Journal: Scientific Reports
Article Title: Dedifferentiation of smooth muscle cells in intracranial aneurysms and its potential contribution to the pathogenesis
doi: 10.1038/s41598-020-65361-x
Figure Lengend Snippet: PDGF-BB as a factor for smooth muscle cells to migrate, dedifferentiate and express pro-inflammatory genes. ( A ) Expression of PDGF-BB or Amphiregulin (AREG) in endothelial cells of intracranial aneurysm (IA) lesions. On the 14 th day after IA induction, IA lesions at the right anterior cerebral-olfactory artery bifurcation were harvested and subjected to the immunohistochemical analyses. The representative images of immunohistochemistry for PDGF-BB or AREG (green), Cadherin 5, a marker for endothelial cells, (red), nuclear staining by DAPI (blue) and merged images are shown. The magnified images corresponding to the square in the upper panels are shown in the lower panels. Bars, 10 µm. ( B ) Chemotactic activity of PDGF-BB or AREG on smooth muscle cells (SMCs). The migration of primary culture of SMCs across matrigel-coated pores via 100 ng/ml PGDF-BB or 100 ng/ml AREG was assessed by a transwell system. The representative images of SMCs migrated are shown. Bars, 10 µm. The number of migrated cells is shown in the lower graph. Data represents the mean ± SEM (n = 4). Statistical analysis was done by a Kruskal–Wallis test. *p < 0.05. ( C ) Dedifferentiation of SMCs by PDGF-BB. Primary culture of SMCs were stimulated with 100 ng/ml PGDF-BB or 100 ng/ml AREG for 72 h and expression of SMA was assessed by western blot analysis using α-tubulin as an internal control. The representative images are shown. ( D,E ) Induction of PTGS2 (COX-2) or IL6 by PDGF-BB in cultured SMCs. Primary culture of SMCs were stimulated with vehicle (V), 100 ng/ml PGDF-BB (P) or 100 ng/ml AREG ( A ) ( D ) or each dose of PDGF-BB ( E ) for 1 h and expression of TNF , CCL2 , PTGS2 or IL6 was examined by quantitative RT-PCR analysis. Data represents the mean ± SEM (n = 4). Statistical analysis was done by a Kruskal–Wallis test. *p < 0.05.
Article Snippet: The following primary antibodies were used; mouse monoclonal anti-smooth muscle alpha actin (SMA) antibody (#M0851, Dako, Agilent, Santa Clara, CA), mouse monoclonal anti-non-muscle Myosin IIB/myosin heavy chain 10 (MYH10) antibody (#ab684, Abcam, Cambridge, UK), rabbit polyclonal anti-tumor necrosis factor (TNF)-alpha antibody (#ab6671, Abcam), rabbit polyclonal anti-monocyte chemoattractant protein-1 (MCP-1) antibody (#ab9779, Abcam), mouse polyclonal anti-cyclooxygenase-2 (COX-2) antibody (#aa570–598, Cayman Chemical, Ann Arbor, MI), rabbit polyclonal anti-IL-6 antibody (#ab6672, Abcam), rabbit polyclonal anti-platelet-derived growth factor (PDGF)-AA antibody (#ab198874, Abcam), rabbit polyclonal anti-PDGF-BB antibody (#ab16829, Abcam), rabbit polyclonal anti-Amphiregulin (AREG) antibody (#bs-3847R, Bioss ANTIBODIES, Boston, MA), rabbit polyclonal anti-VE-cadherin (Cadherin 5) antibody (#36–1900, Thermo Fisher Scientific), mouse monoclonal anti-CD68 antibody (#ab31630, Abcam), rabbit polyclonal anti-NFE2L2 antibody (#16396-1-AP, Thermo Fisher Scientific).
Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry, Marker, Staining, Activity Assay, Migration, Western Blot, Cell Culture, Quantitative RT-PCR
