J Mol Cell Cardiol. 2015 Jul;84:45-51.
Wnt11 gene therapy with adeno-associated virus 9 improves the survival of mice with myocarditis induced by coxsackievirus B3 through the suppression of the inflammatory reaction.
Yutaka Aoyama, M.D.; Koichi Kobayashi, M.D., Ph.D.; Yoshihiro Morishita, M.D.; Kengo Maeda, M.D., Ph.D.; Toyoaki Murohara, M.D., Ph.D.
Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya 466, Japan
The wnt signaling pathway plays important roles in development and in many diseases. Recently several reports suggest that non-canonical Wnt proteins contribute to the inflammatory response in adult animals. However, the effects of Wnt proteins on virus-induced myocarditis have not been explored. Here, we investigated the effect of Wnt11 protein in a model of myocarditis induced by coxsackie virus B3 (CVB3) using recombinant adeno-associated virus 9 (rAAV9). The effect of Wnt11 gene therapy on a CVB3-induced myocarditis model was examined using male BALB/c mice. Mice received a single intravenous injection of either rAAV9-Wnt11 or rAAV9-LacZ two weeks before intraperitoneal administration of CVB3. Intravenous injection of the rAAV9 vector resulted in efficient, durable, and relatively cardiac-specific transgene expression. Survival was significantly greater among rAAV9-Wnt11treated mice than among mice treated with rAAV9-LacZ (87.5% vs. 54.1%, P < 0.05). Wnt11 expression also reduced the infiltration of inflammatory cells, necrosis of the myocardium, and suppressed the mRNA expression of inflammatory cytokines. This is the first report to show that Wnt11 expression improves the survival of mice with CVB3-induced myocarditis. AAV9-mediated Wnt11 gene therapy produces beneficial effects on cardiac function and increases the survival of mice with CVB3-induced myocarditis through the suppression of both infiltration of inflammatory cells and gene expression of inflammatory cytokines.
Recombinant adeno-associated virus has become increasing common as a vector for use in human clinical trials. The popularity of AAV vectors reflects the appreciation of the long-term gene expression in post mitotic cells and the relative lack of deleterious immune response. More than 40 clinical trials with rAAVs have been approved with no serious adverse events attributable to the vector system. rAAV9 has a high natural affinity for myocardium. Our results confirmed that rAAV9-mediated gene delivery is efficient, cardiac-specific, and durable: gene expression was robust in the heart, much lower in the liver, skeletal muscle and kidney. These data show that rAAV9 is suitable vector for delivering genes to the heart.
The Wnt glycolipoproteins are best characterized as regulators of cell proliferation, cell polarity, and cell-fate determination during embryonic development. Within the immune system, the canonical Wnt-b-catenin signaling appears to have an important role in the self-renewal of hematopoietic stem cells and progenitor cells, in T- and B-cell development, in peripheral T-cell activation, and in the maturation of dendritic cells, whereas non-canonical signaling by Wnt5a may counteract canonical Wnt signaling to inhibit B- and T-cell development. Wnt5a also regulates the inflammatory response by modulating interleukin production in monocytes and macrophages. Additionally, Wnt11, which regulates the development of heart and kidney through the non-canonical Wnt signaling passway, suppresses inflammation in intestinal epithelial cells. Our data also showed that Wnt11 signaling suppressed inflammation of coxsackievirus B3 (CVB3)-induced myocarditis and improved survival of myocarditis mice. Moreover, we recently reported that Wnt11 gene therapy improved recovery from myocardial infarction by suppressing the inflammatory response. In this manuscript, we showed Wnt11 condition medium decreased inflammatory cytokines from stimulated Raw cells (macrophage cell line) through the suppression of NF-kB transcriptional factor. Therapies that modulate Wnt11 signaling could be effective therapeutic modality for myocarditis and other diseases that are accompanied by inflammation.
The importance of this study.
The rAAV9 system is a promising method for delivering genes to the heart, and our data reveal a novel function of Wnt11 in the regulation of inflammatory response and provide a rationale for the use of Wnt11 to manipulate human diseases that are mediated by inflammation.
Figure. 3.0 × 1011 GC of AAV9-LacZ expression vector was injected into the tail vein of BALB/c WT mice. LacZ expression was identified in the hearts of mice sacrificed 4 weeks after treatment by 3.0 × 1011 GC of rAAV9-LacZ via X-gal staining
- Wnt11 gene therapy with adeno-associated virus 9 improves the survival of mice with myocarditis induced by coxsackievirus B3 through the suppression of the inflammatory reaction. Aoyama Y, Kobayashi K, Morishita Y, Maeda K, Murohara T. J Mol Cell Cardiol. 2015 Jul;84:45-51.
- Wnt11 Gene Therapy with Adeno-associated Virus 9 Improves Recovery from Myocardial Infarction by Modulating the Inflammatory Response. Morishita Y, Kobayashi K, Klyachko E, Jujo K, Maeda K, Losordo DW, Murohara T. Sci Rep. 2016 Feb 17;6:21705.
We thank Yoko Inoue for her excellent technical assistance, Hiroyuki Nakai (Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, USA) for scientific advice to produce the adeno-associated virus 9, and the University of Pennsylvania Vector Core for providing us with AAV2/9 capsid plasmid. Coxsackie virus B3 (Nancy strain) was a kind gift from Dr. Yoshinori Seko (Division of Cardiovascular Medicine, Institute for Adult Diseases, Asahi Life Foundation, Tokyo, Japan).