Indiana University-Purdue University Indianapolis (IUPUI) / Tissue cells display mechanosensitivity in their ability to discern and respond to changes in the viscoelastic properties of their surroundings. By anchoring and pulling, cells are capable of translating mechanical stimuli into a biological response through a process known as mechanotransduction, a pathway believed to critically impact cell adhesion, morphology and multiple cellular processes from migration to differentiation. While previous studies on polymeric gels have revealed the influence of substrate elasticity on cellular shape and function, a lack of suitable substrates (i.e. with mobile cell-substrate linkers) has hindered research on the role of substrate viscosity. This work presents the successful design and characterization of lipid-bilayer based cell substrates of tunable viscosity affecting cell-substrate linker mobility through changes in viscous drag. Here, two complementary membrane systems were employed to span a wide range of viscosity. Single polymer-tethered lipid bilayers were used to generate subtle changes in substrate viscosity while multiple, polymer-interconnected lipid bilayer stacks were capable of producing dramatic changes in substrate viscosity. The homogeneity and integrity of these novel multibilayer systems in the presence of adherent cells was confirmed using optical microscopy techniques. Profound changes in cellular growth, phenotype and cytoskeletal organization confirm the ability of cells to sense changes in viscosity. Moreover, increased migration speeds coupled with rapid area fluctuations suggest a transition to a different migration mode in response to the dramatic changes in substrate viscosity.

Indiana University-Purdue University Indianapolis (IUPUI) / Cyclin Dependent Kinase regulation is often manipulated by cancer cells to promote unlimited proliferation. P27 is an important regulator of Cyclin E/CDK 2, which has been found in low amounts in many types of malignant cancers. Lovastatin has been shown to cause cell cycle arrest in the G1 phase of the cell cycle by increasing the P27 protein. There has been some question, however, if lovastatin regulates P27 at the transcriptional or translational level. Although it has been claimed that P27 expression regulation is due to an IRES located in its 5’UTR, other studies suggested that P27 expression is regulated at the level of transcription. To further investigate the regulation mechanism of P27 expression, the 5’-UTR of P27 and its deletion mutants were examined using a luciferase reporter gene in HeLa cells following exposure to lovastatin. It was found that lovastatin stimulated a significant 1.4 fold increase in its promoter activity of the full length 5’UTR (575). Deletion of 35 nucleotides from the 5’ end of the UTR eliminated the lovastatin-induced increase in promoter activity. Further mapping analyses of the first 35 bases showed that two regions, M1 (575-559) and M3 (543-527), were less sensitive to lovastatin than the other mutated constructs. Since M1 and M3 still showed some activity, a construct was created with deletions in both the M1 and M3 regions. This showed no increase in luciferase activity when exposed to lovastatin. Looking at RNA levels, there was a 1.5 fold increase in RNA when the full length 5’UTR was inserted into HeLa cells and exposed to 81 µM of lovastatin. In contrast, there was no increase in RNA when M1/M3 (575-559; 543-527) was inserted into HeLa cells and exposed to 81 µM of lovastatin. In addition, there was a 1.6 fold increase in endogenous P27 RNA levels after HeLa cells were exposed to 81 µM of lovastatin. In all of these experiments, there seems to be two promoters that work cooperatively: M1 (575-559) and M3 (543-527).

The development of the cardiovascular system, including the heart and circulating blood within a vascular network, relies on mesoderm-derived cells to contribute to the development of both cardiac and hematopoietic tissues. This dissertation focuses on exploring the roles of crip2, downstream of the transcription factor Nkx2.5 established from an RNA sequencing dataset, in cardiac and hematopoietic development using the zebrafish model. In Chapter 2, we investigate the influence of Crip proteins on the development of the zebrafish heart. Congenital heart defects (CHDs), affecting approximately 1% of live births, arise from structural anomalies during heart development primarily caused by genetic mutations. While there isn’t just one driver of CHDs, transcription factors such as Nkx2.5, play a pivotal role in guiding cardiac morphogenesis. NKX2-5-associated CHDs often involve outflow tract (OFT) malformations. The development of the heart involves two progenitor cell populations, the first heart field (FHF) and second heart field (SHF), contributing to the linear heart tube and subsequent growth. Despite understanding the role of Nkx2.5, the spatiotemporal mechanisms directed by Nkx factors in SHF progenitor specification, proliferation, and identity maintenance remain elusive. This study aims to uncover novel effectors of Nkx transcriptional regulation, using RNA sequencing on dissected wild-type and nkx2.5-/- zebrafish hearts at 26 hours post fertilization (hpf). This work focuses on a LIM domain protein, cysteine rich intestinal protein 2 (crip2), identified as a mis-regulated gene in nkx2.5-deficient embryos, and we explore its role downstream of nkx genes in SHF-derived arterial pole formation. While crip2 is abundantly expressed in the developing heart, the family member crip3 also shows a mild expression pattern. Loss-of-function mutations in crip2 and crip3 (referred to as cripDM) reveal normal cardiac chamber specification. Atrioventricular canal morphology remains unaffected in cripDM embryos. The OFT in cripDM embryos displays a significant dilation, accompanied by increased ltbp3 expression. Surprisingly, the smooth muscle cell population of the OFT does not explain the size increase. This research expands our understanding of OFT development, highlighting the multi-layered contributions of various cell types and factors. In Chapter 3, we further examine the role of crip2 in the development of hematopoietic stem cells given its endothelial expression pattern. Hematopoietic stem and progenitor cells (HSPCs) have multilineage potential and can sustain long-term self-renewal. The ability to derive patient-specific HSCs in culture has immense therapeutic potential to overcome the shortage of compatible donors for HSC transplantations. However, differentiation protocols largely fail to produce long-lived HSCs from human pluripotent stem cells. Understanding the complex genetic networks and signaling pathways required to generate HSCs will facilitate clinical applications in patients. The hemogenic endothelium (HE) is a specialized niche of endothelial cells within the ventral portion of the dorsal aorta that gives rise to HSPCs during the definitive wave of hematopoiesis in the zebrafish embryo. Our data reveal that crip2 has a previously unrecognized function in establishing the proper endothelial cell environment for HSPC specification. CripDM embryos exhibit decreased emergence of HSCs by 26 hpf. Loss of HSPCs in the cripDM results in decreased erythroid, myeloid, and lymphoid lineage production between 30 -72 hpf; these perturbations in the hematopoietic lineages recover by 96 hpf. To decipher the spatiotemporal mechanisms underlying the cripDM phenotype, we performed single cell RNA (scRNA) sequencing of sorted, Kdrl:mCherry+ cells at 30 hpf. Our analysis reveals upregulation of genes essential for vascular development and mis-regulation of Notch signaling pathways in the cripDM embryos. Building on these data, our ongoing studies aim to investigate how crip2 regulates the endothelial niche of the ventral aorta to produce HSCs early in definitive hematopoiesis. We anticipate that our insights will inform potential therapeutic interventions for improvements of human HSC production in vitro.

Indiana University-Purdue University Indianapolis (IUPUI). / The abysmal five year pancreatic cancer survival rate of less than 6% highlights the need for new treatments for this deadly malignancy. Cytotoxic drugs normally target rapidly dividing cancer cells but unfortunately often target stem cells resulting in toxicity. This warrants the development of compounds that selectively target tumor cells. An inhibitor of the mitotic kinase, MPS1, which has been shown to be more selective towards cancer cells than non-tumorigenic cells, shows promise but its effects on stem cells has not been investigated. MPS1 is an essential component of the Spindle Assembly Checkpoint and is proposed to be up-regulated in cancer cells to maintain chromosomal segregation errors within survivable limits. Inhibition of MPS1 kinase causes cancer cell death accompanied by massive aneuploidy. Our studies demonstrate that human adipose stem cells (ASCs) and can tolerate higher levels of a small molecule MPS1 inhibitor than pancreatic cancer cells. In contrast to PANC-1 cancer cells, ASCs and telomerase-immortalized pancreatic ductal epithelial cells did not exhibit elevated chromosome mis-segregation after treatment with the MPS1 inhibitor for 72hrs. In contrast, PANC-1 pancreatic cancer cells exhibited a large increase in chromosomal mis-segregation under similar conditions. Furthermore, growth of ASCs was minimally affected post treatment whereas PANC-1 cells were severely growth impaired suggesting a favorable therapeutic index. Our studies, demonstrate that MPS1 inhibition is selective towards pancreatic cancer cells and that stem cells are less affected in vitro. These data suggest MPS1 inhibition should be further investigated as a new treatment approach in pancreatic cancer.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

Ho, Tik Shun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 88-102). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.vii / Table of Contents --- p.viii / List of Table --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiii / Chapter CHAPTER 1 --- General Introduction --- p.1 / Chapter 1.1 --- Fatty Acid Synthase (FAS) - 7-domain multifunctional enzyme --- p.1 / Chapter 1.1.1 --- Functions --- p.1 / Chapter 1.1.2 --- Structure --- p.2 / Chapter 1.2 --- Fatty Acid biosynthesis reactions --- p.4 / Chapter 1.3 --- Malonyl Coenzyme A - An important mediator in lipogenesis --- p.7 / Chapter 1.4 --- FAS expression in different histotypes --- p.8 / Chapter 1.4.1 --- FAS in normal cells --- p.8 / Chapter 1.4.2 --- FAS in pathological cells --- p.8 / Chapter 1.4.3 --- Tumor-associated FAS (Oncogenic antigen-519) in cancer cells --- p.9 / Chapter 1.5 --- FAS signaling models in breast and prostate cancers --- p.12 / Chapter 1.5.1 --- Association between FAS and PI3K／Akt pathway --- p.12 / Chapter 1.5.2 --- Hypothetical model of FAS hyperactivity in breast and prostate cancer cells --- p.13 / Chapter 1.6 --- FAS inhibition to tackle cancer cell growth --- p.15 / Chapter 1.6.1 --- FAS inhibitors --- p.15 / Chapter 1.6.1.1 --- Cerulenin --- p.16 / Chapter 1.6.1.2 --- C75 --- p.17 / Chapter 1.6.2 --- Small interfering RNA --- p.17 / Chapter 1.7 --- FAS inhibition to enhance chemoresistant cancer cells sensitivity to drugs --- p.19 / Chapter 1.8 --- Hypothesis --- p.20 / Chapter CHAPTER 2 --- Methods and Materials --- p.21 / Chapter 2.1 --- Chemicals and antibodies --- p.21 / Chapter 2.2 --- Cell cultures --- p.21 / Chapter 2.3 --- MTT assay --- p.22 / Chapter 2.4 --- 5-Bromo-2'-deoxyuridine (BrdU)-labeling cell proliferation assay --- p.22 / Chapter 2.5 --- Cytotoxicity detection assay of LDH release --- p.23 / Chapter 2.6 --- DNA flow cytometry --- p.23 / Chapter 2.7 --- Confocal micocropy --- p.24 / Chapter 2.8 --- Immunoblot analysis --- p.24 / Chapter 2.8.1 --- Preparation of protein lysates --- p.24 / Chapter 2.8.2 --- Immunoblotting --- p.25 / Chapter 2.9 --- Caspase inhibitor studies --- p.26 / Chapter 2.10 --- Analysis of mitochondrial membrane potential --- p.26 / Chapter 2.11 --- Determination of caspase activities --- p.27 / Chapter 2.12 --- siRNA transfection --- p.27 / Chapter 2.13 --- Statistical analysis --- p.28 / Chapter CHAPTER 3 --- Results --- p.29 / Chapter 3.1 --- Cytostatic & cytotoxic studies of FAS inhibitors on human cancer cells --- p.29 / Chapter 3.1.1 --- Cerulenin and C75 suppress cell growth of different cancer histotypes --- p.29 / Chapter 3.1.2 --- Cerulenin and C75 suppress cell growth of A-375 dose- and time-dependently --- p.32 / Chapter 3.1.3 --- Cerulenin and C75 exert cytotoxic effect on A-375 but not normal skin HS68 cells --- p.36 / Chapter 3.1.4 --- Cerulenin and C75 arrest cell cycle progression and induce apoptosis with DNA Fragmentation --- p.39 / Chapter 3.2 --- Mechanistic studies of FAS inhibitors in A-375 cells --- p.46 / Chapter 3.2.1 --- Cerulenin and C75 induce caspase-dependent apoptosis --- p.46 / Chapter 3.2.2 --- Cerulenin- and C75-induced apoptosis involve extrinsic death receptor pathway --- p.52 / Chapter 3.2.3 --- Cerulenin- and C75-induced apoptosis involve intrinsic mitochondrial pathway --- p.57 / Chapter 3.2.4 --- Extrinsic death receptor pathway serves as a pioneer and links with intrinsic mitochondrial pathway in cerulenin- and C75-induced apoptosis --- p.65 / Chapter 3.3 --- Small interfering RNA on Fatty Acid Synthase (FAS siRNA) --- p.68 / Chapter 3.3.1 --- FAS siRNA induces PARP cleavage --- p.68 / Chapter 3.3.2 --- FAS siRNA triggers caspase-dependent apoptosis as FAS inhibitors --- p.70 / Chapter CHAPTER 4 --- Discussion --- p.72 / Chapter CHAPTER 5 --- Future Prospect --- p.85 / Chapter CHAPTER 6 --- References --- p.88

Please read the abstract in the section 00front of this document / Thesis (DSc (Oral Pathology))--University of Pretoria, 2005. / Community Dentistry / unrestricted

Calcium is a crucial intracellular messenger controlling a plethora of important intracellular events. Elevated [Ca2+]i levels regulate numerous processes due to the versatility of the Ca2+ signaling in terms of speed, amplitude and spatia-temporal patterning. Depletion of intracellular calcium stores functions as a primary trigger for a message that is translated to the plasma membrane, resulting in the slow activation of plasma membrane Ca2+ influx channels, which allow entry of external calcium. Since these channels depend upon the state of filling of the intracellular Ca2+stores, these influx channels are called store-operated channels (SOCs). It is unclear how empty intracellular stores signal activation of plasma membrane capacitative calcium entry (CCE). In order to bridge the gaps in our understanding of calcium's role in T cell regulation, this project was designed to look at the effects of various pharmacological regulators of T cell calcium stores. At the start, the effort was directed at the regulation of the microsomal calcium A TPases, considering these are perhaps the most essential mediators of intracellular calcium storage. Thapsigargin (TG) and cyclopiazonic acid (CPA) were shown to be the most potent inhibitors of the Ca2+ -ATPase, also a new Ca2+ regulator aaptamine was shown to exert more modest inhibition of the Ca2+ pump. We also characterized a novel compound, gingerol, findings its actions are to stimulate Ca2+ pumps. Cell growth assays revealed an important role for ryanodine receptors (RyRs) in regulating T cell growth. RyR activators CMC and PCB95 dramatically altered T cell growth patterns leading to significantly reduced cell viability. In contrast RyR antagonist dantrolene appeared to induce growth arrest in that cell proliferation was curtailed, yet cells remained viable. Cell viability studies revealed that the Ca2+ pump regulators TG and aaptamine were also observed to reduce cell growth rates, presumably as a result of their ability to deplete Ca2+ stores (100 nM TG was able to decrease cell viability by 90% within 24 hrs of exposure). PCB95 was able to decrease cell viability by 50% within 24 of hrs exposure and CMC decreased cell viability by 75% within 24 hrs and further over a 48 hr period. 2-APB and aaptamine were cytotoxic at higher doses. The inositol 1 ,4,5 -trisphosphate receptor (IP3R) pathway was also found to be critically linked to T cell growth control. We observed that the IP3R modulator, 2- aminoethoxydiphenyl borate (2-APB) induced antigen-like Ca2+ spike that correlates with suppression of T cell growth rates. In this study we have identified two novel T cell Ca2+ store regulators, aaptamine and gingerol. We also find that Ca2+ stores are indeed sensitively linked to T cell growth regulation. Depletion of Ca2+ stores with SERCA inhibitors as well as both RyR and IP3R activators profoundly suppress T cell proliferation most likely via activation of apoptosis.

Thesis (PhD)-- Stellenbosch University, 2014. / ENGLISH ABSTRACT: Exposure to harmful stimuli can render vascular endothelial cells dysfunctional, characterised by reduced nitric oxide (NO) bioavailibility. Endothelial dysfunction (ED) is a reversible precursor of ischaemic heart disease (IHD), and understanding the mechanisms underlying the development of ED could lead to clinical strategies in preventing/treating IHD. Very little is known about the responses of cardiac microvascular endothelial cells (CMECs) to pro-ED stimuli, as most studies are conducted on macrovascular endothelial cells. The current dissertation set out to comprehensively investigate the responses of cultured primary adult rat CMECs to known harmful stimuli, viz. hypoxia and tumor necrosis factor-alpha (TNF-α; proinflammatory cytokine). We were interested to investigate whether this distinct endothelial cell type would develop classical features of ED, and if so, what the underlying mechanisms were. First we aimed to establish a baseline characterization of the CMECs under control conditions. Next, we developed a model of hypoxia-induced cell injury and measured apoptosis/necrosis, intracellular NO and reactive oxygen species (ROS), expression and activation of signalling proteins involved with NObiosynthesis, hypoxia and apoptosis, and differential regulation of proteins. Finally, we characterised CMEC responses to treatment with TNF-α. We assessed apoptosis/necrosis, intracellular NO and ROS levels, NO-biosynthesis pathway proteins and large-scale differential protein regulation. The above measurements were performed by morphological assessment (light and fluorescence microscopy), FACS analysis, western blotting and large-scale proteomic analyses. Data showed that CMECs shared many baseline features with other endothelial cell types, including morphological appearance, LDL-uptake, NO-production, and expression of eNOS protein. In a novel observation, proteomic analysis revealed the expression of 1387 proteins. Another novel finding was the high abundance of structural mitochondrial proteins, suggesting that CMECs require mitochondria for non-respiration purposes as well. High expression of vesicle, glycolytic and RAS signalling proteins were other features of the baseline CMECs. CMECs exposed to hypoxia responded by increased apoptosis/necrosis and expression of the hypoxia-marker, HIF-1α. Interestingly, hypoxic CMECs showed increased eNOS-NO biosynthesis, associated with increased mitochondrial ROS and reduced anti-oxidant systems, suggestive of oxidative stress. In accordance with the literature, several glycolytic proteins were up-regulated. A novel finding was the up-regulation of proteins involved with protein synthesis, not usually described in hypoxic cell studies. The CMECs responded to TNF-α-treatment by exhibiting hallmarks of ED, namely attenuated biosynthesis of PKB/Akt-eNOSderived NO and the development of outspoken response to oxidative stress as indicated by the up-regulation of several anti-oxidant systems. The data showed that TNF-α treatment elicited classical TNF-Receptor 1-mediated signalling characterized by the dual activation of pro-apoptotic pathways (BID and caspase-3) as well as the protective, pro-inflammatory IKB-alpha–NF-KB pathway. In conclusion, this is the first study of its kind to describe a comprehensive characterisation of CMECs under baseline and injury-inducing conditions. On the whole, although it appeared as if the CMECs shared many responses and mechanisms with more frequently researched endothelial cell types, the data also supplied several novel additions to the literature, particularly with the application of proteomics. We believe that this dissertation has provided more insights into endothelial heterogeneity in the vascular system and into the mechanisms adopted by CMECs when exposed to stimuli typically associated with cardiovascular risk. / AFRIKAANSE OPSOMMING: Blootstelling aan skadelike stimuli kan tot disfunksionaliteit van vaskulêre endoteelselle lei wat deur verlaagde biobeskikbaarheid van stikstofoksied (NO) gekenmerk word. Endoteeldisfunksie (ED) is ‘n omkeerbare voorganger van isgemiese hartsiekte (IHD), en ‘n beter begrip van die onderliggende meganismes van ED kan lei tot die ontwikkeling van kliniese strategieë vir die voorkoming/behandeling van IHD. Baie min is bekend oor die respons wat in kardiale mikrovaskulêre endoteelselle (CMECs) uitgelok word na blootstelling aan pro-ED stimuli, omdat meeste studies op makrovaskulêre endoteelselle uitgevoer word. Die huidige proefskrif het daarna gemik om die respons van primêre kulture van volwasse rot CMECs op bekende skadelike stimuli, nl. hipoksie en tumor nekrose faktor-alfa (TNF-α; pro-inflammatoriese sitokien) in diepte te ondersoek. Ons was veral geïnteresseerd om vas te stel of hierdie spesifieke endoteelseltipe die klassieke kenmerke van ED sou ontwikkel, en indien wel, wat die onderliggende meganismes sou wees. Eerstens het ons beoog om ‘n basislyn karaterisering van CMECs onder kontrole toestande daar te stel. Vervolgens het ons ‘n model van hipoksie-geïnduseerde selskade gevestig en apoptose/nekrose, intrasellulêre NO en reaktiewe suurstofspesies (ROS), sowel as die uitdrukking en aktivering van proteine betrokke by NO-biosintese, hipoksie en apoptose en differensiële regulering van proteine gemeet. Laastens het ons die respons van CMECs op behandeling met TNF-α gekarakteriseer. Ons het apoptose/nekrose, intrasellulêre NO en ROS vlakke, NO-biosintese-seintransduksieproteïene en grootskaalse differensiele regulering van proteïene gemeet. Bg. metings is uitgevoer deur gebruik te maak van morfologiese evaluasie (lig -en fluoressensiemikroskopie), vloeisitometriese analises, western blot analises en proteomiese analises. Data het getoon dat die basislyn eienskappe van CMECs grootliks met dié van ander endoteelseltipes ooreenstem, insluitende morfologiese voorkoms, LDL-opname, NO-produksie en die uitdrukking van eNOS proteïen. In ‘n nuwe waarneming, het die proteomiese data die uitdrukking van 1387 proteïene aangetoon. ‘n Ander nuwe bevinding was die voorkoms van ‘n groot aantal strukturele mitokondriale proteïene, wat daarop dui dat die CMECs mitokondria ook vir nie-respiratoriese doeleindes gebruik. ‘n Hoë uitdrukking van vesikulêre, glikolitiese en RAS-seintransduksie proteïene was ook kenmerkend van die basislyn CMECs. CMECS wat aan hipoksie blootgestel is, het reageer met ‘n verhoging in apoptose / nekrose en verhoogde uitdrukking van die hipoksie merker, HIF-1α. ‘n Interressante bevinding was dat eNOS-NO biosintese sterk toegeneem het in die hipoksiese CMECs wat met verhoogde mitokondriale ROS en verlaagde anti-oksidant sisteme (aanduidend van oksidatiewe stres) gepaardgegaan het. In ooreenstemming met die literatuur, is verskeie glikolitiese proteïene opgereguleer. ‘n Nuwe waarneming was die opregulering van proteïene wat betrokke is by proteïensintese, iets wat nie normaalweg in hipoksie-studies beskryf word nie. Die CMECs het op TNF-α behandeling gerespondeer deur tekens van ED te toon, naamlik ‘n afname in die NO afkomstig van PKB/Akt-eNOS biosintese en die ontwikkeling van uitgesproke reaksie op oksidatiewe stres soos aangedui deur die opregulering van verskeie anti-oksidant sisteme. Die data het ook aangedui dat TNF-α behandeling tot klassieke TNF-reseptor 1 bemiddelde seintransduksie gelei het, wat gekenmerk was deur die tweeledige aktivering van pro-apoptotiese seintransduksiepaaie (BID en kaspase-3) sowel as die beskermende, pro-inflammatoriese IKB-alpha-NF-KB seintransduksiepad. Ten slotte: hierdie is die eerste studie van sy soort wat die kenmerke en response van CMECs onder basislyn en pro-besering omstandighede in diepte beskryf. Alhoewel dit oor die algemeen wil voorkom asof die CMECs baie in gemeen het met ander, beter nagevorste endoteelseltipes, het die data egter ook verskeie nuwe bevindinge tot die bestaande literatuur gevoeg, spesifiek die data afkomstig van die proteomiese analises. Ons glo dat hierdie proefskrif meer insig verleen t.o.v. die heterogeniteit van vaskulêre endoteelselle asook t.o.v. die megansimes wat deur CMECs aangewend word wanneer hulle aan skadelike stimuli (geassosieer met kardiovaskulêre risiko) blootgestel word.

Liver progenitor cells (LPCs) play a major role in the regeneration process following chronic liver damage. LPCs can differentiate into hepatocytes and cholangiocytes and thus are capable of replenishing the damaged liver. Due to their plasticity and robust nature in culture systems, they are promising candidates for use in cell therapy. However, to be able to use LPCs as tissue regenerating stem cell-like cells in the clinic, we need to fully understand how they are controlled. Although a strong association between LPCs and inflammation has been shown in many chronic liver diseases, the role of the immune system in LPC-mediated hepatic regeneration is poorly understood. We hypothesise that specific immune cells and mediators are needed to induce the LPC compartment, and that these are common to the LPC response in different injury settings. Therefore, the present study focused on the characterisation of the inflammatory environment in the LPC response, which generates this niche. The aims of this study were (i) to identify the immune cells that are important for the LPC response, (ii) to define the cytokine profile and (iii) to determine the role of the cytokine producing cells during liver regeneration. To study hepatic inflammation following liver injury, a diet-induced model of liver injury (choline-deficient, ethionine-supplemented diet, CDE diet) was compared to two transgenic mouse models of immune-mediated hepatitis (Met-Kb, 178.3). Although all three models are characterised by hepatitis, histological analysis revealed that LPCs were only detectable in the CDE and Met-Kb livers. In the 178.3 model, livers regenerated from proliferating hepatocytes. An LPC response could not be induced in these mice even when liver damage was made more severe. In the other two models, LPC numbers increased over time showing the highest numbers one week after the peak of liver injury. LPCs were often found in close proximity to inflammatory cells, in particular macrophages.