Dr David C. GazeCardiac Research Scientist

Clinical Blood Sciences, St George’s Hospital & Medical School

Visiting Professor of Cardiovascular Biochemistry, Nanjing Tech University ChinaHon. Lecturer, Sports Science & Medicine, Brunel University

Biomarkers of Cardiovascular Disease&

Future Novel Markers of Ischemia

Disclosures

Unrestricted Educational grants

Lecture fees

Consultancy

Not for profit research costs

The Coronary Artery Evolution

Time

Michelangelo di Lodovico Buonarroti Simoni (1475-1564)

Office for National Statistics; Edinburgh (2002)

CHD, 120,891

Stroke, 66,726

Other CVD, 52,650

Lung cancer, 33,509

Colo-rectal cancer,16,155

Breast cancer, 13,011

Other Cancer, 95,848

Respiratory conditions,75,809Injurys and poisoning,

20,120All other causes,

10,1325

0 20000 40000 60000 80000 100000 120000 140000

Number of deaths

Deaths by cause in the UK 2001

IntermediateLesion

FoamCells

AtheromaFattyStreak

FibrousPlaque

ComplicatedLesion/Rupture

Growth mainly by lipid accumulation

From third decade oflife

From fourth decade oflife

Smooth muscleand collagen

Thrombosis,haematoma

From first decade of life

EndothelialDysfunction

Atherothrombosis: With us for life

IrreversibleISCHEMIC CELL DAMAGE

Reversible small area large area

Pro-inflammatoryCytokines Plaque

DestabilisationPlaqueRupture

Acute Phasereactants

Ischemia Necrosis Dysfunction

Atherothrombosis: With us for life

IntermediateLesion

FoamCells

AtheromaFattyStreak

FibrousPlaque

ComplicatedLesion/Rupture

EndothelialDysfunction

Chest Pain Challenge

• Risk Stratification– Must risk-stratify a large patient population

quickly, with limited diagnostic information• Rule In

– Therapy must be administered as soon aspossible to save myocardium

– Problem is identifying these patients in time• Rule Out

– Chest pain patients consume substantial hospitaltime and resources (US $12B year)

– Problem is time/cost to discharge with confidence

Chronological Development ofCardiac Markers

Gaze & Collinson: Exp. Opin. Drug Metab. Toxicol. 2005;1:715-725

1950 19701960 1980 1990 2000

ASTin AMI

CK inAMI

Electrophoresisfor

CK & LD

INHfor

CK-MB

myoglobinRIA

WHODiagnosis

of MI

CK-MBmass

MAbCK-MB

cTnTin AMI

cTnTin UA

cTnI inAMI

cTnrisk

stratification

IMAWB-Ch

Nourin-1

NT-Pro&

BNPESC/ACCdefinition

ofAMI

Faster Recognition Æ Earlier Decisions Æ Better Outcomes

• Time is Muscle

• Therapy must be administered as soon as possible to savemyocardium– Fibrolynitics– Anticoagulants– Antiplatelet Agents (GP IIb/IIIa)– Percutaneous Coronary Intervention (PCI)

All withinminutes

Plaque formationVascular risk markers

• Cholesterol– HDL– LDL– IDL– VLDL– Non HDL cholesterol– Total cholesterol/HDL ratio– Apolipoprotein A1– Apolipoprotein B100

• Lp(a)• Homocysteine

Metabolic markers

• Albumin• Insulin• Gherelin• Leptin• Adiponectin• Resistin

What is Ischemia?

Time

Oxygen

Oxygen supply diminishes withdisease progression

Oxygen demand changesdaily and during life

SA SA UA MI

Ischemia occurs whenO2 demand exceeds supply

Stable Angina (SA) Ischemia due to stable plaque / exertionUnstable Angina (UA) Ischemia due to plaque disruption / thrombusAcute Myocardial Infarction (AMI) Myocardial necrosis due to prolonged ischemiaAcute Coronary Syndromes (ACS) Unstable Angina and Acute Myocardial Infarction

Onset ofnecrosis

PlaqueDisruption

How is Ischaemia Currently Diagnosed

• There is no gold standard for diagnosis of cardiac ischemia

• Diagnosis of ischemia is challenging, and uses multiple imperfect tools

– Clinical Assessment (signs and symptoms)– Presentation ECG (standard of care)– Necrosis Markers (detects consequences of prolonged ischemia)– Other Diagnostic Tools

ê Echocardiogramê Exercise testingê Technetium-99m Sestamibi

• Evaluation of a new diagnostic tool is difficult

Selker et al: Ann. Emerg. Med. 1997

All NecrosisAll Ischemia Some Ischemia, some Necrosis

ACS Sequence and Timing

Plaque

Rupture

OnsetofP

ain

ED

Presentation

Discharge

0-12 to0 hrs

12 to24 hrs

Time

Ischaemiamarker

Necrosismarker

Cardiac Dysfunctionmarker

Am

ountofTissue

Plaque destabilisation: Inflammatory markers

Pentraxins C reactive protein (CRP)

Pentraxin 3

ReactiveOxygenspecies

Surrogate moleculesoxidised low-density lipoproteinmalondialdehyde,myeloperoxidaseIsoprostanes

Uric acid (xanthine oxidase)

Cytokines Tumour necrosis factor (TNF)

Interleukin 6 (IL-6)

Osteoprotegrin

Chemokines Monocyte chemoattractant factor(MCP)

Apoptosis markers FasR

Adhesion molecules CD40

P selectin

E selectin

L selectin

ICAM-1

VCAM-1

Growth factors VEGF

PlGF

HGF

EGF

Soluble CD40 ligand (sCD40L)

• Circulating sCD40L derived from activated platelets.• Triggers inflammatory reaction in vascular endothelial cells via

secretion of cytokines and chemokines

• Increased [sCD40L]

– AMI and UAPAukrust et al: Circulation 1999;100:614-20.Garlichs et al: Heart 2001;86:649-55.

– Following PCICipollone et al: Circulation 2003;108:2776-82

Soluble CD40 ligand (sCD40L)

sCD40L in ACS (OPUS-TIMI 16)

Varo et al: Circulation 2003;108:1049-52

Varo et al: Circulation 2003;108:1049-52

Plaque Rupture I

Ischemia

• WB Choline• Unbound free fatty acids• Ischemia modified albumin

Necrosis

Cytosolic CK-MBMyoglobinHeart fatty acid bindingprotein (HFABP)

Structural Cardiac troponinscardiac troponin Tcardiac troponin IMyosin light chains(MLC)

Whole blood Choline

• Choline is released by cleavage of membrane phospholipidsby phospholipase D to yield plasma choline (PCHO).Choline is then taken by red blood cells.

• Phospholipase activation occurs in a number of processesthought to be involved in plaque destabilisation.

• Ischemic membrane damage produces phospholipidbreakdown and uptake into red blood cells by a cholinetransporter.

• WBCHO was measured by HPLC-mass spectrometry prospectively on

admission in 327 patients.

• Final Dx of AMI by ESC/ACC criteria using cTnT/I

• 30 day follow up as the outcome measure.

• WBCHO was a good indicator of major adverse cardiac events (MACE)

whether used alone or in combination with cardiac troponins.

• It was not a good marker for a subsequent diagnosis of AMI but did

distinguish between high risk and low risk patients without AMI.

Dane O et al: Am. J. Cardiol. 2003;91:1060-1067

Whole blood Choline

Increased risk of death or arrestat 30 days associated withincreasing quartiles of Choline

Unbound Free Fatty Acids (FFAu)

• Ischaemia is associated with the release of fatty acids (FFA) frommuscle tissue, especially cardiac muscle.

• The majority of FFA are bound to albumin but a small amount ofunbound free fatty acids (FFAu) are found in the serum.

• Coronary angioplasty model of ischaemia.• 22 patients• Measurements at 5 minutes pre-procedure and 30 minutes post

procedure.

• Accompanying ST segment changes in the ECG occurred in only 11of the patients, but was associated with the highest FFAu values.

Kleinfeld et al: Am. J. Cardiol. 1996;78:1350-4

Unbound Free Fatty Acids (FFAu)

• Post-PTCA [FFAu] higher (mean =103 nM) than pre-PTCA[FFAu].

• Mean post-PTCA [FFAu] 14-fold higher than the 7.5 nM valueobserved in healthy subjects.

• Accompanying ST segment changes (n=11) associated withthe highest FFAu values.

Kleinfeld et al: Am. J. Cardiol. 1996;78:1350-4

r=0.8

Coronary Angioplasty model

• 88 patients undergoingangioplasty

• Blood taken at– Baseline– Post procedure– 6 hours post– 24 hours post

Collinson et al: Clin. Chem. 2003;49:A38

IMA Values PCI Group

Sample timing peri-PCI

IMA24HIMA6HIMAPOSTIMAPRE

IMA

U/m

l

160

140

120

100

80

60

40

20

P=0.0002

P=0.004

IMA as Aid to Diagnosis of AMI

• Rule Out Myocardial Infarction (ROMI) trial

• >400 Patients, 4 US Hospitals

• IMA + Troponin have higher sensitivity, especially atpresentation

Wu et al: Cardiovascular Toxicology 2001

Matrix metalloproteinasesand their inhibitors

Matrix metalloproteinase 1-27 (MMP1-27)Tissue inhibitors of matrix metalloproteinases 1-4(TIMP1-4)

Collagen peptides Procollagen III aminopropeptide (PIIINP)

Procollagen type I carboxy-terminal peptide (PICP)Procollagen type I amino-terminal peptide (PINP)Type I collagen telopeptide (ICTP)Basement membrane laminin (BML)

Matrix glycoproteins/lectins Tenascin COsteopontinGalectin-3

Extracellular matrix turnover and remodelling

Plaque Rupture II

Biomechanical strain

Natriuretic peptidesAtrial natriuretic peptidepro-B-type natriuretic peptideN-terminal pro-B-type natriuretic peptide

Interleukin 33/ST2 (IL33/ST2)

Growth differentiation factor 15 (GDF 15)

Plaque Rupture III

Neurohormonal activation

AldosteronePro-ADH (Copeptin)

Adrenomedullin

Apelin

Endothelin

Relaxin

Urotensin

Evolution of Troponin Assays

• Better Precision with high sensitivity assays

• Definable Reference population

• Utility as a risk marker rather than eventmarker

Not Measureable

Evolution of Troponin Assays

Troponin Concentration

Freq

uenc

y

99th Percentile

Hig

hSe

nsiti

veLo

D

Not

Mea

sure

able

!

Con

tem

pora

ryLo

D

10% CV

Histological loss of cTn following occlusion

Dog LV 45min total occlusion

a) Patchy hypereosinophilia *b) cTnI loss in region of necrosis *

Left papillary muscle rat 3hr ofocclusion

c) Subtle hypereosinophilia *d) cTnI immunostaining variable but

decreased amounts of staining *

Fishbein et al: Cardiovascular Pathology 2003;12:65-71

Histological loss of cTn following ischemia

Fishbein et al: Cardiovascular Pathology 2003;12:65-71

Dog LV 6hr occlusion no reperfusion

a) Oedema/hypereosinophilia *b) cTnI immunostaining *c) cTnI immunostaining (x60)

Dog LV 1hr occlusion/3hr reperfusion

d) Hypereosinophilia in necrosis zonee) cTnT loss at infarct edgef) cTnI immunostaining (x60)

Your cardiactroponin result

suggestsyou’re in good

health

Cardiac Troponin - The Holy Grail?

What's the Catch? There’s Always a Catch…

Lab tests are not a replacementfor clinical assessment.

AMI remains a clinical diagnosis.

cTn+ does NOT equal AMI butcardiac myocyte injury.

Any troponin elevation requiresexplanation, not dismissal.

cTn elevation in non-ACS

Elevated cTnI in “normals”predicts an adverse outcome

• Predicts an adverse outcome.

• Increased event rate observed in thosepatients with low level but detectable cTnvalues.

• Zethelius, B. et al. Circulation 2006;113:1071-78• Wallace TW, et al Circulation 2006;113:1958-65

Zethelius, B. et al. Circulation 2006;113:1071-1078

All-cause mortality at 10 year follow up in relation to cTnI in men free from CVDat baseline and in men with prevalent CVD at baseline

Elevated cardiac troponin innon-ACS populations

• Secondary and Non-ischemic CardiacInjury

• Pathophysiology: direct myocardialinjury+/- cardiac ischemia

SICI: Secondary Ischaemic Cardiac Injury(NOT Primarily due to a Ruptured Coronary Plaque)

Coronary intervention

Sympathomimetics

Pulmonary embolusCoronary artery spasmCoronary artery embolisationCoronary artery inflammationwith microvasular occlusionEnd stage Renal FailureRhythm disordersAcute Heart failure

Direct coronary traumaExtreme endurance exercise

Primary/Elective Distal embolisation from atheroma debrisPTCA Side branch occlusionsCABG Global ischaemia from inadequate perfusion,

myocardial cell production of anoxia

CocaineCatecholamine Storm Head injury/CVA/intracerebral bleed

Presumed right heart strainJapan - upto 10% of admissionsClot, air or CABG

VasculitidiesConnective tissue disease/damage/SLESevere disease, but 50% normal coronariesProlonged tachycardia/bradycardia

Only if due to IHDStabbing/chest contortionExtreme marathons - Wall motion abnormalitiescTn positive deaths possibly due extreme O2 debt producingischaemia

Collinson & Stubbs: Heart 2003;89:1285-87

Known causes ofmyocarditis

Cardiac Trauma

Metabolic/toxic

Infection BacterialViral

InflammationAutoimmune Polymyositis

SclerodermaSarcoidosis

Drug-induced AlcoholChemotherapyEnvenomation

Direct Road traffic accidentStabbing

Cardiac Surgery

Renal failureMultiple organ failure (MOF)

NICI: Non Ischaemic Cardiac Injury

Collinson & Stubbs: Heart 2003;89:1285-87

Key Take Home Messages• Biochemical investigations are central to the Dx and

Management of CVD.

• Many biomarkers exist targeting different parts of thedisease continuum.

• Markers upstream of necrosis may be sensitive but atthe expense of specificity.

• cTn determined using high sensitivity assays can detectischemia.

• cTn may evolve in time from an acute event marker to arisk marker.

Any Questions?�