Decreased Glucagon-Like Peptide-1 Is Associated With Calcific Aortic Valve Disease: GLP-1 Suppresses the Calcification of Aortic Valve Interstitial Cells

Figures

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  Figure 1. A representative echo image of non-CAVD (upper) and CAVD (bottom).

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  Figure 2. The distribution of GLP-1 in aortic valves with or without calcification. Human aortic valves with calcification (n = 11) that underwent valve replacement operation and without calcification (n = 12) undergoing heart transplantation were assessed by histological and immunochemical analysis. (A) Sections were stained with hematoxylin and eosin, Alizarin Red S, and Masson trichrome staining. IHC stains of GLP-1 and counterstained with hematoxylin. The results of the Non-CAVD valves (left line) are shown at 100 × magnification, and the results of CAVD (right line) are shown at 20 × magnification. (B) The concentration of GLP-1 detected by IHC was determined by assessing its staining with Image-Pro Plus 6.0. The results are shown as the integrated optical density (IOD)/area. The Non-CAVD (n = 12) and CAVD valves (n = 11) are representative of three independent experiments, and five different fields in each section were detected (the P-value was control valves compared with CAVD).

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  Figure 3. GLP-1 inhibits AVIC calcification. In osteogenic medium, the primary AVICs were incubated with increasing doses (12.5, 25, 50, and 100 pmol/L for 21 days) of GLP-1. The group without the GLP-1 treatment was used as a control (Con). (A) Alizarin Red S was used to stain the calcification cells. The pictures were taken using Image-Pro Plus 6.0. Alizarin Red S staining is shown at 400 × magnification. (B) Quantification of Alizarin Red S staining. (C) Quantification of ALP activation in AVICs. The dye was extracted and quantified as described in the Methods section (n = 6, mean ± SD, *P < 0.05, **P < 0.01 compared with Con).

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  Figure 4. GLP-1 postpones the age-induced mineralization of AVICs. To determine the effect of GLP-1 on age-induced mineralization in AVICs, primary AVICs were cultured for 7, 14, and 21 days in osteogenic medium plus GLP-1 (100 pmol/L) or not. The group without the GLP-1 treatment was used as a control (Con). (A) Alizarin Red S stained the calcification cells, and the pictures were taken using Image-Pro Plus 6.0. Alizarin Red S staining is shown at 400 × magnification. (B) Quantification of Alizarin Red S staining. (C) Quantification of ALP activation in AVICs. The dye was extracted and quantified as described in the Methods section (n = 6, mean ± SD, **P < 0.01 compared with Con; #P < 0.05, ##P < 0.01 compared with 7-day culture; @P < 0.05, @@P < 0.01 compared with 14-day culture).

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  Figure 5. GLP-1 regulates the expression of calcification-related genes. The primary AVICs were stimulated with or without GLP-1 (100 pmol/L) for 21 days in osteogenic medium. The group without the GLP-1 treatment was used as a control (Con). (A) RUNX2, MSX2, SOX9, BMP2, and BMP4 were detected by real-time PCR. (B) The protein expression and location of RUNX2, MSX2, SOX9, BMP2, and BMP4 were determined by immunofluorescence analysis. RUNX2, MSX2, and SOX9 were assessed using Alexa Fluor 488-conjugated secondary antibody (Green); BMP2 and BMP4 were assessed using Alexa Fluor 549-conjugated secondary antibody (Red). (C) RUNX2, MSX2, SOX9, BMP2, and BMP4 were detected by western blot. The results are representative of three independent experiments. Real-time PCR (A) and western blot (C) quantified as relative units (genes or proteins/β-actin) (n = 3, mean ± SD, **P < 0.01 compared with Con).