Nox4- and Nox2-dependent oxidant production is required for VEGF-induced SERCA cysteine-674 S-glutathiolation and endothelial cell migration

Abstract

Endothelial cell (EC) migration in response to vascular endothelial growth factor (VEGF) is a critical step in both physiological and pathological angiogenesis. Although VEGF signaling has been extensively studied, the mechanisms by which VEGF-dependent reactive oxygen species (ROS) production affects EC signaling are not well understood. The aim of this study was to elucidate the involvement of Nox2- and Nox4-dependent ROS in VEGF-mediated EC Ca(2+) regulation and migration. VEGF induced migration of human aortic ECs into a scratch wound over 6 h, which was inhibited by overexpression of either catalase or superoxide dismutase (SOD). EC stimulation by micromolar concentrations of H2O2 was inhibited by catalase, but also unexpectedly by SOD. Both VEGF and H2O2 increased S-glutathiolation of SERCA2b and increased Ca(2+) influx into EC, and these events could be blocked by overexpression of catalase or overexpression of SERCA2b in which the reactive cysteine-674 was mutated to a serine. In determining the source of VEGF-mediated ROS production, our studies show that specific knockdown of either Nox2 or Nox4 inhibited VEGF-induced S-glutathiolation of SERCA, Ca(2+) influx, and EC migration. Treatment with H2O2 induced S-glutathiolation of SERCA and EC Ca(2+) influx, overcoming the knockdown of Nox4, but not Nox2, and Amplex red measurements indicated that Nox4 is the source of H2O2. These results demonstrate that VEGF stimulates EC migration through increased S-glutathiolation of SERCA and Ca(2+) influx in a Nox4- and H2O2-dependent manner, requiring Nox2 downstream.

Keywords: Calcium; Endothelial cells; Free radicals; Hydrogen peroxide; Nox2; Nox4; SERCA; VEGF.

Figure 1

Oxidants are required for VEGF-induced EC migration and Ca^2t influx. A. HAEC monolayers overexpressing CuZnSOD and/or catalase were treated with VEGF (50 ng/mL) and migration into a scratch wound was measured over 6 hours (n = 3, *p< 0.05). B. HAEC monolayers overexpressing either MnSOD or CuZnSOD were treated with H₂O₂ (1μM) and migration was measured over 6 hours (n = 3, *p< 0.05). C - E. Catalase was overexpressed in HAEC and VEGF or H₂O₂ was added in the absence of extracellular Ca²⁺. The maximal increase in intracellular Ca²⁺ associated with Ca²⁺ influx upon Ca²⁺ readdition (2 mmol/L) was measured. Data shown are representative VEGF response trace (D) or H₂O₂ response trace (E) with mean ± s.e.m. of Ca²⁺ for measured cells and quantification of change in Fura2 fluorescence ratio between baseline and maximal Ca²⁺ in VEGF and H₂O₂ stimulated cells (C, n = at least 4, ***p< 0.001). G and H. Representative Ca²⁺ influx trace in HAEC overexpressing CuZnSOD and treated with VEGF or H₂O₂ and quantification of maximal Ca²⁺ associated with Ca²⁺ influx (F, n = at least 4, ***p< 0.001).

Figure 2

Nox2 is required for VEGF- and H₂O₂-induced EC Ca²⁺ influx and migration. A. HAEC were treated with specific siRNA to Nox2 and knockdown was confirmed by qRT-PCR for Nox2 mRNA (n = 3, *** p< 0.001). B. Nox2 was knocked down in HAEC and then migration into a scratch wound over 6 hours was measured in response to either VEGF (50 ng/mL) or H₂O₂ (1 μM, n = 3, *p < 0.05, **p < 0.01, *** p< 0.001). C. HAEC were treated with siRNA for Nox2 and the maximal increase in intracellular Ca²⁺ associated with Ca²⁺ entry upon Ca²⁺ readdition was measured (n = at least 4, ***p< 0.001). D and E. Representative Ca²⁺ influx trace in response to VEGF or H₂O₂ in HAEC in which Nox2 was knocked down.

Figure 3

Nox4 is required for VEGF-induced EC Ca²⁺ influx and migration. A. HAEC were treated with adenoviral shRNA specific to Nox4 and knockdown was confirmed by qRT-PCR for Nox4 mRNA (n = 3, *** p< 0.001). B. Nox4 was knocked down in HAEC and then migration into a scratch wound over 6 hours was measured in response to VEGF (50 ng/mL, n = 3, **p < 0.01). C. HAEC were treated with shRNA for Nox4 and the maximal increase in intracellular Ca²⁺ associated with Ca²⁺ entry upon Ca²⁺ readdition was measured (n = at least 4, **p< 0.01, ***p< 0.001). D and E. Representative Ca²⁺ influx trace in response to VEGF or H₂O₂ (1 μM) in HAEC in which Nox4 was knocked down.

Figure 4

Mutation of the SERCA2b reactive cysteine-674 prevents VEGF- or H₂O₂-induced EC migration. A. Either SERCA2b WT or SERCA2b in which cysteine-674 was mutated to a serine was overexpressed in HAEC and migration into a scratch wound over six hours was measured in response to H₂O₂ (1 μM, n = 3, *p< 0.05). Ca²⁺ levels associated with Ca²⁺ influx were measured in HAEC overexpressing either WT SERCA2b or SERCA2b C674S. B. Summary data of change in Fura2 fluorescence ratio between baseline and maximal Ca²⁺ (n = at least 4, **p< 0.01). C. Representative trace of Fura2 fluorescence ratio in HAEC displayed as mean +/− s.e.m.

Figure 5

Nox2 and Nox4 are required for VEGF-induced S-glutathiolation of SERCA2b. A. HAEC were treated with siRNA for Nox2 and VEGF-induced (50 ng/mL) S-glutathiolation of SERCA2b was detected immunochemically following SERCA2b immunoprecipitation (n = 3, *p< 0.05). B. S-glutathiolation of SERCA2b in response to H₂O₂ treatment (1 μM) was assayed in HAEC in which Nox2 was knocked down (n = 3, *p< 0.05). C. Nox4 was knocked down by shRNA in HAEC and S-glutathiolation of SERCA2b in response to either VEGF or H₂O₂ was assessed (n = 3, *p< 0.05).

Figure 6

Proposed scheme of Nox2 and Nox4-dependent VEGF-induced EC migration. Upon stimulation with VEGF, Nox4-derived H₂O₂ works upstream of Nox2 to stimulate production of superoxide (O₂^-•). VEGF also stimulates activation of eNOS to produce ·NO. Together, O₂^-• and ·NO cause S-glutathiolation of SERCA2b cysteine-674, increasing Ca²⁺ uptake into the endoplasmic reticulum and Ca²⁺ influx into the cytosol, stimulating EC migration.