Department of Bioengineering, University of Illinois Urbana Champaign, Urbana, Illinois, United States of America.
PLoS One. 2012;7(9):e44791. doi: 10.1371/journal.pone.0044791. Epub 2012 Sep 12.
VEGFR surface localization plays a critical role in converting extracellular VEGF signaling towards angiogenic outcomes, and the quantitative characterization of these parameters is critical for advancing computational models; however the levels of these receptors on blood vessels is currently unknown. Therefore our aim is to quantitatively determine the VEGFR localization on endothelial cells from mouse hindlimb skeletal muscles. We contextualize this VEGFR quantification through comparison to VEGFR-levels on cells in vitro. Using quantitative fluorescence we measure and compare the levels of VEGFR1 and VEGFR2 on endothelial cells isolated from C57BL/6 and BALB/c gastrocnemius and tibialis anterior hindlimb muscles. Fluorescence measurements are calibrated using beads with known numbers of phycoerythrin molecules. The data show a 2-fold higher VEGFR1 surface localization relative to VEGFR2 with 2,000-3,700 VEGFR1/endothelial cell and 1,300-2,000 VEGFR2/endothelial cell. We determine that endothelial cells from the highly glycolytic muscle, tibialis anterior, contain 30% higher number of VEGFR1 surface receptors than gastrocnemius; BALB/c mice display ~17% higher number of VEGFR1 than C57BL/6. When we compare these results to mouse fibroblasts in vitro, we observe high levels of VEGFR1 (35,800/cell) and very low levels of VEGFR2 (700/cell), while in human endothelial cells in vitro, we observe that the balance of VEGFRs is inverted, with higher levels VEGFR2 (5,800/cell) and lower levels of VEGFR1 (1,800/cell). Our studies also reveal significant cell-to-cell heterogeneity in receptor expression, and the quantification of these dissimilarities ex vivo for the first time provides insight into the balance of anti-angiogenic or modulatory (VEGFR1) and pro-angiogenic (VEGFR2) signaling.
VEGFR 表面定位在将细胞外 VEGF 信号转化为血管生成结果方面起着关键作用，对这些参数进行定量描述对于推进计算模型至关重要；然而，目前尚不清楚这些受体在血管中的水平。因此，我们的目标是定量确定来自小鼠后肢骨骼肌内皮细胞的 VEGFR 定位。我们通过与体外细胞的 VEGFR 水平进行比较来对 VEGFR 定量进行背景化处理。使用定量荧光，我们测量并比较了从 C57BL/6 和 BALB/c 比目鱼肌和胫骨前肌后肢肌肉中分离出的内皮细胞上的 VEGFR1 和 VEGFR2 的水平。荧光测量使用具有已知藻红蛋白分子数的珠子进行校准。数据显示，与 VEGFR2 相比，VEGFR1 的表面定位高 2 倍，2000-3700 个 VEGFR1/内皮细胞和 1300-2000 个 VEGFR2/内皮细胞。我们确定来自高度糖酵解肌肉胫骨前肌的内皮细胞比比目鱼肌含有 30%更高数量的 VEGFR1 表面受体；BALB/c 小鼠比 C57BL/6 显示出约 17%更高数量的 VEGFR1。当我们将这些结果与体外的小鼠成纤维细胞进行比较时，我们观察到高 VEGFR1 水平（35800/细胞）和非常低的 VEGFR2 水平（700/细胞），而在体外人内皮细胞中，我们观察到 VEGFRs 的平衡被颠倒，具有更高水平的 VEGFR2（5800/细胞）和较低水平的 VEGFR1（1800/细胞）。我们的研究还揭示了受体表达的显著细胞间异质性，并且首次对这些差异进行了体外定量，深入了解了抗血管生成或调节（VEGFR1）和促血管生成（VEGFR2）信号的平衡。
