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.

- 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

non-healing over-healing

FibroblastsCollagenfibers

Keratinocytes

Dermis

EpidermisWound

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), - Endomyocardial fibrosis (heart)- Myelofibrosis (bone marrow)- Crohn's Disease (intestine)- Old myocardial infarction (heart) - Scleroderma/systemic sclerosis (skin, lungs)

Upon wound epithelialization, keratinocytes release two sets of ECM 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 fibroblastsObjective 2. Reveal the mechanism for inhibition of type 1 collagen production by KD-CIF

Hypothesis and Objectives

Overall Procedure

- Identifying factors in KCM which are involved in extracellular matrix regulation (ECM)

- Finding out the mechanism of ECM regulation by KCM

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.

pro-collagen1

B-actin

D0, Cnt 12h 24h 36 h 48h 60h 72h 72 h, Cnt

Control Fibroblast KCM treated fibroblasts

Keratinocytes culture

Fibroblasts culture

Keratinocytes Conditioned Media

Keratinocytes For RT-PCR and Western Blot

-2 d d0 +d2 +d4 +d6 +d8

Involucrin

ACTB

+2 d, diff

+6 d, diff

+4d, diff

+8 d, diff

d0, Conflu

ent

-2 dContro

l

ACTB

Pro-Coll

Involucrin

KRT 10

KRT 14

Tbp

+2 d, diff

+6 d, diff

+4d, diff

+8 d, diff

Confluent

-2 d

Collagen Inhibitory Factor (CIF) is released from undifferentiated proliferating keratinocytes.

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.

pro-collagen1

B-actin

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.

A

B

Post- Transcriptional regulation:

Col1a1 Col1a2

6 hr- Contro

l

6 hr- Contro

l+ DRB

6 hr- KCM+ DRB

12 hr- Contro

l

12 hr- Contro

l+ DRB

12hr- KCM+ DRB

6 hr- Contro

l

6 hr- Contro

l+ DRB

6 hr- KCM+ DRB

12 hr- Contro

l

12hr- Contro

l+ DRB

12hr- KCM+ DRB

- Evaluation of Collagen type 1 mRNA Stability

Cyto- control

Cyto- KCM

Nuc- Control

NucKCM

Col1a1

Cyto- control

Cyto- KCM

Nuc- Control

NucKCM

Col1a2

- Cytoplasmic Vs Nuclear RNA

> 30 KD< 50KD < 9KD

retentate

filterate

KCM KCM KCM

> 50 KD >30 KD > 9 KD

At least two sets of factors cooperate to inhibit Collagen production

Size Exclusion Chromatography Apparatus

Fast protein liquid chromatography

pro-collagen1

B-actin

F5 F7 F9 F11 F13 F15 F17 F17 F21 F23 KCM

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

Thanks to … - Dr. Ghahry’s Lab

- Dr. Duronio’s Lab- Peyman Hojabrpour

Thanks for Your Attention

Collagen Synthesis

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