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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!