Syndecan-4 is a major syndecan in primary human endothelial cells in vitro, modulated by inflammatory stimuli and involved in wound healing

Abstract

Syndecans are important cell surface proteoglycans with many functions; yet, they have not been studied to a very large extent in primary human endothelial cells. The purpose of this study was to investigate syndecan-4 expression in cultured human umbilical vein endothelial cells (HUVECs) and assess its role in inflammatory reactions and experimental wound healing. qRT-PCR analysis revealed that syndecan-3 and syndecan-4 were highly expressed in HUVECs, whereas the expression of syndecan-1 and -2 was low. HUVECs were cultured with the inflammatory mediators lipopolysaccharide (LPS) and interleukin 1β (IL-1β). As a result, syndecan-4 expression showed a rapid and strong increase. Syndecan-1 and -2 expressions decreased, whereas syndecan-3 was unaffected. Knockdown of syndecan-4 using siRNA resulted in changes in cellular morphology and focal adhesion sites, delayed wound healing and tube formation, and increased secretion of the pro-inflammatory and angiogenic chemokine, CXCL8. These data suggest functions for syndecan-4 in inflammatory reactions, wound healing and angiogenesis in primary human endothelial cells.

Keywords: angiogenesis; inflammation; primary human endothelial cells; shedding; syndecan-4; wound healing.

Figure 1.

Syndecan mRNA expressions in HUVECs and their response to inflammatory stimuli. (A) Gene expression levels of syndecans (SDC)-1, -2, -3 and -4 were analyzed by qRT-PCR and normalized to the internal control gene RPL30 (n=3). (B) Gene expression of SDC-1, -2, -3 and -4 in untreated cells (Ctr) or after incubation with LPS or IL-1β for 4 and 24 h. Data are presented as the mean ± SEM (n=3–4). A value of p<0.05 is considered statistically significant between treatment and control groups. *p ≤0.05.

Figure 2.

Effect of LPS and IL-1β on syndecan-4 cell-surface expression. HUVECs were cultured with LPS, IL-1β, or left untreated (Ctr) for 24 h. Syndecan-4 cell-surface expression was measured using flow cytometry. Results are presented as mean median fluorescence intensity (MFI) ± SEM. *p<0.05, **p<0.01 indicate statistically significant differences.

Figure 3.

Effect of LPS and IL-1β and matrix metalloproteinase inhibitor, GM6001, on syndecan-4 shedding. (A) HUVECs were exposed to LPS or IL-1β for 4 and 24 h. Supernatants were harvested and the amount of syndecan-4 in the conditioned media was quantified by ELISA (n=6). (B) Syndecan-4 levels in the conditioned media and in cell lysates were determined by ELISA after HUVECs received a 24-h incubation with 5 or 25 µM GM6001 (n=4). A value of p<0.05 is considered a statistically significant difference between the treatment and control. *p<0.05, ***p<0.001.

Figure 4.

Effect of LPS and IL-1β on wound healing in HUVECs. (A) Representative phase contrast images of scratch wounds at 0 and 8 h after wounding in cells pretreated with LPS or IL-1β for 24 h. (B) Percent gap closure of HUVECs upon treatment with LPS and IL-1β after 8 h (n=3). Scale, 200 µm.

Figure 5.

Syndecan-1, -2 and -3 gene expressions in syndecan-4 knockdown HUVECs. Cells were transfected with syndecan-4 siRNA or control siRNA, as described in the Materials & Methods. (A) qRT-PCR analysis of syndecan-4 transcripts in siSDC4 cells (n=9). (B) Syndecan-4 in conditioned media and cell fractions was determined by ELISA (n=3). (C). Cell-surface expression was determined by flow cytometry (n=2). (D) qRT-PCR analysis of the expression of the other syndecan family members after knock down of syndecan-4 (n=10). Results are presented as the mean ± SEM. A value of p<0.05 is considered a statistically significant difference between the treatment and control. *p<0.05 and ***p<0.001.

Figure 6.

Morphology of HUVECs after syndecan-4 knock down. (A) and (B) show light microscopy images of siRNA-transfected cells from two individual cultures. The cells were transfected with control siRNA and either syndecan-4 siRNA from Santa Cruz (A) or syndecan-4 siRNA from Ambion (B), Scale, 200 µm. (C) Quantification of morphological changes, presented as the % of roundness (n=4). (D) Immunostaining of siRNA-transfected cells for the endothelial marker, von Willebrand factor (vWF), showing the same phenotype in both siControl and siSDC4 cells. Scale, 10 µm. The results show one representative out of three donors. A value of p<0.05 is considered a statistically significant difference. **p<0.01.

Figure 7.

Effect of syndecan-4 knockdown on focal adhesions and actin cytoskeleton in HUVECs. HUVECs treated with siRNA against syndecan-4 (siSDC4) or control (siControl) were plated on gelatin-coated chamber slides, fixed, permeabilized and immunostained for vinculin (A) or stained with rhodamine-phalloidin for F-actin (B). Images are representative for stainings of HUVECs from three different donors. (C) Western blot analysis of cell lysates from siSDC4 and siControl for the expression of β-actin. Tubulin was used as a loading control. Scale, 10 µm.

Figure 8.

Migration, tube formation, and proliferation of HUVECs in syndecan-4 knock down cells. (A) Phase contrast images of scratch wound assays on confluent monolayers of cells treated with control siRNA (siControl) or siRNA against syndecan-4 (siSDC4) at 0 h and after 8 h. (B) Bar diagram showing the percent gap closure (n=10) of siControl and siSDC4 cells 8 h after wounding. (C) In vitro tube formation assay with negative control (without growth factors; left panel), siControl and siSDC4 cells. (D) Quantification of tube length (left) and loop numbers (right) of siControl and siSDC4 cells formed in basement membrane extract (BME) gels by Wimasis Image Analysis. Data are expressed as the percent of the control (n=5). (E) Cell proliferation assay. HUVECs were treated with siSDC4 or siControl and cultured in microtiter plates for 24 h. Data are expressed as a percent of the control (n=4). All values are the mean ± SEM. *p<0.05, **p<0.01 and ***p<0.001 indicate a significant difference from the control. Scale, 200 µm.

Figure 9.

Chemokine (C-X-C motif) ligand 1 (CXCL1) and CXCL8 secretion and growth factor expression in syndecan-4 knocked down HUVECs. (A) ELISA analysis of CXCL1 (n=4) and CXCL8 (n=6) levels in the conditioned media from siControl- and siSDC4-treated cells. (B) qRT-PCR determination of various angiogenic growth factors in siControl- and siSDC4-treated cells (n=6–10). Results are presented as the mean ± SEM, and a value of p<0.05 is considered a statistically significant difference between the treatment and control. *p<0.05 and **p<0.01. Abbreviations: VEGF, vascular endothelial growth factor (A and B); FGF2, fibroblast growth factor 2; TGFB2, transforming growth factor beta-2; PDGFA, platelet-derived growth factor-A.