RESUMO
Ectosomes are carriers of proangiogenic factors during cancer progression. This study investigated whether the proangiogenic effect exerted by melanoma-derived ectosomes on recipient endothelial cells is mediated by ectosomal αvß3 and αvß5 integrins. Ectosomes were isolated from the conditioned culture media of four melanoma cell lines and melanocytes. Changes in gene and protein expression of αvß3 and αvß5 integrins, as well as VEGF and TNF-α were assessed in ectosome-treated endothelial cells. To confirm the functional involvement of ectosomal integrins in functional tests (Alamar Blue, wound healing and tube formation assays), ectosomes were also pretreated with anti-integrin antibodies and integrin-blocking peptides echistatin and cilengitide. Melanoma-derived ectosomes induced changes in the expression of αvß3 and αvß5 integrins in recipient endothelial cells, leading to increased viability, migratory properties, and tube formation potential. The extent of proangiogenic stimulation varied depending on the types of cells releasing ectosomes and the recipient cells. The use of anti-integrin antibodies and integrin-blocking peptides revealed a more significant role for the αvß5 integrin/VEGF than the αvß3 integrin/TNF-α pathway in the interactions between ectosomes and endothelial cells. The study demonstrated the functional role of ectosomal αvß3 and αvß5 integrins. It also provided a baseline understanding of ectosome-mediated αvß3 integrin/TNF-α and αvß5 integrin/VEGF signaling in angiogenesis.
Assuntos
Integrina alfaVbeta3 , Melanoma , Receptores de Vitronectina , Humanos , Melanoma/patologia , Melanoma/metabolismo , Integrina alfaVbeta3/metabolismo , Receptores de Vitronectina/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Neovascularização Patológica/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismoRESUMO
INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease of unknown cause with a dismal prognosis. Nintedanib and Pirfenidone are approved worldwide for the treatment of IPF, but they only slow the rate of functional decline and disease progression. Therefore, there is an urgent need for more efficacious and better tolerated drugs. AREAS COVERED: αvß6 and αvß1 are two integrins overexpressed in fibrotic tissue, which play a critical role in the development of lung fibrosis. They act by converting transforming growth factor (TGF)-ß, one of the most important profibrotic cytokine, in its active form. Here, we summarize and critically discuss the potential of a dual αvß6/αvß1 integrin inhibitor for the treatment of IPF. EXPERT OPINION: Bexotegrast, a dual αvß6/αvß1 integrin inhibitor, has the potential to slow or even halt disease progression in IPF. Indeed, the strong pre-clinical rationale and promising early phase clinical trial data have raised expectations.
Assuntos
Progressão da Doença , Fibrose Pulmonar Idiopática , Integrinas , Receptores de Vitronectina , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/fisiopatologia , Humanos , Integrinas/antagonistas & inibidores , Integrinas/metabolismo , Animais , Receptores de Vitronectina/antagonistas & inibidores , Receptores de Vitronectina/metabolismo , Antígenos de Neoplasias , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismoRESUMO
At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence especially because of the propensity of the OC cells to spread in the abdominal cavity leading to peritoneal metastasis. The influence of ascites on the development of pre-metastatic niches, and on the biological mechanisms leading to cancer cell colonization of the mesothelium, remains poorly understood. Here, we show that ascites weakens the mesothelium by affecting the morphology of mesothelial cells and by destabilizing their distribution in the cell cycle. Ascites also causes destabilization of the integrity of mesothelium by modifying the organization of cell junctions, but it does not affect the synthesis of N-cadherin and ZO-1 by mesothelial cells. Moreover, ascites induces disorganization of focal contacts and causes actin cytoskeletal reorganization potentially dependent on the activity of Rac1. Ascites allows the densification and reorganization of ECM proteins of the mesothelium, especially fibrinogen/fibrin, and indicates that it is a source of the fibrinogen and fibrin surrounding OC spheroids. The fibrin in ascites leads to the adhesion of OC spheroids to the mesothelium, and ascites promotes their disaggregation followed by the clearance of mesothelial cells. Both αV and α5ß1 integrins are involved. In conclusion ascites and its fibrinogen/fibrin composition affects the integrity of the mesothelium and promotes the integrin-dependent implantation of OC spheroids in the mesothelium.
Assuntos
Ascite , Fibrina , Fibrinogênio , Integrina alfa5beta1 , Neoplasias Ovarianas , Esferoides Celulares , Microambiente Tumoral , Humanos , Feminino , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Ascite/patologia , Ascite/metabolismo , Integrina alfa5beta1/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Fibrinogênio/metabolismo , Fibrina/metabolismo , Neoplasias Peritoneais/secundário , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/patologia , Carcinoma Epitelial do Ovário/metabolismo , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular Tumoral , Receptores de Vitronectina/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Adesão Celular , Peritônio/patologia , Peritônio/metabolismo , Epitélio/metabolismo , Epitélio/patologia , Caderinas/metabolismo , Células Tumorais CultivadasRESUMO
Neuroinflammation, characterized by microglial activation and the release of multiple inflammatory mediators, is a key factor in acute glaucomatous injury leading to retinal ganglion cell (RGC) death and ultimately irreversible vision loss. Irisin, a novel exercise-induced myokine, has demonstrated anti-inflammatory activity in ischemia/reperfusion injuries across multiple organs and has displayed a significant neuroprotective role in experimental stroke disease models. This study examined the protective impact of irisin and investigated its potential mechanism involved in this process utilizing an acute ocular hypertension (AOH)-induced retinal injury model in mice and a microglia inflammation model induced by lipopolysaccharide (LPS). There was a transient downregulation of irisin in the retina after AOH injury, with parallel emergence of retinal neuroinflammation and RGC death. Irisin attenuated retinal and optic nerve damage and promotes the phenotypic conversion of microglia from M1 to M2. Mechanistically, irisin significantly upregulated the expression of integrin αVß5, p-AMPK, and autophagy-related markers. Integrin αVß5 was highly expressed on microglia but hardly expressed on RGC. The integrin αVß5 inhibitor cilengitide, the AMPK inhibitor dorsomorphin, and the autophagy inhibitor 3-Methyladenine (3-MA) blocked the neuroprotective effects of irisin. Our results suggest irisin attenuates acute glaucoma-induced neuroinflammation and RGC death by activating integrin αVß5/AMPK in microglia and promoting autophagy. It should be considered a potential neuroprotective therapy for acute glaucoma.
Assuntos
Proteínas Quinases Ativadas por AMP , Autofagia , Fibronectinas , Glaucoma , Microglia , Doenças Neuroinflamatórias , Receptores de Vitronectina , Animais , Masculino , Camundongos , Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/efeitos dos fármacos , Modelos Animais de Doenças , Fibronectinas/metabolismo , Glaucoma/tratamento farmacológico , Glaucoma/imunologia , Glaucoma/metabolismo , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/imunologia , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/imunologia , Doenças Neuroinflamatórias/etiologia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Hipertensão Ocular/tratamento farmacológico , Hipertensão Ocular/metabolismo , Receptores de Vitronectina/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologia , Células Ganglionares da Retina/metabolismoRESUMO
Selective inhibition of the RGD (Arg-Gly-Asp) integrin αvß1 has been recently identified as an attractive therapeutic approach for the treatment of liver fibrosis given its function, target expression, and safety profile. Our identification of a non-RGD small molecule lead followed by focused, systematic changes to the core structure utilizing a crystal structure, in silico modeling, and a tractable synthetic approach resulted in the identification of a potent small molecule exhibiting a remarkable affinity for αvß1 relative to several other integrin isoforms measured. Azabenzimidazolone 25 demonstrated antifibrotic efficacy in an in vivo rat liver fibrosis model and represents a tool compound capable of further exploring the biological consequences of selective αvß1 inhibition.
Assuntos
Desenho de Fármacos , Receptores de Vitronectina , Animais , Ratos , Humanos , Receptores de Vitronectina/antagonistas & inibidores , Receptores de Vitronectina/metabolismo , Relação Estrutura-Atividade , Cirrose Hepática/tratamento farmacológico , Modelos Moleculares , Descoberta de Drogas , Ratos Sprague-Dawley , Masculino , Cristalografia por Raios X , Benzimidazóis/farmacologia , Benzimidazóis/química , Benzimidazóis/síntese químicaRESUMO
Integrins have been suggested to be involved in SARS-CoV-2 infection, but the underlying mechanisms remain largely unclear. This study aimed to investigate how integrins facilitate the ACE2-mediated cellular entry of SARS-CoV-2. We first tested the susceptibility of a panel of human cell lines to SARS-CoV-2 infection using the spike protein pseudotyped virus assay and examined the expression levels of integrins in these cell lines by qPCR, western blot and flow cytometry. We found that integrin αvß1 was highly enriched in the SARS-CoV-2 susceptible cell lines. Additional studies demonstrated that RGD (403-405)âAAA mutant was defective in binding to integrin αvß1 compared to its wild type counterpart, and anti-αvß1 integrin antibodies significantly inhibited the entry of SARS-CoV-2 into the cells. Further studies using mouse NIH3T3 cells expressing human ACE2, integrin αv, integrin ß1, and/or integrin αvß1 suggest that integrin αvß1 was unable to function as an independent receptor but could significantly facilitate the cellular entry of SASR-CoV-2. Finally, we observed that the Omicron exhibited a significant increase in the ACE2-mediated viral entry. Our findings may enhance our understanding of the pathogenesis of SARS-CoV-2 infection and offer potential therapeutic target for COVID-19.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Humanos , Camundongos , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/metabolismo , COVID-19/virologia , Células NIH 3T3 , Receptores de Vitronectina/metabolismo , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Internalização do VírusRESUMO
We were the first to report that epithelial ovarian cancer (EOC) cells and tissues express myeloperoxidase (MPO) that is known to play a role in immune surveillance and inflammation by myeloid cells. Additionally, we reported that MPO is colocalized with inducible nitric oxide synthase (iNOS), a key pro-oxidant enzyme, and plays a key role in regulating apoptosis in EOC cells. Whereas myeloid cells express MPO in a dimeric form, intriguingly, here we report the unique expression of only the monomeric form of MPO in EOC cells, tissues, and blood of an ovarian cancer patient. Additionally, we have identified a cell membrane receptor, αV/ß1 integrin, that is uniquely expressed by both chemosensitive and chemoresistant EOC cells with significantly higher expression in chemoresistant EOC cells. More importantly, we have demonstrated that monoclonal antibodies against αV/ß1 integrin induced cytotoxicity in EOC cells, but not in normal cells, that is also synergistic with conventional chemotherapies. Cytotoxicity of αV/ß1 antibodies is due to conformational changes in αV/ß1 integrin which prevents monomeric MPO binding to αV/ß1 integrin inhibiting the activation of MPO, leading to increased apoptosis. Since normal epithelial cells and macrophages lack monomeric MPO and αV/ß1 integrin system, targeting this unique MPO-dependent survival mechanism will selectively eliminate EOC cells and will be the target for developing specific ovarian cancer therapies.
Assuntos
Neoplasias Ovarianas , Receptores de Vitronectina , Feminino , Humanos , Carcinoma Epitelial do Ovário , Células Epiteliais/metabolismo , Neoplasias Ovarianas/metabolismo , Peroxidase/metabolismo , Receptores de Vitronectina/metabolismoRESUMO
Vitronectin (VTN), a multifunctional glycoprotein with various physiological functions, exists in plasma and the extracellular matrix. It is known to be involved in the cell attachment, spreading and migration through binding to the integrin receptor, mainly via the RGD sequence. VTN is also widely used in the maintenance and expansion of pluripotent stem cells, but its effects go beyond that. Recent evidence shows more functions of VTN in the nervous system as it participates in neural differentiation, neuronutrition and neurogenesis, as well as in regulating axon size, supporting and guiding neurite extension. Furthermore, VTN was proved to play a key role in protecting the brain as it can reduce the permeability of the blood-brain barrier by interacting with integrin receptors in vascular endothelial cells. Moreover, evidence suggests that VTN is associated with neurodegenerative diseases, such as Alzheimer's disease, but its function has not been fully understood. This review summarizes the functions of VTN and its receptors in neurons and describes the role of VTN in the blood-brain barrier and neurodegenerative diseases.
Assuntos
Doenças Neurodegenerativas , Vitronectina , Humanos , Vitronectina/metabolismo , Células Endoteliais/metabolismo , Integrinas/metabolismo , Glicoproteínas , Neurônios/metabolismo , Oligopeptídeos , Receptores de Vitronectina/metabolismoRESUMO
Breast cancer cells release a large quantity of biocargo-bearing extracellular vesicles (EVs), which mediate intercellular communication within the tumour microenvironment and promote metastasis. To identify EV-bound proteins related to metastasis, we used mass spectrometry to profile EVs from highly and poorly metastatic breast cancer lines of human and mouse origins. Comparative mass spectrometry indicated that integrins, including αv and ß1 subunits, are preferentially enriched in EVs of highly metastatic origin over those of poorly metastatic origin. These results are consistent with our histopathological findings, which show that integrin αv is associated with disease progression in breast cancer patients. Integrin αv colocalizes with the multivesicular-body marker CD63 at a higher frequency in the tumour and is enriched in circulating EVs of breast cancer patients at late stages when compared with circulating EVs from early-stage patients. With a magnetic bead-based flow cytometry assay, we confirmed that integrins αv and ß1 are enriched in the CD63+ subsets of EVs from both human and mouse highly metastatic cells. By analysing the level of integrin αv on circulating EVs, this assay could predict the metastatic potential of a xenografted mouse model. To explore the export mechanism of integrins into EVs, we performed immunoprecipitation mass spectrometry and identified members of the galectin family as potential shuttlers of integrin αvß1 into EVs. In particular, knockdown of galectin-3, but not galectin-1, causes a reduction in the levels of cell surface integrins ß1 and αv, and decreases the colocalization of these integrins with CD63. Importantly, knockdown of galectin-3 leads to a decrease of integrin αvß1 export into the EVs concomitant with a decrease in the metastatic potential of breast cancer cells. Moreover, inhibition of the integrin αvß1 complex leads to a reduction in the binding of EVs to fibronectin, suggesting that integrin αvß1 is important for EV retention in the extracellular matrix. EVs retained in the extracellular matrix are taken up by fibroblasts, which differentiate into cancer associated fibroblasts. In summary, our data indicate an important link between EV-bound integrin αvß1 with breast cancer metastasis and provide additional insights into the export of integrin αvß1 into EVs in the context of metastasis.
Assuntos
Neoplasias da Mama , Vesículas Extracelulares , Animais , Neoplasias da Mama/metabolismo , Vesículas Extracelulares/metabolismo , Feminino , Galectina 3 , Humanos , Integrina alfaV , Melanoma , Camundongos , Receptores de Vitronectina/metabolismo , Neoplasias Cutâneas , Microambiente Tumoral , Melanoma Maligno CutâneoRESUMO
Rationale: In the glioblastoma (GBM) microenvironment, tumor-associated macrophages (TAMs) are prominent components and facilitate tumor growth. The exact molecular mechanisms underlying TAMs' function in promoting glioma stem cells (GSCs) maintenance and tumor growth remain largely unknown. We found a candidate molecule, transforming growth factor beta-induced (TGFBI), that was specifically expressed by TAMs and extremely low in GBM and GSC cells, and meanwhile closely related to glioma WHO grades and patient prognosis. The exact mechanism of TGFBI linking TAM functions to GSC-driven tumor growth was explored. Methods: Western blot, quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), immunohistochemistry staining (IHC) and public datasets were used to evaluate TGFBI origin and level in GBM. The response of GSCs to recombinant human TGFBI was assessed in vitro and orthotopic xenografts were established to investigate the function and mechanism in vivo. Results: M2-like TAMs infiltration was elevated in high-grade gliomas. TGFBI was preferentially secreted by M2-like TAMs and associated with a poor prognosis for patients with GBM. TGFBI promoted the maintenance of GSCs and GBM malignant growth through integrin αvß5-Src-Stat3 signaling in vitro and in vivo. Of clinical relevance, TGFBI was enriched in the serum and CSF of GBM patients and significantly decreased after tumor resection. Conclusion: TAM-derived TGFBI promotes GSC-driven tumor growth through integrin αvß5-Src-Stat3 signaling. High serum or CSF TGFBI may serve as a potential diagnostic and prognostic bio-index for GBMs.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Glioma/metabolismo , Humanos , Células-Tronco Neoplásicas/metabolismo , Receptores de Vitronectina , Fator de Transcrição STAT3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral , Macrófagos Associados a TumorRESUMO
The arginine-glycine-aspartic acid (RGD) motif is a cell adhesion sequence that binds to integrins. Some RGD-containing peptides promote adhesion of both embryonic stem cells and induced pluripotent stem cells (iPSCs); however, not all such RGD-containing peptides are active. In this study, we elucidated the role of RGD-neighboring sequences on iPSC adhesion using diverse synthetic peptides and recombinant proteins. Our results indicate that iPSC adhesion requires RGDX1 X2 sequences, such as RGDVF and RGDNY, and that the X1 X2 residues are essential for the adhesion via integrin αvß5 but not αvß3. iPSCs express integrin αvß5 but not αvß3; therefore, iPSC adhesion requires the RGDX1 X2 -containing sequences. The importance of the X1 X2 residues was confirmed with both HeLa and A549 cells, which express integrin αvß5 but not αvß3. Analysis of RGD-neighboring sequences provides important insights into ligand-binding specificity of integrins. Identification of integrin αvß5-binding motifs is potentially useful in drug development, drug delivery, cell culture, and tissue engineering.
Assuntos
Células-Tronco Pluripotentes , Receptores de Vitronectina , Adesão Celular/fisiologia , Integrina alfaVbeta3/genética , Oligopeptídeos/química , Peptídeos/química , Células-Tronco Pluripotentes/metabolismo , Receptores de Vitronectina/genética , Receptores de Vitronectina/metabolismoRESUMO
The vitronectin receptor integrin αVß5 can reside in two distinct adhesion structures - focal adhesions (FAs) and flat clathrin lattices (FCLs). Here, we investigate the mechanism that regulates the subcellular distribution of ß5 in keratinocytes and show that ß5 has approximately 7- and 5-fold higher affinity for the clathrin adaptors ARH (also known as LDLRAP1) and Numb, respectively, than for the talin 1 (TLN1); all proteins that bind to the membrane-proximal NPxY motif of the ß5 cytoplasmic domain. Using mass spectrometry, we identified ß5 interactors, including the Rho GEFs p115Rho-GEF and GEF-H1 (also known as ARHGEF1 and ARHGEF2, respectively), and the serine protein kinase MARK2, depletion of which diminishes the clustering of ß5 in FCLs. Replacement of two serine residues (S759 and S762) in the ß5 cytoplasmic domain with phospho-mimetic glutamate residues causes a shift in the localization of ß5 from FAs into FCLs without affecting the interactions with MARK2, p115Rho-GEF or GEF-H1. Instead, we demonstrate that changes in the actomyosin-based cellular contractility by ectopic expression of activated Rho or disruption of microtubules regulates ß5 localization. Finally, we present evidence that ß5 in either FAs or FCLs functions to promote adhesion to vitronectin, cell spreading, and proliferation.
Assuntos
Clatrina , Receptores de Vitronectina , Adesão Celular/fisiologia , Proliferação de Células , Clatrina/metabolismo , Adesões Focais/metabolismo , Receptores de Vitronectina/metabolismo , Serina/metabolismoRESUMO
BACKGROUND: Neuroinflammation is a crucial factor in the development of secondary brain injury after intracerebral hemorrhage (ICH). Irisin is a newly identified myokine that confers strong neuroprotective effects in experimental ischemic stroke. However, whether this myokine can exert neuroprotection effects after ICH remains unknown. This study aimed to investigate the impact of irisin treatment on neuroinflammation and neuronal apoptosis and the underlying mechanism involving integrin αVß5/AMPK pathway after ICH. METHODS: Two hundred and eighty-five adult (8-week-old) male C57BL/6 mice were randomly assigned to sham and ICH surgery groups. ICH was induced via intrastriatal injection of autologous blood. Irisin was administered intranasally at 30 min after ICH. To elucidate the underlying mechanism, cilengitide (a selective integrin αVß5 inhibitor) and dorsomorphin (a selective phosphorylated AMPK inhibitor) were administered before irisin treatment. The short- and long-term neurobehavior tests, brain edema, quantitative-PCR, western blotting, Fluoro-Jade C, TUNEL, and immunofluorescence staining were performed to assess the neurofunctional outcome at the level of molecular, cell, histology, and function. RESULTS: Endogenous irisin and its receptor, integrin αVß5, were increased, peaked at 24 h after ICH. irisin post-treatment improved both short- and long-term neurological functions, reduced brain edema after ICH. Interestingly, integrin αVß5 was mainly located in the microglia after ICH, and irisin post-treatment inhibited microglia/macrophage pro-inflammatory polarization and promoted anti-inflammatory polarization. Moreover, irisin treatment inhibited neutrophil infiltration and suppressed neuronal apoptotic cell death in perihematomal areas after ICH. Mechanistically, irisin post-treatment significantly increased the expression of integrin αVß5, p-AMPK and Bcl-2, and decreased the expression of IL-1ß, TNF-α, MPO, and Bax following ICH. The neuroprotective effects of irisin were abolished by both integrin αVß5 inhibitor cilengitide and AMPK inhibitor dorsomorphin. CONCLUSIONS: This study demonstrated that irisin post-treatment ameliorated neurological deficits, reduced brain edema, and ameliorated neuroinflammation and neuronal apoptosis, at least in part, through the integrin αVß5/AMPK signaling pathway after ICH. Thus, irisin post-treatment may provide a promising therapeutic approach for the early management of ICH.
Assuntos
Hemorragia Cerebral , Fibronectinas , Doenças Neuroinflamatórias , Fármacos Neuroprotetores , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Apoptose , Edema Encefálico/tratamento farmacológico , Edema Encefálico/etiologia , Hemorragia Cerebral/complicações , Hemorragia Cerebral/tratamento farmacológico , Hemorragia Cerebral/patologia , Fibronectinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/farmacologia , Receptores de Vitronectina/metabolismoRESUMO
Contact inhibition of locomotion (CIL) is a process that regulates cell motility upon collision with other cells. Improper regulation of CIL has been implicated in cancer cell dissemination. Here, we identify the cell adhesion molecule JAM-A as a central regulator of CIL in tumor cells. JAM-A is part of a multimolecular signaling complex in which tetraspanins CD9 and CD81 link JAM-A to αvß5 integrin. JAM-A binds Csk and inhibits the activity of αvß5 integrin-associated Src. Loss of JAM-A results in increased activities of downstream effectors of Src, including Erk1/2, Abi1, and paxillin, as well as increased activity of Rac1 at cell-cell contact sites. As a consequence, JAM-A-depleted cells show increased motility, have a higher cell-matrix turnover, and fail to halt migration when colliding with other cells. We also find that proper regulation of CIL depends on αvß5 integrin engagement. Our findings identify a molecular mechanism that regulates CIL in tumor cells and have implications on tumor cell dissemination.
Assuntos
Inibição de Contato , Adesão Celular , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Inibição de Contato/genética , Receptores de Vitronectina , TetraspaninasRESUMO
Lymphangiogenesis is essential for the development of the lymphatic system and is important for physiological processes such as homeostasis, metabolism and immunity. Cellular communication network factor 2 (CCN2, also known as CTGF), is a modular and matricellular protein and a well-known angiogenic factor in physiological and pathological angiogenesis. However, its roles in lymphangiogenesis and intracellular signaling in lymphatic endothelial cells (LECs) remain unclear. Here, we investigated the effects of CCN2 on lymphangiogenesis. In in vivo Matrigel plug assays, exogenous CCN2 increased the number of Podoplanin-positive vessels. Subsequently, we found that CCN2 induced phosphorylation of ERK in primary cultured LECs, which was almost completely inhibited by the blockade of integrin αvß5 and partially decreased by the blockade of integrin αvß3. CCN2 promoted direct binding of ERK to dual-specific phosphatase 6 (DUSP6), which regulated the activation of excess ERK by dephosphorylating ERK. In vitro, CCN2 promoted tube formation in LECs, while suppression of Dusp6 further increased tube formation. In vivo, immunohistochemistry also detected ERK phosphorylation and DUSP6 expression in Podoplanin-positive cells on CCN2-supplemented Matrigel. These results indicated that CCN2 promotes lymphangiogenesis by enhancing integrin αvß5-mediated phosphorylation of ERK and demonstrated that DUSP6 is a negative regulator of excessive lymphangiogenesis by CCN2.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Linfangiogênese/fisiologia , Receptores de Vitronectina/metabolismo , Animais , Movimento Celular/fisiologia , Fator de Crescimento do Tecido Conjuntivo/fisiologia , Fosfatase 6 de Especificidade Dupla/metabolismo , Fosfatase 6 de Especificidade Dupla/fisiologia , Células Endoteliais/metabolismo , Endotélio Linfático/metabolismo , Feminino , Integrinas/genética , Integrinas/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Receptores de Vitronectina/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
RATIONALE: αv integrins, key regulators of transforming growth factor-ß activation and fibrogenesis in in vivo models of pulmonary fibrosis, are expressed on abnormal epithelial cells (αvß6) and fibroblasts (αvß1) in fibrotic lungs. OBJECTIVES: We evaluated multiple αv integrin inhibition strategies to assess which most effectively reduced fibrogenesis in explanted lung tissue from patients with idiopathic pulmonary fibrosis. METHODS: Selective αvß6 and αvß1, dual αvß6/αvß1, and multi-αv integrin inhibitors were characterized for potency, selectivity, and functional activity by ligand binding, cell adhesion, and transforming growth factor-ß cell activation assays. Precision-cut lung slices generated from lung explants from patients with idiopathic pulmonary fibrosis or bleomycin-challenged mouse lungs were treated with integrin inhibitors or standard-of-care drugs (nintedanib or pirfenidone) and analyzed for changes in fibrotic gene expression or TGF-ß signaling. Bleomycin-challenged mice treated with dual αvß6/αvß1 integrin inhibitor, PLN-74809, were assessed for changes in pulmonary collagen deposition and Smad3 phosphorylation. MEASUREMENTS AND MAIN RESULTS: Inhibition of integrins αvß6 and αvß1 was additive in reducing type I collagen gene expression in explanted lung tissue slices from patients with idiopathic pulmonary fibrosis. These data were replicated in fibrotic mouse lung tissue, with no added benefit observed from inhibition of additional αv integrins. Antifibrotic efficacy of dual αvß6/αvß1 integrin inhibitor PLN-74809 was confirmed in vivo, where dose-dependent inhibition of pulmonary Smad3 phosphorylation and collagen deposition was observed. PLN-74809 also, more potently, reduced collagen gene expression in fibrotic human and mouse lung slices than clinically relevant concentrations of nintedanib or pirfenidone. CONCLUSIONS: In the fibrotic lung, dual inhibition of integrins αvß6 and αvß1 offers the optimal approach for blocking fibrogenesis resulting from integrin-mediated activation of transforming growth factor-ß.
Assuntos
Antifibróticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Integrina alfa6beta1/antagonistas & inibidores , Pulmão/efeitos dos fármacos , Receptores de Vitronectina/antagonistas & inibidores , Animais , Bleomicina , Linhagem Celular , Técnicas de Cocultura , Cadeia alfa 1 do Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Integrina alfa6beta1/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Camundongos Endogâmicos C57BL , Fosforilação , Receptores de Vitronectina/metabolismo , Transdução de Sinais , Proteína Smad3/metabolismoRESUMO
Glioblastoma (GBM) is a refractory disease that has a highly infiltrative characteristic. Over the past decade, GBM perivascular niche (PVN) has been described as a route of dissemination. Here, we investigated that trailed membrane structures, namely retraction fibers (RFs), are formed by perivascular extracellular matrix (ECM) proteins. By using the anatomical GBM database, we validated that the ECM-related genes were highly expressed in the cells within the PVN where fibronectin (FN) induced RF formation. By disrupting candidates of FN-binding integrins, integrin α5ß1 was identified as the main regulator of RF formation. De novo RFs were produced at the trailing edge, and focal adhesions were actively localized in RFs, indicating that adhesive force makes RFs remain at the bottom surface. Furthermore, we observed that GBM cells more frequently migrated along the residual RFs formed by preceding cells in microfluidic channels in comparison to those in the channels without RFs, suggesting that the infiltrative characteristics GBM could be attributed to RFs formed by the preceding cells in concert with chemoattractant cues. Altogether, we demonstrated that shedding membrane structures of GBM cells are maintained by FN-integrin α5ß1 interaction and promoted their motility .
Assuntos
Neoplasias Encefálicas/metabolismo , Movimento Celular , Fibronectinas/metabolismo , Glioblastoma/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Vitronectina/metabolismo , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos NusRESUMO
We have previously demonstrated the significance of endothelial cell-expressed α5ß1 integrin in ischemic stroke, having shown that α5ß1 integrin endothelial cell-selective knockout mice are significantly resistance to ischemic stroke injury via preservation of the tight junction protein claudin-5 and subsequent stabilization of the blood-brain barrier (BBB). In addition, inhibition of α5ß1 by the small peptide noncompetitive integrin α5 inhibitor, ATN-161, is beneficial in a mouse model of ischemic stroke through reduction of infarct volume, edema, stabilization of the BBB, and reduced inflammation and immune cell infiltration into the brain. In continuation with our previous findings, we have further evaluated the mechanistic role of ATN-161 in vitro and found that oxygen and glucose deprivation and reperfusion (OGD/R)-induced inflammation, oxidative stress, apoptosis, mitochondrial depolarization, and fibrosis attenuate tight junction integrity via induction of α5, NLRP3, p-FAK, and p-AKT signaling in mouse brain endothelial cells. ATN-161 treatment (10 µM) effectively inhibited OGD/R-induced extracellular matrix (ECM) deposition by reducing integrin α5, MMP-9, and fibronectin expression, as well as reducing oxidative stress by reducing mitochondrial superoxide radicals, intracellular ROS, inflammation by reducing NLRP3 inflammasome, tight junction loss by reducing claudin-5 and ZO-1 expression levels, mitochondrial damage by inhibiting mitochondrial depolarization, and apoptosis via regulation of p-FAK and p-AKT levels. Taken together, our results further support therapeutically targeting α5 integrin with ATN-161, a safe, well-tolerated, and clinically validated peptide, in ischemic stroke.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Encéfalo/irrigação sanguínea , Células Endoteliais/efeitos dos fármacos , AVC Isquêmico/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Doenças Neuroinflamatórias/prevenção & controle , Oligopeptídeos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Junções Íntimas/efeitos dos fármacos , Animais , Hipóxia Celular , Linhagem Celular , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Fibrose , Glucose/deficiência , Mediadores da Inflamação/metabolismo , AVC Isquêmico/metabolismo , AVC Isquêmico/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Vitronectina/antagonistas & inibidores , Receptores de Vitronectina/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Junções Íntimas/metabolismo , Junções Íntimas/patologiaRESUMO
Irisin, the cleaved form of the fibronectin type III domain containing 5 (FNDC5) protein, is involved in metabolism and inflammation. Recent findings indicated that irisin participated in cardiovascular physiology and pathology. In this study, we investigated the effects of FNDC5/irisin on diabetic cardiomyopathy (DCM) in type 2 diabetic db/db mice. Downregulation of myocardial FNDC5/irisin protein expression and plasma irisin levels was observed in db/db mice compared to db/+ controls. Moreover, echocardiography revealed that db/db mice exhibited normal cardiac systolic function and impaired diastolic function. Adverse structural remodeling, including cardiomyocyte apoptosis, myocardial fibrosis, and cardiac hypertrophy were observed in the hearts of db/db mice. Sixteen-week-old db/db mice were intramyocardially injected with adenovirus encoding FNDC5 or treated with recombinant human irisin via a peritoneal implant osmotic pump for 4 weeks. Both overexpression of myocardial FNDC5 and exogenous irisin administration attenuated diastolic dysfunction and cardiac structural remodeling in db/db mice. Results from in vitro studies revealed that FNDC5/irisin protein expression was decreased in high glucose (HG)/high fat (HF)-treated cardiomyocytes. Increased levels of inducible nitric oxide synthase (iNOS), NADPH oxidase 2 (NOX2), 3-nitrotyrosine (3-NT), reactive oxygen species (ROS), and peroxynitrite (ONOO-) in HG/HF-treated H9C2 cells provided evidence of oxidative/nitrosative stress, which was alleviated by treatment with FNDC5/irisin. Moreover, the mitochondria membrane potential (ΔΨm) was decreased and cytochrome C was released from mitochondria with increased levels of cleaved caspase-3 in HG/HF-treated H9C2 cells, indicating the presence of mitochondria-dependent apoptosis, which was partially reversed by FNDC5/irisin treatment. Mechanistic studies showed that activation of integrin αVß5-AKT signaling and attenuation of oxidative/nitrosative stress were responsible for the cardioprotective effects of FNDC5/irisin. Therefore, FNDC5/irisin mediates cardioprotection in DCM by inhibiting myocardial apoptosis, myocardial fibrosis, and cardiac hypertrophy. These findings implicate that FNDC5/irisin as a potential therapeutic intervention for DCM, especially in type 2 diabetes mellitus (T2DM).
Assuntos
Cardiotônicos/administração & dosagem , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Cardiomiopatias Diabéticas/sangue , Cardiomiopatias Diabéticas/tratamento farmacológico , Fibronectinas/administração & dosagem , Estresse Nitrosativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Vitronectina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Cardiomegalia/prevenção & controle , Cardiotônicos/sangue , Modelos Animais de Doenças , Fibronectinas/sangue , Fibronectinas/genética , Masculino , Camundongos , Mitocôndrias/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Proteínas Recombinantes/administração & dosagem , Resultado do Tratamento , Remodelação Ventricular/efeitos dos fármacosRESUMO
OBJECTIVE: The present study aimed to evaluate the protective role of cilengitide (CGT), an integrin αvß3 and αvß5 inhibitor, on doxorubicin (DOX)-induced myocardial fibrosis and cardiac dysfunction in a rat model. Methods. Forty male rats were randomly divided into four groups: DOX (n = 12), intraperitoneal (i.p.) injection of DOX 0.8 â¼ 1.0 mg/kg three times a week for up to 6 weeks, then saline i.p. three times a week for another 3 weeks; CGT (n = 8), CGT 10 mg/kg, i.p. three times a week for 9 weeks; DOX + CGT (n = 12), DOX and CGT co-administration as above for 6 weeks, then CGT alone for another 3 weeks; Control (n = 8), saline i.p. three times a week for 9 weeks. Echocardiography, serum procollagen I C-terminal propeptide (PICP) procollagen III N-terminal propeptide (PIIINP) and C telopeptide type I (CTX-I) were evaluated at baseline and 3, 6 and 9 weeks after initial DOX administration for all surviving rats. The heart tissues were then harvested for myocardial hydroxyproline (HYP) evaluation, qRT-PCR, and western blotting. Results. CGT attenuated DOX-induced eccentric remodeling by improving relative wall thickness at the 9th week. CGT also improved systolic function at the 9th week and diastolic function at the 6th and 9th week. CGT reduced myocardial HYP and serum PICP, PIIINP, CTX-I, and the PICP/PIIINP ratio. RT-PCR and western blot showed that CGT blocked the TGF-ß1/SMAD3 pathway and mitigating extracellular matrix turnover. Conclusions. CGT exerted a cardioprotective effect against doxorubicin-induced fibrosis and improved cardiac function.