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增殖性糖尿病视网膜病变中凝血酶/基质金属蛋白酶-1/蛋白酶激活受体-1链的上调

Upregulation of Thrombin/Matrix Metalloproteinase-1/Protease-Activated Receptor-1 Chain in Proliferative Diabetic Retinopathy.

作者信息

Abu El-Asrar Ahmed M, Alam Kaiser, Nawaz Mohd Imtiaz, Mohammad Ghulam, Van den Eynde Kathleen, Siddiquei Mohammad Mairaj, Mousa Ahmed, De Hertogh Gert, Opdenakker Ghislain

机构信息

a Department of Ophthalmology , College of Medicine, King Saud University , Riyadh , Saudi Arabia.

b Dr. Nasser Al-Rashid Research Chair in Ophthalmology, Department of Ophthalmology, College of Medicine, King Saud University , Riyadh , Saudi Arabia.

出版信息

Curr Eye Res. 2016 Dec;41(12):1590-1600. doi: 10.3109/02713683.2016.1141964. Epub 2016 Jun 3.

DOI:10.3109/02713683.2016.1141964

PMID:27261371

Abstract

PURPOSE

Selective proteolytic activation of protease-activated receptor-1 (PAR1) by thrombin and matrix metalloproteinase-1 (MMP-1) plays a central role in enhancing angiogenesis. We investigated the expression levels of thrombin, MMP-1, and PAR1 and correlated these levels with vascular endothelial growth factor (VEGF) in proliferative diabetic retinopathy (PDR). In addition, we examined the expression of PAR1 and thrombin in the retinas of diabetic rats and PAR1 in human retinal microvascular endothelial cells (HRMEC) following exposure to high-glucose, the proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and the hypoxia mimetic agent cobalt chloride (CoCl).

METHODS

Vitreous samples from 32 PDR and 23 nondiabetic patients, epiretinal membranes from 10 patients with PDR, retinas of rats, and HRMEC were studied by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and Western blot analysis. An assay for in vitro cell migration angiogenesis was performed in HRMEC.

RESULTS

In epiretinal membranes, PAR1 was expressed in vascular endothelial cells, CD45-expressing leukocytes, and myofibroblasts. ELISA and Western blot assays revealed significant increases in the expression levels of thrombin, MMP-1, and VEGF in vitreous samples from PDR patients compared to nondiabetic controls. Significant positive correlations were found between the levels of VEGF and the levels of thrombin (r = 0.41; p = 0.006) and MMP-1 (r = 0.66; p < 0.0001). Significant increases of cleaved PAR1 (approximately 50 kDa) and the proteolytically active thrombin (approximately 50 kDa) were detected in rat retinas after induction of diabetes. The proinflammatory cytokines IL-1β and TNF-α, but not high-glucose and CoCl, induced upregulation of cleaved PAR1 (approximately 30 kDa) in HRMEC. In addition, thrombin and MMP-1 induced VEGF in HRMEC and vorapaxar, a PAR1 inhibitor, inhibited thrombin-induced migration in HRMEC.

CONCLUSIONS

Interactions among thrombin, MMP-1, PAR1, and VEGF might facilitate angiogenesis in PDR.

摘要

目的

凝血酶和基质金属蛋白酶-1（MMP-1）对蛋白酶激活受体-1（PAR1）的选择性蛋白水解激活在促进血管生成中起核心作用。我们研究了增殖性糖尿病视网膜病变（PDR）中凝血酶、MMP-1和PAR1的表达水平，并将这些水平与血管内皮生长因子（VEGF）进行关联。此外，我们检测了糖尿病大鼠视网膜中PAR1和凝血酶的表达，以及人视网膜微血管内皮细胞（HRMEC）在暴露于高糖、促炎细胞因子白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）和缺氧模拟剂氯化钴（CoCl）后PAR1的表达。

方法

通过酶联免疫吸附测定（ELISA）、免疫组织化学和蛋白质印迹分析，对32例PDR患者和23例非糖尿病患者的玻璃体样本、10例PDR患者的视网膜前膜、大鼠视网膜以及HRMEC进行研究。在HRMEC中进行体外细胞迁移血管生成测定。

结果

在视网膜前膜中，PAR1在血管内皮细胞、表达CD45的白细胞和成肌纤维细胞中表达。ELISA和蛋白质印迹分析显示，与非糖尿病对照组相比，PDR患者玻璃体样本中凝血酶、MMP-1和VEGF的表达水平显著升高。VEGF水平与凝血酶水平（r = 0.41；p = 0.006）和MMP-1水平（r = 0.66；p < 0.0001）之间存在显著正相关。糖尿病诱导后，在大鼠视网膜中检测到裂解的PAR1（约50 kDa）和具有蛋白水解活性的凝血酶（约50 kDa）显著增加。促炎细胞因子IL-1β和TNF-α而非高糖和CoCl诱导HRMEC中裂解的PAR1（约30 kDa）上调。此外，凝血酶和MMP-1诱导HRMEC中VEGF的产生，而PAR1抑制剂沃拉帕沙抑制凝血酶诱导的HRMEC迁移。