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.

Expression of apolipoprotein A-I in rabbit carotid endothelium protects against atherosclerosis.

Expression of atheroprotective genes in the blood vessel wall is potentially an effective means of preventing or reversing atherosclerosis. Development of this approach has been hampered by lack of a suitable gene-transfer vector. We used a helper-dependent adenoviral (HDAd) vector to test whether expression of apolipoprotein A-I (apoA-I) in the artery wall could retard the development of atherosclerosis in hyperlipidemic rabbits. Carotid arteries were infused with an HDAd expressing rabbit apoA-I or a "null" HDAd and harvested 2 and 4 weeks later. ApoA-I mRNA and protein were detected only in HDAdApoAI arteries. Lesion size, lipid and macrophage content, and adhesion molecule expression were similar in both groups at 2 weeks. Between 2 and 4 weeks, most of these measures of atherosclerosis increased in HDAdNull arteries, but were stable or decreased in HDAdApoAI arteries (P ≤ 0.04 for all end points in 4-week HDAdApoAI versus HDAdNull arteries). A longer-term study in chow-fed rabbits revealed persistence of HDAd vector DNA and apoA-I expression for ≥48 weeks, with stable vector DNA content and apoA-I expression from 4 to 48 weeks. Expression of apoA-I in arterial endothelium significantly retards atherosclerosis. HDAd provides prolonged, stable expression of a therapeutic transgene in the artery wall.

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.

TGF-[beta]1 limits plaque growth, stabilizes plaque structure, and prevents aortic dilation in apolipoprotein E-null mice.

OBJECTIVE: Impairment of transforming growth factor (TGF)-beta1 signaling accelerates atherosclerosis in experimental mice. However, it is uncertain whether increased TGF-beta1 expression would retard atherosclerosis. The role of TGF-beta1 in aneurysm formation is also controversial. We tested whether overexpression of active TGF-beta1 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-beta1 in their hearts. Compared to littermate controls, these mice had elevated cardiac and plasma TGF-beta1, less aortic root atherosclerosis (P< or =0.002), fewer lesions in the thoracic and abdominal aortae (P< or =0.01), less aortic root dilation (P<0.001), and fewer pseudoaneurysms (P=0.02). Mechanistic studies revealed no effect of TGF-beta1 overexpression on plasma lipids or cytokines, or on peripheral lymphoid organ cells. However, aortae of TGF-beta1-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-beta1 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.

Hormonal regulation of testicular steroid and cholesterol homeostasis.

The male sex steroid, testosterone (T), is synthesized from cholesterol in the testicular Leydig cell under control of the pituitary gonadotropin LH. Unlike most cells that use cholesterol primarily for membrane synthesis, steroidogenic cells have additional requirements for cholesterol, because it is the essential precursor for all steroid hormones. Little is known about how Leydig cells satisfy their specialized cholesterol requirements for steroid synthesis. We show that in mice with a unique hypomorphic androgen mutation, which disrupts the feedback loop governing T synthesis, that genes involved in cholesterol biosynthesis/uptake and steroid biosynthesis are up-regulated. We identify LH as the central regulatory molecule that controls both steroidogenesis and Leydig cell cholesterol homeostasis in vivo. In addition to the primary defect caused by high levels of LH, absence of T signaling exacerbates the lipid homeostasis defect in Leydig cells by eliminating a short feedback loop. We show that T signaling can affect the synthesis of steroids and modulates the expression of genes involved in de novo cholesterol synthesis. Surprisingly, accumulation of active sterol response element-binding protein 2 is not required for up-regulation of genes involved in cholesterol biosynthesis and uptake in Leydig cells.

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.

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. These studies demonstrate that high levels of catalytically active human hepatic lipase (HL) reduce atherosclerosis, whereas high levels of a catalytically inactive HL do not affect atherosclerosis in mice genetically deficient in low-density lipoprotein receptor and mouse HL.

In vivo structure-function studies of human hepatic lipase: the catalytic function rescues the lean phenotype of HL-deficient (hl-/-) mice.

The lean body weight phenotype of hepatic lipase (HL)-deficient mice (hl(-/-)) suggests that HL is required for normal weight gain, but the underlying mechanisms are unknown. HL plays a unique role in lipoprotein metabolism performing bridging as well as catalytic functions, either of which could participate in energy homeostasis. To determine if both the catalytic and bridging functions or the catalytic function alone are required for the effect of HL on body weight, we studied (hl(-/-)) mice that transgenically express physiologic levels of human (h)HL (with catalytic and bridging functions) or a catalytically-inactive (ci)HL variant (with bridging function only) in which the catalytic Serine 145 was mutated to Alanine. As expected, HL activity in postheparin plasma was restored to physiologic levels only in hHL-transgenic mice (hl(-/-)hHL). During high-fat diet feeding, hHL-transgenic mice exhibited increased body weight gain and body adiposity relative to hl(-/-)ciHL mice. A similar, albeit less robust effect was observed in female hHL-transgenic relative to hl(-/-)ciHL mice. To delineate the basis for this effect, we determined cumulative food intake and measured energy expenditure using calorimetry. Interestingly, in both genders, food intake was 5-10% higher in hl(-/-)hHL mice relative to hl(-/-)ciHL controls. Similarly, energy expenditure was ~10% lower in HL-transgenic mice after adjusting for differences in total body weight. Our results demonstrate that (1) the catalytic function of HL is required to rescue the lean body weight phenotype of hl(-/-) mice; (2) this effect involves complementary changes in both sides of the energy balance equation; and (3) the bridging function alone is insufficient to rescue the lean phenotype of hl(-/-)ciHL mice.

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.

Energy balance in congenital generalized lipodystrophy type I.

Congenital generalized lipodystrophy type 1 (CGL-1) is characterized by an absence of adipose tissue and decreased serum leptin levels. Low leptin levels in CGL-1 support the claim that subjects are hypermetabolic and hyperphagic. The present study examines this claim. We determined 24-hour energy expenditure (24-h EE) (kilocalories) (n = 2) and resting metabolic rate (RMR) per kilogram of lean body mass (LBM) (n = 3) in CGL-1 and in 18 healthy control subjects. The 24-h EEs of control and subjects with CGL were compared with respect to kilocalories required per day relative to kilograms of LBM and with respect to RMR relative to kilograms of LBM. Fasting leptin, adiponectin, and 24-hour ghrelin levels were also measured in subjects with CGL-1. The 24-h EE per kilogram of LBM for the subjects with CGL-1 falls on the same regression line observed for this relationship in the controls. The RMR per kilogram of LBM in subjects with CGL-1 also was similar to that in controls. Both 24-h EE and RMR were quite increased when reported per kilogram of total body weight. Subjects with CGL-1 also have decreased fasting leptin and adiponectin hormone levels and no premeal ghrelin rise. People with CGL-1 have similar RMR and daily caloric requirements as healthy controls when these parameters are expressed as a function of LBM. Appetite-regulating hormone levels in CGL-1 suggest that multiple factors act to control appetite in these individuals.

Reduced atherosclerosis in chow-fed mice expressing high levels of a catalytically inactive human hepatic lipase.

Increased expression of catalytically inactive hepatic lipase (ciHL) lowers remnants and low-density lipoproteins (LDL) and may reduce atherosclerosis in mice lacking both LDLreceptors (LDLR) and murine (m) HL. However, in a previous study, ciHL expression failed to reduce atherosclerosis but increased liver fat accumulation after a 3-month high-fat diet, suggesting that diet-induced metabolic changes compromised the antiatherogenic effects of ciHL. Therefore, we hypothesized that reduced dietary fat would reduce atherosclerosis in ciHL expressing mice. Mice lacking both LDLR and mHL, alone, or expressing ciHL were fed a low-fat (chow) diet for 9 months to match the cumulative cholesterol exposure resulting from a 3-month high-fat diet. Plasma lipids and lipoproteins as well as atherosclerosis were determined at sacrifice. Also, liver expression of receptors and proteins contributing to cholesterol delivery including the LDLreceptor related protein (LRP), scavenger receptor (SR)-B1 and apoE were determined. At 9 months, ciHL expression reduced plasma cholesterol by approximately 20% and atherosclerosis by 79% (from 2.67+/-0.61% of aortic surface, Ldlr-/-hl-/-, n=9, to 0.55+/-0.32% of aortic surface, Ldlr-/-hl-/-ciHL, n=7, P=0.01). Also, LRP-expression increased approximately 4-fold, whereas SR-B1 and apoE remained unchanged. These results demonstrate that ciHL expression reduces atherosclerosis. Also, these results demonstrate that ciHL increases LRP expression and suggest increased LRP-mediated lipoprotein clearance as a pathway for ciHL-mediated atherosclerosis reduction.

Attenuated corticosterone response to chronic ACTH stimulation in hepatic lipase-deficient mice: evidence for a role for hepatic lipase in adrenal physiology.

Hepatic lipase (HL), a liver-expressed lipolytic enzyme, hydrolyzes triglycerides and phospholipids in lipoproteins and promotes cholesterol delivery through receptor-mediated whole particle and selective cholesterol uptake. HL activity also occurs in the adrenal glands, which utilize lipoprotein cholesterol to synthesize glucocorticoids in response to pituitary ACTH. It is likely that the role of adrenal HL is to facilitate delivery of exogenous cholesterol for glucocorticoid synthesis. On this basis, we hypothesized that HL deficiency would blunt the glucocorticoid response to ACTH. Furthermore, because exogenous cholesterol also is derived from the LDL receptor (LDLR) pathway, we hypothesized that LDLR deficiency would blunt the response to ACTH. To test these hypotheses, we compared the corticosterone response to eight daily ACTH injections in HL-deficient (hl-/-), LDLR-deficient (Ldlr-/-), and HL- and LDLR-doubly deficient (Ldlr-/- hl-/-) mice with that in wild-type (WT) mice. Plasma corticosterone levels were measured on days 2, 5, and 8. Differences in plasma corticosterone levels between genotypes were analyzed by Kruskal-Wallis one-way ANOVA on ranks and pairwise multiple comparisons by Dunn's test. Our results demonstrate a trend toward reductions in plasma corticosterone levels on day 2 and significant reductions on day 5 and day 8 in the knockout models. Thus, on day 5, plasma corticosterone levels were reduced by 57, 70, and 73% (all P < 0.05) and on day 8 by 76, 59, and 63% (all P < 0.05) in hl-/-, Ldlr-/-, and Ldlr-/- hl-/- mice, respectively. These results demonstrate that HL deficiency, like LDLR deficiency, blunts the adrenal response to chronic ACTH stimulation and suggest a novel role for HL in adrenal physiology.

The bridging function of hepatic lipase clears plasma cholesterol in LDL receptor-deficient "apoB-48-only" and "apoB-100-only" mice.

Hepatic lipase clears plasma cholesterol by lipolytic and nonlipolytic processing of lipoproteins. We hypothesized that the nonlipolytic processing (known as the bridging function) clears cholesterol by removing apoB-48- and apoB-100-containing lipoproteins by whole particle uptake. To test our hypotheses, we expressed catalytically inactive human HL (ciHL) in LDL receptor deficient "apoB-48-only" and "apoB-100-only" mice. Expression of ciHL in "apoB-48-only" mice reduced cholesterol by reducing LDL-C (by 54%, 46 +/- 6 vs. 19 +/- 8 mg/dl, P < 0.001). ApoB-48 was similarly reduced (by 60%). The similar reductions in LDL-C and apoB-48 indicate cholesterol removal by whole particle uptake. Expression of ciHL in "apoB-100-only" mice reduced cholesterol by reducing IDL-C (by 37%, 61 +/- 19 vs. 38 +/- 12 mg/dl, P < 0.003). Apo-B100 was also reduced (by 27%). The contribution of nutritional influences was examined with a high-fat diet challenge in the "apoB-100-only" background. On the high fat diet, ciHL reduced IDL-C (by 30%, 355 +/- 72 vs. 257 +/- 64 mg/dl, P < 0.04) but did not reduce apoB-100. The reduction in IDL-C in excess of apoB-100 suggests removal either by selective cholesteryl ester uptake, or by selective removal of larger, cholesteryl ester-enriched particles. Our results demonstrate that the bridging function removes apoB-48- and apoB-100-containing lipoproteins by whole particle uptake and other mechanisms.

Molecular cloning of nonsecreted endothelial cell-derived lipase isoforms.

To expand our knowledge of factors involved in lipid metabolism in the blood vessel wall, we have cloned unique molecular isoforms of endothelial cell-derived lipase (EDL) (HGMW-approved symbol/LIPG). One isoform encoded a truncated protein (EDL2a) lacking the first 80 amino acid residues of the previously characterized EDL1a isoform, including the signal peptide. A similar second clone (EDL2b) was identified that lacked not only the first 80 amino acids, but also a 74-amino-acid region that encodes a portion of the lid domain. RT-PCR analysis confirmed expression of EDL2a/2b isoforms in several human tissues and cultured cells, including endothelial cells. Western blot and immunofluorescence studies using stable transfectants revealed that EDL2a and EDL2b were localized in the cytosol, while, EDL1a was secreted into the culture medium. Cell extracts of EDL2a/2b transfectants did not have triglyceride or phospholipase activity. Thus endothelial cells express three EDL isoforms, two of which remain intracellular and do not function as lipases.