Extracellular Matrix Glycoprotein Research Articles

Live imaging of Fibronectin 1a-mNeonGreen and Fibronectin 1b-mCherry knock-in alleles during early zebrafish development

Within the developing embryo, cells assemble and remodel their surrounding extracellular matrix during morphogenesis. Fibronectin is an extracellular matrix glycoprotein and is a ligand for several members of the Integrin adhesion receptor family. Here, we compare the expression pattern and loss of function phenotypes of the two zebrafish fibronectin paralogs fn1a and fn1b. We engineered two fluorescently tagged knock-in alleles to facilitate live in vivo imaging of the Fibronectin matrix. Genetic complementation experiments indicate that the knock-in alleles are fully functional. Fn1a-mNeonGreen and Fn1b-mCherry are co-localized in ECM fibers on the surface of the paraxial mesoderm and myotendinous junction. In 5-days old zebrafish larvae, Fn1a-mNeonGreen predominantly localizes to the branchial arches, heart ventricle, olfactory placode and within the otic capsule while Fn1b-mCherry is deposited at the pericardium, proximal convoluted tubule, posterior hindgut and at the ventral mesoderm/cardinal vein. We examined Fn1a-mNeonGreen and Fn1b-mCherry in maternal zygotic integrin α5 mutants and integrin β1a; β1b double mutants and find distinct requirements for these Integrins in assembling the two Fibronectins into ECM fibers in different tissues. Rescue experiments via mRNA injection indicate that the two fibronectins are not fully inter-changeable. Lastly, we examined cross-regulation between the two Fibronectins and find fn1a is necessary for normal Fn1b fibrillogenesis in the presomitic mesoderm, but fn1b is dispensable for the normal pattern of Fn1a deposition.

The extracellular matrix glycoprotein fibrillin-1 in health and disease.

Fibrillin-1 (FBN1) is a large, cysteine-rich, calcium binding extracellular matrix glycoprotein encoded by FBN1 gene. It serves as a structural component of microfibrils and provides force-bearing mechanical support in elastic and nonelastic connective tissue. As such, mutations in the FBN1 gene can cause a wide variety of genetic diseases such as Marfan syndrome, an autosomal dominant disorder characterized by ocular, skeletal and cardiovascular abnormalities. FBN1 also interacts with numerous microfibril-associated proteins, growth factors and cell membrane receptors, thereby mediating a wide range of biological processes such as cell survival, proliferation, migration and differentiation. Dysregulation of FBN1 is involved in the pathogenesis of many human diseases, such as cancers, cardiovascular disorders and kidney diseases. Paradoxically, both depletion and overexpression of FBN1 upregulate the bioavailability and signal transduction of TGF-β via distinct mechanisms in different settings. In this review, we summarize the structure and expression of FBN1 and present our current understanding of the functional role of FBN1 in various human diseases. This knowledge will allow to develop better strategies for therapeutic intervention of FBN1 related diseases.

Loss of the extracellular matrix glycoprotein EMILIN1 accelerates Δ16HER2-driven breast cancer initiation in mice

The extracellular matrix (ECM) is an important component of the tumor microenvironment and undergoes extensive remodeling during both initiation and progression of breast cancer (BC). EMILIN1 is an ECM glycoprotein, whose function has been linked to cancer and metastasis. However, EMILIN1 role during mammary gland and BC development has never been investigated. In silico and molecular analyses of human samples from normal mammary gland and BC showed that EMILIN1 expression was lower in tumors than in healthy mammary tissue and it predicted poor prognosis, particularly in HER2-positive BC. HER2+ BC accounts for 15-20% of all invasive BC and is characterized by high aggressiveness and poor prognosis. The Δ16HER2 isoform, a splice variant with very high oncogenic potential, is frequently expressed in HER2+ BC and correlates with metastatic disease. To elucidate the role of EMILIN1 in BC, we analyzed the phenotype of MMTV-Δ16HER2 transgenic mice, developing spontaneous multifocal mammary adenocarcinomas, crossed with EMILIN1 knock-out (KO) animals. We observed that Δ16HER2/EMILIN1 KO female mice exhibited an accelerated normal mammary gland development and a significantly anticipated appearance of palpable tumors (13.32 vs 15.28 weeks). This accelerated tumor initiation was corroborated by an increased number of tumor foci observed in mammary glands from Δ16HER2/EMILIN1 KO mice compared to the wild-type counterpart. Altogether our results underscore the centrality of ECM in the process of BC initiation and point to a role for EMILIN1 during normal mammary gland development and in protecting from HER2-driven breast tumorigenesis.

Assessment of Tenascin C levels in the serum of patients with bronchial asthma

Asthma is a heterogeneous disease that affects a large proportion of the global population and is distinguished by airway hyperresponsiveness to direct and indirect stimulations. It is a multifactorial disease that is triggered by heredity and environmental causes. Tenascin C (TNC) is an extracellular matrix glycoprotein that promotes inflammatory cell migration from the interstitium to the airways. Stimulation of TNC is through cytokines from T helper 2 (Th2) cells, in addition, it proliferates within basement membranes of the airways in asthmatic patients. This study aimed to determine whether serum TNC can be used as a novel biomarker for asthma diagnosis and to evaluate the association between serum TNC measurement and asthma severity. This case-control study included 64 patients with mild to severe bronchial asthma, diagnosed according to GINA 2022, referred to the Allergy and Clinical Immunology outpatient clinic at Ain Shams University Hospital, and 64 normal subjects as controls. Serum TNC levels were measured by ELISA. Serum TNC levels were significantly higher among bronchial asthma patients than controls (p ˂0.001). The sensitivity of serum TNC measurement in the diagnosis of bronchial asthma was 93.75%, the specificity 60.94%, and the negative predictive value 90.7%. Besides, a significant relation was found between serum TNC levels and the severity of bronchial asthma (p=0.004), as elevated serum TNC levels were the highest among severe asthmatic patients. In conclusion, the results gained in this study revealed that serum TNC level could be proposed as a potential biomarker for the diagnosis of bronchial asthma and a potential predictor of disease severity.

Altered galectin-3 distribution and migratory function in the pre-diabetic non-obese diabetic mouse thymus.

Galectin-3 is an endogenous lectin which binds mainly to β-galactosides on the cell surface and extracellular matrix (ECM) glycoproteins. In the thymus, this lectin is constitutively expressed, being involved in thymocyte adhesion, migration, and death. Galectin-3 has been related to type 1 diabetes, an autoimmune disease characterized by pancreatic β-cell destruction mediated by autoreactive T lymphocytes. Non-obese diabetic (NOD) mice represent a suitable model to study type 1 diabetes, as they develop the disease like humans. We previously described important thymic alterations in these animals such as the development of giant perivascular spaces (PVS), characterized by the retention of T and B cells, intermingled with an ECM network, and associated with a defect in the expression of the fibronectin receptor VLA-5 and reduced sphingosine-1-phosphate receptor expression on developing thymocytes. In order to investigate galectin-3 expression in thymic microenvironmental cells and verify its interaction with cells and ECM molecules in PVS, we performed immunofluorescence following colocalization analysis in the thymic parenchyma of pre-diabetic NOD mice by confocal microscopy. In addition, thymocyte migration assays were performed to evaluate the effect of galectin-3 on NOD thymocyte migration. Herein, we showed a significant enhancement of colocalization with cortical and medullary thymic epithelial cells in NOD mice, as compared to controls. In the giant PVS of these animals, we observed a heterogeneous distribution of galectin-3, predominantly found in clusters of B lymphocytes and dendritic cells. Functionally, NOD thymocyte migratory response towards galectin-3 was impaired and a similar decrease was seen in transendothelial thymocyte migration. Taken together, our data provide the histological and functional background for a potential defective thymocyte migration involving galectin-3, thus placing this molecule as a further player in the intrathymic disturbances observed in pre-diabetic NOD mice.

Evaluation of osteoconductive effect of polycaprolactone (PCL) scaffold treated with fibronectin on adipose-derived mesenchymal stem cells (AD-MSCs).

Replacing damaged organs or tissues and repairing damage by tissue engineering are attracting great interest today. A potentially effective method for bone remodeling involves combining nanofiber scaffolds with extracellular matrix (ECM), and growth factors. Today, electrospun PCL-based scaffolds are widely used for tissue engineering applications. In this study, we used an electrospun polycaprolactone (PCL) scaffold coated with fibronectin (Fn), a ubiquitous ECM glycoprotein, to investigate the induction potential of this scaffold in osteogenesis with adipose-derived mesenchymal stem cells (AD-MSCs). Scanning electron microscopy (SEM) analysis showed that fibronectin, by binding to the membrane receptors of mesenchymal stem cells (MSCs), leads to their attachment and proliferation on the PCL scaffold and provides a suitable environment for osteogenesis. In addition, biochemical tests showed that fibronectin leads to increased calcium deposition. The results also showed that alkaline phosphatase activity was significantly higher in the PCL scaffold coated with fibronectin than in the control groups (PCL scaffold group and tissue culture polystyrene (TCPS) group) (P<0.05). Also, the analysis of quantitative reverse transcription PCR (qRT-PCR) data showed that the relative expression of bone marker genes such as osteonectin (ON), osteocalcin (OC), RUNX family transcription factor 2 (RUNX2), and collagen type I alpha 1 (COL1) was much higher in the cells seeded on the PCL/Fn scaffold than in the other groups (P<0.05). The results show that fibronectin has an increasing effect in accelerating bone formation and promising potential for use in bone tissue engineering.

Fibronectin Conformations after Electrodeposition onto 316L Stainless Steel Substrates Enhanced Early-Stage Osteoblasts' Adhesion but Affected Their Behavior.

The implantation of metallic orthopedic prostheses is increasingly common due to an aging population and accidents. There is a real societal need to implement new metal implants that combine durability, good mechanical properties, excellent biocompatibility, as well as affordable costs. Since the functionalization of low-cost 316L stainless steel substrates through the successive electrodeposition of a polypyrrole film (PPy) and a calcium phosphate deposit doped with silicon was previously carried out by our labs, we have also developed a bio-functional coating by electrodepositing or oxidating of fibronectin (Fn) coating. Fn is an extracellular matrix glycoprotein involved in cell adhesion and differentiation. Impacts of either electrodeposition or oxidation on the structure and functionality of Fn were first studied. Thus, electrodeposition is the technique that permits the highest deposition of fibronectin, compared to adsorption or oxidation. Furthermore, electrodeposition seems to strongly modify Fn conformation by the formation of intermingled long fibers, resulting in changes to the accessibility of the molecular probes tested (antibodies directed against Fn whole molecule and Fn cell-binding domain). Then, the effects of either electrodeposited Fn or oxidized Fn were validated by the resulting pre-osteoblast behavior. Electrodeposition reduced pre-osteoblasts' ability to remodel Fn coating on supports because of a partial modification of Fn conformation, which reduced accessibility to the cell-binding domain. Electrodeposited Fn also diminished α5 integrin secretion and clustering along the plasma membrane. However, the N-terminal extremity of Fn was not modified by electrodeposition as demonstrated by Staphylococcus aureus attachment after 3 h of culture on a specific domain localized in this region. Moreover, the number of pre-osteoblasts remains stable after 3 h culture on either adsorbed, oxidized, or electrodeposited Fn deposits. In contrast, mitochondrial activity and cell proliferation were significantly higher on adsorbed Fn compared with electrodeposited Fn after 48 h culture. Hence, electro-deposited Fn seems more favorable to pre-osteoblast early-stage behavior than during a longer culture of 24 h and 48 h. The electrodeposition of matrix proteins could be improved to maintain their bio-activity and to develop this promising, fast technique to bio-functionalize metallic implants.

Macrophage migration is differentially regulated by fibronectin and laminin through altered adhesion and myosin II localization.

Macrophages are indispensable for proper immune surveillance and inflammatory regulation. They also exhibit dramatic phenotypic plasticity and are highly responsive to their local microenvironment, which includes the extracellular matrix (ECM). This work demonstrates that two fibrous ECM glycoproteins, fibronectin (FN) and laminin (LAM), elicit distinct morphological and migratory responses from macrophages in two-dimensional environments. LAM 111 inhibits macrophage cell spreading, but drives them to migrate rapidly and less persistently compared with cells on FN. Differential integrin engagement and ROCK/myosin II organization helps explain why macrophages alter their morphology and migration character on these two ECM components. This study also demonstrates that LAM 111 exerts a suppressive effect toward FN, as macrophages plated on a LAM/FN mixture adopt a morphology and migratory character almost identical to LAM alone. This suggests that distinct responses can be initiated downstream of receptor-ECM engagement, and that one component of the microenvironment may affect the cell's ability to sense another. Overall, macrophages appear intrinsically poised to rapidly switch between distinct migratory characters based on their ECM environments. The role of ECM composition in dictating motile and inflammatory responses in three-dimensional and in vivo contexts warrants further study.

FAT4 loss initiates hepatocarcinogenesis through the switching of canonical to noncanonical WNT signaling pathways.

Mutation and downregulation of FAT atypical cadherin 4 (FAT4) are frequently detected in HCC, suggesting a tumor suppressor role of FAT4. However, the underlying molecular mechanism remains elusive. CRISPR-Cas9 system was used to knockout FAT4 (FAT4-KO) in a normal human hepatic cell line L02 to investigate the impact of FAT4 loss on the development of HCC. RNA-sequencing and xenograft mouse model were used to study gene expression and tumorigenesis, respectively. The mechanistic basis of FAT4 loss on hepatocarcinogenesis was elucidated using in vitro experiments. We found that FAT4-KO disrupted cell-cell adhesion, induced epithelial-mesenchymal transition, and increased expression of extracellular matrix components. FAT4-KO is sufficient for tumor initiation in a xenograft mouse model. RNA-sequencing of FAT4-KO cells identified PAK6-mediated WNT/β-catenin signaling to promote tumor growth. Suppression of PAK6 led to β-catenin shuttling out of the nucleus for ubiquitin-dependent degradation and constrained tumor growth. Further, RNA-sequencing of amassed FAT4-KO cells identified activation of WNT5A and ROR2. The noncanonical WNT5A/ROR2 signaling has no effect on β-catenin and its target genes (CCND1 and c-Myc) expression. Instead, we observed downregulation of receptors for WNT/β-catenin signaling, suggesting the shifting of β-catenin-dependent to β-catenin-independent pathways as tumor progression depends on its receptor expression. Both PAK6 and WNT5A could induce the expression of extracellular matrix glycoprotein, laminin subunit alpha 4. Laminin subunit alpha 4 upregulation in HCC correlated with poor patient survival. Our data show that FAT4 loss is sufficient to drive HCC development through the switching of canonical to noncanonical Wingless-type signaling pathways. The findings may provide a mechanistic basis for an in-depth study of the two pathways in the early and late stages of HCC for precise treatment.

Unveiling the role of osteosarcoma-derived secretome in premetastatic lung remodelling

BackgroundLung metastasis is the most adverse clinical factor and remains the leading cause of osteosarcoma-related death. Deciphering the mechanisms driving metastatic spread is crucial for finding open therapeutic windows for successful organ-specific interventions that may halt or prevent lung metastasis.MethodsWe employed a mouse premetastatic lung-based multi-omics integrative approach combined with clinical features to uncover the specific changes that precede lung metastasis formation and identify novel molecular targets and biomarker of clinical utility that enable the design of novel therapeutic strategies.ResultsWe found that osteosarcoma-bearing mice or those preconditioned with the osteosarcoma cell secretome harbour profound lung structural alterations with airway damage, inflammation, neutrophil infiltration, and extracellular matrix remodelling with increased deposition of fibronectin and collagens by resident stromal activated fibroblasts, favouring the adhesion of disseminated tumour cells. Systemic-induced microenvironmental changes, supported by transcriptomic and histological data, promoted and accelerated lung metastasis formation. Comparative proteome profiling of the cell secretome and mouse plasma identified a large number of proteins involved in extracellular-matrix organization, cell-matrix adhesion, neutrophil degranulation, and cytokine-mediated signalling, consistent with the observed lung microenvironmental changes. Moreover, we identified EFEMP1, an extracellular matrix glycoprotein exclusively secreted by metastatic cells, in the plasma of mice bearing a primary tumour and in biopsy specimens from osteosarcoma patients with poorer overall survival. Depletion of EFEMP1 from the secretome prevents the formation of lung metastasis.ConclusionsIntegration of our data uncovers neutrophil infiltration and the functional contribution of stromal-activated fibroblasts in ECM remodelling for tumour cell attachment as early pro-metastatic events, which may hold therapeutic potential in preventing or slowing the metastatic spread. Moreover, we identified EFEMP1, a secreted glycoprotein, as a metastatic driver and a potential candidate prognostic biomarker for lung metastasis in osteosarcoma patients.Graphical abstractOsteosarcoma-derived secreted factors systemically reprogrammed the lung microenvironment and fostered a growth-permissive niche for incoming disseminated cells to survive and outgrow into overt metastasis.Daily administration of osteosarcoma cell secretome mimics the systemic release of tumour-secreted factors of a growing tumour in mice during PMN formation;Transcriptomic and histological analysis of premetastatic lungs revealed inflammatory-induced stromal fibroblast activation, neutrophil infiltration, and ECM remodelling as early onset pro-metastatic events;Proteome profiling identified EFEMP1, an extracellular secreted glycoprotein, as a potential predictive biomarker for lung metastasis and poor prognosis in osteosarcoma patients.Osteosarcoma patients with EFEMP1 expressing biopsies have a poorer overall survival.

Epithelial mesenchymal transition induced nuclear localization of the extracellular matrix protein Fibronectin

Fibronectin (FN), an extracellular matrix (ECM) glycoprotein, is a well-known marker for Epithelial Mesenchymal Transition (EMT). In the ECM, FN has been shown to form long fibrils and play critical roles in regulating cellular attachment and migration during EMT associated with physiological processes such as embryonic development, wound healing as well as pathological processes such as tissue fibrosis and cancer. Subsequently, the cytokine, Transforming Growth Factor β (TGFβ), an inducer of EMT, was found to induce FN expression in a c-Jun N-terminal kinase (JNK) dependent manner. Moreover, extracellular FN, by itself, was also shown to induce EMT in breast epithelial cells in serum-free condition. Collectively, all the literature published so far has shown and established the role of extracellular FN during EMT. In this report, we have shown that EMT induced entry of FN into the nucleus of mouse breast epithelial cells. To our knowledge, this is the first report showing nuclear localization of the extracellular matrix protein Fibronectin during EMT and thereby suggests a possible nuclear function for the ECM protein.

One Week of Single-Leg Immobilization Lowers Muscle Connective Protein Synthesis Rates in Healthy, Young Adults.

Short periods of limb immobilization lower myofibrillar protein synthesis rates. Within skeletal muscle, the extracellular matrix of connective proteins is recognized as an important factor determining the capacity to transmit contractile force. Little is known regarding the impact of immobilization and subsequent recovery on muscle connective protein synthesis rates. This study examined the impact of 1 wk of leg immobilization and 2 wk of subsequent ambulant recovery on daily muscle connective protein synthesis rates. Thirty healthy, young (24 ± 5 yr) men were subjected to 7 d of one-legged knee immobilization followed by 14 d of ambulant recovery. Deuterium oxide ingestion was applied over the entire period, and muscle biopsy samples were collected before immobilization, after immobilization, and after recovery to measure muscle connective protein synthesis rates and mRNA expression of key extracellular matrix proteins (collagen I, collagen III), glycoproteins (fibronectin, tenascin-C), and proteoglycans (fibromodulin, and decorin). A two-way repeated-measures (time-leg) ANOVA was used to compare changes in muscle connective protein synthesis rates during immobilization and recovery. During immobilization, muscle connective protein synthesis rates were lower in the immobilized (1.07 ± 0.30%·d -1 ) compared with the nonimmobilized (1.48 ± 0.44%·d -1 ; P < 0.01) leg. When compared with the immobilization period, connective protein synthesis rates in the immobilized leg increased during subsequent recovery (1.48 ± 0.64%·d -1 ; P < 0.01). After recovery, skeletal muscle collagen I, collagen III, fibronectin, fibromodulin, and decorin mRNA expression increased when compared with the postimmobilization time point (all P < 0.001). One week of leg immobilization lowers muscle connective protein synthesis rates. Muscle connective protein synthesis rates increase during subsequent ambulant recovery, which is accompanied by increased mRNA expression of key extracellular matrix proteins.

CILP is a potential pan-cancer marker: combined silico study and in vitro analyses.

CILP (Cartilage intermediate layer protein), an ECM (extracellular matrix) glycoprotein, is found to be associated with intervertebral disc degeneration, chronic heart failure, obese and cardiac fibrosis. However, there are few reports on the role of CILP in tumors. Thus, in this study, we mainly explored the function of CILP in the occurrence and development of tumors and whether it could be a potential pan-cancer marker. Pan-cancer data in this study were obtained from UCSC Xena. Single-cell data were obtained from GSE152938. ROC (Receiver operating characteristic) curves were used to evaluate the accuracy of CILP in predicting the occurrence of different tumor types. The Kaplan-Meier plots were used to assess the relationship between CILP expression and survival prognosis in different tumor types by COX regression analysis. Pseudotime analysis and cell communication analysis were used to further explore the function of CILP at Single cell level. The human RCC (renal cell carcinoma) cell lines ACHN and 786-O were used for further experimental verification. Bulk RNA-seq showed differences in CILP expression in several tumors. ROC curves showed that 14 tumors have AUC > 0.7. Kaplan-Meier plots indicated that CILP is a risk factor for patients in 3 kinds of tumors. ScRNA-seq (Single cell RNA sequencing) suggested that CILP might influence tumors through fibroblasts and cell-cell communication. Finally, we verified the function of CILP at the cellular level by using RCC cell lines ACHN and 786-O and found that knockdown of CILP could significantly inhibit migration and invasion. This finding supports that CILP could be a risk factor as well as a pan-cancer predictor for patients.

Regional levels of tenascin- C in patients with heart failure and atrial fibrillation

Abstract Background Tenascin-C (TNC) is an extracellular matrix glycoprotein that is upregulated at high levels during active tissue remodeling. The serum concentration of TNC has been reported to be increased in patients with several cardiac diseases. The aim of this study was 1) to measure the TNC plasma level in heart failure (HF) and atrial fibrillation (AF) patients compared to patients without structural heart disease and 2) to search for a local production in the heart. Methods This study included 13 patients with HF who underwent CRT- implantation (CRT- group), 13 patients with persistent AF who underwent left atrial catheter ablation (AF- group) and 13 patients without structural heart disease or AF, who underwent electrophysiological study (Control- group). Blood was collected at the beginning of the procedures from the coronary sinus (CS), superior vena cava (SVC) and aorta (Ao). Level of TNC-B (ng/dl) in the CS (TNC-CS), SVC (TNC-SVC) and aorta (TNC-Ao) was measured. Results Patients characteristics: CRT- group: mean age 74y, 8 male, LV- EF 32%, 3 with a history of atrial fibrillation. AF- group: mean age 68y, 9 male, LV- EF 55%. Control- group: mean age 54y, 9 male, LV- EF 65%. The TNC level in the SVC was in the CRT group significant elevated (6125 ± 2030 ng/ml vs 475 ± 238 ng/ml in controls (p&amp;lt;0.01). In the AF group the TNC level in the SVC as well was significantly higher 4983 ± 1814 ng/ml vs 475 ± 238 ng/ml in controls (p&amp;lt;0.01). The TNC level of the CRT- and the AF- group were not significant different (p= 0.219) (Figure 1). No significant differences were noted at the various collection sites (CS, SVC and aorta) between the 3 groups (p = 0.38). Discussion The TNC serum levels in HF- and AF- patients are elevated compared to a control group without structural heart disease and without a history of AF. We could not determinate, the source of serum TNC, differences in the level of TNC between CS and SVC and aorta were not visible. Whether an effective treatment of HF and AF will lower TNC levels and slow the disease progression needs to be evaluated in further studies.

Genome-wide transcriptional responses of osteoblasts to different titanium surface topographies

This is the first genome-wide transcriptional profiling study using RNA-sequencing to investigate osteoblast responses to different titanium surface topographies, specifically between machined, smooth and acid-etched, microrough surfaces. Rat femoral osteoblasts were cultured on machine-smooth and acid-etched microrough titanium disks. The culture system was validated through a series of assays confirming reduced osteoblast attachment, slower proliferation, and faster differentiation on microrough surfaces. RNA-sequencing analysis of osteoblasts at an early stage of culture revealed that gene expression was highly correlated (r = 0.975) between the two topographies, but 1.38 % genes were upregulated and 0.37 % were downregulated on microrough surfaces. Upregulated transcripts were enriched for immune system, plasma membrane, response to external stimulus, and positive regulation to stimulus processes. Structural mapping confirmed microrough surface-promoted gene sharing and networking in signaling pathways and immune system/responses. Target-specific pathway analysis revealed that Rho family G-protein signaling pathways and actin genes, responsible for the formation of stress fibers, cytoplasmic projections, and focal adhesion, were upregulated on microrough surfaces without upregulation of core genes triggered by cell-to-cell interactions. Furthermore, disulfide-linked or -targeted extracellular matrix (ECM) or membranous glycoproteins such as laminin, fibronectin, CD36, and thrombospondin were highly expressed on microrough surfaces. Finally, proliferating cell nuclear antigen (PCNA) and cyclin D1, whose co-expression reduces cell proliferation, were upregulated on microrough surfaces. Thus, osteoblasts on microrough surfaces were characterized by upregulation of genes related to a wide range of functions associated with the immune system, stress/stimulus responses, proliferation control, skeletal and cytoplasmic signaling, ECM-integrin receptor interactions, and ECM-membranous glycoprotein interactions, furthering our knowledge of the surface-dependent expression of osteoblastic biomarkers on titanium.

Hypersensitivity of myelinated A-fibers via toll-like receptor 5 promotes mechanical allodynia in tenascin-X-deficient mice associated with Ehlers–Danlos syndrome

Deficiency of an extracellular matrix glycoprotein tenascin-X (TNX) leads to a human heritable disorder Ehlers–Danlos syndrome, and TNX-deficient patients complain of chronic joint pain, myalgia, paresthesia, and axonal polyneuropathy. We previously reported that TNX-deficient (Tnxb−/−) mice exhibit mechanical allodynia and hypersensitivity to myelinated A-fibers. Here, we investigated the pain response of Tnxb−/− mice using pharmacological silencing of A-fibers with co-injection of N-(2,6-Dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314), a membrane-impermeable lidocaine analog, plus flagellin, a toll-like receptor 5 (TLR5) ligand. Intraplantar co-injection of QX-314 and flagellin significantly increased the paw withdrawal threshold to transcutaneous sine wave stimuli at frequencies of 250 Hz (Aδ fiber responses) and 2000 Hz (Aβ fiber responses), but not 5 Hz (C fiber responses) in wild-type mice. The QX-314 plus flagellin-induced silencing of Aδ- and Aβ-fibers was also observed in Tnxb−/− mice. Co-injection of QX-314 and flagellin significantly inhibited the mechanical allodynia and neuronal activation of the spinal dorsal horn in Tnxb−/− mice. Interestingly, QX-314 alone inhibited the mechanical allodynia in Tnxb−/− mice, and it increased the paw withdrawal threshold to stimuli at frequencies of 250 Hz and 2000 Hz in Tnxb−/− mice, but not in wild-type mice. The inhibition of mechanical allodynia induced by QX-314 alone was blocked by intraplantar injection of a TLR5 antagonist TH1020 in Tnxb−/− mice. These results suggest that mechanical allodynia due to TNX deficiency is caused by the hypersensitivity of Aδ- and Aβ-fibers, and it is induced by constitutive activation of TLR5.

Systematic analysis of integrated bioinformatics to identify upregulated THBS2 expression in colorectal cancer cells inhibiting tumour immunity through the HIF1A/Lactic Acid/GPR132 pathway

BackgroundTHBS2, a member of the extracellular matrix glycoprotein family, can effectively inhibit tumour growth and angiogenesis. This study aimed to investigate the biological role of THBS2 in various types of cancers and the mechanisms underlying the malignant progression of colorectal cancer (CRC).MethodsTHBS2 expression in pan-cancer tissues and cell lines was assessed using the HPA, TISCH and CCLE databases. The CIBERSORT, ESTIMATE, TIMER, xCell and ssGSEA (implemented using the IOBR R package) algorithms were used to calculate the proportion of tumour-infiltrating immune cells based on the expression profile of THBS2 in TCGA-COAD cohort. The clusterprofiler R package was used to implement GO and KEGG pathway enrichm SNVs were compared between the high- and low-THBS2-expression groups using the maftools R package. Additionally, immunotherapy responses were compared between the high- and low-THBS2-expression groups based on immunophenoscores (IPSs). CT26 cells were engineered to overexpress THBS2 (CT26-THBS2) to investigate its regulatory effects on HIF1 and cellular metabolism. The conditioned medium from CT26-THBS2 cells was collected to examine its effect on the M2 polarisation of RAW264.7 macrophages. Subsequently, in vitro experiments were performed to validate the inhibitory effects of M2-polarised macrophages on T-cell proliferation and cytotoxicity. A CT26-THBS2 tumour-bearing mouse model was constructed to validate the impact of high THBS2 expression in tumour cells on the tumour microenvironment in vivo.ResultsTHBS2 expression was upregulated in a majority of tumours, including COAD, and was positively associated with ESTIMATEScore, ImmuneScore and StromalScore. Furthermore, THBS2 expression was positively associated with angiogenesis and epithelial–mesenchymal transition and negatively associated with DNA repair, cell cycle and DNA replication in most tumours. THBS2 expression was considerably associated with progression-free interval (PFI) and positively associated with MSI in COAD. THBS2 methylation levels were remarkably lower in COAD tissues than in healthy tissues. The high expression of THBS2 in CT26 cells remarkably promoted the nuclear translocation of HIF1 and consequently enhanced lactate metabolism in cells. In vitro and in vivo experiments revealed that lactate released by tumour cells promoted M2 polarisation of macrophages, leading to inhibition of T-cell proliferation and cytotoxicity.ConclusionsTHBS2 expression is associated with PFI, immune cell infiltration, immune regulation, cell death, cell migration, epithelial–mesenchymal transition, angiogenesis and genomic variations in COAD. THBS2 may serve as a biomarker for immunotherapy in COAD. Upregulated THBS2 expression in CRC cells inhibits anti-tumour immunity through the HIF1A/lactic acid/GPR132 pathway.

Reelin Signaling in Neurodevelopmental Disorders and Neurodegenerative Diseases.

Reelin is an extracellular matrix glycoprotein involved in neuronal migration during embryonic brain development and synaptic plasticity in the adult brain. The role of Reelin in the developing central nervous system has been extensively characterized. Indeed, a loss of Reelin or a disruption in its signaling cascade leads to neurodevelopmental defects and is associated with ataxia, intellectual disability, autism, and several psychiatric disorders. In the adult brain, Reelin is critically involved in neurogenesis and synaptic plasticity. Reelin's signaling potentiates glutamatergic and GABAergic neurotransmission, induces synaptic maturation, and increases AMPA and NMDA receptor subunits' expression and activity. As a result, there is a growing literature reporting that a loss of function and/or reduction of Reelin is implicated in numerous neurodegenerative diseases. The present review summarizes the current state of the literature regarding the implication of Reelin and Reelin-mediated signaling during aging and neurodegenerative disorders, highlighting Reelin as a possible target in the prevention or treatment of progressive neurodegeneration.

Lung extracellular matrix modulates KRT5+ basal cell activity in pulmonary fibrosis

Aberrant expansion of KRT5+ basal cells in the distal lung accompanies progressive alveolar epithelial cell loss and tissue remodelling during fibrogenesis in idiopathic pulmonary fibrosis (IPF). The mechanisms determining activity of KRT5+ cells in IPF have not been delineated. Here, we reveal a potential mechanism by which KRT5+ cells migrate within the fibrotic lung, navigating regional differences in collagen topography. In vitro, KRT5+ cell migratory characteristics and expression of remodelling genes are modulated by extracellular matrix (ECM) composition and organisation. Mass spectrometry- based proteomics revealed compositional differences in ECM components secreted by primary human lung fibroblasts (HLF) from IPF patients compared to controls. Over-expression of ECM glycoprotein, Secreted Protein Acidic and Cysteine Rich (SPARC) in the IPF HLF matrix restricts KRT5+ cell migration in vitro. Together, our findings demonstrate how changes to the ECM in IPF directly influence KRT5+ cell behaviour and function contributing to remodelling events in the fibrotic niche.

Abstract PO-100: Fusobacterium nucleatum promotes epithelial-mesenchymal transition (EMT) of oral squamous cell carcinoma (OSCC) via INHBA-dependent SMAD/Laminin332/PI3K/AKT pathway

Abstract Introduction The enrichment of Fusobacterium nucleatum has been reported in both oral rinses and lesion samples from oral squamous cell carcinoma (OSCC) patients based on several case-control sequencing analyses, but studies on the potential role of F. nucleatum in OSCC tumorigenesis are still limited. In this study, we sought to elucidate the relationship between F. nucleatum and host transcriptome dysregulation in potentially carcinogenic pathways. Method The association between microbiota dysbiosis and host gene transcriptome was profiled by bacterial 16S rRNA V3-V4 amplicon sequencing and host gene long non-coding RNA sequencing (lncRNA-seq) for tumor and adjacent normal (AN) tissues from OSCC patients. Cell migration and invasion assays were performed using the in vitro bacteria-cell co-culture model. EMT-related molecular mechanisms induced by F. nucleatum were further validated by IncRNA-seq, real-time PCR, and western blot for bacteria-infected cells, respectively, at the genetic and proteomic levels. Result Significant enrichment of Fusobacterium was observed in OSCC tumor samples when compared to the paired AN samples. Correlation analysis between mucosal oral bacteria and host transcriptome in the OSCC tumor microenvironment elucidated the positive relationship between Fusobacterium and EMT pathways including significantly upregulated genes INHBA, SNAI2, and LAMC2. Using in-vitro cell models, we further observed that F. nucleatum significantly promoted cell migration and invasion, and significantly dysregulated the expression of several EMT factors including the increased inducer inhibin beta A (INHBA), core regulators SNAI1 (snai1) and SLUG (snai2), effectors vimentin (VIM) and N-cadherin (CDH2), and decreased E-cadherin (CDH1). Following the INHBA/SMAD pathway induced by F. nucleatum infection, the expression of an essential extracellular matrix glycoprotein, Laminin 332, was dramatically enhanced, which may trigger the phosphorylation of phosphatidylinositol 3-kinase and AKT kinase in the downstream PI3K/AKT pathway for the promoted cell migration and invasion ability. Conclusion Our study confirms the enrichment of Fusobacterium in the OSCC tumor microenvironment and reveals that F. nucleatum infection induces the INHBA/SAMD pathway and subsequently increases Laminin 332 expression, which may further promote the phosphorylation of PI3K/AKT and enhance OSCC migration and invasion. Citation Format: Hengyan Zhu, Liuyang Cai, Zigui Chen, Jason Y. K. Chan. Fusobacterium nucleatum promotes epithelial-mesenchymal transition (EMT) of oral squamous cell carcinoma (OSCC) via INHBA-dependent SMAD/Laminin332/PI3K/AKT pathway [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-100.