Tracking endothelium-dependent NO release in pressurized arteries

Figures

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  FIGURE 1. Three experimental setups for the characterization of NO-dyes. (A) Cell-free setup with no artery present. NO-dye was added to a 2 mL chamber, and fluorescence detected 200 µm above the coverslip. Both PBS and KPS were used at 25°C and KPS also at 37°C. (B) Isolated, cannulated and pressurized artery in KPS at 37°C, with SMCs facing the coverslip. (C) Isolated, opened and stretched artery in KPS at 37°C for en face imaging, with ECs facing the coverslip. 1, a securing device (pipette or micro clamp); 2, an arm to hold the securing device (micromanipulator or caliper) that can be adjusted laterally to stretch the artery held above 3, a coverslip; imaged using an inverted microscope.

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  FIGURE 2. Effect of Cu2+ on artery function. Arteries were mounted in a wire myograph for measuring isometric tension. (A) Effect of 1 µM Cu2+ on concentration-dependent relaxation to SNAP in arteries contracted to PE. Non-parametric paired Wilcoxon test for log EC50 values; *, p = 0.031 vs. Cu2+-free (n = 6). (B) Effect of Cu2+ on concentration-dependent relaxation to ACh in arteries contracted to PE. Non-parametric unpaired KruskalWallis test with Dunn’s multiple comparisons; p = 0.742 and p = 0.051 in the presence of 2 μM and 5 µM Cu2+ vs. Cu2+-free, respectively (n = 4 for each). (C) Effect of Cu2+ on contraction to PE. Parametric paired t-test; *, p = 0.038 and p = 0.002 in the presence of 2 μM and 5 µM Cu2+ vs. Cu2+-free, (n = 8 and n = 5), respectively.

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  FIGURE 3. Cu2FL2E response to SNAP in the cell-free setup. Time course (A) and summary (B) of concentration-dependent increases in Cu2FL2E fluorescence in response to SNAP. Cu2FL2E was used at 1 μM, working ratio 1:2 in PBS at 25°C. SNAP was added to the chamber at t = 0 min. Repeated measures ANOVA with Tukey’s multiple comparisons test (n = 3 for all experiments); *, p < 0.0001 and p = 0.022 for 10 μM and 100 µM vs. 1 µM SNAP, respectively; #, p = 0.047 vs. 10 µM SNAP.

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  FIGURE 4. Effect of Cu2+ on Cu2FL2E response to SNAP in the cell-free setup. Time course (A) and summary (B) of concentration-dependent increases in Cu2FL2E fluorescence in response to 10 µM SNAP. Cu2FL2E was used at 1 μM, working ratios 1:2, 1:5, and 1:10 in PBS at 25°C. SNAP was added to the chamber at t = 0 min. Parametric paired t-test (n = 3); *, p = 0.031 vs. 2 µM Cu2+. (C) Summary of response to 10 µM SNAP when gassed KPS was used (n = 4–6).

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  FIGURE 5. DAR-4M AM response to SNAP in the cell-free setup. (A,B) Effect of 10 µM SNAP and Cu2+ on DAR-4M AM fluorescence. DAR-4M AM was used at 5 μM, in PBS at 25°C. Ordinary one-way ANOVA; p = 0.8390 and p = 0.2382 in the presence of 2 μM and 5 µM Cu2+ vs. Cu2+-free (n = 3). (C) Effect of 10 µM SNAP and Cu2+ on DAR-4M AM fluorescence in gassed KPS at either 25°C or 37°C. In all experiments SNAP was added to the chamber for 10 min. Ordinary one-way ANOVA; p = 0.884 at 37°C vs. 25°C; *, at 37°C in the presence of 1 µM Cu2+ vs. Cu2+-free.

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  FIGURE 6. Cu2FL2E response to ACh and SNAP in the pressurized artery setup. (A) Representative confocal micrographs illustrating the ability of Cu2FL2E to load arteries. While the NO-dye clearly labelled elastin (black holes visible in IEL), SMCs were often visible. Cu2FL2E was used at 2.5 µM, working ratio 1:2 in gassed KPS at 37°C. The elastin was separately labelled with Alexa Fluor® 633 hydrazide (AF, 10 nM). The bottom panels are cross sections through the artery wall at point indicated by magenta line. Merged z-stacks 13.5 µm thick into an x-y image. (B) Time course of changes in NO-dye and AF fluorescence following addition of 1 µM ACh at t = 0 min and 10 µM SNAP at 30 min (Supplementary Movie S1); representative of >5 experiments with slightly modified loading protocols. Time courses are also expressed as either a fluorescence ratio (FR) (C) or FR/FR0 (where t = 0 is always 1, dashed line) (D). There was no clear response above the steady, gradual rise in Cu2FL2E fluorescence observed. Refer to Figure 7 for comparison.

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  FIGURE 7. DAR-4M AM response to ACh and SNAP in the pressurized artery setup. (A) Representative confocal micrographs illustrating the ability of DAR-4M AM to load arteries. While the NO-dye clearly labelled elastin (black holes visible in IEL), SMCs were often visible. DAR-4M AM was used at 5 μM, in gassed KPS at 37°C. The elastin was separately labelled with Alexa Fluor® 633 hydrazide (AF, 10 nM). Merged z-stacks 13.5 µm thick into an x-y image. (B) Time course of changes in NO-dye and AF fluorescence following addition of 1 µM ACh at t = 0 min and 10 µM SNAP at 30 min (Supplementary Movie S2). Time courses are also expressed as either a fluorescence ratio (FR) (C) or FR/FR0 (where t = 0 is always 1, dashed line) (D).

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  FIGURE 8. NO-dye response to ACh and SNAP in the pressurized artery setup. Comparison of summary data for each NO-dye. In all experiments 1 µM ACh was added at t = 0 min, and 10 µM SNAP at 30 min. (A) Summary data expressed as FR/FR0 for Cu2FL2E (n = 3) and DAR-4M (n = 7) loaded arteries. (B) Summary data for Cu2FL2E. Repeated measures one-way ANOVA with Dunnett’s multiple comparisons test; *, p = 0.050, p = 0.019, p = 0.006 for t = −30 min, 30 min, and 60 min vs. 0 min, respectively. (C) Summary data for DAR-4M. Repeated measures one-way ANOVA with Dunnett’s multiple comparisons test; p = 0.838, *, p = 0.017, and *, p = 0.004 for t = −30 min, 30 min, and 60 min vs. 0 min, respectively.

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  FIGURE 9. Summary of DAR-4M AM response to ACh and SNAP in the pressurized artery setup. In these experiments responses to 1 µM ACh (n = 7) were compared to control (basal accumulation without ACh, n = 5) and 1 µM ACh in the presence of L-NAME (L-N, 100 μM, n = 6), each added at t = 0 min. In all experiments 10 µM SNAP was added at 30 min. (A) Summary data expressed as FR/FR0 (A) and then as a percentage of the response to SNAP to better normalize the response between arteries (B). Values at 30 min are summarized in (C). Ordinary one-way ANOVA with Tukey’s multiple comparisons test; p = 0.238 for ACh vs. control; p = 0.576 and #, p = 0.017 vs. L-N + ACh. Add, addition of either nothing (control), ACh or L-N and ACh. (D) Linear regression of individual experiments were averaged to obtain an index of the rate of NO release over the t = 0–30 min period (min−1); shown with 95% confidence intervals. Values are summarized in (E). Ordinary one-way ANOVA with Tukey’s multiple comparisons test; p = 0.286 for ACh vs. control; p = 0.575 and #, p = 0.036 vs. L-N + ACh.

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  FIGURE 10. NO-dye response to ACh and SNAP in the en face artery setup. Representative confocal micrographs illustrating the ability of DAR-4M AM (5 µM) (A) and Cu2FL2E (2.5 µM, working ratio 1:2) (B) to load arteries in gassed KPS at 37°C. The elastin was separately labelled with Alexa Fluor® 633 hydrazide (AF, 10 nM). Merged z-stacks 13.5 µm thick into an x-y image. (C) Time course of changes in NO-dye and AF fluorescence following addition of 1 µM ACh at t = 0 min and 10 µM SNAP at 30 min. This protocol was repeated at least 3 times.

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  FIGURE 11. NO-dye response to ACh and SNAP in the en face artery setup. Comparison of summary data for each NO-dye. In all experiments 1 µM ACh was added at t = 0 min, and 10 µM SNAP at 30 min. (A) Summary data expressed as FR/FR0 for Cu2FL2E (n = 3) and DAR-4M (n = 3) loaded arteries. (B) Summary data for Cu2FL2E. Repeated measures one-way ANOVA with Dunnett’s multiple comparisons test; p = 0.758, *, p = 0.006 and p = 0.010 for t = −30 min, 30 min, and 60 min vs. 0 min, respectively. (C) Summary data for DAR-4M. Repeated measures one-way ANOVA with Dunnett’s multiple comparisons test; p = 0.952, p = 0.277, and p = 0.279 for t = −30 min, 30 min, and 60 min vs. 0 min, respectively.