Journal: Journal of Inflammation (London, England)

Article Title: Microcirculatory disturbance in acute liver injury is triggered by IFNγ-CD40 axis

doi: 10.1186/s12950-024-00387-w

Figure Lengend Snippet: Consequence of IFNγ for microcirculatory disturbance in acute liver injury. A Serum TNFα and IFNγ levels in the SMD and NSMD groups. The data were expressed as mean and SD. ** P < 0.01 when compared to the NSMD group. B RT-qPCR analysis of TNFα and IFNγ gene expression in Con A (15mg/kg) and TNF/GalN (700 mg/kg GalN +15 µg/kg TNFα) livers. The gene expression levels were normalized to those in the untreated mice as NC. The data were presented as mean and SE ( n = 3–5 in each group). ** P < 0.01 vs. NC groups and †† P < 0.01 vs. TNF/GalN group. C Serum ALT levels after 0, 1, 3, 6, and 12 hours of Con A administration. The data were expressed as mean and SD ( n = 6–13 in each group). D Hematoxylin and eosin staining of the livers after 0, 1, 3, 6, and 12 hours of Con A administration. The dashed area indicates necrotic features and arrowheads indicate hemostasis. Scale bars = 50 μm. E RT-qPCR analysis of the genes associated with the inflammatory cytokines, coagulation, and hypoxia in Con A livers (25mg/kg Con A). The gene expression levels were normalized to those at 0 hour. The data were expressed as mean and SE ( n = 3–8 in each group). ALT, alanine aminotransferase; Con A, concanavalin A; GalN, galactosamine; HO-1, heme oxygenase 1. IFNγ, interferon-gamma; LDH, lactate dehydrogenase; NC, normal control; NSMD, non-sinusoidal microcirculatory disturbance; RT-qPCR, quantitative reverse transcription polymerase chain reactions; SMD, sinusoidal microcirculatory disturbance; TNFα, tumor necrosis factor-alpha; VEGF, vascular endothelial growth factor

Article Snippet: Human liver sinusoidal endothelial cells (LSECs) were purchased from LONZA (Walkersville, MD) and cultured on EGMTM-2 BulletKitTM media at 37°C and 5% CO 2 .

Techniques: Quantitative RT-PCR, Gene Expression, Staining, Coagulation, Control, Reverse Transcription

Journal: Journal of Inflammation (London, England)

Article Title: Microcirculatory disturbance in acute liver injury is triggered by IFNγ-CD40 axis

doi: 10.1186/s12950-024-00387-w

Figure Lengend Snippet: Requirement of IFNγ in microcirculatory disturbance. A Serum ALT levels 0, 1, 3, 6, and 12 hours after Con A administration in the WT , Tnf –/– , and Ifng –/– mice. The results were expressed as mean and SD ( n = 6–13 in each group). ** P < 0.01 when compared to the WT mice. B Hematoxylin and eosin staining and PTAH staining of the liver for the WT , Tnf –/– , and Ifng –/– mice. The dashed area indicates necrotic features and arrowheads indicate fibrin depositions in sinusoids. Scale bars = 50 μm. C RT-qPCR analysis of the genes of the inflammatory cytokines, coagulation, and hypoxia in the liver of WT , Tnf –/– , and Ifng –/– mice treated with Con A. The gene expression levels were normalized to those of the WT at 0 hours. The data were expressed as mean and SE. ** P < 0.01 vs. the WT mice ( n = 3–8 in each group). D RT-qPCR analysis of CD40 and Tissue factor in sorted liver sinusoidal endothelial cells. The gene expression levels were normalized to those of the WT at 0 hours. The data were presented as mean and SE ( n = 3 in each group). * P < 0.05, ** P < 0.01 vs. the WT mice. E The distribution of total nonparenchymal cells with c3 re-clustered is presented on the UMAP plot. The feature plots showed the expression of CD40 and CD40L. The heatmap presented the relative strength of the CD40-CD40L signaling network for each cluster. ALT, alanine aminotransferase; CD40L, CD40 ligand; Con A, concanavalin A; IFNγ, interferon-gamma; ILC, innate lymphoid cell; LDH, lactate dehydrogenase; NK cell, natural killer cell; NKT cell, natural killer T cell; PTAH, phosphotungstic acid-hematoxylin; RT-qPCR, quantitative reverse transcription polymerase chain reactions; Tgd cell, gamma delta T cell; TNFα, tumor necrosis factor-alpha; UMAP, uniform manifold approximation and projection; VEGF, vascular endothelial growth factor; WT, wild type

Article Snippet: Human liver sinusoidal endothelial cells (LSECs) were purchased from LONZA (Walkersville, MD) and cultured on EGMTM-2 BulletKitTM media at 37°C and 5% CO 2 .

Techniques: Staining, Quantitative RT-PCR, Coagulation, Gene Expression, Expressing, Reverse Transcription

Journal: Journal of Inflammation (London, England)

Article Title: Microcirculatory disturbance in acute liver injury is triggered by IFNγ-CD40 axis

doi: 10.1186/s12950-024-00387-w

Figure Lengend Snippet: Induction of tissue factor by IFNγ and CD40 ligand in liver endothelial cells. A The LSEC were treated with 1,000 U/ml IFNγ for 12 hours and then incubated for 6 hours with Jurkat cells fixed with 1% paraformaldehyde. B Cell-surface expression of CD40L protein in untreated Jurkat cells by flow cytometry. The histograms depict isotype control (dashed line), unstained control (solid blue line), and CD40L expression (solid red line). The qualitatively identical results were obtained with at least three further batches of cells. C RT-qPCR analysis of CD40 and tissue factor in LSEC cells after IFNγ (1,000 U/ml) and Jurkat cell treatment. The gene expression levels were normalized to those of the IFNγ and Jurkat cell free group. The data were expressed as mean and SE ( n = 5–10 in each group). ** P < 0.01 vs. the IFNγ and Jurkat cell free group and †† P < 0.01 vs. the IFNγ group. CD40L, CD40 ligand; IFNγ, interferon-gamma; LSEC, liver sinusoidal endothelial cell; RT-qPCR, quantitative reverse transcription polymerase chain reactions

Article Snippet: Human liver sinusoidal endothelial cells (LSECs) were purchased from LONZA (Walkersville, MD) and cultured on EGMTM-2 BulletKitTM media at 37°C and 5% CO 2 .

Techniques: Incubation, Expressing, Flow Cytometry, Control, Quantitative RT-PCR, Gene Expression, Reverse Transcription

Journal: Nature Cardiovascular Research

Article Title: Semaphorin-3A regulates liver sinusoidal endothelial cell porosity and promotes hepatic steatosis

doi: 10.1038/s44161-024-00487-z

Figure Lengend Snippet: a , Sema3a-g mRNA expression (log 10 fold change) in LSECs compared to total liver of 14-week-old male C57BL/6 wild-type (wt) mice ( n = 8, n = 7 for Sema3e ). Lines indicate the same mouse. b , Sema3a-g mRNA expression (log 10 fold change, log(0) values are not displayed) in liver tissue of 12-week-old db / db mice, compared to db / + controls (RT–qPCR; n = 8 each). A multiple two-tailed paired ( a ) or unpaired ( b ) Student’s t -test was used to discover significant effects . Discoveries are indicated by q values in a , b . c , Sema3a mRNA expression in liver tissue from standard chow-fed ( n = 4, RT–qPCR) versus HFD-fed ( n = 10) littermates. d , Sema3a mRNA expression of LSECs from 12-week-old male db /+ control versus db/db mice ( n = 7 each). e , Sema3a mRNA expression of LSECs from 12-week-old male wt control versus ob/ob mice ( n = 8 each). f , Relative Sema3a mRNA expression in hepatocytes and LSECs isolated from three and two human donors, respectively (RT–qPCR). g , Graphical overview of SEMA3A and its known receptors. h , Representative immunofluorescent staining for (I and II) neuropilin-1 (red, NRP1), (I and III) LYVE1 (green) and (III) goat IgG isotope control (red) of liver sections of C57BL/6 wt mice ( n = 2 mice). Scale bars, 20 µm. i , Agarose gel with PCR products (RT–PCR) showing the expression of several SEMA3A receptors in primary mouse ( n = 2 LSEC isolation) and human LSECs (male LSEC donor QC-12B15F11). Brightness and contrast have been adjusted to enhance visibility in h , i . j , k , Nrp1 ( j) and Nrp2 ( k ) mRNA expression in LSECs compared to total liver from 14-week-old male C57BL/6 wt mice ( n = 8 each). l , m , Nrp1 and Nrp2 mRNA expression in LSECs from db/db ( l ; n = 7) and ob/ob mice ( m ; n = 8) in comparison to controls. A two-tailed unequal variances t -test was used ( c – e , l , m ) and two-tailed paired t -test ( j , k ). Data are presented as mean ± s.e.m. CD146 + LSECs were isolated by MACS or FACS after MACS ( d , e , l , m ) to get an even higher purity of cells.

Article Snippet: Human LSECs from different donors were purchased from PELOBiotech (PB-CH-153-5511).

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, Control, Isolation, Staining, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Comparison

Journal: Nature Cardiovascular Research

Article Title: Semaphorin-3A regulates liver sinusoidal endothelial cell porosity and promotes hepatic steatosis

doi: 10.1038/s44161-024-00487-z

Figure Lengend Snippet: a , b , SEMA3A-G mRNA expression in primary human LSECs (male donor QC-12B15F11) treated for 18 h with BSA control ( n = 4 wells) versus 0.75 mM palmitic acid ( n = 5 wells) ( a ) or oleic acid versus BSA controls ( n = 6 wells each) ( b ). c – f , Expression of SEMA3A in primary human LSECs after treatment with BSA control ( n = 4 palmitic acid-, n = 6 oleic acid-treated wells) versus 0.25 mM ( n = 5, n = 6 wells), 0.5 mM ( n = 4, n = 6 wells) and 0.75 mM ( n = 5, n = 6 wells) BSA-bound palmitic acid ( c ) or oleic acid ( e ). Expression of SEMA3A in primary human LSECs treated with BSA controls ( n = 4 wells each), 0.5 mM palmitic acid ( d ) or oleic acid ( f ) for 2 h ( n = 5 wells each), 6 h ( n = 6, n = 5 wells), 18 h ( n = 4, n = 5 wells) and 24 h ( n = 5 wells each). A multiple two-tailed unpaired t -test with a two-stage step-up method was used to discover outstanding effects , as indicated by q values in a , b . A one-way ANOVA with Dunnett’s post hoc test was used to test for statistical significance in c – f . In all graphs individual data points and mean ± s.e.m. are presented.

Article Snippet: Human LSECs from different donors were purchased from PELOBiotech (PB-CH-153-5511).

Techniques: Expressing, Control, Two Tailed Test