Journal: bioRxiv

Article Title: 3D-bioprinted, phototunable hydrogel models for studying adventitial fibroblast activation in pulmonary arterial hypertension

doi: 10.1101/2022.11.11.516188

Figure Lengend Snippet: Fibroblast activation in 3D-bioprinted models of pulmonary arterial adventitia. A) Fibrotic activation in soft and stiffened 3D hydrogels measured by αSMA expression. HPAAFs in stiffened constructs were significantly more positive for αSMA than cells in soft constructs. Columns represent mean ± SEM, n = 3. *, p < 0.05, Mann-Whitney U test. B) Representative confocal images of immunostaining for αSMA, actin, and DAPI in soft and stiffened 3D hydrogels. HPAAFs in stiffened constructs showed more prevalent αSMA immunofluorescence than cells in soft constructs. Scale bar = 250 µm. C) Fibroblast proliferation in soft and stiffened 3D bioprinted constructs measured by EdU positivity. HPAAFs in stiffened constructs were significantly more positive for EdU than cells in soft constructs. Columns represent mean ± SEM, n = 3. *, p < 0.05, Mann-Whitney U test. D) Representative confocal images of immunostaining for EdU and Hoechst dye in soft and stiffened 3D hydrogels. HPAAFs in stiffened constructs showed more prevalent EdU immunofluorescence than cells in soft constructs. Scale bar = 300 µm.

Article Snippet: 3D-bioprinted constructs were cultured in complete human pulmonary artery adventitia fibroblast medium (AcceGen) supplemented with 2% FBS, 50 U mL −1 penicillin, 50 × 10 −6 M mL −1 streptomycin, and 0.5 μg mL −1 amphotericin B (Sigma) for metabolic activity experiments.

Techniques: Activation Assay, Expressing, Construct, MANN-WHITNEY, Immunostaining, Immunofluorescence

Journal: The FASEB Journal

Article Title: Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae –induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation

doi: 10.1096/fj.201601045R

Figure Lengend Snippet: TRPV4 is required for TGF-β1–mediated differentiation of NHLFs and DHLFs. A) mLFs isolated from WT and TRPV4-KO mice were treated with TGF-β1 (2 ng/ml) for 24, 48, and 96 h, and expression of α-SMA and FN was analyzed by Western blotting. Note that time-dependent TGF-β1 response seen in WT fibroblasts was attenuated in TRPV4-KO fibroblasts. B) Effect of TGF-β1 treatment on TRPV4-dependent calcium flux in NHLFs. NHLFs were loaded with Fluo-4, and calcium flux was measured as described in Materials and Methods in response to a specific TRPV4 activator, GSK1016790A (100 nM), and quantified. C–E) TRPV4 protein was knocked down in NHLFs by transfecting them with siRNA against TRPV4 (20 nM). NHLFs transfected with nonspecific (NS) siRNA were used as control. Twenty-four hours after transfection, NHLFs were treated with TGF-β1 (2 ng/ml; 48 h). C) TRPV4 transcript was analyzed by real-time PCR. D) α-SMA localization was determined by immunofluorescence (red). E) α-SMA expression was analyzed by Western blotting and quantified. F) TRPV4-mediated calcium flux in NHLFs and DHLFs. Calcium flux, indicative of TRPV4 activity, was analyzed in NHLFs and DHLFs from same passage in response to GSK and quantified. G–I) Differential responses of NHLFs and DHLFs to TGF-β1. G) α-SMA transcript levels both at basal and in response to TGF-β1 (2 ng/ml; 48 h) were analyzed by real-time PCR in NHLFs and DHLFs. H, I) Effect of RN1734, a selective TRPV4 antagonist on TGF-β1–induced fibroblast differentiation in NHLFs and DHLFs. H) Both NHLFs and DHLFs were stimulated with or without TGF-β1 (2 ng/ml; 48 h) in the presence or absence of a TRPV4 inhibitor RN1734 (10, 30, and 50 μM), and α-SMA protein expression was determined by Western blotting. Blots were stripped and reprobed with GAPDH Ab. I) Blots were quantified using AlphaView software. Results are means ± sem from 3 independent experiments (B, C, E–G, I). *P < 0.05; **P < 0.01.

Article Snippet: Validated primary normal human lung fibroblasts (NHLFs; CC-2512) and diseased [asthma; male, 27 yr old, diagnosed at age 7 and on medication (Proventil, albuterol; Merck, Darmstadt, Germany)] human lung fibroblasts (DHLFs; 00194912) were obtained from Lonza (Walkersville, MD, USA).

Techniques: Isolation, Expressing, Western Blot, Transfection, Control, Real-time Polymerase Chain Reaction, Immunofluorescence, Activity Assay, Software

Journal: The FASEB Journal

Article Title: Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae –induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation

doi: 10.1096/fj.201601045R

Figure Lengend Snippet: TRPV4 mediates matrix synthesis in fibroblasts. A, B) NHLFs were treated with TGF-β1 (2 ng/ml; indicated time) with or without TRPV4 inhibitor RN1734 (30 μM preincubation) and collagen 1A1 (A) and FN (B) transcripts were analyzed by real-time PCR. C) FN protein accumulation was analyzed by immunofluorescence in cells without permeabilizing with Triton X-100. D, E) FN protein expression was determined by Western blotting (D) and quantified (E). F–I) DHLFs exhibit higher basal and TGF-β1–induced matrix transcripts and protein levels compared with NHLFs. F, G) NHLFs and DHLFs were treated with TGF-β1 (2 ng/ml; 72 h), and Col1A1 (F) and FN (G) transcripts were analyzed by real-time PCR. H, I) FN protein expression was determined by western blotting (H) and quantified in the presence or absence of RN1734 (30 μM; I). Blots were stripped and reprobed with GAPDH Ab. Blots were quantified using AlphaView software (ProteinSimple). Results are means ± sem from at least 3 independent experiments (A, B, E, G, I). *P < 0.05; **P < 0.01.

Article Snippet: Validated primary normal human lung fibroblasts (NHLFs; CC-2512) and diseased [asthma; male, 27 yr old, diagnosed at age 7 and on medication (Proventil, albuterol; Merck, Darmstadt, Germany)] human lung fibroblasts (DHLFs; 00194912) were obtained from Lonza (Walkersville, MD, USA).

Techniques: Real-time Polymerase Chain Reaction, Immunofluorescence, Expressing, Western Blot, Software

Journal: The FASEB Journal

Article Title: Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae –induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation

doi: 10.1096/fj.201601045R

Figure Lengend Snippet: Hypothetical model depicting TRPV4 in the regulation of fibroblast differentiation and airway remodeling.

Article Snippet: Validated primary normal human lung fibroblasts (NHLFs; CC-2512) and diseased [asthma; male, 27 yr old, diagnosed at age 7 and on medication (Proventil, albuterol; Merck, Darmstadt, Germany)] human lung fibroblasts (DHLFs; 00194912) were obtained from Lonza (Walkersville, MD, USA).

Techniques: