Journal: Scientific Reports

Article Title: Differential responses to thrombospondin-1 and PDGF-BB in smooth muscle cells from atherosclerotic coronary arteries and internal thoracic arteries

doi: 10.1038/s41598-024-66860-x

Figure Lengend Snippet: Morphology and SMC markers in human ITA SMC and ACA SMC. Human SMC derived from 3 distinct ACA donors and 3 distinct ITA donors were plated in T75 flasks with growth medium. Schwann cells and HeLa cells were used as controls. Representative photomicrographs of ACA SMC ( A ) and ITA SMC ( B ) taken at subconfluence are shown. Expression of α-actin ( C ), myh-11 ( D ), calponin ( E ), Sox-10 ( F ) and S100B ( G ) were determined as described in Methods and a representative blot from 3 independent experiments for each protein is shown. Expression of the senescence biomarkers p16INK4A and p21Cip1 were determined by Western blotting as described in Methods for growth arrested ACA SMC and ITA SMC. Expression was quantified and normalized to β-actin expression. Results from 3 independent experiments are shown in H.

Article Snippet: Cell lines of human ACA SMC from 3 distinct donors (Cat# 350q-05a) and cell lines of ITA SMC from 3 distinct donors (Cat# 358-05a) were obtained from Cell Applications (San Diego, CA).

Techniques: Derivative Assay, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Differential responses to thrombospondin-1 and PDGF-BB in smooth muscle cells from atherosclerotic coronary arteries and internal thoracic arteries

doi: 10.1038/s41598-024-66860-x

Figure Lengend Snippet: Proliferative responses of human ITA SMC and ACA SMC to FBS. Human SMC derived from 3 distinct ACA donors and 3 distinct ITA donors were plated at equal seeding density in T25 flasks, grown to subconfluence, growth arrested and then exposed to 10% FBS. Cell counts were performed at 48 h, 72 h, 96 h, 120 h, 144 h, 168 h and 192 h. Results of combining data from the 3 ACA SMC lines and the 3 ITA SMC lines are shown in ( A ) and results from 6 to 10 independent experiments with each cell line are shown in ( B ). [SEM is presented in ( A ) and STD is presented in ( B ); ^— p < 0.05 vs control; *— p < 0.001 vs control; “— p < 0.05 vs ITA SMC].

Article Snippet: Cell lines of human ACA SMC from 3 distinct donors (Cat# 350q-05a) and cell lines of ITA SMC from 3 distinct donors (Cat# 358-05a) were obtained from Cell Applications (San Diego, CA).

Techniques: Derivative Assay, Control

Journal: Scientific Reports

Article Title: Differential responses to thrombospondin-1 and PDGF-BB in smooth muscle cells from atherosclerotic coronary arteries and internal thoracic arteries

doi: 10.1038/s41598-024-66860-x

Figure Lengend Snippet: PDGF-BB induced proliferation is inhibited by anti-TSP1 neutralizing antibody A4.1 and by MiR21-3p inhibitors and MiR21-5p inhibitors in ACA SMC but not in ITA SMC. Human SMC derived from 3 distinct ACA donors ( A , C and D ) and 3 distinct ITA donors ( B , C and E ) were growth arrested as described in Methods. ( A ) and ( B ): The indicated groups were pretreated with vehicle, anti-TSP-1 antibody A4.1 (10 µg/ml), or a non-specific IgG (10 µg/ml) for 30 min prior to treatment with vehicle or PDGF-BB (20 ng/ml). Cell counts were determined 96 h later. [n ≥ 8 independent experiments; #— p < 0.05 compared to Cnt; *— p < 0.05 compared to PDGF-BB + IgG]. ( C ): Total RNA was extracted and quantitative RT-PCR performed in triplicate as described in Methods. ( D ) and ( E ): The indicated groups were pretreated with vehicle, 50 nM miR21-3p inhibitor, 50 nM miR21-5p inhibitor or 50 nM MiRCnt for 24 h prior to treatment with vehicle or PDGF-BB (20 ng/ml). Cell counts were determined 96 h later. [n ≥ 8 independent experiments; *— p < 0.05 compared to cnt; ^— p < 0.05 compared to TSP + MiR cnt].

Article Snippet: Cell lines of human ACA SMC from 3 distinct donors (Cat# 350q-05a) and cell lines of ITA SMC from 3 distinct donors (Cat# 358-05a) were obtained from Cell Applications (San Diego, CA).

Techniques: Derivative Assay, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Differential responses to thrombospondin-1 and PDGF-BB in smooth muscle cells from atherosclerotic coronary arteries and internal thoracic arteries

doi: 10.1038/s41598-024-66860-x

Figure Lengend Snippet: The effects of inhibitors on exogenous TSP1-induced proliferation of ACA SMC and ITA SMC. Human SMC derived from 3 distinct ACA donors and 3 distinct ITA donors were growth arrested and were treated with either vehicle (Cnt) or TSP1 (various concentrations). The results from the different ACA cell lines and different ITA cell lines were consistent and thus the data is presented as an aggregate of the ACA SMC and ITA SMC lines ( A ). In panels ( B ) and ( C ), growth arrested SMC were pretreated with vehicle, anti-TSP-1 antibody A4.1 (10 µg/ml), a non-specific IgG (10 µg/ml), 50 nM miR21-3p inhibitor, 50 nM miR21-5p inhibitor or 50 nM MiRCnt for 30 min prior to treatment with vehicle or TSP1 (12.5 µg/ml). [*— p < 0.05 compared to TSP1 + IgG and #— p < 0.05 compared to TSP1 + MiR cnt].

Article Snippet: Cell lines of human ACA SMC from 3 distinct donors (Cat# 350q-05a) and cell lines of ITA SMC from 3 distinct donors (Cat# 358-05a) were obtained from Cell Applications (San Diego, CA).

Techniques: Derivative Assay

Journal: Scientific Reports

Article Title: Differential responses to thrombospondin-1 and PDGF-BB in smooth muscle cells from atherosclerotic coronary arteries and internal thoracic arteries

doi: 10.1038/s41598-024-66860-x

Figure Lengend Snippet: Expression and secretion of TSP1 in PDGF-BB-treated ACA SMC and ITA SMC. Human SMC derived from 3 distinct ACA donors ( A ) and 3 distinct ITA donors ( B ) were growth arrested as described in Methods and then treated with either vehicle or PDGF-BB (20 ng/ml) for various time periods. TSP1 expression and β-actin expression were determined as described in Methods. Panel A shows a representative blot of lysate from ACA SMC treated with PDGF-BB for 24 h and lysate from ITA SMC treated with PDGF-BB for 24 h run on the same gel. A time course of TSP1 expression is shown in panels B–E. SMC isolated from ACA ( B and C ) and SMC isolated from ITA ( D and E ) were treated with PDGF-BB (20 ng/ml) for various times and representative blots are shown in B and D with results of 6 independent experiments shown in C and E. [*— p < 0.05 compared to basal levels]. In panels F and G, SMC were treated with PDGF-BB (20 ng/ml) for the indicated time points, and then conditioned medium was harvested and concentrated. This was followed by Western blot analysis with anti-TSP1 antibody as described in Methods. There were no statistically significant differences, compared to t = 0, for any of the time points in F or G.

Article Snippet: Cell lines of human ACA SMC from 3 distinct donors (Cat# 350q-05a) and cell lines of ITA SMC from 3 distinct donors (Cat# 358-05a) were obtained from Cell Applications (San Diego, CA).

Techniques: Expressing, Derivative Assay, Isolation, Western Blot