Abstract
Persistence of HIV-1 reservoirs within the Central Nervous System (CNS) remains a significant challenge to the efficacy of potent anti-HIV-1 drugs. The primary human Brain Microvascular Endothelial Cells (HBMVEC) constitutes the Blood Brain Barrier (BBB) which interferes with anti-HIV drug delivery into the CNS. The ATP binding cassette (ABC) transporters expressed on HBMVEC can efflux HIV-1 protease inhibitors (HPI), enabling the persistence of HIV-1 in CNS. Constitutive low level expression of several ABC-transporters, such as MDR1 (a.k.a. P-gp) and MRPs are documented in HBMVEC. Although it is recognized that inflammatory cytokines and exposure to xenobiotic drug substrates (e.g HPI) can augment the expression of these transporters, it is not known whether concomitant exposure to virus and anti-retroviral drugs can increase drug-efflux functions in HBMVEC. Our in vitro studies showed that exposure of HBMVEC to HIV-1 significantly up-regulates both MDR1 gene expression and protein levels; however, no significant increases in either MRP-1 or MRP-2 were observed. Furthermore, calcein-AM dye-efflux assays using HBMVEC showed that, compared to virus exposure alone, the MDR1 mediated drug-efflux function was significantly induced following concomitant exposure to both HIV-1 and saquinavir (SQV). This increase in MDR1 mediated drug-efflux was further substantiated via increased intracellular retention of radiolabeled [3H-] SQV. The crucial role of MDR1 in 3H-SQV efflux from HBMVEC was further confirmed by using both a MDR1 specific blocker (PSC-833) and MDR1 specific siRNAs. Therefore, MDR1 specific drug-efflux function increases in HBMVEC following co-exposure to HIV-1 and SQV which can reduce the penetration of HPIs into the infected brain reservoirs of HIV-1. A targeted suppression of MDR1 in the BBB may thus provide a novel strategy to suppress residual viral replication in the CNS, by augmenting the therapeutic efficacy of HAART drugs.
Highlights
Being a lentivirus, HIV-1 infection shows a long latency period and slow disease progression resulting in severe immune deficiencies and AIDS
To elucidate the transporters which are mainly involved in drug efflux in the human Brain Microvascular Endothelial Cells (HBMVEC), cells were exposed to HIV-1 (0.2 or 1.0 multiplicity of infection (MOI)) for 6 hrs
The drugefflux function in HIV-1 exposed HBMVEC was determined after 24 hrs post virus exposure by calcein-AM retention assay (Fig. 1B)
Summary
HIV-1 infection shows a long latency period and slow disease progression resulting in severe immune deficiencies and AIDS. HIV-1 primarily infects CD4+ Tlymphocytes, but long-term persistence of the virus occurs in monocytes and macrophages, e.g. in microglial cells within the CNS. The sequestered sites within the brain can act as anatomical reservoirs for HIV-1 which prevents complete eradication of the virus despite long-term anti-retroviral therapy [1,2,3]. One of the strategies via which HIV-1 can evade HAART efficacy is due to the presence of drug-efflux transporters on the BBB endothelial cells (EC) [4]. All of the currently available HIV-1 protease inhibitors (HPI) are known to be substrates of different ABC-transporters, and are actively effluxed from the BBB via both MDR1 (P-gp) and the multidrug resistance associated proteins (MRP-1 and MRP-2) [5,6]. A clear understanding of the molecular mechanism of ABC-transporter expression in HIV-infected brain microenvironments may provide novel strategies towards alternative treatment options such as their specific inhibition via gene therapy approaches
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