Shear Stress Attenuates Inward Remodeling in Cultured Mouse Thoracodorsal Arteries in an eNOS-Dependent, but Not Hemodynamic Manner, and Increases Cx37 Expression

Abstract

Background: Arteries chronically constricted in culture remodel to smaller diameters. Conversely, elevated luminal shear stress (SS) promotes outward remodeling of arteries in vivo and prevents inward remodeling in culture in a nitric oxide synthase (NOS)-dependent manner.

Objectives: To determine whether SS-induced prevention of inward remodeling in cultured arteries is specifically eNOS-dependent and requires dilation, and whether SS alters the expression of eNOS and other genes potentially involved in remodeling.

Methods: Female mouse thoracodorsal arteries were cannulated, pressurized to 80 mm Hg, and cultured for 2 days with low SS (<7 dyn/cm2), high SS (≥15 dyn/cm2), high SS + L-NAME (NOS inhibitor, 10-4 M), or high SS in arteries from eNOS-/- mice. In separate arteries cultured 1 day with low or high SS, eNOS and connexin (Cx) 37, Cx40, and Cx43 mRNA were assessed with real-time PCR.

Results: High SS caused little change in passive diameters after culture (-4.7 ± 2.0%), which was less than low SS (-18.9 ± 1.4%; p < 0.0001), high SS eNOS-/- (-18.0 ± 1.5; p < 0.001), or high SS + L-NAME (-12.0 ± 0.6%; nonsignificant) despite similar constriction during culture. Cx37 mRNA expression was increased (p < 0.05) with high SS, but other gene levels were not different.

Conclusions: eNOS is involved in SS-induced prevention of inward remodeling in cultured small arteries. This effect does not require NO-mediated dilation. SS increased Cx37.

Keywords: Connexins; Flow; Gap junctions; Remodeling; eNOS.

Figure 1.. Greater luminal shear stress prevents inward remodeling (% Change in Passive Diameter) in mouse thoracodorsal arteries cultured for 2 days.

Passive diameter was measured at 80 mmHg luminal pressure before culture in MOPS-buffered PSS with addition of papaverine (10⁻⁴ M), and after culture with SNP (10⁻⁴ M) or Ca⁺⁺-free MOPS-buffered PSS with EGTA (1mM). Artery reactivity of each treatment group is shown in Table 1 and culture parameters are shown in Table 2 (first two treatment columns in each table).

Figure 2.. The magnitude of inward remodeling (mean ± SEM) in 2-day cultured arteries is determined by the levels of luminal shear stress and presence of nitric oxide and eNOS.

Artery reactivity of each treatment group is shown in Table 1 and culture parameters are shown in Table 2. *** p < 0.001 vs. Low SS and High SS eNOS −/− (one-way ANOVA with Bonferroni post-hoc tests).

Figure 3.. Passive diameters (mean ± SEM) are smaller post-culture compared to pre-culture in Low SS, High SS + L-NAME, or High SS eNOS −/− groups at various pressures.

There were no differences in pre- vs. post-culture diameters in High SS group at any pressure. Pressures were increased step-wise in ~5 min intervals. * p <0.05. ** p < 0.01, *** p < 0.001 compared to pre-culture passive diameter (2-way repeated measures ANOVA with Bonferroni post-hoc tests).

Figure 4.. The levels of shear stress do not regulate the magnitude of constriction (mean ± SEM) during 2-day culture.

Arteries were then cultured at 80 mmHg luminal pressure in Lebovitz medium + FCS for 2 days. Arteries stably constricted (% of Passive Diameter = diameter / initial passive diameter * 100) in the presence of culture medium throughout the culture period with no differences between the groups. Artery reactivity of each treatment group is shown in Table 1 and culture parameters are shown in Table 2.

Figure 5.. Relative mRNA expression normalized to mRNA of smooth muscle cell marker (SMAA) or endothelial cell marker (PECAM-1) in arteries cultured for 1 day with low or high luminal shear stress (SS).

Ratios (mean ± SEM) are expressed relative to the average value for the Low SS group. * p < 0.05, ** p < 0.01 vs. Low SS (two-tailed Student’s t-test). Artery reactivity in each treatment group is shown in Table 3.

Figure 6.. Responsiveness to acetylcholine at the end of 2-day culture is not correlated with the magnitude of inward remodeling.

Arteries from each treatment group displayed a range of reactivity to acetylcholine at the end of culture (x-axis) that did not correlate with the magnitude of remodeling (y-axis). Linear regression lines shown for each treatment group are not significant.