Endothelial basement membrane laminin 511 is essential for shear stress response.

Shear detection and mechanotransduction by arterial endothelium requires junctional complexes containing PECAM-1 and VE-cadherin, as well as firm anchorage to the underlying basement membrane. While considerable information is available for junctional complexes in these processes, gained largely from in vitro studies, little is known about the contribution of the endothelial basement membrane. Using resistance artery explants, we show that the integral endothelial basement membrane component, laminin 511 (laminin α5), is central to shear detection and mechanotransduction and its elimination at this site results in ablation of dilation in response to increased shear stress. Loss of endothelial laminin 511 correlates with reduced cortical stiffness of arterial endothelium in vivo, smaller integrin ß1-positive/vinculin-positive focal adhesions, and reduced junctional association of actin-myosin II In vitro assays reveal that ß1 integrin-mediated interaction with laminin 511 results in high strengths of adhesion, which promotes p120 catenin association with VE-cadherin, stabilizing it at cell junctions and increasing cell-cell adhesion strength. This highlights the importance of endothelial laminin 511 in shear response in the physiologically relevant context of resistance arteries.

Signals of the Neuropilin-1-MET Axis and Cues of Mechanical Force Exertion Converge to Elicit Inflammatory Activation in Coherent Endothelial Cells.

The neuropilin-1 (NRP1)-MET signaling axis regulates the motility of individual endothelial cells (ECs). It is unknown how this signaling pathway affects the endothelial barrier in coherent ECs forming a tight monolayer. We hypothesized that it is involved both in modulation of the endothelial barrier and in EC activation. To investigate the role of NRP1-MET signaling in inflammatory processes (e.g., systemic inflammatory response syndrome [SIRS] or snakebite-induced SIRS-like conditions), we employed the C-type lectin-related protein rhodocetin-αß (RCαß) as a specific trigger of this signal axis in ECs in vitro. In coherent HUVECs, RCαß reinforced the actin cytoskeleton and increased cell stiffness, thus favoring vascular endothelial cadherin-mediated transmission of intercellular forces. Increased cell stiffness was associated with enhanced activation of RhoA and nuclear translocation of NF-κB. Simultaneously, RCαß-triggered signaling via the NRP1-MET axis increased EC monolayer permeability, induced transcription of proinflammatory genes such as ICAM-1 and, consequently, leukocyte tethering. The RCαß-induced transcriptome differed from that induced by hepatocyte growth factor, although in both cases the same tyrosine kinase, MET, was involved. This was due to RCαß-mediated recruitment of the MET coreceptor NRP1 and additional Rho-mediated activation of the actomyosin system. RCαß induced similar transcriptional and cellular changes if external shear forces were applied. These data highlight the modulatory role of NRP1 as MET coreceptor, and they explain how some snake venoms induce SIRS-like conditions. Additionally, this study demonstrates that inflammatory activation of coherent ECs is triggered by converging signals that are induced by NRP1-MET signaling and influenced by intercellular forces.

Heterotropic modulation of selectin affinity by allosteric antibodies affects leukocyte rolling.

Selectins are a family of adhesion receptors designed for efficient leukocyte tethering to the endothelium under shear. As a key property to resist premature bond disruption, selectin adhesiveness is enhanced by tensile forces that promote the conversion of a bent into an extended conformation of the N-terminal lectin and epidermal growth factor-like domains. Conformation-specific Abs have been invaluable in deciphering the activation mechanism of integrins, but similar reagents are not available for selectins. In this study, we show that the anti-human L-selectin mAbs DREG-55 and LAM1-5 but not DREG-56, DREG-200, or LAM1-1 heterotropically modulate adhesion presumably by stabilizing the extended receptor conformation. Force-free affinity assays, flow chamber, and microkinetic studies reveal a ligand-specific modulation of L-selectin affinity by DREG-55 mAb, resulting in a dramatic decrease of rolling velocity under flow. Furthermore, secondary tethering of polymorphonuclear cells was blocked by DREG-200 but significantly boosted by DREG-55 mAb. The results emphasize the need for a new classification for selectin Abs and introduce the new concept of heterotropic modulation of receptor function.

Extracellular matrix of secondary lymphoid organs impacts on B-cell fate and survival.

We describe a unique extracellular matrix (ECM) niche in the spleen, the marginal zone (MZ), characterized by the basement membrane glycoproteins, laminin α5 and agrin, that promotes formation of a specialized population of MZ B lymphocytes that respond rapidly to blood-borne antigens. Mice with reduced laminin α5 expression show reduced MZ B cells and increased numbers of newly formed (NF) transitional B cells that migrate from the bone marrow, without changes in other immune or stromal cell compartments. Transient integrin α6ß1-mediated interaction of NF B cells with laminin α5 in the MZ supports the MZ B-cell population, their long-term survival, and antibody response. Data suggest that the unique 3D structure and biochemical composition of the ECM of lymphoid organs impacts on immune cell fate.

Direct crosstalk between mast cell-TNF and TNFR1-expressing endothelia mediates local tissue inflammation.

Signaling through tumor necrosis factor receptor 1 (TNFR1) controls bacterial infections and the induction of inflammatory Th1 cell-mediated autoimmune diseases. By dissecting Th1 cell-mediated delayed-type hypersensitivity responses (DTHRs) into single steps, we localized a central defect to the missing TNFR1 expression by endothelial cells (ECs). Adoptive transfer and mast cell knockin experiments into Kit(W)/Kit(W-v), TNF(-/-), and TNFR1(-/-) mice showed that the signaling defect exclusively affects mast cell-EC interactions but not T cells or antigen-presenting cells. As a consequence, TNFR1(-/-) mice had strongly reduced mRNA and protein expression of P-selectin, E-selectin, ICAM-1, and VCAM-1 during DTHR elicitation. In consequence, intravital fluorescence microscopy revealed up to 80% reduction of leukocyte rolling and firm adhesion in TNFR1(-/-) mice. As substitution of TNF(-/-) mice with TNF-producing mast cells fully restored DTHR in these mice, signaling of mast cell-derived TNF through TNFR1-expressing ECs is essential for the recruitment of leukocytes into sites of inflammation.

Role of the extracellular matrix in lymphocyte migration.

The extracellular matrix (ECM) exists in various biochemical and structural forms that can act either as a barrier to migrating leukocytes, in the case of basement membranes, or provide a physical scaffold supporting or guiding migration (interstitial matrix). This review focuses on basement membranes and our current knowledge of the way that leukocytes transmigrate this protein barrier, with emphasis on T lymphocytes. Recent data suggest that the classical concept of cell-matrix adhesion requires revision with respect to leukocyte-ECM interactions. Whereas specific receptors may be required for leukocyte recognition of ECM molecules or three-dimensional structural domains, the role of adhesion in migration as perceived from the traditional studies of adherent cell-ECM interactions is less clear. Further, the indirect effects of ECM such as the binding and presentation of cytokines or chemotactic factors may more profoundly influence the directed migration of normally non-adherent leukocytes than the migration of adherent cells such as epithelial cells or fibroblasts. Proteases (in particular matrix metalloproteinases) released at sites of inflammation can selectively process ECM, cell surface molecules or soluble factors, which may result in the release of bioactive fragments that can function as chemoattractants for different leukocyte subsets or may modulate the activity/function of resident mesenchymal and immune cells. Current findings suggest that different leukocyte types employ different mechanisms to migrate across or through the ECM; this might be determined by the composition and organization of the ECM itself.

Endothelial Basement Membrane Laminins as an Environmental Cue in Monocyte Differentiation to Macrophages.

Monocyte differentiation to macrophages is triggered by migration across the endothelial barrier, which is constituted by both endothelial cells and their underlying basement membrane. We address here the role of the endothelial basement membrane laminins (laminins 411 and 511) in this monocyte to macrophage switch. Chimeric mice carrying CX3CR1-GFP bone marrow were employed to track CCL2-induced monocyte extravasation in a cremaster muscle model using intravital microscopy, revealing faster extravasation in mice lacking endothelial laminin 511 (Tek-cre::Lama5-/- ) and slower extravasation in mice lacking laminin 411 (Lama4-/- ). CX3CR1-GFPlow extravasating monocytes were found to have a higher motility at laminin 511 low sites and to preferentially exit vessels at these sites. However, in vitro experiments reveal that this is not due to effects of laminin 511 on monocyte migration mode nor on the tightness of the endothelial barrier. Rather, using an intestinal macrophage replenishment model and in vitro differentiation studies, we demonstrate that laminin 511, together with the attached endothelium, promote monocyte differentiation to macrophages. Macrophage differentiation is associated with a change in integrin profile, permitting differentiating macrophages to distinguish between laminin 511 high and low areas and to preferentially migrate across laminin 511 low sites. These studies highlight the endothelial basement membrane as a critical site for monocyte differentiation to macrophages, which may be relevant to the differentiation of other cells at vascular niches.

The role of basement membrane laminins in vascular function.

The extracellular matrix is an integral component of the vasculature, contributing to both developmental processes and structural and functional homeostasis. We describe here the types of extracellular matrices that occur in different blood vessel types, ranging from capillaries to veins, venules and arteries, and focus on the endothelial basement membranes and the laminin family of proteins. We summarize data on the molecular composition of endothelial basement membranes, the structure and in vivo expression patterns of the main endothelial laminin isoforms (laminins 411 and 511) and their, to date, deciphered functions in the vasculature. A significant portion of the review focuses on postcapillary venules and leukocyte extravasation and how the endothelial laminins affect adhesion and migration of different leukocyte types, but also how laminins affect endothelial barrier function by modulating expression and localization of endothelial cell-cell junction molecules, and how these effects differ in CNS versus non-CNS tissues. Comparisons are made to small artery dilation in response to shear flow, which has been shown to be dependent on endothelial laminins and junctional complexes. The data discussed support a central role for basement membrane laminins in different aspects of micro- and macro-vessel endothelial function, but also reveal that many open questions remain, including the contribution of perivascular cells which are either embedded or in direct contact with the endothelial cell basement membrane laminins.

Inhibition of hyaluronan export attenuates cell migration and metastasis of human melanoma.

When secreted from malignant cells, hyaluronan facilitates tumor invasion and metastasis, as inhibition of its export by zaprinast inhibited metastasis formation in mice. However, the precise steps of the metastatic cascade, which were influenced by zaprinast, have not been identified as yet. Here we analyzed the cell biological effects of the inhibitor on three human melanoma cell lines that differed in their hyaluronan production and their metastatic capability when xenografted into SCID mice. We measured the influence of zaprinast on cellular hyaluronan export, surface coat formation, proliferation, random migration, colony formation in soft agar, adhesion, and transepithelial resistance. Concentrations of zaprinast not affecting cell proliferation, adhesion and transepithelial resistance, nevertheless reduced hyaluronan export by 50%, surface coat formation, random migration, and colony formation in soft agar. These results indicate that hyaluronan enhances metastasis formation primarily in those steps of the metastatic cascade, which involves tumor cell migration.

The regulation of immune cell trafficking by the extracellular matrix.

The extracellular matrix (ECM) comes in different structural forms and biochemical compositions, which determine both its biophysical properties and its ability to convey specific signals to immune cells encountering or navigating through it. Traditionally, the role of the individual ECM molecules on cell migration has been investigated independent of considerations such as the tension/mechanical strength constituted by the ECM. However, more recently, this aspect has attracted considerable attention and data suggest that rigidity and molecular signals derived from the ECM define the mode of cell migration. We here review the different types of ECM encountered by migrating immune cells in vivo, as well as current information on how both molecular components of the ECM and their supramolecular structure can impact on modes of immune cell migration.

Focal MMP-2 and MMP-9 activity at the blood-brain barrier promotes chemokine-induced leukocyte migration.

Although chemokines are sufficient for chemotaxis of various cells, increasing evidence exists for their fine-tuning by selective proteolytic processing. Using a model of immune cell chemotaxis into the CNS (experimental autoimmune encephalomyelitis [EAE]) that permits precise localization of immigrating leukocytes at the blood-brain barrier, we show that, whereas chemokines are required for leukocyte migration into the CNS, additional MMP-2/9 activities specifically at the border of the CNS parenchyma strongly enhance this transmigration process. Cytokines derived from infiltrating leukocytes regulate MMP-2/9 activity at the parenchymal border, which in turn promotes astrocyte secretion of chemokines and differentially modulates the activity of different chemokines at the CNS border, thereby promoting leukocyte migration out of the cuff. Hence, cytokines, chemokines, and cytokine-induced MMP-2/9 activity specifically at the inflammatory border collectively act to accelerate leukocyte chemotaxis across the parenchymal border.

Endothelial basement membrane laminin alpha5 selectively inhibits T lymphocyte extravasation into the brain.

Specific inhibition of the entry of encephalitogenic T lymphocytes into the central nervous system in multiple sclerosis would provide a means of inhibiting disease without compromising innate immune responses. We show here that targeting lymphocyte interactions with endothelial basement membrane laminins provides such a possibility. In mouse experimental autoimmune encephalomyelitis, T lymphocyte extravasation correlates with sites expressing laminin alpha4 and small amounts of laminin alpha5. In mice lacking laminin alpha4, laminin alpha5 is ubiquitously expressed along the vascular tree, resulting in marked and selective reduction of T lymphocyte infiltration into the brain and reduced disease susceptibility and severity. Vessel phenotype and immune response were not affected in these mice. Rather, laminin alpha5 directly inhibited integrin alpha(6)beta(1)-mediated migration of T lymphocytes through laminin alpha4. The data indicate that T lymphocytes use mechanisms distinct from other immune cells to penetrate the endothelial basement membrane barrier, permitting specific targeting of this immune cell population.

The extracellular matrix of the spleen as a potential organizer of immune cell compartments.

Until recently little information was available on the molecular details of the extracellular matrix (ECM) of secondary lymphoid tissues. There is now growing evidence that these ECMs are unique structures, combining characteristics of basement membranes and interstitial or fibrillar matrices, resulting in scaffolds that are strong and highly flexible and, in certain secondary lymphoid compartments, also forming conduit networks for rapid fluid transport. This review will address the structural characteristics of the ECM of the murine spleen and its potential role as an organizer of immune cell compartments, with reference to the lymph node where relevant.

Arterial remodeling and plasma volume expansion in caveolin-1-deficient mice.

Caveolin-1 (Cav-1) is essential for the morphology of membrane caveolae and exerts a negative influence on a number of signaling systems, including nitric oxide (NO) production and activity of the MAP kinase cascade. In the vascular system, ablation of caveolin-1 may thus be expected to cause arterial dilatation and increased vessel wall mass (remodeling). This was tested in Cav-1 knockout (KO) mice by a detailed morphometric and functional analysis of mesenteric resistance arteries, shown to lack caveolae. Quantitative morphometry revealed increased media thickness and media-to-lumen ratio in KO. Pressure-induced myogenic tone and flow-induced dilatation were decreased in KO arteries, but both were increased toward wild-type (WT) levels following NO synthase (NOS) inhibition. Isometric force recordings following NOS inhibition showed rightward shifts of passive and active length-force relationships in KO, and the force response to alpha(1)-adrenergic stimulation was increased. In contrast, media thickness and force response of the aorta were unaltered in KO vs. WT, whereas lumen diameter was increased. Mean arterial blood pressure during isoflurane anesthesia was not different in KO vs. WT, but greater fluctuation in blood pressure over time was noted. Following NOS inhibition, fluctuations disappeared and pressure increased twice as much in KO (38 +/- 6%) compared with WT (17 +/- 3%). Tracer-dilution experiments showed increased plasma volume in KO. We conclude that NO affects blood pressure more in Cav-1 KO than in WT mice and that restructuring of resistance vessels and an increased responsiveness to adrenergic stimulation compensate for a decreased tone in Cav-1 KO mice.

Modulation of granulocyte-endothelium interactions by antileukoproteinase: inhibition of anti-type II collagen antibody-induced leukocyte attachment to the synovial endothelium.

Antileukoproteinase (ALP) is a physiological inhibitor of granulocytic serine proteases that has been shown to have anti-inflammatory properties in addition to its antiproteolytic activity. On the basis of its potential to block anti-collagen type II (CII) antibody-induced arthritis (CAIA) and to suppress the conformational activation of beta2-integrins in leukocytes, the present study was undertaken to investigate its interference with leukocyte adherence to cytokine-activated endothelium. The potential of recombinant ALP to block the interactions of leukocytes with the endothelial lining was concomitantly investigated in vitro and in vivo. Thus, intravital fluorescence microscopic imaging of leukocyte rolling and firm adhesion to postcapillary venules were performed in the knee joints of DBA1/J mice after intravenous injection of anti-CII mAbs. An IL-1beta-activated endothelial layer formed by a murine glomerular cell line (glEND.2) was used to assay the interaction with human leukocytes in vitro. Electromobility shift and luciferase reporter gene assays permitted the analysis of cytokine-induced activation of the NF-kappaB pathway. Fluorescence-activated cell sorting was applied to determine endothelial E-selectin expression. Leukocyte rolling and firm adhesion to the synovial endothelium in an early response to the anti-CII antibody transfer were significantly decreased in ALP-pretreated mice. Concomitantly, ALP suppressed the IL-1beta-induced NF-kappaB activation and the upregulation of E-selectin expression in glEND.2 cells in vitro. These findings support the notion that the newly uncovered properties of ALP to interfere with cytokine signalling and upregulation of adhesion molecules in endothelial cells are likely to contribute to the therapeutic potential of ALP in immune-complex-induced tissue injury.

Expression and function of laminins in the embryonic and mature vasculature.

Endothelial cells of the blood and lymphatic vasculature are polarized cells with luminal surfaces specialized to interact with inflammatory cells upon the appropriate stimulation; they contain specialized transcellular transport systems, and their basal surfaces are attached to an extracellular basement membrane. In adult tissues the basement membrane forms a continuous sleeve around the endothelial tubes, and the interaction of endothelial cells with basement membrane components plays an important role in the maintenance of vessel wall integrity. During development, the basement membrane of endothelium provides distinct spatial and molecular information that influences endothelial cell proliferation, migration, and differentiation/maturation. Microvascular endothelium matures into phenotypically distinct types: continuous, fenestrated, and discontinuous, which also differ in their permeability properties. Development of these morphological and physiological differences is thought to be controlled by both soluble factors in the organ or tissue environment and by cell-cell and cell-matrix interactions. Basement membranes of endothelium, like those of other tissues, are composed of laminins, type IV collagens, heparan sulfate proteoglycans, and nidogens. However, isoforms of all four classes of molecules exist, which combine to form structurally and functionally distinct basement membranes. The endothelial cell basement membranes have been shown to be unique with respect to their laminin isoform composition. Laminins are a family of glycoprotein heterotrimers composed of an alpha, beta, and gamma chain. To date, 5alpha, 4beta, and 3gamma laminin chains have been identified that can combine to form 15 different isoforms. The laminin alpha-chains are considered to be the functionally important portion of the heterotrimers, as they exhibit tissue-specific distribution patterns and contain the major cell interaction sites. Vascular endothelium expresses only two laminin isoforms, and their expression varies depending on the developmental stage, vessel type, and the activation state of the endothelium. Laminin 8 (composed of laminin alpha4, beta1, and gamma1 chains) is expressed by all endothelial cells regardless of their stage of development, and its expression is strongly upregulated by cytokines and growth factors that play a role in inflammatory events. Laminin 10 (composed of laminin alpha5, beta1, and gamma1 chains) is detectable primarily in endothelial cell basement membranes of capillaries and venules commencing 3-4 wk after birth. In contrast to laminin 8, endothelial cell expression of laminin 10 is upregulated only by strong proinflammatory signals and, in addition, angiostatic agents such as progesterone. Other extracellular matrix molecules, such as BM40 (also known as SPARC/osteonectin), thrombospondins 1 and 2, fibronectin, nidogens 1 and 2, and collagen types VIII, XV, and XVIII, are also differentially expressed by endothelium, varying with the endothelium type and/or pathophysiological state. The data argue for a dynamic endothelial cell extracellular matrix that presents different molecular information depending on the type of endothelium and/or physiological situation. This review outlines the unique structural and functional features of vascular basement membranes, with focus on the endothelium and the laminin family of glycoproteins.

Perivascular cells expressing annexin A5 define a novel mesenchymal stem cell-like population with the capacity to differentiate into multiple mesenchymal lineages.

The annexin A5 gene (Anxa5) was recently found to be expressed in the developing and adult vascular system as well as the skeletal system. In this paper, the expression of an Anxa5-lacZ fusion gene was used to define the onset of expression in the vasculature and to characterize these Anxa5-lacZ-expressing vasculature-associated cells. After blastocyst implantation, Anxa5-lacZ-positive cells were first detected in extra-embryonic tissues and in angioblast progenitors forming the primary vascular plexus. Later, expression is highly restricted to perivascular cells in most blood vessels resembling pericytes or vascular smooth muscle cells. Viable Anxa5-lacZ+ perivascular cells were isolated from embryos as well as adult brain meninges by specific staining with fluorescent X-gal substrates and cell-sorting. These purified lacZ+ cells specifically express known markers of pericytes, but also markers characteristic for stem cell populations. In vitro and in vivo differentiation experiments show that this cell pool expresses early markers of chondrogenesis, is capable of forming a calcified matrix and differentiates into adipocytes. Hence, Anxa5 expression in perivascular cells from mouse defines a novel population of cells with a distinct developmental potential.

A small molecule alpha 4 beta 1 antagonist prevents development of murine Lyme arthritis without affecting protective immunity.

After infection with Borrelia burgdorferi, humans and mice under certain conditions develop arthritis. Initiation of inflammation is dependent on the migration of innate immune cells to the site of infection, controlled by interactions of a variety of adhesion molecules. In this study, we used the newly synthesized compound S18407, which is a prodrug of the active drug S16197, to analyze the functional importance of alpha4beta1-dependent cell adhesion for the development of arthritis and for the antibacterial immune response. S16197 is shown to interfere specifically with the binding of alpha4beta(1 integrin to its ligands VCAM-1 and fibronectin in vitro. Treatment of B. burgdorferi-infected C3H/HeJ mice with the alpha4beta1 antagonist significantly ameliorated the outcome of clinical arthritis and the influx of neutrophilic granulocytes into ankle joints. Furthermore, local mRNA up-regulation of the proinflammatory mediators IL-1, IL-6, and cyclooxygenase-2 was largely abolished. Neither the synthesis of spirochete-specific Igs nor the development of a Th1-dominated immune response was altered by the treatment. Importantly, the drug also did not interfere with Ab-mediated control of spirochete load in the tissues. These findings demonstrate that the pathogenesis, but not the protective immune response, in Lyme arthritis is dependent on the alpha4beta1-mediated influx of inflammatory cells. The onset of inflammation can be successfully targeted by treatment with S18407.