C57 6024

C57BL/6 mouse primary cardiac endothelial cells were obtained from Cell Biologics (C57-6024) and handled following manufacturer’s instructions in complete mouse endothelial cell medium supplemented with VEGF, ECGS, heparin, EGF, hydrocortisone, L-glutamine, antibiotic-antimycotic solution, and FBS (M1168). After cells reached confluency, cells were exposed to normoxia (21% oxygen) or hypoxia (1% oxygen using preequilibrated media for 3 h [37 (link)]) and immediately resuspended in Trizol for miRNA analysis.

Mouse microvascular endothelial cells (Cell Biologics #C57-6024; Chicago, IL, USA; 2.5 × 10⁴ cells/well, n = 2 technical replicates per condition) were seeded in 48-well plates coated with basement membrane extract (Matrigel^® BD #354230; Franklin Lakes, NJ, USA). Cells were stimulated with either basal endothelial cell medium (CellBiologics #M1168), basal medium supplemented with 10% cardiac fibroblast secretome from the same samples used for RNA-Seq (n = 3 biological replicates), or endothelial growth medium (basal medium supplemented with VEGF, endothelial cell growth supplement, heparin, epidermal growth factor, hydrocortisone, l-glutamine, antibiotics, and FBS). Thbs1 was inhibited with a blocking antibody (10 µg/ml; Novus Biologicals #100-2059) as described previously [32 (link)]. As negative controls, endothelial cells were stimulated with the Thbs1 blocking antibody alone, an IgG1 isotype control (R&D #MAB002) alone, or basal medium diluted 1:10 with DMEM + 0.1% FBS. Images were acquired at 10X magnification following 22 h of stimulation. Angiogenesis variables were quantified using the ImageJ Angiogenesis Analyzer feature (http://image.bio.methods.free.fr/ImageJ/?Angiogenesis-Analyzer-for-ImageJ&artpage=2-6).