John P. Cullen

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.

Pulsatile Flow–Induced Angiogenesis

Objective—Angiogenesis plays a key role in the growth and function of normal and pathological tissues. We investigated the effect of pulsatile flow on endothelial cell (EC) in vitro angiogenic activity. Methods and Results—Bovine aortic ECs were exposed to “static” or “flow” (1.2 to 67.0 mL/min, shear stress 1.4 to 19.2 dyne/cm2) conditions for 2 to 24 hours. After exposure, angiogenesis was measured as tubule formation on Matrigel, and EC migration was assessed by filter migration assay. Pulsatile flow increased angiogenesis and EC migration in a temporal and force-dependent manner, with a maximal effect at 16 hours (13.2 dyne/cm2). Pertussis toxin completely inhibited the effect of pulsatile flow on angiogenesis and migration. Transfection of ECs with inhibitory mutants of the &agr; subunit of Gi1 or Gi3, but not Gi2, inhibited the flow-induced angiogenic response by 61±2% and 32±6%, respectively, whereas transfection with constitutively activated mutants of the &agr; subunit of Gi1 or Gi3, but not Gi2, increased the flow-induced response by 202±23% and 70±4%, respectively. In contrast, inhibition of G&bgr;&ggr; by the carboxy terminal fragment of &bgr;-adrenergic receptor kinase overexpression increased the flow-induced response by 82±8%. Conclusions—These results suggest that pulsatile flow stimulates angiogenesis and that this effect is mediated by activation of Gi&agr;1 or Gi&agr;3, but not G&bgr;&ggr;, subunits.

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.

Cyclic Strain Regulates the Notch/CBF-1 Signaling Pathway in Endothelial Cells: Role in Angiogenic Activity

Objective—The purpose of this study was to determine the effect of cyclic strain on Notch signaling in endothelial cells. Methods and Results—Exposure of human endothelial cells (ECs) to cyclic strain (10%) resulted in temporal upregulation of Notch receptors (1 and 4) at the mRNA and protein level. Cyclic strain significantly increased EC network formation on Matrigel (an index of angiogenesis); network AU=775±127 versus 3928±400 for static and strained ECs, respectively. In addition, Angiopoietin 1 (Ang1), Tie1, and Tie2 expression were increased and knockdown of Ang1/Tie1,2 by siRNAs decreased cyclic strain-induced network formation. Knockdown of Notch 1 and 4 by siRNA, or inhibition of Notch mediated CBF-1/RBP-Jk regulated gene expression by RPMS-1, caused a significant decrease in cyclic strain-induced network formation and in Tie1 and Tie2 mRNA expression. Notch 1 or Notch 4 siRNA, but not RPMS-1, inhibited cyclic strain-induced Ang1. Constitutive overexpression of Notch IC resulted in increased network formation, and Ang1 and Tie2 mRNA expression, under both static and strain conditions. Conclusions—These data suggest that cyclic strain-stimulated EC angiogenesis is mediated in part through a Notch-dependent, Ang1/Tie2 signaling pathway. This pathway may represent a novel therapeutic target for disease states in which hemodynamic force-induced angiogenesis occurs.

Ethanol stimulates endothelial cell angiogenic activity via a Notch- and angiopoietin-1-dependent pathway

AIMS Our aims were to determine the effect of alcohol (EtOH) on endothelial angiogenic activity and to delineate the cell signalling mechanisms involved. METHODS AND RESULTS Treatment of human umbilical vein endothelial cells (HUVECs) with EtOH (1-100 mM, 24 h) dose-dependently increased their network formation on Matrigel (an index of angiogenesis) with a maximum response (2.5- to 3-fold increase) at 25 mM. Ethanol also stimulated the proliferation (by cell count and proliferating cell nuclear antigen expression) and migration (by scratch wound assay) of HUVECs. In parallel cultures, EtOH stimulated Notch receptor (1 and 4) and Notch target gene (hrt-1, -2, and -3) mRNA and protein expression and enhanced CBF-1/RBP-Jk promoter activity. EtOH also stimulated, at the mRNA and protein level, the expression of angiopoietin-1 (Ang1) and its Tie2 receptor in these cells. Knockdown of Notch 1 or 4 by siRNA or inhibition of Notch-mediated, CBF-1/RBP-Jk-regulated gene expression by the Epstein-Barr virus-encoded protein RPMS-1 inhibited both ethanol-induced Ang1/Tie2 expression in HUVECs and their network formation on Matrigel. Moreover, knockdown of Ang1 or Tie2 by siRNA inhibited ethanol-induced endothelial network formation. CONCLUSION These data demonstrate that ethanol, at levels consistent with moderate consumption, enhances endothelial angiogenic activity in vitro by stimulating a novel Notch/CBF-1/RBP-JK-Ang1/Tie2-dependent pathway. These actions of ethanol may be relevant to the cardiovascular effects of alcohol consumption purported by epidemiological studies.

Glycogen synthase kinase 3 beta positively regulates Notch signaling in vascular smooth muscle cells: role in cell proliferation and survival

The role of glycogen synthase kinase 3 beta (GSK-3β) in modulating Notch control of vascular smooth muscle cell (vSMC) growth (proliferation and apoptosis) was examined in vitro under varying conditions of cyclic strain and validated in vivo following changes in medial tension and stress. Modulation of GSK-3β in vSMC following ectopic expression of constitutively active GSK-3β, siRNA knockdown and pharmacological inhibition with SB-216763 demonstrated that GSK-3β positively regulates Notch intracellular domain expression, CBF-1/RBP-Jκ transactivation and downstream target gene mRNA levels, while concomitantly promoting vSMC proliferation and inhibiting apoptosis. In contrast, inhibition of GSK-3β attenuated Notch signaling and decreased vSMC proliferation and survival. Exposure of vSMC to cyclic strain environments in vitro using both a Flexercell™ Tension system and a novel Sylgard™ phantom vessel following bare metal stent implantation revealed that cyclic strain inhibits GSK-3β activity independent of p42/p44 MAPK and p38 activation concomitant with reduced Notch signaling and decreased vSMC proliferation and survival. Exposure of vSMC to changes in medial strain microenvironments in vivo following carotid artery ligation revealed that enhanced GSK-3β activity was predominantly localized to medial and neointimal vSMC concomitant with increased Notch signaling, proliferating nuclear antigen and decreased Bax expression, respectively, as vascular remodeling progressed. GSK-3β is an important modulator of Notch signaling leading to altered vSMC cell growth where low strain/tension microenvironments prevail.

Resveratrol, a Polyphenolic Phytostilbene, Inhibits Endothelial Monocyte Chemotactic Protein-1 Synthesis and Secretion

Background/Aims: Resveratrol is a naturally occurring polyphenol phytoestrogen and one of several constituents of red wine thought to be cardioprotective. We investigated the effect of resveratrol on the expression of the atherogenic chemokine, monocyte chemotactic protein-1 (MCP-1). Methods: Human umbilical vein endothelial cells were stimulated with interleukin-1β (IL-1β) in the absence or presence of resveratrol. MCP-1 levels were determined by ELISA and MCP-1 mRNA was measured. Results: Resveratrol (1–100 µM) dose-dependently inhibited IL-1β-stimulated MCP-1 secretion, with ∼45% inhibition at 50 µM resveratrol. This was a Gi-protein- and NO-dependent effect. Resveratrol also significantly inhibited MCP-1 gene expression in a Gi-protein-dependent but NO-independent manner. While resveratrol had no effect on MCP-1 mRNA degradation, it inhibited MCP-1 promoter activity and reduced nuclear factor ĸB and activator protein-1 binding activity induced by IL-1β. Moreover, while hemoxygenase-1 (HO-1) expression was induced by resveratrol in human umbilical vein endothelial cells, neither treatment with the HO-1 inhibitor tin-protoporphyrin IX nor siRNA-directed knockdown of HO-1 had any effect on the inhibition of MCP-1 mRNA or protein secretion by resveratrol. Conclusion: These data demonstrate an inhibitory effect of resveratrol on MCP-1 synthesis and secretion, mediated via distinct signaling pathways. The inhibition of MCP-1 may represent a novel cardioprotective mechanism of resveratrol.

Alcohol Inhibits Smooth Muscle Cell Proliferation via Regulation of the Notch Signaling Pathway

Objective—To determine the role of Notch signaling in mediating alcohols inhibition of smooth muscle cell (SMC) proliferation. Methods and Results—Treatment of human coronary artery SMCs with ethanol (EtOH) decreased Notch 1 mRNA and Notch 1 intracellular domain protein levels, in the absence of any effect on Notch 3. EtOH treatment also decreased C-promoter binding factor-1 (CBF-1)/recombination signal-binding protein (RBP)-jk promoter activity and Notch target gene (hairy related transcription factor [HRT-1] or HRT-2) expression. These effects were concomitant with an inhibitory effect of EtOH on SMC proliferation. Overexpression of constitutively active Notch 1 intracellular domain or human hairy related transcription factor-1 (hHRT-1) prevented the EtOH-induced inhibition of SMC proliferation. In vivo, Notch 1 and HRT-1 mRNA expression was increased after ligation-induced carotid artery remodeling. The vessel remodeling response was inhibited in mice that received “moderate” amounts of alcohol by gavage daily; intimal-medial thickening was markedly reduced, and medial and neointimal SMC proliferating cell nuclear antigen expression was decreased. Moreover, Notch 1 and HRT-1 expression, induced after ligation injury, was inhibited by moderate alcohol consumption. Conclusion—EtOH inhibits Notch signaling and, subsequently, SMC proliferation, in vitro and in vivo. The modulation of Notch signaling in SMCs by EtOH may be relevant to the cardiovascular protective effects of moderate alcohol consumption purported by epidemiological studies.

Aortic anatomic severity grade correlates with resource utilization

BACKGROUND Potential cost effectiveness of endovascular aneurysm repair (EVAR) compared with open aortic repair (OAR) is offset by the use of intraoperative adjuncts (components) or late reinterventions. Anatomic severity grade (ASG) can be used preoperatively to assess abdominal aortic aneurysms, and provide a quantitative measure of anatomic complexity. The hypothesis of this study is that ASG is directly related to the use of intraoperative adjuncts and cost of aortic repair. METHODS Patients who undergo elective OAR and EVAR for abdominal aortic aneurysms were identified over a consecutive 3-year period. ASG scores were calculated manually using three-dimensional reconstruction software by two blinded reviewers. Statistical analysis of cost data was performed using a log transformation. Regression analyses, with a continuous or dichotomous outcome, used a generalized estimating equations approach with the sandwich estimator, being robust with respect to deviations from model assumptions. RESULTS One hundred forty patients were identified for analysis, n = 33 OAR and n = 107 EVAR. The mean total cost (± standard deviation) for OAR was per thousand (k)

Differential expression of Hedgehog/Notch and transforming growth factor-β in human abdominal aortic aneurysms

38.3 ± 49.3, length of stay (LOS) 13.5 ± 14.2 days, ASG score 18.13 ± 3.78; for EVAR, mean total cost was k