A defined clathrin-mediated trafficking pathway regulates sFLT1/VEGFR1 secretion from endothelial cells

FLT1/VEGFR1 negatively regulates VEGF-A signaling and is required for proper vessel morphogenesis during vascular development and vessel homeostasis. Although a soluble isoform, sFLT1, is often mis-regulated in disease and aging, how sFLT1 is trafficked and secreted from endothelial cells is not well understood. Here we define requirements for constitutive sFLT1 trafficking and secretion in endothelial cells from the Golgi to the plasma membrane, and we show that sFLT1 secretion requires clathrin at or near the Golgi. Perturbations that affect sFLT1 trafficking blunted endothelial cell secretion and promoted intracellular mis-localization in cells and zebrafish embryos. siRNA-mediated depletion of specific trafficking components revealed requirements for RAB27A, VAMP3, and STX3 for post-Golgi vesicle trafficking and sFLT1 secretion, while STX6, ARF1, and AP1 were required at the Golgi. Live-imaging of temporally controlled sFLT1 release from the endoplasmic reticulum showed clathrin-dependent sFLT1 trafficking at the Golgi into secretory vesicles that then trafficked to the plasma membrane. Depletion of STX6 altered vessel sprouting in 3D, suggesting that endothelial cell sFLT1 secretion influences proper vessel sprouting. Thus, specific trafficking components provide a secretory path from the Golgi to the plasma membrane for sFLT1 in endothelial cells that utilizes a specialized clathrin-dependent intermediate, suggesting novel therapeutic targets. Supplementary Information The online version contains supplementary material available at 10.1007/s10456-023-09893-6.


Supplementary Table
. Delay differential equations describing intracellular and extracellular sFLT1 amount over time in the mechanistic model.Parameter descriptions and optimal values found in Supplementary Table S4.In the  equation, a conversion factor is applied to the  term to obtain consistent units.

Species Description Units
Rate of change equation

Table S3 .
Input parameters for mechanistic computational model.
Supplementary TableS2.Characteristics of sFLT1 datasets used to build the mechanistic model.Sx, extracellular sFLT1; Si, intracellular sFLT1; +, experimentally measured; -, not measured.* Sx converted to fraction of maximum amount for optimization.† Identification of a system of equations and estimation of secretion parameters.‡ Estimation of biologically relevant production parameter values.

Table S4 .
Optimized parameters for mechanistic computational model.