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.

Clearance of amyloid-beta by circulating lipoprotein receptors.

Low-density lipoprotein receptor-related protein-1 (LRP) on brain capillaries clears amyloid beta-peptide (Abeta) from brain. Here, we show that soluble circulating LRP (sLRP) provides key endogenous peripheral 'sink' activity for Abeta in humans. Recombinant LRP cluster IV (LRP-IV) bound Abeta in plasma in mice and Alzheimer's disease-affected humans with compromised sLRP-mediated Abeta binding, and reduced Abeta-related pathology and dysfunction in a mouse model of Alzheimer disease, suggesting that LRP-IV can effectively replace native sLRP and clear Abeta.

apoE isoform-specific disruption of amyloid beta peptide clearance from mouse brain.

Neurotoxic amyloid beta peptide (Abeta) accumulates in the brains of individuals with Alzheimer disease (AD). The APOE4 allele is a major risk factor for sporadic AD and has been associated with increased brain parenchymal and vascular amyloid burden. How apoE isoforms influence Abeta accumulation in the brain has, however, remained unclear. Here, we have shown that apoE disrupts Abeta clearance across the mouse blood-brain barrier (BBB) in an isoform-specific manner (specifically, apoE4 had a greater disruptive effect than either apoE3 or apoE2). Abeta binding to apoE4 redirected the rapid clearance of free Abeta40/42 from the LDL receptor-related protein 1 (LRP1) to the VLDL receptor (VLDLR), which internalized apoE4 and Abeta-apoE4 complexes at the BBB more slowly than LRP1. In contrast, apoE2 and apoE3 as well as Abeta-apoE2 and Abeta-apoE3 complexes were cleared at the BBB via both VLDLR and LRP1 at a substantially faster rate than Abeta-apoE4 complexes. Astrocyte-secreted lipo-apoE2, lipo-apoE3, and lipo-apoE4 as well as their complexes with Abeta were cleared at the BBB by mechanisms similar to those of their respective lipid-poor isoforms but at 2- to 3-fold slower rates. Thus, apoE isoforms differentially regulate Abeta clearance from the brain, and this might contribute to the effects of APOE genotype on the disease process in both individuals with AD and animal models of AD.

LRP/amyloid beta-peptide interaction mediates differential brain efflux of Abeta isoforms.

LRP (low-density lipoprotein receptor-related protein) is linked to Alzheimer's disease (AD). Here, we report amyloid beta-peptide Abeta40 binds to immobilized LRP clusters II and IV with high affinity (Kd = 0.6-1.2 nM) compared to Abeta42 and mutant Abeta, and LRP-mediated Abeta brain capillary binding, endocytosis, and transcytosis across the mouse blood-brain barrier are substantially reduced by the high beta sheet content in Abeta and deletion of the receptor-associated protein gene. Despite low Abeta production in the brain, transgenic mice expressing low LRP-clearance mutant Abeta develop robust Abeta cerebral accumulations much earlier than Tg-2576 Abeta-overproducing mice. While Abeta does not affect LRP internalization and synthesis, it promotes proteasome-dependent LRP degradation in endothelium at concentrations > 1 microM, consistent with reduced brain capillary LRP levels in Abeta-accumulating transgenic mice, AD, and patients with cerebrovascular beta-amyloidosis. Thus, low-affinity LRP/Abeta interaction and/or Abeta-induced LRP loss at the BBB mediate brain accumulation of neurotoxic Abeta.

IgG-assisted age-dependent clearance of Alzheimer's amyloid beta peptide by the blood-brain barrier neonatal Fc receptor.

The role of blood-brain barrier (BBB) transport in clearance of amyloid beta-peptide (Abeta) by Abeta immunotherapy is not fully understood. To address this issue, we studied the effects of peripherally and centrally administered Abeta-specific IgG on BBB influx of circulating Abeta and efflux of brain-derived Abeta in APPsw(+/-) mice, a model that develops Alzheimer's disease-like amyloid pathology, and wild-type mice. Our data show that anti-Abeta IgG blocks the BBB influx of circulating Abeta in APPsw(+/-) mice and penetrates into the brain to sequester brain Abeta. In young mice, Abeta-anti-Abeta complexes were cleared from brain to blood by transcytosis across the BBB via the neonatal Fc receptor (FcRn) and the low-density lipoprotein receptor-related protein (LRP), whereas in older mice, there was an age-dependent increase in FcRn-mediated IgG-assisted Abeta BBB efflux and a decrease in LRP-mediated clearance of Abeta-anti-Abeta complexes. Inhibition of the FcRn pathway in older APPsw(+/-) mice blocked clearance of endogenous Abeta40/42 by centrally administered Abeta immunotherapy. Moreover, deletion of the FcRn gene in wild-type mice inhibited clearance of endogenous mouse Abeta40/42 by systemically administered anti-Abeta. Our data suggest that the FcRn pathway at the BBB plays a crucial role in IgG-assisted Abeta removal from the aging brain.

Perivascular delivery of Notch 1 siRNA inhibits injury-induced arterial remodeling.

OBJECTIVES: To determine the efficacy of perivascular delivery of Notch 1 siRNA in preventing injury-induced arterial remodeling. METHODS AND RESULTS: Carotid artery ligation was performed to induce arterial remodeling. After 14 days, morphometric analysis confirmed increased vSMC growth and subsequent media thickening and neointimal formation. Laser capture microdissection, quantitative qRT-PCR and immunoblot analysis of medial tissue revealed a significant increase in Notch1 receptor and notch target gene, Hrt 1 and 2 expression in the injured vessels. Perivascular delivery of Notch 1 siRNA by pluronic gel inhibited the injury-induced increase in Notch 1 receptor and target gene expression when compared to scrambled siRNA controls while concomitantly reducing media thickening and neointimal formation to pre-injury, sham-operated levels. Selective Notch 1 knockdown also reversed the injury-induced inhibition of pro-apoptotic Bax expression while decreasing injury-induced anti-apoptotic Bcl-XL expression to sham-operated control levels. In parallel experiments, proliferative cyclin levels, as measured by PCNA expression, were reversed to sham-operated control levels following selective Notch 1 knockdown. CONCLUSION: These results suggest that injury-induced arterial remodeling can be successfully inhibited by localized perivascular delivery of Notch 1 siRNA.