整合素连接激酶（ILK）在视网膜新生血管疾病中的作用研究
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摘要
合并视网膜新生血管性的疾病是世界主要的致盲原因,包括糖尿病性视网膜病变(DRP)、视网膜静脉阻塞、早产儿视网膜病变和特发性视网膜血管炎(Eales病)等多种疾病。其基本病理过程是原发病造成的视网膜血管损伤和视网膜组织缺血,并可诱发视网膜新生血管生成。结构和功能不良的新生血管以及纤维血管增生膜随之引起黄斑水肿、视网膜和玻璃体内出血、对视网膜施加牵拉最终可形成视网膜脱离,损害患者的视力,甚至导致失明。
在促血管生成因子中,血管内皮生长因子(VEGF)是已发现的作用最强的血管生成分子,在它的作用下,视网膜血管内皮细胞增生,形成新生血管。抗VEGF疗法(anti-VEGF,如Bevacizumab,商品名Avastin)的应用是继激光光凝和玻璃体手术后的一个治疗视网膜新生血管的新方法。但是它并不能完全抑制新生血管的发生。在视网膜血管生成中,各种促血管生成和抑制血管生成的因子的平衡是关键,众多的血管作用因子在视网膜新生血管生成中发挥作用。近年的研究显示整合素连接激酶(ILK)参与了肿瘤生长和新生血管生成过程。
视网膜色素上皮细胞(RPE)是一种有紧密连接、参与视网膜外屏障、保持视网膜功能的特化细胞,也已知通过分泌如缺氧诱导因子1α(HIF-1α)、VEGF和色素上皮衍生因子(PEDF)参加许多病理过程,而这几种分子都是与视网膜新生血管有关的重要分子。
ILK是细胞内的一种苏氨酸/丝氨酸(Thr/Ser)激酶,以它的COOH端与跨膜蛋白整合素的β1,β3结合,介导细胞外基质蛋白的信号传递。它是磷脂酰肌醇(-3)激酶(Pi3K)的作用底物,是蛋白激酶B(PKB/Akt)和糖原合成酶激酶3(GSK-3)重要的上游激酶。ILK连接整合素到肌动蛋白细胞骨架,转导从整合蛋白到细胞外基质和各种亚细胞成分的信号以及从细胞外环境到细胞内的信号。它与整合素和生长因子结合,参与了贴壁细胞的生长、增殖、移行,在肿瘤组织中高表达,抑制其活性能减弱肿瘤的侵袭和血管生成。然而在视网膜的新生血管发生过程中是不是同时也有ILK的参与,它和已知的血管生成因子之间的关系如何是我们感兴趣的问题。本实验的目的就是检测ILK在视网膜新生血管的表达,研究ILK在新生血管疾病中可能的作用,期望为治疗视网膜新生血管性疾病提供新的思路。
目的
1.检测增生性糖尿病视网膜病变(PDR)新生血管膜中的血管内皮细胞的数量以及HIF-1α、VEGF、ILK的表达,观察应用玻璃体内注射Avastin后对其的影响,以了解ILK是否参与了DRP的病理过程。
2.检测高糖和缺氧条件下分别诱导的视网膜色素上皮细胞ILK和ICAM-1的表达,以了解RPE是否也能表达ILK和ICAM-1,并参与糖尿病性视网膜病变的病理过程。
3.检测氧致视网膜新生血管的小鼠模型眼内ILK的表达,以了解ILK是否也是参与的分子之一。
方法
1.玻璃体切除术中取出的24例PDR患者的视网膜前新生血管膜,其中术前玻璃体内注射Avastin者12例,未使用Avastin治疗者12例;以10例增生性玻璃体视网膜病变(PVR)的视网膜前增生膜作为对照。HE染色计数增殖膜内的血管内皮细胞数量,免疫组织化学染色检测新生血管膜内HIF-1α、VEGF和ILK的蛋白表达量。
2.培养人RPE细胞,采用免疫荧光染色法、免疫细胞化学染色法和western-blot法检测在高糖条件下、有无曲安奈德(TA)作用时,RPE细胞ILK和ICAM-1的表达,实时荧光定量PCR(real time-PCR)检测两者的mRNA表达,免疫组化检测蛋白表达。
3.建立氧致视网膜新生血管的小鼠模型,活体内荧光素眼底血管造影显示血管病变,如渗漏、血管迂曲和无灌注区形成;在病理切片计数进入玻璃体腔内血管内皮细胞数量;免疫组化法检测ILK、HIF-1、VEGF在视网膜新生血管中和侵入玻璃体的细胞团内的表达。
结果
1.免疫组化半定量检测显示,在所有的24个PDR增生膜中,都有ILK的表达,无论是否使用Avastin,其表达量没有显著性差异(P=0.346)。但在Avastin组,PDR新生血管增殖膜中的血管内皮细胞数明显减少,为21.5±3.94个(n=12),相比之下,未使用Avastin组的细胞数为41.33±7.44个(n=12),P=0.003,具有统计学显著性差异。PVR视网膜前膜中的细胞数为0。在未使用Avastin的PDR增殖膜中,血管内皮表达HIF-1α和VEGF的量明显高于Avastin组,具有统计学显著性差异(PHIF-1=0.023, PVEGF=0.000)。
2.免疫组化、western-blot和免疫荧光染色都显示RPE细胞在高糖培养6 h高表达ILK、ICAM-1蛋白,而实时荧光PCR检测显示在2 h其mRNA表达量开始上调,在6 h下降。高糖对RPE表达ICAM-1的上调作用能被糖皮质激素TA抑制,而ILK的上调不受其抑制。
3.在氧致视网膜新生血管小鼠模型,荧光素血管造影显示视网膜血管迂曲增粗,造影剂渗漏,有无灌注区形成。视网膜切片显示模型眼视网膜前的血管内皮细胞数量增加(23.47±8.43比对0.12±0.35, P<0.01),免疫组化染色显示其高表达HIF-1α、VEGF和ILK。
结论
1.人PDR增殖膜中的新生血管有ILK表达,提示ILK参与了视网膜新生血管的形成过程;玻璃体内注射Avastin治疗PDR,能够减少视网膜新生血管内皮细胞数量,下调HIF-1α、VEGF在增殖膜的表达,但不能下调ILK的表达。在视网膜新生血管发生中,ILK可能是HIF-1α、VEGF的上游因子。
2.RPE细胞在高糖环境下能高表达ILK和ICAM-1,RPE细胞可能通过这两种因子的信号途径而参与DRP的病变。
3.在氧致视网膜新生血管小鼠模型中,新生血管的内皮细胞高表达ILK,提示ILK参与了类似的病理改变。
创新点
1.首次在24例人PDR增生膜中检测到ILK的表达;首次证实抗VEGF疗法(Avastin玻璃体内注射)能明显减少PDR新生血管膜内的血管内皮细胞数量和下调HIF-1α、VEGF的表达,但不能下调ILK的表达。
2.首次在氧致视网膜新生血管小鼠模型中检测到ILK的表达,提示ILK参与了视网膜新生血管的发生过程。
Background
The 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.
Objective
1. 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.
Methods
1. 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.
Results
1. 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.
Conclusions
1. 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.
Novelty
1. 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.
在促血管生成因子中,血管内皮生长因子(VEGF)是已发现的作用最强的血管生成分子,在它的作用下,视网膜血管内皮细胞增生,形成新生血管。抗VEGF疗法(anti-VEGF,如Bevacizumab,商品名Avastin)的应用是继激光光凝和玻璃体手术后的一个治疗视网膜新生血管的新方法。但是它并不能完全抑制新生血管的发生。在视网膜血管生成中,各种促血管生成和抑制血管生成的因子的平衡是关键,众多的血管作用因子在视网膜新生血管生成中发挥作用。近年的研究显示整合素连接激酶(ILK)参与了肿瘤生长和新生血管生成过程。
视网膜色素上皮细胞(RPE)是一种有紧密连接、参与视网膜外屏障、保持视网膜功能的特化细胞,也已知通过分泌如缺氧诱导因子1α(HIF-1α)、VEGF和色素上皮衍生因子(PEDF)参加许多病理过程,而这几种分子都是与视网膜新生血管有关的重要分子。
ILK是细胞内的一种苏氨酸/丝氨酸(Thr/Ser)激酶,以它的COOH端与跨膜蛋白整合素的β1,β3结合,介导细胞外基质蛋白的信号传递。它是磷脂酰肌醇(-3)激酶(Pi3K)的作用底物,是蛋白激酶B(PKB/Akt)和糖原合成酶激酶3(GSK-3)重要的上游激酶。ILK连接整合素到肌动蛋白细胞骨架,转导从整合蛋白到细胞外基质和各种亚细胞成分的信号以及从细胞外环境到细胞内的信号。它与整合素和生长因子结合,参与了贴壁细胞的生长、增殖、移行,在肿瘤组织中高表达,抑制其活性能减弱肿瘤的侵袭和血管生成。然而在视网膜的新生血管发生过程中是不是同时也有ILK的参与,它和已知的血管生成因子之间的关系如何是我们感兴趣的问题。本实验的目的就是检测ILK在视网膜新生血管的表达,研究ILK在新生血管疾病中可能的作用,期望为治疗视网膜新生血管性疾病提供新的思路。
目的
1.检测增生性糖尿病视网膜病变(PDR)新生血管膜中的血管内皮细胞的数量以及HIF-1α、VEGF、ILK的表达,观察应用玻璃体内注射Avastin后对其的影响,以了解ILK是否参与了DRP的病理过程。
2.检测高糖和缺氧条件下分别诱导的视网膜色素上皮细胞ILK和ICAM-1的表达,以了解RPE是否也能表达ILK和ICAM-1,并参与糖尿病性视网膜病变的病理过程。
3.检测氧致视网膜新生血管的小鼠模型眼内ILK的表达,以了解ILK是否也是参与的分子之一。
方法
1.玻璃体切除术中取出的24例PDR患者的视网膜前新生血管膜,其中术前玻璃体内注射Avastin者12例,未使用Avastin治疗者12例;以10例增生性玻璃体视网膜病变(PVR)的视网膜前增生膜作为对照。HE染色计数增殖膜内的血管内皮细胞数量,免疫组织化学染色检测新生血管膜内HIF-1α、VEGF和ILK的蛋白表达量。
2.培养人RPE细胞,采用免疫荧光染色法、免疫细胞化学染色法和western-blot法检测在高糖条件下、有无曲安奈德(TA)作用时,RPE细胞ILK和ICAM-1的表达,实时荧光定量PCR(real time-PCR)检测两者的mRNA表达,免疫组化检测蛋白表达。
3.建立氧致视网膜新生血管的小鼠模型,活体内荧光素眼底血管造影显示血管病变,如渗漏、血管迂曲和无灌注区形成;在病理切片计数进入玻璃体腔内血管内皮细胞数量;免疫组化法检测ILK、HIF-1、VEGF在视网膜新生血管中和侵入玻璃体的细胞团内的表达。
结果
1.免疫组化半定量检测显示,在所有的24个PDR增生膜中,都有ILK的表达,无论是否使用Avastin,其表达量没有显著性差异(P=0.346)。但在Avastin组,PDR新生血管增殖膜中的血管内皮细胞数明显减少,为21.5±3.94个(n=12),相比之下,未使用Avastin组的细胞数为41.33±7.44个(n=12),P=0.003,具有统计学显著性差异。PVR视网膜前膜中的细胞数为0。在未使用Avastin的PDR增殖膜中,血管内皮表达HIF-1α和VEGF的量明显高于Avastin组,具有统计学显著性差异(PHIF-1=0.023, PVEGF=0.000)。
2.免疫组化、western-blot和免疫荧光染色都显示RPE细胞在高糖培养6 h高表达ILK、ICAM-1蛋白,而实时荧光PCR检测显示在2 h其mRNA表达量开始上调,在6 h下降。高糖对RPE表达ICAM-1的上调作用能被糖皮质激素TA抑制,而ILK的上调不受其抑制。
3.在氧致视网膜新生血管小鼠模型,荧光素血管造影显示视网膜血管迂曲增粗,造影剂渗漏,有无灌注区形成。视网膜切片显示模型眼视网膜前的血管内皮细胞数量增加(23.47±8.43比对0.12±0.35, P<0.01),免疫组化染色显示其高表达HIF-1α、VEGF和ILK。
结论
1.人PDR增殖膜中的新生血管有ILK表达,提示ILK参与了视网膜新生血管的形成过程;玻璃体内注射Avastin治疗PDR,能够减少视网膜新生血管内皮细胞数量,下调HIF-1α、VEGF在增殖膜的表达,但不能下调ILK的表达。在视网膜新生血管发生中,ILK可能是HIF-1α、VEGF的上游因子。
2.RPE细胞在高糖环境下能高表达ILK和ICAM-1,RPE细胞可能通过这两种因子的信号途径而参与DRP的病变。
3.在氧致视网膜新生血管小鼠模型中,新生血管的内皮细胞高表达ILK,提示ILK参与了类似的病理改变。
创新点
1.首次在24例人PDR增生膜中检测到ILK的表达;首次证实抗VEGF疗法(Avastin玻璃体内注射)能明显减少PDR新生血管膜内的血管内皮细胞数量和下调HIF-1α、VEGF的表达,但不能下调ILK的表达。
2.首次在氧致视网膜新生血管小鼠模型中检测到ILK的表达,提示ILK参与了视网膜新生血管的发生过程。
Background
The 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.
Objective
1. 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.
Methods
1. 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.
Results
1. 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.
Conclusions
1. 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.
Novelty
1. 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.
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