Lipolytic Modification of LDL by Phospholipase A ₂ Induces Particle Aggregation in the Absence and Fusion in the Presence of Heparin

Abstract

Abstract-One of the first events in atherogenesis is modification of low density lipoprotein (LDL) particles in the arterial wall with ensuing formation of aggregated and fused lipid droplets. The accumulating particles are relatively depleted in phosphatidylcholine (PC). Recently, secretory phospholipase A 2 (PLA 2 ), an enzyme capable of hydrolyzing LDL PC into fatty acid and lysoPC molecules, has been found in atherosclerotic arteries. There is also evidence that both LDL and PLA 2 bind to the glycosaminoglycan (GAG) chains of extracellular proteoglycans in the arterial wall. Here we studied the effect of heparin GAG on the lipolytic modification of LDL by PLA 2 . Untreated LDL, heparin-treated LDL, and heparin-bound LDL were lipolyzed with bee venom PLA 2 . In the presence of albumin, lipolysis resulted in aggregation in all 3 preparations of the LDL particles. Lipolysis of untreated LDL did not result in aggregation if albumin was absent from the reaction medium, and the lipolytic products accumulated in the particles rendering them negatively charged. However, heparin-treated and heparin-bound lipolyzed LDL particles aggregated even in the absence of albumin. Importantly, in the presence of albumin, some of the heparin-treated and heparin-bound lipolyzed LDL particles fused, the proportion of fused particles being substantially greater when LDL was bound to heparin during lipolysis. In summary, lipolysis of LDL PC by PLA 2 under physiological conditions, which allow transfer of the lipolytic degradation products to albumin, leads to fusion of LDL particles in the presence, but not in the absence, of heparin. Thus, it is possible that within the GAG meshwork of the arterial intima, PLA 2 -induced modification of LDL is one source of the lipid droplets during atherogenesis.

Molecular Mechanism for Changes in Proteoglycan Binding on Compositional Changes of the Core and the Surface of Low-Density Lipoprotein–Containing Human Apolipoprotein B100

“…This was performed because lipolysis of LDL with PLA 2 in vitro induces particle aggregation. 22 The results from these studies showed that in vitro modification of recombinant LDL with PLA 2 gave similar results as in vivo modification of LDL, ie, binding affinity increased on PLA 2 digestion and site A was required for the increased interaction of sPLA 2 -modified LDL with glycosaminoglycans (data not shown). However, the intra-assay variations (ie, imprecision) and the inter-assay variations (ie, irreproducibility) were much more pronounced when analyzing the binding of LDL digested with PLA 2 in vitro compared with LDL modified in vivo.…”

“…The reason for this was to achieve similar modification of LDL as in patients with increased plasma levels of sPLA 2 . 13 Prolonged digestion of LDL with PLA 2 in vitro results in hydrolysis of nearly all of the phosphatidylcholines, in pronounced aggregation, and in the presence of glycosaminoglycans fusion of LDL, 22 ie, the modifications of LDL that are more likely to occur within the extracellular matrix of the arterial wall.…”

“…21 Lipolysis of Recombinant and Lion LDL With PLA 2 LDL (1 mg/mL) was incubated for 3 hours in 100 mmol/L HEPES, 5 mmol/L CaCl 2 , 2 mmol/L MgCl 2 , 140 mmol/L NaCl, and 10 mol/L butylated hydroxytoluene, with pH 7.4, with or without 50 ng/mL PLA 2 (from bee venom) in the presence of 2% (weight/ volume) bovine serum albumin. 22 Lipolysis was stopped by adding EDTA to achieve a final concentration of 10 mmol/L as described. 22 Samples of the reaction were separated by fast performance liquid chromatography gel filtration (⌬KTA Explorer; Amersham Pharmacia) with a Superose 6 HR 10/30 column.…”

“…22 Lipolysis was stopped by adding EDTA to achieve a final concentration of 10 mmol/L as described. 22 Samples of the reaction were separated by fast performance liquid chromatography gel filtration (⌬KTA Explorer; Amersham Pharmacia) with a Superose 6 HR 10/30 column. Aliquots of 200 L were injected onto the column and separated with PBS buffer, with pH 7.4, at a flow rate of 0.2 mL/min.…”

PLA 2 -V

“…3 This increase can be explained by the fact that LDL aggregates contain many copies of apoB-100, and, being multivalent, have the potential to interact with proteoglycans via more ionic interactions than their nonaggregated, native-sized counterparts. Interestingly, binding of LDL to proteoglycans promotes PLA 2 -induced aggregation and fusion of LDL particles, 22 and an enhanced interaction of sPLA 2 -V-treated LDL to proteoglycans has been demonstrated by Rosengren et al 15 In terms of atherogenesis, the full repertoire of the ApoB-100 -containing lipoproteins enter the arterial intima and become entrapped within the subendothelial proteoglycan meshwork. A subendothelially located macrophage synthesizes and secretes sPLA 2 -V, which hydrolyzes the phosphatidylcholines on the surface of the entrapped lipoproteins.…”