Vasculoprotective properties of plasma lipoproteins from brown bears (Ursus arctos).

Plasma cholesterol and triglyceride (TG) levels are twice as high in hibernating brown bears (Ursus arctos) than healthy humans. Yet, bears display no signs of early stage atherosclerosis development when adult. To explore this apparent paradox, we analyzed plasma lipoproteins from the same 10 bears in winter (hibernation) and summer using size exclusion chromatography, ultracentrifugation, and electrophoresis. LDL binding to arterial proteoglycans (PGs) and plasma cholesterol efflux capacity (CEC) were also evaluated. The data collected and analyzed from bears were also compared with those from healthy humans. In bears, the cholesterol ester, unesterified cholesterol, TG, and phospholipid contents of VLDL and LDL were higher in winter than in summer. The percentage lipid composition of LDL differed between bears and humans but did not change seasonally in bears. Bear LDL was larger, richer in TGs, showed prebeta electrophoretic mobility, and had 5-10 times lower binding to arterial PGs than human LDL. Finally, plasma CEC was higher in bears than in humans, especially the HDL fraction when mediated by ABCA1. These results suggest that in brown bears the absence of early atherogenesis is likely associated with a lower affinity of LDL for arterial PGs and an elevated CEC of bear plasma.

Proteomics of apolipoproteins and associated proteins from plasma high-density lipoproteins.

Proteomics studies have extended the list of identified apolipoproteins and associated proteins present in HDL and its subclasses. These proteins appear to cluster around specific functions related to lipid metabolism, inflammation, the immune system, hormone-binding, hemostasis, and antioxidant properties. Small studies suggest that there are substantial differences between the HDL proteome from cardiovascular disease patients and that from controls. Furthermore, dyslipidemia therapy shifts the HDL proteome from patients toward the profile observed in healthy controls. In addition, the proteome of HDL and LDL from patients with insulin resistance and peripheral atherosclerosis show significant differences with that of matched healthy controls. The proteome of HDL and LDL density subclasses have apolipoproteins and associated proteins profiles that suggest subclass-specific functions. However, proteomics studies of lipoproteins are few and small and should be interpreted with caution. Nonetheless rapid technical progress in proteomic platforms suggest that soon analysis time will be reduced and precise measurement of identified proteins will be possible. This, combined with controlled purification steps of HDL and its subclasses should provide further information about proteins involved in the particles postulated spectrum of functions, including those believed to be atheroprotective.

Hypoxic regulation of secreted proteoglycans in macrophages.

Macrophages are prominent in hypoxic areas of atherosclerotic lesions, and their secreted proteoglycans (PG), such as versican, can modulate the retention of lipoproteins and the activity of enzymes, cytokines, and growth factors involved in atherogenesis. In this study, we report the effects of hypoxia on PG secreted by human monocyte-derived macrophages (HMDM) and the potential regulation by the transcription factor hypoxia-inducible factor (HIF-1alpha and HIF-2alpha). We found that versican co-localized with HIF-1alpha in macrophage-rich areas in human advanced atherosclerotic lesions. Versican and perlecan mRNA expression increased after exposure to 0.5% O(2) (hypoxia) compared with 21% O(2) (control cells). Using precursors to GAG biosynthesis combined with immunoabsorption with a versican antibody an increased versican synthesis was detected at hypoxia. Furthermore, siRNA knockdown of HIF-1alpha and HIF-2alpha in THP-1 cells showed that the hypoxic induction of versican and perlecan mRNA expression involved HIF signaling. Versican expression was co-regulated by HIF-1alpha and HIF-2alpha but expression of perlecan was influenced only by HIF-1alpha and not by HIF-2alpha knockdown. The results show that oxygen concentration is an important modulator of PG expression in macrophages. This may be a novel component of the complex role of macrophages in atherosclerosis.

In vivo effects of myocardial creatine depletion on left ventricular function morphology and lipid metabolism: study in a mouse model.

BACKGROUND: The failing heart is characterized by disturbed myocardial energy metabolism and creatine depletion. The aims of this study were to evaluate in vivo the effects of creatine (Cr) depletion on 1) left ventricular (LV) function, morphology, and lipid metabolism and 2) to test whether functional, morphologic, and metabolic disturbances induced by Cr depletion are reversible. METHODS AND RESULTS: Male Balb/c mice approximately 20 g were used. Two groups were studied: the mice treated with creatine analogue beta-guanidinopropionic acid (BGP) (n = 30) and controls (n = 30). BGP (1 M) were administered by subcutaneously implanted osmotic minipumps for 4 weeks. The mice were examined in vivo using echocardiography. High-performance liquid chromatography was used for measurements of the myocardial creatine, adenosine nucleotides, and lipids. BGP was discontinued in a subgroup of mice and these animals were followed for an additional 4 weeks, after which echocardiography was performed under resting and stress conditions. Body weight was lower in BGP mice (P < .001) compared with the controls after 4 weeks. The total myocardial Cr pool was approximately 40% lower (P < .001), whereas total nucleotide pool (TAN) was 18% lower (P = n.s.) in the BGP group. LV systolic function was disturbed at rest and stress in the BGP mice (both P < .05). LV dimensions and LV mass were increased in the BGP group (P < .05). There was an accumulation of intracellular triglycerides in the BGP-treated mice (P < .05). Four weeks after BGP discontinuation Cr, TAN and TG content were restored to the normal levels while LV function, dimension, and mass were normalized. CONCLUSIONS: Myocardial Cr depletion results in LV dysfunction, pathologic remodeling, and lipid accumulation. These alterations are completely reversible on normalization of Cr content. Cr metabolism may be an important target for pharmacologic intervention to increase myocardial efficiency and structural integrity of the failing heart.

ApoB-100 Lipoprotein Complex Formation with Intima Proteoglycans as a Cause of Atherosclerosis and Its Possible Ex Vivo Evaluation as a Disease Biomarker.

Experimental and clinical data indicate that the initiation and progress of atherosclerosis and its clinical manifestations are first caused by circulating apoB-100 lipoproteins that enter and are retained in the arterial intima. Extracellular sulfated proteoglycans (PGs) of the intima are the retention agents. The PGs also initiate physical and biochemical lipoprotein degradation with the production of bioactive, lipid products that trigger an inflammatory response that leads to atherosclerosis. There are many simple methods for measuring abnormalities of circulating lipoproteins and their relation to atherosclerotic cardiovascular disease (ACVD). However, limited research aims to evaluate procedures that could report quantitatively about the contribution of the interaction of apoB-100 lipoprotein-arterial intima PGs to clinical manifestation of ACVD. In the present review we discuss observations indicating that simple ex vivo evaluation of the affinity of apoB-100 lipoproteins for arterial PGs and glycosaminoglycans (GAGs) can give an indication of its association with clinical manifestations of atherosclerosis. In addition, we discuss molecular and cellular aspects of the apoB-100 lipoproteins association with arterial PGs that are related to atherogenesis and that support the experimental framework behind the current “Response-to-Retention” hypothesis of atherosclerosis.

Postprandial Hypertriglyceridemia Is Associated with the Variant 54 Threonine FABP2 Gene.

Purpose: Fasting or postprandial hypertriglyceridemia is considered an independent cardiovascular disease (CVD) risk factor. The intestinal fatty acid binding protein (FABP2) is involved in the intracellular transport and metabolism of fatty acids. The presence of the Ala54Thr polymorphism of the FABP2 gene appears to be involved in postprandial hypertriglyceridemia. We explored the possible association of the Ala54Thr polymorphism with fat intolerance in apparently healthy, fasting, normolipidemic subjects with normal body-mass index and without diabetes. Methodology: A total of 158 apparently healthy individuals were classified as fat tolerant (n = 123) or intolerant (n = 35) according to their response (plasma triglycerides) to an oral abbreviated tolerance test with blood samples taken at 0, 2 and 4 h. At 0 h, all subjects ingested 26.3 g of fats. Presence of the Ala54Thr polymorphism of the FABP2 gene was evaluated by polymerase chain reaction⁻restriction fragment length (PCR⁻RFLP). Results: The group with fat intolerance (postprandial hypertriglyceridemia group) showed an increased frequency of the Thr54Thr genotype when compared with the group with normal fat tolerance (control group) (23% vs. 4%, respectively, OR: 16.53, 95% CI: 4.09⁻66.82, p: 0.0001, pc: 0.0003). Carriers of at least one Thr54 allele were up to six times more prevalent in the fat intolerant group than in the non-carriers. (OR: 6.35; 95% CI: 1.86⁻21.59, p: 0.0003, pc: 0.0009). The levels of plasma triglycerides (Tg) at 4 h after the test meal were higher in carriers of at least one 54Thr allele than in carriers of the Ala54 allele (p < 0.05). Conclusions: There is a significant association between postprandial hypertriglyceridemia and the presence of at least one 54Thr allele of the FABP2 gene. In addition, subjects with this genotype showed an increased ratio of Tg/HDL-cholesterol. This parameter is a marker of increased CVD risk and insulin resistance.

Ezetimibe in Combination With Simvastatin Reduces Remnant Cholesterol Without Affecting Biliary Lipid Concentrations in Gallstone Patients.

Background In randomized trials (SHARP [Study of Heart and Renal Protection], IMPROVE -IT [Improved Reduction of Outcomes: Vytorin Efficacy International Trial]), combination of statin and ezetimibe resulted in additional reduction of cardiovascular events. The reduction was greater in patients with type 2 diabetes mellitus (T2 DM ), where elevated remnant cholesterol and high cardiovascular disease risk is characteristic. To evaluate possible causes behind these results, 40 patients eligible for cholecystectomy, randomized to simvastatin, ezetimibe, combined treatment (simvastatin+ezetimibe), or placebo treatment during 4 weeks before surgery, were studied. Methods and Results Fasting blood samples were taken before treatment start and at the end (just before surgery). Bile samples and liver biopsies were collected during surgery. Hepatic gene expression levels were assessed with qPCR . Lipoprotein, apolipoprotein levels, and content of cholesterol, cholesteryl ester, and triglycerides were measured after lipoprotein fractionation. Lipoprotein subclasses were analyzed by nuclear magnetic resonance. Apolipoprotein affinity for human arterial proteoglycans ( PG ) was measured. Biomarkers of cholesterol biosynthesis and intestinal absorption and bile lipid composition were analyzed using mass spectrometry. Combined treatment caused a statistically significant decrease in plasma remnant particles and apolipoprotein B (ApoB)/lipoprotein content of cholesterol, cholesteryl esters, and triglycerides. All treatments reduced ApoB-lipoprotein PG binding. Simvastatin and combined treatment modified the composition of lipoproteins. Changes in biomarkers of cholesterol synthesis and absorption and bile acid synthesis were as expected. No adverse events were found. Conclusions Combined treatment caused atheroprotective changes on ApoB-lipoproteins, remnant particles, bile components, and in ApoB-lipoprotein affinity for arterial PG . These effects might explain the decrease of cardiovascular events seen in the SHARP and IMPROVE - IT trials. Clinical Trial Registration URL : www.clinicaltrialsregister.eu . Unique identifier: 2006-004839-30).

Distinctiveness of secretory phospholipase A2 group IIA and V suggesting unique roles in atherosclerosis.

Clinical observations strongly support an association of circulating levels of secretory phospholipases A(2) (sPLA(2)) in atherosclerotic cardiovascular disease (ACVD). Two modes of action can provide causal support for these statistical correlations. One is the action of the enzymes on circulating lipoproteins and the other is direct action on the lipoproteins once in the arterial extracellular intima. In this review we discuss results suggesting a distinct profile of characteristics related to localization, action on plasma lipoproteins and interaction with arterial proteoglycans for sPLA(2)-IIA and sPLA(2)-V. The differences observed indicate that these enzymes may contribute to atherosclerosis through dissimilar pathways. Furthermore, we comment on recent animal studies from our laboratory indicating that the expression of type V enzyme is up-regulated by genetically and nutritionally-induced dyslipidemias but not the group type IIA enzyme, which is well known to be up-regulated by acute inflammation. The results suggest that if similar up-regulation occurs in humans in response to hyperlipidemia, it may create a distinctive link between the group V enzyme and the disease.

Tesaglitazar, a PPARalpha/gamma agonist, induces interstitial mesenchymal cell DNA synthesis and fibrosarcomas in subcutaneous tissues in rats.

The development of the dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist tesaglitazar as an oral antidiabetic was recently discontinued. Here we present tumor data from a 2-year carcinogenicity study in rats given 0.3, 1, 3, and 10 micromol/kg tesaglitazar is presented with focus on the findings of subcutaneous fibrosarcomas. To investigate the mechanism for induction of fibrosarcomas, replicative DNA synthesis (immunohistochemical detection of BrdU-labeled cells) and expression of PPARgamma (immunohistochemistry and reverse transcription-polymerase chain reaction) in subcutaneous adipose tissues was assessed in rats administered 1 or 10 micromol/kg for 2 weeks or 3 months. Poorly differentiated subcutaneous mesenchymal sarcomas with a predominant spindle cell appearance occurred at the highest dose level of 10 micromol/kg in both sexes, and these tumors were diagnosed as fibrosarcomas. The 10-micromol/kg dose was at or above the maximum tolerated dose and caused considerable cardiovascular mortality. Tesaglitazar stimulated DNA synthesis mainly in subcutaneous interstitial mesenchymal cells. The percentage of BrdU-labeled interstitial cells was increased at 1 and 10 micromol/kg after 2 weeks. The increase in DNA synthesis was still significant at the end of the 12-week treatment at 10 mumol/kg, the dose producing fibrosarcoma. However, at 1 micromol/kg, a dose below the no-observed-effect level for fibrosarcoma, the level of DNA synthesis was similar to control levels at 12 weeks. Immunohistochemical analyses showed no detectable PPARgamma protein in the majority of BrdU-labeled interstitial mesenchymal cells in white and brown fat. This indicates that stimulation of DNA synthesis is not mediated via direct activation of PPARgamma in these cells. The results suggest that the induction of rat fibrosarcoma by tesaglitazar, at exposures 100-fold above the human therapeutic exposure, may involve proliferation of undifferentiated mesenchymal cells in subcutaneous tissues.

Fatty acids cause alterations of human arterial smooth muscle cell proteoglycans that increase the affinity for low-density lipoprotein.

OBJECTIVE: The dyslipidemia of insulin resistance, with high levels of albumin-bound fatty acids, is a strong cardiovascular disease risk. Human arterial smooth muscle cell (hASMC) matrix proteoglycans (PGs) contribute to the retention of apoB lipoproteins in the intima, a possible key step in atherogenesis. We investigated the effects of high NEFA levels on the PGs secreted by hASMCs and whether these effects might alter the PG affinity for low-density lipoprotein. METHODS AND RESULTS: hASMC exposed for 72 hours to high concentrations (800 micromol/L) of linoleate (LO) or palmitate upregulated the core protein mRNAs of the major PGs, as measured by quantitative PCR. Insulin (1 nmol/L) and the PPARgamma agonist rosiglitazone (10 micromol/L) blocked these effects. In addition, high LO increased the mRNA levels of enzymes required for glycosaminoglycan (GAG) synthesis. Exposure to NEFA increased the chondroitin sulfate:heparan sulfate ratio and the negative charge of the PGs. Because of these changes, the GAGs secreted by LO-treated cells had a higher affinity for human low-density lipoprotein than GAGs from control cells. Insulin and rosiglitazone inhibited this increase in affinity. CONCLUSIONS: The response of hASMC to NEFA could induce extracellular matrix alterations favoring apoB lipoprotein deposition and atherogenesis.

Dual PPARalpha/gamma agonist tesaglitazar reduces atherosclerosis in insulin-resistant and hypercholesterolemic ApoE*3Leiden mice.

OBJECTIVE: We investigated whether the dual PPARalpha/gamma agonist tesaglitazar has anti-atherogenic effects in ApoE*3Leiden mice with reduced insulin sensitivity. METHODS AND RESULTS: ApoE*3Leiden transgenic mice were fed a high-fat (HF) insulin-resistance-inducing diet. One group received a high-cholesterol (HC) supplement (1% wt/wt; HC group). A second group received the same HC supplement along with tesaglitazar (T) 0.5 micromol/kg diet (T group). A third (control) group received a low-cholesterol (LC) supplement (0.1% wt/wt; LC group). Tesaglitazar decreased plasma cholesterol by 20% compared with the HC group; cholesterol levels were similar in the T and LC groups. Compared with the HC group, tesaglitazar caused a 92% reduction in atherosclerosis, whereas a 56% reduction was seen in the cholesterol-matched LC group. Furthermore, tesaglitazar treatment significantly reduced lesion number beyond that expected from cholesterol lowering and induced a shift to less severe lesions. Concomitantly, tesaglitazar reduced macrophage-rich and collagen areas. In addition, tesaglitazar reduced inflammatory markers, including plasma SAA levels, the number of adhering monocytes, and nuclear factor kappaB-activity in the vessel wall. CONCLUSIONS: Tesaglitazar has anti-atherosclerotic effects in the mouse model that go beyond plasma cholesterol lowering, possibly caused by a combination of altered lipoprotein profiles and anti-inflammatory vascular effects.

Fat Intolerance in Apparently Healthy Individuals with Normal Fasting Lipoproteins Is Associated with Markers of Cardiovascular Risk.

BACKGROUND: Postprandial increase of triglyceride-rich lipoproteins augments the risk of atherosclerotic cardiovascular disease and all-cause mortality. We explored the hypothesis that a simplified oral fat tolerance test can uncover differences in postprandial triglyceride response associated with potentially atherogenic lipoprotein characteristics, even in a cohort of apparently healthy 31-year-old [mean (SD), 31 (11)] nonobese individuals with normal fasting lipids and lipoproteins. METHODS: We used a fat tolerance test in 96 females and 62 males with blood sampled at 0, 2, and 4 h after a breakfast containing 26.3 g of fats. The postprandial triglyceride response was used to classify the individuals in apparently fat-tolerant and apparently fat-intolerant participants. RESULTS: The intolerant individuals were found to have at 0 h significantly higher body mass index, plasma triglycerides, remnant cholesterol, VLDL cholesterol, and LDL cholesterol and lower apolipoprotein (apo) AI and HDL cholesterol than the tolerant individuals. More than 70% of the variability (r2) of the postprandial response in tolerant and intolerant individuals measured as area under the curve or, at a single point at 4 h after the oral fat load, was linearly correlated with 0-h triglycerides (P < 0001). Fasting lipoprotein parameters, proposed to be markers of cardiovascular risk, as the ratios apo B/apo AI, total cholesterol/HDL cholesterol, and triglycerides/HDL cholesterol, were increased in the intolerant individuals. CONCLUSIONS: A simplified oral fat tolerance test, even when used in an apparently healthy, nonobese, normolipidemic cohort, detected that an increased postprandial triglycerides response was associated with augmented lipoprotein markers of increased cardiovascular risk.

The extracellular matrix on atherogenesis and diabetes-associated vascular disease.

Atherosclerosis is remarkably increased in type 2 diabetes suggesting that mechanisms causing arterial lesion are enhanced by the metabolic disturbances of insulin resistance (IR) and diabetes. Several lines of research suggest that processes taking place in the arterial intima extracellular matrix may be part of a shared pathogenic mechanism. The intima extracellular matrix is where atherogenesis takes place. This layer contains fibrilar macromolecules like collagens, proteoglycans (PGs), hyaluronate, and extracellular multi-domain proteins. Specific interaction of lysine, arginine-rich segments of the apoB-100 lipoproteins, LDL, IDL and Lp (a), with the negatively charged glycosaminoglycans (GAGs) of PGs cause retention of the lipoproteins, one of the initiation process of atherogenesis. Such interactions cause structural modifications of the lipid and protein moieties of the lipoproteins that appear to increase their susceptibility to proteases, phospholipases and free radical-mediated processes. The association of apoB-lipoproteins, specially small and dense LDL, with intima PGs increases their uptake by macrophages and human arterial smooth muscle cells (HASMC) leading to 'foam cell' formation. In vitro, elevated levels of non-esterified fatty acids (NEFA) alter the matrix of endothelial cells basement membrane making them more permeable to macromolecules. NEFA cause changes in the expression of genes controlling the PGs composition of the PGs secreted by HASMC causing formation of a matrix with high affinity for LDL. These results lead us to speculate that an important component of the dyslipidemia of IR and type 2 diabetes, chronic high NEFA, may contribute to cellular alterations that cause changes of the arterial intima extracellular matrix. Such changes may increase the atherogenicity of the retention of apoB lipoproteins in the intima and contribute to the systemic alteration of the arterial wall frequently observed in IR and type 2 diabetes.

PPAR agonists in the treatment of insulin resistance and associated arterial disease.

Augmented release of non-esterified fatty acids (NEFA) from insulin-resistant adipocytes appears to be the main cause of the 'atherogenic lipoprotein profile' associated with insulin resistance and type 2 diabetes. This atherogenic profile is characterised by large very-low-density lipoproteins (VLDL), small, dense low-density lipoproteins (LDL) and low levels of high-density lipoproteins (HDL), resulting in deposition of apo B lipoproteins in the vascular intima and subsequent inhibition of reverse cholesterol transport. This lipoprotein retention also results in a proinflammatory response from the vascular endothelium, which is increased in insulin resistance. Thus the ideal therapy for insulin resistance, and its complications, should both improve its associated dyslipidaemia and ameliorate the vascular atherogenic reaction. Some peroxisome proliferator-activated receptor (PPAR)-gamma and dual PPARalpha/gamma agonists improve insulin resistance and its dyslipidaemia, both in rodents and man, while in animal models they can show clear antiatherosclerotic effects. Nonetheless, it is difficult to evaluate how much of these antiatherosclerotic actions are caused by effects on the dyslipidaemia or by direct effects on vascular cells. Upregulation of PPARgamma and PPARalpha/gamma activity in macrophages can reduce secretion of proinflammatory cytokines and matrix metalloproteases, as well as increase HDL-mediated cholesterol efflux transport--all potentially antiatherosclerotic results. In addition, treatment of smooth muscle cells with PPARgamma agonists can partially revert possible atherogenic changes in the production of matrix proteoglycans induced by exposure to NEFA. Although these findings are still preliminary, and their relevance to human atherosclerosis has not been fully elaborated, these results suggest that improved PPARalpha/gamma agonism may positively modulate several of the metabolic steps connecting insulin resistance with dyslipidaemia and with the atherogenic response.

HDL2 interferes with LDL association with arterial proteoglycans: a possible athero-protective effect.

OBJECTIVES: High levels of large HDL (HDL2) reduce cardiovascular disease risks apparently because it mediates reverse cholesterol transport, and it has anti-inflammatory properties. Here we explored the mechanism behind an additional athero-protective HDL effect related to its capacity to interfere with formation of insoluble LDL-proteoglycans associations, a key step in LDL entrapment in the intima and in atherogenesis. METHODS AND RESULTS: We found that HDL2 levels from type 2 diabetes patients and controls are inversely correlated with complex formation between serum LDL and the arterial proteoglycans versican. Reconstitution experiments indicate that HDL2 was more efficacious inhibitor of the LDL-versican association than the smaller HDL3. This may explain why serum from patients with dyslipidemia of insulin resistance, with low levels of HDL2, have a higher capacity to form insoluble LDL-proteoglycan complex. ApoE enrichment of HDL2 and HDL3 or addition of copies of an apoE peptide with the proteoglycan-binding sequence of this apolipoprotein increased their inhibition of LDL-versican associations. CONCLUSIONS: The inhibitory effect of HDL2 and HDL3 on LDL-versican associations was related to formation of apoE-mediated soluble HDL-versican complexes. We speculate that in the intima large, HDL2 subclasses, by forming reversible soluble associations with proteoglycans can compete with formation of irreversible LDL-proteoglycan aggregates. This can contribute to the HDL2 athero-protective effects. In the dyslipidemia of insulin resistance, associated with low levels of HDL2, this athero-protective property may be compromised.

Macrophages exposed to hypoxia secrete proteoglycans for which LDL has higher affinity.

Macrophages are prominent in hypoxic areas of atherosclerotic lesions. Their secreted proteoglycans (PG) can modulate the retention of lipoproteins as well as the activity of enzymes, cytokines, and growth factors involved in atherogenesis. Versican appears to be one of the main extracellular matrix components binding LDL in the arterial intima. We have recently shown that hypoxia increases versican and perlecan expression in macrophages, and that this increase was regulated by the hypoxia inducible factor (HIF). Here we report effects of hypoxia on human monocyte-derived macrophage (HMDM) secreted glycosaminoglycans (GAG), and its interaction with LDL. After 24 h exposure to 0.5% O2 (hypoxia), metabolically labeled GAG of secreted PG had higher affinity for LDL compared to 21% O2 (control cells). GAG secreted by HMDM in hypoxia were found to be more sulfated and longer which might be responsible for the increased affinity of LDL for these GAG chains. These results indicate that hypoxia induced changes in macrophage GAG biosynthesis have important consequences for the interaction with LDL. If present in vivo, an augmented association of GAG with LDL might contribute to the development of atherosclerosis in hypoxic intima.