Integrin: basement membrane adhesion by corneal epithelial and endothelial cells

Abstract

Integrins mediate adhesion of cells to substrates and maintain tissue integrity by facilitating mechanotransduction between cells, the extracellular matrix, and gene expression in the nucleus. Changes in integrin expression in corneal epithelial cells and corneal endothelial cells impacts their adhesion to the epithelial basement membrane (EpBM) and Descemet’s membrane, respectively. Integrins also play roles in assembly of basement membranes by both activating TGFβ1 and other growth factors. Over the past two decades, this knowledge has been translated into methods to grow corneal epithelial and endothelial cells in vitro for transplantation in the clinic thereby transforming clinical practice and quality of life for patients. Current knowledge on the expression and function of the integrins that mediate adhesion to the basement membrane expressed by corneal epithelial and endothelial cells in health and disease is summarized. This is the first review to discuss similarities and differences in the integrins expressed by both cell types.

1. Introduction

The cornea has a stratified squamous epithelium on its outer apical surface and a simple squamous epithelium on its inner surface exposed to the aqueous humor. Underneath these two epithelia are two basement membranes with distinct compositions. The epithelial basement membrane (EpBM) is produced primarily by secretion of extracellular matrix (ECM) proteins by the basal cells of the corneal epithelium with some ECM contributed by anterior stromal cells. Whereas the corneal epithelial basal cells are highly proliferative, corneal endothelial cells proliferate rarely in the adult human cornea, and over time with aging, there are fewer of these cells present lining the inner surface of the cornea. The endothelial cell basement membrane is called Descemet’s membrane (DM). A schematic showing epithelial and endothelial cell adhesion to their respective basement membranes is presented in Figure 1. The term “Bowman’s layer” refers not to the EpBM but to the acellular layer of ECM under the EpBM which is present in the human and avian but not in the rabbit or mouse cornea (Wilson, 2020).

Figure 1. Schematic comparing corneal epithelium-EpBM and corneal endothelium-DM binding interactions.

(A) Both hemidesmosomes and focal adhesions mediate cell adhesion between the corneal epithelium and EpBM. Type I hemidesmosomes contain integrin α6β4, plectin, BP230, and BP180/type XVII collagen that links laminin-332 (LN332) in the EpBM to keratin cytoskeletal proteins present in the cytoplasm. Focal adhesions at the corneal epithelium-EpBM interface consist of integrin α3β1 or α2β1 bound to fibronectin, laminin, or collagen and associate with cytoplasmic integrin-binding proteins (e.g., talin (TLN), paxillin (PXN), focal adhesion kinase (FAK), and vinculin (VCL)) that are involved in downstream signaling pathways related to cell survival and migration. (B) Focal adhesions formed at the corneal endothelium-DM interface are composed by integrins α3β1 or α6β1 that bind to laminin-511 (LN511) or −411 (LN411) present in the DM. Cytoplasmic integrin-binding proteins localized at the plasma membrane bind to cytoskeletal proteins and influence actin dynamics.

The corneal epithelium is subjected to forces caused by blinking and eye rubbing, and the epithelial cells adhere to the EpBM primarily via intermediate filament based hemidesmosomes. By contrast, the corneal endothelial cells function as pumps to maintain hydration in the cornea and move nutrients from the aqueous humor, into their cytoplasm, and release them into DM where they can remain bound to proteoglycans or diffuse into the corneal stroma. Because they are located inside the anterior chamber, the corneal endothelial cells do not need to provide the same barrier function that the epithelial cells provide at the anterior surface nor are they frequently injured so maintaining strong robust adhesion to DM is not as important as it is for the corneal epithelial cells. The two cell types adhere to their substrates via distinct integrin heterodimers; they secrete different amounts and types of ECM proteins which the integrins on their basal surfaces assemble into basement membranes with distinct properties.

In this review, after a brief introduction to integrins, we describe the current state of knowledge on the role that integrins play in mediating adhesion of corneal epithelial and endothelial cells to their basement membranes. For a discussion of Bowman’s layer and Descemet’s membrane, please see recent reviews by Wilson, et al. (de Oliveira and Wilson, 2020; Wilson, 2020).

2. Integrins: an overview

Bidirectional communication between the ECM and cells is mediated by biophysical cues and mechanotransduction pathways in a process known as ‘dynamic reciprocity’ (Bornstein and McPherson, 1982). This interplay was described by Mina Bissell and colleagues (Bissell et al., 1982) and provided a mechanism for how different cellular populations may arise within a tissue. During development, progenitor cells remain in close proximity to each other and yet may differentiate into completely different cell types depending on the surrounding ECM, hormones, growth factors, and biophysical cues present in the microenvironment that contribute to epigenetic modifications and cell specialization (Bissell et al., 1982; Bissell and Aggeler, 1987; Levin and Stevenson, 2012; Crowder et al., 2016). This mechanism is particularly important during embryonic development and cancer metastasis, which are both associated with significant changes in the ECM that occur in parallel to cellular transformation (Lin and Bissell, 1993). In dynamic reciprocity, conformational changes in integrin structure alter cellular interactions with the ECM and basement membrane proteins, e.g., fibronectin, collagen, laminin, and heparan sulfate proteoglycans, allowing both ‘outside-in’ and ‘inside-out’ signaling (Takagi et al., 2002). Signal transduction pathways associated with cell proliferation, survival, and differentiation, may then be activated by interactions between the cytoskeleton and transcriptional and translational proteins in the nucleus. In turn, as the ECM influences cell signaling, the cell layer may also alter the secretion and deposition of ECM and basement membrane proteins, thus invoking a reciprocity that defines the constantly evolving and aging tissue structure.

The process of ‘dynamic reciprocity’ is fundamental in corneal biology. In a tissue predominately composed by ECM proteins whose organization and maintenance are crucial for tissue transparency and proper refraction, biomechanical signaling via integrins plays a major role in maintaining tissue integrity. The theory of dynamic reciprocity hypothesized in 1982 that proteins with the biomechanical properties of integrins existed; four years later the first integrin was cloned (Tamkun et al., 1986). The laboratories of Richard Hynes at MIT and Martin Hemler and Tim Springer at Harvard Medical School realized after comparing data from chicken β1 integrin with DNA sequences from human immune cells the complexity of the integrin family (for historical perspective see (Hynes, 2004)). Integrins are integral membrane, single pass, αβ heterodimers expressed on the surfaces of both adherent and circulating cells. The β1 integrin subunit would turn out to form heterodimers with over 10 different α chains. The family size would expand dramatically over the next several decades and include another 7 β chains (β2, β3, β4, β5, β6, β7, and β8) complementing 18 α chains. Work in basic cell biology labs would go on to show that integrins function as the major mechanotransducers in tissues to regulate tissue integrity in part by functioning as cell signaling hubs at sites of cell:substrate adhesions.

In the early 1990’s, integrins began to be characterized in the corneal epithelium (Stepp et al., 1990; Grushkin-Lerner and Trinkaus-Randall, 1991; Tervo et al., 1991; Stepp et al., 1993) and stroma (Lauweryns et al., 1991; Garana et al., 1992; Masur et al., 1993). Over time, integrins would also be studied on corneal endothelial cells as well (Okumura et al., 2015), but difficulty growing the cells limited discoveries to organotypic cell cultures (Zieske et al., 1994).

3. Corneal epithelial cell integrins associated with the EpBM

The ECM composition of the EpBM in healthy corneas was compared to that of corneas from patients with diabetes and diabetic retinopathy (Ljubimov et al., 1998). Using newly characterized monoclonal antibodies against individual laminin chains, these researchers showed that the EpBM contains several laminin chains (α1, α5, β1, γ1). LNs are trimers containing α, β, and γ chains; using the revised nomenclature (LNαβγ) these data indicate that the EpBM contains LN111 and LN511. They also showed the presence of LN332 using an antibody that recognizes the LN332 complex. In addition to these LNs, the EpBM also expresses entactin/nidogen, various collagen IV chains (α1, α2, α3, α4, α5, α6), type VII collagen, perlecan, tenascin C, and fibrillin-1. The composition of ECM proteins in basement membranes in the body vary in tissue specific ways; the relative amount of each ECM protein varies with aging and in various pathologies. In a subsequent study, Kabosova and colleagues (2007) compared human infant and adult corneal EpBM and found variations in composition of the EpBM at the center compared to the limbus and between infants and adults (Kabosova et al., 2007). Since the cornea epithelial progenitor and stem cells reside at the limbus, these data show that the limbal niche had a unique ECM composition. Research using model organisms has shown that when basement membranes form during development and when they are regenerated during wound repair, epithelial cells interact directly with LNs; type IV collagen and perlecan assemble on the scaffold formed by integrins and dystrogylcan binding to LNs (Hohenester and Yurchenco, 2013; Pozzi et al., 2017; Pastor-Pareja, 2020).

In the corneal epithelium, several integrins are expressed. In human and mouse central cornea, α2β1, α3β1, αvβ5, and α6β4 all have a polarized localization within the epithelium with expression highest in the basal cells and tapering off so that apical squames do not express integrins. Most integrins localize to the basal aspect of the basal cells where they mediate adhesion to the EpBM. α6β4 mediates adhesion to the EpBM via hemidesmosomes whereas other integrins including α3β1 and αvβ5 mediate adhesion via focal adhesions which are actin based. Integrins are synthesized and assembled into heterodimers in the endoplasmic reticulum and Golgi. They are then transported via endosomes to the basolateral region of basal cells where the endosomes fuse with basolateral membranes. Integrins are then transported within the cell membrane to the basal cell surface.

We know that the integrins expressed at the EpBM are capable of mediating adhesion of corneal epithelial cells to collagens, fibronectins, laminins, and vitronectin. When integrin expression decreases, cells become less proliferative and less adhesive to the underlying basement membrane. Integrins bound to the ECM exert mechanical forces on the matrix. Those forces are transmitted via integrin cytoplasmic domains to the cytoskeleton and the nucleus where they regulate gene expression. By pulling on EpBM proteins as they are secreted by the epithelial cells, integrins induce the unfolding of cryptic sites in the ECM proteins that allow assembly of the ECM proteins with one another to make up the EpBM. As ECM proteins spontaneously assemble into the EpBM, changes in their conformation alter the forces applied to the integrins bound to them. This process both activates the integrin and alters signaling to the cytoskeleton and can expose proteolytically sensitive sites leading to integrin ectodomain cleavage.

Our understanding of the functions of integrins in the EpBM has relied on studies of genetically engineered mice, gene mutations in humans, and various human pathologies. Below, we describe our current understanding focusing on integrins that mediate adhesion to the EpBM. Table 1 lists the integrins that mediate epithelial cell adhesion to the EpBM.

Table 1.

Integrins expressed on corneal epithelial cells

Corneal epithelial cell integrins adhering to the epithelial basement membrane
Integrin Family	Subunit	Gene	Heterodimer	Known Ligands
β4 ITGB4	α6	ITGA6	α6β4	Laminin 332
β1 ITGB1	α2	ITGA2	α2β1	Laminin, Collagen
	α3	ITGA3	α3β1	Laminin, FN, TSP-1, Nidogen, Collagen type IV
αv ITGAV	β1	ITGB1	αvβ1	Laminin, FN
	β5	ITGB5	αvβ5	FN, Laminin, Vitronectin
	β6	ITGB6	αvβ6	Vitronectin, Fibronectin

(Abbreviations: Fibronectin (FN), Thrombospondin-1 (TSP-1)).

α6β4

In 1990, we showed that α6β4 was an integral membrane component of the hemidesmosome (HD) (Stepp et al., 1990). HDs come in two types. Corneal and epidermal HDs are type I HDs. They contain a specific set of components: α6β4, plectin, BP230, and BP180 (type XVII collagen) bound to LN332 in the basement membrane. Type II HDs contain α6β4 and plectin bound to LN332 (Litjens et al., 2006) and are present in simple epithelial cells, myoepithelial cells, and cultured cells. When β4 integrin null mice were generated, pups developed but their skin shed after birth and pups died (Dowling et al., 1996). Later studies showed that loss of β4 integrin also lead to defects in vascular endothelial cells (Wang et al., 2012) and peripheral nerves (Van der Zee et al., 2008). Due to the absence of LN332 at these sites, the α6β4 containing cell substrate adhesions formed by vascular endothelial and neuronal cells are considered a subclass of the type II HD. α6 integrin was also deleted in mice; like β4 null mice, the mice lost their epidermis after birth (Georges-Labouesse et al., 1996). In vitro studies of cells from the epidermis of these mice showed increased TGFβ1 signaling and accelerated terminal differentiation of keratinocytes (Rodius et al., 2007). Mutations in genes that encode α6, β4, or other proteins present in the hemidesmosome cause epidermolysis bullosa- a pathology that can exhibit mild and fatal forms depending on the severity of the mutation and which leads to skin blistering (Hashimoto et al., 2016) and ocular surface defects (Jonsson et al., 2015).

Debridement injury to the mouse cornea can be performed so that the EpBM remains attached to the corneal stroma or the EpBM can be removed at the time of surgery (Pal-Ghosh et al., 2004). When we compared the wound response to these two types of injury, we found that leaving the EpBM behind impaired wound resolution and contributed to the formation of recurrent erosions (Pal-Ghosh et al., 2011b). When the EpBM is left intact, epithelial cells and resident immune cells are forced to degrade it to allow for the formation of new hemidesmosomes with LN332 and type VII collagen. The erosions form, in part, due to proteolytic cleavage of the extracellular domain of β4 integrin by MMPs (Pal-Ghosh et al., 2011a).

Interest in adult stem cells in the skin and treating corneal epithelial stem cell deficiency in the cornea led several groups to show that α6β4 is expressed at high levels on epithelial stem and progenitor cells in the skin (Jones and Watt, 1993; Kaur and Li, 2000; Tani et al., 2000) and cornea (Pajoohesh-Ganji and Stepp, 2005). These discoveries led directly to improvements in the quality of the epidermal and corneal epithelial cells produced in vitro for use in patients with severe burns and treating corneal stem cell deficiencies (Ahmad et al., 2007; Hayashi et al., 2008).

In vivo, corneal epithelial stem cells expressing high levels of α6β4 are located at the limbus (Pajoohesh-Ganji and Stepp, 2005; Polisetti et al., 2016). Differences in EpBM composition at the limbus compared to the central cornea in humans had been shown by (Ljubimov et al., 1995; Ljubimov et al., 1998) and confirmed by Schlötzer-Schrehardt (Schlotzer-Schrehardt et al., 2007). Using this knowledge, optimized ECM coated surfaces are now used to enhance expansion of epithelial stem cells in vitro for transplantation (Polisetti et al., 2017). These types of studies are increasing our understanding of the cell biology mediating corneal epithelial cell adhesion to the EpBM and the role it plays in maintaining the stem and progenitor cells at the limbus.

Recurrent erosions in the mouse can also develop because of a delay in the ability of corneal epithelial basal cells expressing high levels of α6β4 to down regulate their proliferation after debridement injury (Pal-Ghosh et al., 2011b). Inhibiting cell proliferation with a single treatment with mitomycin C restored epithelial integrity and allowed the corneal sensory nerves to reinnervate the tissue (Pal-Ghosh et al., 2016).

Clinical interest in α6β4 increased after studies lead by C. Stephen Foster showed that patients with cicatricial pemphigoid have autoantibodies against β4 integrin (Tyagi et al., 1996; Rashid et al., 2013). These antibodies bind to the basal surface of corneal epithelial cells and prevent them from interacting with the ECM proteins in the EpBM eventually leading to blistering, inflammation, and severe damage to the ocular surface. Treatments aimed at reducing inflammation often improve these symptoms. Bullous pemphigoid is a similar autoimmune disease but instead of autoantibodies against β4 integrin, patients have autoantibodies against BP230 (also called dystonin), BP180 or type XVII collagen. In some cases, patients that present with pemphigoid have inherited mutations in one or more of the hemidesmosomal or desmosomal proteins (Goletz et al., 2017).

α3β1

α3β1 is one of the major integrins in corneal epithelial basal cells. While α6β4 expression increases after corneal epithelial debridement injury, α3β1 expression remains constant (Stepp et al., 1993). Like α6β4, α3β1 adheres to LN332 in the EpBM (Belkin and Stepp, 2000). In vitro studies have shown that α3β1 competes with α6β4 for LN332 via a mechanism referred to as trans-dominant inhibition by competing for cytoskeletal proteins (Diaz-Gonzalez et al., 1996; Hodivala-Dilke et al., 1998). When LN332 is bound to α3β1, it is subjected to mechanical forces exerted primarily via the actin cytoskeleton whereas when LN332 is bound to α6β4, it is subjected to mechanical forces exerted primarily via the intermediate filaments (keratins). While α6β4:LN332 adhesions form hemidesmosomes, α3β1 adhesions are called focal adhesions. Mice engineered to lack α3β1 have defective basement membrane assembly leading to skin and kidney defects (DiPersio et al., 1997). Recently, a mutation in α3 integrin was shown to cause renal developmental defects involving glomerular basement membrane defects (Shukrun et al., 2014). When α3β1 integrin is deleted specifically from mouse skin and mice used in skin tumor studies, α6β4+ positive stem cells left their niche and terminally differentiate leading to smaller tumor sizes (Sachs et al., 2012). These studies together highlight the importance of α3β1 and α6β4 in maintaining cell adhesion within the limbal niche and in response to injury.

Recently, it has been confirmed that limbal epithelial stem/progenitor cells are maintained in the limbal niche by α3β1 and α6β4 integrins and their extracellular ligands LNα1, LNα2, LNα3 and LNα5 (Polisetti et al., 2016). Since LNα5 has been shown to represent a signature component specifically produced by epithelial progenitor cells, recombinant LN511 has been used to enhance in vitro expansion of limbal epithelial stem cells (Polisetti et al., 2017). Consistently, functional blocking of α3β1 and α6β4 integrins suppressed adhesion and proliferation of cells on LN511-coated surfaces.

Figure 2 shows a TEM image of the epithelial cells: EpBM interface, shows hemidesmosomes, and highlights the localization and expression of ECM proteins in the basement membrane and α3 and β4 integrins in the epithelium.

Figure 2. Hemidesmosomes contain LN332 and α6β4 integrin at the EpBM and α3β1 integrin adjacent in focal contacts.

A. i. TEM image showing HDs at the corneal epithelial:basement membrane interface in a control mouse cornea; asterisks highlight HDs. ii and iii: The localization of LN332 and perlecan are shown in the cornea. While LN332 is exclusive to the EpBM, perlecan localizes to both EpBM and DM. Images in A are modified from Sta.Iglesia and Stepp (2000) (Sta Iglesia and Stepp, 2000) with permission from ARVO, as the copyright holder. B. i The localization of α3 (green) and β4 (red) integrin in control corneas is shown; in ii and iii, integrins are shown separately. Note that the localization of α3 integrin is not restricted exclusively to the basal cell layer but extends apically into suprabasal cells. By contrast, β4 integrin is more restricted. Compared to the EpBM, DM does not show any evidence for expression of β4 integrin and minimal expression of α3 integrin. Bar, 50 μm. Image modified from (Pal-Ghosh et al., 2004) with permission from ARVO, as the copyright holder. In Aii and iii and Bi-iii, asterisks indicate the apical surface of the corneal epithelium and arrows indicate DM.

αv family integrins (αvβ1, αvβ5, αvβ6)

αv forms heterodimers with multiple β subunits (β1, β3, β5, β6, and β8). The “v” designation in αv integrins refers to vitronectin which is one of the αv integrin ligands. αv integrins adhere to the tripeptide sequence arginine-glycine-aspartic acid or RGD on extracellular matrix proteins. Vitronectin, abundant in serum, and fibronectin have RGD sequences and bind to αv integrins. In skin and cornea, αvβ5 and αvβ6 are the major forms yet some αvβ1 may also be present.

αv family integrins were shown to regulate the activation of latent TGFβ1 by binding to an RGD sequence in the TGFβ1 latency associated protein (Annes et al., 2004; Munger and Sheppard, 2011). Detailed studies of the mechanism of activation of TGFβ1 by αv integrin have led to numerous insights into fibrosis, epithelial-mesenchymal transition (EMT), and cancer metastasis (Massague, 2012; Meng et al., 2016). While TGFβ1 induces fibrosis in stromal cells, in keratinocytes, TGFβ1 modulates the secretion and assembly of the ECM proteins that make up the EpBM (Stepp et al., 2007).

While αvβ5 has been studied in skin (Janes and Watt, 2004; Jiang et al., 2014) and cornea (Stepp et al., 2002), more research has been performed in retinal pigment epithelial (RPE) cells. Studies using β5 null mice showed that αvβ5 mediates adhesion of RPE cells to Bruch’s membrane as well as the phagocytosis of shed rod and cone outer segments (Nandrot et al., 2004). Mice lacking β5 integrin also fail to regulate shedding of rod and cone outer segments in a circadian manner (Nandrot and Finnemann, 2006). While it hasn’t been studied, it is likely that αvβ5 regulates phagocytosis in corneal epithelial cells that express it.

In normal mice, quiescent epithelial cells switch from expressing αvβ5 to αvβ6 after injury which enhances their migration (Jiang et al., 2014). Mice lacking expression of β6 have delayed wound healing in the skin (Eslami et al., 2009; Duperret et al., 2016) and cornea (Blanco-Mezquita et al., 2011). These mice also show reduced fibrosis after wounding due to impaired activation of latent TGFβ1 (Wu et al., 2019).

Basement membranes have been shown to function as reservoirs or sponges for growth factors secreted by the corneal epithelial cells (Vlodavsky et al., 1991; Dowd et al., 1999). Growth factors bind with variable affinities to heparan binding sites present on many ECM proteins. When the cornea is injured and the EpBM is left intact, TGFβ1 accumulates in the EpBM after corneal injury; if the EpBM has been removed at the time of injury, TGFβ1 diffuses into the stroma (Stramer et al., 2003). By controlling the activation of TGFβ1 secreted by corneal epithelial basal cells, αv family integrins regulate wound resolution and fibrosis.

4. Integrin-mediated Signaling at Descemet’s Membrane

Similar to the anterior cornea, integrins play an essential role in mediating attachment of the endothelium to the posterior elastic lamina known as DM that resides between the endothelial layer and posterior stroma. In humans, the acellular DM is 10–12 μm thick on average and composed of laminin, collagen types IV and VIII, nidogen, fibronectin, and perlecan, which may bind to integrins present on the plasma membrane of corneal endothelial cells. DM is nearly 7 times stiffer than the EpBM with average elastic moduli of 50 kPa (± 17.8 kPa) and 7.5 kPa (± 4.2 kPa), respectively (Last et al., 2009). Visualized by electron microscopy, DM is composed of an anterior banded (fetal) layer, approx. 3 μm in thickness, and a posterior nonbanded (postnatal layer) layer; DM is attached to the corneal stroma by a narrow transitional zone of amorphous ECM termed the interfacial matrix (Murphy et al., 1984; Sawada et al., 1990); a TEM image is presented in Figure 3A. The high abundance of collagen type VIII appears to be specific to the DM compared to other basement membranes found in the body (Kapoor et al., 1988). Contrary to the EpBM which becomes thinner with age, the postnatal DM is known to thicken by 3- to 4-fold from birth to old age due to the accumulation of non-striated collagen fibrils deposited on the striated collagen lamellae with a distinct periodic banding of 110 nm formed in utero (Murphy et al., 1984). Apart from providing structural integrity of the cornea, DM has been suggested to play a role in corneal hydration, endothelial cell differentiation, and maintenance of the corneal curvature. Studies have also suggested that the DM limits exposure of the corneal stroma to pro-fibrotic growth factors that are highly abundant in the aqueous humor, such as TGF-β2, which may promote corneal haze following photorefractive keratectomy or riboflavin-UV-A corneal crosslinking [reviewed in (Wilson, 2019)].

Figure 3. Integrin containing focal adhesions can be visualized at the corneal endothelial: DM interface.

A. TEM image at top showing the ultrastructure of Descemet’s membrane (DM) composed of an interfacial matrix (IFM) mediating attachment to the corneal stroma, anterior banded layer (ABL) and posterior nonbanded layer (PNBL). (Re-produced from (Schlötzer-Schrehardt and Kruse, 2016) with permission). TEM image at bottom showing endothelial-DM interface with electron-dense focal adhesions (arrows). B. Immunofluorescence images of corneal tissue sections showing co-localization of laminin α5 and integrins α3, α6 and β1 along the endothelial-DM interface. Protocol for the immunolabelling method and antibody information available in (Polisetti et al., 2017).

In terms of downstream signaling, activation of integrins by the ECM is dependent on a class of cytoskeletal proteins, known as talins, which function as essential adaptor proteins that mediate binding of the cytoplasmic integrin β-subunit to actin bundles (Tadokoro et al., 2003). Talins contain at least two integrin-binding subunits and may recruit additional cytoskeletal proteins, including vinculin that also binds to actin filaments to increase contractile forces at focal adhesions. Downstream signaling of integrin-activation involves mitogen activated protein kinase, a major regulator of cell proliferation, differentiation, and apoptosis, and focal adhesion kinase, which influences cell survival and migration. Phosphorylated focal adhesion kinase then activates Src, phosphoinositide 3-kinase, and phospholipase C-γ, among others that promote cell survival (via Akt signaling). In addition, the cytoplasmic Rho-GTPases are regulators of cytoskeletal dynamics and often described as molecular switches due to their ability to activate multiple downstream pathways. One notable effector of Rho, Rho-associated kinase (ROCK), has been shown to be an important modulator of actin-myosin dynamics via phosphorylation of myosin light chain phosphatase and LIM kinase, which leads to cofilin deactivation and actin polymerization (Maekawa et al., 1999). An interplay between the Rho/ROCK pathway and TGF-β is evident as cell contraction requires ROCK activity independent of α-smooth muscle actin expression (Miura et al., 2006; Kita et al., 2008). These pathways play an important role in cell migration, adhesion, and contractility, as well as cell survival and proliferation. ROCK-targeted therapeutics aim to selectively modulate these processes in the context of disease (e.g., glaucoma or Fuchs’ endothelial corneal dystrophy), injury (e.g., corneal scarring), or surgery (e.g., Descemet’s Stripping Endothelial Keratoplasty) and will be further discussed in Section 6.

5. Endothelial Cell Integrins

The corneal endothelium is essential in regulating water flux between the aqueous humor and the corneal stroma in order to maintain corneal deturgescence, which limits hydration of the cornea. Primary corneal endothelial cells are suspended in G1-phase of the cell cycle and remain non-proliferative in vivo. Methods for culturing corneal endothelial cells in vitro have shown an ability to promote proliferation using growth factors, in addition to fibronectin or collagen type I or IV coatings to allow cell attachment to traditional polystyrene culture plates or polydimethylsiloxane substrates (Engler et al., 2009; Palchesko et al., 2015). Recent focus has been placed on developing viable substrates for the cultivation and expansion of corneal endothelial cells for transplantation in corneal dystrophies affecting endothelial function, such as Fuch’s endothelial corneal dystrophy, congenital hereditary endothelial dystrophy, and posterior polymorphous corneal dystrophy. The integrins that appear to be relevant for maintaining and promoting maturation of primary human corneal endothelial cells may be inferred from this work. Table 2 shows the integrins expressed by corneal endothelial cells as they interface with DM. Biosynthetic coatings of various polymers and protein-based substrates have shown favorable cellular responses that promote cell attachment, survival, proliferation, and terminal differentiation of corneal endothelial cells. An example is inclusion of the RGD (Arg-Gly-Asp) and DGEA (Asp-Gly-Glu-Ala) α2β1 integrin binding sites on a biosynthetic substratum of poly-ε-lysine hydrogels with octanedioic acid crosslinks, which appears to improve endothelial cell binding, proliferation, and maturation in vitro (Kennedy et al., 2019). Human corneal endothelial cells may also be cultured on other protein or polymer based-approaches, including biocompatible silk protein films that may be surface-coated to promote cell attachment (Ramachandran et al., 2020). Many of these polymers naturally lack cell binding domains, which may be useful in an effort to customize coatings to favor select cell interactions (e.g., endothelial cells), while limiting the binding of other cell types (e.g., lymphocytes) and reduce the likelihood of rejection and promote cell integration.

Table 2.

Integrins expressed on corneal endothelial cells

Corneal endothelial cell integrins adhering to Descemet’s Membrane
Integrin Family	Subunit	Gene	Heterodimer	Known Ligands
β1 ITGB1	α2	ITGA2	α2β1	Laminin, Collagen
	α3	ITGA3	α3β1	Laminin, FN, TSP-1, Nidogen, Collagen type IV
	α4	ITGA4	α4β1	FN-IIIA
	α5	ITGA5	α5β1	Collagen, Laminin
	α6	ITGA6	α6β1	Collagen, Laminin, FN
αv ITGAV	β1	ITGB1	αvβ1	Laminin, FN
	β3	ITGB3	αvβ3	FN, Laminin, Vitronectin
	β5	ITGB5	αvβ5	FN, Laminin, Vitronectin
	β6	ITGB6	αvβ6	FN, Laminin, Vitronectin

(Abbreviations: Fibronectin (FN), thrombospondin-1 (TSP-1)).

Maintenance of the corneal endothelium is also dependent on mechanotransduction pathways mediated by the basement membrane. The corneal endothelium and DM have a direct relationship regulated by the stiffness of the substratum influenced by the biochemical composition of the posterior DM. Mechanotransduction has been shown to favor mesenchymal stem cell differentiation to corneal endothelial cells solely by varying the substratum topography and stiffness (Gutermuth et al., 2019). Micropatterning of cell culture substrates to create surfaces similar to hexagonal structures seen in DMs have also shown to favor corneal endothelial cell binding and maturation based on increased tight junctional marker expression. These studies indicate that topography of the substratum can regulate cell phenotype (Muhammad et al., 2015). These findings may be related to the dynamic spatial orientation of integrins that may adapt based on the composition and organization of the substratum.

In terms of specific integrins involved in cell binding, integrins α3β1 and α6β1 appear to mediate binding interactions between primary human corneal endothelial cells and LN511 in vitro (Okumura et al., 2015). LNα5, LNβ1, LNβ2 and LNγ1 chains were also found along the endothelial-DM interface in vivo, suggesting that LN511 and LN521 represent the major LN forms at the endothelial face of DM. The predominant integrin chains expressed on the mRNA and protein level in endothelial cells in vivo comprise integrin α3, α6, β1 and β5, although integrins α1, α2, α4, α5, α10, α11 and α_v were also weakly expressed (Okumura et al., 2015). In vitro, functional blocking of α3β1 and α6β1 integrins suppressed phosphorylation of focal adhesion kinase (FAK) and cell adhesion to LN511-coated surfaces, indicating that laminins regulate endothelial cell adhesion through binding to integrin α3β1 and α6β1. Figure 3B shows the localization of major integrins α3, α6, and β1 in corneal endothelial cells at DM.

Primary human corneal endothelial cells are known to be susceptible to undergoing EMT. The integrin subunit α2 is upregulated in endothelial cells expressing EMT-markers (Toda et al., 2016). Laminin-411 and −511 also appear important for primary human corneal endothelial cell binding by minimizing the number of cells undergoing EMT (Toda et al., 2016). This phenotypic switching has made transplantations of endothelial cell suspensions more difficult. Research has focused on developing therapeutics to improve endothelial cell binding and maturation post-transplantation.

6. Therapeutics that modulate ROCK

By targeting downstream effectors of integrin-mediated signaling, such as Rho/ROCK, fibrosis can be attenuated (Tada et al., 2001; Yamamoto et al., 2012; Zhou et al., 2013). Inhibition of Rho/ROCK signaling has also been shown to promote retention of stem-like properties and proliferation of retinal pigment epithelial cells cultured in vitro by inhibiting EMT suggesting a direct interplay of signaling pathways involved in proliferation and differentiation (Croze et al., 2014; Croze et al., 2016). This work provides evidence that therapeutic modulation of integrin-mediated signaling may aid in promoting cell viability, adhesion, and recovery, and may translate to improved tissue regeneration following transplantation.

In terms of applications in the cornea, studies in vitro have shown favorable corneal endothelial cell attachment to substrates using a synthetic ROCK inhibitor, Y-27632 (Okumura et al., 2012), which targets both isoforms of ROCK (ROCK1 and ROCK2) (Ishizaki et al., 2000). The improved cell adhesion with ROCK inhibition may be a result of increased cell spreading that is apparent in 2D cultures (Croze et al., 2016) and occurs with a change in distribution of focal adhesions and stress fibers that result from altered cofilin activity. Eyedrops of Y-27632 applied to a primate model with corneal endothelial damage showed improved endothelial cell proliferation and functional recovery based on decreased corneal edema (Okumura et al., 2013). A small clinical study with 8 patients (4 with late-stage Fuchs’ dystrophy) who were given eyedrops 6 times per day showed reduced edema and thickness. This improvement was also seen in some patients with central corneal edema but not in those with diffuse corneal edema (Okumura et al., 2013). Given the potential role for isoform-dependent functions of ROCK on actin dynamics and maintaining stemness in certain cell types, further studies evaluating targeted therapeutics for a select isoform (ROCK1 or ROCK2) may be important for clinical applications (Newell-Litwa et al., 2015). Studies of an approved glaucoma treatment and ROCK1 inhibitor, ripasudil, have also shown favorable clinical results in patients with Fuchs’ dystrophy (Moloney et al., 2017; Macsai and Shiloach, 2019). This work is a significant advance in efforts to identify druggable targets to regulate cell pluripotency, EMT, and adhesion by focusing on integrin-mediated signaling to improve the outcome of corneal endothelial cell transplantations to promote cell attachment and survival. The Rho/ROCK pathway has also been implicated in TGF-β-promoted contraction in fibrotic conditions, such as proliferative diabetic retinopathy and vitreo-retinopathy (Kita et al., 2008).

7. Endothelial Cell Integrins and Disease

Degeneration of the corneal endothelium and underlying DM during Fuchs’ endothelial cell dystrophy leads to corneal edema and haze development. Thickening of DM is a characteristic of Fuchs’ progression and is attributed to elevated EMT by the endothelial layer and the formation of ECM deposits, known as guttae, which range from 5 μm to 50 μm in diameter (Xia et al., 2016). The role of biophysical changes in the DM leading to cellular pathology is apparent in Fuchs’ dystrophy (Ali et al., 2016). Early-onset Fuchs’ is associated with COL8a mutations (L450W and Q455K) that leads to altered collagen type VIII accumulation in the anterior DM (Gottsch et al., 2005a; Gottsch et al., 2005b). These mutations are associated with irregular fibril spacing and softening of the DM that may lead to altered endothelial cell function and precedes the increased ECM deposition and DM-thickening that follows in later stages of Fuchs’ dystrophy. COL8a2 mutant mouse models of early-onset Fuchs’ (COL8a2^L450W/L450W and COL8a2^Q455K/Q455K) have shown a >50% decrease in DM stiffness by 10 months of age which appears to correlate with a loss in endothelial cell counts (Leonard et al., 2019).

Altered integrin expression has been identified in a Fuchs’ tissue-engineered model. A 2-fold reduction in ITGA10 was detected in Fuchs’ corneal endothelial cells in this tissue-engineered system (Goyer et al., 2018). Using real-time PCR array technology, Weller et al. showed upregulation of integrin α1, α3, α4, αL, β1, β3 and β4 in explanted endothelial cell-DM specimens from Fuchs’ patients compared to normal specimens (Weller et al., 2014). A genome-wide association study of Fuchs’ patients and control individuals identified LAMC1 (laminin subunit γ 1) and KANK4 (isoform belonging to the obscure Kank class of proteins involved in stress fiber formation) (Zhu et al., 2008) as potential risk factors for Fuchs’ development (Afshari et al., 2017). Further studies are warranted to characterize integrin expression in the Fuchs’ cornea to determine how interactions between the corneal endothelium and DM affect pathogenesis and progression of the disease.

If we apply the theory of dynamic reciprocity to Fuchs’ dystrophy, differences in the mechanical forces that integrins exert on their substrate would induce guttae formation by altering the forces the cells exert on DM. Integrins, as surface proteins, are druggable targets and there are peptides and antibodies that can be used either block integrin function or induce integrin activation. Select targeting of integrin-mediated signaling to promote tissue regeneration in the absence of EMT or excessive ECM deposition relies primarily on the tissue and disease status. Blocking integrin-activation following corneal wounding leads both to reduced corneal epithelial cell migration following wounding (Blanco-Mezquita et al., 2011) and a parallel delay in stromal scar development (Wu et al., 2019) suggesting that this therapeutic approach may be more beneficial to decrease TGFβ1-driven fibrosis and/or inhibit EMT. During fibrotic conditions, targeting specific integrins has shown some success in certain tissues. For example, global knockout of ανβ6 leads to reduced scar tissue development in chemical injury models or genetic pro-fibrotic models of the lung, kidney, liver, and skin (reviewed in (Henderson and Sheppard, 2013)). In these tissues, development of fibrosis is blunted by inhibiting ανβ6-mediated activation and release of TGFβ1 that occurs following binding of the integrin to the RGD-sequence found in the latency-associated peptide linked to the pro-TGFβ1 form. Likewise, targeting the α3β1 integrin has also shown inhibition of fibrosis in alveolar epithelial cells by preventing EMT and myofibroblast differentiation that requires binding interactions between α3β1 and the formation of a phospho-SMAD2 complex with β-catenin (Kim et al., 2009). This approach targeting select integrins or downstream pathways, such as ROCK, may be a useful means to prevent fibrosis and EMT in various tissues, including the cornea.

8. Summary

Since integrins were first characterized, they have been found to be critical for maintaining tissue architecture in the cornea. Basic and translational research on integrins has contributed new clinical treatments for blinding corneal pathologies and a more complete understanding of the impact of mechanotransduction on corneal homeostasis. Yet, corneal pathology continues to lead to corneal blindness and loss of quality of life; new treatments are needed. Dynamic reciprocity suggests that integrins are involved. While much has been learned about integrins, additional knowledge of the roles of integrins and their adhesion to the EpBM and DMs in health and disease is greatly needed.

Highlights.

Current knowledge on the expression and function of the integrins that mediate adhesion to the basement membranes expressed by corneal epithelial and endothelial cells in health and disease is summarized. This is the first review to discuss similarities and differences in the integrins expressed by both cell types.