Journal: Experimental and therapeutic medicine

Article Title: SPINT2 is involved in the proliferation, migration and phenotypic switching of aortic smooth muscle cells: Implications for the pathogenesis of thoracic aortic dissection.

doi: 10.3892/etm.2023.12245

Figure Lengend Snippet: Figure 1. DEG screening of dataset GSE52093 and bioinformatics analysis. (A) Volcano plot of the gene profiles. (B) The gene network was visualized by heatmap, which indicated that the expression of the identified DEGs could correctly distinguish between the TAD and normal samples. (C) Chord plot of GO analysis. (D) GSEA enrichment in the KEGG pathway of vascular smooth muscle contraction (hsa04270). DEG, differentially expressed gene; SPINT2, serine peptidase inhibitor Kunitz type 2; TAD, thoracic aortic dissection; GO, Gene Ontology; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: The sections (5 μm) of the TAD specimens were incubated overnight at 4 ̊C with the following goat anti‐mouse primary antibodies: SPINT2 (1:50; cat. no. sc‐398119; Santa Cruz Biotechnology, Inc.) and α‐SMA (1:200; cat. no. AF1032; Affinity Biosciences).

Techniques: Expressing, Dissection

Journal: Experimental and therapeutic medicine

Article Title: SPINT2 is involved in the proliferation, migration and phenotypic switching of aortic smooth muscle cells: Implications for the pathogenesis of thoracic aortic dissection.

doi: 10.3892/etm.2023.12245

Figure Lengend Snippet: Figure 2. SPINT2 is expressed at low levels in aorta tissues of TAD specimens. (A) SPINT2 expression was co‑stained with α‑SMA expression in human TAD specimens. (B) The mRNA levels of SPINT2 in aorta tissues of patients with TAD were determined using reverse transcription‑quantitative PCR. (C) SPINT2 protein levels of aorta tissues were analyzed by western blotting. ***P<0.001 compared with the normal group. SPINT2, serine peptidase inhibitor Kunitz type 2; TAD, thoracic aortic dissection; α‑SMA, α‑small nuclear actin; N, normal group; T, TAD group; DAPI, 4',6‑diamidino‑2‑phenylindole.

Article Snippet: The sections (5 μm) of the TAD specimens were incubated overnight at 4 ̊C with the following goat anti‐mouse primary antibodies: SPINT2 (1:50; cat. no. sc‐398119; Santa Cruz Biotechnology, Inc.) and α‐SMA (1:200; cat. no. AF1032; Affinity Biosciences).

Techniques: Expressing, Western Blot, Dissection

Journal: Experimental and therapeutic medicine

Article Title: SPINT2 is involved in the proliferation, migration and phenotypic switching of aortic smooth muscle cells: Implications for the pathogenesis of thoracic aortic dissection.

doi: 10.3892/etm.2023.12245

Figure Lengend Snippet: Figure 3. SPINT2 expression is decreased in PDGF‑BB‑cultured SMCs. SMCs were treated with different concentrations of PDGF‑BB (0, 10, 20 and 40 ng/ml) for 24 h. (A and B) The mRNA and protein levels of SPINT2 were measured using reverse transcription‑quantitative PCR and western blotting analyses. (C) SMC proliferation was detected using a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. *P<0.05, **P<0.01 and ***P<0.001 compared with 0 ng/ml PDGF‑BB group. SPINT2, serine peptidase inhibitor Kunitz type 2; PDGF‑BB, platelet‑derived growth factor BB; SMCs, smooth muscle cells.

Article Snippet: The sections (5 μm) of the TAD specimens were incubated overnight at 4 ̊C with the following goat anti‐mouse primary antibodies: SPINT2 (1:50; cat. no. sc‐398119; Santa Cruz Biotechnology, Inc.) and α‐SMA (1:200; cat. no. AF1032; Affinity Biosciences).

Techniques: Expressing, Western Blot, 5 Diphenyltetrazolium Bromide Assay

Journal: Experimental and therapeutic medicine

Article Title: SPINT2 is involved in the proliferation, migration and phenotypic switching of aortic smooth muscle cells: Implications for the pathogenesis of thoracic aortic dissection.

doi: 10.3892/etm.2023.12245

Figure Lengend Snippet: Figure 4. Overexpression of SPINT2 suppresses viability and proliferation of PDGF‑BB‑treated SMCs. SMCs were infected with NC or SPINT2‑overexpression adenovirus for 24 h and subsequently cultured with 20 ng/ml PDGF‑BB for 24 h. (A and B) The mRNA and protein levels of SPINT2 were verified by reverse transcription‑quantitative PCR and western blotting following PDGF‑BB treatment for 24 h. (C) The SMC viability at 48 h following PDGF‑BB treatment was detected using a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. (D) The expression levels of Ki‑67 were determined by immunofluores‑ cence staining to evaluate the SMC proliferation (scale bar, 50 µm). *P<0.05, **P<0.01 and ***P<0.001 compared with control group. ##P<0.01 and ###P<0.001 compared with the PDGF‑BB + Ad‑vector group. SPINT2, serine peptidase inhibitor Kunitz type 2; PDGF‑BB, platelet‑derived growth factor BB; SMCs, smooth muscle cells; NC, PDGF‑BB + Ad‑vector; DAPI, 4',6‑diamidino‑2‑phenylindole; Ad, adenovirus.

Article Snippet: The sections (5 μm) of the TAD specimens were incubated overnight at 4 ̊C with the following goat anti‐mouse primary antibodies: SPINT2 (1:50; cat. no. sc‐398119; Santa Cruz Biotechnology, Inc.) and α‐SMA (1:200; cat. no. AF1032; Affinity Biosciences).

Techniques: Over Expression, Infection, Cell Culture, Western Blot, 5 Diphenyltetrazolium Bromide Assay, Expressing, Staining, Control

Journal: Experimental and therapeutic medicine

Article Title: SPINT2 is involved in the proliferation, migration and phenotypic switching of aortic smooth muscle cells: Implications for the pathogenesis of thoracic aortic dissection.

doi: 10.3892/etm.2023.12245

Figure Lengend Snippet: Figure 5. Overexpression of SPINT2 inhibits SMC migration and expression of MMPs. (A) Representative images and quantification of the wound healing assay. Scale bar, 200 µm. (B and C) The expression levels of MMP‑2 and MMP‑9 were determined using ELISA and western blotting. **P<0.01 and ***P<0.001 compared with the control group. #P<0.05, ##P<0.01 and ###P<0.001 compared with the PDGF‑BB + Ad‑vector group. SPINT2, serine peptidase inhibitor Kunitz type 2; SMC, smooth muscle cells; MMP, matrix metalloproteinase; PDGF‑BB, platelet‑derived growth factor BB; Ad, adenovirus.

Article Snippet: The sections (5 μm) of the TAD specimens were incubated overnight at 4 ̊C with the following goat anti‐mouse primary antibodies: SPINT2 (1:50; cat. no. sc‐398119; Santa Cruz Biotechnology, Inc.) and α‐SMA (1:200; cat. no. AF1032; Affinity Biosciences).

Techniques: Over Expression, Migration, Expressing, Wound Healing Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Experimental and therapeutic medicine

Article Title: SPINT2 is involved in the proliferation, migration and phenotypic switching of aortic smooth muscle cells: Implications for the pathogenesis of thoracic aortic dissection.

doi: 10.3892/etm.2023.12245

Figure Lengend Snippet: Figure 6. Overexpression of SPINT2 prevents PDGF‑BB‑induced SMC phenotypic switching. SMCs were incubated with 20 ng/ml PDGF‑BB for 24 h following 24 h of adenoviral infection. (A) The expression levels of the synthetic proteins (vimentin and collagen I) were detected by western blotting. (B) The expression levels of the contractile proteins (α‑SMA and SM22α) were detected by western blotting. (C) The expression levels of vimentin were assessed by immunofluorescence analysis. The nuclei were stained with DAPI. Scale bar, 100 µm. ***P<0.001 compared with the control group. ###P<0.001 compared with the PDGF‑BB + Ad‑vector group. SPINT2, serine peptidase inhibitor Kunitz type 2; PDGF‑BB, platelet‑derived growth factor BB; SMCs, smooth muscle cells; α‑SMA, smooth muscle α‑actin; SM22α, smooth muscle protein 22‑α; DAPI, 4',6‑diamidino‑2‑phenylindole; Ad, adenovirus.

Article Snippet: The sections (5 μm) of the TAD specimens were incubated overnight at 4 ̊C with the following goat anti‐mouse primary antibodies: SPINT2 (1:50; cat. no. sc‐398119; Santa Cruz Biotechnology, Inc.) and α‐SMA (1:200; cat. no. AF1032; Affinity Biosciences).

Techniques: Over Expression, Incubation, Infection, Expressing, Western Blot, Immunofluorescence, Staining, Control

Journal: Experimental and therapeutic medicine

Article Title: SPINT2 is involved in the proliferation, migration and phenotypic switching of aortic smooth muscle cells: Implications for the pathogenesis of thoracic aortic dissection.

doi: 10.3892/etm.2023.12245

Figure Lengend Snippet: Figure 7. SPINT2 regulates SMC phenotypic transition via activation of the ERK signaling pathway. (A) SMCs were incubated with 20 ng/ml PDGF‑BB for 24 h following 24 h of adenoviral infection. The expression levels of p‑ERK and ERK in SMCs were detected by western blotting. ***P<0.001 compared with the control group. ###P<0.001 compared with the PDGF‑BB + Ad‑vector group. (B) SMCs were cultured with 100 nmol/l TPA following 24 h of adenoviral infection. SMC proliferation at 48 h following PDGF‑BB treatment was detected using a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. (C) Following 24 h of PDGF‑BB treatment, the expression levels of MMP‑2 and MMP‑9 were determined using ELISA. (D and E) The expression levels of vimentin and SM22α proteins were detected by western blotting. *P<0.05, **P<0.01 and ***P<0.001 compared with the PDGF‑BB + Ad‑SPINT2 group. SPINT2, serine peptidase inhibitor Kunitz type 2; SMCs, smooth muscle cells; PDGF‑BB, platelet‑derived growth factor BB; p, phosphorylated; TPA, tetradecanoylphorbol‑13‑acetate; MMP, matrix metalloproteinase; SM22α, smooth muscle protein 22‑α; Ad, adenovirus.

Article Snippet: The sections (5 μm) of the TAD specimens were incubated overnight at 4 ̊C with the following goat anti‐mouse primary antibodies: SPINT2 (1:50; cat. no. sc‐398119; Santa Cruz Biotechnology, Inc.) and α‐SMA (1:200; cat. no. AF1032; Affinity Biosciences).

Techniques: Activation Assay, Incubation, Infection, Expressing, Western Blot, Control, Cell Culture, 5 Diphenyltetrazolium Bromide Assay, Enzyme-linked Immunosorbent Assay