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1.
PLoS Biol ; 22(9): e3002757, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39231388

RESUMO

Integrins are fundamental for cell adhesion and the formation of focal adhesions (FA). Accordingly, these receptors guide embryonic development, tissue maintenance, and haemostasis but are also involved in cancer invasion and metastasis. A detailed understanding of the molecular interactions that drive integrin activation, FA assembly, and downstream signalling cascades is critical. Here, we reveal a direct association of paxillin, a marker protein of FA sites, with the cytoplasmic tails of the integrin ß1 and ß3 subunits. The binding interface resides in paxillin's LIM3 domain, where based on the NMR structure and functional analyses, a flexible, 7-amino acid loop engages the unstructured part of the integrin cytoplasmic tail. Genetic manipulation of the involved residues in either paxillin or integrin ß3 compromises cell adhesion and motility of murine fibroblasts. This direct interaction between paxillin and the integrin cytoplasmic domain identifies an alternative, kindlin-independent mode of integrin outside-in signalling particularly important for integrin ß3 function.


Assuntos
Paxilina , Ligação Proteica , Paxilina/metabolismo , Animais , Camundongos , Domínios Proteicos , Adesão Celular/fisiologia , Adesões Focais/metabolismo , Humanos , Movimento Celular , Integrina beta3/metabolismo , Integrina beta3/genética , Integrina beta3/química , Fibroblastos/metabolismo , Cadeias beta de Integrinas/metabolismo , Cadeias beta de Integrinas/química , Cadeias beta de Integrinas/genética , Integrina beta1/metabolismo , Transdução de Sinais
2.
Biomolecules ; 14(8)2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-39199276

RESUMO

Several inflammatory cytokines bind to the allosteric site (site 2) and allosterically activate integrins. Site 2 is also a binding site for 25-hydroxycholesterol, an inflammatory lipid mediator, and is involved in inflammatory signaling (e.g., TNF and IL-6 secretion) in addition to integrin activation. FGF2 is pro-inflammatory and pro-thrombotic, and FGF1, homologous to FGF2, has anti-inflammatory and anti-thrombotic actions, but the mechanism of these actions is unknown. We hypothesized that FGF2 and FGF1 bind to site 2 of integrins and regulate inflammatory signaling. Here, we describe that FGF2 is bound to site 2 and allosterically activated ß3 integrins, suggesting that the pro-inflammatory action of FGF2 is mediated by binding to site 2. In contrast, FGF1 bound to site 2 but did not activate these integrins and instead suppressed integrin activation induced by FGF2, indicating that FGF1 acts as an antagonist of site 2 and that the anti-inflammatory action of FGF1 is mediated by blocking site 2. A non-mitogenic FGF1 mutant (R50E), which is defective in binding to site 1 of αvß3, suppressed ß3 integrin activation by FGF2 as effectively as WT FGF1.


Assuntos
Fator 1 de Crescimento de Fibroblastos , Fator 2 de Crescimento de Fibroblastos , Integrina beta3 , Humanos , Integrina beta3/metabolismo , Integrina beta3/genética , Fator 1 de Crescimento de Fibroblastos/metabolismo , Fator 1 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Regulação Alostérica , Anti-Inflamatórios/farmacologia , Sítio Alostérico , Animais , Ligação Proteica , Sítios de Ligação
3.
Int J Mol Sci ; 25(16)2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-39201327

RESUMO

HER2-positive breast cancer, representing 15-20% of all breast cancer cases, often develops resistance to the HER2-targeted therapy trastuzumab. Unfortunately, effective treatments for advanced HER2-positive breast cancer remain scarce. This study aims to investigate the roles of ITGß3, and Hedgehog signaling in trastuzumab resistance and explore the potential of combining trastuzumab with cilengitide as a therapeutic strategy. Quantitative gene expression analysis was performed to assess the transcription of EMT (epithelial-mesenchymal transition) markers Slug, Snail, Twist2, and Zeb1 in trastuzumab-resistant HER2-positive breast cancer cells. The effects of ITGß3 and Hedgehog signaling were investigated. Additionally, the combination therapy of trastuzumab and cilengitide was evaluated. Acquired trastuzumab resistance induced the transcription of Slug, Snail, Twist2, and Zeb1, indicating increased EMT. This increased EMT was mediated by ITGB3 and Hedgehog signaling. ITGß3 regulated both the Hedgehog pathway and EMT, with the latter being independent of the Hedgehog pathway. The combination of trastuzumab and cilengitide showed a synergistic effect, reducing both EMT and Hedgehog pathway activity. Targeting ITGß3 with cilengitide, combined with trastuzumab, effectively suppresses the Hedgehog pathway and EMT, offering a potential strategy to overcome trastuzumab resistance and improve outcomes for HER2-positive breast cancer patients.


Assuntos
Neoplasias da Mama , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Integrina beta3 , Receptor ErbB-2 , Trastuzumab , Humanos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Integrina beta3/metabolismo , Integrina beta3/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-2/genética , Linhagem Celular Tumoral , Transdução de Sinais/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Venenos de Serpentes
4.
J Reprod Immunol ; 165: 104312, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39094215

RESUMO

BACKGROUND: Endometriosis (EMs) is a chronic disease characterized by endometrial-like tissue present outside of the uterus. Macrophages have been confirmed to participate in the development of EMs. Integrin ß3 (ITGB3), a ß-subunit of the integrin family, is crucial in tumor progression. In this study, we investigated the pivotal role of ITGB3 in endometrial stromal cells (ESCs) and its influence on the development of EMs, particularly focusing on the regulatory impact of macrophages. METHODS: In this study, we used western blot, Real-time qPCR, Immunohistochemistry to detected the high expression of ITGB3 in ESCs. ITGB3-overexpression ESCs (ITGB3-OE) was constructed and detected by RNA-seq with normal ESCs. ATP and lactate expression assay, transwell migration assay, wound healing, cell adhesion assay and other molecular biology techniques were used to explore the potential mechanisms. In vivo, we constructed the EMs mouse model and injected with cilengitite to inhibit ITGB3. RESULTS: Here, we found ITGB3 highly expressed in ectopic lesions in EMs. The increasing ITGB3 resulted in activating the glycolysis, which produced more ATP and lactate in ITGB3-OE. After culturing with lactate, the migration, proliferation and invasion ability of ESCs were enhanced, while the result in 2-DG was reversed. In vivo, the results showed that after antagonizing ITGB3, the number of ectopic lesions was decrease. CONCLUSIONS: Our findings indicate that ITGB3 up-regulated by macrophages are able to regulate the glycolysis to promote the development of EMs and lactate enhances the ability of proliferation, migration, invasion and adhesion of EMs iv vivo and in vitro.


Assuntos
Endometriose , Glicólise , Integrina beta3 , Ácido Láctico , Animais , Feminino , Humanos , Camundongos , Movimento Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Endometriose/patologia , Endometriose/metabolismo , Endométrio/patologia , Endométrio/metabolismo , Integrina beta3/metabolismo , Integrina beta3/genética , Ácido Láctico/metabolismo , Macrófagos/metabolismo , Macrófagos/imunologia , Células Estromais/metabolismo , Células Estromais/patologia
5.
J Clin Invest ; 134(16)2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-39145443

RESUMO

The phenotypic switch of vascular smooth cells (VSMCs) from a contractile to a synthetic state is associated with the development and progression of aortic aneurysm (AA). However, the mechanism underlying this process remains unclear. In this issue of the JCI, Song et al. identified SLC44A2 as a regulator of the phenotypic switch in VSMCs. Inhibition of SLC44A2 facilitated the switch to the synthetic state, contributing to the development of AA. Mechanistically, SLC44A2 interacted with NRP1 and ITGB3 to activate the TGF-ß/SMAD signaling pathway, resulting in VSMCs with a contractile phenotype. Furthermore, VSMC-specific SLC44A2 overexpression by genetic or pharmacological manipulation reduced AA in mouse models. These findings suggest the potential of targeting the SLC44A2 signaling pathway for AA prevention and treatment.


Assuntos
Aneurisma Aórtico , Músculo Liso Vascular , Miócitos de Músculo Liso , Transdução de Sinais , Animais , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Aneurisma Aórtico/metabolismo , Aneurisma Aórtico/patologia , Aneurisma Aórtico/genética , Camundongos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Humanos , Fenótipo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/genética , Integrina beta3/metabolismo , Integrina beta3/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Neuropilina-1/metabolismo , Neuropilina-1/genética
6.
J Biol Chem ; 300(8): 107516, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38960036

RESUMO

Focal segmental glomerulosclerosis (FSGS), a common cause of primary glomerulonephritis, has a poor prognosis and is pathologically featured by tubulointerstitial injury. Thrombospondin-1 (TSP-1) is an extracellular matrix protein that acts in combination with different receptors in the kidney. Here, we analyzed the tubular expression of TSP-1 and its receptor integrin ß3 (ITGB3) in FSGS. Previously the renal interstitial chip analysis of FSGS patients with tubular interstitial injury showed that the expression of TSP-1 and ITGB3 were upregulated. We found that the expression of TSP-1 and ITGB3 increased in the tubular cells of FSGS patients. The plasma level of TSP-1 increased and was correlated to the degree of tubulointerstitial lesions in FSGS patients. TSP-1/ITGB3 signaling induced renal tubular injury in HK-2 cells exposure to bovine serum albumin and the adriamycin (ADR)-induced nephropathy model. THBS1 KO ameliorated tubular injury and renal fibrosis in ADR-treated mice. THBS1 knockdown decreased the expression of KIM-1 and caspase 3 in the HK-2 cells treated with bovine serum albumin, while THBS1 overexpression could induce tubular injury. In vivo, we identified cyclo-RGDfK as an agent to block the binding of TSP-1 to ITGB3. Cyclo-RGDfK treatment could alleviate ADR-induced renal tubular injury and interstitial fibrosis in mice. Moreover, TSP-1 and ITGB3 were colocalized in tubular cells of FSGS patients and ADR-treated mice. Taken together, our data showed that TSP-1/ITGB3 signaling contributed to the development of renal tubulointerstitial injury in FSGS, potentially identifying a new therapeutic target for FSGS.


Assuntos
Glomerulosclerose Segmentar e Focal , Integrina beta3 , Trombospondina 1 , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/genética , Animais , Trombospondina 1/metabolismo , Trombospondina 1/genética , Humanos , Camundongos , Integrina beta3/metabolismo , Integrina beta3/genética , Masculino , Camundongos Knockout , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Feminino , Adulto , Transdução de Sinais , Linhagem Celular , Doxorrubicina/farmacologia , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Receptor Celular 1 do Vírus da Hepatite A/genética
7.
Int J Mol Sci ; 25(14)2024 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-39063187

RESUMO

Metastatic melanoma, a deadly form of skin cancer, often develops resistance to the BRAF inhibitor drug vemurafenib, highlighting the need for understanding the underlying mechanisms of resistance and exploring potential therapeutic strategies targeting integrins and TGF-ß signalling. In this study, the role of integrins and TGF-ß signalling in vemurafenib resistance in melanoma was investigated, and the potential of combining vemurafenib with cilengitide as a therapeutic strategy was investigated. In this study, it was found that the transcription of PAI1 and p21 was induced by acquired vemurafenib resistance, and ITGA5 levels were increased as a result of this resistance. The transcription of ITGA5 was mediated by the TGF-ß pathway in the development of vemurafenib resistance. A synergistic effect on the proliferation of vemurafenib-resistant melanoma cells was observed with the combination therapy of vemurafenib and cilengitide. Additionally, this combination therapy significantly decreased invasion and colony formation in these resistant cells. In conclusion, it is suggested that targeting integrins and TGF-ß signalling, specifically ITGA5, ITGB3, PAI1, and p21, may offer promising approaches to overcoming vemurafenib resistance, thereby improving outcomes for metastatic melanoma patients.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Melanoma , Venenos de Serpentes , Vemurafenib , Vemurafenib/farmacologia , Vemurafenib/uso terapêutico , Humanos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melanoma/patologia , Melanoma/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linhagem Celular Tumoral , Venenos de Serpentes/farmacologia , Integrina beta3/metabolismo , Integrina beta3/genética , Fator de Crescimento Transformador beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Integrinas/metabolismo , Integrinas/antagonistas & inibidores , Integrina alfa5/metabolismo , Integrina alfa5/genética , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Indóis/farmacologia , Indóis/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico
8.
Commun Biol ; 7(1): 795, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38951640

RESUMO

The peroxisome is a versatile organelle that performs diverse metabolic functions. PEX3, a critical regulator of the peroxisome, participates in various biological processes associated with the peroxisome. Whether PEX3 is involved in peroxisome-related redox homeostasis and myocardial regenerative repair remains elusive. We investigate that cardiomyocyte-specific PEX3 knockout (Pex3-KO) results in an imbalance of redox homeostasis and disrupts the endogenous proliferation/development at different times and spatial locations. Using Pex3-KO mice and myocardium-targeted intervention approaches, the effects of PEX3 on myocardial regenerative repair during both physiological and pathological stages are explored. Mechanistically, lipid metabolomics reveals that PEX3 promotes myocardial regenerative repair by affecting plasmalogen metabolism. Further, we find that PEX3-regulated plasmalogen activates the AKT/GSK3ß signaling pathway via the plasma membrane localization of ITGB3. Our study indicates that PEX3 may represent a novel therapeutic target for myocardial regenerative repair following injury.


Assuntos
Membrana Celular , Integrina beta3 , Camundongos Knockout , Regeneração , Animais , Masculino , Camundongos , Membrana Celular/metabolismo , Proliferação de Células , Traumatismos Cardíacos/metabolismo , Traumatismos Cardíacos/patologia , Traumatismos Cardíacos/genética , Integrina beta3/metabolismo , Integrina beta3/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Plasmalogênios/metabolismo , Transdução de Sinais
9.
Int J Mol Sci ; 25(12)2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38928286

RESUMO

Integrin αIIbß3 mediates platelet aggregation by binding the Arginyl-Glycyl-Aspartic acid (RGD) sequence of fibrinogen. RGD binding occurs at a site topographically proximal to the αIIb and ß3 subunits, promoting the conformational activation of the receptor from bent to extended states. While several experimental approaches have characterized RGD binding to αIIbß3 integrin, applying computational methods has been significantly more challenging due to limited sampling and the need for a priori information regarding the interactions between the RGD peptide and integrin. In this study, we employed all-atom simulations using funnel metadynamics (FM) to evaluate the interactions of an RGD peptide with the αIIb and ß3 subunits of integrin. FM incorporates an external history-dependent potential on selected degrees of freedom while applying a funnel-shaped restraint potential to limit RGD exploration of the unbound state. Furthermore, it does not require a priori information about the interactions, enhancing the sampling at a low computational cost. Our FM simulations reveal significant molecular changes in the ß3 subunit of integrin upon RGD binding and provide a free-energy landscape with a low-energy binding mode surrounded by higher-energy prebinding states. The strong agreement between previous experimental and computational data and our results highlights the reliability of FM as a method for studying dynamic interactions of complex systems such as integrin.


Assuntos
Simulação de Dinâmica Molecular , Oligopeptídeos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Ligação Proteica , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Humanos , Plaquetas/metabolismo , Sítios de Ligação , Integrina beta3/metabolismo , Integrina beta3/química
10.
Cells ; 13(10)2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38786077

RESUMO

Patients with COVID-19 have coagulation and platelet disorders, with platelet alterations and thrombocytopenia representing negative prognostic parameters associated with severe forms of the disease and increased lethality. METHODS: The aim of this study was to study the expression of platelet glycoprotein IIIa (CD61), playing a critical role in platelet aggregation, together with TRL-2 as a marker of innate immune activation. RESULTS: A total of 25 patients were investigated, with the majority (24/25, 96%) having co-morbidities and dying from a fatal form of SARS-CoV-2(+) infection (COVID-19+), with 13 men and 12 females ranging in age from 45 to 80 years. When compared to a control group of SARS-CoV-2 (-) negative lungs (COVID-19-), TLR-2 expression was up-regulated in a subset of patients with deadly COVID-19 fatal lung illness. The proportion of Spike-1 (+) patients found by PCR and ISH correlates to the proportion of Spike-S1-positive cases as detected by digital pathology examination. Furthermore, CD61 expression was considerably higher in the lungs of deceased patients. In conclusion, we demonstrate that innate immune prolonged hyperactivation is related to platelet/megakaryocyte over-expression in the lung. CONCLUSIONS: Microthrombosis in deadly COVID-19+ lung disease is associated with an increase in the number of CD61+ platelets and megakaryocytes in the pulmonary interstitium, as well as their functional activation; this phenomenon is associated with increased expression of innate immunity TLR2+ cells, which binds the SARS-CoV-2 E protein, and significantly with the persistence of the Spike-S1 viral sequence.


Assuntos
COVID-19 , Pulmão , Megacariócitos , SARS-CoV-2 , Trombose , Receptor 2 Toll-Like , Regulação para Cima , Humanos , COVID-19/patologia , COVID-19/imunologia , COVID-19/metabolismo , Masculino , Feminino , Receptor 2 Toll-Like/metabolismo , Receptor 2 Toll-Like/genética , Megacariócitos/metabolismo , Megacariócitos/patologia , Megacariócitos/virologia , Idoso , Pessoa de Meia-Idade , Idoso de 80 Anos ou mais , Pulmão/patologia , Pulmão/virologia , Pulmão/metabolismo , Regulação para Cima/genética , Trombose/patologia , Integrina beta3/metabolismo , Integrina beta3/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Pneumonia Viral/patologia , Pneumonia Viral/imunologia , Pneumonia Viral/mortalidade , Pneumonia Viral/virologia , Pneumonia Viral/metabolismo , Imunidade Inata , Pandemias
11.
Int Immunopharmacol ; 134: 112272, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38761780

RESUMO

BACKGROUND: A subset of neutrophils isolated from the peripheral blood mononuclear cells (PBMC) layer has recently been described in cancer patients. METHODS: Double-gradient centrifugation was used to separate the neutrophil subsets. Western blotting and immunohistochemical assays were performed to assess CCDC25 expression levels. RESULTS: In this study, we found that low-density neutrophils (LDNs) were more highly enriched in metastatic hepatocellular carcinoma (HCC) patients than in non-metastatic HCC patients. We then showed a CD61+ LDNs subset, which displayed distinct functions and gene expression, when compared with high-density neutrophils (HDNs) and CD61- LDNs. Transcriptomic analysis revealed that the CD61+ LDNs were predominantly enhanced in the transcription of glycolysis and angiogenesis associated gene, HMGB1 associated gene and granulation protein gene. These CD61+ LDNs displayed a prominent ability to trigger metastasis, compared with HDNs and CD61- LDNs. Specifically, CD61+ LDN-derived HMGB1 protein increased the invasion of HCC cells by upregulating CCDC25. Mechanistically, the CD61+ LDN-derived HMGB1 protein enhanced the invasiveness of HCC cells and triggered their metastatic potential, which was mediated by TLR9-NF-κB-CCDC25 signaling. Blocking this signaling pathway reversed the invasion of the CD61+ LDN-induced HCC cells. In vivo, we consistently showed that CD61+ LDN-derived HMGB1 enhances HCC metastasis to the lungs. CONCLUSIONS: Overall, our findings showed that a subset of CD61+ LDNs has pro-metastatic effects on HCC, and may be used to target HCC in the clinical setting.


Assuntos
Carcinoma Hepatocelular , Proteína HMGB1 , Neoplasias Hepáticas , Neutrófilos , Regulação para Cima , Animais , Feminino , Humanos , Masculino , Camundongos , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteína HMGB1/metabolismo , Proteína HMGB1/genética , Integrina beta3 , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Metástase Neoplásica , Neutrófilos/imunologia , Neutrófilos/metabolismo
12.
Structure ; 32(7): 899-906.e3, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38579706

RESUMO

Integrin αIIbß3 is the key receptor regulating platelet retraction and accumulation and a proven drug-target for antithrombotic therapies. Here we resolve the cryo-EM structures of the full-length αIIbß3, which covers three distinct states along the activation pathway. Firstly, we obtain the αIIbß3 structure at 3 Å resolution in the inactive state, revealing the overall topology of the heterodimer with the transmembrane (TM) helices and the ligand-binding domain tucked in a specific angle proximity to the TM region. After the addition of a Mn2+ agonist, we resolve two coexisting structures representing two new states between inactive and active state. Our structures show conformational changes of the αIIbß3 activating trajectory and a unique twisting of the integrin legs, which is required for platelets accumulation. Our structure provides direct structural evidence for how the lower legs are involved in full-length integrin activation mechanisms and offers a new strategy to target the αIIbß3 lower leg.


Assuntos
Microscopia Crioeletrônica , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Humanos , Sítios de Ligação , Manganês/metabolismo , Manganês/química , Modelos Moleculares , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Integrina beta3/química , Integrina alfa2/química
13.
Transfusion ; 64(4): 755-760, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38425280

RESUMO

INTRODUCTION: This case describes passenger lymphocyte syndrome (PLS) generating human platelet antigen 1a (HPA-1a) alloantibodies against the recipient's platelets after liver transplant. Given the rarity of PLS, especially in liver transplant with HPA-1a alloantibodies, disease course and management options are poorly described. METHODS: The patient had cirrhosis secondary to nonalcoholic steatohepatitis complicated by hepatocellular carcinoma, encephalopathy, and severe ascites. The model for end-stage liver disease (MELD) score was 15 at presentation. The patient developed hepatic artery thrombosis after an orthotopic liver transplant and was relisted for transplant with a MELD score of 40. The patient received a hepatitis C virus antibody positive, hepatitis C virus nucleic amplification test positive donor liver on postoperative day (POD) 7 after first transplant. On POD 7 after the second transplant, the patient developed profound thrombocytopenia refractory to platelet infusion. They were found to have serum antibody to HPA-1a based upon serum platelet alloantibody testing. The donor was later found to be negative for HPA-1a by genetic testing. However, the patient's native platelets were HPA-1a positive. The patient was diagnosed with PLS. RESULTS: The patient's treatment course included 57 units of platelets transfused, emergency splenectomy, rituximab, plasma exchange, intravenous immunoglobulin (IVIG), eltrombopag, romiplostim, and efgartigimod. DISCUSSION: The synergistic effect of efgartigimod with eltrombopag and romiplostim most likely resolved the patient's thrombocytopenia. This case represents a novel use of efgartigimod in the treatment of passenger lymphocyte syndrome following liver transplant.


Assuntos
Anemia , Antígenos de Plaquetas Humanas , Benzoatos , Doença Hepática Terminal , Hidrazinas , Transplante de Fígado , Pirazóis , Trombocitopenia , Humanos , Isoanticorpos , Doadores Vivos , Índice de Gravidade de Doença , Trombocitopenia/etiologia , Trombocitopenia/terapia , Linfócitos , Integrina beta3
14.
J Agric Food Chem ; 72(13): 7043-7054, 2024 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-38509000

RESUMO

14-3-3ζ protein, the key target in the regulation and control of integrin ß3 outside-in signaling, is an attractive new strategy to inhibit thrombosis without affecting hemostasis. In this study, 4'-O-methylbavachalconeB (4-O-MB) in Psoraleae Fructus was identified as a 14-3-3ζ ligand with antithrombosis activity by target fishing combined with ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) analysis. The competitive inhibition analysis showed that 4-O-MB targeted 14-3-3ζ and blocked the 14-3-3ζ/integrin ß3 interaction with inhibition constant (Ki) values of 9.98 ± 0.22 µM. Molecular docking and amino acid mutation experiments confirmed that 4-O-MB specifically bound to 14-3-3ζ through LSY9 and SER28 to regulate the 14-3-3ζ/integrin ß3 interaction. Besides, 4-O-MB affected the integrin ß3 early outside-in signal by inhibiting AKT and c-Src phosphorylation. Meanwhile, 4-O-MB could inhibit ADP-, collagen-, or thrombin-induced platelet aggregation function but had no effect on platelet adhesion to collagen-coated surfaces in vivo. Administration of 4-O-MB could significantly inhibit thrombosis formation without disturbing hemostasis in mice. These findings provide new prospects for the antithrombotic effects of Psoraleae Fructus and the potential application of 4-O-MB as lead compounds in the therapy of thrombosis by targeting 14-3-3ζ.


Assuntos
Agregação Plaquetária , Trombose , Camundongos , Animais , Integrina beta3/genética , Integrina beta3/química , Integrina beta3/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/farmacologia , Simulação de Acoplamento Molecular , Trombose/tratamento farmacológico , Trombose/genética , Trombose/metabolismo , Colágeno/metabolismo , Plaquetas/metabolismo
15.
Transfusion ; 64(5): 893-905, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38400657

RESUMO

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a condition during pregnancy, which can lead to thrombocytopenia and a bleeding tendency with intracranial hemorrhage (ICH) being the most concerning complication in the fetus or neonate. An incompatibility between human platelet antigen (HPA)-1a accounts for the majority of FNAIT cases. Binding of HPA-1a-specific alloantibodies to their target on fetal platelets and endothelial cells can induce apoptosis of megakaryocytes, disrupt platelet function, and impair angiogenesis. Currently, there is no screening program to identify pregnancies at risk for severe disease. A better understanding of HPA-1a-specific antibody heterogeneity in FNAIT could aid in identifying pathogenic antibody properties linked to severe disease. STUDY DESIGN AND METHODS: This study aimed to isolate HPA-1a-specific B-cells from an HPA-1a-alloimmunized pregnant woman. Using fluorescently labeled HPA-1a-positive platelets, single B-cells were sorted and cultured for 10 days to stimulate antibody production. Subsequently, supernatants were tested for the presence of antibodies by enzyme-linked immunosorbent assay and their reactivity towards HPA-1a-positive platelets. Amplification and sequencing of variable regions allowed the generation of monoclonal antibodies using a HEK-Freestyle-based expression system. RESULTS: Three platelet-specific B-cells were obtained and cloned of which two were specific for HPA-1a, named D- and M-204, while the third was specific for HLA class I, which was named L-204. DISCUSSION: This study outlined an effective method for the isolation of HPA-1a-specific B-cells and the generation of monoclonal antibodies. Further characterization of these antibodies holds promise for better understanding the pathogenic nature of alloantibodies in FNAIT.


Assuntos
Antígenos de Plaquetas Humanas , Isoanticorpos , Trombocitopenia Neonatal Aloimune , Humanos , Antígenos de Plaquetas Humanas/imunologia , Gravidez , Feminino , Trombocitopenia Neonatal Aloimune/imunologia , Isoanticorpos/imunologia , Integrina beta3/imunologia , Linfócitos B/imunologia , Anticorpos Monoclonais/imunologia , Plaquetas/imunologia , Plaquetas/metabolismo , Recém-Nascido
16.
Cell Death Dis ; 15(2): 113, 2024 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-38321003

RESUMO

Understanding the mechanisms of breast cancer cell communication underlying cell spreading and metastasis formation is fundamental for developing new therapies. ID4 is a proto-oncogene overexpressed in the basal-like subtype of triple-negative breast cancer (TNBC), where it promotes angiogenesis, cancer stem cells, and BRACA1 misfunction. Here, we show that ID4 expression in BC cells correlates with the activation of motility pathways and promotes the production of VEGFA, which stimulates the interaction of VEGFR2 and integrin ß3 in a paracrine fashion. This interaction induces the downstream focal adhesion pathway favoring migration, invasion, and stress fiber formation. Furthermore, ID4/ VEGFA/ VEGFR2/ integrin ß3 signaling stimulates the nuclear translocation and activation of the Hippo pathway member's YAP and TAZ, two critical executors for cancer initiation and progression. Our study provides new insights into the oncogenic roles of ID4 in tumor cell migration and YAP/TAZ pathway activation, suggesting VEGFA/ VEGFR2/ integrin ß3 axis as a potential target for BC treatment.


Assuntos
Neoplasias da Mama , Integrina beta3 , Humanos , Feminino , Integrina beta3/metabolismo , Linhagem Celular Tumoral , Transdução de Sinais , Via de Sinalização Hippo , Fator A de Crescimento do Endotélio Vascular , Proteínas Inibidoras de Diferenciação
17.
Endocrinology ; 165(3)2024 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-38195194

RESUMO

BACKGROUND: Repeated implantation failure (RIF) leads to a waste of high-quality embryos and remains a challenge in assisted reproductive technology. During early human placentation, the invasion of trophoblast cells into the decidua is an essential step for the establishment of maternal-fetal interactions and subsequent successful pregnancy. Bone morphogenetic protein 2 (BMP2) has been reported to regulate endometrial receptivity and promote trophoblast invasion. However, whether there is dysregulation of endometrial BMP2 expression in patients with RIF remains unknown. Additionally, the molecular mechanisms underlying the effects of BMP2 on human trophoblast invasion and early placentation remain to be further elucidated. METHODS: Midluteal phase endometrial samples were biopsied from patients with RIF and from routine control in vitro fertilization followed by quantitative polymerase chain reaction and immunoblotting analyses. Human trophoblast organoids, primary human trophoblast cells, and an immortalized trophoblast cell line (HTR8/SVneo) were used as study models. RESULTS: We found that BMP2 was aberrantly low in midluteal phase endometrial tissues from patients with RIF. Recombinant human BMP2 treatment upregulated integrin ß3 (ITGB3) in a SMAD2/3-SMAD4 signaling-dependent manner in both HTR8/SVneo cells and primary trophoblast cells. siRNA-mediated integrin ß3 downregulation reduced both basal and BMP2-upregulated trophoblast invasion and vascular mimicry in HTR8/SVneo cells. Importantly, shRNA-mediated ITGB3 knockdown significantly decreased the formation ability of human trophoblast organoids. CONCLUSION: Our results demonstrate endometrial BMP2 deficiency in patients with RIF. ITGB3 mediates both basal and BMP2-promoted human trophoblast invasion and is essential for early placentation. These findings broaden our knowledge regarding the regulation of early placentation and provide candidate diagnostic and therapeutic targets for RIF clinical management.


Assuntos
Proteína Morfogenética Óssea 2 , Integrina beta3 , Gravidez , Humanos , Feminino , Integrina beta3/genética , Integrina beta3/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Trofoblastos/metabolismo , Linhagem Celular , Placentação/fisiologia , RNA Interferente Pequeno/metabolismo , Movimento Celular
18.
Oncoimmunology ; 13(1): 2304963, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38235317

RESUMO

Approximately two-thirds of hepatocellular carcinoma (HCC) is considered a "cold tumor" characterized by few tumor-infiltrating T cells and an abundance of immunosuppressive cells. Cilengitide, an integrin αvß3 inhibitor, has failed in clinical trials as a potential anticancer drug. This failure implies that integrin αvß3 may play an important role in immune cells. However, the expression and potential role of integrin αvß3 in T cells of HCC patients remain unknown. Here, we established two HCC models and found that cilengitide had a dual effect on the HCC microenvironment by exerting both antitumor effect and immunosuppressive effect on T cells. This may partly explain the failure of cilengitide in clinical trials. In clinical specimens, HCC-infiltrating T cells exhibited deficient expression and activation of integrin ß3, which was associated with poor T-cell infiltration into tumors. Additionally, integrin ß3 functioned as a positive immunomodulatory molecule to facilitate T-cell infiltration and T helper 1-type immune response in vitro. Furthermore, T cells and platelet-derived microparticles (PMPs) co-culture assay revealed that PMPs adoptively transferred integrin ß3 to T cells and positively regulated T cell immune response. This process was mediated by clathrin-dependent endocytosis and macropinocytosis. Our data demonstrate that integrin ß3 deficiency on HCC-infiltrating T cells may be involved in shaping the immunosuppressive tumor microenvironment. PMPs transfer integrin ß3 to T cells and positively regulate T cell immune response, which may provide a new insight into immune therapy of HCC.


Assuntos
Carcinoma Hepatocelular , Micropartículas Derivadas de Células , Neoplasias Hepáticas , Humanos , Integrina beta3/metabolismo , Integrina beta3/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Integrina alfaVbeta3/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/patologia , Linfócitos T , Microambiente Tumoral
19.
Cells ; 13(2)2024 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-38247863

RESUMO

The soluble urokinase plasminogen activator receptor (suPAR) has been implicated in a wide range of pathological conditions including primary nephrotic syndromes and acute kidney injuries. suPAR can trigger transduction cascades in podocytes by outside-in activation of αVß3-integrin, but there is evidence that the functional cell surface response element is actually a complex of different types of receptors, which may also include the receptor for advanced glycation end-products (RAGE) and formyl peptide receptors (FPRs). Here we observed that ROS accumulation and Src activation could be evoked by continuous 24 h exposure to either suPAR or the FPR agonist fMLF. Responses to suPAR and fMLF were completely blocked by either the FPR antagonist WRW4 or by the αV-integrin inhibitor cilengitide. Moreover, endogenous podocyte mouse Fpr1 co-immunoprecipitates with ß3-integrin, suggesting that these receptors occur as a complex on the cell surface. suPAR- and fMLF-evoked activation of Src and ROS differed in time course. Thus, robust pertussis toxin (PTX)-sensitive responses were evoked by 60 min exposures to fMLF but not to suPAR. By contrast, responses to 24 h exposures to either suPAR or fMLF were PTX-resistant and were instead abolished by knockdown of ß-arrestin-1 (BAR1). FPRs, integrins, and RAGE (along with various Toll-like receptors) can all function as pattern-recognition receptors that respond to "danger signals" associated with infections and tissue injury. The fact that podocytes express such a wide array of pattern-recognition receptors suggests that the glomerular filter is designed to change its function under certain conditions, possibly to facilitate clearance of toxic macromolecules.


Assuntos
Receptores de Formil Peptídeo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , beta-Arrestina 1 , Animais , Camundongos , Integrina beta3 , Podócitos , Espécies Reativas de Oxigênio , Receptor para Produtos Finais de Glicação Avançada , Transdução de Sinais , Integrina alfa5
20.
J Nucl Med ; 65(1): 132-138, 2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-37973184

RESUMO

[68Ga]Ga-NODAGA-Arg-Gly-Asp (RGD) is a PET tracer targeting αvß3 integrin, which is upregulated during angiogenesis soon after acute myocardial infarction (AMI). We prospectively evaluated determinants of myocardial uptake of [68Ga]Ga-NODAGA-RGD and its associations with left ventricular (LV) function in patients after AMI. Methods: Myocardial blood flow and [68Ga]Ga-NODAGA-RGD uptake (60 min after injection) were evaluated by PET in 31 patients 7.7 ± 3.8 d after primary percutaneous coronary intervention for ST-elevation AMI. Transthoracic echocardiography of LV function was performed on the day of PET and at the 6-mo follow-up. Results: PET images showed increased uptake of [68Ga]Ga-NODAGA-RGD in the ischemic area at risk (AAR), predominantly in injured myocardial segments. The SUV in the segment with the highest uptake (SUVmax) in the ischemic AAR was higher than the SUVmean of the remote myocardium (0.73 ± 0.16 vs. 0.51 ± 0.11, P < 0.001). Multivariable predictors of [68Ga]Ga-NODAGA-RGD uptake in the AAR included high peak N-terminal pro-B-type natriuretic peptide (P < 0.001), low LV ejection fraction, low global longitudinal strain (P = 0.01), and low longitudinal strain in the AAR (P = 0.01). [68Ga]Ga-NODAGA-RGD uptake corrected for myocardial blood flow and perfusable tissue fraction in the AAR predicted improvement in global longitudinal strain at follow-up (P = 0.002), independent of peak troponin, N-terminal pro-B-type natriuretic peptide, and LV ejection fraction. Conclusion: [68Ga]Ga-NODAGA-RGD uptake shows increased αvß3 integrin expression in the ischemic AAR early after AMI that is associated with regional and global systolic dysfunction, as well as increased LV filling pressure. Increased [68Ga]Ga-NODAGA-RGD uptake predicts improvement of global LV function 6 mo after AMI.


Assuntos
Integrina beta3 , Infarto do Miocárdio , Humanos , Peptídeo Natriurético Encefálico , Tomografia por Emissão de Pósitrons/métodos , Radioisótopos de Gálio , Infarto do Miocárdio/diagnóstico por imagem , Miocárdio/metabolismo , Oligopeptídeos , Integrina alfaVbeta3/metabolismo
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