Methods

Introduction

Acknowledgements 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 also like to thank Ethan Johnson, Ellen Rounds, Harlie Shaul, Kate-Lyn Skribiski, Chris Gonzalez, Justin Provazza, John Rollins, the University of New Hampshire Hubbard Center for Genome Studies DNA core, and Dartmouth College Molecular Biology Shared Resources Lab for their contributions.

Effects of TGFβ Inhibition on CTGF and Fibrotic Gene Expression

Conclusions

Future Directions

Department of Biological Sciences and Biotechnology Program at Plymouth State University in Plymouth, NH

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861.

Vector Design• TGFβ inhibition decreased wound area invasion by fibroblasts• Inhibiting TGFβ reduces CTGF and fibronectin expression• Vector with CTGF insert was successfully created and tested• A negative feedback pathway may be controlling levels of

CTGF to prevent excess expression • TGFβ or CTGF inhibition may be an effective way of reducing

scar tissue buildup in patients prone to high levels of fibrosis

Characterization of Connective Tissue Growth Factor Gene Expression in a Tissue Culture Model of Wounding

pUC19 with CTGF insert

6,930 bp

Effect of Human CTGF Vector on CTGF Expression

Kimberly Jesseman, Ashley Kennedy, Joel Dufour, Jon Bairam, Alycia Wiggins, Evyn Grimes, Heather Doherty

Effects of TGFβ InhibitionOn Cell Proliferation

Figure 3: pUC19 vector with a CTGF gene insert was designed to transfect CTGF gene variants into NIH/3T3 fibroblast cells.

Results and Discussion: CTGF gene was successfully cut at restriction sites and inserted into pUC19 vector. The size of the band produced from amplification via PCR of exons 3, 4, and 5 of the CTGF gene from vector insert helped confirm that CTGF had been successfully inserted into the vector (data not shown). Analysis of sequence at the ligation junctions, as well as within the gene, confirms that we have successfully created a vector with a CTGF gene insert. This vector was used in experiment 3 and will continue to be used in future work.

A

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D

Fibrosis is a buildup of scar tissue that often results from dysfunctional healing. In several diseases including chronic obstructive pulmonary disorder (COPD), cirrhosis, systemic sclerosis, and kidney disease, fibrosis plays an important role. This scarring has also been detected in the cardiac tissue of many of the Americans who survive a heart attack each year. Buildup of fibrotic tissue can result in cardiac dysfunction, arrhythmia, heart failure and lead to mortality. In animal models, elevated levels of the Connective Tissue Growth Factor (CTGF) gene have been observed in fibrotic heart tissue. This gene along with an inducer of its expression, Transforming Growth Factor Beta (TGFβ), is believed to be a key contributor to fibrotic tissue formation. Together, they are thought to induce wound healing after damage by promoting cell division, cell migration, and the production of extracellular proteins including collagen and fibronectin. Overexpression or prolonged expression of these signaling proteins can result in fibrotic tissue. Like many other conditions, susceptibility to fibrosis varies between individuals. Natural variations in the CTGF gene may play a role in an individual’s risk of developing fibrosis. A number of single nucleotide polymorphisms (SNPs) in the CTGF gene are published, and we have identified several others by sequencing the human CTGF gene of volunteers at Plymouth State University. Though many sequence variations in the CTGF gene have been identified, their effects on human health and susceptibility to fibrosis remain unclear.

The current goal of this research is to examine CTGF expression levels and rates of cell growth and division in mouse embryonic fibroblasts (NIH/3T3) under a number of different conditions. The effects of chemical inhibitors of TGFβ on CTGF gene expression and phenotypic markers of fibrosis are being examined using quantitative PCR (qPCR). After four hours of TGFβ inhibition, CTGF expression and the ability of NIH/3T3 cells to migrate into wound areas were significantly reduced. Inhibition had no significant effect on type I collagen expression, but did significantly decrease fibronectin expression. The effects of vector insertion into NIH/3T3s on CTGF expression was also measured. Insertion of vector containing CTGF decreased expression of CTGF, suggesting a potential negative feedback loop regulating CTGF expression.

Future goals include examining the phenotypic effects of human CTGF SNPs using site directed mutagenesis to insert specific changes into the human CTGF gene. Changes will then be introduced into mouse NIH/3T3 fibroblasts using a plasmid vector. A vector is a small circular piece of DNA naturally found in bacteria that can be inserted into cells. Any genes included in the vector’s genome will be expressed by the cells containing the vector. A vector containing the CTGF gene has been generated and successfully put into NIH/3T3 cells. Understanding the phenotypic effects of altered CTGF expression could lead to novel insights about fibrosis-related molecular pathways and potentially to new anti-fibrotic therapies and improved outcomes for patients suffering from fibrotic diseases.

Cell Environment Mouse embryonic fibroblasts (NIH/3T3s) were used in all tissue culture experiments. They were grown in normal media and were maintained at 37°C and 5% CO2 levels. All experiments were run on 6-well plates when cells were at 50-70% confluence.

Vector Design Full length CTGF was amplified from human male DNA (Promega). The CTGF gene was inserted into the pUC19 vector (New England Biolabs) using BamHI and XbaI restriction enzymes. Gene-vector junctions and individual exons were sequenced to confirm successful vector assembly.

Cell Treatments and HarvestThree experiments were performed:1. Effects of TGFβ Inhibition on Cell Proliferation: NIH/3T3s were treated with 10μM

LY2157299 (Cayman Chemical) and plates were scratched with a pipet tip. Plates were imaged at 0 and 18 hrs.

2. Effects of TGFβ Inhibition on CTGF Gene Expression: NIH/3T3s were treated with two different TGFβ Inhibitors: LY7157299 (10 μM) or GW788388 (2 μM) (Cayman Chemical). Controls were treated with vehicle. After four hours of treatment, cells were harvested.

3. Mouse vs. Human CTGF Expression: NIH/3T3s were transfected with CTGF vector. Vector without the CTGF gene was used as a control. 24 hours after transfection, cells were harvested.

Measurement of Gene Expression Immediately after harvest, RNA was extracted from cells in experiments 2 and 3, and cDNA was synthesized by reverse transcription PCR. qPCR was then used to determine relative amounts of gene expression. In experiment 2, mouse CTGF, Collagen (Col1a1) and Fibronectin (Fn1) expression were measured. In experiment 3, only mouse CTGF gene expression was measured. β-actin expression was used as a control in all experiments to standardize expression levels. qPCR data was analyzed using Microsoft Excel and Student T-tests were used to determine statistical significance. Tests yielding a p-value ≤ 0.05 were considered statistically significant. P-values > 0.05 and < 0.10 were considered to be statistically suggestive.

Figure 1: NIH/3T3s immediately after scratch test and 18 hours later. A) Inhibitor treated cells at 0 hrs. B) Inhibitor treated cells at 18 hr. C) Untreated cells at 0 hrs. D) Untreated cells at 18 hrs.

Results and Discussion: Cells treated with TGFβ inhibitor exhibited decreased wound area invasion when compared to control cells, indicating that TGFβ is important in increasing fibroblast growth and movement. CTGF expression is involved in the signaling pathway that results in cell proliferation and CTGF expression decreased as a result of TGFβ inhibition.

CTGF Col1a1 Fn10

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150 Control GW788388 LY2157299

Gene

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Figure 2: TGFβ inhibitor effect on CTGF, Collagen (Col1a1), and Fibronectin (Fn1) gene expression four hours after treatment. n = 4 for control and each treatment. * Indicates statistical significance, p ≤ 0.05

Results and Discussion: To determine if expression of treated samples was significantly decreased compared to controls, one tailed T-tests were used. A significant decrease in CTGF expression was observed after treatment with each inhibitor (GW788388: p = 0.01; LY2157299: p < 0.01). However, there was no significant effect of TGFB inhibition on collagen expression four hours after treatment (GW788388: p = 0.40; LY2157299: p = 0.50). A longer period of exposure to the TGFβ inhibitor may be necessary before changes in collagen expression become noticeable. A significant decrease in fibronectin expression was observed (GW788388: p = 0.05; LY2157299: p = 0.05), indicating that fibronectin expression is likely influenced by CTGF, TGFβ , or both.

Figure 4: Mouse CTGF expression with CTGF vector relative to expression with empty vector. n = 3 for control and treatment. # indicates results are statistically suggestive (0.05 < P < 0.10).

Results and Discussion: A two tailed T-test was used to determine if differences in expression were statistically significant. Mouse CTGF expression decreased in the presence of vector with the human CTGF insert with a suggestive p value (p = 0.08). This at first seems counterintuitive, as it would be expected that an additional CTGF gene would result in additional CTGF expression. However, the results suggest a feedback loop may regulates CTGF levels. Excess CTGF expression may actually trigger this negative feedback loop to decrease CTGF expression in order to maintain homeostasis.

• Measure expression of target genes at different time points after treatment with inhibitor

• Measure human CTGF expression with vector• Site-directed mutagenesis of vector to add SNPs found in our

sample population• Observe the role of CTGF SNPs in altering wound healing and

fibrosis-related gene expression

* *

* *

#

Human CTGF

Ampicillin Resistance

Gene