Empagliflozin Reverses Oxidized LDL-Induced RECK Suppression, Cardiotrophin-1 Expression, MMP Activation, and Human Aortic Smooth Muscle Cell Proliferation and Migration

Figure 5

Ectopic expression of RECK blunts CT-1-induced SMC proliferation and migration potentially by associating physically with LIFR and gp130. (a, b) Ectopic expression of RECK blunts CT-1-induced SMC proliferation (a) and migration (b). Ad. GFP served as a control. SMC transduced with Ad.RECK or Ad.GFP was made quiescent, exposed to CT-1 for 48 hr (g) or 18 hr (h), and analyzed for proliferation or migration by CyQUANT GR dye assay and Boyden chamber assay, respectively. (c, d) CT-1 promotes RECK physical association with LIFT and gp130. SMCs transduced with Ad.RECK or Ad.GFP was exposed to CT-1 for 15 min and then immunoprecipitated (IP) with anti-RECK antibodies and immunoblotted (IB) with anti-LIFR (c) or gp130 (d) antibodies. Equal loading of immunoprecipitates was confirmed by blotting with anti-RECK antibodies (right-hand panels). (d) RECK (green) and LIFR or gp130 (red) physical association is analyzed by double immunofluorescence and confocal microscopy. The merged images (orange) show RECK, LIFR, or gp130 and nuclei. The omission of primary antibodies in the control panels served as a negative control. DAPI stains the nuclei blue. (a, b) versus untreated, ^† CT-1 or CT-1 + Ad.GFP (n = 5–6).

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