Transcript HDL Cholesterol No Longer Is Good Cholesterol: Emerging Genetic Theories Sunita Dodani & Janice S Dorman University of Pittsburgh (Study proposal)

HDL Cholesterol No Longer Is
Good Cholesterol:
Emerging Genetic Theories
Sunita Dodani & Janice S Dorman
University of Pittsburgh
(Study proposal)
Presentation Overview
• Study background & significance
– Basic description & function of lipids,
lipoproteins and apoproteins
– HDL & apoprotein A-1
– New theories of LDL & HDL role in
atherosclerosis
– Concept of dysfunctional HDL
– Hypothesized causes of dysfunctionalHDL
• Study Rationale
• Study objectives
• Study Design & Methods
Study background & significance
• Fats are triacylglycerols containing
saturated fatty acids
- solid at room temp
- usually from animal source (however,
coconut & palm oil are saturated).
• Oils are triacylglycerols containing monoor polyunsaturated fatty acids
- liquid at room temp
- usually from plant sources (however, fish
oils are polyunsaturated).
• Phospholipids are triacylglycerols that have
a FA replaced with a phosphate linked FA
group.
• The major dietary sterol is cholesterol.
Functions Of Lipids
• Major components of cell membranes.
• Required to solubilise fat soluble vitamins
• Biosynthetic precursors (e.g. steroid
hormones from cholesterol)
• Protection (e.g. kidneys are shielded with
fat in fed state)
• Insulation
Lipid transport in the circulation
Proteins (apoproteins)
Cholesterol
HO
O
R
O
HO
HO
R
Non polar lipids in
core
(TAG and cholesterol
esters)
Lipids are insoluble in plasma. In order to be
transported they are combined with specific
proteins to form lipoproteins
Lipoproteins
• Particles found in plasma that transport
lipids including cholesterol
• Spherical particles with a hydrophobic
core (TG and esterified cholesterol)
• Apolipoproteins on the surface
• large: apoB (b-48 and B-100)
atherogenic
• smaller: apoA-I, apoC-II, apoE
• Classified on the basis of density (NMR
spectroscopy) and electrophoretic
mobility (VLDL; LDL; IDL;HDL; Lp-a)
Five classes of lipoprotein
(all contain characteristic amounts TAG, cholesterol, cholesterol
esters, phospholipids and Apoproteins – NMR Spectroscopy)
Increasing density
Class
Diameter
(nm)
Source and function
Major
apoliproteins
Chylomicrons
(CM)
500
Intestine. Transport
of dietary TAG
A, B48,
C(I,II,III) E
Very low density
lipoproteins
(VLDL)
43
Liver. Transport of
B100,
C(I,II,III) ,
E
Low density
lipoproteins
(LDL)
22
Formed in circulation by
partial breakdown of
IDL. Delivers
cholesterol to peripheral
tissues
B100
High density
lipoproteins
(HDL)
8
Liver. Removes “used”
cholesterol from tissues
and takes it to liver.
Donates apolipoproteins
to CM and VLDL
A,
C(I,II,III),
D, E
endogenously
synthesised TAG
Lipoprotein
class
Density
(g/mL)
Diameter
(nm)
Protein
% of dry
wt
Phospho
lipids %
Triacylglycerols %
of dry wt
HDL
1.0631.21
5 – 15
33
29
8
LDL
1.019 –
1.063
18 – 28
25
21
4
IDL
1.0061.019
25 - 50
18
22
31
VLDL
0.95 –
1.006
30 - 80
10
18
50
Chylomicrons
< 0.95
100 - 500
1 - 2
7
84
Composition and properties of human
lipoproteins
Atherogenic Particles
MEASUREMENTS:
VLDL
VLDLR
TG-rich lipoproteins
Apolipoprotein B
Non-HDL-C
IDL
LDL
Small,
dense
LDL
The Apolipoproteins
• Major components of lipoproteins
• Often referred to as aproteins
• Classified by alphabetical designation
(A thru E)
• The use of roman numeral suffix
describes the order in which the
Apolipoproteins emerge from a
chromatographic column
• Responsible for recognition of particle
by receptors
Apoproteins of human lipoproteins
• A-I (28,300)- principal protein in HDL
• 90 –120 mg% in plasma
• A-II (8,700) – occurs as dimer mainly in HDL
• 30 – 50 mg %; enhances hepatic lipase
activity
• B-48 (240,000) – found only in chylomicrons
– <5 mg %; derived from apo-B-100 gene by
RNA editing; lacks the LDL receptorbinding domain of apo-B-100
• B-100 (500,000) – principal protein in LDL
• 80 –100 mg %; binds to LDL receptor
(Circulation. 2004 Jun 15;109(23 Suppl):III2-7)
Apoproteins of human lipoproteins
• C-I (7,000) – found in chylomicrons, VLDL, HDL
• 4 – 7 mg %; may also activate LCAT
• C-II (8,800) - found in chylomicrons, VLDL, HDL
• 3 – 8 mg %; activates lipoprotein lipase
• C-III (8,800) - found in chylomicrons, VLDL,
IDL, HDL
• 8 15 mg %; inhibits lipoprotein lipase
• D (32,500) - found in HDL
• 8 – 10 mg %; also called cholesterol ester
transfer protein (CETP)
• E (34,100) - found in chylomicrons, VLDL, IDL
HDL
• 3 – 6 mg %; binds to LDL receptor
• H (50,000) – found in chylomicrons; also known as
b-2-glycoprotein I (involved in TG metabolism)
Major lipoprotein classes
Chylomicrons
• Formed through extrusion of
resynthesized triglycerides from the
mucosal cells into the intestinal lacteals
• Flow through the thoracic ducts into
the subclavian veins
• Degraded to remnants by the action of
lipoprotein lipase (LpL) which is located
on capillary endothelial cell surface
• Remnants are taken up by liver
parenchymal cells
Major lipoprotein classes
• VLDL
–
–
–
–
–
–
density >1.006
diameter 30 - 80nm
endogenous triglycerides
apoB-100, apoE, apoC-II/C-III
prebeta in electrophoresis
formed in the liver as nascent
VLDL (contains only triglycerides,
apoE and apoB)
This animation shows how VLDL are metabolised
once they enter the circulation from the liver
Tissues
B100
VLDL
B100
Lipoprotein
LDL lipase
E
CII
Some LDL taken up
by liver (LDL receptors)
Having lost TAG to
tissues LDL contains a
large proportion of
cholesterol/cholesterol
esters
Capillary wall
(endothelial surface)
Some LDL taken up by
other tissues (LDL receptors).
LDL delivers cholesterol and
TAG to the extra hepatic tissues.
LDL membrane receptor
• Found in clathrin coated pits (endocytosis)
• After endocytosis the receptor is recycled
whilst the LDL is degraded to releasing lipid
cargo. Cholesterol uptake down regulates the
cells own production of cholesterol and down
regulates LDL receptor synthesis
• Mutations in LDL receptors causes increased
plasma LDL levels (i.e. increased cholesterol
levels). This accelerates progress of
atherosclerosis (Familial hyperlipedimias).
• The cholesterol in LDL is often called “bad
cholesterol”.
LDL Heterogeneity: Small vs. large LDL
Production of Small Dense LDL
Although size (& lipid content) changes, there is
always 1 molecule apo B protein / particle
Lipoprotein receptors
B
B
B
B
Lipase
TG
CE
VLDL
Oxidation &
modification
(CETP)
VLDL LipidsIDL
largeLDL dense
remnants transferred
1
2
3
Relative Atherogenicity
of Large and Small
LDL Particles
B
B
CE
CE
Tg
Tg
High density lipoprotiens
• Act as a reservoir for apoproteins which can be
donated or received from other lipoproteins.
• Also play a vital role in scavenging “used”
cholesterol (reverse cholesterol transport):
apoproteins
HDL
HDL
HDL receptor mediated
endocytosis by liver
HDL
Liver
“used” cholesterol
transferred to HDL and
converted to cholesterol
ester
some cholesterol
ester transferred to
circulating VLDL
VLDL
LDLreceptor
Peripheral
mediated
LDL
LDL
tissues
endocytosis
Cholesterol can be
converted to bile salts
for excretion or
repackaged in VLDL for
redistribution
High density lipoprotiens HDL
• HDL carries “used” cholesterol (as CE)
back to the liver. Also donate some CE to
circulating VLDL for redistribution to
tissues.
• HDL taken up by liver and degraded. The
cholesterol is excreted as bile salts or
repackaged in VLDL for distribution to
tissues.
• Cholesterol synthesis in the liver is
regulated by the cholesterol arriving
through HDL (and dietary cholesterol
returned by chylomicrons remnants).
• Cholesterol (CE) in HDL is referred to as
“good cholesterol”
Helical Wheel Projection Of A Portion
Of Apolipoprotein A-1
HDL functioning
• HDL may transfer some cholesterol esters to
other lipoproteins.
• Some remain associated with HDL, which may
be taken up by liver & degraded.
• HDL thus transports cholesterol from tissues
& other lipoproteins to the liver, which can
excrete excess cholesterol as bile acids.
• High blood levels of HDL (the "good"
cholesterol) correlate with low incidence of
atherosclerosis
HDL > 40 mg/dl
(NCEP ATP III)
“Independent Predictor of CAD”
Interrelationship between lipoproteins
VLDL
FFA
CE
CETP
LPL
Liver
(LDL receptor)
TG
IDL
TG
CETP
CE
LPL
HDL
TG
FFA
CETP
Liver
(LDL receptor)
CE
LDL
Reverse Cholesterol Transport: Indirect
Extra hepatic tissues
Liver
Cholesterol is reused
or excreted in bile
Cholesterol esters
hydrolysis
Direct
Free cholesterol
ABCA1
Pre-b-HDL
A
LCAT
A
HDL
CETP
Cholesterol to
VLDL, IDL,LDL
Postprandial Changes in Plasma Lipid
Metabolism
Fat storage via LPL
Transfer of cholesterol from cells into plasma
reverse transport of cholesterol from peripheral
tissue to liver
Exchange of cholesterol for VLDL TG in HDL (CETP)
LCAT activity = esterification of free cholesterol (HDL)
These postprandial changes are beneficial in
maintaining whole body homeostasis of glycerides
and cholesterol
1. LCAT deficiency?
2. CETP deficiency?
3. Apo AI deficiency?
LDL
Liver
Dietary fat
Bile salts
small
intestine
chylomicrons
Endogenous
cholesterol
extra hepatic
tissue
Exogenous
cholesterol
chylomicrons
reminants
HDL
VLDL
IDL
capillaries
Lipoprotein Lipase
Lipoprotein Lipase
FFA
Adipose, muscle
FFA
ATHEROSCLEROSIS
Normal
Intima
Atherosclerosis
Proliferation of
intimal
connective
tissue
Medis
Adventitis
Lipid
accumulation
Tissue
breakdown
Factors such as high plasma cholesterol, smoking,
hypertension, diabetes and family history are all
associated with atherosclerosis.
Progression Of Atherosclerosis
Role of LDL in atherosclerosis- oxidation
Monocycle
Cholesterol
ester
LDL
Endothelial
injury
Platelets
Foam
cells
N-LDL
B
OX-LDL
B
Oxidized LDL
LUMEN
Endothelium
LDL, VLDL
Free radicals
INTIMA
?
IEL
Concentration
MEDIA
Residence time in arterial wall
Opportunity to be oxidized, taken up by macrophage,
glycated and be trapped
Modified
protein & lipid =
immunogenicity
Oxidized LDL
B
B
CE
CE
Tg CE
CE
CE
Tg CE
LDL C
Apo B
B
CE
CE
Tg CE
3.5
75
B
B
CE
CE
Tg CE
CE
CE
Tg CE
B
B
CE
CE CE
CE
Tg CE
Tg CE
B
B
CE
CE
CE
CE
Tg CE Tg CE
3.5
150
LDL apoB
Percent
40
30
20
10
0
40
25
small dense LDL is
more toxic (
oxidation etc.) but has
less cholesterol per
particle, measuring
mg/dl LDL cholesterol
doesn’t give
complete picture.
60 80 100 120 140 160 180 200 220 240 260 280
LDL cholesterol
20
15
10
5
0
mmol/l
1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
Controls
CHD patients
Measuring apoB provides a better index of particle number,
and an additional discriminator.
Macrophage induced inflammation
Rupture 7
Endothelium
Fatty streak
Monocyte
LDL Adhesion
1
X-LAM
2
LDL
Lipid
Oxidation
MM-LDL
Oxidation
Ox-LDL
MCP-1
Entry
3
M-CSF
MCP-1
M-CSF
IL-1
5
Differentiation
Modified LDL uptake
ROS
4 Macrophage
Smooth
Muscle cells
Smooth
muscle cell
proliferation
6
Development of
AtherosclerosisLumen
Intima
(Endothelial damage)
Fatty lesion
Risk factors
Regression
fibro lipid plaque formation
Fibrous cap
•Ground substance
•Collagen fibers
•Reticulum fibrils Thrombosis Ulceration
1
2
3
Progressive
Reduction in
blood flow
R
Smooth
Foam
Calcification
Muscle
cells
Intercellular lipid
Lipid core cells Haemorrhage
Fatty streak
Fibrous plaque Complicated lesion
Lumen
Diameter
100%
High LDL Levels
Non-Specific
Receptor Mediated
Pathway
Anti-oxidant therapy not effective
Relationship between HDL/LDL and heart disease
Monocyte (white blood cell)
Cholesterol to liver
LDL
vascular endothelium
(+)
differentiate
Oxidized LDL
Macrophage
LDL (+)
(-) HDL
Foam cells (fatty streak)
Arterial Intima
Role played by Apo A1
HDL Function
•
•
•
•
Removal of CE from LDL
Reverse Cholesterol transport
Apo A-1 prevent seeding of LDL
Apo A-1 prevent oxidized LDL
formation
HDL NO More a Good Cholesterol
Recent theories
 Framingham study of the incidence of
coronary heart disease (CHD) & HDL:44%
of the events occurred in men with HDLcholesterol levels of 40 mg/dl and 43% of
the events occurred in women with HDLcholesterol levels of 50 mg/dl
 A significant number of CHD events occur
in patients with normal LDL-cholesterol
levels and normal HDL-cholesterol levels.
Search for markers with better predictive
value
HDL NO More Good Cholesterol
 Increase CHD on high HDL
 Good HDL becomes bad (Navab M, 2002)
 Conversion of anti-inflammatory HDL
into pro-inflammatory
HDL
Increase risk of atherosclerosis
 Dysfunctional HDL has been detected
by special test of cell-free assay (Navab
M, 2002)
 Non-functioning Apo A1
“What makes HDL dysfunctional”?
HDL NO More Good Cholesterol
Recent hypotheses
1. Products of an inflammatory enzyme,
myeloperoxidase target main Apo A1
Converting HDL into pro-inflammatory
non-functional.
2. Apo A1
macrophages retain increase
cholesterol & cholesterol reverse transport
reduced. Myeperoxidase modify tyrosine
AA
(Fogelman AM
2003)
3. Dysfunctional HDL has increases
hydroperoxidase
This makes it proinflammatory (Van Lenten et al J. Clin. Invest. 96:
2758–2767 )
HDL NO More Good Cholesterol
Role of myeloperoxidase:
 Modify tyrosine AA in Apo A-1 100 X
more than same AA in other protein
 Study: In patients with CHD there
is substantial amount of tyrosine AA
in Apo A1 modified by
myeloperoxidase than in controls
(Zheng et al 2004)
Why this occur ???
Study Rationale
Un answered Questions
1. Which patients are susceptible to
develop dysfunctional HDL
2. What makes myeloperoxidase to
cause change in Apo A1
3. What is the role of
hydroperoxidase in causing
dysfunctional HDL
Study Rationale
Un answered Questions
1. Which patients are susceptible to
develop dysfunctional HDL
2. What makes myeloperoxidase to
cause change in Apo A1
3. What is the role of
hydroperoxidase in causing
dysfunctional HDL
Study Proposal
Objectives are to:
1. Measure the level of functional &
dysfunctional HDL in CHD cases and
controls
2. Assess the risk factors association with
dysfunctional HDL in both cases and
controls
3. Measure the levels of myeloperoxidase,
hydroperoxidase and Apo A-1 protein in
both cases and controls
4. Study the candidate genes for
myeloperoxidase, hydroperoxidase and
Apo A-1in cases and controls
Study Proposal
Study Design: Case-control
Study population:
 South Asian Immigrant population residing
in San Diego, California (total population1500 families)
 Number of Groups
1. Hindus: from India, Nepal & SriLanka
Ethnic groups: Gujarati, Marathi, Hindi
2. Muslims: from Pakistan, India &
Bangladesh
Study Proposal
Cases:
with known CAD
Controls:
without CAD
Risk factors under study
1. Traditional risk factors
2. Emerging risk factors
3. Myeloperoxidase levels
4. Hydroperoxidase levels
5. Apo A-1 levels
Study Proposal
Sample size:
Biostatistician
Data analysis:
Multiple logistic regression