PLCβ3 regulates intracellular Ca²⁺ entry into the cell. (A-C) Human umbilical vein endothelial cells transfected with PLCβ3 (A,C), PLCγ (B), or control shRNA (A-C) were serum starved overnight, loaded with Fura-2 AM, and then stimulated with VEGF (10 ng/mL) at 100 seconds. (C) To distinguish intracellular Ca²⁺ release from the ER from Ca²⁺ entry into the cell, EGTA was added before VEGF stimulation at 50 seconds to measure ER release and CaCl₂ was added at 750 seconds to assess cellular entry. (D) Microangiography using red-permeabilizing tracer and green ISV marker was performed on 3-dpf control (top panel); VEGF-induced, PLCβ3 MO–treated (middle panel); and VEGF-induced, PLCβ3 MO–treated zebrafish with 100μM BAPTA-AM added to the water 24 hours before VEGF induction (bottom panel). Representative images shown were obtained using a Zeiss Apitome microscope equipped with a Fluar 5×, 0.25 numerical aperture lens at room temperature. (E) Schematic of proposed model. VEGF binding to VEGF receptors causes increased intracellular Ca²⁺ (through Ca²⁺ entry into the cell and Ca²⁺ release from the endoplasmic reticulum). Elevated intracellular Ca²⁺ levels promote increased vascular permeability. Activated PLCβ3 inhibits Ca²⁺ entry into the cell, leading to a decrease in VEGF-induced vascular permeability.