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Fluid Shear Stress: a modulator of the vasculo-protective effects of polyphenols?

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HAL Id: hal-02739050

https://hal.inrae.fr/hal-02739050

Submitted on 2 Jun 2020

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Fluid Shear Stress: a modulator of the vasculo-protective effects of polyphenols?

Laurent-Emmanuel Monfoulet

To cite this version:

Laurent-Emmanuel Monfoulet. Fluid Shear Stress: a modulator of the vasculo-protective effects of polyphenols?. 7. International Conference on Polyphenols and Health, Oct 2015, Tours, France. �hal-02739050�

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JOUR / MOIS / ANNEE

Fluid Shear Stress: 

a modulator of the vasculo‐protective 

effects of polyphenols?

Laurent‐Emmanuel Monfoulet

INRA  UMR 1019 Human Nutrition Unit Clermont‐Ferrand ‐ France 

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Preclinical studies Murine Aorta Mauray et al. 2012 (22):72‐80 Coban et al. MNFR 2012, 56: 1270–1281 Clinical Trial  Human PBMCs Morand et al. AJCN 2011; 93:73‐80 Milenkovic et al. PLoSOne. 2011;6(11):e26669 Monocyte  adhered to  endothelium Monocyte  migrating  into intima Arterial  INTIMA Endothelial  Cell Blood Monocyte Polyphenols In vitro studies Endothelial cells Claude et al. MNFR 2014; 58(5):1016‐27 Chanet et al. BJN 2013; 110:587‐598

±

Nutrigenomic approaches: ‐Transcriptomic  ‐Proteomic  ‐Epigenetic

±

±

TransEndothelial Migration (TEM) as a common mode of 

action of polyphenols on vascular health

Hypothesis:

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Coban et al. MNFR 2012,  56, 1270–1281 Control Curcumin MAC‐3 Propidium Iodide

Curcumin intake reduces atherosclerotic lesion development

Macrophages within the lesion Atherosclerotic lesion (aortic sinus) Control Curcumin 0.2% Curcumin  for 16 weeks ApoE‐/‐Mice 8 week‐old

±

Atherosclerotic model:

and recruitment of immune cells

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Underlying mechanisms of action in in vitro studies

Polyphenols   ‐ Circulating molecules ‐ Low plasma concentration (0.1 – 10µM) ‐ Time of exposure (= resident time in the plasma) Milenkovic et al. FRBM 2013 (64):40‐51 Arterial  LUMEN Monocyte  adhered to  endothelium Monocyte  migrating  into intima Arterial  INTIMA Endothelial  Cell Blood Monocyte Atherosclerosis initiation  (Adapted from Jaffer et al JACC 2006  (47)7:1328‐38) Dietary Polyphenols

Reproduce conditions as close as possible to the physiology

Endothelial cells : ‐ Hemodynamic conditions

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Shear Stress: a vascular and physiological parameter

Malek et al. JAMA 1999. 282(21):2035‐2042 Rocca et al. Cordiologie Conferences 2007. XII (8)

Vena cava  < 2 dyne/cm² Veins 2 <  < 5 dyne/cm² Veinules 10 <  < 15 dyne/cm² Capillaries   > 40 dyne/cm² Aorta 5 <  < 10 dyne/cm² ‐ A force due to the blow flow ‐ Pulsatile (Heart beats)   ‐ Continuous ‐ Laminar and unidirectional

Shear Stress is:

 4 μ πR Poiseuille’s law

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‐ To analyse the response of endothelial cells to physiological relevant

concentrations of polyphenols under shear stress conditions

‐ To evaluate how shear stress modulates the response of endothelial

cells to polyphenols regarding recruitment, adhesion and TEM of monocytes in comparison to static conditions

Aims

Curcuma longa Curcumin

A proof of concept 

Response of endothelial cells to physiological relevant concentrations  of CURCUMIN under shear stress conditions 

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After 24h  Before After 48h  200 µm 200 µm 200 µm Flow Vena8 Endothelial+TM Biochips Kima Pump Cellix Flow Laminar Shear Stress  characteristics: 1 Hz pulsatile flow rate Flow rate: 600µL/min  : ~7dyne/cm² (arterial) St atic Shear  Str e ss Actin vCAM vWF Flow

Imposed shear stress on cultured endothelial cells

• are firmly spread and aligned in the flow direction. • have a physiological phenotype (anti‐atherogenic).

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(‐) TNF‐(+) TNF‐Shear Stress  (~7dyne/cm² ‐ 24h) T0 T1 +/‐ TNF (4h) Monocyte (THP1)  adhesion assay T2

Monocyte adhesion induced by inflammatory stress (TNF‐)

Endothelial cells  (HUVECs) TNF‐ Control  200 µm 200 µm 97 monocytes  +/‐ 25 / mm² < 1 monocyte / mm² After 10min Before monocyte perfusion 

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0 20 40 60 80 100 120 Vehicle Vehicle 0.5 µM Cur 1 µM Cur Monocyt e  Adhesion  (%) Static SS

Impact of curcumin on monocyte adhesion

Shear Stress  (~7dyne/cm² ‐ 24h) T0 T1 +/‐ TNF (4h) Monocyte  Adhesion assay T3 Endothelial cells  (HUVECs) T2 +/‐ Curcumin (3h) The reduction of monocyte adhesion by curcumin is slightly  enhanced in shear stress conditions *** $ *** $ §§§ p=0.16 ***p<0.001 compared to Vehicle (‐) TNF‐ $ p<0.05 compared to Vehicle + TNF‐

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0 20 40 60 80 100 120 140 Vehicle Vehicle 0.5 µM Cur 1 µM Cur (‐) TNFa (+) TNFa Tr ansmigr at e Monocyt e (%) Static SS Flow HUVECs Monocytes MCP‐1 Shear Stress  Static

Impact of curcumin on monocyte TransEndothelial Migration 

Preliminary results

The reduction of TEM by curcumin is heightened in shear stress conditions *** $ *** p=0,18 Speed x8

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Blood flow

Mechanisms of action 

of the curcumin

TNFα IKKα IKKβ IKKγ IκBα NFkB IκBα Proteasome NFkB NFkB Transcription ARNm iCAM vCAM In te grin Selectin P P P P P P P P 1.Rolling Induction of  endothelial

dysfunction 2. Adhesion 3. TEM

Monocyte

Endothelial  cell

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Phospho Ser536 Total Cytosol

0.5 µM 1 µM (‐) TNF‐ (+) TNF‐ Curcumin:  Nuclei‐enriched fraction GAPDH H3 vCAM iCAM GAPDH TNFα TNF-R IKKα IKKβ IKKγ IκBα NFkB NFkB NFkB iCAM vCAM P P P P P P P

Mechanisms of action of the curcumin

15 min 4 hours

Static

NFB p65 Phospho Ser536 Total

NFB p65 0 2 4 6 8 10 NF‐kB Ra tio  (Phospho/T ot al/GAPDH) Cytosol 1 2 3 4 NF‐kB Ra tio  (Phospho/T ot al/H3) Nucleus ‐36%

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Conclusion

• We showed that a pre‐exposition of endothelial cells to curcumin

significantly reduces the adhesion of monocytes to endothelial cells and their TEM.

• The reduction of both adhesion and TEM by curcumin are enhanced

under physiological shear stress.

• In static conditions, curcumin modulates the TNF‐R pathway (NF‐B phosphorylation and its nuclear translocation), but does not affect the expression of adhesion molecules.

• Other cellular process can be modulated by curcumin that could explain their inhibitory effect on monocyte infiltration (cytoskeleton & cell junction dynamic?)

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JOUR / MOIS / ANNEE JOUR / MOIS / ANNEE

Human Nutrition Unit ‐ UMR 1019   MicroCard Team  (Clermont‐Fd – France)

Nicolas Gérard Dominique Bayle Séverine Valéro Marie-Anne Verny Edmond Rock André Mazur Christine Morand Dragan Milenkovic Cécile

Gladine Claudine Manach

Sylvie  Mercier

Irena

Krga LebretonAurélien

Nicolas Barber Thomas Sinegre Rémy Bosviel

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