Recently, we showed that platelet phagocytosis occurs in human atherosclerotic plaques and leads to foam cell formation. Platelet phagocytosis, resulting in macrophage activation and iNOS induction, was associated with the formation of amyloid-β peptide (Aβ) via proteolytic cleavage of platelet-derived amyloid precursor protein (APP), possibly by secretases. To test the involvement of γ-secretase in this process, we used indomethacin, ibuprofen, and sulindac sulfide, non-steroidal anti-inflammatory drugs (NSAIDs) known to alter the γ-secretase cleaving site of APP, on their ability to inhibit macrophage activation evoked by platelet phagocytosis. J774 macrophages were incubated with human platelets or lipopolysaccharide (LPS) with or without NSAIDs. Nitrite was quantified as a measure for inducible nitric oxide synthase (iNOS) activity. Indomethacin, ibuprofen, sulindac sulfide, and meloxicam concentration-dependently reduced nitrite production by macrophages incubated with platelets, but did not alter LPS-induced iNOS activity or platelet uptake. However, acetylsalicylic acid and naproxen, two NSAIDs without effect on the γ-secretase cleaving site of APP, did not affect nitrite production in either platelet- or LPS-stimulated macrophages. Surface-enhanced laser desorption/ionization time-of-flight mass-spectrometry demonstrated time-dependent formation of Aβ-containing peptides after platelet phagocytosis, which could be inhibited by indomethacin. In conclusion, these results point to the involvement of γ-secretase in macrophage activation following platelet phagocytosis.
Recently, we demonstrated the presence of amyloid-β (Aβ), which until then had only been studied in Alzheimer's disease, in macrophages around microvessels in advanced human atherosclerotic plaques. 1 In these macrophages, the formation of Aβ was associated with the expression of inducible nitric oxide synthase (iNOS). A source for Aβ in atherosclerotic plaques is blood platelets, which contain amyloid precursor protein (APP) in the α-granules. The platelets can enter atherosclerotic plaques via leaky microvessels and are then phagocytosed by macrophages, 2 which subsequently transform to foam cells. 1 We proposed that during this process Aβ-like peptides are formed, which then activate the macrophage as indicated by iNOS expression. 1 This novel mechanism of macrophage activation in atherosclerotic plaques may favor plaque expansion and/or rupture. Indeed, the massive nitric oxide (NO) release produced by iNOS has been reported to induce apoptotic cell death of smooth muscle cells by enhancing Fas-L/Fas interactions. 3 In the atherosclerotic plaque, smooth muscle cells are the only cells that are able to produce collagen isoforms that contribute to the strength of the plaque. 4 Moreover, activated macrophages can trigger the activation of matrix metalloproteinases in the vascular interstitium, 5 resulting in degradation of the interstitial collagen fibers and decreased strength of the fibrous cap of an atherosclerotic plaque.
The proteolytic enzymes responsible for Aβ formation in macrophages following platelet phagocytosis are not known yet. In Alzheimer's disease the insoluble Aβ1–42 peptide is generated by the cleavage of its precursor APP, by two enzymes designated β- and γ-secretase. Recently, it was shown that indomethacin, ibuprofen, and sulindac sulfide directly affect the γ-secretase cleaving site of APP, thereby altering the amyloid pathology in the brain. 6 The latter effect results in a reduction of the highly amyloidogenic Aβ1–42 levels and an increase in the less harmful Aβ1–38 isoform. The interference with the γ-secretase cleaving site is not seen with all NSAIDs and was not dependent on cyclooxygenase (COX) inhibition. Indeed, acetylsalicylic acid and naproxen do not affect the γ-secretase cleaving site of APP. Since specific γ-secretase inhibitors are not available, we used a series of NSAIDs to investigate the participation of γ-secretase in macrophage activation and the formation of Aβ-like peptides following platelet phagocytosis.
From the *Division of Pharmacology, University of Antwerp, Antwerp, Belgium; †Department of Medicinal Chemistry, University of Liège, Liège, Belgium; and ‡Department of Pathology, General Hospital Middelheim, Antwerp, Belgium.
Received for publication August 18, 2003; accepted December 17, 2003.
Supported in part by the Fund for Scientific Research Belgium (FWO, FNRS) (Nr: 1.5.206.00 and G.0180.01), The Bekales Foundation, University of Antwerp (NOI-BOF), and the Flemish Institute for Improvement of Scientific and Technological Research in Industry (IWT). Dr. M. Kockx is a holder of a fund for fundamental clinical research of the Fund for Scientific Research–Flanders. D. Jans was supported by the Flemish Institute for Improvement of Scientific and Technological Research in Industry (IWT).
Reprints: Dominique Jans, Division of Pharmacology, University of Antwerp (UA), Universiteitsplein 1, B-2610 Antwerp, Belgium (e-mail: email@example.com).