Differential progressive remodeling of coronary and cerebral arteries and arterioles in an aortic coarctation model of hypertension

Abstract

Objectives: Effects of hypertension on arteries and arterioles often manifest first as a thickened wall, with associated changes in passive material properties (e.g., stiffness) or function (e.g., cellular phenotype, synthesis and removal rates, and vasomotor responsiveness). Less is known, however, regarding the relative evolution of such changes in vessels from different vascular beds.

Methods: We used an aortic coarctation model of hypertension in the mini-pig to elucidate spatiotemporal changes in geometry and wall composition (including layer-specific thicknesses as well as presence of collagen, elastin, smooth muscle, endothelial, macrophage, and hematopoietic cells) in three different arterial beds, specifically aortic, cerebral, and coronary, and vasodilator function in two different arteriolar beds, the cerebral and coronary.

Results: Marked geometric and structural changes occurred in the thoracic aorta and left anterior descending coronary artery within 2 weeks of the establishment of hypertension and continued to increase over the 8-week study period. In contrast, no significant changes were observed in the middle cerebral arteries from the same animals. Consistent with these differential findings at the arterial level, we also found a diminished nitric oxide-mediated dilation to adenosine at 8 weeks of hypertension in coronary arterioles, but not cerebral arterioles.

Conclusion: These findings, coupled with the observation that temporal changes in wall constituents and the presence of macrophages differed significantly between the thoracic aorta and coronary arteries, confirm a strong differential progressive remodeling within different vascular beds. Taken together, these results suggest a spatiotemporal progression of vascular remodeling, beginning first in large elastic arteries and delayed in distal vessels.

Keywords: aorta; arterial remodeling; arteriolar function; collagen; elastin; neointima; vascular smooth muscle cell.

Figure 1

Differences in arterial structure and quantification of inner radii over time. The panel on the left shows representative histological images of the descending thoracic aorta, just proximal to the site of the balloon occluder (P-Ao), the left anterior descending (LAD) coronary artery, and the middle cerebral artery (MCA) from a control normotensive mini-pig stained with Verhoeff–Van Gieson (VVG) for elastin in black and collagen/smooth muscle in pink. Inner radius of control (week 0) and hypertensive arteries (weeks 2, 4, 6, and 8), mean ± SEM, for P-Ao (A), LAD (B), and MCA (C). The inner radius was calculated using the circle formula based on the wall area calculated from images showing the entire artery. **0.001 < p < 0.01, two-way ANOVA with Bonferroni post hoc analysis.

Figure 2

Unloaded total wall thickness (mean ± SEM) and its distribution amongst the intima, media, and adventitia in the descending thoracic aorta just proximal to the balloon occluder (P-Ao), the left anterior descending artery (LAD), and the middle cerebral artery (MCA) before creation of the coarctation (week 0) and at 2, 4, 6, and 8 weeks thereafter. (A) The P-Ao thickened significantly in the media at and after 2 weeks. (B) The LAD thickened significantly via both the adventitia at 2 weeks, and media at 4 and 8 weeks. (C) The changes in the MCA at week 4 may have been anomalous. *0.01 < p < 0.05, **0.001 < p < 0.01, ***p < 0.001, two-way ANOVA with Bonferroni post hoc analysis.

Figure 3

Percentage of structurally significant constituents (collagen, smooth muscle, and elastin) in the arterial wall of the proximal aorta (P-Ao), left anterior descending artery, (LAD), and middle cerebral artery (MCA), mean ± SEM. (A) Collagen increased significantly in P-Ao at and after 4 weeks; SMA increased at week 2 (ns) and proceeded to decrease significantly compared to week 2. (B) In the LAD, collagen was significantly decreased at and after 4 weeks while smooth muscle was significantly increased. (C) These constituents did not change significantly in the MCA. *0.01 < p < 0.05, **0.001 < p < 0.01, ***p < 0.001, two-way ANOVA with Bonferroni post hoc analysis.

Figure 4

Macrophages and CD34 positive cells are seen in all three artery types. (A) Representative images of the P-Ao (top) and LAD (bottom) at 4 weeks of hypertension. The zoomed-in images to the left highlight macrophages observed in the adventitia. The percentage of macrophage present was determined as the ratio of the positively stained area compared to the total arterial area. In the P-Ao, macrophage involvement was greatest in the adventitia near the vasa vasorum at week 2. In the LAD, relative macrophage content was greater at week 2 and remained elevated at weeks 6 and 8 although not significantly. (B) Representative images of CD34 stained (red), LAD (left), and MCA (to the right) for the normotensive controls. The zoomed-in images to the left and right highlight the presence of CD34 positive cells in the intima and media of both arteries. *0.01 < p < 0.05, two-way ANOVA with Bonferroni post hoc analysis.

Figure 5

Representative images of birefringent collagen in the P-Ao (left column) and fluorescent αSMA in the LAD (right column) at all observed time points. Arteries have been cropped to the adventitial and luminal boarders.

Figure 6

Diminished nitric oxide-mediated dilation to adenosine at eight weeks of hypertension in coronary arterioles, but not cerebral arterioles. (A) Coronary arterioles isolated from normotensive (NT) pigs dilated in a concentration-dependent manner to adenosine (n = 5). The NOS inhibitor l-NAME (10 μmol/l) attenuated this vasodilator response (n = 5). Vasodilation to adenosine (n = 5) was attenuated in the coronary arterioles isolated from 8-week hypertensive (HT) pigs. l-NAME did not affect this vasodilation (n = 5). (B) Cerebral arterioles isolated from NT (n = 5) and HT (n = 5) pigs dilated in a comparable manner to adenosine. l-NAME attenuated dilation of cerebral arterioles from NT (n = 5) and HT (n = 5) pigs in a comparable manner. *p < 0.05 between groups by two-way ANOVA or within groups by repeated-measures two-way ANOVA. n = number of vessels, excised one per animal.

Figure A1

Algorithm created to determine the area fraction of elastin in the wall. First, a threshold was used to determine the “black” elastin from the VVG stained images. Second, a threshold was added to remove the black nuclei blobs. Then, the ratio of elastin pixels to wall pixels (non-blue portion) was determined.

Figure A2

The number of nuclei were normalized compared to the most dense segment in the radial direction and plotted as a function through the arterial wall going from lumen “0” to adventitia “1” for each artery type [(A) P-Ao, (B) LAD, (C) MCA] at each experimental time point (NT control, and 2, 4, 6, and 8 weeks HT).

Figure A3

The relative percentage of CD34, a marker for hemopeietic progenitor cells, in the P-Ao, LAD and MCA. The change in CD34 was not statistically significant for any artery type over the 8-weeks study period as compared by two-way ANOVA with Bonferroni post hoc analysis.