The importance of laminin at the blood-brain barrier

Abstract

The blood-brain barrier is a unique property of central nervous system blood vessels that protects sensitive central nervous system cells from potentially harmful blood components. The mechanistic basis of this barrier is found at multiple levels, including the adherens and tight junction proteins that tightly bind adjacent endothelial cells and the influence of neighboring pericytes, microglia, and astrocyte endfeet. In addition, extracellular matrix components of the vascular basement membrane play a critical role in establishing and maintaining blood-brain barrier integrity, not only by providing an adhesive substrate for blood-brain barrier cells to adhere to, but also by providing guidance cues that strongly influence vascular cell behavior. The extracellular matrix protein laminin is one of the most abundant components of the basement membrane, and several lines of evidence suggest that it plays a key role in directing blood-brain barrier behavior. In this review, we describe the basic structure of laminin and its receptors, the expression patterns of these molecules in central nervous system blood vessels and how they are altered in disease states, and most importantly, how genetic deletion of different laminin isoforms or their receptors reveals the contribution of these molecules to blood-brain barrier function and integrity. Finally, we discuss some of the important unanswered questions in the field and provide a “to-do” list of some of the critical outstanding experiments.

Introduction

Extracellular matrix (ECM) proteins play an essential role in the establishment and maintenance of tissue architecture (Adams and Watt, 1993). At the cellular level, ECM proteins control many aspects of cell behavior including cell survival, proliferation, migration, and terminal differentiation. One highly specialized ECM-rich structure is the basement membrane (BM), which is essentially a carpet of distinct ECM proteins to which cells tightly adhere (Paulsson, 1992; Jayadev and Sherwood, 2017; Khalilgharibi and Mao, 2021). The BM is absolutely essential, as it not only directs cell organization within the tissue, but it also strongly contributes to the maintenance of tissue integrity. It is interesting to note that all tissues subjected to high levels of physical shear stress, including the skin epidermis, intestinal epithelium, and blood vessels, attach very tightly to a BM. BMs are generated and established early in development and their importance is highlighted by the finding that the absence of key ECM components of BMs results in catastrophic failure of embryonic development (George et al., 1993; Smyth et al., 1999; Pöschl et al., 2004).

While all blood vessels are organized around a BM, blood vessels in different organs are highly specialized according to the local functional requirements of that organ. Vessel permeability is an important issue that differs between different organs, and generally speaking, vessels have high, medium, or low permeability as dictated by the organ. An example of high permeability is the kidney, where blood vessels in the glomerulus have a fenestrated, relatively leaky phenotype that facilitates the easy transport of blood plasma constituents from afferent arterioles into the renal tubule, where they are then selectively reabsorbed at distal stages of the nephron (Satchell and Braet, 2009). Blood vessels of the skin and muscle would be classified as having relatively moderate permeability, whereas those of the central nervous system (CNS) have very low permeability (Rubin and Staddon, 1999; Ballabh et al., 2004; Abbott et al., 2006). Because of these specialized properties, CNS blood vessels allow the transport of only those substrates that CNS cells need, such as glucose and amino acids, while protecting the delicate CNS neurons from any harmful agents present in the blood. These properties of the CNS blood vessels are known as the blood-brain barrier (BBB).

Blood vessels in the CNS are organized around a BM that consists mainly of four ECM proteins, including laminin, collagen IV, fibronectin, and heparan sulfate proteoglycan (Rubin and Staddon, 1999; Ballabh et al., 2004; Abbott et al., 2006). Laminin is of particular interest because it promotes terminal cell differentiation in many different cell types, including neurons, oligodendrocytes, and endothelial cells (Cohen et al., 1986; von der Mark and Ocalan, 1989; Cohen and Johnson, 1991; Buttery and ffrench-Constant, 1999; Wang and Milner, 2006). It is also noteworthy that the degree of vascular integrity of CNS blood vessels correlates with the level of laminin within the BM, such that under mild hypoxic conditions, vascular BM laminin levels are increased, which correlates with greater endothelial tight junction protein expression (Li et al., 2010b; Halder et al., 2018a, b, c), whereas in ischemic stroke, vascular BM laminin levels are decreased, correlating with attenuated BBB integrity (Hallmann et al., 1995; Zalewska et al., 2003; Gu et al., 2005; Li et al., 2012). Taken together, these findings imply that BM laminin positively contributes to BBB integrity and further suggest that improved knowledge of this relationship could lead to the generation of new therapeutic approaches aimed at enhancing BBB integrity in the treatment of various neurological diseases. In this review, we describe the different laminin isoforms and their receptors integrins and dystroglycan, how their expression is regulated under different conditions, and how their genetic deletion affects BBB integrity.

The Blood-Brain Barrier

Blood vessels in the CNS are unique from those in other organs in that they have low permeability and high electrical resistance. Because of these unique properties, the BBB facilitates the transport of substrates that it specifically needs, such as glucose and amino acids, but protects the sensitive neurons of the CNS from the potentially harmful effects of blood components (Huber et al., 2001; Pardridge, 2003; Ballabh et al., 2004; Abbott et al., 2006; Engelhardt et al., 2014; Chow and Gu, 2015). CNS blood vessels are composed of several different cell types, including endothelial cells, pericytes, and the endfeet of astrocytes (Figure 1). Recently, a number of studies have also implicated the role of microglia in contributing to BBB integrity (Lou et al., 2016; Taylor et al., 2018; Halder and Milner, 2019, 2020). At the cellular level, the BBB is found in CNS capillaries, which consist of tightly connected endothelial cells attached to a vascular BM containing various ECM proteins, including laminin (Wolburg and Lippoldt, 2002; Engelhardt et al., 2014; Chow and Gu, 2015). Supporting mural cells called pericytes, located within the vascular BM, maintain close physical contact with endothelial cells, to form an integral part of the capillaries (Armulik et al., 2005; Daneman et al., 2010; Sengillo et al., 2013). CNS blood vessels are also surrounded by an extensive network of endfeet derived from astrocytes within the brain parenchyma (Janzer and Raff, 1987; Abbott et al., 2006; del Zoppo and Milner, 2006; Wolburg-Buchholz et al., 2009). At the molecular level, three main types of mechanisms are responsible for the properties of the BBB. First, endothelial cells are tightly connected to their neighbors by tight junction and adherens proteins. Tight junction proteins include claudins, occludin, and junctional adhesion molecules, which attach to the cellular actin cytoskeleton via zonula occludens proteins such as zonula occludons 1 (Greene and Campbell, 2016; Irudayanathan et al., 2016; Berndt et al., 2019). By creating strong homophilic interactions, adherens proteins such as VE-cadherin further strengthen the connection between neighboring endothelial cells (Brown and Davis, 2002; Stamatovic et al., 2016). Second, both pericytes and astrocytes further enhance BBB integrity (Armulik et al., 2005; Daneman et al., 2010; Sengillo et al., 2013). Third, the vascular BM, to which endothelial cells and other BBB cells are tightly attached, both provides a physical barrier in itself and positively influences the barrier-inducing properties of resident BBB cells (del Zoppo and Milner, 2006).

Figure 1: Schematic representation of the BBB.Endothelial cells (pink) form the innermost layer of blood vessels and are strongly attached to the vascular basement membrane, which is a composite of different ECM proteins (violet), including laminin. Pericytes (red) and their processes closely attach to blood vessels and their cell bodies are located within the vascular basement membrane. Astrocytes within the CNS parenchyma (brown) extend endfeet which also cover the vascular basement membrane, thereby connecting blood vessels to neurons within the brain parenchyma. Microglial cells (green) are highly active motile cells that also form close connections with astrocytes and blood vessels. BBB: Blood-brain barrier; CNS: central nervous system; ECM: extracellular matrix. Created with BioRender.com.

The BBB is formed during embryonic development. In the mouse, it is largely complete around embryonic day 15 (Hallmann et al., 1995). It is noteworthy that BBB disruption is a major component of most common neurological diseases, including meningitis, ischemic stroke, multiple sclerosis, and CNS tumors (Gay and Esiri, 1991; Davies, 2002; Kirk et al., 2003; Bennett et al., 2010; Roberts et al., 2017). Further evidence suggests that it also deteriorates as part of the natural aging process and may be an important trigger in the pathogenesis of vascular dementia by inducing neuronal dysfunction and neurodegeneration (Farkas and Luiten, 2001; Farrall and Wardlaw, 2009; Brown, 2010; Brown and Thore, 2011; Senatorov et al., 2019). Notably, in many of these neurological disorders, BBB disruption is associated with alterations in ECM components of the vascular BM, including laminin (Sobel, 1998; Li et al., 2010a, 2012; Roberts et al., 2018), raising the possibility that aberrant expression of ECM proteins may directly lead to altered vascular cell behavior, resulting in reduced BBB integrity. In this regard, it is clear that a better understanding of the influence of ECM proteins, such as laminin, on BBB integrity may lead to novel therapeutic strategies aimed at enhancing BBB integrity and preventing the onset of neurological disease.

Laminins Are an Integral Component of the Vascular Basement Membrane

The vascular BM of CNS blood vessels contains abundant levels of the ECM proteins laminins, collagen IV, fibronectin and heparan sulphate proteoglycan (Mohan and Spiro, 1986; Timpl, 1989; Yurchenco et al., 2004; Yurchenco and Patton, 2009). The importance of the vascular BM is illustrated by the finding that genetic deletion of key ECM components results in the failure of vascular development (George et al., 1993; Smyth et al., 1999; Pöschl et al., 2004). While it was initially thought that the BM was simply an adhesive structure that vascular cells adhere to form vessel architecture, it’s now understood that the ECM components provide important instructional signals that regulate many aspects of vascular cell behavior, including survival, proliferation, migration and terminal differentiation (Timpl, 1989; Hynes, 1990). Cells bind to laminin and other ECM proteins via a family of cell surface receptors called integrins along with another laminin receptor called dystroglycan (Hynes, 1992, 1996; Hemler, 1999; Moore et al., 2002; Nodari et al., 2008).

Diversity of laminin isoforms

Laminin is one of the most abundant ECM proteins found in the vascular BM. Laminin proteins are heterotrimers consisting of α, β and γ subunits (Figure 2). To date, five different α, four β and three γ subunits have been identified, which combine in a variety of ways to generate up to 20 different laminin isoforms (Kang and Yao, 2022). Because of this diversity, it’s important to realize that laminin isn’t just one molecule but actually, a group of molecules, containing several different laminin isoforms (Edgar, 1989; Timpl and Brown, 1994; Yurchenco et al., 2004). Laminins are named according to their subunit make-up, so for example, laminin-111 is composed of the subunits α1, β1 and γ1 while laminin-411 is composed of the subunits α4, β1 and γ1.

Figure 2: The molecular structure of laminin.Laminin molecules organize as heterotrimers consisting of α (green), β (peach) and γ (orange) subunits of which five α, four β, and three γ have been identified to date. Laminins are named according to their subunit composition; for instance, laminin-111 contains the subunits α1, β1 and γ1. Created with BioRender.com.

Laminin isoforms display unique expression patterns within cerebral blood vessels

Within the adult CNS, laminin expression is found almost exclusively in blood vessels (Sixt et al., 2001; Milner and Campbell, 2002, 2006). The vascular BM laminin contains several different laminin isoforms, each contributed by different cell types. Analyzing laminin expression in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, the Sorokin lab showed that during leukocyte infiltration into the CNS, the two layers of the vascular BM (endothelial and parenchymal) are separated by infiltrating leukocytes which congregate in the space between the two layers, the so-called perivascular space (Sixt et al., 2001). By studying this system, Sixt et al. (2001) demonstrated that the inner (endothelial) layer of BM contains laminin-411 and -511, while the outer (parenchymal) layer of BM contains laminin-211 synthesized by astrocytes, and laminin-111 produced by leptomeningeal cells. From these observations, they deduced that endothelial cells synthesize laminin-411 and -511, and that while laminin-411 was widely expressed in the vascular BM of blood vessels, expression of the laminin-511 isoform was intermittent. Of note, in the EAE model, they also made the important observation that leukocytes tend to breach the BBB at sites expressing laminin-411 but not at laminin-511 expressing locations in the vessel wall, suggesting permissive and inhibitory influences for these laminin isoforms, respectively. It has since been shown that pericytes also express the laminin isoforms laminin-411 and -511 within the vascular BM (Gautam et al., 2020a). In keeping with the distinct regulatory functions of laminin-411 and -511 on regulating leukocyte infiltration, studies of development revealed that while laminin-411 expression can be detected on capillaries as early as embryonic day 11 (Iivanainen et al., 1997), laminin-511 appears at a much later time point, at postnatal 3–4 weeks (Patton et al., 1997; Sorokin et al., 1997). Interestingly, while the laminin isoforms -211, -411 and -511 are expressed on all types of CNS blood vessels (arterioles, capillaries and venules), laminin-111, which is produced by leptomeningeal cells, is expressed only on arterioles and venules, being absent from capillaries (Sixt et al., 2001). This is because laminin-111 is produced by leptomeningeal fibroblasts within meningeal blood vessels, which early in development are present only on the surface of the brain, but during neurodevelopment, these vessels grow into the brain to contribute to the arteriolar and venular circulation.

What do deletion studies tell us about the functions of different laminin isoforms?

To gain a better understanding of the roles that different laminin isoforms play in regulating vascular cell behavior and BBB integrity, several studies have used the Cre-Lox system to delete specific laminin subunits in specific vascular cell types. Because each laminin isoform is composed of the α, β and γ subunits, the phenotypic impact of deletion of any one subunit is largely a function of how widely that subunit is expressed amongst different cell types and tissues (Edgar, 1989; Timpl, 1989; Timpl and Brown, 1994; Yurchenco et al., 2004; Kang and Yao, 2022). For instance, the γ1 laminin subunit is common to 15 of the 20 laminin isoforms defined, so it comes as no surprise that mice globally deficient in this subunit display an embryonic lethal phenotype due to total loss of the ability to develop BMs and thus blood vessels (Smyth et al., 1999).

Global deletion of the laminin α2 subunit

Global deletion of the laminin α2 subunit results in growth retardation and a severe muscular dystrophic phenotype, and mice fail to survive beyond 5 weeks of age (Miyagoe et al., 1997). In the CNS, laminin-211 is a key component of the vascular BM and this laminin is contributed exclusively by astrocytes (Sixt et al., 2001). To gain insight into the potential influence of astrocyte laminin-211 on BBB integrity, laminin α2 knockout (KO) mice were examined prior to death at an earlier (3-week) timepoint. Using Evans Blue tracer, this revealed that α2 laminin KO mice displayed defective BBB integrity, which correlated with delayed vascular maturation, as assessed by prolonged MECA-32 expression (a marker of immature BBB), and reduced levels of VE-cadherin and the tight junction protein claudin-5 (Menezes et al., 2014). The CNS of these globally laminin α2-deficient mice also contained astrocytes with hypertrophic endfeet that expressed higher levels of glial fibrillary acidic protein and lacked polarized aquaporin-4 (AQP-4) channels. In addition, pericyte coverage of cerebral blood vessels was also significantly reduced (Menezes et al., 2014).

Global deletion of the laminin α4 subunit

Mice globally deficient in the α4 laminin subunit, an important component of one of the endothelial laminins (laminin-411) show disrupted vascular development, including vascular BM defects due to reduced synthesis of other ECM components, vessel dilation, aberrant angiogenesis and reduced vessel integrity (Thyboll et al., 2002). Interestingly, while a small number of laminin α4 mutant mice die early, the majority of mice survive, most likely due to compensatory increases in the other endothelial laminin, laminin-511 (Wu et al., 2009). Of note, because CNS blood vessels in wild type mice show continuous expression of the α4 laminin subunit throughout the vascular BM, but a patchy discontinuous expression of the α5 laminin subunit (Sixt et al., 2001), Wu et al. (2009) took advantage of the finding that in α4 subunit-deficient mice, the laminin α5 subunit becomes ubiquitously expressed throughout the BM, by showing that this continuous compensatory expression of α5 laminin correlates with a marked reduction in leukocyte infiltration across the BBB in the EAE model. This observation is consistent with previous findings from the Sorokin’s laboratory that leukocytes tend to breach the BBB at laminin-411 expressing sites but not where laminin-511 is present (Sixt et al., 2001). It should be emphasized that these findings represent a surprising paradox in that while loss of laminin-411 disrupts vessel stability and integrity early during development, in those mice that survive this period, the compensatory upregulation of laminin-511 in α4 subunit deficient mice results in enhanced BBB integrity, thereby reducing leukocyte extravasation in the EAE model.

Endothelial-specific laminin deletion

The global mouse KO of the laminin α5 subunit displays an embryonic lethal phenotype, making it technically challenging to study the role of this subunit in BBB regulation (Miner et al., 1998). Within cells of the BBB, both endothelial cells (Sixt et al., 2001) and pericytes (Gautam et al., 2016) have been shown to express this laminin subunit, making it important to determine the relative contributions of each cell type’s laminin α5 to BBB stability. To answer this question, the Yao lab recently generated transgenic mice in which laminin α5 was specifically deleted in endothelial cells (Gautam et al., 2019). Interestingly, under homeostatic control conditions, loss of endothelial laminin α5 had no obvious effect on BBB integrity, but in an intracerebral hemorrhage model, endothelial cell-laminin α5-KO mice showed enhanced BBB permeability, correlating with greater injury volume, leukocyte intravasation and gliosis (Gautam et al., 2019).

Pericyte-specific laminin deletion

To delete most pericyte laminins, the Yao lab generated transgenic mice by crossing platelet-derived growth factor receptor-β-Cre mice with floxed laminin γ1 subunit mice (Gautam et al., 2016). All the resulting transgenic progeny displayed a severe muscular dystrophy phenotype, resulting in 100% mortality by 4 months of age. At an earlier timepoint (2 weeks postnatal), 11% of this strain displayed hydrocephalus, correlating with BBB disruption, and reduced endothelial expression of the tight junction protein zonula occludons 1 and astrocyte expression of AQP-4, as well as significantly reduced pericyte coverage of blood vessels. Interestingly, when this strain was crossed from a C57BL6-FVB mixed background onto a pure C57BL6 background, the phenotype was much weaker and slower to develop such that by 4 months, BBB integrity was normal and hydrocephalus was absent, but later at 8 months, mild BBB disruption started to develop (Gautam et al., 2020a). When these mice were challenged in a collagenase-induced model of intracerebral hemorrhage, they manifest worse pathology compared to wild type mice, as shown by an increased size of hematoma, worse neurological function, reduced BBB integrity and increased neuronal death (Gautam et al., 2020b). In addition to deleting the majority of pericyte laminins by targeting the ubiquitous laminin γ1 subunit, the same group also knocked out pericyte laminin α5 specifically and found that as with the loss of endothelial laminin α5 this had no effect on BBB integrity under control conditions (Nirwane et al., 2019). However, when challenged in an ischemic stroke model, the PC-laminin α5 laminin KO strain showed attenuated BBB disruption relative to wild type controls, correlating with reduced leukocyte infiltration and infarct volume and improved neurological score (Nirwane et al., 2019). Taken together, these observations suggest that endothelial and pericyte α5 laminin may have opposing influences on BBB integrity, with endothelial laminin α5 enhancing vascular integrity, but pericyte laminin α5 having an antagonistic effect.

Astrocyte-specific laminin deletion

Building on the findings that the predominant astrocyte laminin is laminin-211 and that global α2 laminin KO die around the 4-week timepoint, Chen et al. (2013) deleted laminins specifically in astrocytes by crossing GFAP-Cre mice with floxed laminin γ1 mice, thereby deleting all astrocyte laminin expression. Interestingly, this astrocyte-specific γ1 laminin KO had a milder phenotype than global α2 laminin KO mice because they survived beyond the 4-week timepoint, although when 2–3 months old, they started to manifest spontaneous intracerebral hemorrhage, and by 6 months of age, more than 60% of mice had developed intracerebral hemorrhage. The majority of hemorrhages were located in small arterioles in deep regions of the brain, including the thalamus, hypothalamus and basal ganglia. In contrast to wild type mice, where astrocyte endfeet co-localized strongly with α-smooth muscle actin-positive smooth muscle cells in small arterioles, in the astrocyte-specific laminin γ1 KO strain, the number of smooth muscle cells covering the small arterioles was greatly attenuated. Building on these observations, the authors proposed a model in which the absence of astrocyte laminin reduces proliferation of vascular smooth muscle cells, culminating in the creation of thin and weakened arteriolar walls which eventually rupture (Chen et al., 2013).

In a separate study, the authors examined the impact on the loss of astrocyte laminins on BBB integrity. This showed that astrocyte-specific deletion of laminins results in BBB breakdown, and pericytes showed an altered phenotype (Yao et al., 2014). Based on these findings, they suggest that astrocyte laminin enhances BBB integrity by promoting pericyte differentiation into a BBB-stabilizing phenotype via α2β1 integrin signaling; however, loss of this laminin signaling promotes a contractile BBB-disruptive pericyte phenotype. Concordant with these observations, loss of astrocyte laminin also resulted in reduced astrocyte endfeet AQP-4 expression and reduced endothelial tight junction protein expression (Menezes et al., 2014).

In summary, the consensus of studies performed to date shows that deletion of laminin, either globally or specifically in any of the cells contributing to the BBB, negatively impacts BBB integrity (Chen et al., 2013; Menezes et al., 2014; Gautam et al., 2016, 2019, 2020b). In many of these studies, the reduced vascular integrity was associated with attenuated levels of endothelial tight junction proteins, reduced pericyte coverage, and disordered AQP-4 clustering on astrocyte endfeet. Together, this demonstrates that vascular BM laminin provides positive instructional cues promoting neurovascular integrity. For completeness, however, it should be mentioned that two studies hinted at potential negative effects of some laminins on BBB integrity. The first example is the pericyte-specific laminin α5 KO strain, which demonstrated less BBB breakdown in an ischemic stroke model (Nirwane et al., 2019). The second example is the global α4 laminin subunit KO, which appeared to show both positive and negative influences of laminin α4 on BBB stability because early in development these mice show reduced vascular integrity, but paradoxically, in global α4 laminin subunit KO mice that survive the postnatal period, compensatory upregulation of the laminin α5 subunit in blood vessels, resulted in inhibition of leukocyte infiltration in EAE (Wu et al., 2009).

One clinical situation that is of particular relevance is that of premature neonates, who are born with very fragile immature cerebral blood vessels, that are associated with a high incidence of intraventricular hemorrhage (Ballabh, 2010). Based on our own finding that the expression of laminins and their cognate receptors is upregulated throughout development, it seems likely that insufficient levels of these BBB stabilizing proteins may underpin the fragility of immature blood vessels in premature neonates (Milner and Campbell, 2006). Developmental studies have shown that while developing capillaries express laminin-411 as early as embryonic day 11 (Iivanainen et al., 1997), laminin-511 appears much later, at postnatal 3–4 weeks (Patton et al., 1997; Sorokin et al., 1997), suggesting that it is laminin-411 that confers this vital function during the early stages of cerebrovascular development. Supporting this concept, a previous study showed that blood vessels within the germinal matrix, an area containing a high density of neuronal and glial precursor cells close to the ventricles, has particularly fragile blood vessels, and that laminin expression in these vessels is strongly upregulated in the period from postnatal day 1 to day 4 (Ment et al., 1991). Furthermore, treatment with indomethacin, which has been shown to reduce the incidence of intraventricular hemorrhage (Hanigan et al., 1988), markedly enhanced laminin expression within these blood vessels, suggesting that the protection imparted by indomethacin may at least in part, be mediated by the enhanced levels of BM laminin increasing BBB stability (Ment et al., 1992).

Laminin Receptors

Cells of the BBB bind to laminin and other ECM proteins via cell surface receptors called integrins (Hemler, 1999; Hynes, 2002) and dystroglycan (Moore et al., 2002; Noell et al., 2011). It is now widely accepted that these ECM receptors are not just involved in cell adhesion, but they also play important instructive roles in regulating many aspects of cell behavior including cell proliferation, survival, migration, and terminal differentiation (Hemler, 1999; Hynes, 2002; Silva et al., 2008; Kim et al., 2011; Kechagia et al., 2019). The integrin family of cell surface receptors is expressed as non-covalently linked αβ heterodimers (Figure 3; Hemler, 1999; Hynes, 2002; Kechagia et al., 2019). To date, 16 different α and 8 different β mammalian integrin subunits have been defined, that are capable of dimerizing together to form 24 different integrin heterodimers. The β1 integrins are the largest sub-family, consisting of more than 11 members that include α1β1, α2β1, α3β1, α4β1, etc. Each specific integrin heterodimer has unique ligand specificity, for example, α1β1 is a collagen receptor (Hynes, 2002), while α6β1 and α6β4 integrins are both laminin receptors (Hemler et al., 1988; Sonnenberg et al., 1988; Sorokin et al., 1990; Hogervorst et al., 1993; Niessen et al., 1994; Kikkawa et al., 2000). Integrins perform two main functions (Figure 3). First, they establish a strong physical link between proteins of the ECM and the actin cytoskeleton in which the cytoplasmic domains of integrin β subunits interact with the cytoplasmic adaptor proteins talin, vinculin and α-actinin to form strong physical bonds with the actin cytoskeleton. In this way, they control the adhesive strength and motility of cells, to either instruct the cells to be very strongly attached and fixed in one location, or conversely, to use this adhesive machinery as a means of gaining traction and promoting cell migration. Second, integrin cytoplasmic domains interact with intracellular signaling proteins such as focal adhesion kinase and integrin-linked kinase to trigger intracellular signaling cascades that influence many aspects of cell behavior, including cell death, proliferation, and differentiation. Because integrins provide a unique bridge between the extracellular and intracellular environments, it is now widely accepted that they can transduce bi-directional signals across the cell membrane, both outside-in (where ECM binding to integrin triggers changes in intracellular actin and signaling events) and inside-out (where intracellular signaling events can change the integrin conformation, altering the integrin’s ability to bind ECM ligand (Hemler, 1999; Hynes, 2002; Kechagia et al., 2019). Dystroglycan is another cell surface receptor that binds laminin (Moore et al., 2002; Noell et al., 2011). Unlike the diverse family of integrins, dystroglycan is one entity, consisting of a non-covalently bound distal α subunit that binds laminin, and a transmembrane β subunit that binds to the actin cytoskeleton via the cytoplasmic adaptor protein dystrophin.

Figure 3: Schematic diagram of interactions between laminin and its receptors.Cells of the BBB bind to laminins in the vascular basement membrane via their cognate cell surface receptors, integrins and dystroglycan, which are transmembrane proteins. These receptors form both a transmembrane link between extracellular laminin and the actin cytoskeleton and also promote intracellular signaling events. The cytoplasmic domain of integrins bind to the cytoplasmic adaptor proteins talin, vinculin and α-actinin to form the transmembrane link and bind to several cytoplasmic signaling proteins including FAK and ILK to promote downstream intracellular signaling. The transmembrane β subunit of dystroglycan binds directly to the cytoplasmic adaptor protein dystrophin and thereby links to the actin cytoskeleton. BBB: Blood-brain barrier; FAK: focal adhesion kinase; ILK: integrin-linked kinase. Created with BioRender.com.

Differential expression of integrins and dystroglycan on cerebral blood vessels

The expression of integrins in cerebral blood vessels was first described thirty years ago by Paulus et al. (1993), who performed a histochemical study of human brain. This showed that integrins are strongly expressed by cerebral blood vessels, with the integrin subunits β1, α2, α3, α6 and β4 being described. Many studies have since confirmed these expression patterns and further elaborated on how they are altered in different states of development or disease. Twenty years ago, we showed that in the adult mouse CNS, β1 integrin expression is found at high levels on blood vessels, with negligible signal found in the brain parenchyma, suggesting a critical role for β1 integrins in regulating vascular function (Paulus et al., 1993; Kloss et al., 1999, 2001; Milner and Campbell, 2002). When we studied integrin expression in brain development, we observed an interesting switch in the expression of integrins and their cognate ECM proteins (Milner and Campbell, 2002). At an early stage of postnatal development, a time when cerebral angiogenesis is still very much ongoing, angiogenic blood vessels express high levels of fibronectin and the fibronectin-binding integrins α5β1 and α4β1 integrins, but at later timepoints, when cerebral blood vessels have formed and matured, blood vessels showed reduced expression of fibronectin and its receptors, but upregulation of laminin and its receptor α6β1 integrin and the collagen receptor α1β1 integrin. Subsequent studies have confirmed that cerebral blood vessels express significant levels of the laminin receptors α3β1, α6β1 and α6β4 integrins in addition to the collagen receptor α1β1, consistent with abundant levels of laminin and collagen IV in the vascular BM (Paulus et al., 1993; Haring et al., 1996; Kloss et al., 1999; Tagaya et al., 2001). Recent studies have shown that while α1β1, α3β1 and α6β1 are widely expressed across all cerebral blood vessels, α6β4 expression is largely restricted to arterioles (Welser-Alves et al., 2013), although it is induced on other types of vessel including capillaries during neuroinflammatory disease (Milner and Campbell, 2006; Welser et al., 2017). Exposure to chronic mild hypoxia increases cerebrovascular expression of the α6 and β1 integrin subunits (Halder et al., 2018b), but extreme hypoxia in an ischemic stroke model results in significant loss of these subunits (Li et al., 2010a). It is also interesting to point out that when the CNS is stressed to the point of launching an angiogenic response, such as during hypoxia or ischemia, angiogenic CNS blood vessels show strong upregulation of fibronectin and its receptor α5β1 integrin and also induce de novo expression of the well described pro-angiogenic αvβ3 integrin (Okada et al., 1996; Abumiya et al., 1999; Li et al., 2010a, 2012). Aside from the integrin laminin receptors, dystroglycan is expressed uniformly by all CNS blood vessels from early postnatal development, and studies suggest it is expressed primarily by astrocyte endfeet (Tian et al., 1996; Milner et al., 2008; Welser-Alves et al., 2013). Interestingly, while chronic mild hypoxia has little impact on dystroglycan expression, its expression on blood vessels was markedly reduced by ischemic stroke and at sites of leukocyte infiltration in the EAE model (Agrawal et al., 2006; Milner et al., 2008).

What do deletion studies tell us about the roles of cerebrovascular laminin receptors?

Partial deletion of endothelial β1 integrins leads to increased BBB leak

Total deletion of β1 integrin, either globally or within endothelial cells results in embryonic lethality, highlighting an essential role of these molecules in vascular development (Carlson et al., 2008; Lei et al., 2008). Because this makes it technically challenging to examine the roles of β1 integrins in BBB stability, del Zoppo’s laboratory employed an inducible Cre-Lox approach, to reduce β1 integrin expression by 50%. Interestingly, the lower levels of endothelial β1 integrin in this transgenic strain correlated both with markedly reduced levels of the ECM ligands laminin and collagen IV in the vascular BM, and compromised BBB integrity, as shown by increased IgG permeability (Izawa et al., 2018). These findings reinforced previous observations that intra-cerebral injection of a function-blocking β1 integrin antibody led to enhanced cerebrovascular leak (Osada et al., 2011). In combination, these data point to an important stabilizing role for β1 integrins in the maintenance of BBB integrity.

β4 integrin contributes to BBB integrity under neuroinflammatory conditions

Amongst the integrins, the β4 subunit is unique for two reasons. First, unlike the other β integrin subunits such as β1, β3 and β5, which have a small cytoplasmic domain of ~50 amino acids, the cytoplasmic domain of β4 is ~1000 amino acids long, implying much greater potential for unique molecular interactions with cytoplasmic adaptor proteins and intracellular signaling pathways (Feltri et al., 1997; Hemler, 1999). Second, unlike the endothelial integrin subunits β1, α1, α3, and α6, which are expressed by all types of blood vessels, β4 expression is expressed only by endothelial cells lining arterioles (Welser-Alves et al., 2013). However, during acute neuroinflammation such as that seen in the EAE model or in transgenic mice overexpressing the inflammatory cytokines interleukin-6 and interferon-α in the CNS, α6β4 integrin expression is also induced on other blood vessels including capillaries (Milner and Campbell, 2006; Welser et al., 2017). Taken with the observations that the epidermis also shows very high β4 integrin expression and that global β4 integrin KO mice show perinatal lethality due to severe epidermal blistering (van der Neut et al., 1996), prompted us to speculate that β4 integrin expression in tissues may correlate closely with high levels of mechanical stress. This would indicate why its expression in cerebral blood vessels is restricted to that part of the vascular tree (arterioles) subject to the highest levels of shear stress. If this is true, it would also suggest that its induction in cerebral capillaries in neuroinflammatory disease may represent a protective adaptive mechanism aimed at enhancing BBB integrity at times of vascular insult (Welser et al., 2017). To test this hypothesis, we generated endothelial-specific β4 integrin null (β4-EC-KO) mice, which were viable and fertile and showed no obvious defects in vascular development or BBB integrity under non-challenged conditions (Welser-Alves et al., 2013). To test whether endothelial α6β4 integrin induction in CNS capillaries during inflammatory conditions protects BBB integrity, we compared BBB integrity and EAE severity in β4-EC-KO and wild type littermate mice in the EAE model of MS. This revealed that β4-EC-KO mice developed a worse clinical disease that correlated closely with higher levels of BBB breakdown and leukocyte infiltration, and loss of endothelial tight junction protein expression (Welser et al., 2017). These results support our concept that induction of endothelial α6β4 integrin has an important protective influence on BBB integrity during neuroinflammatory disease.

Deletion of astrocyte dystroglycan has no obvious impact on BBB integrity

All blood vessels in the CNS display robust dystroglycan expression (Welser et al., 2017). While it is well accepted that most dystroglycan associated with the BBB is localized to astrocyte endfeet (Tian et al., 1996; Milner et al., 2008; Welser-Alves et al., 2013), it is still unclear whether it is also expressed by endothelial cells. Despite this robust expression in such a critical location, while mice with astrocyte-specific KO of dystroglycan show defective astrocyte endfeet architecture and loss of orthogonal arrays of the water channel protein AQP-4, surprisingly, these mice show no obvious defect in BBB integrity, at least under control unchallenged conditions (Moore et al., 2002; Noell et al., 2011). In future studies, it will be important to challenge these astrocyte-specific dystroglycan KO mice in neuroinflammatory models such as EAE, to determine whether BBB integrity defects can be uncovered under stressful conditions. In addition, it needs to be clarified if endothelial cells express dystroglycan and then determine if endothelial-specific dystroglycan KO mice show a BBB phenotype, either as a single KO or as a double-KO in which dystroglycan is also deleted in astrocytes.

Conclusions

The BBB plays an essential role in protecting sensitive CNS cells from the potentially harmful impact of blood components as well as precisely regulating the transport of substrates such as glucose and amino acids specifically required (Huber et al., 2001; Pardridge, 2003; Ballabh et al., 2004; Abbott et al., 2006; Engelhardt et al., 2014; Chow and Gu, 2015). The importance of this barrier is highlighted by the observation that neurological disease is often associated with disruption of the BBB (Gay and Esiri, 1991; Davies, 2002; Kirk et al., 2003; Bennett et al., 2010; Roberts et al., 2017) and recent findings suggest it also deteriorates with age (Farkas and Luiten, 2001; Farrall and Wardlaw, 2009; Brown, 2010; Brown and Thore, 2011; Senatorov et al., 2019). The vascular BM is well positioned to provide not only another layer of resistance for the BBB but more importantly, provide important instructive cues to cells of the BBB aimed at enhancing barrier integrity. With this in mind, it comes as no surprise that the family of laminins is abundant components of the vascular BM of CNS blood vessels, and that genetic deletions of specific laminin isoforms in different BBB cell types all tend to confirm the importance of these molecules in contributing to the BBB. Broadly speaking, almost all deletions of laminin isoforms adversely affect BBB integrity, as well as leading to reduced BM composition of other ECM proteins, reduced endothelial expression of tight junction proteins, altered expression of orthogonal AQP-4 clustering on astrocyte endfeet, and reduced pericyte coverage (Chen et al., 2013; Menezes et al., 2014; Yao et al., 2014; Gautam et al., 2016, 2019, 2020b). Consistent with these findings, genetic deletion studies have also highlighted the roles of laminin receptors in contributing to the BBB, including the β1 class of integrins (Izawa et al., 2018), as well as a key role for the α6β4 integrin in supporting greater BBB integrity under inflammatory conditions (Welser-Alves et al., 2013; Welser et al., 2017). While the focus of this review is the contribution of laminin and its influence on BBB properties, one limitation of this review is that we have not included a description of the contributions of other BM ECM components, including collagen IV, fibronectin, and heparan sulphate proteoglycan, all of which show altered expression patterns in different pathological conditions.

While great advances have been made in understanding the contribution of laminin to the maintenance of BBB integrity, many unanswered questions remain. First, because deletion of the laminin α5 subunit appears to have opposite effects on BBB integrity, depending on whether it is deleted in endothelial cells or pericytes (Gautam et al., 2019; Nirwane et al., 2019), it will be instructive to investigate these antagonistic roles by examining both KO strains in the same disease model. Second, based on the observations that laminin-111, which is expressed in the outer (parenchymal) layer of the BM appears to be more effective at inhibiting the infiltration of inflammatory leukocytes (Sixt et al., 2001; Halder et al., 2018c), it will be interesting to determine how forced expression of this laminin isoform in endothelial cells in the inner layer of the BM influences leukocyte infiltration. Third, multiple laminin receptors are expressed at the BBB, including α3β1 and α6β1 integrins, α6β4 integrin under inflammatory conditions, and dystroglycan (Paulus et al., 1993; Welser-Alves et al., 2013), it will be important to determine whether there is a hierarchy in terms of the functional importance of each of these laminin receptors, or whether loss of any one receptor can be adequately compensated for by the others. Fourth, it is important to further investigate the role of dystroglycan in regulating BBB integrity by defining whether endothelial cells express dystroglycan and whether endothelial-specific dystroglycan KO mice or endothelial and astrocyte-specific double-KO mice show defective BBB integrity. Fifth, once we have defined roles for specific laminin receptors, it will be important to ascertain whether enhanced expression of these receptors (e.g., α6β1 can enhance BBB integrity, particularly in animal disease models showing vascular disruption, including hypoxia, ischemia, and inflammatory demyelinating disease.

Author contributions:SKH, AS and RM contributed to writing of this review, and all authors approved the final version of the manuscript.

Conflicts of interest:The authors report no competing interests.

Data availability statement:Not applicable.

C-Editors: Zhao M, Liu WJ, Yu J; T-Editor: Jia Y