H2O2-induced endothelial NO production contributes to vascular cell apoptosis and increased permeability in rat venules

Abstract

Although elevated levels of H(2)O(2) have been implicated to play important roles in the pathogenesis of various cardiovascular diseases, the underlying mechanisms remain unclear. This study aims to examine the effect of H(2)O(2) on endothelial nitric oxide (NO) production in intact venules, and elucidate the role and mechanisms of NO in H(2)O(2)-induced increases in microvessel permeability. Experiments were conducted on individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp), and endothelial [Ca(2+)](i) was measured on fura-2-loaded vessels. Perfusion of H(2)O(2) (10 μM) caused a delayed and progressively increased endothelial [Ca(2+)](i) and Lp, a pattern different from inflammatory mediator-induced immediate and transient response. Under the same experimental conditions, measuring endothelial NO via DAF-2 and the spatial detection of cell apoptosis by fluorescent markers revealed that H(2)O(2) induced two phases of NO production followed by caspase activation, intracellular Ca(2+) accumulation, and vascular cell apoptosis. The initial NO production was correlated with increased endothelial NO synthase (eNOS) Ser(1177) phosphorylation in the absence of elevated endothelial [Ca(2+)](i), whereas the second phase of NO depended on increased [Ca(2+)](i) and was associated with Thr(495) dephosphorylation without increased Ser(1177) phosphorylation. Inhibition of NOS prevented H(2)O(2)-induced caspase activation, cell apoptosis, and increases in endothelial [Ca(2+)](i) and Lp. Our results indicate that H(2)O(2) at micromolar concentration is able to induce a large magnitude of NO in intact venules, causing caspase activation-mediated endothelial Ca(2+) accumulation, cell apoptosis, and increases in permeability. The mechanisms revealed from intact microvessels may contribute to the pathogenesis of oxidant-related cardiovascular diseases.

Fig. 1.

H₂O₂ induces delayed and progressive increases in microvessel hydraulic conductivity (Lp) and EC intracellular calcium concentration ([Ca²⁺]_i). A: representative Lp measurements from one individual experiment after 2-h perfusion with H₂O₂ (10 μM). B: summarized Lp data showing the time-dependent Lp response to H₂O₂. *P < 0.05, significant increase from control. C: the pooled time course of changes in EC [Ca²⁺]_i in H₂O₂ (N = 5) or albumin-Ringer solution (N = 3) perfused vessels.

Fig. 2.

H₂O₂ (10 μM) induces Ca²⁺ influx-independent and -dependent endothelial NO production. A: pooled cumulative FI curve of DAF-2 upon H₂O₂ perfusion in the absence (N = 5) or presence of LaCl₃ (N = 4), AP-CAV (N = 4), and N^G-monomethyl-l-arginine (l-NMMA) (N = 4). B: the time course of NO production rate (df/dt, left Y-axis) superimposed with the changes in EC [Ca²⁺]_i (right Y-axis) in H₂O₂-perfused vessels in the absence or presence of LaCl₃ (pooled data). The shaded areas represent the two phases of NO production. C: representative DAF-2 fluorescence images from one individual experiment, showing the changes in FI_DAF over time after H₂O₂ perfusion. The color scale shows the FI_DAF in arbitrary unit.

Fig. 3.

H₂O₂-induced changes in endothelial nitric oxide synthase (eNOS) phosphorylation at Ser¹¹⁷⁷ and Thr⁴⁹⁵ and the roles of Ca²⁺/CaM in H₂O₂-induced eNOS activation. A: representative confocal images of fluorescent immunostaining of eNOS, phosphorylated eNOS at Ser¹¹⁷⁷ (top panel) and Thr⁴⁹⁵ (bottom panel) in control (albumin-Ringer solution) and H₂O₂ perfused vessels. The time labeled on each image represents the duration of H₂O₂ perfusion. The fluorescence intensity quantifications of each group of images are shown in B–E. The eNOS staining showed no significant change in eNOS expression before and after H₂O₂ perfusion. The last image (far right of bottom panel) is the negative control with second antibody alone. The nuclei were stained with DRAQ5 (red). Each image is the projection of the lower half of the vessel and represents the pattern of multiple segments of three vessels. The scale bar represents 30 μm. B: temporal correlations between the changes in NO production rate (left Y-axis) and eNOS phosphorylation at Ser¹¹⁷⁷ (right Y-axis). A transient increase in Ser¹¹⁷⁷ phosphorylation is correlated with the initial NO production with the peak at 1 min of H₂O₂ perfusion and then declined with time. No increase in Ser¹¹⁷⁷ phosphorylation occurred at the second phase of NO production (25 min of H₂O₂ perfusion). C: changes in eNOS phosphorylation at Thr⁴⁹⁵. Thr⁴⁹⁵ phosphorylation showed a modest decrease from the control level at the initial peak NO (1 min of H₂O₂ perfusion) and a more significant decrease at the second phase of NO (25 min of H₂O₂ exposure). D: effects of AP-CAV (10 μM) on H₂O₂-induced NO production (N = 3). Preperfusion of vessels with AP-CAV attenuated the H₂O₂-induced initial NO and abolished the second phase of NO production. E: effect of AP-CAV on H₂O₂-induced eNOS phosphorylation at Ser¹¹⁷⁷ (N = 3 per group). AP-CAV attenuated the H₂O₂-induced initial increase in Ser¹¹⁷⁷ phosphorylation (a representative image is shown in A, far right top panel). †P < 0.05, significant decrease from control (C); significant decrease from H₂O₂ responses (D and E); N = 3 per group.

Fig. 4.

H₂O₂-induced vascular cell apoptosis in the absence or presence of l-NMMA, z-VAD-FMK, and LaCl₃. A: representative confocal images of FLICA, Alexa488-Annexin-V, Calcein-AM, and Alexa488-Annexin-V with Calcein-AM double staining in each individual experiment. Vessels were perfused with 1% albumin-Ringer solution (first row), H₂O₂ at 10 (second row) and 100 μM (third row) for different time periods as shown on the lower left of each image. Arrows indicate the endothelial cells (ECs) and arrowheads indicate pericytes. *ECs not stained with Calcein-AM. B: inhibitory effect of l-NMMA , z-VAD-FMK, and LaCl₃ on H₂O₂-induced vascular cell apoptosis (N = 6 per group). After preperfusion of l-NMMA, z-VAD-FMK, or LaCl₃ for 30, 30, and 20 min, respectively, each vessel was perfused with H₂O₂ (10 μM) in the presence of each compound. The vessel was double-stained by FLICA (first column) or Annexin-V (second column) with Calcein-AM (third column). Each confocal image is the projection of the bottom half of the vessel. The scale bar represents 30 μm. The dotted line outlines the vascular wall. C: summary of the FLICA staining quantifications in each group. *P < 0.05, significant increase from control (BSA). †P < 0.05, significant decrease from the response to H₂O₂ treatment alone.

Fig. 5.

l-NMMA prevents H₂O₂-induced delayed increases in EC [Ca²⁺]_i and Lp. A and B: H₂O₂-induced changes in Lp and EC [Ca²⁺]_i in the presence of l-NMMA in 2 individual experiments. The application of l-NMMA prevented H₂O₂-induced increases in EC [Ca²⁺]_i and Lp. C: summary of the Lp (N = 5) and EC [Ca²⁺]_i (N = 4) results. †P < 0.05, significant decrease from the Lp and EC [Ca²⁺]_i responses to H₂O₂ in the absence of l-NMMA.

Fig. 6.

Effects of Z-VAD-FMK on H₂O₂-induced NO production, EC Ca²⁺ accumulation, and increases in Lp. A: z-VAD-FMK has no effect on H₂O₂-induced initial NO production, but prevents the second phase of NO production (N = 3). †P < 0.05, significant decrease from responses to H₂O₂ treatment alone. B and C: individual experiments show that z-VAD-FMK prevents the H₂O₂-induced delayed increases in EC [Ca²⁺]_i and Lp. D: summarized EC [Ca²⁺]_i (N = 4) and Lp results (N = 5). *P < 0.05, significant increase from control. †P < 0.05, significant decrease from the responses to H₂O₂ treatment alone.

Fig. 7.

LaCl₃ abolishes H₂O₂-induced endothelial Ca²⁺ accumulation and Lp increases. A: an individual experiment of Lp measurements in the presence of LaCl₃ (50 μM). B: summarized EC [Ca²⁺]_i and Lp results (N = 4 per group). *P < 0.05, significant increase from control. †P < 0.05, significant decrease from H₂O₂-induced response.

Fig. 8.

Schematic diagram demonstrating the underlying mechanisms by which H₂O₂ induces eNOS activation and increases in microvessel permeability. H₂O₂, at near pathological concentration, induces two phases of eNOS activation in ECs of intact venules. The initial eNOS activation is independent of increased EC [Ca²⁺]_i but is associated with increased eNOS phosphorylation at Ser¹¹⁷⁷ and decreased phosphorylation at Thr⁴⁹⁵. This initial NO production leads to the activation of caspase cascade, which in turn disrupts the intracellular Ca²⁺ homeostasis, causing delayed Ca²⁺ accumulation, vascular cell apoptosis, and increases in microvessel permeability. Blockade of NO production by l-NMMA inhibits all the downstream events. Of note, the delayed increase in EC [Ca²⁺]_i also triggers a second pulse of elevated EC [Ca²⁺]_i-dependent eNOS activation which is associated with a decrease in eNOS Thr⁴⁹⁵ phosphorylation and an increase in CaM binding. CAV-1, a CaM binding antagonist, abolishes the second NO production. Caspase activation is further amplified by the delayed increases in EC [Ca²⁺]_i and the second NO production through positive feedback, leading to Ca²⁺ overload and cell apoptosis. Dashed lines indicate the positive-feedback loop. Each inhibitor listed in the shaded box can effectively block the downstream events.