Open access peer-reviewed chapter
Abstract
Increasing evidence implicates the enzyme Heparanase in the development and progression of liver steatosis and fibrosis, where high heparanase expression was demonstrated. Morever, inhibition of heparanase activity significantly attenuated the development of fatty liver in animal models. Non-alcoholic fatty liver disease is the most common liver disease in the western world, with the natural course of a chronic progressive condition that is expected to worsen with time. Potential complications of the disease are steatohepatitis, liver fibrosis, liver cirrhosis and even liver malignancies, such as hepato-cellular carcinoma. As such, non-alcoholic fatty liver disease is considered a leading etiology for liver transplantation in the western world. No effective treatment for fatty liver is available so far, and seeking effective treatment strategies is of great importance. The aim of this chapter is to shed light on the knowledge regarding the involvement of Heparanase in the development and progression of fatty liver, opening the opportunity for future research of potential therapeutic options for treating this common liver pathology.
Keywords
- Heparanase
- lipid uptake
- liver steatosis
- fatty liver
- inflammation
- extracellular matrix
1. Introduction
The liver is the largest solid organ in the body, and plays important roles in metabolism and processing of nutrients and toxins, with cardinal functions in the gastrointestinal system and the digestion process. The liver is composed of two lobes- right and left, encapsulated by the fibrous Gleason capsule. Cells in the liver are mainly hepatocytes- composing about 60% of the liver cells, sinusoidal endothelial cells (18%), Kupffer cells (KCs 13%), hepatic stellate cells (HSCs, 4–10%), and NK cells (2%) [1, 2, 3, 4]. Normally, hepatocytes are arranged in cords surrounding bile canaliculi which drain bile secreted from hepatocytes into bile ducts of the portal triad (Figure 1: normal liver).
Figure 1.
Structure of normal liver. The liver is composed of two lobes. Hepatocytes are the main cell type in the liver, and are arranged in cords surrounding canaliculi which drain bile into bile ducts to help in digestion of nutrients in the gut. Sections of H&E stained liver slides from normal liver (upper picture) and fatty liver showing intra-cellular vaculations and cell ballooning (lower picture), magnified X40.
The extracellular matrix (ECM) is a large network of proteins, glycoseaminoglycans and glycoconjugates and other molecules that surround, support and help to maintain normal structure, function, and integrity of body organs. The ECM helps cells to attach to, and communicate with adjacent cells, and plays important roles in cell growth, cell adhesion, cell movement and migration, and additional cell functions.
Heparan sulfate proteoglycans (HSPG) are the main constituent of the ECM. These macromolecules are composed of glycoseaminoglycan chains covalently bound to a protein core, and are different from each other in their structure and configuration, thus performing different roles in the ECM, which are mainly either adhesive or fibrotic [5]. HSPGs are either embedded in the cell surface or located in the ECM, and play important role in cell-cell and cell-ECM communication and interaction [6, 7, 8], thus serve as mediators in both normal biologic and in pathologic processes, like cell differentiation [9, 10], cell adhesion [11], tissue repair [12, 13], tumor formation and spread [5, 10, 14, 15], autoimmune and inflammatory processes [16, 17, 18], diabetes mellitus and its complications [19], and vessel wall pathologies- like atherosclerosis [20, 21, 22]. Heparanase, an endo-β-D-glucoronidase, is the only enzyme in mammalians that cleaves HS chains in the HSPGs in several specific sites along the polysaccharide chains, thus resulting in modification of structure and function of the HSPGs [21, 23, 24, 25]. In its intracellular role, heparanase participates in degradation and turnover of membrane-associated HSPGs [26, 27]. The extracellular enzyme is involved in HSPG degradation by cleavage of the HS chains, resulting in alteration of the basal membrane and ECM structure, and thus affects the pool of HS-bound ligands, which are released into the surrounding environment. All these actions result in remodeling of the ECM network, and enable the diffusion of different cytokines, growth factors, and lipoproteins, which facilitate cell motility and result in angiogenesis, inflammation, coagulation, fibrosis, and stimulation of autophagy and exosome production [28, 29, 30].
Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the western world, and affects up to 30% of adults [31], with increasing prevalence with age, in line with the global pandemic of obesity and type 2 diabetes mellitus. NAFLD is defined as the intracellular accumulation of fat droplets in the hepatocytes (liver steatosis), as evident by either radiologic or histologic testing, in the absence of alternative etiologies of chronic liver disease or secondary causes of liver steatosis (like the use of various drugs, prolonged alcohol consumption, or inherited or acquired several metabolic pathologies). Isolated NAFLD is characterized by liver steatosis (although could be associated with mild chronic inflammation) in at least 5% of hepatocytes [32]. Natural course of the disease is slow progression to non-alcoholic steatohepatitis (NASH), defined by a pattern of characteristic findings that include liver steatosis, lobular and portal inflammation, and injured liver cells in the form of hepatocyte ballooning. The NAFLD activity score (NAS) is used to assess the degree of hepatic steatosis, lobular inflammation, hepatocellular ballooning, and degree of liver fibrosis [32]. In advanced stages, NAFLD may progress to profound liver fibrosis- as assessed by the METAVIR scoring system, liver cirrhosis, portal hypertension, and end-stage liver diseases with related complications, including liver failure, decompensated liver cirrhosis, and even liver malignancies- like hepatocellular carcinoma (HCC) [33], making NAFLD a leading etiology for liver transplantation in the western world [34, 35, 36].
1.1 Normal lipid uptake by the liver cells
Upon food intake, lipid particles in the form of triglycerides (TGs)- the main form of lipids in the food, free cholesterol, and cholesterol ester, undergo emulsification into small lipid particles by bile, and are then hydrolyzed into free fatty acids (FFAs) and monoglycerides by the pancreatic lipase in the small intestine, where these small particles undergo absorption into the blood circulation. After absorption by the intestinal cells, together with phospholipids, cholesterol and proteins, the small lipid particles form chylomicrons that enter the blood from the lymphatic system. The liver is the main organ in the body in which lipid metabolism occurs, and in the liver cells occur processes of lipid digestion, absorption, synthesis, decomposition, and transport.
Most of the lipids in the body are stored in the form of TGs in adipose tissue. FFAs are the main constituents in TGs in body fat, from which they can be dissolved under different circumstances and enter the blood. For being transported in the blood circulation, FFAs bind to albumin, while cholesterol binds to globulin to form lipoproteins, which may contain more TGs in the form of low density lipoprotein cholesterol (LDL-c), or less TGs forming high density lipoprotein cholesterol (HDL-c), and according to the density of the lipoproteins, plasma lipoproteins are divided into four subgroups: chylomicrons, very low density lipoprotein (v-LDL), LDL, and HDL. Upon binding to lipids, proteins take part in transporting lipids in the plasma, together forming the apolipoproteins (Figure 2).
Figure 2.
Schematic presentation of membrane associated structures involved in lipid uptake and metabolism in the liver. Lipid particles ingested in the intestine/formed by lipolysis from fat tissue enter blood circulation, and undergo uptake into hepatocytes utilizing different transmembrane structures, including the low density lipoprotein receptor (LDLR), LDL receptor-related protein (LRP), CD36 and direct endocytosis by heparan sulfate proteoglycanes (HSPGs). Inside the hepatocytes, lipid particles undergo β-oxidation in intra-cellular organelles. HSPGs play rol in lipid uptake and metabolism by hepatocytes. Focus is given to sites where heparanase is active in processing of lipid particles, as well as sites where heparanase inhibition may occur and cause decreased lipid uptake by the hepatocytes. Hpa = Heparanase. LDL-c = low density lipoprotein cholesterol, HDL-c = high density lipoprotein choleterol, HS = heparan sulfate.
While the main source of fat in the body comes from food ingestion, the body can utilize endogenous fat which is stored mainly in the adipose tissue. In different circumstances, fat in the adipose cells may undergo hydrolyzation into glycerol and FFAs by the enzyme lipase. Upon hydrolysis, glycerol and FFAs are released into the blood and can be used as a source of energy or ingested by the liver cells. HSPGs play important role in lipid uptake by hepatocytes, mainly following removal of the attached lipoprotein particles [37, 38].
1.2 De novo lipogenesis
In addition to fat ingested from diet, hepatocytes can produce fatty acids from the oxidation processes of both glucose and amino acids, and synthesize TGs by acetyl CoA. In fact, hepatocytes can also synthesize endogenous cholesterol by the process of De novo lipogenesis (DNL). Cholesterol biosynthesis in the hepatocytes occurs in the endoplasmic reticulum, and involves more than 30 enzymes, such as acetoacetyl CoA, and may contribute to the excess accumulation of fat droplets inside the hepatocytes.
1.3 Development of NAFLD
The pathogenetic basis of NAFLD is accumulation in the hepatocytes of lipid droplets which occurs secondary to an imbalance of lipid handling by the liver, when one or more of several mechanisms takes place. Possible mechanisms include excess delivery of free fatty acids (FFAs) to the liver from adipose tissue, increased production of lipids by the liver (De novo lipogenesis), decreased oxidation of fatty acids within the hepatocytes, and impaired export of the TG-rich v-LDL particles. Accumulation of fat droplets in hepatocytes results in cell ballooning and derangement of the normal structure of the liver, and thus induces activation of inflammatory reaction, with activation of HSCs and KCs, which are both involved in the initiation of inflammatory and fibrogenic responses, then leading to the release of proinflammatory cytokines, chemokines, and growth factors, that further augment the inflammatory process and result in activation of HSCs. Of the cytokines released by KCs, tumor growth factor-β (TGF-β), which induces fibrogenesis through activation of HSCs and reactive oxygen substances, leading to accelerated inflammation and progressive liver damage [39], where KCs release also the proinflammatory cytokines TNF-α, IL-6, and IL-β, which also contribute to liver damage. Moreover, following liver insult, both KCs and hepatocytes secrete fibroblast growth factor 2 (FGF-2), which stimulates hepatocyte regeneration and growth, as well as the proliferation and activation of HSCs. Higher levels of FGF-2 result in excess ECM deposition by HSCs, and induce hepatic tissue perturbation and disruption [40, 41]. Upon activation, HSCs transform to α-SMA positive myofibroblasts, which secrete large amounts of ECM proteins causing profound alteration of the extracellular micro-environment. As these processes aim to repair damaged liver tissue, prolonged injury to the liver together with prolonged activation of the repair system result in degradation of the regeneration process, and in late stages may result in uncontrolled fibrogenesis, excess ECM accumulation, and disruption of liver structure [42, 43, 44, 45]. Indeed, liver tissue repair is also supported by autophagy, a process by which cells degrade their own components by forming autophagosomes and autolysosomes, and in the liver, autophagy is expected in normal circumstances to result in decreased liver fibrosis [46, 47].
1.4 Development of liver steatosis
While triglycerides are the main lipid component that contributes to the development of fatty liver [48], and account for 60–70% of intrahepatic fat accumulation, also low density lipoprotein (LDL) cholesterol is an important contributor to the development of NAFLD, mainly in cases of high serum LDL levels, where it is known that in subjects with NAFLD, the lipid uptake by the liver is increased, due to enhanced expression on hepatocyte cell surface of the LDL receptor (LDLR), LDL receptor related protein (LRP), and the hepatic fatty acid transporter CD36. In addition, in subjects with high serum levels of lipids, hepatic uptake of lipid particles occurs through direct endocytosis by cell membrane, secondary to increased activity of hepatic lipase and lipoprotein lipase, a process that depends on the activity of HSPGs embedded in the cell wall, in response to augmented insulin resistance that complicates patients with obesity and NAFLD [49, 50].
Following lipid uptake by hepatocytes, lipid particles undergo metabolism and β-oxidation through several intra-cellular metabolic pathways, which include ACAT2, ACAT1, HMGCoA reductase, pPAR-α, pPAR-
The progression of liver steatosis occurs through several stages. Upon lipid uptake by hepatocytes, fatty acids undergo β-oxidation in mitochondria, peroxisomes, and microsomes, still with existing controversy regarding the rate of lipid oxidation (whether increased or decreased) in patients with NAFLD [51, 53], and accumulation of triglyceride-rich lipoproteins in hepatocytes is increased [51, 54]. Likewise, in patients with NAFLD increased production of small dense LDL-C was demonstrated [55].
The endoplasmic reticulum is the main intra-cellular site for lipid synthesis and protein folding and maturation, and is an important participant in the development of NAFLD, especially in the presence of impaired LDL-Triglyceride assembly, which occurs due to activation of intracellular signaling pathways, in response to higher levels of intracellular lipid particles [56]. Lipotoxicity is more prominent under higher endoplasmic reticulum oxidative stress, that occurs more frequently in obese subjects with NAFLD, apparently due to activation of the unfolded protein response (UPR). One more mechanism that contributes to the development and progression of NAFLD is the accelerated formation of extracellular vesicles, that are nano-sized particles which are over-secreted by hepatocytes in response to higher content of toxic lipid particles, and play critical role in the pathogenesis of NAFLD by acting as mediators of paracrine signaling, causing HSCs activation, angiogenesis and activation of macrophages, all leading to liver inflammation and chemotaxis [57, 58].
1.5 The enzyme heparanase
Heparanase is the only enzyme in mammalians that is responsible for cleavage of HS side chains in the HSPGs. The human heparanase gene (heparanase 1) is located on chromosome 4q21.3 [59]. Also heparanase 2 was demonstrated, sharing 40% similarity with heparanase 1, but does not exert similar activity like heparanase 1 [60]. The enzyme heparanase is synthesized in the endoplasmic reticulum, then processed in the Golgi apparatus to 65 kDa proheparanase, and then released to the extra-cellular space, where it interacts with many membrane molecules, of which are membrane HSPGs like syndecans [61], resulting in endocytosis and localization into the lyzosomes, where it undergoes cleavage to its two active forms- 50 and 6 kDa. It was proven that the active enzyme has several final destinations in the cell: it could undergo anchorage on the surface of exosomes, included in autophagosomes, translocated into cell nucleus, or even be secreted to the ECM [62]. The enzyme expresses both enzymatic and non-enzymatic activities, inside the cell and in the ECM. In its enzymatic intracellular role, activity of heparanase is mainly degradation and turnover of membrane-associated HSPGs. The extracellular enzymatic activity is mainly degradation of transmembrane and ECM located HSPGs, leading both to alterations in structure and function of HSPGs in the ECM, and resulting in attenuation of HS-bound ligands and proteins which are released into the surrounding environment, causing diffusion of cytokines, growth factors, and lipoproteins, and facilitating cell motility, angiogenesis, inflammation, coagulation, and stimulating autophagic and exosome production [63, 64, 65, 66, 67].
Non-enzymatic activity of heparanase has been demonstrated, although the receptors that could mediate this activity have not been identified so far. Of the non-enzymatic activities of the enzyme, one can mention that the heparanase proenzyme (65 kDa) was demonstrated to induce signaling cascade towards phosphorylation of several proteins, including those involved in intracellular signaling pathways, such as Akt, ERK, p38, and Src [68]. Of the resultant effects of this activity were noted endothelial cell migration and invasion, which are enhanced by the proheparanase-Akt phosphorylation and activation of PI3K [69]. Moreover, latent heparanase was implicated in induction of tumorogenesis, such as the development of glioma, lymphoma, and T-cell adhesion, apparently due to activation of Akt, PyK2 and ERK, and Akt/PKB phosphorylation [70].
2. Heparanase in liver pathologies
2.1 Heparanase in viral hepatitis
As viruses utilize membrane structure for invading cells, HSPGs are involved in the process of cell infection and inclusion of viruses into host cells [71]. In their study, Gallard et al. demonstrated that hepatitis C virus (HCV) propagation resulted in significant heparanase induction in hepatocytes, while downregulation of heparanase resulted in significant attenuation of HCV infection, suggesting an important role of heparanase in HCV life cycle. Heparanase action was apparently secondary to enhancement of CD63 synthesis and exosomic secretion [72].
2.2 Heparanase in liver fibrosis
In their book chapter, Mazola et al. suggest that the interplay between heparanase and components of the immune and the inflammatory responses activate recruitment, proliferation, and activation of myofibroblasts, the main cell types responsible for deposition of fibrous proteins in the ECM [73]. In a study on mice with chronic Ccl4-mediated chronic induction of liver fibrosis, increased heparanase expression was demonstrated, which was mainly co-localized with macrophages in necro-inflammatory areas, where it seems that heparanase plays a key role in the macrophage-mediated activation of HSCs [74]. Further studies supported additional evidence for the involvement of heparanase in fibrotic processes in different body organs, including liver fibrosis [75].
2.3 Heparanase in malignant diseases of the liver
The expression of heparanase in malignant diseases has been extensively studied, including the effects of several heparanase inhibitors that were evaluated in several malignant diseases. In one study, it was shown that heparanase-1 degrades the HS chains on cells of hepatocellular carcinoma (HCC), a process that resulted in the secretion of vascular endothelial growth factor C (VEGF-C) into the medium of HCC cells, while VEGF-C was shown to promote lymphatic endothelial cell growth leading to facilitating lymphatic metastasis [76]. Chen et al. showed elevated expression of heparanase mRNA and protein in HCC cells, where it accelerates cell adhesion in HCC metastasis, an effect that was significantly attenuated following silencing of the enzyme [77]. In the study of Liu et al., the authors showed that inhibiting heparanase activity by the inhibitor PI-88 exerted favorable effects in patients with HCC. Heparanase inhibition resulted in significant delay in the onset of recurrence of HCC, and provided significant survival benefit for up to three years of follow up [78].
2.4 Heparanase inhibition and liver steatosis
In our former studies, we investigated the effect of heparanase inhibition on hemodynamic, metabolic and histopathologic parameters of body systems in mice, mainly aortic atherosclerosis and fatty liver.
In apolipoprotein E deficient (E0) mice, inhibition of heparanase activity by two inhibitors- PG545 (PIXATIMOD), a HS mimetic with proved inhibitory effect on heparanase, caused significant reduction of serum glucose levels, blood pressure and oxidative stress in serum [79]. In another project, we have shown that the same heparanase inhibitor, used in E0 mice placed on high fat diet, significantly attenuated the development of atherosclerotic lesions in representing sections taken from the aortic arch, along significant reduction of the aortic wall thickness compared to control mice. In addition, we demonstrated in the same study that in comparison to placebo, treatment with PG545 significantly reduced blood pressure and serum levels of TC, TGs, and LDL-C, besides significant reduction of oxidative stress in serum, all of which are considered independent risk factors for the development and progression of atherosclerosis [80].
Recently, we studied the effect of two different heparanase inhibitors on the development of liver steatosis in two kinds of mice. In this study, both E0 mice and C57 Balb-c mice were placed on either chow diet or high fat diet, and treated with either PG545, SST0001- a 100% N-acetylated and glycol split non-anticoagulant heparin which exerts potent anti heparanase activity, or placebo injection (normal saline). Both heparanase inhibitors significantly attenuated the development of liver steatosis. In accordance with our former studies, also in this study serum analyses revealed significant reduction in serum levels of TC and TGs, besides lowering the mRNA expression of key factors involved lipid metabolism, including lipid uptake by the liver, lipolysis, lipogenesis, and beta-oxidation in the liver cells. These beneficial effects seem to heparanase-dependent, as inhibition of heparanase resulted in the favorable effect of attenuating the development of fatty liver [81].
3. Conclusions/concluding remarks
Non-alcoholic fatty liver disease is the most common liver disease in the western world. Yet, the mechanisms underlying the pathogenesis of this formidable is largely vague, thus lacks effective treatment so far. HSPGs are the main constituent of the extracellular matrix, and play important roles in liver pathologies, through cell-cell and cell-ECM interactions, besides their role in uptake and processing of lipid particles by the liver cells. Heparanase is the enzyme that degrades heparan sulfate side chains in HSPGs, involves in remodeling of structure and alter the function of the HSPG macromolecules. Heparanase inhibition was proved to exert favorable results in different pathologies, including malignant diseases, complications of diabetes mellitus, kidney pathologies, inflammatory processes, and vessel wall pathologies like atherosclerosis. Recently we have demonstrated remarkable attenuation in the development of fatty liver in animal models with the use of two different heparanase inhibitors, an additional evidence for the involvement of heparanase in the development of fatty liver, putting heparanase inhibition as a reliable target for future research concerning the development of possible effective treatment for NAFLD. It could be wise to impliment heparanase inhibition as a part of treatment approach for preventing and treating NAFLD, along with restriction of lipid uptake, preventing weight gain and weight, and optimal control of additional conditions that contribute to the development of NAFLD, such insulin resistance and diabetes mellitus.
Acknowledgments
Thanks for Professor Z. Abassi from the Department of Physiology and Biophysics, the Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel, for his valuable assistance.
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