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1.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205118

RESUMO

During metastasis, cancer cells that originate from the primary tumor circulate in the bloodstream, extravasate, and form micrometastases at distant locations. Several lines of evidence suggest that specific interactions between cancer cells and endothelial cells, in particular tumor cell adhesion to the endothelium and transendothelial migration, play a crucial role in extravasation. Here we have studied the role of vascular endothelial (VE)-cadherin which is expressed aberrantly by breast cancer cells and might promote such interactions. By comparing different human breast cancer cell lines, we observed that the number of cancer cells that adhered to endothelium correlated with VE-cadherin expression levels. VE-cadherin silencing experiments confirmed that VE-cadherin enhances cancer cell adhesion to endothelial cells. However, in contrast, the number of cancer cells that incorporated into the endothelium was not dependent on VE-cadherin. Thus, it appears that cancer cell adhesion and incorporation are distinct processes that are governed by different molecular mechanisms. When cancer cells incorporated into the endothelial monolayer, they formed VE-cadherin positive contacts with endothelial cells. On the other hand, we also observed tumor cells that had displaced endothelial cells, reflecting either different modes of incorporation, or a temporal sequence where cancer cells first form contact with endothelial cells and then displace them to facilitate transmigration. Taken together, these results show that VE-cadherin promotes the adhesion of breast cancer cells to the endothelium and is involved in the initial phase of incorporation, but not their transmigration. Thus, VE-cadherin might be of relevance for therapeutic strategies aiming at preventing the metastatic spread of breast cancer cells.


Assuntos
Antígenos CD/genética , Neoplasias da Mama/genética , Caderinas/genética , Adesão Celular/genética , Endotélio Vascular/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Técnicas de Cocultura , Endotélio Vascular/patologia , Endotélio Vascular/ultraestrutura , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Imagem Molecular/métodos , Metástase Neoplásica
2.
Theranostics ; 10(17): 7599-7621, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32685007

RESUMO

Theranostic biomarkers for putative cancer stem-like cells (CSC) in colorectal cancer (CRC) are of particular interest in translational research to develop patient-individualized treatment strategies. Surface proteins still under debate are CD44 and CD133. The structural and functional diversity of these antigens, as well as their plasticity, has only just begun to be understood. Our study aimed to gain novel insight into the plasticity of CD133/CD44, thereby proving the hypothesis of marker-associated tumorigenic and non-tumorigenic phenotypes to be environmentally driven. Methods: CD133/CD44 profiles of 20 CRC cell lines were monitored; three models with distinct surface patterns in vitro were systematically examined. CD133/CD44 subpopulations were isolated by FACS and analyzed upon in vitro growth and/or in limiting dilution engraftment studies. The experimental setup included biomarker analyses on the protein (flow cytometry, Western blotting, immunofluorescence) and mRNA levels (RT-/qPCR) as well as CD44 gene sequencing. Results: In general, we found that (i) the in vitro CD133/CD44 pattern never determined engraftment and (ii) the CD133/CD44 population distributions harmonized under in vivo conditions. The LS1034 cell line appeared as a unique model due to its de novo in vivo presentation of CD44. CD44v8-10 was identified as main transcript, which was stronger expressed in primary human CRC than in normal colon tissues. Biomarker pattern of LS1034 cells in vivo reflected secondary engraftment: the tumorigenic potential was highest in CD133+/CD44+, intermediate in CD133+/CD44- and entirely lost in CD133-/CD44- subfractions. Both CD44+ and CD44- LS1034 cells gave rise to tumorigenic and non-tumorigenic progeny and were convertible - but only as long as they expressed CD133 in vivo. The highly tumorigenic CD133+/CD44(v8-10)+ LS1034 cells were localized in well-oxygenated perivascular but not hypoxic regions. From a multitude of putative modulators, only the direct interaction with stromal fibroblasts triggered an essential, in vivo-like enhancement of CD44v8-10 presentation in vitro. Conclusion: Environmental conditions modulate CD133/CD44 phenotypes and tumorigenic potential of CRC subpopulations. The identification of fibroblasts as drivers of cancer-specific CD44 expression profile and plasticity sheds light on the limitation of per se dynamic surface antigens as biomarkers. It can also explain the location of putative CD133/CD44-positive CRC CSC in the perivascular niche, which is likely to comprise cancer-associated fibroblasts. The LS1034 in vitro/in vivo model is a valuable tool to unravel the mechanism of stromal-induced CD44v8-10 expression and identify further therapeutically relevant, mutual interrelations between microenvironment and tumorigenic phenotype.


Assuntos
Carcinogênese/patologia , Neoplasias Colorretais/patologia , Receptores de Hialuronatos/metabolismo , Células-Tronco Neoplásicas/patologia , Microambiente Tumoral , Antígeno AC133/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Plasticidade Celular , Separação Celular , Feminino , Citometria de Fluxo , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Isoformas de Proteínas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
3.
FASEB J ; 33(2): 1758-1770, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30156910

RESUMO

A hallmark of proliferative retinopathies, such as retinopathy of prematurity (ROP), is a pathological neovascularization orchestrated by hypoxia and the resulting hypoxia-inducible factor (HIF)-dependent response. We studied the role of Hif2α in hematopoietic cells for pathological retina neovascularization in the murine model of ROP, the oxygen-induced retinopathy (OIR) model. Hematopoietic-specific deficiency of Hif2α ameliorated pathological neovascularization in the OIR model, which was accompanied by enhanced endothelial cell apoptosis. That latter finding was associated with up-regulation of the apoptosis-inducer FasL in Hif2α-deficient microglia. Consistently, pharmacological inhibition of the FasL reversed the reduced pathological neovascularization from hematopoietic-specific Hif2α deficiency. Our study found that the hematopoietic cell Hif2α contributes to pathological retina angiogenesis. Our findings not only provide novel insights regarding the complex interplay between immune cells and endothelial cells in hypoxia-driven retina neovascularization but also may have therapeutic implications for proliferative retinopathies.-Korovina, I., Neuwirth, A., Sprott, D., Weber, S., Sardar Pasha, S. P. B., Gercken, B., Breier, G., El-Armouche, A., Deussen, A., Karl, M. O., Wielockx, B., Chavakis, T., Klotzsche-von Ameln, A. Hematopoietic hypoxia-inducible factor 2α deficiency ameliorates pathological retinal neovascularization via modulation of endothelial cell apoptosis.


Assuntos
Apoptose/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Células da Medula Óssea/metabolismo , Medula Óssea/metabolismo , Endotélio Vascular/patologia , Neovascularização Patológica , Vasos Retinianos/patologia , Proteína ADAM17/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Transformada , Modelos Animais de Doenças , Proteína Ligante Fas/metabolismo , Camundongos , Camundongos Knockout , Microglia/metabolismo , Retinopatia da Prematuridade/metabolismo , Retinopatia da Prematuridade/patologia
4.
Cancers (Basel) ; 10(12)2018 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-30563292

RESUMO

Cancer cell proliferation and insufficient blood supply can lead to the development of hypoxic areas in the tumor tissue. The adaptation to the hypoxic environment is mediated by a transcriptional complex called hypoxia-inducible factor (HIF). HIF protein levels are tightly controlled by oxygen-dependent prolyl hydroxylase domain proteins (PHDs). However, the precise roles of these enzymes in tumor progression and their downstream signaling pathways are not fully characterized. Here, we study PHD3 function in murine experimental osteosarcoma. Unexpectedly, PHD3 silencing in LM8 cells affects neither HIF-1α protein levels, nor the expression of various HIF-1 target genes. Subcutaneous injection of PHD3-silenced tumor cells accelerated tumor progression and was accompanied by dramatic phenotypic changes in the tumor vasculature. Blood vessels in advanced PHD3-silenced tumors were enlarged whereas their density was greatly reduced. Examination of the molecular pathways underlying these alterations revealed that platelet-derived growth factor (PDGF)-C signaling is activated in the vasculature of PHD3-deficient tumors. Silencing of PDGF-C depleted tumor growth, increased vessel density and reduced vessel size. Our data show that PHD3 controls tumor growth and vessel architecture in LM8 osteosarcoma by regulating the PDGF-C pathway, and support the hypothesis that different members of the PHD family exert unique functions in tumors.

6.
Histochem Cell Biol ; 149(1): 15-30, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29143117

RESUMO

The cadherin switch has profound consequences on cancer invasion and metastasis. The endothelial-specific vascular endothelial cadherin (VE-cadherin) has been demonstrated in diverse cancer types including breast cancer and is supposed to modulate tumor progression and metastasis, but underlying mechanisms need to be better understood. First, we evaluated VE-cadherin expression by tissue microarray in 392 cases of breast cancer tumors and found a diverse expression and distribution of VE-cadherin. Experimental expression of fluorescence-tagged VE-cadherin (VE-EGFP) in undifferentiated, fibroblastoid and E-cadherin-negative MDA-231 (MDA-VE-EGFP) as well as in differentiated E-cadherin-positive MCF-7 human breast cancer cell lines (MCF-VE-EGFP), respectively, displayed differentiation-dependent functional differences. VE-EGFP expression reversed the fibroblastoid MDA-231 cells to an epithelial-like phenotype accompanied by increased ß-catenin expression, actin and vimentin remodeling, increased cell spreading and barrier function and a reduced migration ability due to formation of VE-cadherin-mediated cell junctions. The effects were largely absent in both MDA-VE-EGFP and in control MCF-EGFP cell lines. However, MCF-7 cells displayed a VE-cadherin-independent planar cell polarity and directed cell migration that both developed in MDA-231 only after VE-EGFP expression. Furthermore, VE-cadherin expression had no effect on tumor cell proliferation in monocultures while co-culturing with endothelial cells enhanced tumor cell proliferation due to integration of the tumor cells into monolayer where they form VE-cadherin-mediated cell contacts with the endothelium. We propose an interactive VE-cadherin-based crosstalk that might activate proliferation-promoting signals. Together, our study shows a VE-cadherin-mediated cell dynamics and an endothelial-dependent proliferation in a differentiation-dependent manner.


Assuntos
Antígenos CD/biossíntese , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caderinas/biossíntese , Diferenciação Celular , Células Endoteliais/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Proliferação de Células , Células Endoteliais/citologia , Feminino , Humanos , Células MCF-7 , Células Tumorais Cultivadas
7.
Thromb Haemost ; 117(7): 1289-1295, 2017 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-28594427

RESUMO

Angiogenesis, literally formation of new blood vessels, is the main process through which the vascular system expands during embryonic and postnatal development. Endothelial cells, which constitute the inner lining of all blood vessels, are typically in a quiescent state in the healthy adult organism. However, in vascular and metabolic diseases, the endothelium becomes unstable and dysfunctional. The resulting tissue hypoxia may thereby induce pathological angiogenesis, which is a hallmark of disease conditions like cancer or diabetic retinopathy. However, recent evidence suggests that angiogenesis is also a major player in the context of further metabolic diseases, especially in obesity. In particular, deregulated angiogenesis is linked with adipose tissue dysfunction and insulin resistance. On the other hand, signalling pathways, such as the PI3K pathway, may regulate metabolic activities in the endothelium. Endothelial cell metabolism emerges as an important regulator of angiogenesis. This review summarises the role of angiogenesis in metabolic-vascular disease, with specific focus on the role of angiogenesis in obesity-related metabolic dysfunction and on signaling pathways, especially PI3K, linking cell metabolism to endothelial function.


Assuntos
Diabetes Mellitus/patologia , Metabolismo Energético , Neovascularização Patológica , Obesidade/patologia , Doenças Vasculares/patologia , Animais , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Obesidade/metabolismo , Obesidade/fisiopatologia , Fosfatidilinositol 3-Quinase/metabolismo , Transdução de Sinais , Doenças Vasculares/metabolismo , Doenças Vasculares/fisiopatologia
8.
J Pathol ; 241(4): 547-558, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27981571

RESUMO

Prolyl hydroxylase domain-containing proteins (PHDs) regulate the adaptation of cells to hypoxia. Pan-hydroxylase inhibition is protective in experimental colitis, in which PHD1 plays a prominent role. However, it is currently unknown how PHD1 targeting regulates this protection and which cell type(s) are involved. Here, we demonstrated that Phd1 deletion in endothelial and haematopoietic cells (Phd1f/f Tie2:cre) protected mice from dextran sulphate sodium (DSS)-induced colitis, with reduced epithelial erosions, immune cell infiltration, and colonic microvascular dysfunction, whereas the response of Phd2f/+ Tie2:cre and Phd3f/f Tie2:cre mice to DSS was similar to that of their littermate controls. Using bone marrow chimeras and cell-specific cre mice, we demonstrated that ablation of Phd1 in haematopoietic cells but not in endothelial cells was both necessary and sufficient to inhibit experimental colitis. This effect relied, at least in part, on skewing of Phd1-deficient bone marrow-derived macrophages towards an anti-inflammatory M2 phenotype. These cells showed an attenuated nuclear factor-κB-dependent response to lipopolysaccharide (LPS), which in turn diminished endothelial chemokine expression. In addition, Phd1 deficiency in dendritic cells significantly reduced interleukin-1ß production in response to LPS. Taken together, our results further support the development of selective PHD1 inhibitors for ulcerative colitis, and identify haematopoietic cells as their primary target. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Colite Ulcerativa/tratamento farmacológico , Macrófagos/metabolismo , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/imunologia , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Colo/efeitos dos fármacos , Colo/patologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Feminino , Deleção de Genes , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , Pró-Colágeno-Prolina Dioxigenase/deficiência , Pró-Colágeno-Prolina Dioxigenase/genética
9.
Mol Cell Biol ; 36(3): 376-93, 2016 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-26572826

RESUMO

Angiogenesis is a central regulator for white (WAT) and brown (BAT) adipose tissue adaptation in the course of obesity. Here we show that deletion of hypoxia-inducible factor 2α (HIF2α) in adipocytes (by using Fabp4-Cre transgenic mice) but not in myeloid or endothelial cells negatively impacted WAT angiogenesis and promoted WAT inflammation, WAT dysfunction, hepatosteatosis, and systemic insulin resistance in obesity. Importantly, adipocyte HIF2α regulated vascular endothelial growth factor (VEGF) expression and angiogenesis of obese BAT as well as its thermogenic function. Consistently, obese adipocyte-specific HIF2α-deficient mice displayed BAT dysregulation, associated with reduced levels of uncoupling protein 1 (UCP1) and a dysfunctional thermogenic response to cold exposure. VEGF administration reversed WAT and BAT inflammation and BAT dysfunction in adipocyte HIF2α-deficient mice. Together, our findings show that adipocyte HIF2α is protective against maladaptation to obesity and metabolic dysregulation by promoting angiogenesis in both WAT and BAT and by counteracting obesity-mediated BAT dysfunction.


Assuntos
Adipócitos/patologia , Tecido Adiposo Marrom/fisiopatologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Deleção de Genes , Obesidade/genética , Obesidade/fisiopatologia , Adipócitos/metabolismo , Tecido Adiposo Marrom/irrigação sanguínea , Tecido Adiposo Marrom/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Inflamação/complicações , Canais Iônicos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/metabolismo , Neovascularização Fisiológica , Obesidade/complicações , Obesidade/metabolismo , Termogênese , Proteína Desacopladora 1 , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
Int J Cancer ; 138(3): 540-54, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-25716346

RESUMO

Inhibiting tumor growth by targeting the tumor vasculature was first proposed by Judah Folkman almost 40 years ago. Since then, different approaches and numerous drugs and agents have been developed to achieve this goal, either with the aim of inhibiting tumor neoangiogenesis or normalizing the tumor vasculature. Among the most promising therapeutic targets are receptor tyrosine kinases (RTKs), some of which are predominantly expressed on tumor endothelial cells, although they are sometimes also present on tumor cells. The majority of RTK inhibitors investigated over the past two decades competes with ATP at the active site of the kinase and therefore block the phosphorylation of intracellular targets. Some of these drugs have been approved for therapy, whereas others are still in clinical trials. Here, we discuss the scientific basis, current status, problems and future prospects of RTK inhibition in anti-tumor therapy.


Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Inibidores da Angiogênese/farmacologia , Animais , Resistencia a Medicamentos Antineoplásicos , Humanos , Camundongos , Metástase Neoplásica
11.
Gastroenterology ; 149(1): 177-189.e10, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25797700

RESUMO

BACKGROUND & AIMS: Senescence prevents cellular transformation. We investigated whether vascular endothelial growth factor (VEGF) signaling via its receptor, VEGFR2, regulates senescence and proliferation of tumor cells in mice with colitis-associated cancer (CAC). METHODS: CAC was induced in VEGFR2(ΔIEC) mice, which do not express VEGFR2 in the intestinal epithelium, and VEGFR2(fl/fl) mice (controls) by administration of azoxymethane followed by dextran sodium sulfate. Tumor development and inflammation were determined by endoscopy. Colorectal tissues were collected for immunoblot, immunohistochemical, and quantitative polymerase chain reaction analyses. Findings from mouse tissues were confirmed in human HCT116 colorectal cancer cells. We analyzed colorectal tumor samples from patients before and after treatment with bevacizumab. RESULTS: After colitis induction, VEGFR2(ΔIEC) mice developed significantly fewer tumors than control mice. A greater number of intestinal tumor cells from VEGFR2(ΔIEC) mice were in senescence than tumor cells from control mice. We found VEGFR2 to activate phosphatidylinositol-4,5-bisphosphate-3-kinase and AKT, resulting in inactivation of p21 in HCT116 cells. Inhibitors of VEGFR2 and AKT induced senescence in HCT116 cells. Tumor cell senescence promoted an anti-tumor immune response by CD8(+) T cells in mice. Patients whose tumor samples showed an increase in the proportion of senescent cells after treatment with bevacizumab had longer progression-free survival than patients in which the proportion of senescent tumor cells did not change before and after treatment. CONCLUSIONS: Inhibition of VEGFR2 signaling leads to senescence of human and mouse colorectal cancer cells. VEGFR2 interacts with phosphatidylinositol-4,5-bisphosphate-3-kinase and AKT to inactivate p21. Colorectal tumor senescence and p21 level correlate with patient survival during treatment with bevacizumab.


Assuntos
Proliferação de Células/genética , Senescência Celular/genética , Colite/genética , Neoplasias Colorretais/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Bevacizumab , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Colite/complicações , Colite/patologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Intervalo Livre de Doença , Feminino , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-akt/metabolismo
12.
Int J Oncol ; 45(6): 2267-77, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25340839

RESUMO

Leiomyosarcomas remain challenging tumors to manage and novel therapy strategies besides radiation and conventional chemotherapy are needed. Targeting angiogenesis by inhibition of vascular endothelial growth factor (VEGF) receptor tyrosine kinases (RTKs) of the tumor vasculature with small molecules is a promising new therapy. It has been shown recently that these receptors are not only expressed on tumor endothelium but also on tumor cells themselves. Thus, we investigated the expression of members of the VEGF receptor (VEGFR) family and corresponding growth factors in leiomyosarcoma tissue specimens and in the leiomyosarcoma cell lines SK-LMS-1 and SK-UT-1. We evaluated the influence of the VEGFR inhibitor PTK787/ZK222584 (PTK787) on cell growth, migration, apoptosis and phosphorylation of intracellular signalling molecules. In human leiomyosarcoma tissue specimens VEGFR­1/-2 and platelet-derived growth factor receptor (PDGFR-ß) were strongly expressed. Both leiomyosarcoma cell lines expressed VEGFR­1/-3 and PDGFR-ß but VEGFR-2 protein expression was positive only in SK-UT-1. SK-LMS-1 and SK-UT-1 cells secreted high and low amounts of VEGF-A, respectively, whereas PDGF-BB secretion was similar in both cell lines. Application of PTK787 led to partial inhibition of PDGF-BB-activated AKT/p90RSK and ERK1/2 signalling pathways. In contrast, protein phosphorylation was not affected by PTK787 in VEGF-A-treated cells. PTK787 turned out to inhibit cell migration even though no effects were observed upon stimulation with VEGF-A or PDGF-BB. In line, cell growth in leiomyosarcoma cell lines remained unchanged upon PTK787 treatment alone and with subsequent VEGF-A- or PDGF-BB-stimulation. However, VEGF-A, but not PDGF-BB-treated cells showed increased cell death upon PTK787 treatment. VEGFR family members are expressed in leiomyosarcomas in vivo and in vitro. Upon receptor stimulation, PTK787 is able to inhibit subsequent phosphorylation events and influences cell survival but not metabolic activity and migration. Thus, the inhibitor is possibly an additional option in the treatment of leiomyosarcomas.


Assuntos
Leiomiossarcoma/tratamento farmacológico , Neovascularização Patológica/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Inibidores da Angiogênese/administração & dosagem , Becaplermina , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Leiomiossarcoma/genética , Leiomiossarcoma/patologia , Neovascularização Patológica/tratamento farmacológico , Ftalazinas/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-sis/administração & dosagem , Proteínas Proto-Oncogênicas c-sis/biossíntese , Piridinas/administração & dosagem , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/biossíntese , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores
13.
Cell Tissue Res ; 355(3): 523-7, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24619142

RESUMO

Cadherins are cell adhesion receptors that play important roles in embryogenesis and tissue homoeostasis. Endothelial cells express various members of the cadherin superfamily, in particular vascular endothelial (VE-) cadherin, which is the main adhesion receptor of endothelial adherens junctions and neural (N-) cadherin, which is normally localized outside the junctions and may mediate adhesion between endothelial cells and non-endothelial cells. Dysregulation of cadherin expression has been implicated in tumor progression, in particular the loss of epithelial (E-) cadherin expression or function and the gain of N-cadherin. Moreover, more recently, aberrant expression of VE-cadherin was observed in certain cancer types. In breast carcinoma, VE-cadherin was shown to promote tumor cell proliferation and invasion through enhancing TGF-ß signaling. Thus, in breast cancer, the cadherin switch involves another player, vascular endothelial cadherin, which is part of an intricate interplay of classical cadherins in breast cancer progression.


Assuntos
Neoplasias da Mama/metabolismo , Caderinas/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Animais , Neoplasias da Mama/patologia , Células Endoteliais/patologia , Endotélio Vascular/patologia , Feminino , Humanos
14.
Mol Cancer Res ; 11(11): 1337-48, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24048703

RESUMO

UNLABELLED: Solid tumor growth is intimately associated with angiogenesis, a process that is efficiently triggered by hypoxia. Therefore, oxygen-sensitive signaling pathways are thought to play a critical role in tumor angiogenesis and progression. Here, the function of prolyl hydroxylase-4 (PHD4), a relative of the prolyl hydroxylase domain proteins 1-3 that promote the degradation of hypoxia-inducible factors (HIF), was interrogated. To test the hypothesis that PHD4 might inhibit tumor angiogenesis, it was overexpressed in osteosarcoma cells, and unexpectedly, this manipulation led to increased tumor blood vessel density. However, the newly formed blood vessels were smaller than normal and appeared to be partially nonfunctional, as indicated by poor vessel perfusion. PHD4 overexpression in tumor cells stimulated the expression of TGF-α, which was necessary and sufficient to promote angiogenic sprouting of endothelial cells. On the other hand, PHD4 overexpression reduced HIF-2α protein levels, which in turn inhibited in vivo tumor growth. Combined, elevated PHD4 levels deregulate angiogenesis via increased TGF-α expression in vitro and in vivo. These data support the hypothesis that tumor growth can be uncoupled from vessel density and that the individual PHD family members exert distinct functions in tumors. IMPLICATIONS: PHD4 influences tumor growth and vascularization through discrete mechanisms and molecular pathways that likely have therapeutic potential.


Assuntos
Neovascularização Patológica/metabolismo , Osteossarcoma/patologia , Prolil Hidroxilases/metabolismo , Sarcoma Experimental/patologia , Fator de Crescimento Transformador alfa/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Células Endoteliais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C3H , Neovascularização Patológica/genética , Osteossarcoma/metabolismo , Prolil Hidroxilases/genética , Sarcoma Experimental/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador alfa/genética
15.
Neurosci Lett ; 553: 142-7, 2013 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-23994060

RESUMO

Oxygen tension is critical for proliferation of human and murine midbrain-derived neural precursor cells (mNPCs). Lack of hypoxia-inducible factor-1α (HIF1α) impairs midbrain dopaminergic neurogenesis which could be rescued by vascular endothelial growth factor (VEGF) via VEGFR-2 signaling. Here, we conditionally inactivated the VEGFR-2, encoded by the fetal liver kinase 1 (Flk1) gene, in murine NPCs to determine its role in proliferation and survival in vitro as well as survival of dopaminergic neurons in vivo. Flk1 conditional knock-out (Flk1 CKO) mice showed no general brain phenotype. There was no midbrain-specific impairment of NPC proliferation as seen in HIF1α CKO mice. In the substantia nigra (SN) of adult Flk1 CKO mice, nonbiased stereological cell counts revealed no reduction of TH-positive neurons of Flk1 CKO mice compared with control Cre/wt mice (in which the wild-type Flk1 allele is expressed in parallel with the Cre recombinase allele). In conclusion, VEGF receptor signaling seems not to be relevant to the development and survival of substantia nigra dopaminergic neurons within the hypoxia-HIF1α signaling pathway.


Assuntos
Substância Negra/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Sobrevivência Celular , Neurônios Dopaminérgicos/citologia , Camundongos , Camundongos Transgênicos , Neurogênese , Transdução de Sinais , Substância Negra/embriologia , Substância Negra/crescimento & desenvolvimento , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
16.
Mol Cell Biol ; 33(17): 3426-38, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23798557

RESUMO

Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible factor (HIF) prolyl hydroxylase 2 (PHD2) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD2 in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD2 in inflammatory, vascular, or epidermal cells. Interestingly, PHD2 deficiency only in keratinocytes and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of ß3-integrin in the keratinocytes around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming growth factor ß signaling. Thus, loss of the central oxygen sensor PHD2 in keratinocytes stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD2 could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds.


Assuntos
Técnicas de Inativação de Genes , Queratinócitos/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Pele/metabolismo , Cicatrização , Animais , Movimento Celular , Proliferação de Células , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia , Integrina beta3/genética , Queratinócitos/citologia , Masculino , Camundongos , Camundongos Knockout , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Pele/citologia , Fenômenos Fisiológicos da Pele , Fator de Crescimento Transformador beta/metabolismo
17.
Hippocampus ; 23(10): 861-72, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23674383

RESUMO

Hypoxia-inducible factors (HIFs) are key transcriptional regulators that play a major role in oxygen homeostasis. HIF activity is tightly regulated by oxygen-dependent hydroxylases, which additionally require iron and 2-oxoglutarate as cofactors. Inhibition of these enzymes has become a novel target to modulate the hypoxic response for therapeutic benefit. Inhibition of prolyl-4-hydroxylase domains (PHDs) have been shown to delay neuronal cell death and protect against ischemic injury in the hippocampus. In this study we have examined the effects of prolyl hydroxylase inhibition on synaptic transmission and plasticity in the hippocampus. Field excitatory postsynaptic potentials (fEPSPs) and excitatory postsynaptic currents (EPSCs) were elicited by stimulation of the Schaffer collateral pathway in the CA1 region of the hippocampus. Treatment of rat hippocampal slices with low concentrations (10 µM) of the iron chelator deferosoxamine (DFO) or the 2-oxoglutarate analogue dimethyloxalyl glycine (DMOG) had no effect on fEPSP. In contrast, application of 1 mM DMOG resulted in a significant decrease in fEPSP slope. Antagonism of the NMDA receptor attenuated the effects of DMOG on baseline synaptic signalling. In rat hippocampal slices pretreated with DMOG and DFO the induction of long-term potentiation (LTP) by tetanic stimulation was strongly impaired. Similarly, neuronal knockout of the single PHD family member PHD2 prevented murine hippocampal LTP. Preconditioning of PHD2 deficient hippocampi with either DMOG, DFO, or the PHD specific inhibitor JNJ-42041935, did not further decrease LTP suggesting that DMOG and DFO influences synaptic plasticity primarily by inhibiting PHDs rather than unspecific effects. These findings provide striking evidence for a modulatory role of PHD proteins on synaptic plasticity in the hippocampus.


Assuntos
Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/enzimologia , Potenciação de Longa Duração/fisiologia , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Aminoácidos Dicarboxílicos/farmacologia , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/enzimologia , Desferroxamina/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp/instrumentação , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Ratos , Ratos Wistar
18.
Blood ; 121(26): 5158-66, 2013 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-23667053

RESUMO

Hypoxia is a prominent feature in the maintenance of hematopoietic stem cell (HSC) quiescence and multipotency. Hypoxia-inducible factor (HIF) prolyl hydroxylase domain proteins (PHDs) serve as oxygen sensors and may therefore regulate this system. Here, we describe a mouse line with conditional loss of HIF prolyl hydroxylase 2 (PHD2) in very early hematopoietic precursors that results in self-renewal of multipotent progenitors under steady-state conditions in a HIF1α- and SMAD7-dependent manner. Competitive bone marrow (BM) transplantations show decreased peripheral and central chimerism of PHD2-deficient cells but not of the most primitive progenitors. Conversely, in whole BM transfer, PHD2-deficient HSCs replenish the entire hematopoietic system and display an enhanced self-renewal capacity reliant on HIF1α. Taken together, our results demonstrate that loss of PHD2 controls the maintenance of the HSC compartment under physiological conditions and causes the outcompetition of PHD2-deficient hematopoietic cells by their wild-type counterparts during stress while promoting the self-renewal of very early hematopoietic progenitors.


Assuntos
Células-Tronco Hematopoéticas/citologia , Hipóxia/fisiopatologia , Células-Tronco Multipotentes/citologia , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Estresse Fisiológico , Animais , Transplante de Medula Óssea , Ciclo Celular , Diferenciação Celular , Células-Tronco Hematopoéticas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia , Prolina Dioxigenases do Fator Induzível por Hipóxia , Integrases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Multipotentes/metabolismo , Proteína Smad7/metabolismo
19.
Nat Cell Biol ; 15(3): 249-60, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23354168

RESUMO

Activities as diverse as migration, proliferation and patterning occur simultaneously and in a coordinated fashion during tissue morphogenesis. In the growing vasculature, the formation of motile, invasive and filopodia-carrying endothelial sprouts is balanced with the stabilization of blood-transporting vessels. Here, we show that sprouting endothelial cells in the retina have high rates of VEGF uptake, VEGF receptor endocytosis and turnover. These internalization processes are opposed by atypical protein kinase C activity in more stable and mature vessels. aPKC phosphorylates Dab2, a clathrin-associated sorting protein that, together with the transmembrane protein ephrin-B2 and the cell polarity regulator PAR-3, enables VEGF receptor endocytosis and downstream signal transduction. Accordingly, VEGF receptor internalization and the angiogenic growth of vascular beds are defective in loss-of-function mice lacking key components of this regulatory pathway. Our work uncovers how vessel growth is dynamically controlled by local VEGF receptor endocytosis and the activity of cell polarity proteins.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Moléculas de Adesão Celular/metabolismo , Endocitose/fisiologia , Endotélio Vascular/citologia , Efrina-B2/metabolismo , Neovascularização Fisiológica , Proteína Quinase C/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/antagonistas & inibidores , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Proteínas Reguladoras de Apoptose , Western Blotting , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/genética , Proteínas de Ciclo Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Endotélio Vascular/metabolismo , Efrina-B2/antagonistas & inibidores , Efrina-B2/genética , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Transgênicos , Morfogênese , Fosforilação , Proteína Quinase C/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Retina/citologia , Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
20.
Blood ; 121(8): 1436-45, 2013 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-23264599

RESUMO

Erythropoiesis must be tightly balanced to guarantee adequate oxygen delivery to all tissues in the body. This process relies predominantly on the hormone erythropoietin (EPO) and its transcription factor hypoxia inducible factor (HIF). Accumulating evidence suggests that oxygen-sensitive prolyl hydroxylases (PHDs) are important regulators of this entire system. Here, we describe a novel mouse line with conditional PHD2 inactivation (cKO P2) in renal EPO producing cells, neurons, and astrocytes that displayed excessive erythrocytosis because of severe overproduction of EPO, exclusively driven by HIF-2α. In contrast, HIF-1α served as a protective factor, ensuring survival of cKO P2 mice with HCT values up to 86%. Using different genetic approaches, we show that simultaneous inactivation of PHD2 and HIF-1α resulted in a drastic PHD3 reduction with consequent overexpression of HIF-2α-related genes, neurodegeneration, and lethality. Taken together, our results demonstrate for the first time that conditional loss of PHD2 in mice leads to HIF-2α-dependent erythrocytosis, whereas HIF-1α protects these mice, providing a platform for developing new treatments of EPO-related disorders, such as anemia.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hematopoese Extramedular/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Policitemia/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Encéfalo/fisiologia , Células Cultivadas , Eritropoetina/genética , Eritropoetina/metabolismo , Feminino , Fibroblastos/citologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Prolina Dioxigenases do Fator Induzível por Hipóxia , Queratinócitos/citologia , Rim/citologia , Rim/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Degeneração Neural/genética , Degeneração Neural/metabolismo , Policitemia/metabolismo , Policitemia/patologia , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Índice de Gravidade de Doença , Trombocitopenia/genética , Trombocitopenia/metabolismo , Trombocitopenia/patologia
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