RESUMO
Making gender bias visible allows to fill the gaps in knowledge and understand health records and risks of women and men. The coronavirus disease 2019 (COVID-19) pandemic has shown a clear gender difference in health outcomes. The more severe symptoms and higher mortality in men as compared to women are likely due to sex and age differences in immune responses. Age-associated decline in sex steroid hormone levels may mediate proinflammatory reactions in older adults, thereby increasing their risk of adverse outcomes, whereas sex hormones and/or sex hormone receptor modulators may attenuate the inflammatory response and provide benefit to COVID-19 patients. While multiple pharmacological options including anticoagulants, glucocorticoids, antivirals, anti-inflammatory agents and traditional Chinese medicine preparations have been tested to treat COVID-19 patients with varied levels of evidence in terms of efficacy and safety, information on sex-targeted treatment strategies is currently limited. Women may have more benefit from COVID-19 vaccines than men, despite the occurrence of more frequent adverse effects, and long-term safety data with newly developed vectors are eagerly awaited. The prevalent inclusion of men in randomized clinical trials (RCTs) with subsequent extrapolation of results to women needs to be addressed, as reinforcing sex-neutral claims into COVID-19 research may insidiously lead to increased inequities in health care. The huge worldwide effort with over 3000 ongoing RCTs of pharmacological agents should focus on improving knowledge on sex, gender and age as pillars of individual variation in drug responses and enforce appropriateness.
Assuntos
Vacinas contra COVID-19/uso terapêutico , COVID-19/prevenção & controle , Equidade em Saúde/tendências , Farmacologia Clínica/tendências , Ensaios Clínicos Controlados Aleatórios como Assunto/métodos , Caracteres Sexuais , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , COVID-19/sangue , COVID-19/imunologia , Hormônios Esteroides Gonadais/antagonistas & inibidores , Hormônios Esteroides Gonadais/sangue , Humanos , Farmacologia Clínica/métodos , Medicina de Precisão/métodos , Medicina de Precisão/tendências , Tratamento Farmacológico da COVID-19RESUMO
PURPOSE: Endothelial-to-mesenchymal transition (EndMT) plays an important role in pathogenesis of a number of inflammatory diseases. Hydroxytyrosol (HT) and, particularly, its major plasma metabolite HT-3O sulfate (HT-3Os) are known olive oil antioxidant and anti-inflammatory polyphenols which exert benefits against vascular diseases by improving endothelial function. However, to date the HT-3Os role in EndMT is not well known. METHODS: To investigate the HT-3Os effects on EndMT in the inflamed endothelium, we used an in vitro model of endothelial dysfunction, challenging endothelial cells (EC), human umbilical EC (HUVEC) and human retinal EC (HREC) with Interleukin-1ß (IL-1ß), an inflammatory agent. HREC were used as a specific model to investigate HT-3Os effects on vascular retinal diseases. RESULTS: We found that IL-1ß treatment-induced EndMT phenotype in both cell models, also changing cell morphology. HT-3Os protected EC against IL-1ß effects, recovering cell morphology and phenotype. Mechanistically, HT-3Os targeting fibroblast growth factor receptor 1 FGFR1 expression and let-7 miRNA, controlled transforming growth factor beta (TGF-ß) signalling in EC, downregulating transcription factors expression (SNAI1 and ZEB2) and gene expression of late EndMT markers (FN1, VIM, NOTCH3, CNN1, MMP2 and MMP9). CONCLUSION: These results demonstrate that HT-3Os blunts pathological EndMT in inflamed EC, maintaining high let-7 miRNA expression and preventing activation of TGF-ß signalling.
Assuntos
Endotélio/efeitos dos fármacos , Endotélio/fisiopatologia , Inflamação/fisiopatologia , Mesoderma/efeitos dos fármacos , Mesoderma/fisiopatologia , Álcool Feniletílico/análogos & derivados , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Células Cultivadas , Técnicas In Vitro , Álcool Feniletílico/farmacologia , SulfatosRESUMO
Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) as gefitinib are standard treatment of non-small cell lung cancer (NSCLC), but resistance often occurs. This study demonstrates that NSCLC cells resistant to gefitinib (GR cells) displayed a significantly higher microsomal prostaglandin E synthase-1 (mPGES-1) expression and activity than parental cells. Overexpression of mPGES-1/prostaglandin E-2 (PGE-2) signaling in GR cells was associated with acquisition of mesenchymal and stem-like cell properties, nuclear EGFR translocation and tolerance to cisplatin. mPGES-1 inhibition reduced mesenchymal and stem-like properties, and nuclear EGFR translocation in GR cells. Consistently, inhibition of mPGES-1 activity enhanced sensitivity to cisplatin and responsiveness to gefitinib in GR cells. We propose the mPGES-1/PGE-2 signaling as a potential target for treating aggressive and resistant lung cancers.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos , Gefitinibe/farmacologia , Neoplasias Pulmonares/patologia , Terapia de Alvo Molecular , Prostaglandina-E Sintases/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Dinoprostona/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptores ErbB/metabolismo , Inativação Gênica , Humanos , Prostaglandina-E Sintases/deficiência , Prostaglandina-E Sintases/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Endocytosis plays a crucial role in receptor signalling. VEGFR2 (also known as KDR) and its ligand VEGFA are fundamental in neovascularisation. However, our understanding of the role of endocytosis in VEGFR2 signalling remains limited. Despite the existence of diverse internalisation routes, the only known endocytic pathway for VEGFR2 is the clathrin-mediated pathway. Here, we show that this pathway is the predominant internalisation route for VEGFR2 only in the absence of ligand. Intriguingly, VEGFA induces a new internalisation itinerary for VEGFR2, the pathway of macropinocytosis, which becomes the prevalent endocytic route for the receptor in the presence of ligand, whereas the contribution of the clathrin-mediated route becomes minor. Macropinocytic internalisation of VEGFR2, which mechanistically is mediated through the small GTPase CDC42, takes place through macropinosomes generated at ruffling areas of the membrane. Interestingly, macropinocytosis plays a crucial role in VEGFA-induced signalling, endothelial cell functions in vitro and angiogenesis in vivo, whereas clathrin-mediated endocytosis is not essential for VEGFA signalling. These findings expand our knowledge on the endocytic pathways of VEGFR2 and suggest that VEGFA-driven internalisation of VEGFR2 through macropinocytosis is essential for endothelial cell signalling and angiogenesis.
Assuntos
Neovascularização Fisiológica , Pinocitose , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Clatrina/metabolismo , Dinaminas/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/ultraestrutura , Humanos , Modelos Biológicos , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Elevated levels of bradykinin (BK) and fibroblast growth factor-2 (FGF-2) have been implicated in the pathogenesis of inflammatory and angiogenic disorders. In angiogenesis, both stimuli induce a pro-inflammatory signature in endothelial cells, activating an autocrine/paracrine amplification loop that sustains the neovascularization process. Here we investigated the contribution of the FGF-2 pathway in the BK-mediated human endothelial cell permeability and migration, and the role of the B2 receptor (B2R) of BK in this cross-talk. BK (1 µM) upregulated the FGF-2 expression and promoted the FGF-2 signaling, both in human umbilical vein endothelial cells (HUVEC) and in retinal capillary endothelial cells (HREC) by the activation of Fibroblast growth factor receptor-1 (FGFR-1) and its downstream signaling (fibroblast growth factor receptor substrate: FRSα, extracellular signalâ»regulated kinases1/2: ERK1/2, and signal transducer and activator of transcription 3: STAT3 phosphorylation). FGFR-1 phosphorylation triggered by BK was c-Src mediated and independent from FGF-2 upregulation. Either HUVEC and HREC exposed to BK showed increased permeability, disassembly of adherens and tight-junction, and increased cell migration. B2R blockade by the selective antagonist, fasitibant, significantly inhibited FGF-2/FGFR-1 signaling, and in turn, BK-mediated endothelial cell permeability and migration. Similarly, the FGFR-1 inhibitor, SU5402, and the knock-down of the receptor prevented the BK/B2R inflammatory response in endothelial cells. In conclusion, this work demonstrates the existence of a BK/B2R/FGFR-1/FGF-2 axis in endothelial cells that might be implicated in propagation of angiogenic/inflammatory responses. A B2R blockade, by abolishing the initial BK stimulus, strongly attenuated FGFR-1-driven cell permeability and migration.
Assuntos
Bradicinina/farmacologia , Células Endoteliais/citologia , Receptor B2 da Bradicinina/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismoRESUMO
The identification of components of the kallikrein-kinin system in the vitreous from patients with microvascular retinal diseases suggests that bradykinin (BK) signaling may contribute to pathogenesis of retinal vascular complications. BK receptor 2 (B2R) signaling has been implicated in both pro-inflammatory and pro-angiogenic effects promoted by BK. Here, we investigated the role of BK/B2R signaling in the retinal neovascularization in the oxygen-induced retinopathy (OIR) model. Blockade of B2R signaling by the antagonist fasitibant delayed retinal vascularization in mouse pups, indicating that the retinal endothelium is a target of the BK/B2R system. In the rabbit cornea assay, a model of pathological neoangiogenesis, the B2 agonist kallidin induced vessel sprouting and promoted cornea opacity, a sign of edema and tissue inflammation. In agreement with these results, in the OIR model, a blockade of B2R signaling significantly reduced retinal neovascularization, as determined by the area of retinal tufts, and, in the retinal vessel, it also reduced vascular endothelial growth factor and fibroblast growth factor-2 expression. All together, these findings show that B2R blockade reduces retinal neovascularization and inhibits the expression of proangiogenic and pro-inflammatory cytokines, suggesting that targeting B2R signaling may be an effective strategy for treating ischemic retinopathy.
Assuntos
Estresse Oxidativo , Receptor B2 da Bradicinina/genética , Doenças Retinianas/etiologia , Doenças Retinianas/metabolismo , Neovascularização Retiniana/genética , Animais , Bradicinina/metabolismo , Antagonistas de Receptor B2 da Bradicinina/farmacologia , Córnea/efeitos dos fármacos , Córnea/metabolismo , Córnea/patologia , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Camundongos , Modelos Biológicos , Ornitina/análogos & derivados , Ornitina/farmacologia , Coelhos , Receptor B2 da Bradicinina/metabolismo , Doenças Retinianas/patologia , Neovascularização Retiniana/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologiaRESUMO
Inflammatory prostaglandin E-2 (PGE-2) favors cancer progression in epithelial tumors characterized by persistent oncogene input. However, its effects on tumor cell stemness are poorly understood at molecular level. Here we describe two epithelial tumor cells A431 and A459, originating from human lung and skin tumors, in which epithelial growth factor (EGF) induces sequential up-regulation of mPGES-1 and iNOS enzymes, producing an inflammatory intracellular milieu. We demonstrated that concerted action of EGF, mPGES-1 and iNOS causes sharp changes in cell phenotype demonstrated by acquisition of stem-cell features and activation of the epithelial-mesenchymal transition (EMT). When primed with EGF, epithelial tumor cells transfected with mPGES-1 or iNOS to ensure steady enzyme levels display major stem-like and EMT markers, such as reduction in E-cadherin with a concomitant rise in vimentin, ALDH-1, CD133 and ALDH activity. Tumorsphere studies with these cells show increased sphere number and size, enhanced migratory and clonogenic capacity and sharp changes in EMT markers, indicating activation of this process. The concerted action of the enzymes forms a well-orchestrated cascade where expression of iNOS depends on overexpression of mPGES-1. Indeed, we show that through its downstream effectors (PGE-2, PKA, PI3K/Akt), mPGES-1 recruits non-canonical transcription factors, thus facilitating iNOS production. In conclusion, we propose that the initial event leading to tumor stem-cell activation may be a leveraged intrinsic mechanism in which all players are either inherent constituents (EGF) or highly inducible proteins (mPGES-1, iNOS) of tumor cells. We suggest that incipient tumor aggressiveness may be moderated by reducing pivotal input of mPGES-1.
Assuntos
Transição Epitelial-Mesenquimal/fisiologia , Receptores ErbB/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Prostaglandina-E Sintases/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/citologia , Humanos , Fenótipo , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Células-Tronco , Células Tumorais CultivadasRESUMO
BACKGROUND: Liposomes, used to improve the therapeutic index of new and established drugs, have advanced with the insertion of active targeting. The lectin from Lotus tetragonolobus (LTL), which binds glycans containing alpha-1,2-linked fucose, reveals surface regionalized glycoepitopes in highly proliferative cells not detectable in normally growing cells. In contrast, other lectins localize the corresponding glycoepitopes all over the cell surface. LTL also proved able to penetrate the cells by an unconventional uptake mechanism. METHODS: We used confocal laser microscopy to detect and localize LTL-positive glycoepitopes and lectin uptake in two cancer cell lines. We then constructed doxorubicin-loaded liposomes functionalized with LTL. Intracellular delivery of the drug was determined in vitro and in vivo by confocal and electron microscopy. RESULTS: We confirmed the specific localization of Lotus binding sites and the lectin uptake mechanism in the two cell lines and determined that LTL-functionalized liposomes loaded with doxorubicin greatly increased intracellular delivery of the drug, compared to unmodified doxorubicin-loaded liposomes. The LTL-Dox-L mechanism of entry and drug delivery was different to that of Dox-L and other liposomal preparations. LTL-Dox-L entered the cells one by one in tiny tubules that never fused with lysosomes. LTL-Dox-L injected in mice with melanoma specifically delivered loaded Dox to the cytoplasm of tumor cells. CONCLUSIONS: Liposome functionalization with LTL promises to broaden the therapeutic potential of liposomal doxorubicin treatment, decreasing non-specific toxicity. GENERAL SIGNIFICANCE: Doxorubicin-LTL functionalized liposomes promise to be useful in the development of new cancer chemotherapy protocols.
Assuntos
Proliferação de Células/efeitos dos fármacos , Fabaceae/metabolismo , Lectinas/administração & dosagem , Lectinas/química , Lipossomos/administração & dosagem , Lipossomos/química , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Citoplasma/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Sistemas de Liberação de Medicamentos/métodos , Epitopos/administração & dosagem , Epitopos/química , Humanos , Lisossomos/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , CamundongosAssuntos
Tratamento Farmacológico da COVID-19 , Idoso , Feminino , Humanos , Masculino , Casas de Saúde , SARS-CoV-2 , Caracteres SexuaisRESUMO
Prostaglandin E2 (PGE2), a key mediator of immunity, inflammation, and cancer, acts through 4 G-protein-coupled E-prostanoid receptors (EPs 1-4). Crosstalk between EPs and receptor tyrosine kinases also occurs. Colony-stimulating factor-1 receptor (CSF-1R) is an RTK that sustains the survival, proliferation, and motility of monocytes/macrophages, which are an essential component of innate immunity and cancer development. The aim of this study was to investigate on a possible crosstalk between EP and CSF-1R. In BAC1.2F5 and RAW264.7 murine macrophages, CSF-1 (EC50 = 18.1 and 10.2 ng/ml, respectively) and PGE2 (EC50 = 1.5 and 5.5 nM, respectively) promoted migration. PGE2 induced rapid CSF-1R phosphorylation that was dependent on Src family kinases (SFKs). CSF-1R inhibition reduced PGE2-elicited ERK1/2 phosphorylation and macrophage migration, indicating that CSF-1R plays a role in PGE2-mediated immunoregulation. EP4 appeared responsible for functional PGE2/CSF-1R crosstalk. Furthermore, PGE2 synergized with CSF-1 in inducing ERK1/2 phosphorylation and macrophage migration. ERK1/2 inhibition completely blocked migration induced by the combination CSF-1/PGE2. CSF-1/PGE2 functional interaction with respect to migration also occurred in bone marrow-derived murine macrophages (EC50 CSF-1, 6.7 ng/ml; EC50 PGE2, 16.7 nM). These results indicated that PGE2 transactivates CSF-1R and synergizes with its signaling at ERK1/2 level in promoting macrophage migration.
Assuntos
Movimento Celular/efeitos dos fármacos , Dinoprostona/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Immunoblotting , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Cardiovascular diseases as atherosclerosis are associated to an inflammatory state of the vessel wall which is accompanied by endothelial dysfunction, and adherence and activation of circulating inflammatory cells. Hydrogen sulfide, a novel cardiovascular protective gaseous mediator, has been reported to exert anti-inflammatory activity. We have recently demonstrated that the SH containing ACE inhibitor zofenoprilat, the active metabolite of zofenopril, controls the angiogenic features of vascular endothelium through H2S enzymatic production by cystathionine gamma lyase (CSE). Based on H2S donor/generator property of zofenoprilat, the objective of this study was to evaluate whether zofenoprilat exerts anti-inflammatory activity in vascular cells through its ability to increase H2S availability. Here we found that zofenoprilat, in a CSE/H2S-mediated manner, abolished all the inflammatory features induced by interlukin-1beta (IL-1ß) in human umbilical vein endothelial cells (HUVEC), especially the NF-κB/cyclooxygenase-2 (COX-2)/prostanoid biochemical pathway. The pre-incubation with zofenoprilat/CSE dependent H2S prevented IL-1ß induced paracellular hyperpermeability through the control of expression and localization of cell-cell junctional markers ZO-1 and VE-cadherin. Moreover, zofenoprilat/CSE dependent H2S reduced the expression of the endothelial markers CD40 and CD31, involved in the recruitment of circulating mononuclear cells and platelets. Interestingly, this anti-inflammatory activity was also confirmed in vascular smooth muscle cells and fibroblasts as zofenoprilat reduced, in both cell lines, proliferation, migration and COX-2 expression induced by IL-1ß, but independently from the SH moiety and H2S availability. These in vitro data document the anti-inflammatory activity of zofenoprilat on vascular cells, reinforcing the cardiovascular protective effect of this multitasking drug.
Assuntos
Anti-Inflamatórios/farmacologia , Captopril/análogos & derivados , Endotélio Vascular/efeitos dos fármacos , Sulfeto de Hidrogênio/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Antígenos CD/metabolismo , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Antígenos CD40/metabolismo , Caderinas/metabolismo , Captopril/farmacologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Cistationina gama-Liase/metabolismo , Endotélio Vascular/metabolismo , Fibroblastos/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Interleucina-1beta/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Miócitos de Músculo Liso/metabolismo , NF-kappa B/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Amyloid ß peptides (Aß1-40 and Aß1-42) cause cerebral degeneration by impairing the activity of angiogenic factors and inducing apoptosis and senescence in the endothelium. Amyloid peptides are known to induce oxidative stress. Impairment of mitochondrial aldehyde dehydrogenase 2 (ALDH2) following oxidative stress, results in accumulation of toxic aldehydes, particularly 4-hydroxynoneal (4-HNE). We sought to determine the role of mitochondrial ALDH2 in Aß-related impairment of angiogenesis. We hypothesized that by increasing the detoxification activity of ALDH2 we would reduce Aß-driven endothelial injuries and restore angiogenesis. We used a selective ALDH2 activator, Alda-1, assessing its ability to repair mitochondrial dysfunction in the endothelium. Treatment of human endothelial cells with Aß1-40 (5-50 µM) induced loss of mitochondrial membrane potential, increased cytochrome c release and ROS accumulation. These events were associated with 4-HNE accumulation and decrease in ALDH2 activity (40%), and resulted in disassembly of endothelial junctions, as evidenced by ß-catenin phosphorylation, disorganization of adherens and tight junctions, and by disruption of pseudocapillary formation. Alda-1 (10-40 µM) abolished Aß-induced 4-HNE accumulation, apoptosis and vascular leakiness, fully restoring the pro-angiogenic endothelial phenotype and responses to FGF-2. Our data document that mitochondrial ALDH2 in the endothelium is a target for the vascular effect of Aß, including loss of barrier function and angiogenesis. ALDH2 activation, by restoring mitochondrial functions in the endothelium, prevents Aß-induced dysfunction and anti-angiogenic effects. Thus, agents activating ALDH2 may reduce endothelial injuries including those occurring in cerebral amyloid angiopathy, preserving the angiogenic potential of the endothelium.
Assuntos
Aldeído Desidrogenase/metabolismo , Peptídeos beta-Amiloides/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas Mitocondriais/metabolismo , Neovascularização Fisiológica , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Aldeído Desidrogenase/genética , Aldeído-Desidrogenase Mitocondrial , Peptídeos beta-Amiloides/metabolismo , Benzamidas/farmacologia , Benzodioxóis/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Proteínas Mitocondriais/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Espécies Reativas de Oxigênio/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Prostaglandin E2 (PGE2) is regarded as the main mediator of inflammatory symptoms. In addition, it also plays an important role in tumor growth and angiogenesis. In this study, we examined the mechanism of PGE2-induced angiogenic response. We show that in the absence of proteoglycan syndecan-4 (Sdc4), PGE2-induced ERK activation is decreased significantly, as is endothelial cell migration and cord formation in a two-dimensional Matrigel assay. In vivo, PGE2-induced angiogenesis is reduced dramatically in Sdc4(-/-) mice. The mechanism was traced to Sdc4-dependent activation of protein kinase Cα (PKCα). Transduction of an Sdc4 S183E mutant (a cytoplasmic domain mutation that blocks Sdc4-dependent PKCα activation) into Sdc4(-/-) endothelial cells was not able to rescue the loss of PGE2-induced ERK activation, whereas a transduction with full-length Sdc4 resulted in full rescue. Furthermore, PGE2-induced angiogenesis was also reduced in PKCα(-/-) mice. Taken together, these results demonstrate that PGE2-induced activation of angiogenesis is mediated via syndecan-4-dependent activation of PKCα.
Assuntos
Dinoprostona/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica/fisiologia , Proteína Quinase C-alfa/metabolismo , Sindecana-4/metabolismo , Animais , Dinoprostona/genética , Ativação Enzimática/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/genética , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Knockout , Proteína Quinase C-alfa/genética , Sindecana-4/genéticaRESUMO
The mitogen-activated protein kinase (MAPK/ERK) pathway is pivotal in controlling the proliferation and survival of melanoma cells. Several mutations, including those in BRAF, exhibit an oncogenic effect leading to increased cellular proliferation. As a result, the combination therapy of a MEK inhibitor with a BRAF inhibitor demonstrated higher efficacy and lower toxicity than BRAF inhibitor alone. This combination has become the preferred standard of care for tumors driven by BRAF mutations. Aldehyde dehydrogenase 1A1 (ALDH1A1) is a known marker of stemness involved in drug resistance in several type of tumors, including melanoma. This study demonstrates that melanoma cells overexpressing ALDH1A1 displayed resistance to vemurafenib and trametinib through the activation of PI3K/AKT signaling instead of MAPK axis. Inhibition of PI3K/AKT signaling partially rescued sensitivity to the drugs. Consistently, pharmacological inhibition of ALDH1A1 activity downregulated the activation of AKT and partially recovered responsiveness to vemurafenib and trametinib. We propose ALDH1A1 as a new potential target for treating melanoma resistant to MAPK/ERK inhibitors.
Assuntos
Família Aldeído Desidrogenase 1 , Resistencia a Medicamentos Antineoplásicos , Melanoma , Células-Tronco Neoplásicas , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-akt , Retinal Desidrogenase , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Melanoma/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Linhagem Celular Tumoral , Família Aldeído Desidrogenase 1/metabolismo , Família Aldeído Desidrogenase 1/genética , Retinal Desidrogenase/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Pirimidinonas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Piridonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Vemurafenib/farmacologia , Aldeído Desidrogenase/metabolismo , Aldeído Desidrogenase/antagonistas & inibidores , Aldeído Desidrogenase/genética , Antineoplásicos/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , FenótipoRESUMO
Protein kinase C epsilon (PKCε) activation controls fibroblast growth factor-2 (FGF-2) angiogenic signaling. Here, we examined the effect of activating PKCε on FGF-2 dependent vascular growth and endothelial activation. ψεRACK, a selective PKCε agonist induces pro-angiogenic responses in endothelial cells, including formation of capillary like structures and cell growth. These effects are mediated by FGF-2 export to the cell membrane, as documented by biotinylation and immunofluorescence, and FGF-2/FGFR1 signaling activation, as attested by ERK1/2-STAT-3 phosphorylation and de novo FGF-2 synthesis. Similarly, vascular endothelial growth factor (VEGF) activates PKCε in endothelial cells, and promotes FGF-2 export and FGF-2/FGFR1 signaling activation. ψεRACK fails to elicit responses in FGF-2(-/-) endothelial cells, and in cells pretreated with methylamine (MeNH2), an exocytosis inhibitor, indicating that both intracellular FGF-2 and its export toward the membrane are required for the ψεRACK activity. In vivo ψεRACK does not induce angiogenesis in the rabbit cornea. However, ψεRACK promotes VEGF angiogenic responses, an effect sustained by endothelial FGF-2 release and synthesis, since anti-FGF-2 antibody strongly attenuates VEGF responses. The results demonstrate that PKCε stimulation promotes angiogenesis and modulates VEGF activity, by inducing FGF-2 release and autocrine signaling.
Assuntos
Células Endoteliais/metabolismo , Exocitose/fisiologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteína Quinase C-épsilon/metabolismo , Animais , Bovinos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Oligopeptídeos/farmacologia , Transporte Proteico , Coelhos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
OBJECTIVE: Endothelium-dependent vasodilation of coronary arterioles is impaired in obese rats and may be improved by a LCD. The aim of this study is to elucidate the mechanism by which this improvement occurs. METHODS: We used four groups of male Zucker rats: lean and obese on either SD or LCD. Coronary arterioles were cannulated and pressurized for diameter measurements during administration of acetylcholine or sodium nitroprusside or during flow. Real-time PCR was performed to quantify mRNA expression of CuZnSOD and catalase. RESULTS: The LCD significantly increased endothelium-dependent dilation in the obese rats. l-NAME and indomethacin reduced responses to flow and acetylcholine in the lean rats without any effect on the obese on either diet. In contrast, TEA and catalase blocked flow-dependent and acetylcholine-induced dilation in the obese on either diet, while no effect was observed on the lean. The LCD in the obese significantly up-regulated catalase mRNA expression and slightly increased CuZnSOD mRNA levels. CONCLUSIONS: A LCD improves endothelium-dependent vasodilation of coronary arterioles in obese rats through the production of H2 O2 which acts as a hyperpolarizing factor, independent of nitric oxide and PGI2 .
Assuntos
Circulação Coronária/efeitos dos fármacos , Carboidratos da Dieta/farmacologia , Endotélio Vascular , Peróxido de Hidrogênio/metabolismo , Obesidade , Vasodilatação/efeitos dos fármacos , Acetilcolina/farmacologia , Animais , Arteríolas/metabolismo , Arteríolas/parasitologia , Arteríolas/patologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Inibidores Enzimáticos/farmacologia , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Nitroprussiato/farmacologia , Obesidade/dietoterapia , Obesidade/metabolismo , Obesidade/patologia , Obesidade/fisiopatologia , Ratos , Ratos Zucker , Vasodilatadores/farmacologiaRESUMO
In this study we investigated the role of CB1 receptor signaling in angiogenesis and the therapeutic exploitation of CB1 inactivation as an antiangiogenic strategy. We started from the observation that CB1 receptor expression is induced during angiogenesis and that the endocannabinoid anandamide stimulated bFGF-induced angiogenesis in the nanomolar physiologic range. To define the functional involvement of CB1 receptor signaling during angiogenesis, 2 different strategies have been carried out: siRNA-mediated knockdown and pharmacologic antagonism of CB1 receptors. CB1 receptors inactivation resulted in the inhibition of bFGF-induced endothelial proliferation, migration, and capillary-like tube formation, through prosurvival and migratory pathways involving ERK, Akt, FAK, JNK, Rho, and MMP-2. To corroborate the potential therapeutic exploitation of CB1 blockade as an antiangiogenic strategy, we performed in vivo assays founding that CB1 blockade was able to inhibit bFGF-induced neovascular growth in the rabbit cornea assay. A relevant finding was the ability to reduce ocular pathologic neo-vascularization in mouse oxygen-induced retinopathy. These results demonstrate that CB1 signaling participates to the proliferative response elicited by proangiogenic growth factors in angiogenesis and that for this reason CB1 receptor could represent a novel target for the treatment of diseases where excessive neoangiogenesis is the underlying pathology.
Assuntos
Neovascularização Fisiológica , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/deficiência , Animais , Sequência de Bases , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Córnea/irrigação sanguínea , Córnea/efeitos dos fármacos , Modelos Animais de Doenças , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Recém-Nascido , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , RNA Interferente Pequeno/genética , Coelhos , Receptor CB1 de Canabinoide/genética , Retinopatia da Prematuridade/tratamento farmacológico , Transdução de Sinais , Quinases Associadas a rho/metabolismoRESUMO
Pediatric and adult cancer patients, following the use of the antitumor drug Doxorubicin develop cardiotoxicity. Pharmacological protection of microvascular endothelium might produce a double benefit: (i) reduction of myocardial toxicity (the primary target of Doxorubicin action) and (ii) maintenance of the vascular functionality for the adequate delivery of chemotherapeutics to tumor cells. This study was aimed to evaluate the mechanisms responsible of the protective effects of the angiotensin converting enzyme inhibitor (ACEI) Zofenoprilat against the toxic effects exerted by Doxorubicin on coronary microvascular endothelium. We found that exposure of endothelial cells to Doxorubicin (0.1-1µM range) impaired cell survival by promoting their apoptosis. ERK1/2 related p53 activation, but not reactive oxygen species, was responsible for Doxorubicin induced caspase-3 cleavage. P53 mediated-apoptosis and impairment of survival were reverted by treatment with Zofenoprilat. The previously described PI-3K/eNOS/endogenous fibroblast growth factor signaling was not involved in the protective effect, which, instead, could be ascribed to cystathionine gamma lyase dependent availability of H2S from Zofenoprilat. Furthermore, considering the tumor environment, the treatment of endothelial/tumor co-cultures with Zofenoprilat did not affect the antitumor efficacy of Doxorubicin. In conclusion the ACEI Zofenoprilat exerts a protective effect on Doxorubicin induced endothelial damage, without affecting its antitumor efficacy. Thus, sulfhydryl containing ACEI may be a useful therapy for Doxorubicin-induced cardiotoxicity.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Antibióticos Antineoplásicos/toxicidade , Captopril/análogos & derivados , Vasos Coronários/efeitos dos fármacos , Doxorrubicina/toxicidade , Endotélio Vascular/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Captopril/farmacologia , Bovinos , Linhagem Celular , Linhagem Celular Tumoral , Vasos Coronários/citologia , Endotélio , Endotélio Vascular/patologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias/tratamento farmacológico , Proteína Supressora de Tumor p53/metabolismoRESUMO
Global repositories of postmarketing safety reports improve understanding of real-life drug toxicities, often not observed in clinical trials. The aim of this scoping review was to map the evidence from spontaneous reporting systems studies (SRSs) of antiangiogenic drugs (AADs) in cancer patients and highlight if the found disproportionality signals of adverse events (AEs) were validated and thus mentioned in the respective Summary of product Characteristics (SmPC). This scoping review was conducted according to PRISMA guidelines for scoping reviews. A knowledge gap on the safety of AADs was found: firstly, several cardiovascular AEs were not mentioned in the SmPCs and no pharmacovigilance studies were conducted despite the well-known safety concerns about these drugs on the cardiovascular system. Second, a disproportionality signal (not validated through causality assessment) of pericardial disease was found in the literature for axitinib with no mention in SmPC of the drug. Despite the exclusion of pharmacoepidemiological studies, we believe that this scoping review, which focuses on an entire class of drugs, could be considered as a novel approach to highlight possible safety concerns of drugs and as a guide for the conduction of a target postmarketing surveillance on AADs.
RESUMO
Introduction: Italian administrative healthcare databases are frequently used for studies on real-world drug utilization. However, there is currently a lack of evidence on the accuracy of administrative data in describing the use of infusive antineoplastics. In this study, we used rituximab as a case study to investigate the validity of the regional administrative healthcare database of Tuscany (RAD) in describing the utilization of infusive antineoplastics. Methods: We identified patients aged 18 years or older who had received ≥1 rituximab administration between 2011 and 2014 in the onco-haematology ward of the University Hospital of Siena. We retrieved this information from the Hospital Pharmacy Database (HPD-UHS) and linked the person-level information to RAD. Patients who had received ≥1dispensing of rituximab, single administration episodes, and patients treated for non-Hodgkin Lymphoma (nHL) or Chronic Lymphocytic Leukemia (CLL) were identified in RAD and validated using HPD-UHS as the reference standard. We identified the indications of use using algorithms based on diagnostic codes (ICD9CM codes, nHL=200*, 202*; CLL=204.1). We tested 22 algorithms of different complexity for each indication of use and calculated sensitivity and positive predictive value (PPV), with 95% confidence intervals (95%CI), as measures of validity. Results: According to HPD-UHS, 307 patients received rituximab for nHL (N=174), CLL (N=21), or other unspecified indications (N=112) in the onco-haematology ward of the University Hospital of Siena. We identified 295 rituximab users in RAD (sensitivity=96.1%), but PPV could not be assessed due to missing information in RAD on dispensing hospital wards. We identified individual rituximab administration episodes with sensitivity=78.6% [95%CI: 76.4-80.6] and PPV=87.6% [95%CI: 86.1-89.2]. Sensitivity of algorithms tested for identifying nHL and CLL ranged from 87.7% to 91.9% for nHL and from 52.4% to 82.7% for CLL. PPV ranged from 64.7% to 66.1% for nHL and from 32.4% to 37.5% for CLL. Discussion: Our findings suggest that RAD is a very sensitive source of information for identifying patients who received rituximab for onco-haematological indications. Single administration episodes were identified with good-to-high accuracy. Patients receiving rituximab for nHL were identified with high sensitivity and acceptable PPV, while the validity for CLL was suboptimal.