Using an in vitro brain endothelial cell model of ischemia in which cells were subjected to an environment of oxygen and glucose deprivation, these investigators showed that tissue plasminogen activator (tPA) induced increased expression and activity of MMP9, a matrix metalloproteinase that targets critical components of the blood-brain barrier. They also studied two rodent models of stroke, one induced by transient occlusion of the middle cerebral artery in mice and one by inducing cerebral thromboembolism in rats, and showed that tPA delivered systemically led to increased MMP9 expression and activity in the infarct zone along with significantly increased cerebral hemorrhage. In both the in vitro and in vivo models, MMP9 expression, infarct volume, and cerebral hemorrhage were dramatically and significantly inhibited by giving the animals or cells activated protein C (APC) along with or a few hours after the tPA. With inhibitory antibodies, short hairpin inhibitory microRNAs, and cells from genetically engineered mice they showed that the damaging effects of tPA required expression of the endothelial cell receptor LRP1 and that the protective effects of APC required expression of the endothelial cell receptors EPCR and PAR1.
In Brief
Very little effective therapy is available to mitigate brain damage and tissue loss once a patient begins to experience symptoms of a stroke. Recombinant human tPA is of benefit, but only if given within a very narrow time window. Furthermore, enthusiasm for its use is tempered by increased risks of cerebral hemorrhage with catastrophic sequelae. These authors have used cell culture and rodent models to define a brain microvascular endothelial signaling pathway activated by tPA that is responsible for some of its untoward effects. They then showed that these effects could be prevented by concomitant delivery of APC. The major “problem” with tPA is apparently not related to clot dissolution or clot instability, but rather to its ability to bind and activate an endothelial cell receptor known as LRP1. This receptor then triggers a cascade of events including activation of the pro-inflammatory transcription factor NFκb, increased expression and activity of matrix metalloproteinase-9 (MMP9), and then MMP9-dependent disruption of the blood-brain barrier with subsequent hemorrhage. APC prevents hemorrhage in this setting by blocking NFκb activation and MMP9 up-regulation. This effect is apparently not due to its ability to slow thrombin generation by proteolytic cleavage of Factor V and VIII, but rather to activation of an alternative signaling pathway mediated by its two endothelial cell surface receptors, PAR1 and EPCR. This is consistent with recent studies from other groups describing the mechanistic basis of sepsis protection by APC and helps define a “new” paradigm by which coagulation and fibrinolytic enzymes mediate systemic and localized effects in the vasculature by acting as paracrine ligands for endothelial receptors, not by “busting” clots or blocking thrombin generation. From these studies emerge several exciting potential avenues for drug development, including simply adding APC (drotrecogin alfa/Xigris®) to tPA infusion during acute stroke. More appealing would be designing drugs that specifically target LRP1 and/or the PAR1/EPCR system without influencing the coagulation and fibrinolytic pathways.
