RESUMO
Our findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective. We also provide additional evidence for the specificity of our anti-PlGF antibody and experiments to suggest that anti-PlGF treatment will not be effective for all tumors and why. Further, we show that PlGF blockage inhibits vessel abnormalization rather than density in certain tumors while enhancing VEGF-targeted inhibition in ocular disease. Our findings warrant further testing of anti-PlGF therapies.
Assuntos
Neovascularização Fisiológica/efeitos dos fármacos , Proteínas da Gravidez/antagonistas & inibidores , Proteínas da Gravidez/metabolismo , Inibidores da Angiogênese/uso terapêutico , Animais , Anticorpos Monoclonais/uso terapêutico , Carcinoma Hepatocelular/irrigação sanguínea , Carcinoma Hepatocelular/prevenção & controle , Corioide/irrigação sanguínea , Modelos Animais de Doenças , Oftalmopatias/patologia , Humanos , Neoplasias Hepáticas Experimentais/irrigação sanguínea , Neoplasias Hepáticas Experimentais/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Papiloma/irrigação sanguínea , Papiloma/induzido quimicamente , Papiloma/prevenção & controle , Fator de Crescimento Placentário , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/prevenção & controleRESUMO
A key function of blood vessels, to supply oxygen, is impaired in tumors because of abnormalities in their endothelial lining. PHD proteins serve as oxygen sensors and may regulate oxygen delivery. We therefore studied the role of endothelial PHD2 in vessel shaping by implanting tumors in PHD2(+/-) mice. Haplodeficiency of PHD2 did not affect tumor vessel density or lumen size, but normalized the endothelial lining and vessel maturation. This resulted in improved tumor perfusion and oxygenation and inhibited tumor cell invasion, intravasation, and metastasis. Haplodeficiency of PHD2 redirected the specification of endothelial tip cells to a more quiescent cell type, lacking filopodia and arrayed in a phalanx formation. This transition relied on HIF-driven upregulation of (soluble) VEGFR-1 and VE-cadherin. Thus, decreased activity of an oxygen sensor in hypoxic conditions prompts endothelial cells to readjust their shape and phenotype to restore oxygen supply. Inhibition of PHD2 may offer alternative therapeutic opportunities for anticancer therapy.
Assuntos
Vasos Sanguíneos/citologia , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Metástase Neoplásica , Neoplasias/irrigação sanguínea , Oxigênio/metabolismo , Animais , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Forma Celular , Proteínas de Ligação a DNA/genética , Células Endoteliais/citologia , Glicólise , Heterozigoto , Hipóxia/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia , Proteínas Imediatamente Precoces/genética , Camundongos , Neoplasias/patologia , Pró-Colágeno-Prolina DioxigenaseRESUMO
Intravitreal (IVT) injection remains the preferred administration route of pharmacological agents intended for the treatment of back of the eye diseases such as diabetic macular edema (DME) and neovascular age-related macular degeneration (nvAMD). The procedure enables drugs to be delivered locally at high concentrations whilst limiting whole body exposure and associated risk of systemic adverse events. Nevertheless, intravitreally-delivered drugs do enter the general circulation and achieving an accurate understanding of systemic exposure is pivotal for the evaluation and development of drugs administered in the eye. We report here the full pharmacokinetic properties of THR-687, a pan RGD integrin antagonist currently in clinical development for the treatment of DME, in both rabbit and minipig. Pharmacokinetic characterization included description of vitreal elimination, of systemic pharmacokinetics, and of systemic exposure following IVT administration. For the latter, we present a novel pharmacokinetic model that assumes clear partition between the vitreous humor compartment itself where the drug is administered and the central systemic compartment. We also propose an analytical solution to the system of differential equations that represent the pharmacokinetic model, thereby allowing data analysis with standard nonlinear regression analysis. The model accurately describes circulating levels of THR-687 following IVT administration in relevant animal models, and we suggest that this approach is relevant to a range of drugs and analysis of subsequent systemic exposure.
Assuntos
Retinopatia Diabética , Edema Macular , Animais , Retinopatia Diabética/tratamento farmacológico , Injeções Intravítreas , Edema Macular/tratamento farmacológico , Coelhos , Suínos , Porco Miniatura , Corpo VítreoRESUMO
Hepatopulmonary syndrome (HPS) is a severe complication of cirrhosis with increased risk of mortality. Pulmonary microvascular alterations are key features of HPS; but underlying mechanisms are incompletely understood, and studies on HPS are limited to rats. Placental growth factor (PlGF), a proangiogenic molecule that is selectively involved in pathological angiogenesis, may play an important role in HPS development; however, its role has never been investigated. In this study, we validated an HPS model by common bile duct ligation (CBDL) in mice, investigated the kinetic changes in pulmonary angiogenesis and inflammation during HPS development, and provide evidence for a novel therapeutic strategy by targeting pathological angiogenesis. Mice with CBDL developed hypoxemia and intrapulmonary shunting on a background of liver fibrosis. Pulmonary alterations included increased levels of proangiogenic and inflammatory markers, which was confirmed in serum of human HPS patients. Increased PlGF production in HPS mice originated from alveolar type II cells and lung macrophages, as demonstrated by immunofluorescent staining. Dysfunctional vessel formation in CBDL mice was visualized by microscopy on vascular corrosion casts. Both prophylactic and therapeutic anti-PlGF (αPlGF) antibody treatment impeded HPS development, as demonstrated by significantly less intrapulmonary shunting and improved gas exchange. αPlGF treatment decreased endothelial cell dysfunction in vivo and in vitro and was accompanied by reduced pulmonary inflammation. Importantly, αPlGF therapy did not affect liver alterations, supporting αPlGF's ability to directly target the pulmonary compartment. CONCLUSION: CBDL in mice induces HPS, which is mediated by PlGF production; αPlGF treatment improves experimental HPS by counteracting pulmonary angiogenesis and might be an attractive therapeutic strategy for human HPS. (Hepatology 2017).
Assuntos
Síndrome Hepatopulmonar/metabolismo , Pulmão/patologia , Neovascularização Patológica/metabolismo , Fator de Crescimento Placentário/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Biomarcadores/metabolismo , Ducto Colédoco/cirurgia , Modelos Animais de Doenças , Endoglina/sangue , Síndrome Hepatopulmonar/fisiopatologia , Humanos , Ligadura/métodos , Fígado/patologia , Cirrose Hepática/patologia , Masculino , Camundongos , Fator de Crescimento Placentário/antagonistas & inibidoresRESUMO
Integrins are associated with various eye diseases such as diabetic retinopathy (DR) and wet age-related macular degeneration (AMD) and implicated in main pathologic disease hallmarks like neovascularization, inflammation, fibrosis and vascular leakage. Targeting integrins has the potential to attenuate these vision-threatening processes, independent of anti-vascular endothelial growth factor (VEGF) responsiveness. The current investigation characterized THR-687 as a novel pan RGD (arginylglycylaspartic acid) integrin receptor antagonist able to compete for binding with the natural ligand with nanomolar potency (e.g. αvß3 (IC50 of 4.4⯱â¯2.7â¯nM), αvß5 (IC50 of 1.3⯱â¯0.5â¯nM) and α5ß1 (IC50 of 6.8⯱â¯3.2â¯nM)). THR-687 prevented the migration of human umbilical vein endothelial cells (HUVECs) into a cell-free area (IC50 of 258⯱â¯113â¯nM) as well as vessel sprouting in an ex vivo mouse choroidal explant model (IC50 of 236⯱â¯173â¯nM), and was able to induce the regression of pre-existing vascular sprouts. Moreover, combined intravitreal and intraperitoneal administration of THR-687 potently inhibited VEGF-induced leakage in the mouse retina. In addition, THR-687 injected intravitreally at 3 different dose levels (0.45â¯mg, 2.25â¯mg or 4.5 mg/eye) potently inhibited neovascularization-induced leakage in the cynomolgus laser-induced choroidal neovascularization (CNV) model. These data suggest that THR-687 is a promising drug candidate for the treatment of vision-threatening retinal vascular eye diseases such as DR and wet AMD.
Assuntos
Neovascularização de Coroide/tratamento farmacológico , Retinopatia Diabética/tratamento farmacológico , Compostos Orgânicos/farmacologia , Receptores Imunológicos/antagonistas & inibidores , Receptores de Peptídeos/antagonistas & inibidores , Vasos Retinianos/efeitos dos fármacos , Degeneração Macular Exsudativa/tratamento farmacológico , Animais , Permeabilidade Capilar/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Angiofluoresceinografia , Células Endoteliais da Veia Umbilical Humana , Humanos , Injeções Intraperitoneais , Injeções Intravítreas , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Compostos Orgânicos/uso terapêutico , Coelhos , Tomografia de Coerência Óptica , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality. We previously showed that the inhibition of placental growth factor (PlGF) exerts antitumour effects and induces vessel normalisation, possibly reducing hypoxia. However, the exact mechanism underlying these effects remains unclear. Because hypoxia and endoplasmic reticulum stress, which activates the unfolded protein response (UPR), have been implicated in HCC progression, we assessed the interactions between PlGF and these microenvironmental stresses. METHODS: PlGF knockout mice and validated monoclonal anti-PlGF antibodies were used in a diethylnitrosamine-induced mouse model for HCC. We examined the interactions among hypoxia, UPR activation and PlGF induction in HCC cells. RESULTS: Both the genetic and pharmacological inhibitions of PlGF reduced the chaperone levels and the activation of the PKR-like endoplasmic reticulum kinase (PERK) pathway of the UPR in diethylnitrosamine-induced HCC. Furthermore, we identified that tumour hypoxia was attenuated, as shown by reduced pimonidazole binding. Interestingly, hypoxic exposure markedly activated the PERK pathway in HCC cells in vitro, suggesting that PlGF inhibition may diminish PERK activation by improving oxygen delivery. We also found that PlGF expression is upregulated by different chemical UPR inducers via activation of the inositol-requiring enzyme 1 pathway in HCC cells. CONCLUSIONS: PlGF inhibition attenuates PERK activation, likely by tempering hypoxia in HCC via vessel normalisation. The UPR, in turn, is able to regulate PlGF expression, suggesting the existence of a feedback mechanism for hypoxia-mediated UPR that promotes the expression of the angiogenic factor PlGF. These findings have important implications for our understanding of the effect of therapies normalising tumour vasculature.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Neovascularização Patológica/genética , Proteínas da Gravidez/biossíntese , eIF-2 Quinase/biossíntese , Animais , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/patologia , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Dietilnitrosamina/toxicidade , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicosaminoglicanos/fisiologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Neovascularização Patológica/patologia , Fator de Crescimento Placentário , Proteínas da Gravidez/genética , Microambiente Tumoral/genética , Resposta a Proteínas não Dobradas/genética , eIF-2 Quinase/genéticaRESUMO
UNLABELLED: The pathophysiology of nonalcoholic steatohepatitis (NASH) should be approached as a multifactorial process. In several stages of NASH, a link between disease progression and hepatic microvasculature changes can be made. In this study we investigated the role of angiogenesis in two mouse models for NASH, and the effect of a preventive and therapeutic antiangiogenic treatment in a diet-induced mouse model for NASH. Protein and RNA levels of angiogenic and inflammatory factors were significantly up-regulated in the liver of C56BL/6 and db/db mice with NASH at different timepoints. To examine the effect of angiogenic factors on the disease progression of NASH, a prevention and treatment study was set up, blocking the placental growth factor (PlGF) or vascular endothelial growth factor receptor 2 (VEGFR2). Our study showed that treatment prevents the progression of NASH by attenuating steatosis and inflammation, both in a preventive and therapeutic setting, thereby confirming the hypothesis that angiogenic factors play an early role in the disease progression from steatosis to NASH. Anti-PlGF (αPlGF) did not significantly improve liver histology. Vascular corrosion casting showed a more disrupted liver vasculature in mice with NASH compared to controls. Treatment with αVEGFR2 showed an improvement of the liver vasculature. Moreover, fat-laden primary hepatocytes treated with αVEGFR2 stored significantly less lipids. CONCLUSION: Our results demonstrate that there is an increased expression of angiogenic factors in the liver in different mouse models for NASH. We found that VEGFR2 blockage attenuates steatosis and inflammation in a diet-induced mouse model for NASH in a preventive and therapeutic setting. Our findings warrant further investigation of the role of angiogenesis in the pathophysiology in NASH.
Assuntos
Modelos Animais de Doenças , Progressão da Doença , Fígado Gorduroso/fisiopatologia , Neovascularização Patológica/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Inibidores da Angiogênese/farmacologia , Animais , Células Cultivadas , Deficiência de Colina/complicações , Diabetes Mellitus Tipo 2/fisiopatologia , Fígado Gorduroso/etiologia , Fígado Gorduroso/prevenção & controle , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Hepatócitos/fisiologia , Técnicas In Vitro , Metabolismo dos Lipídeos/fisiologia , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/fisiopatologia , Metionina/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Mutantes , Hepatopatia Gordurosa não Alcoólica , Fator de Crescimento Placentário , Proteínas da Gravidez/efeitos dos fármacos , Proteínas da Gravidez/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Excessive post-operative wound healing with subsequent scarring frequently leads to surgical failure of glaucoma filtration surgery (trabeculectomy). We investigated the hypothesis that placental growth factor (PlGF) plays a role in post-operative scar formation, and that it therefore may be a target for improvement of filtration surgery outcome. ELISA experiments showed that PlGF levels were significantly increased in aqueous humour of glaucoma patients and after VEGF treatment, which may indicate an important contribution of this growth factor to wound healing after trabeculectomy. Using a mouse model of glaucoma filtration surgery, we were able to show that intracameral injection of a previously characterized anti-PlGF antibody (ThromboGenics NV) significantly improved surgical outcome by increasing bleb survival and bleb area. This was associated with a significant reduction in post-operative proliferation, inflammation and angiogenesis during the first post-operative days after surgery, and with a decrease in collagen deposition at later stages. Furthermore, inhibition of PlGF seemed to be more effective than anti-VEGF-R2 treatment in improving surgical outcome, possibly via its additional effect on inflammation. These results render PlGF an appealing target for ocular wound healing and point to potential therapeutic benefits of PlGF inhibition for the prevention of surgical failure.
Assuntos
Glaucoma/metabolismo , Glaucoma/cirurgia , Proteínas da Gravidez/antagonistas & inibidores , Animais , Anticorpos/administração & dosagem , Anticorpos/farmacologia , Humor Aquoso/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cicatriz/patologia , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Glaucoma/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Placentário , Proteínas da Gravidez/metabolismo , Coelhos , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Cápsula de Tenon/patologia , Trabeculectomia , Resultado do Tratamento , Regulação para Cima/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Placental growth factor (PlGF) and its receptor vascular endothelial growth factor receptor 1 (VEGFR1) play an important role in pathological conditions related to angiogenesis, vascular leakage, and inflammation. This study investigated their contributions to inflammation and the formation of edema in allergic asthma. The expression of PlGF and VEGFR1 was measured in induced sputum of patients with asthma (n = 11) and healthy subjects (n = 11), and in bronchial biopsies of house dust mite (HDM)-allergic patients stimulated with HDM allergens. The effects of the endonasal administration of human PlGF-2 and PlGF deficiency on inflammation and edema were evaluated in a murine model of allergic asthma. The migration of human neutrophils in response to hPlGF-2 was tested in vitro. The expression of PlGF and VEGFR1 was significantly higher in the sputum of patients with asthma, and in Der p 1-induced PlGF in biopsies from HDM-allergic patients. PlGF was increased in the bronchi of ovalbumin (OVA)-challenged mice compared with control mice (65 ± 17 pg/mg versus 18 ± 1 pg/mg, respectively; P < 0.01), and VEGFR1 was expressed in bronchial epithelium, endothelium (control mice), and inflammatory cells (OVA-challenged mice). The endonasal instillation of hPlGF-2 in wild-type, OVA-challenged mice led to an increase in bronchial neutrophils, lung tissue wet/dry ratio, and IL-17. PlGF-deficient mice showed lower numbers of BAL-infiltrating neutrophils, a reduced lung wet/dry ratio, and lower production of IL-17, macrophage inflammatory protein-2, and granulocyte chemotactic protein-2/LPS-induced chemokine compared with wild-type, OVA-challenged mice. hPlGF-2 induced the migration of human neutrophils in vitro in a VEGFR1-dependent way. PlGF and its receptor VEGFR1 are up-regulated in allergic asthma and play a proinflammatory role by inducing tissue edema, and increasing tissue neutrophilia and the production of IL-17.
Assuntos
Asma/imunologia , Brônquios/imunologia , Edema/imunologia , Inflamação/imunologia , Neutrófilos/imunologia , Proteínas da Gravidez/fisiologia , Animais , Feminino , Humanos , Masculino , Camundongos , Fator de Crescimento Placentário , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The transforming and tumor growth-promoting properties of Axl, a member of the Tyro3, Axl, and Mer (TAM) family of receptor tyrosine kinases (TAMRs), are well recognized. In contrast, little is known about the role of the TAMR ligand growth arrest-specific gene 6 (Gas6) in tumor biology. By using Gas6-deficient (Gas6(-/-)) mice, we show that bone marrow-derived Gas6 promotes growth and metastasis in different experimental cancer models, including one resistant to vascular endothelial growth factor inhibitors. Mechanistic studies reveal that circulating leukocytes produce minimal Gas6. However, once infiltrated in the tumor, leukocytes up-regulate Gas6, which is mitogenic for tumor cells. Consistent herewith, impaired tumor growth in Gas6(-/-) mice is rescued by transplantation of wild-type bone marrow and, conversely, mimicked by transplantation of Gas6(-/-) bone marrow into wild-type hosts. These findings highlight a novel role for Gas6 in a positive amplification loop, whereby tumors promote their growth by educating infiltrating leukocytes to up-regulate the production of the mitogen Gas6. Hence, inhibition of Gas6 might offer novel opportunities for the treatment of cancer.
Assuntos
Movimento Celular , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Leucócitos/metabolismo , Leucócitos/patologia , Mitógenos/biossíntese , Neoplasias/patologia , Animais , Transplante de Medula Óssea , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Inflamação/complicações , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Interleucina-10/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Metástase Neoplásica , Neoplasias/irrigação sanguínea , Neoplasias/enzimologia , Neoplasias/genética , Neovascularização Patológica/complicações , Neovascularização Patológica/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Células Estromais/metabolismo , Células Estromais/patologia , Regulação para Cima/genéticaRESUMO
Ocriplasmin (Jetrea®) is a recombinant protease used to treat vitreomacular traction. To gain insight into vitreoretinal observations reported after ocriplasmin treatment, we have developed an in vivo porcine ocriplasmin-induced posterior vitreous detachment (PVD) model in which we investigated vitreoretinal tissues by optical coherence tomography, histology, and cytokine profiling. Eight weeks postinjection, ocriplasmin yielded PVD in 82% of eyes. Subretinal fluid (85%) and vitreous hyperreflective spots (45%) were resolved by week 3. Histological analysis of extracellular matrix (ECM) proteins such as laminin, fibronectin, and collagen IV indicated no retinal ocriplasmin-induced ECM distribution changes. Retinal morphology was unaffected in all eyes. Cytokine profiles of ocriplasmin-treated eyes were not different from vehicle. In cell-based electrical resistance assays, blood-retinal barrier permeability was altered by ocriplasmin concentrations of 6 µg/mL and higher, with all effects being nontoxic, cell-type specific, and reversible. Ocriplasmin was actively taken up by RPE and Müller cells, and our data suggest both lysosomal and transcellular clearance routes for ocriplasmin. In conclusion, transient hyperreflective spots and fluid in a porcine ocriplasmin-induced PVD model did not correlate with retinal ECM rearrangement nor inflammation. Reversible in vitro effects on blood-retinal barrier permeability provide grounds for a hypothesis on the mechanisms behind transient subretinal fluid observed in ocriplasmin-treated patients.
RESUMO
PURPOSE: To evaluate the feasibility of robot-assisted retinal vein cannulation for retinal vein occlusion. METHODS: Prospective experimental study performed in in vivo porcine eyes. A standard three port pars plana vitrectomy was followed by laser-induced branch retinal vein occlusion. Consequently, a retinal vein cannulation with the help of a surgical robot and a microneedle was performed. Complete success was defined as a stable intravenous position of the needle tip confirmed by blood washout for at least 3 min. Secondary outcomes were the occurrence of intra-operative complications and technical failures. RESULTS: Cannulation was successful in 15 of 18 eyes with a complete success rate (duration of infusion of more than 3 min) of 73% after exclusion of two eyes from analysis due to failure in establishing a blood clot. There were no technical failures regarding the robotic device. The intravessel injections of ocriplasmin in two of two eyes led to a clot dissolution. In a subset of five eyes, a second cannulation attempt at the border of the optic disc resulted in a stable intravessel position and infusion during 362 (±138) seconds. CONCLUSION: Robot-assisted retinal vein cannulation with prolonged infusion time is technically feasible. Human experiments are required to analyse the clinical benefit of this new therapy.
Assuntos
Cateterismo/métodos , Oclusão da Veia Retiniana/cirurgia , Veia Retiniana/cirurgia , Robótica/métodos , Vitrectomia/métodos , Animais , Modelos Animais de Doenças , Projetos Piloto , Estudos Prospectivos , Oclusão da Veia Retiniana/diagnóstico , Suínos , Resultado do TratamentoRESUMO
PURPOSE: The complementary effects of mitomycin-C (MMC) and anti-placental growth factor (PlGF) therapy were explored and compared to the combined administration of MMC and aflibercept. Additionally, the effect of PlGF (inhibition) on IOP was investigated, since aqueous PlGF is known to be upregulated in glaucoma patients. METHODS: In the trabeculectomy mouse model, intracameral injection(s) of the PlGF inhibitor (5D11D4) were compared to MMC or aflibercept and to the combination of both compounds. Treatment outcome was studied by bleb investigation and by Sirius Red staining. The effect of subconjunctival PlGF administration and topical 5D11D4 on IOP was investigated in normotensive mice and was compared to topical administration of latanoprost, the gold standard for IOP-lowering. RESULTS: Combination of MMC and 5D11D4 was able to significantly improve surgical outcome compared to monotherapy of MMC or 5D11D4 (n = 20; P < 0.001). Compared to combined treatment of MMC with aflibercept, the simultaneous administration of MMC and 5D11D4 was equally efficacious in improving surgical outcome (n = 15; P = 0.88). In normotensive mice, 5D11D4 was able to significantly reduce the IOP-elevation induced by PlGF (n = 10; P < 0.05), whereas no effect of 5D11D4 was seen in naive mice, which was in contrast to latanoprost. CONCLUSIONS: The current data suggest that application of MMC together with PlGF inhibition may have complementary effects in the improvement of surgical outcome and is equally efficacious as the combined treatment of MMC and aflibercept. Inhibition of PlGF also might open alternative perspectives as IOP-lowering strategy for glaucoma patients with increased aqueous PlGF levels.
Assuntos
Proteínas Sanguíneas/administração & dosagem , Cicatriz/prevenção & controle , Cirurgia Filtrante/efeitos adversos , Glaucoma/cirurgia , Guias como Assunto , Mitomicina/administração & dosagem , Animais , Câmara Anterior , Proteínas Sanguíneas/farmacocinética , Cicatriz/etiologia , Cicatriz/metabolismo , Modelos Animais de Doenças , Glaucoma/metabolismo , Glaucoma/patologia , Injeções , Pressão Intraocular , Camundongos , Camundongos Endogâmicos C57BL , Mitomicina/farmacocinética , Inibidores da Síntese de Ácido Nucleico/administração & dosagem , Inibidores da Síntese de Ácido Nucleico/farmacocinética , Complicações Pós-Operatórias/prevenção & controleRESUMO
PURPOSE: Retinal vein occlusions (RVO) are a major cause of vision loss in people aged 50â years and older. Current therapeutic options limit the consequences of RVO but do not eliminate the cause. Cannulation of the involved vessel and removal of the clot may provide a more permanent solution with a less demanding follow-up. However, cannulation of smaller retinal veins remains challenging. This paper explores the use of ocriplasmin (recombinant plasmin without its kringles) to clear RVO, using a robotic micromanipulator. METHODS: Branch RVO were induced in a porcine model with rose bengal followed by 532â nm endolaser to the superior venous branch of the optic nerve. The vein was cannulated proximal to the occlusion or beyond the first branching vessel from the obstruction. The vein was infused with a physiologic citric acid buffer solution (CAM) or CAM/ocriplasmin. The time of cannulation, number of attempts, and the ability to release the thrombus were recorded. RESULTS: Cannulation and infusion was possible in all the cases. The use of a micromanipulator allowed for a consistent cannulation of the retinal vein and positional stability allowed the vein to remain cannulated for up to 20â min. In none of the attempts (5/5) with CAM did the thrombus dissolve, despite repeat infusion/relaxation cycles. In 7/7 injections of CAM/ocriplasmin near to the point of obstruction, the clot started to dissolve within a few minutes of injection. An infusion, attempted beyond the first venous branch point proximal to the clot, was unsuccessful in 2/3 attempts. CONCLUSIONS: Ocriplasmin is effective in resolving RVO if injected close to the site of occlusion with the use of a micromanipulator.
Assuntos
Fibrinolisina/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Oclusão da Veia Retiniana/tratamento farmacológico , Animais , Modelos Animais de Doenças , Angiofluoresceinografia , Fundo de Olho , Injeções Intravenosas , Veia Retiniana , Oclusão da Veia Retiniana/diagnóstico , Robótica/métodos , SuínosRESUMO
PURPOSE: To develop a methodology for cannulating porcine retinal venules using a robotic assistive arm after inducing a retinal vein occlusion using the photosensitizer rose bengal. METHODOLOGY: Retinal vein occlusions proximal to the first vascular branch point were induced following intravenous injection of rose bengal by exposure to 532nm laser light delivered by slit-lamp or endolaser probe. Retinal veins were cannulated by positioning a glass catheter tip using a robotically controlled micromanipulator above venules with an outer diameter of 80µm or more and performing a preset piercing maneuver, controlled robotically. The ability of a balanced salt (BSS) solution to remove an occlusion by repeat distention of the retinal vein was also assessed. RESULTS: Cannulation using the preset piercing program was successful in 9 of 9 eyes. Piercing using the micromanipulator under manual control was successful in only 24 of 52 attempts, with several attempts leading to double piercing. The best location for cannulation was directly proximal to the occlusion. Infusion of BSS did not result in the resolution of the occlusion. CONCLUSION: Cannulation of venules using a robotic microassistive arm can be achieved with consistency, provided the piercing is robotically driven. The model appears robust enough to allow testing of therapeutic strategies aimed at eliminating a retinal vein thrombus and its evolution over time.
RESUMO
Placental growth factor (PlGF), a homolog of vascular endothelial growth factor (VEGF), exerts pleiotropic functions in cancer by affecting tumor cells as well as endothelial and inflammatory cells. Moreover, PlGF expression correlates with tumor stage, recurrence, metastasis and patient outcome in different types of cancer. Recently, administration of anti-PlGF therapy reduced tumor growth and metastasis in preclinical tumor models. In the present study, we evaluated the diagnostic and prognostic value of systemic and local expression of PlGF in primary endometrial carcinomas. PlGF levels in tumor lysates (n=128) and serum (n=88) of patients with primary endometrial cancer were determined using ELISA. PlGF mRNA expression in endometrial carcinoma tissues was quantified by quantitative qRT-PCR. Results were compared to endometrial cancer stage and grade. Systemic PlGF levels were not altered in patients with endometrial cancer (FIGO stage I-II-III) as compared to healthy controls. Only in FIGO stage IV patients, serum PlGF levels were slightly increased. Local PlGF mRNA and protein expression in endometrial tumors progressively increased with tumor grade. In endometrioid carcinomas, PlGF mRNA expression was significantly increased in endometrioid grade 3 tumors as compared to normal endometrial tissue. PlGF protein expression was significantly increased in endometrioid grade 2 and 3 carcinomas and in serous carcinomas as compared to normal endometrial tissue. Our study showed that systemic/serum PlGF levels cannot be used as a diagnostic or prognostic marker in endometrial cancer. However, the increased local expression of PlGF, primarily in high-grade carcinomas, underscores the possibility for preclinical assessment of anti-PlGF therapy in endometrial cancer.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma/metabolismo , Carcinoma/patologia , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Proteínas da Gravidez/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Biomarcadores Tumorais/sangue , Carcinoma/sangue , Estudos de Casos e Controles , Neoplasias do Endométrio/sangue , Feminino , Humanos , Pessoa de Meia-Idade , Análise Multivariada , Gradação de Tumores , Estadiamento de Neoplasias , Fator de Crescimento Placentário , Proteínas da Gravidez/sangue , RNA Mensageiro/metabolismo , Estatísticas não ParamétricasRESUMO
Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the pharmacologic characterization of the chemical SSR128129E (SSR), which inhibits fibroblast growth factor receptor (FGFR) signaling by binding to the extracellular FGFR domain without affecting orthosteric FGF binding. SSR exhibits allosteric properties, including probe dependence, signaling bias, and ceiling effects. Inhibition by SSR is highly conserved throughout the animal kingdom. Oral delivery of SSR inhibits arthritis and tumors that are relatively refractory to anti-vascular endothelial growth factor receptor-2 antibodies. Thus, orally-active extracellularly acting small-molecule modulators of RTKs with allosteric properties can be developed and may offer opportunities to improve anticancer treatment.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Regulação Alostérica , Animais , Anticorpos Monoclonais/farmacologia , Artrite Experimental/tratamento farmacológico , Reabsorção Óssea/tratamento farmacológico , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/metabolismo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Neovascularização Patológica/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Ocriplasmin contains the active moiety of plasmin enzyme. At a physiologic pH, ocriplasmin is highly proteolytic and autolytic, limiting its duration of activity. Specific inhibitors of plasmin are present in the vitreous under normal and disease conditions and could affect its activity. Each may contribute to its mode of action. METHODS: Degradation characteristics were determined in porcine, human vitreous, and PBS under reducing conditions with different incubation periods between 0 and 24 hours on SDS-PAGE Tris-glycine gels. Residual activity was determined by spectrophotometry of p-nitroaniline release through hydrolysis of L-pyroglutamyl-L-phenylalanyl-L-lysine-p-nitroaniline hydrochloride. The presence of endogenous inactivators of ocriplasmin in human vitreous was determined in a series of vitreous samples using an ELISA specific for alpha(2)-antiplasmin, antithrombin, and antitrypsin. RESULTS: Degradation productions from autolysis are similar between vitreous and PBS with a significant prolongation of the effect in vitreous. Both follow a nonlinear pattern over time. The degradation corresponds best to a second-order kinetic process. The resulting rate constants were 207 ± 60 M(-1) s(-1) in PBS, 81 ± 15 M(-1) s(-1) in porcine vitreous, and 195 M(-1) s(-1) in human vitreous natural inhibitors were identified in samples of donor vitreous. Amounts differed significantly between samples, which may help explain the observed variability in human subjects. CONCLUSIONS: Ocriplasmin is autolytic in vitreous. Biologic activity extends to several days following injection. The exact duration will vary based on the presence and concentration of serine protease inhibitors.
Assuntos
Fibrinolisina/farmacocinética , Fragmentos de Peptídeos/farmacocinética , Corpo Vítreo/metabolismo , Animais , Autólise , Western Blotting , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Sus scrofaRESUMO
Imatinib has revolutionized the treatment of Bcr-Abl1(+) chronic myeloid leukemia (CML), but, in most patients, some leukemia cells persist despite continued therapy, while others become resistant. Here, we report that PlGF levels are elevated in CML and that PlGF produced by bone marrow stromal cells (BMSCs) aggravates disease severity. CML cells foster a soil for their own growth by inducing BMSCs to upregulate PlGF, which not only stimulates BM angiogenesis, but also promotes CML proliferation and metabolism, in part independently of Bcr-Abl1 signaling. Anti-PlGF treatment prolongs survival of imatinib-sensitive and -resistant CML mice and adds to the anti-CML activity of imatinib. These results may warrant further investigation of the therapeutic potential of PlGF inhibition for (imatinib-resistant) CML.