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: Abstract 1537 Poster Board I-560 Sickle cell disease (SCD) is characterized by intravascular hemolysis generating cell-free hemoglobin at concentrations that exceed the scavenging capabilities of heme-binding plasma proteins. Heme is a major source of oxidative stress, which is widely known to increase vascular permeability and cause tissue edema. Barrier disruptive effects of oxidative stress can however be counterbalanced by adaptive antioxidant defenses. Hitherto, the identity of specific antioxidant enzymes and cognate mechanisms protecting individual organs from oxidative stress in SCD remains poorly defined. In this study, microarray analysis was performed using non-toxic concentrations of hemin to analyze mechanisms of antioxidant defense by the endothelium. Multiple candidate antioxidant enzymes were identified each differentially elevated in several major organs in anemic SCD mice compared to non-anemic heterozygote and hemizygote littermates. Remarkably, none of the antioxidants was elevated in the brain of sickle mice. Moreover, the antioxidant phenotype in the kidney, spleen and liver of sickle mice were predominantly acute while the sickle lung was characterized by a predominantly chronic antioxidant phenotype. This latter finding was confirmed in SCD patients with chronic lung disease. Antioxidant enzyme activity was significantly higher in the lungs of adult sickle mice aged 3-6 months than in younger mice aged 4-6 weeks (p=0.004). However, this enhanced antioxidant activity declined significantly in middle-age mice 11-13 months old (p=0.005). Vascular permeability assessed by extravsasation of Evans blue dye was normal in the brain of sickle mice of all ages in agreement with our data indicating absence of oxidative stress in this organ. On the contrary, vascular permeability in the lung, kidney and heart of adult sickle mice was abnormally high. This abnormality deteriorated significantly exclusively in the lung (p= 0.04) but not in the heart or kidney in middle-age mice. Increased lung permeability in middle-age sickle mice was confirmed by overt tissue edema determined by lung wet/dry weight ratios. Our study has shown for the first time that the antioxidant response to the systemic chronic hemolysis of SCD is organ-specific. Furthermore, we have identified decline of antioxidant reserve concomitant with tissue edema as potential age-dependant risk factors for fatal lung complications in SCD. Finally, our data provide a framework to develop targeted antioxidant therapies to preserve organ function in SCD. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1538 Poster Board I-561 Circulating plasma hemoglobin contributes to major vasculopathies including pulmonary hypertension in patients who have sickle cell disease (SCD). There is an emerging concept that such vasculopathies are relatively mild because of activation of several cytoprotective pathways in SCD. The biochemical profile of plasma and the transcriptome of peripheral blood cells in patients who have SCD offer indirect support for this concept. Indeed, heme oxygenase-1 (HO-1), an acute phase enzyme that degrades heme into intermediates and byproducts with vasculoprotective properties is markedly elevated in mononuclear leukocytes of patients who have SCD. Nonetheless, the scope of the cytoprotective mechanisms of the lung and other organs impacted directly by sickle vasculopathies remain poorly appreciated. We previously identified an array of cytoprotective enzymes in lung endothelial cells chronically exposed to non-toxic concentrations of hemin in vitro. In this study, we examined the expression of NAD(P)H oxidase and candidate cytoprotective enzymes in two models of transgenic mice with SCD, and examined HO-1 expression in sickle chronic lung disease. Although NAD(P)H oxidase catalyzes reactive oxygen species generation by heme and is responsible for increased adhesion of leukocytes to the endothelium in SCD mice, there was no elevation of any of its subunits (gp91Phox and p22Phox, p47Phox, p67Phox and p40 Phox) in sickle mice lungs compared to hemizygote control mice lungs. Quantitative RT-PCR analysis revealed unexpectedly no difference in HO-1 mRNA level in sickle and non-sickle control lungs. On the contrary, analysis of the same tissues showed significantly higher NAD(P)H quinone oxidoreductase-1 (NQO1) mRNA level in both Berkeley and Townes knock-in sickle mice compared to controls (p

Description: Emerging evidence indicate the concentration of circulating heme in patients who have sickle cell disease (SCD) is sufficient to contribute to vasculopathies such as pulmonary hypertension. Despite this significance, the identity of specific molecules and pathways responsible for heme-induced pulmonary complications in SCD remains poorly understood. This study was conceived with the idea that whole-genome expression profiling offered a rigorous approach to identify specific molecules involved in both pathological and vasculoprotective mechanisms of sickle chronic lung disease. Human pulmonary artery endothelial cells (PAECs) and pulmonary microvascular endothelial cells (PMVECs) were exposed to a concentration (0–25 mM) range of hemin for seven days and total RNA isolated and interrogated with Affymetrix U133 plus 2.0 Genechips. Microarray data from 24 independent experiments was analyzed using the Bioconductor in the R framework and GeneSpring to generate two unique lists of genes regulated by hemin in PAECs and PMVECs. Multiple genes widely known to be influenced by heme including heme oxygenase-1 (HO-1), ferritin, transferrin receptor, and delta-aminolevulinate synthase were altered as expected thus validating the experimental, statistical and bio-informatics approaches used in this study. The microarray expression data was validated for 26 transcripts in PAECs and 14 transcripts in PMVECs using low-density array multiplex quantitative RT-PCR. Our findings indicate that the cytoprotective response to hemin is markedly more enhanced in PMVECs than in PAECs as determined by the number and the magnitude of differential expression of genes in the oxidative stress response and glutathione metabolism pathways. This finding is supported by a higher basal expression of nuclear factor erythroid 2-related factor 2 (Nrf2) in PMVECs than in PAECs. Heterogeneity of these anti-oxidant phenotypes was confirmed at the protein level in a concentration-dependent manner for multiple enzymes regulated by Nrf2 including NAD(P)H:quinone oxidoreductase 1 (NQO1), which is critical for preventing participation of quinones in redox cycling and generation of reactive oxygen species. Moreover, while NQO1 expression increased 3-fold in PMVECs exposed to hemin for seven days no significant increase in NQO1 expression occurred following shorter periods of hemin treatment. The clinicopathological and pathophysiological relevance of these findings were investigated in post-mortem lung tissues of cases of sickle chronic lung disease and in transgenic mice with SCD. Compared to normal human lung tissues, NQO1 expression increased 3-to 5-fold in the endothelium of small caliber size vessels as well as in both large and small airway epithelium in severe cases of sickle chronic lung disease with extensive pulmonary vascular remodeling. On the contrary, no significant difference in NQO1 expression was detected in the lungs of wild-type mice and transgenic hemizygous or homozygous SCD mice lacking pulmonary vascular remodeling. We conclude that different pulmonary segments and specific anti-oxidant molecules respond uniquely to heme. Unraveling this complex heterogeneity is critical to improving understanding of the pathogenesis and treatment of lung complications in SCD. Induction of NQO1 or its upstream regulator Nrf2 offers a potentially attractive strategy to augment the anti-oxidant phenotype of PAECs to slow the progression of pulmonary vasculopathies in SCD.

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.

Description: Regulation of hematopoietic progenitor cell lineage-commitment, proliferation and differentiation by cell-cell adhesion mechanisms is poorly understood. Activated leukocyte cell adhesion molecule (ALCAM) is a member of the immunoglobulin super family. It is expressed by human hematopoietic stem cells, bone marrow stromal cells, endothelial cells and osteoblasts. Monoclonal anti-ALCAM antibodies inhibit myeloid but not erythroid colony formation, which suggest a lineage-specific role for ALCAM in hematopoiesis. To explore this hypothesis, ALCAM mRNA and protein expression was quantified in human hematopoietic cell lines of myeloid, lymphoid, erythroid, and megakaryocytic lineages by real-time quantitative PCR and western blot analyses. No ALCAM transcripts were detected in K562 and MEG-01 cells, the level of ALCAM mRNA was 2-fold more abundant in HL-60 and THP-1 cells than in U937 and Jurkat cells. This expression pattern was confirmed at the protein level as none of the megakaryocyte-erythroid progenitor cell lines (K562, MEG-01 and HEL) expressed ALCAM. On the contrary, ALCAM was abundantly expressed in THP-1 and HL-60 cells and moderately in U937 and Jurkat cells. GATA-1 was abundantly expressed in megakaryocyte-erythroid progenitor cell lines but not in any of the myeloid cell lines. Thus, there is an inverse relationship between expression of ALCAM and GATA-1 in hematopoietic cells. To test the hypothesis that GATA-1 is involved in silencing ALCAM gene expression, multiple ALCAM-promoter luciferase constructs were studied. A negative regulatory region was identified in the ALCAM promoter containing an inverted GATA-1 cis element at −850 upstream of the translational start site. GATA-1 occupied this canonical element in vivo as determined by chromatin immunoprecipitation experiments. A two-base pair mutation of the −850 GATA-1 cis element increased ALCAM promoter activity 3-fold in K562 and MEG-01 cells, providing direct evidence of GATA-1’s negative regulatory role in ALCAM promoter activity. To test the hypothesis that ALCAM silencing is essential for megakaryocyte-erythroid progenitor cell biology, stable lines of K562 cells were established forcibly expressing ALCAM-GFP or a control GFP. Live cell imaging demonstrated recruitment of ALCAM to sites of cell-cell adhesion in ALCAM-GFP-K562 cells, whereas GFP remained distributed in the cell cytosol in control cells. ALCAM-GFP-K562 cells formed markedly more clusters consisting of significantly more cells than control GFP-K562 cells. Finally, the number of ALCAM-GFP-K562 cells at log-phase growth was significantly higher than GFP-K562 cells over the same time period. Our findings demonstrate for the first time lineage-specific silencing of the cell adhesion molecule ALCAM in megakaryocyte-erythroid progenitor cells, mediated at least in part by GATA-1. That ectopic expression of ALCAM increased proliferation of K562 cells suggests that GATA-1-mediated silencing of ALCAM is essential in slowing down expansion of megakaryocyte-erythroid progenitor cells. Indeed, preliminary studies show an excessive number of erythroid and megakaryocytic cells in the adult spleen of ALCAM-null mice. This model is being used in ongoing studies to confirm our findings in vivo.