Flow-induced arterial remodeling in rat mesenteric vasculature

Abstract

This study was designed to characterize in vivo arterial remodeling of male Wistar rat small mesenteric arteries exposed to varying levels of elevated blood flow in the presence of normal arterial pressure. Through a series of arterial ligations, respective ileal artery and second-order branch blood flows acutely increased approximately 36 and approximately 170% over basal levels. Their respective diameters increased 12 and 38% and their wall area increased 58 and 120% in a time-dependent fashion between 1 and 7 days postlitigation compared with same-animal control vessels. Medical extracellular connective tissue increased concomitantly with medical wall hypertrophy. Immunostaining for proliferating cell nuclear antigen and nuclear profile analyses suggests that both smooth muscle and endothelial cell hyperplasia contribute to flow-induced vascular remodeling. The initial stimulus in this model is flow-mediated shear stress, with possible augmentation by hoop stress, which is increased approximately 7% by the resultant vasodilation. Stable wall thickness-to-lumen diameter ratios at 1, 3, and 7 days, however, suggest chronic hoop stress is tightly regulated and remains constant. The model described herein allows analyses of two arteries with different degrees of flow elevation within the same animal and demonstrates that the magnitude of vessel remodeling in vivo is directly dependent on the duration of flow elevation after abrupt arterial occlusion.

Fig. 1.

Rat mesenteric flow model indicating general mesenteric arterial arrangement, sites of ligation, and placement of pressure cannula and flow probe. Diagram depicts model used for both acute hemodynamic measurements and chronic morphological assessments. In chronic studies, proximal branch of high-flow 6th ileal artery was ligated. Results showed significantly increased blood flows in both the ileal and second-order arteries, with significantly elevated calculated shear rate at wall for high-flow branching artery compared with control preligation levels. No significant changes in mean carotid or local mesenteric arterial pressures were detected. This model allows for same-animal control and high-flow vessels. Control vessels were obtained from 9th ileal artery and 2nd-order branch proximal to ileocecal junction and are not included in illustration.

Fig. 2.

Shear stress-induced changes in lumen diameter (A) and medial wall (B) area for ileal and 2nd-order vessels. No interaction was detected between flow and time for ileal artery luminal enlargement, with same-animal control and high-flow vessels both significantly increasing with time. Significant interactions between flow and time were detected for 2nd-order artery lumen diameter, as well as for changes in ileal and 2nd-order artery wall area. Post hoc independent comparisons of control vs. treatment for each time point are indicated. Data were evaluated with 2-way ANOVA using post hoc Bonferroni-corrected t-tests. Values represent means ± SE; n = 10 for each group. Between 3 and 6 cross sections per vessel were analyzed. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control; †P < 0.05, ††P < 0.01 over time.

Fig. 3.

Medial wall thickness-to-lumen diameter ratios for ileal and 2nd-order arteries show no significant differences between same-animal high-flow and control vessels. Data indicate that chronic hoop stress is tightly regulated and stabilized in presence of normal arterial pressure. Between 3 and 6 cross sections per vessel were analyzed. Values represent means ± SE; n = 10 for each group.

Fig. 4.

A stable percentage of extracellular connective tissue in medial wall indicates that as medial wall hypertrophied in response to elevated flow, increased synthesis of medial connective tissue occurred to maintain a normal medial wall microenvironment. Values represent means ± SE; n = 8 for each group. An average of 8 cross sections per vessel were analyzed.

Fig. 5.

Immunocytochemical results for medial smooth muscle cell (SMC) proliferating cell nuclear antigen (PCNA) in ileal and 2nd-order arteries. Data represent %PCNA-positive medial SMC nuclei for high-flow vessels divided by those for same-animal control vessels for both the ileal artery and 2nd-order branch. Data were best represented in this fashion to account for elevated basal levels of PCNA-positive nuclei, which ranged between 7 and 21%. Data suggest significant DNA synthesis in media of SMCs after exposure to elevated shear rates at wall for 3 and 7 days. Values represent means ± SE; n = 6 for each group. Between 3 and 5 cross sections per vessel were analyzed. *P < 0.05, **P < 0.01 vs. control.

Fig. 6.

A: absolute medial SMC nuclear counts indicate significantly increased cell division after in vivo exposure to flow for 7 days for both ileal and 2nd-order arteries compared with same-animal control vessels. B: normalized medial SMC nuclear counts suggest that, at most time points, increased cell division is proportional to increased medial wall area, maintaining a constant SMC density. The 2nd-order vessel demonstrated a significant decrease in cell density on exposure to elevated flow after 7 days, suggesting that cellular hypertrophy and perhaps nuclear polyploidy may be involved in this vessel at this time point. Alternatively, SMCs in this vessel may be actively engaged in the cell cycle before cytokinesis. Values represent means ± SE; n = 6 for each group. Between 3 and 5 cross sections per vessel were analyzed. *P < 0.05 vs. control.

Fig. 7.

Absolute intimal endothelial cell (EC) nuclear counts (A) and intimal EC nuclear counts normalized to 100-μm luminal perimeter (B). Results indicate that once blood flow is increased, shear stress stimulus is rapidly sensed by intimal endothelium leading to EC replication after 24 h continuing through 7 days. Significant interactions were detected between flow and time for normalized EC counts in both ileal and 2nd-order arteries. Values represent means ± SE; n = 10 for ileal artery group and n = 8 for 2nd-order branch group. Between 3 and 5 cross sections per vessel were analyzed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. control; ⁺P < 0.05, ⁺⁺P < 0.01 over time.