Endothelial Cells from Five Organs Share Comparable Receptor Tyrosine Kinase Levels In Vitro: A Quantitative Flow Cytometry Baseline

Purpose Receptor tyrosine kinase (RTK) concentrations on the plasma membrane regulate angiogenesis. The intracellular RTK pool also regulates the availability of plasma membrane receptors and the activity of signaling pathways. Organs exhibit specialized angiogenic functions, suggesting that RTK concentrations vary across endothelial cells (ECs) from different organs. Methods We measured and statistically analyzed the concentrations of VEGFRs, NRP1, Axl, and PDGFRs across human ECs from five organs: dermis, umbilical vein, kidney, liver, and brain, on the plasma membrane and across the whole-cell using quantitative flow cytometry. Results We quantified VEGFR1, VEGFR2, VEGFR3, NRP1, and Axl, along with PDGFRα and β, across five EC types and found that each receptor remained within the same order of magnitude across five cell types, despite their different organ origins. VEGFR2 levels were the most similar across the cell types, ranging 14,100–22,500 receptors/cell at the whole-cell level and 4800–7500 receptors/cell on the plasma membrane, with no significant differences at both compartments (p ≥ 0.05). Among VEGFRs, VEGFR1 had the lowest plasma membrane concentrations (400–900 receptors/cell) with 9–26% plasma membrane localization, while VEGFR3 showed the highest whole-cell abundance (32,400–62,000 receptors/cell) but minimal plasma membrane localization (2–14%). NRP1 was the most abundant plasma membrane receptor (26,000–39,700 receptors/cell), approximately 5-fold higher than VEGFR2, while Axl levels (6900–12,200 receptors/cell) were comparable to VEGFR2. PDGFR proteins were minimally expressed in all five EC types, particularly negligible on the plasma membrane (< 400 receptors/cell). Single-cell analysis revealed low heterogeneity (quadratic entropy < 0.7) within each EC type, with log-normal distributions for all receptors. Conclusions Our quantitative analysis revealed that RTK protein abundance and subcellular localization were broadly similar and generally remained within the same order of magnitude across cultured EC populations. This similarity suggests that the differential angiogenic signaling responses observed across organ-specific ECs, in vivo, are not primarily reflected in baseline RTK abundance under in vitro monoculture conditions, highlighting the importance of microenvironmental factors in establishing organ-specific vascular phenotypes.