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).