Cultured endothelial cells present organ-specific RTK distributions: advancing receptor measurement and data standardization via quantitative flow cytometry

PurposeReceptor tyrosine kinase (RTK) concentrations on the plasma membrane correlate with angiogenic functions in vitro and in rodent models. The intracellular RTK pool also regulates plasma membrane receptor availability and signaling pathways. Organs have specialized angiogenic functions essential to their distinct roles, supporting the hypothesis that plasma membrane and intracellular RTK concentrations vary across endothelial cells (ECs) from different organs. MethodsUsing quantitative flow cytometry on human ECs derived from dermis, umbilical vein, kidney, liver, and brain, we measured and statistically analyzed the concentrations of selected RTKs within ECs and on their plasma membranes. ResultsVEGFR1 exhibited the lowest concentrations on the plasma membrane (300-900 VEGFR1/cell) among VEGFRs. HDMECs (dermis) showed the lowest VEGFR1 level among the examined EC types. Whole-cell VEGFR1 concentrations were 2500-7500 VEGFR1/cell, with 12-26% located on the plasma membrane. The proportion of VEGFR2 located on the plasma membrane was higher at > 30%, except in HGMECs (kidney) where it was 24%. Plasma membrane VEGFR2 was significantly lower in HDMECs and HGMECs compared with HBMECs (brain), whereas whole-cell VEGFR2 levels were consistently in the range of 14,100-22,500 molecules/cell. VEGFR3 was the least localized to the plasma membrane, from 2% in HGMECs to 14% in HDMECs at the highest level of 4400 VEGFR3/cell. Whole-cell VEGFR3 concentrations ranged from 32,400 in HDMECs to 62,000 VEGFR3/cell in HLiSMECs (liver), with no significant differences among EC types. NRP1 was most abundant on the plasma membrane of HUVECs (umbilical vein) at 39,700 NRP1/cell; other ECs displayed 26,000-29,900 NRP1/cell, approximately 5-fold higher than the numbers of VEGFRs. Across EC types, Axl was present on the plasma membrane at levels (6900-12,200 Axl/cell) similar to those of VEGFR2. ConclusionsWe quantified and statistically analyzed plasma membrane and whole-cell expression of angiogenic RTKs across cultured human ECs from five different organs. Our findings suggest that RTK protein distribution might not fully reflect the differential angiogenic capacities in cultured ECs. In vitro monoculture conditions might reduce EC organ-specific features essential for refining vascular models.