Cell culture and in vitro transfection and angiogenesis assay

Human umbilical vein endothelial cells (HUVEC) were isolated from a donor umbilical as described previously [16] and then grew in endothelial cell growth medium (Cell Applications Inc, San Diego, CA) supplemented with 10% fetal bovine serum (FBS, Gibco, Shanghai, China). To mimic the endothelial cells under ischemic condition in HLI models, HUVECs were exposed to hypoxia (2% oxygen, BioSpherix, Lacona, NY) and serum starvation (HSS). The use of human umbilical vein was approved by Guangzhou Medical University Institutional Review Boards. Normal concentration of glucose (5 mM) or high concentration of D-glucose (25 mM) culture medium was used to mimic nondiabetic or hyperglycemia in vivo.

In vitro transfection of microRNA inhibitor or microRNA mimic was used to knockdown or overexpress miR-133a expression respectively in HUVECs as described previously [17]. Briefly, a reverse transfection protocol using neofx transfection agent (Ambion, Austin, TX) was used to transfect miR-133a inhibitor or miR inhibitor negative control (Life Tech) into HUVECs for 48 h.

In vitro angiogenesis assay was performed as previously described under HSS conditions [17]. Briefly, HUVECs transfected with miR-133a inhibitor, mimic, or control were plated at a density of 1 × 10⁴ cells/well on 96-well dishes, which were coated with growth factor-reduced Matrigel (BD Biosciences, MA, U.S.A) and then exposed to HSS conditions for 6 h to assess tube formation. Each condition was done in six wells. The degree of tube formation was determined by measuring the number of loops from each well under 40× magnifications using ImageJ (National Institute of Health, Bethesda, MD). Each experiment was repeated at least in two different batches of HUVECs. DCH1 inhibitor, 2′,4′-diamino-6-hydroxypyrimidine (DAHP, 1 mM); endothelial NO synthase (eNOS) inhibitor, was used for the pathway study.