First-in-class MKK4 inhibitors enhance liver regeneration and prevent liver failure.

Diminished hepatocyte regeneration is a key feature of acute and chronic liver diseases and after extended liver resections, resulting in the inability to maintain or restore a sufficient functional liver mass. Therapies to restore hepatocyte regeneration are lacking, making liver transplantation the only curative option for end-stage liver disease. Here, we report on the structure-based development and characterization (nuclear magnetic resonance [NMR] spectroscopy) of first-in-class small molecule inhibitors of the dual-specificity kinase MKK4 (MKK4i). MKK4i increased liver regeneration upon hepatectomy in murine and porcine models, allowed for survival of pigs in a lethal 85% hepatectomy model, and showed antisteatotic and antifibrotic effects in liver disease mouse models. A first-in-human phase I trial (European Union Drug Regulating Authorities Clinical Trials [EudraCT] 2021-000193-28) with the clinical candidate HRX215 was conducted and revealed excellent safety and pharmacokinetics. Clinical trials to probe HRX215 for prevention/treatment of liver failure after extensive oncological liver resections or after transplantation of small grafts are warranted.

Sustained perfusion of revascularized bioengineered livers heterotopically transplanted into immunosuppressed pigs.

Implanted bioengineered livers have not exceeded three days of continuous perfusion. Here we show that decellularized whole porcine livers revascularized with human umbilical vein endothelial cells and implanted heterotopically into immunosuppressed pigs whose spleens had been removed can sustain perfusion for up to 15 days. We identified peak glucose consumption rate as a main predictor of the patency of the revascularized bioengineered livers (rBELs). Heterotopic implantation of rBELs into pigs in the absence of anticoagulation therapy led to sustained perfusion for three days, followed by a pronounced immune responses directed against the human endothelial cells. A 10 day steroid-based immunosuppression protocol and a splenectomy at the time of rBEL implantation reduced the immune responses and resulted in continuous perfusion of the rBELs for over two weeks. We also show that the human endothelial cells in the perfused rBELs colonize the liver sinusoids and express sinusoidal endothelial markers similar to those in normal liver tissue. Revascularized liver scaffolds that can maintain blood perfusion at physiological pressures might eventually help to overcome the chronic shortage of transplantable human livers.

Author Correction: Sustained perfusion of revascularized bioengineered livers heterotopically transplanted into immunosuppressed pigs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cold storage of porcine hepatocyte spheroids for spheroid bioartificial liver.

BACKGROUND AND AIMS: Cell-based therapies for liver disease such as bioartificial liver rely on a large quantity and high quality of hepatocytes. Cold storage was previously shown to be a better way to preserve the viability and functionality of hepatocytes during transportation rather than freezing, but this was only proved at a lower density of rat hepatocytes spheroids. The purpose of this study was to optimize conditions for cold storage of high density of primary porcine hepatocyte spheroids. METHODS: Porcine hepatocytes were isolated by a three-step perfusion method; hepatocyte spheroids were formed by a 24 hours rocked culture technique. Hepatocyte cell density was 5 × 106 /mL in 1000 mL spheroid forming medium. Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24, 48 or 72 hours in four different cold storage solutions: histidine-tryptophan-ketoglutarate (HTK) alone; HTK + 1 mM deferoxamine (DEF); HTK + 5 mM N-acetyl-L-cysteine (NAC); and HTK + 1 mM DEF + 5 mM NAC. The viability, ammonia clearance, albumin production, gene expression, and functional activity of cytochrome P450 enzymes were measured after recovery from the cold storage. RESULTS: In this study, we observed that cold-induced injury was reduced by the addition of the iron chelator. Viability of HTK + DEF group hepatocyte spheroids was increased compared with other cold storage groups (P < 0.05). Performance metrics of porcine hepatocyte spheroids cold stored for 24 hours were similar to those in control conditions. The hepatocyte spheroids in control conditions started to lose their ability to clear ammonia while production of albumin was still active at 48 and 72 hours (P < 0.05). In contrast, the viability and functionality of hepatocyte spheroids including ammonia clearance and albumin secretion were preserved in HTK + DEF group at both 48- and 72-hour time points (P < 0.05). CONCLUSIONS: The beneficial effects of HTK supplemented with DEF were more obvious after cold storage of high density of porcine hepatocyte spheroids for 72 hours. The porcine hepatocyte spheroids were above the cutoff criteria for use in a spheroid-based bioartificial liver.

Autologous Gene and Cell Therapy Provides Safe and Long-Term Curative Therapy in A Large Pig Model of Hereditary Tyrosinemia Type 1.

Orthotopic liver transplantation remains the only curative therapy for inborn errors of metabolism. Given the tremendous success for primary immunodeficiencies using ex-vivo gene therapy with lentiviral vectors, there is great interest in developing similar curative therapies for metabolic liver diseases. We have previously generated a pig model of hereditary tyrosinemia type 1 (HT1), an autosomal recessive disorder caused by deficiency of fumarylacetoacetate hydrolase (FAH). Using this model, we have demonstrated curative ex-vivo gene and cell therapy using a lentiviral vector to express FAH in autologous hepatocytes. To further evaluate the long-term clinical outcomes of this therapeutic approach, we continued to monitor one of these pigs over the course of three years. The animal continued to thrive off the protective drug NTBC, gaining weight appropriately, and maintaining sexual fecundity for the course of his life. The animal was euthanized 31 months after transplantation to perform a thorough biochemical and histological analysis. Biochemically, liver enzymes and alpha-fetoprotein levels remained normal and abhorrent metabolites specific to HT1 remained corrected. Liver histology showed no evidence of tumorigenicity and Masson's trichrome staining revealed minimal fibrosis and no evidence of cirrhosis. FAH-immunohistochemistry revealed complete repopulation of the liver by transplanted FAH-positive cells. A complete histopathological report on other organs, including kidney, revealed no abnormalities. This study is the first to demonstrate long-term safety and efficacy of hepatocyte-directed gene therapy in a large animal model. We conclude that hepatocyte-directed ex-vivo gene therapy is a rational choice for further exploration as an alternative therapeutic approach to whole organ transplantation for metabolic liver disease, including HT1.

Randomized Trial of Spheroid Reservoir Bioartificial Liver in Porcine Model of Posthepatectomy Liver Failure.

Acute liver failure (ALF) is a catastrophic condition that can occur after major liver resection. The aim of this study was to determine the effects of the spheroid reservoir bio-artificial liver (SRBAL) on survival, serum chemistry, and liver regeneration in posthepatectomy ALF pigs. Wild-type large white swine (20 kg-30 kg) underwent intracranial pressure (ICP) probe placement followed by 85% hepatectomy. Computed tomography (CT) volumetrics were performed to measure the extent of resection, and at 48 hours following hepatectomy to assess regeneration of the remnant liver. Animals were randomized into three groups based on treatment delivered 24-48 hours after hepatectomy: Group1-standard medical therapy (SMT, n = 6); Group2-SMT plus bio-artificial liver treatment using no hepatocytes (0 g, n = 6); and Group3-SMT plus SRBAL treatment using 200 g of primary porcine hepatocyte spheroids (200 g, n = 6). The primary endpoint was survival to 90 hours following hepatectomy. Death equivalent was defined as unresponsive grade 4 hepatic encephalopathy or ICP greater than 20 mmHg with clinical evidence of brain herniation. All animals in both (SMT and 0 g) control groups met the death equivalent before 51 hours following hepatectomy. Five of 6 animals in the 200-g group survived to 90 hours (P < 0.01). The mean ammonia, ICP, and international normalized ratio values were significantly lower in the 200-g group. CT volumetrics demonstrated increased volume regeneration at 48 hours following hepatectomy in the 200-g group compared with the SMT (P < 0.01) and 0-g (P < 0.01) groups. Ki-67 staining showed increased positive staining at 48 hours following hepatectomy (P < 0.01). Conclusion: The SRBAL improved survival, reduced ammonia, and accelerated liver regeneration in posthepatectomy ALF. Improved survival was associated with a neuroprotective benefit of SRBAL therapy. These favorable results warrant further clinical testing of the SRBAL.

Rapid Deployment Aortic Valves Deliver Superior Hemodynamic Performance In Vitro.

OBJECTIVE: Clinical studies have demonstrated excellent hemodynamic performance of rapid deployment aortic valves; however, few studies have directly compared the performance of these valves with conventional bioprosthetic valves. Thus, the hemodynamic function of the EDWARDS INTUITY valve (rapid deployment valve) was compared with the Edwards Magna Ease valve in vitro (Edwards Lifesciences Corp, Irvine, CA USA). METHODS: Elastomeric material was used to create an aortic root model that included a left ventricular outflow tract and aortic annulus. The model was based on reconstructions from 3-dimensional multislice computed tomography images in patients with aortic stenosis; the aortic root was scaled to a 21-mm effective annulus diameter. EDWARDS INTUITY valves (21-mm diameter) were deployed by stent frame expansion within the aortic root; Edwards Magna Ease valves (21-mm diameter) were sutured to the annulus. The left ventricular outflow tract area index (left ventricular outflow tract area/baseline area) and ellipticity or noncircularity as indexed by Dmax/Dmin were measured under a video microscope after valve placement. Hemodynamic data were collected under pulsatile flow with saline (70 beats per minute, 5 L/min, 100 mm Hg aortic pressure). RESULTS: Compared with the Edwards Magna Ease valve (n = 4), the EDWARDS INTUITY valve (n = 4) had a greater effective orifice area (1.56 ± 0.01 vs 1.85 ± 0.06 cm, P < 0.001) and a lower transvalvular pressure gradient (23.4 ± 0.51 vs 16.8 ± 1.3 mm Hg, P < 0.001). Multiple regression analysis showed that 93% of the variation in the effective orifice area and transvalvular pressure gradient was due to variation in the left ventricular outflow tract area index and ellipticity index. CONCLUSIONS: A clinically relevant aortic root model was developed to evaluate aortic valve performance. The superior performance of the EDWARDS INTUITY valve seemed to be related to both a greater inflow area and a more circular left ventricular outflow tract.

Recent Advances in Decellularization and Recellularization for Tissue-Engineered Liver Grafts.

Liver transplantation from deceased or living human donors remains the only proven option for patients with end-stage liver disease. However, the shortage of donor organs is a significant clinical concern that has led to the pursuit of tissue-engineered liver grafts generated from decellularized liver extracellular matrix and functional cells. Investigative efforts on optimizing both liver decellularization and recellularization protocols have been made in recent decades. In the current review, we briefly summarize these advances, including the generation of high-quality liver extracellular matrix scaffolds, evaluation criteria for quality control, modification of matrix for enhanced properties, and reseeding strategies. These efforts to optimize the methods of decellularization and recellularization lay the groundwork towards generating a transplantable, human-sized liver graft for the treatment of patients with severe liver disease.

Recent Advances in Decellularization and Recellularization for Tissue-Engineered Liver Grafts.

Liver transplantation from deceased or living human donors remains the only proven option for patients with end-stage liver disease. However, the shortage of donor organs is a significant clinical concern that has led to the pursuit of tissue-engineered liver grafts generated from decellularized liver extracellular matrix and functional cells. Investigative efforts on optimizing both liver decellularization and recellularization protocols have been made in recent decades. In the current review, we briefly summarize these advances, including the generation of high-quality liver extracellular matrix scaffolds, evaluation criteria for quality control, modification of matrix for enhanced properties, and reseeding strategies. These efforts to optimize the methods of decellularization and recellularization lay the groundwork towards generating a transplantable, human-sized liver graft for the treatment of patients with severe liver disease.

Concise Review: Liver Regenerative Medicine: From Hepatocyte Transplantation to Bioartificial Livers and Bioengineered Grafts.

Donor organ shortage is the main limitation to liver transplantation as a treatment for end-stage liver disease and acute liver failure. Liver regenerative medicine may in the future offer an alternative form of therapy for these diseases, be it through cell transplantation, bioartificial liver (BAL) devices, or bioengineered whole organ liver transplantation. All three strategies have shown promising results in the past decade. However, before they are incorporated into widespread clinical practice, the ideal cell type for each treatment modality must be found, and an adequate amount of metabolically active, functional cells must be able to be produced. Research is ongoing in hepatocyte expansion techniques, use of xenogeneic cells, and differentiation of stem cell-derived hepatocyte-like cells (HLCs). HLCs are a few steps away from clinical application, but may be very useful in individualized drug development and toxicity testing, as well as disease modeling. Finally, safety concerns including tumorigenicity and xenozoonosis must also be addressed before cell transplantation, BAL devices, and bioengineered livers occupy their clinical niche. This review aims to highlight the most recent advances and provide an updated view of the current state of affairs in the field of liver regenerative medicine. Stem Cells 2017;35:42-50.

Liver transplantation after share 35: Impact on pretransplant and posttransplant costs and mortality.

Share 35 was implemented in 2013 to direct livers to the most urgent candidates by prioritizing Model for End-Stage Liver Disease (MELD) ≥ 35 patients. We aim to evaluate this policy's impact on costs and mortality. Our study includes 834 wait-listed patients and 338 patients who received deceased donor, solitary liver transplants at Mayo Clinic between January 2010 and December 2014. Of these patients, 101 (30%) underwent transplantation after Share 35. After Share 35, 29 (28.7%) MELD ≥ 35 patients received transplants, as opposed to 46 (19.4%) in the pre-Share 35 era (P = 0.06). No significant difference in 90-day wait-list mortality (P = 0.29) nor 365-day posttransplant mortality (P = 0.68) was found between patients transplanted before or after Share 35. Mean costs were $3,049 (P = 0.30), $5226 (P = 0.18), and $10,826 (P = 0.03) lower post-Share 35 for the 30-, 90-, and 365-day pretransplant periods, and mean costs were $5010 (P = 0.41) and $5859 (P = 0.57) higher, and $9145 (P = 0.54) lower post-Share 35 for the 30-, 90-, and 365-day posttransplant periods. In conclusion, the added cost of transplanting more MELD ≥ 35 patients may be offset by pretransplant care cost reduction. Despite shifting organs to critically ill patients, Share 35 has not impacted mortality significantly. Liver Transplantation 23:11-18 2017 AASLD.

Curative ex vivo liver-directed gene therapy in a pig model of hereditary tyrosinemia type 1.

We tested the hypothesis that ex vivo hepatocyte gene therapy can correct the metabolic disorder in fumarylacetoacetate hydrolase-deficient (Fah(-/-)) pigs, a large animal model of hereditary tyrosinemia type 1 (HT1). Recipient Fah(-/-) pigs underwent partial liver resection and hepatocyte isolation by collagenase digestion. Hepatocytes were transduced with one or both of the lentiviral vectors expressing the therapeutic Fah and the reporter sodium-iodide symporter (Nis) genes under control of the thyroxine-binding globulin promoter. Pigs received autologous transplants of hepatocytes by portal vein infusion. After transplantation, the protective drug 2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione (NTBC) was withheld from recipient pigs to provide a selective advantage for expansion of corrected FAH(+) cells. Proliferation of transplanted cells, assessed by both immunohistochemistry and noninvasive positron emission tomography imaging of NIS-labeled cells, demonstrated near-complete liver repopulation by gene-corrected cells. Tyrosine and succinylacetone levels improved to within normal range, demonstrating complete correction of tyrosine metabolism. In addition, repopulation of the Fah(-/-) liver with transplanted cells inhibited the onset of severe fibrosis, a characteristic of nontransplanted Fah(-/-) pigs. This study demonstrates correction of disease in a pig model of metabolic liver disease by ex vivo gene therapy. To date, ex vivo gene therapy has only been successful in small animal models. We conclude that further exploration of ex vivo hepatocyte genetic correction is warranted for clinical use.

Extracorporeal Liver Support and Liver Transplant for Patients with Acute-on-Chronic Liver Failure.

Recognition of acute-on-chronic liver failure (ACLF) as a unique entity is slowly evolving, as are therapies to improve survival of affected patients. Further investigation into its disease process and proper treatments with critical timing are important for improving patient survival. At this time, liver transplant is the only treatment known to improve survival in liver-failure patients. However, liver transplantation has its own disadvantages, such as organ shortage and the need for lifelong immunotherapy. Bridging therapies such as extracorporeal liver-support systems are attractive options to stabilize patients until transplantation or spontaneous recovery. The goals of these liver-support systems are to remove detoxification products, reduce systemic inflammation, and enhance regeneration of the injured liver. These devices have been under development for the past decade; a few are in clinical trials. At this time, there is no proven clearcut survival benefit in these devices, but they may improve the outcome of challenging cases and potentially avoid or postpone liver transplantation in some cases.

Phosphorylation facilitates the integrin binding of filamin under force.

Filamins are actin binding proteins that contribute to cytoskeletal integrity and biochemical scaffolds during mechanochemical signal transductions. Structurally, human filamins are dimers composed of an actin-binding domain with 24 immunoglobulin (Ig)-like repeats. In this study, we focus on the recently solved high-resolution crystal structure of Ig-like repeats 19-21 of filamin-A (IgFLNa-R19-R21). IgFLNa-R19-21 is of marked importance because it contains the binding site for integrins and facilitates the dynamic ability of filamin-A to communicate with the extracellular environment. However, the structure of filamin-A shows an interesting domain arrangement where the integrin binding site on IgFLNa-R21 is hindered sterically by IgFLNa-R20. Thus, a number of hypotheses on the regulation of filamin-A exist. Using molecular dynamics simulations we evaluated the effects of two primary regulators of filamin-A, force and phosphorylation. We find that a tensile force of 40 pN is sufficient to initiate the partial removal of the autoinhibition on the integrin binding site of IgFLNa-R21. Force coupled to phosphorylation at Ser(2152), however, affords complete dissociation of autoinhibition with a decreased force requirement. Phosphorylation seems to decrease the threshold for removing the IgFLNa-R20 beta-strand inhibitor within 300 ps with 40 pN tensile force. Furthermore, the molecular dynamic trajectories illustrate phosphorylation of Ser(2152) without force is insufficient to remove autoinhibition. We believe the results of this study implicate filamin-A as a tunable mechanosensor, where its sensitivity can be modulated by the degree of phosphorylation.

Retrograde ethanol infusion in the vein of Marshall: regional left atrial ablation, vagal denervation and feasibility in humans.

BACKGROUND: The vein of Marshall (VOM) is an attractive target during ablation of atrial fibrillation due to its autonomic innervation, its location anterior to the left pulmonary veins and drainage in the coronary sinus. METHODS AND RESULTS: We studied 17 dogs. A coronary sinus venogram showed a VOM in 13, which was successfully cannulated with an angioplasty wire and balloon. In 5 dogs, electroanatomical maps of the left atrium were performed at baseline and after ethanol infusion in the VOM, which demonstrated a new crescent-shaped scar, extending from the annular left atrium towards the posterior wall and left pulmonary veins. In 4 other dogs, effective refractory periods (ERP) were measured at 3 sites in the left atrium, before and after high-frequency bilateral vagal stimulation. The ERP decreased from 113.6+/-35.0 ms to 82.2+/-25.4 ms (p<0.05) after vagal stimulation. After VOM ethanol infusion, vagally-mediated ERP decrease was eliminated (from 108.6+/-24.1 ms to 96.4 +/-16.9ms, p=NS). The abolition of vagal effects was limited to sites near the VOM (ERP: 104+/-14 ms, vs 98.6+/-12.2 ms post vagal stimulation, p=ns), as opposed to sites remote to VOM (ERP: 107.2+/-14.9 ms, vs 78.6+/-14.7ms post vagal stimulation, p<0.05). To test feasibility in humans, 5 patients undergoing pulmonary vein antral isolation had successful VOM cannulation and ethanol infusion: left atrial voltage maps demonstrated new scar involving the infero-posterior left atrial wall extending towards the left pulmonary veins. CONCLUSIONS: Ethanol infusion in then VOM achieves significant left atrial tissue ablation, abolishes local vagal responses and is feasible in humans.