Neutrophils for Revascularization Require Activation of CCR6 and CCL20 by TNFα.

BACKGROUND: Activation of immune-inflammatory pathways involving TNFα (tumor necrosis factor alpha) signaling is critical for revascularization and peripheral muscle tissue repair after ischemic injury. However, mechanisms of TNFα-driven inflammatory cascades directing recruitment of proangiogenic immune cells to sites of ischemia are unknown. METHODS: Muscle tissue revascularization after permanent femoral artery ligation was monitored in mutant mice by laser Doppler imaging and light sheet fluorescence microscopy. TNFα-mediated signaling and the role of the CCL20 (C-C motif chemokine ligand 20)-CCR6 (C-C chemokine receptor 6) axis for formation of new vessels was studied in vitro and in vivo using bone marrow transplantation, flow cytometry, as well as biochemical and molecular biological techniques. RESULTS: TNFα-mediated activation of TNFR (tumor necrosis factor receptor) 1 but not TNFR2 was found to be required for postischemic muscle tissue revascularization. Bone marrow-derived CCR6+ neutrophil granulocytes were identified as a previously undescribed TNFα-induced population of proangiogenic neutrophils, characterized by increased expression of VEGFA (vascular endothelial growth factor A). Mechanistically, postischemic activation of TNFR1 induced expression of the CCL20 in vascular cells and promoted translocation of the CCL20 receptor CCR6 to the cell surface of neutrophils, essentially conditioning VEGFA-expressing proangiogenic neutrophils for CCL20-dependent recruitment to sites of ischemia. Moreover, impaired revascularization of ischemic peripheral muscle tissue in diabetic mice was associated with reduced numbers of proangiogenic neutrophils and diminished CCL20 expression. Administration of recombinant CCL20 enhanced recruitment of proangiogenic neutrophils and improved revascularization of diabetic ischemic skeletal muscles, which was sustained by sequential treatment with fluvastatin. CONCLUSIONS: We demonstrate that site-specific activation of the CCL20-CCR6 axis via TNFα recruits proangiogenic VEGFA-expressing neutrophils to sites of ischemic injury for initiation of muscle tissue revascularization. The findings provide an attractive option for tissue revascularization, particularly under diabetic conditions.

Concomitant Activation of OSM and LIF Receptor by a Dual-Specific hlOSM Variant Confers Cardioprotection after Myocardial Infarction in Mice.

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) signaling protects the heart after myocardial infarction (MI). In mice, oncostatin M receptor (OSMR) and leukemia inhibitory factor receptor (LIFR) are selectively activated by the respective cognate ligands while OSM activates both the OSMR and LIFR in humans, which prevents efficient translation of mouse data into potential clinical applications. We used an engineered human-like OSM (hlOSM) protein, capable to signal via both OSMR and LIFR, to evaluate beneficial effects on cardiomyocytes and hearts after MI in comparison to selective stimulation of either LIFR or OSMR. Cell viability assays, transcriptome and immunoblot analysis revealed increased survival of hypoxic cardiomyocytes by mLIF, mOSM and hlOSM stimulation, associated with increased activation of STAT3. Kinetic expression profiling of infarcted hearts further specified a transient increase of OSM and LIF during the early inflammatory phase of cardiac remodeling. A post-infarction delivery of hlOSM but not mOSM or mLIF within this time period combined with cardiac magnetic resonance imaging-based strain analysis uncovered a global cardioprotective effect on infarcted hearts. Our data conclusively suggest that a simultaneous and rapid activation of OSMR and LIFR after MI offers a therapeutic opportunity to preserve functional and structural integrity of the infarcted heart.

Long-term single-cell passaging of human iPSC fully supports pluripotency and high-efficient trilineage differentiation capacity.

OBJECTIVES: To establish a straightforward single-cell passaging cultivation method that enables high-quality maintenance of human induced pluripotent stem cells without the appearance of karyotypic abnormalities or loss of pluripotency. METHODS: Cells were kept in culture for over 50 passages, following a structured chronogram of passage and monitoring cell growth by population doubling time calculation and cell confluence. Standard procedures for human induced pluripotent stem cells monitoring as embryonic body formation, karyotyping and pluripotency markers expression were evaluated in order to assess the cellular state in long-term culture. Cells that underwent these tests were then subjected to differentiation into keratinocytes, cardiomyocytes and definitive endoderm to evaluate its differentiation capacity. RESULTS: Human induced pluripotent stem cells clones maintained its pluripotent capability as well as chromosomal integrity and were able to generate derivatives from the three germ layers at high passages by embryoid body formation and high-efficient direct differentiation into keratinocytes, cardiomyocytes and definitive endoderm. CONCLUSIONS: Our findings support the routine of human induced pluripotent stem cells single-cell passaging as a reliable procedure even after long-term cultivation, providing healthy human induced pluripotent stem cells to be used in drug discovery, toxicity, and disease modeling as well as for therapeutic approaches.

Kinin B2-Receptor in human iPSC differentiation into cardiomyocytes / Receptor B2 de cininas na diferenciação de iPSC humanas em cardiomiócitos

Cardiovascular diseases are responsible for almost one third of all global deaths yearly, and therefore are largely studied. Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) have emerged as an exciting technology for cardiac disease modelling and personalised therapy. Nevertheless, issues concerning functional and molecular maturation are still faced. In addition to this, differentiation protocols generally yield a heterogeneous mixed population comprised of nodal, atrial and ventricular-like subtypes, being unsuitable for therapeutic purposes. Bradykinin (BK) is a vasoactive peptide which exerts important physiological roles in the cardiovascular system, having been previously described as important for cellular, keratinocyte and skeletal muscle differentiation. This project performed in cooperation with PluriCell Biotech, a startup specialized in the production and differentiation of hiPSC-CM, has sought (1) characterizing gene and protein expression of molecular markers of maturation and of subtype specification throughout of differentiation; (2) Assessing the electrical functionality of hiPSC-CM through the characterization of subtype-specific action potentials (APs) and (3) Investigating whether the progress of hiPSCCM maturation is regulated by BK through kinin-B2 receptors (B2R). Our results have validated the model that proposes a developmental-dependent switch between skeletal (ssTnI) and cardiac (cTnI) isoforms of troponin I as differentiation progresses, at least to some extent. Furthermore, prolonged time in culture has resulted in higher levels of expression of the ventricular marker MLC2v and in increased rates of ventricular-like action APs. Electrophysiological analysis of hiPSC-CM reveals a mixed population with AP morphologies correspondent to nodal, atrial and ventricular subtypes, all showing pronounced automaticity as well as other features of immature cardiomyocytes, such as low amplitude and depolarization velocity. Such findings are coherent with those from other groups who have attempted to differentiate mature native-like cardiac cells from pluripotent stem cells sources, without fully succeeding. After showing that differentiating hiPSC-CM express a functional and responsive B2R, the receptor was subjected to chronic activation with 10µM BK and 1µM BK or inhibition with 5µM Firazyr+BK. Even though B2R modulation has not interfered negatively with differentiation yields nor cell morphology, analysis of gene andprotein expression of ssTnI or cTnI and of the ventricular marker MLC2v, have revealed no significant results in comparison to untreated controls. This suggests that BK does not interfere on hiPSC-CM maturation nor subtype specification, although we cannot rule out that it could be leading to other unexplored effects. We recommend a closer look into which intracellular signalling pathways become active upon B2R stimulation in hiPSC-CM, in order to narrow down cellular processes for further investigation

Doenças cardiovasculares são responsáveis por quase um terço de todas as mortes globais anualmente, e por isto o sistema cardiovascular é amplamente estudado. Cardiomiócitos derivados a partir de células-tronco pluripotentes induzidas humanas (hiPSCCM) emergiram como uma promissora tecnologia para modelagem de doenças cardíacas e terapia personalizada. No entanto, desafios acerca de sua maturação funcional e molecular ainda são enfrentados. Além disso, protocolos de diferenciação geralmente levam à obtenção de populações heterogêneas contendo células com fenótipos similares aos de cardiomiócitos nodais, atriais e ventriculares sendo, portanto, inapropriadas para fins terapêuticos. A bradicinina (BK) é um peptídio vasoativo que exerce importantes papeis fisiológicos no sistema cardiovascular, além de ter sido previamente descrita como importante para a diferenciação neuronal, de queratinócitos e de músculo esquelético. Este projeto foi realizado em colaboração com a empresa PluriCell Biotech, uma startup especializada na produção e diferenciação de hiPSC-CM, e buscou (1) caracterizar a expressão gênica e proteíca de marcadores moleculares de maturação e de especificação de subtipos cardíacos durante a diferenciação; (2) avaliar a funcionalidade elétrica de hiPSC-CM por meio da caracterização de seus potenciais de ação (PAs) e (3) Investigar se o progresso da diferenciação de hiPSCCM é regulado por bradicinina por meio do receptor B2 (B2R). Nossos resultados validaram o modelo que propõe um switch na expressão das isoformas funcionais de troponina I esquelética (ssTnI) e cardíaca (cTnI), durante o desenvolvimento e diferenciação celular, pelo menos parcialmente. Além disso, tempo prolongado em cultura resultou em maiores níveis de expressão do marcador ventricular MLC2v, assim como maiores frequências de PAs com morfologias similares a de cardiomiócitos ventriculares. Análise eletrofisiológica de hiPSCCM revelam a existência de uma população mista contendo PAs correspondentes aos subtipos nodais, atriais e ventriculares, assim como pronunciada automaticidade e outros atributos típicos de cardiomiócitos imaturos, como baixa amplitude e devagar velocidade de despolarização. Estes resultados são coerentes com os de outros grupos que ainda não foram totalmente bem-sucedidos em diferenciar células cardíacas maduras similares acardiomiócitos nativos a partir de células-troncos pluripotentes. Após mostrar que as hiPSCCM expressam receptores B2 funcionais e responsivos, submetemos o receptor a uma ativação crônica com BK 10µM e BK 1µM ou inibição crônica com Firazyr 5µM + BK. Apesar da modulação do B2R não ter interferido de forma negativa no rendimento da diferenciação ou na morfologia celular, análise de expressão gênica e proteica de ssTnI e cTnI e do marcador ventricular MLC2v não revelou resultados significativos em comparação aos controles não-tratados. Isto sugere que a BK não interfere na maturação e especificação de subtipos cardíacos em hiPSC-CM, apesar de não podermos ignorar o fato de que ela poderia estar desencadeando outros efeitos inexplorados. Nós recomendamos um estudo mais aprofundado acerca de quais vias de sinalização se tornam ativas após estimulação do receptor B2 em hiPSC-CM, com o objetivo de afunilar quais processos celulares poderiam ser investigados em uma próxima etapa deste estudo