Anti-DLL4 monoclonal antibody, clone HMD4-2

BackgroundEthanol (EtOH) inhibits Notch-mediated vascular smooth muscle cell (SMC) proliferation, an event that is key in vessel remodeling and atherogenesis. The object of this study was to determine whether EtOH inhibits Notch signaling in SMC at the level of -secretase, a protease that in concert with -secretase catalyzes the release of the intracellular domain of the Notch receptor necessary for signaling. MethodsHuman coronary artery SMCs (HCASMCs) were treated with a recombinant soluble Notch ligand, Delta-like ligand 4 (DLL4) (2g/ml), or transfected with a constitutively active Notch 1 intracellular domain (N1ICD), in the absence or presence of EtOH. EtOH (25mM) treatment inhibited DLL4-stimulated CBF-1/RBP-Jk-dependent promoter activity (determined by luciferase assay) and downstream target gene HRT-3 mRNA levels. In contrast, EtOH had no effect on N1ICD-driven CBF-1/RBP-Jk-dependent promoter activity or HRT-3 expression. ResultsThese data suggest that EtOH inhibits Notch signaling at, or prior to, Notch intracellular domain (NICD) generation. -Secretase activity was determined in solubilized membrane preparations from HCASMC treated with/without EtOH (25mM) or the -secretase inhibitor DAPT (20M) using (i) a fluorometric assay and (ii) Western blot detection of cleavage products using a Flag-tagged Notch-based substrate, N100Flag. EtOH inhibited basal and DLL4-stimulated -secretase activity, and SMC growth to a similar extent as DAPT, whereas it had no effect on -secretase (TACE/ADAM17) activity also determined by fluorometric assay. Moreover, EtOH treatment inhibited the expression of caveolin-1, a lipid raft protein implicated in regulating -secretase activity, and altered its cellular distribution in HCASMC. ConclusionsEtOH inhibits Notch signaling in vascular SMCs at the level of -secretase activity, possibly by affecting lipid raft function. Such a response might be expected to result in attenuation of pathologic vessel remodeling and thus may contribute to moderate alcohols' cardioprotective effects.

Ticagrelor is a powerful P2Y(12) inhibitor with pleiotropic effects in the cardiovascular system. Consistently, we have reported that in patients with stable coronary artery disease (CAD) and concomitant chronic obstructive pulmonary disease (COPD) who underwent percutaneous coronary intervention (PCI), 1-month treatment with ticagrelor was superior in improving biological markers of endothelial function, compared with clopidogrel. The objective of this study was to investigate the mechanisms underlying these beneficial effects of ticagrelor by conducting molecular analyses of RNA isolated from peripheral blood cells of these patients. We determined mRNAs levels of markers of inflammation and oxidative stress, such as ROR gamma t (T helper 17 cells marker), FoxP3 (regulatory T cells marker), NLRP3, ICAM1, SIRT1, Notch ligands JAG1 and DLL4, and HES1, a Notch target gene. We found that 1-month treatment with ticagrelor, but not clopidogrel, led to increased levels of SIRT1 and HES1 mRNAs. In patients treated with ticagrelor or clopidogrel, we observed a negative correlation among changes in both SIRT1 and HES1 mRNA and serum levels of Epidermal Growth Factor (EGF), a marker of endothelial dysfunction found to be reduced by ticagrelor treatment in our previous study. In conclusion, we report that in stable CAD/COPD patients ticagrelor positively regulates HES1 and SIRT1, two genes playing a protective role in the context of inflammation and oxidative stress. Our observations confirm and expand previous studies showing that the beneficial effects of ticagrelor in stable CAD/COPD patients may be, at least in part, mediated by its capacity to reduce systemic inflammation and oxidative stress.