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Transcriptional and functional adaptations of human endothelial cells to physiological chronic low oxygen.

ABSTRACT: Endothelial cells chronically reside in low-O2 environments in vivo (2%-13% O2), which are believed to be critical for cell homeostasis. To elucidate the roles of this physiological chronic normoxia in human endothelial cells, we examined transcriptomes of human umbilical vein endothelial cells (HUVECs), proliferation and migration of HUVECs in response to fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA), and underlying signaling mechanisms under physiological chronic normoxia. Immediately after isolation, HUVECs were cultured steadily under standard cell culture normoxia (SCN; 21% O2) or physiological chronic normoxia (PCN; 3% O2) up to 25 days. We found that PCN up-regulated 41 genes and down-regulated 21 genes, 90% of which differed from those previously reported from HUVECs cultured under SCN and exposed to acute low O2. Gene ontology analysis indicated that PCN-regulated genes were highly related to cell proliferation and migration, consistent with the results from benchtop assays that showed that PCN significantly enhanced FGF2- and VEGFA-stimulated cell proliferation and migration. Interestingly, preexposing the PCN cells to 21% O2 up to 5 days did not completely diminish PCN-enhanced cell proliferation and migration. These PCN-enhanced cell proliferations and migrations were mediated via augmented activation of MEK1/MEK2/ERK1/ERK2 and/or PI3K/AKT1. Importantly, these PCN-enhanced cellular responses were associated with an increase in activation of VEGFR2 but not FGFR1, without altering their expression. Thus, PCN programs endothelial cells to undergo dramatic changes in transcriptomes and sensitizes cellular proliferative and migratory responses to FGF2 and VEGFA. These PCN cells may offer a unique endothelial model, more closely mimicking the in vivo states.

SUBMITTER: Jiang YZ

PROVIDER: S-EPMC4013893 | biostudies-other | 2013 May

REPOSITORIES: biostudies-other

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Transcriptional and functional adaptations of human endothelial cells to physiological chronic low oxygen.

Jiang Yi-Zhou YZ Wang Kai K Li Yan Y Dai Cai-Feng CF Wang Ping P Kendziorski Christina C Chen Dong-Bao DB Zheng Jing J

Biology of reproduction 20130509 5

Endothelial cells chronically reside in low-O2 environments in vivo (2%-13% O2), which are believed to be critical for cell homeostasis. To elucidate the roles of this physiological chronic normoxia in human endothelial cells, we examined transcriptomes of human umbilical vein endothelial cells (HUVECs), proliferation and migration of HUVECs in response to fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA), and underlying signaling mechanisms under physiological c ...[more]

PMID: 23536375

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Project description:Cultured rat liver endothelial cells were incubated with 1 and 2 mM spermineNONOate at different O2 concentrations in the incubation atmosphere. (Z)-1-{N-[3-Aminopropyl]-N-[4-(3-aminopropylammonio) butyl]-amino}diazen-1-ium-1,2- diolate (spermineNONOate), at 2 mM, was highly cytotoxic at 21% and 95% O2 (as measured by lactate dehydrogenase release); more than 80% of the cells were damaged after 6 h of incubation. Cytotoxicity induced by 2 mM spermineNONOate was significantly decreased at 10%, 5% and 0% O2; cell death was 54%, 36% and 25% respectively after 6 h of incubation. In contrast, 1 mM spermineNONOate was almost non-toxic towards the cells. Only at 95% O2 was a slight damaging effect, of 25%, observed. The nitric oxide (.NO) concentrations released from 1 and 2 mM spermineNONOate were determined as varying between 5 and 12 microM and between 12 and 22 microM respectively as measured by the oxyhaemoglobin and the NO cheletropic spin-trapping methods. The decomposition rate of spermineNONOate and the resulting .NO concentrations were independent of O2 at all applied concentrations. Likewise, the steady-state concentrations of H2O2 remained at approx. 1 nM at all O2 concentrations, as measured with the fluorescent dye scopoletin. L-Tyrosine and L-ascorbate, both of which are known to scavenge reactive nitrogen species, markedly diminished spermineNONOate-induced cytotoxicity at 95% O2. The formation of 3-nitrotyrosine, indicating the reaction of L-tyrosine with nitrogen dioxide (.NO2) and/or peroxynitrite anions, was enhanced in incubations with spermineNONOate at 21% and 95% O2. The results demonstrate that at O2 partial pressures typically found under physiological conditions and at .NO concentrations that can occur in vivo, .NO alone is almost non-toxic towards cultured rat liver endothelial cells. .NO at these concentrations in vivo, however, exerts toxic effects at supraphysiological O2 partial pressures, owing to its oxidation to reactive nitrogen species such as .NO2.

Project description:We previously reported on the role of pericyte-like cells as functional sentinel immune cells in lung injury. However, much about the biological role of pericytes in lung injury remains unknown. Lung pericyte-like cells are well-positioned to sense disruption to the epithelial barrier and coordinate local inflammatory responses due to their anatomic niche within the alveoli. In this report, we characterized transcriptional responses and functional changes in pericyte-like cells following activation by alveolar components from injured and uninjured lungs in a mouse model of acute lung injury (ALI). Purified pericyte-like cells from lung digests using PDGFRβ as a selection marker were expanded in culture as previously described (1). We induced sterile acute lung injury in mice with recombinant human Fas ligand (rhFasL) instillation followed by mechanical ventilation (1). We then collected bronchoalveolar lavage fluid (BALF) from injured and uninjured mice. Purified pericyte-like cells in culture were exposed to growth media only (control), BALF from uninjured mice, and BALF from injured mice for 6 and 24 hours. RNA collected from these treatment conditions were processed for RNAseq. Targets of interest identified by pathway analysis were validated using in vitro and in vivo assays. We observed robust global transcriptional changes in pericyte-like cells following treatment with uninjured and injured BALF at 6 hours, but this response persisted for 24 hours only after exposure to injured BALF. Functional enrichment analysis of pericytes treated with injured BALF revealed the activation of pro-inflammatory, cell migration, and angiogenesis-related pathways, whereas processes associated with tissue development and cell differentiation were down-regulated. We validated select upregulated targets in the inflammatory, angiogenic, and cell migratory pathways using functional biological assays in vitro and in vivo. We conclude that lung pericyte-like cells are highly responsive to alveolar compartment content from both uninjured and injured lungs, but injured BALF elicits a more sustained response. The inflammatory, angiogenic, and migratory changes exhibited by activated pericyte-like cells underscore the phenotypic plasticity of these specialized stromal cells in the setting of acute lung injury.

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Transcriptional and functional adaptations of human endothelial cells to physiological chronic low oxygen.

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Transcriptional and functional adaptations of human endothelial cells to physiological chronic low oxygen.

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