Decrease in pH Strongly Enhances Binding of Native, Proteolyzed, Lipolyzed, and Oxidized Low Density Lipoprotein Particles to Human Aortic Proteoglycans

Abstract

Binding of low density lipoprotein (LDL) to proteoglycans and modification of LDL are key processes in atherogenesis. Recently, it has been demonstrated that during atherogenesis the extracellular pH of atherosclerotic lesions decreases. We have examined the effect of the decreased pH on the binding of LDL to human aortic proteoglycans. The binding of native, oxidized, proteolyzed (␣-chymotrypsin-treated), or lipolyzed (sphingomyelinase-or phospholipase A 2 -treated) LDL particles to proteoglycans were measured in microtiter well assays at pH 5.5-7.5. We found that the lower the pH, the higher the amount of binding of LDL to proteoglycans. At the lowest pH tested (pH 5.5), the amounts of proteoglycan-bound native, proteolyzed, sphingomyelinase-, and phospholipase A 2 -treated LDL were 20-, 23-, 30-, and 37-fold higher, respectively, than at pH 7.5. Interestingly, although oxidized LDL failed to bind to proteoglycans at neutral pH, there was significant binding at acidic pH. Binding of native and modified LDL to proteoglycans at pH 5.5 was blocked by 1 M NaCl, indicating that at neutral pH LDL binds to proteoglycans via ionic interactions. Inhibition of this binding by acetylation and cyclohexanedione treatment of LDL showed that the positively charged amino acids of apolipoprotein B-100, lysine, and arginine, respectively, mediated the ionic interaction. Taken together, our results suggest that in areas of atherosclerotic arterial intima where the extracellular pH decreases, retention of LDL by proteoglycans is enhanced, leading to extracellular accumulation of LDL and progression of the disease.Accumulation of cholesterol in the inner layer of the arterial wall, the intima, leads to the development of atherosclerotic lesions, the lesional cholesterol being mostly derived from circulating low density lipoprotein (LDL) 2 particles (1). Once LDL particles have entered the subendothelial space they are entrapped by the extracellular matrix, particularly by intimal proteoglycans (2-4), which form an organized, negatively charged, tight network (5, 6). The retention of LDL particles by proteoglycans is considered to have key role in the development of atherosclerosis (7,8). A role for LDL retention in experimental atherosclerosis has been directly demonstrated in mice expressing proteoglycan bindingdefective human apoB-100. In these transgenic mice, formation of early atherosclerotic plaques is significantly decreased (9, 10), but this modification does not prevent the development of atherosclerosis at later stages (10).Numerous hydrolytic enzymes and pro-oxidative agents are found in the arterial intima, especially in atherosclerotic lesions, and LDL particles that enter the arterial intima are susceptible to proteolytic, lipolytic, and oxidative modifications, which can lead to aggregation and fusion of the modified LDL particles (11). Indeed, intimal LDL particles show signs of proteolysis, phospholipolysis, and oxidation, and intimal lipid droplets have features that suggest they are formed from modified LDL part...

High binding affinity of electronegative LDL to human aortic proteoglycans depends on its aggregation level

“…Hence, LDL(2) consisted of two subpopulations: one with higher and the other with slightly lower PG binding affinity than LDL (1). On the other hand, it has been described that a decrease in pH enhances the binding of native and aggregated LDL to PGs (26). We observed this acid pH effect in both LDL fractions (Fig.…”

“…Unbound LDL was removed, and wells were washed with 20 mM HEPES, 50 mM NaCl, 2 mM CaCl 2 , 2 mM MgCl 2 , pH 7.4. The amount of bound LDL was determined using an Amplex Red cholesterol kit (Molecular Probes) according to the manufacturerʼs recommendations (26).…”

Conformational changes of apoB-100 in SMase-modified LDL mediate formation of large aggregates at acidic pH

“…Importantly, hypoxic, and therefore also acidic, areas are frequently found in the deep layers of prelesional arteries, where lipids initially accumulate and where the formation of the extracellular lipid core is initiated ( 11,14 ). Because acidity increases the ability of human secretory SMase to hydrolyze LDL ( 9, 10 ), increases the binding of LDL to the extracellular matrix proteoglycans ( 45,46 ), and, as shown here, enhances the extent of aggregation of ceramide-enriched LDL, SMase-induced modifi cation of LDL is likely to be particularly deleterious in the acidic extracellular microenvironments of prelesional arteries and atherosclerotic plaques. exposed segments of apoB-100 are crucial for LDL aggregation.…”

Proteolysis sensitizes LDL particles to phospholipolysis by secretory phospholipase A2 group V and secretory sphingomyelinase

“…It appeared that chymase and cathepsin S led to more extensive release of TCA-soluble products (about 13%) than did plasmin (about 4%) (not shown). Thus, despite the signifi cant difference between the ability of sSMase to lipolyze cathepsin S-treated LDL and chymase-treated LDL, the degrees of proteolysis by these two proteases were comparable when earlier ( 30 ). Wells incubated with only blocking buffer served as controls.…”