Mitsuki Kubo

Therapeutic Neovascularization by Nanotechnology-Mediated Cell-Selective Delivery of Pitavastatin Into the Vascular Endothelium

Objective—Recent clinical studies of therapeutic neovascularization using angiogenic growth factors demonstrated smaller therapeutic effects than those reported in animal experiments. We hypothesized that nanoparticle (NP)-mediated cell-selective delivery of statins to vascular endothelium would more effectively and integratively induce therapeutic neovascularization. Methods and Results—In a murine hindlimb ischemia model, intramuscular injection of biodegradable polymeric NP resulted in cell-selective delivery of NP into the capillary and arteriolar endothelium of ischemic muscles for up to 2 weeks postinjection. NP-mediated statin delivery significantly enhanced recovery of blood perfusion to the ischemic limb, increased angiogenesis and arteriogenesis, and promoted expression of the protein kinase Akt, endothelial nitric oxide synthase (eNOS), and angiogenic growth factors. These effects were blocked in mice administered a nitric oxide synthase inhibitor, or in eNOS-deficient mice. Conclusions—NP-mediated cell-selective statin delivery may be a more effective and integrative strategy for therapeutic neovascularization in patients with severe organ ischemia.

Nanoparticle-mediated endothelial cell-selective delivery of pitavastatin induces functional collateral arteries (therapeutic arteriogenesis) in a rabbit model of chronic hind limb ischemia

OBJECTIVES We recently demonstrated in a murine model that nanoparticle-mediated delivery of pitavastatin into vascular endothelial cells effectively increased therapeutic neovascularization. For the development of a clinically applicable approach, further investigations are necessary to assess whether this novel system can induce the development of collateral arteries (arteriogenesis) in a chronic ischemia setting in larger animals. METHODS Chronic hind limb ischemia was induced in rabbits. They were administered single injections of nanoparticles loaded with pitavastatin (0.05, 0.15, and 0.5 mg/kg) into ischemic muscle. RESULTS Treatment with pitavastatin nanoparticles (0.5 mg/kg), but not other nanoparticles, induced angiographically visible arteriogenesis. The effects of intramuscular injections of phosphate-buffered saline, fluorescein isothiocyanate (FITC)-loaded nanoparticles, pitavastatin (0.5 mg/kg), or pitavastatin (0.5 mg/kg) nanoparticles were examined. FITC nanoparticles were detected mainly in endothelial cells of the ischemic muscles for up to 4 weeks. Treatment with pitavastatin nanoparticles, but not other treatments, induced therapeutic arteriogenesis and ameliorated exercise-induced ischemia, suggesting the development of functional collateral arteries. Pretreatment with nanoparticles loaded with vatalanib, a vascular endothelial growth factor receptor (VEGF) tyrosine kinase inhibitor, abrogated the therapeutic effects of pitavastatin nanoparticles. Separate experiments with mice deficient for VEGF receptor tyrosine kinase demonstrated a crucial role of VEGF receptor signals in the therapeutic angiogenic effects. CONCLUSIONS The nanotechnology platform assessed in this study (nanoparticle-mediated endothelial cell-selective delivery of pitavastatin) may be developed as a clinically feasible and promising strategy for therapeutic arteriogenesis in patients.

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.

Essential Role of Angiotensin II Type 1a Receptors in the Host Vascular Wall, but Not the Bone Marrow, in the Pathogenesis of Angiotensin II–Induced Atherosclerosis

The angiotensin II (Ang II) type 1a (AT1a) receptor is expressed on multiple cell types in atherosclerotic lesions, including bone marrow–derived cells and vascular wall cells, and mediates inflammatory and proliferative responses. Indeed, Ang II infusion accelerates atherogenesis in hyperlipidemic mice by recruiting monocytes and by activating vascular wall cells. Here, we investigated the relative roles of AT1a receptors in the bone marrow vs. the vascular wall in Ang II–induced atherogenesis. Apolipoprotein E–knockout (ApoE−/−) mice with or without bone marrow AT1a receptor were generated by experimental bone marrow transplantation using AT1a+/+ or AT1a−/− recipients. In these mice, 28-d Ang II infusion induced significant atherosclerosis in the aorta, and the severity of plaque formation was not affected by the absence of bone marrow AT1a receptor. We then generated AT1a−/−ApoE−/− mice with or without bone marrow AT1a receptor. Ang II–induced plaque formation was blunted irrespective of the presence of bone marrow AT1a receptor. Host AT1a receptor deficiency was found to suppress Ang II–induced reactive oxygen species production. In addition, AT1a receptor deficiency also impaired monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in the arterial wall 7 d after Ang II initiation. These molecules normally initiate later macrophage-mediated inflammation in the vascular wall. By contrast, AT1a receptor deficiency in the bone marrow did not affect MCP-1–induced monocyte chemotaxis in vitro. In conclusion, AT1a receptors in the host vascular wall, but not in the bone marrow, are essential in Ang II–induced atherogenesis.