3A ). Twenty-four hours after PH, the levels of p-EGFR, p-ERK1/2, and p-AKT appeared to be further elevated in mig-6 knockout mice, indicating that the enhanced Metformin in vivo hepatocyte proliferation at these early time points might be due to amplified EGFR signaling (Fig. 3A,B). Notably, at the 36-hour time point, the levels of p-ERK1/2 declined, whereas EGFR and AKT remained activated in mig-6 knockout livers, suggesting that hepatocyte proliferation might be driven by EGFR-AKT signaling. In addition, we found that total EGFR protein levels were increased in mig-6 knockout mice, suggesting that loss of mig-6 enhances EGFR protein

stability (Fig. 3A,B). Interestingly, EGFR up-regulation seems to occur through a posttranslational mechanism, because EGFR messenger RNA levels were unchanged in mig-6 knockout and wild-type animals (Fig. 3C). In addition, we found increased levels of p-Rb in the regenerating liver of mig-6 knockout mice (Fig.

3A), which might stimulate the expression of genes required for S-phase entry. Furthermore, elevated activity of the activator protein-1 transcription factor c-Jun, which is known to be a key regulator of liver regeneration,21 was detected in mig-6 knockout livers. Interestingly, the EGFR ligand HB-EGF but not TGFα is up-regulated at the transcriptional level at 0, 24, and 36 hours after PH (Fig. 3C), suggesting that HB-EGF may activate the EGFR. Because mig-6 is known to be a negative regulator of all EGF receptors, we examined the expression levels of ErbB2, ErbB3, selleckchem and ErbB4 in regenerating mig-6 knockout and wild-type livers. In line with published data,22 we could not detect ErbB2 nor ErbB4 expression, whereas ErbB3 was weakly expressed (data not shown) suggesting that mig-6 is a specific negative regulator of EGFR signaling in hepatocytes. Notably, 48 hours after PH the activation of the EGFR pathway is comparable between knockout and wild-type control mice

(Fig. 3A-C), suggesting selleck that the EGFR is eventually inactivated by a mig-6–independent mechanism and that mig-6 is dispensable for EGFR regulation at later time points during liver regeneration. To study the effect of mig-6 on EGFR function in human liver cancer cell lines, we stimulated HepG2 and Hs 817.T cells with EGF for the indicated time points (Fig. 4A ). EGF stimulation led to a strong and continuous induction of mig-6 expression (Fig. 4A). Interestingly, mig-6 induction correlates with a rapid decrease in EGFR phosphorylation and expression, as well as a reduction in p-ERK1/2 levels. Importantly, mig-6 is able to bind to the activated form of the EGFR, thereby most likely regulating EGFR activity (Fig. 4B). To better understand the role of mig-6 in human liver cancer cell lines, we down-regulated mig-6 by specific siRNAs in HepG2 cells and examined EGFR signaling. Suppression of mig-6 led to elevated EGFR activity upon EGF stimulation (Fig. 5A ).

3A ). Twenty-four hours after PH, the levels of p-EGFR, p-ERK1/2, and p-AKT appeared to be further elevated in mig-6 knockout mice, indicating that the enhanced learn more hepatocyte proliferation at these early time points might be due to amplified EGFR signaling (Fig. 3A,B). Notably, at the 36-hour time point, the levels of p-ERK1/2 declined, whereas EGFR and AKT remained activated in mig-6 knockout livers, suggesting that hepatocyte proliferation might be driven by EGFR-AKT signaling. In addition, we found that total EGFR protein levels were increased in mig-6 knockout mice, suggesting that loss of mig-6 enhances EGFR protein

stability (Fig. 3A,B). Interestingly, EGFR up-regulation seems to occur through a posttranslational mechanism, because EGFR messenger RNA levels were unchanged in mig-6 knockout and wild-type animals (Fig. 3C). In addition, we found increased levels of p-Rb in the regenerating liver of mig-6 knockout mice (Fig.

3A), which might stimulate the expression of genes required for S-phase entry. Furthermore, elevated activity of the activator protein-1 transcription factor c-Jun, which is known to be a key regulator of liver regeneration,21 was detected in mig-6 knockout livers. Interestingly, the EGFR ligand HB-EGF but not TGFα is up-regulated at the transcriptional level at 0, 24, and 36 hours after PH (Fig. 3C), suggesting that HB-EGF may activate the EGFR. Because mig-6 is known to be a negative regulator of all EGF receptors, we examined the expression levels of ErbB2, ErbB3, selleck compound and ErbB4 in regenerating mig-6 knockout and wild-type livers. In line with published data,22 we could not detect ErbB2 nor ErbB4 expression, whereas ErbB3 was weakly expressed (data not shown) suggesting that mig-6 is a specific negative regulator of EGFR signaling in hepatocytes. Notably, 48 hours after PH the activation of the EGFR pathway is comparable between knockout and wild-type control mice

(Fig. 3A-C), suggesting selleck chemicals that the EGFR is eventually inactivated by a mig-6–independent mechanism and that mig-6 is dispensable for EGFR regulation at later time points during liver regeneration. To study the effect of mig-6 on EGFR function in human liver cancer cell lines, we stimulated HepG2 and Hs 817.T cells with EGF for the indicated time points (Fig. 4A ). EGF stimulation led to a strong and continuous induction of mig-6 expression (Fig. 4A). Interestingly, mig-6 induction correlates with a rapid decrease in EGFR phosphorylation and expression, as well as a reduction in p-ERK1/2 levels. Importantly, mig-6 is able to bind to the activated form of the EGFR, thereby most likely regulating EGFR activity (Fig. 4B). To better understand the role of mig-6 in human liver cancer cell lines, we down-regulated mig-6 by specific siRNAs in HepG2 cells and examined EGFR signaling. Suppression of mig-6 led to elevated EGFR activity upon EGF stimulation (Fig. 5A ).

3A ). Twenty-four hours after PH, the levels of p-EGFR, p-ERK1/2, and p-AKT appeared to be further elevated in mig-6 knockout mice, indicating that the enhanced selleck chemicals llc hepatocyte proliferation at these early time points might be due to amplified EGFR signaling (Fig. 3A,B). Notably, at the 36-hour time point, the levels of p-ERK1/2 declined, whereas EGFR and AKT remained activated in mig-6 knockout livers, suggesting that hepatocyte proliferation might be driven by EGFR-AKT signaling. In addition, we found that total EGFR protein levels were increased in mig-6 knockout mice, suggesting that loss of mig-6 enhances EGFR protein

stability (Fig. 3A,B). Interestingly, EGFR up-regulation seems to occur through a posttranslational mechanism, because EGFR messenger RNA levels were unchanged in mig-6 knockout and wild-type animals (Fig. 3C). In addition, we found increased levels of p-Rb in the regenerating liver of mig-6 knockout mice (Fig.

3A), which might stimulate the expression of genes required for S-phase entry. Furthermore, elevated activity of the activator protein-1 transcription factor c-Jun, which is known to be a key regulator of liver regeneration,21 was detected in mig-6 knockout livers. Interestingly, the EGFR ligand HB-EGF but not TGFα is up-regulated at the transcriptional level at 0, 24, and 36 hours after PH (Fig. 3C), suggesting that HB-EGF may activate the EGFR. Because mig-6 is known to be a negative regulator of all EGF receptors, we examined the expression levels of ErbB2, ErbB3, Selleckchem GDC 973 and ErbB4 in regenerating mig-6 knockout and wild-type livers. In line with published data,22 we could not detect ErbB2 nor ErbB4 expression, whereas ErbB3 was weakly expressed (data not shown) suggesting that mig-6 is a specific negative regulator of EGFR signaling in hepatocytes. Notably, 48 hours after PH the activation of the EGFR pathway is comparable between knockout and wild-type control mice

(Fig. 3A-C), suggesting find more that the EGFR is eventually inactivated by a mig-6–independent mechanism and that mig-6 is dispensable for EGFR regulation at later time points during liver regeneration. To study the effect of mig-6 on EGFR function in human liver cancer cell lines, we stimulated HepG2 and Hs 817.T cells with EGF for the indicated time points (Fig. 4A ). EGF stimulation led to a strong and continuous induction of mig-6 expression (Fig. 4A). Interestingly, mig-6 induction correlates with a rapid decrease in EGFR phosphorylation and expression, as well as a reduction in p-ERK1/2 levels. Importantly, mig-6 is able to bind to the activated form of the EGFR, thereby most likely regulating EGFR activity (Fig. 4B). To better understand the role of mig-6 in human liver cancer cell lines, we down-regulated mig-6 by specific siRNAs in HepG2 cells and examined EGFR signaling. Suppression of mig-6 led to elevated EGFR activity upon EGF stimulation (Fig. 5A ).

1, 2 However, a shortage of available donor organs for transplantation results in the death of many patients awaiting liver transplantation. Hepatocyte transplantation

provides a promising alternative, and numerous experiments have demonstrated that hepatocyte transplantation improves liver function in animals with hepatic failure and innate liver-based metabolic disorders.3, 4 However, hepatocyte transplantation has rarely produced therapeutic effects, because mature hepatocytes cannot be effectively expanded in vitro and the availability of hepatocytes is often limited by shortages of donor organs.5, 6 Thus, previous studies have focused on the development of ITF2357 molecular weight various stem cells that could be readily isolated using noninvasive procedures to yield hepatocytes in vitro and in vivo. Bone marrow mesenchymal stem cells learn more (BMSCs) can differentiate into osteoblasts, adipocytes,

and other mesenchymal cell lineages.7-10 The hepatocyte differentiation capacity of human BMSCs (hBMSCs) has been characterized in vitro and in vivo.11-13 These cells can also be expanded in culture for long periods without any apparent loss of differentiation capacity. Some groups have already started transplanting autologous bone marrow cells into patients with chronic liver fibrosis or cirrhosis.12, 14, 15 However, little is known about the use of hBMSCs to treat fulminant hepatic failure (FHF) in animal models or in human patients with FHF, even though such studies would be clinically important.5 Furthermore, because of difficulties in tracking transplanted hBMSC-derived hepatocytes in patients, and because previous experiments were performed in small animal (mouse or rat) models of chronic liver injury, the roles of BMSCs in liver regeneration have not been fully elucidated.5 FHF-derived BMSCs demonstrate a hepatic transcriptional

profile and express many of the same genes expressed by hepatic progenitor cells,16-18 suggesting that extrahepatic stem cells, especially BMSCs, may be a resource for hepatocyte repopulation and can play an important role in liver regeneration. Thus, we investigated see more whether the intraportal transplantation of hBMSCs is a safe and effective method to prevent FHF in a large animal (pig) model. ALB, albumin; ALT, alanine aminotransferase; BMSC, bone marrow mesenchymal stem cell; D-gal, D-galactosamine; ELISA, enzyme-linked immunosorbent assay; FHF, fulminant hepatic failure; G6PD, glucose-6-phosphate dehydrogenase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; hBMSC, human BMSC; H&E, hematoxylin and eosin; HNF-1α, hepatocyte nuclear factor-1α; HSA, hepatocyte-specific antigen; IPT, intraportal transplantation; PVT, peripheral transplantation; qPCR, quantitative real-time polymerase chain reaction. Human BMSCs were isolated by bone marrow aspiration from the iliac crest of 30 healthy male volunteers.

1, 2 However, a shortage of available donor organs for transplantation results in the death of many patients awaiting liver transplantation. Hepatocyte transplantation

provides a promising alternative, and numerous experiments have demonstrated that hepatocyte transplantation improves liver function in animals with hepatic failure and innate liver-based metabolic disorders.3, 4 However, hepatocyte transplantation has rarely produced therapeutic effects, because mature hepatocytes cannot be effectively expanded in vitro and the availability of hepatocytes is often limited by shortages of donor organs.5, 6 Thus, previous studies have focused on the development of Kinase Inhibitor Library order various stem cells that could be readily isolated using noninvasive procedures to yield hepatocytes in vitro and in vivo. Bone marrow mesenchymal stem cells find more (BMSCs) can differentiate into osteoblasts, adipocytes,

and other mesenchymal cell lineages.7-10 The hepatocyte differentiation capacity of human BMSCs (hBMSCs) has been characterized in vitro and in vivo.11-13 These cells can also be expanded in culture for long periods without any apparent loss of differentiation capacity. Some groups have already started transplanting autologous bone marrow cells into patients with chronic liver fibrosis or cirrhosis.12, 14, 15 However, little is known about the use of hBMSCs to treat fulminant hepatic failure (FHF) in animal models or in human patients with FHF, even though such studies would be clinically important.5 Furthermore, because of difficulties in tracking transplanted hBMSC-derived hepatocytes in patients, and because previous experiments were performed in small animal (mouse or rat) models of chronic liver injury, the roles of BMSCs in liver regeneration have not been fully elucidated.5 FHF-derived BMSCs demonstrate a hepatic transcriptional

profile and express many of the same genes expressed by hepatic progenitor cells,16-18 suggesting that extrahepatic stem cells, especially BMSCs, may be a resource for hepatocyte repopulation and can play an important role in liver regeneration. Thus, we investigated selleck kinase inhibitor whether the intraportal transplantation of hBMSCs is a safe and effective method to prevent FHF in a large animal (pig) model. ALB, albumin; ALT, alanine aminotransferase; BMSC, bone marrow mesenchymal stem cell; D-gal, D-galactosamine; ELISA, enzyme-linked immunosorbent assay; FHF, fulminant hepatic failure; G6PD, glucose-6-phosphate dehydrogenase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; hBMSC, human BMSC; H&E, hematoxylin and eosin; HNF-1α, hepatocyte nuclear factor-1α; HSA, hepatocyte-specific antigen; IPT, intraportal transplantation; PVT, peripheral transplantation; qPCR, quantitative real-time polymerase chain reaction. Human BMSCs were isolated by bone marrow aspiration from the iliac crest of 30 healthy male volunteers.

1, 2 However, a shortage of available donor organs for transplantation results in the death of many patients awaiting liver transplantation. Hepatocyte transplantation

provides a promising alternative, and numerous experiments have demonstrated that hepatocyte transplantation improves liver function in animals with hepatic failure and innate liver-based metabolic disorders.3, 4 However, hepatocyte transplantation has rarely produced therapeutic effects, because mature hepatocytes cannot be effectively expanded in vitro and the availability of hepatocytes is often limited by shortages of donor organs.5, 6 Thus, previous studies have focused on the development of R788 molecular weight various stem cells that could be readily isolated using noninvasive procedures to yield hepatocytes in vitro and in vivo. Bone marrow mesenchymal stem cells http://www.selleckchem.com/products/PF-2341066.html (BMSCs) can differentiate into osteoblasts, adipocytes,

and other mesenchymal cell lineages.7-10 The hepatocyte differentiation capacity of human BMSCs (hBMSCs) has been characterized in vitro and in vivo.11-13 These cells can also be expanded in culture for long periods without any apparent loss of differentiation capacity. Some groups have already started transplanting autologous bone marrow cells into patients with chronic liver fibrosis or cirrhosis.12, 14, 15 However, little is known about the use of hBMSCs to treat fulminant hepatic failure (FHF) in animal models or in human patients with FHF, even though such studies would be clinically important.5 Furthermore, because of difficulties in tracking transplanted hBMSC-derived hepatocytes in patients, and because previous experiments were performed in small animal (mouse or rat) models of chronic liver injury, the roles of BMSCs in liver regeneration have not been fully elucidated.5 FHF-derived BMSCs demonstrate a hepatic transcriptional

profile and express many of the same genes expressed by hepatic progenitor cells,16-18 suggesting that extrahepatic stem cells, especially BMSCs, may be a resource for hepatocyte repopulation and can play an important role in liver regeneration. Thus, we investigated selleck products whether the intraportal transplantation of hBMSCs is a safe and effective method to prevent FHF in a large animal (pig) model. ALB, albumin; ALT, alanine aminotransferase; BMSC, bone marrow mesenchymal stem cell; D-gal, D-galactosamine; ELISA, enzyme-linked immunosorbent assay; FHF, fulminant hepatic failure; G6PD, glucose-6-phosphate dehydrogenase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; hBMSC, human BMSC; H&E, hematoxylin and eosin; HNF-1α, hepatocyte nuclear factor-1α; HSA, hepatocyte-specific antigen; IPT, intraportal transplantation; PVT, peripheral transplantation; qPCR, quantitative real-time polymerase chain reaction. Human BMSCs were isolated by bone marrow aspiration from the iliac crest of 30 healthy male volunteers.

Therefore, it is essential to define in a specialized comprehensive care setting when and which prophylaxis should be given. Introduction  Rare bleeding disorders include the inherited deficiencies of fibrinogen, FII, FV, FV+VIII, FVII, FX, FXI, FXIII and combined deficiency of vitamin-K dependent factors. Recent issues of Haemophilia (November 2008) and Seminars of Thrombosis and Hemostasis (June 2009) have covered the main available treatments

for RBDs. The personal and familial history of each patient needs to be taken into account before choosing the most appropriate therapeutic approach. BAY 57-1293 Dosages and frequency of treatments depend on the minimal haemostatic level of the deficient factor (a matter of controversy), its plasma half-life (which varies with age and even in individuals with the same age and the same level of factor)

and the type of bleeding episode [6]. Replacement therapy is effective in treating bleeding episodes in RBDs. Depending on their availability, patients receive fresh frozen plasma (FFP), cryoprecipitate or factor concentrates. The latter is the treatment of choice because HDAC inhibitor it is safer than FFP or cryoprecipitate (decreased risk of blood-borne pathogen transmission), there is no fluid overload and more precise dosing can be accomplished. If the evidence for the optimal use of products in case of replacement therapy in RBDs is already limited, it is almost non-existent for prophylaxis (FXIII being an exception). Discussion  The conventional treatment (treatment on demand) for most RBD is episodic treatment administered as soon as possible after onset of bleeding. The other approach (prophylaxis) consists of giving either products from an early age to prevent bleeding and, in case of surgery or pregnancy, to prevent bleeding and/or miscarriage (primary prophylaxis) or after bleeding to prevent recurrences (secondary prophylaxis). The UK guidelines on therapeutic products for coagulation disorders provide recommendations about the best treatment options (dosage, management of bleeding, surgery and pregnancy as well as prophylaxis)

for RBDs [7]. In theory, prophylactic administration of factors is the best option for patients with severe RBDs. However, this option has this website to be counter-balanced by the possible transmission of infectious agents, allergic reactions, venous access problems, development of inhibitors, risk of thrombotic complications, Transfusion-Related Acute Lung Injury due to cytotoxic antibodies contained in the infused plasma, and cost. Furthermore, even for some severe RBDs, patients can bleed less than severe haemophiliacs and long asymptomatic periods are not uncommon. For example, in a retrospective survey on patients with afibrinogenaemia (or severe hypofibrinogenaemia), the mean annual incidence of bleeding episodes in patients treated on demand was 0.7 (0–16.5) whereas it was 0.5 (0–2.

Therefore, it is essential to define in a specialized comprehensive care setting when and which prophylaxis should be given. Introduction  Rare bleeding disorders include the inherited deficiencies of fibrinogen, FII, FV, FV+VIII, FVII, FX, FXI, FXIII and combined deficiency of vitamin-K dependent factors. Recent issues of Haemophilia (November 2008) and Seminars of Thrombosis and Hemostasis (June 2009) have covered the main available treatments

for RBDs. The personal and familial history of each patient needs to be taken into account before choosing the most appropriate therapeutic approach. see more Dosages and frequency of treatments depend on the minimal haemostatic level of the deficient factor (a matter of controversy), its plasma half-life (which varies with age and even in individuals with the same age and the same level of factor)

and the type of bleeding episode [6]. Replacement therapy is effective in treating bleeding episodes in RBDs. Depending on their availability, patients receive fresh frozen plasma (FFP), cryoprecipitate or factor concentrates. The latter is the treatment of choice because GDC-0973 chemical structure it is safer than FFP or cryoprecipitate (decreased risk of blood-borne pathogen transmission), there is no fluid overload and more precise dosing can be accomplished. If the evidence for the optimal use of products in case of replacement therapy in RBDs is already limited, it is almost non-existent for prophylaxis (FXIII being an exception). Discussion  The conventional treatment (treatment on demand) for most RBD is episodic treatment administered as soon as possible after onset of bleeding. The other approach (prophylaxis) consists of giving either products from an early age to prevent bleeding and, in case of surgery or pregnancy, to prevent bleeding and/or miscarriage (primary prophylaxis) or after bleeding to prevent recurrences (secondary prophylaxis). The UK guidelines on therapeutic products for coagulation disorders provide recommendations about the best treatment options (dosage, management of bleeding, surgery and pregnancy as well as prophylaxis)

for RBDs [7]. In theory, prophylactic administration of factors is the best option for patients with severe RBDs. However, this option has check details to be counter-balanced by the possible transmission of infectious agents, allergic reactions, venous access problems, development of inhibitors, risk of thrombotic complications, Transfusion-Related Acute Lung Injury due to cytotoxic antibodies contained in the infused plasma, and cost. Furthermore, even for some severe RBDs, patients can bleed less than severe haemophiliacs and long asymptomatic periods are not uncommon. For example, in a retrospective survey on patients with afibrinogenaemia (or severe hypofibrinogenaemia), the mean annual incidence of bleeding episodes in patients treated on demand was 0.7 (0–16.5) whereas it was 0.5 (0–2.

23 However, a recent study from our group demonstrated that absence of blood flow-derived shear stress stimuli per se, which occurs during organ procurement for transplantation, negatively affects the endothelial vasoprotective phenotype inducing acute endothelial dysfunction.11 This pioneering study created the rationale to investigate strategies for organ preservation based on machine perfusion of kidney or liver grafts.24, 25 Furthermore, it allows study of the

molecular mechanisms leading to increased endothelial sensitivity to injury in the absence of shear stress, with the aim of discovery druggable targets to prevent endothelial and tissue damage during organ procurement. For this purpose, we analyzed the effects of shear stress interruption and cold storage on the hepatic endothelial phenotype and function, and developed a pharmacological www.selleckchem.com/products/azd-1208.html strategy to maintain endothelial health in the setting of organ transplantation. We first characterized the hepatic endothelial vasoprotective phenotype during cold storage, both at the tissue and cellular levels, by analyzing the KLF2-derived protective pathways. Our study demonstrates that during cold storage conditions PD0325901 the hepatic endothelial vasoprotective phenotype is rapidly lost. Indeed, the hepatic expression of KLF2 and its target genes eNOS, TM, and HO-1 is significantly reduced

after just 1 hour or 6 hours of cold storage. Reduced expression of KLF2 and its transcriptional target progressively increased throughout cold storage. Although it is well established that within the liver, as well as in the vasculature, the expression

of KLF2 is mainly endothelial,11, 26, 27 we further characterized the effects of shear stress termination and cold storage conditions on the vasoprotective phenotype in freshly isolated HECs. These in vitro experiments confirmed that once flow stimulus is terminated, and cells are preserved under cold-storage selleck compound conditions, hepatic endothelial KLF2-derived vasoprotective pathways are significantly down-regulated. To understand the pathophysiological consequences of an abnormal endothelial phenotype occurring during cold storage, we characterized the hepatic microcirculation status and the hepatic endothelial function during warm reperfusion. These experiments showed that upon reperfusion cold-stored liver grafts exhibit much higher hepatic vascular resistance, as compared to liver grafts not cold stored. Moreover, these liver grafts exhibit acute endothelial dysfunction. These microcirculatory abnormalities were accompanied by significant hepatic injury, as demonstrated by marked increments in: hepatic enzymes release, inflammation, apoptosis, oxidative stress, histological injury, and significant reduction in bile production.

23 However, a recent study from our group demonstrated that absence of blood flow-derived shear stress stimuli per se, which occurs during organ procurement for transplantation, negatively affects the endothelial vasoprotective phenotype inducing acute endothelial dysfunction.11 This pioneering study created the rationale to investigate strategies for organ preservation based on machine perfusion of kidney or liver grafts.24, 25 Furthermore, it allows study of the

molecular mechanisms leading to increased endothelial sensitivity to injury in the absence of shear stress, with the aim of discovery druggable targets to prevent endothelial and tissue damage during organ procurement. For this purpose, we analyzed the effects of shear stress interruption and cold storage on the hepatic endothelial phenotype and function, and developed a pharmacological Doxorubicin strategy to maintain endothelial health in the setting of organ transplantation. We first characterized the hepatic endothelial vasoprotective phenotype during cold storage, both at the tissue and cellular levels, by analyzing the KLF2-derived protective pathways. Our study demonstrates that during cold storage conditions selleck chemicals the hepatic endothelial vasoprotective phenotype is rapidly lost. Indeed, the hepatic expression of KLF2 and its target genes eNOS, TM, and HO-1 is significantly reduced

after just 1 hour or 6 hours of cold storage. Reduced expression of KLF2 and its transcriptional target progressively increased throughout cold storage. Although it is well established that within the liver, as well as in the vasculature, the expression

of KLF2 is mainly endothelial,11, 26, 27 we further characterized the effects of shear stress termination and cold storage conditions on the vasoprotective phenotype in freshly isolated HECs. These in vitro experiments confirmed that once flow stimulus is terminated, and cells are preserved under cold-storage selleck screening library conditions, hepatic endothelial KLF2-derived vasoprotective pathways are significantly down-regulated. To understand the pathophysiological consequences of an abnormal endothelial phenotype occurring during cold storage, we characterized the hepatic microcirculation status and the hepatic endothelial function during warm reperfusion. These experiments showed that upon reperfusion cold-stored liver grafts exhibit much higher hepatic vascular resistance, as compared to liver grafts not cold stored. Moreover, these liver grafts exhibit acute endothelial dysfunction. These microcirculatory abnormalities were accompanied by significant hepatic injury, as demonstrated by marked increments in: hepatic enzymes release, inflammation, apoptosis, oxidative stress, histological injury, and significant reduction in bile production.