Poly (ADP-ribose) polymerase 1 (PARP-1) is a constitutive enzyme, the major isoform of the PARP family, which is involved in the regulation of DNA repair, cell death, metabolism, and inflammatory responses. Pharmacological inhibitors of PARP provide significant therapeutic benefits in various preclinical disease models associated with tissue injury and inflammation. However, our understanding the role of PARP activation in the pathophysiology of liver inflammation and fibrosis is limited. In this study we investigated the role of PARP-1 in liver inflammation and fibrosis using acute and chronic models of carbon tetrachloride (CCl4)-induced liver injury and fibrosis, a model of bile duct ligation (BDL)-induced hepatic fibrosis in vivo, and isolated liver-derived cells ex vivo. Pharmacological inhibition of PARP with structurally distinct inhibitors or genetic deletion of PARP-1 markedly attenuated CCl4-induced hepatocyte death, inflammation, and fibrosis. Interestingly, the chronic CCl4-induced liver injury was also characterized by mitochondrial dysfunction and dysregulation of numerous genes involved in metabolism. Most of these pathological changes were attenuated by PARP inhibitors. PARP inhibition not only prevented CCl4-induced chronic liver inflammation and fibrosis, but was also able to reverse these pathological processes. PARP inhibitors also attenuated the development of BDL-induced hepatic fibrosis in mice. In liver biopsies of subjects with alcoholic or hepatitis B-induced cirrhosis, increased nitrative stress and PARP activation was noted. Conclusion: The reactive oxygen/nitrogen species-PARP pathway plays a pathogenetic role in the development of liver inflammation, metabolism, and fibrosis. PARP inhibitors are currently in clinical trials for oncological indications, and the current results indicate that liver inflammation and liver fibrosis may be additional clinical indications where PARP inhibition may be of translational potential. (Hepatology 2014;59:1998–2009)
Globally, alcohol consumption, viral hepatitis, and steatohepatitis are the leading causes of chronic liver injury and inflammation, resulting in liver fibrosis, cirrhosis, eventually culminating in the development of hepatocellular carcinoma. Liver cirrhosis is the major cause of morbidity and mortality among all chronic liver diseases. Liver fibrosis is characterized by increased deposition of extracellular matrix (ECM), resulting in the disruption of cellular architecture. Derangement of the sinusoidal architecture not only affects hepatocytes, but also exerts adverse effects on other nonparenchymal cell types such as hepatic stellate cells (HSCs), myofibroblasts, and endothelial cells, the function of which would be essential in the maintenance of liver structure and function.[3, 4] Activation of HSCs plays a pivotal role in liver fibrogenesis, since these cells are the primary source of ECM deposition upon liver injury. Although our knowledge on the pathomechanisms of liver fibrosis has expanded over the past decade, the clinical options for the treatment of this devastating condition remain severely limited.
Poly (ADP-ribose) polymerase1 (PARP-1) is a constitutively expressed primarily nuclear enzyme, which plays important physiological roles in regulation of numerous cellular processes, such as DNA repair and the maintenance of chromatin structure. In addition, pathological overactivation of PARP-1, due to reactive oxygen and nitrogen species formation, promotes cell death, and stimulates proinflammatory mediator production. PARP-1 functions as a DNA damage sensor and signaling molecule, binding to both single- and double-stranded DNA breaks. Upon binding to damaged DNA, PARP-1 forms homodimers and catalyzes the cleavage of NAD+ into nicotinamide and ADP-ribose to form long branches of ADP-ribose polymers on target proteins such as histones and PARP-1 itself, which results in cellular energetic depletion, mitochondrial dysfunction, and ultimately necrosis. Numerous transcription factors (e.g., nuclear factor-κB), DNA replication factors, and signaling molecules have also been shown to become poly(ADP-ribosylated) by PARP-1.[6, 7] Modulating these processed PARP inhibitors has been shown to exert tissue protective and antiinflammatory effects in animal models of ischemia-reperfusion injury, circulatory shock, and various forms of inflammation.[5, 8] It has also been recently suggested that PARP-1 and PARP-2 (a minor isoform of the PARP enzyme family) play important roles in regulating important metabolic functions in rodents (e.g., mitochondrial function/biogenesis, and adipogenesis) in various organ systems, including in the liver, at least in part via modulation of NAD+ levels and consequently sirtuin 1 activity.[9-12] Furthermore, PARP inhibition has recently been shown to improve mitochondrial function (respiration, enzyme activity, reactive oxygen species defense) in both Caenorhabditis elegans worms and in AML12 hepatocyte cell line, and promoted longevity in worms.
Recent studies have linked PARP-1 activation and up-regulation to the production of profibrotic markers such as connective tissue growth factor (CTGF) and transforming growth factor β (TGF-β) in kidney tubular epithelial and vascular smooth muscle cells. In this study we investigated the role of PARP-1 in liver inflammation, metabolism, and fibrosis using in vivo models of carbon tetrachloride (CCl4)-induced acute and chronic liver injury, a model of bile duct ligation (BDL)-induced hepatic fibrosis, isolated liver-derived cells, and samples from liver biopsies of cirrhosis human subjects. Our findings unveil a pathogenic role of PARP-1 in liver inflammation, metabolism, and fibrosis and indicate the potential therapeutic utility of PARP inhibitors for liver inflammatory diseases and fibrosis.
The novel findings arising from our study are: 1) pharmacological inhibition of PARP or genetic deletion of PARP1 ameliorates the acute and chronic CCl4 treatment-induced oxidative stress, liver injury, and inflammation in murine models; 2) pharmacological inhibition of PARP or genetic ablation of PARP1 also attenuates liver fibrosis induced by chronic CCl4 exposure; 3) inhibition of PARP or genetic deletion of PARP1 in hepatocytes protects these cells from oxidant-induced cell necrosis; 4) inhibition of PARP or genetic deletion of PARP1 abrogates HSCs activation; 5) PARP inhibitors facilitate the recovery of the liver after established liver fibrosis and also attenuate the development of fibrosis induced by BDL, which is less dependent on parenchymal injury; 6) PARP inhibitors facilitate the recovery of mitochondrial and various metabolic functions; and 7) PARP1 activity and PAR accumulation is markedly enhanced in hepatic tissues obtained from patients with liver cirrhosis. These results indicate that oxidative stress and PARP1 play important pathogenetic roles in liver injury, metabolism, inflammation, and fibrosis.
Shiobara et al. reported increased PARP1 expression, activity, and PAR accumulation in hepatic tissues from patients with liver cirrhosis and hepatocellular carcinoma, which is in agreement with our results. The pathogenetic mechanisms involved in liver fibrogenesis include oxidative stress and death of hepatocytes, involvement of an inflammatory cascade resulting in Kupffer cells and HSCs activation, deposition of extracellular matrix, and infiltration of inflammatory cells.[3, 18] Given the important role of PARP1 in the promotion of oxidative stress-induced cell death, metabolic,[9-12] and inflammatory processes,[5-7] we hypothesized that PARP1 may represent a key checkpoint in liver fibrosis and its inhibition could be of therapeutic potential. To gain functional insights on the role of PARP1 in hepatic inflammation, metabolism, and fibrogenesis, we employed a well-established liver injury models induced by CCl4 and investigated whether PARP inhibition or genetic deletion of PARP1 ameliorates liver inflammation, extracellular matrix deposition, and hepatic cellular injury resulting in fibrosis.
CCl4 is metabolized in the liver by cytochrome P450 to generate corresponding free radicals, which attacks the hepatocytes, induces the necrosis of parenchymal cells, augmenting the inflammatory cascade in the liver. Consistent with previous studies, we observed that CCl4 administration induced marked hepatic injury, oxidative stress, inflammatory cell infiltration, and impaired liver function. In line with the aforementioned role of PARP1 in mediating cell death and proinflammatory responses, here we found that loss of PARP1 or its inhibition was associated with attenuated CCl4-induced hepatic injury and inflammation.
Hepatic fibrogenesis develops on the basis of preexisting and continuous liver injury, in part due to inflammatory cell infiltration. Chronic alterations in hepatic homeostasis are believed to transduce various proinflammatory and prooxidant signals involved in fibrogenesis. Activation of HSC is generally triggered by various cytokines/chemokines and oxidants, including potent mitogens such as platelet-derived growth factor and epidermal growth factor.[21, 22] Activated Kupffer cells also secret TGF-β, which causes HSC activation. Recently, the pivotal role linking PARP1 activation and the profibrotic gene expression such as TGF-β and CTGF has been documented in vascular smooth muscle cells and renal proximal tubular epithelial cells in vitro.[14, 15] Furthermore, PARP1 inhibition has been found to abrogate unilateral ureter obstruction-induced CTGF expression and renal interstitial fibrosis. CTGF and TGF-β have been also shown to be pivotal mediators of hepatic fibrogenesis.[23, 24] Consistent with these observations, we observed that PARP inhibition/genetic deletion abrogated the CCl4-induced HSC activation and fibrogenesis.
Necrosis of hepatic parenchymal cells is both the consequence of liver injury, as well as an active player in the promotion of local proinflammatory processes, which may contribute to the activation of HSCs. Profibrotic responses can also be triggered by hepatocyte cell death. Thus, pharmacological agents, which could selectively block the death of hepatocytes, may also prevent Kupffer cell and HSC activation and consequent fibrosis. In fact, our in vitro and in vivo experiments show that PARP inhibition or genetic deletion of PARP1 attenuates hepatocyte necrosis and inflammatory response. It is likely that the net effect of these processes is the interruption of multiple positive feedforward cycles of proinflammatory mediator production, profibrotic mediator production, cell death, and oxidative and metabolic stress. The attenuation of oxidative and nitrative markers by PARP1 deficiency and by PARP1 inhibitors, in fact, points to the existence of such feedforward cycles, and their interruption in the absence of functional PARP1. However, our results, demonstrating that PARP inhibition also attenuates the BDL-induced hepatic fibrosis in vivo, which is less dependent on parenchymal injury, coupled with attenuation of stellate cell activation by genetic deletion of PARP1 or its pharmacological inhibition in vitro, strongly suggest that PARP1 may also have direct regulatory effects on fibrotic processes, independent on protecting from parenchymal injury and inflammation.
Recent studies also revealed an important role of PARP1, and the significantly less abundant PARP2, in regulating cellular metabolism (e.g., mitochondrial function, mitochondrial biogenesis, adipogenesis, among others) in multiple organ systems (including in the liver) via modulation of cellular NAD+ supply, and consequently NAD+-dependent deacetylase enzyme functions.[9-12] Our observation that PARP inhibition attenuated the CCl4-induced mitochondrial dysfunction, decline in mitochondrial number, dysregulation of various key genes involved in metabolism is also in agreement with the important role of PARP1 in metabolic regulation. Furthermore, these results also provide the in vivo proof of the concept support for an elegant recent study demonstrating that PARP inhibition through increased cellular NAD+ levels led to improved mitochondrial homeostasis in worms and various mammalian cells, which was dependent on the activation of the worm sirtuin homolog sir-2.1 and involved induction of mitonuclear protein imbalance, as well as activation of stress signaling via the mitochondrial unfolded protein response with consequent nuclear translocation and activation of FOXO transcription factor DAF-16, promoting longevity.
PARP inhibitors also exert beneficial effects in preclinical and clinical models of cancers via multiple mechanisms involving attenuation of cancer cell proliferation and migration, decrease of angiogenesis, modulation of the tumor proinflammatory environment, and promotion of cancer cell death. The selective promotion of apoptotic cell death in cancer, but not in normal cells, by PARP inhibitors is based on the novel approach of “synthetic lethality” in cancer therapy, because in certain cancers with selective defects in homologous recombination repair (cancer cells frequently harbor defects in DNA repair pathways leading to genomic instability) inactivation of PARP1, and possibly other minor isoforms of PARP, directly causes cell death. Because of this, several classes of ultrapotent PARP inhibitors are currently in clinical trials for the experimental therapy of various malignancies, including triple-negative breast and ovarian cancers. The initial concern with chronic PARP inhibition was the potential genomic instability and “premature aging” of cells. However, the recent study demonstrating that reduced PARP activity extends lifespan in worms, coupled with improved cardiovascular function and energetics in aging rats chronically treated with PARP inhibitors,[27, 28] argues that PARP inhibitors will be well tolerated even during prolonged use in humans.
Our results, demonstrating the pivotal pathogenetic role of PARP1 in liver fibrosis, and the potential of PARP1 inhibitors in restoring liver function after fibrosis, coupled with the clinical availability of PARP1 inhibitors, suggest that repurposing of PARP1 inhibitors for the treatment of liver diseases associated with injury, inflammation, and fibrosis may be of future potential clinical utility.