Human Smooth Muscle Cell Subpopulations Differentially Accumulate Cholesteryl Ester When Exposed to Native and Oxidized Lipoproteins
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《动脉硬化血栓血管生物学》
From the Vascular Biology Group at the Robarts Research Institute (C.A.A., C.G.S., S.L., Z.N., J.G.P., M.W.H.), and the Departments of Medicine (C.G.S., R.A.H., J.G.P., M.W.H.) and Biochemistry (R.A.H., J.G.P., M.W.H.), University of Western Ontario, London, Ontario, Canada.
ABSTRACT
Background— Vascular smooth muscle cells (SMCs) manifest diverse phenotypes and emerging evidence suggests this is caused by inherently distinct SMC subtypes. Recently, Li et al (Circ Res 2001;89:517–525) successfully cloned 2 uniquely responsive SMC subpopulations from a single human artery and we used this unique resource to test the hypothesis that distinct SMC subtypes are differential precursors of foam cell formation.
Methods and Results— When challenged with human atherogenic native or oxidized hypertriglyceridemic very-low-density lipoprotein (HTG-VLDL), the larger, slower-growing, spindle-shaped HITB5 SMC clone accumulated significantly more cholesteryl ester (CE) and triglyceride (TG) than the smaller, faster-growing epithelioid-shaped HITA2 SMC clone (10 versus 2 μg CE/mg cell protein and 60 versus 7 μg TG/mg PN, P<0.05). Lipoprotein lipase (LPL), a key enzyme involved in lipoprotein uptake, was identified as one differentially expressed protein that altered the predisposition of HITA2 SMCs for lipid accumulation. Although HITB5 SMCs secreted significantly more LPL than did HITA2 SMCs (0.7 versus 0.2 U/mL media, P<0.05), the addition of bovine milk LPL to HITA2 SMCs, significantly increased native and oxidized HTG-VLDL–induced lipid accumulation.
Conclusions— Inherently distinct SMC subsets are differentially predisposed to lipoprotein-induced lipid accumulation. Moreover, the environment can influence the response of SMC subsets to atherogenic lipoproteins.
We tested whether epithelioid and spindle-shaped human SMCs differentially accumulated lipid on exposure to native and oxidized lipoproteins. Spindle-shaped HITB5 cells preferentially accumulated cholesteryl ester and triglyceride, whereas the epithelioid-like HITA2 SMCs required exogenous LPL to accumulate lipid in the presence of lipoproteins. Therefore, SMC subtypes are differentially predisposed to a lipid-accumulating phenotype.
Key Words: atherosclerosis ? lipoproteins ? smooth muscle cells ? foam cells
Introduction
Foam cells are fundamental to the formation, growth, and stability of atherosclerotic plaques.1,2 Considerable data exist regarding the role of macrophage foam cells and the mechanisms by which they form.2 However, smooth muscle cells (SMCs) can also convert to foam cells and contribute to overall foam cell burden in a given lesion.1,3 The mechanism of SMC foam cell formation, however, is incompletely defined and cannot necessarily be assumed to be identical to that for macrophages. Moreover, an understanding of SMC foam cell formation must take into account the well-established diversity in SMC phenotype that can exist in the vessel wall.
The diverse functions manifested by SMCs in the vessel wall have long been considered to reflect plasticity in the differentiation status of SMCs.4 However, emerging evidence suggests that phenotypic diversity of SMCs in diseased arteries may also be a result of the expansion of one or more subsets of inherently distinct SMCs.4–6 SMC subtypes that differ in morphology, growth behavior, and response to growth factors have been identified.7–11 Importantly, it has not been established whether SMC subtypes are distinct with respect to their response to atherogenic lipoproteins.
Recently, Li et al demonstrated the presence of inherently distinct SMCs in human arterial media by cloning, from a single fragment of an adult nonatherosclerotic artery, 12 stable SMC clones.7,12 These segregated into 2 discrete clone types based on morphology: relatively small, epithelioid-like SMCs and larger, elongated, spindle-shaped SMCs. The 2 clone types were not interchangeable and varied somewhat in the expression of SMC differentiation markers. They were strikingly different with respect to growth rates and responsiveness to stimuli. In particular, epithelioid SMCs were faster-growing and more responsive to growth factors such as platelet-derived growth factor and fibroblast growth factor, as compared with the spindle-shaped SMCs.7,12 Other phenotypically distinct SMC clones have been successfully generated from experimental animals, supporting the notion of inherent SMC diversity.8–11 However, isolation of human SMC subtypes has proven more difficult. Therefore, these SMC clones provide a unique opportunity to assess their predisposition to foam cell formation.
SMCs are clearly capable of converting into foam cells1,3 and the true contribution of SMCs to lesion formation may in fact be underestimated. A recent study demonstrated that lesional foam cells, previously assumed to have a macrophage origin based on immunostaining, likely originated in the SMC population.13 Importantly, however, there are still uncertainties with respect to the nature of the atherogenic lipoproteins that induce SMC foam cell formation in vivo and the mechanisms mediating their uptake. Several candidate receptors can contribute to lipoprotein uptake in SMCs: the low-density lipoprotein receptor (LDLR), the very-low-density lipoprotein receptor (VLDLR), the low-density lipoprotein-related protein (LRP), and the scavenger receptors, CD36 and SRAI/II.14
Other factors may influence SMC receptor-mediated uptake of lipoproteins,15,16 including lipoprotein lipase (LPL) and apoE, both of which enhance cellular uptake of native lipoproteins.17–19 In macrophages, LPL and apoE isoform are critical for the uptake of VLDL isolated from hypertriglyceridemic (HTG) subjects.20 Initially, HTG-VLDL interacts with cell surface LPL, resulting in sequestration to the cell surface and hydrolysis of core triglyceride (TG) to free fatty acids (FFA), which are readily taken-up by the cell and re-esterified. Subsequently, the CE-rich remnants are taken-up via LDLR-mediated endocytosis.20 As LPL and apoE are secreted by arterial macrophages and SMCs,16,18 lipoproteins in the arterial wall have the potential to become enriched in apoE, bind LPL, and become better ligands for SMC lipoprotein receptors.
The objectives of the present study were to determine whether homogeneous cultures of inherently distinct SMC subtypes are preferentially predisposed to foam cell formation. The more mature, spindle-shaped HITB5 SMCs preferentially accumulated CE and TG when challenged by either native or oxidized HTG-VLDL when compared with the epithelioid-like HITA2 SMCs. We identified that LPL secretion was substantially higher in HITB5 SMCs and that differential LPL activity accounted for the difference in lipid deposition between the 2 SMC subtypes. Moreover, addition of exogenous LPL to HITA2 SMCs significantly enhanced CE and TG accumulation induced by native and oxidized HTG-VLDL to levels similar to those obtained in HITB5 SMCs. These findings establish that interplay between SMC phenotype and the microenvironment determines the response of SMCs to atherogenic lipoproteins.
Methods
Lipoproteins
Subjects were recruited from the Lipid Clinic at the London Health Sciences Center, University Campus (London, Ontario, Canada). The University of Western Ontario Health Science Standing Committee on Human Research approved the studies. The isolation of HTG-VLDL (Sf 20 to 400) and LDL (Sf 0 to 12), copper oxidation of VLDL and LDL, and LDL acetylation has been described previously.21,22
Cell Culture
Experiments were performed with 2 distinct human SMC clones, the epithelioid-like HITA2 and the spindle-shaped HITB5 cells. In some experiments, the human SMCs clones, HITD6 and HITC6, which are phenotypically similar to the HITA2 and HITB5 clones, respectively, were used. These were cloned from the media of a fragment of distal internal thoracic artery7,12 and have been extensively characterized with respect to SMC differentiation markers, vasoactive responsiveness, cell proliferation and migration rates, and contractibility.7 On recloning of either cell type, the original phenotype is preserved and addition of conditioned medium from one cell type to the other does not cause either cell to change phenotype. The source of fetal bovine serum or lipoprotein-deficient serum, type of media, glucose concentration, or ascorbate did not induce phenotype conversion. Furthermore, when either SMC subtype was transplanted subcutaneously into SCID mice, the morphological differences between the spindle-shaped and epithelioid SMCs were maintained. For details see online Methods and Figure I, available online at http://atvb.ahajournals.org.
For details of culturing cells for incubations with lipoproteins, oil red O staining, cellular lipid mass determinations, and assays for LPL and acyl coenzyme A:cholesterol acyl transferase (ACAT) activities, please see www.ahajournals.org.
Results
Oil Red O Staining and Lipid Accumulation in SMC Subtypes
Unlike primary SMC cultures, the SMC clones, HITA2 and HITB5, have a homogenous appearance and when confluent assume a epithelioid-like, cobblestone appearance and a spindle-shaped, hill-and-valley pattern, respectively,7,12 as illustrated in Figure 1A and 1B. To assess SMC lipid accumulation, HITA2 and HITB5 cells were cultured with or without HTG-VLDL and stained for neutral lipids with Oil Red O. There was little evidence for neutral lipid inclusions in HITA2 cells under control conditions, and only modest increases were observed after incubation with native HTG-VLDL (Figure 1C and 1E). In sharp contrast, there was a marked increase in Oil Red O staining in HITB5 cells incubated with HTG-VLDL compared with cells incubated without lipoproteins (Figure 1D and 1F). To verify that these observations were not a consequence of clonal variation, Oil Red O studies were repeated in 2 other clones, HITD6 and HITC6 (Figure II, available online at http://atvb.ahajournals.org). Similar to HITA2, HITD6 SMCs assume a cobblestone appearance and only stain modestly for neutral lipid in the presence of HTG-VLDL (Figure IIA, IIC, and IIE). Similar to the HITB5 SMCs, HITC6 SMCs assume a hill-and-valley appearance and show marked increases in Oil Red O staining when challenged with HTG-VLDL (Figure IIB, IID, and IIF).
Figure 1. Morphology of HITA2 and HITB5 SMC clones and the effects of HTG-VLDL on lipid accumulation. Cultured HITA2 (A, C, E, and G) and HITB5 (B, D, F, and H) SMCs have a homogenous appearance, and when confluent they assume an epithelioid-like, cobblestone appearance (A) and a spindle-like hill-and-valley pattern (B), respectively. SMC clones were incubated for 16 hours in the absence (C, D) or presence (E, F) of human HTG-VLDL (50 μg cholesterol/mL media) or with HTG-VLDL and bovine milk LPL (G, H) (0.2 U/mL media). SMC preparations were fixed, stained with Oil Red O to detect neutral lipids, and then counterstained with hematoxylin blue as described in Methods.
Because LPL is known to enhance VLDL uptake in both macrophages and SMCs,17–19 we measured lipid accumulation in HIT SMC clones incubated with HTG-VLDL and exogenous bovine milk LPL. Neutral lipid inclusions in HITA2 and HITD6 SMCs were substantially increased compared with HTG-VLDL alone, indicating that LPL enhanced CE and TG accumulation (Figures 1E, 1G, IIE, and IIG). In the HITB5 and HITC6 SMCs, LPL also increased HTG-VLDL-lipid accumulation, demonstrated by the marked intensity in Oil Red O staining compared with HTG-VLDL alone (Figures 1F, 1H, IIF, and IIH). The morphology of these cells was not dramatically affected by the presence of intracellular lipid.
Cellular Lipid Mass Induced by Native and Oxidized HTG-VLDL
The extent of foam cell formation was determined by quantifying cellular lipid accumulation induced by atherogenic lipoproteins. As predicted by Oil Red O staining, incubation of HITA2 cells with HTG-VLDL (50 μg or 150 μg) resulted in only modest accumulations of CE (1.7 and 3.0 μg/mg cell protein, respectively, P<0.05) and TG (7 and 13 μg/mg cell protein, P<0.05; Figure 2A and 2B). In contrast, lipid accumulation was significantly greater in HITB5 cells. HITB5 SMCs incubated with HTG-VLDL (50 μg) induced accumulations of 10.5 μg CE/cell protein (P<0.05) and 60 μg TG/mg cell protein (P<0.05, Figure 2A and 2B). A similar trend was observed with native LDL (150 and 450 μg); CE accumulation in HITA2 SMCs reached only 2 and 6 μg/mg cell protein, respectively, whereas in HITB5 SMCs, CE accumulation increased to 9 and 15 μg/mg cell protein (P<0.05, Figure 2A). Therefore, the spindle-shaped HITB5 SMCs preferentially accumulate CE and TG compared with the epithelioid-shaped HITA2 SMCs.
Figure 2. The effect of native and modified HTG-VLDL and LDL on lipid accumulation in distinct human SMC clones. HITA2 and HITB5 SMCs were incubated for 16 hours with native HTG-VLDL (50 or 150 μg of lipoprotein cholesterol/mL media) or oxidized VLDL (50 μg of lipoprotein cholesterol/mL media), LDL (150 or 450 μg lipoprotein cholesterol/mL media), or oxidized and acetylated LDL (150 μg lipoprotein cholesterol/mL media). Lipids were extracted and cholesteryl ester (CE) (A), triglyceride (TG) (B), and free cholesterol (FC) (C) were measured as described in Methods (n=4 for all except n=8 for 50 μg HTG-VLDL and n=2 for 150 and 450 μg lipoprotein cholesterol/mL media of HTG-VLDL and LDL in HITB5 SMCs). Values represent the mean±SEM. *P<0.05 as compared with control (no lipoproteins) in the same SMC subtype. P<0.05 as compared with the lipid accumulation in HITA2 SMCs induced by the same lipoprotein preparation.
Oxidized HTG-VLDL–Induced Lipid Accumulation in SMC Subtypes
In HITA2 SMCs, oxVLDL, oxLDL, and acLDL all induced CE accumulation (3.5- to 5.4-fold; Figure 2). CE accumulations induced by modified lipoproteins was greater than that induced by their native counterparts (Figure 2), although net CE mass remained low. In HITB5 cells, the effects of modification varied depending on the lipoprotein substrate. OxVLDL induced lipid accumulation in HITB5 cells to the same extent as native HTG-VLDL (11 μg CE and 84 μg TG/mg cell protein, P<0.05; Figure 2). However, oxLDL and acLDL increased CE mass to 4.8 and 5.0 μg CE/mg cell protein (both P<0.05), respectively, markedly less than that induced by their native counterparts (Figure 2).
ACAT Activity in Distinct SMC Clones
Incorporation of oleate into CE is an alternative indicator of foam cell formation. Although a modest enhancement in lipid mass accumulation was observed after 16 hours, there was no detectable increase in oleate incorporation into either CE or TG after a 5-hour incubation of HITA2 cells with HTG-VLDL. In contrast, in HITB5 SMCs, oleate incorporation into CE under control conditions was 3-fold greater than in HITA2 cells, a value that was significantly enhanced a further 2-fold after incubation with HTG-VLDL (Figure III, available online at http://atvb.ahajournals.org). Oleate incorporation into TG under basal conditions was similar for both cell types. However, in HITB5 SMCs, oleate incorporation into TG increased 1.6-fold (P<0.05; Figure III) in the presence of HTG-VLDL, whereas no increment was observed in HITA2 SMCs. Oleate incorporation into CE was induced in both cell types by oxLDL (data not shown) in proportion to the increases in CE mass (Figure 2).
Expression of LPL Activity in Distinct SMC Subpopulations
To determine whether the ability of HITB5 SMCs to preferentially accumulate CE and TG was caused in part by enhanced LPL activity, we measured secreted and total (secreted plus heparin-releasable) activities in the media of HITA2 and HITB5 SMCs. Total and cell surface LPL activities were 3.3-fold higher in HITB5 SMCs than in HITA2 SMCs (Figure 3; P<0.05).
Figure 3. LPL activity secreted by HITA2 and HITB5 SMCs. HITA2 and HITB5 SMCs were incubated with or without heparin (24 hours) in 5% lipoprotein-deficient serum. Media LPL activity was measured as described in Methods. Total LPL activity is measured in the presence of heparin whereas secreted LPL activity is measured in the absence of heparin. Cell surface LPL activity is the difference between these 2 values. Values represent the mean±SEM (n=3). *P<0.05 as compared with HITA2 SMCs.
Exogenous LPL Stimulates Lipid Accumulation in Epithelioid-Like SMC Clones
In vivo, HITA2 SMCs are potentially exposed to LPL derived from lesion macrophages or from neighboring HITB5-like SMCs.18 Therefore, we determined whether exogenous LPL facilitated foam cell formation in HITA2 SMCs when incubated with CE-rich lipoproteins. Increasing concentrations of bovine milk LPL in the presence of HTG-VLDL dose-dependently and significantly increased CE (2.2- to 3.7-fold, P<0.05) and TG (15- to 60-fold, P<0.05) as compared with control (Figure IV, available online at http://atvb.ahajournals.org). The maximal induction of lipid accumulation was observed with 0.2 U/mL of media of LPL, which when combined with endogenous LPL (0.21 U/mg cell protein) is equivalent to LPL activity secreted by HITB5 cells. Similarly, addition of exogenous LPL enhanced oleate incorporation into CE and TG induced by HTG-VLDL in HITA2 SMCs (data not shown), further confirming that LPL can stimulate HITA2 SMC lipid accumulation. Interestingly, the extent of CE induction in HITA2 SMCs incubated with HTG-VLDL plus LPL was similar to that observed in HITB5 SMCs incubated with HTG-VLDL alone. Incubation of HITB5 cells with exogenous LPL did not further increase CE accumulation induced by HTG-VLDL. However, TG accumulation was enhanced, compared with HTG-VLDL alone (Figure 1E and data not shown). Taken together with the LPL activity findings, these data establish that differential expression of LPL contributed to the predisposition of HITB5 SMCs to accumulate lipids. Addition of LPL to HITA2 SMCs in the presence of oxVLDL increased CE accumulation modestly (30%) compared with that observed with oxVLDL alone (data not shown).
LPL Activity Is Required for Lipid Accumulation in Human SMC Subtypes
To determine the role of LPL catalytic activity in foam cell formation, we assessed HTG-VLDL–induced lipid accumulation in the presence of either tetrahydrolipstatin (THL), an inhibitor of LPL catalytic activity,23 or heparin, which disrupts cell surface association of LPL with heparin sulfate proteoglycans (HSPGs). Incubation of either HITA2 or HITB5 SMCs with THL significantly inhibited CE and TG accumulation induced by HTG-VLDL alone (Figure 4). Furthermore, the augmentation in CE and TG accumulation induced by HTG-VLDL plus LPL in the HITA2 SMCs was completely blocked by THL, suggesting that the catalytic activity is absolutely required for HTG-VLDL–induced foam cell formation. Heparin showed a similar trend to decrease CE accumulation (20%) induced by HTG-VLDL in the HITA2 SMCs; however, heparin appeared to enhance TG accumulation (40%) in the presence of HTG-VLDL and LPL (Figure 4). Heparin had no obvious effect on CE or TG accumulation induced by HTG-VLDL in HITB5 SMCs.
Figure 4. The effect of LPL inhibition on cholesteryl ester (CE) (A) and triglyceride (TG) (B) accumulation induced by HTG-VLDL in HITA2 SMCs and HITB5 SMCs. HITA2 and HITB5 SMCs were incubated for 16 hours with HTG-VLDL (50 μg lipoprotein cholesterol/mL media) or with HTG-VLDL plus LPL (0.2 U/mL media) with or without heparin (10 U/mL media) and/or tetrahydrolipstatin (THL) (specific inhibitor of LPL catalytic activity, 1 μmol/L). Cellular CE and TG accumulation was measured as described in Methods. Values represent the mean±SEM expressed as a percent of CE or TG mass induced by HTG-VLDL alone. *P<0.05 as compared with HTG-VLDL alone, P<0.05 as compared with HTG-VLDL plus LPL (n=3).
Discussion
SMCs manifest diverse functions in the vessel wall in response to physiological and pathologic stimuli.4,5 This diversity may arise from shifts in SMC differentiation status.4 However, there are extensive data suggesting that SMC diversity may also be a consequence of inherently distinct SMC lineages that can selectively amplify in the vessel wall.4–6 A hallmark of SMC diversity is the existence of SMCs that, when cultured, assume either an epithelioid or a spindle-shaped morphology.7–11 In this study, we established that the extent to which human SMCs accumulate lipids, and the underlying mechanism is highly dependent on whether the SMC manifests an epithelioid or spindle-shaped phenotype.
We also established that HTG-VLDL is atherogenic to human SMCs. Elevated levels of HTG-VLDL, associated with metabolic syndromes, including obesity and insulin resistance, represent an important risk factor for atherogenesis, because these lipoproteins induce macrophage foam cell formation in both their native or oxidized forms.20,21,24,25 Previous reports have demonstrated in cultured SMCs, presumably heterogeneous populations, that native and modified ?VLDL and LDL are capable of inducing lipid accumulation.26–29 We now demonstrate that in 4 distinct subsets of SMCs the epithelioid-like SMCs and, to a significantly greater extent, spindle-shaped SMCs, accumulate CE and TG mass when challenged with either native or oxidized human HTG-VLDL.
Interestingly, the 2 clones responded differently to extensively modified lipoproteins. Oxidation or acetylation of lipoproteins is typically associated with greater cellular CE accumulation compared with their native counterparts, as demonstrated previously in macrophages and SMCs.21,27 In the present study, the epithelioid-shaped SMCs accumulated more CE in response to oxLDL and acLDL than induced by native LDL, although the net CE accumulation was low. In contrast, CE accumulation in the spindle-shaped SMCs, induced by oxLDL and acLDL, was much less than CE induced by native LDL. This suggests that in both cell types, expression of receptors for modified lipoproteins is relatively low compared with macrophages. In cultures of human aortic SMCs derived from 8 donors, Matsumoto et al showed that SMCs were either CD36+ and SRAI/II– or SRAI/II+ and CD36–, or positive for both scavenger receptors.30 Thus, because of the modest CE accumulation induced in HITB5 cells by acLDL, a lipoprotein whose uptake in macrophages is mainly mediated by SRAI/II, we predict that these spindle-shaped cells express little, if any, SRAI/II.22,31 Because studies are inconsistent with respect to the detection of scavenger receptors in SMCs from atherosclerotic lesions,14,30,32–35 further characterization of the processes distinguishing oxidized lipoprotein uptake in HIT SMC clones is required.
The 2 SMC clones also differed in the extent of lipid accumulation challenged with native HTG-VLDL. In the spindle-shaped HITB5 cells, net CE accumulation by HTG-VLDL was 5- to 8-fold greater than in epithelioid-like HITA2 SMCs. The LDLR contributes to native lipoprotein uptake; however, its expression is either absent or very low in lesion SMCs14 and is therefore unlikely to contribute to SMC-induced foam cell formation in vivo.35 However, the VLDLR and LRP and their lipoprotein ligands, VLDL and VLDL remnants,28,33 have all been detected in lesion SMCs and are therefore predicted to promote SMC foam cell formation.14,33 It remains to be determined the relative contributions of these receptors to SMC foam cell formation28 and whether any differences would contribute to the differential predisposition for lipid accumulation in HITB5 SMCs.
We extensively characterized the mechanism for HTG-VLDL uptake in macrophages, elucidating a 2-step model dependent on LPL activity, apoE isoform, and LDLR expression.20,21 LPL can also enhance the uptake of ?VLDL by LRP and VLDLR,18,19 and oxidation of LDL can enhance its association with LPL and the cell surface.18 The requirement for LPL in the uptake of ?VLDL, isolated from cholesterol-fed rabbits, by bovine aortic SMCs, has been observed previously. Exogenous LPL or addition of macrophage-conditioned media enhanced ?VLDL uptake and CE accumulation in SMCs.36,37 Consistent with the importance of LPL for lipoprotein uptake, its secretion by HITB5 SMCs was >3-fold higher than in HITA2 cells. This explains, in part, why HITB5 SMCs accumulate more CE and TG when challenged by HTG-VLDL compared with HITA2 SMCs. Although HITA2 SMCs secrete very little LPL in vitro, in some circumstances, epithelioid-like SMCs could nonetheless be exposed to LPL in vivo. Macrophages, a prominent cell type in atherosclerotic lesions, secrete LPL.18 Indeed, addition of exogenous LPL dose-dependently increased TG and CE accumulation induced by HTG-VLDL. This demonstrates that in HITA2 SMCs, LPL expression was rate-limiting rather than an expression of lipoprotein receptors. LPL likely induces lipoprotein uptake by at least 2 mechanisms: (1) a bridging effect whereby LPL tethers the lipoprotein to the cell surface; and (2) a remodeling function, whereby epitopes required for lipoprotein uptake are exposed after core TG hydrolysis.18 These findings suggest that although HITA2 SMCs were not inherently predisposed to lipid accumulation, given the appropriate environmental conditions they could overcome the lack of endogenous LPL expression and convert to foam cells.
The predisposition of HITB5 SMCs to accumulate lipid is consistent with a report demonstrating that spindle-shaped SMCs from explants of human pulmonary artery accumulate CE when exposed to native or aggregated LDL.25 These authors did not examine epithelioid-like SMCs. Our observations that SMCs of differing phenotypes differentially accumulate CE and TG when exposed to human lipoproteins is supported by a recent study comparing rabbit ?VLDL uptake between rabbit SMCs cultured from either the intima of atherosclerotic lesions or the normal media.28 Although not homogenous cultures, both sources of cells accumulated CE induced by ?VLDL, but intimal SMCs were significantly more effective. Intimal SMCs are often phenotypically distinct from their medial counterparts; they express fewer contractile proteins, contain less myofilaments, and overall assume a less mature state.4 Although HITA2 SMCs resemble the phenotype of intimal SMCs and HITB5s resemble those of medial SMCs, this is almost certainly an overly simplistic view. Indeed, when cultured in the presence of serum, as in the current study, both clone types have features consistent with lesion SMCs. Furthermore, HITB5 SMCs manifest phenotype plasticity and can assume a synthetic, proliferative phenotype in the presence of serum and a nonproliferative contractile phenotype in its absence.7 The phenotype of SMCs of lesions has also been demonstrated to change depending on environmental stimuli, including altered plasma lipid concentrations.38 Diet-induced lipid-lowering in rabbits promoted maturation of intimal SMCs,38 implicating the phenotype plasticity similar to that demonstrated by human HITB5 cells.
In summary, SMC subtypes are differentially predisposed to lipid accumulation. Both epithelioid and spindle-shaped SMCs are capable of forming foam cells; however, the specifics of the environment will determine whether they do so. We conclude that the relative proportion of SMC subtypes and their local environment will determine the extent to which SMC foam cells develop.
Acknowledgments
This work was supported by a grant (MT 8014 to M.W.H.) and a studentship (to C.A.A.) from the Canadian Institutes for Health Research. M.W.H., R.A.H., and J.G.P. are Career Investigators of the Heart and Stroke Foundation of Ontario, and R.A.H. holds a Canada Research Chair (Tier I) in Human Genetics. We thank Allison Kulchycki for her expert technical assistance.
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Matsumoto K, Hirano K, Nosaki S, Takamoto A, Nishida M, Nakagawa-Toyama Y, Janabi MY, Ohya T, Yamashita S, Matsuzawa Y. Expression of macrophage (Mf) scavenger receptor, CD36, in cultured human aortic smooth muscle cells in association with expression with expression of peroxisome proliferator activated receptor-, which regulates gain of Mf-like phenotype in vitro, and its implication in atherogenesis. Arterioscler Thromb Vasc Biol. 2000; 20: 1027–1032.
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Zingg J, Ricciarelli R, Andorno E, Azzi A. Novel 5' exon of scavenger receptor CD36 is expressed in cultured human vascular smooth muscle cells and atherosclerotic plaques. Arterioscler Thromb Vasc Biol. 2002; 22: 412–417.
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ABSTRACT
Background— Vascular smooth muscle cells (SMCs) manifest diverse phenotypes and emerging evidence suggests this is caused by inherently distinct SMC subtypes. Recently, Li et al (Circ Res 2001;89:517–525) successfully cloned 2 uniquely responsive SMC subpopulations from a single human artery and we used this unique resource to test the hypothesis that distinct SMC subtypes are differential precursors of foam cell formation.
Methods and Results— When challenged with human atherogenic native or oxidized hypertriglyceridemic very-low-density lipoprotein (HTG-VLDL), the larger, slower-growing, spindle-shaped HITB5 SMC clone accumulated significantly more cholesteryl ester (CE) and triglyceride (TG) than the smaller, faster-growing epithelioid-shaped HITA2 SMC clone (10 versus 2 μg CE/mg cell protein and 60 versus 7 μg TG/mg PN, P<0.05). Lipoprotein lipase (LPL), a key enzyme involved in lipoprotein uptake, was identified as one differentially expressed protein that altered the predisposition of HITA2 SMCs for lipid accumulation. Although HITB5 SMCs secreted significantly more LPL than did HITA2 SMCs (0.7 versus 0.2 U/mL media, P<0.05), the addition of bovine milk LPL to HITA2 SMCs, significantly increased native and oxidized HTG-VLDL–induced lipid accumulation.
Conclusions— Inherently distinct SMC subsets are differentially predisposed to lipoprotein-induced lipid accumulation. Moreover, the environment can influence the response of SMC subsets to atherogenic lipoproteins.
We tested whether epithelioid and spindle-shaped human SMCs differentially accumulated lipid on exposure to native and oxidized lipoproteins. Spindle-shaped HITB5 cells preferentially accumulated cholesteryl ester and triglyceride, whereas the epithelioid-like HITA2 SMCs required exogenous LPL to accumulate lipid in the presence of lipoproteins. Therefore, SMC subtypes are differentially predisposed to a lipid-accumulating phenotype.
Key Words: atherosclerosis ? lipoproteins ? smooth muscle cells ? foam cells
Introduction
Foam cells are fundamental to the formation, growth, and stability of atherosclerotic plaques.1,2 Considerable data exist regarding the role of macrophage foam cells and the mechanisms by which they form.2 However, smooth muscle cells (SMCs) can also convert to foam cells and contribute to overall foam cell burden in a given lesion.1,3 The mechanism of SMC foam cell formation, however, is incompletely defined and cannot necessarily be assumed to be identical to that for macrophages. Moreover, an understanding of SMC foam cell formation must take into account the well-established diversity in SMC phenotype that can exist in the vessel wall.
The diverse functions manifested by SMCs in the vessel wall have long been considered to reflect plasticity in the differentiation status of SMCs.4 However, emerging evidence suggests that phenotypic diversity of SMCs in diseased arteries may also be a result of the expansion of one or more subsets of inherently distinct SMCs.4–6 SMC subtypes that differ in morphology, growth behavior, and response to growth factors have been identified.7–11 Importantly, it has not been established whether SMC subtypes are distinct with respect to their response to atherogenic lipoproteins.
Recently, Li et al demonstrated the presence of inherently distinct SMCs in human arterial media by cloning, from a single fragment of an adult nonatherosclerotic artery, 12 stable SMC clones.7,12 These segregated into 2 discrete clone types based on morphology: relatively small, epithelioid-like SMCs and larger, elongated, spindle-shaped SMCs. The 2 clone types were not interchangeable and varied somewhat in the expression of SMC differentiation markers. They were strikingly different with respect to growth rates and responsiveness to stimuli. In particular, epithelioid SMCs were faster-growing and more responsive to growth factors such as platelet-derived growth factor and fibroblast growth factor, as compared with the spindle-shaped SMCs.7,12 Other phenotypically distinct SMC clones have been successfully generated from experimental animals, supporting the notion of inherent SMC diversity.8–11 However, isolation of human SMC subtypes has proven more difficult. Therefore, these SMC clones provide a unique opportunity to assess their predisposition to foam cell formation.
SMCs are clearly capable of converting into foam cells1,3 and the true contribution of SMCs to lesion formation may in fact be underestimated. A recent study demonstrated that lesional foam cells, previously assumed to have a macrophage origin based on immunostaining, likely originated in the SMC population.13 Importantly, however, there are still uncertainties with respect to the nature of the atherogenic lipoproteins that induce SMC foam cell formation in vivo and the mechanisms mediating their uptake. Several candidate receptors can contribute to lipoprotein uptake in SMCs: the low-density lipoprotein receptor (LDLR), the very-low-density lipoprotein receptor (VLDLR), the low-density lipoprotein-related protein (LRP), and the scavenger receptors, CD36 and SRAI/II.14
Other factors may influence SMC receptor-mediated uptake of lipoproteins,15,16 including lipoprotein lipase (LPL) and apoE, both of which enhance cellular uptake of native lipoproteins.17–19 In macrophages, LPL and apoE isoform are critical for the uptake of VLDL isolated from hypertriglyceridemic (HTG) subjects.20 Initially, HTG-VLDL interacts with cell surface LPL, resulting in sequestration to the cell surface and hydrolysis of core triglyceride (TG) to free fatty acids (FFA), which are readily taken-up by the cell and re-esterified. Subsequently, the CE-rich remnants are taken-up via LDLR-mediated endocytosis.20 As LPL and apoE are secreted by arterial macrophages and SMCs,16,18 lipoproteins in the arterial wall have the potential to become enriched in apoE, bind LPL, and become better ligands for SMC lipoprotein receptors.
The objectives of the present study were to determine whether homogeneous cultures of inherently distinct SMC subtypes are preferentially predisposed to foam cell formation. The more mature, spindle-shaped HITB5 SMCs preferentially accumulated CE and TG when challenged by either native or oxidized HTG-VLDL when compared with the epithelioid-like HITA2 SMCs. We identified that LPL secretion was substantially higher in HITB5 SMCs and that differential LPL activity accounted for the difference in lipid deposition between the 2 SMC subtypes. Moreover, addition of exogenous LPL to HITA2 SMCs significantly enhanced CE and TG accumulation induced by native and oxidized HTG-VLDL to levels similar to those obtained in HITB5 SMCs. These findings establish that interplay between SMC phenotype and the microenvironment determines the response of SMCs to atherogenic lipoproteins.
Methods
Lipoproteins
Subjects were recruited from the Lipid Clinic at the London Health Sciences Center, University Campus (London, Ontario, Canada). The University of Western Ontario Health Science Standing Committee on Human Research approved the studies. The isolation of HTG-VLDL (Sf 20 to 400) and LDL (Sf 0 to 12), copper oxidation of VLDL and LDL, and LDL acetylation has been described previously.21,22
Cell Culture
Experiments were performed with 2 distinct human SMC clones, the epithelioid-like HITA2 and the spindle-shaped HITB5 cells. In some experiments, the human SMCs clones, HITD6 and HITC6, which are phenotypically similar to the HITA2 and HITB5 clones, respectively, were used. These were cloned from the media of a fragment of distal internal thoracic artery7,12 and have been extensively characterized with respect to SMC differentiation markers, vasoactive responsiveness, cell proliferation and migration rates, and contractibility.7 On recloning of either cell type, the original phenotype is preserved and addition of conditioned medium from one cell type to the other does not cause either cell to change phenotype. The source of fetal bovine serum or lipoprotein-deficient serum, type of media, glucose concentration, or ascorbate did not induce phenotype conversion. Furthermore, when either SMC subtype was transplanted subcutaneously into SCID mice, the morphological differences between the spindle-shaped and epithelioid SMCs were maintained. For details see online Methods and Figure I, available online at http://atvb.ahajournals.org.
For details of culturing cells for incubations with lipoproteins, oil red O staining, cellular lipid mass determinations, and assays for LPL and acyl coenzyme A:cholesterol acyl transferase (ACAT) activities, please see www.ahajournals.org.
Results
Oil Red O Staining and Lipid Accumulation in SMC Subtypes
Unlike primary SMC cultures, the SMC clones, HITA2 and HITB5, have a homogenous appearance and when confluent assume a epithelioid-like, cobblestone appearance and a spindle-shaped, hill-and-valley pattern, respectively,7,12 as illustrated in Figure 1A and 1B. To assess SMC lipid accumulation, HITA2 and HITB5 cells were cultured with or without HTG-VLDL and stained for neutral lipids with Oil Red O. There was little evidence for neutral lipid inclusions in HITA2 cells under control conditions, and only modest increases were observed after incubation with native HTG-VLDL (Figure 1C and 1E). In sharp contrast, there was a marked increase in Oil Red O staining in HITB5 cells incubated with HTG-VLDL compared with cells incubated without lipoproteins (Figure 1D and 1F). To verify that these observations were not a consequence of clonal variation, Oil Red O studies were repeated in 2 other clones, HITD6 and HITC6 (Figure II, available online at http://atvb.ahajournals.org). Similar to HITA2, HITD6 SMCs assume a cobblestone appearance and only stain modestly for neutral lipid in the presence of HTG-VLDL (Figure IIA, IIC, and IIE). Similar to the HITB5 SMCs, HITC6 SMCs assume a hill-and-valley appearance and show marked increases in Oil Red O staining when challenged with HTG-VLDL (Figure IIB, IID, and IIF).
Figure 1. Morphology of HITA2 and HITB5 SMC clones and the effects of HTG-VLDL on lipid accumulation. Cultured HITA2 (A, C, E, and G) and HITB5 (B, D, F, and H) SMCs have a homogenous appearance, and when confluent they assume an epithelioid-like, cobblestone appearance (A) and a spindle-like hill-and-valley pattern (B), respectively. SMC clones were incubated for 16 hours in the absence (C, D) or presence (E, F) of human HTG-VLDL (50 μg cholesterol/mL media) or with HTG-VLDL and bovine milk LPL (G, H) (0.2 U/mL media). SMC preparations were fixed, stained with Oil Red O to detect neutral lipids, and then counterstained with hematoxylin blue as described in Methods.
Because LPL is known to enhance VLDL uptake in both macrophages and SMCs,17–19 we measured lipid accumulation in HIT SMC clones incubated with HTG-VLDL and exogenous bovine milk LPL. Neutral lipid inclusions in HITA2 and HITD6 SMCs were substantially increased compared with HTG-VLDL alone, indicating that LPL enhanced CE and TG accumulation (Figures 1E, 1G, IIE, and IIG). In the HITB5 and HITC6 SMCs, LPL also increased HTG-VLDL-lipid accumulation, demonstrated by the marked intensity in Oil Red O staining compared with HTG-VLDL alone (Figures 1F, 1H, IIF, and IIH). The morphology of these cells was not dramatically affected by the presence of intracellular lipid.
Cellular Lipid Mass Induced by Native and Oxidized HTG-VLDL
The extent of foam cell formation was determined by quantifying cellular lipid accumulation induced by atherogenic lipoproteins. As predicted by Oil Red O staining, incubation of HITA2 cells with HTG-VLDL (50 μg or 150 μg) resulted in only modest accumulations of CE (1.7 and 3.0 μg/mg cell protein, respectively, P<0.05) and TG (7 and 13 μg/mg cell protein, P<0.05; Figure 2A and 2B). In contrast, lipid accumulation was significantly greater in HITB5 cells. HITB5 SMCs incubated with HTG-VLDL (50 μg) induced accumulations of 10.5 μg CE/cell protein (P<0.05) and 60 μg TG/mg cell protein (P<0.05, Figure 2A and 2B). A similar trend was observed with native LDL (150 and 450 μg); CE accumulation in HITA2 SMCs reached only 2 and 6 μg/mg cell protein, respectively, whereas in HITB5 SMCs, CE accumulation increased to 9 and 15 μg/mg cell protein (P<0.05, Figure 2A). Therefore, the spindle-shaped HITB5 SMCs preferentially accumulate CE and TG compared with the epithelioid-shaped HITA2 SMCs.
Figure 2. The effect of native and modified HTG-VLDL and LDL on lipid accumulation in distinct human SMC clones. HITA2 and HITB5 SMCs were incubated for 16 hours with native HTG-VLDL (50 or 150 μg of lipoprotein cholesterol/mL media) or oxidized VLDL (50 μg of lipoprotein cholesterol/mL media), LDL (150 or 450 μg lipoprotein cholesterol/mL media), or oxidized and acetylated LDL (150 μg lipoprotein cholesterol/mL media). Lipids were extracted and cholesteryl ester (CE) (A), triglyceride (TG) (B), and free cholesterol (FC) (C) were measured as described in Methods (n=4 for all except n=8 for 50 μg HTG-VLDL and n=2 for 150 and 450 μg lipoprotein cholesterol/mL media of HTG-VLDL and LDL in HITB5 SMCs). Values represent the mean±SEM. *P<0.05 as compared with control (no lipoproteins) in the same SMC subtype. P<0.05 as compared with the lipid accumulation in HITA2 SMCs induced by the same lipoprotein preparation.
Oxidized HTG-VLDL–Induced Lipid Accumulation in SMC Subtypes
In HITA2 SMCs, oxVLDL, oxLDL, and acLDL all induced CE accumulation (3.5- to 5.4-fold; Figure 2). CE accumulations induced by modified lipoproteins was greater than that induced by their native counterparts (Figure 2), although net CE mass remained low. In HITB5 cells, the effects of modification varied depending on the lipoprotein substrate. OxVLDL induced lipid accumulation in HITB5 cells to the same extent as native HTG-VLDL (11 μg CE and 84 μg TG/mg cell protein, P<0.05; Figure 2). However, oxLDL and acLDL increased CE mass to 4.8 and 5.0 μg CE/mg cell protein (both P<0.05), respectively, markedly less than that induced by their native counterparts (Figure 2).
ACAT Activity in Distinct SMC Clones
Incorporation of oleate into CE is an alternative indicator of foam cell formation. Although a modest enhancement in lipid mass accumulation was observed after 16 hours, there was no detectable increase in oleate incorporation into either CE or TG after a 5-hour incubation of HITA2 cells with HTG-VLDL. In contrast, in HITB5 SMCs, oleate incorporation into CE under control conditions was 3-fold greater than in HITA2 cells, a value that was significantly enhanced a further 2-fold after incubation with HTG-VLDL (Figure III, available online at http://atvb.ahajournals.org). Oleate incorporation into TG under basal conditions was similar for both cell types. However, in HITB5 SMCs, oleate incorporation into TG increased 1.6-fold (P<0.05; Figure III) in the presence of HTG-VLDL, whereas no increment was observed in HITA2 SMCs. Oleate incorporation into CE was induced in both cell types by oxLDL (data not shown) in proportion to the increases in CE mass (Figure 2).
Expression of LPL Activity in Distinct SMC Subpopulations
To determine whether the ability of HITB5 SMCs to preferentially accumulate CE and TG was caused in part by enhanced LPL activity, we measured secreted and total (secreted plus heparin-releasable) activities in the media of HITA2 and HITB5 SMCs. Total and cell surface LPL activities were 3.3-fold higher in HITB5 SMCs than in HITA2 SMCs (Figure 3; P<0.05).
Figure 3. LPL activity secreted by HITA2 and HITB5 SMCs. HITA2 and HITB5 SMCs were incubated with or without heparin (24 hours) in 5% lipoprotein-deficient serum. Media LPL activity was measured as described in Methods. Total LPL activity is measured in the presence of heparin whereas secreted LPL activity is measured in the absence of heparin. Cell surface LPL activity is the difference between these 2 values. Values represent the mean±SEM (n=3). *P<0.05 as compared with HITA2 SMCs.
Exogenous LPL Stimulates Lipid Accumulation in Epithelioid-Like SMC Clones
In vivo, HITA2 SMCs are potentially exposed to LPL derived from lesion macrophages or from neighboring HITB5-like SMCs.18 Therefore, we determined whether exogenous LPL facilitated foam cell formation in HITA2 SMCs when incubated with CE-rich lipoproteins. Increasing concentrations of bovine milk LPL in the presence of HTG-VLDL dose-dependently and significantly increased CE (2.2- to 3.7-fold, P<0.05) and TG (15- to 60-fold, P<0.05) as compared with control (Figure IV, available online at http://atvb.ahajournals.org). The maximal induction of lipid accumulation was observed with 0.2 U/mL of media of LPL, which when combined with endogenous LPL (0.21 U/mg cell protein) is equivalent to LPL activity secreted by HITB5 cells. Similarly, addition of exogenous LPL enhanced oleate incorporation into CE and TG induced by HTG-VLDL in HITA2 SMCs (data not shown), further confirming that LPL can stimulate HITA2 SMC lipid accumulation. Interestingly, the extent of CE induction in HITA2 SMCs incubated with HTG-VLDL plus LPL was similar to that observed in HITB5 SMCs incubated with HTG-VLDL alone. Incubation of HITB5 cells with exogenous LPL did not further increase CE accumulation induced by HTG-VLDL. However, TG accumulation was enhanced, compared with HTG-VLDL alone (Figure 1E and data not shown). Taken together with the LPL activity findings, these data establish that differential expression of LPL contributed to the predisposition of HITB5 SMCs to accumulate lipids. Addition of LPL to HITA2 SMCs in the presence of oxVLDL increased CE accumulation modestly (30%) compared with that observed with oxVLDL alone (data not shown).
LPL Activity Is Required for Lipid Accumulation in Human SMC Subtypes
To determine the role of LPL catalytic activity in foam cell formation, we assessed HTG-VLDL–induced lipid accumulation in the presence of either tetrahydrolipstatin (THL), an inhibitor of LPL catalytic activity,23 or heparin, which disrupts cell surface association of LPL with heparin sulfate proteoglycans (HSPGs). Incubation of either HITA2 or HITB5 SMCs with THL significantly inhibited CE and TG accumulation induced by HTG-VLDL alone (Figure 4). Furthermore, the augmentation in CE and TG accumulation induced by HTG-VLDL plus LPL in the HITA2 SMCs was completely blocked by THL, suggesting that the catalytic activity is absolutely required for HTG-VLDL–induced foam cell formation. Heparin showed a similar trend to decrease CE accumulation (20%) induced by HTG-VLDL in the HITA2 SMCs; however, heparin appeared to enhance TG accumulation (40%) in the presence of HTG-VLDL and LPL (Figure 4). Heparin had no obvious effect on CE or TG accumulation induced by HTG-VLDL in HITB5 SMCs.
Figure 4. The effect of LPL inhibition on cholesteryl ester (CE) (A) and triglyceride (TG) (B) accumulation induced by HTG-VLDL in HITA2 SMCs and HITB5 SMCs. HITA2 and HITB5 SMCs were incubated for 16 hours with HTG-VLDL (50 μg lipoprotein cholesterol/mL media) or with HTG-VLDL plus LPL (0.2 U/mL media) with or without heparin (10 U/mL media) and/or tetrahydrolipstatin (THL) (specific inhibitor of LPL catalytic activity, 1 μmol/L). Cellular CE and TG accumulation was measured as described in Methods. Values represent the mean±SEM expressed as a percent of CE or TG mass induced by HTG-VLDL alone. *P<0.05 as compared with HTG-VLDL alone, P<0.05 as compared with HTG-VLDL plus LPL (n=3).
Discussion
SMCs manifest diverse functions in the vessel wall in response to physiological and pathologic stimuli.4,5 This diversity may arise from shifts in SMC differentiation status.4 However, there are extensive data suggesting that SMC diversity may also be a consequence of inherently distinct SMC lineages that can selectively amplify in the vessel wall.4–6 A hallmark of SMC diversity is the existence of SMCs that, when cultured, assume either an epithelioid or a spindle-shaped morphology.7–11 In this study, we established that the extent to which human SMCs accumulate lipids, and the underlying mechanism is highly dependent on whether the SMC manifests an epithelioid or spindle-shaped phenotype.
We also established that HTG-VLDL is atherogenic to human SMCs. Elevated levels of HTG-VLDL, associated with metabolic syndromes, including obesity and insulin resistance, represent an important risk factor for atherogenesis, because these lipoproteins induce macrophage foam cell formation in both their native or oxidized forms.20,21,24,25 Previous reports have demonstrated in cultured SMCs, presumably heterogeneous populations, that native and modified ?VLDL and LDL are capable of inducing lipid accumulation.26–29 We now demonstrate that in 4 distinct subsets of SMCs the epithelioid-like SMCs and, to a significantly greater extent, spindle-shaped SMCs, accumulate CE and TG mass when challenged with either native or oxidized human HTG-VLDL.
Interestingly, the 2 clones responded differently to extensively modified lipoproteins. Oxidation or acetylation of lipoproteins is typically associated with greater cellular CE accumulation compared with their native counterparts, as demonstrated previously in macrophages and SMCs.21,27 In the present study, the epithelioid-shaped SMCs accumulated more CE in response to oxLDL and acLDL than induced by native LDL, although the net CE accumulation was low. In contrast, CE accumulation in the spindle-shaped SMCs, induced by oxLDL and acLDL, was much less than CE induced by native LDL. This suggests that in both cell types, expression of receptors for modified lipoproteins is relatively low compared with macrophages. In cultures of human aortic SMCs derived from 8 donors, Matsumoto et al showed that SMCs were either CD36+ and SRAI/II– or SRAI/II+ and CD36–, or positive for both scavenger receptors.30 Thus, because of the modest CE accumulation induced in HITB5 cells by acLDL, a lipoprotein whose uptake in macrophages is mainly mediated by SRAI/II, we predict that these spindle-shaped cells express little, if any, SRAI/II.22,31 Because studies are inconsistent with respect to the detection of scavenger receptors in SMCs from atherosclerotic lesions,14,30,32–35 further characterization of the processes distinguishing oxidized lipoprotein uptake in HIT SMC clones is required.
The 2 SMC clones also differed in the extent of lipid accumulation challenged with native HTG-VLDL. In the spindle-shaped HITB5 cells, net CE accumulation by HTG-VLDL was 5- to 8-fold greater than in epithelioid-like HITA2 SMCs. The LDLR contributes to native lipoprotein uptake; however, its expression is either absent or very low in lesion SMCs14 and is therefore unlikely to contribute to SMC-induced foam cell formation in vivo.35 However, the VLDLR and LRP and their lipoprotein ligands, VLDL and VLDL remnants,28,33 have all been detected in lesion SMCs and are therefore predicted to promote SMC foam cell formation.14,33 It remains to be determined the relative contributions of these receptors to SMC foam cell formation28 and whether any differences would contribute to the differential predisposition for lipid accumulation in HITB5 SMCs.
We extensively characterized the mechanism for HTG-VLDL uptake in macrophages, elucidating a 2-step model dependent on LPL activity, apoE isoform, and LDLR expression.20,21 LPL can also enhance the uptake of ?VLDL by LRP and VLDLR,18,19 and oxidation of LDL can enhance its association with LPL and the cell surface.18 The requirement for LPL in the uptake of ?VLDL, isolated from cholesterol-fed rabbits, by bovine aortic SMCs, has been observed previously. Exogenous LPL or addition of macrophage-conditioned media enhanced ?VLDL uptake and CE accumulation in SMCs.36,37 Consistent with the importance of LPL for lipoprotein uptake, its secretion by HITB5 SMCs was >3-fold higher than in HITA2 cells. This explains, in part, why HITB5 SMCs accumulate more CE and TG when challenged by HTG-VLDL compared with HITA2 SMCs. Although HITA2 SMCs secrete very little LPL in vitro, in some circumstances, epithelioid-like SMCs could nonetheless be exposed to LPL in vivo. Macrophages, a prominent cell type in atherosclerotic lesions, secrete LPL.18 Indeed, addition of exogenous LPL dose-dependently increased TG and CE accumulation induced by HTG-VLDL. This demonstrates that in HITA2 SMCs, LPL expression was rate-limiting rather than an expression of lipoprotein receptors. LPL likely induces lipoprotein uptake by at least 2 mechanisms: (1) a bridging effect whereby LPL tethers the lipoprotein to the cell surface; and (2) a remodeling function, whereby epitopes required for lipoprotein uptake are exposed after core TG hydrolysis.18 These findings suggest that although HITA2 SMCs were not inherently predisposed to lipid accumulation, given the appropriate environmental conditions they could overcome the lack of endogenous LPL expression and convert to foam cells.
The predisposition of HITB5 SMCs to accumulate lipid is consistent with a report demonstrating that spindle-shaped SMCs from explants of human pulmonary artery accumulate CE when exposed to native or aggregated LDL.25 These authors did not examine epithelioid-like SMCs. Our observations that SMCs of differing phenotypes differentially accumulate CE and TG when exposed to human lipoproteins is supported by a recent study comparing rabbit ?VLDL uptake between rabbit SMCs cultured from either the intima of atherosclerotic lesions or the normal media.28 Although not homogenous cultures, both sources of cells accumulated CE induced by ?VLDL, but intimal SMCs were significantly more effective. Intimal SMCs are often phenotypically distinct from their medial counterparts; they express fewer contractile proteins, contain less myofilaments, and overall assume a less mature state.4 Although HITA2 SMCs resemble the phenotype of intimal SMCs and HITB5s resemble those of medial SMCs, this is almost certainly an overly simplistic view. Indeed, when cultured in the presence of serum, as in the current study, both clone types have features consistent with lesion SMCs. Furthermore, HITB5 SMCs manifest phenotype plasticity and can assume a synthetic, proliferative phenotype in the presence of serum and a nonproliferative contractile phenotype in its absence.7 The phenotype of SMCs of lesions has also been demonstrated to change depending on environmental stimuli, including altered plasma lipid concentrations.38 Diet-induced lipid-lowering in rabbits promoted maturation of intimal SMCs,38 implicating the phenotype plasticity similar to that demonstrated by human HITB5 cells.
In summary, SMC subtypes are differentially predisposed to lipid accumulation. Both epithelioid and spindle-shaped SMCs are capable of forming foam cells; however, the specifics of the environment will determine whether they do so. We conclude that the relative proportion of SMC subtypes and their local environment will determine the extent to which SMC foam cells develop.
Acknowledgments
This work was supported by a grant (MT 8014 to M.W.H.) and a studentship (to C.A.A.) from the Canadian Institutes for Health Research. M.W.H., R.A.H., and J.G.P. are Career Investigators of the Heart and Stroke Foundation of Ontario, and R.A.H. holds a Canada Research Chair (Tier I) in Human Genetics. We thank Allison Kulchycki for her expert technical assistance.
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