Journal: Alcoholism, Clinical and Experimental Research

Article Title: TGF β 1 Mediates Alcohol-Induced Nrf2 Suppression in Lung Fibroblasts

doi: 10.1111/acer.12563

Figure Lengend Snippet: Chronic alcohol ingestion decreased nuclear factor (erythroid-derived 2)-like 2 (Nrf2) gene and protein expression in the primary lung fibroblasts. Control-fed and alcohol-fed mice were sacrificed after 8 weeks of feeding and lung fibroblasts were isolated. The lung fibroblasts were cultured and expanded and cells were harvested at passage 4 and analyzed for ( A ) Nrf2 mRNA expression by quantitative polymerase chain reaction and ( B ) Nrf2 protein expression by Western immunoblotting analyses (representative gels shown above summary data). N = 6 per group. * p < 0.05 decreased compared to lungs from control-fed mice.

Article Snippet: In parallel, primary lung fibroblasts from control-fed mice and NIH 3T3 mouse lung fibroblasts (ATCC, Manassas, VA) were cultured in DMEM with 4.5 g/l glucose supplemented with 20% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin.

Techniques: Derivative Assay, Expressing, Control, Isolation, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot

Journal: Alcoholism, Clinical and Experimental Research

Article Title: TGF β 1 Mediates Alcohol-Induced Nrf2 Suppression in Lung Fibroblasts

doi: 10.1111/acer.12563

Figure Lengend Snippet: Alcohol exposure in vitro decreased nuclear factor (erythroid-derived 2)-like 2 (Nrf2) gene and protein expression in primary lung fibroblasts and immortalized lung fibroblasts (NIH 3T3 lung fibroblasts). Primary lung fibroblasts (passage 5 to 8) and NIH 3T3 lung fibroblasts were cultured ± alcohol (60 mM) and mRNA and protein were isolated at 24 and 72 hours respectively. ( A ) Primary lung fibroblasts with in vitro alcohol treatment showed a decrease in Nrf2 gene (as reflected by Nrf2 mRNA levels, normalized to 18s) and Nrf2 protein expression by Western immunoblotting analyses (normalized to β -actin; representative gels shown above summary data). N = 6 per group. ( B ) In parallel, NIH 3T3 lung fibroblasts treatment with alcohol in vitro showed a decrease in Nrf2 gene and Nrf2 protein expression by Western immunoblotting analyses (normalized to β -actin). * p < 0.05 decreased compared to unexposed cells (None).

Article Snippet: In parallel, primary lung fibroblasts from control-fed mice and NIH 3T3 mouse lung fibroblasts (ATCC, Manassas, VA) were cultured in DMEM with 4.5 g/l glucose supplemented with 20% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin.

Techniques: In Vitro, Derivative Assay, Expressing, Cell Culture, Isolation, Western Blot

Journal: Alcoholism, Clinical and Experimental Research

Article Title: TGF β 1 Mediates Alcohol-Induced Nrf2 Suppression in Lung Fibroblasts

doi: 10.1111/acer.12563

Figure Lengend Snippet: Alcohol exposure increased the relative translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein into the nuclear compartment. NIH 3T3 lung fibroblasts were cultured ± alcohol (60 mM) for 72 hours. Total protein were separated into nuclear and cytosolic fractions and analyzed for Nrf2 protein expression by Western immunoblotting analyses (normalized to β -actin in the cytosolic fraction and to histone in the nuclear fraction; representative gels shown above summary data). N = 8 per group. * p < 0.05 decreased compared to cytosolic fractions from unexposed cells (None). ** p < 0.05 increased compared to nuclear fractions from unexposed cells (None).

Article Snippet: In parallel, primary lung fibroblasts from control-fed mice and NIH 3T3 mouse lung fibroblasts (ATCC, Manassas, VA) were cultured in DMEM with 4.5 g/l glucose supplemented with 20% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin.

Techniques: Translocation Assay, Derivative Assay, Cell Culture, Expressing, Western Blot

Journal: Alcoholism, Clinical and Experimental Research

Article Title: TGF β 1 Mediates Alcohol-Induced Nrf2 Suppression in Lung Fibroblasts

doi: 10.1111/acer.12563

Figure Lengend Snippet: Alcohol exposure suppressed Nrf2-ARE activity and downstream induction of antioxidant genes, but this suppression was reversed by treatment with the Nrf2 activator, sulforaphane (SFP). ( A ) NIH 3T3 lung fibroblasts were transfected with ARE-luciferase or renilla-luciferase for 24 hours and then cultured ± alcohol (60 mM) ± SFP (5 μ M) for 20 hours at which time relative Nrf2-ARE activity (Nrf2 luciferase activity normalized to renilla luciferase activity) was quantified. ( B ) NIH 3T3 lung fibroblasts were cultured ± alcohol (60 mM) ± SFP (5 μ M) for 24 hours and then analyzed for GSTT2 and GCLC gene expression by quantitative polymerase chain reaction of mRNA. N = 8 per group. * p < 0.05 increased compared to unexposed cells (None). ** p < 0.05 decreased compared to unexposed cells (None). ARE, antioxidant response element; GCLC, glutamate-cysteine ligase catalytic subunit; GSTT2, glutathione s-transferase theta 2; Nrf2, nuclear factor (erythroid-derived 2)-like 2.

Article Snippet: In parallel, primary lung fibroblasts from control-fed mice and NIH 3T3 mouse lung fibroblasts (ATCC, Manassas, VA) were cultured in DMEM with 4.5 g/l glucose supplemented with 20% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin.

Techniques: Activity Assay, Transfection, Luciferase, Cell Culture, Gene Expression, Real-time Polymerase Chain Reaction, Derivative Assay

Journal: Alcoholism, Clinical and Experimental Research

Article Title: TGF β 1 Mediates Alcohol-Induced Nrf2 Suppression in Lung Fibroblasts

doi: 10.1111/acer.12563

Figure Lengend Snippet: Alcohol exposure decreased Kelch-like ECH-associated protein 1 (Keap1) protein expression in the NIH 3T3 lung fibroblasts. NIH 3T3 lung fibroblasts were cultured ± alcohol (60 mM) for 24 or 72 hours and cells were harvested for mRNA and protein analysis respectively. ( A ) Keap1 mRNA expression by quantitative polymerase chain reaction and ( B ) Keap1 protein expression by Western immunoblot analyses (representative gels shown above summary data). N = 6 per group. * p < 0.05 decreased compared to lungs from control-fed mice.

Article Snippet: In parallel, primary lung fibroblasts from control-fed mice and NIH 3T3 mouse lung fibroblasts (ATCC, Manassas, VA) were cultured in DMEM with 4.5 g/l glucose supplemented with 20% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin.

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Alcoholism, Clinical and Experimental Research

Article Title: TGF β 1 Mediates Alcohol-Induced Nrf2 Suppression in Lung Fibroblasts

doi: 10.1111/acer.12563

Figure Lengend Snippet: (next page) Alcohol exposure induced transforming growth factor beta-1 (TGF β 1) expression in lung fibroblasts, which was mitigated by sulforaphane (SFP) treatment. NIH 3T3 lung fibroblasts were cultured ± alcohol (60 mM) ± SFP (5 μ M) and then analyzed for ( A ) TGF β 1 gene expression (TGF β 1 mRNA levels) and ( B ) protein expression (by flow cytometry) at 6 and 24 hours respectively. The inset above panel B shows representative flow cytometry histograms (histogram for SFP-exposed lung fibroblasts data is not shown) and representative flow cytometry density plot. ( C ) In parallel, cells were treated for 48 hours and harvested for TGF β 1 protein analysis by Western immunoblotting. N = 5 to 6 per group. * p < 0.05 increased compared to unexposed cells (None). ** p < 0.05 decreased compared to alcohol-exposed cells.

Article Snippet: In parallel, primary lung fibroblasts from control-fed mice and NIH 3T3 mouse lung fibroblasts (ATCC, Manassas, VA) were cultured in DMEM with 4.5 g/l glucose supplemented with 20% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin.

Techniques: Expressing, Cell Culture, Gene Expression, Flow Cytometry, Western Blot

Journal: Alcoholism, Clinical and Experimental Research

Article Title: TGF β 1 Mediates Alcohol-Induced Nrf2 Suppression in Lung Fibroblasts

doi: 10.1111/acer.12563

Figure Lengend Snippet: Silencing RNA (siRNA) of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression amplified alcohol-induced transforming growth factor beta-1 (TGF β 1) expression in lung fibroblasts. ( A ) NIH 3T3 lung fibroblasts were transfected with an Nrf2 siRNA vector or a control scrambled vector, and then relative Nrf2 gene expression (Nrf2 mRNA levels) was assessed (* p < 0.05 decreased compared to control). N = 3. ( B ) Transfected fibroblasts were then cultured ± alcohol for 24 hours at which time the percentages of fibroblasts in each group that were positive for TGF β 1 protein expression were quantified by flow cytometry. The inset above panel B shows representative flow cytometry histograms (histogram for unexposed, scrambled vector-transfected lung fibroblasts data is not shown) and representative flow cytometry density plot. ( C ) In parallel, cells were treated for 48 hours and harvested for TGF β 1 protein analysis by Western immunoblotting. ( D ) Culture media from the above conditions were collected and were analyzed for secreted (activated) TGF β 1 by ELISA. Data are expressed as TGF β 1 expression relative to the unexposed, scrambled vector-transfected lung fibroblasts. N = 5 to 6 per group. * p < 0.05 increased compared to cells transfected with the scrambled vector (None) and ** p < 0.05 increased compared to alcohol-exposed cells transfected with the scrambled vector.

Article Snippet: In parallel, primary lung fibroblasts from control-fed mice and NIH 3T3 mouse lung fibroblasts (ATCC, Manassas, VA) were cultured in DMEM with 4.5 g/l glucose supplemented with 20% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin.

Techniques: Derivative Assay, Expressing, Amplification, Transfection, Plasmid Preparation, Control, Gene Expression, Cell Culture, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Alcoholism, Clinical and Experimental Research

Article Title: TGF β 1 Mediates Alcohol-Induced Nrf2 Suppression in Lung Fibroblasts

doi: 10.1111/acer.12563

Figure Lengend Snippet: TGF β 1 treatment independently suppressed Nrf2-ARE activity, and alcohol-suppressed Nrf2-ARE activity was prevented by inhibitors of TGF β 1 signaling. ( A ) NIH 3T3 lung fibroblasts were transfected with ARE-luciferase or renilla-luciferase for 24 hours and then cultured for 20 hours alone or with one of the following: TGF β 1 (5 ng/ml), alcohol (60 mM) and either a TGF β 1 neutralizing antibody (2 μ g/ml) or an isotype control antibody (2 μ g/ml), or alcohol (60 mM) and 1 of 2 ALK5 inhibitors (ALK5 inh 1 is SB431542 and ALK5 inh 2 is A8301; each at 8 μ M) or dimethyl sulfoxide (1:500 dilution) as an ALK5 inhibitor vehicle control. The fibroblasts were then analyzed for relative Nrf2-ARE activity. ( B ) NIH 3T3 lung fibroblasts were cultured for 24 hours either alone, with TGF β 1 (5 ng/ml), with alcohol (60 mM), or with alcohol (60 mM) and an ALK5 inhibitor (ALK5 inh 1 is SB431542). The relative gene expression (mRNA levels) of the ARE-dependent antioxidants GSTT2 and GCLC were then determined. N = 8 per group. * p < 0.05 decreased compared to unexposed, untreated cells (None). ** p < 0.05 increased compared to alcohol-treated cells but still decreased compared to unexposed, untreated cells. ALK5, activin receptor-like kinase 5; ARE, antioxidant response element; GCLC, glutamate-cysteine ligase catalytic subunit; GSTT2, glutathione s-transferase theta 2; Nrf2, nuclear factor (erythroid-derived 2)-like 2; TGF β 1, transforming growth factor beta-1.

Article Snippet: In parallel, primary lung fibroblasts from control-fed mice and NIH 3T3 mouse lung fibroblasts (ATCC, Manassas, VA) were cultured in DMEM with 4.5 g/l glucose supplemented with 20% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin.

Techniques: Activity Assay, Transfection, Luciferase, Cell Culture, Control, Gene Expression, Derivative Assay

Journal: Alcoholism, Clinical and Experimental Research

Article Title: TGF β 1 Mediates Alcohol-Induced Nrf2 Suppression in Lung Fibroblasts

doi: 10.1111/acer.12563

Figure Lengend Snippet: Proposed pathophysiological sequence by which alcohol alters transforming growth factor beta-1 (TGF β 1) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) homeostasis in the lung. The data shown in this manuscript as well as those from previous studies suggests that alcohol induces the expression of TGF β 1 in the lung, including in lung fibroblasts. TGF β 1 in turn suppresses Nrf2 expression and its ability to activate the antioxidant response element (ARE), which then leads to inadequate production of antioxidants including glutathione and subsequent oxidative stress. The decrease in Nrf2-ARE activity and the oxidative stress creates a vicious cycle that feeds back to further increase TGF β 1 expression.

Article Snippet: In parallel, primary lung fibroblasts from control-fed mice and NIH 3T3 mouse lung fibroblasts (ATCC, Manassas, VA) were cultured in DMEM with 4.5 g/l glucose supplemented with 20% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin.

Techniques: Sequencing, Derivative Assay, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Single Cell Transcriptomics of Fibrotic Lungs Unveils Aging-associated Alterations in Endothelial and Epithelial Cell Regeneration

doi: 10.1101/2023.01.17.523179

Figure Lengend Snippet: (A) Volcano plots showing the distribution of differentially expressed genes in gCap EC clusters 1 and 2 compared to other gCap EC clusters. (B) Violin plots showing the expression genes enriched in cluster 1 and 2. (C) Heatmap showing average expression of glycolysis genes in quiescent (Q) versus activated (A) gCap EC clusters. (D, E) Ingenuity pathway analysis shows canonical pathways and upstream regulators enriched in clusters 1 and 2 relative to other gCap EC clusters. P values were generated in IPA using Fisher’s test (log2 FC ≤-0.1 or ≥0.1, p value ≤0.05). P value and activation z-score were used for plotting canonical pathways and activated upstream regulators respectively. (F, G) qPCR analyses of human lung microvascular ECs (HLMECs) treated with the LATS1/2 inhibitor TRULI and the siRNAs targeting YAP and TAZ for 48 hours. YAP activation in these cells partially recapitulates the gene expression signature observed in activated gCap ECs. Values are summarized as mean and SD and analyzed using a two-tailed Student’s t -test. (N = 3). (H) Immunofluorescence images showing genetically labeled gCap ECs (membrane-GFP) in uninjured lungs of Aplnr-CreER(T)-mTmG reporter mouse. Lungs from these mice were harvested ten days following the last dose of tamoxifen (five total doses). An antibody against the pan-endothelial cell marker PECAM-1 was used to visualize all lung ECs. PECAM-1 positive ECs from large vessels showed no GFP expression. (I) Schematic representation of the approach used to detect TrkB-expressing gCap ECs following bleomycin injury. gCap ECs (mGFP) were lineage labeled in Aplnr-CreER(T)-mTmG mice 15 days prior to bleomycin administration (Day 0). Sham and bleomycin-injured lungs were harvested 28 days post bleomycin delivery and subjected to immunofluorescence analysis. An antibody against TrkB was used to detect injured gCap ECs (Red). gCap ECs co-expressing GFP and TrkB (yellow) only emerged in injured lungs (n=2). (J). Immunofluorescence images showing TrkB-expressing cells in the lungs of young and aged mice 37 days post bleomycin challenge (n=2). (K) Schematic showing putative mechanisms implicated in of gCap EC remodeling in response to lung injury and during lung fibrosis resolution.

Article Snippet: Human lung microvascular endothelial cells (HLMECs) were purchased from Cell Applications (San Diego, CA, USA) or ANGIO-PROTEOMIE (Worcester, MA, USA) and maintained in endothelial cell growth basal medium supplemented with microvascular endothelial cell growth kit.

Techniques: Expressing, Generated, Activation Assay, Gene Expression, Two Tailed Test, Immunofluorescence, Labeling, Membrane, Marker