Extra Domain A Research Articles

Targeting Alternative Splicing of Fibronectin (Fn) to Reduce Extra Domain A (EDA)+ Fn Production and Inhibit Kidney Fibrosis

Targeting Alternative Splicing of Fibronectin (Fn) to Reduce Extra Domain A (EDA)+ Fn Production and Inhibit Kidney Fibrosis

Current Approach to Risk Factors and Biomarkers of Intestinal Fibrosis in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD), especially Crohn's disease (CD), characterized by a chronic inflammatory process and progressive intestinal tissue damage, leads to the unrestrained proliferation of mesenchymal cells and the development of bowel strictures. Complications induced by fibrosis are related to high rates of morbidity and mortality and lead to a substantial number of hospitalizations and surgical procedures, generating high healthcare costs. The development of easily obtained, reliable fibrogenesis biomarkers is essential to provide an important complementary tool to existing diagnostic and prognostic methods in IBD management, guiding decisions on the intensification of pharmacotherapy, proceeding to surgical methods of treatment and monitoring the efficacy of anti-fibrotic therapy in the future. The most promising potential markers of fibrosis include cartilage oligomeric matrix protein (COMP), hepatocyte growth factor activator (HGFA), and fibronectin isoform- extra domain A (ED-A), as well as antibodies against granulocyte macrophage colony-stimulating factor (GM-CSF Ab), cathelicidin (LL-37), or circulatory miRNAs: miR-19a-3p and miR-19b-3p. This review summarizes the role of genetic predisposition, and risk factors and serological markers potentially contributing to the pathophysiology of fibrotic strictures in the course of IBD.

Formation of an invasion-permissive matrix requires TGFβ/SNAIL1-regulated alternative splicing of fibronectin

BackgroundAs in most solid cancers, the emergence of cells with oncogenic mutations in the mammary epithelium alters the tissue homeostasis. Some soluble factors, such as TGFβ, potently modify the behavior of healthy stromal cells. A subpopulation of cancer-associated fibroblasts expressing a TGFβ target, the SNAIL1 transcription factor, display myofibroblastic abilities that rearrange the stromal architecture. Breast tumors with the presence of SNAIL1 in the stromal compartment, and with aligned extracellular fiber, are associated with poor survival prognoses.MethodsWe used deep RNA sequencing and biochemical techniques to study alternative splicing and human tumor databases to test for associations (correlation t-test) between SNAIL1 and fibronectin isoforms. Three-dimensional extracellular matrices generated from fibroblasts were used to study the mechanical properties and actions of the extracellular matrices on tumor cell and fibroblast behaviors. A metastatic mouse model of breast cancer was used to test the action of fibronectin isoforms on lung metastasis.ResultsIn silico studies showed that SNAIL1 correlates with the expression of the extra domain A (EDA)-containing (EDA+) fibronectin in advanced human breast cancer and other types of epithelial cancers. In TGFβ-activated fibroblasts, alternative splicing of fibronectin as well as of 500 other genes was modified by eliminating SNAIL1. Biochemical analyses demonstrated that SNAIL1 favors the inclusion of the EDA exon by modulating the activity of the SRSF1 splicing factor. Similar to Snai1 knockout fibroblasts, EDA- fibronectin fibroblasts produce an extracellular matrix that does not sustain TGFβ-induced fiber organization, rigidity, fibroblast activation, or tumor cell invasion. The presence of EDA+ fibronectin changes the action of metalloproteinases on fibronectin fibers. Critically, in an mouse orthotopic breast cancer model, the absence of the fibronectin EDA domain completely prevents lung metastasis.ConclusionsOur results support the requirement of EDA+ fibronectin in the generation of a metastasis permissive stromal architecture in breast cancers and its molecular control by SNAIL1. From a pharmacological point of view, specifically blocking EDA+ fibronectin deposition could be included in studies to reduce the formation of a pro-metastatic environment.

Drug repurposing for targeting fibronectin in treatment of endometriosis and cancers

Increased concentrations of the fibronectin glycoprotein can cause ectopic tissue growth patients with endometriosis and the formation of various cancerous tumors. Furthermore, fibronectin binding to its receptors from the EDA (Extra Domain A) region contributes to promote tumorigenesis, metastasis and vasculogenesis. Thus, the EDA region can be considered a unique target for therapeutic intervention. Therefore, the present study used computational methods to identify the best fibronectin inhibitor(s) among FDA-approved drugs. First, docking-based virtual screening was performed using PyRx 0.8. Next, FDA-approved drugs that obtained favorable results in the docking phase were selected for further studies and analysis using molecular dynamics (MD) simulation. The preliminary findings of the virtual screening showed that 17 FDA-approved drugs (from 2471) had more favorable energy with their binding energy less than −9 kcal/mol. The MD simulation results of these 17 drugs showed that Avapritinib had a lower RMSD value and higher binding energy and hydrogen bonding than the other complexes in the EDA domain. Also, analyses related to the second structure changes displayed that Avapritinib in the EDA domain led to more changes in the second structure. According to the results, the anticancer drug Avapritinib forms a more stable complex with fibronectin than other FDA-approved drugs. Furthermore, this drug leads to more changes in the second EDA structure, which may have more serious potential for inhibiting EDA fibronectin. Communicated by Ramaswamy H. Sarma

Single-cell RNA sequencing and spatial transcriptomics reveal cancer-associated fibroblasts in glioblastoma with protumoral effects.

Cancer-associated fibroblasts (CAFs) were presumed absent in glioblastoma given the lack of brain fibroblasts. Serial trypsinization of glioblastoma specimens yielded cells with CAF morphology and single-cell transcriptomic profiles based on their lack of copy number variations (CNVs) and elevated individual cell CAF probability scores derived from the expression of 9 CAF markers and absence of 5 markers from non-CAF stromal cells sharing features with CAFs. Cells without CNVs and with high CAF probability scores were identified in single-cell RNA-Seq of 12 patient glioblastomas. Pseudotime reconstruction revealed that immature CAFs evolved into subtypes, with mature CAFs expressing actin alpha 2, smooth muscle (ACTA2). Spatial transcriptomics from 16 patient glioblastomas confirmed CAF proximity to mesenchymal glioblastoma stem cells (GSCs), endothelial cells, and M2 macrophages. CAFs were chemotactically attracted to GSCs, and CAFs enriched GSCs. We created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF and TGF-β as mediators of GSC effects on CAFs and osteopontin and HGF as mediators of CAF-induced GSC enrichment. CAFs induced M2 macrophage polarization by producing the extra domain A (EDA) fibronectin variant that binds macrophage TLR4. Supplementing GSC-derived xenografts with CAFs enhanced in vivo tumor growth. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target.

Promotion of Lymphangiogenesis by Targeted Delivery of VEGF-C Improves Diabetic Wound Healing.

Chronic wounds represent a major therapeutic challenge. Lymphatic vessel function is impaired in chronic ulcers but the role of lymphangiogenesis in wound healing has remained unclear. We found that lymphatic vessels are largely absent from chronic human wounds as evaluated in patient biopsies. Excisional wound healing studies were conducted using transgenic mice with or without an increased number of cutaneous lymphatic vessels, as well as antibody-mediated inhibition of lymphangiogenesis. We found that a lack of lymphatic vessels mediated a proinflammatory wound microenvironment and delayed wound closure, and that the VEGF-C/VEGFR3 signaling axis is required for wound lymphangiogenesis. Treatment of diabetic mice (db/db mice) with the F8-VEGF-C fusion protein that targets the alternatively spliced extra domain A (EDA) of fibronectin, expressed in remodeling tissue, promoted wound healing, and potently induced wound lymphangiogenesis. The treatment also reduced tissue inflammation and exerted beneficial effects on the wound microenvironment, including myofibroblast density and collagen deposition. These findings indicate that activating the lymphatic vasculature might represent a new therapeutic strategy for treating chronic non-healing wounds.

EDA-E7 Activated DCs Induces Cytotoxic T Lymphocyte Immune Responses against HPV Expressing Cervical Cancer in Human Setting.

Cervical cancer is a major cause of cancer death in women worldwide. Targeting human papillomavirus (HPV) viral oncoproteins E6 and E7 is a new strategy for cervical cancer immunotherapy and has been associated with resolution of HPV-induced lesions. How to efficiently induce T cell target killing of HPV infected cervical cancer is of great potential benefit for cervical cancer treatment. Fusion protein containing the extra domain A (EDA) from fibronectin, a natural ligand for Toll-like receptor 4 (TLR4), and HPVE7 (EDA-E7) has been shown to efficiently induce dendritic cells maturation and trigger specific antitumor CD8+ T cells response in mice. In this study, we constructed EDA-E7 fusion protein of human origin and tested its function in dendritic cell maturation as well as antitumor T cell response. We found that EDA-E7 could be efficiently captured by human PBMC derived dendritic cells (DCs) in vitro and induce DCs maturation. Importantly, this effect could work in synergy with the TLR ligand anti-CD40 agonist, polyinosinic-polycytidylic acid [poly (I:C)], R848, and CpG2216. EDA-E7 matured DCs could activate T cells and trigger an anti-tumor response in vitro. Single cell RNA sequencing and T cell targeted killing assay confirmed the activation of T cells by EDA-E7 matured DCs. Therefore, therapeutic vaccination with EDA-E7 fusion protein maybe effective for human cervical carcinoma treatment.

Targeting the extra domain A of fibronectin for cancer therapy with CAR-T cells

BackgroundOne of the main difficulties of adoptive cell therapies with chimeric antigen receptor (CAR)-T cells in solid tumors is the identification of specific target antigens. The tumor microenvironment can present...

DP1 (Prostaglandin D2 Receptor 1) Activation Protects Against Vascular Remodeling and Vascular Smooth Muscle Cell Transition to Myofibroblasts in Angiotensin II-Induced Hypertension in Mice.

Vascular smooth muscle cell (VSMC) phenotype transition plays an essential role in vascular remodeling. PGD2 (Prostaglandin D2) is involved in cardiovascular inflammation. In this study, we aimed to investigates the role of DP1 (PGD2 receptor 1) on VSMC phenotype transition in vascular remodeling after Ang II (angiotensin II) infusion in mice. VSMC-specific DP1 knockout mice and DP1flox/flox mice were infused with Ang II for 28 days and systolic blood pressure was measured by noninvasive tail-cuff system. The arterial samples were applied to an unbiased proteome analysis. DP1f/f Myh11 (myosin heavy chain 11) CREERT2 R26mTmG/+ mice were generated for VSMC lineage tracing. Multiple genetic and pharmacological approaches were used to investigate DP1-mediated signaling in phenotypic transition of VSMCs in response to Ang II administration. DP1 knockout promoted vascular media thickness and increased systolic blood pressure after Ang II infusion by impairing Epac (exchange protein directly activated by cAMP)-1-mediated Rap-1 (Ras-related protein 1) activation. The DP1 agonist facilitated the interaction of myocardin-related transcription factor A and G-actin, which subsequently inhibited the VSMC transition to myofibroblasts through the suppression of RhoA (Ras homolog family member A)/ROCK-1 (Rho associated coiled-coil containing protein kinase 1) activity. Moreover, Epac-1 overexpression by lentivirus blocked the progression of vascular fibrosis in DP1 deficient mice in response to Ang II infusion. Our finding revealed a protective role of DP1 in VSMC switch to myofibroblasts by impairing the phosphorylation of MRTF (myocardin-related transcription factor)-A by ROCK-1 through Epac-1/Rap-1/RhoA pathway and thus inhibited the expression of collagen I, fibronectin, ED-A (extra domain A) fibronectin, and vinculin. Thus, DP1 activation has therapeutic potential for vascular fibrosis in hypertension.

Design and Characterization of Novel Antibody-Cytokine Fusion Proteins Based on Interleukin-21.

Interleukin-21 (IL21) is a pleiotropic cytokine involved in the modulation of both innate and adaptive immunity. IL21 is mainly secreted by natural killer (NK) and activated CD4+ T-cells. The biology of this cytokine can be associated to proinflammatory responses reflecting its potent stimulatory activity of NK and CD8+ T-cells. Here we describe four formats of novel IL21-based antibody–cytokine fusion proteins, targeting the extra domain A (EDA) of fibronectin and explore their potential for cancer treatment. The fusion proteins were designed, expressed, and characterized. F8 in single-chain diabody (scDb) format fused to IL21 at its C-terminus exhibited a promising profile in size exclusion chromatography (SEC) and SDS-PAGE. The lead candidate was further characterized in vitro. A cell-based activity assay on murine cytotoxic T-cells showed that human IL21, compared to murine IL21 partially cross-reacted with the murine receptor. The prototype was able to recognize EDA as demonstrated by immunofluorescence analysis on tumor sections. In an in vivo quantitative biodistribution experiment, F8(scDb)-murine IL21 did not preferentially accumulate at the site of disease after intravenous injection, suggesting that additional protein engineering would be required to improve the tumor-homing properties of IL21-based product.

Stabilin-1 mediates beneficial monocyte recruitment and tolerogenic macrophage programming during CVB3-induced viral myocarditis

Pathological innate and adaptive immune response upon viral infection may lead to cardiac injury and dysfunction. Stabilin-1 is a scavenger receptor that regulates several aspects of the innate immunity. Whether stabilin-1 affects the inflammatory response during viral myocarditis (VM) is entirely unknown. Here, we assess the role of stabilin-1 in the pathogenesis of VM and its suitability as a therapeutic target. Genetic loss of stabilin-1 increased mortality and cardiac necrosis in a mouse model of human Coxsackievirus B3 (CVB3)-induced myocarditis. Absence of stabilin-1 significantly reduced monocyte recruitment and strongly reduced the number of alternatively activated anti-inflammatory macrophages in the heart, enhancing a pro-inflammatory cardiac niche with a detrimental T lymphocyte response during VM. Yeast two-hybrid screening, confirmed by affinity chromatography, identified fibronectin as a stabilin-1 interacting partner. Absence of stabilin-1 specifically decreased monocyte adhesion on extracellular fibronectin in vitro. Loss of Type III repeats Extra Domain A (EDA) of fibronectin during VM also increased the mortality and cardiac necrosis as in stabilin-1 knockout mice, with reduced monocytic cardiac recruitment and increased T lymphocyte response.Collectively, stabilin-1 has an immune-suppressive role of limiting myocardial damage during VM, regulating anti-inflammatory monocyte-recruitment to the site of inflammation.

The RNA binding protein Quaking represses splicing of the Fibronectin EDA exon and downregulates the interferon response.

Quaking (QKI) controls RNA metabolism in many biological processes including innate immunity, where its roles remain incompletely understood. To illuminate these roles, we performed genome scale transcriptome profiling in QKI knockout cells with or without poly(I:C) transfection, a double-stranded RNA analog that mimics viral infection. Analysis of RNA-sequencing data shows that QKI knockout upregulates genes induced by interferons, suggesting that QKI is an immune suppressor. Furthermore, differential splicing analysis shows that QKI primarily controls cassette exons, and among these events, we noted that QKI silences splicing of the extra domain A (EDA) exon in fibronectin (FN1) transcripts. QKI knockout results in elevated production and secretion of FN1-EDA protein, which is a known activator of interferons. Consistent with an upregulation of the interferon response in QKI knockout cells, our results show reduced production of dengue virus-2 and Japanese encephalitis virus in these cells. In conclusion, we demonstrate that QKI downregulates the interferon system and attenuates the antiviral state.

Hyaluronan, Transforming Growth Factor β, and Extra Domain A-Fibronectin: A Fibrotic Triad.

Significance: Inflammation is a critical aspect of injury repair. Nonresolving inflammation, however, is perpetuated by the local generation of extracellular matrix-derived damage-associated molecular pattern molecules (DAMPs), such as the extra domain A (EDA) isoform of fibronectin and hyaluronic acid (HA) that promote the eventual acquisition of a fibrotic response. DAMPs contribute to the inflammatory environment by engaging Toll-like, integrin, and CD44 receptors while stimulating transforming growth factor (TGF)-β signaling to activate a fibroinflammatory genomic program leading to the development of chronic disease. Recent Advances: Signaling through TLR4, CD44, and the TGF-β pathways impact the amplitude and duration of the innate immune response to endogenous DAMPs synthesized in the context of tissue injury. New evidence indicates that crosstalk among these three networks regulates phase transitions as well as the repertoire of expressed genes in the wound healing program determining, thereby, repair outcomes. Clarifying the molecular mechanisms underlying pathway integration is necessary for the development of novel therapeutics to address the spectrum of fibroproliferative diseases that result from maladaptive tissue repair. Critical Issues: There is an increasing appreciation for the role of DAMPs as causative factors in human fibroinflammatory disease regardless of organ site. Defining the involved intermediates essential for the development of targeted therapies is a daunting effort, however, since various classes of DAMPs activate different direct and indirect signaling pathways. Cooperation between two matrix-derived DAMPs, HA, and the EDA isoform of fibronectin, is discussed in this review as is their synergy with the TGF-β network. This information may identify nodes of signal intersection amenable to therapeutic intervention. Future Directions: Clarifying mechanisms underlying the DAMP/growth factor signaling nexus may provide opportunities to engineer the fibroinflammatory response to injury and, thereby, wound healing outcomes. The identification of shared and unique DAMP/growth factor-activated pathways is critical to the design of optimized tissue repair therapies while preserving the host response to bacterial pathogens.

Extra Domain A Containing Fibronectin Levels in The Cerebrospinal Fluid in Patients with Meningitis

Background Meningitis is a life-threatening disease because of the inflammation's proximity to the brain and spinal cord. Untreated bacterial meningitis is almost always fatal; therefore it is important to differentiate bacterial meningitis from aseptic meningitis early. Objectives: To evaluate the role of Extradomain A (EDA) Fibronectien in diagnosis of meningitis and differentiation between bacterial and aseptic causes. Methods: This study was conducted on total number of 150 patient aged (18-60years old) suspected to have meningitis, 100 patients were excluded due to trauma, cerebrovascular stroke and CNS tumors. Patients were classified according to the CSF findings into 20 patients as bacterial meningitis group (group1) and 20 patients as aseptic meningitis group (group II). In addition to 10 cases of matched age and sex, free from any CNS diseases serving as control group (group III). All subjects were subjected to full history taking, clinical examination, laboratory investigations, and measurement of plasma and CSF EDA- FN levels using ELISA method. Results: CSF EDA- FN level at a cut off value less than 14.2mg/l have a sensitivity of (90%) and specificity of (85%) to differentiate between bacterial and aseptic meningitis (P-Value <0.001), while plasma EDA- FN level at a cut off value more than 2.45mg/l have a sensitivity of (75%) and specificity of (50%) to differentiate between bacterial and aseptic meningitis (p- value <0.05). Conclusion: CSF and plasma EDA-Fibronectin level can be used as a diagnostic marker in patients with meningitis. It can also differentiate between bacterial and aseptic meningitis.

Extra domain A-containing fibronectin expression in Spin90-deficient fibroblasts mediates cancer-stroma interaction and promotes breast cancer progression.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment play major roles in supporting cancer progression. A previous report showed that SPIN90 downregulation is correlated with CAF activation and that SPIN90-deficient CAFs promote breast cancer progression. However, the mechanisms that mediate cancer-stroma interaction and how such interactions regulate cancer progression are not well understood. Here, we show that extra domain A (EDA)-containing fibronectin (FN), FN(+)EDA, produced by mouse embryonic fibroblasts (MEFs) derived from Spin90-knockout (KO) mice increases their own myofibroblast differentiation, which facilitates breast cancer progression. Increased FN(+)EDA in Spin90-KO MEFs promoted fibril formation in the extracellular matrix (ECM) and specifically interacted with integrin α4β1 as the mediating receptor. Moreover, FN(+)EDA expression by Spin90-KO MEFs increased proliferation, migration, and invasion of breast cancer cells. Irigenin, a specific inhibitor of the interaction between integrin α4β1 and FN(+)EDA, significantly blocked the effects of FN(+)EDA, such as fibril formation by Spin90-KO MEFs and proliferation, migration, and invasion of breast cancer cells. In orthotopic breast cancer mouse models, irigenin injection remarkably reduced tumor growth and lung metastases. It was supported by that FN(+)EDA in assembled fibrils was accumulated in cancer stroma of human breast cancer patients in which SPIN90 expression was downregulated. Our data suggest that SPIN90 downregulation increases FN(+)EDA and promotes ECM stiffening in breast cancer stroma through an assembly of long FN(+)EDA-rich fibrils; moreover, engagement of the Integrin α4β1 receptor facilitates breast cancer progression. Inhibitory effects of irigenin on tumor growth and metastasis suggest the potential of this agent as an anticancer therapeutic.

Antibody-Mediated Delivery of VEGFC Ameliorates Experimental Chronic Colitis.

Crohn's disease (CD) and ulcerative colitis (UC) are two distinct forms of inflammatory bowel disease (IBD) characterized by an expanded lymphatic network with impaired functionality both in mouse models and in human patients. In this study, we investigated whether targeted delivery of the pro-lymphangiogenic vascular endothelial growth factor C (VEGFC) to the site of inflammation may represent a new, clinically feasible strategy for treating IBD. To achieve targeting of inflamed tissue, we developed a fusion protein consisting of human VEGFC fused to the F8 antibody (F8-VEGFC), which specifically binds to the extradomain A (EDA) of fibronectin, a spliced isoform almost exclusively expressed in inflamed tissues. The therapeutic activity of intravenously administered F8-VEGFC, compared to a targeted construct lacking VEGFC (F8-SIP), was investigated in a mouse model of dextran sodium sulfate (DSS)-induced colitis. The presence of EDA fibronectin was detected in both human and mouse inflamed colon tissue. Biodistribution studies of radiolabeled F8-VEGFC revealed a specific accumulation of the antibody in the colon of DSS-administered mice, as compared to an untargeted VEGFC fusion protein (KSF-VEGFC) (binding the irrelevant hen egg lysozyme antigen). Systemic treatment with F8-VEGFC significantly reduced the clinical and histological signs of inflammation, expanded the lymphatic vascular network, reduced the density of immune cells, and also decreased the expression of inflammatory cytokines in the inflamed colon. Overall, these results reveal that administration of F8-VEGFC represents a novel and promising approach for the treatment of IBD.

Design and characterisation of a novel interleukin-15 receptor alpha fusion protein and analysis of interleukin-15 complexation.

Interleukin-15 (IL15) is one of the most important cytokines currently being considered for cancer therapy applications. It is thought that by administering IL15 in complex with its cognate receptor alpha chain (IL15Rα) its biological activity could be increased manifold. We produced a fusion protein of mouse IL15Rα and the F8 antibody, that targets the alternatively-spliced extra-domain A (EDA) of fibronectin, which is overexpressed in many types of cancer. The fusion protein F8IL15Rα was cloned, expressed and characterized in vitro and its ability to bind to mouse IL15 was assessed with both size exclusion chromatography (SEC) and surface plasmon resonance (SPR) experiments. Furthermore, mouse and human IL15 and their corresponding Fc fused IL15Rα subunits were purchased, characterized and used to compare the capacity of F8IL15Rα to generate complexes. Surprisingly, none of the IL15Rα fusion proteins showed IL15 complexation on SEC. However, on SPR, F8IL15Rα displayed the ability to bind IL15. In a cell-based activity assay none of the IL15Rα fusions were able to increase cellular proliferation in combination with IL15 compared to IL15 alone. A better understanding of the molecular requirements for effective IL15 signalling are likely to be important for the development of IL15-based biopharmaceuticals.

Zinc oxide nanoparticles ameliorate collagen lattice contraction in human tenon fibroblasts

Glaucoma is a major cause of irreversible blindness in the world and filtering surgery is commonly carried out to control intraocular pressure. Failure of filtering surgery is usually due to postoperative scarring, and fibroblast proliferation, collagen production and subconjunctival fibrosis play a prominent role in obstructing aqueous humor from the anterior chamber to the subconjunctival space. Zinc oxide (ZnO) nanoparticles have been widely applied in biomedical fields. However, the influence of ZnO nanoparticles on human tenon fibroblasts (HTFs) is still unclear. In the present study, we first explored the effects of various concentrations of ZnO nanoparticles on HTFs proliferation, reactive oxygen species (ROS) generation, cell cycle arrest, and apoptosis. Further, we determined the changes of transforming growth factor-β (TGF-β1), fibronectin (FN) extra domain A (ED-A), and procollagen I carboxyterminal propeptide (PICP) at mRNA and protein levels, explored the effect of ZnO nanoparticles on the collagen lattice contraction in HTFs. The results indicated that ZnO nanoparticles can efficiently inhibit HTFs proliferation, elevate ROS production level, and induce cell cycle arrest at G2/M phase, leading to HTFs apoptosis. ZnO nanoparticles can also decrease the expressions of TGF-β1, ED-A, and PICP at mRNA and protein levels; significantly prevent fibroblast-mediated collagen lattice contraction. Taken together, ZnO nanoparticles can efficiently ameliorate collagen lattice contraction in HTFs, and may be a promising antifibrotic agent in glaucoma filtration surgery. Our findings provide a new insight on anti-scar formation after glaucoma filtration surgery by using ZnO nanoparticles.

Antibody-mediated delivery of VEGF-C potently reduces chronic skin inflammation.

VEGF-C is an important mediator of lymphangiogenesis and has been shown to alleviate chronic inflammation in a variety of disease models. In this study, we investigated whether targeted delivery of VEGF-C to sites of inflammation and site-specific activation of lymphatic vessels would represent a clinically feasible strategy for treating chronic skin inflammation. To this end, we generated a fusion protein consisting of human VEGF-C fused to the F8 antibody (F8-VEGF-C), which is specific for the alternatively spliced, angiogenesis-marking extradomain A (EDA) of fibronectin. In two mouse models of psoriasis-like skin inflammation, mediated by transgenic VEGF-A overexpression or repeated application of imiquimod, intravenous treatment with F8-VEGF-C but not with untargeted VEGF-C significantly reduced ear skin edema and was as effective as the clinically used TNF-α receptor-Fc fusion protein (TNFR-Fc). Treatment with F8-VEGF-C led to a marked expansion of lymphatic vessels in the inflamed skin and significantly improved lymphatic drainage function. At the same time, treatment with F8-VEGF-C significantly reduced leukocyte numbers, including CD4+ and γδ T cells. In sum, our results reveal that targeted delivery of VEGF-C and site-specific induction of lymphatic vessels represent a potentially new and promising approach for the treatment of chronic inflammatory diseases.

A7793 The role of TGF-β1/smad3 pathway on phenotype transformation and collagen synthesis of rat cardiac fibroblasts induced by high sodium

Objectives: In order to research the effects of TGF-β1/smad3 signaling pathway on the phenotype transformation and collagen synthesis of rat cardiac fibroblasts (CFs) induced by high sodium in vitro. Methods: The CCK8 assays were used to determine optimal concentration and action time of smad3 inhibitor (SIS3) and TGF-β1 receptor inhibitor (SB431542) on CFs from Wistar rats. CFs were divided into control group (Na+139 mmol/L), high-sodium group (Na+149 mmol/L- Na+176 mmol/L), high-sodium+SIS3 group (Na+161 mmol/L+SIS3 0.5 μM- 5 μM), high-sodium+SB group (Na+161 mmol/L+SB431542 1 μM- 25 μM), mannitol group (osmotic pressure corresponding to the Na+ 161 mmol/L group), and cultured for 48 h. The mRNA and protein levels of α-SMA, fibronectin extra domain-A (Fn ED-A), Collagen I, Collagen III, TGF-β1, smad3 were detected by real time-PCR, Western blot and immunofluorescence. The content of TGF-β1 in culture supernatant was determined by ELISA. The CFs migration was observed through the cell scratch test. Results: High sodium promoted the proliferation and migration of rat CFs under the exclusion of osmotic pressure. The proliferation of CFs was inhibited in a dose-dependent manner by SIS3 and SB431542. The mRNA and protein levels of α-SMA, Fn ED-A, Collagen I, Collagen III, TGF-β1, p-smad3 were increased in high-sodium group and excluded the influence of osmotic pressure. The secretion of TGF-β1 in culture medium was elevated as well. Meanwhile, the protein levels of α-SMA, Fn ED-A, Collagen I, Collagen III were downregulated by SB431542 and SIS3. Conclusion: High salt can cause the proliferation and phenotype transformation of CFs toward to myofibroblasts and promote collagen synthesis via TGF-β1/ smad3 pathway. TGF-β1/ smad3 may be the key intervention targets for preventing high-salt induced myocardial fibrosis.