Summary of Study ST001252

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000839. The data can be accessed directly via it's Project DOI: 10.21228/M87H7W This work is supported by NIH grant, U2C- DK119886.

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Study IDST001252
Study TitleEicosanoid profiles of dermal fibroblasts (part-I)
Study SummaryThe sphingolipid, ceramide-1-phosphate (C1P), directly binds and activates Group IVA cytosolic phospholipase A2 (cPLA2) to generate eicosanoids. Due to the role of eicosanoids in wound healing, we choose to use our novel genetic mouse model expressing cPLA2 with an ablated C1P interaction site (KI) to examine the cPLA2/C1P interaction in wound healing. Wound closure rate was not affected, but wound maturation was enhanced by loss of the C1P/cPLA2α interaction based on the following findings. Wounds in KI mice displayed: i) increased infiltration of dermal fibroblasts into the wound environment; ii) increased wound tensile strength; and iii) higher Type I/Type III collagen ratios. These findings were recapitulated in vitro as primary dermal fibroblasts (pDFs) from KI mice showed significantly increased collagen deposition and migration velocity compared to WT and KO pDFs. Additionally, the KI showed an altered eicosanoid profile of reduced pro-inflammatory prostaglandins (PGE2) and increased levels of specific HETE species (5-HETE). Elevated 5-HETE levels promoted increased dermal fibroblast migration and collagen deposition. This “gain of function” role for the mutant cPLA2 was also linked to differential cellular localization of cPLA2α and 5-HETE biosynthetic factors. These studies demonstrate regulation of key biological mechanisms by a defined protein:lipid interaction in vivo and provide new insights into cPLA2 function.
Institute
University of South Florida
Last NameChalfant
First NameCharles
Address4202 E Fowler Ave, Tampa, FL 33620
Emailcechalfant@usf.edu
Phone8139747103
Submit Date2019-09-22
Raw Data AvailableYes
Raw Data File Type(s)wiff
Analysis Type DetailMALDI-MS
Release Date2019-10-11
Release Version1
Charles Chalfant Charles Chalfant
https://dx.doi.org/10.21228/M87H7W
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000839
Project DOI:doi: 10.21228/M87H7W
Project Title:Eicosanoid Expression in Dermal Fibroblasts
Project Summary:Quantitative lipidomics studies examining eicosanoid levels in primary dermal fibroblasts
Institute:University of South Florida
Last Name:Chalfant
First Name:Charles
Address:4202 E Fowler Ave, Tampa, FL 33620
Email:cechalfant@usf.edu
Phone:8139747103

Subject:

Subject ID:SU001320
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Genotype Treatment
SA091170Patrick Media 26cPLA2α KI Scratched
SA091171Patrick Media 30cPLA2α KI Scratched
SA091172Patrick Media 27cPLA2α KI Scratched
SA091173Patrick Media 35cPLA2α KI Scratched
SA091174Patrick Media 40cPLA2α KI Scratched
SA091175Patrick Media 36cPLA2α KI Scratched
SA091176Patrick Media 39cPLA2α KI Scratched
SA091177Patrick Media 29cPLA2α KI Scratched
SA091178Patrick Media 20cPLA2α KI Unscratched
SA091179Patrick Media 19cPLA2α KI Unscratched
SA091180Patrick Media 18cPLA2α KI Unscratched
SA091181Patrick Media 24cPLA2α KI Unscratched
SA091182Patrick Media 21cPLA2α KI Unscratched
SA091183Patrick Media 17cPLA2α KI Unscratched
SA091184Patrick Media 22cPLA2α KI Unscratched
SA091185Patrick Media 23cPLA2α KI Unscratched
SA091186Patrick Media 32cPLA2α KO Scratched
SA091187Patrick Media 41cPLA2α KO Scratched
SA091188Patrick Media 42cPLA2α KO Scratched
SA091189Patrick Media 43cPLA2α KO Scratched
SA091190Patrick Media 46cPLA2α KO Scratched
SA091191Patrick Media 47cPLA2α KO Scratched
SA091192Patrick Media 48cPLA2α KO Scratched
SA091193Patrick Media 31cPLA2α KO Scratched
SA091194Patrick Media 11cPLA2α KO Unscratched
SA091195Patrick Media 12cPLA2α KO Unscratched
SA091196Patrick Media 14cPLA2α KO Unscratched
SA091197Patrick Media 10cPLA2α KO Unscratched
SA091198Patrick Media 9cPLA2α KO Unscratched
SA091199Patrick Media 16cPLA2α KO Unscratched
SA091200Patrick Media 15cPLA2α KO Unscratched
SA091201Patrick Media 13cPLA2α KO Unscratched
SA091154Patrick Media 34Wild Type Scratched
SA091155Patrick Media 33Wild Type Scratched
SA091156Patrick Media 37Wild Type Scratched
SA091157Patrick Media 44Wild Type Scratched
SA091158Patrick Media 45Wild Type Scratched
SA091159Patrick Media 28Wild Type Scratched
SA091160Patrick Media 38Wild Type Scratched
SA091161Patrick Media 25Wild Type Scratched
SA091162Patrick Media 1Wild Type Unscratched
SA091163Patrick Media 7Wild Type Unscratched
SA091164Patrick Media 8Wild Type Unscratched
SA091165Patrick Media 5Wild Type Unscratched
SA091166Patrick Media 6Wild Type Unscratched
SA091167Patrick Media 3Wild Type Unscratched
SA091168Patrick Media 2Wild Type Unscratched
SA091169Patrick Media 4Wild Type Unscratched
Showing results 1 to 48 of 48

Collection:

Collection ID:CO001314
Collection Summary:pDFs obtained from WT, KI, and KO mice were plated at a density of 2 x 106 on 10 cm tissue culture plates in high glucose DMEM supplemented with 20% FBS (Gibco) and 2% penicillin/streptomycin. Following the overnight incubation cells were rested in 2% FBS (Gibco) 2% penicillin/streptomycin (Bio Whittaker) high glucose DMEM (Gibco) for 2 hours, then the media was exchanged with 0% FBS 2% penicillin/ high glucose DMEM, and mechanical trauma was induced on the monolayer by performing scratched across the diameter of the plate in an asterisk pattern using 4 x 20 ul pipette tips on a multichannel micro pipettor. Media was taken for lipidomic analysis at 0 h and 2 h.
Sample Type:Fibroblasts

Treatment:

Treatment ID:TR001335
Treatment Summary:Primary Dermal Fibroblasts were plated at a density of 2 x 106 on 10 cm tissue culture plates in high glucose DMEM supplemented with 20% FBS (Gibco) and 2% penicillin/streptomycin. Following the overnight incubation cells were rested in 2% FBS (Gibco) 2% penicillin/streptomycin (Bio Whittaker) high glucose DMEM (Gibco) for 2 hours, then the media was exchanged with 0% FBS 2% penicillin/ high glucose DMEM, and mechanical trauma was induced on the monolayer by performing scratched across the diameter of the plate in an asterisk pattern using 4 x 20 l pipette tips on a multichannel micro pipettor. Media was taken for lipidomic analysis at 0 h and 2 h.

Sample Preparation:

Sampleprep ID:SP001328
Sampleprep Summary:Samples were stored at − 80 °C until the time of analysis. Lipids were extracted for eicosanoids. Eicosanoid Preparation Eicosanoids were extracted using a modified extraction process from previously described. (1, 2). 4 mL of media was combined with an IS mixture comprised of comprised of 10% methanol (400 μl), glacial acetic acid (20 μl), and internal standard (20 μl) containing the following deuterated eicosanoids (1.5 pmol/μl, 30 pmol total) (All standards purchased from Cayman Chemicals): (d4) 6keto-Prostaglandin F1α, (d4) Prostaglandin F2α, (d4) Prostaglandin E2, (d4) Prostaglandin D2, (d8) 5-Hydroxyeicosa-tetranoic acid (5-HETE), (d8) 12-Hydroxyeicosa-tetranoic acid (12-HETE) (d8) 15-Hydroxyeicosa-tetranoic acid (15-HETE), (d6) 20-Hydroxyeicosa-tetranoic acid (20-HETE), (d11) 8,9 Epoxyeicosa-trienoic acid, (d8) 14,15 Epoxyeicosa-trienoic acid, (d8) Arachidonic acid, (d5) Eicosapentaenoic acid, (d5) Docosahexaenoic acid, (d4) Prostaglandin A2, (d4) Leukotriene B4, (d4) Leukotriene C4, (d4) Leukotriene D4, (d4) Leukotriene E4, (d5) 5(S),6(R)-Lipoxin A4, (d11) 5-iPF2α-VI, (d4) 8-iso Prostaglandin F2α, (d11) (±)14,15-DHET, (d11) (±)8,9-DHET, (d11) (±)11,12-DHET, (d4) Prostaglandin E1, (d4) Thromboxane B2, (d6) dihmo gamma linoleic acid, (d5) Resolvin D2, (d5) Resolvin D1, (d5) Maresin 2, and (d5) Resolvin D3. Samples and vial rinses (5% MeOH; 2 ml) were applied to Strata-X SPE columns (Phenomenex), previously washed with methanol (2 ml) and then dH2O (2 ml). Eicosanoids eluted with isopropanol (2 ml), were dried in vacuuo and reconstituted in EtOH:dH2O (50:50;100 μl) prior to UPLC ESI-MS/MS analysis.

Combined analysis:

Analysis ID AN002078
Analysis type MS
Chromatography type Reversed phase
Chromatography system Shimadzu Nexera X2
Column Acentis Express C18 column
MS Type MALDI
MS instrument type Triple quadrupole
MS instrument name ABI Sciex 5500 QTrap
Ion Mode NEGATIVE
Units pmol of lipid

Chromatography:

Chromatography ID:CH001515
Instrument Name:Shimadzu Nexera X2
Column Name:Acentis Express C18 column
Chromatography Type:Reversed phase

MS:

MS ID:MS001929
Analysis ID:AN002078
Instrument Name:ABI Sciex 5500 QTrap
Instrument Type:Triple quadrupole
MS Type:MALDI
MS Comments:Sciex Analyst software was used for analysis
Ion Mode:NEGATIVE
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