Journal: Mediators of Inflammation

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

doi: 10.1155/2023/6112301

Figure Lengend Snippet: Oxidized low-density lipoprotein (OxLDL) suppresses RECK expression in primary human aortic smooth muscle cells (SMC). (a–c) OxLDL, but not nLDL, suppresses RECK mRNA expression in a dose-dependent manner. Quiescent SMCs were exposed to OxLDL at indicated concentrations for 2 hr. RECK expression was analyzed by RT-qPCR using a validated TaqMan™ probe (a; n = 5). Native LDL (nLDL, 45 µ g/ml) served as a control. 18s served as an invariant control. The mRNA expression was presented as a ratio of RECK mRNA to corresponding 18s rRNA. RECK protein levels were analyzed by Western blotting (b, n = 3). Tubulin served as an invariant control. Intensity of immunoreactive bands from three independent experiments was semiquantified by densitometry, and the ratio of RECK to corresponding Tubulin was presented as fold change from nLDL. (d) nLDL up to 60 mg/ml had no significant effects on basal RECK expression. Quiescent SMC incubated with nLDL up to 60 mg/ml for 2 hr were analyzed by western blotting, and a representative image of three independent experiments is shown. (a) ∗ P < 0.05, ∗∗ P < 0.01 versus nLDL ( n = 5), (b) ∗ P < 0.05 versus nLDL ( n = 3).

Article Snippet: The following primary antibodies were used: RECK (1 : 1,000; catalog# 3433, Cell Signaling Technology, Inc./CST), α -Tubulin (1 : 1,000; #2144, CST), NF- κ B p65 (1 : 1,000; #3033; CST), MMP2 (1 : 500; ab97779, Abcam), MMP9 (1 : 1,000; #2270, CST), polyclonal human LIFR (used in neutralization (10 μ g/ml) and western blotting (0.1 μ g/ml)), #AF-249-NA, R&D Systems, Minneapolis, MN), and polyclonal antihuman gp130 (used in neutralization (2.5 μ g/ml) and Western blotting (1 μ g/ml)), #AF-228-NA, R&D Systems, Minneapolis, MN), control IgG (normal goat IgG control, #AF-108-C, R&D Systems).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Incubation

Journal: Mediators of Inflammation

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

doi: 10.1155/2023/6112301

Figure Lengend Snippet: Ectopic expression of RECK blunts OxLDL-induced SMC proliferation and migration. (a, b) Pathophysiological concentrations of OxLDL induce SMC proliferation (a) as analyzed by the CyQUANT GR dye assay and migration (b) analyzed by the Boyden chamber assay. SMCs migrating to the lower surface of the membrane were counted at 20x magnification in 10 independent fields and summarized as mean ± SEM. The inset shows representative images of Matrigel™ transwell invasion. (c) OxLDL induced proinflammatory phenotype changes in SMC. SMC transduced with Ad.RECK or Ad.GFP (moi10 for 24 hr) were exposed to OxLDL (45 mg/ml for 48 hr) and analyzed for SMC markers α SMA and MYH11, and proinflammatory markers ICAM1, Galectin 3, and Olr1. (d) OxLDL does not inhibit ectopically expressed RECK. SMCs transduced with Ad.RECK were made quiescent, exposed to OxLDL for 48 hr, and analyzed for RECK expression by western blotting. (a) ∗ P < 0.01 versus nLDL, † P < 0.05 versus Ad.RECK ( n = 5), (b) ∗ P < 0.01 versus nLDL, † P < 0.05 versus. Ad.RECK ( n = 5), (c) ∗ P < 0.01 versus nLDL, † P < 0.05 versus OxLDL ± Ad.GFP, ( n = 5), (d) ∗ P < 0.05 versus nLDL, † P < 0.05 versus OxLDL or OxLDL + Ad.GFP ( n = 3).

Article Snippet: The following primary antibodies were used: RECK (1 : 1,000; catalog# 3433, Cell Signaling Technology, Inc./CST), α -Tubulin (1 : 1,000; #2144, CST), NF- κ B p65 (1 : 1,000; #3033; CST), MMP2 (1 : 500; ab97779, Abcam), MMP9 (1 : 1,000; #2270, CST), polyclonal human LIFR (used in neutralization (10 μ g/ml) and western blotting (0.1 μ g/ml)), #AF-249-NA, R&D Systems, Minneapolis, MN), and polyclonal antihuman gp130 (used in neutralization (2.5 μ g/ml) and Western blotting (1 μ g/ml)), #AF-228-NA, R&D Systems, Minneapolis, MN), control IgG (normal goat IgG control, #AF-108-C, R&D Systems).

Techniques: Expressing, Migration, CyQUANT Assay, Boyden Chamber Assay, Membrane, Transduction, Western Blot

Journal: Mediators of Inflammation

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

doi: 10.1155/2023/6112301

Figure Lengend Snippet: 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) ∗ P < 0.05 versus untreated, † P < 0.05 CT-1 or CT-1 + Ad.GFP ( n = 5–6).

Article Snippet: The following primary antibodies were used: RECK (1 : 1,000; catalog# 3433, Cell Signaling Technology, Inc./CST), α -Tubulin (1 : 1,000; #2144, CST), NF- κ B p65 (1 : 1,000; #3033; CST), MMP2 (1 : 500; ab97779, Abcam), MMP9 (1 : 1,000; #2270, CST), polyclonal human LIFR (used in neutralization (10 μ g/ml) and western blotting (0.1 μ g/ml)), #AF-249-NA, R&D Systems, Minneapolis, MN), and polyclonal antihuman gp130 (used in neutralization (2.5 μ g/ml) and Western blotting (1 μ g/ml)), #AF-228-NA, R&D Systems, Minneapolis, MN), control IgG (normal goat IgG control, #AF-108-C, R&D Systems).

Techniques: Expressing, Migration, Control, Transduction, CyQUANT Assay, Boyden Chamber Assay, Immunoprecipitation, Immunofluorescence, Confocal Microscopy, Negative Control

Journal: Mediators of Inflammation

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

doi: 10.1155/2023/6112301

Figure Lengend Snippet: 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 α SMA 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).

Article Snippet: The following primary antibodies were used: RECK (1 : 1,000; catalog# 3433, Cell Signaling Technology, Inc./CST), α -Tubulin (1 : 1,000; #2144, CST), NF- κ B p65 (1 : 1,000; #3033; CST), MMP2 (1 : 500; ab97779, Abcam), MMP9 (1 : 1,000; #2270, CST), polyclonal human LIFR (used in neutralization (10 μ g/ml) and western blotting (0.1 μ g/ml)), #AF-249-NA, R&D Systems, Minneapolis, MN), and polyclonal antihuman gp130 (used in neutralization (2.5 μ g/ml) and Western blotting (1 μ g/ml)), #AF-228-NA, R&D Systems, Minneapolis, MN), control IgG (normal goat IgG control, #AF-108-C, R&D Systems).

Techniques: Migration, Expressing, Quantitative RT-PCR, Western Blot, Activity Assay, Fluorimetry Assay