Uptake and Metabolism of Low Density Lipoproteins with Elevated Ceramide Content by Human Microvascular Endothelial Cells

Abstract

Ceramide is a bioactive molecule involved in cellular responses to stress and inflammation. The major pathway for ceramide accumulation is via agonist-induced activation of cellular sphingomyelinases. It has also been shown that the ceramide level in circulating low density lipoprotein (LDL) increases during systemic inflammation, hence it is of importance to understand whether LDL-derived ceramide also contributes to the cellular ceramide homeostasis and affects cell functions. This article provides evidence of uptake of ceramideenriched LDL by human microvascular endothelial cells in a receptor-mediated fashion. This uptake can be competed by native LDL, indicating that the LDL-binding receptor may be involved. Following uptake, part of the LDL-derived ceramide is converted to sphingosine, but more importantly, part of it accumulates inside the cells (approximately 1.44 nmol/mg of cell protein). This accumulation of ceramide correlates with an increased incidence of apoptosis. The addition of tumor necrosis factor-␣ further enhances the accumulation of LDL-derived ceramide and the rate of apoptosis. In contrast, inhibitors of receptor-mediated endocytosis block both, the accumulation of LDL-derived ceramide and the concurrent increase in apoptosis. We also show that LDL-delivered ceramide is a more efficient inducer of apoptosis as compared with ethanol-delivered ceramide, the same apoptotic effect being achieved by substantially smaller increases in intracellular ceramide. Taken together, the presented data indicate that increases in lipoprotein ceramide concentration may result in changes in the bioactive properties of circulating lipoproteins such as the enhanced ability to induce apoptosis in the microvascular endothelium.The uptake and metabolism of modified low density lipoprotein (LDL) 1 are a pre-requisite for the formation of atherosclerotic plaques (1-7). Recent studies suggest that changes in the sphingolipid content of LDL also affect the development of atherosclerosis by promoting aggregation (8 -10) and oxidation (11) of LDL. These studies have focused on the role of secreted, zinc-dependent, sphingomyelinase (S-SMase), which hydrolyzes LDL sphingomyelin (SM) to ceramide. It is secreted mainly by endothelial cells in vitro in response to either TNF-␣ or interleukin-1␤ (12), as well as in response to lipopolysaccharide in vivo (13). Studies using bacterial SMase or recombinant S-SMase have shown that conversion of LDL-SM to ceramide leads to aggregation (8) and fusion (14) of the LDL particles, increases their retention in subintimal space (19), and can facilitate LDL oxidation (11). The possible role of SMase has been further emphasized by data showing that LDL extracted from advanced human aortic plaques has higher ceramide content than LDL extracted from circulation or from normal aorta (19). These findings indicate that changes in the SM/ceramide ratio of LDL affect their atherogenic properties, however, they do not distinguish between the effect of ceramide increase and that of SM depletion. ...

Elevated sphingomyelinase activity and ceramide concentration in serum of patients undergoing high dose spatially fractionated radiation treatment: Implications for endothelial apoptosis

“…Our earlier studies have shown that relatively modest elevation in LDL ceramide content enhances the ability of LDL to induce apoptosis in human microvascular endothelial cells. 20 Furthermore, the potency of the response increases in the presence of the pro-inflammatory cytokine TNF-α. 20 To test whether the increases in serum ceramide during SFGRT could also affect endothelial survival, LDL particles were isolated from patient serum samples taken before (C-LDL) and at 72 h after (SFGRT-LDL) irradiation with the inductive dose of 15 Gy.…”

“…20 Furthermore, the potency of the response increases in the presence of the pro-inflammatory cytokine TNF-α. 20 To test whether the increases in serum ceramide during SFGRT could also affect endothelial survival, LDL particles were isolated from patient serum samples taken before (C-LDL) and at 72 h after (SFGRT-LDL) irradiation with the inductive dose of 15 Gy. LDL was also purified from healthy blood donor (N-LDL).…”

“…The combined eluates were dried in vacuo, and the Cer mass was quantified by HPLC of the long chain bases released after an acid hydrolysis in 0.5 M HCl in methanol at 65˚C for 15 h. Free long chain bases were analyzed as described by Williams et al 28 Assay of apoptosis by terminal deoxynucleotidyltransferase-mediated Nick-end labeling (TUNEL) Assay. Apoptosis in HME-1 cells was detected using the In Situ Cell Death Detection Kit (Roche Diagnostics) 20 Briefly, cells were cultured in 6-well dishes on glass cover slips in the medium described above. Before experiments, cells were serum-deprived and then treated with appropriate reagents and controls for 16 h. Labeling of 3' free hydroxyl ends of the fragmented DNA with fluorescein-conjugated dUTP was catalyzed by terminal deoxynucleotidyl-transferase by following the manufacturer's directions.…”

“…Donor liposomes were prepared as follows. 20 Total lipids were extracted from native LDL (0.5 mg of protein) according to the two-phase extraction procedure of Bligh and Dyer, 27 modified as described previously. 28 After extraction, the lipids from the lower chloroform phase were passed through a sodium sulfate column, and mixed with 10 nmol of NBD-SM (for a final concentration of SM during the transfer of 50 µM).…”

“…The functions of S-SMase are not well understood, however in vitro studies have shown that the enzyme can efficiently hydrolyze More importantly, the apoptotic response to Cer-LDL is greatly augmented by the concurrent addition of TNF-α at doses that are not apoptotic. 20 In addition to LDL, S-SMase can also hydrolyze SM from endothelial cellular membrane in an auto-and paracrine manner. Apparently, this also results in the induction of endothelial cell death and S-SMase has been found to play a pivotal role in endothelium damage in a murine model of acute respiratory syndrome.…”