ABSTRACT
Cardiovascular disease remains a global health concern. Stem cell therapy utilizing human cardiac progenitor cells (hCPCs) shows promise in treating cardiac vascular disease. However, limited availability and senescence of hCPCs hinder their widespread use. To address these challenges, researchers are exploring innovative approaches. In this study, a bioengineered cell culture plate was developed to mimic the natural cardiac tissue microenvironment. It was coated with a combination of extracellular matrix (ECM) peptide motifs and mussel adhesive protein (MAP). The selected ECM peptide motifs, derived from fibronectin and vitronectin, play crucial roles in hCPCs. Results revealed that the Fibro-P and Vitro-P coated plates significantly improved hCPC adhesion, proliferation, migration, and differentiation compared to uncoated plates. Additionally, long-term culture on the coated plates delayed cellular senescence and maintained hCPC stemness. These enhancements were attributed to the activation of integrin downstream signaling pathways. The findings suggest that the engineered ECM peptide motif-MAP-coated plates hold potential for enhancing the therapeutic efficacy of stem cell-based therapies in cardiac tissue engineering and regenerative medicine.
Subject(s)
Fibronectins , Stem Cells , Vitronectin , Vitronectin/metabolism , Humans , Fibronectins/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Cell Differentiation , Cell Culture Techniques/methods , Cells, Cultured , Cell Proliferation , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Cell Adhesion , PeptidesABSTRACT
Cellular senescence plays a role in the development of aging-associated degenerative diseases. Cell therapy is recognized as a candidate treatment for degenerative diseases. To achieve the goal of cell therapy, the quality and good characteristics of cells are concerned. Cell expansion relies on two-dimensional culture, which leads to replicative senescence of expanded cells. This study aimed to investigate the effect of cell culture surface modification using fibronectin (FN) and vitronectin (VN) in adipose-derived stem cells (ADSCs) during long-term expansion. Our results showed that ADSCs cultured in FN and VN coatings significantly enhanced adhesion, proliferation, and slow progression of cellular senescence as indicated by lower SA-ß-gal activities and decreased expression levels of genes including p16, p21, and p53. The upregulation of integrin α5 and αv genes influences phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K), and AKT proteins. FN and VN coatings upregulated AKT and MDM2 leading to p53 degradation. Additionally, MDM2 inhibition by Nutlin-3a markedly elevated p53 and p21 expression, increased cellular senescence, and induced the expression of inflammatory molecules including HMGB1 and IL-6. The understanding of FN and VN coating surface influencing ADSCs, especially senescence characteristics, offers a promising and practical point for the cultivation of ADSCs for future use in cell-based therapies.
Subject(s)
Cellular Senescence , Fibronectins , Proto-Oncogene Proteins c-akt , Proto-Oncogene Proteins c-mdm2 , Signal Transduction , Tumor Suppressor Protein p53 , Vitronectin , Vitronectin/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Tumor Suppressor Protein p53/metabolism , Fibronectins/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Humans , Cells, Cultured , Stem Cells/metabolism , Stem Cells/cytology , Cell Proliferation , Adipose Tissue/cytology , Adipose Tissue/metabolism , Cell Culture Techniques/methodsABSTRACT
Chronic renal failure (CRF) causes a reduction in glomerular filtration rate and damage to renal parenchyma. Fushengong decoction (FSGD) showed improvement in renal function in CRF rats. This study aims to analyze the differentially expressed proteins in CRF patients treated with Western medicine alone or in combination with FSGD. Sixty patients with CRF recruited from Yongchuan Traditional Chinese Medicine Hospital affiliated to Chongqing Medical University were randomly assigned into control (treated with Western medicine alone) and observation groups (received additional FSGD treatment thrice daily for 8 weeks). The clinical efficacy and changes in serum Bun, serum creatinine, Cystatin C, and transforming growth factor beta 1 (TGF-ß1) before and after treatment were observed. We employed isotope relative labeling absolute quantification labeling and liquid chromatography-mass spectrometry to identify differentially expressed proteins and carried out bioinformatics Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. Patients in the observation group showed greater clinical improvement and lower levels of serum Bun, serum creatinine, Cyc-c, and TGF-ß1 than the control group. We identified 32 differentially up-regulated and 52 down-regulated proteins in the observation group. These proteins are involved in the blood coagulation system, protein serine/threonine kinase activity, and TGF-ß, which are closely related to the pathogenesis of CRF. Protein-protein-interaction network analysis indicated that candidate proteins fibronectin 1, fibrinogen alpha chain, vitronectin, and Serpin Family C Member 1 were in the key nodes. This study provided an experimental basis suggesting that FSGD combined with Western medicine could significantly improve renal function and renal fibrosis of CRF patients, which may be through the regulation of fibronectin 1, fibrinogen alpha chain, vitronectin, Serpin Family C Member 1, TGF-ß, and the complement coagulation pathway (see Graphical abstract S1, Supplemental Digital Content, http://links.lww.com/MD/L947).
Subject(s)
Kidney Failure, Chronic , Renal Insufficiency, Chronic , Serpins , Animals , Humans , Rats , Creatinine , Extracellular Matrix Proteins , Fibrinogen , Fibronectins , Kidney Failure, Chronic/drug therapy , Renal Insufficiency, Chronic/drug therapy , Transforming Growth Factor beta , Transforming Growth Factor beta1 , VitronectinABSTRACT
Yersinia pestis, the causative agent of plague, is capable of evading the human immune system response by recruiting the plasma circulating vitronectin proteins, which act as a shield and avoid its lysis. Vitronectin recruitment is mediated by its interaction with the bacterial transmembrane protein Ail, protruding from the Y. pestis outer membrane. By using all-atom long-scale molecular dynamic simulations of Ail embedded in a realistic model of the bacterial membrane, we have shown that vitronectin forms a stable complex, mediated by interactions between the disordered moieties of the two proteins. The main amino acids driving the complexation have also been evidenced, thus favoring the possible rational design of specific peptides which, by inhibiting vitronectin recruitment, could act as original antibacterial agents.
Subject(s)
Bacterial Outer Membrane Proteins , Molecular Dynamics Simulation , Vitronectin , Vitronectin/chemistry , Vitronectin/metabolism , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/metabolism , Humans , Yersinia pestis/chemistry , Yersinia pestis/metabolism , Virulence Factors/chemistry , Virulence Factors/metabolism , Protein Domains , Protein BindingABSTRACT
Candida albicans and other closely related pathogenic yeast-like fungi carry on their surface numerous loosely adsorbed "moonlighting proteins"-proteins that play evolutionarily conserved intracellular functions but also appear on the cell surface and exhibit additional functions, e.g., contributing to attachment to host tissues. In the current work, we characterized this "moonlighting" role for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) of C. albicans and Nakaseomyces glabratus. GAPDH was directly visualized on the cell surface of both species and shown to play a significant part in the total capacity of fungal cells to bind two selected human host proteins-vitronectin and plasminogen. Using purified proteins, both host proteins were found to tightly interact with GAPDH, with dissociation constants in an order of 10-8 M, as determined by bio-layer interferometry and surface plasmon resonance measurements. It was also shown that exogenous GAPDH tightly adheres to the surface of candidal cells, suggesting that the cell surface location of this moonlighting protein may partly result from the readsorption of its soluble form, which may be present at an infection site (e.g., due to release from dying fungal cells). The major dedicated adhesins, covalently bound to the cell wall-agglutinin-like sequence protein 3 (Als3) and epithelial adhesin 6 (Epa6)-were suggested to serve as the docking platforms for GAPDH in C. albicans and N. glabratus, respectively.
Subject(s)
Candida albicans , Fungal Proteins , Glyceraldehyde-3-Phosphate Dehydrogenases , Humans , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Plasminogen/metabolism , Vitronectin/metabolism , Fungal Proteins/metabolismABSTRACT
The low sensitivity of nuclear magnetic resonance (NMR) is a major bottleneck for studying biomolecular structures of complex biomolecular assemblies. Cryogenically cooled probe technology overcomes the sensitivity limitations enabling NMR applications to challenging biomolecular systems. Here we describe solid-state NMR studies of the human blood protein vitronectin (Vn) bound to hydroxyapatite (HAP), the mineralized form of calcium phosphate, using a CryoProbe designed for magic angle spinning (MAS) experiments. Vn is a major blood protein that regulates many different physiological and pathological processes. The high sensitivity of the CryoProbe enabled us to acquire three-dimensional solid-state NMR spectra for sequential assignment and characterization of site-specific water-protein interactions that provide initial insights into the organization of the Vn-HAP complex. Vn associates with HAP in various pathological settings, including macular degeneration eyes and Alzheimer's disease brains. The ability to probe these assemblies at atomic detail paves the way for understanding their formation.
Subject(s)
Durapatite , Vitronectin , Humans , Magnetic Resonance Spectroscopy/methods , Magnetic Resonance Imaging , Nuclear Magnetic Resonance, Biomolecular/methodsABSTRACT
PURPOSE: The purpose of this study was to extensively evaluate the efficacy of integrin αvß3 antagonists for the treatment of experimental dry eye (EDE). METHODS: Vitronectin, an αvß3 ligand, was used to induce tumor necrosis factor-α gene expression in human THP-1 macrophages. To induce EDE, C57BL/6 mice were housed in a low-humidity controlled environment chamber and injected subcutaneously with scopolamine for 7 days. Subsequently, αvß3 antagonists, including RGDfD, c(RGDfD), c(RGDiD), c(RGDfK), ATN-161, SB273005, and cilengitide, were administered topically to EDE animals under controlled environment chamber conditions. Corneal epithelial damage in EDE was assessed by fluorescein staining. The density of conjunctival goblet cells and secretion of tears was measured by period acid-Schiff staining and phenol red-impregnated cotton threads, respectively. Inflammation markers, including tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, IL-17A, and metalloproteinase (MMP)-9, in the pooled cornea and conjunctiva tissues were examined by real-time polymerase chain reaction. RESULTS: The inhibitory effects of αvß3 antagonists on the vitronectin-induced tumor necrosis factor-α gene expression and integrin-mediated inflammatory signaling were validated in THP-1 macrophages. αvß3 antagonists ameliorated the impairment of the corneal epithelial barrier with varying therapeutic efficacies, compared with vehicle-treated mice. c(RGDfD) and c(RGDiD) significantly protected against goblet cell loss, tear reduction, and proinflammatory gene expression in EDE. CONCLUSIONS: Topical applications of αvß3 antagonists yield therapeutic benefits in EDE by promoting corneal epithelial defect healing and reducing inflammation. Antagonistic targeting αvß3 may be a novel promising strategy to treat patients with dry eye disease.
Subject(s)
Dry Eye Syndromes , Integrin alphaVbeta3 , Humans , Animals , Mice , Integrin alphaVbeta3/metabolism , Integrin alphaVbeta3/therapeutic use , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Vitronectin/metabolism , Vitronectin/pharmacology , Vitronectin/therapeutic use , Mice, Inbred C57BL , Dry Eye Syndromes/metabolism , Tears/metabolism , Conjunctiva/pathology , Cornea/pathology , Inflammation/metabolism , Disease Models, AnimalABSTRACT
PURPOSE: Retinal drusen have been described in people with IgA nephropathy. We examined the frequency of drusen in IgA nephropathy and compared their location and composition with those for drusen in age-related macular degeneration. DESIGN: Immunohistological case series of eyes of patients with IgA nephropathy, and a comparison eye with age-related macular degeneration. METHODS: Donor eyes from 4 individuals (3 male, 1 female, aged 40-80 years) with biopsy-proven IgA nephropathy and kidney failure were examined for the presence of drusen, and location and composition using antibodies for vitronectin, IgA, IgM, IgG, C3, and C1q. Results were compared with those for drusen in macular degeneration without IgA nephropathy. RESULTS: All 4 donors had sparse, subretinal pigment epithelium drusen of 55-65 mm diameter that stained for vitronectin but not for IgA or complement. All donors had retinal capillaries and choriocapillaris staining for IgA. The youngest donor (female, 40) had rare deposits in the outer nuclear layer that stained for IgA, but not for vitronectin. The oldest donor (male, 82) had large cystlike spaces in the inner nuclear and plexiform layers, and smaller cysts in the outer nuclear layer, with no staining for IgA or complement. CONCLUSIONS: Retinal drusen are uncommon in IgA nephropathy, even with kidney failure. Drusen in IgA nephropathy resemble drusen found in age-related macular degeneration. IgA-staining deposits in the outer nuclear layer were likely due to systemic deposition of IgA and complement activation. The nature of cystic spaces is unknown. Further analysis of the retinas of people with glomerulonephritis is recommended.
Subject(s)
Glomerulonephritis, IGA , Macular Degeneration , Renal Insufficiency , Retinal Drusen , Humans , Male , Female , Retinal Drusen/diagnosis , Retinal Drusen/pathology , Glomerulonephritis, IGA/complications , Glomerulonephritis, IGA/diagnosis , Vitronectin , Macular Degeneration/pathology , Immunoglobulin AABSTRACT
Human embryonic stem cells (hESCs) have unique abilities that enable their use in cell therapy, disease modeling, and drug development. Their derivation is usually performed using a feeder layer, which is undefined and can potentially cause a contamination by xeno components, therefore there is a tendency to replace feeders with xeno-free defined substrates in recent years. Three hESC lines were successfully derived on the vitronectin with a truncated N-terminus (VTN-N) in combination with E-cadherin in xeno-free conditions for the first time, and their undifferentiated state, hESC morphology, and standard karyotypes together with their potential to differentiate into three germ layers were confirmed. These results support the conclusion that the VTN-N/E-cadherin is a suitable substrate for the xeno-free derivation of hESCs and can be used for the derivation of hESCs according to good manufacturing practices.
Subject(s)
Human Embryonic Stem Cells , Humans , Vitronectin , Cadherins/genetics , Cell- and Tissue-Based Therapy , CommerceABSTRACT
OBJECTIVE: The aim of this study was to analyze the second-trimester levels of vitronectin and plasminogen activator inhibitor-1 in gestational diabetes mellitus. METHODS: This study was conducted between September 2020 and December 2020 at the University of Health Sciences, Bursa Yuksek Ihtisas Research and Training Hospital, Department of Obstetrics and Gynecology. A total of 30 pregnant women with gestational diabetes mellitus and 60 healthy controls between 24 and 27/6 weeks of gestation were included. The inclusion criteria were as follows: being between 18 and 45 years old and 24-27/6 gestational weeks, having singleton pregnancy, diagnosed with gestational diabetes mellitus by using a two-step challenge test. The exclusion criteria of this study were as follows: chronic inflammatory or infectious disease, fasting blood glucose>126 mg/dL, intolerance to glucose tolerance testing, abnormal liver or kidney function tests, as well as pregnancy with pre-gestational diabetes history of adverse perinatal outcomes. Serum vitronectin and plasminogen activator inhibitor-1 levels were measured using the enzyme-linked immunosorbent assay method. RESULTS: Vitronectin and plasminogen activator inhibitor-1 levels were higher in the gestational diabetes mellitus group compared with controls [91.85 (23.08) vs. 80.10 (39.18) ng/mL, for vitronectin and 6.50 (1.05) vs. 4.35(1.0) ng/mL, for plasminogen activator inhibitor-1 (for both p<0.001)]. vitronectin >84.7 ng/mL was found to predict gestational diabetes mellitus with a sensitivity of 70% and specificity of 63.3%. Moreover, vitronectin had a significant positive correlation with fasting blood glucose (r=0.476, p<0.001), postprandial blood glucose (r=0.489, p<0.001), HbA1c (r=0.713, p<0.001), and plasminogen activator inhibitor-1 (r=0.586, p<0.001). CONCLUSION: This study revealed that second-trimester vitronectin and plasminogen activator inhibitor-1 are increased in gestational diabetes mellitus and vitronectin could be a candidate for the prediction of gestational diabetes mellitus.
Subject(s)
Diabetes, Gestational , Pregnancy , Humans , Female , Adolescent , Young Adult , Adult , Middle Aged , Vitronectin , Blood Glucose , Enzyme-Linked Immunosorbent Assay , Exercise TestABSTRACT
For precision in clinical oncology practice, detection of tumor-derived peptides and proteins in urine offers an attractive and noninvasive alternative for diagnostic or screening purposes. In this study, we report comparative quantitative proteomic profiling of urine samples from patients with gastric cancer and healthy controls using tandem mass tags-based multiplexed mass spectrometry approach. We identified 1504 proteins, of which 246 were differentially expressed in gastric cancer cases. Notably, ephrin A1 (EFNA1), pepsinogen A3 (PGA3), sortilin 1 (SORT1), and vitronectin (VTN) were among the upregulated proteins, which are known to play crucial roles in the progression of gastric cancer. We also found other overexpressed proteins, including shisa family member 5 (SHISA5), mucin like 1 (MUCL1), and leukocyte cell derived chemotaxin 2 (LECT2), which had not previously been linked to gastric cancer. Using a novel approach for targeted proteomics, SureQuant, we validated changes in abundance of a subset of proteins discovered in this study. We confirmed the overexpression of vitronectin and sortilin 1 in an independent set of urine samples. Altogether, this study provides molecular candidates for biomarker development in gastric cancer, and the findings also support the promise of urinary proteomics for noninvasive diagnostics and personalized/precision medicine in the oncology clinic.
Subject(s)
Biomarkers, Tumor , Stomach Neoplasms , Humans , Biomarkers, Tumor/metabolism , Stomach Neoplasms/diagnosis , Proteomics/methods , Vitronectin , Proteins , Medical Oncology , Biomarkers , Mucins , Intercellular Signaling Peptides and ProteinsABSTRACT
Delivery of growth factors (GFs) is challenging for regulation of cell proliferation and differentiation due to their rapid inactivation under physiological conditions. Here, a bioactive polyelectrolyte multilayer (PEM) is engineered by the combination of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and glycosaminoglycans to be used as reservoir for GF storage. PNIPAM-grafted-chitosan (PChi) with two degrees of substitution (DS) are synthesized, namely LMW* (DS 0.14) and HMW (DS 0.03), by grafting low (2 kDa) and high (10 kDa) molecular weight of PNIPAM on the backbone of chitosan (Chi) to be employed as polycations to form PEM with the polyanion heparin (Hep) at pH 4. Subsequently, PEMs are chemically crosslinked to improve their stability at physiological pH 7.4. Resulting surface and mechanical properties indicate that PEM containing HMW is responsive to temperature at 20 °C and 37 °C, while LMW is not. More importantly, Hep as terminal layer combined with HMW allows not only a better retention of the adhesive protein vitronectin but also a sustained release of FGF-2 at 37 °C. With the synergistic effect of vitronectin and matrix-bound FGF-2, significant promotion on adhesion, proliferation, and migration of 3T3 mouse embryonic fibroblasts is achieved on HMW-containing PEM compared to Chi-containing PEM and exogenously added FGF-2. Thus, PEM containing PNIPAM in combination with bioactive glycosaminoglycans like Hep represents a versatile approach to fabricate a GF delivery system for efficient cell culture, which can be potentially served as cell culture substrate for production of (stem) cells and bioactive wound dressing for tissue regeneration.
Subject(s)
Chitosan , Heparin , Animals , Mice , Heparin/pharmacology , Heparin/chemistry , Chitosan/chemistry , Chitosan/pharmacology , Fibroblast Growth Factor 2/pharmacology , Vitronectin/pharmacology , Cell Adhesion , Fibroblasts , Glycosaminoglycans/chemistry , Glycosaminoglycans/pharmacologyABSTRACT
Digital pathology and artificial intelligence are promising emerging tools in precision oncology as they provide more robust and reproducible analysis of histologic, morphologic and topologic characteristics of tumor cells and the surrounding microenvironment. This study aims to develop digital image analysis workflows for therapeutic assessment in preclinical in vivo models. For this purpose, we generated pipelines that enable automatic detection and quantification of vitronectin and αvß3 in heterotopic high-risk neuroblastoma xenografts, demonstrating that digital analysis workflows can be used to provide robust detection of vitronectin secretion and αvß3 expression by malignant neuroblasts and to evaluate the possibility of combining traditional chemotherapy (etoposide) with extracellular matrix-targeted therapies (cilengitide). Digital image analysis added evidence for the relevance of territorial vitronectin as a therapeutic target in neuroblastoma, since its expression is modified after treatment, with a mean percentage of 60.44% in combined therapy tumors vs 45.08% in control ones. In addition, the present study revealed the efficacy of cilengitide for reducing αvß3 expression, with a mean αvß3 positivity of 34.17% in cilengitide treated material vs 66.14% in control and with less tumor growth when combined with etoposide, with a final mean volume of 0.04 cm3 in combined therapy vs 1.45 cm3 in control. The results of this work highlight the importance of extracellular matrix-focused therapies in preclinical studies to improve therapeutic assessment for high-risk neuroblastoma patients.
Subject(s)
Neuroblastoma , Tumor Microenvironment , Humans , Etoposide/pharmacology , Etoposide/therapeutic use , Artificial Intelligence , Vitronectin , Workflow , Precision Medicine , Neuroblastoma/drug therapyABSTRACT
Objective: Vitronectin (VTN) has been reported to trigger cell pyroptosis to aggravate inflammation in our previous study. However, the function of VTN in inflammatory bowel disease (IBD) remains to be addressed. Methods: Real-time PCR and western blotting were performed to analyze VTN-regulated intestinal epithelial cell (IEC) differentiation through ferroptosis, and immunofluorescence (IF), luciferase, and chromatin immunoprecipitation were used to identify whether VTN-modulated ferroptosis is dependent on phosphodiesterase 4 (PDE4)/protein kinase A (PKA)/cyclic adenosine monophosphate-response element-binding protein (CREB) cascade pathway. In vivo experiment in mice and a pilot study in patients with IBD were used to confirm inhibition of PDE4-alleviated IECs ferroptosis, leading to cell differentiation during mucosal healing. Results: Herein, we found that caudal-related homeobox transcription factor 2-mediated IECs differentiation was impaired in response to VTN, which was attributed to enhanced ferroptosis characterized by decreased glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 expression. Inhibition of ferroptosis in IECs rescued the inhibitory effect of VTN on cell differentiation. Further analysis showed that VTN triggered phosphorylation of PDE4, leading to inhibit PKA/CREB activation and CREB nuclear translocation, which further reduced GPX4 transactivation. Endogenous PKA interacted with CREB, and this interaction was destroyed in response to VTN stimulation. What is more, overexpression of CREB in CaCO2 cells overcame the promotion of VTN on ferroptosis. Most importantly, inhibition of PDE4 by roflumilast or dipyridamole could alleviate dextran sulfate sodium-induced colitis in mice and in a pilot clinical study confirmed by IF. Conclusions: These findings demonstrated that highly expressed VTN disrupted IECs differentiation through PDE4-mediated ferroptosis in IBD, suggesting targeting PDE4 could be a promising therapeutic strategy for patients with IBD.
Subject(s)
Ferroptosis , Inflammatory Bowel Diseases , Mice , Animals , Vitronectin , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , Pilot Projects , Inflammatory Bowel Diseases/metabolism , Cell DifferentiationABSTRACT
Background: Renal infiltration of inflammatory cells including macrophages is a crucial event in kidney fibrogenesis. However, how macrophage regulates fibroblast activation in the fibrotic kidney remains elusive. In this study, we show that macrophages promoted fibroblast activation by assembling a vitronectin (Vtn)-enriched, extracellular microenvironment. Methods: We prepared decellularized kidney tissue scaffold (KTS) from normal and fibrotic kidney after unilateral ischemia-reperfusion injury (UIRI) and carried out an unbiased quantitative proteomics analysis. NRK-49F cells were seeded on macrophage-derived extracellular matrix (ECM) scaffold. Genetic Vtn knockout (Vtn-/-) mice and chronic kidney disease (CKD) model with overexpression of Vtn were used to corroborate a role of Vtn/integrin αvß5/Src in kidney fibrosis. Results: Vtn was identified as one of the most upregulated proteins in the decellularized kidney tissue scaffold from fibrotic kidney by mass spectrometry. Furthermore, Vtn was upregulated in the kidney of mouse models of CKD and primarily expressed and secreted by activated macrophages. Urinary Vtn levels were elevated in CKD patients and inversely correlated with kidney function. Genetic ablation or knockdown of Vtn protected mice from developing kidney fibrosis after injury. Conversely, overexpression of Vtn exacerbated renal fibrotic lesions and aggravated renal insufficiency. We found that macrophage-derived, Vtn-enriched extracellular matrix scaffold promoted fibroblast activation and proliferation. In vitro, Vtn triggered fibroblast activation by stimulating integrin αvß5 and Src kinase signaling. Either blockade of αvß5 with neutralizing antibody or pharmacological inhibition of Src by Saracatinib abolished Vtn-induced fibroblast activation. Moreover, Saracatinib dose-dependently ameliorated Vtn-induced kidney fibrosis in vivo. These results demonstrate that macrophage induces fibroblast activation by assembling a Vtn-enriched extracellular microenvironment, which triggers integrin αvß5 and Src kinase signaling. Conclusion: Our findings uncover a novel mechanism by which macrophages contribute to kidney fibrosis via assembling a Vtn-enriched extracellular niche and suggest that disrupting fibrogenic microenvironment could be a therapeutic strategy for fibrotic CKD.
Subject(s)
Renal Insufficiency, Chronic , Vitronectin , Mice , Animals , Vitronectin/metabolism , Kidney/pathology , Renal Insufficiency, Chronic/metabolism , src-Family Kinases/metabolism , Macrophages/metabolism , Fibroblasts/metabolism , FibrosisABSTRACT
Riemerella anatipestifer is an important bacterial pathogen in poultry. Pathogenic bacteria recruit host complement factors to resist the bactericidal effect of serum complement. Vitronectin (Vn) is a complementary regulatory protein that inhibits the formation of the membrane attack complex (MAC). Microbes use outer membrane proteins (OMPs) to hijack Vn for complement evasion. However, the mechanism by which R. anatipestifer achieves evasion is unclear. This study aimed to characterise OMPs of R. anatipestifer which interact with duck Vn (dVn) during complement evasion. Far-western assays and comparison of wild-type and mutant strains that were treated with dVn and duck serum demonstrated particularly strong binding of OMP76 to dVn. These data were confirmed with Escherichia coli strains expressing and not expressing OMP76. Combining tertiary structure analysis and homology modelling, truncated and knocked-out fragments of OMP76 showed that a cluster of critical amino acids in an extracellular loop of OMP76 mediate the interaction with dVn. Moreover, binding of dVn to R. anatipestifer inhibited MAC deposition on the bacterial surface thereby enhancing survival in duck serum. Virulence of the mutant strain ΔOMP76 was attenuated significantly relative to the wild-type strain. Furthermore, adhesion and invasion abilities of ΔOMP76 decreased, and histopathological changes showed that ΔOMP76 was less virulent in ducklings. Thus, OMP76 is a key virulence factor of R. anatipestifer. The identification of OMP76-mediated evasion of complement by recruitment of dVn contributes significantly to the understanding of the molecular mechanism by which R. anatipestifer escapes host innate immunity and provides a new target for the development of subunit vaccines.
Subject(s)
Flavobacteriaceae Infections , Poultry Diseases , Animals , Virulence , Ducks , Membrane Proteins , Vitronectin , Bacterial Proteins/metabolism , Flavobacteriaceae Infections/microbiology , Flavobacteriaceae Infections/prevention & control , Immunologic Factors , Complement System Proteins , Poultry Diseases/microbiologyABSTRACT
Peptide sequence periodicity is a simple design tool that can be used to generate functional peptide-based surface coatings. De novo-designed peptide N3-PEG-VK16 is characterized by a hydrophobic periodicity of two that avidly binds to native polystyrene priming its surface for subsequent targeted functionalization via chemical ligation. The peptidic portion of N3-PEG-VK16 is responsible for surface binding, converting polystyrene's hydrophobic surface into a wettable and electrostatically charged environment that facilitates cell attachment. Native polystyrene surfaces are coated by simple peptide adsorption from an aqueous buffered solution, and the resulting primed surface is easily functionalized by cycloaddition chemistry. Herein, we show that ligating a vitronectin-derived peptide to primed polystyrene surfaces enables adhesion, expansion, long-term culture, and phenotype maintenance of human induced pluripotent stem cells. To demonstrate scope, we also show that additional functional ligands can be used, for example, nerve growth factor protein, to control neurite outgrowth.
Subject(s)
Induced Pluripotent Stem Cells , Polystyrenes , Humans , Polystyrenes/chemistry , Cell Adhesion , Peptides/pharmacology , Vitronectin/chemistry , Surface PropertiesABSTRACT
Polyetheretherketone (PEEK) is a thermoplastic polymer that has been recently employed for bone tissue engineering as a result of its biocompatibility and mechanical properties being comparable to human bone. PEEK, however, is a bio-inert material and, when implanted, does not interact with the host tissues, resulting in poor integration. In this work, the surfaces of 3D-printed PEEK disks were functionalized with: (i) an adhesive peptide reproducing [351-359] h-Vitronectin sequence (HVP) and (ii) HVP retro-inverted dimer (D2HVP), that combines the bioactivity of the native sequence (HVP) with the stability toward proteolytic degradation. Both sequences were designed to be anchored to the polymer surface through specific covalent bonds via oxime chemistry. All functionalized PEEK samples were characterized by Water Contact Angle (WCA) measurements, Atomic Force Microscopy (AFM), and X-ray Photoelectron Spectroscopy (XPS) to confirm the peptide enrichment. The biological results showed that both peptides were able to increase cell proliferation at 3 and 21 days. D2HVP functionalized PEEK resulted in an enhanced proliferation across all time points investigated with higher calcium deposition and more elongated cell morphology.
Subject(s)
Polymers , Vitronectin , Humans , Polyethylene Glycols/chemistry , Ketones/chemistry , Peptides , Surface PropertiesABSTRACT
Microvascular immunothrombotic dysregulation is a critical process in the pathogenesis of severe systemic inflammatory diseases. The mechanisms controlling immunothrombosis in inflamed microvessels, however, remain poorly understood. Here, we report that under systemic inflammatory conditions the matricellular glycoproteinvitronectin (VN) establishes an intravascular scaffold, supporting interactions of aggregating platelets with immune cells and the venular endothelium. Blockade of the VN receptor glycoprotein (GP)IIb/IIIa interfered with this multicellular interplay and effectively prevented microvascular clot formation. In line with these experimental data, particularly VN was found to be enriched in the pulmonary microvasculature of patients with non-infectious (pancreatitis-associated) or infectious (coronavirus disease 2019 (COVID-19)-associated) severe systemic inflammatory responses. Targeting the VN-GPIIb/IIIa axis hence appears as a promising, already feasible strategy to counteract microvascular immunothrombotic dysregulation in systemic inflammatory pathologies.