Diabetes reduces aortic endothelial gap junctions in ApoE-deficient mice: simvastatin exacerbates the reduction

Abstract

We examined the endothelial gap junctions in diabetic hyperlipidemic mice. Male apolipoprotein E (apoE)-deficient mice were made diabetic by streptozotocin. Three weeks later, the animals were treated with simvastatin for 2 weeks. The expression of aortic gap junctions in the non-diabetic (n=10), untreated diabetic (n=10), and simvastatin-treated diabetic animals (n=6) was analyzed. There was a >4-fold increase in serum cholesterol level and >50% increase in plaque areas in the diabetic mice, regardless of simvastatin treatment. Western blotting of aortae showed reduced expression of connexin37 (Cx37) and Cx40 in the diabetic mice, which were further decreased in the simvastatin-treated diabetic mice. Immunoconfocal microscopy showed that endothelial gap junctions made of Cx37 and Cx40 were both reduced in the untreated diabetic mice compared with the non-diabetic mice (decrease: Cx37, 41%; Cx40, 42%; both p<0.01). The reduction was greater in the simvastatin-treated mice (decrease in treated diabetic vs non-diabetic: Cx37, 61%; Cx40, 79%; both p<0.01; decrease in treated diabetic vs untreated diabetic: Cx37, 34%; Cx40, 63%; both p<0.01). Cx37 and Cx40 were decreased in the endothelium of plaque surface. Cx43 appeared in the medial layer and inner layer of the intima. All three connexins were rarely expressed in monocytes/macrophages inside the plaques. In conclusion, in apoE-deficient mice, streptozotocin-induced diabetes is associated with downregulation of endothelial Cx37 and Cx40 gap junctions. Short-term treatment with simvastatin exacerbates the downregulation.

Figure 1

Serum glucose (top histogram) and cholesterol (bottom histogram) levels in animals of different groups. See text for details. *p<0.001 for top histogram and p<0.05 for bottom histogram compared with each of the other bars of the same time points. CT, control; STZ, streptozotocin; STZ + SI, streptozotocin plus simvastatin.

Figure 2

Comparison of the burden of atheromatous plaque (in red) between groups 1 (A), 2 (B), and 3 (C) by Sudan IV staining. Note that to better show the anatomical distribution of atherosclerotic plaques, the images were taken before the arches were cut open. See text for details. *p<0.05 compared with each of the other bars. Abbreviations are the same as in Figure 1. Bar = 1 mm.

Figure 3

Comparison of the expression of connexin37 (Cx37) and Cx40 in the aorta by Western blotting. See text for details. (A) HeLa-Cx37, genetically manipulated HeLa cells express Cx37. (B) HeLa-Cx40, HeLa cells express Cx40. Both cells were kindly donated by Professor Klaus Willecke (University of Bonn, Germany). Other abbreviations are the same as in Figure 1.

Figure 4

Confocal images showing the change in expression of aortic endothelial Cx37 (left panel) or Cx40 (right panel) between apolipoprotein E (apoE)-deficient mice (A,B), those made diabetic (C,D), and those made diabetic and treated with simvastatin (E,F), as shown by en face viewing after single labeling. Typical images for Cx37 and Cx40 from the area outside the plaques in each group examined are displayed. The data for analysis are shown in the histograms below each column of images. See text for details. *p<0.01 between the groups. The denotations of all images are as described in Figure 2. Bar = 40 μm.

Figure 5

En face confocal view of the endothelium double labeled for Mac-2 and either Cx37 (A–C) or Cx40 (D–F). In all images, Mac-2 is in red, whereas connexins are in green. Note that, regardless of the different groups, on the areas positive for Mac-2 and at the periphery, connexin spots are less and unevenly distributed. The denotations of all images are as described in Figure 2. Bar = 40 μm.

Figure 6

Section view of the aortic wall double labeled for Mac-2 and Cx37 (A,E,I), Cx40 (C,G), or Cx43 (K). In all color images, Mac-2 is in red, whereas connexins are in green. For each color image, the corresponding phase contrast image (B,D,F,H,J,L) is displayed at the right to show that the whole layer of plaque, if present, is not only made of cells positive for Mac-2. Note that Cx37 and Cx40 are distributed at the luminal surface of the plaques (E,G,I), whereas Cx43 is at the inner layer of intima or in the medial layer (K). Images are oriented with the luminal side up. Bar = 30 μm.