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Differential regulation of angiogenic cellular processes and claudin-5 by histamine and VEGF via PI3K-signaling, transcription factor SNAI2 and interleukin-8

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Abstract

Aims

Histamine and vascular endothelial growth factor A (VEGF) are central regulators in vascular pathologies. Their gene regulation leading to vascular remodeling has remained obscure. In this study, EC regulation mechanisms of histamine and VEGF were compared by RNA sequencing of primary endothelial cells (ECs), functional in vitro assays and in vivo permeability mice model.

Methods and results

By RNA sequencing, similar transcriptional alterations of genes involved in activation of primary ECs, cell proliferation and adhesion were observed between histamine and VEGF. Seventy-six commonly regulated genes were found, representing ~53% of all VEGF-regulated transcripts and ~26% of all histamine-regulated transcripts. Both factors regulated tight junction formation and expression of pro-angiogenic transcription factors (TFs) affecting EC survival, migration and tube formation. Novel claudin-5 upstream regulatory genes were identified. VEGF was demonstrated to regulate expression of SNAI2, whereas pro-angiogenic TFs NR4A1, MYCN and RCAN1 were regulated by both histamine and VEGF. Claudin-5 was shown to be regulated VEGFR2/PI3K-Akt dependently by VEGF and PI3K-Akt independently by histamine. Interleukin-8 was shown to downregulate claudin-5 by histamine. Additionally, SNAI2, NR4A1 and MYCN were shown to mediate EC survival, migration and tube formation and to regulate expression of claudin-5. Further systemic delivery of VEGF and histamine was shown to induce a fast vascular hyperpermeability response in intact vasculature of C57/Bl6 mice followed by regulation of NR4A1 and MYCN.

Conclusions

Our study identifies novel claudin-5 upstream regulatory genes of histamine and VEGF that induce cellular angiogenic processes. Our results increase knowledge of angiogenic EC phenotype and provide novel treatment targets for vascular pathologies.

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Acknowledgements

This study was supported by Academy of Finland (Project No. 250614), CoE of Cardiovascular and Metabolic Disease, ERC Advanced Grant (AdG09-250050) and CARIM PhD and VENI fellowships of the Netherlands Organization of Scientific Research (016.116.017). The personnel of the Kuopio University Hospital maternity ward are thanked for providing the umbilical cords for HUVEC cell isolation.

Author contributions

JPL (Laakkonen) designed, performed research, wrote the paper and provided materials and reagents for the study. JPL (Lappalainen) performed research and wrote the paper. TLT performed research, optimized 2-photon microscopy experiments and edited the paper. PIT and TN contributed VEGF proteins to the study. SJ performed cell experiments and isolated primary human endothelial cells. MUK performed RNA sequencing and data analysis. JCS and SYH edited the paper and provided materials and reagents for the study.

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    Authors and Affiliations

    1. Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland

      Johanna P. Laakkonen, Jari P. Lappalainen, Pyry I. Toivanen, Tiina Nieminen, Suvi Jauhiainen, Minna U. Kaikkonen & Seppo Ylä-Herttuala

    2. Department of Pathology, CARIM, Maastricht University Medical Center, Maastricht, The Netherlands

      Thomas L. Theelen & Judith C. Sluimer

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    1. Johanna P. Laakkonen
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    Correspondence to Johanna P. Laakkonen.

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    Johanna P. Laakkonen and Jari P. Lappalainen have contributed equally to this work.

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    Laakkonen, J.P., Lappalainen, J.P., Theelen, T.L. et al. Differential regulation of angiogenic cellular processes and claudin-5 by histamine and VEGF via PI3K-signaling, transcription factor SNAI2 and interleukin-8. Angiogenesis 20, 109–124 (2017). https://doi.org/10.1007/s10456-016-9532-7

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