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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

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

By: Ali Farrokhi (PhD student)

Supervisor: Dr. Ghahary

25 Jun 2014

introduction
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

slide3

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

slide4

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
slide5

Overall Procedure

  • Identifying factors in KCM which are involved in extracellular matrix regulation (ECM)
  • Finding out the mechanism of ECM regulation by KCM
slide6

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

slide7

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

slide8

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

slide9

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

slide10

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

slide11

Fast protein liquid chromatography

F5 F7 F9 F11 F13 F15 F17 F17 F21 F23 KCM

pro-collagen1

B-actin

Size Exclusion Chromatography Apparatus

future plan
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
slide13

Thanks to …

  • Dr. Duronio’s Lab
  • PeymanHojabrpour

- Dr. Ghahry’s Lab

collagen synthesis
Collagen Synthesis

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