Search

Aim Abdominal aortic aneurysm (AAA) is a lethal and multifactorial disease. To prevent a rupture and dissection of enlarged AAA, prophylactic surgery and stenting are currently available. There are, however, no medical therapies preventing these complications of AAA. Statin is one of the candidates, but its efficacy on AAA formation/progression remains controversial. We have previously demonstrated that nanoparticles (NPs) incorporating pitavastatin (Pitava-NPs)-clinical trials using these nanoparticles have been already conducted-suppressed progression of atherosclerosis in apolipoprotein E-deficient (Apoe－/－) mice. Therefore, we have tested a hypothesis that monocytes/macrophages-targeting delivery of pitavastatin prevents the progression of AAA. Methods Angiotensin II was intraperitoneally injected by osmotic mini-pumps to induce AAA formation in Apoe－/－ mice. NPs consisting of poly(lactic-co-glycolic acid) were used for in vivo delivery of pitavastatin to monocytes/macrophages. Results Intravenously administered Pitava-NPs (containing 0.012 mg/kg/week pitavastatin) inhibited AAA formation accompanied with reduction of macrophage accumulation and monocyte chemoattractant protein-1 (MCP-1) expression. Ex vivo molecular imaging revealed that Pitava-NPs not only reduced macrophage accumulation but also attenuated matrix metalloproteinase activity in the abdominal aorta, which was underpinned by attenuated elastin degradation. Conclusion These results suggest that Pitava-NPs inhibit AAA formation associated with reduced macrophage accumulation and MCP-1 expression. This clinically feasible nanomedicine could be an innovative therapeutic strategy that prevents devastating complications of AAA.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is pathologically characterized by focal fibrinoid necrosis, in which ANCA-mediated neutrophil extracellular trap (NET) formation and subsequent endothelial cell necrosis occur. Cyclophilin D (CypD) plays an important role in mediation of cell necrosis and inflammation via the opening of mitochondrial permeability transition pores. This study was undertaken to examine the role of CypD in AAV pathogenesis.We assessed the role and mechanism of CypD in ANCA-stimulated neutrophils in vitro by immunostaining and electron microscopy observation. We performed a comprehensive RNA-sequencing analysis on ANCA-treated murine neutrophils. To investigate the role of CypD in vivo, we assessed disease features in CypD-knockout mice and wild-type mice using 2 different murine AAV models: anti-myeloperoxidase IgG transfer-induced AAV and spontaneous AAV.In vitro experiments showed that pharmacologic and genetic inhibition of CypD suppressed ANCA-induced NET formation via the suppression of reactive oxygen species and cytochrome c release from the mitochondria. RNA-sequencing analyses in ANCA-treated murine neutrophils revealed the involvement of inflammatory responses, with CypD deficiency reducing ANCA-induced alterations in gene expression. Furthermore, analyses of upstream regulators revealed the relevance of intracellular calcium (CypD activator) and cyclosporin (CypD inhibitor) in ANCA stimulation, indicating that the CypD-dependent opening of mitochondrial permeability transition pores is associated with ANCA-induced neutrophil activation and NETosis. In both AAV mouse models, the genetic deletion of CypD ameliorated crescentic glomerulonephritis via the inhibition of CypD-dependent neutrophil and endothelial necrosis.CypD targeting is a novel and specific therapeutic strategy for AAV via the resolution of necrotizing vasculitis.

Two decades have passed since therapeutic angiogenesis was proposed to promote reparative collateral growth as an alternative therapy for ischemic diseases in patients for whom neither surgical revascularization nor endovascular therapy was suitable. When therapeutic angiogenesis first began, local administration was conducted using recombinant growth factor proteins or gene-encoding growth factors for endothelial cells. Since then, autologous stem cells and endothelial progenitor cell transplantation therapy have been developed. Although many clinical trials have been performed on patients, most therapies have not yet become standard treatments. We have developed a nanoparticle (NP)-mediated, drug-targeting delivery system using bioabsorbable poly-lactic/glycolic acid (PLGA) NPs. In several animal models, pitavastatin-incorporated (Pitava)-NPs showed significant therapeutic effects on critical limb ischemia. Because PLGA NPs are delivered selectively to vascular endothelial cells after intramuscular administration, it is suggested that therapeutic angiogenesis/arteriogenesis plays an important role in the mechanism by which Pitava-NPs exert beneficial therapeutic effects. To translate this to clinical medicine, we have performed studies and produced Pitava-NPs in compliance with good laboratory practice/good manufacturing practice regulations, and completed a phase I/II clinical trial, reporting the safety and efficacy of Pitava-NP intramuscular injection for patients with critical limb ischemia. This review will focus on therapeutic angiogenesis/arteriogenesis for peripheral arterial disease induced by Pitava-NPs.

Background: Myocardial ischemia-reperfusion (IR) injury limits the beneficial effects of early reperfusion therapy for acute myocardial infarction. Emerging evidence suggests that 7-ketocholesterol (7-KC), one of the most common dietary oxysterols, has pro-inflammatory properties and correlates with cardiovascular diseases. However, the underlying mechanisms remain scant. Here we investigated the effects of 7-KC on myocardial IR injury in mice. Methods and Results: Wild type mice were fed either a control high-fat high-cholesterol diet (HFHCD) or HFHCD containing 7-KC (7KC-HFHCD) for three weeks. In a murine model of 30-min myocardial ischemia and subsequent reperfusion, dietary 7KC-HFHCD increased plasma 7-KC level (145 ± 89.7 ng/mL vs 399 ± 175 ng/mL, Phigh inflammatory monocytes to total monocytes increased in 7KC-HFHCD group as assessed by flow cytometric analysis (49 ± 15% vs 61 ± 9.3%, Pin vitro that 7-KC induced ER stress, mitochondrial reactive oxygen species, and nuclear factor-kappa B activation associated with increased mRNA levels of pro-inflammatory cytokines such as MCP-1 and TNF-α in murine peritoneal macrophages. Administration of N-acetyl-L-cysteine (5mM), an antioxidant, decreased 7-KC-induced pro-inflammatory cytokines, and this decrease was not observed in Tlr4 -/- murine macrophages, or in macrophages transfected with liver X receptor siRNA. Conclusions: Dietary 7-KC exacerbated myocardial IR injury through macrophage-mediated inflammation in mice. Oxidative stress is involved in the 7-KC-induced pro-inflammatory response in macrophages. Dietary oxysterols are a promising therapeutic target for IR injury.

Background The opening of mitochondrial permeability transition pore and inflammation cooperatively progress myocardial ischemia‐reperfusion (IR) injury, which hampers therapeutic effects of primary reperfusion therapy for acute myocardial infarction. We examined the therapeutic effects of nanoparticle‐mediated medicine that simultaneously targets mitochondrial permeability transition pore and inflammation during IR injury. Methods and Results We used mice lacking cyclophilin D (CypD, a key molecule for mitochondrial permeability transition pore opening) and C‐C chemokine receptor 2 and found that CypD contributes to the progression of myocardial IR injury at early time point (30–45 minutes) after reperfusion, whereas C‐C chemokine receptor 2 contributes to IR injury at later time point (45–60 minutes) after reperfusion. Double deficiency of CypD and C‐C chemokine receptor 2 enhanced cardioprotection compared with single deficiency regardless of the durations of ischemia. Deletion of C‐C chemokine receptor 2, but not deletion of CypD, decreased the recruitment of Ly‐6C high monocytes after myocardial IR injury. In CypD‐knockout mice, administration of interleukin‐1β blocking antibody reduced the recruitment of these monocytes. Combined administration of polymeric nanoparticles composed of poly‐lactic/glycolic acid and encapsulating nanoparticles containing cyclosporine A or pitavastatin, which inhibit mitochondrial permeability transition pore opening and monocyte‐mediated inflammation, respectively, augmented the cardioprotection as compared with single administration of nanoparticles containing cyclosporine A or pitavastatin after myocardial IR injury. Conclusions Nanoparticle‐mediated simultaneous targeting of mitochondrial injury and inflammation could be a novel therapeutic strategy for the treatment of myocardial IR injury.

Aim To assess the results of a phase I/IIa open-label dose-escalation clinical trial of 5-day repeated intramuscular administration of pitavastatin-incorporated poly (lactic-co-glycolic acid) nanoparticles (NK-104-NP) in patients with chronic limb threatening ischemia (CLTI). Methods NK-104-NP was formulated using an emulsion solvent diffusion method. NK-104-NP at four doses (nanoparticles containing 0.5, 1, 2, and 4 mg of pitavastatin calcium, n=4 patients per dose) was investigated in a dose-escalation manner and administered intramuscularly into the ischemic limbs of 16 patients with CLTI. The safety and therapeutic efficacy of treatment were investigated over a 26-week follow-up period. Results No cardiovascular or other serious adverse events caused by NK-104-NP were detected during the follow-up period. Improvements in Fontaine and Rutherford classifications were noted in five patients (one, three, and one in the 1-, 2-, and 4-mg dose groups, respectively). Pharmacokinetic parameters including the maximum serum concentration and the area under the blood concentration-time curve increased with pitavastatin treatment in a dose-dependent manner. The area under the curve was slightly increased at day 5 compared with that at day 1 of treatment, although the difference was not statistically significant. Conclusions This is the first clinical trial of pitavastatin-incorporated nanoparticles in patients with CLTI. Intramuscular administration of NK-104-NP to the ischemic limbs of patients with CLTI was safe and well tolerated and resulted in improvements in limb function.

Introduction: Macrophage-mediated inflammation promotes various vascular diseases, but how macrophage functions are regulated has not been fully understood. Notch signaling is a fundamental pathway regulating cell differentiation in developmental stages, but its role in adult vascular diseases are largely unknown. Aim: The aim of this study is to explore the role of Notch ligands in macrophage activation and subsequent arteriosclerosis. Methods: 1) To screen the Notch ligands, blocking antibodies of each Notch ligands (Jagged-1, Jagged-2, Dll1, and Dll4) were prepared. After pre-treatment with these antibodies, mouse peritoneal macrophages were stimulated with 10 ng/mL LPS + 10 ng/mL IFN-γ or 10 ng/mL IL-4 for 24 hours. mRNA expressions were quantified by real-time PCR. 2) C57BL/6J mice were subjected to femoral arterial wire injury and blocking antibodies (250 μg/injection) were intraperitoneally injected twice a week for 4 weeks, 3) Apolipoprotein E-deficient mice ( Apoe -/- ) at 8 weeks of age were fed a high-fat diet and blocking antibodies (250 μg/injection) were intraperitoneally injected twice a week for 12 weeks. Results: Blockade of Jagged 2, Dll1 and Dll4 decreased expression of iNOS and IL-1β in macrophages treated with LPS and IFN-γ. In contrast, blockade of Dll1 increased arginase-1 and Ym1 in IL-4 treated macrophages. Immobilized recombinant Dll1 increased prototypical Notch-target gene Hes-1 and induced subsequent IL-1β expression. Therefore, we have tested Dll1 blocking antibodies in in vivo models. After femoral arterial injury, administration of Dll1 blocking antibodies attenuated intimal hyperplasia, perivascular fibrosis, and negative remodeling of injured arteries. In Apoe -/- mice, a high-fat diet feeding increased Dll1 expression in atherosclerotic plaques, especially in Mac-3 positive macrophages. Blockade of Dll1 decreased atherosclerotic plaque area and lipid content determined by oil red O staining. In addition, blockade of Dll1 decreased accumulation of Mac-3 positive macrophages to the plaque. Conclusions: These results indicate that Dll1 promotes macrophage activation and arteriosclerosis. Dll1 may be a potential therapeutic target for vascular diseases promoted by macrophage-mediated inflammation.

Ischemia–reperfusion injury impairs the efficacy of reperfusion therapy after ischemic stroke. Cyclophilin D (CypD)-mediated openings of mitochondrial permeability transition pore (mPTP) and subsequent monocyte-mediated inflammation are considered as major mechanisms of reperfusion injury. However, no medical therapies are currently available. Therefore, we have tested a hypothesis that simultaneous targeting of mPTP and inflammation confers substantial neuroprotection after cerebral ischemia–reperfusion. To address this point, we prepared CypD knockout mice, C–C chemokine receptor 2 (CCR2) knockout mice and CypD/CCR2 double knockout mice. These mice were subjected to 60 min transient cerebral ischemia by occluding middle cerebral arteries. Neurological deficits evaluated 3 days after reperfusion were significantly attenuated in CypD/CCR2 double knockout mice as compared to wild-type mice and other single knockout mice. Then, we have prepared polymeric nanoparticles containing cyclosporine A (CsA-NPs) and pitavastatin (Pitava-NPs), targeting mPTP opening and inflammation, respectively. Simultaneous administration of CsA-NP and Pitava-NP at the time of reperfusion also decreased infarct size and attenuated neurological deficits as compared to control nanoparticles and single administration of CsA-NPs or Pitava-NPs. These results indicate that simultaneous targeting of the mPTP opening and monocyte-mediated inflammation could be a novel strategy for better neurological outcomes in patients with ischemic stroke.

Objective: Mitochondria consistently change their morphology in a process regulated by proteins, including Drp1 (dynamin-related protein 1), a protein promoting mitochondrial fission. Drp1 is involved in the mechanisms underlying various cardiovascular diseases, such as myocardial ischemia/reperfusion injury, heart failure, and pulmonary arterial hypertension. However, its role in macrophages, which promote various vascular diseases, is poorly understood. We therefore tested our hypothesis that macrophage Drp1 promotes vascular remodeling after injury. Method and Results: To explore the selective role of macrophage Drp1, we created macrophage-selective Drp1-deficient mice and performed femoral arterial wire injury. In these mice, intimal thickening and negative remodeling were attenuated at 4 weeks after injury when compared with control mice. Deletion of macrophage Drp1 also attenuated the macrophage accumulation and cell proliferation in the injured arteries. Gain- and loss-of-function experiments using cultured macrophages indicated that Drp1 induces the expression of molecules associated with inflammatory macrophages. Morphologically, mitochondrial fission was induced in inflammatory macrophages, whereas mitochondrial fusion was induced in less inflammatory/reparative macrophages. Pharmacological inhibition or knockdown of Drp1 decreased the mitochondrial reactive oxygen species and chemotactic activity in cultured macrophages. Co-culture experiments of macrophages with vascular smooth muscle cells indicated that deletion of macrophage Drp1 suppresses growth and migration of vascular smooth muscle cells induced by macrophage-derived soluble factors. Conclusions: Macrophage Drp1 accelerates intimal thickening after vascular injury by promoting macrophage-mediated inflammation. Macrophage Drp1 may be a potential therapeutic target of vascular diseases.

Pulmonary hypertension (PH) is a disease with poor prognosis, caused by the obstruction/stenosis of small pulmonary arteries. Statin is known to have vasodilating and anti-inflammatory property and is considered to be a candidate of therapeutic agents for the treatment of PH, but its efficacy has not been verified in clinical trials. We have formulated pitavastatin incorporating nanoparticles composed of poly (lactic-co-glycolic acid) (NK-104-NP) to improve drug delivery to the pulmonary arteries and evaluated their safety and pharmacokinetics in healthy volunteers. To accomplish this purpose, phase I clinical trials were conducted. In the single intravenous administration regimen, 40 healthy subjects were enrolled and PK (pharmacokinetic) parameters in 4 groups (1, 2, 4, and 8 mg as pitavastatin calcium) were as follows: 1.00 hour after the administration, the plasma concentration of pitavastatin reached Cmax (the maximum drug concentration) in all groups. Cmax, AUC0-t (area under the curve from time 0 to the last measurable concentration) and AUC0-∞ (area under the curve from time 0 extrapolated to infinite time) were increased in a dose-dependent manner. Population pharmacokinetic analysis based on these results indicated no accumulation of pitavastatin after repeated administration of NK-104-NP for 7 days. In this 7-day administration trial, the mean Cmax and AUC0-∞ of pitavastatin were not significantly different between days 1 and 7, suggesting that pitavastatin is unlikely to accumulate after repeated administration. In these trials, three adverse events (AEs) were reported, but they were resolved without any complications and judged to have no causal relationships with NK-104-NP. These results indicate that the innovative nanotechnology-based medicine NK-104-NP exhibited dose-dependent pharmacokinetics and was well tolerated with no significant AEs in healthy volunteers.

Administration of drugs and other therapeutic agents has been the central strategy of contemporary medicine for cardiovascular disease. The use of drug delivery systems (DDS) includes micelles, liposomes, polymeric nanoparticles, dendrimers, carbon nanotubes, and crystalline metals. Nano-DDS modify in vivo drug kinetics, depending on (patho)physiological mechanisms such as retard excretion, vascular permeability, and incorporation by mononuclear phagocyte systems, which constitute the 'passive-targeting' property of nano-DDS. These properties of nano-DDS are applicable to inflammatory diseases including atherosclerosis. Atherosclerotic plaque destabilization and rupture account for the majority of acute myocardial infarction, for which inflammatory monocytes and macrophages play critical roles. In our experience, polymeric nanoparticles have been delivered to inflammatory monocytes and macrophages in an atherosclerotic mouse model. Nano-DDS loaded with pioglitazone reduced Ly6Chigh inflammatory monocytes and increased Ly6Clow non-inflammatory monocytes in the peripheral blood, and induced M2 macrophage-associated genes in the aorta. Pioglitazone-nanoparticles finally stabilized atherosclerotic plaques assessed by a decrease in the number of buried fibrous caps in the plaque. Application of nano-DDS is a unique and promising approach to prevent life-threatening cardiovascular events including acute myocardial infarction by regulating inflammation in the cardiovascular system.

The Notch signaling pathway regulates the development of various cell types and organs, and also contributes to disease mechanisms in adults. Accumulating evidence suggests its role in cardiovascular and metabolic diseases. Notch signaling components also control the phenotype of immune cells. Delta-like ligand 4 (Dll4) of the Notch pathway promotes proinflammatory activation of macrophages in vitro and in vivo. Dll4 blockade attenuates chronic atherosclerosis, vein graft disease, vascular calcification, insulin resistance, and fatty liver in mice. The Dll4–Notch axis may, thus, participate in the shared mechanisms for cardiometabolic disorders, serving as a potential therapeutic target for ameliorating these global health problems.

Statins are known to improve pulmonary arterial hypertension (PAH) by their anti-inflammatory and anti-proliferative effects in animal models. However, recent clinical studies have reported that clinically approved statin doses failed to improve clinical outcomes in patients with PAH. We therefore hypothesized that nanoparticle (NP) -mediated targeting of pitavastatin could attenuate the progression of established PAH.We induced PAH by subcutaneously injecting monocrotaline (MCT) in Sprague-Dawley rats. On day 14 after the MCT injection, animals that displayed established PAH on echocardiography were included. On day 17, they were randomly assigned to the following 5 groups: daily intravenous administration of (1) vehicle, (2) fluorescein-isothiocyanate-NP, (3) pitavastatin, (4) pitavastatin-NP, or (5) oral sildenafil. Intravenous NP was selectively delivered to small pulmonary arteries and circulating CD11b-positive leukocytes. On day 21, pitavastatin-NP attenuated the progression of PAH at lower doses than pitavastatin alone. This was associated with the inhibition of monocyte-mediated inflammation, proliferation, and remodeling of the pulmonary arteries. Interestingly, sildenafil attenuated the development of PAH, but had no effects on inflammation or remodeling of the pulmonary arteries. In separate experiments, only treatment with pitavastatin-NP reduced the mortality rate at day 35.NP-mediated targeting of pitavastatin to small pulmonary arteries and leukocytes attenuated the progression of established MCT-induced PAH and improved survival. Therapeutically, pitavastatin-NP was associated with anti-inflammatory and anti-proliferative effects on small pulmonary arteries, which was completely distinct from the vasodilatory effect of sildenafil. Pitavastatin-NP can be a novel therapeutic modality for PAH.

Pulmonary hypertension (PH) is a disease with poor prognosis, caused by the obstruction/stenosis of small pulmonary arteries. Statin is known to have vasodilating and anti-inflammatory property and is considered to be a candidate of therapeutic agents for the treatment of PH, but its efficacy has not been verified in clinical trials. We have formulated pitavastatin incorporating nanoparticles composed of poly (lactic-co-glycolic acid) (NK-104-NP) to improve drug delivery to the pulmonary arteries and evaluated their safety and pharmacokinetics in healthy volunteers. To accomplish this purpose, phase I clinical trials were conducted. In the single intravenous administration regimen, 40 healthy subjects were enrolled and PK (pharmacokinetic) parameters in 4 groups (1, 2, 4, and 8 mg as pitavastatin calcium) were as follows: 1.00 hour after the administration, the plasma concentration of pitavastatin reached C

Aims Monocyte-mediated inflammation is a major mechanism underlying myocardial ischaemia-reperfusion (IR) injury and the healing process after acute myocardial infarction (AMI). However, no definitive anti-inflammatory therapies have been developed for clinical use. Pioglitazone, a peroxisome proliferator-activated receptor-gamma (PPARγ) agonist, has unique anti-inflammatory effects on monocytes/macrophages. Here, we tested the hypothesis that nanoparticle (NP)-mediated targeting of pioglitazone to monocytes/macrophages ameliorates IR injury and cardiac remodelling in preclinical animal models. Methods and results We formulated poly (lactic acid/glycolic acid) NPs containing pioglitazone (pioglitazone-NPs). In a mouse IR model, these NPs were delivered predominantly to circulating monocytes and macrophages in the IR heart. Intravenous treatment with pioglitazone-NPs at the time of reperfusion attenuated IR injury. This effect was abrogated by pre-treatment with the PPARγ antagonist GW9662. In contrast, treatment with a pioglitazone solution had no therapeutic effects on IR injury. Pioglitazone-NPs inhibited Ly6Chigh inflammatory monocyte recruitment as well as inflammatory gene expression in the IR hearts. In a mouse myocardial infarction model, intravenous treatment with pioglitazone-NPs for three consecutive days, starting 6 h after left anterior descending artery ligation, attenuated cardiac remodelling by reducing macrophage recruitment and polarizing macrophages towards the pro-healing M2 phenotype. Furthermore, pioglitazone-NPs significantly decreased mortality after MI. Finally, in a conscious porcine model of myocardial IR, pioglitazone-NPs induced cardioprotection from reperfused infarction, thus providing pre-clinical proof of concept. Conclusion NP-mediated targeting of pioglitazone to inflammatory monocytes protected the heart from IR injury and cardiac remodelling by antagonizing monocyte/macrophage-mediated acute inflammation and promoting cardiac healing after AMI.

Background: Atherothrombosis of a coronary artery with plaque erosion is a major cause of acute coronary syndrome; however, its pathological mechanism and the effects of lipid-lowering therapy are unknown. We have recently reported the CuVIC Trial showing that ezetimibe added to statins improves coronary endothelial function associated with reductions in serum oxysterols in patients after coronary stenting (Takase, et al. ATVB 2017, in press). Methods and Results: We performed balloon injury in iliofemoral arteries in male Japanese white rabbits fed with high cholesterol diet and infused with angiotensin II. We examined the occurrence of atherothrombosis by echography 3 times/week, which were subsequently confirmed by angiography. Histochemical analysis revealed the lack of PECAM1-positive endothelial layer in the atherothrombotic sites. In treatment protocol, animals were divided into 3 groups; 1. Control, 2. Ezetimibe 0.6 mg/kg/day, and 3. Rosuvastatin 1.0 mg/kg/day, resulting total cholesterol 2514+/-179, 1716+/-111, and 1403+/-174, respectively after 8 weeks. Oral treatment with Ezetimibe significantly reduced atherothrombotic occlusion. Although there was no difference in the extent of arterial stenosis among 3 groups, treatment with ezetimibe was associated with better re-endothelialization and less tissue factor (TF)-positive areas in the intima. Serum oxysterols including 7-ketocholesterol (7KC) were lower in the Ezetimibe group. Serum from the model rabbits or 7KC induced TF in cultured SMCs. TF induction by the serum from the Ezetimibe group was significantly less compared with other groups. Conclusions: We established a valid animal model of spontaneous atherothromobotic occlusion in rabbits, which mimicked plaque erosion observed in human coronary arteries. Reduction in serum oxysterols with ezetimibe may prevent atherothrombosis from plaque erosion, by accelerating re-endothelialization and inhibiting TF expression in injured arteries.

Peripheral arterial disease (PAD) causes peripheral tissue ischemia, resulting in skin ulceration, gangrene, and limb amputation. The therapeutic goal is restoration of blood supply, and various treatments including exercise, medical therapy, and surgical revascularization are performed. The prognosis of critical limb ischemia, however, is still unsatisfactory, and there are unmet needs in developing novel therapeutics. We have been developing novel nanotechnology-based drug delivery system aiming efficient angiogenesis by delivering of HMG-CoA reductase inhibitor (statin) to endothelium. In this chapter, we summarized current developing status of nanotechnology-based drug delivery system including animal experiments and ongoing clinical trials (CLINICAL TRIAL REGISTRATION NUMBER: UMIN000008011).

Background Budding uninhibited by benzimidazole‐related 1 (BubR1), a cell cycle–related protein, is an essential component of the spindle checkpoint that regulates cell division. BubR1 insufficiency causes early aging‐associated vascular phenotypes. We generated low‐ BubR1 ‐expressing mutant ( BubR1 L/L ) and apolipoprotein E‐deficient ( ApoE −/− ) mice ( BubR1 L/L ‐ApoE −/− mice) to investigate the effects of BubR1 on atherosclerosis. Methods and Results Eight‐week‐old male BubR1 L/L ‐ApoE −/− mice and age‐matched ApoE −/− mice were used in this study. Atherosclerotic lesion development after being fed a high‐cholesterol diet for 12 weeks was inhibited in BubR1 L/L ‐ApoE −/− mice compared with ApoE −/− mice, and was accompanied by decreased accumulation of macrophages. To address the relative contribution of BubR1 on bone marrow–derived cells compared with non‐bone marrow–derived cells, we performed bone marrow transplantation in ApoE −/− and BubR1 L/L ‐ApoE −/− mice. Decreased BubR1 in bone marrow cells and non‐bone marrow–derived cells decreased the atherosclerotic burden. In vitro assays indicated that decreased BubR1 expression impaired proliferation, but not migration, of bone marrow–derived macrophages. Conclusions BubR1 may represent a promising new target for regulating atherosclerosis.

Background Mitochondria‐mediated cell death plays a critical role in myocardial ischemia‐reperfusion ( IR ) injury. We hypothesized that nanoparticle‐mediated drug delivery of mitochondrial division inhibitor 1 (Mdivi1) protects hearts from IR injury through inhibition of mitochondria outer membrane permeabilization ( MOMP ), which causes mitochondrial‐mediated cell death. Methods and Results We formulated poly (lactic‐co‐glycolic acid) nanoparticles containing Mdivi1 (Mdivi1‐ NP ). We recently demonstrated that these nanoparticles could be successfully delivered to the cytosol and mitochondria of cardiomyocytes under H 2 O 2 ‐induced oxidative stress that mimicked IR injury. Pretreatment with Mdivi1‐ NP ameliorated H 2 O 2 ‐induced cell death in rat neonatal cardiomyocytes more potently than Mdivi1 alone, as indicated by a lower estimated half‐maximal effective concentration and greater maximal effect on cell survival. Mdivi1‐ NP treatment of Langendorff‐perfused mouse hearts through the coronary arteries at the time of reperfusion reduced infarct size after IR injury more effectively than Mdivi1 alone. Mdivi1‐ NP treatment also inhibited Drp1‐mediated Bax translocation to the mitochondria and subsequent cytochrome c leakage into the cytosol, namely, MOMP , in mouse IR hearts. MOMP inhibition was also observed in cyclophilin D knockout (CypD‐ KO ) mice, which lack the mitochondrial permeability transition pore ( MPTP ) opening. Intravenous Mdivi1‐ NP treatment in vivo at the time of reperfusion reduced IR injury in wild‐type and CypD‐ KO mice, but not Bax‐ KO mice. Conclusions Mdivi1‐ NP treatment reduced IR injury through inhibition of MOMP , even in the absence of a CypD/ MPTP opening. Thus, nanoparticle‐mediated drug delivery of Mdivi1 may be a novel treatment strategy for IR injury.

Myocardial ischemia-reperfusion (IR) injury limits the therapeutic effect of early reperfusion therapy for acute myocardial infarction (AMI), in which the recruitment of inflammatory monocytes plays a causative role. Here we develop bioabsorbable poly-lactic/glycolic acid (PLGA) nanoparticles incorporating irbesartan, an angiotensin II type 1 receptor blocker with a peroxisome proliferator-activated receptor (PPAR)γ agonistic effect (irbesartan-NP). In a mouse model of IR injury, intravenous PLGA nanoparticles distribute to the IR myocardium and monocytes in the blood and in the IR heart. Single intravenous treatment at the time of reperfusion with irbesartan-NP (3.0 mg kg−1 irbesartan), but not with control nanoparticles or irbesartan solution (3.0 mg kg−1), inhibits the recruitment of inflammatory monocytes to the IR heart and reduces the infarct size via PPARγ-dependent anti-inflammatory mechanisms and ameliorates left ventricular remodeling 21 days after IR. Irbesartan-NP is a novel approach to treat myocardial IR injury in patients with AMI.

Recent technical innovation has enabled chemical modifications of small materials and various kinds of nanoparticles have been created. In clinical settings, nanoparticle-mediated drug delivery systems have been used in the field of cancer care to deliver therapeutic agents specifically to cancer tissues and to enhance the efficacy of drugs by gradually releasing their contents. In addition, nanotechnology has enabled the visualization of various molecular processes by targeting proteinases or inflammation. Nanoparticles that consist of poly (lactic-co-glycolic) acid (PLGA) deliver therapeutic agents to monocytes/macrophages and function as anti-inflammatory nanoparticles in combination with statins, angiotensin receptor antagonists, or agonists of peroxisome proliferator-activated receptor-γ (PPARγ). PLGA nanoparticle-mediated delivery of pitavastatin has been shown to prevent inflammation and ameliorated features associated with plaque ruptures in hyperlipidemic mice. PLGA nanoparticles were also delivered to tissues with increased vascular permeability and nanoparticles incorporating pitavastatin, injected intramuscularly, were retained in ischemic tissues and induced therapeutic arteriogenesis. This resulted in attenuation of hind limb ischemia. Ex vivo treatment of vein grafts with imatinib nanoparticles before graft implantation has been demonstrated to inhibit lesion development. These results suggest that nanoparticle-mediated drug delivery system can be a promising strategy as a next generation therapy for atherosclerotic vascular diseases.

Atherosclerosis is a chronic inflammatory disorder in arteries, and its complications cause major clinical problems such as acute myocardial infarction and stroke, leading to global health threats. Particularly, activated macrophages participate in multiple steps of atherogenesis from the initiation, to the lesion progression, to the onset of acute complications. To examine its molecular mechanisms and develop new therapies, experimental models of atherosclerosis play important roles. Due to the availability of various genetically altered strains, mouse models of atherosclerosis enormously contribute to testing specific biological hypotheses. Mouse models of atherosclerosis also help to evaluate and monitor the effects of new therapies. This chapter briefly discusses the history of the development of animal models of atherosclerosis and summarizes several mouse strains commonly used in atherosclerosis research. As do clinical trials, preclinical studies should employ multidisciplinary approaches to provide the unambiguous evidence that supports a biological hypothesis or examine the effects of a new therapy from various angles. We thus describe the experimental protocols of atherosclerosis research in mice that covers several disciplines, including ultrasonography, molecular imaging of macrophage activation, histological analyses, and biochemical assays.

Macrophages and smooth muscle cells (SMCs) represent major players in the pathogenesis of atherosclerotic vascular diseases. SMCs often reside in close proximity to macrophage clusters. Activated macrophages may promote pro-atherogenic functions of SMCs. Addressing macrophage-dependent mechanisms of SMC activation may provide new insight into atherogenesis and new therapies for various vascular diseases. Direct evidence for such interplay between atherosclerosis-associated cell types, however, remains scant. While SMC-derived macrophage foam cells have long been reported, recent evidence has also identified SMC-like cells of monocyte origin, suggesting dynamic interchangeability of these cell types. Future efforts may help to understand the interplay between key cell types and offer new paradigms in vascular medicine and pharmacology.

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 −/− 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.

The receptor for advanced glycation end products (RAGE) and the angiotensin II type I receptor (AT1R) have been separately linked to the pathogenesis of diabetic atherosclerosis. However, no prior study has addressed a linkage between RAGE and AT1R in diabetic atherogenesis. Therefore, we tested the hypothesis that upregulation of the ligand-RAGE axis via AT1R is an essential process underlying the disease. Diabetes was induced in apolipoprotein E-deficient (ApoE(-/-)) mice by streptozotocin, and diabetic mice were treated with AT1 receptor blocker (ARB) for 6 weeks. Diabetic ApoE(-/-) mice that were AT1R-deficient (ApoE(-/-)AT1aR(-/-)) were also investigated. In diabetic ApoE(-/-) mice, AT1R was found to increase within 1 week of diabetes induction, before ligand-RAGE pathway activation and other inflammatory changes were observed. Both ARB treatment and AT1aR deficiency suppressed diabetic atherosclerosis, ligand-RAGE expression and inflammatory changes. In contrast, upregulation of the ligand-RAGE pathway was noted in atherosclerotic plaques from non-diabetic ApoE(-/-) mice infused with angiotensin II. In cultured vascular smooth muscle cells, angiotensin II increased RAGE protein levels via AT1R stimulation. Upregulation of the ligand-RAGE pathway via AT1R is an essential mechanism in diabetic atherosclerosis, implying that ARB might decrease diabetic atherogenesis by inhibiting ligand-RAGE signals.

Objectives Chronic renal disease (CRD) accelerates atherosclerosis and cardiovascular calcification. Statins reduce low-density lipoprotein-cholesterol levels in patients with CRD, however, the benefits of statins on cardiovascular disease in CRD remain unclear. This study has determined the effects of pitavastatin, the newest statin, on arterial inflammation and calcification in atherogenic mice with CRD. Methods and Results CRD was induced by 5/6 nephrectomy in cholesterol-fed apolipoprotein E-deficient mice. Mice were randomized into three groups: control mice, CRD mice, and CRD mice treated with pitavastatin. Ultrasonography showed that pitavastatin treatment significantly attenuated luminal stenosis in brachiocephalic arteries of CRD mice. Near-infrared molecular imaging and correlative Mac3 immunostaining demonstrated a significant reduction in macrophage accumulation in pitavastatin-treated CRD mice. Pitavastatin treatment reduced levels of osteopontin in plasma and atherosclerotic lesions in CRD mice, but did not produce a significant reduction in calcification in atherosclerotic plaques as assesses by histology. CRD mice had significantly higher levels of phosphate in plasma than did control mice, which did not change by pitavastatin. In vitro, pitavastatin suppressed the expression of osteopontin in peritoneal macrophages stimulated with phosphate or calcium/phosphate in concentrations similar to those found in human patients with CRD. Conclusion Our study provides in vivo evidence that pitavastatin reduces inflammation within atherosclerotic lesions in CRD mice.

Objective—Despite its large clinical impact, the underlying mechanisms for vein graft failure remain obscure and no effective therapeutic solutions are available. We tested the hypothesis that Notch signaling promotes vein graft disease. Approach and Results—We used 2 biotherapeutics for Delta-like ligand 4 (Dll4), a Notch ligand: (1) blocking antibody and (2) macrophage- or endothelial cell (EC)–targeted small-interfering RNA. Dll4 antibody administration for 28 days inhibited vein graft lesion development in low-density lipoprotein (LDL) receptor-deficient (Ldlr−/−) mice, and suppressed macrophage accumulation and macrophage expression of proinflammatory M1 genes. Dll4 antibody treatment for 7 days after grafting also reduced macrophage burden at day 28. Dll4 silencing via macrophage-targeted lipid nanoparticles reduced lesion development and macrophage accumulation, whereas EC-targeted Dll4 small-interfering RNA produced no effects. Gain-of-function and loss-of-function studies suggested in vitro that Dll4 induces proinflammatory molecules in macrophages. Macrophage Dll4 also stimulated smooth muscle cell proliferation and migration and suppressed their differentiation. Conclusions—These results suggest that macrophage Dll4 promotes lesion development in vein grafts via macrophage activation and crosstalk between macrophages and smooth muscle cells, supporting the Dll4–Notch axis as a novel therapeutic target., United States. National Institutes of Health (R01HL107550), American Heart Association (0655878T), American Heart Association (12GRNT9510001), American Heart Association (12GRNT1207025), Good Samaritan Foundation, Shapiro Family Foundation

Emerging evidence suggests that Notch signaling not only regulates biological processes during development but also participates in the pathogenesis of various diseases in adults, including tumor angiogenesis, hematopoietic malignancies, and cardiometabolic syndromes. Notch signaling involves several ligands and receptors that have unique and overlapping functions. Therefore, blocking function of a ligand or receptor with a neutralizing antibody is a useful approach to examine the specific role of each Notch component. In addition, administration of Notch signaling blocking antibodies in experimental animals offers important insights into clinical translation of Notch biology. In this chapter, we describe examples of in vitro and in vivo loss-of-function experiments with blockade of Notch ligands, particularly Delta-like ligand 4 (Dll4).

Background: Vein graft failure causes devastating complications in patients with peripheral arterial disease or ischemic heart disease, but its underlying mechanisms remain obscure and no effective therapeutic measures are available. Methods and Results: We tested the hypothesis that Notch signaling triggered by its ligand Delta-like 4 (Dll4) promotes macrophage activation and vein graft disease. Vein graft surgery was performed in high-fat fed LDL receptor-deficient (Ldlr-/-) mice by implanting donor inferior vena cava into recipient right carotid arteries. [Approach 1: Dll4 antibody] Dll4 blocking antibody was administered for 28 days. Dll4 blockade inhibited lesion development and macrophage accumulation (Figures) in vein grafts, and suppressed macrophage expression of genes typical of pro-inflammatory M1 macrophages (e.g., IL-1β, TNF-α). In vivo molecular imaging demonstrated that Dll4 antibody treatment suppressed MMP activity in lesional macrophages. Dll4 blockade concomitantly attenuated collagen thinning. [Approach 2: siRNA delivery to macrophages] To address the relative contribution of macrophages to Dll4-mediated vein graft disease in vivo, we delivered Dll4 siRNA oligos encapsulated in macrophage-targeted lipid nanoparticles. In vivo Dll4 silencing in macrophages reduced lesion development and macrophage burden in vein grafts of Ldlr-/- mice to a similar extent as those of Dll4 antibody therapy. In vitro gain-of-function and loss-of-function studies suggested that Dll4 promotes expression of pro-inflammatory molecules in macrophages. Furthermore, macrophage Dll4 stimulated smooth muscle cell (SMC) proliferation and migration and suppressed their differentiation. Conclusions: These results suggest the novel mechanism that macrophage Dll4 promotes vein graft lesion development by exacerbating inflammation and crosstalk between macrophages and SMC, supporting the Dll4-Notch axis as a potential therapeutic target.

Emerging evidence suggests that Notch signaling not only regulates biological processes during development but also participates in the pathogenesis of various diseases in adults, including tumor angiogenesis, hematopoietic malignancies, and cardiometabolic syndromes. Notch signaling involves several ligands and receptors that have unique and overlapping functions. Therefore, blocking function of a ligand or receptor with a neutralizing antibody is a useful approach to examine the specific role of each Notch component. In addition, administration of Notch signaling blocking antibodies in experimental animals offers important insights into clinical translation of Notch biology. In this chapter, we describe examples of in vitro and in vivo loss-of-function experiments with blockade of Notch ligands, particularly Delta-like ligand 4 (Dll4).

Background— Preventing atherosclerotic plaque destabilization and rupture is the most reasonable therapeutic strategy for acute myocardial infarction. Therefore, we tested the hypotheses that (1) inflammatory monocytes play a causative role in plaque destabilization and rupture and (2) the nanoparticle-mediated delivery of pitavastatin into circulating inflammatory monocytes inhibits plaque destabilization and rupture. Methods and Results— We used a model of plaque destabilization and rupture in the brachiocephalic arteries of apolipoprotein E–deficient (ApoE −/− ) mice fed a high-fat diet and infused with angiotensin II. The adoptive transfer of CCR2 +/+ Ly-6C high inflammatory macrophages, but not CCR2 −/− leukocytes, accelerated plaque destabilization associated with increased serum monocyte chemoattractant protein-1 (MCP-1), monocyte-colony stimulating factor, and matrix metalloproteinase-9. We prepared poly(lactic-co-glycolic) acid nanoparticles that were incorporated by Ly-6G − CD11b + monocytes and delivered into atherosclerotic plaques after intravenous administration. Intravenous treatment with pitavastatin-incorporated nanoparticles, but not with control nanoparticles or pitavastatin alone, inhibited plaque destabilization and rupture associated with decreased monocyte infiltration and gelatinase activity in the plaque. Pitavastatin-incorporated nanoparticles inhibited MCP-1–induced monocyte chemotaxis and the secretion of MCP-1 and matrix metalloproteinase-9 from cultured macrophages. Furthermore, the nanoparticle-mediated anti–MCP-1 gene therapy reduced the incidence of plaque destabilization and rupture. Conclusions— The recruitment of inflammatory monocytes is critical in the pathogenesis of plaque destabilization and rupture, and nanoparticle-mediated pitavastatin delivery is a promising therapeutic strategy to inhibit plaque destabilization and rupture by regulating MCP-1/CCR2–dependent monocyte recruitment in this model.