Primary Study On The Effects Of Endothelial Progenitor Cells On Repairment Of The Optic Nerve Damage Induced By Ocular Hypertension

Part1:Changes of vascular endothelial progenitor cell in patients with acute angle-closure glaucomaObjective:To investigate endothelial progenitor cells (EPCs) change in patients with acute primary angle-closure glaucoma (APACG) and explore the effects of EPCs on repairment of the optic nerve damage induced by ocular hypertension in patients with APACG.Methods:A prospective cohort study was designed. Thirty patients with APACG were enrolled in Tianjin Medical University General Hospital as APACG group, and20normal subjects served as control group. Periphery blood samples were obtained from all the patients, and then stained with saturating concentrations of monoclonal antibodies, FITC-conjugated anti-CD34and CD133mAb. EPCs identified by CD34, CD133were enumerated by flow cytometry and the correlation between EPCs change and its relative factors (including degree of optic nerve damage, intraocular pressure after medicine treatment, limosis blood sugar, blood lipid, course of glaucoma, and with cardiovascular disease or not). Informed consent was obtained from each subject before any medical procedure.Results:There was no significant difference in age, gender, vascular-related risk factor, blood biochemical indicators between APACG group and normal control group (P>0.05), but a higher intraocular pressure (IOP) was displayed in APACG group compared with normal control group (P<0.001). The numbers of EPCs was (48±22) cells/ml in APACG group and (65±20) cells/ml in normal control group with a significant difference between them (P=0.004). In APACG group, the EPCs numbers were (60±19) cells/ml and (34±7) cells/ml, respectively in phase1and phase3of optical nerve damage (Z=-3.015, P=0.002). There was a negative correlation between EPCs numbers and baseline IOP within a certain range (r=-0.835, P<0.05). However, no obvious correlations were seen between EPCs numbers and blood lipid Level, blood glucose level or glaucoma course (r=0.343, P=0.227; r=-0.203, P=0.419; r=0.198, P=0.610). The EPCs numbers were in APACG patients with cardiovascular disease were (56±22) cell/ml without ones were (35±15) cells/ml (P=0.005).Conclusion:The numbers of EPCs decrease in APACG patient. These results implied that the pathological factors have some effects on circulating EPCs in APACG occurrence and development.Part2:Changes of retinal ganglion cells apoptosis and circulating endothelial progenitor cells quantity in rat intraocular hypertension model Objective:To observe the changes of circulating EPCs quantity and retinal ganglion cells (RGCs) apoptosis in rat intraocular hypertension model, and to explore the role of EPCs in the pathogenesis of glaucomatous optic nerve damage.Methods:Intraocular hypertension model was induced by argon laser coagulation at limbus vein and superscleral vein. The intraocular pressure (IOP) was measured and recorded before and after coagulation3days,7days,3weeks,2months and3months.20rats’eyes (20rats) were in normal control group,30rats’ eyes (30rats) were in laser control group (with normal IOP), and50rats’eyes (50rats) were in intraocular hypertension model group. Peripheral blood EPCs (CD34+/CD133+) were enumerated by flow cytometry, retinal histopathological changes were detected by HE staining, retinal apoptosis were detected by TUNEL staining, and Bcl-2/Bax were tested by immunohistochemistry in normal control group, laser control group after coagulation3days,7days,3weeks (10rats each group), and intraocular hypertension model group after coagulation3days,7days,3weeks,2months and3months (10rats each group).Results:(1) Before coagulation, the average rats’IOP was9.9±1.2mmHg. IOP began to increase immediately after laser coagulation at limbus vein and superscleral vein. The IOP was37.4±1.5mmHg, and reached top after coagulation3days. Then IOP decreased gradually, and was higher8mmHg more than normal at3months. The successful rate was100%.(2) Circulating EPCs was (62.1±13.1)/ml in normal control group, and was (121.3±22.4)/ml and reached top at intraocular hypertension3days. Then it began decreasing and was (81.3±23.7)/ml at7days, and reached the bottom at3weeks (46.1±15.8)/ml. Then it increased slowly, was (54.4±19.1) and (54.7±15.9)/ml at2months and3months, and had the trend lower than normal.(3) Compared with normal control group, the retinal structure in HE staining had no significance in intraocular hypertension3days,7days, and3weeks. While in intraocular hypertension2months, the retina became thinner, the outer nuclear layer for the most, and the quantity of ganglion cells decreased significantly. While in intraocular hypertension3months, the retina became thinner more, the outer nuclear layer for the most, and the retinal ganglion cells became fewer and more disorganized.(4) Compared with normal control group, the retinal positive cell rate of the Bax immunohistochemical staining had no significance in intraocular hypertension3days and7days, and increased significantly in intraocular hypertension3weeks,2months and3months. The retinal positive cell rate of the Bcl-2immunohistochemical staining had no significance in intraocular hypertension3days,7days, and3weeks, and increased significantly in intraocular hypertension2months and3months. The retinal TUNEL positive cell rate increased gradually along with time of elevated IOP in rat intraocular hypertension model group.Conclusions:The rat intraocular hypertension model is successfully induced by laser coagulation at limbus vein and superscleral vein. Elevated IOP has time-dependent effect on circulating EPCs. Along with time of elevated IOP, the retinal damage became more serious, retinal photoreceptor cell decreased gradually; retinal apoptotic cells increased and apoptotic cells were distributed in RGCs layer, inner molecular layer, inner nuclear layer and outer nuclear layer. These results implied that EPCs might play role during the RGCs apoptosis and optic nerve damage induced by intraocular hypertension.Part3:Changes of retinal HIF-la and peripheral blood VEGF-A, SDF-1, NO, and ET-1and the relationship with circulating endothelial progenitor cells quantity in rat intraocular hypertension modelObjective:To observe the changes of retinal hypoxia-inducible factor-la (HIF-la) expression and peripheral blood vascular endothelial growth factor-A (VEGF-A), stromal cell-derived factor-1(SDF-1), nitric oxide (NO), and endothelin-1(ET-1) and the relationship with circulating EPCs quantity in rat intraocular hypertension model. To explore the effects of HIF-la, VEGF-A and SDF-1on EPCs mobilization and recruitment in intraocular hypertension, and the influences of EPCs to vascular endothelial function.Methods:Intraocular hypertension model was induced by argon laser coagulation at limbus vein and superscleral vein.20rats (20eyes) were in normal control group,20rats (20eyes) were in laser control group (with normal IOP), and150rats (150eyes) were in intraocular hypertension model group. Peripheral blood VEGF-A, SDF-1, NO, and ET-1were detected by ELISA in normal control group (10rats), laser control group (10rats), and intraocular hypertension model group after coagulation3days,7days,3weeks,2months and3months (20rats each group).Retinal HIF-1α protein were detected by western blot in normal control group (10rats), laser control group (10rats), and intraocular hypertension model group after coagulation3days,7days,3weeks,2months and3months (10rats each group).Results:(1) HIF-1α protein expression was low in normal retina. While IOP elevated3days, HIF-1α protein expression rose significantly, and reached top at7days, then began decreasing, but was still above normal control group.(2) Peripheral blood VEGF-A was84.89±4.89ng/L,59.24±6.37ng/L,58.36±5.68ng/L,54.98±5.91ng/L,59.99±5.12ng/L, and64.78±5.94ng/L in normal control group and intraocular hypertension3days,7days,3weeks,2months and3months. Peripheral blood VEGF-A was decreased significantly in intraocular hypertension model group than normal control group (P<0.001). (3) Peripheral blood SDF-1was229.43±11.61pg/ml,218.34±11.33pg/ml,298.51±32.02pg/ml,294.16±11.90pg/ml,301.10±14.61pg/ml, and307.49±20.46pg/ml in normal control group and intraocular hypertension3days,7days,3weeks,2months and3months. Peripheral blood SDF-1was increased significantly in intraocular hypertension7days,3weeks,2months and3months than normal control group and intraocular hypertension3days (P<0.001).(4) Peripheral blood NO was36.74±5.19μmol/L,32.87±4.30μmol/L,45.67±4.48μmol/L,41.29±5.00μmol/L,41.04±5.15μmol/L, and41.45±4.82μmol/L in normal control group and intraocular hypertension3days,7days,3weeks,2months and3months. Peripheral blood NO was decreased significantly in intraocular hypertension3day (P=0.038), and was increased significantly in intraocular hypertension7days,3weeks,2months and3months than normal control group (P<0.05).(5) Peripheral blood ET-1was72.73±6.41ng/L,57.96±4.42ng/L,70.86±12.14ng/L,77.37±9.95ng/L,76.57±5.85μg/L, and77.41±7.43ng/L in normal control group and intraocular hypertension3days,7days,3weeks,2months and3months. Peripheral blood ET-1was decreased significantly in intraocular hypertension3day than normal control group (P<0.001).Conclusions:Elevated IOP makes retina anoxia, retinal HIF-1α protein expression increased, promotes EPCs mobilization from marrow, peripheral blood VEGF-A reduced, SDF-1increased, and then promotes EPCs migration to anoxic retina. Circulating EPCs, NO, and ET-1change in time-dependent manner. These results implied that HIF-1α, VEGF-A and SDF-1might play some role in repairment of the optic nerve damage induced by intraocular hypertension through participating in EPCs mobilization and migration, and influence vascular endothelial function.Part4:Effects of Simvastatin on retinal ganglion cells apoptosis, circulating endothelial progenitor cells quantity and retinal HIF-la, peripheral and retinal VEGF-A, SDF-1, NO, and ET-1in rat intraocular hypertension modelObjective:To observe the effects of Simvastatin on RGCs apoptosis, circulating EPCs quantity, retinal HIF-1α expression, retinal and peripheral blood VEGF-A, SDF-1, NO, and ET-1in rat intraocular hypertension model. To explore the possible role of Simvastatin on glaucoma optic nerve repair by enhancing EPCs mobilization.Methods:10rats (10eyes) were in normal control group,30rats (30eyes) were in intraocular hypertension model group,30rats (30eyes) were in intraocular hypertension with Simvastatin (SV) group, and30rats (30eyes) were in intraocular hypertension with normal saline (NS) group. Rats of SV group were begun to be filled with20mg/kg/d Simvastatin after intraocular pressure (IOP) increased. Rats of NS group were begun to be filled with1ml/d NS after IOP increased. IOP was measured and peripheral blood EPCs (CD34+/CD133+) were enumerated by flow cytometry in SV group after coagulation3days,7days and3weeks. Retinal histopathological changes were detected by HE staining, retinal apoptosis were detected by TUNEL staining, and Bcl-2/Bax were tested by immunohistochemistry in SV group after3weeks. IOP was measured and peripheral blood EPCs (CD34+/CD133+) were enumerated by flow cytometry in NS group after coagulation3days,7days and3weeks.30rats (30eyes) were in normal control group,50rats (50eyes) were in intraocular hypertension model group,50rats (50eyes) were in SV group, and30rats (30eyes) were in NS group. Peripheral blood VEGF-A, SDF-1, NO, and ET-1were detected by ELISA in normal control group (10rats), intraocular hypertension model group (20rats), SV group (20rats) and NS group (10rats) after medicine filling3weeks. Retinal HIF-1α protein was detected by western blot in normal control group (10rats), intraocular hypertension model group (10rats), SV group (10rats) and NS group (10rats) after medicine filling3weeks. Retinal VEGF-A, SDF-1, NO, and ET-1were detected by ELISA in normal control group (10rats), intraocular hypertension model group (20rats), SV group (20rats) and NS group (10rats) after medicine filling3weeks.Results:(1) The average IOP was37.4±1.5mmHg,31.8±4.1mmHg and25.9±2.2mmHg in intraocular hypertension3days,7days and3weeks. The average IOP was38.0±3.7mmHg,33.9±2.9mmHg,24.9±3.2mmHg in SV group after medicine filling3days,7days and3weeks. Simvastatin had no significant effect on IOP of rats (P>0.05).(2) Circulating EPCs was (121.3±22.4),(81.3±23.7) and (46.1±15.8) in rat intraocular hypertension3days,7days and3weeks. Circulating EPCs was (138.5±41.4),(77.9±22.0) and (65.0±13.6) in SV group after medicine filling3days,7days and3weeks. Simvastatin made circulating EPCs significantly increased than intraocular hypertension model group at3days and3weeks (P<0.05).(3) Compared with intraocular hypertension model group, the configuration of all retinal layers in HE staining were more orderly, Bcl-2positive cells rate had no significant change, Bax and TUNEL positive cells rate reduced significantly in SV group.(4) Peripheral blood VEGF-A was83.75±4.76ng/L,53.47±5.36ng/L,80.35±8.62ng/L, and54.29±5.28ng/L in CON, OH, SV, and NS group. Peripheral blood VEGF-A was increased significantly in SV group than OH group (P<0.001). Retinal VEGF-A was43.43±5.33ng/L,23.54±5.38ng/L,32.83±5.64ng/L, and22.60±5.98ng/L in CON, OH, SV, and NS group. Retinal VEGF-A was increased significantly in SV group than OH group (P<0.001).(5) Peripheral blood SDF-1was230.62±11.27pg/ml,296.28±12.14pg/ml,265.74±39.71pg/ml, and291.72±12.21pg/ml in CON, OH, SV, and NS group. Peripheral blood SDF-1was decreased significantly in SV group than OH group (P=0.001). Retinal SDF-1was227.41±18.38pg/ml,187.72±19.19pg/ml,212.14±23.71pg/ml, and185.63±19.26pg/ml in CON, OH, SV, and NS group. Retinal SDF-1was increased significantly in SV group than OH group (P<0.001).(6) Peripheral blood NO was36.26±4.95μmol/L,41.16±4.97μmol/L,44.31±4.14μmol/L, and40.94±4.31μmol/L in CON, OH, SV, and NS group. Peripheral blood NO was increased significantly in SV group than OH group (P=0.044). Retinal NO was38.54±3.53μmol/L,32.20±3.74μmol/L,30.52±3.94μmol/L, and31.16±3.36μmol/L in CON, OH, SV, and NS group. There was no significant difference in retinal NO between SV and OH group (P=0.159).(7) Peripheral blood ET-1was72.68±6.35ng/L,77.84±7.98ng/L,87.62±5.49ng/L, and78.13±7.66ng/L in CON, OH, SV, and NS group. Peripheral blood ET-1was increased significantly in SV group than OH group (P<0.001). Retinal ET-1was53.13±2.07ng/L,28.39±3.33ng/L,39.09±4.81ng/L, and28.20±2.65ng/L in CON, OH, SV, and NS group. Retinal ET-1was increased significantly in SV group than OH group (P<0.001).Conclusions:Simvastatin enhances peripheral blood and retinal VEGF-A through promoting retinal HIF-1α protein expression, accelerates circulating EPCs mobilization, makes retinal SDF-1increased, then promotes EPCs migration to anoxic retina, repairs vascular endothelium, lightens the retinal damage, decreases the retinal apoptotic cells. These results implied that Simvastatin might play a beneficial role on glaucoma optic nerve repair by enhancing EPCs mobilization, and become a new treatment of glaucoma.