Identifying Keratinocyte Derived Collagen Inhibitory Factors to Treat Hypertrophic Scar and Keloid
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Transcript of Identifying Keratinocyte Derived Collagen Inhibitory Factors to Treat Hypertrophic Scar and Keloid
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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
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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
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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)
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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
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Overall Procedure
- Identifying factors in KCM which are involved in extracellular matrix regulation (ECM)
- Finding out the mechanism of ECM regulation by KCM
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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
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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.
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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
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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
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> 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
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Size Exclusion Chromatography Apparatus
Fast protein liquid chromatography
pro-collagen1
B-actin
F5 F7 F9 F11 F13 F15 F17 F17 F21 F23 KCM
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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
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Thanks to … - Dr. Ghahry’s Lab
- Dr. Duronio’s Lab- Peyman Hojabrpour
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Thanks for Your Attention
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Collagen Synthesis
Always cleave up your mess; William McKleroy et al. A J Physiology - Lung Cellular and Molecular Physiology. Published 1 June 2013Vol. 304
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