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.

Objective-Proliferation of vascular smooth muscle cells (VSMCs) contributes to restenosis after coronary intervention.We have shown previously that increased expression of plasminogen activator inhibitor type 1 (PAI-1) limits VSMC apoptosis. Because apoptosis and proliferation appear to be linked, we sought to determine whether increased PAI-1 would affect VSMC proliferation. Methods and Results-VSMCs were explanted from control and transgenic mice (SM22-PAI ϩ ) in which VSMC expression of PAI-1 was increased. Increased growth of SM22-PAI ϩ -VSMCs (2.3Ϯ0.4-fold) reflected, at least partially, increased proliferation. Greater expression of FLICE-like inhibitory protein (FLIP; 2.7-fold) and its cleaved active form were seen in SM22-PAI ϩ -VSMCs. The balance between caspase-8 and FLIP favored proliferation in SM22-PAI ϩ -VSMCs. Increased expression of NF-B and activation of extracellular signal-regulated kinase (ERK) were demonstrated in SM22-PAI ϩ -VSMCs (foldϭNF-Bϭ2.2Ϯ0.1, foldϭphosphorylated-ERKϭ1.6Ϯ0.1). Results were confirmed when expression of PAI-1 was increased by transfection. Inhibition of NF-B and ERK attenuated proliferation in SM22-PAI ϩ -VSMCs. Increased expression of PAI-1 promoted proliferation when VSMCs were exposed to tumor necrosis factor (TNF). Conclusions-Increased expression of PAI-1 is associated with greater activity of FLIP that promotes VSMC proliferation through NF-B and ERK. Thus, when vascular wall expression of PAI-1 is increased, restenosis after coronary intervention is likely to be potentiated by greater proliferation of VSMC and resistance to apoptosis.

Objectives Smad4 is a central mediator of transforming growth factor-β/bone morphogenetic protein signaling that controls numerous developmental processes as well as homeostasis in the adult. The present studies sought to understand the function of Smad4 expressed in vascular smooth muscle cells (VSMC) in vascular development and the underlying mechanisms. Methods and results Breeding of Smad4flox/flox mice with SM22α-Cre mice resulted in no viable offspring with SM22α-Cre;Smad4flox/flox genotype in a total of 165 newborns. Subsequent characterization of 301 embryos between embryonic day 9.5 (E9.5) and E14.5 demonstrated that mice with SM22α-Cre;Smad4flox/flox genotype died between E12.5 and E14.5, due to decreased cell proliferation and increased apoptosis in the embryonic heart and arteries. Additionally, deletion of Smad4 more specifically in SMC with the inducible SMMHC-Cre mice, in which decreased cell proliferation was observed only in the artery but not the heart, also caused lethality of the knockout embryos at E12.5 and E14.5. The Smad4 deficient VSMC lacked smooth muscle α-actin filaments, decreased expression of SMC-specific gene markers, and markedly reduced cell proliferation, migration and attachment. Using specific pharmacological inhibitors and small-interfering RNAs, we demonstrated that inhibition of TGF-β signaling and its regulatory Smad 2/3 decreased VSMC proliferation, migration and expression of SMC-specific gene markers, while inhibition of BMP signaling only affected VSMC migration. Conclusions SMC-specific deletion of Smad4 results in vascular defects that lead to embryonic lethality in mice, which may be attributed to decreased VSMC differentiation, proliferation, migration, as well as cell attachment and spreading. The TGF-β signaling pathway contributes to VSMC differentiation and function; while the BMP signaling pathway regulates VSMC migration. These studies provide important insight into the role of Smad4 and its upstream Smads in regulating smooth muscle cell function and vascular development of mice.

This review focuses on the association between vascular calcification and arterial stiffness, highlighting the important genetic factors, systemic and local microenvironmental signals, and underlying signaling pathways and molecular regulators of vascular calcification. Elevated oxidative stress appears to be a common procalcification factor that induces osteogenic differentiation and calcification of vascular cells in a variety of disease conditions such as atherosclerosis, diabetes mellitus, and chronic kidney disease. Thus, the role of oxidative stress and oxidative stress–regulated signals in vascular smooth muscle cells and their contributions to vascular calcification are highlighted. In relation to diabetes mellitus, the regulation of both hyperglycemia and increased protein glycosylation, by AGEs (advanced glycation end products) and O -linked β-N-acetylglucosamine modification, and its role in enhancing intracellular pathophysiological signaling that promotes osteogenic differentiation and calcification of vascular smooth muscle cells are discussed. In the context of chronic kidney disease, this review details the role of calcium and phosphate homeostasis, parathyroid hormone, and specific calcification inhibitors in regulating vascular calcification. In addition, the impact of the systemic and microenvironmental factors on respective intrinsic signaling pathways that promote osteogenic differentiation and calcification of vascular smooth muscle cells and osteoblasts are compared and contrasted, aiming to dissect the commonalities and distinctions that underlie the paradoxical vascular-bone mineralization disorders in aging and diseases.