Summary of Study ST001254
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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST001254 |
| Study Title | Eicosanoid profiles of dermal fibroblasts (part-II) |
| Study Summary | The 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 Name | Chalfant |
| First Name | Charles |
| Address | 4202 E Fowler Ave, Tampa, FL 33620 |
| cechalfant@usf.edu | |
| Phone | 8139747103 |
| Submit Date | 2019-09-24 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | wiff |
| Analysis Type Detail | MALDI-MS |
| Release Date | 2019-10-11 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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: | SU001322 |
| 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 |
|---|---|---|---|
| SA091224 | Patrick Media 25 | cPLA2α KI | Pyrrophenone (50nM) |
| SA091225 | Patrick Media 33 | cPLA2α KI | Pyrrophenone (50nM) |
| SA091226 | Patrick Media 22 Black | cPLA2α KI | Pyrrophenone (50nM) |
| SA091227 | Patrick Media KI | cPLA2α KI | Untreated |
| SA091228 | Patrick Media 42 | cPLA2α KI | Untreated |
| SA091229 | Patrick Media 41 | cPLA2α KI | Untreated |
| SA091230 | Patrick Media 40 | cPLA2α KI | Untreated |
| SA091231 | Patrick Media 37 | cPLA2α KI | Untreated |
| SA091232 | Patrick Media 28 | cPLA2α KO | CMV control Virus |
| SA091233 | Patrick Media 29 | cPLA2α KO | CMV control Virus |
| SA091234 | Patrick Media 32 | cPLA2α KO | CMV control Virus |
| SA091247 | Patrick Media 2 | cPLA2α KO | cPLA2α KI Virus |
| SA091248 | Patrick Media 4 | cPLA2α KO | cPLA2α KI Virus |
| SA091249 | Patrick Media 3 | cPLA2α KO | cPLA2α KI Virus |
| SA091250 | Patrick Media 5 | cPLA2α KO | cPLA2α KI Virus |
| SA091251 | Patrick Media 9 | cPLA2α KO | cPLA2α KI Virus |
| SA091252 | Patrick Media 11 | cPLA2α KO | cPLA2α KI Virus |
| SA091253 | Patrick Media 1 | cPLA2α KO | cPLA2α KI Virus |
| SA091254 | Patrick Media 10 | cPLA2α KO | cPLA2α KI Virus |
| SA091255 | Patrick Media 12 | cPLA2α KO | cPLA2α KI Virus |
| SA091256 | Patrick Media 7 | cPLA2α KO | cPLA2α KI Virus |
| SA091257 | Patrick Media 8 | cPLA2α KO | cPLA2α KI Virus |
| SA091258 | Patrick Media 6 | cPLA2α KO | cPLA2α KI Virus |
| SA091235 | Patrick Media 24 | cPLA2α KO | Pyrrophenone (50nM) |
| SA091236 | Patrick Media 30 | cPLA2α KO | Pyrrophenone (50nM) |
| SA091237 | Patrick Media 23 Black | cPLA2α KO | Pyrrophenone (50nM) |
| SA091238 | Patrick Media 16 | cPLA2α KO | Wild Type Virus |
| SA091239 | Patrick Media 15 | cPLA2α KO | Wild Type Virus |
| SA091240 | Patrick Media 14 | cPLA2α KO | Wild Type Virus |
| SA091241 | Patrick Media 13 | cPLA2α KO | Wild Type Virus |
| SA091242 | Patrick Media 17 | cPLA2α KO | Wild Type Virus |
| SA091243 | Patrick Media 18 | cPLA2α KO | Wild Type Virus |
| SA091244 | Patrick Media 21 | cPLA2α KO | Wild Type Virus |
| SA091245 | Patrick Media 20 | cPLA2α KO | Wild Type Virus |
| SA091246 | Patrick Media 19 | cPLA2α KO | Wild Type Virus |
| SA091217 | Patrick Media 31 | Wild Type | Pyrrophenone (50nM) |
| SA091218 | Patrick Media 27 | Wild Type | Pyrrophenone (50nM) |
| SA091219 | Patrick Media 26 | Wild Type | Pyrrophenone (50nM) |
| SA091220 | Patrick Media 34 | Wild Type | Untreated |
| SA091221 | Patrick Media 38 | Wild Type | Untreated |
| SA091222 | Patrick Media 43 | Wild Type | Untreated |
| SA091223 | Patrick Media 39 | Wild Type | Untreated |
| Showing results 1 to 42 of 42 |
Collection:
| Collection ID: | CO001316 |
| 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: | TR001337 |
| 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: | SP001330 |
| 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 | AN002083 |
|---|---|
| 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: | CH001520 |
| Instrument Name: | Shimadzu Nexera X2 |
| Column Name: | Acentis Express C18 column |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS001934 |
| Analysis ID: | AN002083 |
| 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 |
