The Endothelial Glycocalyx as a Target of Excess Soluble Fms-like Tyrosine Kinase-1

Abstract

Soluble fms-like tyrosine kinase-1 (sFlt-1) is a secreted protein that binds heparan sulfate expressed on the endothelial glycocalyx (eGC). In this paper we analyze how excess sFlt-1 causes conformational changes in the eGC, leading to monocyte adhesion, a key event triggering vascular dysfunction. In vitro exposure of primary human umbilical vein endothelial cells to excess sFlt-1 decreased eGC height and increased stiffness as determined by atomic force microscopy (AFM). Yet, structural loss of the eGC components was not observed, as indicated by Ulex europaeus agglutinin I and wheat germ agglutinin staining. Moreover, the conformation observed under excess sFlt-1, a collapsed eGC, is flat and stiff with unchanged coverage and sustained content. Functionally, this conformation increased the endothelial adhesiveness to THP-1 monocytes by about 35%. Heparin blocked all these effects, but the vascular endothelial growth factor did not. In vivo administration of sFlt-1 in mice also resulted in the collapse of the eGC in isolated aorta analyzed ex vivo by AFM. Our findings show that excess sFlt-1 causes the collapse of the eGC and favors leukocyte adhesion. This study provides an additional mechanism of action by which sFlt-1 may cause endothelial dysfunction and injury.

Keywords: endothelial dysfunction; endothelial glycocalyx; endothelial injury; heparin; monocyte adhesion; soluble fms-like tyrosine kinase-1 (sFlt-1).

Figure 1

Excess sFlt-1 leads to conformational changes in the eGC in vitro and in vivo. (A,B) HUVECs were exposed to recombinant sFlt-1 (VEGFR1-Fc, 2 µg/mL) or control protein (IgG-Fc, 2 µg/mL) for 24 h in the presence or not of unfractionated heparin (10 µg/mL) after a 30 min preincubation. (C) Schematic representation of the experimental design for sFlt-1 delivery in vivo. Briefly, mice were exposed to continuous administration of recombinant sFlt-1 (300 ng/h; N = 3) or control protein (N = 3) for three days via osmotic minipumps implanted on day 0 (d0). Aortae were isolated on day 3 (d3), and a small patch with the endothelial surface facing upward was analyzed ex vivo (D,E). eGC height (A,D) and stiffness (B,E) were measured by atomic force microscopy (AFM). In (A) and (B), results are expressed as change relative to control. Data are given as mean ± SEM. * p < 0.05; ** p < 0.005. Nested one-way ANOVA was applied along with Tukey’s multiple comparisons test (A,B) or nested t-test (D,E). eGC, endothelial glycocalyx; VEGF, vascular endothelial growth factor.

Figure 2

eGC composition is not altered by excess sFlt-1. HUVECs were exposed to recombinant sFlt-1 (VEGFR1-Fc, 2 µg/mL) or control protein (IgG-Fc, 2 µg/mL) for 24 h in the presence or not of unfractionated heparin (10 µg/mL) after a 30 min preincubation. Changes in the glycocalyx components were measured using the fluorescent eGC markers Ulex europaeus agglutinin-1 (UEA-1) (A) and wheat germ agglutinin (WGA) (B). Data are given as a percentage change from control and are expressed as mean ± SEM. * p < 0.005. Nested one-way ANOVA was applied, along with Tukey’s multiple comparisons test. eGC, endothelial glycocalyx; VEGF, vascular endothelial growth factor.

Figure 3

sFlt-1 increases leukocyte adhesion to the endothelium in vitro and in vivo. (A) HUVECs were treated with recombinant sFlt-1 (VEGFR1-Fc, 2 µg/mL) or control protein (IgG-Fc, 2 µg/mL) in the presence or not of heparin (10 µg/mL) or VEGF (50 ng/mL) for 24 h. The medium was removed, and cells were co-cultured with calcein-labeled THP-1 monocytes for 1 h. The percentage of THP-1 cells adhered to the endothelial monolayer was expressed relative to control cells. (B) Relative THP-1 adhesion to HUVECs treated with sFlt-1 or control protein (1 µg/mL) for 24 h and low dose tumor necrosis factor-α (TNF-α, 0.5 ng/mL) during the last 6 h. Representative results from 3 independent experiments. (C) Surface expression of endothelial adhesion molecules relative to control was measured by flow cytometry 4 h after exposure to sFlt-1 (2 µg/mL). ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1. (D) Schematic representation of the experimental design for sFlt-1 delivery in vivo. Briefly, mice were exposed to continuous administration of recombinant sFlt-1 (300 ng/h) or control protein for seven days via osmotic minipumps implanted on day 0 (d0). (E) Intravital microscopy on postcapillary venules was performed on day 7 (d7) after 2 h of intrascrotal injection of TNF-α. Representative results from 2 independent experiments (N = 7–8 mice/treatment). Data are given as mean ± SEM. * p < 0.05 vs. control; ^# p < 0.05 vs. sFlt-1 + heparin. Nested one-way ANOVA was applied along with Tukey’s multiple comparisons (A) or Mann–Whitney test (B,E). VEGF, vascular endothelial growth factor.

Figure 4

Binding of sFlt-1 to the eGC. (A) Representative immunoblot of HUVECs treated with sFlt-1 or control protein (2 µg/mL) in the presence or not of heparin (10 µg/mL) or exogenous VEGF (50 ng/mL) for 24 h after a 30 min preincubation. Cells were harvested with trypsin-EDTA, lysed under reducing conditions, and protein samples were blotted with an antibody that recognizes the membrane-bound receptor (mFlt-1; higher bands) and its soluble forms (sFlt-1; lower bands). By competing for the same binding site, heparin avoids the attachment of sFlt-1 to the cell membrane, while VEGF does not. Ponceau staining was used to access equal loading. (B) sFlt-1 interactions with VEGF (VEGF-165) through the Ig-like domain 2 and heparan sulfate and heparin through the Ig-like domain 4. Proteins are represented as gray and cyan ribbons, while ligands are orange and magenta sticks. Images were generated using the UCSF Chimera interface. eGC, endothelial glycocalyx; VEGF, vascular endothelial growth factor.

Figure 5

Cell viability under excess sFlt-1. HUVECs were treated with recombinant sFlt-1 (VEGFR1-Fc, 2 µg/mL) or control protein (IgG-Fc, 2 µg/mL) for 24 h in the presence or not of heparin (10 µg/mL) or VEGF (50 ng/mL) after a 30 min preincubation. Cell viability was determined using the MTT assay. Data are given as percentage change from control and expressed as mean ± SEM. Results are based on two independent experiments. * p < 0.05. Nested one-way ANOVA was applied along with Tukey’s multiple comparisons test. VEGF, vascular endothelial growth factor.

Figure 6

Different mechanisms of action of sFlt-1. (A) Depending on its interactions, sFlt-1 acts as a positive [23,25,26] or negative [1,3] regulator of angiogenesis (* based on the literature review). An additional mechanism proposed in this study (light green) is the regulation of endothelial glycocalyx (eGC) conformation through the binding of excess sFlt-1 to heparan sulfate (HS) on the cell surface; (B) Collapse of the eGC (decreased height and increased stiffness) favors leukocyte adhesion to the endothelium and drives endothelial inflammation and dysfunction. HSPG, heparan sulfate proteoglycans; VEGF, vascular endothelial growth factor.