Fig 2: Schematic showing that pretreatment with empagliflozin inhibits OxLDL and CT-1-induced SMC proliferation, migration, and proinflammary phenotype changes. Exposure to OxLDL-induced NF-κB-dependent miR-30b expression and miR-30b-mediated RECK suppression. Pretreatment with empagliflozin reversed these effects. Further, OxLDL induced MMP2 and MMP9 activation, and forced expression of RECK or pretreatment with empagliflozin blunted this response. OxLDL induced CT-1 expression and CT-1-stimulated SMC proliferation and migration in part via LIFR and gp130. Ectopic expression of RECK inhibited these effects by potentially associating with LIFR and gp130. Importantly, empagliflozin blunted CT-1-induced mitogenic and migratory effects. These results suggest the therapeutic potential of RECK overexpression or empagliflozin in vascular proliferative diseases.

Fig 3: OxLDL stimulates SMC migration and proliferation via CT-1 induction. (a, b) OxLDL stimulates CT-1 mRNA expression and secretion. Quiescent SMCs treated with OxLDL for the indicated periods were analyzed for CT-1 mRNA expression by RT-qPCR and its secreted levels in equal amounts of culture supernatants by ELISA. nLDL served as a control. (c–f) CT-1 stimulates SMC migration and proliferation via LIFR and gp130. SMCs were transduced with validated lentiviral LIFR or gp130 shRNA, made quiescent, and exposed to CT-1. Cell proliferation after 48 hr (c) and migration after 18 hr (d) were analyzed by CyQUANT GR dye assay and Boyden chamber assay, respectively. The inset in (d) shows representative images of Matrigel™ transwell invasion. Knockdown of LIFR and gp130 was confirmed by western blotting (e, f), and summarized semiquantification of the intensity of immunoreactive bands is shown in the lower panels. (g, h), Preincubation with neutralizing anti-LIFR or anti-gp130 antibodies blunt CT-1-induced SMC proliferation and migration. The inset in (h) shows representative images of Matrigel™ transwell invasion. (a–d, g, h) ∗0.05, ∗∗P < 0.01 versus nLDL (n = 4 or 5); (e, f) ∗P < 0.05 versus eGFP shRNA (n = 3).

Fig 4: Empagliflozin inhibits OxLDL and CT-1-induced SMC proliferation and migration. (a) Empagliflozin inhibits OxLDL-induced miR-30b expression. Quiescent SMCs were treated with empagliflozin for 15 min followed by the addition of OxLDL or nLDL for 2 hr and analyzed for miR-30b expression by RT-qPCR using a TaqMan® probe. (b, c) Empagliflozin reverses OxLDL-induced RECK suppression. Quiescent SMCs were treated with empagliflozin as in (a), were exposed OxLDL or nLDL for 6 hr and analyzed for RECK expression by western blotting. Semiquantification of the intensity of immunoreactive bands by densitometry is shown in panel (c). (d, e) Empagliflozin inhibits OxLDL- or CT-1-induced MMP2 (d) and MMP9 (e) activity. SMCs made quiescent in medium supplemented with ITS-G 1x and no FBS were exposed to empagliflozin as in (a), followed by OxLDL (black) or CT-1 (red) for 18 hr and analyzed for MMP2 and MMP9 activity using SensoLyte® 520 fluorimetric assay. (f, g) Empagliflozin inhibits OxLDL- or CT-1-induced SMC proliferation and migration. Quiescent SMCs exposed to empagliflozin were treated with OxLDL (black) or CT-1 (red) for 48 hr (f) or 18 hr (g) and then analyzed for proliferation. (h) Empagliflozin reverses OxLDL-induced suppression of the mRNA expression of SMC markers αSMA and MYH11 and the proinflammatory markers ICAM1, Galectin 3, and Olr1 as analyzed by RT-qPCR. (a) ∗P < 0.01 versus nLDL, †P < 0.05 versus OxLDL (n = 5); (c) ∗P < 0.05 versus nLDL, †P < 0.05 versus OxLDL (n = 3); (d–g) P < 0.05 versus nLDL, †P < 0.05 versus OxLDL or CT-1 (n = 4 or 5), (h) ∗P < 0.05 versus nLDL, †P < 0.05 versus OxLDL (n = 5).