What are the two functions of the basement membrane?

IV Summary

The BM is a complex, specialized extracellular matrix that is critically important for the development and maintenance of many tissues in the body. BM ultrastructure has been extensively studied in mammalian tissues; however, there remain many questions to be answered particularly related to BM dysfunction in human disease. Several elegant recent studies in the zebrafish, Danio rerio, shed important new light on the pathogenesis of human disease in a variety of tissues. These studies, utilizing the techniques for analyzing the BM outlined in this chapter, demonstrate the importance and validity of the zebrafish system for BM research. The conservation of BM components, assembly sites, and function between humans and zebrafish, coupled with the multitude of advantages of this lower vertebrate system, including the ease of transgenesis and transplantation studies as well as the suitability for forward genetic screening, makes the zebrafish a valued addition to the future study of BM function.

Introduction

Basement membranes (BMs) are thin sheets of specialized extracellular matrix that were first identified by transmission electron microscopy as continuous ribbon-like structures adjacent to a subset of cells. They are evolutionarily ancient structures, being present even in primitive organisms such as sponges and Hydra. In mammals, BMs underlie endothelial and epithelial cells and surround all muscle cells, fat cells, and peripheral nerves. They play important roles in filtration, in compartmentalization within tissues, and in maintenance of epithelial integrity, and they influence cell proliferation, differentiation, migration, and survival. In the lung, BMs are associated with bronchial and vascular smooth muscle cells, bronchial epithelium, nerve, and pleura, and they are part of the air–blood barrier between microvascular endothelial cells and alveolar epithelial cells (Figure 1).

The biochemical characterization of BM proteins was facilitated by the Engelbreth–Holm–Swarm (EHS) tumor, a transplantable tumor of mouse origin that produces large amounts of BM components that can be easily isolated. Studies of EHS tumor matrix proteins led to the identification of the four major classes of proteins present in all BMs: type IV collagen, laminin, entactin/nidogen, and sulfated proteoglycans. Though all BMs contain these four components, in vertebrates there are different isoforms of each that are differentially distributed in BMs, leading to significant molecular heterogeneity among BMs. This presumably translates into functional heterogeneity. In this article, we focus on the isoforms of type IV collagen and laminin that are expressed in the lung and how they relate to lung function and disease.

Conclusions

TBMN is one of the most common disorders of the kidney, affecting at least 1% of the total population. It appears to be a disease of the adult GBM type IV collagen trimer α3:α4:α5. Genetic evidence indicates that autosomal TBMN is caused by heterozygous mutations in either COL4A3 or COL4A4, while homozygous or combined heterozygous mutations in the same genes lead to autosomal-recessive AD. Thus, individuals with autosomal TBMN are carriers for autosomal AD. In some rare cases, monoallelic mutations can lead to autosomal-dominant AD. Heterozygosity for mutations in COL4A5 in female carriers for X-linked ADcan also mimic a TBMN condition, although some of those individuals will later develop progressive hematuria (Jais et al 2003). It is not clear if the individuals with TBMN and female carriers simulating TBMN that develop progressive disease could have a second mutation in some other of the six alleles that encode the α3:α4:α5 collagen form. The main clinical problem is to differentiate between TBMN and X-linked or autosomal recessive AD, as AD has a severe outcome. While family history and electron microscopy of renal biopsies are helpful for diagnosis, immunohistological examination of expression of the type IV collagen α3, α4, and α5 chains is currently the most informative method. It would be of utmost importance to make sequencing analyses of the COL4A3, COL4A4, and COL4A5 genes available to the clinic as a routine diagnostic method of TBMN and AD.

Basement Membrane and External Lamina

Basement membranes and external lamina are specialized sheets of extracellular matrix that lie between parenchymal cells and support tissues

Basement membranes and external lamina are specialized sheet-like arrangements of extracellular matrix proteins and GAG, and act as an interface between parenchymal cells and support tissues.

They are associated with epithelial cells, muscle cells and Schwann cells, and also form a limiting membrane around the central nervous system. Basement membrane and external lamina have similar structures.

Basement membranes have five major components: type IV collagen (Fig. 4.11), laminin, heparan sulfate, entactin and fibronectin. With the exception of fibronectin, these are synthesized by the parenchymal cells. In addition, there are numerous minor and poorly characterized protein and GAG components.

The general structure of basement membrane has been well characterized (Fig. 4.12). Superimposed on this, minor protein and carbohydrate components are specific to certain tissues. Thus, for example, the renal basement membrane differs from that of the skin.

The main functions of basement membrane are cell adhesion, diffusion barrier and regulation of cell growth

Basement membrane has three main functions:

First, it forms an adhesion interface between parenchymal cells and underlying extracellular matrix, the cells having adhesion mechanisms to anchor them to basement membrane, whereas basement membrane is tightly anchored to the extracellular matrix of support tissues, particularly collagen. Where such an interface occurs in non-epithelial tissues, for example around muscle cells, it is referred to as an external lamina.

Second, the basement membrane acts as a molecular sieve (permeability barrier) with pore size depending on the charge and spatial arrangement of its component GAG. Thus, the basement membrane of blood vessels prevents large proteins leaking into the tissues, that of the kidney prevents protein loss from filtered blood during urine production, and that of the lung permits gaseous diffusion.

Third, basement membrane probably controls cell organization and differentiation by the mutual interaction of cell surface receptors and molecules in the extracellular matrix. These interactions are the subject of intense research, particularly in the investigation of mechanisms that might prevent the spread and proliferation of cancer cells throughout the body.

2.2 Cell Adhesion and Migration

Basement membranes provide an adhesive substrate for cells, and they are linked functionally to the actin cytoskeleton via integrins or other ECM receptors to mediate cell attachment and migration, as well as modulating intracellular signaling pathways. Adhesion to basement membranes via cell surface receptors allows cells to mechanosense local stiffness, stiffness gradients, and other physical cues, which ultimately affect cellular behavior (Hynes, 1992). During Drosophila development, the receptor–ligand interplay between integrins and laminins in the basement membrane regulates follicle cell migration. Whereas integrin levels in follicle cells remain relatively stable, laminin expression in the basement membrane increases over time. The onset and speed of follicle cell migration are determined by this balance between integrin and laminin levels. Laminin-mutant eggs display altered cell migration and disrupted tissue shaping of developing follicles (Diaz de la Loza et al., 2017). Similarly, peripheral nerve establishment in mice requires laminin α5-dependent migration of neural crest cells, which differentiate into the peripheral nervous system and glial cells as they complete migration (Coles, Gammill, Miner, & Bronner-Fraser, 2006).

An in vitro model of cell migration demonstrates the profound effects of ECM dimensionality (2D vs 3D) on cellular behavior (Hakkinen, Harunaga, Doyle, & Yamada, 2011). Human foreskin fibroblasts in a 3D basement membrane extract (Matrigel) lose directionality and fail to migrate, yet the same substrate in a 2D configuration analogous to a basement membrane sheet allows highly efficient migration. These changes in cell migration patterns can be attributed to differences in ECM dimensionality. Even though useful for cell culture of epithelial cells, immersion of cells in a 3D basement membrane extract does not accurately simulate the basement membrane in vivo. A better representation of an in vivo environment is the 2D basement membrane extract model, which allows efficient cell migration. Indeed, cells in vivo migrate efficiently adjacent to the basement membrane. In the mouse submandibular gland, outer bud epithelial cells adjacent to the basement membrane show the highest rates of motility (Daley et al., 2017; Hsu et al., 2013). Motility of the outer bud cells is myosin II- and integrin α6β1 dependent, which suggests cell–ECM interaction. These findings highlight the importance of interactions between basement membranes, cell surface receptors, and cellular processes in regulating cell migratory behavior.

3 Role of Basement Membrane to Epithelial Wound Healing

Basement membrane disassembly can affect reepithelialization in different ways: (1) epithelial cells at the wound edge expose to underlying stromal extracellular matrix. This induces new integrin expression or activation in migrating epithelial cells.14 (2) Upon corneal epithelial debridement, cytokines and growth factors such as basic fibroblast growth factor (bFGF) and transforming growth factor-β (TGF-β) are abundantly present in the basement membrane and bind to the extracellular matrix molecules. They modulate intracellular signaling pathways to impact cell proliferation, differentiation, and/or apoptosis in migrating epithelial cells.15,16 (3) Formation of a stable adhesion complex. The regenerated epithelial cells sit on basement membrane to restore the epithelial barrier function. Therefore, basement membrane plays a central function in the entire process of corneal epithelial wound healing. It is noted that one of the major basement membrane components, laminin-1, regulates a wide variety of cell behaviors including adhesion, proliferation, and differentiation.17,18 Laminin isoforms 1 and 5 can directly associate with integrin α6β4 and facilitate cell migration. Migrating epithelial cells synthesize and deposit laminin-1 and -5 in the basement membrane during epithelial wound.19 Interestingly, approximately at the same time as laminin-1 and -5 syntheses, investigators found an upregulation of connexin and desmoglein-1 and -2. These observations suggested that the basement membrane restoration coincides with the formation of intercellular junctions and the expression of junctional adhesion proteins in corneal epithelial cells.20

Grade 2 Histopathology

Because the basement membrane is no longer completely tethered to the basal cells, it slips farther away with each cycle of weight bearing by the horse. Portions of the lamellar basement membrane are lysed initially between the bases of the secondary epidermal lamellae (see Figure 34-2). The basement membrane retracts from the tips of secondary epidermal lamellae, taking the dermal connective tissue with it. The basement membrane–free epidermal cells appear not to be undergoing necrosis, at least initially, and clump together to form amorphous, basement membrane–free masses on either side of the lamellar axis.1

9.3.4 BASEMENT MEMBRANE

Basement membrane, also referred to as basal laminae, are extracellular sheets of proteins that surround tissues, providing structural support, a filtration function, and a surface for cell attachment, migration, and differentiation (Rohrbach and Timpl, 1993). As described for viruses of medical importance (Romoser et al., 2005), BMs appear to act as a barrier to dissemination of baculoviruses within infected insects. Budded virus (BVs; Fig. 9.4), the baculovirus phenotype that serves to disseminate infection within an infected host, are too large to freely diffuse through the pores in the BM that surround tissues of the host insect (Reddy and Locke, 1990). Co-injection of BVs and clostridial collagenase, a protease known to degrade BM, resulted in enhanced infection of host tissues (Smith-Johannsen et al., 1986). Ultrastructural studies of infection by the baculovirus Cydia pomonella granulovirus (CpGV: Baculoviridae) and AcMNPV revealed a substantial accumulation of BVs in the extracellular spaces between BMs and the plasma membranes of midgut and fat body cells (Hess and Falcon, 1987) (Fig. 9.5). Collectively, these observations suggest that insect BM inhibits the movement of BVs.

The means by which insect viruses negotiate the basement membrane barrier are still unclear. Systemic spread of baculoviruses within the host insect may occur by direct penetration of the basement membrane into the hemocoel, either by an enzymatic process or where the BM is thin (Federici, 1997; Flipsen et al., 1995; Granados and Lawler, 1981). Alternatively, baculoviruses may use the host tracheal system as a conduit to bypass basement membranes and establish systemic infection of host tissues (Engelhard et al., 1994). There is debate over whether one route predominates over the other (Federici, 1997; Volkman, 1997). The identification of virus-encoded proteases with BM-degrading potential in an EPV (Afonso et al., 1999), and granuloviruses (Ko et al., 2000), supports the hypothesis that at least some insect viruses may use enzymatic means to traverse the BM (Liu et al., 2006a). There is also the suggestion that the baculovirus-encoded fibroblast growth factor (fgf), which is common to all baculoviruses that infect multiple tissues within an insect as opposed to being restricted to the gut, may function to attract hemocytes to sites of virus infection by chemotaxis. It is possible that a protease from granular cells, used for BM remodeling (Kurata et al., 1991, 1992; Nardi et al., 2001), may facilitate movement of virus across the BM.

Biological Function

Basement membranes are thin sheet-like extracellular structures that form an anatomical barrier wherever cells meet connective tissues. They provide a substrate for organs and cells and relay important signals for the development of organs and for differentiation and maintenance of the tissue. The GBM has an additional function; it takes an active part in glomerular ultrafiltration of blood. Basement membranes are composed of several specific molecules such as type IV collagen, laminin, proteoglycans and entactin/nidogen and are produced mainly by the endothelial cell layer.

There are six genes encoding the six chains of type IV collagen, α1(IV) through α6(IV). Different basement membranes have a different composition of their α(IV) chains [3]. The α1(IV) and α2(IV) chains are found in most basement membranes, whereas the GBM is composed of α3(IV), α4(IV) and α5(IV) chains (Figure 68.1). The GBM α3(IV), α4(IV) and α5(IV) network provide the strength and flexibility needed for the specialized functions of this basement membrane.

Basement Membranes Are Thin Matrix Layers Specialized for Cell Attachment

Basement membranes are thin (50–100nm), continuous layers of ECM that underlie epithelial and endothelial cell sheets and surround muscle cells, fat cells, and Schwann cells. They form a substratum for cell attachment and a link to the underlying connective tissue. The electron microscope shows two components to basement membranes, a clear or electron lucent layer next to the basal surfaces of the cells and a dark or electron-dense layer beneath this. These layers are called the lamina lucida and the lamina densa, respectively (see Fig. 6-15).

Basement membranes are highly cross-linked complexes of several proteins and proteoglycans (Fig. 6-35). Constitutive components are type IV collagen, laminin, a protein called nidogen/entactin, and heparan sulphate proteoglycan. In total, about 50 basement membrane proteins have been identified, of which collagens, especially type IV, constitute 50% of all basement membranes. The basement membranes of different tissues have specific properties in addition to the general requirement for cell attachment. Specificity is conferred by different isoforms of type IV collagen, laminin, and heparan sulphate proteoglycan. Thus, 7 different type IV collagens and 12 different laminins are known. Such specificity is important for regulating the varied functions of different tissues and organs. Of the major basement membrane components, laminin and type IV collagen have the ability to self-assemble into sheetlike structures, whereas the other components do not. Studies on basement membrane assembly by cultured cells indicate that laminin first forms a network that associates with the cell surfaces through integrin adhesion receptors (especially β1) and dystroglycan, a transmembrane proteoglycan. Type IV collagen forms an independent but associated network, the interaction between the two being facilitated by nidogen/entactin. This complex then forms a scaffolding for the binding of other basement membrane constituents.

Basement membranes are the targets of a number of human diseases. Mutations in the gene for the α5 chain of type IV collagen are associated with Alport syndrome, a disease that involves nephritis and deafness. Junctional EB, a severe blistering disease of the skin that results in early infantile death, is associated with mutations in the genes that encode the laminin-5 chains, whereas dystrophic EB, a severely debilitating blistering disease that results in syndactyly, is due to mutations in collagen VII genes and consequent absence of anchoring fibrils. Autoantibodies to type IV collagen α3 chain, which is present in the glomerular basement membrane of the kidney, are associated with Goodpasture syndrome.

A key component of tumor growth is angiogenesis, the elaboration of new blood vessels. Growing tumors cannot exceed a few millimeters in diameter without acquiring a new blood supply, or they will die of anoxia. Tumor cells produce growth factors that promote angiogenesis. The basement membrane inhibits the proliferation and migration of endothelial cells, thus preventing them from branching out to produce new vessels. During tumor growth, inflammatory and stromal cells within the matrix near the tumor produce matrix metalloproteinases that degrade the vascular basement membrane, thus enabling the endothelial cells to proliferate, migrate, and form new blood vessels to supply the tumor. Research on this process offers hope that inhibition of angiogenesis may be used to prevent tumor growth.