- Email: [email protected]
Induction and Maintenance of Cellular Hypometabolsm for Improved Tissue Survival for Reconstructive Procedures
S142
Surgical Forum Abstracts
lead to postoperative hepatic dysfunction. Kupffer cells (KC) are resident macrophages that play a critical role in the inflammatory response during hepatic IRI. The Nrf2-antioxidant response element (ARE) pathway is a cellular defense pathway against oxidative stress. We have previously shown that global Nrf2 deficiency leads to increased hepatic IRI. We hypothesize that overexpression of Nrf2 in KCs leads to decreased cellular damage after hepatic IRI. METHODS: Nrf2 wild type (LysMcre+/caNrf2-, n¼7) and KC specific Nrf2 overexpressers (LysMcre+/caNrf2+, n¼5) C57BL/6 mice underwent partial hepatic ischemia surgery, with 60 minutes of ischemia followed by 6 hours of reperfusion. After reperfusion, the mice were sacrificed, and blood and liver samples were collected for analyses. Serum ALT was measured to determine hepatocellular damage. H&E was performed to evaluate areas of necrosis. Quantitative RT-PCR was performed to determine transcript levels of inflammatory cytokines. Hepatic 8-Isoprostanes levels were measured to assess oxidative stress. Student’s t-test was performed for comparison between groups. P<0.05 for statistical significance. RESULTS: After IR, LysMcre+/caNrf2+ mice demonstrated significantly decreased serum ALT and decreased areas of necrosis as compared to controls. On qRT-PCR, LysMcre+/caNrf2+ animals had significantly decreased gene expression of proinflammatory cytokines including IL-6, CCL2, and CXCL10. LysMcre+/ caNrf2+ animals also had significantly decreased levels of 8isoprostanes. CONCLUSIONS: In our model, Nrf2 overexpression in KCs leads to decreased hepatocellular damage, necrosis, inflammation, and oxidative stress. Pharmacologic targeting of the Nrf2-ARE pathway in KCs may be a novel strategy to mitigate hepatic IRI. Induction and Maintenance of Cellular Hypometabolsm for Improved Tissue Survival for Reconstructive Procedures John R Jackson, PhD, NaJung Kim, PhD, Jaehyun Kim, PhD, Karl-Erik Andersson, James Yoo, MD, PhD, Anthony Atala, MD, FACS Wake Forest School of Medicine, Winston-Salem, NC INTRODUCTION: Implantation of tissue grafts often confront low oxygen environment until host vascularization is established. Insufficient diffusion of oxygen levels leads to cell death followed by failure of tissue graft. We previously used adenosine that decreases oxygen consumption by reducing cellular metabolism. However, adenosine is irreversibly converted into inosine and loses its activity by an enzyme called adenosine deaminase (ADA). In this study we tested the effectiveness of adenosine derivatives on cell survival under hypoxic conditions. METHODS: We used three known ADA inhibitors, cladribine, pentostatin, and fludarabine phosphate. Metabolic rate of cells was analyzed in the presence or absence of adenosine derivatives and ADA. Cellular metabolism was evaluated under hypoxic
J Am Coll Surg
conditions. Intracellular ATP content was measured. The effects of metabolic downregulation of cladribine were further validated in the rat ischemic tibialis anterior (TA) muscle. RESULTS: Among the three adenosine derivatives tested, cladribine was able to downregulate cellular metabolism regardless of ADA activity. In contrast, adenosine, pentostatin, and fludarabine phosphate were inactivated upon ADA treatment. Cladribinetreated cells were able to recover their normal metabolic state upon removal of cladribine. Injection of cladribine into ischemic TA muscle maintained muscle fiber structures and cell viability for 3 days, whereas adenosine treated muscle showed disintegrated fibers and cell death. CONCLUSIONS: Among the adenosine derivatives tested, cladribine showed the most promising potential as an ADA inhibitor. Our results show that prolonged cell viability can be maintained by the use of adenosine derivatives under hypoxic conditions and this approach may improve the outcomes of tissue survivability in reconstructive surgery. Upregulation of the Pro-Apoptotic Protein XIAPAssociated Factor-1 (XAF1) Contributes to Liver Damage in Liver Ischemia/Reperfusion through the Apoptotic Pathway Remealle A How, MD, John R Klune, MD, Shoko Kimura, MD, Shinchiro Yokota, Anthony V Spadaro, BS, Qiang Du, MD, Allan Tsung, MD, FACS, David A Geller, MD, FACS University of Pittsburgh Medical Center, Pittsburgh, PA INTRODUCTION: Interferon Regulatory Factor-1 (IRF-1), a transcription factor that plays a key role in liver transplant ischemia/ reperfusion (I/R) injury, induces XIAP-associated factor-1 (XAF1), a pro-apoptotic protein inhibiting the anti-apoptotic effects of the X-linked inhibitor of apoptosis protein (XIAP). Previously, we’ve shown that XAF1 is induced in an IRF-1 dependent manner in orthotopic liver transplant. Here, we hypothesize that XAF1 overexpression contributes to liver damage by increasing apoptosis and that XAF1 inhibition leads to apoptosis downregulation. METHODS: In vitro, wild-type (WT) mouse hepatocytes (MHCs) were transfected with adenovirus control, adenovirus with XAF1 expression plasmid (AdXAF1), or adenovirus with shRNA-XAF1 plasmid (AdshRNA-XAF1). MHCs were exposed to hypoxia for 15 hours. Apoptosis was induced using Actinomycin D + TNFalpha. In vivo, AdXAF1 (2x10e8 pfu) was injected in mice penile vein and the liver harvested in 2 days. XAF1 expression and apoptosis were measured by XAF1 and cleaved caspase-3 Western Blot. RESULTS: In vitro, AdXAF1 transfection increased XAF1 and cleaved caspase 3 levels. Inhibition by AdshRNA-XAF1 significantly decreased XAF1 expression and cleaved caspase-3 levels compared to AdLacZ control in MHCs treated with hypoxia and ActD+TNF-alpha. In vivo, injection of AdXAF1 overexpressed XAF1 and induced apoptosis with increased levels of cleaved caspase-3 in the mice liver (Table).
Surgical Forum Abstracts
lead to postoperative hepatic dysfunction. Kupffer cells (KC) are resident macrophages that play a critical role in the inflammatory response during hepatic IRI. The Nrf2-antioxidant response element (ARE) pathway is a cellular defense pathway against oxidative stress. We have previously shown that global Nrf2 deficiency leads to increased hepatic IRI. We hypothesize that overexpression of Nrf2 in KCs leads to decreased cellular damage after hepatic IRI. METHODS: Nrf2 wild type (LysMcre+/caNrf2-, n¼7) and KC specific Nrf2 overexpressers (LysMcre+/caNrf2+, n¼5) C57BL/6 mice underwent partial hepatic ischemia surgery, with 60 minutes of ischemia followed by 6 hours of reperfusion. After reperfusion, the mice were sacrificed, and blood and liver samples were collected for analyses. Serum ALT was measured to determine hepatocellular damage. H&E was performed to evaluate areas of necrosis. Quantitative RT-PCR was performed to determine transcript levels of inflammatory cytokines. Hepatic 8-Isoprostanes levels were measured to assess oxidative stress. Student’s t-test was performed for comparison between groups. P<0.05 for statistical significance. RESULTS: After IR, LysMcre+/caNrf2+ mice demonstrated significantly decreased serum ALT and decreased areas of necrosis as compared to controls. On qRT-PCR, LysMcre+/caNrf2+ animals had significantly decreased gene expression of proinflammatory cytokines including IL-6, CCL2, and CXCL10. LysMcre+/ caNrf2+ animals also had significantly decreased levels of 8isoprostanes. CONCLUSIONS: In our model, Nrf2 overexpression in KCs leads to decreased hepatocellular damage, necrosis, inflammation, and oxidative stress. Pharmacologic targeting of the Nrf2-ARE pathway in KCs may be a novel strategy to mitigate hepatic IRI. Induction and Maintenance of Cellular Hypometabolsm for Improved Tissue Survival for Reconstructive Procedures John R Jackson, PhD, NaJung Kim, PhD, Jaehyun Kim, PhD, Karl-Erik Andersson, James Yoo, MD, PhD, Anthony Atala, MD, FACS Wake Forest School of Medicine, Winston-Salem, NC INTRODUCTION: Implantation of tissue grafts often confront low oxygen environment until host vascularization is established. Insufficient diffusion of oxygen levels leads to cell death followed by failure of tissue graft. We previously used adenosine that decreases oxygen consumption by reducing cellular metabolism. However, adenosine is irreversibly converted into inosine and loses its activity by an enzyme called adenosine deaminase (ADA). In this study we tested the effectiveness of adenosine derivatives on cell survival under hypoxic conditions. METHODS: We used three known ADA inhibitors, cladribine, pentostatin, and fludarabine phosphate. Metabolic rate of cells was analyzed in the presence or absence of adenosine derivatives and ADA. Cellular metabolism was evaluated under hypoxic
J Am Coll Surg
conditions. Intracellular ATP content was measured. The effects of metabolic downregulation of cladribine were further validated in the rat ischemic tibialis anterior (TA) muscle. RESULTS: Among the three adenosine derivatives tested, cladribine was able to downregulate cellular metabolism regardless of ADA activity. In contrast, adenosine, pentostatin, and fludarabine phosphate were inactivated upon ADA treatment. Cladribinetreated cells were able to recover their normal metabolic state upon removal of cladribine. Injection of cladribine into ischemic TA muscle maintained muscle fiber structures and cell viability for 3 days, whereas adenosine treated muscle showed disintegrated fibers and cell death. CONCLUSIONS: Among the adenosine derivatives tested, cladribine showed the most promising potential as an ADA inhibitor. Our results show that prolonged cell viability can be maintained by the use of adenosine derivatives under hypoxic conditions and this approach may improve the outcomes of tissue survivability in reconstructive surgery. Upregulation of the Pro-Apoptotic Protein XIAPAssociated Factor-1 (XAF1) Contributes to Liver Damage in Liver Ischemia/Reperfusion through the Apoptotic Pathway Remealle A How, MD, John R Klune, MD, Shoko Kimura, MD, Shinchiro Yokota, Anthony V Spadaro, BS, Qiang Du, MD, Allan Tsung, MD, FACS, David A Geller, MD, FACS University of Pittsburgh Medical Center, Pittsburgh, PA INTRODUCTION: Interferon Regulatory Factor-1 (IRF-1), a transcription factor that plays a key role in liver transplant ischemia/ reperfusion (I/R) injury, induces XIAP-associated factor-1 (XAF1), a pro-apoptotic protein inhibiting the anti-apoptotic effects of the X-linked inhibitor of apoptosis protein (XIAP). Previously, we’ve shown that XAF1 is induced in an IRF-1 dependent manner in orthotopic liver transplant. Here, we hypothesize that XAF1 overexpression contributes to liver damage by increasing apoptosis and that XAF1 inhibition leads to apoptosis downregulation. METHODS: In vitro, wild-type (WT) mouse hepatocytes (MHCs) were transfected with adenovirus control, adenovirus with XAF1 expression plasmid (AdXAF1), or adenovirus with shRNA-XAF1 plasmid (AdshRNA-XAF1). MHCs were exposed to hypoxia for 15 hours. Apoptosis was induced using Actinomycin D + TNFalpha. In vivo, AdXAF1 (2x10e8 pfu) was injected in mice penile vein and the liver harvested in 2 days. XAF1 expression and apoptosis were measured by XAF1 and cleaved caspase-3 Western Blot. RESULTS: In vitro, AdXAF1 transfection increased XAF1 and cleaved caspase 3 levels. Inhibition by AdshRNA-XAF1 significantly decreased XAF1 expression and cleaved caspase-3 levels compared to AdLacZ control in MHCs treated with hypoxia and ActD+TNF-alpha. In vivo, injection of AdXAF1 overexpressed XAF1 and induced apoptosis with increased levels of cleaved caspase-3 in the mice liver (Table).