A small molecule PAI-1 functional inhibitor attenuates neointimal hyperplasia and vascular smooth muscle cell survival by promoting PAI-1 cleavage

Abstract

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of urokinase-and tissue-type plasminogen activators (uPA and tPA), is an injury-response gene implicated in the development of tissue fibrosis and cardiovascular disease. PAI-1 mRNA and protein levels were elevated in the balloon catheter-injured carotid and in the vascular smooth muscle cell (VSMC)-enriched neointima of ligated arteries. PAI-1/uPA complex formation and PAI-1 antiproteolytic activity can be inhibited, via proteolytic cleavage, by the small molecule antagonist tiplaxtinin which effectively increased the VSMC apoptotic index in vitro and attenuated carotid artery neointimal formation in vivo. In contrast to the active full-length serine protease inhibitor (SERPIN), elastase-cleaved PAI-1 (similar to tiplaxtinin) also promoted VSMC apoptosis in vitro and similarly reduced neointimal formation in vivo. The mechanism through which cleaved PAI-1 (CL-PAI-1) stimulates apoptosis appears to involve the TNF-α family member TWEAK (TNF-α weak inducer of apoptosis) and it’s cognate receptor, fibroblast growth factor (FGF)-inducible 14 (FN14). CL-PAI-1 sensitizes cells to TWEAK-stimulated apoptosis while full-length PAI-1 did not, presumably due to its ability to down-regulate FN14 in a low density lipoprotein receptor-related protein 1 (LRP1)-dependent mechanism. It appears that prolonged exposure of VSMCs to CL-PAI-1 induces apoptosis by augmenting TWEAK/FN14 pro-apoptotic signaling. This work identifies a critical, anti-stenotic, role for a functionally-inactive (at least with regard to its protease inhibitory function) cleaved SERPIN. Therapies that promote the conversion of full-length to cleaved PAI-1 may have translational implications.

Pharmacological Targeting of Plasminogen Activator Inhibitor-1 Decreases Vascular Smooth Muscle Cell Migration and Neointima Formation

“…These experimental conditions, while informative regarding the potential of PAI-039 to inhibit PAI-1, probably did not adequately reproduce the physiological conditions under which SMCs migrate in vivo. A recent study showed that PAI-039 induces apoptosis of SMCs, thereby producing an anti-proliferative effect, 49 , which is consistent with the known anti-apoptotic properties of PAI-1. 20, 22 In addition, another recent study demonstrated that PAI-039 improved dermal wound closure in diabetic mice, 50 which is intriguing because vascular remodeling has been considered as a form of wound repair.…”

Inhibition of SERPINE1 Function Attenuates Wound Closure in Response to Tissue Injury: A Role for PAI-1 in Re-Epithelialization and Granulation Tissue Formation

“…Since it is known that tiplaxtinin and TM5275 promote PAI-1 substrate behavior, however, these inhibitors may prevent the conformational change in β-sheet A of PAI-1 necessary to accommodate insertion of the PAI-1 RCL during the protease inhibition reaction [25]. If tiplaxtinin or TM5275 were to complex with PAI-1 at the vitronectin-binding site, it has been suggested that these inhibitors would not be effective since the majority of circulating PAI-1 is bound to vitronectin; nevertheless, the available data confirm significant in vivo efficacy for these drugs [13][14][15][16][17][18][19][20]27]. Figure 1.…”

“…TWEAK-FN14 interaction in ApoE −/− pro-atherosclerotic mice, furthermore, stimulates PAI-1 expression in aortic lesions with co-localization of both PAI-1 and FN14 expression [49]. Coincidentally, full-length PAI-1 but not the cleaved SERPIN down-regulates surface FN14 expression via LRP1-mediated endocytosis [27]. It appears, therefore, that PAI-1 modulates not only cellular migration but also survival by cell surface receptor titration.…”

“…PAI-1 expression driven by the SM22α promoter potently inhibits apoptosis by binding to, and preventing, caspase-3 activation in VSMC [41]. Expression alone, however, may not dictate functionality, since the small-molecule PAI-1 functional inhibitors, tiplaxtinin and TM5275, stimulate apoptosis in several cell types [24,27,42,43].…”

“…Under normal circumstances, myofibroblasts decrease by apoptosis; pathological scars, in contrast, display persistent contracture due to myofibroblast persistence with continued matrix contractility (D). Since inhibition of PAI-1 function attenuates vascular pathological remodeling via induction of smooth muscle cell apoptosis [27], reduced PAI-1 expression in the latter stages of wound healing may contribute to decreased myofibroblast numbers. Since elevated levels of PAI-1 are evident in keloids, targeted PAI-1 inhibition may be a novel therapeutic approach to resolve pathophysiological scars.…”