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Higher cardiovascular morbidity in patients with a wide range of autoimmune diseases highlights the importance of autoimmunity in promoting atherosclerosis. Our purpose was to investigate the mechanisms of accelerated atherosclerosis and identified vascular autoantigens targeted by autoimmunity. We created a mouse model of autoimmunity-associated atherosclerosis by transplanting bone marrow from FcγRIIB knockout (FcRIIB(-/-)) mice into LDL receptor knockout mice. We characterized the cellular and molecular mechanisms of atherogenesis and identified specific aortic autoantigens using serologic proteomic studies. En face lesion area analysis showed more aggressive atherosclerosis in autoimmune mice compared with control mice (0.64 ± 0.12 vs 0.32 ± 0.05 mm(2); P < 0.05, respectively). At the cellular level, FcRIIB(-/-) macrophages showed significant reduction (46-72%) in phagocytic capabilities. Proteomic analysis revealed circulating autoantibodies in autoimmune mice that targeted 25 atherosclerotic lesion proteins, including essential components of adhesion complex, cytoskeleton, and extracellular matrix, and proteins involved in critical functions and pathways. Microscopic examination of atherosclerotic plaques revealed essential colocalization of autoantibodies with endothelial cells, their adherence to basement membranes, the internal elastica lamina, and necrotic cores. The new vascular autoimmunosome may be a useful target for diagnostic and immunotherapeutic interventions in autoimmunity-associated diseases that have accelerated atherosclerosis.-Merched, A. J., Daret, D., Li, L., Franzl, N., Sauvage-Merched, M. Specific autoantigens in experimental autoimmunity-associated atherosclerosis.

International audience; Mesenchymal stem cells (MSC) are multipotent postnatal stem cells, involved in the treatment of ischemic vascular diseases. We investigate the ability of MSC, exposed to short-term hypoxic conditions, to participate in vascular and tissue regeneration in an in vivo model of hindlimb ischemia. Transplantation of hypoxic preconditioned murine MSC (HypMSC) enhanced skeletal muscle regeneration at day 7, improved blood flow and vascular formation compared to injected nonpreconditioned MSC (NormMSC). These observed effects were correlated with an increase in HypMSC engraftment and a putative role in necrotic skeletal muscle fiber clearance. Moreover, HypMSC transplantation resulted in a large increase in Wnt4 (wingless-related MMTV integration site 4) expression and we demonstrate its functional significance on MSC proliferation and migration, endothelial cell (EC) migration, as well as myoblast differentiation. Furthermore, suppression of Wnt4 expression in HypMSC, abrogated the hypoxia-induced vascular regenerative properties of these cells in the mouse hindlimb ischemia model. Our data suggest that hypoxic preconditioning plays a critical role in the functional capabilities of MSC, shifting MSC location in situ to enhance ischemic tissue recovery, facilitating vascular cell mobilization, and skeletal muscle fiber regeneration via a paracrine Wnt-dependent mechanism.

Our aim was to investigate by in vivo biopanning the lesions developed early in atherosclerosis and identify human antibodies that home to diseased regions. We have designed a two-step approach for a rapid isolation of human Monoclonal phage-display single-chain antibodies (MoPhabs) reactive with proteins found in lesions developed in an animal model of atherosclerosis. After a single round of in vivo biopanning, the MoPhabs were eluted from diseased sections of rabbit aorta identified by histology and NMR microscopy. MoPhabs expressed in situ were selected by subtractive colony filter screening for their capacity to recognize atherosclerotic but not normal aorta. MoPhabs selected by our method predominantly bind atherosclerotic lesions. Two of them, B3.3G and B3.GER, produced as scFv fragments, recognized an epitope present on the surface in early atherosclerotic lesions and within the intimal thickness in more complex plaques. These human MoPhabs homed to atherosclerotic lesions in ApoE(-/-) mice after in vivo injection. A protein of approximately 56 kDa recognized by B3.3G was affinity-purified and identified by mass spectrometry analysis as vitronectin. This is the first time that single round in vivo biopanning has been used to select human antibodies as candidates for diagnostic imaging and for obtaining insight into targets displayed in atherosclerotic plaques.

Phosphorylation and subsequent inactivation of glycogen synthase kinase (GSK)-3beta via the Akt/PI3-Kinase pathway during ischemic preconditioning (PC) has been shown to be cardioprotective. As FrzA/sFRP-1, a secreted antagonist of the Wnt/Frizzled pathway, is expressed in the heart and is able to decrease the phosphorylation of GSK-3beta in vitro on vascular cells, we examined its effect during PC using transgenic mouse overexpressing FrzA in cardiomyocytes (alpha-MHC promoter) under a conditional transgene expression approach (tet-off system). Overexpression of FrzA inhibited the increase in GSK-3beta phosphorylation as well as protein kinase C (PKC) epsilon activation in transgenic mice after PC as compared with littermates. Phospho-Akt (P-Akt), phospho-JNK, or the cytoplasmic beta-catenin levels were not modified, phospho-p38 (P-p38) was slightly increased in transgenic mice after PC as compared with littermates. FrzA transgenic mice displayed a larger infarct size and a greater worsening of cardiac function compared with littermates. All these differences were reversed by the addition of doxycycline. This study demonstrates for the first time that disruption of a beta-catenin independent Wnt/Frizzled pathway induces the activation of GSK-3beta and reverses the benefit of preconditioning.

Lysyl oxidases (Lox), which are members of the amine oxidase family, are involved in the maturation of elastic lamellae and collagen fibers. Modifications of amine oxidases in idiopathic annulo-aortic ectasia disease (IAAED) have never been investigated. Our aim was to examine the expression of several proteins that might interfere with elastic fiber organization in control ( n = 10) and IAAED ( n = 18) aortic tissues obtained at surgery. Expression of amine oxidases and semicarbazide-sensitive amine oxidase (SSAO), and cellular phenotypic markers were examined by immunohistopathology and confocal microscopy. The expression of these proteins was assessed in relation to clinical and histomorphological features of the arterial wall. In control aorta, SSAO staining was expressed along elastic lamellae, whereas in aneurysmal areas of IAAED, SSAO was markedly decreased, in association with severe disorganization of elastic lamellae. Smooth muscle myosin heavy chain was also decreased in IAAED compared with controls, indicating smooth muscle cell dedifferentiation. Multiple regression analysis showed that elastic lamellar thickness (ELT) was correlated positively with the SSAO: elastin ratio and negatively with the Lox: elastin ratio, and that the clinical features of IAAED (aneurysm, thoracic aorta diameter, and aortic insufficiency) were positively correlated with ELT but not with SSAO. The relationship between SSAO expression and ELT suggests that this amine oxidase may be involved in elastic fiber organization. However, in advanced IAAED, the deficit in SSAO expression could be secondary to the decrease and fragmentation of elastic fibers and/or to vascular smooth muscle cell dedifferentiation.

Background Myocardial infarction results in irreversible myocyte loss. In a murine model, we tested the feasibility of a novel repair technique combining bone marrow cell (BMC) transplantation and cardiomyoplasty. Methods Myocardial infarction was induced cryogenically in backcrossed ROSA 26 transgenic × C57BL/6J mice (n = 75). Thirty days later, surviving mice (n = 69) were randomized to sham treatment (rethoracotomy only; n=11), patch only treatment (n = 29), or patch + BMC treatment (n = 29). Abdominal muscle patches were harvested from donor littermates not expressing the β-galactosidase reporter gene and sutured on the epicardium directly above the infarct zone. Patch only–treated mice received uncoated patches. Patch + BMC–treated mice received patches coated with 5 × 10 6 β-galactosidase-expressing BMCs embedded in a collagen-rich three-dimensional matrix. Results Mortality rate was 52% after muscle patch implantation. Bone marrow cells were able to migrate from muscle patch into the infarct zone, as demonstrated by β-galactosidase immunostaining, and ultimately constituted 8% of all cells in scar tissue (mean ± standard deviation, 219 ± 111/mm 2 ). Angiogenesis and cell survival in the scar were improved by patch + BMC treatment. Left ventricular geometry and cardiac function were improved by patch treatment, with or without BMC, although the effects were stronger after patch + BMC treatment. Conclusions Epicardial deposition of a BMC-coated muscle patch is a promising approach to restoring cardiac function after myocardial infarction.

Objective: FrzA, a member of the group of secreted frizzled related proteins (sFRP) that is expressed in the cardiovascular system, has been shown to antagonize the Wnt/frizzled signaling pathway. We have recently demonstrated its role in vascular cell growth control in vitro. In this study, we aimed to examine the mechanisms by which FrzA exerts its antiproliferative effect on vascular cells in vitro and its potential effect in vivo. Methods and results: On synchronized, growth-arrested endothelial cells (EC) and smooth muscle cells (SMC) treated with the recombinant purified FrzA protein, flow cytometry analysis showed that the recombinant FrzA protein delayed G1 phase and entry into S-phase. Western blot experiments demonstrated that the treatment of EC or SMC with FrzA was associated with a decrease in the level of the cyclins and cyclin-dependent kinases and an increase in cytosolic phospho-β-catenin levels. The FrzA-induced cell cycle delay was resolved by 24 h. C57BL/6J mice underwent surgery to produce unilateral hindlimb ischemia and empty adenoviruses (AdE) or adenoviruses coding for FrzA (AdFrzA) were injected at the time of the surgery. In AdFrzA-treated mice in the 7 days following surgery, we showed a decrease in cell proliferation, capillary density, and blood flow recovery and a reduced expression of cyclin and cdk activity in the ischemic muscle compared to that in the AdE-treated ischemic muscle. To gain insight into the pathway activated by FrzA overexpression, we showed an increase in the level of cytosolic phospho-β-catenin, a marker of β-catenin degradation, in AdFrzA-treated ischemic muscle compared to that in control AdE-treated ischemic muscle. Conclusion: We provided the first evidence that an impairment of the Wnt-Frizzled pathway, via FrzA overexpression, controlled proliferation and neovascularization after muscle ischemia.

Background— The secreted frizzled related proteins (sFRP) are soluble proteins thought to interfere with the Wnt signaling. Our group previously demonstrated that one of these members, sFRP-1/FrzA, is strongly expressed during early phases of the vascularization process in embryonic vasculature and in the endothelium of arteries and capillaries in adults and modulated vascular cell proliferation. Methods and Results— Analysis of the expression of sFRP-1 during cyclic ovarian angiogenesis revealed that sFRP-1 is expressed during the formation of neovessels and becomes undetectable when the vasculature is fully maturated. We then studied the role of FrzA in several distinct angiogenic models. FrzA induced angiogenesis in a chick chorioallantoic membrane model. Moreover, gene transfer of AdFrzA in grafted mesenchymal and glioma cells increased vessel density and tumor growth. FrzA induced formation of vessels, which were enlarged, longer, and appeared to be more mature compared with vessels formed under control treatments. In vitro, FrzA increased migration and tube formation of endothelial cells and seemed to protect them from apoptosis. FrzA-angiogenic effect in vitro was independent of vascular endothelial growth factor, fibroblast growth factor-2, or angiopiotin-1 induction and Akt activation. In contrast, FrzA decreased glycogen synthase kinase-3 phosphorylation. Conclusions— These results showed that FrzA has proangiogenic effects and suggest that Wnt signaling may be involved in normal differentiation as well as in the pathological development of vasculature.

Discrepancies between success in experimental animals with a variety of pharmacologic strategies and failure with such agents in clinical trials have raised questions concerning the mechanism of restenosis. Recent observations suggest a potential implication for the adventitial (Adv) layer in neointimal formation.The purpose of this study was to examine the Adv changes in the rat carotid artery subjected to balloon injury. These changes were characterized by morphometric, immunohistochemical, and electron microscopy analyses, with special attention devoted to early time-points post-injury.We report that the most important adventitial changes occurred in the first 48 h post-injury. Within 2 h there was extensive cell-loss by apoptosis and oncosis in the Adv and in the media; this was followed by the rapid onset of proliferation and a parallel slow increase in Adv thickening, reaching a maximum at 7 days. We further demonstrate an early migration of these Adv cells to the media and neointima. Moreover, we characterize the Adv cell phenotype with a panel of antibodies. Within 48 h after injury, a population of Adv cells expressed alpha-actin and vinculin with a maximum expression 7 days post-injury. At that time, these Adv cells started to express smooth muscle myosin heavy chain, a specific marker of smooth muscle cells. In parallel, we report an impaired production of elastic fibres in the Adv and medial layer.We reported a detailed time-course of adventitial changes after rat carotid injury (cell death, proliferation, migration and differentiation) that supports an important role of adventitia in neointima formation.

Because tissue freeze-drying is an excellent way to preserve antigenic conformation, we have tested the feasibility of this technique to reveal nonradioactive in situ hybridization (ISH) of tissue mRNA. We have compared mRNA detection after different methods of tissue preservation, freeze-drying, cryosectioning, and formaldehyde or methanol fixation. Our results show that nonradioactive ISH is more sensitive for tissues preserved by freeze-drying than for other tissue preparations. We have demonstrated that freeze-drying allows combination of ISH and immunohistochemistry for simultaneous detection of mRNA and antigen because with this technique of tissue preservation ISH does not affect the sensitivity or the amount of the detected antigens. This work underscores the fact that tissue freeze-drying is an easy, convenient, and reliable technique for both ISH and immunohistochemistry and achieves excellent structural conditions for nonradioactive detection.

Abstract —Vitronectin (VN) is a plasma glycoprotein that promotes cell attachment and induces migration of human smooth muscle cells (SMCs) in culture. VN has been observed to accumulate in human atherosclerotic plaques, although its origin and role in atherosclerosis are not yet established. In the present experiments, synthesis of VN by intimal cells and its colocalization with receptors, αvβ3 and αvβ5, were studied by in situ hybridization and immunohistochemistry on 15 human atherosclerotic plaques from carotid arteries obtained after surgery. Strong VN protein and mRNA expression was observed in the intima and in the media. In the intima, VN mRNA expression was colocalized with SMCs, indicating that these cells produce VN, which may account for its accumulation in atherosclerotic plaques. In SMCs in culture, immunoprecipitation after metabolic labeling demonstrated that human SMCs do synthesize vitronectin. Confocal microscopic examination showed that VN colocalized with its receptors, αvβ3 and αvβ5, in the atherosclerotic intima. However, the distribution of the VN receptors on SMCs in culture in contact with VN was different. These observations suggest that VN plays various parts in atherogenesis via different SMC membrane receptors. ( Arterioscler Thromb Vasc Biol. 1998;18:168-176.)

Abstract Extracellular nucleotides, particularly ATP, are involved in the modulation of arterial vasomotricity via P2 purinoceptors present on smooth muscle and endothelial cells. These nucleotides could also be implicated in the smooth muscle cell hyperplasia observed in intimal lesions. In this study, we tried to define the potential role of the P2Y 2 (P 2u ) purinoceptor by studying its expression in normal and balloon-injured rat aortas. The cloning of a rat P2Y 2 cDNA from a rat smooth muscle cell cDNA library made it possible to study P2Y 2 expression both by Northern blot and in situ hybridization. Northern blot experiments indicated that P2Y 2 mRNA was present in rat medial aortic smooth muscle and in cultured rat aortic smooth muscle cells. In situ hybridization indicated that P2Y 2 mRNA was present in endothelial cells of the intima and in some smooth muscle cells scattered throughout the media of adult rat aortas, while almost all medial smooth muscle cells of rat embryo aorta expressed this receptor. In contrast with adult aortic media, the majority of neointimal smooth muscle cells found in aortic intimal lesions either 8 or 20 days after balloon injury were positive for P2Y 2 mRNA. Moreover, a subpopulation of neointimal cells localized at the luminal surface could be identified by a higher P2Y 2 expression than the underlying neointimal smooth muscle cells. These data showing a strong expression of the P2Y 2 purinoceptor in the neointima of injured arteries suggest that extracellular nucleotides may be involved, via this receptor, in the intimal hyperplasia and/or chronic constriction observed at the lesion site, and consequently in the restenotic process.

Abstract The aim of the present study was to investigate the effect of synthetic glucocorticoid dexamethasone (Dex) on cholesterol esterification in cultured human smooth muscle cells (SMC). In labeled SMC, Dex stimulated the esterification of [ 3 H]cholesterol in a dose-dependent manner. This effect was specific for glucocorticoid hormones and could be inhibited by cycloheximide (3 ng/mL), actinomycin D (10 −5 mol/L), and the specific glucocorticoid antagonist RU 486 (10 −8 mol/L). When plasma membrane was selectively labeled with trace quantities of [ 3 H]cholesterol (0.25 μCi/mL, 1 hour, 10°C), Dex (10 −8 mol/L) caused a net flux of free [ 3 H]cholesterol into the cells. Moreover, Dex (10 −8 mol/L, 24 hours) stimulated the esterification of sterols, newly synthesized from [ 14 C]mevalonate (10 μCi/mL, 4 hours) and lowered the amount of [ 14 C]sterols susceptible for cholesterol oxidase. The incorporation of [ 14 C]oleic acid into cholesteryl esters was markedly higher in Dex-pretreated SMC than in the control cells (2.1±0.07 and 1.4±0.1 pmol/h/μg protein, respectively, P 3 -mediated [ 3 H]cholesterol efflux was also inhibited in Dex-treated cells; moreover, HDL 3 (40 μg/mL, 24 hours) had practically no effect on [ 3 H]cholesteryl ester content in Dex-treated SMC but caused a 50% reduction of [ 3 H]cholesteryl esters in the control cells. Thus, in human SMC glucocorticoids alter the redistribution of cholesterol between the pools of free and esterified cholesterol, paralleled by the change in acyl coenzyme A:cholesteryl acyltransferase and neutral cholesteryl ester hydrolase activities, leading to the impaired HDL 3 -mediated cholesterol efflux.

Basal vasomotor tone in coronary vessels is, in part, maintained by nitric oxide (NO) production by endothelial constitutive NO synthase (ecNOS). Alteration of coronary circulation observed in left ventricular hypertrophy secondary to hypertension could be associated with a decrease in NO production. The aim of this study was to measure: (1) coronary flow in the Langendorff-perfused heart model at baseline, after maximum vasodilation in response to adenosine (10(-5) M), after endothelium-dependent vasodilation in response to bradykinin (10(-8) M) and after ecNOS inhibition by nitro-L-arginine methyl ester (L-NAME) (10(-4) M); (2) medial thickening of coronary microvessels and perivascular collagen on histological heart sections; and (3) ecNOS expression by immunohistochemical staining in these vessels using 20-week-old spontaneously hypertensive (SHR) and Wistar-Kyoto control rats (WKY). These measurements were determined by computer-directed color analysis. When SHR were compared with WKY rats, we found: (1) a decrease in basal flow (10.1+/-0.6 v 15.3+/-1.2 ml/min/g, n=10, P0.0001), in maximum flow (15.4+/-0.7 v 24.3+/-1.3 ml/min/g, n=10, P0.001), in bradykinin-induced flow increment (1.5+/-0.3 v 2.6+/-0.3 ml/min/g, n=5, P0.05) and in L-NAME-sensitive flow (3.3+/-0.6 v 6.3+/-0.9 ml/min/g, n=7, P0.05); (2) an increase in medial thickness (9.4+/-0.6 v 5.4+/-0.3 microm, n=8, P0.001) and in perivascular collagen area (1509+/-311 v 462+/-120 microm2, n=8, P0.01) of coronary arterioles; and (3) a decrease in ecNOS expression in the endothelium (ecNOS-stained cross-sectional area in arterioles: 40.0+/-9.1 v 84.6+/-9.0 microm2, n=7, PO.005). These results suggest that in SHR the decrease in basal coronary flow can be related to a structural alteration of the microvessels with an increase of perivascular collagen but also to a decrease in ecNOS expression which might be associated with reduced NO production.

To determine the extent and origin of the stimulation of 15-lipoxygenase activity in atherosclerotic aortas, formation of hydroxy-derivatives from arachidonic acid was measured by HPLC-analysis and 15-lipoxygenase mRNA expression was investigated by RNA blot and in situ hybridization in atherosclerotic and normal rabbit aortic tissues. The synthesis of hydroxy-eicosatetraenoic acids (HETE) from exogenously added [ 14 C]arachidonic acid was unchanged in atherosclerotic aortas in comparison with healthy aortas, but pretreatment with indomethacin demonstrated that 15-HETE production resulted essentially (75%) from cyclooxygenase activity in healthy aorta and from lipoxygenase activity in atherosclerotic aorta. The RNA blot and in situ hybridization with radiolabelled oligonucleotide probe demonstrated that 15-lipoxygenase mRNA was strictly localized in intimal thickening of atherosclerotic aortas. The immunostaining using anti-α smooth muscle actin, revealed that smooth muscle cell rich areas of the intimal thickening expressed 15-lipoxygenase mRNA. In addition, RNA blot hybridization indicated that cultured smooth muscle cells from atherosclerotic aortas expressed strongly 15-lipoxygenase mRNA. These results demonstrate that augmentation of 15-lipoxygenase activity in atherosclerotic aortas is correlated with 15-lipoxygenase mRNA expression in atherosclerotic plaque, and that intimal smooth muscle cells were involved, in addition to macrophages, in the expression of 15-lipoxygenase.

We have investigated whether cellular metabolism of linoleic acid (18:2) can influence prostacyclin (PGI2) production by cultured rat aortic smooth muscle cells (SMC) and tissues. Incubation of rat SMC homogenates with [1-14C]18:2 results in the enzymatic synthesis of [14C]13-HODE (hydroxyoctadecadienoic acid) and to a lesser extent [14C]9-HODE as defined by gas-liquid chromatography-mass spectrometry (GLC-MS). The observed changes, in percent enzymatically synthesized 13-HODE in the presence of indomethacin, aspirin, metyrapone, 15-HPETE (hydroperoxyeicosatetraenoic acid), and NDGA, suggest that it is formed from the PGH (prostaglandin endoperoxide) synthase pathway. Incubation of intact adherent SMC with [14C]linoleic acid demonstrates that the monohydroxylated compounds are predominantly esterified within the membrane phospholipids and not released into the incubation medium. The simultaneous incubation or a short-term preincubation of 18:2 and arachidonic acid (20:4) do not modify the enzymatic profile of 20:4 transformation. By contrast, long-term preincubation of cells with 18:2 or 13-HODE stimulates the transformation of exogenously added [14C]20:4 to [14C]6-keto PGF1 alpha. However, exogenous 13-HODE does not enhance [14C]6-keto PGF1 alpha recovery from [14C]20:4 prelabeled SMCs. Our results demonstrate that 18:2 is a substrate for PGH-synthase in rat aortic SMC and tissues. The 13-HODE formed is essentially esterified in cell phospholipids and remains without any significant effects on the release of [14C]6-keto PGF1 alpha from [14C]20:4 prelabeled SMC.

Background— Inflammatory processes play a critical role in myocarditis, dilated cardiomyopathy, and heart failure. The expression of the inflammatory chemokine osteopontin (OPN) is dramatically increased in cardiomyocytes and inflammatory cells during myocarditis and heart failure in human and animals. However, its role in the development of heart diseases is not known. Methods and Results— To understand whether OPN is involved in cardiomyopathies, we generated a transgenic mouse (MHC-OPN) that specifically overexpresses OPN in cardiomyocytes with cardiac-specific promoter-directed OPN expression. Young MHC-OPN mice were phenotypically indistinguishable from their control littermates, but most of them died prematurely with a half-life of 12 weeks of age. Electrocardiography revealed conduction defects. Echocardiography showed left ventricular dilation and systolic dysfunction. Histological analysis revealed cardiomyocyte loss, severe fibrosis, and inflammatory cell infiltration. Most of these inflammatory cells were activated T cells with Th1 polarization and cytotoxic activity. Autoantibodies against OPN, cardiac myosin, or troponin I, were not found in the serum of MHC-OPN mice. Conclusions— These data show that OPN expression in the heart induces in vivo T-cell recruitment and activation leading to chronic myocarditis, the consequence of which is myocyte destruction and hence, dilated cardiomyopathy. Thus, OPN might therefore constitute a potential therapeutic target to limit heart failure.

Purpose Sonic Hedgehog (Shh), a morphogen involved in embryonic development, stimulates repair of ischemic myocardium and skeletal muscle (Pola et al 2003 and Kusano et al 2005). Shh was shown to induce both the formation of new vessels and their muscularization. However the mechanisms involved in these processes are still poorly understood. We investigated the hypothesis that the angiogenic and chemotactic protein osteopontin (OPN) was involved in the angiogenic effect of Shh. Methods and Results We used a plasmid transfection strategy to increase Shh expression in the ischemic region of the limb and we compared the effect of Shh overexpression in wild type (OPN+/+) and OPN deficient (OPN-/-) 12-weeks-old mice transfected either with Shh plasmid (pShh) or control plasmid (β-gal). Shh overexpression induced a significant increase in the number of new vessels (+ 22 %± 4.2) in OPN+/+ mice in comparaison with β-gal transfected wild type mice, moreover the Shh effect was lost in OPN-/- mice. Analysis of vessel muscularization showed that Shh mainly increased the number of small non-muscularized vessels. In parallel, we observed an increased number of OPN expressing cells in the ischemic lesion mainly localized close to or inside debasing/regenerating muscle fibres. These cells expressed Mac3 and F4/80 markers suggesting a macrophage phenotype. We suggested that shh could increase the migration of macrophages in an OPN-dependent manner in order to accelerate the repair of the ischemic tissue. The experiments related to the function of OPN in ischemic limb stimulated or not by Shh is currently under investigation. Conclusion Our results demonstrated that OPN is required for the Shh angiogenic effect in the ischemic tissue and suggest that osteopontin expression may be associated with a better migration of macrophages in the ischemic lesion under Shh stimulation. Kusano KF et al. Nat Med. 2005 ; 11:1197-1204. Pola R et al. Circulation. 2003 ; 108:479-485.

Objective— Estradiol (E 2 ) is known to accelerate reendothelialization and thus prevent intimal thickening and in-stent restenosis after angioplasty. Transplantation experiments with ERα −/− mice have previously shown that E 2 acts through local and bone marrow cell compartments to enhance endothelial healing. However, the downstream mechanisms induced by E 2 to mediate endothelial repair are still poorly understood. Methods and Results— We show here that after endovascular carotid artery injury, E 2 -enhanced endothelial repair is lost in osteopontin-deficient mice (OPN −/− ). Transplantation of OPN −/− bone marrow into wild-type lethally irradiated mice, and vice versa, suggested that osteopontin plays a crucial role in both the local and the bone marrow actions of E 2 . In the vascular compartment, using transgenic mice expressing doxycyclin regulatable-osteopontin, we show that endothelial cell specific osteopontin overexpression mimics E 2 -enhanced endothelial cell migration and proliferation in the regenerating endothelium. In the bone marrow cell compartment, we demonstrate that E 2 enhances bone marrow–derived mononuclear cell adhesion to regenerating endothelium in vivo, and that this effect is dependent on osteopontin. Conclusions— We demonstrate here that E 2 acceleration of the endothelial repair requires osteopontin, both for bone marrow–derived cell recruitment and for endothelial cell migration and proliferation.

Mesenchymal stem cell (MSC) transplantation offers a great angiogenic opportunity in vascular regenerative medicine. The canonical Wnt/β-catenin signaling pathway has been demonstrated to play an essential role in stem cell fate. Recently, genetic studies have implicated the Wnt/Frizzled (Fz) molecular pathway, namely Wnt7B and Fz4, in blood growth regulation. Here, we investigated whether MSC could be required in shaping a functional vasculature and whether secreted Frizzled-related protein-1 (sFRP1), a modulator of the Wnt/Fz pathway, could modify MSC capacities, endowing MSC to increase vessel maturation. In the engraftment model, we show that murine bone marrow-derived MSC induced a beneficial vascular effect through a direct cellular contribution to vascular cells. MSC quickly organized into primitive immature vessel tubes connected to host circulation; this organization preceded host endothelial cell (EC) and smooth muscle cell (SMC) recruitment to later form mature neovessel. MSC sustained neovessel organization and maturation. We report here that sFRP1 forced expression enhanced MSC surrounding neovessel, which was correlated with an increase in vessel maturation and functionality. In vitro, sFRP1 strongly increased platelet-derived growth factor-BB (PDGF-BB) expression in MSC and enhanced β-catenin-dependent cell-cell contacts between MSC themselves and EC or SMC. In vivo, sFRP1 increased their functional integration around neovessels and vessel maturation through a glycogen synthase kinase 3 beta (GSK3β)-dependent pathway. sFRP1-overexpressing MSC compared with control MSC were well elongated and in a closer contact with the vascular wall, conditions required to achieve an organized mature vessel wall. We propose that genetically modifying MSC to overexpress sFRP1 may be potentially effective in promoting therapeutic angiogenesis/arteriogenesis processes.Disclosure of potential conflicts of interest is found at the end of this article.

Our aim was to investigate by in vivo biopanning the lesions developed early in atherosclerosis and identify human antibodies that home to diseased regions. We have designed a two-step approach for a rapid isolation of human Monoclonal phage-display single-chain antibodies (MoPhabs) reactive with proteins found in lesions developed in an animal model of atherosclerosis. After a single round of in vivo biopanning, the MoPhabs were eluted from diseased sections of rabbit aorta identified by histology and NMR microscopy. MoPhabs expressed in situ were selected by subtractive colony filter screening for their capacity to recognize atherosclerotic but not normal aorta. MoPhabs selected by our method predominantly bind atherosclerotic lesions. Two of them, B3.3G and B3.GER, produced as scFv fragments, recognized an epitope present on the surface in early atherosclerotic lesions and within the intimal thickness in more complex plaques. These human MoPhabs homed to atherosclerotic lesions in ApoE(-/-) mice after in vivo injection. A protein of approximately 56 kDa recognized by B3.3G was affinity-purified and identified by mass spectrometry analysis as vitronectin. This is the first time that single round in vivo biopanning has been used to select human antibodies as candidates for diagnostic imaging and for obtaining insight into targets displayed in atherosclerotic plaques.

SUMMARY 1 Smooth muscle cells (SMC) participate in both inflammatory and dedifferentiation processes during atherosclerosis, as well as during mechanical injury following angioplasty. In the latter, we studied medial SMC differentiation and inflammation processes implicated early after de-endothelialization in relation to mechanical stresses. We hypothesized that activation of a subpopulation of SMC within the media plays a crucial role in the early phase of neointimal formation. 2 For this purpose, we used a rabbit model of balloon injury to study activation and differentiation of medial SMC in the early time after denudation and just before neointima thickening. Inflammation was evaluated by the expression of vascular cell adhesion molecule (VCAM)-1, integrin a4b1 and nuclear factor (NF)-kB. Myosin isoforms and 2P1A2 antigen, a membrane protein expressed by rabbit dedifferentiated SMC, were used as markers of differentiation. 3 On day 2 after de-endothelialization, VCAM-1, a4b1 and NF-kB were coexpressed by a well-defined subpopulation of SMC of the internal part of the media, in the vicinity of the blood stream. At the same time, the majority of SMC throughout the media expressed non-muscle myosin heavy chain-B (nm-MHC-B) and 2P1A2 antigen. On day 7, when intimal thickening appeared, SMC of the media were no longer activated, whereas some intimal SMC expressed the activation markers. Thus, after de-endothelialization, early dedifferentiation occurs in most of the medial SMC, whereas activation concerned only a subpopulation of SMC located in the internal media. Using the T-type voltage-operated calcium channel blocker mibefradil (0.1–1 mmol/L) in SMC culture, we showed that this agent exhibited an antiproliferative effect in a dose-dependant manner only on undifferentiated cells. 4 In conclusion, the results suggest that the activated SMC represent cells that are potentially able to migrate and participate in the intimal thickening process. Thus, the medial SMC inflammatory process, without any contribution of inflammatory cells, may represent a major mechanism underlying the development of intimal thickening following mechanical stress. In humans, inhibition of T-type calcium channels may be a tool to prevent the early proliferation step leading to neointimal formation.

Our objective was to investigate the Escherichia coli localization (such as supernatant, cytoplasm and inclusion bodies) of an anti-alphaIIb-beta3 (alphaIIbbeta3) scFv fragment referred to as scFv[EBB3] produced in batch fermentation. Immobilized metal affinity chromatography (IMAC) purification was performed on supernatant using expanded bed absorbed technology (EBA) and on sonicated cells in native conditions over an immobilized copper-ion affinity column. Inclusion bodies were solubilized before IMAC purification and the refolding procedure was performed on the column. The majority of scFv[EBB3] were present as inclusion bodies (55%), whereas 36% were found in the cytoplasm and only 9% secreted in the supernatant. The scFv activity was assessed by enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunohistochemistry analyses performed on a thrombus induced in vivo on an atherosclerotic rabbit model.

Background and methods Hepatic lipase (HL) is an enzyme which hydrolyzes triglycerides from plasma lipoproteins and thus takes part in the metabolism of triglyceride-rich lipoprotein remnants and high density lipoproteins. The search described here concentrated on the description of the double invalidation of the HL and LDL receptor genes in mice in order to better understand the possible role of HL in combined hyperlipidemia/hyperalphalipoproteinemia and development of atherosclerosis. Results We show here that mice lacking both endogenous HL and LDL receptor (HL−/−:LDLR−/−) dramatically increased their plasma triglyceride-rich lipoproteins and their remnants as a consequence of reduced liver uptake. This result is strenghthened by the fact that HL−/−:LDLR−/− were found to overexpress LRP, LSR, and apoE genes. Interestingly, HL−/−:LDLR−/− mice showed premature spontaneous atherosclerosis and aortic lesions from 1-year-old animals were two-fold larger than those of LDLR−/− single mutants. We confirmed that HL−/− and wild-type mice did not develop atherosclerosis lesion even 1 year after birth. Conclusions Analysis of this double HL–LDLR knockout mouse model provides in vivo evidence that HL has a major role in the clearance of TRL remnants when LDLR is deficient and in the reduction of the development of atherosclerosis.

Myocardial infarction involves scar-formation mechanisms in which inflammation, proliferation, cell differentiation, apoptosis and angiogenesis all play a role. Better knowledge of the scar-formation process would be helpful in developing new therapies. The authors have generated a mouse model for infarction because its possible application in transgenic mice would allow the role of target genes in postinfarction scar-formation mechanisms to be studied. An infarction is caused by ligating the descending branch of the left coronary artery. At various times after ligation, the mice are sacrificed to determine the size of the infarction, left ventricular function and the overall myocardial scar-formation process. Early mortality was 10%. Between the fourth and sixth day postsurgery, 25% of mice died of a ruptured, infarcted left ventricle. The size of the infarctions diminished with time, while the surface of the left ventricle increased. In hemodynamics, 15 and 30 days after infarction, left ventricle telediastolic pressure was higher, telesystolic pressure was lower and contractility in indexes had collapsed. After an inflammatory phase in which polynuclear neutrophils colonized the scar, granulation tissue set in with a proliferation of myofibroblasts and growth of new blood vessels. These cells disappeared from the scar gradually, leaving behind a matrix rich in collagen and devoid of any contractile properties. The authors have characterized a murine model of myocardial infarction, with applications in transgenic mice and in view of establishing new agents in postmyocardial infarction repair.

Objective: Frizzled A is a very recent protein expressed in the cardiovascular hood by cardiomyocytes and by endothelial cells. This protein plays key roles in vitro in vascular cell proliferation and is able to induce an in vivo angiogenic response. Regarding these properties, we assess the hypothesis that Frizzled A could act in the healing process after myocardial infarction. Methods: To investigate the role of Frizzled A, we established a transgenic mouse line overexpressing the protein and developed a model of myocardial infarction by coronary artery ligation. Results: The incidence of cardiac rupture after myocardial infarction was reduced in transgenic mice (6.5 versus 26.4% in controls, n ¼ 165; P , 0:01). Infarct sizes were smaller in transgenic mice (18% of left ventricle circumference versus 28.1% in control at day 30; P , 0:001; n ¼ 6) and the cardiac function was improved (3800 ^ 370 versus 2800 ^ 840 mmHg/s dp/dtmax in controls, 2 2800 ^ 440 versus 21800 ^ 211 dp/dtmin in controls at day 15; P , 0:001; n ¼ 6). Early leukocyte infiltration had decreased in transgenic mice during the first week (103 ^ 59 versus 730 ^ 463 cells/mm 2 in controls at day 7; P , 0:001; n ¼ 6) and the apoptotic index was decreased by 50% at day 7. Capillary density in the scar was higher in transgenic mice (290 ^ 103 versus 104 ^ 43 vessels/mm 2 in control at day 15; P , 0:001) and vessels were more muscularized and mean lumen area was 3-fold higher (952 ^ 902 versus 313 ^ 350 mm 2 in control; P , 0:001). Conclusion: Overexpression of Frizzled A reduced the infarct size, improved cardiac recovery, modified inflammatory response and amplified angiogenesis. For these reasons, this protein would be of interest for cardiac surgeons using angiogenic therapy (as gene or protein injection) in ischemic heart diseases in non-revascularizable patients. q 2003 Elsevier B.V. All rights reserved.

Smooth muscle cell (SMC) migration involves interactions with extracellular matrix (ECM) and is an important process in response to arterial wall injury. We investigated the expression and the functional role of vitronectin (VN) in the response after vascular injury.VN and alpha v beta 3/beta 5 integrin expressions were investigated after balloon carotid injury of Sprague-Dawley rats. Adventitial delivery of blocking antibodies to VN, alpha v beta 5 and beta 3 integrins were performed to assess their roles in neointima formation. In vitro, migration assays were carried out on human SMC.Immunohistochemistry and in situ hybridization for VN showed an upregulation of VN during the early time points of intima formation. alpha v beta 3/beta 5 integrins expression correlated with VN expression. After 7 days, blocking antibodies to VN, alpha v beta 5 and beta 3 induced a significant decrease on intimal area associated with a decrease in intimal cell counts. A slight decrease in intimal cell proliferation without any effect on apoptosis was observed after VN blockade. In vitro, migrating SMC strongly expressed VN after injury and neutralizing anti-VN antibody inhibited SMC migration. Blocking experiment with anti-alpha v beta 5 and -alpha v beta 3 integrin antibodies showed that not only VN-alpha v beta 3 but also VN-alpha v beta 5 interactions are required for SMC migration.This study characterizes the VN-ECM interaction in SMC and supports the role of VN in mediating SMC migration and neointimal formation in response to injury.

International audience; The aim of this study was to investigate mitochondrial alterations in an animal model of chronic myocardial ischemia in rats obtained by surgical constriction of the left coronary artery. Resting coronary blood flow was measured using the fluorescent microsphere technique. Contractile function, defined by rate-pressure product, and myocardial oxygen consumption were measured in a Langendorff preparation. The mitochondrial function was evaluated on permeabilized skinned fibers. Three weeks after surgery, ischemic hearts showed a significant decrease in coronary blood flow compared with sham. Hemodynamic measurements showed a significant systolic and diastolic dysfunction. Alterations in mitochondrial function in ischemic hearts were mainly characterized by a significant decrease in the maximal velocity and apparent half-saturation constant for ADP, loss of the stimulatory effect of creatine, and a stimulatory effect of exogenous cytochrome c. These functional alterations were supported by structural alterations characterized by mitochondrial clustering and swelling associated with membrane rupture. We conclude that the alterations in systolic function after chronic ischemia are supported by severe modifications of mitochondrial structure and function.

Although mineral deposits have long been described to be a prominent feature of atherosclerosis, the mechanisms of arterial calcification are not well understood. However, accumulation of the non-collagenous matrix bone-associated proteins, osteopontin, osteocalcin, and osteonectin, has been demonstrated in atheromatous plaques. The aim of this study was to evaluate the role of these proteins in arterial calcification and, more precisely, during the initiation of this process. A model of rapid aortic calcification was developed in rabbits by an oversized balloon angioplasty. Calcification was followed using von Kossa staining and osteopontin, osteocalcin, and osteonectin were identified using immunohistochemistry. The aortic injury was rapidly followed by calcified deposits that appeared in the media as soon as 2 days after injury and then accumulated in zipper-like structures. Osteonectin was not detected in calcified deposits at any time after injury. In contrast, osteopontin and osteocalcin were detected in 8- and 14-day calcified structures, respectively, but not in the very early 2-day mineral deposits. These results suggest that these matrix proteins, osteopontin, osteocalcin, and osteonectin, are not involved in the initiation step of the aortic calcification process and that the former two might play a role in the regulation of arterial calcification.

Objectives: The histological nature and characteristics of aortic coarctation are not clearly defined, the aim of this study is to analyse intimal thickening in aortic coarctation. Methods : In order to characterize the components of intimal thickening in coarctation, narrowed segments of aorta obtained after surgery from ten children were examined immunocytochemically and by electron microscopy. Results : Histological analysis of aortic coarctation demonstrated a widened subendothelial region with separation of endothelial cells from the internal elastic lamina. Masson’s trichrome staining showed a marked increase in extracellular matrix and cell numbers in the intimal thickening compared with normal aorta. Cellular component analysis demonstrated invagination of the intima by smooth muscle actin-positive cells, with a fragmentation of the internal elastic lamina. No proliferating smooth muscle and inflammatory cells were identified in the intima. In order to characterize the smooth muscle cell phenotypes, various smooth muscle cell markers were sought using specific monoclonal antibodies: α-smooth muscle actin, smooth muscle-myosin heavy chain, heavy caldesmon, desmin. In moderate coarcted aorta, at least two distinct smooth muscle phenotypes were identified. In the juxtamedial part of the intima smooth muscle, cells were differentiated and expressed all smooth muscle markers; in the subendothelial part of the intimal thickening, the majority of smooth muscle cells expressed only α-smooth muscle actin and appeared dedifferentiated. In regions of marked stenosis, a strong expression of smooth muscle-myosin heavy chain, and heavy caldesmon in the intimal thickening pointed to the presence of redifferentiated smooth muscle cells, not still expressing desmin. Electron microscopic examination also revealed a variety of smooth muscle cell phenotypes in the intimal thickening. In the superficial layer, smooth muscle cells appeared to be in the synthetic state, while in the deeper part, both synthetic and contractile components were identified. Conclusions : These observations indicated that human coarctation was characterized by intimal recruitment of non-proliferating smooth muscle cells with dedifferentiated phenotype. However, the presence of smooth muscle cells with an intermediate phenotype in the narrowest part of the coarctation suggest that the redifferentiation process could participate in the pathogenesis of aortic coarctation.

Summary. We describe the preparation of a stable human lymphoblastoid cell line obtained during ex vivo studies in which peripheral blood lymphocytes of a Glanzmann's thrombasthenia patient were transformed with Epstein-Barr virus. Somatic hybrids secreted an IgM monoclonal antibody (B7) that reacted with the myosin heavy chain of human platelets by immunoblotting. Flow cytometry showed that B7 barely recognized unstimulated intact platelets, but bound abundantly after permeabilization of fixed cells with Triton X-100. The reactivity of the antibody on thin sections of human myocardium and aorta was studied by immunohistochemistry. B7 specifically stained myosin of myocytes, but there was no labelling of aortic smooth muscle cells. The epitope was conserved in cardiac or skeletal myosin prepared from pig or rabbit. Measurement of the dissociation constant in a competitive ELISA showed that B7 bound with high affinity (10−8 M). Purified Fab fragments retained their ability to bind to myosin, suggesting that B7 may be useful in the imaging of myocardial necrosis after myocardial infarction, myocarditis, cardiac drug toxicosis or graft rejection. This work also shows that EBV transformation of B cells may uncover naturally occurring autoantibodies which under normal circumstances are inhibited by the immune surveillance system.

Cultured rat aortic smooth muscle cells (SMC) metabolize 12(S)hydroxyeicosatetraenoic acid (12(S)HETE) by two different pathways: β-oxidation leading to 16:3(8-OH), and 10–11 reductase activity producing 20:3(12-OH) which is β-oxidized to 16:2(8-OH). In this work, we demonstrate that 10–11 reductase activity is modulated in cultured rat aortic SMC as a function of cell state (proliferating vs quiescent) and stimulated by serum. Most of the 20:3(12-OH) is recovered in the incubation medium but a significant part is esterified into phospholipids. By comparison with its parent compound, 12(S)HETE, 20:3 (12-OH) is mainly incorporated into phosphatidyl-choline and phosphatidyl-ethanolamine, suggesting that it may affect cellular functions. Taken together, these findings may be relevant to the effects of 12(S)HETE on vascular SMC functions related to atherosclerotic development.

12(S)-HETE, a major lipoxygenase-derived compound from arachidonic acid is incorporated and metabolized by vascular smooth muscle cells via beta-oxidation. We have now identified for the first time in this cell type 12(S)-HETE metabolites formed by a combination of reductase and oxidation pathways. HPLC and GC-MS analysis of time-course experiments allow us to characterize two different metabolic pathways: a direct peroxisomal beta-oxidation of 12(S)-HETE leading to the formation of 16:3 (8-OH) which accumulates first and a reduction of one of the conjugated double bonds of 12(S)-HETE giving the dihydro-intermediate 20:3(12-OH) that transiently accumulates before being converted itself by peroxisomal beta-oxidation to 16:2(8-OH). Taken together these results may suggest that the transient accumulation of 20:3(12-OH) through transcellular metabolism of 12(S)-HETE may represent a part of the modulatory effect of 12(S)-HETE on vascular function.

Osteopontin (OPN) is not expressed in healthy heart while, its expression is dramatically increased in cardiomyocytes and inflammatory cells during cardiomyopathies and heart failure. However its role in the development of heart diseases is not known.To understand whether OPN is involved in cardiomyopathy, we created a transgenic mouse (MHC-OPN) expressing recombinant OPN (rOPN) specifically in cardiomyocytes using aMHC promoter-directed OPN expression and tTA technology. In these mice, rOPN expression could be regulated by doxycyclin oral administration.After birth, MHC-OPN young mice were phenotypically indistinguishable from their littermate controls, but most of them died early between the 8 th and 15 th weeks after birth with a half life of 12 weeks. However, less than 10 % MHC-OPN mice survived and were still alive 30 weeks after birth. Inhibition of recombinant OPN expression by doxycyclin at the beginning of T cell infiltration (5 weeks after birth) or when DCM was initiated (11 weeks after birth) reduced myocarditis and thus avoided the early death.Electrocardiography demonstrated atrio-ventricular and intra ventricular defects. Moreover, echocardiography showed left ventricular dilation without hypertrophy and a systolic dysfunction, as indicated by reduced left ventricular fractional shortening (control mice: 29,8±2,0 %, n=13 ; and MHC-OPN mice: 13,7±4,0 %, n=5 ; T test p

Twenty salicylate derivatives were tested for their antagonistic activity on the inhibitory effect of aspirin on platelet aggregation. The blocking effect was not limited to the salicylate but also characterised some of its substituted compounds. The substituant influence did not seem to be related to electronic or size parameters. This antagonistic activity of these derivatives decreased as concentrations increased, owing to the emergence of their own inhibitory activity: several salicylate derivatives showed dual inhibitory and inhibition antagonistic activity, with both properties present at the same concentration. A mechanism involving dissociated activities on the two enzymatic sites of cyclooxygenase is proposed.

Prostaglandin synthesis in aortic smooth muscle cells originating from healthy an atherosclerotic rabbits was studied by incubating [14C]arachidonic acid with intact confluent cells and cell homogenates. In spite of a reduced 6-keto prostaglandin F1 alpha formation, no potentiating effect on the prostaglandin E2 generation occurred. Indeed, both cyclooxygenase and prostaglandin I2 synthetase activities appear to be reduced. These results suggest that an impaired arachidonic acid utilisation in aortic smooth muscle cells may be involved in the course of the atherosclerotic process.

Angiotensin converting enzyme (ACE) inhibitors were evaluated from their effects on human arterial smooth muscle cells in culture. [3H]ramipril binding has been determined in two different cell lines exhibiting ACE(+) and ACE(-) phenotypes. Specific binding occurred only in ACE(+) cells, was saturable, and displayed a Kd of 1 nM with a beta max value of 3.5 fmol/10(6) cells. Ramipril specific binding did not significantly modulate PGI2 synthesis in ACE(+) cells. By contrast, ramipril and, to a lesser extent, captopril appeared as weak inhibitors of PGI2 synthesis in ACE(-) cells. These data indicate that human arterial SMCs may express several phenotypes of the renin-angiotensin system under culture conditions and that ACE inhibitors may exert a non-renin-angiotensin-mediated pharmacological effect on eicosanoid synthesis.

Aortic explants obtained from normal adult rats and composed of all 3 tunics were cultured in a semi-synthetic gelosed medium supplemented by 10% serum. Explants cultured with normocholesterolaemic serum kept their in vivo characteristics for more than 12 days at electron microscope and histometabolic levels. However, explants showed considerable differences when cultured in a medium with hypercholesterolaemic serum. Cell proliferation in the tunica media and enhanced synthesis of macromolecular components of the extracellular matrix were observed. These alterations occurring inside the explants, which had kept all their tissular relationships, suggest that this culture system may be valuable for further studies on atherosclerotic processes.

The metabolism of linoleic acid by washed human platelets was investigated. [1.14C] linoleic acid was converted to [1.14C] hydroxy octadecadienoic acids (HODEs) at about the same rate with which [1.14C] 12-HETE was produced from [1.14C] arachidonic acid. The total radioactivity in HODEs was distributed among two isomers: 13-HODE (85%) and 9-HODE (15%) as defined by CG-MS. The production of HODEs by intact washed platelets was inhibited by indomethacin (IC50:5 x 10(-7) M) which suggest that hydroxy fatty acids were produced by PGH-synthase. By contrast, the production of HODEs by platelet cytosolic fractions was not modified under indomethacin treatment but completely abolished by NDGA (10(-3) M) and inhibited by the platelet lipoxygenase inhibitors 15-HETE (2.10(-5) M) and baicalein (10(-5) M). Platelets thus contain two different active systems which may convert linoleic acid to hydroxy fatty acids. Since these compounds remained essentially associated with the platelets, their presence may significantly participate in the mechanisms of platelet activation.

Low (LDL) and high (HDL) density lipoproteins stimulate prostacyclin (PGI 2 ) synthesis in cultured rabbit and human aortic smooth muscle cells. In this respect, the efficacy of HDL exceeded that of LDL, HDL 3 being the most effective. HDL 3 obtained from hypoalphacholesterolemic patients' serum had a lesser stimulative effect on PGI 2 synthesis as compared with HDL 3 of normolipidemic subjects. Partially purified apoprotein A-1 stimulates the metabolism of 14 C-arachidonic acid accompanied with enhanced formation of prostaglandins, especially 6-keto-PGI 1α . Within a 24 h incubation in the fetal calf serum-free medium, prostaglandins I 2 and E 1 (1 × 10 −7 m ) reduce the intracellular cholesterol level in human aortic smooth muscle cells by 30%. Total HDL fraction as well as HDL 3 and HDL 2b applied in combination with prostaglandins have a synergistic effect resulting in a 50% fall in intracellular cholesterol. Hypothetically, the uptake of cholesterol by HDL may include the following stages: (1) HDL interacts with the cell and activates the intracellular PGI 2 synthesis probably via apo A-1 modulatory action on arachidonic acid metabolism; (2) newly synthesized PGI 2 activates cholesteryl ester hydrolase leading to the formation of free cholesterol; (3) HDL takes up free cholesterol.

Prostacyclin (PGI2) synthesis seems to be one of the major physiological mechanisms involved in regulating platelet and vessel wall interactions. PGI2 is produced in large amounts by vascular endothelial cells, and vascular smooth muscle cells (SMC) also produce significant quantities. The capacity of SMC to produce PGI2, especially after endothelial injury, seems to be of importance. It is probably this type of situaton that is involved in the atherosclerotic process: experimental atherosclerosis in rabbits has been associated with a severe decrease in PGI, synthesis by arteries. Lipid peroxide accumulation within the arterial wall or in the plasma may also be involved in this process. Using arterial SMC in culture, we demonstrate here that, in comparison with healthy cultured cells, cells originating from atherosclerotic aorta have a decreased capacity to produce PGI2. The results were obtained using biological and radiochemical techniques and were confirmed by GC-MS. They suggest a potential role for PGI2 in inhibiting the atherosclerotic process.

The capacity of arterial SMCs to produce PGI 2 when stimulated by exogenous AA was studied in proliferative and confluent cultured cells and at different periods following endothelial denudation in vivo. PGI 2 production per cell was doubled during the exponential growth-phase in culture. By contrast, increased PGI 2 formation did not correlate with mitotic activity in intimal regeration tissue but with the presence of SMCs in a synthetic phenotype. The present results suggest a potential role of PGI 2 in SMC differentiation and proliferation.

Aortic tissues consisting of all three tunics were removed from normal adult rabbits and cultured in a semisynthetic gelosed medium supplemented by 10% serum obtained either from normal or hypercholesterolemic rabbits. Fibrillar cross-striated aggregates appeared with a high frequency (50%) in the extracellular space of explants cultured from four to eight days in medium supplemented by serum from hypercholesterolemic rabbits, but did not appear in explants cultured in serum from control animals (3%). The electron-dense segment was ruthenium red positive and digested by testicular hyaluronidase. The electron-lucent segment, composed of ruthenium red negative thin filaments, was not modified after hyaluronidase treatment but was strongly digested after collagenase treatment. It is believed that this material was fibrous long spacing collagen synthetized under culture conditions, as shown after tritiated proline incorporation.

In addition to the well established cyclooxygenase pathway, cultured aortic smooth muscle cells convert arachidonic acid to several polar metabolites identified by high performance liquid chromatography and gaz chromatography-mass spectrometry. 15-Hydroxyeicosatetraenoic acid, 12-Hydroxyeicosatetraenoic acid and 5-Hydroxyeicosatetraenoic acid are the major products formed. These observations indicate that the rabbit aortic smooth muscle cells are a potential source of lipoxygenase products and raise the possibility that this pathway of arachidonic acid metabolism can influence the biological functions of arterial myocytes under normal and pathological conditions.