Journal: bioRxiv

Article Title: IL-11 neutralising therapies target hepatic stellate cell-induced liver inflammation and fibrosis in NASH

doi: 10.1101/470062

Figure Lengend Snippet: a , IL-11 is upregulated in hepatic stellate cells (HSCs) stimulated with TGFβ1 (n=3). b , IL-11 protein is secreted from HSCs stimulated with TGFβ1 (ELISA, n=3). c , Human precision cut liver slices were stimulated with TGFβ1 and IL-11 protein was measured in supernatant (ELISA, n=3). d , IL6R, gp130, and IL11RA expression in HSCs (TPM, transcripts per million). e, f , Representative fluorescence images (scale bars, 200 µm) of HSCs and automated fluorescence quantification for ( e ) ACTA2 +ve cells and ( f ) Collagen I immunostaining following incubation without stimulus (-), with TGFβ1, PDGF, or IL-11. g , Collagen secretion supernatants of HSC stimulated with TGFβ1, PDGF, or IL-11 (Sirius red assay. n≥7). h , Dose-dependent matrigel invasion of HSCs induced by IL-11(n=3). i , Hyper IL-11 induces IL-11 protein secretion from HSCs (ELISA, n=3). a-c, e-g, i , TGFβ1 (5 ng ml -1 ), Hyper IL-11 (0.2 ng ml -1 ), PDGF (20 ng ml -1 ), IL-11 (5 ng ml -1 ); 24 h stimulation; h , 48 h stimulation. j , Schematic of mice receiving daily subcutaneous injection of either saline (control) or rmIl-11 (100 µg kg -1 ). k , Relative liver hydroxyproline content, l , mRNA expression of pro-fibrotic markers, and m , serum ALT levels (k, l , control, n=7; rmIl-11, n=10; m , control n=8; rmIl-11, n=11). a, b, g, h, i, k, m Data are represented as mean ±s.d; c-f, l , Box-and-whisker plots show median (middle line), 25th–75th percentiles (box) and min-max percentiles (whiskers). a-d, i, k-m , Two-tailed Student’s t -test; e-h , two-tailed Dunnett’s test. FC: fold change; I/A: intensity/area.

Article Snippet: Total secreted collagen in the cell culture supernatant was quantified using Sirius red collagen detection kit (9062, Chondrex).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Fluorescence, Immunostaining, Incubation, Injection, Whisker Assay, Two Tailed Test

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Modulation of Cancer-Associated Fibrotic Stroma by An Integrin α v β 3 Targeting Protein for Pancreatic Cancer Treatment

doi: 10.1016/j.jcmgh.2020.08.004

Figure Lengend Snippet: ProAgio depletes CAPaSC by targeting integrin α V β 3 and resorbs tumor collagen. ( A and B ) Mean tumor volume of ( A ) subcutaneous xenograft of Panc1 with ( solid lines ) or without ( dotted lines ) co-implantation of PaSC and mean tumor weight ( B ) of Panc1 subcutaneous xenograft with or without co-implantation of PaSC upon treatment with vehicle ( black lines in panel A , grey dots in panel B ) or 10 mg/kg ProAgio (12 daily doses, red lines in panel A , red dots in panel B ) (n = 6). ( C and E ) Representative images of immunofluorescence staining of α-SMA (red) ( C ) and Masson trichrome (blue) staining for collagen ( E ). ( D and F ) Quantitative analyses of immunofluorescence staining of α-SMA ( D ) and Masson trichrome ( F ) staining in tumor sections of Panc1 xenograft (with or without PaSC co-implantation) mice treated with vehicle or 10 mg/kg ProAgio. ( G and I ) Representative images of immunofluorescence staining of Ki67 ( G , red) and CD31 ( I , red). ( H and J ) Quantitative analyses of immunofluorescence staining of Ki67 ( H ) and quantification of mean vessel density, branch points, and vessel length based on CD31 staining ( J ) in tumor sections of Panc1 xenograft (with or without PaSC co-implantation) mice treated with vehicle or ProAgio. Quantification of Ki67 staining is presented as the percentage of Ki67 + nuclei per view field (upper 3 panels, with PaSC co-implantation; bottom 3 panels, without PaSC co-implantation). ( C , G , and I ) Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). ( A and B ) Error bars represent means ± SEM. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001. n.s., denotes not significant.

Article Snippet: Sirius red, Masson trichrome, and Oil red O staining were performed using kits obtained from IHC WORLD (Woodstock, MD) by following the vendor’s instructions.

Techniques: Immunofluorescence, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Modulation of Cancer-Associated Fibrotic Stroma by An Integrin α v β 3 Targeting Protein for Pancreatic Cancer Treatment

doi: 10.1016/j.jcmgh.2020.08.004

Figure Lengend Snippet: ProAgio depletes CAPaSC by targeting integrin α V β 3 and resorbs tumor collagen. ( A ) Endogenous alleles of KrasG12D and Trp53R172H are conditionally activated in the pancreata of LSL-KrasG12D/+ ; LSL-Trp53R172H/+ ; Pdx-1-Cre (triple mutant) mice. ( B ) Specific polymerase chain reaction analysis of genomic DNA of generated GEM-KPC mice. ( C and D ) Kaplan–Meier survival analysis of GEM-KPC (median survival: vehicle, 20 days; ProAgio, 51 days) ( A ), and OrKPC ( B ) mice treated with vehicle or 10 mg/kg ProAgio (median survival: vehicle, 31 days; ProAgio, 39 days) (10 daily doses + 10 alternate day doses). Treatment was started after 13 weeks of age in GEM-KPC mice in panel C , or 30 days after tumor inoculation in panel D . The numbers in parentheses indicate the group size. ( E ) Representative images of Sirius red (red) and Masson trichrome (blue) staining and IHC staining of α-SMA of tumor sections from GEM-KPC mice treated with vehicle or 10 mg/kg ProAgio at different time points (R x 20, at end of 20 doses; End stage, end of survival experiments). ( F and G ) Quantitative analyses of Sirius red staining (presented as Sirius red + area %) ( F ) and αSMA + staining area % ( G ) in tumor sections of vehicle or ProAgio-treated (10 mg/kg) GEM-KPC mice. A higher staining of Sirius red was observed in vehicle-treated tumors on day 80 (R x 20 doses) than ProAgio-treated tumors on day 110 (end stage) in GEM-KPC mice ( P < .001; n = 7–9). ( H ) Representative images of Sirius red (Sirius red) and IHC staining of α-SMA in tumor sections of vehicle or ProAgio-treated (10 mg/kg) OrKPC mice at the end of 20 doses (R x 20 doses). ( I ) mRNA levels of α-SMA ( left panel ) and FAP ( right panel ) in the tumor tissue lysates of GEM-KPC mice treated with vehicle or ProAgio (n = 4) were analyzed by quantitative reverse-transcription polymerase chain reaction. The mRNA levels in the tumor extracts were normalized to mRNA level of β-actin. ( G ) Error bars represent means ± SEM. ∗ P < .05, ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001. n.s., denotes not significant; WT, wild-type.

Article Snippet: Sirius red, Masson trichrome, and Oil red O staining were performed using kits obtained from IHC WORLD (Woodstock, MD) by following the vendor’s instructions.

Techniques: Mutagenesis, Polymerase Chain Reaction, Generated, Staining, Immunohistochemistry, Reverse Transcription

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: (Pro)renin Receptor Knockdown Attenuates Liver Fibrosis Through Inactivation of ERK/TGF-β1/SMAD3 Pathway

doi: 10.1016/j.jcmgh.2021.05.017

Figure Lengend Snippet: Hepatic PRR knockdown ameliorated TAA-induced liver fibrosis with suppression of ERK/TGF-β1/Smad3 pathway. Vehicle or TAA-administered mice were treated with lentivirus-delivered scramble shRNA or PRR shRNA (n = 6–8 in each group). ( A ) Western blot of hepatic PRR levels ( left ) and expression of hepatic renin mRNA ( right ) in the 4 groups. ( B ) Representative images of liver sections after Sirius red staining for the quantification of positively stained area. Scale bar : 100 μm. ( C ) Hepatic hydroxyproline levels. ( D ) Western blot of α-SMA with quantitative analysis. ( E ) Hepatic transcript expression of Col1a1 , PAI-1 ( Serpine1 ), Timp1 , and Fn1 . The expression levels in mice treated with vehicle and scramble virus were assigned arbitrarily as 1. ( F ) Western blot analysis of phosphorylated and total ERK1/2 (p/t-ERK1/2), TGF-β1, phosphorylated Smad3 (p-Smad3), and Smad3 in mouse livers. ∗ P < .05 vs mice treated with vehicle and scramble virus. # P < .05 vs mice treated with TAA and scramble virus.

Article Snippet: Sirius red staining was performed using the Sirius Red Staining Kit (Polysciences, Inc, Warrington, PA).

Techniques: shRNA, Western Blot, Expressing, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: (Pro)renin Receptor Knockdown Attenuates Liver Fibrosis Through Inactivation of ERK/TGF-β1/SMAD3 Pathway

doi: 10.1016/j.jcmgh.2021.05.017

Figure Lengend Snippet: Hepatic PRR knockdown attenuated MCD diet–induced liver fibrosis. ( A ) PRR mRNA expression in quiescent HSCs from normal chow (NC)-fed mice and in vivo–activated HSCs isolated from mice fed MCD diet for 12 weeks (n = 6 in each group). ∗ P < .05 vs the quiescent HSC group. ( B ) Hepatic dual immunofluorescence of PRR and α-SMA in mice fed with NC or MCD diet. Scale bar : 100 μm. ( C–H ) MCD diet–fed mice were treated with lentiviral scramble shRNA or PRR shRNA (n = 6–8 in each group). Mice fed the NC diet (vehicle) and that received lentiviral-scramble shRNA served as controls (n = 6). ( C ) Western blot analysis of hepatic PRR levels and transcript expression of hepatic renin. ( D ) Sirius red staining of liver sections and quantification of the positively stained areas. Scale bar : 200 μm. ( E ) Hepatic hydroxyproline levels. ( F ) Western blot for α-SMA with quantitative analysis. ( G ) Hepatic transcript expression of profibrotic genes. The expression levels in the mice treated with vehicle and lentiviral scramble shRNA were assigned arbitrarily as 1. ( H ) Western blot analysis of phosphorylated and total ERK1/2, TGF-β1, p-Smad3, and Smad3 in mouse livers. ∗ P < .05 vs mice treated with vehicle and scramble virus. # P < .05 vs mice fed with MCD diet and scramble virus. DAPI, diamidino-2-phenylindole; NV, normal mice treated with vehicle; p-ERK, phosphorylated extracellular signal regulated kinase; p-SMAD3, phosphorylated SMAD3; t-ERK, total extracellular signal regulated kinase.

Article Snippet: Sirius red staining was performed using the Sirius Red Staining Kit (Polysciences, Inc, Warrington, PA).

Techniques: Expressing, In Vivo, Isolation, Immunofluorescence, shRNA, Western Blot, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: (Pro)renin Receptor Knockdown Attenuates Liver Fibrosis Through Inactivation of ERK/TGF-β1/SMAD3 Pathway

doi: 10.1016/j.jcmgh.2021.05.017

Figure Lengend Snippet: Myofibroblast-specific knockdown of PRR mitigated liver fibrosis in TAA-injured mice. Mice administered TAA randomly received injections of lentivirus expressed PRR shRNA (n = 11) or scrambled sequences (n = 9) under α-SMA promoter (labeled as SMA-miR-shPRR or SMA-miR-scramble, respectively). ( A ) Structure of lentiviral SMA-EGFP-miR-PRR shRNA vector. ( B ) Myofibroblast-specific delivery was confirmed in vivo. Hepatic immunofluorescence of GFP, α-SMA, and diamidino-2-phenylindole (DAPI). Scale bar : 100 μm. ( C ) HSCs isolated after lentiviral injection, arrowheads indicate GFP (+) cells and arrows indicate GFP (-) cells. Scale bars : 50 μm. Western blot analysis of isolated HSCs confirmed successful knockdown of PRR. ( D ) Sirius red staining of liver sections along with the quantification of positively stained area. Scale bar : 100 μm. ( E ) Hepatic hydroxyproline levels. ( F ) Western blot analysis of hepatic α-SMA expression. ( G ) Hepatic transcript levels of Col1a1 , PAI-1 ( Serpine1 ), Timp1 , and Fn1 . The expression levels in mice treated with vehicle and SMA-miR-scramble virus were assigned arbitrarily as 1. ( H ) Western blot analysis of PRR, phosphorylated and total ERK1/2, TGF-β1, p-Smad3, and Smad3 in mouse livers. ∗ P < .05 vs mice treated with TAA and SMA-miR-scramble virus.

Article Snippet: Sirius red staining was performed using the Sirius Red Staining Kit (Polysciences, Inc, Warrington, PA).

Techniques: shRNA, Labeling, Plasmid Preparation, In Vivo, Immunofluorescence, Isolation, Injection, Western Blot, Staining, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: (Pro)renin Receptor Knockdown Attenuates Liver Fibrosis Through Inactivation of ERK/TGF-β1/SMAD3 Pathway

doi: 10.1016/j.jcmgh.2021.05.017

Figure Lengend Snippet: Attenuation of liver fibrosis by myofibroblast-specific knockdown of PRR in the MCD diet mouse model. Mice fed with a MCD diet for 6 weeks randomly received injections of lentivirus vector-expressed PRR shRNA (n = 6) or scrambled sequences (n = 7) under α-SMA promoter (labeled as SMA-miR-shPRR or SMA-miR-scramble, respectively). All mice were killed after 12 weeks on a MCD diet. ( A ) Sirius red staining of liver sections with the quantification of the positively stained area. Scale bar : 100 μm. ( B ) Hepatic hydroxyproline contents. ( C ) Western blot analysis of hepatic α-SMA levels. ( D ) Western blot analysis of hepatic the PRR-ERK1/2-TGF-β1-Smad3 pathway. ∗ P < .05 between groups. miR, microRNA; p-ERK, phosphorylated extracellular signal regulated kinase; p-SMAD, phosphorylated SMAD; t-ERK, total extracellular signal regulated kinases.

Article Snippet: Sirius red staining was performed using the Sirius Red Staining Kit (Polysciences, Inc, Warrington, PA).

Techniques: Plasmid Preparation, shRNA, Labeling, Staining, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: (Pro)renin Receptor Knockdown Attenuates Liver Fibrosis Through Inactivation of ERK/TGF-β1/SMAD3 Pathway

doi: 10.1016/j.jcmgh.2021.05.017

Figure Lengend Snippet: Myofibroblast-specific overexpression of PRR aggravated liver fibrosis in TAA-injured mice. ( A ) Structure of lentiviral SMA-PRR complementary DNA (cDNA)-His tag vector. ( B ) HSCs isolated after lentiviral injection. Left : Flow cytometric analysis of the expression of His and PRR in HSCs isolated from TAA mice that received SMA-scramble or SMA-PRR-His virus (n = 6 in each group). Right : Western blot of the isolated HSCs. ( C–G ) Vehicle or TAA-administered mice randomly received injections of lentivirus expressing PRR or scrambled sequences (n = 5−9) under the α-SMA promoter (labeled as SMA-PRR-His or SMA-scramble, respectively). ( C ) Western blot analysis of hepatic PRR levels and hepatic transcript expression of renin. ( D ) Hepatic hydroxyproline levels. ( E ) Sirius red staining of liver sections along with the quantification of positively stained areas. Scale bar : 100 μm. ( F ) Hepatic transcript levels of Col1a1 , PAI-1 ( Serpine1 ), Timp1 , and Fn1 . The expression levels in mice treated with vehicle and SMA-scramble virus were assigned arbitrarily as 1. ( G ) Western blot analysis of α-SMA, phosphorylated and total ERK1/2, TGF-β1, p-Smad3, and Smad3 in mouse livers. ∗ P < .05 vs mice treated with vehicle and SMA-scramble virus. # P < .05 vs mice treated with TAA and SMA-scramble virus. p-ERK, phosphorylated extracellular signal regulated kinases; p-SMAD, phosphorylated SMAD; t-ERK, total extracellular signal regulated kinases.

Article Snippet: Sirius red staining was performed using the Sirius Red Staining Kit (Polysciences, Inc, Warrington, PA).

Techniques: Over Expression, Plasmid Preparation, Isolation, Injection, Expressing, Western Blot, Labeling, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: (Pro)renin Receptor Knockdown Attenuates Liver Fibrosis Through Inactivation of ERK/TGF-β1/SMAD3 Pathway

doi: 10.1016/j.jcmgh.2021.05.017

Figure Lengend Snippet: Lentiviral vectors did not cause liver inflammation or fibrosis in mice liver. Serum ALT levels, Sirius red staining, and H&E staining of liver in the vehicle-administered mice treated with ( A ) normal saline, lentivirus-scramble, or lentivirus-PRR-shRNA (killed 4 weeks after injections); ( B ) normal saline, lentivirus-SMA-miR-scramble, or lentivirus-SMA-miR-shPRR (killed 4 weeks after vector injections); and ( C ) normal saline, lentivirus-SMA-scramble, or lentivirus-SMA-PRR-His (killed 4 weeks after vector injections). n = 5–6 in each group. Scale bars : 100 μm.

Article Snippet: Sirius red staining was performed using the Sirius Red Staining Kit (Polysciences, Inc, Warrington, PA).

Techniques: Staining, shRNA, Plasmid Preparation