Smooth Muscle Cell-derived Foam Cells Research Articles

An update on ox-LDL-inducing vascular smooth muscle cell-derived foam cells in atherosclerosis.

Excess cholesterol accumulation induces the accumulation of foam cells, eventually accelerating atherosclerosis progress. Historically, the mechanisms of macrophage-derived foam cells have attracted attention because of their central role in plaque development, which was challenged by lineage tracing in union with single-cell sequencing (sc-seq). Accumulated studies have uncovered how vascular smooth muscle cells (VSMCs) proliferate and migrate to the vascular intima and accumulate, then transform into foam cells induced by surplus lipids, finally accounting for 30% to 70% of the total foam cells within the plaque of both mice and humans. Therefore, the mechanisms of VSMC-derived foam cells have received increasing attention. The review intends to summarize the transformation mechanism of VSMCs into foam cells induced by oxidized low-density lipoproteins (ox-LDL) in atherosclerosis.

TLR4 promotes smooth muscle cell-derived foam cells formation by inducing receptor-independent macropinocytosis.

Foam cells are primarily formed through scavenger receptors that mediate the uptake of various modified low-density lipoproteins (LDL) into cells. In addition to the receptor-dependent pathway, macropinocytosis is an essential non-receptor endocytic pathway for vascular smooth muscle cells (VSMCs) to take up lipids. However, the molecular mechanisms underlying this process remain unclear. Primary cultured VSMCs were stimulated with 200 ng/ml lipopolysaccharide (LPS) and 200 μg/ml native LDL (nLDL). We observed a significant increase in TLR4 protein expression and a significant activation of macropinocytosis, which correlated with the highest uptake of nLDL and intracellular lipid deposition in WT VSMCs. However, macropinocytosis was inhibited and lipid accumulation decreased after treatment with macropinocytosis inhibitors and Syk inhibitors in WT VSMCs. Consistently, TLR4 knockout significantly suppressed macropinocytosis and lipid droplets accumulation in VSMCs. Taken together, our findings suggest a critical role of TLR4/Syk signaling in promoting receptor-independent macropinocytosis leading to VSMC-derived foam cells formation.

Abstract 3066: Pro-Atherogenic Macrophages Induce The Upregulation Of MLKL And Cell Death In Vascular Smooth Muscle Cells During Atherogenic Stimulation

Background: A hallmark of advanced, rupture-prone atherosclerotic plaques is the presence of a necrotic core - a dense mass of pro-inflammatory and cellular debris. Traditionally, it has been thought that most of the foam cells within this core are derived from macrophages. However, recent advances in single cell technologies have revealed that vascular smooth muscle cells have the capacity to transdifferentiate into "macrophage-like foam cells", accounting for almost half of the cell content in advanced plaques. Our lab and others have discovered that a primary cause of the necrotic core is necroptotic cell death, where the mixed lineage kinase domain like pseudokinase MLKL is a key executioner of this process. While the contribution of necroptotic macrophage-derived foam cells to atherosclerotic lesion growth and instability has been fairly well-studied, the role of necroptotic vascular smooth muscle cell-derived foam cells in this process remains unclear. Hypothesis: MLKL regulates cell death differently in macrophages and vascular smooth muscle cells when challenged with pro-atherogenic stimuli. Methods and Results: Bone marrow-derived macrophages (BMDMs) and aortic vascular smooth muscle cells (VSMCs) were isolated from wildtype mice and treated with pro-atherogenic stimuli in the presence or absence of apoptotic and necroptotic inhibitors, zVAD and Necrostatin-1 (Nec1). BMDMs underwent cell death in response to treatment with oxLDL or LPS plus zVAD within 24 hours (as measured by LDH release and SYTOX assays) while VSMCs survive up to 72 hours. However, when VSMCs are treated with BMDM conditioned media in addition to pro-atherogenic stimuli, VSMCs begin to show higher relative rates of cell death compared to controls. Confocal microscopy and Western blots revealed that VSMCs treated with cell death ligands in conjunction with BMDM conditioned media showed an increase in relative expression of total and pMLKL- a hallmark of necroptosis, compared to VSMCs without conditioned media. Conclusions: Overall, this work suggests VSMCs are inherently resistant to necroptosis unless treated with conditioned media from BMDMs, which may suggest that macrophages have the capacity to prime necroptosis in VSMCs, likely via secreted factors.

Millet shell polyphenols ameliorate atherosclerosis development by suppressing foam cell formation

Polyphenols are bioactive compounds that occur naturally in plants, and they are widely used for disease prevention and health maintenance. In present study, the effects of millet shell polyphenols (MSPs) in thwarting atherosclerosis were explored. The results found that MSPs effectively inhibited the ability of macrophages to phagocytose lipids, and reduced the secretion of inflammatory factors IL-1β and TNF-α by obstructing the expression of STAT3 and NF-κB in macrophages. Eventually, MSPs hindered the formation of macrophage-derived foam cells. On the other hand, MSPs promoted the transformation of HASMCs from synthesis to contraction by regulating the gene expression levels of smooth muscle myosin heavy chain (SMMHC), desmin (DES), smoothelin (SMTN) and elastin (ELN). Lipid phagocytosis inhibited along with this process, thereby reducing the formation of smooth muscle cell-derived foam cells. In addition, experiments in ApoE−/− mice also showed that MSPs increased high-density lipoprotein cholesterol (HDL-C). Collectively, MSPs play a role in preventing atherosclerosis by impeding foam cell production. This study offers an integrative strategy for thwarting plaque formation in the early stages of atherosclerosis in cardiovascular disease.

Nϵ-Carboxymethyl-Lysine Deteriorates Vascular Calcification in Diabetic Atherosclerosis Induced by Vascular Smooth Muscle Cell-Derived Foam Cells.

Nϵ-carboxymethyl-lysine (CML), an advanced glycation end product, is involved in vascular calcification (VC) in diabetic atherosclerosis. This study aimed to investigate the effects of CML on VC in diabetic atherosclerosis induced by vascular smooth muscle cell (VSMC)–derived foam cells. Human studies, animal studies and cell studies were performed. The human study results from 100 patients revealed a poor blood glucose and lipid status and more severe coronary lesions and stenosis in patients with coronary artery disease and diabetes mellitus. Intraperitoneal injection of streptozotocin combined with a high-fat diet was used to build a diabetic atherosclerosis model in ApoE−/− mice. The animal study results indicated that CML accelerated VC progression in diabetic atherosclerosis by accelerating the accumulation of VSMC-derived foam cells in ApoE−/− mice. The cell study results illustrated that CML induced VSMC-derived foam cells apoptosis and aggravated foam cells calcification. Consistent with this finding, calcium content and the expression levels of alkaline phosphatase, bone morphogenetic protein 2 and runt-related transcription factor 2 were significantly elevated in A7r5 cells treated with oxidation-low-density lipoprotein and CML. Thus, we concluded that CML promoted VSMC-derived foam cells calcification to aggravate VC in diabetic atherosclerosis, providing evidence for the contribution of foam cells to diabetic VC.

Impaired SIRT1 promotes the migration of vascular smooth muscle cell-derived foam cells.

The formation of fat-laden foam cells, contributing to the fatty streaks of the plaques of atheroma, is the critical early process in atherosclerosis. The previous study demonstrated that vascular smooth muscle cells (VSMCs) contain a much larger burden of the excess cholesterol in comparison with monocyte-derived macrophages in human coronary atherosclerosis, as the main origin of foam cells. It is noteworthy that VSMC-derived foam cells are deposited in subintima but not media, where VSMCs normally deposit in. Therefore, migration from media to intima is an indispensable step for a VSMC to accrue neutral lipids and form foam cell. Whether this migration occurs paralleled with or prior to the formation of foam cell is still unclear. Herein, the present study was designed to test the VSMC migratory capability in the process of foam cell formation induced by oxidized low-density lipoprotein (oxLDL). In conclusion, we provide evidence that oxLDL induces the VSMC-derived foam cells formation with increased migration ability and MMP-9 expression, which were partly attributed to the impaired SIRT1 and enhanced nuclear factor-kappa B (NF-κB) activity. As activation of transient receptor potential vanilloid type 1 (TRPV1) has been reported to have anti-atherosclerotic effects, we investigated its role in oxLDL-treated VSMC migration. It is found that activating TRPV1 by capsaicin inhibits VSMC foam cell formation and the accompanied migration through rescuing the SIRT1 and suppressing NF-κB signaling. The present study provides evidence that SIRT1 may be a promising intervention target of atherosclerosis, and raises the prospect of TRPV1 in prevention and treatment of atherosclerosis.

Genistein suppresses smooth muscle cell-derived foam cell formation through tyrosine kinase pathway

PurposeGenistein, as a protein tyrosine kinase inhibitor, has been shown to possess anti-atherosclerotic effects. Since the smooth muscle cell-derived foam cells are key components of atherosclerotic plaques. The aim of this study is to investigate the influence of genistein on foam cell transformation from vascular smooth muscle cells and possible mechanisms contributing to these effects. Methods and ResultsVascular smooth muscle cells exposed to ox-LDL developed into foam cell, as demonstrated by Oil Red O staining and cholesterol content analysis. Ox-LDL induced phenotype transformation of smooth muscle cells, decreased expression of α-actin and increased expression of CD68 (a specific marker for monocytes, can also function as a subtype of scavenger receptors). The expression of scavenger receptors CD36 and LOX-1 was measured, and their role in foam cell formation in the presence of genistein, daidzein (a structurally similar analogue of genistein) and herbimycin A (a commonly tyrosine kinase inhibitor). The results showed that foam cell formation was markedly reduced by genistein and herbimycin A, as well as the expression of CD68, CD36 and LOX-1. However, daidzein had no such effect. In addition, genistein-induced down-regulation of CD68, CD36 and LOX-1 could be reversed by sodium orthovanadate (a membrane-permeable protein tyrosine phosphatase inhibitor). ConclusionThe results showed that ox-LDL induce smooth muscle cell-derived foam cell formation and transform the phenotype of smooth muscle cell. While tyrosine kinase inhibitor, genistein could suppress smooth muscle cell-derived foam cell formation through inhibiting the protein expressions of CD68, CD36 and LOX-1.

Characterization and classification of psittacine atherosclerotic lesions by histopathology, digital image analysis, transmission and scanning electron microscopy

Atherosclerosis is a degenerative and inflammatory vascular disease characterized in mammals and birds by the accumulation of inflammatory cells, lipids, calcium, and formation of large fibrofatty lesions within the intima of arteries resulting in the disorganization of the arterial wall and stenosis of the lumen. Despite the high incidence of atherosclerosis in parrots and the high number of case reports, there are few pathologic investigations and the ultrastructural study of the lesions has not been documented. Sixty-three major arteries were collected from 24 psittacine birds of 11 species during routine post-mortem examinations. Samples from the major arteries were fixed in 2% paraformaldehyde and 1.25% glutaraldehyde, and processed for transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Additional samples were fixed in 10% formalin and embedded in paraffin for histological examination. Additional histochemical stains for calcium, elastic fibres, and lipid were performed. Toluidine blue-stained 0.5 µm-thick resin sections were also obtained. Digital image analysis was performed to provide objective quantitative information on the different lesions. The histopathology and ultrastructure of psittacine atherosclerosis were found to be similar to other avian and mammalian species. Seven lesion types could be described, which were similar to the human classification system. Digital image analysis, TEM, and SEM helped to further describe the lesions and refine the classification system. TEM findings were similar to other avian and mammalian species with the notable presence of macrophage-derived and smooth muscle cell-derived foam cells and extracellular lipid. SEM revealed various stages of endothelial surface defects and, occasionally, adherent blood cells.

Histological changes and risk factor associations in type 2 atherosclerotic lesions (fatty streaks) in young adults

ObjectivesThe purpose of this study was to investigate which histological changes associated with risk factors could contribute to the progression from the initial atherosclerotic lesions including fatty streaks to the advanced lesions. MethodsWe examined the associations of histomorphometric findings in the determined anatomical sites of mid-thoracic aortas (TAs) and left anterior descending coronary arteries (LADs) with major risk factors for atherosclerosis, using a young autopsied series from the the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study. The histological classification by the American Heart Association was graded for 1013 TAs and 1009 LADs. Histometric study, including immunohistochemistry, was performed in type 2 lesions (fatty streaks) of TAs from 59 subjects and LADs from 45 ones. ResultsFor the progression from the initial lesions into the advanced atherosclerotic lesions, the most effective lipid profiles were low plasma HDL-C in TA and elevated serum non-HDL-C in LAD. This lipid profile of each artery correlated with number or density of intimal smooth muscle cell-derived foam cells, respectively. The serum concentration of non-HDL-C correlated with macrophage foam cells in TAs. Hypertension and hyperglycemia were associated with increase of intimal area and/or collagen content in both arteries, but not with either types of foam cell proliferation. Smoking correlated with increased collagen content in TAs. ConclusionThere were histologically different ways of progressing from fatty streaks to advanced atherosclerotic lesions depending on the risk factors. For the atherosclerosis progression from type 2 lesions to advanced lesions, increase in number of smooth muscle cell-derived foam cells could be an important indicator.

Lipid Peroxidation Modification of Protein Generates Nϵ-(4-Oxononanoyl)lysine as a Pro-inflammatory Ligand

4-Oxo-2(E)-nonenal (ONE), a peroxidation product of ω-6 polyunsaturated fatty acids, covalently reacts with lysine residues to generate a 4-ketoamide-type ONE-lysine adduct, N(ε)-(4-oxononanoyl)lysine (ONL). Using an ONL-coupled protein as the immunogen, we raised the monoclonal antibody (mAb) 9K3 directed to the ONL and conclusively demonstrated that the ONL was produced during the oxidative modification of a low density lipoprotein (LDL) in vitro. In addition, we observed that the ONL was present in atherosclerotic lesions, in which an intense immunoreactivity was mainly localized in the vascular endothelial cells and macrophage- and vascular smooth muscle cell-derived foam cells. Using liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for quantification of the ONL and confirmed that the ONL was indeed formed during the lipid peroxidation-mediated modification of protein in vitro and in vivo. To evaluate the biological implications for ONL formation, we examined the recognition of ONL by the scavenger receptor lectin-like oxidized LDL receptor-1 (LOX-1). Using CHO cells stably expressing LOX-1, we evaluated the ability of ONL to compete with the acetylated LDL and found that both the ONE-modified and ONL-coupled proteins inhibited the binding and uptake of the modified LDL. In addition, we demonstrated that the ONL-coupled protein was incorporated into differentiated THP-1 cells via LOX-1. Finally, we examined the effect of ONL on the expression of the inflammation-associated gene in THP-1 and observed that the ONL-coupled proteins significantly induced the expression of atherogenesis-related genes, such as the monocyte chemoattractant protein-1 and tumor necrosis factor-α, in a LOX-1-dependent manner. Thus, ONL was identified to be a potential endogenous ligand for LOX-1.

CD36, serves as a receptor for advanced glycation endproducts (AGE)

Interaction of advanced glycation endproducts (AGE) with AGE receptors induces several cellular phenomena relating potentially to diabetic complications. Five AGE receptors identified so far are receptor for AGE (RAGE), 80 K-H, OST-48, galectin-3, and macrophage scavenger receptor, types I and II (SR-A) [Eur. J. Biochem. 230 (1995) 408; Nature 386 (1997) 292.]. Since SR-A is known to belong to the class A scavenger receptor family and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36 belonging to class B scavenger receptor family (SR-B) can recognize AGE proteins as a ligand. This was tested in the present study at the cellular level by using Chinese hamster ovary (CHO) cells overexpressing human CD36 (CHO-CD36 cells). 125I-AGE-bovine serum albumin (BSA) was endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CHO-CD36, but not wild-type CHO cells. Endocytic uptake of 125I-AGE-BSA by these cells was inhibited 50% by oxidized low-density lipoprotein (Ox-LDL) and 60% by FA6-152, an anti-CD36 antibody inhibiting cellular binding of Ox-LDL. Our results indicate that CD36 expressed by these cells mediates endocytic uptake and subsequent intracellular degradation of AGE proteins. Since CD36 is one of the major Ox-LDL receptors and is up-regulated in macrophage- and smooth muscle cell-derived foam cells in human atherosclerotic lesions, the present results suggest that, like Ox-LDL, AGE proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.

CD36, a member of class B scavenger receptor family, is a receptor for advanced glycation end products.

Interaction of advanced glycation end products (AGE) with AGE-receptors induces several cellular phenomena relating potentially to diabetic complications. Five AGE-receptors identified so far are RAGE (receptor for AGE), 80 K-H, OST-48, galectin-3, and SR-A (macrophage scavenger receptor type I and II). Since SR-A belongs to the class A scavenger receptor family and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36 belonging to the class B scavenger receptor family (SR-B) can recognize AGE-proteins as a ligand. This was tested in the present study at the cellular level using CHO (Chinese hamster ovary) cells overexpressing human CD36 (CHO-CD36 cells). 125I-AGE-BSA (bovine serum albumin) was endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CHO-CD36 but not wild-type CHO cells. Endocytic uptake of 125I-AGE-BSA by these cells was inhibited 50% by oxidized LDL (Ox-LDL) and 60% by FA6-152, an anti-CD36 antibody inhibiting cellular binding of Ox-LDL. Our results indicate that CD36 expressed by these cells mediates endocytic uptake and subsequent intracellular degradation of AGE-proteins. Because CD36 is one of the major Ox-LDL receptors and is upregulated in macrophage- and smooth muscle cell-derived foam cells in human atherosclerotic lesions, the present results suggest that, like Ox-LDL, AGE-proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.

CD36, a Member of the Class B Scavenger Receptor Family, as a Receptor for Advanced Glycation End Products

Interaction of advanced glycation end products (AGE) with AGE receptors induces several cellular phenomena potentially relating to diabetic complications. Five AGE receptors identified so far are RAGE (receptor for AGE), galectin-3, 80K-H, OST-48, and SRA (macrophage scavenger receptor class A types I and II). Since SRA is known to belong to the class A scavenger receptor family, and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36, although belonging to the class B scavenger receptor family, can recognize AGE proteins as ligands. This was tested at the cellular level in this study using Chinese hamster ovary (CHO) cells overexpressing human CD36 (CD36-CHO cells). Cellular expression of CD36 was confirmed by immunoblotting and immunofluorescent microscopy using anti-CD36 antibody. Upon incubation at 37 degrees C, (125)I-AGE-bovine serum albumin (AGE-BSA) and (125)I-oxidized low density lipoprotein (LDL), an authentic ligand for CD36, were endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CD36-CHO cells, but not wild-type CHO cells. In binding experiments at 4 degrees C, (125)I-AGE-BSA exhibited specific and saturable binding to CD36-CHO cells (K(d) = 5.6 microg/ml). The endocytic uptake of (125)I-AGE-BSA by these cells was inhibited by 50% by oxidized LDL and by 60% by FA6-152, an anti-CD36 antibody inhibiting cellular binding of oxidized LDL. Our results indicate that CD36 expressed by these cells mediates the endocytic uptake and subsequent intracellular degradation of AGE proteins. Since CD36 is one of the major oxidized LDL receptors and is up-regulated in macrophage- and smooth muscle cell-derived foam cells in human atherosclerotic lesions, these results suggest that, like oxidized LDL, AGE proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.

Propionyl-L-carnitine prevents the progression of atherosclerotic lesions in aged hyperlipemic rabbits

We have characterized the extent and the phenotype of total and proliferating cell population of aortic plaques in aged rabbits receiving a long-term low-dose cholesterol hyperlipemic diet, which represents an experimental model of atherosclerosis. For nine months, rabbits received the hypercholesterolemic diet alone or in addition to a treatment with propionyl-L-carnitine (PLC), a derivative of carnitine, an intramitochondrial carrier of fatty acids present in most cell types. We observed that, in both PLC-treated and control hyperlipemic rabbits, the ratio between proliferating macrophage-derived and smooth muscle cells was 2:1. PLC in addition to the hypercholesterolemic diet induced a marked lowering of plasma triglycerides, very low density lipoprotein (VLDL) and intermediate density lipoprotein (IDL) triglycerides, while plasma cholesterol was slightly and transiently reduced. Moreover, PLC-treated hyperlipemic rabbits exhibited a reduction of plaque thickness and extent, a slight but significant reduction of the percentage of macrophage-derived cells as compared to control hyperlipemic animals and a reduction of the number of both proliferating macrophage- and smooth muscle cell-derived foam cells. Finally, both proliferating and non-proliferating plaque cells expressed large amounts of macrophage colony-stimulating factor protein, in particular macrophage-derived foam cells. These results indicate that a modification of plasma lipemic pattern obtained by a long-term oral administration of PLC was associated with a decrease of plaque cell proliferation and severity of aortic atherosclerotic lesions.

Immunohistochemical detection of macrophage‐derived foam cells and macrophage colony‐stimulating factor in pulmonary atherogenesis of cholesterol‐fed rabbits

In order to investigate the role of monocyte/macrophages and their relationship to the expression of macrophage colony-stimulating factor (MCSF) in pulmonary atherosclerosis, lungs were excised from rabbits that had been fed for 60 and 90 days on a diet containing 0.5% cholesterol. In the lungs, fatty streaks and elevated foam cell lesions predominated in the large or medium-sized elastic pulmonary arteries, while massive accumulation of foam cells in the intima of muscular arteries produced marked luminal narrowing and nearly complete occlusion. In these lesions, most of the foam cells were reactive with RbM2, a monoclonal antibody (mAb) against rabbit macrophages, while smooth muscle cell-derived foam cells were detected by mAb against smooth muscle actin in the deeper area of elevated foam cell lesions of elastic arteries. Ultrastructural observation confirmed the presence of monocytes in the intima, their differentiation into macrophages, and their transformation into foam cells in the atherosclerotic lesions. Immunohistochemical expression of MCSF was demonstrated in the endothelial cells, smooth muscle cells and foam cells. A minor macrophage-derived foam cell population was demonstrated to possess a proliferative capacity. These data suggest that MCSF is involved in the differentiation of monocytes into macrophages, their transformation into foam cells, and their proliferation during pulmonary atherogenesis.

Eicosanoid metabolism in cholesterol-enriched arterial smooth muscle cells. Evidence for reduced posttranscriptional processing of cyclooxygenase I and reduced cyclooxygenase II gene expression.

Eicosanoid biosynthetic activity by the cyclooxygenase pathway is reduced in smooth muscle cell-derived foam cells [Pomerantz, K.B., & Hajjar, D.P. (1989) J. Lipid Res. 30, 1219-1231; Pomerantz, K.B., & Hajjar, D.P. (1990) Biochemistry 29, 1892-1899]. The present study identifies those mechanisms which contribute to reduced production of cyclooxygenase products following cholesterol enrichment of arterial smooth muscle cells. Cyclooxygenase activity, as assessed by the conversion of exogenous arachidonate to 6-keto-PGF1 alpha, was reduced approximately 8-fold in intact lipid-laden cells relative to untreated cells. Microsomes from cholesterol-enriched cells also converted less [3H]arachidonic acid to 6-keto-PGF1 alpha and PGE2 relative to microsomes from untreated cells. The reduction in cyclooxygenase activity paralleled the reduced mass of the constitutive form of cyclooxygenase (COX-1) and PGI2 synthase by approximately 80% and 33%, respectively. Northern blot hybridization analyses of COX-1 mRNA steady-state levels revealed no differences between normal and cholesterol-enriched cells under basal conditions, indicating that cholesterol enrichment did not alter COX-1 gene expression. Furthermore, cholesterol enrichment did not alter the relative levels of COX-1 mRNA expression over time following exposure of the cells to actinomycin D, indicating that cholesterol enrichment did not significantly alter the rate of COX-1 mRNA degradation. Recovery of PGI2 biosynthesis in untreated cells exposed to serum following the inactivation of COX occurred within 12 h, while the recovery of COX activity in lipid-enriched cells did not return to levels observed in untreated cells even after up to 48 h, suggesting that the induction of COX-2 (inducible form of cyclooxygenase) synthesis by growth factors or cytokines is impaired. Indeed, cholesterol enrichment attenuated IL-1 beta-, PDGF-, and TNF alpha-induced PGI2 synthesis relative to controls and was consistent with the results of in vitro labeling experiments demonstrating that cholesterol enrichment reduced the incorporation of [35S]methionine into immunoprecipitable COX-1 and COX-2 following induction by PDGF. Cholesterol enrichment also reduced the induction of COX-2 mRNA steady-state levels following exposure to PDGF. Taken together, these data demonstrate that reduced eicosanoid synthesis in smooth muscle-derived foam cells is due, in part, to impaired transcription of mRNA for COX-1 and COX-2 as well as fatty acid remodeling in membrane phospholipids. These findings support the hypothesis that cholesterol enrichment alters posttranscriptional processing of COX-1 expression, as well as altering COX-2 gene expression.

Induction of basic fibroblast growth factor mRNA and protein synthesis in smooth muscle cells by cholesteryl ester enrichment and 25-hydroxycholesterol.

Basic fibroblast growth factor (bFGF) is a potent smooth muscle cell mitogen. Smooth muscle cell and macrophage-derived foam cells, resulting from cholesteryl ester accretion, are hallmark characteristics of atherosclerosis. We wanted to determine if bFGF synthesis is altered during cholesteryl ester accumulation in smooth muscle cells. Cholesteryl ester enrichment causes a 3-fold increase in bFGF in cellular lysates and a 3-fold increase in steady state mRNA levels for bFGF, as compared with control cells. Conditioned media from cholesteryl ester-enriched smooth muscle cells contains 6 times more mitogenic activity than conditioned media from control cells; this activity is neutralized by an antibody directed against bFGF but not by an antibody directed against platelet-derived growth factor. These results suggest that cholesteryl ester enrichment also enhances bFGF release. Since oxysterols have been implicated in the pathogenesis of atherosclerosis, we determined if oxysterols could affect bFGF production and release. 25-Hydroxycholesterol also increases the release of bFGF-like mitogens from smooth muscle cells, as well as increasing mRNA transcript levels for bFGF. Cholesteryl ester enrichment and 25-hydroxycholesterol did not promote bFGF release secondary to cell injury. In conclusion, these data define a basic mechanism for smooth muscle cell hyperplasia during atherogenesis involving the generation of bFGF by smooth muscle cell-derived foam cells.

Etoposide treatment suppresses atherosclerotic plaque development in cholesterol-fed rabbits.

To study the mechanisms by which monocytes/macrophages and smooth muscle cells contribute to atherosclerotic lesions, we studied atherosclerotic plaque formation in cholesterol-fed rabbits treated with etoposide, a drug that has been shown to have several effects that could interfere with the proposed interactions between these two cell types (M.W. Aarnoudes et al, Virchows Arch B 1984;47:211-216 and M. Rozencweig et al, Cancer 1977;40:334-342). Our results show that long-term etoposide treatment of New Zealand White rabbits maintained on a high-cholesterol diet decreases the extent of fatty streak formation in the aortic intima. Moreover, the plaques formed in the presence of etoposide are thinner and at least focally have less fibrous tissue and fewer smooth muscle cell-derived foam cells than do plaques in control rabbits. These effects are independent of the extent of the diet-induced hyperlipemia or an effect of etoposide on blood cell count and may be related to the inhibition of intimal cell proliferation by etoposide.

High-density lipoprotein-induced cholesterol efflux from arterial smooth muscle cell-derived foam cells: functional relationship of the cholesteryl ester cycle and eicosanoid biosynthesis

Eicosanoids have been implicated in the regulation of arterial smooth muscle cell (SMC) cholesteryl ester (CE) metabolism. These eicosanoids, which include prostacyclin (PGI2), stimulate CE hydrolytic activities. High-density lipoproteins (HDL), which promote cholesterol efflux, also stimulate PGI2 production, suggesting that HDL-induced cholesterol efflux is modulated by eicosanoid biosynthesis. To ascertain the role of endogenously synthesized eicosanoids produced by arterial smooth muscle cells in the regulation of CE metabolism, we examined the effects of cyclooxygenase inhibition on CE hydrolytic enzyme activities, cholesterol efflux, and cholesterol content in normal SMC and SMC-derived foam cells following exposure to HDL and another cholesterol acceptor protein, serum albumin. Alterations of these activities were correlated with cholesterol efflux in response to HDL or bovine serum albumin (BSA) in the presence or absence of aspirin. HDL stimulated PGI2 synthesis and CE hydrolases in a dose-dependent manner. Eicosanoid dependency was established by demonstrating that HDL-induced acid cholesteryl ester hydrolase (ACEH) activity was blocked by aspirin. CE enrichment essentially abrogated HDL-induced PGI2 production in cells which also exhibited decreased lysosomal and cytoplasmic CE hydrolase activities. In CE-enriched cells whose cytoplasmic CE pool was metabolically labeled with [3H]oleate or cLDL containing [3H]cholesteryl linoleate, aspirin did not alter HDL- or BSA-induced net CE hydrolysis or efflux, respectively. Finally, aspirin treatment did not alter the mass of either free or esterified cholesterol content of untreated or CE-enriched SMC following exposure to acceptor proteins. These data demonstrated that CE enrichment significantly reduced HDL-induced activation of CE hydrolytic activity via inhibition of endogenous PGI2 production.(ABSTRACT TRUNCATED AT 250 WORDS)

動脈硬化巣内平滑筋細胞のphenotypeに関する研究

We have performed Immunocytochemical analysis on the intimal smooth muscle cells using monoclonal antibodies specific to: muscle actins [HHF35 (H)], smooth muscle actins [CGA7 (C)], vimentin [43βH8 (V)] and desmin (D). All studies were performed on methanol-Carnoy's-fixed, paraffin-embedded tissues. Intimal smooth muscle cells were divided into 3 major groups, based on qualitative aspects of immunoreactivities with antibodies. Group I (V+, H+, C+, D+): They were few in number, and mostly recognized in the fibrous thickening lesions of arterioles. Group II (V+, H+, C+, D-): Most of the intimal smooth muscle cells, including smooth muscle cell-derived foam cells, expressed this phenotype. Group III (V+, H+, C-, D-): Approximately 20 to 30% of HHF35-positive cells were non reactive with CGA7. This study demonstrates the application of monoclonal antibody technology to the study of the phenotypic state of intimal smooth muscle cells.