Cellular and molecular basis of fibrogenesis in NASH

Inflammation 

The fibrogenic progression of NASH is almost invariably associated with inflammation and/or hepatocellular damage. A pivotal profibrogenic mechanism operated by infiltrating cells, such as inflammatory cells, is the synthesis and release of soluble factors playing a biological role with respect to HSCs, including platelet-derived growth factor (PDGF) and transforming growth factor-β1 (TGF-ß1) [1]. In addition, these and other soluble factors, including chemokines, are also expressed by activated Kupffer cells [2]. Some types of leucocytes have been shown to counter-regulate the development of fibrosis. Depletion of natural killer (NK) cells worsens matrix accumulation, through induction of programmed cell death of activated HSC [3].

Oxidative stress 

Oxidative stress has been documented in the liver of patients with NASH and in animal models of steatohepatitis. Compelling evidence accumulated in the last decade indicates that oxidative stress-related molecules, such as reactive oxygen intermediates and reactive aldehydes, act as mediators responsible for the progression of liver fibrosis, inducing different biological actions in HSCs, including type-I procollagen expression [4]. Inflammation and oxidative stress are closely linked, as observed in monocyte chemo-attractant protein-1 knock-out mice, where interference with inflammatory cell recruitment limits the generation of intrahepatic oxidative stress [5].

Apoptosis 

Recent experimental evidence shows that generation of apoptotic hepatocytes is a relevant pro-fibrogenic stimulus, as indicated by direct actions of apoptotic bodies on HSCs [6]. IDN-6556, a general caspase inhibitor, has also been shown to reduce fibrosis in a bile duct ligation model [7].

Factors associated with altered glucose metabolism 

In clinical studies, the presence of diabetes has been recognized as a potent predictor of the presence and progression of fibrosis. Elevated glucose levels up-regulate the expression of TGF-β and connective tissue growth factor (CTGF) by stellate cells [8]. In addition, insulin and insulin-like growth factor-1 also behave as weak growth factors for HSCs, and may induce cell migration [9]. More recently, receptors for advanced glycation end-products were shown to be expressed by HSCs and to be up-regulated by TGF-β [10].

Nuclear hormone receptors 

A number of studies have recently suggested that antidiabetic thiazolidinediones (TZDs), or ‘glitazones’ may represent a possible novel pharmacological treatment for liver fibrosis. TZDs are employed for the treatment of insulin resistance in patients with type-2 diabetes and are selective ligands for the nuclear transcription factor peroxisome proliferator-activated receptor (PPAR)γ [11]. PPARγ is expressed in quiescent HSCs and its abundance and/or transcriptional activity decreases along the activation process that accompanies the acquisition of fibrogenic properties [12], [13], [14], and exposure of HSCs to PPARγ ligands reverts most features of the activated phenotype of HSCs. Daily intragastric administration of rosiglitazone or pioglitazone, started at the same time as injury, leads to a marked reduction of fibrotic tissue accumulation and fibrogenic cell proliferation [15]. These data make appealing the possible therapeutical use of this group of compounds, which have been proposed as a possible treatment of NASH [16].

Endogenous cannabinoid system 

Cannabinoids are a group of molecules with interesting modulatory effects on liver fibrogenesis. There are at least two different cannabinoid receptors, CB1 and CB2. Recent investigations indicate that, while CB2 mediates antifibrogenic actions, activation of CB1 positively modulates the development of fibrosis [17]. Moreover, a CB1 receptor antagonist has been shown to reduce obesity and to ameliorate metabolic parameters in patients with the metabolic syndrome [18].

Renin–angiotensin system 

The renin–angiotensin system is another pivotal player in the pathogenesis of liver fibrosis that might have particular relevance in patients with NASH [1]. Blockade of the renin–angiotensin system or use of aldosterone antagonists attenuates the fibrosis development in different experimental models of chronic liver injury, suggesting that interference with the renin–angiotensin system is a promising strategy to prevent fibrosis progression [1]. This approach might be appropriated particularly in patients with the metabolic syndrome and NASH, where hypertension is often a clinical problem that requires pharmacologic treatment. In addition, local activation of the renin–angiotensin system has been shown to take place in adipose tissue, where it might contribute to the abnormalities related to the metabolic syndrome.

Pattern-recognition receptors 

Several studies have demonstrated the presence of a ‘leaky gut’ in patients with obesity, leading to increased bacterial translocation to mesenteric lymph nodes and to elevation of plasma endotoxin levels, especially in the portal circulation. This phenomenon has been extensively investigated in alcoholic liver disease, where it has been recognized as a relevant mechanism for Kupffer cell activation. More recently, HSCs have been shown to express different membrane receptors belonging to this family, including several members of the toll-like receptor subgroups. These pattern-recognition receptors are bound and activated by products of the bacterial cell wall, such as endotoxin, peptidoglycan and lipoteichoic acid. Interaction of these receptors with their cognate ligands leads to increased expression of pro-inflammatory cytokines and amplification or pro-fibrogenic signals [19], [20].

Adipokines 

This group of cytokines produced by adipose tissue is believed to play a role in NASH, which develops in the presence of an excess of fat. Leptin is a hormone produced by adipocytes, which regulate food intake via actions on the hypothalamus. Different groups have provided compelling in vivo evidence for the pro-fibrogenic action of leptin in rodents [21]. The pro-fibrogenic action of leptin depends, at least in part, on a direct effect on HSCs, which express functional leptin receptors and are directly responsive to leptin. A recently identified activity of leptin on fibrogenic cells is the induction of vascular endothelial growth factor, one of the most potent inducers of neovessel formation [22].

Adiponectin is considered to be a major determinant of sensitivity to insulin, acting at different sites of glucose metabolism, including liver, muscle, and fat itself [23]. Experimental data also show that administration of recombinant adiponectin ameliorates the metabolic derangements and liver damage in mouse models of alcoholic and nonalcoholic hepatitis [24]. A direct antifibrogenic action of adiponectin has also been demonstrated in animals undergoing toxic liver damage, and a balance between the biology of leptin and that of adiponectin seems to take place in stellate cells [25], [26]. Adiponectin’s effects are mediated by two receptors, known as AdipoR1 and AdipoR2 [27], and at least some of the metabolic effects of adiponectin are dependent on the receptor-mediated activation of AMP-dependent protein kinase (AMPK). We recently demonstrated that activation of AMPK by adiponectin or antidiabetic drugs reverts many of the biological features of activated HSCs [28]. The emerging biology of adiponectin makes this molecule a very appealing target for future studies in NASH and other liver diseases.

Other adipokines are possibly implicated in the fibrogenic process. Resistin contributes to insulin resistance in rodents, but its metabolic effects in humans are still uncertain [29]. Evidence obtained in our laboratory indicates that resistin modulates the biology of human HSCs inducing a pro-inflammatory phenotype, and resistin expression is detectable in liver tissue, especially in conditions of damage [30].

In conclusion, a more detailed knowledge of the molecular mechanisms specifically operating in NASH-related fibrogenesis will be instrumental for the identification of novel therapeutic approaches. From such a standpoint, strategies targeting most, if not all, of the aforementioned pathways are currently being evaluated for possible utilization in humans.