RESUMO
Coronavirus disease 2019 (COVID-19) is a highly contagious disease that has claimed the lives of millions of people worldwide in the last 2 years. Because of the disease's rapid spread, it is critical to diagnose it at an early stage in order to reduce the rate of spread. The images of the lungs are used to diagnose this infection. In the last 2 years, many studies have been introduced to help with the diagnosis of COVID-19 from chest X-Ray images. Because all researchers are looking for a quick method to diagnose this virus, deep learning-based computer controlled techniques are more suitable as a second opinion for radiologists. In this article, we look at the issue of multisource fusion and redundant features. We proposed a CNN-LSTM and improved max value features optimization framework for COVID-19 classification to address these issues. The original images are acquired and the contrast is increased using a combination of filtering algorithms in the proposed architecture. The dataset is then augmented to increase its size, which is then used to train two deep learning networks called Modified EfficientNet B0 and CNN-LSTM. Both networks are built from scratch and extract information from the deep layers. Following the extraction of features, the serial based maximum value fusion technique is proposed to combine the best information of both deep models. However, a few redundant information is also noted; therefore, an improved max value based moth flame optimization algorithm is proposed. Through this algorithm, the best features are selected and finally classified through machine learning classifiers. The experimental process was conducted on three publically available datasets and achieved improved accuracy than the existing techniques. Moreover, the classifiers based comparison is also conducted and the cubic support vector machine gives better accuracy.
Assuntos
COVID-19 , Aprendizado Profundo , Mariposas , Animais , Humanos , Redes Neurais de Computação , Raios XRESUMO
Structural mutants of p53 induce global p53 protein destabilization and misfolding, followed by p53 protein aggregation. First evidence indicates that p53 can be part of protein condensates and that p53 aggregation potentially transitions through a condensate-like state. We show condensate-like states of fluorescently labeled structural mutant p53 in the nucleus of living cancer cells. We furthermore identified small molecule compounds that interact with the p53 protein and lead to dissolution of p53 structural mutant condensates. The same compounds lead to condensation of a fluorescently tagged p53 DNA-binding mutant, indicating that the identified compounds differentially alter p53 condensation behavior depending on the type of p53 mutation. In contrast to p53 aggregation inhibitors, these compounds are active on p53 condensates and do not lead to mutant p53 reactivation. Taken together our study provides evidence for structural mutant p53 condensation in living cells and tools to modulate this process.
RESUMO
The angiopoietin (Angpt)-TIE signaling pathway controls vascular maturation and maintains the quiescent phenotype of resting vasculature. The contextual agonistic and antagonistic Tie2 ligand ANGPT2 is believed to be exclusively produced by endothelial cells, disrupting constitutive ANGPT1-TIE2 signaling to destabilize the microvasculature during pathologic disorders like inflammation and cancer. However, scattered reports have also portrayed tumor cells as a source of ANGPT2. Employing ISH-based detection of ANGPT2, we found strong tumor cell expression of ANGPT2 in a subset of patients with melanoma. Comparative analysis of biopsies revealed a higher fraction of ANGPT2-expressing tumor cells in metastatic versus primary sites. Tumor cell-expressed Angpt2 was dispensable for primary tumor growth, yet in-depth analysis of primary tumors revealed enhanced intratumoral necrosis upon silencing of tumor cell Angpt2 expression in the absence of significant immune and vascular alterations. Global transcriptional profiling of Angpt2-deficient tumor cells identified perturbations in redox homeostasis and an increased response to cellular oxidative stress. Ultrastructural analyses illustrated a significant increase of dysfunctional mitochondria in Angpt2-silenced tumor cells, thereby resulting in enhanced reactive oxygen species (ROS) production and downstream MAPK stress signaling. Functionally, enhanced ROS in Angpt2-silenced tumor cells reduced colonization potential in vitro and in vivo. Taken together, these findings uncover the hitherto unappreciated role of tumor cell-expressed ANGPT2 as an autocrine-positive regulator of metastatic colonization and validate ANGPT2 as a therapeutic target for a well-defined subset of patients with melanoma. SIGNIFICANCE: This study reveals that tumor cells can be a source of ANGPT2 in the tumor microenvironment and that tumor cell-derived ANGPT2 augments metastatic colonization by protecting tumor cells from oxidative stress.
Assuntos
Angiopoietina-2/metabolismo , Melanoma/secundário , Nevo/patologia , Neoplasias Cutâneas/patologia , Angiopoietina-2/genética , Animais , Comunicação Autócrina , Biópsia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Estimativa de Kaplan-Meier , Sistema de Sinalização das MAP Quinases , Melanoma/mortalidade , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Pele/patologia , Neoplasias Cutâneas/mortalidade , Análise Serial de Tecidos , Microambiente TumoralRESUMO
BACKGROUND: To assess antivascular effects, and evaluate clinically translatable magnetic resonance imaging (MRI) biomarkers of tumour response in vivo, following treatment with vanucizumab, a bispecific human antibody against angiopoietin-2 (Ang-2) and vascular endothelial growth factor-A (VEGF-A). METHODS: Colo205 colon cancer xenografts were imaged before and 5 days after treatment with a single 10 mg kg(-1) dose of either vanucizumab, bevacizumab (anti-human VEGF-A), LC06 (anti-murine/human Ang-2) or omalizumab (anti-human IgE control). Volumetric response was assessed using T2-weighted MRI, and diffusion-weighted, dynamic contrast-enhanced (DCE) and susceptibility contrast MRI used to quantify tumour water diffusivity (apparent diffusion coefficient (ADC), × 10(6) mm(2) s(-1)), vascular perfusion/permeability (K(trans), min(-1)) and fractional blood volume (fBV, %) respectively. Pathological correlates were sought, and preliminary gene expression profiling performed. RESULTS: Treatment with vanucizumab, bevacizumab or LC06 induced a significant (P<0.01) cytolentic response compared with control. There was no significant change in tumour ADC in any treatment group. Uptake of Gd-DTPA was restricted to the tumour periphery in all post-treatment groups. A significant reduction in tumour K(trans) (P<0.05) and fBV (P<0.01) was determined 5 days after treatment with vanucizumab only. This was associated with a significant (P<0.05) reduction in Hoechst 33342 uptake compared with control. Gene expression profiling identified 20 human genes exclusively regulated by vanucizumab, 6 of which are known to be involved in vasculogenesis and angiogenesis. CONCLUSIONS: Vanucizumab is a promising antitumour and antiangiogenic treatment, whose antivascular activity can be monitored using DCE and susceptibility contrast MRI. Differential gene expression in vanucizumab-treated tumours is regulated by the combined effect of Ang-2 and VEGF-A inhibition.
Assuntos
Adenocarcinoma/tratamento farmacológico , Inibidores da Angiogênese/uso terapêutico , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Perfilação da Expressão Gênica , Imageamento por Ressonância Magnética/métodos , Terapia de Alvo Molecular , Neovascularização Patológica/tratamento farmacológico , Adenocarcinoma/irrigação sanguínea , Adenocarcinoma/diagnóstico por imagem , Adenocarcinoma/patologia , Inibidores da Angiogênese/imunologia , Angiopoietina-2/antagonistas & inibidores , Angiopoietina-2/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Bevacizumab/uso terapêutico , Linhagem Celular Tumoral , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/diagnóstico por imagem , Neoplasias do Colo/patologia , Replicação do DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoglobulina E/imunologia , Camundongos , Neovascularização Patológica/diagnóstico por imagem , Neovascularização Patológica/patologia , Omalizumab/uso terapêutico , Carga Tumoral , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Vascular endothelial growth factor (VEGF)-A blockade has been validated clinically as a treatment for human cancers. Angiopoietin-2 (Ang-2) is a key regulator of blood vessel remodeling and maturation. In tumors, Ang-2 is up-regulated and an unfavorable prognostic factor. Recent data demonstrated that Ang-2 inhibition mediates anti-tumoral effects. We generated a tetravalent bispecific antibody (Ang-2-VEGF-TAvi6) targeting VEGF-A with 2 arms based on bevacizumab (Avastin®), and targeting Ang-2 with 2 arms based on a novel anti-Ang-2 antibody (LC06). The two Ang-2-targeting single-chain variable fragments are disulfide-stabilized and fused to the C-terminus of the heavy chain of bevacizumab. Treatment with Ang-2-VEGF-A-TAvi6 led to a complete abrogation of angiogenesis in the cornea micropocket assay. Metastatic spread and tumor growth of subcutaneous, orthotopic and anti-VEGF-A resistant tumors were also efficiently inhibited. These data further establish Ang-2-VEGF bispecific antibodies as a promising anti-angiogenic, anti-metastatic and anti-tumor agent for the treatment of cancer.
Assuntos
Angiopoietina-2/antagonistas & inibidores , Anticorpos Biespecíficos , Anticorpos Antineoplásicos , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Experimentais , Neovascularização Patológica , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Anticorpos Antineoplásicos/imunologia , Anticorpos Antineoplásicos/farmacologia , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Metástase Neoplásica , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/imunologia , Neovascularização Patológica/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Angiogenesis, the process by which new vessels form from existing ones, plays an important role in many developmental processes and pathological conditions. We study angiogenesis in the context of a highly controllable experimental environment: the cornea micropocket assay. Using a multidisciplinary approach that combines experiments, image processing and analysis, and mathematical modelling, we aim to provide mechanistic insight into the action of two angiogenic factors, vascular endothelial growth factor A (VEGF-A) and basic fibroblast growth factor (bFGF). We use image analysis techniques to extract quantitative data, which are both spatially and temporally resolved, from experimental images, and we develop a mathematical model, in which the corneal vasculature evolves in response to both VEGF-A and bFGF. The experimental data are used for model parametrization, while the mathematical model is used to assess the utility of the cornea micropocket assay and to characterize proposed synergies between VEGF-A and bFGF.
Assuntos
Neovascularização da Córnea , Fator 2 de Crescimento de Fibroblastos/metabolismo , Modelos Cardiovasculares , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The blockade of VEGF pathway has been clinically validated as an initial treatment for renal cell carcinoma (RCC). Angiopoietin-2 (Ang-2) has been indicated as a key regulator for angiogenesis escape. The effect of a novel bispecific antibody (A2V CrossMab) against both Ang-2 and VEGF was investigated in comparison with either factor. A2V CrossMab significantly reduced tumor volume, vessel density, and interstitial fluid pressure compared to either monotherapy of anti-VEGF or anti-Ang-2. Host-derived angiogenesis-related genes have been significantly down-regulated in A2V CrossMab group. These data demonstrate that A2V CrossMab has additive anti-tumor effect for the treatment of RCC.
Assuntos
Angiopoietina-2/imunologia , Anticorpos Biespecíficos/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Neoplasias Renais/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/imunologia , Angiopoietina-2/antagonistas & inibidores , Angiopoietina-2/metabolismo , Animais , Anticorpos Biespecíficos/imunologia , Carcinoma de Células Renais/genética , Análise por Conglomerados , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Renais/genética , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Primary cutaneous large B-cell lymphomas, leg type (PCLBCL/LT) are primary cutaneous B-cell lymphoma (PCBCL) with an intermediate prognosis. Therefore, antracycline-based polychemotherapy combined with rituximab has been recommended as first-line treatment. Yet, despite this regimen, the 5-year survival rate remains 50-66% only. Angiogenesis, the formation of a vascular network, is essential for the pathogenesis of nodal lymphomas. So far, no study has analysed angiogenesis and its key factors in PCLBCL/LT. The present study was aimed at characterizing angiogenesis in PCLBCL/LT to identify the angiogenic molecules as potential therapeutic targets. The intra-tumoral microvessel density (MVD) was assessed by immunohistochemical studies of CD20 and CD31. The MVD was higher in PCLBCL/LT compared with indolent PCBCL. Analyses of open-source microarray data showed correlation between the angiogenic molecule angiopoietin-2 (Ang-2) and pan-endothelial cell markers. ELISA studies determined a shift between Ang-2 and Ang-1 towards Ang-2 in the peripheral blood of PCLBCL/LT patients. Immunofluorescence costainings against the Ang receptor Tie2/angiogenic integrins/CD34 revealed that the vasculature in both aggressive and indolent PCBCL tumors harbours an endothelial cell subpopulation with reduced expression of Tie2. In contrast, the alternative Ang-2 binding partners, angiogenic integrins, are strongly expressed in PCBCL. In line with these findings, downstream targets of Ang-2-integrin signalling, that is phosphorylation of focal adhesion kinase at Tyr397, and sprouting angiogenesis are enhanced in PCLBCL/LT. Our data present Ang-2 as a promising therapeutic target and anti-angiogenic therapy as a new line in treatment of PCLBCL/LT as a hitherto intractable disease.
Assuntos
Angiopoietina-2/metabolismo , Linfoma de Células B/metabolismo , Neovascularização Patológica/metabolismo , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/metabolismo , Angiopoietina-2/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Integrinas/metabolismo , Linfoma de Células B/genética , Microvasos/patologia , Fosforilação , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Neoplasias Cutâneas/genéticaRESUMO
Despite the development of novel therapies, the therapy of malignant melanoma remains challenging. Various studies have shown the vascular system to be pivotal for metastasis in melanoma. Consequently, the effect of various antiangiogenic therapies has been and is being investigated in preclinical and clinical trials. While most studies focus on inhibition of vascular endothelial growth factor (VEGF) signaling, others are aimed at determining the effect of multikinase inhibitors or the inhibition of angiogenic integrin activity. However, overall survival rates have not significantly improved in clinical trials with antiangiogenic agents. Resistance to anti-VEGF monotherapy has been observed in several studies, especially in malignant melanoma. Angiopoietin-2 (Ang-2) represents a promising candidate molecule for antiangiogenic therapy and the effect of Ang-2 inhibitors is currently being explored in first trials. In melanoma, Ang-2 has been shown to be a marker for metastasis formation and represents an interesting therapeutic target molecule. Future studies are required to analyze the effect of a combined approach, using anti-VEGF and anti-Ang-2, as therapy for malignant melanoma.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Melanoma/irrigação sanguínea , Melanoma/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/tratamento farmacológico , Angiopoietina-2/antagonistas & inibidores , Animais , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Quimioterapia Combinada , Humanos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Antiangiogenic tumor therapy has failed in the adjuvant setting. Here we show that inhibition of the Tie2 ligand angiopoietin-2 (Ang2) effectively blocks metastatic growth in preclinical mouse models of postsurgical adjuvant therapy. Ang2 antibody treatment combines well with low-dose metronomic chemotherapy (LDMC) in settings in which maximum-dose chemotherapy does not prove effective. Mechanistically, Ang2 blockade could be linked to quenching the inflammatory and angiogenic response of endothelial cells (ECs) in the metastatic niche. Reduced EC adhesion molecule and chemokine expression inhibits the recruitment of tumor-promoting CCR2(+)Tie2(-) metastasis-associated macrophages. Moreover, LDMC contributes to therapeutic efficacy by inhibiting the recruitment of protumorigenic bone marrow-derived myeloid cells. Collectively, these data provide a rationale for mechanism-guided adjuvant tumor therapies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias da Mama/tratamento farmacológico , Metástase Neoplásica/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Adjuvantes Farmacêuticos/administração & dosagem , Adjuvantes Farmacêuticos/efeitos adversos , Administração Metronômica , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/efeitos adversos , Animais , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Mamárias Experimentais , Dose Máxima Tolerável , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/patologia , Paclitaxel/administração & dosagem , Paclitaxel/efeitos adversos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Liver regeneration requires spatially and temporally precisely coordinated proliferation of the two major hepatic cell populations, hepatocytes and liver sinusoidal endothelial cells (LSECs), to reconstitute liver structure and function. The underlying mechanisms of this complex molecular cross-talk remain elusive. Here, we show that the expression of Angiopoietin-2 (Ang2) in LSECs is dynamically regulated after partial hepatectomy. During the early inductive phase of liver regeneration, Ang2 down-regulation leads to reduced LSEC transforming growth factor-ß1 production, enabling hepatocyte proliferation by releasing an angiocrine proliferative brake. During the later angiogenic phase of liver regeneration, recovery of endothelial Ang2 expression enables regenerative angiogenesis by controlling LSEC vascular endothelial growth factor receptor 2 expression. The data establish LSECs as a dynamic rheostat of liver regeneration, spatiotemporally orchestrating hepatocyte and LSEC proliferation through angiocrine- and autocrine-acting Ang2, respectively.
Assuntos
Angiopoietina-2/metabolismo , Proliferação de Células , Endotélio Vascular/metabolismo , Hepatócitos/fisiologia , Regeneração Hepática/fisiologia , Angiopoietina-2/genética , Animais , Hepatectomia , Hepatócitos/citologia , Regeneração Hepática/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
PURPOSE: VEGF-A blockade has been clinically validated as a treatment for human cancers. Angiopoietin-2 (Ang-2) expression has been shown to function as a key regulator of tumor angiogenesis and metastasis. EXPERIMENTAL DESIGN: We have applied the recently developed CrossMab technology for the generation of a bispecific antibody recognizing VEGF-A with one arm based on bevacizumab (Avastin), and the other arm recognizing Ang-2 based on LC06, an Ang-2 selective human IgG1 antibody. The potency of Ang-2-VEGF CrossMab was evaluated alone and in combination with chemotherapy using orthotopic and subcutaneous xenotransplantations, along with metastasis analysis by quantitative real-time Alu-PCR and ex vivo evaluation of vessels, hypoxia, proliferation, and apoptosis. The mechanism of action was further elucidated using Western blotting and ELISA assays. RESULTS: Ang-2-VEGF-A CrossMab showed potent tumor growth inhibition in a panel of orthotopic and subcutaneous syngeneic mouse tumors and patient or cell line-derived human tumor xenografts, especially at later stages of tumor development. Ang-2-VEGF-A CrossMab treatment led to a strong inhibition of angiogenesis and an enhanced vessel maturation phenotype. Neoadjuvant combination with chemotherapy resulted in complete tumor regression in primary tumor-bearing Ang-2-VEGF-A CrossMab-treated mice. In contrast to Ang-1 inhibition, anti-Ang-2-VEGF-A treatment did not aggravate the adverse effect of anti-VEGF treatment on physiologic vessels. Moreover, treatment with Ang-2-VEGF-A CrossMab resulted in inhibition of hematogenous spread of tumor cells to other organs and reduced micrometastatic growth in the adjuvant setting. CONCLUSION: These data establish Ang-2-VEGF-A CrossMab as a promising antitumor, antiangiogenic, and antimetastatic agent for the treatment of cancer.
Assuntos
Angiopoietina-2/imunologia , Anticorpos Biespecíficos/administração & dosagem , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/imunologia , Angiopoietina-2/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Bevacizumab , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/imunologia , Camundongos , Metástase Neoplásica , Neoplasias/imunologia , Neovascularização Patológica/imunologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Genetic experiments (loss-of-function and gain-of-function) have established the role of Angiopoietin/Tie ligand/receptor tyrosine kinase system as a regulator of vessel maturation and quiescence. Angiopoietin-2 (Ang-2) acts on Tie2-expressing resting endothelial cells as an antagonistic ligand to negatively interfere with the vessel stabilizing effects of constitutive Ang-1/Tie-2 signaling. Ang-2 thereby controls the vascular response to inflammation-inducing as well as angiogenesis-inducing cytokines. This study was aimed at assessing the role of Ang-2 as an autocrine (i.e. endothelial-derived) regulator of rapid vascular responses (within minutes) caused by permeability-inducing agents. Employing two independent in vivo assays to quantitatively assess vascular leakage (tracheal microsphere assay, 1-5 min and Miles assay, 20 min), the immediate vascular response to histamine, bradykinin and VEGF was analyzed in Ang-2-deficient (Ang-2(-/-)) mice. In comparison to the wild type control mice, the Ang2(-/-) mice demonstrated a significantly attenuated response. The Ang-2(-/-) phenotype was rescued by systemic administration (paracrine) of an adenovirus encoding Ang-2. Furthermore, cytokine-induced intracellular calcium influx was impaired in Ang-2(-/-) endothelioma cells, consistent with reduced phospholipase activation in vivo. Additionally, recombinant human Ang-2 (rhAng-2) alone was unable to induce vascular leakage. In summary, we report here in a definite genetic setting that Ang-2 is critical for multiple vascular permeability-inducing cytokines.
Assuntos
Angiopoietina-2/metabolismo , Citocinas/farmacologia , Angiopoietina-2/genética , Animais , Western Blotting , Bradicinina/genética , Bradicinina/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/genética , Células Cultivadas , Feminino , Células Endoteliais da Veia Umbilical Humana , Pulmão/citologia , Pulmão/metabolismo , Camundongos , Camundongos Knockout , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Septic shock is characterized by increased vascular permeability and hypotension despite increased cardiac output. Numerous vasoactive cytokines are upregulated during sepsis, including angiopoietin 2 (ANG2), which increases vascular permeability. Here we report that mice engineered to inducibly overexpress ANG2 in the endothelium developed sepsis-like hemodynamic alterations, including systemic hypotension, increased cardiac output, and dilatory cardiomyopathy. Conversely, mice with cardiomyocyte-restricted ANG2 overexpression failed to develop hemodynamic alterations. Interestingly, the hemodynamic alterations associated with endothelial-specific overexpression of ANG2 and the loss of capillary-associated pericytes were reversed by intravenous injections of adeno-associated viruses (AAVs) transducing cDNA for angiopoietin 1, a TIE2 ligand that antagonizes ANG2, or AAVs encoding PDGFB, a chemoattractant for pericytes. To confirm the role of ANG2 in sepsis, we i.p. injected LPS into C57BL/6J mice, which rapidly developed hypotension, acute pericyte loss, and increased vascular permeability. Importantly, ANG2 antibody treatment attenuated LPS-induced hemodynamic alterations and reduced the mortality rate at 36 hours from 95% to 61%. These data indicate that ANG2-mediated microvascular disintegration contributes to septic shock and that inhibition of the ANG2/TIE2 interaction during sepsis is a potential therapeutic target.
RESUMO
Bispecific antibodies are considered as a promising class of future biotherapeutic molecules. They comprise binding specificities for two different antigens, which may provide additive or synergistic modes of action. There is a wide variety of design alternatives for such bispecific antibodies, including the "CrossMab" format. CrossMabs contain a domain crossover in one of the antigen-binding (Fab) parts, together with the "knobs-and-holes" approach, to enforce the correct assembly of four different polypeptide chains into an IgG-like bispecific antibody. We determined the crystal structure of a hAng-2-binding Fab in its crossed and uncrossed form and show that CH1-CL-domain crossover does not induce significant perturbations of the structure and has no detectable influence on target binding.
Assuntos
Angiopoietina-2/imunologia , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/metabolismo , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estabilidade Proteica , Estrutura Terciária de Proteína , Eletricidade Estática , Relação Estrutura-Atividade , TemperaturaRESUMO
There is increasing experimental evidence for an important role of Angiopoietin-2 (Ang-2) in tumor angiogenesis and progression. In addition, Ang-2 is up-regulated in many cancer types and correlated with poor prognosis. To investigate the functional role of Ang-2 inhibition in tumor development and progression, we generated novel fully human antibodies that neutralize specifically the binding of Ang-2 to its receptor Tie2. The selected antibodies LC06 and LC08 recognize both rodent and human Ang-2 with high affinity, but LC06 shows a higher selectivity for Ang-2 over Ang-1 compared to LC08 which can be considered an Ang-2/Ang-1 cross-reactive antibody. Our data demonstrate that Ang-2 blockade results in potent tumor growth inhibition and pronounced tumor necrosis in subcutaneous and orthotopic tumor models. These effects are attended with a reduction of intratumoral microvessel density and tumor vessels characterized by fewer branches and increased pericyte coverage. Furthermore, anti-Ang-2 treatment strongly inhibits the dissemination of tumor cells to the lungs. Interestingly, in contrast to the Ang-2/Ang-1 cross-reactive antibody LC08 that leads to a regression of physiological vessels in the mouse trachea, the inhibition with the selective anti-Ang-2 antibody LC06 appears to be largely restricted to tumor vasculature without obvious effects on normal vasculature. Taken together, these data provide strong evidence for the selective Ang-2 antibody LC06 as promising new therapeutic agent for the treatment of various cancers.
Assuntos
Angiopoietina-1/antagonistas & inibidores , Angiopoietina-2/antagonistas & inibidores , Angiopoietina-2/imunologia , Anticorpos Neutralizantes/farmacologia , Antineoplásicos/farmacologia , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/tratamento farmacológico , Angiopoietina-1/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos , Antineoplásicos/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Córnea/efeitos dos fármacos , Córnea/imunologia , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Microvasos/efeitos dos fármacos , Microvasos/imunologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/imunologia , Fosforilação , Distribuição Aleatória , Receptor TIE-2/antagonistas & inibidores , Receptor TIE-2/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The tumor-initiating capacity of primary human breast cancer cells is maintained in vitro by culturing these cells as spheres/aggregates. Inoculation of small cell numbers derived from these non-adherent cultures leads to rapid xenograft tumor formation in mice. Accordingly, injection of more differentiated monolayer cells derived from spheres results in significantly decelerated tumor growth. For our study, two breast cancer cell lines were generated from primary tumors and cultured as mammospheres or as their adherent counterparts. We examined the in vivo tumorigenicity of these cells by injecting serial dilutions into immunodeficient mice. Inoculation of 106 cells per mouse led to rapid tumor formation, irrespective of cell line or culture conditions. However, after injection of only 103 cells, solely sphere cells were highly tumorigenic. In vitro, we investigated differentiation markers, established breast CSC markers and conducted mRNA profiling. Cytokeratin 5 and 18 were increased in both monolayer cell types, indicating a more differentiated phenotype. All cell lines were CD24(-)/CD44(+) and did not express CD133, CD326 or E-cadherin. ALDH1 activity was not detectable in any cell line. A verapamilsensitive Hoechst side population was present in sphere cells, but there was no correlation with tumorigenicity in vivo. mRNA profiling did not reveal upregulation of relevant transcription factors. In vitro cell cycle kinetics and in vivo tumor doubling times displayed no difference between sphere and monolayer cultures. Our data indicate that intrinsic genetic and functional markers investigated are not indicative of the in vivo tumori-genicity of putative breast tumor-initiating cells.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica/metabolismo , Células-Tronco Neoplásicas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Ciclo Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imunofenotipagem , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Esferoides Celulares , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Angiopoietin-2 (ANG-2) is a key regulator of angiogenesis that exerts context-dependent effects on ECs. ANG-2 binds the endothelial-specific receptor tyrosine kinase 2 (TIE2) and acts as a negative regulator of ANG-1/TIE2 signaling during angiogenesis, thereby controlling the responsiveness of ECs to exogenous cytokines. Recent data from tumors indicate that under certain conditions ANG-2 can also promote angiogenesis. However, the molecular mechanisms of dual ANG-2 functions are poorly understood. Here, we identify a model for the opposing roles of ANG-2 in angiogenesis. We found that angiogenesis-activated endothelium harbored a subpopulation of TIE2-negative ECs (TIE2lo). TIE2 expression was downregulated in angiogenic ECs, which abundantly expressed several integrins. ANG-2 bound to these integrins in TIE2lo ECs, subsequently inducing, in a TIE2-independent manner, phosphorylation of the integrin adaptor protein FAK, resulting in RAC1 activation, migration, and sprouting angiogenesis. Correspondingly, in vivo ANG-2 blockade interfered with integrin signaling and inhibited FAK phosphorylation and sprouting angiogenesis of TIE2lo ECs. These data establish a contextual model whereby differential TIE2 and integrin expression, binding, and activation control the role of ANG-2 in angiogenesis. The results of this study have immediate translational implications for the therapeutic exploitation of angiopoietin signaling.
Assuntos
Angiopoietina-2/metabolismo , Regulação para Baixo , Integrinas/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Angiopoietina-2/genética , Animais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Humanos , Integrinas/genética , Masculino , Melanoma/genética , Melanoma/patologia , Camundongos , Proteínas de Neoplasias/genética , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fosforilação/genética , Receptores Proteína Tirosina Quinases/genética , Receptor TIE-2 , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
OBJECTIVE: Angiogenesis, a critical contributor to ocular as well as neoplastic diseases, is stimulated by endothelial production of angiopoietin-2 (Ang2). Our objective was to determine the requirement of ocular angiogenesis for Ang2 in animal models of disease. METHODS: We developed and compared the effect of a novel human Ang2 antibody with a pan-angiopoietin strategy on angiogenesis in ocular angiogenesis in animal models of oxygen-induced retinopathy, and laser photocoagulation and confirmed its efficacy in xenografted human colorectal tumors. RESULTS: Human anti-Ang2 and anti-angiopoietin1(Ang1)/Ang2 antibodies blocked colorectal carcinoma growth in immuno-compromised mice (p < 0.001, n = 6). Injection of 1 µg of Ang2 or Ang2/Ang1 antibody-inhibited angiogenesis in models of retinal (p < 0.001, n = 6), and choroidal neovascularization (p < 0.001, n = 11-13 per group) to levels similar to that with anti-VEGF antibodies. There was no difference between Ang2 specific and Ang1/Ang2 bi-specific antibodies. In vitro, Ang2 antibodies showed no cytotoxicity and did not inhibit endothelial cell migration or proliferation. CONCLUSION: Thus, human Ang2 antibodies are potentially therapeutic agents for ocular neovascularization in models of retinal and choroidal neovascularization, in the absence of VEGF inhibition.
Assuntos
Angiopoietina-2/antagonistas & inibidores , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Neovascularização Patológica/tratamento farmacológico , Ribonuclease Pancreático/antagonistas & inibidores , Animais , Neovascularização de Coroide/tratamento farmacológico , Neovascularização de Coroide/patologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Doenças Retinianas/induzido quimicamente , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/patologia , Transplante HeterólogoRESUMO
We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This "crossover" retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible "CrossMab" formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by standard techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMab(CH1-CL) was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.