Gelatinase A expression in endothelial cells is regulated by at least two cis-acting promoter elements.

Increased expression of gelatinase A is associated with both angiogenesis and alterations in blood vessel structure. Heart-derived endothelial cells derived from spontaneously hypertensive rats (SHR) were found to express significantly more gelatinase A in culture, both at the protein and mRNA level, than endothelial cells from normotensive Wistar-Kyoto (WKY) rats. Other matrix metalloproteinases, as well as their tissue inhibitors, were not differentially regulated. A 1683 bp gelatinase A promoter fragment linked to a luciferase reporter demonstrated up to 40-fold more activity when transfected into SHR-derived cells versus WKY-derived cells. The promoter region between -1324 and -1272, previously termed RE1, contributed up to a five-fold increase in basal promoter activity in both cells, but contributed only 12% of the promoter activity in SHR-derived cells compared to 85% in WKY-derived cells. In SHR-derived cells, but not in WKY-derived cells, a second region between -1435 and -1375, termed RE2, contributed 60% of the total activity of the 1683 bp promoter fragment. Both electrophoretic mobility shift assays and Southwestern blots demonstrated differences in RE2-specific binding factors in nuclear extracts derived from the two cell types. SHR-derived endothelial cells thus represent a new model system to study the regulation of gelatinase A expression, which itself may contribute to the abnormal vascular structure seen in the SHR.

Gelatinase A activation is regulated by the organization of the polymerized actin cytoskeleton.

Gelatinase A (GL-A) is a matrix metalloproteinase (MMP) involved in both connective tissue remodeling and tumor invasion. GL-A activation is mediated by a membrane-type MMP (MT-MMP) that cleaves the GL-A propeptide. In this study, we examined the role of the actin cytoskeleton in regulating GL-A activation and MT-MMP-1 expression. Human palmar fascia fibroblasts and human fetal lung fibroblasts were cultured on a planar substratum or within different types of collagen lattices. Fibroblasts that formed stress fibers, either on a planar substratum or within an attached collagen lattice, showed reduced GL-A activation compared with fibroblasts lacking stress fibers, within either floating or stress-released collagen lattices. To determine whether changes in the organization of the actin cytoskeleton could promote GL-A activation, fibroblasts with stress fibers were treated with cytochalasin D. Within 24 h after treatment, GL-A activation was dramatically increased. Associated with this GL-A activation was an increase in MT-MMP-1 mRNA as determined by Northern blot analysis. Treatment with nocodazole, which induced microtubule depolymerization and cell shape changes without affecting stress fibers, did not promote GL-A activation. These results suggest that the extracellular matrix and the actin cytoskeleton transduce signals that modulate GL-A activation and regulate tissue remodeling.

Tissue inhibitor of metalloproteinases-1 is decreased and activated gelatinases are increased in chronic wounds.

The balance between matrix deposition and tissue turnover is fundamental in wound healing. It is likely that the balance between proteolytic enzymes and their inhibitors contributes to this balance. Matrix metalloproteinases are clearly important in tissue turnover, but their roles in wound healing are poorly understood. To investigate this, fluid from healing wounds resulting from mastectomies was collected from 1 h to 10 d post-surgery, and was analyzed for tissue inhibitor of metalloproteinases-1 concentrations. In all cases, tissue inhibitor of metalloproteinases-1 levels were initially comparable to those in serum, but increased rapidly to significantly higher levels within two days, with a tenfold average increase for five patients. On the other hand, zymography revealed that gelatinase A (72 kDa) levels increased moderately, whereas gelatinase B levels (92 kDa) decreased an average of twofold within 4 d. In contrast, fluid from chronic wounds had significantly more gelatinolytic activity, including lower-molecular-weight proteinase species that may represent activated or superactivated gelatinase fragments, as suggested by immunoprecipitation with specific antibodies. Tissue inhibitor of metalloproteinases-1 levels were lower in chronic than in healing wounds. These data may indicate that excess proteolysis in chronic wounds retards successful healing, and results from an imbalance of proteinase and inhibitors, as well as the presence of higher levels of activated metalloproteinases.