FIGURE 1.

Schematic of experimental methods and design. (a) Primary human monocytes were cultured with macrophage colony stimulating factor (MCSF) for 5 days (including a media change on day 3) to differentiate the cells into unactivated (M0) macrophages. M0 macrophages were then cultured with MCSF supplemented with LPS and IFNG for 2 days according to previous methods⁴⁷; (b) M1 macrophages were cultured in four different treatment groups each containing M1 macrophage culture medium (cRPMI-M1, detailed in the methods): M1 Control; M1 macrophages seeded onto intact hCVAM (hCVAM); M1 macrophages co-cultured with hCVAM separated by a semi-permeable transwell insert (Soluble Factors); and hCVAM without any seeded macro-phages (hCVAM-only Control). All treatment groups containing hCVAM (hCVAM, Soluble Factors and hCVAM-only Control) were performed with n = 3 donors of tissue. All macrophages were obtained from one donor; (c) the experiment was initiated after 7 days of macrophage culture when M1 polarization was complete, which was considered day 0 for subsequent experiments. Samples for RNA extraction and gene expression analysis were collected on days 1 and 6 (denoted with ∼), while conditioned media samples were collected on days 1, 3 (during media change), and 6 (denoted with +).