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1.
Ann N Y Acad Sci ; 1470(1): 31-43, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32166759

RESUMO

Loss of vascular elasticity results from progressive degeneration of the extracellular matrix of elastic arteries under the effect of aging and certain diseases, including atherosclerosis. To investigate the influence of vessel wall stiffening on endothelial cell (EC) function, we seeded human umbilical vein ECs onto variably compliant polydimethylsiloxane substrates. When plated on the more compliant substrate, ECs assembled into capillary-like structures. By contrast, they failed to form a network on stiff substrates, even in the presence of vascular endothelial growth factor (VEGF). Cell proliferation and migration increased with stiffness, while ECs released more nitric oxide (NO) on the soft substrate. Treatment with VEGF increased migration and NO release in a stiffness-dependent manner. Atomic force microscopy measurement of cell elasticity along with actin fiber analysis revealed that ECs plated on the more compliant surface were mechanically softer, with mostly diffuse actin arrangement. Our results demonstrate that matrix stiffening induces actin reorganizations, reflected by cortical stiffening in ECs, which may lead to a decrease in their angiogenic capacity and NO release. Hence, the mechanical properties of ECs display a prognostic and therapeutic potential and might serve as a reliable biomarker of vascular function.


Assuntos
Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica/fisiologia , Óxido Nítrico/metabolismo , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia
2.
Acta Diabetol ; 57(8): 947-958, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32130518

RESUMO

AIMS: Excessive glucose serum concentration, endothelial dysfunction and microangiopathy are key features of diabetes mellitus, being both diagnostic parameters and pathogenetic mechanisms. Vascular endothelial growth factor (VEGF) is importantly implicated in the physiology and pathology of blood vessels, including diabetic vascular damage. METHODS: These factors certainly affect endothelial cells, and to evaluate mechanisms involved, we took advantage of telomerase-immortalized human microvascular endothelial (TIME) cells. TIME cells were exposed to different glucose concentrations and to VEGF treatments. Culture conditions also included the use of basement membrane extract, as an in vitro differentiation model. Cell morphology was then evaluated in the different conditions, and cellular proteins were extracted to analyze specific protein products by Western blot. RESULTS: High glucose concentrations and VEGF did substantially affect neither morphology nor growth of cultured TIME cells, while both considerably increased differentiation into "capillary-like" structures when cells were cultured on basement membrane extract. CONCLUSIONS: Under these conditions, high glucose concentration and VEGF also produced a short-term increase in pERK1/2 and p85 proteins, while total and phosphorylated AKT were not affected. These data suggest a direct angiogenetic effect of glucose, affecting intracellular transduction mechanisms with an action similar to that of VEGF. This effect on endothelial cell proliferation and differentiation could be part of pathogenetic mechanisms producing diabetic microvascular alterations.


Assuntos
Indutores da Angiogênese/metabolismo , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Glucose/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Indutores da Angiogênese/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
3.
Int J Nanomedicine ; 15: 587-599, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32095072

RESUMO

Introduction: As heterologous islets or islet-like stem cells become optional sources for islet transplantation, the subcutaneous site appears to be an acceptable replacement of the intrahepatic site due to its graft retrievability. The device-less (DL) procedure improves the feasibility; however, some limitations such as fibrotic overgrowth or immunodeficiency still exist. Nanofibers could mimic the extracellular matrix to improve the vitality of transplanted islets. Therefore, we designed a vascular endothelial growth factor (VEGF)-modified polyvinyl alcohol (PVA)/silicone nanofiber (SiO2-VEGF) to optimize the DL procedure. Methods: SiO2-VEGF nanofibers were designed by nano-spinning and characterized the physical-chemical properties before subcutaneous islet transplantation. Cell viability, vessel formation, and glucose-stimulated insulin secretion were tested in vitro to ensure biocompatibility; and blood glucose level (BGL), transplanted islet function, and epithelial-mesenchymal transition (EMT)-related biomarker expression were analyzed in vivo. Results: The intensity of inflammatory reaction induced by SiO2 nanofibers was between nylon and silicone, which did not bring out excessive fibrosis. The vascularization could be enhanced by VEGF functionalization both in vitro and in vivo. The BGL control was better in the DL combined with SiO2-VEGF group. The percentage of recipients that achieved normoglycemia was higher and earlier (71% at day 57), and the intraperitoneal glucose tolerance test (IPGTT) also confirmed better islet function. The expressions of vimentin, α-SMA, and twist-1 were upregulated, which indicated that SiO2-VEGF nanofibers might promote islet function by regulating the EMT pathway. Discussion: In summary, our new SiO2-VEGF combined with DL procedure might improve the feasibility of subcutaneous islet transplantation for clinical application.


Assuntos
Diabetes Mellitus Experimental/terapia , Transplante das Ilhotas Pancreáticas/métodos , Nanofibras/química , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Glicemia/análise , Glicemia/metabolismo , Diabetes Mellitus Experimental/sangue , Células Endoteliais da Veia Umbilical Humana , Humanos , Injeções Subcutâneas , Insulina/metabolismo , Transplante das Ilhotas Pancreáticas/efeitos adversos , Transplante das Ilhotas Pancreáticas/instrumentação , Masculino , Camundongos Endogâmicos ICR , Neovascularização Fisiológica/efeitos dos fármacos , Álcool de Polivinil/química , Dióxido de Silício/química , Silicones/química , Fator A de Crescimento do Endotélio Vascular/química
4.
Oncol Rep ; 43(2): 625-634, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31894333

RESUMO

While exploring new angiogenesis inhibitors from microbial metabolites, we recently isolated ahpatinins C, E, and G from a soil­derived Streptomyces sp. 15JA150. Ahpatinins C, E and G are known to have pepsin and renin inhibitory activities; however, their antiangiogenic activities and underlying molecular mechanisms have not been fully elucidated. In the present study, the antiangiogenic properties of ahpatinins C, E and G were investigated. The results revealed that the natural compounds significantly inhibited the vascular endothelial growth factor (VEGF)­induced proliferation, invasion, adhesion, and tube formation of human umbilical vein endothelial cells (HUVECs) without exhibiting any cytotoxicity. It was also revealed that ahpatinin E effectively suppressed the neovascularization of the chorioallantoic membranes in growing chick embryos. Notably, ahpatinins C, E, and G led to the downregulation of VEGF­induced activation of VEGF receptor 2 (VEGFR2) and its downstream signaling mediators, including AKT, ERK1/2, JNK, p38, and NF­κB, in HUVECs. Moreover, they reduced the expression of matrix metalloproteinase (MMP)­2 and MMP­9 in the HUVECs following stimulation with VEGF. Furthermore, ahpatinins C, E, and G reduced the tumor cell­induced invasion and tube forming abilities of HUVECs, as well as the expression of VEGF, by suppressing hypoxia­inducible factor­1α (HIF­1α) activity in U87MG glioblastoma cells. Collectively, the present findings indicated that ahpatinins C, E, and G may be used in anticancer therapy by targeting tumor angiogenesis through the inhibition of both VEGFR2 and HIF­1α pathways.


Assuntos
Inibidores da Angiogênese/farmacologia , Fatores Biológicos/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Streptomyces/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Adesão Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pepstatinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
5.
Diabetes ; 69(4): 736-748, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31915155

RESUMO

Diabetic retinopathy is the most common microvascular complication of diabetes, and in the advanced diabetic retinopathy appear vitreal fibrovascular membranes that consist of a variety of cells, including vascular endothelial cells (ECs). New therapeutic approaches for this diabetic complication are urgently needed. Here, we report that in cultured human retinal microvascular ECs, high glucose induced expression of p110δ, which was also expressed in ECs of fibrovascular membranes from patients with diabetes. This catalytic subunit of a receptor-regulated PI3K isoform δ is known to be highly enriched in leukocytes. Using genetic and pharmacological approaches, we show that p110δ activity in cultured ECs controls Akt activation, cell proliferation, migration, and tube formation induced by vascular endothelial growth factor, basic fibroblast growth factor, and epidermal growth factor. Using a mouse model of oxygen-induced retinopathy, p110δ inactivation was found to attenuate pathological retinal angiogenesis. p110δ inhibitors have been approved for use in human B-cell malignancies. Our data suggest that antagonizing p110δ constitutes a previously unappreciated therapeutic opportunity for diabetic retinopathy.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Retinopatia Diabética/metabolismo , Células Endoteliais/metabolismo , Neovascularização Retiniana/metabolismo , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Endoteliais/efeitos dos fármacos , Fatores de Crescimento Endotelial/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Glucose/farmacologia , Imuno-Histoquímica , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/farmacologia
6.
DNA Cell Biol ; 39(2): 167-176, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31808715

RESUMO

Angiogenesis, a process of new blood vessel formation from existing blood vessels, plays an important role in tumor growth and the tissue repair process. It is generally acknowledged that angiogenesis might contribute two both processes. In tumor growth, angiogenesis often increases oncogenic signaling, and in tissue repair, it decreases the stiffness of wound tissue and potentially exacerbates scar formation, resulting in pain and poor function. These poor outcomes are due to an increase in the expression of important genes involved in angiogenesis, such as hypoxia-inducible factor-1 alpha (HIF-1α) and its transcriptional target vascular endothelial growth factor (VEGF). Therefore, this adverse effect of angiogenesis should be taken into consideration. Limiting vessel growth instead of boosting growth may be beneficial for favorable long-term healing outcomes. Posttranslational modifications, including acetylation, which is mediated by histone acetyltransferases, and deacetylation, which is mediated by histone deacetylases (HDACs), are critical to HIF-1α function. Most studies have indicated that HDAC inhibitors (HDACIs) show great promise as antiangiogenic agents in the early phase of clinical trials. In this review, we discuss the role of the HDACs HIF-1α and VEGF in angiogenesis. Furthermore, we also discuss the molecular and cellular underpinnings of the effects of HDACIs on antiangiogenesis, which creates new avenues for anticancer therapeutics and the repair of wounded tissue.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Neovascularização Patológica/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/farmacologia , Indutores da Angiogênese/farmacologia , Animais , Hipóxia Celular/efeitos dos fármacos , Histona Desacetilases/efeitos dos fármacos , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismo
7.
Biochem Pharmacol ; 172: 113771, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31863779

RESUMO

The inhibition of angiogenesis is suggested to be an attractive strategy for cancer therapeutics. Heat shock protein 90 (Hsp90) is closely related to tumorigenesis as it regulates the stabilization and activated states of many client proteins that are essential for cell survival and tumor growth. Here, we investigated the mechanism whereby AT-533, a novel Hsp90 inhibitor, inhibits breast cancer growth and tumor angiogenesis. Based on our results, AT-533 suppressed the tube formation, cell migration, and invasion of human umbilical vein endothelial cells (HUVECs), and was more effective than the Hsp90 inhibitor, 17-AAG. Furthermore, AT-533 inhibited angiogenesis in the aortic ring, Matrigel plug, and chorioallantoic membrane (CAM) models. Mechanically, AT-533 inhibited the activation of VEGFR-2 and the downstream pathways, including Akt/mTOR/p70S6K, Erk1/2 and FAK, in HUVECs, and the viability of breast cancer cells and the HIF-1α/VEGF signaling pathway under hypoxia. In vivo, AT-533 also inhibited tumor growth and angiogenesis by inducing apoptosis and the HIF-1α/VEGF signaling pathway in breast cancer cells. Taken together, our findings indicate that the Hsp90 inhibitor, AT-533, suppresses breast cancer growth and angiogenesis by blocking the HIF-1α/VEGF/VEGFR-2 signaling pathway. AT-533 may thus be a potentially useful drug candidate for breast cancer therapy.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Indazóis/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Indazóis/uso terapêutico , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
8.
Mol Cell Endocrinol ; 499: 110590, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31550517

RESUMO

During pregnancy, uterine vascular vasodilation is enhanced through adapted Ca2+ signaling, facilitated through increased endothelial connexin 43 (Cx43) gap junctional communication (GJC). In preeclampsia (PE), this adaptive response is missing. Of note, the angiogenic factor VEGF can also act via Src and ERK to close Cx43 gap junctions. While VEGFR2 is necessary for such closure, a role VEGFR1 is less clear. We reasoned if VEGFR2 is acting alone, then substituting another growth factor receptor with VEGFR2-like signaling should have the same effect. In uterine artery endothelial cells derived from pregnant sheep (P-UAEC), endogenous EGFR expression is very low. When we used adenovirus to raise EGFR, we also dose-dependently induced EGF-sensitive Cx43 phosphorylation mainly via ERK, and corresponding loss of Ca2+ bursts, but eliminated VEGF effects on phosphorylation of Cx43 or loss of Ca2+ bursting. This surprising observation suggests that while activated EGFR may indeed substitute for VEGFR2, it also sequesters a limited pool of effector molecules needed for VEGFR2 to phosphorylate Cx43. Thus, low endogenous EGFR expression in P-UAEC may be a necessary strategy to allow VEGFR-2 control of GJC, a first step in initiating angiogenesis in healthy pregnancy. Of further note, trophoblasts are rich in EGFR, and we have demonstrated shed PLAP+/EGFR + extracellular vesicles in maternal circulation in first trimester plasma samples using nanoscale high resolution flow cytometry. Collectively our data suggest that placenta derived exosomes positive for EGFR should be further considered as a possible cause of endothelial dysfunction in women with PE.


Assuntos
Células Endoteliais/citologia , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/genética , Útero/irrigação sanguínea , Fator A de Crescimento do Endotélio Vascular/farmacologia , Adulto , Animais , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Conexina 43/metabolismo , Dependovirus/genética , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Idade Gestacional , Humanos , Idade Materna , Fosforilação , Gravidez , Ovinos , Transdução Genética , Útero/citologia , Útero/metabolismo
9.
Int J Mol Sci ; 21(1)2019 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-31877924

RESUMO

BACKGROUND: Vascular endothelial growth factor (VEGF) is upregulated by hypoxia and is a crucial stimulator for choroidal neovascularization (CNV) in age-related macular degeneration and pathologic myopia, as well as retinal neovascularization in proliferative diabetic retinopathy. Retinal and choroidal endothelial cells play key roles in the development of retinal and CNV, and subsequent fibrosis. At present, the effects of gold nanoparticles (AuNPs) on the VEGF-induced choroid-retina endothelial (RF/6A) cells are still unknown. In our study, we investigated the effects of AuNPs on RF/6A cell viabilities and cell adhesion to fibronectin, a major ECM protein of fibrovascular membrane. Furthermore, the inhibitory effects of AuNPs on RF/6A cell migration induced by VEGF and its signaling were studied. METHODS: The cell viability assay was used to determine the viability of cells treated with AuNPs. The migration of RF/6A cells was assessed by the Transwell migration assay. The cell adhesion to fibronectin was examined by an adhesion assay. The VEGF-induced signaling pathways were determined by western blotting. RESULTS: The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay revealed no cytotoxicity of AuNPs on RF/6A cells. AuNPs inhibited VEGF-induced RF/6A cell migration in a concentration-dependent manner but showed no significant effects on RF/6A cell adhesion to fibronectin. Inhibitory effects of AuNPs on VEGF-induced Akt/eNOS were found. CONCLUSIONS: These results suggest that AuNPs are an effective inhibitor of VEGF-induced RF/6A cell migration through the Akt/eNOS pathways, but they have no effects on their cell viabilities and cell adhesion to fibronectin.


Assuntos
Movimento Celular/efeitos dos fármacos , Corioide/metabolismo , Células Endoteliais/metabolismo , Ouro , Nanopartículas Metálicas/química , Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Linhagem Celular , Corioide/citologia , Células Endoteliais/citologia , Ouro/química , Ouro/farmacologia , Macaca mulatta , Retina/citologia
10.
Medicine (Baltimore) ; 98(44): e17599, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31689763

RESUMO

This study compares 2 methods of macular function evaluation: the microperimetric examination (mean central retinal sensitivity and fixation stability) and the distance best-corrected visual acuity (BCVA) examination, which is the most frequently used method of assessing macular function in patients with newly diagnosed wet age-related macular degeneration (AMD) who have been treated with anti-vascular endothelial growth factor (VEGF) drug (aflibercept).Prospective analysis was conducted on 44 eyes of 44 patients treated with intravitreal injection of anti-VEGF (aflibercept) because of newly diagnosed neovascular AMD. According to the research protocol, all patients had a 6-month follow-up. The response to treatment was monitored functionallybyMP-1 microperimetry, fixation, and distance BCVA assessment after injection. Improvement of retinal sensitivity and BCVA was found under aflibercept treatment. There was statistically significant improvement in retinal sensitivity in the MP-1 study 3 and 6 months from the beginning of anti-VEGF therapy. Moreover, a significant improvement in retinal sensitivity between 3 and 6 months of observation was demonstrated. At the same time, up to 3 months from the beginning of treatment, BCVA improved significantly compared to the baseline value. In the 6th month of the study BCVA remained stable without further significant improvement.Microperimetric examination with medium sensitivity and fixation stability assessment is a very valuable test determining the retinal function. It is clear that examining the macular morphology itself in modern diagnostics is not enough to assess retinal function. Microperimetry technique is a valuable tool for functional long-term evaluation of retinal function (also for a period of more than 3 months).


Assuntos
Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Retina/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Degeneração Macular Exsudativa/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Injeções Intravítreas , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Receptores de Fatores de Crescimento do Endotélio Vascular/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacologia , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/farmacologia , Acuidade Visual
11.
Mol Vis ; 25: 636-644, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31700228

RESUMO

Purpose: Conbercept is a novel recombinant fusion protein designed as a decoy receptor for vascular endothelial growth factor (VEGF) and placental growth factor. The primary purpose was to investigate the effect and safety of conbercept, based on a practical protocol, in the eyes of patients with diabetic macular edema (DME), and the secondary aim was to evaluate the efficacy of low-dose triamcinolone acetonide in patients with refractory DME who had little response to conbercept. Methods: In this retrospective clinical study, 89 treatment eyes from 76 patients with clinically significant DME were initially treated with one to three consecutive monthly intravitreal conbercept (IVC) injections, followed by retreatment with conbercept or switch therapy to triamcinolone acetonide (TA) based on a 6-month observation of the curative effect of IVC. Results: Sixty eyes were initiated on conbercept treatment for DME throughout the entire 1-year assessment period. After at least three consecutive monthly IVC treatments, 29 eyes further received intravitreal triamcinolone acetonide (IVTA) injections at month 6. From baseline to 1 year, the mean number of conbercept injections in the IVC group (n=60) was 4.5±1.0, and the mean number of conbercept injections in the IVC plus IVTA group (n=29) was 3.1±0.3. The mean best-corrected visual acuity (BCVA) and central macular thickness (CMT) were statistically significantly improved at 1 and 3 months after IVC treatments in the IVC group, and gradually improved at 9 months after IVTA treatments in the IVC plus IVTA group. There were no severe complications or conbercept-related adverse ocular and systemic side effects. Conclusions: Conbercept could be effective for visual and anatomic improvements in DME eyes with relatively fewer intravitreal injections and longer treatment intervals in clinical practice. Low-dose TA may be useful for patients with refractory DME resistant to anti-VEGF therapy.


Assuntos
Retinopatia Diabética/complicações , Edema Macular/tratamento farmacológico , Edema Macular/etiologia , Proteínas Recombinantes de Fusão/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Catarata/fisiopatologia , Retinopatia Diabética/fisiopatologia , Feminino , Humanos , Pressão Intraocular/efeitos dos fármacos , Injeções Intravítreas , Macula Lutea/efeitos dos fármacos , Macula Lutea/patologia , Macula Lutea/fisiopatologia , Edema Macular/fisiopatologia , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/efeitos adversos , Proteínas Recombinantes de Fusão/farmacologia , Fator A de Crescimento do Endotélio Vascular/efeitos adversos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Acuidade Visual/efeitos dos fármacos
12.
Proc Natl Acad Sci U S A ; 116(47): 23551-23561, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31685607

RESUMO

Angiogenesis frequently occurs in the context of acute or persistent inflammation. The complex interplay of proinflammatory and proangiogenic cues is only partially understood. Using an experimental model, permitting exposure of developing blood vessel sprouts to multiple combinations of diverse biochemical stimuli and juxtacrine cell interactions, we present evidence that a proinflammatory cytokine, tumor necrosis factor (TNF), can have both proangiogenic and antiangiogenic effects, depending on the dose and the presence of pericytes. In particular, we find that pericytes can rescue and enhance angiogenesis in the presence of otherwise-inhibitory high TNF doses. This sharp switch from proangiogenic to antiangiogenic effect of TNF observed with an escalating dose of this cytokine, as well as the effect of pericytes, are explained by a mathematical model trained on the biochemical data. Furthermore, this model was predictive of the effects of diverse combinations of proinflammatory and antiinflammatory cues, and variable pericyte coverage. The mechanism supports the effect of TNF and pericytes as modulating signaling networks impinging on Notch signaling and specification of the Tip and Stalk phenotypes. This integrative analysis elucidates the plasticity of the angiogenic morphogenesis in the presence of diverse and potentially conflicting cues, with immediate implications for many physiological and pathological settings.


Assuntos
Células Endoteliais/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Pericitos/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Comunicação Celular , Técnicas de Cultura de Células , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação , Lisofosfolipídeos/farmacologia , Modelos Biológicos , Neovascularização Patológica/patologia , Pericitos/efeitos dos fármacos , Receptores Notch/fisiologia , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Engenharia Tecidual , Fator de Necrose Tumoral alfa/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/farmacologia , Fator A de Crescimento do Endotélio Vascular/fisiologia
13.
BMC Cancer ; 19(1): 958, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619190

RESUMO

BACKGROUND: We analyzed the changes in permeability of endothelial cell layers after photon irradiation, with a focus on the metalloproteases ADAM10 and ADAM17, and on VE-cadherin, components crucial for the integrity of endothelial intercellular junctions, and their roles in the transmigration of cancer cells through endothelial cell monolayers. METHODS: Primary HUVEC were irradiated with 2 or 4 Gy photons at a dose rate of 5 Gy/min. The permeability of an irradiated endothelial monolayer for macromolecules and tumor cells was analyzed in the presence or absence of the ADAM10/17 inhibitors GI254023X and GW280264X. Expression of ADAM10, ADAM17 and VE-Cadherin in endothelial cells was quantified by immunoblotting and qRT. VE-Cadherin was additionally analyzed by immunofluorescence microscopy and ELISA. RESULTS: Ionizing radiation increased the permeability of endothelial monolayers and the transendothelial migration of tumor cells. This was effectively blocked by a selective inhibition (GI254023X) of ADAM10. Irradiation increased both, the expression and activity of ADAM10, which led to increased degradation of VE-cadherin, but also led to higher rates of VE-cadherin internalization. Increased degradation of VE-cadherin was also observed when endothelial monolayers were exposed to tumor-cell conditioned medium, similar to when exposed to recombinant VEGF. CONCLUSIONS: Our results suggest a mechanism of irradiation-induced increased permeability and transendothelial migration of tumor cells based on the activation of ADAM10 and the subsequent change of endothelial permeability through the degradation and internalization of VE-cadherin.


Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Células Endoteliais/efeitos da radiação , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Proteínas de Membrana/metabolismo , Proteólise/efeitos da radiação , Radiação Ionizante , Migração Transendotelial e Transepitelial/efeitos da radiação , Proteína ADAM10/antagonistas & inibidores , Proteína ADAM10/genética , Proteína ADAM17/antagonistas & inibidores , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Linhagem Celular Tumoral , Dipeptídeos/farmacologia , Células Endoteliais/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Permeabilidade/efeitos da radiação , Radioterapia/efeitos adversos , Transdução de Sinais/efeitos da radiação , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia
14.
Nanoscale ; 11(42): 19943-19958, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31602446

RESUMO

The crosstalk between vascularization and nerve regeneration in the peripheral nervous system has recently been suggested to play an important role in the treatment of peripheral nerve injury. Regenerative strategies via synergistic delivery of multiple biochemical cues have received growing attention, especially the combination of pro-angiogenic factors and neurotrophic factors. Here we developed a self-assembling peptide nanofiber hydrogel dual-functionalized with vascular endothelial growth factor (VEGF)- and brain-derived neurotrophic factor (BDNF)-mimetic peptide epitopes for peripheral nerve reconstruction. It could simultaneously present VEGF- and BDNF-mimetic peptide epitopes and provides a three-dimensional (3D) neurovascular microenvironment for endothelial cell and neural cell growth. In vitro cellular experiments showed that the functionalized peptide hydrogel scaffold effectively promoted the pro-myelination of Schwann cell, as well as the adhesion and proliferation of endothelial cell compared with scaffolds presenting VEGF- or BDNF-mimetic peptide epitope alone. When implanted in a rat model to bridge a critical-size sciatic nerve gap in vivo, the functionalized peptide hydrogel significantly improved the number of newly formed blood vessels, the density of regenerating axons, the morphometric analysis of the regenerated muscles and the electrophysiological findings, indicating the synergistic effect of the two bioactive motifs on peripheral nerve regeneration. Collectively, constructing an artificial neurovascular microenvironment in the lesion area by using the functionalized self-assembling peptide nanofiber hydrogel may have a great potential for promoting nerve tissue engineering and regeneration in other tissues.


Assuntos
Fator Neurotrófico Derivado do Encéfalo , Hidrogéis , Regeneração Nervosa/efeitos dos fármacos , Peptídeos , Nervos Periféricos/fisiologia , Fator A de Crescimento do Endotélio Vascular , Animais , Fator Neurotrófico Derivado do Encéfalo/química , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Masculino , Neovascularização Fisiológica/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Nervos Periféricos/irrigação sanguínea , Ratos , Ratos Sprague-Dawley , Células de Schwann/metabolismo , Tecidos Suporte/química , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/farmacologia
15.
Cancer Sci ; 110(12): 3773-3787, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31578782

RESUMO

Vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed in tumor-associated endothelial cells, where it modulates tumor-promoting angiogenesis, and it is also found on the surface of tumor cells. Currently, there are no Ab therapeutics targeting VEGFR2 approved for the treatment of prostate cancer or leukemia. Therefore, development of novel efficacious anti-VEGFR2 Abs will benefit cancer patients. We used the Institute of Cellular and Organismic Biology human Ab library and affinity maturation to develop a fully human Ab, anti-VEGFR2-AF, which shows excellent VEGFR2 binding activity. Anti-VEGFR2-AF bound Ig-like domain 3 of VEGFR2 extracellular region to disrupt the interaction between VEGF-A and VEGFR2, neutralizing downstream signaling of the receptor. Moreover, anti-VEGFR2-AF inhibited capillary structure formation and exerted Ab-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity in vitro. We found that VEGFR2 is expressed in PC-3 human prostate cancer cell line and associated with malignancy and metastasis of human prostate cancer. In a PC-3 xenograft mouse model, treatment with anti-VEGFR2-AF repressed tumor growth and angiogenesis as effectively and safely as US FDA-approved anti-VEGFR2 therapeutic, ramucirumab. We also report for the first time that addition of anti-VEGFR2 Ab can enhance the efficacy of docetaxel in the treatment of a prostate cancer mouse model. In HL-60 human leukemia-xenografted mice, anti-VEGFR2-AF showed better efficacy than ramucirumab with prolonged survival and reduced metastasis of leukemia cells to ovaries and lymph nodes. Our findings suggest that anti-VEGFR2-AF has strong potential as a cancer therapy that could directly target VEGFR2-expressing tumor cells in addition to its anti-angiogenic action.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Leucemia/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos/uso terapêutico , Linhagem Celular Tumoral , Epitopos de Linfócito B , Humanos , Masculino , Camundongos , Fosforilação , Fator A de Crescimento do Endotélio Vascular/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Mol Carcinog ; 58(12): 2316-2326, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31553086

RESUMO

Primary tumor can induce the formation of premetastatic niche. The hyperpermeability of the vessels in the premetastatic niche is the first step in the development of metastasis. However, the cellular and molecular mechanisms of vascular hyperpermeability remain to be elucidated. In this study, 4T1 breast cells were injected into the breasts of mice to establish a tumor model. Our results showed that primary tumors induced hyperpermeability of the vessels in the premetastatic lung. Subsequent studies showed that the level of vascular endothelial growth factor (VEGF) was elevated in the tumor-bearing mice serum and the levels of tight junction (TJ) proteins occludin and ZO-1 were decreased in the premetastatic lung. In vitro studies demonstrated that VEGF increased the permeability of dextran and decreased the levels of occludin and ZO-1 in human umbilical vein endothelial cells. Moreover, the hyperpermeability of vessels and the degradation of occludin was blocked by bevacizumab. Overexpression of occludin alleviated the VEGF-induced hyperpermeability. Further investigations revealed that VEGF-induced occludin phosphorylation at Ser-490 and ubiquitination. Finally, we showed that VEGF accelerated the process of occludin degradation through the ubiquitin-proteasome system. In conclusion, primary tumor-secrete VEGF induce the occludin phosphorylation/ubiquitination and downregulation, resulting in the disruption of TJs and hyperpermeability of vessels in premetastatic lung. The occludin phosphorylation/ubiquitination pathway may be the mechanism of VEGF-induced vascular hyperpermeability in the lung premetastatic niche.


Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Pulmão/metabolismo , Neoplasias Experimentais/metabolismo , Ocludina/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Pulmão/irrigação sanguínea , Pulmão/patologia , Camundongos Endogâmicos BALB C , Mutação de Sentido Incorreto , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Ocludina/genética , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Ubiquitinação/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo
17.
Hypertension ; 74(5): 1113-1123, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31542966

RESUMO

Chronic kidney disease (CKD) universally associates with renal microvascular rarefaction and inflammation, but whether a link exists between these 2 processes is unclear. We designed a therapeutic construct of VEGF (vascular endothelial growth factor) fused to an ELP (elastin-like polypeptide) carrier and show that it improves renal function in experimental renovascular disease. We test the hypothesis that ELP-VEGF therapy will improve CKD, and that recovery will be driven by decreasing microvascular rarefaction partly via modulation of macrophage phenotype and inflammation. CKD was induced in 14 pigs, which were observed for 14 weeks. At 6 weeks, renal blood flow and filtration were quantified using multidetector computed tomography, and then pigs received single intrarenal ELP-VEGF or placebo (n=7 each). Renal function was quantified again 4 and 8 weeks later. Pigs were euthanized and renal microvascular density, angiogenic and inflammatory markers, fibrosis, macrophage infiltration, and phenotype were quantified. Loss of renal hemodynamics in CKD was progressively recovered by ELP-VEGF therapy, accompanied by improved renal microvascular density, fibrosis, and expression of inflammatory mediators. Although renal macrophage infiltration was similar in both CKD groups, ELP-VEGF therapy distinctly shifted their phenotype from proinflammatory M1 to VEGF-expressing M2. Our study unravels potential mechanisms and feasibility of a new strategy to offset progression of CKD using drug-delivery technologies. The results indicate that renal recovery after ELP-VEGF therapy was largely driven by modulation of renal macrophages toward VEGF-expressing M2 phenotype, restoring VEGF signaling and sustaining improvement of renal function and microvascular integrity in CKD.


Assuntos
Elastina/farmacologia , Macrófagos/efeitos dos fármacos , Circulação Renal/efeitos dos fármacos , Insuficiência Renal Crônica/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Biópsia por Agulha , Células Cultivadas , Modelos Animais de Doenças , Portadores de Fármacos , Imuno-Histoquímica , Injeções Intralesionais , Testes de Função Renal , Macrófagos/citologia , Microcirculação/efeitos dos fármacos , Tomografia Computadorizada Multidetectores/métodos , Distribuição Aleatória , Recuperação de Função Fisiológica , Insuficiência Renal Crônica/diagnóstico por imagem , Insuficiência Renal Crônica/patologia , Sensibilidade e Especificidade , Sus scrofa , Coleta de Tecidos e Órgãos , Resultado do Tratamento
18.
Chem Biol Interact ; 313: 108826, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31545954

RESUMO

BACKGROUND: Despite of the most effective surgical removal of malignant tumors, metastasis makes cancer treatment difficult. The studies on natural compounds to inhibit this metastasis have been actively performed until now. However, the effect of tomatidine on metastasis remains unclear. METHOD: The effect of tomatidine on antioxidative activity was measured with DPPH radical assay and reducing power assay. After treatment with tomatidine, the viability of human fibrosarcoma cells (HT1080 cells) was evaluated with MTT assay. The effect of tomatidine on the inhibition of matrix metalloproteinase-2 (MMP-2) and MMP-9, gelatinases related to metastasis, was analyzed using gelatin zymography, western blot and immunofluorescence staining. Cell invasion assay was used to investigate anti-metastasis activity of tomatidine. RESULT: Tomatidine showed no DPPH radical scavenging effect and showed 8% of reduction power at 8 µM. Furthermore, tomatidine below 8 µM showed more than 80% of cell viability in MTT assay. The inhibition of tomatidine on MMP-2 activity and its protein expression levels were observed by gelatin zymography, western blot and immunofluorescence. It was observed that tomatidine inhibited not only p38 and ERK but also cell invasion. CONCLUSION: Above results suggest that tomatidine could use as a potential candidate for cancer prevention and metastasis through the inhibitory effect on gelatinase.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Tomatina/análogos & derivados , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Depuradores de Radicais Livres/farmacologia , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Tomatina/farmacologia , Fator de Transcrição AP-1/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia
19.
Protein J ; 38(6): 693-703, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31564024

RESUMO

The vascular endothelial growth factor (VEGF) is an essential factor to pathologic angiogenesis. Disruption of VEGF/VEGF receptor interaction in cancer patients inhibits the development of new and pre-existing tumor blood vessels. Consequently, VEGF becomes an important therapeutic target for handling solid tumors. In this work, human VEGF was produced in the culture supernatant of SiHa cells transduced with a replication-defective adenoviral vector (pAdhVEGF121) encoding this molecule. The 35 kDa VEGF121 homodimer was obtained from clarified culture media as a glycosylated protein. VEGF121 expression levels were strictly dependent on the adenoviral viral load used. VEGF121 was produced with purity over 98% after a single step chromatography by immobilized metal affinity chromatography. Additionally, VEGF121 binds Bevacizumab antibody with a KD of 7 nM. Biological characterization by mitogenic assay in HUVEC and ECV-304 cells showed that VEGF121 stimulates cell proliferation in a dose-dependent manner in both cells. Finally, the neovascularization activity of VEGF121 was demonstrated by vascular permeability assays in matrigel plug-bearing mice, showing significantly increased vasculature leakage after treatment with VEGF121. Consequently, transduction of SiHa cells with adenovirus is a suitable alternative for manufacture heterologous proteins of therapeutic interest.


Assuntos
Fator A de Crescimento do Endotélio Vascular/isolamento & purificação , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos
20.
In Vitro Cell Dev Biol Anim ; 55(9): 756-765, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31420803

RESUMO

Vascular endothelial growth factor (VEGF) inhibition forms the basis for anti-angiogenic therapies. With the methods based on the monoclonal antibody-mediated typical VEGF blockade, pathological angiogenesis in the tumor microenvironment is inhibited and the limitation of tumor growth is provided; however, the existing tumor tissue cannot be intervened. In this study, the anti-angiogenic effects of Semaphorin (SEMA) 3F, which has frequently been reported to have tumor suppressive properties, on a chick chorioallantoic membrane model as well as in vitro cell-cell interactions were investigated and comparatively assessed using anti-VEGF antibody. Vascular endothelial cells and chick embryos were stimulated with 10-16 ng/mL VEGF165 prior to SEMA 3F administration in order to generate pathological vascularization conditions. Both in vitro and in ovo results revealed that SEMA 3F suppressed VEGF165-induced abnormal vascularization more effectively than anti-VEGF. Moreover, the required dose of SEMA 3F was significantly lower than that of anti-VEGF (103 times less under in ovo conditions). In light of these results, SEMA 3F is recommended as an important therapeutic agent for the prevention of pathological angiogenesis. SEMA 3F may offer an effective and efficient anti-angiogenic intervention that can be administered at a lower dose alternative to typical VEGF blocking agents.


Assuntos
Proteínas de Membrana/genética , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Proteínas do Tecido Nervoso/genética , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Anticorpos Monoclonais/farmacologia , Embrião de Galinha , Membrana Corioalantoide/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas de Membrana/farmacologia , Morfogênese/efeitos dos fármacos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Neovascularização Patológica/genética , Neovascularização Patológica/imunologia , Neovascularização Patológica/patologia , Proteínas do Tecido Nervoso/farmacologia , Microambiente Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia
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