Moderate dose alcohol protects against serum amyloid protein A1-induced endothelial dysfunction via both notch-dependent and notch-independent pathways.

BACKGROUND: Arterial endothelium plays a critical role in maintaining vessel homeostasis and preventing atherosclerotic cardiovascular disease (CVD). Low-to-moderate alcohol (EtOH) consumption is associated with reduced atherosclerosis and stimulates Notch signaling in endothelial cells. The aim of this study was to determine whether EtOH protects the endothelium against serum amyloid A1 (SAA1)-induced activation/injury, and to determine whether this protection is exclusively Notch-dependent. METHODS AND RESULTS: Human coronary artery endothelial cells (HCAEC) were stimulated or not with "pro-atherogenic" SAA1 (1 µM) in the absence or presence of EtOH (25 mM), the Notch ligand DLL4 (3 µg/ml), or the Notch inhibitor DAPT (20 µM). EtOH stimulated Notch signaling in HCAEC, as evidenced by increased expression of the Notch receptor and hrt target genes. Treatment with EtOH alone or stimulation of Notch signaling by DLL4 increased eNOS activity and enhanced HCAEC barrier function as assessed by trans-endothelial electrical resistance. Moreover, EtOH and DLL4 both inhibited SAA1-induced monolayer leakiness, cell adhesion molecule (ICAM, VCAM) expression, and monocyte adhesion. The effects of EtOH were Notch-dependent, as they were blocked with DAPT and by Notch receptor (N1, N4) knockdown. In contrast, EtOH's inhibition of SAA1-induced inflammatory cytokines (IL-6, IFN-γ) and reactive oxygen species (ROS) was Notch-independent, as these effects were unaffected by DAPT or by N1 and/or N4 knockdown. CONCLUSIONS: EtOH at moderate levels protects against SAA1-induced endothelial activation via both Notch-dependent and Notch-independent mechanisms. EtOH's maintenance of endothelium in a nonactivated state would be expected to preserve vessel homeostasis and protect against atherosclerosis development.

Moderate Alcohol Consumption Targets S100ß+ Vascular Stem Cells and Attenuates Injury-Induced Neointimal Hyperplasia.

BACKGROUND: Stem cells present in the vessel wall may be triggered in response to injurious stimuli to undergo differentiation and contribute to vascular disease development. Our aim was to determine the effect of moderate alcohol (EtOH) exposure on the expansion and differentiation of S100 calcium-binding protein B positive (S100ß+ ) resident vascular stem cells and their contribution to pathologic vessel remodeling in a mouse model of arteriosclerosis. METHODS AND RESULTS: Lineage tracing analysis of S100ß+ cells was performed in male and female S100ß-eGFP/Cre/ERT2-dTomato transgenic mice treated daily with or without EtOH by oral gavage (peak BAC: 15 mM or 0.07%) following left common carotid artery ligation for 14 days. Carotid arteries (ligated or sham-operated) were harvested for morphological analysis and confocal assessment of fluorescent-tagged S100 ß + cells in FFPE carotid cross sections. Ligation-induced carotid remodeling was more robust in males than in females. EtOH-gavaged mice had less adventitial thickening and markedly reduced neointimal formation compared to controls, with a more pronounced inhibitory effect in males compared to females. There was significant expansion of S100ß+ -marked cells in vessels postligation, primarily in the neointimal compartment. EtOH treatment reduced the fraction of S100ß+ cells in carotid cross sections, concomitant with attenuated remodeling. In vitro, EtOH attenuated Sonic Hedgehog-stimulated myogenic differentiation (as evidenced by reduced calponin and myosin heavy chain expression) of isolated murine S100ß+ vascular stem cells. CONCLUSIONS: These data highlight resident vascular S100ß+ stem cells as a novel target population for alcohol and suggest that regulation of these progenitors in adult arteries, particularly in males, may be an important mechanism contributing to the antiatherogenic effects of moderate alcohol consumption.

Differential effects of alcohol and its metabolite acetaldehyde on vascular smooth muscle cell Notch signaling and growth.

Alcohol (EtOH) consumption can variously affect cardiovascular disease. Our aim was to compare the effects of EtOH and its primary metabolite acetaldehyde (ACT) on vascular smooth muscle Notch signaling and cell growth, which are important for atherogenesis. Human coronary artery smooth muscle cells (HCASMCs) were treated with EtOH (25 mM) or ACT (10 or 25 µM). As previously reported, EtOH inhibited Notch signaling and growth of HCASMCs. In contrast, ACT treatment stimulated HCASMC proliferation (cell counts) and increased proliferating cell nuclear antigen expression, concomitant with stimulation of Notch signaling, as determined by increased Notch receptor (N1 and N3) and target gene (Hairy-related transcription factor 1-3) mRNA levels. Interaction of the ligand with the Notch receptor initiates proteolytic cleavage by α- and γ-secretase, resulting in the release of the active Notch intracellular domain. Neither EtOH nor ACT had any significant effect on α-secretase activity. A fluorogenic peptide cleavage assay demonstrated almost complete inhibition by EtOH of Delta-like ligand 4-stimulated γ-secretase activity in solubilized HCASMCs (similar to the effect of the control inhibitor DAPT) but no effect of ACT treatment. EtOH, but not ACT, affected the association and distribution of the γ-secretase catalytic subunit presenilin-1 with lipid rafts, as determined by dual fluorescent labeling and confocal microscopic visualization. In conclusion, ACT stimulates vascular smooth muscle cell Notch signaling and growth, effects opposite to those of EtOH. These differential actions on vascular smooth muscle cells of EtOH and its metabolite ACT may be important in mediating the ultimate effects of drinking on cardiovascular disease. NEW & NOTEWORTHY Acetaldehyde stimulates, in a Notch-dependent manner, the vascular smooth muscle cell growth that contributes to atherogenesis; effects opposite to those of ethanol. These data suggest that in addition to ethanol itself, its metabolite acetaldehyde may also mediate some of the effects of alcohol consumption on vascular cells and, thus, cardiovascular health.

Alcohol Reduces Arterial Remodeling by Inhibiting Sonic Hedgehog-Stimulated Stem Cell Antigen-1 Positive Progenitor Stem Cell Expansion.

BACKGROUND: Cell and molecular mechanisms mediating the cardiovascular effects of alcohol are not fully understood. Our aim was to determine the effect of moderate ethanol (EtOH) on sonic hedgehog (SHh) signaling in regulating possible stem cell antigen-1 positive (Sca1+ ) progenitor stem cell involvement during pathologic arterial remodeling. METHODS: Partial ligation or sham operation of the left carotid artery was performed in transgenic Sca1-enhanced green fluorescent protein (eGFP) mice gavaged with or without "daily moderate" EtOH. RESULTS: The EtOH group had reduced adventitial thickening and less neointimal formation, compared to ligated controls. There was expansion of eGFP-expressing (i.e., Sca1+ ) cells in remodeled vessels postligation (day 14), especially in the neo intima. EtOH treatment reduced the number of Sca1+ cells in ligated vessel cross-sections concomitant with diminished remodeling, compared to control ligated vessels. Moreover, EtOH attenuated SHh signaling in injured carotids as determined by immunohistochemical analysis of the target genes patched 1 and Gli2, and RT-PCR of whole-vessel Gli2 mRNA levels. Intraperitoneal injection of ligated Sca1-eGFP mice with the SHh signaling inhibitor cyclopamine diminished SHh target gene expression, reduced the number of Sca1+ cells, and ameliorated carotid remodeling. EtOH treatment of purified Sca1+ adventitial progenitor stem cells in vitro inhibited SHh signaling, and their rSHh-induced differentiation to vascular smooth muscle cells. CONCLUSIONS: EtOH reduces SHh-responsive Sca1+ progenitor cell myogenic differentiation/expansion in vitro and during arterial remodeling in response to ligation injury in vivo. Regulation of vascular Sca1+ progenitor cells in this way may be an important novel mechanism contributing to alcohol's cardiovascular protective effects.

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

OBJECTIVE: The molecular mechanisms leading to the development of abdominal aortic aneurysms (AAAs) remain poorly understood. The aim of this study was to determine the expression of Sonic Hedgehog (SHh), transforming growth factor ß (TGF-ß), and Notch signaling components in human aneurysmal and nonaneurysmal aorta in vivo. METHODS: Paired tissue samples were obtained from aneurysmal and nonaneurysmal (control) segments of the aortic wall of eight patients with suitable anatomy undergoing open repair of infrarenal AAAs. Protein and messenger RNA (mRNA) expression levels were determined by Western blot and quantitative real-time polymerase chain reaction analysis. RESULTS: Aneurysm development resulted in a significant reduction in vascular smooth muscle (vSMC) differentiation genes α-actin and SMC22α at both mRNA and protein levels. In parallel experiments, an 80.0% ± 15% reduction in SHh protein expression was observed in aneurysmal tissue compared with control. SHh and Ptc-1 mRNA levels were also significantly decreased, by 82.0% ± 10% and 75.0% ± 5%, respectively, in aneurysmal tissue compared with nonaneurysmal control tissue. Similarly, there was a 50.0% ± 9% and 60.0% ± 4% reduction in Notch receptor 1 intracellular domain and Hrt-2 protein expression, respectively, in addition to significant reductions in Notch 1, Notch ligand Delta like 4, and Hrt-2 mRNA expression in aneurysmal tissue compared with nonaneurysmal tissue. There was no change in Hrt-1 expression observed in aneurysmal tissue compared with control. In parallel experiments, we found a 2.2 ± 0.2-fold and a 5.6 ± 2.2-fold increase in TGF-ß mRNA and protein expression, respectively, in aneurysmal tissue compared with nonaneurysmal tissue. In vitro, Hedgehog signaling inhibition with cyclopamine in human aortic SMCs resulted in decreased Hedgehog/Notch signaling component and vSMC differentiation gene expression. Moreover, cyclopamine significantly increased TGF-ß1 mRNA expression by 2.6 ± 0.9-fold. CONCLUSIONS: These results suggest that SHh/Notch and TGF-ß signaling are differentially regulated in aneurysmal tissue compared with nonaneurysmal tissue. Changes in these signaling pathways and the resulting changes in vSMC content may play a causative role in the development of AAAs.

Ethanol inhibits γ-secretase proteolytic activity in vascular smooth muscle cells.

BACKGROUND: Ethanol (EtOH) inhibits Notch-mediated vascular smooth muscle cell (SMC) proliferation, an event that is key in vessel remodeling and atherogenesis. The object of this study was to determine whether EtOH inhibits Notch signaling in SMC at the level of γ-secretase, a protease that in concert with α-secretase catalyzes the release of the intracellular domain of the Notch receptor necessary for signaling. METHODS: Human coronary artery SMCs (HCASMCs) were treated with a recombinant soluble Notch ligand, Delta-like ligand 4 (DLL4) (2 µg/ml), or transfected with a constitutively active Notch 1 intracellular domain (N1ICD), in the absence or presence of EtOH. EtOH (25 mM) treatment inhibited DLL4-stimulated CBF-1/RBP-Jk-dependent promoter activity (determined by luciferase assay) and downstream target gene HRT-3 mRNA levels. In contrast, EtOH had no effect on N1ICD-driven CBF-1/RBP-Jk-dependent promoter activity or HRT-3 expression. RESULTS: These data suggest that EtOH inhibits Notch signaling at, or prior to, Notch intracellular domain (NICD) generation. γ-Secretase activity was determined in solubilized membrane preparations from HCASMC treated with/without EtOH (25 mM) or the γ-secretase inhibitor DAPT (20 µM) using (i) a fluorometric assay and (ii) Western blot detection of cleavage products using a Flag-tagged Notch-based substrate, N100Flag. EtOH inhibited basal and DLL4-stimulated γ-secretase activity, and SMC growth to a similar extent as DAPT, whereas it had no effect on α-secretase (TACE/ADAM17) activity also determined by fluorometric assay. Moreover, EtOH treatment inhibited the expression of caveolin-1, a lipid raft protein implicated in regulating γ-secretase activity, and altered its cellular distribution in HCASMC. CONCLUSIONS: EtOH inhibits Notch signaling in vascular SMCs at the level of γ-secretase activity, possibly by affecting lipid raft function. Such a response might be expected to result in attenuation of pathologic vessel remodeling and thus may contribute to moderate alcohols' cardioprotective effects.

Adult vascular smooth muscle cells in culture express neural stem cell markers typical of resident multipotent vascular stem cells.

Differentiation of resident multipotent vascular stem cells (MVSCs) or de-differentiation of vascular smooth muscle cells (vSMCs) might be responsible for the SMC phenotype that plays a major role in vascular diseases such as arteriosclerosis and restenosis. We examined vSMCs from three different species (rat, murine and bovine) to establish whether they exhibit neural stem cell characteristics typical of MVSCs. We determined their SMC differentiation, neural stem cell marker expression and multipotency following induction in vitro by using immunocytochemistry, confocal microscopy, fluorescence-activated cell sorting analysis and quantitative real-time polymerase chain reaction. MVSCs isolated from rat aortic explants, enzymatically dispersed rat SMCs and rat bone-marrow-derived mesenchymal stem cells served as controls. Murine carotid artery lysates and primary rat aortic vSMCs were both myosin-heavy-chain-positive but weakly expressed the neural crest stem cell marker, Sox10. Each vSMC line examined expressed SMC differentiation markers (smooth muscle α-actin, myosin heavy chain and calponin), neural crest stem cell markers (Sox10(+), Sox17(+)) and a glia marker (S100ß(+)). Serum deprivation significantly increased calponin and myosin heavy chain expression and decreased stem cell marker expression, when compared with serum-rich conditions. vSMCs did not differentiate to adipocytes or osteoblasts following adipogenic or osteogenic inductive stimulation, respectively, or respond to transforming growth factor-ß1 or Notch following γ-secretase inhibition. Thus, vascular SMCs in culture express neural stem cell markers typical of MVSCs, concomitant with SMC differentiation markers, but do not retain their multipotency. The ultimate origin of these cells might have important implications for their use in investigations of vascular proliferative disease in vitro.

Flk-1/KDR mediates ethanol-stimulated endothelial cell Notch signaling and angiogenic activity.

UNLABELLED: We previously reported that ethanol (EtOH) stimulates endothelial angiogenic activity mediated via a notch- and angiopoietin-1 (Ang-1) pathway. As crosstalk exists between notch and vascular endothelial growth factor (VEGF) signaling, we examined whether the VEGF receptor (VEGFR) Flk-1 (fetal liver kinase 1) mediates EtOH-stimulated notch signaling and angiogenic activity. METHODS AND RESULTS: Treatment of human coronary artery endothelial cells (HCAECs) with EtOH (1-50 mM, 24 h) dose-dependently increased Flk-1 expression with a maximum increase observed at 25 mM EtOH. Ethanol treatment activated both Flk-1 and Flt-1 (FMS-like tyrosine kinase 1) as indicated by their phosphorylation, and subsequent stimulation of Akt. EtOH activation of Flk-1 was inhibited by the VEGFR inhibitor SU5416. Gene silencing of Flk-1 using small interfering RNA inhibited the EtOH-induced increase in notch receptors 1 and 4 and notch target gene (hairy enhancer of split-related transcription factor 1) mRNA. Knockdown of Flk-1 inhibited EtOH-induced Ang-1/Tie-2 mRNA expression and blocked EtOH-induced HCAEC network formation on Matrigel, a response that was restored by notch ligand, notch ligand delta-like ligand 4, treatment. In vivo, moderate alcohol feeding increased vascular remodeling in mouse ischemic hindlimbs. CONCLUSIONS: These data demonstrate that EtOH activates Flk-1 and Flt-1 receptors in HCAECs and promotes angiogenic activity via an Flk-1/notch pathway. These effects of EtOH may be relevant to the influence of moderate alcohol consumption on cardiovascular health.

Inhibition of patched-1 prevents injury-induced neointimal hyperplasia.

OBJECTIVE: To determine the role of patched receptor (Ptc)-1 in mediating pulsatile flow-induced changes in vascular smooth muscle cell growth and vascular remodeling. APPROACH AND RESULTS: In vitro, human coronary arterial smooth muscle cells were exposed to normal or pathological low pulsatile flow conditions for 24 hours using a perfused transcapillary flow system. Low pulsatile flow increased vascular smooth muscle cell proliferation when compared with normal flow conditions. Inhibition of Ptc-1 by cyclopamine attenuated low flow-induced increases in Notch expression while concomitantly decreasing human coronary arterial smooth muscle cell growth to that similar under normal flow conditions. In vivo, ligation injury-induced low flow increased vascular smooth muscle cell growth and vascular remodeling, while increasing Ptc-1/Notch expression. Perivascular delivery of Ptc-1 small interfering RNA by pluronic gel inhibited the pathological low flow-induced increases in Ptc-1/Notch expression and markedly reduced the subsequent vascular remodeling. CONCLUSIONS: These results suggest that pathological low flow stimulates smooth muscle cell growth in vitro and vascular remodeling in vivo via Ptc-1 regulation of Notch signaling.

Caveolin-1 inhibition mediates the opposing effects of alcohol on γ-secretase activity in arterial endothelial and smooth muscle cells.

Notch is important to vessel homeostasis. We investigated the mechanistic role of caveolin-1 (Cav-1) in mediating the effects of alcohol (Ethanol/EtOH) on the γ-secretase proteolytic activity necessary for Notch signaling in vascular cells. Human coronary artery endothelial cells (HCAEC) were treated with EtOH (0-50 mM), Notch ligand delta-like ligand 4 (Dll4), and the γ-secretase inhibitor DAPT. EtOH stimulated Notch signaling in HCAEC as evidenced by increased Notch receptor (N1, N4) and target gene (hrt2, hrt3) mRNA levels with the most robust response achieved at 25 mM EtOH. Ethanol (25 mM) stimulated γ-secretase proteolytic activity, to the same extent as Dll4, in HCAEC membranes. Ethanol inhibited Cav-1 mRNA and protein levels in HCAEC. Caveolin-1 negatively regulated γ-secretase activity in HCAEC as Cav-1 knockdown stimulated it, while Cav-1 overexpression inhibited it. Moreover, Cav-1 overexpression blocked the stimulatory effect of EtOH on γ-secretase activity in HCAEC. Although EtOH also inhibited Cav-1 expression in human coronary artery smooth muscle cells (HCASMC), EtOH inhibited γ-secretase activity in HCASMC in contrast to its effect in HCAEC. The inhibitory effect of EtOH on γ-secretase in HCASMC was mimicked by Cav-1 knockdown and prevented by Cav-1 overexpression, suggesting that in these cells Cav-1 positively regulates γ-secretase activity. In conclusion, EtOH differentially regulates γ-secretase activity in arterial EC and SMC, being stimulatory and inhibitory, respectively. These effects are both mediated by caveolin-1 inhibition which itself has opposite effects on γ-secretase in the two cell types. This mechanism may underlie, in part, the effects of moderate drinking on atherosclerosis.

Alcohol and vascular endothelial function: Biphasic effect highlights the importance of dose.

BACKGROUND: Alcohol (ethanol) consumption has different influences on arterial disease, being protective or harmful depending on the amount and pattern of consumption. The mechanisms mediating these biphasic effects are unknown. Whereas endothelial cells play a critical role in maintaining arterial health, this study compared the effects of moderate and high alcohol concentrations on endothelial cell function. METHODS: Human coronary artery endothelial cells (HCAEC) were treated with levels of ethanol associated with either low-risk/moderate drinking (i.e., 25 mM) or high-risk/heavy drinking (i.e., 50 mM) after which endothelial function was assessed. The effect of ethanol's primary metabolite acetaldehyde (10 and 25 µM) was also determined. RESULTS: Moderate ethanol exposure (25 mM) improved HCAEC barrier integrity as determined by increased transendothelial electrical resistance (TEER), inhibited cell adhesion molecule (CAM) mRNA expression, decreased inflammatory cytokine (interferon-γ and interleukin 6) production, inhibited monocyte chemotactic protein-1 (MCP-1) expression and monocyte adhesion, and increased homeostatic Notch signaling. In contrast, exposure to high-level ethanol (50 mM) decreased TEER, increased CAM expression and inflammatory cytokine production, and stimulated MCP-1 and monocyte adhesion, with no effect on Notch signaling. Reactive oxygen species (ROS) generation and endothelial nitric oxide synthase activity were increased by both alcohol treatments, and to a greater extent in the 50 mM ethanol group. Acetaldehyde-elicited responses were generally the same as those of the high-level ethanol group. CONCLUSIONS: Ethanol has biphasic effects on several endothelial functions such that a moderate level maintains the endothelium in a nonactivated state, whereas high-level ethanol causes endothelial dysfunction, as does acetaldehyde. These data show the importance of dose when considering ethanol's effects on arterial endothelium, and could explain, in part, the J-shaped relationship between alcohol concentration and atherosclerosis reported in some epidemiologic studies.

N-Glycans on the extracellular domain of the Notch1 receptor control Jagged-1 induced Notch signalling and myogenic differentiation of S100ß resident vascular stem cells.

Notch signalling, critical for development and postnatal homeostasis of the vascular system, is highly regulated by several mechanisms including glycosylation. While the importance of O-linked glycosylation is widely accepted, the structure and function of N-glycans has yet to be defined. Here, we take advantage of lectin binding assays in combination with pharmacological, molecular, and site-directed mutagenetic approaches to study N-glycosylation of the Notch1 receptor. We find that several key oligosaccharides containing bisecting or core fucosylated structures decorate the receptor, control expression and receptor trafficking, and dictate Jagged-1 activation of Notch target genes and myogenic differentiation of multipotent S100ß vascular stem cells. N-glycans at asparagine (N) 1241 and 1587 protect the receptor from accelerated degradation, while the oligosaccharide at N888 directly affects signal transduction. Conversely, N-linked glycans at N959, N1179, N1489 do not impact canonical signalling but inhibit differentiation. Our work highlights a novel functional role for N-glycans in controlling Notch1 signalling and differentiation of vascular stem cells.

Endothelial Grb2-associated binder 1 is crucial for postnatal angiogenesis.

OBJECTIVE: Grb2-associated binder 1 (Gab1), a scaffolding adaptor protein, plays an important role in transmitting key signals that control cell growth, differentiation, and function from multiple tyrosine kinase receptors. The study was designed to investigate the role of endothelial Gab1 in angiogenesis and its underlying molecular mechanisms. METHODS AND RESULTS: Using Cre-Lox recombination technology, we generated endothelial-specific Gab1 knockout (Gab1-ecKO) mice. Gab1-ecKO mice are viable and showed no obvious developmental defects in the vascular system. To analyze the role of Gab1 in postnatal angiogenesis, we used hindlimb ischemia and Matrigel plug models. We found that loss of endothelial Gab1 in mice dramatically impaired postnatal angiogenesis. Gab1-ecKO mice had impaired ischemia-initiated blood flow recovery, exhibited reduced angiogenesis, and were associated with marked limb necrosis. We further observed significant endothelial cell (EC) death in the ischemic hindlimb of Gab1-ecKO mice. Matrigel plug assay showed that hepatocyte growth factor (HGF)-mediated angiogenesis was inhibited in Gab1-ecKO mice. In vitro studies showed that Gab1 was required for HGF-induced EC migration, tube formation, and microvessel sprouting. Mechanistically, HGF stimulated Gab1 tyrosine phosphorylation in ECs, leading to activation of extracellular regulated MAP kinase 1/2 and Akt, which are angiogenic and survival signaling. CONCLUSIONS: Gab1 is essential for postnatal angiogenesis through mediating angiogenic and survival signaling.

Exosomal Composition, Biogenesis and Profiling Using Point-of-Care Diagnostics-Implications for Cardiovascular Disease.

Arteriosclerosis is an important age-dependent disease that encompasses atherosclerosis, in-stent restenosis (ISR), pulmonary hypertension, autologous bypass grafting and transplant arteriosclerosis. Endothelial dysfunction and the proliferation of vascular smooth muscle cell (vSMC)-like cells is a critical event in the pathology of arteriosclerotic disease leading to intimal-medial thickening (IMT), lipid retention and vessel remodelling. An important aspect in guiding clinical decision-making is the detection of biomarkers of subclinical arteriosclerosis and early cardiovascular risk. Crucially, relevant biomarkers need to be good indicators of injury which change in their circulating concentrations or structure, signalling functional disturbances. Extracellular vesicles (EVs) are nanosized membraneous vesicles secreted by cells that contain numerous bioactive molecules and act as a means of intercellular communication between different cell populations to maintain tissue homeostasis, gene regulation in recipient cells and the adaptive response to stress. This review will focus on the emerging field of EV research in cardiovascular disease (CVD) and discuss how key EV signatures in liquid biopsies may act as early pathological indicators of adaptive lesion formation and arteriosclerotic disease progression. EV profiling has the potential to provide important clinical information to complement current cardiovascular diagnostic platforms that indicate or predict myocardial injury. Finally, the development of fitting devices to enable rapid and/or high-throughput exosomal analysis that require adapted processing procedures will be evaluated.

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-3ß) 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-3ß in vSMC following ectopic expression of constitutively active GSK-3ß, siRNA knockdown and pharmacological inhibition with SB-216763 demonstrated that GSK-3ß 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-3ß 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-3ß 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-3ß 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-3ß is an important modulator of Notch signaling leading to altered vSMC cell growth where low strain/tension microenvironments prevail.

Alcohol inhibits smooth muscle cell proliferation via regulation of the Notch signaling pathway.

OBJECTIVE: To determine the role of Notch signaling in mediating alcohol's 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. CONCLUSIONS: 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.

The calcium binding protein S100ß marks hedgehog-responsive resident vascular stem cells within vascular lesions.

A hallmark of subclinical atherosclerosis is the accumulation of vascular smooth muscle cell (SMC)-like cells leading to intimal thickening. While medial SMCs contribute, the participation of hedgehog-responsive resident vascular stem cells (vSCs) to lesion formation remains unclear. Using transgenic eGFP mice and genetic lineage tracing of S100ß vSCs in vivo, we identified S100ß/Sca1 cells derived from a S100ß non-SMC parent population within lesions that co-localise with smooth muscle α-actin (SMA) cells following iatrogenic flow restriction, an effect attenuated following hedgehog inhibition with the smoothened inhibitor, cyclopamine. In vitro, S100ß/Sca1 cells isolated from atheroprone regions of the mouse aorta expressed hedgehog signalling components, acquired the di-methylation of histone 3 lysine 4 (H3K4me2) stable SMC epigenetic mark at the Myh11 locus and underwent myogenic differentiation in response to recombinant sonic hedgehog (SHh). Both S100ß and PTCH1 cells were present in human vessels while S100ß cells were enriched in arteriosclerotic lesions. Recombinant SHh promoted myogenic differentiation of human induced pluripotent stem cell-derived S100ß neuroectoderm progenitors in vitro. We conclude that hedgehog-responsive S100ß vSCs contribute to lesion formation and support targeting hedgehog signalling to treat subclinical arteriosclerosis.

Disease-Relevant Single Cell Photonic Signatures Identify S100ß Stem Cells and their Myogenic Progeny in Vascular Lesions.

A hallmark of subclinical atherosclerosis is the accumulation of vascular smooth muscle cell (SMC)-like cells leading to intimal thickening and lesion formation. While medial SMCs contribute to vascular lesions, the involvement of resident vascular stem cells (vSCs) remains unclear. We evaluated single cell photonics as a discriminator of cell phenotype in vitro before the presence of vSC within vascular lesions was assessed ex vivo using supervised machine learning and further validated using lineage tracing analysis. Using a novel lab-on-a-Disk(Load) platform, label-free single cell photonic emissions from normal and injured vessels ex vivo were interrogated and compared to freshly isolated aortic SMCs, cultured Movas SMCs, macrophages, B-cells, S100ß+ mVSc, bone marrow derived mesenchymal stem cells (MSC) and their respective myogenic progeny across five broadband light wavelengths (λ465 - λ670 ± 20 nm). We found that profiles were of sufficient coverage, specificity, and quality to clearly distinguish medial SMCs from different vascular beds (carotid vs aorta), discriminate normal carotid medial SMCs from lesional SMC-like cells ex vivo following flow restriction, and identify SMC differentiation of a series of multipotent stem cells following treatment with transforming growth factor beta 1 (TGF- ß1), the Notch ligand Jagged1, and Sonic Hedgehog using multivariate analysis, in part, due to photonic emissions from enhanced collagen III and elastin expression. Supervised machine learning supported genetic lineage tracing analysis of S100ß+ vSCs and identified the presence of S100ß+vSC-derived myogenic progeny within vascular lesions. We conclude disease-relevant photonic signatures may have predictive value for vascular disease.

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.

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 mug/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, Gli(2), VEGF-A, Notch 1, and Hrts. CONCLUSIONS: Hedgehog promotes a coordinate regulation of Notch target genes in adult SMCs via VEGF-A.