RESUMO
Despite whole-genome sequencing (WGS), many cases of single-gene disorders remain unsolved, impeding diagnosis and preventative care for people whose disease-causing variants escape detection. Since early WGS data analytic steps prioritize protein-coding sequences, to simultaneously prioritize variants in non-coding regions rich in transcribed and critical regulatory sequences, we developed GROFFFY, an analytic tool that integrates coordinates for regions with experimental evidence of functionality. Applied to WGS data from solved and unsolved hereditary hemorrhagic telangiectasia (HHT) recruits to the 100,000 Genomes Project, GROFFFY-based filtration reduced the mean number of variants/DNA from 4,867,167 to 21,486, without deleting disease-causal variants. In three unsolved cases (two related), GROFFFY identified ultra-rare deletions within the 3' untranslated region (UTR) of the tumor suppressor SMAD4, where germline loss-of-function alleles cause combined HHT and colonic polyposis (MIM: 175050). Sited >5.4 kb distal to coding DNA, the deletions did not modify or generate microRNA binding sites, but instead disrupted the sequence context of the final cleavage and polyadenylation site necessary for protein production: By iFoldRNA, an AAUAAA-adjacent 16-nucleotide deletion brought the cleavage site into inaccessible neighboring secondary structures, while a 4-nucleotide deletion unfolded the downstream RNA polymerase II roadblock. SMAD4 RNA expression differed to control-derived RNA from resting and cycloheximide-stressed peripheral blood mononuclear cells. Patterns predicted the mutational site for an unrelated HHT/polyposis-affected individual, where a complex insertion was subsequently identified. In conclusion, we describe a functional rare variant type that impacts regulatory systems based on RNA polyadenylation. Extension of coding sequence-focused gene panels is required to capture these variants.
Assuntos
Proteína Smad4 , Telangiectasia Hemorrágica Hereditária , Humanos , Sequência de Bases , DNA , Leucócitos Mononucleares/patologia , Nucleotídeos , Poliadenilação/genética , RNA , Proteína Smad4/genética , Telangiectasia Hemorrágica Hereditária/genética , Sequenciamento Completo do GenomaRESUMO
The immunomodulatory imide drug (IMiD) class, which includes the founding drug member thalidomide and later generation drugs, lenalidomide and pomalidomide, has dramatically improved the clinical treatment of specific cancers, such as multiple myeloma, and it combines potent anticancer and anti-inflammatory actions. These actions, in large part, are mediated by IMiD binding to the human protein cereblon that forms a critical component of the E3 ubiquitin ligase complex. This complex ubiquitinates and thereby regulates the levels of multiple endogenous proteins. However, IMiD-cereblon binding modifies cereblon's normal targeted protein degradation towards a new set of neosubstrates that underlies the favorable pharmacological action of classical IMiDs, but also their adverse actions-in particular, their teratogenicity. The ability of classical IMiDs to reduce the synthesis of key proinflammatory cytokines, especially TNF-α levels, makes them potentially valuable to reposition as drugs to mitigate inflammatory-associated conditions and, particularly, neurological disorders driven by an excessive neuroinflammatory element, as occurs in traumatic brain injury, Alzheimer's and Parkinson's diseases, and ischemic stroke. The teratogenic and anticancer actions of classical IMiDs are substantial liabilities for effective drugs in these disorders and can theoretically be dialed out of the drug class. We review a select series of novel IMiDs designed to avoid binding with human cereblon and/or evade degradation of downstream neosubstrates considered to underpin the adverse actions of thalidomide-like drugs. These novel non-classical IMiDs hold potential as new medications for erythema nodosum leprosum (ENL), a painful inflammatory skin condition associated with Hansen's disease for which thalidomide remains widely used, and, in particular, as a new treatment strategy for neurodegenerative disorders in which neuroinflammation is a key component.
Assuntos
Mieloma Múltiplo , Doenças Neurodegenerativas , Humanos , Talidomida/farmacologia , Talidomida/uso terapêutico , Agentes de Imunomodulação , Doenças Neuroinflamatórias , Mieloma Múltiplo/tratamento farmacológico , Ubiquitina-Proteína Ligases/metabolismo , Doenças Neurodegenerativas/tratamento farmacológicoRESUMO
This review article provides a comprehensive overview of thalidomide upper limb embryopathy including updates about its pathogenesis, a historical account of the management of the paediatric thalidomide patient, experience with management of the adult patient, as well as creating awareness about early onset age-related changes associated with limb differences. Despite its withdrawal from the market in November 1961, novel discoveries have meant thalidomide is licensed again and currently still in use to treat a variety of conditions, including inflammatory disorders and some cancers. Yet, if not used safely, thalidomide still has the potential to cause damage to the embryo. Recent work identifying thalidomide analogues that retain clinical benefits yet without the harmful effects are showing great promise. Understanding the problems thalidomide survivors face as they age can allow surgeons to support their unique healthcare issues and translate these principles of care to other congenital upper limb differences.
Assuntos
Anormalidades Múltiplas , Doenças Fetais , Adulto , Humanos , Criança , Feminino , Talidomida/efeitos adversos , Extremidade SuperiorRESUMO
Due to its antiangiogenic and anti-immunomodulatory activity, thalidomide continues to be of clinical interest despite its teratogenic actions, and efforts to synthesize safer, clinically active thalidomide analogs are continually underway. In this study, a cohort of 27 chemically diverse thalidomide analogs was evaluated for antiangiogenic activity in an ex vivo rat aorta ring assay. The protein cereblon has been identified as the target for thalidomide, and in silico pharmacophore analysis and molecular docking with a crystal structure of human cereblon were used to investigate the cereblon binding abilities of the thalidomide analogs. The results suggest that not all antiangiogenic thalidomide analogs can bind cereblon, and multiple targets and mechanisms of action may be involved.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Inibidores da Angiogênese/farmacologia , Aorta/efeitos dos fármacos , Simulação de Acoplamento Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Talidomida/análogos & derivados , Talidomida/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Inibidores da Angiogênese/química , Animais , Simulação por Computador , Humanos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Cereblon (CRBN) is a substrate recruiter element of the E3 cullin 4-RING ubiquitin ligase complex, and a binding target of immunomodulatory agents (IMiDs). CRBN is responsible for the pleiotropic effects of IMiDs, yet its function in angiogenesis and in mediating the antiangiogenic effects of IMiDs remains unclear. We investigated the role of CRBN in the angiogenic process and in propagating the antiangiogenic effects of IMiDs in vitro. siRNA-mediated CRBN knock down in human endothelial cells (HUVEC and HMVEC-L), did not affect endothelial cell proliferation, migration, or tube formation. Using CRBN-deficient mice, we further demonstrated that microvessal formation can occur independently of cereblon in the ex vivo mouse aortic ring model. The cereblon E3 ubiquitin ligase complex can recruit endothelial cell-specific factors, AGO2 (associated with angiogenesis), and SALL4 (associated with embryogenesis/angiogenesis), for ubiquitin-mediated degradation. Knockdown of CRBN caused a corresponding increase in AGO2 and SALL4 protein expression and IMiD treatment was able to rescue the siCRBN effect to increase the CRBN expression. These findings suggest one potential mechanism of action that likely involves a tightly coordinated regulation of CRBN with endothelial cell targets and highlight the need to further elucidate the mechanism(s), which could include cereblon-independent pathways, through which IMiDs exert their antiangiogenic effects.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Inibidores da Angiogênese/farmacologia , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Lenalidomida/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Interferência de RNA , Ubiquitina-Proteína Ligases/genéticaRESUMO
VGLL proteins are transcriptional co-factors that bind TEAD family transcription factors to regulate events ranging from wing development in fly, to muscle fibre composition and immune function in mice. Here, we characterise Vgll3 in skeletal muscle. We found that mouse Vgll3 was expressed at low levels in healthy muscle but that its levels increased during hypertrophy or regeneration; in humans, VGLL3 was highly expressed in tissues from patients with various muscle diseases, such as in dystrophic muscle and alveolar rhabdomyosarcoma. Interaction proteomics revealed that VGLL3 bound TEAD1, TEAD3 and TEAD4 in myoblasts and/or myotubes. However, there was no interaction with proteins from major regulatory systems such as the Hippo kinase cascade, unlike what is found for the TEAD co-factors YAP (encoded by YAP1) and TAZ (encoded by WWTR1). Vgll3 overexpression reduced the activity of the Hippo negative-feedback loop, affecting expression of muscle-regulating genes including Myf5, Pitx2 and Pitx3, and genes encoding certain Wnts and IGFBPs. VGLL3 mainly repressed gene expression, regulating similar genes to those regulated by YAP and TAZ. siRNA-mediated Vgll3 knockdown suppressed myoblast proliferation, whereas Vgll3 overexpression strongly promoted myogenic differentiation. However, skeletal muscle was overtly normal in Vgll3-null mice, presumably due to feedback signalling and/or redundancy. This work identifies VGLL3 as a transcriptional co-factor operating with the Hippo signal transduction network to control myogenesis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Muscular , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos Knockout , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Neoplasias/metabolismo , Ligação Proteica , Fatores de Transcrição de Domínio TEA , Transcriptoma/genéticaRESUMO
Thalidomide remains notorious as a result of the damage it caused to children born to mothers who used it to treat morning sickness between 1957 and 1961. The re-emergence of the drug to treat a range of conditions including erythema nodosum leprosum (a complication of leprosy) has led to a new generation of thalidomide damaged children being born in Brazil. Although thalidomide affects most of the developing tissues and organs of the body, the damage to the limbs is striking. Indeed phocomelia, the severe reduction or loss of the proximal long bones with retention of the distal hand/foot plate remains the stereotypical image of thalidomide. This review focuses on the type and range of damage thalidomide caused to the limbs, reviews current understanding of the mechanisms underlying thalidomide-induced limb malformations and outlines some of the challenges remaining in elucidating its teratogenicity.
Assuntos
Deformidades Congênitas dos Membros/induzido quimicamente , Teratogênicos/farmacologia , Talidomida/efeitos adversos , Anormalidades Induzidas por Medicamentos/história , Extremidades/embriologia , Feminino , História do Século XX , História do Século XXI , Humanos , Botões de Extremidades/embriologia , Gravidez , Teratogênese/efeitos dos fármacos , Talidomida/farmacologiaRESUMO
Vascular anomalies are common in the upper extremities, but there continues to be a relative paucity of information about them in publications dealing with surgery in the hands and upper limbs. The wide spectrum of pathology and an inconsistent use of terminology make vascular anomalies susceptible to incorrect diagnosis and as a result, to misdirected management. This article aims to provide an update on vascular anomalies relevant to the upper limbs, focusing on significant advances in pathogenesis and genetics, classification systems, diagnosis and treatment.
Assuntos
Neoplasias de Tecido Vascular/diagnóstico , Neoplasias de Tecido Vascular/terapia , Extremidade Superior/irrigação sanguínea , Malformações Vasculares/diagnóstico , Malformações Vasculares/terapia , Classe I de Fosfatidilinositol 3-Quinases/genética , Humanos , Anormalidades Musculoesqueléticas/genética , Mutação , Malformações Vasculares/classificaçãoRESUMO
Limbal epithelial stem cells (LESCs) are believed to be responsible for corneal epithelial maintenance and repair after injury, but their activity has never been properly quantified in aging or wounded eyes. In this study, labelling with thymidine analogues, 5-iodo-2'-deoxyuridine (IdU), 5-chloro-2'-deoxyuridine (CldU) and 5-ethynyl-2'-deoxyuridine (EdU), was used to estimate cell-cycle time of the corneal and limbal epithelia in wild-type eyes, comparing aging (12â¯months) and young adult (8â¯week) mice. In C57BL/6 mice, cells cycled significantly faster in the central corneal epithelium of aging eyes (3.24⯱â¯0.2â¯days) compared to 10â¯week old mice (4.97⯱â¯0.5â¯days). Long-term labelling with IdU was used to detect slow-cycling stem cells, followed by CldU or EdU labelling to quantify the proliferative dynamics of LESCs during corneal wound healing. In unwounded eyes, 4.52⯱â¯1.4% of LESCs were shown to enter S phase in a 24â¯h period and were estimated to divide every 2-3â¯weeks. Within 24â¯h of corneal injury this rose significantly to 32.8⯱â¯10.0% of stem cells indicating a seven-fold increase in activation. In contrast, no comparable increase in LESC activation was observed in aging mice after wounding. In the 24-48â¯h period after wounding in young adults, LESC activation continued to increase (86.5⯱â¯8.2% of label-retaining cells in wounded eye were in S-phase) but surprisingly, 46.0⯱â¯9.4% of LESCs were observed to reenter S-phase in the contralateral unwounded eye. These data imply an unsuspected systemic effect of corneal wounding on LESC activation suggesting that injury to one eye elicits a regenerative response in both.
Assuntos
Células Epiteliais/metabolismo , Epitélio Corneano/metabolismo , Limbo da Córnea/metabolismo , Células-Tronco/metabolismo , Animais , Humanos , CamundongosRESUMO
Persistent hyperactivity of the Hippo effector YAP in activated satellite cells is sufficient to cause embryonal rhabdomyosarcoma (ERMS) in mice. In humans, YAP is abundant and nuclear in the majority of ERMS cases, and high YAP expression is associated with poor survival. However, YAP1 is rarely mutated in human ERMS. Instead, the most common mutations in ERMS are oncogenic RAS mutations. First, to compare YAP1 S127A and KRAS G12V-driven rhabdomyosarcomas, we re-analysed gene expression microarray datasets from mouse rhabdomyosarcomas caused by these genes. This revealed that only 20% of the up or downregulated genes are identical, suggesting substantial differences in gene expression between YAP and KRAS-driven rhabdomyosarcomas. As oncogenic RAS has been linked to YAP in other types of cancer, we also tested whether KRAS G12V alone or in combination with loss of p53 and p16 activates YAP in myoblasts. We found that neither KRAS G12V alone nor KRAS G12V combined with loss of p53 and p16 activated Yap or Yap/Taz-Tead1-4 transcriptional activity in C2C12 myoblasts or U57810 cells. In conclusion, whilst oncogenic KRAS mutation might activate Yap in other cell types, we could find no evidence for this in myoblasts because the expression of KRAS G12V expression did not change Yap/Taz activity in myoblasts and there was a limited overlap in gene expression between KRAS G12V and YAP1 S127A-driven tumours.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Oncogenes , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Rabdomiossarcoma Embrionário/genética , Animais , Linhagem Celular , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Camundongos Transgênicos , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
Primodos was a hormone pregnancy test used between 1958-1978 that has been implicated with causing a range of birth defects ever since. Though Primodos is no longer used, it's components, Norethisterone acetate and Ethinyl estradiol, are used in other medications today including treatments for endometriosis and contraceptives. However, whether Primodos caused birth defects or not remains controversial, and has been little investigated. Here we used the developing zebrafish embryo, a human cell-line and mouse retinal explants to investigate the actions of the components of Primodos upon embryonic and tissue development. We show that Norethisterone acetate and Ethinyl estradiol cause embryonic damage in a dose and time responsive manner. The damage occurs rapidly after drug exposure, affecting multiple organ systems. Moreover, we found that the Norethisterone acetate and Ethinyl estradiol mixture can affect nerve outgrowth and blood vessel patterning directly and accumulates in the forming embryo for at least 24 hrs. These data demonstrate that Norethisterone acetate and Ethinyl estradiol are potentially teratogenic, depending on dose and embryonic stage of development in the zebrafish. Further work in mammalian model species are now required to build on these findings and determine if placental embryos also are affected by synthetic sex hormones and their mechanisms of action.
Assuntos
Embrião não Mamífero/efeitos dos fármacos , Etinilestradiol/toxicidade , Hormônios/química , Acetato de Noretindrona/toxicidade , Testes de Gravidez/efeitos adversos , Testes de Toxicidade , Peixe-Zebra/embriologia , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Embrião não Mamífero/citologia , Embrião não Mamífero/inervação , Desenvolvimento Embrionário/efeitos dos fármacos , Etinilestradiol/análise , Humanos , Camundongos , Sistema Nervoso/efeitos dos fármacos , Sistema Nervoso/crescimento & desenvolvimento , Acetato de Noretindrona/análise , Fatores de TempoRESUMO
A library of 15 novel and heretofore uncharacterized adamantyl and noradamantyl phthalimidines was synthesized and evaluated for neuroprotective and anti-angiogenic properties. Phthalimidine treatment in LPS-challenged cells effected reductions in levels of secreted TNF-α and nitrite relative to basal amounts. The primary SAR suggests nitration of adamantyl phthalimidines has marginal effect on TNF-α activity but promotes anti-nitrite activity; thioamide congeners retain anti-nitrite activity but are less effective reducing TNF-α. Site-specific nitration and thioamidation provided phthalimidine 24, effecting an 88.5% drop in nitrite concurrent with only a 4% drop in TNF-α. Notable anti-angiogenesis activity was observed for 20, 21 and 22.
Assuntos
Inibidores da Angiogênese/farmacologia , Desenho de Fármacos , Fármacos Neuroprotetores/farmacologia , Nitritos/antagonistas & inibidores , Ftalimidas/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Estrutura Molecular , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Nitritos/metabolismo , Ftalimidas/síntese química , Ftalimidas/química , Células RAW 264.7 , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Thalidomide notoriously caused severe birth defects, particularly to the limbs, in those exposed in utero following maternal use of the drug to treat morning sickness. How the drug caused these birth defects remains unclear. Many theories have been proposed including actions on the forming blood vessels. However, thalidomide survivors also have altered nerve patterns and the drug is known for its neurotoxic actions in adults following prolonged use. We have previously shown that CPS49, an anti-angiogenic analog of thalidomide, causes a range of limb malformations in a time-sensitive manner in chicken embryos. Here we investigated whether CPS49 also is neurotoxic and whether effects on nerve development impact upon limb development. We found that CPS49 is neurotoxic, just like thalidomide, and can cause some neuronal loss late developing chicken limbs, but only when the limb is already innervated. However, CPS49 exposure does not cause defects in limb size when added to late developing chicken limbs. In contrast, in early limb buds which are not innervated, CPS49 exposure affects limb area significantly. To investigate in more detail the role of neurotoxicity and its impact on chicken limb development we inhibited nerve innervation at a range of developmental timepoints through using ß-bungarotoxin. We found that neuronal inhibition or ablation before, during or after limb outgrowth and innervation does not result in obvious limb cartilage patterning or number changes. We conclude that while CPS49 is neurotoxic, given the late innervation of the developing limb, and that neuronal inhibition/ablation throughout limb development does not cause similar limb patterning anomalies to those seen in thalidomide survivors, nerve defects are not the primary underlying cause of the severe limb patterning defects induced by CPS49/thalidomide.
Assuntos
Inibidores da Angiogênese/toxicidade , Padronização Corporal/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Deformidades Congênitas dos Membros/induzido quimicamente , Crescimento Neuronal/efeitos dos fármacos , Teratogênicos/toxicidade , Talidomida/análogos & derivados , Animais , Bungarotoxinas/farmacologia , Embrião de Galinha , Extremidades/embriologia , Extremidades/inervação , Feminino , Botões de Extremidades/inervação , Camundongos , Camundongos Endogâmicos C57BL , Síndromes Neurotóxicas , Talidomida/toxicidadeRESUMO
Visual information is relayed from the eye to the brain via retinal ganglion cell (RGC) axons. Mice lacking NRP1 or NRP1-binding VEGF-A isoforms have defective RGC axon organisation alongside brain vascular defects. It is not known whether axonal defects are caused exclusively by defective VEGF-A signalling in RGCs or are exacerbated by abnormal vascular morphology. Targeted NRP1 ablation in RGCs with a Brn3bCre knock-in allele reduced axonal midline crossing at the optic chiasm and optic tract fasciculation. In contrast, Tie2-Cre-mediated endothelial NRP1 ablation induced axon exclusion zones in the optic tracts without impairing axon crossing. Similar defects were observed in Vegfa120/120 and Vegfa188/188 mice, which have vascular defects as a result of their expression of single VEGF-A isoforms. Ectopic midline vascularisation in endothelial Nrp1 and Vegfa188/188 mutants caused additional axonal exclusion zones within the chiasm. As in vitro and in vivo assays demonstrated that vessels do not repel axons, abnormally large or ectopically positioned vessels are likely to present physical obstacles to axon growth. We conclude that proper axonal wiring during brain development depends on the precise molecular control of neurovascular co-patterning.
Assuntos
Axônios/metabolismo , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Neuropilina-1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Padronização Corporal , Diencéfalo/embriologia , Diencéfalo/metabolismo , Células Endoteliais/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/metabolismo , Camundongos Endogâmicos C57BL , Mutação/genética , Neovascularização Fisiológica , Quiasma Óptico/embriologia , Quiasma Óptico/metabolismo , Células Ganglionares da Retina/metabolismo , Fator de Transcrição Brn-3B/metabolismo , Vias Visuais/metabolismoRESUMO
The development of new angiogenic inhibitors highlights a need for robust screening assays that adequately capture the complexity of vessel formation, and allow for the quantitative evaluation of the teratogenicity of new anti-angiogenic agents. This review discusses the use of screening assays in vertebrate embryos, specifically focusing upon chicken and zebrafish embryos, for the detection of anti-angiogenic agents.
Assuntos
Inibidores da Angiogênese/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Embrião de Mamíferos/efeitos dos fármacos , Embrião não Mamífero/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Embrião de Mamíferos/irrigação sanguínea , Embrião não Mamífero/irrigação sanguínea , Humanos , Modelos AnimaisRESUMO
Angiogenesis, the formation of new blood vessels, is essential for tumor growth, stabilization and progression. Angiogenesis inhibitors are now widely used in the clinic; however, there are relatively few published studies on the mechanism of their presumed teratogenic effects. To address this issue, we screened a variety of angiogenesis inhibitors in developing zebrafish and chicken embryo models to assess for developmental defects and potential teratogenic effects. We confirmed previous reports that sunitinib, sorafenib and TNP-470 are teratogenic and demonstrate that axitinib, pazopanib, vandetanib, and everolimus are also teratogens in these models. A dose response study identified the drugs inhibit HUVEC cell proliferation in vitro, and also target the developing blood vessels of embryos in vivo. This provides further evidence for the potential risk of fetal toxicity when using these drugs in a clinical setting, and emphasizes the importance of the development and maintenance of the vasculature in the embryo. We conclude that angiogenesis inhibitors, regardless of the molecular target, are teratogenic when exposed to chicken embryos.
Assuntos
Inibidores da Angiogênese/efeitos adversos , Teratogênese , Teratogênicos/farmacologia , Animais , Células Cultivadas , Embrião de Galinha , Relação Dose-Resposta a Droga , Humanos , Peixe-Zebra/embriologiaRESUMO
Thalidomide, a drug known for its teratogenic side-effects, is used successfully to treat a variety of clinical conditions including leprosy and multiple myeloma. Intense efforts are underway to synthesize and identify safer, clinically relevant analogs. Here, we conduct a preliminary in vivo screen of a library of new thalidomide analogs to determine which agents demonstrate activity, and describe a cohort of compounds with anti-angiogenic properties, anti-inflammatory properties and some compounds which exhibited both. The combination of the in vivo zebrafish and chicken embryo model systems allows for the accelerated discovery of new, potential therapies for cancerous and inflammatory conditions.
Assuntos
Inibidores da Angiogênese/farmacologia , Anti-Inflamatórios/farmacologia , Embrião de Galinha/efeitos dos fármacos , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Talidomida/farmacologia , Peixe-Zebra/embriologia , Anormalidades Induzidas por Medicamentos/etiologia , Inibidores da Angiogênese/toxicidade , Animais , Animais Geneticamente Modificados , Anti-Inflamatórios/toxicidade , Relação Dose-Resposta a Droga , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Neovascularização Fisiológica/efeitos dos fármacos , Infiltração de Neutrófilos/efeitos dos fármacos , Medição de Risco , Talidomida/análogos & derivados , Talidomida/toxicidade , Fluxo de Trabalho , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Thalidomide has demonstrated clinical activity in various malignancies affecting immunomodulatory and angiogenic pathways. The development of novel thalidomide analogs with improved efficacy and decreased toxicity is an ongoing research effort. We recently designed and synthesized a new class of compounds, consisting of both tetrafluorinated thalidomide analogues (Gu973 and Gu998) and tetrafluorobenzamides (Gu1029 and Gu992). In this study, we demonstrate the antiangiogenic properties of these newly synthesized compounds. We examined the specific antiangiogenic characteristics in vitro using rat aortic rings with carboxyamidotriazole as a positive control. In addition, further in vitro efficacy was evaluated using human umbilical vein endothelial cells (HUVEC) and PC3 cells treated with 5 and 10 µmol/L doses of each compound. All compounds were seen to reduce microvessel outgrowth in rat aortic rings as well as to inhibit HUVECs to a greater extent, at lower concentrations than previously tested thalidomide analogs. The antiangiogenic properties of the compounds were also examined in vivo in fli1:EGFP zebrafish embryos, where all compounds were seen to inhibit the extent of outgrowth of newly developing blood vessels. In addition, Gu1029 and Gu973 reduced the anti-inflammatory response in mpo:GFP zebrafish embryos, whereas Gu998 and Gu992 showed no difference. The compounds' antitumor effects were also explored in vivo using the human prostate cancer PC3 xenograft model. All four compounds were also screened in vivo in chicken embryos to investigate their teratogenic potential. This study establishes these novel thalidomide analogues as a promising immunomodulatory class with anticancer effects that warrant further development to characterize their mechanisms of action.
Assuntos
Inibidores da Angiogênese/farmacologia , Hidrocarbonetos Fluorados/farmacologia , Neovascularização Patológica/prevenção & controle , Talidomida/análogos & derivados , Talidomida/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Concentração Inibidora 50 , Masculino , Ratos Sprague-Dawley , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
Nearly 60 years ago thalidomide was prescribed to treat morning sickness in pregnant women. What followed was the biggest man-made medical disaster ever, where over 10,000 children were born with a range of severe and debilitating malformations. Despite this, the drug is now used successfully to treat a range of adult conditions, including multiple myeloma and complications of leprosy. Tragically, a new generation of thalidomide damaged children has been identified in Brazil. Yet, how thalidomide caused its devastating effects in the forming embryo remains unclear. However, studies in the past few years have greatly enhanced our understanding of the molecular mechanisms the drug. This review will look at the history of the drug, and the range and type of damage the drug caused, and outline the mechanisms of action the drug uses including recent molecular advances and new findings. Some of the remaining challenges facing thalidomide biologists are also discussed.
Assuntos
Anormalidades Induzidas por Medicamentos/história , Teratogênese/efeitos dos fármacos , Teratogênicos/farmacologia , Talidomida/efeitos adversos , Anormalidades Induzidas por Medicamentos/etiologia , Adulto , Animais , Feminino , História do Século XX , História do Século XXI , Humanos , Masculino , GravidezRESUMO
Thalidomide and its analog, Lenalidomide, are in current use clinically for treatment of multiple myeloma, complications of leprosy and cancers. An additional analog, Pomalidomide, has recently been licensed for treatment of multiple myeloma, and is purported to be clinically more potent than either Thalidomide or Lenalidomide. Using a combination of zebrafish and chicken embryos together with in vitro assays we have determined the relative anti-inflammatory activity of each compound. We demonstrate that in vivo embryonic assays Pomalidomide is a significantly more potent anti-inflammatory agent than either Thalidomide or Lenalidomide. We tested the effect of Pomalidomide and Lenalidomide on angiogenesis, teratogenesis, and neurite outgrowth, known detrimental effects of Thalidomide. We found that Pomalidomide, displays a high degree of cell specificity, and has no detectable teratogenic, antiangiogenic or neurotoxic effects at potent anti-inflammatory concentrations. This is in marked contrast to Thalidomide and Lenalidomide, which had detrimental effects on blood vessels, nerves, and embryonic development at anti-inflammatory concentrations. This work has implications for Pomalidomide as a treatment for conditions Thalidomide and Lenalidomide treat currently.