alpha-chymotrypsin and Arteriosclerosis

Topics: Amyloidosis; Animals; Arteriosclerosis; Arthritis, Rheumatoid; Blood Coagulation Disorders; Blood Coagulation Factors; Chymotrypsin; Chymotrypsinogen; Diabetes Mellitus; Emphysema; Fibrinolysis; Gastrointestinal Diseases; Humans; Kallikreins; Kidney Diseases; Muscular Dystrophies; Oligopeptides; Peptide Hydrolases; Substrate Specificity; Swine

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.

Topics: Animals; Arteriosclerosis; Cells, Cultured; Cholesterol Esters; Chymotrypsin; Female; Humans; Hydrolysis; Lipoproteins, LDL; Macrophages; Mice; Particle Size; Pronase; Trypsin

Lipid droplets resembling those seen in the extracellular space of the arterial intima were generated in vitro when granule proteases of rat serosal mast cells degraded the apolipoprotein B-100 (apoB-100) component of granule-bound low density lipoprotein (LDL), and the particles fused on the granule surface (Paanenen, K., and Kovanen, P. T. (1994) J. Biol. Chem. 269, 2023-2031). Moreover, the binding of the fused particles to the heparin proteoglycan component of the granules was found to be strengthened. We have now treated LDL particles with alpha-chymotrypsin and examined the strength with which the proteolytically modified LDL binds to human aortic proteoglycans on an affinity column. We found that chymotryptic degradation of the LDL particles triggered particle fusion. The higher the degree of proteolytic degradation, the higher were the degree of fusion and the strength of binding to the aortic proteoglycans. Separation of the proteolyzed particles by size exclusion chromatography into two fractions, unfused and fused particles, and analysis of their binding strengths revealed that not only the fused but also the unfused proteolyzed particles bound more tightly to the proteoglycans than did the native LDL particles. To investigate the mechanism underlying this increase in binding strength, we attached [13C]dimethyl groups to the lysines and used NMR spectroscopy to quantify the active lysine residues of apoB-100, which are thought to be located in basic areas of apoB-100 and involved in binding of LDL to proteoglycans. Analysis of the 13C-labeled particles showed that, despite loss of apoB-100 fragments from the particles, the number of active lysine residues in the unfused proteolyzed particles had not decreased. In the fused proteolyzed particles, the number of active lysine residues was markedly increased. Thus, proteolytic fusion appears to increase the number of basic domains of apoB-100, which would explain the observed increase in the strength of binding of the modified LDL particles to arterial proteoglycans. Since the fused particles resemble the small lipid droplets found in the atherosclerotic arterial intima, this LDL modification offers a plausible mechanism for the focal accumulation of lipid droplets in the extracellular proteoglycan matrix during atherogenesis.

Topics: Aorta; Apolipoprotein B-100; Apolipoproteins B; Arteriosclerosis; Chromatography, Affinity; Chymotrypsin; Humans; Lipid Metabolism; Lipoproteins, LDL; Magnetic Resonance Spectroscopy; Particle Size; Protein Binding; Proteoglycans

Topics: Adolescent; Adult; Aortitis; Arteriosclerosis; Chymotrypsin; Clinical Enzyme Tests; Diagnosis, Differential; Female; Humans; Male; Middle Aged; Pancreatic Elastase; Severity of Illness Index

Topics: Anti-Bacterial Agents; Arteriosclerosis; Burns; Chymotrypsin; Debridement; Deoxyribonucleases; Endarteritis; Enzyme Therapy; Female; Humans; Male; Nervous System Diseases; Skin Transplantation; Streptodornase and Streptokinase; Streptokinase; Transplantation, Autologous; Trypsin; Ulcer; Varicose Ulcer; Veins; Wounds and Injuries

Topics: Arteriosclerosis; Blindness; Chymotrypsin; Diabetes Complications; Eye Diseases; Hemorrhage; Humans; Hypertension; Phlebitis; Visual Fields; Vitreous Body

Topics: Alveolar Process; Angiography; Anti-Inflammatory Agents; Arteriosclerosis; Arteriovenous Anastomosis; Capillary Permeability; Chymotrypsin; Endarteritis; Gingiva; Gingivitis; Humans; Maxillary Artery; Periodontal Diseases; Periodontium; Plethysmography; Protein Deficiency; Sodium Isotopes; Trypsin; Vascular Diseases

Topics: Arteriosclerosis; Chymotrypsin; Diabetic Retinopathy; Drug Therapy; Geriatrics; Humans; Retinal Diseases; Retinal Vessels; Thrombosis

Topics: Arteriosclerosis; Blood Vessels; Chymotrypsin; Disease; Hematologic Tests; Infusions, Intra-Arterial; Vascular Diseases