Tissue-type plasminogen activator crosses the intact blood-brain barrier by LRP-mediated transcytosis: A phenomenon that is switched during oxygen glucose deprivation to an increased and LRP-independent process

Abstract

Despite uncontroversial benefit from its thrombolytic activity, the documented neurotoxic effect of tissue plasminogen activator (tPA) raises an important issue: the current emergency stroke treatment might not be optimum, if exogenous tPA can enter the brain and thus add to the deleterious effects of endogenous tPA within the cerebral parenchyma. Here, we aimed at determining whether vascular tPA crosses the blood-brain barrier (BBB) during cerebral ischemia, and if so, by which mechanism. We have first evidenced that brain lesions induced by intra-striatal injection of NMDA were enhanced either when tPA was co-infused in the striatum or when intravenously injected. In addition, co-infusion of PAI-1 with NMDA in the striatum prevented the deleterious effect of intravenous injection of tPA. No BBB alteration was observed in any of these conditions, as assessed by confocal microscopy analysis of FITC-Dextran (77 kDa) extravasation. Altogether, these data suggest that tPA (69 kDa) has to cross the intact BBB to potentiate neuronal death within the parenchyma. By using biotinylated tPA, we have shown by immunohistochemistry, that tPA crossed the BBB in vivo in control and NMDA-injected animals. Moreover, tPA can be detected in the cerebrospinal fluid after its intravenous injection. In order to characterize the mechanism of this passage, we used a cellular model of BBB. Although tPA did not influence the integrity of the BBB by itself, tPA was capable to cross the BBB. Its passage was blocked at 4 C and was saturable, suggesting a transendothelial and receptor–mediated mechanism. As the low-density lipoprotein receptor related protein (LRP) and mannose receptor have been already shown to bind tPA, we investigated whether one of these two receptors could be involved in this passage. Although mannose had no effect on the passage of tPA, RAP (receptor–associated protein, an antagonist of LRP) blocked the passage of tPA. We next investigated whether oxygen and glucose deprivation (OGD) could influence the mechanism of the passage of tPA. OGD led to an exacerbation of tPA passage, and switched the mechanism to a LRP-independent process. These data suggest two mechanisms by which tPA can cross the BBB, a moderate receptor-mediated transcytosis in control conditions and an exacerbated and unsaturable passage involving an unspecific transcellular pathway after ischemic conditions. Preventing the interaction of tPA with LRP could thus be an interesting strategy to block the deleterious effect of tPA in vivo, but only as long as the integrity of the BBB is not altered. These data show the importance of taking the side effects of blood derived tPA into account, and offer bases to improve the current thrombolytic strategy for stroke treatment.