Lipoprotein (a)-mediated vascular calcification: population-based and in vitro studies.

BACKGROUND: Lipoprotein (a) [Lp(a)] is a causal risk factor for cardiovascular diseases, while its role in vascular calcification has not been well-established. Here, we investigated an association of Lp(a) with vascular calcification using population-based and in vitro study designs. METHODS: A total of 2806 patients who received coronary computed tomography were enrolled to assess the correlation of Lp(a) with the severity of coronary artery calcification (CAC). Human aortic smooth muscle cells (HASMCs) were used to explore mechanisms of Lp(a)-induced vascular calcification. RESULTS: In the population study, Lp(a) was independently correlated with the presence and severity of CAC (all p < 0.05). In vitro study showed that cell calcific depositions and alkaline phosphatase (ALP) activity were increased and the expression of pro-calcific proteins, including bone morphogenetic protein-2 (BMP2) and osteopontin (OPN), were up-regulated by Lp(a) stimulation. Interestingly, Lp(a) activated Notch1 signaling, resulting in cell calcification, which was inhibited by the Notch1 signaling inhibitor, DAPT. Lp(a)-induced Notch1 activation up-regulated BMP2-Smad1/5/9 pathway. In contrast, Noggin, an inhibitor of BMP2-Smad1/5/9 pathway, significantly blocked Lp(a)-induced HASMC calcification. Notch1 activation also induced translocation of nuclear factor-κB (NF-κB) accompanied by OPN overexpression and elevated inflammatory cytokines production, while NF-κB silencing alleviated Lp(a)-induced vascular calcification. CONCLUSIONS: Elevated Lp(a) concentrations are independently associated with the presence and severity of CAC and the impact of Lp(a) on vascular calcification is involved in the activation of Notch1-NF-κB and Notch1-BMP2-Smad1/5/9 pathways, thus implicating Lp(a) as a potential novel therapeutic target for vascular calcification.

Combined Detection of Plasma Tumor Necrosis Factor-α Converting Enzyme and Notch1 is Valuable in Screening Endoleak After Endovascular Abdominal Aortic Aneurysms Repair.

OBJECTIVES: Endoleaks may be present in up to 25% of patients after endovascular abdominal aortic aneurysm repair (EVAR) and there is no clear consensus on valuable biomarkers to determine endoleak presence. The aim of this study was to examine the potential value of plasma tumor necrosis factor-α converting enzyme (TACE) and Notch1 concentrations in determining endoleak presence after EVAR. METHODS: A total of 110 patients with abdominal aortic aneurysm who underwent EVAR were enrolled in our study, and plasma TACE and Notch1 concentrations were measured prior to and 6 months after EVAR. Logistic regression was performed to assess the association of postoperative plasma TACE and Notch1 concentrations with endoleak after adjusting for potential confounders. The ability of plasma TACE and Notch1 concentrations to determine endoleak presence was assessed using receiver operating characteristic curves and area under the curve (AUC). RESULTS: Twenty-four patients developed endoleaks 6 months after EVAR. Both postoperative plasma TACE and Notch1 concentrations were higher in patients with endoleak than in those without endoleak (2376.4 ± 28.1 pg/ml vs. 2094.1 ± 27.3 pg/ml, P < 0.01; 218.6 ± 1.9 pg/ml vs. 195.0 ± 2.1 pg/ml, P < 0.01, respectively). The AUCs from receiver operating characteristic curve analysis of plasma TACE and Notch1 concentrations in determining endoleak presence were 0.844 (95% CI 0.771 to 0.918, P < 0.01) and 0.860 (95% CI 0.791 to 0.930, P < 0.01), respectively. Combining the detection of plasma Notch1 and TACE concentrations could improve the accuracy in determining endoleak presence (AUC 0.930, 95% CI 0.883 to 0.978, P < 0.01). The predicted probability cutoff of 0.22 yielded a sensitivity of 95.8% and a specificity of 82.6% for endoleak presence. CONCLUSIONS: Plasma TACE and Notch1 levels can discriminate patients with and without endoleak 6 months after EVAR, and have a potential role in screening patients requiring computed tomography angiography.

Altered expression of Notch1 in Alzheimer's disease.

Notch signaling is an evolutionarily conserved pathway that regulates cell-cell interactions through binding of Notch family receptors to their cognate ligands. Notch signaling has an essential role in vascular development and angiogenesis. Recent studies have reported that Notch may be implicated in Alzheimer's disease (AD) pathophysiology. We measured the levels of soluble Notch1 (sNotch1) in the plasma samples from 72 dementia patients (average age 75.1 y), 89 subjects with amnestic mild cognitive impairment (MCI) (average age 73.72 y), and 150 cognitively normal controls (average age 72.34 y). Plasma levels of sNotch1 were 25.27% lower in dementia patients as compared to healthy control subjects. However, the level of Notch1 protein was significantly increased in human brain microvascular endothelial cells (HBMECs) after amyloid-beta treatment. Also, Notch1 mRNA level was significantly increased in HBMECs and iPSC-derived neuronal cells from AD patient compared to normal control. These results indicate that altered expression of Notch1 might be associated with the risk of Alzheimer's disease.

Candidate early predictive plasma protein markers of doxorubicin-induced chronic cardiotoxicity in B6C3F1 mice.

Cardiotoxicity is a serious adverse effect of doxorubicin (DOX) treatment in cancer patients. Currently, there is a lack of sensitive biomarkers to predict the risk of DOX-induced cardiotoxicity. Using SOMAmer-based proteomic technology, 1129 proteins were profiled to identify potential early biomarkers of cardiotoxicity in plasma from male B6C3F1 mice given a weekly intravenous dose of 3 mg/kg DOX or saline (SAL) for 2, 3, 4, 6, or 8 weeks (6, 9, 12, 18, or 24 mg/kg cumulative DOX doses, respectively). Also, a group of mice received the cardio-protectant, dexrazoxane (DXZ; 60 mg/kg; intraperitoneal) 30 min before a weekly DOX or SAL dose. Proteomic analysis in plasma collected a week after the last dose showed a significant ≥1.2-fold change in level of 18 proteins in DOX-treated mice compared to SAL-treated counterparts during 8-week exposure. Of these, neurogenic locus notch homolog protein 1 (NOTCH1), von Willebrand factor (vWF), mitochondrial glutamate carrier 2, Wnt inhibitory factor 1, legumain, and mannan-binding lectin serine protease 1 were increased in plasma at 6 mg/kg cumulative DOX dose, prior to the release of myocardial injury marker, cardiac troponin I at 12 mg/kg and higher cumulative doses. These six proteins also remained significantly elevated following myocardial injury or pathology at 24 mg/kg. Pretreatment of mice with DXZ significantly attenuated DOX-induced elevated levels of only NOTCH1 and vWF with mitigation of cardiotoxicity. This suggests NOTCH1 and vWF as candidate early biomarkers of DOX cardiotoxicity, which may help in addressing a clinically important question of identifying cancer patients at risk for cardiotoxicity.

Proteomic analysis of heart failure hospitalization among patients with chronic kidney disease: The Heart and Soul Study.

BACKGROUND: Patients with chronic kidney disease (CKD) are at increased risk for heart failure (HF). We aimed to investigate differences in proteins associated with HF hospitalizations among patients with and without CKD in the Heart and Soul Study. METHODS AND RESULTS: We measured 1068 unique plasma proteins from baseline samples of 974 participants in The Heart and Soul Study who were followed for HF hospitalization over a median of 7 years. We sequentially applied forest regression and Cox survival analyses to select prognostic proteins. Among participants with CKD, four proteins were associated with HF at Bonferroni-level significance (p<2.5x10(-4)): Angiopoietin-2 (HR[95%CI] 1.45[1.33, 1.59]), Spondin-1 (HR[95%CI] 1.13 [1.06, 1.20]), tartrate-resistant acid phosphatase type 5 (HR[95%CI] 0.65[0.53, 0.78]) and neurogenis locus notch homolog protein 1 (NOTCH1) (HR[95%CI] 0.67[0.55, 0.80]). These associations persisted at p<0.01 after adjustment for age, estimated glomerular filtration and history of HF. CKD was a significant interaction term in the associations of NOTCH1 and Spondin-1 with HF. Pathway analysis showed a trend for higher representation of the Cardiac Hypertrophy and Complement/Coagulation pathways among proteins prognostic of HF in the CKD sub-group. CONCLUSIONS: These results suggest that markers of heart failure differ between patients with and without CKD. Further research is needed to validate novel markers in cohorts of patients with CKD and adjudicated HF events.

Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia.

Activating mutations of NOTCH1 (a well-known oncogene in T-cell acute lymphoblastic leukemia) are present in ∼4-13% of chronic lymphocytic leukemia (CLL) cases, where they are associated with disease progression and chemorefractoriness. However, the specific role of NOTCH1 in leukemogenesis remains to be established. Here, we report that the active intracellular portion of NOTCH1 (ICN1) is detectable in ∼50% of peripheral blood CLL cases lacking gene mutations. We identify a "NOTCH1 gene-expression signature" in CLL cells, and show that this signature is significantly enriched in primary CLL cases expressing ICN1, independent of NOTCH1 mutation. NOTCH1 target genes include key regulators of B-cell proliferation, survival, and signal transduction. In particular, we show that NOTCH1 transactivates MYC via binding to B-cell-specific regulatory elements, thus implicating this oncogene in CLL development. These results significantly extend the role of NOTCH1 in CLL pathogenesis, and have direct implications for specific therapeutic targeting.

MicroRNA-101 regulates T-cell acute lymphoblastic leukemia progression and chemotherapeutic sensitivity by targeting Notch1.

The present study aimed to investigate the role of microRNA (miR)-101 in acute lymphoblastic leukemia progression and chemoresistance. Furthermore, a novel target gene of miR-101 was identified. Here, we confirmed that miR-101 was significantly downregulated in the blood samples of patients with T-cell acute lymphoblastic leukemia (T-ALL) compared with the healthy controls, as determined by reverse transcription quantitative polymerase chain reaction (RTqPCR) analysis. The in vitro experiments demonstrated that miR-101 significantly repressed the proliferation and invasion, and induced potent apoptosis in Jurkat cells, as determined by CCK-8, flow cytometer and cell invasion assays. Luciferase assay confirmed that Notch1 was a target gene of miR-101, and western blotting showed that miR-101 suppressed the expression of Notch1 at the protein level. Moreover, functional restoration assays revealed that Notch1 mediates the effects of miR-101 on Jurkat cell proliferation, apoptosis and invasion. miR-101 enhanced the sensitivity of Jurkat cells to the chemotherapeutic agent adriamycin. Taken together, our results show for the first time that miR-101 acts as a tumor suppressor in T-cell acute lymphoblastic leukaemia and it could enhance chemotherapeutic sensitivity. Furthermore, Notch1 was identified to be a novel target of miR-101. This study indicates that miR-101 may represent a potential therapeutic target for T-cell acute lymphoblastic leukemia intervention.

Preliminary comparison of plasma notch-associated microRNA-34b and -34c levels in drug naive, first episode depressed patients and healthy controls.

BACKGROUND: Major depressive disorder (MDD) is a common debilitating disease of unknown etiology. The expression of miRNA is closely related to depression and efficacy of antidepressant therapy. However, whether Notch-associated miRNAs expressions involved in first-episode of MDD are still unknown. METHODS: In this study, the expression levels of Notch1, Hes1 mRNA and 5 miRNAs (miR-369-3p, miR-34b-5p, miR-34c-5p, miR-381 and miR-107) in peripheral blood leukocytes of 32 MDD patients and 32 healthy controls were detected using qRT-PCR method. We also assessed the severity of depressive symptom, suicide risk level, negative life events and event-related potential P300. RESULTS: The expression levels of miR-34b-5p (62.49 as the median of cases group and 38.62 as median of control group) and miR-34c-5p (7.17 as the median of cases group and 5.45 as median of control group) in MDD patients were significantly higher than these in control subjects. NOTCH1 gene were significantly lower in MDD patients (5.35 as the median of cases group and 6.80 as median of control group), and was negatively correlated with the expression miR-34c-5p and miR-34b-5p. The expression level of miR-34b-5p and miR-369-3p were significantly lower in patients with suicide idea. N1 latency of P300 were positive correlated with miR-34c-5p, miR-107 and miR-381, and P2 latency of P300 were positive correlated with miR-34c-5p, miR-107 and miR-381. LIMITATIONS: The sample size was small and the role of candidate miRNAs in the regulation of Notch1 gene and cognitive function are still need to be further investigated. CONCLUSIONS: Differentially Notch-associated miRNAs expressions in peripheral blood might be involved in MDD, and the miR-34b-5p and miR-34c-5p levels in peripheral blood leukocytes are closely related to MDD, suicide idea and cognitive function, further studies with large sample size are warranted to test the feasibility of these miRNAs serving as biomarkers for MDD.

Increased expression of Notch 1 in a rat liver transplantation model.

Liver transplantation is the standard treatment for end­stage liver failure; however, rejection can result in allograft failure. In order to investigate the role of Notch 1 during rejection, the present study evaluated Notch 1 expression, as well as the levels of immune reactivity, in rat liver allografts. A heterotopic liver transplantation model was established using Dark Agouti (DA) rats as donors and Lewis rats as recipients (DA/Lewis), with DA recipient rats serving as controls (DA/DA). The concentration levels of immune reactivity markers and serum Notch 1 were measured on days 3, 5, and 7. The overall survival was significantly shorter (<10 days) in the DA/Lewis group, as compared with the DA/DA group (P<0.0001). The concentration levels of serum alanine aminotransferase and total bilirubin were significantly higher 5 and 7 days following transplantation in the DA/Lewis group, as compared with the DA/DA group (P<0.001). The concentration levels of serum Notch 1 were significantly higher in the DA/Lewis group, as compared with the DA/DA group on days 3, 5, and 7 following transplantation (P<0.0001). These results indicate that the expression levels of serum Notch 1 significantly increase during liver allograft rejection, suggesting that Notch 1 is involved in the mechanism underlying liver allograft rejection. Notch 1 may serve as a marker of acute rejection in a rat liver transplantation model.

Incidence and Expression of Circulating Cell Free p53-Related Genes in Acute Myocardial Infarction Patients.

AIM: The circulating RNA levels are predictive markers in several diseases. We determined the levels of circulating p53-related genes in patients with acute ST-segment elevation myocardial infarction (STEMI), indicating major heart muscle damage. METHODS: Plasma RNA was extracted from the patients (n=45) upon their arrival to the hospital (STEMI 0h) and at four hours post-catheterization (STEMI 4h) as well as from controls (n=34). RESULTS: Of 18 circulating p53-related genes, nine genes were detectable. A significantly lower incidence of circulating p21 (p ＜ 0.0001), Notch1 (p=0.042) and BTG2 (p ＜ 0.0001) was observed in the STEMI 0h samples in comparison to the STEMI 4h and control samples. Lower expression levels (2.1-fold) of circulating BNIP3L (p=0.011), p21 (3.4-fold, p=0.005) and BTG2 (6.3-fold, p=0.0001) were observed in the STEMI 0h samples in comparison to the STEMI 4h samples, with a 7.4-fold lower BTG2 expression (p ＜ 0.001) and 2.6-fold lower p21 expression (p=0.034) compared to the control samples. Moreover, the BNIP3L expression (borderline significance, p=0.0655) predicted the level of peak troponin, a marker of myocardial infarction. In addition, the BNIP3L levels on admission (p=0.0025), at post-catheterization (p=0.020) and the change between the groups (p=0.0079) were inversely associated with troponin. The BNIP3L (p=0.0139) and p21 levels (p=0.0447) were also associated with a longer time to catheterization. CONCLUSIONS: Our results suggest that circulating downstream targets of p53 are inhibited during severe AMI and subsequently re-expressed after catheterization, uncovering possible novel death-or-survival decisions regarding the fate of p53 in the heart and the potential use of its target genes as prognostic biomarkers for oxygenation normalization.

Combining detection of Notch1 and tumor necrosis factor-α converting enzyme is a reliable biomarker for the diagnosis of abdominal aortic aneurysms.

AIMS: Although many markers were associated with abdominal aortic aneurysm (AAA), there is no clear consensus on which marker is of the most value. Studies have implicated the role of Notch signaling in the pathogenesis of AAA. We investigate the value of plasma Jagged1, Notch receptors and tumor necrosis factor-α converting enzyme (TACE) in identifying AAA. MAIN METHODS: 42 patients with AAA and 36 controls were enrolled in our study. The concentrations of plasma Jagged1, Notch receptors and TACE were measured by enzyme-linked immunosorbent assay (ELISA). The diagnostic value of plasma Notch1 and TACE was assessed by logistic regression and receiver operator characteristic (ROC) curve. Double immunofluorescence staining was used to investigate the distribution of Notch1 and TACE in AAA tissue specimens. KEY FINDINGS: The concentrations of plasma Notch1 and TACE were significantly higher in AAA than in the controls, respectively (Notch1: P < 0.001; TACE: P = 0.0001). The area under the curve (AUC) from ROC curve of plasma Notch1 and TACE in determining the presence of AAA was 0.878 and 0.804, respectively. Combining detection of plasma Notch1 and TACE could improve the accuracy in detecting AAA (AUC 0.984, P < 0.0001). The predicted probability cutoff of 0.70 gave a sensitivity of 90.5% and a specificity of 100% for combining detection of plasma Notch1 and TACE in predicting AAA. SIGNIFICANCE: This is the first report revealing that plasma Notch1 and TACE are highly expressed in AAA. Combining detection of plasma Notch1 and TACE may be reliable for identifying the presence of AAA.

The role of HIF-1, angiopoietin-2, Dll4 and Notch1 in bleeding gastrointestinal vascular malformations and thalidomide-associated actions: a pilot in vivo study.

OBJECTIVE: To investigate plasma levels of hypoxia inducible factor-1 (HIF-1), angiopoietin-2 (Ang-2), Delta-like ligand 4 (Dll4) and Notch1 in patients with recurrent gastrointestinal bleeding due to gastrointestinal vascular malformation (GIVM) with or without thalidomide treatment. METHODS: Ten eligible patients with recurrent gastrointestinal bleeding due to GIVM, who received thalidomide 100 mg/d for 4 months, were followed up for 1 year. The effective response was the proportions of patients with yearly bleeding episodes reduced by ≥50% at 1 year after treatment. Plasma levels of HIF-1, Ang-2, Dll4 and Notch1 were measured using enzyme-linked immunosorbent assay in the GIVM thalidomide treatment group before and after treatment (10 patients), the GIVM non-thalidomide treatment group (25 patients) and the control group (18 participants). RESULTS: In the GIVM thalidomide treatment group, eight patients (8/10) achieved effective response and five (5/10) displayed complete cessation of bleeding. Mean plasma levels of HIF-1, Ang-2, Dll4 and Notch1 were all higher in the GIVM thalidomide and non-thalidomide treatment groups than in the control group (all P < 0.001). However, Ang-2 decreased more significantly in the effective subgroups (P = 0.003) and no-bleeding patients (P = 0.008). CONCLUSIONS: HIF-1, Ang-2, Dll4 and Notch1 might participate in the formation of GIVM. Thalidomide is an effective and safe treatment agent for GIVM and its associated bleeding. The reduction degree of Ang-2 after a 4-month treatment of thalidomide may offer values for evaluating its prognosis and efficacy.

[Expression of Notch1 on peripheral lymphocytes before and after acute graft rejection following renal transplantation].

OBJECTIVE: To study the changes in Notch1 expression on peripheral lymphocytes after acute graft rejection after renal transplantation. METHODS: Twenty renal transplant recipients experiencing acute graft rejection and 20 without acute rejection were enrolled in this study. Flow cytometry was used to detect the expression of Notch1 on peripheral lymphocytes of the patients before operation, at the occurrence of acute rejection and after anti-rejection therapy. The rates of Notch1-positive lymphocytes measured at different time points were compared between the two groups. RESULT: In patients with acute graft rejection, Notch1 expression at the time of rejection onset was significantly higher than that before operation (t=4.245, P=0.000) and that of patients with graft rejection (t=3.839, P=0.000), and was obviously decreased after anti-rejection therapy (t=3.102, P=0.004). Patients without graft rejection showed no significant changes in Notch1 expression after the transplantation (P=0.409). Notch1 expression was comparable between the recipients receiving Tac therapy and those with CsA therapy (P>0.05). CONCLUSION: Monitoring Notch1 expression on the peripheral lymphocytes after renal transplantation may help in the diagnosis of acute graft rejection and prediction of the effect of an anti-rejection therapy.