Age-Related Impairment of Structure and Function of Iliac Artery Endothelium in Rats Is Improved by Elevated Fluid Shear Stress

Abstract

BACKGROUND Aging plays an important role in endothelial dysfunction. Fluid shear stress (FSS) can activate endothelial cells (ECs). Herein, we tested the hypothesis that this endothelial impairment could be improved by elevated FSS (EFSS) in aged rats. MATERIAL AND METHODS EFSS was created through ligation of the unilateral common iliac artery in 20-month-old rats, evaluated by measuring blood flow velocity with Doppler spectrum. The effect of FSS on aged ECs was examined by senescence-associated ß-galactosidase (SA-ß-Gal) staining, ultrastructural observation, and immunostaining and qPCR analysis of eNOS and SIRT1 expression on both the mRNA and protein levels. RESULTS (1) FSS was significantly increased in the right common iliac artery (RCIA) in rats with the ligation of the left common iliac artery (LCIA). (2) SA-ß-Gal staining was significantly attenuated by EFSS in the RCIA of aged rats. (3) Ultrastructural observation showed that ECs in the RCIA of normal aged rats became irregular and enlarged, with increasingly polypoid nuclei and fewer mitochondria, whereas ECs in the RCIA of aged rats with LCIA ligation became more prominent and contained more mitochondria. (4) eNOS and SIRT1 expression in the RCIA of aged rats with LCIA ligation was significantly upregulated compared with that in control group rats. CONCLUSIONS The present study for the first time shows that EFSS has the ability to improve age-related impairment of endothelial structure and functions.

Figure 1

Blood flow velocity and diameter of RCIA in aged rats. (A) Blood flow velocity of RCIA before and after ligation of LCIA in aged rats. (B) HE staining of RCIA before and after ligation of LCIA in aged rats. (C) Quantitative analysis of blood flow velocity and lumen diameter of RCIA before and after ligation of LCIA in aged rats. *** p<0.001; NS – not significant. n=10 per group.

Figure 2

SA-β-gal staining in RCIA in rats. (A) RCIA of normal adult rats; (B) RCIA of normal aged rats; (C) RCIA of aged rats with ligation of LCIA; (D) quantitative analysis of SA-β-gal positive staining in 3 groups. ** p<0.01, *** p<0.001. n=3 per group.

Figure 3

TEM image showing ultrastructural of the endothelium in RCIA in rats. (A, A1, A2): RCIA of normal adult rats; (B, B1, F, F1, F2): RCIA of normal aged rats; (C, D, D1, E): RCIA of aged rats with ligation of LCIA. EC – endothelial cells; N – nuclei; SMC – smooth muscle cells; EI – elastica interna; M – mitochondria; rER – rough endoplasmic reticulum; Lu – lumen; SE – subendothelial layer; G – Golgi complex. n=3 per group.

Figure 4

The expression of eNOS and SIRT1 on protein and mRNA levels in RCIA. (A) The expression of eNOS protein and mRNA in RCIA; blue: DAPI; red: eNOS; (B) The expression of SIRT1 protein and mRNA in RCIA; blue: DAPI; red: SIRT1. * p<0.05; ** p<0.01; *** p<0.001. For immunofluorescence: n=3 per group; for RNA analysis: n=4 per group.