ASSESSMENT OF IN VITRO ISCHEMIC STROKE-LIKE INJURY AND MATRIX METALLOPROTEINASE 9 RESPONSES IN HUMAN BRAIN VASCULAR CELL MODELS

Abstract

Loss of vascular integrity and successive opening of the blood-brain barrier (BBB) are key compromising events that lead to a vicious cascade of secondary injuries following acute ischemic stroke (AIS). Matrix metalloproteinase 9 (MMP-9) has been strongly implicated as a potential culprit in mediating the proteolytic breakdown of components integral such as tight junctional proteins to the BBB and inflammatory response with adhesion molecules post-stroke; however, studies have mainly focused on neurons as a therapeutic target, rather than the structural and biochemical barrier of the cerebrovasculature. Therefore, the focus of this thesis was then to investigate the expression of adherence and junctional proteins, as well as levels and activity of MMP-9 in male human brain microvascular endothelial cells (HBMECs) following normoxia (21% O2) or hypoxia plus glucose deprivation (HGD, 1% O2) exposure at P7 for 3 h, timepoint determined from our previous TEER studies revealing deterioration of endothelial barrier as early as 3 h. Concomitantly, MMP-9 levels and activity were assessed in human brain vascular smooth muscle cells (HBVSMCs) following the same exposure conditions as well as treatment to MMP- 9 inhibitor JNJ0966 (0.5 μM), to determine if HGD induced MMP-9 activity could be altered using this novel compound following in vitro ischemic-like injury. Following HGD exposure, cell density and morphology were altered. In addition, a simultaneous decrease in tight junction proteins claudin-5 and zonula occludens 1 (ZO-1) was observed, while HGD exposure resulted in an increase in vascular cell adhesion molecule 1 (VCAM-1) levels in HBMECs, as observed via standard immunoblotting. In both HBMECs and HBVSMCs, HGD also increased MMP-9 protein levels and increased MMP-9 activity assessed via western blot and zymography respectively. However, although there was a trend, the MMP-9 inhibitor, JNJ0966, did not alter HGD-induced MMP-9 activity in HBVSMCs. In conclusion, HGD induced barrier integrity loss occurred by mediating decreased tight junction expression and concomitant MMP-9 activation in human cerebrovasculature. Mechanism(s) by which HGD regulates enzymatic activity of MMP-9 following ischemic-like injury warrants further investigation.