Journal: Scientific Reports

Article Title: Role of PAI-1 in hepatic steatosis and dyslipidemia

doi: 10.1038/s41598-020-79948-x

Figure Lengend Snippet: Pharmacological inhibition of PAI-1 with TM5614 regulates Pcsk9 . 20 week-old wild-type C57BL/6 J male mice fed SC were administered a PAI-1 inhibitor, TM5614, given orally (20 mg/kg/day) for 10 days. At the endpoint hepatic mRNA and murine plasma were analyzed. ( A ) Hierarchical clustering of expression changes seen by RNA-seq. Heat map created using Morpheus ( https://software.broadinstitute.org/morpheus/ ). ( B ) Volcano plot representation of all gene expression changes. Blue points represent statistically significant changes and red points represent non-significant changes. ( C ) Gene ontology analysis of statistical differentially expressed genes. Data generated using Metascape ( https://metascape.org/gp/index.html#/main/step1 ). ( D ) Murine plasma PCSK9 levels. ( E ) Total Cholesterol levels in mouse plasma. Values are expressed as mean ± SEM (n = 5), * p < 0.05, ** p < 0.01.

Article Snippet: Human PAI-1 (Molecular Innovations, Cat# HPAIKT-TOT), PCSK9 levels (R&D Systems, Cat# DPC900), and FGF21 (R&D, DF2100) were quantified by ELISA.

Techniques: Inhibition, Expressing, RNA Sequencing Assay, Software, Generated

Journal: Scientific Reports

Article Title: Role of PAI-1 in hepatic steatosis and dyslipidemia

doi: 10.1038/s41598-020-79948-x

Figure Lengend Snippet: Lipid metabolism is differentially regulated in PAI-1 heterozygosity. RNA-SEQ was performed on hepatic mRNA from 20 week-old Pai-1 +/− and littermate WT control C57BL/6 J male mice fed SC (n = 3). ( A ) Hierarchical clustering of expression changes seen by RNA-seq. Heat map created using Morpheus ( https://software.broadinstitute.org/morpheus/ ). ( B ) Volcano plot representation of all gene expression changes. Blue points represent statistically significant changes and red points represent non-significant changes. ( C ) Gene ontology analysis of statistical differentially expressed genes. Data generated using Metascape ( https://metascape.org/gp/index.html#/main/step1 ). ( D ) Murine PCSK9 plasma levels. ( E ) Total cholesterol plasma levels. Values are express as mean ± SEM (n = 7).

Article Snippet: Human PAI-1 (Molecular Innovations, Cat# HPAIKT-TOT), PCSK9 levels (R&D Systems, Cat# DPC900), and FGF21 (R&D, DF2100) were quantified by ELISA.

Techniques: RNA Sequencing Assay, Expressing, Software, Generated

Journal: Scientific Reports

Article Title: Role of PAI-1 in hepatic steatosis and dyslipidemia

doi: 10.1038/s41598-020-79948-x

Figure Lengend Snippet: Long-term TM5614 treatment leads to a reduction in circulatory cholesterol and PCSK9 in mice fed a HFHS diet. WT C57BL/6 J male mice fed HFHS diet with and without TM5614. ( A ) Active PAI-1 levels (ng/mL). ( B ) Cholesterol distribution on plasma lipoprotein fractions resolved by FPLC from pooled samples (n = 2 for HFHS, n = 3 for HFHS + TM5614) (2-way ANOVA p = 0.03). ( C ) Quantification of cholesterol in each lipoprotein fraction calculated using area under the curve from the FPLC profile. ( D ) Hepatic mRNA expression of cholesterol regulatory pathway nodes. ( E ) Plasma PCSK9 levels (ng/mL). ( F ) LDLR protein levels from liver lysates (ng LDLR/mg Liver Protein). Values are express as mean ± SEM (n = 7), * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Human PAI-1 (Molecular Innovations, Cat# HPAIKT-TOT), PCSK9 levels (R&D Systems, Cat# DPC900), and FGF21 (R&D, DF2100) were quantified by ELISA.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Role of PAI-1 in hepatic steatosis and dyslipidemia

doi: 10.1038/s41598-020-79948-x

Figure Lengend Snippet: PAI-1 heterozygosity is associated with lower Pcsk9 and cholesterol profiles in HFHS fed mice. 20 week-old wild-type or Pai-1 +/− C57BL/6 J male mice fed HFHS diet. At the endpoint murine hepatic mRNA and plasma were analyzed. ( A ) Plasma PAI-1 levels. ( B ) Cholesterol content profile of the lipoprotein fractions resolved by FPLC from pooled samples (n = 6 for WT, n = 7 for Pai-1 +/− ). ( C ) Plasma total cholesterol. ( D ) Hepatic PCSK9 mRNA expression normalized to GAPDH. Values are expressed as mean ± SEM (n = 6–7), * p < 0.05.

Article Snippet: Human PAI-1 (Molecular Innovations, Cat# HPAIKT-TOT), PCSK9 levels (R&D Systems, Cat# DPC900), and FGF21 (R&D, DF2100) were quantified by ELISA.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Role of PAI-1 in hepatic steatosis and dyslipidemia

doi: 10.1038/s41598-020-79948-x

Figure Lengend Snippet: Plasma PCSK9 levels correlate with PAI-1 in humans. Characterization of the Amish cohort human plasma [( A ) PAI-1 levels, ( B ) PCSK9 levels, ( C ) correlation between plasma PAI-1 vs. plasma PCSK9 levels, and ( D ) FGF21 levels] (values are express as mean ± SEM (n = 7 affected, 16 carriers , 17 unaffected). ( E ) Relationship between PCSK9 and PAI-1 levels in a separate cohort of HFpEF patients, values are express as mean ± SEM (n = 147). ( F ) PAI-1 levels in human plasma from a cohort of patients with hyperlipidemia before and during treatment with a PCSK9 inhibitor (values are express as mean ± SEM, n = 28).

Article Snippet: Human PAI-1 (Molecular Innovations, Cat# HPAIKT-TOT), PCSK9 levels (R&D Systems, Cat# DPC900), and FGF21 (R&D, DF2100) were quantified by ELISA.

Techniques:

Journal: Scientific Reports

Article Title: Role of PAI-1 in hepatic steatosis and dyslipidemia

doi: 10.1038/s41598-020-79948-x

Figure Lengend Snippet: PAI-1 inhibition triggers a lipid-lowering effect mediated by PCSK9 downregulation. As TM5614 inhibits PAI-1 activity, hepatic PCSK9 is downregulated at the transcriptional level (1). Consequently, plasma levels of PCSK9 decrease (2). Lower plasma PCSK9 levels exert a lower impact on catabolism of the LDL receptor (3), enabling the receptor to clear larger amounts of LDL-C from the circulation (4). The increase in intracellular cholesterol triggers the translocation of cholesterol sensing factors to the nucleus (5) to downregulate the transcription of genes involved in lipid metabolism (6). LRP-1, c-MET or FGFR/KlothoB are potential mediators for the observed effect. Animation created with https://biorender.com/ .

Article Snippet: Human PAI-1 (Molecular Innovations, Cat# HPAIKT-TOT), PCSK9 levels (R&D Systems, Cat# DPC900), and FGF21 (R&D, DF2100) were quantified by ELISA.

Techniques: Inhibition, Activity Assay, Translocation Assay

Journal: Nature Communications

Article Title: SARS-CoV-2 infection of human lung epithelial cells induces TMPRSS-mediated acute fibrin deposition

doi: 10.1038/s41467-023-42140-6

Figure Lengend Snippet: A The average overlap among all BALF samples decreases with identified protein abundance. Most abundant proteins exhibit greater than 80% overlap and are observed in all BALF samples whereas low abundance proteins show less overlap and more unique to each sample. B Pearson correlation coefficient between pairwise samples calculated based on the abundances of 92 common proteins among acute COVID (C), recovered COVID (R), and healthy (H) samples. C Heatmap showing differential protein abundance in mass spectrometry identified proteins among various BALF samples. Plasma proteins, complement components, and coagulation factors are upregulated in acute COVID BALF sample. D ) Heatmap displaying the differential protein abundance for each coagulation factor. n.d. stands for not detected in the BALF sample. Panels ( C ) and ( D ) use the same color scheme. Statistical analyses were performed using two-way ANOVA with p -values significant for column analysis with p = 0.0017 (**), p = 0.0002 (***). E Concentrations of total IgG, fibrinogen, and prothrombin present in healthy, acute COVID and recovered COVID BALF samples as measured by ELISA. Data are presented as mean values +/−SD. Statistical analyses were performed using two-sided, unpaired t-tests, with p = 0.0135 (*), p = 0.0003 (***), p < 0.0001 (****).

Article Snippet: ELISA assays were used to determine the levels of fibrinogen (Abcam, ab108841), total IgG (Abcam, ab195215), and prothrombin (Molecular Innovations, HPTKT-TOT) present in human BALF samples.

Techniques: Mass Spectrometry, Coagulation, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: SARS-CoV-2 infection of human lung epithelial cells induces TMPRSS-mediated acute fibrin deposition

doi: 10.1038/s41467-023-42140-6

Figure Lengend Snippet: A , B Expression of members of TMPRSS genes, ST14 and TMPRSS11D, in various cells as measured by A counts per 10 million total reads (TPM) from RNAseq, and B western blot. The expressions of TMPRSS genes in HEK293 and Vero cells are derived from GSE153744 and GSE83900 datasets from NCBI GEO databases. C pSARS-2 infection resulted in the release of soluble ST14 in the culture supernatant. D Metalloproteinase inhibitors, BB-94 and prinomastat, but not others inhibited infected NHBE cells induced fibrin clotting. The inhibitors were added during the viral infection but not during fibrin clotting assay. E Enzymatic cleavage of the prothrombin peptide, Thrb-324, by recombinant matriptase and HAT. F Recombinant matriptase and HAT cleaved prothrombin for fibrin clot formation similar to factor Xa. G Infection of 25 ng ST14 or 50 ng TMPRSS11D transfected ACE2-293T cells induced fibrin clot formation. Infected (I) or uninfected (UI) ACE2-293T cells without transfection did not form fibrin clots. All data are presented as mean values ± SD and unless stated all statistical analyses are performed using two-sided unpaired student t-test with p -values < 0.05 (*), <0.01 (**), <0.0001 (****).

Article Snippet: ELISA assays were used to determine the levels of fibrinogen (Abcam, ab108841), total IgG (Abcam, ab195215), and prothrombin (Molecular Innovations, HPTKT-TOT) present in human BALF samples.

Techniques: Expressing, Western Blot, Derivative Assay, Infection, Coagulation, Recombinant, Transfection