Abeta42 generation is toxic to endothelial cells and inhibits eNOS function through an Akt/GSK-3beta signaling-dependent mechanism.

The application of beta-amyloid (Abeta) is cytotoxic to endothelial cells, promotes vasoconstriction and impairs nitric oxide (NO) generation or action. However, there is no information on the effect of intracellular Abeta on endothelial cell biology, although recent studies indicate that neuronal Abeta drives Alzheimer's disease pathogenesis. Since the serine-threonine kinase Akt is crucial to both neuronal and endothelial cell survival as well as eNOS activation, we investigated the effects of Abeta expression on Akt-signaling in cultured endothelial cells. Virally-encoded Abeta42 was proapoptotic and inhibitory to Akt phosphorylation in human umbilical vein endothelial cells (HUVECs). Toxicity was characterized by mitochondrial dysfunction, DNA condensation and activation of caspase-3. Substrates downstream of Akt action, GSK-3beta and eNOS, are underphosphorylated in the presence of Abeta. Constitutive activation of Akt reversed Abeta-induced toxicity and stimulated caspase-3 activity, suggesting that inhibition of Akt signaling is functionally significant. These Abeta effects were mediated, in part, through the derepression of GSK-3beta activation and correlated with reductions in NO production. We conclude that intracellular production of Abeta42 is cytotoxic to endothelial cells and that disruption of the Akt/GSK-3beta cell signaling pathway is involved.

Dissociation of ERK and Akt signaling in endothelial cell angiogenic responses to beta-amyloid.

Cerebrovascular deposits of beta-amyloid (Abeta) peptides are found in Alzheimer's disease and cerebral amyloid angiopathy with stroke or dementia. Dysregulations of angiogenesis, the blood-brain barrier and other critical endothelial cell (EC) functions have been implicated in aggravating chronic hypoperfusion in AD brain. We have used cultured ECs to model the effects of beta-amyloid on the activated phosphorylation states of multifunctional serine/threonine kinases since these are differentially involved in the survival, proliferation and migration aspects of angiogenesis. Serum-starved EC cultures containing amyloid-beta peptides underwent a 2- to 3-fold increase in nuclear pyknosis. Under growth conditions with sublethal doses of beta-amyloid, loss of cell membrane integrity and inhibition of cell proliferation were observed. By contrast, cell migration was the most sensitive to Abeta since inhibition was significant already at 1 muM (P = 0.01, migration vs. proliferation). In previous work, intracellular Abeta accumulation was shown toxic to ECs and Akt function. Here, extracellular Abeta peptides do not alter Akt activation, resulting instead in proportionate decreases in the phosphorylations of the MAPKs: ERK1/2 and p38 (starting at 1 microM). This inhibitory action occurs proximal to MEK1/2 activation, possibly through interference with growth factor receptor coupling. Levels of phospho-JNK remained unchanged. Addition of PD98059, but not LY294002, resulted in a similar decrease in activated ERK1/2 levels and inhibition of EC migration. Transfection of ERK1/2 into Abeta-poisoned ECs functionally rescued migration. The marked effect of extracellular Abeta on the migration component of angiogenesis is associated with inhibition of MAPK signaling, while Akt-dependent cell survival appears more affected by cellular Abeta.

NERF2, a member of the Ets family of transcription factors, is increased in response to hypoxia and angiopoietin-1: a potential mechanism for Tie2 regulation during hypoxia.

Vascular endothelial growth factor (VEGF) and angiopoietins regulate endothelial cell survival and migration and are essential for angiogenesis. Considerable progress has been made towards understanding hypoxia-mediated regulation of VEGF and its receptors. In contrast, little is known about the regulation of angiopoietins and their receptors in hypoxic cells. Using RT-PCR, RNAase protection assay, and Western blotting, we found that Tie1 and Tie2 mRNA and protein levels increased in response to hypoxia in human umbilical vein endothelial cells. Previously, we have shown that NERF2, a member of Ets family of transcription factors that is specifically expressed in endothelial cells, binds to the promoter region of Tie2 and transactivates Tie2 expression. In this study, we show that expression of NERF2 was increased under hypoxia and that this increase temporally correlated with the increase in Tie2 expression. Hypoxia-induced expression of NERF2 and Tie2 was blocked by angiopoietin-2, a competitive inhibitor of angiopoietin-1, and by recombinant soluble extracellular domain of Tie2 but not by VEGF-neutralizing antibodies. In addition, angiopoietin-1 directly induced expression of NERF2 in quiescent cells. These novel findings suggest that angiopoietin-1 regulates expression of NERF2 and its own receptor in hypoxic cells.

Calcium dyshomeostasis in beta-amyloid and tau-bearing skeletal myotubes.

The relative scarcity of inclusion-affected muscle cells or markers of cell death in inclusion body myositis (IBM) is in distinction to the specific and early intracellular deposition of several Alzheimer's Disease (AD)-related proteins. The current study examined the possible correlation between myotube beta-amyloid and/or Tau accumulations and a widespread mishandling of intracellular muscle calcium concentration that could potentially account for the unrelenting weakness in affected patients. Cultured myogenic cells (C(2)C(12)) expressed beta-amyloid-42 (Abeta(42)) and fetal Tau peptides, as human transgenes encoded by herpes simplex virus, either individually or concurrently. Co-expression of Abeta(42) in C(2)C(12) myotubes resulted in hyperphosphorylation of Tau protein that was not observed when Tau was expressed alone. Resting calcium concentration and agonist-induced RyR-mediated Ca(2+) release were examined using calcium-specific microelectrodes and Fluo-4 epifluorescence, respectively. Co-expression of Abeta(42) and Tau cooperatively elevated basal levels of myoplasmic-free calcium, an effect that was accompanied by depolarization of the plasma membrane. Sarcoplasmic reticulum (SR) calcium release, induced by KCl depolarization, was not affected by Abeta(42) or Tau. In contrast, expression of Abeta(42), Tau, or Abeta(42) together with Tau resulted in enhanced sensitivity of ryanodine receptors to activation by caffeine. Notably, expression of beta-amyloid, alone, was sufficient to result in an increased sensitivity to direct activation by caffeine. Current results indicate that amyloid proteins cooperate to raise resting calcium levels and that these effects are associated with a passive SR Ca(2+) leak and Tau hyperphosphorylation in skeletal muscle.