Germán Camejo

Association of apo B lipoproteins with arterial proteoglycans: Pathological significance and molecular basis

Retention of apo B-100 lipoproteins, low density lipoprotein (LDL) and probably lipoprotein(a), Lp(a), by intima proteoglycans (PGs) appears to increase the residence time needed for their structural, hydrolytic and oxidative modifications. If the rate of LDL entry exceeds the tissue capacity to eliminate the modified products, this process may be a contributor to atherogenesis and lesion advancement. LDL binds to PGs of the intima, by association of specific positive segments of the apo B-100 with the negatively-charged glycosaminoglycans (GAGs) made of chondroitin sulfate (CS), dermatan sulfate (DS) and probably heparan sulfate (HS). Small, dense LDL has a higher affinity for CS-PGs than large buoyant particles, probably because they expose more of the segments binding the GAGs than larger LDL. PGs cause irreversible structural alterations of LDL that potentiate hydrolytic and oxidative modifications. These alterations also increase LDL uptake by macrophages and smooth muscle cells. These in vitro data suggest that part of the atherogenicity of LDL may depend on its tendency to form complexes with arterial PGs in vivo. Ex vivo results support this hypothesis. Subjects with coronary heart disease have LDL with significantly higher affinity for arterial PGs. This is also a characteristic of subjects with the atherogenic lipoprotein phenotype, with high levels of small, dense LDL. The LDL-PG affinity, however can be modified by dietary or pharmacological interventions that change the composition and size of LDL. Lesion-prone intima contain PGs with a high affinity for LDL. Increased LDL entrapment at these sites may be a key step in a cyclic atherogenic process.

Effect of arterial proteoglycans and glycosaminoglycans on low density lipoprotein oxidation and its uptake by human macrophages and arterial smooth muscle cells.

The reversible interaction of low density lipoprotein (LDL) with arterial chondroitin sulfate proteoglycans (CSPGs) or glycosaminoglycans (GAGs) selects LDL particles with a high affinity for sulfated GAGs and also induces modifications in apolipoprotein B (apo B) and the lipid organization of the lipoprotein. In the present work we studied the effect that the reversible interaction with sulfated polysaccharides has on the susceptibility of LDL to in vitro oxidation. For this purpose soluble, nonaggregated CSPG- or GAG-treated LDL was subjected to oxidation in the presence of 5 microM CuSO4 for as long as 48 hours. The rate of formation of thiobarbituric acid-reactive substances, the decrease in isoelectric point, the increase in relative electrophoretic mobility of LDL, the higher degradation rate by human macrophages, and the lower degradation rate by human arterial smooth muscle cells showed that LDLs exposed to CSPGs and GAGs were significantly more susceptible to oxidation than native LDL. Results from competition experiments indicate that C6S-treated LDL after 4 hours of oxidation is taken up via the acetylated LDL receptor in human macrophages. Coincubation of lipoproteins with human macrophages or human arterial smooth muscle cells for 24 hours also indicated that C6S-treated LDL was more susceptible to cell-induced modifications than native LDL. The occurrence in vivo of similar processes may contribute to focal retention, increased rate oxidation of LDL in the arterial intima, and foam cell formation during atherogenesis.

Cellular Consequences of the Association of ApoB Lipoproteins With Proteoglycans: Potential Contribution to Atherogenesis

Many of the discussed results come from empirical experiments performed with in vitro models whose relevance to the complex environment of the intima is limited. However, they are consistent with the line of reasoning that intima PGs interact specifically with apoB lipoproteins and contribute to their retention. This could provide the residence time and the initial alterations of the lipoproteins that favor their further modifications by oxidative processes and hydrolytic enzymes. Products of such modifications, and the modified particles, may be stimuli for changes in the functionality of endothelium, smooth muscle cells, and macrophages. The focal synthesis of PGs with high affinity for apoB lipoproteins could make the phenomena chronic. Clinical and laboratory studies indicate that dense LDL, poor in surface polar lipids, is associated with an atherogenic phenotype. Particles with these properties may contribute to the disease via its high affinity for arterial PGs. This affinity can be modulated by diet, lifestyle, and lipid-lowering drugs.

Possible Functional Interactions of Apolipoprotein B-100 Segments That Associate With Cell Proteoglycans and the ApoB/E Receptor

The interaction of apoE lipoproteins with cells appears to be mediated by an association with basic sequences of proteoglycans and the apoB/E receptor. ApoB-100 has basic sequences, homologous with those of apoE, that form part of the apoB/E receptor-binding domain. These sequences of apoB-100 also interact with proteoglycans. We investigated whether such segments, in analogy with apoE, could act cooperatively on LDL interactions with proteoglycans and the receptor. As a model we used the two most basic regions of apoB-100, 3147 through 3157 and 3359 through 3367, connected by three glycines (3145-3157-GGG-3359-3367). Such segments may be proximal in LDL by the presence of a disulfide bridge between Cys(3167) and Cys(3297). The apoB heterodimer but not the separated monomers inhibited 125I-LDL degradation in fibroblasts and THP-1 cells by 50% at approximately 11 mumol/L. The heterodimer affinity with arterial proteoglycans was closer to that of LDL and higher than that of the individual peptides. The heterodimer appears to bind specifically to THP-1 cells, with a Kd of 6.2 x 10(-8) mol/L and a Bmax of 1.3 x 10(6) molecules/cell. Monoclonal antibody C-7, which recognizes the apoB receptor, inhibited the binding to cells. Treatment of fibroblasts with chondroitinase ABC or chlorate decreased 125I-LDL degradation markedly. Hydrolysis of pericellular proteoglycans of fibroblasts by chondroitinases reduced mostly the low-affinity, high-capacity component of LDL binding. This compartment appears to hold 70% of the cell-associated LDL when internalization is inhibited at 4 degrees C. Therefore, cell-surface chondroitin sulfate/dermatan sulfate proteoglycans appear to modulate binding and receptor-mediated internalization of LDL. This may be caused, at least in part, by the association of proteoglycans with the apoB-100 segments 3145 through 3157 and 3359 through 3367.

Modifications of low-density lipoprotein induced by arterial proteoglycans and chondroitin-6-sulfate

Association of low-density lipoproteins (LDL) with arterial chondroitin sulfate proteoglycans (CSPG) appears to contribute to their deposition in the extracellular intimal compartment and to its internalization by macrophages. CSPG and LDL interact by ionic bridges with formation of soluble and insoluble complexes. We studied the alterations on LDL structure induced by its association with arterial CSPG and other glycosaminoglycans (GAG). In soluble complexes, at low and at physiological ionic strength, arterial CSPG and sulfated GAG modify the kinetics of apoB-100 proteolysis by trypsin. However, less marked alterations in the peptide patterns were observed with proteinase V8 and almost none with thermolysin. This is indirect evidence that the presence of CSPG and GAG modified the exposure of polar regions of apoB-100 in LDL. Competitive binding experiments with agarose-bound heparin and soluble GAG also suggest that after formation of insoluble complexes with arterial CSPG and resolubilization the exposure of Lys, Arg-rich segments of apoB-100 is increased. Results from differential scanning calorimetry and differential thermal spectrophotometry showed that the CSPG and GAG-induced modifications reduced the thermal stability of the surface and core in LDL. If present in vivo, the structural alterations of polar segments of the LDL protein moiety may influence the outcome of its interaction with the arterial mesenchyma.

CD44, a Cell Surface Chondroitin Sulfate Proteoglycan, Mediates Binding of Interferon-γ 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-γ (IFN-γ) binds to GAG; however, the relevance of this interaction for the biological activity of IFN-γ 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-γ. We found that treatment of HASMC with chondroitinase ABC, an enzyme that degrades chondroitin sulfate GAG, reduced IFN-γ binding by more than 50%. This treatment increased the affinity of 125I-IFN-γ for cells from a K d value of about 93 nm to aK d value of about 33 nm. However, the total binding was reduced from 9.3 ± 0.77 pmol/μg to 3.0 ± 0.23 pmol/mg (n = 4). Interestingly, pretreatment with chondroitinase ABC reduced significantly the cellular response toward IFN-γ. The interaction of IFN-γ with chondroitin sulfate GAG was confirmed by affinity chromatography of isolated cell-associated35S-, 3H-labeled PG on a column with immobilized IFN-γ. The cell-associated PG that binds to IFN-γ 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-γ 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-γ. These results indicate that IFN-γ 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-γ at the cell surface, thus facilitating its binding to high affinity receptors and modulating the ability of IFN-γ to signal a cellular response.

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 &mgr;mol/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 PPAR&ggr; agonist rosiglitazone (10 &mgr;mol/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.

Structure of low density lipoprotein in complexes formed with arterial matrix components

Abstract Temperature dependent techniques (differential scanning calorimetry, polarizing light microscopy and n.m.r. spectroscopy) were used to study the physico-chemical state of low density lipoprotein (LDL) in LDL-aorta glycosaminoglycan and LDL aorta proteoglycan complexes. Complex formation between LDL and glycosaminoglycans (GAGs) resulted in a reversible liquid-liquid crystalline reorganization of the core lipids within the LDL molecule. In the proteoglycan-LDL complexes, prepared by the addition of porcine arterial proteoglycans or of human ‘lipoprotein complexing proteoglycan’, the formation of liquid crystals was an irreversible process, suggesting that the protein moiety of the proteoglycans also participates in the interaction with LDL. Arterial specimens were also examined. In atheromatous intima samples, spherulites with the above characteristics could be identified at the sites of lipoprotein deposition. Since proteoglycans are present in the arterial intima media, the phenomena observed in the present model system may also take place in vivo . The formation of proteoglycan-LDL complexes may result in the establishment of a liquid crystalline LDL structure and may thus play a role in the immobilization of LDL and in the development of the atherosclerotic lesion.

Hydrolytic Enzymes Released From Resident Macrophages and Located in the Intima Extracellular Matrix as Agents That Modify Retained Apolipoprotein B Lipoproteins

Identification of the molecular mechanisms causing the focal response of arterial cells that induce atherosclerotic lesions may open new therapeutic venues for prevention of this disease. Many of the products that appear to trigger the tissue reaction originate from apolipoprotein B (apoB)-lipoproteins retained in the intima extracellular matrix. These may be key phenomena contributing to the initial and late phases of atherosclerotic plaque development.1–4⇓⇓⇓ The preferential retention of apoB-lipoproteins, especially LDL, can lead to direct modifications of the labile structure of these complex particles and furthermore could provide the time required for enzymatic and nonenzymatic more profound alterations of their lipid and protein moieties.3,4⇓ Such structural changes may produce lipid and peptide neo-epitopes, lipid hydrolytic, and oxidative products with potent biological effects. Degradation of the apoB-100 and the polar lipid surface components can also lead to aggregation and fusion of lipoprotein particles that eventually form part of the complex array of extracellular lipid aggregates that grow with the progress of lesions.5 The structural and enzymatic agents that could contribute to such alterations in the extracellular intima may pre-exist there, like the proteoglycans, or could be secreted by macrophages and other intima-residing cells. These modifications may be part of a physiological scavenging process for removal of undesirable lipoprotein components. However, with elevated circulating levels of apoB-lipoproteins, especially at sites where intimal thickening occurs by matrix expansion, the beneficial scavenging process may become insufficient …

Cationic polypeptides modulate in vitro association of low density lipoprotein with arterial proteoglycans, fibroblasts, and arterial tissue.

Polymers of lys (plys) and arg (parg) were found to be efficient inhibitors of the formation of complexes between low density lipoprotein (LDL) and human chondroitin-6-sulfate-rich proteoglycans. Displacement curves indicate that efficiency was dependent on molecular weight. Inclusion of alanine in the polymer up to a 1:1 molar ratio (plys,ala) has a moderate effect on displacing capacity. Poly-L-lys (plys) and poly-L-arg (parg) exhibited similar displacing ability. Inclusion of tryptophan in plys and parg diminished their effect, whereas inclusion of serine in plys,ser (3:1) improved it. Plys (18.3 kD) stimulates LDL binding to human fibroblasts. This may be due to the association of polylysine to LDL, leading to an increase in its positive charge. These more positively charged LDL may have an increased association with the negatively charged region of the apolipoprotein B/E receptor. Perfusion experiments on rabbit aortic segments were used to measure the influx of 125I-LDL into the intima and to study the effect of basic polypeptides. Plys in a 10:1 molar ratio decreased the LDL uptake by approximately 25% when added to the system together with the LDL or in experiments in which the tissue segments were pre-perfused with plys, and LDL was added after elimination of the plys. The results suggest that polycationic polypeptides, due to their strong affinity for sulfated proteoglycans, interfere with the interactions of LDL with components of the arterial extracellular matrix.