Staphylococcus aureus β-Toxin Exerts Anti-angiogenic Effects by Inhibiting Re-endothelialization and Neovessel Formation

Abstract

Staphylococcus aureus causes severe, life-threatening infections that often are complicated by severe local and systemic pathologies with non-healing lesions. A classic example is S. aureus infective endocarditis (IE), where the secreted hemolysin β-toxin potentiates the disease via its sphingomyelinase and biofilm ligase activities. Although these activities dysregulate human aortic endothelial cell activation, β-toxin effect on endothelial cell function in wound healing has not been addressed. With the use of the ex vivo rabbit aortic ring model, we provide evidence that β-toxin prevents branching microvessel formation, highlighting its ability to interfere with tissue re-vascularization and vascular repair. We show that β-toxin specifically targets both human aortic endothelial cell proliferation and cell migration and inhibits human umbilical vein endothelial cell rearrangement into capillary-like networks in vitro. Proteome arrays specific for angiogenesis-related molecules provided evidence that β-toxin promotes an inhibitory profile in endothelial cell monolayers, specifically targeting production of TIMP-1, TIMP-4, and IGFBP-3 to counter the effect of a pro-angiogenic environment. Dysregulation in the production of these molecules is known to result in sprouting defects (including deficient cell proliferation, migration, and survival), vessel instability and/or vascular regression. When endothelial cells are grown under re-endothelialization/wound healing conditions, β-toxin decreases the pro-angiogenic molecule MMP-8 and increases the anti-angiogenic molecule endostatin. Altogether, the data indicate that β-toxin is an anti-angiogenic virulence factor and highlight a mechanism where β-toxin exacerbates S. aureus invasive infections by interfering with tissue re-vascularization and vascular repair.

Keywords: Staphylococcus aureus; angiogenesis; endothelial cell; sphingomyelinase (SMase); β-toxin.

FIGURE 1

β-toxin inhibits production of angiogenic proteins from human aortic endothelial cells in monolayer growth. Immortalized human aortic endothelial cells (iHAECs) grown to near confluency on gelatin-coated plates were treated with either VEGF (10 ng mL^–1), axitinib (30 μM), or β-toxin (50 μg mL^–1) for 24 h. Protein production was assessed by Proteome Profiler Human Angiogenesis Array Kit. Results are the mean fold change over untreated cells of three independent experiments conducted in duplicate. Angiogenic-related factors with a 50% increase (>1.5-fold change) or decrease (<0.5-fold change) from media control.

FIGURE 2

β-toxin inhibits wound healing. Time course analysis of iHAECs grown to confluency in silicone inserts that create uniform gaps upon removal and treated with either VEGF (10 ng mL^–1), axitinib (10 μM), or β-toxin (50 μg mL^–1) for 24 h. Images captured every 30 min. (A) Phase-contrast microscopy at Time 0 (representative image) and at 24 h for all conditions tested. Scale bar = 200 μm. (B) iHAECs treated with VEGF or axitinib. (C) iHAECs treated with β-toxin. (D) iHAECs pretreated overnight with β-toxin prior to gap formation and thereafter. (B–D) All results are mean ± SEM of five independent experiments with four replicates each. ^∗p < 0.0332, ^∗∗p < 0.0021, ^∗∗∗p < 0.002; two-way repeated measures ANOVA with Tukey’s multiple comparisons test.

FIGURE 3

β-toxin modulates production of angiogenic proteins from iHAECs during wound healing. iHAECs grown to confluency in silicone inserts that create uniform gaps upon removal and treated with either VEGF (10 ng mL^–1), axitinib (10 μM), or β-toxin (50 μg mL^–1) for 24 h. Angiogenesis proteome arrays were determined from culture supernatants collected at 24 h. Results shown are the mean fold change from untreated cells of five independent experiments. Angiogenic-related factors with a 50% increase (>1.5-fold change) or decrease (<0.5-fold change) from media control.

FIGURE 4

β-toxin inhibits migration and proliferation. (A) Percent metabolic activity. iHAECs grown to near confluency on 1% gelatin-coated plates and treated for 24 h with mitomycin C (MMC) in the absence or presence of β-toxin (50 μg mL^–1). ^∗∗∗∗p ≤ 0.0001; two-way repeated measures ANOVA. Unpaired, two-tailed t-test was used to compare individual MMC treatments to media control (0 μg mL^–1). (B) Phase-contrast microscopy at Time 0 (representative image) and at 24 h for all conditions tested. Images captured every 30 min. Scale bar = 200 μm. (C) Percent wound closure over time of iHAECs treated with MMC (2 μg mL^–1) ± β-toxin (50 μg mL^–1). All results are mean ± SEM of five independent experiments with four replicates each. ^∗∗∗p < 0.002; two-way repeated measures ANOVA. (D) Cell proliferation of iHAECs seeded at 7,000 cells/well and treated with MMC (2 μg mL^–1) or β-toxin (50 μg mL^–1) over a 20-h period. Cells counted every 30 min for the first 5 h then every 5 h thereafter. Results represent the change in cell count (mean ± SEM) of three independent experiments conducted in triplicate. ^∗p < 0.0332, Unpaired, two-tailed t-test at 20 h.

FIGURE 5

β-toxin has differential effects on tube formation. iHAECs seeded on GFR-Matrigel were treated with either axitinib (30 μM) or β-toxin (50 μg mL^–1) and tube formation imaged every 1 h for 12 h. (A) Phase-contrast microscopy at 3 h. Scale bar = 200 μm. (B) Average tube length over time of iHAECs ± axitinib. (C) Average loop count over time of iHAECs ± axitinib. (D) Average tube length over time of iHAECs ± β-toxin. (E) Average loop count over time of iHAECs ± β-toxin. (F) Average tube length over time of HUVECs ± β-toxin. (G) Average loop count over time of HUVECs ± β-toxin. (B–G) Results are means ± SD for at least 6 independent experiments with five replicates each. Statistical significance determined by two-way repeated measures ANOVA.

FIGURE 6

β-toxin inhibits sprout formation. Thoracic and abdominal aortas were collected and sectioned from 2 to 3 kg New Zealand white rabbits. Rings were cultured on GFR-Matrigel in the presence or absence of β-toxin (50 μg mL^–1). Scale bar = 500 μm. (A) Phase-contrast microscopy of thoracic aortic rings. (B) Phase-contrast microscopy of abdominal aortic rings.