Role of heparan sulfate in mediating CXCL8-induced endothelial cell migration

Abstract

CXCL8 (Interleukin-8, IL-8) plays an important role in angiogenesis and wound healing by prompting endothelial cell migration. It has been suggested that heparan sulfate (HS) could provide binding sites on endothelial cells to retain and activate highly diffusible cytokines and inflammatory chemokines. In the present study, we aimed to test the hypothesis that HS is essential for enhancement of endothelial cell migration by CXCL8, and to explore the underlying mechanism by detecting the changes in expression and activity of Rho GTPases and in the organization of actin cytoskeleton after enzymatic removal of HS on human umbilical vein endothelial cells (HUVECs) by using heparinase III. Our results revealed that the wound healing induced by CXCL8 was greatly attenuated by removal of HS. The CXCL8-upregulated Rho GTPases including Cdc42, Rac1, and RhoA, and CXCL8-increased Rac1/Rho activity were suppressed by removal of HS. The polymerization and polarization of actin cytoskeleton, and the increasing of stress fibers induced by CXCL8 were also abolished by heparinase III. Taken together, our results demonstrated an essential role of HS in mediating CXCL8-induced endothelial cell migration, and highlighted the biological importance of the interaction between CXCL8 and heparan sulfate in wound healing.

Keywords: CXCL8; Cell migration; HUVEC; Heparan sulfate; Rho GTPases.

Figure 1. Heparinase III attenuated the CXCL8-induced wound healing.

HUVEC monolayers were scratched by a yellow tip after confluence in 6-well plates, followed by twice-washed to remove the cell debris, and then were treated with 15 mU/ml heparinase III (HepIII), 100 ng/ml CXCL8, or both for indicated times (4, 8, and 12 h), respectively. Normal cell without treatment was set as control (CT). At each time point, photographs were taken (A), and the wound areas were calculated to evaluate the cell migration capacity (B). CXCL8 induced wound healing suggesting CXCL8 promotes cell migration, and this could be attenuated by heparinase III. Scale bar: 400 µm; n = 4; ^∗P < 0.05, ^∗∗P < 0.01 vs. control; #P < 0.05 vs. CXCL8 at each time point.

Figure 2. Effect of heparinase III on the CXCL8-modulated expression of Rho GTPases.

HUVECs were treated with 15 mU/ml heparinase III (HepIII), 100 ng/ml CXCL8, or both for indicated times (4 and 12 h), respectively. Normal cell without treatment was set as control (CT). The expression of Rho-GTPases was detected by western blot, and quantitative data were obtained using ImageJ: (A and B) Cdc42; (C and D) Rac1; (E and F) RhoA. ^∗P < 0.05 vs. CT at 12 h; #P < 0.05.

Figure 3. Rho GTPases activity.

After treatments, the Rho GTPase activity in HUVECs was detected by using the active Rho/Rac1 pulldown assay, and quantitative data were obtained. (A and B) CXCL8 increased the Rac1 activity at 12 h, and this was abolished by heparinase III; (C and D) Heparinase III significantly reduced the CXCL8-induced Rho activity at both 4 h and 12 h. ^∗aP < 0.05 vs. CT at 4 h; ^∗bP < 0.05 vs. CT at 12 h; #P < 0.05.

Figure 4. Heparinase III inhibited the CXCL8-induced actin cytoskeleton reorganization.

After treatments, cells were fixed in 2% paraformaldehyde, permeabilized with 1% Triton X-100, and stained with BODIPY^® FL phallacidin to visualize the actin cytoskeleton (Green) by confocal microscopy (A). Blue indicates cell nuclei stained by DAPI. Red and yellow arrow heads indicate stress fibers and stress bands, respectively. Scale bar: 50 µm. (B) Relaxation rate of stress fiber, which defined as ratio of actual length of a stress fiber to shortest distance between both ends (as shown in the rectangle box). n = 30 stress fibers. (C) Thickness of stress fiber. n = 30 stress fibers. (D) Width of stress bands. n = 30 stress bands. ^∗aP < 0.05 vs. CT at 4 h; ^∗bP < 0.05 vs. CT at 12 h; #P < 0.05.