Switch in Laminin ß2 to Laminin ß1 Isoforms During Aging Controls Endothelial Cell Functions-Brief Report.

OBJECTIVE: Endothelial cells play important roles in tissue homeostasis and vascularization, a function that is impaired by aging. Here, we aim to decipher the role of the microenvironment underlying the impairment of endothelial cell functions by aging. APPROACH AND RESULTS: RNA sequencing of isolated cardiac endothelial cells derived from young and 18-month-old mouse hearts revealed that aging affects the endothelial expression of genes encoding extracellular matrix proteins, specifically the laminin ß1 (Lamb1) and laminin ß2 (Lamb2) chains. Whereas Lamb1 was upregulated, Lamb2 was decreased in endothelial cells in old mice compared with young controls. A similar change in expression patterns was observed after induction of acute myocardial infarction. Mimicking aging and injury conditions by plating endothelial cells on laminin ß1-containing laminin 411 matrix impaired endothelial cell adhesion, migration, and tube formation and augmented endothelial-to-mesenchymal transition and endothelial detachment compared with laminin 421, which contains the laminin ß2 chain. Because laminins can signal via integrin receptors, we determined the activation of ITGB1 (integrin ß1). Laminin 421 coating induced a higher activation of ITGB1 compared with laminin 411. siRNA-mediated silencing of ITGB1 reduced laminin ß2-dependent adhesion, suggesting that laminin ß2 more efficiently activates ITGB1. CONCLUSIONS: Mimicking age-related modulation of laminin ß1 versus ß2 chain expression changes the functional properties and phenotype of endothelial cells. The dysregulation of the extracellular matrix during vascular aging may contribute to age-associated impairment of organ function and fibrosis.

Intrathecal antibody distribution in the rat brain: surface diffusion, perivascular transport and osmotic enhancement of delivery.

KEY POINTS: It is unclear precisely how macromolecules (e.g. endogenous proteins and exogenous immunotherapeutics) access brain tissue from the cerebrospinal fluid (CSF). We show that transport at the brain-CSF interface involves a balance between Fickian diffusion in the extracellular spaces at the brain surface and convective transport in perivascular spaces of cerebral blood vessels. Intrathecally-infused antibodies exhibited size-dependent access to the perivascular spaces and tunica media basement membranes of leptomeningeal arteries. Perivascular access and distribution of full-length IgG could be enhanced by intrathecal co-infusion of hyperosmolar mannitol. Pores or stomata present on CSF-facing leptomeningeal cells ensheathing blood vessels in the subarachnoid space may provide unique entry sites into the perivascular spaces from the CSF. These results illuminate new mechanisms likely to govern antibody trafficking at the brain-CSF interface with relevance for immune surveillance in the healthy brain and insights into the distribution of therapeutic antibodies. ABSTRACT: The precise mechanisms governing the central distribution of macromolecules from the cerebrospinal fluid (CSF) to the brain and spinal cord remain poorly understood, despite their importance for physiological processes such as antibody trafficking for central immune surveillance, as well as several ongoing intrathecal clinical trials. In the present study, we clarify how IgG and smaller single-domain antibodies (sdAb) distribute throughout the whole brain in a size-dependent manner after intrathecal infusion in rats using ex vivo fluorescence and in vivo three-dimensional magnetic resonance imaging. Antibody distribution was characterized by diffusion at the brain surface and widespread distribution to deep brain regions along the perivascular spaces of all vessel types, with sdAb accessing a four- to seven-fold greater brain area than IgG. Perivascular transport involved blood vessels of all caliber and putative smooth muscle and astroglial basement membrane compartments. Perivascular access to smooth muscle basement membrane compartments also exhibited size-dependence. Electron microscopy was used to show stomata on leptomeningeal coverings of blood vessels in the subarachnoid space as potential access points allowing substances in the CSF to enter the perivascular space. Osmolyte co-infusion significantly enhanced perivascular access of the larger antibody from the CSF, with intrathecal 0.75 m mannitol increasing the number of perivascular profiles per slice area accessed by IgG by ∼50%. The results of the present study reveal potential distribution mechanisms for endogenous IgG, which is one of the most abundant proteins in the CSF, as well as provide new insights with respect to understanding and improving the drug delivery of macromolecules to the central nervous system via the intrathecal route.

Blockade of MCAM/CD146 impedes CNS infiltration of T cells over the choroid plexus.

BACKGROUND: Very late antigen 4 (VLA-4; integrin α4ß1) is critical for transmigration of T helper (TH) 1 cells into the central nervous system (CNS) under inflammatory conditions such as multiple sclerosis (MS). We have previously shown that VLA-4 and melanoma cell adhesion molecule (MCAM) are important for trans-endothelial migration of human TH17 cells in vitro and here investigate their contribution to pathogenic CNS inflammation. METHODS: Antibody blockade of VLA-4 and MCAM is assessed in murine models of CNS inflammation in conjunction with conditional ablation of α4-integrin expression in T cells. Effects of VLA-4 and MCAM blockade on lymphocyte migration are further investigated in the human system via in vitro T cell transmigration assays. RESULTS: Compared to the broad effects of VLA-4 blockade on encephalitogenic T cell migration over endothelial barriers, MCAM blockade impeded encephalitogenic T cell migration in murine models of MS that especially depend on CNS migration across the choroid plexus (CP). In transgenic mice lacking T cell α4-integrin expression (CD4::Itga4-/-), MCAM blockade delayed disease onset. Migration of MCAM-expressing T cells through the CP into the CNS was restricted, where laminin 411 (composed of α4, ß1, γ1 chains), the proposed major ligand of MCAM, is detected in the endothelial basement membranes of murine CP tissue. This finding was translated to the human system; blockade of MCAM with a therapeutic antibody reduced in vitro transmigration of MCAM-expressing T cells across a human fibroblast-derived extracellular matrix layer and a brain-derived endothelial monolayer, both expressing laminin α4. Laminin α4 was further detected in situ in CP endothelial-basement membranes in MS patients' brain tissue. CONCLUSIONS: Our findings suggest that MCAM-laminin 411 interactions facilitate trans-endothelial migration of MCAM-expressing T cells into the CNS, which seems to be highly relevant to migration via the CP and to potential future clinical applications in neuroinflammatory disorders.

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.

The neurovascular unit as a selective barrier to polymorphonuclear granulocyte (PMN) infiltration into the brain after ischemic injury.

The migration of polymorphonuclear granulocytes (PMN) into the brain parenchyma and release of their abundant proteases are considered the main causes of neuronal cell death and reperfusion injury following ischemia. Yet, therapies targeting PMN egress have been largely ineffective. To address this discrepancy we investigated the temporo-spatial localization of PMNs early after transient ischemia in a murine transient middle cerebral artery occlusion (tMCAO) model and human stroke specimens. Using specific markers that distinguish PMN (Ly6G) from monocytes/macrophages (Ly6C) and that define the cellular and basement membrane boundaries of the neurovascular unit (NVU), histology and confocal microscopy revealed that virtually no PMNs entered the infarcted CNS parenchyma. Regardless of tMCAO duration, PMNs were mainly restricted to luminal surfaces or perivascular spaces of cerebral vessels. Vascular PMN accumulation showed no spatial correlation with increased vessel permeability, enhanced expression of endothelial cell adhesion molecules, platelet aggregation or release of neutrophil extracellular traps. Live cell imaging studies confirmed that oxygen and glucose deprivation followed by reoxygenation fail to induce PMN migration across a brain endothelial monolayer under flow conditions in vitro. The absence of PMN infiltration in infarcted brain tissues was corroborated in 25 human stroke specimens collected at early time points after infarction. Our observations identify the NVU rather than the brain parenchyma as the site of PMN action after CNS ischemia and suggest reappraisal of targets for therapies to reduce reperfusion injury after stroke.

The extracellular matrix of lymph node reticular fibers modulates follicle border interactions and germinal center formation.

Germinal center (GC) formation and antibody production in lymph node follicles require coordinated interactions between B-cells, T-cells and dendritic cells (DCs), orchestrated by the extracellular matrix-rich reticular fiber (RF) network. We describe a unique laminin 523-containing RF network around and between follicles that associates with PDGFrecßhighCCL19lowgp38low fibroblastic reticular cells (FRC). In the absence of FRC expression of laminin α5 (pdgfrb-cre:Lama5fl/fl), pre-Tfh-cells, B-cells and DCs are displaced from follicle borders, correlating with fewer Tfh-cells and GC B-cells. Total DCs are not altered in pdgfrb-cre:Lama5fl/fl mice, but cDC2s, which localize to laminin α5 in RFs at follicle borders, are reduced. In addition, PDGFrecßhighCCL19lowgp38low FRCs show lower Ch25h expression, required for 7α,25-dihydroxycholesterol synthesis that attracts pre-Tfh-cells, B-cells and DCs to follicle borders. We propose that RF basement membrane components represent a type of tissue memory that guides the localization and differentiation of both specialized FRC and DC populations, required for normal lymph node function.

Endothelial basement membrane laminins - new players in mouse and human myoendothelial junctions and shear stress communication.

Basement membranes (BMs) are critical but frequently ignored components of the vascular system. Using high-resolution confocal imaging of whole-mount-stained mesenteric arteries, we identify integrins, vinculin, focal adhesion kinase (FAK) and several BM proteins including laminins as novel components of myoendothelial junctions (MEJs), anatomical microdomains that are emerging as regulators of cross-talk between endothelium and smooth muscle cells (SMCs). Electron microscopy revealed multiple layers of the endothelial BM that surround endothelial projections into the smooth muscle layer as structural characteristics of MEJs. The shear-responsive calcium channel TRPV4 is broadly distributed in endothelial cells and occurs in a proportion of MEJs where it localizes to the tips of the endothelial projections that are in contact with the underlying SMCs. In mice lacking the major endothelial laminin isoform, laminin 411 (Lama4-/-), which we have previously shown over-dilate in response to shear and exhibit a compensatory laminin 511 upregulation, localization of TRPV4 at the endothelial-SMC interface in MEJs was increased. Endothelial laminins do not affect TRPV4 expression, rather in vitro electrophysiology studies using human umbilical cord arterial endothelial cells revealed enhanced TRPV4 signalling upon culturing on an RGD-motif containing domain of laminin 511. Hence, integrin-mediated interactions with laminin 511 in MEJ structures unique to resistance arteries modulate TRPV4 localization at the endothelial-smooth muscle interface in MEJs and signalling over this shear-response molecule.

Loss of astrocyte polarization upon transient focal brain ischemia as a possible mechanism to counteract early edema formation.

Brain edema is the main cause of death from brain infarction. The polarized expression of the water channel protein aquaporin-4 (AQP4) on astroglial endfeet surrounding brain microvessels suggests a role in brain water balance. Loss of astrocyte foot process anchoring to the basement membrane (BM) accompanied by the loss of polarized localization of AQP4 to astrocytic endfeet has been shown to be associated with vasogenic/extracellular edema in neuroinflammation. Here, we asked if loss of astrocyte polarity is also observed in cytotoxic/intracellular edema following focal brain ischemia after transient middle cerebral artery occlusion (tMCAO). Upon mild focal brain ischemia, we observed diminished immunostaining for the BM components laminin α4, laminin α2, and the proteoglycan agrin, in the core of the lesion, but not in BMs in the surrounding penumbra. Staining for the astrocyte endfoot anchorage protein ß-dystroglycan (DG) was dramatically reduced in both the lesion core and the penumbra, and AQP4 and Kir4.1 showed a loss of polarized localization to astrocytic endfeet. Interestingly, we observed that mice deficient for agrin expression in the brain lack polarized localization of ß-DG and AQP4 at astrocytic endfeet and do not develop early cytotoxic/intracellular edema following tMCAO. Taken together, these data indicate that the binding of DG to agrin embedded in the subjacent BM promotes polarized localization of AQP4 to astrocyte endfeet. Reduced DG protein levels and redistribution of AQP4 as observed upon tMCAO might therefore counteract early edema formation and reflect a beneficial mechanism operating in the brain to minimize damage upon ischemia.

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.

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.

Molecular characterization of perivascular drainage pathways in the murine brain.

Perivascular compartments surrounding central nervous system (CNS) vessels have been proposed to serve key roles in facilitating cerebrospinal fluid flow into the brain, CNS waste transfer, and immune cell trafficking. Traditionally, these compartments were identified by electron microscopy with limited molecular characterization. Using cellular markers and knowledge on cellular sources of basement membrane laminins, we here describe molecularly distinct compartments surrounding different vessel types and provide a comprehensive characterization of the arachnoid and pial compartments and their connection to CNS vessels and perivascular pathways. We show that differential expression of plectin, E-cadherin and laminins α1, α2, and α5 distinguishes pial and arachnoid layers at the brain surface, while endothelial and smooth muscle laminins α4 and α5 and smooth muscle actin differentiate between arterioles and venules. Tracer studies reveal that interconnected perivascular compartments exist from arterioles through to veins, potentially providing a route for fluid flow as well as the transport of large and small molecules.

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.

GPI-specific phospholipase D mRNA expression in tumor cells of different malignancy.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is a highly specific enzyme whose only known substrate is the GPI anchor of cell surface proteins. GPI-PLD measurements, however, are technically difficult since the enzyme is expressed at low levels in cells and tissues, and serum contains large amounts of inactive, latent GPI-PLD interfering with protein-based assays. We have therefore developed a semi-quantitative RT-PCR method to measure mRNA expression of all known GPI-PLD isoforms in cells and tissues. In human ovarian cancer cell lines, GPI-PLD mRNA expression correlated with GPI-PLD enzyme activity and with the shedding of the GPI-anchored tumor and prognostic markers, urokinase receptor and CA125, from the cell surface. This supports a potential role for this enzyme in the generation of circulating prognostic markers in malignant tumors. Similarly, in human epithelial cells of the skin, GPI-PLD mRNA expression increased with tumor progression. Whereas normal keratinocytes did not express significant amounts of GPI-PLD mRNA, expression was dramatically induced by serum in immortalized HaCaT keratinocytes and constitutively high and independent of serum in tumorigenic A431 epidermoid carcinoma cells. In addition, GPI-PLD expression was significantly increased in highly malignant. H-ras-transfected murine bladder carcinoma cells as compared to the low malignant, non-transfected parental cells. The competitive RT-PCR described here represents the first quantitative assay specific for cellular GPI-PLD isoforms, and our in vitro analyses suggest that GPI-PLD expression might be associated with tumor malignancy.