Immunologic parameters of spleen cells from normal or IL-6-deficient mice bearing orthotopic aortic allografts.
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Animals↗
Biomedical subjects
J C Thiers
Publications and source records attributed to J C Thiers.
An orthotopic aortic graft mouse model to study the immunopathology of chronic vascular rejection.
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Animals↗
Simultaneous orthotopic transplantation of carotid and aorta in the rat by the sleeve technique.
Graft vascular disease (GVD) remains the major limitation to long-term survival after solid organ transplantation. Aortic or carotid allografts in rats have been shown to be useful models because similar changes to those observed in man develop within weeks. Both immunological and non-immunological factors influence the process of GVD and a method that could permit rapid multiple arterial allotransplantation in the rat would be of great value. We performed simultaneous orthotopic aortic and carotid allotransplantations in 25 rats. The vessels were anastomosed using a sleeve technique. No immunosuppression was given. The animals were killed at 15, 30, or 60 days and histological analyses of the grafts were performed. The overall survival rate was 80% and the incidence of technical failure was very low. The histopathological aspect revealed typical progressive GVD. In conclusion, we have developed a new model of simultaneous aortic and carotid transplantation in rats. This model, which incorporates a modification of the sleeve anastomosis, is rapid and yields an easy tool to investigate immunological and non-immunological processes driving GVD.
Anastomosis, Surgical↗
Alteration of plasmalemmal caveolae mimics endothelial dysfunction observed in atheromatous rabbit aorta.
OBJECTIVE: In endothelial cells, nitric oxide (NO) is produced by the endothelial isoform of nitric oxide synthase (eNOS), which is localized in the cholesterol-rich plasmalemmal microdomains involved in signal transduction, known as caveolae. The present study was undertaken to evaluate the effect of hypercholesterolemia and fatty streak formation on the endothelial caveolae and on endothelial function, and attempted to determine to what extent the caveolae were involved in endothelium-derived NO production. METHODS AND RESULTS: We first studied the effect of atheroma on endothelial NO production. Fatty streak infiltrated aorta of cholesterol-fed New Zealand White rabbits demonstrated an impairment of acetylcholine-induced relaxation and nearly normal calcium ionophore A23187-induced maximal relaxation. The abundance of caveolae in the endothelium covering the fatty streak, as well as their 'grape-like' clustering, appeared to be decreased. We therefore investigated the effect, on endothelial NO production, of the cholesterol-binding agents 2-hydroxypropyl-beta-cyclodextrin (hp-beta-CD) and filipin, known to alter caveolae structure and/or function. Treatment with either hp-beta-CD (2%) or filipin (4 microg/ml) did not affect contraction to phenylephrine or relaxant responses to A23187 or to the NO donor sodium nitroprusside. In contrast, both treatments impaired acetylcholine-induced relaxation. Cultured bovine aortic endothelial cells (BAEC) similarly treated with hp-beta-CD demonstrated a 50% decrease of total cellular cholesterol and a decreased abundance of caveolae as well as their 'grape-like' clustering. Cholesterol depletion decreased the bradykinin-induced transient peak of free intracellular calcium and subsequent receptor-stimulated NO production (assessed using reporter cells rich in soluble guanylyl cyclase), whereas that elicited by A23187 remained unaltered. CONCLUSION: Fatty streak deposit is associated with a decrease in caveolae 'transductosomes' abundance which appears to represent a novel mechanism of endothelial dysfunction.
2-Hydroxypropyl-beta-cyclodextrin↗
Orthotopic aortic transplantation in mice: a new model of allograft arteriosclerosis.
BACKGROUND: Graft arteriosclerosis is a major cause of death after allotransplantation of organs such as the heart or the kidney. Aortic allotransplantation in mice is a useful experimental model to study the mechanisms of this pathology. However, the conventional heterotopic aortic model is limited by a high morbidity and is technically difficult to perform. We developed a new simple method for aortic transplantation in mice. METHODS: The infrarenal aorta from the donor mouse was anastomosed to the recipient's aorta at the same position using a sleeve technique. Orthotopic aortic transplantation was performed in 45 mice, 5 isografts and 40 allografts. No immunosuppression was given, and the mice were killed at day 15 or 30. The graft was examined macroscopically, and several histologic sections were made. RESULTS: The overall survival rate was 78%. The incidence of thrombosis was low (4 cases) compared with previously published series. Histology of aortas revealed typical aspects of rejection in the allografts with a chronic picture at day 30. No significant lesion was observed in isografts. CONCLUSIONS: We have developed a model of orthotopic aortic transplantation in mice. This new model is easy to carry out and has a low incidence of thrombosis, probably because there is no size discrepancy between donor and recipient aortic segment.
Anastomosis, Surgical↗
Bcl-2 alters the balance between apoptosis and necrosis, but does not prevent cell death induced by oxidized low density lipoproteins.
Oxidized low density lipoproteins (oxLDL) participate in atherosclerosis plaque formation, rupture, and subsequent thrombosis. Because oxLDL are toxic to cultured cells and Bcl-2 protein prevents apoptosis, the present work aimed to study whether Bcl-2 may counterbalance the toxicity of oxLDL. Two experimental model systems were used in which Bcl-2 levels were modulated: 1) lymphocytes in which the (high) basal level of Bcl-2 was reduced by antisense oligonucleotides; 2) HL60 and HL60/B (transduced by Bcl-2) expressing low and high Bcl-2 levels, respectively. In cells expressing relatively high Bcl-2 levels (lymphocytes and HL60/B), oxLDL induced mainly primary necrosis. In cells expressing low Bcl-2 levels (antisense-treated lymphocytes, HL60 and ECV-304 endothelial cells), the rate of oxLDL-induced apoptosis was higher than that of primary necrosis. OxLDL evoked a sustained calcium rise, which is a common trigger to necrosis and apoptosis since both types of cell death were blocked by the calcium chelator EGTA. Conversely, a sustained calcium influx elicited by the calcium ionophore A23187 induced necrosis in cells expressing high Bcl-2 levels and apoptosis in cells expressing low Bcl-2 levels. This suggests that Bcl-2 acts downstream from the calcium peak and inhibits only the apoptotic pathway, not the necrosis pathway, thus explaining the apparent shift from oxLDL-induced apoptosis toward necrosis when Bcl-2 is overexpressed.
Apoptosis↗
[Fibrocytes and activated fibrocytes in the healing of an open skin wound].
The microscopic, electron microscopic and immunohistochemical observation of biopsy specimens taken at an early stage, at close and regular intervals (every 4 hours), from open skin wounds created in the pig and the monkey, together with quantitative analysis of the various cell types in the granulation tissue, supports the conception that the activated fibrocyte (fibroblast) originates from the fibrocyte of the wound edges and thus completes some earlier experimental studies. We describe here the various stages of the differentiation of the wound edge fibrocyte into an activated fibrocyte and its proliferation and migration from the edges to the site of the wound. This does not exclude the possibility that local mesenchymal cells take part in the formation of activated fibrocytes. The activated fibrocyte build the collagen of the granulation tissue and then remodel and ensure wound contraction by becoming fibroclasts and myofibroblasts. This article defines the signification of the terms fibrocyte, activated fibrocyte, fibroblast and activated fibroblast.
Animals↗
Potential role for ceramide in mitogen-activated protein kinase activation and proliferation of vascular smooth muscle cells induced by oxidized low density lipoprotein.
Proliferation of vascular smooth muscle cells (SMC) is a hallmark in the pathogenesis of atherosclerotic lesions. Mildly oxidized low density lipoproteins (UV-oxLDL), which are mitogenic to cultured AG-08133A SMC, activate the sphingomyelin (SM)-ceramide pathway. We report here the following. (i) UV-oxLDL elicited a biphasic and sustained activation of MBP kinase activity, phosphorylation and nuclear translocation of p44/42 mitogen-activated protein kinase (MAPK), and [3H]thymidine incorporation, which were inhibited by PD-098059, a MAPK kinase inhibitor. (ii) The use of preconditioned media (from SMC pre-activated by UV-oxLDL) transferred to native SMC and blocking antibodies against growth factors suggest that UV-oxLDL-induced activation of MAPK and [3H]thymidine incorporation seem to be independent of any autocrine secretion of growth factors. (iii) UV-oxLDL-induced activation of a neutral sphingomyelinase, SM hydrolysis, ceramide production, and [3H]thymidine incorporation were inhibited by two serine-protease inhibitors (serpins), suggesting that a serpin-sensitive proteolytic pathway is involved in the activation of the SM-ceramide signaling pathway. (iv) UV-oxLDL-induced MAPK activation and [3H]thymidine incorporation were mimicked by ceramide generated in the plasma membrane by bacterial sphingomyelinase treatment or by addition of the permeant C2-ceramide. Serpins did not inhibit the MAPK activation and [3H]thymidine incorporation induced by C2-ceramide, indicating that activation of the MAPK and [3H]thymidine incorporation is subsequent to the stimulation of the SM-ceramide pathway. Taken together, these data suggest that mitogenic concentrations of UV-oxLDL are able to stimulate the SM-ceramide pathway through a protease-dependent mechanism and activate p44/42 MAPK, leading to proliferation of vascular SMC.
Animals↗
Differential effects of interleukin-1 receptor antagonist and tumor necrosis factor binding protein on fatty-streak formation in apolipoprotein E-deficient mice.
BACKGROUND: The cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) are secreted by the different cell populations of the vascular wall and have been suggested to promote atherosclerosis. METHODS AND RESULTS: Their respective roles in fatty-streak formation in apolipoprotein E-deficient mice were investigated by use of IL-1 receptor antagonist and TNF binding protein. Estradiol-17beta was used as a positive control. Blocking TNF seemed to be active in female animals but not in males. IL-1 receptor antagonist was as effective as or more effective than estradiol in both sexes. CONCLUSIONS: IL-1 plays a crucial role in the initial step of the atherosclerotic process in this animal model, and blocking the activity of this cytokine should be considered as a therapeutic possibility.
Animals↗
Establishment and characterization of a human T-lymphocyte cell line immortalized by SV40 and with abnormal expression of TCR/CD3.
Human lymphocytes derived from the peripheral blood of a healthy woman were transfected with a plasmid carrying the simian virus 40 (SV40) large T antigen. The successfully transformed cells contained SV40 large T DNA and were negative for Epstein-Barr virus (EBV) and human T-cell leukaemia virus (HTLV)-1 genomes. The immortalized cell line was assigned to the T-lymphocyte lineage on the basis of morphological, immunological and cytochemical criteria. While the cells expressed CD1a and CD4 at the cell surface, the CD3 complex was solely intracytoplasmic. Immunoprecipitation studies indicated that these cells lacked T-cell receptor (TCR) alpha-chains but not beta-chains. They were negative for activation markers such as CD25, CD69 and major histocompatibility (MHC) class II molecules. In addition, the transformed cells exhibited a complete growth independency towards interleukin-2 (IL-2). However, after phorbol ester stimulation, CD25 and CD69 markers were expressed and IL-2 was secreted. This new human immortalized T-lymphocytic cell line, which is cell-surface TCR/CD3-negative, may be useful as an in vitro model for studying TCR/CD3 assembly, expression and signal transduction.
Antigens, Polyomavirus Transforming↗
Model SV40-transformed fibroblast lines for metabolic studies of human prosaposin and acid ceramidase deficiencies.
Skin fibroblasts from patients with Farber disease (acid ceramidase deficiency) and from two siblings of the only known family affected with prosaposin deficiency were transformed by transfection with a plasmid carrying the SV40 large T antigen. The prosaposin-deficient transformed cell lines conserved their original metabolic defects, and in particular they were free of detectable immunoreactivity when using anti-saposin B and anti-saposin C antisera. Ultrastructurally, the cells contained heterogeneous lysosomal storage products. As found for their parental cell lines, the SV40-transformed fibroblasts exhibited deficient in vitro activities of lysosomal ceramidase and beta-galactosylceramidase, but a normal activity of acid sphingomyelinase. As observed for SV40-transformed fibroblasts from Farber disease, degradation of radioactive glucosylceramide or low density lipoprotein-associated radiolabelled sphingomyelin by the prosaposin-deficient cells in situ showed a clear impairment in the turnover of lysosomal ceramide. Ceramide storage in prosaposin-deficient cells was also demonstrated by ceramide mass determination. In contrast to acid ceramidase deficient cells, both the accumulation of ceramide and the reduced in vitro activity of acid ceramidase in cells from prosaposin deficiency could be corrected by addition of purified saposin D. The data confirm that prosaposin is required for lysosomal ceramide degradation, but not for sphingomyelin turnover. The SV40-transformed fibroblasts will be useful for pathophysiological studies on human prosaposin deficiency.
Acid Ceramidase↗
The sphingomyelin-ceramide signaling pathway is involved in oxidized low density lipoprotein-induced cell proliferation.
Development of atherosclerosis is believed to involve proliferation of smooth muscle cells (SMC). Our laboratory previously demonstrated that the growth of bovine aortic SMC was stimulated by mildly oxidized low density lipoproteins (oxLDL) and that the mitogenic effect of oxLDL was greater than that induced by native LDL (Augé, N., Pieraggi, M. T., Thiers, J. C., Nègre-Salvayre, A., and Salvayre R.(1995) Biochem. J. 309, 1015-1020). Since the lipid mediator ceramide has been described to be proliferative, the present work aimed at studying the potential involvement of the so-called sphingomyelin-ceramide pathway in the signal transduction cascade induced by oxLDL. Incubation of SMC with UV-oxidized LDL induced sphingomyelin hydrolysis (32%), which peaked at 60 min and was accompanied by a concomitant increase of intracellular ceramide level. The effect of oxidized LDL on sphingomyelin turnover exhibited the same LDL dose dependence as their mitogenic effect. Exogenous bacterial sphingomyelinase induced sphingomyelin hydrolysis and ceramide generation and also stimulated cell growth, in contrast to exogenous phospholipases A2, C, or D. This mitogenic effect was reproduced by incubating the cells with the cell-permeant ceramides, N-acetyl- and N-hexanoylsphingosines. Altogether, these data strongly suggest for the first time that activation of the sphingomyelin-ceramide pathway may play a pivotal role in the oxLDL-induced SMC proliferation and atherogenesis.
Animals↗
An atypical aortic atherosclerotic lesion in cynomolgus monkeys during hypercholesterolemia: a protection by smooth muscle cells against advanced lesions?
This study focuses on the fortuitous discovery of an atypical atherosclerotic lesion in four of 49 male adult cynomolgus monkeys (macacus fascicularis) which were maintained for a long time at a high level of hypercholesterolemia, and in seven of 19 female cynomolgus monkeys examined from the second to the 24th week of hypercholesterolemic diet: this lesion was in formation or already mature during this period of diet. This atypical lesion was formed by a collagen and elastic network surrounding synthetic smooth muscle cells without fibrofatty or fibrous plaques. Lipids were occasionally seen in the inner intima. The lesion appeared early (from the third week of diet). Once established, its morphology did not change. It became more extensive, but was not complicated by lipid overload in spite of prolonged, permanent hypercholesterolemia. This response to hypercholesterolemia is interesting because the activity of the smooth muscle cells differs from that observed in the classic lesion: they intervene earlier, their replication is very marked and rapid, their elastin secretion is greater and remains constant over time, and their phagocytic properties are reduced. This experimental study examines the installation and the maintenance of this lesion and raises the problem of its origin.
Animals↗
The turnover of cytoplasmic triacylglycerols in human fibroblasts involves two separate acyl chain length-dependent degradation pathways.
Cultured fibroblasts from patients affected with the genetic metabolic disorder named neutral lipid storage disease (NLSD) exhibit a dramatic accumulation of cytoplasmic triacylglycerols (Radom, J., Salvayre, R., Nègre, A., Maret, A., and Douste-Blazy, L. (1987) Eur. J. Biochem. 164, 703-708). We compared here the metabolism of radiolabeled short-, medium- and long-chain fatty acids in these cells. Short/medium-chain fatty acids (C4-C10) were incorporated into polar lipids (60-80%) and triacylglycerols (20-40%) at a lower rate (5-10 times lower) than long-chain fatty acids. Pulse-chase experiments allowed to evaluate the degradation rate of cytoplasmic triacylglycerols in normal and NLSD fibroblasts and to discriminate between two catabolic pathways of cytoplasmic triacylglycerols. Short/medium-chain (C4-C10) triacylglycerols were degraded at a normal rate in NLSD fibroblasts, whereas long-chain (C12 and longer) triacylglycerols remained undegraded. These data are confirmed by mass analysis. The use of diethylparanitrophenyl phosphate (E600) and parachloromercuribenzoate (PCMB) inhibitors allows to discriminate between the two triacylglycerol degradation pathways. E600 inhibited selectively the in situ degradation of short/medium-chain triacylglycerols without inhibition of the degradation of long-chain triacylglycerols, whereas PCMB inhibited selectively the in situ hydrolysis of long-chain triacylglycerols without affecting the degradation of long-chain triacylglycerols. This was correlated with the in vitro properties of cellular triacylglycerol-hydrolyzing enzymes characterized by their substrate specificity and their susceptibility to inhibitors; the neutral lipase specific to long-chain triacylglycerols is inhibited by PCMB, but not by E600, in contrast to short/medium-chain lipase, which is inhibited by E600 but not by PCMB. The data of in vitro and in situ experiments suggest the existence in fibroblasts of two separate acyl chain length-dependent pathways involved in the degradation of cytoplasmic triacylglycerols, one mediated by a neutral long-chain lipase and another one mediated by a short/medium-chain lipase.
Cells, Cultured↗
Proliferative and cytotoxic effects of mildly oxidized low-density lipoproteins on vascular smooth-muscle cells.
We have investigated the role of low-density lipoprotein (LDL) oxidation in the proliferative effect of LDLs on cultured bovine aortic smooth-muscle cells and compared it with their effect on bovine aortic endothelial cells. The following conclusions were reached. (1) Non-toxic doses of mildly oxidized LDLs elicit a proliferative effect on smooth-muscle cells significantly higher than that of native LDLs or lipoprotein-depleted serum. The proliferative effect is dependent on time (relatively slow), dose (high doses are cytotoxic) and the level of LDL oxidation. (2) The proliferative effect on smooth-muscle cells is counterbalanced at high concentrations of mildly oxidized LDLs (or at high oxidation levels) by their cytotoxic effect. (3) The same dose of mildly oxidized LDLs exhibits no proliferative effect on endothelial cells but rather a cytotoxic one. Endothelial cells may therefore be intrinsically more susceptible to the cytotoxic effect of mildly oxidized LDLs than are smooth-muscle cells. (4) The proliferative effect of native LDLs on smooth-muscle cells results (at least in part) from cell-induced LDL oxidation during cell culture as suggested by (i) the progressive LDL oxidation over the 3 days of contact between LDLs and smooth-muscle cells and (ii) the concomitant inhibition of LDL oxidation and proliferative effect by butylated hydroxytoluene. The hypothetical mechanisms and potential involvement in atherogenesis are discussed.
Animals↗
A delayed and sustained rise of cytosolic calcium is elicited by oxidized LDL in cultured bovine aortic endothelial cells.
Bovine aortic endothelial cells (BAEC) pulsed for 5 h with mildly oxidized low density lipoproteins (LDL), exhibited a broad, sustained and high peak of [Ca2+]i occurring several hours after the end of the pulse and reaching very high [Ca2+]i values (around 2500-3000 nmol/l) and a concomitant drop of cytosolic pH (around 0.2-0.3 pH units) without any loss of cell viability. When BAEC were continuously pulsed with oxidized LDL, the peak of [Ca2+]i was more sustained than in short pulse experiments and was associated with irreversible morphological changes usually associated with cytotoxic events (blebbing) and with a marked loss of viability. The potential involvement of these biochemical and morphological changes in atherogenesis are discussed.
Animals↗
[Non-atherosclerotic aorta in Cynomolgus after a hypercholesterolemic regimen].
The authors present a study on atherosclerotic and non atherosclerotic lesions of aortas of Cynomolgus (Macaca Fascicularis) on high fat diet (HFD) (6-12-24 months), after regression and after resumption. At 6 months 2 aortic responses are seen: an edema, deep and superficial clumps of foam cells, few synthetic smooth muscle cells (SSMC) few collagenic fibers--an edema, few superficial foam cells, many SSMC, secreting collagen and elastin. At 12-24 months, after regression and resumption, two aortic lesions are observed: a pronounced atheroma (47 animals) and no atheroma (14 animals). In this case, in the inner part of the aortic wall there are a fibrosis and an elastogenesis, SSMC and just some superficial lipids deposits. These aortic responses of SSMC are certainly the consequence of the environment (the diet) but also the possible consequence of the genetic determinism of SSMC since some animals only present this early, constant fibro-elastic response during this experimental follow up even though all the animals have been subjected to the same lipidic stress.
Animals↗
Age-related changes in the elastic tissue of the human thoracic aorta.
Thirty human aortas with varying degrees of atheroma graded macroscopically according to the WHO classification were taken at autopsy from subjects of different ages (24-86 years). Study by light microscopy showed aortas with an intact wall (4 subjects, 25-46 years) with a thin intima and regular elastic layers, and aortas with varying degrees of modification of the wall, where the intima was of varying thickness and the elastic fibers showed varying degrees of damage (moderate lesions: 5 subjects, 35-52 yrs; severe lesions: 21 subjects, 26-86 yrs). From each aorta, a 4-cm segment from the tunica media, free of atheromatous lesions, was defatted and subjected to successive treatment with EDTA-Tris, 6 M guanidine-HCl-Tris, 6 M guanidine-HCl-Tris-DTE and collagenase. The residues (EP residues) were subjected to amino acid (AA) analysis and transmission electron microscopy (TEM) study. In the young subject, the AA composition was similar to that of elastin and the TEM images were characteristic of this substance. In the aging subject, an increase in polar AA and a parallel decrease in apolar AA and crosslinks was noted. By TEM, the elastin was seen to be associated with abundant fibrillar material. Trypsin treatment of EP residues gave E residues, whose composition and TEM appearance were similar in all samples, corresponding to the standard composition of elastin and its classic appearance by electron microscopy. We suggest that the fibrillar material removed by trypsin is the morphological reflection of the chemical variations observed in the EP residues. These correspond to contamination of the elastin by a polar protein fraction. This contamination is closely correlated with age but not with the degree of atheroma. Thus the age-related chemical changes in elastin appear to be independent of the onset and evolution of atheromatous lesions. The 10-15 nm diameter of the contaminating fibrillar material suggests that may be the microfibrillar fraction of elastic tissue.
Adult↗