Abstract
Cocaine use is associated with breach in the blood brain barrier (BBB) and increased HIV-1 neuro-invasion. We show that the cellular enzyme “Prolidase” plays a key role in cocaine-induced disruption of the BBB. We established a barrier model to mimic the BBB by culturing human brain microvascular endothelial cells (HBMECs) in transwell inserts. In this model, cocaine treatment enhanced permeability of FITC-dextran suggesting a breach in the barrier. Interestingly, cocaine treatment increased the activity of matrix metallo-proteinases that initiate degradation of the BBB-associated collagen. Cocaine exposure also induced prolidase expression and activity in HBMECs. Prolidase catalyzes the final and rate-limiting step of collagen degradation during BBB remodeling. Knock-down of prolidase abrogated cocaine-mediated increased permeability suggesting a direct role of prolidase in BBB breach. To decipher the mechanism by which cocaine regulates prolidase, we probed the inducible nitric oxide synthase (iNOS) mediated phosphorylation of prolidase since mRNA levels of the protein were not altered upon cocaine treatment. We observed increased iNOS expression concurrent with increased prolidase phosphorylation in cocaine treated cells. Subsequently, inhibition of iNOS decreased prolidase phosphorylation and reduced cocaine-mediated permeability. Finally, cocaine treatment increased transmigration of monocytic cells through the HBMEC barrier. Knock-down of prolidase reduced cocaine-mediated monocyte transmigration, establishing a key role of prolidase in cocaine-induced breach in endothelial cell barrier.
Highlights
Cocaine use is associated with breach in the blood brain barrier (BBB) and increased HIV-1 neuroinvasion
To probe the effects of cocaine on BBB integrity, we employed an in vitro BBB model composed of a monolayer of human brain microvascular endothelial cells (HBMECs, Fig. 1A)
HBMECs have been widely used to study the molecular and cellular details of BBB because they are a key component of the BBB and possess unique features that are distinct from the peripheral endothelial cells[51,52]
Summary
Cocaine use is associated with breach in the blood brain barrier (BBB) and increased HIV-1 neuroinvasion. We established a barrier model to mimic the BBB by culturing human brain microvascular endothelial cells (HBMECs) in transwell inserts In this model, cocaine treatment enhanced permeability of FITC-dextran suggesting a breach in the barrier. Knock-down of prolidase reduced cocaine-mediated monocyte transmigration, establishing a key role of prolidase in cocaineinduced breach in endothelial cell barrier. Cocaine’s ability to alter the expression of intracellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and endothelial-leukocyte adhesion molecule (ELAM or selectin-1) has been postulated as a key contributory factor for the ensuing BBB breach[17,18,19] These biochemical alterations have been associated with increased leukocyte migration across the BBB, elevated levels of pro-inflammatory cytokines and chemokines such as TNF-α, nuclear factor kappa B (NF-kB), IL-6, and others, resulting in neuro-inflammation[18,25]. Cocaine treatment has been shown to increase transcription of membrane type (MT)-MMP-1 in HBMECs48
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