Journal: Frontiers in Allergy

Article Title: Matricellular Protein Periostin Promotes Pericyte Migration in Fibrotic Airways

doi: 10.3389/falgy.2021.786034

Figure Lengend Snippet: Periostin expression is increased following chronic aeroallergen exposure in mice. (A) Schematic diagram of HDM-induced allergic airway inflammation in mice. (B) Female C57/Bl6 mice (6–8 weeks old) were exposed to either sterile PBS (10 μl intranasally) or house dust mite extract (HDM; 25 μg in 10 μL) 5 days a week for five consecutive weeks. Hematoxylin and eosin stained lung sections from PBS and HDM-exposed mice. (C) Bronchoalveolar lavage fluid total cell counts and percentage of eosinophils. (D) At the end of the allergen exposure protocol, lung sections obtained from PBS control and HDM-exposed mice were stained with an anti-periostin antibody (green) and DAPI to stain nuclei (blue). Images were taken at 400x and 630x magnification as stated. Arrows indicate periostin positive cells. (E) Expression of periostin was calculated in the area of interest around each airway using ImageJ. ** p < 0.01, n = 3 representative of two independent experiments. (F) Bronchoalveolar lavage samples were collected and the periostin content was assessed by ELISA. n = 4 representative of two independent experiments. (G,H) Lung sections were stained for periostin (green), the pericyte marker PDGFRβ (red), and cell nuclei (DAPI, blue) to demonstrate the presence of periostin-expressing pericytes around the airways and blood vessels of HDM-exposed mice. (I) Tracheobronchial whole mounts were stained for the mesenchymal cell marker α-smooth muscle actin (α-SMA; red), the endothelial cell marker CD31 (cyan), and periostin (green) and imaged at 400x magnification; arrow indicate periostin-positive pericytes. (J) The expression of periostin and α-SMA in tracheobronchial whole mounts was calculated using ImageJ. AW, airway; BV, blood vessel. ** p < 0.01, n = 3–5 representative of two independent experiments.

Article Snippet: HPLPCs (human placental pericytes) from Promocell (Heidelberg, Germany) were cultured until between passage 5–10 in pericyte-specific medium (Promocell) with 1% antibiotic/antimycotic (ThermoFisher, Massachusetts, USA).

Techniques: Expressing, Staining, Enzyme-linked Immunosorbent Assay, Marker

Journal: Frontiers in Allergy

Article Title: Matricellular Protein Periostin Promotes Pericyte Migration in Fibrotic Airways

doi: 10.3389/falgy.2021.786034

Figure Lengend Snippet: Type 2 inflammatory mediators stimulate periostin expression by pericytes. (A,B) Immunostaining performed on pericytes grown in pericyte medium and treated with 10 ng/ml of TGF-β or 100 ng/ml of periostin for 24 h. Cells were stained with an anti-periostin antibody (green) and the nuclear stain DAPI (blue). Images were taken at 400x magnification and intensity of periostin stain was calculated with ImageJ. Intensity of stain per field of view was divided by the number of cells in order to determine periostin expression per cell. * p < 0.05, n = 5 representative of two independent experiments. (C) Cultured pericytes were treated with 10 ng/ml TGF-β, 10 ng/ml EGF, 10 ng/ml VEGF, 100 ng/ml IL-13 or 100 ng/ml Periostin in pericyte medium for 7 days before the supernatant was harvested. The periostin content was assessed using an anti-periostin ELISA kit. **** p < 0.0001, n = 3 representative of two independent experiments. (D,E) Cultured pericytes were treated with 10 ng/ml TGF-β or 100 ng/ml IL-13 for either 7 or 9 days. Cells were stained with an anti-periostin antibody and an anti-αSMA antibody. The images were taken at 400x magnification and quantifications were made using ImageJ. * p < 0.05, n = 3–4 representative of two independent experiments.

Article Snippet: HPLPCs (human placental pericytes) from Promocell (Heidelberg, Germany) were cultured until between passage 5–10 in pericyte-specific medium (Promocell) with 1% antibiotic/antimycotic (ThermoFisher, Massachusetts, USA).

Techniques: Expressing, Immunostaining, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Allergy

Article Title: Matricellular Protein Periostin Promotes Pericyte Migration in Fibrotic Airways

doi: 10.3389/falgy.2021.786034

Figure Lengend Snippet: Type 2 inflammatory mediators stimulate pericyte migration. (A) Scratch assay performed on pericytes that were grown in pericyte medium and treated with 10 ng/ml of TGF-β, 100 ng/ml of IL-13 or 100 ng/ml of periostin for 48 h. Cells were transferred into serum-free medium and a scratch was made in each monolayer using a p200 pipette tip. (B) Images were taken at 100x magnification immediately after scratching and 24 h later. The scratch width was determined using ImageJ and the average distance of cell migration was calculated. n = 6 representative of two independent experiments, * p < 0.05 vs. untreated cells.

Article Snippet: HPLPCs (human placental pericytes) from Promocell (Heidelberg, Germany) were cultured until between passage 5–10 in pericyte-specific medium (Promocell) with 1% antibiotic/antimycotic (ThermoFisher, Massachusetts, USA).

Techniques: Migration, Wound Healing Assay, Transferring

Journal: Frontiers in Allergy

Article Title: Matricellular Protein Periostin Promotes Pericyte Migration in Fibrotic Airways

doi: 10.3389/falgy.2021.786034

Figure Lengend Snippet: Cinnamaldehyde treatment suppresses periostin expression and pericyte migration. (A) Cultured pericytes were treated with 100 ng/ml IL-13 in pericyte media for 7 days with the addition of cinnamaldehyde for the last 3 days at the concentration stated in (A) or only cinnamaldehyde at the stated concentration for 3 days. The supernatants were harvested and the periostin content was assessed by ELISA. ** p < 0.01, *** p < 0.001 vs. the same cinnamaldehyde concentration without IL-13, n = 2 representative of two independent experiments. (B) Scratch assay performed on pericytes that were grown in pericyte medium and treated with 10 ng/ml of TGF-β, 100 ng/ml of IL-13 or 100 ng/ml of periostin for 7 days with 1 mM cinnamaldehyde added for the last 3 days. Cells were transferred into media lacking serum and a scratch was made in each monolayer using a p200 pipette tip. Images were taken at 100x magnification immediately after scratching and 24 h later. The scratch width was determined using ImageJ and the average distance of cell migration was calculated. n = 3 representative of two independent experiments, Δ = p < 0.05 vs. the same growth factor treatment without cinnamaldehyde, * p < 0.05, ** p < 0.01 vs. control treatment without cinnamaldehyde. (C) Schematic diagram of the putative signaling pathways regulating periostin expression in pericytes (created using Biorender).

Article Snippet: HPLPCs (human placental pericytes) from Promocell (Heidelberg, Germany) were cultured until between passage 5–10 in pericyte-specific medium (Promocell) with 1% antibiotic/antimycotic (ThermoFisher, Massachusetts, USA).

Techniques: Expressing, Migration, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Wound Healing Assay, Transferring