Eileen M. Redmond

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.

Increased endothelial nitric oxide synthase activity in the hyperemic vessels of portal hypertensive rats

BACKGROUND/AIM Portal hypertension is characterized by splanchnic hyperemia due to a reduction in mesenteric vascular resistance. Mediators of this hyperemia include nitric oxide. This is based on several reports indicating a marked splanchnic hyporesponsiveness in portal hypertension to vasoconstrictor stimuli both in vitro and in vivo, and a subsequent reversal using specific inhibitors of nitric oxide synthase. The objective of this study was to determine firstly whether the functional activity and/or expression of nitric oxide synthase is altered in portal hypertensive vasculature and secondly which isoenzyme form was responsible for the preferential response to nitric oxide blockade in these animals. METHODS We compared nitric oxide synthase functional activity in the hyperemic vasculature of sham and portal hypertensive rats (following partial portal vein ligation). Nitric oxide synthase activities were determined by measuring the conversion of L-arginine to citrulline using ion-exchange chromatography and the amount of immunodetectable nitric oxide synthase in sham and portal hypertensive vessels was determined by Western blot. RESULTS Ca(2+)-dependent nitric oxide synthase activity was significantly elevated (p < 0.05) in portal hypertensive particulate fractions from the superior mesenteric artery, thoracic aorta and portal vein. Vascular tissue cGMP levels and plasma nitrite levels were both significantly elevated in portal hypertension. Immunodetection with specific antisera raised against the inducible nitric oxide synthase demonstrated a lack of induction within the hyperemic vasculature. Immunodetection with antisera against endothelial nitric oxide synthase showed a significant increase in portal hypertensive portal vein only. These results demonstrate enhanced calcium-dependent nitric oxide synthase activity in portal hypertension hyperemic vessels concurrent with elevated tissue cGMP levels. CONCLUSION We conclude that enhanced endothelial nitric oxide synthesis may in part contribute to the hyperdynamic circulation of portal hypertension.

Endothelial dysfunction in cirrhosis and portal hypertension.

Portal hypertension (PHT) is a common clinical syndrome associated with chronic liver diseases; it is characterized by a pathological increase in portal pressure. Pharmacotherapy for PHT is aimed at reducing both intrahepatic vascular tone and elevated splanchnic blood flow. Due to the altered hemodynamic profile in PHT, dramatic changes in mechanical forces, both pressure and flow, may play a pivotal role in controlling endothelial and vascular smooth muscle cell signaling, structure, and function in cirrhotics. Nitric oxide, prostacyclin, endothelial-derived contracting factors, and endothelial-derived hyperpolarizing factor are powerful vasoactive substances released from the endothelium in response to both humoral and mechanical stimuli that can profoundly affect both the function and structure of the underlying vascular smooth muscle. This review will examine the contributory role of hormonal- and mechanical force-induced changes in endothelial function and signaling and the consequence of these changes on the structural and functional response of the underlying vascular smooth muscle. It will focus on the pivotal role of hormonal and mechanical force-induced endothelial release of vasoactive substances in dictating the reactivity of the underlying vascular smooth muscle, i.e., whether hyporeactive or hyperreactive, and will examine the extent to which these substances may exert a protective and/or detrimental influence on the structure of the underlying vascular smooth muscle in both a normal hemodynamic environment and following hemodynamic perturbations typical of PHT and cirrhosis. Finally, it will discuss the intracellular processes that regulate the release/expression of these vasoactive substances and that control the transformation of this normally protective cell to one that may promote the development of vasculopathy in PHT.

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.

Endothelial Cells Inhibit Flow-Induced Smooth Muscle Cell Migration Role of Plasminogen Activator Inhibitor-1

Background —The endothelium may play a pivotal role in hemodynamic force–induced vascular remodeling. We investigated the role of endothelial cell (EC) plasminogen activator inhibitor-1 (PAI-1) in modulating flow-induced smooth muscle cell (SMC) migration. Methods and Results —Human SMCs cocultured with or without human ECs were exposed to static (0 mL/min) or flow (26 mL/min; shear stress 23 dyne/cm2) conditions for 24 hours in a perfused capillary culture system. SMC migration was then assessed with a Transwell migration assay. In the absence but not in the presence of ECs, pulsatile flow significantly increased the migration of SMCs (264±26%) compared with SMCs under static conditions, concomitant with a 3- and 4-fold increase in PAI-1 mRNA and protein, respectively, in cocultured ECs. In the presence of PAI-1−/− ECs, flow increased wild-type SMC migration (226±25%), an effect that was reversed by exogenous PAI-1. To determine whether the antimigratory activity of PAI-1 was dependent primarily on inhibition of PAs or its association with vitronectin, experiments were conducted with PAI-1R (a mutant PAI-1 that binds to vitronectin but does not inhibit PA) and PAI-1K (a mutant that inhibits PA but has reduced affinity for vitronectin). PAI-1R inhibited both basal and flow-induced migration, whereas PAI-1K inhibited flow-induced migration in the absence of any effect on baseline migration. Conclusions —Flow-induced EC PAI-1 inhibits flow-induced SMC migration in vitro. EC PAI-1 expression may be one of the predominant mechanisms responsible for controlling the process of vascular remodeling.

Regulation of endothelin receptors by nitric oxide in cultured rat vascular smooth muscle cells

Two important mediators of endothelium‐dependent regulation of vascular smooth muscle tone and proliferation are nitric oxide (NO) and endothelin (ET‐1). An imbalance between NO and ET‐1 may contribute to the alterations in vascular tone characteristic of cardiovascular disease. The objective of this study was to determine whether NO regulates ET receptors in cultured rat superior mesenteric artery vascular smooth muscle cells (RVSMC). Chronic treatment of quiescent RVSMC with any one of three chemically dissimilar NO‐generating drugs, S‐nitroso‐N‐acetyl penicillamine (SNAP), sodium nitroprusside (SNP), and isosorbide dinitrate (ISDN) produced a significant dose‐ and time‐dependent increase in the number of ET‐A receptors, while concomitantly increasing the affinity of ET‐1 for this receptor. This effect was mimicked by both 8‐bromo‐cGMP and 8‐bromo‐cAMP. The requirement of both protein and RNA synthesis and activation of a cAMP‐dependent protein kinase (A‐kinase) was demonstrated following inhibition of this regulation by cycloheximide, actinomycin D and KT5720 (a specific A‐kinase inhibitor), respectively. In addition, the cytokine interleukin 1β (IL‐1β) which induced NOS activity with subsequent NO synthesis in vascular smooth muscle, also caused a similar upregulation of ET receptors. This effect was attenuated in the presence of the specific NOS inhibitor, L‐NAME. To assess the possible functional consequences of this NO‐mediated upregulation, the effect of SNAP pretreatment on isolated vessel reactivity was determined. In both superior mesenteric artery and thoracic aorta rings, SNAP pretreatment caused a significant increase in the maximal force of contraction to ET‐1. Collectively, these data suggest that NO regulates ET‐A receptors in vitro through a cGMP‐dependent mechanism via activation of the cAMP‐dependent protein kinase. We conclude that a similar interaction between NO and ET‐1 may be operational in vivo.

Perfused transcapillary smooth muscle and endothelial cell co-culture—a novelin vitro model

SummaryAs mostin vitro endothelial cell (EC)-vascular smooth muscle cell (SMC) co-culture studies have been performed utilizing static culture conditions, none have successfully mimicked the physical environment of these cellsin vivo. EC covering the inner surface of blood vessels are continuously exposed to a hemodynamically imposed mechanical stress resulting from the flow of blood, while SMC are affected by pressure, a flow-related force acting perpendicular to the surface. We have developed a perfused transcapillary co-culture system that permits the chronic exposure of EC and SMC to physiological shear stresses and pressures.SMC and EC co-cultures were successfully established and maintained in long-term culture (7 wk) on an enclosed perfused bundle of semipermeable polypropylene capillaries. By altering flow rate and/or viscosity, shear stresses of 0.07–20 dyn/cm2 can be readily achieved in this system. Electron microscopic analysis revealed that SMC formed multilayers around the outside of the capillaries, whereas EC, subjected to 3 dyn/cm2 shear stress, formed an intact closely adherent monolayer lining the capillary lumen. EC and SMC exhibited characteristic ultrastructural and gross morphology. EC were separated from SMC by the capillary wall (pore size 0.5 µm, width 150 µM) and while no direct cell-cell contact was evident some cells were seen to migrate into the capillary wall. Both EC and SMC are exposed to the same culture medium, allowing the interaction of substances released in both directions. Yet separate populations of cells are maintained and can be individually harvested for further analysis. This co-culture system that mimics the architecture and physical environment of the vessel wall should have many potential applications in vascular biology.

Nitric oxide regulates angiotensin II receptors in vascular smooth muscle cells

The objective of this study was to determine whether an enhanced generation of nitric oxide (NO) causes regulation of angiotensin II receptors in vitro using rat vascular smooth muscle cells in culture. Chronic treatment of cells with a series of NO-generating drugs, sodium nitroprusside, S-nitroso-N-acetylpenicillamine and isosorbide dinitrate for 18h dose and time-dependently decreased [125I]-angiotensin II binding to cells without any significant change in affinity. Induction of nitric oxide synthase following lipopolysaccharide (10 and 100 ng/ml) treatment of cells for 18 h increased basal nitric oxide synthase activity with a concomitant increase of nitrite and cyclic cGMP levels in the conditioned media. LPS treatment significantly (P < 0.05) decreased [125I]-angiotensin II binding to these cells, an effect that was significantly (P < 0.05) attenuated in the presence of NG-nitro-L-arginine methyl ester. In contrast, treatment of cells with atrial natriuretic factor, dibutyryl cGMP, 8-bromo-cGMP, NaNO2 or NaNO3 failed to significantly alter the affinity or number of [125I]-angiotensin II binding sites. These results suggest that NO regulates angiotensin II receptors in vitro through a cGMP-independent mechanism.

The role of nitric oxide synthase isoforms in extrahepatic portal hypertension: studies in gene-knockout mice

BACKGROUND & AIMS Considerable debate exists concerning which isoform of nitric oxide synthase (NOS) is responsible for the increased production of NO in PHT. We used the portal vein ligation model of PHT in wild-type and eNOS- or iNOS-knockout mice to definitively determine the contribution of these isoforms in the development of PHT. METHODS The portal vein of wild-type mice, or those with targeted mutations in the nos2 gene (iNOS) or the nos3 gene (eNOS), was ligated and portal venous pressure (Ppv), abdominal aortic blood flow (Qao), and portosystemic shunt determined 2 weeks later. RESULTS In wild-type mice, as compared with sham-operated controls, portal vein ligation (PVL) resulted in a time-dependent increase in Ppv (7.72 +/- 0.37 vs 17.57 +/- 0.51 cmH(2)O, at 14 days) concomitant with a significant increase in Qao (0.12 +/- 0.003 vs 0.227 +/- 0.005 mL/min/g) and portosystemic shunt (0.47% +/- 0.01% vs 84.13% +/- 0.09% shunt). Likewise, PVL in iNOS-deficient mice resulted in similar increases in Ppv, Qao, and shunt development. In contrast, after PVL in eNOS-deficient animals, there was no significant change in Ppv (7.52 +/- 0.22 vs 8.07 +/- 0.4 cmH(2)0) or Qao (0.111 +/- 0.01 vs 0.14 +/-.023 mL/min/g). However, eNOS (-/-) mice did develop a substantial portosystemic shunt (0.33% +/- 0.005% vs 84.53% +/- 0.19% shunt), comparable to that seen in wild-type animals after PVL. CONCLUSIONS These data support a key role for eNOS, rather than iNOS, in the pathogenesis of PHT.