Contributions
Abstract: EP572
Type: E-Poster Presentation
Session title: Bleeding disorders (congenital and acquired)
Background
Intraventricular hemorrhage (IVH) is a severe complication of prematurity that results in an increased risk of neonatal mortality and neurodevelopmental impairment. After preterm IVH, subsequent destruction of red blood cells (RBC) and oxidation of extracellular hemoglobin (Hb) with the release of heme may cause a local inflammation with cellular dysfunction in the subarachnoid space. We have recently reported elevated expression of pro-inflammatory cell-free microRNA (miRNA) in cerebrospinal fluid (CSF) samples obtained from preterm infants with grade III or IV IVH in correlation with high levels of oxidized Hb forms and soluble adhesion molecules.
Aims
In this study, our aim was to investigate the IVH-induced inflammatory cellular events in human choroid plexus epithelial cells (HCPEpiC) among in vitro conditions.
Methods
Primary HCPEpiC cell cultures were first exposed to CSF samples of preterm infants with IVH vs. clinical control samples (10 v/v %) for 24 hours. To observe the direct effect of hemorrhage, cells were treated with heme (25 µM) or TNF-α (100 ng/mL) as a positive pro-inflammatory agonist for 1-24 hours. After treatment, total RNA was extracted using TRI reagent, and HMOX1, IL8, IL1B, VCAM1 and ICAM1 messenger RNA (mRNA) expression were quantified by RT-qPCR. miR-155 and miR-223 were also analyzed intracellularly and in the supernatants of the cells using normalization to “spike-in” cel-miR-39. Concentrations of soluble IL-8, VCAM-1 and ICAM-1 were measured by ELISA. Nuclear translocation of the NF-κB p65 subunit was investigated with fluorescence microscopy. Cell viability was evaluated using MTT assay and via the measurement of LDH leakage in cell culture medium.
Results
Exposure to IVH CSF samples a substantial cell activation was triggered in HCPEpiCs with significant elevation in HMOX1, IL8, IL1B and ICAM1 mRNA expression in comparison to controls. Interestingly, VCAM1 mRNA level did not change after 24 hours. When cells were preincubated with heme, there was a time-dependent up-regulation in IL8, IL1B and ICAM1 mRNAs in the presence of induced HMOX1 expression. Furthermore, significantly elevated protein levels of IL-8 and ICAM-1 were observed similar to TNF-α. Importantly, both mediators induced the nuclear translocation of the NF-κB p65 subunit. The expression of miR-155 and miR-223 were elevated in the cell culture supernatants after treatment with heme and TNF-α that underlines our former results in IVH CSF. In contrast, intracellular miR-223 was decreased, which may be related to up-regulated ICAM1 expression. Based on the results of cell viability assay and LDH leakage, we did not find any substantial heme- or TNF-α-induced cellular damage in our experimental conditions.
Conclusion
In IVH, choroid plexus epithelial cells are highly affected by the consequences of bleeding that may contribute to the development of different complications of the disease.
Keyword(s): Heme, Hemolysis, Hemorrhage, Inflammation
Abstract: EP572
Type: E-Poster Presentation
Session title: Bleeding disorders (congenital and acquired)
Background
Intraventricular hemorrhage (IVH) is a severe complication of prematurity that results in an increased risk of neonatal mortality and neurodevelopmental impairment. After preterm IVH, subsequent destruction of red blood cells (RBC) and oxidation of extracellular hemoglobin (Hb) with the release of heme may cause a local inflammation with cellular dysfunction in the subarachnoid space. We have recently reported elevated expression of pro-inflammatory cell-free microRNA (miRNA) in cerebrospinal fluid (CSF) samples obtained from preterm infants with grade III or IV IVH in correlation with high levels of oxidized Hb forms and soluble adhesion molecules.
Aims
In this study, our aim was to investigate the IVH-induced inflammatory cellular events in human choroid plexus epithelial cells (HCPEpiC) among in vitro conditions.
Methods
Primary HCPEpiC cell cultures were first exposed to CSF samples of preterm infants with IVH vs. clinical control samples (10 v/v %) for 24 hours. To observe the direct effect of hemorrhage, cells were treated with heme (25 µM) or TNF-α (100 ng/mL) as a positive pro-inflammatory agonist for 1-24 hours. After treatment, total RNA was extracted using TRI reagent, and HMOX1, IL8, IL1B, VCAM1 and ICAM1 messenger RNA (mRNA) expression were quantified by RT-qPCR. miR-155 and miR-223 were also analyzed intracellularly and in the supernatants of the cells using normalization to “spike-in” cel-miR-39. Concentrations of soluble IL-8, VCAM-1 and ICAM-1 were measured by ELISA. Nuclear translocation of the NF-κB p65 subunit was investigated with fluorescence microscopy. Cell viability was evaluated using MTT assay and via the measurement of LDH leakage in cell culture medium.
Results
Exposure to IVH CSF samples a substantial cell activation was triggered in HCPEpiCs with significant elevation in HMOX1, IL8, IL1B and ICAM1 mRNA expression in comparison to controls. Interestingly, VCAM1 mRNA level did not change after 24 hours. When cells were preincubated with heme, there was a time-dependent up-regulation in IL8, IL1B and ICAM1 mRNAs in the presence of induced HMOX1 expression. Furthermore, significantly elevated protein levels of IL-8 and ICAM-1 were observed similar to TNF-α. Importantly, both mediators induced the nuclear translocation of the NF-κB p65 subunit. The expression of miR-155 and miR-223 were elevated in the cell culture supernatants after treatment with heme and TNF-α that underlines our former results in IVH CSF. In contrast, intracellular miR-223 was decreased, which may be related to up-regulated ICAM1 expression. Based on the results of cell viability assay and LDH leakage, we did not find any substantial heme- or TNF-α-induced cellular damage in our experimental conditions.
Conclusion
In IVH, choroid plexus epithelial cells are highly affected by the consequences of bleeding that may contribute to the development of different complications of the disease.
Keyword(s): Heme, Hemolysis, Hemorrhage, Inflammation