Yifei Lv

Attenuation of CCl4-Induced Hepatic Fibrosis in Mice by Vaccinating against TGF-β1

Transforming growth factor β1 (TGF-β1) is the pivotal pro-fibrogenic cytokine in hepatic fibrosis. Reducing the over-produced expression of TGF-β1 or blocking its signaling pathways is considered to be a promising therapeutic strategy for hepatic fibrosis. In this study, we evaluated the feasibility of attenuating hepatic fibrosis by vaccination against TGF-β1 with TGF-β1 kinoids. Two TGF-β1 kinoid vaccines were prepared by cross-linking TGF-β1-derived polypeptides (TGF-β125–[41-65] and TGF-β130–[83-112]) to keyhole limpet hemocyanin (KLH). Immunization with the two TGF-β1 kinoids efficiently elicited the production of high-levels of TGF-β1-specific antibodies against in BALB/c mice as tested by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The antisera neutralized TGF-β1-induced growth-inhibition on mink lung epithelial cells (Mv1Lu) and attenuated TGF-β1-induced Smad2/3 phosphorylation, α-SMA, collagen type 1 alpha 2 (COL1A2), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression in the rat hepatic stellate cell (HSC) line, HSC-T6. Vaccination against TGF-β1 with the kinoids significantly suppressed CCl4-induced collagen deposition and the expression of α-SMA and desmin, attenuated hepatocyte apoptosis and accelerated hepatocyte proliferation in BALB/c mice. These results demonstrated that immunization with the TGF-β1 kinoids efficiently attenuated CCl4-induced hepatic fibrosis and liver injury. Our study suggests that vaccination against TGF-β1 might be developed into a feasible therapeutic approach for the treatment of chronic fibrotic liver diseases.

Oral Administration of Recombinant Adeno-associated Virus-mediated Bone Morphogenetic Protein-7 Suppresses CCl4-induced Hepatic Fibrosis in Mice

Fibrogenesis and hepatocyte degeneration are the main pathological processes in chronic liver diseases. Transforming growth factor-β1 (TGF-β1) is the key profibrotic cytokine in hepatic fibrosis. Bone morphogenetic protein-7 (BMP-7) is a potent antagonist of TGF-β1 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 (CCl4)-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 CCl4-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.Fibrogenesis and hepatocyte degeneration are the main pathological processes in chronic liver diseases. Transforming growth factor-β1 (TGF-β1) is the key profibrotic cytokine in hepatic fibrosis. Bone morphogenetic protein-7 (BMP-7) is a potent antagonist of TGF-β1 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.

Downregulated expression of CIAPIN1 may contribute to gastric carcinogenesis by accelerating cell proliferation and promoting cell cycle progression

Our previous studies revealed that cytokine induced apoptosis inhibitor 1 (CIAPIN1), which was reported to be essential in mouse definitive hematopoiesis, was related to multidrug resistance in gastric cancer cells and that the distribution of CIAPIN1 in normal human tissues was similar to the distribution of Ras. This study aimed to explore whether CIAPIN1 plays a role in gastric carcinogenesis. Expression of CIAPIN1 in normal, inflammatory gastric mucosa, gastric precancerous lesions and gastric adenocarcinoma was detected by immunohistochemistry and Western blotting and, influence of CIAPIN1 on the proliferation of gastric cancer cells was investigated by ectopic expression of CIAPIN1 and RNA interference (RNAi). Our immunohistochemical results demonstrated that the expression of CIAPIN1 in gastric antral mucosa was progressively reduced along the sequence of normal/inflammatory gastric mucosa-atrophy-intestinal metaplasia-dysplasia-adenocarcinoma. The downregulation of CIAPIN1 in cancerous tissues was further confirmed by Western blotting. No relationship between the expression level of CIAPIN1 and the clinicopathological parameters such as age, gender, differentiation, TNM stage and the existence of metastasis was found in gastric cancer patients. In in vitro cellular experiments, ectopic expression of CIAPIN1 by cDNA transfection resulted in suppression of cell proliferation and inhibition of cell cycle progression while knockdown of CIAPIN1 with siRNA accelerated cell proliferation and promoted cell cycle progression in SGC7901 and MKN28 gastric cancer cells. These results suggest that downregulated CIAPIN1 expression may contribute to gastric carcinogenesis by accelerating cell proliferation and promoting cell cycle progression.

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.

Adenovirus-mediated hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein suppresses dextran sulfate sodium-induced acute ulcerative colitis in rats

Background: Although increased expression of hepatocarcinoma‐intestine‐pancreas/pancreatitis‐associated protein (HIP/PAP) has been reported in ulcerative colitis (UC), its role in UC remains unclear. This study was designed to assess the function of HIP/PAP in experimental UC and further to explore its underlying mechanisms. Methods: Recombinant adenovirus was prepared to mediate ectopic expression of HIP/PAP in the colon of rats. The effect of HIP/PAP on dextran sodium sulfate (DSS)‐induced colitis was assessed by disease activity index (DAI), macroscopic, and histological evaluations. Superoxide dismutase (SOD) and myeloperoxidase (MPO) activities, malondialdehyde (MDA) content, and tumor necrosis factor &agr; (TNF‐&agr;) and interleukin‐6 (IL‐6) production were determined in colonic mucosa. Proliferation cell nuclear antigen (PCNA) was immunostained to reflect the proliferation of colonic epithelia. The effects of HIP/PAP on proliferation and H2O2‐induced apoptosis of SW480 and LoVo colonic adenocarcinoma cells were also determined. Gene expression profiles in SW480 after HIP/PAP overexpression were analyzed by microarray analysis. Results: The protective effect of HIP/PAP against DSS‐induced colitis in rats was confirmed. Ectopic expression of HIP/PAP resulted in attenuation of oxidative damage, reduction of TNF‐&agr; and IL‐6 expression, and elevation of epithelial proliferation in colonic mucosa and led to decreased apoptosis and increased proliferation in colonic adenocarcinoma cells. Microarray analysis revealed altered expression of inflammation‐related molecules, growth factors, proliferation‐related molecules, and antioxidant enzymes under overexpression of HIP/PAP. Conclusions: HIP/PAP has a protective effect against DSS‐induced colitis in rats via inhibiting inflammation, alleviating oxidative damage, and promoting colonic epithelium regeneration. HIP/PAP might represent a new promising therapeutic strategy in UC. (Inflamm Bowel Dis 2012)

Intracolonically administered adeno‐associated virus‐bone morphogenetic protein‐7 ameliorates dextran sulphate sodium‐induced acute colitis in rats

The current treatment of ulcerative colitis (UC) is less than ideal and has room for improvement. Bone morphogenetic protein‐7 (BMP‐7) exerts a protective effect on experimental UC. Hence, we considered that intracolonically (i.c.) administered adeno‐associated virus (AAV) delivering BMP‐7 might have therapeutic potential for UC.

Recombinant adeno-associated virus carrying thymosin β4 suppresses experimental colitis in mice

AIM To investigate the protective effect of a recombinant adeno-associated virus carrying thymosin β4 (AAV-Tβ4) on murine colitis via intracolonic administration. METHODS AAV-Tβ4 was prepared and intracolonically used to mediate the secretory expression of Tβ4 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-Tβ4 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)-1β 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 Tβ4 into the colonic tissues of the mice, and AAV-Tβ4 led to a strong expression of Tβ4 in mouse colons. In both the DSS and TNBS colitis models, AAV-Tβ4-treated mice displayed distinctly attenuated colon injuries and reduced apoptosis rate of colonic mucosal epithelia. AAV-Tβ4 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-Tβ4 also modulated colonic TNF-α, IL-1β and IL-10 levels and suppressed the compensatory proliferation of colonic epithelial cells in DSS- and TNBS-treated mice. CONCLUSION Tβ4 exerts a protective effect on murine colitis, indicating that AAV-Tβ4 could potentially be developed into a promising agent for the therapy of inflammatory bowel diseases.

Adenovirus-mediated expression of p33ING1b induces apoptosis and inhibits proliferation in gastric adenocarcinoma cells in vitro

BackgroundInhibitor of growth 1b (ING1b) is considered to be a class II tumor suppressor gene. Although reduced expression of p33ING1b has been reported in many human malignancies, including gastric cancers, the effect of p33ING1b on gastric cancer cells has yet to be investigated.MethodsExpression of p33ING1b in gastric adenocarcinoma tissues and their adjacent non-malignant gastric mucosa, as well as in gastric adenocarcinoma cell lines and normal gastric epithelial cells, was detected by using Western blotting. Recombinant adenoviruses were prepared to mediate the ectopic expression of p33ING1b (Ad-ING1b) and green fluorescent protein (GFP)(Ad-GFP) in the gastric adenocarcinoma cell lines, SGC-7901, MKN28, and MKN45 and the normal gastric epithelial cell line GES-1. Alterations in the proliferation and apoptosis of the cells after adenoviral infection were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively, and cell cycle distribution was analyzed in a fluorescence-activated cell sorter.ResultsWestern blotting confirmed the reduced expression of p33ING1b in gastric adenocarcinoma tissues and gastric adenocarcinoma cell lines. The ectopic expression of p33ING1b mediated by Ad-ING1b resulted in decreased growth, increased apoptosis, and cell cycle arrest at the G1 phase in both benign and malignant gastric epithelial cells regardless of their p53 status. Addition of a p53 inhibitor, pifithrin-α, did not abolish the pro-apoptotic and cell cycle-arresting effects of p33ING1b in p53 wild-type cells.ConclusionsDown-regulation of p33ING1b might play an important role in the development of gastric adenocarcinoma. Targeted local expression of p33ING1b may offer a promising alternative therapeutic measure for gastric cancer.

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-β1, 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.

In vitro selection and characterization of deoxyribonucleic acid aptamers against connective tissue growth factor

Connective tissue growth factor (CTGF) is a secreted matricellular protein possessing complex biological functions. CTGF modulates a number of signaling pathways that are involved in cell adhesion, migration, angiogenesis, myofibroblast activation, extracellular matrix deposition and tissue remodeling. Aptamers are oligonucleic acid chains or polypeptides that bind with specific target molecules hence have the potential to be used in the detection and blockade of the targets. In this study, we selected CTGF-targeting DNA aptamers by using systematic evolution of ligands by exponential enrichment (SELEX). After 8 iterative rounds of selection, cloning, DNA sequencing and affinity determination, six aptamers with high affinities to CTGF were obtained. Among them, one (C-ap17P) binds with the N-terminal region (aa 1-190) and the other five (C-ap11, 12, 14, 15 and 18) bind with the C-terminal region (aa 191-350) of hCTGF specifically. The biological stability assay indicated that a representative aptamer, C-ap17P, could keep its integrity at a rather high level for at least 24 h in complete DMEM cell culture medium. These CTGF aptamers might be used as a easy and fast detection tool for CTGF and be developed as CTGF-specific inhibitors for both research works and clinical applications.