Inhibition of apoptosis and caspase‐3 in vascular smooth muscle cells by plasminogen activator inhibitor type‐1

Abstract

Increased expression of plasminogen activator inhibitor type 1 (PAI-1) is associated with decreased apoptosis of neoplastic cells. We sought to determine whether PAI-1 alters apoptosis in vascular smooth muscle cells (VSMC) and, if so, by what mechanisms. A twofold increase in the expression of PAI-1 was induced in VSMC from transgenic mice with the use of the SM-22alpha gene promoter (SM22-PAI+). Cultured VSMC from SM22-PAI+ mice were more resistant to apoptosis induced by tumor necrosis factor plus phorbol myristate acetate or palmitic acid compared with VSMC from negative control littermates. Both wild type (WT) and a stable active mutant form of PAI-1 (Active) inhibited caspase-3 amidolytic activity in cell lysates while a serpin-defective mutant (Mut) PAI-1 did not. Similarly, both WT and Active PAI-1 decreased amidolytic activity of purified caspase-3, whereas Mut PAI-1 did not. WT but not Mut PAI-1 decreased the cleavage of poly-[ADP-ribose]-polymerase (PARP), the physiological substrate of caspase-3. Noncovalent physical interaction between caspase-3 and PAI-1 was demonstrable with the use of both qualitative and quantitative in vitro binding assays. High affinity binding was eliminated by mutations that block PAI-1 serpin activity. Accordingly, attenuated apoptosis resulting from elevated expression of PAI-1 by VSMC may be attributable, at least in part, to reversible inhibition of caspase-3 by active PAI-1.

Augmentation of Proliferation of Vascular Smooth Muscle Cells by Plasminogen Activator Inhibitor Type 1

“…8,20 In the present studies, greater proliferation was demonstrated in VSMCs from PAI-1 transgenic mice (SM22-PAI ϩ ). After 6 days in culture, the average number of SM22-PAI ϩ VSMCs was 2.3Ϯ0.4-fold greater than control VSMCs ( Figure 1A …”

“…Previously we have reported that increased expression of PAI-1 renders VSMCs resistant to apoptosis. 20 The resistance to apoptosis and the induction of increased proliferation appear to be linked. PAI-1 inhibits directly the activity of caspase-3 but not caspase-8.…”

“…20 The identity of smooth muscle cells was confirmed by Western blot and flow cytometry with smooth muscle cell specific ␣-actin antibody. 20 Experiments were performed with cells in DMEM with Hams nutrient mixture F12 (DME/F12; Gibco-BRL). Tumor necrosis factor (TNF) was purchased from Sigma.…”

“…VSMCs were obtained by explantation from the aortas of SM22-PAI ϩ mice that exhibit a 3-fold increased expression of PAI-1 8,20 and negative control littermates, and grown in Dulbecco Modified Eagle Medium (DMEM; Gibco-BRL) supplemented with 20% fetal bovine serum as we described previously. 20 The identity of smooth muscle cells was confirmed by Western blot and flow cytometry with smooth muscle cell specific ␣-actin antibody.…”

“…19 We have shown previously that increased expression of PAI-1 inhibits apoptosis of VSMCs by directly inhibiting caspase-3. 20 Because inhibition of caspase activity was found to increase expression of FLIP, 21,22 we hypothesized that inhibition of caspase-3 by PAI-1 would increase expression or activation of FLIP. FLIP has been demonstrated to lead to activation of nuclear factor B (NF-B) and extracellular signal-regulated kinase (ERK) that promote proliferation.…”