begm  (Thermo Fisher)

Journal: Molecular Therapy. Nucleic Acids

Article Title: ACE-2 blockade and TMPRSS2 inhibition mitigate SARS-CoV-2 severity following cigarette smoke exposure in airway epithelial cells in vitro

doi: 10.1016/j.omtn.2025.102580

Figure Lengend Snippet: ACE-2 and TMPRSS2 expression are elevated in cigarette-smoke-exposed ferret lung (A) Dot plot shown from RNA-seq analysis of SARS-CoV-2 receptor and associated gene expression in lung tissue of ferrets exposed to cigarette smoke for 6 months. (B) ACE-2 and (C) TMPRSS2 mRNA expression in lung tissue from smoke-exposed vs. air control ferrets as assessed by real-time qPCR. (D) Representative western blot and (E) quantification of ACE-2 expression in smoke-exposed ferret lung. (F) Quantification of TMPRSS2 expression in smoke-exposed ferret lung. (G) Representative immunofluorescence images of smoke-exposed and air-control ferret lung sections stained with ACE-2 antibody (red) and DAPI (blue). Scale bars, 50 μM. Areas of magnification (magnification 40X) are outlined by the red dashed line. (H) Representative immunofluorescence images of smoke-exposed and air-control ferret lung sections stained with TMPRSS-2 antibody (Green), Muc5AC (red), and DAPI (blue). Scale bars, 50 μM. Areas of magnification (magnification 40X) are outlined by the red dashed line. Real-time qPCR analysis of (I) ACE2 and (J) TMPRSS2 mRNA expression in terminally differentiated ferret tracheal epithelial cells (FTECs) exposed to cigarette smoke extract (CSE) or vehicle control. ∗ p < 0.05, ∗∗ p < 0.005,∗∗∗ p < 0.0005.

Article Snippet: Primary HBE cells were isolated from lung explants at the time of organ transplantation from COPD patients, healthy donors without lung disease, and healthy donors, expanded in submerged culture for one or two passages in bronchial epithelial growth medium (BEGM, Lonza, Walksville, MD), and then seeded on Transwell membranes (Corning, New York, NY) as described previously., , Cells were grown at ALI in PneumaCult-ALI media (Stem Cell Technology) for 3–4 weeks until terminally differentiated.

Techniques: Expressing, RNA Sequencing, Gene Expression, Control, Western Blot, Immunofluorescence, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: ACE-2 blockade and TMPRSS2 inhibition mitigate SARS-CoV-2 severity following cigarette smoke exposure in airway epithelial cells in vitro

doi: 10.1016/j.omtn.2025.102580

Figure Lengend Snippet: ACE-2 and TMPRSS2 expression are elevated in CSE-exposed human airway cells Real-time qPCR measurement of (A) ACE-2 and (B) TMPRSS2 mRNA expression in Calu-3 cells exposed to cigarette smoke extract (CSE) or vehicle control for 48 h Real-time qPCR measurement of (C) ACE-2 and (D) TMPRSS2 mRNA expression in primary human bronchial epithelial (HBE) cells derived from healthy control donors and treated with CSE or vehicle control for 48 h. N = 3 monolayers/condition across 3–4 different donors. Western blot image HBE cells treated with cigarette smoke extract for 24 h (E) and quantitation of western blot for ACE2 (F) and TMPRSS2 (G). Real-time qPCR measurement of (H) ACE-2 and (I) TMPRSS2 mRNA expression in bronchial epithelial cells derived from patients with COPD (COPD HBE) or healthy non-smoker donors. N = 3 monolayers/condition across 3–4 different donors. Western blot analysis of lung tissue from COPD and healthy non-smoker donors (J), with corresponding quantification of TMPRSS2 protein expression (K). (G) Representative western blot for ACE2 (L), with quantification of ACE2 protein expression comparing non-smokers vs. COPD (M) and non-smokers vs. smokers (N). N = 3–4 monolayers/conditions derived from 3 to 4 different donors. ∗ p < 0.05, ∗∗ p < 0.005.

Article Snippet: Primary HBE cells were isolated from lung explants at the time of organ transplantation from COPD patients, healthy donors without lung disease, and healthy donors, expanded in submerged culture for one or two passages in bronchial epithelial growth medium (BEGM, Lonza, Walksville, MD), and then seeded on Transwell membranes (Corning, New York, NY) as described previously., , Cells were grown at ALI in PneumaCult-ALI media (Stem Cell Technology) for 3–4 weeks until terminally differentiated.

Techniques: Expressing, Control, Derivative Assay, Western Blot, Quantitation Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: ACE-2 blockade and TMPRSS2 inhibition mitigate SARS-CoV-2 severity following cigarette smoke exposure in airway epithelial cells in vitro

doi: 10.1016/j.omtn.2025.102580

Figure Lengend Snippet: SARS-CoV-2 infection is increased in CSE-exposed FTECs and Calu-3 cells (A) Schematic outline for experiments evaluating the relationship between cigarette smoking and SARS-CoV-2 infection in ferret tracheal epithelial cells (FTECs) and Calu-3 cells. Cells were treated with cigarette smoke extract (CSE) or vehicle control for 48 h prior to inoculation with SARS-CoV-2 (MOI-3). After 72 h of SARS-CoV-2 infection with concomitant CSE exposure, cells were harvested for analysis. (B) Quantification of viral load by real-time qPCR of SARS-CoV-2 mRNA at 3 days post-infection in FTECs exposed to CSE or vehicle control, with (C) visualization and (D) quantification of SARS-CoV-2-infected cells by foci forming assay in Vero-E6 cells. N = 3–5 well per conditions. (E) Quantification of viral load by real-time qPCR at 3 days after SARS-CoV-2 infection in Calu-3 cells exposed to CSE or vehicle control, with (F) visualization and (G) quantification of SARS-CoV-2-infected cells by foci forming assay in Vero-E6 cells. N = 3–5 well per conditions. ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Primary HBE cells were isolated from lung explants at the time of organ transplantation from COPD patients, healthy donors without lung disease, and healthy donors, expanded in submerged culture for one or two passages in bronchial epithelial growth medium (BEGM, Lonza, Walksville, MD), and then seeded on Transwell membranes (Corning, New York, NY) as described previously., , Cells were grown at ALI in PneumaCult-ALI media (Stem Cell Technology) for 3–4 weeks until terminally differentiated.

Techniques: Infection, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: ACE-2 blockade and TMPRSS2 inhibition mitigate SARS-CoV-2 severity following cigarette smoke exposure in airway epithelial cells in vitro

doi: 10.1016/j.omtn.2025.102580

Figure Lengend Snippet: SARS-CoV-2 genomic replication is increased in CSE-exposed normal HBE cells and HBE cells derived from COPD patients (A) Transepithelial electrical resistance (TEER) measurements in vehicle-treated, cigarette smoke extract (CSE)-treated, and COPD human bronchial epithelial (HBE) monolayer cultures infected with SARS-CoV-2 or mock infection. The data were obtained by averaging three independent Transwell reads, each of which represented the mean of three separate readings. ( N = 3–5) HBE. Each line corresponds to a distinct donor (3–5 donors in total), and each donor is represented by 3–5 replicates. (B) Quantification of viral load by real-time qPCR of SARS-CoV-2 mRNA at 72 h post-infection with SARS-CoV-2 in CSE- or vehicle-exposed HBE cells. N = 3 different donors, with each line representing a separate donor. (C) Quantification of viral load by real-time qPCR of SARS-CoV-2 mRNA at 72 h post-infection with SARS-CoV-2 in healthy non-smoker or COPD HBE cells. N = monolayers/condition derived from 3 to 5 different donors. (D and E) Representative images using RNAscope in situ hybridization for comparable detection of genomic RNA with only the SARS-CoV-2-specific S probe. Images show CSE- or vehicle-exposed HBE cells, and healthy non-smoker or COPD HBE cells, at 72 h post-inoculation with (D) mock infection or (E) SARS-CoV-2. SARS-CoV-2 (red), TMPRSS2 (green), ACE-2 (white), nuclei (blue). ∗∗ p < 0.01.

Article Snippet: Primary HBE cells were isolated from lung explants at the time of organ transplantation from COPD patients, healthy donors without lung disease, and healthy donors, expanded in submerged culture for one or two passages in bronchial epithelial growth medium (BEGM, Lonza, Walksville, MD), and then seeded on Transwell membranes (Corning, New York, NY) as described previously., , Cells were grown at ALI in PneumaCult-ALI media (Stem Cell Technology) for 3–4 weeks until terminally differentiated.

Techniques: Derivative Assay, Infection, RNAscope, In Situ Hybridization

Journal: Molecular Therapy. Nucleic Acids

Article Title: ACE-2 blockade and TMPRSS2 inhibition mitigate SARS-CoV-2 severity following cigarette smoke exposure in airway epithelial cells in vitro

doi: 10.1016/j.omtn.2025.102580

Figure Lengend Snippet: Simultaneous ACE2 blockade and TMPRSS2 inhibition reduces SARS-CoV-2 infection after CSE exposure in FTECs (A) Scheme depicting the approach to ACE2 antisense oligonucleotide (ASO) treatment, cigarette smoke extract (CSE) exposure, and SARS-CoV-2 infection in ferret tracheal epithelial cells (FTECs). Seven days of treatment with ACE2 ASO (20 μM) or control ASO was followed by a 48-h incubation period in CSE or vehicle control prior to inoculation with SARS-CoV-2 or no virus for 72 h of infection. Assessment of mRNA expression of (B) ACE-2 and (C) TMPRSS2 following the scheme depicted in (A). (D) Scheme depicting the approach to ACE2 ASO and camostat mesylate treatment, CSE exposure, and SARS-CoV-2 infection in FTECs. Camostat mesylate (100 mM) was added for 2 h on day 9, prior to inoculation with SARS-CoV-2. Assessment of (E) viral load and mRNA expression of (F) ACE-2 and (G) SARS-Cov2 infection for a shorter duration following the scheme depicted in (D). Viral load following shorter duration of infection (H) and (I). Real-time qPCR was used for mRNA quantification in all studies. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Primary HBE cells were isolated from lung explants at the time of organ transplantation from COPD patients, healthy donors without lung disease, and healthy donors, expanded in submerged culture for one or two passages in bronchial epithelial growth medium (BEGM, Lonza, Walksville, MD), and then seeded on Transwell membranes (Corning, New York, NY) as described previously., , Cells were grown at ALI in PneumaCult-ALI media (Stem Cell Technology) for 3–4 weeks until terminally differentiated.

Techniques: Inhibition, Infection, Control, Incubation, Virus, Expressing