Hypoxia-inducible factor 1 recruits FACT and RNF20/40 to mediate histone ubiquitination and transcriptional activation of target genes.

Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator that mediates cellular adaptation to decreased oxygen availability. HIF-1 recruits chromatin-modifying enzymes leading to changes in histone acetylation, citrullination, and methylation at target genes. Here, we demonstrate that hypoxia-inducible gene expression in estrogen receptor (ER)-positive MCF7 and ER-negative SUM159 human breast cancer cells requires the histone H2A/H2B chaperone facilitates chromatin transcription (FACT) and the H2B ubiquitin ligase RING finger protein 20/40 (RNF20/40). Knockdown of FACT or RNF20/40 expression leads to decreased transcription initiation and elongation at HIF-1 target genes. Mechanistically, FACT and RNF20/40 are recruited to hypoxia response elements (HREs) by HIF-1 and stabilize binding of HIF-1 (and each other) at HREs. Hypoxia induces the monoubiquitination of histone H2B at lysine 120 at HIF-1 target genes in an HIF-1-dependent manner. Together, these findings delineate a cooperative molecular mechanism by which FACT and RNF20/40 stabilize multiprotein complex formation at HREs and mediate histone ubiquitination to facilitate HIF-1 transcriptional activity.

Nitric oxide favours tumour-promoting inflammation through mitochondria-dependent and -independent actions on macrophages.

Production of nitric oxide (NO) has been demonstrated in several malignancies, however its role remains not fully understood, specifically in relation to the metabolic and functional implications that it may have on immune cells participating in tumorigenesis. Here, we show that inducible NO synthase (iNOS) is expressed in cancers of the colon and the prostate, mainly by tumour cells, and NO generation is evidenced by widespread nitrotyrosine (NT) staining in tumour tissue. Furthermore, presence of NT is observed in the majority of tumour-associated macrophages (TAMs), despite low iNOS expression by these cells, suggesting that NO from the tumour microenvironment affects TAMs. Indeed, using a co-culture model, we demonstrate that NO produced by colon and prostate cancer cells is sufficient to induce NT formation in neighbouring macrophages. Moreover, exposure to exogenous NO promotes mitochondria-dependent and -independent changes in macrophages, which orientate their polarity towards an enhanced pro-inflammatory phenotype, whilst decreasing antigen-presenting function and wound healing capacity. Abrogating endogenous NO generation in murine macrophages, on the other hand, decreases their pro-inflammatory phenotype. These results suggest that the presence of NO in cancer may regulate TAM metabolism and function, favouring the persistence of inflammation, impairing healing and subverting adaptive immunity responses.

Enhanced Function of Induced Pluripotent Stem Cell-Derived Endothelial Cells Through ESM1 Signaling.

The mortality rate for (cardio)-vascular disease is one of the highest in the world, so a healthy functional endothelium is of outmost importance against vascular disease. In this study, human induced pluripotent stem (iPS) cells were reprogrammed from 1 ml blood of healthy donors and subsequently differentiated into endothelial cells (iPS-ECs) with typical EC characteristics. This research combined iPS cell technologies and next-generation sequencing to acquire an insight into the transcriptional regulation of iPS-ECs. We identified endothelial cell-specific molecule 1 (ESM1) as one of the highest expressed genes during EC differentiation, playing a key role in EC enrichment and function by regulating connexin 40 (CX40) and eNOS. Importantly, ESM1 enhanced the iPS-ECs potential to improve angiogenesis and neovascularisation in in vivo models of angiogenesis and hind limb ischemia. These findings demonstrated for the first time that enriched functional ECs are derived through cell reprogramming and ESM1 signaling, opening the horizon for drug screening and cell-based therapies for vascular diseases. Therefore, this study showcases a new approach for enriching and enhancing the function of induced pluripotent stem (iPS) cell-derived ECs from a very small amount of blood through ESM1 signaling, which greatly enhances their functionality and increases their therapeutic potential. Stem Cells 2019;37:226-239.

Quaking Is a Key Regulator of Endothelial Cell Differentiation, Neovascularization, and Angiogenesis.

The capability to derive endothelial cell (ECs) from induced pluripotent stem cells (iPSCs) holds huge therapeutic potential for cardiovascular disease. This study elucidates the precise role of the RNA-binding protein Quaking isoform 5 (QKI-5) during EC differentiation from both mouse and human iPSCs (hiPSCs) and dissects how RNA-binding proteins can improve differentiation efficiency toward cell therapy for important vascular diseases. iPSCs represent an attractive cellular approach for regenerative medicine today as they can be used to generate patient-specific therapeutic cells toward autologous cell therapy. In this study, using the model of iPSCs differentiation toward ECs, the QKI-5 was found to be an important regulator of STAT3 stabilization and vascular endothelial growth factor receptor 2 (VEGFR2) activation during the EC differentiation process. QKI-5 was induced during EC differentiation, resulting in stabilization of STAT3 expression and modulation of VEGFR2 transcriptional activation as well as VEGF secretion through direct binding to the 3' UTR of STAT3. Importantly, mouse iPS-ECs overexpressing QKI-5 significantly improved angiogenesis and neovascularization and blood flow recovery in experimental hind limb ischemia. Notably, hiPSCs overexpressing QKI-5, induced angiogenesis on Matrigel plug assays in vivo only 7 days after subcutaneous injection in SCID mice. These results highlight a clear functional benefit of QKI-5 in neovascularization, blood flow recovery, and angiogenesis. Thus, they provide support to the growing consensus that elucidation of the molecular mechanisms underlying EC differentiation will ultimately advance stem cell regenerative therapy and eventually make the treatment of cardiovascular disease a reality. The RNA binding protein QKI-5 is induced during EC differentiation from iPSCs. RNA binding protein QKI-5 was induced during EC differentiation in parallel with the EC marker CD144. Immunofluorescence staining showing that QKI-5 is localized in the nucleus and stained in parallel with CD144 in differentiated ECs (scale bar = 50 µm). Stem Cells 2017 Stem Cells 2017;35:952-966.

Biological and structural analyses of bovine heparin fractions of intermediate and high molecular weight.

Low molecular weight heparin, which is generally obtained by chemical and enzymatic depolymerization of unfractionated heparin, has high bioavailability and can be subcutaneously injected. The aim of the present investigation was to fractionate bovine heparin using a physical method (ultrafiltration through a 10kDa cut-off membrane), avoiding structural modifications that can be caused by chemical or enzymatic treatments. Two fractions with different molecular weights were obtained: the first had an intermediate molecular weight (B-IMWH; Mn=9587Da) and the other had a high molecular weight (B-HMWH; 22,396Da). B-IMWH and B-HMWH have anticoagulant activity of 103 and 154IU/mg respectively, which could be inhibited by protamine. Both fractions inhibited α-thrombin and factor Xa in vitro and showed antithrombotic effect in vivo. Moreover, ex vivo aPTT assay demonstrated that B-IMWH is absorbed by subcutaneous route. The results showed that ultrafiltration can be used to obtain two bovine heparin fractions, which differ on their molecular weights, structural components, anticoagulant potency, and administration routes.

Metabolic switches during the first steps of adipogenic stem cells differentiation.

The understanding of metabolism during cell proliferation and commitment provides a greater insight into the basic biology of cells, allowing future applications. Here we evaluated the energy and oxidative changes during the early adipogenic differentiation of human adipose tissue-derived stromal cells (hASCs). hASCs were maintained under differentiation conditions during 3 and 7days. Oxygen consumption, mitochondrial mass and membrane potential, reactive oxygen species (ROS) generation, superoxide dismutase (SOD) and catalase activities, non-protein thiols (NPT) concentration and lipid peroxidation were analyzed. We observed that 7days of adipogenic induction are required to stimulate cells to consume more oxygen and increase mitochondrial activity, indicating organelle maturation and a transition from glycolytic to oxidative energy metabolism. ROS production was only increased after 3days and may be involved in the differentiation commitment. ROS source was not only the mitochondria and we suggest that NOX proteins are related to ROS generation and therefore adipogenic commitment. ROS production did not change after 7days, but an increased activity of catalase and NPT concentration as well as a decreased lipid peroxidation were observed. Thus, a short period of differentiation induction is able to change the energetic and oxidative metabolic profile of hASCs and stimulate cytoprotection processes.