Identification of genetic profile and biomarkers involved in acute respiratory distress syndrome.

PURPOSE: The purpose of this study was to profile genetic causal factors of acute respiratory distress syndrome (ARDS) and early predict patients at high ARDS risk. METHODS: We performed a phenome-wide Mendelian Randomization analysis through summary statistics of an ARDS genome-wide association study (1250 cases and 1583 controls of European ancestry) and 33,150 traits. Transcriptomic data from human blood and lung tissues of a preclinical mouse model were used to validate biomarkers, which were further used to construct a prediction model and nomogram. RESULTS: A total of 1736 traits, including 1223 blood RNA, 159 plasma proteins, and 354 non-gene phenotypes (classified by Biochemistry, Anthropometry, Disease, Nutrition and Habit, Immunology, and Treatment), exhibited a potentially causal relationship with ARDS development, which were accessible through a user-friendly interface platform called CARDS (Causal traits for Acute Respiratory Distress Syndrome). Regarding candidate blood RNA, four genes were validated, namely TMEM176B, SLC2A5, CDC45, and VSIG8, showing differential expression in blood of ARDS patients compared to controls, as well as dynamic expression in mouse lung tissues. Importantly, the addition of four blood genes and five immune cell proportions significantly improved the prediction performance of ARDS development, with 0.791 of the area under the curve from receiver-operator characteristic, compared to 0.725 for the basic model consisting of Acute Physiology and Chronic Health Evaluation (APACHE) III Score, sex, body mass index, bacteremia, and sepsis. A model-based nomogram was also developed for the clinical practice. CONCLUSION: This study identifies a wide range of ARDS relevant factors and develops a promising prediction model, enhancing early clinical management and intervention for ARDS development.

HKDC1 reprograms lipid metabolism to enhance gastric cancer metastasis and cisplatin resistance via forming a ribonucleoprotein complex.

As essential modulators of transcription and translation, RNA-binding proteins (RBPs) are frequently dysregulated in cancer. Bioinformatics study reveals that the RNA-binding protein hexokinase domain component 1 (HKDC1) is overexpressed in gastric cancer (GC). As HKDC1 plays a role in lipid homeostasis in the liver and glucose metabolism in certain cancers, the exact mechanism of action of HKDC1 in GC remains largely unknown. Upregulation of HKDC1 correlates with chemoresistance and poor prognosis in GC patients. HKDC1 enhances invasion, migration and resistance to cisplatin (CDDP) in GC cells in vitro and in vivo. Comprehensive transcriptomic sequencing and metabolomic analysis reveal that HKDC1 mediates abnormal lipid metabolism in GC cells. Herein, we identify a number of HKDC1-binding endogenous RNAs in GC cells, including protein kinase, DNA-activated, catalytic subunit (PRKDC) mRNA. We further validate that PRKDC is a crucial downstream effector of HKDC1 induced-GC tumorigenesis depends on lipid metabolism. Interestingly, G3BP1, a well-known oncoprotein, can be bound by HKDC1. HKDC1 cooperates with G3BP1 to enhance the stability of PRKDC transcript. Our results reveal a novel HKDC1/G3BP1-PRKDC regulatory axis that induces GC metastasis and chemoresistance via reprogramming lipid metabolism, which may provide an effective therapeutic strategy for a subset of GC with HKDC1 overexpression.

Oxygenated Static Preservation of Donation after Cardiac Death Liver Grafts Improves Hepatocyte Viability and Function.

BACKGROUND: Cell therapy, such as hepatocyte transplantation (HTx), is promising for the treatment of metabolic liver diseases or as a bridge to orthotopic liver transplantation in patients with fulminant liver failure. However, one of the limitations of this therapy is the shortage of donors. The present study aims to investigate whether the two-layer method (TLM) of cold preservation with oxygenation improves the viability and activity of hepatocytes from rat donation after cardiac death (DCD) donors compared with results obtained with the University of Wisconsin (UW) solution. Moreover, we evaluated the hepatocyte function after culture or transplantation into the spleen. MATERIALS AND METHODS: We used male Sprague-Dawley rats for this study. The DCD model was induced by phrenotomy after injecting heparin. We assigned rats based on warm ischemia times of 15 and 30 min to groups S and L, respectively. Each group (n = 5) was then subdivided as follows: (1) group S: not preserved (S/N), preserved by TLM for 3 h (S/TLM3) and 12 h (S/TLM12), and in the UW solution for 3 h (S/UW3) and 12 h (S/UW12), and (2) group L: not preserved (L/N), preserved by TLM for 3 h (L/TLM3) and 12 h (L/TLM12), and in the UW solution for 3 h (L/UW3) and 12 h (L/UW12). The cell viability and function of isolated DCD hepatocytes were analyzed for culture or HTx into the spleen. RESULTS: The viability and ATP levels of DCD hepatocytes significantly improved after TLM compared with the values after preservation in cold UW solution in group S/N (p < 0.059). The levels of albumin production and urea synthesis by hepatocytes after culture were significantly higher in groups S/TLM3 and S/TLM12 than in groups S/UW3 and S/UW12 (p < 0.05), respectively. Further, serum albumin levels after HTx were also markedly higher in groups S/TLM3 and S/TLM12 than in groups S/UW3 and S/UW12. The morphological features revealed that cultured and transplanted hepatocytes remained clearly viable and maintained an expression for specific hepatic function, such as the production of albumin and glycogen. CONCLUSION: This novel method of oxygenated cold preservation of DCD livers can expand the hepatocyte donor pool for HTx and establish a wider application of this developing technique.

Cold preservation of the liver with oxygenation by a two-layer method.

BACKGROUND: The two-layer method (TLM) has recently been found to be superior to simple cold storage in University of Wisconsin (UW) solution as a means of pancreas preservation for islet transplantation. In this study, we investigated whether TLM would result in better hepatocyte function over UW cold storage and if it could be applied to hepatocyte transplantation. MATERIALS AND METHODS: Hepatocytes from male Sprague Dawley rat livers were isolated and divided into three groups: a non-preservation group (group 1), a 10-h preservation group (group 2), and a 24-h preservation group (group 3). Groups 2 and 3 were then divided into three subgroups: a group preserved by the TLM (subgroup a), a group preserved in UW solution (subgroup b), and a group preserved in water (subgroup c). Isolated hepatocytes were evaluated for cell yield, viability, and adenosine triphosphate level after preservation. Hepatocytes were either cultured or transplanted. RESULTS: Although no differences in cell yield or morphological findings were observed between any of the groups, TLM significantly improved hepatocyte viability and adenosine triphosphate levels in comparison with UW cold storage. Albumin production or urea synthesis were significantly higher in subgroup 3a than in subgroup 3b at almost all time points. Surprisingly, after hepatocyte transplantation, the serum albumin level in subgroup 2a was significantly higher than in subgroup 2b at every time point. CONCLUSIONS: The results of this study demonstrated that liver preservation by the TLM before hepatocyte isolation might be beneficial and will be useful in the field of hepatotocyte transplantation.

Microencapsule technique protects hepatocytes from cryoinjury.

AIM: Hepatocyte transplantation is a potential alternative to whole organ liver transplantation. To realize this procedure, a hepatocyte bank system capable of supplying large numbers of hepatocytes must be established. We previously reported an easy method for cryopreserving hepatocytes using a microencapsulation technique. Here, we investigated how cryoinjury to microencapsulated hepatocytes could be avoided during cryopreservation. METHODS: Hepatocytes from Sprague-Dawley rats were harvested in situ using a two-step ethylenediaminetetraacetic acid (EDTA)/collagenase digestion protocol. The cells were microencapsulated using alginate-poly L-lysine. The microencapsulated hepatocytes were put into vials and immediately immersed in liquid nitrogen. The growth of ice crystals in the vials containing the microencapsulated hepatocytes was observed using cryomicroscopy. The microencapsulated hepatocytes were sectioned for ultrastructural examination to investigate their intracellular conditions. Finally, total RNA was isolated from the cryopreserved microencapsulated hepatocytes and analyzed for hepatocyte nuclear factor (HNF) using reverse transcriptase polymerase chain reaction (RT-PCR) analysis. RESULTS: Cryomicroscopy showed that the alginate microencapsulation technique protected the hepatocytes from physical damage caused by the growth of extracellular ice crystals. Ultrastructural examination revealed that the intracellular environment of the microencapsulated hepatocytes was maintained. The RT-PCR analysis additionally suggested that the alginate gel also maintained the HNF level. CONCLUSION: Our microencapsulation technique protects hepatocytes from cryoinjury. This novel technique could be utilized by hepatocyte banks.

Hepatocyte transplantation from steatotic liver in a rat model.

BACKGROUND: Hepatocyte transplantation (HTx) has progressed significantly, but widespread application remains slow because of the shortage of donor hepatocytes. Many sources of hepatic cells have been proposed as alternatives to isolated hepatocytes, but primary isolated hepatocytes continue to be the best source for liver cell-based therapy. To expand the donor pool, we focused on steatotic liver as a new cell source for HTx because numerous steatotic livers are discarded as unsuitable for orthotopic liver transplantation. This study investigated the efficacy of steatotic hepatocyte transplantation (SHTx) using steatotic liver in a rat model. MATERIALS AND METHODS: Hepatocytes were isolated from obese and lean Zucker rats. Hepatocytes from each group were cultured to analyze the function of steatotic hepatocytes. Hepatocytes from each group were also transplanted into the spleens of Nagase analbuminemic rats (NARs) to investigate the efficacy of SHTx. RESULTS: In the in vitro experiment, a real-time reverse-transcription polymerase chain reaction assay showed that albumin and several hepatocyte nuclear factors were highly expressed in both groups. Morphologically, the steatotic hepatocytes were positive for albumin, and an enzyme-linked immunosorbent assay showed no significant differences between the two groups except for albumin production after 5 d of culture. In the in vivo experiment, the transplanted steatotic hepatocytes in the spleens of Nagase analbuminemic rats were positive for albumin and periodic acid-Schiff staining. Surprisingly, an enzyme-linked immunosorbent assay showed no significant differences in the serum albumin levels between the two groups throughout the study period. CONCLUSIONS: We have demonstrated that steatotic hepatocytes are a potential new cell source for HTx therapy.

Long-term maintenance of the drug transport activity in cryopreservation of microencapsulated rat hepatocytes.

Transplantation of isolated hepatocytes has been proposed to compensate for essential functions lacking in liver failure or for genetic defects that alter a specific liver metabolic pathway. Hepatocyte utilization for these purposes would be facilitated with a reliable, reproducible, and effective method of long-term hepatocyte storage. We have recently developed a simple new system for cryopreservation of hepatocytes that encapsulates alginate microspheres and maintains liver-specific function. The aim of this study was to elucidate the transport and drug-metabolizing enzyme activities of cryopreserved microencapsulated hepatocytes stored for a long time. Morphological examinations showed there is no apparent injury of the hepatocytes during cryopreservation processes. A drug-metabolizing enzyme (testosterone 6beta-hydroxylase, a specific probe for CYP3A2) and drug transport activities [salicylate, allopurinol, and prostaglandin E2 (PGE2), typical substrates of rOat2] in cryopreserved microencapsulated hepatocytes were maintained up to 120 days. Our results thus demonstrate for the first time that cryopreservation of primary rat hepatocytes by the encapsulation technique allows long-term retention of drug metabolism and drug transport activities.

Intrasplenic transplantation of encapsulated hepatocytes decreases mortality and improves liver functions in fulminant hepatic failure from 90% partial hepatectomy in rats.

BACKGROUND: Encapsulated cell therapy might be a promising approach to enable cell transplantation without immunosuppression. This study investigates the viability and hepatic function of hepatocytes encapsulated with alginate/poly-L-lysine in vitro and the effect of the intrasplenic transplantation of cultured encapsulated hepatocytes on survival in 90% hepatectomized rats as a preliminary step toward allogeneic hepatocyte transplantation without immunosuppression. MATERIALS AND METHODS: Rat hepatocytes were isolated and encapsulated using alginate/poly-L-lysine. Encapsulated hepatocytes were cultured for 28 days to measure cell viability, liver function, and morphology. Rats were treated with a 90% partial hepatectomy and then immediately underwent the intrasplenic transplantation of the cultured encapsulated hepatocytes, the capsule alone, or the allogeneic hepatocytes without the capsule. The survival rate, liver function, and cell morphology were assessed after transplantation. RESULTS: The cultured encapsulated hepatocytes maintained their viability and showed better metabolic activity than day 0 cultured encapsulated hepatocytes. The encapsulated cells strongly expressed albumin and were positive for periodic acid-Schiff staining. Electron microscopy demonstrated that the microencapsulated hepatocytes retained the structural elements of hepatic cytoplasm and nuclei. Intrasplenic transplantation of the encapsulated hepatocytes increased the survival rate and improved the hepatic function. Encapsulated hepatocytes transplanted into rat spleen survived well and retained their hepatic function. Moreover, dramatic liver regeneration was observed 48 hr after transplantation in the group that received intrasplenic transplantations of encapsulated hepatocytes. CONCLUSIONS: The intrasplenic transplantation of cultured encapsulated hepatocytes improved the survival rate of an acute liver failure rat model induced by a 90% partial hepatectomy.

A novel method of cryopreservation of rat and human hepatocytes by using encapsulation technique and possible use for cell transplantation.

Encapsulated hepatocyte transplantation is a promising approach to cell transplantation without immunosuppression as an alternative to whole organ liver transplantation. However, the shortage of donor cells for hepatocyte transplantation has not been resolved, and at this critical point, it seems necessary to establish a method of hepatocyte cryopreservation to allow clinical application of hepatocyte transplantation and the development of a bioartificial liver system in the near future. In this study we demonstrated that cryopreserved microencapsulated rat and human hepatocytes can retain their hepatic function and that cryopreserved microencapsulated human hepatocytes transplanted into rat spleen remain viable without immunosuppression. Rat and human hepatocytes were isolated by a collagenase digestion method, and they were microencapsulated with poly-L-lysine. The microencapsulated rat hepatocytes were transferred to culture medium (DMEM containing 10% FBS and 10% DMSO) and immediately frozen in liquid nitrogen. A warm water bath (37 degrees C) was used to thaw the microencapsulated hepatocytes. Hepatic function, drug metabolism, and cell morphology were assessed after 90 days of cryopreservation. After 1 week of cryopreservation, microencapsulated hepatocytes were cultured for up to 2 weeks to assess their hepatic function and morphology. The morphology of human hepatocytes was assessed after 30 days of cryopreservation. Cryopreserved human hepatocytes were transplanted into rat spleen to assess their morphology. Cryopreserved microencapsulated hepatocytes retained their viability and were strongly positive for expression of albumin, OAT2, CYP3A2, and CYP3A9. Two weeks after cultivation, the cryopreserved microencapsulated rat hepatocytes had retained their hepatic function (urea synthesis). Cryopreserved microencapsulated human hepatocytes also mainly survived and retained their hepatic function for at least 30 days after cryopreservation. Moreover, entrapped cryopreserved human hepatocytes also survived and expressed albumin in rat spleen after transplantation. We demonstrated a novel method of long-term cryopreservation of rat and human hepatocytes by using an encapsulation technique, with retention of biological activity and excellent survival of the cryopreserved microencapsulated human hepatocytes transplanted into rat spleen. We believe that this novel approach to hepatocytes cryopreservation provides a new direction in encapsulated cell therapy with the goal of clinical application in the near future.