Effects of substrate stiffness on the proliferation of primary human umbilical vein endothelial cells and the release of NO and ET-1 during dengue virus infection / 中华微生物学和免疫学杂志

ABSTRACT

Objective To investigate the effects of substrate stiffness on the proliferation of human umbilical vein endothelial cells ( HUVEC) during dengue virus infection.Methods Polyacrylamide gels were prepared for cell culture [(0±4) kPa].The proliferation of HUVEC cultured on substrates with differ-ent stiffness was determined by using 3-(4,5-diethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-etrazolium,inner salt ( MTS) assay.The cycle and apoptosis of HUVEC were determined by flow cytometry analysis.Dengue virus serotype 2 (DENV-2) strains were propagated and identified by con-ventional assays.The HUVEC were infected with DENV-2 strains at a MOI of 10 and cultured on traditional plastic and hydrogel substrates, respectively.The levels of nitric oxide (NO) and endothelin-1 (ET-1) were detected by nitric acid reductase assay and double antibody sandwich ELISA.Results Young′s modulus E value of the hydrogels was (3030 ±0.44) Pa.The proliferation of HUVEC and the expression of NO and ET-1 were enhanced along the increased substrate stiffness.However, no significant differences with the cell cycle and apoptosis were observed between cells cultured on different substrates.Conclusion The stiffness of substrates affected not only the proliferation of HUVEC, but also the release of cytokines during DENV-2 infection.The development of dengue fever was associated with the decreased secretion of vascular active substances as a result of blood vessel injury.The establishment of hydrogel substrates as the model of vascu-lar basement membranes might provide a new way for the in vitro investigation of the pathogenesis of DENV infection.