Lysophosphatidic acid triggers mast cell-driven atherosclerotic plaque destabilization by increasing vascular inflammation

Abstract

Lysophosphatidic acid (LPA), a bioactive lysophospholipid, accumulates in the atherosclerotic plaque. It has the capacity to activate mast cells, which potentially exacerbates plaque progression. In this study, we thus aimed to investigate whether LPA contributes to plaque destabilization by modulating mast cell function. We here show by an imaging mass spectrometry approach that several LPA species are present in atherosclerotic plaques. Subsequently, we demonstrate that LPA is a potent mast cell activator which, unlike other triggers, favors release of tryptase. Local perivascular administration of LPA to an atherosclerotic carotid artery segment increases the activation status of perivascular mast cells and promotes intraplaque hemorrhage and macrophage recruitment without impacting plaque cell apoptosis. The mast cell stabilizer cromolyn could prevent intraplaque hemorrhage elicited by LPA-mediated mast cell activation. Finally, the involvement of mast cells in these events was further emphasized by the lack of effect of perivascular LPA administration in mast cell deficient animals. We demonstrate that increased accumulation of LPA in plaques induces perivascular mast cell activation and in this way contributes to plaque destabilization in vivo. This study points to local LPA availability as an important factor in atherosclerotic plaque stability.

Fig. 1.

A: TOF-SIMS spectra recorded from an atherosclerotic artery segment in negative mode with tentative peak assignments, showing LPA species. B: HE staining and C: MOMA-2⁺ macrophage staining of a mouse carotid artery lesion. Negative ion micrographs of a flanking lesion analyzed by TOF-SIMS. Color intensity corresponds to signal strength. D: Total ion current (TIC). E: Intraplaque distribution patterns of a selection of relevant ions including phosphatidic acids, triglycerides, and cholesterol. F: Several LPA species. Scale bar indicates 100 μm.

Fig. 2.

LPA induces tryptase release from mast cells and induces vascular leakage. A: After intraperitoneal LPA challenge tryptase activity was increased by 2.3-fold in the C57Bl/6 mice, which did not occur in mast cell deficient Kit(W^−sh/W^−sh) mice. *P < 0.05 compared with C57Bl/6 at baseline. B: LPA, compound 48/80, and nonactivated MC/9 cells induced minor to moderate vascular leakage as judged by Evans Blue spot size, probably due to activation of locally residing mast cells. LPA-activated MC/9 cells significantly induced vascular leakage, similarly as compound 48/80-activated MC/9 cells, which were used as positive control. **P < 0.01, ***P < 0.001 compared with PBS control, ^#P < 0.05, ^##P < 0.01 compared with MC/9. C: Quantification of activated MC/9 cells in skins of mast cell deficient Kit(W^−sh/W^−sh) mice, illustrating the mast cell absence in PBS, compound 48/80, and LPA injected skins. Injection of unstimulated MC/9 cells resulted in spontaneous activation of 47 ± 6% possibly due to shear stress, a number that was significantly increased by prior mast cell priming with compound 48/80 and LPA. ^#P < 0.05, ^##P < 0.01 compared with unstimulated MC/9 cells.

Fig. 3.

Adventitial mast cell content of atherosclerotic carotid artery lesions after focal LPA administration. A: Plaque size is not changed at three days after local LPA challenge as compared with control mice or mice that had received the mast cell stabilizer cromolyn. B: Total adventitial mast cell contents of control, LPA-treated, and LPA/cromolyn-treated mice were essentially similar. C: Adventitial mast cell degranulation was increased at three days after local LPA challenge as compared with control mice (*P < 0.05). Cromolyn treatment normalized the levels of mast cell activation in the LPA-challenged animals.

Fig. 4.

Effects of LPA on macrophages and hemorrhage. A: Intimal macrophage content in control, LPA-treated, and LPA/cromolyn-treated animals. Intimal macrophage content significantly increased in the LPA-treated group, an effect that was prevented by cromolyn treatment. The right panels are representative MOMA-2 stained cryosections of control (upper panel), LPA-treated (middle panel), and LPA/cromolyn-treated animals (lower panel); *P < 0.05 compared to control; ^##P < 0.01 compared to LPA. B: Effect of LPA on murine RAW 264.7 proliferation. A dose-dependent increase in proliferation is seen in RAW 264.7 murine macrophages. **P < 0.01, ***P < 0.001 versus untreated cells (control). C: Quantification of the number of plaques containing intraplaque hemorrhages in control, LPA-treated, and LPA/cromolyn-treated animals suggesting an increased frequency of hemorrhages after LPA challenge. Cromolyn treatment normalized the increase in intraplaque hemorrhage in LPA-challenged animals. A representative hematoxylin/eosin stained cryosection of a plaque from an LPA-challenged mouse is displayed on the right demonstrating intraplaque hemorrhages and erythrocyte extravasation (arrows) in the intima. D: Quantification of the number of Perl's iron positive plaques in control, LPA-treated, and LPA/cromolyn-treated animals suggesting an increased frequency of iron deposits after LPA treatment. Cromolyn treatment normalized the frequency of plaques with iron deposits. On the right is a representative Perl's iron staining revealing large areas with iron deposits (arrows).

Fig. 5.

LPA in mast cell-deficient apoE^−/−Kit(W^−sh/W^−sh) mice. A: LPA did not affect lesion size in apoE^−/−Kit(W^−sh/W^−sh) mice, while also intimal macrophage content was not significantly changed after LPA challenge (B). C: LPA did not elicit any intraplaque hemorrhages in the mast cell-deficient apoE^−/−Kit(W^−sh/W^−sh) mice (P < 0.05 as compared with LPA treatment in apoE^−/− mice), suggesting that LPA induces intraplaque hemorrhage in a mast cell-dependent fashion.