Epidermal Growth Factor Family Research Articles

MSCs-IL33-ILC2s-AREG-Tecs Axis Promotes the Remodeling of Thymic Function in Acute Gvhd

Background: Previous studies suggested that mesenchymal stromal cells (MSCs) administrated at the acute graft versus host disease (aGVHD) phase effectively decreased the incidence and severity of chronic GVHD (cGVHD) by boosting thymic regeneration. Our research has found that MSCs expressing interleukin 33 (IL33) may participate in thymus regeneration from aGVHD damage through type 2 innate lymphoid cells (ILC2s)-mediated tissue recovery. However, the mechanism for ILC2s repairing the damaged thymus caused by aGVHD is unclear. Here, we further explored the mechanisms on how ILC2s promote thymus recovery post-BMT. Methods: We carried out studies in a aGVHD mice model of fully MHC-mismatched myeloablative bone marrow transplantation (C57BL/6 to BALB/c). IL33 knocked-down (MSC/IL33 -) and empty-load MSCs (MSC/Control) were generated by lentivirus transfection, and then administrated intravenously at day 7 post-BMT to intervene in aGVHD and assess the effect of IL33 from MSCs on ILC2s (Lin-CD45+iCD3-CD127+GATA3+) in the thymus by flow cytometery at day 14 post-BMT. Clinical scores and survival were recorded once every days. Furthermore, we isolated, purified and amplified ILC2s from C57BL/6 mouse thymus by immunomagnetic bead sorting and flow cytometry for aGVHD intervention and studied whether ILC2s can home to thymus by fluorescence tracing and the effects of ILC2s on TECs by immunofluorescence. To study the potential mechanism of ILC2s affecting TECs by using transcriptome sequencing (RNA-seq) and related molecular biology experiments. Finally, the potential target amphiregulin (AREG) was found from RNA-seq analyses and the murine recombinant AREG was used as an intervention to evaluate its effect on aGVHD mouse. Results: In this study, we found that knocking down the expression of IL33 in MSCs partially blocked the repair effect of MSCs on thymus and decreased the expansion of thymic ILC2s in aGVHD mice compared with the MSC/Control group. Then, we found that exogenous infusion of thymic ILC2s is very effective in treating thymic tissue injury, and can home to the recipient thymus and further migrate to the thymic medulla region. Furthermore, we found that ILC2s may home to the thymus and migrate to the thymic medulla region through CCR7 and MSCs promote the high expression of AREG in ILC2s through RNA-seq. Importantly, we found that the expression level of intrathymic AREG in No-GVHD, MSCs-GVHD and ILC2s-GVHD group was significantly increased than aGVHD group on day 14 post-BMT and exogenous administration of AREG after transplantation can significantly increase the proliferation of thymic epithelial cells, especially the regeneration of thymic medulla and the generation of new thymus-derived CD4/CD8 double-positive T cells. Conclusion: In conclusion, we demonstrate that MSCs infusion may rely on the MSCs-IL33-ILC2s-AREG-TECs axis mediated by ILC2s to remodeling the thymic microenvironment homeostasis, thus promoting the reconstruction of T cell immune homeostasis. Importantly, the results presented in this report identify a previously unrecognized role for the epidermal growth factor family member AREG in promoting the remodeling of thymus tissue. and providing new insights for the mechanism of MSCs treatment for GVHD.

Preferential expression of SCN1A in GABAergic neurons improves survival and epileptic phenotype in a mouse model of Dravet syndrome

The SCN1A gene encodes the alpha subunit of a voltage-gated sodium channel (Nav1.1), which is essential for the function of inhibitory neurons in the brain. Mutations in this gene cause severe encephalopathies such as Dravet syndrome (DS). Upregulation of SCN1A expression by different approaches has demonstrated promising therapeutic effects in preclinical models of DS. Limiting the effect to inhibitory neurons may contribute to the restoration of brain homeostasis, increasing the safety and efficacy of the treatment. In this work, we have evaluated different approaches to obtain preferential expression of the full SCN1A cDNA (6 Kb) in GABAergic neurons, using high-capacity adenoviral vectors (HC-AdV). In order to favour infection of these cells, we considered ErbB4 as a surface target. Incorporation of the EGF-like domain from neuregulin 1 alpha (NRG1α) in the fiber of adenovirus capsid allowed preferential infection in cells lines expressing ErbB4. However, it had no impact on the infectivity of the vector in primary cultures or in vivo. For transcriptional control of transgene expression, we developed a regulatory sequence (DP3V) based on the Distal-less homolog enhancer (Dlx), the vesicular GABA transporter (VGAT) promoter, and a portion of the SCN1A gene. The hybrid DP3V promoter allowed preferential expression of transgenes in GABAergic neurons both in vitro and in vivo. A new HC-AdV expressing SCN1A under the control of this promoter showed improved survival and amelioration of the epileptic phenotype in a DS mouse model. These results increase the repertoire of gene therapy vectors for the treatment of DS and indicate a new avenue for the refinement of gene supplementation in this disease.Key messagesAdenoviral vectors can deliver the SCN1A cDNA and are amenable for targeting.An adenoviral vector displaying an ErbB4 ligand in the capsid does not target GABAergic neurons.A hybrid promoter allows preferential expression of transgenes in GABAergic neurons.Preferential expression of SCN1A in GABAergic cells is therapeutic in a Dravet syndrome model.

NOTCH1 and PIK3CA mutation are related to HPV-associated vulvar squamous cell carcinoma

NOTCH1 and PIK3CA are members of important cell signalling pathways that are deregulated in squamous cell carcinomas of various organs. Vulvar squamous cell carcinomas (vulvSCC) are classically divided into two pathways, HPV-associated or HPV-independent, but the effect of NOTCH1 and PIK3CA mutations in both groups is unclear. We analysed two different cohorts of vulvSCC using Hybrid Capture-based Comprehensive Genomic Profiling and identified NOTCH1 and PIK3CA mutations in 35% and 31% of 48 primary vulvSCC. In this first cohort, PIK3CA and NOTCH1 mutations were significantly correlated with HPV infection (p < 0.01). Furthermore, mutations in both genes were associated with an advanced tumor stage and poorly differentiated status (p < 0.05). PIK3CA and NOTCH1 mutations were also associated with shorter patient survival which did not reach significance. In the second cohort of 735 advanced vulvSCC from metastatic site biopsies or from sites of unresectable loco-regional disease, NOTCH1 and PIK3CA mutations were reported in 14% and 20.3%, respectively. 4 of 48 (8%) and 22 of 735 vulvSCC (3.0%) featured genomic alterations (short variants and/or copy number changes and/or rearrangements) in both NOTCH1 and PIK3CA. NOTCH1 mutations were mostly located in the extracellular EGF-like domains, were inactivating and indicated that NOTCH1 functions predominantly as a tumor suppressor gene in vulvSCC. In contrast, PIK3CA mutations favored hotspot codons 1624 and 1633 of the gene, indicating that PIK3CA acts as an oncogene in vulvar carcinogenesis. In conclusion, NOTCH1 and PIK3CA mutations are detectable in a substantial proportion of vulvSCC and are related to HPV infection and more aggressive tumor behaviour.

OC 41.5 Two Positively Charged Patches in the EGF-1 Domain of Factor XII Synergistically Contribute to Surface Binding and Activation

OC 41.5 Two Positively Charged Patches in the EGF-1 Domain of Factor XII Synergistically Contribute to Surface Binding and Activation

Integrating plasma proteomes with genome-wide association data for causal protein identification in multiple myeloma

BackgroundMultiple myeloma (MM) is a severely debilitating and fatal B-cell neoplastic disease. The discovery of disease-associated proteins with causal genetic evidence offers a chance to uncover novel therapeutic targets.MethodsFirst, we comprehensively investigated the causal association between 2994 proteins and MM through two-sample mendelian randomization (MR) analysis using summary-level data from public genome-wide association studies of plasma proteome (N = 3301 healthy individuals) and MM (598 cases and 180,756 controls). Sensitivity analyses were performed for these identified causal proteins. Furthermore, we pursued the exploration of enriched biological pathways, prioritized the therapeutic proteins, and evaluated their druggability using the KEGG pathway analysis, MR-Bayesian model averaging analysis, and cross-reference with current databases, respectively.ResultsWe identified 13 proteins causally associated with MM risk (false discovery rate corrected P < 0.05). Six proteins were positively associated with the risk of MM, including nicotinamide phosphoribosyl transferase (NAMPT; OR [95% CI]: 1.35 [1.18, 1.55]), tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1; 1.14 [1.06, 1.22]), neutrophil cytosol factor 2 (NCF2; 1.27 [1.12, 1.44]), carbonyl reductase 1, cAMP-specific 3',5'-cyclic phosphodiesterase 4D (PDE4D), platelet-activating factor acetylhydrolase IB subunit beta (PAFAH1B2). Seven proteins were inversely associated with MM, which referred to suppressor of cytokine signaling 3 (SOCS3; 0.90 [0.86, 0.94]), Fc-gamma receptor III-B (FCGR3B; 0.75 [0.65,0.86]), glypican-1 (GPC1; 0.69 [0.58,0.83]), follistatin-related protein 1, protein tyrosine phosphatase non-receptor type 4 (PTPN4), granzyme B, complement C1q subcomponent subunit C (C1QC). Three of the causal proteins, SOCS3, FCGR3B, and NCF2, were enriched in the osteoclast differentiation pathway in KEGG enrichment analyses while GPC1 (marginal inclusion probability (MIP):0.993; model averaged causal effects (MACE): − 0.349), NAMPT (MIP:0.433; MACE: − 0.113), and NCF2 (MIP:0.324; MACE:0.066) ranked among the top three MM-associated proteins according to MR-BMA analyses. Furthermore, therapeutics targeting four proteins are currently under evaluation, five are druggable and four are future breakthrough points.ConclusionsOur analysis revealed a set of 13 novel proteins, including six risk and seven protective proteins, causally linked to MM risk. The discovery of these MM-associated proteins opens up the possibility for identifying novel therapeutic targets, further advancing the integration of genome and proteome data for drug development.

Membranous Expression of Heart Development Protein with EGF-like Domain 1 Is Associated with a Good Prognosis in Patients with Bladder Cancer.

To investigate the correlation between total protein expression of heart development protein with EGF-like domain 1 (HEG1) and clinicopathological characteristics in patients with bladder cancer (BC) after radical cystectomy (RC). We retrospectively analyzed data from 110 patients who underwent RC at Kitasato University Hospital. And we prepared an anti-HEG1 monoclonal antibody W10B9, which can detect total HEG1 protein. HEG1 protein expression in tumor cells was evaluated separately for membrane and cytoplasmic staining using immunohistochemistry. Membranous HEG1 expression was associated with absent lymphovascular invasion (p < 0.01) and low pT stage (p < 0.01). Kaplan-Meier analysis revealed that the membranous HEG1-positive group had significantly long recurrence-free survival (RFS) (p < 0.01) and cancer-specific survival (p = 0.01). Expression of membranous HEG1 was identified as an independent prognostic factor for RFS (p = 0.04). There were no significant differences between cytoplasmic HEG1 expression and clinicopathologic factors including prognosis. The expression of membranous HEG1 could serve as a favorable prognostic indicator in patients with BC treated with RC.

Synergistic regulation of Notch signaling by different O-glycans promotes hematopoiesis.

Glycosylation of Notch receptors by O-fucose glycans regulates Notch ligand binding and Notch signaling during hematopoiesis. However, roles in hematopoiesis for other O-glycans that modify Notch receptors have not been determined. Here we show that the EGF domain specific GlcNAc transferase EOGT is required in mice for the optimal production of lymphoid and myeloid cells. The phenotype of Eogt null mice was largely cell-autonomous, and Notch target gene expression was reduced in T cell progenitors. Moreover, EOGT supported residual Notch signaling following conditional deletion of Pofut1 in hematopoietic stem cells (HSC). Eogt : Pofut1 double mutant HSC had more severe defects in bone marrow and in T and B cell development in thymus and spleen, compared to deletion of Pofut1 alone. The combined results show that EOGT and O-GlcNAc glycans are required for optimal hematopoiesis and T and B cell development, and that they act synergistically with POFUT1 and O-fucose glycans to promote Notch signaling in lymphoid and myeloid differentiation.

Red sea bream iridovirus infection downregulates inflammation-related genes in the spleen of rock bream (Oplegnathus fasciatus).

This study investigated the kinetics of red sea bream iridovirus and host gene expression during infection in rock bream (Oplegnathus fasciatus), a species highly sensitive to the virus. After intraperitoneal injection of the viral solution at 104 TCID50/fish, the viral genome copy number in the spleen was 104.7 ± 0.2 and 105.9 ± 0.4 copies/μg DNA at 3 and 5 days post-injection (dpi), respectively. Using transcriptomic analyses via MiSeq, viral gene transcripts were detected at 3 and 5 dpi. Six genes including RING-finger domain-containing protein and laminin-type epidermal growth factor-like domain genes were significantly expressed at 5 dpi. Further, 334 host genes were differentially expressed compared with those before infection. Genes were clustered into four groups based on their expression profiles. Interferon-stimulated genes were more prevalent in groups showing upregulation at 5 dpi and 3 and 5 dpi. In contrast, the group showing downregulation at 3 dpi included inflammation-related genes, such as granzyme and eosinophil peroxidase genes. Downregulation of certain inflammation-related genes may contribute to the susceptibility of this fish to the virus.

Angiopoietin4 (ANGPT4) expression and potential mechanisms in carcinogenesis: current achievements and perspectives.

Angiopoietin4(ANGPT4) which plays a significant role in endothelial cell proliferation, survival, angiogenesis and expansion in tumors and other pathological states is a significant regulator of tumor angiogenesis. ANGPT4 expression is enhanced in many cancer cells. For example, the overexpression of ANGPT4 promotes the formation, development and progress of lung adenocarcinoma, glioblastoma and ovarian cancer. Related studies show that ANGPT4 encourages the proliferation, survival and invasion of tumor cells, while promoting the expansion of the tumor vascular system and affecting the tumor immune microenvironment. ANGPT4 can also promote carcinogenesis by affecting the ERK1/2, PI3K/AKT and other signal pathways downstream of tyrosine kinase with immunoglobulin-like and EGF-like domains 2(TIE2) and TIE2. Therefore, ANGPT4 may be a potential and significant biomarker for predicting malignant tumor progression and adverse outcomes. In addition, inhibition of ANGPT4 may be a meaningful cancer treatment. This paper reviews the latest research results of ANGPT4 in preclinical research, and emphasizes its role in carcinogenesis. Additional research on the carcinogenic function of ANGPT4 could provide new insights into cancer biology and novel methods for cancer diagnosis and treatment.

Organization of Purkinje cell development by neuronal MEGF11 in cerebellar granule cells

As cerebellar granule cells (GCs) coordinate the formation of regular cerebellar networks during postnatal development, molecules in GCs are expected to be involved. Here, we test the effects of the knockdown (KD) of multiple epidermal growth factor-like domains protein 11 (MEGF11), which is a homolog of proteins mediating astrocytic phagocytosis but is substantially increased at the later developmental stages of GCs on cerebellar development. MEGF11-KD in GCs of developing mice results in abnormal cerebellar structures, including extensively ectopic Purkinje cell (PC) somas, and in impaired motor functions. MEGF11-KD also causes abnormally asynchronous synaptic release from GC axons, parallel fibers, before the appearance of abnormal cerebellar structures. Interestingly, blockade of this abnormal synaptic release restores most of the cerebellar structures. Thus, apart from phagocytic functions of its related homologs in astrocytes, MEGF11 in GCs promotes proper PC development and cerebellar network formation by regulating immature synaptic transmission.

The promoting effect of modified Dioscorea pills on vascular remodeling in chronic cerebral hypoperfusion via the Ang/Tie signaling pathway.

The objective of this study was to investigate the effect of modified Dioscorea pills (MDP) on microcirculatory remodeling in the hippocampus of rats with chronic cerebral hypoperfusion (CCH) through the angiopoietin (Ang)/tyrosine kinase receptor tyrosine kinase with immunoglobulin-like and EGF-like domains (Ang receptor) 2 (Tie-2) signaling pathways, which may underlie the cognitive improvement observed in CCH rats. Forty male Sprague-Dawley rats raised under specific pathogen-free conditions were randomly divided into three groups: control group (10 rats), model group (15 rats), and MDP group (15 rats). The rats in the model group and MDP group underwent bilateral common carotid artery occlusion using the 2-vessel occlusion (2-VO) method to induce CCH. Rats in the control group underwent the same surgical procedures as those in the model group, except for ligation and occlusion of the carotid arteries. After 1 week of 2-VO, rats in the MDP group were administered MDP condensed decoction intragastrically at a dose of 1 ml/100 g body weight (prepared by the Preparation Room of Hubei Provincial Hospital of Traditional Chinese Medicine) for 45 days, while rats in the other two groups received normal saline intragastrically with the same dose and duration as the MDP group. After the intervention, all rats were euthanized, and brain perfusion was performed to obtain the hippocampal tissue for analysis. Immunohistochemical staining for CD43 was performed to assess microvessel density (MVD); western blot and the reverse transcription-polymerase chain reaction (RT-PCR) were used to analyze the expression of proteins and genes in angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), Tie-2, and vascular endothelial growth factor (VEGF) proteins and genes in the hippocampal tissue and compute the Ang-1/Ang-2 ratio. MDP treatment reduced neuronal loss and promoted restoration of the damaged hippocampal structure in CCH rats. The model group showed significantly higher MVD (14.93 ± 1.92) compared to the control group (5.78 ± 1.65) (P < 0.01), whereas MDP treatment further increased MVD (21.19 ± 2.62). Western blot and RT-PCR analysis revealed that CCH significantly increased the expression of Ang-1, Ang-2, Tie-2, and VEGF proteins and genes, while MDP treatment further significantly upregulated the expression of these proteins and genes. In addition, MDP significantly elevated the gene and protein expression of the Ang-1/Ang-2 ratio compared to the control group (P = 0.041, P = 0.029). CCH induces microvascular neogenesis in the hippocampus, and MDP promotes angiogenesis and microcirculation remodeling in CCH rats via the Ang/Tie signaling pathway, which may be an important mechanism for its restorative effects on hippocampal perfusion and improvement of cognitive function in CCH rats.

MiR-33a-3p regulates METTL3-mediated AREG stability and alters EMT to inhibit pancreatic cancer invasion and metastasis

Recent studies have shown that amphoteric regulatory protein (AREG), a member of the epidermal growth factor (EGF) family, is expressed in many cancers and is an independent prognostic indicator for patients with pancreatic cancer, but whether AREG is regulated at the epigenetic level to promote the development of pancreatic cancer (PC) has not been elucidated. Our results support the notion that AREG is overexpressed in pancreatic cancer tissues and cell lines. Functionally, the deletion of AREG impedes pancreatic cancer (PC) cell proliferation, migration, and invasion. In addition, we identified and validated that methyltransferase-like 3 (METTL3) induced the m6A modification on AREG and facilitated the stability of AREG mRNA after sequencing. Additionally, we obtained experimental evidence that miR-33a-3p targets and inhibits METTL3 from taking action, as predicted by using the miRDB and RNAinter. Remediation experiments showed that miR-33a-3p inhibits PC progression through METTL3. In summary, this research reveals that miR-33a-3p inhibits m6A-induced stabilization of AREG by targeting METTL3, which plays a key role in the aggressive progression of PC. AREG could be a potential target for PC treatment.

Genome-wide association study on abdomen depth, head width, hip width, and withers height in native cattle of Guilan (Bos indicus).

Native breeds in any country are a national capital, and their preservation is of great importance. Native Cattle of Guilan (NCG) is one of the few pure native breeds in Iran and the West Asia region. During the last decade, NCG population has decreased by more than 40%. This study aimed to identify significant single nucleotide polymorphisms (SNPs) and candidate genes associated with meat production traits in NCG using a genome-wide association study (GWAS). The blood and hair samples were collected from 72 NCG individuals and genotyped using the Illumina Bovine SNP50 chip. The results of the genomic scan showed that several SNPs were associated with abdominal depth, head width, hip width, and withers height in NCG. Several candidate genes were identified, including multiple epidermal growth factor-like domains 11 (MEGF11), Methionine Sulfoxide Reductase A (MSRA), chondroitin sulfate synthase 3 (CHSY3), Cyclin-Dependent Kinase 7 (CDK7), and Parkin (PRKN) genes, which are involved in muscle growth, meat tenderness, differentiation of fat cells, fat metabolism, and adipogenesis. These genes can contribute to meat quantity and quality in NCG. This study provided valuable insights into the genetics of NCG and the identification of effective genes associated with meat production traits. The results of this study could be used for the preservation and sustainable use of this breed of native cattle, as an important genetic resource in Iran.

Abstract P1083: Retinoid Regulation Of Cardiac Inflammation

Survivors of acute cardiac events (e.g., myocardial infarction, MI) often recover poorly due to pathological remodeling and progressive heart failure. Cell-based therapies hold potential to improve recovery but have had limited success. Our lab has found that infusion of highly proliferative cardiac cell clones from patient biopsies can improve recovery from MI in mice, but with variable efficacy stemming from unknown factors. Because Vitamin A (VA) stores are known to be mobilized to the heart in response to MI, we investigated the effect of VA on patient-derived cardiac cells. Clones were cultured and treated with 500 nanomolar retinoic acid (RA, bioactive metabolite of VA) for 48 hours. Protein was extracted and digested for proteomic analysis using liquid chromatography and mass spectrometry. To investigate interaction of RA with pro-regenerative epidermal growth factor (EGF) family receptor signaling, cells were treated with 50ng/ml recombinant neuregulin (NRG1) alone or in co-treatment with RA. Treatment with RA caused significant increase in protein expression of inflammatory mediators including IL1A/B, ICAM1, prostaglandin synthase, and NFkB2, and less studied targets including differentiation factor latexin (LXN) and orphan G-protein coupled receptor GPRC5A. Expression was validated at the transcript level. NRG1 treatment significantly modulated expression of metabolic regulators carnitine-palmitoyl transferase, phosphofructokinase, and dipeptidyl peptidase but was inhibited by co-treatment with RA. Our data suggest that RA may induce a pro-inflammatory phenotype in proliferative cardiac cells and that RA inhibits pro-regenerative NRG1/EGF receptor signaling, perhaps as part of an emergency inflammatory response to injury. While the biology underlying limited successes of cell therapies to treat heart disease is incompletely understood, managing inflammation is one strategy that has shown benefits in treatment of heart failure and reprogramming cells to therapeutic ends. Further experiments are called for to investigate an inflammatory role of VA during MI in vivo, and to determine how the interaction of VA and NRG1 may regulate proliferative cardiac cell fate and function after injury.

The role of disulfide bonds in the formation of the spatial structure of the human epidermal growth factor

The epidermal growth factor (EGF) is a peptide of the EGF-like growth factor family with a common conserved EGF-like domain formed by three intramolecular disulfide bonds. This article describes changes in the spatial structure of EGF and its mutant form with the D46G substitution in its C-terminal fragment observed upon disulfide bonds reduction in the corresponding synthetic peptides in a 0.01 M phosphate buffer (pH = 7.4). The structure was analyzed using circular dichroism spectroscopy, spectrofluorimetry, native polyacrylamide gel electrophoresis, and centrifugal ultrafiltration. It was shown that disulfide bonds reduction changes the geometry of the EGF-like domain towards an increase in the content of the beta-structure, while these peptides remain in dimeric form. According to the molecular modeling results, this can lead to the elongation of the main beta-hairpin of the EGF-like domain, to the elongation of the intermolecular beta-structure, or to the formation of a new beta-structure between the N- and C-terminal fragments of each molecule, which will change the intermolecular interface in dimeric form. Disulfide bonds reduction prevents EGF dimer dissociation to monomers. Under physiological conditions, this can lead to the inability of EGF to form binding sites for EGFR (epidermal growth factor receptor) and to cause its activation.

Hepatocellular carcinoma cells remodel the pro-metastatic tumour microenvironment through recruitment and activation of fibroblasts via paracrine Egfl7 signaling

BackgroundThe tumour microenvironment consists of a complex and dynamic milieu of cancer cells, including tumour-associated stromal cells (leukocytes, fibroblasts, vascular cells, etc.) and their extracellular products. During invasion and metastasis, cancer cells actively remodel the tumour microenvironment and alterations of microenvironment, particularly cancer-associated fibroblasts (CAFs), can promote tumour progression. However, the underlying mechanisms of the CAF formation and their metastasis-promoting functions remain unclear.MethodsPrimary liver fibroblasts and CAFs were isolated and characterized. CAFs in clinical samples were identified by immunohistochemical staining and the clinical significance of CAFs was also analysed in two independent cohorts. A transwell coculture system was used to confirm the role of HCC cells in CAF recruitment and activation. qRT-PCR, western blotting and ELISA were used to screen paracrine cytokines. The role and mechanism of Egfl7 in CAFs were explored via an in vitro coculture system and an in vivo mouse orthotopic transplantation model.ResultsWe showed that CAFs in hepatocellular carcinoma (HCC) are characterized by the expression of α-SMA and that HCC cells can recruit liver fibroblasts (LFs) and activate them to promote their transformation into CAFs. High α-SMA expression, indicating high CAF infiltration, was correlated with malignant characteristics. It was also an independent risk factor for HCC survival and could predict a poor prognosis in HCC patients. Then, we demonstrated that EGF-like domain multiple 7 (Egfl7) was preferentially secreted by HCC cells, and exhibited high potential to recruit and activate LFs into the CAF phenotype. The ability of Egfl7 to modulate LFs relies upon increased phosphorylation of FAK and AKT via the receptor ανβ3 integrin. Strikingly, CAFs activated by paracrine Egfl7 could further remodel the tumour microenvironment by depositing fibrils and collagen and in turn facilitate HCC cell proliferation, invasion and metastasis.ConclusionOur data highlighted a novel role of Egfl7 in remodelling the tumour microenvironment: it recruits LFs and activates them to promote their transformation into CAFs via the ανβ3 integrin signaling pathway, which further promotes HCC progression and contributes to poor clinical outcomes in HCC patients.3o7rdHzTehVwN25qa8k-ATVideo

A potent anticoagulant hybrid of snake venom derived FIX-binding protein and anti-factor IX RNA aptamer: Assessed by in-silico and electrochemical analyses

Anticoagulant therapies are crucial in the management of surgical complications as well as the prophylaxis of thrombosis. Many studies are being conducted on the Habu snake-venom anticoagulant, FIX-binding protein (FIX-Bp), for its greater potency and strong affinity to FIX clotting factor. On the other hand, the capacity to promptly reverse such acute anticoagulation is equally important. Combining a reversible anticoagulant with FIX-Bp may be advantageous in maintaining the balance between adequate anticoagulation and repealing when necessary. In this study, authors integrated FIX-Bp and RNA aptamer-based anticoagulants into a single target, FIX clotting factor, in order to achieve a robust anticoagulant effect. An in-silico and electrochemical approach were used to investigate the combination of FIX-Bp and RNA aptamers as a bivalent anticoagulant and to verify the competing or predominant binding sites of each anticoagulant. The in-silico analysis discovered that both the venom- and aptamer-anticoagulant had a strong affinity for the FIX protein at the Gla-domain and EGF-1 domain by holding 9 conventional hydrogen bonds with the binding energy of −34.859 kcal/mol. The electrochemical technique verified that both anticoagulants had different binding sites. The impedance load upon RNA aptamer binding to FIX protein was 14 %, whereas the addition of FIX-Bp caused a significant impedance rise of 37 %. This indicates that the addition of aptamers prior to FIX-Bp is a promising strategy for the conception of a hybrid anticoagulant.

A MASP-like functions as PRR to regulate the mRNA expressions of inflammatory factors in the Pacific oyster Crassostrea gigas.

Mannose-binding lectin-associated serine protease (MASP) is a type of central serine protease in the complement lectin pathway. In the present study, a MASP-like was identified from the Pacific oyster Crassostrea gigas, defined as CgMASPL-2. The cDNA sequence of CgMASPL-2 was of 3399 bp with an open reading frame of 2757 bp and encoded a polypeptide of 918 amino acids containing three CUB domains, an EGF domain, two IG domains, and a Tryp_SPC domain. In the phylogenetic tree, CgMASPL-2 was firstly clustered with Mytilus californianus McMASP-2-like, and then assigned into the invertebrate branch. CgMASPL-2 shared similar domains with M. californianus McMASP-2-like and Littorina littorea LlMReM1. CgMASPL-2 mRNA was expressed in all the tested tissues with the highest expression in haemolymph. CgMASPL-2 protein was mainly distributed in the cytoplasm of haemocytes. The mRNA expression of CgMASPL-2 increased significantly in haemocytes after Vibrio splendidus stimulation. The recombinant 3 × CUB-EGF domains of CgMASPL-2 displayed binding activities to diverse polysaccharides (lipopolysaccharide, peptidoglycan and mannose) and microbes (Staphylococcus aureus, Micrococcus luteus, Pichia pastoris, Vibrio anguillarum, V. splendidus and Escherichia coli). In anti-CgMASPL-2 treated oysters, the mRNA expressions of CgIL17-1 and CgIL17-2 in haemocytes decreased significantly after V. splendidus stimulation. The results indicated that CgMASPL-2 could directly sense microbes and regulate the mRNA expressions of inflammatory factors.

EGFL6 promotes bone metastasis of lung adenocarcinoma by increasing cancer cell malignancy and bone resorption.

Lung adenocarcinoma is the most common and aggressive type of lung cancer with the highest incidence of bone metastasis. Epidermal growth factor-like domain multiple 6 (EGFL6) is an exocrine protein, and the expression of EGFL6 is correlated with survival of patient with lung adenocarcinoma. However, the association between EGFL6 expression in lung adenocarcinoma and bone metastasis has not been investigated. In this study, we found that EGFL6 levels in lung adenocarcinoma tissues correlate with bone metastasis and TNM stages in surgical patients. In vitro, overexpression of EGFL6 in lung adenocarcinoma cells promoted their proliferation, migration, and invasion ability compared with control by enhancing EMT process and activating Wnt/β-catenin and PI3K/AKT/mTOR pathways. In the nude mouse model, overexpression of EGFL6 enhanced tumor growth and caused greater bone destruction. Moreover, the exocrine EGFL6 of human lung adenocarcinoma cells increased osteoclast differentiation of bone marrow mononuclear macrophages (BMMs) of mice via the NF-κB and c-Fos/NFATc1 signaling pathways. However, exocrine EGFL6 had no effect on osteoblast differentiation of bone marrow mesenchymal stem cells (BMSCs). In conclusion, high expression of EGFL6 in lung adenocarcinomas is associated with bone metastasis in surgical patients. The underlying mechanism may be the increased metastatic properties of lung adenocarcinoma cells with high EGFL6 level and the enhanced osteoclast differentiation and bone resorption by exocrine EGFL6 from tumors. Therefore, EGFL6 is a potential therapeutic target to reduce the ability of lung adenocarcinomas to grow and metastasize and to preserve bone mass in patients with bone metastases from lung adenocarcinomas.

Semaphorin 4D Induces Vasculogenic Differentiation of Dental Pulp Stem Cells.

This work aimed to evaluate the effect of Semaphorin 4D (SEMA4D) signaling through Plexin B1 on the vasculogenic differentiation of dental pulp stem cells. We assessed the protein expression of SEMA4D and Plexin B1 in dental pulp stem cells (DPSC) from permanent human teeth and stem cells from human exfoliated deciduous (SHED) teeth using Western blots. Their expression in human dental pulp tissues and DPSC-engineered dental pulps was determined using immunofluorescence. We then exposed dental pulp stem cells to recombinant human SEMA4D (rhSEMA4D), evaluated the expression of endothelial cell differentiation markers, and assessed the vasculogenic potential of rhSEMA4D using an in vitro sprouting assay. Lastly, Plexin B1 was silenced to ascertain its role in SEMA4D-mediated vasculogenic differentiation. We found that SEMA4D and Plexin B1 are expressed in DPSC, SHED, and human dental pulp tissues. rhSEMA4D (25-100 ng/mL) induced the expression of endothelial markers, i.e., vascular endothelial growth factor receptor (VEGFR)-2, cluster of differentiation (CD)-31, and tyrosine kinase with immunoglobulin-like and EGF-like domains (Tie)-2, in dental pulp stem cells and promoted capillary-like sprouting in vitro (p < 0.05). Furthermore, Plexin B1 silencing abrogated the vasculogenic differentiation of dental pulp stem cells and significantly inhibited capillary sprouting upon exposure to rhSEMA4D. Collectively, these data provide evidence that SEMA4D induces vasculogenic differentiation of dental pulp stem cells through Plexin B1 signaling.