Dyslipidaemia and lipoprotein pattern in systemic lupus erythematosus (SLE) and SLE-related cardiovascular disease.

OBJECTIVE: This study focused on lipoprotein composition and properties in systemic lupus erythematosus (SLE). METHODS: The size distribution of plasma lipoproteins was studied by nuclear magnetic resonance (NMR). Cholesteryl ester transfer protein (CETP) activity was determined by enzyme-linked immunosorbent assay (ELISA). The affinity of low density lipoprotein (LDL) for proteoglycans was assayed. Twenty-six women (aged 52+/-8.2 years) with SLE and a history of cardiovascular disease (CVD) (SLE cases) were compared with 26 age-matched women with SLE and without CVD (SLE controls) and 26 age-matched population-based control women (controls). RESULTS: Very low density lipoprotein (VLDL) particles (nmol/L) were more prevalent among SLE cases than SLE controls (0.039) and tended to be more common in SLE cases than in controls (p = 0.073). By contrast, high density lipoprotein (HDL) particles (nmol/L) were more prevalent among controls than SLE cases (p = 0.024) whereas the number of LDL particles (nmol/L) did not differ significantly. Small dense (sd)LDL (nmol/L) were more common in controls and tended to be more common in SLE cases than in SLE controls (p = 0.036 and 0.086, respectively). Small high density lipoproteins (sHDL) (nmol/L) were more prevalent in controls than in SLE controls and SLE cases (p = 0.002 and p<0.001, respectively). VLDL or LDL size (nm) did not differ significantly between groups (data not shown) whereas HDL size (nm) was increased among SLE controls as compared to controls (p = 0.024) and tended to be increased among SLE cases as compared to controls (p = 0.070). The affinity of LDL for proteoglycans or CETP activity did not differ between groups (data not shown). CONCLUSIONS: sdLDL was not increased and SLE cases and SLE controls had decreased levels of sHDL. VLDL differentiates between SLE cases and SLE controls. The lipid pattern in SLE-related CVD was thus not similar to the pattern seen in diabetes or in CVD in general.

Increased lipolysis by secretory phospholipase A(2) group V of lipoproteins in diabetic dyslipidaemia.

BACKGROUND: Lipolysis of lipoproteins by secretory phospholipase A(2) group V (sPLA(2)-V) promotes inflammation, lipoprotein aggregation and foam cell formation--all considered as atherogenic mechanisms. OBJECTIVE: In this study, we compared the susceptibility to sPLA(2)-V lipolysis of VLDL and LDL from individuals with type 2 diabetes and the metabolic syndrome (T2D-MetS) and from healthy controls. Design. VLDL and LDL were isolated from 38 T2D-MetS subjects and 38 controls, treated pair-wise. Extent of sPLA(2)-V lipolysis was measured as release of nonesterified free fatty acids (NEFA). In a subset of the subjects, lipoprotein composition was determined as a relationship between lipid and apolipoprotein components. RESULTS: Mean paired increase in sPLA(2)-V lipolysis after 1 h for T2D-MetS versus control was 2.0 micromol NEFA l(-1) for VLDL (P = 0.004) and 0.75 micromol NEFA l(-1) for LDL (P = 0.001). There were also substantial differences in lipoprotein composition between the groups. T2D-MetS VLDL had higher triglyceride and cholesterol contents than control VLDL. T2D-MetS LDL was smaller and contained more triglycerides and less cholesterol than control LDL. Both VLDL and LDL from T2D-MetS subjects also contained more apolipoprotein CIII per particle. CONCLUSION: VLDL and LDL from T2D-MetS individuals were more susceptible to sPLA(2)-V lipolysis than those from control individuals. This may result in elevated levels of NEFA and lysophosphatidylcholine, both in circulation and in LDL, possibly contributing to the elevated inflammatory state and increased risk of cardiovascular diseases seen in these individuals.

Reduced cholesterol accumulation by leptin deficient (ob/ob) mouse macrophages.

OBJECTIVE: Although presenting many aspects of the metabolic syndrome, leptin deficient (ob/ob) mice do not spontaneously develop atherosclerosis. To examine the role of leptin in foam cell formation we analyzed ob/ob leukocyte inflammation markers and macrophage cholesterol accumulation. METHODS: Resident and thioglycollate (TG) elicited peritoneal cells of ob/ob and wildtype mice were studied. Activation markers, scavenger receptors (SR) and cholesterol accumulation were analyzed using flow cytometry and Taqman analysis. Cytokines, haptoglobin, adiponectin and amyloid A levels were analyzed with ELISA. RESULTS: Macrophages of ob/ob mice had reduced expression of MHC class II, CD11b, CD40, SR-A and CD36 and reduced cholesterol accumulation in vitro. Plasma haptoglobin was increased and T-cell IFNgamma was reduced in ob/ob mice. Peritoneal TG instillation induced an unexpectedly weak inflammatory response in ob/ob mice. CONCLUSIONS: The ob/ob mice had a reduced inflammatory response and reduced macrophage cholesterol accumulation in vitro. The data suggest decreased foam cell formation and atherosclerosis development in ob/ob mice.

Low-grade inflammation, endothelial activation and carotid intima-media thickness in type 2 diabetes.

OBJECTIVES: The objective of this study was to assess the relationship between inflammation, endothelial activation and incipient atherosclerosis in type 2 diabetes. DESIGN: Cross-sectional study. Setting and subjects. We studied 239 type 2 diabetic patients [71 with clinical cardiovascular disease (CVD)] and 78 healthy control subjects, aged 50-75 in a single research centre. METHODS: Carotid intima-media thickness (IMT) was determined by ultrasound. Circulating intracellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, ultra-sensitive C-reactive protein, human serum amyloid A, interleukin-6, monocyte colony-stimulating factor, secretory nonpancreatic phospholipase A(2) type IIA, glucose, HbA1c, and lipid/lipoprotein variables were measured. RESULTS: Carotid IMT was significantly thicker in diabetic patients than healthy controls across the whole age range. IMT was also thicker in diabetic patients with, than without, CVD, but this difference disappeared after controlling for confounding factors. Concentrations of the inflammatory and endothelial markers except IL-6 were significantly higher in the diabetic patients than in healthy controls, but comparable in diabetic patients with and without CVD. The main determinants of IMT in the diabetic patients were blood pressure, age and diabetes duration. CONCLUSIONS: Low-grade inflammation and endothelial activation are increased in diabetic patients but do not associate with IMT or clinical CVD. The inflammatory reaction seems to be rather a feature of the metabolic syndrome than a direct determinant of atherosclerosis.

Effects of simvastatin and atorvastatin on inflammation markers in plasma.

OBJECTIVES: To study the effect of statins on plasma markers for inflammation. DESIGN: Patients with hypercholesterolemia were randomized in one of the following treatments: Simvastatin (S) + placebo: S 40 mg for 6 weeks - S 80 mg for 6 weeks - S 80 mg for 24 weeks and Atorvastatin (A) + placebo: A 20 mg for 6 weeks - A 40 mg for 6 weeks - A 80 mg for 24 weeks. SUBJECTS: Forty-seven patients with hypercholesterolemia were recruited in four different outpatient clinics. MAIN OUTCOME MEASURES: Samples were obtained at randomization after 6, 12 and 36 weeks. Plasma or serum was analysed for lipids and for inflammation markers: C-reactive protein (CRP), serum amyloid A (SAA), soluble phospholipase A2 (SPLA2), intercellular adhesion molecule-1 (ICAM-1) and interleukin-6 (IL-6). RESULTS: The reduction in LDL was similar for the two statins, except at the highest dose of atorvastatin (41 vs. 47%). The increase in HDL tended to be more pronounced in the simvastatin group, significantly so on the highest dose of atorvastatin (P < 0.05). CRP and SAA was significantly reduced by atorvastatin, whilst no reduction was seen for simvastatin. There was a significant difference in treatment effects between the two statins. Both statins caused a reduction in SPLA2. For IL-6 and ICAM-1 only small and inconsistent reductions were observed for both statins. CONCLUSION: Atorvastatin reduced the liver-derived acute-phase reactants, CRP and SAA, whilst the effect of simvastatin was small or absent. Small and inconsistent effects were seen for both statins on plasma levels of IL-6 and ICAM-1.

Intravascular hemolysis increases atherogenicity of diet-induced hypercholesterolemia in rabbits in spite of heme oxygenase-1 gene and protein induction.

Free radical mediated oxidation of apoB lipoproteins in the arterial intima appears to contribute to atherogenicity of the entrapped particles. A plausible pathogenic mechanism for oxidation is the one induced by heme leaking from erythrocytes that is then carried into the arterial wall by its high affinity for lipoproteins. In the intima, in the presence of H(2)O(2) secreted by macrophages, heme can be a potent oxidant. To study the role of heme as a promoter of oxidative stress damage in vivo we used a model of intravascular hemolysis (IVH) caused by phenylhydrazine in rabbits with and without diet-induced moderate hypercholesterolemia (MHC). Evaluation of the antioxidant status of plasma indicated that at the end of the treatment period this was compromised by the MHC-IVH. After 10 weeks the animals with combined MHC-IVH showed more of the aorta surface covered by lesions (27%+/-8, mean (SD) than the animals with only MHC (11%+/-7), in spite of having similar plasma levels of VLDL+LDL lipoproteins. The animals with only IVH, as well as the controls, showed minimal lesions (<1%). Heme oxygenase (HO-1) expression in aorta and other tissues was markedly increased in the group with MHC-IVH and it was correlated with the extent of IVH. The data suggest that the oxidative stress associated with IVH potentiates the atherogenicity of moderate hypercholesterolemia and that in spite of a strong induction of HO-1 this is not sufficient to counteract the atherogenicity of the combined condition.

Phospholipase A(2) in vascular disease.

Secretory phospholipase A(2) (PLA(2)) can be proatherogenic both in the circulation and in the arterial wall. In blood plasma, PLA(2) can modify the circulating lipoproteins and so induce formation of small dense LDL particles, which are associated with increased risk for cardiovascular disease. In the arterial wall, PLA(2) can hydrolyze lipoproteins. The PLA(2)-modified lipoproteins bind tightly to extracellular proteoglycans, which may lead to their enhanced retention in the arterial wall. The modified lipoproteins may also aggregate and fuse, which can lead to accumulation of their lipids within the extracellular matrix. The PLA(2)-modified particles are more susceptible to further modifications by other enzymes and agents and can be taken up by macrophages, leading to accumulation of intracellular lipids. In addition, lysophospholipids and free fatty acids, the hydrolysis products of PLA(2), promote atherogenesis. Thus, these lipid mediators can be carried, either by the PLA(2)-modified lipoproteins themselves or by albumin, into the arterial cells, which then undergo functional alterations. This may, in turn, lead to specific changes in the extracellular matrix, which increase the retention and accumulation of lipoproteins within the matrix. In the present article, we discuss the possible actions of PLA(2) enzymes, especially PLA(2)-IIA, in the arterial wall during atherogenesis.

[Why are plasmatic apoB lipoproteins atherogenic? The hypothesis of response to retention]. / Por qué son aterogénicas las lipoproteínas apoB del plasma: la hipótesis de respuesta a la retención.

The development of atherosclerotic lesions can be described as a tissular response to deposition of apoB-100 containing lipoproteins (LpApoB) in the arterial intima. These particles that circulate in blood are the low-density lipoproteins (LDL), the very low and intermediate density lipoproteins (VLDL and IDL) and the Lp(a). To initiate the tissue response is critic that LpApoB are retained in the subendothelial space. This occurs by the interaction of specific positive segments of the apoB-100 with negative glycosaminoglycan chains of the proteoglycans of the intima extracellular matrix. The inflammatory response involve macrophages and other immuno-competent cells, smooth muscle cells and endothelial cells. The direct agents that induce this reaction appear to be products of hydrolytic and oxidative modifications of the phospholipids, triglycerides and sterols of the retained LpApoB. The enzymes that cause these modifications are secreted by arterial cells stimulated by pro-inflammatory cytokines. The sequence of LpApoB retention and modification in the extracellular matrix followed by proliferation and inflammation seems to be a cyclic process that leads to the chronic progress of atherosclerotic lesions. These concepts constitute the base of a new hypothesis to explain atherosclerosis: the "response to retention hypothesis".

Lipolysis of LDL by human secretory phospholipase A(2) induces particle fusion and enhances the retention of LDL to human aortic proteoglycans.

The first morphological sign of atherogenesis is the accumulation of extracellular lipid droplets in the proteoglycan-rich subendothelial layer of the arterial intima. Secretory nonpancreatic phospholipase A(2) (snpPLA(2)), an enzyme capable of lipolyzing LDL particles, is found in the arterial extracellular matrix and in contact with the extracellular lipid droplets. We have recently shown that in the presence of heparin, lipolysis of LDL with bee venom PLA(2) induces aggregation and fusion of the particles. Here, we studied the effect of human snpPLA(2) on the integrity of LDL particles and on their interaction with human aortic proteoglycans. In addition, the capacity of the proteoglycans to retain PLA(2)-lipolyzed LDL particles was tested in a microtiter well assay. We found that lipolysis of LDL induced fusion of proteoglycan-bound LDL particles, which increased their binding strength to the proteoglycans. Moreover, lipolysis of LDL with snpPLA(2) under physiological salt and albumin concentrations induced a 3-fold increase in the amount of LDL bound to proteoglycans. The results imply a role for PLA(2) in the retention and accumulation of LDL to the proteoglycan matrix in atherosclerosis.

Cell adhesion molecules and secretory type II phospholipase A2 in relation to carotid atherosclerosis in patients with hypercholesterolaemia.

OBJECTIVE: There has been no previous study investigating the relationship between subclinical atherosclerosis, as measured by ultrasound, and circulating levels of cell-adhesion molecules and snpPLA2 in patients with hypercholesterolaemia. The main aims of the present study were therefore to investigate the relationship between subclinical atherosclerosis, cell-adhesion molecules and snpPLA2 in a group of subjects with hypercholesterolaemia and also in a healthy control group; and to investigate the relationship between conventional risk factors, cell-adhesion molecules and snpPLA2 in these groups. METHODS: Study subjects were recruited from the general population (50 subjects with hypercholesterolaemia and all 105 controls) and from the lipid clinic of Sahlgrens Hospital, Gothenburg (55 subjects with hypercholesterolaemia). Cell-adhesion molecules and snpPLA2 were analyzed by ELISA. RESULTS: The levels of sICAM turned out to be positively and significantly associated with plaque occurrence and plaque size in the carotid artery in the patient group, but not in the control group. There were no significant relationships between sVCAM, sE-selectin or snpPLA2 and atherosclerosis, as measured by ultrasound. However, sICAM was significantly associated with sVCAM, sE-selectin and snpPLA2 both in the patient and in the control groups. In the patient group sICAM was positively and significantly associated with triglyceride levels and also negatively associated with high density lipoprotein (HDL) levels. CONCLUSIONS: The present study showed that sICAM-1 was significantly associated with atherosclerosis, as measured by ultrasound, in the carotid artery in patients with hypercholesterolaemia. Furthermore, sICAM-1 levels were associated with plasma levels of snpPLA2 (r=0.44, P < 0.001), which recently have been shown to predict coronary events. Finally, high sICAM-1 levels were associated with an atherogenic lipid profile including high triglyceride levels and low HDL levels.

Low-density lipoprotein (LDL) binds to a G-protein coupled receptor in human platelets. Evidence that the proaggregatory effect induced by LDL is modulated by down-regulation of binding sites and desensitization of its mediated signaling.

We present evidence of a link between low-density lipoprotein (LDL) receptor binding and activation of a platelet G-coupled protein. LDL stimulation induced cytosolic [Ca2+]i mobilization, increase in inositol 1,4,5-triphosphate (IP3) formation and a rapid cytosol-to-membrane translocation of protein kinase C (PKC) enzymatic activity. Pertussis toxin inhibited all the stimulatory effects, whereas cholera toxin had no effect. Using ligand-binding assays, we demonstrated that exposing platelet LDL receptors to high concentrations of LDL (1.5 g/l) caused a rapid down-regulation and desensitization, as shown by the reduction in the Bmax, intracellular [Ca2+]i mobilization and IP3 formation to 65, 73 and 63%, respectively. The inhibitory effects were reversible and dose and time dependent. Furthermore, VLDL (0.2 g/l) and IDL (0.07 g/l) induced similar desensitization effects. However, HDL3 (up to 1.5 g/l), chylomicrons (up to 0.5 g/l) and cyclohexandione-modified LDL (which does not bind to platelets) had no significant effects. Protein kinase C inhibitors (150 nmol/l staurosporine, 100 micromol/l H-7, and 10 nmol/l bisindolylmaleimide) inhibited desensitization to 71%, on average. Sequestration blocking agents (0.30 g/l, concanavalin A) had no significant effect if phosphorylation was operative. However, there was a complete blockade with the concurrent inhibition of both pathways. In contrast, cAMP-dependent protein kinase inhibitors (PKI, 1 micromol/l) or beta2-adrenergic receptor kinase inhibitors (100 nmol/l, heparin), had no effect. Overall results indicate that LDL binds to a pertussis sensitive G-protein coupled receptor and that high levels of lipoproteins down-regulate the number of receptors and desensitize its mediated response by a mechanism that involves PKC-phosphorylation and sequestration of binding sites. This new regulatory mechanism may have implications for the thrombogenicity in hyperlipidemia and for effects of lipid lowering therapy.

Antibodies to oxidized LDL in relation to carotid atherosclerosis, cell adhesion molecules, and phospholipase A(2).

-The role of the humoral immune response to oxidized low density lipoprotein (Ox-LDL) in atherogenesis is unclear and available studies are contradictory. The aims of the present study were (1) to compare antibody titers to modified LDL in a group of patients with hypercholesterolemia (n=102) with those in matched controls (n=102), (2) to analyze whether these titers were related to atherosclerosis development as measured by ultrasound, and (3) to analyze whether these titers were related to soluble cell adhesion molecules and secretory type II phospholipase A(2) in plasma. The results showed that male patients with hypercholesterolemia had lower immunoglobulin G (IgG) titers compared with those in healthy controls. In the control group, there was an inverse correlation between intima-media thickness of the carotid artery bulb and IgM titers against Ox-LDL and malondialdehyde-LDL (r=-0.35, P:=0.001; and r=-0.31, P:=0.003, respectively). In the patient group, however, only weak associations were seen. IgG titers were positively associated with soluble intercellular adhesion molecule-1, soluble E-selectin, and secretory type II phospholipase A(2). Taken together, the results of this study support the concept that the humoral immune response against Ox-LDL may be protective in early atherosclerosis. The pattern, however, is complex, and the role of the immune response may differ in different patient groups as well as at different stages of the disease.

Elevated levels of small, low-density lipoprotein with high affinity for arterial matrix components in patients with rheumatoid arthritis: possible contribution of phospholipase A2 to this atherogenic profile.

OBJECTIVE: This work studied the presence of inflammatory and atherogenic lipoprotein markers that could explain the high incidence of cardiovascular disease (CVD) reported in rheumatoid arthritis (RA) patients. METHODS: Inflammatory markers were 1) soluble adhesion molecules (intercellular adhesion molecule [ICAM] and vascular cell adhesion molecule [VCAM]), 2) C-reactive protein (CRP), 3) fibrinogen (Fb), 4) cytokines (interferon-gamma [IFNgamma], tumor necrosis factor alpha [TNFalpha]), and 5) secretory group IIA phospholipase A2 (sPLA2-IIA). Atherogenic lipoprotein markers were 1) the size distribution of plasma lipoprotein subclasses, and 2) the binding affinity of low-density lipoprotein (LDL) to chondroitin 6-sulfate glycosaminoglycan (GAG). RESULTS: RA patients (n = 31) and matched controls (n = 28) had similar plasma concentrations of total cholesterol, triglycerides, Apo B, Apo A-I, very low-density lipoprotein, intermediate-density lipoprotein, and high-density lipoprotein (HDL). RA patients had significantly higher plasma levels of sPLA2-IIA, ICAM, CRP, Fb, TNFalpha, and IFNgamma compared with controls. RA patients also had significantly higher levels of small, dense LDL-1 (P < 0.05) and lower levels of small HDL-2 particles (P < 0.001) compared with controls. In addition, LDL from RA patients had a significantly higher binding affinity (Kd) to GAG (mean +/- SD Kd 204+/-22.4 nM Apo B) than did LDL from control subjects (Kd 312+/-36 nM Apo B) (P < 0.05). This Kd value showed a significant negative correlation with the plasma levels of LDL-1 (r = -0.566, P < or = 0.004). In RA patients, a significant positive correlation was obtained between sPLA2-IIA and CRP, ICAM, and LDL-1. HDL-2 showed a negative correlation with sPLA2-IIA. CONCLUSION: These atherogenic lipoprotein factors combined with the presence of chronic inflammation may contribute to the high CVD-related mortality in RA patients.

Phospholipase A2 and small, dense low-density lipoprotein.

High levels of small, dense LDL in plasma are associated with increased risk for cardiovascular disease. There are some biochemical characteristics that may render small, dense LDL particles more atherogenic than larger, buoyant LDL particles. First, small, dense LDL particles contain less phospholipids and unesterified cholesterol in their surface monolayer than do large, buoyant LDL particles. This difference in lipid content appears to induce changes in the conformation of apolipoprotein B-100, leading to more exposure of proteoglycan-binding regions. This may be one reason for the high-affinity binding of small, dense LDL to arterial proteoglycans. Reduction of the phospholipid content in the surface monolayer LDL by treatment with secretory phospholipase A2 (sPLA2) forms small, dense LDL with an enhanced tendency to interact with proteoglycans. Circulating levels of sPLA2-IIA appears to be an independent risk factor for coronary artery disease and a predictor of cardiovascular events. In addition, in-vivo studies support the hypothesis that sPLA2 proteins contribute to atherogenesis and its clinical consequences. These data suggest that modification of LDL by sPLA2 in the arterial tissue or in plasma may be a mechanism for the generation of atherogenic lipoprotein particles in vivo, with a high tendency to be entrapped in the arterial extracellular matrix.

Platelet function in patients with familial hypertriglyceridemia: evidence that platelet reactivity is modulated by apolipoprotein E content of very-low-density lipoprotein particles.

We evaluated platelet function in patients with familial hypertriglyceridemia (FHTG). Compared with healthy gender-matched controls, platelets from patients showed lower aggregation (P < .01) and thromboxane formation (P < .01) in response to collagen. Very-low-density lipoprotein (VLDL) particles obtained from the patients inhibited collagen-induced aggregation, whereas VLDL particles from controls had opposite effects. The VLDL-induced effect was regulated by its apolipoprotein E (apoE) content. Indeed, apoE-VLDL-rich fractions caused antiaggregative effects, whereas apoE-VLDL-poor fractions produced a strong proaggregative response. Since we have recently demonstrated that VLDL particles may regulate the activity of platelet low-density lipoprotein (LDL) receptor by a phenomenon of downregulation and desensitization, in this study, we have investigated the effect of prolonged exposure to circulating VLDL levels on the activity of platelet LDL receptor by a double-blind controlled study with gemfibrozil (600 mg twice daily) in 18 subjects with FHTG. Platelets from patients exhibited fewer platelet LDL receptors and 125I-LDL binding was saturable at a lower protein concentration. After 6 months, gemfibrozil therapy versus placebo had a marked lipid-lowering effect, significantly decreased triglycerides (61%), VLDL cholesterol (72%), apoB (28%), and apoE (55%), and increased high-density lipoprotein (44%) and apoA-I (18%). Furthermore, gemfibrozil affected the apoprotein composition of VLDL: total protein increased by 28%, the molar ratio of apoE to apoB decreased 64%, and apoE content decreased 55%. However, no differences in phospholipid, triglyceride, or total cholesterol were detected. Moreover, platelet function was markedly altered by gemfibrozil therapy. Collagen-induced aggregation and thromboxane formation were significantly enhanced (P < .01). The initial antiaggregative pattern of VLDL particles was changed to a significant proaggregative effect (P < .01), and the number of LDL binding sites was markedly upregulated (P < .001). Both receptor upregulation and the change in the aggregative effect of VLDL particles were associated with the reduction of apoE content in VLDL particles (P < .05). The overall results indicate that in the regulation of platelet reactivity in hypertriglyceridemic patients, apoE content of VLDL particles and their interaction with the platelet LDL receptor are involved.

Interferon-gamma induces secretory group IIA phospholipase A2 in human arterial smooth muscle cells. Involvement of cell differentiation, STAT-3 activation, and modulation by other cytokines.

Increased expression of secretory non-pancreatic phospholipase A(2) (sPLA(2)-IIA) could be part of the inflammatory reaction in atherosclerosis. However, the factors controlling sPLA(2)-IIA production in human vascular cells are unknown. We investigated regulation of sPLA(2)-IIA expression and secretion by human arterial smooth muscle cells in culture (HASMC). SPLA(2)-IIA was induced after 3-14 days of culture in non-proliferating conditions. SPLA(2)-IIA was co-expressed with heavy caldesmon, a cytoskeleton protein, and p27, a G(1) cyclin inhibitor, proteins characteristically expressed by differentiated cells. Further incubation with 50-500 units/ml of interferon (IFN)-gamma significantly increased sPLA(2)-IIA mRNA and secretion. IFN-gamma-induced sPLA(2)-IIA was found to be active in cell media and associated with cell membrane proteoglycans. IFN-gamma induced sPLA(2)-IIA expression was antagonized by tumor necrosis factor (TNF)-alpha and interleukin (IL)-10. TNF-alpha added individually induced a significant but transient (4 h) increase in sPLA(2)-IIA secretion. IL-10 by itself did not affect sPLA(2)-IIA expression and secretion. IFN-gamma-stimulated sPLA(2)-IIA transcription involved STAT-3 protein. Interestingly, IL-6 but not IFN-gamma up-regulated the sPLA(2)-IIA expression in HepG2 cells, thus sPLA(2)-IIA induction by IFN-gamma response appears to be cell specific. In summary, conditions leading to cell differentiation induced sPLA(2)-IIA expression in HASMC and further exposure to IFN-gamma can up-regulate sPLA(2)-IIA transcription and secretion. This IFN-gamma stimulatory effect can be modulated by other cytokines.

Molecular basis for the association of group IIA phospholipase A(2) and decorin in human atherosclerotic lesions.

Group IIA secretory nonpancreatic phospholipase A(2) (snpPLA(2)) is associated with collagen fibers in the extracellular matrix of human atherosclerotic plaques. Decorin, a small proteoglycan (PG) carrying chondroitin/dermatan sulfate glycosaminoglycans (GAGs), forms part of the collagen network in human arteries. To explore whether snpPLA(2) may be associated with collagen fibers via interaction with decorin, we performed (1) immunohistochemistry to compare the relative in vivo localization of snpPLA(2) and decorin in human atherosclerotic tissue and (2) in vitro experiments to study the interaction between snpPLA(2) and decorin. In atherosclerotic lesions, decorin was detected within the snpPLA(2)-positive part of the intima close to the media. Electrophoretic mobility shift assay showed that snpPLA(2) binds to decorin synthesized by human fibroblasts. Native and GAG-depleted decorin enhanced the association of snpPLA(2) to collagen types I and VI in a solid-phase binding assay. Furthermore, snpPLA(2) bound efficiently to a recombinant decorin core protein fragment B/E (Asp45-Lys359). This binding was competed with soluble decorin and inhibited at NaCl concentrations >150 mmol/L. The decorin core protein fragment B/E competed better than dermatan sulfate for binding of snpPLA(2) to decorin-coated microtiter wells. The enzymatic activity of snpPLA(2) increased 2- to 3-fold in the presence of decorin or GAG-depleted decorin. The results show that snpPLA(2) binds preferentially to the decorin protein core rather than to the GAG chain and that this interaction enhances snpPLA(2) activity. As a consequence, this active extracellular enzyme may contribute to the pathogenesis of atherosclerosis by modifying lipoproteins and releasing inflammatory lipid mediators at places of lipoprotein retention in the arterial wall.

Endogenously produced glycosaminoglycans affecting the release of lipoprotein lipase from macrophages and the interaction with lipoproteins.

Macrophages are intimately involved in the pathogenesis of atherosclerotic diseases. A key feature of this process is their uptake of various lipoproteins and subsequent transformation to foam cells. Since lipoprotein lipase (LPL) is believed to play a role in foam cell formation, we investigated if endogenously produced proteoglycans (PGs) affect the release of this enzyme from macrophages. The human leukaemic cell line THP-1 which differentiates into macrophages by treatment with phorbol ester (phorbol 12-myristate 13-acetate) served as a model. The differentiation of THP-1 macrophages promoted the release of PGs into the cell medium which caused the detachment of LPL activity from the cell surface, and prevented LPL re-uptake and inactivation. These PGs were mainly composed of chondroitin sulfate type and exerted a heparin-like effect on LPL release. LPL is known to increase the cell association of lipoproteins by the well known bridging function. Exogenous bovine LPL at a concentration of 1 microg/ml enhanced low density lipoprotein (LDL)-binding 10-fold. Endogenously produced PGs reduced LPL-mediated binding of LDL. It is proposed that the differentiation-dependent increase in the release of PGs interferes with binding of LPL and reduces lipoprotein-binding to macrophages.

Phospholipase A(2) modification of low density lipoproteins forms small high density particles with increased affinity for proteoglycans and glycosaminoglycans.

The presence of a lipoprotein profile with abundance of small, dense low density lipoproteins (LDL), low levels of high density lipoproteins (HDL), and elevated levels of triglyceride-rich very low density lipoproteins is associated with an increased risk for coronary heart disease. The atherogenicity of small, dense LDL is believed to be one of the main reasons for this association. This particle contains less phospholipids (PL) and unesterified cholesterol than large LDL, and the apoB-100 appears to occupy a more extensive area at its surface. Although there are experiments that suggest a metabolic pathway leading to the overproduction of small, dense LDL, no clear molecular model exists to explain its association with atherogenesis. A current hypothesis is that small, dense LDL, because of its higher affinity for proteoglycans, is entrapped in the intima extracellular matrix and is more susceptible to oxidative modifications than large LDL. Here we describe how a specific reduction of approximately 50% of the PL of a normal buoyant LDL by immobilized phospholipase A(2) (PLA(2)) (EC 3.1.1.4) produces smaller and denser particles without inducing significant lipoprotein aggregation (<5%). These smaller LDL particles display a higher tendency to form nonsoluble complexes with proteoglycans and glycosaminoglycans than the parent LDL. Binding parameters of LDL and glycosaminoglycans and proteoglycans produced by human arterial smooth muscle cells were measured at near to physiological conditions. The PLA(2)-modified LDL has about 2 times higher affinity for the sulfated polysaccharides than control LDL. In addition, incubation of human plasma in the presence of PLA(2) generated smaller LDL and HDL particles compared with the control plasma incubated without PLA(2). These in vitro results indicate that the reduction of surface PL characteristic of small, dense LDL subfractions, besides contributing to its small size and density, may enhance its tendency to be retained by proteoglycans.

CD44, a cell surface chondroitin sulfate proteoglycan, mediates binding of interferon-gamma and some of its biological effects on human vascular smooth muscle cells.

Several cytokines and growth factors act on cells after their association with the glycosaminoglycan (GAG) moiety of cell surface proteoglycans (PGs). Interferon-gamma (IFN-gamma) binds to GAG; however, the relevance of this interaction for the biological activity of IFN-gamma on human cells remains to be established. Human arterial smooth muscle cells (HASMC), the main cells synthesizing PG in the vascular wall, respond markedly to IFN-gamma. We found that treatment of HASMC with chondroitinase ABC, an enzyme that degrades chondroitin sulfate GAG, reduced IFN-gamma binding by more than 50%. This treatment increased the affinity of 125I-IFN-gamma for cells from a Kd value of about 93 nM to a Kd value of about 33 nM. However, the total binding was reduced from 9. 3 +/- 0.77 pmol/microg to 3.0 +/- 0.23 pmol/mg (n = 4). Interestingly, pretreatment with chondroitinase ABC reduced significantly the cellular response toward IFN-gamma. The interaction of IFN-gamma with chondroitin sulfate GAG was confirmed by affinity chromatography of isolated cell-associated 35S-, 3H-labeled PG on a column with immobilized IFN-gamma. The cell-associated PG that binds to IFN-gamma was a chondroitin sulfate PG (CSPG). This CSPG had a core protein of approximately 110 kDa that was recognized by anti-CD44 antibodies on Western blots. High molecular weight complexes between IFN-gamma and chondroitin 6-sulfate were observed in gel exclusion chromatography. Additions of chondroitin 6-sulfate to cultured HASMC antagonized the antiproliferative effect and expression of major histocompatibility complex II antigens induced by IFN-gamma. These results indicate that IFN-gamma binds with low affinity to the chondroitin sulfate GAG moiety of the cell surface CSPG receptor CD44. This interaction may increase the local concentration of IFN-gamma at the cell surface, thus facilitating its binding to high affinity receptors and modulating the ability of IFN-gamma to signal a cellular response.