Recovery after spinal cord injury is in part attributed to secondary events that govern both detrimental and reparative processes. Our general hypothesis is that matrix metalloproteinase (MMP)-9 has an evolving role in the injured spinal cord, initially serving as a mediator of posttraumatic hemorrhagic necrosis (PTHN) and emerging thereafter as a proangiogenic factor. As we advance our understanding of this MMP, we will be better positioned to develop MMP-targeted therapeutics for the spinal cord injured patient. PTHN evolves within hours after injury and is characterized by hemorrhages that begin in the grey matter and extend thereafter along the axis of the cord.
Aim 1 will test the hypothesis that infiltrating leukocytes mediate MMP-9 dependent PTHN. We will determine if mice deficient in MMP-9, overexpress tissue inhibitor of matrix metallproteinase -1 (TIMP-1), or depleted of leukocytes are resistant to PTHN. Indices of vascular- related catastrophic failure, including abnormal permeability, and hemorrhage will be evaluated. We will determine if PTHN is enhanced upon complementation of MMP-9 null mice with MMP-9 expressing leukocytes or in mice deficient in TIMP-1, and if transient blockade of MMP-9 improves long-term functional recovery. Although MMP-9 is active during wound healing, its contribution to angiogenesis is unknown. We will determine if macrophages, including bone marrow derived macrophages (BMDMs), are sources of MMP-9 during wound healing and if this protease supports angiogenesis.
Aims 2 and 3 will examine synergism between chemokine stromal-derived factor-1 (SDF-1), its receptor, CXCR4, and MMP-9 in the homing and transendothelial migration of BMDMs to the injured cord.
Aim 2 will test the hypothesis that SDF-1 is elevated in the injured cord and signals the homing of CXCR4+BMDMs. RT-PCR and immunochemistry will measure mRNA and identify cells that express this chemokine and its receptor. To determine its effect on homing of BMDMs, SDF-1 will be delivered to injured mice that have been reconstituted with bone marrow from eGFP-expressing wild-type (WT) mice and in mice, administered quantum dot-labeled, BMDMs, i.v. Loss-of-function studies will be conducted in injured animals treated with a CXCR4 receptor antagonist.
Aim 3 will test the hypothesis that CXCR4+BMDMs utilize MMP-9 to migrate across the endothelium into the injured cord. CXCR4+macrophages and gelatinolytic activity will be quantified in injured MMP-9 WT and null mice and MMP-9 null mice, reconstituted with bone marrow from MMP-9 nulls.
Aim 4 will test the hypothesis that MMP-9 supports angiogenesis. Angiogenesis will be compared in WTs, MMP-9 and TIMP-1 null mice, TIMP-1 overexpressing mice, and mice treated with an MMP-9 antagonist. RT-PCR and immunocytochemistry will assay angiogenesis-related genes and VEGF bioavailability. To determine if angiogenesis is governed by infiltrating, MMP-9 expressing BMDMs, we will compare revascularization in MMP-9 null mice reconstituted with bone marrow from WT and MMP-9 null animals and in VEGF-treated, MMP-9 null mice.
Matrix metalloproteinases constitute a large family of proteins with diverse biologic functions in normal, diseased, and injured tissues. In the proposed studies we focus on matrix metalloproteinase (MMP)-9, a member of this family, that governs both damaging and reparative processes in the injured spinal cord. A long- term goal of these studies is to develop MMP-targeted therapeutic interventions for the spinal cord injured patient that are designed to block MMP-mediated adverse events without compromising those functions that may facilitate recovery processes.
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