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BACKGROUND: Cisplatin (CDDP) is a mainstay treatment for advanced head and neck squamous cell carcinomas (HNSCC) despite a high frequency of innate and acquired resistance. We hypothesised that tumours acquire CDDP resistance through an enhanced reductive state dependent on metabolic rewiring. METHODS: To validate this model and understand how an adaptive metabolic programme might be imprinted, we performed an integrated analysis of CDDP-resistant HNSCC clones from multiple genomic backgrounds by whole-exome sequencing, RNA-seq, mass spectrometry, steady state and flux metabolomics. RESULTS: Inactivating KEAP1 mutations or reductions in KEAP1 RNA correlated with Nrf2 activation in CDDP-resistant cells, which functionally contributed to resistance. Proteomics identified elevation of downstream Nrf2 targets and the enrichment of enzymes involved in generation of biomass and reducing equivalents, metabolism of glucose, glutathione, NAD(P), and oxoacids. This was accompanied by biochemical and metabolic evidence of an enhanced reductive state dependent on coordinated glucose and glutamine catabolism, associated with reduced energy production and proliferation, despite normal mitochondrial structure and function. CONCLUSIONS: Our analysis identified coordinated metabolic changes associated with CDDP resistance that may provide new therapeutic avenues through targeting of these convergent pathways.
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Antineoplásicos , Neoplasias de Cabeza y Cuello , Humanos , Cisplatino/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello , Proteína 1 Asociada A ECH Tipo Kelch/genética , Factor 2 Relacionado con NF-E2/genética , Resistencia a Antineoplásicos/genética , Línea Celular Tumoral , Glucosa , Antineoplásicos/farmacologíaRESUMEN
Steroid hormones are pivotal modulators of pathophysiological processes in many organs, where they interact with nuclear receptors to regulate gene transcription. However, our understanding of hormone action at the single cell level remains incomplete. Here, we focused on estrogen stimulation of the well-characterized GREB1 and MYC target genes that revealed large differences in cell-by-cell responses, and, more interestingly, between alleles within the same cell, both over time and hormone concentration. We specifically analyzed the role of receptor level and activity state during allele-by-allele regulation and found that neither receptor level nor activation status are the determinant of maximal hormonal response, indicating that additional pathways are potentially in place to modulate cell- and allele-specific responses. Interestingly, we found that a small molecule inhibitor of the arginine methyltransferases CARM1 and PRMT6 was able to increase, in a gene specific manner, the number of active alleles/cell before and after hormonal stimulation, suggesting that mechanisms do indeed exist to modulate hormone receptor responses at the single cell and allele level.
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Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteína-Arginina N-Metiltransferasas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Transcripción Genética , Estrógenos/metabolismo , Hormonas Esteroides Gonadales/genética , Histona Acetiltransferasas/genética , Humanos , Conformación Molecular , Proteínas Nucleares/antagonistas & inhibidores , Unión Proteica/genética , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Análisis de la Célula IndividualRESUMEN
BACKGROUND: Triple-negative breast cancers (TNBCs), which lack receptors for estrogen, progesterone, and amplification of epidermal growth factor receptor 2, are highly aggressive. Consequently, patients diagnosed with TNBCs have reduced overall and disease-free survival rates compared to patients with other subtypes of breast cancer. TNBCs are characterized by the presence of cancer cells with mesenchymal properties, indicating that the epithelial to mesenchymal transition (EMT) plays a major role in the progression of this disease. The EMT program has also been implicated in chemoresistance, tumor recurrence, and induction of cancer stem cell (CSC) properties. Currently, there are no targeted therapies for TNBC, and hence, it is critical to identify the novel targets to treat TNBC. METHODS: A library of compounds was screened for their ability to inhibit EMT in cells with mesenchymal phenotype as assessed using the previously described Z-cad reporters. Of the several drugs tested, GSK3ß inhibitors were identified as EMT inhibitors. The effects of GSK3ß inhibitors on the properties of TNBC cells with a mesenchymal phenotype were assessed using qRT-PCR, flow cytometry, western blot, mammosphere, and migration and cell viability assays. Publicly available datasets also were analyzed to examine if the expression of GSK3ß correlates with the overall survival of breast cancer patients. RESULTS: We identified a GSK3ß inhibitor, BIO, in a drug screen as one of the most potent inhibitors of EMT. BIO and two other GSK3ß inhibitors, TWS119 and LiCl, also decreased the expression of mesenchymal markers in several different cell lines with a mesenchymal phenotype. Further, inhibition of GSK3ß reduced EMT-related migratory properties of cells with mesenchymal properties. To determine if GSK3ß inhibitors target mesenchymal-like cells by affecting the CSC population, we employed mammosphere assays and profiled the stem cell-related cell surface marker CD44+/24- in cells after exposure to GSK3ß inhibitors. We found that GSK3ß inhibitors indeed decreased the CSC properties of cell types with mesenchymal properties. We treated cells with epithelial and mesenchymal properties with GSK3ß inhibitors and found that GSK3ß inhibitors selectively kill cells with mesenchymal attributes while sparing cells with epithelial properties. We analyzed patient data to identify genes predictive of poor clinical outcome that could serve as novel therapeutic targets for TNBC. The Wnt signaling pathway is critical to EMT, but among the various factors known to be involved in Wnt signaling, only the higher expression of GSK3ß correlated with poorer overall patient survival. CONCLUSIONS: Taken together, our data demonstrate that GSK3ß is a potential target for TNBCs and suggest that GSK3ß inhibitors could serve as selective inhibitors of EMT and CSC properties for the treatment of a subset of aggressive TNBC. GSK3ß inhibitors should be tested for use in combination with standard-of-care drugs in preclinical TNBC models.
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Transición Epitelial-Mesenquimal/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Neoplasias de la Mama Triple Negativas/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Conjuntos de Datos como Asunto , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Humanos , Concentración 50 Inhibidora , Cloruro de Litio/farmacología , Cloruro de Litio/uso terapéutico , Células Madre Neoplásicas/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Pirroles/farmacología , Pirroles/uso terapéutico , Análisis de Supervivencia , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/mortalidad , Vía de Señalización WntRESUMEN
Cystic fibrosis (CF) is a single-gene disorder that affects the lung, digestive system, and other organs. Mutations in the CF transmembrane conductance regulator (CFTR) gene are classified into several classes based on their pathogenic mechanism and clinical severity. The distinct and heterogeneous clinical behavior of each CF class and the respective CFTR mutations have made the development of a durable therapy for all CF patients extremely challenging. While the FDA-approved drug elexacaftor/tezacaftor/ivacaftor (Trikafta) benefits CF patients carrying at least one F508del mutation in CFTR, it's not effective for many CF patients carrying a variety of other CFTR mutations. To establish a better understanding of CF pathophysiology and aid the development of novel therapeutics for different classes of CF patients, we have created four CF-mutation-specific cell models that recapitulate respectively four distinct CF classes and disease phenotypes, as confirmed by sequencing, CFTR mRNA and protein quantification. The channel function of each cell model was first validated using a well-established FLIPR (Fluorescent Imaging Plate Reader) membrane potential assay and then assessed by the YFP-based functional assay. Integrated with a halide-sensitive fluorescent reporter, these CF cell models can be used for high-throughput drug screening, as demonstrated by a proof-of-concept study using Trikafta. These cell models have the potential to advance CFTR mutation-specific therapies thus addressing the unmet needs of CF patients with rare mutations.
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The epithelial-mesenchymal transition (EMT) has been implicated in conferring stem cell properties and therapeutic resistance to cancer cells. Therefore, identification of drugs that can reprogram EMT may provide new therapeutic strategies. Here, we report that cells derived from claudin-low mammary tumors, a mesenchymal subtype of triple-negative breast cancer, exhibit a distinctive organoid structure with extended "spikes" in 3D matrices. Upon a miR-200 induced mesenchymal-epithelial transition (MET), the organoids switch to a smoother round morphology. Based on these observations, we developed a morphological screening method with accompanying analytical pipelines that leverage deep neural networks and nearest neighborhood classification to screen for EMT-reversing drugs. Through screening of a targeted epigenetic drug library, we identified multiple class I HDAC inhibitors and Bromodomain inhibitors that reverse EMT. These data support the use of morphological screening of mesenchymal mammary tumor organoids as a platform to identify drugs that reverse EMT.
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Antineoplásicos/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Neoplasias Mamarias Animales/patología , Mesodermo/patología , Organoides/patología , Animales , Azacitidina/farmacología , Benzamidas/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica , Procesamiento de Imagen Asistido por Computador , Neoplasias Mamarias Animales/genética , Ratones Endogámicos BALB C , MicroARNs/genética , MicroARNs/metabolismo , Proteínas de Neoplasias/metabolismo , Organoides/efectos de los fármacos , Pirimidinas/farmacología , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
BACKGROUND: Despite the use of aggressive multimodality treatment, most anaplastic thyroid carcinoma (ATC) patients die within a year of diagnosis. Although the combination of BRAF and MEK inhibitors has recently been approved for use in BRAF-mutated ATC, they remain effective in a minority of patients who are likely to develop drug resistance. There remains a critical clinical need for effective systemic agents for ATC with a reasonable toxicity profile to allow for rapid translational development. MATERIAL AND METHODS: Twelve human thyroid cancer cell lines with comprehensive genomic characterization were used in a high-throughput screening (HTS) of 257 compounds to select agents with maximal growth inhibition. Cell proliferation, colony formation, orthotopic thyroid models, and patient-derived xenograft (PDX) models were used to validate the selected agents. RESULTS: Seventeen compounds were effective, and docetaxel, LBH-589, and pralatrexate were selected for additional in vitro and in vivo analysis as they have been previously approved by the US Food and Drug Administration for other cancers. Significant tumor growth inhibition (TGI) was detected in all tested models treated with LBH-589; pralatrexate demonstrated significant TGI in the orthotopic papillary thyroid carcinoma model and 2 PDX models; and docetaxel demonstrated significant TGI only in the context of mutant TP53. CONCLUSIONS: HTS identified classes of systemic agents that demonstrate preferential effectiveness against aggressive thyroid cancers, particularly those with mutant TP53. Preclinical validation in both orthotopic and PDX models, which are accurate in vivo models mimicking tumor microenvironment, may support initiation of early-phase clinical trials in non-BRAF mutated or refractory to BRAF/MEK inhibition ATC.
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Antineoplásicos/farmacología , Ensayos Analíticos de Alto Rendimiento , Inhibidores de Proteínas Quinasas/farmacología , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/tratamiento farmacológico , Animales , Pruebas de Carcinogenicidad , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Proteínas Proto-Oncogénicas B-raf/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacosRESUMEN
Leukocyte inflammatory responses require integrin cell-adhesion molecule signaling through spleen tyrosine kinase (Syk), a non-receptor kinase that binds directly to integrin ß-chain cytoplasmic domains. Here, we developed a high-throughput screen to identify small molecule inhibitors of the Syk-integrin cytoplasmic domain interactions. Screening small molecule compound libraries identified the ß-lactam antibiotics cefsulodin and ceftazidime, which inhibited integrin ß-subunit cytoplasmic domain binding to the tandem SH2 domains of Syk (IC50 range, 1.02-4.9 µM). Modeling suggested antagonist binding to Syk outside the pITAM binding site. Ceftazidime inhibited integrin signaling via Syk, including inhibition of adhesion-dependent upregulation of interleukin-1ß and monocyte chemoattractant protein-1, but did not inhibit ITAM-dependent phosphorylation of Syk mediated by FcγRI signaling. Our results demonstrate a novel means to target Syk independent of its kinase and pITAM binding sites such that integrin signaling via this kinase is abrogated but ITAM-dependent signaling remains intact. As integrin signaling through Syk is essential for leukocyte activation, this may represent a novel approach to target inflammation.
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Antiinflamatorios/farmacología , Cefsulodina/farmacología , Ceftazidima/farmacología , Cadenas beta de Integrinas/efectos de los fármacos , Leucocitos/efectos de los fármacos , Quinasa Syk/antagonistas & inhibidores , Antiinflamatorios/química , Cefsulodina/química , Ceftazidima/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Cadenas beta de Integrinas/química , Cadenas beta de Integrinas/metabolismo , Leucocitos/enzimología , Masculino , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , Bibliotecas de Moléculas Pequeñas , Quinasa Syk/química , Quinasa Syk/metabolismo , Células THP-1RESUMEN
To address the unmet need for effective biomarker-driven targeted therapy for human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) and cervical cancer, we conducted a high-throughput drug screen using 1122 compounds in 13 HPV-positive and 11 matched HPV-negative cell lines. The most effective drug classes were inhibitors of polo-like kinase, proteasomes, histone deacetylase, and Aurora kinases. Treatment with a pan-Aurora inhibitor, danusertib, led to G2M arrest and apoptosis in vitro. Furthermore, danusertib decreased tumor size compared with controls in patient derived xenograft models of HNSCC. To identify biomarkers predicting response, we determined associations between mutations and drug sensitivity. Our data and the Genomics of Drug Sensitivity in Cancer database showed that cancer cells with KMT2D mutations were more sensitive to Aurora kinase inhibitors than were cells without mutations. Knockdown of KMT2D in wild-type cells led to increased Aurora kinase inhibitor-induced apoptosis. We identified Aurora kinase inhibitors as effective and understudied drugs in HNSCC and CESC. This is the first published study to demonstrate that mutations in KMT2D, which are common in many cancers, correlate with drug sensitivity in two independent datasets.
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Aurora Quinasa A/antagonistas & inhibidores , Carcinoma de Células Escamosas/genética , Proteínas de Unión al ADN/genética , Mutación , Proteínas de Neoplasias/genética , Infecciones por Papillomavirus/genética , Animales , Apoptosis , Área Bajo la Curva , Benzamidas/farmacología , Biomarcadores/metabolismo , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/virología , Ciclo Celular , Línea Celular , Proliferación Celular , Evaluación Preclínica de Medicamentos , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Trasplante de Neoplasias , Papillomaviridae , Infecciones por Papillomavirus/tratamiento farmacológico , Farmacogenética , Pirazoles/farmacología , Neoplasias del Cuello UterinoRESUMEN
To identify cellular and molecular changes that driver pediatric low grade glioma (PLGG) progression, we analyzed putative cancer stem cells (CSCs) and evaluated key biological changes in a novel and progressive patient-derived orthotopic xenograft (PDOX) mouse model. Flow cytometric analysis of 22 PLGGs detected CD133+ (<1.5%) and CD15+ (20.7 ± 28.9%) cells, and direct intra-cranial implantation of 25 PLGGs led to the development of 1 PDOX model from a grade II pleomorphic xanthoastrocytoma (PXA). While CSC levels did not correlate with patient tumor progression, neurosphere formation and in vivo tumorigenicity, the PDOX model, IC-3635PXA, reproduced key histological features of the original tumor. Similar to the patient tumor that progressed and recurred, IC-3635PXA also progressed during serial in vivo subtransplantations (4 passages), exhibiting increased tumor take rate, elevated proliferation, loss of mature glial marker (GFAP), accumulation of GFAP-/Vimentin+ cells, enhanced local invasion, distant perivascular migration, and prominent reactive gliosis in normal mouse brains. Molecularly, xenograft cells with homozygous deletion of CDKN2A shifted from disomy chromosome 9 to trisomy chromosome 9; and BRAF V600E mutation allele frequency increased (from 28% in patient tumor to 67% in passage III xenografts). In vitro drug screening identified 2/7 BRAF V600E inhibitors and 2/9 BRAF inhibitors that suppressed cell proliferation. In summary, we showed that PLGG tumorigenicity was low despite the presence of putative CSCs, and our data supported GFAP-/Vimentin+ cells, CDKN2A homozygous deletion in trisomy chromosome 9 cells, and BRAF V600E mutation as candidate drivers of tumor progression in the PXA xenografts.
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Separase is an endopeptidase that cleaves cohesin subunit Rad21, facilitating the repair of DNA damage during interphase and the resolution of sister chromatid cohesion at anaphase. Separase activity is negatively regulated by securin and Cdk1-cyclin B in vivo. Separase overexpression is reported in a broad range of human tumors, and its overexpression in mouse models results in tumorigenesis. To elucidate further the mechanism of separase function and to test if inhibition of overexpressed separase can be used as a strategy to inhibit tumor-cell proliferation, small-molecule inhibitors of separase enzyme are essential. Here, we report a high-throughput screening for separase inhibitors (Sepins). We developed a fluorogenic separase assay using rhodamine 110-conjugated Rad21 peptide as substrate and screened a small-molecule compound library. We identified a noncompetitive inhibitor of separase called Sepin-1 that inhibits separase enzymatic activity with a half maximal inhibitory concentration (IC50) of 14.8 µM. Sepin-1 can inhibit the growth of human cancer cell lines and breast cancer xenograft tumors in mice by inhibiting cell proliferation and inducing apoptosis. The sensitivity to Sepin-1 in most cases is positively correlated to the level of separase in both cancer cell lines and tumors.
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Antineoplásicos/química , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Separasa/antagonistas & inhibidores , Animales , Anticuerpos/química , Apoptosis , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Daño del ADN , Proteínas de Unión al ADN , Modelos Animales de Enfermedad , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Hidrólisis , Concentración 50 Inhibidora , Dosis Máxima Tolerada , Ratones , Microscopía Fluorescente , Trasplante de Neoplasias , Neoplasias/patología , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Péptidos/química , Fosfoproteínas/química , Rodaminas/químicaRESUMEN
Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that affects multiple organs. Treatment is mainly surgical, and effective systemic therapies are needed. We developed a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point-mutated VHL protein. The 786-0 cell line was infected with full-length W117A-mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of functional readouts, including hypoxia-inducible factor 2α (HIF2α) and glucose transporter 1 (Glut1) levels, were performed. We found that bortezomib, MG132, and the Prestwick compounds 8-azaguanine, thiostrepton, and thioguanosine upregulated VHL-W117A-Venus in 786-0 cells. 8-Azaguanine downregulated HIF2α levels and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate posttranslational processing. Nuclear-cytoplasmic localization of VHL-W117A-Venus varied among the different compounds. In conclusion, a 786-0 cell line containing VHL-W117A-Venus was successfully used to identify compounds that upregulate VHL levels, with differential effect on VHL intracellular localization and posttranslational processing. Further screening efforts will broaden the number of pharmacophores available to develop therapeutic agents that will upregulate and refunctionalize mutated VHL.
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Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Línea Celular , Inhibidores de Cisteína Proteinasa/farmacología , Descubrimiento de Drogas/métodos , Humanos , Leupeptinas/farmacología , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma , Estabilidad Proteica/efectos de los fármacos , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas/farmacología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
BACKGROUND: In the current situation of weak drug pipelines, impending patent expiration of several blockbuster drugs, industry consolidation and changing business models that target special diseases like cancer, diabetes, Alzheimer's and obesity, the pharmaceutical industry is under intense pressure to generate a strong drug pipeline distinguished by better productivity, diversity and cost effectiveness. The goal is discovering high-quality leads in the initial stages of the development cycle, to minimize the costs associated with failures at later ones. OBJECTIVE: Thus, there is a great amount of interest in further developing and optimizing high-throughput screening and in silico screening, the two methods responsible for generating most of the lead compounds. Although high-throughput screening is the predominant starting point for discovery programs, in silico methods have gradually made inroads by their more rational approach, to expedite the drug discovery and development process. CONCLUSION: Modern drug discovery strategies include both methods in tandem or in an iterative way. This review primarily provides a succinct overview and comparison of experimental and in silico screening techniques, selected case studies where both methods were used in concert to investigate their performance and complementary nature and a statement on the developments in experimental and in silico approaches in the near future.
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Long-term peritoneal dialysis is associated with the development of functional and structural alterations of the peritoneal membrane. Long-term exposure to the high glucose concentrations in conventional peritoneal dialysate has been implicated in the pathogenesis of peritoneal hyperpermeability and neoangiogenesis. Vascular endothelial growth factor (VEGF) is an endothelial-specific growth factor that potently stimulates microvascular permeability and proliferation. High glucose exposure upregulates VEGF expression in various cell types and tissues. This study investigated whether VEGF plays a pathogenetic role in hyperglycemia-induced microvascular dysfunction in the peritoneal membrane. The peritoneal microcirculation of streptozotocin-induced diabetic rats and age-matched controls was studied in vivo with a combination of functional and morphologic techniques. The diabetic microcirculation was characterized by an elevated transport of small solutes, indicating the presence of an increased effective vascular surface area. The leakage of FITC-albumin was more rapid in diabetic vessels, suggesting hyperpermeability for macromolecules. Structurally, an increased vascular density with focal areas of irregular capillary budding was found in the diabetic peritoneum. The hyperglycemia-induced structural and functional microvascular alterations were prevented by long-term treatment with neutralizing anti-VEGF monoclonal antibodies, whereas treatment with isotype-matched control antibodies had no effect. VEGF blockade did not influence microvascular density or macromolecular leakage in control rats, demonstrating specificity for the hyperglycemia-induced alterations. The present results thus support an causative link among high glucose exposure, upregulation of VEGF, and peritoneal microvascular dysfunction.
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Factores de Crecimiento Endotelial/fisiología , Hiperglucemia/fisiopatología , Linfocinas/fisiología , Diálisis Peritoneal , Peritoneo/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Transporte Biológico , Vasos Sanguíneos/patología , Permeabilidad Capilar , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/patología , Factores de Crecimiento Endotelial/antagonistas & inhibidores , Factores de Crecimiento Endotelial/inmunología , Femenino , Fluoresceína-5-Isotiocianato/farmacocinética , Linfocinas/antagonistas & inhibidores , Linfocinas/inmunología , Microcirculación , Peritoneo/irrigación sanguínea , Ratas , Ratas Wistar , Valores de Referencia , Albúmina Sérica/farmacocinética , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Vascular endothelial growth factor (VEGF) is a cytokine that potently stimulates angiogenesis, microvascular hyperpermeability, and endothelium-dependent vasodilation, effects that are largely mediated by endothelial nitric oxide synthase (eNOS). The expression of VEGF is pronounced in glomerular visceral epithelial cells, but its function in renal physiology and pathophysiology is unknown. VEGF expression is upregulated by high ambient glucose concentrations in several cell types in vitro and in glomeruli of diabetic rats. To assess the role of VEGF in the pathophysiology of early renal dysfunction in diabetes, monoclonal anti-VEGF antibodies (Ab) were administered to control and streptozotocin-induced diabetic rats for 6 wk after induction of diabetes. Based on in vitro binding studies, an adequate serum VEGF inhibitory activity was achieved during the entire course of anti-VEGF Ab administration. Anti-VEGF Ab treatment but not administration of isotype-matched control Ab decreased hyperfiltration, albuminuria, and glomerular hypertrophy in diabetic rats. VEGF blockade also prevented the upregulation of eNOS expression in glomerular capillary endothelial cells of diabetic rats. Antagonism of VEGF had no effect on GFR and glomerular volume in control rats. These results identify VEGF as a pathogenetic link between hyperglycemia and early renal dysfunction in diabetes. Targeting VEGF may prove useful as a therapeutic strategy for the treatment of early diabetic nephropathy.