Adenosine Receptors in Health and Disease

1
Increased Cardiovascular Risk with Pain-relieving
Coxib Medications Disruption of Cholesterol
Metabolism
Allison B. Reiss, M.D. Vascular Biology
Institute Winthrop University Hospital
2
The Scope of the Current Problem for Coxibs and
NSAIDs and CV/GI Risk

• Given the amount of recent information in the lay
  press and medical journals, patients are confused
  about both the CV and GI effects of NSAIDs vs.
  coxibs and reluctant to follow physician
  recommendations
• Physicians are reluctant to prescribe newer
  medication for their patients, as it takes a lot
  of time to evaluate the scientific evidence,
  data are sometimes contradictory, and they are
  concerned about potential legal issues, real or
  imagined

NSAIDsnonsteroidal anti-inflammatory drugs
GIgastrointestinal CVcardiovascular.
3
Pathological Stages of Atherogenesis

• Endothelial injury
• Monocyte recruitment by injured endothelium
• Fatty streak formation
• Fibrous plaque maturation

4
Cholesterol is Metabolized in the Artery Wall

• Not just a pipe, but a metabolically active
  organ system that maintains vascular homeostasis
• An active participant in the atherosclerotic
  process
• Endothelial dysfunction may contribute to
  development and clinical expression of
  atherosclerosis
• 1989 cholesterol 27-hydroxylase, a cholesterol
  metabolizing enzyme, is sequenced and cloned
• 27-Hydroxylase previously thought to be a liver
  enzyme, but

5
1994 Cholesterol is Not Just Metabolized in the
Liver

• Reiss AB, Martin KO, Javitt NB, Martin DW, Grossi
  EA, Galloway AC. Sterol 27-hydroxylase high
  levels of activity in vascular endothelium. J
  Lipid Res. 1994351026-1030.
• Bjorkhem I, Andersson O, Diczfalusy U, Sevastik
  B, Xiu R-J, Duan C, Lund E. Atherosclerosis and
  sterol 27-hydroxylase evidence for a role of
  this enzyme in elimination of cholesterol from
  human macrophages. Proc Natl Acad Sci U S A.
  1994918592-8596.

6
Endothelial Defense MechanismsAgainst Plaque
Formation
Cholesterol 27-Hydroxylase
27-Hydroxycholesterol
Cholesterol
7
Cholesterol Metabolism by the P450 Cholesterol
27-Hydroxylase
Cholesterol 27-hydroxylase
27-Hydroxycholesterol
Cholesterol
8
Reverse Cholesterol Transport
Circulating Cholesterol (bound by plasma
lipoproteins)
Cholesterol
ABCA1
ABCA1
Cholesterol
27-Hydroxylase
LXR
Cholesterol (bound by plasma lipoproteins)
27-Hydroxycholesterol
Macrophage
cholesterol
27-Hydroxycholesterol
27-Hydroxycholesterol
Excreted as Bile Acids from the Liver
9
Anti-Atherogenic Effects of
27-Hydroxycholesterol

• Inhibition of cholesterol synthesis
• Inhibition of foam cell formation
• Inhibition of smooth muscle proliferation
• Solubilization of cholesterol for transport to
  the liver
• Induces expression of the cholesterol efflux
  molecule ABCA1

Cholesterol 27-Hydroxylase
27-Hydroxycholesterol
Cholesterol
10
Cerebrotendinous Xanthomatosis
Genetically determined absence of cholesterol
27-hydroxylase leads, invariably, to the
development of premature coronary artery disease
Cholesterol 27-Hydroxylase
Cholesterol
27-Hydroxycholesterol
11
What Controls Vascular Expression of Cholesterol
27-Hydroxylase?

• Not the obvious bile acids, cholesterol or
  steroids
• A hint from the immune system autoimmune
  diseases such as lupus and rheumatoid arthritis
  are associated with an increased risk of
  atherosclerosis

12
Studying Human Gene Expression in the Laboratory
THP-1 Monocytes/Macrophages

• A human monocytic cell line with distinct
  monocytic markers isolated circa 1980
• Matures into macrophage-like adherent cells
  following stimulation with phorbol 12-myristate
  13-acetate or phorbol dibutyrate
• Derived from the peripheral blood of a 1 year old
  male with acute monocytic leukemia.
• Widely used as in vitro model for human
  monocytes/macrophages

13
IFN-g Downregulates Cholesterol27-Hydroxylase in
THP-1 Cells
27-OH mRNA
GAPDH
Cholesterol 27-OH mRNA ( control)
Control
IL-1
TNF-?
IFN-g
27-OH Protein
14
IC-C1q Diminish 27-Hydroxylase Message in
Peripheral Blood Mononuclear Cells
27-Hydroxylase
GAPDH
Control mRNA
Control
IC-C1q
15
In Vitro Foam Cell Formation
Murine Peritoneal Macrophages Or THP-1 Monocytoid
Cells
Foam Cells Oil Red O
16
IFN-? Increases Foam Cell Formation in
Lipid-loaded THP- 1cells
Control
IFN-?
17
ATP-Binding Cassette Transporter 1 (ABCA1)

• ABCA1 is an integral membrane protein that
  utilizes ATP as a source of energy for
  transporting lipids and other metabolites across
  membranes, where they are removed from cells by
  apolipoproteins such as apoA-I
• ABCA1 is a key regulator of cellular cholesterol
  and phospholipid transport.
• Patients with mutations in the ABCA1 gene have
  Tangier Disease with low levels of HDL, high
  levels of triglycerides and cardiovascular
  disease.

18
Immune Complexes Downregulate ABCA-1 Gene
Expression in THP-1 Cells
Control
IC
19
Cyclooxygenase-2 (COX-2) Inhibitors an Overview

• Selective inhibitors of COX-2 are highly
  effective anti-inflammatory and analgesic drugs
  that exert their action by preventing the
  formation of prostanoids.
• Used to relieve the symptoms of osteoarthritis
  and rheumatoid arthritis, and to treat
  dysmenorrhea.

20
The APPROVe Trial

• APPROVe trial (Adenomatous Polyp Prevention on
  Vioxx)
• Trial on benign sporadic colonic adenomas showed
  a significant increase in the incidence of
  cardiovascular events in rofecoxib-treated
  subjects compared with placebo (relative risk
  1.92, 95 confidence interval 1.19 to 3.11)
• Vioxx (rofecoxib) was withdrawn from the market
  by its manufacturer, Merck, on September 30,
  2004.
• Decreased utilization of this highly effective
  class of pain relieving medications in clinical
  practice.

21
COX-2 Inhibitors Elevate Heart Attack and Stroke
Incidence Why?

• COX-2-derived prostacyclins exert a
  cardioprotective effect.
• When drugs inhibit COX-2 activity, prostacyclin
  levels fall, leaving arteries more vulnerable to
  clotting.
• COX-2 inhibitors suppress biosynthesis of PGI2, a
  potent vasodilator and platelet inhibitor without
  a concomitant change in the production of the
  prothrombotic product, thromboxane (Tx)A2.
• COX-2 inhibitors raise BP more frequently than
  NSAIDs or placebo.

22
Prostanoid Biosynthesis
23
A New Theory

• There is intense interest in understanding the
  mechanism(s) that lead to increased CV
  consequences of COX-2 inhibition.
• COX-2 is widely expressed in atherosclerotic
  plaques and in the arterial wall.
• Concentrations of the COX-2 protein are increased
  in endothelial cells, smooth muscle cells, and
  macrophages in human atherosclerotic lesions.
• Does COX-2 inhibition affect cholesterol
  accumulation?
• HYPOTHESIS Pro-atherogenic mechanisms of
  selective COX-2 inhibition include compromise of
  cholesterol balance in the artery wall.

24
Dose-Dependent Decrease in 27-Hydroxylase mRNA
with COX-2 Inhibition in THP-1
25

• 1 2 3
  4
• Beta-Actin
• 27-OHase
• Control NS-398 NS-398 NS-398
• 10mM 50mM 100mM
• Cultured THP-1 cells were untreated or exposed
  to NS-398 for 18 hours. Total cell protein was
  isolated and 27-OHase detected with specific
  antibody with Beta-actin as an internal standard.

COX-2 Inhibition Decreases 27-Hydroxylase Protein
in THP-1 Cells
26
COX-2 Inhibition Decreases ABCA1 mRNA in THP-1
27
Dose-Dependent Decrease in ABCA1 Protein with
COX-2 Inhibition in THP-1 Macrophages
NS398
Control
50 mM
10 mM
100 ?M
ABCA1
ß-actin
28
NS-398 Increases Foam Cell Transformation of
Lipid-loaded THP-1Macrophages
a
b
NS-398
Control
Photomicrographs at 40X magnification of lipid
laden THP-1macrophages stained with oil-red-O.
Figure 1 . Photomicrographs at 40X magnification
of lipid laden macrophages stained with
oil-red-O. a) Minimal foam cell formation of
THP-1 macrophages treated with acetylated LDL
alone (control), compared with, b) THP-1
macrophages treated with NS-398 showing
significant increase in foam cell transformation
as seen by greater propensity to stain with
oil-red O.
Figure 1 . Photomicrographs at 40X magnification
of lipid laden macrophages stained with
oil-red-O. a) Minimal foam cell formation of
THP-1 macrophages treated with acetylated LDL
alone (control), compared with, b) THP-1
macrophages treated with NS-398 showing
significant increase in foam cell transformation
as seen by greater propensity to stain with
oil-red O.
Figure 1 . Photomicrographs at 40X magnification
of lipid laden macrophages stained with
oil-red-O. a) Minimal foam cell formation of
THP-1 macrophages treated with acetylated LDL
alone (control), compared with, b) THP-1
macrophages treated with NS-398 showing
significant increase in foam cell transformation
as seen by greater propensity to stain with
oil-red O.
Figure 1 . Photomicrographs at 40X magnification
of lipid laden macrophages stained with
oil-red-O. a) Minimal foam cell formation of
THP-1 macrophages treated with acetylated LDL
alone (control), compared with, b) THP-1
macrophages treated with NS-398 showing
significant increase in foam cell transformation
as seen by greater propensity to stain with
oil-red O.
Figure 1 . Photomicrographs at 40X magnification
of lipid laden macrophages stained with
oil-red-O. a) Minimal foam cell formation of
THP-1 macrophages treated with acetylated LDL
alone (control), compared with, b) THP-1
macrophages treated with NS-398 showing
significant increase in foam cell transformation
as seen by greater propensity to stain with
oil-red O.
29
Prostanoid Biosynthesis
30
27-OHase and ABCA1 mRNA are Decreased by NS398
and the Decrease is Reversed by PGE2 or PGD2, but
Not TXA2
A)
Control
PGE2
PGD2
TXA2
NS-398
B)
Control
PGE2
PGD2
TXA2
NS-398
31
Atheroma-promoting Effect of NS398 is Magnified
by SLE Plasma

• COX-2 inhibition in the presence of 50 SLE
  plasma increases THP-1 macrophage foam cell
  transformation significantly compared to SLE
  plasma alone (82.7 2.8 vs. 42.8 5.3,
  plt0.001).
• Addition of PGD2 or PGE2 significantly reversed
  NS398-induced foam cell transformation in the
  presence of SLE plasma.
• Exaggerated risk to SLE patients treated with
  COX-2 inhibitors, perhaps due to circulating
  inflammatory mediators.

32
COX-2 RNA Silencing Decreases ABCA1
a.

• Transfection with COX-2 siRNA significantly
  diminished ABCA1 message, 22.9 4.9 decrease
  vs. mock, p0.03.

33
COX-2 RNA Silencing Promotes Foam Cell Formation

• THP-1 macrophages transfected with COX-2 gene
  silencer had greater propensity to form lipid
  laden foam cells than mock transfected cells,
  58.3 1.6 increase, p0.003.

34
Acetaminophen Exposure Does Not Affect RCT Genes
35
SUMMARY COX Inhibition and Reverse Cholesterol
Transport

• COX inhibition decreases expression of
  cholesterol 27-OHase and ABCA1.
• COX inhibition also increases foam cell
  transformation in THP-1 macrophages.
• Observed increases in cardiovascular risk with
  COX inhibition may be ascribed at least in part
  to altered cholesterol metabolism.
• Notably, disrupted cholesterol balance may be
  corrected by specific PGs, suggesting possibile
  future therapeutic modalities for COX inhibition
  that would support an anti-atherogenic PG state.
• Signal transduction pathways involved in
  prostaglandin-mediated effects on 27-OHase levels
  remain to be elucidated.

36
Acknowledgements
Kamran Anwar, Ph.D. Sari D. Edelman, D.O. Edwin
S. L. Chan, M.D. Steven Carsons M.D. Arthritis
Foundation, S.L.E. Foundation, AHA, NIH