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Background: During wound healing, fibroblast to myofibroblast transition is required for wound contraction and remodeling. While hypoxia is an important biophysical factor in wound microenvironment, the exact regulatory mechanism underlying hypoxia and fibroblast-to-myofibroblast transition remains unclear. We previously found that tetraspanin CD9 plays an important role in oxygen sensing and wound healing. Herein, we investigated the effects of physiological hypoxia on fibroblast-to-myofibroblast transition and the biological function and mechanism of CD9 in it. Methods: Human skin fibroblasts (HSF) and mouse dermis wounds model were established under physiological hypoxia (2% O2). The cell viability and contractility of HSF under hypoxia were evaluated by CCK8 and collagen gel retraction, respectively. The expression and distribution of fibroblast-to-myofibroblast transition markers and CD9 in HSF were detected by Western blotting and immunofluorescence. CD9 slicing and overexpressing HSFs were constructed to determine the role of CD9 by small interfering RNA and recombinant adenovirus vector. The association of TβR2 and TβR1 was measured by immunoprecipitation to explore the regulatory mechanism. Additionally, further validation was conducted on mouse dermis wounds model through histological analysis. Results: Enhanced fibroblast-to-myofibroblast transition and upregulated CD9 expression was observed under hypoxia in vitro and in vivo. Besides, reversal of fibroblast-to-myofibroblast transition under hypoxia was observed when silencing CD9, suggesting that CD9 played a key role in this hypoxia-induced transition. Moreover, hypoxia increased fibroblast-to-myofibroblast transition by activating TGF-β1/Smad2/3 signaling, especially increased interaction of TβR2 and TβR1. Ultimately, CD9 was determined to directly affect TβR1–TβR2 association in hypoxic fibroblast. Conclusion: Collectively, these findings suggest that CD9 promotes TβR2–TβR1 association, thus driving the transition of human dermal fibroblasts to myofibroblast under hypoxia. [ABSTRACT FROM AUTHOR]

Introduction: Osteoclasts determine bone tissue turnover. Their increased activity causes osteoporosis, their dysfunction osteopetrosis. Methods and Results: Murine monocytic ER-Hoxb8 cells differentiate into OCs upon treatment with M-CSF and RANKL and upregulate the collagen-binding integrin α2β1 distinctly earlier than other OC markers, such as the OC-associated receptor, OSCAR. Integrin α2β1 promotes OC differentiation at multiple levels by stimulating differentiation-relevant genes, by regulating cell matrix adhesion and the formation of adhesion-promoting protrusions, and by the upregulation of proteins involved in precursor cell fusion. The two key factors in osteoclastogenesis, RANK and NFATc1, were essentially unaffected after knocking out the ITGA2 gene encoding integrin α2 subunit. However, compared to integrin α2β1 expressing ER-Hoxb8 cells, ITGA2-deficient cells adhered differently with more branched filopodia and significantly longer tunneling nanotubes. Despite the higher number of fusion-relevant TNTs, they form fewer syncytia. They also resorb less hydroxyapatite, because integrin α2β1 regulates expression of lacuna proteins necessary for bone matrix resorption. The impaired syncytia formation of ITGA2-deficient OC precursor cells also correlated with reduced gene activation of fusion-supporting DC-STAMP and with an almost abolished transcription of tetraspanin CD9. CD9 only partially colocalized with integrin α2β1 in TNTs and filopodia of integrin α2β1-expressing OC precursors. [ABSTRACT FROM AUTHOR]

Chronic liver injury leads to decreased liver function and increased fibrosis. Fibrosis is not only associated with the development of portal hypertension and carcinogenesis, but with the occurrence of events and a poor prognosis, highlighting the importance of non‐invasive fibrosis assessment in patients. In the present study, we searched for markers related to liver fibrosis via proteomic analysis of small extracellular vesicles (sEVs). In the discovery cohort, proteomic analysis was carried out in the sEVs extracted from the sera of 5 patients with decompensated cirrhosis, 5 patients with compensated cirrhosis, and 5 controls without liver disease. Interestingly, in this cohort, fibulin‐4 was significantly associated with cirrhosis while in the validation cohort [formed by 191 patients: 7 patients without disease, 16 patients without liver disease (other diseases), 38 patients with chronic liver disease (CLD), 75 patients with cirrhosis of Child–Pugh class A (36 without hepatocellular carcinoma [HCC], 29 with HCC), and 65 patients with cirrhosis of Child–Pugh class B–C (39 without HCC, 26 with HCC)], fibulin‐4/CD9 levels increased with cirrhosis progression. Furthermore, the fibulin‐4/CD9 ratio was significantly higher in patients with varices. Immunostaining also revealed strong fibulin‐4 expression in cholangiocytes within the fibrous areas and mesothelial cells in liver tissue blood vessels. Taken together, our results suggest that fibulin‐4, essential for lysyl oxidase activation, might be a new liver fibrosis marker found in the sEVs of patients with cirrhosis. [ABSTRACT FROM AUTHOR]

Abstract Background During wound healing, fibroblast to myofibroblast transition is required for wound contraction and remodeling. While hypoxia is an important biophysical factor in wound microenvironment, the exact regulatory mechanism underlying hypoxia and fibroblast-to-myofibroblast transition remains unclear. We previously found that tetraspanin CD9 plays an important role in oxygen sensing and wound healing. Herein, we investigated the effects of physiological hypoxia on fibroblast-to-myofibroblast transition and the biological function and mechanism of CD9 in it. Methods Human skin fibroblasts (HSF) and mouse dermis wounds model were established under physiological hypoxia (2% O2). The cell viability and contractility of HSF under hypoxia were evaluated by CCK8 and collagen gel retraction, respectively. The expression and distribution of fibroblast-to-myofibroblast transition markers and CD9 in HSF were detected by Western blotting and immunofluorescence. CD9 slicing and overexpressing HSFs were constructed to determine the role of CD9 by small interfering RNA and recombinant adenovirus vector. The association of TβR2 and TβR1 was measured by immunoprecipitation to explore the regulatory mechanism. Additionally, further validation was conducted on mouse dermis wounds model through histological analysis. Results Enhanced fibroblast-to-myofibroblast transition and upregulated CD9 expression was observed under hypoxia in vitro and in vivo. Besides, reversal of fibroblast-to-myofibroblast transition under hypoxia was observed when silencing CD9, suggesting that CD9 played a key role in this hypoxia-induced transition. Moreover, hypoxia increased fibroblast-to-myofibroblast transition by activating TGF-β1/Smad2/3 signaling, especially increased interaction of TβR2 and TβR1. Ultimately, CD9 was determined to directly affect TβR1–TβR2 association in hypoxic fibroblast. Conclusion Collectively, these findings suggest that CD9 promotes TβR2–TβR1 association, thus driving the transition of human dermal fibroblasts to myofibroblast under hypoxia.

The adenohypophysis is composed of the anterior and intermediate lobes (AL and IL, respectively), and secretes hormones that play an important role in reproduction. CD9- and SOX2-double (CD9/SOX2) positive cells located in the marginal cell layer (MCL) facing the Rathke’s cleft in the AL and IL form the primary stem cell niche in the adult adenohypophysis of rats. In this study, we successfully obtained 3-dimensional (3D) cell aggregates that closely resembled the primary niche of MCL in vivo. After incubation in a Matrigel containing several growth factors, approximately 20% of the cells in the CD9/SOX2-positive cell aggregates were differentiated into hormone-producing cells. The cell aggregates generated in this study may provide insight into the regulation of the pituitary stem/progenitor cell niche and the turnover of hormone-producing cells.

Chronic liver injury leads to decreased liver function and increased fibrosis. Fibrosis is not only associated with the development of portal hypertension and carcinogenesis, but with the occurrence of events and a poor prognosis, highlighting the importance of non‐invasive fibrosis assessment in patients. In the present study, we searched for markers related to liver fibrosis via proteomic analysis of small extracellular vesicles (sEVs). In the discovery cohort, proteomic analysis was carried out in the sEVs extracted from the sera of 5 patients with decompensated cirrhosis, 5 patients with compensated cirrhosis, and 5 controls without liver disease. Interestingly, in this cohort, fibulin‐4 was significantly associated with cirrhosis while in the validation cohort [formed by 191 patients: 7 patients without disease, 16 patients without liver disease (other diseases), 38 patients with chronic liver disease (CLD), 75 patients with cirrhosis of Child–Pugh class A (36 without hepatocellular carcinoma [HCC], 29 with HCC), and 65 patients with cirrhosis of Child–Pugh class B–C (39 without HCC, 26 with HCC)], fibulin‐4/CD9 levels increased with cirrhosis progression. Furthermore, the fibulin‐4/CD9 ratio was significantly higher in patients with varices. Immunostaining also revealed strong fibulin‐4 expression in cholangiocytes within the fibrous areas and mesothelial cells in liver tissue blood vessels. Taken together, our results suggest that fibulin‐4, essential for lysyl oxidase activation, might be a new liver fibrosis marker found in the sEVs of patients with cirrhosis.

Hepatic encephalopathy (HE) is a debilitating neurological disorder associated with liver failure and characterized by impaired brain function. Decade-long studies have led to significant advances in our understanding of HE; however, effective therapeutic management of HE is lacking, and HE continues to be a significant cause of morbidity and mortality in patients, underscoring the need for continued research into its pathophysiology and treatment. Accordingly, the present study provides a comprehensive overview aimed at elucidating the molecular underpinnings of HE and identifying potential therapeutic targets. A moderate-grade HE model was induced in rats using thioacetamide, which simulates the liver damage observed in patients, and its impact on cognitive function, neuronal arborization, and cellular morphology was also evaluated. We employed label-free LC–MS/MS proteomics to quantitatively profile hippocampal proteins to explore the molecular mechanism of HE pathogenesis; 2175 proteins were identified, 47 of which exhibited significant alterations in moderate-grade HE. The expression of several significantly upregulated proteins, such as FAK1, CD9 and Tspan2, was further validated at the transcript and protein levels, confirming the mass spectrometry results. These proteins have not been previously reported in HE. Utilizing Metascape, a tool for gene annotation and analysis, we further studied the biological pathways integral to brain function, including gliogenesis, the role of erythrocytes in maintaining blood–brain barrier integrity, the modulation of chemical synaptic transmission, astrocyte differentiation, the regulation of organ growth, the response to cAMP, myelination, and synaptic function, which were disrupted during HE. The STRING database further elucidated the protein‒protein interaction patterns among the differentially expressed proteins. This study provides novel insights into the molecular mechanisms driving HE and paves the way for identifying novel therapeutic targets for improved disease management. [ABSTRACT FROM AUTHOR]

Extracellular vesicle (EV) secretion is mediated by purinergic receptor P2X7 (P2RX7), an ATP‐gated cation channel highly expressed in microglia. We have previously shown that administration of GSK1482160, a P2RX7 selective inhibitor, suppresses EV secretion from murine microglia and prevents tauopathy development, leading to the recovery of the hippocampal function in PS19 mice, expressing P301S tau mutant. It is yet unknown, however, whether the effect of GSK1482160 on EV secretion from glial cells is specifically regulated through P2RX7. Here we tested GSK1482160 on primary microglia and astrocytes isolated from C57BL/6 (WT) and P2rx7–/– mice and evaluated their EV secretion and phagocytotic activity of aggregated human tau (hTau) under ATP stimulation. GSK1482160 treatment and deletion of P2rx7 significantly reduced secretion of small and large EVs in microglia and astrocytes in both ATP stimulated or unstimulated condition as determined by nanoparticle tracking analysis, CD9 ELISA and immunoblotting of Tsg101 and Flotilin 1 using isolated EVs. GSK1482160 treatment had no effect on EV secretion from P2rx7–/– microglia while we observed significant reduction in the secretion of small EVs from P2rx7–/– astrocytes, suggesting its specific targeting of P2RX7 in EV secretion except small EV secretion from astrocytes. Finally, deletion of P2rx7 suppressed IL‐1β secretion and phagocytosed misfolded tau from both microglia and astrocytes. Together, these findings show that GSK1482160 suppresses EV secretion from microglia and astrocytes in P2RX7‐dependment manner, and P2RX7 critically regulates secretion of IL‐1β and misfolded hTau, demonstrating as the viable target of suppressing EV‐mediated neuroinflammation and tau propagation. [ABSTRACT FROM AUTHOR]

Viruses are obligate parasites that depend on the cellular machinery for their propagation. Several viruses also incorporate cellular proteins that facilitate viral spread. Defining these cellular proteins is critical to decipher viral life cycles and delineate novel therapeutic strategies. While numerous studies have explored the importance of host proteins in coronavirus spread, information about their presence in mature virions is limited. In this study, we developed a protocol to highly enrich mature HCoV-OC43 virions and characterize them by proteomics. Recognizing that cells release extracellular vesicles whose content is modulated by viruses, and given our ability to separate virions from these vesicles, we also analyzed their protein content in both uninfected and infected cells. We uncovered 69 unique cellular proteins associated with virions including 31 high-confidence hits. These proteins primarily regulate RNA metabolism, enzymatic activities, vesicular transport, cell adhesion, metabolite interconversion, and translation. We further discovered that the virus had a profound impact on exosome composition, incorporating 47 novel cellular proteins (11 high confidence) and excluding 92 others (61 high confidence) in virus-associated extracellular vesicles compared to uninfected cells. Moreover, a dsiRNA screen revealed that 11 of 18 select targets significantly impacted viral yields, including proteins found in virions or extracellular vesicles. Overall, this study provides new and important insights into the incorporation of numerous host proteins into HCoV-OC43 virions, their biological significance, and the ability of the virus to modulate extracellular vesicles. IMPORTANCE In recent years, coronaviruses have dominated global attention, making it crucial to develop methods to control them and prevent future pandemics. Besides viral proteins, host proteins play a significant role in viral propagation and offer potential therapeutic targets. Targeting host proteins is advantageous because they are less likely to mutate and develop resistance compared to viral proteins, a common issue with many antiviral treatments. In this study, we examined the protein content of the less virulent biosafety level 2 HCoV-OC43 virus as a stand-in for the more virulent SARS-CoV-2. Our findings reveal that several cellular proteins incorporated into the virion regulate viral spread. In addition, we report that the virus extensively modulates the content of extracellular vesicles, enhancing viral dissemination. This underscores the critical interplay between the virus, host proteins, and extracellular vesicles. [ABSTRACT FROM AUTHOR]

CD9 is a crucial regulator of cell adhesion in the immune system and plays important physiological roles in hematopoiesis, blood coagulation or viral and bacterial infections. It is involved in the transendothelial migration of leukocytes which might also be hijacked by cancer cells during their invasion and metastasis. CD9 is found at the cell surface and the membrane of exosomes affecting cancer progression and therapy resistance. High expression of CD9 is mostly associated with good patients outcome, with a few exceptions. Discordant findings have been reported for breast, ovarian, melanoma, pancreatic and esophageal cancer, which might be related to using different antibodies or inherent cancer heterogeneity. According to in vitro and in vivo studies, tetraspanin CD9 is not clearly associated with either tumor suppression or promotion. Further mechanistic experiments will elucidate the role of CD9 in particular cancer types and specific conditions. [ABSTRACT FROM AUTHOR]

Primary Sjogren's syndrome (pSS) is a complex chronic autoimmune disease in which local tissue damage in exocrine glands is combined with broader systemic involvement across the body in tissues including the skin. These combined manifestations negatively impact patient health and quality of life. While studies have previously reported differences in immune cell composition in the peripheral blood of pSS patients relative to healthy control subjects, a detailed immune cell landscape of the damaged exocrine glands of these patients remains lacking. Through single-cell transcriptomics and repertoire sequencing of immune cells in paired peripheral blood samples and salivary gland biopsies, we present here a preliminary picture of adaptive immune response in pSS. We characterize a number of points of divergence between circulating and glandular immune responses that have been hitherto underappreciated, and identify a novel population of CD8+ CD9+ cells with tissue-residential properties that are highly enriched in the salivary glands of pSS patients. Through comparative analyses with other sequencing data, we also observe a potential connection between these cells and the tissue-resident memory cells found in cutaneous vasculitis lesions. Together, these results indicate a potential role for CD8+ CD9+ cells in mediating glandular and systemic effects associated with pSS and other autoimmune disorders. [ABSTRACT FROM AUTHOR]

IntroductionOsteoclasts determine bone tissue turnover. Their increased activity causes osteoporosis, their dysfunction osteopetrosis.Methods and ResultsMurine monocytic ER-Hoxb8 cells differentiate into OCs upon treatment with M-CSF and RANKL and upregulate the collagen-binding integrin α2β1 distinctly earlier than other OC markers, such as the OC-associated receptor, OSCAR. Integrin α2β1 promotes OC differentiation at multiple levels by stimulating differentiation-relevant genes, by regulating cell matrix adhesion and the formation of adhesion-promoting protrusions, and by the upregulation of proteins involved in precursor cell fusion. The two key factors in osteoclastogenesis, RANK and NFATc1, were essentially unaffected after knocking out the ITGA2 gene encoding integrin α2 subunit. However, compared to integrin α2β1 expressing ER-Hoxb8 cells, ITGA2-deficient cells adhered differently with more branched filopodia and significantly longer tunneling nanotubes. Despite the higher number of fusion-relevant TNTs, they form fewer syncytia. They also resorb less hydroxyapatite, because integrin α2β1 regulates expression of lacuna proteins necessary for bone matrix resorption. The impaired syncytia formation of ITGA2-deficient OC precursor cells also correlated with reduced gene activation of fusion-supporting DC-STAMP and with an almost abolished transcription of tetraspanin CD9. CD9 only partially colocalized with integrin α2β1 in TNTs and filopodia of integrin α2β1-expressing OC precursors.DiscussionOur findings define integrin α2β1 as an early marker of OC differentiation.

AbstractCoagulation disturbances are major contributors to COVID-19 pathogenicity, but limited data exist on the involvement of extracellular vesicles (EVs) and residual cells (RCs). Fifty hospitalized COVID-19 patients stratified by their D-dimer levels into high (>1.5 mg/L, n = 15) or low (≤1.5 mg/l, n = 35) and 10 healthy controls were assessed for medium-sized EVs (mEVs; 200–1000 nm) and large EVs/RCs (1000–4000 nm) by high sensitivity flow cytometry. EVs were analyzed for CD61, CD235a, CD45, and CD31, commonly used to detect platelets, red blood cells, leukocytes or endothelial cells, respectively, whilst phosphatidyl serine EVs/RCs were detected by lactadherin-binding implicating procoagulant catalytic surface. Small EV detection (sEVs; 50–200 nm) and CD41a (platelet integrin) colocalization with general EV markers CD9, CD63, and CD81 were performed by single particle interferometric reflectance imaging sensor. Patients with increased D-dimer exhibited the highest number of RCs and sEVs irrespective of cell origin (p

Tetraspanin CD81 is a transmembrane protein used as a co-receptor by different viruses and implicated in some cancer and inflammatory diseases. The design of therapeutic small molecules targeting CD81 lags behind monoclonal antibodies and peptides but different synthetic and natural products binding to CD81 have been identified. We have investigated the interaction between synthetic compounds and CD81, considering both the cholesterol-bound full-length receptor and a truncated protein corresponding to the large extracellular loop (LEL) of the tetraspanin. They represent the closed and open conformations of the protein, respectively. Stable complexes were characterized with bi-aryl compounds (notably the quinolinone-benzothiazole 6) and atypical molecules bearing a 1-amino-boraadamantane scaffold well adapted to interact with CD81 (5a-d). In each case, the mode of binding to CD81 was analyzed, the binding sites identified and the molecular contacts determined. The narrow intra-LEL binding site of CD81 can accommodate the elongated bi-aryl 6 but not a series of isosteric compounds with a bis(bicyclic) scaffold. The bora-adamantane derivatives appeared to bind well to CD81, but essentially to the external surface of the protein loop. The binding selectivity of the compounds was assessed comparing binding to the LEL of tetraspanins CD81, CD9 and Tspan15. A net preference for CD81 over CD9 was evidenced, but the LEL of Tspan15 also provided a suitable binding site for the compounds, notably for the bora-adamantane derivatives. This work provides an aid to the identification and design of tetraspanin-binding small molecules, underlining the distinct behavior of the open and closed conformation of the protein for drug binding. [ABSTRACT FROM AUTHOR]

Exosomes continue to attract interest as a promising nanocarrier drug delivery technology. They are naturally derived nanoscale extracellular vesicles with innate properties well suited to shuttle proteins, lipids, and nucleic acids between cells. Nonetheless, their clinical utility is currently limited by several major challenges, such as their inability to target tumor cells and a high proportion of clearance by the mononuclear phagocyte system (MPS) of the liver and spleen. To overcome these limitations, we developed "Smart Exosomes" that co-display RGD and CD47 p110–130 through CD9 engineering (Exo Smart ). The resultant Exo Smart demonstrates enhanced binding capacity to αvβ3 on pancreatic ductal adenocarcinoma (PDAC) cells, resulting in amplified cellular uptake in in vitro and in vivo models and increased chemotherapeutic efficacies. Simultaneously, Exo Smart significantly reduced liver and spleen clearance of exosomes by inhibiting macrophage phagocytosis via CD47 p110–130 interaction with signal regulatory proteins (SIRPα) on macrophages. These studies demonstrate that an engineered exosome drug delivery system increases PDAC therapeutic efficacy by enhancing active PDAC targeting and prolonging circulation times, and their findings hold tremendous translational potential for cancer therapy while providing a concrete foundation for future work utilizing novel peptide-engineered exosome strategies. Schematic illustration of ExoSmart enhances pancreatic cancer (PDAC) targeting while increase of evasion of mononuclear phagocyte system (MPS) clearance. (A) Diagram of ExoSmart; (B) Schematic diagram of ExoSmart working model; (C) Fluorescence imaging of liver, spleen and tumors of PDAC orthotopic mice that receive DiR stained exosomes via i.v. injection; (D) Immunofluorescence analysis of exosome in liver, spleen and tumors using anti-hemagglutinin (HA) antibody. [Display omitted] • A "Smart Exosome" that co-display RGD and CD47 p110–130 through CD9 engineering (Exo Smart ) was created. • The Exo Smart enhanced binding capacity to αvβ3 on PDAC cells, resulting in amplified cellular uptake in PDAC. • The Exo Smart significantly reduced liver and spleen clearance of exosomes by inhibiting macrophage phagocytosis. • The Exo Smart drug delivery system significantly increased PDAC therapeutic efficacy. [ABSTRACT FROM AUTHOR]

Tongue cancer is distinguished by aggressive behavior, a high risk of recurrence, lymph, and distant metastases. Hypoxia-Induced Factor 1 α functions as a CD9 transcription factor. CD9 is a transmembrane protein that may be found on the cell membrane. It can modulate the expression of the Epidermal Growth Factor Receptor (EGFR) pathway. ELISA was used to measure serum CD9, p-EGFR, and p-Akt levels in 70 tongue cancer patients and 35 healthy controls. RT-PCR was used to analyze the gene expression of the related genes. The gene as well as protein expression of CD9, EGFR/p-EGFR, and Akt/p-Akt was significantly higher in case subjects when compared with the controls. The expression of CD9 was higher in case subjects who were smokers/alcoholics when to control subjects who were smokers/alcoholics. Overexpression of CD9 due to hypoxic conditions leads to the activation of EGFR-signaling pathway resulting in cancer progression, resistance to chemotherapy. Hence, CD9 could be a potential target to suppress cancer progression. [ABSTRACT FROM AUTHOR]

Extracellular vesicles produced by tumor cells (TEVs) influence all stages of cancer development and spread, including tumorigenesis, cancer progression, and metastasis. TEVs can trigger profound phenotypic and functional changes in target cells through three main general mechanisms: (i) docking of TEVs on target cells and triggering of intra-cellular signaling; (ii) fusion of TEVs and target cell membranes with release of TEVs molecular cargo in the cytoplasm of recipient cell; and (iii) uptake of TEVs by recipient cells. Though the overall tumor-promoting effects of TEVs as well as the general mechanisms involved in TEVs interactions with, and uptake by, recipient cells are relatively well established, current knowledge about the molecular determinants that mediate the docking and uptake of tumor-derived EVs by specific target cells is still rather deficient. These molecular determinants dictate the cell and organ tropism of TEVs and ultimately control the specificity of TEVs-promoted metastases. Here, we will review current knowledge on selected specific molecules that mediate the tropism of TEVs towards specific target cells and organs, including the integrins, ICAM-1 Inter-Cellular Adhesion Molecule), ALCAM (Activated Leukocyte Cell Adhesion Molecule), CD44, the metalloproteinases ADAM17 (A Disintegrin And Metalloproteinase member 17) and ADAM10 (A Disintegrin And Metalloproteinase member 10), and the tetraspanin CD9. [ABSTRACT FROM AUTHOR]

Introduction: Parkinson's disease (PD) is a neurodegenerative disorder characterized by dopaminergic dysfunction and associated with abnormalities in the cholinergic system. However, the relationship between PD and cholinergic dysfunction, particularly in exosomes, is not fully understood. Methods: We enrolled 37 patients with PD and 44 healthy controls (HC) to investigate acetylcholinesterase (AChE) activity in CD9-positive and L1CAMpositive exosomes. Exosomes were isolated from plasma using antibodycoupled magnetic beads, and their sizes and concentrations were assessed using transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Subsequently, the AChE activity in these exosomes was analyzed in relation to various clinical parameters. Results: A significant decrease in AChE activity was observed in CD9-positive exosomes derived from patients with PD, whereas no significant differences were found in L1CAM-positive exosomes. Further analysis with a larger sample size confirmed a substantial reduction in AChE activity in CD9-positive exosomes from the PD plasma, with moderate diagnostic accuracy. The decrease in AChE activity of CD9-positive exosomes did not show an association with cognitive impairment but displayed a trend toward correlation with PD progression. Discussion: The reduction in AChE activity in CD9-positive exosomes suggests potential peripheral cholinergic dysfunction in PD, independent of the central cholinergic system. The observed alterations in AChE activity provide valuable insights into the association between cholinergic dysfunction and the pathogenesis of PD. [ABSTRACT FROM AUTHOR]

Abstract Extracellular vesicle (EV) secretion is mediated by purinergic receptor P2X7 (P2RX7), an ATP‐gated cation channel highly expressed in microglia. We have previously shown that administration of GSK1482160, a P2RX7 selective inhibitor, suppresses EV secretion from murine microglia and prevents tauopathy development, leading to the recovery of the hippocampal function in PS19 mice, expressing P301S tau mutant. It is yet unknown, however, whether the effect of GSK1482160 on EV secretion from glial cells is specifically regulated through P2RX7. Here we tested GSK1482160 on primary microglia and astrocytes isolated from C57BL/6 (WT) and P2rx7–/– mice and evaluated their EV secretion and phagocytotic activity of aggregated human tau (hTau) under ATP stimulation. GSK1482160 treatment and deletion of P2rx7 significantly reduced secretion of small and large EVs in microglia and astrocytes in both ATP stimulated or unstimulated condition as determined by nanoparticle tracking analysis, CD9 ELISA and immunoblotting of Tsg101 and Flotilin 1 using isolated EVs. GSK1482160 treatment had no effect on EV secretion from P2rx7–/– microglia while we observed significant reduction in the secretion of small EVs from P2rx7–/– astrocytes, suggesting its specific targeting of P2RX7 in EV secretion except small EV secretion from astrocytes. Finally, deletion of P2rx7 suppressed IL‐1β secretion and phagocytosed misfolded tau from both microglia and astrocytes. Together, these findings show that GSK1482160 suppresses EV secretion from microglia and astrocytes in P2RX7‐dependment manner, and P2RX7 critically regulates secretion of IL‐1β and misfolded hTau, demonstrating as the viable target of suppressing EV‐mediated neuroinflammation and tau propagation.

This editorial underscores the role of CD9+ tissue-resident memory (Trm) cells in autoimmune diseases, specifically in Sjogren's syndrome, and points to potential implications for better understanding the nature of recurrent autoimmune flares. New findings from Chang et al. highlight the presence and functional role of CD8+ Trm cells in inflamed labial glands in Sjogren's syndrome patients. This, together with the noted expression of CD9 in these Trm cells, opens a new research avenue into the mechanistic understanding of autoimmune diseases. The editorial also emphasizes the need for further studies to answer pressing questions related to CD9+ Trm cell function and their role in autoimmune diseases. [ABSTRACT FROM AUTHOR]

Intervertebral disc degeneration is a leading cause of chronic low back pain. Cell‐based strategies that seek to treat disc degeneration by regenerating the central nucleus pulposus (NP) hold significant promise, but key challenges remain. One of these is the inability of therapeutic cells to effectively mimic the performance of native NP cells, which are unique amongst skeletal cell types in that they arise from the embryonic notochord. In this study, we use single cell RNA sequencing to demonstrate emergent heterogeneity amongst notochord‐derived NP cells in the postnatal mouse disc. Specifically, we established the existence of progenitor and mature NP cells, corresponding to notochordal and chondrocyte‐like cells, respectively. Mature NP cells exhibited significantly higher expression levels of extracellular matrix (ECM) genes including aggrecan, and collagens II and VI, along with elevated transforming growth factor‐beta and phosphoinositide 3 kinase‐protein kinase B signaling. Additionally, we identified Cd9 as a novel surface marker of mature NP cells, and demonstrated that these cells were localized to the NP periphery, increased in numbers with increasing postnatal age, and co‐localized with emerging glycosaminoglycan‐rich matrix. Finally, we used a goat model to show that Cd9+ NP cell numbers decrease with moderate severity disc degeneration, suggesting that these cells are associated with maintenance of the healthy NP ECM. Improved understanding of the developmental mechanisms underlying regulation of ECM deposition in the postnatal NP may inform improved regenerative strategies for disc degeneration and associated low back pain. [ABSTRACT FROM AUTHOR]

Coagulation disturbances are major contributors to COVID-19 pathogenicity, but limited data exist on the involvement of extracellular vesicles (EVs) and residual cells (RCs). Fifty hospitalized COVID-19 patients stratified by their D-dimer levels into high (>1.5 mg/L, n = 15) or low (≤1.5 mg/l, n = 35) and 10 healthy controls were assessed for medium-sized EVs (mEVs; 200-1000 nm) and large EVs/ RCs (1000-4000 nm) by high sensitivity flow cytometry. EVs were analyzed for CD61, CD235a, CD45, and CD31, commonly used to detect platelets, red blood cells, leukocytes or endothelial cells, respectively, whilst phosphatidyl serine EVs/RCs were detected by lactadherin-binding implicating procoagulant catalytic surface. Small EV detection (sEVs; 50-200 nm) and CD41a (platelet integrin) colocalization with general EV markers CD9, CD63, and CD81 were performed by single particle interferometric reflectance imaging sensor. Patients with increased D-dimer exhibited the highest number of RCs and sEVs irrespective of cell origin (p < .05). Platelet activation, reflected by increased CD61+ and lactadherin+ mEV and RC levels, associated with coagulation disturbances. Patients with low D-dimer could be discriminated from controls by tetraspanin signatures of the CD41a+ sEVs, suggesting the changes in the circulating platelet sEV subpopulations may offer added prognostic value during COVID progression. [ABSTRACT FROM AUTHOR]

It is critical to find an alternative therapeutic approach to combat Pseudomonas aeruginosa (P. aeruginosa) that can simultaneously reduce the occurrence of bacterial resistance. The tetraspanin CD9, a highly expressed membrane protein in melanocytes was chosen for this study because it is highly expressed in keratinocytes and has been implicated in the pathogenesis of bacterial infections in a previous study. The antimicrobial activity of CD9 peptides against the standard strain P. aeruginosa (ATCC 27853) and a clinical multidrug-resistant P. aeruginosa (MDR- P. aeruginosa) was studied using the disc diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of CD9 peptides were determined by broth microdilution assays with concentrations ranging from 1 mg/mL to 4.88×10-4 mg/mL. The antibiofilm activity of the CD9 peptides was also determined. CD9 peptides showed an 11.75 ± 2.36 mm inhibition zone against the standard P. aeruginosa strain but none against the MDR- P. aeruginosa. Both isolates had the same MIC value, 0.25 mg/mL. The MBC for the standard strain P. aeruginosa was 0.5 mg/mL, while for the MDR- P. aeruginosa strain, it was 1 mg/mL. CD9 peptides significantly inhibited up to 70% biofilm against both P. aeruginosa isolates. CD9 peptides showed a modest inhibitory effect against the standard strain P. aeruginosa but not against MDR- P. aeruginosa. Interestingly, CD9 peptides were found to be a good anti-biofilm treatment against both P. aeruginosa isolates. This study demonstrated that CD9 peptides have the potential to be an alternative antimicrobial treatment against P. aeruginosa.

It is critical to find an alternative therapeutic approach to combat Pseudomonas aeruginosa (P. aeruginosa) that can simultaneously reduce the occurrence of bacterial resistance. The tetraspanin CD9, a highly expressed membrane protein in melanocytes was chosen for this study because it is highly expressed in keratinocytes and has been implicated in the pathogenesis of bacterial infections in a previous study. The antimicrobial activity of CD9 peptides against the standard strain P. aeruginosa (ATCC 27853) and a clinical multidrug-resistant P. aeruginosa (MDR- P. aeruginosa) was studied using the disc diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of CD9 peptides were determined by broth microdilution assays with concentrations ranging from 1 mg/mL to 4.88x10-4 mg/mL. The antibiofilm activity of the CD9 peptides was also determined. CD9 peptides showed an 11.75 ± 2.36 mm inhibition zone against the standard P. aeruginosa strain but none against the MDR- P. aeruginosa. Both isolates had the same MIC value, 0.25 mg/mL. The MBC for the standard strain P. aeruginosa was 0.5 mg/mL, while for the MDR- P. aeruginosa strain, it was 1 mg/mL. CD9 peptides significantly inhibited up to 70% biofilm against both P. aeruginosa isolates. CD9 peptides showed a modest inhibitory effect against the standard strain P. aeruginosa but not against MDR- P. aeruginosa. Interestingly, CD9 peptides were found to be a good anti-biofilm treatment against both P. aeruginosa isolates. This study demonstrated that CD9 peptides have the potential to be an alternative antimicrobial treatment against P. aeruginosa. [ABSTRACT FROM AUTHOR]

The tetraspanins CD9, CD81 and CD63 are major components of extracellular vesicles (EVs). Yet, their impact on EV composition remains under‐investigated. In the MCF7 breast cancer cell line CD63 was as expected predominantly intracellular. In contrast CD9 and CD81 strongly colocalized at the plasma membrane, albeit with different ratios at different sites, which may explain a higher enrichment of CD81 in EVs. Absence of these tetraspanins had little impact on the EV protein composition as analysed by quantitative mass spectrometry. We also analysed the effect of concomitant knock‐out of CD9 and CD81 because these two tetraspanins play similar roles in several cellular processes and associate directly with two Ig domain proteins, CD9P‐1/EWI‐F/PTGFRN and EWI‐2/IGSF8. These were the sole proteins significantly decreased in the EVs of double CD9‐ and CD81‐deficient cells. In the case of EWI‐2, this is primarily a consequence of a decreased cell expression level. In conclusion, this study shows that CD9, CD81 and CD63, commonly used as EV protein markers, play a marginal role in determining the protein composition of EVs released by MCF7 cells and highlights a regulation of the expression level and/or trafficking of CD9P‐1 and EWI‐2 by CD9 and CD81. [ABSTRACT FROM AUTHOR]

ABSTRACT Epithelial colonization is a critical first step in bacterial pathogenesis. Staphylococcus aureus can utilize several host factors to associate with cells, including α5β1 integrin and heparan sulfate proteoglycans, such as the syndecans. Here, we demonstrate that a partner protein of both integrins and syndecans, the host membrane adapter protein tetraspanin CD9, is essential for syndecan-mediated staphylococcal adhesion. Fibronectin is also essential in this process, while integrins are only critical for post-adhesion entry into human epithelial cells. Treatment of epithelial cells with CD9-derived peptide or heparin caused significant reductions in staphylococcal adherence, dependent on both CD9 and syndecan-1. Exogenous fibronectin caused a CD9-dependent increase in staphylococcal adhesion, whereas blockade of β1 integrins did not affect adhesion but did reduce the subsequent internalization of adhered bacteria. CD9 disruption or deletion increased β1 integrin-mediated internalization, suggesting that CD9 coordinates sequential staphylococcal adhesion and internalization. CD9 controls staphylococcal adhesion through syndecan-1, using a mechanism that likely requires CD9-mediated syndecan organization to correctly display fibronectin at the host cell surface. We propose that CD9-derived peptides or heparin analogs could be developed as anti-adhesion treatments to inhibit the initial stages of staphylococcal pathogenesis. IMPORTANCE Staphylococcus aureus infection is a significant cause of disease and morbidity. Staphylococci utilize multiple adhesion pathways to associate with epithelial cells, including interactions with proteoglycans or β1 integrins through a fibronectin bridge. Interference with another host protein, tetraspanin CD9, halves staphylococcal adherence to epithelial cells, although CD9 does not interact directly with bacteria. Here, we define the role of CD9 in staphylococcal adherence and uptake, observing that CD9 coordinates syndecan-1, fibronectin, and β1 integrins to allow efficient staphylococcal infection. Two treatments that disrupt this action are effective and may provide an alternative to antibiotics. We provide insights into the mechanisms that underlie staphylococcal infection of host cells, linking two known adhesion pathways together through CD9 for the first time.

Abstract The tetraspanins CD9, CD81 and CD63 are major components of extracellular vesicles (EVs). Yet, their impact on EV composition remains under‐investigated. In the MCF7 breast cancer cell line CD63 was as expected predominantly intracellular. In contrast CD9 and CD81 strongly colocalized at the plasma membrane, albeit with different ratios at different sites, which may explain a higher enrichment of CD81 in EVs. Absence of these tetraspanins had little impact on the EV protein composition as analysed by quantitative mass spectrometry. We also analysed the effect of concomitant knock‐out of CD9 and CD81 because these two tetraspanins play similar roles in several cellular processes and associate directly with two Ig domain proteins, CD9P‐1/EWI‐F/PTGFRN and EWI‐2/IGSF8. These were the sole proteins significantly decreased in the EVs of double CD9‐ and CD81‐deficient cells. In the case of EWI‐2, this is primarily a consequence of a decreased cell expression level. In conclusion, this study shows that CD9, CD81 and CD63, commonly used as EV protein markers, play a marginal role in determining the protein composition of EVs released by MCF7 cells and highlights a regulation of the expression level and/or trafficking of CD9P‐1 and EWI‐2 by CD9 and CD81.

Juno and CD9 protein, expressed in oolemma, are known to be essential for sperm-oocyte binding and fusion. Although evidence exists that these two proteins cooperate, their interaction has not yet been demonstrated. Here in, we present Juno and CD9 mutual localization over the surface of mouse metaphase II oocytes captured using the 3D STED super-resolution technique. The precise localization of examined proteins was identified in different compartments of oolemma such as the microvillar membrane, planar membrane between individual microvilli, and the membrane of microvilli-free region. Observed variance in localization of Juno and CD9 was confirmed by analysis of transmission and scanning electron microscopy images, which showed a significant difference in the presence of proteins between selected membrane compartments. Colocalization analysis of super-resolution images based on Pearson’s correlation coefficient supported evidence of Juno and CD9 mutual position in the oolemma, which was identified by proximity ligation assay. Importantly, the interaction between Juno and CD9 was detected by co-immunoprecipitation and mass spectrometry in HEK293T/17 transfected cell line. For better understanding of experimental data, mouse Juno and CD9 3D structure were prepared by comparative homology modelling and several protein-protein flexible sidechain dockings were performed using the ClusPro server. The dynamic state of the proteins was studied in real-time at atomic level by molecular dynamics (MD) simulation. Docking and MD simulation predicted Juno-CD9 interactions and stability also suggesting an interactive mechanism. Using the multiscale approach, we detected close proximity of Juno and CD9 within microvillar oolemma however, not in the planar membrane or microvilli-free region. Our findings show yet unidentified Juno and CD9 interaction within the mouse oolemma protein network prior to sperm attachment. These results suggest that a Juno and CD9 interactive network could assist in primary Juno binding to sperm Izumo1 as a prerequisite to subsequent gamete membrane fusion.

Natural killer cells (NKs) and T-lymphocytes with NK functions (NKTs) are the major effectors of the maternal immune tolerance to a semi-allogeneic fetus, which additionally fulfil a feto-trophic function during physiological pregnancy. Tim-3 (T-cell Ig and mucin domain-containing protein 3) and CD9 molecules play a critical role in the implementation of immunoregulatory and feto-trophic functions of NKs and NKTs, however, their expression in peripheral blood cells has not been studied. The aim of this work was to study Tim-3 and CD9 expression in peripheral blood NK and NKT subpopulations during physiological pregnancy. The object of the study was the peripheral blood of healthy women in the first and third trimesters of pregnancy. The control group included healthy non-pregnant women in the first phase of the menstrual cycle. Tim-3 and CD9 expression was analyzed by flow cytometry in regulatory CD16–CD56bright NKs and CD16–CD56+ NKTs, as well as cytotoxic NK CD16+CD56dim/– NKs and CD16+CD56+ NKTs. It was found that in the first trimester of pregnancy, the total number and ratio of regulatory and cytotoxic NKs and NKTs did not change. Tim-3 expression increased in all NK and NKT subpopulations, except for cytotoxic CD16+CD56dim NKs. CD9 expression increased in all NK subpopulations, but in NKTs, it was indistinguishable from that in non-pregnant women. At the same time, a direct correlation between CD9 and Tim-3 expressions was revealed in regulatory NKs and NKTs in the first trimester of pregnancy. In the third trimester, the number of regulatory CD16–CD56bright NKs increased, while that of cytotoxic CD16+CD56dim NKs and regulatory CD16–CD56+ NKTs decreased compared to non-pregnant women. The number of CD16+CD56– NKs did not change in the first and third trimesters of pregnancy. Tim-3 expression was upregulated in all NK and cytotoxic NKT subpopulations, while CD9 was only upregulated in regulatory NKs. Thus, Tim-3 and CD9 expressions in different NK and NKT subpopulations changed in the first and third trimesters, which may play an important role in the regulation of their phenotype and functions during pregnancy. [ABSTRACT FROM AUTHOR]

Membrane-bound vesicles such as extracellular vesicles (EVs) can function as biochemical effectors on target cells. Docking of the vesicles onto recipient plasma membranes depends on their interaction with cell-surface proteins, but a generalizable technique that can quantitatively observe these vesicle-protein interactions (VPIs) is lacking. Here, we describe a fluorescence microscopy that measures VPIs between single vesicles and cell-surface proteins, either in a surface-tethered or in a membrane-embedded state. By employing cell-derived vesicles (CDVs) and intercellular adhesionmolecule-1 (ICAM-1) as amodel system, we found that integrin-driven VPIs exhibit distinct modes of affinity depending on vesicle origin. Controlling the surface density of proteins also revealed a strong support from a tetraspanin protein CD9, with a critical dependence on molecular proximity. An adsorption model accounting for multiple protein molecules was developed and captured the features of density-dependent cooperativity. We expect that VPI imaging will be a useful tool to dissect the molecularmechanisms of vesicle adhesion and uptake, and to guide the development of therapeutic vesicles. [ABSTRACT FROM AUTHOR]

CD9 is a crucial regulator of cell adhesion in the immune system and plays important physiological roles in hematopoiesis, blood coagulation or viral and bacterial infections. It is involved in the transendothelial migration of leukocytes which might also be hijacked by cancer cells during their invasion and metastasis. CD9 is found at the cell surface and the membrane of exosomes affecting cancer progression and therapy resistance. High expression of CD9 is mostly associated with good patients outcome, with a few exceptions. Discordant findings have been reported for breast, ovarian, melanoma, pancreatic and esophageal cancer, which might be related to using different antibodies or inherent cancer heterogeneity. According to in vitro and in vivo studies, tetraspanin CD9 is not clearly associated with either tumor suppression or promotion. Further mechanistic experiments will elucidate the role of CD9 in particular cancer types and specific conditions. [ABSTRACT FROM AUTHOR]

The molecular mechanisms of opium action with regard to coronary artery disease (CAD) have not yet been determined. The aim of this study was to evaluate the effect of opium on the expression of scavenger receptors including CD36, CD68, and CD9 tetraspanin in monocytes and the plasma levels of tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), malondialdehyde (MDA), and nitric oxide metabolites (NOx) in CAD patients with and without opium addiction. This case–control study was conducted on three groups: (1) opium-addicted CAD patients (CAD + OA, n = 30); (2) CAD patients with no opium addiction (CAD, n = 30); and (3) individuals without CAD and opium addiction as the control group (Ctrl, n = 17). The protein and mRNA levels of CD9, CD36, and CD68 were evaluated by the flow cytometry and quantitative polymerase chain reaction (RT-qPCR) methods, respectively. The consumption of atorvastatin, aspirin, and glyceryl trinitrate was found be higher in the CAD groups compared with the control group. The plasma level of TNF-α was significantly higher in the CAD + OA group than in the CAD and Ctrl groups (p = 0.001 and p = 0.005, respectively). MDA levels significantly increased in CAD and CAD + OA patients in comparison with the Ctrl group (p = 0.010 and p = 0.002, respectively). No significant differences were found in CD9, CD36, CD68, IFN-γ, and NOx between the three groups. The findings demonstrated that opium did not have a significant effect on the expression of CD36, CD68, and CD9 at gene and protein levels, but it might be involved in the development of CAD by inducing inflammation through other mechanisms. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) naturally carry cargo from producer cells, such as RNA and protein, and can transfer these messengers to other cells and tissue. This ability provides an interesting opportunity for using EVs as delivery vehicles for therapeutic agents, such as for gene therapy. However, endogenous loading of cargo, such as microRNAs (miRNAs), is not very efficient as the copy number of miRNAs per EV is quite low. Therefore, new methods and tools to enhance the loading of small RNAs is required. In the current study, we developed fusion protein of EV membrane protein CD9 and RNA-binding protein AGO2 (hCD9.hAGO2). We show that the EVs engineered with hCD9.hAGO2 contain significantly higher levels of miRNA or shRNA (miR-466c or shRNA-451, respectively) compared to EVs that are isolated from cells that only overexpress the desired miRNA or shRNA. These hCD9.hAGO2 engineered EVs also transfer their RNA cargo to recipient cells more efficiently. We were not able to detect changes in gene expression levels in recipient cells after the EV treatments, but we show that the cell viability of HUVECs was increased after hCD9.hAGO2 EV treatments. This technical study characterizes the hCD9.hAGO2 fusion protein for the future development of enhanced RNA loading to EVs. [ABSTRACT FROM AUTHOR]

Synthetic peptides, including tetraspanin CD9 peptides, are increasingly coming into focus as new treatment strategies against various organisms, including bacteria, that cause underarm odour. The use of deodorants and antiperspirants is associated with side effects. Therefore, it is critical to find an alternative therapeutic approach to combat underarm odour. The aim of this study is to investigate the antibacterial effect of tetraspanin CD9 peptides against the skin microbiota that cause malodour in the underarms. The antimicrobial activity of CD9 peptides against Micrococcus luteus (M. luteus), Bacillus subtilis (B. subtilis), Staphylococcus epidermidis (S. epidermidis), and Corynebacterium xerosis (C. xerosis) was investigated by the disc diffusion method. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by broth microdilution assays using CD9 peptide concentrations ranging from 1 mg/mL to 0.0078 mg/mL. In addition, the anti-biofilm activity of the CD9 peptides was determined. The CD9 peptides showed different antibacterial activity with an inhibition zone of 7.67, 9.67, 7.00, and 6.00 mm for S. epidermidis, M. luteus, C. xerosis, and B. subtilis, respectively. All bacteria had the same MBC value of 1 mg/mL. A high MIC of CD9 peptides was observed for S. epidermidis and M. luteus at 0.5 mg/mL. The MIC values of B. subtilis and C. xerosis were 0.125 mg/mL and 0.25 mg/mL, respectively. CD9 peptides significantly inhibited biofilm development of S. epidermidis, B. subtilis, and C. xerosis isolates. The CD9 tetraspanin peptide has excellent antibacterial activity against bacteria that cause underarm odour. Therefore, the CD9 tetraspanin peptide is a promising alternative to deodorants and antiperspirants to combat commensal bacteria of the skin that cause underarm odour. [ABSTRACT FROM AUTHOR]

Sex-determining region Y-box 2 (SOX2)-positive cells are stem/progenitor cells in the adenohypophysis, comprising the anterior and intermediate lobes (AL and IL, respectively). The cells are located in the marginal cell layer (MCL) facing Rathke’s cleft (primary niche) and the parenchyma of the AL (secondary niche). We previously demonstrated in vitro that the tetraspanin superfamily CD9 and SOX2 double-positive (CD9/SOX2-positive) cells in the IL-side MCL migrate to the AL side and differentiate into hormone-producing and endothelial cells in the AL parenchyma. Here, we performed in vivo studies to evaluate the role of IL-side CD9/SOX2-positive cells in pregnancy, lactation, and treatment with diethylstilbestrol (DES; an estrogen analog) when an increased population of prolactin (PRL) cells was observed in the AL of the rat pituitary. The proportions of CD9/SOX2-, CD9/Ki67-, and PRL/TUNEL-positive cells decreased in the primary and secondary niches during pregnancy and DES treatment. In contrast, the number of CD9/PRL-positive cells increased in the AL-side MCL and AL parenchyma during pregnancy and during DES treatment. The proportion of PRL/Ki67-positive cells increased in the AL-side MCL and AL parenchyma in response to DES treatment. Next, we isolated CD9-positive cells from the IL-side MCL using an anti-CD9 antibody. During cell culture, the cells formed free-floating three-dimensional clusters (pituispheres). Furthermore, CD9-positive cells in the pituisphere differentiated into PRL cells, and their differentiation potential was promoted by DES. These findings suggest that CD9/SOX2-positive cells in the IL-side MCL may act as adult stem cells in the AL parenchyma that supply PRL cells under the influence of estrogen.

CD9 is a crucial regulator of cell adhesion in the immune system and plays important physiological roles in hematopoiesis, blood coagulation or viral and bacterial infections. It is involved in the transendothelial migration of leukocytes which might also be hijacked by cancer cells during their invasion and metastasis. CD9 is found at the cell surface and the membrane of exosomes affecting cancer progression and therapy resistance. High expression of CD9 is mostly associated with good patients outcome, with a few exceptions. Discordant findings have been reported for breast, ovarian, melanoma, pancreatic and esophageal cancer, which might be related to using different antibodies or inherent cancer heterogeneity. According to in vitro and in vivo studies, tetraspanin CD9 is not clearly associated with either tumor suppression or promotion. Further mechanistic experiments will elucidate the role of CD9 in particular cancer types and specific conditions.

Fertilization proteins JUNO and CD9 play vital roles in sperm‐egg fusion, but little is known about their expression patterns during in vitro maturation (IVM) and their function during in vitro fertilization (IVF) of bovine oocytes. In this study, qRT‐PCR and immunofluorescence staining were used to detect the mRNA and protein expression levels of JUNO and CD9 genes in bovine oocytes and cumulus cells. Then, fertilization rate of MII oocytes treated with (i) JUNO antibody (1, 5 and 25 μg/ml) or (ii) CD9 antibody (1, 5 and 25 μg/ml) or (iii) CD9 antibody (5 μg/ml) + JUNO antibody (5 μg/ml) were recorded. Our results showed that the mRNA and protein expression levels of JUNO and CD9 genes significantly increased from bovine GV oocytes to MII oocytes, and similar mRNA expression patterns of JUNO and CD9 were also detected in cumulus cells. All groups of oocytes treated with CD9 antibody or JUNO antibody showed significantly decreased fertilization rates (p <.05). Particularly, the fertilization ability of oocytes treated with CD9 antibody (5 μg/ml) + JUNO antibody (5 μg/ml) sharply decreased to 3.48 ± 0.11%. In conclusion, our study revealed the expression levels of JUNO and CD9 genes in oocytes and cumulus cells increased during IVM of bovine oocytes, with JUNO protein playing a major role in the fertilization of bovine oocytes. [ABSTRACT FROM AUTHOR]

Exosomes are nanovesicles with a 40–150 nm diameter and are essential for communication between cells. Literature data suggest that exosomes obtained from different sources (cell cultures, blood plasma, urea, saliva, tears, spinal fluid, milk) using a series of centrifugations and ultracentrifugations contain hundreds and thousands of different protein and nucleic acid molecules. However, most of these proteins are not an intrinsic part of exosomes; instead, they co-isolate with exosomes. Using consecutive ultracentrifugation, gel filtration, and affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we isolated highly purified vesicle preparations from 18 horse milk samples. Gel filtration of the initial preparations allowed us to remove co-isolating proteins and their complexes and to obtain highly purified vesicles morphologically corresponding to exosomes. Using affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we obtained extra-purified CD9+ and CD63+ exosomes, which simultaneously contain these two tetraspanins, while the CD81 tetraspanin was presented in a minor quantity. SDS-PAGE and MALDI analysis detected several major proteins with molecular masses over 10 kDa: CD9, CD63, CD81, lactadherin, actin, butyrophilin, lactoferrin, and xanthine dehydrogenase. Analysis of extracts by trifluoroacetic acid revealed dozens of peptides with molecular masses in the range of 0.8 to 8.5 kDa. Data on the uneven distribution of tetraspanins on the surface of horse milk exosomes and the presence of peptides open new questions about the biogenesis of these extracellular vesicles. [ABSTRACT FROM AUTHOR]

Background: Human cell-secreted extracellular vesicles (EVs) are versatile nanomaterials suitable for disease-targeted drug delivery and therapy. Native EVs, however, usually do not interact specifically with target cells or harbor therapeutic drugs, which limits their potential for clinical applications. These functions can be introduced to EVs by genetic manipulation of membrane protein scaffolds, although the efficiency of these manipulations and the impacts they have on the properties of EVs are for the most part unknown. In this study, we quantify the effects of genetic manipulations of different membrane scaffolds on the physicochemical properties, molecular profiles, and cell uptake of the EVs. Methods: Using a combination of gene fusion, molecular imaging, and immuno-based on-chip analysis, we examined the effects of various protein scaffolds, including endogenous tetraspanins (CD9, CD63, and CD81) and exogenous vesicular stomatitis virus glycoprotein (VSVG), on the efficiency of integration in EV membranes, the physicochemical properties of EVs, and EV uptake by recipient cells. Results: Fluorescence imaging and live cell monitoring showed each scaffold type was integrated into EVs either in membranes of the endocytic compartment, the plasma membrane, or both. Analysis of vesicle size revealed that the incorporation of each scaffold increased the average diameter of vesicles compared to unmodified EVs. Molecular profiling of surface markers in engineered EVs using on-chip assays showed the CD63-GFP scaffold decreased expression of CD81 on the membrane surface compared to control EVs, whereas its expression was mostly unchanged in EVs bearing CD9-, CD81-, or VSVG-GFP. The results from cell uptake studies demonstrated that VSVG-engineered EVs were taken up by recipient cells to a greater degree than control EVs. Conclusion: We found that the incorporation of different molecular scaffolds in EVs altered their physicochemical properties, surface protein profiles, and cell-uptake functions. Scaffold-induced changes in the physical and functional properties of engineered EVs should therefore be considered in engineering EVs for the targeted delivery and uptake of therapeutics to diseased cells. [ABSTRACT FROM AUTHOR]

Background: Controversial evidence exists regarding the metastasis-suppressor or promoting activity of motility-related protein-1 (MRP-1/CD9) in breast cancer (BC). Objectives: we aimed at investigating the possible correlation of CD9 with a metastasis marker matrix metalloproteinase 9 (MMP9) in human BC tissues. Methods: A total of 19 BC and 5 normal breast tissues were analyzed. The mRNA expression of CD9 and MMP9 was measured by quantitative PCR. The correlation of genes' expression with each other and clinicopathological features (i. e. tumor pathology, grade, estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), P53, and KI67) was tested by relevant statistical analysis methods. Results: There was no significant difference between patients with BC and the control group regarding the expression of MMP9 (P = 0.394) and CD9 (P = 0.887). A significant strong positive correlation was observed between CD9 and MMP-9 expressions (r = 0.761 and P = 0.0002). The MMP9 expression was significantly higher in ER or PR positive compared to ER or PR negative tumors (P = 0.046). Conclusions: Given the strong correlation with MMP9, it seems that CD9 might have a role in metastasis in human BC. However, much more studies are required to further support this hypothesis. [ABSTRACT FROM AUTHOR]

Advancing age is a negative prognostic factor for cutaneous melanoma. However, the role of extracellular vesicles (EVs) within the melanoma tumor microenvironment (TME) has remained unexplored in the context of aging. While the size and morphology of the EVs isolated from young vs. aged fibroblasts remained unaltered, the contents of the protein cargo were changed. Aging reduced the expression of the tetraspanin CD9 in both the dermal fibroblasts and released EVs. CD9 is a crucial regulator of EV cargo sorting. Modulating the CD9 expression in fibroblasts was sufficient to alter its levels in EVs. Mass spectrometry analysis of EVs released by CD9 knockdown (KD) vs. control cells revealed a significant increase in angiopoietin-like protein 2 (ANGPTL2), an angiogenesis promoter. Analysis of primary endothelial cells confirmed increased sprouting under CD9 KD conditions. Together, our data indicate that aged EVs play an important role in promoting a tumor-permissive microenvironment. [Display omitted] • Aging promotes a decline in CD9 expression in dermal fibroblast extracellular vesicles • Loss of CD9 drives secretion of the angiogenesis promotor ANGPTL2 in extracellular vesicles • Extracellular vesicles increase angiogenesis in melanoma in an age-dependent manner Hüser et al. show that an age-associated decline in CD9 expression in dermal fibroblasts promotes the secretion of CD9-low and ANGPTL2-high extracellular vesicles. The loss of CD9 promotes angiogenesis and presents an age-related mechanism, which could drive melanoma progression. [ABSTRACT FROM AUTHOR]

Background: Extracellular vesicles (EV) released from neurons into the blood can reflect the state of nervous tissue. Measurement of neuron derived EV (NDE) may serve as an indicator of brain injury. Methods: A sandwich immunoassay was established to measure plasma NDE using anti-neuron CD171 and anti-EV CD9 ([CD171 + CD9+]). Plasma samples were obtained from commercial sources, cross-country (n = 9), football (n = 22), soccer (n = 19), and rugby (n = 18) athletes over time. Plasma was also collected from patients undergoing total aortic arch replacement (TAR) with selective cerebral perfusion during cardiopulmonary bypass before and after surgery (n = 36). Results: The specificity, linearity, and reproducibility of NDE assay (measurement of [CD171 + CD9+]) were confirmed. By scanning electron microscopy and nanoparticle tracking, spherical vesicles ranging in size from 150 to 300 nm were confirmed. Plasma levels of NDE were widely spread over 2 to 3 logs in different individuals with a significant age-dependent decrease. However, NDE were very stable in each individual within a ± 50% change over time (cross-country, football, soccer), whereas rugby players were more variable over 4 years. In patients undergoing TAR, NDE increased rapidly in days post-surgery and were significantly (P =.0004) higher in those developing postoperative delirium (POD) (n = 13) than non-delirium patients (n = 23). Conclusions: The blood test to determine plasma levels of NDE was established by a sandwich immunoassay using 2 antibodies against neuron (CD171) and exosomes (CD9). NDE levels varied widely in different individuals and decreased with age, indicating that NDE levels should be considered as a normalizer of NDE biomarker studies. However, NDE levels were stable over time in each individual, and increased rapidly after TAR with greater increases associated with patients developing POD. This assay may serve as a surrogate for evaluating and monitoring brain injuries. [ABSTRACT FROM AUTHOR]

Intercellular communication between cancer cells themselves or with healthy cells in the tumor microenvironment and/or pre-metastatic sites plays an important role in cancer progression and metastasis. In addition to ligand–receptor signaling complexes, extracellular vesicles (EVs) are emerging as novel mediators of intercellular communication both in tissue homeostasis and in diseases such as cancer. EV-mediated transfer of molecular activities impacting morphological features and cell motility from highly metastatic SW620 cells to non-metastatic SW480 cells is a good in vitro example to illustrate the increased malignancy of colorectal cancer leading to its transformation and aggressive behavior. In an attempt to intercept the intercellular communication promoted by EVs, we recently developed a monovalent Fab fragment antibody directed against human CD9 tetraspanin and showed its effectiveness in blocking the internalization of melanoma cell-derived EVs and the nuclear transfer of their cargo proteins into recipient cells. Here, we employed the SW480/SW620 model to investigate the anti-cancer potential of the anti-CD9 Fab antibody. We first demonstrated that most EVs derived from SW620 cells contain CD9, making them potential targets. We then found that the anti-CD9 Fab antibody, but not the corresponding divalent antibody, prevented internalization of EVs from SW620 cells into SW480 cells, thereby inhibiting their phenotypic transformation, i.e., the change from a mesenchymal-like morphology to a rounded amoeboid-like shape with membrane blebbing, and thus preventing increased cell migration. Intercepting EV-mediated intercellular communication in the tumor niche with an anti-CD9 Fab antibody, combined with direct targeting of cancer cells, could lead to the development of new anti-cancer therapeutic strategies. [ABSTRACT FROM AUTHOR]

Background: Prostasomes, extracellular vesicles (EVs) abundantly present in seminal plasma, express distinct tetraspanins (TS) and galectin-3 (gal-3), which are supposed to shape their surface by an assembly of different molecular complexes. In this study, detergent-sensitivity patterns of membrane-associated prostasomal proteins were determined aiming at the solubilization signature as an intrinsic multimolecular marker and a new parameter suitable as a reference for the comparison of EVs populations in health and disease. Methods: Prostasomes were disrupted by Triton X-100 and analyzed by gel filtration under conditions that maintained complete solubilization. Redistribution of TS (CD63, CD9, and CD81), gal-3, gamma-glutamyltransferase (GGT), and distinct N-glycans was monitored using solid-phase lectin-binding assays, transmission electron microscopy, electrophoresis, and lectin blot. Results: Comparative data on prostasomes under normal physiology and conditions of low sperm count revealed similarity regarding the redistribution of distinct N-glycans and GGT, all presumed to be mainly part of the vesicle coat. In contrast to this, a greater difference was found in the redistribution of integral membrane proteins, exemplified by TS and gal-3. Accordingly, they were grouped into two molecular patterns mainly consisting of overlapped CD9/gal-3/wheat germ agglutinin-reactive glycoproteins and CD63/GGT/concanavalin A-reactive glycoproteins. Conclusions: Solubilization signature can be considered as an all-inclusive distinction factor regarding the surface properties of a particular vesicle since it reflects the status of the parent cell and the extracellular environment, both of which contribute to the composition of spatial membrane arrangements.

Abstract Background CD9 is implicated in cancer progression and metastasis by its role in suppressing cancer cell proliferation and survival. However, the prognostic and clinicopathological significance of CD9 expression is controversial. Therefore, the current meta-analysis was conducted to determine the prognostic and clinicopathological significance of CD9 expression in cancer patients. Methods Eligible studies were selected through database search of PubMed, Embase and Cochrane library up to April 5 2020. The necessary data were extracted from the included studies. Pooled hazard ratio (HR) and odds ratio (OR) with 95% confidence interval (CI) were calculated to evaluate the prognostic and clinicopathological significance of CD9 expression in cancer patients. Results A total of 17 studies consisting of 3456 cancer patients were included in this meta-analysis. An increased CD9 expression was significantly associated with a more favorable overall survival (OS) (HR 0.47, 95% CI 0.31–0.73, p = 0.001) and disease-free survival (DFS) (HR 0.48, 95% CI 0.30–0.79, p = 0.003). In subgroup analysis of cancer type, an increased CD9 expression was associated with increased OS in breast cancer and digestive system cancer, and with increased DFS in head and neck cancer and leukemia/lymphoma. Additionally, an increased CD9 expression significantly correlated with lower overall stage (OR 0.45, 95% CI 0.29–0.72, p = 0.001). Conclusion An increased CD9 expression was associated with favorable survival in cancer patients suggesting that CD9 expression could be a valuable survival factor in cancer patients.

The adenohypophysis consists of the anterior and intermediate lobes (AL and IL). The marginal cell layer (MCL), including the ventral region of the IL and the dorsal region of the AL lining the Rathke's cleft, acts as the primary stem/progenitor cell niches in adult adenohypophysis. The cells of the MCL on the IL side consisted of cluster of differentiation 9 (CD9)-positive stem/progenitor cells with or without motile cilia. However, any additional cellular properties of multiciliated CD9-positive cells are not known. The present study aimed to identify the character of the multiciliated cells in stem cell niche of the pituitary gland. We observed the fine structure of the multiciliated cells in the MCL of male Wistar rats at an early stage after birth and in adulthood (P60) using scanning electron microscopy. Since the previous study showed that the MCL cells of adult rats synthesize retinoic acid (RA), the present study determined whether the multiciliated cells are involved in RA regulation by the expression of retinal aldehyde dehydrogenase 1 (RALDH1) and CYP26A1, an enzyme synthesizing and degrading RA, respectively. Results showed that 96% of multiciliated cells in adult male rats expressed CYP26A1, while 60% expressed RALDH1. Furthermore, the isolated CD9-positive cells from the IL side MCL responded to RA and activated the degradation system of RA by increasing Cyp26a1 expression. These findings indicated that multiciliated cells are involved in RA metabolism in the MCL. Our observations provide novel insights regarding the stem cell niche of the adult pituitary. [ABSTRACT FROM AUTHOR]

Enteric glia regulate gut functions in health and disease through diverse interactions with neurons and immune cells. Intracellular localization of traditional markers of enteric glia such as GFAP, s100b, and Sox10 makes them incompatible for studies that require antigen localization at the cell surface. Thus, new tools are needed for probing the heterogeneous roles of enteric glia at the protein, cell, and functional levels. Here we selected several cell surface antigens including Astrocyte Cell Surface Marker 2 (ACSA2), Cluster of differentiation 9 (CD9), lysophosphatidic acid receptor 1 (LPAR1), and Proteolipid protein 1 (PLP1) as potential markers of enteric glia. We tested their specificity for enteric glia using published single-cell/-nuclei and glia-specific translating mRNA enriched transcriptome datasets, immunolabeling, and flow cytometry. The data show that ACSA2 is a specific marker of mucosal and myenteric glia while other markers are suitable for identifying all subpopulations of enteric glia (LPAR1), glia and immune cells (CD9), or are not suitable for cell-surface labeling (PLP1). These new tools will be useful for future work focused on understanding specific glial functions in health and disease. Summary Statement This study identifies astrocyte cell surface antigen 2 as a novel marker of myenteric glia in the intestine. This, in combination with other markers identified in this study, could be used for selective targeting of enteric glia. [ABSTRACT FROM AUTHOR]

Abstract Background Respiratory diseases are a major cause of morbidity and mortality in the horses of all ages including foals. There is limited understanding of the expression of immune molecules such as tetraspanins and surfactant proteins (SP) and the regulation of the immune responses in the lungs of the foals. Therefore, the expression of CD9, SP-A and SP-D in foal lungs was examined. Results Lungs from one day old (n = 6) and 30 days old (n = 5) foals were examined for the expression of CD9, SP-A, and SP-D with immunohistology and Western blots. Western blot data showed significant increase in the amount of CD9 protein (p = 0.0397) but not of SP-A and SP-D at 30 days of age compared to one day. Immunohistology detected CD9 in the alveolar septa and vascular endothelium but not the bronchiolar epithelium in the lungs of the foals in both age groups. SP-A and SP-D expression was localized throughout the alveolar septa including type II alveolar epithelial cells and the vascular endothelium of the lungs in all the foals. Compared to one day old foals, the expression of SP-A and SP-D appeared to be increased in the bronchiolar epithelium of 30 day old foals. Pulmonary intravascular macrophages were also positive for SP-A and SP-D in 30 days old foals and these cells are not developed in the day old foals. Conclusions This is the first data on the expression of CD9, SP-A and SP-D in the lungs of foals.

Abstract CD9 is a glycoprotein of the transmembrane 4 superfamily that is involved in various cellular processes. Studies related to the immune functions and activities of CD9 in teleost fish are limited. In this study, we characterized two CD9 homologs, PoCD9.1 and PoCD9.3, from Japanese flounder (Paralichthys olivaceus). Sequence analysis showed that PoCD9.1 and PoCD9.3 possess characteristic transmembrane 4 superfamily (TM4SF) structures. PoCD9.1 shares 70.61% sequence identity with PoCD9.3. The expression of PoCD9.1 and PoCD9.3 in the three main immune tissues was significantly induced in a time-dependent manner by extracellular and intracellular pathogen infection, which indicates that the two CD9 homologs play an important role in the response to pathogenic infection. Following infection with the extracellular pathogen Vibrio anguillarum, the expression profiles of both PoCD9.1 and PoCD9.3 were similar. After infection with the intracellular pathogen Edwardsiella piscicida, the expression levels of PoCD9.1 and PoCD9.3 were different at different stages of infection, especially in the spleen. The spleen was the most important tissue for the PoCD9.1 and PoCD9.3 responses to pathogen infection among the three examined immune tissues. Knockdown of PoCD9.1 and PoCD9.3 attenuated the ability of host cells to eliminate pathogenic bacteria, and PoCD9.1 knockdown was more lethal than PoCD9.3 knockdown for host cells with E. piscicida infection. Overexpression of PoCD9.1 and PoCD9.3 promoted host or host cell defence against E. piscicida infection. These findings suggest that PoCD9.1 and PoCD9.3 serve as immune-related factors, play an important role in the immune defence system of Japanese flounder, and display different functions in response to different pathogens at different stages of infection.

Blood samples are minimally invasive and can be collected repeatedly, but they are far from the site of disease and the target molecules are diluted by the large amount of blood. Therefore, we performed lipidomics using immunoprecipitation as a method to enrich specific fractions of serum. In this study, a CD9 antibody was immobilized on magnetic beads to enrich CD9-containing components in the serum for lipidomics. The percentages of phospholipids recovered from serum by methanol and isopropanol extractions were not significantly different, but triglycerides were barely recovered from serum by methanol extraction, requiring the use of isopropanol. However, once the serum was enriched with CD9 magnetic beads, triglycerides, and phospholipids were recovered at similar levels in both methanol and isopropanol extractions. Therefore, it is possible that the triglyceride fraction of the whole serum and the triglyceride fraction were enriched in CD9 magnetic beads differ in localization and properties. In addition, the variation per disease was small in general serum lipidomics; however, the difference per disease appeared larger when CD9 magnetic bead enrichment was employed. [ABSTRACT FROM AUTHOR]