HLA class I antibodies provoke graft arteriosclerosis in human arteries transplanted into SCID/beige mice.

Antibodies toward HLA class I and/or MICA are commonly observed in transplanted patients suffering from allograft arteriosclerosis, also called chronic vascular rejection (CVR). The relative importance of cellular versus humoral alloreactivity for CVR is still disputed. We demonstrate that antibodies toward HLA class I provoke lesions typical for CVR in human arteries in vivo in the absence of cellular immunity. To show this, we grafted segments of human mesenteric arteries from 8 deceased organ donors into 36 immunodeficient SCID/beige mice in the infrarenal aortic position. Three mice died postoperatively. The remaining 33 mice received weekly i.v. injections of either a monoclonal antibody toward HLA class I, toward MICA or an irrelevant monoclonal antibody. At sacrifice after 6 weeks, mice receiving the HLA antibody showed a significant neointimal thickening in the grafted artery due to smooth muscle cell (SMC) proliferation while control mice receiving anti-MICA or irrelevant antibody showed little or no thickening. Whereas antibodies toward HLA class I were mitogenic to SMC in vitro, those directed toward MICA did not have any effect. Humoral alloreactivity toward HLA may thus play a causal role for the development of CVR and this opens new possibilities for the treatment of CVR.

Oxygen-regulated protein-150 prevents calcium homeostasis deregulation and apoptosis induced by oxidized LDL in vascular cells.

Oxidized LDLs (oxLDLs) induce apoptosis, which contributes to the pathogenesis of atherosclerosis. The 150 kDa oxygen-regulated protein (ORP150), an endoplasmic reticulum (ER)-resident chaperone, is upregulated by hypoxia and prevents ischemia-induced cell death. The aim of this work was to investigate whether and how ORP150 can prevent apoptosis induced by oxLDLs in vascular cells. OxLDLs induced ORP150 expression in the ER of human microvascular endothelial cell line (HMEC-1). ORP150 expression was blocked by antioxidants, by the permeant calcium chelator BAPTA-AM, and by inhibitors of the inositol-1,4,5 trisphosphate (IP3) receptors, 2-aminoethyl diphenylborinate (2-APB) and xestospongin C. ORP150 silencing by siRNA-enhanced oxLDL-induced apoptosis, while forced ORP150 expression increased the resistance of cells via an inhibition of the oxLDL-induced calcium rise, and of subsequent calpain activation, cytochrome c release, caspase 3 activation and apoptosis. A similar protective effect was achieved by BAPTA-AM, 2-APB and xestospongin C. Altogether, these data indicate that (i)ORP150 inhibits oxLDL-induced apoptosis by blocking calcium signaling and subsequent apoptosis, (ii)calcium released from ER stores through IP3 channels is involved in the oxLDL-induced calcium rise and apoptosis, and is inhibited by ORP150. Finally, ORP150 is expressed in advanced atherosclerotic lesions, where it may locally participate to reduce the apoptotic effect of oxLDLs and the subsequent risk of plaque rupture.

Effect of FTY720 on apoptosis of smooth muscle cells.

BACKGROUND: Hyperproliferation of smooth muscle cells (SMCs) plays a key role in allograft arteriosclerosis. This prompted us to investigate the effect of the novel immune modulator and synthetic sphingolipid FTY720 on apoptosis of SMCs. METHODS: Rabbit SMC cultures were treated with FTY720 and apoptosis and necrosis were detected by fluorescence microscopy. RESULTS: We investigated dose- and time-dependent effects of FTY720 and found that clinically relevant low doses of FTY720 (<1 micromol/L) did not induce apoptosis, whereas 10 micromol/L FTY720 induced apoptosis after 48 hours incubation. CONCLUSION: At doses of FTY720 used in clinics for treatment of renal allografts and multiple sclerosis. FTY720 did not induce SMC apoptosis.

Chronic vascular rejection: histologic comparison between two murine experimental models.

BACKGROUND: We previously developed an experimental model to study chronic vascular rejection (CVR) in mice, the orthotopic aortic allograft. More recently we performed human arterial grafts into SCID/Beige mice reconstituted with human spleen cells. We report herein the differences in CVR lesions. MATERIAL AND METHODS: In the first model, recipient mice were C57BL/6 (H-2b), and donor mice were DBA/2 (H-2d). In the second model, terminal branches of the human superior mesenteric artery were transplanted into SCID/Beige mice in the infrarenal aorta. Human immune reconstitution was achieved by a single intraperitoneal injection of 30 x 10(6) human spleen cells. The presence of human lymphocytes and IgG was verified weekly. In both models, the vascular grafts were inserted in the infrarenal aortic position using the sleeve technique. The transplanted mice were sacrificed at 35 days after the operation. The grafts were analyzed by histology and morphometry. The mean intimal thickening was calculated based on transverse sections at 0.1-mm intervals. RESULTS: Typical CVR lesions developed with neointimal thickening, T-cell infiltration, and smooth muscle cell (SMC) proliferation in both models. In the mouse aortic model, disappearance of SMC in the media was noted in contrast to human arterial transplants, where the media remained intact. CONCLUSION: Other groups have noted that arteries conserve their media in clinical organ transplants. From this point of view, the lesions in the second experimental model (human arteries) better reflect the pathology of CVR in clinical transplantation than the murine aortic transplant model.

Human immune reconstitution with spleen cells in SCID/Beige mice.

BACKGROUND: We developed an original experimental model to study chronic vascular rejection (CVR) consisting of a graft of human mesenteric artery followed by human immune reconstitution into CB.17 SCID/Beige mice. Human immune reconstitution achieved after human PBMC injection has often been variable and incomplete. The aim of this work was to develop an alternative method to achieve a complete, functional human immune reconstitution. METHOD: After institutional authorizations, spleen cells were recovered from cadaveric organ donors. Single intraperitoneal injections of various doses of spleen cells were made into 70 CB.17 SCID/Beige mice. Reconstitution of the human immune system was monitored by flow cytometry (circulating human cells) and ELISA (human IgG). Colonization of murine lymphoid organs by human cells was studied by immunohistochemistry and flow cytometry. Evaluation of the immune function consisted of examination of CVR lesions in human arterial grafts. The animals were humanely killed at day 28. RESULTS: After injection of 30 to 40 x 10(6) spleen cells, the mice showed significant human CD3(+), CD19(+), and CD56(+) populations in peripheral blood. The mean human cells levels were, respectively, 8.2% +/- 5.4%, 2.9% +/- 1.2%, and 5.3% +/- 5.1%. Murine spleen and mesenteric lymph nodes were colonized by human T and B cells, while the murine thymus was only colonized by human T cells. Human IgG was detected in murine serum (65.9 +/- 63.3 mg/L) and typical CVR lesions were observed within the allogeneic grafts. CONCLUSION: Intraperitoneal injection of 30 to 40 x 10(6) human spleen cells into CB.17 SCID/Beige mice induces complete and functional human immune reconstitution allowing the study of CVR under human allogeneic conditions.

Use of human mesenteric arteries to study chronic vascular rejection in SCID/beige mice reconstituted with human spleen cells.

We wanted to establish a preclinical model of chronic vascular rejection (CVR) by transplanting small arteries from the mesentery of cadaveric organ donors by the rapid "sleeve" technique into SCID/beige mice reconstituted with human allogeneic spleen cells. After institutional authorization and with informed consent from relatives, we obtained tissues and cells from cadaveric organ donors. A piece of mesentery was recovered from the donor and kept in buffered solution at 4 degrees C until use. After dissection of the mesentery, small arteries of suitable size were transplanted in place of the infrarenal aorta of the mice. Cells for the immunological reconstitution of the mice were spleen cells from the same or other organ donors. Twenty-three suitable arterial segments were obtained from the mesentery of three cadaveric donors. Ten of the mice received 3 x 10(7) human spleen cells intraperitoneally 1 week after the arterial graft and they all showed circulating human CD3+ and CD19+ cells 2 weeks after injection. The mice were sacrificed 5 weeks after the arterial graft. SCID/beige mice reconstituted with allogeneic spleen cells showed a typical CVR, whereas mice that received no cells had a normal vascular anatomy. We believe our model is well suited for the study of treatment of CVR under human allograft conditions.

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.

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.

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.

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.

[Fibrocytes and activated fibrocytes in the healing of an open skin wound]. / Fibrocytes et fibrocytes activés dans la cicatrisation d'une plaie cutanée ouverte.

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.

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.

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.

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.

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.

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.

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.

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.