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RATIONALE: Intercellular tight junctions are crucial for correct regulation of the endothelial barrier. Their composition and integrity are affected in pathological contexts, such as inflammation and tumor growth. JAM-A (junctional adhesion molecule A) is a transmembrane component of tight junctions with a role in maintenance of endothelial barrier function, although how this is accomplished remains elusive. OBJECTIVE: We aimed to understand the molecular mechanisms through which JAM-A expression regulates tight junction organization to control endothelial permeability, with potential implications under pathological conditions. METHODS AND RESULTS: Genetic deletion of JAM-A in mice significantly increased vascular permeability. This was associated with significantly decreased expression of claudin-5 in the vasculature of various tissues, including brain and lung. We observed that C/EBP-α (CCAAT/enhancer-binding protein-α) can act as a transcription factor to trigger the expression of claudin-5 downstream of JAM-A, to thus enhance vascular barrier function. Accordingly, gain-of-function for C/EBP-α increased claudin-5 expression and decreased endothelial permeability, as measured by the passage of fluorescein isothiocyanate (FITC)-dextran through endothelial monolayers. Conversely, C/EBP-α loss-of-function showed the opposite effects of decreased claudin-5 levels and increased endothelial permeability. Mechanistically, JAM-A promoted C/EBP-α expression through suppression of ß-catenin transcriptional activity, and also through activation of EPAC (exchange protein directly activated by cAMP). C/EBP-α then directly binds the promoter of claudin-5 to thereby promote its transcription. Finally, JAM-A-C/EBP-α-mediated regulation of claudin-5 was lost in blood vessels from tissue biopsies from patients with glioblastoma and ovarian cancer. CONCLUSIONS: We describe here a novel role for the transcription factor C/EBP-α that is positively modulated by JAM-A, a component of tight junctions that acts through EPAC to up-regulate the expression of claudin-5, to thus decrease endothelial permeability. Overall, these data unravel a regulatory molecular pathway through which tight junctions limit vascular permeability. This will help in the identification of further therapeutic targets for diseases associated with endothelial barrier dysfunction. Graphic Abstract: An graphic abstract is available for this article.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Permeabilidade Capilar , Moléculas de Adesão Celular/metabolismo , Claudina-5/metabolismo , Células Endoteliais/metabolismo , Receptores de Superfície Celular/metabolismo , Junções Íntimas/metabolismo , Adulto , Idoso , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proteínas Estimuladoras de Ligação a CCAAT/genética , Moléculas de Adesão Celular/genética , Linhagem Celular , Claudina-5/genética , Feminino , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Neovascularização Patológica , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Receptores de Superfície Celular/genética , Transdução de Sinais , Junções Íntimas/genética , Regulação para CimaRESUMO
We investigated the clinical significance of a vascular growth pattern of hepatocellular carcinoma (HCC), the vessels that encapsulate tumor clusters (VETC), previously linked to HCC metastatic dissemination. VETC was assessed in a large multi-institutional cohort of 541 resected HCCs from Italy, Korea and Japan, and matched against a full spectrum of clinical and pathological variables. The VETC phenotype (defined as ≥ 55% tumor area by CD34 immunostaining) was easily reproducible and reliably detectable in whole sections and small-sized tissues of tissue microarray. VETC HCCs represented 18.9% of the whole series, the lowest proportion occurring in the cohort with smallest tumors (8.7%, Japanese series). VETC was significantly associated with several clinical and pathological features such as high alfa-fetoprotein (AFP) level, tumor size greater than 5 cm, poor differentiation, macrotrabecular pattern, less compact pattern, less inflammatory infiltrates, and frequent microvascular invasion. VETC was associated with early recurrence (hazard ratio [HR]: 1.52 [1.06-2.19], P = 0.023), disease-free survival (HR: 1.66 [1.21-2.27], P = 0.002), and overall survival (HR: 2.26 [1.37-3.72], P = 0.001) at multivariable analysis. VETC affected the survival in HCC patients stratified for etiology (hepatitis C virus/hepatitis B virus), vascular invasion, and specific molecular phenotypes (ß-catenin/GS+). This distinct vascular pattern was enriched in the recently reported macrotrabecular massive HCC subtype, which was seen in 7.8% (42 of 541) of patients and associated with high AFP levels and poor differentiation. Conclusion: The VETC pattern was found to be easily detectable in a consistent fraction of HCC and a powerful pathological finding affecting survival. This study suggests that the heterogeneous pattern of angiogenesis is involved in HCC behavior.
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Carcinoma Hepatocelular/irrigação sanguínea , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/irrigação sanguínea , Neoplasias Hepáticas/patologia , Neovascularização Patológica/patologia , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos RetrospectivosRESUMO
Cerebral cavernous malformation (CCM) is a vascular dysplasia, mainly localized within the brain and affecting up to 0.5% of the human population. CCM lesions are formed by enlarged and irregular blood vessels that often result in cerebral haemorrhages. CCM is caused by loss-of-function mutations in one of three genes, namely CCM1 (also known as KRIT1), CCM2 (OSM) and CCM3 (PDCD10), and occurs in both sporadic and familial forms. Recent studies have investigated the cause of vascular dysplasia and fragility in CCM, but the in vivo functions of this ternary complex remain unclear. Postnatal deletion of any of the three Ccm genes in mouse endothelium results in a severe phenotype, characterized by multiple brain vascular malformations that are markedly similar to human CCM lesions. Endothelial-to-mesenchymal transition (EndMT) has been described in different pathologies, and it is defined as the acquisition of mesenchymal- and stem-cell-like characteristics by the endothelium. Here we show that endothelial-specific disruption of the Ccm1 gene in mice induces EndMT, which contributes to the development of vascular malformations. EndMT in CCM1-ablated endothelial cells is mediated by the upregulation of endogenous BMP6 that, in turn, activates the transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) signalling pathway. Inhibitors of the TGF-ß and BMP pathway prevent EndMT both in vitro and in vivo and reduce the number and size of vascular lesions in CCM1-deficient mice. Thus, increased TGF-ß and BMP signalling, and the consequent EndMT of CCM1-null endothelial cells, are crucial events in the onset and progression of CCM disease. These studies offer novel therapeutic opportunities for this severe, and so far incurable, pathology.
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Progressão da Doença , Transição Epitelial-Mesenquimal , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Animais , Proteína Morfogenética Óssea 6/antagonistas & inibidores , Proteína Morfogenética Óssea 6/metabolismo , Proteína Morfogenética Óssea 6/farmacologia , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Humanos , Proteína KRIT1 , Camundongos , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismo , Regulação para CimaRESUMO
AIMS: Nodule-in-nodule (N/N) hepatocellular carcinoma (HCC) is a convincing proof of multistep hepatocarcinogenesis. In this lesion, an inner HCC develops within an outer, more differentiated, tumour, which can be rapidly taken over by the former so that N/N HCC is rarely detected. METHODS AND RESULTS: Ten resected N/N HCCs arising in cirrhotic background and characterized: (i) as outer lesions by early (n = 3) and G1 (n = 7) HCC; (ii) as inner lesions by G1 (n = 3) and G2 (n = 7) HCC. The largest/smallest diameters of outer and inner nodules were, respectively, 20/6 mm and 16/4 mm. We investigated vascular (CD34 and endocan), hepatocellular VEGF, GS, GPC3, HSP70 and CHC) and molecular (TERT promoter and ß-catenin) changes taking place from the outer neoplastic compartment to the inner neoplastic compartment (INC). A diffuse pattern of CD34+ capillarized vessels and focal endocan immunoreactivity were major distinctive features acquired in the INC; VEGF immunoreactivity was inversely related to CD34 staining. A gain in the number of cells immunoreactive for GPC3, HSP70, and CHC, but not of GS-immunoreactive cells, also occurred in the INC. TERT promoter mutations were seen in half of the cases in both compartments, whereas ß-catenin mutations were more rarely detectable. CONCLUSIONS: Major phenotypic changes take place in the INC of N/N HCC. TERT promoter mutations take place frequently and very early, and, in contrast to ß-catenin mutations, do not appear to be acquired during N/N growth. These findings suggest that inner nodules represent a step further along the pathway of tumour progression, in contrast to earlier, simply initiated, lesions, and that complete neovascularisation predicts a change in HCC biology.
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Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Biomarcadores Tumorais/análise , Feminino , Humanos , Masculino , Mutação , Neovascularização Patológica/patologia , Fenótipo , Telomerase/genética , beta Catenina/genéticaRESUMO
Cerebral cavernous malformation (CCM) is a disease of the central nervous system causing hemorrhage-prone multiple lumen vascular malformations and very severe neurological consequences. At present, the only recommended treatment of CCM is surgical. Because surgery is often not applicable, pharmacological treatment would be highly desirable. We describe here a murine model of the disease that develops after endothelial-cell-selective ablation of the CCM3 gene. We report an early, cell-autonomous, Wnt-receptor-independent stimulation of ß-catenin transcription activity in CCM3-deficient endothelial cells both in vitro and in vivo and a triggering of a ß-catenin-driven transcription program that leads to endothelial-to-mesenchymal transition. TGF-ß/BMP signaling is then required for the progression of the disease. We also found that the anti-inflammatory drugs sulindac sulfide and sulindac sulfone, which attenuate ß-catenin transcription activity, reduce vascular malformations in endothelial CCM3-deficient mice. This study opens previously unidentified perspectives for an effective pharmacological therapy of intracranial vascular cavernomas.
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Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Hemangioma Cavernoso do Sistema Nervoso Central/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Sulindaco/análogos & derivados , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Proteínas Reguladoras de Apoptose , Neoplasias do Sistema Nervoso Central/genética , Neoplasias do Sistema Nervoso Central/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sulindaco/farmacologia , Fator de Crescimento Transformador beta/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
PURPOSE OF REVIEW: Endothelial cells dysfunctions are crucial determinants of several human diseases. We review here the most recent reports on endothelial cell defects in cerebral cavernous malformations (CCMs), particularly focusing on adherens junctions. CCM is a vascular disease that affects specifically the venous microvessels of the central nervous system and which is caused by loss-of-function mutation in any one of the three CCM genes (CCM1, 2 or 3) in endothelial cells. The phenotypic result of these mutations are focal vascular malformations that are permeable and fragile causing neurological symptoms and occasionally haemorrhagic stroke. RECENT FINDINGS: CCM is still an incurable disease, as no pharmacological treatment is available, besides surgery. The definition of the molecular alterations ensuing loss of function mutation of CCM genes is contributing to orientate the testing of targeted pharmacological tools.Several signalling pathways are altered in the three genotypes in a similar way and concur in the acquisition of mesenchymal markers in endothelial cells. However, also genotype-specific defects are reported, in particular for the CCM1 and CCM3 mutation. SUMMARY: Besides the specific CCM disease, the characterization of endothelial alterations in CCM has the potentiality to shed light on basic molecular regulations as the acquisition and maintenance of organ and vascular site specificity of endothelial cells.
Assuntos
Células Endoteliais/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/etiologia , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Junções Aderentes/metabolismo , Angiopoietinas/metabolismo , Animais , Biomarcadores , Exocitose , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Humanos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Pesquisa Translacional BiomédicaRESUMO
BACKGROUND AND PURPOSE: Cerebral cavernous malformation (CCM) is characterized by multiple lumen vascular malformations in the central nervous system that can cause neurological symptoms and brain hemorrhages. About 20% of CCM patients have an inherited form of the disease with ubiquitous loss-of-function mutation in any one of 3 genes CCM1, CCM2, and CCM3. The rest of patients develop sporadic vascular lesions histologically similar to those of the inherited form and likely mediated by a biallelic acquired mutation of CCM genes in the brain vasculature. However, the molecular phenotypic features of endothelial cells in CCM lesions in sporadic patients are still poorly described. This information is crucial for a targeted therapy. METHODS: We used immunofluorescence microscopy and immunohistochemistry to analyze the expression of endothelial-to-mesenchymal transition markers in the cavernoma of sporadic CCM patients in parallel with human familial cavernoma as a reference control. RESULTS: We report here that endothelial cells, a cell type critically involved in CCM development, undergo endothelial-to-mesenchymal transition in the lesions of sporadic patients. This switch in endothelial phenotype has been described only in genetic CCM patients and in murine models of the disease. In addition, TGF-ß/p-Smad- and ß-catenin-dependent signaling pathways seem activated in sporadic cavernomas as in familial ones. CONCLUSIONS: Our findings support the use of common therapeutic strategies for both sporadic and genetic CCM malformations.
Assuntos
Neoplasias do Sistema Nervoso Central/patologia , Endotélio Vascular/patologia , Transição Epitelial-Mesenquimal , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Adolescente , Adulto , Idoso , Neoplasias do Sistema Nervoso Central/cirurgia , Criança , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Hemangioma Cavernoso do Sistema Nervoso Central/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
VE-cadherin is an endothelial-specific transmembrane protein concentrated at cell-to-cell adherens junctions. Besides promoting cell adhesion and controlling vascular permeability, VE-cadherin transfers intracellular signals that contribute to vascular stabilization. However, the molecular mechanism by which VE-cadherin regulates vascular homoeostasis is still poorly understood. Here, we report that VE-cadherin expression and junctional clustering are required for optimal transforming growth factor-beta (TGF-beta) signalling in endothelial cells (ECs). TGF-beta antiproliferative and antimigratory responses are increased in the presence of VE-cadherin. ECs lacking VE-cadherin are less responsive to TGF-beta/ALK1- and TGF-beta/ALK5-induced Smad phosphorylation and target gene transcription. VE-cadherin coimmunoprecipitates with all the components of the TGF-beta receptor complex, TbetaRII, ALK1, ALK5 and endoglin. Clustered VE-cadherin recruits TbetaRII and may promote TGF-beta signalling by enhancing TbetaRII/TbetaRI assembly into an active receptor complex. Taken together, our data indicate that VE-cadherin is a positive and EC-specific regulator of TGF-beta signalling. This suggests that reduction or inactivation of VE-cadherin may contribute to progression of diseases where TGF-beta signalling is impaired.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Receptores de Activinas Tipo II/metabolismo , Alantoide/citologia , Alantoide/efeitos dos fármacos , Alantoide/metabolismo , Animais , Caderinas/deficiência , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Dimerização , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Células Endoteliais/citologia , Humanos , Cinética , Camundongos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Little is known about the molecular mechanisms that regulate the organization of vascular lumen. In this paper we show that lumen formation correlates with endothelial polarization. Adherens junctions (AJs) and VE-cadherin (VEC, encoded by CDH5) are required for endothelial apicobasal polarity in vitro and during embryonic development. Silencing of CDH5 gene expression leads to abrogation of endothelial polarity accompanied by strong alterations in lumenal structure. VEC co-distributes with members of the Par polarity complex (Par3 and PKCzeta) and is needed for activation of PKCzeta. CCM1 is encoded by the CCM1 gene, which is mutated in 60% of patients affected by cerebral cavernous malformation (CCM). The protein interacts with VEC and directs AJ organization and AJ association with the polarity complex, both in cell-culture models and in human CCM1 lesions. Both VEC and CCM1 control Rap1 concentration at cell-cell junctions. We propose that VEC, CCM1 and Rap1 form a signaling complex. In the absence of any of these proteins, AJs are dismantled, cell polarity is lost and vascular lumenal structure is severely altered.
Assuntos
Neoplasias Encefálicas/genética , Células Endoteliais/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neovascularização Fisiológica , Proteínas Proto-Oncogênicas/metabolismo , Junções Aderentes/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Neoplasias Encefálicas/patologia , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Polaridade Celular/genética , Células Endoteliais/patologia , Predisposição Genética para Doença , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Humanos , Proteína KRIT1 , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Complexos Multiproteicos/metabolismo , Polimorfismo Genético , Ligação Proteica/genética , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genética , Transdução de Sinais , Proteínas rap1 de Ligação ao GTP/genética , Proteínas rap1 de Ligação ao GTP/metabolismoRESUMO
Ewing sarcoma (ES) is the second most common malignant bone tumor in children and has also been described in adults with highly aggressive behavior. ES belongs to the small round blue cell tumor family and presents the distinctive translocation of FET-ETS family genes (85% with EWSR1), generating gene fusions. Extraskeletal ES mainly occurs in soft tissues; the urogenital tract is rarely affected, and ureteral localization is an exceptional event with only 4 cases described in the literature. Here we report the first Italian case of primary ES of the ureter, a 24-year-old young man with lower back pain and a narrowed left ureteral lumen on CT scan. ES of the urogenital tract is an almost unique condition with a nonspecific clinical presentation and a challenging diagnosis for pathologists. We encourage awareness of these exceptional events in the differential diagnosis of ureteral lesions in young patients.
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Renal medullary carcinoma (RMC) is a rare entity with poor prognosis bearing inactivating genomic alterations in SMARCB1/INI1 resulting in the loss of expression of INI1 and occurring in young patients with sickle cell trait or sickle cell disease. Recently, rare examples with histological characteristics of RMC have been described in older patients without hemoglobinopathies and provisionally termed "Renal cell carcinoma unclassified with medullary phenotype" (RCCU-MP). Fluorescence in situ Hybridization (FISH) can detect alterations in SMARCB1/INI1 consisting mostly in inactivating translocation of one allele and deletion of the second. To date, only seven further cases of RCCU-MP have been described in the literature. Here we report the second Italian case of RCCU-MP, a 62-year-old man presenting with persistent dull back pain and incidentally discovering a 13 cm mass in the right kidney. The nomenclature of this entity is still debated and might be updated as a variant of medullary carcinoma in the upcoming WHO classification. In the meantime, we encourage awareness of these extraordinarily rare neoplasms with poor outcomes.
RESUMO
We retrospectively investigated in women treated with fulvestrant for HR+/HER2 negative advanced breast cancer clinical, pathological and molecular features associated with long-term benefit from treatment defined as being progression-free at 18 months. Specifically, we analyzed on formalin-fixed paraffin-embedded tumor samples ESR1 and PI3KCA mutations and miRNAs profiles. 59 patients were evaluable (median age of 67 years, range 32-92). 18-month PFS rate was 27%; the lack of visceral metastases significantly predicted the likelihood of being progression-free at 18 months, while PI3KCA mutations, found in 36% of patients, were not associated with 18-month PFS. As of miRNAs, miR-549a, miR-644a, miR-16-5p were negatively while let-7c-5p was positively associated with 18-month PFS. In addition, miR-520d-3p and miR-548g-3p values were significantly lower while miR-603, miR-181a-5p and miR-199a-miR-199b-3p values were significantly higher in patients achieving 18-month PFS. In silico analysis of targets modulated by these two latter groups of miRNAs show that in patients achieving 18-month PFS the Hippo and Wnt signaling pathways were predicted to be upregulated while endocrine resistance was potentially repressed by miR-603, miR-181a-5p and miR-199a-miR-199b-3p. Our results provide additional clues on the molecular mechanisms involved in fulvestrant activity and resistance. Underlying pathways should be further elucidated and confirmed in larger cohorts.
Assuntos
Neoplasias da Mama , MicroRNAs , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Fulvestranto/farmacologia , Fulvestranto/uso terapêutico , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
In recent years, immunohistochemical protein expression was studied as a surrogate to the molecular classification of bladder cancer, although no tissue biomarkers are available for clinical use to predict survival or the response to neoadjuvant chemotherapy (CT) in UC, as the literature produced conflicting results. This retrospective study included TURB specimens harboring foci of HG pT2 muscle-invasive bladder carcinoma (MIBC) from 251 patients who subsequently underwent radical cystectomy. We performed immunohistochemical analysis on tumor samples, for relevant gene-expression-based markers for basal type (CD44, CK5/6) and luminal type (CK20 and pPARγ). Piescore, investigated in both non-muscle-invasive (NMI) and muscle-invasive (MI) components of the tumor, divided basal and luminal UC-types when at least three of the four markers were consistent with a specific phenotype, mixed types if one/two luminal and basal markers were present simultaneously, and neu-like types when all four markers investigated were negative. Eighteen selected cases were also investigated with RT-PCR to validate, and to increase the specificity of, the immunohistochemical results. We observe an immunophenotypical difference in the NMI and MI components in 96/251 UC patients (38.25%): half of tumors (44/96 cases) have a transition to basal, 36.46% (35/96 cases) to neu-like, 12.5% (12/96 cases) to mixed, and 5.2% (5/96 cases) to luminal phenotypes. Mixed tumors in the NMI component are more likely to change phenotype than other groups, particularly compared with basal tumors, which demonstrate greater stability (only 8/96 cases, p < 0.00001). The transition of luminal tumors to basal display a better OS compared with the transition toward neu-like tumors (p = 0.027). Overall, the phenotypical switch does not affect lymphovascular invasion, pT, DFS, or OS compared with non-switched cases. In the MI component, the presence of CD44 expression, irrespective of score-related phenotype, shows a protective effect in papillary-type UC (OS p = 0.008, HR 0.453, PFS p = 0.07, HR 0.599), and in UC naïve for CT (p = 0.0479). Piescore immunophenotyping reveals an intratumoral phenotypical transition between the NMI and MI components of the same tumor. The molecular change is a common event in the mixed and luminal categories, but not in basal tumors, which show better phenotypical stability. This phenomenon could partially explain the sensitivity of a subset of luminal UC to chemotherapy: good responders could be "non-real" luminal UC, which acquire nasal markers, such as CD44.
RESUMO
During heart development endocardial cells within the atrio-ventricular (AV) region undergo TGFbeta-dependent epithelial-mesenchymal transformation (EMT) and invade the underlying cardiac jelly. This process gives rise to the endocardial cushions from which AV valves and part of the septum originate. In this paper we show that in mouse embryos and in AV explants TGFbeta induction of endocardial EMT is strongly inhibited in mice deficient for endothelial beta-catenin, leading to a lack of heart cushion formation. Using a Wnt-signaling reporter mouse strain, we demonstrated in vivo and ex vivo that EMT in heart cushion is accompanied by activation of beta-catenin/TCF/Lef transcriptional activity. In cultured endothelial cells, TGFbeta2 induces alpha-smooth muscle actin (alphaSMA) expression. This process was strongly reduced in beta-catenin null cells, although TGFbeta2 induced smad phosphorylation was unchanged. These data demonstrate an involvement of beta-catenin/TCF/Lef transcriptional activity in heart cushion formation, and suggest an interaction between TGFbeta and Wnt-signaling pathways in the induction of endothelial-mesenchymal transformation.
Assuntos
Diferenciação Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Endotélio/metabolismo , Septos Cardíacos/embriologia , Mesoderma/metabolismo , Transativadores/metabolismo , Animais , Antígenos CD , Caderinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/metabolismo , Camundongos , Receptor Notch1 , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , beta CateninaRESUMO
Cerebral cavernous malformations (CCMs) are vascular malformations located within the central nervous system often resulting in cerebral hemorrhage. Pharmacological treatment is needed, since current therapy is limited to neurosurgery. Familial CCM is caused by loss-of-function mutations in any of Ccm1, Ccm2, and Ccm3 genes. CCM cavernomas are lined by endothelial cells (ECs) undergoing endothelial-to-mesenchymal transition (EndMT). This switch in phenotype is due to the activation of the transforming growth factor beta/bone morphogenetic protein (TGFß/BMP) signaling. However, the mechanism linking Ccm gene inactivation and TGFß/BMP-dependent EndMT remains undefined. Here, we report that Ccm1 ablation leads to the activation of a MEKK3-MEK5-ERK5-MEF2 signaling axis that induces a strong increase in Kruppel-like factor 4 (KLF4) in ECs in vivo. KLF4 transcriptional activity is responsible for the EndMT occurring in CCM1-null ECs. KLF4 promotes TGFß/BMP signaling through the production of BMP6. Importantly, in endothelial-specific Ccm1 and Klf4 double knockout mice, we observe a strong reduction in the development of CCM and mouse mortality. Our data unveil KLF4 as a therapeutic target for CCM.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central/genética , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Fatores de Transcrição Kruppel-Like/metabolismo , Animais , Proteína Morfogenética Óssea 6/antagonistas & inibidores , Proteína Morfogenética Óssea 6/genética , Proteína Morfogenética Óssea 6/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Progressão da Doença , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células HEK293 , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Humanos , Proteína KRIT1 , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Mutação , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Transdução de Sinais , Proteína Smad1/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
While tumor blood vessels share many characteristics with normal vasculature, they also exhibit morphological and functional aberrancies. For example, the neural adhesion molecule L1, which mediates neurite outgrowth, fasciculation, and pathfinding, is expressed on tumor vasculature. Here, using an orthotopic mouse model of pancreatic carcinoma, we evaluated L1 functionality in cancer vessels. Tumor-bearing mice specifically lacking L1 in endothelial cells or treated with anti-L1 antibodies exhibited decreased angiogenesis and improved vascular stabilization, leading to reduced tumor growth and metastasis. In line with these dramatic effects of L1 on tumor vasculature, the ectopic expression of L1 in cultured endothelial cells (ECs) promoted phenotypical and functional alterations, including proliferation, migration, tubulogenesis, enhanced vascular permeability, and endothelial-to-mesenchymal transition. L1 induced global changes in the EC transcriptome, altering several regulatory networks that underlie endothelial pathophysiology, including JAK/STAT-mediated pathways. In particular, L1 induced IL-6-mediated STAT3 phosphorylation, and inhibition of the IL-6/JAK/STAT signaling axis prevented L1-induced EC proliferation and migration. Evaluation of patient samples revealed that, compared with that in noncancerous tissue, L1 expression is specifically enhanced in blood vessels of human pancreatic carcinomas and in vessels of other tumor types. Together, these data indicate that endothelial L1 orchestrates multiple cancer vessel functions and represents a potential target for tumor vascular-specific therapies.
Assuntos
Células Endoteliais/citologia , Endotélio Vascular/patologia , Neoplasias/irrigação sanguínea , Neovascularização Patológica , Molécula L1 de Adesão de Célula Nervosa/fisiologia , Animais , Vasos Sanguíneos , Permeabilidade Capilar , Carcinoma/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Hemangioma/metabolismo , Humanos , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Metástase Neoplásica , Molécula L1 de Adesão de Célula Nervosa/genética , Neoplasias Pancreáticas/metabolismo , Permeabilidade , Fenótipo , Interferência de RNA , Receptor TIE-2/genética , Fator de Transcrição STAT3/metabolismoRESUMO
Reproducing the characteristics and the functional responses of the blood-brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize ß-catenin, or they are infected with a transcriptionally active form of ß-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation.
Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Células Endoteliais/metabolismo , Proteínas Wnt/metabolismo , Animais , Camundongos , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta Catenina/metabolismoRESUMO
Cerebral cavernous malformations (CCM) are vascular malformations of the central nervous system (CNS) that lead to cerebral hemorrhages. Familial CCM occurs as an autosomal dominant condition caused by loss-of-function mutations in one of the three CCM genes. Constitutive or tissue-specific ablation of any of the Ccm genes in mice previously established the crucial role of Ccm gene expression in endothelial cells for proper angiogenesis. However, embryonic lethality precluded the development of relevant CCM mouse models. Here, we show that endothelial-specific Ccm2 deletion at postnatal day 1 (P1) in mice results in vascular lesions mimicking human CCM lesions. Consistent with CCM1/3 involvement in the same human disease, deletion of Ccm1/3 at P1 in mice results in similar CCM lesions. The lesions are located in the cerebellum and the retina, two organs undergoing intense postnatal angiogenesis. Despite a pan-endothelial Ccm2 deletion, CCM lesions are restricted to the venous bed. Notably, the consequences of Ccm2 loss depend on the developmental timing of Ccm2 ablation. This work provides a highly penetrant and relevant CCM mouse model.