Helén L. Dichek

CLONING OF A UNIQUE LIPASE FROM ENDOTHELIAL CELLS EXTENDS THE LIPASE GENE FAMILY

A new lipoprotein lipase-like gene has been cloned from endothelial cells through a subtraction methodology aimed at characterizing genes that are expressed with in vitrodifferentiation of this cell type. The conceptual endothelial cell-derived lipase protein contains 500 amino acids, including an 18-amino acid hydrophobic signal sequence, and is 44% identical to lipoprotein lipase and 41% identical to hepatic lipase. Comparison of primary sequence to that of lipoprotein and hepatic lipase reveals conservation of the serine, aspartic acid, and histidine catalytic residues as well as the 10 cysteine residues involved in disulfide bond formation. Expression was identified in cultured human umbilical vein endothelial cells, human coronary artery endothelial cells, and murine endothelial-like yolk sac cells by Northern blot. In addition, Northern blot and in situ hybridization analysis revealed expression of the endothelial-derived lipase in placenta, liver, lung, ovary, thyroid gland, and testis. A c-Myc-tagged protein secreted from transfected COS7 cells had phospholipase A1 activity but no triglyceride lipase activity. Its tissue-restricted pattern of expression and its ability to be expressed by endothelial cells, suggests that endothelial cell-derived lipase may have unique functions in lipoprotein metabolism and in vascular disease.

Heparan Sulfate Proteoglycans Participate in Hepatic Lipaseand Apolipoprotein E-mediated Binding and Uptake of Plasma Lipoproteins, Including High Density Lipoproteins

High density lipoprotein (HDL) particles and HDL cholesteryl esters are taken up by both receptor-mediated and non-receptor-mediated pathways. Here we show that cell surface heparan sulfate proteoglycans (HSPG) participate in hepatic lipase (HL)- and apolipoprotein (apo) E-mediated binding and uptake of mouse and human HDL by cultured hepatocytes. The HL secreted by HL-transfected McA-RH7777 cells enhanced both HDL binding at 4 °C (∼2–4-fold) and HDL uptake at 37 °C (∼2–5-fold). The enhanced binding and uptake of HDL were partially inhibited by the 39-kDa protein, an inhibitor of low density lipoprotein receptor-related protein (LRP), but were almost totally blocked by heparinase, which removes the sulfated glycosaminoglycan chains from HSPG. Therefore, HL may mediate the uptake of HDL by two pathways: an HSPG-dependent LRP pathway and an HSPG-dependent but LRP-independent pathway. The HL-mediated binding and uptake of HDL were only minimally reduced when catalytically inactive HL or LRP binding-defective HL was substituted for wild-type HL, indicating that much of the HDL uptake required neither HL binding to the LRP nor lipolytic processing. To study the role of HL in facilitating the selective uptake of cholesteryl esters, we used HDL into which radiolabeled cholesteryl ether had been incorporated. HL increased the selective uptake of HDL cholesteryl ether; this enhanced uptake was reduced by more than 80% by heparinase but was unaffected by the 39-kDa protein. Like HL, apoE enhanced the binding and uptake of HDL (∼2-fold) but had little effect on the selective uptake of HDL cholesteryl ether. In the presence of HL, apoE did not further increase the uptake of HDL, and at a high concentration apoE impaired or decreased the HL-mediated uptake of HDL. Therefore, HL and apoE may utilize similar (but not identical) binding sites to mediate HDL uptake. Although the relative importance of cell surface HSPG in the overall metabolism of HDL in vivo remains to be determined, cultured hepatocytes clearly displayed an HSPG-dependent pathway that mediates the binding and uptake of HDL. This study also demonstrates the importance of HL in enhancing the binding and uptake of remnant and low density lipoproteins via an HSPG-dependent pathway.

Overexpression of Hepatic Lipase in Transgenic Mice Decreases Apolipoprotein B-containing and High Density Lipoproteins EVIDENCE THAT HEPATIC LIPASE ACTS AS A LIGAND FOR LIPOPROTEIN UPTAKE

To determine the mechanisms by which human hepatic lipase (HL) contributes to the metabolism of apolipoprotein (apo) B-containing lipoproteins and high density lipoproteins (HDL)in vivo, we developed and characterized HL transgenic mice. HL was localized by immunohistochemistry to the liver and to the adrenal cortex. In hemizygous (hHLTg +/0) and homozygous (hHLTg +/+) mice, postheparin plasma HL activity increased by 25- and 50-fold and plasma cholesterol levels decreased by 80% and 85%, respectively. In mice fed a high fat, high cholesterol diet to increase endogenous apoB-containing lipoproteins, plasma cholesterol decreased 33% (hHLTg +/0) and 75% (hHLTg +/+). Both apoB-containing remnant lipoproteins and HDL were reduced. To extend this observation, the HL transgene was expressed in human apoB transgenic (huBTg) and apoE-deficient (apoE −/−) mice, both of which have high plasma levels of apoB-containing lipoproteins. (Note that thehuBTg mice that were used in these studies were all hemizygous for the human apoB gene.) In both thehuBTg,hHLTg +/0 mice and theapoE −/−,hHLTg +/0mice, plasma cholesterol decreased by 50%. This decrease was reflected in both the apoB-containing and the HDL fractions. To determine if HL catalytic activity is required for these decreases, we expressed catalytically inactive HL (HL-CAT) in apoE −/−mice. The postheparin plasma HL activities were similar in theapoE −/− and theapoE −/−,HL-CAT +/0mice, reflecting the activity of the endogenous mouse HL and confirming that the HL-CAT was catalytically inactive. However, the postheparin plasma HL activity was 20-fold higher in theapoE −/−,hHLTg +/0mice, indicating expression of the active human HL. Immunoblotting demonstrated high levels of human HL in postheparin plasma of bothapoE −/−,hHLTg +/0and apoE −/−,HL-CAT +/0mice. Plasma cholesterol and apoB-containing lipoprotein levels were ∼60% lower inapoE −/−,HL-CAT +/0mice than in apoE −/− mice. However, the HDL were only minimally reduced. Thus, the catalytic activity of HL is critical for its effects on HDL but not for its effects on apoB-containing lipoproteins. These results provide evidence that HL can act as a ligand to remove apoB-containing lipoproteins from plasma.

Macrophage-Targeted Overexpression of Urokinase Causes Accelerated Atherosclerosis, Coronary Artery Occlusions, and Premature Death

Background—Human atherosclerotic lesions contain elevated levels of urokinase plasminogen activator (uPA), expressed predominantly by macrophages. Methods and Results—To test the hypothesis that macrophage-expressed uPA contributes to the progression and complications of atherosclerosis, we generated transgenic mice with macrophage-targeted overexpression of uPA. The uPA transgene was bred into the apolipoprotein E–null background, and transgenic mice and nontransgenic littermate controls were fed an atherogenic diet. uPA-transgenic mice had significantly elevated uPA activity in the atherosclerotic artery wall, of a magnitude similar to elevations reported in atherosclerotic human arteries. Compared with littermate controls, uPA-transgenic mice had accelerated atherosclerosis, dilated aortic roots, occlusive proximal coronary artery disease, myocardial infarcts, and early mortality. Conclusions—These data support the hypothesis that overexpression of uPA by artery wall macrophages is atherogenic and suggest that uPA inhibitors might be therapeutically useful.

TGF-β1 Limits Plaque Growth, Stabilizes Plaque Structure, and Prevents Aortic Dilation in Apolipoprotein E-Null Mice

Objective—Impairment of transforming growth factor (TGF)-&bgr;1 signaling accelerates atherosclerosis in experimental mice. However, it is uncertain whether increased TGF-&bgr;1 expression would retard atherosclerosis. The role of TGF-&bgr;1 in aneurysm formation is also controversial. We tested whether overexpression of active TGF-&bgr;1 in hyperlipidemic mice affects atherogenesis and aortic dilation. Methods and Results—We generated apolipoprotein E–null mice with transgenes that allow regulated overexpression of active TGF-&bgr;1 in their hearts. Compared to littermate controls, these mice had elevated cardiac and plasma TGF-&bgr;1, less aortic root atherosclerosis (P≤0.002), fewer lesions in the thoracic and abdominal aortae (P≤0.01), less aortic root dilation (P<0.001), and fewer pseudoaneurysms (P=0.02). Mechanistic studies revealed no effect of TGF-&bgr;1 overexpression on plasma lipids or cytokines, or on peripheral lymphoid organ cells. However, aortae of TGF-&bgr;1–overexpressing mice had fewer T-lymphocytes, more collagen, less lipid, lower expression of inflammatory cytokines and matrix metalloproteinase-13, and higher expression of tissue inhibitor of metalloproteinase-2. Conclusions—When overexpressed in the heart and plasma, TGF-&bgr;1 is an antiatherogenic, vasculoprotective cytokine that limits atherosclerosis and prevents aortic dilation. These actions are associated with significant changes in cellularity, collagen and lipid accumulation, and gene expression in the artery wall.

Cardiovascular Disease Risk Factors in Youth With Diabetes Mellitus A Scientific Statement From the American Heart Association

The rates of both type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) are increasing in youth.1 In the past 10 years, guidelines for the identification and management of cardiovascular disease (CVD) risk factors in youth with diabetes mellitus have been published by multiple professional organizations, including the American Diabetes Association (ADA),2,3 the American Heart Association (AHA),4,5 the American Academy of Pediatrics,6 the International Society of Pediatric and Adolescent Diabetes (ISPAD),7 and the Pediatric Cardiovascular Risk Reduction Initiative.8 This scientific statement summarizes and interprets these guidelines and new developments in the field in the past decade and outlines future research and clinical needs to improve cardiovascular health and risk factor management in youth with diabetes mellitus. Additional goals for this statement are to increase awareness of CVD risk factors and their identification, prevention, and treatment and to improve cardiovascular health in youth with diabetes mellitus by encouraging advancement in research and clinical care, including understanding and implementing current CVD guidelines. Improving cardiovascular health in youth with diabetes mellitus has important public health implications; therefore, this statement aims to reach healthcare providers in diabetes mellitus, cardiology, and related fields. (Note: The sections within this scientific statement are organized by diabetes mellitus type, when possible, with brief summary statements concluding each section. Multiple definitions for CVD are used in the cited articles. Readers of this statement should include risk factors, surrogate markers, and end-organ damage under this umbrella term of CVD.) ### Type 1 Diabetes Mellitus Multiple studies document an increase of 2% to 5% annually in the incidence of T1DM worldwide.9 The SEARCH for Diabetes in Youth (SEARCH) study estimated that there were 166 018 to 179 388 youth with T1DM in the United States in 2010.1 Worldwide, rates of T1DM differ …

Mechanisms of Urokinase Plasminogen Activator (uPA)-mediated Atherosclerosis ROLE OF THE uPA RECEPTOR AND S100A8/A9 PROTEINS

Data from clinical studies, cell culture, and animal models implicate the urokinase plasminogen activator (uPA)/uPA receptor (uPAR)/plasminogen system in the development of atherosclerosis and aneurysms. However, the mechanisms through which uPA/uPAR/plasminogen stimulate these diseases are not yet defined. We used genetically modified, atherosclerosis-prone mice, including mice with macrophage-specific uPA overexpression and mice genetically deficient in uPAR to elucidate mechanisms of uPA/uPAR/plasminogen-accelerated atherosclerosis and aneurysm formation. We found that macrophage-specific uPA overexpression accelerates atherosclerosis and causes aortic root dilation in fat-fed Ldlr−/− mice (as we previously reported in Apoe−/− mice). Macrophage-expressed uPA accelerates atherosclerosis by stimulation of lesion progression rather than initiation and causes disproportionate lipid accumulation in early lesions. uPA-accelerated atherosclerosis and aortic dilation are largely, if not completely, independent of uPAR. In the absence of uPA overexpression, however, uPAR contributes modestly to both atherosclerosis and aortic dilation. Microarray studies identified S100A8 and S100A9 mRNA as the most highly up-regulated transcripts in uPA-overexpressing macrophages; up-regulation of S100A9 protein in uPA-overexpressing macrophages was confirmed by Western blotting. S100A8/A9, which are atherogenic in mice and are expressed in human atherosclerotic plaques, are also up-regulated in the aortae of mice with uPA-overexpressing macrophages, and macrophage S100A9 mRNA is up-regulated by exposure of wild-type macrophages to medium from uPA-overexpressing macrophages. Macrophage microarray data suggest significant effects of uPA overexpression on cell migration and cell-matrix interactions. Our results confirm in a second animal model that macrophage-expressed uPA stimulates atherosclerosis and aortic dilation. They also reveal uPAR independence of these actions and implicate specific pathways in uPA/Plg-accelerated atherosclerosis and aneurysmal disease.

Mice Lacking Hepatic Lipase Are Lean and Protected against Diet-Induced Obesity and Hepatic Steatosis

Hepatic lipase (HL)-mediated lipoprotein hydrolysis provides free fatty acids for energy, storage, and nutrient signaling and may play a role in energy homeostasis. Because HL-activity increases with increased visceral fat, we hypothesized that increased HL-activity favors weight gain and obesity and consequently, that HL deficiency would reduce body fat stores and protect against diet-induced obesity. To test this hypothesis, we compared wild-type mice (with endogenous HL) and mice genetically deficient in HL with respect to daily body weight and food intake, body composition, and adipocyte size on both chow and high-fat (HF) diets. Key determinants of energy expenditure, including rate of oxygen consumption, heat production, and locomotor activity, were measured by indirect calorimetry. HL-deficient mice exhibited reduced weight gain on both diets (by 32%, chow; by 50%, HF; both P < 0.0001, n = 6-7 per genotype), effects that were associated with reduced average daily food intake (by 22-30% on both diets, P < 0.0001) and a modest increase in the rate of oxygen consumption (by 25%, P < 0.003) during the light cycle. Moreover, in mice fed the HF diet, HL deficiency reduced both body fat (by 30%, P < 0.0001) and adipocyte size (by 53%, P < 0.01) and fully prevented the development of hepatic steatosis. Also, HL deficiency reduced adipose tissue macrophage content, consistent with reduced inflammation and a lean phenotype. Our results demonstrate that in mice, HL deficiency protects against diet-induced obesity and its hepatic sequelae. Inhibition of HL-activity may therefore have value in the prevention and/or treatment of obesity.

Level of Macrophage uPA Expression Is an Important Determinant of Atherosclerotic Lesion Growth in Apoe −/− Mice

Objective—Enhanced plasminogen activation, mediated by overexpression of urokinase-type plasminogen activator (uPA), accelerates atherosclerosis in apolipoprotein E–null mice. However, the mechanisms through which uPA acts remain unclear. In addition, although elevated uPA expression can accelerate murine atherosclerosis, there is not yet any evidence that decreased uPA expression would retard atherosclerosis. Methods and Results—We used a bone marrow transplant (BMT) approach and apolipoprotein E–deficient (Apoe−/−) mice to investigate cellular mechanisms of uPA-accelerated atherosclerosis, aortic dilation, and sudden death. We also used BMT to determine whether postnatal loss of uPA expression in macrophages retards atherosclerosis. BMT from uPA-overexpressing mice yielded recipients with macrophage-specific uPA overexpression; whereas BMT from uPA knockout mice yielded recipients with macrophage-specific loss of uPA expression. Recipients of uPA-overexpressing BM acquired all the vascular phenotypes (accelerated atherosclerosis, aortic medial destruction and dilation, severe coronary stenoses) as well as the sudden death phenotype of uPA-overexpressing mice. Moreover, fat-fed 37-week-old recipients of uPA-null BM had significantly less atherosclerosis than recipients of uPA wild-type marrow (40% less aortic surface lesion area; P=0.03). Conclusions—The level of uPA expression by macrophages—over a broad range—is an important determinant of atherosclerotic lesion growth in Apoe−/− mice.

Divergent Effects of the Catalytic and Bridging Functions of Hepatic Lipase on Atherosclerosis

Objective—Increased expression of human hepatic lipase (HL) or a catalytically inactive (ci) HL clears plasma cholesterol in mice deficient in low-density lipoprotein receptors (LDLr) and murine HL. We hypothesized that increased expression of both HL and ciHL reduces atherosclerosis in these mice. Methods and Results—Mice deficient in both LDLr and murine HL, alone or transgenically expressing similar levels of either human HL or ciHL, were fed a high-fat, cholesterol-enriched “Western” diet for 3 months to accelerate the development of atherosclerosis. Levels of plasma lipids, insulin, glucose, and liver enzymes were measured monthly, and aortic atherosclerosis was quantitated after 3 months. Plasma insulin, glucose, and liver enzyme levels did not differ significantly from controls. After 3 months, expression of HL reduced plasma cholesterol by 55% to 65% and reduced atherosclerosis by 40%. Surprisingly, expression of ciHL did not reduce plasma cholesterol or atherosclerosis. Conclusions—High levels of HL, but not ciHL, delay the development of atherosclerosis in mice deficient in LDLr and mHL.