Administration of the KCa channel activator SKA-31 improves endothelial function in the aorta of atherosclerosis-prone mice

Figures

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  FIGURE 1. Representative wire myography tracings for cumulative concentration-dependent responses to acetylcholine (ACh) for abdominal aortic rings isolated from WT Normal Diet mice (A), Apoe−/− HFD vehicle-treated mice (B), Apoe−/− HFD SKA-31-treated mice (C), and Apoe−/− HFD senicapoc-treated mice (D). Note that in the representative curves B, C, and D from Apoe−/− mice, a modest contractile response to 10 µM acetylcholine can be seen after the initial relaxation response. W/O: washout of ACh and PE in the vessel bath by replacing the existing bath volume with fresh Krebs-Henseleit buffer. Individual washouts were indicated with black circles. Tension of the force transducer was adjusted to zero after a final washing step if the force recording registered <0 g. Note that 1 μM PE was added to the vessel bath at the end of the main protocol to verify that the acetylcholine-induced relaxation was reversible, and the aortic ring remained viable.

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  FIGURE 2. Acetylcholine-evoked endothelium-dependent relaxation responses in abdominal aortic rings isolated from WT mice and Apoe−/− HFD mice treated with either vehicle or drug. Graph, concentration-response curves for increasing concentrations of acetylcholine (ACh) from 1 nM to 10 µM. Relaxation is reported as % decrease of phenylephrine (PE)-induced contraction (1 µM), with 100% relaxation being the point at which active tone returned to the pre-contraction level (i.e., in the absence of PE). Solid lines in the graph were generated by fitting data points with a non-linear regression equation, as described in the Materials and Methods section. Table, Emax and logEC50 values for the four experimental groups, which were obtained from the non-linear regression fits shown in panel A. The N values reported in the Table represent the number of individual animals within a given experimental group. Data are presented as mean ± SD and were analyzed with a one-way ANOVA, followed by a Tukey’s post-hoc test; ****P < 0.0001 vs. WT Normal Diet. †††† P < 0.0001 vs. Apoe−/− HFD Vehicle.

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  FIGURE 3. Representative wire myography tracings for cumulative concentration-dependent responses to sodium nitroprusside (SNP) for abdominal aortic rings isolated from WT Normal Diet mice (A), Apoe−/− HFD Vehicle-treated mice (B), Apoe−/− HFD SKA-31-treated mice (C), and Apoe−/− HFD senicapoc-treated mice (D). W/O: washout of SNP and PE in the vessel bath by replacing the bath volume with fresh Krebs’ buffer. Individual washouts are indicated with black circles. Tension of the force transducer was re-adjusted to zero after a final washing step if the force recording was <0 g. Note that 1 μM PE was added to the vessel bath at the end of the main protocol to verify the reversibility of the SNP-induced relaxation.

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  FIGURE 4. Sodium nitroprusside-evoked relaxation responses in abdominal aortic rings isolated from WT mice and Apoe−/− HFD mice treated with either vehicle or drug. Graph, concentration-response curves for increasing concentrations of sodium nitroprusside (SNP) from 0.1 nM–1 µM. Relaxation is reported as the % reduction of phenylephrine (PE)-induced contraction (1 µM), with 100% relaxation being the point at which active tone returned to the baseline level (i.e., in the absence of PE). Solid lines in the graph were generated by fitting data points with a non-linear regression equation, as described in the Materials and Methods section. Table, Emax and logEC50 values for the four experimental groups, which were obtained from the non-linear regression fits displayed in panel A. Data were analyzed with a one-way ANOVA, followed by a Tukey’s post-hoc test and are presented as mean ± SD; ****P < 0.0001 vs. WT Normal Diet. The N values reported in the Table represent the number of individual animals within a given experimental group.

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  FIGURE 5. Representative wire myography tracings for cumulative concentration-dependent responses to phenylephrine (PE) for abdominal aortic rings isolated from WT Normal Diet mice (A), Apoe−/− HFD Vehicle-treated mice (B), Apoe−/− HFD SKA-31-treated mice (C), and Apoe−/− HFD senicapoc-treated mice (D). W/O denotes washout of KCl and PE by replacing the bath volume with fresh Krebs’ buffer. Individual washouts are indicated with black circles. Tension of the force transducer was re-adjusted to zero after a final washing step if the force reading registered <0 g.

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  FIGURE 6. Phenylephrine-evoked contractile responses in abdominal aortic rings isolated from WT mice and Apoe−/− HFD mice treated with either vehicle or drug. (A) Concentration-response curve for increasing concentrations of phenylephrine (PE) from 0.1 nM–10 µM. Contraction is reported as % of KCl-induced contraction (60 mM), with 100% contraction being the point at which PE-induced contraction matched the contraction induced by 60 mM KCl. Solid lines in the graph were generated by fitting data points with a non-linear regression equation, as described in the Materials and Methods section. (B) Maximal contraction to 60 mM KCl in the same abdominal aortic rings presented in A, with units in grams of force (g). Table, Emax and logEC50 values for the four experimental groups, which were obtained from the non-linear regression fits displayed in (A). The N values reported in the Table represent the number of individual animals within a given experimental group. Data were analyzed with a one-way ANOVA, followed by a Tukey’s post-hoc test and are presented as mean ± SD. No statistically significant differences were observed.

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  FIGURE 7. Investigation of key endothelial signaling molecules in abdominal aorta by q-PCR analyses. The mRNA levels of the selected molecular targets are plotted relative to the mRNA expression of GAPDH (internal reference). Plotted symbols represent individual animals in the wild-type (WT) normal diet group (n = 9) and Apoe−/− HFD mice treated with vehicle (n = 11–12), 10 mg/kg SKA-31 (n = 11–12) or 40 mg/kg senicapoc (n = 9–12). The asterisk (*) indicates a statistical difference between the WT normal diet mice and Apoe−/− + Vehicle treated mice (P < 0.02). No differences in the expression level of a given mRNA target were noted among the 3 groups of treated Apoe−/− HFD mice (P > 0.2, Kruskal–Wallis one-way ANOVA and Dunn’s post-hoc test).

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  FIGURE 8. Fatty plaque measurement in the thoracic aorta from WT and Apoe−/− HFD mice. (A) Representative en face images of the thoracic aorta from mice in the four experimental groups. The area labelled (1) encompasses the total thoracic aorta (i.e., the areas of (2) + (3) combined). The area labelled (2) consists of the aortic arch and superior vessels. The area labelled (3) consists of the descending thoracic aorta. WT Normal Diet aortae showed very little Oil Red O staining for fatty plaques (red staining made up <1% of total thoracic aorta area) and were excluded from lesion area analyses. Scale bar displayed in the lower righthand corner of images in panel A represents 1 mm. (B) Quantification of lesion area, shown as % of the aortic area covered by lesions. Lesion areas were analyzed with a one-way ANOVA, followed by a Tukey’s post-hoc test; no statistically significant differences were observed. Data are presented as mean ± SD and N values are stated in the panel legend.

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  FIGURE 9. Lesion area measurement in the aortic sinuses of WT and Apoe−/− HFD mice. (A) Representative H&E-stained aortic sinus sections from WT Normal Diet mice (n = 3), Apoe−/− HFD Vehicle-treated mice (n = 9), Apoe−/− HFD SKA-31-treated mice (n = 11), and Apoe−/− HFD senicapoc-treated mice (n = 9). In representative images for WT Normal Diet and Apoe−/− HFD vehicle treated mice, the border of the aortic sinus is outlined in black, and lesion areas inside the aortic sinus are outlined in red. The scale bar displayed in the lower righthand corner of images represents 0.2 mm. (B) Lesion area quantified as a percentage of aortic sinus area. Data in panel B are presented as mean ± SD, and were analyzed with a one-way ANOVA, followed by a Tukey’s post-hoc test; ****P < 0.0001.

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  FIGURE 10. Macrophage content in the aortic sinuses of WT and Apoe−/− HFD mice. (A) Representative anti-Mac-2 immuno-stained aortic sinus sections from WT Normal Diet (n = 3), Apoe−/− HFD Vehicle-treated (n = 10), Apoe−/− HFD SKA-31-treated (n = 10), and Apoe−/− HFD senicapoc-treated (n = 10) mice. The Apoe−/− HFD Vehicle representative image shows a black outline representing the border of the aortic sinus, while the anti-Mac-2-stained areas within the aortic sinus are outlined in red. The scale bar displayed in the lower righthand corner of each image in (A) represents 0.2 mm. (B) Mac-2-stained area plotted as a percentage of the visible aortic sinus area. Data in (B) are presented as mean ± SD and were analyzed with a one-way ANOVA, followed by a Tukey’s post-hoc test; **P < 0.01.

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  FIGURE 11. Detection and quantification of collagen staining in the aortic sinus. (A) shows representative thin histological sections of fixed aortic sinus from wild-type mice (n = 5) and Apoe−/− mice on a high fat diet (HFD) treated with either drug vehicle (n = 9), 10 mg/kg SKA-31 (n = 6–7) or 40 mg/kg senicapoc (n = 9–10). The upper row displays aortic sinus sections viewed under brightfield illumination using differential interference contrast (DIC) filtering, while the lower row shows adjacent sections from the same heart stained with Picrosirius Red (PSR) and illuminated with a linear polarized lens and captured with DIC filtering. All aortic sinus sections displayed in (A) were recorded using a ×4 objective. Brightfield illumination was used to quantify the total lesion area in mm2 per 1 mm2 of aortic lumen area (B), and the percent of total lumen area occupied by atherosclerotic lesion (C). Arrows in the upper and lower images in panel A indicate plaque-like lesions protruding into the aortic lumen. The amount of collagen detected per mm2 of atherosclerotic lesion is displayed in (D) and the percentage of PSR staining observed in total lesion areas is plotted in (E). The scale bar…

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  FIGURE 12. Detection and quantification of alpha-smooth muscle actin (α-SMA) staining in the aortic sinus. (A) shows representative thin histological sections of immune-stained aortic sinus from wild-type (WT) mice (n = 7) and Apoe−/− HFD mice treated with drug vehicle (n = 11), 10 mg/kg SKA-31 (n = 8) or 40 mg/kg senicapoc (n = 7). The upper row displays aortic sinus sections immuno-stained with a rat anti-mouse α-SMA antibody followed by staining with a biotinylated rabbit anti-rat secondary antibody, while the lower row shows adjacent sections from the same heart stained with the biotinylated rabbit secondary antibody alone. Black arrows indicate plaque-like lesions protruding into the aortic lumen. All aortic sinus sections displayed in panel F were recorded using a ×10 objective. Brightfield illumination was used to quantify the percent of total lesion area exhibiting α-SMA staining; these data are quantified in panel (B). The scale bar shown in the lower righthand corner of images in (A) represents 200 μm. Statistical analyses were performed using one-way ANOVA and a Tukey’s post-hoc test; *P < 0.05.