Profiling Lipid Mediators of Inflammation in Microenvironment of Chronic Venous Leg Ulcers

Jodi C. McDaniel, PhD Assistant ProfessorThe Ohio State University College of Nursing

Agenda

BackgroundCurrent study Future directionClinical relevance

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

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.

Stages of Wound Healing

Key Players By Stage

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.

Topical Solutions

•Dressings have been designed to absorb excessive proteases.

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.

N-6, N-3 Metabolic Pathways

EPA & DHA from fish, fish oil, fish oil

supplementation

Omega-6 Fatty Acids

Omega-3 fatty Acids

Linoleic Acid (LA) 18:2n6 (Corn, Soy, Safflower oils)

AA 20: 4n-6

COX

AT-LXs LXA4 LXB4

LO

4 series leukotrienes

2 series prostaglandins;

2 series thromboxanes

LO COX

COX

Alpha-Linolenic Acid (ALA)18:3n3 (Flaxseed, walnuts, green

vegetables)

DHA 22: 6n-3

LO EPA

20: 5n-3

Δ6 desaturase

Δ5 desaturase

18: 3n-6

20: 3n-6

18: 4n-3

20: 4n-3

RvE1; RvE2; 5 series leukotrienes

COX

RvD1 PD1

AT- RvDs

LO

AT- RvEs; 3 series

prostaglandins; 3 series

thromboxanes

DIET

More inflammatory Less inflammatory

Hypothesis

EPA + DHA

lipid mediators that excessive neutrophilactivity

healing

EPA+DHA

EPA+DHA 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.

…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.

EPA & DHA from fish, fish oil, fish oil

supplementation

Omega-6 Fatty Acids

Omega-3 fatty Acids

Linoleic Acid (LA) 18:2n6 (Corn, Soy, Safflower oils)

AA 20: 4n-6

COX

AT-LXs LXA4 LXB4

LO

4 series leukotrienes

2 series prostaglandins;

2 series thromboxanes

LO COX

COX

Alpha-Linolenic Acid (ALA)18:3n3 (Flaxseed, walnuts, green

vegetables)

DHA 22: 6n-3

LO EPA

20: 5n-3

Δ6 desaturase

Δ5 desaturase

18: 3n-6

20: 3n-6

18: 4n-3

20: 4n-3

RvE1; RvE2; 5 series leukotrienes

COX

RvD1 PD1

AT- RvDs

LO

AT- RvEs; 3 series

prostaglandins; 3 series

thromboxanes

DIET

FADS2

FADS1

FADS1,FADS2 gene cluster

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

Current Study - CVLUs

* CCTS funded project

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

Current Study

•Determine dietary intake of PUFAs via food frequency questionnaire (FFQ)

Current Study

•Determine plasma levels of PUFAs

•Determine lipid mediator levels in plasma and wound fluid

Fluid Collection – Occlusive Dressing

Current Study

• Determine variants in FADS gene cluster

Preliminary DataLipid Mediators    pg/µl fluidPGE2 6.30 (3.67)

PGD2 0.52 (0.55)

PGF2α 2.78 (0.60)

PGE1 1.29 (0.53)

PGF1α 1.30 (0.82)

TXB2 4.87 (3.92)

LTB4 0.45 (0.41)

9-HODE 2.47 (2.40)13-HODE 10.83 (15.36)5-HETE 6.03 (7.00)8-HETE 0.18 (0.08)11-HETE 0.25 (0.23)15-HETE 6.68 (11.74)12-HETE 2.35 (2.11)15-HETrE 0.82 (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

Preliminary Data

SNPs

 Allele

associated with increased

PUFA*

Genotype% Frequency

 

rs174537 GGG GT TT UD**0.22 0.56 0.22 0

rs102275 ACC CT TT UD0.56 0.33 0 0.11

rs174546 CCC CT TT UD0.11 0.56 0 0.33

rs174556 CCC CT TT UD0.11 0 0.22 0.67

rs1535 AAA AG GG UD0.78 0 0.11 0.11

rs174576 CAA AC CC UD0.67 0.22 0 0.1

rs174579 CCC CT TT UD0 0 0.22 0.78

N=9 * Mathias, BMC Genet. 2011** UD = undetermined

Preliminary Data

Clinical Relevance

EPA + DHA Supplementation ?

Goal: Facilitate Wound Healing!

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

Questions/Comments

Conceptual model for diet–gene interactions

Thank you