Vascular occlusion is the major cause of morbidity and mortality in sickle cell disease but its mechanisms are poorly understood. We demonstrate by using intravital microscopy in mice expressing human sickle hemoglobin (SS) that SS red blood cells (RBCs) bind to adherent leukocytes in inflamed venules, producing vasoocclusion of cremasteric venules. SS mice deficient in P-and E-selectins, which display defective leukocyte recruitment to the vessel wall, are protected from vasoocclusion. These data uncover a previously unsuspected paradigm for the pathogenesis of sickle cell vasoocclusion in which adherent leukocytes play a direct role and suggest that drugs targeting SS RBC-leukocyte or leukocyte-endothelial interactions may prevent or treat the vascular complications of this debilitating disease. Sickle cell disease, the first molecular disease identified in humans, is among the most common inherited hematological disorder in the United States. It results from a single amino acid substitution in the ␤-chain of hemoglobin (Hb␤ S ). Hb␤ S polymerizes on deoxygenation, producing less deformable sickle red blood cells (SS RBCs) that can obstruct blood vessels (1). More than 20 years ago, it was demonstrated that SS RBCs displayed increased adherence to endothelial cells (2, 3). Subsequent studies recognized the importance of several adhesion pathways in these interactions (4-10), and revealed that young (lowdensity) SS RBC were more adherent to the endothelium than dense (often irreversibly sickled) SS RBCs (11-13). Collectively, these observations led to the current multistep model for sickle cell vasoocclusion in which light-density cells first adhere in postcapillary venules and secondary trapping of dense cells produces vascular obstruction and ischemia.To test this model in vivo, we applied the technique of intravital microscopy, which allows direct visualization of the microvasculature, to mice genetically engineered to exclusively express human (h) Hb␤ S . Such mice have circulating irreversibly sickled cells and manifest several of the cardinal features of sickle cell disease, including anemia, reticulocytosis, and organ damage (14, 15). To overcome the limitations imposed by the high perinatal mortality and low breeding efficiency, we generated sickle cell mice by transplantation of sickle cell bone marrow precursors into lethally irradiated normal adult recipients. Unexpectedly, we observed that SS RBCs interacted primarily with adherent leukocytes in postcapillary and collecting venules rather than the endothelial surface, and that these interactions led to vascular occlusion. These data indicate a direct role for leukocytes in the pathogenesis of sickle cell vasoocclusion and provide a potential explanation for the clinical association between leukocytosis and poor prognosis in sickle cell disease. Bone Marrow Transplantation. Femoral bone marrow nucleated cells from WT C57BL͞6, SA, and SS mice were harvested and injected (1.5 ϫ 10 6 cells͞recipient) via the lateral tail vein of lethally irradiated (1,2...

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Intussusceptive angiogenesis is a morphogenetic process that forms new blood vessels by the division of a single blood vessel into two lumens. Here, we show that this process of intraluminal division participates in the inflammation-induced neovascularization associated with chemically induced murine colitis. In studies of both acute (4-7 days) and chronic (28-31 days) colitis, intravital microscopy of intravascular tracers demonstrated a twofold reduction in blood flow velocity. In the acute colitis model, the decreased velocity was associated with marked dilatation of the mucosal plexus. In contrast, chronic inflammation was associated with normal caliber vessels and duplication (and triplication) of the quasi-polygonal mucosal plexus. Scanning electron microscopy (SEM) of intravascular corrosion casts suggested that pillar formation and septation, previously linked to the morphogenetic process of intussusceptive angiogenesis, were present within days of the onset of inflammation. Four weeks after the onset of inflammation, SEM of vascular corrosion casts demonstrated replication of the mucosal plexus without significant evidence of sprouting angiogenesis. These data suggest that mucosal capillaries have comparable aggregate cross-sectional area in acute and chronic colitis; however, there is a significant increase in functional capillary density in chronic colitis. We conclude that intussusceptive angiogenesis is a fundamental mechanism of microvascular adaptation to prolonged inflammation. Anat Rec, 293:849-857, 2010. Key words: angiogenesis; corrosion casting; intravital microscopy; microcirculation; scanning electron microscopyThe microvascular adaptations to local inflammation depend upon the structure of the preexisting capillary reserve. In skeletal muscle, the increased metabolic demand of exercise or inflammation results in a rapid threefold increase in the number of perfused capillaries (Chambers and Zweifach, 1942;Clark and Clark, 1943; Abbreviations used: DSS = dextran sodium sulfate; SEM = scanning electron microscopy; TEM = transmission electron microscopy; TNBS = 2,4,6-trinitrobenzenesulfonic acid.