Gina M. Coudriet

Human-Scale Whole-Organ Bioengineering for Liver Transplantation: A Regenerative Medicine Approach

At this time, the only definitive treatment of hepatic failure is liver transplantation. However, transplantation has been limited by the severely limited supply of human donor livers. Alternatively, a regenerative medicine approach has been recently proposed in rodents that describe the production of three-dimensional whole-organ scaffolds for assembly of engineered complete organs. In the present study, we describe the decellularization of porcine livers to generate liver constructs at a scale that can be clinically relevant. Adult ischemic porcine livers were successfully decellularized using a customized perfusion protocol, the decellularization process preserved the ultrastructural extracellular matrix components, functional characteristics of the native microvascular and the bile drainage network of the liver, and growth factors necessary for angiogenesis and liver regeneration. Furthermore, isolated hepatocytes engrafted and reorganized in the porcine decellularized livers using a human-sized organ culture system. These results provide proof-of-principle for the generation of a human-sized, three-dimensional organ scaffold as a potential structure for human liver grafts reconstruction for transplantation to treat liver disease.

FoxO1 Links Insulin Resistance to Proinflammatory Cytokine IL-1β Production in Macrophages

OBJECTIVE Macrophages play an important role in the pathogenesis of insulin resistance via the production of proinflammatory cytokines. Our goal is to decipher the molecular linkage between proinflammatory cytokine production and insulin resistance in macrophages. RESEARCH DESIGN AND METHODS We determined cytokine profiles in cultured macrophages and identified interleukin (IL)-1β gene as a potential target of FoxO1, a key transcription factor that mediates insulin action on gene expression. We studied the mechanism by which FoxO1 mediates insulin-dependent regulation of IL-1β expression in cultured macrophages and correlated FoxO1 activity in peritoneal macrophages with IL-1β production profiles in mice with low-grade inflammation or insulin resistance. RESULTS FoxO1 selectively promoted IL-1β production in cultured macrophages. This effect correlated with the ability of FoxO1 to bind and enhance IL-1β promoter activity. Mutations of the FoxO1 binding site within the IL-1β promoter abolished FoxO1 induction of IL-1β expression. Macrophages from insulin-resistant obese db/db mice or lipopolysaccharide-inflicted mice were associated with increased FoxO1 production, correlating with elevated levels of IL-1β mRNA in macrophages and IL-1β protein in plasma. In nonstimulated macrophages, FoxO1 remained inert with benign effects on IL-1β expression. In response to inflammatory stimuli, FoxO1 activity was augmented because of an impaired ability of insulin to phosphorylate FoxO1 and promote its nuclear exclusion. This effect along with nuclear factor-κB acted to stimulate IL-1β production in activated macrophages. CONCLUSIONS FoxO1 signaling through nuclear factor-κB plays an important role in coupling proinflammatory cytokine production to insulin resistance in obesity and diabetes.

Hepatocyte Growth Factor Modulates Interleukin-6 Production in Bone Marrow Derived Macrophages: Implications for Inflammatory Mediated Diseases

The generation of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β fuel the acute phase response (APR). To maintain body homeostasis, the increase of inflammatory proteins is resolved by acute phase proteins via presently unknown mechanisms. Hepatocyte growth factor (HGF) is transcribed in response to IL-6. Since IL-6 production promotes the generation of HGF and induces the APR, we posited that accumulating HGF might be a likely candidate for quelling excess inflammation under non-pathological conditions. We sought to assess the role of HGF and how it influences the regulation of inflammation utilizing a well-defined model of inflammatory activation, lipopolysaccharide (LPS)-stimulation of bone marrow derived macrophages (BMM). BMM were isolated from C57BL6 mice and were stimulated with LPS in the presence or absence of HGF. When HGF was present, there was a decrease in production of the pro-inflammatory cytokine IL-6, along with an increase in the anti-inflammatory cytokine IL-10. Altered cytokine production correlated with an increase in phosphorylated GSK3β, increased retention of the phosphorylated NFκB p65 subunit in the cytoplasm, and an enhanced interaction between CBP and phospho-CREB. These changes were a direct result of signaling through the HGF receptor, MET, as effects were reversed in the presence of a selective inhibitor of MET (SU11274) or when using BMM from macrophage-specific conditional MET knockout mice. Combined, these data provide compelling evidence that under normal circumstances, HGF acts to suppress the inflammatory response.

Lack of the Receptor for Advanced Glycation End-Products Attenuates E. coli Pneumonia in Mice

Background The receptor for advanced glycation end-products (RAGE) has been suggested to modulate lung injury in models of acute pulmonary inflammation. To study this further, model systems utilizing wild type and RAGE knockout (KO) mice were used to determine the role of RAGE signaling in lipopolysaccharide (LPS) and E. coli induced acute pulmonary inflammation. The effect of intraperitoneal (i.p.) and intratracheal (i.t.) administration of mouse soluble RAGE on E. coli injury was also investigated. Methodology/Principal Findings C57BL/6 wild type and RAGE KO mice received an i.t. instillation of LPS, E. coli, or vehicle control. Some groups also received i.p. or i.t. administration of mouse soluble RAGE. After 24 hours, the role of RAGE expression on inflammation was assessed by comparing responses in wild type and RAGE KO. RAGE protein levels decreased in wild type lung homogenates after treatment with either LPS or bacteria. In addition, soluble RAGE and HMGB1 increased in the BALF after E. coli instillation. RAGE KO mice challenged with LPS had the same degree of inflammation as wild type mice. However, when challenged with E. coli, RAGE KO mice had significantly less inflammation when compared to wild type mice. Most cytokine levels were lower in the BALF of RAGE KO mice compared to wild type mice after E. coli injury, while only monocyte chemotactic protein-1, MCP-1, was lower after LPS challenge. Neither i.p. nor i.t. administration of mouse soluble RAGE attenuated the severity of E. coli injury in wild type mice. Conclusions/Significance Lack of RAGE in the lung does not protect against LPS induced acute pulmonary inflammation, but attenuates injury following live E. coli challenge. These findings suggest that RAGE mediates responses to E. coli-associated pathogen-associated molecular pattern molecules other than LPS or other bacterial specific signaling responses. Soluble RAGE treatment had no effect on inflammation.

Forkhead Box O6 (FoxO6) Depletion Attenuates Hepatic Gluconeogenesis and Protects against Fat-induced Glucose Disorder in Mice

Background: Excessive endogenous glucose production is a major contributing factor for fasting hyperglycemia in diabetes. Results: FoxO6 deficiency attenuates hepatic gluconeogenesis and protects against fat-induced glucose disorder in mice. Conclusion: FoxO6 plays a significant role in regulating gluconeogenesis in the liver. Significance: FoxO6 is a potential therapeutic target for improving glucose metabolism in diabetes. Excessive endogenous glucose production contributes to fasting hyperglycemia in diabetes. FoxO6 is a distinct member of the FoxO subfamily. To elucidate the role of FoxO6 in hepatic gluconeogenesis and assess its contribution to the pathogenesis of fasting hyperglycemia in diabetes, we generated FoxO6 knock-out (FoxO6-KO) mice followed by determining the effect of FoxO6 loss-of-function on hepatic gluconeogenesis under physiological and pathological conditions. FoxO6 depletion attenuated hepatic gluconeogenesis and lowered fasting glycemia in FoxO6-KO mice. FoxO6-deficient primary hepatocytes were associated with reduced capacities to produce glucose in response to glucagon. When fed a high fat diet, FoxO6-KO mice exhibited significantly enhanced glucose tolerance and reduced blood glucose levels accompanied by improved insulin sensitivity. These effects correlated with attenuated hepatic gluconeogenesis in FoxO6-KO mice. In contrast, wild-type littermates developed fat-induced glucose intolerance with a concomitant induction of fasting hyperinsulinemia and hyperglycemia. Furthermore, FoxO6-KO mice displayed significantly diminished macrophage infiltration into liver and adipose tissues, correlating with the reduction of macrophage expression of C-C chemokine receptor 2 (CCR2), a factor that is critical for regulating macrophage recruitment in peripheral tissues. Our data indicate that FoxO6 depletion protected against diet-induced glucose intolerance and insulin resistance by attenuating hepatic gluconeogenesis and curbing macrophage infiltration in liver and adipose tissues in mice.

Treatment with a Catalytic Superoxide Dismutase (SOD) Mimetic Improves Liver Steatosis, Insulin Sensitivity, and Inflammation in Obesity-Induced Type 2 Diabetes

Oxidative stress and persistent inflammation are exaggerated through chronic over-nutrition and a sedentary lifestyle, resulting in insulin resistance. In type 2 diabetes (T2D), impaired insulin signaling leads to hyperglycemia and long-term complications, including metabolic liver dysfunction, resulting in non-alcoholic fatty liver disease (NAFLD). The manganese metalloporphyrin superoxide dismustase (SOD) mimetic, manganese (III) meso-tetrakis (N-ethylpyridinium-2-yl) porphyrin (MnP), is an oxidoreductase known to scavenge reactive oxygen species (ROS) and decrease pro-inflammatory cytokine production, by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. We hypothesized that targeting oxidative stress-induced inflammation with MnP would assuage liver complications and enhance insulin sensitivity and glucose tolerance in a high-fat diet (HFD)-induced mouse model of T2D. During 12 weeks of feeding, we saw significant improvements in weight, hepatic steatosis, and biomarkers of liver dysfunction with redox modulation by MnP treatment in HFD-fed mice. Additionally, MnP treatment improved insulin sensitivity and glucose tolerance, while reducing serum insulin and leptin levels. We attribute these effects to redox modulation and inhibition of hepatic NF-κB activation, resulting in diminished ROS and pro-inflammatory cytokine production. This study highlights the importance of controlling oxidative stress and secondary inflammation in obesity-mediated insulin resistance and T2D. Our data confirm the role of NF-κB-mediated inflammation in the development of T2D, and demonstrate the efficacy of MnP in preventing the progression to disease by specifically improving liver pathology and hepatic insulin resistance in obesity.

Administration of a negative vaccination induces hyporesponsiveness to islet allografts.

As a result of less than optimal outcomes the use of islet allografts as a standard insulin replacement therapy is limited to adults with a history of extreme glucose dysregulation and hypoglycemia unawareness. In this study, we examined the use of prophylactic immunotherapy to prevent islet allograft rejection in the absence of antirejection drugs. Our protocol to achieve allograft acceptance used a negative vaccination strategy that is comprised of apoptotic donor cells delivered in Incomplete Freunds Adjuvant (IFA) 1 week prior to islet transplantation. The goal of this new protocol is to elicit hyporesponsiveness to alloantigen prior to islet transplantation. First, we examined our protocol without islet allograft transplants and determined that the negative vaccination was not globally immunosuppressive or immunostimulatory. Islet allograft experiments using fully MHC-mismatched islet donors and recipients demonstrated that the negative vaccination strategy induced long-term islet allograft acceptance. Upon rechallenge with alloantigen, the negative vaccination protocol successfully achieved hyporesponsiveness. In addition, the microenvironment at the site of the tolerant allograft revealed a decrease in proinflammatory mediators (IFN-γ, TNF-α) and an increase in the anti-inflammatory mediator IL-10, as well as increased expression of the master regulator of T-regulatory cells, FOXP3. Our data suggest that pretreating allograft recipients with apoptotic donor alloantigen delivered in IFA induced long-term islet allograft acceptance and glycemic control by introducing alloantigen to the recipient immune system in a nonimmunostimulatory manner prior to transplant.

Using Metalloporphyrins to Preserve β Cell Mass and Inhibit Immune Responses in Diabetes

As of 2012, 29.1 million Americans are living with diabetes [1], creating a significant healthcare concern for the United States. Diabetes is a disease highly driven by inflammation and reactive oxygen species (ROS). Therefore, agents that affect secondary inflammation as a result of oxidative stress like metalloporphyrins are a desirable approach to alleviate the inflammation associated with disease. Here, we will discuss the role ROS play in the immunopathology of diabetes, the work that has elucidated the efficacy of metalloporphyrins in the disease, and their future utility in the clinic.