Thrombin and Protease-Activated Receptors
Thrombin is a serine protease that plays a critical role in coagulation in distal microvessels. In addition, thrombin has pleiotropic extravascular effects. It can induce protection at low doses but act as a neurotoxin at high doses, killing cells via the protease-activated receptors (PARs). In preliminary experiments, using a protease-activatable cell-penetrating probe, we have shown directly that thrombin protease activity participates in damaging the neurovascular unit. We did this by comparing quantified vascular leakage and cell death markers with the quantity of thrombin activation (probe fluorescence) in the parenchyma after middle cerebral artery occlusion (MCAo). We blocked thrombin with a direct thrombin inhibitor, argatroban, and greatly reduced ischemic edema and tissue damage at realistic therapeutic time window (delay dose three hours after reperfusion).
Thrombin activity associated with neurons. Photomicrograph showing thrombin activatable cell penetrating peptides (ACPP) in parenchymal tissue with severe vascular disruption (FITC-dextran leakage). ACPP positive cells colocalize with neuronal cellular marker (NeuN). Scale Bar — 3mm.
We also exacerbated tissue damage by infusing thrombin during ischemia. The results suggested a critical role for thrombin in mediating brain injury in focal ischemia. We think thrombin partially mediates edema and cell death during stroke via activity at the PAR-1 receptor. We have made significant progress in studying this mechanism of neuroprotection post-MCAo with different APC mutants selective for PAR-1 receptors (3K3A-APC mutant in phase I clinical trial). We have made significant progress in understanding the role of PAR-1 by using lentivirus-mediated shRNA knockdown of PAR-1 in rats and by studying the effect of stroke in aged PAR-1, PAR-3 and PAR-4 knockout animals. In addition, we currently are developing conditional knockout animal models to further investigate the function of thrombin and thrombin receptors in the central nervous system.
Stereotaxic injection of PAR-1 shRNA lentivirus transfects neurons and GFAP positive cells. Lentivirus was injected into rat striatum, and animals were subjected to MCAo after one week. Post-MCAo, they survived for 24 hours. Large number of GFP positive cells can be detected following injection. Confocal analysis of images shows GFP labeled cells colocalize with NeuN (neuronal marker), GFAP (astrocytes) and Nissl body (neurons). GFP positive cells do not colocalize with IBA1 (microglia marker). Scale bars (B) 100μm; (C, D, E) 50μm; (F) 20μm.
Stem Cell Therapy in Stroke
The Lyden Laboratory is focused on developing novel neural stem cell therapies in ischemic stroke animal models by developing optimally preconditioned neural progenitor cells to treat ischemic stroke. Our lab has developed effective methods for intra-arterial infusion of neural progenitor cells in our MCAo stroke model. Furthermore, we have been able to transduce rat neural progenitor cells with CXCR4-GFP lentiviral vectors. CXCR4 has been shown to enhance cell homing for neural progenitor cells after ischemic stroke. We are also working on additional strategies to precondition neural progenitor cells to enable immune invasion after cell transplantation.
Rat neural progenitor cell grafts in the ischemic hemisphere seven days after middle cerebral artery occlusion (MCAo). Representative images of stereotaxic injections of rNPCs in the ischemic hemisphere (N=5) (A, B). Intra-arterially delivered rNPCs also can reside in the ischemic hemisphere seven days after MCAo (N=3) (D, E).