aβ vasoactivity: an inflammatory reaction

97

A� Vasoactivity: An Inflammatory Reaction

DANIEL PARIS, TERRENCE TOWN, TIMOTHY PARKER, JAMES HUMPHREY, AND MICHAEL MULLANa

The Roskamp Institute, University of South Florida, Tampa, Florida 33613, USA

ABSTRACT: Mounting evidence from in vitro and in vivo studies in transgenicmice overproducing �-amyloid peptides (A�) suggests that A� can induce vas-oconstriction and decrease cerebral blood flow. In this report, we describe thevasoactive properties of A�, in particular the enhancement of endothelin-1-induced vasoconstriction and A�’s induction of a long-lasting vasoconstrictiveevent. Furthermore, we show that low doses (as low as 50 nM) of freshly solu-bilized A� similar to those observed in the plasma of patients suffering fromAlzheimer’s disease are vasoactive. By using various inhibitors and activatorsof the phospholipase A2 (PLA2)/arachidonic acid (AA) cascade, we demon-strate that A� vasoactivity is dependent on activation of this intracellular sig-naling pathway, resulting in stimulation of downstream cyclooxygenase-2 and5-lipoxygenase, which mediate production of proinflammatory eicosanoids.Taken together, our data show that A� directly activates an intracellular proin-flammatory pathway, which is responsible for its vasoactive properties.

BACKGROUND

Vascular pathology frequently co-occurs with Alzheimer’s disease (AD), and, inparticular, cerebral amyloid angiopathy (CAA) is one of the commonest abnormali-ties detected at autopsy in carefully standardized examination (83% of AD cases asassessed by the Consortium to Establish a Registry for Alzheimer’s Disease(CERAD)1). It is becoming increasingly accepted that such vascular pathology maymodify the risk of developing AD, may alter clinical presentation, and may modifythe progression of the disease. However, we are concerned here not with the effectsof gross anatomic pathological changes associated with AD, but, rather, with the hy-pothesis that β-amyloid peptides (Aβ) may induce functional abnormalities in themicrovasculature of AD patients, and that these functional changes may contributeto the clinical picture, pathology, and progression of the disease. This hypothesisarises from mounting evidence that soluble forms of Aβ peptides have vasoactive ef-fects in isolated mammalian vessels and in transgenic animal models where Aβ pep-tides are overexpressed.2–4 At the clinical level, it is possible that these“physiologic” effects of soluble Αβ mediate hypoperfusion and perhaps ischemia inAD brains, thereby amplifying the AD pathological process. This hypothesis in-cludes the tenet that Aβ peptides exert these effects in their soluble form, and thatdeposition of insoluble amyloid is not required to produce these effects. With regard

aAddress for correspondence: Dr. Michael Mullan, The Roskamp Institute, University ofSouth Florida, 3515 E. Fletcher Ave., Tampa, FL 33613. Tel.: (813) 974-3722; fax: (813) 974-3915.

e-mail: [email protected]

98 ANNALS NEW YORK ACADEMY OF SCIENCES

to hypoperfusion, both single photon emission computed tomography (SPECT) andpositron emission tomography (PET) studies confirm reduction in cerebral bloodflow in AD,5–6 but the concomitant decrease in glucose metabolism has promptedthe interpretation that the former observation is a consequence of reduced neuronalmetabolic demand.7

Although the potential vasoactive effects of Aβ have not been demonstrated inhumans, data from transgenic animal models of AD and CAA are highly supportiveof the hypothesis that soluble Aβ peptides can induce vasoconstriction and opposenormal vasorelaxation. For instance, transgenic mice that overexpress Aβ peptidesexhibit decreased cerebral blood flow in response to vasodilators4 prior to the for-mation of amyloid plaques, suggesting that the isolated vessel bath system may bean accurate model of the preclinical effects of soluble Aβ levels on the microcere-brovasculature in AD. Other transgenic models of AD also overexpressing Aβ do notrespond well after vascular insult compared to their nontransgenic littermates. Inparticular, after middle cerebral artery occlusion there is enlarged infarct size, re-duced blood flow in the penumbra of the infarct, and reduced response to vasodila-tors in the same area.8 Again, this is consistent with a vasoactive role for soluble Aβpeptides, as their levels reach supraphysiologic amounts around the microcere-brovasculature in the brain parenchyma.

However, in persons affected with AD, there has been no direct testing of the ex-istence or clinical importance of any Aβ-induced vasoactive effects similar to thoseobserved in vitro or in transgenic animals. Indeed, a primary role of hypoperfusionis not established and, as mentioned, thought to be largely consequent upon reducedmetabolic demand of dysfunctional neurons. For instance, in a small study conduct-ed by Jagust and colleagues,9 it was suggested that there was no evidence to proposethat the prominent perfusion changes seen in AD were primary and that AD caseshad similar increases in perfusion compared to normals when challenged by hypo-capnia. By contrast, Nagata and colleagues10 observed increased oxygen extractionin AD cases compared to controls, suggesting a primary role for the vasculature inlimiting perfusion to the brain. Interestingly, the same study suggested no change invascular reactivity, at least in relation to changes in PaCO2. Already, then, there areimportant differences in studies of blood flow in AD cases and transgenic models ofAD. Some of these differences may be due to the different experimental paradigmsbetween human and animal studies and, in particular, the restrictions inherent in hu-man studies that preclude challenge of the appropriate vasoactive mechanisms trig-gered by Aβ. For instance, although vascular reactivity is maintained in AD inrelation to hypercapnia, it may not be maintained in relation to compounds that di-rectly relate to the proposed mechanism of Aβ vasoactivity. The experimental de-sign of studies that would contribute such information is not obvious, but muchneeded. As a first step to elucidating the contribution of Aβ’s vasoactive effects intransgenic models of AD and in AD itself, we must be clear on exactly how Aβ me-diates its vasoactive effects, and this is the subject of this report.

MATERIALS AND METHODS

Freshly dissected aortae were prepared from normal male Sprague-Dawley rats(7–8 months old, purchased from Zivic Miller, Zelienople, PA) as previously de-

99PARIS et al.: A� VASOACTIVITY

scribed.3 Rat aortae were segmented into rings and suspended in Kreb’s buffer onhooks connected to a tensiometer linked to a MacLab system. Aortic rings wereequilibrated for 2 h, in 7 ml tissue baths thermoregulated to 37°C containing Kreb’sbuffer oxygenated with 95% O2 : 5% CO2. A baseline tension of 2 g was applied toeach ring, and the first set of aortic rings was pretreated with various inhibitors oractivators of the phopholipase A2 (PLA2)/arachidonic acid (AA) cascade, eitheralone or in combination with Aβ peptides. After 5 min of incubation in the presenceor absence of Aβ, vessels were subjected to a dose range of endothelin-1 (ET-1, from1 nM to 5 nM). The second set of vessels was treated with 1 µM of Aβ peptides priorto the addition of ET-1. A third set received only ET-1 treatment (control). Each ET-1dose was added only after the constriction response to the previous dose had reacheda plateau. In all cases the means ± 1 standard error (SE) of the percentage vasocon-striction increase over baseline were determined for each dose of ET-1 used.

FIGURE 1. Freshly solubilized Aβ1–40 or Aβ1–42 enhance ET-1-induced vasoconstric-tion. Certain aortic rings were treated with Aβ1–40 or Aβ1–42 5 min prior to the addition ofa dose range of ET-1. Analysis of variance (ANOVA) showed significant main effects of ET-1dose (p <0.001), Aβ1–40 ( p <0.001), and Aβ1-42 ( p <0.001). ANOVA also revealed signifi-cant interactive terms between ET-1 dose and either Aβ1–40 ( p <0.001) or Aβ1–42 ( p = 0.001),indicating ET-1 dose-dependent enhancement of vasoconstriction by Aβ.


RESULTS AND DISCUSSION

Aβ1–40 has previously been shown to exhibit vasoactive properties, and it hasbeen suggested that this effect is mediated by free radical production.2 However, wehave excluded the involvement of free radical and reactive oxygen species as possi-ble contributors to the vasoactive properties of Aβ peptides, since Mn(111)tet-rakis(4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide mimetic andperoxynitrite scavenger, catalase, and Trolox were unable to reduce or oppose the ef-fect of Aβ in rat aortae.11 Interestingly, Aβ vasoactivity appears to be related to theconformation adopted by the peptide in solution, and is observable with soluble, butnot with aggregated forms of Aβ.12 To determine the impact of Aβ on endogenousvasoconstrictors, we examined the effect of ET-1 on vessels pretreated with Aβ. ET-1

FIGURE 2. Dose-response curve showing Aβ1–40 vasoactivity. Certain aortic ringswere treated with a dose range of Aβ1–40 5 min prior to the addition of a dose range of ET-1.ANOVA revealed significant main effects of ET-1 dose (p <0.001), Aβ dose ( p <0.001), andan interactive term between them ( p <0.001). One-way ANOVA across ET-1 doses revealedsignificant between-groups differences ( p <0.001), and Bonferonni’s post-hoc comparisonacross the 2.5 nM and 5 nM doses of ET-1 showed a significant difference between the 50 nMdose of Aβ and the Aβ-free condition ( p = 0.001).


is one of the most potent cerebral vasoconstrictors known in the brain, and, togetherwith NO, controls cerebral vasoregulation.13 Our data show that Aβ peptides (1 µM,1–40 or 1–42) synergistically enhance ET-1-induced vasoconstriction to a similarextent (FIG. 1). Furthermore, Aβ’s response to ET-1 is much more potent than withphenylephrine (PE2), and is observable within minutes. This observation led us tofocus on the ET-1 system as an assay for Aβ vasoactivity.

The initial experiments showing Aβ’s vasoactive response employed relativelyhigh doses of Aβ. However, it is entirely possible that such elevated doses of Aβ arenot physiologically relevant. In order to approach physiologic levels of Aβ, we as-sessed the vasoconstrictive effect of low doses of Aβ. Furthermore, we employedfreshly solubilized Aβ, as this form of the peptide may exert its bioactivity prior toclinical presentation of the disease. Data show that Aβ’s vasoactivity is observablewithin minutes with nanomolar doses of freshly solubilized Aβ (as low as 50 nM,plateauing at 250 nM), which are physiologic (FIG. 2). Specifically, it has beenshown that circulating levels of Aβ range between 30 and 150 nM in AD blood.14

This dose-response curve shows that the vasculature is extremely sensitive to the ef-fects of soluble Aβ, suggesting that, in AD and CAA where perivascular Aβ levels

FIGURE 3. Aβ induces a long-lasting vasoconstriction. Certain aortic rings were treat-ed with 1 µM Aβ1–40 5 min prior to the addition of a dose range of ET-1 (1, 2.5, and 5 nM).Following the 5 nM dose of ET-1, maximum tension was taken as the t = 0 time point (andstandardized to 100% both in control and Aβ-treated vessels), with vasotension assessed foreach following minute until t = 10 min. n = 8 for control and Aβ-treated vessels. ANOVArevealed significant main effects of Aβ ( p <0.001), time (p <0.001), and an interaction be-tween them ( p <0.001). Post-hoc t test for independent samples across time points revealeda significant difference ( p <0.001) between control and Aβ-treated rat aortae.


are substantially increased, the cerebrovasculature may play an important role in thepathogenesis of the disease prior to the formation of Aβ aggregates.

Interestingly, Aβ not only increases the magnitude of contraction induced by ET-1,but also enhances the sustained phase of ET-1-induced vasoconstriction at doses aslow as 20 nM of freshly solubilized Aβ (FIG. 3). Long-lasting vasoconstriction in-duced by Aβ is evident with other vasoconstrictors, such as phenylepherine or athromboxane A2 analogue (U-46619), indicating that long-lasting vasoconstrictionis not specific to the vasoconstrictor used. However, Aβ’s enhancement of maximumvasoconstriction was observable only with ET-1, and not with these other vasocon-strictors (named above).

Although considerable information is available regarding the mechanism bywhich vasoconstrictors mediate their effects, less is known about the intracellularevents that lead to a sustained contraction. It has been suggested that activation ofmitogen-activated protein kinase (MAPK) may be associated with sustained smoothmuscle contraction.15 Interestingly, activation of the MAPK module has been shownto be a critical player in mediating inflammatory reactions in various cell types, in-cluding smooth muscle cells.16–18 Inflammation is becoming increasingly substan-tiated as a contributor to AD pathogenesis, and the possible involvement of Aβ in theinduction of this inflammatory process has been suggested. For example, the oc-

FIGURE 4. Effect of cPLA2 inhibition on Aβ-induced vasoactivity. Certain aorticrings were treated with 1 µM freshly solubilized Aβ1–40, 1 µM MAFP, or MAFP + Aβ 5 minprior to the addition of a dose range of ET-1. There were significant main effects by ANOVAof ET-1 dose (p <0.001), Aβ ( p <0.001), but not MAFP ( p <0.001). There were also signif-icant interactive terms between ET-1 dose and Aβ ( p <0.01), and among ET-1, Aβ, andMAFP (p <0.01). One-way ANOVA across ET-1 doses revealed significant between-groupsdifferences (p <0.001), and post-hoc testing showed significant differences between controland Aβ ( p <0.001), and Aβ and Aβ + MAFP ( p = 0.001).


currence of immune system proteins, activated microglia, and astrocytes inperivascular senile plaques suggests that Aβ may play a substantial role inneuroinflammation.19–22 Based on such circumstantial evidence for a proinflamma-tory role of Aβ peptides in AD brains, we asked whether Aβ might mediate vasoac-tivity through activation of an inflammatory response involving stimulation of theMAPK module. AA release and production of eicosanoids are prerequisites for in-flammation, and PLA2s are key enzymes that initiate the AA cascade, which leadsto the generation of multiple eicosanoid products during both acute and chronicinflammation. Thus, we first investigated the effect of blocking PLA2 on Aβvasoactivity.

Data show that inhibition of either cytosolic PLA2 (cPLA2) by MAFP (a specificcPLA2 inhibitor, FIG. 4) or secretory PLA2 (sPLA2) by oleyloxyphosphorylcholine(type I sPLA2 inhibitor, FIG. 5) is sufficient to completely abolish Aβ vasoactivity,suggesting that Aβ mediates its effects via an activation of sPLA2 and cPLA2.Melittin and mastoparan, two small peptides known to stimulate cPLA2, are able tomimic the effects of Aβ by enhancing ET-1-induced vasoconstriction to a similar ex-tent as Aβ. Interestingly, when melittin or mastoparan are used in combination withAβ, a statistical interaction is observed among ET-1, Aβ, and either peptide, further

FIGURE 5. Interaction among oleyloxyethylphosphocholine (oleylox.), Aβ, and ET-1on vasoconstriction. Certain aortic rings were treated with 1 µM freshly solubilized Aβ1–40,1 µM oleylox., or oleylox. + Aβ 5 min prior to the addition of a dose range of ET-1. Therewere significant main effects by ANOVA of ET-1 dose ( p <0.001), Aβ (p <0.001), and ole-ylox. (p <0.001). There were also significant interactive terms between ET-1 dose and eitherAβ ( p <0.001) or oleylox. (p <0.001), and among ET-1, Aβ and oleylox. ( p <0.001). One-way ANOVA across ET-1 doses revealed significant between-groups differences (p <0.001),and post-hoc testing showed significant differences between control and Aβ ( p <0.001), andAβ and Aβ + oleylox. ( p = 0.001).


confirming that Aβ vasoactivity is mediated via activation of cPLA2 (data notshown). Low doses of purified type I sPLA2 from porcine pancreas also mimic Aβ’svasoactive effect by enhancing ET-1-induced vasoconstriction to a similar extent asAβ. Furthermore, as with Aβ, statistical interaction is observed among sPLA2, Aβ,and ET-1 (data not shown). Interestingly, not all of the PLA2 isoforms are involvedin this process. In particular, inactivation of type II sPLA2 with quercetin or inhibi-tion of calcium-independent PLA2 (type VI) with haloenol lactone suicide substrate(HELSS) does not inhibit Aβ vasoactivity. Moreover, we have confirmed that Aβvasoactivity is selectively mediated by an activation of the PLA2 cascade, but not byan activation of phospholipase C, phospholipase D, or protein kinase C. Thus, ourdata show that the vasoactive effects exerted by Aβ are specifically mediated by anactivation of the PLA2 pathway.

Taken together, our data show that both sPLA2 and cPLA2 are necessary to me-diate Aβ vasoactivity, leaving the possibility open that sPLA2 may activate cPLA2.This effect has recently been shown, and involves a stimulation of p38 MAPK, aswell as p44/42 MAPK, both of which result in the phosphorylation and concomitantactivation of cPLA2.23–25 Thus, we investigated the effect of inhibition of the MAPK

FIGURE 6. Effect of MEK1/2 inhibition on Aβ-enhancement of ET-1-induced vaso-constriction. Certain aortic rings were treated with 1 µM freshly solubilized Aβ1–40, 25 µMPD98059, PD98059 + Aβ, or untreated (control) 5 min prior to the addition of a dose rangeof ET-1. ANOVA revealed significant main effects of ET-1 dose (p <0.001), Aβ ( p <0.001),and PD98059 (p <0.001). There were also significant interactive terms between ET-1 doseand either Aβ ( p <0.001) or PD98059 ( p <0.001), and among ET-1 dose, Aβ, and PD98059( p <0.001). One-way ANOVA across ET-1 doses revealed significant between-groups dif-ferences (p <0.001), and post-hoc testing showed significant differences between controland Aβ ( p <0.001), Aβ and Aβ + PD98059 (p <0.001), and control and Aβ + PD98059( p <0.001), but not between control and PD98059 ( p = 1.00), or PD98059 and Aβ +PD98059 (p = 0.880).


module in our vessel bath system. Vessels were pretreated with PD 98059, a highlyspecific MEK1/2 (MAPK kinase) inhibitor that has been shown to completely blockthe activation of p44/42 MAPK.25 We observed complete blockade of Aβ vasoactiv-ity by PD 98059, showing that MEK1/2 activity is necessary to mediate Aβ vasoac-tivity (FIG. 6). Interestingly, PD 98059 was also able to completely inhibit sPLA2enhancement of ET-1-induced vasoconstriction, showing that sPLA2 induction ofvasoconstriction, like Aβ, is essentially mediated via MEK1/2 (data not shown).Moreover, inhibition of p38 MAPK by SB 202190 resulted in complete blockade ofAβ vasoactivity (data not shown), further confirming that the MAPK module is nec-essary to ensure the activation of cPLA2 via Aβ.

AA will give rise to various eicosanoids, via the cyclooxygenase (COX) andlipoxygenase (LOX) pathways, which are proinflammatory compounds. We showthat both COX-2 (FIG. 7) and 5-LOX (data not shown) mediate Aβ vasoactivity,since specific inhibition of COX-2 with NS-398 or 5-LOX by MK-886 results in par-tial blockade of Aβ vasoactivity. Furthermore, when added in combination, NS-398and MK-886 completely abolish Aβ’s vasoconstrictive effect (data not shown). Inthe final analysis, we have dissected the intracellular signaling events which leads tothe vasoactive properties induced by Aβ peptides. Specifically, our data show that

FIGURE 7. Effect of COX-2 inhibition on Aβ-enhancement of ET-1-induced vasocon-striction. Certain aortic rings were treated with 1 µM freshly solubilized Aβ1–40, 5 µM NS-398,NS-398 + Aβ, or untreated (control) 5 min prior to the addition of a dose range of ET-1.ANOVA revealed significant main effects of ET-1 dose ( p <0.001), Aβ ( p <0.001), and NS-398( p <0.001). There were also significant interactive terms between ET-1 dose and either Aβ( p <0.001) or NS-398 ( p <0.001), and among ET-1 dose, Aβ, and NS-398 ( p <0.001). One-wayANOVA across ET-1 doses revealed significant between-groups differences ( p <0.001), andpost-hoc testing showed significant differences between control and Aβ ( p <0.001), and Aβand Aβ + NS-398 (p = 0.001).


freshly solubilized Aβ induces vasoconstriction by initially stimulating sPLA2,which, in turn, results in activation of the MAPK module, which ensures the stimu-lation of cPLA2, thus inducing the release of AA. COX-2 and 5-LOX then convertAA into various proinflammatory eicosanoids, which ultimately bring about Aβ’svasoconstrictive effect. These events are summarized in FIGURE 8.

We have previously shown that cyclic guanosine monophosphate (cGMP)-elevating agents, in particular the type V cGMP phosphodiesterase inhibitor dipy-ridamole, are able to block the Aβ-induced proinflammatory response in microglialcells.11 Also, these agents are able to inhibit the release of tumor necrosis factor-α(TNF-α) from microglia which are activated by lipopolysaccharide (LPS, data notshown). Interestingly, we have also shown that dipyridamole can block Aβ vasoac-tivity.11 We now suggest that dipyridamole and other cGMP-elevating compoundsmay influence the PLA2 pathway described above, since it has been shown thatcGMP can inhibit the MAPK module,26,27 and, via this mechanism, cGMP-elevatingagents may block the induction of cPLA2, thereby inhibiting Aβ’s vasoactive effect.Another effect of cGMP is to lower levels of intracellular calcium, by blocking the

FIGURE 8. Aβ’s signaling pathway.


release of Ca++ from intracellular stores as well as the entry of extracellular calcium.Since Ca++ is necessary for cPLA2 translocation to the nuclear membrane, whereCOX-2 and 5-LOX are located, cGMP-elevating agents may inhibit cPLA2 translo-cation via decreasing intracellular Ca++.

CONCLUSION

We have shown that low doses (in the nM range similar to the level of circulatingAβ found in AD) of freshly solubilized Aβ peptides are able to potentiate ET-1-induced vasoconstriction, and are also able to increase the duration of the constric-tion induced by various vasoconstrictors. We have dissected the signal transductionpathway that is responsible for the vasoactive properties of soluble Aβ peptides, andfind that this effect is mediated via the activation of a proinflammatory pathway.Therefore, our data suggest that soluble Aβ peptides initiate an inflammatory cas-cade, and, at the clinical level, may be responsible for the initiation of AD-type neu-roinflammation prior to Aβ deposition. Since we have shown that specific inhibitorsof the PLA2 pathway are efficient blockers of Aβ’s proinflammatory vasoactivity,this provides a basis for novel therapies aimed at blocking Aβ’s bioactivity.

ACKNOWLEDGMENTS

This work was supported by the generosity of Mr. and Mrs. Robert Roskamp.M. Mullan is the recipient of a Veteran’s Administration Merit Award.

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aβ vasoactivity: an inflammatory reaction

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