RESUMEN
The number of cancer drugs is increasing as new chemical entities are developed to target molecules, often protein kinases, driving cancer progression. In 2009, Fedorov et al. identified that of the protein kinases in the human kinome, most of the focus has been on a small subset. They highlighted that many poorly investigated protein kinases were cancer drivers, but there was no relationship between publications and involvement in cancer development or progression. Since 2009, there has been a doubling in the number of publications, patents, and drugs targeting the kinome. To determine whether this was an expansion in knowledge of well-studied targets-searching in the light under the lamppost-or an explosion of investigations into previously poorly investigated targets, we searched the literature for publications on each kinase, updating Federov et al.'s assessment of the druggable kinome. The proportion of papers focusing on the 50 most-studied kinases had not changed, and the makeup of those 50 had barely changed. The majority of new drugs (80%) were against the same group of 50 kinases identified as targets 10 years ago, and the proportion of studies investigating previously poorly investigated kinases (<1%) was unchanged. With three exceptions [p38 mitogenactivated protein kinase (p38a), AMP-activated protein kinase catalytic α-subunit 1,2, and B-Raf proto-oncogene (BRAF) serine/threonine kinase], >95% of publications addressing kinases still focused on a relatively small proportion (<50%) of the human kinome independently of their involvement as cancer drivers. There is, therefore, still extensive scope for discovery of therapeutics targeting different protein kinases in cancer and still a bias toward well-characterized targets over the innovative searchlight into the unknown. SIGNIFICANCE STATEMENT: This study presents evidence that drug discovery efforts in cancer are still to some extent focused on a narrow group of well-studied kinases 10 years after the identification of multiple novel cancer targets in the human kinome. This suggests that there is still room for researchers in academia, industry, and the not-for-profit sector to develop new and diverse therapies targeting kinases for cancer.
Asunto(s)
Antineoplásicos , Neoplasias , Proteínas Quinasas Activadas por AMP , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Humanos , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas B-raf , SerinaRESUMEN
BACKGROUND: Diabetes mellitus is a chronic disease which is detrimental to cardiovascular health, often leading to secondary microvascular complications, with huge global health implications. Therapeutic interventions that can be applied to multiple vascular beds are urgently needed. Diabetic retinopathy (DR) and diabetic kidney disease (DKD) are characterised by early microvascular permeability changes which, if left untreated, lead to visual impairment and renal failure, respectively. The heparan sulphate cleaving enzyme, heparanase, has previously been shown to contribute to diabetic microvascular complications, but the common underlying mechanism which results in microvascular dysfunction in conditions such as DR and DKD has not been determined. METHODS: In this study, two mouse models of heparan sulphate depletion (enzymatic removal and genetic ablation by endothelial specific Exotosin-1 knock down) were utilized to investigate the impact of endothelial cell surface (i.e., endothelial glycocalyx) heparan sulphate loss on microvascular barrier function. Endothelial glycocalyx changes were measured using fluorescence microscopy or transmission electron microscopy. To measure the impact on barrier function, we used sodium fluorescein angiography in the eye and a glomerular albumin permeability assay in the kidney. A type 2 diabetic (T2D, db/db) mouse model was used to determine the therapeutic potential of preventing heparan sulphate damage using treatment with a novel heparanase inhibitor, OVZ/HS-1638. Endothelial glycocalyx changes were measured as above, and microvascular barrier function assessed by albumin extravasation in the eye and a glomerular permeability assay in the kidney. RESULTS: In both models of heparan sulphate depletion, endothelial glycocalyx depth was reduced and retinal solute flux and glomerular albumin permeability was increased. T2D mice treated with OVZ/HS-1638 had improved endothelial glycocalyx measurements compared to vehicle treated T2D mice and were simultaneously protected from microvascular permeability changes associated with DR and DKD. CONCLUSION: We demonstrate that endothelial glycocalyx heparan sulphate plays a common mechanistic role in microvascular barrier function in the eye and kidney. Protecting the endothelial glycocalyx damage in diabetes, using the novel heparanase inhibitor OVZ/HS-1638, effectively prevents microvascular permeability changes associated with DR and DKD, demonstrating a novel systemic approach to address diabetic microvascular complications.
Asunto(s)
Diabetes Mellitus Tipo 2 , Angiopatías Diabéticas , Nefropatías Diabéticas , Glucuronidasa , Animales , Ratones , Glicocálix/metabolismo , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/prevención & control , Heparitina Sulfato/metabolismo , Heparitina Sulfato/farmacología , Albúminas/farmacología , Angiopatías Diabéticas/etiología , Angiopatías Diabéticas/prevención & control , Angiopatías Diabéticas/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismoRESUMEN
BACKGROUND: Regulation of alternative splicing is a new therapeutic approach in cancer. The programmed cell death receptor 1 (PD-1) is an immunoinhibitory receptor expressed on immune cells that binds to its ligands, PD-L1 and PD-L2 expressed by cancer cells forming a dominant immune checkpoint pathway in the tumour microenvironment. Targeting this pathway using blocking antibodies (nivolumab and pembrolizumab) is the mainstay of anti-cancer immunotherapies, restoring the function of exhausted T cells. PD-1 is alternatively spliced to form isoforms that are either transmembrane signalling receptors (flPD1) that mediate T cell death by binding to the ligand, PD-L1 or an alternatively spliced, soluble, variant that lacks the transmembrane domain. METHODS: We used PCR and western blotting on primary peripheral blood mononuclear cells (PBMCs) and Jurkat T cells, IL-2 ELISA, flow cytometry, co-culture of melanoma and cholangiocarcinoma cells, and bioinformatics analysis and molecular cloning to examine the mechanism of splicing of PD1 and its consequence. RESULTS: The soluble form of PD-1, generated by skipping exon 3 (∆Ex3PD1), was endogenously expressed in PBMCs and T cells and prevents cancer cell-mediated T cell repression. Multiple binding sites of SRSF1 are adjacent to PD-1 exon 3 splicing sites. Overexpression of phosphomimic SRSF1 resulted in preferential expression of flPD1. Inhibition of SRSF1 phosphorylation both by SRPK1 shRNA knockdown and by a selective inhibitor, SPHINX31, resulted in a switch in splicing to ∆Ex3PD1. Cholangiocarcinoma cell-mediated repression of T cell IL-2 expression was reversed by SPHINX31 (equivalent to pembrolizumab). CONCLUSIONS: These results indicate that switching of the splicing decision from flPD1 to ∆Ex3PD1 by targeting SRPK1 could represent a potential novel mechanism of immune checkpoint inhibition in cancer.
Asunto(s)
Empalme Alternativo , Colangiocarcinoma , Humanos , Fosforilación , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Arginina/genética , Arginina/metabolismo , Serina/química , Serina/genética , Serina/metabolismo , Agotamiento de Células T , Interleucina-2/genética , Leucocitos Mononucleares/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Factores de Empalme Serina-Arginina/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , InmunoterapiaRESUMEN
Angiogenic VEGF isoforms are upregulated in diabetic retinopathy (DR), driving pathological growth and fluid leakage. Serine-arginine-rich protein kinase-1 (SRPK1) regulates VEGF splicing, and its inhibition blocks angiogenesis. We tested the hypothesis that SRPK1 is activated in diabetes, and an SRPK1 inhibitor (SPHINX31) switches VEGF splicing in DR and prevents increased vascular permeability into the retina. SRPK1 was activated by high glucose (HG), in a PKC-dependent manner, and was blocked by SPHINX31. HG induced release of SRSF1 from the nuclear speckles, which was also SRPK1 dependent, and increased retinal pigment epithelial (RPE) monolayer admittance, which was reversed by SRPK1 inhibition (P < 0.05). Diabetes increased retinal permeability and thickness after 14 days which was blocked by treatment with SPHINX31 eye drops (P < 0.0001). These results show that SRPK1 inhibition, administered as an eye drop, protected the retinal barrier from hyperglycemia-associated loss of integrity in RPE cells in vitro and in diabetic rats in vivo. A clinical trial of another SRPK1 inhibitor has now been initiated in patients with diabetic macular edema.NEW & NOTEWORTHY VEGF-A165b splicing is induced by hyperglycemia through PKC-mediated activation of SRPK1 in RPE cells, increasing their permeability and angiogenic capability. SRPK1 inhibitors can be given as eye drops to reduce retinal permeability and edema in diabetic retinopathy.
Asunto(s)
Diabetes Mellitus Experimental , Retinopatía Diabética , Hiperglucemia , Edema Macular , Animales , Arginina , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/tratamiento farmacológico , Retinopatía Diabética/tratamiento farmacológico , Humanos , Soluciones Oftálmicas , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas , Ratas , Serina , Factores de Empalme Serina-Arginina , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Asthma is a complex disease with multiple phenotypes that may differ in disease pathobiology and treatment response. IL33 single nucleotide polymorphisms (SNPs) have been reproducibly associated with asthma. IL33 levels are elevated in sputum and bronchial biopsies of patients with asthma. The functional consequences of IL33 asthma SNPs remain unknown. OBJECTIVE: This study sought to determine whether IL33 SNPs associate with asthma-related phenotypes and with IL33 expression in lung or bronchial epithelium. This study investigated the effect of increased IL33 expression on human bronchial epithelial cell (HBEC) function. METHODS: Association between IL33 SNPs (Chr9: 5,815,786-6,657,983) and asthma phenotypes (Lifelines/DAG [Dutch Asthma GWAS]/GASP [Genetics of Asthma Severity & Phenotypes] cohorts) and between SNPs and expression (lung tissue, bronchial brushes, HBECs) was done using regression modeling. Lentiviral overexpression was used to study IL33 effects on HBECs. RESULTS: We found that 161 SNPs spanning the IL33 region associated with 1 or more asthma phenotypes after correction for multiple testing. We report a main independent signal tagged by rs992969 associating with blood eosinophil levels, asthma, and eosinophilic asthma. A second, independent signal tagged by rs4008366 presented modest association with eosinophilic asthma. Neither signal associated with FEV1, FEV1/forced vital capacity, atopy, and age of asthma onset. The 2 IL33 signals are expression quantitative loci in bronchial brushes and cultured HBECs, but not in lung tissue. IL33 overexpression in vitro resulted in reduced viability and reactive oxygen species-capturing of HBECs, without influencing epithelial cell count, metabolic activity, or barrier function. CONCLUSIONS: We identify IL33 as an epithelial susceptibility gene for eosinophilia and asthma, provide mechanistic insight, and implicate targeting of the IL33 pathway specifically in eosinophilic asthma.
Asunto(s)
Asma , Regulación de la Expresión Génica/inmunología , Predisposición Genética a la Enfermedad , Interleucina-33 , Polimorfismo de Nucleótido Simple , Adulto , Asma/genética , Asma/inmunología , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Interleucina-33/genética , Interleucina-33/inmunología , Masculino , Persona de Mediana EdadRESUMEN
Many potential causes for painful diabetic neuropathy have been proposed including actions of cytokines and growth factors. High mobility group protein B1 (HMGB1) is a RAGE (also known as AGER) agonist whose levels are increased in diabetes and that contributes to pain by modulating peripheral inflammatory responses. HMGB1 enhances nociceptive behaviour in naïve animals through an unknown mechanism. We tested the hypothesis that HMGB1 causes pain through direct neuronal activation of RAGE and alteration of nociceptive neuronal responsiveness. HMGB1 and RAGE expression were increased in skin and primary sensory (dorsal root ganglion, DRG) neurons of diabetic rats at times when pain behaviour was enhanced. Agonist-evoked TRPV1-mediated Ca2+ responses increased in cultured DRG neurons from diabetic rats and in neurons from naïve rats exposed to high glucose concentrations. HMGB1-mediated increases in TRPV1-evoked Ca2+ responses in DRG neurons were RAGE- and PKC-dependent, and this was blocked by co-administration of the growth factor splice variant VEGF-A165b. Pain behaviour and the DRG RAGE expression increases were blocked by VEGF-A165b treatment of diabetic rats in vivo Hence, we conclude that HMGB1-RAGE activation sensitises DRG neurons in vitro, and that VEGF-A165b blocks HMGB-1-RAGE DRG activation, which may contribute to its analgesic properties in vivo.
Asunto(s)
Neuropatías Diabéticas/metabolismo , Glucosa/metabolismo , Proteína HMGB1/metabolismo , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Células Receptoras Sensoriales/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Calcio/metabolismo , Neuropatías Diabéticas/genética , Femenino , Ganglios Espinales/metabolismo , Proteína HMGB1/genética , Humanos , Masculino , Nociceptores/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Receptor para Productos Finales de Glicación Avanzada/genética , Canales Catiónicos TRPV/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: The gold standard for measuring blood-retinal barrier permeability is the Evans blue assay. However, this technique has limitations in vivo, including non-specific tissue binding and toxicity. This study describes a non-toxic, high-throughput, and cost-effective alternative technique that minimizes animal usage. METHODS: Sodium fluorescein fundus angiography was performed in non-diabetic and diabetic Brown Norway rats on days 0, 7, 14, 21, and 28. Sodium fluorescein intensity in the retinal interstitium and a main retinal vessel were measured over time. The intensity gradients were used to quantify retinal vascular permeability. Post-study eyes were fixed, dissected, and stained (isolectin B4) to measure required parameters for permeability quantification including total vessel length per retinal volume, radius, and thickness. RESULTS: In the non-diabetic cohort retinal permeability remained constant over the 28-day study period. However, in the diabetic cohort there was a significant and progressive increase in retinal permeability from days 14-28 (P < .01, P < .001, P < .0001). CONCLUSIONS: This novel imaging methodology in combination with mathematical quantification allows retinal permeability to be non-invasively and accurately measured at multiple time points in the same animal. In addition, this technique is a non-toxic, rapid, sensitive, and cost-effective alternative to the Evans blue assay.
Asunto(s)
Barrera Hematorretinal , Permeabilidad Capilar , Diabetes Mellitus Experimental , Retinopatía Diabética , Animales , Barrera Hematorretinal/metabolismo , Barrera Hematorretinal/fisiopatología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatología , Retinopatía Diabética/metabolismo , Retinopatía Diabética/fisiopatología , Masculino , RatasRESUMEN
Seasonal changes in mammalian physiology, such as those affecting reproduction, hibernation, and metabolism, are controlled by pituitary hormones released in response to annual environmental changes. In temperate zones, the primary environmental cue driving seasonal reproductive cycles is the change in day length (i.e., photoperiod), encoded by the pattern of melatonin secretion from the pineal gland. However, although reproduction relies on hypothalamic gonadotrophin-releasing hormone output, and most cells producing reproductive hormones are in the pars distalis (PD) of the pituitary, melatonin receptors are localized in the pars tuberalis (PT), a physically and functionally separate part of the gland. How melatonin in the PT controls the PD is not understood. Here we show that melatonin time-dependently acts on its receptors in the PT to alter splicing of vascular endothelial growth factor (VEGF). Outside the breeding season (BS), angiogenic VEGF-A stimulates vessel growth in the infundibulum, aiding vascular communication among the PT, PD, and brain. This also acts on VEGF receptor 2 (VEGFR2) expressed in PD prolactin-producing cells known to impair gonadotrophin secretion. In contrast, in the BS, melatonin releases antiangiogenic VEGF-Axxxb from the PT, inhibiting infundibular angiogenesis and diminishing lactotroph (LT) VEGFR2 expression, lifting reproductive axis repression in response to shorter day lengths. The time-dependent, melatonin-induced differential expression of VEGF-A isoforms culminates in alterations in gonadotroph function opposite to those of LTs, with up-regulation and down-regulation of gonadotrophin gene expression during the breeding and nonbreeding seasons, respectively. These results provide a mechanism by which melatonin can control pituitary function in a seasonal manner.
Asunto(s)
Neovascularización Fisiológica , Hipófisis/irrigación sanguínea , Hipófisis/fisiología , Ovinos/fisiología , Animales , Cruzamiento , Femenino , Gonadotrofos/metabolismo , Masculino , Melatonina/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Reproducción , Estaciones del Año , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
More than 95% of genes in the human genome are alternatively spliced to form multiple transcripts, often encoding proteins with differing or opposing function. The control of alternative splicing is now being elucidated, and with this comes the opportunity to develop modulators of alternative splicing that can control cellular function. A number of approaches have been taken to develop compounds that can experimentally, and sometimes clinically, affect splicing control, resulting in potential novel therapeutics. Here we develop the concepts that targeting alternative splicing can result in relatively specific pathway inhibitors/activators that result in dampening down of physiologic or pathologic processes, from changes in muscle physiology to altering angiogenesis or pain. The targets and pharmacology of some of the current inhibitors/activators of alternative splicing are demonstrated and future directions discussed.
Asunto(s)
Empalme Alternativo/efectos de los fármacos , Descubrimiento de Drogas/métodos , Terapia Molecular Dirigida , ARN/metabolismo , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , ARN/genética , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
The Notch ligand delta-like ligand 4 (Dll4), upregulated by VEGF, is a key regulator of vessel morphogenesis and function, controlling tip and stalk cell selection during sprouting angiogenesis. Inhibition of Dll4 results in hypersprouting, nonfunctional, poorly perfused vessels, suggesting a role for Dll4 in the formation of mature, reactive, functional vessels, with low permeability and able to restrict fluid and solute exchange. We tested the hypothesis that Dll4 controls transvascular fluid exchange. A recombinant protein expressing only the extracellular portion of Dll4 [soluble Dll4 (sDll4)] induced Notch signaling in endothelial cells (ECs), resulting in increased expression of vascular-endothelial cadherin, but not the tight junctional protein zonula occludens 1, at intercellular junctions. sDll4 decreased the permeability of FITC-labeled albumin across EC monolayers, and this effect was abrogated by coculture with the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. One of the known molecular effectors responsible for strengthening EC-EC contacts is PKA, so we tested the effect of modulation of PKA on the sDll4-mediated reduction of permeability. Inhibition of PKA reversed the sDll4-mediated reduction in permeability and reduced expression of the Notch target gene Hey1. Knockdown of PKA reduced sDLL4-mediated vascular-endothelial cadherin junctional expression. sDll4 also caused a significant decrease in the hydraulic conductivity of rat mesenteric microvessels in vivo. This reduction was abolished upon coperfusion with the PKA inhibitor H89 dihydrochloride. These results indicate that Dll4 signaling through Notch activation acts through a cAMP/PKA pathway upon intercellular adherens junctions, but not tight junctions, to regulate endothelial barrier function. NEW & NOTEWORTHY Notch signaling reduces vascular permeability through stimulation of cAMP-dependent protein kinase A.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/farmacología , Proteínas de Unión al Calcio/farmacología , Permeabilidad Capilar/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Mesenterio/irrigación sanguínea , Receptores Notch/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacos , Uniones Adherentes/efectos de los fármacos , Uniones Adherentes/enzimología , Animales , Antígenos CD/metabolismo , Cadherinas/metabolismo , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Masculino , Inhibidores de Proteínas Quinasas/farmacología , Ratas Wistar , Vénulas/efectos de los fármacos , Vénulas/enzimologíaRESUMEN
INTRODUCTION: Arteriolargenesis can be induced by concomitant stimulation of nitric Oxide (NO)-Angiopoietin receptor (Tie)-Vascular Endothelial Growth Factor (VEGF) signaling in the rat mesentery angiogenesis assay. We hypothesized that the same combination of exogenously added growth factors would also have a positive impact on arteriolargenesis and, consequently, the recovery of blood flow in a model of unilateral hindlimb ischemia. RESULTS AND METHODS: NO-Tie mice had faster blood flow recovery compared to control mice, as assessed by laser speckle imaging. There was no change in capillary density within the ischemic muscles, but arteriole density was higher in NO-Tie mice. Given the previously documented beneficial effect of VEGF signaling, we tested whether NO-Tie-VEGF mice would show further improvement. Surprisingly, these mice recovered no differently from control, arteriole density was similar and capillary density was lower. Dll4 is a driver of arterial specification, so we hypothesized that Notch1 expression would be involved in arteriolargenesis. There was a significant upregulation of Notch1 transcripts in NO-Tie-VEGF compared with NO-Tie mice. Using soluble Dll4 (sDll4), we stimulated Notch signaling in the ischemic muscles of mice. NO-Tie-sDll4 mice had significantly increased capillary and arteriole densities, but impaired blood flow recovery. CONCLUSION: These results suggest that Dll4 activation early on in revascularization can lead to unproductive angiogenesis and arteriolargenesis, despite increased vascular densities. These results suggest spatial and temporal balance of growth factors needs to be perfected for ideal functional and anatomical revascularisation.
Asunto(s)
Angiopoyetinas/metabolismo , Isquemia , Músculo Esquelético , Neovascularización Fisiológica , Óxido Nítrico/metabolismo , Receptor Notch1/metabolismo , Receptores TIE/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Arteriolas/metabolismo , Arteriolas/patología , Células CHO , Proteínas de Unión al Calcio/metabolismo , Capilares/metabolismo , Capilares/patología , Cricetulus , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Isquemia/metabolismo , Isquemia/patología , Ratones , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The abundant, nuclear-retained, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been associated with a poorly differentiated and aggressive phenotype of mammary carcinomas. This long non-coding RNA (lncRNA) localizes to nuclear speckles, where it interacts with a subset of splicing factors and modulates their activity. In this study, we demonstrate that oncogenic splicing factor SRSF1 bridges MALAT1 to mutant p53 and ID4 proteins in breast cancer cells. Mutant p53 and ID4 delocalize MALAT1 from nuclear speckles and favor its association with chromatin. This enables aberrant recruitment of MALAT1 on VEGFA pre-mRNA and modulation of VEGFA isoforms expression. Interestingly, VEGFA-dependent expression signatures associate with ID4 expression specifically in basal-like breast cancers carrying TP53 mutations. Our results highlight a key role for MALAT1 in control of VEGFA isoforms expression in breast cancer cells expressing gain-of-function mutant p53 and ID4 proteins.
Asunto(s)
Neoplasias de la Mama/fisiopatología , Proteínas Inhibidoras de la Diferenciación/metabolismo , ARN Largo no Codificante/genética , Proteína p53 Supresora de Tumor/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Neoplasias de la Mama/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Inhibidoras de la Diferenciación/genética , Mutación , Neovascularización Patológica , Isoformas de Proteínas/metabolismo , Empalme del ARN , Factores de Empalme Serina-Arginina/genética , Proteína p53 Supresora de Tumor/genética , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
Objective- The NTs (neurotrophins), BDNF (brain-derived neurotrophic factor) and NT-3 promote vascular development and angiogenesis. This study investigated the contribution of endogenous NTs in embryonic stem cell (ESC) vascular differentiation and the potential of exogenous BDNF to improve the process of ESC differentiation to endothelial cells (ECs). Approach and Results- Mouse ESCs were differentiated into vascular cells using a 2-dimensional embryoid body (EB) model. Supplementation of either BDNF or NT-3 increased EC progenitors' abundance at day 7 and enlarged the peripheral vascular plexus with ECs and SM22α+ (smooth muscle 22 alpha-positive) smooth muscle cells by day 13. Conversely, inhibition of either BDNF or NT-3 receptor signaling reduced ECs, without affecting smooth muscle cells spread. This suggests that during vascular development, endogenous NTs are especially relevant for endothelial differentiation. At mechanistic level, we have identified that BDNF-driven ESC-endothelial differentiation is mediated by a pathway encompassing the transcriptional repressor EZH2 (enhancer of zeste homolog 2), microRNA-214 (miR-214), and eNOS (endothelial nitric oxide synthase). It was known that eNOS, which is needed for endothelial differentiation, can be transcriptionally repressed by EZH2. In turn, miR-214 targets EZH2 for inhibition. We newly found that in ESC-ECs, BDNF increases miR-214 expression, reduces EZH2 occupancy of the eNOS promoter, and increases eNOS expression. Moreover, we found that NRP-1 (neuropilin 1), KDR (kinase insert domain receptor), and pCas130 (p130 Crk-associated substrate kinase), which reportedly induce definitive endothelial differentiation of pluripotent cells, were increased in BDNF-conditioned ESC-EC. Mechanistically, miR-214 mediated the BDNF-induced expressional changes, contributing to BDNF-driven endothelial differentiation. Finally, BDNF-conditioned ESC-ECs promoted angiogenesis in vitro and in vivo. Conclusions- BDNF promotes ESC-endothelial differentiation acting via miR-214.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/fisiología , Diferenciación Celular , Células Madre Embrionarias/fisiología , Células Endoteliales/fisiología , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , MicroARNs/metabolismo , Neovascularización Fisiológica , Óxido Nítrico Sintasa de Tipo III/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proteína Sustrato Asociada a CrK/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Inmunofilinas/metabolismo , Ratones , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Factores de Crecimiento Nervioso/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
To investigate human glomerular structure under conditions of physiological perfusion, we have analyzed fresh and perfusion-fixed normal human glomeruli at physiological hydrostatic and oncotic pressures using serial resin section reconstruction, confocal, multiphoton, and electron microscope imaging. Afferent and efferent arterioles (21.5 ± 1.2 µm and 15.9 ± 1.2 µm diameter), recognized from vascular origins, lead into previously undescribed wider regions (43.2 ± 2.8 µm and 38.4 ± 4.9 µm diameter) we have termed vascular chambers (VCs) embedded in the mesangium of the vascular pole. Afferent VC (AVC) volume was 1.6-fold greater than efferent VC (EVC) volume. From the AVC, long nonbranching high-capacity conduit vessels ( n = 7) (Con; 15.9 ± 0.7 µm diameter) led to the glomerular edge, where branching was more frequent. Conduit vessels have fewer podocytes than filtration capillaries. VCs were confirmed in fixed and unfixed specimens with a layer of banded collagen identified in AVC walls by multiphoton and electron microscopy. Thirteen highly branched efferent first-order vessels (E1; 9.9 ± 0.4 µm diameter) converge on the EVC, draining into the efferent arteriole (15.9 ± 1.2 µm diameter). Banded collagen was scarce around EVCs. This previously undescribed branching topology does not conform to the branching of minimum energy expenditure (Murray's law), suggesting that even distribution of pressure/flow to the filtration capillaries is more important than maintaining the minimum work required for blood flow. We propose that AVCs act as plenum manifolds possibly aided by vortical flow in distributing and balancing blood flow/pressure to conduit vessels supplying glomerular lobules. These major adaptations to glomerular capillary structure could regulate hemodynamic pressure and flow in human glomerular capillaries.
Asunto(s)
Hemodinámica , Glomérulos Renales/irrigación sanguínea , Microcirculación , Microvasos/fisiología , Circulación Renal , Humanos , Presión Hidrostática , Microscopía Confocal , Microscopía Electrónica de Transmisión , Microscopía de Fluorescencia por Excitación Multifotónica , Microvasos/ultraestructura , Modelos Biológicos , Podocitos/fisiología , Fijación del TejidoRESUMEN
The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.
Asunto(s)
Bioensayo/métodos , Neoplasias , Neovascularización Patológica , Animales , Bioensayo/instrumentación , Guías como Asunto , Humanos , Ratones , Neoplasias/irrigación sanguínea , Neoplasias/metabolismo , Neoplasias/patología , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patologíaRESUMEN
Dysregulation of VEGF-A bioavailability has been implicated in the development of lung injury/fibrosis, exemplified by Idiopathic Pulmonary Fibrosis (IPF). VEGF-A is a target of the hypoxic response via its translational regulation by HIF-1α. The role of hypoxia and hyperoxia in the development and progression of IPF has not been explored. In normal lung (NF) and IPF-derived fibroblasts (FF) VEGF-Axxxa protein expression was upregulated by hypoxia, mediated through activation of VEGF-Axxxa gene transcription. VEGF-A receptors and co-receptors were differentially expressed by hypoxia and hyperoxia. Our data supports a potential role for hypoxia, hyperoxia and VEGF-Axxxa isoforms as drivers of fibrogenesis.
Asunto(s)
Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Aerobiosis/fisiología , Hipoxia de la Célula/fisiología , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/patología , Expresión Génica , Humanos , Fibrosis Pulmonar Idiopática/patología , Pulmón/patología , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
RATIONALE: Fibrosis after lung injury is related to poor outcome, and idiopathic pulmonary fibrosis (IPF) can be regarded as an exemplar. Vascular endothelial growth factor (VEGF)-A has been implicated in this context, but there are conflicting reports as to whether it is a contributory or protective factor. Differential splicing of the VEGF-A gene produces multiple functional isoforms including VEGF-A165a and VEGF-A165b, a member of the inhibitory family. To date there is no clear information on the role of VEGF-A in IPF. OBJECTIVES: To establish VEGF-A isoform expression and functional effects in IPF. METHODS: We used tissue sections, plasma, and lung fibroblasts from patients with IPF and control subjects. In a bleomycin-induced lung fibrosis model we used wild-type MMTV mice and a triple transgenic mouse SPC-rtTA+/-TetoCre+/-LoxP-VEGF-A+/+ to conditionally induce VEGF-A isoform deletion specifically in the alveolar type II (ATII) cells of adult mice. MEASUREMENTS AND MAIN RESULTS: IPF and normal lung fibroblasts differentially expressed and responded to VEGF-A165a and VEGF-A165b in terms of proliferation and matrix expression. Increased VEGF-A165b was detected in plasma of progressing patients with IPF. In a mouse model of pulmonary fibrosis, ATII-specific deficiency of VEGF-A or constitutive overexpression of VEGF-A165b inhibited the development of pulmonary fibrosis, as did treatment with intraperitoneal delivery of VEGF-A165b to wild-type mice. CONCLUSIONS: These results indicate that changes in the bioavailability of VEGF-A sourced from ATII cells, namely the ratio of VEGF-Axxxa to VEGF-Axxxb, are critical in development of pulmonary fibrosis and may be a paradigm for the regulation of tissue repair.
Asunto(s)
Expresión Génica/genética , Fibrosis Pulmonar/genética , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Modelos Animales de Enfermedad , Humanos , Pulmón/fisiopatología , Ratones , Ratones Endogámicos C57BL , Isoformas de Proteínas , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/fisiopatología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
KEY POINTS: Combining nitric oxide (NO)-mediated increased blood flow with angiopoietin-1-Tie2 receptor signalling induces arteriolargenesis - the formation of arterioles from capillaries - in a model of physiological angiogenesis. This NO-Tie-mediated arteriolargenesis requires endogenous vascular endothelial growth factor (VEGF) signalling. Inhibition of VEGF signalling increases pericyte coverage in microvessels. Together these findings indicate that generation of functional neovasculature requires close titration of NO-Tie2 signalling and localized VEGF induction, suggesting that the use of exogenous VEGF expression as a therapeutic for neovascularization may not be successful. ABSTRACT: Signalling through vascular endothelial growth factor (VEGF) receptors and the tyrosine kinase with IgG and EGF domains-2 (Tie2) receptor by angiopoietins is required in combination with blood flow for the formation of a functional vascular network. We tested the hypothesis that VEGF and angiopoietin-1 (Ang1) contribute differentially to neovascularization induced by nitric oxide (NO)-mediated vasodilatation, by comparing the phenotype of new microvessels in the mesentery during induction of vascular remodelling by over-expression of endothelial nitric oxide synthase in the fat pad of the adult rat mesentery during inhibition of angiopoietin signalling with soluble Tie2 (sTie2) and VEGF signalling with soluble Fms-like tyrosine kinase receptor-1 (sFlt1). We found that NO-mediated angiogenesis was blocked by inhibition of VEGF with sFlt1 (from 881 ± 98% increase in functional vessel area to 279 ± 72%) and by inhibition of angiopoietin with sTie2 (to 337 ± 67%). Exogenous angiopoietin-1 was required to induce arteriolargenesis (8.6 ± 1.3% of vessels with recruitment of vascular smooth muscle cells; VSMCs) in the presence of enhanced flow. sTie2 and sFlt1 both inhibited VSMC recruitment (both 0%), and VEGF inhibition increased pericyte recruitment to newly formed vessels (from 27 ± 2 to 54 ± 3% pericyte ensheathment). We demonstrate that a fine balance of VEGF and angiopoietin signalling is required for the formation of a functional vascular network. Endogenous VEGF signalling prevents excess neovessel pericyte coverage, and is required for VSMC recruitment during increased nitric oxide-mediated vasodilatation and angiopoietin signalling (NO-Tie-mediated arteriogenesis). Therapeutic vascular remodelling paradigms may therefore require treatments that modulate blood flow to utilize endogenous VEGF, in combination with exogenous Ang1, for effective neovascularization.
Asunto(s)
Angiopoyetina 1/fisiología , Neovascularización Fisiológica/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Masculino , Mesenterio/irrigación sanguínea , Mesenterio/fisiología , Ratas Wistar , Receptor TIE-2/fisiología , Flujo Sanguíneo Regional , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 1 de Factores de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
KEY POINTS: Progressive depletion of all vascular endothelial growth factor A (VEGF-A) splice isoforms from the kidney results in proteinuria and increased glomerular water permeability, which are both rescued by over-expression of VEGF-A165 b only. VEGF-A165 b rescues the increase in glomerular basement membrane and podocyte slit width, as well as the decrease in sub-podocyte space coverage, produced by VEGF-A depletion. VEGF-A165 b restores the expression of platelet endothelial cell adhesion molecule in glomerular endothelial cells and glomerular capillary circumference. VEGF-A165 b has opposite effects to VEGF-A165 on the expression of genes involved in endothelial cell migration and proliferation. ABSTRACT: Chronic kidney disease is strongly associated with a decrease in the expression of vascular endothelial growth factor A (VEGF-A). However, little is known about the contribution of VEGF-A splice isoforms to kidney physiology and pathology. Previous studies suggest that the splice isoform VEGF-A165 b (resulting from alternative usage of a 3' splice site in the terminal exon) is protective for kidney function. In the present study, we show, in a quad-transgenic model, that over-expression of VEGF-A165 b alone is sufficient to rescue the increase in proteinuria, as well as glomerular water permeability, in the context of progressive depletion of all VEGF-A isoforms from the podocytes. Ultrastructural studies show that the glomerular basement membrane is thickened, podocyte slit width is increased and sub-podocyte space coverage is reduced when VEGF-A is depleted, all of which are rescued in VEGF-A165 b over-expressors. VEGF-A165 b restores the expression of platelet endothelial cell adhesion molecule-1 in glomerular endothelial cells and glomerular capillary circumference. Mechanistically, it increases VEGF receptor 2 expression both in vivo and in vitro and down-regulates genes involved in migration and proliferation of endothelial cells, otherwise up-regulated by the canonical isoform VEGF-A165 . The results of the present study indicate that manipulation of VEGF-A splice isoforms could be a novel therapeutic avenue in chronic glomerular disease.
Asunto(s)
Riñón/metabolismo , Proteinuria/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Línea Celular , Humanos , Riñón/patología , Ratones , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Podocitos/metabolismo , Podocitos/ultraestructura , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteinuria/genética , Proteinuria/patología , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/genética , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
KEY POINTS: We have developed novel techniques for paired, direct, real-time in vivo quantification of endothelial glycocalyx structure and associated microvessel permeability. Commonly used imaging and analysis techniques yield measurements of endothelial glycocalyx depth that vary by over an order of magnitude within the same vessel. The anatomical distance between maximal glycocalyx label and maximal endothelial cell plasma membrane label provides the most sensitive and reliable measure of endothelial glycocalyx depth. Sialic acid residues of the endothelial glycocalyx regulate glycocalyx structure and microvessel permeability to both water and albumin. ABSTRACT: The endothelial glycocalyx forms a continuous coat over the luminal surface of all vessels, and regulates multiple vascular functions. The contribution of individual components of the endothelial glycocalyx to one critical vascular function, microvascular permeability, remains unclear. We developed novel, real-time, paired methodologies to study the contribution of sialic acids within the endothelial glycocalyx to the structural and functional permeability properties of the same microvessel in vivo. Single perfused rat mesenteric microvessels were perfused with fluorescent endothelial cell membrane and glycocalyx labels, and imaged with confocal microscopy. A broad range of glycocalyx depth measurements (0.17-3.02 µm) were obtained with different labels, imaging techniques and analysis methods. The distance between peak cell membrane and peak glycocalyx label provided the most reliable measure of endothelial glycocalyx anatomy, correlating with paired, numerically smaller values of endothelial glycocalyx depth (0.078 ± 0.016 µm) from electron micrographs of the same portion of the same vessel. Disruption of sialic acid residues within the endothelial glycocalyx using neuraminidase perfusion decreased endothelial glycocalyx depth and increased apparent solute permeability to albumin in the same vessels in a time-dependent manner, with changes in all three true vessel wall permeability coefficients (hydraulic conductivity, reflection coefficient and diffusive solute permeability). These novel technologies expand the range of techniques that permit direct studies of the structure of the endothelial glycocalyx and dependent microvascular functions in vivo, and demonstrate that sialic acid residues within the endothelial glycocalyx are critical regulators of microvascular permeability to both water and albumin.