Ruian Xu

rAAV-mediated stable expression of heme oxygenase-1 in stellate cells: A new approach to attenuate liver fibrosis in rats†

Liver fibrosis is the consequence of activation of hepatic stellate cells mediated by persistent or recurrent liver injury, where oxidative stress or inflammatory response resulting from immune cells and cytokines are involved. Targeting of hepatic stellate cells could be an important strategy for the therapy of liver fibrosis. In this study, we showed a tropism of recombinant adeno‐associated virus (rAAV, serotype 2) with high efficiency in transduction of a homeostatic gene, heme oxygenase‐1 (HO‐1), to activated stellate cells. The binding of rAAVs to stellate cells increased significantly after serum‐stimulated activation compared with quiescent status. Portal injection of rAAVs to normal or carbon tetrachloride (CCl4)‐induced liver fibrosis showed a distinct distribution of rAAV binding. The majority of injected rAAVs bound to the cells in fibrotic areas that were associated with higher expression levels of fibroblast growth factor receptor‐1α at 2 hours after administration. Isolation of different types of cells from CCl4‐induced fibrotic livers showed predominant expression of transgene in stellate cells after rAAV/HO‐1 administration on day 3 and remained stable for 12 weeks. In addition, HO‐1–transduced stellate cells showed reduced transcript levels of type 1 collagen and impaired proliferative ability compared with controls. With this approach, the severity of established micronodular cirrhosis was markedly reduced. In conclusion, these findings suggest a new approach for the treatment of liver fibrosis using adeno‐associated virus‐mediated gene transfer. (HEPATOLOGY 2005;42:335–342.)

The Structure and Pharmacological Functions of Coumarins and Their Derivatives

Coumarins are of many different structures. They constitute an important class of pharmacological agents possessing a range of different physiological activities including anti-cancer, anti-oxidant, anti- inflammation, anti-HIV, anti-coagulant, anti-bacterial, analgesic and comparative immune-modulation. Recently, coumarins have attracted intense research interest. Of great interest is the possibility that this class of molecules could be a source of drugs for the therapy of several diseases. These include recent insights into inhibiting cell proliferation by interfering with mitotic spindle microtubule function, decrease Matrix Metalloproteinase (MMP) activity, block the cell cycle in the S or G2/M phases to interfere with processes of cell division, suppress O2(-) generation in leukocytes, inhibit different protein kinases, modulate the signalings, induce carcinogen-detoxifying enzymes glutathione S-transferases (GSTs) and/or NAD(P)H quinine oxidoreductase (NQO1), suppress the phosphorylation of Akt/PKB as a mechanism inhibiting inflammation, progress in structure modification to increase in anti-fungal action, to broaden against bacteria spectrum, to enhance inhibiting activities of nitric oxide synthase (NOS) and cyclooxygenase (COX), to strengthen anti-oxidant activity and to exhibite a much higher cytotoxicity against human umbilical vein endothelial cell (HUVEC). With fewer non-hemorrhagic side effects than the indanedione derivatives, they can be applied as an oral anticoagulant commonly for preventing venous thromboembolism following orthopedic surgery, recurrent myocardial infarction and the treatment of systemic embolism in atrial fibrillation, together with the significant advances in the basis of drug action. It is therefore useful to build up some correlations with the data available in order to better explore the molecular and cellular mechanism of coumarin action in the treatment of diseases. This review will focus on recent advances in molecular and cellular mechanisms of coumarin action involved with the relationship between structure and activity.

Overexpression of Soluble TRAIL Induces Apoptosis in Human Lung Adenocarcinoma and Inhibits Growth of Tumor Xenografts in Nude Mice

Recombinant adeno-associated virus 2/5 (rAAV2/5), a hybrid rAAV-2 with AAV-5 capsid, seems to be a very efficient delivery vector for the transduction of the lung adenocarcinoma cell line A549. Infection of the A549 cell line with a rAAV2/5 vector encoding the extracellular domain of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, amino acids 114-281) resulted in secretion of soluble TRAIL (sTRAIL) and induction of apoptosis in these cells. rAAV2/5-sTRAIL mediated delivery and stable expression of sTRAIL resulted in the presence of the trimeric form of sTRAIL in sera of nude mice that were implanted with s.c. or orthotopic A549 tumors. The rAAV2/5-sTRAIL transduction of the tumors resulted in a statistically significant reduction in tumor growth and prolonged survival of the tumor-bearing animals. Primary cell culture, histologic examination of the tumors, and serum analyses showed the absence of detectable TRAIL-induced toxicity in normal tissues including the liver. The successful inhibition of lung cancer growth and the absence of detectable toxicity suggest a putative role for rAAV2/5-sTRAIL(114-281) in the therapy of lung cancer.

Differential regulation of neurogenesis by the two Xenopus GATA-1 genes.

Previously, we have shown that the ventralizing factor bone morphogenetic protein 4 (BMP-4) can inhibit Xenopus neurogenesis. The erythroid transcription factor GATA-1 functions downstream of the BMP-4 signaling pathway and mediates BMP-4-induced erythropoiesis. We have found that similar to BMP-4, GATA-1b inhibits neuralization of Xenopus animal cap (AC) cells. The neural inhibition is not seen with GATA-1a, although both GATA-1a and GATA-1b RNAs are translated at the same efficiency and induce globin expression equally in AC cells. GATA-1b RNA injection into AC cells neither induces expression of Xbra (a general mesoderm marker) nor affects expression of XK81 (epidermal keratin) or BMP-4 and Xvent-1 (two ventral markers). These data suggest that GATA-1b retains the epidermal fate of the AC. Intact GATA-1b protein is required for both inhibition of neurogenesis and induction of globin expression. Our findings indicate that GATA-1b can function in ectoderm to specifically regulate neural inducing mechanisms, apparently related to the expression of chordin, a neuralizing gene. Furthermore, tadpole stage embryos injected with GATA-1b are devoid of all dorsoanterior structures including neural tissue. This report provides evidence that the two transcription factors, derived from a recent genome duplication, share a common biological activity (stimulation of erythropoiesis) while also exhibiting a distinct function (inhibition of neurogenesis).

Suppression of Lung Tumor Growth and Metastasis in Mice by Adeno-Associated Virus-Mediated Expression of Vasostatin

Purpose: Angiogenesis inhibitors have strong therapeutic potential as antitumor agents in suppressing tumor growth and metastatic progression. Vasostatin, the N-terminal domain of calreticulin, is a potent angiogenesis inhibitor. In this study, we determined the effectiveness of vasostatin delivered by recombinant pseudotype adeno-associated virus 2/5 (rAAV2/5-VAS) as a gene therapy approach for lung cancer treatment. Experimental Design: We used rAAV2/5 to deliver vasostatin intratumorally or systemically in different mouse lung tumor models — subcutaneous, orthotopic xenograft, and spontaneous metastasis lung tumor models. The therapeutic efficacy of rAAV2/5-VAS was determined by monitoring tumor volume, survival rate, and degree of neovascularization after treatment in these models. Results: Mice bearing subcutaneous tumor of rAAV2/5-VAS pretreated Lewis lung carcinoma cells showed >50% reduction in primary tumor volume and reduced spontaneous pulmonary metastases. The tumor-suppressive action of rAAV2/5-VAS in subcutaneous human lung tumor A549 xenograft correlated with a reduced number of capillary vessels in tumors. In the orthotopic xenograft model, rAAV2/5-VAS suppressed metastasis of A549 tumors to mediastinal lymph nodes and contralateral lung. Furthermore, treatment of immunocompetent mice in the spontaneous lung metastases model with rAAV2/5-VAS after primary tumor excision prolonged their median survival from 21 to 51.5 days. Conclusion: Our results show the effectiveness of rAAV2/5-VAS as an angiogenesis inhibitor in suppressing tumor growth during different stages of tumor progression, validating the application of rAAV2/5-VAS gene therapy in treatment against lung cancer.

Potential of DNMT and its Epigenetic Regulation for Lung Cancer Therapy

Lung cancer, the leading cause of mortality in both men and women in the United States, is largely diagnosed at its advanced stages that there are no effective therapeutic alternatives. Although tobacco smoking is the well established cause of lung cancer, the underlying mechanism for lung tumorigenesis remains poorly understood. An important event in tumor development appears to be the epigenetic alterations, especially the change of DNA methylation patterns, which induce the most tumor suppressor gene silence. In one scenario, DNA methyltransferase (DNMT) that is responsible for DNA methylation accounts for the major epigenetic maintenance and alternation. In another scenario, DNMT itself is regulated by the environment carcinogens (smoke), epigenetic and genetic information. DNMT not only plays a pivotal role in lung tumorigenesis, but also is a promising molecular bio-marker for early lung cancer diagnosis and therapy. Therefore the elucidation of the DNMT and its related epigenetic regulation in lung cancer is of great importance, which may expedite the overcome of lung cancer.

Overexpression of von Hippel-Lindau tumor suppressor protein and antisense HIF-1alpha eradicates gliomas

The von Hippel–Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the α subunits of hypoxia-inducible-factors responsible for stimulating tumor angiogenesis and glycolysis, and targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to tumorigenesis and development of sporadic renal cell carcinomas and central nervous system hemangioblastomas. In the present study, we investigated whether engineered overexpression of pVHL in C6 glioma cells, which already express endogenous pVHL, would suppress the tumorigenicity of this particular tumor cell type. C6 cells overexpressing VHL displayed a reduced growth rate (70% inhibition) compared to the parental cell line when subcutaneously implanted in athymic (nu/nu) mice. Growth inhibition was associated with a 50% reduction in the number of tumor vessels and a 60% increase in tumor cell apoptosis, due in part to downregulation of HIF-1, VEGF, and the antiapoptotic factor Bcl-2, respectively. Gene transfer of VHL suppressed the growth of established C6 gliomas, and synergized with antisense HIF-1 to completely eradicate tumors. The data suggest that VHL gene therapy and/or agents that increase VHL expression could have utility in the treatment of gliomas, particularly when combined with agents that inhibit the expression or function of HIF-1.

Recombinant adeno‐associated virus‐mediated TRAIL gene therapy suppresses liver metastatic tumors

To evaluate the tumoricidal activity of tumor necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL) on disseminated liver metastatic tumors, we constructed a recombinant adeno‐associated virus (rAAV) expressing the extracellular domain (95–281aa) of human TRAIL (TRAIL95–281, and the recombinant virus was designated as rAAV‐TRAIL) using the 3‐plasmid, helper‐virus‐free, packaging system. Transduction of mouse lymphoma EL‐4 cells and Jurkat T cells lead to the expression of TRAIL95–281 protein in both virus‐transduced cells and the culture media, along with apoptosis of these cells in vitro. The therapeutic potential of rAAV‐TRAIL was then evaluated in an orthotopic transplanted mouse model mimicking liver cancer metastasis, which was established by injection of EL‐4 cells into the liver of C57BL/6 mice via the hepatic portal veins. Subsequent intraportal vein injection of rAAV‐TRAIL, not the control virus, into the liver of these mice resulted in significant suppression of tumor growth and prolonged survival, while normal hepatocyte toxicity is undetectable. Histological and biochemical analysis in tumor tissue and serum confirmed that TRAIL95–281 was stably expressed in relatively high level in hepatocytes and was secreted into the serum in active trimeric form. Futhermore, the mechanism for rAAV‐TRAIL to inhibit tumor growth was by inducing apoptosis of the tumor cells metastasizing to the livers. These results strongly suggest that the rAAV‐TRAIL‐mediated gene delivery could be a promising approach for the treatment of liver metastasis cancer.

Oral adeno‐associated virus‐sTRAIL gene therapy suppresses human hepatocellular carcinoma growth in mice

The extracellular domain of the tumor necrosis factor‐related apoptosis‐inducing ligand (sTRAIL) may function as a soluble cytokine to selectively kill various cancer cells without toxicity to most normal cells. We constructed a series of recombinant adeno‐associated virus (AAV) vectors expressing the extracellular domain of human TRAIL fused with signal peptides of human insulin, interferon, human growth hormone, and serum albumin and designated them as AAV‐ISN‐T, AAV‐IFN‐T, AAV‐HGH‐T, and AAV‐Alb‐T, respectively. Transduction of human SMMC‐7721 liver cancer cells with AAV‐ISN‐T led to higher levels of TRAIL95‐281 protein expression in the cell culture media and produced more apoptosis of the cells in vitro than those with AAV‐IFN‐T, AAV‐HGH‐T, and AAV‐Alb‐T. The therapeutic potential of AAV‐ISN‐T was then evaluated in a transplanted mouse model established by injection of human liver cancer SMMC‐7721 cells subcutaneously. Subsequent oral or intraperitoneal administration of AAV‐ISN‐T resulted in a rapid, high level and long time expression of soluble TRAIL in the sera and livers of the animals, as well as effective suppression of tumor growth, with no toxicity to normal hepatocytes. These data strongly suggest that it is possible to increase soluble TRAIL expression to make full use of tumoricidal activity of TRAIL as a therapeutic strategy. In conclusion, we provide evidence that oral administration of AAV‐TRAIL might be an important alternative route with practical significance for cancer gene therapy. (HEPATOLOGY 2005;42:1355–1363.)

Diabetes Gene Therapy: Potential and Challenges

Gene therapy, developing rapidly as a result of advances in molecular biology and the Human Genome Project, is now highlighted as a most hopeful technology of the 21st century. The major goal of gene therapy in diabetes mellitus (DM) is to maintain euglycemia in face of wide variations in dietary intake. Although some obstacles remain to be overcome, the risk-benefit ratio of gene therapy in DM is better than that of lifelong injections of insulin, and islet transplantation, which faces the problems of donor shortage and rejection. This review focuses on the recent advances in gene therapy of insulin-requiring diabetes, with particular emphasis on 1. the gene delivery systems by viral vectors, since most gene therapy approaches for DM involve the use of viral vectors, paying special attention to current efforts to overcome the disadvantages of adenovirus, adenovirus-associated virus and retrovirus vectors and targeting gene delivery for optimal efficiency of gene expression; 2. coupling the synthesis and release of the transgene insulin to serum glucose concentrations, especially with reference to the current promoters controlling at transcriptional level the ectopic insulin expression in autologous hepatocytes; 3. beta-cell replacement strategies: engineering of beta-cells, especially those derived from pluripotent stem cells, non beta-cells, and on a new comer, the K cells. Recent advances in the use of stem cells for potential application in diabetes gene therapy are also discussed.