First effects of rising amyloid-ß in transgenic mouse brain: synaptic transmission and gene expression.

Detecting and treating Alzheimer's disease, before cognitive deficits occur, has become the health challenge of our time. The earliest known event in Alzheimer's disease is rising amyloid-ß. Previous studies have suggested that effects on synaptic transmission may precede plaque deposition. Here we report how relative levels of different soluble amyloid-ß peptides in hippocampus, preceding plaque deposition, relate to synaptic and genomic changes. Immunoprecipitation-mass spectrometry was used to measure the early rise of different amyloid-ß peptides in a mouse model of increasing amyloid-ß ('TASTPM', transgenic for familial Alzheimer's disease genes APP/PSEN1). In the third postnatal week, several amyloid-ß peptides were above the limit of detection, including amyloid-ß40, amyloid-ß38 and amyloid-ß42 with an intensity ratio of 6:3:2, respectively. By 2 months amyloid-ß levels had only increased by 50% and although the ratio of the different peptides remained constant, the first changes in synaptic currents, compared to wild-type mice could be detected with patch-clamp recordings. Between 2 and 4 months old, levels of amyloid-ß40 rose by ∼7-fold, but amyloid-ß42 rose by 25-fold, increasing the amyloid-ß42:amyloid-ß40 ratio to 1:1. Only at 4 months did plaque deposition become detectable and only in some mice; however, synaptic changes were evident in all hippocampal fields. These changes included increased glutamate release probability (P < 0.001, n = 7-9; consistent with the proposed physiological effect of amyloid-ß) and loss of spontaneous action potential-mediated activity in the cornu ammonis 1 (CA1) and dentate gyrus regions of the hippocampus (P < 0.001, n = 7). Hence synaptic changes occur when the amyloid-ß levels and amyloid-ß42:amyloid-ß40 ratio are still low compared to those necessary for plaque deposition. Genome-wide microarray analysis revealed changes in gene expression at 2-4 months including synaptic genes being strongly affected but often showing significant changes only by 4 months. We thus demonstrate that, in a mouse model of rising amyloid-ß, the initial deposition of plaques does not occur until several months after the first amyloid-ß becomes detectable but coincides with a rapid acceleration in the rise of amyloid-ß levels and the amyloid-ß42:amyloid-ß40 ratio. Prior to acceleration, however, there is already a pronounced synaptic dysfunction, reflected as changes in synaptic transmission and altered gene expression, indicating that restoring synaptic function early in the disease progression may represent the earliest possible target for intervention in the onset of Alzheimer's disease.

Heme oxygenase-1 expression enhances vascular endothelial resistance to complement-mediated injury through induction of decay-accelerating factor: a role for increased bilirubin and ferritin.

Catabolism of free heme by heme oxygenase-1 (HO-1) generates carbon monoxide, biliverdin, and free iron (Fe). These end-products are responsible for much of the biologic activity of HO-1, including anti-inflammatory, antiapo-ptotic, antiproliferative, and antioxidant effects. We have identified an additional cytoprotective action, the regulation of complement activation, mediated via induction of decay-accelerating factor (DAF). Pharmacologic inhibition or short-interfering RNA (siRNA) depletion of HO-1 prevented induction of DAF expression in human endothelial cells. In contrast, HO-1 agonists hemin and cobalt protoporphyrin IX significantly increased DAF protein expression, reflecting an increase in transcription and steady-state mRNA. Adenoviral-mediated overexpression of HO-1 increased DAF expression, enhancing protection against C3 deposition and complement-mediated lysis, and this was reversed by DAF inhibitory monoclonal antibody (mAb) 1H4. Likewise, bilirubin, Fe chelation, and overexpression of heavy-chain ferritin all induced DAF expression in endothelial cells (EC). Analysis of cardiac endothelial cells isolated from Hmox1(-/-) mice revealed a 60% reduction in DAF expression compared with Hmox1(+/+) EC, and Hmox1(-/-) cells showed enhanced sensitivity to complement. We propose that modulation of complement activation through induction of DAF represents an important component of the cytoprotective effects of HO-1 against vascular injury, such as that associated with posttransplant vasculopathy, allograft rejection, and ischemia reperfusion.

A protein kinase Cepsilon-anti-apoptotic kinase signaling complex protects human vascular endothelial cells against apoptosis through induction of Bcl-2.

Endothelial cell apoptosis is associated with vascular injury and predisposes to atherogenesis. Endothelial cells express anti-apoptotic genes including Bcl-2, Bcl-XL and survivin, which also contribute to angiogenesis and vascular remodeling. We report a central role for protein kinase Cepsilon (PKCepsilon) in the regulation of Bcl-2 expression and cytoprotection of human vascular endothelium against apoptosis. Using myristoylated inhibitory peptides, a predominant role for PKCepsilon in vascular endothelial growth factor-mediated endothelial resistance to apoptosis was revealed. Immunoblotting of endothelial cells infected with an adenovirus expressing a constitutively active form of PKCepsilon (Adv-PKCepsilon-CA) or control Adv-beta-galactosidase demonstrated a 3-fold, PKCepsilon-dependent increase in Bcl-2 expression, with no significant change in Bcl-XL, Bad, Bak, or Bax. The induction of Bcl-2 inhibited apoptosis induced by serum starvation or etoposide, and PKCepsilon activation attenuated etoposide-induced caspase-3 cleavage. The functional role of Bcl-2 was confirmed with Bcl-2 antagonist HA-14-1. Inhibition of phosphoinositide 3-kinase attenuated vascular endothelial growth factor-induced protection against apoptosis, and this was rescued by overexpression of constitutively active PKCepsilon, suggesting PKCepsilon acts downstream of phosphoinositide 3-kinase. Co-immunoprecipitation studies demonstrated a physical interaction between PKCepsilon and Akt, which resulted in formation of a signaling complex, leading to optimal induction of Bcl-2. This study reveals a pivotal role for PKCepsilon in endothelial cell cytoprotection against apoptosis. We demonstrate that PKCepsilon forms a signaling complex and acts co-operatively with Akt to protect human vascular endothelial cells against apoptosis through induction of the anti-apoptotic protein Bcl-2 and inhibition of caspase-3 cleavage.

Statin-induced expression of CD59 on vascular endothelium in hypoxia: a potential mechanism for the anti-inflammatory actions of statins in rheumatoid arthritis.

Hypoxia, which leads to dysfunctional cell metabolism, and complement activation both play central roles in the pathogenesis of rheumatoid arthritis (RA). Recent studies have reported that mice deficient for the complement-inhibitory protein CD59 show enhanced susceptibility to antigen-induced arthritis and reported that statins have anti-inflammatory effects in RA. We hypothesized that the anti-inflammatory effect of statins in RA relates in part to their ability to increase CD59 expression in hypoxic conditions and therefore to reduce complement activation. Flow-cytometric analysis showed that CD59 expression on endothelial cells (EC) was unaffected by atorvastatin in normoxia (21% O2), whereas in hypoxic conditions (1% O2) an up to threefold dose-dependent increase in CD59 expression was seen. This effect of hypoxia was confirmed by treatment of EC with chemical mimetics of hypoxia. The upregulation of CD59 protein expression in hypoxia was associated with an increase in steady-state mRNA. L-Mevalonate and geranylgeraniol reversed the response, confirming a role for inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase and geranylgeranylation. Likewise, inhibition by NG-monomethyl-L-arginine and NG-nitro-L-arginine methyl ester confirmed that CD59 upregulation in hypoxia was nitric oxide dependent. The expression of another complement-inhibitory protein, decay-accelerating factor (DAF), is known to be increased by atorvastatin in normoxia; this response was also significantly enhanced under hypoxic conditions. The upregulation of CD59 and DAF by atorvastatin in hypoxia prevented the deposition of C3, C9 and cell lysis that follows exposure of reoxygenated EC to serum. This cytoprotective effect was abrogated by inhibitory anti-CD59 and anti-DAF mAbs. The modulation of EC CD59 and DAF by statins under hypoxic conditions therefore inhibits both early and late complement activation and may contribute to the anti-inflammatory effects of statins in RA.

A role for proteinase-activated receptor 2 and PKC-epsilon in thrombin-mediated induction of decay-accelerating factor on human endothelial cells.

Thrombin, an important mediator of thrombosis and inflammation, may also enhance vascular cytoprotection. Thus thrombin induces expression of the complement-inhibitory protein decay-accelerating factor (DAF) in human umbilical vein endothelial cells (HUVECs), thus increasing protection against complement-mediated injury. Using PKC isozyme-specific peptide antagonists and adenoviral constructs, we have shown in the present study that PKC-epsilon is the primary isozyme involved in DAF induction by thrombin. Experiments with proteinase-activated receptor-1 (PAR1) and PAR2 activating peptides (APs) showed that DAF expression induced by PAR1-AP was PKC-alpha-dependent; in contrast, PAR2-AP induction of DAF required activation of PKC-epsilon. PAR1-AP and PAR2-AP in combination exerted an additive effect on DAF protein expression, which was equivalent to that observed with thrombin alone. These data implied a specific role for PAR2 in DAF induction, which was supported by the observation that upregulation of endothelial cell (EC) PAR2-enhanced DAF induction by thrombin. ERK1/2, p38, and JNK MAPK were also involved in thrombin-induced DAF upregulation, with evidence of interdependence between ERK1/2 and JNK. A role for transactivation of PAR2 by PAR1 was suggested by partial inhibition of thrombin-induced DAF expression by the PAR1 signaling antagonists BMS-200261 and SCH79797, whereas inhibition of thrombin-induced cleavage of PAR1 by specific MAbs or hirudin completely abrogated the response. Together, these data imply that the predominant pathway for thrombin-induced DAF expression involves transactivation of PAR2 by PAR1 and signaling via PKC-epsilon/MAPK. This may represent an important, novel pathway for endothelial cytoprotection during inflammation and angiogenesis and suggests that PAR2 may play a central role in some thrombin-induced responses.

Decay-accelerating factor induction on vascular endothelium by vascular endothelial growth factor (VEGF) is mediated via a VEGF receptor-2 (VEGF-R2)- and protein kinase C-alpha/epsilon (PKCalpha/epsilon)-dependent cytoprotective signaling pathway and is inhibited by cyclosporin A.

Decay-accelerating factor (DAF), a membrane-bound complement regulatory protein, is up-regulated on endothelial cells (ECs) following treatment with vascular endothelial growth factor (VEGF), providing enhanced protection from complement-mediated injury. We explored the signaling pathways involved in this response. Incubation of human umbilical vein ECs with VEGF induced a 3-fold increase in DAF expression. Inhibition by flk-1 kinase inhibitor SU1498 and failure of placental growth factor (PlGF) to up-regulate DAF confirmed the role of VEGF-R2. The response was also blocked by pretreatment with phospholipase C-gamma (PLCgamma) inhibitor U71322 and protein kinase C (PKC) antagonist GF109203X. In contrast, no effect was seen with nitric oxide synthase inhibitor N(G)-monomethyl-l-arginine (l-NMMA). Use of PKC agonists and isozyme-specific pseudosubstrate peptide antagonists suggested a role for PKCalpha and -epsilon in VEGF-mediated DAF up-regulation. This was confirmed by transfection of ECs with PKCalpha and -epsilon dominant-negative constructs, which in combination completely abrogated induction of DAF by VEGF. In contrast, LY290042, a phosphoinositide 3-kinase (PI3K) inhibitor, significantly augmented DAF expression, suggesting a negative regulatory role for phosphoinositide 3-kinase. The widely used immunosuppressive drug cyclosporin A (CsA) inhibited DAF induction by VEGF in a dose-dependent manner. The VEGF-induced DAF expression was functionally effective, significantly reducing complement-mediated EC lysis, and this cytoprotective effect was reversed by CsA. These data provide evidence for a VEGF-R2-, phospholipase C-gamma-, and PKCalpha/epsilon-mediated cytoprotective pathway in ECs. This may represent an important mechanism for the maintenance of vascular integrity during chronic inflammation involving complement activation. Moreover, inhibition of this pathway by CsA may play a role in CsA-mediated vascular injury.