Hypoxia-inducible factor 1␣ (HIF1␣) induction in adipocytes is a critical component of the "fibrotic response," directly linked to metabolic dysfunction in adipose tissues under hypoxic conditions. We reasoned that inhibition of HIF1␣ may ameliorate the negative aspects of the obesity-associated fat pad expansion. We used the selective HIF1␣ inhibitor PX-478, whose effectiveness has previously been established in tumor models. We demonstrate that PX-478 treatment effectively suppresses the high-fat-diet (HFD)-induced HIF1␣ activation in adipose tissue. HIF1␣ inhibition causes a reduction of weight gain in mice on an HFD but not on a chow diet. Treatment increases energy expenditure and prompts resistance to HFD-mediated deterioration of metabolic parameters. Moreover, PX-478-treated mice have reduced fibrosis and fewer inflammatory infiltrates in their adipose tissues. We confirm the metabolic effects obtained with PX-478 treatment using an adipose tissue-specific, doxycycline-inducible dominant negative HIF1␣ mutant (dn-HIF1␣). Consistent with the pharmacological results, genetic inhibition of endogenous HIF1␣ activity prompts similar metabolic improvements in HFD-fed mice. Collectively, our results demonstrate that HIF1␣ inhibition in the adipocyte leads to significant metabolic improvements, suggesting that selective HIF1␣ inhibition in adipose tissue may be an effective therapeutic avenue in the context of metabolic dysfunction.

Our group and others have determined that immune effector cells from patients with advanced cancers exhibit reduced activation of IFN signaling pathways. We hypothesized that increases in immune regulatory cells termed myeloid derived suppressor cells (MDSC) could interfere with the host immune response to tumors by inhibiting immune cell responsiveness to interferons. The C26 murine adenocarcinoma model was employed to study immune function in advanced malignancy. C26 bearing mice had significantly elevated levels of GR1+CD11b+ MDSC as compared to control mice, and splenocytes from tumor bearing mice exhibited reduced phosphorylation of STAT1 (P-STAT1) on Tyr 701 in response to IFN alpha or IFN gamma. This inhibition was seen in splenic CD4+ and CD8+ T cells as well as NK cells. In vitro co culture experiments revealed that MDSC inhibited the IFN responsiveness of splenocytes from normal mice. Treatment of C26 bearing mice with gemcitabine or an anti-GR1 antibody led to depletion of MDSC and restored splenocyte IFN responsiveness. Spleens from C26 bearing animals displayed elevated levels of iNOS protein and nitric oxide (NO). In vitro treatment of splenocytes with a nitric oxide donor led to a decreased STAT1 IFN response. The elevation in NO in C26 bearing mice was associated with increased levels of nitration on STAT1. Finally, splenocytes from iNOS knockout mice bearing C26 tumors exhibited a significantly elevated IFN-response as compared to control C26 tumor bearing mice. These data suggest that NO produced by MDSC can lead to reduced interferon responsiveness in immune cells.

Tumor-educated macrophages facilitate tumor metastasis and angiogenesis. We discovered that granulocyte macrophage colony-stimulating factor (GM-CSF) blocked macrophages vascular endothelial growth factor (VEGF) activity by producing soluble VEGF receptor-1 (sVEGFR-1) and determined the effect on tumor-associated macrophage behavior and tumor growth. We show GM-CSF treatment of murine mammary tumors slowed tumor growth and slowed metastasis. These tumors had more macrophages, fewer blood vessels, and lower oxygen concentrations. This effect was sVEGFR-1 dependent. In situ hybridization and flow cytometry identified macrophages as the primary source of sVEGFR-1. These data suggest that GM-CSF can re-educate macrophages to reduce angiogenesis and metastases in murine breast cancer. [Cancer Res 2009;69(5):2133-40]

preconditioning induces the expression of prosurvival and proangiogenic markers in mesenchymal stem cells. Am J Physiol Cell Physiol 299: C1562-C1570, 2010. First published September 22, 2010; doi:10.1152/ajpcell.00221.2010.-Stem cells transplanted to the ischemic myocardium usually encounter massive cell death within a few days of therapy. Hypoxic preconditioning (HPC) is currently employed as a strategy to prepare stem cells for increased survival and engraftment in the heart. However, HPC of stem cells has provided varying results, supposedly due to the differences in the oxygen concentration, duration of exposure, and passage conditions. In the present study, we determined the effect of HPC on rat mesenchymal stem cells (MSCs) exposed to 0.5% oxygen concentration for 24, 48, or 72 h. We evaluated the expression of prosurvival, proangiogenic, and functional markers such as hypoxia-inducible factor-1␣, VEGF, phosphorylated Akt, survivin, p21, cytochrome c, caspase-3, caspase-7, CXCR4, and c-Met. MSCs exposed to 24-h hypoxia showed reduced apoptosis on being subjected to severe hypoxic conditions. They also had significantly higher levels of prosurvival, proangiogenic, and prodifferentiation proteins when compared with longer exposure (72 h). Cells taken directly from the cryopreserved state did not respond effectively to the 24-h HPC as those that were cultured under normoxia before HPC. Cells cultured under normoxia before HPC showed decreased apoptosis, enhanced expression of connexin-43, cardiac myosin heavy chain, and CD31. The preconditioned cells were able to differentiate into the cardiovascular lineage. The results suggest that MSCs cultured under normoxia before 24-h HPC are in a state of optimal expression of prosurvival, proangiogenic, and functional proteins that may increase the survival and engraftment in the infarct heart. These results could provide further insights into optimal preparation of MSCs which would greatly influence the effectiveness of cell therapy in vivo. myocardial infarction; cell therapy THE OXYGEN CONCENTRATION in the microenvironment of stem cells plays an important role in controlling stem cell potency, proliferation, and differentiation ability (1,26,28). Stem cells that are normally cultured at ambient air in an in vitro environment differ in their exposure to the concentration of oxygen, compared with their natural physiological niches where they reside and function (7, 9). During cell transplantation procedures to treat conditions such as myocardial infarction (MI), the cultured stem cells encounter a sudden shortage in oxygen availability when transplanted into an ischemic heart tissue. A growing body of evidence attributes failure of stem cell therapy to the extensive loss of transplanted stem cells upon introducing them to such a harsh ischemic environment, which is high in inflammation factors and free radicals generated by oxidative stress (14, 38). Varying the oxygen exposure level while culturing the stem cells may play a major role in determining the survival of...

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The generation of reactive oxygen species and mitochondrial dysfunction has been implicated in doxorubicin (DOX)-induced cardiotoxicity. The aim of the present study was to determine whether Spirulina, a blue-green algae, could serve as a cardioprotective agent during DOX treatment in a mouse model. Mice were treated with DOX (4 mg/kg bw, intraperitoneally), weekly, for 4 weeks. Spirulina was administered orally for 3 days twice daily, then for 7 weeks along with the four equal injections of DOX. Cardiotoxicity was assessed, at 3 weeks after the end of the DOX-treatment period, by mortality, volume of ascites, liver congestion, oxidative stress and ultrastructural changes of heart tissue. The DOX-treated animals showed higher mortality (53%) and more ascites. Myocardial damage, as assessed by ultrastructural changes, showed loss of myofibrils, cytoplasmic vacuolization and mitochondrial swelling. Myocardial superoxide dismutase and glutathione peroxidase activities were decreased and lipid peroxidation was increased. Pretreatment with Spirulina significantly protected the mice from DOX-induced cardiotoxic effects as evidenced from lower mortality (26%), less ascites, lower levels of lipid peroxidation, normalization of antioxidant enzymes and ultrastructural studies showing minimal damage to the heart. In vitro cytotoxic studies using ovarian cancer cells demonstrated that Spirulina did not compromise the anti-tumor activity of doxorubicin. These results suggest that Spirulina has a protective effect against cardiotoxicity induced by DOX and it may, therefore, improve the therapeutic index of DOX.

Chronic HF causes alterations in ion channel expression and ion currents, resulting in attenuation of the APD and atrial contractility and a substrate for persistent AF.

IntroductionDilated cardiomyopathy is a major cause of progressive heart failure. Utilization of stem cell therapy offers a potential means of regenerating viable cardiac tissue. However, a major obstacle to stem cell therapy is the delivery and survival of implanted stem cells in the ischemic heart. To address this issue, we have developed a biomimetic aligned nanofibrous cardiac patch and characterized the alignment and function of human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) cultured on this cardiac patch. This hiPSC-CMs seeded patch was compared with hiPSC-CMs cultured on standard flat cell culture plates.MethodshiPSC-CMs were cultured on; 1) a highly aligned polylactide-co-glycolide (PLGA) nanofiber scaffold (~50 microns thick) and 2) on a standard flat culture plate. Scanning electron microscopy (SEM) was used to determine alignment of PLGA nanofibers and orientation of the cells on the respective surfaces. Analysis of gap junctions (Connexin-43) was performed by confocal imaging in both the groups. Calcium cycling and patch-clamp technique were performed to measure calcium transients and electrical coupling properties of cardiomyocytes.ResultsSEM demonstrated >90% alignment of the nanofibers in the patch which is similar to the extracellular matrix of decellularized rat myocardium. Confocal imaging of the cardiomyocytes demonstrated symmetrical alignment in the same direction on the aligned nanofiber patch in sharp contrast to the random appearance of cardiomyocytes cultured on a tissue culture plate. The hiPSC-CMs cultured on aligned nanofiber cardiac patches showed more efficient calcium cycling compared with cells cultured on standard flat surface culture plates. Quantification of mRNA with qRT-PCR confirmed that these cardiomyocytes expressed α-actinin, troponin-T and connexin-43 in-vitro.ConclusionsOverall, our results demonstrated changes in morphology and function of human induced pluripotent derived cardiomyocytes cultured in an anisotropic environment created by an aligned nanofiber patch. In this environment, these cells better approximate normal cardiac tissue compared with cells cultured on flat surface and can serve as the basis for bioengineering of an implantable cardiac patch.