| BackgroundCorneal vascularizationIn the absence of disease, the vasculature of the mammalian eye is quiescent in part because of the action of angiogenic inhibitors that prevent vessels from invading the cornea and vitreous. Here, an inhibitor responsible for the avascularity of these ocular compartments is identified as pigment epithelium derived factor (PEDF), a protein previously shown to have neurotrophic activity. The amount of inhibitory PEDF produced by retinal cells was positively correlated with oxygen concentrations, suggesting that its loss plays a permissive role in ischemia-driven retinal neovascularization. These results suggest that PEDF may be of therapeutic use, especially in retinopathies where pathological neovascularization compromises vision and leads to blindness.Angiogenesis, the growth of new blood vessels from preexisting ones, is under tight regulation in most healthy tissues, in part because of the influence of naturally occurring inhibitors that prevent new vessel growth. The disruption of such control mechanisms plays an essential role in the development of a variety of diseases, from arthritis to cancer. In the healthy mammalian eye, vessels are normally excluded from the cornea and the vitreous, compartments that have been shown to have antiangiogenic activity. Failure to exclude vessels from the cornea is associated with loss of visual acuity, opacification, and abnormal healing. In the retina, excessive neovascularization underlies ischemic retinopathies such as proliferative diabetic retinopathy and age-related macular degeneration,currently the leading causes of blindness in the western world.Corneal neovascularization (NV) refers to the growth of blood vessels into physiologically avascular corneal tissue. It can lead to the loss of the immunologic privilege of the cornea and severe visual impairment.Corneal NV is capable of occurring secondary to infection, inflammation, trauma, and postsurgical complications. Typically, ingrowth of the new vessels originates from the limbus and is accompanied by inflammatory responses. Also, another feature of corneal NV is that the blood vessels that grow into the cornea can regress into what are referred to as ghost vessels. Therefore, preventing the occurrence and arresting the progression of the corneal NV is of significance in preventing vision loss.The regulation of angiogenesis has not been fully understood. It is thought that the regulation of angiogenesis involves a complex interplay between proangiogenic and antiangiogenic factors. Accumulating evidence strongly suggests that NV occurs in response to a disturbed balance between proangiogenic factors such as vascular endothelial growth factor (VEGF) and antiangiogenic factors such as pigment epithelium-derived factor (PEDF).The unbalanced expression of these2counterbalancing systems because of increased proangiogenic factors and decreased antiangiogenic factors has been implicated in some neovascular diseases, such as retinopathy of prematurity and proliferative diabetic retinopathy. Our previous study has established the role of the disturbed balance between angiogenic stimulators and inhibitors in the development of pterygia, a corneal NV disease. Therefore, restoration of the balance by either increase of angiogenic inhibitors or suppression of angiogenic stimulators, or both, should lead to the quiescence of NV, which represents a strategy in the prevention and treatment of ocular neovascular diseasesThe function of VEGF in NV has been well characterized. VEGF has been shown to be a key mediator of angiogenesis in many models. PEDF has been identified as a potent endogenous antiangiogenic factor that inhibits the migration and proliferation of vascular endothelial cells. Our previous studies have demonstrated that the normal cornea contains high levels of PEDF, and the PEDF levels are decreased in pterygia, a neovascular disease, suggesting that decreased PEDF levels contribute to NV in pterygia. The present study has evaluated the therapeutic potential of recombinant PEDF on corneal NV induced by chemical cautery in a rat model.The treatment of corneal NV remains a clinical challenge. Laser treatment or photodynamic therapy induces only temporary closure of new vessels. Antiangiogenic therapies, using natural peptides or compounds, are believed to have potential to prevent the onset or arrest the progression of NV. PEDF is a potent natural inhibitor of angiogenesis.It is expressed at high levels in ocular tissues and is considered a key factor responsible for maintaining the avascular status of the cornea, vitreous, and outer retinal layer of the eye. It has been proposed that PEDF is a major antiangiogenic factor essential for corneal avascularity.The estimated human blood concentration of PEDF is about100nM. These findings suggest that the concentration of PEDF used in our experiments was within a physiological range (10mg/mL), which is consistent with a previous study reported by Takenaka. VEGF is an angiogenic stimulator and promotes proliferation and migration of the vascular endothelial cells and enhances vascular permeability. It has been shown to play a key role in pathological NV. Our previous results showed that in pterygia, decreased PEDF levels and increased VEGF levels result in an increased angiogenic stimulator to inhibitor ratio and thus are responsible for the NV in pterygia. The present study demonstrates that there are high expression levels of endogenous PEDF and low levels of VEGF in the normal cornea. The low VEGF: PEDF ratio in the cornea correlates with the avascular status in the normal cornea.Pigment epithelium-derived factor (PEDF), an angiogenesis inhibitor with multiple other functions, balances angiogenesis in the eye and blocks tumor progression. Retinoblastoma, an angiogenesis dependent tumor, is the most common ocular cancer in children without effective treatment.Huasheng Yang’s research suggested that PEDF suppressed tumor growth by blocking angiogenesis instead of a direct cytotoxic effect on tumor cells. Vascular endothelial growth factor (VEGF), a major angiogenic stimulator, was down-regulated by PEDF in both SO-Rb50cells and retinoblastoma xenografts. Hypoxia-inducible factor (HIF)-1a, a crucial transcriptional factor for VEGF expression, was also down-regulated by PEDF both in vitro and in vivo. PEDF reduced HIF-1a nuclear translocation, which may be responsible for the down-regulation of VEGF. Down-regulation of VEGF expression in tumor cells through inhibiting HIF-la, thus attenuating the paracrine effect of VEGF on endothelial cell proliferation and vascular permeability in tumor tissues, may represent a mechanism for the anti-angiogenic activity of PEDF.The role of Fas-FasL in macrophage-related inflammatory reactions was recently discussed in other systems, and, interestingly, several reports demonstrated that inflammatory cells such as macrophages play an important role in laser-induced CNV. Our current results, however, do not directly support the inflammatory hypothesis but merely suggest a role for RPE cells in the modulation of choroidal neovascularization.This finding is in agreement with Kaplan and coworkers, who showed that Fas-deficient(lpr) and FasL-defective (gld) mice had an increased incidence of neovascularization compared with normal mice in a model of laser-induced damage. Here, the massive subretinal neovascularization with uncontrolled growth of vessels was not due to inflammatory cell defects since chimeric mice with FasL-(gld) RPE cells and normal lymphoid (inflammatory) cells developed neovascularization at the same rate and severity as did nonchimeric gld mice.Thus, although our model is closely related to inflammation, it is more likely that the Fas-mediated effect on choroidal neovascularization is mediated by endothelial apoptosis.It is now believed that RPE senescence is an important event in the development of AMD. Under normal conditions, FasL, expressed in the RPE, inhibits any abnormal growth of new vessels from the choroid into the retina. With age, the expression on FasL may be altered or reduced and subretinal neovascularization associated with vision loss occurs. Our study indicates the importance of the FasL-Fas pathway in controlling pathological vessel growth. Besides its relevance as a potential therapeutic target, these results suggest that subretinal neovascularization in AMD patients may result from a lack of an inhibitory function of FasL positive RPE on angiogenesis.Part I Function of PEDF on rat corneal neovascularization and effect of PEDF on the expression of vegf、pedfObjectiveTo explore the inhibitory effect of PEDF on rat corneal neovacularization and observe its effect on the expression of VEGF and PEDF on rat cornea.Method 36healthy SPF SD (Sprague-Dawley) rats were induced corneal neovascularization models by alkali burn. The rats were divided randomly into three groups, group A(normal saline group), group B(PEDF group) and group C (normal rats). The growth of cornea neovascularization(NV) was observed by slit lamp microscope every day and the area of NV were recorded on4th、7th、10th、14th day after the rat cornea alkali burn model was established. After the record, every three rats of each group were euthanized and the whole cornea were removed for immunohistochemistry analyses to measure expression levels of PEDF and VEGF.SPSS13.0statistical package was used for data analysis. The data was recorded by mean±standard deviation. Compare the area of neovascularization at each time point in two groups with repeated measure analysis of variance. Statistical tests were considered significant when P values were less than0.05.ResultOn the4th、7th、10th、14th day, the neovascularization area of group A(normal saline group) were (5.31±0.46) mm2.(16.90±0.06) mm2、(23.45±0.09) mm2、(28.26±0.02) mm2; while the group B(PEDF group) were (2.60±0.15) mm2、(10.07±0.25)mm2,(20.92±0.05)mm2,(25.97±0.04)mm2;in group C (normal rats) no neovascularization was founded. Comparision between groups was statistically significantly different, P<0.05.On the7th day and10th day, by light microscope, there was a large amount of neovascularization and many inflammatory cells in cornea of group B. In contrast, the number of neovascularization in group A was less than that of group B with weaker inflammatory reaction. The amout of new corneal vessels was most on the10th day in both groups,, P<0.05.In normal corneal tissue, VEGF was not expressed in the epithelial basement membrane; the7th day of two groups, the expression of VEGF was mostly intense, P<0.05,the positive reaction to light brown or brown intracytoplasmic granules, and its expression mainly in parts of the formation of new blood vessel. The NV area of PEDF group were markedly reduced compared with normal saline group, P<0.05, and the expression of VEGF was always less intense in group A than group B, P<0.05.In normal corneal tissue, PEDF was weak expression in the epithelial basement membrane; the10th day of two groups, the expression of PEDF was mostly intense, the positive reaction to light brown or brown intracytoplasmic granules, and its expression mainly in parts of the formation of new blood vessel. The NV area of PEDF group were markedly reduced compared with normal saline group, and the expression of PEDF was always much more intense in group A than group B, P<0.05.From the experiment, we con observe that the new vessels grew in blossom from7th to10th day, and then begain to decay,and VEGF expressed most intensely on the7th day,and follows a upward expression tendency from0th to7th day,while a downward expression tendency from7th to14th day after the alkaline burn model was built. Coincidently, the expression of PEDF reached the peak value on the10th day and followed a same up and down variation tendency. And the turning point was precisely the10th day.The VEGF/PEDF ratio kept rising from0th to10th day while was less than1all along with new vessels kept growing from limbus to the centra of rat cornea to a maximum on the10th day,while afterwards, reversion occurred,the ratio came to be morn than1with the new vessels begain to decline.ConclusionThe contrast of NV area in two groups shows that PEDF by the local application is effective in inhibition of corneal neovascularization induced by alkali burn.The correlation of expression variation of PEDF and VEGF might underly that the mecanlism of PEDF in inhibition of corneal neovasculation can be associated with suppressing the expression of VEGF and promoting the autocrine of PEDF. The ratio of VEGF/PEDF decided the generation and closure of corneal neovasculariation.Part Ⅱ Effect of PEDF on the expression of Fas/FasL and apoptosis of new vessel endothelial cells in rat CNV model induced by alkali burnObjectiveTo detect the effects of topical application of PEDF on the expression of Fas/FasL and apoptosis of new vessel endothelial cells(determined by TUNEL) on rat NV model. To explore the possible mechanisms of PEDF in inhibiting corneal neovascularization..Method36healthy SPF SD (Sprague-Dawley) rats were induced corneal neovascularization by alkali burn. The rats were divided randomly into three groups, group A(saline group), group B(PEDF group) and group C (normal rats). After the record, every three rats of each group were euthanized and the whole cornea were removed for immunohistochemistry analyses to measure expression levels of Fas and FasL. And the sample was also used to analysis the apoptosis of new vessel endothelial cell by TUNEL method.SPSS13.0statistical package was used for data analysis. The data was recorded by mean±standard deviation. Fas、FasL expression of groups and amount of apoptosis cells use independent sample t test at each time point in two groups.The apoptosis index use rank sum test. Statistical tests were considered significant when P values were less than0.05.Result In normal corneal tissue, Fas was weak expression in the epithelial basement membrane; on the10th day of two groups, the expression of Fas was mostly intense,P<0.05,with the positive reaction to light brown or brown intracytoplasmic granules, and its expression mainly in parts of the formation of new blood vessel. The NV area of Fas group were significantly smaller than normal saline group, P<0.05, and the expression of Fas was always much more intense in group A than group B, P<0.05.The expression of Fas kept rising to a maximum on the10th day and began to decline afterwards to the14th day.In normal corneal tissue, FasL was weakly expressed in the epithelial basement membrane; on the10th day of two groups, the expression of FasL was mostly intense, P<0.05,with the positive reaction to light brown or brown intracytoplasmic granules, and its expression mainly in parts of the formation of new blood vessel. The expression of FasL was always much more intense in group A than group B, P<0.05.The expression of Fas also kept rising to a maximum on the10th day and began to decline afterwards to the14th day.In normal corneal tissue, no apoptotic new vessel endothelial cells could be detected. After the alkali burn model was bulilt, apoptotic cells could be detected in both groups,but there was always more new vessel endothelial cells found to be in apoptosis status in group A than in group B at each time point, P<0.05.The apoptosis index of two groups analysed by rank sum test proved that the AI of group A is always smaller than group B, P<0.05. The apoptotic endothelial cells counted to be maximum on the10th day in both groups, P<0.05.ConclusionThe growth of corneal neovascularition run agagainst with the existence of Fas and FasL, which may be the result of induction on the apoptosis of new vessel endothelial cells in the alkaline burn cornea.The expression trend of Fas/FasL is the same with PEDF and opposite with VEGF might underly that the topical application of PEDF in corneal neovascularization may be related with promoting the expression of Fas/FasL resulted in the apoptosis of new vessel endothelial cells. |
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