Development of a Serum-Free Culture Method for Endothelial Cells of the Stria Vascularis and Their Pro-inflammatory Secretome Changes Induced by Oxidative Stress
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Objectives. Reactive oxygen species in the stria vascularis (SV) of the cochlea may be involved in the pathogenesis of sen-sorineural hearing loss. However, the effects of oxidative stress on SV endothelial cells (SV-ECs) remain largely un-known, and no feasible in vitro cell culture model exists for the functional study of SV-ECs. Methods. We isolated primary SV-ECs from the SV of neonatal mice. The apoptosis-reducing effects of fibronectin in SV-ECs cultured with serum-free medium were determined using β-galactosidase staining and flow cytometry. SV-ECsincubated in serum-free medium were treated with various H2O2 concentrations to evaluate the effects of H2O2 ontheir viability. The secretome of SV-ECs treated with or without H2O2 (100 μM or 500 μM) was analyzed using high-resolution mass spectrometry. The function of the SV-EC secretome was evaluated by a macrophage assay. Results. We successfully isolated and characterized the SV-ECs. Treatment with H2O2 at concentrations up to 500 μM for2 hours and further incubation with serum-free medium in plates precoated with fibronectin showed no significanteffect on apoptosis. Compared to the control SV-ECs, the amount of differential proteins in the secretome of SV-ECsstimulated with 500 μM H2O2 was much higher than in those treated with 100 μM H2O2. Kyoto Encyclopedia ofGenes and Genomes and Gene Ontology analyses suggested that the proteins differentially expressed in SV-ECstreated with 500 μM H2O2 were involved in the regulation of multiple signaling pathways and cellular processes. Thesecretome of H2O2-stimulated SV-ECs exhibited significant pro-inflammatory effects on macrophages. Conclusion. We successfully established an in vitro serum-free culture method, identified the differential proteins releasedby oxidative stress-induced ECs and their functions, and revealed the pro-inflammatory effects of the secretome ofH2O2-stimulated SV-ECs. Therefore, SV-ECs might elicit immunoregulatory effects on bystander cells in the microen-vironment of oxidative stress-induced cochlea, especially cochlear macrophages.
Abstract
Objectives. Reactive oxygen species in the stria vascularis (SV) of the cochlea may be involved in the pathogenesis of sen-sorineural hearing loss. However, the effects of oxidative stress on SV endothelial cells (SV-ECs) remain largely un-known, and no feasible in vitro cell culture model exists for the functional study of SV-ECs. Methods. We isolated primary SV-ECs from the SV of neonatal mice. The apoptosis-reducing effects of fibronectin in SV-ECs cultured with serum-free medium were determined using β-galactosidase staining and flow cytometry. SV-ECsincubated in serum-free medium were treated with various H2O2 concentrations to evaluate the effects of H2O2 ontheir viability. The secretome of SV-ECs treated with or without H2O2 (100 μM or 500 μM) was analyzed using high-resolution mass spectrometry. The function of the SV-EC secretome was evaluated by a macrophage assay. Results. We successfully isolated and characterized the SV-ECs. Treatment with H2O2 at concentrations up to 500 μM for2 hours and further incubation with serum-free medium in plates precoated with fibronectin showed no significanteffect on apoptosis. Compared to the control SV-ECs, the amount of differential proteins in the secretome of SV-ECsstimulated with 500 μM H2O2 was much higher than in those treated with 100 μM H2O2. Kyoto Encyclopedia ofGenes and Genomes and Gene Ontology analyses suggested that the proteins differentially expressed in SV-ECstreated with 500 μM H2O2 were involved in the regulation of multiple signaling pathways and cellular processes. Thesecretome of H2O2-stimulated SV-ECs exhibited significant pro-inflammatory effects on macrophages. Conclusion. We successfully established an in vitro serum-free culture method, identified the differential proteins releasedby oxidative stress-induced ECs and their functions, and revealed the pro-inflammatory effects of the secretome ofH2O2-stimulated SV-ECs. Therefore, SV-ECs might elicit immunoregulatory effects on bystander cells in the microen-vironment of oxidative stress-induced cochlea, especially cochlear macrophages.
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