Phospholipid Transfer Protein Augments Apoptosis in THP-1–Derived Macrophages Induced by Lipolyzed Hypertriglyceridemic Plasma
Objective— Lipolysis of triglyceride-rich lipoproteins (TGRLPs) generates phospholipid-rich surface remnants and induces cytotoxic effects in adjacent vascular cells. We hypothesized that by integrating surface remnants into HDL, phospholipid transfer protein (PLTP) alleviates cytotoxicity.
Methods and Results— To test this hypothesis and gain insight into cytotoxicity during the postprandial phase in vivo, we injected normo-TG and hyper-TG human volunteers after a standardized fat meal (postprandial sample) with heparin, thereby stimulating lipolysis (postprandial heparinized sample). Incubation of (primary) human macrophages and primary human endothelial cells with postprandial heparinized hyper-TG plasma induced pronounced cytotoxic effects that were dose dependent on the TG content of the sample. No such effects were seen with normo-TG and postprandial hyper-TG plasma. In vitro lipolysis of VLDL and chylomicrons indicated that both lipoprotein fractions can cause cytotoxicity. Interestingly, in experiments with THP-1–derived macrophages stably transfected with PLTP, PLTP substantially augmented both net phospholipid uptake and apoptotic cell death due to postprandial heparinized hyper-TG plasma. We observed that activation of caspase-3/7, poly-ADP-ribose polymerase, and enhanced bioactivity of acid sphingomyelinase may all contribute to this augmented apoptosis.
Conclusions— Our data show that lipolysis of TGRLPs and their remodelling by PLTP interact to disturb cellular phospholipid flux and intracellular signaling processes, ultimately leading to apoptosis in human macrophages and endothelial cells.
- phospholipid transfer protein
- human macrophages
- in vivo lipolysis
- triglyceride-rich lipoproteins
Hypertriglyceridemia (hyper-TG), in both the fasting and postprandial state, promotes atherosclerosis.1,2 The mechanisms underlying these relations are complex and include decreased HDL levels,3 increased triglyceride-rich remnant particles,4,5 and insulin resistance.6 TG hydrolysis of TG-rich lipoproteins (TGRLPs) by lipoprotein lipase (LPL) bound to the endothelium produces nonesterified fatty acids and represents a crucial step in the metabolic fate of ingested fat.7 Depletion of the hydrophobic core of TGRLPs by LPL generates redundant surface remnants rich in phospholipid,8 which in turn are acted on by phospholipases and phospholipid transfer protein (PLTP).9 Lipoprotein remodelling by postprandial phospholipases (eg, hepatic lipase and, less pronounced, by LPL itself) increases potentially cytotoxic phospholipid species, eg, lysolecithin.10 PLTP is an abundant plasma protein synthesized by the liver and adipose tissue9,11 and in addition, is expressed locally in macrophages of the atherosclerotic plaque.12 PLTP is exclusively responsible for protein-mediated transfer of phospholipids from surface remnants to HDL,13 thereby increasing HDL mass and altering its subfraction distribution.14 Much less is known about the role of PLTP in transfer between lipoproteins and cells. PLTP has clearly been demonstrated to deliver vitamin E from lipoproteins to endothelial cells, thereby ameliorating endothelial dysfunction,15 and from LDL and HDL to red blood cells.16 Whether PLTP transfers phospholipids to cells is not well established; however, by mechanisms yet to be defined, PLTP alters phospholipid content and phospholipid composition in hepatocytes17 and ATP binding cassette transporter A1–dependent efflux of cholesterol from macrophage foam cells.18 In this study, we hypothesized that transfer of cytotoxic phospholipids to cells by PLTP might lead to vascular damage. Observations linking lipolytic products to vascular pathobiology include increased endothelial permeability,19 increased TGRLP binding,20 decreased viability of endothelial cells and macrophages,8 endothelial dysfunction,21 and atherosclerosis.22,23 To the best of our knowledge, no information linking lipolytic products or PLTP to apoptotic processes is available.
Apoptosis is a well-regulated physiological process that allows the efficient removal of damaged vascular cells.24,25 Appropriately targeted apoptosis limits undesirable inflammatory processes,26 thereby facilitating vascular remodelling and regeneration. However, excessive and inappropriate apoptosis leads to vascular dysfunction and ultimately, vascular pathology.27 Enzymes and transcription factors involved in the regulation of this complex cascade include caspases,28 poly (ADP-ribose) polymerase (PARP), sphingomyelinases (SMases),29 and nuclear factor-κB (NF-κB).30
In the present study, we assessed apoptosis and necrosis induced by lipolyzed postprandial plasma and its lipoprotein fractions in human macrophages and endothelial cells; the role of TGRLPs, LPL, nonesterified fatty acids, and PLTP in these processes; and intracellular signaling pathways in THP-1–derived macrophages relevant to apoptosis including caspases, PARP, acid SMases, and NF-κB.
For a complete and more detailed methodology, please see http://atvb.ahajournals.org.
Oral Fat Load and Lipolysis In Vivo
After an overnight fast study subjects ingested a standardized oral fat load, and blood samples were collected before (baseline) and 4 hours after the procedure (postprandial). Then unfractionated heparin (50 IU/kg body weight) was injected intravenously, and 30 minutes later after in vivo lipolysis, another blood sample was taken (postprandial heparinized). The 2 study groups comprised normotriglyceridemic (normo-TG) probands free of the metabolic syndrome (n=4) and hyper-TG subjects fulfilling the Adult Treatment Panel III criteria for the metabolic syndrome (n=5).
Human Cell Culture
Human THP-1 cells were obtained from the American Type Culture Collection (Manassas, Va) and were cultivated by standard procedures. Differentiation into macrophages was achieved in supplemented RPMI-1640 cell culture medium (Biochrom, Berlin, Germany) containing 50 nmol/L phorbol 12-myristate 13-acetate (Promega, Madison, Wis).
Peripheral blood mononuclear cells were prepared from forearm venous blood of healthy volunteers by Biocoll density gradient centrifugation. Primary human monocytes were selected by use of paramagnetic beads (CD14+) according to the manufacturer’s instructions (Miltenyi Biotech, Bergisch Gladbach, Germany). Further differentiation into primary human monocyte–derived macrophages was achieved with RPMI-1640 medium containing 10% homologous or type AB serum (Sigma, St. Louis, Mo).
Primary human umbilical vein endothelial cells were isolated from umbilical cords kindly donated by the Gynaecology and Obstetrics Department, Innsbruck Medical University, Innsbruck, Austria. Primary human arterial endothelial cells were cultivated from tissue specimens from patients who underwent operation for aneurysms.
Fluorescence-Activated Cell Sorting Analysis for Determination of Cell Viability
Fluorescein isothiocyanate– or phycoerythrin-conjugated annexin V and propidium iodide (Alexis, Gruenberg, Germany) were used for flow cytometric determination of apoptotic and necrotic cells 24 hours after incubation of the cells with 5% fetal calf serum (control), normo-TG plasma samples, and hyper-TG plasma samples.
Stable Transfection of THP-1 Cells
pcDNA3.1-PLTP (PLTP transfection) and pcDNA3.1-GFP (mock transfection) were transfected into THP-1 cells with use of the Nucleofector Kit V from Amaxa Biosystems (Gaithersburg, Md). Stable transformants were selected with 1 mg/mL G418 (Invitrogen, Paisley, UK) in RPMI-1640 supplemented with 10% fetal calf serum–gold (PAA Laboratories, Pasching, Austria). Individual colonies were chosen and expanded for analysis of PLTP activity.
Cellular [3H]DPPC Uptake
Phospholipid uptake in THP-1–derived macrophages was performed according to McCormack et al30a with minor modifications. In brief, purified postprandial and postprandial heparinized TGRLPs from a normo-TG individual (after ingestion of a standardized oral fat meal) were labeled with [3H]dipalmitoylphosphatidylcholine ([3H]DPPC; Amersham, Uppsala, Sweden) from phosphatidylcholine vesicles. Labeled TGRLPs (normalized for TG content to 400 μmol/L) were added to the cells for 0, 30, 60, or 180 minutes. Then supernatants and cells were collected and radiation was counted in a scintillation counter (Beckman, Krefeld, Germany). After adjustment of cellular protein content, net uptake of [3H]DPPC from postprandial TGRLPs was calculated in percent according to the equation: 3H in cells/(3H in cells+3H in medium)×100.
Determination of Acid SMase Activity
Determination of acid SMase activity was performed as described in detail elsewhere.31
Cytotoxicity of In Vivo Lipolyzed Postprandial Plasma to THP-1 and Primary Human Cells (Monocytes, Monocyte-Derived Macrophages, and Umbilical Vein and Arterial Endothelial Cells)
Previous studies have shown that postprandial plasma lipolyzed in vitro is toxic to mouse peritoneal macrophages.8 We confirmed those findings in THP-1– and monocyte-derived macrophages after incubation of the cells with 5% native or in vitro lipolyzed plasma for 24 hours (data not shown). In vitro incubation of LPL with postprandial plasma may lead to accumulation of lipolytic products that in vivo are rapidly metabolized or taken up by cells. To test more stringently whether lipolysis also leads to cytotoxicity in vivo, we stimulated postprandial lipolysis in humans by intravenous injection of heparin 4 hours after a fat meal. By displacement of lipases from their endothelial binding sites, this procedure results in a pronounced, physiological stimulation of lipolysis.
In normo-TG patients, the viability of THP-1 and primary human cells exposed to postprandial and postprandial heparinized plasma was similar (Figure 1A–1F, group I). In hyper-TG patients, however, the viability of these cells after incubation with postprandial heparinized plasma was significantly decreased when compared with postprandial plasma (Figure 1A–1F, group II). Mechanistically, cytotoxicity in THP-1 and primary human cells was mainly due to increases in apoptotic cells (please see online data supplement Figure I, top, available at http://atvb.ahajournals.org). Compared with postprandial heparinized normo-TG samples, incubation with postprandial heparinized hyper-TG and chylomicronemic samples led to an obvious increase in shrunken, rounded, and gleamy cells, with an increase in cellular granularities, which are all indicators of cell death (please see online data supplement Figure I, bottom, available at http://atvb.ahajournals.org).
Generation of Stable PLTP-Transfected THP-1 Cell Clones
We hypothesized that stable expression of PLTP by macrophages might ameliorate the cytotoxicity of postprandial heparinized plasma, eg, by accelerating the integration of surface remnants of TGRLPs into HDL. To test this hypothesis, we generated 3 THP-1 cell clones stably expressing differing amounts of PLTP. PLTP mRNA levels were very low in control cells, somewhat higher in PLTP-transfected clone 1, and very substantial in PLTP-transfected clones 2 and 3 (please see online data supplement Figure IIIA, available at http://atvb.ahajournals.org).
PLTP activity in serum-free cellular supernatants from nontransfected and mock-transfected THP-1–derived macrophages averaged 6.7% and 4.4%, respectively, of normal human plasma PLTP activity (Figure 2A). In comparison, PLTP activity in serum-free cellular supernatants from PLTP-transfected THP-1–derived macrophages (clones 2 and 3) averaged 64.3% and 93.5%, respectively, of normal human plasma PLTP activity (Figure 2A). The amount of active PLTP in supernatants from primary human macrophages was similar to that in nontransfected and mock-transfected THP-1–derived macrophages (data not shown). In contrast, no appreciable amounts of active PLTP were found in supernatants from THP-1 monocytes and primary human monocytes (data not shown).
Cytotoxicity of In Vivo Lipolyzed Postprandial Plasma Is Increased in THP-1–Derived Macrophages Expressing PLTP
As expected, exposure of nontransfected and mock-transfected THP-1 macrophages to hyper-TG postprandial heparinized plasma resulted in an increase in annexin V–positive cells, ie, increased apoptosis (Figure 2B). Remarkably and contrary to our original hypothesis, expression of PLTP led to a pronounced and dose-dependent stimulation of the apoptotic process (Figure 2B and please see online data supplement Figure IV, available at http://atvb.ahajournals.org). These experiments demonstrate that PLTP actually promotes apoptosis in THP-1 macrophages exposed to postprandial heparinized plasma.
Net Uptake of [3H]DPPC From Postprandial TGRLPs Is Increased in THP-1–Derived Macrophages Expressing PLTP
Normal transfer of native phospholipids between lipoproteins requires active PLTP. We hypothesized that PLTP also transfers phospholipids between lipoproteins and cells and that deranged transport of phospholipids due to overexpression of PLTP may induce apoptosis in these cells. To directly test a major component of this hypothesis, we exposed nontransfected, mock-transfected, and PLTP-transfected THP-1 macrophages to [3H]DPPC transported with postprandial TGRLPs (Figure 3A and B). Indeed, [H]DPPC content was clearly and time-dependently increased in both PLTP-overexpressing clones. In parallel experiments, cellular phospholipid mass (as well as cholesterol and TG mass), however, was not appreciably different between cell lines (please see online data supplement Figure V, available at http://atvb.ahajournals.org). Taken together, these experiments strongly suggest that PLTP promotes the uptake of native lipoprotein-derived phospholipids into macrophages.
Concentrations of Caspase-3 and PARP Are Increased in Apoptotic THP-1–Derived Macrophages
To further explore the mechanism of apoptosis augmented by PLTP, we performed immunoblot analysis of active, cleaved caspase-3 subunits and of active PARP, proteins involved in the regulation of apoptosis. In both control and PLTP-transfected cells, signal intensities of caspase-3 and PARP were similar after exposure to fetal calf serum (control serum) and hyper-TG postprandial plasma (Figure 4A). However, exposure to hyper-TG postprandial heparinized plasma led to a marked increase in caspase-3 and PARP in nontransfected and mock-transfected cells (Figure 4A). In addition, PLTP-transfected clone 3 obviously showed the most intense bands, again demonstrating an augmentation of cytotoxicity of postprandial plasma by cellular PLTP expression (Figure 4A).
Bioactivity of Caspase-3/7 Is Increased in Apoptotic THP-1–Derived Macrophages
To evaluate whether increased intracellular concentrations of caspase-3 are associated with increased bioactivity of this enzyme, we measured caspase-3/7 activity after 6 and 24 hours in THP-1 cells exposed to hyper-TG postprandial heparinized plasma (Figure 4B). After 24 hours, caspase activity in postheparin samples was somewhat higher in nontransfected and low PLTP–expressing clones (clone 1; Figure 4B). However, a very pronounced increase in caspase activity was observed in clone 3, corroborating an augmentation of the cytotoxicity of postprandial plasma by cellular PLTP expression (Figure 4B).
Bioactivity of Acid SMase Is Increased in Apoptotic THP-1–Derived Macrophages
Lipoprotein-derived oxidized phospholipids have previously been shown to increase acid SMase activity, thereby promoting apoptosis.29 Therefore, to further explore the mechanisms of apoptosis augmented by PLTP, we measured the enzymatic activity of acid SMase in non-, mock-, and PLTP-transfected THP-1 macrophages exposed to postprandial and postprandial heparinized plasma (Figure 5A) and in separate experiments, to native postprandial VLDL and postprandial VLDL after in vitro lipolysis (Figure 5B). No appreciable changes were seen after exposure of postprandial plasma and native postprandial VLDL. However, in PLTP-transfected clone 3, but not in non- and mock-transfected cells, a substantial increase in acid SMase activity was observed after exposure to postprandial heparinized plasma (Figure 5A) and to postprandial VLDL after in vitro lipolysis (Figure 5B). Augmentation of acid SMase activity in PLTP-overexpressing cells may represent an early step in induction of apoptosis by lipolyzed TGRLPs. For additional results, please see http://atvb.ahajournals.org.
Hyper-TG causes atherosclerosis both directly by atherogenic TGRLP remnant particles5 and indirectly by disturbing the antiatherogenic functions of HDL.3 An additional mechanism contributing to the detrimental effects of hyper-TG on the vasculature may be the lipolytic process itself.4,8 Indeed, lipolytic products produced both by lipolysis in vitro and by purification from human postprandial plasma ex vivo have been shown to compromise the viability of murine macrophages in cell culture.8 In addition to these in vitro findings, lipolytic products substantially increased the endothelial permeability of the perfused hamster aorta, strongly suggesting that the cell culture findings are also applicable to the in vivo situation.19 Our article provides 4 major new findings regarding the role of lipolytic products and vascular pathobiology. First, we extended previous data to human macrophages and to human venous and arterial endothelial cells, indicating that all cell types studied so far are subject to the cytotoxicity of hyper-TG postprandial heparinized plasma. Second, we observed that both the circulating postprandial TG levels and induction of lipolysis by heparin injection were independently directly related to cytotoxicity. In contrast, circulating nonesterified fatty acid levels were unrelated to cytotoxicity. Third, we observed that in vitro lipolysis of TGRLPs (ie, postprandial VLDL and chylomicrons) from normo-TG subjects caused cytotoxicity in a dose-dependent manner. This demonstrates that cytotoxicity is not due to specific qualitative disturbances of hyper-TG lipoproteins but rather reflects the lipolytic process itself. In addition, we have shown that lipolytic VLDL and chylomicrons are necessary and sufficient to explain the cytotoxicity of lipolytic postprandial plasma. Taken together, our experimental results support the concept that lipolysis of TGRLPs, brought about chiefly by the actions of LPL and hepatic lipase, has the potential to adversely affect vascular integrity. In line with this concept, chylomicronemia in the absence of LPL activity, such as in type I hyperlipoproteinemia, does not induce atherosclerosis, despite excessive hyper-TG.32
Lipolysis of TGRLPs, particularly in the postprandial phase, is intricately linked to subsequent remodelling by PLTP,33 which results in the integration of lipolytic surface remnants into HDL,9 thereby profoundly altering HDL speciation.14 Because injection of heparin into humans substantially increases circulating LPL but does not change the plasma concentration of active PLTP, the ensuing imbalance between lipolysis and remodelling may contribute to the toxicity of lipolytic TGRLPs. We therefore assessed whether stable overexpression of PLTP by macrophages might ameliorate cellular cytotoxicity by transferring toxic phospholipids into HDL, thereby diverting them from cells. We have performed experiments in THP-1–derived macrophages that exhibited similar basal amounts of active PLTP when compared with primary human macrophages.12 Those experiments clearly demonstrated that overexpression of PLTP, which is naturally upregulated in macrophage-derived foam cells,12 substantially augmented cytotoxicity of macrophages induced by lipolyzed TGRLPs.
Based on our studies, a plausible explanation for this result is an increased transfer of native and/or oxidized phospholipids from lipolytic surface remnants to cells by PLTP. Transfer of lipoprotein components to cells by PLTP has been directly demonstrated previously (eg, vitamin E),15 and mice genetically lacking PLTP exhibit striking deficiencies of vitamin E in the liver,34 brain,35 and testes.36 Because PLTP is strongly expressed in macrophages of atherosclerotic lesions12 and in endothelial cells,11 we propose that local expression of PLTP may compromise vascular structure and function. In support of these findings, in genetically modified mice, the net metabolic effect of PLTP appears to promote atherogenesis. In PLTP−/− mice, atherosclerosis is decreased by 50%.37 Pronounced but not moderate overexpression of PLTP in mice increases atherosclerosis.38–40 However, in these models, major changes in circulating lipoproteins and vitamin E likely account for much of the proatherogenic effect of PLTP. To specifically investigate the role of macrophage PLTP in atherosclerosis, bone marrow transplantations from PLTP−/− into LDL receptor−/− mice have been performed by 2 study groups, yielding contradictory results.41,42 Valenta et al41 observed an increase in atherosclerosis after transplantation of PLTP−/− bone marrow, suggesting an atheroprotective potential for macrophage PLTP. In contrast, Vikstedt et al42 observed a decrease in atherosclerosis after transplantation of PLTP−/− bone marrow, suggesting an atherogenic potential of macrophage PLTP. ApoE−/− mice, transplanted with PLTP−/− macrophages, showed a pronounced increase in atherosclerosis, which, however, mainly reflects lower secretion of apoE by macrophages and consequently, much higher plasma cholesterol levels in this particular model.43 In humans, interestingly, cross-sectional studies appear to associate increased PLTP activity with coronary and carotid atherosclerosis.44,45
Fourth, a major novel finding of this article is a more precise definition of postprandial cytotoxicity. In THP-1–derived macrophages, human monocyte—derived macrophages, human umbilical vein endothelial cells, and human arterial endothelial cells, we have consistently demonstrated that cell damage is predominantly due to apoptosis. Apoptosis is crucial for vascular remodelling and regeneration.46 However, it must be tightly regulated to achieve a balance between removal of damaged cells on the one hand and proliferation and influx of cells into the vessel wall on the other.47 A number of signaling cascades are involved in the regulation of apoptosis.26 Interestingly, a recent report identified oxidized phospholipids derived from minimally modified LDL as a major factor directly inducing apoptosis in arterial smooth muscle cells.29 Mechanistically, within minutes, oxidized phospholipids activate acid SMase,29 which produces ceramide, thereby triggering downstream signal transduction cascades responsible for apoptosis.29,30 In addition to oxidized phospholipid, lysophosphatidylcholine has been shown to promote apoptosis as well.48 In line with these data, in our study PLTP and lipolytic VLDL interact to activate acid SMase. We have demonstrated that an influx of phospholipid derivatives originating from the redundant surface coat of TGRLPs after lipolysis is accelerated in PLTP-overexpressing cells, which may well account for augmented apoptosis. Furthermore, we have demonstrated that caspase-3/7 activity and mass are increased when cells are exposed to lipolytic VLDL and PLTP. Taken together, activated acid SMase and increased caspase-3/7 readily explain the induction of apoptosis by lipolytic products and PLTP.
In summary, active lipolysis in postprandial and hyper-TG plasma leads to apoptosis of human macrophages, which is further augmented by active PLTP. Mechanistically, overexpression of PLTP in macrophages augments cellular uptake of lipoprotein-derived phospholipids and activates intracellular acid SMase and caspase-3/7, thereby triggering apoptotic cell death. Our data support the notion that remodelling of TGRLPs by lipases and PLTP has the potential to acutely regulate the cellular composition of healthy and diseased vessels, thereby affecting the stability of atherosclerotic plaques, for example.
We thank Karin Salzmann for her excellent assistance with zonal ultracentrifugation and analytical work.
Sources of Funding
This study was supported by grant P16121-B07 from the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (FWF), by grant No. 9890 from the Jubiläumsfonds der Österreichischen Nationalbank (ÖNB), and by grant No. 48 from the Fonds zur Förderung der Forschung an den Universitätskliniken Innsbruck (MFF; all to B.F.).
Original received June 20, 2006; final version accepted January 17, 2007.
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