A key role for matrix metalloproteinases and neutral sphingomyelinase-2 in transplant vasculopathy triggered by anti-HLA antibody.

BACKGROUND: Outcomes for organ transplantation are constantly improving because of advances in organ preservation, surgical techniques, immune clinical monitoring, and immunosuppressive treatment preventing acute transplant rejection. However, chronic rejection including transplant vasculopathy still limits long-term patient survival. Transplant vasculopathy is characterized by progressive neointimal hyperplasia leading to arterial stenosis and ischemic failure of the allograft. This work sought to decipher the manner in which the humoral immune response, mimicked by W6/32 anti-HLA antibody, contributes to transplant vasculopathy. METHODS AND RESULTS: Studies were performed in vitro on cultured human smooth muscle cells, ex vivo on human arterial segments, and in vivo in a model consisting of human arterial segments grafted into severe combined immunodeficiency/beige mice injected weekly with anti-HLA antibodies. We report that anti-HLA antibodies are mitogenic for smooth muscle cells through a signaling mechanism implicating matrix metalloproteinases (MMPs) (membrane type 1 MMP and MMP2) and neutral sphingomyelinase-2. This mitogenic signaling and subsequent DNA synthesis are blocked in smooth muscle cells silenced for MMP2 or for neutral sphingomyelinase-2 by small interfering RNAs, in smooth muscle cells transfected with a vector coding for a dominant-negative form of membrane type 1 MMP, and after treatment by pharmacological inhibitors of MMPs (Ro28-2653) or neutral sphingomyelinase-2 (GW4869). In vivo, Ro28-2653 and GW4869 reduced the intimal thickening induced by anti-HLA antibodies in human mesenteric arteries grafted into severe combined immunodeficiency/beige mice. CONCLUSIONS: These data highlight a crucial role for MMP2 and neutral sphingomyelinase-2 in vasculopathy triggered by a humoral immune response and open new perspectives for preventing transplant vasculopathy with the use of MMP and neutral sphingomyelinase inhibitors, in addition to conventional immunosuppression.

Lipid-induced peroxidation in the intestine is involved in glucose homeostasis imbalance in mice.

BACKGROUND: Daily variations in lipid concentrations in both gut lumen and blood are detected by specific sensors located in the gastrointestinal tract and in specialized central areas. Deregulation of the lipid sensors could be partly involved in the dysfunction of glucose homeostasis. The study aimed at comparing the effect of Medialipid (ML) overload on insulin secretion and sensitivity when administered either through the intestine or the carotid artery in mice. METHODOLOGY/PRINCIPAL FINDINGS: An indwelling intragastric or intracarotid catheter was installed in mice and ML or an isocaloric solution was infused over 24 hours. Glucose and insulin tolerance and vagus nerve activity were assessed. Some mice were treated daily for one week with the anti-lipid peroxidation agent aminoguanidine prior to the infusions and tests. The intestinal but not the intracarotid infusion of ML led to glucose and insulin intolerance when compared with controls. The intestinal ML overload induced lipid accumulation and increased lipid peroxidation as assessed by increased malondialdehyde production within both jejunum and duodenum. These effects were associated with the concomitant deregulation of vagus nerve. Administration of aminoguanidine protected against the effects of lipid overload and normalized glucose homeostasis and vagus nerve activity. CONCLUSIONS/SIGNIFICANCE: Lipid overload within the intestine led to deregulation of gastrointestinal lipid sensing that in turn impaired glucose homeostasis through changes in autonomic nervous system activity.

Stress-induced sphingolipid signaling: role of type-2 neutral sphingomyelinase in murine cell apoptosis and proliferation.

BACKGROUND: Sphingomyelin hydrolysis in response to stress-inducing agents, and subsequent ceramide generation, are implicated in various cellular responses, including apoptosis, inflammation and proliferation, depending on the nature of the different acidic or neutral sphingomyelinases. This study was carried out to investigate whether the neutral Mg(2+)-dependent neutral sphingomyelinase-2 (nSMase2) plays a role in the cellular signaling evoked by TNFalpha and oxidized LDLs, two stress-inducing agents, which are mitogenic at low concentrations and proapoptotic at higher concentrations. METHODOLOGY AND PRINCIPAL FINDINGS: For this purpose, we used nSMase2-deficient cells from homozygous fro/fro (fragilitas ossium) mice and nSMase2-deficient cells reconstituted with a V5-tagged nSMase2. We report that the genetic defect of nSMase2 (in fibroblasts from fro/fro mice) does not alter the TNFalpha and oxidized LDLs-mediated apoptotic response. Likewise, the hepatic toxicity of TNFalpha is similar in wild type and fro mice, thus is independent of nSMase2 activation. In contrast, the mitogenic response elicited by low concentrations of TNFalpha and oxidized LDLs (but not fetal calf serum) requires nSMase2 activation. CONCLUSION AND SIGNIFICANCE: nSMase2 activation is not involved in apoptosis mediated by TNFalpha and oxidized LDLs in murine fibroblasts, and in the hepatotoxicity of TNFalpha in mice, but is required for the mitogenic response to stress-inducing agents.

Integrin alpha(v)beta(3), metalloproteinases, and sphingomyelinase-2 mediate urokinase mitogenic effect.

Plasminogen activators are implicated in the pathogenesis of several diseases such as inflammatory diseases and cancer. Beside their serine-protease activity, these agents trigger signaling pathways involved in cell migration, adhesion and proliferation. We previously reported a role for the sphingolipid pathway in the mitogenic effect of plasminogen activators, but the signaling mechanisms involved in neutral sphingomyelinase-2 (NSMase-2) activation (the first step of the sphingolipid pathway) are poorly known. This study was carried out to investigate how urokinase plasminogen activator (uPA) activates NSMase-2. We report that uPA, as well as its catalytically inactive N-amino fragment ATF, triggers the sequential activation of MMP-2, NSMase-2 and ERK1/2 in ECV304 cells that are required for uPA-induced ECV304 proliferation, as assessed by the inhibitory effect of Marimastat (a MMP inhibitor), MMP-2-specific siRNA, MMP-2 defect, and NSMase-specific siRNA. Moreover, upon uPA stimulation, uPAR, MT1-MMP, MMP-2 and NSMase-2 interacted with integrin alpha(v)beta(3), evidenced by co-immunoprecipitation and immunocytochemistry experiments. Moreover, the alpha(v)beta(3) blocking antibody inhibited the uPA-triggered MMPs/uPAR/integrin alpha(v)beta(3) interaction, NSMase-2 activation, Ki67 expression and DNA synthesis in ECV304. In conclusion, uPA triggers interaction between integrin alpha(v)beta(3), uPAR and MMPs that leads to NSMase-2 and ERK1/2 activation and cell proliferation. These findings highlight a new signaling mechanism for uPA, and suggest that, upon uPA stimulation, uPAR, MMPs, integrin alpha(v)beta(3) and NSMase-2 form a signaling complex that take part in mitogenic signaling in ECV304 cells.

Interleukin-6 deficiency fails to prevent chronic rejection after aortic allografts in apolipoprotein E-deficient mice.

BACKGROUND: Chronic vascular rejection (CVR) is characterized by an intimal thickening in the arteries of allografts due to immunoinflammatory reactions and smooth muscle cell proliferation. Interleukin 6 (IL-6) levels are increased in patients with graft rejection, however the role of IL-6 in CVR is not known. We investigated if IL-6 deficiency in the recipient could prevent CVR after an aortic allograft. METHODS: Donor aortas from wild-type DBA/2 mice were transplanted into C57BL/6 recipients, either wild-type mice or mice deficient for IL-6 (IL-6(-/-)), apolipoprotein E (ApoE(-/-)), or both (IL-6(-/-)ApoE(-/-)). Alloantibody titers were determined at Day 30, 60, or 90 after grafting. The grafts were examined for CVR lesions by morphometry and immunohistology. RESULTS: All recipient allografts displayed lesions typical for CVR. The lesions were larger in IL-6-deficient strains, and significantly so in IL-6(-/-)ApoE(-/-) recipients. Early immunoglobulin (Ig) G1 alloantibody deposits were observed in the grafts of ApoE-deficient strains and late IgG2a deposits in the grafts of IL-6-deficient strains. A rapid and sustained type 1 helper T cell (Th1; IgG2a) alloresponse in IL-6(-/-) mice, and a strong type 2 helper T cell (Th2; IgG1) response in ApoE(-/-) mice were observed. IL-6(-/-)ApoE(-/-) mice displayed the highest alloantibody titer, with a Th1 dominance. CONCLUSIONS: Unexpectedly, IL-6 deficiency in the recipient mice did not prevent CVR lesions but even aggravated them in IL-6(-/-)ApoE(-/-) recipients. This was associated with increased local and systemic alloresponses.

Oxidized low-density lipoproteins trigger endoplasmic reticulum stress in vascular cells: prevention by oxygen-regulated protein 150 expression.

Oxidized low-density lipoproteins (oxLDLs) trigger various biological responses potentially involved in atherogenesis. Disturbing endoplasmic reticulum (ER) function results in ER stress and unfolded protein response, which tends to restore ER homeostasis but switches to apoptosis when ER stress is prolonged. We aimed to investigate whether ER stress is induced by oxLDLs and can be prevented by the ER-associated chaperone ORP150 (150-kDa oxygen-regulated protein). oxLDLs and the lipid oxidation products 7-ketocholesterol and 4-hydroxynonenal induce ER stress in human endothelial cells (HMEC-1), characterized by the activation of ER stress sensors (phosphorylation of Ire1alpha and PERK, nuclear translocation of ATF6) and of their subsequent pathways (eukaryotic initiation factor 2alpha phosphorylation, expression of XBP1/spliced XBP1, CHOP, and KDEL chaperones GRP78, GRP94, ORP150). ER stress was inhibited by the antioxidant N-acetylcysteine. In advanced atherosclerotic lesions, phospho-Ire1alpha, KDEL, and ORP150 staining were localized in lipid-rich areas with 4-hydroxynonenal adducts and CD68-positive macrophagic cells. By comparison, staining for 4-hydroxynonenal, phospho-Ire1alpha, KDEL, and ORP were faint and more diffuse in intimal hyperplasia. ER stress takes part in the apoptotic effect of oxLDLs, through the Ire1alpha/c-Jun N-terminal kinase pathway, as assessed by the protective effect of specific small interfering RNAs and c-Jun N-terminal kinase inhibitor. Forced expression of the chaperone ORP150 reduced both oxLDL-induced ER stress and apoptosis. ER stress markers and ORP150 chaperone are expressed in areas containing oxLDLs in atherosclerotic lesions and are induced by oxLDLs and oxidized lipids in cultured cells. The forced expression of ORP150 highlights its new protective role against oxLDL-induced ER stress and subsequent apoptosis.

Carbonyl scavenger and antiatherogenic effects of hydrazine derivatives.

Reactive carbonyl compounds (RCC) generated by polyunsaturated fatty acid oxidation alter progressively cellular and tissular proteins by forming adducts on free amino groups and thiol residues (carbonyl stress). Carbonyl scavengers may neutralize RCC, but their protective effect in atherosclerosis has not been extensively studied. We report the carbonyl scavenger and antiatherogenic properties of hydrazine derivatives, namely hydralazine, an antihypertensive drug, isoniazid, an antituberculosis agent, and two antidepressants, phenelzine and iproniazid. These drugs were poorly efficient in preventing the oxidation of LDL mediated by smooth muscle cells (SMCs), but inhibited the toxicity of UV-oxidized LDL (oxLDL) and of 4-hydroxynonenal (4-HNE). Hydrazine derivatives prevented the formation of foam cells resulting from LDL oxidation in human macrophagic U937 cells, and blocked the carbonyl stress in SMCs, by inhibiting the decrease in free amino group content, the increase in carbonylated proteins, and the formation of 4-HNE adducts on PDGFR. Experimental studies carried out on apoE-/- mice supplemented with drugs (30 mg/L in drinking water) showed a significant carbonyl stress inhibition correlated with a net reduction of atherosclerotic lesion development. In conclusion, these data indicate that hydrazine derivatives exhibit carbonyl scavenger and antiatherogenic properties, which opens novel therapeutical approaches for atherosclerosis and its cardiovascular complications.

E-cadherin/beta-catenin/T-cell factor pathway is involved in smooth muscle cell proliferation elicited by oxidized low-density lipoprotein.

The E-cadherin/beta-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 beta-catenin, as assessed by the dissociation of the beta-catenin/cadherin complex, and the concomitant rise of active beta-catenin in the cytosol. The oxidized LDL-induced rise of active beta-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-beta phosphorylation induced the inhibition of the proteasomal degradation of beta-catenin. Then active beta-catenin associated with Tcf4 and translocated into the nucleus. This enhanced the expression of the cell cycle activator cyclin D1. This crucial role of beta-catenin in the mitogenic effect of oxidized LDL was confirmed by silencing beta-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 beta-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 beta-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/beta-catenin/Tcf signaling pathway in atherosclerosis.

Caveolin-1 sensitizes vascular smooth muscle cells to mildly oxidized LDL-induced apoptosis.

Oxidized low-density lipoprotein (oxLDL)-induced apoptosis of vascular cells may participate to plaque instability and rupture. Caveolin-1 has emerged as an important regulator of several signal transduction pathways and processes that play a role in atherosclerosis. In this study we examined the potential role of caveolin-1 in the regulation of oxLDL-induced Ca(2+) signaling and apoptosis in vascular smooth muscle cells (VSMC). Cells expressing caveolin-1 were more susceptible to oxLDL-induced apoptosis, and this was correlated with enhanced Ca(2+) entry and pro-apoptotic events. Moreover, caveolin-1 silencing by small interfering RNA decreased the level of apoptotic cells after oxLDL treatment. These findings provide new insights about the potential role of caveolin-1 in the regulation of oxLDL-induced apoptosis in vascular cells and its contribution to the instability of the plaque.

Methylglyoxal induces advanced glycation end product (AGEs) formation and dysfunction of PDGF receptor-beta: implications for diabetic atherosclerosis.

PURPOSE: Low molecular weight carbonyl compounds, such as the alpha-ketoaldehydes methylglyoxal (MGO) and glyoxal (GO), are formed under hyperglycemic conditions and behave as advanced glycation end product (AGE) precursors. They form adducts on proteins, thereby inducing cellular dysfunctions involved in chronic complications of diabetes. METHODS AND MAIN FINDINGS: Nontoxic concentrations of GO or MGO altered the PDGF-induced PDGFRbeta-phosphorylation, ERK1/2-activation, and nuclear translocation, and the subsequent proliferation of mesenchymal cells (smooth muscle cells and skin fibroblasts). This resulted mainly from inhibition of the intrinsic tyrosine kinase of PDGFRbeta and in part from altered PDGF-BB binding to PDGFRbeta. Concomitantly, the formation of AGE adducts (N(epsilon)carboxymethyl-lysine and N(epsilon)carboxyethyl-lysine) was observed on immunoprecipitated PDGFRbeta. Arginine and aminoguanidine, used as carbonyl scavengers, reversed the inhibitory effect and the formation of AGE adducts on PDGFRbeta. AGE-PDGFRbeta adducts were also detected by anti-AGE antibodies in PDGFRbeta immunopurified from aortas of diabetic (streptozotocin-treated) compared to nondiabetic apolipoprotein E-null mice. Mass spectrometry analysis of aortas demonstrated increased AGE formation in diabetic specimens. CONCLUSIONS: These data indicate that MGO and GO induce desensitization of PDGFRbeta that helps to reduce mesenchymal cell proliferation.

Multiple human mesenteric arterial grafts from the same donor to study human chronic vascular rejection in humanized SCID/beige mice.

BACKGROUND: Chronic vascular rejection (CVR) is a major problem in clinical transplantation. Studies in experimental animals have been important to understand some of its mechanisms, but they are hampered by the difficulty of extrapolating the results into clinical practice. METHODS: We created a new experimental model for the study of human CVR by grafting multiple human mesenteric arteries from the same human donor into different SCID/beige mice in the infrarenal aortic position. Twenty-seven different mice were successfully grafted with a human artery from 6 donors. One week later, 23 of the mice received an intraperitoneal injection of 40 million human spleen cells, either from the same donor (autologous) or from another donor (allogeneic). RESULTS: In 81% of the mice an immune reconstitution was obtained, shown by the presence of human T, B and NK cells and IgG in circulating blood. At the time of sacrifice, 5 weeks after the arterial transplantation, a typical CVR with infiltration of human immune cells and deposit of human immunoglobulin was observed in the reconstituted mice that received allogeneic cells, whereas only minor lesions were noted in autologous combinations. No CVR was observed without injection of human splenocytes. We did not observe lymphoma or graft-vs-host reactions during the experiment. CONCLUSIONS: We show that it is feasible to graft multiple human arteries from the same donor into SCID/beige mice, and that a specific and typical CVR is observed after reconstitution with allogeneic spleen cells. Our method allows for pre-clinical testing of new therapeutics in controlled series.

Activation of the {beta}-catenin/T-cell-specific transcription factor/lymphoid enhancer factor-1 pathway by plasminogen activators in ECV304 carcinoma cells.

Besides its involvement in clot lysis, the plasminogen activator (PA) system elicits various cellular responses involved in cell migration, adhesion, and proliferation and plays a key role in the progression of cancers. beta-Catenin interacts with E-cadherins and functions as transcriptional coactivator of the Wnt-signaling pathway, which is implicated in tumor formation when aberrantly activated. We report that tissue-type plasminogen activator (tPA) elicited tyrosine phosphorylation and cytosolic accumulation of an active (non-serine-threonin phosphorylated, nonubiquitinated) form of beta-catenin in ECV304 carcinoma cells. tPA-dependent beta-catenin activation is mediated through epidermal growth factor receptor (EGFR) transactivation (via Src), suggested by the inhibitory effects of AG1478 and PP2 (specific inhibitors of EGFR and Src, respectively) and by the lack of beta-catenin activation in EGFR-negative B82 fibroblasts. EGFR phosphorylation and beta-catenin activation were inhibited by plasminogen activator inhibitor 1 and pertussis toxin, two inhibitors of the urokinase-type plasminogen activator (uPA)/uPA receptor system. beta-Catenin activation was correlated with the phosphorylation of glycogen synthase kinase-3beta through a phosphatidylinositol 3-kinase/Akt-dependent mechanism. Gel shift experiments revealed the activation of beta-catenin/T-cell-specific transcription factor (Tcf)/lymphoid enhancer factor-1 (Lef) transcriptional complex, evidenced by an increased binding of nuclear extracts to oligonucleotides containing the cyclin D1 Lef/Tcf site. beta-Catenin silencing through small interfering RNA and antisense oligonucleotides inhibited both the tPA-mediated cyclin D1 expression and cell proliferation. A similar activation of the beta-catenin pathway was triggered by amino-terminal fragment, the NH(2)-terminal catalytically inactive fragment of tPA, thus suggesting that this effect was independent of the proteolytic activity of plasminogen activators. In conclusion, the beta-catenin/Lef/Tcf pathway is activated by tPA and is involved in cell cycle progression and proliferation.

Engraftment of human T, B and NK cells in CB.17 SCID/beige mice by transfer of human spleen cells.

Models of severe combined immuno-deficient (SCID) mice reconstituted with a competent human immune system represent a valuable tool for the study of human immune responses in vivo. Reconstitution with human cells can be achieved using large numbers of peripheral blood lymphocytes, but levels of engraftment are poor and graft versus host disease (GVHD) frequently occurs. SCID/beige mice are at the same time deficient for adaptive and innate immunity and the objective of this study was to develop a safe and efficient way to achieve human lymphocyte engraftment in these mice using human spleen cells. After institutional authorisations and informed consent of relatives, a piece of spleen was obtained from cadaveric organ donors and the splenocytes were isolated and cryopreserved for later use. Single intraperitoneal injections of 5-100 x10(6) splenocytes were performed into SCID/beige mice. Reconstitution of a human immune system was monitored weekly by the presence of human cells and IgG in peripheral blood. The mice were sacrificed 4 weeks after the injection and the engraftment in lymphoid organs was studied. A reproducible reconstitution was obtained with intraperitoneal injection of 30-40 x10(6) spleen cells. Human T, B and NK cells as well as human IgG were present in peripheral blood. In lymphoid tissues, the same lymphocytic subpopulations were detected and in addition some antigen presenting cells. The reconstitution was functional because graft rejection was observed after transplantation of human allogeneic tissues. When less than 30 x10(6) cells were injected, the reconstitution was variable. When more than 40 x10(6) cells were injected, GVHD occurred with increasing frequency. In conclusion, we show that intraperitoneal injection of 30-40 x10(6) human splenocytes into SCID/beige mice induces a quick and functional engraftment of human T, B and NK cells with no risk of GVHD. This model may be used to study human transplantation immunobiology in vivo.

Two distinct calcium-dependent mitochondrial pathways are involved in oxidized LDL-induced apoptosis.

OBJECTIVE: Oxidized low-density lipoprotein (oxLDL)-induced apoptosis of vascular endothelial cells may contribute to plaque erosion and rupture. We aimed to clarify the relationship between the oxLDL-induced calcium signal and induction of apoptotic pathways. METHODS AND RESULTS: Apoptosis was evaluated by biochemical methods, including studies of enzyme activities, protein processing, release of proapoptotic factors, chromatin cleavage, and especially by morphological methods that evaluate apoptosis/necrosis by SYTO-13/propidium iodide fluorescent labeling. The oxLDL-induced sustained calcium rise activated 2 distinct calcium-dependent mitochondrial apoptotic pathways in human microvascular endothelial cells. OxLDLs induced calpain activation and subsequent Bid cleavage and cytochrome C release, which were blocked by calpeptin. Cyclosporin-A inhibited cytochrome C release, possibly by inhibiting the opening of the mitochondrial permeability transition pore (mPTP). Calcineurin, another cyclosporin-sensitive step, was not implicated, because oxLDLs inhibited calcineurin and FK-506 treatment was ineffective. Cytochrome C release in turn induced caspase-3 activation. In addition, oxLDLs triggered release and nuclear translocation of mitochondrial apoptosis-inducing factor through a mechanism dependent on calcium but independent of calpains, mPTP, and caspases. CONCLUSIONS: OxLDL-induced apoptosis involves 2 distinct calcium-dependent pathways, the first mediated by calpain/mPTP/cytochrome C/caspase-3 and the second mediated by apoptosis-inducing factor, which is cyclosporin-insensitive and caspase-independent.

Role for matrix metalloproteinase-2 in oxidized low-density lipoprotein-induced activation of the sphingomyelin/ceramide pathway and smooth muscle cell proliferation.

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-/- 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-/- 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.

Proliferation and wound healing of vascular cells trigger the generation of extracellular reactive oxygen species and LDL oxidation.

Cell proliferation of vascular cells is a key feature in vascular biology, wound healing, and pathophysiological processes such as atherosclerosis and restenosis. In atherosclerotic intima, cell proliferation colocalizes with oxidized LDL that indicate a local oxidative stress. This study aims to investigate whether cell proliferation is causally related with extracellular ROS generation and subsequent LDL oxidation. Sparse proliferating endothelial and smooth muscle cells generate higher levels of extracellular ROS (O2*- and H2O2) and LDL oxidation than confluent contact-inhibited cells. During wound healing of confluent cell layer, cell proliferation associated with healing also induced enhanced extracellular ROS generation and LDL oxidation. Proliferation-associated extracellular ROS generation is mediated through mitogenic signaling pathways, involving ERK1/2 and PKC, but is independent of de novo DNA synthesis, gene expression and protein synthesis. Data obtained with inhibitors of oxidases suggest that proliferation-associated extracellular ROS are not generated by a single ROS-generating system and are not essential for cell proliferation. In conclusion, our data show that proliferating vascular cells (in sparse culture or during wound healing) generate high levels of extracellular ROS and LDL oxidation through regulation of ROS-generating systems by mitogenic signaling. This constitutes a link between proliferative events and oxidative stress/LDL oxidation in atherosclerotic lesions and restenosis.

Pancreatic bile salt-dependent lipase induces smooth muscle cells proliferation.

BACKGROUND: Because bile salt-dependent lipase (BSDL), an enzyme secreted by the pancreatic acinar cells and associated with LDL in circulating blood, also locates with smooth muscle cells (SMCs) in atherosclerotic lesions, we aimed to investigate its effects on SMCs. METHODS AND RESULTS: Immunohistochemical experiments allowed us to detect an expression of BSDL in atherosclerotic lesions from hypercholesterolemic monkeys and from human arteries. BSDL was found to be associated with SMCs but not with macrophages. BSDL was significantly mitogenic for cultured SMCs. This effect was inhibited by heparin and anti-BSDL antibodies, whereas heat-denaturated and diisopropylfluorophosphate-treated BSDL were inefficient. The mitogenic effect of BSDL was associated with an activation of the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase pathway, which was inhibited by heparin, and involved several mechanisms, among them diacylglycerol and oleic acid production as well as a rapid basic fibroblast growth factor release. CONCLUSIONS: Circulating BSDL is associated with SMCs within the intimal arteria and may trigger SMC proliferation, which could contribute to the development of atherosclerotic lesions.