Integrin beta 3-overexpressing mesenchymal stromal cells display enhanced homing and can reduce atherosclerotic plaque

Figure 3 Integrin subunit beta 3 enhances mesenchymal stem cell migration in vitro and in vivo. A: In vitro migration of integrin subunit beta 3 (ITGB3)-overexpressing mesenchymal stem cell (MSC) (MSCs^ITGB3) toward lower Raw264.7, MVSMC and PMVEC stimulated by tumor necrosis factor α. Scale bars = 100 μm; B: Quantification of migrated cells. Data are presented as the mean ± SEM; C: Experimental scheme of chemotactic assays with mouse atherosclerotic vascular samples. The blood vessel was divided into three parts. For each of the experiments, each part was cultured for 24 h in medium. To perform the assay, a suspension of 2 × 10⁵ MSCs, 2 × 10⁵ MSCs^GFP, or 2 × 10⁵ MSCs^ITGB3 in medium were seeded in the upper chamber. Following incubation for 24 h, non-migrated cells were removed; D: Representative image and quantification of migrated cells stuck in the porous membrane or the lower layer of the transwell stained with 0.1% crystal violet; E: MSCs^ITGB3 and MSCs^GFP, both expressing green fluorescence, were intravenously injected into mice. The expression of green fluorescent protein (GFP) and CD31 was examined by frozen staining. GFP-positive cells were quantified per microscopic field of vascular staining in triplicate mice; F: The expression of GFP and SMA were examined by frozen staining. GFP-positive cells were quantified per microscopic field of vascular staining in triplicate mice. Scale bars = 100 μm; G: Expression levels of GFP in atherosclerosis analyzed using quantitative real-time polymerase chain reaction of mRNA samples extracted from MSC^GFP and MSC^ITGB3 vessels of three mice. Data are presented as the mean ± SEM for each group. The data are representative of three independent experiments. ^aP < 0.05, ^bP < 0.001. GFP: Green fluorescent protein; ITGB3: Integrin subunit beta 3; MSC: Mesenchymal stem cell; qRT-PCR: Quantitative real-time polymerase chain reaction; SEM: Standard error of the mea; TNF: Tumor necrosis factor.