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/genéticaRESUMO
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 terapêuticoRESUMO
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 SinaisRESUMO
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/químicaRESUMO
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 , PandemiasRESUMO
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/metabolismoRESUMO
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 DiferenciaçãoRESUMO
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 TumoralRESUMO
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 CelularRESUMO
ABSTRACT: Aging leads to a decline in function of hematopoietic stem cells (HSCs) and increases susceptibility to hematological disease. We found CD61 to be highly expressed in aged murine HSCs. Here, we investigate the role of CD61 in identifying distinct subpopulations of aged HSCs and assess how expression of CD61 affects stem cell function. We show that HSCs with high expression of CD61 are functionality superior and retain self-renewal capacity in serial transplantations. In primary transplantations, aged CD61High HSCs function similarly to young HSCs. CD61High HSCs are more quiescent than their CD61Low counterparts. We also show that in aged bone marrow, CD61High and CD61Low HSCs are transcriptomically distinct populations. Collectively, our research identifies CD61 as a key player in maintaining stem cell quiescence, ensuring the preservation of their functional integrity and potential during aging. Moreover, CD61 emerges as a marker to prospectively isolate a superior, highly dormant population of young and aged HSCs, making it a valuable tool both in fundamental and clinical research.
Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , Animais , Camundongos , Envelhecimento , Células-Tronco Hematopoéticas/metabolismo , Integrina beta3/metabolismoRESUMO
BACKGROUND: Glanzmann thrombasthenia (GT) is an autosomal recessive platelet aggregation disorder caused by mutations in ITGA2B or ITGB3. OBJECTIVES: We aimed to assess the phenotype and investigate the genetic etiology of a GT pedigree. METHODS: A patient with bleeding manifestations and mild mental retardation was enrolled. Complete blood count, coagulation, and platelet aggregation tests were performed. Causal mutations were identified via whole exome and genome sequencing and subsequently confirmed through polymerase chain reaction and Sanger sequencing. The transcription of ITGB3 was characterized using RNA sequencing and reverse transcription polymerase chain reaction. The αâ ¡b and ß3 biosynthesis was investigated via whole blood flow cytometry and in vitro studies. RESULTS: GT was diagnosed in a patient with defective platelet aggregation. Novel compound heterozygous ITGB3 variants were identified, with a maternal nonsense mutation (c.2222G>A, p.Trp741∗) and a paternal SINE-VNTR-Alu (SVA) retrotransposon insertion. The 5' truncated SVA element was inserted in a sense orientation in intron 11 of ITGB3, resulting in aberrant splicing of ITGB3 and significantly reducing ß3 protein content. Meanwhile, both the expression and transportation of ß3 were damaged by the ITGB3 c.2222G>A. Almost no αâ ¡b and ß3 expressions were detected on the patient's platelets surface. CONCLUSION: Novel compound heterozygous ITGB3 mutations were identified in the GT pedigree, resulting in defects of αâ ¡bß3 biosynthesis. This is the first report of SVA retrotransposon insertion in the genetic pathogenesis of GT. Our study highlights the importance of combining multiple high-throughput sequencing technologies for the molecular diagnosis of genetic disorders.
Assuntos
Trombastenia , Humanos , Trombastenia/diagnóstico , Trombastenia/genética , Retroelementos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Fenótipo , Plaquetas/metabolismo , Integrina beta3/genética , Integrina beta3/metabolismoRESUMO
Due to its key roles in malignant tumor progression and reprograming of the tumor microenvironment, integrin ß3 has attracted great attention as a new target for tumor therapy. However, the structure-function relationship of integrins ß3 remains incompletely understood, leading to the shortage of specific and effective targeting probes. This work uses a purified extracellular domain of integrin ß3 and integrin ß3-positive cells to screen aptamers, specifically targeting integrin ß3 in the native conformation on live cells through the SELEX approach. Following meticulous truncation and characterization of the initial aptamer candidates, the optimized aptamer S10yh2 was produced, exhibiting a low equilibrium dissociation constant (Kd) in the nanomolar range. S10yh2 displays specific recognition of cancer cells with varying levels of integrin ß3 expression and demonstrates favorable stability in serum. Subsequent analysis of docking sites revealed that S10yh2 binds to the seven amino acid residues located in the core region of integrin ß3. The S10yh2 aptamer can downregulate the level of integrin heterodimer αvß3 on integrin ß3 overexpressed cancer cells and partially inhibit cell migration behavior. In summary, S10yh2 is a promising probe with a small size, simple synthesis, good stability, high binding affinity, and selectivity. It therefore holds great potential for investigating the structure-function relationship of integrins.
Assuntos
Aptâmeros de Nucleotídeos , Neoplasias , Humanos , Integrina beta3/química , Integrina beta3/metabolismo , Aptâmeros de Nucleotídeos/farmacologia , Integrina alfaVbeta3/metabolismo , Movimento Celular , Microambiente TumoralRESUMO
Tumor tissues often exhibit unique integrin receptor presentation during development, such as high exposures of αvß3 and αIIbß3 integrins. These features are not present in normal tissues. The induction of selective thrombosis and infarction in the tumor-feeding vessels, as well as specific antagonism of αvß3 integrin on the surface of tumor endothelial cells, is a potential novel antitumor strategy. The Echistatin-Annexin V (EAV) fusion protein is a novel Annexin V (ANV) derivative that possesses a high degree of αvß3 and αIIbß3 integrin receptor recognition and binding characteristics while retaining the specific binding ability of the natural ANV molecule for phosphatidylserine (PS). We systematically investigated the biological effects of this novel molecule with superimposed functions on mouse melanoma. We found that EAV inhibited the viability and migration of B16F10 murine melanoma cells in a dose-dependent manner, exhibited good tumor suppressive effects in a xenograft mouse melanoma model, strongly induced tumor tissue necrosis in mice, and targeted the inhibition of angiogenesis in mouse melanoma tumor tissue. EAV exhibited stronger biological effects than natural ANV molecules in inhibiting melanoma in mice. The unique biological effects of EAV are based on its high ß3-type integrin receptor-specific recognition and binding ability, as well as its highly selective binding to PS molecules. Based on these findings, we propose that EAV-mediated tumor suppression is a novel and promising antitumor strategy that targets both PS- and integrin ß3-positive tumor neovascularization and the tumor cells themselves, thus providing a possible mechanism for the treatment of melanoma.
Assuntos
Integrina beta3 , Melanoma , Humanos , Camundongos , Animais , Integrina beta3/metabolismo , Anexina A5/metabolismo , Células Endoteliais/metabolismo , Melanoma/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Integrina alfaVbeta3/metabolismoRESUMO
Platelet integrin αIIbß3 is maintained in a bent inactive state (low affinity to physiologic ligand), but can rapidly switch to a ligand-competent (high-affinity) state in response to intracellular signals ("inside-out" activation). Once bound, ligands drive proadhesive "outside-in" signaling. Anti-αIIbß3 drugs like eptifibatide can engage the inactive integrin directly, inhibiting thrombosis but inadvertently impairing αIIbß3 hemostatic functions. Bidirectional αIIbß3 signaling is mediated by reorganization of the associated αIIb and ß3 transmembrane α-helices, but the underlying changes remain poorly defined absent the structure of the full-length receptor. We now report the cryo-EM structures of full-length αIIbß3 in its apo and eptifibatide-bound states in native cell-membrane nanoparticles at near-atomic resolution. The apo form adopts the bent inactive state but with separated transmembrane α-helices, and a fully accessible ligand-binding site that challenges the model that this site is occluded by the plasma membrane. Bound eptifibatide triggers dramatic conformational changes that may account for impaired hemostasis. These results advance our understanding of integrin structure and function and may guide development of safer inhibitors.
Assuntos
Plaquetas , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Eptifibatida , Ligantes , Microscopia Crioeletrônica , Plaquetas/metabolismo , Integrina beta3/metabolismo , LipídeosRESUMO
BACKGROUND: HIV-infected individuals are known to be at higher risk for thrombotic cardiovascular disease (CVD), which may also be differentially affected by components of anti-HIV drugs. To identify the effects of a series of FDA-approved anti-HIV drugs on platelet aggregation in humans, focusing on the novel pharmacological effects of rilpivirine (RPV), a reverse transcriptase inhibitor, on platelet function both in vitro and in vivo and the mechanisms involved. METHODS AND RESULTS: In vitro studies showed that RPV was the only anti-HIV reagent that consistently and efficiently inhibited aggregation elicited by different agonists, exocytosis, morphological extension on fibrinogen, and clot retraction. Treatment of mice with RPV significantly prevented thrombus formation in FeCl3-injured mesenteric vessels, postcava with stenosis surgery, and ADP -induced pulmonary embolism models without defects in platelet viability, tail bleeding, and coagulation activities. RPV also improved cardiac performance in mice with post-ischemic reperfusion. A mechanistic study revealed that RPV preferentially attenuated fibrinogen-stimulated Tyr773 phosphorylation of ß3-integrin by inhibiting Tyr419 autophosphorylation of c-Src. Molecular docking and surface plasmon resonance analyses showed that RPV can bind directly to c-Src. Further mutational analysis showed that the Phe427 residue of c-Src is critical for RPV interaction, suggesting a novel interaction site for targeting c-Src to block ß3-integrin outside-in signaling. CONCLUSION: These results demonstrated that RPV was able to prevent the progression of thrombotic CVDs by interrupting ß3-integrin-mediated outside-in signaling via inhibiting c-Src activation without hemorrhagic side effects, highlighting RPV as a promising reagent for the prevention and therapy of thrombotic CVDs.
Assuntos
Fármacos Anti-HIV , Trombose , Humanos , Camundongos , Animais , Integrina beta3/metabolismo , Fosforilação , Rilpivirina/metabolismo , Rilpivirina/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Reposicionamento de Medicamentos , Simulação de Acoplamento Molecular , Plaquetas/metabolismo , Trombose/tratamento farmacológico , Trombose/prevenção & controle , Trombose/metabolismo , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacologia , Fibrinogênio/metabolismoRESUMO
The platelet specific integrin αIIbß3 mediates platelet adhesion, aggregation and plays a central role in thrombosis and hemostasis. In resting platelets, αIIbß3 is expressed on the membrane surface and in intracellular compartments. Upon activation, the number of surface-expressed αIIbß3 is increased by the translocation of internal granule pools to the plasma membrane. The WASH complex is the major endosomal actin polymerization-promoting complex and has been implicated in the generation of actin networks involved in endocytic trafficking of integrins in other cell types. The role of the WASH complex and its subunit Strumpellin in platelet function is still unknown. Here, we report that Strumpellin-deficient murine platelets display an approximately 20% reduction in integrin αIIbß3 surface expression. While exposure of the internal αIIbß3 pool after platelet activation was unaffected, the uptake of the αIIbß3 ligand fibrinogen was delayed. The number of platelet α-granules was slightly but significantly increased in Strumpellin-deficient platelets. Quantitative proteome analysis of isolated αIIbß3-positive vesicular structures revealed an enrichment of protein markers, which are associated with the endoplasmic reticulum, Golgi complex and early endosomes in Strumpellin-deficient platelets. These results point to a so far unidentified role of the WASH complex subunit Strumpellin in integrin αIIbß3 trafficking in murine platelets.
Assuntos
Integrina alfa2 , Integrina beta3 , Peptídeos e Proteínas de Sinalização Intracelular , Animais , Camundongos , Plaquetas/metabolismo , Integrina alfa2/metabolismo , Integrina beta3/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Megacariócitos/metabolismo , Camundongos KnockoutRESUMO
The relationship between autism spectrum disorder (ASD) and dendritic spine abnormalities is well known, but it is unclear whether the deficits relate to specific neuron types and brain regions most relevant to ASD. Recent genetic studies have identified a convergence of ASD risk genes in deep layer pyramidal neurons of the prefrontal cortex. Here, we use retrograde recombinant adeno-associated viruses to label specifically two major layer V pyramidal neuron types of the medial prefrontal cortex: the commissural neurons, which put the two cerebral hemispheres in direct communication, and the corticopontine neurons, which transmit information outside the cortex. We compare the basal dendritic spines on commissural and corticopontine neurons in WT and KO mice for the ASD risk gene Itgb3, which encodes for the cell adhesion molecule ß3 integrin selectively enriched in layer V pyramidal neurons. Regardless of the genotype, corticopontine neurons had a higher ratio of stubby to mushroom spines than commissural neurons. ß3 integrin affected selectively spine length in corticopontine neurons. Ablation of ß3 integrin resulted in corticopontine neurons lacking long (> 2 µm) thin dendritic spines. These findings suggest that a deficiency in ß3 integrin expression compromises specifically immature spines on corticopontine neurons, thereby reducing the cortical territory they can sample. Because corticopontine neurons receive extensive local and long-range excitatory inputs before relaying information outside the cortex, specific alterations in dendritic spines of corticopontine neurons may compromise the computational output of the full cortex, thereby contributing to ASD pathophysiology.
Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Camundongos , Animais , Espinhas Dendríticas/metabolismo , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Integrina beta3/genética , Integrina beta3/metabolismo , Células Piramidais/fisiologiaRESUMO
Purpose: To investigate the effect of acupuncture for improving the pregnancy rate of COH rats from the viewpoint of regulating the opening time of the implantation window and endometrial receptivity. Methods: Experimental rats were randomly divided into normal group (N), model group (M) and acupuncture group(A), and samples were collected on Day 4, 5 and 6 after mating. COH rats were treated with acupuncture at SP6, LR3, and ST36 once a day for 7 times. The pinopodes were observed under a scanning electron microscope. Serum estrogen and progesterone levels were measured via ELISA. The protein and mRNA levels of estrogen receptor (ER), progesterone receptor (PR), leukemia inhibitory factor (LIF), integrin ß3, vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF-2) in the endometrium were evaluated via West-blot, immunohistochemistry, and PCR. Results: Compared with group N, the pregnancy rate of group M was significantly decreased (P<0.05), and the abnormal serum hormone levels and implantation window advancement were observed. Compared with group M, the pregnancy rate of group A was significantly increased (P<0.05), the supraphysiological serum progesterone levels were restored to normalcy (P<0.05), and the advanced implantation window was restored to a certain extent. Further, the abnormal ER, PR, LIF, integrin ß3, VEGF, and FGF-2 expression levels of the endometrium got recovered to varying degrees. Conclusion: Acupuncture may restore the estrogen and progesterone balance in COH rats and the forward shift of the implantation window to a certain extent, improving the endometrial receptivity and finally improving the pregnancy rate of COH rats.
Assuntos
Terapia por Acupuntura , Síndrome de Hiperestimulação Ovariana , Gravidez , Humanos , Feminino , Ratos , Animais , Progesterona , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Integrina beta3/genética , Integrina beta3/metabolismo , Integrina beta3/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Endométrio , Estrogênios/metabolismoRESUMO
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by memory loss and personality changes that ultimately lead to dementia. Currently, 50 million people worldwide suffer from dementia related to AD, and the pathogenesis underlying AD pathology and cognitive decline is unknown. While AD is primarily a neurological disease of the brain, individuals with AD often experience intestinal disorders, and gut abnormalities have been implicated as a major risk factor in the development of AD and relevant dementia. However, the mechanisms that mediate gut injury and contribute to the vicious cycle between gut abnormalities and brain injury in AD remain unknown. In the present study, a bioinformatics analysis was performed on the proteomics data of variously aged AD mouse colon tissues. We found that levels of integrin ß3 and ß-galactosidase (ß-gal), two markers of cellular senescence, increased with age in the colonic tissue of mice with AD. The advanced artificial intelligence (AI)-based prediction of AD risk also demonstrated the association between integrin ß3 and ß-gal and AD phenotypes. Moreover, we showed that elevated integrin ß3 levels were accompanied by senescence phenotypes and immune cell accumulation in AD mouse colonic tissue. Further, integrin ß3 genetic downregulation abolished upregulated senescence markers and inflammatory responses in colonic epithelial cells in conditions associated with AD. We provide a new understanding of the molecular actions underpinning inflammatory responses during AD and suggest integrin ß3 may function as novel target mediating gut abnormalities in this disease.
Assuntos
Doença de Alzheimer , Camundongos , Animais , Doença de Alzheimer/genética , Integrina beta3/metabolismo , Inteligência Artificial , Senescência Celular/genética , Inflamação/complicaçõesRESUMO
BACKGROUND: Yishen Tongbi (YSTB) decoction is a patented herbal formula that is used in China to treat rheumatoid arthritis (RA); however, the exact mechanism of its anti-synovial hyperplasia efficacy has not been fully elucidated. PURPOSE: Based on our previous proteomics study, we aimed to reveal whether YSTB inhibits the proliferation and migration of RA-FLSs through the SLC3A2/integrin ß3 pathway in vivo and in vitro. STUDY DESIGN: The study design consists of three parts, a comparison of the expression of SLC3A2 and integrin ß3 in synovial tissues of RA and OA patients; an animal experiment to verify the pharmacodynamic effect of YSTB, and in vitro experiment to elucidate the specific mechanism of YSTB. METHODS: The expression of SLC3A2 and integrin ß3 in the synovial tissues of patients with RA and osteoarthritis (OA) patients were detected by immunohistochemistry (IHC). In vitro, firstly, the proliferation and migration abilities of HFLS (human fibroblast-like synoviocytes) and HFLS-RA (human fibroblast-like synoviocytes-RA) cells were compared by EdU staining and wound healing assays, respectively, and the differences in the expression and localization of SLC3A2, integrin ß3, p-FAK and p-Src between HFLS and HFLS-RA cells were detected by IF and WB. In vivo, DBA/1 mice were injected with bovine collagen II to construct a CIA mouse model. Paw swelling, body weight and the arthritis index (AI) were used as basic treatment evaluation indicators for YSTB. Micro-CT and histopathological analyses of the knee and ankle joints were also performed. In addition, the expression of SLC3A2, integrin ß3, p-FAK and p-Src in the synovial tissue of mice was detected by IHC. Subsequently, CCK-8 was used to screen for suitable concentrations of YSTB for use in HFLS-RA cells. EdU staining and transwell migration assays were performed to evaluate the inhibitory effect of YSTB on cell proliferation and migration, and WB was conducted to assess whether YSTB inhibited HFLS-RA migration through downregulation of the SLC3A2/integrin ß3 pathways. RESULTS: IHC showed that the expression of SLC3A2 and integrin ß3 was higher in RA synovial tissues than in OA tissues. In vivo experiments showed that YSTB inhibited synovial hyperplasia, prevented bone destruction, and reduced the expression of SLC3A2, integrin ß3, p-FAK and p-Src. In vitro experiments showed that YSTB inhibited HFLS-RA migration and proliferation by inhibiting the expression of SLC3A2/integrin ß3 and downstream signaling molecules. CONCLUSION: YSTB inhibits the proliferation and migration of synovial fibroblasts in RA by downregulating the SLC3A2/integrin ß3 pathways.