Akito Maeda

Mesenchymal stem cells (MSCs) are capable of differentiating into a limited number of diverse cells and secrete regenerative factors that contribute to the repair of damaged tissue. In response to signals emitted by tissue damage, MSCs migrate from the bone marrow and area surrounding blood vessels within tissues into the circulating blood, and accumulate at the site of damage. Hence, MSC transplantation therapy is beginning to be applied to the treatment of various intractable human diseases. Recent medicinal plants studies have shown that plant-derived components can activate cell functions. For example, several plant-derived components activate cell signaling pathways, such as phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK), enhance expression of the CXCL12/CXCR4 axis, stimulate extracellular matrix remodeling, and consequently, promote cell migration of MSCs. Moreover, plant-derived components have been shown to promote recruitment of MSCs to damaged tissues and enhance healing in disease models, potentially advancing their therapeutic use. This article provides a comprehensive review of several plant-derived components that activate MSC migration and homing to damaged sites to promote tissue repair.

Substances that enhance the migration of mesenchymal stem cells to damaged sites have the potential to improve the effectiveness of tissue repair. We previously found that ethanol extracts of Mallotus philippinensis bark promoted migration of mesenchymal stem cells and improved wound healing in a mouse model. We also demonstrated that bark extracts contain cinnamtannin B-1, a flavonoid with in vitro migratory activity against mesenchymal stem cells. However, the in vivo effects of cinnamtannin B-1 on the migration of mesenchymal stem cells and underlying mechanism of this action remain unknown. Therefore, we examined the effects of cinnamtannin B-1 on in vivo migration of mesenchymal stem cells and wound healing in mice. In addition, we characterized cinnamtannin B-1-induced migration of mesenchymal stem cells pharmacologically and structurally. The mobilization of endogenous mesenchymal stem cells into the blood circulation was enhanced in cinnamtannin B-1-treated mice as shown by flow cytometric analysis of peripheral blood cells. Whole animal imaging analysis using luciferase-expressing mesenchymal stem cells as a tracer revealed that cinnamtannin B-1 increased the homing of mesenchymal stem cells to wounds and accelerated healing in a diabetic mouse model. Additionally, the cinnamtannin B-1-induced migration of mesenchymal stem cells was pharmacologically susceptible to inhibitors of phosphatidylinositol 3-kinase, phospholipase C, lipoxygenase, and purines. Furthermore, biflavonoids with similar structural features to cinnamtannin B-1 also augmented the migration of mesenchymal stem cells by similar pharmacological mechanisms. These results demonstrate that cinnamtannin B-1 promoted mesenchymal stem cell migration in vivo and improved wound healing in mice. Furthermore, the results reveal that cinnamtannin B-1-induced migration of mesenchymal stem cells may be mediated by specific signaling pathways, and the flavonoid skeleton may be relevant to its effects on mesenchymal stem cell migration.

The γ‐secretase complex (which contains presenilins, nicastrin, anterior pharynx defective‐1, and presenilin enhancer‐2) cleaves type I transmembrane proteins, including Notch and amyloid precursor protein. Dysregulated γ‐secretase activity has been implicated in the pathogenesis of Alzheimer's disease, stroke, atherosclerosis, and cancer. Tight regulation of γ‐secretase activity is required for normal physiology. Here, we isolated HIG1 (hypoxia inducible gene 1, domain member 1A) from a functional screen of γ‐secretase inhibitory genes. HIG1 was highly expressed in the brain. Interestingly, HIG1 was localized to the mitochondria and was directly bound to γ‐secretase components on the mitochondrial membrane in SK‐N‐SH neuroblastoma cells. Overexpresssion of HIG1 attenuated hypoxia‐induced γ‐secretase activation on the mitochondrial membrane and the accumulation of intracellular amyloid β. This accumulation was accompanied by hypoxia‐induced mitochondrial dysfunction. The latter half domain of HIG1 was required for binding to the γ‐secretase complex and suppression of γ‐secretase activity. Moreover, depletion of HIG1 increased γ‐secretase activation and enhanced hypoxia‐induced mitochondrial dysfunction. In summary, HIG1 is a novel modulator of the mitochondrial γ‐secretase complex, and may play a role in the maintenance of normal mitochondrial function.—Hayashi, H., Nakagami, H., Takeichi, M., Shimamura, M., Koibuchi, N., Oiki, E., Sato, N., Koriyama, H., Mori, M., Gerardo Araujo, R., Maeda, A., Morishita, R., Tamai, K., Kaneda, Y. HIG1, a novel regulator of mitochondrial γ‐secretase, maintains normal mitochondrial function. FASEB J. 26, 2306‐2317 (2012). www.fasebj.org

We previously identified a novel angiogenic peptide, AG30, with antibacterial effects that could serve as a foundation molecule for the design of wound‐healing drugs. Toward clinical application, in this study we have developed a modified version of the AG30 peptide characterized by improved antibacterial and angiogenic action, thus establishing a lead compound for a feasibility study. Because AG30 has an α‐helix structure with a number of hydrophobic and cationic amino acids, we designed a modified AG30 peptide by replacing several of the amino acids. The replacement of cationic amino acids (yielding a new molecule, AG30/5C), but not hydrophobic amino acids, increased both the angiogenic and the antimicrobial properties of the peptide. AG30/5C was also effective against methicillin‐resistant Staphylococcus aureus (MRSA) and antibiotic‐resistant Pseudomonas aeruginosa. In a diabetic mouse wound‐healing model, the topical application of AG30/5C accelerated wound healing with increased angiogenesis and attenuated MRSA infection. To facilitate the eventual clinical investigation/application of these compounds, we developed a large‐scale procedure for the synthesis of AG30/5C that employed the conventional solution method and met Good Manufacturing Practice guidelines. In the evaluation of stability of this peptide in saline solution, RP‐HPLC analysis revealed that AG30/5C was fairly stable under 5°C for 12 months. Therefore, we propose the use of AG30/5C as a wound‐healing drug with antibacterial and angiogenic actions.

The utility of various synthetic peptides has been investigated in clinical trials of the treatment of cancers, infectious diseases and endocrine diseases. In the process of functional gene screening with in silico analysis for molecules with angiogenic properties, we generated a small peptide, angiogenic peptide (AG)‐30, that possesses both antimicrobial and pro‐inflammatory activities. AG‐30 has an α‐helix structure with a number of hydrophobic or net positively charged amino acids and a propensity to fold into amphipathic structures. Indeed, AG‐30 exhibited antimicrobial activity against various bacteria, induced vascular endothelial cell growth and tube formation in a dose‐dependent manner and increased neovascularization in a Matrigel plug assay. As a result, AG‐30 up‐regulated expression of angiogenesis‐related cytokines and growth factors for up to 72 hrs in human aortic endothelial cells. To further evaluate the angiogenic effect of AG‐30 in vivo, we developed a slow‐release AG‐30 system utilizing biodegradable gelatin microspheres. In the ischaemic mouse hind limb, slow‐release AG‐30 treatment results in an increase in angiogenic score, an increase in blood flow (as demonstrated by laser Doppler imaging) and an increase in capillary density (as demonstrated by immunostaining with anti‐CD31 antibody). These data suggest that the novel peptide, AG‐30, may have therapeutic potential for ischaemic diseases.

Isolation of effective therapeutic genes is critical for the advancement of gene therapy for various diseases, including vascular diseases and cancers. The goal of the present study was to screen a human cDNA library, using a hemagglutinating virus of Japan envelope (HVJ-E) vector, to isolate candidate genes with potent therapeutic potential. The advantages of a high-throughput functional screening system based on the HVJ-E vector include (1) rapid preparation of the vector containing the DNA library, (2) effective fusion-mediated transfer of the plasmids to various cells with minimal toxicity, and (3) easy cloning of candidate genes by transformation of Escherichia coli. These advantages resulted in a lower probability of damage to isolated clones and in minimization of the time needed to screen for candidate genes. Screening of a human heart library for candidate genes to regulate endothelial cell growth identified three growth-stimulating genes, as evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and c-fos promoter activity, the products of which were more potent than vascular endothelial growth factor. Similarly, two growth-inhibiting genes were identified, the effects of which were similar to angiostatin. Overall, this novel system will help advance our understanding of cell biology and promote the utility of human gene therapy.

The small GTPase Rap1 is a potent activator of leukocyte integrin. However, the regulatory mechanism involved is unknown. Here, we identify the Rap1 effector, RAPL, as an essential regulator in this activation. RAPL was enriched in mouse lymphoid tissues and associated with Rap1 after stimulation by the T cell receptor and with chemokine CXCL12. Human RAPL stimulated lymphocyte polarization and the patch-like redistribution of lymphocyte-function-associated antigen 1 (LFA-1) to the leading edge, resulting in enhanced adhesion to intercellular adhesion molecule 1 (ICAM-1). Triggered by activated Rap1, RAPL associated with LFA-1 and rapidly relocated to the leading edge and accumulated at immunological synapses. Thus, RAPL regulates lymphocyte adhesion through the spatial distribution of LFA-1.