Clinical biomarkers focus on personalising health

Jesus Egido MD,PhD.Professor of Medicine. Autonoma University

Chief, Division of Nephrology and Hypertension Director of Vascular , Renal and Diabetes Labora tory

IIS-Fundacion Jimenez Díaz .Madrid

Proteomic and metabolomic strategiesin the search of new biomarkers in CVD

Searching for novel biomarkers of vascular complica tions

Diagnostic/ Pronostic

Pathogenetic Therapeutic

LDL-c

CD40LHSP27

Troponin

Endothelial cellsactivation:

vWF, sFasL, sICAM-1,sVCAM-1, sE-Selectin

Oxidative stress:Ox-LDL, Lp-LPA2, Myeloperoxidase,NADPH oxidase

Metaloproteinases:MMP-1, -2, -9, -10,

TIMP-1, -2PAPP-A Others:

TGF-β, Leukotrienes,COX-2,

PPAR receptors

Platelets activation:sCD40L, P-selectin,Platelet-monocyte

aggregates

Angiogenic factors:VEGF, PIGF, HGF

Acute phase: hs-CRP,

Serum amyloid A

Cytokines:IL-6, MCP-1,

TNF-α, IL-18, IL-10

Inflammation

Circulating biomarkers associated with atherosclerosis under investigation.

Traditional approach

Blanco-Colio LM et al. Expert Rev Proteomics 2009;6 :461-4

Cardiovascular proteomics , a translational approac h

Searching for novel biomarkers by proteomics in atherothrombotic disease

Cholesterol deposits in the vessel wall Plaque growth Complicated plaque

with trombos

MALDI-Imaging (proteic maps)

SIMS-Imaging (metaboloma)

LCM(Laser Microdisection)

Individual cells 2D-DIGE

Secretome(Differential Expression of proteins)

2D-E

Monocytes, RBC, plateletsPlasma

Tissue

Tuñón et al. J Am Coll Cardiol 2010;55:2009–16

33 out of1400 proteins analyzed were differentially expressed in ACS

Dardé M et al. Journal of Proteome Research 2010, 9, 4420–4432

Analysis of the Plasma Proteome Associated with Acu te Coronary Syndrome

2-DE

DIGE

4 novel proteins not previously identified

Protein Biomarkers of New -Onset Cardiovascular DiseaseA prospective Study

Yin X et al. Arterioscler Thromb Vasc Biol. 2014;34:939-945

Discovery MS , 861 proteins; Targeted MS 59 protei ns in 336 patients

Proteomics profiling identified a protein panel tha t are associated with new-onset of CVD

IGFBP-1

AAA C

*

*

pg

/ml

Plasma profiling by a protein array approach identifies IGFBP1 (insulin- like growth factor Binding protein1) as a novel biomar ker

of abdominal aortic aneurysm.

Ramos-Mozo P et al. Atherosclerosis 2012 ;544-550

Several proteins related to blood cells identified by proteomic approaches

Martinez-Pinna R et al.Curr Atheroscler Rep. 2010 May;12:202-8

CAROTID ATHEROSCLEROSIS

*

ASYMPTOMATICMONOCYTES

PERIPHERAL ARTERY DISEASE

Madrigal-Matute J et al. JAHA 2014 (accepted)

Galectin-3, a biomarker linking oxidative stress and i nflammation with the clinical outcomes of patients with atheroth rombosis

N=199

N=188

Combination of MCP -1, galectin -3 and proBNP predict CV events in patients with CAD

Tuñón J, et al. Am J Cardiol 2014:113;434-440

Acute thrombotic events, heart failure or death

706 patients with CAD followed for 2.2 ± 1 yr

Searching for novel biomarkers by proteomics in atherothrombotic disease

Cholesterol deposits in the vessel wall Plaque growth Complicated plaque

with trombos

MALDI-Imaging (proteic maps)

SIMS-Imaging (metaboloma)

LCM(Laser Microdisection)

Individual cells 2D-DIGE

Secretome(Differential Expression of proteins)

2D-E

Monocytes, RBC, plateletsPlasma

Tissue

Tuñón et al. J Am Coll Cardiol 2010;55:2009–16

A Proteomic Focus on the AlterationsOccurring at the Human AtheroscleroticCoronary Intima

Ferritin light chain

De la Cuesta F. MCP. 2011;10:M110.003517.

13 proteins altered (7 , 6 )

Preatherosclerosis

CAD

3 novel proteins

Identification of Heat Shock Protein 27 as a potential biomarker of atherosclerosis by 2DE -MALDI TOF

Martin-Ventura JL et al. Circulation 2004; 110:2216-2219

0

2500

5000

†*

Mammary NCP CP

* p<0.005

† p<0.0001

HS

P2

7 (

ng

/ mg

pr o

t )

0

10

20

30

40

50

60

70

80

HEALTHY

(n=26)

CAROTID

(n=26)

*H

SP

27

(n

g/ m

l)

Plasma

P<0.0001

Carotid plaque SupernatantsMammary

Martín-Ventura et al. ATVB, 2006; Atherosclerosis 2007.

Normal artery CP = complicated plaque(unstable)

Proteases low levels Proteases high levels

HSP70HSP27

Proteolytic degradation

Inflammation Apoptosis

The diminution of HSPs favors the unstability o f atherosclerotic plaque

(eg. Plasmin)

Serum HSP27 levels are lower in patients with CAD and are predictive of future CV events

Seibert et al .J Am Coll Cardiol 2013;62:1446–54

Administration of rHSP27 Attenuates Atherogenesis i n Female ApoE/ Mice

rHSP27 Attenuates the Progression of Established Atherosclerotic Lesions and Promotes Morphological Features of plaque stability

Seibert et al .J Am Coll Cardiol 2013;62:1446–54

% lesion area of aortic wall % lesion area of ao rtic sinus

Total serum cholesterol

Raizman JE Biochim Biophys Acta. 2013 Dec;1831(12):1721-8

Heat shock protein 27 attenuates neointima formation and accelerates reendothelialization after arterial inj ury and stentimplantation.

Ma X et al FASEB J. 2014 Feb;28(2):594-602.

0

2.5

5

7.5

0

2.5

5

7.5

0

2.5

5

7.5

0

2.5

5

7.5

0

2.5

5

7.5

0

2.5

5

7.5

Mammary 1

Mammary 2

Mammary 3

Carotid Plaque 1

Carotid Plaque 2

Carotid Plaque 3

5000 10000 15000 20000

5000 10000 15000 20000

Blanco-Colio et al. Arterioscler Thromb Vasc Biol 2007;27:916-922

Identification by SELDI TOF-MS protein chip ® of a 18.4 kda peak as soluble TWEAK (TNF-like weak inducer of apoptosis)

Supernatans of cultured human atherosclerotic plaqu es

18250 18500 18750 19000

M2

M1

M3

C1

C2

C3

18.4 kDa

0

0,2

0,4

0,6

0,8

1

Mammary artery(N=30)

Carotid Plaques(N=30)

Log

Nor

mal

ized

Inte

nsity

p<0.001

sTWEAK

IMT ThicknessIMT Thickness Endothelial dysfunction

Carotid AtherosclerosisCarotid Atherosclerosis

Peripheral Artery DiseasePeripheral Artery Disease

Abdominal Aortic

Aneurysm

Coronary Artery Disease

Diagnostic and Prognostic Biomarker

Genetic deletion or TWEAK blocking antibody administration reduces atherosclerosis

and enhances plaque stability in mice. Sastre et al; J Cell Mol Med 2014;18:721-34

L

A ILT layers isolation & separatelyincubated inprotein free medium

DIGElabeling

Thrombus (ILT)

Identification of peroxiredoxin-1 as a novel biomarker of abdominal aortic aneurysm.

Proteomic analysis of intraluminal thrombus

Proteins

MALDI-MS Analysis

Av.

Rat

io

*

PRX-1 is increased in luminal supernatants

Martinez-Pinna et al. ArteriosclerThrombVasc Biol. 2011; 31: 935-43

(32 Differentialproteins)

Luminal

Abluminal

AAA

*

PR

X-1

(n

g/m

l)

AAA diameter (mm)

r= 0,6; p<0,01; n= 83

Circulating Peroxiredoxin 1 levels correlate with AAA size & growth

Application of Metabolomics to Cardiovascular Biomarke r andPathway Discovery

Lewis GD et al. J Am Coll Cardiol. 2008 8; 52: 117–123

Integration of metabolomics with other omics approa ches and relationship to phenotype

Translating Metabolomics to Cardiovascular Biomarkers

• Metabolomics is the systematic study of the unique chemical fingerprints of small-molecules (metabolites) related to a variety of cellular metabolic processes in a cell, org an, or organism

• Metabolomics has mainly focused on CV risk factors su ch as diabetes mellitus, obesity, and metabolic syndrome

• Other metabolic studies in CVD – Myocardial Ischemia, Infarction, and Cardiogenic Shock– Atherosclerosis– Atrial Fibrillation

Types of metabolites detectable by mass spectrometry techniques based on degree of hydrophobicity and molecular weight s.

Senn T et al. Prog Cardiovasc Dis. 2012 ; 55: 70–76

Metabolite Profiling Identifies Pathways Associated withMetabolic Risk in Humans

Cheng et al. Circulation. 2012 ; 125: 2222–2231

LC/MS focused on 45 metabolites in 1,015 subjects from Framingham Heart Study

Strong association with insulin resistance

Glutamic acid GlutamineGlutamine/glutamate ratio Glycine

Metabolite profiles and the risk of developing dia betes2,422normoglycemic individuals followed for 12yr; 2 01 developed diabetes

5 amino acids had highly significant association with future diabetes:-isoleucine-leucine-valine-tyrosine-phenylalanine

Wang TJ et al Nature Medicine . 2011

A combination of three causeda fivefold higher risk in top quartile

2,023 patients undergoing cardiac catheterization followed for 3.1 yrs Mass spectrometry profiling of 69 metabolites were performed in fasting plasma

Five of 13 metabolite factors were independently as sociated with mortality

-Medium-chain acylcarnitines -Short-chain dicarboxylacylcarnitines-Long-chain dicarboxylacylcarnitines

-Branched-chain amino acids -Fatty acids

Shah et al. Am Heart J 2012;163:844-850

Metabolomic studies in plama of patients after acute coronary syndrome

Teul J et al. Journal of Pharmaceutical and Biomedical Analysis 56 (2011) 343– 351

Time 0 4 days 2 mo 6 mo controls

Non target approach with gas chromatography mass sp ectrometry: 27 statistically significant metabolit es

Targeted analysis of 21 fatty acid profile in plasma revealed the hig hest valuesin patients with ACS. Five out of 21 FA comprised the 85% of total

Mas S et al. Diabetes. 2010;59:1292-301

Lipid cartography of human atherosclerotic plaque b y cluster-TOF-SIMS imaging.Local non-esterified fatty acids correlate with inf lammation

Lipidomics Profiling and Risk of Cardiovascular Dis ease inthe Prospective Population-Based Bruneck Study

Stegemann C et al. Circulation. 2014 ;129:1821-31

685 plasmas; 135 lipid species from 8 different lipid classes were study by mass spectometry –based lipidomics profiling

3 lipid species most consistently associated with incident CVD

Triacylglycerol

Cholesteryl ester

phosphatidylethanolamine

Differences in the secretion of identified metaboli tes in human atherosclerotic abdominal aneurisms

Comparison of secretomes from aneurysm and healthy arteries .

Comparison of secretomes from aneurysm artery, as well as luminaland abluminal part of the thrombus

Ciborowski M et al. J. Proteome Res. 2011, 10, 1374–1382

Around 350 metabolites were secreted by AAA thrombus

Metabolomic with LC-QTOF-MS Permits the Prediction of Disease Stage in Aortic Abdominal Aneurysm Based on Plasma Metabolic Fingerprint

Small AAA

Large AAAControl AAA

Ciborowski M et al. PLoS One. 2012;7:e31982

-Sphingolipids, lysophospholipids, cholesterol metab olites, and acylcarnitines have a role in the development and progression of AAA. -Guanidinosuccinic acid was found as a strong marker of large AAA.

Potential future biomarkers for personalized med icine in CVD and Cancer

Aging is a common feature in the two conditions

• “Traditional biomarkers”

•Emerging biomarkers

-Proteomics/metabolomics-Gene expressions signatures-Epigenetic changes -Circulating and tissue microRNA-Extracellular vesicles

Schema of biomarker discovery, validation, and impl ementation.A long and tough way

Klein J Diabetes 2012;61:3072-3073

Looking at the future

• The emerging use of biomarkers may enable physicians to make treatment decisions based on the specific characteristics of individual patients and the particular signatures in pathological samples(eg, tumors) instead of population statistics.

• Unraveling complex molecular interactions and networks and incorporating clinical information in the modelling will present a paradigm shift in molecular medicine and personalized therapy

Renal, Vascular and Diabetes Research Lab.

University Hospital Fundación Jim énez Díaz .

Autónoma University. Madrid.

José LuisMartín-Ventura, PhD