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

ObjectivesWe 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.MethodsChronic 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.ResultsTreatment 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.ConclusionsThe 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.Clinical RelevanceRestoration of tissue perfusion in patients with critical limb ischemia is a major therapeutic goal. Recent clinical trials designed to induce neovascularization by administering exogenous angiogenic growth factors or cells failed to demonstrate a decisive clinical benefit. A controlled drug delivery system for a new approach to therapeutic neovascularization therefore would be more favorable. In the present study, we applied nanoparticle-mediated delivery system and report that endothelial cell-selective delivery of pitavastatin increased the development of collateral arteries and improved exercise-induced ischemia in a rabbit model of chronic hind limb ischemia. This nanotechnology platform is a promising strategy for the treatment of patients with severe organ ischemia and represents a significant advance in therapeutic arteriogenesis over current approaches

A New Therapeutic Modality for Acute Myocardial Infarction: Nanoparticle-Mediated Delivery of Pitavastatin Induces Cardioprotection from Ischemia-Reperfusion Injury via Activation of PI3K/Akt Pathway and Anti-Inflammation in a Rat Model.

There is an unmet need to develop an innovative cardioprotective modality for acute myocardial infarction (AMI), for which the effectiveness of interventional reperfusion therapy is hampered by myocardial ischemia-reperfusion (IR) injury. Pretreatment with statins before ischemia is shown to reduce MI size in animals. However, no benefit was found in animals and patients with AMI when administered at the time of reperfusion, suggesting insufficient drug targeting into the IR myocardium. Here we tested the hypothesis that nanoparticle-mediated targeting of pitavastatin protects the heart from IR injury.In a rat IR model, poly(lactic acid/glycolic acid) (PLGA) nanoparticle incorporating FITC accumulated in the IR myocardium through enhanced vascular permeability, and in CD11b-positive leukocytes in the IR myocardium and peripheral blood after intravenous treatment. Intravenous treatment with PLGA nanoparticle containing pitavastatin (Pitavastatin-NP, 1 mg/kg) at reperfusion reduced MI size after 24 hours and ameliorated left ventricular dysfunction 4-week after reperfusion; by contrast, pitavastatin alone (as high as 10 mg/kg) showed no therapeutic effects. The therapeutic effects of Pitavastatin-NP were blunted by a PI3K inhibitor wortmannin, but not by a mitochondrial permeability transition pore inhibitor cyclosporine A. Pitavastatin-NP induced phosphorylation of Akt and GSK3β, and inhibited inflammation and cardiomyocyte apoptosis in the IR myocardium.Nanoparticle-mediated targeting of pitavastatin induced cardioprotection from IR injury by activation of PI3K/Akt pathway and inhibition of inflammation and cardiomyocyte death in this model. This strategy can be developed as an innovative cardioprotective modality that may advance currently unsatisfactory reperfusion therapy for AMI

Prognostic Accuracy of the qSOFA Score for In-Hospital Mortality in Elderly Patients with Obstructive Acute Pyelonephritis: A Multi-Institutional Study

Prognostic accuracy of the quick sequential organ failure assessment (qSOFA) score for mortality may be limited in elderly patients. Using our multi-institutional database, we classified obstructive acute pyelonephritis (OAPN) patients into young and elderly groups, and evaluated predictive performance of the qSOFA score for in-hospital mortality. qSOFA score ≥ 2 was an independent predictor for in-hospital mortality, as was higher age, and Charlson comorbidity index (CCI) ≥ 2. In young patients, the area under the curve (AUC) of the qSOFA score for in-hospital mortality was 0.85, whereas it was 0.61 in elderly patients. The sensitivity and specificity of qSOFA score ≥ 2 for in-hospital mortality was 80% and 80% in young patients, and 50% and 68% in elderly patients, respectively. For elderly patients, we developed the CCI-incorporated qSOFA score, which showed higher prognostic accuracy compared with the qSOFA score (AUC, 0.66 vs. 0.61, p < 0.001). Therefore, the prognostic accuracy of the qSOFA score for in-hospital mortality was high in young OAPN patients, but modest in elderly patients. Although it can work as a screening tool to determine therapeutic management in young patients, for elderly patients, the presence of comorbidities should be considered at the initial assessment

Effects of Pitavastatin-NP on cell death after IR.

<p><b>(A)</b>, Effects of Pitavastatin-NP at the time of reperfusion after pretreatment with Cyclosporine A (CsA) (10 mg/kg) every 12 hours starting 36 hours before ischemia on MI size. N = 7 per group. Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests. <b>(B)</b>, Effects of Pitavastatin-NP at the time of reperfusion after pretreatment with CsA (10 mg/kg) every 12 hours starting 36 hours before ischemia on cytosolic cytochrome C in IR myocardium 30 minutes after reperfusion. N = 4 per group. Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests. <b>(C)</b>, Effects of Pitavastatin-NP at the time of reperfusion after pretreatment with Cyclosporine A (CsA) (10 mg/kg) every 12 hours starting 36 hours before ischemia on mitochondrial cytochrome C in IR myocardium 30 minutes after reperfusion. Data are mean±SEM (n = 4 per group). Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests. <b>(D)</b>, Representative photomicrographs of cross-sections from IR myocardium stained with ED-1 in AAR. Scale bar: 20 μm. <b>(E),</b> Effects of Pitavastatin-NP at the time of reperfusion after pretreatment with CsA (10 mg/kg) every 12 hours starting 36 hours before ischemia on ED-1-positive leukocyte (monocytes) infiltration in IR myocardium 24-hour after reperfusion. N = 7 per group. Data are compared using one-way ANOVA followed by Dunnett’s multiple comparison tests.</p

Effects of Pitavastatin-NP on MI size.

<p><b>(A)</b>, Representative stereomicrographs of heart sections double-stained with Evans blue and TTC 24 hours after reperfusion. Scale bar: 5 mm. <b>(B),</b> Effects of Pitavastatin-NP and pitavastatin alone on MI size at the time of reperfusion. N = 6–10 per group. Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests. <b>(C)</b>, Quantification of Area at risk in the group treated with pitavastatin-NP or pitavastatin alone. Data are mean are (n = 6–10 per group) Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests.</p

Experimental protocols.

<p>Adult male Sprague-Dawley (SD) rats, 8 weeks of age were used. Experimental protocol 1: At the time of reperfusion, animals were divided into 3 groups receiving intravenous injection of the following drugs; 1) vehicle (saline 3.3 mL/kg), 2) FITC alone (FITC 0.33 mg in saline 3.3 mL/kg), or 3) FITC-NP (PLGA 8.3 mg containing 0.33 mg FITC in saline 3.3 mL/kg). Three hours after reperfusion, animals were sacrificed. The left lower panel shows representative stereomicrographs of heart sections double-stained with Evans blue and TTC: the MI area (TTC negative, white), non-MI area within AAR (TTC positive/Evans blue negative, red), non-ischemic area (TTC positive/Evans blue positive, purple) and AAR (Evans blue negative). Experimental protocol 2: At the time of reperfusion, animals were divided into 4 groups receiving intravenous injection of the following drugs; 1) vehicle (saline 3.3 mL/kg), 2) FITC-NP (PLGA 8.3 mg/kg in saline 3.3 mL/kg), 3) pitavastatin (1.0 and 10 mg/kg in saline 3.3 mL/kg), or 4) pitavastatin-NP (PLGA containing of 0.1 and 1.0 mg/kg pitavastatin in saline 3.3 mL/kg). Twenty-four hours after reperfusion, animals were sacrificed and infarct size was measured. Experimental protocol 3: Animals were divided into 3 groups receiving administration of the following drugs; 1) vehicle (saline 3.3 mL/kg), 2) vehicle (saline 3.3 mL/kg) after pretreatment with Cyclosporine A (CsA) (10 mg/kg) every 12 hours starting 36 hours before ischemia, 3) pitavastatin-NP (PLGA containing of 1.0 mg/kg pitavastatin in saline 3.3 mL/kg) after pretreatment with CsA (10 mg/kg) every 12 hours starting 36 hours before ischemia. Twenty-four hours after reperfusion, animals were sacrificed and infarct size was measured. Experimental protocol 4: To examine the effects of Pitavastatin-NP on left ventricular function after IR, animals were divided into 3 groups that received intravenous injection of the following drugs at the time of reperfusion: 1) vehicle (saline 3.3 mL/kg), 2) pitavastatin alone (1.0 mg/kg in saline 3.3 mL/kg) or 3) Pitavastatin-NP (PLGA containing 1.0 mg/kg pitavastatin in saline 3.3 mL/kg). Echocardiography and measurement of systolic blood pressure and heart rate by using tail-cuff method were performed at baseline and 2-day, 1-week, 2-weeks and 4-weeks after reperfusion.</p

Effects of Pitavastatin-NP on cell death after IR.

<p><b>(A)</b>, Effects of Pitavastatin-NP at the time of reperfusion after pretreatment with Cyclosporine A (CsA) (10 mg/kg) every 12 hours starting 36 hours before ischemia on MI size. N = 7 per group. Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests. <b>(B)</b>, Effects of Pitavastatin-NP at the time of reperfusion after pretreatment with CsA (10 mg/kg) every 12 hours starting 36 hours before ischemia on cytosolic cytochrome C in IR myocardium 30 minutes after reperfusion. N = 4 per group. Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests. <b>(C)</b>, Effects of Pitavastatin-NP at the time of reperfusion after pretreatment with Cyclosporine A (CsA) (10 mg/kg) every 12 hours starting 36 hours before ischemia on mitochondrial cytochrome C in IR myocardium 30 minutes after reperfusion. Data are mean±SEM (n = 4 per group). Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests. <b>(D)</b>, Representative photomicrographs of cross-sections from IR myocardium stained with ED-1 in AAR. Scale bar: 20 μm. <b>(E),</b> Effects of Pitavastatin-NP at the time of reperfusion after pretreatment with CsA (10 mg/kg) every 12 hours starting 36 hours before ischemia on ED-1-positive leukocyte (monocytes) infiltration in IR myocardium 24-hour after reperfusion. N = 7 per group. Data are compared using one-way ANOVA followed by Dunnett’s multiple comparison tests.</p

Leukocyte counts and Plasma biomarker profile 24 hours after IR.

<p>Data are expressed as the mean ± SEM (N = 6 each).</p><p>*<i>P</i><0.05 versus vehicle group. IR: ischemia-reperfusion.</p><p>Leukocyte counts and Plasma biomarker profile 24 hours after IR.</p

Effects of Pitavastatin-NP on inflammation and apoptosis in IR myocardium 24 hours after reperfusion.

<p><b>(A)</b>, Representative photomicrographs of cross-sections from IR myocardium stained with NF-B (p65 subunit), MCP-1, ED-1 and TUNEL. Scale bar: 20 μm. <b>(B)</b>, Quantification of the number of NF-B (p65 subunit) positive cells, the MCP-1-positive area, ED-1-positive leukocytes (monocytes) in IR myocardium and the number of TUNEL-positive cells in infarct border myocardium 24 hours after reperfusion. Data are mean±SEM (n = 5–8 per group). Data are compared using one-way ANOVA followed by Dunnett’s multiple comparison tests.</p