The Notch signaling pathway is critical for cell fate determination during embryonic development, including many aspects of vascular development. An emerging paradigm suggests that the Notch gene regulatory network is often recapitulated in the context of phenotypic modulation of vascular smooth muscle cells (VSMC), vascular remodeling, and repair in adult vascular disease following injury. Notch ligand receptor interactions lead to cleavage of receptor, translocation of the intracellular receptor (Notch IC), activation of transcriptional CBF-1/RBP-Jκ–dependent and –independent pathways, and transduction of downstream Notch target gene expression. Hereditary mutations of Notch components are associated with congenital defects of the cardiovascular system in humans such as Alagille syndrome and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Recent loss- or gain-of-function studies have provided insight into novel Notch-mediated CBF-1/RBP-Jκ–dependent and –independent signaling and cross-regulation to other molecules that may play a critical role in VSMC phenotypic switching. Notch receptors are critical for controlling VSMC differentiation and dictating the phenotypic response following vascular injury through interaction with a triad of transcription factors that act synergistically to regulate VSMC differentiation. This review focuses on the role of Notch receptor ligand interactions in dictating VSMC behavior and phenotype and presents recent findings on the molecular interactions between the Notch components and VSMC-specific genes to further understand the function of Notch signaling in vascular tissue and disease.

Notch signaling has been shown recently to regulate vascular cell fate in adult cells. By applying a uniform equibiaxial cyclic strain to vascular smooth muscle cells (SMCs), we investigated the role of strain in modulating Notch-mediated growth of SMCs in vitro. Rat SMCs cultured under conditions of defined equibiaxial cyclic strain (0% to 15% stretch; 60 cycles/min; 0 to 24 hours) exhibited a significant temporal and force-dependent reduction in Notch 3 receptor expression, concomitant with a significant reduction in Epstein Barr virus latency C promoter-binding factor-1/recombination signal-binding protein of the Jκ immunoglobulin gene–dependent Notch target gene promoter activity and mRNA levels when compared with unstrained controls. The decrease in Notch signaling was Gi-protein– and mitogen-activated protein kinase–dependent. In parallel cultures, cyclic strain inhibited SMC proliferation (cell number and proliferating cell nuclear antigen expression) while significantly promoting SMC apoptosis (annexin V binding, caspase-3 activity and bax/bcl-x _L ratio). Notch 3 receptor overexpression significantly reversed the strain-induced changes in SMC proliferation and apoptosis to levels comparable to unstrained control cells, whereas Notch inhibition further potentiated the changes in SMC apoptosis and proliferation. These findings suggest that cyclic strain inhibits SMC growth while enhancing SMC apoptosis, in part, through regulation of Notch receptor and downstream target gene expression.

Vascular smooth muscle cell (SMC) fate decisions (cell growth, migration, and apoptosis) are fundamental features in the pathogenesis of vascular disease. We investigated the role of Notch 1 and 3 receptor signaling in controlling adult SMC fate in vitro by establishing that hairy enhancer of split ( hes ‐ 1 and ‐ 5 ) and related hrt 's ( hrt ‐ 1 , ‐ 2 , and ‐ 3 ) are direct downstream target genes of Notch 1 and 3 receptors in SMC and identified an essential role for nuclear protein CBF‐1/RBP‐Jk in their regulation. Constitutive expression of active Notch 1 and 3 receptors (Notch IC) resulted in a significant up‐regulation of CBF‐1/RBP‐Jk‐dependent promoter activity and Notch target gene expression concomitant with significant increases in SMC growth while concurrently inhibiting SMC apoptosis and migration. Moreover, inhibition of endogenous Notch mediated CBF‐1/RBP‐Jk regulated gene expression with a non‐DNA binding mutant of CBF‐1, a Notch IC deleted of its delta RAM domain and the Epstein‐Barr virus encoded RPMS‐1, in conjunction with pharmacological inhibitors of Notch IC receptor trafficking (brefeldin A and monensin), resulted in a significant decrease in cell growth while concomitantly increasing SMC apoptosis and migration. These findings suggest that endogenous Notch receptors and downstream target genes control vascular cell fate in vitro. Notch signaling, therefore, represents a novel therapeutic target for disease states in which changes in vascular cell fate occur in vivo.

Epstein-Barr virus (EBV) infection of primary B cells causes B-cell activation and proliferation. Activation of B cells requires binding of antigen to the B-cell receptor and a survival signal from ligand-bound CD40, signals that are provided by the EBV LMP1 and LMP2A latency proteins. Recently, Toll-like receptor (TLR) signaling has been reported to provide a third B-cell activation stimulus. The interaction between the EBV and TLR pathways was therefore investigated. Both UV-inactivated and untreated EBV upregulated the expression of TLR7 and downregulated the expression of TLR9 in naive B cells. UV-inactivated virus transiently stimulated naive B-cell proliferation in the presence of the TLR7 ligand R837, while addition of the TLR7 antagonist IRS 661 impaired cell growth induced by untreated EBV. Interferon regulatory factor 5 (IRF-5) is a downstream mediator of TLR7 signaling. IRF-5 was induced following EBV infection, and IRF-5 was expressed in B-cell lines with type III latency. Expression of IRF-5 in this setting is surprising since IRF-5 has tumor suppressor and antiviral properties. B-cell proliferation assays provided evidence that EBV modulates TLR7 signaling responses. Examination of IRF-5 transcripts identified a novel splice variant, V12, that was induced by EBV infection, was constitutively nuclear, and acted as a dominant negative form in IRF-5 reporter assays. IRF-4 negatively regulates IRF-5 activation, and IRF-4 was also present in type III latently infected cells. EBV therefore initially uses TLR7 signaling to enhance B-cell proliferation and subsequently modifies the pathway to regulate IRF-5 activity.

Vascular smooth muscle cell (VSMC) phenotypic modulation is a key factor in vascular pathology. We have investigated the role of Notch receptor signaling in controlling human vascular smooth muscle cell (hVSMC) differentiation in vitro and established a role for cyclic strain-induced changes in Notch signaling in promoting this phenotypic response. The expression of α-actin, calponin, myosin, and smoothelin was examined by performing immunocytochemistry, Western blot analysis, and quantitative real-time PCR in hVSMCs cultured under static conditions after forced overexpression of constitutively active Notch 1 and 3 receptors, inhibition of endogenous Cp-binding factor 1 (CBF-1)/recombination signal sequence-binding protein-Jκ (RBP-Jκ) signaling, and exposure to cyclic strain using a Flexercell Tension Plus unit. Overexpression of constitutively active Notch intracellular (IC) receptors (Notch 1 IC and Notch 3 IC) resulted in a significant downregulation of α-actin, calponin, myosin, and smoothelin expression, an effect that was significantly attenuated after inhibition of Notch-mediated, CBF-1/RBP-Jκ-dependent signaling by coexpression of RPMS-1 (Epstein-Barr virus-encoded gene product) and selective knockdown of basic helix-loop-helix factors [hairy enhancer of split ( HES) gene and Hes-related transcription (Hrt) factors Hrt-1, Hrt-2, and Hrt-3] using targeted small interfering RNA. Cells cultured under conditions of defined equibiaxial cyclic strain (10% strain, 60 cycles/min, 24 h) exhibited a significant reduction in Notch 1 IC and Notch 3 IC expression concomitant with a significant increase in VSMC differentiation marker expression. Moreover, this cyclic strain-induced increase was further enhanced after inhibition of CBF-1/RBP-Jκ-dependent signaling with RPMS-1. These findings suggest that Notch promotes changes in hVSMC phenotype via activation of CBF-1/RBP-Jκ-dependent pathways in vitro and contributes to the phenotypic response of VSMCs to cyclic strain-induced changes in VSMC differentiation.