Cyclic stretch stimulates vascular smooth muscle cell alignment by redox-dependent activation of Notch3

Abstract

Mice deficient in Notch3 have defects in arterial vascular smooth muscle cell (VSMC) mechanosensitivity, including impaired myogenic responses and autoregulation, and inappropriate VMSC orientation. Experiments were performed to determine if Notch3 is activated by mechanical stimulation and contributes to mechanosensitive responses of VSMCs, including cell realignment. Cyclic, uniaxial stretch (10%, 1 Hz) of human VSMCs caused Notch3 activation, demonstrated by a stretch-induced increase in hairy and enhancer of split 1/hairy-related transcription factor-1 expression, translocation of Notch3 to the nucleus, and a decrease in the Notch3 extracellular domain. These effects were prevented by inhibiting the expression [small interfering (si)RNA] or proteolytic activation of Notch3 {N-(R)-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-l-naphthylalanyl-l-alanine-2-aminoethyl amide (TAPI-1; 50 μmol/l) to inhibit TNF-α-converting enzyme (TACE) or N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT; 20 μmol/l) to inhibit γ-secretase}. Stretch increased the activity of ROS within VSMCs, determined using dichlorodihydrofluorescein fluorescence. Catalase (1,200 U/ml), which degrades H₂O₂, inhibited the stretch-induced activation of Notch3, whereas in nonstretched cells, increasing H₂O₂ activity [H₂O₂ or manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin] caused activation of Notch3. Stretch increased the activity of TACE, which was prevented by catalase. Stretch-induced activation of p38 MAPK in VSMCs was inhibited either by catalase or by inhibiting Notch3 expression (siRNA). Stretch caused VSMCs to realign perpendicular to the direction of the mechanical stimulus, which was significantly inhibited by catalase or by inhibiting the expression (siRNA) or activation of Notch3 (TAPI-1 or DAPT). Therefore, cyclic uniaxial stretch activates Notch3 signaling through a ROS-mediated mechanism, and the presence of Notch3 is necessary for proper stretch-induced cell alignment in VSMCs. This mechanism may contribute to the physiological role of Notch3 in mediating developmental maturation of VSMCs.

Gliotoxin is a potent NOTCH2 transactivation inhibitor and efficiently induces apoptosis in chronic lymphocytic leukaemia (CLL) cells

“…Keeping in mind that NOTCH signalling is sensitive to redox regulation (Coant et al, 2010;Zhu et al, 2011), it is possible to speculate that gliotoxin may directly interfere with the assembly of the NOTCH2 transcription factor complex on DNA. This hypothesis is strengthened by the fact that gliotoxin inhibits the formation of DNA-bound NOTCH2 complexes even when directly added to the EMSA reaction, as demonstrated in this report.…”

Biomechanical regulation of vascular smooth muscle cell functions: from in vitro to in vivo understanding

“…Similarly, by comparing the ex vivo artery model and in vitro co-culture model, differences have been observed in the migration distance of ECs between the ex vivo and in vitro models, with ECs migration being slower in the ex vivo artery model [108]. The difference could be mostly attributable to stronger EC-ECM binding in the natural arterial structure than [72,74], notch [61,64], luminal structures such as ion channel (K þ and Ca 2þ ) [68] and tyrosine-protein kinase [66]. Transmembrane integrins bind to specific ECM protein and associate with a variety of FAK proteins to trigger downstream RhoA/ROCK signalling cascades [75], and regulate the migration of SMC.…”

Notch Activation of Ca2+ Signaling in the Development of Hypoxic Pulmonary Vasoconstriction and Pulmonary Hypertension

“…HIF-1a can stabilize NICD under normoxic and hypoxic conditions, resulting in transcriptional up-regulation of Notch target genes (40). Several studies have demonstrated that reactive oxygen species can activate Notch signaling (41)(42)(43). Given that hypoxia increases reactive oxygen species, it is possible that Notch signaling is activated in response by reactive oxygen species in hypoxic conditions (44).…”