Adenoviral Overexpression and Small Interfering RNA Suppression Demonstrate That Plasminogen Activator Inhibitor-1 Produces Elevated Collagen Accumulation in Normal and Keloid Fibroblasts

Keloids are tumor-like skin scars that grow as a result of the aberrant healing of skin injuries, with no effective treatment. We provide new evidence that both overexpression of plasminogen activator inhibitor-1 (PAI-1) and elevated collagen accumulation are intrinsic features of keloid fibroblasts and that these characteristics are causally linked. Using seven strains each of early passage normal and keloid fibroblasts, the keloid strains exhibited inherently elevated collagen accumulation and PAI-1 expression in serumfree, 0.1% ITS+ culture; larger increases in these parameters occurred when cells were cultured in 3% serum. To demonstrate a causal relationship between PAI-1 overexpression and collagen accumulation, normal fibroblasts were infected with PAI-1-expressing adenovirus. Such cells exhibited a two- to fourfold increase in the accumulation of newly synthesized collagen in a viral dose-dependent fashion in both monolayers and fibrin gel, provisional matrix-like cultures. Three different PAI-1-targeted small interfering RNAs, alone or in combination, produced greater than an 80% PAI-1 knockdown and reduced collagen accumulation in PAI-1-overexpressing normal or keloid fibroblasts. A vitronectin-binding mutant of PAM was equipotent with wild-type PAI-1 in inducing collagen accumulation, whereas a complete protease inhibitor mutant retained approximately 50% activity. Thus, PAM may use more than its protease inhibitory activity to control keloid collagen accumulation. PAI-targeted interventions, such as small interfering RNA and lentiviral short hairpin RNA-containing microRNA sequence suppression reported here, may have therapeutic utility in the prevention of keloid scarring. (Am J Pathol 2008, 173:1311-1325; DOI: 10.2353/ajpath.2008.080272)