PDGF-BB induces VEGFR1 phosphorylation and endothelial cell proliferation

Introduction:: Over 70 vascular diseases are attributed to dysregulated angiogenesis^{1, 2}. Current therapies targeting vascular endothelial growth factors (VEGFs), the primary pro-angiogenic factors, have demonstrated limited efficacy³. This limitation suggests the involvement of other molecules in the pathological process beyond the commonly recognized uni-family VEGF-VEGF receptor (VEGFR) signaling axis. Recently, cross-family binding between platelet-derived growth factors (PDGFs) and VEGFR2 was discovered (Figure 1A), offering an additional mechanism for addressing angiogenesis dysregulation⁴. However, the downstream functional effects of this cross-family PDGF-to-VEGFR binding have not been fully elucidated. In this study, we seek to investigate the role of PDGF-BB in regulating endothelial signaling and function.

Materials and Methods:: Human brain microvascular endothelial cells (HBMECs) were purchased from Cell Systems (ACBRI 376 V). XTT assay was performed to measure PDGF-induced cell proliferation. The cells were incubated in low-serum (1% FBS) media overnight and stimulated with VEGF-A or PDGF-BB at specific concentrations for 24 before XTT reading. To detect PDGF-induced VEGFR1 phosphorylation, both Enzyme-linked immunosorbent assay (ELISA) and immunoblotting were employed. The cells were stimulated with VEGF-A or PDGF-BB for 30 min. The protocols of Human Phospho-VEGFR1/Flt-1 DuoSet IC ELISA kit (R&D Systems, DYC4170) and Human Phospho-VEGFR2/KDR DuoSet IC ELISA kit (R&D Systems, DYC1766) were followed for ELISA. Statistical analysis was performed using one-way ANOVA and multi-comparison post-hoc Tukey's test.

Results, Conclusions, and Discussions:: We first confirm the absence of PDGFRα and β on the plasma membrane of HBMECs, obtaining less than 400 receptors/cell, using flow cytometry. To identify the effects of cell proliferation, we treat HBMECs with PDGF-BB concentrations ranging from 20 pM to 8 nM and observe that 20 pM PDGF-BB induces the highest proliferation, which was 240% greater than untreated cells (p< 0.001) (Figure 2B). We further show PDGF-BB activation of VEGFR1 on endothelial cells, not expressing PDGFRs on the plasma membrane: ~20 pM of PDGF-BB induces a 2-fold increase in VEGFR1 phosphorylation (p< 0.001), but no VEGFR2 phosphorylation is observed (Figure 2C and 2D). We further investigate the possible PDGF-BB-mediated VEGFR1 phosphorylation site by immunoblotting (Figure 2E and 2F). We focus on the lowest concentrations (~2 pM and ~20 pM) since they correspond to the concentrations that yield both significant cell proliferation and VEGFR1 phosphorylation. We observe that neither PDGF-BB concentrations, ~2 pM nor ~20 pM, significantly stimulate VEGFR1 phosphorylation at tyrosine site 1213 (p=0.34 and 0.30, respectively).

Our findings of PDGF-BB-mediated phosphorylation of VEGFR1 suggest an alternate mechanism of VEGFR1 activation exists, since PDGF-BB--VEGFR1 binding was not previously observed, but PDGF-BB--VEGFR2 binding was seen (Figure 1A)⁶. It is well-established that VEGFR1 primarily heterodimerizes with VEGFR2 to regulate VEGF activity (Figure 1B)^{7, 8}, suggesting the need to explore VEGFR1-VEGFR2 heterodimerization in PDGF-mediated cross-family interactions. The tyrosine site 1213 of VEGFR1 is likely to be less important in the signaling pathway since VEGFR1 phosphorylation was not observed. Further studies should screen all intracellular VEGFR1 tyrosine sites to determine potential phosphorylation, while also determining if PDGF-BB induces additional angiogenic hallmarks on endothelial cells, such as cell migration and tube formation. By uncovering the angiogenic functions of PDGF-VEGFR interactions, we can gain a more comprehensive understanding of the cross-family angiogenic pathway, which may reveal alternate mechanisms for addressing vascular dysregulation.


Acknowledgements (Optional): : This material is based upon work supported by the National Science Foundation under Grant No. 1923151. Research reported in this publication was also supported by the National Institute Of Heart, Lung, and Blood of the National Institutes of Health under Award Number 7R01HL159946-02. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation or the National Institutes of Health.

References (Optional): : [1] Weddell, J. et al. Ency Cardiovasc Res & Med. 2018. [2] Egginton, S. Jour Phys. 2010. [3] Francia, G. et al. Cancer Cell. 2009. [4] Mamer, S. et al. Sci Rep. 2017. [5] Colciago, A. et al. Int J Biomed Sci. 2009. [6] Mamer, S. et al. Sci. Rep. 2017. [7] Koch, S. et al. Biochem J. 2011. [8] Cudmore, M. et al. Nat Commun. 2012.