SDF-1α in coculture medium were examined byELISA, and the VEGF and SDF-1mRNA in RPE cells were examined byqRT-PCR. Results The CNV severity in DM group significantly increased comparedwith that in control group and DM+INS group (p <0.05). Insulin treatment couldpartly lessen the CNV severity caused by hyperglycemia (p <0.05). However,The CNV severity in DM+INS group was still greater than control group (p<0.05). Compared with the control group and DM+INS group, the expression ofVEGF and SDF-1detected by ELISA and qRT-PCR increased in DM group (p<0.05). Insulin treatment could partly lessen the high level of VEGF and SDF-1caused by hyperglycemia (p <0.05). However, the expression of VEGF andSDF-1in DM+INS group was still higher than control group (p <0.05).The chemeric rate was higher than85%in chemeric mice. The chemericmice in DM group demonstrated increased GFP cells area, ratio of GFP cellsarea/CNV area, GFP cells, expression of VEGF and SDF-1and ratio of BMCsexpressing CD31+and F4/80+in CNV (p <0.05). However, the ratio of BMCsexpressing α-SMA changed weakly.Success in culturing the mouse and human BMSCs are confirmed byplastic-adherent BMCs and capacity to differentiate into the adipogenic andosteogenic lineages in vitro. The mouse BMSCs transduction efficiency ofAd-GFP at24hours is approximately70%. More BMSCs recruited into CNVlesions in DM group than that in control group (p <0.05). The migration ofBMSCs increased in co-culturing system under high glucose media, and VEGFand SDF-1protein and mRNA production were enhanced under high glucose invitro (p <0.05).Conclusions Hyperglycemia of diabetes could aggravate CNV severityand increase the recruitment and incorporation of bone marrow derived cells into CNV. The increase of VEGF and SDF-1by RPE cells may be attributed tothe aggravation of CNV development. |
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