RESUMO
Endothelial cells, forming a monolayer along blood vessels, intricately regulate vascular hemostasis, inflammatory responses, and angiogenesis. A key determinant of these functions is the controlled secretion of Weibel-Palade bodies (WPBs), which are specialized endothelial storage organelles housing a presynthesized pool of the hemostatic protein von Willebrand factor and various other hemostatic, inflammatory, angiogenic, and vasoactive mediators. This review delves into recent mechanistic insights into WPB biology, including the biogenesis that results in their unique morphology, the acquisition of intraluminal vesicles and other cargo, and the contribution of proton pumps to organelle acidification. Additionally, in light of a number of proteomic approaches to unravel the regulatory networks that control WPB formation and secretion, we provide a comprehensive overview of the WPB exocytotic machinery, including their molecular and cellular mechanisms.
Assuntos
Células Endoteliais , Exocitose , Corpos de Weibel-Palade , Fator de von Willebrand , Corpos de Weibel-Palade/metabolismo , Humanos , Fator de von Willebrand/metabolismo , Animais , Células Endoteliais/metabolismo , Proteômica/métodos , HemostasiaRESUMO
BACKGROUND: Weibel-Palade bodies (WPBs) are endothelial cell-specific cigar-shaped secretory organelles containing various biologically active molecules. WPBs play crucial roles in thrombosis, hemostasis, angiogenesis, and inflammation. The main content of WPBs is the procoagulant protein vWF (von Willebrand factor). Physical contacts and functional cross talk between mitochondria and other organelles have been demonstrated. Whether an interorganellar connection exists between mitochondria and WPBs is unknown. METHODS: We observed physical contacts between mitochondria and WPBs in human umbilical vein endothelial cells by electron microscopy and living cell confocal microscopy. We developed an artificial intelligence-assisted method to quantify the duration and length of organelle contact sites in live cells. RESULTS: We found there existed physical contacts between mitochondria and WPBs. Disruption of mitochondrial function affected the morphology of WPBs. Furthermore, we found that Rab3b, a small GTPase on the WPBs, was enriched at the mitochondrion-WPB contact sites. Rab3b deficiency reduced interaction between the two organelles and impaired the maturation of WPBs and vWF multimer secretion. CONCLUSIONS: Our results reveal that Rab3b plays a crucial role in mediating the mitochondrion-WPB contacts, and that mitochondrion-WPB coupling is critical for the maturation of WPBs in vascular endothelial cells.
Assuntos
Corpos de Weibel-Palade , Fator de von Willebrand , Humanos , Corpos de Weibel-Palade/metabolismo , Fator de von Willebrand/metabolismo , Inteligência Artificial , Exocitose , Células Endoteliais da Veia Umbilical Humana/metabolismo , Mitocôndrias/metabolismo , Células CultivadasRESUMO
BACKGROUND: Von Willebrand factor (VWF) and VWF propeptide (VWFpp) are stored in eccentric nanodomains within platelet alpha-granules. VWF and VWFpp can undergo differential secretion following Weibel-Palade body exocytosis in endothelial cells; however, it is unclear if the same process occurs during platelet alpha-granule exocytosis. Using a high-throughput 3-dimensional super-resolution imaging workflow for quantification of individual platelet alpha-granule cargo, we studied alpha-granule cargo release in response to different physiological stimuli. OBJECTIVES: To investigate how VWF and VWFpp are released from alpha-granules in response to physiological stimuli. METHODS: Platelets were activated with protease-activated receptor 1 (PAR-1) activating peptide (PAR-1 ap) or collagen-related peptide (CRP-XL). Alpha-tubulin, VWF, VWFpp, secreted protein acidic and cysteine rich (SPARC), and fibrinogen were imaged using 3-dimensional structured illumination microscopy, followed by semiautomated analysis in FIJI. Uptake of anti-VWF nanobody during degranulation was used to identify alpha-granules that partially released content. RESULTS: VWFpp overlapped with VWF in eccentric alpha-granule subdomains in resting platelets and showed a higher degree of overlap with VWF than SPARC or fibrinogen. Activation of PAR-1 (0.6-20 µM PAR-1 ap) or glycoprotein VI (GPVI) (0.25-1 µg/mL CRP-XL) signaling pathways caused a dose-dependent increase in alpha-granule exocytosis. More than 80% of alpha-granules remained positive for VWF, even at the highest agonist concentrations. In contrast, the residual fraction of alpha-granules containing VWFpp decreased in a dose-dependent manner to 23%, whereas SPARC and fibrinogen were detected in 60% to 70% of alpha-granules when stimulated with 20 µM PAR-1 ap. Similar results were obtained using CRP-XL. Using an extracellular anti-VWF nanobody, we identified VWF in postexocytotic alpha-granules. CONCLUSION: We provide evidence for differential secretion of VWF and VWFpp from individual alpha-granules.
Assuntos
Células Endoteliais , Fator de von Willebrand , Humanos , Fator de von Willebrand/metabolismo , Células Endoteliais/metabolismo , Corpos de Weibel-Palade/metabolismo , Plaquetas/metabolismo , Fibrinogênio/metabolismo , ExocitoseRESUMO
BACKGROUND: The von Willebrand factor (VWF) is a multimeric plasma glycoprotein essential for hemostasis, inflammation, and angiogenesis. The majority of VWF is synthesized by endothelial cells (ECs) and stored in Weibel-Palade bodies (WPB). Among the range of proteins shown to co-localize to WPB is angiopoietin-2 (Angpt-2), a ligand of the receptor tyrosine kinase Tie-2. We have previously shown that VWF itself regulates angiogenesis, raising the hypothesis that some of the angiogenic activity of VWF may be mediated by its interaction with Angpt-2. METHODS: Static-binding assays were used to probe the interaction between Angpt-2 and VWF. Binding in media from cultured human umbilical vein ECs s and in plasma was determined by immunoprecipitation experiments. Immunofluorescence was used to detect the presence of Angpt-2 on VWF strings, and flow assays were used to investigate the effect on VWF function. RESULTS: Static-binding assays revealed that Angpt-2 bound to VWF with high affinity (KD,app â¼3 nM) in a pH and calcium-dependent manner. The interaction was localized to the VWF A1 domain. Co-immunoprecipitation experiments demonstrated that the complex persisted following stimulated secretion from ECs and was present in plasma. Angpt-2 was also visible on VWF strings on stimulated ECs. The VWF-Angpt-2 complex did not inhibit the binding of Angpt-2 to Tie-2 and did not significantly interfere with VWF-platelet capture. CONCLUSIONS: Together, these data demonstrate a direct binding interaction between Angpt-2 and VWF that persists after secretion. VWF may act to localize Angpt-2; further work is required to establish the functional consequences of this interaction.
Assuntos
Corpos de Weibel-Palade , Fator de von Willebrand , Humanos , Fator de von Willebrand/metabolismo , Corpos de Weibel-Palade/metabolismo , Angiopoietina-2/metabolismo , Exocitose , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células CultivadasRESUMO
An in-frame heterozygous large deletion of exons 4 through 34 of the von Willebrand factor (VWF) gene was identified in a type 3 von Willebrand disease (VWD) index patient (IP), as the only VWF variant. The IP exhibited severe bleeding episodes despite prophylaxis treatment, with a short VWF half-life after infusion of VWF/factor VIII concentrates. Transcript analysis confirmed transcription of normal VWF messenger RNA besides an aberrant deleted transcript. The IP endothelial colony-forming cells (ECFCs) exhibited a defect in the VWF multimers and Weibel-Palade bodies (WPBs) biogenesis, although demonstrating normal VWF secretion compared with healthy cells. Immunostaining of IP-ECFCs revealed subcellular mislocalization of WPBs pro-inflammatory cargos angiopoietin-2 (Ang2, nuclear accumulation) and P-selectin. Besides, the RNA-sequencing (RNA-seq) analysis showed upregulation of pro-inflammatory and proangiogenic genes, P-selectin, interleukin 8 (IL-8), IL-6, and GROα, copackaged with VWF into WPBs. Further, whole-transcriptome RNA-seq and subsequent gene ontology (GO) enrichment analysis indicated the most enriched GO-biological process terms among the differentially expressed genes in IP-ECFCs were regulation of cell differentiation, cell adhesion, leukocyte adhesion to vascular endothelial, blood vessel morphogenesis, and angiogenesis, which resemble downstream signaling pathways associated with inflammatory stimuli and Ang2 priming. Accordingly, our functional experiments exhibited an increased endothelial cell adhesiveness and interruption in endothelial cell-cell junctions of the IP-ECFCs. In conclusion, the deleted VWF has a dominant-negative impact on multimer assembly and the biogenesis of WPBs, leading to altered trafficking of their pro-inflammatory cargos uniquely, which, in turn, causes changes in cellular signaling pathways, phenotype, and function of the endothelial cells.
Assuntos
Selectina-P , Fator de von Willebrand , Células Endoteliais/metabolismo , Humanos , Selectina-P/metabolismo , Fenótipo , Corpos de Weibel-Palade/metabolismo , Fator de von Willebrand/metabolismoRESUMO
The plasma glycoprotein von Willebrand factor (VWF) is exclusively synthesized in endothelial cells (ECs) and megakaryocytes, the precursor cells of platelets. Its primary function lies in hemostasis. However, VWF is much more than just a "fishing hook" for platelets and a transporter for coagulation factor VIII. VWF is a true multitasker when it comes to its many roles in cellular processes. In ECs, VWF coordinates the formation of Weibel-Palade bodies and guides several cargo proteins to these storage organelles, which control the release of hemostatic, inflammatory and angiogenic factors. Leukocytes employ VWF to assist their rolling on, adhesion to and passage through the endothelium. Vascular smooth muscle cell proliferation is supported by VWF, and it regulates angiogenesis. The life cycle of platelets is accompanied by VWF from their budding from megakaryocytes to adhesion, activation and aggregation until the end in apoptosis. Some tumor cells acquire the ability to produce VWF to promote metastasis and hide in a shell of VWF and platelets, and even the maturation of osteoclasts is regulated by VWF. This review summarizes the current knowledge on VWF's versatile cellular functions and the resulting pathophysiological consequences of their dysregulation.
Assuntos
Fator de von Willebrand/metabolismo , Animais , Apoptose/fisiologia , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Hemostasia/fisiologia , Humanos , Leucócitos/metabolismo , Miócitos de Músculo Liso/metabolismo , Corpos de Weibel-Palade/metabolismoRESUMO
von Willebrand factor (VWF) is an essential hemostatic protein that is synthesized and secreted by endothelial cells and stored in Weibel-Palade bodies (WPBs). The secretory Rab GTPases Rab27A, Rab3B, and Rab3D have been linked with WPB trafficking and secretion. How these Rabs are activated and recruited to WPBs remains elusive. In this study, we identified MAP kinase-activating death domain (MADD) as the guanine nucleotide exchange factor for Rab27A and both Rab3 isoforms in primary human endothelial cells. Rab activity assays revealed a reduction in Rab27A, Rab3B, and Rab3D activation upon MADD silencing. Rab activation, but not binding, was dependent on the differentially expressed in normal and neoplastic cells (DENN) domain of MADD, indicating the potential existence of 2 Rab interaction modules. Furthermore, immunofluorescent analysis showed that Rab27A, Rab3B, and Rab3D recruitment to WPBs was dramatically decreased upon MADD knockdown, revealing that MADD drives Rab membrane targeting. Artificial mistargeting of MADD using a TOMM70 tag abolished Rab27A localization to WPB membranes in a DENN domain-dependent manner, indicating that normal MADD localization in the cytosol is crucial. Activation of Rab3B and Rab3D was reduced upon Rab27A silencing, suggesting that activation of these Rabs is enhanced through previous activation of Rab27A by MADD. MADD silencing did not affect WPB morphology, but it did reduce VWF intracellular content. Furthermore, MADD-depleted cells exhibited decreased histamine-evoked VWF release, similar to Rab27A-depleted cells. In conclusion, MADD acts as a master regulator of VWF secretion by coordinating the activation and membrane targeting of secretory Rabs to WPBs.
Assuntos
Corpos de Weibel-Palade , Proteínas rab de Ligação ao GTP , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte , Células Endoteliais/metabolismo , Exocitose , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato , Humanos , Corpos de Weibel-Palade/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The generation of cell-penetrating peptides as cargo-delivery systems has produced an immense number of studies owing to the importance of these systems as tools to deliver molecules into the cells, as well as due to the interest to shed light into a yet unclear mechanism of the entrance of these peptides into the cells. However, many cell-penetrating peptides might present drawbacks due to causing cellular toxicity, or due to being entrapped in endosomes, or as a result of their degradation before they meet their target. In this work, a cargo transporting molecule, the Cell Penetrating Sequential Oligopeptide Carrier (CPSOC), formed by the repetitive -Lys-Aib-Cys- moiety, was tested for its ability to penetrate the cell membrane and transport the conjugated peptides into the cells. The cysteine residue anchors bioactive molecules through a stable thioether bond. The lysine supplies the positive charge to the construct, whereas the α-amino isobutyric acid is well known to induce helicoid conformation to the peptide backbone and protects from enzymatic degradation. The present study demonstrates that CPSOC penetrates the membrane transporting the conjugated cargo into the cell. When we tested CPSOC-conjugated peptides carrying critical domains of Cdc42, a small GTPase implicated in exocytosis, the internalized peptides were found to be functional because they inhibited exocytosis of von Willebrand factor from endothelial Weibel-Palade bodies a trafficking event depending on the Cdc42 protein. The data suggest that the carrier can deliver efficiently functional peptides into the cells, and thus, it can be used as a multiple-cargo transporting molecule.
Assuntos
Peptídeos Penetradores de Células , Corpos de Weibel-Palade , Exocitose , Fator de von WillebrandRESUMO
Estrogen therapy is used to treat patients with post-menopausal symptoms, such as hot flashes and dyspareunia. Estrogen therapy also decreases the risk of fractures from osteoporosis in post-menopausal women. However, estrogen increases the risk of venous thromboembolic events, such as pulmonary embolism, but the pathways through which estrogen increase the risk of thromboembolism is unknown. Here, we show that estrogen elicits endothelial exocytosis, the key step in vascular thrombosis and inflammation. Exogenous 17ß-estradiol (E2) stimulated endothelial exocytosis of Weibel-Palade bodies (WPBs), releasing von Willebrand factor (vWF) and interleukin-8 (IL-8). Conversely, the estrogen antagonist ICI-182,780 interfered with E2-induced endothelial exocytosis. The ERα agonist propyl pyrazole triol (PPT) but not the ERß agonist diarylpropionitrile (DPN) induced vWF release, while ERα silencing counteracted vWF release by E2, suggesting that ERα mediates this effect. Exocytosis triggered by E2 occurred rapidly within 15 min and was not inhibited by either actinomycin D or cycloheximide. On the contrary, it was inhibited by the pre-treatment of U0126 or SB203580, an ERK or a p38 inhibitor, respectively, suggesting that E2-induced endothelial exocytosis is non-genomically mediated by the MAP kinase pathway. Finally, E2 treatment enhanced platelet adhesion to endothelial cells ex vivo, which was interfered with the pre-treatment of ICI-182,780 or U0126. Taken together, our data show that estrogen activates endothelial exocytosis non-genomically through the ERα-MAP kinase pathway. Our data suggest that adverse cardiovascular effects such as vascular inflammation and thrombosis should be considered in patients before menopausal hormone treatment.
Assuntos
Células Endoteliais/efeitos dos fármacos , Estradiol/efeitos adversos , Exocitose/efeitos dos fármacos , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Terapia de Reposição de Estrogênios/efeitos adversos , Exocitose/fisiologia , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Adesividade Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/fisiologia , Pós-Menopausa/efeitos dos fármacos , Pós-Menopausa/fisiologia , Fatores de Risco , Tromboembolia/etiologia , Corpos de Weibel-Palade/efeitos dos fármacos , Corpos de Weibel-Palade/patologia , Corpos de Weibel-Palade/fisiologiaRESUMO
Ca2+ regulates a variety of cellular processes that are essential to maintain cell integrity and function. Different methods have been used to study these processes by increasing intracellular Ca2+ levels. Here, we describe a protocol to initiate Ca2+-dependent membrane-related events, using laser ablation by near-infrared irradiation. This creates a rupture in the plasma membrane that allows the extracellular Ca2+ to enter the cell and thereby induce a receptor-independent Ca2+ increase. We report laser ablation protocols to study two different Ca2+-induced processes in human endothelial cells-membrane resealing and exocytosis of secretory granules called Weibel-Palade bodies (WPBs). Thus, laser ablation represents a technique that permits the analysis of different Ca2+-regulated processes at high spatiotemporal resolution in a controlled manner.
Assuntos
Células Endoteliais/metabolismo , Exocitose/genética , Terapia a Laser/métodos , Fator de von Willebrand/genética , Cálcio/metabolismo , Membrana Celular/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Corpos de Weibel-Palade/genéticaRESUMO
Osteoprotegerin (OPG) regulates bone metabolism by reducing the activation of osteoclasts, but may also be involved in blood vessel calcification and atherosclerosis. Within endothelial cells OPG is stored in Weibel-Palade bodies (WPBs). Blood kinetics of OPG are essentially unknown. We aimed to assess these using two distinct in vivo models; one after stimulation with desmopressin (DDAVP) and another after stimulation with lipopolysaccharide (LPS). Both clinical trials were conducted at the Department of Clinical Pharmacology at the Medical University of Vienna, Austria. Participants received desmopressin (0.3 µg/kg), LPS (2 ng/kg), or placebo (sodium chloride 0.9%) with subsequent blood sampling at time points up to 24 hours after administration. The primary objective of this study was to investigate the plasma kinetics of OPG after stimulation with desmopressin and LPS. Secondary analyses included the release of other WPB contents including von Willebrand factor (vWF). This analysis included 31 healthy volunteers (n = 16 for desmopressin and placebo, n = 15 for LPS). Infusion of desmopressin did not increase OPG concentrations compared with placebo, while LPS infusion significantly increased OPG levels, both compared with desmopressin (p < 0.0001) and to placebo (p = 0.004), with a maximum of â¼twofold increase in OPG levels â¼6 hours after infusion. von Willebrand factor levels increased after both desmopressin and LPS infusion (p < 0.0001), with a maximum of â¼threefold increase 2 hours after desmopressin and a maximum of â¼twofold increase 6 hours after LPS administration. In conclusion, we report that, in contrast to vWF, OPG is not released upon stimulation with desmopressin, but increases significantly during experimental endotoxemia.
Assuntos
Desamino Arginina Vasopressina/farmacologia , Células Endoteliais/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Osteoprotegerina/sangue , Corpos de Weibel-Palade/efeitos dos fármacos , Áustria , Biomarcadores/sangue , Estudos Cross-Over , Método Duplo-Cego , Células Endoteliais/metabolismo , Voluntários Saudáveis , Humanos , Cinética , Projetos Piloto , Corpos de Weibel-Palade/metabolismo , Fator de von Willebrand/metabolismoRESUMO
Objective Weibel-Palade bodies (WPBs) are endothelial cell (EC)-specific organelles formed by vWF (von Willebrand factor) polymerization and that contain the proangiogenic factor Ang-2 (angiopoietin-2). WPB exocytosis has been shown to be implicated for vascular repair and inflammatory responses. Here, we investigate the role of WPBs during angiogenesis and vessel stabilization. Approach and Results WPB density in ECs decreased at the angiogenic front of retinal vascular network during development and neovascularization compared with stable vessels. In vitro, VEGF (vascular endothelial growth factor) induced a VEGFR-2 (vascular endothelial growth factor receptor-2)-dependent exocytosis of WPBs that contain Ang-2 and consequently the secretion of vWF and Ang-2. Blocking VEGF-dependant WPB exocytosis and Ang-2 secretion promoted pericyte migration toward ECs. Pericyte migration was inhibited by adding recombinant Ang-2 or by silencing Ang-1 (angiopoietin-1) or Tie2 (angiopoietin-1 receptor) in pericytes. Consistently, in vivo anti-VEGF treatment induced accumulation of WPBs in retinal vessels because of the inhibition of WPB exocytosis and promoted the increase of pericyte coverage of retinal vessels during angiogenesis. In tumor angiogenesis, depletion of WPBs in vWF knockout tumor-bearing mice promoted an increase of tumor angiogenesis and a decrease of pericyte coverage of tumor vessels. By another approach, normalized tumor vessels had higher WPB density. Conclusions We demonstrate that WPB exocytosis and Ang-2 secretion are regulated during angiogenesis to limit pericyte coverage of remodeling vessels by disrupting Ang-1/Tie2 autocrine signaling in pericytes.
Assuntos
Neovascularização Patológica/fisiopatologia , Neovascularização Fisiológica/fisiologia , Pericitos/fisiologia , Corpos de Weibel-Palade/fisiologia , Angiopoietina-2/fisiologia , Animais , Células Cultivadas , Células Endoteliais/fisiologia , Exocitose , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/irrigação sanguínea , Retina/fisiologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Essentials Endothelial colony forming cells (ECFCs) are a powerful tool to study vascular diseases ex vivo. Separate ECFC lines show variations in morphology and von Willebrand factor-related parameters. Maximum cell density is correlated with von Willebrand factor expression in ECFCs. Variations in ECFC lines are dependent on the age and mesenchymal state of the cells. ABSTRACT: Background Endothelial colony forming cells (ECFCs) are cultured endothelial cells derived from peripheral blood. ECFCs are a powerful tool to study pathophysiological mechanisms underlying vascular diseases, including von Willebrand disease. In prior research, however, large variations between ECFC lines were observed in, among others, von Willebrand factor (VWF) expression. Objective Understand the relation between phenotypic characteristics and VWF-related parameters of healthy control ECFCs. Methods ECFC lines (n = 16) derived from six donors were studied at maximum cell density. Secreted and intracellular VWF antigen were measured by ELISA. The angiogenic capacity of ECFCs was investigated by the Matrigel tube formation assay. Differences in expression of genes involved in angiogenesis, aging, and endothelial to mesenchymal transition (EndoMT) were measured by quantitative PCR. Results Different ECFC lines show variable morphologies and cell density at maximum confluency and cell lines with a low maximum cell density show a mixed and more mesenchymal phenotype. We identified a significant positive correlation between maximum cell density and VWF production, both at protein and mRNA level. Also, significant correlations were observed between maximum cell density and several angiogenic, aging and EndoMT parameters. Conclusions We observed variations in morphology, maximum cell density, VWF production, and angiogenic potential between healthy control ECFCs. These variations seem to be attributable to differences in aging and EndoMT. Because variations correlate with cell density, we believe that ECFCs maintain a powerful tool to study vascular diseases. It is however important to compare cell lines with the same characteristics and perform experiments at maximum cell density.
Assuntos
Células Progenitoras Endoteliais/metabolismo , Fator de von Willebrand/metabolismo , Contagem de Células , Forma Celular , Transdiferenciação Celular , Células Cultivadas , Senescência Celular , Colágeno , Meios de Cultivo Condicionados/química , Combinação de Medicamentos , Células Progenitoras Endoteliais/ultraestrutura , Expressão Gênica , Humanos , Laminina , Mesoderma/citologia , Neovascularização Fisiológica , Proteoglicanas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Corpos de Weibel-Palade/química , Fator de von Willebrand/análise , Fator de von Willebrand/genéticaRESUMO
Endothelial cells selectively release cargo stored in Weibel-Palade bodies (WPBs) to regulate vascular function, but the underlying mechanisms are poorly understood. Here we show that histamine evokes the release of the proinflammatory ligand, P-selectin, while diverting WPBs carrying non-inflammatory cargo away from the plasma membrane to the microtubule organizing center. This differential trafficking is dependent on Rab46 (CRACR2A), a newly identified Ca2+-sensing GTPase, which localizes to a subset of P-selectin-negative WPBs. After acute stimulation of the H1 receptor, GTP-bound Rab46 evokes dynein-dependent retrograde transport of a subset of WPBs along microtubules. Upon continued histamine stimulation, Rab46 senses localized elevations of intracellular calcium and evokes dispersal of microtubule organizing center-clustered WPBs. These data demonstrate for the first time that a Rab GTPase, Rab46, integrates G protein and Ca2+ signals to couple on-demand histamine signals to selective WPB trafficking.
Assuntos
Sinalização do Cálcio/genética , Proteínas de Ligação ao Cálcio/genética , Receptores Histamínicos H1/genética , Corpos de Weibel-Palade/genética , Membrana Celular/genética , Dineínas/genética , Exocitose/genética , Proteínas de Ligação ao GTP/genética , Histamina/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Microtúbulos/genética , Selectina-P/genética , Transporte Proteico/genética , Transdução de Sinais/genética , Corpos de Weibel-Palade/metabolismoRESUMO
Weibel-Palade bodies (WPBs) are secretory granules that contain von Willebrand factor and P-selectin, molecules that regulate hemostasis and inflammation, respectively. The presence of CD63/LAMP3 in the limiting membrane of WPBs has led to their classification as lysosome-related organelles. Many lysosome-related organelles contain intraluminal vesicles (ILVs) enriched in CD63 that are secreted into the extracellular environment during cell activation to mediate intercellular communication. To date, there are no reports that WPBs contain or release ILVs. By light microscopy and live-cell imaging, we show that CD63 is enriched in microdomains within WPBs. Extracellular antibody recycling studies showed that CD63 in WPB microdomains can originate from the plasma membrane. By cryo-electron tomography of frozen-hydrated endothelial cells, we identify internal vesicles as novel structural features of the WPB lumen. By live-cell fluorescence microscopy, we directly observe the exocytotic release of EGFP-CD63 ILVs as discrete particles from individual WPBs. WPB exocytosis provides a novel route for release of ILVs during endothelial cell stimulation.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Exocitose/fisiologia , Corpos de Weibel-Palade/metabolismo , Micropartículas Derivadas de Células/ultraestrutura , Células Cultivadas , Microscopia Crioeletrônica , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Humanos , Tetraspanina 30/metabolismo , Corpos de Weibel-Palade/ultraestruturaRESUMO
OBJECTIVE: Endothelial cells store VWF (von Willebrand factor) in rod-shaped secretory organelles, called Weibel-Palade bodies (WPBs). WPB exocytosis is coordinated by a complex network of Rab GTPases, Rab effectors, and SNARE (soluble NSF attachment protein receptor) proteins. We have previously identified STXBP1 as the link between the Rab27A-Slp4-a complex on WPBs and the SNARE proteins syntaxin-2 and -3. In this study, we investigate the function of syntaxin-3 in VWF secretion. APPROACH AND RESULTS: In human umbilical vein endothelial cells and in blood outgrowth endothelial cells (BOECs) from healthy controls, endogenous syntaxin-3 immunolocalized to WPBs. A detailed analysis of BOECs isolated from a patient with variant microvillus inclusion disease, carrying a homozygous mutation in STX3(STX3-/-), showed a loss of syntaxin-3 protein and absence of WPB-associated syntaxin-3 immunoreactivity. Ultrastructural analysis revealed no detectable differences in morphology or prevalence of immature or mature WPBs in control versus STX3-/- BOECs. VWF multimer analysis showed normal patterns in plasma of the microvillus inclusion disease patient, and media from STX3-/- BOECs, together indicating WPB formation and maturation are unaffected by absence of syntaxin-3. However, a defect in basal as well as Ca2+- and cAMP-mediated VWF secretion was found in the STX3-/- BOECs. We also show that syntaxin-3 interacts with the WPB-associated SNARE protein VAMP8 (vesicle-associated membrane protein-8). CONCLUSIONS: Our data reveal syntaxin-3 as a novel WPB-associated SNARE protein that controls WPB exocytosis.
Assuntos
Células Endoteliais/metabolismo , Exocitose , Síndromes de Malabsorção/metabolismo , Microvilosidades/patologia , Mucolipidoses/metabolismo , Proteínas Qa-SNARE/metabolismo , Corpos de Weibel-Palade/metabolismo , Fator de von Willebrand/metabolismo , Cálcio/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Células Endoteliais/ultraestrutura , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Síndromes de Malabsorção/diagnóstico , Síndromes de Malabsorção/genética , Microvilosidades/genética , Microvilosidades/metabolismo , Mucolipidoses/diagnóstico , Mucolipidoses/genética , Mutação , Proteínas Qa-SNARE/genética , Proteínas R-SNARE/metabolismo , Via Secretória , Transdução de Sinais , Corpos de Weibel-Palade/ultraestruturaRESUMO
The synaptic protein Neuroligin 2, similarly to its isoform Neuroligin 1, is produced by endothelial cells, but its activity in the vascular context remains unknown. This study aimed at verifying the hypothesis that Neuroligin 2, in parallel with its extraneuronal involvement in pancreatic beta cells exocytosis, modulated cytokine release from endothelial cells and consequently angiogenesis. We used in vitro approaches to modulate Neuroligin 2 expression and Neuroligin 2 null mice to test our hypotheses. In vitro, upon VEGF stimulation, Neuroligin 2 silencing strongly reduces Angiopoietin 2 release in the medium and increases the endothelial cell retention of Weibel Palade Bodies, the specialized organelles that store Angiopoietin 2 and various other cytokines. On the contrary, Neuroligin 2 overexpression almost depletes cells of Weibel Palade Bodies, independent of VEGF. In vivo, both the retina and tumor xenografts grown in NLGN2- null mice display an immature vasculature, with lower pericyte coverage and lower Tie2 phosphorylation. At the molecular level NLGN2 colocalizes with its neuronal partner collibystin, a CDC42 guanine nucleotide exchange factor, which is also expressed by endothelial cells and in turn modulates Angiopoietin 2 release. Neuroligin 2, an inhibitory synaptic protein, modulates a peculiar aspect of vascular function and could represent a novel target of therapy in various fields, from tumor angiogenesis to vascular diseases.
Assuntos
Angiopoietina-2/metabolismo , Moléculas de Adesão Celular Neuronais/fisiologia , Neovascularização Fisiológica , Proteínas do Tecido Nervoso/fisiologia , Animais , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Vasos Retinianos/citologia , Vasos Retinianos/fisiologia , Fatores de Troca de Nucleotídeo Guanina Rho/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Corpos de Weibel-Palade/fisiologia , Fator de von Willebrand/metabolismoRESUMO
The use of propranolol for the treatment of infantile hemangioma (IH) has been widely investigated in recent years. However, the underlying therapeutic mechanism of propranolol for the treatment of IH remains poorly understood. The aim of the present study was to investigate the expression of proteins regulated by cellular tumor antigen p53 (p53) in associated apoptosis pathways in IH endothelial cells (HemECs) treated with propranolol. Furthermore, the present study aimed to investigate the exact apoptotic pathway underlying the therapeutic effect of propranolol against IH. In the present study, HemECs were subcultured and investigated using an inverted phase contrast microscope, immunocytochemical staining and a scanning electron microscope (SEM). Experimental groups and blank control groups were prepared. All groups were subjected to drug treatment. A high p53 expression model of HemECs was successfully established via transfection, and a low p53 expression model of HemECs was established using pifithrinα. The apoptosis rate of each group was determined using Annexin Vfluorescein isothiocyanate/propidium iodide double staining and flow cytometry. The expression levels of downstream proteins regulated by p53 [tumour necrosis factor receptor superfamily member 6 (FAS), p53induced death domaincontaining protein (PIDD), death receptor 5 (DR5), BH3interacting domain death agonist (BID), apoptosis regulator BAX (BAX), p53 unregulated modulator of apoptosis (PUMA), phosphatidylinositolglycan biosynthesis class S protein (PIGS), and insulinlike growth factorbinding protein 3 (IGFBP3)] were revealed in the experimental and control groups via western blotting. Microscopic observation revealed the growth of an adherent monolayer of cells, which were closely packed and exhibited contact inhibition. Immunocytochemical staining demonstrated increased expression of clotting factor VIII. SEM analysis revealed presence of WeibelPalade bodies. The results of the analyses verified that the cultured cells were HemECs. The staining of the samples resulted in a significantly increased rate of apoptosis in experimental groups compared with the blank control group. This result suggested that there is an association between p53 expression and the rate of apoptosis of propranololtreated HemECs. The results of the western blot analysis demonstrated an upregulation of BAX expression and a downregulation of IGFBP3 expression in the HemECs treated with propranolol. There were no significant differences in the expression levels of FAS, DR5, PIDD, BID, PUMA and PIGS between experimental and control groups. This result suggests that p53 has an important role in HemEC apoptosis. The results of the present study additionally suggest that the propranololinduced HemEC apoptosis pathway is a mitochondrial apoptosis pathway and is regulated by p53BAX signaling.
Assuntos
Apoptose/efeitos dos fármacos , Hemangioma/tratamento farmacológico , Hemangioma/metabolismo , Propranolol/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismo , Apoptose/genética , Células Endoteliais , Feminino , Hemangioma/genética , Hemangioma/patologia , Humanos , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Propranolol/farmacologia , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética , Corpos de Weibel-Palade/genética , Corpos de Weibel-Palade/metabolismo , Corpos de Weibel-Palade/patologia , Proteína X Associada a bcl-2/genéticaRESUMO
Telocytes (TCs) are stromal cells defined by the presence of long and slender prolongations (telopodes). They are a biologically and functionally heterogeneous population that has not been previously investigated in the sclera. The purpose of this study is to investigate the presence and characteristics of scleral telocytes through a combined immunohistochemical and transmission electron microscopy (TEM) study using samples from ten adult patients. Stromal cells with a TC-like morphology expressed CD34, CD45, CD105, vimentin and occasionally CD68 but were negative for collagen III, CD31, CD133, and CD146. Conjunctival epithelial cells expressed CD45, CD105, CD146, and vimentin. These phenotypes support a scleral niche with immune TCs and haematopoietic stem cells (HSCs). In TEM, we often found spindle-shaped stromal cells projecting telopodes or filopodes, with extremely long nuclei extended even within those prolongations. We separated these cells into a light subtype, which contained a complete set of organelles, and a dark subtype, consisting of undifferentiated stem/progenitor cells. The light cells contained dense vesicles, Weibel-Palade bodies, and rounded α-granule-like structures. These storage areas for the von Willebrand factor (vWF) are known to express selectins that are critically involved in HSC homing and could also indicate endothelial progenitors. The dark cells were scarcely myoid, populated the episcleral perivascular niches and the scleral stroma, and were equipped with lipid storage areas such as lamellar bodies and lipid droplets (LDs). Previously, unreported intranuclear LDs were found in these cells, which is characteristic of an HSC population. It appears that the human scleral stroma is a niche harbouring TC-like cells with immune and HSC phenotypes, and the mere presence or characteristics of telopodes are not enough to differentiate them.