1. Profiling Lipid Mediators of Inflammation in Microenvironment of Chronic Venous Leg Ulcers Jodi C. McDaniel, PhD Assistant Professor The Ohio State University College of Nursing

2. Agenda  Background  Current study  Future direction  Clinical relevance

3. Major Chronic Wound Types Venous leg ulcers Pressure ulcers Diabetic foot ulcers  Chronic wounds affect 6.5 million U.S. patients  $25 billion spent annually for treatment Singer & Clark. N Engl J Med. 1999; Crovetti et al. Transfus Apher Sci. 2004; Brem at al. Mol Med. 2007

4. Chronic venous leg ulcers (CVLUs)  ~1.69% of U.S. population ≥ 65 years affected  ~ 600,000 cases annually  ~ 24 weeks to heal  ~ 15% never heal  up to 71% of cases recur  up to $5 billion annually for treatment expenses  significant social and economic cost Valencia et al. J Am Acad Dermatol. 2001; Coleridge-Smith. Leg ulcers. Diagnosis and management. 2005; Abbade & Lastoria. Int J Dermatol. 2005; Heit et al. J Vasc Surg.2001.

5. Stages of Wound Healing

6. Key Players By Stage

7. Neutrophils Neutrophil influx into wound site & release of proteases important for healing, BUT prolonged, excessive levels detrimental: - destroy growth factors, receptors and extracellular matrix essential for healing Majority of proteases associated with chronic wounds compared to healing wounds are primarily of neutrophil origin Moor et al. Wound Repair Regen. 2009; Yager et al. Int J Low Extrem Wounds. 2007; Smith. Int J Low Extrem Wounds. 2006.

8. Topical Solutions Dressings have been designed to absorb excessive proteases.

9. Systemic Solutions? Endogenously generated lipid mediators derived from n-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic (EPA) & docosahexaenoic (DHA)  neutrophil influx & activity These lipid mediators include certain eicosanoids (e.g. PGE3), and resolvins & protectins. Norling & Serhan. J Intern Med. 2010; Arita et al. Journal of Immunology. 2007; Dona et al. Blood. 2008.

10. N-6, N-3 Metabolic Pathways More inflammatory Less inflammatory

11. Hypothesis  EPA + DHA  lipid mediators that  excessive neutrophil activity  healing

12. EPA+DHA

13.  Balanced EPA:AA ratios associated with  risk of heart disease & improvements in inflammatory diseases (e.g. rheumatoid arthritis)  Minimal amts. synthesized in body, so require dietary sources - primarily from oily fish or fish oil supplements Calder. Eur J Pharmacol. 2011; Simopoulos. Exp Biol Med. 2008; Cleland et al. J Rheumatol. 2006.

14. …But Genetic Variations Affect PUFA Metabolism Activity of desaturases affected by genetic polymorphisms Delta-5 & delta-6 desaturases encoded by fatty acid desaturase (FADS)1 & FADS2, respectively Located on desaturase gene cluster on chromosome 11 (11q12-13.1) Rzehak et al. J Nutr. 2009; Martinelli et al. Am J Clin Nutr. 2008; Schaeffer et al. Hum Mol Genet. 2006; Tanaka et al. PLoS Genet. 2009.

15. FADS2 FADS1

16. FADS1,FADS2 gene cluster

17. FADS Polymorphisms Certain ancestry groups have ↑ frequency of alleles in FADS cluster associated with higher levels of AA Genetically predisposed to more efficient conversion of LA to AA, systemic inflammation & inflammatory conditions Individuals having genotypes associated with higher LA to AA conversion rates & diets high in LA & AA may be at ↑ risk of developing CVLUs Mathias et al. BMC Genet. 2011; Sergeant et al. Br J Nutr. 2011

18. Current Study - CVLUs * CCTS funded project

19. Current Study  Profiling lipid mediators in plasma & wound fluid of patients with CVLUs  Determining frequency of genetic variants in FADS gene The OSU Clinical Research Center

20. Current Study Determine dietary intake of PUFAs via food frequency questionnaire (FFQ)

21. Current Study Determine plasma levels of PUFAs Determine lipid mediator levels in plasma and wound fluid

22. Fluid Collection – Occlusive Dressing

23. Current Study Determine variants in FADS gene cluster

24. Preliminary Data Lipid Mediators pg/µl fluid PGE 2 6.30 (3.67) PGD 2 0.52 (0.55) PGF 2α 2.78 (0.60) PGE 1 1.29 (0.53) PGF 1α 1.30 (0.82) TXB 2 4.87 (3.92) LTB 4 0.45 (0.41) 9-HODE2.47 (2.40) 13-HODE10.83 (15.36) 5-HETE6.03 (7.00) 8-HETE0.18 (0.08) 11-HETE0.25 (0.23) 15-HETE6.68 (11.74) 12-HETE2.35 (2.11) 15-HETrE0.82 (1.29) Lipid mediators in chronic wound fluid exclusively products of n-6 PUFA metabolism No detectable levels of products of n-3 PUFA metabolism Suggesting that microenvironment of chronic leg wounds exhibits an n-6 PUFA lipid mediator profile N = 5

25. Preliminary Data SNPs Allele associated with increased PUFA* Genotype % Frequency rs174537G GGGTTTUD** 0.220.560.220 rs102275A CCCTTTUD 0.560.3300.11 rs174546C CCCTTTUD 0.110.5600.33 rs174556C CC CTTTUD 0.1100.220.67 rs1535A AAAGGGUD 0.780 0.11 rs174576C AAACCCUD 0.670.2200.1 rs174579C CCCTTTUD 000.220.78 N=9 * Mathias, BMC Genet. 2011 ** UD = undetermined

26. Preliminary Data

27. Clinical Relevance EPA + DHA Supplementation ?

28. Goal: Facilitate Wound Healing!

29. Team Sashwati Roy, PhD – OSU College of Medicine Martha Belury, PhD – OSU College of Education and Human Ecology Anna Nicolaou, PhD – University of Bradford, U.K. Christopher Holloman, PhD – OSU Dept. of Stat. OSU Clinical Research Center Staff

30. Questions/Comments Conceptual model for diet–gene interactions

31. Thank you