RESUMO
Anti-vascular endothelial growth factor (VEGF) therapies have improved clinical outcomes for patients with cancers and retinal vascular diseases. Three anti-VEGF agents, pegaptanib, ranibizumab, and aflibercept, are approved for ophthalmic indications, while bevacizumab is approved to treat colorectal, lung, and renal cancers, but is also used off-label to treat ocular vascular diseases. The efficacy of bevacizumab relative to ranibizumab in treating neovascular age-related macular degeneration has been assessed in several trials. However, questions persist regarding its safety, as bevacizumab can form large complexes with dimeric VEGF165, resulting in multimerization of the Fc domain and platelet activation. Here, we compare binding stoichiometry, Fcγ receptor affinity, platelet activation, and binding to epithelial and endothelial cells in vitro for bevacizumab and aflibercept, in the absence or presence of VEGF. In contrast to bevacizumab, aflibercept forms a homogenous 1:1 complex with each VEGF dimer. Unlike multimeric bevacizumab:VEGF complexes, the monomeric aflibercept:VEGF complex does not exhibit increased affinity for low-affinity Fcγ receptors, does not activate platelets, nor does it bind to the surface of epithelial or endothelial cells to a greater degree than unbound aflibercept or control Fc. The latter finding reflects the fact that aflibercept binds VEGF in a unique manner, distinct from antibodies not only blocking the amino acids necessary for VEGFR1/R2 binding but also occluding the heparin-binding site on VEGF165.
Assuntos
Bevacizumab/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Inibidores da Angiogênese/efeitos adversos , Inibidores da Angiogênese/metabolismo , Inibidores da Angiogênese/uso terapêutico , Animais , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/metabolismo , Bevacizumab/efeitos adversos , Bevacizumab/uso terapêutico , Linhagem Celular , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Degeneração Macular/imunologia , Degeneração Macular/metabolismo , Degeneração Macular/terapia , Camundongos , Camundongos Transgênicos , Ativação Plaquetária , Ligação Proteica , Multimerização Proteica , Receptores de IgG/genética , Receptores de IgG/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Proteínas Recombinantes de Fusão/efeitos adversos , Proteínas Recombinantes de Fusão/uso terapêutico , Trombocitopenia/etiologia , Trombose/etiologia , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
Blood vessel remodeling is crucial to the formation of the definitive vasculature, but little is known about the mechanisms controlling this process. We show that Delta-like ligand 4 (Dll4)/Notch pathway regulates vessel regression in normal pathologic conditions. Genetic and pharmacologic inhibition of Dll4/Notch prevented retinal capillary regression in the oxygen-induced retinopathy (OIR) model and during normal development. Deletion of the Notch-regulated ankyrin repeat protein, a negative regulator of the Notch pathway, produced an opposite phenotype. Inhibition of Dll4/Notch reduced vessel occlusion, maintaining blood flow that is essential for survival of microvessels. Dll4/Notch inhibition up-regulated the expression of vasodilators adrenomedullin and suppressed the expression of vasoconstrictor angiotensinogen. Angiotensin II induced rapid nonperfusion and regression of developing retinal capillaries, whereas Ace1 and AT1 inhibitors and adrenomedullin attenuated vasoobliteration in OIR, indicating that both pathways are involved in modulating vessel remodeling. In contrast, inhibition of vascular endothelial growth factor-A (VEGF-A) did not result in a pervasive loss of retinal capillaries, demonstrating that reduced expression of VEGF-A is not the proximate cause of capillary regression in OIR. Modulation of VEGF-A and Dll4/Notch signaling produced distinct changes in blood vessel morphology and gene expression, indicating that these pathways can have largely independent functions in vascular remodeling.
Assuntos
Vasos Sanguíneos/patologia , Vasos Sanguíneos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Receptor Notch1/fisiologia , Fluxo Sanguíneo Regional/genética , Vasoconstrição/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Animais Recém-Nascidos , Atrofia/genética , Vasos Sanguíneos/metabolismo , Células CHO , Proteínas de Ligação ao Cálcio , Células Cultivadas , Cricetinae , Cricetulus , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , Receptor Notch1/genética , Receptor Notch1/metabolismo , Regeneração/genética , Regeneração/fisiologia , Fluxo Sanguíneo Regional/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Vasoconstrição/fisiologiaRESUMO
Genetic ablation of Inppl1, which encodes SHIP2 (SH2-domain containing inositol 5-phosphatase 2), was previously reported to induce severe insulin sensitivity, leading to early postnatal death. In the previous study, the targeting construct left the first eighteen exons encoding Inppl1 intact, generating a Inppl1(EX19-28-/-) mouse, and apparently also deleted a second gene, Phox2a. We report a new SHIP2 knockout (Inppl1(-/-)) targeted to the translation-initiating ATG, which is null for Inppl1 mRNA and protein. Inppl1(-/-) mice are viable, have normal glucose and insulin levels, and normal insulin and glucose tolerances. The Inppl1(-/-) mice are, however, highly resistant to weight gain when placed on a high-fat diet. These results suggest that inhibition of SHIP2 would be useful in the effort to ameliorate diet-induced obesity, but call into question a dominant role of SHIP2 in modulating glucose homeostasis.
Assuntos
Gorduras na Dieta/metabolismo , Obesidade/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Análise Química do Sangue , Peso Corporal , Éxons , Feminino , Deleção de Genes , Genes Reporter , Glucose/metabolismo , Homeostase , Inositol Polifosfato 5-Fosfatases , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , Transdução de Sinais , Distribuição TecidualRESUMO
Vascular remodeling is a feature of chronic inflammation during which capillaries transform into venules that expand the region of the vasculature in which leakage and leukocyte emigration both occur. Recently, we found that angiopoietin/Tie2 receptor signaling drives the transformation of capillaries into venules at an early stage of the sustained inflammatory response in the airways of mice infected with Mycoplasma pulmonis. However, the precise contributions of both angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) are not clear. In this study, we sought to determine the contribution of Ang2 to this vascular remodeling. Ang2 mRNA expression levels increased and phosphorylated Tie2 immunoreactivity in mucosal blood vessels decreased, indicative of diminished receptor signaling after infection. Selective inhibition of Ang2 throughout the infection by administration of either of two distinct function-blocking antibodies reduced the suppression of Tie2 phosphorylation and decreased the remodeling of mucosal capillaries into venules, the amount of leukocyte influx, and disease severity. These findings are consistent with Ang2 acting as an antagonist of Tie2 receptors and the reduction of Tie2 phosphorylation in endothelial cells rendering the vasculature more responsive to cytokines that promote both vascular remodeling and the consequences of inflammation after M. pulmonis infection. By blocking such changes, Ang2 inhibitors may prove beneficial in the treatment of sustained inflammation in which vascular remodeling, leakage, and leukocyte influx contribute to its pathophysiology.
Assuntos
Angiopoietina-2/fisiologia , Vasos Sanguíneos/fisiologia , Neovascularização Fisiológica/genética , Sistema Respiratório/irrigação sanguínea , Doenças Respiratórias/genética , Angiopoietina-2/genética , Angiopoietina-2/imunologia , Angiopoietina-2/metabolismo , Animais , Vasos Sanguíneos/metabolismo , Inflamação/genética , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Mycoplasma/complicações , Infecções por Mycoplasma/genética , Infecções por Mycoplasma/metabolismo , Mycoplasma pulmonis/fisiologia , Neovascularização Fisiológica/fisiologia , Pneumonia/etiologia , Pneumonia/genética , Pneumonia/metabolismo , Pneumonia/patologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/fisiologia , Receptor TIE-2 , Sistema Respiratório/metabolismo , Sistema Respiratório/patologia , Doenças Respiratórias/metabolismo , Doenças Respiratórias/patologiaRESUMO
Endogenous modulators of the central melanocortin system, such as the agouti-related protein (AgRP), should hold a pivotal position in the regulation of energy intake and expenditure. Despite this, AgRP-deficient mice were recently reported to exhibit normal food intake, body weight gain, and energy expenditure. Here we demonstrate that 2- to 3-month-old Agrp null mice do in fact exhibit subtle changes in response to feeding challenges (fasting and MCR agonists) but, of more significance and magnitude, exhibit reduced body weight and adiposity after 6 months of age. This age-dependent lean phenotype is correlated with increased metabolic rate, body temperature, and locomotor activity and increased circulating thyroid hormone (T4 and T3) and BAT UCP-1 expression. These results provide further proof of the importance of the AgRP neuronal system in the regulation of energy homeostasis.
Assuntos
Proteínas/genética , Proteínas/fisiologia , Tecido Adiposo/metabolismo , Glândulas Suprarrenais/metabolismo , Envelhecimento , Proteína Relacionada com Agouti , Animais , Composição Corporal , Temperatura Corporal , Peso Corporal , Encéfalo/metabolismo , Calorimetria , Comportamento Alimentar , Regulação da Expressão Gênica , Vetores Genéticos , Peptídeos e Proteínas de Sinalização Intercelular , Óperon Lac , Camundongos , Camundongos Transgênicos , Modelos Genéticos , Neurônios/metabolismo , Fenótipo , Hormônios Tireóideos/metabolismo , Fatores de Tempo , beta-Galactosidase/metabolismoRESUMO
VEGF is the best characterized mediator of tumor angiogenesis. Anti-VEGF agents have recently demonstrated impressive efficacy in human cancer trials, but the optimal dosing of such agents must still be determined empirically, because biomarkers to guide dosing have yet to be established. The widely accepted (but unverified) assumption that VEGF production is quite low in normal adults led to the notion that increased systemic VEGF levels might quantitatively reflect tumor mass and angiogenic activity. We describe an approach to determine host and tumor production of VEGF, using a high-affinity and long-lived VEGF antagonist now in clinical trials, the VEGF Trap. Unlike antibody complexes that are usually rapidly cleared, the VEGF Trap forms inert complexes with tissue- and tumor-derived VEGF that remain stably in the systemic circulation, where they are readily assayable, providing unprecedented capability to accurately measure VEGF production. We report that VEGF production is surprisingly high in non-tumor-bearing rodents and humans, challenging the notion that systemic VEGF levels can serve as a sensitive surrogate for tumor load; tumor VEGF contribution becomes significant only with very large tumor loads. These findings have the important corollary that anti-VEGF therapies must be sufficiently dosed to avoid diversion by host-derived VEGF. We further show that our assay can indicate when VEGF is optimally blocked; such biomarkers to guide dosing do not exist for other anti-VEGF agents. Based on this assay, VEGF Trap doses currently being assessed in clinical trials are in the efficacious range.
Assuntos
Inibidores da Angiogênese/farmacologia , Fatores de Crescimento do Endotélio Vascular/biossíntese , Envelhecimento/fisiologia , Inibidores da Angiogênese/imunologia , Animais , Anticorpos/imunologia , Biomarcadores , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos SCID , Ligação Proteica , Fatores de Crescimento do Endotélio Vascular/sangue , Fatores de Crescimento do Endotélio Vascular/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Displasia do Colo do Útero/metabolismo , Displasia do Colo do Útero/patologiaRESUMO
PURPOSE: To determine the effect of vascular endothelial growth factor (VEGF) TrapR1R2 on bFGF-induced experimental corneal neovascularization (NV). METHODS: Control pellets or pellets containing 80 ng bFGF were surgically implanted into wild-type C57BL/6 and VEGF-LacZ mouse corneas. The corneas were photographed, harvested, and the percentage of corneal NV was calculated. The harvested corneas were evaluated for VEGF expression. VEGF-LacZ mice received tail vein injections of an endothelial-specific lectin after pellet implantation to determine the temporal and spatial relationship between VEGF expression and corneal NV. Intraperitoneal injections of VEGF TrapR1R2 or a human IgG Fc domain control protein were administered, and bFGF pellet-induced corneal NV was evaluated. RESULTS: NV of the corneal stroma began on day 4 and was sustained through day 21 following bFGF pellet implantation. Progression of vascular endothelial cells correlated with increased VEGF-LacZ expression. Western blot analysis showed increased VEGF expression in the corneal NV zone. Following bFGF pellet implantation, the area of corneal NV in untreated controls was 1.05+/-0.12 mm2 and 1.53+/-0.27 mm2 at days 4 and 7, respectively. This was significantly greater than that of mice treated with VEGF Trap (0.24+/-0.11 mm2 and 0.35+/-0.16 mm2 at days 4 and 7, respectively; p<0.05). CONCLUSIONS: Corneal keratocytes express VEGF after bFGF stimulation and bFGF-induced corneal NV is blocked by intraperitoneal VEGF TrapR1R2 administration. Systemic administration of VEGF TrapR1R2 may have potential therapeutic applications in the management of corneal NV.
Assuntos
Neovascularização da Córnea/prevenção & controle , Modelos Animais de Doenças , Receptores de Fatores de Crescimento/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Animais , Western Blotting , Córnea/metabolismo , Córnea/patologia , Neovascularização da Córnea/induzido quimicamente , Neovascularização da Córnea/patologia , Fator 2 de Crescimento de Fibroblastos/toxicidade , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
VEGF and Angiopoietin-1 requisitely collaborate during blood vessel development. While Angiopoietin-1 obligately activates its Tie2 receptor, Angiopoietin-2 can activate Tie2 on some cells, while it blocks Tie2 activation on others. Our analysis of mice lacking Angiopoietin-2 reveals that Angiopoietin-2 is dispensable for embryonic vascular development but is requisite for subsequent angiogenic remodeling. Unexpectedly, mice lacking Angiopoietin-2 also exhibit major lymphatic vessel defects. Genetic rescue with Angiopoietin-1 corrects the lymphatic, but not the angiogenesis, defects, suggesting that Angiopoietin-2 acts as a Tie2 agonist in the former setting, but as an antagonist in the latter setting. Our studies define a vascular growth factor whose primary role is in postnatal angiogenic remodeling and also demonstrate that members of the VEGF and Angiopoietin families collaborate during development of the lymphatic vasculature.
Assuntos
Indutores da Angiogênese/fisiologia , Padronização Corporal , Sistema Linfático/crescimento & desenvolvimento , Glicoproteínas de Membrana/fisiologia , Neovascularização Fisiológica/fisiologia , Angiopoietina-1 , Angiopoietina-2 , Animais , Ascite Quilosa/genética , Ascite Quilosa/patologia , DNA Complementar/genética , Edema/genética , Edema/patologia , Olho/irrigação sanguínea , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Homozigoto , Sistema Linfático/patologia , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Vasos Retinianos/patologiaRESUMO
In response to hypoxia, hypoxia-inducible factors act as the primary proangiogenic triggers by regulating transcription levels of target genes, including VEGF. However, little is known about the specific factors that control other components of the angiogenic process, particularly formation of matrix scaffolds that promote adhesion and migration of endothelial cells. We show that in the postnatal mouse retina, the orphan nuclear receptor tailless (Tlx) is strongly expressed in the proangiogenic astrocytes, which secrete VEGF and fibronectin. Tlx expression by retinal astrocytes is controlled by oxygen concentration and rapidly downregulated upon contact with blood vessels. In mice null for Tlx, retinal astrocytes maintain VEGF expression; however, the extracellular assembly of fibronectin matrices by astrocytes is severely impaired, leading to defective scaffold formation and a complete failure of normal retinal vascular development. This work identifies Tlx as an essential component of the molecular network involved in the hypoxia-inducible proangiogenic switch in retinal astrocytes.
Assuntos
Astrócitos/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Neovascularização Fisiológica , Receptores Citoplasmáticos e Nucleares/metabolismo , Retina/citologia , Animais , Astrócitos/citologia , Colágeno Tipo IV/metabolismo , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Knockout , Oxigênio/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Despite extensive literature on vascular endothelial growth factor (VEGF) expression and regulation by steroid hormones, the lack of clear understanding of the mechanisms of angiogenesis in the endometrium is a major limitation for use of antiangiogenic therapy targeting endometrial vessels. In the current work, we used the rhesus macaque as a primate model and the decidualized mouse uterus as a murine model to examine angiogenesis during endometrial breakdown and regeneration. We found that blockade of VEGF action with VEGF Trap, a potent VEGF blocker, completely inhibited neovascularization during endometrial regeneration in both models but had no marked effect on preexisting or newly formed vessels, suggesting that VEGF is essential for neoangiogenesis but not survival of mature vessels in this vascular bed. Blockade of VEGF also blocked reepithelialization in both the postmenstrual endometrium and the mouse uterus after decidual breakdown, evidence that VEGF has pleiotropic effects in the endometrium. In vitro studies with a scratch wound assay showed that the migration of luminal epithelial cells during repair involved signaling through VEGF receptor 2-neuropilin 1 (VEGFR2-NP1) receptors on endometrial stromal cells. The leading front of tissue growth during endometrial repair was strongly hypoxic, and this hypoxia was the local stimulus for VEGF expression and angiogenesis in this tissue. In summary, we provide novel experimental data indicating that VEGF is essential for endometrial neoangiogenesis during postmenstrual/postpartum repair.
Assuntos
Endométrio/irrigação sanguínea , Endométrio/fisiologia , Menstruação/fisiologia , Neovascularização Fisiológica , Regeneração , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Movimento Celular , Endométrio/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Feminino , Macaca mulatta , Menstruação/metabolismo , Camundongos , Camundongos Endogâmicos , Neovascularização Fisiológica/efeitos dos fármacos , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão/farmacologia , Regeneração/efeitos dos fármacos , Células Estromais/fisiologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
The present studies explore the roles of vascular endothelial growth factor (VEGF) and estradiol on angiogenesis and stromal and epithelial cell proliferation in the marmoset endometrium during the proliferative phase of the ovulatory cycle. At the start of the proliferative phase, marmosets were 1) treated with vehicle, 2) treated with a VEGF inhibitor (VEGF Trap, aflibercept), 3) ovariectomized, 4) ovariectomized and given replacement estradiol, or 5) treated with VEGF Trap and given replacement estradiol. The uterus was examined 10 d later in the late proliferative phase. Changes in endothelial and epithelial cell proliferation were quantified using a volumetric density method after immunohistochemistry for bromodeoxyuridine to localize proliferating cells, CD31 to visualize endothelial cells, and dual staining to distinguish endothelial cell proliferation. Endothelial proliferation was elevated in late proliferative controls but virtually absent after VEGF Trap. Ovariectomy had a similar inhibitory effect, whereas angiogenesis was restored by estrogen replacement. Estradiol replacement in VEGF Trap-treated marmosets resulted in only a small increase in endothelial cell proliferation that remained significantly below control values. VEGF Trap treatment and ovariectomy also markedly reduced stromal cell proliferation but resulted in increased stromal cell density associated with a reduction in overall endometrial volume. Estrogen replacement in both ovariectomized and VEGF Trap-treated animals restored stromal proliferation rates and cell density. These results show that endometrial angiogenesis and stromal proliferation during the proliferative phase are driven by estradiol and that the effect of estrogen on angiogenesis is mediated largely by VEGF.
Assuntos
Proliferação de Células , Endométrio/irrigação sanguínea , Estradiol/fisiologia , Neovascularização Fisiológica/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Callithrix , Proliferação de Células/efeitos dos fármacos , Endométrio/citologia , Endométrio/fisiologia , Estradiol/farmacologia , Feminino , Neovascularização Fisiológica/efeitos dos fármacos , Tamanho do Órgão , Ovariectomia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
To assess whether there is a link between estrogen, vascular endothelial growth factor (VEGF), and early aspects of uterine angiogenesis, an acute temporal study was conducted in which ovariectomized baboons were pretreated with VEGF Trap, which sequesters endogenous VEGF, and administered estradiol at time 0 h. Serum estradiol levels approximated 500 pg/ml 4-6 h after estradiol administration. VEGF mRNA levels in endometrial glandular epithelial and stromal cells were increased to values 6 h after estradiol that were 3.74 +/- 0.99-fold (mean +/- se) and 5.70 +/- 1.60-fold greater (P < 0.05), respectively, than at 0 h. Microvessel interendothelial cell tight junctions, which control paracellular permeability, were present in the endometrium at time 0 h, but not evident 6 h after estradiol administration. Thus, microvessel paracellular cleft width increased (P < 0.01, ANOVA) from 5.03 +/- 0.22 nm at 0 h to 7.27 +/- 0.48 nm 6 h after estrogen. In contrast, tight junctions remained intact, and paracellular cleft widths were unaltered in estradiol/VEGF Trap and vehicle-treated animals. Endometrial microvessel endothelial cell mitosis, i.e. percent Ki67+/Ki67- immunolabeled endothelial cells, increased (P < 0.05) from 2.9 +/- 0.3% at 0 h to 21.4 +/- 7.0% 6 h after estrogen treatment but was unchanged in estradiol/VEGF Trap and vehicle-treated animals. In summary, the estrogen-induced disruption of endometrial microvessel endothelial tight junctions and increase in endothelial cell proliferation were prevented by VEGF Trap. Therefore, we propose that VEGF mediates the estrogen-induced increase in microvessel permeability and endothelial cell proliferation as early steps in angiogenesis in the primate endometrium.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células Endoteliais/metabolismo , Estradiol/farmacologia , Junções Íntimas/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Permeabilidade Capilar/efeitos dos fármacos , Endométrio/citologia , Células Endoteliais/citologia , Células Endoteliais/ultraestrutura , Estradiol/administração & dosagem , Estradiol/sangue , Feminino , Imuno-Histoquímica , Antígeno Ki-67/análise , Microscopia Eletrônica de Transmissão , Microvasos/citologia , Microvasos/fisiologia , Ovariectomia , Papio , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Junções Íntimas/ultraestrutura , Fator de von Willebrand/análise , Fator de von Willebrand/imunologiaRESUMO
OBJECTIVE: To evaluate whether hemangiogenesis, lymphangiogenesis, and concomitant invasion of mononuclear phagocytes occurring after high-risk corneal transplantation in already vascularized high-risk recipient corneal beds increase the risk for subsequent immune rejection. METHODS: Three intrastromal sutures were left in place for 6 weeks in the corneas of BALB/c mice, causing neovascularization. Three weeks after suture removal, keratoplasty was performed (donors C57BL/6 mice). The treatment group received a vascular endothelial growth factor A (VEGF-A)-neutralizing cytokine trap at 0, 4, 7, and 14 days postoperatively (Fc protein was used as the control treatment). Morphometry was performed in corneal flat mounts using lymphatic endothelial hyaluronan receptor-1 (a specific lymphatic endothelial marker), CD31 (a panendothelial marker), and F4/80 (a marker for mononuclear phagocytes). RESULTS: After corneal transplantation, significant additional hemangiogenesis (mean area covered by vessels [SD], 68% [18%] postoperatively vs 40% [18%] preoperatively; P = .03) and lymphangiogenesis (12% [1.3%] postoperatively vs 9% [2.8%] preoperatively; P = .03) were observed. Postoperative neutralization of VEGF-A inhibited operation-induced hemangiogenesis (35% [8%]; P = .007) and lymphangiogenesis (6% [1.6%]; P = .03) and decreased the recruitment of mononuclear phagocytes into the graft (mean [SD], 501 cells/mm(2) [152] in treated mice vs 684 cells/mm(2) [35] in Fc controls; P = .03). After 8 weeks, 23% of the treated corneas were not rejected, whereas all control corneas were rejected after 21 days (P = .007). CONCLUSIONS: Neutralization of VEGF-A after high-risk corneal transplantation effectively inhibits postoperative hemangiogenesis, lymphangiogenesis, and recruitment of antigen-presenting cells and improves corneal graft survival. CLINICAL RELEVANCE: Blocking of VEGF-A after high-risk corneal transplantation may be a novel approach to improve graft survival.
Assuntos
Neovascularização da Córnea/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Ceratoplastia Penetrante , Proteínas Recombinantes de Fusão/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Células Apresentadoras de Antígenos/fisiologia , Antígenos de Diferenciação/metabolismo , Neovascularização da Córnea/etiologia , Neovascularização da Córnea/metabolismo , Modelos Animais de Doenças , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Sobrevivência de Enxerto/fisiologia , Receptores de Hialuronatos/metabolismo , Linfangiogênese/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fagócitos/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Receptores de Fatores de Crescimento do Endotélio VascularRESUMO
Purpose: We establish and characterize the chronic retinal neovascularization (RNV) induced by intravitreal (IVT) injection of DL-α-aminoadipic acid (AAA) in a rabbit model and investigate the extent and duration of inhibitory actions induced by IVT aflibercept on the RNV. Methods: Rabbits received a single IVT injection of AAA, with weekly follow-up fundus photography, fluorescein angiography (FA), and optical coherence tomography (OCT). After 10 weeks, they received a single IVT aflibercept or control injection. RNV leakage was quantified from FA by image analysis with Photoshop. Some eyes were collected for histologic analysis. Results: IVT AAA produced neuronal degeneration over a large fraction of the retina. RNV formed in the damaged area and by 10 weeks exhibited stable morphology and leakage, which persisted for at least 65 weeks. Control IVT injections did not affect RNV leakage, but IVT aflibercept completely blocked RNV leakage. The inhibition was reversible (i.e., the leakage returned as the drug cleared), and the duration of antileak effects with 500 µg aflibercept was approximately 8 weeks. Partial regression of the pathologic vasculature also occurred with aflibercept, with reestablishment as the drug cleared. Conclusions: This model mimics a chronic human disease in its stability and persistence, and the antileak action of aflibercept is fully reversible with a dose-dependent duration. Therefore, this large eye model is uniquely suitable for investigations into the efficacy and duration of action of novel formulations and pharmacotherapies for retinal vascular diseases, and for studying the underlying pathobiology of retinal angiogenesis.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Modelos Animais de Doenças , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Neovascularização Retiniana/tratamento farmacológico , Vasos Retinianos/efeitos dos fármacos , Inibidores da Angiogênese/uso terapêutico , Animais , Barreira Hematorretiniana/efeitos dos fármacos , Doença Crônica , Relação Dose-Resposta a Droga , Angiofluoresceinografia , Imuno-Histoquímica , Injeções Intravítreas , Masculino , Coelhos , Neovascularização Retiniana/diagnóstico por imagem , Neovascularização Retiniana/fisiopatologia , Fatores de Tempo , Tomografia de Coerência Óptica , Fator A de Crescimento do Endotélio Vascular/metabolismo , Corpo Vítreo/metabolismoAssuntos
Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Células Endoteliais/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator B de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Bevacizumab , Células Endoteliais/imunologia , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Ranibizumab , Receptores de Fatores de Crescimento do Endotélio Vascular , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator B de Crescimento do Endotélio Vascular/imunologia , Fator B de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/imunologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Lymphangiogenesis, an important initial step in tumor metastasis and transplant sensitization, is mediated by the action of VEGF-C and -D on VEGFR3. In contrast, VEGF-A binds VEGFR1 and VEGFR2 and is an essential hemangiogenic factor. We re-evaluated the potential role of VEGF-A in lymphangiogenesis using a novel model in which both lymphangiogenesis and hemangiogenesis are induced in the normally avascular cornea. Administration of VEGF Trap, a receptor-based fusion protein that binds and neutralizes VEGF-A but not VEGF-C or -D, completely inhibited both hemangiogenesis and the outgrowth of LYVE-1(+) lymphatic vessels following injury. Furthermore, both lymphangiogenesis and hemangiogenesis were significantly reduced in mice transgenic for VEGF-A(164/164) or VEGF-A(188/188) (each of which expresses only one of the three principle VEGF-A isoforms). Because VEGF-A is chemotactic for macrophages and we demonstrate here that macrophages in inflamed corneas release lymphangiogenic VEGF-C/VEGF-D, we evaluated the possibility that macrophage recruitment plays a role in VEGF-A-mediated lymphangiogenesis. Either systemic depletion of all bone marrow-derived cells (by irradiation) or local depletion of macrophages in the cornea (using clodronate liposomes) prior to injury significantly inhibited both hemangiogenesis and lymphangiogenesis. We conclude that VEGF-A recruitment of monocytes/macrophages plays a crucial role in inducing inflammatory neovascularization by supplying/amplifying signals essential for pathological hemangiogenesis and lymphangiogenesis.
Assuntos
Neovascularização da Córnea/metabolismo , Linfangiogênese/fisiologia , Macrófagos/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Camundongos , Camundongos Transgênicos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Interactions between endothelial cells (ECs) and perivascular mural cells (MCs) via signaling molecules or physical contacts are implicated both in vascular remodeling and maintenance of vascular integrity. However, it remains unclear how MCs regulate the morphogenic activity of ECs to form an organized vascular architecture, comprising distinct artery, vein, and capillary, from a simple mesh-like network. A clear elucidation of this question requires an experimental model system in which ECs are separated from MCs and yet form vascular structures. Here we report that injection of an antagonistic mAb against PDGFR-beta into murine neonates provides such an experimental system in the retina by completely blocking MC recruitment to developing vessels. While a vascular network was formed even in the absence of MCs, it was poorly remodeled and leaky. Using this vascular system ideal for direct assessment of the activities of MC-derived molecules, we show that addition of recombinant modified angiopoietin-1 restored a hierarchical vasculature, and also rescued retinal edema and hemorrhage in the complete absence of MCs. These observations demonstrate the potential of Ang1 as a new therapeutic modality for MC dropout in diseases such as diabetic retinopathies.
Assuntos
Indutores da Angiogênese/farmacologia , Vasos Sanguíneos/fisiologia , Glicoproteínas de Membrana/farmacologia , Neovascularização Fisiológica/fisiologia , Angiopoietina-1 , Animais , Vasos Sanguíneos/citologia , Vasos Sanguíneos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Neovascularização Fisiológica/efeitos dos fármacos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/fisiologia , Proteínas Recombinantes/farmacologia , Vasos Retinianos/citologia , Vasos Retinianos/efeitos dos fármacosRESUMO
Acute intensive insulin therapy is an independent risk factor for diabetic retinopathy. Here we demonstrate that acute intensive insulin therapy markedly increases VEGF mRNA and protein levels in the retinae of diabetic rats. Retinal nuclear extracts from insulin-treated rats contain higher hypoxia-inducible factor-1alpha (HIF-1alpha) levels and demonstrate increased HIF-1alpha-dependent binding to hypoxia-responsive elements in the VEGF promoter. Blood-retinal barrier breakdown is markedly increased with acute intensive insulin therapy but can be reversed by treating animals with a fusion protein containing a soluble form of the VEGF receptor Flt; a control fusion protein has no such protective effect. The insulin-induced retinal HIF-1alpha and VEGF increases and the related blood-retinal barrier breakdown are suppressed by inhibitors of p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol (PI) 3-kinase, but not inhibitors of p42/p44 MAPK or protein kinase C. Taken together, these findings indicate that acute intensive insulin therapy produces a transient worsening of diabetic blood-retinal barrier breakdown via an HIF-1alpha-mediated increase in retinal VEGF expression. Insulin-induced VEGF expression requires p38 MAPK and PI 3-kinase, whereas hyperglycemia-induced VEGF expression is HIF-1alpha-independent and requires PKC and p42/p44 MAPK. To our knowledge, these data are the first to identify a specific mechanism for the transient worsening of diabetic retinopathy, specifically blood-retinal barrier breakdown, that follows the institution of intensive insulin therapy.
Assuntos
Barreira Hematorretiniana/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus/fisiopatologia , Fatores de Crescimento Endotelial/metabolismo , Insulina/farmacologia , Linfocinas/metabolismo , Proteínas Nucleares/metabolismo , Retina/metabolismo , Animais , Barreira Hematorretiniana/fisiologia , Núcleo Celular/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados , Retinopatia Diabética/fisiopatologia , Modelos Animais de Doenças , Implantes de Medicamento , Fatores de Crescimento Endotelial/genética , Glucose/metabolismo , Glucose/farmacologia , Humanos , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Insulina/uso terapêutico , Linfocinas/genética , Masculino , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Ratos Long-Evans , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Retina/citologia , Fatores de Transcrição/metabolismo , Ativação Transcricional/fisiologia , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Nerve growth factor (NGF) plays a role in sympathetic neuron integrity and survival. Brain-derived neurotrophic factor (BDNF) also has trophic effects on sympathetic neurons. We report here the serendipitous finding that co-treatment of hippocampus with BDNF and the NGF antagonist TrkA-Fc leads to perivascular inflammation and marked vasoconstriction. This effect is not observed with either reagent alone or in combination with other control proteins. Because NGF supports sympathetic neuron health, we tested the hypothesis that BDNF combined with sympathetic compromise caused this effect. Superior cervical ganglia were removed bilaterally with concurrent BDNF infusion into hippocampus. Perivascular inflammation was observed at 3 days, but not 12 days post treatment, when sympathetic terminals had receded, suggesting that the presence of these terminals was necessary for inflammation. Since sympathetic dysfunction may lead to compensatory overactivity of norepinephrine (NE) signaling, we co-infused BDNF with NE in the hippocampus and observed perivascular inflammation. In humans, sympathetic overactivity has been reported in a variety of vascular diseases. Some of these diseases, e.g. primary Raynaud's, are not accompanied by serious inflammatory disease whereas others, such as scleroderma and systemic lupus, are. We speculate that BDNF may contribute to the transformation of sympathetic dysfunction to inflammatory disease.
Assuntos
Doenças do Sistema Nervoso Autônomo/imunologia , Doenças do Sistema Nervoso Autônomo/metabolismo , Edema Encefálico/imunologia , Edema Encefálico/metabolismo , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Animais , Doenças do Sistema Nervoso Autônomo/patologia , Doenças do Sistema Nervoso Autônomo/fisiopatologia , Edema Encefálico/patologia , Fator Neurotrófico Derivado do Encéfalo/administração & dosagem , Hipocampo/metabolismo , Inflamação/metabolismo , Inflamação/fisiopatologia , Bombas de Infusão , Neurônios/imunologia , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: The rate of reendothelialization is critical in neointima formation after arterial injury. Vascular endothelial growth factor (VEGF), a potent endothelial mitogen, has been advocated for accelerating endothelial repair and preventing intimal hyperplasia after percutaneous coronary interventions. However, the precise mechanism of action of VEGF treatment and the physiologic role of endogenous VEGF after arterial injury are not well described. To better understand the role of VEGF in arterial repair, we overexpressed both VEGF and a soluble, chimeric VEGF receptor (VEGF-trap), which binds free VEGF with high affinity, in a mouse model of arterial injury. METHODS AND RESULTS: Four groups of C57BL/6 mice underwent denuding endothelial injury 1 day after systemic injection of recombinant adenovirus expressing (1) VEGF, (2) VEGF-trap, (3) VEGF plus VEGF-trap, or (4) control adenovirus. Circulating levels of adenovirus-encoded proteins were significantly elevated after gene transfer. VEGF overexpression accelerated reendothelialization and increased luminal endothelial cell proliferation 2 weeks after arterial injury (P<0.05), resulting in decreased neointima formation at 4 weeks compared with control (P<0.01). Cotreatment with VEGF-trap completely sequestered free VEGF and abrogated the beneficial effect of VEGF overexpression. Interestingly, sequestration of endogenous VEGF by VEGF-trap overexpression alone also led to delayed reendothelialization at 2 weeks (P<0.01) and increased neointima formation at 4 weeks (P<0.01). CONCLUSIONS: VEGF overexpression accelerated endothelial repair and inhibited neointima formation after arterial injury. Conversely, sequestration of exogenous and/or endogenous VEGF by VEGF-trap delayed reendothelialization and significantly increased neointima size. This demonstrates the therapeutic potential of VEGF but also emphasizes the important physiologic role of endogenous VEGF in vascular repair.