Eiko Iwata

DOCK2 Is Essential for Antigen-Induced Translocation of TCR and Lipid Rafts, but Not PKC-θ and LFA-1, in T Cells

DOCK2 is a mammalian homolog of Caenorhabditis elegans CED-5 and Drosophila melanogaster Myoblast City which are known to regulate actin cytoskeleton. DOCK2 is critical for lymphocyte migration, yet the role of DOCK2 in TCR signaling remains unclear. We show here that DOCK2 is essential for TCR-mediated Rac activation and immunological synapse formation. In DOCK2-deficient T cells, antigen-induced translocation of TCR and lipid rafts, but not PKC-theta and LFA-1, to the APC interface was severely impaired, resulting in a significant reduction of antigen-specific T cell proliferation. In addition, we found that the efficacy of both positive and negative selection was reduced in DOCK2-deficient mice. These results suggest that DOCK2 regulates T cell responsiveness through remodeling of actin cytoskeleton via Rac activation.

Nanoparticle-Mediated Delivery of Nuclear Factor κB Decoy Into Lungs Ameliorates Monocrotaline-Induced Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is an intractable disease of the small pulmonary artery that involves multiple inflammatory factors. We hypothesized that a redox-sensitive transcription factor, nuclear factor &kgr;B (NF-&kgr;B), which regulates important inflammatory cytokines, plays a pivotal role in PAH. We investigated the activity of NF-&kgr;B in explanted lungs from patients with PAH and in a rat model of PAH. We also examined a nanotechnology-based therapeutic intervention in the rat model. Immunohistochemistry results indicated that the activity of NF-&kgr;B increased in small pulmonary arterial lesions and alveolar macrophages in lungs from patients with PAH compared with lungs from control patients. In a rat model of monocrotaline-induced PAH, single intratracheal instillation of polymeric nanoparticles (NPs) resulted in delivery of NPs into lungs for ≤14 days postinstillation. The NP-mediated NF-&kgr;B decoy delivery into lungs prevented monocrotaline-induced NF-&kgr;B activation. Blockade of NF-&kgr;B by NP-mediated delivery of the NF-&kgr;B decoy attenuated inflammation and proliferation and, thus, attenuated the development of PAH and pulmonary arterial remodeling induced by monocrotaline. Treatment with the NF-&kgr;B decoy NP 3 weeks after monocrotaline injection improved the survival rate as compared with vehicle administration. In conclusion, these data suggest that NF-&kgr;B plays a primary role in the pathogenesis of PAH and, thus, represent a new target for therapeutic intervention in PAH. This nanotechnology platform may be developed as a novel molecular approach for treatment of PAH in the future.

Nanoparticle-Mediated Delivery of Pitavastatin Into Lungs Ameliorates the Development and Induces Regression of Monocrotaline-Induced Pulmonary Artery Hypertension

Pulmonary artery hypertension (PAH) is an intractable disease of the small PAs in which multiple pathogenic factors are involved. Statins are known to mitigate endothelial injury and inhibit vascular remodeling and inflammation, all of which play crucial roles in the pathogenesis of PAH. We tested the hypothesis that nanoparticle (NP)-mediated delivery of pitavastatin into the lungs can be a novel therapeutic approach for the treatment of PAH. Among the marketed statins, pitavastatin was found to have the most potent effects on proliferation of PA smooth muscle cells in vitro. We formulated pitavastatin-NP and found that pitavastatin-NP was more effective than pitavastatin alone in inhibiting cellular proliferation and inflammation in vitro. In a rat model of monocrotaline-induced PAH, a single intratracheal instillation of NP resulted in the delivery of NP into alveolar macrophages and small PAs for up to 14 days after instillation. Intratracheal treatment with pitavastatin-NP, but not with pitavastatin, attenuated the development of PAH and was associated with a reduction of inflammation and PA remodeling. NP-mediated pitavastatin delivery was more effective than systemic administration of pitavastatin in attenuating the development of PAH. Importantly, treatment with pitavastatin-NP 3 weeks after monocrotaline injection induced regression of PAH and improved survival rate. This mode of NP-mediated pitavastatin delivery into the lungs is effective in attenuating the development of PAH and inducing regression of established PAH, suggesting potential clinical significance for developing a new treatment for PAH.

Local Delivery of Imatinib Mesylate (STI571)-Incorporated Nanoparticle Ex Vivo Suppresses Vein Graft Neointima Formation

Background— Clinical outcome of surgical revascularization using autologous vein graft is limited by vein graft failure attributable to neointima formation. Platelet-derived growth factor (PDGF) plays a central role in the pathogenesis of vein graft failure. Therefore, we hypothesized that nanoparticle (NP)-mediated drug delivery system of PDGF-receptor (PDGF-R) tyrosine kinase inhibitor (imatinib mesylate: STI571) could be an innovative therapeutic strategy. Methods and Results— Uptake of STI571-NP normalized PDGF-induced cell proliferation and migration. Excised rabbit jugular vein was treated ex vivo with PBS, STI571 only, FITC-NP, or STI571-NP, then interposed back into the carotid artery position. NP was detected in many cells in the neointima and media at 7 and 28 days after grafting. Significant neointima was formed 28 days after grafting in the PBS group; this neointima formation was suppressed in the STI571-NP group. STI571-NP treatment inhibited cell proliferation and phosphorylation of the PDGF-R-β but did not affect inflammation and endothelial regeneration. Conclusions— STI571-NP–induced suppression of vein graft neointima formation holds promise as a strategy for preventing vein graft failure.

Soluble Flt-1 Gene Transfer Ameliorates Neointima Formation After Wire Injury in flt-1 Tyrosine Kinase–Deficient Mice

Objective—We have demonstrated that vascular endothelial growth factor (VEGF) expression is upregulated in injured vascular wall, and blockade of VEGF inhibited monocyte infiltration and neointima formation in several animal models. In the present study, we aimed to clarify relative role of two VEGF receptors, flt-1 versus flk-1/KDR, in neointima formation after injury using flt-1 tyrosine kinase-deficient (Flt-1 TK−/−) mice and soluble Flt-1(sFlt-1) gene transfer. Methods and Results—Neointima formation was comparable between wild-type and Flt-1 TK−/− mice 28 days after intraluminal wire injury in femoral arteries. By contrast, neointima formation was significantly suppressed by sFlt-1 gene transfer into Flt-1 TK−/− mice that blocks VEGF action on flk-1 (intima/media ratio: 2.8±0.4 versus 1.4±0.4, P<0.05). The inhibition of neointima formation was preceded by significant reduction of monocyte chemoattractant protein (MCP-1) expression in vascular smooth muscle cells (VSMCs) and monocyte infiltration 7 days after injury. Gene transfer of sFlt-1 or treatment of flk-1–specific antibody significantly inhibited VEGF-induced MCP-1 expression determined by RT-PCR in cultured aortic tissue and VSMCs. MCP-1–induced chemotaxis was equivalent between wild-type and Flt-1 TK−/− mice. Conclusions—These results suggest that endogenous VEGF accelerates neointima formation through flk-1 by regulating MCP-1 expression in VSMCs and macrophage-mediated inflammation in injured vascular wall in murine model of wire injury.