Heart and Vascular disease are

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

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Atherosclerosis Progression
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Cholesterol-filled foam cells are major component
of thickened wall
Foam Cells
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In clinically important lesions, much of
cholesterol is within Lysosomes
Endothelial Cells
Lipid-engorged Lysosome
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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.

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Foam Cell Formed by Internalization of
Cholesterol, derived primarily from modified Low
Density Lipoproteins (LDL)
Modified LDL
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Cellular Cholesterol Metabolisim
Lipoprotein
Macrophage
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Cholesterol accumulation in lysosomes implies a
failure of normal metabolism
Lysosome
Lysosome
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Why does cholesterol accumulate in late
endosomes/lysosomes?
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Tissue Culture Experiments(mildly oxidized LDL)
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Bi-phasic Accumulation
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Is oxidation required?
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Atherosclerotic Lesion

• Besides Ox-
• LDL, lesion
• Contains
• Extracellular lipid
• Aggregates of LDL

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No significant difference in phenotype with
different types of loading.
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Lyosomal Accumulation
/
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• Oxidation not required.

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What is the mechanism of Inhbition of Hydrolysis?
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• Possibilities
• Lack of hydrolase
• Direct inhibition of enzyme by sustrate
• Wrong environment

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Lysosensor Yellow/Blue
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Lysosensor Yellow/Blue
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Day 1 Agg-LDL

• After 3d, number of lysosomes remained steady
• After 3d, size of lysosomes increased

Day 7 Agg-LDL
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• Oxidation not required.
• Cholesterol accumulation inhibits ability of
  lysosomes to maintain acidic environment.

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

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Why does pH increase?
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Cholesterol Accumulation from Agg-LDL
FC accumulation precedes CE
FC
CE
300
200
µg cholesterol/mg cell protein
100
0
0
2
6
Days
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Filipin Staining for FC(AggLDL,THP-1 Days 2 and
6)
At both 2 and 6 days there is significant FC in
Lysosomes
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Is FC accumulation the culprit?
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Cholesterol from acetylated LDL does not
accumulate in Lysosomes
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Progesterone inhibits FC movement out of Lysosomes
Ac-LDL
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Cells loaded via ac-LDL in presence of
progesterone fail to acidify lysosomes
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Change in of Acidic Lysosomes
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• 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.

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Suggested Mechanism
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Is lysosomal inhibition long lived?
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Sequential Incubation
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No effect of 6 hr OxLDL Pretreatment
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Sequential Study Design
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3 day OxLDL treatment inhibits subsequent AcLDL
metabolism
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Free Cholesterol Accumulation
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• 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.

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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.

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Conclusions

• Foam cells of advanced lesions exhibit an
  acquired lysosomal storage disease

• Reducing lysosomal cholesterol could reestablish
  lysosome function

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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 Patr
ician Yancey, Ph.D. Hydrolysis Vanderbilt
University Larry Swift, Ph.D. Isolation of
Lysosomes with Vanderbilt University
varying cholesterol levels Funding NHLBI,
AHA, Vanderbilt University