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.

• 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