Doxorubicin (DOX) is an effective antineoplastic agent used for the treatment of a variety of cancers. Unfortunately, its use is limited as this drug induces cardiotoxicity and heart failure as a side effect. There is no report which describes whether transplanted embryonic stem (ES) cells or their conditioned medium (CM) in DOX induced cardiomyopathy (DIC) can repair and regenerate myocardium. Therefore, we transplanted ES cells or CM in DIC to examine apoptosis, fibrosis, cytoplasmic vacuolization and myofibrillar loss and their associated Akt and ERK pathway. Moreover, we also determined activation of endogenous c-kit+ve cardiac stem cells (CSCs), levels of HGF and IGF-1, growth factors required for c-kit cell activation, and their differentiation into cardiac myocytes, which also contributes in cardiac regeneration and improved heart function. We generated DIC in C57Bl/6 mice (cumulative dose of DOX 12mg/kg body weight, i.p), and animals were treated with ES cells, CM or cell culture medium in controls. Two weeks post-DIC, ES cells or CM transplanted hearts showed a significant (p<0.05) decrease in cardiac apoptotic nuclei and their regulation with Akt and ERK pathway. Cardiac fibrosis observed in the ES cell or CM groups was significantly less compared with DOX and cell culture medium groups (p<0.05). Next, cytoplasmic vacuolization and myofibrillar loss was reduced (p<0.05) following treatment with ES cells or CM. Moreover, our data also demonstrated increased levels of c-kit+ve CSCs in ES cells or CM hearts and differentiated cardiac myocytes from these CSCs, suggesting endogenous cardiac regeneration. Importantly, the levels of HFG and IGF-1 were significantly increased in ES cells or CM transplanted hearts. In conclusion, we reported that transplanted ES cells or CM in DIC hearts significantly decreases various adverse pathological mechanisms as well as enhances cardiac regeneration that effectively contributes to improved heart function.

Cardiac myocyte differentiation reported thus far is from iPS cells generated from mouse and human fibroblasts. However, there is no article on the generation of iPS cells from cardiac ventricular specific cell types such as H9c2 cells. Therefore, whether transduced H9c2 cells, originally isolated from embryonic cardiac ventricular tissue, will be able to generate iPS cells and have the potential to repair and regenerate infarcted myocardium remains completely elusive. We transduced H9c2 cells with four stemness factors, Oct3/4, Sox2, Klf4, and c-Myc, and successfully reprogrammed them into iPS cells. These iPS cells were able to differentiate into beating cardiac myocytes and positively stained for cardiac specific sarcomeric α-actin and myosin heavy chain proteins. Following transplantation in the infarcted myocardium, there were newly differentiated cardiac myocytes and formation of gap junction proteins at 2 weeks post-myocardial infarction (MI), suggesting newly formed cardiac myocytes were integrated into the native myocardium. Furthermore, transplanted iPS cells significantly (p < 0.05) inhibited apoptosis and fibrosis and improved cardiac function compared with MI and MI+H9c2 cell groups. Moreover, our iPS cell derived cardiac myocyte differentiation in vitro and in vivo was comparable to embryonic stem cells in the present study. In conclusion we report for the first time that we have H9c2 cell-derived iPS cells which contain the potential to differentiate into cardiac myocytes in the cell culture system and repair and regenerate infarcted myocardium with improved cardiac function in vivo.

We recently reported that embryonic stem cells-conditioned medium (ES-CM) contains antiapoptotic factors that inhibit apoptosis in the cardiac myoblast H9c2 cells. However, the mechanisms of inhibited apoptosis remain elusive. In this report, we provide evidence for the novel mechanisms involved in the inhibition of apoptosis provided by ES-CM. ES-CM from mouse ES cells was generated. Apoptosis was induced after exposure with H(2)O(2) (400 mum) in H9c2 cells followed by the replacement with ES-CM or culture medium. H9c2 cells treated with H(2)O(2) were exposed to ES-CM, and ES-CM plus cell survival protein phosphatidylinositol 3-kinase/Akt inhibitor, LY-294002, or extracellular signal-regulated kinase (ERK1/2) inhibitor, PD-98050. After 24 h, H9c2 cells treated with ES-CM demonstrated a significant increase in cell survival. ES-CM significantly inhibited (P < 0.05) apoptosis determined by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining, apoptotic ELISA, and caspase-3 activity. Importantly, enhanced cell survival and inhibited apoptosis with ES-CM was abolished with LY-294002. In contrast, PD-98050 shows no effect on ES-CM-increased cell survival. Furthermore, H(2)O(2)-induced apoptosis is associated with decreased levels of phosphorylated (p)Akt activity. Following treatment with ES-CM, we observed a decrease in apoptosis with an increase in pAkt, and the increased activity was attenuated with the Akt inhibitor, suggesting that the Akt pathway is involved in the decreased apoptosis and cell survival provided by ES-CM. In contrast, we observed no change in ES-CM-decreased apoptosis or pERK with PD-98050. In conclusion, we suggest that ES-CM inhibited apoptosis and is mediated by Akt but not the ERK pathway.

It was hypothesized that monocyte treatment with bone morphogenetic protein 7 (BMP7) would significantly enhance monocyte polarization into M2 macrophages as well as increasing the levels of anti-inflammatory cytokines. In a cell culture system using monocytes (human acute monocytic leukemia cell line THP-1), we studied the effects of BMP7 on monocytes polarizing into M2 macrophages. The data demonstrate that THP-1 cells contain a BMP type II receptor (BMPR2), and that its activation is significantly (p < 0.05) increased following treatment with BMP7. Furthermore, there was an increase of M2 macrophages, BMPR2, and anti-inflammatory cytokines interleukin (IL)-10 and IL-1ra compared with the respective controls. Moreover, treatment with BMP7 caused a significant (p < 0.05) decrease in the levels of pro-inflammatory cytokines IL-6, tumour necrosis factor (TNF-α), and monocyte chemotactic protein-1 (MCP-1), compared with the controls. In conclusion, we suggest for the first time that BMP7 has a unique potential to polarize monocytes into M2 macrophages, required for tissue repair, which will have significant applications for the treatment of atherosclerosis.

Inflammation plays a fundamental role in the inception and development of atherosclerosis (ATH). Mechanisms of inflammation include the infiltration of monocytes into the injured area and subsequent differentiation into either pro-inflammatory M1 macrophages or anti-inflammatory M2 macrophages. We have previously published data suggesting bone morphogenetic protein-7 (BMP-7) enhances M2 macrophage differentiation and anti-inflammatory cytokine secretion in vitro. In this regard, we hypothesized BMP-7 would inhibit plaque formation in an animal model of ATH through monocytic plasticity mediation. ATH was generated in male and female Apo E-/- mice via partial left carotid artery (PLCA) ligation and mice were divided into 3 groups: Sham, PLCA, and PLCA+BMP-7 (200ug/kg; i.v.). Our data suggest that BMP-7 inhibits plaque formation and increases arterial systolic velocity. Furthermore, we report inhibition of monocyte infiltration and a decrease in associated pro-inflammatory cytokines (MCP-1, TNF-α, and IL-6) in the PLCA+BMP-7 mice. In contrast, our data suggest a significant (p<0.05) increase in M2 macrophage populations with consequential enhanced anti-inflammatory cytokine (IL-1RA, IL-10, and Arginase 1) expression following BMP-7 treatment. We have also observed that mechanisms promoting monocyte into M2 macrophage differentiation by BMP-7 involve the upregulation and activation of the BMP-7 receptor (BMP-7RII). In conclusion, we report that BMP-7 has the potential to mediate cellular plasticity and mitigate the inflammatory immune response, which results in decreased plaque formation and improved blood velocity.

We examined whether factors released from embryonic stem (ES) cells inhibit cardiac and vascular cell apoptosis and stimulate endogenous progenitor cells that enhance neovascularization with improved cardiac function. We generated and transplanted ES-conditioned medium (CM) in the infarcted heart to examine effects on cardiac and vascular apoptosis, activation of endogenous c-kit and FLK-1 +ve cells, and their role in cardiac neovascularization. TUNEL, caspase-3 activity, immunohistochemistry, H&E, and Masson's trichrome stains were used to determine the effect of transplanted ES-CM on cardiac apoptosis and neovascularization. TUNEL staining and caspase-3 activity confirm significantly ( p < 0.05) reduced apoptosis in MI+ES-CM compared with MI+ cell culture medium. Immunohistochemistry demonstrated increased ( p < 0.05, 53%) c-kit +ve and FLK-1 +ve positive cells, as well as increased ( p < 0.05, 67%) differentiated CD31-positive cells in ES-CM groups compared with respective controls. Furthermore, significantly ( p < 0.05) increased coronary artery vessels were observed in ES-CM transplanted hearts compared with control. Heart function was significantly improved following ES-CM transplantation. Next, we observed significantly increased ( p < 0.05) levels of c-kit activation proteins (HGF and IGF-1), anti-apoptosis factors (IGF-1 and total antioxidants), and neovascularization protein (VEGF). In conclusion, we suggest that ES-CM following transplantation in the infarcted heart inhibits apoptosis, activates cardiac endogenous c-kit and FLK-1 +ve cells, and differentiates them into endothelial cells (ECs) that enhances neovascularization with improved cardiac function.

Macrophage polarization is emerging as an important area of research for the development of novel therapeutics to treat inflammatory diseases. Within the current study, the role of Notch1R in macrophage differentiation was investigated as well as downstream effects in THP-1 monocytes cultured in "inflammation mimicry" media. Interference of Notch signaling was achieved using either the pharmaceutical inhibitor DAPT or Notch1R small interfering RNA (siRNA), and Notch1R expression, macrophage phenotypes, and anti- and proinflammatory cytokine expression were evaluated. Data presented show that Notch1R expression on M1 macrophages as well as M1 macrophage differentiation is significantly elevated during cellular stress (P < 0.05). However, under identical culture conditions, interference to Notch signaling via Notch1R inhibition mitigated these results as well as promoted M2 macrophage differentiation. Moreover, when subjected to cellular stress, macrophage secretion of proinflammatory cytokines was significantly heightened (P < 0.05). Importantly, Notch1R inhibition not only diminished proinflammatory cytokine secretion but also enhanced anti-inflammatory protein release (P < 0.05). Our data suggest that Notch1R plays a pivotal role in M1 macrophage differentiation and heightened inflammatory responses. Therefore, we conclude that inhibition of Notch1R and subsequent downstream signaling enhances monocyte to M2 polarized macrophage outcomes and promotes anti-inflammatory mediation during cellular stress.

Inflammation has been implicated as a perpetrator of diabetes and its associated complications. Monocytes, key mediators of inflammation, differentiate into pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages upon infiltration of damaged tissue. However, the inflammatory cell types, which propagate diabetes progression and consequential adverse disorders, remain unclear. The current study was undertaken to assess monocyte infiltration and the role of fibroblast growth factor-9 (FGF-9) on monocyte to macrophage differentiation and cardioprotection in the diabetic infarcted heart. Db/db diabetic mice were assigned to sham, myocardial infarction (MI), and MI+FGF-9 groups. MI was induced by permanent coronary artery ligation and animals were subjected to 2D transthoracic echocardiography two weeks post-surgery. Immunohistochemical and immunoassay results from heart samples collected suggest significantly increased infiltration of monocytes (Mean ± SEM; MI: 2.02% ± 0.23% vs. Sham 0.75% ± 0.07%; p<0.05) and associated pro-inflammatory cytokines (TNF-α, MCP-1, and IL-6), adverse cardiac remodeling (Mean ± SEM; MI: 33% ± 3.04% vs. Sham 2.2% ± 0.33%; p<0.05), and left ventricular dysfunction (Mean ± SEM; MI: 35.4% ± 1.25% vs. Sham 49.19% ± 1.07%; p<0.05) in the MI group. Importantly, treatment of diabetic infarcted myocardium with FGF-9 resulted in significantly decreased monocyte infiltration (Mean ± SEM; MI+FGF-9: 1.39% ± 0.1% vs. MI: 2.02% ± 0.23%; p<0.05), increased M2 macrophage differentiation (Mean ± SEM; MI+FGF-9: 4.82% ± 0.86% vs. MI: 0.85% ± 0.3%; p<0.05) and associated anti-inflammatory cytokines (IL-10 and IL-1RA), reduced adverse remodeling (Mean ± SEM; MI+FGF-9: 11.59% ± 1.2% vs. MI: 33% ± 3.04%; p<0.05), and improved cardiac function (Fractional shortening, Mean ± SEM; MI+FGF-9: 41.51% ± 1.68% vs. MI: 35.4% ± 1.25%; p<0.05). In conclusion, our data suggest FGF-9 possesses novel therapeutic potential in its ability to mediate monocyte to M2 differentiation and confer cardiac protection in the post-MI diabetic heart.