Background-Oxidized LDLs (oxLDLs) and matrix metalloproteinases (MMPs) are present in atherosclerotic lesions.OxLDLs activate various signaling pathways potentially involved in atherogenesis. OxLDLs induce smooth muscle cell (SMC) proliferation mediated by the activation of the sphingomyelin/ceramide pathway and tyrosine kinase receptors. MMPs are also able to induce SMC migration and proliferation in addition to extracellular matrix degradation. The present study was designed to investigate whether MMPs play a role in the mitogenic effect of oxLDLs. Methods and Results-OxLDLs induce the release of activated MMP-2 in SMC culture medium. MMP-2 was identified by its 65-kDa gelatinase activity on zymography and by using specific blocking antibodies and MMP-2 Ϫ/Ϫ cells. MMP inhibitors (batimastat and Ro28-2653) and the blocking antibodies anti-MMP-2 and anti-membrane type 1-MMP inhibited the oxLDL-induced sphingomyelin/ceramide pathway activation and subsequent activation of ERK1/2 and DNA synthesis but did not inhibit the oxLDL-induced epidermal growth factor receptor and platelet-derived growth factor receptor activation. Exogenously added activated MMP-2 or membrane type 1-MMP-1 triggered the activation of both sphingomyelin/ceramide and ERK1/2 pathways and DNA synthesis. Conversely, suppression of MMP-2 expression in MMP-2 Ϫ/Ϫ cells or in SMCs treated by small-interference RNA also blocked both sphingomyelin/ceramide signaling and DNA synthesis. Conclusions-Together, these data demonstrate that MMP-2 plays a pivotal role in oxLDL-induced activation of the sphingomyelin/ceramide signaling pathway and subsequent SMC proliferation. These pathways may constitute a potential therapeutic target for modulating the oxLDL-induced proliferation of SMCs in atherosclerosis or restenosis.

Objective-Oxidized low-density lipoprotein (oxLDL)-induced smooth muscle cell (SMC) proliferation requires the coactivation of various signaling pathways, namely sphingomyelin/ceramide/sphingosine-1-phosphate, epithelial growth factor receptor (EGFR), and phosphoinositide 3-kinase (PI-3K) pathways. This study aimed to clarify the respective role and the hypothetical cross-talk between sphingomyelin/ceramide/sphingosine-1-phosphate, EGFR, and PI-3K/Akt pathways in the balance between mitogenic and cytotoxic effects elicited by oxLDL. Methods and Results-Coimmunoprecipitation experiments and the use of inhibitors and dominant-negative mutantshowed that oxLDL-induced PI-3K activation is dependent on EGFR. PI-3K activation is independent of the sphingomyelin/ceramide/sphingosine-1-phosphate pathway, because PI-3K inhibition by LY294002 or dominantnegative ⌬p85 mutant does not abrogate sphingomyelin hydrolysis, and, conversely, the use of permeant C2-ceramide and of N,N-dimethyl-sphingosine, a sphingosine kinase inhibitor, does not alter PI-3K activity. Activation of Akt/PKB by oxLDL requires PI-3K, as shown by the inhibition by LY204002 and in ⌬p85 SMC. The inhibition of Akt/PKB by PI-3K inhibitor LY204002 or by overexpression of kinase-dead Akt shifted the mitogenic effect of oxLDL toward apoptosis, thus suggesting that the PI-3K/Akt pathway acts as a survival pathway. Conclusions-SMC proliferation elicited by moderate concentrations of oxLDL involves the sphingomyelin/ceramide/ sphingosine-1-phosphate pathway, which leads to extracellular regulated kinase 1/2 activation and DNA synthesis, and the EGFR/PI-3K/Akt pathway, which prevents the apoptotic effect of oxLDL. Key Words: proliferation Ⅲ survival Ⅲ oxidized low-density lipoprotein Ⅲ phosphoinositide 3-kinase Ⅲ epithelial growth factor receptor Ⅲ ceramide S mooth muscle cell (SMC) proliferation plays a critical role in the formation of the fibrous cap and plaque stability, whereas toxic events are potentially involved in necrotic core formation, endothelial cell injury, and plaque rupture. 1,2 Oxidized low-density lipoproteins (oxLDLs) exert various biological effects potentially involved in atherosclerosis development, such as chemotaxis, cell proliferation, or cytotoxicity. [3][4][5][6][7] OxLDL-induced SMC proliferation involves the activation of the sphingomyelin-ceramide pathway that results in the generation of the mitogenic mediator sphingosine-1-phosphate. 3,8 On the other hand, oxLDLs elicit activation of tyrosine kinase receptors such as epithelial growth factor receptor (EGFR), 9 which could play a role in cell proliferation. Recent studies provided evidence that phosphatidylinositol 3-kinase (PI-3K) is implicated in macrophage proliferation induced by oxLDL. 5 PI-3K is activated by growth factors and peptide hormones 10 -12 and regulates multiple cellular processes required for cell survival and proliferation. 13 A cross-talk between the sphingomyelinceramide and PI-3K pathways has been reported, but the data are relatively conflicting, because the ...

The understanding of the role, genetic regulation and coordinated function of proteins mediating intestinal cholesterol transport may lead to novel ways of treating cardiovascular disease.

Abstract-The E-cadherin/␤-catenin/T-cell factor (Tcf) signaling pathway plays a crucial role in embryogenesis and carcinogenesis and has recently emerged in atherosclerosis. The aim of this work was to investigate whether this signaling pathway is involved in smooth muscle cell proliferation induced by oxidized low-density lipoprotein (LDL). In human aortic smooth muscle cells, mitogenic concentration of mildly oxidized LDL induced the activation of ␤-catenin, as assessed by the dissociation of the ␤-catenin/cadherin complex, and the concomitant rise of active ␤-catenin in the cytosol. The oxidized LDL-induced rise of active ␤-catenin required metalloproteinase activation, as well as epidermal growth factor receptor and Src signaling, as assessed by the use of pharmacological inhibitors and cells overexpressing a SrcK-inactive form. The concomitant phosphatidylinositol 3-kinase/Akt activation and glycogen synthase kinase 3-␤ phosphorylation induced the inhibition of the proteasomal degradation of ␤-catenin. Then active ␤-catenin associated with Tcf4 and translocated into the nucleus. This enhanced the expression of the cell cycle activator cyclin D1. This crucial role of ␤-catenin in the mitogenic effect of oxidized LDL was confirmed by silencing ␤-catenin by specific small interfering RNA that blocked DNA synthesis. Immunohistochemistry staining of stable and disrupted plaques from carotid endarterectomy sections showed a correlation between active ␤-catenin and Ki67, a proliferation marker, and a more intense staining in the smooth muscle cell layer surrounding the lipid core of disrupted plaques. In conclusion, the ␤-catenin pathway is required for the mitogenic effect of oxidized LDL on human aortic smooth muscle cells. This study highlights the putative important role of the E-cadherin/␤-catenin/Tcf signaling pathway in atherosclerosis.