Francisco Leyva

Associations of smoking, alcohol and physical activity with risk factors for coronary heart disease and diabetes in the first follow‐up cohort of the Heart Disease and Diabetes Risk Indicators in a Screened Cohort study (HDDRISC‐1)

Godsland IF, Levya F, Walton C, Worthington M, Stevenson JC (Imperial College School of Medicine, London, UK). Associations of smoking, alcohol and physical activity with risk factors for coronary heart disease and diabetes in the first follow‐up cohort of the Heart Disease and Diabetes Risk Indicators in a Screened Cohort study. J Intern Med 1998; 244: 33–41.

Factors of the Metabolic Syndrome: Baseline Interrelationships in the First Follow-up Cohort of the HDDRISC Study (HDDRISC-1)

Syndromes of risk factor disturbance may contribute to the development of coronary heart disease and non-insulin-dependent diabetes mellitus, but their definition and quantification remain problematic. Using factor analysis, constellations of risk factor variables that could indicate distinct syndromes of metabolic disturbance were explored in the baseline data of the first follow-up cohort of 742 men from the Heart Disease and Diabetes Risk Indicators in a Screened Cohort (HDDRISC) study. The primary analysis considered 16 intercorrelated variables measured in more than 90% of cohort participants. A missing-values estimation routine was used to ensure inclusion of all participants in the analysis. Subanalyses were undertaken, including a repeat of the primary analysis on the 522 individuals who had received measurement of HDL cholesterol, an oblique rather than orthogonal factor rotation procedure performed on primary and HDL subset analyses, a repeat of these two primary and HDL subset analyses using only those participants with complete measurements, and a repeat of these six analyses including only the seven variables conventionally associated with the metabolic syndrome. The principal factor that emerged in all analyses undertaken comprised oral glucose tolerance test insulin and glucose response, serum uric acid, and body mass index. Fasting serum triglyceride concentration was included in this factor in 11 of the 12 analyses undertaken, fasting plasma insulin in 8, fasting plasma glucose in 5, and mean arterial pressure in 3. HDL cholesterol factored in isolation from insulin in all analyses undertaken. These findings provide strong support for a core metabolic cluster, which is unlikely to include blood pressure and does not include HDL. The factor scores relating to this cluster will provide a means of assessing its quantitative importance in prospective analysis of the development of CHD and diabetes in this cohort.

The glycolytic pathway to coronary heart disease: A hypothesis

Coronary heart disease (CHD) is pathogenetically linked to numerous metabolic disturbances. These are inextricably interrelated, constituting identifiable clusters or syndromes of cardiovascular risk. Prominent among these is the insulin resistance syndrome, whose components, including hyperuricemia, have all been linked to CHD pathogenesis. Many mechanisms have been put forward to account for the emergence of this syndrome, but none offer a satisfactory explanation for the involvement of hyperuricemia. Possible explanations relate to the observation of glycolytic disturbances in insulin-resistant and hyperuricemic states. This might be expected from the fact that uric acid production is linked to glycolysis and that glycolysis is controlled by insulin. Phosphoribosylpyrophosphate (PPRP) is an important metabolite in this respect. Its availability depends on ribose-5-phosphate (R-5-P), the production of which is governed by glycolytic flux. Diversion of glycolytic intermediates toward R-5-P, PPRP, and uric acid will follow if there is diminished activity of glyceraldehyde-3-phosphate dehydrogenase (GA3PDH), which is regulated by insulin. Serum triglyceride concentrations may also increase, as might be expected from accumulation of glycerol-3-phosphate. Thus, intrinsic defects in GA3PDH and a loss of its responsiveness to insulin, by causing accumulation of glycolytic intermediates, may explain the association between insulin resistance, hyperuricemia, and hypertriglyceridemia. This scenario raises the possibility that disturbances of a single glycolytic enzyme may be pivotal in the modulation of metabolic risk factors for CHD.

Native Low-Density Lipoprotein Uptake by Macrophage Colony-Stimulating Factor–Differentiated Human Macrophages Is Mediated by Macropinocytosis and Micropinocytosis

Objective—To examine the pinocytotic pathways mediating native low-density lipoprotein (LDL) uptake by human macrophage colony-stimulating factor-differentiated macrophages (the predominant macrophage phenotype in human atherosclerotic plaques). Methods and Results—We identified the kinase inhibitor SU6656 and the Rho GTPase inhibitor toxin B as inhibitors of macrophage fluid-phase pinocytosis of LDL. Assessment of macropinocytosis by time-lapse microscopy revealed that both drugs almost completely inhibited macropinocytosis, although LDL uptake and cholesterol accumulation by macrophages were only partially inhibited (approximately 40%) by these agents. Therefore, we investigated the role of micropinocytosis in mediating LDL uptake in macrophages and identified bafilomycin A1 as an additional partial inhibitor (approximately 40%) of macrophage LDL uptake that targeted micropinocytosis. When macrophages were incubated with both bafilomycin A1 and SU6656, inhibition of LDL uptake was additive (reaching 80%), showing that these inhibitors target different pathways. Microscopic analysis of fluid-phase uptake pathways in these macrophages confirmed that LDL uptake occurs through both macropinocytosis and micropinocytosis. Conclusion—Our findings show that human macrophage colony-stimulating factor-differentiated macrophages take up native LDL by macropinocytosis and micropinocytosis, underscoring the importance of both pathways in mediating LDL uptake by these cells.

Extracellular cholesterol-rich microdomains generated by human macrophages and their potential function in reverse cholesterol transport.

Previous studies have shown that cholesterol in atherosclerotic plaques is present in both intracellular and extracellular forms. In the current study, we investigated a mechanism for extracellular cholesterol accumulation and examined the capacity of this pool of cholesterol to be removed by cholesterol acceptors, a step in reverse cholesterol transport. Human monocyte-derived macrophages differentiated with macrophage-colony stimulating factor were incubated with acetylated LDL to allow cholesterol enrichment and processing. These macrophages were subsequently labeled with a monoclonal antibody that specifically detects ordered cholesterol arrays, revealing the presence of unesterified cholesterol-rich microdomains on the cell surfaces and in the extracellular matrix. Similar unesterified cholesterol-rich microdomains were present in human atherosclerotic plaques. Actin microfilaments functioned in microdomain deposition or maintenance, and Src family kinases regulated transfer of these microdomains from the cell surface onto the extracellular matrix. Mediators of reverse cholesterol transport, apolipoprotein A-I (apoA-I), and HDL were capable of removing these extracellular un-esterified cholesterol-rich microdomains. However, apoA-I removed the microdomains only when macrophages were present. ApoA-I removal of microdomains was blocked by glyburide and inhibitor of ATP-binding cassette transporter A1 (ABCA1) function. In summary, cultures of cholesterol-enriched human monocyte-derived macrophages generate extracellular unesterified cholesterol-rich microdomains, which can subsequently be removed by cholesterol acceptors and therefore potentially function in reverse cholesterol transport.

Evaluation of transduction efficiency in macrophage colony-stimulating factor differentiated human macrophages using HIV-1 based lentiviral vectors

BackgroundMonocyte-derived macrophages contribute to atherosclerotic plaque formation. Therefore, manipulating macrophage function could have significant therapeutic value. The objective of this study was to determine transduction efficiency of two HIV-based lentiviral vector configurations as delivery systems for the transduction of primary human blood monocyte-derived macrophages.ResultsHuman blood monocytes were transduced using two VSV-G pseudotyped HIV-1 based lentiviral vectors containing EGFP expression driven by either native HIV-LTR (VRX494) or EF1α promoters (VRX1090). Lentiviral vectors were added to cultured macrophages at different times and multiplicities of infection (MOI). Transduction efficiency was assessed using fluorescence microscopy and flow cytometry. Macrophages transduced between 2 and 120 hours after culturing showed the highest transduction efficiency at 2-hours transduction time. Subsequently, cells were transduced 2 hours after culturing at various vector concentrations (MOIs of 5, 10, 25 and 50) to determine the amount of lentiviral vector particles required to maximally transduce human monocyte-derived macrophages. On day 7, all transduced cultures showed EGFP-positive cells by microscopy. Flow cytometric analysis showed with all MOIs a peak shift corresponding to the presence of EGFP-positive cells. For VRX494, transduction efficiency was maximal at an MOI of 25 to 50 and ranged between 58 and 67%. For VRX1090, transduction efficiency was maximal at an MOI of 10 and ranged between 80 and 90%. Thus, transductions performed with VRX1090 showed a higher number of EGFP-positive cells than VRX494.ConclusionsThis report shows that VSV-G pseudotyped HIV-based lentiviral vectors can efficiently transduce human blood monocyte-derived macrophages early during differentiation using low particle numbers that do not interfere with differentiation of monocytes into macrophages.

Physiological hyperinsulinemia is not associated with alterations in venous plasma levels of endothelin-1 in healthy individuals

Elevations in circulating levels of both endothelin-1 (ET-1) and insulin are found in coronary heart disease and chronic heart failure. Although several studies have shown that insulin can stimulate ET-1 release from endothelial cell cultures, in vivo studies have yielded equivocal results. We sought to determine whether endogenous insulin at physiological concentrations leads to alterations in venous plasma ET-1 levels in healthy subjects. In addition, we investigated the effects of physiological and supraphysiological doses of insulin on the release of ET-1 from human umbilical vein endothelial cells (HUVECs) in vitro. In the in vitro experiment, ET-1 and insulin levels were measured during an intravenous glucose tolerance test (IVGTT) in 10 healthy subjects. In the in vitro experiment, HUVECs were incubated in the absence of serum and with different concentrations of insulin (25 pmol/L to 1 mumol/L) for 4 hours before measurement of secreted ET-1. The in vivo study showed no significant alterations in venous plasma ET-1 levels during IVGTTs (maximum plasma insulin, 616.9 +/- 147.0 pmol/L [mean +/- SEM]). In the in vitro experiment, increases in ET-1 release were observed under serum-free conditions at 100 pmol/L (physiological) and 1 mumol/L (supraphysiological) insulin (ET-1, 22.4% and 46.4% higher than control cultures, respectively, both P < .05). Our results show that insulin at physiological concentrations does not alter plasma ET-1 levels in healthy individuals, but does stimulate its secretion from vascular endothelial cells in vitro. This may have implications for the study of elevated ET-1 in hyperinsulinemic states.

The Metabolic Syndrome

The association between coronary heart disease (CHD), non-insulin dependent diabetes mellitus (NIDDM), hypertension and obesity has long been recognized by both clinicians and epidemiologists. In 1988, Reaven coined the term “Syndrome X” to denote the association of these disorders with metabolic disturbances such as low plasma high density lipoprotein (HDL) cholesterol and hypertriglyceridemia, hyperinsulinemia, hyperglycemia and insulin resistance [1]. However, the term “syndrome X”, had been previously coined by Kemp in 1973 to denote the cardiological syndrome of angina and normal coronary arteries [2]. Regarding Reaven’s syndrome X, further objection to the inclusion of insulin resistance in its description is that insulin resistance has not confidently been shown to be the key metabolic disturbance, despite the plausibility of the proposed mechanism in this respect. For now, the syndrome is best referred to as the “metabolic syndrome”.