Quantitative Analysis of Structural and Signaling Lipids

A networks approach towards investigating the human skin lipidome

Dayanjan Wijesinghe, Ph.D.

The human skin

Largest organ of the body

Protective barrier function against Allergens Irritants Microorganisms Water loss

These functions are primarily through the SC Cells (corneocytes) embedded in a highly organized lipid matrix Primary lipids are

CeramidesCholesterols Free Fatty Acids

There are times when wounds heal…

There are times when they don't…

102.821.4

9.8

7.4

11.0

11.3

1.0

Worldwide Wound Incidence( In Millions !!!)

Surgical Lacerations Burns Pressure UlcerVenous Ulcer Diabetic Ulcer Other

1. Snowballing national and international health problem!2. Very little focused research3. Externally accessible4. Treatments easily testable5. Very fast translatability6. Multiple research opportunities

a) Diagnosticsb) Treatmentsc) Dressings

7. Multiple avenues of fundinga) NIH – NIGMS, NIDDK, NHLBI, NIAb) DoD – DMRDP(CDMRP) “to advance medical research and

development (R&D) for wounded warriors ..”c) Department of Veterans Affairs – PU, DFU. d) Public-Private partnerships

Why study wound healing?

An area where the combination of analytical chemistry, nanotechnology, and medicine will yield outstanding translational results!

Importance of the lipidome

Lipidome

Structural and Barrier

Signaling

Energetics

Membrane protein

Interactions

Genome

Transcriptome

ProteomeLipidome

Metabolome

•Total metabolic status

• Inflammatory status

• Healing potential

• Wound nutritional status

• Redox balance

• Bacterial load

• Hormone status

•Perfusion status

Why Study the lipidome?

MicrobesEnvironmentLifestyle

Phenome

a) Dysregulation of biochemical networks that prevent healing?b) How can those networks be modified to enable healing?c) Is what I am doing actually working?

Diversity and Variability of the Skin Lipidome and

Inherent Problems in their Analysis

Janssens et al, 2012

Structure of the skin ceramides

Lamellar and lateral lipid organization in human SC

Janssens et al, 2012

Imokawa et al, 2008

Tape Stripping for studying the skin lipidome

Diversity of the lipidome (>180,000)

Isobaric Overlapment

Experiment: EMS scan of Bovine Heart Extract (BHE)

• Lipidomic spectra are incredibly complex

• MS spectra alone is not sufficient for iidentification

• MS/MS spectra generated on precursors in zones of isobaric overlap will contain product ions form other isobaric species

The need for analytical specificity

Prostaglandin E2Prostaglandin D2

SLIDE COURTESY: Dr. Greg Schultz, Ph.D., Director Wound Healing Institute, University of Florida

Chemometrics(data reduction)

Identifying Chemicals of

Interest

Understanding Biochemical Relevance

UnderstandingTherapeutic

Requirements

IdentifyingDiagnosticMolecules

Mass Spectrometric Characterization of Wounds

Analytical CharacterizationGenome

Transcriptome

Proteome

LipidomeMetabolome

•Metabolic status

• Inflammatory status

• Healing potential

• Redox balance

•Perfusion status

•Wound nutrition

•Bacterial load

•Hormone status

•Signaling status

Lipidomic changes skin wound healing

Dayanjan S Wijesinghe, Charles E Chalfant. (2013) Systems level lipid analysis methodologies forqualitative and quantitative investigation of lipid signaling events during wound healing. Advances inWound Care.

Lipids during human wound healing

Wijesinghe DS, Brentnall M, Mietla JA, Hoeferlin LA, Diegelmann RF,Boise LH, Chalfant CE. (2014) Ceramide kinase is required for a normaleicosanoid response and the subsequent orderly migration of fibroblasts.Journal of Lipid Research.

What happens when you remove C1P

Wijesinghe DS, Brentnall M, Mietla JA, Hoeferlin LA, Diegelmann RF,Boise LH, Chalfant CE. (2014) Ceramide kinase is required for a normaleicosanoid response and the subsequent orderly migration of fibroblasts.Journal of Lipid Research.

Changing C1P alters eicosanoids

Wijesinghe DS, Brentnall M, Mietla JA, Hoeferlin LA, Diegelmann RF,Boise LH, Chalfant CE. (2014) Ceramide kinase is required for a normaleicosanoid response and the subsequent orderly migration of fibroblasts.Journal of Lipid Research.

Changing C1P alters eicosanoids

Wijesinghe DS, Brentnall M, Mietla JA, Hoeferlin LA, Diegelmann RF,Boise LH, Chalfant CE. (2014) Ceramide kinase is required for a normaleicosanoid response and the subsequent orderly migration of fibroblasts.Journal of Lipid Research.

How does this affect wound healing

Wijesinghe DS, Brentnall M, Mietla JA, Hoeferlin LA, Diegelmann RF,Boise LH, Chalfant CE. (2014) Ceramide kinase is required for a normaleicosanoid response and the subsequent orderly migration of fibroblasts.Journal of Lipid Research.

CERK ablation affects fibroblast migration

Wijesinghe DS, Brentnall M, Mietla JA, Hoeferlin LA, Diegelmann RF,Boise LH, Chalfant CE. (2014) Ceramide kinase is required for a normaleicosanoid response and the subsequent orderly migration of fibroblasts.Journal of Lipid Research.

Phenotype rescued by exogenous eicosanoids

Wijesinghe DS, Brentnall M, Mietla JA, Hoeferlin LA, Diegelmann RF,Boise LH, Chalfant CE. (2014) Ceramide kinase is required for a normaleicosanoid response and the subsequent orderly migration of fibroblasts.Journal of Lipid Research.

Can Lipidomics Be Used to Predict Successful Wound

Healing?

Short answer, YES!

Analytical CharacterizationChemometrics

(data reduction)

Identifying Chemicals of

Interest

Understanding Biochemical Relevance

UnderstandingTherapeutic

Requirements

Generating synergistic drug lipid combinations as

nanoliposomes

Delivery of the therapeutic agent/s and monitoring the

biochemical response

IdentifyingDiagnosticMolecules

LCMS Based Diagnostics

Genome

Transcriptome

Proteome

LipidomeMetabolome

Research Interests - Summary

•Metabolic status

• Inflammatory status

• Healing potential

• Redox balance

•Perfusion status

•Wound nutrition

•Bacterial load

•Hormone status

•Signaling status

Research Strategy - Future DirectionsGenerating synergistic

drug lipid combinations as nanoliposomes

Delivery of the therapeutic agent/s and monitoring the

biochemical response

IdentifyingDiagnosticMolecules

LCMS Based Diagnostics

Analytical CharacterizationChemometrics

(data reduction)

Identifying Chemicals of

Interest

Understanding Biochemical Relevance

UnderstandingTherapeutic

Requirements

Genome

Transcriptome

Proteome

LipidomeMetabolome

Funding and Salary Support

2014 AB SCIEX Young Investigator Award: “Development of MassSpectrometry Applications for Clinical Lipidomic Research”

U01 – NIH “The utilization of photonics technology to rapidly detectbioactive lipids associated with preeclampsia development” co-PI(2015-2019)

VCU Lipidomics/Metabolomics Core Facility (VLMC) – Deputy Director

VA CDA1 “Development of Mass Spectrometry Applications for theInvestigation of Wound Healing” 2010-2012

PI – NRSA T32 Mass spectrometry application for investigatingsignaling in tissue injury and repair” 2009-2010

DoD – “Pre-hospital use of plasma for the treatment of traumahemorrhagic shock”

Thank You!