human primary vascular smooth muscle cells (vsmc)  (BioResource International Inc)

Journal: iScience

Article Title: Treatment of calcific arterial disease via enhancement of autophagy using GSK343

doi: 10.1016/j.isci.2023.108360

Figure Lengend Snippet: Impaired autophagy is a determinant of VSMC calcification (A) RNA sequencing differential gene expression analysis for calcified versus healthy human VSMCs. Calcified VSMCs were grown in osteogenic media for 3 days. (B) Analysis of canonical pathways impacted by global transcriptomic differential gene expression in calcified VSMCs. (C) Heat map of mRNA expression of autophagy-related genes in healthy versus calcified VSMCs. (D) Quantitative western blot analysis of calcification (RUNX2), autophagy initiation proteins (ULK1, ULK2, ATG13 and p62), and VSMC contractile (CNN1, SM22α) markers along with alizarin red stain for calcium. (E) Quantitative western blot analysis of autophagy flux markers in calcified versus healthy VSMCs in the presence or absence of chloroquine (CQ). (F) Alizarin red staining of human VSMCs under osteogenic conditions indicates calcification is inhibited in cells treated with rapamycin (autophagy activator) and is increased in cells treated with a ULK1 inhibitor (SBI-0206965; autophagy inhibition). (G) (Left panel) Ex vivo treatment of mouse aortas with ULK1 inhibitor shows increased calcification (OsteoSense, red) and inflammation as measured by MMP activity (MMPsense, green). Right panel shows increased calcium deposition in aortas treated with ULK1 inhibitor (alizarin red staining) under osteogenic conditions and increased nuclear Runx2 signal (red) as assessed by immunofluorescence. (H) Alizarin red staining and in situ hybridization for ULK1 mRNA (green) in fixed human tissue from aortic calcification and calciphylaxis patients were compared with healthy tissue, demonstrating reduced ULK1 mRNA levels in the setting of large and small vessel calcification. Significance was calculated using 1-way ANOVA with a Tukey’s multi-comparisons test. Scale bars = 50 um.

Article Snippet: Primary human aortic vascular smooth muscle cells (VSMC) were purchased from Cell Applications Inc. (354K-05a), California, USA.

Techniques: RNA Sequencing, Gene Expression, Expressing, Western Blot, Staining, Inhibition, Ex Vivo, Activity Assay, Immunofluorescence, In Situ Hybridization

Journal: iScience

Article Title: Treatment of calcific arterial disease via enhancement of autophagy using GSK343

doi: 10.1016/j.isci.2023.108360

Figure Lengend Snippet: Differential expression of autophagy initiation genes is associated with human coronary artery disease, and therapeutic activation of autophagy inhibits vascular calcification (A) Volcano plot of differentially expressed genes in normal (n = 24) versus ischemic (n = 36) human coronary artery tissues as detected using DESeq2. Significant differentially expressed genes were considered using FDR adjusted p value <0.05, as indicated by the green points. Autophagy genes are labeled with human gene symbols. (B) Violin plots of differentially regulated autophagy genes in normal versus ischemic coronary artery tissues. Values represent log 10 normalized gene expression levels quantified using a collapsed gene model and RNA-SeQC. p values shown are derived from DESeq2 analysis in normal (n = 24) versus ischemic (n = 36) human coronary artery samples. (C) Immunoblot analysis of RUNX2 from total lysates of human VSMCs under normal or osteogenic conditions with or without treatment of autophagy activator (either GSK343 or rapamycin). (D) Treatment with either GSK343 (10 μM) or rapamycin (100 nM), activators of autophagy, inhibited the calcification of human VSMCs grown in calcification media for 10 days, as evidenced by alizarin red stain. (E) Collagen gel contraction assay was performed demonstrating that autophagy activation with either GSK343 or rapamycin restores the contractility of VSMCs that is disrupted when grown in osteogenic media and decreases cellular calcium content (mg/dL) by colorimetric cresolphthalein method. (F) Heat map of the fold change in mRNA levels of autophagy-related genes from human VSMCs grown under osteogenic media treated without or with GSK343 at low (5 μM) or high (10 μM) concentrations compared with VSMCs in normal media. (G) Immunoblot analysis of ULK1, ULK2, p62, and LC3BI/II from total lysates of human VSMCs under normal or osteogenic conditions and treated with GSK343 (10 μM with three replicates). (H) Quantitative western blot analysis of autophagy flux markers in calcified versus healthy VSMCs shows decreased p62 protein in GSK343-treated cells. (I) Pie chart showing the genomic distribution of the differentially accessible chromatin regions from human healthy VSMCs (top panel, left chart), calcified VSMCs (middle chart), and calcified VSMCs treated with GSK343 (right chart). (J) Overlapping ATAC-seq tracks of the promoter regions of the autophagy-initiation genes (ULK1, ATG13A, BECN1), the contractile gene (CNN1), the calcification marker (RUNX2), and the lysosome marker (LAMP1). ATAC-seq peaks for healthy VSMCs are presented in green and for calcified VSMCs in red. The ATAC-seq data have been normalized to take depth into account, and the scale on the y axis was chosen for optimal visualization of peaks. Also, representative ATAC-seq tracks of the autophagy-initiation genes (ULK1 and ATG13A) from VSMCs under osteogenic conditions treated with DMSO (red peaks) or GSK343 (yellow peaks) are presented. Shown are a portion of the promoter regions (1-2Kb). Significance was calculated using 1-way ANOVA with a Tukey’s multi-comparisons test. Scale bars = 50 um.

Article Snippet: Primary human aortic vascular smooth muscle cells (VSMC) were purchased from Cell Applications Inc. (354K-05a), California, USA.

Techniques: Quantitative Proteomics, Activation Assay, Labeling, Gene Expression, Derivative Assay, Western Blot, Staining, Collagen Gel Contraction Assay, Marker

Journal: iScience

Article Title: Treatment of calcific arterial disease via enhancement of autophagy using GSK343

doi: 10.1016/j.isci.2023.108360

Figure Lengend Snippet: Therapeutic activation of autophagy by GSK343 inhibits calcification in ex vivo calcification (A) Immunofluorescence staining showing reduced ULK1 (green) and CNN1 (magenta) levels in fresh-fixed tissue of human calcified aortas (left panel) and dermal biopsies from calciphylaxis (right panel) compared with healthy controls. (B) Ex vivo calcification assays were performed with fresh surgical biopsies from patients with either aortic calcification (left panel) or (C) calciphylaxis (right panel). Alizarin red staining shows the calcification levels of surgical biopsies in normal or osteogenic media treated for 21 days with DMSO or GSK343 (20 μM). Treatment with GSK343 inhibited the ex vivo calcification of aortic and calciphylaxis tissues (middle panels) and was associated with increased ULK1 and CNN1 expression (lower panel). (D) In our summative model of autophagy in VSMC calcification, defects in the autophagy initiation machinery at the transcriptional level impair autophagy flux and exacerbate calcification of VSMCs. GSK343 pharmacologically activates autophagy by relaxing chromatin and restoring expression of autophagy initiation genes (e.g., ULK1, BECN1, ATG13) to favor the formation of autolysosomes, which associates with inhibition of VSMC calcification. p values determined as compared with the corresponding control, by one-sample t test. Scale bars = 50 um.

Article Snippet: Primary human aortic vascular smooth muscle cells (VSMC) were purchased from Cell Applications Inc. (354K-05a), California, USA.

Techniques: Activation Assay, Ex Vivo, Immunofluorescence, Staining, Expressing, Inhibition, Control