ALBERT

Description: Migration of endothelial cells as a sheet in fully differentiated blood vessels is essential for reducing vascular permeability during wound healing. Indeed, loss of collective endothelial sheet migration contributes to increased vascular permeability in tumor angiogenesis and several vascular proliferative disorders. Despite this significance, mechanisms responsible for keeping migrating endothelial cells in a monolayer, sheet or tube are poorly understood. To unravel the basis for collective endothelial cell migration, we used time-lapse video microscopy to study early events of wound closure in confluent monolayers of primary microvascular endothelial cells in a live cell chamber. Immediately after wounding (0– 20 min), endothelial cells at the margin of the wound (marginal cells) retracted away from the wounded area and showed no visible lamellipodia extensions. The next phase of early wound healing (20 min – 6 hours) revealed extensive lamellipodia formation and migration of marginal cells into the wounded region. Remarkably, sub-marginal endothelial cells that were several microns away from the wound edge protruded lamellipodia that formed dynamic cell-cell contacts with the substratum of marginal cells at the wound edge. In several instances sub-marginal cells physically and coordinately pulled back endothelial cells at wound edge to maintain regularity of the endothelial sheet front. Cell-tracking measurements revealed autonomous and yet coordinated migration of marginal and sub-marginal endothelial cells culminating in net protrusion of the endothelial sheet into the wound. This study provides in real-time evidence of retraction of endothelial cells at the wound edge by several microns prior to the initiation of forward migration. In addition, we show for the first time that endothelial cells several microns away from the wound edge actively participate in sheet migration through the extention of lamellipodia into the substratum of cells at the wound edge. These findings highlight an important role in endothelial sheet migration for the Rho family of GTPases given their intimate control of cell retraction and lamelipodia extensions. Future studies will directly evaluate the influence of Rac, Cdc42 and RhoA in retraction of marginal cells and formation of lamellipodia by sub-marginal cells in endothelial sheet migration.

Description: Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a member of the immunoglobulin cell adhesion super family, which has been implicated in diverse physiological and pathophysiological events involving cell migration. Hitherto, ALCAM’s role in inflammation has not been determined. In this study, we show ALCAM is involved in controlling migration of mononuclear leukocytes across the pulmonary endothelium. We demonstrated that ALCAM is localized at intercellular junctions in pulmonary microvascular endothelial cells in vitro and in vivo. ALCAM co-localized with multiple adherens junction molecules including cadherins, catenins and Dlg, as determined by confocal microscopy, and these observations were confirmed by co-immunoprecipitation and co-distribution assays. Treatment of endothelial cultures with EGTA and cytochalasin D translocated ALCAM from intercellular junctions to the cytosol indicating a requirement for homotypic cadherin adhesion and an intact endothelial cytoskeleton for maintaining ALCAM at endothelial cell junctions. Collectively, these data supports the conclusion that ALCAM contributes to the adherens junction complex in endothelial cells. To determine ALCAM’s role in leukocyte-endothelial cell interactions, adult Sprague Dawley rats were intratracheally instilled with macrophage inflammatory protein-1, and this treatment caused acute expression of ALCAM exclusively in newly recruited mononuclear but not polymorphonuclear leukocytes in the alveolar airway. Given that no ALCAM reactivity was observed in peripheral blood leukocytes, we concluded ALCAM is activated as part of the phenotypic switch by mononuclear leukocytes transitioning from circulation to interstitial tissue compartments. To determine the physiological relevance of this finding we examined whether ALCAM was required for transendothelial migration using monocyte chemoattractant protein 1 (MCP-1). MCP-1 dose- and time-dependently increased the number of transmigrated THP-1 monocytes across pulmonary microvascular endothelial monolayers. Recombinant soluble ALCAM dose-dependently reduced the number of transmigrated THP-1 monocytes, whereas in control experiments recombinant soluble vascular endothelial cadherin had no effect on transmigration. This study shows for the first time that ALCAM is located at endothelial cell junctions where it is intimately involved in controlling the number of monocytes that pass through endothelial barriers. ALCAM may therefore play an essential role in the response to inflammation by enhancing recruitment of mononuclear leukocytes by inflamed tissues.

Description: Pulmonary vascular occlusion is a major cause of morbidity and mortality in sickle cell disease. Adhesion between sickle erythrocytes, leukocytes and the endothelium are intimately involved in pulmonary vascular occlusion, which contributes to the pathogenesis of the acute chest syndrome. Despite this significance, specific molecules and cognate mechanisms responsible for trapping sickle erythrocytes in the lung microcirculation are poorly understood. In this study, we show for the first time that products released by activated neutrophils act through the phosphatidyserine and phosphatidylserine receptor axis to promote retention of sickle erythrocytes in the lung microcirculation. We demonstrated that co-incubation with autologous activated neutrophils increased 2-fold the proportion of phosphatidylserine exposed sickle erythrocytes. This effect was abrogated by prior treatment of neutrophils with zileuton a pharmacological inhibitor of 5-lipoxygenase. We next determined that primary endothelial cells derived from the pulmonary vasculature expressed the stereospecific receptor for phosphatidylserine. Transcripts for phosphatidylserine receptor (PSR) were 2-fold more abundant in endothelial cells from the pulmonary microvasculature compared to those from the pulmonary artery. To determine the relevance of neutrophil activation in PSR expression, cultures of PMVECs were treated with supernatants of activated neutrophils from patients with sickle cell anemia, and this resulted in 2-fold increase in PSR transcripts. To unravel the pathophysiological relevance of these findings, isolated rat lungs were perfused with 51Cr labeled sickle erythrocytes pre-incubated with annexin V, and retention of sickle erythrocytes quantified by 51Cr radioactivity. The number of sickle erythrocytes trapped in isolated lungs was 2-fold lower compared to the value in control experiments performed with untreated sickle erythrocytes. In agreement with our previous findings activated neutrophils significantly increased sickle erythrocyte retention in the lung (P=0.0004), however cloaking of sickle erythrocytes with annexin V reduced retention by 3-fold. Collectively, these data show that activation of neutrophils increases expression of PSR and its cognate ligand on endothelial cells and sickle erythrocytes respectively, providing the molecular basis for an adhesion complex that increases retention of sickle erythrocytes in the pulmonary endothelium. The PSR-phosphatidylserine adhesion complex therefore offers a novel therapeutic target to reduce pulmonary vascular occlusive events in sickle cell disease.

Description: The vascular system is a complex network of conduit and microvascular vessels exposed to different microenvironments that imprints unique phenotypic traits on individual endothelial cell populations. Endothelial cells in fully differentiated blood vessels in adult tissues have a quiescent phenotype characterized by an increased resistance to proliferate, migrate or undergo apoptosis. The intrinsic capacity of endothelial cells to switch from a quiescent to angiogenic phenotype however plays an important role in wound healing, several vascular proliferative disorders and tumor angiogenesis. Endothelial cells revert to an angiogenic phenotype as isolated cells in culture nonetheless they are contact inhibited at confluence reflecting their in vivo phenotype in the endothelium. In this study, we used time-lapse video microscopy to study early events in endothelial sheet migration in confluent monolayers of primary endothelial cells derived from conduit (PAECs) and microvascular (PMVECs) blood vessels. Recordings were made in a live cell chamber and were restricted to six hours to minimize the effect of proliferation on sheet migration. PMVECs at the wound edge were significantly highly spread and squamous in appearance compared to PAECs, which had a distinctly more cuboidal morphology. Majority (〉95%) of PMVECs at the wound edge produced extensive lamellipodia based on morphology and dynamics that measured 25 μm ± 4 μm. By contrast, PAECs formed significantly smaller lamelipodia which extended by 8 μm ± 4 μm. On average PMVEC sheets migrated at a speed of 12.5 μm per hour covering a total distance of 75 μm ± 15 μm (n=6). Sheet migration rate in PMVECs was 3-fold faster than in PAECs (3.8 μm per hour), which covered a total distance of 23 μm ± 10 μm in the same time period. To unravel the molecular basis for this functional diversity, gene micro array analysis was performed. We identified unique transcriptional profiles for cell-cell adhesion molecules, integrins and disintegrin-metalloproteases each with a distinct role in collective cell migration. In particular, integrin alpha 7, which is a major regulator of lamellipodia formation was found to be 20-fold more abundant in PMVECs than in PAECs. This study provides molecular and functional evidence for heterogeneity of endothelial sheet migration. This central finding highlights variability in angiogenic plasticity in fully differentiated endothelial cells, which may have important ramifications for anti-angiogenesis therapy.