Clinical BiomarkerDefined Biomarkers in Every Disease in Human Blood

Clinical Biomarker

F.rezaee@erasmusmc.nlF.rezaee@ACTA.nlF.rezaee@UMCG.nlFarhad.Rezaee@clinicabiomarkers.comwww.clinicabiomarkers.com

Contact with Dr. F. Rezaee (associated professor in Erasmus Medical Center)

www.clinicabiomarkers.com

Biomarkers

1- Clinical Biomarkers (Disease state)2- Cellular Biomarkers (Cell function)

What is a Clinical Biomarker?

An indicator for the presence of a disease state in Human

Clinical Biomarkers

1- Diagnostic Clinical Biomarkers (Indicator or predictive of existed Diseases)2- Prognostic Clinical Biomarkers (provide knowledge about the cause or development of a disease or even recovery of a disease)

Identification of Clinical Biomarkers in Human Body

1- Human Tissues (e.g. Liver, Spleen, Pancreas, etc.)2- Human Primary Cells (e.g. Adipocytes, Macrophages,

Platelets,etc.)3- Human Body Fluids (e.g. Cerebrospinal fluid (CSF),

Blood (Plasma, Serum), Saliva (extracellular fluid, etc.)

Identification of Clinical Biomarkers in Human Plasma versus other Human Materials

Simple 1- Hard Intervention2- High Risk3- Pain4- Blood Contamination5- Unknown Source 6- No clear Biomarker?

1- Defined Biomarkers2- Source independet3- No Intervention 4- No Risk5- No Pain

Advantages DisadvantagesDisadvantagesAdvantages

1- Very Complex2- ≈ 15000 Proteins3- 40 Proteins (≈95% of plasma protein Concentrations)

Other Human Materials Human Plasma

PlasmaBuffy Coat

Red Cells

Syst

ems B

iolo

gy

Transcriptomics

Proteomics

Phospholipidomics

Imageomics

Metabolomics

Glycomics

Kinomics

NanoBioMedicine

Conventionaltechnology

Human Plasma of patients

Human Plasma of Controls

Standard Systems Biology: Identification of Biomarkers for different diseases in

Human Plasma

S ystem s B io lo g yD e f in it io n

In a scientifically context, Systems Biology can be referred as “the use of a combination of all techniques and knowledge (or develop novel

ones) to find answer to any biological and medical questions

Standard Systems Biology: Identification of Biomarkers for different diseases in

Human Plasma?

1- Biological Knowledge

2- Molecular Knowledge

3- Cellular Knowledge

Advantages Disadvantages

1- Not suitable to find clinical biomarkers (proteins) for diseases in blood at present

2- Not suitable for the detection of very low abundant proteins at present

This indicates that an innovative and original strategy is essential to detect the disease at a very early stage in the patients for the

therapy

Systems Biology: Identification of Biomarkers for different diseases in Human Plasma

Syst

ems B

iolo

gy

Transcriptomics

Proteomics

Phospholipidomics

Imageomics

Metabolomics

Glycomics

Kinomics

NanoBioMedicine

Conventionaltechnology

I have developed a breakthrough method, which make it possible to detect and identify the defined clinical Biomarkers for diseases in human plasma (100x more coverage)

The transfer of basic science knowledge to the clinic to prevent and recover the diseases and/or extend healthy aging via generation of

defined clinical biomarkers for different diseases for the development of new therapeutics.

Transfer Basic Science to the Clinic

T h e im p ac t o f C lin ic al B io m arkers o n 1-Pu b lic H ealth , 2- E c o n o m y, 3-

S o c ie ty

1- T he d iagno sis o f the d isease at early stage by physic ian2- T he therapy start o n tim e3- T he therapy will be effic ient and m o re effec tive4- T he m o rb id ity and m o rtality are extrem ely reduc ed5- T he health insuranc e c o sts go do wn6- T o prevent the d isease, find new drug target and extend healthy ag ing .

Pu b lic H ealth

T h e im p ac t o f C lin ic al B io m arkers o n 1-Pu b lic H ealth , 2- E c o n o m y, 3-

S o c ie ty

1- C enter o f d iagno stic2- Sell the Bio m arkers3- C ap ital stream s to c o untry and C ity (m o no po ly)4- H ealth c are budget go do wn 5- Pro sperity6- Investm ent o n o ther issues

Ec o n o m y

The impact of Clinical Biomarkers on 1-Public Health, 2- Economy, 3- Society

Society

1- Prosperity2- Economy growth3- Healthy aging4- Excellent education

Markets of clinical Biomarkers

The Compound Annual Growth Rate (CAGR)

1- Biomarkers Market worth $45.55 Billion by 2020(Markets and Markets) and will reach over $78.2 Billion by

2024 (PharmExec.com)

2- For many decades Clinical Biomarkers will be major focus of medical science and pharmaceutical Companies

http://www.pharmexec.com/global-biomarkers-market-reach-over-782-billion-2024

http://www.marketsandmarkets.com/Market-Reports/biomarkers-advanced-technologies-and-global-Market-43.html

7.5 billion inhabitants 9% T2D affected.

USA: 30.3 million T2DEU: 66 million T2DWorldwide: 700 million T2D The global cost of diabetes $825 billion per year Diagnosis diabetes in the U.S.A. $245 billion per year Diagnosis diabetes in EU $120 billion per year

Global Market

Glo b al Marke t

1 trillion dollars T2D diagnosis per year, €1500 per patient per year

Novel Biomarker for T2D found by BIO-CLINICA BIOMARKERS will lead to a very high profitable investment

BIO-CLINICA BIOMARKERS Company (CEO; Dr. F. Rezaee)

UK Researchers Identify 750 Biomarkers for Potential Early Cancer Screening

Is correct or not correct to use the word Biomarker ?

NO

Approximately 100 biomarkers for Cardiovascular Diseases (CVDs) NO

Approximately 40 biomarkers for Diabetes (T2D) NO

WHY THE ANSWER IS NO?

Based on defined clinical Biomarkers, it is not allowed to preselect for discovery of clinical Biomarkers. It is not allowed to remove any fraction from whole proteome samples. Clinical Biomarkers Discovery has to be executed in whole intact System.

The best situation 1-4 Clinical Biomarkers for one specific disease, Multifactorial diseases are exceptions from this potential law.

EXAMPLES

EXAMPLES

Systems Biology of Aged-Induced Diseases

FISH EYE DISEASE (FED)

LCAT mutation (T123→I),

Two heterozygotes with mutation (V309→M )

One Compound heterozygote patient with the both mutations (T123→I and V309→M)

FLD (familial LCAT deficiency)

FED (Fish Eye Disease)

Lecithin cholesterol acyltransferase (LCAT)

DIGE (Diffrences in gel electrophoresis)

800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000M/z0

100

%

Farhad8AMC0668 Center 11 (Cen,2, 80.00, Ht) TOF (800:4000) LD+

831.22

1283.61

832.19

1012.59

896.46

1031.53

1226.58

1724.03

1284.621453.77

1380.77

1454.77

1707.00

1468.86

1469.85

1725.03

1726.03

1815.93

1817.933594.01

3034.542645.341879.18 2963.73 3184.74 3592.063344.153721.13 3881.61

APO A-I

1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 2100 2150 2200 2250m/z0

100

%

far0030 25 (0.896) Cn (Cen,2, 80.00, Ht); Sb (20,40.00 ); Sm (SG, 2x6.00); Cm (1:28) TOF LD+ 4021497.69

1313.60

1292.96

1276.98

1173.461244.47

1314.60

1335.58

1336.58

1351.55

1352.56

1373.49

1411.521416.60

1498.69

1499.70

1753.76

1520.67

1730.701663.661642.681535.63

1536.63

1557.601558.61

1665.67

1755.77

1775.72

1776.742248.891792.67 2233.89

1819.672250.85

FISH EYE DISEASE

Adipose tissue and inflammation, adipocytes and Insulin regulation adipocytes and plasma lipoproteins

Adipose tissue and inflammation, adipocytes and Insulin regulation adipocytes and plasma lipoproteins

F.Rezaee@med.umcg.nlFarhad Rezaee

In this seminar:

Aadipose tissue

Eye catcher

Obesity is a problem of epidemic-pandemic proportions and is related to poor health

Body needs fat

Abdominal Obesity is a major cause of Abdominal Aortic Aneurysm (AAA)

Adipose Tissue Structure

Adipose tissue (1)

1- Storage of energy in the form of triglycerides (TG) for lean times

2- The largest endocrine organ

What is the main cause of obesity?

An overload of energy (TG) in the adipose tissue and in particular adipocyte-associated LDs is the

main cause of obesity

What is main function of adipose tissue?

Obesity is defined as BMI (kg/m2) determined by the increasing mass of adipose tissue (BMI above 30 is obese)

Adipose tissue (2)

An inflammatory state of adipose tissue contributes to the development of insulin resistance (IR) and type 2 diabetes (T2D)

Obesity promotes an inflammatory state

What is the link between obesity and pathophysiological diseases?

Obesity

Inflammation

Cancer

Infection

Cachexia

Insulin ResistanceCoronary Heart Disease

Hypertension

Stroke

Atherosclerosis

Adipose tissue (3)Metabolic disorder Obesity or Cachexia pathophysiological states

Regional fat metabolismTwo main fat types:

LIPID DROPLETS

ADIPOCYTES

Plasma lipoproteins

Lipoprotein structure VLDL HDL

Disturbances in plasma lipoprotein homeostasis result in dyslipidemia, e.g. hypertriglyceridemia, hypercholesterolemia and etc., which in turn have serious pathophysiological consequences like CVDs

Lipoproteins are classified by density and size, which are inversely related

VLDL/CM

LDLIDL

Lp(a)

HDL2-RichHDL3-Rich

VHDL

PLFF

Plasma Lipoproteins

Den

sity

(g/m

l)

Size (nm)

HDL Lipid Droplet

Similarities between LP and LDLP LD

Challenges

Inflammation

Insulin RegulationLipoproteins

?

Cardivascular diseases

Adipose tissue is considered as an immune organ

Objective

Why do I make this statement?

Conventional wisdom: immune functionality is the result of adipose tissue macrophages (ATM).

My research challenges this notion: Adipose tissue adipocytes (ATA) exhibit immune cell function

No body measured such function in human primary adipocytes or other human AT-associtaed cells alone

1-Classical hypothesis: necrotic AT-adipocytes (ATA) in obese state activate AT-macrophages (ATM) that then lead to a sustained chronic inflammation in AT (Only in AT).

2-The link between human adipocytes and the source of inflammation in AT has not been systematically studied.

3-AT is considered as an immune organ

3-Our hypothesis: human primary adipocytes are able to prime inflammation in AT.

Classical hypothesis versus my research challenges

Measure to know?

Challenges

1-the adipose tissue adipocytes (ATA) exhibit immune cell function

2-Adipose tissue can be potentially considered as a new organ to compensate hormone/enzyme disorders

3-Mitochondria is inactive in obese adipocytes and LDs are “the only boss” in adipocytes

4-A homeostatic balance between lipoproteins may guarantee a regulated function of lipoproteins, -and not the quantity of a specific lipoprotein

5-Human primary adipocytes produce and secrete Insulin

Macrophages Characteristic production1-Cytokines

2-Chemokines

3-MHC-II molecules

4-CDs

5-Adhesion Molecules

We must show that adipocytes are also able to produce similar products!

RESULTS 1

Differentiation of human preadipocytes to adipocytes

Pre-adipocytes Adipocytes

AdipogenesisTo establish our research model!

ADIPOQ LEPLIPE

PLIN

PNPLA2LPL

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

Preadipocytes Adipocytes

Adipocytes differentiation mRNA markers

1000x

Differentiation markersR

elat

ive

inte

nsity

( 2

log

)

Rel

ativ

e in

tens

ity (

2lo

g )

Cytokine / Chemokine Ligand and Receptor Genes

Adipocytes express cytokines and cytokine receptors

IL1BIL1R

1IL1R

2 IL3IL3R

A IL4IL4R IL5

IL5RA IL6

IL6RA

IL6RB IL7

IL7R IL2IL19 IL26

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

Preadipocytes Adipocytes

Cytokine and cytokine receptors

Adipocytes express cytokines and cytokine receptors R

elat

ive

inte

nsity

( 2

log

)

Adipocytes-associated Chemokine and Chemokine Receptor GenesR

elat

ive

inte

nsity

( 2

log

)

Chemokine and Chemokine Receptor Genes

Adhesion, MHC-I, MHC-II and CDs Genes

Genes

Confocal analysis of C-RP in adipocytes

CRP

merge

Fat organelle Nuclei

NegativeMerge

Immunoelectron microscopy of C-RP in adipocytes

Negative C-RP

Are these adipocytes-associated immune components biologically active?

We have measured 50 proteins treated with and without LPS

Adipocytes treated with LPS (1)

Proteins

Adipocytes treated with LPS (2)

Proteins

Adipocytes treated with LPS (3)

Proteins

Adipocytes-associated components are biologically active

Are adipocytes-associated components biologically functional?

We performed a migration assay according to Boyden chamber to measure adipocyte

functionality

Migration assay according to Boyden Chamber

Are adipocytes-associated components biologically functional?

YES

Summary

Preadipocytes and adipocytes synthesize immune-associated components (genes and proteins)

Since preadipocytes and adipocytes express immune-associated genes, expression of these genes suggested to be a bona fide property of this cell type

Since these genes and proteins response to LPS, They are biologically active

Since adipocytes-associated MCP-1 and IP-10 are able to activate CD4+, They are biologically functional.

Human primary adipocytes exhibit immune cell function.

Conclusion

Adipocytes are able to prime inflammation

Adipocytes Produce Insulin

the role of adipocytes behind the link between obesity and insulin regulation in human is unknown

The overload of lipid droplets with triglycerides in adipocytes is the main cause of obesity and its afflictions

such as insulin resistance

Adipocytes Produce Insulin?

RESULTS 2

Hypothesis: Fat determines the concentration of insulin

insulin concentration and human patients

Bariatric Surgery Heals Type 2 Diabetes

Assumption: the production of insulin by human adipocytes is not from pancreas

Insulin stain in pancreas of prediabetes BB rats

Insulin in control RATs

Insulin in other human primary cell types

The novel knowledge obtained by the present study could help to re-design future studies related to insulin and therapy of diabetes.

Human adipocytes constitutively synthesize and secrete insulin and that is biologically functional.

Human primary adipocytes secretes insulin but preadipocytes do not and that suggested to be due to the lipid droplets

Conclusion

Insulin expression in a variety of human cell types including the adipocytes is an evidence for a ubiquitous insulin expression

Data suggested a cross talk between AT and pancreas to tune up energy metabolic system

A homeostatic balance between lipoproteins may guarantee a regulated function of lipoproteins, and not the quantity of a

specific lipoprotein?

PLASMA LIPOPROTEINS

RESULTS 3

Phospholipidomic analysis of all defined plasma lipoproteins

VLDL/CM

LDLIDL

Lp(a)

HDL2-RichHDL3-Rich

VHDL

PLFF

Plasma Lipoproteins

Den

sity

(g/m

l)

Size (nm)

High Performance Thin Layer Chromatography

Proteomics analysis of plasmaVLDL, LDL, Lp (a), and HDL

95 proteins

51 proteins

55 proteins

Proteomic analysis of high-density lipoproteinRezaee F, Casetta B, Levels JH, Speijer D, Meijers JC (Proteomics 2006) AMC

Phospholipidomic analysis of all defined human plasma lipoproteinsDashti M, Kulik W, Hoek F, Veerman EC, Peppelenbosch MP, Rezaee F

(Nature Scientific Reports 2011) UMCG, AMC, VU, ERASMUSMC

Human plasma very low density lipoprotein carries Indian hedgehogQueiroz KC, Tio RA, Zeebregts CJ, Bijlsma MF, Zijlstra F, Badlou B, de Vries M, Ferreira CV, Spek CA, Peppelenbosch MP, Rezaee F ( J Proteome Research 2010) UMCG, AMC

Human plasma Proteome of Very Low-Density Lipoprotein and Low-Density Lipoprotein exhibits a link with coagulation and lipid metabolism

Dashty M, Motazacker MM, Levels J, de Vries M, Mahmoudi M, Peppelenbosch MP, Rezaee F (Thromb Haemost. 2014) UMCG, AMC, STANFORD, ERASMUSMC

Proteome analysis of defined human plasma Lipoprotein a (LP (a))(Manuscript will be submitted today, 2019) UMCG, ICL, VU, STANFORD,

ERASMUSMC

Papers related to proteome and lipidome of Plasma lipoprotein

Ernst Schaefer Citated VLDL/LDL paper as “Of outstanding interest”

The paper have been recommended on PUBADVANCED by A Director at ROCHE

The paper have been also recommended for Science Global Discovery

Human plasma Proteome Very Low-Density Lipoprotein and Low-Density Lipoprotein exhibits a link with coagulation and lipid metabolism

Dashty M, Motazacker MM, Levels J, de Vries M, Mahmoudi M, Peppelenbosch MP, Rezaee F (Thromb Haemost. 2014) UMCG, AMC, STANFORD, ERASMUSMC

Found very interesting paper by Head of Mass Spectrometry of OCDEM (University of Oxford)

The majority of apolipoproteins and proteins involved in lipid metabolism are conserved between the different lipoproteins.

The known phospholipids are also conserved between the different lipoproteins.

Human diseases related to proteins carried by VLDL and LDL can be divided in three major categories: 1 – dyslipidaemia, 2 –atherosclerosis and vascular disease, and 3 – coagulation disorders.

A variety of proteins present on LDL and VLDL strongly supports that protein shuttling through lipoproteins may be involved in the regulation of biological processes

SUMMARY

CONCLUSION

A homeostatic balance between lipoproteins may guarantee a regulated function of lipoproteins, and not the quantity of a

specific lipoprotein

SYSTEMS BIOLOGY

Present and future studies

Quantitative proteomics of patients with CVDs versus control

HDL; PILOT STUDY

A NOVEL METHOD FOR QUANTITATIVE PROTEOMICS

I developed a novel method, which increases the number of quantified proteins from 20 to 300 or even more in

plasma research and you do not need to run a gel (That is a revolution contribution in proteomics).

This approach is applicable to any good samples

With this approach, we are able to find easily the candidate protein(s) (diagnostic or prognostic markers) in any

patients (CVDs, T1D, T2D, Obesity, Coagulation Disorders)

This approach is applicable to all spieces (e.g. Bacteria, Viruses)

PROTEOMICS

1800 proteins

Visceral Adipocytes proteome

Cytoplasma (560)

Mitochondria (388)

Nucleus (332)

Extracelluar (198)

ER (164)

Golgi (99)

Lysosome (49)

Peroxisome (26)

Lipid droplets (7)

Distribution of proteins

0.4 %

MITOCHONDRIA

Mitochondria

Distribution of genes

NanoBioTechnology

Personalized protein coronas: a “key” factor at the nanobiointerface

MJ. Hajipour, S. Laurent, A. Aghaie, F. Rezaee* and Morteza Mahmoudi**

Composition-dependent effects of nanoparticles on blood coagulation

Kamran Bakhtiari, Joost C.M. Meijers, Sophie Laurent, Svetlana Mintova, Eng-Poh Ng, Hussein Awala, Somayyeh Mirsadeghi, Morteza Mahmoudi, Farhad Rezaee

STEM CELL BASED THERAPY

*Stem Cell Therapy: Stem Cell based Therapy including Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (IPSCs) for metabolic diseases such as T1D and CVDs.

Thank you