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

Post-annealed graphite carbon nitride nanoplates obtained by sugar-assisted exfoliation with improved visible-light photocatalytic performance

Two-dimensional (2D) graphitic carbon nitride (g-C3N4) nanoplates (CNNP) have become a hot research topic in photocatalysis due to their small thickness and large specific surface area that favors charge transport and catalytic surface reactions. However, the wide application of 2D g-C3N4 nanoplates prepared by ordinary methods suffers from increased band gaps with a poor solar harvesting capability caused by the strong quantum confinement effect and reduced conjugation distance. In this paper, a facile approach of exfoliation and the following fast thermal treatment of the bulk g-C3N4 is proposed to obtain a porous few-layered g-C3N4 with nitrogen defects. Due to the preferable crystal, textural, optical and electronic structures, the as-obtained porous CNNP demonstrated a significantly improved photocatalytic activity towards water splitting than the bulk g-C3N4 and even the 3 nm-thick CNNP obtained by sugarassisted exfoliation of the bulk g-C3N4. The difference in the enhancement factors between the H2O splitting and organic decomposition has revealed the effect of N defects. This study offers insightful outlooks on the scalable fabrication of a porous few-layered structure with a promoted photocatalytic performance. (C) 2020 Elsevier Inc. All rights reserved

Sugar-assisted mechanochemical exfoliation of graphitic carbon nitride for enhanced visible-light photocatalytic performance

A simple co-grinding treatment with fructose is introduced for the efficient and scalable preparation of g-C3N4 nanoplates. The results revealed that these g-C3N4 nanoplates still preserved the basic framework of carbon nitride and even displayed superior morphological properties and electronic structures. With respect to the pristine carbon nitride, the few-layered g-C3N4 impressively demonstrated an enhanced photocatalytic activity towards hydrogen generation and the degradation of Rhodamine B (RhB) under visible light illumination, emphasizing the vital roles of the morphology and electronic structures on the photocatalytic performance. This study provided sustainable and cost-effective tactics for the delamination of g-C3N4 for efficient energy conversion. (C) 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved

Possible monitoring of mesophotic scleractinian corals using an underwater mini-ROV to sample coral eDNA

Mesophotic coral ecosystems (MCEs) are light-dependent tropical or subtropical communities occurring at depths of 30–150 m. Broader surveys of MCEs are needed to better understand stony corals, the keystone species of coral-reef ecosystems. While MCEs have been studied by professional SCUBA divers and with deep-sea robots, comprehensive surveys of MCEs are required. An eDNA metabarcoding method has recently been used to survey scleractinian corals in shallow reefs. We tested whether MCEs might be more comprehensively surveyed by collecting seawater samples using an underwater mini-remote operated vehicle (mini-ROV). Seawater was collected 1–2 m above reef tops at depths of 20–80 m at 24 sites in six locations around the Zamami Islands (Okinawa, Japan). Water samples were then subjected to coral-specific eDNA amplification. Metabarcoding analyses of amplicons showed that except for one site, coral-specific eDNA from approximately 0.5 l seawater samples was sufficient to identify genera. The proportion of Acropora eDNA was higher at shallow reefs and upper ridges of slopes, while the proportion of Porites increased at mesophotic sites. Although further technical improvements are required, this study suggests that it may be possible to monitor mesophotic corals to the generic level using eDNA collected using mini-ROVs

Monitoring mesophotic scleractinian corals using an underwater mini-ROV to sample eDNA

Mesophotic coral ecosystems (MCEs) are light-dependent tropical or subtropical communities occurring at depths of 30 to 150 m. We recently devised a coral-specific environmental DNA (eDNA) barcoding method that can identify 36 scleractinian genera in shallow reefs by sampling ~1L of surface seawater. If eDNA barcoding is combined with sampling using underwater mini-Remote Operated Vehicles (mini-ROVs), it may be possible to survey mesophotic corals more easily and broadly. Around the Zamami Islands, in Okinawa, Japan, seawater was collected 1–2 m above the bottom at six locations 20–80 m below the surface and subjected to coral-specific eDNA amplification. Metabarcoding analyses showed that (a) eDNA from ~0.5 L seawater was sufficient to identify genera and to yield comparative ratios of genera at these sites; (b) Acropora dominates shallow reefs and upper ridges of slopes, while other genera including Porites, Pocillopora and Polyphyllia are more abundant at mesophotic sites; (c) one site showed a gradient in which Acropora was replaced by Plesiastrea at increasing depths. Although further technical improvements are required, the use of eDNA and underwater mini-ROVs may permit monitoring of mesophotic corals more broadly and easily.Funding provided by: JST COI-NEXT project*Crossref Funder Registry ID: Award Number: Funding provided by: Okinawa Institute of Science and Technology Graduate UniversityCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100004199Award Number:Seawater samples were collected at reefs around the Zamami Islands on 9-10 March and 23-25 May 2022. Mini-ROV used in this study was FIFISH V6Plus. On the boat, seawater collected by the sampler was moved to 1L bottle and filtered promptly through 0.45-μm Sterivex filters (Merck) using peristaltic pomp. 1 mL of RNAlater (Qiagen) was added to the filtrate to prevent DNA degradation and was maintained at 4℃ before transfer to a -20℃ freezer in the laboratory. eDNA in Sterivex filters was extracted following the Environmental DNA Sampling and Experiment Manual v. 2.1. Extracted eDNA samples were PCR-amplified using primers, Scle_12S_Fw (5'-CCAGCMGACGCGGTRANACTTA-3') and Scle_12S_Rv (5'-AAWTTGACGACGGCCATGC-3'), for mitochondrial 12S rRNA genes of scleractinian corals. PCR amplification was carried out Tks Gflex DNA Polymerase (Takara) under cycling conditions of 1 min at 94°C, followed by 35 cycles of 10 s at 98°C, 15 s at 60°C and 30 s at 68°C, with an extension of 5 min at 68°C in the final cycle. PCR products were extracted and cleaned with a FastGene Gel/PCR Extraction Kit (NIPPON Genetics Co., Ltd.). Amplicon sequencing libraries of cleaned PCR products were prepared using a KAPA Hyper Prep Kit (NIPPON Genetics) without fragmentation. Libraries were multiplexed and 300-bp paired-end reads were sequenced on a MiSeq platform (Illumina) using a MiSeq Reagent kit v3 (600 cycles)

Supplementary Table S2B. from Possible monitoring of mesophotic scleractinian corals using an underwater mini-ROV to sample coral eDNA

Number of detected sites in relation to each genus

Movie 1 from Possible monitoring of mesophotic scleractinian corals using an underwater mini-ROV to sample coral eDNA

Mesophotic coral ecosystems (MCEs) are light-dependent tropical or subtropical communities occurring at depths of 30–150 m. Broader surveys of MCEs are needed to better understand stony corals, the keystone species of coral-reef ecosystems. While MCEs have been studied by professional SCUBA divers and with deep-sea robots, comprehensive surveys of MCEs are required. An eDNA metabarcoding method has recently been used to survey scleractinian corals in shallow reefs. We tested whether MCEs might be more comprehensively surveyed by collecting seawater samples using an underwater mini-remote operated vehicle (mini-ROV). Seawater was collected 1–2 m above reef tops at depths of 20–80 m at 24 sites in six locations around the Zamami Islands (Okinawa, Japan). Water samples were then subjected to coral-specific eDNA amplification. Metabarcoding analyses of amplicons showed that except for one site, coral-specific eDNA from approximately 0.5 l seawater samples was sufficient to identify genera. The proportion of Acropora eDNA was higher at shallow reefs and upper ridges of slopes, while the proportion of Porites increased at mesophotic sites. Although further technical improvements are required, this study suggests that it may be possible to monitor mesophotic corals to the generic level using eDNA collected using mini-ROVs

Supplementary Table S1. from Possible monitoring of mesophotic scleractinian corals using an underwater mini-ROV to sample coral eDNA

Summary of sequence results