Study Of Effect Of The Integrin-linked Kinase In The Retinal Diseases Of Neovascularization

BackgroundThe diseases complicated with retinal neovascularization are major causes for blindness worldwide, including various diseases such as diabetic retinopathy (DRP), retinal vein occlusion (RVO), retinopathy of prematurity (ROP), and idiopathic retinal vasculitis (Eales disease). Vascular damage and ischemia of the retinal tissues are thought to be the basis of the pathological process and then retinal neovascularization may be induced. With immature of structure and poor function, the newly formed blood vessels and proliferating fibrovascular membranes subsequentally result in macular edema and hemorrhage in the retina and vitreous, and exert traction on the retina which can finally be detached. That impairs severely the patient vision, and even causes total loss of vision.Vascular endothelial growth factor (VEGF) is the strongest angiogenic molecule in all specific growth factors known to induce neovascularzition. With its stimulation, retinal vascular endothelial cells proliferate and form new vessels. The application of anti-VEGF therapy, such as Bevacizumab (Avastin ), recently has becom a new method for treatment of retinal and choroidal neovascularzition in addition to laser photocoagulation and vitreous surgery. However, it can not entirely inhibit the occurrence of new blood vessels. In the formation of retinal neovascularzition, the balance of pro- and anti-angiogenic factors is critical, and various molecules may play a role in the process. Recent studies showed that intergrin-linked kinase (ILK) invloves in the angiogenesis in tumor growth and new vessels.The retinal pigment epithelium (RPE) cell is a kind of characterized ones participating in the external barrier of the retina with its tight junctions to maintain the function of the retina, and is also known to be involved in many pathology by secretion of factors, such as hypoxia induced factor-1α(HIF-1α), VEGF and pigment epithelium derived factor (PEDF). These factors are all of importance in retinal neovascularization.Integrin-linked kinase (ILK) is an ankyrin-repeat containing serine/ threonine protein kinase that interacts with the cytoplasmic domain ofβ1 andβ3 integrins. ILK is a downstream substrate of phosphoinositide 3-kinase (PI3K), and an important upstream kinase for the regulation of protein kinase B (PKB/ Akt) and glycogen synthase kinase 3 (GSK-3). It regulates integrin-dependent functions as a crucial regulator of cell responding to signaling mediated by integrins and growth factor. ILK has been implicated in the regulation of cell growth and survival, cell cycle progression, invasion and migration, cell motility and contraction, vascular development, and tumor angiogenesis. The expression and activity of ILK are increased in a range of cancers, and small-molecule inhibitors of ILK activity have been identified and shown to inhibit tumor growth, invasion and angiogenesis. But the expression of ILK in retinal neovascularization has not been documented, and we are interested in weather ILK play a role in DRP and ROP, and the relation between ILK and other angiogenesis factor such as VEGF and HIF-1α. Maybe ILK can be a potential target for treatment of retinal neovescularization.Objective1. To investigate whether ILK is involved in the pathogenesis of PDR by analyzing the expression and the activity of ILK in the preretinal neovascular membranes obtained from patients with PDR during vitrectomy.2. To detect the expression of ILK and intercellular adhesion molecule-1 (ICAM-1) in the RPE cells cultured with high glucose and treated with or without triamcinolone acetonide (TA), and to investigate the possible roles of RPE secreted ILK in the progression of DRP.3. To investigate whether ILK are involved in pathology of oxygen induced retinal neovascularzition model in mice, by analyzing the expression of ILK in the preretinal new vessels and new vessels in the vitreous body.Methods1. Twenty-four preretinal fibrovascular membranes were obtained from the patients with PDR during vitrectomy, of them 12 received Avastin intravitreally before surgery and the other 12 did not. Ten preretinal membranes were obtained from patients with proliferative vitreoretinopathy (PVR) served as controls. The numbers of the nucleus of vascular endothelial cells in the membranes stained with hematoxylin and eosin were counted, and the expressions of ILK, VEGF and HIF-1αin the membranes were detected by immunohistochemical methods.2. The expression of ILK and CAM-1 protein levels in the RPE cells at 1, 2, 6, 12 and 24 h after cultured with high glucose and treated with TA was detected by immunohistochemistry (IHC), immunofluorescence and Western blot. The mRNA levels of ILK and ICAM-1 were also examined by Real-time quantitative reverse transcription polymerase chain reaction (Real-time RT-PCR) in the same time.3. The model of oxygen-induced retinial neovascularzition in mice was established. Fluorescein fundus angiography (FFA) was performed in vivo to identify the pathology, such as permeability, circuity of retinal vessels and nonperfusion area. And the numbers of cells invading into the vitreous were counted in each slice of the model; the protein level of ILK, HIF-1 and VEGF in the retina and the cells in the vitreous were detected by IHC. All of these result compared with the controls which were fed in normal atmosphere.Results1. The immunohistochemical staining revealed that there was positive for ILK expression in all 24 PDR membranes regardless whether or not use of avastin treatment. There was no statistically significance in the amount of expression of ILK between two groups (P=0.346). In the group with avastin, however, the number of endothelial cells in the membranes did reduce (21.5±3.94, n=12), compared to that (41.33±7.44, n=12) of the group without avastin. There was statistically significance in the number of endothelial cells in the membranes between two groups (P=0.003). And the number of cells in PVR membrances was O±1. The levels of ILK and HIF-1αprotein in the group without avastin were higher than that in avastin group (PHIF-1=0.023, PVEGF=0.000).2. IHC, western-blot and immunofluorescence staning revealed that the levels of ILK and ICAM-1 up-regulated 6 h after RPE cells cultured with high glucose (30 mmol/L). Real time RT-PCR analysis showed that the levels of ILK mRNA increased at 2 h, and then decreased at 6 h. The up-regulation of ICAM-1 expression in RPE cells cultured with high glucose could be inhibited by TA. However, TA did not inhibit the up-regulation of ILK expression.3. In the mice model with oxygen induced retinal neovascularzition, FFA showed permeable on retinal vessels, circuity of retinal vessels and nonperfusion areas around the optic disc. The number of endothelial cells in the vitrous of the model was 23.47±8.43, higher than that of the control group (0.12±0.35, P<0.01). Immunohistochemical staining showed increasing expression of ILK, VEGF and HIF-1α.Conclusions1. The expression of ILK in the preretinal membranes in PDR indicate that ILK involves in the pathological process of neovascularization in PDR. Intravitreal injection of Avastin can decrease the numbers of endothelial cells in the proliferative fibrovascular membranes, and it also can down-regulate the expression of VEGF and HIF-1αprotein, but it can not affect the expression of ILK protein. The results suggest that ILK may be the upstream factor of VEGF and HIF-1αduring the process of PDR.2. RPE cell with high glucose condition can up-regulate the expression ILK and ICAM-1, and with the two factors, RPE cells may play a role in the pathology of DRP.3. Strong expression of ILK in the endothelial cells of the new vessels in the vitreous was found in mice model of oxygen induced retinal neovascularzition, indicating ILK invlovement in the pathological process.Novelty1. We first found that ILK was expressed in the 24 examples of preretinal membranes from PDR patients. And first showed that anti-VEGF therapy (Avastin) can significantly reduce the number of endothelial cells in the membranes and down-regulate the expression of HIF-1αand VEGF except ILK.2. We found in the mice model, the ILK was expressed in the neovascularzition pre-retinal and in the vitreous.