Ryuji Tominaga

Formulation of nanoparticle-eluting stents by a cationic electrodeposition coating technology: efficient nano-drug delivery via bioabsorbable polymeric nanoparticle-eluting stents in porcine coronary arteries.

OBJECTIVES The objective of this study was to formulate a nanoparticle (NP)-eluting drug delivery stent system by a cationic electrodeposition coating technology. BACKGROUND Nanoparticle-mediated drug delivery systems (DDS) are poised to transform the development of innovative therapeutic devices. Therefore, we hypothesized that a bioabsorbable polymeric NP-eluting stent provides an efficient DDS that shows better and more prolonged delivery compared with dip-coating stent. METHODS We prepared cationic NP encapsulated with a fluorescence marker (FITC) by emulsion solvent diffusion method, succeeded to formulate an NP-eluting stent with a novel cation electrodeposition coating technology, and compared the in vitro and in vivo characteristics of the FITC-loaded NP-eluting stent with dip-coated FITC-eluting stent and bare metal stent. RESULTS The NP was taken up stably and efficiently by cultured vascular smooth muscle cells in vitro. In a porcine coronary artery model in vivo, substantial FITC fluorescence was observed in neointimal and medial layers of the stented segments that had received the FITC-NP-eluting stent until 4 weeks. In contrast, no substantial FITC fluorescence was observed in the segments from the polymer-based FITC-eluting stent or from bare metal stent. The magnitudes of stent-induced injury, inflammation, endothelial recovery, and neointima formation were comparable between bare metal stent and NP-eluting stent groups. CONCLUSIONS Therefore, this NP-eluting stent is an efficient NP-mediated DDS that holds as an innovative platform for the delivery of less invasive nano-devices targeting cardiovascular disease.

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.

The effects of pulsatile and nonpulsatile systemic perfusion on renal sympathetic nerve activity in anesthetized dogs.

It is still controversial whether to pulse or not to pulse for the establishment of ideal extracorporeal circulation. We directly measured the renal sympathetic nerve activity in mongrel dogs (n = 10, weighing from 13 to 21 kg) to determine the effects of pulsatile and nonpulsatile systemic perfusion on the control of the sympathetic nerve activity during left ventricular assistance. Pulsatile perfusion was generated with an air-driven, diaphragm-type blood pump, and nonpulsatile perfusion was generated with a centrifugal pump. Renal sympathetic nerve activity and the blood flow of the descending aorta were then recorded during pulsatile and nonpulsatile systemic perfusion. Other variables, such as mean arterial pressure, central venous pressure, left atrial pressure, and blood gas levels, were kept constant. At the same mean arterial pressure, renal sympathetic nerve activity during pulsatile perfusion decreased significantly to 80% of renal sympathetic nerve activity during nonpulsatile perfusion (26.8 +/- 2.4 vs 33.4 +/- 2.9 spikes/sec, p < 0.01). Total systemic vascular resistance during pulsatile perfusion decreased significantly to 85% of that during nonpulsatile perfusion (5700 +/- 580 vs 6667 +/- 709 dynes.sec.cm-5, p < 0.05). These results suggest that pulsatile systemic perfusion, compared with nonpulsatile systemic perfusion, reduces sympathetic nerve activity and peripheral vascular resistance and thus may improve both microcirculation and organ function.

Effects of design geometry of intravascular endoprostheses on stenosis rate in normal rabbits

To investigate the effects of gaps between the individual wire coils of a shape memory Nitinol alloy intravascular endoprosthesis, 20 stents with and without gaps were implanted transluminally into the infrarenal abdominal aortas of 10 normal rabbits after balloon angioplasty. Digital subtraction angiography (DSA) was done at 4, 12, 16, 20, and 24 weeks after stent implantation to examine the stenosis rate and major side branch patency. Stenosis rate within stents with gaps were significantly lower than those without gaps: 8.1 +/- 5.0% versus 15.0 +/- 6.8% at 12 weeks, 13.6 +/- 6.0% versus 26.0 +/- 9.4% at 24 weeks by DSA, p greater than 0.005 and p less than 0.01 respectively. The maximum and mean neointimal thickness measured histologically at the time of animal death correlated significantly to the narrowest diameter obtained from the DSA studies, (r = 0.84 and r = 0.79, respectively, p less than 0.01). Greater hyperplasia of the neointima was evident in the stented arterial segments with stents without gaps compared with those with gaps (83 +/- 22 versus 187 +/- 46 microns mean thickness, p less than 0.001). The patency rate of the side branches in the stented arterial segment was significantly (p less than 0.05) higher in stents having gaps. These results suggest that the placement of gaps between wire pitches reduced the neointimal thickness within stents and prevented the obstruction of arterial side branches.

Mammalian formin fhod3 regulates actin assembly and sarcomere organization in striated muscles.

Actin filament assembly in nonmuscle cells is regulated by the actin polymerization machinery, including the Arp2/3 complex and formins. However, little is known about the regulation of actin assembly in muscle cells, where straight actin filaments are organized into the contractile unit sarcomere. Here, we show that Fhod3, a myocardial formin that localizes to thin actin filaments in a striated pattern, regulates sarcomere organization in cardiomyocytes. RNA interference-mediated depletion of Fhod3 results in a marked reduction in filamentous actin and disruption of the sarcomeric structure. These defects are rescued by expression of wild-type Fhod3 but not by that of mutant proteins carrying amino acid substitution for conserved residues for actin assembly. These findings suggest that actin dynamics regulated by Fhod3 are critical for sarcomere organization in striated muscle cells.

Local Delivery of Anti-Monocyte Chemoattractant Protein-1 by Gene-Eluting Stents Attenuates In-Stent Stenosis in Rabbits and Monkeys

Objective—We have previously shown that the intramuscular transfer of the anti–monocyte chemoattractant protein-1 (MCP-1) gene (called 7ND) is able to prevent experimental restenosis. The aim of this study was to determine the in vivo efficacy and safety of local delivery of 7ND gene via the gene-eluting stent in reducing in-stent neointima formation in rabbits and in cynomolgus monkeys. Methods and Results—We here found that in vitro, 7ND effectively inhibited the chemotaxis of mononuclear leukocytes and also inhibited the proliferation/migration of vascular smooth muscle cells. We then coated stents with a biocompatible polymer containing a plasmid bearing the 7ND gene, and deployed these stents in the iliac arteries of rabbits and monkeys. 7ND gene-eluting stents attenuated stent-associated monocyte infiltration and neointima formation after one month in rabbits, and showed long-term inhibitory effects on neointima formation when assessments were carried out at 1, 3, and 6 months in monkeys. Conclusions—Strategy of inhibiting the action of MCP-1 with a 7ND gene-eluting stent reduced in-stent neointima formation with no evidence of adverse effects in rabbits and monkeys. The 7ND gene-eluting stent could be a promising therapy for treatment of restenosis in humans.

Coenzyme Q10: The Prophylactic Effect on Low Cardiac Output Following Cardiac Valve Replacement

A randomized, prospective study of the effectiveness of preoperative administration of coenzyme Q10 on the prophylaxis of postoperative low cardiac output state was performed in 50 patients with acquired valvular diseases necessitating valve replacement. There were 25 patients in the treatment group and 25 in the control group. Patients in the treatment group received 30 to 60 mg of coenzyme Q10 orally for six days before operation. Preoperative clinical variables, operative procedures, total cardiopulmonary bypass time, and aortic cross-clamping time were similar for the two groups. Postoperatively, mild to severe low cardiac output state developed in 28 of 50 patients (56%) and necessitated the administration of considerable amounts of inotropic agent. The treatment group showed a significantly lower incidence of low cardiac output state during the recovery period than the control group (p less than 0.05). These results suggest that preoperative administration of coenzyme Q10 will increase the tolerance of human hearts to ischemia during aortic cross-clamping.

Stent-Based Local Delivery of Nuclear Factor-κB Decoy Attenuates In-Stent Restenosis in Hypercholesterolemic Rabbits

Background— Nuclear factor-&kgr;B (NF-&kgr;B) plays a critical role in the vascular response to injury. However, the role of NF-&kgr;B in the mechanism of in-stent restenosis remains unclear. We therefore tested the hypothesis that blockade of NF-&kgr;B by stent-based delivery of a cis-element “decoy” of NF-&kgr;B reduces in-stent neointimal formation. Methods and Results— Stents were coated with a polymer containing or not containing NF-&kgr;B decoy, which represented a fast-release formulation (<7 days). Bare, polymer-coated, and NF-&kgr;B decoy–eluting stents were implanted in iliac arteries of hypercholesterolemic rabbits. Increased NF-&kgr;B activity was noted at early stages after stenting, which was suppressed by stent-based delivery of NF-&kgr;B decoy. NF-&kgr;B decoy–eluting stents also reduced monocyte infiltration and monocyte chemoattractant protein-1 expression and suppressed CD14 activation on circulating leukocytes. Importantly, NF-&kgr;B decoy–eluting stents attenuated neointimal formation on day 28. There was no evidence of an incomplete healing process (persistent inflammation, hemorrhage, fibrin deposition, impaired endothelial regeneration) at the site of NF-&kgr;B decoy–eluting stents. Transfection of NF-&kgr;B decoy suppressed proliferation of human coronary artery smooth muscle cells in vitro. No systemic adverse effects of NF-&kgr;B decoy were detected. Conclusions— Stent-based local delivery of NF-&kgr;B decoy reduced in-stent neointimal formation with no evidence of incomplete healing. These data suggest that this strategy may be a practical and promising means for prevention of in-stent restenosis in humans.

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.

Comparative study of retrograde and selective cerebral perfusion with transcranial Doppler

BACKGROUND Retrograde cerebral perfusion (RCP) is a simple technique and is expected to provide cerebral protection. However, its optimum management and limitations remain unclear. Transcranial Doppler has been used to monitor cerebral perfusion. Using this Doppler technique, we compared cerebral blood flow for RCP with that for selective cerebral perfusion. METHODS Thirty-two consecutive patients underwent elective surgical repair of an aortic aneurysm involving the aortic arch at Kyushu University Hospital. Retrograde cerebral perfusion was used in 15 patients and selective cerebral perfusion, in 17 patients. Continuous measurement of middle cerebral artery blood flow velocities was performed by transcranial Doppler technique. RESULTS Retrograde middle cerebral artery blood flow velocities during RCP could be measured in only 3 patients, whereas middle cerebral artery blood flow velocities during selective cerebral perfusion could be measured in all but 1 woman. The increase in middle cerebral artery blood flow velocities after RCP was significantly greater than that after selective cerebral perfusion. CONCLUSIONS The measurement of middle cerebral artery blood flow velocities with transcranial Doppler technique is practicable during selective cerebral perfusion but difficult during RCP. The increase in middle cerebral artery blood flow velocities after RCP indicates reactive hyperemia and reflects the critical decrease in cerebral blood flow during this type of perfusion.