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Heart and Vascular disease are #1 cause of morbidity and mortality in devel-oped countries.

Heart and Vascular disease are #1 cause of morbidity and mortality in devel-oped countries. Atherosclerosis is underlying cause of most of this. Atherosclerosis Progression. Cholesterol-filled foam cells are major component of thickened wall. Foam Cells.

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Heart and Vascular disease are #1 cause of morbidity and mortality in devel-oped countries.

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  1. Heart and Vascular disease are #1 cause of morbidity and mortality in devel-oped countries. • Atherosclerosis is underlying cause of most of this.

  2. Atherosclerosis Progression

  3. Cholesterol-filled foam cells are major component of thickened wall Foam Cells

  4. In clinically important lesions, much of cholesterol is within Lysosomes Endothelial Cells Lipid-engorged Lysosome

  5. Key Points • Lysosomal dysfunction associated with atherosclerosis is an age-related acquired deficiency with the potential to be reversed. • Lysosomal dysfunction is result of cholesterol accumulation in lysosomes.

  6. Foam Cell Formed by Internalization of Cholesterol, derived primarily from modified Low Density Lipoproteins (LDL) Modified LDL

  7. Cellular Cholesterol Metabolisim Lipoprotein Macrophage

  8. Cholesterol accumulation in lysosomes implies a failure of normal metabolism Lysosome Lysosome

  9. Why does cholesterol accumulate in late endosomes/lysosomes?

  10. Tissue Culture Experiments(mildly oxidized LDL)

  11. Bi-phasic Accumulation

  12. Is oxidation required?

  13. Atherosclerotic Lesion • Besides Ox- • LDL, lesion • Contains: • Extracellular lipid • Aggregates of LDL

  14. No significant difference in phenotype with different types of loading.

  15. Lyosomal Accumulation /

  16. Oxidation not required.

  17. What is the mechanism of Inhbition of Hydrolysis?

  18. Possibilities: • Lack of hydrolase • Direct inhibition of enzyme by sustrate • Wrong environment

  19. Lysosensor Yellow/Blue

  20. Lysosensor Yellow/Blue

  21. Day 1 Agg-LDL • After 3d, number of lysosomes remained steady • After 3d, size of lysosomes increased Day 7 Agg-LDL

  22. Oxidation not required. • Cholesterol accumulation inhibits ability of lysosomes to maintain acidic environment.

  23. Oxidation not required. • Cholesterol accumulation inhibits ability of lysosomes to maintain acidic environment. • Increased pH inhibits both lipolysis and proteolysis.

  24. Why does pH increase?

  25. FC accumulation precedes CE Cholesterol Accumulation from Agg-LDL FC CE 300 200 µg cholesterol/mg cell protein 100 0 0 2 6 Days

  26. Nile Red LAMP-1 Filipin Overlap D2 D6 Filipin Staining for FC(AggLDL,THP-1 Days 2 and 6) At both 2 and 6 days there is significant FC in Lysosomes

  27. Is FC accumulation the culprit?

  28. Cholesterol from acetylated LDL does not accumulate in Lysosomes

  29. Progesterone inhibits FC movement out of Lysosomes Ac-LDL

  30. Cells loaded via ac-LDL in presence of progesterone fail to acidify lysosomes

  31. 24 hours 72 hours 100 100 90 90 80 80 70 70 60 60 % of Lysosomes % of Lysosomes 50 50 40 40 30 30 20 20 10 10 0 0 AcLDL AcLDL Prog+ Prog+ AcLD AcLD Prog Prog +Prog +Prog Wash Wash +Prog +Prog +Wash +Wash Change in % of Acidic Lysosomes

  32. Oxidation not required. • Cholesterol accumulation inhibits ability of lysosomes to maintain acidic environment. • Increased pH inhibits both lipolysis and proteolysis. • Accumulation of FC in lysosomes seems to mediate failure to acidify.

  33. Suggested Mechanism

  34. Is lysosomal inhibition long lived?

  35. Sequential Incubation

  36. FC CE Lys Inc 150 30 25 100 20 % of Cell Volume 15 µg cholesterol/mg protein 50 10 5 0 0 Ac Ox/Ac Ac Ox/Ac Treatment Treatment No effect of 6 hr OxLDL Pretreatment

  37. Sequential Study Design

  38. Lys Inc FC CE 400 50 40 300 30 µg /mg Cell Protein % of Cell Volume 200 20 100 10 0 0 Ac/Ac Ac/Ox Ox/Ox Ox/Ac Ac/Ac Ac/Ox Ox/Ox Ox/Ac 3 day OxLDL treatment inhibits subsequent AcLDL metabolism

  39. Free Cholesterol Accumulation

  40. Oxidation not required. • Cholesterol accumulation inhibits ability of lysosomes to maintain acidic environment. • Increased pH inhibits both lipolysis and proteolysis. • Accumulation of FC in lysosomes seems to mediate failure to acidify. • Once inhibited, the lysosomal dysfunction is maintained.

  41. Summary • CE phase is result of failure of lysosomes to maintain acidity. • Cholesterol accumulation (FC?) appears to be the culprit. • Inhibition of lysosome function is long lived and perhaps related to FC level.

  42. Conclusions • Foam cells of advanced lesions exhibit an acquired lysosomal storage disease • Reducing lysosomal cholesterol could reestablish lysosome function

  43. Acknowledgements Jerome Lab Evelyn Griffin, M.S.: Agg-LDL & DISP Loading, hydrolysis Jody Ullery, B.S.: Sequential Ox-LDL/Ac-LDL Brian Cox, B.S.: Lysosomal Acidification Collaborators Patrician Yancey, Ph.D.: Hydrolysis Vanderbilt University Larry Swift, Ph.D.: Isolation of Lysosomes with Vanderbilt University varying cholesterol levels Funding: NHLBI, AHA, Vanderbilt University

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