Neutrophils in atherosclerosis

Atherosclerosis is a complex inflammatory disease localized in medium-sized and large arteries and it is the most important contributor to cardiovascular disease. Complications of atherosclerosis such as myocardial infarction and stroke are leading causes of mortality in many countries. Leukocyte recruitment to the arterial intima is crucial for the development of atherosclerotic lesions. The roles of macrophages and T-lymphocytes in promoting atherosclerotic plaque development and destabilization have been extensively studied. However, the most abundant white blood cell in the circulation, the neutrophil, has until recently rarely been associated with disease pathogenesis. The work of this thesis aimed at investigating the potential presence and roles of neutrophils in atherosclerosis. Previous in vivo studies of leukocyte recruitment in atherogenesis have not been able to selectively detect individual subpopulations of leukocytes. In order to study neutrophils more specifically, we aimed at introducing a system, by which we could selectively study the roles of monocytes and neutrophils by microscopy. By crossing mice deficient in apolipoprotein E (ApoE-/- mice) with mice homozygous for a knock-in mutation for enhanced green fluorescent protein (EGFP) in the lysozyme M (lys-M) locus, we generated lysozyme M-deficient atherosclerosis-prone mice with endogenously fluorescent neutrophils and monocytes (ApoE-/-/LysEGFP/EGFP mice, Paper I). In order to address whether absence of lys-M and replacement with EGFP influence atherogenesis, we compared the generated mice with their littermate ApoE-/- mice and found no differences in white blood cell count, cholesterol profile, plaque composition or lesion area between the two strains. The generated mouse strain enabled us to use intravital microscopy to efficiently detect fluorescent monocytes and neutrophils that were interacting with atherosclerotic endothelium in vivo, and to use confocal microscopy to observe individual cells within lesions. In order to specifically study neutrophil presence in, and recruitment to, atherosclerotic lesions, we used ApoE-/-/LysEGFP/EGFP mice in several experiments. By use of intravital microscopy we showed that a vast majority of leukocytes interacting with endothelium on lesion shoulders are neutrophils, suggesting a significant recruitment of these cells to plaque (Paper II). Furthermore, flow cytometry and confocal microscopy showed that neutrophils make up for 1.8% of CD45+ leukocytes in the aortic wall of ApoE-/-/LysEGFP/EGFP mice and that their contribution relative to monocyte/macrophages within lesions is approximately 1:3. Interestingly, we could show that neutrophils accumulate at sites of high density of monocytes and preferentially in shoulder regions of plaques. In some regions of plaque neutrophils actually outnumber monocytes/macrophages. Atherosclerosis is known to aggravate during systemic inflammatory diseases, and common infections can trigger acute cardiovascular events. In Paper III, we investigate the potential for systemic inflammatory stimuli to induce recruitment of leukocytes to the walls of large arteries in normal and atherosclerotic mice. ApoE-/- and control C57Bl/6 mice were challenged with cytokines (TNF-a and IL-1b), LPS or infection with Influenza A in order to induce a systemic inflammatory response. The stimulation triggered a rapid systemic cytokine release and an increase in the relative number of peripheral neutrophils. Interestingly, there was a significant increase in the number of leukocytes adherent to atherosclerotic endothelium as detected with scanning electron microscopy on aortic endothelium. Furthermore, flow cytometry on aortic cells revealed a marked recruitment of neutrophils following inflammatory challenge. Altogether, this thesis demonstrates that neutrophils are recruited to atherosclerotic lesions and that neutrophils represent the principal subset of leukocytes that interact with atherosclerotic endothelium. Furthermore, neutrophils invade lesions in significant numbers under baseline conditions and are found especially in shoulder regions and at sites of high inflammatory activity. Neutrophil invasion is significantly increased during systemic inflammation. These findings establish neutrophils as potentially important players in the pathogenesis of atheroscleri

Deletion of L-Selectin Increases Atherosclerosis Development in ApoE−/− Mice

Atherosclerosis is an inflammatory disease characterized by accumulation of leukocytes in the arterial intima. Members of the selectin family of adhesion molecules are important mediators of leukocyte extravasation. However, it is unclear whether L-selectin (L-sel) is involved in the pathogenesis of atherosclerosis. In the present study, mice deficient in L-selectin (L-sel−/−) animals were crossed with mice lacking Apolipoprotein E (ApoE−/−). The development of atherosclerosis was analyzed in double-knockout ApoE/L-sel (ApoE−/− L-sel−/−) mice and the corresponding ApoE−/− controls fed either a normal or a high cholesterol diet (HCD). After 6 weeks of HCD, aortic lesions were increased two-fold in ApoE−/− L-sel−/− mice as compared to ApoE−/− controls (2.46%±0.54% vs 1.28%±0.24% of total aortic area; p<0.05). Formation of atherosclerotic lesions was also enhanced in 6-month-old ApoE−/− L-sel−/− animals fed a normal diet (10.45%±2.58% vs 1.87%±0.37%; p<0.05). In contrast, after 12 weeks of HCD, there was no difference in atheroma formation between ApoE−/− L-sel−/− and ApoE−/− mice. Serum cholesterol levels remained unchanged by L-sel deletion. Atherosclerotic plaques did not exhibit any differences in cellular composition assessed by immunohistochemistry for CD68, CD3, CD4, and CD8 in ApoE−/− L-sel−/− as compared to ApoE−/− mice. Leukocyte rolling on lesions in the aorta was similar in ApoE−/− L-sel−/− and ApoE−/− animals. ApoE−/− L-sel−/− mice exhibited reduced size and cellularity of peripheral lymph nodes, increased size of spleen, and increased number of peripheral lymphocytes as compared to ApoE−/− controls. These data indicate that L-sel does not promote atherosclerotic lesion formation and suggest that it rather protects from early atherosclerosis

Contribution of endothelial injury and inflammation in early phase to vein graft failure: the causal factors impact on the development of intimal hyperplasia in murine models.

OBJECTIVES: Autologous veins are preferred conduits in by-pass surgery. However, long-term results are hampered by limited patency due to intimal hyperplasia. Although mechanisms involved in development of intimal hyperplasia have been established, the role of inflammatory processes is still unclear. Here, we studied leukocyte recruitment and intimal hyperplasia in inferior vena cava grafts transferred to abdominal aorta in mice. METHODS AND RESULTS: Several microscopic techniques were used to study endothelium denudation and regeneration and leukocyte recruitment on endothelium. Scanning electron microscopy demonstrated denudation of vein graft endothelium 7 days post-transfer and complete endothelial regeneration by 28 days. Examination of vein grafts transferred to mice transgenic for green fluorescent protein under Tie2 promoter in endothelial cells showed regeneration of graft endothelium from the adjacent aorta. Intravital microscopy revealed recruitment of leukocytes in vein grafts at 7 days in wild type mice, which had tapered off by 28 days. At 28 and 63 days there was significant development of intimal hyperplasia. In contrast; no injury, leukocyte recruitment nor intimal hyperplasia occurred in arterial grafts. Leukocyte recruitment was reduced in vein grafts in mice deficient in E- and P-selectin. In parallel, intimal hyperplasia was reduced in vein grafts in mice deficient in E- and P-selectin and in wild type mice receiving P-selectin/E-selectin function-blocking antibodies. CONCLUSION: The results show that early phase endothelial injury and inflammation are crucial processes in intimal hyperplasia in murine vein grafts. The data implicate endothelial selectins as targets for intervention of vein graft disease

IH in vascular grafts.

<p>Morphologic assessment of vascular grafts stained with Hematoxylin-eosin. (A) Upper panel demonstrates VGs in WT mice and in EP^−/− mice at different time point. Bar graphs represent IH area in AGs and VGs from WT and VGs from EP^−/− mice. (B) Lower panel demonstrates VGs in WT mice without or with treatment with combination of function-blockage antibodies against P- and E-selectin or rat IgG₁ λ isotype control in cuff-assisted vein grafting technique. Bar graph represents IH area. Error bars represent mean±SEM. *p<0.05. Scale bar = 100 µm. Arrows indicated IH of VGs.</p

Endothelial structure in vascular grafts.

<p>Scanning electron microscopy images of endothelium in AGs and VGs (magnification 1K to 1.9K, scale bar = 50 µm). Images demonstrate endothelium in native aorta, native IVC (A), VGs and AGs (B) at different time point in WT mice. Bar graph shows endothelial coverage area change in AGs and VGs (C). Arrow = red blood cells, Triangle arrow = leukocytes, ** = denudated endothelium. Error bars represent mean±SEM. *p<0.05.</p

Leukocyte rolling and adhesion in native vessels and vascular grafts.

<p>Intravital microscopic data on leukocyte recruitment. Bar graphs represent (A) leukocyte rolling and adhesion in native vessels in WT and EP^−/−mice, (B) leukocyte adhesion in AGs and VGs in WT and EP^−/− mice and (C) leukocyte rolling in AGs and VGs in WT mice at different time points. Bar graphs represent number of leukocytes that were visible in a 100×100 µm square during 30 seconds. Error bars represent mean±SEM. *p<0.05.</p

Distinct Infiltration of Neutrophils in Lesion Shoulders in ApoE−/− Mice

Inflammation and activation of immune cells are key mechanisms in the development of atherosclerosis. Previous data indicate important roles for monocytes and T-lymphocytes in lesions. However, recent data suggest that neutrophils also may be of importance in atherogenesis. Here, we use apolipoprotein E (ApoE)-deficient mice with fluorescent neutrophils and monocytes (ApoE−/−/LysEGFP/EGFP mice) to specifically study neutrophil presence and recruitment in atherosclerotic lesions. We show by flow cytometry and confocal microscopy that neutrophils make up for 1.8% of CD45+ leukocytes in the aortic wall of ApoE−/−/LysEGFP/EGFP mice and that their contribution relative to monocyte/macrophages within lesions is approximately 1:3. However, neutrophils accumulate at sites of monocyte high density, preferentially in shoulder regions of lesions, and may even outnumber monocyte/macrophages in these areas. Furthermore, intravital microscopy established that a majority of leukocytes interacting with endothelium on lesion shoulders are neutrophils, suggesting a significant recruitment of these cells to plaque. These data demonstrate neutrophilic granulocytes as a major cellular component of atherosclerotic lesions in ApoE−/− mice and call for further study on the roles of these cells in atherogenesis