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1.
Methods Mol Biol ; 2206: 39-46, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754809

RESUMO

During angiogenesis, endothelial cells must undergo a coordinated set of morphological changes in order to form a new vessel. There is a need for endothelial cells to communicate with each other in order to take up different identities in the sprout and to migrate collectively as a connected chord. Endothelial cells must also interact with a wide range of other cells that contribute to vessel formation. In ischemic disease, hypoxic cells in tissue will generate proangiogenic signals that promote and guide angiogenesis. In solid tumors, this function is co-opted by tumor cells, which make a complex range of interactions with endothelial cells, even integrating into the walls of vessels. In vessel repair, cells from the immune system contribute to the promotion and remodeling of new vessels. The coculture angiogenesis assay is a long-term in vitro protocol that uses fibroblasts to secrete and condition an artificial stromal matrix for tubules to grow through. We show here how the assay can be easily adapted to include additional cell types, facilitating the study of cellular interactions during neovascularization.

2.
Small GTPases ; : 1-5, 2020 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-32857689

RESUMO

Angiogenesis is the formation of new blood vessels from pre-existing ones. Angiogenesis requires endothelial cells to change shape and polarity, as well as acquire the ability to directionally migrate ‒ processes that are classically regulated by the Rho family of GTPases. RhoJ (previously TCL) is an endothelium enriched Rho GTPase with a 78% amino acid similarity to the ubiquitously expressed Cdc42. In our recent publication, we demonstrate that α5ß1 integrin co-traffics with RhoJ. RhoJ specifically represses the internalization of the active α5ß1 conformer, leading to a reduced ability of endothelial cells to form fibronectin fibrils. Surprisingly, this function of RhoJ is in opposition to the role of Cdc42, a known driver of fibrillogenesis. Intriguingly, we discovered that the competition for limiting amounts of the shared effector, PAK3, could explain the ability of these two Rho GTPases to regulate fibrillogenesis in opposing directions. Consequently, RhoJ null mice show excessive fibronectin deposition around retinal vessels, possibly due to the unopposed action of Cdc42. Our work suggests that the functional antagonism between RhoJ and Cdc42 could restrict fibronectin remodelling to sites of active angiogenesis to form a provisional matrix for vessel growth. One correlate of our findings is that RhoJ dependent repression of fibronectin remodelling could be atheroprotective in quiescent vessels.

3.
Diabetologia ; 63(10): 2205-2217, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32728894

RESUMO

AIMS/HYPOTHESIS: Treatment of vascular complications of diabetes remains inadequate. We reported that muscle pericytes (MPs) from limb muscles of vascular patients with diabetes mellitus display elevated levels of oxidative stress causing a dysfunctional phenotype. Here, we investigated whether treatment with dimethyl-2-oxoglutarate (DM-2OG), a tricarboxylic acid cycle metabolite with antioxidant properties, can restore a healthy metabolic and functional phenotype. METHODS: MPs were isolated from limb muscles of diabetes patients with vascular disease (D-MPs) and from non-diabetic control participants (ND-MPs). Metabolic status was assessed in untreated and DM-2OG-treated (1 mmol/l) cells using an extracellular flux analyser and anion-exchange chromatography-mass spectrometry (IC-MS/MS). Redox status was measured using commercial kits and IC-MS/MS, with antioxidant and metabolic enzyme expression assessed by quantitative RT-PCR and western blotting. Myogenic differentiation and proliferation and pericyte-endothelial interaction were assessed as functional readouts. RESULTS: D-MPs showed mitochondrial dysfunction, suppressed glycolytic activity and reduced reactive oxygen species-buffering capacity, but no suppression of antioxidant systems when compared with ND-MP controls. DM-2OG supplementation improved redox balance and mitochondrial function, without affecting glycolysis or antioxidant systems. Nonetheless, this was not enough for treated D-MPs to regain the level of proliferation and myogenic differentiation of ND-MPs. Interestingly, DM-2OG exerted a positive effect on pericyte-endothelial cell interaction in the co-culture angiogenesis assay, independent of the diabetic status. CONCLUSIONS/INTERPRETATION: These novel findings support the concept of using DM-2OG supplementation to improve pericyte redox balance and mitochondrial function, while concurrently allowing for enhanced pericyte-endothelial crosstalk. Such effects may help to prevent or slow down vasculopathy in skeletal muscles of people with diabetes. Graphical abstract.

4.
Angiogenesis ; 23(3): 371-383, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32274611

RESUMO

BACKGROUND: VEGFR2 (vascular endothelial growth factor receptor 2) is the major pro-angiogenic receptor in endothelial cells. Compared to other members of the receptor tyrosine kinase family, we know relatively few VEGFR2 signaling partners. Our objective was to use mass spectrometry-based proteomics to identify novel binding partners of activated VEGFR2. METHODS: We created an endothelial cell line stably expressing GFP-tagged VEGFR2 and isolated activated receptor complexes. Analysis by mass spectrometry identified raftlin as a novel binding partner of VEGFR2. RESULTS: We found that raftlin is recruited to the activated VEGFR2 complex via the co-receptor Nrp1 (neuropilin-1). We show that raftlin regulates the surface levels of Nrp1 in endothelial cells, controlling the availability of Nrp1 for VEGFR2 interaction. Raftlin stabilizes active VEGFR2 at the cell surface by inhibiting endocytosis of the activated receptor. Raftlin also promotes recycling of internalized VEGFR2 to the cell surface. Raftlin alters the signaling outcomes of VEGFR2 activation, inhibiting the activation of p38 and FAK (focal adhesion kinases) specifically. Both pathways are linked to cell migration in endothelial cells, and raftlin inhibits endothelial cell migration in response to VEGF. CONCLUSION: Nrp1 is an important co-receptor for VEGFR2; however, its functions are still only partially understood. We show that raftlin works with Nrp1 in endothelial cells to control intracellular trafficking of the activated VEGFR2. This modulates the response to VEGF and controls endothelial cell migration.

5.
Curr Biol ; 30(11): 2146-2155.e5, 2020 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-32302585

RESUMO

Rho guanosine triphosphatases (GTPases) are master regulators of cell shape and cell movement [1]. The archetypal family members RhoA, Rac1, and Cdc42 arose early in eukaryotic evolution and coordinate a diverse range of cell morphologies and migrations. Evolution of the vertebrates was paralleled by expansion of this family through gene duplication. Emergence of an adaptive immune system and more complex neural systems presented new roles for Rho GTPases, filled by new family members. Cdc42 underwent gene duplication to produce two related proteins-RhoQ and RhoJ [2]. RhoQ is active in neural dynamics; however, RhoJ is highly expressed in endothelial cells under control of the endothelial-specific promoter ERG [3, 4]. RhoJ is required for angiogenesis [5, 6] and has multiple roles in this process [7, 8]. We recently demonstrated that RhoJ regulates the endosomal trafficking of podocalyxin during angiogenesis to control lumen formation [9]. Here, we use vesicle purification and proteomic analysis to identify the endothelial targets of RhoJ-mediated trafficking. We identify α5ß1 integrin as a major RhoJ cargo and show that RhoJ regulates the intracellular trafficking of active α5ß1 integrin in endothelial cells to repress fibronectin fibrillogenesis. Accordingly, mice lacking RhoJ show deregulated deposition of fibronectin around vessels during developmental angiogenesis. Intriguingly, we show that RhoJ acts in opposition to Cdc42 in this process through competition for a shared partner, PAK3. These studies identify a critical role for RhoJ in matrix remodeling during blood vessel formation and demonstrate a functional interrelationship between RhoJ and its evolutionary parent.

6.
FASEB J ; 33(11): 12277-12287, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31431053

RESUMO

Endothelial cells (ECs) form an active barrier between the circulation and the body. In addition to controlling transport of molecules between these 2 compartments, the endothelium is a major secretory organ, releasing proteins both into the circulation and into the vascular matrix. Although it is clearly important that proteins are correctly sorted into these 2 spaces, we currently know little of the polarization of this secretion or how it is controlled. Here, we present an optimized system for the analysis of polarized secretion and show that it allows the derivation of deep, robust proteomes from small numbers of primary ECs. We present the first endothelial apically and basolaterally secreted proteomes, demonstrating that ECs polarize the secretion of extracellular vesicle cargoes to the apical surface. Conversely, we find that protein secretion at the basolateral surface is focused on components of the extracellular matrix (ECM). Finally, we examine the role of liprin-α1 in secretion toward the basolateral compartment and identify a subset of ECM components that share this route with fibronectin.-Wei, H., Sundararaman, A., Dickson, E., Rennie-Campbell, L., Cross, E., Heesom, K. J., Mellor, H. Characterization of the polarized endothelial secretome.

7.
EMBO J ; 37(13)2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29866703

RESUMO

Wound angiogenesis is an integral part of tissue repair and is impaired in many pathologies of healing. Here, we investigate the cellular interactions between innate immune cells and endothelial cells at wounds that drive neoangiogenic sprouting in real time and in vivo Our studies in mouse and zebrafish wounds indicate that macrophages are drawn to wound blood vessels soon after injury and are intimately associated throughout the repair process and that macrophage ablation results in impaired neoangiogenesis. Macrophages also positively influence wound angiogenesis by driving resolution of anti-angiogenic wound neutrophils. Experimental manipulation of the wound environment to specifically alter macrophage activation state dramatically influences subsequent blood vessel sprouting, with premature dampening of tumour necrosis factor-α expression leading to impaired neoangiogenesis. Complementary human tissue culture studies indicate that inflammatory macrophages associate with endothelial cells and are sufficient to drive vessel sprouting via vascular endothelial growth factor signalling. Subsequently, macrophages also play a role in blood vessel regression during the resolution phase of wound repair, and their absence, or shifted activation state, impairs appropriate vessel clearance.


Assuntos
Macrófagos/fisiologia , Neovascularização Fisiológica , Cicatrização/fisiologia , Animais , Animais Geneticamente Modificados , Células Cultivadas , Diagnóstico por Imagem , Fibroblastos , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos Endogâmicos C57BL , Peixe-Zebra/genética
8.
Antioxid Redox Signal ; 28(2): 110-130, 2018 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-28793782

RESUMO

AIMS: Deoxyribose-1-phosphate (dRP) is a proangiogenic paracrine stimulus released by cancer cells, platelets, and macrophages and acting on endothelial cells. The objective of this study was to clarify how dRP stimulates angiogenic responses in human endothelial cells. RESULTS: Live cell imaging, electron paramagnetic resonance, pull-down of dRP-interacting proteins, followed by immunoblotting, gene silencing of different NADPH oxidases (NOXs), and their regulatory cosubunits by small interfering RNA (siRNA) transfection, and experiments with inhibitors of the sugar transporter glucose transporter 1 (GLUT1) were utilized to demonstrate that dRP acts intracellularly by directly activating the endothelial NOX2 complex, but not NOX4. Increased reactive oxygen species generation in response to NOX2 activity leads to redox-dependent activation of the transcription factor nuclear factor kappa B (NF-κB), which, in turn, induces vascular endothelial growth factor receptor 2 (VEGFR2) upregulation. Using endothelial tube formation assays, gene silencing by siRNA, and antibody-based receptor inhibition, we demonstrate that the activation of NF-κB and VEGFR2 is necessary for the angiogenic responses elicited by dRP. The upregulation of VEGFR2 and NOX2-dependent stimulation of angiogenesis by dRP were confirmed in excisional wound and Matrigel plug vascularization assays in vivo using NOX2-/- mice. INNOVATION: For the first time, we demonstrate that dRP acts intracellularly and stimulates superoxide anion generation by direct binding and activation of the NOX2 enzymatic complex. CONCLUSIONS: This study describes a novel molecular mechanism underlying the proangiogenic activity of dRP, which involves the sequential activation of NOX2 and NF-κB and upregulation of VEGFR2. Antioxid. Redox Signal. 28, 110-130.


Assuntos
NADPH Oxidase 2/metabolismo , NF-kappa B/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Ribosemonofosfatos/farmacologia , Linhagem Celular , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
9.
BMC Cancer ; 17(1): 145, 2017 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-28219369

RESUMO

BACKGROUND: RhoBTB1 and 2 are atypical members of the Rho GTPase family of signaling proteins. Unlike other Rho GTPases, RhoBTB1 and 2 undergo silencing or mutation in a wide range of epithelial cancers; however, little is known about the consequences of this loss of function. METHODS: We analyzed transcriptome data to identify transcriptional targets of RhoBTB2. We verified these using Q-PCR and then used gene silencing and cell imaging to determine the cellular function of these targets downstream of RhoBTB signaling. RESULTS: RhoBTB1 and 2 regulate the expression of the methyltransferases METTL7B and METTL7A, respectively. RhoBTB1 regulates the integrity of the Golgi complex through METTL7B. RhoBTB1 is required for expression of METTL7B and silencing of either protein leads to fragmentation of the Golgi. Loss of RhoBTB1 expression is linked to Golgi fragmentation in breast cancer cells. Restoration of normal RhoBTB1 expression rescues Golgi morphology and dramatically inhibits breast cancer cell invasion. CONCLUSION: Loss of RhoBTB1 expression in breast cancer cells leads to Golgi fragmentation and hence loss of normal polarity.


Assuntos
Neoplasias da Mama/metabolismo , Complexo de Golgi/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Transporte , Linhagem Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Proteínas rho de Ligação ao GTP
10.
Methods Mol Biol ; 1430: 159-66, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27172952

RESUMO

During angiogenesis, endothelial cells invade into the stromal matrix: a complex, structured array of extracellular matrix proteins. This three-dimensional deformable substrate also contains a mixture of angiogenic factors as well as embedded stromal cells. Interactions between endothelial cells and the stromal tissue make complex and important contributions to the process of angiogenesis; however, the composition of the stromal matrix is hard to replicate in vitro. The coculture angiogenesis assay is a long-term assay that uses fibroblasts to secrete and condition a stromal matrix that more closely mimics tissue than a simple collagen gel. Like all in vitro assays of angiogenesis, it has both strengths and weaknesses. Here we give protocols for the two of the most useful applications of the assay: screening for regulators of angiogenesis and high-resolution imaging.


Assuntos
Técnicas de Cocultura/métodos , Células Epiteliais/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Neovascularização Fisiológica , Imagem Óptica
11.
Curr Biol ; 25(17): 2325-31, 2015 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-26299518

RESUMO

Angiogenesis is the fundamental process by which new blood vessels form from pre-existing vasculature. It plays a critical role in the formation of the vasculature during development and is triggered in response to tissue hypoxia in adult organisms. This process requires complex and coordinated regulation of the endothelial cell cytoskeleton to control cell shape and polarity. In our previous work, we showed that the cytoskeletal regulator FMNL3/FRL2 controls the alignment of stabilized microtubules during polarized endothelial cell elongation and that depletion of FMNL3 retards elongation of the intersegmental vessels in zebrafish. Recent work has shown that FMNL3 is also needed for vascular lumen formation, a critical element of the formation of functional vessels. Here, we show that FMNL3 interacts with Cdc42 and RhoJ, two Rho family GTPases known to be required for lumen formation. FMNL3 and RhoJ are concentrated at the early apical surface, or AMIS, and regulate the formation of radiating actin cables from this site. In diverse biological systems, formins mediate polarized trafficking through the generation of similar actin filaments tracks. We show that FMNL3 and RhoJ are required for polarized trafficking of podocalyxin to the early apical surface--an important event in vascular lumenogenesis.


Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas/genética , Sialoglicoproteínas/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/genética , Citoesqueleto/fisiologia , Fibroblastos , Forminas , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Transporte Proteico , Proteínas/metabolismo , Sialoglicoproteínas/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
12.
Biochem J ; 466(3): 431-42, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25748676

RESUMO

Rho GTPases are critical for platelet function. Although the roles of RhoA, Rac and Cdc42 are characterized, platelets express other Rho GTPases, whose activities are less well understood. This review summarizes our understanding of the roles of platelet Rho GTPases and focuses particularly on the functions of Rif and RhoG. In human platelets, Rif interacts with cytoskeleton regulators including formins mDia1 and mDia3, whereas RhoG binds SNARE-complex proteins and cytoskeletal regulators ELMO and DOCK1. Knockout mouse studies suggest that Rif plays no critical functions in platelets, likely due to functional overlap with other Rho GTPases. In contrast, RhoG is essential for normal granule secretion downstream of the collagen receptor GPVI. The central defect in RhoG-/- platelets is reduced dense granule secretion, which impedes integrin activation and aggregation and limits platelet recruitment to growing thrombi under shear, translating into reduced thrombus formation in vivo. Potential avenues for future work on Rho GTPases in platelets are also highlighted, including identification of the key regulator for platelet filopodia formation and investigation of the role of the many Rho GTPase regulators in platelet function in both health and disease.


Assuntos
Plaquetas/enzimologia , Plaquetas/metabolismo , Proteínas rho de Ligação ao GTP/fisiologia , Animais , Humanos , Ativação Plaquetária/fisiologia , Agregação Plaquetária/fisiologia , Transdução de Sinais/fisiologia
13.
Neurosci Lett ; 590: 178-83, 2015 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-25668492

RESUMO

The small GTPase Rif is required for the early stages of dendritic spine formation in neurons, acting through the formin mDia2 to control actin polymerization. Rif is expressed at high levels in the brain, suggesting broader roles in neuronal function. We screened a yeast two-hybrid cDNA library to identify additional binding partners for Rif of potential relevance to neuronal function. We found that Rif interacts with FARP1, a neuronal activator of the RhoA GTPase. We show that Rif has two separate roles in FARP1 regulation-in controlling its association with plexinA4, and in releasing active RhoA from a plexinA4/FARP1 complex. The regulation of FARP1 by Rif promotes neurite retraction in cells stimulated with the semaphorin Sema6A.


Assuntos
Citoesqueleto/metabolismo , Neuritos/fisiologia , Receptores de Superfície Celular/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Células HEK293 , Humanos , Células Jurkat , Células PC12 , Ratos , Semaforinas/metabolismo , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo
14.
Methods Mol Biol ; 1214: 265-70, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25468611

RESUMO

Angiogenesis is a complex process involving the interactions of endothelial cells not only with pro-angiogenic factors but also with stromal cells and stromal matrix components. Modeling this process in vitro is challenging, and many different assays have been described, each with their own particular strengths and weaknesses. The coculture assay is a long-term assay of angiogenesis that uses fibroblasts to secrete and condition a stromal matrix that more closely mimics tissue than a simple collagen gel. The assay is particularly suited to screening for angiogenic regulators and also for high-resolution imaging of endothelial cells undergoing angiogenic morphogenesis.


Assuntos
Técnicas de Cocultura/métodos , Neovascularização Fisiológica , Imunofluorescência , Inativação Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Microscopia de Fluorescência , Morfogênese , RNA Interferente Pequeno/genética , Coloração e Rotulagem
15.
Methods Cell Biol ; 118: 15-34, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24295298

RESUMO

The ability to deplete specific proteins from cells has transformed cell biology. Targeting of gene transcripts using RNA interference has allowed for a highly refined approach to the analysis of gene function that has been applied to all aspects of cell biology. Developments of the technology have reached a point where it is now a relatively trivial task to assess the role of an individual protein in a particular cell function. RNAi also allows for genome-wide screening as a discovery step toward the identification of new components of cellular pathways and machines. The technique has been applied extensively to the analysis of Golgi complex function, leading to significant insight into the biology of this complex organelle. Here, we describe the commonly used options for targeting individual genes for both transient and stable knockdown. We consider the alternative methods for introducing these reagents into cells and outline methods that we and others have used widely for validation of specificity and efficacy of gene targeting.


Assuntos
Técnicas de Silenciamento de Genes , Complexo de Golgi/fisiologia , Animais , Técnicas de Cultura de Células , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Proteínas da Matriz do Complexo de Golgi , Células HEK293 , Humanos , Lentivirus/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas/genética , Proteínas/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , Tubulina (Proteína) , Cultura de Vírus
16.
J Biol Chem ; 288(47): 34217-29, 2013 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-24106270

RESUMO

Rho GTPases such as Rac, RhoA, and Cdc42 are vital for normal platelet function, but the role of RhoG in platelets has not been studied. In other cells, RhoG orchestrates processes integral to platelet function, including actin cytoskeletal rearrangement and membrane trafficking. We therefore hypothesized that RhoG would play a critical role in platelets. Here, we show that RhoG is expressed in human and mouse platelets and is activated by both collagen-related peptide (CRP) and thrombin stimulation. We used RhoG(-/-) mice to study the function of RhoG in platelets. Integrin activation and aggregation were reduced in RhoG(-/-) platelets stimulated by CRP, but responses to thrombin were normal. The central defect in RhoG(-/-) platelets was reduced secretion from α-granules, dense granules, and lysosomes following CRP stimulation. The integrin activation and aggregation defects could be rescued by ADP co-stimulation, indicating that they are a consequence of diminished dense granule secretion. Defective dense granule secretion in RhoG(-/-) platelets limited recruitment of additional platelets to growing thrombi in flowing blood in vitro and translated into reduced thrombus formation in vivo. Interestingly, tail bleeding times were normal in RhoG(-/-) mice, suggesting that the functions of RhoG in platelets are particularly relevant to thrombotic disorders.


Assuntos
Coagulação Sanguínea , Plaquetas/enzimologia , GTP Fosfo-Hidrolases/metabolismo , Vesículas Secretórias/metabolismo , Trombose/enzimologia , Difosfato de Adenosina/farmacologia , Animais , Plaquetas/patologia , Proteínas de Transporte/farmacologia , Feminino , GTP Fosfo-Hidrolases/genética , Hemostáticos/farmacologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Peptídeos/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/genética , Vesículas Secretórias/genética , Trombina/metabolismo , Trombina/farmacologia , Trombose/genética , Trombose/patologia
17.
PLoS One ; 8(1): e54663, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23359340

RESUMO

BACKGROUND: Formation of filopodia and other shape change events are vital for platelet hemostatic function. The mechanisms regulating filopodia formation by platelets are incompletely understood however. In particular the small GTPase responsible for initiating filopodia formation by platelets remains elusive. The canonical pathway involving Cdc42 is not essential for filopodia formation in mouse platelets. The small GTPase Rif (RhoF) provides an alternative route to filopodia generation in other cell types and is expressed in both human and mouse platelets. HYPOTHESIS/OBJECTIVE: We hypothesized that Rif might be responsible for generating filopodia by platelets and generated a novel knockout mouse model to investigate the functional role of Rif in platelets. METHODOLOGY/PRINCIPAL FINDINGS: Constitutive RhoF(-/-) mice are viable and have normal platelet, leukocyte and erythrocyte counts and indices. RhoF(-/-) platelets form filopodia and spread normally on various agonist surfaces in static conditions and under arterial shear. In addition, RhoF(-/-) platelets have normal actin dynamics, are able to activate and aggregate normally and secrete from alpha and dense granules in response to collagen related peptide and thrombin stimulation. CONCLUSIONS: The small GTPase Rif does not appear to be critical for platelet function in mice. Functional overlap between Rif and other small GTPases may be responsible for the non-essential role of Rif in platelets.


Assuntos
Plaquetas/citologia , Pseudópodes , Proteínas de Ligação a Telômeros/fisiologia , Animais , Sequência de Bases , Primers do DNA , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Telômeros/genética
18.
Biochem Soc Trans ; 40(1): 268-72, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22260703

RESUMO

The Rif GTPase is a recent addition to small Rho GTPase family; it shares low homology with other members in the family and evolutionarily parallels with the development of vertebrates. Rif has the conserved Rho GTPase domain structures and cycles between a GDP-bound inactive form and a GTP-bound active form. In its active form, Rif signals through multiple downstream effectors. In the present review, our aim is to summarize the current information about the Rif effectors and how Rif remodels actin cytoskeleton in many aspects.


Assuntos
Fibras de Estresse/metabolismo , Proteínas rho de Ligação ao GTP/fisiologia , Animais , Forma Celular , Humanos , Linfócitos/metabolismo , Linfócitos/fisiologia , Morfogênese , Sistema Nervoso/citologia , Sistema Nervoso/enzimologia , Sistema Nervoso/crescimento & desenvolvimento , Pseudópodes/enzimologia , Pseudópodes/fisiologia , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo
19.
J Cell Sci ; 125(Pt 6): 1420-8, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22275430

RESUMO

The process of angiogenesis requires endothelial cells (ECs) to undergo profound changes in shape and polarity. Although this must involve remodelling of the EC cytoskeleton, little is known about this process or the proteins that control it. We used a co-culture assay of angiogenesis to examine the cytoskeleton of ECs actively undergoing angiogenic morphogenesis. We found that elongation of ECs during angiogenesis is accompanied by stabilisation of microtubules and their alignment into parallel arrays directed at the growing tip. In other systems, similar microtubule alignments are mediated by the formin family of cytoskeletal regulators. We screened a library of human formins and indentified formin-like 3 (FMNL3; also known as FRL2) as a crucial regulator of EC elongation during angiogenesis. We showed that activated FMNL3 triggers microtubule alignment and that FMNL3 is required for this alignment during angiogenic morphogenesis. FMNL3 was highly expressed in the ECs of zebrafish during development and embryos that were depleted for FMNL3 showed profound defects in developmental angiogenesis that were rescued by expression of the human gene. We conclude that FMNL3 is a new regulator of endothelial microtubules during angiogenesis and is required for the conversion of quiescent ECs into their elongated angiogenic forms.


Assuntos
Citoesqueleto/fisiologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Proteínas de Membrana/genética , Neovascularização Fisiológica/genética , Proteínas/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Técnicas de Cocultura , Forminas , Células Endoteliais da Veia Umbilical Humana , Humanos , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
20.
Biochem Soc Trans ; 39(6): 1597-600, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22103493

RESUMO

Angiogenesis is a complex process that involves multiple cellular events. In addition to receiving inputs from a range of stimulatory and inhibitory factors, endothelial cells undergoing angiogenesis make multiple interactions with the extracellular matrix and with other cell types in the stroma. Recreating angiogenesis in vitro is probably an impossible goal; however, a number of assays have been developed that recapitulate many of the key events of the process. These assays are indispensible tools for investigating the signalling pathways that control the formation of new blood vessels. In the present paper, we review the organotypic co-culture assay of angiogenesis - until recently, a comparatively underemployed assay, but one with a number of powerful advantages for angiogenesis research. We give a set of optimized protocols for its use, including protocols for siRNA (small interfering RNA)-based screens, and we discuss appropriate methods for obtaining quantitative data from the assay.


Assuntos
Bioensaio/métodos , Técnicas de Cocultura/métodos , Células Endoteliais/citologia , Fibroblastos/citologia , Neovascularização Fisiológica , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Inativação Gênica , Humanos
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