Yvonne A. Birney

Notch 1 and 3 receptor signaling modulates vascular smooth muscle cell growth, apoptosis, and migration via a CBF-1/RBP-Jk dependent pathway

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 hrts (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.

Notch and Vascular Smooth Muscle Cell Phenotype

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-Jkappa-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-Jkappa-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.

Cyclic Strain Inhibits Notch Receptor Signaling in Vascular Smooth Muscle Cells In Vitro

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 J&kgr; 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-xL 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.

Influence of basolateral condition on the regulation of brain microvascular endothelial tight junction properties and barrier function

Basolateral condition of the brain microvascular endothelium is believed to influence blood-brain barrier (BBB) phenotype, although the precise transcriptional and post-translational mechanisms involved are poorly defined. In vivo, the basolateral surface of the blood-brain endothelium is bathed in serum-free interstitial fluid and encompassed by astrocytic end-feet. We hypothesized that these conditions impact on BBB function by directly modulating expression and biochemical properties of tight junctions. To investigate this, an in vitro transwell culture model was employed to selectively modify the basolateral environment of bovine brain microvascular endothelial cells (BBMvECs). In the absence of basolateral (but not apical) serum, we observed higher levels of expression, association and plasma membrane localization for the tight junction proteins, occludin and zonula occludens-1 (ZO-1), in parallel with elevated transendothelial electrical resistance (TEER) and reduced (14)[C]-sucrose permeability of BBMvEC monolayers. We further examined the effects of non-contact co-culture with basolateral astrocytes (C6 glioma) on indices of BBMvEC barrier function in both the presence and absence of serum. Astrocyte co-culture with serum led to enhanced occludin protein expression, occludin/ZO-1 association, and ZO-1 membrane localization, in parallel with increased TEER of BBMvEC monolayers. Astrocyte co-culture in the absence of serum (i.e. basolateral conditions most consistent with in vivo BBB physiology) however, gave the highest increases in BBMvEC barrier indices. Thus, we can conclude that factors influencing condition of the basolateral environment of the brain microvasculature can directly, and independently, modify BBB properties by regulating the expression and biochemical properties of the tight junction proteins, occludin and ZO-1.

Sonic Hedgehog Induces Notch Target Gene Expression in Vascular Smooth Muscle Cells via VEGF-A

Objective—Notch, VEGF, and components of the Hedgehog (Hh) signaling pathway have been implicated in vascular morphogenesis. The role of Notch in mediating hedgehog control of adult vascular smooth muscle cell (SMC) growth and survival remains unexplored. Methods and Results—In cultured SMCs, activation of Hh signaling with recombinant rShh (3.5 &mgr;g/mL) or plasmid encoded Shh increased Ptc1 expression, enhanced SMC growth and survival and promoted Hairy-related transcription factor (Hrt) expression while concomitantly increasing VEGF-A levels. These effects were significantly reversed after Hh inhibition with cyclopamine. Shh-induced stimulation of Hrt-3 mRNA and SMC growth and survival was attenuated after inhibition of Notch-mediated CBF-1/RBP-Jk–dependent signaling with RPMS-1 while siRNA knockdown of Hrt-3 inhibited SMC growth and survival. Recombinant VEGF-A increased Hrt-3 mRNA levels while siRNA knockdown abolished rShh stimulated VEGF-A expression while concomitantly inhibiting Shh-induced increases in Hrt-3 mRNA levels, proliferating cell nuclear antigen (PCNA), and Notch 1 IC expression, respectively. Hedgehog components were expressed within intimal SMCs of murine carotid arteries after vascular injury concomitant with a significant increase in mRNA for Ptc1, Gli2, VEGF-A, Notch 1, and Hrts. Conclusion—Hedgehog promotes a coordinate regulation of Notch target genes in adult SMCs via VEGF-A.

Modulation of Nitric Oxide and 6-keto-Prostaglandin F1α Production in Bovine Aortic Endothelial Cells by Conjugated Linoleic Acid

Conjugated linoleic acid (CLA) refers to a group of polyunsaturated fatty acids that exist as positional (18:2) and stereo (cis/trans) isomers of conjugated dienoic octadecadienoate. Reports consistently indicate that CLA may inhibit both the onset and progression of atherosclerosis, via an as yet unknown mechanism(s). In an effort to identify the putative biochemical effects of CLA on bovine aortic endothelial cells (BAECs), the authors examined both the temporal and dose-dependent effects of a commercial CLA isomeric mixture on the expression and enzymatic function of endothelial nitric oxide synthase (eNOS) and cyclooxygenase-I/II (COX-I/II) in these cells. Initial investigations indicated that CLA mix (0 to 10 microg/mL, 0 to 24 h) failed to regulate either the expression or activity of eNOS in BAECs under basal conditions. Pretreatment of BAECs with CLA mix (10 microg/mL) for either 3 or 24 h, followed by incubation with 5 microM bradykinin (BK) for 3 h, however, increased BK-stimulated nitrite release by 2.4 +/- 0.6- and 3.0 +/- 0.4-fold, respectively, more than control cells (BK-stimulation without CLA pretreatment). Under basal conditions, CLA mix (10 microg/mL, 0 to 24 h) had no significant effect on either COX-I or COX-II expression, genes that could be readily induced in response to hemodynamic stimuli. CLA could, however, significantly attenuate BAEC release of 6-keto-prostaglandin F(1alpha) (6k-PGF(1alpha)), a stable breakdown product of prostaglandin I2 (PGI2) within the cyclooxygenase pathway, in a dose- and time-dependent manner. In conclusion, therefore, the results suggest that CLA may potentiate agonist-stimulated eNOS activation whilst attenuating COX-dependent PGI2 synthesis in BAECs. This ability to increase agonist-stimulated nitric oxide (NO) levels, whilst reducing production of inflammatory mediators within vascular ECs, supports a putative atheroprotective role for CLA and provides an important biochemical insight into its purported ability to modulate endothelium-mediated vascular homeostasis.

Eicosanoids in cirrhosis and portal hypertension

In the last decade, the knowledge of the pathogenesis of portal hypertension and cirrhosis has increased dramatically. In portal hypertension, almost all the known vasoactive systems/substances are activated or increased and the most recent studies have stressed the importance of the endothelial factors, in particular, prostaglandins. Prostaglandins are formed following the oxygenation of arachidonic acid by the cyclooxygenase (Cox) pathway. An important consideration in portal hypertension and cirrhosis in the periphery is the altered hemodynamic profile and its contributory role in controlling endothelial release of these vasoactive substances. Prostaglandins are released from the endothelium in response to both humoral and mechanical stimuli and can profoundly affect both intrahepatic and peripheral vascular resistance. Within the liver, intrahepatic resistance is altered due to a diminution in sinusoidal responsiveness to vasodilators and an increase in prostanoid vasoconstrictor responsiveness. This review will examine the contributory role of both hormonal and/or hemodynamic force-induced changes in prostaglandin production and signaling in cirrhosis and portal hypertension and the consequence of these changes on the structural and functional response of both the vasculature and the liver.

Pulse pressure-induced transmural fluid flux increases bovine aortic smooth muscle cell apoptosis in a mitogen activated protein kinase dependent manner.

Background: Mechanical forces associated with blood flow are critical in the regulation of vascular smooth muscle cell (VSMC) growth, migration, differentiation and apoptosis as fundamental features in the pathogenesis of vascular disease. We investigated the effect of pulse pressure on VSMC apoptosis. Methods: Using a perfused transcapillary co-culture system, bovine thoracic aortic SMC (BASMC) were exposed to increases in pulsatile flow (0.3–17 ml/min) and hence pulse pressure (amplitude of pulse 6–50 mm Hg) in the absence or presence of bovine aortic endothelial cells (BAEC). The extent of apoptosis was determined by measuring caspase-3 activity, the levels of pro- and anti-apoptotic Bcl-2 family proteins, FasL and cellular apoptosis susceptibility (CAS) protein expression and the extent of DNA fragmentation. Results: Changes in pulse pressure resulted in a significant force- and time-dependent increase in caspase-3 activity in BASMC. This effect was maximal after 6 h, independent of BAEC presence, and attenuated following inhibition of mitogen-activated protein kinase (MAPK) activity with PD98059. In parallel cultures, there was a significant increase in Bad and Bax expression, concomitant with an increase in DNA fragmentation, and a significant decrease in Bcl-2 and Bcl-XL expression. The pro-apoptotic effects of pulse pressure were specific for differentiated cells but independent of p53, inasmuch as FasL and CAS expression were enhanced in differentiated adult cells but decreased in de-differentiated embryonic cells in response to flow. Conclusions: These results suggest that pulse pressure promotes phenotypically distinct VSMC apoptosis in vitro in an endothelial-independent, MAPK-dependent, manner.

Regulation of Endopeptidases EC3.4.24.15 and EC3.4.24.16 in Vascular Endothelial Cells by Cyclic Strain: Role of Gi Protein Signaling

Objective—Endopeptidase EC3.4.24.15 (EP24.15)- and EC3.4.24.16 (EP24.16)-specific peptide hydrolysis plays an important role in endothelium-mediated vasoregulation. Given the significant influence of hemodynamic forces on vascular homeostasis and pathology, we postulated that these related peptidases may be mechanosensitive. The objective of this study, therefore, was to investigate the putative role of cyclic strain in regulating the expression and enzymatic activity of EP24.15 and EP24.16 in bovine aortic endothelial cells (BAECs). Methods and Results—BAECs were cultured under conditions of defined cyclic strain (0% to 10% stretch, 60 cycles/min, 0 to 24 hours). Strain significantly increased EP24.15 and EP24.16 soluble activity in a force- and time-dependent manner, with elevations of 2.3±0.4- and 1.9±0.3-fold for EP24.15 and EP24.16, respectively, after 24 hours at 10% strain. Pharmacological agents and dominant-negative G protein mutants used to selectively disrupt Gi&agr;- and G&bgr;&ggr;-mediated signaling pathways attenuated strain-dependent (24 hours, 5%) increases for both enzymes. Differences in the inhibitory profile for both enzymes were also noted, with EP24.15 displaying greater sensitivity to Gi&agr;2/3 inhibition and EP24.16 exhibiting greater sensitivity to Gi&agr;1/2 and G&bgr;&ggr; inhibition. Cyclic strain also increased levels of secreted EP24.15 and EP24.16 activity by 2.6±0.02- and 3.6±0.2-fold, respectively, in addition to mRNA levels for both enzymes (EP24.15 +42%, EP24.16 +56%). Conclusions—Our findings suggest that cyclic strain putatively regulates both the mRNA expression and enzymatic function of EP24.15 and EP24.16 in BAECs via alternate Gi protein signaling pathways.