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Morphine-treated Primary Brain Endothelial Cells LC-MS/MS DIA

ABSTRACT: This project aimed at investigating the impact of morphine exposure on brain endothelial cells via an in vitro monoculture of primary human brain microvascular endothelial cells (HBMECs) and state of the art mass spectrometry-based proteomics, using a data-independent acquisition (DIA) MS-based quantitative approach. HBMECS were exposed to morphine at three different concentrations (1, 10, 100 uM) and for two different time points (24h and 48h) to explore dose- and time-dependent effects.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain Microvascular Endothelial Cell, Cell Culture

SUBMITTER: Sandrine Reymond

LAB HEAD: Jean-Charles Sanchez

PROVIDER: PXD024351 | Pride | 2022-03-21

REPOSITORIES: Pride

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			Action	DRS
	20190718_LU50CM158_POOL24A.raw	Raw
	20190718_LU50CM159_C24A.raw	Raw
	20190718_LU50CM160_M124A.raw	Raw
	20190718_LU50CM161_M1024A.raw	Raw
	20190718_LU50CM162_M10024A.raw	Raw

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Project description:Morphine, a commonly used antinociceptive drug in hospitals, is known to cross the blood-brain barrier (BBB) by first passing through brain endothelial cells. Despite its pain-relieving effect, morphine also has detrimental effects, such as the potential induction of redox imbalance in the brain. However, there is still insufficient evidence of these effects on the brain, particularly on the brain endothelial cells and the extracellular vesicles that they naturally release. Indeed, extracellular vesicles (EVs) are nanosized bioparticles produced by almost all cell types and are currently thought to reflect the physiological state of their parent cells. These vesicles have emerged as a promising source of biomarkers by indicating the functional or dys-functional state of their parent cells and, thus, allowing a better understanding of the biological processes involved in an adverse state. However, there is very little information on the mor-phine effect on human brain microvascular endothelial cells (HBMECs), and even less on their released EVs. Therefore, the current study aimed at unraveling the detrimental mechanisms of morphine exposure (at 1, 10, 25, 50 and 100 µM) for 24 h on human brain microvascular endothe-lial cells as well as on their associated EVs. Isolation of EVs was carried out using an affini-ty-based method. Several orthogonal techniques (NTA, western blotting and proteomics analy-sis) were used to validate the EVs enrichment, quality and concentration. Data-independent mass spectrometry (DIA-MS)-based proteomics was applied in order to analyze the proteome modu-lations induced by morphine on HBMECs and EVs. We were able to quantify almost 5500 pro-teins in HBMECs and 1500 proteins in EVs, of which 256 and 148, respectively, were found to be differentially expressed in at least one condition. Pathway enrichment analysis revealed that the “cell adhesion and extracellular matrix remodeling” process and the “HIF1 pathway”, a pathway related to oxidative stress responses, were significantly modulated upon morphine exposure in HBMECs and EVs. Altogether, the combination of proteomics and bioinformatics findings high-lighted shared pathways between HBMECs exposed to morphine and their released EVs. These results put forward molecular signatures of morphine-induced toxicity in HBMECs that were also carried by EVs. Therefore, EVs could potentially be regarded as a useful tool to investigate brain endothelial cells dysfunction, and to a different extent, the BBB dysfunction in patient cir-culation using these “signature pathways”.

Dataset Information

Morphine-treated Primary Brain Endothelial Cells LC-MS/MS DIA

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