Hdmec

RA fibroblast–like synoviocytes (RA-FLS) were purchased from Cell Applications (San Diego, CA, United States), and human dermal microvascular endothelial cells (HDMECs) were obtained from ScienCell Research Laboratories (6076 Corte Del Cedro, Carlsbad, CA, United States). Cells at passages three to six were used for experiments. The co-culture system of RA-FLS and HDMECs was established as described previously (Zhang et al., 2017 (link)). Briefly, RA-FLS (4×10⁵ cells/ml) and HDMECs (ranging from 2×10⁵ cells/ml to 3×10⁵ cells/ml) were seeded in the lower and upper chambers of a transwell apparatus (Costar), respectively, and incubated in 2.0 μM arsenic trioxide (ATO; As₂O₃; Yitaida Pharmaceutical Factory, Harbin, Heilongjiang, China) with or without TNF-α (50 ng/ml, Peprotech, Rocky Hill, United States). After being cultured in 1% FBS DMEM for 48 h, the supernatants were harvested. Fresh supernatants were taken out for the subsequent transwell assays, tube formation tests, and ex vivo aortic ring angiogenesis assays. Remaining supernatants were frozen at -20°C until analysis by commercial enzyme-linked immunosorbent assay (ELISA) kit for VEGF (DVE00, R&D Systems). The mRNA and protein expression in RA-FLS co-cultured were, respectively, determined by quantitative real-time PCR (qRT-PCR) and Western blot analysis.

RA-FLS and NH-FLS were purchased from Cell Applications (San Diego, CA, USA) and were maintained in synoviocyte growth medium (Cell Applications). HDMECs were purchased from ScienCell Research Laboratories (6076 Corte Del Cedro, Carlsbad, CA, USA) and were maintained in Endothelial cell Growth Medium (EGM)-2 from the same company. All cells were maintained in a humidified atmosphere of 5% CO₂ at 37^°C and cells of passages from three to five were used for the experiment.

Three microvascular endothelial cell lines including primary human dermal microvascular endothelial cells (HDMEC) (Sciencell, Carlsbad, CA, USA), primary human pulmonary microvascular endothelial cells (HPMEC) (Sciencell) and primary human retinal microvascular endothelial cells (HRtMEC) (CSC, Tysons, VA, USA) were cultured as in vitro models for dengue virus infection. The cells were maintained at 37°C with 5% CO₂ supplemented with endothelial cell growth medium (ECM) (Sciencell) for HDMEC and HPMEC, and CSC complete medium (CSC) for HRtMEC.

Primary HDF (Cat # 2320), HDMEC (Cat # 2020), and HEK (Cat # 2110) were obtained from ScienCell and cultured in their special medium provided by ScienCell. Primary HDFs were cultured in a fibroblast medium (8 mM glucose), supplemented with 2% fetal bovine serum (FBS) and 1% fibroblast growth supplement. Primary HDMECs were cultured in an endothelial cell medium (5.55 mM glucose), supplemented with 5% FBS and 1% endothelial cell growth supplement. Primary HEKs were cultured in a fibroblast medium (6 mM glucose) supplemented with 1% keratinocyte growth supplement. All media were supplemented with 100 IU/ml penicillin. All types of the cells were maintained at 37°C in a 5% CO₂ humidified incubator, and cells from the fourth to ninth passages were used for experiments. In the experiments, cells were initially plated on 3-cm plates in their special medium. We uniformly define the glucose concentration of their special medium as normal glucose (NG). At 80% confluence, the cells were switched to HG (30 Mm) medium for 24 h. For each condition, three biological replicates were generated.

Human dermal microvascular endothelial cells (HDMEC) and umbilical vein endothelial cells (HUVEC) were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA) and cultured in endothelial cell medium supplemented with 5% fetal bovine serum, 1% endothelial cell growth supplement, and 1% penicillin/streptomycin solution (ScienCell Research Laboratories, Carlsbad, CA, USA). Vascular smooth muscle cells (VSMC) from the human umbilical arteries (HUASMC, ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in smooth muscle cell medium supplemented with 2% fetal bovine serum, 1% smooth muscle cell growth supplement (ScienCell Research Laboratories, Carlsbad, CA, USA), and 1% penicillin/streptomycin solution.