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
Problem: Hypertrophic scar and Keloid
Keratinocytes
Collagen
fibers
Wound
Epidermis
Fibroblasts
Dermis
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)
Hypothesis and Objectives
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 (KDCIF) for fibroblasts
Objective 2. Reveal the mechanism for inhibition of type 1 collagen production by KD-CIF
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.
D0, Cnt
12h
24h
36 h
48h
60h
72h
72 h, Cnt
pro-collagen1
B-actin
Control Fibroblast
KCM treated fibroblasts
Collagen Inhibitory Factor (CIF) is released from undifferentiated proliferating keratinocytes.
Fibroblasts culture
Pro-Coll
ACTB
Keratinocytes Conditioned Media
-2 d
Keratinocytes culture
Keratinocytes For
RT-PCR and
Western Blot
Involucrin
KRT 10
KRT 14
Tbp
Involucrin
ACTB
d0
+d2
+d4
+d6
+d8
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
D3, Cnt
pro-collagen1
B-actin
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:
- Evaluation of Collagen type 1 mRNA Stability
Col1a1
Col1a2
- Cytoplasmic Vs Nuclear RNA
Col1a1
Col1a2
At least two sets of factors cooperate to inhibit Collagen production
KCM
KCM
KCM
retentate
> 50 KD
>30 KD
filterate
< 50KD
> 30 KD
> 9 KD
< 9KD
Fast protein liquid chromatography
F5
pro-collagen1
B-actin
Size Exclusion Chromatography Apparatus
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