Effect of Chemical Modification on tPA on the Integrity of HMBEC Monolayer In Vitro

Nova Southeastern University, Davie, Florida, USA

Objective. The purpose of this study is to evaluate effect of chemically modified tPA on the HMBEC monolayer integrity. Background. Tissue-type plasminogen activator (tPA) has been the only FDA-approved thrombolytic agent for ischemic stroke (IS). However, thrombolytic therapy for IS inherently suffers from intracranial hemorrhage (ICH). Literature evidence pointed out the ability of tPA to stimulate matrix metalloproteinase enzymes (MMP-9) through its interactions with endothelial cell surface moieties, such as LRP-1. The elevated MMP-9 may compromise the integrity of the blood-brain barrier, significantly potentiating the risk of ICH. Previously we have shown that chemical modification of tPA attenuated MMP-9 generation from HBMEC culture in vitro. Methods. Low-molecular weight heparin (LMWH) was covalently conjugated to tPA, as reported previously. Human brain microvascular endothelial cells (HBMEC) was cultured on 24-well transwell system, in endothelial growth media supplemented with 10% fetal bovine serum (FBS) at 37 C under 5% carbon dioxide. Trans-epithelial electrical resistance (TEER) was monitored under different seeding conditions over a period of ~10 days. Both TEER and Permeability of fluorescein-labelled bovine serum albumin (FITC-BSA) was evaluated when the cells were treated with tPA, the conjugate or vehicle. Results. Treating HBMEC with tPA caused significant increase in FITC-BSA permeability and decrease in TEER compared to the control, which was not observed in the LMWH-tPA treatment group. Conclusion. Selective chemical modification of tPA may attenuate the MMP-9 generation from HBMEC and may pave a way to developing safer thrombolytic agents. Grants. The work was supported in part by NSU President’s Faculty Research and Development Grant (PFRDG).