ABSTRACT: Transcriptional profiling of lung tissue of rat pretreated with cobalt in normoxic and hypoxic conditions and comapring with saline treated normoxic and hypoxic lung tissue in different time interval
Project description:The goal of the study was to examine the effect of hypoxic preconditioning with cobalt on global gene profiling in lung tissues. Agilent two-color experiment,Organism: Rat ,Slides, Agilentâs Whole Rat Genome Microarray 4x44k GPL4135 with 44000 features, Labeling kit: Agilents Low input RNA linear amplification Kit Cat # 5184-3523, Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA Rat lung tissue: cobalt + Hypoxia (0h, 6h, 12h, 24h, 48h) vs. Hypoxia (0h, 6h, 12h, 24, 48h)
Project description:Background Extracellular vesicles (EVs) secreted by mesenchymal stromal cells (MSCs) provide significant protection against renal ischemia-reperfusion injury (IRI). Hypoxia is considered an important method for enhancing the tissue repair capabilities of MSCs. However, the specific effects of hypoxia on MSCs and MSC-EVs, as well as their therapeutic potential for renal IRI, remain unclear. In this study, we investigated the alterations in MSCs and the production of MSC-EVs following hypoxia pre-treatment, and further explored the key intrinsic mechanisms by which hypoxic MSC-EVs treat renal IRI. Methods Human umbilical cord MSCs were cultured under normoxic and hypoxic conditions. Proliferation and related pathways were measured, and RNA sequencing was used to detect changes in the transcription profile. MSC-EVs from both normoxic and hypoxic conditions were isolated and characterized. In vivo, the localization and therapeutic effects of MSC-EVs were assessed in a rat renal IRI model. Histological examinations were employed to assess the structure, proliferation, and apoptosis of IRI kidney tissue respectively. Renal function was measured by analyzing serum creatinine and blood urea nitrogen levels. In vitro, the therapeutic potential of MSC-EVs were measured in renal tubular epithelial cells injured by antimycin A. Protein sequencing analysis of hypoxic MSC-EVs was conducted, and the depletion of Glutathione S-Transferase Omega 1 (GSTO1) in hypoxic MSC-EVs was performed to verify its key role in alleviating renal injury. Results Hypoxia alters MSCs transcription, promotes their proliferation, and increases the production of EVs. Hypoxia-pretreated MSC-EVs exhibited a superior ability to mitigate renal IRI, enhancing proliferation and reducing apoptosis of renal tubular epithelial cells both in vivo and in vitro. Protein profiling of the EVs revealed an accumulation of numerous anti-oxidative stress proteins, with GSTO1 being particularly prominent. GSTO1 knock down was significantly reduced the antioxidant and therapeutic effects in renal IRI of hypoxic MSC-EVs. Conclusions Hypoxia significantly promotes MSC-EVs generation and enhances the therapeutic effect of EVs on renal IRI. The effect of antioxidant stress induced by GSTO1 is one of the most important underlying mechanisms. Our findings underscore that hypoxia-pretreated MSC-EVs represent a novel and promising therapeutic intervention for renal IRI.
Project description:Proteome characterization of mesenchymal stem cells (MSC) and exosomes.
MSCs were cultured in normoxic, hypoxic and in presence of FBS. Exosomes were prepared from normoxic and hypoxic conditions.
Project description:This study investigated the role of astrocyte-derived EVs in neuroprotection and tissue recovery post-ischemia. Cortical astrocytes were cultured under normoxic and hypoxic conditions to harvest extracellular vesicles (EVs). The EVs were then administered to rats that had undergone experimental stroke. We assessed infarct volume, neuronal survival, and neurological function, and utilized label-free mass spectrometry-based proteomics to evaluate the hypoxia-induced changes in the EV proteome. Administration of astrocyte-derived EVs significantly reduced infarct volume, improved neurological function, and mitigated blood-brain barrier (BBB) permeability. Proteomic analysis revealed a differential expression of 67 upregulated proteins under hypoxic conditions and 37 downregulated proteins under normoxic conditions, highlighting the protective protein signatures elicited by astrocytes. Our findings underscore the role of astrocyte-derived EVs in fostering a conducive environment for neuronal survival under hypoxic stress. (Supported by DGAPA-PAPIIT IN207020 and IN214723 and CONACYT A1-S-13219).
Project description:In vivo profiling of hypoxic gene expression in gliomas using the hypoxia marker EF5 and laser-capture microdissection We have employed the hypoxia marker EF5 coupled with laser capture microdissection to isolate RNA from viable hypoxic and normoxic regions of 9L experimental gliomas. We report the identification of the hypoxic gene expression profile in a solid tumor using a prototypical hypoxia marker EF5, Laser Capture Microdissection (LCM) and Microarray Gene Expression Profiling (EF5-LCM-MGEP). We have utilized LCM to isolate total RNA from normoxic and hypoxic regions in the rat 9L glioma tumor model grown in its isogenic host (Fischer rat). Microarray analysis of the isolated RNA generated an in vivo hypoxia expression profile. For comparison, we also analyzed RNA samples isolated from rat 9L glioma cells in culture exposed to hypoxia or normoxia.
Project description:Exosomes derived from NSCLC cells under normoxic or hypoxic conditions were compared to identify hypoxic associated enrichment of proteins in exosomes.
