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Iñaki González-‐Foruria, Pietro Santulli, Sandrine Chouzenoux, Francisco Carmona, Charles Chapron, Frédéric Ba@eux
Dysregula+on of the ADAM17/Notch signaling pathways in endometriosis correlates with disease severity and
fibrosis
Introduc+on
• Benign gynaecological disease
• Chronic inflammatory condi+on (Bulun, 2009)
• Overproduc+on of prostaglandins, metalloproteases, cytokines and chemokines (Santulli, 2014)
• Oxida+ve stress plays an essen+al role in the onset and progression (Ngo, 2009; Santulli, 2015)
Introduc+on
• Oxida+ve stress induces the synthesis of inflammatory mediators
• ADAM family metalloproteases, such as ADAM17 (Zhang, 2001)
Introduc+on
• Oxida+ve stress induces the synthesis of inflammatory mediators
• ADAM family metalloproteases, such as ADAM17 (Zhang, 2001)
• ADAM family members are proteoly+cally ac+ve over target proteins, such as Notch (Milleri, 2013)
• Notch are transmembrane receptors that regulate many cell processes (Hori, 2013)
Introduc+on
• Notch ac+vates trancrip+on of nuclear target genes related to fibrosis
• Notch promotes fibroblasts prolifera+on and fibrosis (Kavian, 2014)
ADAM-‐17
Notch complex
Transcrip+onal ac+va+on
Mul+ple cell
func+ons
FIBROSIS
ROS
Nuclear transloca+on
Introduc+on
• Notch ac+vates trancrip+onal regula+on of nuclear target genes related to fibrosis
• Notch promotes fibroblasts prolifera+on and fibrosis (Kavian, 2014)
• No data evaluates ADAM17/Notch signaling pathway in endometriosis
• Hyperac+va+on of ADAM/Notch in other immunological diseases (Kavian, 2010)
Methods
• 202 women, younger than 42 years-‐old, excluding cancer
• Laparoscopy for gynaecological reasons
• Endometriosis rAFS classifica+on
• Endometriosis 3 phenotypes: – Superficial (SUP) – Endometrioma (OMA)
– Deep infiltra+ng endometriosis (DIE)
Methods
• Peritoneal fluid was obtained (n= 202)
• Stromal endometrial cells collec+on and culture: – Controls (n=8): endometrial biopsy (Cs)
– DIE pa+ents (n=8): endometrial biopsy (Es) + DIE biopsy (Ps)
Methods
• Advanced Oxida+on Protein Products –AOPP-‐ measurement in PF (Witko-‐Sarsat, 1998)
• ADAM17 metalloprotease ac+vity in PF (ELISA) and cultured stromal endometrial cells (Cs, Es, Ps) (WB)
• Notch ac+va+on (Notch intracellular domains -‐NICD-‐) in Cs, Es and Ps (WB)
• Fibroblasts ac+va+on (α-‐SMA and type-‐I collagen) in Cs, Es and Ps with/without DAPT (WB)
Patient characteristics! Endometriosis (N=121) Controls (N=81)
P!Age (years) a 30.8 ± 5.1! 31.7± 5.36! 0.267t!Height (cm) a 167.5 ± 6.1! 164.1 ± 6.0! <0.001t!Weight (kg) a 59.5 ± 8.1! 61.5 ± 9.9! 0.162t!BMI (kg/m2) a 21.2 ± 2.5! 22.8 ± 3.4! 0.001t!Parity a 0.2 ± 0.5! 0.4 ± 0.7! 0.093t!Gravidity a 0.4 ± 0.7! 0.6 ± 1.0! 0.123t! rAFS Classification:!
Mean implants score rAFS a, d 11.3 ± 11.2! NA! !Mean adhesions score rAFS a, d 9.6 ± 16.3! NA!Mean total score rAFS a, d 21.2 ± 23.1! NA!rAFS stage (n, %): d
!
NA!
I
37( 30.6%)
II
16 (13.2%)
III
26 (21.5%)
IV 42 (34.7%)
Surgical classification: !Superficial endometriosis (n, %) 41 (33.9%)! NA! !Endometrioma (n,%) 32 (26.4%)! NA!
Endometrioma size (cm): a
!
!
Right
4.9 ± 2.8
NA
Left 4.8 ± 3.1
Endometrioma laterality (n, %): !
NA !
Bilateral
7/32 (21.9%)
Right
11/32 (34.4%)
Left 14/32 (43.7%)
DIE lesions (n, %) e 48 (39.7%)! NA! Mean number of DIE lesions a 2.3 ± 1.5! NA!
Total number of DIE lesions (n, %):
!
NA!1
13/48 (27.1%)
2
11/48 (22.9%)
≥3 24/48 (50.0%)
Anatomical distribution of DIE (n, %): e, b
!USL
35/48 (72.9%)
NA
Vagina
15/48 (31.2%)
Bladder
7/48 (14.6%)
Intestine
23/48 (47.9%)
Ureter 2/48 (4.2%)
Results
Patient characteristics! Endometriosis (N=121) Controls (N=81)
P!Age (years) a 30.8 ± 5.1! 31.7± 5.36! 0.267t!Height (cm) a 167.5 ± 6.1! 164.1 ± 6.0! <0.001t!Weight (kg) a 59.5 ± 8.1! 61.5 ± 9.9! 0.162t!BMI (kg/m2) a 21.2 ± 2.5! 22.8 ± 3.4! 0.001t!Parity a 0.2 ± 0.5! 0.4 ± 0.7! 0.093t!Gravidity a 0.4 ± 0.7! 0.6 ± 1.0! 0.123t! rAFS Classification:!
Mean implants score rAFS a, d 11.3 ± 11.2! NA! !Mean adhesions score rAFS a, d 9.6 ± 16.3! NA!Mean total score rAFS a, d 21.2 ± 23.1! NA!rAFS stage (n, %): d
!
NA!
I
37( 30.6%)
II
16 (13.2%)
III
26 (21.5%)
IV 42 (34.7%)
Surgical classification: !Superficial endometriosis (n, %) 41 (33.9%)! NA! !Endometrioma (n,%) 32 (26.4%)! NA!
Endometrioma size (cm): a
!
!
Right
4.9 ± 2.8
NA
Left 4.8 ± 3.1
Endometrioma laterality (n, %): !
NA !
Bilateral
7/32 (21.9%)
Right
11/32 (34.4%)
Left 14/32 (43.7%)
DIE lesions (n, %) e 48 (39.7%)! NA! Mean number of DIE lesions a 2.3 ± 1.5! NA!
Total number of DIE lesions (n, %):
!
NA!1
13/48 (27.1%)
2
11/48 (22.9%)
≥3 24/48 (50.0%)
Anatomical distribution of DIE (n, %): e, b
!USL
35/48 (72.9%)
NA
Vagina
15/48 (31.2%)
Bladder
7/48 (14.6%)
Intestine
23/48 (47.9%)
Ureter 2/48 (4.2%)
Results
Results
• The use of DAPT to prevent NICD release, reduces fibrosis markers in Ps cells, but not in Cs cells
Conclusions
• AOPP and ADAM17 increase and correlaYon in endometriosis
• ADAM17 up-‐regulated in DIE
• ADAM17 and Notch are hyperacYvated in stromal endometrial cells from DIE compared to control endometrial cells
Conclusions
• Increased fibrosis in endometrial cells from DIE
• Notch inhibi+on diminishes fibrosis only in endometrial cells from DIE pa+ents, but not control cells
• ADAM17/Notch signaling pathway is up-‐regulated leading to major fibrosis in endometriosis
*This work was supported by grants from University Paris Descartes, INSERM and Fundación Alfonso Maran Escudero.
Thank you for your a@enYon
ADAM-‐17
Notch complex
Nuclear transloca+on
Transcrip+onal ac+va+on
FIBROSIS
ROS
Metalloproteinase ADAM-‐17 cleavage
NICD γ-‐Secretase complex