Goichi Matsumoto

Cationized gelatin delivery of a plasmid DNA expressing small interference RNA for VEGF inhibits murine squamous cell carcinoma

Double‐stranded RNA (dsRNA) plays a major role in RNA interference (RNAi), a process in which segments of dsRNA are initially cleaved by the Dicer into shorter segments (21–23 nt) called small interfering RNA (siRNA). These siRNA then specifically target homologous mRNA molecules causing them to be degraded by cellular ribonucleases. RNAi downregulates endogenous gene expression in mammalian cells. Vascular endothelial growth factor (VEGF) is a key molecule in vasculogenesis as well as in angiogenesis. Tumor growth is an angiogenesis‐dependent process, and therapeutic strategies aimed at inhibiting angiogenesis are theoretically attractive. To investigate the feasibility of using siRNA for VEGF in the specific knockdown of VEGF mRNA, thereby inhibiting angiogenesis, we have performed experiments with a DNA vector based on a siRNA system that targets VEGF (siVEGF). It almost completely inhibited the expression of three different isoforms (VEGF120, VEGF164 and VEGF188) of VEGF mRNA and the secretion of VEGF protein in mouse squamous cell carcinoma NRS‐1 cells. The siVEGF released from cationized gelatin microspheres suppressed tumor growth in vivo. A marked reduction in vascularity accompanied the inhibition of a siVEGF‐transfected tumor. Fluorescent microscopic study showed that the complex of siVEGF with cationized gelatin microspheres was still present around the tumor 10 days after injection, while free siVEGF had vanished by that time. siVEGF gene therapy increased the fraction of vessels covered by pericytes and induced expression of angiopoietin‐1 by pericytes. These data suggest that cationized‐gelatin microspheres containing siVEGF can be used to normalize tumor vasculature and inhibit tumor growth in a NRS‐1 squamous cell carcinoma xenograft model. (Cancer Sci 2006; 97: 313 – 321)

Essential Role of LFA-1 in Activating Th2-Like Responses by α-Galactosylceramide-Activated NKT Cells

NKT cells produce large amounts of cytokines associated with both the Th1 (IFN-γ) and Th2 (IL-4) responses following stimulation of their invariant Vα14 Ag receptor. The role of adhesion molecules in the activation of NKT cells by the Vα14 ligand α-galactosylceramide (α-GalCer) remains unclear. To address this issue, LFA-1−/− (CD11a−/−) mice were used to investigate IL-4 and IFN-γ production by NKT cells following α-GalCer stimulation. Intriguingly, LFA-1−/− mice showed increased IL-4, IL-5, and IL-13 production and polarized Th2-type responses in response to α-GalCer in vitro and in vivo. Furthermore, the Th2-specific transcription factor GATA-3 was up-regulated in α-GalCer-activated NKT cells from LFA-1−/− mice. These results provide the first genetic evidence that the adhesion receptor LFA-1 has a crucial role in Th2-polarizing functions of NKT cells.

Enhanced Regeneration of Critical Bone Defects Using a Biodegradable Gelatin Sponge and β-Tricalcium Phosphate with Bone Morphogenetic Protein-2

We examine the osteogenicity of a sponge biomaterial consisting of a biodegradable mixture of gelatin and β-tricalcium phosphate (βTCP) that bound bone morphogenetic protein 2 (BMP-2) in critical-sized bone defects in rats. Gelatin-βTCP sponges containing either phosphate buffered saline or incorporating BMP-2 are implanted into 5 mm diameter bone defects created in rat mandibles. We assess the defects biweekly for 8 weeks following implantation. There is significantly higher osteoinductive activity and significantly more Gla-osteocalcin content at bone-defect healing sites treated with gelatin-βTCP sponges incorporating BMP-2 than there is in those treated with sponges that did not contain BMP-2. Histologically, new bone that contains bone marrow and that is connected to the original bone almost entirely replaces the regenerated bone. These results show that biodegradable gelatin-βTCP incorporating BMP-2 is osteogenic enough to promote healing in large bone defects.

Cold shock domain protein A (CSDA) overexpression inhibits tumor growth and lymph node metastasis in a mouse model of squamous cell carcinoma

Cancer cells metastasize by entering the lymphatic system. Regional lymph-node dissemination is the first detectable step in the metastasis of oral squamous cell carcinoma (SCC) and is highly correlated to the prognosis of the disease. Cold shock domain protein A (CSDA) is a DNA-binding protein that represses angiogenesis and lymphangiogenesis by directly binding to hypoxia response element (HRE) and serum response element (SRE). In our study we used the cell line NR-S1M, a mouse SCC model with a high rate of lymph-node metastasis. Into these cells we transfected the expression-plasmid coding for full-length mouse CSDA. Of importance, we showed that overexpression of CSDA significantly inhibits the production of VEGF-A and VEGF-C in NR-S1M cells. The overexpression of CSDA in NR-S1M cells inhibited tumor growth, inhibited regional lymph-node metastasis, and reduced the density of blood vessels and lymphatic vessels in the primary tumors in vivo. Our results support the hypothesis that VEGF-A and VEGF-C are crucial regulators of angiogenesis and lymphangiogenesis in NR-S1M cells. Therefore, they are promising targets for CSDA overexpression gene therapy to inhibit tumor growth and lymph-node metastasis in SCC.

Bone regeneration by polyhedral microcrystals from silkworm virus.

Bombyx mori cypovirus is a major pathogen which causes significant losses in silkworm cocoon harvests because the virus particles are embedded in micrometer-sized protein crystals called polyhedra and can remain infectious in harsh environmental conditions for years. But the remarkable stability of polyhedra can be applied on slow-release carriers of cytokines for tissue engineering. Here we show the complete healing in critical-sized bone defects by bone morphogenetic protein-2 (BMP-2) encapsulated polyhedra. Although absorbable collagen sponge (ACS) safely and effectively delivers recombinant human BMP-2 (rhBMP-2) into healing tissue, the current therapeutic regimens release rhBMP-2 at an initially high rate after which the rate declines rapidly. ACS impregnated with BMP-2 polyhedra had enough osteogenic activity to promote complete healing in critical-sized bone defects, but ACS with a high dose of rhBMP-2 showed incomplete bone healing, indicating that polyhedral microcrystals containing BMP-2 promise to advance the state of the art of bone healing.

Gelatin powders accelerate the resorption of calcium phosphate cement and improve healing in the alveolar ridge.

The aim of this study was to show the effectiveness of combining calcium phosphate cement and gelatin powders to promote bone regeneration in the canine mandible. We mixed gelatin powders with calcium phosphate cement to create a macroporous composite. In four beagle dogs, two saddle-type bone defects were created on each side of the mandible, and calcium phosphate cement alone or calcium phosphate cement containing composite gelatin powders was implanted in each of the defects. After a healing period of six months, mandibles were removed for µCT and histological analyses. The µCT and histological analyses showed that at experimental sites at which calcium phosphate cement alone had been placed new bone had formed only around the periphery of the residual calcium phosphate cement and that there had been little or no ingrowth into the calcium phosphate cement. On the other hand, at experimental sites at which calcium phosphate cement containing composite gelatin powders had been placed, we observed regenerated new bone in the interior of the residual calcium phosphate cement as well as around its periphery. The amount of resorption of calcium phosphate cement and bone regeneration depended on the mixing ratio of gelatin powders to calcium phosphate cement. New bone replacement was significantly better in the sites treated with calcium phosphate cement containing composite gelatin powders than in those treated with calcium phosphate cement alone.

Control of angiogenesis by VEGF and endostatin-encapsulated protein microcrystals and inhibition of tumor angiogenesis

Encapsulation of cytokines within protein microcrystals (polyhedra) is a promising approach for the stabilization and delivery of therapeutic proteins. Here, we investigate the influence of vascular endothelial growth factor (VEGF) microcrystals and endostatin microcrystals on angiogenesis. VEGF was successfully encapsulated into microcrystals derived from insect cypovirus with overexpression of protein disulfide bond isomerase. VEGF microcrystals were observed to increase the phosphorylation of p42/p44 MAP kinase and to stimulate the proliferation, migration, and network and tube formation of human umbilical vein endothelial cells (HUVECs). Endostatin was also successfully encapsulated into microcrystals. Endostatin microcrystals showed antiangiogenesis activities and inhibited the migration, and network and tube formation of HUVECs. Local administration of endostatin microcrystals in mice inhibited both angiogenesis and tumor growth with clear significant differences between treatment and control groups. Endostatin microcrystals only affected angiogenesis, but had no significant effect on lymphangiogenesis compared to controls. Local therapy using endostatin microcrystals offers a potential approach to achieve sustained therapeutic release of antiangiogenic molecules for cancer treatment.

Polyhedral microcrystals encapsulating bone morphogenetic protein 2 improve healing in the alveolar ridge

Atelocollagen sponges incorporating polyhedra encapsulating bone morphogenetic protein 2 (BMP-2) were implanted into lateral bone defects in the mandible. Half of the bone defects on the left side were treated with atelocollagen sponges containing 1.8 × 107 BMP-2 polyhedra, and half were treated with sponges containing 3.6 × 106 BMP-2 polyhedra. As controls, we treated the right-side bone defects in each animal with an atelocollagen sponge containing 5 µg of recombinant human BMP-2 (rhBMP-2) or 1.8 × 107 empty polyhedral. After a healing period of six months, whole mandibles were removed for micro-computed tomography (CT) and histological analyses. Micro-CT images showed that more bone had formed at all experimental sites than at control sites. However, the density of the new bone was not significantly higher at sites with an atelocollagen sponge containing BMP-2 polyhedra than at sites with an atelocollagen sponge containing rhBMP-2 or empty polyhedra. Histological examination confirmed that the BMP-2 polyhedra almost entirely replaced the atelocollagen sponges and connected the original bone with the regenerated bone. These results show that the BMP-2 delivery system facilitates the regeneration of new bone in the mandibular alveolar bone ridge and has an advance in the technology of bone regeneration for implant site development.

Osteochondroma on the Coronoid Process of the Mandible

Abstract A 38-year-old man with severely limited mouth opening caused by unilateral coronoid process enlargement due to osteochondroma is reported.

3D co-cultures of keratinocytes and melanocytes and cytoprotective effects on keratinocytes against reactive oxygen species by insect virus-derived protein microcrystals.

Stable protein microcrystals called polyhedra are produced by certain insect viruses. Cytokines, such as fibroblast growth factors (FGFs), can be immobilized within polyhedra. Here, we investigated three-dimensional (3D) co-cultures of keratinocytes and melanocytes on collagen gel containing FGF-2 and FGF-7 polyhedra. Melanocytes were observed to reside at the base of the 3D cell culture and melanin was also typically observed in the lower layer. The 3D cell culture model with FGF-2 and FGF-7 polyhedra was a useful in vitro model of the epidermis due to effective melanogenesis, proliferation and differentiation of keratinocytes. FGF-7 polyhedra showed a potent cytoprotective effect when keratinocytes were treated with menadione, which is a generator of reactive oxygen species. The cytoprotective effect was activated by the inositol triphosphate kinase-Akt pathway leading to upregulation of the antioxidant enzymes superoxide dismutase and peroxiredoxin 6.