Development of a Serum-Free Culture Method for Endothelial Cells of the Stria Vascularis and Their Pro-inflammatory Secretome Changes Induced by Oxidative Stress

Abstract

Objectives

Reactive oxygen species in the stria vascularis (SV) of the cochlea may be involved in the pathogenesis of sensorineural hearing loss. However, the effects of oxidative stress on SV endothelial cells (SV-ECs) remain largely unknown, 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-ECs incubated in serum-free medium were treated with various H ₂O ₂ concentrations to evaluate the effects of H ₂O ₂ on their viability. The secretome of SV-ECs treated with or without H ₂O ₂ (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 H ₂O ₂ at concentrations up to 500 μM for 2 hours and further incubation with serum-free medium in plates precoated with fibronectin showed no significant effect on apoptosis. Compared to the control SV-ECs, the amount of differential proteins in the secretome of SV-ECs stimulated with 500 μM H ₂O ₂ was much higher than in those treated with 100 μM H ₂O ₂. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses suggested that the proteins differentially expressed in SV-ECs treated with 500 μM H ₂O ₂ were involved in the regulation of multiple signaling pathways and cellular processes. The secretome of H ₂O ₂-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 released by oxidative stress-induced ECs and their functions, and revealed the pro-inflammatory effects of the secretome of H ₂O ₂-stimulated SV-ECs. Therefore, SV-ECs might elicit immunoregulatory effects on bystander cells in the microenvironment of oxidative stress-induced cochlea, especially cochlear macrophages.