HAL Id: hal-02739050
https://hal.inrae.fr/hal-02739050
Submitted on 2 Jun 2020
HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come from teaching and research institutions in France or
L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires
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�
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 ‐ FrancePreclinical 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:
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
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 PolyphenolsReproduce conditions as close as possible to the physiology
Endothelial cells : ‐ Hemodynamic conditionsShear 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‐ 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
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).
(‐) 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 perfusion0 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‐0 20 40 60 80 100 120 140 Vehicle Vehicle 0.5 µM Cur 1 µM Cur (‐) TNFa (+) TNFa Tr ansmigr at e d Monocyt e s (%) 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 x8Blood 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 endothelialdysfunction 2. Adhesion 3. TEM
Monocyte
Endothelial cell
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 PMechanisms of action of the curcumin
15 min 4 hoursStatic
NFB p65 Phospho Ser536 Total‐
NFB 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%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?)
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