SFlt‐1 Production in Endothelial Cells is Regulated in Part by VEGF Receptor Signaling

Abstract

Preeclampsia (PE) is a pregnancy‐specific hypertensive disorder complicating 2–8% of pregnancies worldwide. Although the etiology of PE is still unknown, inadequate perfusion of the placenta and the resulting placental ischemia are believed to be the initiating pathological factors of the disease. The ischemic placenta releases a number of factors that drive the disorder in the mother, including soluble fms‐like tyrosine kinase‐1 (sFlt‐1), a soluble splice variant of vascular endothelial growth factor receptor (VEGFR‐1), containing only the extracellular domain of the receptor. Because sFlt‐1 binds and sequesters vascular endothelial growth factor (VEGF), it acts as an antiangiogenic factor, causing angiogenic imbalance and endothelial dysfunction. While sFlt‐1 in PE is thought to originate primarily in the placenta, another proposed source is the maternal endothelium, although the factors which could cause upregulation of sFlt‐1 in these tissues is unclear. Here we hypothesized that VEGF could directly regulate production of sFlt‐1 in vascular endothelial cells. To test our hypothesis, we treated Human Umbilical Vein Endothelial Cells (HUVECs) with various VEGFR inhibitors to discern whether VEGFRs contribute to sFlt‐1 production. Cells were cultured in a 24 well plate and were treated with the VEGFR1/2 antagonist Vatalanib (Vat), VEGFR2 Kinase Inhibitor (V2KI), the neuropilin inhibitor EG00229, or left untreated as controls. After 24 hours media and cells were collected for ELISA and RNA isolation. sFlt‐1 exists as three main splice variants in human: V2, V3, and V4, with V2 being the predominant subtype. Because of this, expression levels of all three variants, as well as full length Flt‐1 receptor, were measured via qRT‐PCR and normalized to β‐actin. sFlt‐1 V2 expression was reduced by almost 70% (1.25 ± 0.36% vs 0.39 ± 0.04% fold change; p<0.05) in the EG00229 treated group but was not significantly different than controls in Vat and V2KI groups. sFlt‐1 V4 expression levels also showed a downward trend with EG00229 treatment, although this did not reach significance (p=0.065). sFlt‐1 V4 expression levels were not significantly different between Vat and V2KI treatment and the control group. sFlt‐1 V3 expression levels did not significantly change between any of the treated groups and the control group. No effect was observed in any experimental group on full length Flt‐1 expression levels compared to control. Surprisingly, when measured with ELISA, only treatment with Vat significantly decreased media sFlt‐1 levels (6145 ± 411.2pg/ml vs 3264 ± 666.5pg/ml; p<0.01). However, non‐significant trends to decrease media sFlt‐1 in V2KI (6145 ± 411.2pg/ml vs 5220 ± 111.8pg/ml; p=0.1043) and EG00229 (6145 ± 411.2pg/ml vs 5258 ± 378.8pg/ml; p=0.1237) were also observed. While EG00229 treatment changed sFlt‐1 expression levels the most, only pan‐antagonism of VEGFR1 and R2 significantly decreased media sFlt‐1 levels. From this data we concluded that VEGFR1 and R2 signaling could partially sFlt‐1 release through non‐genomic mechanisms. Future studies will look to elucidate the mechanisms through which VEGFR1 and R2 control sFlt‐1 production.Support or Funding InformationThis work was supported in part by NIH grants P01HL51971, P20GM104357, T32HL105324, and R01HL137791This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.