RESUMO
BACKGROUND: Arteriovenous fistula (AVF) is currently the preferred vascular access for hemodialysis patients. However, the low maturation rate of AVF severely affects its use in patients. A more comprehensive understanding and study of the mechanisms of AVF maturation is urgently needed. METHODS AND RESULTS: In this study, we downloaded the publicly available datasets (GSE119296 and GSE220796) from the Gene Expression Omnibus (GEO) and merged them for subsequent analysis. We screened 84 differentially expressed genes (DEGs) and performed the functional enrichment analysis. Next, we integrated the results obtained from the degree algorithm provided by the Cytohubba plug-in, Molecular complex detection (MCODE) plug-in, weighted gene correlation network analysis (WGCNA), and Least absolute shrinkage and selection operator (LASSO) logistic regression. This integration allowed us to identify CTSG as a hub gene associated with AVF maturation. Through the literature search and Pearson's correlation analysis, the genes matrix metalloproteinase 2 (MMP2) and MMP9 were identified as potential downstream effectors of CTSG. We then collected three immature clinical AVF vein samples and three mature samples and validated the expression of CTSG using immunohistochemistry (IHC) and double-immunofluorescence staining. The IHC results demonstrated a significant decrease in CTSG expression levels in the immature AVF vein samples compared to the mature samples. The results of double-immunofluorescence staining revealed that CTSG was expressed in both the intima and media of AVF veins. Moreover, the expression of CTSG in vascular smooth muscle cells (VSMCs) was significantly higher in the mature samples compared to the immature samples. The results of Masson's trichrome and collagen I IHC staining demonstrated a higher extent of collagen deposition in the media of immature AVF veins compared to the mature. By constructing an in vitro CTSG overexpression model in VSMCs, we found that CTSG upregulated the expression of MMP2 and MMP9 while downregulating the expression of collagen I and collagen III. Furthermore, CTSG was found to inhibit VSMC migration. CONCLUSIONS: CTSG may promote AVF maturation by stimulating the secretion of MMP2 and MMP9 from VSMCs and reducing the extent of medial fibrosis in AVF veins by inhibiting the secretion of collagen I and collagen III.
Assuntos
Fístula Arteriovenosa , Derivação Arteriovenosa Cirúrgica , Humanos , Derivação Arteriovenosa Cirúrgica/efeitos adversos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Catepsina G , Diálise Renal/métodos , Colágeno , Colágeno Tipo I , Fístula Arteriovenosa/etiologiaRESUMO
Heart failure (HF) is a complex clinical syndrome resulting from various cardiac diseases and a significant medical issue worldwide. Although the role of inflammation in HF pathogenesis is well-known, the specific cell types and regulatory molecules involved remain poorly understood. Here, we identified key cell types and novel biomarkers via an analysis of single-cell and bulk RNA sequencing data obtained from patients with two major HF types of ischemic cardiomyopathy and dilated cardiomyopathy. Myeloid cells were identified as the primary cell population involved in HF through cellular fraction and gene set enrichment analysis. Additionally, differential analysis of myeloid cells revealed crosstalk between cellular communication and cytokine-regulated immune responses in HF, with the MIF pathway emerging as a crucial immune regulatory pathway. The CD74/CXCR4 receptor complex in myeloid cell subgroup Mφ2 was significantly upregulated, potentially acting as a crucial regulator in HF. Upon receiving the MIF signal molecule, the CD74/CXCR4 receptor can activate NF-κB signaling to produce chemokines and thereby enhance the inflammatory response. CD74 and CXCR4 may serve as biomarkers and treatment targets for HF.
Assuntos
Insuficiência Cardíaca , Fatores Inibidores da Migração de Macrófagos , Humanos , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , RNA , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/metabolismo , Insuficiência Cardíaca/genética , Análise de Sequência de RNARESUMO
The interaction between integrin α4ß7 and mucosal vascular addressin cell-adhesion molecule-1 (MAdCAM-1) facilitates the adhesion of circulating lymphocytes to the surface of high endothelial venules in inflammatory bowel diseases (IBDs). Lymphocyte adhesion is a multistep cascade involving the tethering, rolling, stable adhesion, crawling, and migration of cells, with integrin α4ß7 being involved in rolling and stable adhesions. Targeting the integrin α4ß7-MAdCAM-1 interaction may help decrease inflammation in IBDs. This interaction is regulated by force; however, the underlying mechanism remains unknown. Here, we investigate this mechanism using a parallel plate flow chamber and atomic force microscopy. The results reveal an initial increase in the lifetime of the integrin α4ß7-MAdCAM-1 interaction followed by a decrease with an increasing force. This was manifested in a two-state curve regulated via a catch-bond-slip-bond conversion regardless of Ca2+ and/or Mg2+ availability. In contrast, the mean rolling velocity of cells initially decreased and then increased with the increasing force, indicating the flow-enhanced adhesion. Longer tether lifetimes of single bonds and lower rolling velocities mediated by multiple bonds were observed in the presence of Mg2+ rather than Ca2+. Similar results were obtained when examining the adhesion to substrates co-coated with chemokine CC motif ligand 25 and MAdCAM-1, as opposed to substrates coated with MAdCAM-1 alone. In conclusion, the integrin α4ß7-MAdCAM-1 interaction occurs via ion- and cytokine-dependent flow-enhanced adhesion processes and is regulated via a catch-bond mechanism.
Assuntos
Imunoglobulinas , Integrinas , Adesão Celular , Imunoglobulinas/química , LinfócitosRESUMO
Bruton's tyrosine kinase (BTK) is a key protein from the TEC family and is involved in B-cell lymphoma occurrence and development. Targeting BTK is therefore an effective strategy for B-cell lymphoma treatment. Since previous studies on BTK have been limited to structure-function analyses of static protein structures, the dynamics of conformational change of BTK upon inhibitor binding remain unclear. Here, molecular dynamics simulations were conducted to investigate the molecular mechanisms of association and dissociation of a reversible (ARQ531) and irreversible (ibrutinib) small-molecule inhibitor to/from BTK. The results indicated that the BTK kinase domain was found to be locked in an inactive state through local conformational changes in the DFG motif, and P-, A-, and gatekeeper loops. The binding of the inhibitors drove the outward rotation of the C-helix, resulting in the upfolded state of Trp395 and the formation of the salt bridge of Glu445-Arg544, which maintained the inactive conformation state. Met477 and Glu475 in the hinge region were found to be the key residues for inhibitor binding. These findings can be used to evaluate the inhibitory activity of the pharmacophore and applied to the design of effective BTK inhibitors. In addition, the drug resistance to the irreversible inhibitor Ibrutinib was mainly from the strong interaction of Cys481, which was evidenced by the mutational experiment, and further confirmed by the measurement of rupture force and rupture times from steered molecular dynamics simulation. Our results provide mechanistic insights into resistance against BTK-targeting drugs and the key interaction sites for the development of high-quality BTK inhibitors. The steered dynamics simulation also offers a means to rapidly assess the binding capacity of newly designed inhibitors.
Assuntos
Linfoma de Células B , Simulação de Dinâmica Molecular , Humanos , Tirosina Quinase da Agamaglobulinemia/metabolismo , Inibidores de Proteínas Quinases/química , Conformação Molecular , Linfoma de Células B/tratamento farmacológicoRESUMO
Diabetic nephropathy (DN) is a type of kidney injuries associated with diabetes mellitus and the prevalence of DN has increased dramatically. However, DN still pose problems in therapy, and prognosis. Identifying new DN biomarkers would be helpful in reducing morbidity and mortality from DN and developing novel preventive approaches. In the study, from GSE36336 dataset with DN glomeruli samples, we screened for 238 differentially expressed genes. Enrichment analysis were performed to find out biological function and diseases of DEGs. Next, depended on protein-protein interaction network, We identified top 10 hub genes (Serpine1, Cxcl10, Cfd, Ppbp, Retn, Socs2, Ccr5, Mmp8, Pf4, Cxcl9) may played potential roles in DN. Meanwhile, transcriptome sequencing on podocyte were performed to reconfirm the reliability of Ppbp. To verify the efficiency of the selected genes as biomarkers, several experiments like qRT-PCR, renal histologic analysis and immunofluorescence were conducted to validate. Our results showed that PPBP have the potential to become a novel biomarker for DN podocyte injury.
Assuntos
Quimiocinas CXC/genética , Biologia Computacional/métodos , Nefropatias Diabéticas/genética , Perfilação da Expressão Gênica/métodos , Podócitos/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem Celular , Quimiocinas CXC/metabolismo , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Masculino , Camundongos Endogâmicos C57BL , Mapas de Interação de Proteínas/genética , Reprodutibilidade dos TestesRESUMO
Human urinary induced pluripotent stem cells (hUiPSCs) produced from exfoliated renal epithelial cells present in urine may provide a non-invasive source of endothelial progenitors for the treatment of ischaemic diseases. However, their differentiation efficiency is unsatisfactory and the underlying mechanism of differentiation is still unknown. Gremlin1 (GREM1) is an important gene involved in cell differentiation. Therefore, we tried to elucidate the roles of GREM1 during the differentiation and expansion of endothelial progenitors. HUiPSCs were induced into endothelial progenitors by three stages. After differentiation, GREM1 was obviously increased in hUiPSC-induced endothelial progenitors (hUiPSC-EPs). RNA interference (RNAi) was used to silence GREM1 expression in three stages, respectively. We demonstrated a stage-specific effect of GREM1 in decreasing hUiPSC-EP differentiation in the mesoderm induction stage (Stage 1), while increasing differentiation in the endothelial progenitors' induction stage (Stage 2) and expansion stage (Stage 3). Exogenous addition of GREM1 recombinant protein in the endothelial progenitors' expansion stage (Stage 3) promoted the expansion of hUiPSC-EPs although the activation of VEGFR2/Akt or VEGFR2/p42/44MAPK pathway. Our study provided a new non-invasive source for endothelial progenitors, demonstrated critical roles of GREM1 in hUiPSC-EP and afforded a novel strategy to improve stem cell-based therapy for the ischaemic diseases.
Assuntos
Diferenciação Celular/genética , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Linhagem Celular , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Imunofenotipagem , Modelos Biológicos , RNA Interferente Pequeno/genéticaRESUMO
The PSGL-1-actin cytoskeleton linker proteins ezrin/radixin/moesin (ERM), an adaptor between P-selectin glycoprotein ligand-1 (PSGL-1) and spleen tyrosine kinase (Syk), is a key player in PSGL-1 signal, which mediates the adhesion and recruitment of leukocytes to the activated endothelial cells in flow. Binding of PSGL-1 to ERM initials intracellular signaling through inducing phosphorylation of Syk, but effects of tensile force on unligation and phosphorylation site exposure of ERM bound with PSGL-1 remains unclear. To answer this question, we performed a series of so-called "ramp-clamp" steered molecular dynamics (SMD) simulations on the radixin protein FERM domain of ERM bound with intracellular juxtamembrane PSGL-1 peptide. The results showed that, the rupture force of complex pulled with constant velocity was over 250 pN, which prevented the complex from breaking in front of pull-induced exposure of phosphorylation site on immunoreceptor tyrosine activation motif (ITAM)-like motif of ERM; the stretched complex structure under constant tensile forces <100 pN maintained on a stable quasi-equilibrium state, showing a high mechano-stabilization of the clamped complex; and, in consistent with the force-induced allostery at clamped stage, increasing tensile force (<50 pN) would decrease the complex dissociation probability but facilitate the phosphorylation site exposure, suggesting a force-enhanced biophysical connectivity of PSGL-1 signaling. These force-enhanced characters in both phosphorylation and unligation of ERM bound with PSGL-1 should be mediated by a catch-slip bond transition mechanism, in which four residue interactions on binding site were involved. This study might provide a novel insight into the transmembrane PSGL-1 signal, its biophysical connectivity and molecular structural basis for cellular immune responses in mechano-microenvironment, and showed a rational SMD-based computer strategy for predicting structure-function relation of protein under loads.
Assuntos
Proteínas do Citoesqueleto , Domínios FERM , Glicoproteínas de Membrana , Proteínas de Membrana , Animais , Sítios de Ligação , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Simulação de Dinâmica Molecular , Ligação ProteicaRESUMO
Prostate cancer (PCa) is the third most common reason of cancer-related deaths in men. Accumulating evidence has shown that dysregulation of long noncoding RNAs (lncRNAs) is closely related to cancer initiation and development. Although large numbers of lncRNAs have been discovered, knowledge regarding their function and physiological/pathological significance remains limited. In this study, we aimed to reveal functional lncRNAs and identify prognosis-related RNAs in PCa by analyzing data from The Cancer Genome Atlas (TCGA). To achieve this, an lncRNA-mRNA coexpression network was constructed by weighted correlation network analysis. Additionally, a subnetwork was extracted from this weighted correlation network, and seven lncRNAs were identified as core nodes. Further Kaplan-Meier survival analysis showed that three lncRNAs (LINC00683, LINC00857, and FENDRR) were significantly downregulated in PCa samples, and there was a strong positive correlation with patient survival. Importantly, LINC00683 has not been fully reported as related with PCa. Additionally, gene set enrichment analysis indicated that LINC00683 might be involved in cancer-related pathways such as the Wnt pathway. Based on the findings of this study, lncRNA LINC00683 is likely to provide a new diagnostic biomarker and therapeutic target for future PCa treatments.
Assuntos
Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , RNA Longo não Codificante/genética , Humanos , Masculino , Prognóstico , Mapas de Interação de Proteínas/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Regulação para Cima/genética , Via de Sinalização Wnt/genéticaRESUMO
We in this study measured the site density of E-selectin in order to explore the practical pliability using radionuclide labeling method and γ-imaging of single photon emission computer tomography (SPECT). This method required labeling of antibody with 125I using Indogen method and binding of the labeled antibody to E-selectin. Labeled E-selectin was separated and purified in a Sephadex G25 column. The different fractions of the eluants were imaged, analyzed and quantified with SPECT method. For measuring the saturation curve of E-selectin, 130 µL of E-selectin solution with different concentrations were added in a 48-well plate and incubated overnight at 4â. After incubation, 130 µL of labeled antibody solution were added and kept incubated for 30 min. The resulted mixture was washed, and the radioactivity in each sample was detected by SPECT. The levels of radioactivity were translated to site densities, and were used to plot a standard curve. The labeled product was quantitatively analyzed with SPECT. The labeling rate of E-selectin was 78%. The saturation curve of different concentration samples showed that when the concentration was in the concentration range of 0-1 mg/mL, the standard curve was y=6 045.7 x-51.166, R2=0.997 9. Based on this finding, it could be concluded that γ-imaging is an important tool for analysis of radiolabeled product and determination of site density.
RESUMO
Selectin-ligand interactions mediate tethering and rolling of circulating leukocytes on the vessel wall during inflammation. Extensive study has been devoted to elucidating the kinetic and mechanical constraints of receptor-ligand-interaction-mediated leukocyte adhesion, yet many questions remain unanswered. Here, we describe our design of an inverted flow chamber to compare adhesions of HL-60 cells to E-selectin in the upright and inverted orientations. This new, to our knowledge, design allowed us to evaluate the effect of gravity and to investigate the mechanisms of flow-enhanced adhesion. Cell rolling in the two orientations was qualitatively similar, and the quantitative differences can be explained by the effect of gravity, which promotes free-flowing cells to tether and detached cells to reattach to the surface in the upright orientation but prevents such attachment from happening in the inverted orientation. We characterized rolling stability by the lifetime of rolling adhesion and detachment of rolling cells, which could be easily measured in the inverted orientation, but not in the upright orientation because of the reattachment of transiently detached cells. Unlike the transient tether lifetime of E-selectin-ligand interaction, which exhibited triphasic slip-catch-slip bonds, the lifetime of rolling adhesion displayed a biphasic trend that first increased with the wall shear stress, reached a maximum at 0.4 dyn/cm(2), and then decreased gradually. We have developed a minimal mathematical model for the probability of rolling adhesion. Comparison of the theoretical predictions to data has provided model validation and allowed evaluation of the effective two-dimensional association on-rate, kon, and the binding affinity, Ka, of the E-selectin-ligand interaction. kon increased with the wall shear stress from 0.1 to 0.7 dyn/cm(2). Ka first increased with the wall shear stress, reached a maximum at 0.4 dyn/cm(2), and then decreased gradually. Our results provide insights into how the interplay between flow-dependent on-rate and off-rate of E-selectin-ligand bonds determine flow-enhanced cell rolling stability.
Assuntos
Adesão Celular , Selectina E/metabolismo , Migração e Rolagem de Leucócitos , Leucócitos/citologia , Células HL-60 , Humanos , Cinética , Modelos BiológicosRESUMO
Atherosclerosis is a complex disease characterized by lipid accumulation in the vascular wall and influenced by multiple genetic and environmental factors. To understand the mechanisms of molecular regulation related to atherosclerosis better, a protein interaction network was constructed in the present study. Genes were collected in nucleotide database and interactions were downloaded from Biomolecular Object Network Database (BOND). The interactional data were imported into the software Cytoscape to construct the interaction network, and then the degree characteristics of the network were analyzed for Hub proteins. Statistical significance pathways and diseases were figured out by inputting Hub proteins to KOBAS2. 0. The complete pathway network related to atherosclerosis was constructed. The results identified a series of key genes related to atherosclerosis, which would be the potential promising drug targets for effective prevention.
Assuntos
Aterosclerose/genética , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Bases de Dados Factuais , Humanos , SoftwareRESUMO
Endogenous viral elements (EVEs) are widely distributed throughout eukaryotic genomes, and their evolution and potential function have attracted a lot of interest. Draft genome sequences for Schistosoma mansoni, Schistosoma japonicum and Schistosoma haematobium are now available; however, information about EVEs in blood flukes of the genus schistosoma is scanty. Here, genome-wide survey into the putative EVE sequences of the three key schistosome genomes were present. Totally 4, 117 gene sequences were identified, including retrovirus-like gypsy elements, RNA viruses and dsDNA viruses. Compared with S. japonicum and S. haematobium, S. mansoni appeared to greatly out numbered by gypsy members. Phylogenetic analysis revealed one novel endogenous retrovirus element in S. mansoni. This initial characterization of schistosomes showed that schistosomes harbour distinct EVEs that may have played an important evolutionary role. Studies of schistosomes' endogenous viruses helped us to glance at an earlier viral event in the class Trematoda, greatly broadening the field of palaeovirology.
Assuntos
Vírus de DNA/isolamento & purificação , Retrovirus Endógenos/isolamento & purificação , Vírus de RNA/isolamento & purificação , Schistosoma/virologia , Animais , Humanos , Schistosoma/genética , Schistosoma haematobium/virologia , Schistosoma japonicum/virologia , Schistosoma mansoni/virologiaRESUMO
Gliomas are the most prevalent primary malignant tumors affecting the brain, with high recurrence and mortality rates. Accurate diagnoses and effective treatment challenges persist, emphasizing the need for identifying new biomarkers to guide clinical decisions. Long noncoding RNAs (lncRNAs) hold potential as diagnostic and therapeutic biomarkers in cancer. However, only a limited subset of lncRNAs in gliomas have been explored. Therefore, this study aims to identify lncRNA signatures applicable to patients with gliomas across all grades and explore their clinical significance and potential biological mechanisms. Data used in this study were obtained from TCGA, CGGA, and GEO datasets to identify key lncRNA signatures in gliomas through differential and survival analyses and machine learning algorithms. We examined their associations with the clinical characteristics, gene mutations, diagnosis, and prognosis of gliomas. Functional enrichment analysis was employed to elucidate the potential biological mechanisms associated with these significant lncRNA signatures. We explored competing endogenous RNA (ceRNA) regulatory networks. We found that NDUFA6-DT emerged as a significant lncRNA signature in gliomas, with reduced NDUFA6-DT expression associated with a worse prognosis in gliomas. Nomogram analysis incorporating NDUFA6-DT expression levels exhibited excellent prognostic and predictive capabilities. Functional annotation suggested that NDUFA6-DT might influence immunological responses and synaptic transmission, potentially modifying glioma initiation and progression. The associated ceRNA network revealed the possible presence of the NDUFA6-DT-miR-455-3p-YWHAH/YWHAG axis in low-grade glioma (LGG) and glioblastoma multiforme (GBM), regulating the PI3K-AKT signaling pathway and influencing glioma cell survival and apoptosis. We believe that NDUFA6-DT is a novel lncRNA linked to glioma diagnosis and prognosis, potentially becoming a pivotal biomarker for glioma.
Assuntos
Biomarcadores Tumorais , Neoplasias Encefálicas , Regulação Neoplásica da Expressão Gênica , Glioma , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , Glioma/genética , Glioma/patologia , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Prognóstico , Redes Reguladoras de GenesRESUMO
E-selectin-mediated rolling on vascular surface of circulating leukocyte on vascular surface is a key initial event during inflammatory response and lymphocyte homing. This event depends not only on the specific interactions of adhesive molecules but also on the hemodynamics of blood flow. Little is still understood about whether wall shear stress or shear rate regulates the rolling. With flow chamber techniques, we here measured the effects of transport, shear stress and cell deformation on rolling of both unfixed and fixed HL-60 cells on E-selectin either in the absence or in the presence of 3% Ficoll in medium at various wall shear stresses from 0.05 to 0.7 dyn/cm(2). The results demonstrated a triphasic force-dependent rolling, that is, as increasing of force, the rolling would be accelerated firstly, then followed a decelerating phase occurred at the initial shear threshold of about 0.1 dyn/cm(2), and lastly returned to an accelerating process starting at the optimal shear threshold of 0.35 dyn/cm(2) approximately. The catch bond regime was completely reflected to rolling behaviors, such as tether lifetime, cell stop time and rolling velocity, meaning that the dominant factor to govern rolling is force. The initial shear threshold might be the minimum level of wall shear stress to sustain a stationary rolling, and the optimal shear threshold would make rolling to the most stable and regular. These findings strongly elucidate the catch bond mechanism for flow-enhanced rolling through E-selectin since longer bond lifetimes led to slower and stabler rolling.
Assuntos
Selectina E/metabolismo , Anticorpos Monoclonais/farmacologia , Sítios de Ligação/efeitos dos fármacos , Ácido Edético/farmacologia , Células HL-60 , Humanos , LigantesRESUMO
MAdCAM-1 binds to integrin α4ß7, which mediates the rolling and arrest of circulating lymphocytes upon the vascular endothelia during lymphocytic homing. The calcium response by adhered lymphocytes is a critical event for lymphocyte activation and subsequent arrest and migration under flow. However, whether the interaction of integrin α4ß7 /MAdCAM-1 can effectively trigger the calcium response of lymphocytes remains unclear, as well as whether the fluid force affects the calcium response. In this study, we explore the mechanical regulation of integrin α4ß7-induced calcium signaling under flow. Flou-4 AM was used to examine the calcium response under real-time fluorescence microscopy when cells were firmly adhered to a parallel plate flow chamber. The interaction between integrin α4ß7 and MAdCAM-1 was found to effectively trigger calcium signaling in firmly adhered RPMI 8226 cells. Meanwhile, increasing fluid shear stress accelerated the cytosolic calcium response and enhanced signaling intensity. Additionally, the calcium signaling of RPMI 8226 activated by integrin α4ß7 originated from extracellular calcium influx instead of cytoplasmic calcium release, and the signaling transduction of integrin α4ß7 was involved in Kindlin-3. These findings shed new light on the mechano-chemical mechanism of calcium signaling in RPMI 8226 cells induced by integrin α4ß7.
Assuntos
Cálcio , Integrinas , Cálcio/metabolismo , Sinalização do Cálcio , Integrinas/metabolismo , Linfócitos/metabolismo , Fenômenos Mecânicos , HumanosRESUMO
It is crucial for hemostasis that platelets are rapidly recruited to the site of vascular injury by the adhesive ligand von Willebrand factor (VWF) multimers. The metalloproteinase ADAMTS13 regulates this hemostatic activity by proteolytically reducing the size of VWF and its proteolytic kinetics has been investigated by biochemical and single-molecule biophysical methods. However, how ADAMTS13 cleaves VWF in flowing blood remains poorly defined. To investigate the force-induced VWF cleavage, VWF A1A2A3 tridomains were immobilized and subjected to hydrodynamic forces in the presence of ADAMTS13. We demonstrated that the cleavage of VWF A1A2A3 by ADAMTS13 exhibited biphasic kinetics governed by shear stress, but not shear rate. By fitting data to the single-molecule Michaelis-Menten equation, the proteolytic constant kcat of ADAMTS13 had two distinct states. The mean proteolytic constant of the fast state (kcat-fast) was 0.005 ± 0.001 s-1, which is >10-fold faster than the slow state (kcat-slow = 0.0005 ± 0.0001 s-1). Furthermore, proteolytic constants of both states were regulated by shear stress in a biphasic manner, independent of the solution viscosity, indicating that the proteolytic activity of ADAMTS13 was regulated by hydrodynamic force. The findings provide new insights into the mechanism underlying ADAMTS13 cleaving VWF under flowing blood.
Assuntos
Hemostasia , Fator de von Willebrand , Plaquetas , Proteína ADAMTS13RESUMO
All-trans retinoic acid (ATRA)-induced differentiation of acute promyelocytic leukemia (APL) toward granulocytes may trigger APL differentiation syndrome (DS), but there is less knowledge about the mechano-chemical regulation mechanism of APL DS under the mechano-microenvironment. We found that ATRA-induced changes in proliferation, morphology, and adhesive molecule expression levels were either dose or stimulus time dependent. An optimal ATRA stimulus condition for differentiating HL60 cells toward neutrophils consisted of 1 × 10-6 M dose and 120 h of stimulus time. Under wall shear stresses, catch-slip bond transition governs P-selectin-mediated rolling for neutrophils and untreated or ATRA-treated (1 × 10-6 M, 120 h) HL60 cells. The ATRA stimuli slowed down the rolling of HL60 cells on immobilized P-selectin no matter whether ICAM-1 was engaged. The ß2 integrin near the PSGL-1/P-selectin axis would be activated within sub-seconds for each cell group mentioned above, thus contributing to slow rolling. A faster ß2 integrin activation rate and the higher expression levels of PSGL-1 and LFA-1 were assigned to induce the over-enhancement of ATRA-treated HL60 adhesion in flow, causing APL DS development. These findings provided an insight into the mechanical-chemical regulation for APL DS development via ATRA treatment of leukemia and a novel therapeutic strategy for APL DS through targeting the relevant adhesion molecules.
Assuntos
Leucemia Promielocítica Aguda , Selectina-P , Humanos , Células HL-60 , Antígenos CD18 , Tretinoína/farmacologia , Tretinoína/uso terapêutico , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismoRESUMO
Coronary artery disease (CAD) is considered one of the leading causes of death worldwide. Although dysregulation of long non-coding RNAs (lncRNAs) has been reported to be associated with the initiation and progression of CAD, the knowledge regarding their specific functions as well their physiological/pathological significance in CAD is very limited. In this study, we aimed to systematically analyze immune-related lncRNAs in CAD and explore the relationship between key immune-related lncRNAs and the immune cell infiltration process. Based on differential expression analysis of mRNAs and lncRNAs, an immune-related lncRNA-mRNA weighted gene co-expression network containing 377 lncRNAs and 119 mRNAs was constructed. LINC01480 and AL359237.1 were identified as the hub immune-related lncRNAs in CAD using the random forest-recursive feature elimination and least absolute shrinkage and selection operator logistic regression. Furthermore, 93 CAD samples were divided into two subgroups according to the expression values of LINC01480 and AL359237.1 by consensus clustering analysis. By performing gene set enrichment analysis, we found that cluster 2 enriched more cardiovascular risk pathways than cluster 1. The immune cell infiltration analysis of ischemic cardiomyopathy (ICM; an advanced stage of CAD) samples revealed that the proportion of macrophage M2 was upregulated in the LINC01480 highly expressed samples, thus suggesting that LINC01480 plays a protective role in the progression of ICM. Based on the findings of this study, lncRNA LINC01480 may be used as a novel biomarker and therapeutic target for CAD.
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Diabetic nephropathy (DN) is the leading cause of end-stage renal disease and has become a serious medical issue globally. Although it is known to be associated with glomerular injury, tubular injury has been found to participate in DN in recent years. However, mechanisms of diabetic renal tubular injury remain unclear. Here, we investigated the differentially expressed genes in the renal tubules of patients with DN by analyzing three RNA-seq datasets downloaded from the Gene Expression Omnibus database. Gene set enrichment analysis and weighted gene co-expression network analysis showed that DN is highly correlated with the immune system. The immune-related gene SERPINA3 was screened out with lasso regression and Kaplan-Meier survival analyses. Considering that SERPINA3 is an inhibitor of mast cell chymase, we examined the expression level of SERPINA3 and chymase in human renal tubular biopsies and found that SERPINA3 was upregulated in DN tubules, which is consistent with the results of the differential expression analysis. Besides, the infiltration and degranulation rates of mast cells are augmented in DN. By summarizing the biological function of SERPINA3, chymase, and mast cells in DN based on our results and those of previous studies, we speculated that SERPINA3 is a protective immune-related molecule that prevents renal tubular injury by inhibiting the proliferation and activation of mast cells and downregulating the activity of chymase.
Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Serpinas , Humanos , Nefropatias Diabéticas/patologia , Quimases/metabolismo , Rim/patologia , Túbulos Renais/patologia , Biomarcadores/metabolismo , Diabetes Mellitus/patologia , Serpinas/genética , Serpinas/metabolismoRESUMO
Accumulating evidence indicates that the N6-methyladenosine (m6A) modification plays a critical role in human cancers. Given the current understanding of m6A modification, this process is believed to be dynamically regulated by m6A regulators. Although the discovery of m6A regulators has greatly enhanced our understanding of the mechanism underlying m6A modification in cancers, the function and role of m6A in the context of prostate cancer (PCa) remain unclear. Here, we aimed to establish a comprehensive diagnostic scoring model that can act as a complement to prostate-specific antigen (PSA) screening. To achieve this, we first drew the landscape of m6A regulators and constructed a LASSO-Cox model using three risk genes (METTL14, HNRNP2AB1, and YTHDF2). Particularly, METTL14 expression was found to be significantly related to overall survival, tumor T stage, relapse rate, and tumor microenvironment of PCa patients, showing that it has important prognostic value. Furthermore, for the sake of improving the predictive ability, we presented a comprehensive diagnostic scoring model based on a novel 6-gene panel by combining with genes found in our previous study, and its application potential was further validated by the whole TCGA and ICGC cohorts. Our study provides additional clues and insights regarding the treatment and diagnosis of PCa patients.