Abstract-In human occluded saphenous vein grafts, we previously demonstrated cytotoxic foam cells, presumably derived from macrophages engulfing platelets. In the present study, we investigated whether platelet phagocytosis occurs in human atherosclerotic plaques, whether this activates macrophages, and whether the platelet constituent, amyloid precursor protein (APP), was involved. Immunohistochemistry documented the presence of APP, ␤-amyloid peptide (A␤, cleaved from APP), and platelets (CD9), along with inducible NO synthase (iNOS) and cyclooxygenase-2, two markers of macrophage activation, around microvessels in advanced human carotid artery plaques (nϭ18). A␤ colocalized with iNOS-expressing macrophages that were often surrounded by platelets. In vitro, murine J774 and human THP-1 macrophages were incubated with or without washed human platelets. Coincubation of macrophages and platelets led to platelet phagocytosis (electron and confocal microscopy) and formation of lipid-, APP-, and A␤-containing foam cells. These expressed iNOS mRNA (reverse transcription-polymerase chain reaction) and protein and produced nitrite and tumor necrosis factor-␣ (ELISA). Macrophage pretreatment with 4-(2-aminoethyl)benzenesulfonyl fluoride, a protease inhibitor, reduced APP processing and inhibited NO biosynthesis induced by platelet phagocytosis but not by lipopolysaccharides. Human atherosclerotic plaques and J774 and THP-1 macrophages contained mRNA of the APP-cleaving enzyme ␤-secretase. This is the first demonstration of A␤, a peptide extensively studied in Alzheimer's disease, in human atherosclerotic plaques. It was present in activated iNOSexpressing perivascular macrophages that had phagocytized platelets. In vitro studies indicate that platelet phagocytosis leads to macrophage activation and suggest that platelet-derived APP is proteolytically processed to A␤, resulting in iNOS induction. This represents a novel mechanism for macrophage activation in atherosclerosis. T he composition of an atherosclerotic plaque is an important determinant of plaque stability. Unstable ruptureprone plaques are characterized by a thin fibrous cap that contains few smooth muscle cells. 1,2 Several lines of evidence suggest that macrophage activation in the vulnerable shoulder of the plaque could contribute to plaque rupture. 3,4 We have previously postulated the release of factors toxic to smooth muscle cells, possibly NO, from activated macrophages in human atherosclerotic plaques. 5,6 It has been reported that foam cell formation can be induced by platelet phagocytosis. 7-10 Moreover, in human (sub)occluded saphenous vein grafts, the formation of toxic foam cells within mural thrombi is presumably the result of platelet phagocytosis by macrophages. 11 Therefore, we questioned whether in human atherosclerotic plaques platelet phagocytosis evokes macrophage activation and whether proteolytic processing of amyloid precursor protein (APP), present in platelet ␣-granules, 12-15 is involved in this process.To test this hypothesis, we fi...

Macrophage activation in atherosclerotic plaques plays a role in plaque destabilization, rupture and subsequent atherothrombosis. Platelet phagocytosis that occurs within human atherosclerotic plaques can activate macrophages and it has been suggested that the platelet constituent amyloid precursor protein (APP) is involved. Recent studies show that amyloid beta (Abeta), a peptide extensively studied in Alzheimer's disease and that is cleaved from APP by beta- and gamma-secretase, and/or Abeta-like peptides are also present in human atherosclerotic plaques, in particular in activated, inducible nitric oxide synthase (iNOS) expressing perivascular macrophages that had phagocytized platelets. In vitro studies confirm that platelet phagocytosis leads to macrophage activation and suggest that platelet-derived APP is proteolytically processed to Abeta-like peptides, resulting in iNOS induction. In addition, non-steroidal anti-inflammatory drugs (NSAIDs) and HMG-CoA reductase inhibitors (statins), two classes of drugs reported to affect APP processing and Abeta formation in Alzheimer's disease, have been evaluated for their capacity to inhibit macrophage activation evoked by platelet phagocytosis. Remarkably, the same NSAIDs reported to alter gamma-secretase activity in Alzheimer's disease also reduce macrophage activation after platelet phagocytosis and inhibit formation of Abeta-containing peptides. From the statins investigated (fluvastatin, atorvastatin, simvastatin, pravastatin, lovastatin and rosuvastatin) only fluvastatin and atorvastatin selectively inhibit macrophage activation after platelet phagocytosis, possibly through inhibition of Rho activity. Taken together, these new findings point to the involvement of platelet-derived APP in macrophage activation in atherosclerosis and suggest a biochemical link between atherosclerosis and Alzheimer's disease. Accordingly, drugs interfering with APP processing might have an impact on both diseases.

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-beta peptide (Abeta) via proteolytic cleavage of platelet-derived amyloid precursor protein (APP), possibly by secretases. To test the involvement of gamma-secretase in this process, we used indomethacin, ibuprofen, and sulindac sulfide, non-steroidal anti-inflammatory drugs (NSAIDs) known to alter the gamma-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 gamma-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 Abeta-containing peptides after platelet phagocytosis, which could be inhibited by indomethacin. In conclusion, these results point to the involvement of gamma-secretase in macrophage activation following platelet phagocytosis.

Statins, through HMGCoA reductase inhibition, are potent inhibitors of cholesterol synthesis and are widely used in cardiovascular disease. Recent evidence suggests that the beneficial effects of statins extend beyond their action on serum cholesterol levels. The so called pleiotropic effects can be attributed to reduced production of isoprenoid intermediates such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). We investigated the effects of statins on macrophage activation after platelet phagocytosis. Platelet phagocytosis is known as an alternative mechanism for foam cell formation and macrophage activation in human atherosclerotic plaques. Processing of amyloid precursor protein (APP), present in platelets, to amyloid‐β (Aβ) might be responsible for induction of the inducible nitric oxide synthase (iNOS) in macrophages. It has been reported that statins can affect APP processing not only through reduction of cholesterol levels, but also via other mechanisms such as reduced isoprenylation of small GTP binding proteins. Interferon‐γ primed murine J774 macrophages were incubated with washed human blood platelets for 18 h with or without different statins. Nitrite was quantified in the supernatant as a measure for iNOS activity. Macrophage activation following platelet phagocytosis was reduced in the presence of fluvastatin and atorvastatin, but not with simvastatin and rosuvastatin. Besides 30 μM of simvastatin, there was no effect of statins on LPS‐stimulated macrophages, indicating that LPS activates macrophages via a different pathway than platelets. The effect of fluvastatin could be reversed by mevalonic acid, FPP and GGPP. Our results show that some statins decrease macrophage activation after platelet phagocytosis. This effect may be explained by interference with cholesterol synthesis or through reduced isoprenylation of small GTP binding proteins. Diminished macrophage activation in an atherosclerotic plaque can contribute to plaque stabilisation.