Anti-CX3CL1 (fractalkine) monoclonal antibody attenuates lung and skin fibrosis in sclerodermatous graft-versus-host disease mouse model.

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular injury and inflammation, followed by excessive fibrosis of the skin and other internal organs, including the lungs. CX3CL1 (fractalkine), a chemokine expressed on endothelial cells, supports the migration of macrophages and T cells that express its specific receptor CX3CR1 into targeted tissues. We previously reported that anti-CX3CL1 monoclonal antibody (mAb) treatment significantly inhibited transforming growth factor (TGF)-ß1-induced expression of type I collagen and fibronectin 1 in human dermal fibroblasts. Additionally, anti-mouse CX3CL1 mAb efficiently suppressed skin inflammation and fibrosis in bleomycin- and growth factor-induced SSc mouse models. However, further studies using different mouse models of the complex immunopathology of SSc are required before the initiation of a clinical trial of CX3CL1 inhibitors for human SSc. METHODS: To assess the preclinical utility and functional mechanism of anti-CX3CL1 mAb therapy in skin and lung fibrosis, a sclerodermatous chronic graft-versus-host disease (Scl-cGVHD) mouse model was analyzed with immunohistochemical staining for characteristic infiltrating cells and RNA sequencing assays. RESULTS: On day 42 after bone marrow transplantation, Scl-cGVHD mice showed increased serum CX3CL1 level. Intraperitoneal administration of anti-CX3CL1 mAb inhibited the development of fibrosis in the skin and lungs of Scl-cGVHD model, and did not result in any apparent adverse events. The therapeutic effects were correlated with the number of tissue-infiltrating inflammatory cells and α-smooth muscle actin (α-SMA)-positive myofibroblasts. RNA sequencing analysis of the fibrotic skin demonstrated that cGVHD-dependent induction of gene sets associated with macrophage-related inflammation and fibrosis was significantly downregulated by mAb treatment. In the process of fibrosis, mAb treatment reduced cGVHD-induced infiltration of macrophages and T cells in the skin and lungs, especially those expressing CX3CR1. CONCLUSIONS: Together with our previous findings in other SSc mouse models, the current results indicated that anti-CX3CL1 mAb therapy could be a rational therapeutic approach for fibrotic disorders, such as human SSc and Scl-cGVHD.

Live Cell Monitoring of Separase Activity, a Key Enzymatic Reaction for Chromosome Segregation, with Chimeric FRET-Based Molecular Sensor upon Cell Cycle Progression.

Separase is a key cysteine protease in the separation of sister chromatids through the digestion of the cohesin ring that inhibits chromosome segregation as a trigger of the metaphase-anaphase transition in eukaryotes. Its activity is highly regulated by binding with securin and cyclinB-CDK1 complex. These bindings prevent the proteolytic activity of separase until the onset of anaphase. Chromosome missegregation and aneuploidy are frequently observed in malignancies. However, there are some difficulties in biochemical examinations due to the instability of separase in vitro and the fact that few spatiotemporal resolution approaches exist for monitoring live separase activity throughout mitotic processes. Here, we have developed FRET-based molecular sensors, including GFP variants, with separase-cleavable sequences as donors and covalently attached fluorescent dyes as acceptor molecules. These are applicable to conventional live cell imaging and flow cytometric analysis because of efficient live cell uptake. We investigated the performance of equivalent molecular sensors, either localized or not localized inside the nucleus under cell cycle control, using flow cytometry. Synchronized cell cycle progression rendered significant separase activity detections in both molecular sensors. We obtained consistent outcomes with localized molecular sensor introduction and cell cycle control by fluorescent microscopic observations. We thus established live cell separase activity monitoring systems that can be used specifically or statistically, which could lead to the elucidation of separase properties in detail.

Diastereoselective Synthesis of trans-anti-Hydrophenanthrenes via Ti-mediated Radical Cyclization and Total Synthesis of Kamebanin.

Ent-kaurenes consist of an ABC-ring based on a trans-anti-hydrophenanthrene skeleton and a D ring with an exomethylene. Highly oxygen-functionalized ent-kauren-15-ones have promising antiinflammatory pharmacological activity. In this study, we developed a novel diastereoselective synthesis of trans-anti-hydrophenanthrenes via a Ti-mediated reductive radical cyclization. We also demonstrated the applicability of this method by developing the first total synthesis of (±)-kamebanin (longest linear sequence; 17 steps, overall yield; 6.5 %). Furthermore, this synthesis provided a formal semi-pinacol rearrangement for the construction of the quaternary carbon at C8 and a novel Thorpe-Ziegler-type reaction for the construction of the D-ring.

Cell-matrix interface regulates dormancy in human colon cancer stem cells.

Cancer relapse after chemotherapy remains a main cause of cancer-related death. Although the relapse is thought to result from the propagation of resident cancer stem cells1, a lack of experimental platforms that enable the prospective analysis of cancer stem cell dynamics with sufficient spatiotemporal resolution has hindered the testing of this hypothesis. Here we develop a live genetic lineage-tracing system that allows the longitudinal tracking of individual cells in xenotransplanted human colorectal cancer organoids, and identify LGR5+ cancer stem cells that exhibit a dormant behaviour in a chemo-naive state. Dormant LGR5+ cells are marked by the expression of p27, and intravital imaging provides direct evidence of the persistence of LGR5+p27+ cells during chemotherapy, followed by clonal expansion. Transcriptome analysis reveals that COL17A1-a cell-adhesion molecule that strengthens hemidesmosomes-is upregulated in dormant LGR5+p27+ cells. Organoids in which COL17A1 is knocked out lose the dormant LGR5+p27+ subpopulation and become sensitive to chemotherapy, which suggests that the cell-matrix interface has a role in the maintenance of dormancy. Chemotherapy disrupts COL17A1 and breaks the dormancy in LGR5+p27+ cells through FAK-YAP activation. Abrogation of YAP signalling prevents chemoresistant cells from exiting dormancy and delays the regrowth of tumours, highlighting the therapeutic potential of YAP inhibition in preventing cancer relapse. These results offer a viable therapeutic approach to overcome the refractoriness of human colorectal cancer to conventional chemotherapy.

Biomimetic Total Syntheses of (+)-Chloropupukeananin, (-)-Chloropupukeanolide D, and Chloropestolides.

Chloropupukeananin, chloropupukeanolides, and chloropestolides are a family of structurally complex bioactive natural products that possess highly functionalized tricyclo[4.3.1.03,7]decane or bicyclo[2.2.2]octane skeletons. Biosynthesis of the chloropupukeananin family is triggered by the intermolecular heterodimeric Diels-Alder reaction between maldoxin and iso-A82775C; however, the enzymes involved have not yet been identified. We herein report the one-pot biomimetic synthesis of chloropupukeananin and chloropupukeanolide D. Moreover, the effect of the solvent on the intermolecular Diels-Alder reaction of siccayne and maldoxin suggested that the biosynthesis of the chloropupukeananin family involves a Diels-Alderase-catalyzed heterodimeric Diels-Alder reaction.

Inhibition of the Progression of Skin Inflammation, Fibrosis, and Vascular Injury by Blockade of the CX3 CL1/CX3 CR1 Pathway in Experimental Mouse Models of Systemic Sclerosis.

OBJECTIVE: To assess the preclinical efficacy and mechanism of action of an anti-CX3 CL1 monoclonal antibody (mAb) in systemic sclerosis (SSc). METHODS: Cultured human dermal fibroblasts were used to evaluate the direct effect of anti-CX3 CL1 mAb on fibroblasts. In addition, bleomycin-induced and growth factor-induced models of SSc were used to investigate the effect of anti-CX3 CL1 mAb on leukocyte infiltration, collagen deposition, and vascular damage in the skin. RESULTS: Anti-CX3 CL1 mAb treatment significantly inhibited Smad3 phosphorylation (P < 0.05) and expression of type I collagen and fibronectin 1 (P < 0.01) in dermal fibroblasts stimulated with transforming growth factor ß1 (TGFß1). In the bleomycin model, daily subcutaneous bleomycin injection increased serum CX3 CL1 levels (P < 0.05) and augmented lesional CX3 CL1 expression. Simultaneous administration of anti-CX3 CL1 mAb or CX3 CR1 deficiency significantly suppressed the dermal thickness, collagen content, and capillary loss caused by bleomycin (P < 0.05). Injection of bleomycin induced expression of pSmad3 and TGFß1 in the skin, which was inhibited by anti-CX3 CL1 mAb. Further, the dermal infiltration of CX3 CR1+ cells, macrophages (inflammatory and alternatively activated [M2-like] subsets), and CD3+ cells significantly decreased following anti-CX3 CL1 mAb therapy (P < 0.05), as did the enhanced skin expression of fibrogenic molecules, such as thymic stromal lymphopoietin and secreted phosphoprotein 1 (P < 0.05). However, the treatment did not significantly reduce established skin fibrosis. In the second model, simultaneous anti-mCX3 CL1 mAb therapy significantly diminished the skin fibrosis induced by serial subcutaneous injection of TGFß and connective tissue growth factor (P < 0.01). CONCLUSION: Anti-CX3 CL1 mAb therapy may be a novel approach for treating early skin fibrosis in inflammation-driven fibrotic skin disorders such as SSc.

Blockade of the fractalkine-CX3CR1 axis ameliorates experimental colitis by dislodging venous crawling monocytes.

Chemokine systems modulate inflammatory and immune responses in inflammatory bowel disease (IBD). The colons of IBD patients show increased levels of fractalkine (FKN) and high numbers of FKN receptor-positive (CX3CR1+) cells; however, the FKN-CX3CR1 axis's role in intestinal inflammation, especially in intravascular leukocyte behaviors, still remains unclear. Here, we show that interruption of the FKN-CX3CR1 axis by anti-FKN monoclonal antibody (mAb) ameliorates murine colitis through regulation of intravascular monocyte behaviors in murine colitis models. FKN expression was detectable in vascular endothelium and CX3CR1+ macrophages accumulated in the mucosal lamina propria and submucosa of the inflamed colons. CD115+ monocytes tethered to the venous endothelium and expressed pro-inflammatory mediators. The anti-FKN mAb improved colitis symptoms, markedly reduced pro-inflammatory factors in the colon, maintained blood vessel integrity and reduced tethered monocytes in the inflamed veins. Intravital imaging revealed that CD115+Gr-1low/- monocytes crawled on the apical surfaces of venous endothelium, and anti-FKN mAb rapidly dislodged the crawling monocytes and inhibited their patrolling behavior. These findings suggest that the FKN-CX3CR1 axis triggers the patrolling behavior of crawling monocytes on the venous endothelium of inflamed colons, and accelerates the subsequent leukocyte activation and infiltration by locally producing inflammatory cytokines and chemokines. The mAb also ameliorated symptoms in another IBD model, T-cell-transferred colitis. Blocking the FKN-CX3CR1 axis with an anti-FKN mAb considerably inhibits the colitis-triggered inflammatory cascades, which may be an alternative strategy to treat IBD.

Role of Anti-Fractalkine Antibody in Suppression of Joint Destruction by Inhibiting Migration of Osteoclast Precursors to the Synovium in Experimental Arthritis.

OBJECTIVE: To elucidate the role of the fractalkine (FKN)/CX3 CR1 pathway in joint destruction in rheumatoid arthritis. METHODS: We examined the effect of treatment with anti-mouse FKN (anti-mFKN) monoclonal antibody (mAb) on joint destruction and the migration of osteoclast precursors (OCPs) into the joint, using the collagen-induced arthritis (CIA) model. DBA/1 mice were immunized with bovine type II collagen to induce arthritis, and then treated with anti-mFKN mAb. Disease severity was monitored by arthritis score, and joint destruction was evaluated by soft x-ray and histologic analyses. Plasma levels of joint destruction markers were assessed by enzyme-linked immunosorbent assay. FKN expression on endothelial cells was detected by immunohistochemistry. Bone marrow-derived OCPs were labeled with fluorescein and transferred to mice with CIA, and the migration of the OCPs to the joints was then analyzed. RESULTS: Both prophylactic and therapeutic treatment with anti-mFKN mAb significantly decreased the arthritis and soft x-ray scores. Plasma levels of cartilage oligomeric matrix protein and matrix metalloproteinase 3 decreased after treatment with anti-mFKN mAb. Histologic analysis revealed that anti-mFKN mAb inhibited synovitis, pannus formation, and cartilage destruction, as well as suppressed bone damage, with a marked reduction in the number of tartrate-resistant acid phosphatase-positive osteoclasts. Anti-mFKN mAb strongly inhibited the migration of bone marrow-derived OCPs into the affected synovium. CONCLUSION: Anti-mFKN mAb notably ameliorates arthritis and joint destruction in the CIA model, as well as inhibits migration of OCPs into the synovium. These results suggest that inhibition of the FKN/CX3 CR1 pathway could be a novel strategy for treatment of both synovitis and joint destruction in rheumatoid arthritis.

Cataract-preventing contact lens for in vivo imaging of mouse retina.

In vivo imaging of mouse retinas using two-photon or confocal laser scanning fluorescence microscopy is a powerful tool for time-lapse analyses of the dynamic movements of cell populations. However, acute and reversible opacification of the crystalline lenses of mouse eyes under anesthesia decreases the visibility of the ocular fundus. Therefore, we developed a customized contact lens for preventing cataract during continuous retinal imaging in anesthetized mice. This experimental approach will aid in the elucidation of cellular and molecular dynamics in the CNS under physiological and pathological conditions.

Obesity Suppresses Cell-Competition-Mediated Apical Elimination of RasV12-Transformed Cells from Epithelial Tissues.

Recent studies have revealed that newly emerging transformed cells are often eliminated from epithelial tissues via cell competition with the surrounding normal epithelial cells. This cancer preventive phenomenon is termed epithelial defense against cancer (EDAC). However, it remains largely unknown whether and how EDAC is diminished during carcinogenesis. In this study, using a cell competition mouse model, we show that high-fat diet (HFD) feeding substantially attenuates the frequency of apical elimination of RasV12-transformed cells from intestinal and pancreatic epithelia. This process involves both lipid metabolism and chronic inflammation. Furthermore, aspirin treatment significantly facilitates eradication of transformed cells from the epithelial tissues in HFD-fed mice. Thus, our work demonstrates that obesity can profoundly influence competitive interaction between normal and transformed cells, providing insights into cell competition and cancer preventive medicine.

Imaging Window Device for Subcutaneous Implantation Tumor.

Most of preclinical cancer studies use xenograft models established from human cell lines or patient-derived cancer cells subcutaneously implanted into the flank of immunocompromised mice. These models are often assumed to represent the original diseases and are valuable tools, at least to some extent, for understanding both the basic biology of cancer and for proof-of-concept studies of molecularly targeted therapies. However, analyzing the cellular behavior of individual components within xenografts, including tumor cells, stromal cells, immune cells, and blood vessels, is challenging. In particular, it has been difficult and urgently required to trace the whole process of heterogeneous tumor microenvironment formation mediated by various components described above. Here we demonstrate a method for monitoring this process using a window device system that we have recently developed and a subcutaneous xenograft model that accurately recapitulates the histology of human lung adenocarcinoma. Use of our imaging window device and a multiphoton laser scanning microscope provides a powerful tool for investigating tumor heterogeneity and responses to drug treatments in an in vivo live imaging system.

Sustained inflammation after pericyte depletion induces irreversible blood-retina barrier breakdown.

In the central nervous system, endothelial cells (ECs) and pericytes (PCs) of blood vessel walls cooperatively form a physical and chemical barrier to maintain neural homeostasis. However, in diabetic retinopathy (DR), the loss of PCs from vessel walls is assumed to cause breakdown of the blood-retina barrier (BRB) and subsequent vision-threatening vascular dysfunctions. Nonetheless, the lack of adequate DR animal models has precluded disease understanding and drug discovery. Here, by using an anti-PDGFRß antibody, we show that transient inhibition of the PC recruitment to developing retinal vessels sustained EC-PC dissociations and BRB breakdown in adult mouse retinas, reproducing characteristic features of DR such as hyperpermeability, hypoperfusion, and neoangiogenesis. Notably, PC depletion directly induced inflammatory responses in ECs and perivascular infiltration of macrophages, whereby macrophage-derived VEGF and placental growth factor (PlGF) activated VEGFR1 in macrophages and VEGFR2 in ECs. Moreover, angiopoietin-2 (Angpt2) upregulation and Tie1 downregulation activated FOXO1 in PC-free ECs locally at the leaky aneurysms. This cycle of vessel damage was shut down by simultaneously blocking VEGF, PlGF, and Angpt2, thus restoring the BRB integrity. Together, our model provides new opportunities for identifying the sequential events triggered by PC deficiency, not only in DR, but also in various neurological disorders.

Nectin and junctional adhesion molecule are critical cell adhesion molecules for the apico-basal alignment of adherens and tight junctions in epithelial cells.

Tight junctions (TJs) and adherens junctions (AJs) form an apical junctional complex at the apical side of the lateral membranes of epithelial cells, in which TJs are aligned at the apical side of AJs. Many cell adhesion molecules (CAMs) and cell polarity molecules (CPMs) cooperatively regulate the formation of the apical junctional complex, but the mechanism for the alignment of TJs at the apical side of AJs is not fully understood. We developed a cellular system with which epithelial-like TJs and AJs were reconstituted in fibroblasts and analyzed the cooperative roles of CAMs and CPMs. We exogenously expressed various combinations of CAMs and CPMs in fibroblasts that express negligible amounts of these molecules endogenously. In these cells, the nectin-based cell-cell adhesion was formed at the apical side of the junctional adhesion molecule (JAM)-based cell-cell adhesion, and cadherin and claudin were recruited to the nectin-3- and JAM-based cell-cell adhesion sites to form AJ-like and TJ-like domains, respectively. This inversed alignment of the AJ-like and TJ-like domains was reversed by complementary expression of CPMs Par-3, atypical protein kinase C, Par-6, Crb3, Pals1 and Patj. We describe the cooperative roles of these CAMs and CPMs in the apico-basal alignment of TJs and AJs in epithelial cells.

Binding between the junctional proteins afadin and PLEKHA7 and implication in the formation of adherens junction in epithelial cells.

Adherens junction (AJ) is a specialized cell-cell junction structure that plays a role in mechanically connecting adjacent cells to resist strong contractile forces and to maintain tissue structure, particularly in the epithelium. AJ is mainly comprised of cell adhesion molecules cadherin and nectin and their associating cytoplasmic proteins including ß-catenin, α-catenin, p120(ctn), and afadin. Our series of studies have revealed that nectin first forms cell-cell adhesion and then recruits cadherin to form AJ. The recruitment of cadherin by nectin is mediated by the binding of α-catenin and p120(ctn) to afadin. Recent studies showed that PLEKHA7 binds to p120(ctn), which is associated with E-cadherin, and maintains the integrity of AJ in epithelial cells. In this study, we showed that PLEKHA7 bound to afadin in addition to p120(ctn) and was recruited to the nectin-3α-based cell-cell adhesion site in a manner dependent on afadin, but not on p120(ctn). The binding of PLEKHA7 to afadin was required for the proper formation of AJ, but not for the formation of tight junction, in EpH4 mouse mammary gland epithelial cells. These results indicate that PLEKHA7 plays a cooperative role with nectin and afadin in the proper formation of AJ in epithelial cells.

Rab13 small G protein and junctional Rab13-binding protein (JRAB) orchestrate actin cytoskeletal organization during epithelial junctional development.

During epithelial junctional development, both vesicle transport and reorganization of the actin cytoskeleton must be spatiotemporally regulated. Coordination of these cellular functions is especially important, but the precise mechanism remains elusive. Previously, we identified junctional Rab13-binding protein (JRAB)/molecules interacting with CasL-like 2 (MICAL-L2) as an effector of the Rab13 small G protein, and we found that the Rab13-JRAB system may be involved in the formation of cell-cell adhesions via transport of adhesion molecules. Here, we showed that JRAB interacts with two actin-binding proteins, actinin-1 and -4, and filamentous actin via different domains and regulates actin cross-linking and stabilization through these interactions. During epithelial junctional development, JRAB is prominently enriched in the actin bundle at the free border; subsequently, JRAB undergoes a Rab13-dependent conformational change that is required for maturation of cell-cell adhesion sites. These results suggest that Rab13 and JRAB regulate reorganization of the actin cytoskeleton throughout epithelial junctional development from establishment to maturation of cell-cell adhesion.

Interaction of nectin-like molecule 2 with integrin alpha6beta4 and inhibition of disassembly of integrin alpha6beta4 from hemidesmosomes.

In normal epithelial cells, integrin α(6)ß(4) is abundantly expressed and forms hemidesmosomes, which is a cellular structure that mediates cell-extracellular matrix binding. In many types of cancer cells, integrin α(6)ß(4) is up-regulated, laminin is cleaved, and hemidesmosomes are disrupted, eventually causing an enhancement of cancer cell movement and facilitation of their invasion. We previously showed that the immunoglobulin-like cell adhesion molecule Necl-2 (Nectin-like molecule 2), known as a tumor suppressor, inhibits cancer cell movement by suppressing the ErbB3/ErbB2 signaling. We show here that Necl-2 interacts in cis with integrin α(6)ß(4). The binding of Necl-2 with integrin ß(4) was mediated by its extracellular region. In human colorectal adenocarcinoma Caco-2 cells, integrin α(6)ß(4) was localized at hemidesmosomes. Small interfering RNA-mediated suppression of Necl-2 expression enhanced the phorbol ester-induced disruption of the integrin α(6)ß(4) complex at hemidesmosomes, whereas expression of Necl-2 suppressed the disruption of this structure. These results indicate that tumor-suppressive functions of Necl-2 are mediated by the stabilization of the hemidesmosome structure in addition to the inhibition of the ErbB3/ErbB2 signaling.

The active site structure of nitrided and oxynitrided graphite as a cathode catalyst in a fuel cell.

The first-principles studies were performed for the modified graphites with various edge conformations, which revealed that the NH edge facilitates easy transfer of an electron into the adsorbed O(2).

Necl-5/PVR enhances PDGF-induced attraction of growing microtubules to the plasma membrane of the leading edge of moving NIH3T3 cells.

Microtubules (MTs) search for and grow toward the leading edge of moving cells, followed by their stabilization at a specific structure at the rear site of the leading edge. This dynamic re-orientation of MTs is critical to directional cell movement. We previously showed that Necl-5/poliovirus receptor (PVR) interacts with platelet-derived growth factor (PDGF) receptor and integrin α(v) ß(3) at the leading edge of moving NIH3T3 cells, resulting in an enhancement of their directional movement. We studied here the role of Necl-5 in the PDGF-induced attraction of growing MTs to the leading edge of NIH3T3 cells. Necl-5 enhanced the PDGF-induced growth of MTs and attracted them near to the plasma membrane of the leading edge of NIH3T3 cells in an integrin α(v) ß(3) -dependent manner. Furthermore, Necl-5 enhanced the PDGF-induced attraction of the plus-end-tracking proteins (+TIPs), including EB1, CLIP170, an intermediate chain subunit of cytoplasmic dynein, and p150(Glued) , a subunit of dynactin, near to the plasma membrane of the leading edge. Thus, Necl-5 plays a role in the attraction of growing MTs to the plasma membrane of the leading edge of moving cells.