Rac1 activates HIF-1 in retinal pigment epithelium cells under hypoxia

Background  Upregulation of vascular endothelial growth factor (VEGF) in hypoxic retinal pigment epithelium (RPE) cells, mediated by hypoxia-inducible factor-1 (HIF-1) is responsible for choroidal neovascularization (CNV). HIF-1α is the inducible subunit of HIF-1, but the underlying mechanisms by which RPE cells sense a decrease in oxygen concentration and transduce this signal to HIF-1α are largely unknown. Rho family small GTPase Rac1, as a potential intermediate, possibly plays a pivotal role in activating HIF-1α in RPE cells under hypoxia. Aims  To further define Rac1 playing an essential role in the induction of HIF-1α expression in RPE cells under hypoxia. Methods  In this study, we examined the expression of HIF-1α and Rac1 in human RPE cells under hypoxia for 0, 1, 2, 4, 8, 12 and 24 h by RT-PCR and Western blot. To elucidate whether Rac1 is responsible for activating the expression of hypoxia-induced HIF-1α, human RPE cells were treated with Rac1 inhibitor NSC23766 under hypoxia for 0, 1, 2, 4, 8, 12 and 24 h, and expression of HIF-1α and Rac1 measured by RT-PCR and Western blot. Results  The mRNA expression of HIF-1α and Rac1 in RPE cells significantly increased in a time-dependent manner, reaching the maximum at 4 h, and thereafter slowly declined. HIF-1α protein induction in human RPE cells was found after 1 h of hypoxia, reaching the maximum at 8 h, and then slowly declined. In response to hypoxia, the levels of Rac1 protein significantly increased, reaching the maximum at 4 h, and then slowly declined. After treatment with NSC23766, both HIF-1α and Rac1 expression were significantly inhibited in hypoxic RPE cells. Conclusions  Rac1 is crucial to activate HIF-1 in RPE cells under hypoxia, which may be a novel target other than VEGF and HIF-1 in developing CNV inhibitors.     

脉络膜新生血管相关信号通路的研究进展

脉络膜新生血管(choroidal neovascularization,CNV)与许多眼底病变有关,是引起视力障碍的重要原因之一.细胞内信号转导通路的调节异常参与了CNV的发生和发展.新近的研究发现,PI3K/Akt、Wnt、STAT3、Notch等信号通路在CNV的形成中发挥了重要的作用.这些信号通路的研究对于更加深入地揭示CNV的发生发展机制及寻找新的治疗靶点具有重要意义.     

Heparanase-1 activities in the development of laser induced choroidal neovascularization

AIM:To investigate the role of heparanase-1 in laser-induced choroidal neovascularization (CNV).METHODS:Experimental CNV was induced by krypton laser photocoagulation in 15 male Brown Norway rats. Fundus fluorescein angiography and histopathological examination were performed in observing the CNV development. The expression and distribution of heparanase-1 protein in the laser lesions were determined by immunohistochemistry and western blotting analysis.RESULTS:The success rate of laser induced CNV was approximately 75% on 3-4 weeks after laser photocoagulation. The protein levels of heparanase-1 increased significantly in the retina-choroidal complex of CNV models when compared to normal rat eyes (P<0.01). Immunostaining confirmed strong heparanase-1 expressions in all laser lesions, and it displayed to be highest at the newly formed blood vessels within the fibrovascular complex in the subretinal space.CONCLUSION:Heparanase-1 is closely involved in the development of laser induced CNV.     

脉络膜新生血管基因工程小鼠模型研究

随着基因工程技术的不断成熟,现有多种针对脉络膜新生血管(CNV)发展的关键因素和过程的基因工程小鼠模型,以适应针对CNV过程不同研究要点的需求。例如针对CNV过程中关键因素血管内皮生长因子(VEGF)的VEGF₁₆₄ RPE65转基因、Tet/VMD2/VEGF等; ApoE过表达小鼠是年龄相关性黄斑病变中自发性CNV形成机制的重要模型; 与视网膜色素上皮(RPE)变化相关的Ccl2/Cx3cr1缺陷小鼠; 脉络膜新生血管与视网膜新生血管吻合过程可见于SOD1^{-/ -}老化、Vldlr ^-/-定向突变等; 继发于脉络膜新生血管的视网膜新生血管可见于Cp^-/-Heph^-/Y敲除小鼠等。这些基因工程小鼠的主要优点为诱导快,发生时间短; 与CNV病理生理学关联强,可比较CNV各种生物学成分,便于对其发生机制的研究; 与人类CNV关联密切,为人类CNV治疗评估提供研究手段等。但其也有不足,如诱导率低、发生CNV眼的百分率低、面积小; 常发视网膜增生性瘤性病变,对CNV研究造成了一定干扰。研究者可根据自己的需求选择适合的模型并适当修改相应实验参数。     

Focal adhesion kinase signaling pathway participates in the formation of choroidal neovascularization and regulates the proliferation and migration of choroidal microvascular endothelial cells by acting through HIF-1 and VEGF expression in RPE cells

Choroidal neovascularization (CNV) is one of the most frequent causes of severe and progressive vision loss, while its pathogenesis is still poorly understood. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, plays a crucial role in linking signals initiated by both the extracellular matrix (ECM) and soluble signaling factors and controls essential cellular processes. Extensive evidence has shown that FAK is activated in angiogenic response. This study aims to investigate the effect of FAK on CNV formation. The Brown-Norway (BN) rats underwent laser rupture of Bruch's membrane to induce CNV and were then killed at 1, 3, 7, and 14 days following laser injury. Immunofluorescence and Western blot were processed to detect FAK protein. Retinal pigment epithelial (RPE) cells were cultured under hypoxia and RNA interference (RNAi) technique was used to knock down the FAK gene in RPE cells. Expression of hypoxia inducible factor-1 (HIF-1α) and vascular endothelial growth factor (VEGF) in RPE cells were investigated by RT-PCR and Western blot. Two kinds of coculture models were used to observe the effects of specific blockade of FAK in RPE cells on the proliferation and migration of choroidal microvascular endothelial cells (CECs), respectively. FAK was highly expressed in the rat RPE-choroid tissue after photocoagulation. In vitro experiment showed that FAK was involved in hypoxia signaling in cultured RPE cells. The absence of FAK effectively reduced the expression of hypoxia-induced HIF-1α and VEGF in RPE cells, resulting in the inhibition of proliferation and migration of CECs. Our results suggest that FAK pathway activation plays a role in the development of CNV, and regulates the proliferation and migration of CECs by acting through HIF-1 and then up-regulating the expression of the angiogenic factor VEGF in RPE cells. It is reasonable to propose that FAK siRNA will potentially provides a means to attenuate the strong stimuli for neovascularization in CNV-dependent disorders, which could present a therapeutically relevant strategy for the inhibition of CNV.     

环氧化酶-2与脉络膜新生血管

脉络膜新生血管（CNV）是引起视力严重损害的常见原因之一,其发生发展是一个多因子调控、多细胞参与的复杂过程。环氧化酶-2（COX-2）是诱导型环氧化酶,是前列腺素生物合成途径的关键酶。COX-2通过调节血管内皮生长因子（VEGF）的合成及生理功能的发挥、内皮细胞的移行和凋亡,在新生血管的形成过程中起重要作用。近期研究证实,COX-2参与了CNV的形成过程,其抑制剂能够抑制CNV的发生发展,为预防和治疗CNV性疾病提供了新的方向。     

Celastrol inhibits laser-induced choroidal neovascularization by decreasing VEGF induced proliferation and migration

AIM: To evaluate celastrol’s effect on choroidal neovascularization (CNV). METHODS: In this study, neovascular formation in vitro (tube formation and aortic ring culture) and in vivo (laser induced neovascular in mice) was treated with celastrol to evaluate this natural compound’s impact on CNV. Western blot was applied to explore the possible mechanism for it. For in vitro assay, triplicate for each group was repeated at least three times. For in vivo assay, each group contains 5 mice. RESULTS: Celastrol supressed tube formation and aortic ring sprout neovascularization. In vitro assay exhibited that celastrol inhibiting vascular endothelial growth factor (VEGF)-induced proliferation and migration of human umbilical vein endothelial cells and human choroidal endothelial cells, and by blocking VEGF signaling. Furthermore, intraperitoneal administration of celastrol significantly reduced the area of laser-induced CNV in an in vivo mouse model. By day 14, the area of CNV had decreased by 49.15% and 80.26% in the 0.1 mg/kg celastrol-treated group (n=5) and in the 0.5 mg/kg celastrol treated group (n=5), respectively, compared to the vehicle-treated group (n=5). CONCLUSION: Celastrol inhibits CNV by inhibiting VEGF-induced proliferation and migration of vascular endothelial cells, indicating that celastrol is a potent, natural therapeutic compound for the prevention of CNV.     

碱性成纤维细胞生长因子及其受体FGFR1在脉络膜新生血管中的表达

目的　研究碱性成纤维细胞生长因子 (bFGF)及其受体FGFR1在氪激光诱导的棕色挪威 (BN)大鼠脉络膜新生血管 (CNV)形成中的作用机制。方法　应用氪激光 ( 64 7nm ,3 60mW ,5 0 μm ,0 .0 5s)对 3 0只雄性BN大鼠实验眼进行视网膜光凝 ,分别于光凝后 3、7、14、2 1、2 8和 5 6d摘除眼球进行原位杂交检测bFGFmRNA ,免疫组织化学检测bFGF和FGFR1。结果　光凝 3d后 ,光凝区视网膜内bFGFmRNA和FGFR1蛋白表达水平逐渐下降 (P <0 .0 1)。 7d后视网膜下出现bFGFmRNA和FGFR1染色阳性的CNV ,此后其阳性染色面积及密度逐渐增加 (P <0 .0 1)。结论　bFGF和FGFR1通过自分泌和旁分泌途径在新生血管形成部位结合 ,引起FGFR1自磷酸化 ,诱导血管内皮细胞分化和CNV形成     

血管内皮生长因子与病理性脉络膜新生血管

脉络膜新生血管(choroidal neovascularization,CNV)是造成许多眼底疾病和视力严重下降的主要原因。而血管内皮生长因子(vascular endothelial growth factor,VEGF)与CNV的形成密切相关,VEGF的过度表达将导致CNV形成。抗VEGF治疗能有效抑制CNV形成,从而达到治疗效果。本文就VEGF在眼内的分布及其在病理性CNV生成中的表达,和病理性CNV抗VEGF治疗的研究现状做一综述。     

色素家兔脉络膜新生血管中结缔组织生长因子表达

目的:通过激光诱导色素家兔脉络膜新生血管(choroidalneovascularization,CNV)模型,探讨结缔组织生长因子(connectivetissuegrowthfactor,CTGF)在CNV形成中的作用。方法:用高强度半导体泵浦的倍频Nd:YAG532nm绿激光光凝兔视网膜后极部,诱发CNV。术后1,3,4wk用原位杂交方法检测CTGFmRNA在CNV中的表达。结果:光凝后1wk,CNV膜开始形成,CTGFmRNA主要表达于CNV及其附近;光凝后3,4wk,CTGFmRNA仍明显表达,并有增强的趋势。结论:结缔组织生长因子可能参与了实验性CNV的发生发展。     

康柏西普玻璃体内注射治疗特发性脉络膜新生血管

目的探究康柏西普玻璃体内注射治疗特发性脉络膜新生血管(idiopathic choroidal neovascularization,ICNV)的疗效。方法选择2016年1月至2018年1月我院眼科收治的ICNV患者146例(146眼)纳入研究,所有患者均行玻璃体内注射康柏西普治疗,测量治疗前后最佳矫正视力(BCVA)、眼压、黄斑中心凹脉络膜厚度(subfoveal choroidal thickness,SFCT)及上侧脉络膜厚度(superior choroidal thickness,SCT)、下侧脉络膜厚度(inferior choroidal thickness,ICT)、鼻侧脉络膜厚度(nasal choroidal thickness,NCT)、颞侧脉络膜厚度(temporal choroidal thickness,TCT),观察并评价脉络膜新生血管(CNV)渗漏情况,比较患眼治疗前后BCVA、眼压及各区域脉络膜厚度,以及患眼及对侧眼脉络膜厚度差异。结果与治疗前比较,患眼治疗后1 d、1个月时BCVA均显著提高(均为P<0.05)。患眼治疗前后眼压比较差异均无统计...     

Expression of cell adhesion molecules and vascular endothelial growth factor in experimental choroidal neovascularisation in the rat

AIMS—To investigate the longevity and reproducibility of choroidal neovascularisation (CNV) induced by krypton laser photocoagulation in the rat. The presence of cell adhesion molecules (CAMs) and vascular endothelial growth factor (VEGF) during the development of CNV was also studied.
METHODS—67 pigmented rats underwent retinal photocoagulation by krypton laser. The eyes were examined by either single or serial fluorescein angiography at 3 days, 1, 2-3, 4-5, 7-8, and 12 weeks post photocoagulation. The expression of CAMs (ICAM-1, E-selectin, and CD44) and VEGF post photocoagulation was studied by immunohistochemistry.
RESULTS—CNV related fluorescein leakage appeared in 46.4% of 766 laser spots delivered to the 58 eyes that were tested at 2-3 weeks post treatment. The ratio of hyperfluorescent laser sites did not change significantly at 8 weeks post laser. The number of leaky spots was independent of the total number of lesions delivered to each eye (at 2-3 weeks post laser 10-15 spots/eye: 44% and 25-30 spots/eye: 49%; t=0.7673; p=0.3903). Nine eyes were followed by serial angiography between 2 and 12 weeks. The laser spots with fluorescein leakage at 2 weeks (51.5%) remained leaky at 12 weeks (51.5%). Histopathologically, macrophage accumulation peaked at 5 days and CNV was firstly observed at 1 week post photocoagulation. ICAM-1, E-selectin, CD44, and VEGF were maximally induced at 3-5 days post laser photocoagulation, and were localised to RPE, choroidal vascular endothelial, and inflammatory cells. VEGF was also detected in intravascular leucocytes at the sites of laser lesions.
CONCLUSIONS—These studies demonstrated that krypton laser photocoagulation can be successfully used to produce lesions similar to those of human CNV. The response induced remained present for an extended period of time (12 weeks), thus offering a potential model to screen candidate CNV inhibitory agents. In addition, it is proposed that the expression of ICAM-1, E-selectin, CD44, and VEGF before new vessel formation might be linked to the initiation of CNV.

Keywords: choroidal neovascularisation; cell adhesion molecules; vascular endothelial growth factor     

Gender and estrogen supplementation increases severity of experimental choroidal neovascularization

Observational clinical studies suggest that post-menopausal women may be at risk for more severe age-related macular degeneration, and that estrogen loss due to menopause may contribute. We sought to determine the effect of gender and estrogen status on the severity of choroidal neovascularization (CNV) in a mouse model for experimental choroidal neovascularization. Laser-induced CNV was performed in mice with or without estrogen supplementation. At various times, eyes were removed for analysis of severity of CNV lesions or for extraction of choroidal mRNA to evaluate iNOS, TNF-alpha, MMP-9, and ER-alpha expression, which are molecules relevant to angiogenic processes. Also, splenic macrophages were analysed for iNOS to determine the effect of estrogen treatment in vitro. Finally, laser-induced CNV was performed in iNOS -/- mice. Our result showed that aged female mice had significantly larger CNV than age-matched males. Ovariectomy in adult mice did not increase severity, but paradoxically estrogen supplementation after ovariectomy did increase CNV severity. More severe CNV were associated with a significant decrease in choroidal iNOS mRNA. Splenic macrophages from estrogen supplemented mice showed a significant increased in TNF-alpha mRNA expression (eight fold difference compared to the control) but only a mild change in iNOS mRNA levels (2-3 fold difference). In vitro data further showed that nitric oxide production in splenic macrophages at different estrogen levels was not different from controls. Finally, CNV severity was significantly more severe in iNOS -/- mice, compared to iNOS +/+ mice after laser treatment. In conclusion, aged female mice developed more severe CNV than do males. Estrogen replacement seems to increase severity, possibly by suppressing the upregulation of choroidal iNOS and activating macrophages. The putative beneficial or detrimental role of estrogen biology in age-related macular degeneration must be more carefully evaluated and may vary with the stage of age-related macular degeneration (atrophic or neovascular) as well as with the specific target cell type (monocytes vs. endothelial cell or vascular smooth muscle cell).     

PEDF和VEGF mRNA在实验性脉络膜新生血管组织中的表达

目的 研究血管内皮生长因子(vessel endothelial growth factor，VEGF)和色素上皮衍生因子(pigment epithelium derived factor，PEDF)在实验性小鼠脉络膜新生血管(choroidal neowascularization,CNV)组织中的表达情况。探讨二者在CNV形成过程中所起的作用。方法 用半导体激光诱导小鼠CNV模型。分别于激光后1、3、7d、2和3周时取出眼球，采用原位杂交方法检测CNV组织中VEGF和PEDF mRNA的表达情况。结果 VEGF和PEDF mRNA在激光诱导的小鼠CNV组织形成过程中均有显著表达。激光光凝早期二者的表达均增高，但VEGF mRNA的表达升高更显著。激光照射后3和7d时．VEGF mRNA的表达即达到高峰，阳性率分别为26．05％和27．92％，而PEDF mRNA的阳性表达率分别为21．13％和23．55％．2周时，VEGF mRNA表达开始下降，约为23．95％，而PEDF mRNA的表达则达到高峰,为29．19％,光凝后3周时，二者的表达均下降，但PEDF mRNA的表达仍高于VEGF mRNA的表达，分别为24．87％和21．93％．结论 VEGF和PEDF mRNA明显表达于实验性小鼠CNV组织中。2者表达失衡可能在CNV的形成过程中起到调控作用。     

Optical coherence tomography characteristics of responses to intravitreal bevacizumab in idiopathic choroidal neovascularization

AIM: To investigate factors associated with responses to intravitreal bevacizumab (IVB) in naive idiopathic choroidal neovascularization (iCNV) by high domain optical coherence tomography (OCT). METHODS: We retrospectively reviewed clinical data of 40 eyes of iCNV patients who received a single or multiple IVB on an as-needed basis (1.25 mg/0.05 mL). One month after the first injection, subretinal fluid (SRF) volume was evaluated and the eyes were divided into 3 groups based on responses to IVB. Good, moderate, and poor responses were defined as 61%-99%, 30%-60%, and <30% resolution of SRF on OCT after IVB in iCNV, respectively. OCT findings were analyzed to find factors associated with difference in response levels. Comparisons were made using Wilcoxon’s matched-pairs signed-rank test, the Mann-Whitney U test for means with continuous data and Fisher’s exact test for categorical data. RESULTS: The mean number of IVB was 1.28±1.50 and mean follow up time was 3.60±1.20mo. At postoperative 1mo, there were 8 (20%) eyes in good response, 20 (50%) in moderate response and 12 (30%) eyes in poor response group and at last visit there were 28 good responders (70%), 8 (20%) moderate responders and 4 (10%) poor responders. Statistically significant difference was detected between good responders and non good responders in choroidal neovessels thickness (P=0.029), SRF height (P=0.049) and SRF volume (P=0.031) at post treatment 1mo. CONCLUSION: OCT is a valuable diagnostic tool. Decrease in choroidal neovessels thickness, SRF height and volume predicts favorable response of iCNV to IVB therapy.     

Notch信号通路在脉络膜新生血管中的研究进展

脉络膜新生血管可见于多种眼部疾病,是引起视力损害的重要原因之一.近期多项研究表明,Notch信号在脉络膜新生血管的发生中起着关键性作用.作为相邻细胞间的信号转导通路,Notch信号途径可参与血管形成中的多个步骤,并与多种细胞及多种信号通路发生交互作用.因此,揭示Notch信号在脉络膜新生血管中的作用及其机制,对该类疾病的发生机制和靶向治疗具有重要意义.     

Ranibizumab治疗脉络膜新生血管的研究现状

脉络膜新生血管(choroidal neovascularization,CNV)是引起多种眼底疾病、导致视力丧失的重要原因之一。CNV的发生机制尚不清楚,目前普遍认为血管生成因子与抑制因子之间平衡的破坏具有关键作用,而血管内皮生长因子(vascular endothelial growth factor,VEGF)是其重要的始动因素。Ranibizumab是一种重组的人源化抗VEGF单克隆抗体片段,能够结合并抑制VEGF,阻止血管渗漏和新生血管的形成,抑制CNV的发生,进而阻碍CNV相关疾病的病程进展。随着对CNV发生机制的深入研究,针对CNV发生过程进行治疗,有可能从根本上治愈CNV相关疾病。     

生长因子在脉络膜新生血管生成的微环境中的多重作用

脉络膜新生血管(choroidal neovascularization,CNV)可见于多种眼底疾病,是引起视力障碍的重要原因之一。虽然人们对生长因子在CNV生成中的作用已有共识,但深入了解CNV所处微环境中生长因子、细胞和细胞外基质之间的相互作用,对阐明CNV发生机制具有重要的意义。     

脉络膜新生血管形成的模型构建与选择

脉络膜新生血管形成（choroid neovascularization,CNV）已成为眼科界难题之一,制备一种可靠、稳定的CNV动物模型对于深入研究CNV的发病机制及治疗方法有深远的意义.目前,CNV动物模型主要有3种：激光诱导、生长因子诱导、基因缺陷动物模型.其理论基础主要建立于击破Bruch膜或产生过量的VEGF.常用的实验模型动物包括鼠科类、兔、猪和灵长类动物.各种模型动物具有不同的生理特性,不同动物模型具有不同病理变化,应根据不同的实验要求选择最合适的实验动物.