Chi-Keung Lau

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.)

Prevention of Chronic Deterioration of Heart Allograft by Recombinant Adeno-Associated Virus-Mediated Heme Oxygenase-1 Gene Transfer

Background—Allograft deterioration is the major obstacle to organ transplantation as a long-term treatment of end-stage heart failure. In this study, we transduced the antioxidant gene, heme oxygenase-1 (HO-1), to heart grafts using a recombinant adeno-associated viral vector (rAAV) in a rat heart transplantation model and investigated its potentiality in prevention of chronic graft deterioration. Methods and Results—rAAV/HO-1 was administered to heart grafts through the coronary arteries during cold preservation. We investigated the expression patterns and activities of transgene, graft survival, graft histomorphology, and relevance of HO-1 expression on graft survival and chronic graft deterioration by itself. Long-term allograft survival can be achieved by rAAV/HO-1-mediated stable transgene expression. The development of graft arteriosclerosis and interstitial fibrosis was prevented in rAAV/HO-1–transduced allografts on day 100. rAAV/HO-1–mediated long-term graft protection was accompanied by remarkable downregulation of the intragraft mRNA level of macrophage migration inhibitory factor, tumor necrosis factor-&agr;, and transforming growth factor-&bgr;1. Blockage of HO activities by zinc protoporphyrin IX at different posttransplant phases showed that the stable expression of HO-1 is a prerequisite for both survival of grafts and prevention of graft arteriosclerosis. Conclusions—rAAV/HO-1 gene transfer represents a novel therapeutic approach to prevent chronic allograft deterioration in clinical heart transplantation.

Brain-Derived Neurotrophic Factor Promotes Tumorigenesis via Induction of Neovascularization: Implication in Hepatocellular Carcinoma

Purpose: Brain-derived neurotrophic factor (BDNF) has emerged as a novel angiogenic factor, and yet its impact on tumorigenesis is unclear. This study aimed at investigating the roles of BDNF in angiogenesis and tumor development. Experimental Design: BDNF was overexpressed in a mouse endothelial cell (EC) line by stable transfection, and angiogenic properties of the transfectants were assessed. Microarray analysis was employed to explore the molecular pathways. The impact of modulating BDNF levels in two mouse EC lines on tumorigenic potential of a transformed mouse liver cell line was evaluated by an in vivo cotransplantation model. BDNF and tropomyosin receptor kinase B (TrkB) protein levels were determined in 50 pairs of human hepatocellular carcinoma (HCC) tissues by Western blotting and immunohistochemistry. Survival analysis was carried out to determine their clinical significance. Results: Overexpression of BDNF could promote EC proliferation, migration, invasion, and survival. Microarray and molecular studies showed that RhoA, caspase-9, caspase-3, growth arrest specific 6, and VEGF could mediate BDNF/TrkB-induced angiogenesis. The cotransplantation experiment showed that high BDNF-expressing ECs could facilitate tumor angiogenesis and growth, whereas knockdown of BDNF by short hairpin RNAs impaired such effects. Furthermore, examination on human HCC tissues revealed upregulation of BDNF and TrkB protein levels in 46.0% and 33.3% of the cases studied, respectively. Immunohistochemistry disclosed strong BDNF reactivity in both tumor and endothelial cells. High TrkB expression was associated with shorter overall survival. Conclusions: BDNF/TrkB system was crucial for tumor angiogenesis and growth, which may represent a potential target for antiangiogenic therapy in HCC. Clin Cancer Res; 17(10); 3123–33. ©2011 AACR.

Insulin in University of Wisconsin solution exacerbates the ischemic injury and decreases the graft survival rate in rat liver transplantation.

Background. Insulin keeps the liver in a metabolically vigorous state. However, organ preservation aims to decrease the metabolic rate. The objective of this study was to clarify the effect of insulin used in University of Wisconsin (UW) preservation solution on the liver graft. Methods. The liver grafts were preserved by UW solution with or without insulin for 7, 9, and 24 hr, respectively. The influence of insulin was studied by 7‐day survival rate, liver function, morphology, and intragraft gene expression 24 hr after transplantation. Morphology was studied on the preserved grafts. Results. The morphology of the graft in the insulin group showed more severe ischemia‐reperfusion injury. The 7‐day graft survival rates of the 7‐hr subgroups with and without insulin were 55% and 93%, respectively (P = 0.02). In the 9‐hr subgroups, the survival rates were 0% and 78%, respectively (P =0.002). The serum levels of aspartate aminotransferase (AST) (P =0.008) and alanine aminotransferase (ALT) (P =0.032) were higher in the 7‐hr subgroup with insulin. The same trend was found in the 9‐hr subgroups (AST, P =0.016; ALT, P =0.016). The expression level of 215 genes were much lower at 24 hr after transplantation in the grafts preserved with insulin than in those preserved without insulin, and most of the genes were related to metabolic activities. Conclusions. Insulin in UW solution may exacerbate graft ischemic injury and decrease the graft survival rate in rat liver transplantation. Insulin, in the absence of glucose in UW solution, may exhaust the metabolic activity of the liver graft. It is harmful rather than helpful for isolated rat liver grafts preserved in UW solution.

A cell penetrating heme oxygenase protein protects heart graft against ischemia/reperfusion injury

Ischemia/reperfusion (I/R) injury is an unavoidable barrier that significantly affects outcome of solid organ transplantation. Here, we establish a protein transduction system to extend graft preservation time and to prevent I/R injury in heart transplantation. We generated a recombinant heme oxygenase-1 (HO-1) protein containing a modified protein transduction domain (PTD). PTD could cross cover cell membrane and carry target molecule to parenchymal cells of cold-preserved heart grafts. The newly generated PTD-HO-1 protein localized mainly in subcellular membrane organelle and nucleus after delivery that significantly prolonged cold preservation of heart grafts. This effect was associated with significantly less endothelial cell activation, less neutrophil and macrophage infiltration in PTD-HO-1-transduced heart grafts after reperfusion as compared with controls. In addition, transduction of PTD-HO-1 protein to heart graft significantly suppressed the I/R injury-associated myocardiocyte apoptosis. The infarct areas of heart graft after I/R injury were significantly reduced after PTD-HO-1 protein treatment. We show here for the first time that PTD can maintain its biological activities during cold preservation. Transduction of cell penetrating HO-1 protein significantly prolongs the cold preservation time and protects the graft from the I/R injury. This approach represents a novel method for the improvement of the overall outcome of organ transplantation.

Targeting of interleukin-10 is superior to cytotoxic T-lymphocyte associated antigen 4 with human immunoglobulin G 1 for the prevention of chronic allograft deterioration in organ transplantation

Genetic manipulation of the allograft is an attractive approach to prevent the graft against chronic deterioration through stable expression of immunomodulatory or protective genes. However, the best strategy for prevention of chronic allograft deterioration remains unclear.

Abstract 2554: Synergistic antitumor effect of AUY922 in combination with sorafenib in hepatocellular carcinoma cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnPURPOSE: Molecularly targeted therapy is of particular importance for HCC due to a lack of efficacy with currently approved chemo-toxic therapies. It is of interest to investigate the combined effect of two molecular targeted drugs, sorafenib (a multi-kinase inhibitor with anti-angiogenic, pro-apoptotic and Raf kinase inhibitory activity) and a highly potent non-geldamycin HSP90 inhibitor, NVP-AUY922 on HCC treatment. EXPERIMENTAL DESIGNS: Cell viability after different treatments was measured by both 3,[4,5-dimethylthiazol-2-yl] -2,5-diphenyl-tetrazolium bromide (MTT) assay and cytofluorometric annexin-V apoptotic assay to compare the apoptotic cells induced by single versus combined treatment. Cell cycle analysis was also performed, and Western blotting was employed to determine the effects on MEK signaling, and in vivo efficacy was determined in nude mice with PLC/PRF/5 xenografts. RESULTS: Combined drugs treatment produced a synergistic effect on decreased HCC cell viability shown by MTT assay. An increase in the number of apoptotic cells were observed by apoptotic assay in combined drugs group when compared with single agents sorafenib or NVP-AUY922. Also, sorafenib combined with NVP-AUY922 induced S-phase arrest which was demonstrated by cell cycle analysis. Sorafenib alone decreases phospho-Erk1/2 expression, and this effect was further enhanced in combination treatment with NVP-AUY922. Finally, sorafenib plus NVP-AUY922 significantly suppressed PLC/PRF/5 xenograft tumor growth when compared with single (NVP-AUY922 or Sorafenib) treatment alone. CONCLUSION: The combination therapy of sorafenib and NVP-AUY922 can be a new and promising therapeutic approach to the treatment of HCC.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2554. doi:10.1158/1538-7445.AM2011-2554

Abstract 5377: Disruption of akt/hif1 signaling can enhance the therapeutic efficacy of ischemic hypoxia and chemotherapy

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnPURPOSE: This study investigates the possible molecular basis leading to failure in a treatment that is composed of hypoxia and chemotherapy in a rat orthotopic hepatoma model. EXPERIMENTAL DESIGNS: Cell viability after cisplatin treatments under normoxia and hypoxia would be measured by 3,[4,5-dimethylthiazol-2-yl] −2,5-diphenyl-tetrazolium bromide (MTT) assay and cytofluorometric annexin-V apoptotic assay would be used to compare the apoptotic cells induced by cisplatin under normoxia and hypoxia. Western blotting would be employed to determine the akt/hif1 signaling. In vivo hypoxic condition was induced by hepatic artery ligation, whereas chemotherapeutic effect was achieved by intraportal injection of cisplatin. Hif1 inhibitor was administered to blockade Hif-1α expression both in vitro and in vivo. RESULTS: Cell viability was higher under hypoxic than normoxic conditions shown by MTT assay and decreased apoptotic cells could be observed by apoptotic assay under hypoxia. Hif-1α and akt were regulated each other under hypoxic conditions to confer cisplatin resistance in vitro. In an rat orthotopic hepatoma model, combining blockade of Hif-1α activity with ischemic hypoxia significantly enhanced the efficacy of chemotherapy, leading to suppression of tumor growth and prolongation of animal survival. CONCLUSION: Combined Hif1 inhibitor with hypoxia and chemotherapy can be a new and promising therapeutic approach to the treatment of HCC.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5377.