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Identifying Keratinocyte Derived Collagen Inhibitory Factors to Treat Hypertrophic Scar and Keloid

Identifying Keratinocyte Derived Collagen Inhibitory Factors to Treat Hypertrophic Scar and Keloid. By: Ali Farrokhi (PhD student) Supervisor: Dr. Ghahary 25 Jun 2014. Introduction. The history of wound healing is, in a sense, the history of humankind.

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Identifying Keratinocyte Derived Collagen Inhibitory Factors to Treat Hypertrophic Scar and Keloid

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  1. Identifying Keratinocyte Derived Collagen Inhibitory Factors to Treat Hypertrophic Scar and Keloid By: Ali Farrokhi (PhD student) Supervisor: Dr. Ghahary 25 Jun 2014

  2. Introduction • The history of wound healing is, in a sense, the history of humankind. • One of the oldest medical manuscripts known to man is a clay tablet that dates back to 2200 B.C, describes the “three healing gestures”—washing the wounds, making the plasters, and bandaging the wound. wound healing over-healing non-healing

  3. Problem: Hypertrophic scar and Keloid Over healing such as post-burn hypertrophic scarring (HSc) and keloid are disfiguring and devastating, resulting in bulky, itchy and inelastic scars that pose serious functional and cosmetic problems for recovering burn patients. Fibrosis: • Cirrhosis (liver), • Endomyocardialfibrosis (heart) • - Myelofibrosis(bone marrow) • - Crohn'sDisease (intestine) • - Old myocardial infarction (heart) • - Scleroderma/systemic sclerosis (skin, lungs) Wound Keratinocytes Epidermis Collagen fibers Fibroblasts Dermis

  4. Hypothesis and Objectives • Upon wound epithelialization, keratinocytes release two sets ofECM regulating factors: • The 1st set: stimulate the expression of Matrix Metalo-Proteinase (MMP-1, 3) • The 2nd set: suppresses the expression of key ECM components like type I and III collagen. • To test this hypothesis, we propose the following Specific Objectives: • Objective 1. Identify and characterize Keratinocyte Derived Collagen Inhibitory Factors (KD-CIF) for fibroblasts • Objective 2. Reveal the mechanism for inhibition of type 1 collagen production by KD-CIF

  5. Overall Procedure • Identifying factors in KCM which are involved in extracellular matrix regulation (ECM) • Finding out the mechanism of ECM regulation by KCM

  6. Keratinocytes Conditioned Media (KCM) inhibits collagen type 1 production in fibroblasts. Western blot analysis of intracellular proteins showed the inhibition of Collagen type 1 synthesis in time dependent manner. D0, Cnt 12h 24h 36 h 48h 60h 72h 72 h, Cnt KCM treated fibroblasts Control Fibroblast pro-collagen1 B-actin

  7. Collagen Inhibitory Factor (CIF) is released from undifferentiated proliferating keratinocytes. d0, Confluent -2 d Control +2 d, diff +6 d, diff +4d, diff +8 d, diff Fibroblasts culture Pro-Coll ACTB KRT 14 Keratinocytes Conditioned Media KRT 10 Involucrin +d4 +d8 -2 d +d6 d0 +d2 Tbp Keratinocytes culture Keratinocytes For RT-PCR and Western Blot Involucrin -2 d +2 d, diff +6 d, diff +4d, diff Confluent +8 d, diff ACTB

  8. Keratinocytes Conditioned Media (KCM) inhibits collagen type 1 production in fibroblasts. Western blot analysis of intracellular proteins showed the inhibition of Collagen type 1 synthesis in time dependent manner. A D0, Cnt 12h 24h 36 h 48h 60h 72h D3, Cnt KCM inhibits collagen type 1 expression post-transcriptionally. After treatment of fibroblasts with KCM, cells were harvested at different time points to evaluate Col1a1 (A) and Col1a2 (B) genes expression by qRT-PCR. B pro-collagen1 B-actin

  9. Post- Transcriptional regulation: - Evaluation of Collagen type 1 mRNA Stability Col1a1 Col1a2 6 hr- Control 12 hr- Control 6 hr- Control+ DRB 6 hr- KCM+ DRB 6 hr- Control 12hr- Control+ DRB 12hr- KCM+ DRB 12 hr- Control 6 hr- Control+ DRB 6 hr- KCM+ DRB 12 hr- Control+ DRB 12hr- KCM+ DRB - Cytoplasmic Vs Nuclear RNA Col1a1 Col1a2 NucKCM Nuc- Control Cyto- control Cyto- KCM NucKCM Cyto- control Nuc- Control Cyto- KCM

  10. At least two sets of factors cooperate to inhibit Collagen production KCM KCM KCM > 9 KD retentate >30 KD > 50 KD < 50KD filterate > 30 KD < 9KD

  11. Fast protein liquid chromatography F5 F7 F9 F11 F13 F15 F17 F17 F21 F23 KCM pro-collagen1 B-actin Size Exclusion Chromatography Apparatus

  12. Future Plan • Find out which step of post-transcriptional regulation is involved in Collagen type 1 regulation by KCM • Which specific factors are in KCM that do Collagen production inhibition

  13. Thanks to … • Dr. Duronio’s Lab • PeymanHojabrpour - Dr. Ghahry’s Lab

  14. Thanks for Your Attention

  15. Collagen Synthesis Always cleave up your mess; William McKleroyet al. A J Physiology - Lung Cellular and Molecular Physiology. Published 1 June 2013Vol. 304

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