Elevating Endogenous Sphingosine-1-Phosphate (S1P) Levels Improves Endothelial Function and Ameliorates Atherosclerosis in Low Density Lipoprotein Receptor-Deficient (LDL-R-/-) Mice.

BACKGROUND: Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid and a constituent of high-density lipoprotein (HDL) exerting several atheroprotective effects in vitro. However, the few studies addressing anti-atherogenic effects of S1P in vivo have led to disparate results. We here examined atherosclerosis development in low-density lipoprotein receptor (LDL-R)-deficient (LDL-R-/-) mice with elevated endogenous S1P levels. METHODS AND RESULTS: Sub-lethally irradiated LDL-R-/- mice were transplanted with bone marrow deficient in sphingosine kinase 2 (SphK2), which led to the elevation of S1P concentrations in erythrocytes, plasma and HDL by approximately 1.5- to 2.0-fold in SphK2-/-/LDL-R-/- mice. Afterwards, mice were fed a Western diet for 14 weeks. Elevation of endogenous S1P significantly reduced atherosclerotic lesion formation by approximately half without affecting the plasma lipid profile. Furthermore, the macrophage content of atherosclerotic lesions and lipopolysaccharide-induced monocyte recruitment to the peritoneal cavity were reduced in SphK2-/-/LDL-R-/- mice. Studies using intra-vital microscopy revealed that endogenous S1P lowered leukocyte adhesion to capillary wall and decreased endothelial permeability to fluorescently labelled LDL. Moreover, SphK2-/-/LDL-R-/- mice displayed decreased levels of vascular cell adhesion molecule 1 in atherosclerotic lesions and in plasma. Studies in vitro demonstrated reduced monocyte adhesion and transport across an endothelial layer exposed to increasing S1P concentrations, murine plasma enriched in S1P or plasma obtained from SphK2-deficient animals. In addition, decreased permeability to fluorescence-labelled dextran beads or LDL was observed in S1P-treated endothelial cells. CONCLUSION: We conclude that raising endogenous S1P levels exerts anti-atherogenic effects in LDL-R-/- mice that are mediated by favourable modulation of endothelial function.

Autophagy facilitates secretion and protects against degeneration of the Harderian gland.

The epithelial derived Harderian gland consists of 2 types of secretory cells. The more numerous type A cells are responsible for the secretion of lipid droplets, while type B cells produce dark granules of multilamellar bodies. The process of autophagy is constitutively active in the Harderian gland, as confirmed by our analysis of LC3 processing in GFP-LC3 transgenic mice. This process is compromised by epithelial deletion of Atg7. Morphologically, the Atg7 mutant glands are hypotrophic and degenerated, with highly vacuolated cells and pyknotic nuclei. The mutant glands accumulate lipid droplets coated with PLIN2 (perilipin 2) and contain deposits of cholesterol, ubiquitinated proteins, SQSTM1/p62 (sequestosome 1) positive aggregates and other metabolic products such as porphyrin. Immunofluorescence stainings show that distinct cells strongly aggregate both proteins and lipids. Electron microscopy of the Harderian glands reveals that its organized structure is compromised, and the presence of large intracellular lipid droplets and heterologous aggregates. We attribute the occurrence of large vacuoles to a malfunction in the formation of multilamellar bodies found in the less abundant type B Harderian gland cells. This defect causes the formation of large tertiary lysosomes of heterologous content and is accompanied by the generation of tight lamellar stacks of endoplasmic reticulum in a pseudo-crystalline form. To test the hypothesis that lipid and protein accumulation is the cause for the degeneration in autophagy-deficient Harderian glands, epithelial cells were treated with a combination of the proteasome inhibitor and free fatty acids, to induce aggregation of misfolded proteins and lipid accumulation, respectively. The results show that lipid accumulation indeed enhanced the toxicity of misfolded proteins and that this was even more pronounced in autophagy-deficient cells. Thus, we conclude autophagy controls protein and lipid catabolism and anabolism to facilitate bulk production of secretory vesicles of the Harderian gland.

KRP-203, sphingosine 1-phosphate receptor type 1 agonist, ameliorates atherosclerosis in LDL-R-/- mice.

OBJECTIVE: Sphingosine 1-phosphate (S1P) partly accounts for antiatherogenic properties of high-density lipoproteins. We previously demonstrated that FTY720, a synthetic S1P analog targeting all S1P receptors but S1P receptor type 2, inhibits murine atherosclerosis. Here, we addressed the identity of S1P receptor mediating atheroprotective effects of S1P. APPROACH AND RESULTS: Low-density lipoprotein receptor-deficient mice on cholesterol-rich diet were given selective S1P receptor type 1 agonist KRP-203 (3.0 mg/kg per day; 6 and 16 weeks). KRP-203 substantially reduced atherosclerotic lesion formation without affecting plasma lipid concentrations. However, KRP-203 induced lymphopenia, reduced total (CD4(+), CD8(+)) and activated (CD69(+)/CD8(+), CD69(+)/CD4(+)) T cells in peripheral lymphoid organs, and interfered with lymphocyte function, as evidenced by decreased T-cell proliferation and interleukin-2 and interferon-γ production in activated splenocytes. Cyto- and chemokine (tumor necrosis factor-α, regulated and normal T cell expressed and secreted) levels in plasma and aortas were reduced by KRP-203 administration. Moreover, macrophages from KRP-203-treated mice showed reduced expression of activation marker MCH-II and poly(I:C)-elicited production of tumor necrosis factor-α, monocyte chemoattractant protein-1, and interleukin-6. In vitro studies demonstrated that KRP-203 reduced tumor necrosis factor-α, interleukin-6, and interferon-γ-induced protein-10 production; IκB and signal transducer and activator of transcription-1 phosphorylation; and nuclear factor κB and signal transducer and activator of transcription-1 activation in poly(I:C)-, lipopolysaccharide-, or interferon-γ-stimulated bone marrow macrophages, respectively. CONCLUSIONS: Present results demonstrate that activation of S1P signaling pathways inhibit atherosclerosis by modulating lymphocyte and macrophage function and suggest that S1P receptor type 1 at least partially mediates antiatherogenic effects of S1P.

Connexin expression patterns in arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inheritable myocardial disease accounting for ventricular tachycardia and sudden death in the young and arising from areas of fibrofatty replacement of predominantly right ventricular myocardium. That some patients manifest life-threatening ventricular tachycardia in the absence of substantial myocardial replacement suggests that gap junction remodeling might be acting synergistically to ventricular remodeling to promote arrhythmogenesis. Hence, we sought to verify gap junction composition and distribution by analyzing the expression and occurrence of specific gap junction proteins (connexins [Cxs]) in patients with ARVC. Right ventricular endomyocardial biopsy specimens were taken from 16 patients with definite ARVC (age 48 ± 16 years) and analyzed for Cx40, Cx43, and Cx45 messenger ribonucleic acid expression (relative to glyceraldehyde-3-phosphate-dehydrogenase messenger ribonucleic acid expression). The results were compared to those obtained from nondiseased donor hearts (n = 6; age 32 ± 11 years). The patients with ARVC showed a significant reduction in the messenger ribonucleic acid expression of Cx40 (p <0.0001) and Cx45 (p <0.0001) compared to that of the controls. The expression of Cx43 was similar in patients with ARVC and controls (p = 0.098). Mutations in plakophilin-2 were identified in 7 of 16 patients (25%). The Cx expression levels were comparable between the mutation carriers and noncarriers (p = NS). In conclusion, ARVC features alterations in the expression of Cxs and their distribution at cardiac intercalated discs. Apart from the deposition of extracellular matrix, the potential loss of gap junctions and shift in the composition of gap junctional Cxs in the ventricular conduction system might further contribute to the development of ventricular arrhythmias in patients with ARVC.

Npp1 promotes atherosclerosis in ApoE knockout mice.

Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) generates inorganic pyrophosphate (PP(i)), a physiologic inhibitor of hydroxyapatite deposition. In a previous study, we found NPP1 expression to be inversely correlated with the degree of atherosclerotic plaque calcification. Moreover, function-impairing mutations of ENPP1, the gene encoding for NPP1, are associated with severe, artery tunica media calcification and myointimal hyperplasia with infantile onset in human beings. NPP1 and PP(i) have the potential to modulate atherogenesis by regulating arterial smooth muscle cell (SMC) differentiation and function, including increase of pro-atherogenic osteopontin (OPN) expression. Hence, this study tested the hypothesis that NPP1 deficiency modulates both atherogenesis and atherosclerotic intimal plaque calcification. Npp1/ApoE double deficient mice were generated by crossing mice bearing the ttw allele of Enpp1 (that encodes a truncation mutation) with ApoE null mice and fed with high-fat/high-cholesterol atherogenic diet. Atherosclerotic lesion area and calcification were examined at 13, 18, 23 and 28 weeks of age. The aortic SMCs isolated from both ttw/ttw ApoE(-/-) and ttw/+ ApoE(-/-) mice demonstrated decreased Opn expression. The 28-week-old ttw/ttw ApoE(-/-) and ttw/+ ApoE(-/-) had significantly smaller atherosclerotic lesions compared with wild-type congenic ApoE(-/-) mice. Only ttw/ttw but not ttw/+ mice developed artery media calcification. Furthermore in ttw/+ mice, there was a tendency towards increased plaque calcification compared to ApoE(-/-) mice without Npp1 deficiency. We conclude that Npp1 promotes atherosclerosis, potentially mediated by Opn expression in ApoE knockout mice.

Expression of NPP1 is regulated during atheromatous plaque calcification.

Mutations of the ENPP1 gene encoding ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) are associated with medial calcification in infancy. While the inhibitory role of matrix proteins such as osteopontin (OPN) with respect to atherosclerotic plaque calcification has been established, the role of NPP1 in plaque calcification is not known. We assessed the degree of plaque calcification (computed tomography), NPP1 and OPN localization (immunohistochemistry) and expression (RT-PCR) in a cohort of 45 patients undergoing carotid endatherectomy for significant stenosis of the internal carotid artery and in normal arteries (N= 50). We correlated NPP1 and OPN expression levels to the degree of plaque calcification, to pro-atherogenic factors and statin therapy. NPP1 was demonstrated in the base and in the shoulder of atherosclerotic plaques. Compared to normal arteries and non-calcified plaques, in calcified plaques NPP1 mRNA was decreased (P < 0.0001). OPN mRNA levels were up-regulated in carotid atheroma. NPP1 and OPN expression levels positively correlated with the degree of plaque calcification (R= 0.54, P= 0.00019 and R= 0.46, P= 0.017, respectively) and with risk factors of atherosclerosis. Expression of the calcification inhibitor NPP1 is down-regulated in calcified atherosclerotic plaques. Our correlation data point to a counter-active mechanism, which in the end turns out to be insufficient to prevent further progression of calcification.

Aortic dissection associated with Cogans's syndrome: deleterious loss of vascular structural integrity is associated with GM-CSF overstimulation in macrophages and smooth muscle cells.

BACKGROUND: Cogan's syndrome is a rare disorder of unknown origin characterized by inflammatory ocular disease and vestibuloauditory symptoms. Systemic vasculitis is found in about 10% of cases. CASE PRESENTATION: A 46-year-old female with Cogans's syndrome and a history of arterial hypertension presented with severe chest pain caused by an aneurysm of the ascending aorta with a dissection membrane located a few centimeters distal from the aortic root. After surgery, histopathological analysis revealed that vascular matrix integrity and expression of the major matrix molecules was characterized by elastolysis and collagenolysis and thus a dramatic loss of structural integrity. Remarkably, exceeding matrix deterioration was associated with massively increased levels of granulocyte macrophage colony stimulating factor (GM-CSF). CONCLUSION: Our data suggest that the persistently increased secretion of the inflammatory mediator GM-CSF by resident inflammatory cells but also by SMC may be the trigger of aortic wall structural deterioration.

Damage of guinea pig heart and arteries by a trioleate-enriched diet and of cultured cardiomyocytes by oleic acid.

BACKGROUND: Mono-unsaturated fatty acids (MUFAs) like oleic acid have been shown to cause apoptosis of cultured endothelial cells by activating protein phosphatase type 2C alpha and beta (PP2C). The question arises whether damage of endothelial or other cells could be observed in intact animals fed with a trioleate-enriched diet. METHODOLOGY/PRINCIPAL FINDINGS: Dunkin-Hartley guinea pigs were fed with a trioleate-enriched diet for 5 months. Advanced atherosclerotic changes of the aorta and the coronary arteries could not be seen but the arteries appeared in a pre-atherosclerotic stage of vascular remodelling. However, the weight and size of the hearts were lower than in controls and the number of apoptotic myocytes increased in the hearts of trioleate-fed animals. To confirm the idea that oleic acid may have caused this apoptosis by activation of PP2C, cultured cardiomyocytes from guinea pigs and mice were treated with various lipids. It was demonstrable that oleic acid dose-dependently caused apoptosis of cardiomyocytes from both species, yet, similar to previous experiments with cultured neurons and endothelial cells, stearic acid, elaidic acid and oleic acid methylester did not. The apoptotic effect caused by oleic acid was diminished when PP2C alpha and beta were downregulated by siRNA showing that PP2C was causally involved in apoptosis caused by oleic acid. CONCLUSIONS/SIGNIFICANCE: The glycerol trioleate diet given to guinea pigs for 5 months did not cause marked atherosclerosis but clearly damaged the hearts by activating PP2C alpha and beta. The diet used with 24% (wt/wt) glycerol trioleate is not comparable to human diets. The detrimental role of MUFAs for guinea pig heart tissue in vivo is shown for the first time. Whether it is true for humans remains to be shown.

Endocytotic segregation of gliadin peptide 31-49 in enterocytes.

OBJECTIVE: Coeliac disease (CD) is a multisystemic autoimmune inflammation of the intestinal tract induced by wheat gluten and related cereals in human leucocyte antigen (HLA)-DQ2/8-positive individuals. The molecular mechanisms relevant to oral tolerance induction towards toxic cereals such as gliadin remain poorly understood. Enterocytes, which express predominantly HLA-DR proteins, are capable of processing, transcytosing and presenting food antigens from the intestinal lumen to T lymphocytes of the lamina propria. METHODS: Epitope-specific monoclonal antigliadin antibodies are utilised to unravel the intraepithelial transport processes of gliadin peptides in human duodenal biopsy specimens from patients with CD and reconstitute the transepithelial and endocytic pathways of gliadin in intestinal epithelial HT29 cells. RESULTS: The gliadin peptide AA 31-49 is segregated from the peptides AA 56-68 and AA 229-246 along the endosomal pathway. Thus, AA 31-49 bypasses HLA-DR-positive late endosomes in intestinal cells and in biopsy specimens of patients with untreated CD. Further, it is localised in early endosomes and consequently escapes antigen presentation at the basolateral membrane, unlike peptides AA 56-68 and AA 229-246 that reach HLA-DR-positive late endosomes. Strikingly, forms of gliadin peptide AA 31-49 conjugated to cholera toxin B are sorted into late endosomes of HT29 cells. CONCLUSIONS: Endocytic segregation of gliadin peptide AA 31-49 seems to be a constitutive process. It explains why this peptide cannot stimulate gluten-sensitive T cells. Presentation of gliadin peptides by HLA-DR proteins via late endosomes within enterocytes might induce a tolerogenic effect and constitutes a potentially promising therapeutic approach for induction of tolerance towards gliadin.

Transgenic model of cardiac rhabdomyosarcoma formation.

OBJECTIVES: Cardiac rhabdomyosarcomas are rare, and the pathogenesis of this detrimental disease is widely unknown. Most data are obtained from case reports or small series, and models for systematic pathogenetic studies are lacking. We aimed to establish a transgenic mouse model of cardiac rhabdomyosarcoma formation. METHODS: Standard techniques were used to construct a minigene comprised of the 5' region of the 1.4-kb SM22alpha gene (expressed in embryonic cardiac muscle) and the 2.7-kb SV40 T antigen early region. This T antigen fragment includes the coding sequences for the binding sites of p53 and the proteins of the pRb family. Genotyping of transgenic mice was performed by means of polymerase chain reaction, and phenotypic expression was evaluated by means of immunohistochemistry. RESULTS: Transgenic mice were studied at the age of approximately 8 to 12 weeks. Cardiac tumors were found of variable size in the left or right sides of the heart and were associated with T antigen expression. Histologic analysis revealed a 3.1-fold enhanced cell density, enlarged cell nuclei, and a 3.4-fold enhanced DNA content. Phenotypic characterization of cardiac tumors resulted in positive staining for desmin, smooth muscle alpha-actin, troponin C, and Myo D1, which met the criteria for rhabdomyosarcomas. CONCLUSIONS: To the best of our knowledge, the present study is the first description of a mouse model of cardiac rhabdomyosarcoma formation based on genetic modulation. Our model will be a valuable tool for illuminating the pathogenesis of cardiac rhabdomyosarcomas and will allow the testing of new therapeutic approaches to fight this dreadful disease.

Mechanisms of arterial calcification: spotlight on the inhibitors.

Similarities in the mechanisms of vascular calcification and the processes of bone and cartilage mineralization have come to light in recent years. Although formerly thought to be an inactive process of hydroxyapatite crystal precipitation, presently, vascular calcification is considered a regulated type of tissue mineralization. Moreover, different pathways of tissue mineralization are discussed. Pathological types of calcification are correlated with aging, metabolic disorders, chronic low-grade inflammation, and with genetic and acquired dysregulation of inorganic pyrophosphate (PPi) metabolism. This chapter focuses on recent developments in understanding the mechanisms of vascular calcification with special emphasis on the particular calcification pathway and the impact of deficient inhibition of calcification.

Freeze-fracture replica immunolabelling reveals caveolin-1 in the human cardiomyocyte plasma membrane.

Mutations in or ablation of the gene encoding caveolin-3, a protein of muscle cell caveolae, result in forms of muscular dystrophy and cardiomyopathy. Another member of the caveolin gene family, caveolin-1, is widely considered not to be expressed in myocytes, yet ablation of the gene encoding this protein in mice also results in cardiomyopathy. By applying the high-resolution electron-microscopical imaging technique of freeze-fracture replica immunolabelling, we report here evidence that caveolin-1 is expressed in human cardiac myocytes, localized to both caveolae and non-caveolar domains in the plasma membrane. Disorders of the myocyte resulting from defects in caveolin-1 may thus arise directly, at the level of the myocyte, rather than via other cell types as previously proposed.

Transgenic model of smooth muscle cell cycle reentry: expression pattern of the collageneous matrix.

BACKGROUND: We have previously shown that genetically induced smooth muscle cell (SMC) cycle reentry in transgenic mouse models expressing the SV40 T antigen (TAg) resulted in adaptive arterial remodeling. The present investigation targeted the in vitro expression pattern of the collageneous matrix associated with TAg-induced SMC cycle modulation. METHODS: SMC cultures were established from the transgenic model expressing temperature-sensitive TAg. This allowed inducible transgene expression at the permissive temperature of 33 degrees C compared with the restrictive temperature of 39.5 degrees C. To distinguish a transgene effect from a temperature effect, SMCs with constitutively expressed TAg were used as controls. Data were obtained using array technology, Northern blotting, reverse transcription polymerase chain reaction, and zymography. RESULTS: TAg-induced SMC cycle reentry resulted in significant down-regulation of matrix metalloproteinase (MMP)-3, whereas MMP-2, -9, and -11 were not influenced. In addition, SMC cycle reentry resulted in significantly increased RNA levels of procollagen alpha2(IV), procollagen alpha2(V), and procollagen alpha1(XI), whereas procollagen alpha1(III) and procollagen alpha1(VIII) were down-regulated. Studies of the RNA expression levels of granulocyte-macrophage colony-stimulating factor revealed an up-regulation of this proinflammatory and matrix-modulating cytokine. CONCLUSIONS: This transgenic model provides evidence that TAg-induced cell cycle reentry is associated with a complex modulation of the collageneous matrix. Factors identified in this in vitro study reveal a comprehensive expression pattern of candidates, which might allow the vessel to undergo adaptive arterial remodeling under in vivo conditions. Our results will give rise to further investigations to elaborate on this hypothesis and to improve understanding of the role of such factors in vascular diseases.

Low-energy electromagnetic fields promote proliferation of vascular smooth muscle cells.

The rationale was to investigate the effects of low-energy electromagnetic fields (EMF) on the proliferation of bovine coronary and murine aortic smooth muscle cells (SMC). EMF were applied to SMC at field frequencies of 25, 50, or 100 Hz, and exposure time was set to 5, 15, or 30 minutes. Significant increases in SMC-counts compared with sham exposed controls were found for all EMF-frequencies tested. The effect was most pronounced for 50 Hz fields with maximum increases of 1.2-fold over controls. Sequential double exposure of mouse aortic SMC to 50 Hz fields revealed significantly enhanced cell proliferation by 1.2 fold compared with single exposure (p < 0.05). Experiments performed on bovine SMC also revealed significant increases in cell proliferation. The results demonstrate that EMF are capable of significantly enhancing the proliferation of vascular SMC. These results rise the question whether EMF would qualify as supportive means to angio-/arteriogenic approaches.

Granulocyte macrophage colony-stimulating factor deficiency affects vascular elastin production and integrity of elastic lamellae.

BACKGROUND: Granulocyte macrophage colony-stimulating factor (GM-CSF) deficiency affects the production and fiber assembly/organization of the vascular collagenous matrix; structural alterations to the elastic system were observed. The present study elaborates the effect of GM-CSF deficiency on the vascular elastin system. METHODS AND RESULTS: Histological examination of the aorta of GM-CSF-deficient mice revealed structurally altered elastic fibers. The elastic fiber area was significantly enhanced, whereas the remaining medial area was not affected. Aortic size was significantly increased. Reverse transcription polymerase chain reaction demonstrated decreased expression levels of tropoelastin, lysyl oxidase and bone morphogenetic protein 1 (BMP-1). Cell culture studies on vascular smooth muscle cells showed that after clearance of GM-CSF with GM-CSF antibodies, the tropoelastin mRNA expression was markedly reduced. Concomitantly, lysyl oxidase and BMP-1 mRNA levels were decreased. Treatment with GM-CSF stimulated the expression of these mRNAs. CONCLUSIONS: Our studies demonstrate that disorganization of elastic lamellae as induced by GM-CSF deficiency is associated with adaptive vascular remodeling. The decreased tropoelastin expression observed is associated with elastic fiber hypertrophy. This paradox effect may be explained by decreased expression levels of lysyl oxidase and BMP-1, both mediating cross-linkage and thus assembly and organization of elastic fibers. From our data, we conclude that GM-CSF is a prerequisite for the maintenance of structural integrity of the vessel wall.

Hypophosphatemia, hyperphosphaturia, and bisphosphonate treatment are associated with survival beyond infancy in generalized arterial calcification of infancy.

BACKGROUND: Generalized arterial calcification of infancy has been reported to be frequently lethal, and the efficiency of any therapy, including bisphosphonates, is unknown. A phosphate-poor diet markedly increases survival of NPP1 null mice, a model of generalized arterial calcification of infancy. METHODS AND RESULTS: We performed a multicenter genetic study and retrospective observational analysis of 55 subjects affected by generalized arterial calcification of infancy to identify prognostic factors. Nineteen (34%) patients survived the critical period of infancy. In all 8 surviving patients tested, hypophosphatemia due to reduced renal tubular phosphate reabsorption developed during childhood. Eleven of 17 (65%) patients treated with bisphosphonates survived. Of 26 patients who survived their first day of life and were not treated with bisphosphonates only 8 (31%) patients survived beyond infancy. Forty different homozygous or compound heterozygous mutations, including 16 novel mutations in ENPP1, were found in 41 (75%) of the 55 patients. Twenty-nine (71%) of these 41 patients died in infancy (median, 30 days). Seven of the 14 (50%) patients without ENPP1 mutations died in infancy (median, 9 days). When present on both alleles, the mutation p.P305T was associated with death in infancy in all 5 cases; otherwise, no clear genotype-phenotype correlation was seen. CONCLUSION: ENPP1 coding region mutations are associated with generalized arterial calcification of infancy in approximately 75% of subjects. Except for the p.P305T mutation, which was universally lethal when present on both alleles, the identified ENPP1 mutations per se have no discernable effect on survival. However, survival seems to be associated with hypophosphatemia linked with hyperphosphaturia and also with bisphosphonate treatment.

Function of scavenger receptor class A type I/II is not important for smooth muscle foam cell formation.

Macrophages (MPhi) and smooth muscle cells (SMC) are transformed into foam cells by massive accumulation of modified lipoproteins during atherogenesis. It is known that class AI/II scavenger receptors participate in the foam cell formation of MPhi. The mechanism of lipid accumulation in SMC is however unknown. Therefore, we investigated if class AI/II scavenger receptors mediate the uptake of modified lipoproteins in SMC. Additionally, we examined the influence of MPhi and proinflammatory cytokines in this process. Our flow cytometric experiments revealed significant uptake of DiI-AcLDL in SMC. This uptake was markedly enhanced by IL-1alpha and TNF-alpha, whereas cocultured MPhi decreased the uptake of DiI-AcLDL in SMC. Competition and blocking experiments were performed to enlighten the role of class AI/II scavenger receptors. The competition experiments showed that surplus NatLDL, a ligand not known to interact with class AI/II scavenger receptors, caused a drastically decreased uptake of DiI-AcLDL in SMC. Additionally, blocking of class AI/II scavenger receptors with antibody 2F8 did not influence the uptake of DiI-AcLDL in SMC. Furthermore, fluorescence microscopic double staining of human coronary arteries with early, intermediate and advanced atherosclerotic lesions showed no colocalization of class AI scavenger receptors with SMC. These results indicate that class AI/II scavenger receptors play only a minor role in the uptake of modified lipoproteins in SMC. We suggest that SMC foam cell formation is mainly mediated by other receptors than class AI/II scavenger receptors.