Hypoxia-inducible factor 1α (HIF1α) 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 HIF1α may ameliorate the negative aspects of the obesity-associated fat pad expansion. We used the selective HIF1α 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 HIF1α activation in adipose tissue. HIF1α 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 HIF1α mutant (dn-HIF1α). Consistent with the pharmacological results, genetic inhibition of endogenous HIF1α activity prompts similar metabolic improvements in HFD-fed mice. Collectively, our results demonstrate that HIF1α inhibition in the adipocyte leads to significant metabolic improvements, suggesting that selective HIF1α inhibition in adipose tissue may be an effective therapeutic avenue in the context of metabolic dysfunction.

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-1α, 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.

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 IFNs. 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 with control mice, and splenocytes from tumor-bearing mice exhibited reduced phosphorylation of STAT1 (P-STAT1) on Tyr⁷⁰¹ in response to IFN-α or IFN-γ. This inhibition was seen in splenic CD4⁺ and CD8⁺ T cells as well as natural killer cells. In vitro coculture 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. In vitro treatment of splenocytes with a nitric oxide donor led to a decreased STAT1 IFN response. The elevation in nitric oxide 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 with control C26 tumor-bearing mice. These data suggest that nitric oxide produced by MDSC can lead to reduced IFN responsiveness in immune cells. Cancer Res; 71(15); 5101–10. ©2011 AACR.

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]