A Central Regulator of Chronic Inflammation and Tissue Fibrosis

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Chapter 22

Galectin-3: A Central Regulator of Chronic Inflammation and Tissue Fibrosis Neil C. Henderson,*,1 Alison C. Mackinnon,1 Claire Rooney,2 and Tariq Sethi*,2 1MRC

Centre for Inflammation Research, The Queen’s Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, United Kingdom 2Department of Respiratory Medicine and Allergy, King’s College London, London, United Kingdom *E-mail: [email protected] (N.C.H); [email protected] (T.S.)

Fibrosis is a major cause of morbidity and mortality worldwide. Tissue injury, with chronic inflammation and fibrogenesis, results in disruption of tissue architecture and eventually organ failure. Currently, the therapeutic options for tissue fibrosis are severely limited and organ transplantation is often the only effective treatment for end-stage fibrotic diseases. However, demand for donor organs greatly outstrips supply, and so effective anti-fibrotic treatments are urgently required. In recent years, Galectin-3 has gained prominence as a central regulator of chronic inflammation and fibrogenesis. Tissue fibrosis models in multiple organs have demonstrated that galectin-3 has profound effects on organ fibrogenesis and scarring. In this review we will examine the ways in which galectin-3 regulates these processes through both direct effects on tissue myofibroblasts and also by mediating cross talk between the innate immune system and fibroblast populations in various organs. Additionally, we will discuss how the manipulation of galectin-3 using small molecule inhibitors may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.

© 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.


Introduction Fibrosis represents a massive health care burden worldwide. Chronic tissue injury and fibrogenesis results in disruption of tissue architecture, organ dysfunction and eventually organ failure. Our therapeutic repertoire for the treatment of tissue fibrosis is severely limited and organ transplantation is currently the only effective treatment in end-stage fibrotic disease. However, the disparity between donor organ availability and organ demand means that many patients die. Therefore effective anti-fibrotic treatments are urgently required. Galectin-3 is a β-galactoside-binding lectin of ~30kDa. It is unique among galectins in that it is a chimeric protein with a C-terminal carbohydrate recognition domain (CRD) linked to a proline, glycine, and tyrosine rich additional N-terminal non-CRD with multiple homologue repeats, which are involved in higher order oligomerization (1). Galectin-3 is pleiotropic and it’s localisation within the tissue micro-environment may be extracellular, cytoplasmic or nuclear. This imparts great flexibility on galectin-3 as a key regulator of a wide range of biological processes such as adhesion (2–4), proliferation (5, 6) and cell survival (7, 8). In recent years, there has been increasing interest in the role of galectin-3 in chronic inflammation and tissue fibrogenesis. A variety of different murine models of organ fibrosis have demonstrated that galectin-3 has profound effects on the fibrotic process, mediating it’s effects on multiple different cell lineages during tissue scarring (Figure 1). In this review we will examine how galectin-3 regulates chronic inflammation and fibrosis in different organs, and highlight the progress which has been made in identifying galectin-3 as an attractive therapeutic target in the search for effective anti-fibrotic therapies.

Hepatic Fibrosis Increased hepatic galectin-3 expression has been demonstrated in cirrhotic human liver secondary to a wide range of etiologies (9). Although galectin-3 was not present in normal hepatocytes, immunohistochemistry of liver biopsies from both hepatitis B induced cirrhosis of the liver and hepatocellular carcinoma complicating hepatitis B infection demonstrated increased expression of galectin-3. Galectin-3 was highly expressed in cirrhotic liver in a peripheral distribution within regenerating nodules. In a further study of galectin-3 expression in human liver cirrhosis (10) galectin-3 expression was found to be increased in liver cirrhosis secondary to a variety of different causes with varying mechanisms of liver injury including viral-induced liver disease (Hepatitis B and C), autoimmune, copper or iron overload, primary biliary cirrhosis and alcohol-induced liver disease. Galectin-3 expression was negligible in the normal liver and increased in the cirrhotic nodules of hepatocytes, particularly at the nodule periphery. These data suggest that galectin-3 up-regulation within human liver is a basic response to liver injury, irrespective of the initiating agent or disease process. 378 In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 1. Galectin-3 regulates tissue fibrosis in multiple organs. a) Picrosirius red staining (collagen staining) in wild type and galectin-3-/- mouse liver following chronic CCl4-induced liver fibrosis (10). b) Sirius red staining in wild type and galectin-3-/- mouse kidney following unilateral ureteric obstruction (UUO) induced renal fibrosis (20). c) Masson’s trichrome staining (collagen staining) in wild type and galectin-3-/- mouse lung following adenoviral TGFβ1 (ad-TGFβ1) induced lung fibrosis (41). d) Masson’s trichrome staining in wild type and galectin-3-/- mouse lung following bleomycin-induced lung fibrosis (41). e) Representative sections through the brachiocephalic artery of ApoE-/- and ApoE-/-/G-3-/- mice (fed high cholesterol diet for 12 weeks) stained with Masson’s trichrome (MacKinnon et al., unpublished data). (see color insert)

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


Galectin-3 expression has also been examined in murine models of hepatic fibrosis (10). After eight weeks of CCl4 treatment galectin-3 expression was increased in the periportal areas and areas of bridging fibrosis in mouse liver. Dense hepatocyte galectin-3 staining was also present at the periphery of the hepatocyte nodules. Furthermore, in a rat model of reversible CCl4 induced liver fibrosis galectin-3 expression was temporally and spatially associated with liver fibrosis, with expression minimal in normal rat liver, maximal at peak liver fibrosis, and then virtually absent again at 24 weeks (recovery from fibrosis) (10). Hepatic stellate cells (Ito cells, liver specific pericytes) are the major source of extracellular matrix proteins during hepatic fibrogenesis (11, 12), and therefore represent an important target in the development of anti-fibrotic therapies for liver fibrosis. Galectin-3 has been shown to stimulate the proliferation of rat and mouse hepatic stellate cells (10, 13) and a number of in vivo studies have shown an important role for galectin-3 in the regulation of hepatic fibrosis. Disruption of the galectin-3 gene blocked hepatic stellate cell activation and collagen expression in a CCl4-induced model of liver fibrosis (10). The decrease in liver fibrosis observed in the galectin-3-/- mouse occurred despite equivalent liver injury and inflammation, and similar tissue expression of the pro-fibrotic cytokine TGF-β. TGF-β failed to transactivate galectin-3-/- hepatic stellate cells, in contrast with wildtype hepatic stellate cells. However TGF-β stimulated Smad2 and -3 phosphorylation was equivalent, suggesting that galectin-3 is required for TGF-β mediated myofibroblast activation and extracellular matrix production. Furthermore, deletion of the galectin-3 gene has also recently been shown to have anti-fibrotic effects in murine models of non-alcoholic steatohepatitis (NASH) (14) and biliary fibrosis (15) further highlighting the important role of galectin-3 in the regulation of hepatic fibrosis. These data suggest that pharmacological targeting of galectin-3 may have clinical utility in the treatment of patients with liver fibrosis.

Renal Fibrosis Kidney fibrosis represents a major cause of morbidity and mortality worldwide. End stage renal failure secondary to renal fibrosis results in large numbers of patients becoming dialysis dependent or having to undergo kidney transplantation. At present alternative treatment options are severely limited. The expression and subcellular distribution of galectin-3 alters with cellular differentiation. In light of this there are numerous functional roles attributed to galectin-3 in the context of the renal system. These include development, where galectin-3 plays a role in both mouse and human foetal collecting duct morphogenesis (16, 17), and the addition of exogenous galectin-3 to embryonic mouse explants inhibits branching of the ureteric bud (16). Similarly, functional adaptation of the kidney to different physiological conditions occurs through galectin-3 dependent mechanisms. In immortalised cell lines, metabolic acidosis precipitates cellular remodelling whereby B-intercalated (bicarbonate secreting) cells are transformed into A-intercalated (acid secreting) cells, mediated by a galectin-3-hensin interaction (18). Mechanistically the supposition is that 380 In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


galectin-3 plays a role in the terminal differentiation of epithelial cells. This is also seen in examples of aberrant renal physiology: for example in acute kidney injury, a high dose folic acid nephropathy model demonstrates tubular necrosis and apoptosis with incomplete kidney healing, interstitial fibrosis, loss of peritubular capillaries and macrophage infiltration. Modified citrus pectin, which binds to and antagonises galectin-3, has been shown to be protective in this experimental model (19). It has long been recognised that macrophage infiltration into the kidney is an early hallmark of many forms of renal injury. Indeed in human kidney biopsy studies there is a significant association between degree of tubulointerstial macrophage infiltration, severity of fibrosis and progression to end stage renal failure. Recently the role of galectin-3 in renal fibrosis has been examined. Using a mouse model of renal fibrosis (unilateral ureteric obstruction, UUO) galectin-3 expression was shown to be upregulated following UUO, and deletion of galectin-3 protected against renal myofibroblast accumulation/activation and fibrogenesis (20). Furthermore, specific depletion of macrophages using CD11b-DTR transgenic mice reduced fibrosis severity following UUO demonstrating that macrophages are key cells in the pathogenesis of renal fibrosis. Absence of galectin-3 did not affect macrophage recruitment following UUO, or macrophage proinflammatory cytokine profiles in response to IFN-γ/LPS (21). Adoptive transfer of wildtype but not galectin-3-/-macrophages did, however, restore the fibrotic phenotype in galectin-3 knockout mice. In vitro, cross-over experiments using wildtype and galectin-3-/- macrophage supernatants and renal fibroblasts confirmed that secretion of galectin-3 by macrophages is a critical step in the activation of renal fibroblasts to a profibrotic phenotype (20). Therefore, these data demonstrate that galectin-3 expression and secretion by macrophages is a major mechanism linking macrophages to the promotion of renal fibrosis. Recently, a second model of kidney injury has examined the role of galectin-3 in chronic allograft injury (CAI) in murine renal transplants (22). Chronic allograft injury (CAI) is characterized by interstitial fibrosis and tubular atrophy and results in a progressive decline in graft function, resulting in the loss of 5% of renal transplants per annum. This study used a mouse model of CAI, characterized by a single class II mismatch between BM12 donor and C57/BL6 recipient strains. Transplantation of BM12 kidneys into C57/BL6 mice was associated with interstitial fibrosis, tubular atrophy and increased galectin-3 expression compared with syngeneic controls. However, transplantation of BM12 kidneys into galectin-3-/- mice resulted in significant preservation of tubules and decreased interstitial fibrosis, with a reduction in myofibroblast activation and collagen I expression compared with wild type controls (22). Furthermore, infiltrating leukocytes numbers were unaltered by abrogation of galectin-3, but reduced expression of YM1 (a marker of alternative macrophage activation) coupled with a reduction in the number of circulating CD4-positive T cells and reduced expression of interleukin-4 in galectin-3-/- mice suggest possible mechanisms by which galectin-3 may promote renal transplant fibrosis. These data suggest that galectin-3 may represent a novel therapeutic target in chronic allograft injury. 381 In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


These studies highlight the importance of macrophages in two diverse models of renal fibrosis. However there remains some debate regarding the mechanism of this effect. In the UUO model, as renal fibrosis progresses, macrophage infiltration increases and galectin-3 remains upregulated. Moreover, depletion of macrophages using CD11b-DTR transgenic mice decreased fibrosis severity following UUO demonstrating that macrophages are key regulators of renal fibrosis. This is supported by other studies which show that inhibition of macrophage recruitment following UUO reduces the severity of renal fibrosis (23–25). However the studies in this area are not all in agreement, as adoptive transfer of macrophages at later time-points during UUO-induced renal injury have also been shown to ameliorate renal fibrosis (26). This may reflect the fact that macrophages play a varied and time- dependent role in both inflammatory injury and repair mechanisms, or that differentiated macrophages may fall further into as yet poorly defined sub-populations with distinct functional roles. In the studies examining the role of galectin-3 in renal fibrosis (20, 22) galectin-3 is identified as the link between macrophages, fibroblasts and the pro-fibrotic phenotype. It is known that galectin-3 mediates IL-4 induced alternative macrophage activation (21) and that IL-4/IL-13 activated macrophages upregulate profibrotic genes, stimulating matrix production and enhancing fibrosis (27–29). The concept of a galectin-3 / macrophage / fibroblast ‘axis’ is supported by the UUO adoptive transfer studies whereby wild type but not galectin-3-/macrophages lead to myofibroblast activation and collagen deposition. Cross over supernatant experiments additionally confirmed that galectin-3 secretion by macrophages is required to activate quiescent renal fibroblasts (20). In keeping with this the galectin-3 knockout allograft model shows reduced levels of alternatively activated macrophages, reduced myofibroblast accumulation and fibrosis compared to controls (22). Therefore the results from both these models indicate a key role for galectin-3 in the pathogenesis of renal fibrosis. A better understanding of the mechanisms regulating this process and targeted strategies to inhibit galectin-3 in the kidney may allow the development of new treatments for patients with renal fibrosis.

Pulmonary Fibrosis Galectin-3 is highly expressed on lung macrophages and to a lesser extent on bronchial epithelium and is markedly upregulated in type II alveolar epithelial cells following irradiation-induced lung injury in rats (30). Galectin-3 has also been shown to have an important role in the development of asthma where expression is upregulated in areas of fibroproliferation following allergen challenge (31, 32). Deletion of galectin-3 results in reduced airway inflammation and interstitial fibrosis associated with reduced expression of the major pro-fibrotic cytokine TGFβ (31). However, overexpression of galectin-3 using adenoviral delivery itself reduced hyper-responsiveness and eosinophil accumulation in rats (33, 34). This highlights that non-targeted overexpression may lead to functional effects that are quite distinct from those observed endogenously. 382 In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Galectin-3 immunostaining is also increased in the small airways of COPD patients when compared with nonsmokers and smokers (35) establishing an important role for this galectin in chronic lung disease. There is also convincing evidence that galectin-3 functions as an anti-apoptotic factor in lung carcinoma and is expressed within and around non-small cell lung cancer (NSCLC) but not small cell lung cancer (SCLC) in human biopsy specimens (36) and its presence has been shown to correlate with poor response to chemotherapy. Furthermore, genetic polymorphisms in galectin-3 have been associated with platinum-based chemotherapy response and prognosis of NSCLC (37). With regard to chronic inflammatory disorders of the lung, idiopathic pulmonary fibrosis (IPF) is a chronic condition of unknown etiology with repeated acute lung injury causing progressive fibrosis and architectural distortion resulting in deterioration of lung function and ultimately respiratory failure and death. The median time to death from diagnosis of this devastating condition is 2.5 years (38) and the incidence of IPF continues to rise (39). At present no specific therapy is available. Corticosteroids alone or in combination with immunosuppressive drugs have been used with limited success (40) and there are no reliable prognostic factors or biomarkers to predict disease progression. Galectin-3 is expressed in the lungs of mice exposed to the fibrotic agent bleomycin and in response to adenoviral mediated increased TGFβ, the major profibrotic cytokine in the lung (41). TGFβ1 induces the fibrogenic response through receptor-mediated phosphorylation of members of the Smad family of cytoplasmic transcription factors Smad2 and Smad3 (42) and Smad3 deficient mice are protected from TGF β1-induced fibrosis (43, 44). There is also increasing evidence for a role of the wnt/β-catenin pathway in regulating TGFβ1-dependent signalling (45) and this pathway is aberrantly activated in IPF (46). Pulmonary fibrosis in two well characterized rodent models of lung fibrosis (induced by TGFβ and bleomycin), was dramatically reduced in mice deficient in galectin-3 (41). This was manifest by reduced TGFβ1-induced EMT and myofibroblast activation and collagen production in vitro and in vivo. Galectin-3 deletion reduced retention of TGFβ receptors at the cell surface and reduced phosphorylation and nuclear translocation of β-catenin but had no effect on Smad2/3 phosphorylation. Moreover, a novel inhibitor of galectin-3, TD139, blocked TGFβ-induced β-catenin activation in vitro and in vivo and attenuated the late stage progression of lung fibrosis following bleomycin-induced lung injury (41). The regulation of myofibroblast activation by galectin-3 is summarized schematically in Figure 2. In patients with IPF there is increased lung expression of galectin-3 (47) and levels are elevated in bronchoalveolar lavage fluid and serum from patients with stable IPF compared to non-specific interstitial pneumonitis and controls. Moreover, serum galectin-3 rises sharply during an acute exacerbation suggesting that galectin-3 may be a marker of active fibrosis in IPF (41). Therefore this study identifies galectin-3 as an important regulator of lung fibrosis and provides a proof of principle for galectin-3 inhibition as a potential novel therapeutic strategy for IPF.



Figure 2. Schematic showing a potential mechanism for galectin-3-mediated regulation of myofibroblast activation. Galectin-3 binding to n-acetyl lactosamine residues (l-nac) on TGFβ-RII causes association with TGFβ-RI and potentiation of TGFβ signalling;- smad2/3 and Akt activation. Liberation of beta-catenin from adherens junction during myofibroblast activation is sequestered in the cytosol where its nuclear translocation is inhibited by GSK3-β. Nuclear galectin-3 complexes with beta-catenin in a transcription complex that mediates expression of mesenchymal genes and galectin-3. TD139 inhibits extracellular galectin-3 binding and function. (see color insert)

Atherosclerosis Atherosclerosis is a major cause of cardiovascular disease (CVD) and stroke. In the U.S. it is estimated that there are around 2000 deaths from CVD each day, more each year than from cancer, respiratory disease, and accidents combined (48). Atherosclerosis is recognized as being a chronic inflammatory with macrophages playing a dominant role (49, 50). Macrophages recruited to the vascular intima engulf lipoprotein particles and differentiate into foam cells which orchestrate lesion development by promoting inflammation, smooth muscle cell proliferation and extracellular matrix deposition (50). Galectin-3 is highly expressed in human atherosclerotic plaques and in mouse models of atheroma (51, 52) and is commonly used as a marker for plaque macrophages. There is currently growing interest in galectin-3 as a validated biomarker for heart failure (53–55) and as a predictor of response to statin therapy in heart failure (56) and galectin-3 undoubtedly plays an important role in atherosclerotic disease development. However, previous studies have yielded conflicting results regarding the role of galectin-3 in plaque development. The first study, using C57/Bl6 mice, showed that deletion of galectin-3 increased 384 In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


lesion formation in mice fed a high fat diet for 8 months (57). However, high fat feeding in mice without a defect in lipoprotein metabolism (e.g. ApoE-/or LDLR-/-) does not cause development of advanced lesions, and does not induce the same high levels of serum cholesterol. Therefore, this study could not address the role of galectin-3 on the later, and arguably more relevant, stages of atherosclerosis. The second study showed that in an atherosclerosis prone model (ApoE-/- but on a mixed 129sv/C57/Bl6 background), galectin-3 deletion reduced plaque size in older mice (50 weeks) fed a normal diet (58). Differences in strain and/or serum cholesterol could be the main factor responsible for this paradox. A third study using high cholesterol feeding in ApoE-/-C57/Bl6 mice. shows that galectin-3 deletion in atherosclerosis-prone mice fed a high cholesterol diet results in smaller and less complex plaques with reduced plaque collagen and smaller lipid core and no adverse effects on plaque vulnerability (Mackinnon et al., unpublished). The reduced plaque burden was not due to reduced serum cholesterol but was associated with reduced weight gain in galectin-3-/- mice. The mechanism of this reduced weight gain is unclear but it may be due to a previously described function of galectin-3 to stimulate adipocyte proliferation (59). Indeed, high levels of circulating galectin-3 have been associated with obesity (60). Galectin-3-/- macrophages have a specific defect in M2 alternative macrophage activation (21) and there is reduced M2 marker expression in later lesions in galectin-3-/-/ApoE-/-mice. In humans, M2 macrophages predominate in diseased versus normal intima of carotid arteries, and patients with a predisposition towards an M2 phenotype may be more susceptible to atherosclerotic disease (61). Our previous work has shown that galectin-3 mediates alternative/M2 macrophage activation via activation of the dimeric transmembrane protein CD98 (21), a disulfide-linked 125 kDa heterodimeric type II transmembrane glycoprotein (62) which is highly expressed on macrophages (63) (summarized schematically in Figure 3). Deletion of CD98 or inhibition of CD98 function with pharmacological inhibitors prevents M2 activation (21). CD98 may mediate the effects of galectin-3 on M2 activation within plaques and may provide another target for therapeutic intervention. Reduced plaque stability and rupture leading to thrombosis is the most common fatal consequence of atherosclerosis and it will be essential to address this issue in order to fully evaluate the potential of galectin-3 inhibitors as a novel therapy or as an adjunct to statin therapy. Modified citrus pectin is a naturally-occurring pectin found in the peel and pulp of citrus fruits. In the United States of America, MCP is registered as a food supplement and is generally regarded as safe (GRAS). MCP inhibits galectin-3 binding and function in vitro and in vivo (64–66). Administration of 1% MCP in the drinking water to ApoE-/- mice for 4 weeks at the end of a 10 week high cholesterol feeding regimen reduced plaque burden in the descending aorta compared to vehicle control. Therefore, strategies to inhibit galectin-3 function may reduce plaque progression and potentially represent a novel therapeutic strategy in the treatment of atherosclerotic disease.



Figure 3. Schematic showing mechanism for the regulation of alternative macrophage activation by galectin-3. Galectin-3 binding to n-acetyl lactosamine residues (l-nac) on IL-4Ra and CD98 potentiates IL-4-induced STAT6 activation and CD98 mediated activation of PI3-kinase augmenting transcription of alternative activation genes arginase-1 and mannose receptor and expression of galectin-3. (see color insert)

Conclusion In recent years it has become clear that galectin-3 has profound effects on tissue fibrosis. The majority of the in vivo data demonstrates a pro-fibrotic role for galectin-3, mediating myofibroblast activation and extracellular matrix deposition with disruption of tissue architecture and organ dysfunction. However, the component parts of tissue fibrogenesis are exquisitely complex and newer data has highlighted the important cross-talk between cells of the immune system and tissue myofibroblasts in the evolution and resolution of fibrosis. Galectin-3 represents an excellent example of a molecule which can exert potent effects on multiple cell types during the fibrotic process, both through direct effects on scar-secreting myofibroblasts and by altering the behaviour of innate immune cells such as macrophages. Strategies to manipulate galectin-3, such as the emerging small molecule inhibitors, will hopefully result in the development of effective anti-fibrotic therapies.

Acknowledgments N.C.H., A.C.M. and T.S. acknowledge the support of the Wellcome Trust.


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