Fibronectin Binding Research Articles

Anti–Integrin αvβ6 Antibody as a Diagnostic Marker for Pediatric Patients With Ulcerative Colitis

Anti–Integrin αvβ6 Antibody as a Diagnostic Marker for Pediatric Patients With Ulcerative Colitis

Streptococcus pneumoniae binds collagens and C1q via the SSURE repeats of the PfbB adhesin.

The binding of Streptococcus pneumoniae to collagen is likely an important step in the pathogenesis of pneumococcal infections, but little is known of the underlying molecular mechanisms. Streptococcal surface repeats (SSURE) are highly conserved protein domains present in cell wall adhesins from different Streptococcus species. We find here that SSURE repeats of the pneumococcal adhesin plasminogen and fibronectin binding protein B (PfbB) bind to various types of collagen. Moreover, deletion of the pfbB gene resulted in a significant impairment of the ability of encapsulated or unencapsulated pneumococci to bind collagen. Notably, a PfbB SSURE domain is also bound to the complement component C1q that bears a collagen‐like domain and promotes adherence of pneumococci to host cells by acting as a bridge between bacteria and epithelial cells. Accordingly, deletion of PfbB or pre‐treatment with anti‐SSURE antibodies markedly decreased pneumococcal binding to C1q as well as C1q‐dependent adherence to epithelial and endothelial cells. Further data indicated that C1q promotes pneumococcal adherence by binding to integrin α2β1. In conclusion, our results indicate that the SSURE domains of the PfbB protein promote interactions of pneumococci with various types of collagen and with C1q. These repeats may be useful targets in strategies to control S. pneumoniae infections.

Fibronectin binding protein B binds to loricrin and promotes corneocyte adhesion by Staphylococcus aureus.

Colonisation of humans by Staphylococcus aureus is a major risk factor for infection, yet the bacterial and host factors involved are not fully understood. The first step during skin colonisation is adhesion of the bacteria to corneocytes in the stratum corneum where the cornified envelope protein loricrin is the main ligand for S. aureus. Here we report a novel loricrin-binding protein of S. aureus, the cell wall-anchored fibronectin binding protein B (FnBPB). Single-molecule force spectroscopy revealed both weak and ultra-strong (2 nN) binding of FnBPB to loricrin and that mechanical stress enhanced the strength of these bonds. Treatment with a peptide derived from fibrinogen decreased the frequency of strong interactions, suggesting that both ligands bind to overlapping sites within FnBPB. Finally, we show that FnBPB promotes adhesion to human corneocytes by binding strongly to loricrin, highlighting the relevance of this interaction to skin colonisation.

Oral Vaccination of Mice With Trichinella spiralis Putative Serine Protease and Murine Interleukin-4 DNA Delivered by Invasive Lactiplantibacillus plantarum Elicits Protective Immunity.

Trichinellosis is a serious zoonotic parasitic disease caused by Trichinella spiralis (T. spiralis) that causes considerable economic losses for the global pig breeding and food industries. As such, there is an urgent need for a vaccine that can prevent T. spiralis infection. Previous studies have reported that recombinant invasive Lactococcus lactis (LL) expressing Staphylococcus aureus fibronectin binding protein A (LL-FnBPA+) can transfer DNA vaccines directly to dendritic cells (DCs) across an epithelial cell monolayer, leading to significantly higher amounts of heterologous protein expression compared to non-invasive Lactococcus lactis. In this study, the invasive bacterium Lactiplantibacillus plantarum (L. plantarum) expressing FnBPA was used as a carrier to deliver a novel oral DNA vaccine consisting of T. spiralis adult putative serine protease (Ts-ADpsp) and murine interleukin (IL)-4 DNA to mouse intestinal epithelial cells. Experimental mice were orally immunized 3 times at 10-day intervals. At 10 days after the last vaccination, mice were challenged with 350 T. spiralis infective larvae by oral inoculation. Immunization with invasive L. plantarum harboring pValac-Ts-ADpsp/pSIP409-FnBPA induced the production of anti-Ts-ADpsp-specific IgG of serum, type 1 and 2 helper T cell cytokines of mesenteric lymph node (MLN) and spleen, secreted (s) IgA of intestinal lavage, and decreased T. spiralis burden and intestinal damage compared to immunization with non-invasive L. plantarum expressing Ts-ADpsp (pValac-Ts-ADpsp/pSIP409). Thus, invasive L. plantarum expressing FnBPA and IL-4 stimulates both mucosal and cellular immune response to protect against T. spiralis infection, highlighting its therapeutic potential as an effective DNA vaccine for trichinellosis.

Hope toward potent drug target using tertiary topological instances of Mycobacterial transmembrane protein

Integral membrane proteins are important for various cellular processes. They are the major part involved in the transportation of different proteins across the membrane and thus might also be helpful in maintaining the charge potential. Rv0882 was predicted to be an integral membrane protein by various bioinformatics analyses. TBpred servers characterize the presence of this protein in the transmembrane region. SOSUI, TMHMM and HMMTOP servers found various transmembrane regions of this protein at various positions. TOPCONS web server provides the presence of an alpha-helical region in this protein. Secondary structure prediction and Three-dimensional (3D) modeling of this protein were done by using PSIPRED and QUARK respectively. Prediction of ligand binding site shows that various residues of this protein bind to different compounds whose results are confirmed by molecular docking of these compounds with Rv0882 protein. This protein also comprises a Fibronectin binding motif (RWFV) which also suggests that this protein might also act as a Fibronectin binding protein (FnBp). Previous studies proved that integral membrane proteins are important for transportation and FnBp are involved in host-pathogen interaction. Thus, studying this gene might be beneficial for stretching information about an untouched site of this bacterium which might be helpful in declining this infection.

Recent Insights into Human Endometrial Peptidases in Blastocyst Implantation via Shedding of Microvesicles.

Blastocyst implantation involves multiple interactions with numerous molecules expressed in endometrial epithelial cells (EECs) during the implantation window; however, there is limited information regarding the molecular mechanism underlying the crosstalk. In blastocysts, fibronectin plays a major role in the adhesion of various types of cells by binding to extracellular matrix proteins via the Arg-Gly-Asp (RGD) motif. In EECs, RGD-recognizing integrins are important bridging receptors for fibronectin, whereas the non-RGD binding of fibronectin includes interactions with dipeptidyl peptidase IV (DPPIV)/cluster of differentiation (CD) 26. Fibronectin may also bind to aminopeptidase N (APN)/CD13, and in the endometrium, these peptidases are present in plasma membranes and lysosomal membranes. Blastocyst implantation is accompanied by lysosome exocytosis, which transports various peptidases and nutrients into the endometrial cavity to facilitate blastocyst implantation. Both DPPIV and APN are released into the uterine cavity via shedding of microvesicles (MVs) from EECs. Recently, extracellular vesicles derived from endometrial cells have been proposed to act on trophectoderm cells to promote implantation. MVs are also secreted from embryonal stem cells and may play an active role in implantation. Thus, crosstalk between the blastocyst and endometrium via extracellular vesicles is a new insight into the fundamental molecular basis of blastocyst implantation.

An Extracellular Matrix-Liposome Composite, a Novel Extracellular Matrix Delivery System for Accelerated Tissue Regeneration.

The unfolded states of fibronectin (FN) subsequently induce the formation of an extracellular matrix (ECM) fibrillar network, which is necessary to generate new substitutive tissues. Here, the authors demonstrate that negatively charged small unilamellar vesicles (SUVs) qualify as candidates for FN delivery due to their remarkable effects on the autonomous binding and unfolding of FN, which leads to increased tissue regeneration. In vitro experiments revealed that the FN-SUV complex remarkably increased the attachment, differentiation, and migration of fibroblasts. The potential utilization of this complex in vivo to treat inflammatory colon diseases is also described based on results obtained for ameliorated conditions in rats with ulcerative colitis (UC) that had been treated with the FN-SUV complex. Their findings provide a new ECM-delivery platform for ECM-based therapeutic applications and suggest that properly designed SUVs may be an unprecedented FN-delivery system that is highly effective in treating UC and inflammatory bowel diseases.

Abstract 10717: Role of Integrin Signaling in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is characterized by progressive obstruction and decreased compliance of pulmonary arteries (PA), leading to right ventricular (RV) failure and premature death. It is now recognized that, like cancer cells, PA smooth muscle cells (PASMCs) exhibit exaggerated proliferation and resistance to apoptosis in response to increased PA stiffness caused by extracellular matrix (ECM) remodeling. Integrins (members of the cell adhesion receptors superfamily) are known to promote cell proliferation, survival, hypertrophic growth and fibrosis, which are key elements in the progression of PAH. Therefore, we explored integrins expression and its effects on PA remodeling and RV failure. By RNA sequencing, we demonstrated a significant upregulation of integrin signaling pathways. Among all integrins, the fibronectin binding integrin (FiBI) family was the most abundant in PASMCs and RV fibroblasts (RVFbs) isolated from PAH patients (n=5). By western blot (WB) we showed that integrin expression is significantly changed in distal PAs, PASMCs and RV from PAH patients (n=6-8) compared to controls (n=5-8). Similarly, increased expression was found in monocrotaline (MCT) and pulmonary artery banding (PAB) rats (n=20, p<0.05.) In vitro , pharmacological inhibition of FiBI decreases PAH-PASMCs proliferation (WB: PCNA and Ki67 labeling; p<0.05) and resistance to apoptosis (WB: Survivin and Annexin V labeling; p<0.05). These effects were associated with a decreased activation of the AKT/mTOR pathway. In cardiomyocytes and human RVFbs, FiBI inhibition decreased phenylephrine-induced hypertrophy (Phalloidin labeling; p<0.001) and TGFβ1-induced RVFbs activation (WB: αSMA, CTGF, FN and Col1; p<0.05) and proliferation (Ki67; p<0.05). In vivo , pharmacological FiBI inhibition improved PAs remodeling (EVG staining) and RV function (SV, CO, TAPSE, S-Wave, as assessed by echocardiography and right heart catheterization) in MCT rats with established PAH (n=20; p<0.05). We demonstrated that upregulation of fibronectin binding integrins and broad spectrum integrin inhibition resulted in improvements in distal PAs remodeling and RV failure. These results suggest that integrins play an important role in the pathophysiology of PAH.

Candesartan prevents arteriopathy progression in cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy model.

Cerebral small vessel disease (CSVD) causes dementia and gait disturbance due to arteriopathy. Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a hereditary form of CSVD caused by loss of high-temperature requirement A1 (HTRA1) serine protease activity. In CARASIL, arteriopathy causes intimal thickening, smooth muscle cell (SMC) degeneration, elastic lamina splitting, and vasodilation. The molecular mechanisms were proposed to involve the accumulation of matrisome proteins as substrates or abnormalities in transforming growth factor β (TGF-β) signaling. Here, we show that HTRA1-/- mice exhibited features of CARASIL-associated arteriopathy: intimal thickening, abnormal elastic lamina, and vasodilation. In addition, the mice exhibited reduced distensibility of the cerebral arteries and blood flow in the cerebral cortex. In the thickened intima, matrisome proteins, including the hub protein fibronectin (FN) and latent TGF-β binding protein 4 (LTBP-4), which are substrates of HTRA1, accumulated. Candesartan treatment alleviated matrisome protein accumulation and normalized the vascular distensibility and cerebral blood flow. Furthermore, candesartan reduced the mRNA expression of Fn1, Ltbp-4, and Adamtsl2, which are involved in forming the extracellular matrix network. Our results indicate that these accumulated matrisome proteins may be potential therapeutic targets for arteriopathy in CARASIL.

High-impact FN1 mutation decreases chondrogenic potential and affects cartilage deposition via decreased binding to collagen type II.

Osteoarthritis is the most prevalent joint disease worldwide, yet progress in development of effective disease-modifying treatments is slow because of lack of insight into the underlying disease pathways. Therefore, we aimed to identify the causal pathogenic mutation in an early-onset osteoarthritis family, followed by functional studies in human induced pluripotent stem cells (hiPSCs) in an in vitro organoid cartilage model. We demonstrated that the identified causal missense mutation in the gelatin-binding domain of the extracellular matrix protein fibronectin resulted in significant decreased binding capacity to collagen type II. Further analyses of formed hiPSC-derived neo-cartilage tissue highlighted that mutated fibronectin affected chondrogenic capacity and propensity to a procatabolic osteoarthritic state. Together, we demonstrate that binding of fibronectin to collagen type II is crucial for fibronectin downstream gene expression of chondrocytes. We advocate that effective treatment development should focus on restoring or maintaining proper binding between fibronectin and collagen type II.

P68.11 Correlation of CD26 With Pro-Fibrotic Mediators in Lung Tumors

CD26/dipeptidyl peptidase 4 (CD26) is a multifunctional enzyme containing collagen and fibronectin binding sites. CD26 is not only expressed on various malignancies, but also associated with the progression of organ fibrosis of the heart, liver, kidney, and lung. We previously showed a high expression of CD26 in lung adenocarcinoma and a significant association between CD26 expression and a worse survival of patients showing an active epithelial-mesenchymal transition status. Here, we extended our analysis on the correlation between CD26, fibrosis and inflammation mediators on patient lung tumors.

Adaptation of Staphylococcus aureus to the Human Skin Environment Identified Using an ex vivo Tissue Model.

The healthy human epidermis provides physical protection and is impenetrable for pathogenic microbes. Nevertheless, commensal and pathogen bacteria such as Staphylococcus aureus are able to colonize the skin surface, which may subsequently lead to infection. To identify and characterize regulatory elements facilitating adaptation of S. aureus to the human skin environment we used ex vivo tissue explants and quantified S. aureus gene transcription during co-culture. This analysis provided evidence for a significant downregulation of the global virulence regulator agr upon initial contact with skin, regardless of the growth phase of S. aureus prior to co-culture. In contrast, the alternative sigma factor B (sigB) and the antimicrobial peptide-sensing system (graRS) were expressed during early colonization. Consistently, sigB target genes such as the clumping factor A (clfA) and fibrinogen and fibronectin binding protein A (fnbA) were strongly upregulated upon skin contact. At later timepoints of the adhesion process, wall teichoic acid (WTA) synthesis was induced. Besides the expression of adhesive molecules, transcription of molecules involved in immune evasion were increased during late colonization (staphylococcal complement inhibitor and staphylokinase). Similar to nasal colonization, enzymes involved in cell wall metabolism (sceD and atlA) were highly transcribed. Finally, we detected a strong expression of proteases from all three catalytic classes during the entire colonization process. Taken together, we here present an ex vivo skin colonization model that allows the detailed characterization of the bacterial adaptation to the skin environment.

Probing tissue transglutaminase mediated vascular smooth muscle cell aging using a novel transamidation-deficient Tgm2-C277S mouse model

Tissue transglutaminase (TG2), a multifunctional protein of the transglutaminase family, has putative transamidation-independent functions in aging-associated vascular stiffening and dysfunction. Developing preclinical models will be critical to fully understand the physiologic relevance of TG2’s transamidation-independent activity and to identify the specific function of TG2 for therapeutic targeting. Therefore, in this study, we harnessed CRISPR-Cas9 gene editing technology to introduce a mutation at cysteine 277 in the active site of the mouse Tgm2 gene. Heterozygous and homozygous Tgm2-C277S mice were phenotypically normal and were born at the expected Mendelian frequency. TG2 protein was ubiquitously expressed in the Tgm2-C277S mice at levels similar to those of wild-type (WT) mice. In the Tgm2-C277S mice, TG2 transglutaminase function was successfully obliterated, but the transamidation-independent functions ascribed to GTP, fibronectin, and integrin binding were preserved. In vitro, a remodeling stimulus led to the significant loss of vascular compliance in WT mice, but not in the Tgm2-C277S or TG2−/− mice. Vascular stiffness increased with age in WT mice, as measured by pulse-wave velocity and tensile testing. Tgm2-C277S mice were protected from age-associated vascular stiffening, and TG2 knockout yielded further protection. Together, these studies show that TG2 contributes significantly to overall vascular modulus and vasoreactivity independent of its transamidation function, but that transamidation activity is a significant cause of vascular matrix stiffening during aging. Finally, the Tgm2-C277S mice can be used for in vivo studies to explore the transamidation-independent roles of TG2 in physiology and pathophysiology.

Pangenome analysis and virulence profiling of Streptococcus intermedius

BackgroundStreptococcus intermedius, a member of the S. anginosus group, is a commensal bacterium present in the normal microbiota of human mucosal surfaces of the oral, gastrointestinal, and urogenital tracts. However, it has been associated with various infections such as liver and brain abscesses, bacteremia, osteo-articular infections, and endocarditis. Since 2005, high throughput genome sequencing methods enabled understanding the genetic landscape and diversity of bacteria as well as their pathogenic role. Here, in order to determine whether specific virulence genes could be related to specific clinical manifestations, we compared the genomes from 27 S. intermedius strains isolated from patients with various types of infections, including 13 that were sequenced in our institute and 14 available in GenBank.ResultsWe estimated the theoretical pangenome size to be of 4,020 genes, including 1,355 core genes, 1,054 strain-specific genes and 1,611 accessory genes shared by 2 or more strains. The pangenome analysis demonstrated that the genomic diversity of S. intermedius represents an “open” pangenome model. We identified a core virulome of 70 genes and 78 unique virulence markers. The phylogenetic clusters based upon core-genome sequences and SNPs were independent from disease types and sample sources. However, using Principal Component analysis based on presence/ absence of virulence genes, we identified the sda histidine kinase, adhesion protein LAP and capsular polysaccharide biosynthesis protein cps4E as being associated to brain abscess or broncho-pulmonary infection. In contrast, liver and abdominal abscess were associated to presence of the fibronectin binding protein fbp54 and capsular polysaccharide biosynthesis protein cap8D and cpsB.ConclusionsBased on the virulence gene content of 27 S. intermedius strains causing various diseases, we identified putative disease-specific genetic profiles discriminating those causing brain abscess or broncho-pulmonary infection from those causing liver and abdominal abscess. These results provide an insight into S. intermedius pathogenesis and highlights putative targets in a diagnostic perspective.

Fibronectin binding to von Willebrand factor occurs via the A1 domain

BackgroundCollagen interactions with von Willebrand factor (VWF) perform an important role in initiation of hemostasis. ObjectivesWe hypothesized that in addition to collagen, other extracellular matrix (ECM) proteins such as fibronectin can bind VWF. MethodsFibronectin‐VWF interactions were measured by ELISA using both plasma‐derived and recombinant VWF–containing variants in specific domains. Inhibition was measured by antibody competition using antibodies directed against both VWF and fibronectin. Binding affinities were measured by the Octet Biosensor for fibronectin and collagen IV. ResultsFibronectin was able to bind both plasma‐derived and recombinant wild‐type VWF. This interaction was inhibited by both anti‐VWF antibodies and collagen types III and IV. Several VWF A1 domain variants in the region of the collagen IV binding site also demonstrated absent fibronectin binding, as did variants with defects in high‐molecular‐weight multimers. Binding affinity testing showed fibronectin has a strong affinity for VWF, in a range similar to that of collagen IV. Fibronectin binds VWF via a restricted region of the A1 domain. This interaction requires high‐molecular‐weight multimers and is similar to that seen with vascular collagens. ConclusionsTherefore, VWF would appear to be the common factor linking platelet adhesion to various ECM proteins and facilitating hemostasis under conditions of ECM exposure. [Display omitted]

Critical Extracellular Matrix Proteins Besides Collagen Bind Von Willebrand Factor

Introduction: Vascular endothelial injury exposes blood to many extracellular matrix proteins including collagen. Von Willebrand Factor (VWF) is a coagulation factor that binds platelets to collagen in the process of normal hemostasis. Collagen IV has been shown to bind VWF through the A1 domain. Many adhesive glycoproteins found in the extracellular matrix of vascular endothelium have been shown to induce hemostasis by binding coagulation factors. We hypothesized that in addition to collagen, other extracellular matrix proteins can bind VWF as well. We investigated VWF's interactions with four other extracellular matrix (ECM) proteins: laminin, fibronectin, thrombospondin, and vitronectin.Methods: Enzyme linked immunoassay (ELISA) was performed to measure the ability of laminin, fibronectin, thrombospondin, and vitronectin to bind to VWF. Each ECM protein was coated individually on a maleic anhydride plate. Detection of VWF:ECM complexes was performed using a combination of two biotinylated monoclonal anti-VWF antibodies. Inhibition of VWF:ECM interactions was tested using monoclonal and polyclonal antibodies against VWF and the individual ECM proteins. We also tested inhibition of VWF:ECM interactions by competing with collagen IV, laminin, fibronectin, thrombospondin, and vitronectin. ELISA was also used to test laminin, fibronectin, thrombospondin, and vitronectin binding to recombinant VWF (rVWF) constructs to determine a specific binding region and to determine the requirement for high molecular weight multimers with each ECM protein. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on all ECM proteins to investigate purity.Results: Results for the four ECM proteins generally paralleled each other and will be referred to together unless otherwise specified. SDS-PAGE, however, showed independent bands for each ECM protein preparation, confirming that these interactions were with the individual proteins and not with a single protein contaminating all preparations. ELISA showed binding of laminin, fibronectin, thrombospondin, and vitronectin to wild type (WT) rVWF and plasma from heathy individuals with normal VWF. No binding was detected for individuals with type 3 VWD. Polyclonal anti-VWF (DAKO) inhibited all VWF:ECM interactions (average 99% inhibition), indicating that binding is VWF specific. Monoclonal anti-VWF A1 domain (AVW-3) also inhibited all VWF:ECM interactions (average 98% inhibition), confirming that the A1 domain of VWF is crucial for this interaction. Several rVWF variants were used to test the importance of VWF functional domains. Compared to WT rVWF, VWF:ECM binding was reduced or undetectable with all ECM proteins for 1395A (undetectable) and 1399H (average 4%) but normal for 1786D, 1392A and 1402A, which were shown to abrogate VWF binding to collagen III and collagen IV, respectively. An 11 amino acid deletion from 1392 to 1402 also showed undetectable binding. Reduced binding with these constructs indicate the critical nature of the 1395 to 1399 amino acid region of the A1 domain to the VWF:ECM interactions. Compared to WT rVWF, binding was decreased or undetectable on all ECM proteins for 1597W (average 26%), 2773R (undetectable) and Y87S (undetectable) rVWF constructs. The 1597W, 2773R, and Y87S rVWF constructs contain defects in domains of VWF crucial to multimerization. Reduced binding with these constructs indicate that the VWF:ECM interactions are multimer dependent. All VWF:ECM interactions were significantly inhibited by collagen IV (90% inhibition for thrombospondin, complete inhibition for all other ECMs). The VWF:ECM interactions were also partially inhibited by thrombospondin (average 46% inhibition) and vitronectin (average 62% inhibition). Laminin and fibronectin did not noticeably inhibit any VWF:ECM interactions.Conclusions: In addition to collagen, VWF binds other ECM proteins found in vascular endothelium including laminin, fibronectin, thrombospondin, and vitronectin. These four ECM proteins appear to bind to VWF in a restricted region of the A1 domain as competitive binding suggests differences between collagen IV and other ECM proteins. The role and importance of VWF's interaction with these ECM proteins offer alternative binding partners that may represent a unique contribution to hemostasis. DisclosuresFlood:CSL Behring: Consultancy; Shire: Consultancy.

Pharmacological inhibition of PAI-1 alleviates cardiopulmonary pathologies induced by exposure to air pollutants PM2.5

Numerous studies have established that acute or chronic exposure to environmental pollutants like particulate matter (PM) leads to the development of accelerated aging related pathologies including pulmonary and cardiovascular diseases, and thus air pollution is one of the major global threats to human health. Air pollutant particulate matter 2.5 (PM2.5)-induced cellular dysfunction impairs tissue homeostasis and causes vascular and cardiopulmonary damage. To test a hypothesis that elevated plasminogen activator inhibitor-1 (PAI-1) levels play a pivotal role in air pollutant-induced cardiopulmonary pathologies, we examined the efficacy of a drug-like novel inhibitor of PAI-1, TM5614, in treating PM2.5-induced vascular and cardiopulmonary pathologies. Results from biochemical, histological, and immunohistochemical studies revealed that PM2.5 increases the circulating levels of PAI-1 and thrombin and that TM5614 treatment completely abrogates these effects in plasma. PM2.5 significantly augments the levels of pro-inflammatory cytokine interleukin-6 (IL-6) in bronchoalveolar lavage fluid (BALF), and this also can be reversed by TM5614, indicating its efficacy in amelioration of PM2.5-induced increases in inflammatory and pro-thrombotic factors. TM5614 reduces PM2.5-induced increased levels of inflammatory markers cluster of differentiation 107 b (Mac3) and phospho-signal transducer and activator of transcription-3 (pSTAT3), adhesion molecule vascular cell adhesion molecule 1 (VCAM1), and apoptotic marker cleaved caspase 3. Longer exposure to PM2.5 induces pulmonary and cardiac thrombosis, but TM5614 significantly ameliorates PM2.5-induced vascular thrombosis. TM5614 also reduces PM2.5-induced increased blood pressure and heart weight. In vitro cell culture studies revealed that PM2.5 induces the levels of PAI-1, type I collagen, fibronectin (Millipore), and sterol regulatory element binding protein-1 and 2 (SREBP-1 and SREBP-2), transcription factors that mediate profibrogenic signaling, in cardiac fibroblasts. TM5614 abrogated that stimulation, indicating that it may block PM2.5-induced PAI-1 and profibrogenic signaling through suppression of SREBP-1 and 2. Furthermore, TM5614 blocked PM2.5-mediated suppression of nuclear factor erythroid related factor 2 (Nrf2), a major antioxidant regulator, in cardiac fibroblasts. Pharmacological inhibition of PAI-1 with TM5614 is a promising therapeutic approach to control air pollutant PM2.5-induced cardiopulmonary and vascular pathologies.

In-Silico Analysis of Key Regulatory Pathways and Hub Genes From Peripheral Blood in Type 2 Diabetes Mellitus

Background: Type II diabetes mellitus (T2DM), characterized by fasting hyperglycemia and impaired insulin secretion and action, is a global health burden. Despite the advances in this field, the mechanism underlying T2DM is far from clear. Objective: The present study sheds light upon a systematic evaluation of the genes, pathways, and interaction networks underlying T2DM with the aid of bioinformatics. Methods: Two Gene Expression Omnibus microarray datasets: GSE148961 and GSE26168 were selected for this study. The common differentially expressed genes (DEG) were sorted by the cutoff |logFC|≥1.0 for the first dataset and |logFC|≥0.263 for the second. Gene Ontology (GO), functional enrichment, and protein-protein interaction (PPI) network were analyzed in Search Tool for the Retrieval of Interacting Genes (STRING). The MCODE and CytoHubba plugins in Cytoscape (v3.7.2) were used to identify gene clusters and top hub genes, respectively. Top 10 nodes were ranked in CytoHubba according to MCC, DMNC, MNC, Degree, and EPC methods, and genes common in at least 3 methods were selected as top nodes. Results: 88 common DEGs were identified by Venn diagram (http://bioinformatics.psb.ugent.be/cgi-bin/liste/Venn/calculate_venn.htpl). GO analysis had 91 significantly enriched biological processes, including regulated exocytosis, secretion, vesicle mediated transport, antibacterial humoral response, and neutrophil degranulation. 4 molecular functions- fibrinogen binding, fibronectin binding, lipopolysaccharide binding, and Extracellular matrix binding; and 38 cellular components, including secretory vesicle, endomembrane system, and adherens junction were significant. The PPI network was highly significant (p-value < 0.001) at medium confidence (0.400) with 88 nodes, 140 edges, and an average node degree of 3.18. The MCODE plugin revealed two clusters, the former with 14 nodes, 73 edges, and a score of 9.733, and the latter with 6 nodes, 14 edges, and a score of 4.000. 9 candidate genes: ELANE, DEFA4, BPI, MPO, LTF, CAMP, OLFM4, LCN2, and VCL were identified, amongst which ELANE, LCN2, and MPO are associated with T2DM pathogenesis, while BPI and LTF have protective effects. OLFM4 deletion has been observed to improve glucose tolerance in mice models. Conclusion: This study provides a comprehensive analysis of genes, pathways, and functions which may be pivotal in T2DM pathogenesis and may represent potential therapeutic targets.

“Microfluidics Studies of the Regulation of Myoblast Migration and Differentiation Behaviour – Possible Application in Wound Healing”

Natural extracellular matrices made from cultured cells or tissues, are tissue and cell specific, and critical in tissue engineering and cellular applications. This study focused on the effects of a more naturally produced substrate, made by myotubes differentiated within a device channel, on migration and proliferation (haptotaxis) of a second set of cells (set2) loaded into the device. First, four different microfluidic devices were designed with pillars in either an offset pattern or aligned rows along the channel to investigate the myotube formation. C2C12 cells preloaded with Hoechst stain to label DNA (set1 cells), were loaded into device channels in medium with 2% serum to induce differentiation. Results showed that in devices with aligned rows, cells were more concentrated toward pillars, and their distance from a pillar was smaller than for cells in devices with offset rows of pillars. An average of 20-30 set2 cells was tracked over 10 hours, by image capture every 2 hours in the 3 or 4 channels of each device. Results were compiled in Excel and analyzed by multi-way ANOVAs using Jamovi software. Minimum distance to a pillar occurred rapidly, at day 0 for all four devices and increased over time. After allowing set1 cells to differentiate for 5 days, prestained set2 cells were loaded. The nucleus position of set2 cells was categorized as located nearest to one of four places in the migration channel: the nucleus of a set1 cell, an extension of a set1 cell, a pillar, or the device-channel wall. Results showed that a 3-channel device with offset rows of pillars was best able to lead set1 cells to form and align myotubes in the channel and then attract the most set2 cells to nuclei of those set1 cells. Since cellular proximity is critical to myotube formation and set 2 cells were closer to the set1 nuclei than to device pillars over 10 hours imaging. Differences in flow rate among the four devices suggest that the pillars’ orientation, channel dimensions, and initial velocity are factors that influenced behavioural variations among set1 cells from the time of loading to the end of the 5-day differentiation period. Experiments in devices that were precoated with a fibronectin substrate showed that fibronectin shortened the time to confluency of set1 cells by about 2 days. Perfusion of set1 cells after 5 days of differentiation with an RGD inhibitor ((Arg-Gly-Asp) peptide) of fibronectin binding to integrins, induced a significant shift in the behavior of set2 cells toward proximity to set1 nuclei, whereas without RGD, set2 cells moved toward a device pillar. Additional experiments targeting processes during the fibronectin signaling in migration are in progress to further explore mechanism targeted by variations in device design that affect cell behavior or movement. Further study of cellular taxis should provide clues to identifying a device that would best create a muscle by promoting muscle fiber growth (by fusion of set2 to set1 cells) or in the longer term, induce set2 cells to become quiescent satellite (stem) cells.

Blockade of checkpoint ILT3/LILRB4/gp49B binding to fibronectin ameliorates autoimmune disease in BXSB/Yaa mice

Abstract Extracellular matrix (ECM) not only provides a supporting framework for each tissue, but also elicits cellular responses by providing an inductive substrate. Fibronectin (FN), a major ECM macromolecule, is composed of multiple modules that interact with ECM-associated proteins, plasma proteins, and cell-surface proteins to ensure its modulatory roles. Here we revealed that FN is identified as a physiological ligand of the immunoglobulin (Ig)-like transcript 3/leukocyte Ig-like receptor B4 (ILT3/LILRB4) which serves as an immune checkpoint for autoimmune and cancer diseases. Human LILRB4 and the murine ortholog, gp49B, bound FN with sub-micromolar affinities as assessed by bio-layer interferometry. The major LILRB4-binding site in FN was located at the N-terminal 30-kDa module (FN30). Blockade of B4–FN binding with gp49 antibody or a recombinant FN30-Fc fusion protein reduced the secretion of pathogenic IgG autoantibodies, thereby reducing PAS-positive glomerular deposition in lupus-prone BXSB/Yaa mice. Compared with the control group, activation-induced cytidine deaminase (AID) expression in splenic activated B cells of FN30-Fc-treated BXSB/Yaa mice was decreased, indicating gp49B-FN30 signaling contributes in the class switch recombination (CSR) of Ig genes. These results suggested that gp49B–FN interplay is critical for pathogenic autoantibodies-mediated glomerulonephritis in the BXSB/Yaa lupus mouse model.