RESUMEN
AIMS: TP53 alterations have a significant prognostic effect in myeloid neoplasms. Our objective was to investigate the TP53 gene mutation status, p53 protein expression and their relationship in dysplasia-related myeloid neoplasms with varying levels of myeloblast counts. METHODS AND RESULTS: A total of 76 bone marrow biopsy samples with different blast counts were analysed. Total and strong (3+) p53 expression was determined. Dual immunohistochemical staining was performed to determine the cell population associated with p53 expression. NGS analysis was performed using the Accel-Amplicon Comprehensive TP53 panel. Both p53 expression and TP53 VAF showed a significant correlation with the myeloblast ratio (P < 0.0001); however, p53 expression was also present in other cell lineages. The VAF value exhibited a significant correlation with p53 expression. A high specificity (0.9800) was observed for TP53 mutation using the ≥ 10% strong (3+) p53 cut-off value, although the sensitivity (0.4231) was low. CONCLUSIONS: Strong (3+) p53 expression using a ≥ 10% cut-off value accurately predicts TP53 mutation but does not reveal the allelic state. The p53 expression is significantly influenced by myeloblast count, and histological interpretation should consider the presence of intermixed non-neoplastic marrow cells with varying physiological p53 expression.
Asunto(s)
Mutación , Síndromes Mielodisplásicos , Proteína p53 Supresora de Tumor , Humanos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/patología , Síndromes Mielodisplásicos/metabolismo , Masculino , Persona de Mediana Edad , Femenino , Adulto , Anciano , Anciano de 80 o más Años , Médula Ósea/patología , Médula Ósea/metabolismo , Adulto JovenRESUMEN
Using immunohistochemistry, we examined a large cohort of 135 ovarian tumors, made up of 96 low-grade serous carcinomas (LGSCs) and 39 serous borderline tumors (micropapillary variant, mSBT), with the aim of exploring their HER2 status (overexpression). We followed with comprehensive genomic analyses on this sample set from our previous study, which revealed HER2 mutation in 5% (4/75) of LGSC and 10% (3/29) of mSBT. No cases were evaluated as HER2-positive, but 6 LGSCs and 1 mSBT were scored as HER2 1+, and 2 LGSCs and 1 mSBT showed the so-called HER2 "ultra-low" phenotype. This could be of clinical value as a potential therapeutical target concerning emerging therapeutic treatments (antibody conjugates). However, the clinical significance of this expression still needs to be established.
RESUMEN
Expression of neuroendocrine (NE) markers in primary ovarian non-NE epithelial tumors has rarely been evaluated. The aim of our study was to evaluate the expression of the most widely used NE markers in these neoplasms and to determine any prognostic significance of NE marker expression. The cohort consisted of 551 primary ovarian tumors, including serous borderline tumors, low-grade serous carcinomas, high-grade serous carcinomas (HGSC), clear cell carcinomas, endometroid carcinomas, mucinous borderline tumors, and mucinous carcinomas. Immunohistochemical analysis was performed using antibodies against INSM1, synaptophysin, chromogranin, and CD56 on tissue microarray. Positivity for INSM1, synaptophysin, chromogranin, and CD56 was most frequently observed in mucinous tumors (48.7%, 26.0%, 41.5%, and 100%, respectively). The positivity for these NE markers was mostly restricted to nonmucinous elements distributed throughout the tumor. The mucinous borderline tumor and mucinous carcinomas groups had similar proportions of positivity (mucinous borderline tumor: 53%, mucinous carcinomas: 39%). In the other tumor types, except for HGSC, there was only focal expression (5%-10%) or negativity for NE markers. HGSC showed high CD56 expression (in 26% of cases). Survival analysis was only performed for CD56 in HGSC as this was the only group with sufficient positive cases, and it showed no prognostic significance. Except for mucinous tumors, expression of NE markers in non-NE ovarian epithelial tumors is low. CD56 expression in HGSC occurs frequently but is without diagnostic or prognostic value.
Asunto(s)
Adenocarcinoma Mucinoso , Tumores Neuroendocrinos , Neoplasias Ováricas , Femenino , Humanos , Sinaptofisina/metabolismo , Biomarcadores de Tumor/metabolismo , Cromograninas , Tumores Neuroendocrinos/patología , Neoplasias Ováricas/patología , Adenocarcinoma Mucinoso/diagnóstico , Proteínas Represoras/metabolismoRESUMEN
The clinical impact of therapy-related acute leukemias is increasing with the extension of cancer-related survival; however, the origins remain largely unknown. Acute erythroleukemia (AEL), a rare unfavorable type of myeloid neoplasia, may also develop secondary to cytotoxic therapy. The disorder is featured by specific genetic alterations, most importantly multi-allelic mutations of the TP53 gene. While AEL might appear as a part of the therapy-related MDS/AML, spectrum information regarding the genetic complexity and progression is largely missing. We present two AEL cases arising after cytotoxic therapy and melphalan-based myeloablation/autologous peripheral stem cell transplantation due to multiple myeloma (MM). As stated, multiple pathogenic TP53 variants were present unrelated to preexisting MM, in parallel with uninvolved/wild-type hemopoiesis. Potential mechanisms of leukemic transformation are discussed, which include (1) preexisting preneoplastic hemopoietic stem cells (HSC) serving as the common origin for both MM and AEL, (2) the generation and intramedullary survival of p53-deficient post-chemotherapy HSCs, (3) reinoculation of mobilized autologous TP53 mutated HSCs, and (4) melphalan treatment-related late-onset myelodysplasia/leukemia with newly acquired TP53 mutations.
Asunto(s)
Leucemia Eritroblástica Aguda , Mieloma Múltiple , Trasplante Autólogo , Mieloma Múltiple/terapia , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Humanos , Persona de Mediana Edad , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/patología , Leucemia Eritroblástica Aguda/terapia , Masculino , Proteína p53 Supresora de Tumor/genética , Trasplante de Células Madre Hematopoyéticas/métodos , Transformación Celular Neoplásica/genética , Mutación , Femenino , Melfalán/uso terapéutico , Melfalán/administración & dosificación , Anciano , Quimioradioterapia/métodos , Neoplasias Primarias Secundarias/etiología , Neoplasias Primarias Secundarias/terapia , Neoplasias Primarias Secundarias/genéticaRESUMEN
BACKGROUND: Recent genomic studies revealed enhancer of zeste homolog 2 (EZH2) gain-of-function mutations, representing novel therapeutic targets in follicular lymphoma (FL) in around one quarter of patients. However, these analyses relied on single-site tissue biopsies and did not investigate the spatial heterogeneity and temporal dynamics of these alterations. OBJECTIVES: We aimed to perform a systematic analysis of EZH2 mutations using paired tissue (tumor biopsies [TB]) and liquid biopsies (LB) collected prior to treatment within the framework of a nationwide multicentric study. METHODS: Pretreatment LB and TB samples were collected from 123 patients. Among these, 114 had paired TB and LB, with 39 patients characterized with paired diagnostic and relapse samples available. The EZH2 mutation status and allele burden were assessed using an in-house-designed, highly sensitive multiplex droplet digital PCR assay. RESULTS: EZH2 mutation frequency was found to be 41.5% in the entire cohort. In patients with paired TB and LB samples, EZH2 mutations were identified in 37.8% of the patients with mutations exclusively found in 5.3% and 7.9% of TB and LB samples, respectively. EZH2 mutation status switch was documented in 35.9% of the patients with paired diagnostic and relapse samples. We also found that EZH2 wild-type clones may infiltrate the bone marrow more frequently compared to the EZH2 mutant ones. CONCLUSION: The in-depth spatio-temporal analysis identified EZH2 mutations in a considerably higher proportion of patients than previously reported. This expands the subset of FL patients who most likely would benefit from EZH2 inhibitor therapy.
Asunto(s)
Linfoma Folicular , Humanos , Linfoma Folicular/diagnóstico , Linfoma Folicular/genética , Linfoma Folicular/tratamiento farmacológico , Proteína Potenciadora del Homólogo Zeste 2/genética , Recurrencia Local de Neoplasia , Mutación , Biopsia , Biopsia Líquida , RecurrenciaRESUMEN
Primary ovarian mucinous tumors represent a heterogeneous group of neoplasms, and their diagnosis may be challenging. We analyzed 124 primary ovarian mucinous tumors originally diagnosed as mucinous borderline tumors (MBTs) or mucinous carcinomas (MCs), with an emphasis on interobserver diagnostic agreement and the potential for diagnostic support by molecular profiling using a next-generation sequencing targeted panel of 727 DNA and 147 RNA genes. Fourteen experienced pathologists independently assigned a diagnosis from preset options, based on a review of a single digitized slide from each tumor. After excluding 1 outlier participant, there was a moderate agreement in diagnosing the 124 cases when divided into 3 categories (κ = 0.524, for mucinous cystadenoma vs MBT vs MC). A perfect agreement for the distinction between mucinous cystadenoma/MBT as a combined category and MC was found in only 36.3% of the cases. Differentiating between MBTs and MCs with expansile invasion was particularly problematic. After a reclassification of the tumors into near-consensus diagnostic categories on the basis of the initial participant results, a comparison of molecular findings between the MBT and MC groups did not show major and unequivocal differences between MBTs and MCs or between MCs with expansile vs infiltrative pattern of invasion. In contrast, HER2 overexpression or amplification was found only in 5.3% of MBTs and in 35.3% of all MCs and in 45% of MCs with expansile invasion. Overall, HER2 alterations, including mutations, were found in 42.2% of MCs. KRAS mutations were found in 65.5% and PIK3CA mutations in 6% of MCs. In summary, although the diagnostic criteria are well-described, diagnostic agreement among our large group of experienced gynecologic pathologists was only moderate. Diagnostic categories showed a molecular overlap. Nonetheless, molecular profiling may prove to be therapeutically beneficial in advanced-stage, recurrent, or metastatic MCs.
Asunto(s)
Adenocarcinoma Mucinoso , Cistoadenoma Mucinoso , Neoplasias Quísticas, Mucinosas y Serosas , Neoplasias Ováricas , Humanos , Femenino , Cistoadenoma Mucinoso/patología , Reproducibilidad de los Resultados , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Adenocarcinoma Mucinoso/diagnóstico , Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/patologíaRESUMEN
Mitogen-activated protein kinase 8-interacting protein 3 gene (MAPK8IP3) encodes the c-Jun-amino-terminal kinase-interacting protein 3 (JIP3) and is involved in retrograde axonal transport. Heterozygous de novo pathogenic variants in MAPK8IP3 result in a neurodevelopmental disorder with or without brain abnormalities and possible axonal peripheral neuropathy. Whole-exome sequencing was performed on an individual presenting with severe congenital muscle hypotonia of neuronal origin mimicking lethal spinal muscular atrophy. Compound heterozygous rare variants (a splice and a missense) were detected in MAPK8IP3, inherited from the healthy parents. Western blot analysis in a muscle biopsy sample showed a more than 60% decrease in JIP3 expression. Here, we suggest a novel autosomal recessive phenotype of a lower motor neuron disease caused by JIP3 deficiency.
Asunto(s)
Atrofia Muscular Espinal , Enfermedades Musculares , Anomalías Musculoesqueléticas , Humanos , Hipotonía Muscular/diagnóstico , Hipotonía Muscular/genética , Atrofia Muscular Espinal/diagnóstico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patología , Fenotipo , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas Adaptadoras Transductoras de Señales/genéticaRESUMEN
Biological heterogeneity is a key feature of malignancies that significantly contributes to disease progression and therapy resistance. Residual/relapsed tumor foci may represent genetically divergent subclones, which remain uncovered as repeated and multiple tumor sampling is usually limited. The analysis of circulating free DNA (cfDNA) from the peripheral blood plasma (also called a liquid biopsy, LB) is a new achievement that provides an effective tool for follow-up monitoring of cancer-related genetic status. The present study highlights the phenomenon of mutational variability observed in patients with metastatic KRAS mutant colorectal cancer (mCRC) during treatment with bevacizumab in combination in a longitudinal fashion. The prospective study included 490 mCRC patients evaluated between 2020 and 2022 in our institution. Out of the 211 KRAS mutant cases (43.06%) 12 tumors were identified with multiple KRAS gene variants (5.68%). Detailed follow-up investigations were possible in 3 of these patients including the genotyping of the primary and available metastatic tumors, and the peripheral blood cfDNA. cfDNA was collected from three different time points before and between cycles of combined treatment with bevacizumab chemotherapy. KRAS gene variants were identified using reverse-hybridization strips, and next-generation sequencing (NGS), and confirmed by conventional Sanger sequencing. Interestingly, surgery and multiple treatment cycles reorganized the mutational profiles in the selected cases. The effect of the treatments resulted either in the overrepresentation of one of the pre-existing gene variants or in the appearance of new KRAS variants absent in the primary sample, according to the plasma cfDNA findings. Besides the KRAS variants demonstrated by targeted analysis, NGS mutational profiling identified some additional pathogenic variants from the cfDNA samples (including NRAS and MET alterations). In conclusion, plasma cfDNA sampling enables the monitoring of mutational heterogeneity and subclonal dynamics of the actual metastatic tumor mass in mCRC. The pattern of molecular profile potentially reflects a differential drug response determining further progression.
Asunto(s)
Adenocarcinoma , Ácidos Nucleicos Libres de Células , Neoplasias del Colon , Neoplasias Colorrectales , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Estudios Prospectivos , Bevacizumab/genética , Bevacizumab/uso terapéutico , Neoplasias Colorrectales/genética , Análisis de Secuencia de ADN , Mutación , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
The overexpression of the pH regulator carbonic anhydrase IX (CAIX) due to hypoxic/metabolic stress was reported in various tumors as an adverse prognostic feature. Our retrospective study aimed to investigate the general pattern and dynamics of CAIX expression in rectal adenocarcinoma following preoperative neoadjuvant therapy (NAT) in matched initial biopsy and surgical resection samples. A total of 40/55 (72.72%) of the post-treatment samples showed partial CAIX expression, frequently in the proximity of hypoxic tumor areas. CAIX expression showed a significant increase in post-treatment tumors (mean% 21.8 ± 24.9 SD vs. 39.4 ± 29.4 SD, p < 0.0001), that was not obvious in untreated tumors (mean% 15.0 ± 21.3 SD vs. 20 ± 23.02, p = 0.073). CAIXhigh phenotype was associated with mutant KRAS status and lack of pathological regression (WHO Tumor Regression Grade 4 and 5). However, the adverse effect of CAIX on overall or progression-free survival could not be statistically confirmed. In conclusion, the dynamic upregulation of CAIX expression is a general feature of rectal adenocarcinoma following neoadjuvant chemo-radiotherapy indicating therapy-induced metabolic reprogramming and cellular adaptation. A synergism of the CAIX-associated regulatory pathways and the mutant KRAS oncogenic signaling most likely contributes to therapy resistance and survival of residual cancer.
Asunto(s)
Adenocarcinoma , Neoplasias del Recto , Humanos , Anhidrasa Carbónica IX/genética , Anhidrasa Carbónica IX/metabolismo , Terapia Neoadyuvante , Estudios Retrospectivos , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Biomarcadores de Tumor/metabolismo , Antígenos de Neoplasias/metabolismo , Neoplasias del Recto/tratamiento farmacológico , Neoplasias del Recto/genética , Adenocarcinoma/genética , Adenocarcinoma/terapia , QuimioradioterapiaRESUMEN
Stimulation of thermogenesis by inducing uncoupling protein 1 (UCP1) expression in adipocytes is thought to promote weight loss by increasing energy expenditure, and it is postulated that the human newborn has thermogenic subcutaneous fat depots. However, it remains unclear whether a relevant number of UCP1-expressing (UCP1+) adipocytes exist in the early postnatal life. Here we studied the distribution of UCP1 and the expression of thermogenic genes in the subcutaneous adipose tissues of the human fetus, infant and child. We show that the deep layer of human fetal and neonatal subcutaneous fat, particularly the abdominal wall, is rich in UCP1+ adipocytes. These adipocytes develop in the late third trimester and persist throughout childhood, expressing a panel of genes linked to mitochondrial biogenesis and thermogenesis. During the early childhood adiposity rebound-a critical phase that determines obesity risk later in life-the absence of adipose tissue UCP1 expression in children with normal body mass index (BMI) correlates with an obesity-associated gene expression signature. Finally, UCP1 expression is negatively correlated with BMI z-score and adipocyte size in infants and children. Overall, our results show that the absence of UCP1 expression in adipose tissue is an early indicator of adipose tissue expansion in children.
Asunto(s)
Obesidad Infantil , Niño , Preescolar , Humanos , Recién Nacido , Tejido Adiposo/metabolismo , Obesidad Infantil/genética , Obesidad Infantil/metabolismo , Grasa Subcutánea/metabolismo , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMEN
Upon the discovery of frequent oncogenic histone alterations in paediatric diffuse high-grade gliomas, the epigenetic and transcriptional landscapes of tumours have become increasingly important aspects of diagnostic and prognostic analysis. The replacement of lysine 27 with methionine in H3 histone variants - H3 p.K28M (K27M) - was the first reported histone mutation associated with human malignancies, seen in up to 80% of paediatric diffuse midline gliomas. This discovery contributed to the updated 2021 World Health Organization (WHO) classification of central nervous system (CNS) tumours in which paediatric diffuse high-grade gliomas were classified into molecular-based categories. Therefore, molecular analysis of tumour cells has become increasingly necessary for determining disease prognosis and potential therapeutic strategies. Although detection of histone alterations is crucial for the diagnosis of specific glioma subtypes, several studies have identified them in other CNS tumours, which may be misleading during routine diagnostic work. While traditional biopsies remain the standard for diagnosis of gliomas, they pose a high risk for surgical complications and patient morbidity. Consequently, this review highlights the importance of the H3 K27-alterations in paediatric gliomas and several other CNS tumours. We also discuss the potential of liquid biopsies as a minimally invasive and highly effective alternative for confirming the diagnosis and potential targeted epigenetic therapies which may improve the survival of patients.
Asunto(s)
Neoplasias del Sistema Nervioso Central , Glioma , Humanos , Niño , Histonas/genética , Neoplasias del Sistema Nervioso Central/diagnóstico , Neoplasias del Sistema Nervioso Central/genética , Glioma/diagnóstico , Glioma/genética , Lisina , MetioninaRESUMEN
Body cavity fluids accumulating in progressive malignancies are potential subjects of regular clinical testing for cancer-related features. Besides the cellular component, the supernatant of the fluid proved to gain diagnostic impact as the cell-free DNA (cfDNA) fraction ideally reflects general molecular features of the related tumorous process, e.g. in lung carcinoma. Thus, malignant pleural effusions can be used for lung cancer genetic profiling and this might remain the only source for testing in critical cases. The cfDNA concentration of the pleural effusion depends on many factors in both benign and malignant conditions. Further to direct pleural metastatic spread, the redirection of tissue lymphatic circulation, tumor angiogenesis, inflammatory processes and other variables may contribute to or enhance the enrichment of the effusion tumor DNA from the earliest stages of carcinogenesis. Our review addresses the traffic of cfDNA in the pleural space and the diagnostic utility of effusion cfDNA from the perspective of the complex pleural pathophysiology.
Asunto(s)
Ácidos Nucleicos Libres de Células , ADN Tumoral Circulante , Neoplasias Pulmonares , Derrame Pleural Maligno , Ácidos Nucleicos Libres de Células/genética , ADN Tumoral Circulante/genética , ADN de Neoplasias/genética , Genotipo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Derrame Pleural Maligno/diagnóstico , Derrame Pleural Maligno/genética , Derrame Pleural Maligno/patologíaRESUMEN
Duchenne muscular dystrophy (DMD) is the most common inherited muscle dystrophy. Patients are characterized by muscle weakness, gross motor delay, and elevated serum creatinine kinase (CK) levels. The disease is caused by mutations in the DMD gene located on the X chromosome. Due to the X-linked recessive inheritance pattern, DMD most commonly affects males, who are generally diagnosed between the age of 3-5 years. Here we present an ultra-rare manifestation of DMD in a female patient. Cytogenetic examination showed that she has a t(X;10)(p21.1;p12.1) translocation, which turned out to affect the DMD gene with one of the breakpoints located in exon 54 (detected by genome sequencing). The X-inactivation test revealed skewed X-inactivation (ratio 99:1). Muscle histology and dystrophin immunohistochemistry showed severe dystrophic changes and highly reduced dystrophin expression, respectively. These results, in accordance with the clinical picture and a highly elevated serum CK, led to the diagnosis of DMD. In conclusion, although in very rare cases, DMD can manifest in female patients as well. In this case, a balanced X-autosome reciprocal translocation disrupts the DMD gene and skewed X-inactivation leads to the manifestation of the DMD phenotype.
Asunto(s)
Distrofina , Distrofia Muscular de Duchenne , Masculino , Femenino , Humanos , Distrofina/genética , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Inactivación del Cromosoma X , Cromosoma X , MutaciónRESUMEN
The chemical milieu, microbiota composition, and immune activity show prominent differences in distinct healthy skin areas. The objective of the current study was to compare the major permeability barrier components (stratum corneum and tight junction (TJ)), investigate the distribution of (corneo)desmosomes and TJs, and measure barrier function in healthy sebaceous gland-rich (SGR), apocrine gland-rich (AGR), and gland-poor (GP) skin regions. Molecules involved in cornified envelope (CE) formation, desquamation, and (corneo)desmosome and TJ organization were investigated at the mRNA and protein levels using qRT-PCR and immunohistochemistry. The distribution of junction structures was visualized using confocal microscopy. Transepidermal water loss (TEWL) functional measurements were also performed. CE intracellular structural components were similarly expressed in gland-rich (SGR and AGR) and GP areas. In contrast, significantly lower extracellular protein levels of (corneo)desmosomes (DSG1 and CDSN) and TJs (OCLN and CLDN1) were detected in SGR/AGR areas compared to GP areas. In parallel, kallikrein proteases were significantly higher in gland-rich regions. Moreover, gland-rich areas were characterized by prominently disorganized junction structures ((corneo)desmosomes and TJs) and significantly higher TEWL levels compared to GP skin, which exhibited a regular distribution of junction structures. According to our findings, the permeability barrier of our skin is not uniform. Gland-rich areas are characterized by weaker permeability barrier features compared with GP regions. These findings have important clinical relevance and may explain the preferred localization of acantholytic skin diseases on gland-rich skin regions (e.g., Pemphigus foliaceus, Darier's disease, and Hailey-Hailey disease).
Asunto(s)
Acantólisis/metabolismo , Epidermis/metabolismo , Glándulas Sebáceas/metabolismo , Uniones Estrechas/metabolismo , Acantólisis/patología , Adulto , Anciano , Epidermis/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Permeabilidad , Glándulas Sebáceas/patología , Uniones Estrechas/patologíaRESUMEN
Infiltration of red blood cells into atheromatous plaques and oxidation of hemoglobin (Hb) and lipoproteins are implicated in the pathogenesis of atherosclerosis. α1-microglobulin (A1M) is a radical-scavenging and heme-binding protein. In this work, we examined the origin and role of A1M in human atherosclerotic lesions. Using immunohistochemistry, we observed a significant A1M immunoreactivity in atheromas and hemorrhaged plaques of carotid arteries in smooth muscle cells (SMCs) and macrophages. The most prominent expression was detected in macrophages of organized hemorrhage. To reveal a possible inducer of A1M expression in ruptured lesions, we exposed aortic endothelial cells (ECs), SMCs and macrophages to heme, Oxy- and FerrylHb. Both heme and FerrylHb, but not OxyHb, upregulated A1M mRNA expression in all cell types. Importantly, only FerrylHb induced A1M protein secretion in aortic ECs, SMCs and macrophages. To assess the possible function of A1M in ruptured lesions, we analyzed Hb oxidation and heme-catalyzed lipid peroxidation in the presence of A1M. We showed that recombinant A1M markedly inhibited Hb oxidation and heme-driven oxidative modification of low-density lipoproteins as well plaque lipids derived from atheromas. These results demonstrate the presence of A1M in atherosclerotic plaques and suggest its induction by heme and FerrylHb in the resident cells.
Asunto(s)
alfa-Globulinas/metabolismo , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Hemo/metabolismo , Hemoglobinas/metabolismo , Peroxidación de Lípido , Oxidación-Reducción , Aterosclerosis/patología , Biomarcadores , Enfermedades de las Arterias Carótidas/etiología , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Células Cultivadas , Progresión de la Enfermedad , Susceptibilidad a Enfermedades , Hemorragia/metabolismo , Hemorragia/patología , Humanos , Inmunohistoquímica , Miocitos del Músculo Liso/metabolismo , Placa Aterosclerótica/etiología , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologíaRESUMEN
The lysis of red blood cells was shown to occur in human ruptured atherosclerotic lesions and intraventricular hemorrhage (IVH) of the brain. Liberated cell-free hemoglobin was found to undergo oxidation in both pathologies. We hypothesize that hemoglobin-derived peptides are generated during hemoglobin oxidation both in complicated atherosclerotic lesions and IVH of the brain, triggering endothelial cell dysfunction. Oxidized hemoglobin and its products were followed with spectrophotometry, LC-MS/MS analysis and detection of the cross-linking of globin chains in complicated atherosclerotic lesions of the human carotid artery and the hemorrhaged cerebrospinal liquid of preterm infants. The vascular pathophysiologic role of oxidized hemoglobin and the resultant peptides was assessed by measuring endothelial integrity, the activation of endothelial cells and the induction of proinflammatory genes. Peptide fragments of hemoglobin (VNVDEVGGEALGRLLVVYPWTQR, LLVVYPWTQR, MFLSFPTTK, VGAHAGEYGAELERMFLSFPTTK, and FLASVSTVLTSKYR) were identified in ruptured atherosclerotic lesions and in IVH of the human brain. Fragments resulting from the oxidation of hemoglobin were accompanied by the accumulation of ferryl hemoglobin. Similar to complicated atherosclerotic lesions of the human carotid artery, a high level of oxidized and cross-linked hemoglobin was observed in the cerebrospinal fluid after IVH. Haptoglobin inhibited hemoglobin fragmentation provoked by peroxide. The resultant peptides failed to bind haptoglobin or albumin. Peptides derived from hemoglobin oxidation and ferryl hemoglobin induced intercellular gap formation, decreased junctional resistance in the endothelium, and enhanced monocyte adhesion to endothelial cells. Enhanced expression of TNF and the activation of NLRP3 and CASP1 followed by the increased generation of IL-1ß and nuclear translocation of the NF-κß transcription factor occurred in response to hemoglobin-derived peptides, and ferryl hemoglobin in endothelium was upregulated in both pathologies. We conclude that the oxidation of hemoglobin in complicated atherosclerotic lesions and intraventricular hemorrhage of the brain generates peptide fragments and ferryl hemoglobin with the potential to trigger endothelial cell dysfunction.
Asunto(s)
Enfermedades de las Arterias Carótidas/metabolismo , Hemorragia Cerebral Intraventricular/metabolismo , Endotelio Vascular/fisiopatología , Hemoglobinas , Encéfalo/metabolismo , Encéfalo/patología , Enfermedades de las Arterias Carótidas/patología , Células Cultivadas , Hemorragia Cerebral Intraventricular/patología , Cromatografía Liquida , Hemoglobinas/química , Hemoglobinas/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Oxidación-Reducción , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Wolcott-Rallison Syndrome (WRS) is a rare autosomal recessive disease that is the most common cause of neonatal diabetes in consanguineous families. WRS is caused by various genetic alterations of the Eukaryotic Translation Initiation Factor 2-Alpha Kinase 3 (EIF2AK3) gene. METHODS: Genetic analysis of a consanguineous family where two children were diagnosed with WRS was performed by Sanger sequencing. The altered protein was investigated by in vitro cloning, expression and immunohistochemistry. RESULTS: The first cases in Hungary, - two patients in one family, where the parents were fourth-degree cousins - showed the typical clinical features of WRS: early onset diabetes mellitus with hyperglycemia, growth retardation, infection-induced multiple organ failure. The genetic background of the disease was a novel alteration in the EIF2AK3 gene involving the splice site of exon 11- intron 11-12 boundary: g.53051_53062delinsTG. According to cDNA sequencing this created a new splice site and resulted in a frameshift and the development of an early termination codon at amino acid position 633 (p.Pro627AspfsTer7). Based on in vitro cloning and expression studies, the truncated protein was functionally inactive. Immunohistochemistry revealed that the intact protein was absent in the islets of pancreas, furthermore insulin expressing cells were also dramatically diminished. Elevated GRP78 and reduced CHOP protein expression were observed in the liver. CONCLUSIONS: The novel genetic alteration causing the absence of the EIF2AK3 protein resulted in insufficient handling of severe endoplasmic reticulum stress, leading to liver failure and demise of the patients.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Epífisis/anomalías , Mutación INDEL , Osteocondrodisplasias/genética , Sitios de Empalme de ARN/genética , eIF-2 Quinasa/genética , Preescolar , Consanguinidad , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/patología , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/genética , Epífisis/patología , Resultado Fatal , Femenino , Mutación del Sistema de Lectura , Humanos , Hungría , Lactante , Fallo Hepático/complicaciones , Fallo Hepático/genética , Fallo Hepático/patología , Osteocondrodisplasias/diagnóstico , Osteocondrodisplasias/patología , Linaje , Hermanos , Virosis/complicaciones , Virosis/patologíaRESUMEN
Poly(ADP-ribose) polymerase-1 (PARP1) is a pro-inflammatory protein, whose pro-inflammatory properties were demonstrated in human. The pro-inflammatory properties of PARP1 were shown in Th1- and Th2-mediated inflammatory pathologies, but not Th17-mediated inflammation. Thus, we studied the role of PARP1 in the imiquimod-induced model of psoriasis. To our surprise, in imiquimod-induced psoriasis, PARP1 acted as an anti-inflammatory factor and its genetic deletion exacerbated symptoms. We showed that in the absence of PARP1, the epidermis thickened and the number of TUNEL-positive cells decreased in the epidermis. These data indicate programmed cell death is decreased in keratinocytes. Changes in involucrin expression suggest that keratinocyte differentiation is hampered. Furthermore, epidermal expression of IL6 increased in the psoriasiform lesions of PARP1 knockout mice, suggesting that the inflammatory response is also derailed in the absence of PARP1. Finally, we showed that PARP1 expression is reduced in human psoriatic lesions compared with control skin samples. In imiquimod-treated HPV-KER keratinocytes, PARP inhibition recapitulated the in vivo findings, namely keratinocyte hyperproliferation; furthermore, the mRNA expression of psoriasis-associated cytokines (IL6, IL1ß, IL8, IL17 and IL23A) was also induced. The inhibition of TRPV1 abrogated the effects of the combined imiquimod + PARP inhibitor treatment.
Asunto(s)
Citocinas/genética , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Psoriasis/fisiopatología , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Humanos , Imiquimod/farmacología , Inflamación/genética , Interleucina-6/metabolismo , Queratinocitos , Masculino , Ratones , Ratones Noqueados , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Psoriasis/inducido químicamente , Psoriasis/patología , ARN Mensajero/metabolismo , Índice de Severidad de la Enfermedad , Canales Catiónicos TRPV/antagonistas & inhibidores , Células Th17RESUMEN
Objective- Vascular calcification is associated with high risk of cardiovascular events and mortality. Osteochondrogenic differentiation of vascular smooth muscle cells (VSMCs) is the major cellular mechanism underlying vascular calcification. Because tissue hypoxia is a common denominator in vascular calcification, we investigated whether hypoxia per se triggers osteochondrogenic differentiation of VSMCs. Approach and Results- We studied osteochondrogenic differentiation of human aorta VSMCs cultured under normoxic (21% O2) and hypoxic (5% O2) conditions. Hypoxia increased protein expression of HIF (hypoxia-inducible factor)-1α and its target genes GLUT1 (glucose transporter 1) and VEGFA (vascular endothelial growth factor A) and induced mRNA and protein expressions of osteochondrogenic markers, that is, RUNX2 (runt-related transcription factor 2), SOX9 (Sry-related HMG box-9), OCN (osteocalcin) and ALP (alkaline phosphatase), and induced a time-dependent calcification of the extracellular matrix of VSMCs. HIF-1 inhibition by chetomin abrogated the effect of hypoxia on osteochondrogenic markers and abolished extracellular matrix calcification. Hypoxia triggered the production of reactive oxygen species, which was inhibited by chetomin. Scavenging reactive oxygen species by N-acetyl cysteine attenuated hypoxia-mediated upregulation of HIF-1α, RUNX2, and OCN protein expressions and inhibited extracellular matrix calcification, which effect was mimicked by a specific hydrogen peroxide scavenger sodium pyruvate and a mitochondrial reactive oxygen species inhibitor rotenone. Ex vivo culture of mice aorta under hypoxic conditions triggered calcification which was inhibited by chetomin and N-acetyl cysteine. In vivo hypoxia exposure (10% O2) increased RUNX2 mRNA levels in mice lung and the aorta. Conclusions- Hypoxia contributes to vascular calcification through the induction of osteochondrogenic differentiation of VSMCs in an HIF-1-dependent and mitochondria-derived reactive oxygen species-dependent manner.
Asunto(s)
Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Factor 1 Inducible por Hipoxia/genética , Hipoxia/complicaciones , Especies Reactivas de Oxígeno/metabolismo , Calcificación Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Disulfuros/farmacología , Femenino , Regulación de la Expresión Génica , Humanos , Alcaloides Indólicos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , ARN Mensajero/genética , Distribución Aleatoria , Valores de Referencia , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Calcificación Vascular/fisiopatologíaRESUMEN
Objective- Calcific aortic valve disease is a prominent finding in elderly and in patients with chronic kidney disease. We investigated the potential role of iron metabolism in the pathogenesis of calcific aortic valve disease. Approach and Results- Cultured valvular interstitial cells of stenotic aortic valve with calcification from patients undergoing valve replacement exhibited significant susceptibility to mineralization/osteoblastic transdifferentiation in response to phosphate. This process was abrogated by iron via induction of H-ferritin as reflected by lowering ALP and osteocalcin secretion and preventing extracellular calcium deposition. Cellular phosphate uptake and accumulation of lysosomal phosphate were decreased. Accordingly, expression of phosphate transporters Pit1 and Pit2 were repressed. Translocation of ferritin into lysosomes occurred with high phosphate-binding capacity. Importantly, ferritin reduced nuclear accumulation of RUNX2 (Runt-related transcription factor 2), and as a reciprocal effect, it enhanced nuclear localization of transcription factor Sox9 (SRY [sex-determining region Y]-box 9). Pyrophosphate generation was also increased via upregulation of ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase-2). 3H-1, 2-dithiole-3-thione mimicked these beneficial effects in valvular interstitial cell via induction of H-ferritin. Ferroxidase activity of H-ferritin was essential for this function, as ceruloplasmin exhibited similar inhibitory functions. Histological analysis of stenotic aortic valve revealed high expression of H-ferritin without iron accumulation and its relative dominance over ALP in noncalcified regions. Increased expression of H-ferritin accompanied by elevation of TNF-α (tumor necrosis factor-α) and IL-1ß (interleukin-1ß) levels, inducers of H-ferritin, corroborates the essential role of ferritin/ferroxidase via attenuating inflammation in calcific aortic valve disease. Conclusions- Our results indicate that H-ferritin is a stratagem in mitigating valvular mineralization/osteoblastic differentiation. Utilization of 3H-1, 2-dithiole-3-thione to induce ferritin expression may prove a novel therapeutic potential in valvular mineralization.