Thrombospondin 2 (TSP2) is a matricellular protein controlling the apoptosis-proliferation balance in endothelial cells. Little is known about its transcriptional regulation compared with that of TSP1. We found that overexpression of a constitutively active mutant of Rac (Rac V12 ) specifically increases TSP2 mRNA levels without affecting TSP1 in human aortic endothelial cells (HAEC). Moreover, TSP2 induction by Rac V12 is dependent upon reactive oxygen species (ROS) production, as gp91ds-tat peptide, an inhibitor of NADPH oxidase, and the flavoprotein inhibitor diphenylene iodinium (DPI) block TSP2 synthesis. Furthermore, we found that increasing Rac V12 expression results in a biphasic proliferative curve, with proliferation initially increasing as Rac V12 expression increases and then returning to levels less than that of control cells at higher expression. This growth inhibition is mediated by TSP2, as either DPI treatment, which blocks TSP2 synthesis, or pan-TSP blocking antibodies restore the proliferative ability of HAEC with high expression. Mechanistically, we show that the effect of TSP2 on cell proliferation is independent of the antiangiogenic TSP2 Hep1 sequence, which is capable of altering actin cytoskeletal reorganization but not proliferation in our experimental conditions. Finally, we show in vivo that Rac-induced TSP2 expression is observed in the aorta of transgenic mice selectively expressing Rac V12 in smooth muscle cells. These results identify Rac-induced ROS as a new pathway involved in the regulation of TSP2 expression.

Background — Plaque disruption is the inciting event for coronary thrombosis and acute coronary syndromes. Multiple factors influence plaque rupture, including the loss of vascular smooth muscle cells (VSMCs). We hypothesized that monocytes/macrophages (MMs) activated by macrophage colony-stimulating factor (M-CSF) are responsible for VSMC death. Methods and Results — VSMC apoptosis was markedly increased in the presence of both M-CSF and MMs (58.8±3.3%) compared with VSMCs plus M-CSF without MMs (15.7±1.5%, P ≤0.00005), VSMCs plus MMs without M-CSF (22.7±3.7%, P ≤0.0001), or control VSMCs alone (13.2±2.1%, P ≤0.0001). MM cell contact was required for M-CSF–stimulated killing of VSMCs, and MMs displayed an M-CSF concentration-dependent killing effect. Abciximab binds Mac-1 (CD11b/CD18) on MMs. When added to VSMCs exposed to MMs and M-CSF, abciximab (7 μg/mL) significantly reduced VSMC apoptosis (19.1±2.2%, P ≤0.0003). Therapeutic doses of tirofiban (0.35 μg/mL) and eptifibatide (5 μg/mL), which inhibit platelet glycoprotein (GP) IIb/IIIa but not Mac-1, did not block activated MM-induced VSMC apoptosis (65.0±3.4% and 51.3±2.5%, respectively). A recombinant anti–CD-18 antibody had an effect similar to that of abciximab (16.5±0.4%). Conclusions — These data suggest that monocytes and physiological concentrations of M-CSF trigger VSMC apoptosis. Abciximab and specific inhibitors of the Mac-1 receptor can antagonize this process.

Background-The killing of vascular cells by activated macrophages is an important step in the process of destabilization of the arterial wall. The death receptor Fas is implicated in vascular cell death. Hence, we extended our studies in a rat aortic allograft model, using adenovirus-mediated overexpression of soluble Fas (sFas) to block Fas binding to Fas ligand (Fas-L). The contribution of Fas to vascular cell injury and consequent transplant arteriosclerosis was investigated. Methods and Results-Activated monocytes in the presence of macrophage colony-stimulating factor induce endothelial cell apoptosis in vitro, which was significantly inhibited by adenovirus-mediated sFas overexpression. Next, donor rat abdominal aortas were either untreated or transduced with adenoviruses encoding (1) rat soluble Fas (Ad3rsFas), (2) no insert (Ad3Null), and (3) ␤-galactosidase (Ad3nBg). A total of 175 aortic grafts were harvested 2 to 90 days after transplantation. Vascular cell apoptosis and CD45ϩ cell infiltration were significantly reduced in Ad3rsFas-transduced aortas, as compared with control allografts. Moreover, the control allografts developed marked intimal thickening, whereas Ad3rsFas-transduced allografts had significantly less neointima until the 90-day time point. Key Words: arteriosclerosis Ⅲ apoptosis Ⅲ endothelium Ⅲ inflammation Ⅲ gene therapy C ardiac allograft vasculopathy (CAV), characterized by the development of transplant arteriosclerosis, remains the leading cause of late graft failure and patient death in heart transplantation. The archetypal pathological findings of CAV are diffuse concentric intimal proliferation and intense mononuclear cell infiltration, including T cells and macrophages, in most graft vessels. 1,2 Although the precise molecular and cellular mechanisms are not yet elucidated, endothelial cell (EC) injury and recipient inflammatory response are believed to be crucial in the development of CAV. 1,3,4 Increased apoptosis, including vascular EC and smooth muscle cell (SMC), has been observed in acute and chronic rejection; such apoptosis probably is mediated through the Fas/Fas-Ligand (Fas-L) pathway. 5-8 Fas-Lϩ T cells and macrophages bind to Fasϩ vascular cells, inducing apoptosis. One of earliest features of CAV is the adherence of monocytes to the endothelium, followed by their migration into the intima, which may enhance the development of atherosclerotic lesions. 9 Numerous investigations have focused on the molecular mechanism of monocyte attraction and migration into vascular tissues; little attention, however, has been paid to the actual role of infiltrating macrophages in the mediation of CAV. Conclusions-sFasMacrophage colony-stimulating factor (MCSF), which is elevated within inflammatory atherosclerotic lesions, 9 was shown to block spontaneous monocyte apoptosis and to stimulate monocyte differentiation, proliferation, and activation. 10 We have previously reported that monocytes, in the presence of MCSF, switched to a phenotype capable of inducing SMC apoptosis. 11 In ...