Agata Szade

Cellular and molecular mechanisms of inflammation‐induced angiogenesis

Blood vessel formation is a fundamental process for the development of organism and tissue regeneration. Of importance, angiogenesis occurring during postnatal development is usually connected with inflammation. Here, we review how molecular and cellular mechanisms underlying inflammatory reactions regulate angiogenesis. Inflamed tissues are characterized by hypoxic conditions and immune cell infiltration. In this review, we describe an interplay of hypoxia‐inducible factors (HIFs), HIF1 and HIF2, as well as NF‐κB and nitric oxide in the regulation of angiogenesis. The mobilization of macrophages and the differential role of M1 and M2 macrophage subsets in angiogenesis are also discussed. Next, we present the current knowledge about microRNA regulation of inflammation in the context of new blood vessel formation. Finally, we describe how the mechanisms involved in inflammation influence tumor angiogenesis. We underlay and discuss the role of NF‐E2‐related factor 2/heme oxygenase‐1 pathway as crucial in the regulation of inflammation‐induced angiogenesis.

Murine Bone Marrow Lin−Sca-1+CD45− Very Small Embryonic-Like (VSEL) Cells Are Heterogeneous Population Lacking Oct-4A Expression

Murine very small embryonic-like (VSEL) cells, defined by the Lin−Sca-1+CD45− phenotype and small size, were described as pluripotent cells and proposed to be the most primitive hematopoietic precursors in adult bone marrow. Although their isolation and potential application rely entirely on flow cytometry, the immunophenotype of VSELs has not been extensively characterized. Our aim was to analyze the possible heterogeneity of Lin−Sca+CD45− population and investigate the extent to which VSELs characteristics may overlap with that of hematopoietic stem cells (HSCs) or endothelial progenitor cells (EPCs). The study evidenced that murine Lin−Sca-1+CD45− population was heterogeneous in terms of c-Kit and KDR expression. Accordingly, the c-Kit+KDR−, c-Kit−KDR+, and c-Kit−KDR− subpopulations could be distinguished, while c-Kit+KDR+ events were very rare. The c-Kit+KDR− subset contained almost solely small cells, meeting the size criterion of VSELs, in contrast to relatively bigger c-Kit−KDR+ cells. The c-Kit−KDR−FSClow subset was highly enriched in Annexin V-positive, apoptotic cells, hence omitted from further analysis. Importantly, using qRT-PCR, we evidenced lack of Oct-4A and Oct-4B mRNA expression either in whole adult murine bone marrow or in the sorted of Lin−Sca-1+CD45−FSClow population, even by single-cell qRT-PCR. We also found that the Lin−Sca-1+CD45−c-Kit+ subset did not exhibit hematopoietic potential in a single cell-derived colony in vitro assay, although it comprised the Sca-1+c-Kit+Lin− (SKL) CD34−CD45−CD105+ cells, expressing particular HSC markers. Co-culture of Lin−Sca-1+CD45−FSClow with OP9 cells did not induce hematopoietic potential. Further investigation revealed that SKL CD45−CD105+ subset consisted of early apoptotic cells with fragmented chromatin, and could be contaminated with nuclei expelled from erythroblasts. Concluding, murine bone marrow Lin−Sca-1+CD45−FSClow cells are heterogeneous population, which do not express the pluripotency marker Oct-4A. Despite expression of some hematopoietic markers by a Lin−Sca-1+CD45−c-Kit+KDR− subset of VSELs, they do not display hematopoietic potential in a clonogenic assay and are enriched in early apoptotic cells.

Heme Oxygenase-1 Controls an HDAC4-miR-206 Pathway of Oxidative Stress in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is an aggressive soft tissue cancer characterized by disturbed myogenic differentiation. Here we report a role for the oxidative stress response factor HO-1 in progression of RMS. We found that HO-1 was elevated and its effector target miR-206 decreased in RMS cell lines and clinical primary tumors of the more aggressive alveolar phenotype (aRMS). In embryonal RMS (eRMS), HO-1 expression was induced by Pax3/7-FoxO1, an aRMS hallmark oncogene, followed by a drop in miR-206 levels. Inhibition of HO-1 by tin protoporphyrin (SnPP) or siRNA downregulated Pax3/7-FoxO1 target genes and induced a myogenic program in RMS. These effects were not mediated by altered myoD expression; instead, cells with elevated HO-1 produced less reactive oxygen species, resulting in nuclear localization of HDAC4 and miR-206 repression. HO-1 inhibition by SnPP reduced growth and vascularization of RMS tumors in vivo accompanied by induction of miR-206. Effects of SnPP on miR-206 expression and RMS tumor growth were mimicked by pharmacologic inhibition of HDAC. Thus, HO-1 inhibition activates an miR-206-dependent myogenic program in RMS, offering a novel therapeutic strategy for treatment of this malignancy. Cancer Res; 76(19); 5707-18. ©2016 AACR.

Effect of crossing C57BL/6 and FVB mouse strains on basal cytokine expression.

C57BL/6 is the most often used laboratory mouse strain. However, sometimes it is beneficial to cross the transgenic mice on the C57BL/6 background to the other strain, such as FVB. Although this is a common strategy, the influence of crossing these different strains on homeostatic expression of cytokines is not known. Here we have investigated the differences in the expression of selected cytokines between C57BL/6J and C57BL/6JxFVB mice in serum and skeletal muscle. We have found that only few cytokines were altered by crossing of the strains. Concentrations of IL5, IL7, LIF, MIP-2, and IP-10 were higher in serum of C57BL/6J mice than in C57BL/6JxFVB mice, whereas concentration of G-CSF was lower in C57BL/6J. In the skeletal muscle only the concentration of VEGF was higher in C57BL/6J mice than in C57BL/6JxFVB mice. Concluding, the differences in cytokine expression upon crossing C57BL/6 and FVB strain in basal conditions are not profound.

Spheroid-plug model as a tool to study tumor development, angiogenesis, and heterogeneity in vivo

Subcutaneous injection of the tumor cell suspension is a simple and commonly used tool for studying tumor development in vivo. However, subcutaneous models poorly resemble tumor complexity due to the fast growth not reflecting the natural course. Here, we describe an application of the new spheroid-plug model to combine the simplicity of subcutaneous injection with improved resemblance to natural tumor progression. Spheroid-plug model relies on in vitro formation of tumor spheroids, followed by injection of single tumor spheroid subcutaneously in Matrigel matrix. In spheroid-plug model, tumors grow slower in comparison to tumors formed by injection of cell suspension as assessed by 3D ultrasonography (USG) and in vivo bioluminescence measurements. The slower tumor growth rate in spheroid-plug model is accompanied by reduced necrosis. The spheroid-plug model ensures increased and more stable vascularization of tumor than classical subcutaneous tumor model as demonstrated by 3D USG Power Doppler examination. Flow cytometry analysis showed that tumors formed from spheroids have enhanced infiltration of endothelial cells as well as hematopoietic and progenitor cells with stem cell phenotype (c-Kit+ and Sca-1+). They also contain more tumor cells expressing cancer stem cell marker CXCR4. Here, we show that spheroid-plug model allows investigating efficiency of anticancer drugs. Treatment of spheroid-plug tumors with known antiangiogenic agent axitinib decreased their size and viability. The antiangiogenic activity of axitinib was higher in spheroid-plug model than in classical model. Our results indicate that spheroid-plug model imitates natural tumor growth and can become a valuable tool for cancer research.

Heme oxygenase‐1 affects generation and spontaneous cardiac differentiation of induced pluripotent stem cells

Cellular stress can influence efficiency of iPSCs generation and their differentiation. However, the role of intracellular cytoprotective factors in these processes is still not well known. Therefore, we investigated the effect of HO‐1 (Hmox1) or Nrf2 (Nfe2l2), two major cytoprotective genes. Hmox1–/– fibroblasts demonstrated decreased reprogramming efficiency in comparison to Hmox1+/+ cells. Reversely, pharmacological enhancement of HO‐1 resulted in higher number of iPSCs colonies. Importantly, elevated level of both p53 and p53‐regulated miR‐34a and 14‐3‐3σ was observed in HO‐1‐deficient fibroblasts whereas downregulation of p53 in these cells markedly increased their reprogramming efficiency. In human fibroblasts HO‐1 silencing also induced p53 expression and affected reprogramming outcome. Hmox1+/+ and Hmox1–/– iPSCs similarly differentiated in vitro to cells originating from three germ layers, however, lower number of contracting cells was observed during this process in HO‐1‐deficient cells indicating attenuated cardiac differentiation. Importantly, silencing of Hmox1 in murine ESC using CRISPR/Cas‐9 editing also impaired their spontaneous cardiac differentiation. Decreased reprogramming efficiency was also observed in Nrf2‐lacking fibroblasts. Reversely, sulforaphane, a Nrf2 activator, increased the number of iPSCs colonies. However, both Nfe2l2+/+ and Nfe2l2–/– iPSCs showed similar pluripotency and differentiation capacity. These results indicate that regulation of HO‐1 expression can further optimize generation and cardiac differentiation of iPSCs.

Heme oxygenase 1 affects granulopoiesis in mice through control of myelocyte proliferation

Heme oxygenase-1 (HO-1) is stress-inducible, cytoprotective enzyme degrading heme to carbon monoxide (CO), biliverdin and Fe2+. We showed that HO-1 knock-out mice (HO-1-/-) have a twofold higher level of granulocytes than wild type (WT) mice, despite decreased concentration of granulocyte colony-stimulating factor (G-CSF) in the blood and reduced surface expression of G-CSF receptor on the hematopoietic precursors. This suggests the effect of HO-1 on granulopoiesis. Here we aimed to determine the stage of granulopoiesis regulated by HO-1. The earliest stages of hematopoiesis were not biased toward myeloid differentiation in HO-1-/- mice. Within committed granulocytic compartment, in WT mice, HO-1 was up-regulated starting from myelocyte stage. This was concomitant with up-regulation of miR-155, which targets Bach1, the HO-1 repressor. In HO-1-/- mice granulopoiesis was accelerated between myelocyte and metamyelocyte stage. There was a higher fraction of proliferating myelocytes, with increased nuclear expression of pro-proliferative C/EBPβ (CCAAT/enhancer binding protein beta) protein, especially its active LAP (liver-enriched activator proteins) isoform. Also our mathematical model confirmed shortening the myelocyte cyclic-time and prolonged mitotic expansion in absence of HO-1. It seems that changes in C/EBPβ expression and activity in HO-1-/- myelocytes can be associated with reduced level of its direct repressor miR-155 or with decreased concentration of CO, known to reduce nuclear translocation of C/EBPs. Mature HO-1-/- granulocytes were functionally competent as determined by oxidative burst capacity. In conclusion, HO-1 influences granulopoiesis through regulation of myelocyte proliferation. It is accompanied by changes in expression of transcriptionally active C/EBPβ protein. As HO-1 expression vary in human and is up-regulated in response to chemotherapy, it can potentially influence chemotherapy-induced neutropenia.

Reprint of: Heme oxygenase 1 affects granulopoiesis in mice through control of myelocyte proliferation

Heme oxygenase-1 (HO-1) is stress-inducible, cytoprotective enzyme degrading heme to carbon monoxide (CO), biliverdin and Fe2+. We showed that HO-1 knock-out mice (HO-1-/-) have a twofold higher level of granulocytes than wild type (WT) mice, despite decreased concentration of granulocyte colony-stimulating factor (G-CSF) in the blood and reduced surface expression of G-CSF receptor on the hematopoietic precursors. This suggests the effect of HO-1 on granulopoiesis. Here we aimed to determine the stage of granulopoiesis regulated by HO-1. The earliest stages of hematopoiesis were not biased toward myeloid differentiation in HO-1-/- mice. Within committed granulocytic compartment, in WT mice, HO-1 was up-regulated starting from myelocyte stage. This was concomitant with up-regulation of miR-155, which targets Bach1, the HO-1 repressor. In HO-1-/- mice granulopoiesis was accelerated between myelocyte and metamyelocyte stage. There was a higher fraction of proliferating myelocytes, with increased nuclear expression of pro-proliferative C/EBPβ (CCAAT/enhancer binding protein beta) protein, especially its active LAP (liver-enriched activator proteins) isoform. Also our mathematical model confirmed shortening the myelocyte cyclic-time and prolonged mitotic expansion in absence of HO-1. It seems that changes in C/EBPβ expression and activity in HO-1-/- myelocytes can be associated with reduced level of its direct repressor miR-155 or with decreased concentration of CO, known to reduce nuclear translocation of C/EBPs. Mature HO-1-/- granulocytes were functionally competent as determined by oxidative burst capacity. In conclusion, HO-1 influences granulopoiesis through regulation of myelocyte proliferation. It is accompanied by changes in expression of transcriptionally active C/EBPβ protein. As HO-1 expression vary in human and is up-regulated in response to chemotherapy, it can potentially influence chemotherapy-induced neutropenia.

Erratum to: Spheroid-plug model as a tool to study tumor development, angiogenesis, and heterogeneity in vivo

1 Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland 2 Centre for Molecular Biophysics, Cell Recognition and Glycobiology, UPR4301-CNRS, Orleans, France 3 Malopolska Centre of Biotechnology, Krakow, Poland 4 Present address: Skaggs School of Pharmacy and Pharmaceutical Sciences, Laboratory of Bioresponsive Materials, University of California, San Diego, CA, USA Tumor Biol. (2016) 37:12901 DOI 10.1007/s13277-016-5099-6