Vascular surgical stretch injury leads to activation of P2X7 receptors and impaired endothelial function

Figures

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  Fig 1. Decreased endothelial function in a subfailure overstretch RA model was restored with apyrase and P2X7R antagonists FCF and A740003. A. RA was nonstretched (Ctrl), longitudinally stretched to twice its ex vivo length (Stretch), longitudinally stretched before treatment with FCF (100 μM, Stretch+FCF) or stretched before treatment with apyrase (4units/mL, Stretch+Apy). Vessels were pre-contracted with phenylephrine (0.5 μM) and relaxed with carbachol (0.5 μM), n = 7–15, * p < 0.05 compared to control, (paired t-test). B, RA was unstretched (Ctrl) or stretched and incubated with A740003 [19, 26] (100 μM, Stretch+A74) and the vessels were pre-contracted with phenylephrine (0.5 μM) and treated with carbachol (0.5 μM). n = 12 segments from different rats, * p<0.05, (paired t-test).

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  Fig 2. eATP stimulation of RA decreased endothelial function. A. RA rings were treated with eATP (2,5,10, 20 and 25 mM) for 1hour, suspended in the muscle bath, pre-contracted with phenylephrine (0.5 μM) and treated with carbachol (0.5 μM), n = 5–10, * p < 0.05 compared to control, ns = not significant compared to control. B. RA rings were suspended in a muscle bath and either untreated or treated with 2’(3’)-O-(Benzoylbenzoyl) adenosine 5’–triphosphate (BzATP, 0.5, 1, 2, and 5 mM) for 1 hr, and the vessels were pre-contracted with phenylephrine (0.5 μM) and treated with carbachol (0.5 μM). n = 4–7 segments from different rats, * p<0.05 compared to control (paired t-test).

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  Fig 3. Treatment with eATP reduces nitric oxide production in HSV endothelial cells. HSVEC (>70% confluence, passages 2–4) were either untreated (Ctrl) or treated with ATP (2 mM) or ATP and FCF (100 μM) for 2 hours. Cells were stimulated with carbachol (1 μM, CCH) for 10 min at 37°C, and nitric oxide (NO) generated by the cells was measured as nitrite. NO generated with CCH was set as 100%, n = 4 passages, *p < 0.05, (One way ANOVA).

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  Fig 4. eATP induced phosphorylation of p38 MAPK in HSV endothelial cells. HSVEC were untreated (Ctrl), or treated with ATP (2 mM) for 30 minutes. Some cells were pre-treated with FCF (100 μM), or SB203580 (SB, 20 μM) for 1 hour. Phospho-p38 MAPK and total p38 MAPK proteins were quantitated with immunoblotting (adjusted to the loading control GAPDH). (A) Representative western blots of phospho-p38 MAPK and p38 MAPK. (B) Cumulative data showing the relative percent phosphorylation of p38 MAPK. Phosphorylation with eATP alone was set as 100%, n = 4 passages,* p < 0.05, (One way ANOVA).

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  Fig 5. Time course of phosphorylation of p-ERK1/2 MAPK in ATP treated HSVEC. HSVEC were untreated (0) treated with ATP (2 mM) for various time points (10, 30, and 60 minutes) and phospho p44/42 MAPK (P-ERK1/2) and total p44/42 (ERK1/2) proteins were quantitated with immunoblotting (adjusted to the loading control GAPDH). (A) Representative western blot of phospho ERK1/2, (B) Cumulative data showing the phosphorylation (fold) of p ERK1/2 MAPK, * p < 0.05, N = 4, (One way ANOVA).

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  Fig 6. PD 98059 decreases the phosphorylation of ERK in HSVEC, but does not restore ATP-induced decrease in endothelial function in RA. A. HSVEC were untreated (Ctrl), treated with ATP (2 mM), or ATP with 10 μM PD 98059 (PD+ATP) for 20 min and phospho p44/42 MAPK (P-ERK1/2) and total p44/42 (ERK1/2) proteins were quantitated with immunoblotting (adjusted to the loading control GAPDH). Cumulative data showing the phosphorylation (fold) of p ERK1/2 MAPK, * p < 0.05, N = 3, paired t-test. B. RA rings were untreated (Ctrl), treated with eATP (20 mM, ATP) or ATP with 20 μM PD 98059 (PD+ATP) for 1hr, then suspended in the muscle bath and the vessels were pre-contracted with phenylephrine (5 x 10−7 M) and treated with carbachol (5 x 10−7 M). N = 5 segments from different rats, * p<0.05, ns = nonsignificant, (1 way ANOVA).

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  Fig 7. Effect of arginase inhibitor and L-arginine on eATP-induced reduction of nitric oxide production in HSV endothelial cells. HSVEC were either untreated (Ctrl), treated with ATP (2 mM), L-NAME, (100 μM), ATP with L-arginine (L-Arg, 2 mM), or ATP with NOHA (10 μM), for 2 hours. The cells were then stimulated with carbachol (CCH, 1 μM) for 10 minutes and the nitric oxide generated was measured as nitrite using the NO assay kit and relative percent of NO generated was calculated. NO generated with CCH was set as 100%, n = 6 passages, *p < 0.05, (One way ANOVA).

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  Fig 8. Stretch injury of RA decreased endothelial function that was restored with SB 203580. RA was unstretched (Ctrl), longitudinally stretched to twice its length (Stretch), or longitudinally stretched and treated with SB 203580 (SB, 20 μM), NOHA (50 μM) or L-Arginine (L-Arg, 2 mM) and endothelial relaxation was determined as described in Fig.1. n = 7 different rats, * p < 0.05, (paired t-test).

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  Fig 9. Harvest traction stretch injury led to ATP release, activation of P2X7R and endothelial dysfunction. During endoscopic harvest of human saphenous vein (HSV), traction stretch injury leads to release of ATP from vascular cells. The high concentrations of extracellular ATP (eATP) activate P2X7 receptors (P2X7R) which leads to further release of ATP from P2X7R channels and activation of p38MAPK. Brilliant blue FCF (FCF) is an inhibitor of P2X7R and SB203580 is a small molecule inhibitor of p38 MAPK. P38 MAPK leads to arginase activation which decreases nitric oxide (NO) resulting in endothelial dysfunction.