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Experimental and Therapeutic Medicine Jan 2022Neurogenic bladder (NGB) is an important complication of urinary tract dysfunction after spinal cord injury (SCI). However, using urodynamics and urography to guide...
Neurogenic bladder (NGB) is an important complication of urinary tract dysfunction after spinal cord injury (SCI). However, using urodynamics and urography to guide therapy remains invasive and complicated. Therefore, the present study aimed to identify potential noninvasive biomarkers from urinary exosomes that can facilitate diagnosis and guide prognosis of patients with NGB subsequent to SCI. Urinary exosomes were isolated, and their proteome profile was analyzed by mass spectrometry. Transmission electron microscopy and Nanoparticle Tracking Analysis confirmed the size and morphological characteristics of urinary exosomes. In addition, bioinformatics analysis and parallel reaction monitoring (PRM) were used to screen candidate biomarkers. The selected biomarkers were validated using western blotting and ELISA. Mass spectrometry identified 134 upregulated proteins and 99 downregulated proteins between the vesicoureteral reflux (VUR) and non-VUR groups. A total of 18 candidate proteins were selected for PRM validation, but only vitronectin (VTN) and α-1 type I collagen (COL1A1) demonstrated significant differences. In the validation experiments using western blotting and ELISA, VTN was exclusively highly expressed in VUR patients compared with non-VUR patients. However, the ELISA results of COL1A1 revealed no significant difference when a larger sample size was used. Furthermore, a receiver operating characteristic curve of ELISA-based VTN demonstrated an area under the curve of 0.795 and 80% sensitivity at a threshold set to give 82.9% specificity. Collectively, these results suggested that VTN in urinary exosomes may be used as a biomarker to predict the progression and guide the prognosis of NGB.
PubMed: 34934436
DOI: 10.3892/etm.2021.10988 -
Journal of Cerebral Blood Flow and... Oct 2022We found that blood vitronectin (VTN) leaks into the brain and exacerbates tissue loss after stroke by increasing pro-inflammatory IL-6 expression in female, but not...
We found that blood vitronectin (VTN) leaks into the brain and exacerbates tissue loss after stroke by increasing pro-inflammatory IL-6 expression in female, but not male, mice. VTN signals through integrins and downstream focal adhesion kinase (FAK). Here, a two day systemic treatment with a small molecule FAK inhibitor starting 6 h after middle cerebral artery occlusion reduced ipsilateral brain injury size by ∼40-45% at 7 and 14 d, as well as inflammation and motor dysfunction in wild-type female, but not male, mice. FAK inhibition also reduced IL-6 expression in the injured female striatum at 24 h by 62%. Inducible selective gene deletion of FAK in astrocytes also reduced acute IL-6 expression by 72% only in females, and mitigated infarct size by ∼80% and inflammation at 14 d after stroke. Lastly, VTN-/- females had better outcomes, but FAK inhibitor treatment had no additional protective or anti-inflammatory effects. Altogether, this suggests that VTN is detrimental in females primarily through FAK and that FAK inhibition provides neuroprotection (cerebroprotection) by reducing VTN-induced IL-6 expression in astrocytes. Thus, VTN signaling can be targeted to mitigate harmful inflammation with relevance to treatments for women with ischemic stroke, who often have worse outcomes than men.
Topics: Animals; Anti-Inflammatory Agents; Brain Ischemia; Female; Focal Adhesion Protein-Tyrosine Kinases; Humans; Inflammation; Integrins; Interleukin-6; Ischemic Stroke; Mice; Mice, Inbred C57BL; Neuroprotection; Stroke; Vitronectin
PubMed: 35702047
DOI: 10.1177/0271678X221107871 -
Cells Aug 2021Male human fetal germ cells (hFGCs) give rise to spermatogonial stem cells (SSCs), which are the adult precursors of the male gametes. Human SSCs are a promising...
Male human fetal germ cells (hFGCs) give rise to spermatogonial stem cells (SSCs), which are the adult precursors of the male gametes. Human SSCs are a promising (autologous) source of cells for male fertility preservation; however, in contrast to mouse SSCs, we are still unable to culture them in the long term. Here, we investigated the effect of two different culture media and four substrates (laminin, gelatin, vitronectin and matrigel) in the culture of dissociated second trimester testes, enriched for hFGCs. After 6 days in culture, we quantified the presence of POU5F1 and DDX4 expressing hFGCs. We observed a pronounced difference in hFGC number in different substrates. The combination of gelatin-coated substrate and medium containing GDNF, LIF, FGF2 and EGF resulted in the highest percentage of hFGCs (10% of the total gonadal cells) after 6 days of culture. However, the vitronectin-coated substrate resulted in a comparable percentage of hFGCs regardless of the media used (3.3% of total cells in Zhou-medium and 4.8% of total cells in Shinohara-medium). We provide evidence that not only the choices of culture medium but also choices of the adequate substrate are crucial for optimizing culture protocols for male hFGCs. Optimizing culture conditions in order to improve the expansion of hFGCs will benefit the development of gametogenesis assays in vitro.
Topics: Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Collagen; Culture Media; DEAD-box RNA Helicases; Drug Combinations; Gelatin; Gene Expression Profiling; Germ Cells; Humans; Laminin; Male; Octamer Transcription Factor-3; Proteoglycans; RNA-Seq; Single-Cell Analysis; Stem Cells; Testis; Vitronectin
PubMed: 34440801
DOI: 10.3390/cells10082033 -
Frontiers in Cell and Developmental... 2021Chemotherapy resistance to glioblastoma (GBM) remains an obstacle that is difficult to overcome, leading to poor prognosis of GBM patients. Many previous studies have...
Chemotherapy resistance to glioblastoma (GBM) remains an obstacle that is difficult to overcome, leading to poor prognosis of GBM patients. Many previous studies have focused on resistance mechanisms intrinsic to cancer cells; the microenvironment surrounding tumor cells has been found more recently to have significant impacts on the response to chemotherapeutic agents. Extracellular matrix (ECM) proteins may confer cell adhesion-mediated drug resistance (CAMDR). Here, expression of the ECM proteins laminin, vitronectin, and fibronectin was assessed in clinical GBM tumors using immunohistochemistry. Then, patient-derived GBM cells grown in monolayers on precoated laminin, vitronectin, or fibronectin substrates were treated with cilengitide, an integrin inhibitor, and/or carmustine, an alkylating chemotherapy. Cell adhesion and viability were quantified. Transcription factor (TF) activities were assessed over time using a bioluminescent assay in which GBM cells were transduced with lentiviruses containing consensus binding sites for specific TFs linked to expression a firefly luciferase reporter. Apoptosis, mediated by p53, was analyzed by Western blotting and immunocytofluorescence. Integrin α activation of the FAK/paxillin/AKT signaling pathway and effects on expression of the proliferative marker Ki67 were investigated. To assess effects of integrin α activation of AKT and ERK pathways, which are typically deregulated in GBM, and expression of epidermal growth factor receptor (EGFR), which is amplified and/or mutated in many GBM tumors, shRNA knockdown was used. Laminin, vitronectin, and fibronectin were abundant in clinical GBM tumors and promoted CAMDR in GBM cells cultured on precoated substrates. Cilengitide treatment induced cell detachment, which was most pronounced for cells cultured on vitronectin. Cilengitide treatment increased cytotoxicity of carmustine, reversing CAMDR. ECM adhesion increased activity of NFκB and decreased that of p53, leading to suppression of p53-mediated apoptosis and upregulation of multidrug resistance gene 1 (MDR1; also known as ABCB1 or P-glycoprotein). Expression of Ki67 was correlative with activation of the integrin α -mediated FAK/paxillin/AKT signaling pathway. EGFR expression increased with integrin α knockdown GBM cells and may represent a compensatory survival mechanism. These results indicate that ECM proteins confer CAMDR through integrin α in GBM cells.
PubMed: 33834020
DOI: 10.3389/fcell.2021.616580 -
Journal of Phycology Apr 2018Vitronectin (Vn) is a glycoprotein that serves a wide variety of roles in multicellular organisms. It was first identified in multicellular animals but has also been...
Vitronectin (Vn) is a glycoprotein that serves a wide variety of roles in multicellular organisms. It was first identified in multicellular animals but has also been isolated from land plants and some algae, where it appears to serve as an extracellular adhesive molecule. In order to further elucidate presence and localization of a Vn-like protein and its potential role in algae, we surveyed different morphological regions of 24 species of macro- and microalgae and three species of cyanobacteria for the presence of a Vn-like protein. Vn-like proteins were not detected in any of the species of cyanobacteria, microalgae or Rhodophyta investigated. They were detected in several species of the Phaeophyceae and Chlorophyta where their localization was limited to the holdfast and rhizoids of these organisms, respectively. Detection of a Vn-like protein (between 0.0125 and 0.097 μg · μL protein extract) was therefore limited to locations associated with substrate attachment.
Topics: Algal Proteins; Bacterial Proteins; Cyanobacteria; Microalgae; Seaweed; Vitronectin
PubMed: 29250784
DOI: 10.1111/jpy.12613 -
Applied Bionics and Biomechanics 2017Vitronectin is a matricellular protein that plays an important role in both coagulation and angiogenesis through its effects on cell adhesion and the plasminogen system....
Vitronectin is a matricellular protein that plays an important role in both coagulation and angiogenesis through its effects on cell adhesion and the plasminogen system. Vitronectin is known to bind to endothelial cells upon integrin activation. However, the effect of integrin activation by shear stress and growth factors on cell-associated vitronectin and plasminogen system activity has not yet been studied. We therefore exposed human umbilical vein endothelial cells to steady laminar flow, oscillating disturbed flow, or fibroblast growth factor-2 (FGF-2) for 24 hours. We then measured cell-associated vitronectin by Western blot and plasminogen system activity using a Chromozym assay. Steady laminar flow, oscillating disturbed flow, and FGF-2 all increased cell-associated vitronectin, although the vitronectin molecular weight varied among the different conditions. FGF-2 also increased cell-associated vitronectin in microvascular endothelial cells and vascular smooth muscle cells. The increase in cell-associated vitronectin increased plasminogen system activity. Confocal microscopy showed that vitronectin was primarily located in the basal and intracellular regions. integrin inhibition via genistein, an anti- antibody, or siRNA knockdown abrogated the FGF-2-induced increase in cell-associated vitronectin and increased plasminogen system activity. These data show that shear stress and growth factors increase cell-associated vitronectin through integrin activation, which may affect coagulation and angiogenesis.
PubMed: 28659710
DOI: 10.1155/2017/9040161 -
American Journal of Physiology. Lung... Jun 2016Transforming growth factor-β (TGF-β) is a critical driver of acute lung injury and fibrosis. Injury leads to activation of TGF-β, which regulates changes in the...
Transforming growth factor-β (TGF-β) is a critical driver of acute lung injury and fibrosis. Injury leads to activation of TGF-β, which regulates changes in the cellular and matrix makeup of the lung during the repair and fibrosis phase. TGF-β can also initiate alveolar epithelial cell (AEC) apoptosis. Injury leads to destruction of the laminin-rich basement membrane, which is replaced by a provisional matrix composed of arginine-glycine-aspartate (RGD) motif-containing plasma matrix proteins, including vitronectin and fibronectin. To determine the role of specific matrix proteins on TGF-β-induced apoptosis, we studied primary AECs cultured on different matrix conditions and utilized mice with deletion of vitronectin (Vtn(-/-)) or mice in which the vitronectin RGD motif is mutated to nonintegrin-binding arginine-glycine-glutamate (RGE) (Vtn(RGE/RGE)). We found that AECs cultured on fibronectin and vitronectin or in wild-type mouse serum are resistant to TGF-β-induced apoptosis. In contrast, AECs cultured on laminin or in serum from Vtn(-/-) or Vtn(RGE/RGE) mice undergo robust TGF-β-induced apoptosis. Plasminogen activator inhibitor-1 (PAI-1) sensitizes AECs to greater apoptosis by disrupting AEC engagement to vitronectin. Inhibition of integrin-associated signaling proteins augments AEC apoptosis. Mice with transgenic deletion of PAI-1 have less apoptosis after bleomycin, but deletion of vitronectin or disruption of the vitronectin RGD motif reverses this protection, suggesting that the proapoptotic function of PAI-1 is mediated through vitronectin inhibition. Collectively, these data suggest that integrin-matrix signaling is an important regulator of TGF-β-mediated AEC apoptosis and that PAI-1 functions as a natural regulator of this interaction.
Topics: Alveolar Epithelial Cells; Amino Acid Motifs; Animals; Apoptosis; Cells, Cultured; Integrins; Mice, Inbred C57BL; Mice, Knockout; Protein Interaction Domains and Motifs; Signal Transduction; Transforming Growth Factor beta; Vitronectin
PubMed: 27106291
DOI: 10.1152/ajplung.00424.2015 -
BioMed Research International 2018Platelet-rich fibrin (PRF) clots and membranes are autologous blood concentrates widely used in oral surgical procedures; less is known, however, about the liquid...
OBJECTIVE
Platelet-rich fibrin (PRF) clots and membranes are autologous blood concentrates widely used in oral surgical procedures; less is known, however, about the liquid formulations of such products. The aim of this in vitro study is to assess the behavior of different implant surfaces when in contact with two liquid leucocyte- and platelet-rich fibrin (L-PRF) products.
METHODS
Six commercial pure titanium discs, of 9.5 mm diameter and 1.5 mm thickness, were used. Three of these samples had a micro/nano-rough surface; three were machined. Three different protocols were tested. Protocols involved the immersion of the samples in (1) a platelets, lymphocytes, and fibrinogen liquid concentrate (PLyF) for 10 minutes, (2) an exudate obtained from L-PRF clots rich in fibronectin and vitronectin for 5 minutes, and (3) the fibronectin/vitronectin exudate for 2 minutes followed by immersion in the PLyF concentrate for further 8 minutes. After these treatments, the samples were fixed and observed using a scanning electron microscope (SEM).
RESULTS
Under microscopic observation, (1) the samples treated with the PLyF concentrate revealed a dense fibrin network in direct contact with the implant surface and a significant number of formed elements of blood; (2) in the samples treated with the fibronectin/vitronectin exudates, only a small number of white and red blood cells were detectable; and (3) in samples exposed to the combined treatment, there was an apparent increase in the thickness of the fibrin layer. When compared to the machined surface, the micro/nano-rough samples showed an overall increased retention of fibrin, leading to a thicker coating.
CONCLUSIONS
Liquid L-PRF products promote the formation of a dense fibrin clot on micro/nano-rough implant surfaces in vitro. The adjunctive treatment of surfaces with the fibronectin/vitronectin exudate could provide support to contact of the fibrin with the surface, though it is not essential for the clot formation. Further studies are necessary to better elucidate the properties and benefits of liquid L-PRF products.
Topics: Biomimetics; Blood Coagulation; Blood Platelets; Fibrin; Fibronectins; Humans; Leukocytes; Lymphocytes; Platelet-Rich Fibrin; Prostheses and Implants; Vitronectin
PubMed: 29854805
DOI: 10.1155/2018/9031435 -
International Journal of Molecular... Aug 2021Cell signaling mediated by the αv integrin plays a pivotal role in macrophage activation in various inflammatory processes, but its involvement in the pathogenesis of...
Cell signaling mediated by the αv integrin plays a pivotal role in macrophage activation in various inflammatory processes, but its involvement in the pathogenesis of dry eye disease (DED) remains unclear. In a murine model of DED, we found increased αv integrin expression in ocular surface macrophages. The αv integrins inhibitor c(RGDfK) ameliorated the corneal damage caused by DED, suggesting a pathogenic role for αv integrin. Because tear hyperosmolarity induces ocular inflammation in DED, a hyperosmolar culture of murine bone marrow-derived macrophages (BMDMs) is used to reproduce inflammation in vitro. However, the expression of proinflammatory cytokine mRNA was minimal, even though αv integrin was induced. In searching for components that are involved in αv integrin-mediated inflammation but that are missing from the culture model, we showed that the levels of vitronectin (VTN), a binding ligand of αv integrins, were increased in the tear fluid and conjunctival stroma of DED animals. The addition of VTN prominently enhanced hyperosmolarity-induced inflammation in BMDMs. Mechanistically, we showed that VTN/αv integrins mediated NF-κB activation to induce inflammatory gene expression in the BMDMs. Our findings indicate that interaction the of VTN with αv integrins is a crucial step in the inflammatory process in DED and suggests a novel therapeutic target.
Topics: Animals; Cell Line; Cytokines; Dry Eye Syndromes; Eye; Female; Gene Expression Regulation; Humans; Inflammation; Integrin alphaV; Macrophages; Mice; Mice, Inbred C57BL; Signal Transduction; THP-1 Cells; Tears; Vitronectin
PubMed: 34445121
DOI: 10.3390/ijms22168410 -
PLoS Pathogens Jul 2022During hematogenously disseminated candidiasis, blood borne fungi must invade the endothelial cells that line the blood vessels to infect the deep tissues. Although...
During hematogenously disseminated candidiasis, blood borne fungi must invade the endothelial cells that line the blood vessels to infect the deep tissues. Although Candida albicans, which forms hyphae, readily invades endothelial cells, other medically important species of Candida are poorly invasive in standard in vitro assays and have low virulence in immunocompetent mouse models of disseminated infection. Here, we show that Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei can bind to vitronectin and high molecular weight kininogen present in human serum. Acting as bridging molecules, vitronectin and kininogen bind to αv integrins and the globular C1q receptor (gC1qR), inducing human endothelial cells to endocytose the fungus. This mechanism of endothelial cell invasion is poorly supported by mouse endothelial cells but can be restored when mouse endothelial cells are engineered to express human gC1qR or αv integrin. Overall, these data indicate that bridging molecule-mediated endocytosis is a common pathogenic strategy used by many medically important Candida spp. to invade human vascular endothelial cells.
Topics: Animals; Candida; Candida albicans; Candidiasis; Endothelial Cells; Humans; Mice; Vitronectin
PubMed: 35797411
DOI: 10.1371/journal.ppat.1010681