This post reviews some key research findings regarding liver regeneration and discusses what is known about the mechanisms involved. It turns out that a bunch of my favorite blog topics are involved: telomerase, stem/progenitor cells, mTOR signaling, MAPK signaling, NOTCH signaling and NF-kappaB activation.
“The liver is the largest and most metabolically complex organ in humans(ref),” it has a complex internal structure(ref), and it performs many diverse tasks required for the health of other body systems(ref). Further, the human liver has a remarkable capacity for self-regeneration when damaged or when even up to 80% of it is removed by surgery(ref). Unlike almost any other organ in humans, it can simply grow back. If a section of the liver is removed, as part of regeneration “normally quiescent hepatocytes undergo one or two rounds of replication to restore the liver mass by a process of compensatory hyperplasia(ref).” Hepatocytes are the central cells involved in normal liver functioning making up 70% to 80% of the mass of the liver. Compensatory hyperplasia simply means extra fast proliferation of cells to replace a lost part of the liver. The amazing thing is how fast this happens. In a study of regeneration in pig livers, maximum regenerative response occurred three days after up to 70% partial hepatectomy(ref). “The timing and events of the regenerative response in the pig compared favorably with other animal models and the maximum regenerative response occurred on the third postoperative day, irrespective of the size of the partial hepatectomy.”
Liver regeneration is not always possible depending on the nature and extent of damage, and the age of the patient involved. According to the American Liver Foundation, around 17,000 Americans are currently on a waiting list for a liver transplant. Understanding of what is involved in liver regeneration might assist in facilitating regeneration beyond that normally possible and possibly in developing regeneration strategies for other human organs.
· Telomerase activation is centrally involved in liver regeneration
Since telomere shortening has long been known to limit organ regeneration(ref), it can be concluded a-priori that liver regeneration must involve the expression of telomerase. The publication Regeneration in pig livers by compensatory hyperplasia induces high levels of telomerase activity tells an important part of the story. In simple language, liver regeneration is accompanied by a burst of telomerase activation. “Quiescent human hepatocytes exhibit very low or undetectable levels of telomerase activity. However, hepatocytes display a remarkable proliferative capability following liver injury. To investigate whether liver regeneration by compensatory hyperplasia is associated with telomerase activation, we measured telomerase activity in pig livers after 70 to 80% partial hepatectomy using a fully quantitative real-time telomeric repeat amplification protocol. In contrast to commonly studied inbred laboratory mouse strains, telomere length and telomerase activity in porcine tissues are comparable to humans. RESULTS: Following partial hepatectomy, histology revealed mitotic hepatocytes as marker for compensatory hyperplasia. As expected, there was no induction of inflammation. Telomerase activity increased significantly showing the highest levels (5-fold upregulation) in pigs treated with partial hepatectomy and hepatic decompression. Moreover, telomerase activity significantly correlated to the number of mitotic hepatocytes. CONCLUSION: Our data demonstrate telomerase activation in liver regeneration by compensatory hyperplasia in a large animal model with telomere biology comparable to humans. Telomerase activation may constitute a mechanism to protect proliferating liver cells against telomere shortening and oxidative stress.” [Some of the research reported here is based on working with pig livers, good models of human livers. “Moreover, pig telomeres are comparable to those of humans regarding length and shortening during aging (ref)(ref). Because of these similarities, pigs have been utilized as model system to investigate telomerase regulation and telomere dynamics in mammalians(ref)(ref).”]
· Telomerase activation possibly allows extensive mitosis (cell division) of hepatocytes
The publication Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential provides data with respect to fetal hepatocytes that may or may not apply to the mature working hepatocytes that remain in the working part of a partially destroyed liver. The study was of fetal hepatocytes in-vitro. “Telomerase-reconstituted cells were capable of preserving elongated telomeres, propagated in culture beyond replicative senescence for more than 300 cell doublings (to date), and maintained their liver-specific nature, as analyzed by a panel of hepatic growth factors, growth factor receptors, and transcription factors as well as albumin, glucose-6-phosphatase, glycogen synthesis, cytochrome P450 (CYP) expression profiles, and urea production. Moreover, the immortalized cells exhibited no oncogenicity, and no up-regulation of c-Myc was detected. The cells engrafted and survived in the liver of immunodeficient mice with hepatocellular gene expression.” The extent to which this finding applies to mature hepatocytes in-vivo subject to niche signaling is unknown.
· The differentiation of hepatic progenitor cells is involved.
An important question is “where do the new hepatocytes come from? There are two possibilities: mitosis (cell division) of the hepatocytes already in the liver, and differentiation of hepatic progenitor cells to make new hepatocytes. “Hepatic progenitor cells are immature epithelial cells that reside in the smallest ramifications of the biliary tree in human liver. These cells are capable of differentiating toward the biliary and the hepatocytic lineages. — an increased number of progenitor cells (referred to as “activation”) and differentiation of the same toward hepatocytes or bile duct epithelial cells, or both, is a component of virtually all human liver diseases. The extent of progenitor cell activation and the direction of differentiation are correlated with the severity of the disease and the type of mature epithelial cell (hepatocyte or bile duct epithelial cell), respectively, that is damaged(ref).”
These findings are interesting but raised my thirst for answers to several other questions: 1. What cells are making the telomerase (e.g. hepatic progenitor cells or hepatocytes)? 2. What is the role of the telomerase in liver regeneration? 3. To what extent is the liver regeneration due to mitosis of existing hepatocytes and to what extent is it due to differentiation of hepatic progenitor cells? 4. What other stem cells might be involved, such as for renewing the supply of hepatic progenitor cells? 5. What kind of signaling is articulating the whole regeneration process? 6. What hope does all this offer for longevity? So, I went on a literature quest for a couple of days. I found a number of research papers that cast slivers and rays of light on some of the questions.
· Normally by themselves, hepatocytes do not express telomerase
In fact the publication In vitro expansion of human hepatocytes is restricted by telomere-dependent replicative aging says “As expected, untransduced PHH (proliferating human hepatocytes ) progressively lost telomeric repeats and arrested after 30-35 cell divisions with telomeres of less than 5 kilo bases. In comparison, telomerase-reconstituted PHH maintained elongated telomeres and continued to proliferate as shown by colorimetric assays and cell counts. In this study, telomere stabilization extended the proliferative capacity of in vitro proliferating human neonatal hepatocytes. Therefore, telomere attrition needs to be addressed when developing techniques to expand human hepatocytes.”
· Liver regeneration capability declines with the age of the animal or person
An explanation is given in a publication entitled Aging Reduces Proliferative Capacities of Liver by Switching Pathways of C/EBPÎ± Growth Arrest. “The liver is capable of completely regenerating itself in response to injury and after partial hepatectomy. In liver of old animals, the proliferative response is dramatically reduced, the mechanism for which is unknown. The liver specific protein, C/EBPÎ±, normally arrests proliferation of hepatocytes through inhibiting cyclin dependent kinases (cdks). We present evidence that aging switches the liver-specific pathway of C/EBPÎ± growth arrest to repression of E2F transcription. We identified an age-specific C/EBPÎ±-Rb-E2F4 complex that binds to E2F-dependent promoters and represses these genes. The C/EBPÎ±-Rb-E2F4 complex occupies the c-myc promoter and blocks induction of c-myc in livers of old animals after partial hepatectomy. Our results show that the age-dependent switch from cdk inhibition to repression of E2F transcription causes a loss of proliferative response in the liver because of an inability to induce E2F target genes after partial hepatectomy providing a possible mechanism for the age-dependent loss of liver regenerative capacity.” No surprise since many studies show that c-myc plays critical roles in cell proliferation and differentiation.
· Liver regeneration involves complex signaling
This signaling has been studied for some time. I have already mentioned the roles of E2F transcription and c-myc. A 1998 paper Signal transduction during liver regeneration states “Following partial hepatectomy (PH), there is a rapid and highly orchestrated series of biochemical events which occur prior to cellular proliferation. Some of these events are presumably intimately linked with the eventual regeneration of the liver, whereas others are likely to be stress related or required for the continued differentiated function of the liver while regeneration is occurring. The regulation of the AP-1 transcription factor c-Jun during hepatic regeneration has been studied here. — It is concluded that the stimulation of one-third or two-thirds PH (partial hepatectomy) activates JNK through a mechanism that requires TNFalpha, which phosphorylates the c-Jun activation domain in hepatocytes, resulting in enhanced transcription of AP-1-dependent genes. — the induction of NFkappaB during liver regeneration following PH appears to be a required event to prevent apoptosis and to allow for normal cell cycle progression.” I comment that the induction of NF-kappaB is not surprising since such translocation of NF-kappaB into the cell nucleus is required to activate the expression of a number of growth-related genes.
· MAPK, mTOR and Notch pathways are involved in liver regeneration, suggesting that stem/progenitor cell differentiation plays an important role.
The publication PI3K-FRAP/mTOR pathway is critical for hepatocyte proliferation whereas MEK/ERK supports both proliferation and survival suggests intimate involvement in liver regeneration of two pathways that have been discussed in this blog: mTOR and MAPK/ERK, and lends further weight to the argument that differentiation of stem/progenitor cells may be critically involved. The blog post More mTOR links to aging theories links mTOR expression to the quiescence and proliferation of hematopoietic stem cells. I made the point “Effective mTORC1 negative regulation is essential for keeping the stem cell supply chain working well, at least insofar as hematopoietic stem cells are concerned.” That is, the negative regulation is needed to keep rate of differentiation of hematopoietic stem cells under control so as not to exhaust the pools of such cells. When the body’s task is regenerating a liver, on the other hand the need is for increased differentiation of stem/progenitor cells to create new hepatocytes, that is activation of the P13K-FRAP/mTOR pathway as described in the above-mentioned publication. At least, that is my conjecture.
The same publication mentions activation of the MAPK/ERK pathway. “–partial hepatectomy induces a rapid but transient activation of mitogenic signal transduction pathways, in particular phosphoinositide 3-kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)(ref).” The role of the MAPK/ERK and Notch pathways in the stem cell supply chain are discussed in my recent blog post Niche, Notch and Nudge. MAPK/ERK appears to be very important regulator of Notch activity, and Notch activity in turn is essential for the inter-cellular communication required to form a complete organ. You can’t get a complicated highly-structured organ like the liver without the cells doing a lot of talking to each other.
· Progenitor cell rejuvenation can reverse age-related decline in liver renewal by restoring Notch signaling
The “letter to nature” Rejuvenation of aged progenitor cells by exposure to a young systemic environment discusses the role of Notch signaling with respect to liver progenitor cells. “The decline in hepatic progenitor cell proliferation owing to the formation of a complex involving cEBP-a and the chromatin remodeling factor brahma (Brm) inhibits the regenerative capacity of aged liver. — The exposure of satellite cells from old mice to young serum enhanced the expression of the Notch ligand (Delta), increased Notch activation, and enhanced proliferation in vitro. Furthermore, heterochronic parabiosis (a fancy term for pairing up old mice with young mice) increased aged hepatocyte proliferation and restored the cEBPa complex to levels seen in young animals. These results suggest that the age-related decline of progenitor cell activity can be modulated by systemic factors that change with age.” Further, similar pairing of old mice did not result in comparable cell rejuvenation. “The cEBP- Î±–Brm protein complex was detected in liver extracts from old isochronic parabionts but not in young isochronic parabionts. Notably, the formation of the cEBP- Î± –Brm complex was diminished in liver extracts from old heterochronic parabionts. The complex was present at elevated levels in young heterochronic parabionts as compared to young controls. This was also consistent with the modest inhibition of hepatocyte proliferation in young heterochronic parabionts. A young systemic environment restores a young molecular signaling profile to aged progenitor cells in the liver and also appears to enhance their proliferation(ref).”
· The research that I read leaves me unclear as to the primary actions and locations of telomerase activation and the relationship of telomerase activation to hepatocyte proliferation. I hypothesize that one important role of the increased telomerase expression is to accelerate the differentiation of hepatic progenitor cells into new hepatocytes.
The pig-liver researchers stated “Based on our results, we propose that telomerase activation in proliferating hepatocytes is the main cause for increased telomerase activity in regenerative liver nodules. This conclusion is supported by the significant correlation of telomerase activity to the number of mitotic hepatocytes in our study(ref).” However this statement does not say whether telomerase activation is a cause of the proliferation or a byproduct of it. And, if there is a causal relationship it does not say what it is. Telomerase activation could possibly a) increase proliferation by promoting the rate of differentiation of hepatic progenitor cells into hepatocytes or b) by the rate of mitosis of such progenitor cells or c) by the rate of mitosis of hepatocytes or by c) delay of senescence of hepatocytes, or by d) any combination of these factors. To my knowledge telomerase activation is not normally associated with increased mitosis of cells but is known to be able to significantly delay cell senescence and to promote differentiation of stem and progenitor cells, so my guess is that of the above choices all are likely to apply.
· Research in liver-related stem cells is recent and partial
The 2005 article Which stem cells for adult liver? demonstrated a lack of concrete knowledge about liver stem cells. “While hepatocytes can be considered conceptually as unipotent stem cells, the presence of true stem or progenitor cells within adult livers has been largely debated.” The article goes on to discuss a number of possibilities that might be used for stem cell therapy in livers. Some earlier reports suggested evidence for the presence of hematopoietic stem cells in adult livers(ref). A November 2008 research report indicates some progress. “A novel protein marker has been found that identifies rare adult liver stem cells, whose ability to regenerate injured liver tissue has the potential for cell-replacement therapy. For the first time, researchers at the University of Pennsylvania School of Medicine led by Linda Greenbaum, MD, Assistant Professor of Medicine in the Division of Gastroenterology, have demonstrated that cells expressing the marker can differentiate into both liver cells and cells that line the bile duct.” The marker is the protein Foxl1.
· Hepatic progenitor and stem cells are possibly implicated in liver cancers
The understanding of cancer stem cells is fairly new and the existence of liver cancer stem cells is an area being investigated actively. According to a 2009 review paper Liver stem cells and hepatocellular carcinoma. “Constant proliferation of stem cells is a vital component in liver tissues. In these renewing tissues, mutations will most likely result in expansion of the altered stem cells, perpetuating and increasing the chances of additional mutations and tumor progression. However, many details about hepatocellular cancer stem cells that are important for early detection remain poorly understood” A September 2009 article reports the isolation of liver cancer stem cells that appear before tumor formation.
· Telomerase activity may telegraph the possibility of liver cancer
Again, this has been known for a relatively long time. The 1998 publication Telomerase activity in precancerous hepatic nodules states “These findings suggest that telomerase activation is an early event in large nodule formation in cirrhosis, which may facilitate the action of other factors in the process of carcinogenesis. Telomerase activity in large hepatic nodules is not always indicative of malignant transformation.” Another publication comes to the conclusions “These results suggest that the induction of hTERT mRNA is an important early event and that its measurement by real-time quantitative RT-PCR is a useful tool to detect premalignant/malignant tendencies in hepatic nodules. However, hTERT gene dosage and c-myc expression are not the main mechanisms regulating hTERT expression in hepatocarcinogenesis.” And a third 2002 study states “This study shows that hTERT re-expression takes place both in hepatic regeneration occurring in cirrhosis and in the early steps of hepatocarcinogenesis, and involves mainly the beta-splice variant of this molecule. Additional regulatory mechanisms may be required for the expression of the full-length hTERT transcript.”
The ambivalent attitude to telomerase activation that has been widespread among cancer researchers for many years applies also to some of those concerned with liver regeneration. The 2007 publication Telomerase activation in liver regeneration and hepatocarcinogenesis: Dr. Jekyll or Mr. Hyde? concludes “At present, it is unclear, whether telomerase activation preserves the non-malignant phenotype and replicative longevity of liver cells or constitutes an early alteration obligatory for an unlimited proliferation and malignant transformation.” I wonder why this is posed as an either-or choice instead of accepting the overwhelming evidence that both are the case and which one is present depends on circumstances.
· Progress is being made in decoding the signaling leading to liver cancers
The 2008 paper Hepatocellular cancer arises from loss of transforming growth factor beta signaling adaptor protein embryonic liver fodrin through abnormal angiogenesis suggests a specific mechanism. “Loss of ELF (embryonic liver fodrin) in the liver leads the cancer formation by deregulated hepatocyte proliferation and stimulation of angiogenesis in early cancers.” This paper builds on the argument in a 2007 paper Disruption of transforming growth factor-beta signaling through beta-spectrin ELF leads to hepatocellular cancer through cyclin D1 activation. “Thus, we show that TGF-beta signaling and Smad adaptor ELF suppress human hepatocarcinogenesis, potentially through cyclin D1 deregulation. Loss of ELF could serve as a primary event in progression toward a fully transformed phenotype and could hold promise for new therapeutic approaches in human HCCs.”
Another 2008 publication CpG island methylator phenotype association with upregulated telomerase activity in hepatocellular carcinoma looks at cancer gene activation in terms of DNA methylation: “CpG island methylator phenotype (CIMP) involves the targeting of multiple genes by promoter hypermethylation. — We examined the promoter methylation status of 9 genes associated with telomerase activity in 120 HCC, 120 cirrhosis tissues and 10 normal liver tissues by methylation-specific PCR. — CIMP lead to high levels of expression of telomerase activity through the simultaneous inactivation of multiple genes associated with telomerase activity by concordant methylation.” Another interesting study concludes “The results of this study suggest that HBx expression may play a role in hepatocellular carcinogenesis by interfering with telomerase activity during hepatocyte proliferation.”
So, in the light of all of the above the bottom-line guesses to answer my questions are:
1. What cells are making the telomerase? I found no direct answers. I presume both hepatic progenitor cells and hepatocytes are doing so in response to chain of distress signaling present in damaged livers that is only partially understood.
2. What is the role of the telomerase in liver regeneration? I hypothesize that there are two important roles: a) enhancing the proliferation potential of hepatocytes and hepatic progenitor cells by allowing a large number of additional population doublings before cell senescence sets in and b) is promoting the health and differentiation of hepatic progenitor cells into hepatocytes.
3. To what extent is the liver regeneration due to mitosis of existing hepatocytes and to what extent is it due to differentiation of hepatic progenitor cells? Again I found no direct answer but there seems to be strong evidence that both division of existing cells and differentiation of hepatic progenitor cells play critical roles.
4. What other stem cells might be involved, such as for renewing the supply of hepatic progenitor cells? Again there is no simple answer based on experiment. Several kinds of multipotent stem cells are capable of transforming into hepatocytes and possibly into hepatic progenitor cells. I conjecture that when liver regeneration is taking place several levels of the stem cell supply chain may be activated.
5. What kind of signaling is articulating the whole regeneration process? A number of kinds of signaling are identified in various papers. Among those of importance that I have discussed before are mTOR, MAPK, and Notch.
6. What hope does all this offer for longevity? A great deal, I would say. First, it appears that the age-related decline in liver regeneration capability can be reversed and that the “heterochronic parabiosis” techniques mentioned above might possibly be adopted so they work in clinical practice. If they can be made to work they would apply to stem cell proliferation and differentiation in general and could be broadly applied across other organ systems for life extension. Second, knowledge of the biochemical and genetic pathways active in liver regeneration will probably be of high relevance in other areas of regenerative medicine.