RESUMO
Subretinal fibrosis is a major untreatable cause of poor outcomes in neovascular age-related macular degeneration. Mouse models of subretinal fibrosis all possess a degree of invasiveness and tissue damage not typical of fibrosis progression. This project characterises JR5558 mice as a model to study subretinal fibrosis. Fundus and optical coherence tomography (OCT) imaging was used to non-invasively track lesions. Lesion number and area were quantified with ImageJ. Retinal sections, wholemounts and Western blots were used to characterise alterations. Subretinal lesions expand between 4 and 8 weeks and become established in size and location around 12 weeks. Subretinal lesions were confirmed to be fibrotic, including various cell populations involved in fibrosis development. Müller cell processes extended from superficial retina into subretinal lesions at 8 weeks. Western blotting revealed increases in fibronectin (4 wk and 8 wk, p < 0.001), CTGF (20 wks, p < 0.001), MMP2 (12 wks and 20 wks p < 0.05), αSMA (12 wks and 20 wks p < 0.05) and GFAP (8 wk and 12 wk, p ≤ 0.01), consistent with our immunofluorescence results. Intravitreal injection of Aflibercept reduced subretinal lesion growth. Our study provides evidence JR5558 mice have subretinal fibrotic lesions that grow between 4 and 8 weeks and confirms this line to be a good model to study subretinal fibrosis development and assess treatment options.
Assuntos
Modelos Animais de Doenças , Fibrose , Retina , Tomografia de Coerência Óptica , Animais , Camundongos , Tomografia de Coerência Óptica/métodos , Retina/patologia , Retina/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fibronectinas/metabolismo , Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Degeneração Macular/patologia , Degeneração Macular/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Injeções Intravítreas , Proteína Glial Fibrilar Ácida/metabolismo , Actinas/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de FusãoRESUMO
Vascular endothelial growth factor (VEGF) is a pleiotropic growth factor that binds a broad spectrum of cell types and regulates diverse cellular processes, including angiogenesis, growth and survival. However, it is technically difficult to quantify VEGF-cell binding activity because of reversible nature of ligand-receptor interactions. Here we used T7 bacteriophage display to quantify and compare binding activity of three human VEGF-A (hVEGF) isoforms, including hVEGF111, 165 and 206. All three isoforms bound equally well to immobilized aflibercept, a decoy VEGF receptor. hVEGF111-Phage exhibited minimal binding to immobilized heparan sulfate, whereas hVEGF206-Phage and hVEGF165-Phage had the highest and intermediate binding to heparan, respectively. In vitro studies revealed that all three isoforms bound to human umbilical vein endothelial cells (HUVECs), HEK293 epithelial and SK-N-AS neuronal cells. hVEGF111-Phage has the lowest binding activity, while hVEGF206-Phage has the highest binding. hVEGF206-Phage was the most sensitive to detect VEGF-cell binding, albeit with the highest background binding to SK-N-AS cells. These results suggest that hVEGF206-Phage is the best-suited isoform to quantify VEGF-cell binding even though VEGF165 is the most biologically active. Furthermore, this study demonstrates the utility of T7 phage display as a platform for rapid and convenient ligand-cell binding quantification with pros and cons discussed.
Assuntos
Células Endoteliais da Veia Umbilical Humana , Ligação Proteica , Fator A de Crescimento do Endotélio Vascular , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células HEK293 , Isoformas de Proteínas/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Bacteriófago T7/metabolismo , Bacteriófago T7/genética , Técnicas de Visualização da Superfície Celular/métodos , Heparitina Sulfato/metabolismo , Proteínas Recombinantes de FusãoRESUMO
PURPOSE: Pathological angiogenesis and vascular instability are observed in diabetic retinopathy (DR), diabetic macular edema (DME), and wet age-related macular degeneration (wAMD). Many receptor tyrosine kinases (RTKs) including vascular endothelial growth factor receptors (VEGFRs) contribute to angiogenesis, whereas the RTK TIE2 is important for vascular stability. Pan-VEGFR tyrosine kinase inhibitors (TKIs) such as vorolanib, sunitinib, and axitinib are of therapeutic interest over current antibody treatments that target only one or two ligands. This study compared the anti-angiogenic potential of these TKIs. METHODS: A kinase HotSpot™ assay was conducted to identify TKIs inhibiting RTKs associated with angiogenesis and vascular stability. Half-maximal inhibitory concentration (IC50) for VEGFRs and TIE2 was determined for each TKI. In vitro angiogenesis inhibition was investigated using a human umbilical vein endothelial cell sprouting assay, and in vivo angiogenesis was studied using the chorioallantoic membrane assay. Melanin binding was assessed using a melanin-binding assay. Computer modeling was conducted to understand the TIE2-axitinib complex as well as interactions between vorolanib and VEGFRs. RESULTS: Vorolanib, sunitinib, and axitinib inhibited RTKs of interest in angiogenesis and exhibited pan-VEGFR inhibition. HotSpot™ assay and TIE2 IC50 values showed that only axitinib potently inhibited TIE2 (up to 89%). All three TKIs effectively inhibited angiogenesis in vitro. In vivo, TKIs were more effective at inhibiting VEGF-induced angiogenesis than the anti-VEGF antibody bevacizumab. Of the three TKIs, only sunitinib bound melanin. TKIs differ in their classification and binding to VEGFRs, which is important because type II inhibitors have greater selectivity than type I TKIs. CONCLUSIONS: Vorolanib, sunitinib, and axitinib exhibited pan-VEGFR inhibition and inhibited RTKs associated with pathological angiogenesis. Of the three TKIs, only axitinib potently inhibited TIE2 which is an undesired trait as TIE2 is essential for vascular stability. The findings support the use of vorolanib for therapeutic inhibition of angiogenesis observed in DR, DME, and wAMD.
Assuntos
Inibidores da Angiogênese , Axitinibe , Células Endoteliais da Veia Umbilical Humana , Imidazóis , Indazóis , Indóis , Inibidores de Proteínas Quinases , Pirróis , Receptores de Fatores de Crescimento do Endotélio Vascular , Sunitinibe , Axitinibe/farmacologia , Humanos , Sunitinibe/farmacologia , Inibidores da Angiogênese/farmacologia , Imidazóis/farmacologia , Pirróis/farmacologia , Indóis/farmacologia , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Indazóis/farmacologia , Animais , Inibidores de Proteínas Quinases/farmacologia , Receptor TIE-2/metabolismo , Receptor TIE-2/antagonistas & inibidores , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismoRESUMO
The high-grade serous ovarian cancer (HG-SOC) tumor microenvironment (TME) is constellated by cellular elements and a network of soluble constituents that contribute to tumor progression. In the multitude of the secreted molecules, the endothelin-1 (ET-1) has emerged to be implicated in the tumor/TME interplay; however, the molecular mechanisms induced by the ET-1-driven feed-forward loops (FFL) and associated with the HG-SOC metastatic potential need to be further investigated. The tracking of the patient-derived (PD) HG-SOC cell transcriptome by RNA-seq identified the vascular endothelial growth factor (VEGF) gene and its associated signature among those mostly up-regulated by ET-1 and down-modulated by the dual ET-1R antagonist macitentan. Within the ligand-receptor pairs concurrently expressed in PD-HG-SOC cells, endothelial cells and activated fibroblasts, we discovered two intertwined FFL, the ET-1/ET-1R and VEGF/VEGF receptors, concurrently activated by ET-1 and shutting-down by macitentan, or by the anti-VEGF antibody bevacizumab. In parallel, we observed that ET-1 fine-tuned the tumoral and stromal secretome toward a pro-invasive pattern. Into the fray of the HG-SOC/TME double and triple co-cultures, the secretion of ET-1 and VEGF, that share a common co-regulation, was inhibited upon the administration of macitentan. Functionally, macitentan, mimicking the effect of bevacizumab, interfered with the HG-SOC/TME FFL-driven communication that fuels the HG-SOC invasive behavior. The identification of ET-1 and VEGF FFL as tumor and TME actionable vulnerabilities, reveals how ET-1R blockade, targeting the HG-SOC cells and the TME simultaneously, may represent an effective therapeutic option for HG-SOC patients.
Assuntos
Endotelina-1 , Neoplasias Ovarianas , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/tratamento farmacológico , Endotelina-1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Sulfonamidas/farmacologia , Pirimidinas/farmacologia , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Células Estromais/metabolismo , Células Estromais/patologia , Linhagem Celular Tumoral , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Gradação de Tumores , Receptor de Endotelina A/metabolismo , Receptor de Endotelina A/genéticaRESUMO
This review article explores the intricate correlation between growth factors and bone metastases, which play a crucial role in the development of several types of malignancies, namely breast, prostate, lung, and renal cancers. The focal point of our discussion is on crucial receptors for growth factors, including Epidermal Growth Factor Receptor (EGFR), Transforming Growth Factor-ß (TGFß), Vascular Endothelial Growth Factor Receptor (VEGFR), and Fibroblast Growth Factor Receptor (FGFR). These receptors, which are essential for cellular activities including growth, differentiation, and survival, have important involvement in the spread of cancer and the interactions between tumors and the bone environment. We discuss the underlying mechanisms of bone metastases, with a specific emphasis on the interaction between growth factor receptors and the bone microenvironment. EGFR signaling specifically enhances the process of osteoclast development and the formation of osteolytic lesions, especially in breast and lung malignancies. TGFß receptors have a role in both osteolytic and osteoblastic metastases by releasing TGFß, which attracts cancer cells and promotes bone remodeling. This is a crucial element in the spread of prostate cancer to the bones. The functions of FGFR and VEGFR in the processes of bone formation and tumor angiogenesis, respectively, highlight the complex and diverse nature of these interactions. The review emphasizes the possibility of targeted therapeutics targeting these receptors to interrupt the cycle of tumor development and bone degradation. Therapeutic approaches include focusing on the VEGF/VEGFR, EGF/EGFR, FGF/FGFR, and TGFß/TGFßR pathways. These include a variety of compounds, such as small molecule inhibitors and monoclonal antibodies, which have shown potential to interfere with tumor-induced alterations in bone. The text discusses clinical trials and preclinical models, offering insights into the effectiveness and constraints of various treatments. Ultimately, this study provides a succinct but thorough summary of the present knowledge and treatment strategies focused on growth factor receptors in bone metastases. This highlights the significance of comprehending the signaling of growth factor receptors in the microenvironment where tumors spread to the bones, as well as the possibility of using targeted therapies to enhance the results for cancer patients with bone metastases. The advancement of treating bone metastases hinges on the development of treatments that specifically target the intricate relationships between malignancies and bone.
Assuntos
Neoplasias Ósseas , Humanos , Neoplasias Ósseas/secundário , Neoplasias Ósseas/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Receptores ErbB/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
"A journey of mixed emotions" is a quote that best describes the progress chart of vascular endothelial growth factor receptor (VEGFR) inhibitors as cancer therapeutics in the last decade. Exhilarated with the Food and Drug Administration (FDA) approvals of numerous VEGFR inhibitors coupled with the annoyance of encountering the complications associated with their use, drug discovery enthusiasts are on their toes with an unswerving determination to enhance the rate of translation of VEGFR inhibitors from preclinical to clinical stage. The recently crafted armory of VEGFR inhibitors is a testament to their growing dominance over other antiangiogenic therapies for cancer treatment. This review perspicuously underscores the earnest attempts of the researchers to extract the antiproliferative potential of VEGFR inhibitors through the design of mechanistically diverse structural assemblages. Moreover, this review encompasses sections on structural/molecular properties and physiological functions of VEGFR, FDA-approved VEGFR inhibitors, and hurdles restricting the activity range/clinical applicability of VEGFR targeting antitumor agents. In addition, tactics to overcome the limitations of VEGFR inhibitors are discussed. A clear-cut viewpoint transmitted through this compilation can provide practical directions to push the cart of VEGFR inhibitors to advanced-stage clinical investigations in diverse malignancies.
Assuntos
Antineoplásicos , Neoplasias , Inibidores de Proteínas Quinases , Receptores de Fatores de Crescimento do Endotélio Vascular , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Estrutura MolecularRESUMO
Vascular endothelial growth factor (VEGF) and VEGF reporter (VEGFR) are essential molecules in VEGF signalling pathway. Although the functions of VEGF and VEGFR have been well reported in vertebrates, their functions are still poorly understood in invertebrates. In this study, the open reading frame sequences of EsVEGF1 and EsVEGFR4 were cloned from Eriocheir sinensis, and their corresponding proteins shared typical structure characteristics with their counterparts in other species. EsVEGF1 were predominantly expressed in hepatopancreas and muscle while EsVEGFR4 mainly expressed in hemocytes and intestine. The expression levels of EsVEGF1 in hemocytes were rapidly induced by Staphylococcus aureus and Vibrio parahaemolyticus, and it also increased rapidly in hepatopancreas after being challenged with V. parahaemolyticus. The expression levels of EsVEGFR4 only increased in hepatopancreas of crabs injected with S. aureus. The extracellular immunoglobulin domain of EsVEGFR4 could bind with Gram-negative and Gram-positive bacteria as well as lipopolysaccharide and peptidoglycan. EsVEGF1 could act as the ligand for EsVEGFR4 and Toll-like receptor and regulate the expression of crustins and lysozyme with a tissue-specific manner, while have no regulatory function on that of anti-lipopolysaccharide factors. This study will provide new insights into the immune defense mechanisms mediated by VEGF and VEGFR in crustaceans.
Assuntos
Braquiúros , Receptores de Fatores de Crescimento do Endotélio Vascular , Fator A de Crescimento do Endotélio Vascular , Animais , Braquiúros/metabolismo , Braquiúros/microbiologia , Braquiúros/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Peptídeos Antimicrobianos/metabolismo , Peptídeos Antimicrobianos/genética , Peptídeos Antimicrobianos/química , Sequência de Aminoácidos , Staphylococcus aureus , Regulação da Expressão Gênica , Vibrio parahaemolyticus , Filogenia , Hepatopâncreas/metabolismo , Hemócitos/metabolismoRESUMO
Worldwide, cancer is a major public health concern. It is a well-acknowledged life-threatening disease. Despite numerous advances in the understanding of the genetic basis of cancer growth and progression, therapeutic challenges remain high. Human tumors exhibited mutation or overexpression of several tyrosine kinases (TK). The vascular endothelial growth factor receptor (VEGFR) is a TK family member and is well known for tumor growth and progression. Therefore, VEGF/VEGFR pathway inhibition is an appealing approach for cancer drug discovery. This review will discuss the structure-based optimization of thienopyrimidines incorporating the aryl urea moiety to develop scaffolds of potent anticancer activity via VEGFR inhibition published between 2013 and 2023. Increasing knowledge of probable scaffolds that can act as VEGFR inhibitors might spur the hunt for novel anticancer medications that are safer, more effective, or both.
Assuntos
Antineoplásicos , Inibidores de Proteínas Quinases , Pirimidinas , Receptores de Fatores de Crescimento do Endotélio Vascular , Ureia , Humanos , Pirimidinas/farmacologia , Pirimidinas/química , Pirimidinas/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Ureia/farmacologia , Ureia/química , Ureia/análogos & derivados , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Relação Estrutura-Atividade , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Estrutura Molecular , AnimaisRESUMO
Amauroderma rugosum (AR), also known as "Blood Lingzhi" in Chinese, is a basidiomycete belonging to the Ganodermataceae family. Four polysaccharide fractions were systematically isolated and purified from AR. Subsequently, their compositions were examined and analyzed via high-performance gel permeation chromatography (HPGPC), analysis of the monosaccharide composition, Fourier-transform infrared spectroscopy (FT-IR), and 1H nuclear magnetic resonance (NMR). The zebrafish model was then used to screen for proangiogenic activities of polysaccharides by inducing vascular insufficiency with VEGF receptor tyrosine kinase inhibitor II (VRI). The third fraction of AR polysaccharides (PAR-3) demonstrated the most pronounced proangiogenic effects, effectively ameliorating VRI-induced intersegmental vessel deficiency in zebrafish. Concurrently, the mRNA expression levels of vascular endothelial growth factor (VEGF)-A and VEGF receptors were upregulated by PAR-3. Moreover, the proliferation, migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs) were also stimulated by PAR-3, consistently demonstrating that PAR-3 possesses favorable proangiogenic properties. The activation of the Akt, ERK1/2, p38 MAPK, and FAK was most likely the underlying mechanism. In conclusion, this study establishes that PAR-3 isolated from Amauroderma rugosum exhibits potential as a bioresource for promoting angiogenesis.
Assuntos
Células Endoteliais da Veia Umbilical Humana , Peixe-Zebra , Animais , Humanos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Polissacarídeos/farmacologia , Polissacarídeos/química , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Neovascularização Fisiológica/efeitos dos fármacos , Indutores da Angiogênese/farmacologia , Indutores da Angiogênese/química , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Basidiomycota/química , Polissacarídeos Fúngicos/farmacologia , Polissacarídeos Fúngicos/químicaRESUMO
PURPOSE: This systematic review summarizes evidence of VEGFR gene mutations and VEGF/VEGFR protein expression in glioblastoma multiforme (GBM) patients, alongside the efficacy and safety of anti-VEGFR tyrosine kinase inhibitors (TKIs) for GBM treatment. METHODS: A comprehensive literature review was conducted using PubMed up to August 2023. Boolean operators and MeSH term "glioma," along with specific VEGFR-related keywords, were utilized following thorough examination of existing literature. RESULTS: VEGFR correlates with glioma grade and GBM progression, presenting a viable therapeutic target. Regorafenib and axitinib show promise among studied TKIs. Other multi-targeted TKIs (MTKI) and combination therapies exhibit potential, albeit limited by blood-brain barrier penetration and toxicity. Combining treatments like radiotherapy and enhancing BBB penetration may benefit patients. Further research is warranted in patient quality of life and biomarker-guided selection. CONCLUSION: While certain therapies hold promise for GBM, future research should prioritize personalized medicine and innovative strategies for improved treatment outcomes.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Inibidores de Proteínas Quinases , Receptores de Fatores de Crescimento do Endotélio Vascular , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologiaRESUMO
Vascular endothelial growth factor (VEGF) targeted therapy serves as an important therapeutic approach for renal cancer, but its clinical effectiveness is unsatisfactory. Moreover, there is a lack of reliable biomarkers for preoperative assessment of tumor VEGF expression. This study aimed to explore the potential for further applications of 177Lu/89Zr-labeled aflibercept (Abe), a VEGF-binding agent, in imaging visualization of VEGF expression and therapy for renal cancer. To determine specificity uptake in renal cancer, BALB/c mice with VEGF-expressing Renca tumor were intravenously injected with [89Zr]Zr-Abe, [177Lu]Lu-Abe, or Cy5.5-Abe and the blocking group was designed as a control group. PET, SPECT, and fluorescence images were acquired, and the biodistribution of [89Zr]Zr-Abe and [177Lu]Lu-Abe was performed. Additionally, the [177Lu]Lu-Abe, [177Lu]Lu-Abe-block, 177Lu only, Abe only, and PBS groups were compared for evaluation of the therapeutic effect. To assess the safety, we monitored and evaluated the body weight, blood biochemistry analysis, and whole blood analysis and major organs were stained with hematoxylin and eosin after [177Lu]Lu-Abe treatment. DOTA-Abe was successfully labeled with 177Lu and Df-Abe with 89Zr in our study. The uptake in tumor of [89Zr]Zr-Abe was significantly higher than that of [89Zr]Zr-Abe-block (P < 0.05) and provided excellent tumor contrast in PET images. [177Lu]Lu-Abe demonstrated promising tumor-specific targeting capability with a high and persistent tumor uptake. The standardized tumor volume of [177Lu]Lu-Abe was significantly smaller than those of other treatment groups (P < 0.05). [177Lu]Lu-Abe also had smaller tumor volumes and reduced expression of VEGF and CD31 compared to those of the control groups. Fluorescence images demonstrate higher tumor uptake in the Cy5.5-Abe group compared to the Cy5.5-Abe-block group (P < 0.05). In conclusion, [89Zr]Zr-Abe enables noninvasive analysis of VEGF expression, serving as a valuable tool for assessing the VEGF-targeted therapy effect. Additionally, all of the findings support the enhanced therapeutic efficacy and safety of [177Lu]Lu-Abe, making it a viable option for clinical practice in renal cancer.
Assuntos
Neoplasias Renais , Lutécio , Camundongos Endogâmicos BALB C , Radioisótopos , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão , Zircônio , Animais , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacocinética , Zircônio/química , Camundongos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/diagnóstico por imagem , Neoplasias Renais/metabolismo , Distribuição Tecidual , Humanos , Linhagem Celular Tumoral , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/química , Nanomedicina Teranóstica/métodos , Feminino , Tomografia por Emissão de Pósitrons/métodos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The vascular endothelial growth factor (VEGF) family of cytokines are key drivers of blood vessel growth and remodeling. These ligands act via multiple VEGF receptors (VEGFR) and co-receptors such as Neuropilin (NRP) expressed on endothelial cells. These membrane-associated receptors are not solely expressed on the cell surface, they move between the surface and intracellular locations, where they can function differently. The location of the receptor alters its ability to 'see' (access and bind to) its ligands, which regulates receptor activation; location also alters receptor exposure to subcellularly localized phosphatases, which regulates its deactivation. Thus, receptors in different subcellular locations initiate different signaling, both in terms of quantity and quality. Similarly, the local levels of co-expression of other receptors alters competition for ligands. Subcellular localization is controlled by intracellular trafficking processes, which thus control VEGFR activity; therefore, to understand VEGFR activity, we must understand receptor trafficking. Here, for the first time, we simultaneously quantify the trafficking of VEGFR1, VEGFR2, and NRP1 on the same cells-specifically human umbilical vein endothelial cells (HUVECs). We build a computational model describing the expression, interaction, and trafficking of these receptors, and use it to simulate cell culture experiments. We use new quantitative experimental data to parameterize the model, which then provides mechanistic insight into the trafficking and localization of this receptor network. We show that VEGFR2 and NRP1 trafficking is not the same on HUVECs as on non-human ECs; and we show that VEGFR1 trafficking is not the same as VEGFR2 trafficking, but rather is faster in both internalization and recycling. As a consequence, the VEGF receptors are not evenly distributed between the cell surface and intracellular locations, with a very low percentage of VEGFR1 being on the cell surface, and high levels of NRP1 on the cell surface. Our findings have implications both for the sensing of extracellular ligands and for the composition of signaling complexes at the cell surface versus inside the cell.
Assuntos
Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Transdução de Sinais , Fosforilação , Neuropilina-1/metabolismoRESUMO
OBJECTIVE: Tuberous sclerosis complex (TSC) is a genetic disorder, characterized by tumor formation in the brain and other organs, and severe neurological symptoms, such as epilepsy. Abnormal vascular endothelial growth factor (VEGF) expression may promote angiogenesis in kidney and lung tumors in TSC and has been identified in brain specimens from TSC patients, but the role of VEGF and vascular abnormalities in neurological manifestations of TSC is poorly defined. In this study, we investigated abnormalities in brain VEGF expression, cerebral blood vessel anatomy, and blood-brain barrier (BBB) structure and function in a mouse model of TSC. METHODS: Tsc1GFAP CKO mice were used to investigate VEGF expression and vascular abnormalities in the brain by Western blotting and immunohistochemical analysis of vascular and BBB markers. In vivo two-photon imaging was used to assess BBB permeability to normally impenetrable fluorescently labeled compounds. The effect of mechanistic target of rapamycin (mTOR) pathway inhibitors, VEGF receptor antagonists (apatinib), or BBB stabilizers (RepSox) was assessed in some of these assays, as well as on seizures by video-electroencephalography. RESULTS: VEGF expression was elevated in cortex of Tsc1GFAP CKO mice, which was reversed by the mTOR inhibitor rapamycin. Tsc1GFAP CKO mice exhibited increased cerebral angiogenesis and vascular complexity in cortex and hippocampus, which were reversed by the VEGF receptor antagonist apatinib. BBB permeability was abnormally increased and BBB-related tight junction proteins occludin and claudin-5 were decreased in Tsc1GFAP CKO mice, also in an apatinib- and RepSox-dependent manner. The BBB stabilizer (RepSox), but not the VEGF receptor antagonist (apatinib), decreased seizures and improved survival in Tsc1GFAP CKO mice. SIGNIFICANCE: Increased brain VEGF expression is dependent on mTOR pathway activation and promotes cerebral vascular abnormalities and increased BBB permeability in a mouse model of TSC. BBB modulation may affect epileptogenesis and represent a rational treatment for epilepsy in TSC.
Assuntos
Epilepsia , Esclerose Tuberosa , Humanos , Camundongos , Animais , Barreira Hematoencefálica , Fator A de Crescimento do Endotélio Vascular/metabolismo , Esclerose Tuberosa/complicações , Esclerose Tuberosa/genética , Proteínas Supressoras de Tumor/genética , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Convulsões , Serina-Treonina Quinases TOR/genética , Sirolimo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Kidney organoids derived from human pluripotent stem cells (HiPSCs) hold huge applications for drug screening, disease modeling, and cell transplanting therapy. However, these applications are limited since kidney organoid cannot maintain complete morphology and function like human kidney. Kidney organoids are not well differentiated since the core of the organoid lacked oxygen, nutrition, and vasculature, which creates essential niches. Hypoxia-inducible factor-1 α (HIF-1α) serves as a critical regulator in vascularization and cell survival under hypoxia environment. Less is known about the role of HIF-1α in kidney organoids in this regard. This study tried to investigate the effect of HIF-1α in kidney organoid vascularization and related disease modeling. METHODS: For the vascularization study, kidney organoids were generated from human induced pluripotent stem cells. We overexpressed HIF-1α via plasmid transfection or treated DMOG (Dimethyloxallyl Glycine, an agent for HIF-1α stabilization and accumulation) in kidney progenitor cells to detect the endothelium. For the disease modeling study, we treated kidney organoid with cisplatin under hypoxia environment, with additional HIF-1α transfection. RESULT: HIF-1α overexpression elicited kidney organoid vascularization. The endothelial cells and angiotool analysis parameters were increased in HIF-1α plasmid-transfected and DMOG-treated organoids. These angiogenesis processes were partially blocked by VEGFR inhibitors, semaxanib or axitinib. Cisplatin-induced kidney injury (Cleaved caspase 3) was protected by HIF-1α through the upregulation of CD31 and SOD2. CONCLUSION: We demonstrated that HIF-1α elicited the process of kidney organoid vascularization and protected against cisplatin-induced kidney organoid injury in hypoxia environment.
Assuntos
Angiogênese , Subunidade alfa do Fator 1 Induzível por Hipóxia , Rim , Modelos Biológicos , Organoides , Organoides/irrigação sanguínea , Organoides/metabolismo , Rim/metabolismo , Células-Tronco Multipotentes , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Humanos , Plasmídeos/genética , Expressão Gênica , Células-Tronco/citologia , Células-Tronco/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Inibidores da Angiogênese/farmacologia , Angiogênese/efeitos dos fármacos , Angiogênese/fisiopatologia , Axitinibe/farmacologia , Células Cultivadas , Cisplatino/farmacologia , Hipóxia Celular , Nefropatias/fisiopatologiaRESUMO
Tumor endothelial cells (TECs) are key stromal components of the tumor microenvironment, and are essential for tumor angiogenesis, growth and metastasis. Accumulating evidence has shown that small single-stranded non-coding microRNAs (miRNAs) act as powerful endogenous regulators of TEC function and blood vessel formation. This systematic review provides an up-to-date overview of these endothelial miRNAs. Their expression is mainly regulated by hypoxia, pro-angiogenic factors, gap junctions and extracellular vesicles, as well as long non-coding RNAs and circular RNAs. In preclinical studies, they have been shown to modulate diverse fundamental angiogenesis-related signaling pathways and proteins, including the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) pathway; the rat sarcoma virus (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway; the phosphoinositide 3-kinase (PI3K)/AKT pathway; and the transforming growth factor (TGF)-ß/TGF-ß receptor (TGFBR) pathway, as well as krüppel-like factors (KLFs), suppressor of cytokine signaling (SOCS) and metalloproteinases (MMPs). Accordingly, endothelial miRNAs represent promising targets for future anti-angiogenic cancer therapy. To achieve this, it will be necessary to further unravel the regulatory and functional networks of endothelial miRNAs and to develop safe and efficient TEC-specific miRNA delivery technologies.
Assuntos
MicroRNAs , Neoplasias , Humanos , MicroRNAs/metabolismo , Células Endoteliais/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias/terapia , Neoplasias/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Microambiente Tumoral/genéticaRESUMO
The aim of this work was to determine endometrial mRNA expression and uterine protein localization of vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 during the estrous cycle and peri-implantation period in sows. Uterine tissues were collected from pregnant sows on days 12, 14, 16, and 18 after artificial insemination and from non-pregnant animals on days 2 and 12 of the estrous cycle (day 0 = day of estrus). Using immunohistochemistry, a positive signal for VEGF and its receptor VEGFR2 was found in uterine luminal epithelial cells, endometrial glands, stroma, blood vessels, and myometrium. A VEGFR1 signal was only found in endometrial and myometrial blood vessels and stroma. By day 18 of gestation, the mRNA expression levels of VEGF, VEGFR1, and VEGFR2 were higher than those observed on days 2 and 12 of the estrous cycle and on days 12, 14, and 16 of gestation. Then, a primary culture of sow endometrial epithelial cells was established to define the potential of the selective inhibition of VEGFR2 after treatment with inhibitor SU5416 and determine its effects on the expression pattern of the VEGF system. The endometrial epithelial cells treated with SU5416 showed a dose-dependent decrease in VEGFR1 and VEGFR2 mRNA expression. The present study provides additional evidence on the importance of the VEGF system during peri-implantation, as well as on the specific inhibitory activity of SU5416 in epithelial cells, which, as demonstrated, express the protein and mRNA of VEGF and its receptors VEGFR1 and VEGFR2.
Assuntos
Inibidores da Angiogênese , Fator A de Crescimento do Endotélio Vascular , Animais , Suínos , Feminino , Fator A de Crescimento do Endotélio Vascular/metabolismo , Inibidores da Angiogênese/metabolismo , Útero/metabolismo , Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Inflammatory pleuritis often causes pleural effusions, which are drained through lymphatic vessels (lymphatics) in the parietal pleura. The distribution of button- and zipper-like endothelial junctions can identify the subtypes of lymphatics, the initial, pre-collecting, and collecting lymphatics. Vascular endothelial growth factor receptor (VEGFR)-3 and its ligands VEGF-C/D are crucial lymphangiogenic factors. Currently, in the pleura covering the chest walls, the anatomy of the lymphatics and connecting networks of blood vessels are incompletely understood. Moreover, their pathological and functional plasticity under inflammation and the effects of VEGFR inhibition are unclear. This study aimed to learn the above-unanswered questions and immunostained mouse chest walls as whole-mount specimens. Confocal microscopic images and their 3-dimensional reconstruction analyzed the vasculatures. Repeated intra-pleural cavity lipopolysaccharide challenge induced pleuritis, which was also treated with VEGFR inhibition. Levels of vascular-related factors were evaluated by quantitative real-time polymerase chain reaction. We observed the initial lymphatics in the intercostals, collecting lymphatics under the ribs, and pre-collecting lymphatics connecting both. Arteries branched into capillaries and gathered into veins from the cranial to the caudal side. Lymphatics and blood vessels were in different layers with an adjacent distribution of the lymphatic layer to the pleural cavity. Inflammatory pleuritis elevated expression levels of VEGF-C/D and angiopoietin-2, induced lymphangiogenesis and blood vessel remodeling, and disorganized the lymphatic structures and subtypes. The disorganized lymphatics showed large sheet-like structures with many branches and holes inside. Such lymphatics were abundant in zipper-like endothelial junctions with some button-like junctions. The blood vessels were tortuous and had various diameters and complex networks. Stratified layers of lymphatics and blood vessels were disorganized, with impaired drainage function. VEGFR inhibition partially maintained their structures and drainage function. These findings demonstrate anatomy and pathological changes of the vasculatures in the parietal pleura and their potential as a novel therapeutic target.
Assuntos
Vasos Linfáticos , Pleurisia , Camundongos , Animais , Pleura/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Vasos Linfáticos/metabolismo , Linfangiogênese , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Inflamação/metabolismo , Pleurisia/metabolismo , Pleurisia/patologiaRESUMO
Necroptosis is a mode of programmed, lytic cell death that is executed by the mixed lineage kinase domain-like (MLKL) pseudokinase following activation by the upstream kinases, receptor-interacting serine/threonine protein kinase (RIPK)-1 and RIPK3. Dysregulated necroptosis has been implicated in the pathophysiology of many human diseases, including inflammatory and degenerative conditions, infectious diseases and cancers, provoking interest in pharmacological targeting of the pathway. To identify small molecules impacting on the necroptotic machinery, we performed a phenotypic screen using a mouse cell line expressing an MLKL mutant that kills cells in the absence of upstream death or pathogen detector receptor activation. This screen identified the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) tyrosine kinase inhibitor, ABT-869 (Linifanib), as a small molecule inhibitor of necroptosis. We applied a suite of cellular, biochemical and biophysical analyses to pinpoint the apical necroptotic kinase, RIPK1, as the target of ABT-869 inhibition. Our study adds to the repertoire of established protein kinase inhibitors that additionally target RIPK1 and raises the prospect that serendipitous targeting of necroptosis signalling may contribute to their clinical efficacy in some settings.
Assuntos
Proteínas Quinases , Humanos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Necroptose , Fator A de Crescimento do Endotélio Vascular/metabolismo , Apoptose , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
Currently, renal cell carcinoma (RCC) is the most prevalent type of kidney cancer. Targeted therapy has replaced radiation therapy and chemotherapy as the main treatment option for RCC due to the lack of significant efficacy with these conventional therapeutic regimens. Sunitinib, a drug used to treat gastrointestinal tumors and renal cell carcinoma, inhibits the tyrosine kinase activity of a number of receptor tyrosine kinases, including vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), c-Kit, rearranged during transfection (RET) and fms-related receptor tyrosine kinase 3 (Flt3). Although sunitinib has been shown to be efficacious in the treatment of patients with advanced RCC, a significant number of patients have primary resistance to sunitinib or acquired drug resistance within the 6-15 months of therapy. Thus, in order to develop more efficacious and long-lasting treatment strategies for patients with advanced RCC, it will be crucial to ascertain how to overcome sunitinib resistance that is produced by various drug resistance mechanisms. In this review, we discuss: 1) molecular mechanisms of sunitinib resistance; 2) strategies to overcome sunitinib resistance and 3) potential predictive biomarkers of sunitinib resistance.