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

Tai-Lan Tuan^*, Paul Hwu^*, Wendy Ho^*, Peter Yiu^*, Richard Chang^*, Annette Wysocki and Paul D. Benya^¶

From The Saban Research Institute of Childrens Hospital,^* Los Angeles, California; the Department of Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California; the University of Mississippi Medical Center, Jackson, Mississippi; the J. Vernon Luck, Sr., M.D. Research Center of Orthopaedic Hospital, Los Angeles, California, and the UCLA-Orthopaedic Hospital Department of Orthopaedic Surgery,^¶ David Geffen School of Medicine, University of California, Los Angeles, California

Keloids are tumor-like skin scars that grow as a result of theaberrant healing of skin injuries, with no effective treatment.We provide new evidence that both overexpression of plasminogenactivator inhibitor-1 (PAI-1) and elevated collagen accumulationare intrinsic features of keloid fibroblasts and that thesecharacteristics are causally linked. Using seven strains eachof early passage normal and keloid fibroblasts, the keloid strainsexhibited inherently elevated collagen accumulation and PAI-1expression in serum-free, 0.1% ITS+ culture; larger increasesin these parameters occurred when cells were cultured in 3%serum. To demonstrate a causal relationship between PAI-1 overexpressionand collagen accumulation, normal fibroblasts were infectedwith PAI-1-expressing adenovirus. Such cells exhibited a two-to fourfold increase in the accumulation of newly synthesizedcollagen in a viral dose-dependent fashion in both monolayersand fibrin gel, provisional matrix-like cultures. Three differentPAI-1-targeted small interfering RNAs, alone or in combination,produced greater than an 80% PAI-1 knockdown and reduced collagenaccumulation in PAI-1-overexpressing normal or keloid fibroblasts.A vitronectin-binding mutant of PAI-1 was equipotent with wild-typePAI-1 in inducing collagen accumulation, whereas a completeprotease inhibitor mutant retained approximately 50% activity.Thus, PAI-1 may use more than its protease inhibitory activityto control keloid collagen accumulation. PAI-1-targeted interventions,such as small interfering RNA and lentiviral short hairpin RNA-containingmicroRNA sequence suppression reported here, may have therapeuticutility in the prevention of keloid scarring.