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Objective— Uptake of lipids by macrophages (MΦ) leads to lipid droplet accumulation and foam cell formation. The PAT family proteins are implicated in lipid droplet formation, but the precise function of the 47-kDa tail interacting protein (TIP47), a member of this family, is poorly defined. The present study was performed to determine the function of TIP47 in MΦ lipid metabolism. Methods and Results— Freeze-fracture cytochemistry demonstrates that TIP47 is present in the plasma membrane of MΦ and is aggregated into clusters when the cells are incubated with oleate. Suppression of adipophilin levels using siRNA knockdown leads to migration of TIP47 from a cytoplasmic pool to the lipid droplet. Further, reduction of TIP47 decreases triglyceride levels, whereas raising TIP47 levels by expression of EGFP-TIP47 shows the opposite effect. Conclusion— Our results show that the TIP47 protein levels directly correlate with triglyceride levels. We propose that TIP47 may act as a carrier protein for free fatty acids and in this way participates in conversion of MΦ into foam cells.

Objective— We sought to clarify the response of endothelial connexins to hyperlipidemia and lipid-lowering therapy. Methods and Results— Aortic endothelial gap junctions were analyzed by en face immunoconfocal microscopy and electron microscopy in C57BL/6 mice subjected to the following regimens: (1) normal chow (NC) for 3 months (3 mo), (2) NC for 9 mo, (3) NC for 3 mo, followed by a cholesterol-enriched diet (CED) for 6 mo, (4) NC for 3 mo and CED for 6 mo, with simvastatin in the final week, and (5) (in apoprotein E [apoE]-deficient mice) NC and examined at 3 mo and 7 to 9 mo. In wild-type mice, connexin37 (Cx37) and Cx40 were markedly downregulated in the CED-fed animals compared with those fed NC (CED vs 9-mo NC, 77% reduction in Cx37 and 65% reduction in Cx40; both P P P Conclusions— Mouse aortic endothelial gap junctions and connexins are downregulated during long-term hyperlipidemia. Short-term treatment with simvastatin leads to recovery of Cx37 expression but not Cx40 expression.

Abstract —Upregulation of connexin43-gap junctions is associated with transition of contractile vascular smooth muscle cells (SMCs) to the synthetic state. To determine whether phenotypically distinct subpopulations of medial SMCs differentially express connexin43, we investigated the human distal internal mammary artery, a structurally heterogeneous vessel with features ranging from elastic to elastomuscular to muscular. Immunoconfocal microscopy combined with quantitative analysis and complemented by in situ hybridization showed that SMCs in the elastic medial regions expressed high levels of connexin43 but low levels of desmin, whereas those of muscular medial regions expressed low levels of connexin43 but high levels of desmin. Ultrastructurally, SMCs of both regions were of the contractile phenotype, but the former cells were irregular in shape with relatively prominent synthetic organelles whereas the latter were spindle shaped with fewer synthetic organelles. Vimentin, smooth muscle α-actin, calponin, h-caldesmon, and myosin heavy chains (SM1 and SM2) were equally highly expressed by most cells in both subpopulations. The connexin43/desmin expression pattern of SMCs in regions of intimal thickening resembled those of elastic medial regions. These findings refine the view suggested from previous studies that high levels of connexin43 expression are associated with SMCs of a less contractile/more synthetic phenotype. In the internal mammary artery, the 2 subpopulations of SMCs with markedly different connexin43 expression levels both represent a differentiated contractile phenotype, but the subpopulation showing high levels of connexin43-gap junctions is characterized by low levels of desmin and structural features that reflect a more synthetic tendency.

Abstract Interactions between cells form the framework for understanding the pathogenesis of atherosclerosis, but little information is available on the role of direct intercellular communication via gap junctions in this process. To investigate gap junction expression in the pathogenesis of human atherosclerosis, lesions representing different stages of the disease were obtained from coronary arteries of hearts removed from patients undergoing cardiac transplantation. Twelve hearts, each providing 1 to 3 segments of artery, were used in the study. Sections were examined by confocal laser scanning microscopy after immunofluorescent labeling with a specific antibody against connexin43, the major gap-junctional protein of smooth muscle cells, to permit high-definition visualization of immunolabeled gap junctions through the depth of the specimen. Double labeling using anti-connexin43 and cell type–specific antibodies demonstrated colocalization of gap junctions with smooth muscle cells but not with macrophages, a relationship confirmed by electron microscopy. Regions of intimal thickening and early atheromatous lesions showed markedly increased expression of connexin43 gap junctions between intimal smooth muscle cells compared with the undiseased vessel. This increase in gap junctions was most marked in regions of intimal thickening, semiquantitative analysis of the confocal digital images revealing a >10-fold increase compared with the undiseased vessel. The quantity of labeled gap junctions in early atheromatous lesions, although higher than that of the undiseased vessel, was lower than that of intimal thickenings, and this trend toward reduced levels of gap junction immunolabeling with lesion progression continued, the value observed in the most advanced atheromatous lesions being lower than that of the undiseased vessel. As the quantity of gap junctions declined, their distribution became more patchy and the sizes of individual junctions larger. The results suggest that enhanced expression of gap junctions between smooth muscle cells may play a role in maintaining the synthetic phenotype during early growth of the atherosclerotic plaque.

Abstract Macrophage scavenger receptors mediate the uptake of chemically modified LDL in an unregulated manner, leading to massive intracellular accumulation of lipid and thus a foamy cellular morphology. In atherosclerotic lesions, foam cells originate not only from macrophages but also from smooth muscle cells, yet smooth muscle cells do not normally express scavenger receptors, and when exposed to chemically modified LDL in vitro, lipid accumulation does not occur. The mechanism of conversion of smooth muscle cells into foam cells in the arterial wall is thus still under discussion. To investigate whether direct interaction between macrophages and smooth muscle cells may be involved and to explore the effects of components of the two cell types on the expression of scavenger receptors, we report here experiments using somatic cell hybrids formed by fusion of the two cell types. Immunofluorescent labeling and confocal microscopic techniques were applied to investigate and measure (1) lipid accumulation (using Nile Red staining), (2) the binding and uptake of acetylated LDL (using 1,1′-dioctadecyl-1-3,3,3′,3′-tetramethylindocarbocyanine perchlorate–labeled acetylated LDL), and (3) receptor expression (assessed using a specific anti-receptor antibody) in smooth muscle cell–macrophage heterokaryons, macrophage-macrophage homokaryons, smooth muscle cell–smooth muscle cell homokaryons, and unfused macrophages and smooth muscle cells. The results demonstrate that scavenger receptor expression becomes repressed in macrophage–smooth muscle cell heterokaryons but not in macrophage-macrophage homokaryons. One possible explanation for the observed repression would be the existence of a negative regulatory cytoplasmic factor produced by smooth muscle cells.

Background— LOX-1, a receptor for oxidized low-density lipoprotein (OxLDL), seems to play a critical role in foam cell formation of macrophages (Mφs) and smooth muscle cells (SMC). Inhibition of LOX-1 expression reduces foam cell formation and might influence lipid core formation in atherosclerotic lesions. Because statins are able to downregulate LOX-1 expression in vitro, we examined if pravastatin can be used to reduce LOX-1 expression and lipid core formation in lesions of Watanabe heritable hyperlipidemic (WHHL) rabbits. Methods and Results— Pravastatin downregulated LOX-1 expression in cultured human Mφs and in cultured human aortic SMCs. Homozygous WHHL rabbits were treated with 50 mg kg −1 d −1 pravastatin for 32 weeks. Immunohistochemical studies revealed that LOX-1 was expressed in intimal Mφs and SMCs of atherosclerotic lesions. The pravastatin-treated rabbits showed, compared with untreated rabbits, a significantly reduced LOX-1 protein and mRNA expression in the aortic arch. Lipid labeling of this aorta region also demonstrated a strong reduction of the ratio of lipid core area/total lesion area in pravastatin-treated rabbits. Conclusions— The in vivo inhibition of LOX-1 expression by pravastatin demonstrated here represents a new pleiotropic effect of pravastatin. This in vivo inhibition of LOX-1 might be one mechanism for the lipid core reducing effect of pravastatin in atherogenesis.

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type, serine protease inhibitor with inhibitory activity toward activated factor XI, plasma kallikrein, plasmin, certain matrix metalloproteinases, and the tissue factor:activated factor VII complex. In this study, we investigated TFPI-2 expression and localization in normal and atherosclerotic human arteries by using in situ hybridization and immunohistochemical techniques. In healthy human blood vessels, TFPI-2 was detected in the vascular endothelium alone. In human atherosclerotic tissues, TFPI-2 expression was assigned to macrophages, T cells, endothelial cells, and smooth muscle cells. Western blot analysis for TFPI-2 confirmed its production by cultured human aortic smooth muscle cells, U937 cells (monocytes), and Jurkat (T cell) cell lines. Reverse transcription–polymerase chain reaction revealed similar TFPI-2 expression levels in both monocytes and macrophages in culture. Electron microscopic study with immunogold labeling revealed the association of TFPI-2 antigen with both the extracellular matrix and plasma membranes. TFPI-2 antigen was detected in some areas of atheroma that also stained positively for both tissue factor and factor VII. Moreover, detection of TFPI-2 in close spatial proximity to plasmin/plasminogen on macrophages, on endothelial cells, and in matrix-rich areas highlighted its possible functional significance in the regulation of plasmin activity and downstream proteolytic mechanisms that occur in the atherosclerotic lesion.

Abstract —The gap-junctional protein, connexin43, is differentially expressed in vascular smooth muscle cells (SMCs) according to phenotype. Previous studies suggest that desmin-negative SMCs are characterized by high levels of connexin43, whereas desmin-positive SMCs (of a more contractile phenotype) typically have low connexin43 levels. In this study, we examine systematically the inverse relationship between connexin43 and desmin in SMCs of defined regions of the rat aortic media and determine whether additional connexin isotypes are expressed and contribute to this relationship. Immunoconfocal microscopy demonstrated that (1) the inverse relationship between connexin43 and desmin expression holds true for the media of sequential aortic zones, with 1 exception, the ascending aorta, and (2) an additional vascular connexin, connexin45, is expressed by aortic SMCs. Examination of connexin43, connexin45, and desmin expression in sequential aortic zones reveals 3 SMC subpopulations. The first, predominating in the aortic arch and thoracic aorta, is desmin negative and contains high connexin43 levels; the second, predominating in the abdominal aorta and iliac artery, is desmin positive and contains low connexin43 levels; and the third, which is restricted to the ascending aorta, is desmin positive and expresses high connexin43 levels. Connexin45 levels are high in the ascending aorta but low in the other aortic segments. In para-aortic veins, a fourth SMC subpopulation appears, one that is desmin positive and contains connexin45 but not connexin43. These results demonstrate that a diversity of connexin expression patterns characterizes distinctive subpopulations of medial SMCs in situ with a potential to contribute to regional differentiation of vascular function.

Abstract —Endothelial cells form gap junctions that, according to vessel type, may be composed of up to 3 types of connexin, connexin37, connexin40, and connexin43. Although changes in connexin expression have been linked to growth and injury in cultured endothelial cells, information on connexin expression in regenerating endothelium in situ is lacking. We investigated gap junction distribution and expression of all 3 endothelial connexins during healing in rat carotid artery after denudation injury. En face viewing of the vascular luminal surface by means of immunoconfocal microscopy was used to examine the spatial and temporal expression pattern of the endothelial connexins. Gap junction spots labeled by specific antisera against connexin37, connexin40, and connexin43 were quantified 7, 14, and 28 days after injury, and the relations among the connexins were examined by using colocalization analysis. Complementary electron microscopy was also conducted. After injury, the regenerating endothelium initially expressed small, sparse gap junctions, the numbers of which progressively increased to values equivalent to those of controls. Although connexin40 gap-junctional spot size and area returned to uninjured levels by 28 days after injury, connexin37 and connexin43 spot size and area exceeded those of the uninjured artery ( P 80% in the intact artery at postinjury day 28 and P

Abstract —Tissue factor (TF) pathway inhibitor (TFPI) is the major downregulator of the procoagulant activity of the TF–factor VIIa (FVIIa) complex (TF · FVII). The active TF present in the atherosclerotic vessel wall is proposed to be responsible for the major complication of primary atherosclerosis, namely, acute thrombosis after plaque rupture, but our knowledge of the sites of TFPI expression in relation to TF remains fragmentary. The aim of this study was to investigate the expression, localization, and activity of TFPI and its relation to the activity and distribution of TF in the normal and atherosclerotic vessel wall. We applied a novel approach in which serial cross sections of human vascular segments were used to perform a complete set of assays: immunolabeling for TFPI and/or TF, in situ hybridization for the expression of TFPI mRNA, ELISA for the determination of TFPI antigen, and functional assay for the activity of TFPI and TF. In healthy vessels, TFPI protein and mRNA are present in luminal and microvascular endothelial cells (ECs) and in the medial smooth muscle cells (SMCs). In atherosclerotic vessels, TFPI protein and mRNA frequently colocalized with TF in ECs overlying the plaque and in microvessels, as well as in the medial and neointimal SMCs, and in macrophages and T cells in areas surrounding the necrotic core. At the ultrastructural level, immunogold electron microscopy confirmed the localization of TFPI in ECs, macrophages/foam cells, and SMCs. In ECs and SMCs, the gold particles decorated the plasmalemma proper and the caveolae. ELISA on cross sections revealed that atherosclerotic tissues contain more TFPI than do the healthy vessels. TFPI was functionally active against TF · FVIIa-induced coagulation, and its activity was higher in those tissues that display less TF. The largest amount of TFPI and TF were detected in complicated arterial plaques. By immunofluorescence, TFPI colocalized with platelet- and fibrin-rich areas within the organized thrombi. Atherosclerotic vessel sections promote activation of factor X, which is dependent on the presence of TF and enhanced by preincubation of the sections with anti-TFPI IgG. Taken altogether, our results suggest that TFPI is largely expressed in the normal vessel wall and enhanced in the atherosclerotic vessel, in a manner suggesting a significant role of TFPI in the regulation of TF activity.

Abstract Granulocyte-macrophage colony-stimulating factor (GM-CSF), one of a family of cytokines that regulate proliferation in macrophages and other types of cells, has been implicated in the inflammatory-fibroproliferative response of atherosclerosis. However, previous studies have been restricted to cultured cells and animal models. In the present study, we investigated GM-CSF expression in undiseased and atherosclerotic human coronary arteries at both the mRNA and protein levels. Dual in situ hybridization/cell-marking experiments demonstrated that subpopulations of intimal smooth muscle cells (SMCs) and endothelial cells express the cytokine in the histologically normal human coronary artery and that augmented expression occurs at these sites, and in macrophage accumulations and medial SMCs, in the atherosclerotic vessel. Corresponding data were obtained by in situ hybridization and reverse transcription–polymerase chain reaction and Northern analyses of cultured cells. Cultured human coronary arterial SMCs showed constitutive expression of GM-CSF in cells that had adopted an activated synthetic phenotype. Electron microscope immunocytochemistry revealed that GM-CSF is a protein localized in the cytoplasmic matrix of SMCs of both the undiseased and atherosclerotic vessel wall; extracellular matrix was largely unlabeled, with only occasional small patches of amorphous immunopositive material. The expression of GM-CSF by subpopulations of intimal SMCs in the undiseased artery and the marked upregulation of GM-CSF apparent in atherosclerotic lesions suggest roles for the cytokine in the cellular events underlying initiation and progression of the human atherosclerotic lesion.