Clinical and epidemiological profiles from a case series of 26 Brazilian CADASIL patients / Perfil clínico e epidemiológico de uma série de casos de 26 pacientes com diagnóstico de CADASIL

Abstract Background Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a genetic cause of ischemic stroke and the most common form of non-atherosclerotic stroke. Despite being the most prevalent vascular hereditary disease, clinical data regarding the Brazilian population are scarce. Considering that the Brazilian population has one of the most heterogeneous genetic constitutions in the world, knowledge about genetic and epidemiological profiles is mandatory. The present study aimed to elucidate the epidemiological and clinical features of CADASIL in Brazil. Methods We performed a case series study comprising 6 rehabilitation hospitals in Brazil and reported the clinical and epidemiological data from the medical records of patients admitted from 2002 to 2019 with genetic confirmation. Results We enrolled 26 (16 female) patients in whom mutations in exons 4 and 19 were the most common. The mean age at the onset of the disease was of 45 years. Ischemic stroke was the first cardinal symptom in 19 patients. Cognitive impairment, dementia, and psychiatric manifestations were detected in 17, 6, and 16 patients respectively. In total, 8 patients had recurrent migraines, with aura in 6 (75%) of them. White matter hyperintensities in the temporal lobe and the external capsule were found in 20 (91%) and 15 patients (68%) respectively. The median Fazekas score was of 2. Lacunar infarcts, microbleeds, and larger hemorrhages were observed in 18 (82%), 9, and 2 patients respectively. Conclusion The present is the most extensive series of Brazilian CADASIL patients published to date, and we have reported the first case of microbleeds in the spinal cord of a CADASIL patient. Most of our clinical and epidemiological data are in accordance with European cohorts, except for microbleeds and hemorrhagic strokes, for which rates fall in between those of European and Asian cohorts.

Resumo Antecedentes Arteriopatia cerebral autossômica dominante com enfartes subcorticais e leucoencefalopatia (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy, CADASIL, em inglês) é uma causa genética de acidente vascular cerebral (AVC) isquêmico e a forma mais comum de acidente vascular cerebral não aterosclerótico. Apesar de ser a doença vascular hereditária mais prevalente que há, os dados clínicos para a população brasileira são escassos. Considerando que o Brasil tem uma das constituições genéticas mais heterogêneas do mundo, o conhecimento sobre perfis genéticos e epidemiológicos é obrigatório. Este estudo teve como objetivo elucidar as características clínicas e epidemiológicas de pacientes com CADASIL no Brasil. Métodos Apresentamos uma série de casos envolvendo 6 hospitais de reabilitação no Brasil, e relatamos dados clínicos e epidemiológicos de prontuários de pacientes admitidos entre 2002 e 2019 com confirmação genética. Resultados incluímos 26 pacientes (16 mulheres) em que as mutações nos éxons 4 e 19 eram as mais comuns. A idade média de início da doença foi de 45 anos. O AVC isquêmico foi o primeiro sintoma cardinal em 19 pacientes. Comprometimento cognitivo, demência e manifestações psiquiátricas foram detectados em 17, seis e 16 pacientes, respectivamente. Ao todo, 8 pacientes apresentavam enxaqueca, sendo com aura em 6 (75%) pacientes. Hiperintensidades de substância branca no polo temporal e na cápsula externa foram encontradas em 20 (91%) e 15 pacientes (68%), respectivamente. A pontuação mediana na escala de Fazekas foi de 2. Infartos lacunares, microssangramentos e macro-hemorragias foram observadas em 18 (82%), 9 (41%) e 2 (9%) pacientes, respectivamente. Conclusão O presente estudo representa a mais extensa série de pacientes brasileiros com CADASIL publicada até o momento, e relatamos o primeiro caso de micro-hemorragia na medula espinhal de um paciente com CADASIL. A maior parte dos nossos dados clínicos e epidemiológicos está de acordo com as coortes europeias, exceto para micro-hemorragias e macro-hemorragias, para as quais as taxas se enquadram entre as das coortes europeias e asiáticas.

CADASIL caused by mutation of NOTCH3 gene c.697T>A: a report on a Chinese family / 国际脑血管病杂志

Objective:To report the clinical features, imaging findings and gene mutation features of a Chinese family with cerebral autosomal dominant arteriopathy with subcritical infarcts and leukoencephalopathy (CADASIL).Methods:We summarized the clinical and imaging features of a CADASIL family confirmed by gene sequencing. NOTCH3 gene sequencing was conducted for the proband, and the structure of the protein encoded by the mutant gene was predicted. Results:The patients in this family mainly presented with recurrent lacunar infarction and hypertension, without headache and emotional disorders such as anxiety or depression. Head MRI of the proband showed multiple lacunar infarctions and extensive white matter degeneration. Susceptibility-weighted imaging showed multiple small intracranial hemorrhages. The analysis of NOTCH3 gene showed that the proband had c.697T>A mutation. The 3D structure prediction of the protein encoded by this mutation locus showed that this locus could lead to the conversion of cysteine to serine at the 233rd position. Conclusions:The patients of this CADASIL family have a c.697T>A mutation of NOTCH3 gene. This mutation may cause the change of amino acid in the structure of the wild type Notch3 protein, which may lead to increased formation of β-folding structures in the surrounding region, thus changing the structure and function of protein and causing disease.

Mechanism study on regulation of glioblastoma cell proliferation and apoptosis by sciadopitysin combined with CX-4945 through Notch1 pathway / 国际肿瘤学杂志

Objective:To investigate the effects and mechanism of sciadopitysin combined with CK2 inhibitor CX-4945 on proliferation and apoptosis of glioblastoma U87 cells.Methods:Glioblastoma U87 cells were cultured in vitro, and treated with 0.01, 0.10, 1.00, 10.00, 100.00 μmol/L of sciadopitysin respectively. U87 cells were treated with 1.25, 2.50, 5.00, 10.00, 20.00 μmol/L of CX-4945. U87 cells were divided into control group (without any treatment), sciadopitysin group (100.00 μmol/L of sciadopitysin), CX-4945 group (5.00 μmol/L of CX-4945), sciadopitysin combined with CX-4945 group (100.00 μmol/L of sciadopitysin plus 5.00 μmol/L of CX-4945). MTT method was used to detect cell viability, Caspase3/7 activity assay and Annexin Ⅴ/ PI double staining were used to detect cell apoptosis, and Western blotting was used to detect the expressions of Notch1 pathway related proteins ICN1, HES1 and DLL3. Results:The cell viabilities of U87 cells treated with 0, 0.01, 0.10, 1.00, 10.00, 100.00 μmol/L of sciadopitysin were (100.00±6.30) %, (112.02±7.63) %, (140.84±6.73) %, (113.92±7.92) %, (102.60±7.12) % and (73.16±2.74) % respectively, and there was a statistically significant difference ( F=55.21, P<0.001). There were statistically significant differences in the cell viabilities of U87 cells between 0 μmol/L and 0.01, 0.10, 1.00, 100.00 μmol/L of sciadopitysin treatment ( P=0.009; P<0.001; P=0.003; P<0.001). The cell viability of U87 cells was inhibited by 100.00 μmol/L of sciadopitysin, while sciadopitysin at other low concentrations manifested as enhancement or no obvious effect. The cell viabilities of U87 cells treated with 0, 1.25, 2.50, 5.00, 10.00, 20.00 μmol/L of CX-4945 were (100.00±5.53) %, (108.70±10.24) %, (93.14±2.82) %, (81.46±4.92) %, (56.92±3.99) % and (31.24±2.67) % respectively, and there was a statistically significant difference ( F=135.18, P<0.001). There were statistically significant differences in the cell viabilities of U87 cells between 0 μmol/L and 1.25, 5.00, 10.00, 20.00 μmol/L of CX-4945 treatment ( P=0.022; P<0.001; P<0.001; P<0.001). Low concentration (1.25 μmol/L) of CX-4945 enhanced the cell viability of U87 cells, however higher concentrations (5.00, 10.00, 20.00 μmol/L) of CX-4945 shown inhibitory effect. The cell viabilities of U87 cells in the control group, sciadopitysin group, CX-4945 group and sciadopitysin combined with CX-4945 group were (100.00±5.53) %, (71.96±2.10) %, (77.66±4.12) % and (42.56±4.22) % respectively, and there was a statistically significant difference ( F=160.56, P<0.001). There were statistically significant differences between the control group and each treatment groups (all P<0.001). There were statistically significant differences between the sciadopitysin combined with CX-4945 group and sciadopitysin group, CX-4945 group (both P<0.001). The Caspase3/7 activities of U87 cells in the above four groups were 2.34±0.47, 4.02±0.22, 3.67±0.32 and 5.85±0.28 respectively, and there was a statistically significant difference ( F=55.80, P<0.001). The apoptosis rates of each groups were (0.40±0.10) %, (17.37±0.57) %, (3.00±0.66) % and (33.47±0.87) % respectively, and there was a statistically significant difference ( F=1 822.18, P<0.001). Further pairwise comparison showed that there were statistically significant differences in Caspase3/7 activities and apoptosis rates between the control group and each treatment groups ( P<0.001, P=0.001, P<0.001; P<0.001, P=0.001, P<0.001). There were statistically significant differences in Caspase3/7 activities and apoptosis rates between the sciadopitysin combined with CX-4945 group and sciadopitysin group, CX-4945 group (all P<0.001). The protein expression levels of Notch 1 pathway related proteins ICN1 (0.55±0.07 vs. 1.01±0.09), HES1 (0.66±0.08 vs. 1.00±0.06) and DLL3 (0.74±0.04 vs. 1.01±0.09) in U87 cells decreased significantly after treatment with 100.00 μmol/L of sciadopitysin ( t=5.94, P=0.004; t=5.15, P=0.007; t=4.00, P=0.016) . Conclusion:Sciadopitysin can synergize with CK2 inhibitor CX-4945 to inhibit the proliferation and promote apoptosis of glioblastoma U87 cells by inhibiting Notch1 signaling pathway.

Mutations in the EGFr region of NOTCH3 gene and CADASIL phenotype / 国际脑血管病杂志

CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is a hereditary small vessel disease originated from adult onset, which is caused by the mutation of NOTCH3 gene located in the region of chromosome 19p13. Its clinical features include recurrent ischemic stroke, progressive cognitive impairment, migraine and mental disorders. Recent studies have shown that the mutations in the EGFr region of NOTCH3 gene are associated with the course, clinical manifestations and imaging features of CADASIL. This article reviews the research progress of the NOTCH3 gene EGFr region mutation genotype, clinical phenotype of CADASIL and their correlation, hoping to provide ideas for the early diagnosis and pathogenesis of CADASIL.

Neonatal lateral meningocele syndrome: report of a case and literature review / 中华围产医学杂志

Objective:To investigate the clinical characteristics and pathogenic gene mutation of lateral meningocele syndrome(LMS).Methods:We retrospectively collected the clinical manifestations, laboratory examination, imaging examinations, and genetic analysis of a neonate with LMS which was diagnosed at the Department of Neonatology of the Second Affiliated Hospital of Wenzhou Medical University in May 2020. Relevant literature up to February 2021, retrieved from PubMed, OMIM, CNKI, Wanfang, and CQVIP database with the terms of "lateral meningocele syndrome", " NOTCH3", were reviewed to summarize the clinical characteristics, pathogenesis, and genetic etiology of this disease. Results:A full-term male newborn was admitted to our hospital due to feeding difficulty 7 d after birth. The clinical characteristics included hypotonia, dysphagia, hypertension, lateral spinal meningocele, craniofacial anomaly, and cryptorchidism. Abnormal spinal MRI and brainstem evoked potential were also observed. Whole exome sequencing revealed a heterozygous frameshift variation c.6667_6686del(p.Ala2223Profs*12) of NOTCH3 gene located in 19p13.12, which was not detected in the parents. Only 12 English literature were retrieved, with 17 patients from 15 pedigrees. Out of the 18 patients including the index case, 10 were genetically diagnosed as LMS. The age at diagnosis ranged from 15 d to 55 years. Regarding the clinical features, multiple lateral thoracolumbar spinal meningoceles (18/18) was the most common one, followed by retrognathia and low-set ears (16/18), eyelid ptosis and down slanting palpebral fissures (15/18), hypotonia (13/18), hypertension (11/18), developmental delay (9/18), mixed or conductive hearing loss (9/18), cardiovascular dysplasia (7/18), and cryptorchidism (7/10). A total of nine NOTCH3 gene variants were detected, all were heterozygous variants, including six frameshift and three nonsense variants. Conclusions:LMS is caused by NOTCH3 gene mutation with the clinical characteristics including multiple lateral thoracolumbar spinal meningoceles, craniofacial dysmorphisms, hypotonia, hypertension, developmental delay, difficulty in feeding, cryptorchidism, etc.

Role of Notch1/Hes1 signaling pathway in high glucose and hypoxia-reoxygenation injury to cardiomyocytes / 中华麻醉学杂志

Objective:To evaluate the role of Notch1/hairy and enhancer of split homolog1(Hes1) signaling pathway in high glucose and hypoxia-reoxygenation (H/R) injury to cardiomyocytes.Methods:H9c2 cardiomyocytes were cultured in low-glucose DMEM culture medium supplemented with 10% fetal bovine serum.The cells were divided into 6 groups ( n=12 each) using a random number table method: control group (group C), H/R group, H/R+ Jagged-1 group (group H/R+ J), high glucose group (group HG), high glucose+ H/R group (group HG+ H/R) and high glucose+ H/R+ Jagged-1 group (group HG+ H/R+ J). The cells were incubated in low-glucose culture medium for 72 h in group C. After incubated in low-glucose culture medium for 72 h, the cells were exposed to 24-h hypoxia in an incubator filled with 95% N 2-5% CO 2 at 37℃, immediately followed by 6-h reoxygenation in an incubator filled with 95% O 2-5% CO 2 at 37℃ in group H/R.In group H/R+ J, Jagged-1 (Notch1/Hes1 signaling pathway specific activator) 5μg/ml was added to low-glucose culture medium and the cells were incubated for 72h before H/R.In group HG, H9c2 cardiomyocytes were incubated in high-glucose culture medium containing 33 mmol/L glucose for 72 h. In group HG+ H/R, the cells were incubated in high-glucose medium for 72 h before H/R.In group HG+ H/R+ J, Jagged-1 5μg/ml was added to high-glucose culture medium, and the cells were incubated for 72 h before H/R.At 6 h of reoxygenation, the supernatant of the culture medium was collected for detection of the activities of superoxide dismutase (SOD) and lactic dehydrogenase (LDH), the cell viability (by CCK-8 assay) and the cell apoptosis rate (by flow cytometry) and for determination of expression of Notch1, Hes1 and c-caspase-3 (by Western blot). Results:Compared with group C, the cell survival rate and SOD activity were significantly decreased, and apoptosis rate and LDH activity were increased in H/R, H/R+ J and HG groups, expression of Notch1, Hes1 and c-caspase-3 was up-regulated in H/R and H/R+ J groups, and the expression of Notch1 and Hes1 was down-regulated and c-caspase-3 expression was up-regulated in group HG ( P<0.05). Compared with group H/R, the cell survival rate and SOD activity was significantly increased, apoptosis rate and LDH activity were decreased, expression of Notch1 and Hes1 was up-regulated, and c-caspase-3 expression was down-regulated in group H/R+ J, and the cell survival rate and SOD activity were significantly decreased, apoptosis rate and LDH activity were increased, expression of Notch1 and Hes1 was down-regulated, and c-caspase-3 expression was up-regulated in group HG+ H/R ( P<0.05). Compared with group HG, the cell survival rate and SOD activity were significantly decreased, and apoptosis rate and LDH activity were increased in HG+ H/R and HG+ H/R+ J groups ( P<0.05), and expression of Notch1 and Hes1 was down-regulated, and c-caspase-3 expression was up-regulated in group HG+ H/R ( P<0.05). Compared with group HG+ H/R, the cell survival rate and SOD activity were significantly increased, apoptosis rate and LDH activity were decreased, expression of Notch1 and Hes1 was up-regulated, and c-caspase-3 expression was down-regulated in group HG+ H/R+ J ( P<0.05). Compared with group H/R+ J, the cell survival rate and SOD activity were significantly decreased, apoptosis rate and LDH activity were increased, expression of Notch1 and Hes1 was down-regulated, and c-caspase-3 expression was up-regulated in group HG+ H/R+ J ( P<0.05). Conclusion:Activation of Notch1/Hes1 signaling pathway is the endogenous protective mechanism of high glucose and H/R injury to cardiomyocytes.

Notch1 in Tumor Microvascular Endothelial Cells and Tumoral miR-34a as Prognostic Markers in Locally Advanced Triple-Negative Breast Cancer / 한국유방암학회지

Effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells / 国际肿瘤学杂志

Objective To investigate the effect of targeted silencing Notch1 on proliferation and apoptosis of human non-small cell lung cancer stem cells.Methods Lung cancer A549 cells and SPC-A-1 cells were selected and divided into control group,Nc-shRNA group and Notch1-shRNA group.The Nc-shRNA group was a negative control RNAi lentivirus group,and the Notch1-shRNA group was a Notch1 inhibitory RNAi lentivirus group.The lentiviral-mediated shRNA interference technology was used to target the silencing of Notch1.The silencing effect of Notch1 gene was verified by quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting.Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) and sarcosphere formation assay.Apoptosis was detected by Annexin V/7-AAD double staining.Western blotting was used to detect the expression of proliferating cell nuclear antigen (PCNA),B-cell lymphoma-2 (Bcl-2) and Notch1 downstream gene Hes-1.Results The results of qRT-PCR showed that the relative expression levels of Notch1 in control group,Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1 cells were 1.000 ± 0.000,0.937 ± 0.025,0.490 ± 0.036 and 1.000 ± 0.000,1.077 ± 0.070,0.373± 0.038,with statistically significant differences (F =359.707,P ＜0.001;F =210.455,P ＜0.001),further paired comparison,the relative expression of Notch1 in Notch1-shRNA group was significantly lower than that in Nc-shRNA group (all P ＜ 0.05).Western blotting showed that the expressions of Notch1 protein in A549 cells and SPC-A-1 cells were consistent with the mRNA results.MTT assay showed that the 24 h A values of A549 cells in control group,Nc-shRNA group and Notch1-shRNA group were 0.209 ± 0.005,0.219 ± 0.009,0.159 ±0.006,48 h A values were 0.293 ± 0.004,0.302 ± 0.004,0.205 ± 0.005,72 h A values were 0.450 ± 0.003,0.430 ± 0.012,0.348 ± 0.017,with statistically significant differences (F =79.487,P＜0.001;F =508.664,P ＜0.001;F =57.156,P ＜0.001),further paired comparison,the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 24,48,72 h (all P ＜ 0.05).The 48 h A values of SPC-A-1 cells in control group,Nc-shRNA group and Notch1-shRNA group were 0.438 ±0.022,0.412 ± 0.015,0.364 ± 0.010,72h A values were 0.540 ± 0.016,0.519 ± 0.009,0.438 ± 0.019,with statistically significant differences (F =15.667,P =0.004;F =37.299,P ＜ 0.001),further paired comparison,the proliferation ability of Notch1-shRNA group was significantly lower than that of Nc-shRNA group at 48 h and 72 h (all P ＜ 0.05).The sphere sizes of control group,Nc-shRNA group and Notch1-shRNA group in A549 cells were (149.667 ± 6.506) μm,(136.667 ± 7.095) μm,(86.676 ± 7.638) μm,with statistically significant difference (F =65.940,P ＜ 0.001).The sphere sizes of the three groups in SPC-A-1 cells were (118.667 ± 6.658) μm,(128.000 ± 7.000) μm,(60.675 ± 4.509) μm,with statistically significant difference (F =105.372,P ＜0.001).Further paired comparison,the sphere size of Notch1shRNA group was significandy smaller than that of Nc-shRNA group in the two kinds of cells (all P ＜ 0.05).The apoptosis rates of control group,Nc-shRNA group and Notch1-shRNA group in A549 cells and SPC-A-1cells were (0.489 ± 0.014)％,(0.633 ± 0.021)％,(1.683 ± 0.221)％ and (1.323 ± 0.194)％,(1.690 ± 0.188) ％,(3.017 ± 0.356) ％,with statistically significant differences (F =77.660,P ＜ 0.001;F=32.200,P =0.001),further paired comparison,the apoptosis rate of Notch1-shRNA group was significantly higher than that of Nc-shRNA group in the two kinds of cells (all P ＜ 0.05).Western blotting showed that the expressions of PCNA,Bcl-2 and Hes-1 in control group,Nc-shRNA group and Notch1-shRNA group in A549 cells were statistically significant (F =155.343,P ＜ 0.001;F =22.576,P =0.002;F =70.108,P＜0.001),and the expressions of PCNA,Bcl-2 and Hes-1 in the three groups in SPC-A-1 cells were statistically significant (F =49.419,P ＜0.001;F =28.090,P =0.001;F =12.040,P =0.007).Further paired comparison,the expressions of PCNA,Bcl-2 and Hes-1 in Notch1-shRNA group were significantly lower than those in Nc-shRNA group in the two kinds of cells,and the differences were statistically significant (all P ＜0.05).Conclusion Targeted silencing of Notch1 can reduce the proliferation activity of lung cancer stem cells and promote apoptosis,which may be related to the down-regulation of its downstream gene Hes-1.

The expression and role of Notch1/RBP-Jκ/Msx2 signaling pathway in the aorta of chronic kidney disease rats with vascular calcification / 中华肾脏病杂志

Objective To observe the expression of Notch1 signaling pathway in the aorta of chronic kidney disease (CKD) rats with vascular calcification and to explore the role of this signaling pathway activation in aortic calcification of CKD.Methods A total of 40 male SD rats were randomly divided into normal group (Nor group) and CKD with vascular calcification group (CKD+VC group).Rats in each group were sacrificed at 4 weeks,6 weeks and 8 weeks respectively after the success of modeling.Their 24-hour urine was reserved to test 24 hour urine protein (24 h Upro);blood sample was collected from abdominal aorta to test blood urea nitrogen (BUN),serum creatinine (Scr),Ca and P.The histopathology of renal was detected by HE staining.The aortic calcification was detected by alizarin red S staining.Immunohistochemistry (IHC) was used to test the protein expressions of alpha-smooth muscle actin (or-SMA),Runt-related transcription factor 2 (Runx2),Notchl,recombination signal-binding protein for immunoglobulin kappa J region (RBP-Jκ),Msh homeobox 2 (Msx2),Jagged1 and Notch1 intracellular domain (N1-ICD) in the aorta.Real time PCR was applied to detect the mRNA expressions of α-SMA,smooth muscle 22 alpha (SM22α),Runx2,alkaline phosphatase (ALP),Notch1,RBP-Jκ,Msx2 and Jagged1.Results Compared with those in Nor group,24 h Upro,BUN and Scr increased in the CKD+VC group at 4th,6th and 8th weeks (all P ＜ 0.05).Numerous continuous calcified nodules were detected in the vascular wall of the CKD+VC group,but none in Nor group.As compared with Nor group,the expression of α-SMA was low,while the expression of Runx2 was relatively high in the CKD+VC rats at each time point (all P ＜ 0.05).The expressions of Notchl,RBP-Jκ,Msx2,Jaggedl and NI-ICD in the Nor group were slightly appeared in the aortic wall,while in CKD+VC group these signal protein expressions increased relatively during the experimental period (all P ＜ 0.05).As compared with Nor group,the expressions of α-SMA and SM22α mRNA were low,yet the expressions of Runx2 and ALP mRNA were high in the CKD+VC rats at each time point (all P ＜ 0.05).The mRNA levels of Notch1,RBP-Jκ,Msx2 and Jagged1 in the CKD+VC group at each time point were significantly up-regulated as compared with the Nor group (all P ＜ 0.05).Conclusions There exist phenotypic changes in smooth muscle cells and activations of Notch1/RBP-Jκ/Msx2 signaling pathway in CKD rats with vascular calcification.It may be one of the important signal transduction pathways.

Notch signaling pathway regulates osteosarcoma stem cell characteristics by inhibiting Eph pathway / 中华骨科杂志

Objective To investigate the role of Notch signaling pathway to maintain the stem cell-like characteristics of osteosarcoma and its underlying mechanism.Methods Lentiviral NICD1 or Numb-shRNA was transduced into MG63 osteosarcoma cells to activate Notch activity in vitro.The impact of Notch on osteosarcoma stem cells were assessed by the tumor sphere formation assay and flow cytometry analysis of cell surface markers STRO-1/CD117.The expression of stem cell related genes (Sox2,Oct4) were evaluated by Western blot and qPCR.The nude mice were randomly divided into 3 groups:the NICD1 overexpression (NICD-OE) group,the DAPT group and the control (CON) group.The tumor growth was monitored for 8 weeks and the tumor volume and weight were recorded weekly.To investigate whether Notch regulates Eph pathway,Eph pathway related protein EphB,pEphB was measured by Western blot.The impact of ephrinB 1 on NICD overexpression cell were assessed by tumor sphere formation assay.The expression of Sox2 and Oct4 was evaluated by Western blot.Results NICD1 overexpression or Numb-shRNA increased the activity of Notch pathway.The Notch-activated osteosarcoma showed enhanced in vitro tumor spheroid formation capacity,increased Stro-1/CD117double positive ratio,and upregulated expression of Sox2 and Oct4 in vitro.In animal experiments,it was found that activation of Notch pathway promoted tumor formation in vivo and Notch inhibition decreased it.The primary osteosarcoma cells were obtained from mice xenograft treated with DAPT and its tumor sphere formation capacity was significantly reduced.Finally,The Notch pathway inhibits the phosphorylation of EphB,as well as the downstream signal pathway of EphB,but there is no significant change in total EphB.The activation of Eph pathway inhibited Notch induced up-regulation of tumor sphere formation and Sox2 and Oct4 expression.Conclusion Notch signaling pathway maintains the stem cell-like characteristics of osteosarcoma probably by inhibiting the Eph pathway.

The predicting value of Notch1 expressed on peripheral blood mononuclear cell for coronary artery calcification / 中国医师杂志

Objective To investigate the predicting value of Notch1 levels expressed in peripheral blood mononuclear cell (PBMC) for coronary artery calcification.Methods 300 consecutive patients with coronary artery disease (CAD) who hospitalizing in the department of cardiology in Guangzhou first people's hospital from January 2016 to June 2017 were enrolled.All Patients received 320-slice multi-detector row computed tomography scanning and coronary artery calcium sore(CCS)were measured.Patients were divided into three groups:control group (CCS =0),Low CCS group (CCS ＜97.6) and high CCS group (CCS ≥97.6) according to the mean value of CCS (CCS =97.6).Notch1 expressed in PBMC,serum interlekin-6 (IL-6) and high sensitivity C reactive protein (hs-CRP)of patients were examined and compared among three groups.Results The levels of Notch1 in PBMC and serum IL-6,hs-CRP of patients in high CCS group were significant higher than the other two groups [Notch1:7.02 ± 0.86 vs 6.32 ± 0.78 vs 5.49 ± 0.71;IL-6:(133.66 ± 10.18) μg/L vs (127.49 ± 10.79) μg/L vs (111.62 ± 9.87) μg/L;hs-CRP:(3.98 ± 1.02) mg/L vs (3.11 ±0.95)mg/L vs (2.56 ±0.76)mg/L] (P ＜0.05).The Spearman correlation analysis showed that the levels of Notch1 in PBMC were positive correlated with the levels of serum IL-6 and hs-CRP in enrolled patients with coronary calcification (P ＜ 0.05).Multivariate logistic regression analysis showed that the levels of Notch1 in PBMC and serum IL-6 were two strong independent risk factors for severity of coronary calcification in patients with CAD (P ＜ 0.05).Conclusions Notch1 expression in PBMC of patients with CAD was valuable to predicate the severity of coronary calcification.That the Notch1 signal path regulating the inflammation conditions in patients may be one of the most important mechanisms in the formation and progress of coronary calcification.

Association of notch 1 signal pathway with lymph node and distant metastases of papillary thyroid carcinoma: A meta analysis / 肿瘤

Objective: To analyze the associations of Notch 1 expression with the lymph node metastasis and distant metastasis of papillary thyroid carcinoma (PTC) by Meta analysis. Methods: Computer retrieve was conducted in PubMed (MEDLINE), Cochrane Library, EMBASE, China Journal Full-text Database (CNKI), China Biology Medicine disc (CBMdisc) to search the studies which were about the association of Notch1 signal with the lymph node and distant metastases of PTC, and published from 2010 to 2017. The literatures were screened and evaluated, then the information was extracted independently by 2 researchers according to the literature selection criteria. Meta analysis was performed using RevMan 5.3 and STATA 12.0 software. The odds ratio (OR) and 95% confidence interval (CI) were calculated. The sensitivity analysis and publication bias test were performed. Results: A total of 7 clinical case-control studies involving 743 patients with PTC were selected. Meta-analysis showed that the expression of Notch1 was significantly positively correlated with lymph node metastasis of PTC (OR = 4.68, 95% CI: 3.00-7.30), furthermore the test for overall effect showed that Z = 6.80 and P < 0.000 01. However, there was no significant correlation between Notch1 expression and the distant metastasis of PTC (OR = 1.59, 95% CI: 0.88-2.89), the test for overall effect showed that Z and P values were 1.53 and 0.1 3 respectively. Conclusion: The Notch1 signaling pathway plays a promoting role in the lymph node metastasis of PTC, which suggests that the expression of Notch1 has a certain predictive value for the clinical prognosis of PTC.

Papel de la vía de señalización Notch en la diferenciación de las células inmunes / Role of Notch signaling pathway in the differentiation of immune cells

Resumen La vía de señalización Notch es una vía conservada evolutivamente y está involucrada en el control de diversos eventos durante el desarrollo de las células eucariotas. Esta vía se ha relacionado con la expresión y la diferenciación de las células inmunes; por lo tanto, su activación es fundamental en la expresión de la respuesta inmune innata y adquirida, que permite a los mamíferos defenderse frente antígenos externos.

Abstract The Notch signaling pathway is evolutionarily preserved and is involved in the control of several events during the development of eukaryotic cells. This pathway has been linked to expression and differentiation of immune cells, therefore it´s activation is critical in the expression of the innate immune and acquired response that allows mammals to defend themselves against external antigens.

The effect of Notch1 and Delta-like ligand 4 in the neovascularization of the proliferative diabetic retinopathy / 中华眼底病杂志

Objective o observe the expression of Notch1 and Delta-like ligand 4 (Dll4) on the fibrovascular membranes in proliferative diabetic retinopathy (PDR),and investigate its relationship with vascular endothelial growth factor receptor 2 (VEGFR2).Methods Fifty-seven PDR patients (60 eyes) who underwent vitrectomy were enrolled in this study.The PDR patients were divided into non-injection group (30 patients,32 eyes) and injection group (27 patients,28 eyes).The eyes in injection group received intravitreal injection with ranibizumab at 2 to 7 days before surgery.The preretinal fibrovascular membranes were obtained from the PDR patients during vitrectomy.Eighteen epiretinal membranes were obtained from the non-diabetic patients was served as controls.The real-time polymerase chain reaction (RT-PCR) and immunohistochemical methods were used to detecting the expression ofNotch1,Dll4 and VEGFR2.In the meantime,the numbers of the nucleus of vascular endothelial cells in the membranes stained with hematoxylin were counted.Results The immunohistochemical staining revealed that there were positive expression ofNotch1,Dll4 and VEGFR2 in all PDR membranes,regardless of the injection of the ranibizumab.The levels ofNotch1,Dll4 and VEGFR2 protein in non-injection group were higher than those of injection group (t=3.45,6.01,4.08;P=0.030,0.008,0.023).In injection group,the number of endothelial cells in the membranes reduced (17.17 ± 2.48) compared with that of the non-injection group (41.50± 5.57).There was significant difference in the number of endothelial cells in the membranes between the two groups (t=9.58,P＜0.05).RT-PCR showed that the differences of the mRNA expression ofNotch1,Dll4 and VEGFR2 were all statistically significant among the PDR group and control group (H=12.50,12.50,12.02;P＜0.05).The expression ofNotch1,Dll4 and VEGFR2 in the PDR membranes was higher than that of epiretinal membranes from non-diabetic patients.In the PDR group,the expression of Notch1,Dll4 and VEGFR2 of non-injection group was higher than that of injection group.Spearman correlation analysis showed that the expression of mRNA between VEGFR2 and Dll4 (r=0.83),VEGFR2 and Notch1 (r=0.81),Notch1 and Dll4 (r=0.87) were all significantly correlated (P＜0.05).Conclusions The expression of Notch1 and Dll4 in the PDR membranes are higher than that of the control group,and it is positively correlated with the expression of the VEGFR2.Notchl and Dll4 play a regulatory rule in the neovascularization in PDR,the acting way may be correlated with VEGFR2.

The Role of Notch1 Signaling in Anaplastic Thyroid Carcinoma / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The Notch signaling pathway is widely expressed in normal, reactive, and neoplastic tissues; however, its role in thyroid tissues has not been fully elucidated. Therefore, this study was conducted to characterize the expression of the Notch signaling pathway in papillary thyroid cancer (PTC) cells and anaplastic thyroid cancer (ATC) cells. MATERIALS AND METHODS: Expression of activated Notch1 in ATC and PTC paraffin-embedded tissues was determined by immunohistochemistry. The small interfering RNA techniquewas employed to knock down Notch1 expression in ATC and PTC cell lines. RESULTS: The expression of activated Notch1 was higher in ATC cases than in PTC cases. Inhibition of Notch1 significantly reduced proliferation and migration of ATC cells, but not PTC cells. In addition, inhibition of Notch1 in ATC cells significantly reduced the expression of key markers of epithelial-mesenchymal transition and cancer stem cells. Conversely, changes in the expression of these proteins were not observed in PTC cells. CONCLUSION: The results of this study suggest that Notch1 expression plays different roles in tumor progression in ATC and PTC cells. We also found that Notch1 expression was significantly related to the highly invasive or proliferative activity of ATC cells.

Clinical significance of S100A6 and Notch1 in multiple myeloma patients / 中华血液学杂志

Objective@#To investigate the expression levels of S100A6, Notch1 in multiple myeloma (MM) patients and its clinical significance.@*Mathods@#The expression levels of S100A6, Notch1 in 28 MM cases and 20 healthy controls were determined by real time quantitative PCR (RQ-PCR) , and their relationships with clinical features and outcomes were analyzed. Immunohistochemical was used to analysis the levels of S100A6 and Notch1 in bone marrow biopsy samples and intramedullary metastases soft tissues. RQ-PCR and Western blot were used to test the changes of Notch1 mRNA and Notch1 protein in U266 MM cells after S100A6 silenced by siRNA.@*Results@#①The expression levels of S100A6, Notch1 in primary MM patients was 2.19±1.25, 2.98±0.64, significantly higher than those in controls (0.71±0.20, 0.58±0.39, P<0.05) and patients in platform status (0.85±0.26, 0.72±0.40, P<0.05) . 8 cases with intramedullary metastasis had significantly higher levels of S100A6 (3.36±1.23) and Notch1 (5.71±3.96) , as compared to those without extra medullary metastases. ②S100A6 expression was positive correlation with Notch1 (r=0.505, P=0.007) . ③S100A6 and Notch1 proteins were positive in plasma cells of bone marrow biopsy samples and intramedullary metastases soft tissues. ④The Notch1 mRNA and Notch1 expression decreased significantly after 48 hours treatment by S100A6 siRNA in U266 cells.@*Conclusion@#S100A6 and Notch1 were closely associated with MM progress and intramedullary metastasis. They have significant correlation and might be as two prognostic molecular markers in MM.

Microglia mediated inflammatory signaling pathways after ischemic stroke / 国际脑血管病杂志

Microglia is the most important immune cell in the central nervous system.It plays a key role in mediating the immune response in the central nervous system.Sterile inflammation is the key factor in the pathophysiological process following ischemic stroke.Microglia is activated and induces a series of inflammatory signals through binding of injuring related ligands to their corresponding receptors.This article introduces the activation of microglia and inflammatory response after ischemic stroke from Toll-like receptors,inflammasome,cytokine receptors,Notch signaling and other signaling pathways.

Clinical and imaging features of CADASIL / 国际脑血管病杂志

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic cerebral small vessel disease.Migraine,recurrent subcortical ischemia,progressive cognitive impairment,and emotional disorders are the main features.The diagnosis of CADASIL depends on typical clinical symptoms and neuroimaging findings,and it is confirmed by skin biopsyz and gene testing.In recent years,some new insights have been obtained in the clinical and imaging features of CADASIL This article reviews the research progress in this field.

Endogenous neural stem cells promotes Notch-1 expression after spinal cord injury in rats / 中国医师杂志

Objective To explore the expressions and changes of endogenous neural stem cells (ENSCs) and Notch-1 after acute spinal cord injury in adult rats,and to explore its role in the nerve regeneration process.Methods The 30 Sprague Dawley (SD) female rats (3-6 months) (300-350 g) were divided into control groups (n =5) and experimental group (n =25) by digital random method.The control group only accepted lamina decompression,and the experimental group was the spinal cord injury group.Five rats were drawn when at 1 d,3 d,1 w,2 w,and 4 w,then behavior,histology,immunohistochemistry and immunofluorescence method were used to detect the proliferation and expression of endogenous neural stem cells and Notch-1 protein.Results Behavioral observation showed the experimental group rats were disfunction.Histological observation showed nerve fiber structure turbulence,edema and denaturation in the experimental group.Immunohistochemistry staining showed the Notch-1 expression every experimental group.Notch-1 positive cell peaked at 3 days.Immunofluorescence test showed,in the experimental group damage to segment the area surrounding the BrdU positive staining cells was significantly higher than the control group.Using three-dimensional quantitative study method detected in 1 w after spinal cord injury was the number of newborn cells mitosis,most about was about 75 times in the control group.Linear regression method was used to analyze the different time points after BrdU and Notch-1 protein expression positive area,the area of the results found that both into linear correlation.Conclusions The new born neurons after spinal cord injury in rats Notch-1 expression has a certain relevance.In addition,the expression of signal protein Notch-1,might be associated with early proliferation of ENSCs in rats.

Endogenous neural stem cells promotes Notch-1 expression after spinal cord injury in rats / 中国医师杂志

Objective To explore the expressions and changes of endogenous neural stem cells (ENSCs) and Notch-1 after acute spinal cord injury in adult rats,and to explore its role in the nerve regeneration process.Methods The 30 Sprague Dawley (SD) female rats (3-6 months) (300-350 g) were divided into control groups (n =5) and experimental group (n =25) by digital random method.The control group only accepted lamina decompression,and the experimental group was the spinal cord injury group.Five rats were drawn when at 1 d,3 d,1 w,2 w,and 4 w,then behavior,histology,immunohistochemistry and immunofluorescence method were used to detect the proliferation and expression of endogenous neural stem cells and Notch-1 protein.Results Behavioral observation showed the experimental group rats were disfunction.Histological observation showed nerve fiber structure turbulence,edema and denaturation in the experimental group.Immunohistochemistry staining showed the Notch-1 expression every experimental group.Notch-1 positive cell peaked at 3 days.Immunofluorescence test showed,in the experimental group damage to segment the area surrounding the BrdU positive staining cells was significantly higher than the control group.Using three-dimensional quantitative study method detected in 1 w after spinal cord injury was the number of newborn cells mitosis,most about was about 75 times in the control group.Linear regression method was used to analyze the different time points after BrdU and Notch-1 protein expression positive area,the area of the results found that both into linear correlation.Conclusions The new born neurons after spinal cord injury in rats Notch-1 expression has a certain relevance.In addition,the expression of signal protein Notch-1,might be associated with early proliferation of ENSCs in rats.