Breaking the symmetry of cell contractility drives tubulogenesis via CXCL1 polarization.

The intricate interplay between biomechanical and biochemical pathways in modulating morphogenesis is an interesting research topic. How biomechanical force regulates epithelial cell tubulogenesis remains poorly understood. Here, we established a model of tubulogenesis by culturing renal proximal tubular epithelial cells on a collagen gel while manipulating contractile force. Epithelial cells were dynamically self-organized into tubule-like structures by augmentation of cell protrusions and cell-cell association. Reduction and asymmetric distribution of phosphorylated myosin light chain 2, the actomyosin contractility, in cells grown on soft matrix preceded tube connection. Notably, reducing matrix stiffness via sonication of collagen fibrils and inhibiting actomyosin contractility with blebbistatin promoted tubulogenesis, whereas inhibition of cytoskeleton polymerization suppressed it. CXC chemokine ligand 1 (CXCL1) expression was transcriptionally upregulated in cells undergoing tubulogenesis. Additionally, inhibiting actomyosin contractility facilitated CXCL1 polarization and cell protrusions preceding tube formation. Conversely, inhibiting the CXCL1-CXC receptor 1 pathway hindered cell protrusions and tubulogenesis. Mechanical property asymmetry with cell-collagen fibril interaction patterns at cell protrusions and along the tube structure supported the association of anisotropic contraction with tube formation. Furthermore, suppressing the mechanosensing machinery of integrin subunit beta 1 reduced CXCL1 expression, collagen remodeling, and impaired tubulogenesis. In summary, symmetry breaking of cell contractility on a soft collagen gel promotes CXCL1 polarization at cell protrusions which in turn facilitates cell-cell association and thus tubule connection.

TEM1/endosialin/CD248 promotes pathologic scarring and TGF-ß activity through its receptor stability in dermal fibroblasts.

BACKGROUND: Pathologic scars, including keloids and hypertrophic scars, represent a common form of exaggerated cutaneous scarring that is difficult to prevent or treat effectively. Additionally, the pathobiology of pathologic scars remains poorly understood. We aim at investigating the impact of TEM1 (also known as endosialin or CD248), which is a glycosylated type I transmembrane protein, on development of pathologic scars. METHODS: To investigate the expression of TEM1, we utilized immunofluorescence staining, Western blotting, and single-cell RNA-sequencing (scRNA-seq) techniques. We conducted in vitro cell culture experiments and an in vivo stretch-induced scar mouse model to study the involvement of TEM1 in TGF-ß-mediated responses in pathologic scars. RESULTS: The levels of the protein TEM1 are elevated in both hypertrophic scars and keloids in comparison to normal skin. A re-analysis of scRNA-seq datasets reveals that a major profibrotic subpopulation of keloid and hypertrophic scar fibroblasts greatly expresses TEM1, with expression increasing during fibroblast activation. TEM1 promotes activation, proliferation, and ECM production in human dermal fibroblasts by enhancing TGF-ß1 signaling through binding with and stabilizing TGF-ß receptors. Global deletion of Tem1 markedly reduces the amount of ECM synthesis and inflammation in a scar in a mouse model of stretch-induced pathologic scarring. The intralesional administration of ontuxizumab, a humanized IgG monoclonal antibody targeting TEM1, significantly decreased both the size and collagen density of keloids. CONCLUSIONS: Our data indicate that TEM1 plays a role in pathologic scarring, with its synergistic effect on the TGF-ß signaling contributing to dermal fibroblast activation. Targeting TEM1 may represent a novel therapeutic approach in reducing the morbidity of pathologic scars.

CD248 Regulates Inflammation and Encapsulation in Silicone-Related Capsule Formation.

BACKGROUND: Capsular contracture is the most common reason for having a secondary breast implant operation. The failure of the implanted device and discomfort are related to foreign body response, which involves a pathologic encapsulation. An up-regulated expression of CD248 was previously demonstrated to modulate inflammation and fibrosis. The authors hypothesized that CD248 contributes to foreign body reaction and contracture during silicone-stimulated capsule formation. METHODS: A murine capsular contracture model was established to correlate CD248 with capsular contracture. The timing and site of CD248 expression were characterized by protein analysis and histologic examination. The capsules between wild-type mice and CD248 knockout mice were compared in this model to verify the possible role of CD248 in silicone-related capsule formation. RESULTS: CD248 was expressed in the peri-silicone implant capsule by stromal fibroblast and perivascular fibroblast. CD248 was overexpressed on day 4 and down to a constant level, but it was still up-regulated through day 21 to day 56 after silicone implantation. The CD248 knockout mice showed a prolonged inflammation period, whereas the wild-type mice developed a thinner but more collagenous capsule. CONCLUSIONS: In conclusion, an effective murine capsular contracture model was established to study the relationship between CD248 and capsular contracture. CD248 may play a role in inflammation and encapsulation during silicone implantation. CD248 deletion in mice contributed to a loose and irregular collagen bundle in a capsule area, implying a decrease in contracture. Therefore, CD248 could be a potential therapeutic target in capsular contracture. CLINICAL RELEVANCE STATEMENT: CD248 may play a role in inflammation and encapsulation during silicone implantation. It could be a potential therapeutic target in clinical capsular contracture.

CD248 promotes insulin resistance by binding to the insulin receptor and dampening its insulin-induced autophosphorylation.

BACKGROUND: In spite of new treatments, the incidence of type 2 diabetes (T2D) and its morbidities continue to rise. The key feature of T2D is resistance of adipose tissue and other organs to insulin. Approaches to overcome insulin resistance are limited due to a poor understanding of the mechanisms and inaccessibility of drugs to relevant intracellular targets. We previously showed in mice and humans that CD248, a pre/adipocyte cell surface glycoprotein, acts as an adipose tissue sensor that mediates the transition from healthy to unhealthy adipose, thus promoting insulin resistance. METHODS: Molecular mechanisms by which CD248 regulates insulin signaling were explored using in vivo insulin clamp studies and biochemical analyses of cells/tissues from CD248 knockout (KO) and wild-type (WT) mice with diet-induced insulin resistance. Findings were validated with human adipose tissue specimens. FINDINGS: Genetic deletion of CD248 in mice, overcame diet-induced insulin resistance with improvements in glucose uptake and lipolysis in white adipose tissue depots, effects paralleled by increased adipose/adipocyte GLUT4, phosphorylated AKT and GSK3ß, and reduced ATGL. The insulin resistance of the WT mice could be attributed to direct interaction of the extracellular domains of CD248 and the insulin receptor (IR), with CD248 acting to block insulin binding to the IR. This resulted in dampened insulin-mediated autophosphorylation of the IR, with reduced downstream signaling/activation of intracellular events necessary for glucose and lipid homeostasis. INTERPRETATION: Our discovery of a cell-surface CD248-IR complex that is accessible to pharmacologic intervention, opens research avenues toward development of new agents to prevent/reverse insulin resistance. FUNDING: Funded by Canadian Institutes of Health Research (CIHR), Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Foundations for Innovation (CFI), the Swedish Diabetes Foundation, Family Ernfors Foundation and Novo Nordisk Foundation.

Soluble CD93 lectin-like domain sequesters HMGB1 to ameliorate inflammatory diseases.

Rationale: CD93, a C-type lectin-like transmembrane glycoprotein, can be shed in a soluble form (sCD93) upon inflammatory stimuli. sCD93 effectively enhances apoptotic cell clearance and has been proposed as an inflammatory disease biomarker. The function of sCD93 involved directly in inflammation remains to be determined. Herein, we attempted to examine the hypothesis that sCD93 might sequester proinflammatory high-mobility group box 1 protein (HMGB1), exerting anti-inflammatory properties. Methods: Different forms of soluble recombinant human CD93 (rCD93) were prepared by a mammalian protein expression system. rCD93-HMGB1 interaction was assessed using co-immunoprecipitation and solid-phase binding assays. Effects of soluble rCD93 were evaluated in HMGB1-induced macrophage and vascular smooth muscle cells (VSMC) activation and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, CaCl2-induced and angiotensin II-infused abdominal aortic aneurysm (AAA) formation and ovariectomized-induced osteoporosis in mice. Results: Protein binding studies revealed that soluble rCD93, via the lectin-like domain (D1), can bind to HMGB1 and intercept HMGB1-receptor interaction. Soluble rCD93 containing D1 inhibited HMGB1-induced proinflammatory cytokine production and intracellular mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB activation in macrophages and VSMCs, thereby attenuating CaCl2-induced and angiotensin II-infused AAA models. During osteoclastogenesis, RANKL stimulated HMGB1 secretion that promoted RANKL-induced osteoclastogenesis in return. Soluble rCD93 containing D1 impeded RANKL-induced osteoclastogenic marker gene expression and intracellular MAPK/NF-κB signaling, thereby mitigating ovariectomized-induced osteoporosis. Conclusion: These findings demonstrate the therapeutic potential of soluble recombinant CD93 containing D1 in inflammatory diseases. Our study highlights a novel anti-inflammatory mechanism, i.e., sequestration of HMGB1, through which sCD93 prevents HMGB1-receptor interaction on effector cells and alleviates inflammation.

Chondrocyte Thrombomodulin Protects against Osteoarthritis.

Osteoarthritis (OA) is a prevalent form of arthritis that affects over 32.5 million adults worldwide, causing significant cartilage damage and disability. Unfortunately, there are currently no effective treatments for OA, highlighting the need for novel therapeutic approaches. Thrombomodulin (TM), a glycoprotein expressed by chondrocytes and other cell types, has an unknown role in OA. Here, we investigated the function of TM in chondrocytes and OA using various methods, including recombinant TM (rTM), transgenic mice lacking the TM lectin-like domain (TMLeD/LeD), and a microRNA (miRNA) antagomir that increased TM expression. Results showed that chondrocyte-expressed TM and soluble TM [sTM, like recombinant TM domain 1 to 3 (rTMD123)] enhanced cell growth and migration, blocked interleukin-1ß (IL-1ß)-mediated signaling and protected against knee function and bone integrity loss in an anterior cruciate ligament transection (ACLT)-induced mouse model of OA. Conversely, TMLeD/LeD mice exhibited accelerated knee function loss, while treatment with rTMD123 protected against cartilage loss even one-week post-surgery. The administration of an miRNA antagomir (miR-up-TM) also increased TM expression and protected against cartilage damage in the OA model. These findings suggested that chondrocyte TM plays a crucial role in counteracting OA, and miR-up-TM may represent a promising therapeutic approach to protect against cartilage-related disorders.

The influences of finite aperture size in photoacoustic computed tomography.

In photoacoustic computed tomography (PACT), the "finite aperture effect" is often characterized as a tangential resolution that increases proportionally with the distance from the rotation center. However, this conclusion is based on the inaccurate point-detector assumption used in image reconstruction. In this study, we appropriately modeled the finite size of the acoustic detector in the back-projection (BP) based image reconstruction to improve the accuracy of the time delay calculation and systematically investigated its effects. Our results showed that the main effect of the finite aperture size is the creation of a limited high-quality imaging region (HQIR) around the scanning center, due to the directional sensitivity of the detector. We also demonstrated that the "finite aperture effect" can reduce the optimal number of detectors required for spatial anti-aliasing. These new findings provide novel perspectives for optimizing PACT systems and corresponding reconstruction methods.

The application of the PDCA cycle in the nutritional management of patients with nasopharyngeal carcinoma.

OBJECTIVE: This study is to explore the effect of the Plan-Do-Check-Act (PDCA) cycle on the nutritional management of patients with nasopharyngeal carcinoma (NPC). METHODS: A total of 100 NPC patients were randomly divided into a control group and a PDCA group, with 50 patients in each group. The control group adopted a routine nutritional management strategy, and the PDCA group adopted a PDCA cycle management strategy. The body weight, body mass index (BMI), hemoglobin, serum prealbumin, serum albumin, the Patient-Generated Subjective Global Assessment (PG-SGA) score, the Nutrition Risk Screening 2002 (NRS-2002) score, the incidence rate of nutritional risk, the grade of malnutrition, and the grade of oral mucositis were compared between the two groups. RESULTS: The body weight, BMI, and serum prealbumin in the PDCA group were higher than those in the control group, and the difference was statistically significant (p < 0.05). The NRS2002 score and PG-SGA score in the PDCA group were lower than those in the control group, and the differences were statistically significant (p < 0.05). The incidence of nutritional risk, the grade of malnutrition, and the grade of oral mucositis were less in the PDCA group than those in the control group (p < 0.05). There was no significant difference in hemoglobin and serum albumin between the two groups (p > 0.05). CONCLUSION: The PDCA cycle can improve body weight, BMI, and serum prealbumin in NPC patients. It can reduce the NRS2002 score, the PG-SGA score, the incidence of nutritional risk, the severity of malnutrition, and the severity of oral mucositis in NPC patients.

Interference in melanoma CD248 function reduces vascular mimicry and metastasis.

BACKGROUND: Tumor vascular mimicry is an emerging issue that affects patient survival while having no treatment at the current moment. Despite several factors implicated in vascular mimicry, little is known about stromal factors that modulate tumor microenvironment and shape malignant transformation. CD248, a type-I transmembrane protein dominantly expressed in stromal cells, mediates the interaction between cells and extracellular matrix proteins. CD248 protein expression is associated with the metastatic melanoma phenotype and promotes tumor progression in the stromal cells. This study aimed to explore the cell-autonomous effects of CD248 in melanoma vascular mimicry to aid cancer therapy development. METHODS: Loss-of-function approaches in B16F10 melanoma cells were used to study the cell-autonomous effects of CD248 on cell adhesion, migration, proliferation, and vascular mimicry. A solid-phase binding assay was performed to identify the interaction between CD248 and fibronectin. Horizontal and vertical cell migration assays were performed to analyze cell migration activity, and cell-patterned network formation on Matrigel was used to evaluate vascular mimicry activity. Recombinant CD248 (rCD248) proteins were generated, and whether rCD248 interfered with melanoma CD248 functions was evaluated in vitro. An experimental lung metastasis mouse model was used to investigate the effect of rCD248 treatment in vivo. RESULTS: CD248 protein expression in melanoma cells was increased by a fibroblast-conditioned medium. Knockdown of CD248 expression significantly decreased cell adhesion to fibronectin, cell migration, and vascular mimicry in melanoma cells. The lectin domain of CD248 was directly involved in the interaction between CD248 and fibronectin. Furthermore, rCD248 proteins containing its lectin domain inhibited cell adhesion to fibronectin and slowed down cell migration and vascular mimicry. Treatment with rCD248 protein could reduce pulmonary tumor burden, accompanied by a reduction in vascular mimicry in mice with melanoma lung metastasis. CONCLUSION: CD248 expression in melanoma cells promotes malignant transformation by increasing the activity of cell adhesion, migration, and vascular mimicry, whereas rCD248 protein functions as a molecular decoy interfering with tumor-promoting effects of CD248 in melanoma cells.

CD248 Regulates Wnt Signaling in Pericytes to Promote Angiogenesis and Tumor Growth in Lung Cancer.

The tumor microenvironment plays a central role in cancer initiation and progression. CD248 is expressed in tumor-associated stromal cells, particularly fibroblasts and pericytes. Exploring the function of CD248 has the potential to provide biological insights into tumor-supportive stroma and potential therapeutic targets. Here, we investigated the role of stromal CD248 in lung cancer. In orthotopic lung cancer transplantation models, tumor volume, density of vessels and pericytes, and functionality of tumor vessels were all lower in mice lacking Cd248 (Cd248LacZ/LacZ) compared with Cd248 wild-type or haploinsufficient mice. Two angiogenic factors, OPN and SERPINE1, were decreased in Cd248LacZ/LacZ pericytes, and supplementation with both factors rescued their proliferation and endothelial cell tube formation-promoting ability. Mechanistically, Wnt/ß-catenin signaling induced Opn and Serpine1 expression and was suppressed in Cd248LacZ/LacZ pericytes. CD248 interacted with Wnt pathway repressors IGFBP4 and LGALS3BP, leading to increased Wnt/ß-catenin signaling. Correspondingly, administration of a ß-catenin inhibitor in Cd248+/LacZ mice mimicked the effect of Cd248 loss and blocked the growth of transplanted lung tumor cells that were resistant to this inhibitor in vitro. In addition, CD248+ pericytes coexpressed OPN and SERPINE1 and correlated with increased tumor size in human lung cancer. Additionally, high expression of CD248, OPN, and SERPINE1 was associated with poor survival in lung cancer patients. In summary, CD248 derepresses Wnt signaling and upregulates OPN and SERPINE1 in pericytes, resulting in enhanced angiogenesis and lung cancer growth. This novel axis of CD248-Wnt signaling-angiogenic factors in pericytes provides a potential target for lung cancer therapy. SIGNIFICANCE: These findings demonstrate that CD248 maintains pericyte function in lung cancer through the Wnt signaling pathway and present CD248 as a potential therapeutic target.

Absence of the lectin-like domain of thrombomodulin reduces HSV-1 lethality of mice with increased microglia responses.

BACKGROUND: Herpes simplex virus 1 (HSV-1) can induce fatal encephalitis. Cellular factors regulate the host immunity to affect the severity of HSV-1 encephalitis. Recent reports focus on the significance of thrombomodulin (TM), especially the domain 1, lectin-like domain (TM-LeD), which modulates the immune responses to bacterial infections and toxins and various diseases in murine models. Few studies have investigated the importance of TM-LeD in viral infections, which are also regulated by the host immunity. METHODS: In vivo studies comparing wild-type and TM-LeD knockout mice were performed to determine the role of TM-LeD on HSV-1 lethality. In vitro studies using brain microglia cultured from mice or a human microglia cell line to investigate whether and how TM-LeD affects microglia to reduce HSV-1 replication in brain neurons cultured from mice or in a human neuronal cell line. RESULTS: Absence of TM-LeD decreased the mortality, tissue viral loads, and brain neuron apoptosis of HSV-1-infected mice with increases in the number, proliferation, and phagocytic activity of brain microglia. Moreover, TM-LeD deficiency enhanced the phagocytic activity of brain microglia cultured from mice or of a human microglia cell line. Co-culture of mouse primary brain microglia and neurons or human microglia and neuronal cell lines revealed that TM-LeD deficiency augmented the capacity of microglia to reduce HSV-1 replication in neurons. CONCLUSIONS: Overall, TM-LeD suppresses microglia responses to enhance HSV-1 infection.

Omnibearing Interpretation of External Ions Passivated Ion Migration in Mixed Halide Perovskites.

Fundamental understanding of ion migration inside perovskites is of vital importance for commercial advancements of photovoltaics. However, the mechanism for external ions incorporation and its effect on ion migration remains elusive. Herein, taking K+ and Cs+ co-incorporated mixed halide perovskites as a model, the impact of external ions on ion migration behavior has been interpreted via multiple dimensional characterization aspects. The space-effect on phase segregation inhibition has been revealed by the photoluminescence evolution and in situ dynamic cathodoluminescence behaviors. The plane-effect on current suppression along grain boundary has been evidenced via visualized surface current mapping, local current hysteresis, and time-resolved current decay. And the point-effect on activation energy incremental for individual ions has been also probed by cryogenic electronic quantification. All these results sufficiently demonstrate the passivated ion migration results in the eventually improved phase stability of perovskite, of which the origin lies in various ion migration energy barriers.

Interpretation of Rubidium-Based Perovskite Recipes toward Electronic Passivation and Ion-Diffusion Mitigation.

Rubidium cation (Rb+ ) addition is witnessed to play a pivotal role in boosting the comprehensive performance of organic-inorganic hybrid perovskite solar cells. However, the origin of such success derived from irreplaceable superiorities brought by Rb+ remains ambiguous. Herein, grain-boundary-including atomic models are adopted for the accurate theoretical analysis of practical Rb+ distribution in perovskite structures. The spatial distribution, covering both the grain interiors and boundaries, is thoroughly identified by virtue of synchrotron-based grazing-incidence X-ray diffraction. On this basis, the prominent elevation of the halogen vacancy formation energy, improved charge-carrier dynamics, and the electronic passivation mechanism in the grain interior are expounded. As evidenced by the increased energy barrier and suppressed microcurrent, the critical role of Rb+ addition in blocking the diffusion pathway along grain boundaries, inhibiting halide phase segregation, and eventually enhancing intrinsic stability is elucidated. Hence, the linkage avalanche effect of occupied location dominated by subtle changes in Rb+ concentration on electronic defects, ion migration, and phase stability is completely investigated in detail, shedding a new light on the advancement of high-efficiency cascade-incorporating strategies and perovskite compositional engineering.

Microglia Reduce Herpes Simplex Virus 1 Lethality of Mice with Decreased T Cell and Interferon Responses in Brains.

Herpes simplex virus 1 (HSV-1) infects the majority of the human population and can induce encephalitis, which is the most common cause of sporadic, fatal encephalitis. An increase of microglia is detected in the brains of encephalitis patients. The issues regarding whether and how microglia protect the host and neurons from HSV-1 infection remain elusive. Using a murine infection model, we showed that HSV-1 infection on corneas increased the number of microglia to outnumber those of infiltrating leukocytes (macrophages, neutrophils, and T cells) and enhanced microglia activation in brains. HSV-1 antigens were detected in brain neurons, which were surrounded by microglia. Microglia depletion increased HSV-1 lethality of mice with elevated brain levels of viral loads, infected neurons, neuron loss, CD4 T cells, CD8 T cells, neutrophils, interferon (IFN)-ß, and IFN-γ. In vitro studies demonstrated that microglia from infected mice reduced virus infectivity. Moreover, microglia induced IFN-ß and the signaling pathway of signal transducer and activator of transcription (STAT) 1 to inhibit viral replication and damage of neurons. Our study reveals how microglia protect the host and neurons from HSV-1 infection.

Recombinant thrombomodulin domain 1 rescues pathological angiogenesis by inhibition of HIF-1α-VEGF pathway.

Pathological angiogenesis (PA) contributes to various ocular diseases, including age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity, which are major causes of blindness over the world. Current treatments focus on anti-vascular endothelial growth factor (VEGF) therapy, but persistent avascular retina, recurrent intravitreal neovascularization, and general adverse effects are reported. We have previously found that recombinant thrombomodulin domain 1 (rTMD1) can suppress vascular inflammation. However, the function of rTMD1 in VEGF-induced PA remains unknown. In this study, we found that rTMD1 inhibited VEGF-induced angiogenesis in vitro. In an oxygen induced retinopathy (OIR) animal model, rTMD1 treatment significantly decreased retinal neovascularization but spared normal physiological vessel growth. Furthermore, loss of TMD1 significantly promoted PA in OIR. Meanwhile, hypoxia-inducible factor-1α, the transcription factor that upregulates VEGF, was suppressed after rTMD1 treatment. The levels of interleukin-6, and intercellular adhesion molecule-1 were also significantly suppressed. In conclusion, our results indicate that rTMD1 not only has dual effects to suppress PA and inflammation in OIR, but also can be a potential HIF-1α inhibitor for clinical use. These data bring forth the possibility of rTMD1 as a novel therapeutic agent for PA.

Nanostructured Lipid Carrier Gel Formulation of Recombinant Human Thrombomodulin Improve Diabetic Wound Healing by Topical Administration.

Recombinant human thrombomodulin (rhTM), an angiogenesis factor, has been demonstrated to stimulate cell proliferation, keratinocyte migration and wound healing. The objective of this study was to develop nanostructured lipid carrier (NLC) formulations encapsulating rhTM for promoting chronic wound healing. RhTM-loaded NLCs were prepared and characterized. Encapsulation efficiency was more than 92%. The rate of rhTM release from different NLC formulations was influenced by their lipid compositions and was sustained for more than 72 h. Studies on diabetic mouse wound model suggested that rhTM-NLC 1.2 µg accelerated wound healing and was similar to recombinant human epidermal growth factor-NLC (rhEGF-NLC) 20 µg. By incorporating 0.085% carbopol (a highly crosslinked polyacrylic acid polymer) into rhTM NLC, the NLC-gel presented similar particle characteristics, and demonstrated physical stability, sustained release property and stability within 12 weeks. Both rhTM NLC and rhTM NLC-gel improved wound healing of diabetic mice and cell migration of human epidermal keratinocyte cell line (HaCaT) significantly. In comparison with rhTM solution, plasma concentrations of rhTM post applications of NLC and NLC-gel formulations were lower and more sustained in 24 h. The developed rhTM NLC and rhTM NLC-gel formulations are easy to prepare, stable and convenient to apply to the wound with reduced systemic exposure, which may warrant potential delivery systems for the care of chronic wound patients.

VEGF-Induced Endothelial Podosomes via ROCK2-Dependent Thrombomodulin Expression Initiate Sprouting Angiogenesis.

[Figure: see text].

Role of tumor endothelial marker 1 (Endosialin/CD248) lectin-like domain in lipopolysaccharide-induced macrophage activation and sepsis in mice.

Deleterious hyper-inflammation resulting from macrophage activation may aggravate sepsis and lead to lethality. Tumor endothelial marker 1 (TEM1), a type I transmembrane glycoprotein containing six functional domains, has been implicated in cancer and chronic sterile inflammatory disorders. However, the role of TEM1 in acute sepsis remains to be determined. Herein we explored the functional significance of the TEM1 lectin-like domain (TEM1D1) in monocyte/macrophage activation and sepsis using TEM1D1-deleted (TEM1LeD/LeD) transgenic mice and recombinant TEM1D1 (rTEM1D1) protein. Under stimulation with lipopolysaccharides (LPS) or several other toll-like receptor agonists, TEM1LeD/LeD macrophages produced lower levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 than wild-type TEM1wt/wt macrophages. Compared with TEM1wt/wt macrophages, LPS-macrophage binding and intracellular mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB activation were suppressed in TEM1LeD/LeD macrophages. In vivo, TEM1D1 deletion improved survival in LPS-challenged mice with reduction of circulating TNF-α and IL-6 and alleviation of lung injury and pulmonary leukocyte accumulation. In contrast, rTEM1D1 could bind to LPS and markedly suppress LPS-macrophage binding, MAPK/NF-κB signaling in macrophages and proinflammatory cytokine production. Treatment with rTEM1D1 improved survival and attenuated circulating TNF-α and IL-6, lung injury and pulmonary accumulation of leukocytes in LPS-challenged mice. These findings demonstrated differential roles for the TEM1 lectin-like domain in macrophages and soluble TEM1 lectin-like domain in sepsis. TEM1 in macrophages mediates LPS-induced inflammation via its lectin-like domain, whereas rTEM1D1 interferes with LPS-induced macrophage activation and sepsis.

Dual function of clock component OsLHY sets critical day length for photoperiodic flowering in rice.

Circadian clock, an endogenous time-setting mechanism, allows plants to adapt to unstable photoperiod conditions and induces flowering with proper timing. In Arabidopsis, the central clock oscillator was formed by a series of interlocked transcriptional feedback loops, but little is known in rice so far. By MutMap technique, we identified the candidate gene OsLHY from a later flowering mutant lem1 and further confirmed it through genetic complementation, RNA interference knockdown, and CRISPR/Cas9-knockout. Global transcriptome profiling and expression analyses revealed that OsLHY might be a vital circadian rhythm component. Interestingly, oslhy flowered later under ≥12 h day length but headed earlier under ≤11 h day length. qRT-PCR results exhibited that OsLHY might function through OsGI-Hd1 pathway. Subsequent one-hybrid assays in yeast, DNA affinity purification qPCR, and electrophoretic mobility shift assays confirmed OsLHY could directly bind to the CBS element in OsGI promoter. Moreover, the critical day length (CDL) for function reversal of OsLHY in oslhy (11-12 h) was prolonged in the double mutant oslhy osgi (about 13.5 h), indicating that the CDL set by OsLHY was OsGI dependent. Additionally, the dual function of OsLHY entirely relied on Hd1, as the double mutant oslhy hd1 showed the same heading date with hd1 under about 11.5, 13.5, and 14 h day lengths. Together, OsLHY could fine-tune the CDL by directly regulating OsGI, and Hd1 acts as the final effector of CDL downstream of OsLHY. Our study illustrates a new regulatory mechanism between the circadian clock and photoperiodic flowering.

Recombinant thrombomodulin lectin-like domain attenuates Porphyromonas gingivalis lipopolysaccharide-induced osteoclastogenesis and periodontal bone resorption.

BACKGROUND: Evidence demonstrates that the thrombomodulin (TM) lectin domain (TMD1) exerts anti-inflammatory functions. Lipopolysaccharides derived from Porphyromonas gingivalis (Pg-LPS) are considered a major pathogenic factor for chronic periodontitis, promoting inflammation, osteoclastogenesis and alveolar bone resorption. Herein, we aimed to evaluate the potential therapeutic effect of recombinant TMD1 (rTMD1) in suppression of Pg-LPS-induced osteoclastogenesis and periodontal bone loss. METHODS: In vitro, the effects of Pg-LPS, tumor necrosis factor (TNF)-α and rTMD1 on osteoclast differentiation were investigated using receptor activator of nuclear factor-κB ligand (RANKL)-stimulated RAW 264.7 macrophages. In vivo, the effects of rTMD1 treatment were evaluated in a model of experimental periodontitis induced by direct injection of Pg-LPS into the vestibular gingiva. RESULTS: Administration of Pg-LPS to RANKL-stimulated RAW 264.7 macrophages resulted in upregulation of CD86 and osteoclast marker (eg, Dc-stamp and Trap) gene expression and increase of pro-inflammatory cytokine production (e.g., TNF-α) during osteoclast differentiation, and rTMD1 can attenuate these effects. Also, rTMD1 inhibited Pg-LPS-enhanced in vitro bone resorption in a dose-dependent manner. Moreover, TNF-α promoted phosphorylation of p38 and ERK during osteoclast differentiation, and the signal activation can be inhibited by rTMD1. Finally, treatment with rTMD1 hindered Pg-LPS-induced alveolar bone loss in experimental periodontitis in mice. CONCLUSION: Our study demonstrated that rTMD1 attenuates Pg-LPS-enhanced M1 macrophage polarization, osteoclastogenesis and periodontal bone resorption and thus holds therapeutic promise for periodontitis.