Role of Galectins in Wound Healing - ACS Symposium Series (ACS

Chapter 26

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Role of Galectins in Wound Healing Noorjahan Panjwani, Ph.D.* New England Eye Center, Departments of Ophthalmology and Biochemistry, Tufts University School of Medicine, 136 Harrison Ave., Boston, Massachusetts 02111 * E-mail: [email protected]. Tel: 617-636-6776. Fax: 617-636-0348.

Impaired or delayed re-epithelialization underlies serious disorders of wound healing that are painful, difficult to treat, and affect a variety of human tissues. Recent studies have provided evidence that members of the galectin class of β-galactoside-binding proteins play a crucial role in modulating re-epithelialization of wounds by novel carbohydrate-based recognition systems. Galectins constitute a family of widely distributed carbohydrate-binding proteins characterized by their affinity for β-galactoside-containing glycans found on many cell surface and extracellular matrix glycoproteins. In mammals, 15 members of the galectin family have been identified to date. Studies aimed at characterization of the role of galectins in wound healing have shown that galectin-3 promotes re-epithelialization of corneal and skin wounds, galectin-7 promotes re-epithelialization of corneal, skin and kidney wounds, and galectins-2 and-4 promote re-epithelialization of intestinal wounds. Molecular mechanisms by which galectins promote wound healing have also begun to be elucidated. Galectin-3 promotes re-epithelialization of wounds by: (i) activating α3β1-integrin–Rac1 signaling to promote formation of lamellipodia in epithelial cells, and (ii) interacting with N-glycans of laminin-332. Findings that galectins stimulate the re-epithelialization of corneal, dermal, intestinal and kidney wounds have broad implications for developing novel therapeutic strategies for the treatment of nonhealing wounds.

© 2012 American Chemical Society In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


I. Re-Epithelialization of Wounds Wound healing presents therapeutic challenges in a variety of clinical scenarios. Organ systems as different as cornea, skin and gastrointestinal (GI) may be the site of healing disorders that are related in their biological basis (1–6). These are but three of the many human organ systems in which impaired or delayed re-epithelialization may result in persistent epithelial defects which define a condition with serious medical implications. A unified hypothesis to comprehensively explain the failure of some, and not other, wounds to heal within a normal course of time has yet to be articulated. Meanwhile, patients with debilitations caused by a range, from relatively obscure to unfortunately commonplace, disease-associated, accidental, surgical or inflicted wounds (for example of combat) rely on what we know to guide their treatment. Attempts at resolution of chronic wounds of various etiologies can be frustrating and may not have a positive outcome. Millions of individuals are affected worldwide. In the U.S. alone, combat-related and other traumatic wounds cause over 300,000 hospitalizations annually (7, 8) . In the cornea, epithelial defects may persist and threaten the integrity of the anterior stroma, causing ulceration and in the worst cases perforation of the stromal tissue resulting in significant visual loss. Chronic wounds in the elderly, decubitus ulcer, and venous statis ulcer of the skin also are attributable to delayed re-epithelialization and resultant persistent epithelial defects. Damage and impairment of the intestinal surface barrier are commonly observed in a variety of GI diseases including inflammatory bowel diseases (IBDs). The treatment goal is prompt re-epithelialization of the wound, essential for rapid resealing of the epithelial surface barrier to control inflammation and to restore intestinal homeostasis. When re-epithelialization of intestinal wounds in IBDs is delayed, uncontrolled intestinal inflammation and general immune responses become inevitable (9, 10). Failure to re-epithelialize is generally caused not by inadequate cell proliferation but is due to a reduced potential of the epithelium to migrate across the wound bed (11–13). Cell migration involves sequential adhesion to and release from the substrate, a complex process of cell-matrix interactions (14–17). Results of recent studies suggest that members of the galectin class of β-galactoside-binding proteins have a critical role in modulating cell-matrix interactions and re-epithelialization of wounds through novel carbohydrate-based recognition systems (18–25).

II. Galectins Galectins constitute a family of widely distributed carbohydrate-binding proteins characterized by their affinity for β-galactoside-containing glycans found on many cell surface and extracellular matrix (ECM) glycoproteins (26, 27). In mammals, there are currently 15 identified members of the galectin family. They all range in subunit size from 14- to 39-kDa. Each galectin contains a canonical carbohydrate recognition domain (CRD) of ~130 amino acids. Galectins can be expressed both intracellularly and extracellularly. Galectins do not contain a 416 In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


classical signal sequence or a transmembrane domain and are secreted from the cell via nonclassical pathways. Some galectins such as galectins-1, -3, -8 and -9 have wide tissue distribution, whereas others, such as galectins-4, -5 and -6, exhibit tissue specificity. There is currently intense interest in characterizing the function of galectins because so many important cellular responses such as cell adhesion (28–30), migration (18, 31), immune response (32, 33) and angiogenesis (34–40) are regulated by this class of lectins. Carbohydrate-Binding Specificity of Galectins Although all galectins specifically recognize galactose-containing glycans, each galectin has unique, fine specificity for more complex galactose-containing oligosaccharides, which occurs as a consequence of variability in the CRD sequence. Due to differences in the carbohydrate-binding specificities, each galectin associates with certain types of glycans for signaling (41, 42). The differences in the sugar-binding specificity of different members of the galectin family can be profound. For example, Gal1 (Gal1) recognizes α2-3 sialylated glycans, but not α2-6 sialylated glycans; Gal2 does not bind glycans sialylated with either linkage; Gal3 binds internal N-acetyllactosamine (LacNAc) within polyLacNAc (41); and depending on cellular microenvironment, sialylation may also impact Gal3 binding and signaling (43). In summary, due to fine differences in carbohydrate-binding specificities, each galectin may interact with a discrete spectrum of glycoprotein receptors, with consequent specific downstream effects. For example, (i) the affinity of Gal1 for the blood group A tetrasaccharide is about 100-fold lower than that for Gal3 (44), and (ii) only Gal8, but not Gal1, Gal2, Gal3, or Gal7, interact with the glycans of a well-known lymphatic vessels glycoprotein, podoplanin (45). Galectin-Glycan Lattices All lectins are either dimers or oligomers. This multivalency allows the formation of lectin-carbohydrate lattices to cross-link and clusterize cell surface receptors including growth factor receptors and integrins. The diverse functions of galectins are thought to result from the formation of galectin–glycan lattice (46–48), by which the glycoprotein receptors are trapped, and thereby, precluded from undergoing endocytosis (49). It is by this mechanism that the interactions between galectins and N-glycans of the cell surface receptors regulate the density and distribution of cell surface receptors as well as cell responsiveness to the receptor ligand (46–49). For example, Gal3 interacts with the N-glycans of the epidermal growth factor (EGF) receptor in a carbohydrate-dependent manner; this delays its constitutive endocytic removal and promotes EGF signaling (49). Recent studies in our laboratory have revealed that Gal3 promotes cell migration and formation of lamellipodia by activating α3β1-integrin-Rac1 signaling in epithelial cells, and that carbohydrate-mediated interaction between Gal3 and complex N-glycans on the α3β1 integrin is involved in Gal3-induced lamellipodia formation and cell migration (18). 417 In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

III. Role of Galectins in Wound Healing


A. Galectin-3 Gal3 is expressed in inflammatory cells and in epithelia and fibroblasts of various tissues (26). It is found on the cell surface, within ECM, and in the cytoplasm of cells, and is thought to influence cell-matrix adhesion by binding to the ECM and cell surface-glycosylated counter receptors (e.g. growth factor receptors, integrins, certain isoforms of laminin, fibronectin and vitronectin). In addition, this lectin is found in the nucleus of the cells and may influence cell-matrix interactions indirectly by influencing the expression of well-known cell adhesion molecules (e.g.α6β1 and α4β7integrins) and cytokines (e.g. IL-1).

1. Role of Gal3 in Corneal Wound Healing In corneas, Gal3 is located in high density at sites of corneal epithelial cellmatrix adhesion (25), an ideal location for influencing cell-matrix interactions and cell migration. To determine whether Gal3 plays a role in re-epithelialization of corneal wounds, experiments were conducted to determine whether the rate of wound closure rate is impaired in Gal3-deficient mice. In this study, two different models of corneal wound healing were used. Corneas with excimer laser ablations or alkali-burn wounds were allowed to partially heal in vivo or in vitro for up to 22 h. At the end of the healing period, remaining wound areas were quantitated and compared among different groups. Regardless of whether the corneas were injured by excimer laser or by alkali treatment and whether the corneas were allowed to heal in vivo or in vitro, corneal epithelial wound closure rate (expressed as mm2/h) was significantly slower in gal3−/−mice compared with gal3+/+ mice (Figure 1A1E) (25). In contrast, no differences were found in the wound closure rates between Gal1+/+ and Gal1−/−groups (Figure 1F). To determine whether delayed re-epithelialization of corneal wounds in Gal3−/− mice was because of a deficiency in the rate of corneal epithelial cell proliferation, normal and healing Gal3+/+ and Gal3−/− corneas were labeled with BrdUrd to identify cells undergoing DNA synthesis. There was no significant difference in the number BrdUrd-labeled cells between Gal3+/+ and Gal3−/− corneas (25), suggesting that the rate of corneal epithelial cell proliferation is not perturbed in Gal3−/− mice and that delayed re-epithelialization of corneal wounds in Gal3−/−mice is most likely to be due to an impairment in the process of cell migration. The next set of experiments was conducted to determine whether exogenous Gal3 would stimulate re-epithelialization of corneal wounds. In this study, the corneas of Gal3+/+ mice with alkali-burn wounds were incubated in serum-free media in the presence and absence of varying amounts of recombinant Gal3. After a 20–22-h healing period, the remaining wound areas were quantified. The exogenous Gal3 stimulated the rate of wound closure in a concentration-dependent manner in Gal3+/+ mice (Figure 2). Overall, the extent of acceleration of re-epithelialization of wounds was 43 and 71% in the presence of 10 and 20 μg/ml Gal3, respectively. It was further demonstrated that the stimulatory 418 In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


effect of Gal3 on the rate of corneal epithelial wound closure can be almost completely abrogated by a competing disaccharide, β-lactose, but not by an irrelevant disaccharide, sucrose. This finding suggested that the lectin CRD was directly involved in the beneficial effect of the exogenous lectin on the wound closure. In corresponding experiments, recombinant Gal1 did not stimulate the corneal epithelial wound closure rate. Subsequent studies have shown that exogenous Gal3 promotes re-epithelialization of wounds in rat corneas(Yano C, et al. IOVS2010; 51:ARVO E-Abstract 371), monkey corneas (Fujii A, et al. IOVS2012; 53:ARVO E-Abstract 3540) as well as in a rat model of dry eye(Sasaki A, et al.IOVS2012; 53:ARVO E-Abstract 2359).

Figure 1. Corneal epithelial wound closure rate is significantly slower in gal3-/mice. Corneas of gal3+/+ and gal3-/- mice with 2-mm transepithelial excimer laser ablations or alkali-burn wounds were allowed to partially heal in vivo for 16–18 h or in vitro for 20–22 h in serum-free media. At the end of the healing period, wound areas were quantified. Regardless of injury by excimer laser (A and B) or by alkali treatment (C and D) or whether corneas were allowed to heal in vivo (A and C) or in vitro (B and D), corneal epithelial wound closure rate 419 In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


expressed in mm2/h was significantly slower in gal3-/- mice compared with that in the gal3+/+ mice. A value of 1.0 was assigned to the healing rate of gal3+/+ corneas. The values for gal3-/- corneas are expressed as a change in healing rate with respect to gal3+/+ corneas. Wound closure rates expressed as mm2/h among different groups were: (a) excimer laser in vivo: gal3+/+, 0.076±0.003; gal3-/-, 0.060±0.004; (b) excimer laser in vitro: gal3+/+, 0.051±0.003; gal3-/-, 0.035±0.005;(c) alkali injury in vivo: gal3+/+, 0.182±0.003; gal3-/-, 0.150±0.008; and (d) alkali injury in vitro: gal3+/+, 0.106±0.005; gal3-/-, 0.081±0.004. Panel E shows outlines of remaining wound areas from one of the experiments (group: alkali injury, healing in vivo). There was no difference in wound closure rate between galectin-1+/+ and galectin-1-/- mice corneas (F). Mean±S.E. of two or more experiments are shown. *, p

Role of Galectins in Wound Healing - ACS Symposium Series (ACS

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