The Role of Matrix Metalloproteinase 2 (MMP2) In Diabetes‐induced Loss of PECAM‐1 in The Retina: Direct and Indirect Mechanisms

Abstract

ObjectiveMatrix metalloproteinases (MMPs) are calcium‐dependent, zinc‐containing endopeptidases that are responsible for degrading extracellular matrix proteins, and for cleavage of various substrates such as cell surface receptors, proteoglycans, chemokines and cytokines. The aim of this study was to investigate the molecular mechanisms by which MMP2 may lead to PECAM‐1 loss in the diabetic retina, either directly through cleavage, or indirectly by modulating tumor necrosis factor‐α (TNF‐α) levels and activity.BackgroundPlatelet endothelial cell adhesion molecule‐1 (PECAM‐1) is a cell surface protein that is heavily expressed on vascular endothelial cells, and has major roles in barrier function, endothelial cell‐endothelial cell adhesion, and cell signaling. We have previously found a significant decrease (50–90%, P<0.05) in PECAM‐1 levels in the diabetic retina, and in cultured rat retinal microvascular endothelial cells (RRMECs) grown under high glucose conditions (30–40%, P<0.05). The decrease in PECAM‐1 levels was accompanied by a significant increase in MMP2 levels and activity in the diabetic plasma (72% and 78.5% respectively, P<0.05), and in RRMECs grown under high glucose conditions (12‐fold increase, P<0.05).Materials and MethodsFor our in vitro studies, primary RRMECs were grown in normal glucose (NG, 5 mM glucose) or high glucose (HG, 25 mM) media for six days, with treatments added 6–24 hrs prior to harvesting cells for analysis. For our animal studies, age‐matched male Wistar rats were injected with either vehicle (control), or Streptozotocin (STZ) (diabetic, 30 mg/kg/day) for three consecutive days in a model of type I diabetes. GM6001, a broad spectrum MMP inhibitor, was started at week six post‐STZ injection for two weeks (4 mM, 25 μl/eye, in sterile eye drops), and retinas were collected eight weeks post‐STZ injection for analysis. PECAM‐1, MMP2, and TNF‐α levels and interactions were assessed using western blotting, zymography, immunofluorescence, immunoprecipitation, or ELISA assays. In silico analysis for possible protease cleavage sites was done using PROSPER, provided by Monash University, Australia.ResultsSeveral PECAM‐1 cleavage sites by MMP2 were identified using in silico analysis. Moreover, we confirmed PECAM‐1/MMP2 interactions using immunoprecipitation. PECAM‐1 levels were significantly decreased in RRMECs treated with MMP2 (81%, P<0.05), with a significant increase in TNF‐α levels (118.2%, P<0.05). Moreover, RRMECs treated with TNF‐α had a significant decrease in PECAM‐1 levels (34%, P<0.05). Furthermore, TNF‐α levels were significantly increased in diabetic STZ plasma, and in RRMECs grown under HG conditions (31%, 74.4% respectively, P<0.05). To investigate the protective effect of MMP2 inhibition on PECAM‐1 levels, we treated diabetic rats with GM6001 eye drops, and saw an increase of PECAM‐1 levels (77.2%, P<0.05) when compared to untreated rats.ConclusionThese results indicate a possible role of MMP2 in the diabetes‐induced loss of PECAM‐1 in the retina, which can be via direct cleavage of PECAM‐1, or indirectly via upregulation of TNF‐α, with the two mechanisms evaluated in future studies.Support or Funding InformationNIH EY025632, and AHA Predoctoral Fellowship 16PRE30080003This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.