Characterizing VEGFR-2 Phosphorylation Patterns Due to Strain and Anti-Angiogenic Therapies

Introduction:: Angiogenesis, the development of new blood vessels off pre-existing vasculature, is an essential step of tumor progression. When small, a tumor can rely on diffusion to receive vital nutrients and oxygen; however, to grow, the tumor signals to nearby vessels to vascularize the mass [1]. Key players in this process are vascular endothelial growth factor (VEGF), which is secreted by tumor and stromal cells, and the VEGF receptor-2 (VEGFR-2), which is present on endothelial cells (ECs). Certain cancer therapies target this pathway to stop tumor growth by preventing new blood vessels from growing, but many of these anti-angiogenic therapies have proven ineffective in preventing tumor progression or increasing survival rates [2]. We hypothesize that other factors besides chemical or ligand signaling, such as mechanical stimulation, may be promoting angiogenesis. The tumor microenvironment (TME) is known to be mechanically different compared to normal tissue; it has a stiffer extracellular matrix, irregular sheer stress, and increased strain [1]. This project focuses on how strain, which is commonly caused by highly contractile cancer-associated fibroblasts (CAFs), can promote angiogenesis [3]. Previous research has demonstrated that certain VEGFR-2 tyrosine residues, particularly Y1214, are differentially phosphorylated due to mechanical forces such as stiffness and VEGF anchorage [4, 5]. In this study, we demonstrate that ECs exposed to strain develop increased VEGFR-2 phosphorylation and downstream activity, even in the presence of inhibitors, and show increased angiogenic activity.


Materials and Methods:: Human microvascular endothelial cells (HMECs) were treated with combinations of 3µM SU5416, a VEGFR-2 inhibitor, and 25ng/mL VEGF. Cells were lysed, and a Western Blot was performed quantifying VEGFR-2 pY1054/Y1059, pY1214, and total VEGFR-2 levels. Later, HMECs were plated on Flexcell plates (Uniaxial, Collagen-I coated) and strained for 0-15min (10% elongation, 0.3Hz) with some groups being treated with VEGF or SU5416. A Western Blot quantified total VEGFR-2. Microfluidic devices with three connected chambers in series were utilized to quantify angiogenesis [6]. Human umbilical vein endothelial cells (HUVECs) passage 3-7 and normal human lung fibroblasts (NHLFs) passage 3-7 were loaded into the center chamber. For one set of devices, side chambers were filled with either highly contractile CAFs or normal breast fibroblasts (NBFs), which do not have high levels of contractile behaviors. Devices were treated with SU5416 or a vehicle control. Another set of devices had side chambers filled with fibrin and magnetic Dynabeads (ThermoFisher, 14013), and the other side was filled with a blank fibrin gel. Some devices were grown above a rotating magnet, causing an oscillatory strain on the bead chamber to mimic strains caused by CAFs in a cell-free manner. Devices were grown for 7d, then fixed and stained for VE-cadherin. Devices were imaged using fluorescent microscopy, then vessel growth was measured using ImageJ and AngioTool.

Results, Conclusions, and Discussions:: When HMECs were treated with SU5416, both Y1054/Y1059 and Y1214 show decreased phosphorylation, even with the addition of VEGF (Figure 1A). Y1054/Y1059 phosphorylation is necessary for full kinase activity, and Y1214 is involved with the ERK1/2 pathway and expresses prolonged phosphorylation with ligand anchorage [5, 7, 8]. Further studies will analyze how phosphorylation in the presence of VEGFR-2 inhibition is affected by the addition of strain. Total VEGFR-2 was analyzed on strained ECs. When VEGFR-2 is activated, it is internalized and degraded to promote downstream activity, resulting in decreased VEGFR-2 expression when active [4]. Regarding our study, strain with SU5416 causes a decrease in total VEGFR-2 expression, particularly at 15min; however, the addition of VEGF increases expression (Figure 1B). This decrease in VEGFR-2 with SU5416 and strain suggests that VEGFR-2 is being activated and degraded even with inhibition. Further testing and analysis of specific phosphorylated residues will aid in characterizing how strain, VEGF, and SU5416 interact to promote VEGFR-2 activity.

To analyze functional effects of strain and VEGFR-2 inhibition, we utilized microfluidic devices to study angiogenesis. Devices with CAFs and NBFs show increased vessel growth in the chamber with CAFs even when treated with SU5416 (Figure 2A-B). This suggests that even with anti-angiogenic therapy, CAFs can promote angiogenesis, likely in part due to the strain they place on surrounding ECs. Growth factors produced by CAFs are unlikely to have much effect on these results because flow is moving from the center to the sides, effectively removing any soluble factors produced by stromal cells. Furthermore, this means that vessels are growing with the flow, which is opposite to what normally occurs [9, 10]. However, to ensure that growth factors produced by CAFs did not have an effect, we performed a similar test using magnetic beads in place of CAFs. The strain produced by the beads is sufficient to increase angiogenesis compared to beads that were not moved by a circling magnet (Figure 2C-D). This suggests that the addition of strain without any growth factors is sufficient to promote increased vascular growth.


Acknowledgements (Optional): : The authors wish to thank the following funding sources: R00-CA230202 (M.K.S.L.), IMPACT Award (O’Neal Comprehensive Cancer Center, M.K.S.L.), the UAB Graduate School Blazer Fellowships (B.M.).


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