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 (alpha-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.
Highlights
Numerous hydrolytic enzymes and pro-oxidative agents are found in the arterial intima, especially in atherosclerotic lesions, and low density lipoprotein (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)
The high concentration of LDL particles in the arterial intima results from their selective retention in the intima (33–35), proteoglycans playing an important role in this process by binding LDL particles that have entered the intimal space (10)
We have observed that proteolytic and phospholipolytic modifications of LDL enhance the binding of LDL to proteoglycans, especially if the modified LDL particles are aggregated/fused (reviewed by Oorni et al (11))
Summary
Isolation and Modifications of LDL—Human LDL (d ϭ 1.019 –1.050 g/ml) was isolated from plasma of healthy volunteers (n ϭ 8) by sequential ultracentrifugation in the presence of 3 mM EDTA (20, 21). The amounts of LDL are expressed in terms of their protein concentrations, which were determined by the method of Lowry et al (22) with bovine serum albumin as a standard. Each experiment was performed with LDL from at least two donors
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