Delayed wound repair and impaired angiogenesis in mice lacking syndecan-4

The syndecans make up a family of transmembrane heparan sulfate proteoglycans that act as coreceptors with integrins and growth factor tyrosine kinase receptors. Syndecan-4 is upregulated in skin dermis after wounding, and, in cultured fibroblasts adherent to the ECM protein fibronectin, this proteoglycan signals cooperatively with beta1 integrins. In this study, we generated mice in which the syndecan-4 gene was disrupted by homologous recombination in embryonic stem cells to test the hypothesis that syndecan-4 contributes to wound repair. Mice heterozygous or homozygous for the disrupted syndecan-4 gene are viable, fertile, and macroscopically indistinguishable from wild-type littermates. Compared with wild-type littermates, mice heterozygous or homozygous for the disrupted gene have statistically significant delayed healing of skin wounds and impaired angiogenesis in the granulation tissue. These results indicate that syndecan-4 is an important cell-surface receptor in wound healing and angiogenesis and that syndecan-4 is haplo-insufficient in these processes.     

Reverse cholesterol transport: relationship between free cholesterol uptake and HDL3 in normolipidaemic and hyperlipidaemic subjects

Abstract. High density lipoproteins (HDL) are responsible for the Reverse Cholesterol Transport (RCT). The role of the composition of the HDL particle in RCT, involving free cholesterol (chol) uptake from cell membranes, is not completely understood. We have therefore studied the uptake capacity from subjects with a wide variety of plasma HDL cholesterol concentrations in an HDL-receptor free model consisting of bovine heart mitochondrial membranes labeled with [¹⁴C]cholesterol. HDL were isolated by molecular sieve chromatography from fresh plasma samples of eight subjects with low plasma HDL chol concentrations (≤ 1.0 mmol L-¹) and 15 subjects with normal HDL chol concentrations. The latter were subdivided into an intermediate (HDL chol: 1.0–1.4 mmol L^-1; n= 9) and a high HDL chol group (>1.4 mmol L-¹; n= 6). In the HDL fractions isolated by chromatography (cHDL), total chol and apolipopro-tein (apo) A1 were measured. Free chol uptake was significantly decreased by 32% in the tertile with the lowest plasma HDL chol (49 1 ± 15.8 arbitrary units; mean ± SD), compared to the tertile with high HDL chol (72.1 ± 16.6 au). Linear regression analysis showed a positive correlation between the free choi uptake and plasma HDL3 concentrations (r= 0.61; P<0.01), HDL chol (r= 0.56; P<0.01), HDL associated apo A1 (r = 0.46; P<0.05), cHDL apo AT (r = 0.56; P<0.05) and cHDL chol (r = 0.46; P<0.05) in all subjects combined. Stepwise multiple-regression analysis confirmed the association of [¹⁴C]cholesterol uptake with plasma HDL3 concentrations (β, 061; P= 0.004). No correlations were found between free chol uptake and total plasma apoAI (r = 0.26; ns) or HDL2 (r = 0.27; ns). After an oral fat load in four FCH patients, free chol uptake parallelled the changes in plasma HDL3 chol concentrations. We conclude that HDL3 is involved in the early steps of RCT and low HDL3 levels may result in less efficient RCT in hypertriglyceridemia.     

Serum paraoxonase-1 activity is more closely related to HDL particle concentration and large HDL particles than to HDL cholesterol in Type 2 diabetic and non-diabetic subjects

Objectives

We determined relationships of the anti-oxidative enzyme, paraoxonase-1 (PON-1), with high density lipoprotein (HDL) subfractions, and tested whether these relationships are stronger than those with HDL cholesterol and apolipoprotein A-I (apoA-I) in subjects with and without type 2 diabetes mellitus (T2DM).

Design and methods

Serum PON-1 (arylesterase activity) and HDL subfractions (nuclear magnetic resonance spectroscopy) were determined in 67 T2DM patients and in 56 non-diabetic subjects.

Results

PON-1 activity, HDL cholesterol and apoA-I were decreased in T2DM (all p < 0.05). The HDL particle concentration was unaltered, but large HDL particles, medium HDL particles and HDL particle size were decreased, whereas small HDL particles were increased in T2DM (all p < 0.05). PON-1 was more closely related to HDL cholesterol than to apoA-I (p = 0.001). In turn, the positive relationship of PON-1 with the HDL particle concentration and with large HDL particles was stronger than that with HDL cholesterol (both p < 0.01). The inverse relationship of PON-1 with T2DM was only modestly attenuated by HDL cholesterol or HDL particle characteristics.

Conclusions

PON-1 activity is more closely related to the HDL particle concentration or large HDL particles than to HDL cholesterol. Impaired PON-1 activity in T2DM is not to a considerable extent explained by altered HDL subfraction levels.     

Hypertension, hypertriglyceridemia, and impaired endothelium-dependent vascular relaxation in mice lacking insulin receptor substrate-1.

Insulin resistance is often associated with atherosclerotic diseases in subjects with obesity and impaired glucose tolerance. This study examined the effects of insulin resistance on coronary risk factors in IRS-1 deficient mice, a nonobese animal model of insulin resistance. Blood pressure and plasma triglyceride levels were significantly higher in IRS-1 deficient mice than in normal mice. Impaired endothelium-dependent vascular relaxation was also observed in IRS-1 deficient mice. Furthermore, lipoprotein lipase activity was lower than in normal mice, suggesting impaired lipolysis to be involved in the increase in plasma triglyceride levels under insulin-resistant conditions. Thus, insulin resistance plays an important role in the clustering of coronary risk factors which may accelerate the progression of atherosclerosis in subjects with insulin resistance.     

Low HDL cholesterol concentration is associated with increased intima-media thickness independent of arterial stiffness in healthy subjects from families with low HDL cholesterol

BACKGROUND: Low high-density lipoprotein cholesterol (HDL-C) is associated with increased risk for developing coronary artery disease. Cardiovascular disease is characterized by increased intima-media thickness (IMT) and arterial stiffness, but the effect of low HDL on these measurements has not been reported. MATERIALS AND METHODS: We studied 18 apparently healthy subjects from families with low HDL-C and 18 control subjects, which were pair-matched to maximize statistical power. Intima-media thickness was assessed using ultrasound examination of the carotid arteries. Arterial stiffness was measured using applanation tonometry on the radial artery and pulse-wave analysis to obtain central aortic pulse-pressure waveform, from which the augmentation index, a measure of global large artery stiffness, was calculated. RESULTS: Low HDL subjects (age 41 +/- 3 years, BMI 26.6 +/- 1.0 kg m(-2) had significantly lower HDL-C than the control subjects (age 41 +/- 3 years, BMI 26.5 +/- 1.0 kg m-2; 1.00 +/- 0.05 vs. 1.49 +/- 0.09 mmol L-1, low HDL vs. control subjects, P < 0.0001). Subjects with low HDL-C had significantly thicker mean IMTs than the control subjects (0.77 +/- 0.03 vs. 0.70 +/- 0.02 mm, low HDL vs. control subjects, P < 0.01). The maximal (0.99 +/- 0.04 vs. 0.89 +/- 0.03 mm, P < 0.01), far wall (0.76 +/- 0.04 vs. 0.69 +/- 0.02 mm, P < 0.05) and carotid bulb (1.11 +/- 0.06 vs. 0.97 +/- 0.04 mm) IMTs were also significantly increased, whereas the mean common carotid and the internal artery IMT were not. The age-related increase in mean IMT was more pronounced in the low HDL subjects than the control subjects (P < 0.01 for difference between elevations of age vs. IMT slopes). There were no differences in central pressure augmentation, the augmentation index, peripheral or central blood pressures between the groups. CONCLUSIONS: A low HDL-C concentration is associated with thickening of carotid IMT independent of other risk factors in healthy affected members of low HDL families.     

The ability of HDL to inhibit VCAM-1 expression and oxidized LDL uptake is impaired in renal patients

ObjectivesThis study examines the ability of HDL from hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) patients to suppress the expression of adhesion molecules in endothelial cells (ICAM-1, VCAM-1) and in monocytes (LFA-1, VLA-4) and to inhibit the uptake of oxidized LDL by macrophages.Design and methodsGene expression and the uptake of oxidized LDL were determined in 12 HD patients, 12 CAPD patients and 14 healthy volunteers.ResultsHDL from renal patients were less effective than control lipoproteins in reducing VCAM-1 expression. HDL from CAPD patients inhibited LFA-1 expression to the highest extent. The ability of HDL from renal patients to reduce oxidized LDL uptake was lower compared to control group.ConclusionsDecreased ability of HDL to suppress expression of VCAM-1 in endothelial cells and the uptake of oxidized LDL by macrophages can be one of the risk factors for atherosclerosis development in patients with renal failure.     

Nociception in pigeons is not impaired by capsaicin

In order to examine the sensitivity of the nociceptive system of birds to capsaicin, the algesic potency of the drug was compared with that of veratridine and mustard oil by instillation into the eye of conscious pigeons and guinea-pigs. In guinea-pigs, 10(-6) g/ml capsaicin provoked severe protective reactions, but even high concentrations of 10(-2) g/ml were insufficient in pigeons. Veratridine and mustard oil induced similar reactions in both species. Close arterial injections of algesic substances revealed that the threshold dose of capsaicin for cardiovascular and nocifensive reactions was 10,000-fold higher in pigeons (200 micrograms) than in guinea-pigs (0.02 micrograms). All other algesic substances tested (bradykinin, 5-HT, veratridine and KCl) had similar thresholds in both species. Slow infusion of a total dose of 600 mg/kg capsaicin into the radial artery of pigeons did not alter the sensitivity to any of the algesic substances tested, which demonstrates that even high concentrations of capsaicin have no desensitizing effect. The demonstrated insensitivity of pigeons to the algesic effect of capsaicin is discussed in the context of the inability of the drug to deplete substance P (SP) from afferent terminals in the spinal cord of the pigeon.     

Diminished rate of mouse peritoneal macrophage cholesterol efflux is not related to the degree of HDL glycation in diabetes mellitus

The efflux of (14)C-cholesterol from mouse peritoneal macrophages mediated by in vivo and in vitro glycation of intact HDL(3) and by HDL(3) apolipoproteins was investigated. Cholesterol-laden cells were incubated a long time with HDL(3) from control subjects (C), poorly controlled diabetes mellitus patients (D) and with HDL C submitted to in vitro glycation (G), as well as with all their respectively isolated apolipoproteins. A diminished cholesterol efflux rate occurred in incubations with intact HDL(3) D but not with intact HDL(3)G or with apoHDL(3)C, G or D. The specific binding of (125)I-HDL(3)G to the cell receptor, obtained upon incubation in the absence and in the presence of excess unlabelled HDL(3), was lower than the control. The role of apoE secretion by cholesterol-laden macrophages on cholesterol efflux was analyzed by incubating apoE knockout and control mice macrophages with HDL C or HDL G: a lower cholesterol efflux was observed from apoE knockout macrophages but glycation of HDL(3) did not influence this process either. The diminished capacity to remove cholesterol by the HDL drawn from diabetic subjects must be attributed to other modifications of the lipoproteins, except for non enzymatic glycation. Thus, events that impair the cell cholesterol removal in diabetes mellitus are multifaceted.     

A low-fat diet decreases high density lipoprotein (HDL) cholesterol levels by decreasing HDL apolipoprotein transport rates.

Diets that reduce atherosclerosis risk lower levels of HDL cholesterol (HDL-C), but the significance of this is unclear. To better understand the mechanism of this phenomenon we studied the turnover of HDL apolipoproteins A-I and A-II in 13 subjects on two contrasting metabolic diets. Upon changing from high to low intake of saturated fat and cholesterol the mean HDL-C decreased 29% from 56 +/- 13 (SD) to 40 +/- 10 mg/dl, while apo A-I levels fell 23% from 139 +/- 22 to 107 +/- 22 mg/dl (both P less than 0.001). Mean apo A-II levels did not change. The fractional catabolic rate (FCR) of apo A-I increased 11% from 0.228 +/- 0.048 to 0.254 +/- 0.063 pools/d, while its absolute transport rate (TR) decreased 14% from 12.0 +/- 2.7 to 10.3 +/- 3.4 mg/kg per d (both P = 0.005). The decrease in HDL-C and apo A-I levels correlated with the decrease in apo A-I TR (r = 0.79 and 0.83, respectively; P less than 0.001), but not with the increase in apo A-I FCR (r = -0.04 and -0.02, respectively). In contrast, within each diet the HDL-C and apo A-I levels were inversely correlated with apo A-I FCR both on the high-fat (r = -0.85 and -0.77, P less than 0.001 and = 0.002, respectively) and low-fat diets (r = -0.67 and -0.48, P = 0.012 and 0.098, respectively) but not with apo A-I TR. In summary, diet-induced changes in HDL-C levels correlate with and may result from changes in apo A-I TR. In contrast, differences in HDL-C levels between people on a given diet correlate with and may result from differences in apo A-I FCR. Therefore, the mechanism of dietary effects on HDL levels differs substantially from the mechanism explaining the differences in levels between individuals on a fixed diet. In assessing coronary heart disease risk, it may be inappropriate to conclude that diet-induced decreases in HDL are equivalent to low HDL within a given diet.     

Ratio of triglycerides to HDL cholesterol is an indicator of LDL particle size in patients with type 2 diabetes and normal HDL cholesterol levels

OBJECTIVE: In patients with type 2 diabetes, a normal HDL cholesterol level does not rule out that LDL particles may be small. Although techniques for analyzing LDL subfractions are not likely to be used in clinical practice, a prediction of LDL size based on a regular lipid profile may be useful for assessment of cardiovascular risk. RESEARCH DESIGN AND METHODS: Sixty patients with type 2 diabetes with acceptable glycemic control and an HDL cholesterol level > or = 1 mmol/l were recruited after cessation of lipid-altering treatments. LDL size was determined by 2-20% PAGE; patients having small LDL (n = 30) were compared with those having intermediate or large LDL (n = 30). RESULTS: Clinical characteristics, pharmacological therapies, lifestyle, and prevalence of diabetes-related complications were similar in both patient groups. LDL size correlated negatively with plasma triglycerides (TGs) (R2 = 0.52) and positively with HDL cholesterol (R2 = 0.14). However, an inverse correlation between the TG-to-HDL cholesterol molar ratio and LDL size was even stronger (R2 = 0.59). The ratio was > 1.33 in 90% of the patients with small LDL particles (95% CI 79.3-100) and 16.5% of those with larger LDL particles. A cutoff point of 1.33 for the TG-to-HDL cholesterol ratio distinguishes between patients having small LDL values better than TG cutoff of 1.70 and 1.45 mmol/l. CONCLUSIONS: The TG-to-HDL cholesterol ratio may be related to the processes involved in LDL size pathophysiology and relevant with regard to the risk of clinical vascular disease. It may be suitable for the selection of patients needing an earlier and aggressive treatment of lipid abnormalities.     

Massive xanthomatosis and altered composition of atherosclerotic lesions in hyperlipidemic mice lacking acyl CoA:cholesterol acyltransferase 1

Inhibitors of acyl CoA:cholesterol acyltransferase (ACAT) have attracted considerable interest as a potential treatment for atherosclerosis. Currently available inhibitors probably act nonselectively against the two known ACATs. One of these enzymes, ACAT1, is highly expressed in macrophages in atherosclerotic lesions, where it contributes to foam-cell formation. In this study, we examined the effects of selective ACAT1 deficiency in two mouse models of atherosclerosis. In the setting of severe hypercholesterolemia caused by deficiency in apoE or the LDL receptor (LDLR), total ACAT1 deficiency led to marked alterations in cholesterol homeostasis and extensive deposition of unesterified cholesterol in the skin and brain. Bone marrow transplantation experiments demonstrated that ACAT1 deficiency in macrophages was sufficient to cause dermal xanthomas in hyperlipidemic LDLR-deficient mice. ACAT1 deficiency did not prevent the development of atherosclerotic lesions in either apoE-deficient or LDLR-deficient mice, despite causing relatively lower serum cholesterol levels. However, the lesions in ACAT1-deficient mice were atypical in composition, with reduced amounts of neutral lipids and a paucity of macrophages in advanced lesions. Although the latter findings may be associated with increased lesion stability, the marked alterations in cholesterol homeostasis indicate that selectively inhibiting ACAT1 in the setting of severe hyperlipidemia may have detrimental consequences.     

Altered hepatic transport of immunoglobulin A in mice lacking the J chain

We have created J chain knockout mice to define the physiologic role of the J chain in immunoglobulin synthesis and transport. The J chain is covalently associated with pentameric immunoglobulin (Ig) M and dimeric IgA and is also expressed in most IgG-secreting cells. J chain- deficient mice have normal serum IgM and IgG levels but markedly elevated serum IgA. Although polymeric IgA was present in the mutant mice, a larger proportion of their serum IgA was monomeric than was found in wild-type mouse serum. Bile and fecal IgA levels were decreased in J chain-deficient mice compared with wild-type mice, suggesting inefficient transport of J chain-deficient IgA by hepatic polymeric immunoglobulin receptors (pIgR). The pIgR-mediated transport of serum-derived IgA from wild-type and mutant mice was assessed in Madin-Darby canine kidney (MDCK) cells transfected with the pIgR. These studies revealed selective transport by pIgR-expressing MDCK cells of wild-type IgA but not J chain-deficient IgA. We conclude that although the J chain is not required for IgA dimerization, it does affect the efficiency of polymerization or have a role in maintaining IgA dimer stability. Furthermore, the J chain is essential for efficient hepatic pIgR transport of IgA.     

Age and residual cholesterol efflux affect HDL cholesterol levels and coronary artery disease in ABCA1 heterozygotes

We and others have recently identified mutations in the ABCA1 gene as the underlying cause of Tangier disease (TD) and of a dominantly inherited form of familial hypoalphalipoproteinemia (FHA) associated with reduced cholesterol efflux. We have now identified 13 ABCA1 mutations in 11 families (five TD, six FHA) and have examined the phenotypes of 77 individuals heterozygous for mutations in the ABCA1 gene. ABCA1 heterozygotes have decreased HDL cholesterol (HDL-C) and increased triglycerides. Age is an important modifier of the phenotype in heterozygotes, with a higher proportion of heterozygotes aged 30-70 years having HDL-C greater than the fifth percentile for age and sex compared with carriers less than 30 years of age. Levels of cholesterol efflux are highly correlated with HDL-C levels, accounting for 82% of its variation. Each 8% change in ABCA1-mediated efflux is predicted to be associated with a 0.1 mmol/l change in HDL-C. ABCA1 heterozygotes display a greater than threefold increase in the frequency of coronary artery disease (CAD), with earlier onset than unaffected family members. CAD is more frequent in those heterozygotes with lower cholesterol efflux values. These data provide direct evidence that impairment of cholesterol efflux and consequently reverse cholesterol transport is associated with reduced plasma HDL-C levels and increased risk of CAD.     

Macrophage ABCA1 and ABCG1, but not SR-BI, promote macrophage reverse cholesterol transport in vivo

Macrophage ATP-binding cassette transporter A1 (ABCA1), scavenger receptor class B type I (SR-BI), and ABCG1 have been shown to promote cholesterol efflux to extracellular acceptors in vitro and influence atherosclerosis in mice, but their roles in mediating reverse cholesterol transport (RCT) from macrophages in vivo are unknown. Using an assay of macrophage RCT in mice, we found that primary macrophages lacking ABCA1 had a significant reduction in macrophage RCT in vivo, demonstrating the importance of ABCA1 in promoting macrophage RCT, however substantial residual RCT exists in the absence of macrophage ABCA1. Using primary macrophages deficient in SR-BI expression, we found that macrophage SR-BI, which was shown to promote cholesterol efflux in vitro, does not contribute to macrophage RCT in vivo. To investigate whether macrophage ABCG1 is involved in macrophage RCT in vivo, we used ABCG1-overexpressing, -knockdown, and -knockout macrophages. We show that increased macrophage ABCG1 expression significantly promoted while knockdown or knockout of macrophage ABCG1 expression significantly reduced macrophage RCT in vivo. Finally, we show that there was a greater decrease in macrophage RCT from cells where both ABCA1 and ABCG1 expression were knocked down than from ABCG1-knockdown cells. These results demonstrate that ABCA1 and ABCG1, but not SR-BI, promote macrophage RCT in vivo and are additive in their effects.