Wound healing consists of three phases: inflammation, proliferation, and remodeling. During proliferation and remodeling, new extracellular matrix (ECM) is laid down by cells called fibroblasts. ECM consists of proteins that provide structural support in tissues. ECM production and fibroblast migration and proliferation are stimulated by connective tissue growth factor (CTGF). During wound healing, CTGF is induced by transforming growth factor beta (TGF-beta) to stimulate this response. When TGF-beta and CTGF are overexpressed, excessive amounts of ECM proteins are laid down, forming scar tissue, also known as fibrosis. Fibrotic tissue in organs can impair healthy function, leading to organ failure. To better understand how CTGF functions in fibrotic tissue, cells with varying levels of CTGF expression will be examined in a cell model of wounding. We will measure gene expression and observe cells for changes in fibrosis-related phenotypes. We hypothesize that increased CTGF expression will lead to enhanced cell growth, motility, and ECM production, as compared to controls. Results will provide a foundation for further research into the effects of CTGF variations on fibrosis-related phenotypes.

1. 0
0.02
0.04
0.06
0.08
No Vector pSV B-Gal &
pUC19
pSV B-Gal &
CTGF pUC19
Absorbance(405nm)
Methods
Acknowledgements
Successful Insertion of CTGF into Vector
Conclusions
Future Directions
Department of Biological Sciences at Plymouth State University in Plymouth, NH
References
CTGF and TGFβ Treatment Accelerates Scratch Area Infiltration
• CTGF and TGFβ accelerate wound area invasion by
fibroblast cells
• The effect of CTGF on wound closure is TGFβ
dependent
• We successfully inserted the CTGF gene into our
pUC19 vector
• NIH/3T3s were successfully transfected with vector
• pSV β-Gal can be used to quantify relative
transfection efficiencies
Effects of CTGF Overexpression on Fibrosis-Related Phenotypes
Kimberly Jesseman, Lorna Smith, Kathryn Kahrhoff, Stacy Peterson, Ashley Kennedy and Heather Doherty PhD
Introduction
The development of scar tissue, also known as fibrosis, can alter the healthy
function of organs, eventually leading to organ failure (Wynn, 2008). Diseases that
manifest excess scar tissue are a leading cause of mortality (Gurtner et al., 2008).
Excessive scarring can occur in all tissues and organ systems of the body (Wynn,
2007), including the liver (Bataller et al., 2005), the heart (Krenning et al., 2010), and
the lungs (Phan, 2002). Scar tissue is caused by excess extracellular matrix (ECM)
production during wound healing. To aid in the healing process, expression of
connected tissue growth factor (CTGF) is increased. CTGF is a signaling molecule
secreted by fibroblast cells (Wynn, 2008) and is important in cell migration,
attachment, survival, proliferation, and ECM production (Blom et al., 2002). The
signaling molecule transforming growth factor (TGFβ) is known to stimulate CTGF
expression during the wound healing process. Increased expression of CTGF and
TGFβ is associated with susceptibility and severity of fibrotic diseases (Wynn, 2007;
Bataller et al., 2005; Krenning et al., 2010; Phan, 2002).
In order to increase our understanding of scarring, overexpression of CTGF was
investigated in a cell culture wound model. Mouse embryonic fibroblast cells (ATCC
NIH/3T3) were used in this model because of their importance and involvement in
fibrosis. The cells were treated with CTGF and TGFβ, to induce overexpression of
CTGF (Frazier et al., 1996). Cells treated with CTGF and TGFβ were hypothesized to
show enhanced cell growth and motility compared to other treatment groups. In
contrast, cells treated with CTGF, TGFβ, and TGFβ inhibitor were hypothesized to
show similar growth to untreated cells. By treating with TGFβ and the inhibitor, we
were able to observe the importance of TGFβ on CTGF-mediated cell motility and
growth in a cell model of wounding. In order to further examine the overexpression
of CTGF, a vector including the CTGF gene was constructed to transfect into
fibroblast cells. The transfection procedure was tested and validated by
cotransfecting CTGF vector with a control plasmid. Gene expression from this control
can be quantified with a colorimetric test to determine relative transfection
efficiencies between wells. Future directions will include transfecting the vectors
into NIH/3T3 cells to observe how overexpressing CTGF using this vector will
influence the growth and motility of the fibroblasts.
Control(untreated)CTGFandTGFβCTGFandTGFβWith
TGFβInhibition
D E F
A B C J
G H II
0 hours 12 hours 24 hours
Successful Transfection of NIH/3T3 Cells
Ampr
0
20
40
60
80
100
0 6 12 18 24
%ScratchAreaRemaining
Time Post-Scratch (Hours)
Control CTGF and TGFB CTGF TGFB and Inhibitor
Post-Hoc Comparison (24 H) P-Value
Control v CTGF and TGFβ *0.050
Control v CTGF, TGFβ and Inhibitor 0.233
CTGF and TGFβ v CTGF, TGFβ and Inhibitor **0.001
K
B
CTGF pUC19
Empty pUC19
Ladder
3000
6000
A
B
Ampr pSV β-Gal
6820 bp
LacZ
SV40 PromoterA
1
2000
400040004000
6000
CTGF
pUC19 with
CTGF insert
6934 bp
XbaI
1
2000
6000
4000
BamHI
Cells and Cell Environment:
Mouse embryonic fibroblasts (NIH/3T3s) were maintained at 37C and 5% CO2 in
Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum and
Pen/Strep/Glut.
Scratch Tests:
Scratch tests were performed in order to simulate cell response in wound healing.
Wells were scratched using a p10-100 pipet tip. After scratching, all wells received
fresh media with one of three treatments: 1) water and DMSO (control), 2) TGF and
CTGF or, 3) TGF, CTGF, and the TGF inhibitor GW788388 (Sigma-Aldrich). Scratch
test area was imaged at 40x total magnification. Images were taken at 0, 6, 12, 18
and 24 hours post scratch. ImageJ software (NIH) was used to measure remaining
scratch test area from each picture. Factorial analysis of variance (ANOVA) was used
to determine whether hour or treatment played a significant role in fibroblast
infiltration into the scratch area. Post-hoc Student’s t-tests were used to test for
significant differences in remaining scratch area of each treatment at 24 hours
(p<0.05 was considered statistically significant). Minitab 17 (Minitab Inc.) was used
for all statistical analyses.
Vector Construction:
In order to create the pUC19/CTGF vector, CTGF was amplified and then double
digested along with empty pUC19 using restriction enzymes BamHI and XbaI. The
gene and plasmid were ligated and then transformed into E. coli cells. Colonies
positive for vector insertion were picked and expanded. pUC19/CTGF vector
candidates were digested with BamHI along with empty vector and run on a gel to
verify size differences. Successful insertion was further verified with sequencing of
ligation junctions and the entire CTGF gene. Sequencing was done at Dartmouth
Molecular Biology Core Facility.
β-Gal Assay Testing:
The pSV-beta-Galactosidase (pSV -Gal) vector was obtained from Promega. Cells
were split onto 6 well plates and once they reached 70% confluence, vector was
transfected into cells using lipofectamine 2000 (Invitrogen). Treatments included a
control with no vector and experimental cotransfections of pSV -Gal and
pUC19/CTGF or pSV -Gal and empty pUC19. Cells were harvested 24 hours post-
transfection and a β-galactosidase assay was completed to measure transfection
efficiency.
Figure 1: Representative NIH/3T3 pictures at 0, 12, and 24 hours after scratch testing and quantitation of scratch closure. A-C) Untreated cells at 0, 12, and 24 hours, respectively.
D-F) CTGF and TGFβ treated cells at 0, 12, and 24 hours, respectively. G-I) CTGF, TGFβ, and inhibitor treated cells at 0, 12, and 24 hours, respectively. J) Percentage of original scratch
area remaining for each treatment over time. K) Results of post-hoc Student’s t-tests at the 24 hour timepoint. p<0.05 was considered significant for all analyses (n=3).
Results: A-I) Cells treated with CTGF and TGFβ proteins show greater wound invasion than untreated cells, suggesting increased motility and mitogenic potential of the fibroblast
cells. Cells treated with both proteins and the TGFβ inhibitor appeared similar to the control. J) After 24 hours, CTGF and TGFβ treatment resulted in the least scratch wound area
remaining. Time (p<0.001), treatment (p=0.001), and the combination of both (p=0.002) significantly influenced the percentage of scratch area remaining as determined by factorial
ANOVA. K) At 24 hours, CTGF and TGFβ treatment resulted in significantly less scratch area remaining than the control (p=0.05) and inhibitor treatments (p=0.001). The controls and
inhibitor treated cells were not significantly different from one another after 24 hours (p=0.233). Overall, TGF and CTGF treatment increased the speed at which cells infiltrated the
scratch area as compared to controls, and addition of a TGF inhibitor reversed this effect.
Figure 2: A) Diagram of pUC19 vector with CTGF gene insert. B) CTGF vector and empty
pUC19 vector digested with BamHI and run on a gel to check for proper insert size.
Results: A) pUC19 vector with a CTGF gene insert was designed to transfect into
NIH/3T3 fibroblast cells. B) The size of the band produced after digestion helps confirm
that CTGF has been successfully inserted into the vector. Additional analysis by
sequencing at the ligation junctions, as well as within the entire gene (not shown),
verifies that we have successfully created a vector with the CTGF gene insert.
Figure 3: A) Diagram of pSV β-Gal control vector. This vector includes an ampicillin
resistance gene and the LacZ gene encoding the β-Galactosidase protein. Expression of
this gene is induced in our fibroblasts via an SV40 promoter. B) Absorbance at 405nm of
β-Gal assay reactions from cells without vector, cells transfected with pSV β-Gal and
pUC19 vector, and cells transfected with pSV β-Gal and CTGF vector (n=1).
Results: B) pSV β-Gal control vector was cotransfected with pUC19/CTGF vector or
empty pUC19 as a transfection efficiency control. Cells with pSV β-Gal and empty pUC19
or with pSV β-Gal and pUC19/CTGF vector had an absorbance about 7 times and 5.5
times greater than controls, respectively. The LacZ gene is expressed robustly when the
pSV β-Gal control vector is transfected with the pUC19 or pUC19/CTGF vector, and both
result in an absorbance that is much higher than that of the controls. This suggests that
the vectors were successfully transfected into our NIH/3T3 cells.
• Compare phenotypes of cells transfected with empty
versus CTGF-containing vector
• Measure expression of fibrosis-related genes
including CTGF, fibronectin, and type III and IV
collagens
• Perform scratch test to examine differences in
wound healing
• Examine how CTGF genetic variations impact fibrosis-
related phenotypes
We would like to thank Plymouth State University, the PSU Research Advisory Council,
the PSU Student Research Advisory Council, and the New Hampshire Idea Network of
Biological Research Excellence for funding support. We would like to thank the
Dartmouth College Molecular Biology Shared Resources Lab for sequencing. We would
also like to thank Kimberly Amerson, Jon Bairam, Joel Dufour, Hailey Gentile, Zachary
Stevens, Amed Torres, and Alycia Wiggins.
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