Cholesterol efflux and reverse cholesterol transport assays in vivo

Question 1

RCT, including cholesterol efflux, can be
studied in

Answer 1

vivo

Question 2

Cholesterol efflux in vivo involves

Answer 2

the monitoring of cholesterol from cells to bloodstream

• The RCT assay is straight forward,
  however it can be unpleasant

Question 3

Summary of cholesterol efflux
and RCT assay in vivo

Answer 3

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

Question 4

how much cholesterol does ur brain have

Answer 4

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)

Question 5

how are lipids trasnproted to the CNS

Answer 5

by lipoproteins

Question 6

what Lp does the CNS not contain

Answer 6

LpB

Question 7

ApoA-I in the brain

Answer 7

ApoA-I is present at 0.5% of bloodstream
levels via some blood brain barrier transfer

Question 8

ApoE in the brain

Answer 8

ApoE is synthesized and secreted in the
CNS by astrocytes and microglial cells
– Is used to produce apoE-HDL

Question 9

ApoE-HDL what is it and where is it found

Answer 9

is a gamma-migrating
lipoprotein found almost exclusively in the
brain

Question 10

what are the three sizes of ApoE HDL

Answer 10

– 8 nm, 11 nm, 14 nm
– The 8 nm and 11 nm particles can also apoJ

Question 11

Synthesis of apoE-HDL

Answer 11

• 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)

Question 12

function of 8nm apoE

Answer 12

• 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

Question 13

In ABCA1-ko mice

Answer 13

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

Question 14

ApoE isoforms

Answer 14

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?

Question 15

Immunoblot analyses for apoA-I under
non-reducing conditions

Answer 15

revealed 3 bands:
– ApoA-I monomer
– ApoA-I dimer
– ApoA-I / apoA-II heterodimer

Question 16

Immunoblot analyses for apoA-I under
reducing conditions

Answer 16

Only an apoA-I monomer was detected
under reducing conditions
– Indicated that an amino acid on apoA-I was
likely substituted with cysteine

Question 17

ApoA-IMilano vs. apoA-I

Answer 17

Protein and DNA sequencing ultimately
identified Arg 173 from apoA-I being
substituted with Cys in apoA-IMilano

Question 18

HDL in ApoA-IMilano vs. apoA-I

Answer 18

Low mature HDL is observed in patients
with apoA-IMilano, but no cardiovascular
diseases are observed

Question 19

function of ApoA-IMilano vs. apoA-I

Answer 19

ApoA-I Milano appears to function better than
apoA-I

Question 20

Compared to apoA-I, apoA-IMilano

Answer 20

– Increased ability to scavenge oxidized lipids
– Inhibits platelet aggregation
– Leads to atherosclerotic lesion regression in
animal models
– Exhibits greater cholesterol efflux and RCT

Question 21

Age-related macular
degeneration (ARMD)

Answer 21

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

Question 22

what is ARMD caused by

Answer 22

Caused by damage to the retina that leads
to a distorted visual field in the center of
the eye (or macula)

Question 23

what is Drusen

Answer 23

• 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

Question 24

Drusen composition

Answer 24

• 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

Question 25

drusen lipoproteins

Answer 25

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)

Question 26

RPE cells synthesize

Answer 26

VLDL with unusual lipid content

Question 27

GWAS studies and ARMD

Answer 27

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

Question 28

Hepatic lipase -514 C/T HL activity

Answer 28

highest for CC middle for CT and lowest for TT

Question 29

Hepatic lipase -514 C/T T allele

Answer 29

• 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