[Matrix metalloproteinases inhibitor doxycycline inhibits TGF-beta 1-induced RPE cell migration].

OBJECTIVE: To investigate the effects of matrix metalloproteinases (MMP) inhibitor (Doxycycline) on retinal pigment epithelial (RPE) cell migration induced by transforming growth factor beta 1 (TGF-beta 1). METHODS: The third to sixth passage human RPE cells cultures were treated with TGF-beta 1 at different concentrations (0.01, 0.10, 1.00, 10.00 microg/L), and the conditioned media were collected after 36 h of TGF-beta 1 exposure. Gelatinase activities in conditioned media were analyzed by zymography. Migration assay was performed in a Boyden chamber with addition of different concentrations of Doxycycline. The number of migrated cells was counted under light microscopy. RESULTS: RPE cells migration were stimulated by TGF-beta 1 significantly, the number of migrated RPE cells increased about 27% compared with the control (P < 0.01). In the presence of Doxycycline, the RPE cells migration induced by TGF-beta 1 was inhibited and the number of migrated cells was reduced to 50% - 70% compared with control (P < 0.01). TGF-beta 1 also stimulated the secretion of MMP-2 in dose-dependent manner. CONCLUSIONS: This study indicates that Doxycycline can inhibit migration of RPE cells stimulated by TGF-beta 1. RPE migration induced by TGF-beta 1 may act partially through the stimulation of MMP production.

[The change of the level of the vascular endothelial growth factor in aqueous humor of patients with neovascular glaucoma before and after anterior retinal cryotherapy].

OBJECTIVE: To compare the change of the level of the vascular endothelial growth factor (VEGF) in aqueous humor of patients with neovascular glaucoma (NVG) before and after anterior retinal cryotherapy and investigate the effects of the fluctuation of VEGF on the neovascularization of iris. METHODS: 28 patients with neovascular glaucoma were undergone iris fluorescent angiography to identify the area and amount of new vessels before and after anterior retinal cryotherapy. The neovascularization of iris was observed from 7 to 14 days by iris fluorescent angiography to confirm the regression of new vessels in iris before trabeculectomy. Samples of aqueous humor were obtained before anterior retinal cryotherapy and trabeculectomy, and 30 samples of aqueous humor from patients with senile cataract were collected as normal group. The concentrations of VEGF were measured using enzyme linked immunosorbent assay. RESULTS: the concentration of VEGF (2.096 +/- 0.512) ng/ml in aqueous humor from patients with NVG were much higher than the specimen from the patients pre-trabeculectomy (0.478 +/- 0.312) ng/ml, There was a significant difference between the two groups (t = 17.994, P < 0.01). The mean VEGF concentration of the aqueous humor from patients with senile cataract was (0.198 +/- 0.045) ng/ml which was much lower compared with the samples from patients of pre-trabeculectomy (t = 18.453, P < 0.01). CONCLUSIONS: The concentration of VEGF decline after the regression of new vessels in iris. The results suggest that VEGF play an important role in formation of iris neovascularization. Blockage the release of VEGF might reduce the occurrence of neovascular glaucoma.

[Down-regulation of vascular endothelial growth factor in human retinal pigment epithelial cells by small hairpin loop RNA targeting hypoxia inducible factor-1 alpha under hypoxia condition].

OBJECTIVE: To explore the effect of hypoxia inducible factor-1 alpha (HIF-1 alpha) gene on the expression of vascular endothelial growth factor (VEGF) in human retinal pigment epithelial (hRPE) cells under hypoxia conditions by using small hairpin loop RNA (shRNA) to silence HIF-1 alpha. METHODS: CoCl(2) (150 micromol/L) was used to simulate the hypoxia environment for hRPE cells. After choosing a target site of HIF-1 alpha mRNA, shRNA was designed and synthesized by this target site. hRPE cells were transfected by this shRNA in vitro. Then, these cells were cultured under hypoxia conditions (150 micromol/L CoCl(2)). The mRNA expression of HIF-1 alpha and VEGF was measured by semi-quantitative reverse transcription PCR (RT-PCR). The protein level of HIF-1 alpha and VEGF was studied by western blot analysis. RESULTS: After hRPE cells were transfected by HIF-1 alpha-specific shRNA, RT-PCR showed that the expression of HIF-1 alpha mRNA was inhibited by 77.1%, and western blot analysis showed that the level of HIF-1 alpha protein was significantly decreased in hRPE cells under hypoxia conditions. Moreover, the expression of VEGF mRNA was inhibited by 27.8% and the level of VEGF protein was also significantly decreased in transfected hRPE cells under hypoxia conditions. CONCLUSIONS: Under hypoxia conditions, HIF-1 alpha-specific shRNA effectively keeps HIF-1 alpha gene silenced, and consequently down-regulates VEGF expression against hypoxia. These results suggest that HIF-1 alpha is one of the most important cytokines for retinal neovascularization.

[The inhibitory effects of angiostatin on retinal neovascularization induced by oxygen].

OBJECTIVE: To observe the inhibitory effects of angiostatin on microvascular endothelial cells of mouse retina and test the efficacy of native angiostatin in suppressing experimental retinal neovascularization induced by oxygen. METHODS: Angiostatin was purified with L-lysine Sepharose 4B from human plasma. The primary microvascular endothelial cells from rat retina were cultured. Microvascular endothelial cells growth inhibition assay was carried out with MTT method. Mouse models of hyperoxia-induced ischemic retinopathy were established. Angiostatin or normal saline (NS) were injected into the vitreous in 5 groups: normal, control and various doses. The nuclei of new vessel buds extending from the retina into the vitreous in different groups were counted and compared under the light microscope. RESULTS: Angiostatin could inhibit the growth of microvascular endothelial cells from rat retina in vitro. There were plenty of new vessel buds in the eyes of all mice in hyperoxic condition. The number of the nuclei of new vesselbuds in the murine eyes with injection of angiostatin. They were reduced by 42% (P < 0.01), 57% (P < 0.01) and 82% (P < 0.01) respectively. CONCLUSION: Angiostatin can powerfully inhibit growth of microvascular endothelial cells. The proliferation of retinal vessel may be suppressed by using angiostatin.

[Posterior vitreous detachment induced by intravitreal injection of the combination of chondroitinase ABC and matrix metalloproteinase-3].

OBJECTIVE: To investigate the occurrence of enzymatic induction of posterior vitreous detachment (PVD) by the combination of Chondroitinase ABC (CA) and matrix metalloproteinase-3 (MMP-3), so as to seek a noninvasive and effective pharmacologic approach to facilitate and eventually replace the present mechanical vitreous surgery. METHODS: Twenty-four pigmented rabbits were randomly assigned to two groups of twelve each, the experimental group was treated with CA (0.2 U) and MMP-3 (10 ng) combination, the control group was received equivalent dose of balanced salt solution (BSS). Clinical examinations and electroretinography were performed before and after injection. Over a different period of time, the rabbits were euthanized and killed and their eyes were examined histologically. RESULTS: The foci of partial synchisis and clinically named PVD were recognized for the first time three days after injection. Complete liquefaction was found in every eye of experimental group, and in which, 5/8 eyes developed clinically detected PVD one week after injection Histologic section showed PVD with various extent in 3/7, 7/7, 7/7 eyes of experimental group 30 minutes, 60 minutes and one week after injection respectively, and complete PVD in 0/7, 1/7, 5/7 eyes of experimental group at the same periodic intervals as above. By contrast, no vitreous liquefaction was found and only one eye showed confined partial PVD in the control group. Clinic, electrophysiologic and histologic evaluation in all rabbits revealed no evidence of ocular toxicity. CONCLUSIONS: Vitreous 1iquefaction and PVD can be produced shortly after intravitreal injection using a combination of CA and MMP-3, and the two enzymes were cooperative. Synchisis and weakening of vitreoretinal adherence were almost simultaneously. The dose of 0.2 U CA and 10 ng MMP-3 combination proved to be safe and ideal selection to induce PVD.