[Partial Arch Repair for Reruptured Aortic Arch Aneurysm due to Endoleak After 2-debranching Thoracic Endovascular Aortic Repair:Report of a Case].

A 67-year-old male was admitted to our hospital for sudden onset chest pain and hoarseness. He underwent 2-debranching thoracic endovascular aortic repair for a ruptured aortic arch aneurysm four years prior. However, computed tomography (CT) revealed an aneurysmal rerupture due to a typeⅠa endoleak. We performed partial arch replacement with uncovered stent removal under intermittent hypothermic circulatory arrest. We needed to be more careful than usual open heart surgery because a non-anatomical bypass procedure was performed. The surgery was successful without any major complications, and the patient was discharged on the 23th postoperative day. Reinterventions post-endovascular repair are sometimes difficult;thus, open surgery could be useful for arch replacement.

Dual-color live imaging unveils stepwise organization of multiple basal body arrays by cytoskeletons.

For mucociliary clearance of pathogens, tracheal multiciliated epithelial cells (MCCs) organize coordinated beating of cilia, which originate from basal bodies (BBs) with basal feet (BFs) on one side. To clarify the self-organizing mechanism of coordinated intracellular BB-arrays composed of a well-ordered BB-alignment and unidirectional BB-orientation, determined by the direction of BB to BF, we generated double transgenic mice with GFP-centrin2-labeled BBs and mRuby3-Cep128-labeled BFs for long-term, high-resolution, dual-color live-cell imaging in primary-cultured tracheal MCCs. At early timepoints of MCC differentiation, BB-orientation and BB-local alignment antecedently coordinated in an apical microtubule-dependent manner. Later during MCC differentiation, fluctuations in BB-orientation were restricted, and locally aligned BB-arrays were further coordinated to align across the entire cell (BB-global alignment), mainly in an apical intermediate-sized filament-lattice-dependent manner. Thus, the high coordination of the BB-array was established for efficient mucociliary clearance as the primary defense against pathogen infection, identifying apical cytoskeletons as potential therapeutic targets.

Enhanced Tumor Targeting and Antitumor Activity of Methylated ß-Cyclodextrin-Threaded Polyrotaxanes by Conjugating Cyclic RGD Peptides.

We previously reported that acid-degradable methylated ß-cyclodextrins (Me-ß-CDs)-threaded polyrotaxanes (Me-PRXs) can induce autophagic cell death through endoplasmic reticulum (ER) stress-related autophagy, even in apoptosis-resistant cells. Hence, Me-PRXs show great potential as anticancer therapeutics. In this study, peptide-supermolecule conjugates were designed to achieve the targeted delivery of Me-PRX to malignant tumors. Arg-Gly-Asp peptides are well-known binding motifs of integrin αvß3, which is overexpressed on angiogenic sites and many malignant tumors. The tumor-targeted cyclic Arg-Gly-Asp (cRGD) peptide was orthogonally post-modified to Me-PRX via click chemistry. Surface plasmon resonance (SPR) results indicated that cRGD-Me-PRX strongly binds to integrin αvß3, whereas non-targeted cyclic Arg-Ala-Glu (cRGE) peptide conjugated to Me-PRX (cRGE-Me-PRX) failed to interact with integrins αvß3. In vitro, cRGD-Me-PRX demonstrated enhanced cellular internalization and antitumor activity in 4T1 cells than that of unmodified Me-PRX and non-targeted cRGE-Me-PRX, due to its ability to recognize integrin αvß3. Furthermore, cRGD-Me-PRX accumulated effectively in tumors, leading to antitumor effects, and exhibited excellent biocompatibility and safety in vivo. Therefore, cRGD conjugation to enhance selectivity for integrin αvß3-positive cancer cells is a promising design strategy for Me-PRXs in antitumor therapy.

Lysosomal cholesterol overload in macrophages promotes liver fibrosis in a mouse model of NASH.

Accumulation of lipotoxic lipids, such as free cholesterol, induces hepatocyte death and subsequent inflammation and fibrosis in the pathogenesis of nonalcoholic steatohepatitis (NASH). However, the underlying mechanisms remain unclear. We have previously reported that hepatocyte death locally induces phenotypic changes in the macrophages surrounding the corpse and remnant lipids, thereby promoting liver fibrosis in a murine model of NASH. Here, we demonstrated that lysosomal cholesterol overload triggers lysosomal dysfunction and profibrotic activation of macrophages during the development of NASH. ß-cyclodextrin polyrotaxane (ßCD-PRX), a unique supramolecule, is designed to elicit free cholesterol from lysosomes. Treatment with ßCD-PRX ameliorated cholesterol accumulation and profibrotic activation of macrophages surrounding dead hepatocytes with cholesterol crystals, thereby suppressing liver fibrosis in a NASH model, without affecting the hepatic cholesterol levels. In vitro experiments revealed that cholesterol-induced lysosomal stress triggered profibrotic activation in macrophages predisposed to the steatotic microenvironment. This study provides evidence that dysregulated cholesterol metabolism in macrophages would be a novel mechanism of NASH.

Supermolecule-Drug Conjugates Based on Acid-Degradable Polyrotaxanes for pH-Dependent Intracellular Release of Doxorubicin.

Doxorubicin (DOX)-conjugated acid-degradable polyrotaxanes (PRXs) were designed as supramolecular drug carriers capable of releasing drugs in acidic cellular environments. Acid-degradable PRXs composed of α-cyclodextrin (α-CD) as a cyclic molecule, poly(ethylene glycol) (PEG) as a polymer axis, and N-triphenylmethyl (N-Trt) groups as an acid-labile stopper molecules were synthesized and DOX was conjugated with the threaded α-CDs in the PRXs. Because the acid-induced cleavage of N-Trt groups in PRXs leads to PRX dissociation, the DOX-modified α-CDs were released under acidic conditions (pH 5.0). The cytotoxicity of DOX-conjugated PRXs in colon-26 cells revealed significant cell death for DOX-conjugated PRXs after 48 h of treatment. Confocal laser scanning microscopy (CLSM) analysis revealed that the fluorescence signals derived from DOX-conjugated PRXs were observed in cellular nuclei after 48 h, suggesting that the DOX-modified α-CDs were released and accumulated in cellular nuclei. These results confirmed that acid-degradable PRXs can be utilized as drug carriers capable of releasing drug-modified α-CDs in acidic lysosomes and eliciting cytotoxicity. Overall, acid-degradable PRXs represent a promising supramolecular framework for the delivery and intracellular release of drug-modified α-CDs, and PRX-drug conjugates are expected to contribute to the development of pH-responsive drug carriers for cancer therapy.

Activated cholesterol metabolism is integral for innate macrophage responses by amplifying Myd88 signaling.

Recent studies have shown that cellular metabolism is tightly linked to the regulation of immune cells. Here, we show that activation of cholesterol metabolism, involving cholesterol uptake, synthesis, and autophagy/lipophagy, is integral to innate immune responses in macrophages. In particular, cholesterol accumulation within endosomes and lysosomes is a hallmark of the cellular cholesterol dynamics elicited by Toll-like receptor 4 activation and is required for amplification of myeloid differentiation primary response 88 (Myd88) signaling. Mechanistically, Myd88 binds cholesterol via its CLR recognition/interaction amino acid consensus domain, which promotes the protein's self-oligomerization. Moreover, a novel supramolecular compound, polyrotaxane (PRX), inhibited Myd88­dependent inflammatory macrophage activation by decreasing endolysosomal cholesterol via promotion of cholesterol trafficking and efflux. PRX activated liver X receptor, which led to upregulation of ATP binding cassette transporter A1, thereby promoting cholesterol efflux. PRX also inhibited atherogenesis in Ldlr-/- mice. In humans, cholesterol levels in circulating monocytes correlated positively with the severity of atherosclerosis. These findings demonstrate that dynamic changes in cholesterol metabolism are mechanistically linked to Myd88­dependent inflammatory programs in macrophages and support the notion that cellular cholesterol metabolism is integral to innate activation of macrophages and is a potential therapeutic and diagnostic target for inflammatory diseases.

Mitigating RANKL-induced cholesterol overload in macrophages with ß-cyclodextrin-threaded polyrotaxanes suppresses osteoclastogenesis.

Free cholesterol acts as an endogenous agonist for estrogen-related receptor α (ERRα), a nuclear receptor that regulates osteoclastogenesis. Because stimulation of macrophages with receptor activator of nuclear factor κB ligand (RANKL) induces an overload of free cholesterol and activates ERRα, we hypothesized that direct removal of cellular cholesterol would suppress osteoclastogenesis. In this study, the effect of 2-hydroxypropyl ß-cyclodextrin (HP-ß-CD), a highly water-soluble cyclic glucopyranose, and ß-CD-threaded polyrotaxanes (PRXs), supramolecular polymers designed to release threaded ß-CDs in acidic lysosomes, on RANKL-induced cholesterol overload and osteoclast differentiation of murine macrophage-like RAW264.7 cells were investigated. PRXs suppressed RANKL-induced cholesterol overload. Additionally, RANKL-induced osteoclast differentiation of RAW264.7 cells was inhibited by PRXs. In contrast, HP-ß-CD did not reduce cholesterol levels or inhibit osteoclast differentiation in RAW264.7 cells. Gene expression analysis of osteoclast markers suggested that PRXs suppress only the early stage of osteoclast differentiation, as PRXs cannot be internalized into multinucleated osteoclasts. However, modification of PRXs with cell-penetrating peptides facilitated their cellular uptake into multinucleated osteoclasts and inhibited osteoclast maturation. Thus, PRXs are promising candidates for inhibiting osteoclast differentiation by suppressing cholesterol overload and may be useful for treating osteoporosis or other bone defects caused by the overactivity of osteoclasts.

Cografting of Zwitterionic Sulfobetaines and Cationic Amines on ß-Cyclodextrin-Threaded Polyrotaxanes Facilitates Cellular Association and Tissue Accumulation with High Biocompatibility.

ß-Cyclodextrins (ß-CDs) and ß-CD-containing polymers have attracted considerable attention as potential candidates for the treatment of cholesterol-related metabolic and intractable diseases. We have advocated the use of ß-CD-threaded acid-degradable polyrotaxanes (PRXs) as intracellular delivery carriers for ß-CDs. As unmodified PRXs are insoluble in aqueous solutions, chemical modification of PRXs is an essential process to improve their solubility and impart novel functionalities. In this study, we investigated the effect of the modification of zwitterionic sulfobetaines on PRXs due to their excellent solubility, biocompatibility, and bioinert properties. Sulfobetaine-modified PRXs were synthesized by converting the tertiary amino groups of precursor 2-(N,N-dimethylamino)ethyl carbamate-modified PRXs (DMAE-PRXs) using 1,3-propanesultone. The resulting sulfobetaine-modified PRXs showed high solubility in aqueous solutions and no cytotoxicity, while their intracellular uptake levels were low. To further improve this system, we designed PRXs cografted with zwitterionic sulfobetaine and cationic DMAE groups via partial betainization of the DMAE groups. Consequently, the interaction with proteins, intracellular uptake levels, and liver accumulation of partly betainized PRXs were found to be higher than those of completely betainized PRXs. Additionally, partly betainized PRXs showed no toxicity in vitro or in vivo despite the presence of residual cationic DMAE groups. Furthermore, partly betainized PRXs ameliorated the abnormal free cholesterol accumulation in Niemann-Pick type C disease patient-derived cells at lower concentrations than ß-CD derivatives and previously designed PRXs. Overall, the cografting of sulfobetaines and amines on PRXs is a promising chemical modification for therapeutic applications due to the high cholesterol-reducing ability and biocompatibility of such modified PRXs. In addition, modification with both zwitterionic and cationic groups can be used for the design of various polymeric materials exhibiting both bioinert and bioactive characteristics.

Calcium phosphate-adsorbable and acid-degradable carboxylated polyrotaxane consisting of ß-cyclodextrins suppresses osteoclast resorptive activity.

Recently, the potential of ß-cyclodextrin-thread acid-degradable polyrotaxane (AdPRX) has been emphasized as a therapeutic agent for cholesterol-related metabolic disorders. In this study, we investigated whether carboxymethyl carbamate-modified AdPRX (CMC-AdPRX) can be used for adsorption to calcium phosphate to treat bone diseases. We first synthesized CMC-AdPRX and used it to coat the calcium phosphate plate. RAW264.7 cells were then differentiated into osteoclasts via a receptor activator of nuclear factor-κB ligand, and the number of osteoclasts and the area of absorption lacunae were determined. The number of tartrate-resistant acid phosphatase-positive multinucleated cells was reduced on the CMC-AdPRX-coated plate. The area of the absorption lacunae was smaller with CMC-AdPRX than with AdPRX, which was not carboxy-modified. Our results suggest that CMC-AdPRX can adsorb to calcium phosphate and act on differentiated osteoclasts to suppress their functional expression.

[Total Aortic Arch Replacement for Typeâ Endoleak after Thoracic Endovascular Aortic Repair using the Chimney Graft Technique:Report of a Case].

Here we report a case of total aortic arch replacement for typeⅠendoleak after thoracic endovascular aortic repair( TEVAR) using the concomitant chimney graft technique. An 81-year-old man was admitted with sudden back pain. Six years prior, he had undergone TEVAR for treatment of a distal aortic arch aneurysm. Preoperative computed tomography revealed an 80-mm-diameter arch aneurysm and typeⅠendoleak. The back pain was caused by impending aneurysmal rupture;therefore, urgent total arch replacement was performed. One stent was cut from the main endograft and anastomosed to its distal side. The bare metal stent in the left common carotid artery was removed and reconstructed at a healthy distal artery. Postoperative computed tomography revealed no endoleak of the aneurysm, and the patient's postoperative course was uneventful.

Hyperhomocysteinemia-related lung disease and hemolytic anemia with bone marrow features masquerading as myelodysplasia.

Hyperhomocysteinemia is linked to TMA-related clinical symptoms such as apparent thromboembolism, microangiopathic hemolytic anemia (MAHA), and various types of end-organ damage due to microvascular thrombi; this is because high plasma levels of homocysteine impair the vascular endothelium. However, the association between hyperhomocysteinemia and pulmonary involvement is unclear. Here, we describe a 63-year-old male who was hospitalized with respiratory failure and MAHA with MDS-like features in the bone marrow. Plasma homocysteine levels were elevated significantly with 199.4 µmol/L (reference: 6.3-18.9) due to a homozygous (T/T) polymorphism for the 677C>T mutation within the MTHFR gene associated with chronic alcoholism-induced folate deficiency. Pulmonary lesions showed ground-glass opacity and there was pleural effusion. The patient was managed successfully with a combination of folate/mecobalamin supplementation, plasma exchange, and a methylprednisolone pulse, followed by oral prednisolone. Clinical symptoms, lung disease, MAHA, and bone marrow abnormalities improved as plasma homocysteine levels normalized.

Planar cell polarity induces local microtubule bundling for coordinated ciliary beating.

Multiciliated cells (MCCs) in tracheas generate mucociliary clearance through coordinated ciliary beating. Apical microtubules (MTs) play a crucial role in this process by organizing the planar cell polarity (PCP)-dependent orientation of ciliary basal bodies (BBs), for which the underlying molecular basis remains elusive. Herein, we found that the deficiency of Daple, a dishevelled-associating protein, in tracheal MCCs impaired the planar polarized apical MTs without affecting the core PCP proteins, causing significant defects in the BB orientation at the cell level but not the tissue level. Using live-cell imaging and ultra-high voltage electron microscope tomography, we found that the apical MTs accumulated and were stabilized by side-by-side association with one side of the apical junctional complex, to which Daple was localized. In vitro binding and single-molecule imaging revealed that Daple directly bound to, bundled, and stabilized MTs through its dimerization. These features convey a PCP-related molecular basis for the polarization of apical MTs, which coordinate ciliary beating in tracheal MCCs.

Surface-tethering of methylated polyrotaxanes with 4-vinylbenzyl groups onto poly(ether ether ketone) substrates for improving osteoblast compatibility.

Poly(ether ether ketone) (PEEK) is a high-performance thermoplastic used for several industrial applications due to its excellent mechanical properties. However, the use of PEEK is limited to dental materials because of its poor implant-bone integration. In the present study, methylated polyrotaxanes (MePRXs) with 4-vinylbenzyl groups, which are supermolecules composed of methylated α-cyclodextrins and poly(ethylene glycol) chains end-capped with 4-vinylbenzyl groups, were covalently tethered onto PEEK surfaces using photo-induced polymerization to improve their osteoblast compatibility. The surface-tethering of MePRXs onto PEEK surfaces was confirmed by analyzing their attenuated total reflectance Fourier transform infrared spectra and contact angles. When mouse preosteoblasts were cultured on the MePRX-PEEK and bare PEEK surfaces, the MePRX-PEEK surfaces showed significantly better proliferation and osteoblast differentiation than the bare PEEK surfaces. These results suggest that surface modification of PEEKs using MePRXs improves their osteoblast compatibility.

A microtubule-LUZP1 association around tight junction promotes epithelial cell apical constriction.

Apical constriction is critical for epithelial morphogenesis, including neural tube formation. Vertebrate apical constriction is induced by di-phosphorylated myosin light chain (ppMLC)-driven contraction of actomyosin-based circumferential rings (CRs), also known as perijunctional actomyosin rings, around apical junctional complexes (AJCs), mainly consisting of tight junctions (TJs) and adherens junctions (AJs). Here, we revealed a ppMLC-triggered system at TJ-associated CRs for vertebrate apical constriction involving microtubules, LUZP1, and myosin phosphatase. We first identified LUZP1 via unbiased screening of microtubule-associated proteins in the AJC-enriched fraction. In cultured epithelial cells, LUZP1 was found localized at TJ-, but not at AJ-, associated CRs, and LUZP1 knockout resulted in apical constriction defects with a significant reduction in ppMLC levels within CRs. A series of assays revealed that ppMLC promotes the recruitment of LUZP1 to TJ-associated CRs, where LUZP1 spatiotemporally inhibits myosin phosphatase in a microtubule-facilitated manner. Our results uncovered a hitherto unknown microtubule-LUZP1 association at TJ-associated CRs that inhibits myosin phosphatase, contributing significantly to the understanding of vertebrate apical constriction.

Iqgap3-Ras axis drives stem cell proliferation in the stomach corpus during homoeostasis and repair.

OBJECTIVE: Tissue stem cells are central regulators of organ homoeostasis. We looked for a protein that is exclusively expressed and functionally involved in stem cell activity in rapidly proliferating isthmus stem cells in the stomach corpus. DESIGN: We uncovered the specific expression of Iqgap3 in proliferating isthmus stem cells through immunofluorescence and in situ hybridisation. We performed lineage tracing and transcriptomic analysis of Iqgap3 +isthmus stem cells with the Iqgap3-2A-tdTomato mouse model. Depletion of Iqgap3 revealed its functional importance in maintenance and proliferation of stem cells. We further studied Iqgap3 expression and the associated gene expression changes during tissue repair after tamoxifen-induced damage. Immunohistochemistry revealed elevated expression of Iqgap3 in proliferating regions of gastric tumours from patient samples. RESULTS: Iqgap3 is a highly specific marker of proliferating isthmus stem cells during homoeostasis. Iqgap3+isthmus stem cells give rise to major cell types of the corpus unit. Iqgap3 expression is essential for the maintenance of stem potential. The Ras pathway is a critical partner of Iqgap3 in promoting strong proliferation in isthmus stem cells. The robust induction of Iqgap3 expression following tissue damage indicates an active role for Iqgap3 in tissue regeneration. CONCLUSION: IQGAP3 is a major regulator of stomach epithelial tissue homoeostasis and repair. The upregulation of IQGAP3 in gastric cancer suggests that IQGAP3 plays an important role in cancer cell proliferation.

Correction: An antibody-supermolecule conjugate for tumor-specific targeting of tumoricidal methylated ß-cyclodextrin-threaded polyrotaxanes.

Correction for 'An antibody-supermolecule conjugate for tumor-specific targeting of tumoricidal methylated ß-cyclodextrin-threaded polyrotaxanes' by Kei Nishida et al., J. Mater. Chem. B, 2020, DOI: 10.1039/d0tb00575d.

Multiparametric assessment of the impact of opsin expression and anesthesia on striatal cholinergic neurons and auditory brainstem activity.

Recent developments in genetic engineering have established murine models that permit the selective control of cholinergic neurons via optical stimulation. Despite copious benefits granted by these experimental advances, the sensory physiognomy of these organisms has remained poorly understood. Therefore, the present study evaluates sensory and neuronal response properties of animal models developed for the study of optically induced acetylcholine release regulation. Auditory brainstem responses, fluorescence imaging, and patch clamp recording techniques were used to assess the impact of viral infection, sex, age, and anesthetic agents across the ascending auditory pathway of ChAT-Cre and ChAT-ChR2(Ai32) mice. Data analyses revealed that neither genetic configuration nor adeno-associated viral infection alters the early stages of auditory processing or the cellular response properties of cholinergic neurons. However, anesthetic agent and dosage amount profoundly modulate the response properties of brainstem neurons. Last, analyses of age-related hearing loss in virally infected ChAT-Cre mice did not differ from those reported in wild type animals. This investigation demonstrates that ChAT-Cre and ChAT-ChR2(Ai32) mice are viable models for the study of cholinergic modulation in auditory processing, and it emphasizes the need for prudence in the selection of anesthetic procedures.

Reciprocal Association between the Apical Junctional Complex and AMPK: A Promising Therapeutic Target for Epithelial/Endothelial Barrier Function?

Epithelial/endothelial cells adhere to each other via cell-cell junctions including tight junctions (TJs) and adherens junctions (AJs). TJs and AJs are spatiotemporally and functionally integrated, and are thus often collectively defined as apical junctional complexes (AJCs), regulating a number of spatiotemporal events including paracellular barrier, selective permeability, apicobasal cell polarity, mechano-sensing, intracellular signaling cascades, and epithelial morphogenesis. Over the past 15 years, it has been acknowledged that adenosine monophosphate (AMP)-activated protein kinase (AMPK), a well-known central regulator of energy metabolism, has a reciprocal association with AJCs. Here, we review the current knowledge of this association and show the following evidences: (1) as an upstream regulator, AJs activate the liver kinase B1 (LKB1)-AMPK axis particularly in response to applied junctional tension, and (2) TJ function and apicobasal cell polarization are downstream targets of AMPK and are promoted by AMPK activation. Although molecular mechanisms underlying these phenomena have not yet been completely elucidated, identifications of novel AMPK effectors in AJCs and AMPK-driven epithelial transcription factors have enhanced our knowledge. More intensive studies along this line would eventually lead to the development of AMPK-based therapies, enabling us to manipulate epithelial/endothelial barrier function.