Cocoa shell extract supplementation in hypertensive rats induces outward hypertrophic remodelling in mesenteric resistance arteries, reducing fibrosis

Figures

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  FIGURE 1. Experimental design. Abbreviations: CSE, cocoa shell extract; DAPI, 4′,6‐diamidino‐2‐phenylindole; MUN, maternal undernutrition; VEH, vehicle.

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  FIGURE 2. Pressure–diameter curves. Data were obtained by pressure myography in MRA from control and MUN adult rats after 3 weeks of supplementation with a neutral gelatine (VEH) or CSE. The p‐value and Cohen's d index were obtained from the AUC by a linear mixed‐effects model considering supplementation as a fixed effect and dam as a random effect. Each experimental group includes one or two segments from six rats and three different dams. Abbreviations: AUC, area under the curve; CSE, cocoa shell extract; CTRL, control; MRA, mesenteric resistance artery; MUN, maternal undernutrition; VEH, vehicle.

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  FIGURE 3. Pressure–WT, pressure–CSA and wall‐to‐lumen ratio. Data were obtained by pressure myography in MRA from control and MUN adult rats after 3 weeks of supplementation with a neutral gelatine (VEH) or CSE. The p‐value and Cohen's d index were obtained from the AUC by a linear mixed‐effects model considering supplementation as a fixed effect and dam as a random effect. Each experimental group includes one or two segments from six rats and three different dams. Abbreviations: AUC, area under the curve; CSA, cross‐sectional area; CSE, cocoa shell extract; CTRL, control; MUN, maternal undernutrition; VEH, vehicle; WT, wall thickness.

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  FIGURE 4. Stress–strain relationship and calculated β‐values. Data were obtained by pressure myography in MRA from control and MUN adult rats after 3 weeks of supplementation with a neutral gelatine (VEH) or CSE. Left panels show the adjusted stress–strain curves and right panels the median with interquartile range (Q1; Q3) of the β‐values obtained from them. The p‐value and Cohen's d index were obtained by a linear mixed‐effects model considering supplementation as a fixed effect and dam as a random effect. Each experimental group includes one or two segments from six rats and three different dams. Abbreviations: CSE, cocoa shell extract; CTRL, control; MRA, mesenteric resistance artery; MUN, maternal undernutrition; VEH, vehicle.

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  FIGURE 5. Area of fenestrae in the IEL. Data were obtained by confocal microscopy in pressure‐fixed MRA segments from control and MUN adult rats after 3 weeks of supplementation with a neutral gelatine (VEH) or CSE. The p‐value and Cohen's d index were obtained by a linear mixed‐effects model considering supplementation as a fixed effect and dam as a random effect. Each experimental group includes one or two segments from six rats and three different dams. Representative image projections were reconstructed from serial images taken with a ×40 zoom × 4 at 488 nm/500–600 nm (elastin autofluorescence). Image size 512 pixels × 512 pixels. Scale bars: 25 µm. Abbreviations: CSE, cocoa shell extract; CTRL, control; IEL, internal elastic lamina; MRA, mesenteric resistance artery; MUN, maternal undernutrition; VEH, vehicle.

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  FIGURE 6. Adventitial collagen content. Data were obtained by confocal microscopy in pressure‐fixed MRA segments stained with collagen antibody, from control and MUN adult rats after 3 weeks of supplementation with a neutral gelatine (VEH) or CSE. The p‐value and Cohen's d index were obtained by a linear mixed‐effects model considering supplementation as a fixed effect and dam as a random effect. Each experimental group includes one or two segments from six rats and three different dams. Representative image projections were reconstructed from serial images taken with a ×40 zoom × 4 at 555 nm/569–600 nm (Cy3 secondary anti‐collagen antibody). Image size 512 pixels × 512 pixels. Scale bars: 25 µm. Abbreviations: CSE, cocoa shell extract; CTRL, control; MRA, mesenteric resistance artery; MUN, maternal undernutrition; VEH, vehicle.

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  FIGURE 7. Adventitia and media layer characteristics. Data were obtained by confocal microscopy in pressure‐fixed MRA segments from control and MUN adult rats after 3‐weeks of supplementation with a neutral gelatine (VEH) or CSE. Arteries were visualized at 359 nm excitation/457 nm emission wavelengths (4′,6‐diamidino‐2‐phenylindole staining). The p‐value and Cohen's d index were obtained by a linear mixed‐effects model considering supplementation as a fixed effect and dam as a random effect. Each experimental group includes one or two segments from six rats and three different dams. Abbreviations: CSE, cocoa shell extract; CTRL, control; MRA, mesenteric resistance artery; MUN, maternal undernutrition; VSMC, vascular smooth muscle cell.

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  FIGURE 8. Contractile responses. Data were assessed by wire myography in control and MUN adult rats before and after 3 weeks of supplementation with a neutral gelatine (VEH) or CSE. The p‐value and Cohen's d index were obtained from the AUC by a linear mixed‐effects model considering supplementation as a fixed effect and dam as a random effect. Each experimental group includes one or two segments from six rats and three different dams. n = 6 rats per group. Abbreviations: AUC, area under the curve; CSE, cocoa shell extract; CTRL, control; MUN, maternal undernutrition; NA, noradrenaline; VEH, vehicle.

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  FIGURE 9. Summary of effects of CSE supplementation on mesenteric resistance artery structure and mechanical function in male rats. Mesenteric resistance artery from MUN hypertensive males exhibits inward hypotrophic remodelling and increased stiffness, related to elevated collagen content. CSE supplementation enlarged the lumen size and the medial layer through smooth muscle cell hypertrophy in both control and MUN males. In MUN hypertensive males, supplementation also improved elasticity through reduction of collagen content and increasing area of fenestrae. Abbreviations: CSE, cocoa shell extract; MUN, maternal undernutrition; VEH, vehicle.