During atherogenesis, lipid droplets appear in the extracellular space of the arterial intima. We previously observed generation of lipid droplets on the surface of exocytosed mast cell granules when granule neutral proteases degraded the granule-bound LDL particles and the particles became unstable and fused [Kovanen, P.T., & Kokkonen, J.O. (1991) J. Biol. Chem. 266, 4430-4436]. We have now extended our studies to the fluid phase and examined the effects of several proteases (trypsin, alpha-chymotrypsin, Pronase, plasmin, kallikrein, and thrombin) all known for their ability to cleave the apolipoprotein B-100 component (apoB-100) of LDL. The fused LDL particles were separated from unfused particles by gel filtration or by density gradient ultracentrifugation. Proteolytic degradation of LDL with trypsin, alpha-chymotrypsin, or Pronase led to fragmentation of apoB-100 and release of the fragments from the LDL particles and triggered particle fusion. In contrast, proteolytic degradation of LDL with plasmin, kallikrein, or thrombin, which also led to fragmentation of apoB-100 but not to release of fragments, did not trigger particle fusion. With advancing degradation of apoB-100, particles having progressively lower densities and larger sizes were generated. Thus, after incubation for 24 h with alpha-chymotrypsin (apoB-100:alpha-chymotrypsin mass ratio 10:1) 40% of the apoB-100 was degraded and about 30% of the LDL particles had fused and reached diameters of up to 70 nm and densities ranging from 1.020 to < 1.005 g/mL. When the proteolyzed LDL particles, both unfused and fused, were incubated with macrophages, only those particles that had undergone fusion were ingested and converted into intracellular cholesteryl ester droplets. Thus proteolysis of LDL with release of apoB-100 fragments renders the particles sufficiently unstable to fuse and thus to become liable to ingestion by macrophages. Since the fused LDL particles resemble the extracellular lipid droplets in the atherosclerotic arterial intima and generate foam cells in vitro, these findings support the idea that proteolytic fusion of LDL is an atherogenic process.

Degranulated mast cells are present in human fatty streaks. Chymase in granules released from degranulated rat serosal mast cells, i.e., in granule remnants, proteolyzes human high density lipoprotein 3 (HDL 3 ), and so reduces its ability to induce cholesterol efflux from macrophage foam cells in vitro. In this study we found that remnant chymase, by proteolyzing human serum and human aortic intimal fluid, prevents these two physiologic fluids from effectively inducing cholesterol efflux from cultured macrophage foam cells. Inhibition was strongest when remnants were added to apolipoprotein AI (apoAI)-containing lipoproteins; the remnants had no effect on the weaker efflux produced by apoAI-deficient serum. Western blot analysis showed that granule remnants degrade apoAI in serum and in intimal fluid. When released from remnants, chymase lost its ability to proteolyze HDL 3 in the presence of serum. Thus, remnant chymase (but not isolated chymase) was able to resist the natu-

Matrix metalloproteinases (MMPs) have been suggested to function in remodeling of the arterial wall, but no information is available on their possible role in early atherogenesis, when cholesterol accumulates in the cells of the arterial intima, forming foam cells. Here, we incubated the major component responsible for efflux of cholesterol from foam cells, high density lipoprotein 3 (HDL 3 ), with MMP-1, -3, -7, -9, or -12 at 37°C before adding it to cholesterol-loaded human monocyte-derived macrophages. After incubation with MMP-3, -7, or -12, the ability of HDL 3 to induce the high affinity component of cholesterol efflux from the macrophage foam cells was strongly reduced, whereas preincubation with MMP-1 reduced cholesterol efflux only slightly and preincubation with MMP-9 had no effect. These differential effects of the various MMPs were reflected in their differential abilities to degrade the small pre-␤ migrating particles present in the HDL 3 fraction. NH 2 -terminal sequence and mass spectrometric analyses of the apolipoprotein (apo) A-I fragments generated by MMPs revealed that those MMPs that strongly reduced cholesterol efflux (MMPs-3, -7, and -12) cleaved the COOH-terminal region of apoA-I and produced a major fragment of about 22 kDa, whereas MMPs-1 and -9, which had little and no effect on cholesterol efflux, degraded apoA-I only slightly and not at all, respectively. These results show, for the first time, that some members of the MMP family can degrade the apoA-I of HDL 3 , so blocking cholesterol efflux from macrophage foam cells. This expansion of the substrate repertoire of MMPs to include apoA suggests that these proteinases are directly involved in the accumulation of cholesterol in atherosclerotic lesions.

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