Dalla ricerca di base alle applicazioni cliniche:

Cellfood TM migliora il metabolismo respiratorio delle cellule

endoteliali ed inibisce la generazione di ROS indotta da ipossia

Dr. Elisabetta Ferrero

DIBIT-Division of Molecular Oncology - San Raffaele Scientific Institute, Italy

WORKSHOP

"Inquinamento e danno da stress

ossidativo

NOVITA' dalla ricerca dal mondo

della nutraceutica»

Endothelium is the inner cellular lining that covers all blood and lymphatic vessels

lumen

tissue

3-dimensionality

growth factors, cytokines

chemical composition

physical characteristics

(stiffness, pH, O2 tension)

cell-cell interactions

cell-matrix interactions

mechanical stress fluid flow

pressure

AFFECT CELL BEHAVIOUR

AFFECT RESPONSE TO DRUG

Microenvironments

Different microenvironments determine spatial EC heterogeneity

Under physiological conditions, “quiescent” Endothelial Cells (EC) are as an always active input–output device

None

Hypoxia

+ Drug

HHV-8

+TNF

•contractile forces •pressure •drugs

TNF-a

CD-31 PECAM

NONE

ICAM-1 expression

NONE

Ferrero E, Belloni D

to maintain a balance in the regulation of

Dilatation Constriction Growth inhibition Growth promotion Antithrombosis Prothrombosis Anti-inflammation Pro-inflammation

Endothelial Disfunction Hypoxia

ROS

TNF

VEGF

Dilatation Constriction Growth inhibition Growth promotion Antithrombosis Prothrombosis Anti-inflammation Pro-inflammation

Endothelial Disfunction Hypoxia

ROS

TNF

VEGF

Dilatation Constriction Growth inhibition Growth promotion Antithrombosis Prothrombosis Anti-inflammation Pro-inflammation

Endothelial Disfunction Hypoxia

ROS

TNF

VEGF

need for compounds that tightly regulate endothelial

activation

REVIEW ARTICLE

Cardiology Journal

2011, Vol. 18, No. 4, pp. 352–363

Novel therapeutic targets for preserving a healthy

endothelium: Strategies for reducing the risk

of vascular and cardiovascular disease Joseph Ramli Pedro CalderonArtero, Robert C. Block, Shaker A. Mousa1

BE Leone, E.Ferrero

HUVEC ultrastructure

HUVEC model

Rat cerebral artery

(M Frontczak-Baniewiczi)

Normal cerebral artery

(JM Fain)

Heterotypic cells

(tumor cells)

GF, cytokines

Hypoxia Patients’sera E. Ferrero and B.E. Leone

Hypoxia is common finding in tumors, pulmonary disorders, occlusive vascular disease, and septic shock

ROS production

Endothelial activation/dysfunction/death

Angiogenic switch

Veschini L, Ferrero E. Blood 2007 Veschini l, Ferrero E, Faseb J 2011

EC are fine sensors of O2 activation

and are equipped with mechanisms that adjust metabolism to O2

fluctuation

HIF-1a/DAPI

Normoxia Hypoxia (2%O2)

Specific aim

role of CellfoodTM on EC respiratory metabolism, ROS

generation and HIF-1alpha pathway

Anti-oxidants limitations

poor solubility

inability to cross membrane barriers, poor delivery

rapid clearance from cells

Cellfood TM does not affect EC viability and morphology

24 hrs

CF increases O2 consumption and mitochondrial activity

Brief-time exposure Long-time exposure

T5 T1

T0

T0

T1 T5

NT

NT

Mit

oT

rak

er/

acri

din

e

ora

ng

e

Mit

oT

rak

er

NT

NT

1d

8d

8d

8d NT

O2

(%

)

CF single administration CF daily administration

…. without affecting EC viability.

to

t1 t5

nt

nt to

t1 t5

nt

1d

8d

nt 8d

nt 8d

CellfoodTM sustains ATP production

But not LDH production:

Metabolic shift from glycolisis to mytochondrial pathway?

Oxidative stress results from imbalance

Reductants

(antioxidants)

antioxidant supplementation could represent a common potential therapeutic strategy in conditions associated with

“increased oxidative stress”

Oxidants

(ROS)

Hormones, growth factors, proinflammatory cytokines

Proteins, lipids, DNA structural and functional

damage

1h

r 2

4 h

rs

nox hypo nox+CF hypo+CF

nt

Time 24 hrs

CellfoodTM down-regulates expression of hypoxia-induced HIF-1alpha

nox nox+CF hypo

nt hypo Hypo+CF nox nox+CF

and of glucose transporter Glut-1

thus CF interferes with the hypoxic response

and of glucose transporter Glut-1

thus CF interferes with the hypoxic response

HIF-1alpha/DAPI

nox nox+CF hypo+CF hypo

1 h

r

1 h

r

1 - CellfoodTM preserves mitochondrial activity and increases O2 consumption 2 - CellfoodTM sustains ATP generation 3 - CellfoodTM inhibits hypoxia induced ROS generation 4 - CellfoodTM interferes with HIF1-a pathway 5 - CellfoodTM up-regulates MnSod expression as an adaptive mechanism

Specific Conclusions Specific Conclusions

hypo

Specific Conclusions

General Conclusion

CellfoodTM preserves EC from hypoxia-driven activation

Endothelial Activation/ Disfunction

Relevance

CellfoodTM

Dilatation Constriction Growth inhibition Growth promotion Antithrombosis Prothrombosis Anti-inflammation Pro-inflammation

CFTM maintains proper EC

homeostasis ?

Dilatation Constriction Growth inhibition Growth promotion Antithrombosis Prothrombosis Anti-inflammation Pro-inflammation

CFTM maintains proper EC

CellfoodTM could be useful against hypoxia induced angiogenesis?

HUVEC proliferation

0

2000

4000

6000

8000

10000

12000

48h 72h

NT

CF

Hyp

Hyp

+C

F

NT

CF

Hyp

Hyp

+C

F

n=2

......and about other angiogenic hallmarks? -EC adhesion molecules expression and organization (VE-cadherin) -EC permeability -EC migration

perspectives

EC response is not an all-or-nothing phenomenon

Spectrum of response depends on:

the stimulus

the spatio-temporal network

the concentration

CONCLUSIONS

Specific

HIF-1alpha activation leads HUVEC activation/angiogenesis

General

The primordial function of HIF-1alpha was to mediate adaptive responses

that allow cells to survive oxygen deprivation

ReactiveOxygenSpecies (ROS) are normal products of aerobic metabolism and participate in

physiological and pathophysiological processes

Bashan N, Physiol Rev, 89; 2009

Antioxidants

Mitochondrial hypothesis

Mitochondria must generate ATP If the set-point of a mitochondrion is low, it must work harder,

which will produce more ROS

All these works are energy-dependent

Vander Heiden MG, SCIENCE 324 2009

Mitochondrial respiration

Anaerobic glycolysis

Aerobic glycolysis

need for anti-oxidant and for compounds that tightly regulate endothelial

activation

interfering with HIF-1-alpha: HIF inhibitors (S.Galban, 2009; G. Melillo 2007)

down-modulating the cellular response to hypoxia recalibrating EC to their quiescent state

(P. Carmeliet, 2009)

General AIM

treatments that can increase mitochondrial function and/or

eliminate ROS could be effective therapies?