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Peter Gal et al.

East-Slovak Institute of Cardiovascular Diseases Pavol Jozef Safarik University. Pharmacological targeting of estrogen receptors-α and -β differently modulates the phenotype of keratinocytes with impact on skin wound healing. Peter Gal et al. Introduction.

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Peter Gal et al.

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  1. East-Slovak Institute of Cardiovascular Diseases Pavol Jozef Safarik University Pharmacological targeting of estrogen receptors-α and -β differently modulates the phenotype of keratinocytes with impact on skin wound healing Peter Gal et al.

  2. Introduction increasing average age of (female) population = poor wound healing • USA over 10 billions USD / year - postmenopausalperiod is characterized by: • decrease of estrogen levels • decrease of TGF-β1 levels • increase of MIF levels

  3. Introduction / Aims Estrogen Replacement Therapy (17β estradiol) – various effects on tissue repair → we used specific ER agonists • in vitro:roles of ER-α and -βon fibroblasts, endothelial cells, and keratinocytes (these cells do express ERs) • in vivo part:roles of ER-α and -βin wound healing (excisions = histology + incisions = wound tensile strength measurement)

  4. Methods – in vitro • Human dermal fibroblasts (HDF) / keratinocytes (HaCaT) • isolated from skin residues (DMEM + 10% FBS, ATB, 5%CO2) • ICC: fibronectin+SMA+DAPI / panel of keratins + Ki67 • Human umbilical vein endothelial cells (HUVEC) • isolated from umbilical cords • (M199 + 10% HS + 10% hi NBCS, ECGF, heparin, ATB) • gelatin zymography: MMP-2 a -9 (expresia / aktivita) • PPT / DPN –10nM

  5. Results – in vitro ERs HDFs: HUVECs:

  6. Results – in vitro ERs

  7. Results – in vitro keratinocytes

  8. Results – in vitro fibroblasts SMA Fibronectin a – PPT b – DPN c – control d – TGF-β1

  9. Results – in vitro endothelial cells

  10. Methods – in vivo • female rats • Sprague-Dawley (n=64), OVX – 3 months prior wounding • groups (5 d n=8, 10 d n=8) • NOVX – sham surgery • OVX-C – ovariectomizedvehicle treated • OVX-α – ovariektomized PPT treated (1 mg/kg) • OVX-β – ovariektomized DPN treated (1 mg/kg)

  11. Methods – in vivo histology • left round wound – HE + VG • light microscope (analyzed by QuickPHOTO Micro 2.2) • right wound – FIBR/SMA, CK/VIM, nuclei DAPI • fluorescence mikroscope(analyzed by Lucia 5.1) • 3 channels – RGB:blue – DAPI, green – FITC, red – TRITC

  12. Methods – in vivo TS measurement • both incisions – 2 x 1 cm skin/wound strip • TS=MBS/A, [g/mm2] • wound tensile strength (TS) measuring device

  13. Results – in vivoHE + VG a – OVX-α b – OVX-β c – NOVX-C d – OVX-C

  14. Results – in vivoIHC a – OVX-α b – OVX-β c – NOVX-C d – OVX-C

  15. Results – in vivowound TS

  16. Discussion: incision vs. excision: different treatment ? TGF-β1↑ fb > myofb;mmp13 ↑ wound contraction / scaring ↑ wound TS ↓

  17. Discussion / Conclussion - targeting ER-α(PPT) – excision - targeting ER-β (DPN) – incision + epithelization (open wound) PPT DPN ECM formation + epithelization↑ TGF-β1 fb > myofb;↑ mmp13? / 2 / 9 wound contraction ↑ wound TS ↓ Wound type specific approach !/?

  18. Acknowledgement team post-docs: • Dr. Martin Novotný • Dr. Tomáš Vasilenko • Dr. Martin Slezák • Dr. Ivan Kováč • Dr. Pavol Szabo • Dr. Lenka Varinská colleagues from Prague: • prof. Karel Smetana ml. • Dr. Barbora Dvořánková technician: • Magdaléna Majnušová Ph.D. and M.S. students: Martin Slezak, Martina Polakova, Jan Jakubco, Martina Kostelnikova

  19. Thank you for your attantion!

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