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β−Amyloid Vasoactivity and Proinflammation in Microglia Can Be Blocked by cGMP-Elevating Agents

DANIEL PARIS,a TERRENCE TOWN, TIMOTHY PARKER, JAMES HUMPHREY, AND MICHAEL MULLAN

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

BACKGROUND

Vascular damage and reactive gliosis are often found colocalized with amyloiddeposits in Alzheimer’s disease (AD) brain,1 suggesting that the cerebrovasculaturemay be a clinically relevant site of AD pathology and may contribute to the neuroin-flammatory process in AD. Using intact rat aortae in a tissue bath system, we havepreviously shown that freshly solubilized β-amyloid (Aβ) peptides are able to impairrelaxation in response to acetylcholine2 or the nitric oxide (NO) donor sodium nitro-prusside (SNP).3 Furthermore, we have demonstrated that Aβ peptides are able topotentiate the vasoconstriction induced by endothelin-1 (ET-1),4,5 which is one ofthe most potent cerebral vasoconstrictors in the brain.6 Interestingly, Aβ’s enhance-ment of ET-1-induced vasoconstriction has been shown to be mediated independent-ly of the presence of an intact endothelium,5 suggesting that the ability of vascularsmooth muscle cells to determine the dynamic caliber of the blood vessel may be di-rectly impacted by Aβ.

The molecular mechanism of Aβ vasoactivity has been suggested to be mediatedvia free radicals,2 because superoxide dismutase appears to partially oppose Aβ’svasoconstrictive effect.5 We have recently shown, however, that the pathway to Aβvasoactivity is not impacted by the superoxide dismutase mimic and peroxynitritescavenger MnTBAP,4 catalase, or the water-soluble vitamin E analogue Trolox (datanot shown). Furthermore, we have shown that Aβ vasoactivity is not due to an inhi-bition of nitric oxide synthase (NOS) activity, since inactivation of NOS does notmimic or modulate the vasoactive properties of Aβ.3 Collectively, these results showthat Aβ vasoactivity is not mediated by reactive oxygen species, and they promptedus to search for an alternative mechanism.

Soluble guanylyl cyclase (sGC) is responsible for the synthesis of cGMP, and NOstimulates sGC in underlying vascular smooth muscle cells, resulting in vasorelax-ation. This effect is antagonized by ET-1, which has been shown to impede increasesin cGMP levels in vessels stimulated with the NO donor SNP. This balance betweenNO and ET-1 provides one mechanism for control of cerebral vasotonus, and, sincewe showed that Aβ can oppose NO-induced vasorelaxation and enhance ET-1-induced vasoconstriction, we evaluated the possibility that Aβ may oppose the NO/

aAddress for correspondence: Daniel Paris, The Roskamp Institute, University of South Flor-ida, 3515 E. Fletcher Ave., Tampa, FL 33613.

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cGMP pathway, thereby potentiating ET-1-induced vasoconstriction. Cyclic GMPlevels are modulated essentially by two mechanisms: sGC stimulation leads to in-creased cGMP production, and activation of cGMP phosphodiesterases (cGMP-PDEs) results in degradation of cGMP. Our data show that, whereas Aβ does not me-diate its vasoconstrictive effect via inhibition of sGC, activation of cGMP-PDE is in-volved, because a specific type V cGMP-PDE inhibitor (dipyridamole) is able toblock Aβ’s vasoactive effect in a statistically interactive manor.

We then asked the question whether the signal transduction pathway mediatingAβ’s vasoconstrictive effect might generally mediate Aβ bioactivity in different celltypes. To evaluate this possibility, we investigated Aβ’s effect on cultured microglia,a cell type that has been implicated as a mediator of neuroinflammation in AD brain.We show that various cGMP-elevating agents are able to attenuate Aβ-induced leu-kotriene B4 (LTB4) (a proinflammatory eicosanoid) production in microglia. Takentogether, our data show that Aβ’s effects on both isolated vessels and cultured mi-croglia can be modulated by cGMP, and suggest that Aβ’s bioactivity can be broughtabout via a common signal transduction pathway in different systems.

FIGURE 1. Effect of Aβ on SNP-induced relaxation. Control vessels and vessels pre-treated with 1 mM of freshly solubilized Aβ1–40 were constricted with 3.5 nM of PE. Furtherrelaxation was induced with the doses of SNP shown. Data were then standardized such thatthe maximum value for PE-induced tension was 100% for both Aβ and control channels. Aβ( p < 0.001) and SNP (p < 0.001) were significant factors in the ANOVA, although there wasno significant interaction between them (p = 0.890). Aβ decreases the induced relaxation at0.1 mM SNP ( p < 0.02), at 1 mM SNP ( p < 0.01), and at 2 mM SNP (p < 0.01).

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RESULTS AND DISCUSSION

Vessels were treated with phenylephrine (PE, 3.5 nM) to obtain a stable long-last-ing constriction. We observed, in vessels co-treated with Aβ and PE, that the relax-ation induced by SNP was reduced in comparison to the relaxation induced by thesame amount of SNP in control vessels (FIG. 1). These data suggested two possibil-ities: either Aβ was able to inhibit the stimulation of soluble guanylyl cyclase (sGC)by NO, or it increased the degradation of cGMP. We investigated the role of sGC inthe vasoactivity mediated by Aβ by using a highly selective inhibitor of sGC, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). These data showed that the vas-

FIGURE 2. Effect of dipyridamole on Aβ-enhancement of ET-1-induced vasoconstric-tion. Certain aortic rings were treated with 1 µM of freshly solubilized Aβ1–40, 10 µM ofdipyridamole (dipyr.), or Aβ + dipyr 5 min before the addition of a dose range of ET-1. Re-sults are expressed as the mean ± 1 SE of the percentage vasoconstriction increase over base-line. There were significant main effects of ET-1 ( p < 0.001), Aβ ( p < 0.001), and dipyr ( p< 0.001), as well as significant interactive terms between ET-1 and either Aβ (p < 0.001) ordipyr (p < 0.001). Further, there was significant interaction among ET-1, Aβ, and dipyr (p< 0.05). One-way ANOVA revealed significant between-treatment group differences acrossall ET-1 doses (p < 0.001), and post-hoc comparison between control vessels and Aβ-treatedvessels revealed a significant difference ( p < 0.01), as did Aβ-treated vessels compared toAβ + dipry-treated aortae ( p < 0.01).

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oconstriction induced by ET-1 was synergistically enhanced after ODQ treatment,but was only additive, as opposed to statistically interactive, with Aβ and ODQ co-treatment.3 This result suggested that Aβ vasoactivity was not mediated via inhibi-tion of sGC. In this same report, we demonstrated that stimulation of sGC with YC-1, a NO-independent activator of sGC, was only able to reduce Aβ-induced vasocon-striction in an additive manner, further supporting that Aβ vasoactivity was not dueto modulation of sGC activity. Moreover, inhibition of NOS activity by L-NAME-enhanced ET-1 induced vasoconstriction, yet this effect was merely additive in con-junction with Aβ, showing that Aβ vasoactivity does not result from an alteration ofNOS activity or NO production.

Having established that Aβ vasoactivity was not due to an alteration of the NO-mediated synthesis of cGMP, we went on to examine whether cGMP hydrolysis wasinvolved. We used dipyridamole (dipyr), a specific inhibitor of type V cGMP-PDE,to test the possible involvement of cGMP-PDE in mediating Aβ vasoactivity. We ob-served that dipyridamole was able to interactively block Aβ-enhancement of ET-1-induced vasoconstriction, suggesting that Aβ stimulates cGMP degradation (FIG. 2).

FIGURE 3. Effect of cGMP elevation on Aβ-induced microglial LTB4 release. Foreach condition presented, n = 6. ANOVA revealed significant main effects of Aβ ( p < 0.01),dipyridamole (10 µM, p = 0.01), 8-Br cGMP ( p < 0.001), and SNP ( p < 0.001). Significantinteractive terms were noted between Aβ and each drug used (p < 0.01). Post-hoc testingdid not reveal significant differences between control and each drug used or control and drug+ Aβ ( p > 0.05). This shows that each drug used alone does not significantly affect consti-tutive LTB4 release, and administration of each drug in combination with Aβ results in totalblockade of Aβ-induced microglial LTB4 release.

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In order to further confirm this hypothesis, we measured cGMP levels in Aβ-treatedaortae and found that cGMP levels were reduced in Aβ-treated vessels compared tountreated controls.3 Data thus far had suggested that Aβ’s vasoactivity is mediatedthrough a specific signal transduction pathway impacting cGMP levels. We hypoth-esized that Aβ may exert its biological activity via similar mechanisms in variouscell types. Thus, we investigated the effects of cGMP-elevating agents on Aβ’s acti-vation of a proinflammatory pathway in N9 microglial cells (kindly provided by Dr.Ricciardi-Castagnoli, Cellular Pharmacology Center, Milan, Italy).

We choose leukotriene B4 (LTB4), which is a proinflammatory eicosanoid end-product of the classical arachidonic acid/5-lipoxygenase pathway, as a marker of mi-croglial proinflammation. Data showed that treatment of N9 microglia with Aβresulted in marked LTB4 release. This effect was significantly attenuated by co-treat-ment of microglia with Aβ and the cGMP-elevating agents dipyridamole, SNP, or acell-permeable analogue of cGMP, 8-bromo cGMP (8-Br cGMP, FIG. 3). Taken to-gether, these data show that cGMP-elevating agents display antiinflammatory proper-ties, and, specifically, are able to block Aβ vasoactivity and proinflammatory responsein microglial cells. Furthermore, these results suggest that the intracellular signalingpathway triggered by Aβ is conserved across different cellular systems, because drugsthat oppose Aβ vasoactivity also block Aβ-induced microglial inflammation.

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.

REFERENCES

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