Journal: Biochemical pharmacology

Article Title: S-adenosylmethionine attenuates angiotensin II-induced aortic dissection formation by inhibiting vascular smooth muscle cell phenotypic switch and autophagy.

doi: 10.1016/j.bcp.2023.115967

Figure Lengend Snippet: Fig. 4. SAM alleviates autophagy activation in AngII-induced VSMCs in vitro. (A) The molecular formula of SAM. (B) Cytotoxicity assay was performed to detect the effect of SAM on the viability of VSMCs (n = 5). (C) Representative western blotting bands of expression of autophagy-associated marker proteins (Beclin1, p62, and LC3II/LC3I) in VSMCs by gradient concentration of SAM (n = 3). The results of statistical analysis of expression of Beclin1 (D), p62 (E) and LC3II/LC3I (F). (G) Representative western blotting bands of the expression of gradient concentration of AngII on autophagy-related marker proteins (Beclin1, p62, and LC3II/LC3I) in VSMCs (n = 3). The results of statistical analysis of the protein expression levels of Beclin1 (H), p62 (I) and LC3II/LC3I (J). (K) Representative pictures of autophagic lysosomes of VSMCs in four groups detected by MDC staining (Scale bars: 50 μm, n = 6), and the four groups are Control, SAM, AngII, and AngII + SAM groups. (L) Results of statistical analysis of MDC fluorescence intensity. Representative pictures of LC3B (M) and p62 (O) IF staining in four groups of VSMCs (Scale bars: 50 μm, n = 5). Results of statistical analysis of mean fluorescence intensity of LC3B (N) and p62 (P)in four groups of VSMCs. The statistical analyses between multiple groups were performed by one-way ANOVA and Tukey’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: VSMCs were stimulated with AngII (MedChemExpress, #HY-13948, China) for 48 h. Previous studies have demonstrated that adding exogenously administered SAM of medium increases intracellular SAM levels [20,23].

Techniques: Activation Assay, In Vitro, Cytotoxicity Assay, Western Blot, Expressing, Marker, Concentration Assay, Staining, Control, Fluorescence, Comparison

Journal: Biochemical pharmacology

Article Title: S-adenosylmethionine attenuates angiotensin II-induced aortic dissection formation by inhibiting vascular smooth muscle cell phenotypic switch and autophagy.

doi: 10.1016/j.bcp.2023.115967

Figure Lengend Snippet: Fig. 5. SAM abolishes AngII-induced phenotypic switch of VSMCs in vitro. (A) The western blot experiment was performed to detect the expression of contractile marker protein SM22α and synthetic marker proteins (OPN and MMP2) in four groups of VSMCs (n = 3). The four groups contain the Control group, SAM group, AngII group, and AngII + SAM group. Results of statistical analysis of protein expression levels of SM22α (B), OPN (C), and MMP2 (D). The representative pictures of the IF staining of VSMCs OPN (E) in the four groups (Scale bars: 50 μm, n = 5). Representative pictures of IF staining of VSMCs MMP2 (G) in the four groups (Scale bars: 20 μm, n = 5). Results of statistical analysis of mean fluorescence intensity of VSMCs OPN (F) and MMP2 (H) in 4 groups. The statistical analyses between multiple groups were performed by one-way ANOVA and Tukey’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: VSMCs were stimulated with AngII (MedChemExpress, #HY-13948, China) for 48 h. Previous studies have demonstrated that adding exogenously administered SAM of medium increases intracellular SAM levels [20,23].

Techniques: In Vitro, Western Blot, Expressing, Marker, Control, Staining, Fluorescence, Comparison

Journal: Biochemical pharmacology

Article Title: S-adenosylmethionine attenuates angiotensin II-induced aortic dissection formation by inhibiting vascular smooth muscle cell phenotypic switch and autophagy.

doi: 10.1016/j.bcp.2023.115967

Figure Lengend Snippet: Fig. 7. SAM affects AngII-induced dysregulation of autophagy and phenotypic switch in VSMCs in vitro, mainly through activation of the PI3K/AKT signaling pathway. Results of statistical analysis of protein expression levels of p-PI3K (A), PI3K (B), p-AKT (C), AKT (D), p-mTOR (E), and mTOR (F). The four groups were Control, SAM, AngII, and AngII + SAM groups (n = 3). Results of statistical analysis of protein expression levels of p-PI3K (G), PI3K (H), p-AKT (I), AKT (J), p-mTOR (K), and mTOR (L). The four groups were Control group, AngII group, AngII + SAM group, and AngII + SAM + LY294002 group (n = 3). The statistical analyses between multiple groups were performed by one-way ANOVA and Tukey’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: VSMCs were stimulated with AngII (MedChemExpress, #HY-13948, China) for 48 h. Previous studies have demonstrated that adding exogenously administered SAM of medium increases intracellular SAM levels [20,23].

Techniques: In Vitro, Activation Assay, Expressing, Control, Comparison

Journal: Hypertension

Article Title: Pharmacological Characterization of SAR407899, a Novel Rho-Kinase Inhibitor

doi: 10.1161/hypertensionaha.109.134353

Figure Lengend Snippet: Figure 1. In vitro characterization of SAR407899. A, SAR407899 exhibits a Ki value of 364 nM against human ROCK-2 vs a Ki value of 27114 nM for fasudil. B, SAR407899, Y-27632, and fasudil, as well as 13 compounds structurally related to SAR407899, were not selective for human ROCK-2 vs human ROCK-1. C through E, Detection of ROCK-mediated phosphorylation of MYPTT696. C, SAR407899 (0.1, 0.3, 1.0, and 3.0 mol/L) specifically inhibits the ROCK-mediated phosphorylation of MYPTT696 in HeLa cells stimu- lated with PHEN (5 mol/L). D, Primary rat aortic smooth muscle cells were stimulated with PHEN (5 mol/L), and the ROCK-mediated phosphorylation of MYPTT696 was inhibited by SAR407899 at 1 mol/L and 10 mol/L. E, ROCK-mediated phosphorylation of MYPTT696 in thoracic aorta of SHRs was inhibited by an IV infusion of SAR407899 at 3 mg/kg. The presence of MYPT in thoracic aorta of SHRs is shown on the right.

Article Snippet: Cell culture and primary rat vascular smooth muscle cells HeLa cells (American Type Culture Collection) were maintained in DMEM (Gibco, Germany) containing 10% FCS.

Techniques: In Vitro, Phospho-proteomics

Journal: European Journal of Clinical Investigation

Article Title: Phenylacetylglutamine and trimethylamine N‐oxide: Two uremic players, different actions

doi: 10.1111/eci.14074

Figure Lengend Snippet: In vitro experiments investigating the effect of TMAO and PAG in VSMC calcification and oxidative stress. (A) TMAO‐induced calcification assay using BoneTag Optical Dye ( n = 6 per group). (B) PAG‐induced calcification assay using BoneTag Optical Dye. Aortic VSMCs were incubated with uremic toxins for 7 days in the background of high phosphate (2.5 mM phosphate). For both experiments, readouts were normalised for protein content using BCA assay ( n = 4 per group). (C) mRNA expression analysis of osteogenic marker Runx2. Relative gene expression was normalised to DMEM control group ( n = 4 per group). (D) Representative images of alizarin red staining for specified conditions. (E) Reactive oxygen species assay for specified conditions using DCFDA/H2DCFDA assay ( n = 4 per group). Readout taken 4 h after addition of toxins. Statistics: data presented as mean ± SEM. Groups were compared using one‐way ANOVA followed by group‐wise comparisons using Tukey's post hoc test. p‐ values <.05 were deemed statistically significant. * p < .05, ** p < .01, *** p < .001, **** p < .0001. PAG, phenylacetylglutamine; ROS, reactive oxygen species; TMAO, trimethylamine N‐oxide.

Article Snippet: Human aortic VSMCs (ATCC) from a healthy male donor were incubated between passages 4–7 with one of the following conditions: control (DMEM), high phosphate (2.5 mmol/L sodium phosphate), TMAO (2.5 mmol/L sodium phosphate + 100/300 μM TMAO), or PAG (2.5 mmol/L sodium phosphate + 100/300 μM PAG).

Techniques: In Vitro, Incubation, BIA-KA, Expressing, Marker, Gene Expression, Control, Staining