INDICATE ALTERATION OF THE SKIN BARRIER Pascale MILANI, Anna - PowerPoint PPT Presentation

CORNEOCYTES NANO-MECHANICAL PROPERTIES CHANGES INDICATE ALTERATION OF THE SKIN BARRIER Pascale MILANI, Anna Drillat, Julien CHLASTA, Rawad ABDAYEM, Sanja KEZIC, and Marek HAFTEK

Stratum corneum: an essential physical barrier Physical barrier – mechanical resistance • Chemical barrier • Water barrier • Photoprotective barrier • … •

Stratum corneum: an essential physical barrier Desquamation Mature Corneodesmosome Lipid envelope Immature Cornified envelope Keratin & Intercellular lipids NMF

Evaluation of barrier condition in a minimally invasive manner Tape stripping + Atomic force microscopy

AFM operation x y z

AFM operation

Evaluation of corneocytes mechanical properties Milani et al, J. Mol. Rec. 2017

Evaluation of corneocytes mechanical properties 140 350 *** Ceramide 120 300 Involucrine 100 250 Elastic Modulus (MPa) *** Transglutaminase 80 200 Loricrine 60 150 Keratin 100 40 filament 20 50 NMF 0 0 Lipid Cornified keratin envelope envelope fibers Milani et al, J. Mol. Rec. 2017

Exploration of the maturation process profound superficial immature mature L2 L7 L14 mature L2 L7 Stratum Lipid envelope corneum L14 immature Cornified envelope Keratin fibers E XISTENCE OF DEPTH AND MATURATION - RELATED MECHANICAL CHANGES WITHIN THE SC 50 MPa 500 MPa Milani et al, J. Mol. Rec. 2017

Exploration of the maturation process T HE RELATIVE STIFFNESS : AN INDICATION OF CORNEOCYTE MATURITY Cornified envelope Intracellular matrix 1.2 Intracellular stiffness 1.0 Relative stiffness 0.8 0.6 0.4 CE stiffness 0.2 0.0 N2 N7 N14 profound superficial immature mature Milani et al, J. Mol. Rec. 2017

Exploration of the maturation process T HE RELATIVE STIFFNESS : AN INDICATION OF CORNEOCYTE MATURITY Cornified envelope Keratin fibers 1.2 Intracellular stiffness 1.0 Relative stiffness 1. Moisture gradient 0.8 0.6 0.4 0.2 0.0 N2 N7 N14 profound superficial immature mature Milani et al, J. Mol. Rec. 2017

Exploration of the maturation process T HE RELATIVE STIFFNESS : AN INDICATION OF CORNEOCYTE MATURITY Cornified envelope Keratin fibers 1.2 Intracellular stiffness 1.0 Relative stiffness 1. Moisture gradient 0.8 0.6 0.4 0.2 0.0 N2 N7 N14 profound superficial immature mature Milani et al, J. Mol. Rec. 2017

Exploration of the maturation process T HE RELATIVE STIFFNESS : AN INDICATION OF CORNEOCYTE MATURITY Cornified envelope Keratin fibers 1.2 Intracellular stiffness 1.0 Relative stiffness 1. Moisture gradient 0.8 0.6 2. Water holding capacity 0.4 ❖ Ongoing enzymatic processing of filaggrin 0.2 0.0 ❖ Folding and cross-linking of keratin N2 N7 N14 filaments profound superficial immature mature Milani et al, J. Mol. Rec. 2017

Exploration of the maturation process T HE RELATIVE STIFFNESS : AN INDICATION OF CORNEOCYTE MATURITY 1. Cross-linking during maturation CE stiffness JA Segre et al, Nat Genet, 1999

Exploration of the maturation process OPTIMAL DESQUAMATION Rigid – « stronger » Mechanical 1. Physical-Mechanical barrier resistance Mechanical constraint 2. Mechanical constraint likely Soft – « fragile » contributes to proneness to desquamation

Understanding age-induced mechanical alterations of the barrier Structural and Rate of turnover is molecular decreased modification Aging … Non-optimal Biomechanical desquamation properties ?

Understanding age-induced mechanical alterations of the barrier Subcellular scale measurement Overall mesure of the SC N = 6 N = 3 N = 2 N = 2 N = 2 800 ♀ 700 600 Ea (Mpa) 500 400 300 200 Biniek et al. J of derm. 2015 100 0 20-30yrs 30-40yrs 40-50yrs 50-60yrs 60-70yrs C ORNEOCYTE STIFFNESS INCREASES WITH AGE

Understanding age-induced mechanical alterations of the barrier N = 6 N = 3 N = 2 N = 2 N = 2 800 ❑ Cross-linking of keratin filaments ♀ 700 ❖ Oxydation 600 ❖ Glycation Ea (Mpa) 500 ❑ Decreasing water holding capacity 400 300 ❖ Less NMFs ❖ Increased intermolecular forces 200 100 0 20-30yrs 30-40yrs 40-50yrs 50-60yrs 60-70yrs C ORNEOCYTE STIFFNESS INCREASES WITH AGE

Understanding age-induced mechanical alterations of the barrier L2/L7 1.2 1 Relative stiffness 0.8 ❑ Less turnover ❑ Non-optimal desquamation 0.6 ❖ Accumulation of mature 0.4 corneocytes 0.2 0 20-30yrs 30-40yrs 40-50yrs 50-60yrs 60-70yrs I NTERNAL STIFFNESS GRADIENT IS DISRUPTED

Understanding age-induced mechanical alterations of the barrier OPTIMAL DESQUAMATION Higher fracture stress SC undergoes significant alterations Rigid 1. Stiffer with higher frature stress 2. Non-optimal desquamation Rigid Mechanical constraint Soft

Characterization of barrier dysfunction: atopic dermatitis ❑ Disrupted epidermal terminal differentiation ❑ Deficiency of structurale proteins ❑ Reduced lipids ❑ Abnormal skin barrier ❑ Inflammation ❖ GENETIC (FLG) ❖ ENVIRONNEMENT ❖ IMMUNOLOGIC FACTORS…

Characterization of barrier dysfunction: atopic dermatitis Haftek et al, in preparation

Characterization of barrier dysfunction: atopic dermatitis N = 50 N = 30 N = 80 N = 50 N = 49 *** 800 700 600 500 AD CORNEOCYTES ARE SOFTER Ea (MPa) 400 300 200 100 0 FLG+/+ FLG-/+ FLG+/+ FLG-/+ FLG-/- AD HEALTHY Haftek et al, in preparation

Characterization of barrier dysfunction: atopic dermatitis NORMAL SKIN AD SKIN OPTIMAL OPTIMAL DESQUAMATION DESQUAMATION Rigid – « stronger » Soft – « fragile » Mechanical Mechanical resistance resistance Mechanical Mechanical constraint constraint Soft – « fragile » Soft – « fragile »

Conclusions • The relative stiffness of corneocytes appears to be linked to their maturity level • The rigidity of superficial corneocytes may likely contribute to their proneness to optimal desquamation • Age induced accumulation of stiff corneocytes at the surface • Corneocytes from AD patients presented a lower stiffness than healthy subjects • This approach can be used for minimally invasive evaluation of various skin conditions and exploring the mode of action of pharmacological or cosmetic ingredients.

Thanks you for attention AD samples were collected in Dublin , Ireland, in Prof. Alan Irvine’s Dept .

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Characterization of barrier dysfunction: atopic dermatitis

Supplemental data S IMULATION BY FINITE ELEMENTS Extracted from Roduit et al.,2009 Extracted from Milani et al.,2011 CONFIDENTIAL

cutometer AFM Indenter Micro indenter Traction,Tension and Suction assay nm µm m cm CONFIDENTIAL