Adjuvant role of macrophages in stem cell-induced cardiac repair in rats

Fig. 1. Expression of inflammation-related mediators in rBMDMs is altered by coculture with rBMMSCs. a The mRNA levels of factors such as iNOS, IL-1β, TNF-α, and Arg1 were assessed in LPS-stimulated BMDMs with or without rBMMSC coculture. b The mRNA levels of factors such as Arg1, CD206, and IL-10 were assessed in IL-4/IL-13-treated BMDMs with or without rBMMSC coculture. c The iNOS mRNA to Arg1 mRNA ratio was calculated to show the skewed macrophage polarization toward the anti-inflammatory phenotype. The Arg1 and iNOS protein expression patterns in LPS-stimulated rBMDMs (d) or IL-4/IL-13-treated rBMDMs (e) with or without rBMMSC coculture. rBMDMs Rat bone marrow-derived macrophages; rBMMSCs Rat bone marrow-derived mesenchymal stem cells

Fig. 2. Antibody array showing that the soluble mediator profiles from BMDMs are altered by coculture with BMMSCs. rBMDM (Veh) Vehicle-treated rat BMDMs; rBMDM (LPS) LPS-stimulated rat BMDMs; rBMDM (LPS) + rBMMSCs, LPS stimulation of rBMDMs followed by coculture with rBMMSCs

Fig. 3. Therapeutic efficacies of BMCs and MIX in a rat myocardial infarction (MI) model. a Representative gross images (upper) and trichrome-stained heart tissues (lower) from the MI model rats injected with PBS, rBMMSCs, or MIX. Cardiac fibrosis was quantified (right). b Representative echocardiograms of the non-MI, PBS, rBMMSC, and MIX groups. c The cardiac function indexes were measured by echocardiography

Fig. 4. Angiogenesis and macrophage infiltration in the infarcted myocardium. a The infarcted myocardium was immunostained for vWF to analyze angiogenesis in the PBS, rBMMSC, and MIX groups, and vWF-positive cells were counted for quantification. b Immunostaining with CD68 showed macrophage infiltration, and the number of CD68-positive cells was quantified

Fig. 5. Representative images showing that anti-inflammatory macrophages are more abundant in the MIX group than in the BMMSC group. a Anti-inflammatory macrophages were detected by double staining with the macrophage marker CD68 and the anti-inflammatory marker CD206 in confocal images. b The number of anti-inflammatory macrophages identified by double-positive staining for CD68 and CD206 was higher in the MIX group than in the rBMMSC group

Fig. 6. IRF5 is essential in BMCs and exerts an anti-inflammatory effect on LPS-stimulated macrophages. a mRNA expression patterns of IRF family members were examined in hBMMSCs after coculture with THP-1-derived macrophages stimulated with vehicle or LPS. b IRF5 protein expression was upregulated in hBMMSCs cocultured with LPS-stimulated macrophages. c IRF5 protein expression also increased in rBMMSCs cocultured with rBMDMs. d LPS-induced upregulation of the proinflammatory markers CXCL10 and CXCL11 was apparently inhibited by coculture with hBMMSCs transfected with the siRNA control. Conversely, coculture with hBMMSCs transfected with the IRF5 siRNA failed to attenuate the upregulation of CXCL10 and CXCL11. e CXCL10 protein expression was measured in media from THP-1-differentiated macrophages cocultured with hBMMSCs transfected with the siRNA control or IRF5 siRNA

Fig. 7. Schematic representation of the contribution of BMMSC-primed BMDMs to the overall improvement in BMC-induced cardiac repair