Increased Cardiovascular Risk Increased Cardiovascular Risk with Pain-relieving Coxibwith Pain-relieving Coxib

Medications: Disruption of Medications: Disruption of Cholesterol MetabolismCholesterol Metabolism

Increased Cardiovascular Risk Increased Cardiovascular Risk with Pain-relieving Coxibwith Pain-relieving Coxib

Medications: Disruption of Medications: Disruption of Cholesterol MetabolismCholesterol Metabolism

Allison B. Reiss, M.D.Allison B. Reiss, M.D.Vascular Biology InstituteVascular Biology Institute

Winthrop University HospitalWinthrop University Hospital

The Scope of the Current Problem The Scope of the Current Problem for Coxibs and NSAIDs and CV/GI Riskfor Coxibs and NSAIDs and CV/GI Risk

Given the amount of recent information in the lay Given the amount of recent information in the lay press and medical journals, patients are confused press and medical journals, patients are confused about both the CV and GI effects of NSAIDs vs. about both the CV and GI effects of NSAIDs vs. coxibs and reluctant to follow physician coxibs and reluctant to follow physician recommendationsrecommendations

Physicians are reluctant to prescribe newer Physicians are reluctant to prescribe newer medication for their patients, as it takes a lot medication for their patients, as it takes a lot of time to evaluate the scientific evidence, data are of time to evaluate the scientific evidence, data are sometimes contradictory, and they are concerned sometimes contradictory, and they are concerned about potential legal issues, real or imaginedabout potential legal issues, real or imagined

NSAIDs=nonsteroidal anti-inflammatory drugs; GI=gastrointestinal; CV=cardiovascular.

Pathological Stages of Pathological Stages of AtherogenesisAtherogenesis

Endothelial injuryEndothelial injury

Monocyte recruitment by Monocyte recruitment by injured endotheliuminjured endothelium

Fatty streak formationFatty streak formation

Fibrous plaque maturationFibrous plaque maturation

Cholesterol is Metabolized in the Cholesterol is Metabolized in the Artery WallArtery Wall

Not just a “pipe,” but a metabolically active organ Not just a “pipe,” but a metabolically active organ system that maintains vascular homeostasis system that maintains vascular homeostasis

An active participant in the atherosclerotic processAn active participant in the atherosclerotic process Endothelial dysfunction may contribute to Endothelial dysfunction may contribute to

development and clinical expression of development and clinical expression of atherosclerosisatherosclerosis

1989 cholesterol 27-hydroxylase, a cholesterol 1989 cholesterol 27-hydroxylase, a cholesterol metabolizing enzyme, is sequenced and clonedmetabolizing enzyme, is sequenced and cloned

27-Hydroxylase previously thought to be a liver 27-Hydroxylase previously thought to be a liver enzyme, but…enzyme, but…

1994: Cholesterol is Not Just 1994: Cholesterol is Not Just Metabolized in the LiverMetabolized in the Liver

Reiss AB, Martin KO, Javitt NB, Martin DW, Grossi Reiss AB, Martin KO, Javitt NB, Martin DW, Grossi EA, Galloway AC. EA, Galloway AC. Sterol 27-hydroxylase: high levels Sterol 27-hydroxylase: high levels of activity in vascular endothelium.of activity in vascular endothelium. J Lipid Res.J Lipid Res. 1994;35:1026-1030. 1994;35:1026-1030.

Bjorkhem I, Andersson O, Diczfalusy U, Sevastik B, Bjorkhem I, Andersson O, Diczfalusy U, Sevastik B, Xiu R-J, Duan C, Lund E. Xiu R-J, Duan C, Lund E. Atherosclerosis and sterol Atherosclerosis and sterol 27-hydroxylase: evidence for a role of this enzyme in 27-hydroxylase: evidence for a role of this enzyme in elimination of cholesterol from human macrophages.elimination of cholesterol from human macrophages. Proc Natl Acad Sci U S A.Proc Natl Acad Sci U S A. 1994;91:8592-8596. 1994;91:8592-8596.

Endothelial Defense MechanismsEndothelial Defense MechanismsAgainst Plaque FormationAgainst Plaque Formation

27-Hydroxycholesterol27-HydroxycholesterolCholesterol

Cholesterol 27-Hydroxylase

Cholesterol27-hydroxylase

Cholesterol 27-Hydroxycholesterol

OHOH

CH2OH

Cholesterol Metabolism by the P450 Cholesterol Metabolism by the P450 Cholesterol 27-HydroxylaseCholesterol 27-Hydroxylase

Circulating CholesterolCirculating Cholesterol(bound by plasma lipoproteins)(bound by plasma lipoproteins)

27-Hydroxycholesterol27-Hydroxycholesterol

cholesterolcholesterol27-Hydroxycholesterol27-Hydroxycholesterol

Excreted as Bile AcidsExcreted as Bile Acidsfrom the Liverfrom the Liver

CholesterolCholesterol

27-Hydroxylase27-Hydroxylase

27-Hydroxycholesterol27-Hydroxycholesterol

ReverseReverseCholesterolCholesterol

TransportTransport

MacrophageMacrophage

LXRLXR

ABCA1ABCA1 ABCA1ABCA1

CholesterolCholesterol

(bound by plasma (bound by plasma lipoproteins)lipoproteins)

CholesterolCholesterol

Anti-Atherogenic Effects of Anti-Atherogenic Effects of 27-Hydroxycholesterol 27-Hydroxycholesterol

Inhibition of cholesterol synthesisInhibition of cholesterol synthesis Inhibition of foam cell formationInhibition of foam cell formation Inhibition of smooth muscle proliferationInhibition of smooth muscle proliferation Solubilization of cholesterol for transport to the liverSolubilization of cholesterol for transport to the liver Induces expression of the cholesterol efflux Induces expression of the cholesterol efflux

molecule ABCA1molecule ABCA1

Cholesterol 27-Hydroxycholesterol

Cholesterol27-Hydroxylase

Cerebrotendinous XanthomatosisCerebrotendinous Xanthomatosis

Genetically determined absence of Genetically determined absence of cholesterol 27-hydroxylase leads, cholesterol 27-hydroxylase leads, invariably,invariably, to the development of premature coronary to the development of premature coronary

artery diseaseartery disease

Cholesterol 27-Hydroxycholesterol

Cholesterol27-Hydroxylase

What Controls Vascular Expression What Controls Vascular Expression of Cholesterol 27-Hydroxylase?of Cholesterol 27-Hydroxylase?

Not the obvious: bile acids, Not the obvious: bile acids, cholesterol or steroidscholesterol or steroids

A hint from the immune system: A hint from the immune system: autoimmune diseases such as autoimmune diseases such as lupus and rheumatoid arthritis are lupus and rheumatoid arthritis are associated with an increased risk of associated with an increased risk of atherosclerosisatherosclerosis

Studying Human Gene Expression in Studying Human Gene Expression in the Laboratory: THP-1 the Laboratory: THP-1

Monocytes/MacrophagesMonocytes/Macrophages A human monocytic cell line with distinct A human monocytic cell line with distinct

monocytic markers isolated circa 1980monocytic markers isolated circa 1980 Matures into macrophage-like adherent cells Matures into macrophage-like adherent cells

following stimulation with phorbol 12-following stimulation with phorbol 12-myristate 13-acetate or phorbol dibutyratemyristate 13-acetate or phorbol dibutyrate

Derived from the peripheral blood of a 1 year Derived from the peripheral blood of a 1 year old male with acute monocytic leukemia. old male with acute monocytic leukemia.

Widely used as in vitro model for human Widely used as in vitro model for human monocytes/macrophagesmonocytes/macrophages

IFN-IFN- Downregulates Cholesterol Downregulates Cholesterol27-Hydroxylase in THP-1 Cells27-Hydroxylase in THP-1 Cells

0

20

40

60

80

100

120

Control IL-1 TNF- IFN-

Ch

ole

ster

ol

27-O

H m

RN

A (

% c

on

tro

l)27-OH mRNA

27-OH Protein

GAPDH

IC-C1q Diminish 27-Hydroxylase Message IC-C1q Diminish 27-Hydroxylase Message in Peripheral Blood Mononuclear Cellsin Peripheral Blood Mononuclear Cells

27-HydroxylaseGAPDH

0

20

40

60

80

100

120

Control IC-C1q

% C

ontr

ol m

RN

A

In VitroIn Vitro Foam Cell Formation Foam Cell Formation

PMA

Acetylated LDL

Murine Peritoneal MacrophagesOr THP-1 Monocytoid Cells

Foam CellsOil Red O

IFN-IFN- Increases Foam Cell Formation in Increases Foam Cell Formation in

Lipid-loaded THP- 1cellsLipid-loaded THP- 1cells

Control IFN-

ATP-Binding Cassette Transporter 1ATP-Binding Cassette Transporter 1 (ABCA1)(ABCA1)

ABCA1 is an integral membrane protein that utilizes ABCA1 is an integral membrane protein that utilizes ATP as a source of energy for transporting lipids and ATP as a source of energy for transporting lipids and other metabolites across membranes, where they are other metabolites across membranes, where they are removed from cells by apolipoproteins such as apoA-I removed from cells by apolipoproteins such as apoA-I

ABCA1 is a key regulator of cellular cholesterol and ABCA1 is a key regulator of cellular cholesterol and phospholipid transport. phospholipid transport.

Patients with mutations in the ABCA1 gene have Patients with mutations in the ABCA1 gene have Tangier Disease with low levels of HDL, high levels of Tangier Disease with low levels of HDL, high levels of triglycerides and cardiovascular disease. triglycerides and cardiovascular disease.

Immune Complexes Downregulate ABCA-1 Gene Expression in THP-1 Cells

0

20

40

60

80

100

120%

cont

rol A

BCA1

mRN

A

Control IC

Cyclooxygenase-2 (COX-2) Cyclooxygenase-2 (COX-2) Inhibitors: an OverviewInhibitors: an Overview

Selective inhibitors of COX-2 are highly Selective inhibitors of COX-2 are highly effective anti-inflammatory and analgesic effective anti-inflammatory and analgesic drugs that exert their action by preventing drugs that exert their action by preventing the formation of prostanoids. the formation of prostanoids.

Used to relieve the symptoms of Used to relieve the symptoms of osteoarthritis and rheumatoid arthritis, and osteoarthritis and rheumatoid arthritis, and to treat dysmenorrhea. to treat dysmenorrhea.

The APPROVe TrialThe APPROVe Trial

APPROVe trial (Adenomatous Polyp Prevention on APPROVe trial (Adenomatous Polyp Prevention on Vioxx)Vioxx)

Trial on benign sporadic colonic adenomas showed Trial on benign sporadic colonic adenomas showed a significant increase in the incidence of a significant increase in the incidence of cardiovascular events in rofecoxib-treated subjects cardiovascular events in rofecoxib-treated subjects compared with placebo (relative risk 1.92, 95% compared with placebo (relative risk 1.92, 95% confidence interval 1.19 to 3.11) confidence interval 1.19 to 3.11)

Vioxx (rofecoxib) was withdrawn from the market by Vioxx (rofecoxib) was withdrawn from the market by its manufacturer, Merck, on September 30, 2004. its manufacturer, Merck, on September 30, 2004.

Decreased utilization of this highly effective class of Decreased utilization of this highly effective class of pain relieving medications in clinical practice.pain relieving medications in clinical practice.

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

COX-2-derived prostacyclins exert a cardioprotective COX-2-derived prostacyclins exert a cardioprotective effect. effect.

When drugs inhibit COX-2 activity, prostacyclin levels When drugs inhibit COX-2 activity, prostacyclin levels fall, leaving arteries more vulnerable to clotting.fall, leaving arteries more vulnerable to clotting.

COX-2 inhibitors suppress biosynthesis of PGI2, a COX-2 inhibitors suppress biosynthesis of PGI2, a potent vasodilator and platelet inhibitor without a potent vasodilator and platelet inhibitor without a concomitant change in the production of the concomitant change in the production of the prothrombotic product, thromboxane (Tx)A2. prothrombotic product, thromboxane (Tx)A2.

COX-2 inhibitors raise BP more frequently than COX-2 inhibitors raise BP more frequently than NSAIDs or placebo.NSAIDs or placebo.

Prostanoid BiosynthesisProstanoid Biosynthesis

A New TheoryA New Theory There is intense interest in understanding the There is intense interest in understanding the

mechanism(s) that lead to increased CV consequences of mechanism(s) that lead to increased CV consequences of COX-2 inhibition. COX-2 inhibition.

COX-2 is widely expressed in atherosclerotic plaques and COX-2 is widely expressed in atherosclerotic plaques and in the arterial wall.in the arterial wall.

Concentrations of the COX-2 protein are increased in Concentrations of the COX-2 protein are increased in endothelial cells, smooth muscle cells, and macrophages endothelial cells, smooth muscle cells, and macrophages in human atherosclerotic lesions. in human atherosclerotic lesions.

Does COX-2 inhibition affect cholesterol accumulation? Does COX-2 inhibition affect cholesterol accumulation? HYPOTHESIS: Pro-atherogenic mechanisms of HYPOTHESIS: Pro-atherogenic mechanisms of

selective COX-2 inhibition include compromise of selective COX-2 inhibition include compromise of cholesterol balance in the artery wall.cholesterol balance in the artery wall.

Dose-Dependent Decrease in 27-Dose-Dependent Decrease in 27-Hydroxylase mRNA with COX-2 Hydroxylase mRNA with COX-2

Inhibition in THP-1Inhibition in THP-1

Control 10 M 50 M 100 M0

25

50

75

100

**

**

**

% 2

7 H

yd

roxyla

se m

RN

A

exp

ressio

n

NS-398

11 2 3 4 2 3 4

Beta-ActinBeta-Actin

27-OHase27-OHase

Control NS-398 NS-398 NS-398Control NS-398 NS-398 NS-398 1010M 50M 50M 100M 100MM

Cultured THP-1 cells were untreated or exposed to Cultured THP-1 cells were untreated or exposed to NS-398 for 18 hours. Total cell protein was isolated NS-398 for 18 hours. Total cell protein was isolated and 27-OHase detected with specific antibody with and 27-OHase detected with specific antibody with Beta-actin as an internal standard.Beta-actin as an internal standard.

COX-2 Inhibition Decreases 27-Hydroxylase Protein in THP-1 Cells

COX-2 Inhibition Decreases COX-2 Inhibition Decreases ABCA1 mRNA in THP-1ABCA1 mRNA in THP-1

Control 10 M 50 M 100 M0

25

50

75

100

** ****

NS-398

% A

BC

A1 m

RN

Aexp

ressio

n

ABCA1

ß-actin

NS398Control

50 M10 M 100 M

Dose-Dependent Decrease in ABCA1 Protein with COX-2 Inhibition in THP-1

Macrophages

Photomicrographs at 40X magnification of lipid laden THP-1macrophages stained with oil-red-O.

Control

a b

NS-398

NS-398 Increases Foam Cell Transformation of Lipid-loaded THP-1Macrophages

Prostanoid BiosynthesisProstanoid Biosynthesis

27-OHase and ABCA1 mRNA are Decreased by NS398 and the 27-OHase and ABCA1 mRNA are Decreased by NS398 and the Decrease is Reversed by PGE2 or PGD2, but Not TXA2Decrease is Reversed by PGE2 or PGD2, but Not TXA2

0

50

100

150%

27 H

yd

ro

xylase m

RN

A

exp

ressio

n

A)

B)

Control PGE2 PGD2 TXA2

+ NS-398

Control PGE2 PGD2 TXA2

+ NS-398

0

50

100

150

200

250

300

350

% A

BC

A1 m

RN

A

exp

ressio

n

Atheroma-promoting Effect of NS398 Atheroma-promoting Effect of NS398 is Magnified by SLE Plasmais Magnified by SLE Plasma

COX-2 inhibition in the presence of 50% SLE plasma COX-2 inhibition in the presence of 50% SLE plasma increases THP-1 macrophage foam cell increases THP-1 macrophage foam cell transformation significantly compared to SLE transformation significantly compared to SLE plasma alone (82.7 ± 2.8 % vs. 42.8 ± 5.3%, p<0.001). plasma alone (82.7 ± 2.8 % vs. 42.8 ± 5.3%, p<0.001).

Addition of PGD2 or PGE2 significantly reversed Addition of PGD2 or PGE2 significantly reversed NS398-induced foam cell transformation in the NS398-induced foam cell transformation in the presence of SLE plasma. presence of SLE plasma.

Exaggerated risk to SLE patients treated with COX-2 Exaggerated risk to SLE patients treated with COX-2 inhibitors, perhaps due to circulating inflammatory inhibitors, perhaps due to circulating inflammatory mediators.mediators.

COX-2 RNA Silencing COX-2 RNA Silencing Decreases ABCA1Decreases ABCA1

• Transfection with COX-2 siRNA Transfection with COX-2 siRNA significantly diminished ABCA1 message, significantly diminished ABCA1 message, 22.9 ± 4.9% decrease vs. mock, p=0.03.22.9 ± 4.9% decrease vs. mock, p=0.03.

a.

COX-2 RNA Silencing COX-2 RNA Silencing Promotes Foam Cell FormationPromotes Foam Cell Formation

• THP-1 macrophages transfected THP-1 macrophages transfected with COX-2 gene silencer had with COX-2 gene silencer had greater propensity to form lipid greater propensity to form lipid laden foam cells than mock laden foam cells than mock transfected cells, 58.3% ± 1.6 transfected cells, 58.3% ± 1.6 increase, p=0.003. increase, p=0.003.

Acetaminophen Exposure Does Acetaminophen Exposure Does Not Affect RCT GenesNot Affect RCT Genes

Acetaminophen Effects on RCT genes

100 uM Ty1mM Ty

5mM Ty

Control

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Op

tica

l In

ten

sity

CD36

27-OHase

ABCA1

SUMMARY: COX Inhibition and Reverse SUMMARY: COX Inhibition and Reverse Cholesterol TransportCholesterol Transport

COX inhibition decreases expression of cholesterol 27-OHase COX inhibition decreases expression of cholesterol 27-OHase and ABCA1. and ABCA1.

COX inhibition also increases foam cell transformation in COX inhibition also increases foam cell transformation in THP-1 macrophages. THP-1 macrophages.

Observed increases in cardiovascular risk with COX Observed increases in cardiovascular risk with COX inhibition may be ascribed at least in part to altered inhibition may be ascribed at least in part to altered cholesterol metabolism. cholesterol metabolism.

Notably, disrupted cholesterol balance may be corrected by Notably, disrupted cholesterol balance may be corrected by specific PGs, suggesting possibile future therapeutic specific PGs, suggesting possibile future therapeutic modalities for COX inhibition that would support an anti-modalities for COX inhibition that would support an anti-atherogenic PG state.atherogenic PG state.

Signal transduction pathways involved in prostaglandin-Signal transduction pathways involved in prostaglandin-mediated effects on 27-OHase levels remain to be elucidated.mediated effects on 27-OHase levels remain to be elucidated.

AcknowledgementsAcknowledgements

Kamran Anwar, Ph.D.Kamran Anwar, Ph.D.

Sari D. Edelman, D.O.Sari D. Edelman, D.O.

Edwin S. L. Chan, M.D.Edwin S. L. Chan, M.D.

Steven Carsons M.D.Steven Carsons M.D.

Arthritis Foundation, Arthritis Foundation,

S.L.E. Foundation, AHA, NIHS.L.E. Foundation, AHA, NIH