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Endothelial sphingosine kinase/SPNS2 axis is critical for vessel-like formation by human mesoangioblasts

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Abstract

The interaction between endothelial cells and pericytes is crucial for the stabilization of newly formed vessels in angiogenesis. The comprehension of the mechanisms regulating pericyte recruitment might open therapeutical perspectives on vascular-related pathologies. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that derives from sphingomyelin catabolism and regulates biological functions in cell survival, proliferation, and differentiation. In this study, we aimed to identify the role of S1P axis in the intercellular communication between human mesenchymal progenitor mesoangioblasts (MAB) and endothelial cells (human microvascular endothelial cells (H-MVEC)) in the formation of capillary-like structures. We demonstrated that the S1P biosynthetic pathway brought about by sphingosine kinases (SK) SK1 and SK2 as well as spinster homolog 2 (SPNS2) transporter in H-MVEC is crucial for MAB migration measured by Boyden chambers and for the formation and stabilization of capillary-like structures in a 3D Matrigel culture. Moreover, the conditioned medium (CM) harvested from H-MVEC, where SK1, SK2, and SPNS2 were down-regulated, exerted a significantly diminished effect on MAB capillary morphogenesis and migration. Notably, we demonstrated that S1P1 and S1P3 receptors were positively involved in CM-induced capillary-like formation and migration, while S1P2 exerted a negative role on CM-induced migratory action of MAB. Finally, SK inhibition as well as MAB S1P1 and S1P3 down-regulation impaired H-MVEC-MAB cross-talk significantly reducing in vivo angiogenesis evaluated by Matrigel plug assay. These findings individuate novel targets for the employment of MAB in vascular-related pathologic conditions.

Key message

• Down-regulation of SK1/2 in H-MVEC impaired vessel formation when cultured with MAB.

• H-MVEC SPNS2 is critical for morphogenesis and migration induced by H-MVEC CM of MAB.

• CM from SK1- and SK2-siRNA H-MVEC impaired morphogenesis and migration of MAB.

• S1P1/3 were involved on CM-induced morphogenesis and migration of MAB.

• Matrigel plug assay showed the role of S1P axis in MAB-endothelial cell interaction.

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Abbreviations

SK:

Sphingosine kinase

S1P:

Sphingosine 1-phosphate

FCS:

Fetal calf serum

siRNA:

Short-interfering RNA

SCR:

Scrambled

CM:

Conditioned medium

PBS:

Phosphate-buffered saline

DMEM:

Dulbecco’s modified Eagle’s medium

BSA:

Bovine serum albumin

TGFβ:

Transforming growth factor beta

SPNS2:

Spinster homolog 2

bFGF:

Basic fibroblast growth factor

VEGF:

Vascular endothelial growth factor

MAPK:

Mitogen-activated protein kinase

PI3K:

Phosphatidylinositol 3-kinase

SCID:

Severe combined immunodeficiency

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Acknowledgments

The authors are indebted to Prof. Giulio Cossu and Dr. Rossana Tonlorenzi, Stem Cell Research Institute, Istituto Scientifico H San Raffaele, 20132 Milano, Italy, for providing human MAB and to K. L. Lynch and T. L. MacDonald (University of Virginia, Charlottesville, VA, USA) for providing VPC96047 and VPC96091.

This work was supported by Fondi di Ateneo (ex 60 %) to CD; Ente Cassa di Risparmio di Firenze to GF; Associazione Italiana Ricerca sul Cancro (AIRC) (Grant IG 2013 N. 14266 to MDR).

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Author notes

  1. Simona Serratì

    Present address: Department of Experimental Oncology, Hematology Unit, Advanced Cellular Therapy Centre, Bari, Italy

Authors and Affiliations

  1. Dipartimento di Scienze Biomediche Sperimentali e Cliniche “Mario Serio”, Università di Firenze, Viale G.B. Morgagni 50, 50134, Florence, Italy

    Anna Laurenzana, Francesca Cencetti, Simona Serratì, Gennaro Bruno, Francesca Bianchini, Eugenio Torre, Gabriella Fibbi, Mario Del Rosso, Paola Bruni & Chiara Donati

  2. Faculty of Mathematics and Natural Science, Institute of Nutritional Science, Department of Toxicology, University of Potsdam, Arthur-Scheunert Allee 114-116, 14558, Potsdam, Nuthetal, Germany

    Lukasz Japtok

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  1. Anna Laurenzana
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Correspondence to Chiara Donati.

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Laurenzana, A., Cencetti, F., Serratì, S. et al. Endothelial sphingosine kinase/SPNS2 axis is critical for vessel-like formation by human mesoangioblasts. J Mol Med 93, 1145–1157 (2015). https://doi.org/10.1007/s00109-015-1292-0

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