The pathogenic remodeling of blood vessels often involves smooth muscle proliferation and migration. As in other instances of cell movement, smooth muscle cell migration into the neointima involves a regulated series of adhesion and de-adhesion events regulated by cell-surface receptors and surface-associated proteases. The matrix metalloproteinase (MMP), gelatinase A, plays a significant role in this process by enabling cells to overcome matrix barriers, and also regulates cell-matrix adhesion during migration. It does this through its interaction with its specific activator, MT1-MMP, a membrane-associated proteinase that initiates cleavage of the gelatinase A pro-peptide in a complex process involving multiple interactions with cell-surface proteins. One such interaction involves MT1-MMP itself, which bindings gelatinase A through a bridge with the MMP inhibitor, TIMP-2, a critical mediator of gelatinase A activation. TIMP-2 binds the active site of MT1-MMP and the carboxyl domain of gelatinase A, forming a triplex which positions gelatinase A appropriate for subsequent activation by another MT1-MMP molecule. The carboxyl domain of gelatinase A also binds the integrin, alphavbeta3, which also plays a role in the activation process. Finally, cell bind TIMP-2 to other, as yet unknown sites which may also be key elements of gelatinase A activation. Interestingly, fibroblasts and smooth muscle cells rapidly respond to changes in cell shape by activating and binding gelatinase A in a process independent of new protein synthesis, suggesting that the components of the activation mechanism are present on cells but preventing from interacting appropriately. In this study, the role of TIMP-2 in both the cell binding and activation of gelatinase A will be explored. First, the biochemical basis for the role of TIMP-2 in both the cell binding and activation of gelatinase A will be explored. First, the biochemical basis for TIMP-2's specificity for MT1-MMP will be deduced using chimeric TIMP molecules in which TIMP-2 sequence will be exchanged for sites within TIMP-2, a highly homologous inhibitor with virtually no specificity for MT1-MMP. Net, the non-MMP cell-surface binding sites for TIMP-2 will be characterized, and the critical amino acid sequences within TIMP-2 that confer this binding will be determined. Finally, the contribution of TIMP-2 to gelatinase A binding and activation will be modeled. Understanding this mechanism will be important in defining the biochemical events that occur during vascular remodeling in both normal and disease processes.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL062268-03
Application #
6390276
Study Section
Pathobiochemistry Study Section (PBC)
Program Officer
Wassef, Momtaz K
Project Start
1999-09-01
Project End
2003-08-31
Budget Start
2001-09-01
Budget End
2002-08-31
Support Year
3
Fiscal Year
2001
Total Cost
$228,847
Indirect Cost
Name
University of Oklahoma Health Sciences Center
Department
Pathology
Type
Schools of Medicine
DUNS #
937727907
City
Oklahoma City
State
OK
Country
United States
Zip Code
73117
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Phelps, Eric D; Updike, Dawn L; Bullen, Elizabeth C et al. (2006) Transcriptional and posttranscriptional regulation of angiopoietin-2 expression mediated by IGF and PDGF in vascular smooth muscle cells. Am J Physiol Cell Physiol 290:C352-61