Cell therapy improves cardiac function. Few cells have been investigated more extensively or consistently shown to be more effective than c-kit sorted cells; however, c-kit expression is easily lost during passage. Here, our primary goal was to develop an improved method to isolate c-kitpos cells and maintain c-kit expression after passaging. Cardiac mesenchymal cells (CMCs) from wild-type mice were selected by polystyrene adherence properties. CMCs adhering within the first hours are referred to as rapidly adherent (RA); CMCs adhering subsequently are dubbed slowly adherent (SA). Both RA and SA CMCs were c-kit sorted. SA CMCs maintained significantly higher c-kit expression than RA cells; SA CMCs also had higher expression endothelial markers. We subsequently tested the relative efficacy of SA versus RA CMCs in the setting of post-infarct adoptive transfer. Two days after coronary occlusion, vehicle, RA CMCs, or SA CMCs were delivered percutaneously with echocardiographic guidance. SA CMCs, but not RA CMCs, significantly improved cardiac function compared to vehicle treatment. Although the mechanism remains to be elucidated, the more pronounced endothelial phenotype of the SA CMCs coupled with the finding of increased vascular density suggest a potential pro-vasculogenic action. This new method of isolating CMCs better preserves c-kit expression during passage. SA CMCs, but not RA CMCs, were effective in reducing cardiac dysfunction. Although c-kit expression was maintained, it is unclear whether maintenance of c-kit expression per se was responsible for improved function, or whether the differential adherence property itself confers a reparative phenotype independently of c-kit.

Despite the well-documented capacity of embryonic stem cells (ESCs) to differentiate into cardiomyocytes, transplantation of ESCs or ESC-derived cells is plagued by several formidable problems, including graft rejection, arrhythmias, and potential risk of teratomas. Life-long immunosuppression is a disease in itself. Transplantation of human ESC-derived cells in primates causes life-threatening arrhythmias, and the doses used to show efficacy are not clinically relevant. In contemporary clinical research, the margin of tolerance for such catastrophic effects as malignancies is zero, and although the probability of tumours can be reduced by ESC differentiation, it is unlikely to be completely eliminated, particularly when billions of cells are injected. Although ESCs and ESC-derived cells were touted as capable of long-term regeneration, these cells disappear rapidly after transplantation and there is no evidence of long-term engraftment, let alone regeneration. There is, however, mounting evidence that they act via paracrine mechanisms—just like adult cells. To date, no controlled clinical trial of ESC-derived cells in cardiovascular disease has been conducted or even initiated. In contrast, adult cells have been used in thousands of patients with heart disease, with no significant adverse effects and with results that were sufficiently encouraging to warrant Phase II and III trials. Furthermore, induced pluripotent stem cells offer pluripotency similar to ESCs without the need for lifelong immunosuppression. After two decades, the promise that ESC-derived cells would regenerate dead myocardium has not been fulfilled. The most reasonable interpretation of current data is that ESC-based therapies are not likely to have clinical application for heart disease.

Rhabdomyosarcoma (RMS) is the most common soft‐tissue sarcoma of adolescence and childhood. Because RMS tumors are highly vascularized, we sought to determine which factors secreted by RMS cells are crucial in stimulating angiogenesis in response to hypoxia. To address this issue, we evaluated expression of several proangiogenic factors [interleukin (IL)‐8, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)‐2, stromal‐derived factor (SDF)‐1, hepatocyte growth factor (HGF) and leukemia inhibitory factor (LIF)] in 8 human RMS cell lines in both normal steady‐state and hypoxic conditions. We found by real‐time quantitative polymerase chain reaction (RQ‐PCR) and confirmed by enzyme‐linked immunosorbent assay (ELISA) that from all the factors evaluated, IL‐8, whose expression is very low in normoxia, had been very highly expressed and secreted by RMS cells lines during hypoxic conditions (∼40–170 times). Interestingly, this upregulation was not affected by knocking down hypoxia‐inducible factor (HIF)‐1α, but was inhibited by mitogen‐activated protein kinase (MAPK)p42/44 and phosphatidylinositaol 3‐kinase (PI3K)/AKT pathway inhibitors. This suggests that IL‐8 expression is regulated in an activating protein (AP)‐1‐ and nuclear factor (NF)‐κB‐dependent manner. Furthermore, we found that conditioned media (CM) harvested from RMS cells exposed to hypoxia activated and stimulated chemotactic responses in human umbilical vein endothelial cells (HUVECs) and that IL‐8 was responsible for hypoxia‐related effects. Finally, by employing shRNA, the expression of IL‐8 in human RH‐30 cells was downregulated. We noticed that such RMS cells, if injected into skeletal muscles of immunodeficient mice, have a reduced ability for tumor formation. We conclude that IL‐8 is a pivotal proangiogenic factor released by human RMS cells in hypoxic conditions and that the targeting of IL‐8 may prove to be a novel and efficient strategy for inhibiting RMS growth.

We observed that human rhabdomyosarcoma (RMS) cells highly express a tissue factor that promotes thrombin formation, which indirectly and directly affects RMS progression. First, we found that thrombin activates platelets to generate microvesicles (PMV), which transfer to RMS cells' α2β3 integrin and increase their adhesiveness to endothelial cells. Accordingly, RMS cells covered with PMVs showed higher metastatic potential after i.v. injection into immunodeficient mice. Furthermore, PMVs activate mitogen-activated protein kinase (MAPK)p42/44 and AKT to chemoattract RMS cells. We also found that RMS cells express functional protease-activated receptor-1 (PAR1) and PAR3 and respond to thrombin stimulation by MAPKp42/44 and MAPKp38 phosphorylation. To our surprise, thrombin did not affect RMS proliferation or survival; it inhibited the chemotactic and adhesive properties of RMS cells. However, when PAR1-specific agonist thrombin receptor–activating peptide 6 was used, which does not activate PAR3, selective PAR1 stimulation enhanced RMS proliferation. To learn more on the role of PAR1 and PAR3 antagonism in RMS proliferation and metastasis, we knocked down both receptors by using a short hairpin RNA strategy. We found that although thrombin does not affect growth of PAR1^−/− cells, it stimulated the proliferation of PAR3^−/− cells. More importantly, PAR3^−/− cells, in contrast to PAR1^−/− ones, formed larger tumors in immunodeficient mice. We conclude that thrombin is a novel underappreciated modulator of RMS metastasis and that we have identified a novel role for PAR3 in thrombin signaling. Mol Cancer Res; 8(5); 677–90. ©2010 AACR.