Satoshi Nagano

Local overexpression of HB-EGF exacerbates remodeling following myocardial infarction by activating noncardiomyocytes

Insulin-like growth factor (IGF), hepatocyte growth factor (HGF), and heparin-binding epidermal growth factor-like growth factor (HB-EGF) are cardiogenic and cardiohypertrophic growth factors. Although the therapeutic effects of IGF and HGF have been well demonstrated in injured hearts, it is uncertain whether natural upregulation of HB-EGF after myocardial infarction (MI) plays a beneficial or pathological role in the process of remodeling. To answer this question, we conducted adenoviral HB-EGF gene transduction in in vitro and in vivo injured heart models, allowing us to highlight and explore the HB-EGF-induced phenotypes. Overexpressed HB-EGF had no cytoprotective or additive death-inducible effect on Fas-induced apoptosis or oxidative stress injury in primary cultured mouse cardiomyocytes, although it significantly induced hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. Locally overexpressed HB-EGF in the MI border area in rabbit hearts did not improve cardiac function or exhibit an angiogenic effect, and instead exacerbated remodeling at the subacute and chronic stages post-MI. Namely, it elevated the levels of apoptosis, fibrosis, and the accumulation of myofibroblasts and macrophages in the MI area, in addition to inducing left ventricular hypertrophy. Thus, upregulated HB-EGF plays a pathophysiological role in injured hearts in contrast to the therapeutic roles of IGF and HGF. These results imply that regulation of HB-EGF may be a therapeutic target for treating cardiac hypertrophy and fibrosis.

An efficient construction of conditionally replicating adenoviruses that target tumor cells with multiple factors

Despite the enormous potential of conditionally replicating adenoviruses (CRAs), the time-consuming and laborious methods required to construct CRAs have hampered both the development of CRAs that can specifically target tumors with multiple factors (m-CRA) and the efficient analysis of diverse candidate CRAs. Here, we present a novel method for efficiently constructing diverse m-CRAs. Elements involving viral replication, therapeutic genes, and adenoviral backbones were separately introduced into three plasmids of P1, P2, and P3, respectively, which comprised different antibiotic resistant genes, different ori, and a single loxP (H) sequence. Independently constructed plasmids were combined at 100% accuracy by transformation with originally prepared Cre and specific antibiotics in specific Escherichia coli; transfection of the resulting P1+2+3 plasmids into 293 cells efficiently generated m-CRAs. Moreover, the simultaneous generation of diverse m-CRAs was achieved at 100% accuracy by handling diverse types of P1+2 and P3. Alternatively, co-transfection of P1+3 and P2 plasmids into Cre-expressing 293 cells directly generated m-CRA with therapeutic genes. Thus, our three-plasmid system, which allows unrestricted construction and efficient fusion of individual elements, should expedite the process of generating, modifying, and testing diverse m-CRAs for the development of the ideal m-CRA for tumor therapy.

Diffuse encephaloventriculitis and substantial leukoencephalopathy after intraventricular administration of recombinant adenovirus

Abstract Objectives: The use of recombinant adenovirus as a vehicle for gene transfer into ependymal cells is a potential therapeutic tool for the treatment of various neural disorders. However, gene transfer into the ependymal cells of the ventricular wall is associated with high-level expression of the transferred gene, which declines rapidly. The purpose of this study is to understand the cause of this early decline in gene expression. Methods: Different doses of adenovirus-expressing β-galactosidase (Ad-β-gal) were injected into the lateral brain ventricle of C57BL/6 mice, and the brains were observed histologically and with magnetic resonance (MR) imaging for a month. Results: Inoculation of the lateral ventricle with more than 1×108 viral particles (2.6×106 pfu) resulted in a rapid decline ofβ -gal expression. MR imaging indicated gradual ventriculomegaly and histological analysis showed the loss of the ependymal cells from the ventricular wall, lymphocytes infiltration near the wall, degeneration of myelinated fibers and apoptosis in the external capsule. Reactive astrocytes proliferated in the external capsule 17 days following inoculation. To avoid this irreversible brain atrophy, the inoculated adenovirus should be reduced to less than 1×107 particles (2.6×105 pfu) in mice. Discussion: Our results indicate the presence of a unique and diffuse immune response of the brain; therefore, the clinical use of recombinant virus for intraventricular gene transfer must be carefully evaluated.

446. A Rapid, Efficient and Feasible Method for Constructing Conditionally-Replicating Adenoviral Vectors that Specifically Target Cancer Cells with Multiple Factors

Conditionally-replicating adenoviral vectors (CRAs) have potentials to circumvent the limitation of current vector systems, i.e., the impossibility to deliver therapeutic gene to every cancer cell in the whole body. However, previously reported CRAs have some fundamental problems. First, cancer-specificity of these CRAs is insufficient or uncertain because they are regulated by only a single or a couple of factors. Second, it is time-consuming and laborious to construct CRAs by previous systems; it is almost impossible to efficiently construct numbers of diverse CRAs having differently combined multiple factors. As a result, there has been no systematic analysis to assess virological and biological properties of numbers of diverse CRAs simultaneously in various cancer and normal cells. In this respect, neither actual therapeutic potential nor clinical utility remains uncertain even though previous reports represented a drastic phenomenon of sole CRA in limited type of cancer cell or of animal model.