Leukocyte-endothelial interactions in arteries in vivo : a new concept in the study of physiology and disease in large blood vessels

Abstract

Pathophysiological events in large blood vessels contribute to immense morbidity and mortality in our society. A major clinical problem is a disease known as atherosclerosis. It involves a slow inflammatory deposition of lipid within the walls of large arteries that with time will weaken the arterial wall. Because leukocytes are key players in the development of the atherosclerotic disease, the mechanisms responsible for leukocyte invasion in atherogenesis have been the focus of intensive research. However, so far no experimental models have been able to explore the dynamic events underlying leukocyte recruitment in large arteries in vivo. Therefore, data on the dynamic events responsible for leukocyte accumulation in atherosclerosis have for long been sought for.

This thesis describes the first direct observations of the dynamic events underlying leukocyte recruitment in large arteries and atherosclerosis in vivo. By use of intravital fluorescence microscopy on large arteries in the mouse, dynamic interactions between leukocytes and endothelial cells were observed and characterized. In arteries of healthy mice, interactions between leukocytes and endothelial cells are virtually absent. However, upon stimulation with cytokines TN17oc and IL-1 beta OBS 1 P, as well as in unstimulated atherosclerotic mice, interactions are abundant. Adhesive contacts between leukocytes and endothelium in arteries are transient compared with venules in inflammation, a fact that restricts the ability for rolling leukocytes to adhere firmly and invade the arterial wall. Nonetheless, upon cytokine-stimulation, leukocyte arrest and transmigration caused accumulation of leukocytes in the subendothelial space. Leukocyte arrest was prominent in regions of low or fluctuating shear and the distribution of leukocyte arrest after cytokine treatment showed a striking anatomical correlation with lesion formation in atherosclerotic mice. In areas of low shear, dynamic interactions between leukocytes and endothelium were more stable than in regions exposed to high shear inasmuch as rolling velocity of leukocytes was lower and the time leukocytes were in contact with the endothelium was longer in low shear areas. These stable interactions likely contributed to the enhanced arrest of leukocytes in regions exposed to low shear stress.

The molecular mechanisms underlying interactions between leukocytes and endothelium were investigated by intravenous injections of saturating doses of monoclonal antibodies. P-selectin was found to be the principal receptor for leukocyte rolling in atherosclerosis as well as after cytokine treatment. E-selectin stabilized rolling interactions in both situations. Interestingly, no role in leukocyte recruitment in atherosclerosis was detected for the alpha4 integrin/VCAM-1 pathway. However, because leukocytes interacting with arterial endothelium were mainly neutrophils, subclasses of leukocytes other than neutrophils may still in part use the alpha4 integrin/VCAM-1 pathway to invade the arterial wall.

In conjunction with the studies on large arteries, the mechanisms by which leukocytes initiate contact with vessel wall in various situations of leukocyte recruitment were investigated. Capture of leukocytes from the free flow was, contrary to previous published data in the literature, found to be of importance in the recruitment of leukocytes in many situations. Capture mediated by L-selectin dependent interactions between rolling and freely flowing leukocytes (secondary capture) increased recruitment of leukocytes to the rolling cell pool. This was evident in all arterial vessels and in large, but not small, venules. Secondary capture mediated part of the capture of leukocytes from the free flow in atherosclerosis. This provides the first evidence for a role of L-selectin in leukocyte accumulation in atherogenesis.

In summary, the data presented in this thesis provide important insights into the dynamics of leukocyte recruitment in large vessels in vivo. The results display and explain some of the characteristics of leukocyte accumulation in arteries and atherosclerosis, but also contribute to a better understanding of mechanisms involved in leukocyte accumulation in a wide range of pathological processes. What is more, the novel techniques provide new prospects for the study of large vessel physiology and disease.