Osteoblasts Regulate Angiogenesis in Response to Mechanical Unloading
During mechanical unloading, endothelial cells reduce osteogenesis and increase bone resorption. Here we describe the feedback response of endothelial cells to unloaded osteoblasts. Primary endothelial cells, ex vivo mouse aortic rings and chicken egg yolk membranes were incubated with conditioned medium from mouse primary osteoblasts (OB-CM) subjected to unit gravity or simulated microgravity, to assess its effect on angiogenesis. In vivo injection of botulin toxin A (Botox) in the quadriceps and calf muscles of C57BL/6J mice was performed to mimic disuse osteoporosis. Unloaded osteoblasts showed strong upregulation of the pro-angiogenic factor, VEGF, and their conditioned medium increased in vitro endothelial cell viability, Cyclin D1 expression, migration and tube formation, ex vivo endothelial cell sprouting from aortic rings, and in ovo angiogenesis. Treatment with the VEGF blocker, avastin, prevented unloaded OB-CM-mediated in vitro and ex vivo enhancement of angiogenesis. Bone mechanical unloading by Botox treatment, known to reduce bone mass, prompted the overexpression of VEGF in osteoblasts. The cross talk between osteoblasts and endothelial cells plays a pathophysiologic role in the response of the endothelium to unloading during disuse osteoporosis. In this context, VEGF represents a prominent osteoblast factor stimulating angiogenesis.
KeywordsOsteoblast Mechanical unloading Angiogenesis VEGF Endothelial cells
This work was supported by the European Commission grants—Program “PEOPLE”—Call identifier: FP7-PEOPLE-2011-IRSES—Proposal No. 295181—Acronym: INTERBONE, Program “H2020”—Call identifier: H2020-MSCA-RISE-2015—Proposal No. 690850—Acronym RUBICON, and Program “Collaborative Project—Large-scale integrating project”—Call identifier: FP7-HEALTH.2012.2.1.1-1-C Proposal No. 602300—Acronym: SYBIL. V.V. and M.C. were recipients of Marie Curie fellowships from the INTERBONE project. V.V. was also recipient of Marie Curie fellowship from the RUBICON project. We are indebted with Dr. Rita Di Massimo for the editing of the manuscript.
AT coordinated the study, discussed the experimental plan and the results, and prepared the final version of the paper. VV performed the experiments. RP performed the histomorphometric analysis. SC discussed the experimental plan and the results. MC supervised the study, discussed the experimental plan and the results, contributed to the evaluation of the results, drafted and organized the paper. All reviewed and approved the manuscript.
Compliance with Ethical Standards
Conflict of interest
Vimal Veeriah, Riccardo Paone, Suvro Chatterjee, Anna Teti, Mattia Capulli declare that they have no conflict of interest to disclose.
Human and Animal Rights and Informed Consent
All procedures involving animals and their care were conducted in conformity with national and international laws and policies (European Economic Community Council Directive 86/609, OJ L 358, 1, December 12, 1987; Italian Legislative Decree 4.03.2014, n.26, Gazzetta Ufficiale della Repubblica Italiana no. 61, March 4, 2014). The animal experimentation shown in this article was approved by the Italian Ministry of Health, authorization number: 1012/2016; and was done in adherence to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.
- 3.Clarkin CE, Garonna E, Pitsillides AA, Wheeler-Jones CPD (2008) Heterotypic contact reveals a COX-2-mediated suppression of osteoblast differentiation by endothelial cells: a negative modulatory role for prostanoids in VEGF-mediated cell: cell communication? Exp Cell Res 314:3152–3161. https://doi.org/10.1016/j.yexcr.2008.07.027 CrossRefGoogle Scholar
- 7.Capulli M, Rufo A, Teti A, Rucci N (2009) Global transcriptome analysis in mouse calvarial osteoblasts highlights sets of genes regulated by modeled microgravity and identifies A “mechanoresponsive osteoblast gene signature”. J Cell Biochem 107:240–252. https://doi.org/10.1002/jcb.22120 CrossRefGoogle Scholar
- 13.Miller WJ, Kayton ML, Patton A et al (2004) A novel technique for quantifying changes in vascular density, endothelial cell proliferation and protein expression in response to modulators of angiogenesis using the chick chorioallantoic membrane (CAM) assay. J Transl Med 2:4. https://doi.org/10.1186/1479-5876-2-4 CrossRefGoogle Scholar
- 16.Atik OS, Uslu MM, Eksioglu F, Satana T (2006) Etiology of senile osteoporosis: a hypothesis. Clin Orthop Relat Res 443:25–27. https://doi.org/10.1097/01.blo.0000200235.76565.c8 CrossRefGoogle Scholar
- 20.Carlsson SIM, Bertilaccio MTS, Ascari I et al (2002) Modulation of human endothelial cell behaviour in simulated microgravity. J Gravit Physiol 9:P273–P274Google Scholar
- 25.Radek KA, Baer LA, Eckhardt J et al (2008) Mechanical unloading impairs keratinocyte migration and angiogenesis during cutaneous wound healing. J Appl Physiol 104Google Scholar