Critical role of lipid raft redox signaling platforms in endostatin-induced coronary endothelial dysfunction - PubMed (original) (raw)

Comparative Study

Critical role of lipid raft redox signaling platforms in endostatin-induced coronary endothelial dysfunction

Si Jin et al. Arterioscler Thromb Vasc Biol. 2008 Mar.

Abstract

Objective: Endostatin (EST) was found to initiate a redox signaling cascade associated with activation of NADPH oxidase in endothelial cells (ECs). The present study tested whether EST stimulates clustering of ceramide-enriched lipid rafts (LRs), which assembles and activates NADPH oxidase to form redox signaling platforms.

Methods and results: Using confocal microscopy, we first demonstrated a colocalization of LR clusters with NADPH oxidase subunits, gp91(phox) and p47(phox) in the ECs membrane on EST stimulation. Immunoblot analysis of floated detergent-resistant membrane fractions found that in LR fractions NADPH oxidase subunits gp91(phox) and p47(phox) are enriched and that the activity of this enzyme increased dramatically, as measured by electron spin resonance (ESR) spectrometry. This EST-increased LR platform formation was shown to be attenuated by inhibition or RNA interference of acid sphingomyelinase (A-SMase). Functionally, EST pretreatment significantly impaired bradykinin or A23187-induced vasodilation in isolated small coronary arteries, which could be partially reversed by LR disruptors.

Conclusions: The early injury effect of EST on the vascular endothelium is associated with the formation of redox signaling platforms via lipid raft clustering.

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Figures

Figure 1

Figure 1. Representative confocal images of LR clusters (Alexa488-CTX) and gp91_phox_(A), P_47phox_(B) labeling (Texas red-conjugated)

The overlay images exhibited yellow spots or patches (right), which represented co-localization of gp91_phox_ or P47_phox_ and LR component, ganglioside GM1.2. The figure presents representative images from experiments done using 6 batches of ECs cultures.

Figure 2

Figure 2. Distribution and localization of gp91phox and p47phox in floated membrane fractions from ECs

A: using LR marker protein flotillin-1, B: using anti-gp91phox, and C: using anti-p47_phox_ (all were representative gels from 4 separated experiments).

Figure 3

Figure 3. Effects of LRs disruption on EST-induced increase of NADPH oxidase activity in LR-enriched fractions and non-LR fractions

*P < 0.05 vs. vehicle; & p<0.05 vs. EST; # P<0.05 vs. vehicle; n=6.

Figure 4

Figure 4. Effects of LRs disruption on EST-induced O2.− production in CAECs

Summarized data depicting changes in O2.− production in CAECs with different treatments. * indicates P<0.05 vs. control; # indicates P<0.05 vs. EST group; n = 5.

Figure 5

Figure 5. Confocal microscopic analysis of A-SMase in LR clusters in ECs stimulated with EST

Texas-red conjugated anti-A-SMase in the left and LRs markers (Al488-CTX in the middle) were used to stain cells. Desip = desipramine; A-SMase siRNA was A-SMase siRNA transfection. The images are representatives from 6 separated experiments.

Figure 6

Figure 6. Effects of LR disruptors on EST-induced impairment of the vasodilator responses

A: Nyst, responses to BK; D: Nyst, responses to A23187. B: MCD, responses to BK; E: MCD, responses to A23187. C: DPI, apocynin, responses to BK; F: DPI, apocynin, responses to A23187. n=7 or 6, *P<0.05 vs. control.

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