Methods

Abstract

AcknowledgementsWe 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 (NH-INBRE) for funding support. We would also like to thank Hailey Gentile, Chris Gonzalez, Justin Provazza, and Joel Dufour.

Cytokine Treatment Alters Fibrosis-Related Gene Expression Conclusions

Future Directions

Department of Biological Sciences at Plymouth State University in Plymouth, NH

References 1. Broughton G, Janis JE, Attinger CE (2006) The Basic Science of Wound Healing. Plastic and Reconstructive

Surgery 117: 12S-34S.2. Chen, Chih-Chiun and Lau, Lester F. 2010. Functions and Mechanisms of Action of CCN Matricellular Proteins.

Int J Biochem Cell Biol. Apr 2009; 41(4): 771–783. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2668982/3. Brigstock, D. R. (1999). The Connective Tissue Growth Factor/Cysteine-Rich 61/Nephroblastoma

Overexpressed (CCN) Family 1. Endocrine reviews, 20(2), 189-206.4. Frazier, K. et al. (1996). Stimulation of fibroblast cell growth, matrix production, and granulation tissue

formation by connective tissue growth factor. Journal of Investigative Dermatology, 107(3), 404-411.5. Sonnylal, S., et al. (2010). Selective expression of connective tissue growth factor in fibroblasts in vivo

promotes systemic tissue fibrosis. Arthritis & Rheumatism, 62(5), 1523-1532. 6. Igarashi, A., Nashiro, K., Kikuchi, K., Sato, S., Ihn, H., Grotendorst, G. R., & Takehara, K. (1995). Significant

Correlation Between Connective Tissue Growth Factor Gene Expression and Skin Sclerosis in Tissue Sections from Patients with Systemic Sclerosis. J Invest Dermatol, 105, 280-284.

7. Ponticos, M. et al. (2009). Pivotal role of connective tissue growth factor in lung fibrosis: MAPK dependent ‐transcriptional activation of type I collagen. Arthritis & Rheumatism, 60(7), 2142-2155

8. Gressner, O. A., & Gressner, A. M. (2008). Connective tissue growth factor: a fibrogenic master switch in fibrotic liver diseases. Liver international, 28(8), 1065-1079.

9. Ito, Y., Aten, J., Bende, R. J., Oemar, B. S., Rabelink, T. J., Weening, J. J., & Goldschmeding, R. (1998). Expression of connective tissue growth factor in human renal fibrosis. Kidney international, 53(4), 853-861.

10. Gupta, S., Clarkson, M. R., Duggan, J., & Brady, H. R. (2000). Connective tissue growth factor: potential role in glomerulosclerosis and tubulointerstitial fibrosis. Kidney international, 58(4), 1389-1399.

11. Chen, M. et al. (2000). CTGF expression is induced by TGF-β in cardiac fibroblasts and cardiac myocytes: a potential role in heart fibrosis. Journal of molecular and cellular cardiology, 32(10), 1805-1819.

12. Doherty, H. The Role of Quantitative Variations in Connective Tissue Growth Factor Gene Expression in Cardiac Hypertrophy and Fibrosis. Chapel Hill. (2010):11-12

13. Grotendorst, G. R., Okochi, H., & Hayashi, N. (1996). A novel transforming growth factor beta response element controls the expression of the connective tissue growth factor gene. Cell Growth & Differentiation, 7(4), 469-480.

14. Igarashi, A., Okochi, H., Bradham, D. M., & Grotendorst, G. R. (1993). Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. Molecular biology of the cell, 4(6), 637-645.

15. Mori, T. et al. (1999). Role and interaction of connective tissue growth factor with transforming growth factor‐β in persistent fibrosis: A mouse fibrosis model. Journal of cellular physiology, 181(1), 153-159.

16. Shi-Wen X, Leask A, Abraham D (2008) Regulation and function of connective tissue growth factor/CCN2 in tissue repair, scarring and fibrosis. Cytokine & growth factor reviews 19: 133-144.

Cells and Cell Environment• Mouse embryonic fibroblasts (NIH/3T3s) were used in all experiments• Cells were maintained in 5% CO2 at 37°C in Dulbecco’s Modified Eagle Medium

with 10% fetal bovine serum and Pen/Strep/Glut

Cell Treatments and Harvest:Experiment 1: CTGF and TGFβ Treatments• Cells were serum starved for 12 hours before treatment• At ~70% confluence sterile water (control), TGFβ (1ng/mL), CTGF (20ng/mL), or

both CTGF and TGFβ were added to media• Cells were harvested for RNA extraction at 1, 2, 4, 6, and 12 hours post-treatment Experiment 2: TGFβ Inhibition• Wells were scratched using a pipet tip then fresh media was added with water

and DMSO (control), TGFβ and CTGF, or TGFβ, CTGF, and TGFβ-inhibitor (GW788388)

• The scratch area was imaged at a 40X magnification with a Motic microscope and camera at 0, 3, 6, 9, 12, 15, 18, and 24 hours post-scratch

Gene Expression• RNA extractions were completed post-harvest using RNeasy kit (Qiagen)• Relative gene expression of fibrosis-related genes was measured using

quantitative polymerase chain reaction (qPCR) and normalized with -actin• qPCR data was analyzed using Microsoft Excel• Student T-test was used to test for statistical significance (p<0.05 was considered

statistically significant)• Additional T-tests for comparisons between cytokine treatments were Bonferroni

corrected by multiplying p-values by 2

CTGF and TGFβ Increase Fibroblast Motility and Mitogenic Potential

Cytokine Treatment• Measure expression of other fibrosis-related genes

including Fibronectin and type III and IV collagens• Measure gene expression at other time points including 0,

18, and 24 hoursScratch Test• Examine treatment with only CTGF or TGFβ• Perform assays to determine differences in rate of cell

division or motility• Examine how CTGF genetic variations impact fibrosis-

related phenotypes in fibroblast cells

• TGFβ alone seems to have a greater impact on fibrotic phenotype than CTGF alone

• The action of TGFβ and CTGF together seems primarily attributable to TGFβ

• Inhibition of TGFβ may be a method to reduce fibrotic tissue buildup after injury

• Col1a1 expression in response to cytokine treatment needs further study

• CTGF’s role in inducing fibrotic gene expression remains unclear

Effects of Inflammatory Cytokines on Fibrosis-Related Gene Expression in FibroblastsA. E. Kennedy*, K. M. Jesseman*, L. L. Smith, and H. E. Doherty PhD

Introduction• Wound healing occurs in three basic phases: inflammation, proliferation, and

maturation• Connective Tissue Growth Factor (CTGF) is thought to regulate wound invasion by

fibroblasts, including cell proliferation and motility, as well as induce extracellular matrix (ECM) turnover

• Overexpression of CTGF has been implicated in several fibrotic diseases including systemic sclerosis, cirrhosis, kidney disease, and heart disease

• Fibrotic diseases occur when excess extracellular matrix (ECM) proteins are produced during wound healing, negatively impacting tissue function

• Fibrotic gene expression in fibroblasts may be influenced by CTGF and Transforming Growth Factor Beta (TGFβ)

• TGFβ is an inflammatory cytokine known to be an inducer of CTGF gene expression

• Whether CTGF, TGFβ, or a combination of the two are required for pro-fibrotic gene expression is currently unresolved

• The current goal of this research is to examine the impact of TGFβ and CTGF related gene expression in models of wound healing in fibroblasts

• Long term goals of this research include: • Understanding the roles that CTGF and TGFβ play in wound healing, both

independently and synergistically• Improving anti-fibrotic therapies by elucidating fibrotic pathways

*Equal Contributions

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Figure 1: Fibrotic gene expression at 1, 2, 4, 6 and 12 hours after cytokine treatment. A) CTGF, B) TGFβ, and C) Collagen type I alpha 1 (Col1a1) expression.

Results for Figure 1:Cytokine Treated vs. ControlCTGF expression was significantly different than controls in cells treated with CTGF at 4 hours (p=0.028). CTGF expression in cells treated with TGFβ at 1 (p=0.005), 2 (p=0.013), and 12 hours (p=0.025) was significantly different. Expression in cells treated with both CTGF and TGFβ was significantly different than the control at all time points (1 hour, p=0.031; 2 hours, p=0.004; 4 hours, p<0.001; 6 hours, p=0.015; and 12 hours, p=0.011). TGFβ expression was significantly increased in cells treated with both CTGF and TGFβ at 4 hours (p=0.025). Col1a1 expression was significantly decreased after CTGF treatment for 6 (p=0.031) and 12 hours (p=0.042). TGFβ treatment increased Col1a1 expression significantly at 6 hours (p=0.029). Overall, CTGF treatment alone does not significantly increase fibrosis-related gene expression in NIH/3T3 fibroblasts. However, TGFβ treatment alone or a combination of the two cytokines does increase expression.

Individual vs. Combination TreatmentCompared to TGFβ alone, treatment with both CTGF and TGFβ did not significantly increase fibrotic gene expression. Compared to CTGF alone, treatment with both CTGF and TGFβ significantly increased CTGF expression at 2 (p=0.008), 4 (p<0.001), and 12 hours (p=0.023). Compared to CTGF alone, treatment with both cytokines significantly increased TGFβ expression at 1 hour (p=0.042). Treatment with both cytokines differs from CTGF alone, but it does not substantially differ from TGFβ alone.

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A CBThe normal healing process is broken up into three steps: inflammation, proliferation, and maturation. Two proteins vital to normal, healthy wound healing are Connective Tissue Growth Factor (CTGF) and Transforming Growth Factor Beta (TGFβ). Overexpression of these genes during healing can result in an excessive deposition of extracellular matrix (ECM) by fibroblasts. Buildup of this ECM is known as fibrosis and is observed in numerous diseases including cirrhosis, systemic sclerosis, kidney disease, and heart disease. In this study, we examined the effects of CTGF and TGFβ on fibroblasts to better understand how these two cytokines impact fibrosis-related phenotypes. We hypothesized that treating fibroblasts with CTGF and TGFβ together would yield a greater fibrotic response than treatment with either cytokine alone. To model post-wound inflammation, cells were treated with CTGF, TGFβ, or a combination of the two. Changes in gene expression were measured with quantitative polymerase chain reaction (qPCR). A scratch test assay, which models wounding, was also performed to examine fibroblast wound invasion. Cytokine treatment did impact fibrosis-related gene expression, but the combination of CTGF and TGFβ did not always have the greatest impact. Invasion of fibroblasts into the wound area was strongly influenced by CTGF and TGFβ. Addition of a TGFβ inhibitor eliminated the effect of CTGF and TGFβ treatment. Overall, the addition of cytokines increased fibrotic gene expression and fibroblast wound invasion, demonstrating their importance in the fibrotic response after wounding.

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Figure 2: NIH/3T3s at 0, 12, 18, and 24 hours after scratch testing. A-C) Untreated cells, CTGF and TGFβ treated cells, and CTGF, TGFβ, and inhibitor treated cells at 0 hours, respectively. D-F) Untreated cells, CTGF and TGFβ treated cells, and CTGF, TGFβ, and inhibitor treated cells at 12 hours, respectively. G-I) Untreated cells, CTGF and TGFβ treated cells, and CTGF, TGFβ, and inhibitor treated cells at 18 hours, respectively. J-L) Untreated cells, CTGF and TGFβ treated cells, and CTGF, TGFβ, and inhibitor treated cells at 24 hours, respectively. Results for Figure 2: Cells treated with CTGF and TGFβ show greater wound invasion than untreated cells, suggesting increased motility and mitogenic potential of cytokine treated fibroblast cells. Cells treated with CTGF, TGFβ, and a TGFβ inhibitor appeared similar to the control, suggesting that TGFβ inhibition mitigates the effect of cytokine treatment.

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