Journal: Medical research archives

Article Title: Effects of Empagliflozin on Intermittent Hypoxia-Induced TRAF3IP2-Dependent Human Aortic Smooth Muscle Cell Proliferation

doi: 10.18103/mra.v10i10.3237

Figure Lengend Snippet: A, IH upregulates TRAF3IP2 expression. At 70–80% confluency, SMC were made quiescent, and exposed to IH for the indicated number of cycles. Nx represents time equivalent to 50 cycles of IH. TRAF3IP2 expression was analyzed by Western blotting using 20 μg of cleared whole cell lysates (n=3). Tubulin served as a loading control. B, IH stimulates superoxide generation via TRAF3IP2 and Nox2. Quiescent SMC exposed to 50 cycles of IH were analyzed for superoxide generation by the lucigenin-enhanced chemiluminescence assay. In a subset of experiments, SMC were exposed to NAC or gp91 ds-tat or transduced with Ad.TRAF3IP2-shRNA prior to IH. sgp91 ds-tat or GFP shRNA served as controls (n=6). Knockdown of TRAF3IP2 was confirmed by Western blotting as shown on the right. The adapter molecule MyD88 served as an off target. C, IH stimulated H 2 O 2 production via TRAF3IP2 and Nox4. SMC were treated as in C , but with GKT137831 or NAC or transduced with Ad.TRAF3IP2-shRNA, and then analyzed for H 2 O 2 production by Amplex Red assay (n=6). D, IH stimulated nitric oxide generation. Quiescent SMC exposed to IH as in C , but pretreated with 1400W or AMT, or transduced with Ad.TRAF3IP2-shRNA were analyzed for nitric oxide generation by the Greiss reaction, and the data were presented as cumulative nitrite production in μM (n=6). E, IH upregulates TRAF3IP2 expression via nitroxidative stress. Quiescent SMC treated as in C , D and E were analyzed for TRAF3IP2 expression by Western blotting (n=3). F, IH stimulates SMC proliferation via TRAF3IP2 and nitroxidative stress. Quiescent SMC treated as in C - E were analyzed for proliferation by the CyQUANT ™ assay after 48h (n=6) G. The pharmacological inhibitors and the shRNA used did not compromise cell viability as analyzed by a colorimetric LDH release assay and activation of caspase-3 by Western blotting. A, though a representative Western blot is shown, changes in target protein expression from three independent experiments was semi-quantified by densitometry and presented on the right as fold change over Nx, which was set at a value of 1. The numbers at the bottom in panels F and G denote lane numbers. *P

Article Snippet: The following primary antibodies were used: TRAF3IP2 (#NB100–56740, Novus Biologicals, Centennial, CO), Tubulin (#2144, Cell Signaling Technology, Inc, Danvers, MA; CST), p-p65 (#3033, CST), Lamin A/C (#4777, CST), p-STAT3 (CST), STAT3 (#9132, CST), cleaved caspase-3 (#ab32040, abcam, Waltham, MA), caspase-3 (#ab90347, abcam), HIF-1α (#ab179483, abcam), IL-6 (#ab233706, abcam), gp130 (#ab217671, abcam), IL-6R (#AF-228-NA, R & D Systems), SGLT2 (#sc-393350, Santa Cruz Biotechnology, Inc., Dallas, TX; SCB) and MyD88 (#sc-74532, SCB).

Techniques: Expressing, Western Blot, Control, Chemiluminescence Immunoassay, Transduction, shRNA, Knockdown, Amplex Red Assay, CyQUANT Assay, Lactate Dehydrogenase Assay, Activation Assay

Journal: Medical research archives

Article Title: Effects of Empagliflozin on Intermittent Hypoxia-Induced TRAF3IP2-Dependent Human Aortic Smooth Muscle Cell Proliferation

doi: 10.18103/mra.v10i10.3237

Figure Lengend Snippet: A, IH activates NF-κB via TRAF3IP2 and nitroxidative stress. Quiescent SMC were treated with NAC, gp91 ds-tat, GKT137831, 1400W or AMT prior to IH (50 cycles), and analyzed for NF-κB activation by Western blotting using equal amounts of nuclear protein extracts (10 μg) and activation-specific p65 antibodies (Ser 536 ). Lamin A/C served as a loading control. In a subset of experiments, SMC were transduced with Ad.TRAF3IP2 shRNA, made quiescent, and then exposed to IH (right hand panel). B, IH activates HIF-1α via TRAF3IP2 and nitroxidative stress. Quiescent SMC treated as in A were analyzed for HIF-1α activation by Western blotting using cleared whole cell lysates (20 μg). Tubulin served as a loading control. C, IH induces HIF-1α activation via NF-κB. Quiescent SMC were treated with the NF-κB inhibitor SN-50 or the proteasomal inhibitor MG-132 prior to IH and analyzed for HIF-1α activation as in B . Inhibition of NF-κB activation was confirmed by Western blotting (right hand panel) as in A . D, Targeting NF-κB and HIF-1α inhibit IH-induced SMC proliferation without affecting cell viability. SMC transduced with lentiviral shRNA against NF-κBp65 or HIF-1α were made quiescent and exposed to IH. Cell proliferation was analyzed by the CyQUANT ™ assay after 48h (D; n=6). Cell viability was analyzed by a colorimetric LDH release assay and activation of caspase-3 by Western blotting (E). A,B,C,E, though a representative Western blot is shown, changes in target protein expression from three independent experiments were semi-quantified by densitometry, and presented at the bottom of respective panels as a fold change over control, which was set at a value of 1. *P

Article Snippet: The following primary antibodies were used: TRAF3IP2 (#NB100–56740, Novus Biologicals, Centennial, CO), Tubulin (#2144, Cell Signaling Technology, Inc, Danvers, MA; CST), p-p65 (#3033, CST), Lamin A/C (#4777, CST), p-STAT3 (CST), STAT3 (#9132, CST), cleaved caspase-3 (#ab32040, abcam, Waltham, MA), caspase-3 (#ab90347, abcam), HIF-1α (#ab179483, abcam), IL-6 (#ab233706, abcam), gp130 (#ab217671, abcam), IL-6R (#AF-228-NA, R & D Systems), SGLT2 (#sc-393350, Santa Cruz Biotechnology, Inc., Dallas, TX; SCB) and MyD88 (#sc-74532, SCB).

Techniques: Activation Assay, Western Blot, Control, Transduction, shRNA, Inhibition, CyQUANT Assay, Lactate Dehydrogenase Assay, Expressing

Journal: Medical research archives

Article Title: Effects of Empagliflozin on Intermittent Hypoxia-Induced TRAF3IP2-Dependent Human Aortic Smooth Muscle Cell Proliferation

doi: 10.18103/mra.v10i10.3237

Figure Lengend Snippet: A-C, IH upregulates IL-6 mRNA and protein expression via TRAF3IP2, NF-κB and HIF-1α. SMC were transduced with NF-κBp65, HIF-1α or TRAF3IP2 shRNA using viral vectors, made quiescent, and exposed to 50 cycles of IH. GFP shRNA served as a control. IL-6 mRNA was analyzed by RT-qPCR (A) and protein expression by Western blotting (B). Knockdown of NF-κBp65 and HIF-1α was confirmed by Western blotting (C). ASK1 served as a non-targeting control. Tubulin served as a loading control. D, IL-6 activates STAT3 in a concentration- and time-dependent manner. Quiescent SMC treated with the indicated concentrations of IL-6 (left hand panel) and for up to 120 min at 30 ng/ml (middle panel) were analyzed for total and p-STAT3 levels by Western blotting using cleared whole cell lysates (20 μg). In a subset of experiments, SMC were exposed to Polymyxin B sulphate for 2 h prior to incubation with IL-6 (30 ng/ml for 15 min). STAT3 activation was analyzed by Western blotting (right hand panel). E, F, IL-6 induces STAT3 phosphorylation via IL-6R, gp130 and JAK. Quiescent SMC treated with the gp130 inhibitor SC144, JAK inhibitor HO-3867 and the STAT3 inhibitor Tofacitinib prior to IH were analyzed for STAT3 phosphorylation as in D . In a subset of experiments, SMC were transduced with IL-6R or gp130 shRNA, made quiescent, and then exposed to IH (right hand panel). Knockdown of IL-6R and gp130 was confirmed by Western blotting (F). B-F, though a representative Western blot is shown, changes in target protein expression from three independent experiments were semi-quantified by densitometry and presented at the bottom or side of respective panels as a fold change over control, which was set at a value of 1. The numbers at the bottom of panels denote lane numbers. *P

Article Snippet: The following primary antibodies were used: TRAF3IP2 (#NB100–56740, Novus Biologicals, Centennial, CO), Tubulin (#2144, Cell Signaling Technology, Inc, Danvers, MA; CST), p-p65 (#3033, CST), Lamin A/C (#4777, CST), p-STAT3 (CST), STAT3 (#9132, CST), cleaved caspase-3 (#ab32040, abcam, Waltham, MA), caspase-3 (#ab90347, abcam), HIF-1α (#ab179483, abcam), IL-6 (#ab233706, abcam), gp130 (#ab217671, abcam), IL-6R (#AF-228-NA, R & D Systems), SGLT2 (#sc-393350, Santa Cruz Biotechnology, Inc., Dallas, TX; SCB) and MyD88 (#sc-74532, SCB).

Techniques: Expressing, Transduction, shRNA, Control, Quantitative RT-PCR, Western Blot, Knockdown, Concentration Assay, Incubation, Activation Assay, Phospho-proteomics

Journal: Medical research archives

Article Title: Effects of Empagliflozin on Intermittent Hypoxia-Induced TRAF3IP2-Dependent Human Aortic Smooth Muscle Cell Proliferation

doi: 10.18103/mra.v10i10.3237

Figure Lengend Snippet: A, Quiescent SMC treated with the gp130, JAK or STAT3 inhibitor prior to IL-6 addition were analyzed for proliferation at 48 h by the CyQUANT ™ assay. In a subset of experiments, SMC were transduced with IL-6 or gp130 or TRAF3IP2 shRNA, made quiescent, and then exposed to IL-6 (n=6). B, Pharmacological inhibitors or shRNA-mediated knockdown of targets described in A did not negatively impact SMC viability. Cell viability was analyzed by LDH release by the LDH-Glo ™ Cytotoxicity Assay (n=6) and activation of caspase-3 by Western blotting (bottom panel; n=3). In LDH cytotoxicity assay, treatment of SMC with Triton X-100 served as a positive control and was set to 100% LDH release. H 2 O 2 (100 μM) served as a positive control in Western blotting. *P

Article Snippet: The following primary antibodies were used: TRAF3IP2 (#NB100–56740, Novus Biologicals, Centennial, CO), Tubulin (#2144, Cell Signaling Technology, Inc, Danvers, MA; CST), p-p65 (#3033, CST), Lamin A/C (#4777, CST), p-STAT3 (CST), STAT3 (#9132, CST), cleaved caspase-3 (#ab32040, abcam, Waltham, MA), caspase-3 (#ab90347, abcam), HIF-1α (#ab179483, abcam), IL-6 (#ab233706, abcam), gp130 (#ab217671, abcam), IL-6R (#AF-228-NA, R & D Systems), SGLT2 (#sc-393350, Santa Cruz Biotechnology, Inc., Dallas, TX; SCB) and MyD88 (#sc-74532, SCB).

Techniques: CyQUANT Assay, Transduction, shRNA, Knockdown, Cytotoxicity Assay, Activation Assay, Western Blot, LDH Cytotoxicity Assay, Positive Control, Saline

Journal: Medical research archives

Article Title: Effects of Empagliflozin on Intermittent Hypoxia-Induced TRAF3IP2-Dependent Human Aortic Smooth Muscle Cell Proliferation

doi: 10.18103/mra.v10i10.3237

Figure Lengend Snippet: A, IL-6 upregulates SGLT2 expression. Quiescent SMC exposed to IL-6 (30 ng/ml for 4h) were analyzed for SGLT2 expression by Western blotting using equal amounts of whole cell lysates (20 μg). Cell lysates from the human proximal tubule epithelial cells HK-2 and human kidney extracts served as positive controls (20 μg). B, IL-6 upregulates SGLT2 expression via STAT3. Quiescent SMC exposed to IL-6 (30 ng/ml) for up to 6h (left hand panel) were analyzed for SGLT2 expression as in A. In a subset of experiments, quiescent SMC were treated with the STAT3 inhibitor HO-3867 prior to IL-6 addition (30 mg/ml for 4 h). C, Empagliflozin inhibits IL-6-induced STAT3 phosphorylation via SGLT2. Quiescent SMC were exposed to empagliflozin (1 μM for 15 min) prior to IL-6 addition (30 ng/ml for 15 min). Total and phospho-STAT3 levels were analyzed by Western blotting using whole cell lysates. In a subset of experiments, SMC were transduced with SGLT2 shRNA by lentiviral transduction, made quiescent, and then treated with IL-6. Knockdown of SGLT2 was confirmed by Western blotting. TRAF3IP2 served as an off target. D, Empagliflozin or SGLT2 knockdown failed to affect cell viability. Cell viability was analyzed by LDH-Glo ™ Cytotoxicity Assay (n=6) and activation of caspase-3 by Western blotting as in . A-D, though a representative Western blot is shown, changes in target protein expression from three independent experiments were semi-quantified by densitometry, and presented at the bottom or side of respective panels as a fold change over control, which was set at a value of 1. The numbers at the bottom of panels denote lane numbers. *P

Article Snippet: The following primary antibodies were used: TRAF3IP2 (#NB100–56740, Novus Biologicals, Centennial, CO), Tubulin (#2144, Cell Signaling Technology, Inc, Danvers, MA; CST), p-p65 (#3033, CST), Lamin A/C (#4777, CST), p-STAT3 (CST), STAT3 (#9132, CST), cleaved caspase-3 (#ab32040, abcam, Waltham, MA), caspase-3 (#ab90347, abcam), HIF-1α (#ab179483, abcam), IL-6 (#ab233706, abcam), gp130 (#ab217671, abcam), IL-6R (#AF-228-NA, R & D Systems), SGLT2 (#sc-393350, Santa Cruz Biotechnology, Inc., Dallas, TX; SCB) and MyD88 (#sc-74532, SCB).

Techniques: Expressing, Western Blot, Phospho-proteomics, Transduction, shRNA, Knockdown, Cytotoxicity Assay, Activation Assay, Control, Saline

Journal: Medical research archives

Article Title: Effects of Empagliflozin on Intermittent Hypoxia-Induced TRAF3IP2-Dependent Human Aortic Smooth Muscle Cell Proliferation

doi: 10.18103/mra.v10i10.3237

Figure Lengend Snippet: A, IH upregulates SGLT2, and targeting SGLT2 by empagliflozin inhibits IH-induced TRAF3IP2 expression. Quiescent SMC were exposed to IH or Nx for 50 cycles, and analyzed for SGLT2 expression by Western blotting. Human kidney homogenate served as a positive control. In a subset of experiments, quiescent SMC were exposed to empagliflozin (1 μM for 15 min) prior to IH (50 cycles), and then analyzed for TRAF3IP2 expression by Western blotting (n=3). B, Empagliflozin inhibits IH-induced nitrooxidative stress. Quiescent SMC exposed to empagliflozin as in A were analyzed for superoxide, H 2 O 2 and nitrate+nitrite levels by lucigenin-enhanced chemiluminescence assay, Amplex Red assay and Greiss reaction (n=6). C, Empagliflozin inhibits IH-induced NF-κB and HIF-1α activation. Quiescent SMC exposed to empagliflozin and IH as in A were analyzed for NF-κB activation by Western blotting using equal amounts of nuclear protein extracts and activation-specific anti-p65 antibodies. Total and phospho-HIF-1α levels were analyzed in whole cell lysates by Western blotting. D, empagliflozin inhibits IH-induced IL-6 expression and STAT3 phosphorylation. Quiescent SMC exposed to empagliflozin followed by IH were analyzed for IL-6 expression and phospho-STAT3 levels by Western blotting using cleared whole cell lysates. E, Empagliflozin inhibits IH-induced SMC proliferation without affecting cell viability. Quiescent SMC treated as in A were analyzed for proliferation by the CyQUANT ™ assay after 48h (n=6). Cell viability was analyzed by LDH-Glo ™ Cytotoxicity Assay (n=6) and activation of caspase-3 by Western blotting as in . *P

Article Snippet: The following primary antibodies were used: TRAF3IP2 (#NB100–56740, Novus Biologicals, Centennial, CO), Tubulin (#2144, Cell Signaling Technology, Inc, Danvers, MA; CST), p-p65 (#3033, CST), Lamin A/C (#4777, CST), p-STAT3 (CST), STAT3 (#9132, CST), cleaved caspase-3 (#ab32040, abcam, Waltham, MA), caspase-3 (#ab90347, abcam), HIF-1α (#ab179483, abcam), IL-6 (#ab233706, abcam), gp130 (#ab217671, abcam), IL-6R (#AF-228-NA, R & D Systems), SGLT2 (#sc-393350, Santa Cruz Biotechnology, Inc., Dallas, TX; SCB) and MyD88 (#sc-74532, SCB).

Techniques: Expressing, Western Blot, Positive Control, Chemiluminescence Immunoassay, Amplex Red Assay, Activation Assay, Phospho-proteomics, CyQUANT Assay, Cytotoxicity Assay

Journal: Medical research archives

Article Title: Effects of Empagliflozin on Intermittent Hypoxia-Induced TRAF3IP2-Dependent Human Aortic Smooth Muscle Cell Proliferation

doi: 10.18103/mra.v10i10.3237

Figure Lengend Snippet: IH induces primary human aortic SMC proliferation via the crosstalk between TRAF3IP2 and nitrooxidative stress, NF-κB and HIF-1α activation, and induction of IL-6 and activation of its downstream signaling. Moreover, IL-6 induced SGLT2 expression in part via STAT3 and targeting SGLT2 by empagliflozin attenuated STAT3 phosphorylation and SMC proliferation without affecting cell viability. IH also upregulated SGLT2 expression and targeting SGLT2 by empagliflozin attenuated IH-induced TRAF3IP2 expression, NF-κB and HIF-1α activation, IL-6 expression, STAT3 phosphorylation and SMC proliferation. Together, these mechanistic in vitro results suggest the therapeutic potential of empagliflozin in vascular proliferative diseases.

Article Snippet: The following primary antibodies were used: TRAF3IP2 (#NB100–56740, Novus Biologicals, Centennial, CO), Tubulin (#2144, Cell Signaling Technology, Inc, Danvers, MA; CST), p-p65 (#3033, CST), Lamin A/C (#4777, CST), p-STAT3 (CST), STAT3 (#9132, CST), cleaved caspase-3 (#ab32040, abcam, Waltham, MA), caspase-3 (#ab90347, abcam), HIF-1α (#ab179483, abcam), IL-6 (#ab233706, abcam), gp130 (#ab217671, abcam), IL-6R (#AF-228-NA, R & D Systems), SGLT2 (#sc-393350, Santa Cruz Biotechnology, Inc., Dallas, TX; SCB) and MyD88 (#sc-74532, SCB).

Techniques: Activation Assay, Expressing, Phospho-proteomics, In Vitro