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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?