Fig. 4

Sclerostin expression at vascular tissue. (A) Schematics of the immunohistochemistry microphotographs (2X) of the control subject vessel (left) and T2D patient vessel (right). All the microphotographs were captured following a medio-lateral axis in order to cover the two layers of the artery. The dashed boxes represent the schematic location of the microphotographs captured for the intima-media and the adventitia layer. (B) Representative microphotographs obtained at 20X magnification of the artery for the intima-media layer (top panel) and the adventitia layer of the artery (bottom panel) for T2D and control groups. Scalebars: 100µm. The red arrows show examples of sclerostin location. (C) Sclerostin count in calcified lower limb arteries of T2D patients (n = 7) and non-calcified arteries of control subjects (n = 3) by immunohistochemistry. Data are presented as the mean ± standard error of the mean of sclerostin labeled proteins in the intima-media and the adventitia layer of the artery and the total average for both groups. The p-values were determined by the unpaired Student´s t-test. * = p < 0.05 vs. control subjects’ vessels. # = p < 0.05 vs. intima-media layer of T2D patients’ vessels. (D) Representative microphotographs of immunofluorescence of sclerostin (green color), counterstained with DAPI (blue color) of control subject vessel (left) and T2D patient vessel (right). The red arrows show examples of sclerostin location. (E) Quantification of the immunofluorescence images of calcified lower limb arteries of T2D patients (n = 7) and non-calcified arteries of control subjects (n = 3). Data are presented as the mean ± standard error of the mean of sclerostin labeled proteins and DAPI in the total count of both groups. The p-values were determined by the unpaired Student´s t-test. * = p < 0.05 vs. control subjects’ vessels. (F) Sclerostin relative expression in both groups by RT-qPCR using the 2^−ΔΔCt method (fold-change). All data are presented as the mean ± standard error and Student´s t-test was used for the comparison. * = p < 0.05. T2D, type 2 diabetes; DAPI, 4’,6-diamidino-2-306 phenylindole; IF, immunofluorescence; SOST, sclerostin.