James Humphrey

Soluble Alzheimers β-amyloid constricts the cerebral vasculature in vivo

Abstract Bilateral temporoparietal hypoperfusion has been frequently observed early in the Alzheimers disease (AD) process. An increased β-amyloid (Aβ) peptide is believed to play a central role in the pathogenesis of AD. In vitro experiments have shown that freshly solubilized Aβ enhances constriction of cerebral and peripheral vessels. We propose that in vivo the Aβ vasoactive property may contribute to cerebral hypoperfusion of AD patients. To test this hypothesis, we intra-arterially infused freshly solubilized Aβ1–40 in rats and observed changes in cerebral blood flow and cerebrovascular resistance using fluorescent microspheres. We found that infusion of Aβ in vivo resulted in a decreased blood flow and increased vascular resistance specifically in cerebral cortex but not in heart or kidneys. These data suggest that Aβ has a direct and specific constrictive effect on cerebral vessels in vivo, which may contribute to the cerebral hypoperfusion observed early in the AD process.

Vasoactive effects of Aβ in isolated human cerebrovessels and in a transgenic mouse model of Alzheimer's disease: Role of inflammation

Abstract Aβ peptides are the major protein constituents of Alzheimers disease (AD) senile plaques and also form some deposits in the cerebrovasculature leading to cerebral amyloid angiopathy and hemorrhagic stroke. Functional vascular abnormalities are one of the earlier clinical manifestations in both sporadic and familial forms of AD. Most of the cardiovascular risk factors (for instance, diabetes, hypertension, high cholesterol levels, atherosclerosis and smoking) constitute risk factors for AD as well, suggesting that functional vascular abnormalities may contribute to AD pathology. We studied the effect of Aβ on endothelin-1 induced vasoconstriction in isolated human cerebral arteries collected following rapid autopsies. We report that freshly solubilized Aβ enhances endothelin-1 induced vasoconstriction in isolated human middle cerebral and basilar arteries. The vasoactive effect of Aβ in these large human cerebral arteries is inhibited by NS-398, a selective cyclooxygenase-2 inhibitor and by SB202190, a specific p38 Mitogen Activated Protein Kinase inhibitor suggesting the involvement of a pro-inflammatory pathway. Using a scanner laser Doppler imager, we observed that cerebral blood flow is decreased in the double transgenic APPsw Alzheimer mouse (PS1/APPsw) compared to PS1 littermates and can be improved by chronic treatment with either NS-398 or SB202190. Altogether, our data suggest a link between inflammation and the compromised cerebral hemodynamics in AD.

Activation of microglial cells by the CD40 pathway: relevance to multiple sclerosis.

It is well known that microglial cells perform a key role in mediating inflammatory processes, which are associated with neurodegenerative diseases such as multiple sclerosis (MS). In this study, we report that CD40 expression on microglia is greatly enhanced by a low dose (10 U/ml) of IFN-gamma. We also find that ligation of microglial CD40 by CD40L triggers a significant production of TNF-alpha. Activation of microglia by ligation of CD40 in the presence of IFN-gamma results in cultured cortical neuronal injury, which is markedly attenuated by blockade of the CD40 pathway or neutralization of TNF-alpha. Finally, we find significant levels of IFN-gamma and TNF-alpha in the medium of co-cultured activated CD4+ T cells and microglial cells, showing that microglia can supply the CD40 receptor to activated CD4+ T cells and suggesting that this cellular interaction is a key event in MS pathophysiology.

Inhibition of Alzheimer's β-Amyloid Induced Vasoactivity and Proinflammatory Response in Microglia by a cGMP-Dependent Mechanism

beta-amyloid (Abeta) peptides are the major protein components of senile plaques in Alzheimers disease (AD) brains. Vascular damage and reactive gliosis are found colocalized with amyloid deposits in AD brains, suggesting that the vasculature may be a clinically significant site of AD pathology. Our results show that freshly solubilized Abeta1-40 enhances the vasoconstriction induced by endothelin-1 (ET-1) and increases resistance to relaxation triggered by nitric oxide (NO), suggesting that Abeta may oppose the NO/cGMP pathway. Using specific inhibitors and activators of the NO/cGMP pathway, we show that Abeta vasoactivity is not due to a modulation of nitric oxide synthase (NOS) or soluble guanylyl cyclase (sGC). However, we find that a selective cGMP phosphodiesterase (cGMP-PDE) inhibitor (dipyridamole) is able to interactively block the enhanced vasoconstriction as well as the opposition to relaxation induced by Abeta, suggesting that Abeta could effect the activity of this enzyme. Cyclic GMP levels, but not cAMP concentrations, are reduced after Abeta treatment of rat aortic rings, further substantiating this hypothesis. Moreover, in examination of this pathway in another cell type pertinent to AD, we find that Abeta induces a proinflammatory response in microglia as evidenced by increased leukotriene B4 release. We show that both dipyridamole and compounds which increase cGMP levels prevent Abeta-induced microglial inflammation. Our results suggest that therapeutic intervention aimed at reduction of microglial-mediated inflammation via inhibition of cGMP-PDE or elevation of cGMP may be beneficial in the treatment of AD.

Soluble β-amyloid peptides mediate vasoactivity via activation of a pro-inflammatory pathway

Freshly solubilized β-amyloid (Aβ) peptides display vasoactive properties, increasing both the magnitude and the duration of endothelin-1-induced vasoconstriction. We show that Aβ vasoactivity is mediated by the stimulation of a pro-inflammatory pathway involving activation of secretory phospholipase A2 (PLA2), mitogen activated protein kinase (MAPK) kinase (MEK1/2), p38 MAPK, cytosolic PLA2, and the release of arachidonic acid. Ultimately, arachidonic acid is metabolized into proinflammatory eicosanoids via the 5-lipoxygenase and cyclooxygenase-2 (COX-2) enzymes, both of which we show to be required for Aβ vasoactivity. Accordingly, p38 MAPK activity is higher in the brains of transgenic mice that overproduce Aβ, and COX-2 immunoreactivity is increased in the cerebrovasculature of these transgenic animals. Taken together, our data show that freshly solubilized Aβ peptides can trigger a pro-inflammatory reaction in the vasculature that can be blocked by inhibiting specific target molecules, providing the basis for novel therapeutic intervention.

Induction of CD40 on human endothelial cells by Alzheimer’s β-amyloid peptides

Growing evidence suggests that β-amyloid (Aβ) peptides play a central role in mediating vascular endothelium dysfunction, but the extent to which immune mechanisms are involved in this process remains unclear. To explore such mechanisms, we incubated cultured human aortic endothelial cells (HAEC) with freshly solublized Aβ and examined expression of a central immunoregulatory molecule, CD40, in these cells using reverse transcriptase-polymerase chain reaction, Western immunoblotting, and Flow cytometry. Our results show that treatment of endothelial cells with Aβ1-40, Aβ1-42 or gamma interferon (IFN-γ) results in a dose-dependent induction of endothelial CD40 expression. Furthermore, ligation of endothelial CD40 and simultaneous treatment of human endothelial cells with IFN-γ or Aβ peptides leads to a significant release of interleukin-1β (IL-1β), a marker for endothelial cell activation. Since IL-1β is an important inflammatory response mediator, these findings suggest that the functional role of Aβ-induced endothelial CD40 may be promotion of the inflammatory cascade in vascular endothelial cells.

Aβ Vasoactivity: An Inflammatory Reaction

Abstract: Mounting evidence from in vitro and in vivo studies in transgenic mice overproducing β‐amyloid peptides (Aβ) suggests that Aβ can induce vasoconstriction and decrease cerebral blood flow. In this report, we describe the vasoactive properties of Aβ, in particular the enhancement of endothelin‐1‐induced vasoconstriction and Aβs induction of a long‐lasting vasoconstrictive event. Furthermore, we show that low doses (as low as 50 nM) of freshly solubilized Aβ similar to those observed in the plasma of patients suffering from Alzheimers disease are vasoactive. By using various inhibitors and activators of the phospholipase A2 (PLA2)/arachidonic acid (AA) cascade, we demonstrate that Aβ vasoactivity is dependent on activation of this intracellular signaling pathway, resulting in stimulation of downstream cyclooxygenase‐2 and 5‐lipoxygenase, which mediate production of proinflammatory eicosanoids. Taken together, our data show that Aβ directly activates an intracellular proinflammatory pathway, which is responsible for its vasoactive properties.

Isoform-specific vasoconstriction induced by Apolipoprotein E and modulation of this effect by Alzheimer's β-amyloid peptide

Abeta peptides are thought to be centrally involved in Alzheimers disease (AD) pathogenesis, although Abetas pathophysiological mechanisms remain to be elucidated. We previously showed that soluble beta-amyloid1-40 (Abeta) and Abeta1-42 exhibit vasoactive properties, and are able to promote vasoconstriction in rat aortae induced by an endogenous vasoconstrictor, endothelin-1. It is well established that the APOE epsilon4 allele confers risk for both familial and sporadic AD, as well as for hypertension. We now report that physiologic amounts (10 nM) of specific human recombinant apoE isoforms are vasoactive (E4 > E3, and not E2) in isolated rat aortae. In order to investigate if various apoE isoforms could modulate Abeta vasoactivity, we co-incubated Abeta1-40 with various isoforms of apoE in our tissue bath system. Our results show that, while none of the APOE isoforms are able to affect the maximum constriction induced by Abeta; the apoE E4 isoform synergistically enhances the rate of vasoconstriction induced by Abeta. Our data suggest that apoE may promote hypertension and contribute to AD pathogenesis via enhancement of vasoconstriction, and support a link between hypertension, cerebral amyloid angiopathy and AD.

Role of Peroxynitrite in the Vasoactive and Cytotoxic Effects of Alzheimer's β-Amyloid1–40Peptide

Increasing evidence implicates oxidative stress as partially responsible for the neurodegenerative process of Alzheimers disease (AD). Recent reports show an increased production of nitrotyrosine in AD brains, suggesting that peroxynitrite is produced in excess in this disease. Furthermore, incidence of cerebral amyloid angiopathy in AD cases is very frequent (83%), strongly suggesting a vascular component of AD pathogenesis. We have evaluated the hypothesis that peroxynitrite could be responsible for mediating the cytotoxicity and vasoactivity induced by the amyloid-beta1-40 (Abeta) peptide. Rat brain endothelial cells (RBE-4) appear to be sensitive to Abeta-induced toxicity but not to the cytotoxicity induced by peroxynitrite. Addition of Cu/Zn superoxide dismutase to cell culture media, which is only able to clear extracellular superoxide, was not effective in blocking Abeta-induced toxicity. However, we were able to partially block Abeta-induced cytotoxicity by using Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) which dismutes superoxide intracellularily. Yet, MnTBAP was not able to prevent the vasoactivity triggered by Abeta. Moreover, addition of peroxynitrite to rat aortae did not modulate the vasotension induced by Abeta. We conclude that intracellular superoxide radicals may contribute to Abeta-induced cytotoxicity. Our results also indicate that peroxynitrite does not significantly contribute to Abeta-induced cytotoxicity in rat brain endothelial cells (RBE-4) or vasoactivity in rat aortae. These results suggest that therapeutic efforts aimed at removal of reactive oxygen species with SOD is unlikely to be beneficial for treatment of Abeta-induced endothelial dysfunction. However, compounds that clear free radicals intracellularly may well be beneficial.

Pro-inflammatory effect of freshly solubilized β-amyloid peptides in the brain

Abstract It has recently been shown that the level of soluble β-amyloid (Aβ) peptides correlates well with the severity of synaptic loss and the density of neurofibrillary tangles observed in Alzheimer’s disease (AD) brain. However, the biological activity of soluble forms of Aβ peptides in the brain remains to be determined. We have investigated ex vivo the effect of freshly solubilized Aβ 1–40 peptides (fsAβ) on prostaglandin E 2 (PGE 2 ) production in rat brain slices. PGE 2 levels increased rapidly following treatment with fsAβ, an effect that was prevented by SB202190, a selective inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), and by NS-398, which preferentially inhibits cyclooxygenase-2 (COX-2) compared to COX-1. In an attempt to determine the cellular systems of the brain responsible for prostaglandin production in response to fsAβ, the effect of fsAβ was tested on isolated brain microvessels, primary cultures of brain smooth muscle cells/pericytes and endothelial cells, and a human neuron-like cell line (IMR32). Our data show that fsAβ ex vivo can stimulate prostaglandin accumulation in incubates of isolated rat brain microvessels. In addition, fsAβ appears to cause a concentration-dependent enhancement of prostaglandin accumulation in primary cultures of brain microvessel-derived smooth muscle cells/pericytes but not of brain endothelial cells. Finally, fsAβ also stimulated PGF 2α accumulation in cultures of differentiated IMR32 cells, but to a lesser extent than in brain smooth muscle cell/pericyte cultures. Deposition of aggregated forms of Aβ in the brain has been thought to trigger an inflammatory response which accompanies the neuropathologic events of AD. Our data provide evidence that fsAβ triggers a pro-inflammatory reaction in rat brain, and suggest that the cerebrovasculature may constitute an important source of pro-inflammatory eicosanoids.