Effects of Axial Stretch on Cell Proliferation and Intimal Thickness in Arteries in Organ Culture

Abstract

Intimal hyperplasia (IH) remains the major cause of intermediate and long-term failure of vascular grafts and endovascular interventions. Arteries are subjected to a significant longitudinal stress in addition to the shear stress and tensile stress from the blood flow. The aim of this study was to determine the effect of axial stretch on cell proliferation and IH in arteries. Porcine carotid arteries, intact or endothelial cell (EC) denudated, were maintained ex vivo at different stretch ratios (1.3, 1.5, and 1.8) and flow rates (16 or 160 mL/min) while remaining at physiologic pressure for 7 days. The viability of the arteries was verified with norepinephrine, carbachol, and sodium nitroprusside stimulations, and the cell proliferation was detected using bromodeoxyuridine labeling and immunostaining. Our results showed that the axial stretch ratio did not significantly affect intimal thickness and cell proliferation in normal arteries. However, axial stretch increased the neointimal thickness in EC denudated arteries cultured under low flow conditions. The cell proliferation increased significantly in the intima and inner half of the media of the EC denudated arteries under normal or elevated axial stretch in comparison to intact arteries at the same stretch ratio. These results demonstrated that axial stretch with EC denudation and low flow increases neointimal formation and cell proliferation in the arteries.

FIGURE 1

Top panels: Typical temporal diameter changes in response to norepinephrine (NE), carbachol (CCh), and sodium nitroprusside (SNP) in normal (a) and EC denudated (b) arteries. Bottom panels: The averaged percent diameter changes of normal (c) and EC denudated (d) arteries. Values are mean ± SD with n = 4 for each group.

FIGURE 2

Micrographs of H&E stained cross sections of fresh arteries and arteries cultured for 7 days. First row: fresh arteries, second row: arteries cultured under normal axial stretch, third row: arteries cultured under elevated axial stretch, and fourth row: arteries cultured under relaxed axial stretch. Left column: EC intact arteries, right column: EC denudated arteries.

FIGURE 3

Comparison of intimal thickness in intact arteries cultured under normal flow for 7 days and EC denudated arteries cultured under low flow for 7 days. Arteries were cultured at different stretch ratios: relaxed (1.3), control (1.5), and stretched (1.8). Values are mean ± SD. n = 3, 5, and 4 for the relaxed, normal, and stretched groups of intact arteries; n = 6, 4, and 5 for the relaxed, normal, and stretched groups of EC denudated groups. ^#p < 0.05 vs. EC denudated, relaxed group.

FIGURE 4

Micrographs (10×) of BrdU stained arterial cross sections of EC denudated arteries cultured for 7 days. First row: arteries cultured under normal axial stretch, second row: arteries cultured under elevated axial stretch, and third row: arteries cultured under relaxed axial stretch. Left column: BrdU-positive nuclei, right column: Hoechst counterstaining.

FIGURE 5

Comparison of BrdU index in intact arteries and EC denudated arteries cultured under normal or low flow for 7 days. Values are mean ± SD. n = 3, 8, 8 for the relaxed, normal, and stretched groups of intact arteries; n = 4 for all EC denudated groups. ** p < 0.01, *** p < 0.001 vs. control EC denu group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. relaxed EC denu group.

FIGURE 6

Micrographs (40×) of smooth muscle α-actin stained arterial cross sections of EC denudated arteries and EC intact arteries. EC denudated arteries were cultured under relaxed (a), normal (b), and stretched (c) axial stretch ratios. Arrows on panels (a) and (b) indicate the internal elastic lamina; intimal hyperplasia is significantly more developed in panel (c) when compared to panel (a). A representative image of an intact artery cultured under relaxed stretch ratio is shown (d). (e) is a fresh artery and (f) is a negative control stain. Negative stain is seen in the intima of EC intacted arteries (d and e).

FIGURE 7

Micrographs of H&E stained cross sections of arteries after 14 days in organ culture (a: EC intact, b: EC denudated).