Mesenchymal stem cells (MSCs) have been widely studied for their applications in stem cell-based regeneration. During myocardial infarction (MI), infiltrated macrophages have pivotal roles in inflammation, angiogenesis and cardiac remodeling. We hypothesized that MSCs may modulate the immunologic environment to accelerate regeneration. This study was designed to assess the functional relationship between the macrophage phenotype and MSCs. MSCs isolated from bone marrow and bone marrow-derived macrophages (BMDMs) underwent differentiation induced by macrophage colony-stimulating factor. To determine the macrophage phenotype, classical M1 markers and alternative M2 markers were analyzed with or without co-culturing with MSCs in a transwell system. For animal studies, MI was induced by the ligation of the rat coronary artery. MSCs were injected within the infarct myocardium, and we analyzed the phenotype of the infiltrated macrophages by immunostaining. In the MSC-injected myocardium, the macrophages adjacent to the MSCs showed strong expression of arginase-1 (Arg1), an M2 marker. In BMDMs co-cultured with MSCs, the M1 markers such as interleukin-6 (IL-6), IL-1β, monocyte chemoattractant protein-1 and inducible nitric oxide synthase (iNOS) were significantly reduced. In contrast, the M2 markers such as IL-10, IL-4, CD206 and Arg1 were markedly increased by co-culturing with MSCs. Specifically, the ratio of iNOS to Arg1 in BMDMs was notably downregulated by co-culturing with MSCs. These results suggest that the preferential shift of the macrophage phenotype from M1 to M2 may be related to the immune-modulating characteristics of MSCs that contribute to cardiac repair.

The activity-guided fractionation of the extract of the herb of Prunella vulgaris (Labiatae) led to the isolation of four triterpenes, i.e., betulinic acid, ursolic acid, 2 alpha,3 alpha-dihydroxyurs-12-en-28-oic acid, and 2 alpha-hydroxyursolic acid. One of these compounds, 2 alpha,3 alpha-dihydroxyursolic acid, demonstrated significant inhibition on the release of beta-hexosaminidase from the cultured RBL-2H3 cells in a dose-dependent manner; the IC50 value was calculated to be 57 microM. When the isolated compounds were tested for their effects on the production of nitric oxide from cultured murine macrophages, RAW 264.7 cells, ursolic acid and 2 alpha-hydroxyursolic acid exhibited strong inhibitory activities (IC50 values, 17 and 27 microM, respectively).

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Although mesenchymal stem cells (MSC) have been shown to be safe in preclinical studies of cardiovascular disease, multiple meta-analyses have debated whether functional improvement is significant or not. The cardiac differentiation from MSC is achievable using cardiogenic factors, however, the high cost and long culture period may limit the applications. Here, we developed a novel method to optimize the therapeutic outcome for myocardial infarction (MI). Treatment of MSC with apicidin, a histone deacetylase inhibitor, dramatically increased the expressions of cardiac markers such as GATA4, Nkx2.5, and cardiac troponin I (cTnI). In AC/MSC, stemness-related genes and yes-associated protein (YAP), a potent oncogene that drives cell proliferation, were significantly suppressed. Furthermore apicidin treatment or YAP knockdown downregulated miR-130a expression followed by induction of cardiac markers in MSC. In the comparison study, we found that both cardiac gene induction and angiogenesis were most prominent in the mixture of non-treated MSC and AC/MSC (Mix). Using mouse MI model, we show that application of Mix was strongly associated with cardiac differentiation of injected MSC and improved cardiac performance. Our results suggest that suppression of YAP/miR-130a shifts MSC cell fate toward cardiac lineage and identify apicidin as a potential pharmacological target for therapeutic development.

Bone marrow-derived mesenchymal stem cells (BMMSCs) are used extensively for cardiac repair and interact with immune cells in the damaged heart. Macrophages are known to be modulated by stem cells, and we hypothesized that priming macrophages with BMMSCs would enhance their therapeutic efficacy. Rat bone marrow-derived macrophages (BMDMs) were stimulated by lipopolysaccharide (LPS) with or without coculture with rat BMCs. In the LPS-stimulated BMDMs, induction of the inflammatory marker iNOS was attenuated, and the anti-inflammatory marker Arg1 was markedly upregulated by coculture with BMMSCs. Myocardial infarction (MI) was induced in rats. One group was injected with BMMSCs, and a second group was injected with MIX (a mixture of BMMSCs and BMDMs after coculture). The reduction in cardiac fibrosis was greater in the MIX group than in the BMC group. Cardiac function was improved in the BMMSC group and was substantially improved in the MIX group. Angiogenesis was better in the MIX group, and anti-inflammatory macrophages were more abundant in the MIX group than in the BMMSC group. In the BMMSCs, interferon regulatory factor 5 (IRF5) was exclusively induced by coculture with macrophages. IRF5 knockdown in BMMSCs failed to suppress inflammatory marker induction in the macrophages. In this study, we demonstrated the successful application of BMDMs primed with BMMSCs as an adjuvant to cell therapy for cardiac repair.

Atherosclerosis, the leading cause of death, is a vascular disease of chronic inflammation. We recently showed that angiopoietin-like 4 (ANGPTL4) promotes cardiac repair by suppressing pathological inflammation. Given the fundamental contribution of inflammation to atherosclerosis, we assessed the role of ANGPTL4 in the development of atherosclerosis and determined whether ANGPTL4 regulates atherosclerotic plaque stability. We injected ANGPTL4 protein twice a week into atherosclerotic Apoe−/− mice and analyzed the atherosclerotic lesion size, inflammation, and plaque stability. In atherosclerotic mice, ANGPTL4 reduced atherosclerotic plaque size and vascular inflammation. In the atherosclerotic lesions and fibrous caps, the number of α-SMA(+), SM22α(+), and SM-MHC(+) cells was higher, while the number of CD68(+) and Mac2(+) cells was lower in the ANGPTL4 group. Most importantly, the fibrous cap was significantly thicker in the ANGPTL4 group than in the control group. Smooth muscle cells (SMCs) isolated from atherosclerotic aortas showed significantly increased expression of CD68 and Krüppel-like factor 4 (KLF4), a modulator of the vascular SMC phenotype, along with downregulation of α-SMA, and these changes were attenuated by ANGPTL4 treatment. Furthermore, ANGPTL4 reduced TNFα-induced NADPH oxidase 1 (NOX1), a major source of reactive oxygen species, resulting in the attenuation of KLF4-mediated SMC phenotypic changes. We showed that acute myocardial infarction (AMI) patients with higher levels of ANGPTL4 had fewer vascular events than AMI patients with lower levels of ANGPTL4 (p < 0.05). Our results reveal that ANGPTL4 treatment inhibits atherogenesis and suggest that targeting vascular stability and inflammation may serve as a novel therapeutic strategy to prevent and treat atherosclerosis. Even more importantly, ANGPTL4 treatment inhibited the phenotypic changes of SMCs into macrophage-like cells by downregulating NOX1 activation of KLF4, leading to the formation of more stable plaques.

Resistin-like alpha (Retnla) is a member of the resistin family and known to modulate fibrosis and inflammation. Here, we investigated the role of Retnla in the cardiac injury model. Myocardial infarction (MI) was induced in wild type (WT), Retnla knockout (KO), and Retnla transgenic (TG) mice. Cardiac function was assessed by echocardiography and was significantly preserved in the KO mice, while worsened in the TG group. Angiogenesis was substantially increased in the KO mice, and cardiomyocyte apoptosis was markedly suppressed in the KO mice. By Retnla treatment, the expression of p21 and the ratio of Bax to Bcl2 were increased in cardiomyocytes, while decreased in cardiac fibroblasts. Interestingly, the numbers of cardiac macrophages and unsorted bone marrow cells (UBCs) were higher in the KO mice than in the WT mice. Besides, phosphorylated histone H3(+) cells were more frequent in bone marrow of KO mice. Moreover, adiponectin in UBCs was notably higher in the KO mice compared with WT mice. In an adoptive transfer study, UBCs were isolated from KO mice to transplant to the WT infarcted heart. Cardiac function was better in the KO-UBCs transplanted group in the WT-UBCs transplanted group. Taken together, proliferative and adiponectin-rich bone marrow niche was associated with substantial cardiac recovery by suppression of cardiac apoptosis and proliferation of cardiac fibroblast.

Intensive research has focused on minimizing the infarct area and stimulating endogenous regeneration after myocardial infarction. Our group previously elucidated that apicidin, a histone deacetylase (HDAC) inhibitor, robustly accelerates the cardiac commitment of naïve mesenchymal stem cells (MSCs) through acute loss of YAP1. Here, we propose the novel regulation of YAP1 in MSCs. We found that acute loss of YAP1 after apicidin treatment resulted in the mixed effects of transcriptional arrest and proteasomal degradation. Subcellular fractionation revealed that YAP1 was primarily localized in the cytoplasm. YAP1 was acutely relocalized into the nucleus and underwent proteasomal degradation. Interestingly, phosphor-S127 YAP1 was shuttled into the nucleus, suggesting that a mechanism other than phosphorylation governed the subcellular localization of YAP1. Apicidin successfully induced acetylation and subsequent dissociation of YAP1 from 14-3-3, an essential molecule for cytoplasmic restriction. HDAC6 regulated both acetylation and subcellular localization of YAP1. An acetylation-dead mutant of YAP1 retarded nuclear redistribution upon apicidin treatment. We failed to acquire convincing evidence for polyubiquitination-dependent degradation of YAP1, suggesting that a polyubiquitination-independent regulator determined YAP1 fate. Nuclear PSME4, a subunit of the 26 S proteasome, recognized and degraded acetyl YAP1 in the nucleus. MSCs from PSME4-null mice were injected into infarcted heart, and aberrant sudden death was observed. Injection of immortalized human MSCs after knocking down PSME4 failed to improve either cardiac function or the fibrotic scar area. Our data suggest that acetylation-dependent proteasome subunit PSME4 clears acetyl-YAP1 in response to apicidin treatment in the nucleus of MSCs.