Hypoxia Upregulates the Notch3 Signaling Pathway to Promote Epithelial-Mesenchymal Transition in Pulmonary Artery Cells.

Objective: To investigate the role and mechanism of Notch3 in a hypoxia-induced model of pulmonary hypertension, specifically pulmonary artery hypertension. Methods: A pulmonary artery hypertension rat model was induced using monocrotaline, and hepatic encephalopathy staining was used to observe the pathomorphological changes in pulmonary artery tissue. Primary isolation and extraction of rat pulmonary artery endothelial cells were performed, and a pulmonary artery hypertension cell model was established through hypoxia induction. Notch3 overexpression lentivirus (LV-Notch3) was used for intervention, and the expression of the Notch3 gene was detected using a real-time polymerase chain reaction. Western blotting was conducted to assess the expression of vascular endothelial growth factor, matrix metalloproteinase-2, and matrix metalloproteinase-9 proteins. Cell proliferation levels were measured using a medical training therapy assay. Results: Compared to the control group, the model group showed significant thickening of the pulmonary artery membrane, increased pulmonary angiogenesis, and endothelial cell damage. After Notch3 overexpression, the LV-Notch3 group showed further thickening of the pulmonary artery tunica media, increased pulmonary angiogenesis, and significantly improved endothelial cell injury. Compared to control cells, the model group showed a significant decrease in Notch3 expression (P < .05), while the expression levels of vascular endothelial growth factor, MMP-2, and MMP-9 proteins and cell proliferation ability increased significantly (P < .05). Following Notch3 overexpression, there was a significant increase in Notch3 expression (P < .05), and the expression levels of vascular endothelial growth factor, MMP-2, and MMP-9 proteins, as well as cell proliferation ability decreased significantly (P < .05). Conclusions: Notch3 can potentially reduce angiogenesis and proliferation in pulmonary artery endothelial cells and improve hypoxia-induced pulmonary artery hypertension in rats.

Total flavonoids of Litchi seed attenuate stem cell-like properties in breast cancer by regulating Notch3 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Breast cancer has been the most commonly-diagnosed cancer worldwide, and the treatment and prognosis of which are often limited by breast cancer stem cells (BCSCs). Litchi seeds have shown good anti-cancer activity in various cancers including prostate cancer, lung cancer and breast cancer. However, the activity and underlying mechanism of Litchi seeds against BCSCs remain unknown. AIM OF THE STUDY: To investigate the activity and mechanism of total flavonoids of litchi seed (TFLS) against BCSCs in vitro and in vivo. MATERIALS AND METHODS: Two orthotopic xenograft mouse models were established using HCC1806 cells pretreated or untreated with TFLS to determine whether TFLS could target BCSCs in vivo. Mammosphere formation and flow cytometry assays were employed to evaluate the effect of TFLS on BCSCs in vitro. The underlying mechanism was investigated using RT-qPCR, Western blot, immunohistochemistry and immunofluorescence experiments. RESULTS: TFLS could significantly inhibit the viability of HCC1806, MCF-7 and HCC1937 cells in vitro and suppress the growth of HCC1806 cells in vivo. TFLS attenuated stem cell-like properties of breast cancer through reducing the percentage of CD44+CD24-/low cells, inhibiting the mammospheres formation and down-regulating the mRNA and protein levels of cancer stem cells related markers (Oct4, Nanog, Sox2) in MCF-7 and HCC1806 cells. Meanwhile, TFLS suppressed the tumor-initiating ability of BCSCs via reducing the percentage of CD44+CD24-/low cells in tumor and lowering tumor incidence rate in orthotopic xenograft mice. In addition, TFLS treatments restricted the expression and nuclear translocation of Notch3, subsequently down-regulated Hes1 and Runx2 expressions. CONCLUSIONS: TFLS could suppress the growth of breast cancer and eliminate breast cancer stem cells by inhibiting the Notch3 signaling pathway.

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.

Omega-3 fatty acids impair miR-1-3p-dependent Notch3 down-regulation and alleviate sepsis-induced intestinal injury.

BACKGROUND: Sepsis is a troublesome syndrome that can cause intestinal injury and even high mortality rates. Omega-3 fatty acids (FAs) are known to protect against intestinal damage. Accordingly, the current study set out to explore if omega-3 FAs could affect sepsis-induced intestinal injury with the involvement of the microRNA (miR)-1-3p/Notch3-Smad axis. METHODS: First, cecal ligation and perforation (CLP) was performed to establish septic mouse models in C57BL/6J mice, and mouse intestinal epithelial MODE-K cells were induced by lipopolysaccharide (LPS) to establish sepsis cell models. The CLP-induced septic mice or LPS-exposed cells were subjected to treatment with Omega-3 FAs and activin (Smad signaling activator), miR-1-3p inhibitor and over-expressed/short hairpin RNA (oe-/sh)-Notch3 to explore their roles in inflammation, intestinal oxidative stress and cell apoptosis. A dual-luciferase reporter gene assay was further performed to verify the regulatory relationship between miR-1-3p and Notch3. RESULTS: Omega-3 FAs inhibited CLP-induced intestinal injury and ameliorated LPS-induced intestinal epithelial cell injury by down-regulating miR-1-3p, as evidenced by decreased levels of tumor necrosis factor-α, interleukin-1ß (IL-1ß) and IL-6, in addition to diminished levels of reactive oxygen species, malondialdehyde levels and superoxide dismutase activity. Furthermore, miR-1-3p could down-regulate Notch3, which inactivated the Smad pathway. CONCLUSION: Collectively, our findings indicated that omega-3 FAs elevate the expression of Notch3 by down-regulating miR-1-3p, and then blocking the Smad pathway to alleviate intestinal epithelial inflammation and oxidative stress injury caused by sepsis.

Vitamin C alleviates the senescence of periodontal ligament stem cells through inhibition of Notch3 during long-term culture.

Periodontal ligament stem cells (PDLSCs), as potential "seed cells" for periodontal tissue repair and regeneration, require to be expanded in vitro for a large scale. Senescence of PDLSCs occurred during long-term culture may compromise the therapeutic effects of PDLSCs. Medium supplements may be useful in antisenescence. However, the effects and mechanisms of vitamin C (Vc) treatment on PDLSCs during long-term culture are still unclear. In this study, we identified that Vc-treated PDLSCs cells maintained a slender morphology, higher growth rate and migration capacity, stemness, and osteogenic differentiation capability during a long-term culture. Moreover, we also identified that Notch3 was significantly upregulated during the cell senescence, and Vc treatment alleviated the senescence of PDLSCs through inhibition of Notch3 during long-term culture. In summary, Vc treatment suppressed PDLSCs senescence by reducing the expression of Notch3 and might be a simple and useful strategy to inhibit cellular senescence during the cell long-term culture.

Notch signaling pathway mediates the immunomodulatory mechanism of Yangfei Huoxue decoction alleviating bleomycin-induced pulmonary fibrosis in rats.

OBJECTIVE: To investigate the immunomodulatory mechanism by which Yangfei Huoxue decoction (YHD) alleviates bleomycin (BLM)-induced pulmonary fibrosis (PF) in rats. METHODS: Rats were randomly divided into two time-point groups (day 14 and 28), and each time-point group comprised the following six subgroups: control, BLM, dexamethasone (DXM), YHD high dose (YHD-H), YHD middle dose (YHD-M), and YHD low dose (YHD-L). Haematoxylin and eosin and Masson staining, flow cytometry, enzyme-linked immunosorbent assay, Western blotting and UPLC-QT of analyses were examined. RESULTS: The results showed that YHD reduced the degree of alveolar inflammation and fibrosis; downregulated the expression of CD28, CD80, CD86, Delta-like 1, Notch2, and Notch3; and upregulated the proportions of Th1/Th2 and Tc1/Tc2. The seven components of YHD were detected. CONCLUSION: The current study indicates that YHD mainly functions by regulating the immune system and that the molecular mechanism may be related to the regulation of the Notch signaling pathway.

Activation of notch 3/c-MYC/CHOP axis regulates apoptosis and promotes sensitivity of lung cancer cells to mTOR inhibitor everolimus.

The mammalian target of rapamycin (mTOR) pathway converges diverse environmental cues to support the lung cancer growth and survival. However, the mTOR-targeted mono-therapy does not achieve expected therapeutic effect. Here, we revealed that fangchinoline (FCL), an active alkaloid that purified from the traditional Chinese medicine Stephania tetrandra S. Moore, enhanced the anti-lung cancer effect of mTOR inhibitor everolimus (EVE). The combination of EVE and FCL was effective to activate Notch 3, and subsequently evoked its downstream target c-MYC. The blockage of Notch 3 signal by the molecular inhibitor of γ-secretase or siRNA of Notch 3 reduced the c-MYC expression and attenuated the combinational efficacy of EVE and FCL on cell apoptosis and proliferation. Moreover, the c-MYC could bind to the C/EBP homologous protein (CHOP) promoter and facilitate CHOP transcription. The conditional genetic deletion of CHOP reduced the apoptosis on lung cancer cells to the same degree as blockage of Notch 3/c-MYC axis, providing further evidence for that the Notch 3/c-MYC axis regulates the transcription of CHOP and finally induces apoptosis upon co-treatment of FCL and EVE in lung cancer cells. Overall, our findings, to the best of our knowledge, firstly link CHOP to Notch 3/c-MYC axis-dependent apoptosis and provide the Notch 3/c-MYC/CHOP activation as a promising strategy for mTOR-targeted combination therapy in lung cancer treatment.

Evodiamine suppresses Notch3 signaling in lung tumorigenesis via direct binding to γ-secretases.

BACKGROUND: Notch activation requires proteolytic cleavage of the receptor by γ-secretase protein complex. Inhibition of Notch receptor activation (e.g. Notch3) with γ-secretase inhibitor is a potential new therapeutic approach for the targeted therapy of non-small cell lung cancer (NSCLC). However, only a few safe and effective γ-secretase inhibitors have been discovered. Evodiamine (EVO), a compound derived from Euodiae Fructus (Chinese name, Wu-Zhu-Yu), exhibits remarkable anti-NSCLC activities. However, the underlying mechanisms of action have yet to be fully elucidated. PURPOSE: We sought to determine the involvement of Notch3 signaling in the anti-NSCLC effects of EVO, and to explore whether EVO suppressed Notch3 signaling by inhibiting γ-secretase in cultured A549 and H1299 NSCLC cells and in urethane-induced lung cancer FVB mouse model. METHODS: Cell viability, migration, stemness and cell cycle distribution of EVO were examined by the MTT assay, wound healing assay, soft agar colony assay and flow cytometry analysis, respectively. The binding affinity of EVO and γ-secretase complex was analyzed by molecular docking. Cellular thermal shift assay (CETSA) was performed to study the drug-target interactions in NSCLC cells. Protein levels were determined by Western blotting. RESULTS: EVO dramatically inhibited cell viability, induced G2/M cell cycle arrest, suppressed cell migration, and reduced stemness in NSCLC cells. Mechanistic studies indicated that EVO prevented the γ-secretase cleavage of Notch3 at the cell surface and hence inhibited Notch3 activation. Moreover, EVO notably reduced tumor growth in the mouse model and inhibited Notch3 activity in the tumors. CONCLUSION: This study provides new insights into the anti-NSCLC action of EVO, and suggests that suppressing Notch3 signaling by inhibiting γ-secretase is a mechanism of action underlying the anti-NSCLC effect of EVO.

Electroacupuncture Reduced Apoptosis of Hippocampal Neurons in Mice with Cerebral Infarction by Regulating the Notch3 Signaling Pathway.

This study is designed to explore the effect of electric acupuncture on the apoptosis of hippocampal neurons through the Notch3 signaling pathway. A middle cerebral artery occlusion (MCAO) mouse model was generated by thread embolization. A Y-maze was used to test cognitive function (learning and memory). The apoptosis factor caspase-3 was identified through an enzyme-linked immunosorbent assay (ELISA). TTC staining was used to evaluate the cerebral infarction area. Apoptosis of hippocampal neurons was identified by TUNEL staining and flow cytometry. The expression levels of genes and proteins related to the Notch3 signaling pathway were assessed by qRT-PCR and Western blot. It was determined that 7 days of electroacupuncture treatment improved recovery from cerebral infarction. Electroacupuncture therapy reduced the cerebral infarction area in mice and decreased the apoptosis of hippocampal neurons. The apoptosis rate of hippocampal neurons and the area of cerebral infarction were significantly lower in the electroacupuncture group than in the MCAO group (P < 0.01), while the apoptosis rate of hippocampal neurons and the area of cerebral infarction were significantly higher in the inhibitor group than in the electroacupuncture group (P < 0.01). The expression levels of genes related to the Notch3 signaling pathway were significantly increased in the electric acupuncture treatment group compared with the MCAO group (P < 0.01). The results showed that electroacupuncture inhibited the apoptosis of hippocampal neurons through the Notch3 signaling pathway, while the expression levels of genes related to the Notch3 signaling pathway were significantly higher in the inhibitor group than in the electroacupuncture group (P < 0.01). Electroacupuncture inhibited the apoptosis of hippocampal neurons in mice with cerebral infarction by stimulating the Notch3 signaling pathway and triggering corresponding protein expression.

Hdac1 Regulates Differentiation of Bipotent Liver Progenitor Cells During Regeneration via Sox9b and Cdk8.

BACKGROUND & AIMS: Upon liver injury in which hepatocyte proliferation is compromised, liver progenitor cells (LPCs), derived from biliary epithelial cells (BECs), differentiate into hepatocytes. Little is known about the mechanisms of LPC differentiation. We used zebrafish and mouse models of liver injury to study the mechanisms. METHODS: We used transgenic zebrafish, Tg(fabp10a:CFP-NTR), to study the effects of compounds that alter epigenetic factors on BEC-mediated liver regeneration. We analyzed zebrafish with disruptions of the histone deacetylase 1 gene (hdac1) or exposed to MS-275 (an inhibitor of Hdac1, Hdac2, and Hdac3). We also analyzed zebrafish with mutations in sox9b, fbxw7, kdm1a, and notch3. Zebrafish larvae were collected and analyzed by whole-mount immunostaining and in situ hybridization; their liver tissues were collected for quantitative reverse transcription polymerase chain reaction. We studied mice in which hepatocyte-specific deletion of ß-catenin (Ctnnb1flox/flox mice injected with Adeno-associated virus serotype 8 [AAV8]-TBG-Cre) induces differentiation of LPCs into hepatocytes after a choline-deficient, ethionine-supplemented (CDE) diet. Liver tissues were collected and analyzed by immunohistochemistry and immunoblots. We performed immunohistochemical analyses of liver tissues from patients with compensated or decompensated cirrhosis or acute on chronic liver failure (n = 15). RESULTS: Loss of Hdac1 activity in zebrafish blocked differentiation of LPCs into hepatocytes by increasing levels of sox9b mRNA and reduced differentiation of LPCs into BECs by increasing levels of cdk8 mRNA, which encodes a negative regulator gene of Notch signaling. We identified Notch3 as the receptor that regulates differentiation of LPCs into BECs. Loss of activity of Kdm1a, a lysine demethylase that forms repressive complexes with Hdac1, produced the same defects in differentiation of LPCs into hepatocytes and BECs as observed in zebrafish with loss of Hdac1 activity. Administration of MS-275 to mice with hepatocyte-specific loss of ß-catenin impaired differentiation of LPCs into hepatocytes after the CDE diet. HDAC1 was expressed in reactive ducts and hepatocyte buds of liver tissues from patients with cirrhosis. CONCLUSIONS: Hdac1 regulates differentiation of LPCs into hepatocytes via Sox9b and differentiation of LPCs into BECs via Cdk8, Fbxw7, and Notch3 in zebrafish with severe hepatocyte loss. HDAC1 activity was also required for differentiation of LPCs into hepatocytes in mice with liver injury after the CDE diet. These pathways might be manipulated to induce LPC differentiation for treatment of patients with advanced liver diseases.

SOX2OT variant 7 contributes to the synergistic interaction between EGCG and Doxorubicin to kill osteosarcoma via autophagy and stemness inhibition.

BACKGROUND: Doxorubicin is the preferred chemotherapeuticdrug for osteosarcoma treatment of which clinical efficacy is limited because of its chemo-resistance and cardiac toxicity. It is necessary to develop the combination regimen with complementary molecular mechanisms to reduce the side effects and enhance sensitivity of Doxorubicin. EGCG is a polyphenol in green tea with antitumor bioactivity,which has been found that its combination with certain chemotherapeutic drugs could improve the antitumor efficiency. METHODS: In this study, MTT assay was used to detect the cell growth inhibition The CD133+/CD44+ cells were isolated from U2OS and SaoS2 cell lines using magnetic-activated cell sorting and identified by flow cytometry analysis. qRT-PCR was used for determining the relative mRNA levels of key genes. Immunofluorescence was performed to evaluate the autophagy flux alterations. Self-renewal ability was accessed by sphere-forming assay. Tumorigenicity in nude mice was preformed to evaluate tumorigenicity in vivo. RESULTS: We found that EGCG targeting LncRNA SOX2OT variant 7 produced synergistic effects with Doxorubicin on osteosarcoma cell growth inhibition. On the one hand, EGCG could reduce the Doxorubicin-induced pro-survival autophagy through decreasing SOX2OT variant 7 to improve the growth inhibition of Doxorubicin. On the other hand, EGCG could partially inactivate Notch3/DLL3 signaling cascade targeting SOX2OT variant 7 to reduce the stemness then abated drug-resistance of osteosarcoma cells. CONCLUSIONS: This study will help to reveal the molecular mechanisms of synergistic effects of EGCG and Doxorubicin on OS chemotherapy and improve the clinical efficacy of chemotherapy as well as provide a basis for developing antitumor drugs targeting osteosarcoma stem cells.

DLL4 expression is a prognostic marker and may predict gemcitabine benefit in resected pancreatic cancer.

BACKGROUND: There is an increasing interest for Notch signalling pathway and particularly Delta-like ligand 4 (DLL4) as potential therapeutic target to improve outcome for patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: Using immunohistochemistry (IHC) and tissue microarray (TMA), we assessed the expression patterns of DLL4, Notch1 and Notch3 in 151 patients from two independent cohorts of resected PDAC. We investigated the prognostic and the predictive significance of these proteins. RESULTS: High IHC DLL4 expression in cancer cells was associated with worse overall survival (OS) and disease-free survival (DFS) than low DLL4 expression (median OS: 12.9 vs 30.4 months, P=0.004 and median DFS: 8.8 vs 17.4 months, P=0.02). High DLL4 expression remained a significant negative prognostic factor in multivariate analysis (HR for OS: 2.1, P=0.02 and HR for DFS: 2.0, P=0.02). Low DLL4 abundance was associated with a longer OS-only for patients who received an adjuvant gemcitabine-based chemotherapy (P<0.001) but not for patients who did not receive gemcitabine (P=0.72). Furthermore, the interaction test for adjuvant gemcitabine therapy was statistically significant (P<0.001). The validating cohort recapitulated the findings of the training cohort. CONCLUSIONS: Low DLL4 abundance in tumour cells may predict the benefit from adjuvant gemcitabine therapy after PDAC resection.

[Effect of triptolide on expressions of Notch receptors and ligands in rats with adjuvant- induced arthritis and reduced pulmonary function].

OBJECTIVE: To investigate the effects of triptolide on Notch receptor and ligand expressions in rats with adjuvant-induced arthritis (AA). METHODS: Forty rats were randomly divided into normal control (NC) group, model (MC) group, methotrexate group and triptolide groups. Rat models of AA were established by an intradermal injection of 0.1 mL Freund's complete adjuvant into the right paw. Twelve days after the injection, the rats were treated with corresponding drugs for 30 days; the rats in NC group and MC group were given saline only. Paw edema volume (E), arthritis index (AI), pulmonary function, histomorphologies, and Notch receptor/ ligand expression in the lung tissue were analyzed after the treatments. RESULTS: Compared with the NC group, E, AI, Notch3, Notch4, and Delta1 expressions in the lung tissues significantly increased while pulmonary function and pulmonary expressions of Notch1, Jagged1, and Jagged2 significantly decreased the model rats (P<0.01). Compared with the MC group, triptolide-treated rats showed significantly improved pulmonary functions, increased expressions of Notch1, Jagged1, and Jagged2 and decreased expressions of Notch3, Notch4, and Delta1 in the lungs (P<0.05, P<0.01); the therapeutic effect of triptolide was better than that of methotrexate. CONCLUSION: Triptolide can reduce inflammatory reaction and immune complex deposition to improve joint and pulmonary symptoms in rats with AA possibly by up-regulating the expressions of Notch3, Notch4, and Delta1 and down-regulating the expressions of Jagged1, Jagged2, and Notch1.

Effect of triptolide on expressions of Notch receptors and ligands in rats with adjuvant- induced arthritis and reduced pulmonary function / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of triptolide on Notch receptor and ligand expressions in rats with adjuvant-induced arthritis (AA).</p><p><b>METHODS</b>Forty rats were randomly divided into normal control (NC) group, model (MC) group, methotrexate group and triptolide groups. Rat models of AA were established by an intradermal injection of 0.1 mL Freund's complete adjuvant into the right paw. Twelve days after the injection, the rats were treated with corresponding drugs for 30 days; the rats in NC group and MC group were given saline only. Paw edema volume (E), arthritis index (AI), pulmonary function, histomorphologies, and Notch receptor/ ligand expression in the lung tissue were analyzed after the treatments.</p><p><b>RESULTS</b>Compared with the NC group, E, AI, Notch3, Notch4, and Delta1 expressions in the lung tissues significantly increased while pulmonary function and pulmonary expressions of Notch1, Jagged1, and Jagged2 significantly decreased the model rats (P<0.01). Compared with the MC group, triptolide-treated rats showed significantly improved pulmonary functions, increased expressions of Notch1, Jagged1, and Jagged2 and decreased expressions of Notch3, Notch4, and Delta1 in the lungs (P<0.05, P<0.01); the therapeutic effect of triptolide was better than that of methotrexate.</p><p><b>CONCLUSION</b>Triptolide can reduce inflammatory reaction and immune complex deposition to improve joint and pulmonary symptoms in rats with AA possibly by up-regulating the expressions of Notch3, Notch4, and Delta1 and down-regulating the expressions of Jagged1, Jagged2, and Notch1.</p>

Activity-dependent Notch signalling in the hypothalamic-neurohypophysial system of adult mouse brains.

Notch signalling has a key role in cell fate specification in developing brains; however, recent studies have shown that Notch signalling also participates in the regulation of synaptic plasticity in adult brains. In the present study, we examined the expression of Notch3 and Delta-like ligand 4 (DLL4) in the hypothalamic-neurohypophysial system (HNS) of the adult mouse. The expression of DLL4 was higher in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) compared to adjacent hypothalamic regions. Double-labelling immunohistochemistry using vesicular GABA transporter and glutamate transporter revealed that DLL4 was localised at a subpopulation of excitatory and inhibitory axonal boutons against somatodendrites of arginine vasopressin (AVP)- and oxytocin (OXT)-containing magnocellular neurones. In the neurohypophysis (NH), the expression of DLL4 was seen at OXT- but not AVP-containing axonal terminals. The expression of Notch3 was seen at somatodendrites of AVP- and OXT-containing magnocellular neurones in the SON and PVN and at pituicytes in the NH. Chronic physiological stimulation by salt loading, which remarkably enhances the release of AVP and OXT, decreased the number of DLL4-immunoreactive axonal boutons in the SON and PVN. Moreover, chronic and acute osmotic stimulation promoted proteolytic cleavage of Notch3 to yield the intracellular fragments of Notch3 in the HNS. Thus, the present study demonstrates activity-dependent reduction of DLL4 expression and proteolytic cleavage of Notch3 in the HNS, suggesting that Notch signalling possibly participates in synaptic interaction in the hypothalamic nuclei and neuroglial interaction in the NH.

Cerebral hemorrhages in CADASIL: report of four cases and a brief review.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited cerebral small vessel disease, clinically characterized by migraine, recurrent transient ischemic attacks or strokes, psychiatric disorders and cognitive decline. Strokes are typically ischemic, while hemorrhagic events have been only sporadically described. However, cerebral microbleeds have been found in 31-69% of CADASIL patients. METHODS: We describe four unrelated CADASIL patients who had hemorrhagic strokes. We also briefly review the literature on intracerebral hemorrhage (ICH) in CADASIL. RESULTS: Three patients had a thalamo-capsular hemorrhage (age at onset: 54, 67, 77) and one of these had a second hemispheric cerebellar hemorrhage. Another patient experienced an interpeduncular cistern subarachnoid hemorrhage when he was 39. None of these patients was receiving antiplatelets, anticoagulants or statins at the time of hemorrhage; all were hypertensive. NOTCH3 gene analysis revealed mutations on exons 14, 22 (two patients presenting the same mutation), and 24. MRI signs of previous hemorrhages were present in all these patients. CONCLUSIONS: Hemorrhagic stroke can occur in CADASIL similarly to sporadic cerebral small vessel diseases; this finding expands the phenotype of the disease. A diagnosis of CADASIL should probably be considered also in patients with ICH. These data bear potential implications in terms of need of better control of risk factors, particularly hypertension, and raise relevant questions about the use of antiplatelets as prevention measures in CADASIL patients.

CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a dominantly inherited small artery disease that leads to dementia and disability in mid-life. The clinical presentation of CADASIL is variable between and within affected families and is characterized by symptoms including migraine with aura, subcortical ischemic events, mood disturbances, apathy, and cognitive impairment. The mean age at onset of symptoms is 45 years, with variable duration of the disease ranging from 10 to 40 years. In 1996, linkage studies mapped and identified mutations in the NOTCH3 gene on chromosome 19 as causative in CADASIL. Head magnetic resonance imaging (MRI) is always abnormal in participants with NOTCH3 mutations after age 35. Magnetic resonance imaging shows on T2-weighted images or fluid attenuation inversion recovery (FLAIR) sequence, widespread areas of increased signal in the white matter associated with focal hyperintensities in basal ganglia, thalamus, and brainstem. The pathologic hallmark of CADASIL is the presence of electron-dense granules in the media of arterioles that can be identified by electron microscopic evaluation of skin biopsies.

Expression of Notch signalling-related genes in normal and differentiating rat dental pulp cells.

Notch signalling is of fundamental importance to various processes during embryonic development and in adults. The possible role of Hey1, an important Notch signalling component, in odontoblast differentiation was evaluated in this study. Primary cultured dental pulp cells, derived from upper incisors of 5-week-old Wistar rats, were placed in alpha-modification of Eagle's minimal essential medium supplemented with 10% Fetal Bovine Serum (FBS), and ascorbic acid (AA) and beta-glycerophosphate (beta-GP), with or without dexamethasone, and cultured on dishes coated with collagen type IA for 7 days. Conventional and real-time Polymerase Chain Reaction (PCR) was performed to determine the expression of Notch-related genes and dentin sialophosphoprotein as a marker of odontoblast differentiation. Dentin sialophosphoprotein and Hey1 expression was significantly increased and decreased in the presence of AA + beta-GP compared with controls, respectively. These findings suggest that Hey1 may be a negative regulator in odontoblast differentiation.