Contribution of endothelial injury and inflammation in early phase to vein graft failure: the causal factors impact on the development of intimal hyperplasia in murine models

Abstract

Objectives: Autologous veins are preferred conduits in by-pass surgery. However, long-term results are hampered by limited patency due to intimal hyperplasia. Although mechanisms involved in development of intimal hyperplasia have been established, the role of inflammatory processes is still unclear. Here, we studied leukocyte recruitment and intimal hyperplasia in inferior vena cava grafts transferred to abdominal aorta in mice.

Methods and results: Several microscopic techniques were used to study endothelium denudation and regeneration and leukocyte recruitment on endothelium. Scanning electron microscopy demonstrated denudation of vein graft endothelium 7 days post-transfer and complete endothelial regeneration by 28 days. Examination of vein grafts transferred to mice transgenic for green fluorescent protein under Tie2 promoter in endothelial cells showed regeneration of graft endothelium from the adjacent aorta. Intravital microscopy revealed recruitment of leukocytes in vein grafts at 7 days in wild type mice, which had tapered off by 28 days. At 28 and 63 days there was significant development of intimal hyperplasia. In contrast; no injury, leukocyte recruitment nor intimal hyperplasia occurred in arterial grafts. Leukocyte recruitment was reduced in vein grafts in mice deficient in E- and P-selectin. In parallel, intimal hyperplasia was reduced in vein grafts in mice deficient in E- and P-selectin and in wild type mice receiving P-selectin/E-selectin function-blocking antibodies.

Conclusion: The results show that early phase endothelial injury and inflammation are crucial processes in intimal hyperplasia in murine vein grafts. The data implicate endothelial selectins as targets for intervention of vein graft disease.

Figure 1. Endothelial structure in vascular grafts.

Scanning electron microscopy images of endothelium in AGs and VGs (magnification 1K to 1.9K, scale bar = 50 µm). Images demonstrate endothelium in native aorta, native IVC (A), VGs and AGs (B) at different time point in WT mice. Bar graph shows endothelial coverage area change in AGs and VGs (C). Arrow = red blood cells, Triangle arrow = leukocytes, ** = denudated endothelium. Error bars represent mean±SEM. *p<0.05.

Figure 2. Endothelial regeneration in VGs.

Confocal microscopy images of endothelium in IVC and in VGs. Endothelium of IVC in a Tie2-GFP mouse (A). Photomontage of confocal images of a FVB VG grafted into a Tie2-GFP recipient at 14 days (B) and at 28 days (C). The GFP-labeled endothelial cells located at center of a FVB VG at 28 days (C). Migration of GFP-labeled endothelial cells from aorta is evident by sequent time point. Zoomed images show photos in higher magnification. Isolated GFP-positive cells in center of grafts are also visible. Scale bar = 100 µm. Dotted line = Anastomosis between aorta and VG, Triangle head = isolated endothelial cells in middle of VG, Arrow = direction of blood flow.

Figure 3. Leukocyte rolling and adhesion in native vessels and vascular grafts.

Intravital microscopic data on leukocyte recruitment. Bar graphs represent (A) leukocyte rolling and adhesion in native vessels in WT and EP^−/−mice, (B) leukocyte adhesion in AGs and VGs in WT and EP^−/− mice and (C) leukocyte rolling in AGs and VGs in WT mice at different time points. Bar graphs represent number of leukocytes that were visible in a 100×100 µm square during 30 seconds. Error bars represent mean±SEM. *p<0.05.

Figure 4. IH in vascular grafts.

Morphologic assessment of vascular grafts stained with Hematoxylin-eosin. (A) Upper panel demonstrates VGs in WT mice and in EP^−/− mice at different time point. Bar graphs represent IH area in AGs and VGs from WT and VGs from EP^−/− mice. (B) Lower panel demonstrates VGs in WT mice without or with treatment with combination of function-blockage antibodies against P- and E-selectin or rat IgG₁ λ isotype control in cuff-assisted vein grafting technique. Bar graph represents IH area. Error bars represent mean±SEM. *p<0.05. Scale bar = 100 µm. Arrows indicated IH of VGs.