Gundumi A. Upadhya

Saline-Linked Surface Radiofrequency Ablation: Factors Affecting Steam Popping and Depth of Injury in the Pig Liver

Summary Background Data:Saline-linked surface radiofrequency (RF) ablation is a new technique for applying RF energy to surfaces. The surface is cooled, which prevents charring and results in deeper coagulation. However, subsurface heating may lead to steam formation and a form of tissue disruption called steam popping. We determined parameters that predict steam popping and depth of tissue destruction under nonpopping conditions. A commercially available saline-linked surface RF cautery device (Floating Ball 3.0, TissueLink, Inc.) was used. Methods:One hundred eighty circular lesions were created varying in lesion diameter, duration, power, and inflow occlusion. Variables affecting popping were determined. Then factors influencing lesion depth were studied at fixed nonpopping diameter/power combinations (1 cm/10W, 2 cm/15W, 4 cm/60W). Tissue viability was determined in selected samples by staining of tissue NADH. Results:The probability of steam popping was directly related to power level and inflow occlusion, and indirectly related to lesion diameter. Depth of injury under safe nonpopping conditions was directly related to power, lesion size, and inflow occlusion. Maximum depth in excess of 20 mm was achieved using a 4 cm diameter at 60W with inflow occlusion. Microscopy of NADH-stained tissues showed a complete cell killing in the macroscopically visible coagulated area. Conclusions:Steam popping can be avoided by selecting power level/lesion diameter combinations. Tissue destruction to 20 mm can be safely achieved with short periods of inflow occlusion. The device has promise as a treatment of superficial tumors and close resection margins.

Endoplasmic reticulum stress is a mediator of posttransplant injury in severely steatotic liver allografts

Hepatic steatosis continues to present a major challenge in liver transplantation. These organs have been shown to have increased susceptibility to cold ischemia/reperfusion (CIR) injury in comparison with otherwise comparable lean livers; the mechanisms governing this increased susceptibility to CIR injury are not fully understood. Endoplasmic reticulum (ER) stress is an important link between hepatic steatosis, insulin resistance, and metabolic syndrome. In this study, we investigated ER stress signaling and blockade in the mediation of CIR injury in severely steatotic rodent allografts. Steatotic allografts from genetically leptin‐resistant rodents had increased ER stress responses and increased markers of hepatocellular injury after liver transplantation into strain‐matched lean recipients. ER stress response components were reduced by the chemical chaperone taurine‐conjugated ursodeoxycholic acid (TUDCA), and this resulted in an improvement in the allograft injury. TUDCA treatment decreased nuclear factor kappa B activation and the proinflammatory cytokines interleukin‐6 and interleukin‐1β. However, the predominant response was decreased expression of the ER stress cell death mediator [CCAAT/enhancer‐binding protein homologous protein (CHOP)]. Furthermore, activation of inflammation‐associated caspase‐11 was decreased, and this linked ER stress/CHOP to proinflammatory cytokine production after steatotic liver transplantation. These data confirm ER stress in steatotic allografts and implicate this as a mediating mechanism of inflammation and hepatocyte death in the steatotic liver allograft. Liver Transpl 17:189–200, 2011.

Ischemia–reperfusion injury in rat steatotic liver is dependent on NFκB P65 activation☆

BACKGROUND Steatotic liver grafts tolerate ischemia-reperfusion (I/R) injury poorly, contributing to increased primary graft nonfunction following transplantation. Activation of nuclear factor kappa-B (NFκB) following I/R injury plays a crucial role in activation of pro-inflammatory responses leading to injury. METHODS We evaluated the role of NFκB in steatotic liver injury by using an orthotopic liver transplant (OLT) model in Zucker rats (lean to lean or obese to lean) to define the mechanisms of steatotic liver injury. Obese donors were treated with bortezomib to assess the role of NF-κB in steatotic liver I/R injury. Hepatic levels of NF-κB and pro-inflammatory cytokines were analyzed by ELISA. Serum transaminase levels and histopathological analysis were performed to assess associated graft injury. RESULTS I/R injury in steatotic liver results in significant increases in activation of NF-κB (40%, p<0.003), specifically the p65 subunit following transplantation. Steatotic donor pretreatment with proteasome inhibitor bortezomib (0.1mg/kg) resulted in significant reduction in levels of activated NF-κB (0.58±0.18 vs. 1.37±0.06O.D./min/10 μg protein, p<0.003). Bortezomib treatment also reduced expression of pro-inflammatory cytokines MIP-2 compared with control treated steatotic and lean liver transplants respectively (106±17.5 vs. 443.3±49.9 vs. 176±10.6 pg/mL, p=0.02), TNF-α (223.8±29.9 vs. 518.5±66.5 vs. 264.5±30.1 pg/2 μg protein, p=0.003) and IL-1β (6.0±0.91 vs. 19.8±5.2 vs. 5±1.7 pg/10 μg protein, p=0.02) along with a significant reduction in ALT levels (715±71 vs. 3712.5±437.5 vs. 606±286 U/L, p=0.01). CONCLUSION These results suggest that I/R injury in steatotic liver transplantation are associated with exaggerated activation of NFκB subunit p65, leading to an inflammatory mechanism of reperfusion injury and necrosis. Proteasome inhibition in steatotic liver donor reduces NFκB p65 activation and inflammatory I/R injury, improving transplant outcomes of steatotic grafts in a rat model.

Antibody mediated therapy targeting CD47 inhibits tumor progression of hepatocellular carcinoma

Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival times. The efficacy of current systemic therapies for HCC is limited. In this study, we used xenograft tumor models to investigate the use of antibodies that block CD47 and inhibit HCC tumor growth. Immunostaining of tumor tissue and HCC cell lines demonstrated CD47 over-expression in HCC as compared to normal hepatocytes. Macrophage phagocytosis of HCC cells was increased after treatment with CD47 antibodies (CD47mAbs) that block CD47 binding to SIRPα. Further, CD47 blockade inhibited tumor growth in both heterotopic and orthotopic models of HCC, and promoted the migration of macrophages into the tumor mass. Our results demonstrate that targeting CD47 by specific antibodies has potential immunotherapeutic efficacy in human HCC.

Novel strategy to decrease reperfusion injuries and improve function of cold‐preserved livers using normothermic ex vivo liver perfusion machine

Normothermic extracorporeal liver perfusion (NELP) can decrease ischemia/reperfusion injury to the greatest degree when cold ischemia time is minimized. Warm perfusion of cold‐stored livers results in hepatocellular damage, sinusoidal endothelial cell (SEC) dysfunction, and Kupffer cell activation. However, the logistics of organ procurement mandates a period of cold preservation before NELP. The aim of this study was to determine the beneficial effects of gradual rewarming of cold‐stored livers by placement on NELP. Three female porcine livers were used for each group. In the immediate NELP group, procured livers were immediately placed on NELP for 8 hours. In the cold NELP group, livers were cold‐stored for 4 hours followed by NELP for 4 hours. In rewarming groups, livers were cold‐stored for 4 hours, then gradually rewarmed in different durations to 38°C and kept on NELP for an additional 4 hours. For comparison purposes, the last 4 hours of NELP runs were considered to be the evaluation phase. Immediate NELP livers had significantly lower concentrations of liver transaminases, hyaluronic acid, and β‐galactosidase and had higher bile production compared to the other groups. Rewarming livers had significantly lower concentrations of hyaluronic acid and β‐galactosidase compared to the cold NELP livers. In addition, there was a significant decline in international normalized ratio values, improved bile production, reduced biliary epithelial cell damage, and improved cholangiocyte function. Thus, if a NELP machine is not available at the procurement site and livers will need to undergo a period of cold preservation, a gradual rewarming protocol before NELP may greatly reduce damages that are associated with reperfusion. In conclusion, gradual rewarming of cold‐preserved livers upon NELP can minimize the hepatocellular damage, Kupffer cell activation, and SEC dysfunction. Liver Transpl 22:333–343, 2016.

Cold preservation of isolated sinusoidal endothelial cells in MMP 9 knockout mice: Effect on morphology and platelet adhesion

Cold preservation of rat sinusoidal endothelial cells causes actin disassembly, cell rounding, matrix metalloproteinase (MMP) secretion, and platelet adhesiveness. Studies in rats suggest that gelatinases MMP2 and MMP9 are the key mediators of the injury. We created a model of cold preservation injury in mouse sinusoidal endothelial cell (MSEC) to examine the effect of cold on MSEC, specifically on MSEC from genetically deleted mice (MMP9/KO) mice. MSEC were isolated from wild‐type and MMP9/KO mice and cold preserved for up to 24 hours. MMP activity was measured in culture supernatants and in effluents from preserved whole mouse livers. Cellular and actin morphology were studied by light and fluorescence microscopy. A platelet–MSEC adhesion assay was performed. Yield, growth, and appearance of MSEC were similar in wild‐type and MMP9/KO mice. Cold‐preserved wild‐type MSEC exhibited actin disassembly and cell rounding as in the rat but at a much slower rate. These morphologic cell changes were attenuated in MSEC from MMP9/KO mice. Both MMP2 and MMP9 were present in liver effluents of wild‐type mice, but MMP9 was absent in effluents from MMP9/KO mice. Total MMP activity in culture supernatants was greater after preservation in wild‐type than in MMP9/KO mice. There was significantly more platelet adhesion to wild‐type MSEC than to MSEC from MMP9/KO mice. In conclusion, MSEC is an excellent model system for the study of cold preservation injury. Injury is similar to rat sinusoidal endothelial cells but delayed. MMP9 is a key mediator of the cold preservation injury. (Liver Transpl 2004;10:1041–1048.)

Normothermic extracorporeal liver perfusion for donation after cardiac death (DCD) livers.

BACKGROUND The susceptibility of extended criteria livers to ischemia reperfusion injury is a major obstacle in organ cold preservation. Normothermic extracorporeal liver perfusion (NELP) has been investigated to reduce ischemic damage, restore physiologic function, and assess viability of the liver prior to transplant. The goal of this study is to compare physiological parameters of livers maintained continuously on NELP to those preserved in cold solution. METHODS Livers from 9 female landrace pigs were subjected to either 20 minutes (W20-NELP), 40 minutes (W40-NELP), or 60 minutes (W60-NELP) of warm ischemia followed by 6 hours of NELP followed by a 2-hour NELP evaluation phase. This was compared with 3 livers subjected to 40 minutes of warm ischemia time followed by 6 hours of cold storage (W40-Cold) and a 2-hour NELP evaluation phase. Groups were compared with the 2-way analysis of variance test. RESULTS NELP stabilized transaminases accompanied by significant improvement in bile production and decline in lactate and INR values in all W-NELP groups. Histologic analysis demonstrated significant improvement from 0 hour (mild-to-moderate sinusoidal dilation and zone 3 necrosis) to the end of the NELP run (minimal necrosis and mild IRI). Comparison of W40-NELP and W40-Cold revealed greater bile production and oxygen extraction ratio in W40-NELP. In contrast, markers of cellular and functional damage were increased in the W40-Cold group. CONCLUSION NELP improves metabolic and functional parameters of livers with either short or extended warm ischemia times compared with livers subjected to comparable cold ischemia times.

CD47 blockade reduces ischemia/reperfusion injury and improves survival in a rat liver transplantation model.

Orthotopic liver transplantation (OLT) remains the standard treatment option for nonresponsive liver failure. Because ischemia/reperfusion injury (IRI) is an important impediment to the success of OLT, new therapeutic strategies are needed to reduce IRI. We investigated whether blocking the CD47/thrombospondin‐1 inhibitory action on nitric oxide signaling with a monoclonal antibody specific to CD47 (CD47mAb400) would reduce IRI in liver grafts. Syngeneic OLT was performed with Lewis rats. Control immunoglobulin G or CD47mAb400 was administered to the donor organ at procurement or to both the organ and the recipient at the time of transplant. Serum transaminases, histological changes of the liver, and animal survival were assessed. Oxidative stress, inflammatory responses, and hepatocellular damage were also quantified. A significant survival benefit was not achieved when CD47mAb400 was administered to the donor alone. However, CD47mAb400 administration to both the donor and the recipient increased animal survival afterward. The CD47mAb400‐treated group showed lower serum transaminases, bilirubin, oxidative stress, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick‐end labeling staining, caspase‐3 activity, and proinflammatory cytokine expression of tumor necrosis factor α, interleukin‐1β, and interleukin‐6. Thus, CD47 blockade with CD47mAb400 administered both to the donor and the recipient reduced liver graft IRI in a rat liver transplantation model. This may translate to decreased liver dysfunction and increased survival of liver transplant recipients. Liver Transpl 21:468–477, 2015.

CD47 blockade reduces ischemia-reperfusion injury and improves outcomes in a rat kidney transplant model.

Background Ischemia-reperfusion injury (IRI) significantly contributes to delayed graft function and inflammation, leading to graft loss. Ischemia-reperfusion injury is exacerbated by the thrombospondin-1-CD47 system through inhibition of nitric oxide signaling. We postulate that CD47 blockade and prevention of nitric oxide inhibition reduce IRI in organ transplantation. Methods We used a syngeneic rat renal transplantation model of IRI with bilaterally nephrectomized recipients to evaluate the effect of a CD47 monoclonal antibody (CD47mAb) on IRI. Donor kidneys were flushed with CD47mAb OX101 or an isotype-matched control immunoglobulin and stored at 4°C in University of Wisconsin solution for 6 hr before transplantation. Results CD47mAb perfusion of donor kidneys resulted in marked improvement in posttransplant survival, lower levels of serum creatinine, blood urea nitrogen, phosphorus and magnesium, and less histological evidence of injury. In contrast, control groups did not survive more than 5 days, had increased biochemical indicators of renal injury, and exhibited severe pathological injury with tubular atrophy and necrosis. Recipients of CD47mAb-treated kidneys showed decreased levels of plasma biomarkers of renal injury including Cystatin C, Osteopontin, Tissue Inhibitor of Metalloproteinases-1 (TIMP1), &bgr;2-Microglobulin, Vascular Endothelial Growth Factor A (VEGF-A), and clusterin compared to the control group. Furthermore, laser Doppler assessment showed higher renal blood flow in the CD47mAb-treated kidneys. Conclusion These results provide strong evidence for the use of CD47 antibody–mediated blockade to reduce IRI and improve organ preservation for renal transplantation.

Protective role of bortezomib in steatotic liver ischemia/reperfusion injury through abrogation of MMP activation and YKL-40 expression.

Steatotic liver grafts tolerate ischemia-reperfusion (I/R) injury poorly, contributing to poor survival following transplantation. However the molecular mechanisms leading to I/R injury still remain to be defined. We have previously reported that the protective effect of bortezomib towards inhibiting cold induced I/R injury in obese rat liver transplant model is through NF-κB down modulation. In this report using an orthotopic liver transplant (OLT) model in Zucker rats (from obese, leptin deficient donor, to lean recipient) we defined the mechanisms of steatotic liver injury, and characterized the role of bortezomib in inhibiting MMP activation and YKL-40, both of which are involved in extracellular matrix deposition and fibrosis, the key pathological features of liver allograft failure. Obese donor rats were treated with bortezomib (i.v., 0.1mg/kg immediately prior to liver procurement) to assess the role of MMP and YKL-40 in steatotic liver I/R injury. I/R injury in steatotic livers resulted in significant increases in expression of YKL-40 (9 fold), and activation of MMP-2 (15 fold)/MMP-9 (12 fold). Bortezomib treatment reduced the expression of YKL-40 and MMP to basal levels. Bortezomib also inhibited the pro-fibrotic (VEGF, HGF, bFGF, TGF-β) and pro-inflammatory (IL-1β, TNF-α and IFN-γ) cytokines significantly in comparison to untreated animals with I/R injury. These results demonstrate that I/R injury in steatotic livers following transplantation are associated with MMP activation and YKL-40 upregulation resulting in pro-fibrotic and pro-inflammatory cytokine release. Administration of the proteosomal inhibitor, bortezomib, effectively attenuated the I/R injury by inhibiting MMP and YKL-40 expression and therefore support the clinical utility of this drug in donor management for preventing I/R injury and its sequelae.