n-(n-(3-5-difluorophenacetyl)alanyl)phenylglycine-tert-butyl-ester and Hypoxia

Hypoxia-induced pulmonary hypertension (HPH) is a clinical syndrome associated with high morbidity and mortality. However, the underlying mechanisms remain unclear. Both the mammalian target of rapamycin (mTOR) and the Notch3 signaling pathways have been reported to be involved in HPH; however, it is unknown whether there is a connection between these two signaling pathways in HPH. This study was designed to investigate the relationship between mTOR and Notch3 in HPH. After treatment with 10% O2 for 4 weeks, male C57BL/6 mice developed HPH with gradually increased right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and pulmonary arteriolar remodeling accompanied by the activation of mTOR complex 1 (mTORC1) and Notch3 in the lung tissue and pulmonary arterioles. Pretreatment with the mTORC1 inhibitor rapamycin not only alleviated pulmonary arterial pressure and pulmonary arteriolar remodeling but also suppressed hypoxia-induced mTORC1 and Notch3 activation. Prophylactic N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) administration, a Notch signaling inhibitor, protected against the effects of hypoxia. These in vivo data were confirmed by in vitro experiments on human pulmonary arterial smooth muscle cell (PASMC) exposed to 3% O2 . Furthermore, overexpression of Notch3 intracellular domain partially abrogated the inhibitory effects of rapamycin on human PASMC proliferation. These data indicate that both mTORC1 and Notch3 signaling are involved in HPH and the downstream effects of mTORC1 activation in HPH are partially dependent on the activation of Notch3 signaling.

Topics: Animals; Cell Proliferation; Dipeptides; Humans; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Myocytes, Smooth Muscle; Oxygen; Receptor, Notch3; Receptors, Notch; Signal Transduction; TOR Serine-Threonine Kinases

Neuroinflammation mediated by the activated microglia is suggested to play a pivotal role in the pathogenesis of hypoxic brain injury; however, the underlying mechanism of microglia activation remains unclear. Here, we show that the canonical Notch signaling orchestrates microglia activation after hypoxic exposure which is closely associated with multiple pathological situations of the brain. Notch-1 and Delta-1 expression in primary microglia and BV-2 microglial cells was significantly elevated after hypoxia. Hypoxia-induced activation of Notch signaling was further confirmed by the concomitant increase in the expression and translocation of intracellular Notch receptor domain (NICD), together with RBP-Jκ and target gene Hes-1 expression. Chemical inhibition of Notch signaling with N-[N-(3,5-difluorophenacetyl)-1-alany1- S-phenyglycine t-butyl ester (DAPT), a γ-secretase inhibitor, effectively reduced hypoxia-induced upregulated expression of most inflammatory mediators. Notch inhibition also reduced NF-κB/p65 expression and translocation. Remarkably, Notch inhibition suppressed expression of TLR4/MyD88/TRAF6 pathways. In vivo, Notch signaling expression and activation in microglia were observed in the cerebrum of postnatal rats after hypoxic injury. Most interestingly, hypoxia-induced upregulation of NF-κB immunoexpression in microglia was prevented when the rats were given DAPT pretreatment underscoring the interrelationship between Notch signaling and NF-κB pathways. Taken together, we conclude that Notch signaling is involved in regulating microglia activation after hypoxia partly through the cross talk between TLR4/MyD88/TRAF6/NF-κB pathways. Therefore, Notch signaling may serve as a prospective target for inhibition of microglia activation known to be implicated in brain damage in the developing brain.

Topics: Amyloid Precursor Protein Secretases; Animals; Animals, Newborn; Basic Helix-Loop-Helix Transcription Factors; Cell Hypoxia; Cell Line, Tumor; Dipeptides; Enzyme Inhibitors; Gene Expression Regulation, Developmental; Homeodomain Proteins; Hypoxia; Immunoglobulin J Recombination Signal Sequence-Binding Protein; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Microglia; Myeloid Differentiation Factor 88; NF-kappa B; Rats; Receptor, Notch1; Signal Transduction; TNF Receptor-Associated Factor 6; Toll-Like Receptor 4; Transcription Factor HES-1

Pulmonary hypertension (PH) is a fatal disease that lacks an effective therapy. Notch signaling pathway plays a crucial role in the angiogenesis and vascular remodeling. However, its roles in vascular remodeling in PH have not been well studied. In the current study, using hypoxia-induced PH model in rat, we examined the expression of Notch and its downstream factors. Then, we used vessel strip culture system and γ-secretase inhibitor DAPT, a Notch signaling inhibitor to determine the effect of Notch signaling in vascular remodeling and its potential therapeutic value. Our results indicated that Notch 1-4 were detected in the lung tissue with variable levels in different cell types such as smooth muscle cells and endothelial cells of pulmonary artery, bronchia, and alveoli. In addition, following the PH induction, all of Notch1, Notch3, Notch4 receptor, and downstream factor, HERP1 in pulmonary arteries, mRNA expressions were increased with a peak at 1-2 weeks. Furthermore, the vessel wall thickness from rats with hypoxia treatment increased after cultured for 8 days, which could be decreased approximately 30% by DAPT, accompanied with significant increase of expression level of apoptotic factors (caspase-3 and Bax) and transformation of vascular smooth muscle cell (VSMC) phenotype from synthetic towards contractile. In conclusion, the current study suggested Notch pathway plays an important role in pulmonary vascular remodeling in PH and targeting Notch signaling pathway could be a valuable approach to design new therapy for PH.

Topics: Animals; Apoptosis; Blood Vessels; Cell Proliferation; Cells, Cultured; Dipeptides; Disease Models, Animal; Gene Expression Regulation; Hypertension, Pulmonary; Hypoxia; Immunohistochemistry; In Vitro Techniques; Lung; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phenotype; Rats; Rats, Wistar; Receptors, Notch; Reproducibility of Results; Signal Transduction; Staining and Labeling

Through long-term laboratory selection (over 200 generations), we have generated Drosophila melanogaster populations that tolerate severe, normally lethal, levels of hypoxia. Because of initial experiments suspecting genetic mechanisms underlying this adaptation, we compared the genomes of the hypoxia-selected flies with those of controls using deep resequencing. By applying unique computing and analytical methods we identified a number of DNA regions under selection, mostly on the X chromosome. Several of the hypoxia-selected regions contained genes encoding or regulating the Notch pathway. In addition, previous expression profiling revealed an activation of the Notch pathway in the hypoxia-selected flies. We confirmed the contribution of Notch activation to hypoxia tolerance using a specific γ-secretase inhibitor, N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), which significantly reduced adult survival and life span in the hypoxia-selected flies. We also demonstrated that flies with loss-of-function Notch mutations or RNAi-mediated Notch knockdown had a significant reduction in hypoxia tolerance, but those with a gain-of-function had a dramatic opposite effect. Using the UAS-Gal4 system, we also showed that specific overexpression of the Notch intracellular domain in glial cells was critical for conferring hypoxia tolerance. Unique analytical tools and genetic and bioinformatic strategies allowed us to discover that Notch activation plays a major role in this hypoxia tolerance in Drosophila melanogaster.

Topics: Adaptation, Physiological; Amyloid Precursor Protein Secretases; Animals; Chromosomes, Insect; Dipeptides; DNA; Drosophila melanogaster; Drosophila Proteins; Hypoxia; Mutation; Protein Structure, Tertiary; Receptors, Notch; Selection, Genetic; X Chromosome

Notch receptor signaling is implicated in controlling smooth muscle cell proliferation and in maintaining smooth muscle cells in an undifferentiated state. Pulmonary arterial hypertension is characterized by excessive vascular resistance, smooth muscle cell proliferation in small pulmonary arteries, leading to elevation of pulmonary vascular resistance, right ventricular failure and death. Here we show that human pulmonary hypertension is characterized by overexpression of NOTCH3 in small pulmonary artery smooth muscle cells and that the severity of disease in humans and rodents correlates with the amount of NOTCH3 protein in the lung. We further show that mice with homozygous deletion of Notch3 do not develop pulmonary hypertension in response to hypoxic stimulation and that pulmonary hypertension can be successfully treated in mice by administration of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a gamma-secretase inhibitor that blocks activation of Notch3 in smooth muscle cells. We show a mechanistic link from NOTCH3 receptor signaling through the Hairy and enhancer of Split-5 (HES-5) protein to smooth muscle cell proliferation and a shift to an undifferentiated smooth muscle cell phenotype. These results suggest that the NOTCH3-HES-5 signaling pathway is crucial for the development of pulmonary arterial hypertension and provide a target pathway for therapeutic intervention.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Proliferation; Dipeptides; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation; Humans; Hypertension, Pulmonary; Hypoxia; In Vitro Techniques; Lung; Mice; Mice, Knockout; Microscopy, Electron, Transmission; Myocytes, Smooth Muscle; Pulmonary Artery; Rats; Receptor, Notch3; Receptors, Notch; Repressor Proteins; RNA, Messenger; Signal Transduction; Time Factors