Oral administration of recombinant adeno-associated virus-mediated bone morphogenetic protein-7 suppresses CCl(4)-induced hepatic fibrosis in mice.

Fibrogenesis and hepatocyte degeneration are the main pathological processes in chronic liver diseases. Transforming growth factor-ß1 (TGF-ß1) is the key profibrotic cytokine in hepatic fibrosis. Bone morphogenetic protein-7 (BMP-7) is a potent antagonist of TGF-ß1 and an antifibrotic factor. In this study, we generated a recombinant adeno-associated virus carrying BMP-7 (AAV-BMP-7) and tested its ability to suppress carbon tetrachloride (CCl(4))-induced hepatic fibrosis when orally administered to mice. Our results show that the ectopic expression of BMP-7 in gastrointestinal (GI) mucosa due to the AAV-BMP-7 administration led to the long-term elevation of serum BMP-7 concentrations and resulted in the drastic amelioration of CCl(4)-induced hepatic fibrosis in BALB/c mice. Immunostaining for α-smooth muscle actin (α-SMA) and desmin demonstrated that AAV-BMP-7 inhibited the activation of hepatic stellate cells (HSCs) in the fibrotic mouse liver. Moreover, the ectopic expression of BMP-7 promoted hepatocyte proliferation, as confirmed by an increase in the amount of proliferating cell nuclear antigen (PCNA)-positive hepatocytes in the mice that received AAV-BMP-7. Our results clearly indicate that BMP-7 is capable of inhibiting hepatic fibrosis and promoting hepatocyte regeneration. We suggest that oral AAV-BMP-7 could be developed into a safe, simple, and effective therapy for hepatic fibrosis.

Vaccination with platelet-derived growth factor B kinoids inhibits CCl4-induced hepatic fibrosis in mice.

Platelet-derived growth factor B (PDGF-B) plays an essential role in hepatic fibrosis. Inhibition of the PDGF-B signaling in chronically injured livers might represent a potential therapeutic measure for hepatic fibrosis. In this study, we assessed the effects of vaccination against PDGF-B on CCl4-induced liver fibrosis in BALB/c mice. The PDGF-B kinoid immunogens were prepared by cross-linking two PDGF-B-derived B-cell epitope peptides [PDGF-B¹6-(23-38) and PDGF-B¹6-(72-83)] to ovalbumin and keyhole limpet hemocyanin, respectively. Enzyme-linked immunosorbent assay, Western blotting, and NIH3T3 cell proliferation assay verified that immunization with the PDGF-B kinoids elicited the production of high levels of neutralizing anti-PDGF-B autoantibodies. The vaccination markedly alleviated CCl4-induced hepatic fibrosis, as indicated by the lessened morphological alternations and reduced hydroxyproline contents in the mouse livers. Moreover, immunohistochemical staining for proliferating cell nuclear antigen, α-smooth muscle actin, and desmin demonstrated that neutralization of PDGF-B inhibited both the proliferation and the activation of hepatic stellate cells in the fibrotic mouse livers. Taken together, this study demonstrated that vaccination with PDGF-B kinoids significantly suppressed CCl4-induced hepatic fibrosis in mice. Our results suggest that vaccination against PDGF-B might be developed into an effective, convenient, and safe therapeutic measure for the treatment of hepatic fibrosis.

Regulation of Nrf2 by phosphorylation: Consequences for biological function and therapeutic implications.

The transcription factor nuclear factor erythroid-derived 2-like 2 (NRF2) participates in the activation of the antioxidant cytoprotective pathway and other important physiological processes to maintain cellular homeostasis. The dysregulation of NRF2 activity plays a role in various diseases, such as cardiovascular diseases, neurodegenerative diseases, and cancer. Thus, NRF2 activity is tightly regulated through multiple mechanisms, among which phosphorylation by kinases is critical in the posttranslational regulation of NRF2. For instance, PKC, casein kinase 2, and AMP-activated kinase positively, while GSK-3 negatively regulates NRF2 activity through phosphorylation of different sites. Here, we provide an overview of the phosphorylation regulation pattern of NRF2 and discuss the therapeutic potential of interventions targeting NRF2 phosphorylation.

Discovery of 3,5-Dimethyl-4-Sulfonyl-1H-Pyrrole-Based Myeloid Cell Leukemia 1 Inhibitors with High Affinity, Selectivity, and Oral Bioavailability.

Myeloid cell leukemia 1 (Mcl-1) protein is a key negative regulator of apoptosis, and developing Mcl-1 inhibitors has been an attractive strategy for cancer therapy. Herein, we describe the rational design, synthesis, and structure-activity relationship study of 3,5-dimethyl-4-sulfonyl-1H-pyrrole-based compounds as Mcl-1 inhibitors. Stepwise optimizations of hit compound 11 with primary Mcl-1 inhibition (52%@30 µM) led to the discovery of the most potent compound 40 with high affinity (Kd = 0.23 nM) and superior selectivity over other Bcl-2 family proteins (>40,000 folds). Mechanistic studies revealed that 40 could activate the apoptosis signal pathway in an Mcl-1-dependent manner. 40 exhibited favorable physicochemical properties and pharmacokinetic profiles (F% = 41.3%). Furthermore, oral administration of 40 was well tolerated to effectively inhibit tumor growth (T/C = 37.3%) in MV4-11 xenograft models. Collectively, these findings implicate that compound 40 is a promising antitumor agent that deserves further preclinical evaluations.

Elevated interleukin-35 suppresses liver inflammation by regulation of T helper 17 cells in acute hepatitis B virus infection.

Interleukin (IL)-35 is a responsive anti-inflammatory cytokine implicated in different diseases processes. It has been reported that elevated IL-35 contributed to immunosuppression in chronic hepatitis by modulation of T helper 17 (Th17) and regulatory T cells. However, the role of IL-35 in acute hepatitis B (AHB) was still not completely elucidated. Thus, in the present study, we analyzed the expression and regulatory activity of IL-35 to Th17 cells and inflammatory response during acute hepatitis B virus (HBV) infection in both peripheral blood cells isolated from AHB patients and in hydrodynamic induced HBV-infected mouse model. Plasma IL-35 level and circulating HBV peptides-induced Th17 frequency was significantly elevated in AHB patients, and IL-35 expression negatively correlated with liver inflammation. In vitro IL-35 stimulation to CD4+ T cells purified from AHB patients down-regulated HBV peptides-induced Th17-phenotype, which presented as reduced IL-17 and IL-22 production. In vivo IL-35 administration dampened liver inflammation in HBV plasmid injected mice, however, did not affect HBV antigens production. This process was accompanied by suppression of natural killer cells and down-regulation of HBV peptides-induced Th17 cells in the liver, but did not affect total intrahepatic lymphocytes and other cell subsets numbers or chemokines expression in the liver. In conclusion, the current data indicated that IL-35 might be a novel mediator associated with hepatocytes damage and liver inflammation by regulating HBV peptides-induced Th17 cells during acute HBV infection. The potential anti-inflammatory property of IL-35 might be pivotal for developing new therapeutic approaches for hepatitis B.

Recombinant adeno-associated virus carrying thymosin ß4 suppresses experimental colitis in mice.

AIM: To investigate the protective effect of a recombinant adeno-associated virus carrying thymosin ß4 (AAV-Tß4) on murine colitis via intracolonic administration. METHODS: AAV-Tß4 was prepared and intracolonically used to mediate the secretory expression of Tß4 in mouse colons. Dextran sulfate sodium (DSS) was applied to induce the murine ulcerative colitis, and 2,4,6-trinitrobenzene sulfonic acid (TNBS) was used to establish a mouse colitis model resembling Crohn's disease. The disease severity and colon injuries were observed and graded to reveal the effects of AAV-Tß4 on colitis. The activities of myeloperoxidase (MPO) and superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were determined using biochemical assays. Colonic levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-10 were measured using ELISA, and mucosal epithelial cell apoptosis and proliferation were detected by TUNEL assay and immunochemistry, respectively. RESULTS: Recombinant AAVs efficiently delivered LacZ and Tß4 into the colonic tissues of the mice, and AAV-Tß4 led to a strong expression of Tß4 in mouse colons. In both the DSS and TNBS colitis models, AAV-Tß4-treated mice displayed distinctly attenuated colon injuries and reduced apoptosis rate of colonic mucosal epithelia. AAV-Tß4 significantly reduced inflammatory cell infiltrations and relieved oxidative stress in the inflamed colons of the mice, as evidenced by decreases in MPO activity and MDA content and increases in SOD activity. AAV-Tß4 also modulated colonic TNF-α, IL-1ß and IL-10 levels and suppressed the compensatory proliferation of colonic epithelial cells in DSS- and TNBS-treated mice. CONCLUSION: Tß4 exerts a protective effect on murine colitis, indicating that AAV-Tß4 could potentially be developed into a promising agent for the therapy of inflammatory bowel diseases.

A virus-like particle-based connective tissue growth factor vaccine suppresses carbon tetrachloride-induced hepatic fibrosis in mice.

Connective tissue growth factor (CTGF) has been recognized as a central mediator and promising therapeutic target in hepatic fibrosis. In this study, we generated a novel virus-like particle (VLP) CTGF vaccine by inserting the 138-159 amino acid (aa) fragment of CTGF into the central c/e1 epitope of C-terminus truncated hepatitis B virus core antigen (HBc, aa 1-149) using a prokaryotic expression system. Immunization of BALB/c mice with the VLP vaccine efficiently elicited the production of anti-CTGF neutralizing antibodies. Vaccination with this CTGF vaccine significantly protected BALB/c mice from carbon tetrachloride (CCl4)-induced hepatic fibrosis, as indicated by decreased hepatic hydroxyproline content and lower fibrotic score. CCl4 intoxication-induced hepatic stellate cell activation was inhibited by the vaccination, as indicated by decreased α-smooth muscle actin expression and Smad2 phosphorylation. Vaccination against CTGF also attenuated the over-expression of some profibrogenic factors, such as CTGF, transforming growth factor-ß1, platelet-derived growth factor-B and tissue inhibitor of metalloproteinase-1 in the fibrotic mouse livers, decreased hepatocyte apoptosis and accelerated hepatocyte proliferation in the fibrotic mouse livers. Our results clearly indicate that vaccination against CTGF inhibits fibrogenesis, alleviates hepatocyte apoptosis and facilitate hepatic regeneration. We suggest that the vaccine should be developed into an effective therapeutic measure for hepatic fibrosis.