International Journal of Molecular Sciences · 9 authors, 1 centre
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Excess sFlt-1 causes collapse of the endothelial glycocalyx (eGC), reducing its height and increasing stiffness without loss of components, leading to a 35% increase in monocyte adhesion. This effect is blocked by heparin but not by VEGF, indicating a VEGF-independent mechanism. The findings reveal a novel pathway by which sFlt-1 contributes to endothelial dysfunction and vascular inflammation.
**Background:** Soluble fms-like tyrosine kinase-1 (sFlt-1) is a secreted VEGF receptor that binds heparan sulfate (HS) on the endothelial glycocalyx (eGC). Elevated sFlt-1 levels are associated with endothelial dysfunction in diseases such as preeclampsia, heart failure, sepsis, and chronic kidney disease. While sFlt-1's antiangiogenic effects via VEGF neutralization are well known, its direct impact on the eGC structure and function is poorly understood. The eGC is a gel-like layer that regulates vascular permeability, leukocyte adhesion, and mechanotransduction. This study investigates whether excess sFlt-1 induces conformational changes in the eGC and promotes monocyte adhesion.
**Methods:** Primary human umbilical vein endothelial cells (HUVECs) and EA.hy926 cells were treated with recombinant human sFlt-1 (2 µg/mL for 24 h, or 1 µg/mL for dose-response). eGC height and stiffness were measured by atomic force microscopy (AFM) nanoindentation. eGC components were assessed by Ulex europaeus agglutinin I (UEA-1) and wheat germ agglutinin (WGA) lectin staining, and heparan sulfate immunofluorescence. Monocyte adhesion was quantified using calcein-AM-labeled THP-1 cells. Adhesion molecule expression (ICAM-1, VCAM-1, E-Selectin, P-Selectin) was analyzed by flow cytometry. In vivo, C57BL/6 mice received recombinant sFlt-1 (300 ng/h) or control IgG-Fc via osmotic minipump for 3–7 days; eGC was analyzed ex vivo in aorta by AFM, and leukocyte adhesion was assessed by intravital microscopy of TNF-α-stimulated cremaster muscle. Heparin (10 µg/mL) and VEGF (50 ng/mL) pretreatments were used to probe mechanism.
**Key Results:** In vitro, sFlt-1 (2 µg/mL, 24 h) significantly decreased eGC height and increased stiffness (p < 0.05 vs. control), indicating a collapsed, flat, and stiff conformation. These effects were dose-dependent. UEA-1 and WGA staining showed no significant loss of eGC components, confirming a conformational rather than degradative change. Heparin completely blocked sFlt-1-induced eGC collapse, while VEGF had no effect. sFlt-1 increased THP-1 monocyte adhesion by approximately 35% (p < 0.05 vs. control). This effect was also blocked by heparin but not by VEGF or α5β1 integrin blockade. Adhesion molecule expression (ICAM-1, VCAM-1, E-Selectin, P-Selectin) remained unchanged. sFlt-1 further enhanced TNF-α-induced monocyte adhesion. In vivo, sFlt-1 infusion reduced eGC height and increased stiffness in mouse aorta (p < 0.05 vs. control) and increased leukocyte adhesion in TNF-α-stimulated cremaster venules (p < 0.05). Heparin did not protect against sFlt-1-induced loss of cell viability (MTT assay).
**Clinical Implications:** This study identifies a novel, VEGF-independent mechanism by which excess sFlt-1 induces endothelial dysfunction: collapse of the eGC with increased stiffness and enhanced monocyte adhesion. This may contribute to vascular inflammation in diseases with elevated sFlt-1, such as preeclampsia, heart failure, and sepsis. The ability of heparin to block eGC collapse and monocyte adhesion suggests potential therapeutic targeting of sFlt-1-HS interactions. However, heparin did not prevent sFlt-1-induced cell death, indicating that multiple pathways are involved. The findings highlight the eGC as a critical mediator of sFlt-1's pro-inflammatory effects and suggest that preserving eGC integrity could mitigate vascular injury.