Zhiming Suo

Characteristics of thein VitroVasoactivity of β-Amyloid Peptides

The β-amyloid (Aβ1–40) peptide has previously been shown to enhance phenylephrine contraction of aortic ringsin vitro.We have employed a novel observation, that Aβ peptides enhance endothelin-1 (ET-1) contraction, to examine the relationship between vasoactivity and potential amyloidogenicity of Aβ peptides, the role played by free radicals and calcium in the vasoactive mechanism, and the requirement of an intact endothelial layer for enhancement of vasoactivity. Rings of rat aortae were constricted with ET-1 before and after addition of amyloid peptide and/or other compounds, and a comparison was made between post- and pretreatment contractions. In this system, vessel constriction is consistently dramatically enhanced by Aβ1–40, is enhanced less so by Aβ1–42, and is not enhanced by Aβ25–35. The endothelium isnotrequired for Aβ vasoactivity, and calcium channel blockers have a greater effect than antioxidants in blocking enhancement of vasoconstriction by Aβ peptides. In contrast to Aβ-induced cytotoxicity, Aβ-induced vasoactivity is immediate, occurs in response to low doses of freshly solubilized peptide, and appears to be inversely related to the amyloidogenic potential of the Aβ peptides. We conclude that the mechanism of Aβ vasoactivity is distinct from that of Aβ cytotoxicity. Although free radicals appear to modulate the vasoactive effects, the lack of requirement for endothelium suggests that loss of the free radical balance (between NO and O−2) may be a secondary influence on Aβ enhancement of vasoconstriction. These effects of Aβ on isolated vessels, and reported effects of Aβ in cells of the vasculature, suggest that Aβ-induced disruption of vascular tone may be a factor in the pathogenesis of cerebral amyloid angiopathy and Alzheimers disease. Although the mechanism of enhanced vasoconstriction is unknown, it is reasonable to propose thatin vivocontact of Aβ peptides with small cerebral vessels may increase their tendency to constrict and oppose their tendency to relax. The subclinical ischemia resulting from this would be expected to up-regulate βAPP production in and around the vasculature with further increase in Aβ formation and deposition. The disruptive and degenerative effects of such a cycle would lead to the complete destruction of cerebral vessels and consequently neuronal degeneration in the affected areas.

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.

Alzheimer's β-amyloid peptides induce inflammatory cascade in human vascular cells: the roles of cytokines and CD40

Accumulating evidence suggests that beta-amyloid (Abeta)-induced inflammatory reactions may partially drive the pathogenesis of Alzheimers disease (AD). Recent data also implicate similar inflammatory processes in cerebral amyloid angiopathy (CAA). To evaluate the roles of Abeta in the inflammatory processes in vascular tissues, we have tested the ability of Abeta to trigger inflammatory responses in cultured human vascular cells. We found that stimulation with Abeta dose-dependently increased the expression of CD40, and secretion of interferon-gamma (IFN-gamma) and interleukin-1beta (IL-1beta) in endothelial cells. Abeta also induced expression of IFN-gamma receptor (IFN-gammaR) both in endothelial and smooth muscle cells. Characterization of the Abeta-induced inflammatory responses in the vascular cells showed that the ligation of CD40 further increased cytokine production and/or the expression of IFN-gammaR. Moreover, IL-1beta and IFN-gamma synergistically increased the Abeta-induced expression of CD40 and IFN-gammaR. We have recently found that Abeta induces expression of adhesion molecules, and that cytokine production and interaction of CD40-CD40 ligand (CD40L) further increase the Abeta-induced expression of adhesion molecules in these same cells. These results suggest that Abeta can function as an inflammatory stimulator to activate vascular cells and induces an auto-amplified inflammatory molecular cascade, through interactions among adhesion molecules, CD40-CD40L and cytokines. Additionally, Abeta1-42, the more pathologic form of Abeta, induces much stronger effects in endothelial cells than in smooth muscle cells, while the reverse is true for Abeta1-40. Collectively, these findings support the hypothesis that the Abeta-induced inflammatory responses in vascular cells may play a significant role in the pathogenesis of CAA and AD.

Superoxide free radical and intracellular calcium mediate Aβ1–42 induced endothelial toxicity

The 39-42 amino acid residue amyloid beta peptide (A beta), the major protein component in senile plaques and cerebrovascular amyloidosis in the brain in Alzheimers disease (AD), has been shown to be neurotoxic in vitro. Accumulating data from several areas suggest that cerebrovascular dysfunction and damage may also play a significant role in the AD process. For instance, we have recently demonstrated enhanced vasoconstriction and resistance to relaxation in intact rat aorta treated with A beta [Thomas et al., beta-Amyloid-mediated vasoactivity and vascular endothelial damage, Nature, 380 (1996) 168-171]. Significant vessel damage occurred after thirty minutes of exposure, but could be prevented with superoxide dismutase. To further investigate the role of A beta toxicity on endothelial cells, we have applied A beta peptides to cultures of human aortic endothelial cells (HAEC). Our results show that both A beta(1-42) and A beta(25-35) are toxic to HAEC in a time- and dose-dependent manner, and that this toxicity can be partially prevented by the calcium channel blocker, verapamil, and the antioxidant, superoxide dismutase. The common form of A beta, A beta(1-40), which has been shown to be neurotoxic, is much less toxic to HAEC. A beta toxicity to HAEC occurs within 30 min of treatment with relatively lower doses than those usually observed in primary cultured neurons and vascular smooth muscle cells. It was recently reported that a variety of mutations in the beta-amyloid protein precursor gene and the Presenilin-1 and -2 genes linked to early-onset familial AD cause an increase in the plasma concentration of A beta(1-42) in mutation carriers [Scheuner et al., Secreted amyloid beta-protein similar to that in the senile plaques of Alzheimers disease is increased in vitro by the presenilin 1 and 2 and APP mutations linked to familial Alzheimers disease, Nature Med., 2 (1996) 864-870]. Human aortic endothelial cells are more sensitive to A beta(1-42) than A beta(1-40), via a pathway involving an excess of superoxide free radicals and influx of extracellular calcium. Finally, we have evidence that both apoptotic and necrotic processes are activated by the A beta peptides in these endothelial cells.

Gender-specific association of the angiotensin converting enzyme gene with Alzheimer's disease

Epidemiological studies have demonstrated that risk factors for vascular disease are also risk factors for Alzheimers disease (AD). The gene for the angiotensin converting enzyme (ACE) has recently been reported to be associated with risk for AD. We have investigated the possibility of such an association in 98 clinic-based and 73 community-based AD cases versus 175 community-based controls and find a gender-specific association of ACE genotype with AD in the female clinic population. These data suggest that gender may interact with genetic factors to influence risk for AD. Gender-specific risk for AD has been previously reported, and a biological rationale for involvement of ACE in the AD process is supported by studies exploring the relationship between AD and vascular risk factors such as hypertension. However, the results may also be a consequence of the known anomalies that arise in genetic association studies as a consequence of sample selection.

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.

The butyrylcholinesterase gene is neither independently nor synergistically associated with late-onset AD in clinic-and community-based populations

The K variant of the butyrylcholinesterase gene (BChE) was recently found to occur at an increased frequency in a late onset Alzheimers disease (AD) population, specifically in individuals carrying the epsilon4 allele of the apolipoprotein E (APOE) gene. This suggested synergy between these two genes resulting in an increased risk of late-onset AD. We have genotyped 62 community-based and 329 clinic-based AD cases, and 201 community-based controls at BChE and APOE and find no independent association between BChE and AD nor interaction with APOE in risk for AD in either our clinic or community-based samples.

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.

Alzheimer's β‐amyloid vasoactivity: identification of a novel β‐amyloid conformational intermediate

The β‐amyloid (Aβ) peptide has previously been shown to enhance phenylephrine or endothelin‐1 induced constriction of aortic rings in vitro. The characteristics of Aβ vasoactivity (dose, fragment length, timing) suggest that the mechanism is distinct from Aβ cytotoxicity. To identify which properties of Aβ determine its biological activity on vessels, we investigated a number of Aβ analogues and fragments, individually and in combination, including those that are known to be associated with Alzheimers disease (Aβ1–42) and hereditary cerebral hemorrhage with amyloidosis – Dutch type (Aβ(22Q)1–40). The vasoactivity appears to be related to the conformation adopted by the peptide in solution. The β‐pleated sheet rich Aβ1–42 and Aβ(22Q)1–40 were each less vasoactive than the mainly random coil wild type Aβ1–40. However, the most vasoactive Aβ peptides were combinations which contain mixtures of random coil and β‐sheet structure. The finding that peptides containing low or high levels of β‐pleated conformation are less vasoactive than those containing intermediate amounts of this structural motif allows us to propose the existence of a transitional form between random coil and β‐pleated that is the vasoactive species of Aβ. This is the first time that Aβ conformational intermediates have been identified and a biological activity associated with them.

The Vasoactivity of Aβ Peptides

ABSTRACT: We have demonstrated that freshly solubilized Aβ peptides can enhance vasoconstriction by phenylephrine or endothelin of isolated rat aorta. Concentrations of peptide producing these effects (100 nM—1 μM) are much lower than those requiring toxicity to endothelial cells in culture, and effects are immediate, not requiring the prolonged time periods for aggregation necessary in Aβ cell culture toxicity experiments. Pre‐treatment with SOD diminishes the enhancement of vasoconstriction by Aβ peptides, suggesting that the effects are partly mediated via a decrease in the nitric oxide/superoxide ratio. Enhancement of endothelin vasoconstriction is observed with Aβ1‐40 and Aβ1‐42, but not with Aβ25‐35 even at 5 μM, again suggesting the mechanism of Aβ vasoactivity is distinct from that of Aβ cytotoxicity. These observations raise the possibility that Aβ peptides in contact with the cerebrovasculature could result in vasoconstriction, hypoperfusion and oxygen free radical imbalance contributing to the neurodegeneration of AD.