Novel molecular targets in hepatic stellate cells for the treatment of liver fibrosis Smith Cortínez, Natalia
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10.33612/diss.128130988
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Chapter 6.
GENERAL CONCLUSIONS AND FUTURE
PERSPECTIVES
I. General conclusions and discussion
Despite the fact that the molecular mechanisms driving liver fibrosis progression have been extensively investigated for decades now, there is still no effective therapy available for treating fibrosis and/or cirrhosis in patients with chronic liver disease. Liver transplantation is the last resort patients for end-stage liver disease. Thus, it is imperative that we discover novel targets to develop innovative drugs to treat liver fibrosis. In this thesis, we focused on the main cell type involved in liver fibrosis progression, the hepatic stellate cell (HSC). HSC produce collagen and other ECM proteins that accumulate in the fibrotic liver, causing stiffness and increasing portal tension, both severe complications for patients. We investigated novel molecular pathways that control HSC activation and collagen release, to find new targets to treat fibrogenesis in patients with chronic liver diseases. The contribution of mitochondrial
metabolism to HSC activation was studied in Chapter 2 and Chapter 3, and the molecular mechanisms driving collagen-I export by HSC was studied in Chapter 4 and
Chapter 5. A graphical summary of the main objectives and results in this thesis is
Figure 1: Graphical summary of main results obtained in this thesis. The main objective of this thesis was to discover novel molecular targets to treat liver fibrosis by preventing -or reversing- HSC activation in vitro. For this, we investigated two main mechanisms: 1. mitochondrial – and glycolytic metabolism and its contribution to HSC activation (Chapter 2 and Chapter 3), and 2. Collagen release by HSC (Chapter 4 and chapter 5). In the figure, we have summarized the model we used, the general aim and the main findings on each experimental chapter. Figure created with BioRender.com.
Contribution of mitochondrial metabolism to HSC activation
Upon liver injury, non-proliferative qHSC lose their lipid content and become proliferative, migratory and ECM-producing myofibroblasts, also referred to as activated HSC (1,2). This transdifferentiation process demands a lot of energy and recent studies support the hypothesis that HSC activation is associated with a strong increase in glycolysis and glutaminolysis to provide the this energy (3–5). In Chapter
2, we analyzed the contribution of mitochondrial oxidative phosphorylation (OXPHOS)
metabolism to HSC transdifferentiation, in direct comparison to glycolysis. As we found that OXPHOS is even more increased during HSC activation compared to glycolysis, we next analyzed the anti-fibrotic properties of a novel IP3R inhibitor (dmXeB) in
mitochondria and thereby strongly affects Ca2+-requiring enzymes of the mitochondrial
TCA cycle (6,7).
In Chapter 2, we analyzed mitochondrial and glycolytic metabolism by functional assays that allowed us to quantify in real-time oxygen consumption, as a measure of mitochondrial metabolism, and extracellular acidification rate, as a measure of glycolysis. In these studies, we used primary rat HSC, because it allowed us to adequately compare quiescent and activated HSC. We demonstrated that rat HSC not only upregulate glycolysis, but also increase mitochondrial respiration to support activation. We showed that the induction of mitochondrial respiration was accompanied by extensive mitochondrial fusion, but did not increase mitochondrial biogenesis or mass. Previous reports showed that both glycolytic and glutaminolytic enzymes were upregulated during HSC activation (3,4). However, no functional assays were performed to characterize the contribution of mitochondrial metabolism towards HSC activation or perpetuation. Thus, this is the first study that shows the functional contribution of both glycolysis and mitochondrial respiration to rat HSC activation. In fact, the functional metabolic assays revealed that mitochondrial metabolism is even more enhanced (5-fold) during activation of HSC, when compared to glycolysis (3-fold). In line, the extensive mitochondrial fusion observed in aHSC is a clear indicator that mitochondria are highly active (8). Surprisingly, we did not observe an increase in individual components of the mitochondrial transport chain (ECT), despite the 5-fold increased mitochondrial OXPHOS activity. Recently, it was shown that mitochondria assemble their ETC proteins into super-complexes upon enhanced energy demands, thereby generating a more efficient ATP-producing machinery without changing mitochondrial mass (9,10). This may also happen in activating HSC. We used specific inhibitors of glycolysis, glutaminolysis and OXPHOS to assess their respective contribution to HSC activation. To target glycolysis, we used 2-deoxyglucose (2DG), a widely used hexokinase 1 (HX1, the first enzyme in glycolysis) competitive inhibitor. To target mitochondrial metabolism we used two different inhibitors: 1. CB-839 to inhibit glutaminase, which blocks the conversion of glutamine to the TCA cycle-intermediate α-ketoglutarate (αKG), or 2. Rotenone or metformin to inhibiting the complex I of the ETC (Figure 2). We found that targeting mitochondrial metabolism by CB-839, rotenone or metformin, had a greater impact on HSC activation than targeting glycolysis with 2DG.
Even though inhibition of glycolysis with 2DG has been a strategy used by many researchers to target cancer cell proliferation, and its use has reached phaseI/II clinical trials, the use of 2DG was discontinued in clinical trials due to lack of therapeutic effects (11). It may not come as a surprise that targeting glycolysis in a systemic approach may case (severe) adverse effects. Indeed, patients that received the glycolysis inhibitor 3-bromopyruvate in an alternative medicine clinic actually died (12). This suggests that, even though 2DG has anti-proliferative and anti-fibrotic properties in
vitro in human aHSC (this thesis) and anti-fibrotic properties in vivo (3), the use of 2DG,
or other glycolysis inhibitors are not suitable for systemic application in patients with liver fibrosis.
CB-839, a recently developed glutaminase inhibitor, strongly inhibited HSC proliferation and suppressed the expression of activation markers Col1a1 and Acta2. CB-839 shows promise for the treatment of for fast-growing cancers since it decreases proliferation of cancer cells in vitro and reduces tumor growth in vivo (13–15) and does so by decreasing glutamine consumption, glutamate production, oxygen consumption, glutathione levels and several tricarboxylic acid cycle (TCA) intermediates. It is thought that the decrease of TCA cycle-derived intermediates in CB-839-treated cells is the main contributor to the reduced proliferation observed in vitro (11,16). This suggests the mechanism of action in CB-839-treated HSC could be the same, however further experiments are needed to confirm this. If adverse effects of CB-839 therapy in these clinical trial remain low/acceptable, this drug may also be tested for the treatment of liver fibrosis. Moreover, given the anti-tumor effects of CB-839, it is tempting to speculate that this drug may also reduce the risk of cirrhosis-associated hepatocellular carcinoma.
Metformin is the recommended first-line oral therapy for type 2 diabetes, as it is very safe and has been in clinical use for over 50 years (17). Metformin inhibits complex I of the ETC, which lowers energy availability, activating the energy sensor adenosine monophosphate kinase (AMPK) and thereby inhibition gluconeogenesis and glycogen synthesis in the liver. Moreover, it stimulates insulin signaling and glucose transport in muscles. These processes help to reduce hypoglycaemia in diabetic patients (17,18). Interestingly, metformin was shown to improve liver histology and serum alanine aminotransferase (ALT) levels in NASH patients (19,20). Moreover,
Figure 2: Inhibitors and its targets used in Chapter 2. Targeting glycolysis was done by using 2-Deoxyglucose to inhibit the first enzyme in glycolysis, hexokinase 1 (HX 1). Targeting mitochondrial metabolism was done by using metformin to inhibit complex I of the electron transport chain and by using CB-839 to inhibit the conversion of glutamine to alpha ketoglutarate (a-KG) by inhibiting glutaminase.
type 2 diabetes patients that were diagnosed with cirrhosis and continued with metformin treatment showed a 57% reduced mortality rate compared to cirrhotic type 2 diabetes patients that discontinued metformin treatment. This may suggest that metformin indeed has beneficial effects liver fibrosis/cirrhosis.
Another potent regulator of HSC activation, as well as mitochondrial respiration, is calcium (Ca+2) (21–25). Ca+2 plays a key role in regulating HSC proliferation,
migration and contraction. The activity of several enzymes in the TCA depends on Ca+2
and, as a consequence, mitochondrial respiration is impaired when calcium concentrations are reduced in mitochondria (6,26,27). Following these observations, in Chapter 3 we targeted ER-to-mitochondria transfer of Ca+2 in HSC, by using a novel,
chemically-synthesized compound, desmethyl Xestospongin B (dmXeB). DmXeB is a
novel variant of the natural compound Xestospongin B that is produced by a marine sponge (6). XeB is a specific inhibitor of IP3R and prevents the transfer of Ca+2 from
ER to mitochondria (6), thereby creating a bioenergetic crisis that selectively kills cancer cells (7,25). Acquiring the natural compound involves scuba diving for
0.0084% of Xestospongin B from the dry weight of the sponge (28,29). Given its therapeutic potential, efforts have been made to chemically synthesize XeB and generate XeB-derivatives that may be more potent and/or specific. DmXeB is one of several XeB-derivatives synthesized so far, and shows similar inhibition propertiesas XeB (Armen et al. 2020, unpublished). In Chapter 3, we observed that dmXeB has anti-fibrotic properties in vitro, as it prevented and reversed HSC activation by reducing mitochondrial metabolism. This compound retained the vitamin A-storing properties of HSC, while they were cultured in vitro. In the context of finding novel drugs to treat liver fibrosis, it is of key importance that such compounds are not only highly specific, but also not toxic to other (liver) cells, tissues or organs. To analyze this for dmXeB, we tested its possible toxicity in primary rat hepatocytes, the most abundant cell type in liver, and in human liver organoids, as representative of liver-resident stem-cells. Concentrations of dmXeB (5 µmol/L) that strongly reduced HSC proliferation, migration and activation, caused little or no toxicity in primary rat hepatocytes or primary human liver organoids. Higher dmXeB concentrations (10 µmol/L) were highly toxic for primary rat aHSC, but not for primary rat hepatocytes or primary human liver organoids. Moreover, dmXeB did not show systemic toxicity in a physiological context, since tail vain injection with high concentrations of dmXeB did not cause lethality nor evident toxicity to major organs in mice (Cardenas et al., unpublished data). These encouraging results suggest that dmXeB may have potential to be used as specific drug to prevent and/or reverse liver fibrosis without causing severe (hepatic) side effects. To further confirm the anti-fibrotic properties in vitro, experiments using dmXeB as treatment in animal models of liver fibrosis, such as CCL4 in mice, should be
performed next. Also, to make the translation to human liver fibrosis, dmXeB could be used to analyze its effect on treat primary human HSC and/or LX-2 cells, or precision-cut human liver slices (PCLS). PCLS allow to test anti-fibrotic compounds ex vivo in the context of the complex cellular composition and architecture of the (human) liver (30).
Molecular mechanisms driving collagen-I release by HSC
The key hallmark in HSC activation that strongly contributes to liver fibrosis progression is the excessive production and deposition of ECM proteins. The main components of fibrotic ECM in the liver are collagen-I and –III. Chapters 4 and 5 focused on analyzing the molecular mechanisms involved in collagen-I production and
secretion, specifically for human HSC. Remarkably, the mechanisms driving collagen-I export in human HSC are actually far from clear. Specifically, the factors that are required for procollagen-I to leave the ER (hydroxylation) and the vesicle-mediated traffic from ER to the plasma membrane were studied. For this, we used primary human (activated) HSC and the human HSC cell line LX-2 cells. Moreover, we analyzed healthy and cirrhotic human liver tissue for fibrosis-associated regulation of selected genes.
In Chapter 4, we investigated the role of ascorbic acid, e.g. Vitamin C, in collagen production and secretion. Ascorbic acid is a key vitamin for humans (and guinea pigs), since these two species carry a deleterious mutation in a gene (GULO) that is required for synthesizing ascorbic acid. Ascorbic acid is an essential co-factor for hydroxylases involved in collagen biosynthesis and, as a consequence, it is well-known to be necessary for collagen-I production and release in fibroblasts from various tissues. However, this had not been established for human HSC yet. In fact, the role of ascorbic acid in liver fibrosis is highly controversial, as some reports claim that its anti-oxidant properties suppress proliferation of rat HSC (31) and progression of liver fibrosis in rats (32), while others observed pro-fibrotic effects of ascorbic acid in in vivo and in vitro models (33–35). Since the laboratory animals can synthesize ascorbic acid themselves, it is crucial to study this in human models. In Chapter 4, we showed that ascorbic acid is necessary for collagen maturation and release in primary human HSC and LX-2 cells. Although this is in line with observations made with other types of fibroblasts, it is an important finding as culture methods for primary human HSC do not routinely include ascorbic acid supplementation. Thus, this key feature of HSC, e.g. collagen production, maturation and release, is actually hardly happening in standard
in vitro culture conditions of human HSC. Interestingly, human HSC only express one
of the 2 dedicated ascorbic acid transporters e.g. SLC23A2/SVCT2, which is upregulated during HSC activation and in human cirrhotic livers, suggesting that activating HSC increase the capacity to take up ascorbic acid to support efficient collagen production and deposition. Surprisingly, LX-2 cells died when we genetically suppressed SLC23A2/SVCT2 expression, indicating that vitamin C is actually a primary requirement for HSC viability. Since collagen export is highly dependent on hydroxylase activity, we tested a novel and safe hydroxylase inhibitor, dimethyloxalylglycine (DMOG), which is currently being tested in clinical trials for
various diseases (36), and found that it effectively suppressed collagen release and also HSC activation. These results suggest that DMOG could also be a good candidate to treat liver fibrosis and could be implemented soon as its use in humans has already been approved. DMOG has been used to prevent intestinal fibrosis in animal models, and it was shown to inhibit the TGFβ signaling in intestinal fibroblasts, in addition to reducing collagen-I deposition (37). To validate anti-fibrotic actions in vivo, DMOG should be applied in animal models of liver fibrosis, such as CCl4 in mice. As for
dmXeB, translation to human can also be made by exposing human PCLS to DMOG in the absence or presence of TGFβ and/or ascorbic acid. Ideally, such studies would result in a combination therapy of DMOG and vitamin C, in order to prevent collagen deposition (by DMOG) and at the same time supply other cell types with the anti-oxidant actions of vitamin C.
Following ascorbic acid-mediated hydroxylation, collagens are self-assembled into triple helixes and loaded into COPII-positive vesicles (38,39). Traffic through the trans-golgi network (TGN) of secretory proteins is well-described for 60-90 nm-sized COPII vesicles (38). However, collagen-I assembles into 300-400 nm structures already in the ER (38,40,41). The identification of TANGO1 (Transport ANd Golgi Organization 1) helped to further describe collagen-IV traffic through the TGN, since it allows COPII vesicles to accommodate oversized cargo at ER exit sites (39). However, this pathway is not used by collagen-I, as it was efficiently exported in the absence of TANGO1 (39). P4-ATPases are a family of 14 different ATPases that control lipid asymmetry in biological membranes and participate in the biogenesis of intracellular transport vesicles in biosynthetic and endocytic pathways (42). In Chapter 5, we inhibited P4-ATPase activity in LX-2 cells to reveal its possible contribution to intracellular collagen-I trafficking and secretion. We used also CRcollagen-ISPR/Cas technology to completely block CDC50A expression, an essential binding partner for most P4-ATPases, in LX-2 cells. Collagen-I accumulated in large intracellular vesicles in LX-2-CDC50A knock-out cells, which was accompanied by reduced collagen-I secretion. Interestingly, a similar phenotype was observed when the expression of only one selected P4-ATPase, e.g. ATP10D, was genetically suppressed, while this was not observed in ATP8B1 knock-out LX-2 cells. Genetic variants of ATP10D are associated with metabolic disease and to elevated sphingolipids in human plasma, which are known to activate TGFβ signaling and potentiate liver fibrosis progression. However, it is unlikely that ATP10D
affects collagen-I production by dysregulated sphingolipids production and downstream transcriptional regulation, since we did not observe a strong effect on mRNA levels of COL1A1 in ATP10D or CDC50A-knockdown cells. Interestingly, the common laboratory mouse strain C57BL/6 has an Atp10d-gene inactivating nonsense mutation, which leads to the expression of a truncated inactive form of ATP10D (43). These mice are known to be less susceptible to CCl4-induced liver fibrosis compared
to other mice strains, which may thus be related (in part) to the absence of functional ATP10D in these mice (44). Transgenic C57BL/6 mice have been generated in which functional ATP10D is reintroduced (45), and it is interesting now to analyze whether these mice are more susceptible to fibrosis-inducing conditions compared to the “wild type” C57BL/6 strain. Mutations in several other P4-ATPases have also been linked to human disease, as it is the case for mutations in ATP8B1 that cause progressive familial intrahepatic cholestasis type 1 (PFIC1) (46). Collagen I production and secretion was normal after gene-inactivation of ATP8B1 in LX-2 cells, indicating that a role in fibrogenesis is not a general feature of P4-ATPases in HSC. Still, human HSC express variable levels of 10 other P4-ATPases, so it cannot be excluded that one or more family members also assists in ECM production and secretion.
II. Future
perspectives
In Chapter 2 we compared the role of mitochondrial and glycolytic metabolism in HSC activation, and targeted each one to discover novel molecular targets for possible treatment of liver fibrosis. Inhibiting mitochondrial metabolism with metformin (inhibiting complex I of the ETC) or with CB-839 (inhibiting glutaminolysis) showed promising results to suppress the activation in human HSC. Metformin is an FDA-approved drug used for decades in clinical practice for the treatment of metabolic disease, in particular type 2 diabetes (T2D)(18). Due to its safety and effect on cell bioenergetics, metformin is also used to treat gestational diabetes, polycystic ovary syndrome, colon and breast cancer (18). The main side effect of metformin is lactic acidosis, which usually occurs in patients with underlying diseases, like liver disease. However, these observations are currently being questioned and some authors even suggest that there is evidence that metformin may protect against lactic acidosis in liver disease (47). Interestingly,T2D patients typically suffer from fatty liver disease and associated liver fibrosis also. Liver fibrosis may improve as a secondary effect of the effective treatment of T2D, but our results suggest that this may actually also be a primary effect of metformin. Thus, metformin could be considered as a drug to treat liver fibrosis, irrespective of the underlying cause on chronic liver disease. Importantly, recent data show that metformin is safe for use in T2D patients with cirrhotic liver disease (48). Furthermore, CB-839 effectively suppressed activation of human HSC. The therapeutic potential of CB-839 is currently being tested in several clinical trials in patients with advanced metastatic solid tumors, non-small cell lung cancer, myelodysplastic syndrome or colorectal cancer (ClinicalTrials.gov IDs: NCT03875313, NCT03831932, NCT03047993, NCT02861300, NCT03263429Our data suggest that CB-839 may also have therapeutic potential for the treatment of liver fibrosis. Moreover, given its anti-cancer properties, CB-839 may also prevent the progression of cirrhosis to hepatocellular carcinoma (HCC).
The aim of Chapter 2 in this thesis was to test the hypothesis that HSC activation could be prevent or reversed by using mitochondrial metabolism inhibitors. As we confirmed this hypothesis, it is tempting to speculate that other FDA-approved drugs that target mitochondrial metabolism could also have beneficial effects in treating liver fibrosis. Many FDA-approved drugs target the mitochondrial electron transport chain complexes, oxidative phosphorylation (OXPHOS), or the entry of anions to
mitochondria (49). For an excellent overview of available FDA-approved drugs that target mitochondrial metabolism we refer to Aminzadeh-Goharis et al. (49). For example, the use of tigecycline (a common antibiotic) has been shown to have anti-carcinogenic properties in vitro and in vivo in acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, large B cell lymphoma, lung and ovarian cancer, renal cell carcinoma and retinoblastoma (49). Tigecycline acts on cancer cells by inhibiting mitochondrial respiration, leading to a bioenergetic crisis, oxidative stress and cellular damage (50). Another example is the compound VLX600, which has been validated for the safety and tolerability in patients with refractory advanced solid tumors (51), and it has been shown to inhibit the growth of colon carcinoma cells in vitro and
in vivo as well as growth of breast cancer cells in vitro (49). Thus, repurposing these
available drugs for the treatment of liver fibrosis could be a rewarding approach. In Chapter 3 we tested the efficacy of a novel IP3R inhibitor to target
ER-to-mitochondrial calcium transfer and reverse HSC activation in vitro. This showed a strong inhibition of HSC activation with little or no toxicity towards primary rat hepatocytes or primary human liver organoids. Many other calcium inhibitors have been used in vitro and in vivo to prevent HSC fibrogenesis (21,52–54). These reduce HSC activation by lowering mitochondrial metabolism. Ca+2 has a wide spectrum of
functions, acting not only as second messenger in signaling pathways, but also as cofactor for many enzymes (27,55). Moreover, it is known to play a key role in HSC activation mechanisms (22). Unfortunately, many Ca+2 channel blockers used for
hypertension, angina pectoris, arrhythmias and heart failure have been linked to drug-induced liver disease, causing metabolic injury (56). For this reason, it is of crucial importance to study the selectivity of dmXeB to target HSC in a pathophysiologic context, e.g. in in vivo experiments using laboratory animals and/or using PCLS that allow the complex interaction between hepatic cells that occurs in liver disease and is needed for proper drug testing. Another strategy to assure selectivity and avoid off-target effects in other organs is to deliver dmXeB (or other drugs) into HSC in vivo in targeting devices, such as liposomes. Liposomes are closed spherical vesicles, consist of a bilayer lipid membrane that encloses an aqueous phase where drugs can be stored (57). Such liposomes can be further modified so that they specifically target to activated HSC via the mannose-6-phosphate/insulin-like growth factor receptor (M6P/IGFII receptor) (58). Combining the HSC targeted-delivery of liposomes and the
specific anti-fibrotic effects of dmXeB could be a promising strategy for treating liver fibrosis.
In Chapter 4, we describe the need of ascorbic acid for human HSC to efficiently produce and secrete collagen. Moreover, we showed that human HSC express only one (SLC23A2/SVCT2) of the two known ascorbic acid transporters, while hepatocytes express them both. SLC23A2 expression is enhanced in human cirrhotic liver as well as in activated HSC in vitro. SLC23A2 appeared to be an essential gene in LX-2 cells, as all LX-2 cells died 7 days after transduction with the SLC23A2-targeting lentivirus. Thus, SLC23A2/SCVT2 may be direct drug target to treat liver fibrosis. Flavonoids have been described to inhibit ascorbic acid uptake in cells by inhibiting the active transporters SVCT1 and SVCT2 (59,60), as well as the passive ascorbic acid transporters GLUT1 and GLUT3 (61). Furthermore, the flavonoid quercetin ameliorates liver fibrosis in CCL4-treated mice (62). Though the therapeutic actions of
quercetin appear mostly linked to its anti-oxidant capacity, a potential role in controlling vitamin C uptake by HSC, and thereby suppressing collagen deposition, can also be hypothesized, in particular in chronic liver disease. To continue on this line, the direct effect of quercetin on SCVT2-mediated ascorbic acid transport should be studied.
The work presented in chapter 4 also hinted for another therapeutic strategy to suppress excessive ECM production in liver disease. The hydroxylase inhibitor DMOG efficiently suppressed collagen-I production and release in activated HSC, both in normoxic and hypoxic conditions. Hypoxia, e.g. oxygen shortage, is a typical condition in diseased organs, including the fibrotic liver, which further activates HSC and induces angiogenesis (63). Hydroxylases inhibitors are currently being tested for therapeutic efficacy in anemia (36), and appears to be well-tolerated by patients. Thus, also this class of drugs, e.g. hydroxylase inhibitors like DMOG, are interesting leads to test for their anti-fibrotic properties in treating liver disease.
In Chapter 5, we investigated the role of P4-ATPases in collagen-I release by human HSC. We found that the ATP10D-CDC50A complex is involved in collagen-I secretion, since the absence of either ATP10D or CDC50A, caused intracellular collagen-I accumulation in LX-2 cells. Gene variants of ATP10D gene are associated with increased sphingolipid concentration in plasma, contributing to diseases like diabetes, obesity, myocardial infarction and atherosclerosis (64–66). It is interesting
now to assess whether such ATP10D variants also associate with (liver) fibrogenesis. Since we tested the role ATP10D only in HSC, we cannot rule that it may function similarly in (myo)fibroblasts from other tissues. Future research may therefore focus on the role of ATP10D and/or CDC50A in 1) the release of other types of fibrillar collagens (types II or III) and 2) the release of collagen-I in skin, lung, or intestinal myofibroblasts. In order to translate this findings into a therapeutic approach, more research needs to be conducted to clarify the mechanism by which ATP10D-CDC50A-collagen-loaded vesicles leave the ER and exit the plasma membrane, especially to determine if COPII and/or TANGO proteins participate in the vesicle formation. An interesting approach could be the use of small inhibitors to target CDC50A or ATP10D, specifically targeted to HSC. Although there are currently no inhibitors of ATP10D available, inhibitor screening tools are available for other P4-ATPase family members that could be adapted for ATP10D (United States Patent Application 20170023548).
In summary, the studied molecular and cellular processes that lead to HSC activation and ECM production revealed several high-potential targets for the treatment of liver fibrosis. Their therapeutic impact should be further studied in pre-clinical models to be translated to patient treatment.
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