Janus kinase 2 inhibition by pacritinib as potential therapeutic target for liver fibrosis

anus kinase 2 (JAK2) signaling is increased in human and experimental liver fibrosis with portal hypertension. JAK2 inhibitors, such as pacritinib, are already in advanced clinical development for other indications and might also be effective in liver fibrosis. Here, we investigated the antifibrotic role of the JAK2 inhibitor pacritinib on activated hepatic stellate cells (HSCs) in vitro and in two animal models of liver fibrosis in vivo.Jonel Trebicka is supported by the German Research Foundation project ID 403224013–SFB 1382 (A09); by the German Federal Ministry of Education and Research (BMBF) for the DEEP‐HCC project; by the Hessian Ministry of Higher Education, Research, and the Arts (HMWK) for the ENABLE cluster project; and by Eurostars (Grant ID 12350). The MICROB‐PREDICT (project ID 825694), DECISION (project ID 847949), GALAXY (project ID 668031), LIVERHOPE (project ID 731875), and IHMCSA (project ID 964590) projects have received funding from the European Union's Horizon 2020 research and innovation program. The manuscript reflects only the authors' views, and the European Commission is not responsible for any use that may be made of the information it contains. The funders had no influence on study design, data collection and analysis, decision to publish, or preparation of the manuscript

Serum free sulfhydryl status is associated with patient and graft survival in renal transplant recipients

Oxidative stress contributes significantly to graft failure, morbidity and mortality in renal transplant recipients (RTR). In cells, free sulfhydryl groups (reduced thiols, R-SH) are the transducers of redox-regulated events; their oxidation status is modulated by interaction with reactive oxygen and nitrogen oxide species and thought to be in equilibrium with the circulating pool. We hypothesized that high levels of serum free thiols are a reflection of a favorable redox status and therefore positively associated with cardiovascular risk parameters, patient and graft survival in RTR. To test this, reactive free thiol groups (R-SH; corrected for total protein) were quantified in serum of 695 RTR (57% male, 53±13yr, functioning graft ?1yr) using Ellman's Reagent, and R-SH determinants were evaluated with multivariable linear regression models. Associations between R-SH and mortality or graft failure were assessed using multivariable Cox regression analyses. In multivariable models, male gender, estimated glomerular filtration rate and serum thiosulfate positively associated with R-SH while BMI, HbA1c, corrected calcium and NT-pro-BNP inversely associated with R-SH (model R(2)=0.26). During follow-up (3.1 [2.7-3.9] yrs), 79 (11%) patients died and 45 (7%) patients developed graft failure. R-SH correlated inversely with all-cause mortality (HR 0.58 [95% CI 0.45-0.75] per SD increase) and graft failure (HR 0.42 [0.30-0.59]; both P<0.001), independent of parameters with which R-SH significantly associated in the multivariable regression analyses, except for NT-pro-BNP. Serum R-SH are associated with a beneficial cardiovascular risk profile and better patient and graft survival in RTR, suggesting potential usefulness as low-cost, high-throughput screening tool for whole-body redox status in translational studies. Whether R-SH modification improves long-term outcome of RTR warrants further exploration

Y-27632 and Y-33075 treatments reduce contraction in human TWNT-4 and primary murine HSCs.

(A) Schematic depiction of the RhoA-ROCK pathway including mechanism of action of the ROCK inhibitors Y-27632 and Y-33075. (B) Chemical structure of ROCK inhibitors Y-27632 and Y33075. (C) Effect of Y-27632 and Y-33075 on collagen gel contraction of activated human TWNT-4 cells (n = 3). (D) Representative images of the contraction assay in TWNT-4 cells after incubation with the ROCK inhibitors for 24 hours. (E) Effect of Y-27632 and Y-33075 on collagen gel contraction of culture-activated primary murine FVB/NJ cells (n = 3). (F) Representative images of gel contraction in FVB/NJ cells 24 hours after treatment with the ROCK inhibitors. (G) Protein expression levels of phospho-MLC and MLC in TWNT-4 after 24 hour incubation with the ROCK inhibitors. (H) Ratio of phospho-MLC to MLC in TWNT-4 cell lysates after treatment with ROCK inhibitors for 24 hours (n = 2). Results are expressed as the mean ± standard error of the mean (SEM); *p<0.05, **p<0.01 and ***p<0.001.</p

Antibodies.

(DOCX)</p

Y-27632 and Y-33075 treatments increase migration in human TWNT-4 and primary murine HSCs.

(A) Effect of ROCK inhibitors Y-27632 and Y-33075 on wound closure in activated human TWNT-4 cells measured at 4, 8 and 24 hours (n = 3). (B) Representative images of the wound healing assay in TWNT-4 cells at 4, 8 and 24 hours after treatment with the ROCK inhibitors. The scale bars apply for all images and the length represents 200μm. (C) Effect of ROCK inhibitors Y-27632 and Y-33075 on wound closure in primary culture-activated FVB/NJ cells measured at 4, 8 and 24 hours (n = 3). (D) Representative images of the wound healing assay taken 4, 8 and 24 hours after treatment of FVB/NJ cells with ROCK inhibitors (scale bars, 200μm). Results are expressed as the mean ± standard error of the mean (SEM); *p<0.05, **p<0.01 and ***p<0.001.</p

S1 File -

(PDF)</p

Y-27632 and Y-33075 treatments reduce fibrosis in human TWNT-4 and primary mouse HSCs.

(A) Western blots of Col1a1, p-Moesin, Moesin and GAPDH using TWNT-4 cell lysates after 24 hour incubation with the ROCK inhibitors. (B) Quantification of protein expression of Col1a1 after treatment of TWNT-4 cells with ROCK inhibitors for 24 hours (n = 3). (C) Quantification of protein expression of Moesin after treatment of TWNT-4 cells with ROCK inhibitors for 24 hours (n = 3). (D) Quantification of protein expression of pMoesin after treatment of TWNT-4 cells with ROCK inhibitors for 24 hours (n = 3). (E) Western Blots of Col1a1, p-Moesin, Moesin and GAPDH using cell lysates of primary FVB/NJ cells after incubation with ROCK inhibitors for 24 hours. (F). Quantification of protein expression of Col1a1 after treatment of FVB/NJ cells with ROCK inhibitors for 24 hours (n = 3). (G) Quantification of protein expression of Moesin after treatment of FVB/NJ cells with ROCK inhibitors for 24 hours (n = 3). (H) Quantification of protein expression of p-Moesin after treatment of FVB/NJ cells with ROCK inhibitors for 24 hours (n = 3). (I) TWNT-4 mRNA expression levels of the pro-fibrotic marker Col1a1 and Tgfβ (corrected to 18s as a housekeeping gene) after 24h incubation with ROCK inhibitors Y-27632 and Y-33075 (n = 3). (J) FVB/NJ mRNA expression levels of the pro-fibrotic marker Col1a1 and Tgfβ (corrected to 18s as a housekeeping gene) after 24h incubation with ROCK inhibitors Y-27632 and Y-33075 (n = 3). Results are expressed as the mean ± standard error of the mean (SEM); *p<0.05, **p<0.01 and ***p<0.001.</p

Y-27632 and Y-33075 treatments decrease proliferation in human TWNT-4 and primary mouse HSCs.

(A) Colorimetric BrdU evaluation of proliferation levels in TWNT-4 cells after 24h treatment with ROCK inhibitors (n = 3). (B) mRNA expression of Pcna as a marker for proliferation in TWNT-4 cells (corrected against 18s as a housekeeping gene) after 24 hour treatment with ROCK inhibitors. (C) Effect of 24 hour ROCK inhibitor treatments on proliferation in murine FVB/NJ cells assessed by colorimetric BrdU-Assay (n = 3). (D) mRNA expression of Pcna as a marker for proliferation in FVB/NJ cell lysates (corrected against 18s as a housekeeping gene) after 24 hour treatment with ROCK inhibitors (n = 3). (E) Protein expression of α-SMA as a marker for the activation of HSCs using cell lysates of human TWNT-4 cells after 24 hour treatment with Y-27632 and Y-33075. (F) Quantification of α-SMA protein expression after treatment of TWNT-4 cells with ROCK inhibitors for 24 hours (n = 3). (G) α-SMA protein expression in murine FVB/NJ cell lysates as a marker for activation in HSCs after 24 hour treatment with ROCK inhibitors. (H) Quantification of α-SMA protein expression after treatment of TWNT-4 cells with ROCK inhibitors for 24 hours (n = 3). Results are expressed as the mean ± standard error of the mean (SEM); *p<0.05, **p<0.01 and ***p<0.001.</p

The sspecific NLRP3 antagonist IFM-514 decreases fibrosis and inflammation in experimental murine non-alcoholic steatohepatitis

Background and Aims: Activation of the inflammasome NLRP3 (NOD-, LRR- and pyrin domain containing 3) contributes to the development of non-alcoholic fatty liver disease (NAFLD) and progression to non-alcoholic steatohepatitis (NASH). Therefore, this study explored the therapeutic effects of a novel and selective NLRP3 antagonist in a murine dietary model of NASH. Methods: Groups of 12-week-old ApoE-/- mice were fed ad lib for 7 weeks with a methionine/choline deficient (MCD) and western diet (WD). After 3 weeks of diet-induced injury, mice were injected i. p. with the NLRP3 antagonist IFM-514 (100 mg/kg body weight) or vehicle (0.5% carmellose) every day, 5 days/week for a further 4 weeks. Several markers of inflammation, fibrosis and steatosis were evaluated. Whole transcriptome sequencing and panel RNA expression analysis (NanoString) were performed. Results: IFM-514 inhibited IL-1β production in mice challenged with 20 mg/kg lipopolysaccharide, and in mouse and human inflammatory cells in vitro. IFM-514 inhibited hepatic inflammation in the in vivo non-alcoholic steatohepatitis model assessed by H&E staining and in the hepatic gene expression of inflammasome-related proinflammatory cytokines. This effect was associated with significant reduction in caspase-1 activation. Similarly, IFM-514 was efficacious in vivo in MDC-fed ApoE-/- mice, markedly reducing portal pressure, Sirius red staining and 4-hydroxyproline content compared to vehicle-treated mice. Moreover, IFM-514 significantly reduced hepatic steatosis in MCD-fed ApoE-/- mice, as evidenced by NAFLD scores, oil red O staining, hepatic triglycerides and gene expression. In WD treated animals, similar trends in inflammation and fibrosis were observed, although not sufficient IFM-514 levels were reached. Conclusion: Overall, IFM-514 reduced liver inflammation and fibrosis, with mild effects on liver steatosis in experimental murine NASH. Blocking of NLRP3 may be an attractive therapeutic approach for NASH patients