Proteomics to identify novel biomarkers and therapeutic targets in cardiovascular disease Markus Kubicek Laboratorios de Biologia Vascular y Proteínómica Estructural, Instituto de Biomedicina de Valencia
ardiovascular disease - the killer number one CVD accounted for 38.5 percent of all deaths or 1 of every 2.6 deaths in the United States in 2001. CVD mortality was about 60 percent of total mortality. Source:CDC/NCHS CVD cause about a death a minute among females claiming nearly half a million female lives every year. That s more lives than the next 7 causes of death combined. Starting at age 75, the prevalence of CVD among women is higher than among men. The harsh fact is, cardiovascular diseases are the No. 1 killer of women and men
MORTALITY RATES IN SPAIN Digestive Disease Cancer 5% 22% 35% CARDIOVASCULAR DISEASES Respiratory Disease 10% 27% Others Cardiac Insuficiency 19% 5% Others 39% (INE, Dic. 2003) 37% Stroke Iscemic Cardiopathy
Coronary Atherosclerosis and Myocard Infarct Myocard Infarct Thrombus
Atherosclerosis:a slowly progressing disease lacking early diagnosis Adventitia (Fibroblasts) External Elastic Media (SMCs) Internal Elastic Intima (ECs) Advanced lesion Healthy artery Minor arterial cell proliferation Reduced leukocyte adhesion ATHEROGENIC STIMULI Onset atherogenesis Endothelial dysfunction Leukocyte recruitment Abundant production of inflammatory cytokines/chemokines Fatty streak (early lesion) Foam cell / lipid core formation Leukocyte proliferation SMC activation (proliferation/migration, extracellular matrix production) Fibrous cap formation Necrotic core formation Plaque instability Plaque rupture and thrombus formation ISCHEMIC EVENT (i.e, myocardial infarction, stroke)
Genetic Factors Envinromental Factors antiatherogenic proatherogenic Individual risk for cardiovascular disease
The strength and limitations of proteomics in the study of vascular disease Transcription GENE GENOME stress Exon pre mrna 1 2 3 4 5 metabolism temperature Extracellular signaling Alternative splicing culture condition I II IIIa IIIb IIIc IV Intron I II IIIa IV I II IIIb IV I II IIIc IV Translation Translation P Protein IIIa Protein IIIb Protein IIIc mature mrna TRANSCRIPTOME Expressed proteins Post-translational modifications (Phosphorylation, glycation, proteolysis, etc) PROTEOME PHENOTYPE
Separation of proteins by two-dimensional Gel-electrophoresis (2D-PAGE) TISSUE SAMPLING Control Pathologic Protein extraction and solubilization 1st DIMENSION: Isoelectric Focussing (IEF) 3 5 7 9 11 ph - + kda Strip equilibration Control............... kda 200. 115 80 50 35 27 18 Pathologic........... Excision of differentially expressed proteins....... 2nd DIMENSION: SDS-PAGE (molecular weight) Protein visualisation
Proteolysis (i.e. trypsin) I II III IV Ion source MS1 colission cell MS2 Detector Peptide digestion mix V time of flight time of flight Intesnsity V III I II IV MS or MS/MS Mass spectrum (fingerprits) m/z I 695.3 LGEYGL II 720.3 LSQTFPL III 575.3 LPCVEDYL IV 841.3 LVQEVTDFAK V 550.8 KQTAL MTHSDSDILTNIEDPSSHVPEFSSSSKLSQTFPLNADFAEITKLKQT ALAELVKLPCVEDYLTNIEDPSSHVPEFSSSSKLGEYGLFQNAILKV QEVTKFAKKLGMRAC: Serum Albumin A05103 Data base comparison Protein Identification
PROTEOMIC APPROACH FOR THE IDENTIFICATION OF ATHEROSCLEROSIS-BIOMARKERS FROM HUMAN PATIENT SAMPLES Hypercholesteremia Protein identification Angina pectoris Hypertension 115 82 63 48 37 total blood plasma Protein extraction 26 19 15 lymphoprep mononuclear cells erythrocytes and platelets 2D-PAGE
Proteiomic alterations in human angina pectoris patients c29 I) Angina c4 pectoris p31 p32 p33 p30 In collaboration with Dr. Panayotis Fantidis Hospital Clinico San Carlos, Madrid Protein AP-4 c29 c4 p31 p32 p33 p30 c29 c4 p31 p32 p33 p30 among all changes observed in angina patients 2 yet not described proteins have been found to be differentially expressed in MNCs of > 80% of ~60 patients analyzed Protein AP-33 antibody production c29 c4 p31 p32 p33 p30
Proteiomic alterations in human hypertension patients Patient suffering hypertension II) Hypertension C5 abbrv SSP all kda pi 234-2 vs. -1 comment A1a 0402-45 4,1 spec. A1b 1403-60 4,2 spec. A2a 3706 n.a. 98 4,6 d x 10 A2b 3707 n.a. 98 4,6 d x 5 A2c 3805 4802 100 4,6 d x 5 up in Pts In collaboration with Dr. Javier Chaves and Dr. Josep Redón A1 C3 D3 C1 A2 After treatment of hypertension Z1 C5 C3 C2 Hospital Clinico Universitario de Valencia D3 B1 5503 6501 80 5 d (sat) less in cj B2 8603 8703 80 6,4 spec only in 230-1 C1 5103 5102 30 5,5 missing C2 5105 5102 28 5,5 spec all expt. 231-1!!! C3 5101 5003 28 5,4 d x 1,5 C4 5302 5302 40 4,5 up 2,6 up in Pts C5a 5402 5402 40 6,2 d x 2 C5b 5405 5405 40 6,3 d 1,25 C5c 5406 5404 40 6,4 d x 2 C5d 5410 6405 40 6,5 d x 4 C6 4001 D1 9202 9202 32 6,8 up 2,5 up in some pts D2 9001 9005 26 6,8 up 1,8? D3 6102 6102 26 6,6 d x 3,8 up in pts D4 6203 7203 28 6,6 d x 5 d in pts Z1 2403 n.d. 44 4,4 spec. Z2 2103 2103 28 4,4 up 4,4 faint Z3 2104 2104 28 4,4 d x 2,5 faint Z4 3103 n.d. 28 4,5 spec faint Z5 3101 3002 25 4,5 up 1,92 faint Z6a 3506 3504 115 4,5 d 2,5 Z6b 3403 3505 115 4,5 d 2
Proteomic alteration of protein A4c is reversed after medical treatment cj ct 233-1 234-1 234-1 234-2 II) Hypertension In collaboration with Dr. Javier Chaves and Dr. Josep Redón Hospital Clinico Universitario de Valencia Among proteomic alterations found in MNCs from 25 A4c A4c A4c (n2) 1.2 3.5patients 5 differentially expressed proteins 3.5 have been 1 3 found so far to be reversed after treatment. 3 2.5 0.8 2.5 2 1.5 1 0.5 0.6 0.4 0.2 2 1.5 1 0.5 0 cj ct 233-1 234-1 0 234-1 234-2 0 contr. pts treated
Proteiomic alterations associated to hypercholestermia III) Hypercholesteremia ATHEROSCLEROSIS apoe-null mice + p< 0.0001 vs 2 control + 3000 * Aortic human plasma bank of hereditary * + p< 0.0001 vs 2 days hypercholesteremia * [cholesterol] (mg/dl) 2000 1000 Low Fat Diet n=5 * 2 days n=10 7 days30 days n=11 n=11 High Fat Diet Oil Red O In collaboration with Dr. M. Pocoví Universidad de Zaragoza MK and Kiko Verdeguer arch Thoracic aorta Instituto de Biomedicina Valencia ApoE deficient mouse model of atherosclerotic disease Abdomina aorta
lymphoprep PROTEOMIC APPROACH FOR THE IDENTIFICATION OF ATHEROSCLEROSIS-BIOMARKERS FROM APOE-/- MICE AORTA supersize me 115 82 Mass Spectrometry 63 48 BLOOD Protein extraction 37 26 19 2 dimensional gel electrophoresis plasma 15 MNC
2D-ELECTROPHORESIS COMPARISON FOR DISCOVERY OF BIOMARKERS CONTROL DIET apoe KO MICE ATHEROGENIC DIET apoe KO MICE AORTA DISECTION (TORACICAL SECTION) AORTA DISECTION (TORACICAL SECTION) TISSUE HOMOGENIZATION (PROTEIN EXTRACTION) TISSUE HOMOGENIZATION (PROTEIN EXTRACTION) Molecular Mass (KDa) 115 82 63 48 37 26 19 15 2D-ELECTROPHORESIS CONTROL DIET Molecular Mass (KDa) 115 82 63 48 37 26 19 15 2D-ELECTROPHORESIS ATHEROGENIC DIET IMAGE COMPARISON WITH BIOINFORMATIC ANALYSIS
Proteiomic alterations of mononucleated cells derived from control and ATG diet fed mice A1 A4 Control diet B1 B4 D4 abbrv SSP kda pi ATG vs CTR A1a 0501 55 4,2 down 1,3 A1b 0502 53 4,2 up 1,5 A2 1501 55 4,8 down x 2,5 A3 1503 50 5 spec. Atg A4 1401 48 5,1 down x 7 30 days atherogenic diet B4 A1 B1 B1a 4401 49 5,7 down 1,5 B1b 4402 49 5,8 down 1,6 B1c 5401 49 5,9 down 2 B1d 6401 49 6 down 1,8 B2 7501 60 6,1 down 2 B3 7502 50 6,15 down 2 B4 7504 50 6,2 down 3 B5 8301 45 6,5 down 2,3 B6 8501 50 6,6 down 2,9 B7 8502 50 6.7 down 1,9 A4 D4 D1 4103 21 5,7 up 1,9 D2 5201 35 5,6 down 1,8 D3 6001/TLN 20 6 up 1,6 D4a 7301 35 6,4 down 1,8 D4b 7302 36 6,4 up 1,7 D5 9201 30 6,8 down 2,3 Z1 1 16 5 up 2,2
Proteiomic alterations in mouse plasma from control versus high-fat diet fed mice Control diet control high fat Elevated in high fat 2104 4203 6401 7303 x4 x4 4203 x1 Nombre SSP1 ATG vs CT ATG vs CT ATG vs CT B1 2104 3,83 2,76 2,49 A8(C1) 4203 3,66 10,62 2,71 x1 5007 2,65 2,33 2,28 x4 6201 4,79 2,45 5,83 2103 5002 2104 Reduced in high fat x1 high-fat diet 5002 6401 7303 1602 2103 Nombre SSP1 ATG vs CT ATG vs CT ATG vs CT C3 5002-2,94-5,00-4,00 A4 6401-1,67-2,04-1,79 A1 7303-3,23-4,17-2,56 C4 2103-1,49-1,75-1,10 6401 7303 6205x4 4203 1201 Absent in control 6203 Nombre 2103 1201 2104 x1 1602 SSP1 A7 6203-8202 B5 1201 B9 1602
Proteiomic Proteiomic alterations alterations in mouse associated plasma from to control versus hypercholestermia high-fat diet fed mice III) Hypercholesteremia 8,00 In collaboration 6,00 with Dr. M. Pocoví Universidad de Zaragoza 4,00 human plasma bank of hereditary hypercholesteremia 2,00 project in progress! C3 0,00 B1 C1 x1 x4 A4 A1 C4-2,00 Ratio aterog/control -4,00 MK and Kiko Verdeguer -6,00 Instituto de Biomedicina Valencia ApoE deficient mouse model of atherosclerotic disease Proteomic alterations in ApoA E minus mice after 30 days of atherogenic diet: alterations found in 3 indepenent experiments, each carried out in tripcicates. Reproducible proteomic alteration of 14 proteins in mouse serum from which interestingly 3 have been shown to be also differentially expressed in mouse aorta.
ACKNOWLEDGEMENTS Vicente Andres Kiko Verdeguer Juanjo Calvete (JJ-TOF) The group Collaborations: Dr. Javier Chaves Dr. Josep Redón Hospital Clinico Universitario de Valencia Dr. Panayotis Fantidis Hospital Clinico San Carlos, Madrid Dr. M. Pocoví Universidad de Zaragoza