RESUMO
Angiogenesis is regulated by complex interactions between endothelial cells and support cells of the vascular microenvironment, such as tissue myeloid cells and vascular mural cells. Multicellular interactions during angiogenesis are difficult to study in animals and challenging in a reductive setting. We incorporated stromal cells into an established bead-based capillary sprouting assay to develop assays that faithfully reproduce major steps of vessel sprouting and maturation. We observed that macrophages enhance angiogenesis, increasing the number and length of endothelial sprouts, a property we have dubbed "angiotrophism." We found that polarizing macrophages toward a pro-inflammatory profile further increased their angiotrophic stimulation of vessel sprouting, and this increase was dependent on macrophage Notch signaling. To study endothelial/pericyte interactions, we added vascular pericytes directly to the bead-bound endothelial monolayer. These pericytes formed close associations with the endothelial sprouts, causing increased sprout number and vessel caliber. We found that Jagged1 expression and Notch signaling are essential for the growth of both endothelial cells and pericytes and may function in their interaction. We observed that combining endothelial cells with both macrophages and pericytes in the same sprouting assay has multiplicative effects on sprouting. These results significantly improve bead-capillary sprouting assays and provide an enhanced method for modeling interactions between the endothelium and the vascular microenvironment. Achieving this in a reductive in vitro setting represents a significant step toward a better understanding of the cellular elements that contribute to the formation of mature vasculature.
Assuntos
Comunicação Celular , Microambiente Celular , Células Endoteliais da Veia Umbilical Humana/citologia , Macrófagos/citologia , Modelos Biológicos , Neovascularização Fisiológica , Pericitos/citologia , Receptores Notch/metabolismo , Animais , Linhagem Celular , Polaridade Celular , Sobrevivência Celular , Técnicas de Cocultura , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos , Células Mieloides/citologia , Células Mieloides/metabolismo , Pericitos/metabolismo , Proteínas Serrate-Jagged/metabolismo , Transdução de SinaisRESUMO
UNLABELLED: A proangiogenic role for Jagged (JAG)-dependent activation of NOTCH signaling in the endothelium has yet to be described. Using proteins that encoded different NOTCH1 EGF-like repeats, we identified unique regions of Delta-like ligand (DLL)-class and JAG-class ligand-receptor interactions, and developed NOTCH decoys that function as ligand-specific NOTCH inhibitors. N110-24 decoy blocked JAG1/JAG2-mediated NOTCH1 signaling, angiogenic sprouting in vitro, and retinal angiogenesis, demonstrating that JAG-dependent NOTCH signal activation promotes angiogenesis. In tumors, N110-24 decoy reduced angiogenic sprouting, vessel perfusion, pericyte coverage, and tumor growth. JAG-NOTCH signaling uniquely inhibited expression of antiangiogenic soluble (s) VEGFR1/sFLT1. N11-13 decoy interfered with DLL1-DLL4-mediated NOTCH1 signaling and caused endothelial hypersprouting in vitro, in retinal angiogenesis, and in tumors. Thus, blockade of JAG- or DLL-mediated NOTCH signaling inhibits angiogenesis by distinct mechanisms. JAG-NOTCH signaling positively regulates angiogenesis by suppressing sVEGFR1-sFLT1 and promoting mural-endothelial cell interactions. Blockade of JAG-class ligands represents a novel, viable therapeutic approach to block tumor angiogenesis and growth. SIGNIFICANCE: This is the first report identifying unique regions of the NOTCH1 extracellular domain that interact with JAG-class and DLL-class ligands. Using this knowledge, we developed therapeutic agents that block JAG-dependent NOTCH signaling and demonstrate for the first time that JAG blockade inhibits experimental tumor growth by targeting tumor angiogenesis.
Assuntos
Fragmentos Fc das Imunoglobulinas/administração & dosagem , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias/irrigação sanguínea , Neoplasias/terapia , Receptor Notch1/administração & dosagem , Receptores Notch/antagonistas & inibidores , Proteínas Recombinantes de Fusão/administração & dosagem , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/química , Inibidores da Angiogênese/genética , Animais , Feminino , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/terapia , Ligação Proteica , Receptor Notch1/química , Receptor Notch1/genética , Receptores Notch/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Transdução de SinaisRESUMO
17ß-Estradiol can promote the growth and development of several estrogen receptor (ER)-negative breast cancers. The effects are rapid and non-genomic, suggesting that a membrane-associated ER is involved. ERα36 has been shown to mediate rapid, non-genomic, membrane-associated effects of 17ß-estradiol in several cancer cell lines, including triple negative HCC38 breast cancer cells. Moreover, the effect is anti-apoptotic. The aim of this study was to determine if ERα36 mediates this anti-apoptotic effect, and to elucidate the mechanism involved. Taxol was used to induce apoptosis in HCC38 cells, and the effect of 17ß-estradiol pre-treatment was determined. Antibodies to ERα36, signal pathway inhibitors, ERα36 deletion mutants, and ERα36-silencing were used prior to these treatments to determine the role of ERα36 in these effects and to determine which signaling molecules were involved. We found that the anti-apoptotic effect of 17ß-estradiol in HCC38 breast cancer cells is in fact mediated by membrane-associated ERα36. We also showed that this signaling occurs through a pathway that requires PLD, LPA, and PI3K; Gαs and calcium signaling may also be involved. In addition, dynamic palmitoylation is required for the membrane-associated effect of 17ß-estradiol. Exon 9 of ERα36, a unique exon to ERα36 not found in other identified splice variants of ERα with previously unknown function, is necessary for these effects. This study provides a working model for a mechanism by which estradiol promotes anti-apoptosis through membrane-associated ERα36, suggesting that ERα36 may be a potential membrane target for drug design against breast cancer, particularly triple negative breast cancer.
RESUMO
Traditionally, steroid hormones such as the vitamin D3 metabolites, testosterone and dihydrotesterone, and 17ß-estradiol act through cytosolic and nuclear receptors that directly interact with DNA to alter gene transcription and regulate cellular development. However, recent studies focused on rapid and membrane effects of steroid hormones have given invaluable insight into their non-classical mechanisms of action. In some cases, the traditional receptors were implicated as acting also in the plasma membrane as membrane-associated receptors. However, recent data have demonstrated the presence of an alternative splicing variant to traditional estrogen receptor α known as ERα36, which is present in the plasma membranes of several different cell types including several cancer cell types and even in some normal cells including cartilage and bone cells. The physiological effects that result from the membrane activation of ERα36 may vary from one cell type to another, but the mechanism of action appears to use similar pathways such as the activation of various protein kinases and phospholipases leading to the activation of signaling cascades that result in rapid, non-genomic responses. These rapid responses can affect cell proliferation and apoptotic signaling, indirectly activate downstream genomic signaling through phosphorylation cascades of transcription factors, and crosstalk with classical pathways via interaction with classical receptors. This review describes the data from the last several years and discusses the non-classical, rapid, and membrane-associated cellular responses to steroid hormones, particularly 17ß-estradiol, through the classical receptors ERα and ERß and various non-classical receptors, especially estrogen receptor-α36 (ERα36).
Assuntos
Membrana Celular/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Processamento Alternativo , Animais , Estradiol/metabolismo , Feminino , Humanos , Masculino , Fosfolipase D/metabolismo , Proteína Quinase C/metabolismo , Receptores de Estrogênio/metabolismo , Caracteres Sexuais , Transdução de SinaisRESUMO
17ß-estradiol (E2) plays a key role in tumorigenesis by enhancing cell survivability and metastasis through its cytoplasmic receptors. Recently, a variant of estrogen receptor alpha, ERα36 has been implicated as a substantial mediator of E2's proliferative and antiapoptotic effects through rapid membrane-associated signaling, and cancers previously regarded as hormone-independent due to the absence of traditional receptors, may in fact be susceptible to E2. Despite rising from a secondary sex organ and having a clear gender disposition, laryngeal cancer is not uniformly accepted as hormone dependent, even in the face of compelling evidence of E2 responsiveness. The aim of this study was to further elucidate the role of E2 in the tumorigenesis of laryngeal cancer, both in vitro and in vivo. ERα36 presence was evaluated in membranes of the laryngeal carcinoma cell line, Hep2, as well as in laryngeal tumor samples. In vitro ERα36 was found to mediate rapid activation of protein kinase C and phospholipase D by E2, leading to increased proliferation and protection against chemotherapy-induced apoptosis. Furthermore, in response to E2 activation of ERα36, an upregulation of angiogenic and metastatic factors was observed. Clinical analysis of laryngeal tumors revealed a similar association between the amount of ERα36 and VEGF and indicated a role in lymph node metastasis. These findings present compelling evidence of ERα36-dependent E2 signaling in laryngeal cancer. Thus, targeting ERα36 may reduce the deleterious effects of E2 in laryngeal cancer, ultimately suggesting the importance of antiestrogen therapy or the production of novel drugs that specifically target ERα36.
Assuntos
Carcinoma/metabolismo , Receptor alfa de Estrogênio/metabolismo , Neoplasias Laríngeas/metabolismo , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/metabolismo , Antineoplásicos/farmacologia , Apoptose , Carcinogênese , Carcinoma/tratamento farmacológico , Carcinoma/patologia , Proliferação de Células , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos , Estradiol/metabolismo , Receptor alfa de Estrogênio/antagonistas & inibidores , Feminino , Células HeLa , Humanos , Neoplasias Laríngeas/tratamento farmacológico , Neoplasias Laríngeas/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Estadiamento de Neoplasias , Neovascularização Patológica , Fosfolipase D/metabolismo , Proteína Quinase C/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Protein kinase C (PKC) signaling can be activated rapidly by 17ß-estradiol (E(2)) via nontraditional signaling in ERα-positive MCF7 and ERα-negative HCC38 breast cancer cells and is associated with tumorigenicity. Additionally, E(2) has been shown to elicit anti-apoptotic effects in cancer cells counteracting pro-apoptotic effects of chemotherapeutics. Supporting evidence suggests the existence of a membrane-associated ER that differs from the traditional receptors, ERα and ERß. Our aim was to identify the ER responsible for rapid PKC activation and to evaluate downstream effects, such as proliferation, apoptosis, and metastasis. RT-PCR, Western blot, and immunofluorescence were used to determine the presence of ER splice variants in multiple cell lines. E(2) effects on PKC activity were measured with and without ER-blocking antibodies. Cell proliferation was determined by [(3)H]thymidine incorporation, and cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, (MTT) whereas apoptosis was determined by DNA fragmentation and TUNEL. Quantitative RT-PCR and sandwich ELISA were used to determine the effects on metastatic factors. The role of membrane-dependent signaling in cancer cell invasiveness was examined using an in vitro assay. The results indicate the presence of an ERα splice variant, ERα36, in ERα-positive MCF7 and ERα-negative HCC38 breast cancer cells, which localized to plasma membranes and rapidly activated PKC in response to E(2), leading to deleterious effects such as enhancement of proliferation, protection against apoptosis, and enhancement of metastatic factors. These findings propose ERα36 as a novel target for the development of therapies that can prevent progression of breast cancer in the primary tumor as well as during metastasis.
Assuntos
Processamento Alternativo , Neoplasias da Mama/metabolismo , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Proteínas de Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Neutralizantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Células COS , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/patologia , Proliferação de Células/efeitos dos fármacos , Chlorocebus aethiops , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Feminino , Células HeLa , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/genética , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismoRESUMO
BACKGROUND: Osseointegration depends on the implant surface, bone quality and the local and systemic host environment, which can differ in male and female patients. This study was undertaken in order to determine if male and female cells respond differently to titanium surfaces that have micron-scale roughness and if interactions of calciotropic hormones [1α,25(OH)2D3 and 17ß-oestradiol (E2)] and microstructured surfaces on osteoblasts are sex dependent. METHODS: Osteoblasts from 6-week old Sprague-Dawley rats were cultured on tissue culture polystyrene (TCPS) or on titanium (Ti) disks with two different surface topographies, a smooth pretreated (PT) surface and a coarse grit-blasted/acid-etched (SLA) surface, and treated with 1α,25(OH)2D3, E2, or E2 conjugated to bovine serum albumin (E2-BSA). RESULTS: Male and female cells responded similarly to Ti microstructure with respect to cell number and levels of osteocalcin, transforming growth factor-ß1, osteoprotegerin and prostaglandin E2 in their conditioned media, exhibiting a more differentiated phenotype on SLA than on PT or TCPS. E2 and E2-BSA increased differentiation and local factor production, an effect that was microstructure dependent and found only in female osteoblasts. 1α,25(OH)2D3 increased osteoblast differentiation and local factor production in female and male cells, but the effect was more robust in male cells. CONCLUSIONS: Male and female rat osteoblasts respond similarly to surface microstructure but exhibit sexual dimorphism in substrate-dependent responses to systemic hormones. Oestrogen affected only female cells while 1α,25(OH)2D3 had a greater effect on male cells. These results suggest that successful osseointegration in males and females may depend on the implant surface design and correct levels of calciotropic hormones.