ECTS Abstracts (2015) 1 P142

VEGF-Dependent Control of Osteoblast/Adipocyte Differentiation

Agnes Berendsen & Bjorn Olsen

Harvard School of Dental Medicine, Boston, MA, USA.

Vascular endothelial growth factor A (VEGF) functions as a key factor in angiogenesis but also plays essential roles in cellular survival, cartilage and bone development, and bone maintenance. Apart from coupling angiogenesis and osteogenesis, VEGF regulates osteoblast progenitor cell fate by controlling the balance between osteoblast and adipocyte differentiation. Conditional deletion of VEGF in Osterix-expressing osteoblast progenitor cells in mice, carrying floxed Vegfa alleles and the Osx-Cre transgene, leads to age-related osteopenia characterised by loss of bone mass with increased marrow fat. Both in vivo and in vitro studies indicated that VEGF knockdown induces differentiation of mouse bone marrow stem cells (BMSCs) into adipocytes at the expense of osteoblasts. Experiments aimed at rescuing differentiation defects revealed that addition of exogenous recombinant VEGF had no effect on BMSC fate, suggesting that VEGF functions via intracrine rather than paracrine mechanisms. A role for intracellular VEGF was supported by detection of VEGF and VEGF receptors in nucleus and cytoplasm using immunostaining and western blotting of BMSC subcellular fractions. To assess the effect of modulating intracellular VEGF levels on BMSC differentiation, we generated a cell-permeable VEGF protein consisting of VEGF fused to TAT, a cell-penetrable peptide, followed by a nuclear localisation sequence. This cell-permeable VEGF proved to be effective in entering cells and translocating to their nucleus. Remarkably, cell-permeable VEGF, but not paracrine VEGF, stimulated expression of osteoblast marker genes in BMSCs. VEGF protein levels in nucleus and cytoplasm appeared to be regulated by mechanisms involving proteasome activity. In summary, we have identified a novel function for VEGF in controlling the fate of BMSCs involving intracrine mechanisms that are independent of its role as secreted growth factor. (Animal experiments were approved by Harvard Medical Area Standing Committee and in agreement with U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals.)

Disclosure: The authors declared no competing interests. This work was supported by a NIH grant 5R01AR036819 (to B.R.O.) and an HSDM Dean’s Scholarship (to A.D.B.)

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