Telocytes in Pregnancy-Induced Physiological Liver Growth

Liver has an extraordinary ability to regenerate after injury and surgical resection, principally mediated by the proliferation of remaining hepatocytes and the differentiation of liver stem cells [1,2,3]. However, liver regenerative capacity can be grievously altered upon severe and chronic liver injury [4,5,6], and is negatively affected by aging [7,8,9]. Other than liver regeneration after injury and resection, pregnancy-induced liver growth is a physiological hepatic adaption to meet the enhanced nutritional and metabolic demands for developing placenta and fetus [10,11,12,13]. It has previously been shown that pregnancy is able to increase liver regenerative capacity in aged liver, suggesting its potential therapeutic value in liver failure [14]. However, the mechanisms underlying maternal hepatic adaptions to pregnancy are poorly elucidated.

Telocytes (TCs), a novel type of interstitial cell population firstly identified by Popescu's group, are characterized by a small cell body and extremely long prolongations named telopodes (Tps) with alternating thin segments (podomers) and dilated segments (podoms) [15] (see http://www.telocytes.com). FIB-SEM tomography, the most advanced and powerful technique to visualize cells confirmed the existence of TCs [16]. Since their identification in 2010, TCs have been found in various mammalian organs and tissues and contribute to form a complex interstitial network for the maintenance of tissue homeostasis, such as heart [17,18,19,20,21], lung [22,23,24], placenta [25], pancreas [26,27], skin [28,29], skeletal muscle [30,31,32], uterus [33,34,35,36,37,38], urinary system [39,40,41], and others [42,43,44,45,46,47,48,49,50,51,52,53,54,55]. Furthermore, increasing evidence has demonstrated the presence of TCs within stem cell niches [23,29,31,49,56,57] and indicated the potential involvement of TCs in tissue regeneration/repair after injury [32,38,58,59,60,61,62,63,64]. Our group has recently identified the presence of TCs in liver [54], and further demonstrated a close relationship between TCs and the cells (hepatocytes/stem cells) essentially involved in liver regeneration using a murine model of partial hepatectomy [65]. The aim of the present study was to likewise investigate the possible roles of TCs in hepatic adaptions to pregnancy.

Eight-week-old female C57BL/6 mice, purchased from Shanghai SLAC Laboratory Animal Center, were maintained in a temperature-controlled room on a 12 h light/dark cycle, with ad libitum access to food and water. Mice were mated and the presence of a copulatory plug in the vagina was considered as gestation day 0.5. Pregnant mice were then designed to be sacrificed at different time points (pregnancy day 0.5, 4.5, 8.5, 10.5, 12.5, 14.5, 16.5, and 18.5) (n=8 per group). The liver weight was measured to evaluate liver growth during pregnancy. Frozen liver sections were used for immunofluroscent staining. This study was approved by the local ethical committees and all animal experiments were conducted under the guidelines on the use and care of laboratory animals for biomedical research published by National Institutes of Health (No. 85-23, revised 1996).

Mice were intraperitoneally injetected with 50 mg/kg of 5-ethynyl-2'-deoxyuridine (EdU) 1 h before sacrificed. The frozen sections (6 um) were fixed with 4% paraformaldehyde for 30 min after washed with PBS, and then incubated with Albumin primary antibody (1:100, BS6520, Bioworld) in diluted by PBS containing 0.25% Triton X-100 overnight at 4°C. After that, sections were incubated with anti-rabbit Rhodamine-conjugated secondary antibody (1:200, Sc-362262, Santa Cruz) for 1 h at room temperature. After wash with PBS, staining with anti-EdU working solution was performed at room temperature for 30 min according to the manual of a EdU detection kit (Click-iT Plus EdU Alexa Fluor 647 Imaging Kit, Life Technology). Finnally, sections were stained with DAPI (Prolong® Gold, Life Technology) and observed under a confocal laser scanning microscope (LSM 710, Carl Zeiss MicroImaging GmbH). The results were expressed as EdU and Albumin double positive cell number per mm².

Double immunolabeling for CD34/PDGFR-α, considered as the specific markers for TCs, was used for detection of TCs in the present study ⁶⁶. Briefly, 6 μm-thick frozen liver sections were fixed in 4% paraformaldehyde for 15 min, washed with PBS for three times, pre-incubated in PBS supplemented with 10% goat serum for 1 h, and then incubated overnight at 4°C with rabbit polyclonal anti-PDGFR-α (Abcam, ab61219) and rat monoclonal anti-CD34 (Abcam, ab8158) primary antibodies diluted by 1:100 in PBS with 0.25% Triton X-100. After that, sections were exposed for 1 h to goat anti-rat labeled with FITC (Santa Cruz, sc-2011) and goat anti-rabbit labeled with rhodamine (Santa Cruz, sc-362262) secondary antibodies diluted by 1:200 in the same buffer. Finally, sections were stained with DAPI (ProLong® Gold, Life technology). The images were analyzed with confocal laser scanning microscope (LSM 710, Carl Zeiss MicroImaging GmbH, Germany) under an amplification of 400×.

EdU and CD34 double labelled staining was used to determine the proliferative TCs. In brief, 6 μm-thick frozen liver sections were fixed in 4% paraformaldehyde for 15 min, washed with PBS for three times, pre-incubated in PBS supplemented with 10% goat serum for 1 h, and then incubated overnight at 4°C with rat monoclonal anti-CD34 (Abcam, ab8158) primary antibodies diluted by 1:100 in PBS with 0.25% Triton X-100. After that, sections were exposed for 1 h to goat anti-rat labeled with FITC (Santa Cruz, sc-2011) secondary antibodies diluted by 1:200 in the same buffer. After wash with PBS, staining with anti-EdU working solution was performed at room temperature for 30 min according to the manual of a EdU detection kit (Click-iT Plus EdU Alexa Fluor 647 Imaging Kit, Life Technology). Finally, sections were stained with DAPI (ProLong® Gold, Life technology). The images were analyzed with confocal laser scanning microscope (LSM 710, Carl Zeiss MicroImaging GmbH, Germany) under an amplification of 400×.

Data are expressed as mean ± SEM. All analyses were performed using SPSS 19.0. Statistical significance was determined with one-way ANOVA test followed by two-tailed Student's t test. Significance is defined as P-value less than 0.05.

Liver weight continued to increase which appeared significant from pregnancy day 10.5 as compared to day 0.5 in pregnant mice (Fig. 1), indicating that pregnancy induces liver growth.

EdU and Albumin double immunostaining was conducted to investigate the proliferative effect of pregnancy-induced liver growth. As compared to pregnancy day 0.5, EdU positive hepatocytes number per mm² of liver tissues was significantly increased in early stage (day 4.5 and 8.5) and late stage (day 14.5 and 16.5) of pregnancy (Fig. 2).

As labeled by double immunofluorescent staining for CD34 and PDGFR-α, the number of CD34/PDGFR-α positive cells (TCs) per mm² of liver tissues was significantly increased at pregnancy day 4.5 and 14.5 (Fig. 3A and B), which was in accordance with the time points when high level of hepatocytes proliferation rate occurred during pregnancy. However, as indicated by immunofluorescent staining for CD34 and EdU (Fig. 4), we did not observe significant difference among the time points we have checked, which might be due to the fact that the rate of proliferative TCs was extremely low or proliferative endothelial cells might cover up the true changes of proliferative TCs.

The present study shows that pregnancy-induced liver growth preceded via increased proliferation of hepatocytes. Furthermore, the number of TCs in liver detected by double immunolabeling for CD34/PDGFR-α was significantly increased at pregnancy day 4.5 and day 14.5, that was coincident with the occurrence of two peaks of hepatocytes proliferation during pregnancy. These results suggest the potential involvements of TCs in hepatic proliferative adaptions to pregnancy.

To date, the mechanisms underlying hepatic adaptions to pregnancy are largely unknown [10,11,13,14,67,68,69]. In the present study, we demonstrated two peaks of hepatocytes proliferation occurring at early stages and late stages of pregnancy as shown by EdU and Albumin double immunostaining. The increased proliferation of hepatocytes related to pregnancy has previously been documented [13,14,67,69], while the occurrence of two proliferative peaks of hepatocytes during pregnancy in the present study was firstly reported here. We hypothesized that the possible reasons may be related to different species (rat vs. mouse) [13], ages (old vs. young) [14], and animal models (pregnancy vs. ovariectomy or pseudopregnancy) [14,67] applied in our study and in others. In addition, the metabolic and hormonal mechanisms underlying hepatocyte proliferation during pregnancy need to be further explored.

Previously, our group documented the presence of TCs in liver and further indicated the potential roles of TCs in liver regeneration after hepatectomy, probably by influencing hepatocyte proliferation and/or liver stem cell differentiation [65]. In the present study, we further demonstrated the increased number of CD34/PDGFR-α positive TCs in pregnant liver at the same time points (pregnancy day 4.5 and day 14.5) when the two proliferative peaks of hepatocytes appeared, suggesting a possible relationship between TCs and hepatocytes proliferation in pregnancy-induced liver growth. Increasing evidence has shown that TCs are critically implicated in tissue regeneration/repair by forming a complicated network with tissue/organ-specific cells, immunoreactive cells, other interstitial cells and stem cells, and thus actively contribute to intercellular signaling coordination either by minute intercellular junctions or by paracrine effect via ectovesicles [23,29,30,56,57,59,70]. However, the exact mechanisms how TCs might affect the proliferative capacity of hepatocytes in pregnancy-induced liver growth remain to be further studied.

A major limitation of the present study is that the data presented here does not fully demonstrated a functional relation between TCs and hepatocyte proliferation in pregnancy-induced liver growth. We have also conducted CD34 plus EdU immunostaining, however, no significant difference was observed among the time points we had checked. We speculated that this was due to the fact that the rate of proliferative TCs was extremely low because pregnancy-induced physiological liver growth was a weak physiological stimuli. Besides that, as CD34 is also a marker for endothelial cells, thus proliferative endothelial cells might cover up the true changes of proliferative TCs. It would be interesting to check the rate of proliferative TCs using CD34/PDGFR-α/EdU three immunostainings in other liver regeneration models such as partial hepatectomy (PH) models. Nevertheless, considering the fact that pregnancy-induced liver growth preceded via increased proliferation of hepatocytes at pregnancy day 4.5, 8.5, 14.5 and 16.5 while the number of TCs in liver was significantly increased at pregnancy day 4.5 and day 14.5, we think that TCs proliferate first (or at least at the same time) compared with hepatocytes and therefore a possible relationship between TCs and hepatocyte proliferation in pregnancy-induced liver growth might exist.

In conclusion, the present study demonstrates an increase of CD34/PDGFR-α positive TCs in pregnant liver accompanied by high level of hepatocyte proliferation. Considering that liver regeneration capacity is usually far from ideal in certain circumstances like severe liver injury or surgical resection, understanding how TCs take effect in pregnancy-induced physiological liver growth might raise bright prospect for the treatment of liver failure.

The authors declare there are no conflicts of interest.

This work was supported by the grants from National Natural Science Foundation of China (81070343 and 81370559 to C. Yang; 81200169 to J. Xiao; 81400635 to F. Wang; 81400647 to Y. Bei), funds from Shanghai Innovation Program (12431901002 to C. Yang), Innovation Program of Shanghai Municipal Education Commission (13YZ014 to J. Xiao), Foundation for University Young Teachers by Shanghai Municipal Education Commission (year 2012, to J. Xiao), Innovation fund from Shanghai University (sdcx2012038 to J. Xiao), and Program for the integration of production, teaching and research for University Teachers supported by Shanghai Municipal Education Commission (year 2014, to J. Xiao), Jonit Projects in Major Diseases funding from Shanghai Municipal Commission of Health and Family Planning (2014ZYJB0201 to C. Yang), Jonit Projects for Novel Frontier Technology in Shanghai Municipal Hospital from Shanghai Municipal Commission of Health and Family Planning (SHDC1204122 to C. Yang), Shanghai Medical Guide Project from Shanghai Science and Technology Committee (14411971500 to F. Wang), grants from Chinese Foundation for Hepatitis Prevention and Control (TQGB20140141 to F. Wang) and funds from Shanghai Innovation Program (12431901002 to C. Yang), and China Postdoctoral Science Foundation (2014M561456 to Y. Bei).

F. Wang, Y. Bei and Y. Zhao contributed equally to this work.