Comparison of artery organ culture and co-culture models for studying endothelial cell migration and its effect on smooth muscle cell proliferation and migration

Abstract

Arterial restenosis associated with intimal hyperplasia is the major cause of long-term failure of vascular interventions. Endothelium injury and the proliferation and migration of smooth muscle cells (SMC) are key events in the development of intimal hyperplasia. The objectives of this study were to develop an ex vivo artery injury model for studying endothelial cell (EC) migration and to compare it with an in vitro co-culture arterial wall injury model in terms of the effect of flow on EC migration and its effect on SMC migration and proliferation. Our results demonstrated that shear flow improves reendothelialization in the injured area by promoting EC migration. The migration distance of ECs is much smaller in the arteries than in an in vitro cell culture model (3.57+/-1.29 mm vs. 5.2+/-1.4 cm, p<0.001). SMC proliferation was significantly less in the EC intact and reendothelialization areas than in the EC denuded areas indicating that reendothelialization suppresses SMC proliferation. Our models provide a new approach to study techniques to enhance endothelium healing.

Figure 1

Photographs of the retractable injury device (a) and the lumen of an artery stained with 0.5% Evans blue dye (b) illustrating a band of the EC injured area in the middle of the artery created by the retractable device. Schematics of ex-vivo artery organ culture system (c) and in-vitro co-culture system (d)

Figure 1

Photographs of the retractable injury device (a) and the lumen of an artery stained with 0.5% Evans blue dye (b) illustrating a band of the EC injured area in the middle of the artery created by the retractable device. Schematics of ex-vivo artery organ culture system (c) and in-vitro co-culture system (d)

Figure 2

Micrographs of arterial longitudinal sections at EC intact (a) and EC denuded (b) areas after being cultured under low flow conditions for 7 days. The internal elastic lamina is intact in both areas. Verhoff stain, scale: 10μm.

Figure 3

Top: Comparison of the width of endothelium denuded area in arteries (mean ± SD, n = 4) after being cultured for 7 days under low and normal arterial shear stress (at low shear stress of 0.2 Pa and arterial shear stress of 1.5 Pa, respectively). ** p<0.01 versus Fresh, # p<0.05 versus Low Shear. Bottom: Comparison of the migration distances of EC in the in vitro co-culture model after being cultured for 7 days under flows at a low shear stress of 0.2 Pa and an arterial shear stress of 1.6 Pa, respectively, ***p<0.001.

Figure 4

Comparison of BrdU index (mean ± SD) in the intima in the EC intact area upstream EC denuded area and in normal arteries without EC denudation after 7 days in organ culture. * p<0.05.

Figure 5

Top panels: Fluorescent micrographs of BrdU-stained arterial longitudinal sections of an EC intact area (a) and an EC denuded area (b) in an artery after being cultured under arterial shear condition for 7 days. Bottom panels: Comparisons of BrdU indices in the EC intact and EC denuded areas of arteries cultured under low shear and arterial shear conditions for 7 days (mean ± SD, n = 4, *p<0.05).

Figure 6

Upper panels: Smooth muscle alpha-actin staining of: a) an upstream EC intact area, b) an EC denuded area, and c) a downstream EC intact area. Bottom panel: Comparison of the medial wall thickness between EC intact and EC denuded areas (mean ± SD, n = 4, *p<0.05)

Figure 7

Distribution of medial SMC proliferation (BrdU index, mean ± SD, n = 4) along the flow direction in arteries cultured under arterial shear for 7 days. Locations 1-3: fully reendothelialized areas, 4-6: incomplete reendothlialization areas, and noEC: un-endothelialized areas. *** p<0.001 versus EC intact.

Figure 8

Distribution of the number of proliferating SMC per section (3 cm in length by 10cm in width) along the flow direction in EC-SMC co-culture model after cultured for 7 days under low shear and arterial shear conditions. Dotted lines with arrows indicate the ranges the EC migrated. n = 4, * p < 0.05, ** p<0.005 versus Arterial Shear.

Figure 9

Top: Smooth muscle alpha-actin staining at EC intact (a) and EC denuded (b) areas after being cultured under arterial shear conditions for 7 days. Scale: 10μm. Middle: Comparisons of areas and aspect ratios (c and d, respectively) of SMCs near the lumen in the EC intact and EC denuded areas (mean ± SD, n = 4, *p<0.05). Bottom: Distributions of the number of migrated SMC per section (2 cm in length by 10 cm in width) along the flow direction in the co-culture model under low shear and arterial shear conditions. n = 3, * p < 0.05, ** p<0.005 versus Arterial Shear.