Angptl2 induces TGF-β1/Smad signaling in LF fibroblasts.

<p><b>A</b>: Changes in <i>TGF-β1</i> mRNA expression in LF fibroblasts (n = 4) in response to Angptl2 protein applied at the indicated concentration for 6 h. <b>B</b>: Changes in <i>TGF-β1</i> mRNA expression in LF fibroblasts (n = 4) at the indicated time after administration of 5 µg/ml Angptl2 protein. <b>A</b>, <b>B</b>: Expression of <i>TGF-β1</i> mRNA in LF fibroblasts without Angptl2 stimulation was set to 1. Data represent the mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01 versus control (without Angptl2 stimulation). <b>C</b>: TGF-β1 protein concentration in the supernatants of cultures incubated without or with Angptl2 (5 µg/ml) for 24 h. Data represent the mean ± SEM. **<i>P</i><0.01 versus control (without Angptl2 stimulation). <b>D</b>: Changes in <i>TGF-βR1</i> mRNA expression in LF fibroblasts (n = 3) at 6 h after administration of 5 µg/ml Angptl2 protein. **<i>P</i><0.01. <b>E</b>: Changes in <i>TGF-βR2</i> mRNA expression in LF fibroblasts (n = 3) at 6 h after administration of 5 µg/ml Angptl2 protein. **<i>P</i><0.01. <b>F</b>: Representative data of western blot analysis of p-Smad3 expression (upper), Samd2/3 (middle), and Hsc70 (lower) in LF fibroblasts with or without 5 µg/ml Angptl2 protein for 24 h. <b>G</b>: Quantitative evaluation of <b>F</b> (n = 3). Data represent the mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01.</p

Angptl2 expression is positively correlated with TGF-β1 expression in LF tissues.

<p><b>A</b>: Comparison of the expression of <i>TGF-β1</i> mRNA for the LSCS patient group (n = 43) and the non-LSCS group (n = 15). Data represent the mean ± SEM. The value of the non-LSCS was set to 1. **<i>P</i><0.01. <b>B</b>: Correlation between LF thickness and <i>TGF-β1</i> mRNA expression in LF tissues. <b>C</b>: Correlation between <i>Angptl2</i> mRNA expression and <i>TGF-β1</i> mRNA expression in LF tissues. The minimum value for <i>TGF-β1</i> and <i>Angptl2</i> expression in the samples analyzed (<b>B</b>, <b>C</b>) was set to 1. The correlation coefficient (R) and probability (P) value obtained by regression analysis are shown. <b>D</b>: Double immunofluorescence staining for TGF-β1 and vimentin. Nuclei were stained with DAPI. Scale bar represents 50 µm in each panel. <b>F</b>: Immunohistochemistry for p-Smad3 in LF tissue from the LSCS group (left panels) and the non-LSCS group (right panels). The inset in the left panel shows a higher magnification of the area surrounded by the dashed line. Arrowheads indicate p-Smad3-positive cells. Scale bar represents 50 µm in each panel. <b>G</b>: Quantitative evaluation of <b>F</b> (n = 3). Regions of interest (ROI) were selected from nine sites (cranial, middle, and caudal sides of the dorsal, middle, and dural layers) in each sample. Images magnified ×100 were used for the measurements, and the average number of p-Smad3-positive cells as a percentage of the total number of cells was culculated. Data represent the mean ± SEM. **<i>P</i><0.01.</p

Angptl2 contributes to stretching stimulation-mediated TGF-β1 expression.

<p><b>A</b>: Alterations of <i>TGF-β1</i> mRNA expression in LF fibroblasts (n = 3) treated with or without FK506 after stretching stimulation (elongation ratio of 10%, 10 cycles/m) for 24 h. As a control, <i>TGF-β1</i> expression in LF fibroblasts without stretching stimulation was set to 1. <b>B</b>: Downregulation of <i>Angptl2</i> mRNA in LF fibroblasts after Angptl2 siRNA treatment for 24 h (n = 3). Expression of <i>Angptl2</i> mRNA in LF fibroblasts treated with siRNA was set to 1. <b>C</b>: Changes in <i>TGF-β1</i> mRNA expression in LF fibroblasts at 48 h after Angptl2 siRNA treatment (n = 3). Expression of <i>TGF-β1</i> mRNA in LF fibroblasts treated with siRNA was set to 1. <b>D</b>: Alterations of <i>TGF-β1</i> mRNA expression in LF fibroblasts treated with or without Angptl2 siRNA (n = 3) after stretching stimulation (elongation ratio of 10%, 10 cycles/m) for 24 h. As a control, <i>TGF-β1</i> expression in LF fibroblasts with or without Angptl2 siRNA was set to 1. <b>E</b>: TGF-β1 protein concentration in the culture medium of LF fibroblasts with or without Angptl2 siRNA (n = 3) following stretching stimulation (elongation ratio of 10%, 10 cycles/m) for 24 h and 24 h incubation. Data represent the mean ± SEM. *<i>P</i><0.05, **<i>P</i><0.01.</p