Regression of liver fibrosis: evidence and challenges.

It has been reported that liver fibrosis could be reversed after eliminating liver injuries. This article systematically summarizes the evidence of fibrosis regression based on histology, liver stiffness, and serum biomarkers, and discusses several clinically relevant challenges. Evidence from liver biopsy has been regarded as the gold standard in the assessment of fibrosis regression. Semi-quantitative staging and grading systems are traditionally and routinely used to define regression. Recently, the predominantly regressive, indeterminate, and predominantly progressive score was proposed, based on the regressive features from "hepatic repair complex", to provide additional information regarding the quality of fibrosis. For non-invasive assessment, although liver stiffness and serum biomarkers could be applied to reflect the dynamic changes of liver fibrosis, other confounding factors such as liver inflammation have to be considered. In conclusion, both histology and non-invasive methods can provide evidence regarding fibrosis regression. The predictive value of fibrosis regression in long-term prognosis warrants further investigation.

SIRT1 Protects Against Oxidative Stress-Induced Endothelial Progenitor Cells Apoptosis by Inhibiting FOXO3a via FOXO3a Ubiquitination and Degradation.

Cell loss due to apoptosis induced by oxidative stress is a major hurdle for endothelial progenitor cells (EPCs)-based therapy. Sirtuin 1 (SIRT1) plays important roles in many pathophysiological processes by deacetylating various substrates, including forkhead transcription factor (FOXO). However, after deacetylation, the fate of FOXO protein remains to be explored. In the present study, we investigated whether SIRT1 exerted a protective effect on hydrogen peroxide (H(2)O(2))-induced EPCs apoptosis and, if so, what the underlying mechanism might be. EPCs were isolated and obtained from human umbilical cord blood by density gradient centrifugation and identified by morphology, tube formation ability, cell surface markers, and the ability to take up acetylated low-density lipoprotein (Dil-Ac-LDL) and bind ulex europaeus agglutinin 1 (FITC-UEA-1). Immunofluorescence showed that SIRT1 is localized in the nucleus of EPCs in the presence or absence of H(2)O(2). SIRT1 protein level in EPCs was increased by the treatment with H(2)O(2) for 24 h. Incubation of EPCs with H(2)O(2) dose dependently induced EPCs apoptosis. SIRT1 overexpression reduced the rate of EPCs apoptosis induced by H(2)O(2), whereas SIRT1 downregulation and EX527, a specific SIRT1 inhibitor, exerted the opposite effect. SIRT1 overexpression decreased the total FOXO3a protein expression, whereas SIRT1 downregulation and EX527 increased the amount of FOXO3a protein. SIRT1 reduced FOXO3a transcriptional activity according to Bim expression. Co-immunoprecipitation assay showed that SIRT1 could bind to FOXO3a, reduce its acetylation level and increase its ubiquitination level. To sum up, our work demonstrated that SIRT1 had a pivotally protective role in the regulation of EPCs apoptosis induced by H(2)O(2) and that SIRT1 protected against apoptosis by inhibiting FOXO3a via FOXO3a ubiquitination and subsequent degradation.

Hydrogen peroxide induced impairment of endothelial progenitor cell viability is mediated through a FoxO3a dependant mechanism.

OBJECTIVES: Increased oxidative stress has been suggested to contribute to the functional impairment of endothelial progenitor cells (EPCs). The Forkhead box O transcription factors (FoxOs) are critical regulators involved in various cellular processes including cell apoptosis. Here, we investigated whether FoxOs are required in oxidative stress induced EPC apoptosis. METHODS AND RESULTS: EPCs were cultured from cord blood derived mononuclear cells and treated with hydrogen peroxide (H2O2) for induction of oxidative stress. Incubation with H2O2 dose dependently reduced viability and increased apoptosis in EPCs. Western blotting showed that EPCs predominantly expressed FoxO3a and the expression was markedly increased upon H2O2 treatment. Transduction with adenoviral vectors expressing either a wide-type or a non-phosphorylatable, constitutively active mutant of FoxO3a led to further increased apoptosis of EPCs after H2O2 treatment. Conversely, FoxO3a silencing rescued EPCs from these H2O2 induced deleterious effects. Overexpression of FoxO3a also increased the level of the pro-apoptotic protein Bim, whereas FoxO3a silencing downregulated H2O2 induced Bim expression. Furthermore, Matrigel assay demonstrated that FoxO3a overexpression significantly impaired the tube forming ability of EPCs, whereas its silencing completely protected EPCs from H2O2 induced decrease of capillary formation. CONCLUSIONS: These data suggest that oxidative stress induced impairment of EPC survival is mediated through a FoxO3a dependant mechanism, possibly by transcriptional regulation of Bim. Our data indicate FoxO3a as a potential therapeutic target for improvement of EPC number and function in patients with ischemic heart disease.

Delivery of therapeutic AGT shRNA by PEG-Bu for hypertension therapy.

Gene silencing by RNA interference (RNAi) is a promising approach for gene therapy. However, up to today, it is still a major challenge to find safe and efficient non-viral vectors. Previously, we reported PEI-Bu, a small molecular weight PEI derivative, as an efficient non-viral carrier. However, like many PEI-based polymers, PEI-Bu was too toxic. In order to reduce cytotoxicity while maintain or even enhance transfecion efficiency, we modified PEI-Bu with poly(ethylene glycol) (PEG) to obtain PEG-Bu, and used it to delivery a theraputic short hairpin RNA (shRNA) targeting angiotensinogen (AGT) to normal rat liver cells (BRL-3A), which was a key target for the treatment of hypertension. The structure of PEG-Bu was confirmed by proton nuclear magnetic resonance ((1)H-NMR). Gel permeation chromatography (GPC) showed that the weight average molecular weight (Mw) of PEG-Bu was 5880 Da, with a polydispersity of 1.58. PEG-Bu could condense gene cargo into spherical and uniform nanoparticles with particle size (65-88 nm) and zeta potential (7.3-9.6 mV). Interestingly and importantly, PEG-Bu displayed lower cytotoxicity and enhanced tranfection efficiency than PEI-Bu after PEGylation in both normal cells BRL-3A and tumor cells HeLa. Moreover, PEG-Bu could efficiently delivery AGT shRNA to knockdown the AGT expression. To sum up, PEG-Bu would be a promising non-viral vector for delivering AGT shRNA to BRL-3A cells for hypertension therapy.

Autologous transplantation of EPCs encoding FGF1 gene promotes neovascularization in a porcine model of chronic myocardial ischemia.

OBJECTIVES: The functional impairment of endothelial progenitor cells (EPCs) constitutes an important barrier for therapeutic angiogenesis. Here, we tested the hypotheses that a secreted version of acidic fibroblast growth factor (sp-FGF1) gene transfer may achieve functional improvement of EPCs and that autologous transplantation of sp-FGF1-modified EPCs can facilitate neovascularization in a porcine model of chronic myocardial ischemia. METHODS AND RESULTS: EPCs were cultured from porcine peripheral blood and transduced with a recombinant adeno-associated virus encoding the sp-FGF1 gene (Td/FGF1-EPC). EPC function was evaluated 3 days after the gene transfer. In vitro, the sp-FGF1-modified EPCs displayed enhanced migration, tube formation and survival. Chronic myocardial ischemia was induced by the placement of an ameroid constrictor around the left circumflex coronary artery (LCx). Four weeks after ameroid placement, coronary angiography was performed and the cells were administered through the stenotic LCx. Myocardial perfusion defects were significantly reduced in animals transplanted with Td/FGF1-EPC compared to animals that received non-transduced EPCs or PBS (P<0.05) as assessed by SPECT 4 weeks after cell transplantation. Furthermore, the vascular density of ischemic myocardium was significantly increased in Td/FGF1-EPC transplanted animals (P<0.05). In addition, FGF1 protein expression was only detected in Td/FGF1-EPC transplanted animals. CONCLUSIONS: The functional activities of EPCs were enhanced by sp-FGF1 gene transfer. Transplantation of this gene modified EPCs promoted neovascularization in a porcine model of chronic myocardial ischemia, indicating the therapeutic potential of this cell-based gene therapy strategy for the treatment of ischemic diseases.