Hideki Tomioka

OPG/RANKL/RANK axis is a critical inflammatory signaling system in ischemic brain in mice

Significance Although a high-serum osteoprotegerin (OPG) level is associated with an unfavorable outcome in ischemic stroke, it is unclear whether OPG is a culprit or an innocent bystander. Here we show that the deletion of OPG and enhanced RANKL/RANK signaling contribute to the reduction of infarct volume with lower brain edema, whereas infarct volume is increased by reduced RANKL/RANK signaling in OPG−/− mice and WT mice treated with anti-RANKL neutralizing antibody. OPG, RANKL, and RANK mRNA were increased in ischemic brain and were expressed in activated microglia and macrophages. Enhanced RANKL/RANK signaling showed neuroprotective effects with reduced expression in inflammatory cytokines in LPS-stimulated neuron-glia mixed culture. Our findings propose anti-inflammatory roles for RANKL/RANK signaling in ischemic brains. Osteoprotegerin (OPG) is a soluble secreted protein and a decoy receptor, which inhibits a receptor activator of nuclear factor κB (NF-κB) ligand (RANKL)/the receptor activator of NF-κB (RANK) signaling. Recent clinical studies have shown that a high-serum-OPG level is associated with unfavorable outcome in ischemic stroke, but it is unclear whether OPG is a culprit or an innocent bystander. Here we demonstrate that enhanced RANKL/RANK signaling in OPG−/− mice or recombinant RANKL-treated mice contributed to the reduction of infarct volume and brain edema via reduced postischemic inflammation. On the contrary, infarct volume was increased by reduced RANKL/RANK signaling in OPG−/− mice and WT mice treated with anti-RANKL neutralizing antibody. OPG, RANKL, and RANK mRNA were increased in the acute stage and were expressed in activated microglia and macrophages. Although enhanced RANKL/RANK signaling had no effects in glutamate, CoCl2, or H2O2-stimulated neuronal culture, enhanced RANKL/RANK signaling showed neuroprotective effects with reduced expression in inflammatory cytokines in LPS-stimulated neuron-glia mixed culture, suggesting that RANKL/RANK signaling can attenuate inflammation through a Toll-like receptor signaling pathway in microglia. Our findings propose that increased OPG could be a causal factor of reducing RANKL/RANK signaling and increasing postischemic inflammation. Thus, the OPG/RANKL/RANK axis plays critical roles in controlling inflammation in ischemic brains.

Novel anti-microbial peptide SR-0379 accelerates wound healing via the PI3 kinase/Akt/mTOR pathway.

We developed a novel cationic antimicrobial peptide, AG30/5C, which demonstrates angiogenic properties similar to those of LL-37 or PR39. However, improvement of its stability and cost efficacy are required for clinical application. Therefore, we examined the metabolites of AG30/5C, which provided the further optimized compound, SR-0379. SR-0379 enhanced the proliferation of human dermal fibroblast cells (NHDFs) via the PI3 kinase-Akt-mTOR pathway through integrin-mediated interactions. Furthermore SR-0379 promoted the tube formation of human umbilical vein endothelial cells (HUVECs) in co-culture with NHDFs. This compound also displays antimicrobial activities against a number of bacteria, including drug-resistant microbes and fungi. We evaluated the effect of SR-0379 in two different would-healing models in rats, the full-thickness defects under a diabetic condition and an acutely infected wound with full-thickness defects and inoculation with Staphylococcus aureus. Treatment with SR-0379 significantly accelerated wound healing when compared to fibroblast growth factor 2 (FGF2). The beneficial effects of SR-0379 on wound healing can be explained by enhanced angiogenesis, granulation tissue formation, proliferation of endothelial cells and fibroblasts and antimicrobial activity. These results indicate that SR-0379 may have the potential for drug development in wound repair, even under especially critical colonization conditions.

Therapeutic vaccine against DPP4 improves glucose metabolism in mice

Significance Type 2 diabetes mellitus (T2DM) has become a common disease, and long-term effective drugs have become a necessity. In recent years, dipeptidyl peptidase 4 (DPP4) inhibitors have been commercialized due to its ability to inhibit glucagon-like peptide 1 degradation, a hormone important for enhancing insulin secretion. Despite the availability of efficient drugs, the success of treatment is limited by patients’ inconsistent drug intake and the economic burden included in a lifelong treatment required for T2DM. To alleviate these limitations, in this study an affordable and effective immunotherapeutic method was developed and assessed for T2DM treatment. We selected and designed the appropriate peptide sequences that induce the anti-DPP4 antibody that effectively improves the diabetic phenotype without an adverse autoimmune response. The increasing prevalence of type 2 diabetes mellitus is associated with a significant economic burden. We developed a dipeptidyl peptidase 4 (DPP4)-targeted immune therapy to increase glucagon-like peptide 1 hormone levels and improve insulin sensitivity for the prevention and treatment of type 2 diabetes mellitus. Immunization with the DPP4 vaccine in C57BL/6J mice successfully increased DPP4 titer, inhibited plasma DPP4 activity, and induced an increase in the plasma glucagon-like peptide 1 level. Moreover, this elevated titer was sustained for 3 mo. In mice fed a high-fat diet, DPP4 vaccination resulted in improved postprandial glucose excursions and insulin sensitivity and, in the diabetic KK-Ay and db/db mice strains, DPP4 vaccination significantly reduced glucose excursions and increased both plasma insulin and pancreatic insulin content. Importantly, T cells were not activated following challenge with DPP4 itself, which suggests that this vaccine does not induce cell-mediated autoimmunity. Additionally, no significant immune-mediated damage was detected in cells and tissues where DPP4 is expressed. Thus, this DPP4 vaccine may provide a therapeutic alternative for patients with diabetes.

Development of novel DNA vaccine for VEGF in murine cancer model

We developed DNA vaccine for vascular endothelial growth factor (VEGF), which may provide the therapeutic option instead of anti-VEGF antibody, bevacizumab. Plasmid containing VEGF mini-gene was constructed in the insertion of B-cell epitope of Hepatitis B core protein [HBc-VEGF], which was an epitope carrier. High titer of anti-VEGF antibody was observed in BALB/c mice which were intramuscularly immunized with HBc-VEGF by electropolator. In mice inoculated with colon 26 cells, tumor volume and microvessel density was decreased in HBc-VEGF with a significant prolonged survival. Co-treatment of purified IgG from immunized mice with HBc-VEGF showed in vitro neutralizing activity for VEGF-induced ERK phosphorylation and tube formation in cultured endothelial cells. Furthermore, intravitreally injection of this purified IgG reduced the neovessel formation in the mouse oxygen-induced retinopathy and laser-induced choroidal neovascularization models. These results first provided that DNA vaccine against VEGF possessed the anti-angiogenic effect, leading to prolonged survival in mouse cancer model.

Long-term expression of periostin during the chronic stage of ischemic stroke in mice.

Periostin is an extracellular matrix glycoprotein and has various cellular effects. Previously, we demonstrated the neuroprotective effects of periostin during the acute stage of cerebral ischemia. However, its expression during the chronic stage remains unknown. Herein, we examined the expression of full-length periostin (periostin 1; Pn1) and its splicing variant lacking exon 17 (periostin 2; Pn2) during the 28 days following transient middle cerebral artery occlusion in mice. Real-time reverse transcription-PCR showed that the expression of Pn2 was dramatically upregulated between days 3 and 28, and the highest expression was observed on day 7. The expression of Pn1 was also increased, but delayed compared with Pn2. Immunohistochemistry showed that periostin was weakly expressed in reactive astrocytes in the peri-infarct region and in microglia/macrophages in infarct regions, on days 3 and 7. Periostin was also expressed around CD31-positive cells in both the peri-infarct and the sub-ventricular zone (SVZ) on days 3 and 7. SOX-2 positive cells, which are neural stem cells, also expressed periostin on day 7. The highest periostin immunoreactivity that occurred co-localized with collagen I and fibronectin in the peri-infarct region between days 7 and 28. Thus, the expression pattern of periostin mRNA was dependent on the splicing variant, and it continued to be expressed up to 28 days after cerebral ischemia. As periostin was expressed in various cells, such as reactive astrocytes/microglia, fibroblasts and neuronal progenitor cells, periostin might be associated with pathophysiology in post-ischemic inflammation and neurogenesis.

Inhibition of Neointima Formation through DNA Vaccination for Apolipoprotein(a): A New Therapeutic Strategy for Lipoprotein(a)

Lipoprotein(a) [Lp(a)] is an unique lipoprotein consisting of the glycoprotein apolipoprotein(a) [apo(a)] in low-density lipoprotein. Although Lp(a) is a well-known independent risk factor for cardiovascular disease; however, there is no drugs to decrease plasma Lp(a) level. Thus, to inhibit the biological activity of Lp(a), we developed DNA vaccine for apo(a) by the targeting to the selected 12 hydrophilic amino acids in the kringle-4 type 2 domain of apo(a). Hepatitis B virus core protein was used as an epitope carrier to enhance the immunogenicity. Intramuscular immunization with apo(a) vaccine resulted in the significant inhibition of neointima formation in carotid artery ligation model using Lp(a) transgenic mice, associated with anti-apo(a) antibody and decrease in vascular Lp(a) deposition. Overall, this study provided the first evidence that the pro-atherosclerotic actions of Lp(a) could be prevented by DNA vaccine directed against apo(a), suggesting a novel therapeutic strategy to treat cardiovascular diseases related to high Lp(a).

Beperminogene perplasmid for the treatment of critical limb ischemia

Therapeutic angiogenesis for the treatment of ischemic disease can be attained through the delivery of recombinant growth factor proteins, through gene transfer or cell transplantation. Gene transfer associated with adenovirus or naked plasmid DNAs has been extensively studied in clinical trials. An investigational product, beperminogene perplasmid, is the naked plasmid DNA encoding the cDNA of human HGF, which has potent angiogenic activity. In several clinical trials, beperminogene perplasmid showed favorable safety and efficacy profile in the treatment of critical limb ischemia. This article reviews the results of pre-clinical and clinical studies of beperminogene perplasmid in the treatment of critical limb ischemia caused by peripheral arterial disease and Buerger’s disease.