Cholesterol efflux and reverse cholesterol transport assays in vivo Flashcards
RCT, including cholesterol efflux, can be
studied in
vivo
Cholesterol efflux in vivo involves
the monitoring of cholesterol from cells to bloodstream
- The RCT assay is straight forward,
however it can be unpleasant
Summary of cholesterol efflux
and RCT assay in vivo
RCT assays share the same labeling and
equilibration steps as for cholesterol efflux in vitro
* Macrophages are injected into the
intraperitoneum (of a small animal)
* Blood and feces are collected over 48h
* Blood, bile, & tissues collected after 48h
* All collected materials are assessed for
labeled cholesterol
how much cholesterol does ur brain have
The brain is only 2% of your total body
weight, but the brain holds 25% of the total mass your entire body cholesterol content
therefore Cholesterol and other lipids need to be
transported in the central nervous system
(CNS)
how are lipids trasnproted to the CNS
by lipoproteins
what Lp does the CNS not contain
LpB
ApoA-I in the brain
ApoA-I is present at 0.5% of bloodstream
levels via some blood brain barrier transfer
ApoE in the brain
ApoE is synthesized and secreted in the
CNS by astrocytes and microglial cells
– Is used to produce apoE-HDL
ApoE-HDL what is it and where is it found
is a gamma-migrating
lipoprotein found almost exclusively in the
brain
what are the three sizes of ApoE HDL
– 8 nm, 11 nm, 14 nm
– The 8 nm and 11 nm particles can also apoJ
Synthesis of apoE-HDL
- All forms of apoE-HDL contain unesterified
cholesterol and phospholipid (PL)
– 75% of PL is phosphatidylethanolamine
(which differs from plasma lipoproteins, where the bulk of PL is phosphatidylcholine)
function of 8nm apoE
- Currently thought that the 8 nm apoE-HDL is secreted intact from cells, and brain ABCA1 is responsible for transfering lipids to make larger apoE-HDL
In ABCA1-ko mice
apoE is only 20% of
normal levels in the brain and microglial
cells accumulate lipid
– Suggests that small (8 nm) apoE-HDL is
rapidly removed from the CNS or not secreted
-The LDLR is reduced in microglial cells
from ABCA1-ko mice, and ~80% reduction
of apoE-HDL observed in LDLR-ko mice
– LDLR is essential for synthesizing apoE-HDL
ApoE isoforms
ApoE has isoforms that are beneficial (E2)
or detrimental (E4) in Alzheimer’s disease
– Could apoE isoforms be linked with LDLR association for apoE-HDL synthesis?
– Could apoE isoforms result in different
“functionality’ for apoE-HDL?
Immunoblot analyses for apoA-I under
non-reducing conditions
revealed 3 bands:
– ApoA-I monomer
– ApoA-I dimer
– ApoA-I / apoA-II heterodimer
Immunoblot analyses for apoA-I under
reducing conditions
Only an apoA-I monomer was detected
under reducing conditions
– Indicated that an amino acid on apoA-I was
likely substituted with cysteine
ApoA-IMilano vs. apoA-I
Protein and DNA sequencing ultimately
identified Arg 173 from apoA-I being
substituted with Cys in apoA-IMilano
HDL in ApoA-IMilano vs. apoA-I
Low mature HDL is observed in patients
with apoA-IMilano, but no cardiovascular
diseases are observed
function of ApoA-IMilano vs. apoA-I
ApoA-I Milano appears to function better than
apoA-I
Compared to apoA-I, apoA-IMilano
– Increased ability to scavenge oxidized lipids
– Inhibits platelet aggregation
– Leads to atherosclerotic lesion regression in
animal models
– Exhibits greater cholesterol efflux and RCT
Age-related macular
degeneration (ARMD)
A major cause of blindness or visual
impairment in adults over 50 yrs. age
Is a progressive disease that is initiated
from the presence of drusen
what is ARMD caused by
Caused by damage to the retina that leads
to a distorted visual field in the center of
the eye (or macula)
what is Drusen
- Drusen (singular druse), is the German
term for a geode (or a glittering rock) - The composition of drusen was originally
thought to consist of cellular debris that
accumulated over time - Only very recently (~10 yrs.), it was found
that this is not the case
Drusen composition
- ApoE (in 100% of cases)
- ApoB100 (in 100% of cases)
- ApoA-I (in 60% of cases)
- ApoC-I (in 93% of cases)
- ApoC-II (% not known)
- ApoC-III (in 17% of cases)
- Except for apoC-III, all of these are
produced by retinal pigment epithelial cells
drusen lipoproteins
Drusen contain VLDL
* Lipoproteins were first identified and
isolated from drusen 8 years ago
* The lipoproteins were VLDL (based on
density), with a diameter of 60-100 nm
(which is similar to plasma VLDL)
RPE cells synthesize
VLDL with unusual lipid content
GWAS studies and ARMD
Two years ago, a genome-wide
association study identified a single
nucleotide polymorphism in the promoter
of HL to be directly linked with ARMD
– Polymorphism #rs10468017 (-514 C/T)
* Follow-up analyses of human eyes with
ARMD post-mortem showed a very high
level of HL expression in RPE cells
Hepatic lipase -514 C/T HL activity
highest for CC middle for CT and lowest for TT
Hepatic lipase -514 C/T T allele
- Subjects with the T-allele have larger HDL,
higher HDL-TG, higher HDL-C (specifically
within HDL 2), and higher apoA-I - The effects of the T-allele are more
pronounced in females than males - Subjects with the T-allele have reduced
incidence of ARMD
