Fig 2.

A, Human primary vascular smooth muscle cells (VSMCs) treated with cholesterol or oxidized phospholipids (OxPAPC) demonstrated structural changes compared with control (CTRL) cells grown in normal media using bright field microscopy. Immunofluorescence staining showed migration of HDAC9 to the nucleus and increased HDAC9 expression in treated VSMCs. B, Treated VSMCs had significantly increased expression of HDAC9, CD68, and LGALS3 and decreased expression of ACTA2, SM22, and MYH11 compared with the control cells. Inhibition of HDAC9 in the control cells demonstrated efficacy of HDAC9 silencing, also confirmed by Western blot (Supplementary Fig 2). In the treated cells, HDAC9 silencing reduced expression of HDAC9, CD68, and LGALS3 and restored expression of ACTA2, SM22, and MYH11. P values reported for comparisons between control and cholesterol and control and OXPAPC groups and cholesterol and OXPAPC with small interfering HDAC9 (siHDAC9) treatment. C1, Transwell experiments using supernatant from treated VSMCs and VSMCs grown in coculture with THP-1 (MC) cells incubated for 4 and 12 hours. Treated VSMCs and their supernatant increased migration of macrophages (circled in red) across the 3-μm-pore Transwell plates. C2, The number of MCs that migrated to the bottom well were counted after 4 and 12 hours; each group was repeated in quadruplicate. P values reflect comparisons between the counts of the migrated MCs between the control group and each of the three treatment groups at 4 and 12 hours. The VSMCs that underwent silencing for HDAC9 showed no difference in the migration of monocytes compared with untreated, or control, group VSMCs, despite treatment with cholesterol. C3, Immunofluorescence staining of the Transwell plates depicts the interaction of macrophages and VSMCs showing increased staining of LGALS3 and ACTA2 in VSMCs that had undergone treatment, especially at the macrophage–VSMC interface.