Myeloid-related protein-8/14 is critical for the biological response to vascular injury

Abstract

Background: Myeloid-related protein (MRP)-8 (S100A8) and MRP-14 (S100A9) are members of the S100 family of calcium-modulated proteins that regulate myeloid cell function and control inflammation, in part, through activation of Toll-like receptor-4 and the receptor for advanced glycation end products. A transcriptional profiling approach in patients with acute coronary syndromes identified MRP-14 as a novel predictor of myocardial infarction. Further studies demonstrated that elevated plasma levels of MRP-8/14 heterodimer predict increased risk of first and recurrent cardiovascular events. Beyond its serving as a risk marker, whether MRP-8/14 participates directly in vascular inflammation and disease remains unclear.

Methods and results: We evaluated vascular inflammation in wild-type and MRP-14-deficient (MRP-14(-/-)) mice that lack MRP-8/14 complexes with experimental arterial injury, vasculitis, or atherosclerosis. After femoral artery wire injury, MRP-14(-/-) mice had significant reductions in leukocyte accumulation, cellular proliferation, and neointimal formation compared with wild-type mice. In a cytokine-induced local Shwartzman-like reaction that produces thrombohemorrhagic vasculitis, MRP-14(-/-) mice had significant reductions in neutrophil accumulation, lesion severity, and hemorrhagic area. In response to high-fat feeding, mice doubly deficient in apolipoprotein E and MRP-8/14 complexes had attenuation in atherosclerotic lesion area and in macrophage accumulation in plaques compared with mice deficient in apolipoprotein E alone.

Conclusions: This study demonstrates that MRP-8/14 broadly regulates vascular inflammation and contributes to the biological response to vascular injury by promoting leukocyte recruitment.

Figure 1. Photomicrographs of MRP-8 and MRP-14 immunostaining of normal and injured femoral arteries from WT and MRP-14^−/− mice

A-B, MRP-8 or MRP-14 immunostaining of an uninjured femoral artery from a WT mouse. C–D, MRP-8 or MRP-14 immunostaining of a femoral artery from a WT mouse 7d post injury. E–F, MRP-8 or MRP-14 immunostaining of a femoral artery from a MRP-14^−/− mouse 7d post injury (original magnification 10X).

Figure 2. Photomicrographs of injured femoral arteries from WT and MRP-14^−/− mice after wire injury

A–B, CD45 immunostaining 7d after injury. C–D, Mac-3 immunostaining 7d after injury. E–F, 7/4 immunostaining 7d after injury. G–H, BrdU immunostaining 7d after injury. I–J, VerHoeff elastin stain 28d after injury. Arrows delineate the internal elastic lamina (original magnification 10X).

Figure 3. Deficiency of MRP-14 attenuates smooth muscle cell proliferation in vitro

Basal and PDGF-stimulated proliferative activity of cultured mouse aortic smooth muscle cells derived from WT or MRP-14^−/− mice. Proliferation was measured 18 hours following addition of PDGF. Representative experiment, mean ± standard deviation, n= 3 (■ WT, ○ MRP-14^−/−)(P<0.0001 by ANOVA).

Figure 4. Deficiency of MRP-14 attenuates lesion severity during cytokine-induced thrombohemorrhagic vasculitis

A, Necropsy images showing representative vasculitis lesions in WT and MRP-14^−/− mice. B, Scoring of vasculitic lesions from WT and MRP-14^−/− mice. C, Quantification of skin thickness (dermis plus epidermis) from histologic section of vasculitic lesions. Data represent mean ± standard deviation, n=13 mice per group.

Figure 5. Photomicrographs of skin lesions from WT and MRP-14^−/− mice during cytokine-induced thrombohemorrhagic vasculitis

A–B, MRP-8 or MRP-14 immunostaining of normal skin from a WT mouse. C–D, MRP-8 or MRP-14 immunostaining of vasculitic lesion from a WT mouse. E–F, MRP-8 or MRP-14 immunostaining of vasculitic lesion from a MRP-14^−/− mouse. G–H, Hematoxylin and eosin staining of representative vasculitic lesions demonstrating hemorrhage (original magnification 40X). I, Quantification of % hemorrhage area. J–K, Immunohistochemical staining for neutrophils in vasculitic lesions (mAb7/4-positive cells)(20X). L, Quantification of % neutrophil-positive area. Data represent mean ± SD, n=13 mice per group.

Figure 6. Deficiency of MRP-14 attenuates atherosclerotic lesion formation and plaque inflammation

A, Sudan IV staining of intact closed and longitudinally opened and pinned thoracoabdominal aortas harvested from ApoE^−/− and ApoE^−/−MRP-14^−/− mice after 20 weeks of high-fat feeding. B, Quantification of % lesion area as assessed by Sudan IV staining and computer-assisted imaging analysis. C, Quantification of plaque macrophage content (% Mac-3-positive area) in longitudinal sections from the descending aorta and the lesser curvature of the aortic arch. D–G, Representative photomicrographs staining for macrophages (Mac-3-positive cells) within atherosclerotic lesions in the descending aorta (D, E) and aortic arch (F, G) (original magnification 20X). For paired samples in the atherosclerotic lesion area analysis, comparison of the % lesion area in closed vs. open analysis resulted in a correlation value of 0.63. For macrophage staining, comparison of the percent macrophage area in the descending aorta vs. the aortic arch resulted in a correlation value of 0.41.

Figure 7. MRP-8/14 regulates macrophage cytokine production and migration

ELISA analysis of LPS-induced (5μg/ml) cytokine production by cultured macrophages derived from WT and MRP-14^−/− mice. Twelve hour stimulation, representative experiment of n=3 per cytokine. A, TNF-α, MCP-1, Il-1β, IL-12 production. B, Transwell assay of MCP-1-stimulated peripheral blood mononuclear cell migration.

Figure 8. MRP-8/14 is expressed in human carotid atheroma

A–C, MRP-8 staining of carotid atheroma or normal carotid artery. D–F, MRP-14 staining of carotid atheroma or normal carotid artery. G–I, MRP-8/14 complex-specific staining of carotid atheroma or normal carotid artery. J–K, CD68 staining of carotid atheroma or normal carotid artery. B,E,H,K, Adjacent tissue sections demonstrating MRP-8, MRP-14, and MRP-8/14 association with lesional CD68-positive macrophages (original magnification 10x or 40x as indicated).