heart and vascular disease are #1 cause of morbidity and mortality in devel-oped countries
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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. Atherosclerosis Progression. Cholesterol-filled foam cells are major component of thickened wall. Foam Cells. - PowerPoint PPT PresentationTRANSCRIPT
• Heart and Vascular disease are #1 cause of morbidity and mortality in devel-oped countries.
• Atherosclerosis is underlying cause of most of this.
• Heart and Vascular disease are #1 cause of morbidity and mortality in devel-oped countries.
• Atherosclerosis is underlying cause of most of this.
Atherosclerosis ProgressionAtherosclerosis Progression
Cholesterol-filled foam cells are major component of thickened wall
Cholesterol-filled foam cells are major component of thickened wall
FoamCells
In clinically important lesions, much of cholesterol is within Lysosomes
In clinically important lesions, much of cholesterol is within Lysosomes
EndothelialCells
Lipid-engorgedLysosome
Key PointsKey Points
1. Lysosomal dysfunction associated with atherosclerosis is an age-related acquired deficiency with the potential to be reversed.
2. Lysosomal dysfunction is result of cholesterol accumulation in lysosomes.
1. Lysosomal dysfunction associated with atherosclerosis is an age-related acquired deficiency with the potential to be reversed.
2. Lysosomal dysfunction is result of cholesterol accumulation in lysosomes.
Foam Cell Formed by Internalization of Cholesterol, derived primarily from
modified Low Density Lipoproteins (LDL)
Foam Cell Formed by Internalization of Cholesterol, derived primarily from
modified Low Density Lipoproteins (LDL)
ModifiedLDL
ModifiedLDL
Cellular Cholesterol MetabolisimCellular Cholesterol Metabolisim
LipoproteinLipoprotein
MacrophageMacrophage
Cholesterol accumulation in lysosomes implies a failure of normal metabolism
Cholesterol accumulation in lysosomes implies a failure of normal metabolism
LysosomeLysosome
LysosomeLysosome
Why does cholesterol accumulatein late endosomes/lysosomes?
Tissue Culture Experiments(mildly oxidized LDL)
Tissue Culture Experiments(mildly oxidized LDL)
Bi-phasic AccumulationBi-phasic Accumulation
Is oxidation required?
Atherosclerotic LesionAtherosclerotic Lesion
Besides Ox-LDL, lesionContains:• Extracellular
lipid• Aggregates
of LDL
Besides Ox-LDL, lesionContains:• Extracellular
lipid• Aggregates
of LDL
No significant difference in phenotype with different types of loading.
No significant difference in phenotype with different types of loading.
//
Lyosomal Accumulation
1. Oxidation not required.1. Oxidation not required.
What is the mechanism of Inhbition of Hydrolysis?
Possibilities:
• Lack of hydrolase
• Direct inhibition of enzyme by sustrate
• Wrong environment
Possibilities:
• Lack of hydrolase
• Direct inhibition of enzyme by sustrate
• Wrong environment
Lysosensor Yellow/BlueLysosensor Yellow/Blue
Lysosensor Yellow/BlueLysosensor Yellow/Blue
Day 1 Agg-LDLDay 1 Agg-LDL
Day 7 Agg-LDLDay 7 Agg-LDL
• After 3d, number of lysosomes remained steady
• After 3d, size of lysosomes increased
1. Oxidation not required.
2. Cholesterol accumulation inhibits ability of lysosomes to maintain acidic environment.
1. Oxidation not required.
2. Cholesterol accumulation inhibits ability of lysosomes to maintain acidic environment.
1. Oxidation not required.
2. Cholesterol accumulation inhibits ability of lysosomes to maintain acidic environment.
3. Increased pH inhibits both lipolysis and proteolysis.
1. Oxidation not required.
2. Cholesterol accumulation inhibits ability of lysosomes to maintain acidic environment.
3. Increased pH inhibits both lipolysis and proteolysis.
Why does pH increase?
Cholesterol Accumulation from Agg-LDL
0 2 6Days
0
100
200
300µ
g c
ho
lest
ero
l/mg
cel
l pro
tein
FC CE
FC accumulation precedes CEFC accumulation precedes CE
Filipin Staining for FC(AggLDL,THP-1 Days 2 and 6)
OverlapOverlapLAMP-1LAMP-1FilipinFilipinNile RedNile Red
D2D2
D6D6
At both 2 and 6 days there is significant FC in Lysosomes
At both 2 and 6 days there is significant FC in Lysosomes
Is FC accumulationthe culprit?
Cholesterol from acetylated LDL does not accumulate in Lysosomes
Cholesterol from acetylated LDL does not accumulate in Lysosomes
Progesterone inhibits FC movement out of Lysosomes
Ac-LDL
Cells loaded via ac-LDL in presence of progesterone fail to acidify
lysosomes
24 hours24 hours 72 hours72 hours
0
10
20
30
40
50
60
70
80
90
100
% o
f L
yso
som
es
Prog Prog+Wash
AcLD+Prog
AcLDL+Prog+Wash
0
10
20
30
40
50
60
70
80
90
100
% o
f L
yso
som
es
Prog Prog+Wash
AcLD+Prog
AcLDL+Prog+Wash
Change in % of Acidic Lysosomes
1. Oxidation not required.
2. Cholesterol accumulation inhibits ability of lysosomes to maintain acidic environment.
3. Increased pH inhibits both lipolysis and proteolysis.
4. Accumulation of FC in lysosomes seems to mediate failure to acidify.
1. Oxidation not required.
2. Cholesterol accumulation inhibits ability of lysosomes to maintain acidic environment.
3. Increased pH inhibits both lipolysis and proteolysis.
4. Accumulation of FC in lysosomes seems to mediate failure to acidify.
Suggested Mechanism
Is lysosomal inhibitionlong lived?
Sequential Incubation
Ac Ox/Ac
Treatment
0
50
100
150
µg
ch
ole
ster
ol/m
g p
rote
in
FC CE
Ac Ox/Ac
Treatment
0
5
10
15
20
25
30
% o
f C
ell V
olu
me
Lys Inc
No effect of 6 hr OxLDL Pretreatment
Sequential Study Design
3 day OxLDL treatment inhibits subsequent AcLDL metabolism
Ac/Ac Ac/Ox Ox/Ox Ox/Ac0
100
200
300
400
µg
/m
g C
ell
Pro
tein
FC CE
Ac/Ac Ac/Ox Ox/Ox Ox/Ac0
10
20
30
40
50
% o
f C
ell
Vo
lum
e
Lys Inc
Free Cholesterol Accumulation
1. Oxidation not required.
2. Cholesterol accumulation inhibits ability of lysosomes to maintain acidic environment.
3. Increased pH inhibits both lipolysis and proteolysis.
4. Accumulation of FC in lysosomes seems to mediate failure to acidify.
5. Once inhibited, the lysosomal dysfunction is maintained.
1. Oxidation not required.
2. Cholesterol accumulation inhibits ability of lysosomes to maintain acidic environment.
3. Increased pH inhibits both lipolysis and proteolysis.
4. Accumulation of FC in lysosomes seems to mediate failure to acidify.
5. Once inhibited, the lysosomal dysfunction is maintained.
SummarySummary• CE phase is result of
failure of lysosomes to maintain acidity.
• CE phase is result of failure of lysosomes to maintain acidity.
• Cholesterol accumulation (FC?) appears to be the culprit.
• Cholesterol accumulation (FC?) appears to be the culprit.
• Inhibition of lysosome function is long lived and perhaps related to FC level.
• Inhibition of lysosome function is long lived and perhaps related to FC level.
ConclusionsConclusions• Foam cells of
advanced lesions exhibit an acquired lysosomal storage disease
• Foam cells of advanced lesions exhibit an acquired lysosomal storage disease
• Reducing lysosomal cholesterol could reestablish lysosome function
• Reducing lysosomal cholesterol could reestablish lysosome function
AcknowledgementsAcknowledgements
Jerome LabEvelyn Griffin, M.S.: Agg-LDL & DISP Loading,
hydrolysisJody Ullery, B.S.: Sequential Ox-LDL/Ac-LDLBrian Cox, B.S.: Lysosomal Acidification
CollaboratorsPatrician Yancey, Ph.D.: Hydrolysis Vanderbilt UniversityLarry Swift, Ph.D.: Isolation of Lysosomes with Vanderbilt University varying cholesterol levels
Funding: NHLBI, AHA, Vanderbilt University
Jerome LabEvelyn Griffin, M.S.: Agg-LDL & DISP Loading,
hydrolysisJody Ullery, B.S.: Sequential Ox-LDL/Ac-LDLBrian Cox, B.S.: Lysosomal Acidification
CollaboratorsPatrician Yancey, Ph.D.: Hydrolysis Vanderbilt UniversityLarry Swift, Ph.D.: Isolation of Lysosomes with Vanderbilt University varying cholesterol levels
Funding: NHLBI, AHA, Vanderbilt University