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The Effect of Ex Vivo Human Serum from Liver Disease Patients on Cellular Protein Synthesis and Growth.

ABSTRACT: Sarcopenia is a common complication affecting liver disease patients, yet the underlying mechanisms remain unclear. We aimed to elucidate the cellular mechanisms that drive sarcopenia progression using an in vitro model of liver disease. C2C12 myotubes were serum and amino acid starved for 1-h and subsequently conditioned with fasted ex vivo serum from four non-cirrhotic non-alcoholic fatty liver disease patients (NAFLD), four decompensated end-stage liver disease patients (ESLD) and four age-matched healthy controls (CON) for 4- or 24-h. After 4-h C2C12 myotubes were treated with an anabolic stimulus (5 mM leucine) for 30-min. Myotube diameter was reduced following treatment with serum from ESLD compared with CON (-45%) and NAFLD (-35%; p < 0.001 for both). A reduction in maximal mitochondrial respiration (24% and 29%, respectively), coupling efficiency (~12%) and mitophagy (~13%) was identified in myotubes conditioned with NAFLD and ESLD serum compared with CON (p < 0.05 for both). Myostatin (43%, p = 0.04) and MuRF-1 (41%, p = 0.03) protein content was elevated in myotubes treated with ESLD serum compared with CON. Here we highlight a novel, experimental platform to further probe changes in circulating markers associated with liver disease that may drive sarcopenia and develop targeted therapeutic interventions.

SUBMITTER: Allen SL

PROVIDER: S-EPMC8997893 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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The Effect of Ex Vivo Human Serum from Liver Disease Patients on Cellular Protein Synthesis and Growth.

Allen Sophie L SL Seabright Alex P AP Quinlan Jonathan I JI Dhaliwal Amritpal A Williams Felicity R FR Fine Nicholas H F NHF Hodson David J DJ Armstrong Matthew J MJ Elsharkaway Ahmed M AM Greig Carolyn A CA Lai Yu-Chiang YC Lord Janet M JM Lavery Gareth G GG Breen Leigh L

Cells 20220324 7

Sarcopenia is a common complication affecting liver disease patients, yet the underlying mechanisms remain unclear. We aimed to elucidate the cellular mechanisms that drive sarcopenia progression using an in vitro model of liver disease. C2C12 myotubes were serum and amino acid starved for 1-h and subsequently conditioned with fasted ex vivo serum from four non-cirrhotic non-alcoholic fatty liver disease patients (NAFLD), four decompensated end-stage liver disease patients (ESLD) and four age-ma ...[more]

PMID: 35406665

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Project description:IntroductionPatients with cirrhosis undergoing liver transplantation frequently exhibit systemic inflammation, coagulation derangements, and edema, indicating endothelial dysfunction. This syndrome may worsen after ischemia-reperfusion injury of the liver graft, coincident with organ dysfunction that worsens patient outcomes. Little is known about changes in endothelial permeability during liver transplantation. We hypothesized that sera from these patients would increase permeability in cultured human endothelial cells ex vivo.MethodsAdults with cirrhosis presenting for liver transplantation provided consent for blood collection during surgery. Sera were prepared at five time points spanning the entire operation. The barrier function of human pulmonary microvascular endothelial cells in culture was assessed by transendothelial resistance measured using the ECIS ZΘ system. Confluent cells from two different endothelial cell donors were stimulated with human serum from liver transplant patients. Pooled serum from healthy men and purified inflammatory agonists served as controls. The permeability response to serum was quantified as the area under the normalized resistance curve. Responses were compared between time points and analyzed for associations with clinical characteristics of liver transplant patients and their grafts.ResultsLiver transplant sera from all time points during surgery-induced permeability in both endothelial cell lines. The magnitude of permeability change was heterogeneous between patients, and there were differences in the effects of sera on the two endothelial cell lines. In one of the cell lines, the severity of liver disease was associated with greater permeability at the start of surgery. In the same cell line, serum collected 15 min after liver reperfusion induced significantly more permeability as compared to that collected at the start of surgery. Early postreperfusion sera from patients undergoing living donor transplants induced more permeability than sera from deceased donor transplants. Sera from two exemplary cases of patients on preoperative dialysis, and one patient with an unexpectedly long warm ischemia time of the liver graft, induced exaggerated and prolonged endothelial permeability.DiscussionSerum from patients with cirrhosis undergoing liver transplantation induces permeability of cultured human pulmonary microvascular endothelial cells. Increased endothelial permeability during liver transplantation may contribute to organ injury and present a target for future therapeutics.

Project description:IntroductionNormothermic ex vivo liver perfusion (NEVLP) is an organ preservation method that allows liver graft functional assessment prior to transplantation. One key component of normothermic perfusion solution is an oxygen carrier to provide oxygen to the liver to sustain metabolic activities. Oxygen carriers such as red blood cells (RBCs) or hemoglobin-based oxygen carriers have an unknown effect on the liver-resident immune cells during NEVLP. In this study, we assessed the effects of different oxygen carriers on the phenotype and function of liver-resident immune cells.MethodsAdult Lewis rat livers underwent NEVLP using three different oxygen carriers: human packed RBCs (pRBCs), rat pRBCs, or Oxyglobin (a synthetic hemoglobin-based oxygen carrier). Hourly perfusate samples were collected for downstream analysis, and livers were digested to isolate immune cells. The concentration of common cytokines was measured in the perfusate, and the immune cells underwent phenotypic characterization with flow cytometry and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The stimulatory function of the liver-resident immune cells was assessed using mixed lymphocyte reactions.ResultsThere were no differences in liver function, liver damage, or histology between the three oxygen carriers. qRT-PCR revealed that the gene expression of nuclear factor κ light chain enhancer of activated B cells (NF-kB), Interleukin (IL-1β), C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 7 (CCL7), and CD14 was significantly upregulated in the human pRBC group compared with that in the naive, whereas the rat pRBC and Oxyglobin groups were not different from that of naive. Flow cytometry demonstrated that the cell surface expression of the immune co-stimulatory protein, CD86, was significantly higher on liver-resident macrophages and plasmacytoid dendritic cells perfused with human pRBC compared to Oxyglobin. Mixed lymphocyte reactions revealed increased allogeneic T-cell proliferation in the human and rat pRBC groups compared to that in the Oxyglobin group.ConclusionsLiver-resident immune cells are important mediators of rejection after transplantation. In this study, we show that the oxygen carrier used in NEVLP solutions can affect the phenotype of these liver-resident immune cells. The synthetic hemoglobin-based oxygen carrier, Oxyglobin, showed the least amount of liver-resident immune cell activation and the least amount of allogeneic proliferation when compared to human or rat pRBCs. To mitigate liver-resident immune cell activation during NEVLP (and subsequent transplantation), Oxyglobin may be an optimal oxygen carrier.

Project description:Osteocytes are deeply embedded in the mineralized matrix of bone and are nonproliferative, making them a challenge to isolate and maintain using traditional in vitro culture methods without sacrificing their inimitable phenotype. We studied the synergistic effects of two microenvironmental factors that are vital in retaining, ex vivo, the phenotype of primary human osteocytes: hypoxia and three-dimensional (3D) cellular network. To recapitulate the lacunocanalicular structure of bone tissue, we assembled and cultured primary human osteocytic cells with biphasic calcium phosphate microbeads in a microfluidic perfusion culture device. The 3D cellular network was constructed by the following: (1) the inhibited proliferation of cells entrapped by microbeads, biomimetically resembling lacunae, and (2) the connection of neighboring cells by dendrites through the mineralized, canaliculi-like interstitial spaces between the microbeads. We found that hypoxia synergistically and remarkably upregulated the mature osteocytic gene expressions of the 3D-networked cells, SOST (encoding sclerostin) and FGF23 (encoding fibroblast growth factor 23), by several orders of magnitude in comparison to those observed from two-dimensional and normoxic culture controls. Intriguingly, hypoxia facilitated the self-assembly of a nonproliferating, osteoblastic monolayer on the surface of the 3D-networked cells, replicating the osteoblastic endosteal cell layer found at the interface between native bone and bone marrow tissues. Our ability to replicate, with hypoxia, the strong expressions of these mature osteocytic markers, SOST and FGF23, is important since these (1) could not be significantly produced in vitro and (2) are new important targets for treating bone diseases. Our findings are therefore expected to facilitate ex vivo studies of human bone diseases using primary human bone cells and enable high-throughput evaluation of potential bone-targeting therapies with clinical relevance.

Dataset Information

The Effect of Ex Vivo Human Serum from Liver Disease Patients on Cellular Protein Synthesis and Growth.

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The Effect of Ex Vivo Human Serum from Liver Disease Patients on Cellular Protein Synthesis and Growth.

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