Journal: Cells

Article Title: EF24, a Curcumin Analog, Reverses Interleukin-18-Induced miR-30a or miR-342-Dependent TRAF3IP2 Expression, RECK Suppression, and the Proinflammatory Phenotype of Human Aortic Smooth Muscle Cells

doi: 10.3390/cells13201673

Figure Lengend Snippet: Interleukin-18 (IL-18) upregulates TRAF3IP2 but suppresses RECK expression in primary human aortic smooth muscle cells (ASMC). ( A – C ) IL-18 upregulates TRAF3IP2 expression. ASMCs were grown in complete media, and at 70–80% confluency, made quiescent for 48 h, and then incubated with rhIL-18 (10 ng/mL) for up to 12 h (experimental design in ( A )). TRAF3IP2 mRNA expression was analyzed by RT-qPCR using a TaqMan™ probe ( B ) and its protein levels by Western blotting ( C ), with GAPDH and Tubulin serving as loading controls, respectively. * p < 0.05, ** at least p < 0.01 vs. untreated controls ( n = 4). ( D , E ) Quiescent ASMCs were incubated with IL-18 as in (A) and were analyzed for RECK mRNA expression by RT-qPCR using a TaqMan™ probe ( D ) and protein levels by Western blotting ( E ). * p < 0.05, ** at least p < 0.01 vs. untreated controls ( n = 4). ( F – H ) Specificity of IL-18 on TRAF3IP2 induction ( G ) and RECK suppression ( H ) was verified by incubating with neutralizing IL-18R1 antibody or IL-18BP-Fc chimera for 1 h prior to IL-18 addition for 2 ( G ) or 6 h ( H ), with normal goat IgG or Fc serving as controls. mRNA expressions of TRAF3IP2 and RECK were analyzed by RT-qPCR. ( C , E ) While a representative immunoblot is shown, the intensities of immunoreactive bands from 4 independent experiments were semiquantified by densitometry and are summarized on the right. ( G ) * p < 0.05, ** p < 0.01 vs. untreated controls, † p < 0.01 versus IL-18 (n = 6); ( H ) * p < 0.05, ** p < 0.01 vs. untreated controls, † p < 0.01 versus IL-18 ( n = 4).

Article Snippet: Human primary aortic SMC (ASMC) was purchased from LONZA (#CC-2571, Basel, Switzerland) and cultured in SmGMTM-2 Smooth Muscle Cell Growth Medium-2 with SingleQuotsTM supplements containing growth factors (#CC-4149) as described previously [ , ].

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

Journal: Cells

Article Title: EF24, a Curcumin Analog, Reverses Interleukin-18-Induced miR-30a or miR-342-Dependent TRAF3IP2 Expression, RECK Suppression, and the Proinflammatory Phenotype of Human Aortic Smooth Muscle Cells

doi: 10.3390/cells13201673

Figure Lengend Snippet: Targeting TRAF3IP2 or RECK overexpression blunts IL-18-induced ASMC proliferation and migration. ( A – E ) Silencing TRAF3IP2 inhibits IL-18-induced ASMC proliferation ( B ) and migration ( C ). ASMC were transduced with adenovirus-expressing shRNA targeting human TRAF3IP2 (moi 10 for 48 h), made quiescent, and then exposed to IL-18 (10 ng/mL; experimental design in ( A )). ASMC proliferation was assessed after 48 h of IL-18 addition using the CyQUANT Cell proliferation assay ( B ), and migration after 18 h using Boyden chamber assay ( C ). ASMCs migrating to the lower surface of the membrane were counted in 10 different fields and summarized as mean ± SEM. ( B , C ) * p < at least 0.01 vs. Untreated; † p < 0.01 vs. IL-18 or IL-18+GFP (n = 6). Knockdown of TRAF3IP2 was confirmed by RT-qPCR using a TaqMan™ probe ( D ) and Western blotting ( E ). ( D , E ) * p < 0.01 vs. untreated (n = 3). ( F , G ) Dose-dependent effects of Ad.RECK on RECK expression (experimental design in ( F )). Induction of RECK following adenoviral transduction was confirmed by Western blotting with tubulin serving as an internal control ( G ). ( H – K ) Forced expression of RECK inhibits IL-18-stimulated ASMC proliferation and migration. ASMCs were transduced with adenovirus-expressing human RECK cDNA (moi 10 for 24 h), made quiescent, and then treated with IL-18 (experimental design in ( H )) and analyzed for proliferation ( I ) and migration ( J ) as in ( B , C ). ( C , J ) The insets show representative images of Matrigel™ Transwell invasion. Scale bar: 20 μM. ( E , G , K ) While a representative immunoblot is shown, the intensities of immunoreactive bands from three ( E ), four ( G ) and three ( K ) independent experiments were semiquantified by densitometry and are summarized on the right. ( I ) * p < at least 0.01 vs. Untreated; † p < 0.01 vs. IL-18 or IL-18+GFP (n = 6); ( J ) * p < at least 0.01 vs. Untreated; † p < 0.01 vs. IL-18 or IL-18+eGFP (n = 4); ( K ) * p < 0.01 vs. Untreated; † p < 0.05 vs. IL-18 or IL-18+eGFP (n = 3).

Article Snippet: Human primary aortic SMC (ASMC) was purchased from LONZA (#CC-2571, Basel, Switzerland) and cultured in SmGMTM-2 Smooth Muscle Cell Growth Medium-2 with SingleQuotsTM supplements containing growth factors (#CC-4149) as described previously [ , ].

Techniques: Over Expression, Migration, Transduction, Expressing, shRNA, CyQUANT Assay, Proliferation Assay, Boyden Chamber Assay, Membrane, Knockdown, Quantitative RT-PCR, Western Blot, Control

Journal: Cells

Article Title: EF24, a Curcumin Analog, Reverses Interleukin-18-Induced miR-30a or miR-342-Dependent TRAF3IP2 Expression, RECK Suppression, and the Proinflammatory Phenotype of Human Aortic Smooth Muscle Cells

doi: 10.3390/cells13201673

Figure Lengend Snippet: TRAF3IP2 knockdown restores SMC marker expression and inhibits ASMC proinflammatory phenotype without affecting cell viability. ( A – G ) Silencing TRAF3IP2 restores IL-18-mediated suppression in SMC markers, but inhibits the expression of proinflammatory phenotype markers, without significantly modulating cell viability. ASMCs were transduced with adenoviral TRAF3IP2 shRNA (moi10 for 48 h), made quiescent and then treated with IL-18 (10 ng/mL for 48 h; experimental design in ( A )). Expressions of the SMC markers ACTA2 ( B , C ) and MYH11 ( D , E ) were analyzed by both RT-qPCR ( B , D ) and Western blotting ( C , E ). The proinflammatory phenotype markers Galectin 3, Olr1, VCAM, CCL2, IL-6, IL-8, and TNF-α were analyzed by RT-qPCR using TaqMan™ probes ( F ). Cell viability was assessed by analyzing cleaved caspase-3 levels using a commercially available Caspase-3 (Cleaved) Human ELISA ( G ). H 2 O 2 (100 μM for 18 h) served as a positive control and induced a significant increase in cleaved capase-3 levels. ( C , E ) While a representative immunoblot is shown, the intensities of immunoreactive bands from three independent experiments were semiquantified by densitometry and are summarized on the right. ( B , D , F , G ) * p < at least 0.01 vs. Untreated; † p < 0.01 vs. IL-18 or IL-18+GFP (n = 6 or 7). ( C , E ) * p < 0.05 vs. Untreated; † p < 0.05 vs. IL-18 or IL-18+GFP (n = 3).

Article Snippet: Human primary aortic SMC (ASMC) was purchased from LONZA (#CC-2571, Basel, Switzerland) and cultured in SmGMTM-2 Smooth Muscle Cell Growth Medium-2 with SingleQuotsTM supplements containing growth factors (#CC-4149) as described previously [ , ].

Techniques: Knockdown, Marker, Expressing, Transduction, shRNA, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: Cells

Article Title: EF24, a Curcumin Analog, Reverses Interleukin-18-Induced miR-30a or miR-342-Dependent TRAF3IP2 Expression, RECK Suppression, and the Proinflammatory Phenotype of Human Aortic Smooth Muscle Cells

doi: 10.3390/cells13201673

Figure Lengend Snippet: IL-18 inhibits miR-30a and miR-342 expression via stress-activated kinases. ( A – C ) IL-18 inhibits miR-30a and miR-342 expression via stress-activated kinases. Quiescent ASMCs were treated with inhibitors of either p38 MAPK (SB239063, 10 μM in DMSO for 1 h), ERK1/2 (SCH772984 10 μM in DMSO for 1 h) or JNK (SP600125, 20 μM in DMSO for 1 h) prior to IL-18 addition at 10 ng/mL for 30 min (experimental design in ( A )). ( B , C ) Fresh DMSO (0.1%) served as a solvent control. miR-30a and miR-342 expressions were analyzed by TaqMan ® Advanced miRNA assays, with U6 serving as a loading control. ( B ) * p < 0.001 vs. untreated, † p < at least 0.01 vs. IL-18 or IL-18+DMSO (n = 11), ( C ) * p < 0.01 vs. untreated, † p < at least 0.05 vs. IL-18 or IL-18+DMSO (n = 5). ( D – F ) miR-30a mimic inhibits IL-18-induced TRAF3IP2 expression. ASMC were transfected with miR-30a mimic (80 nM), made quiescent and then exposed to IL-18 at 10 ng/mL for 2 h (experimental design in ( D )). TRAF3IP2 mRNA expression was analyzed by RT-qPCR (E) and its protein levels by Western blotting ( F ). ( G – I ) miR-342 mimic restores IL-18-induced RECK suppression. ASMCs were transfected with miR-342 mimic (80 nM), made quiescent and then exposed to IL-18 at 10 ng/mL for 6 h (experimental design in ( G )). RECK mRNA expression was analyzed by RT-qPCR ( H ) and its protein levels by Western blotting ( I ). ( F , I ) While a representative immunoblot is shown, the intensities of immunoreactive bands from 4 independent experiments were semiquantified by densitometry and are summarized as mean ± SEM on the right. ( E , F , H , I ) * p < 0.05, † at least p < 0.01 vs. untreated controls (n = 4).

Article Snippet: Human primary aortic SMC (ASMC) was purchased from LONZA (#CC-2571, Basel, Switzerland) and cultured in SmGMTM-2 Smooth Muscle Cell Growth Medium-2 with SingleQuotsTM supplements containing growth factors (#CC-4149) as described previously [ , ].

Techniques: Expressing, Solvent, Control, Transfection, Quantitative RT-PCR, Western Blot

Journal: Cells

Article Title: EF24, a Curcumin Analog, Reverses Interleukin-18-Induced miR-30a or miR-342-Dependent TRAF3IP2 Expression, RECK Suppression, and the Proinflammatory Phenotype of Human Aortic Smooth Muscle Cells

doi: 10.3390/cells13201673

Figure Lengend Snippet: EF24 inhibits IL-18-induced ASMC proliferation and migration, without affecting cell viability. ( A ) Chemical structure of EF24, a curcumin analog. ( B – F ) EF24 is not cytotoxic to ASMC at the concentrations used. Quiescent ASMCs were exposed to EF24 at the indicated concentrations for 48 h (experimental design in ( B )). Cell viability was analyzed by trypan blue dye exclusion ( C ), cleaved caspase-3 levels by ELISA ( D ) with H 2 O 2 (100 μM) serving as a positive control, ELISA of mono-oligonucleosomal fragmented DNA ( E ) with H 2 O 2 (100 μM) serving as a positive control, and LDH release with LDH release in response to 0.2% Triton-X100 being considered as 100% ( F ). DMSO (0.1%) alone served as a solvent control (depicted as “0”). Cells without any treatment served as a control ( C ). ( D – F ) * p < 0.01 vs. untreated controls or treated with DMSO alone. ( G – H ) EF24 inhibits IL-18-induced ASMC proliferation and migration. Quiescent ASMCs were incubated with EF24 at various concentrations ranging from 1 to 10 μM in DMSO for 1 h, followed by the addition of IL-18 at 10 ng/mL for 48 h (experimental design in ( G )). Cell proliferation was analyzed by the CyQUANT Cell proliferation assay ( H ). Cell migration was analyzed by Boyden chamber assay after 18 h (( I ) The insets show representative images of Matrigel™ transwell invasion). The combination of IL-18 and EF24 did not affect cell viability ( J ). ( C , I ) Scale bars, 20 μM. ( H , I ) * p < at least 0.05 vs. untreated controls, † p < 0.05 vs. IL-18 without EF24 (n = 6). ( J ) * p < 0.01 vs. untreated controls (n = 12).

Article Snippet: Human primary aortic SMC (ASMC) was purchased from LONZA (#CC-2571, Basel, Switzerland) and cultured in SmGMTM-2 Smooth Muscle Cell Growth Medium-2 with SingleQuotsTM supplements containing growth factors (#CC-4149) as described previously [ , ].

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Positive Control, Solvent, Control, Incubation, CyQUANT Assay, Proliferation Assay, Boyden Chamber Assay

Journal: Cells

Article Title: EF24, a Curcumin Analog, Reverses Interleukin-18-Induced miR-30a or miR-342-Dependent TRAF3IP2 Expression, RECK Suppression, and the Proinflammatory Phenotype of Human Aortic Smooth Muscle Cells

doi: 10.3390/cells13201673

Figure Lengend Snippet: EF24 reverses ASMC proinflammatory phenotype without affecting cell viability. ( A – F ) Pretreatment with EF24 restores the IL-18-induced suppression of SMC markers and inhibits the expression of proinflammatory phenotype markers, without significantly modulating cell viability. Quiescent ASMCs were treated with EF24 (2.5 μM for 1 h in DMSO) prior to the addition of IL-18 at 10 ng/mL for 48 h (experimental design in ( A )). The expression levels of SMC markers ACTA2 (( B ) mRNA, ( C ) protein levels) and MYH11 (( D ) mRNA, ( E ) protein levels) were analyzed by RT-qPCR and Western blotting, and those of proinflammatory phenotype markers Galectin 3, Olr1, VCAM, CCL2, IL-6, IL-8, and TNF-α ( F ) were analyzed by RT-qPCR using TaqMan™ probes. Cell viability was assessed by analyzing cleaved caspase-3 levels using a Caspase-3 (Cleaved) Human ELISA kit ( G ). H 2 O 2 (100 μM) for 24 h served as a positive control and induced a significant increase in cleaved capase-3 levels. ( C , E ) While a representative immunoblot is shown, the intensities of immunoreactive bands from three independent experiments were semiquantified by densitometry and are summarized on the right. ( B , D , F , G ) * p < at least 0.01 vs. Untreated; † p < at least 0.05 vs. IL-18 or IL-18+DMSO (n = 5–6), ( C – E ) * p < 0.05 vs. Untreated; † p < 0.05 vs. IL-18 or IL-18+DMSO (n = 3).

Article Snippet: Human primary aortic SMC (ASMC) was purchased from LONZA (#CC-2571, Basel, Switzerland) and cultured in SmGMTM-2 Smooth Muscle Cell Growth Medium-2 with SingleQuotsTM supplements containing growth factors (#CC-4149) as described previously [ , ].

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: Cells

Article Title: EF24, a Curcumin Analog, Reverses Interleukin-18-Induced miR-30a or miR-342-Dependent TRAF3IP2 Expression, RECK Suppression, and the Proinflammatory Phenotype of Human Aortic Smooth Muscle Cells

doi: 10.3390/cells13201673

Figure Lengend Snippet: EF24 inhibits IL-18-induced MMP2 and MMP9 expressions. ( A – D ) Silencing MMPs 2 and 9 inhibits IL-18-induced ASMC migration. ASMCs were transduced with adenoviral vector-expressing human MMP2 or MMP9 shRNA (moi10 for 48 h), made quiescent in basal medium containing ITS-G 1X supplement and then treated with IL-18 at 10 ng/mL for 18 h (experimental design in ( A )). Ad.siGFP at moi 10 for 48 h served as a control. Cell migration was analyzed by the Boyden chamber assay ( B ). ( C , D ) Knockdown of MMPs 2 and 9 was analyzed by RT-qPCR and their protein levels by Western blotting (insets). ( C , D ) While a representative immunoblot is shown, the intensities of immunoreactive bands from 3 independent experiments were semiquantified by densitometry and are summarized as mean ± SEM on the right. ( E – G ) EF24 inhibits IL-18-induced MMP expression. SMCs made quiescent in basal medium containing ITS-G 1X supplement for 48 h were incubated with EF24 (2.5 μM) for 1 h followed by IL-18 at 10 ng/mL for 2 h (experimental design in ( E )). MMP2 ( F ) and MMP9 ( G ) mRNA expressions were analyzed by RT-qPCR. ( B , F , G ) * p < at least 0.01 vs. Untreated; † p < at least 0.05 vs. IL-18 or IL-18+siGFP (n = 3–6); (( C , D ) left) * p < 0.001 vs. Untreated; (( C , D ) right) * p < 0.05 vs. Untreated.

Article Snippet: Human primary aortic SMC (ASMC) was purchased from LONZA (#CC-2571, Basel, Switzerland) and cultured in SmGMTM-2 Smooth Muscle Cell Growth Medium-2 with SingleQuotsTM supplements containing growth factors (#CC-4149) as described previously [ , ].

Techniques: Migration, Transduction, Plasmid Preparation, Expressing, shRNA, Control, Boyden Chamber Assay, Knockdown, Quantitative RT-PCR, Western Blot, Incubation

Journal: Cells

Article Title: EF24, a Curcumin Analog, Reverses Interleukin-18-Induced miR-30a or miR-342-Dependent TRAF3IP2 Expression, RECK Suppression, and the Proinflammatory Phenotype of Human Aortic Smooth Muscle Cells

doi: 10.3390/cells13201673

Figure Lengend Snippet: Schematic showing that EF24, a curcumin analog with better bioavailability and biologic activity, inhibits the proinflammatory IL-18-induced primary human aortic smooth muscle cells’ (ASMCs) proliferation, migration, and proinflammatory phenotype changes. EF24 inhibits the stress-activated kinase-dependent miR-30a and miR-342 inhibition, TRAF3IP2 upregulation and RECK suppression. While miR-30a mimic reverses IL-18-induced TRAF3IP2 upregulation, the miR-342 mimic restores IL-18-mediated RECK suppression, potentially via reduced DNMT1 expression and promoter demethylation (dashed purple box). While IL-18 promotes ASMC migration and proliferation, these effects were reversed by TRAF3IP2 knockdown or the ectopic expression of RECK. Further, while IL-18 induced ASMC migration in part via induction of the gelatinases MMP2 and MMP9, these effects were inhibited by EF24. Moreover, EF24 restores IL-18-mediated suppression in SMC markers and blunts the expression of the proinflammatory phenotype markers. These results suggest that the curcumin analog EF24 reverses IL-18-induced ASMC proliferation and migration and proinflammatory phenotypic changes by targeting TRAF3IP2 and restoring RECK expression. These results suggest the therapeutic potential of EF24 in vascular inflammatory and proliferative diseases, including atherosclerosis.

Article Snippet: Human primary aortic SMC (ASMC) was purchased from LONZA (#CC-2571, Basel, Switzerland) and cultured in SmGMTM-2 Smooth Muscle Cell Growth Medium-2 with SingleQuotsTM supplements containing growth factors (#CC-4149) as described previously [ , ].

Techniques: Activity Assay, Migration, Inhibition, Expressing, Knockdown

Journal: Journal of vascular research

Article Title: Aberrant soluble epoxide hydrolase and oxylipin levels in a porcine arteriovenous graft stenosis model

doi: 10.1159/000365251

Figure Lengend Snippet: CYP2J2 and sEH expression in SMC and effect of EETs and/or sEH inhibitor on PDGF-induced proliferation of human SMC or fibroblasts. Panel A. Cell lysates from porcine SMC were size fractionated, transferred then immunostained with anti-sEH antibody in the absence (-) or presence (+) of an sEH-specific blocking peptide (left blot). Lysates from murine liver or human SMC were immunostained with anti-human CYP2J2 antibody which detected a protein of ∼57 kD (right blot). Panel B. SMC were serum-starved and then induced to proliferate with PDGF (50 ng/ml). The addition of EETs (0.1 μM-10 μM) in the absence or presence of the sEH inhibitor AUDA (0.1 μM) did not inhibit proliferation. Panel B. AUDA (0.1 μM-10 μM) had no significant effect on fibroblast proliferation induced by 10% FBS. Each bar represents the mean ± SD of 3 or 4 observations.

Article Snippet: [ 51 ] Human aortic SMC and fibroblasts (Cambrex Bio Science, Chicago, IL) as well as porcine venous SMC and fibroblasts (passages 5-8) were seeded into 96-well tissue culture plates at subconfluence and made quiescent by incubation in media with no serum for 48 hr.

Techniques: Expressing, Blocking Assay

Journal: Journal of vascular research

Article Title: Aberrant soluble epoxide hydrolase and oxylipin levels in a porcine arteriovenous graft stenosis model

doi: 10.1159/000365251

Figure Lengend Snippet: Effect of sEH inhibitors on PDGF-stimulated migration of human SMC and fibroblasts through a porous membrane. Panel A. Human aortic SMC were seeded in the upper chamber of the migration apparatus after pretreatment with one of two concentrations of either AUDA or t-AUCB. Migration is shown as a percent of that seen with PDGF alone without inhibitors. Panel B. Human adventitial fibroblasts were seeded into the upper chamber after pretreatment with one of the two inhibitors. *p<0.05 vs. PDGF alone.

Article Snippet: [ 51 ] Human aortic SMC and fibroblasts (Cambrex Bio Science, Chicago, IL) as well as porcine venous SMC and fibroblasts (passages 5-8) were seeded into 96-well tissue culture plates at subconfluence and made quiescent by incubation in media with no serum for 48 hr.

Techniques: Migration, Membrane