Hiroko Fujii

Ultrasound-Targeted Gene Delivery Induces Angiogenesis After a Myocardial Infarction in Mice

OBJECTIVESnThis study evaluated the capacity of ultrasound-targeted microbubble destruction (UTMD) to deliver angiogenic genes, improve perfusion, and recruit progenitor cells after a myocardial infarction (MI) in mice.nnnBACKGROUNDnAngiogenic gene therapy after an MI may become a clinically relevant approach to improve the engraftment of implanted cells if targeted delivery can be accomplished noninvasively. The UTMD technique uses myocardial contrast echocardiography to target plasmid gene delivery to the myocardium and features low toxicity, limited immunogenicity, and the potential for repeated application.nnnMETHODSnEmpty plasmids (control group) or those containing genes for vascular endothelial growth factor (VEGF), stem cell factor (SCF), or green fluorescent protein (to visualize gene delivery) were incubated with perflutren lipid microbubbles. The microbubble-deoxyribonucleic acid mixture was injected intravenously into C57BL/6 mice at 7 days after coronary artery ligation (MI). The UTMD technique facilitated transgene release into the myocardium. Twenty-one days after MI, myocardial perfusion and function were assessed by contrast echocardiography. Protein expression was quantified by Western blot and enzyme-linked immunosorbent assay. Flow cytometry quantified progenitor cell recruitment to the heart. Blood vessel density was evaluated immunohistochemically.nnnRESULTSnGreen fluorescent protein expression in the infarcted myocardium demonstrated gene delivery. Myocardial VEGF and SCF levels increased significantly in the respective groups (p < 0.05). The physiologic impact of VEGF and SCF gene delivery was confirmed by increased myocardial recruitment of VEGF receptor 2- and SCF receptor (c-kit)-expressing cells, respectively (p < 0.05). Consequently, capillary and arteriolar density (Factor VIII and alpha-smooth muscle actin staining), myocardial perfusion, and cardiac function were all enhanced (p < 0.01 relative to control group) in recipients of VEGF or SCF.nnnCONCLUSIONSnNoninvasive UTMD successfully delivered VEGF and SCF genes into the infarcted heart, increased vascular density, and improved myocardial perfusion and ventricular function. The UTMD technique may be an ideal method for noninvasive, repeated gene delivery after an MI.

c-Kit Dysfunction Impairs Myocardial Healing After Infarction

Background— We hypothesized that c-kit receptor function in the bone marrow is important for facilitating healing, leading to efficient cardiac repair after myocardial infarction (MI). Methods and Results— We used KitW/KitW-v c-kit mutant mice and their wild-type littermates to assess the importance of c-kit function in cardiac remodeling after coronary ligation. We found that mutant mice developed 1.6-fold greater ventricular dilation (P=0.008) attributable to a 1.3-fold greater infarct expansion by day 14 after MI (P=0.01). The number of proliferating smooth muscle α-actin expressing cells was 1.8-fold lower in mutant mice at day 3 (P<0.01), resulting in a 1.6 to 1.8-fold reduction in total regional nonvascular smooth muscle α-actin expressing cells by both microscopy and flow cytometry (P<0.001 for both). This decrease was accompanied by a 1.4-fold reduction in the number of CD31 expressing blood vessels (P<0.05). Prior transplantation of wild-type bone marrow cells into mutant mice rescued the efficient establishment of vessel-rich repair tissue by inducing a 1.5-fold increase in nonvascular smooth muscle α-actin expressing cells and CD31 expressing blood vessels (P<0.05 for both). The increased recruitment of cells into the infarct region in the chimeric mice was associated with reduced infarct expansion (P<0.03) compared to wild-type levels. Conclusions— Bone marrow c-kit function critically impacts the myofibroblast repair response in infarcted hearts. Interventions that increase the infiltration of c-kit+ cells to the infarcted heart may potentiate this endogenous repair response, prevent infarct expansion, and improve the recovery of cardiac function after MI.

Molecular imaging of endothelial progenitor cell engraftment using contrast-enhanced ultrasound and targeted microbubbles

AIMSnImaging methods to track the fate of progenitor cells after their delivery would be useful in assessing the efficacy of cell-based therapies. We hypothesized that contrast-enhanced ultrasound (CEU) using microbubbles targeted to a genetically engineered cell-surface marker on endothelial progenitor cells (EPCs) would allow the targeted imaging of vascular engraftment.nnnMETHODS AND RESULTSnRodent bone marrow-derived EPCs were isolated, cultured, and transfected to express the marker protein, H-2Kk, on the cell surface. Non-transfected EPCs and EPCs transfected with either null plasmid or Firefly luciferase served as controls. Control microbubbles (MB(C)) and microbubbles targeted to H-2Kk expressed on EPCs (MB(H-2Kk)) were constructed. Binding of targeted microbubbles to EPCs was assessed in vitro using a parallel plate flow chamber system. CEU imaging of EPC-targeted microbubbles was assessed in vivo using subcutaneously implanted EPC-supplemented Matrigel plugs in rats. In flow chamber experiments, there was minimal attachment of microbubbles to plated control EPCs. Although numbers of adhered MB(C) were also low, there was greater and more diffuse attachment of MB(H-2Kk) to plated H-2Kk-transfected EPCs. Targeted CEU demonstrated marked contrast enhancement at the periphery of the H-2Kk-transfected EPC-supplemented Matrigel plug for MB(H-2Kk,) whereas contrast enhancement was low for MB(C). Contrast enhancement was also low for both microbubbles within control mock-transfected EPC plugs. The signal intensity within the H-2Kk-transfected EPC plug was significantly greater for MB(H-2Kk) when compared with MB(C).nnnCONCLUSIONnMicrobubbles targeted to a genetically engineered cell-surface marker on EPCs exhibit specific binding to EPCs in vitro. These targeted microbubbles bind to engrafted EPCs in vivo within Matrigel plugs and can be detected by their enhancement on CEU imaging.

Treatment with a highly selective β1 antagonist causes dose-dependent impairment of cerebral perfusion after hemodilution in rats

BACKGROUND:Acute &bgr;-blockade has been associated with a dose-dependent increase in adverse outcomes, including stroke and mortality. Acute blood loss contributes to the incidence of these adverse events. In an attempt to link the risks of acute blood loss and &bgr;-blockade, animal studies have demonstrated that acute &bgr;-blockade impairs cerebral perfusion after hemodilution. We expanded on these findings by testing the hypothesis that acute &bgr;-blockade with a highly &bgr;1-specific antagonist (nebivolol) causes dose-dependent cerebral hypoxia during hemodilution. METHODS:Anesthetized rats and mice were randomized to receive vehicle or nebivolol (1.25 or 2.5 mg/kg) IV before hemodilution to a hemoglobin concentration near 60 g/L. Drug levels, heart rate (HR), cardiac output (CO), regional cerebral blood flow (rCBF, laser Doppler), and microvascular brain PO2 (PBrO2, G2 Oxyphor) were measured before and after hemodilution. Endothelial nitric oxide synthase (NOS), neuronal NOS (nNOS), inducible NOS, and hypoxia inducible factor (HIF)-1&agr; were assessed by Western blot. HIF-&agr; expression was also assessed using an HIF-(ODD)-luciferase mouse model. Data were analyzed using analysis of variance with significance assigned at P < 0.05, and corrected P values are reported for all post hoc analyses. RESULTS:Nebivolol treatment resulted in dose-specific plasma drug levels. In vehicle-treated rats, hemodilution increased CO and rCBF (P < 0.010) whereas PBrO2 decreased to 45.8 ± 18.7 mm Hg (corrected P < 0.001; 95% CI 29.4–69.7). Both nebivolol doses comparably reduced HR and attenuated the CO response to hemodilution (P < 0.012). Low-dose nebivolol did not impair rCBF or further reduce PBrO2 after hemodilution. High-dose nebivolol attenuated the rCBF response to hemodilution and caused a further reduction in PBrO2 to 28.4 ± 9.6 mm Hg (corrected P = 0.019; 95% CI 17.4–42.7). Both nebivolol doses increased brain endothelial NOS protein levels. Brain HIF-1&agr;, inducible NOS, and nNOS protein levels and brain HIF-luciferase activity were increased in the high-dose nebivolol group after hemodilution (P < 0.032). CONCLUSIONS:Our data demonstrate that nebivolol resulted in a dose-dependent decrease in cerebral oxygen delivery after hemodilution as reflected by a decrease in brain tissue PO2 and an increase in hypoxic protein responses (HIF-1&agr; and nNOS). Low-dose nebivolol treatment did not result in worsened tissue hypoxia after hemodilution, despite comparable effects on HR and CO. These data support the hypothesis that acute &bgr;-blockade with a highly &bgr;1-specific antagonist causes a dose-dependent impairment in cerebral perfusion during hemodilution.

Optimization of Ultrasound-mediated Anti-angiogenic Cancer Gene Therapy

Ultrasound-targeted microbubble destruction (UTMD) can be used to deliver silencing gene therapy to tumors. We hypothesized that UTMD would be effective in suppressing angiogenesis within tumors, and that modulation of the ultrasound pulsing intervals (PI) during UTMD would affect the magnitude of target knockdown. We performed UTMD of vascular endothelial growth factor receptor-2 (VEGFR2) short hairpin (sh)RNA plasmid in an heterotopic mammary adenocarcinoma model in rats, evaluating PIs of 2, 5, 10, and 20 seconds. We demonstrated that UTMD with a PI of 10 seconds resulted in the greatest knockdown of VEGFR2 by PCR, immunostaining, western blotting, smaller tumor volumes and perfused areas, and lower tumor microvascular blood volume (MBV) and flow by contrast-enhanced ultrasound (CEU) compared with UTMD-treated tumors at 2, 5, and 20 seconds, control tumors, tumors treated with intravenous shRNA plasmid and scrambled plasmid. CEU perfusion assessment using the therapeutic probe demonstrated that tumors were fully replenished with microbubbles within 10 seconds, but incompletely replenished at PI-2 and PI-5 seconds. In conclusion, for anti-VEGFR2 cancer gene therapy by UTMD, PI of 10 seconds results in higher target knockdown and a greater anti-angiogenic effect. Complete replenishment of tumor vasculature with silencing gene-bearing microbubbles in between destructive pulses of UTMD is required to maximize the efficacy of anti-angiogenic cancer gene therapy.

Docosahexaenoic Acid, But Not Eicosapentaenoic Acid, Supplementation Reduces Vulnerability to Atrial Fibrillation

Background—The potential health benefits of &ohgr;-3 polyunsaturated fatty acids (PUFAs) usually are studied using a combination of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). This combination reduces vulnerability to experimentally induced atrial fibrillation (AF). It is unknown whether EPA and DHA have differential effects when taken alone. Using a model of pacing-induced atrial hemodynamic overload, we investigated the individual effects of EPA and DHA on vulnerability to AF and atrial remodeling. Methods and Results—Thirty-four dogs were randomized into 3 groups, all of which underwent simultaneous atrial and ventricular pacing at 220 beats per minute for 14 days. One group received purified DHA (≈1 g/d) orally for 21 days beginning 7 days before pacing began. Similarly, 1 group received ≈1 g/d purified EPA. In a third (control) group (No-PUFAs), 8 dogs received ≈1 g/d olive oil, and 12 were unsupplemented. Electrophysiological and echocardiographic measurements were taken at baseline and 21 days. Atrial tissue samples were collected at 21 days for histological and molecular analyses. Persistent AF inducibility was significantly reduced by DHA compared with No-PUFAs median [25–75 percentiles], 0% [0%–3%] for DHA versus 3.1% [2.2%–11%] for No-PUFAs; P=0.007) but not by EPA (3.4% [1.9%–8.9%]). DHA also reduced atrial fibrosis compared with No-PUFAs (11±6% versus 20±4%, respectively; P<0.05), whereas EPA did not (15±5%; P>0.05). Conclusions—DHA is more effective than EPA in attenuating AF vulnerability and atrial remodeling in structural remodeling–induced AF.

N-3 polyunsaturated fatty acid supplementation does not reduce vulnerability to atrial fibrillation in remodeling atria

BACKGROUNDnProphylactic supplementation with omega-3 polyunsaturated fatty acids (PUFAs) reduce vulnerability to atrial fibrillation (AF). The effect of PUFAs given after cardiac injury has occurred is unknown.nnnOBJECTIVEnTo investigate using a model of pacing-induced cardiac injury, the time course of development of injury and whether it was altered by postinjury PUFAs.nnnMETHODSnSixty-five dogs were randomized to undergo simultaneous atrial and ventricular pacing (SAVP, 220 beats/min) for 0, 2, 7, or 14 days. Twenty-two dogs received PUFAs (850 mg/d) either prophylactically or after some pacing had occurred (postinjury). Electrophysiologic and echocardiographic measurements were taken at baseline and sacrifice. Atrial tissue samples were collected at sacrifice for histologic and molecular analyses.nnnRESULTSnWith no PUFAs, the inducibility of AF increased with pacing duration (P < .001). Postinjury PUFAs (started after 7 days of pacing) did not reduce the inducibility of AF after 14 days of pacing (9.3% ± 8.8% no PUFAs vs 9.7% ± 9.9% postinjury PUFAs; P = .91). Atrial myocyte size and fibrosis increased with pacing duration (P < .05). Postinjury PUFAs did not significantly attenuate the cell size increase after 14 days of pacing (no PUFAs 38% ± 30% vs postinjury PUFAs 19% ± 28%; P = .11). Similarly, postinjury PUFAs did not attenuate the increase in fibrosis after 14 days of pacing (no PUFAs 66% ± 51% vs postinjury PUFAs 63% ± 76%; P = .90).nnnCONCLUSIONnPUFA supplementation begun after cardiac injury has already occurred does not reduce atrial structural remodeling or vulnerability to AF.

Human angiogenic cell precursors restore function in the infarcted rat heart: a comparison of cell delivery routes.

We recently isolated angiogenic cell precursors (ACPs) from human blood, which can induce angiogenesis in vitro.

Statin therapy prevents expansive remodeling in venous bypass grafts

BACKGROUNDnVenous grafts (VG) have high failure rates by 10 years in aortocoronary bypass surgery. We have previously shown that expansive remodeling followed by increased LDL retention are early atherosclerotic changes in experimental VG placed in the arterial circulation. The objective of this study was to determine whether statin therapy prevents these expansive remodeling changes.nnnMETHODS AND RESULTSnReversed jugular vein-to-common carotid artery interposition graft was constructed in 27 cholesterol-fed (0.5%) rabbits. Rabbits were randomized either to control or atorvastatin (5 mg/kg/day) groups, starting two weeks prior to vein graft implantation and continuing until sacrifice at 1 or 12 weeks post-surgery. Ultrasound measurements of arterial luminal cross-sectional area (CSA) were done at day 3 and at 4, 8 and 12 weeks post-surgery. Histomorphometric measurements were performed following sacrifice at 12 weeks. Atorvastatin treatment significantly decreased total plasma cholesterol levels at 4, 8 and 12 weeks (12 weeks: 6.7 ± 4.2 mmol/L versus control 38.7 ± 10.6 mmol/L, p<0.0002). Atorvastatin significantly reduced expansive remodeling at 4, 8 and 12 weeks (lumen CSA: 44.6 ± 6.6 mm(2) versus control 77.6 ± 10.7 mm(2), p<0.0001). Intimal CSA by histomorphometry was also significantly reduced by atorvastatin at 12 weeks (5.59 ± 2.19 mm(2) versus control 9.57 ± 2.43 mm(2), p<0.01). VG macrophage infiltration, MMP-2 activity and metalloelastase activity were reduced in the atorvastatin treated group.nnnCONCLUSIONnAtorvastatin inhibits both expansive remodeling and intimal hyperplasia in arterialized VG, likely through inhibition of macrophage infiltration and reduction of tissue proteolytic activity. The mechanism proposed above may be important for preventing VG atherosclerosis and late VG failure.

Dronedarone and Captisol-enabled amiodarone in an experimental cardiac arrest.

Objective: To compare the energy required for defibrillation and postshock outcomes after the administration of dronedarone, amiodarone, and placebo in a porcine model of cardiac arrest. Methods: Forty-two pigs were randomized to amiodarone, dronedarone, or control treatments. After induction of ventricular fibrillation, compressions and ventilations were performed for 3 minutes and treatment was administered over 30 seconds. If defibrillation was unsuccessful, cardiopulmonary resuscitation continued and repeated shocks were administered every 2 minutes with continual hemodynamic monitoring for a total duration of 30 minutes. Results: The cumulative energy required for defibrillation was 570 ± 422 J for dronedarone, 441 ± 365 J for amiodarone, and 347 ± 281 J for control (P = not significant). Survival at 30 minutes was 1 (7.1%) for dronedarone compared with 11 (78.6%) for control (P = 0.001). Mortality in the dronedarone group was because of refibrillation in 3 (21.4%) cases, atrioventricular block in 1 (7.1%) case, and hypotension not because of bradycardia in 9 (64.3%) cases. Two minutes after successful defibrillation, systolic aortic pressure was lower in dronedarone versus control (86.6 ± 26.9 vs. 110 ± 15.1 mm Hg; P = 0.035). Conclusions: The administration of dronedarone resulted in a significant reduction in survival and both systolic aortic and coronary perfusion pressure compared with control.