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Background: The protective role of high-density lipoprotein (HDL) in the cardiovascular system is related to its role in the reverse transport of cholesterol from the arterial wall to the liver for subsequent excretion via the bile. Scavenger receptor class B type I (SR-BI) binds HDL and mediates selective uptake of cholesterol ester and cellular efflux of cholesterol to HDL. The role of SR-BI in atherosclerosis has been well established in murine models but it remains unclear whether SR-BI plays an equally important role in atherosclerosis in humans. The aim of this study was to investigate the expression of SR-BI and its isoforms in human macrophages and atherosclerotic plaques., Methods: The effect of hypoxia and minimally modified low-density lipoprotein (mmLDL), two proatherogenic stimuli, on SR-BI expression was studied in human monocyte-derived macrophages from healthy subjects using real-time PCR. In addition, SR-BI expression was determined in macrophages obtained from subjects with atherosclerosis (n = 15) and healthy controls (n = 15). Expression of SR-BI isoforms was characterized in human atherosclerotic plaques and macrophages using RT-PCR and DNA sequencing., Results: SR-BI expression was decreased in macrophages after hypoxia (p < 0.005). In contrast, SR-BI expression was increased by exposure to mmLDL (p < 0.05). There was no difference in SR-BI expression in macrophages from patients with atherosclerosis compared to controls. In both groups, SR-BI expression was increased by exposure to mmLDL (p < 0.05). Transcripts corresponding to SR-BI and SR-BII were detected in macrophages. In addition, a third isoform, referred to as SR-BIII, was discovered. All three isoforms were also expressed in human atherosclerotic plaque. Compared to the other isoforms, the novel SR-BIII isoform was predicted to have a unique intracellular C-terminal domain containing 53 amino acids., Conclusion: We conclude that SR-BI is regulated by proatherogenic stimuli in humans. However, we found no differences between subjects with atherosclerosis and healthy controls. This indicates that altered SR-BI expression is not a common cause of atherosclerosis. In addition, we identified SR-BIII as a novel isoform expressed in human macrophages and in human atherosclerotic plaques.

Objective: Atherosclerosis is an inflammatory disease. Several chemokines are important for monocyte/macrophage and T-cell recruitment to the lesion. CXCL16 is a recently discovered chemokine that is expressed in soluble and transmembrane forms, ligates CXCR6 chemokine receptor, and guides migration of activated Th1 and Tc1 cells. It is identical to scavenger receptor SR-PSOX, which mediates uptake of oxidized low-density lipoprotein. We investigated whether CXCL16 expression is controlled by interferon-gamma (IFN-gamma)-cytokine abundant in atherosclerotic lesions., Methods and Results: CXCL16 and CXCR6 expression was identified by polymerase chain reaction and histochemistry in atherosclerotic lesions from humans and apolipoprotein-E-deficient mice. In vitro IFN-gamma induced CXCL16 in human monocytic THP-1 cells and primary human monocytes, which led to increased uptake of oxidized low-density lipoprotein in THP-1 cells, which could be blocked by peptide antibodies against CXCL16. In vivo IFN-gamma induced CXCL16 expression in murine atherosclerotic lesions., Conclusions: We demonstrate a novel role of IFN-gamma in foam cell formation through upregulation of CXCL16/SR-PSOX. CXCR6 expression in the plaque confirms the presence of cells able to respond to CXCL16. Therefore, this chemokine/scavenger receptor could serve as a molecular link between lipid metabolism and immune activity in the atherosclerotic lesion.

A method for the quantitative determination of specific mRNAs in small numbers of cells, freshly isolated from tissues or early cell cultures, was developed by combining quantitative reverse transcription-polymerase chain reaction (RT-PCR) and quantitative flow cytometry. Freshly isolated umbilical vein endothelial cells were sorted by flow cytometry and then lysed. The number of cells in the lysate was determined by counting of nuclei after propidium iodide staining using flow cytometry. The number of plasminogen activator inhibitor-1 (PAI-1) mRNA copies per cell was determined by quantitative RT-PCR using point-mutated PAI-1 cRNA as an internal standard. The cells were shown to contain 400-900 copies of PAI-1 mRNA molecules per cell which confirms that endothelial cells in vivo express PAI-1. PAI-1 mRNA expression was also analyzed in small numbers of endothelial cells in primary culture in basal conditions and after incubation with different interleukins. The method allowed reliable and reproducible estimation of the number of mRNA copies per cell from original cell samples containing less than 1000 cells. This method can be used for the quantitative determination of various mRNA species in specified cell populations from small tissue samples or cultured cells.

Objectives: the preventive effect of acetylsalicylic acid in cardiovascular disease may be due to inhibition of platelet aggregation mediated by COX-1, but may in addition be due to anti-inflammatory effects by inhibition of COX-2. The objective of this study was to analyse the expression of COX-1 and COX-2 in atherosclerotic and healthy vascular walls., Design: the expression COX-1 and COX-2 was analysed in biopsies from human atherosclerotic carotid arteries and from healthy mammary arteries and saphenous veins., Materials: vascular biopsies were obtained from patients undergoing carotid endarterectomy or coronary bypass surgery., Methods: RT-PCR was used for mRNA analysis and for localization of proteins we used immunohistochemistry., Results: COX-2 was found in atherosclerotic plaques: in macrophages, in some smooth muscle cells and in endothelial cells of small vessels in the lesions. In non-atherosclerotic blood vessels, COX-2 was detected in the endothelium of the vasa vasorum in the adventitia. COX-1 was found in the endothelium in healthy and in atherosclerotic vessels., Conclusions: the expression of COX-2 by inflammatory and vascular cells in atherosclerotic arteries suggests that products of this enzyme may be important in the pathogenesis of atherosclerosis., (Copyright 2000 Harcourt Publishers Ltd.)

High plasma plasminogen activator inhibitor-1 (PAI-1) activity is a frequent finding in obesity and adipose tissue has recently been suggested to be a source of circulating PAI-1 in humans. In the present study, differences in adipose tissue gene expression and protein secretion rate of PAI-1 between subcutaneous and visceral adipose tissue was analysed in specimens obtained from 22 obese individuals. The secretion rate of PAI-1 was two-fold higher in subcutaneous adipose tissue than in visceral adipose tissue (292 +/- 50 vs 138 +/- 24 ng PAI-1/10(7) cells, P <0.05). In accordance with the secretion data, subcutaneous adipose tissue contained about three-fold higher levels of PAI-1 mRNA than visceral adipose tissue (2.43 +/- 0.37 vs 0.81 +/- 0.12 attomole PAI-1 mRNA/microg total RNA, P <0.00 ). PAI-1 secretion from subcutaneous but not from visceral adipose tissue correlated significantly with cell size (r = 0.43, P<0.05). In summary, subcutaneous adipose tissue secreted greater amounts of PAI-1 and had a higher PAI-1 gene expression than visceral adipose tissue from the same obese individuals. Bearing in mind that subcutaneous adipose tissue is the largest fat depot these finding may be important for the coagulation abnormalities associated with obesity.

High plasma plasminogen activator inhibitor-1 (PAI-1) activity is a frequent finding in obesity, and both PAI-1 and obesity are risk factors for cardiovascular disease. To study the mechanisms underlying increased PAI-1 levels in obese individuals, gene expression and secretion of PAI-1 were measured in human abdominal subcutaneous adipose tissue. A total of 32 obese, otherwise healthy subjects and 10 never-obese healthy subjects with a body mass index (BMI) of 42.6 +/- 1.2 and 24.3 +/- 1.9 kg/m2 (mean +/- SEM), respectively, were investigated. Plasma PAI-1 activity, adipose tissue PAI-1 secretion and adipocyte PAI-1 mRNA levels were increased seven-fold (p < 0.0001), sixfold (p < 0.0001) and twofold (p < 0.05), respectively, in the obese group. There were clear associations between adipose tissue secretion of PAI-1 and PAI-1 mRNA levels on the one hand and fat cell volume on the other (r = 0.68, p < 0.0001 and r = 0.51, p < 0.01, respectively, in the obese group). PAI-1 mRNA levels were also related to the amount of PAI-1 secreted among obese individuals (r = 0.31, p = 0.09). It is concluded that adipose tissue secretes significant amounts of PAI-1, that PAI-1 secretion from adipose tissue is increased in obesity, and that PAI-1 secretion is related to the lipid content and cell volume of fat cells. Plasma PAI-1 activity is elevated in obesity, at least in part due to increased gene expression in adipocytes, which, in turn, enhances PAI-1 secretion from adipose tissue.