Monica Serra

Caloric restriction delays early phases of carcinogenesis via effects on the tissue microenvironment

Caloric restriction (CR) is an effective and consistent means to delay aging and the incidence of chronic diseases related to old age, including cancer. However, the precise mechanisms responsible for the beneficial effect of CR on carcinogenic process are yet to be identified. In the present studies the hypothesis was tested that the CR might delay carcinogenesis via modulatory effects exerted on the age-associated, neoplastic-prone tissue microenvironment. Using a well characterized, orthotopic cell transplantation (Tx) system in the rat, preneoplastic hepatocytes isolated from liver nodules were injected into either old syngeneic rats fed ad libitum (AL) or animals of the same age given a CR diet (70% of AL feeding). Analysis of donor-derived cell clusters performed at 10 weeks post-Tx revealed a significant shift towards smaller class sizes in the group receiving CR diet. Clusters comprising more than 50 cells, including large hepatic nodules, were thrice more frequent in AL vs. CR animals. Incidence of spontaneous endogenous nodules was also decreased by CR. Markers of cell senescence were equally expressed in the liver of AL and CR groups. However, higher levels of SIRT1 and FOXO1 proteins were detected in CR-exposed livers, while expression of HDAC1 and C/EBPβ were decreased. These results are interpreted to indicate that CR delays the emergence of age-associated neoplastic disease through effects exerted, at least in part, on the tissue microenvironment. Nutrient-sensing pathways might mediate such modulatory effect.

Caloric restriction promotes rapid expansion and long-lasting increase of Lactobacillus in the rat fecal microbiota

ABSTRACT Previous studies indicated that caloric restricted diet enables to lower significantly the risk of cardiovascular and metabolic diseases. In experimental animal models, life-long lasting caloric restriction (CR) was demonstrated to induce changes of the intestinal microbiota composition, regardless of fat content and/or exercise. To explore the potential impact of short and long-term CR treatment on the gut microbiota, we conducted an analysis of fecal microbiota composition in young and adult Fisher 344 rats treated with a low fat feed under ad libitum (AL) or CR conditions (70%). We report here significant changes of the rat fecal microbiota that arise rapidly in young growing animals after short-term administration of a CR diet. In particular, Lactobacillus increased significantly after 8 weeks of CR treatment and its relative abundance was significantly higher in CR vs AL fed animals after 36 weeks of dietary intervention. Taken together, our data suggest that Lactobacillus intestinal colonization is hampered in AL fed young rats compared to CR fed ones, while health-promoting CR diet intervention enables the expansion of this genus rapidly and persistently up to adulthood.

Differentiation of amniotic epithelial cells into various liver cell types and potential therapeutic applications

The aim of Regenerative Medicine is to replace or regenerate human cells, tissues or organs in order to restore normal function. Among all organs, the liver is endowed with remarkable regenerative capacity. Nonetheless, there are conditions in which this ability is impaired, and the use of isolated cells, including stem cells, is being considered as a possible therapeutic tool for the management of chronic hepatic disease. Placenta holds great promise for the field of regenerative medicine. It has long been used for the treatment of skin lesions and in ophthalmology, due to its ability to modulate inflammation and promote healing. More recently, cells isolated from the amniotic membrane are being considered as a possible resource for tissue regeneration, including in the context liver disease. Two cell types can be easily isolated from human amnion: epithelial cells (hAEC) and mesenchymal stromal cells (hAMSC). However only the first cell population has been demonstrated to be a possible source of proficient hepatic cells. This review will summarize current knowledge on the differentiation of hAEC into liver cells and their potential therapeutic application.

Aging promotes neoplastic disease through effects on the tissue microenvironment

A better understanding of the complex relationship between aging and cancer will provide important tools for the prevention and treatment of neoplasia. In these studies, the hypothesis was tested that aging may fuel carcinogenesis via alterations imposed in the tissue microenvironment. Preneoplastic hepatocytes isolated from liver nodules were orthotopically injected into either young or old syngeneic rats and their fate was followed over time using the dipeptidyl-peptidase type IV (DPPIV) system to track donor-derived-cells. At 3 months post-Tx, the mean size of donor-derived clusters was 11±3 cells in young vs. 42±8 in old recipients. At 8 months post-Tx, no visible lesion were detected in any of 21 young recipients, while 17/18 animals transplanted at old age displayed hepatic nodules, including 7 large tumors. All tumors expressed the DPPIV marker enzyme, indicating that they originated from transplanted cells. Expression of senescence-associated β-galactosidase was common in liver of 18-month old animals, while it was a rare finding in young controls. Finally, both mRNA and IL6 protein were found to be increased in the liver of aged rats compared to young controls. These results are interpreted to indicate that the microenvironment of the aged liver promotes the growth of pre-neoplastic hepatocytes.

Hyperplasia vs hypertrophy in tissue regeneration after extensive liver resection

AIM To address to what extent hypertrophy and hyperplasia contribute to liver mass restoration after major tissue loss. METHODS The ability of the liver to regenerate is remarkable on both clinical and biological grounds. Basic mechanisms underlying this process have been intensively investigated. However, it is still debated to what extent hypertrophy and hyperplasia contribute to liver mass restoration after major tissue loss. We addressed this issue using a genetically tagged system. We were able to follow the fate of single transplanted hepatocytes during the regenerative response elicited by 2/3 partial surgical hepatectomy (PH) in rats. Clusters of transplanted cells were 3D reconstructed and their size distribution was evaluated over time after PH. RESULTS Liver size and liver DNA content were largely recovered 10 d post-PH, as expected (e.g., total DNA/liver/100 g b.w. was 6.37 ± 0.21 before PH and returned to 6.10 ± 0.36 10 d after PH). Data indicated that about 2/3 of the original residual hepatocytes entered S-phase in response to PH. Analysis of cluster size distribution at 24, 48, 96 h and 10 d after PH revealed that about half of the remnant hepatocytes completed at least 2 cell cycles. Average size of hepatocytes increased at 24 h (248.50 μm2 ± 7.82 μm2, P = 0.0015), but returned to control values throughout the regenerative process (up to 10 d post-PH, 197.9 μm2 ± 6.44 μm2, P = 0.11). A sizeable fraction of the remnant hepatocyte population does not participate actively in tissue mass restoration. CONCLUSION Hyperplasia stands as the major mechanism contributing to liver mass restoration after PH, with hypertrophy playing a transient role in the process.

Caloric restriction promotes functional changes involving short-chain fatty acid biosynthesis in the rat gut microbiota

Caloric restriction (CR) is known to promote health and longevity, likely via modification of the gut microbiota (GM). However, functional and metabolic changes induced in the GM during CR are still unidentified. Here, we investigated the short- and long-term effects of CR on the rat GM using a metaproteogenomic approach. We show that a switch from ad libitum (AL) low fat diet to CR in young rats is able to induce rapid and deep changes in their GM metaproteomic profile, related to a reduction of the Firmicutes/Bacteroidetes ratio and an expansion of lactobacilli. Specifically, we observed a significant change in the expression of the microbial enzymes responsible for short-chain fatty acid biosynthesis, with CR boosting propionogenesis and limiting butyrogenesis and acetogenesis. Furthermore, these CR-induced effects were maintained up to adulthood and started to be reversed after a short-term diet change. We also found that CR alters the abundance of an array of host proteins released in stool, mainly related to epithelial barrier integrity and inflammation. Hence, our results provide thorough information about CR-induced modifications to GM and host functional activity, and might constitute the basis for novel GM-based approaches aimed at monitoring the effectiveness of dietary interventions.

Evidence of Amniotic Epithelial Cell Differentiation toward Hepatic Sinusoidal Endothelial Cells

Amniotic epithelial cells (AECs) represent a useful and noncontroversial source for liver-based regenerative medicine, as they can differentiate into hepatocytes upon transplantation into the liver. However, the possibility that AECs can differentiate into other liver cell types, such as hepatic sinusoidal endothelial cells (HSECs), has never been assessed. In order to test this hypothesis, rat- and human-derived AECs (rAECs and hAECs, respectively) were subjected to endothelial cell tube formation assay in vitro. Moreover, to evaluate differentiation in vivo, the retrorsine (RS) model of liver repopulation was used. Pyrrolizidine alkaloids (including RS) are known to target both hepatocytes and endothelial cells, inducing cell enlargement and inhibition of cell cycle progression. rAECs and hAECs were able to form capillary-like structures when cultured under proangiogenic conditions. For in vivo experiments, rAECs were obtained from dipeptidyl peptidase type IV (DPP-IV, CD26) donors and were transplanted into the liver of recipient CD26 negative animals pretreated with RS. rAEC-derived cells were engrafted in between hepatocytes and resembled HSECs as assessed by morphological analysis and the pattern of expression of CD26. Donor-derived CD26+ cells coexpressed HSEC markers RECA-1 and SE-1, while they lacked expression of typical hepatocyte markers (i.e., cytochrome P450, hepatocyte nuclear factor 4α). As such, these results provide the first evidence that AECs can respond to proangiogenic signals in vitro and differentiate into HSECs in vivo. Furthermore, they support the conclusion that AECs possesses great plasticity and represents a promising tool in the field of regenerative medicine both in the liver and in other organs.

A GC–MS untargeted metabolomics analysis in the plasma and liver of rats lacking dipeptidyl-peptidase type IV enzyme activity

This study was achieved with the aim to find metabolic changes between Fischer rats with different dipeptidyl peptidase-type 4 (DPPIV) expression. The DPPIV is an enzyme expressed in several tissues and is critically involved in the regulation of meal-related insulin secretion in healthy individuals. The metabolic consequences of chronic DPPIV inhibition were analyzed in a surrogate animal model of genetic enzyme deficiency. Hyphenated gas chromatography–mass spectrometry (GC–MS) and multivariate data analysis techniques were used to study the metabolic aqueous fraction profile of 18 plasma and liver samples in two syngeneic rat strains differing in DPPIV activity (DPPIV+ vs. DPPIV−). The hyperglycemic response following oral glucose administration was attenuated in DPPIV− rats, as expected. Statistical significant differences between the two strains were observed among the low molecular weight polar metabolites analyzed from plasma and liver.These included a decrease in malic acid and glutamine and an increase in pyroglutamic acid, serine, and alanine in plasma of DPPIV− rats. In addition, palmitic acid, l-proline, and ribitol were decreased in the liver of DPPIV− strain. Such alterations were compatible with a normal phenotype. These results suggest that long-term exposure to DPPIV inhibitors looks compatible with an overall balanced metabolism.