TKF, a mexicanolide-type limonoid derivative, suppressed hepatic stellate cells activation and liver fibrosis through inhibition of the YAP/Notch3 pathway.

BACKGROUND: Liver fibrosis is a common scarring response and may ultimately lead to liver cancer, unfortunately, there is currently no effective antifibrotic drug approved for human use. Limonoids exhibit a broad spectrum of biological activities; however, the potential role of limonoids against fibrosis is largely unknown. PURPOSE: This study investigates the antifibrotic activities and potential mechanisms of TKF (3-tigloyl-khasenegasin F), a natural mexicanolide-type limonoid derivative. STUDY DESIGN/METHODS: Two well-established mouse models (CCl4 challenge and bile duct ligation) were used to assess anti-fibrotic effects of TKF in vivo. Human hepatic stellate cell (HSC) line LX-2 and mouse primary hepatic stellate cells (pHSCs) also served as in vitro liver fibrosis models. RESULT: TKF administration significantly attenuated hepatic histopathological injury and collagen accumulation and suppressed fibrogenesis-associated gene expression including Col1a1, Acta2, and Timp1. In LX-2 cells and mouse pHSCs, TKF dose-dependently suppressed HSC activation and the expression levels of fibrogenic markers. Mechanistic studies showed that TKF inhibited Notch3-Hes1 and YAP signalings in vivo and in vitro. Furthermore, YAP inhibition or knockdown downregulated the Notch3 expression; however, Notch3 inhibition or knockdown did not affect the level of YAP in activated HSC. We revealed that TKF inhibited Notch3-Hes1 activation and downregulated hepatic fibrogenic gene expression via inhibiting YAP. CONCLUSION: The therapeutic benefit of TKF against liver fibrosis results from inhibition of YAP and Notch3-Hes1 pathways, indicating that TKF may be a novel therapeutic candidate for liver fibrosis.

Global Cardiovascular Risk Profile and Cerebrovascular Abnormalities in Presymptomatic Individuals with CADASIL or Autosomal Dominant Alzheimer's Disease.

BACKGROUND: Cardiovascular risk factors increase the risk of developing dementia, including Alzheimer's disease and vascular dementia. OBJECTIVE: Studying individuals with autosomal dominant mutations leading to the early onset of dementia, this study examines the effect of the global cardiovascular risk profile on early cognitive and neuroimaging features of Alzheimer's disease and vascular dementia. METHODS: We studied 85 non-demented and stroke-free individuals, including 20 subjects with Presenilin1 (PSEN1) E280A mutation leading to the early onset of autosomal dominant Alzheimer's disease (ADAD), 20 subjects with NOTCH3 mutations leading to cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and to the early onset of vascular dementia, and 45 non-affected family members (non-carriers). All subjects underwent clinical and neuropsychological evaluations and an MRI. The global cardiovascular risk profile was estimated using the office-based Framingham Cardiovascular Risk Profile (FCRP) score. RESULTS: In individuals with CADASIL, a higher FCRP score was associated with a reduced hippocampal volume (B = -0.06, p < 0.05) and an increased severity of cerebral microbleeds (B = 0.13, p < 0.001), lacunes (B = 0.30, p < 0.001), and perivascular space enlargement in the basal ganglia (B = 0.50, p < 0.05). There was no significant association between the FCRP score and neuroimaging measures in ADAD or non-carrier subjects. While the FCRP score was related to performance in executive function in non-carrier subjects (B = 0.06, p < 0.05), it was not significantly associated with cognitive performance in individuals with CADASIL or ADAD. CONCLUSION: Our results suggest that individuals with CADASIL and other forms of vascular cognitive impairment might particularly benefit from early interventions aimed at controlling cardiovascular risks.

Revealing the BRD4-NOTCH3 fusion: A novel hill in the cancer landscape.

OBJECTIVES: Gene fusions are becoming more evident in cancer scenario for either being the driver alterations, or for the great therapeutic target potential in many cases. Our aim was to characterize the BRD4-NOTCH3 fusion correlating with clinical features, and to determine the frequency of this fusion in the oncological population. MATERIAL AND METHODS: One patient diagnosed with lung adenocarcinoma at Hospital Sírio-Libanês (Brazil) was included. Foundation Medicine database was searched for all BRD4-NOTCH3 fusions among 233,804 specimens. RESULTS: A 76-year-old male patient was diagnosed with lung adenocarcinoma. Molecular assessments demonstrated negative ALK and EGFR, with PD-L1 expression positive by 60 %. He was treated with first line chemotherapy and second line immunotherapy. Subsequent treatments resume re-exposures to chemotherapy with poor responses. A next-generation sequencing (NGS) based assay was performed in the tumor biopsy, revealing mainly mutation in STK11, microsatellite stability, TMB-intermediate, MYC amplification and a BRD4-NOTCH3 fusion. The breakpoint analysis of this fusion indicates that BRD4 active domains are preserved, suggesting that it maintained DNA binding activity, as well as its capacity to be halt by BET inhibitors. Foundation Medicine database was searched for all BRD4-NOTCH3 fusions among more than 230-thousand specimens and it was found in 87 new cases in a rate of 0.04 % occurrence in solid tumors, predominately in gynecological cancers. The same rate was found when we analyzed a different dataset. CONCLUSION: In conclusion, this is the first report of the BRD4-NOTCH3 gene fusion associated with clinical characterization and, although rare, the occurrence of this fusion is constant in different population. Our data suggest that this fusion has great potential to targeted-therapy.

NOTCH3 Gene Mutation in a Chilean Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy Family.

INTRODUCTION: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare hereditary stroke disorder caused by mutations in the NOTCH3 gene. We report the first Chilean CADASIL family with complete radiological and histological studies. METHODS: The family tree was constructed from an autopsy-confirmed confirmed patient, and includes 3 generations. We performed clinical, pathologic, genetic, and radiologic examinations on members of a family with CADASIL. RESULTS: In the second generation, findings compatible with CADASIL were identified in 6 individuals, all of whom had a missense mutation in exon 3 (c.268C>T) resulting in an arginine to cysteine amino acid substitution at position 90 (R90C). In the third generation, a missense mutation was detected in one of the 4 asymptomatic individuals. CONCLUSIONS: There are similarities in clinical presentation between this family and previously described Asian and European series with R90C mutations. Detecting genotypes with a gain or loss of cysteine residues opens the door to future gene transfection-based therapies.

Protein Expression Analysis in Uterine Cervical Cancer for Potential Targets in Treatment.

Specific markers in lesions of the human uterine cervix cancer (UCC) are still needed for prognostic, diagnostic and/or therapeutic purposes. In this study we evaluated key molecules at protein level between normal epithelium, cervical intraepithelial neoplasia (CIN1-3) and invasive cancer of a group of molecules previously reported at mRNA level. For that purpose, human formalin-fixed paraffin embedded tissue microarrays (TMAs) were constructed containing 205 Mexican tissue core specimens. Immunohistochemistry and quantitative analysis of histological staining was performed against twenty-two distinct proteins for each core and the processing platform ImageJ. In the progression of the disease we found key statistical differences for the proteins SEL1, Notch3 and SOCS3. High expressions of SEL1L, Notch3 and SOCS3 have potential value to increase the prognostic of UCC in combination with markers such as p16INK4a. This study identified key drivers in cervical carcinogenesis that should be evaluated for the development of UCC therapies.

Ischemic Stroke and Six Genetic Variants in CRP, EPHX2, FGA, and NOTCH3 Genes: A Meta-Analysis.

BACKGROUND: Ischemic stroke (IS) is a leading cause of death and disability worldwide. As genetic heritability for IS is estimated at about 35%-40%, the identification of genetic variants associated with IS risk is of great importance. The main objective of this study was to carry out a meta-analysis for polymorphisms in CRP, EPHX2, FGA, and NOTCH3 genes and the risk for IS. METHODS: Literature search for 6 candidate polymorphisms and IS was conducted using HuGE Navigator, PubMed, and Google Scholar databases. Meta-Analyst program was used to calculate pooled odds ratios (ORs) with a random effects model. RESULTS: Twenty-five published studies for 6 candidate polymorphisms were included: CRP-rs1800947 (5 studies), CRP-rs1205 (3 studies), EPHX2-rs751141 (5 studies), FGA-rs6050 (6 studies), NOTCH3-rs3815188 (3 studies), and NOTCH3-rs1043994 (3 studies), for a total number of 7,825 IS cases and 56,532 control subjects. We did not find significant pooled ORs (P values > .05) for any of the genetic variants evaluated in this work. CONCLUSIONS: Our meta-analysis results did not show significant associations between these 6 polymorphisms in 4 candidate genes and IS, despite the functional role of some of these single nucleotide polymorphisms (e.g., rs6050 in FGA gene). Future studies are needed to identify additional main genetic risk factors for IS in different populations.

Cadasil - genetic and ultrastructural diagnosis: case report / Cadasil - diagnóstico genético e ultraestrutural: relato de caso

ABSTRACT Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is a hereditary disorder which affects the cerebral vasculature due to mutations in the NOTCH 3 gene. The diagnosis may be established through genetic testing for detection of these mutations and/or by skin biopsy. We report a case of the disorder in a female patient, who presented recurrent transient ischemic attacks that evolved to progressive subcortical dementia. Neuroimaging disclosed extensive leukoaraiosis and lacunar infarcts. The genetic analysis for NOTCH 3 was confirmatory. The ultrastructural examination of the skin biopsy sample, initially negative, confirmed the presence of characteristic changes (presence of granular osmiophilic material inclusions [GOM]), after the analysis of new sections of the same specimen. The present findings indicate that negative findings on ultrastructural examinations of biopsy should not exclude the diagnosis of the disease and that further analyses of the sample may be necessary to detect the presence of GOM.

ABSTRACT Arteriopatia Cerebral Autossômica Dominante com Infartos Subcorticais e Leucoencefalopatia (CADASIL) é uma desordem hereditária que acomete a vasculatura cerebral devido a mutações no gene do NOTCH 3. O diagnóstico pode ser estabelecido através de testes genéticos para a detecção das mutações e/ou de biópsia de pele. É relatado um caso da desordem em paciente feminino, que apresentou ataques isquêmicos transitórios recorrentes, evoluindo para um quadro progressivo de demência subcortical. A neuroimagem mostrou extensa leucoaraiose e infartos lacunares. A análise genética para NOTCH 3 foi confirmatória. O exame ultraestrutural de biópsia de pele, inicialmente negativa, confirmou a presença de alterações características (presença de material granular osmiofílico [GOM]), após a análise de novos cortes do mesmo material. Os presentes achados indicam que achados negativos de exames ultraestruturais de biópsia não deveriam excluir o diagnóstico da doença e que análise adicional da amostra pode ser necessária para detectar a presença de GOM.

Identification of differential expressed transcripts in cervical cancer of Mexican patients.

The aim of this study was to identify the gene expression profile in biopsies of patients with cervical intraepithelial neoplasia (CIN) 1, CIN 2, CIN 3, and microinvasive cancer by suppression subtractive hybridization and Southern blotting. After analyzing 1,800 cDNA clones, we found 198 upregulated genes, 166 downregulated, and no significant change of gene expression in 86 clones (p = 0.005). These results were validated by Northern blot analysis (p = 0.0001) in the identification of 28 overexpressed and 7 downregulated transcripts. We observed a set of genes related to the Notch signaling pathway that may be involved in the transformation of cervical cells and in the development to malignancy. The differentially expressed genes may provide useful information about the molecular mechanisms involved in human cervical carcinoma and as diagnostic markers.

[Monitoring cognitive characteristics in a population with hereditary cerebrovascular disease (CADASIL) in Colombia]. / Seguimiento de las características cognitivas en una población con enfermedad cerebrovascular hereditaria (CADASIL) en Colombia.

INTRODUCTION: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary disease that affects small vessels and presents with vascular episodes, neuropsychiatric disorders, migraine and cognitive impairment. The cognitive disorder varies according to the time elapsed since onset. It is a condition with a subcortical origin related to executive dysfunction, slowing, attention-related disorders and memory disorders. AIM: To define the cognitive characteristics in two neuropsychological evaluations of carriers of Notch3 gene mutations as compared to non-carriers belonging to Colombian families with CADASIL. SUBJECTS AND METHODS: The study followed a longitudinal, retrospective design with 140 participants, including both carriers and non-carriers of the mutation. Cognitive performance was analysed by comparing the first and the last neuropsychological evaluation carried out on each subject at a four-year interval. RESULTS: There were statistically significant differences (p < 0.05) between the two groups in the last evaluation, but only in some tests. Carriers and non-carriers did not display any significant changes between the first and the last evaluation. CONCLUSIONS: No differences were found between both groups in the two evaluations. Cognitive impairment is not observed with the passage of time in carriers, probably owing to the fact that most of them were young, asymptomatic subjects. We believe that four years' follow-up is not enough time to observe a significant progression in the alterations affecting the cognitive functions in carriers of mutations in the Notch3 gene, which causes CADASIL. We also consider that more sensitive cognitive tools are needed to perform the neuropsychological evaluation.

CADASIL presenting with a movement disorder: a clinical study of a Chilean kindred.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary vascular disease that usually begins with migraine, followed by repeated strokes and progressive dementia. We describe an unusual clinical presentation of this condition in members of a Chilean family with an established NOTCH3 mutation. We report early clinical, neuropsychological, transcranial ultrasound, magnetic resonance imaging (MRI), cerebral blood flow, and skin biopsy findings on these patients. Of the patients, 2 presented with facial dystonia, 1 of whom had abnormal single photon emission computed tomography and transcranial ultrasound studies after normal brain MRI scans. Our report emphasizes that CADASIL must be considered in the study of patients with secondary dystonia.

[Vascular hereditary dementia CADASIL type in Colombia. III. Linkage analysis to notch3 gene]. / Demencia vascular hereditaria tipo CADASIL en Colombia. III. Análisis de ligamiento a Notch3.

OBJECTIVE: To perform linkage analysis between the Short Tandem Repeats (STR) microsatellite markers D19S923, D19S929, D19S22, which are in strong genetic linkage to Notch3 gene in order to contrast the hypothesis that the vascular hereditary dementia phenotype described in a multigenerational extended pedigree from Colombia correspond to CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy). Even we know that using techniques as the Single Strand Conformational Polymorphisms (SSCP) could determine mutations in Notch3, the rationality of this approach is that intronic variations could not be defined and that we are interested in determine if some forms of the clinical presentation and its phenotypic variability make part of CADASIL. INTRODUCTION: The CADASIL phenotype is caused by mutations in the Notch3 gene. Clinical features of CADASIL are: 1. Recurrent cerebra-vascular episodes; 2. Migraine history; 3. History of transitory ischemic attack and, 4. Behavior changes and dementia. MATERIAL AND METHODS: By using SIMLINK we showed that the extended genealogy had the enough power to detect significant LOD (logarithm of oods) score values when Notch3 was considered the disorder cause. Linkage analysis was carried out by using parametric and non parametrical methods. The Elston-Stewart general method was used as the parametrical analysis and the sib pair method as the non-parametrical one. We perform simulations changing the affection status codification by including as affected or not including those individuals with migraine. Furthermore, in order to detect the stability of the results, we changed the penetrance values, the genetic frequencies on both, the marker loci and the affection locus. RESULTS: The maximum pair-wise LOD score was 2.04 which was detected at the marker D19S23 with q= 0.11cM. This distance correspond exactly with the Notch3 location. That is 100 times more probable that there is linkage that there is not. In other words this probability could be explained as if the phenotype correspond to CADASIL than to other vascular dementia. The non parametric results were compatibles with the parametric ones. When the migraine symptom was considered as a part of the affected status, the LOD score values showed not linkage. CONCLUSIONS: The results of the linkage analysis to these STR microsatellite markers suggest that the vascular hereditary dementia phenotype described in this family correspond to CADASIL caused by a polymorphism on the Notch3 gene. On the contrary, these same results suggest that the migraine phenotype is not a part of the progressive dementia.

[Analysis of complex segregation in a large family with hereditary cerebrovascular disease in Antioquia, Colombia]. / Análisis de segregación compleja de una familia numerosa con enfermedad cerebrovascular hereditaria en Antioquia (Colombia).

INTRODUCTION: Among different kinds of cerebrovascular diseases, few of them are caused by genetic disturbances, such as CADASIL (caused by Notch3 mutations), CARASIL, mitochondrial encephalopathy, MELAS and dementia typed Binswanger. However, to describe these type of cerebrovascular diseases related with genetic mutations could permit to determinate the causes of both hereditary and sporadic cerebrovascular diseases and then lead solutions. OBJECTIVE: To describe the genetic, environmental and cohort factors that determinate the presence of many affected people by a several cerebrovascular diseases in the pedigree of a large family from Antioquia (Colombia). PATIENTS AND METHODS: We performed one pedigree (268 individuals), through singular recruit and then complex segregation analysis with POINTER program. RESULTS: The model that more close to data is autosomal dominant mayor locus without influence of environmental factors. Frequency of allele of susceptibility to develop stroke or subcortical vascular dementia was 0.0006. Mayor gene is over epistatic effects or interactions with other gene. CONCLUSIONS: Described an autosomal dominant hereditary model through complex segregation analysis in a pedigree of patients with hereditary cerebral vascular diseases characterized by recurrent strokes, early onset subcortical dementia, hearing loss, antecedent of migraine and MRI signal abnormalities, subcortical infarcts and leukoencephalopathy. In this family the parameter calculated, autosomal dominant model, and clinical feature strongly support the diagnostic of CADASIL, linkage analysis and sequentiation will be performed to determinate if mutant gene is Notch3.