Effects of Losartan on expression of connexins at the early stage of atherosclerosis in rabbits

Abstract

Aim: to investigate effects of Losartan on expression of connexin 40 and 43 (Cx40 and Cx43), in arteries at the early stage of atherosclerosis in a rabbit model.

Methods: A total of 28 male New Zealand white rabbits were divided into following groups: control group, high fat diet group, and Losartan group (10 mg/kg/day). Losartan was administrated in food for two weeks. Iliac arteries were obtained for immunohistochemistry, transmission electron microscopy, Western blot, and reverse transcriptase-polymerase chain reaction (RT-PCR).

Results: Transmission electron microscopy revealed abundant gap junctions between neointimal smooth muscle cells (SMCs), which were markedly reduced by treatment. RT-PCR and Western blot assay showed that the mRNA and protein expression of Cx40 and Cx43 were elevated in the neointimal area at the early stage of atherosclerosis. The mRNA and protein expression of Cx43 were significantly down-regulated by losartan treatment but those of Cx40 were not markedly changed.

Conclusion: Cx40 and Cx43 in the neointimal SMCs were up-regulated at the early stage of atherosclerosis. Losartan (an angiotensin-converting enzyme inhibitor) could reduce neointima proliferation and down-regulate the elevated protein expression of Cx43, suggesting the rennin-angiotensin system (RAS) plays an important role in the remodeling of gap junction between ventricular myocytes under pathological conditions.

Keywords: balloon angioplasty; connexin; gap junction; statin; vascular proliferation.

Figure 1

Hematoxylin/eosin staining of the iliac arteries in the normal group (panel A), high fat diet group (panel B), and high fat diet plus losartan group (panel C). In panel A, the elastic lamina was clear and intact; endothelia and smooth muscle cells (arrow) arranged regularly. In panel B, the elastic lamina was incomplete and neointima was exposed. Numerous smooth muscle cells near the internal elastic lamina arranged irregularly and extended to the intima (arrow). After treatment with losartan as shown in panel C, less neointima was markedly exposed.

Figure 2

Immunohistochemistry for connexin 40 (Cx40) and connexin 43 (Cx43) in the rabbit iliac arteries. Cx40 expression was shown in panel A, B, and C and Cx43 expression in panel D, E, and F. Panel A: Cx40 staining (brown spots, arrow) between the smooth muscle cells in the tunica media of iliac arteries from the normal group. Panel B: Cx40 staining was distributed in both the tunica media and neointima with more prominent staining in the latter. Panel C: After losartan treatment, Cx40 staining was markedly reduced in the neointima of arteries. The distribution of Cx43 was similar to that of Cx40.

Figure 3

Thin-section electron micrographs indicated gap junctions between smooth muscle cells in the rabbit iliac arteries of the normal group (panel A), high fat diet group (panel B), and high fat diet plus losartan group (panel C). In panel B, neointimal smooth muscle cells showed abundant and large gap junction when compared with normal group and losartan treated group. Bar=0.2 μm for panels A, B, C and D, × 65,000.

Figure 4

Protein expression of Cx43 (panel A) and Cx40 (panel B) in the rabbit iliac arteries of different groups. Top panels independently represented the expression of Cx43 and Cx40 from different groups and bottom panels are Actin. 1) normal control (n =7); 2) high fat diet group (n =7); 3) high fat diet + losartan (n = 7). *P<0.05, **P<0.01 vs normal group, ^##P<0.01 vs high fat diet group.

Figure 5

mRNA expression of Cx40 (panel A) and Cx43 (panel B) in the rabbit iliac arteries of different groups by RT-PCR. Top panels represented mRNA expression of Cx40 and Cx43 from each of the groups and bottom panels are Actin. 1) normal control (n =7); 2) high fat diet group (n =7); 3) high fat diet group + losartan (n =7). *P <0.05, **P<0.01 vs normal group, ^##P<0.01 vs high fat diet group.