VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis.

CD99 is a long-known leukocyte antigen that does not belong to any of the known protein families. It was recently found on endothelial cells, where it mediates transendothelial migration of human monocytes and lymphocyte recruitment into inflamed skin in the mouse. Here, we show that CD99L2, a recently cloned, widely expressed antigen of unknown function with moderate sequence homology to CD99, is expressed on mouse leukocytes and endothelial cells. Using antibodies, we found that CD99L2 and CD99 are involved in transendothelial migration of neutrophils in vitro and in the recruitment of neutrophils into inflamed peritoneum. Intravital and electron microscopy of cremaster venules revealed that blocking CD99L2 inhibited leukocyte transmigration through the vessel wall (diapedesis) at the level of the perivascular basement membrane. We were surprised to find that, in contrast to CD99, CD99L2 was not relevant for the extravasation of lymphocytes into inflamed tissue. Although each protein promoted cell aggregation of transfected cells, endothelial CD99 and CD99L2 participated in neutrophil extravasation independent of these proteins on neutrophils. Our results establish CD99L2 as a new endothelial surface protein involved in neutrophil extravasation. In addition, this is the first evidence for a role of CD99 and CD99L2 in the process of leukocyte diapedesis in vivo. IntroductionLeukocytes are recruited into inflamed tissue via endothelial adhesion molecules and chemoattractants. [1][2][3][4] Upon docking to the endothelial surface, which is mediated mainly via selectins and leukocyte integrins, leukocytes transmigrate through the vessel wall, a process called diapedesis. 5,6 Leukocytes have been reported to traverse the endothelial cell layer on either a transcellular or a paracellular junctional route. 7,8 Almost all known endothelial membrane and adhesion molecules that participate in the diapedesis process are located at endothelial cell contacts and are therefore likely to participate in the junctional migration of leukocytes.Vascular/endothelial cadherin (VE-cadherin) represents a barrier on this route, because adhesion-blocking antibodies against VE-cadherin accelerate recruitment of leukocytes into inflamed tissue. 9 In contrast to VE-cadherin, all other endothelial cell contact proteins assist in the diapedesis process. All of these proteins except one are members of the immunoglobulin supergene family (Ig-SF), such as platelet-endothelial cell adhesion molecule (PE-CAM)-1, 10,11 members of the junctional adhesion molecule (JAM) family, 12-14 endothelial cell-selective adhesion molecule (ESAM), 15 intercellular adhesion molecule (ICAM)-2 16 and polio virus receptor (PVR), 17 a member of the nectin family. It is not yet known how they function in detail and it will be a challenging goal for the future to elucidate potential cascades and the interplay of these endothelial cell contact proteins during leukocyte diapedesis.One of the most recently identified proteins participating in leukocyte diapedesis is CD...

Actin-binding proteins are essential for linear and branched actin filament dynamics that control shape change, cell migration, and cell junction remodeling in vascular endothelium (endothelial cells [ECs]). The epithelial protein lost in neoplasm (EPLIN) is an actin-binding protein, expressed as EPLIN-a and EPLIN-b by alternative promoters; however, the isoform-specific functions are not yet understood. Aortic compared to cava vein ECs and shear stress-exposed cultured ECs express increased EPLIN-b levels that stabilize stress fibers. In contrast, EPLIN-a expression is increased in growing and migrating ECs, is targeted to membrane protrusions, and terminates their growth via interaction with the Arp2/3 complex. The data indicate that EPLIN-a controls protrusion dynamics while EPLIN-b has an actin filament stabilizing role, which is consistent with FRAP analyses demonstrating a lower EPLIN-b turnover rate compared to EPLIN-a. Together, EPLIN isoforms differentially control actin dynamics in ECs, essential in shear stress responses, cell migration, and barrier function.

CD99 is a crucial regulator of the transmigration (diapedesis) of leukocytes through the blood vessel wall. Here, we report that CD99 acts at 2 different steps in the extravasation process. In agreement with previous antibody-blocking experiments, we found that CD99 gene inactivation caused neutrophil accumulation between venular endothelial cells and the basement membrane in the inflamed cremaster. Unexpectedly, we additionally found that leukocyte attachment to the luminal surface of the venular endothelium was impaired in the absence of CD99. Intravital video microscopy revealed that CD99 supported rapid chemokine-induced leukocyte arrest. Inhibition of leukocyte attachment and extravasation were both solely due to the absence of CD99 on endothelial cells, whereas CD99 on leukocytes was irrelevant. Therefore, we searched for heterophilic ligands of endothelial CD99 on neutrophils. We found that endothelial cells bind to the paired immunoglobulinlike receptors (PILRs) in a strictly CD99-dependent way. In addition, endothelial CD99 was coprecipitated with PILRs from neutrophils that adhered to endothelial cells. Furthermore, soluble CD99 carrying a transferable biotin tag could transfer this tag covalently to PILR when incubated with intact neutrophils. Binding of neutrophils under flow to a surface coated with P-selectin fragment crystallizable (Fc) and intercellular adhesion molecule 1 (ICAM-1) Fc became more shear resistant if CD99 Fc was coimmobilized. This increased shear resistance was lost if neutrophils were preincubated with anti-PILR antibodies. We concluded that endothelial CD99 promotes leukocyte attachment to endothelium in inflamed vessels by a heterophilic ligand. In addition, CD99 binds to PILRs on neutrophils, an interaction that leads to increased shear resistance of the neutrophil attachment to ICAM-1.

CD99-like 2 (CD99L2) is a membrane protein with moderate sequence homology to CD99, which initiates cell aggregation of transfected cells and that is strongly expressed on endothelial cells, neutrophils, and lymphocytes. We showed recently that Abs against CD99L2 inhibit neutrophil, but not T lymphocyte, recruitment into inflamed tissues. In this study, we have generated conditional gene–deficient mice for CD99L2 and show by analyzing them in various inflammation models several results. First, gene ablation of CD99L2 impairs neutrophil recruitment into inflamed cremaster and peritoneum. Second, despite the strong expression of CD99L2 on peripheral neutrophils, only gene ablation on endothelial cells but not on myeloid cells affects neutrophil extravasation. Third, in contrast to our previous Ab-based results, recruitment of activated T cells into inflamed skin was impaired in mice lacking CD99L2 on endothelial cells. We conclude that CD99L2 is an essential endothelial Ag for leukocyte extravasation, which does not require homophilic interactions with CD99L2 on leukocytes.