Isolation and culture of corneal neovascular endothelial cells and expression of chemokine receptors

Background

The pathogenesis and mechanism research of corneal neovascularization is of important significance for the prevention and management of corneal neovascularization. Some relative researches are being performed on non-corneal neovascularization-derived vascular endothelial cells, so the results are affected to a certain extent.

Objective

This study was to isolate and culture vascular endothelial cells from experimental corneal neovascularization tissue and detect the expression of chemokine receptors in vitro.

Methods

Corneal neovascularization models were established on 10 SPF male BALB/c mice with the age of 7-8 weeks by sticking the filter papers with NaOH on the central corneas, and then the immunofluorescence technique was use to assay the CD31 expression in corneal flatmount 2 weeks after modeling. Corneal pieces were made in 2 weeks after alkali burn and then were digested by collagenase type D. Vascular endothelial cells were isolated from neovascularized tissue by affinity purification using magnetic beads coated with anti-CD31. The cells were cultured on fibronectin-coated walls and then identified by immunocytochemistry. Reverse transcription-PCR was employed to detect the expressions of chemokine receptors in the cells. The use and care of the animals complied with ARVO Statement and this experimental procedure was approved by Soochow University Animal Care Committee.

Results

Corneal neovascularization occurred at 7 days and peaked at 2 weeks after modeling, and immunofluorescence exhibited the green network-like fluorescence for CD31 antibody in corneas. The cells grew against the wall 2 hours after culture with the polygon shape and large dimension, and the growth obviously quickened after passage. The cultured cells showed the positive response for CD31 antibody, showing the brown dye in cytoplasm, in contrast, the expression of CD31 was absent in corneal stromal cells. Chemokine receptors were positively expressed in the cells with the strongest expression levels in CCR1, CCR2, CCR3 and CCR4 mRNA and the weakest expression levels in CCR9, CXCR4 and CXCR5 mRNA, while CXCR3, CCR6, CCR10 and CX3CR1 mRNA were expressed with the moderate intensity.

Conclusions

Vascular endothelial cells can be obtained from experimental neovascularized corneas by affinity purification and express chemokine receptors, which facilitate the study of their biological properties.

[Key words]Endothelial cells/corneal neovascularization-derived; Cells, cultured; Corneal neovascularization/chemically induced; Receptors, chemokine; Diseases models, animal; Mice, inbred BALB/c