Tight junction membrane proteins regulate the mechanical resistance of the apical junctional complex.

Epithelia must be able to resist mechanical force to preserve tissue integrity. While intercellular junctions are known to be important for the mechanical resistance of epithelia, the roles of tight junctions (TJs) remain to be established. We previously demonstrated that epithelial cells devoid of the TJ membrane proteins claudins and JAM-A completely lack TJs and exhibit focal breakages of their apical junctions. Here, we demonstrate that apical junctions fracture when claudin/JAM-A-deficient cells undergo spontaneous cell stretching. The junction fracture was accompanied by actin disorganization, and actin polymerization was required for apical junction integrity in the claudin/JAM-A-deficient cells. Further deletion of CAR resulted in the disruption of ZO-1 molecule ordering at cell junctions, accompanied by severe defects in apical junction integrity. These results demonstrate that TJ membrane proteins regulate the mechanical resistance of the apical junctional complex in epithelial cells.

Force-dependent remodeling of cytoplasmic ZO-1 condensates contributes to cell-cell adhesion through enhancing tight junctions.

The physiological importance of biomolecular condensates is widely recognized, but how it is controlled in time and space during development is largely unknown. Here, we show that a tight junction protein ZO-1 forms cytoplasmic condensates in the trophectoderm (TE) of the mouse embryo before E4.0. These disappear via dissolution, and ZO-1 accumulates at the cell junction as the blastocyst cavity grows and internal pressure on TE cells increases. In contrast, this dissolution was less evident in TE cells attached to the inner cell mass because they receive weaker tensile forces. Furthermore, analyses using MDCK cells demonstrated that the ZO-1 condensates are generated and maintained by liquid-liquid phase separation. Our study also highlights that the dynamics of these condensates depends on the physical environment via an interaction between ZO-1 and F-actin. We propose that the force-dependent regulation of ZO-1 condensation contributes to the establishment of robust cell-cell adhesion during early development.

Optogenetic relaxation of actomyosin contractility uncovers mechanistic roles of cortical tension during cytokinesis.

Actomyosin contractility generated cooperatively by nonmuscle myosin II and actin filaments plays essential roles in a wide range of biological processes, such as cell motility, cytokinesis, and tissue morphogenesis. However, subcellular dynamics of actomyosin contractility underlying such processes remains elusive. Here, we demonstrate an optogenetic method to induce relaxation of actomyosin contractility at the subcellular level. The system, named OptoMYPT, combines a protein phosphatase 1c (PP1c)-binding domain of MYPT1 with an optogenetic dimerizer, so that it allows light-dependent recruitment of endogenous PP1c to the plasma membrane. Blue-light illumination is sufficient to induce dephosphorylation of myosin regulatory light chains and a decrease in actomyosin contractile force in mammalian cells and Xenopus embryos. The OptoMYPT system is further employed to understand the mechanics of actomyosin-based cortical tension and contractile ring tension during cytokinesis. We find that the relaxation of cortical tension at both poles by OptoMYPT accelerated the furrow ingression rate, revealing that the cortical tension substantially antagonizes constriction of the cleavage furrow. Based on these results, the OptoMYPT system provides opportunities to understand cellular and tissue mechanics.

Mechanical Stress Regulates Epithelial Tissue Integrity and Stiffness through the FGFR/Erk2 Signaling Pathway during Embryogenesis.

Physical forces generated by tissue-tissue interactions are a critical component of embryogenesis, aiding the formation of organs in a coordinated manner. In this study, using Xenopus laevis embryos and phosphoproteome analyses, we uncover the rapid activation of the mitogen-activated protein (MAP) kinase Erk2 upon stimulation with centrifugal, compression, or stretching force. We demonstrate that Erk2 induces the remodeling of cytoskeletal proteins, including F-actin, an embryonic cadherin C-cadherin, and the tight junction protein ZO-1. We show these force-dependent changes to be prerequisites for the enhancement of cellular junctions and tissue stiffening during early embryogenesis. Furthermore, Erk2 activation is FGFR1 dependent while not requiring fibroblast growth factor (FGF) ligands, suggesting that cell/tissue deformation triggers receptor activation in the absence of ligands. These findings establish previously unrecognized functions for mechanical forces in embryogenesis and reveal its underlying force-induced signaling pathways.

Mechanical Force Induces Phosphorylation-Mediated Signaling that Underlies Tissue Response and Robustness in Xenopus Embryos.

Mechanical forces are essential drivers of numerous biological processes, notably during development. Although it is well recognized that cells sense and adapt to mechanical forces, the signal transduction pathways that underlie mechanosensing have remained elusive. Here, we investigate the impact of mechanical centrifugation force on phosphorylation-mediated signaling in Xenopus embryos. By monitoring temporal phosphoproteome and proteome alterations in response to force, we discover and validate elevated phosphorylation on focal adhesion and tight junction components, leading to several mechanistic insights into mechanosensing and tissue restoration. First, we determine changes in kinase activity profiles during mechanoresponse, identifying the activation of basophilic kinases. Pathway interrogation using kinase inhibitor treatment uncovers a crosstalk between the focal adhesion kinase (FAK) and protein kinase C (PKC) in mechanoresponse. Second, we find LIM domain 7 protein (Lmo7) as upregulated upon centrifugation, contributing to mechanoresponse. Third, we discover that mechanical compression force induces a mesenchymal-to-epithelial transition (MET)-like phenotype.

A Prospective Multicenter Study Using a Virtual 3 Fr Percutaneous Coronary Intervention System: The V3 Registry.

OBJECTIVES: To evaluate the safety and feasibility of virtual 3 Fr (V3), sheathless 5 Fr percutaneous coronary intervention (PCI). BACKGROUND: A small-diameter guiding catheter (GC) makes less-invasive PCI possible. The V3 is an extremely slender PCI system; however, the outcome of using this system has not yet been determined. METHODS: The V3 registry is a prospective, multicenter, non-randomized study that enrolled patients who underwent elective V3-PCI. The primary endpoint was clinical success rate, and the secondary endpoints were PCI success rate in all cases, major adverse cardiac and cerebrovascular event (MACCE) at 30 days, and access-site complications. RESULTS: A total of 260 patients with 321 lesions were enrolled. Of this group, 70% were male and the mean age was 70.8 ± 10.0 years. Type B2/C lesions comprised 50.7% of the total. The clinical success rate was 95.8%, and the PCI success rate was 99.2%. PCI failure was reported in 2 chronic total occlusion cases. No MACCE was reported. Although there was no major bleeding, hematoma occurred at the puncture site in 12.7% of cases. There was a single radial artery occlusion (0.4%) without symptoms. CONCLUSIONS: PCI with the V3 was safe and feasible. Radial artery occlusion and major bleeding complications were extremely low. However, access-site hematoma frequently complicated catheter exchange.

Indications and outcomes of excimer laser coronary atherectomy: Efficacy and safety for thrombotic lesions-The ULTRAMAN registry.

BACKGROUND: Excimer laser coronary atherectomy (ELCA) recently became available in Japan, but ELCA's effectiveness and safety are not clear. METHODS AND RESULTS: We enrolled consecutive patients who underwent ELCA and were registered in the Utility of Laser for Transcatheter Atherectomy-Multicenter Analysis around Naniwa (ULTRAMAN) registry comprising six Japanese medical centers around Naniwa in Japan with patients registered from April 2006 to June 2015. We evaluated the catheter sizes used and compared the success rate, thrombolysis in myocardial infarction (TIMI) flow, blush score, and complications between the rich-thrombus (RT) group [acute coronary syndrome (ACS) and saphenous vein graft (SVG)] and the poor-thrombus (PT) group [in-stent restenosis (ISR), chronic total occlusion (CTO), calcification, and long or bifurcation (L&B) lesions]. Of the 328 patients, 6 (1.8%) were treated for an SVG, 175 (53.4%) were treated for ACS, 18 (5.5%) for CTO, 106 (32.4%) for ISR, 8 (2.4%) for calcification, and 15 for L&B lesions (4.6%). A 1.7-mm (concentric)-diameter ELCA catheter was used most frequently (59.4%). High success rates were achieved in both the RT and PT groups, but the TIMI flow grade and blush score were significantly lower and the complications rate was significantly higher in the RT group (n=181). CONCLUSIONS: In Japan, the major indications for ELCA have been ACS and ISR. ELCA can provide a safe and effective treatment even for RT lesions.

Phosphorylation-dependent ubiquitination of paraxial protocadherin (PAPC) controls gastrulation cell movements.

Paraxial protocadherin (PAPC) has been shown to be involved in gastrulation cell movements during early embryogenesis. It is first expressed in the dorsal marginal zone at the early gastrula stage and subsequently restricted to the paraxial mesoderm in Xenopus and zebrafish. Using Xenopus embryos, we found that PAPC is also regulated at the protein level and is degraded and excluded from the plasma membrane in the axial mesoderm by the late gastrula stage. Regulation of PAPC requires poly-ubiquitination that is dependent on phosphorylation. PAPC is phosphorylated by GKS3 in the evolutionarily conserved cytoplasmic domain, and this in turn is necessary for poly-ubiquitination by an E3 ubiquitin ligase ß-TrCP. We also show that precise control of PAPC by phosphorylation/ubiquitination is essential for normal Xenopus gastrulation cell movements. Taken together, our findings unveil a novel mechanism of regulation of a cell adhesion protein and show that this system plays a crucial role in vertebrate embryogenesis.

A successful treatment for a lesion of chronic total occlusion with contralateral angiography in a single radial access.

A 50-year-old man was admitted due to effort chest pain. Coronary angiogram showed a total occlusion of LAD. The 5-French JL 3.5 was engaged into the left coronary artery. The XT-A guidewire was advanced to the distal of the occluded lesion. Contralateral angiography was performed using JL 3.5. The guiding catheter was pullback from the left coronary orifice leaving the guidewire at LAD, and the catheter tip was rotated clockwise to right coronary cusp for right coronary angiography. We could confirm that the guidewire was in the true lumen vessel.

Transgenic Xenopus laevis for live imaging in cell and developmental biology.

The stable transgenesis of genes encoding functional or spatially localized proteins, fused to fluorescent proteins such as green fluorescent protein (GFP) or red fluorescent protein (RFP), is an extremely important research tool in cell and developmental biology. Transgenic organisms constructed with fluorescent labels for cell membranes, subcellular organelles, and functional proteins have been used to investigate cell cycles, lineages, shapes, and polarity, in live animals and in cells or tissues derived from these animals. Genes of interest have been integrated and maintained in generations of transgenic animals, which have become a valuable resource for the cell and developmental biology communities. Although the use of Xenopus laevis as a transgenic model organism has been hampered by its relatively long reproduction time (compared to Drosophila melanogaster and Caenorhabditis elegans), its large embryonic cells and the ease of manipulation in early embryos have made it a historically valuable preparation that continues to have tremendous research potential. Here, we report on the Xenopus laevis transgenic lines our lab has generated and discuss their potential use in biological imaging.

A successful treatment for a lesion of chronic total occlusion using a virtual 3 Fr guiding catheter.

A 43 year-old man was admitted due to effort related chest squeezing for 3 months. Coronary angiogram showed a total occlusion of the proximal right coronary artery (RCA) with collateral vessels from left anterior descending artery. The 5F sheathless guiding catheter was engaged into the RCA and the 3F JL3.5 catheter was inserted into left coronary artery via left radial artery for simultaneous contra lateral angiography. We advanced the wire through the lesion with 2 wire technique. Three XIENCE stents (2.5 mm × 28 mm) was inserted from distal to mid RCA, and a 3.0 mm × 15 mm XIENCE stent was inserted to proximal RCA. The final angiographic result showed well expanded stent. The treatment of chronic total occlusion could be possible even slender device by getting hold of the characteristics of the device and evaluating an objective assessment of lesion characteristics.

[A research of letter color visibility in package insert information using simulator].

Package insert of pharmaceutical drug is one of the most prioritized information for pharmacists to secure safety of patients. However, the color of character, size, font and so on are various company by company product to product from a viewpoint of visibility. It may be cause a serious accident in case visibility is unclear, although it is the most important information. Moreover, package insert with high visibility is required for color vision defectives from a viewpoint of a universal design. Then, the authors selected the package insert which has the boxed warning in the ethical pharmaceutical currently stored mostly in the present health insurance pharmacy and quantified the red color using the color meter. We advocate the state of a suitable package insert from a viewpoint of a universal design, whether the red color is high visible or not for color vision defectives using simulator.

Long-term angiographic outcomes of post-sirolimus-eluting stent restenosis in Japanese patients.

Though restenosis after drug-eluting stent implantation is still observed, the factors affecting post-sirolimus-eluting stent restenosis (re-restenosis) have not been fully determined. We evaluated the long-term angiographic outcomes and examined background factors affecting re-restenosis. We enrolled 51 patients with 68 sirolimus-eluting stent (SES) restenosis lesions who underwent target lesion revascularization (TLR) and angiographic follow-up studies. Re-restenosis was observed in 29 of 68 restenosis lesions, and the rate was 42.6%. Study subjects were divided into two groups: a re-restenosis (Re-R) group (20 patients) with 29 lesions and a restenosis (R) group (31 patients) with 39 lesions with no re-restenosis. There were no differences in age, sex, coronary risk factors, past history, or medications between the two groups. Re-restenosis was observed more frequently in the right coronary artery (Re-R group vs. R group; 65.5 vs. 33.3%, P = 0.009). The incidence of stent fracture was higher in the Re-R group (Re-R group vs. R group; 48.3 vs. 12.8%, P = 0.003). QCA results showed that the initial lesion length at the time of first coronary intervention was significantly longer in the Re-R group (Re-R group vs. R group; 21.6 ± 3.37 vs. 12.6 ± 4.98 mm, P = 0.049). The rate of re-restenosis was 47.1% when treated with POBA alone, while it was 36.7% with SES treatment. In multivariate analysis, the initial lesion length at the time of first coronary intervention (odds ratio = 1.64, 95% CI 1.29-2.06, P < 0.001) and stent fracture (odds ratio = 12.42, 95% CI 1.89-81.4, P = 0.009) were independent predictors of re-restenosis. This study demonstrates that recurrent restenosis with SES treatment is associated with lesion length and stent fracture, a finding that is beneficial in the management of restenosis after SES implantation.

A successful treatment for in-stent restenosis using a 4-French guiding catheter with optical coherence tomography guidance.

An 83-year-old woman was admitted due to effort related chest squeezing over the duration of 5 months. A coronary angiogram revealed significant in-stent restenosis at proximal left anterior descending artery. Optical coherence tomography (OCT) image acquisition was successfully performed using a non-occlusive technique. Restenotic tissue consisting of eccentric layers was observed by the OCT. The only imaging method of intracoronary evaluation using a 4F guiding catheter is an OCT. An OCT assessment was helpful for the percutaneous coronary intervention using a 4F guiding catheter.

Erythropoietin in patients with acute coronary syndrome and its cardioprotective action after percutaneous coronary intervention.

BACKGROUND: Erythropoietin (EPO) has been shown to have effects beyond hematopoiesis, such as prevention of cardiac apoptosis. The purpose of the current study is to examine the influence of the time-course change in the serum concentration of endogenous EPO on cardiac functions in the chronic phase in patients with acute coronary syndrome, who successfully achieved reperfusion by primary percutaneous coronary intervention (PCI). METHODS AND RESULTS: The prospective study included 34 patients with angiographically documented coronary artery disease, including 24 patients with acute myocardial infarction (AMI) and 10 patients with unstable angina pectoris (UAP) who underwent successful PCI within 24 h from the onset. Serum EPO concentration significantly increased at Day 3 and Day 7 compared with that at Day 1 in the AMI group, and the level at Day 3 was significantly higher in the AMI group than in the UAP group. There were significant correlations between DeltaEPO and Delta left ventricular ejection fraction (LVEF) or Delta left ventricular end-diastolic volume index and between peak EPO concentration and DeltaLVEF. CONCLUSIONS: These data showed the time-dependent increase of serum EPO in AMI patients after primary PCI, indicating its possible contribution to cardioprotective effect in the chronic phase.

XRab40 and XCullin5 form a ubiquitin ligase complex essential for the noncanonical Wnt pathway.

Rab GTPases are key regulators of intracellular membrane trafficking. We sought to elucidate the roles of Rab GTPases in Xenopus gastrulation, and found that a Xenopus homolog of Rab40 (XRab40) is required for normal gastrulation. XRab40 is localized at the Golgi apparatus and interacts with ElonginB/C and Cullin5 to form a ubiquitin ligase. XRab40/XCullin5 functions cooperatively and regulates the ubiquitination and localization of Rap2 GTPase. Furthermore, XRab40/XCullin5 regulates the membrane localization of Dishevelled (Dsh), a key signaling molecule in the Wnt pathway, through Rap2 and its effector Misshapen/Nck-interacting kinase (XMINK). XMINK interacts with Dsh, and is translocated to the plasma membrane by Wnt activation. We propose a novel signaling cascade consisting of XRab40/XCullin5, Rap2 and XMINK, which plays a crucial role in the regulation of the noncanonical Wnt pathway.

Wnt signalling regulates paxillin ubiquitination essential for mesodermal cell motility.

Gastrulation movements are critical for establishing the three germ layers and the architecture of vertebrate embryos. During Xenopus laevis gastrulation, mesodermal tissue migrates on the blastocoel roof and elongates along the antero-posterior axis. During this process, cells in the dorsal mesoderm are polarized and intercalate with each other, which is defined as convergent extension and is known to be regulated by the non-canonical Wnt pathway. Here, we show that paxillin plays an essential role in this process. Paxillin is a focal-adhesion associated protein implicated in the regulation of actin cytoskeletal organization and cell motility, but its role in Xenopus embryogenesis has not yet been clarified. We demonstrate that the Wnt pathway controls the ubiquitination and stability of paxillin, and that this regulatory mechanism is essential for convergent extension movements. We identified a RING finger protein XRNF185, which physically binds to paxillin and the proteasome. XRNF185 destabilizes paxillin at focal adhesions and promotes mesodermal cell migration during convergent extension. We propose a mechanism to regulate gastrulation movements that involves paxillin ubiquitination and stability controlled by Wnt signalling.

[Coronary artery plaque assessment using Volcano Therapeutics' Virtual Histology intravascular ultrasound and temperature guide wire].

OBJECTIVES: This study investigated the tissue characterization of human coronary artery plaque using Virtual Histology intravascular ultrasound (VH-IVUS) and temperature guide wire. METHODS AND RESULTS: Between August 2005 and December 2005, VH-IVUS examination and guide wire based measurement of coronary temperature were performed in 66 patients: 39 with stable angina pectoris (SAP), and 27 with unstable angina pectoris(UAP). Patients in the acute myocardial infarction with necrotic tissue and thrombus were excluded. After measurement of distal coronary pressure and coronary temperature, the following tissue types of atherosclerotic plaque were evaluated by VH-IVUS: fibrotic, fibro-fatty, dense calcium and necrotic core. Occurrence of fibro-fatty tissue was significantly higher in UAP patients than in SAP patients (17.4 +/- 7.0% vs 11.2 +/- 5.2%, p = 0.04), and coronary temperature was significantly higher in UAP patients than in SAP patients (0.08 +/- 0.05 vs 0.03 +/- 0.02 degrees C, p = 0.01). CONCLUSIONS: These results suggest that the combination of VH-IVUS analysis and measurement of coronary temperature may be able to predict the progression of lesions.

[Clinical usefulness of 201Tl/99mTc-PYP dual myocardial quantitative gated SPECT program using low-dose dobutamine loading in assessment of myocardial viability in patient with acute myocardial infarction--a case report].

An 86-year-old man with chest pain was admitted to our hospital. Coronary angiography revealed 99% stenosis of the mid segment of the left anterior descending coronary artery, therefore, a coronary stent was implanted. Immediately after the stent implantation, 99% stenosis occurred at the proximal site of the 1st diagonal artery because of stent jeal. On the 4th hospital day, ECG-gated 201TL/99mTc-PYP dual myocardial quantitative gated SPECT was performed at rest and during low-dose dobutamine loading. The 201Tl scintigraphy revealed moderately reduced uptake in the anterior, septal and apical walls, and 99mTc-PYP uptake was observed in the mid-anterior wall. A three-dimensional surface display of gated 201Tl SPECT images showed severe hypokinesis in the anterior, septal and apical walls at rest. On the other hand, during low-dose dobutamine loading, improved wall motion was observed in the basal anterior and septal walls, while no change was observed in the midanterior and apical wall movements. Three-dimensional surface display of gated 201Tl/99mTc-PYP dual SPECT images revealed similar patterns of wall motion as those of gated 201Tl SPECT images at rest. During low-dose dobutamine loading, on the other hand, a three-dimensional surface display of gated 201Tl/99mTc-PYP dual SPECT images revealed improved wall motion in the basal anterior, septal and apical walls, but worsened wall motion of the mid-anterior wall. After 6 months, a follow-up coronary angiography revealed no re-stenosis of the stent, but 99% stenosis at the proximal aspect of the 1st diagonal artery. Left ventriculography revealed improved wall motion in the apex and akinesis of the mid-anterior wall. These wall motion findings were similar to those visualized in the three-dimensional surface display of gated 201Tl/99mTc-PYP dual SPECT images during low-dose dobutamine loading in the acute phase. These results suggest that 201Tl/99mTc-PYP dual myocardial quantitative gated SPECT using low-dose dobutamine loading could be useful for the assessment of myocardial viability after reperfusion therapy in patients with acute myocardial infarction.