Mesenchymal Stem Cell Migration and Proliferation Are Mediated by Hypoxia-Inducible Factor-1 alpha Upstream of Notch and SUMO Pathways

Mesenchymal stem cells (MSCs) are effective in treating several pathologies. We and others have demonstrated that hypoxia or hypoxia-inducible factor 1 alpha (HIF-1 alpha) stabilization improves several MSC functions, including cell adhesion, migration, and proliferation, thereby increasing their therapeutic potential. To further explore the mechanisms induced by HIF-1 alpha in MSCs, we studied its relationship with Notch signaling and observed that overexpression of HIF-1 alpha in MSCs increased protein levels of the Notch ligands Jagged 1-2 and Delta-like (Dll) 1, Dll3, and Dll4 and potentiated Notch signaling only when this pathway was activated. Crosstalk between HIF and Notch resulted in Notch-dependent migration and spreading of MSCs, which was abolished by gamma-secretase inhibition. However, the HIF-1-induced increase in MSC proliferation was independent of Notch signaling. The ubiquitin family member, small ubiquitin-like modifier (SUMO), has important functions in many cellular processes and increased SUMO1 protein levels have been reported in hypoxia. To investigate the potential involvement of SUMOylation in HIF/Notch crosstalk, we measured general SUMOylation levels and observed increased SUMOylation in HIF-1-expressing MSCs. Moreover, proliferation and migration of MSCs were reduced in the presence of a SUMOylation inhibitor, and this effect was particularly robust in HIF-MSCs. Immunoprecipitation studies demonstrated SUMOylation of the intracellular domain of Notch1 (N1ICD) in HIF-1-expressing MSCs, which contributed to Notch pathway activation and resulted in increased levels of N1ICD nuclear translocation as assessed by subcellular fractionation. SUMOylation of N1ICD was also observed in HEK293T cells with stabilized HIF-1 alpha expression, suggesting that this is a common mechanism in eukaryotic cells. In summary, we describe, for the first time, SUMOylation of N1ICD, which is potentiated by HIF signaling. These phenomena could be relevant for the therapeutic effects of MSCs in hypoxia or under conditions of HIF stabilization.This work was supported, in part, by grants from the Instituto de Salud Carlos III PI13/00414, PI16/0107, RE-TICS RD12/0019/0025 to P.S. and RETICS RD12/0019/0003 (TERCEL) to J.L.D.L.P cofunded by FEDER ``una manera de hacer Europa. It was also supported by the Regenerative Medicine Program of Instituto de Salud Carlos III and Valencian Community to Centro de Investigacion Principe Felipe. The authors are grateful to Dr. A. Dorronsoro for critical review of the work and Dr. K McCreath for manuscript editing.S

Hydrogen Sulfide Improves Cardiomyocyte Function in a Cardiac Arrest Model

[EN] Background: Cardioplegic arrest is a common procedure for many types of cardiac surgery, and different formulations have been proposed to enhance its cardio-protective effect. Hydrogen sulfide is an important signaling molecule that has cardio-protective properties. We therefore studied the cardio-protective effect of hydrogen sulfide in cardiac cell culture and its potential therapeutic use in combination with cardioplegia formulations. Materials/Methods: We added hydrogen sulfide donor GYY4137 to HL-1 cells to study its protective effect in nutrient starved conditions. In addition, we tested the potential use of GYY4137 when it is added into two different cardioplegia formulations: Cardi-Braun (R) solution and del Nido solution in an ex vivo Langendorff perfused rat hearts model. Results: We observed that eight-hour pre-treatment with GYY4137 significantly suppressed apoptosis in nutrient-starved HL-1 cells (28% less compared to untreated cells; p<0.05), maintained ATP content, and reduced protein synthesis. In ex vivo experiments, Cardi-Braun (R) and del Nido cardioplegia solutions supplemented with GYY4137 significantly reduced the pro-apoptotic protein caspase-3 content and preserved ATP content. Furthermore, GYY4137 supplemented cardioplegia solutions decreased the S-(5-adenosyl)-L-methionine/S-(adenosyl)-L-homocysteine ratio, reducing the oxidative stress in cardiac tissue. Finally, heart beating analysis revealed the preservation of the inter-beat interval and the heart rate in del Nido cardioplegia solution supplemented with GYY4137. Conclusions: GYY4137 preconditioning preserved energetic state during starved conditions, attenuating the cardiomyocytes apoptosis in vitro. The addition of GYY4137 to cardioplegia solutions prevented apoptosis, ATP consumption, and oxidative stress in perfused rat hearts, restoring its electrophysiological status after cardiac arrest. These findings suggested that GYY4137 sulfide donor may improve the cardioplegia solution performance during cardiac surgery.Instituto de Salud Carlos III grants (PI10/743, PI13/0414) and RETICS RD12/0019/0025 co-funded by FEDER "Una Manera de Hacer Europa". AG. acknowledges a fellowship from Erasmus Mundus Eurotango Program. ADJ acknowledges support from the Ramon y Cajal program (RYC-2008-02378). P.S. acknowledges support from PI10/743, PI13/414 grants and the Miguel Servet I3SNS and RETICS Program (RD12/0025). We thank Dr Kenneth McCreath for helpful comments on the manuscript.Garcia, NA.; Moncayo-Arlandi, J.; Vázquez Sánchez, A.; Genoves, P.; Calvo Saiz, CJ.; Millet Roig, J.; Martí, N.... (2017). Hydrogen Sulfide Improves Cardiomyocyte Function in a Cardiac Arrest Model. Annals of Transplantation. 22:285-295. https://doi.org/10.12659/AOT.901410S2852952

Hypoxia-inducible factor 1 alpha contributes to cardiac healing in mesenchymal stem cells-mediated cardiac repair

12 p.-6 fig.-1 tab.Mesenchymal stem cells (MSC) are effective in treating myocardial infarction (MI) and previous reports demonstrated that hypoxia improves MSC self-renewal and therapeutics. Considering that hypoxia-inducible factor- 1 alpha (HIF-1a) is a master regulator of the adaptative response to hypoxia, we hypothesized that HIF-1a overexpression in MSC could mimic some of the mechanisms triggered by hypoxia and increase their therapeutic potential without hypoxia stimulation. Transduction of MSC with HIF-1a lentivirus vectors (MSC-HIF) resulted in increased cell adhesion and migration, and activation of target genes coding for paracrine factors. When MSC-HIF were intramyocardially injected in infarcted nude rats, significant improvement was found (after treatment of infarcted rats with MSC-HIF) in terms of cardiac function, angiogenesis, cardiomyocyte proliferation, and reduction of fibrotic tissue with no induction of cardiac hypertrophy. This finding provides evidences for a crucial role of HIF-1a on MSC biology and suggests the stabilization of HIF-1a as a novel strategy for cellular therapies.This work was supported in part by grants from the Instituto de Salud Carlos III for the Regenerative Medicine Program of Valencian Community to Centro de Investigación Principe Felipe, KUTXA founding and from the FIS (PI07/784, CP08/80 and PI10/00743).Peer reviewe