RESUMO
Previously we showed that neuropeptide Y (NPY), a sympathetic vasoconstrictor neurotransmitter, stimulates endothelial cell migration, proliferation, and differentiation in vitro. Here, we report on NPY's actions, receptors, and mediators in ischemic angiogenesis. In rats, hindlimb ischemia stimulates sympathetic NPY release (attenuated by lumbar sympathectomy) and upregulates NPY-Y2 (Y2) receptor and a peptidase forming Y2/Y5-selective agonist. Exogenous NPY at physiological concentrations also induces Y5 receptor, stimulates neovascularization, and restores ischemic muscle blood flow and performance. NPY-mediated ischemic angiogenesis is not prevented by a selective Y1 receptor antagonist but is reduced in Y2(-/-) mice. Nonischemic muscle vascularity is also lower in Y2(-/-) mice, whereas it is increased in NPY-overexpressing rats compared with their WT controls. Ex vivo, NPY-induced aortic sprouting is markedly reduced in Y2(-/-) aortas and spontaneous sprouting is severely impaired in NPY(-/-) mice. NPY-mediated aortic sprouting, but not cell migration/proliferation, is blocked by an antifetal liver kinase 1 antibody and abolished in mice null for eNOS. Thus, NPY mediates neurogenic ischemic angiogenesis at physiological concentrations by activating Y2/Y5 receptors and eNOS, in part due to release of VEGF. NPY's effectiveness in revascularization and restoring function of ischemic tissue suggests its therapeutic potential in ischemic conditions.
Assuntos
Isquemia/tratamento farmacológico , Músculo Esquelético/irrigação sanguínea , Neovascularização Patológica/induzido quimicamente , Neuropeptídeo Y/farmacologia , Neuropeptídeo Y/fisiologia , Animais , Dipeptidil Peptidase 4/fisiologia , Fatores de Crescimento Endotelial/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Isquemia/patologia , Isquemia/fisiopatologia , Linfocinas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase/deficiência , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores de Neuropeptídeo Y/agonistas , Receptores de Neuropeptídeo Y/deficiência , Receptores de Neuropeptídeo Y/genética , Receptores de Neuropeptídeo Y/fisiologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The progressive growth of most neoplasms is dependent upon the establishment of new blood vessels, a process regulated by tumor-secreted factors and matrix proteins. We examined the in vitro and in vivo angiogenic ability of conditioned media obtained from fibrosarcoma, carcinoma, and osteosarcoma cells and their decorin-transfected counterparts. Human endothelial cells were investigated in vitro by evaluating three essential steps of angiogenesis: migration, attachment, and differentiation. On the whole, wild-type tumor cell-secretions enhanced endothelial cell attachment, migration, and differentiation, whereas their decorin-expressing forms inhibited these processes. Similarly, decorin-containing media suppressed endothelial cell sprouting in an ex vivo aortic ring assay. Since angiogenesis is an important component of tumor expansion, the growth rate of these cells as tumor xenografts was examined by implantation in nude mice. In vivo, the decorin-expressing tumor xenografts grew at markedly lower rates and showed a significant suppression of neovascularization. Immunohistochemical, Northern and Western blot analyses indicated that the decorin-expressing cells produced vascular endothelial growth factor (VEGF) at markedly reduced rates vis-á-vis their wild-type counterparts. Specificity of this process was confirmed by experiments where addition of recombinant decorin to the wild-type tumor cells caused 80-95% suppression of VEGF mRNA and protein. These results provide a novel mechanism of action for decorin, and indicate that decorin could adversely affect in vivo tumor growth by suppressing the endogenous tumor cell production of a powerful angiogenic stimulus.
Assuntos
Neoplasias/irrigação sanguínea , Neovascularização Patológica , Proteoglicanas/fisiologia , Animais , Capilares/efeitos dos fármacos , Capilares/crescimento & desenvolvimento , Adesão Celular/efeitos dos fármacos , Divisão Celular , Células Cultivadas , Quimiotaxia , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Técnicas de Cultura , Decorina , Relação Dose-Resposta a Droga , Fatores de Crescimento Endotelial/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Proteínas da Matriz Extracelular , Humanos , Laminina/metabolismo , Linfocinas/metabolismo , Camundongos , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Proteoglicanas/análise , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Sympathetic nerve activation often accompanies tissue ischemia, which in turn stimulates angiogenesis, but whether the nerves regulate vascular functions beyond vasoconstriction (i.e., by promoting new vessel formation) has never been established. Neuropeptide Y (NPY) is a sympathetic cotransmitter preferentially released during intense or prolonged stress, which causes vasoconstriction and vascular smooth muscle cell proliferation by activating multiple Gi/o-coupled receptors, Y1 and Y5. At nonvasoconstrictive concentrations and through non-Y1 receptors, NPY also stimulates endothelial cell adhesion to matrix, migration, proliferation, capillary tube formation on matrigel, and aortic sprouting. Recent studies also indicate that NPY and its non-Y1 receptors exert powerful angiogenic effects in peripheral limb ischemia, promising a new way of treatment for revascularization of ischemic tissues.
Assuntos
Neuropeptídeo Y/fisiologia , Animais , Medicina Baseada em Evidências , Humanos , Músculo Liso Vascular/irrigação sanguínea , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatologia , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/fisiopatologia , Neuropeptídeo Y/metabolismo , Sistema Nervoso Simpático/irrigação sanguínea , Sistema Nervoso Simpático/metabolismo , Sistema Nervoso Simpático/fisiopatologiaRESUMO
Angiostatin inhibits angiogenesis by binding to endothelial cells (ECs) lining the vasculature of growing tumors. These cells are in a dynamic state during angiogenesis and are thus not firmly attached to the extracellular matrix. This makes them more vulnerable to anoikis, a process resulting in cell death initiated by or promoted by loss of attachment. Another potential source of EC vulnerability during tumor angiogenesis is that tumor extracellular pH is typically lower than in normal tissues. This presents an additional challenge to ECs in terms of maintaining ionic homeostasis. We report here that the lethality of angiostatin is significantly enhanced both by reduced matrix attachment during exposure and lowered extracellular pH (pH(e)). Another effect of angiostatin at reduced pH(e) is a decreased intracellular pH (pH(i)). These effects were observed in three model systems: aortic ring sprouts, ECs during tube formation, and ECs in a scratch/migration assay. In these three dynamic assays, angiostatin-induced cell death and intracellular acidification were clearly seen when pH(e) was reduced to 6.7. The intracellular acidification was far greater than that induced by pH(e) reduction alone. In contrast, the effect of angiostatin on pH(i) and on viability were not observed in a subconfluent monolayer in which the cells were allowed to attach to substrate for 48 h prior to exposure to angiostatin. These data suggest that low pH(e) and reduced adhesion to matrix play a role in the specificity of angiostatin for tumor neovasculature in contrast to wound healing and other normal angiogenic processes. The results also implicate roles for both pH(e) and pH(i) regulation in the mechanism of angiostatin action.
Assuntos
Anoikis/fisiologia , Endotélio Vascular/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Plasminogênio/farmacologia , Acidose/metabolismo , Angiostatinas , Animais , Células Cultivadas , Endotélio Vascular/química , Guanidinas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Líquido Intracelular/química , Neoplasias , Ratos , Sulfonas/metabolismoRESUMO
Which of Y1-Y5 receptors (Rs) mediate NPY's angiogenic activity was studied using Y2R-null mice and R-specific antagonists. In Y2R-null mice, NPY-induced aortic sprouting and in vivo Matrigel capillary formation were decreased by 50%; Y1R-antagonist blocked the remaining response. NPY-induced sprouting was equally inhibited by Y2R- (and Y5R- but less by Y1R-) antagonists in wild type mice. Spontaneous and NPY-induced revascularization of ischemic gastrocnemius muscles were similarly reduced in Y2R-null mice. Thus, NPY-induced angiogenesis, spontaneous and ischemic, is primarily mediated by Y2Rs. However, Y5Rs and, to a lesser degree Y1Rs, also may play a role in NPY-mediated angiogenesis.
Assuntos
Neovascularização Fisiológica/fisiologia , Receptores de Neuropeptídeo Y/fisiologia , Animais , Capilares/crescimento & desenvolvimento , Camundongos , Camundongos Knockout , Receptores de Neuropeptídeo Y/genéticaRESUMO
Tumor growth requires a competent vascular supply and angiogenesis has been considered as a potential target for the treatment of several cancers. The two clinically approved taxanes, paclitaxel and docetaxel, are novel antimitotic agents that are under extensive investigation in clinical trials. Both taxanes have demonstrated significant activity against many solid tumors, but little is known about the effect of paclitaxel and docetaxel on endothelial cell function and angiogenic processes. The purpose of our study was to examine and compare the effects of these drugs on angiogenic processes in vitro and in vivo. These processes include: proliferation, migration and differentiation of cultured human umbilical vein endothelial cells (HUVEC) (in vitro), capillary sprouting of rat aortic ring explants (ex vivo) and HT1080 tumor growth in vivo. Our results demonstrate that endothelial cells are 10-100-fold more sensitive to these drugs than tumor cells. Additionally, comparison of the taxanes demonstrated that angiogenesis is blocked by both drugs primarily via inhibition of proliferation and differentiation (tube assay) and induction of cell death. Docetaxel, however, appears to be more potent at inhibiting angiogenesis, with an IC(50) concentration 10x less than that of paclitaxel. We conclude that these important findings should be taken in account in clinical trials where tumor angiogenesis is being targeted.
Assuntos
Inibidores da Angiogênese/farmacologia , Endotélio Vascular/efeitos dos fármacos , Paclitaxel/análogos & derivados , Paclitaxel/farmacologia , Taxoides , Inibidores da Angiogênese/uso terapêutico , Animais , Aorta/citologia , Apoptose/efeitos dos fármacos , Capilares/efeitos dos fármacos , Capilares/ultraestrutura , Carcinoma Pulmonar de Células não Pequenas/patologia , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Docetaxel , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Endotélio Vascular/citologia , Fibrossarcoma/irrigação sanguínea , Fibrossarcoma/tratamento farmacológico , Fibrossarcoma/patologia , Glioblastoma/patologia , Humanos , Neoplasias Pulmonares/patologia , Masculino , Melanoma/patologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Paclitaxel/uso terapêutico , Ratos , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/transplante , Veias Umbilicais/citologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor growth and angiogenesis are interdependent. Paclitaxel and radiation therapy are commonly used in the clinic, in a number of disease sites, requiring high dosages of both drug and radiation for cure. Paclitaxel (Taxol) is a diterpenoid with antitumor activity against a variety of human neoplasms and can amplify the cytotoxic effect of ionizing radiation in vitro, presumably by inducing arrest at metaphase, known to be a very radiosensitive phase of the cell cycle. Little is known about how angiogenesis is affected by paclitaxel when the combination of paclitaxel and radiation are used. We have evaluated the combination of paclitaxel and radiation at various concentrations, on cytokine-induced angiogenesis in vitro with the goal of determining whether reduction of radiation and paclitaxel doses is possible without sacrificing efficacy. We have found that paclitaxel inhibited endothelial cell proliferation, migration, and tube formation (differentiation) at one-tenth the concentration needed to achieve a similar effect on tumor cell lines. In combination with radiation, inhibition of endothelial cell function was additive and increased twofold. The combination of low-dose paclitaxel and radiation suggests a complementary strategy with potential clinical ramifications to target angiogenesis-dependent malignancies.