Hepatocyte regeneration is driven by embryo-like DNA methylation reprogramming.

As a result of partial hepatectomy, the remaining liver tissue undergoes a process of renewed proliferation that leads to rapid regeneration of the liver. By following the early stages of this process, we observed dramatic programmed changes in the DNA methylation profile, characterized by both de novo and demethylation events, with a subsequent return to the original adult pattern as the liver matures. Strikingly, these transient alterations partially mimic the DNA methylation state of embryonic hepatoblasts (E16.5), indicating that hepatocytes actually undergo epigenetic dedifferentiation. Furthermore, Tet2/Tet3-deletion experiments demonstrated that these changes in methylation are necessary for carrying out basic embryonic functions, such as proliferation, a key step in liver regeneration. This implies that unlike tissue-specific regulatory regions that remain demethylated in the adult, early embryonic genes are programmed to first undergo demethylation, followed by remethylation as development proceeds. The identification of this built-in system may open targeting opportunities for regenerative medicine.

Chronic Intermittent Hypoxia during Sleep Causes Browning of Interscapular Adipose Tissue Accompanied by Local Insulin Resistance in Mice.

Obstructive sleep apnea (OSA) is a highly prevalent condition, characterized by intermittent hypoxia (IH), sleep disruption, and altered autonomic nervous system function. OSA has been independently associated with dyslipidemia, insulin resistance, and metabolic syndrome. Brown adipose tissue (BAT) has been suggested as a modulator of systemic glucose tolerance through adaptive thermogenesis. Reductions in BAT mass have been associated with obesity and metabolic syndrome. No studies have systematically characterized the effects of chronic IH on BAT. Thus, we aimed to delineate IH effects on BAT and concomitant metabolic changes. C57BL/6J 8-week-old male mice were randomly assigned to IH during sleep (alternating 90 s cycles of 6.5% FIO2 followed by 21% FIO2) or normoxia (room air, RA) for 10 weeks. Mice were subjected to glucose tolerance testing and 18F-FDG PET-MRI towards the end of the exposures followed by BAT tissues analyses for morphological and global transcriptomic changes. Animals exposed to IH were glucose intolerant despite lower total body weight and adiposity. BAT tissues in IH-exposed mice demonstrated characteristic changes associated with "browning"-smaller lipids, increased vascularity, and a trend towards higher protein levels of UCP1. Conversely, mitochondrial DNA content and protein levels of respiratory chain complex III were reduced. Pro-inflammatory macrophages were more abundant in IH-exposed BAT. Transcriptomic analysis revealed increases in fatty acid oxidation and oxidative stress pathways in IH-exposed BAT, along with a reduction in pathways related to myogenesis, hypoxia, and IL-4 anti-inflammatory response. Functionally, IH-exposed BAT demonstrated reduced absorption of glucose on PET scans and reduced phosphorylation of AKT in response to insulin. Current studies provide initial evidence for the presence of a maladaptive response of interscapular BAT in response to chronic IH mimicking OSA, resulting in a paradoxical divergence, namely, BAT browning but tissue-specific and systemic insulin resistance. We postulate that oxidative stress, mitochondrial dysfunction, and inflammation may underlie these dichotomous outcomes in BAT.

Interleukin 6-dependent genomic instability heralds accelerated carcinogenesis following liver regeneration on a background of chronic hepatitis.

Liver cancer, which typically develops on a background of chronic liver inflammation, is now the second leading cause of cancer mortality worldwide. For patients with liver cancer, surgical resection is a principal treatment modality that offers a chance of prolonged survival. However, tumor recurrence after resection, the mechanisms of which remain obscure, markedly limits the long-term survival of these patients. We have shown that partial hepatectomy in multidrug resistance 2 knockout (Mdr2-/- ) mice, a model of chronic inflammation-associated liver cancer, significantly accelerates hepatocarcinogenesis. Here, we explore the postsurgical mechanisms that drive accelerated hepatocarcinogenesis in Mdr2-/- mice by perioperative pharmacological inhibition of interleukin-6 (IL6), which is a crucial liver regeneration priming cytokine. We demonstrate that inhibition of IL6 signaling dramatically impedes tumorigenesis following partial hepatectomy without compromising survival or liver mass recovery. IL6 blockade significantly inhibited hepatocyte cell cycle progression while promoting a hypertrophic regenerative response, without increasing apoptosis. Mdr2-/- mice contain hepatocytes with a notable persistent DNA damage response (γH2AX, 53BP1) due to chronic inflammation. We show that liver regeneration in this microenvironment leads to a striking increase in hepatocytes bearing micronuclei, a marker of genomic instability, which is suppressed by IL6 blockade. CONCLUSION: Our findings indicate that genomic instability derived during the IL6-mediated liver regenerative response within a milieu of chronic inflammation links partial hepatectomy to accelerated hepatocarcinogenesis; this suggests a new therapeutic approach through the usage of an anti-IL6 treatment to extend the tumor-free survival of patients undergoing surgical resection. (Hepatology 2017;65:1600-1611).

Impaired liver regeneration after hepatectomy and bleeding is associated with a shift from hepatocyte proliferation to hypertrophy.

Extensive liver resections are common, and bleeding is frequent in these operations. Impaired regeneration after partial hepatectomy (PHx) may contribute to liver failure. We thus assessed the impact of acute bleeding on the liver regeneration progress after PHx and explored possible contributing molecular mechanisms. In rats, the regeneration progress was delayed and attenuated with PHx and bleeding and was not restored with colloid resuscitation. Livers restored their initial volume by postoperative day (POD) 2 after PHx through hepatocyte proliferation vs. POD 4 in the PHx and bleeding group, primarily by hepatocyte hypertrophy. With bleeding, hepatocyte proliferation was hindered in two mechanisms: by inhibiting cells from starting proliferation and by causing hindrance in G1/S progression. Liver hypoxia was prominent, with significant prolonged up-regulation of hypoxia-inducible factors (HIF) and HIF-targeted genes only in the PHx and bleeding group. Gene expression profiling revealed alterations in numerous genes that belong to critical pathways, including cell cycle, DNA replication, PI3K-Akt, purine, and pyrimidine metabolism. Because liver surgery is frequently performed in patients with a predamaged liver, an improper regenerative process after PHx and bleeding might lead to decompensation. The results hint at specific pathways to target in order to improve liver regeneration during PHx and bleeding.-Matot, I., Nachmansson, N., Duev, O., Schulz, S., Schroeder-Stein, K., Frede, S., Abramovitch, R. Impaired liver regeneration after hepatectomy and bleeding is associated with a shift from hepatocyte proliferation to hypertrophy.

Pregnancy restores the regenerative capacity of the aged liver via activation of an mTORC1-controlled hyperplasia/hypertrophy switch.

Regenerative capacity is progressively lost with age. Here we show that pregnancy markedly improved liver regeneration in aged mice concomitantly with inducing a switch from proliferation-based liver regeneration to a regenerative process mediated by cell growth. We found that the key mediator of this switch was the Akt/mTORC1 pathway; its inhibition blocked hypertrophy, while increasing proliferation. Moreover, pharmacological activation of this pathway sufficed to induce the hypertrophy module, mimicking pregnancy. This treatment dramatically improved hepatic regenerative capacity and survival of old mice. Thus, cell growth-mediated mass reconstitution, which is relatively resistant to the detrimental effects of aging, is employed in a physiological situation and holds potential as a therapeutic strategy for ameliorating age-related functional deterioration.

Endogenous plasminogen activators mediate progressive intracerebral hemorrhage after traumatic brain injury in mice.

Persistent intracerebral hemorrhage (ICH) is a major cause of death and disability after traumatic brain injury (TBI) for which no medical treatment is available. Delayed bleeding is often ascribed to consumptive coagulopathy initiated by exposed brain tissue factor. We examined an alternative hypothesis, namely, that marked release of tissue-type plasminogen activator (tPA) followed by delayed synthesis and release of urokinase plasminogen activator (uPA) from injured brain leads to posttraumatic bleeding by causing premature clot lysis. Using a murine model of severe TBI, we found that ICH is reduced in tPA(-/-) and uPA(-/-) mice but increased in PAI-1(-/-) mice compared with wild-type (WT) mice. tPA(-/-), but not uPA(-/-), mice developed a systemic coagulopathy post-TBI. Tranexamic acid inhibited ICH expansion in uPA(-/-)mice but not in tPA(-/-) mice. Catalytically inactive tPA-S(481)A inhibited plasminogen activation by tPA and uPA, attenuated ICH, lowered plasma d-dimers, lessened thrombocytopenia, and improved neurologic outcome in WT, tPA(-/-), and uPA(-/-) mice. ICH expansion was also inhibited by tPA-S(481)A in WT mice anticoagulated with warfarin. These data demonstrate that protracted endogenous fibrinolysis induced by TBI is primarily responsible for persistent ICH and post-TBI coagulopathy in this model and offer a novel approach to interrupt bleeding.

Mutant KRAS is a druggable target for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) represents an unmet therapeutic challenge. PDA is addicted to the activity of the mutated KRAS oncogene which is considered so far an undruggable therapeutic target. We propose an approach to target KRAS effectively in patients using RNA interference. To meet this challenge, we have developed a local prolonged siRNA delivery system (Local Drug EluteR, LODER) shedding siRNA against the mutated KRAS (siG12D LODER). The siG12D LODER was assessed for its structural, release, and delivery properties in vitro and in vivo. The effect of the siG12D LODER on tumor growth was assessed in s.c. and orthotopic mouse models. KRAS silencing effect was further assessed on the KRAS downstream signaling pathway. The LODER-encapsulated siRNA was stable and active in vivo for 155 d. Treatment of PDA cells with siG12D LODER resulted in a significant decrease in KRAS levels, leading to inhibition of proliferation and epithelial-mesenchymal transition. In vivo, siG12D LODER impeded the growth of human pancreatic tumor cells and prolonged mouse survival. We report a reproducible and safe delivery platform based on a miniature biodegradable polymeric matrix, for the controlled and prolonged delivery of siRNA. This technology provides the following advantages: (i) siRNA is protected from degradation; (ii) the siRNA is slowly released locally within the tumor for prolonged periods; and (iii) the siG12D LODER elicits a therapeutic effect, thereby demonstrating that mutated KRAS is indeed a druggable target.

Practical Applications of in Vivo and ex Vivo MRI in Toxicologic Pathology Using a Novel High-performance Compact MRI System.

Magnetic resonance imaging (MRI) is widely used in preclinical research and drug development and is a powerful noninvasive method for assessment of phenotypes and therapeutic efficacy in murine models of disease. In vivo MRI provides an opportunity for longitudinal evaluation of tissue changes and phenotypic expression in experimental animal models. Ex vivo MRI of fixed samples permits a thorough examination of multiple digital slices while leaving the specimen intact for subsequent conventional hematoxylin and eosin (H&E) histology. With the advent of new compact MRI systems that are designed to operate in most conventional labs without the cost, complexity, and infrastructure needs of conventional MRI systems, the possibility of MRI becoming a practical modality is now viable. The purpose of this study was to investigate the capabilities of a new compact, high-performance MRI platform (M2™; Aspect Imaging, Israel) as it relates to preclinical toxicology studies. This overview will provide examples of major organ system pathologies with an emphasis on how compact MRI can serve as an important adjunct to conventional pathology by nondestructively providing 3-dimensional (3-D) digital data sets, detailed morphological insights, and quantitative information. Comparative data using compact MRI for both in vivo and ex vivo are provided as well as validation using conventional H&E.

Inflammation-induced hepatocellular carcinoma is dependent on CCR5 in mice.

UNLABELLED: Human hepatocellular carcinoma (HCC) is an inflammation-induced cancer, which is the third-leading cause of cancer mortality worldwide. We investigated the role of the chemokine receptors, CCR5 and CCR1, in regulating inflammation and tumorigenesis in an inflammation-induced HCC model in mice. Multidrug resistance 2 gene (Mdr2)-knockout (Mdr2-KO) mice spontaneously develop chronic cholestatic hepatitis and fibrosis that is eventually followed by HCC. We generated two new strains from the Mdr2-KO mouse, the Mdr2:CCR5 and the Mdr2:CCR1 double knockouts (DKOs), and set out to compare inflammation and tumorigenesis among these strains. We found that in Mdr2-KO mice lacking the chemokine receptor, CCR5 (Mdr2:CCR5 DKO mice), but not CCR1 (Mdr2:CCR1 DKO), macrophage recruitment and trafficking to the liver was significantly reduced. Furthermore, in the absence of CCR5, reduced inflammation was also associated with reduced periductal accumulation of CD24(+) oval cells and abrogation of fibrosis. DKO mice for Mdr2 and CCR5 exhibited a significant decrease in tumor incidence and size. CONCLUSIONS: Our results indicate that CCR5 has a critical role in both the development and progression of liver cancer. Therefore, we propose that a CCR5 antagonist can serve for HCC cancer prevention and treatment.

Assessment with unenhanced MRI techniques of renal morphology and hemodynamic changes during acute kidney injury and chronic kidney disease in mice.

BACKGROUND/AIMS: Changes in renal oxygenation and perfusion have been identified as common pathways to the development and progression of renal disease. Recently, the sensitivity of hemodynamic response imaging (HRI) was demonstrated; this is a functional magnetic resonance imaging (MRI) method combined with transient hypercapnia and hyperoxia for the evaluation of renal perfusion and vascular reactivity. The aim of this study was to utilize HRI for the noninvasive evaluation of changes in renal hemodynamics and morphology during acute, chronic and acute-on-chronic renal failures. METHODS: Renal-HRI maps and true fast imaging with steady-state precession (True-FISP) images were used to evaluate renal perfusion, morphology and corticomedullary differentiation (CMD). MR images were acquired on two mouse models of kidney injury: adenine-induced chronic kidney disease (CKD) and rhabdomyolysis-induced acute kidney injury (AKI). Serum urea was measured from these mice in order to determine renal function. RESULTS: Renal-HRI maps revealed a blunted response to hypercapnia and hyperoxia with evolving kidney dysfunction in both models, reflecting hampered renal vascular reactivity and perfusion. True-FISP images showed a high sensitivity to renal morphological changes, with different patterns characterizing each model. Calculated data obtained from HRI and True-FISP during the evolution of renal failure and upon recovery, with and without protective intervention, closely correlated with the degree of renal impairment. CONCLUSIONS: This study suggests the potential combined usage of two noninvasive MRI methods, HRI and True-FISP, for the assessment of renal dysfunction without the potential risk associated with contrast-agents administration. HRI may also serve as a research tool in experimental settings, revealing the hemodynamic changes associated with kidney dysfunction.

Quantitative functional MRI biomarkers improved early detection of colorectal liver metastases.

PURPOSE: To implement and evaluate the performance of a computerized statistical tool designed for robust and quantitative analysis of hemodynamic response imaging (HRI) -derived maps for the early identification of colorectal liver metastases (CRLM). MATERIALS AND METHODS: CRLM-bearing mice were scanned during the early stage of tumor growth and subsequently during the advanced-stage. Three experienced radiologists marked various suspected-foci on the early stage anatomical images and classified each as either highly certain or as suspected tumors. The statistical model construction was based on HRI maps (functional-MRI combined with hypercapnia and hyperoxia) using a supervised learning paradigm which was further trained either with the advanced-stage sets (late training; LT) or with the early stage sets (early training; ET). For each group of foci, the classifier results were compared with the ground-truth. RESULTS: The ET-based classification significantly improved the manual classification of the highly certain foci (P < 0.05) and was superior compared with the LT-based classification (P < 0.05). Additionally, the ET-based classification, offered high sensitivity (57-63%), accompanied with high positive predictive value (>94%) and high specificity (>98%) for suspected-foci. CONCLUSION: The ET-based classifier can strengthen the radiologist's classification of highly certain foci. Additionally, it can aid in classifying suspected-foci, thus enabling earlier intervention which can often be lifesaving.

Induced muscle and liver absence of Gne in postnatal mice does not result in structural or functional muscle impairment.

Background: GNE Myopathy is a unique recessive neuromuscular disorder characterized by adult-onset, slowly progressive distal and proximal muscle weakness, caused by mutations in the GNE gene which is a key enzyme in the biosynthesis of sialic acid. To date, the precise pathophysiology of the disease is not well understood and no reliable animal model is available. Gne KO is embryonically lethal in mice. Objective: To gain insights into GNE function in muscle, we have generated an inducible muscle Gne KO mouse. To minimize the contribution of the liver to the availability of sialic acid to muscle via the serum, we have also induced combined Gne KO in liver and muscle. Methods: A mouse carrying loxp sequences flanking Gne exon3 was generated by Crispr/Cas9 and bred with a human skeletal actin (HSA) promoter driven CreERT mouse. Gne muscle knock out was induced by tamoxifen injection of the resulting homozygote GneloxpEx3loxp/HSA Cre mouse. Liver Gne KO was induced by systemic injection of AAV8 vectors carrying the Cre gene driven by the hepatic specific promoter of the thyroxine binding globulin gene. Results: Characterization of these mice for a 12 months period showed no significant changes in their general behaviour, motor performance, muscle mass and structure in spite of a dramatic reduction in sialic acid content in both muscle and liver. Conclusions: We conclude that post weaning lack of Gne and sialic acid in muscle and liver have no pathologic effect in adult mice. These findings could reflect a strong interspecies versatility, but also raise questions about the loss of function hypothesis in Gne Myopathy. If these findings apply to humans they have a major impact on therapeutic strategies.

Resuscitation with aged blood exacerbates liver injury in a hemorrhagic rat model.

OBJECTIVE: Blood loss and transfusion are frequent among patients undergoing liver surgery. Concerns have been raised about the safety and efficacy of transfusing stored blood. The influence of transfusing fresh vs. stored blood on the liver has not been studied to date. We tested the hypothesis that transfusion of stored, but not fresh blood, adversely affects liver outcome in vivo following acute hemorrhage. Additionally, possible mechanisms linking adverse liver outcome with increased storage duration were evaluated. DESIGN: Prospective, controlled, animal study. SETTING: University research laboratory. SUBJECTS: Adult male Sprague-Dawley rats INTERVENTIONS: Anesthetized rats were randomized to control, hemorrhagic and shock group (acute bleeding; HSG), or hemorrhagic and blood resuscitation groups (BR) (with fresh blood [BR-d0], blood stored for 4 [BR-d4] or 7 [BR-d7] days, or packed RBCs stored for 7 days [packed RBC-d7]). MEASUREMENTS AND MAIN RESULTS: Administration of blood or packed RBC stored for 7 days exacerbated liver injury as reflected by liver necrosis and enhanced apoptosis (p < 0.001). Functional MRI analysis of the liver demonstrated significant improvement in liver perfusion with fresh blood (% change in functional MRI signal intensity due to hyperoxia was 16% ± 3% in BR-d0 vs. 4% ± 3% in hemorrhagic group, p < 0.001) but not with stored blood (12% ± 2% and 9% ± 5% for BR-d4 and BR-d7, respectively). Analysis of stored blood showed reduction in RBC deformability at 7 days of storage, reflecting a five-fold increase in the number of undeformable cells. CONCLUSION: Liver injury is exacerbated by the transfusion of stored blood, primarily due to the change in the rheological properties of RBC. This data call for clinical studies in patients undergoing liver resection or transplantation.

Hemodynamic response magnetic resonance imaging: application for renal hemodynamic characterization.

BACKGROUND: The clinical use of iodinated radiocontrast agents or gadolinium for renal perfusion imaging is limited in the presence of renal dysfunction. We have previously demonstrated the feasibility of hemodynamic response imaging (HRI), a functional magnetic resonance imaging (MRI) method combined with hypercapnia and hypercapnic-hyperoxia, for monitoring changes in liver perfusion and hemodynamics. The aim of the present study was to evaluate the utility of HRI for monitoring changes in renal perfusion and hemodynamics. METHODS: Renal HRI maps were acquired during graded hypercapnia (95% air + 5% CO2) and hypercapnic-hyperoxia (95% O2 + 5% CO2) in control mice. The utility of HRI for monitoring changes in renal perfusion and oxygenation was evaluated using pharmacological inhibition of nitric oxide synthase and cycloxygenase as well as in rhabdomyolysis-induced acute kidney injury (AKI) in mice. HRI results were further interpreted using Doppler ultrasound (US). RESULTS: Renal HRI maps revealed pronounced signal-intensity changes in response to both hypercapnia and hypercapnic-hyperoxia, reflecting intense vascular reactivity. These changes were significantly attenuated following the pharmacological intervention and during AKI, corresponding with hampered perfusion dynamics, as confirmed by Doppler US. CONCLUSIONS: The applicability of the non-invasive HRI method suggests its potential use for the evaluation of renal perfusion and vascular reactivity, excluding the need for contrast-agent administration.

Accelerated carcinogenesis following liver regeneration is associated with chronic inflammation-induced double-strand DNA breaks.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide and is considered to be the outcome of chronic liver inflammation. Currently, the main treatment for HCC is surgical resection. However, survival rates are suboptimal partially because of tumor recurrence in the remaining liver. Our aim was to understand the molecular mechanisms linking liver regeneration under chronic inflammation to hepatic tumorigenesis. Mdr2-KO mice, a model of inflammation-associated cancer, underwent partial hepatectomy (PHx), which led to enhanced hepatocarcinogenesis. Moreover, liver regeneration in these mice was severely attenuated. We demonstrate the activation of the DNA damage-response machinery and increased genomic instability during early liver inflammatory stages resulting in hepatocyte apoptosis, cell-cycle arrest, and senescence and suggest their involvement in tumor growth acceleration subsequent to PHx. We propose that under the regenerative proliferative stress induced by liver resection, the genomic unstable hepatocytes generated during chronic inflammation escape senescence and apoptosis and reenter the cell cycle, triggering the enhanced tumorigenesis. Thus, we clarify the immediate and long-term contributions of the DNA damage response to HCC development and recurrence.

AN1284 attenuates steatosis, lipogenesis, and fibrosis in mice with pre-existing non-alcoholic steatohepatitis and directly affects aryl hydrocarbon receptor in a hepatic cell line.

Non-alcoholic steatohepatitis (NASH) is an aggressive form of fatty liver disease with hepatic inflammation and fibrosis for which there is currently no drug treatment. This study determined whether an indoline derivative, AN1284, which significantly reduced damage in a model of acute liver disease, can reverse steatosis and fibrosis in mice with pre-existing NASH and explore its mechanism of action. The mouse model of dietary-induced NASH reproduces most of the liver pathology seen in human subjects. This was confirmed by RNA-sequencing analysis. The Western diet, given for 4 months, caused steatosis, inflammation, and liver fibrosis. AN1284 (1 mg or 5 mg/kg/day) was administered for the last 2 months of the diet by micro-osmotic-pumps (mps). Both doses significantly decreased hepatic damage, liver weight, hepatic fat content, triglyceride, serum alanine transaminase, and fibrosis. AN1284 (1 mg/kg/day) given by mps or in the drinking fluid significantly reduced fibrosis produced by carbon tetrachloride injections. In human HUH7 hepatoma cells incubated with palmitic acid, AN1284 (2.1 and 6.3 ng/ml), concentrations compatible with those in the liver of mice treated with AN1284, decreased lipid formation by causing nuclear translocation of the aryl hydrocarbon receptor (AhR). AN1284 downregulated fatty acid synthase (FASN) and sterol regulatory element-binding protein 1c (SREBP-1c) and upregulated Acyl-CoA Oxidase 1 and Cytochrome P450-a1, genes involved in lipid metabolism. In conclusion, chronic treatment with AN1284 (1mg/kg/day) reduced pre-existing steatosis and fibrosis through AhR, which affects several contributors to the development of fatty liver disease. Additional pathways are also influenced by AN1284 treatment.

Premature Macrophage Activation by Stored Red Blood Cell Transfusion Halts Liver Regeneration Post-Partial Hepatectomy in Rats.

Liver resection is a common treatment for various conditions and often requires blood transfusions to compensate for operative blood loss. As partial hepatectomy (PHx) is frequently performed in patients with a pre-damaged liver, avoiding further injury is of paramount clinical importance. Our aim was to study the impact of red blood cell (RBC) resuscitation on liver regeneration. We assessed the impact of RBC storage time on liver regeneration following 50% PHx in rats and explored possible contributing molecular mechanisms using immunohistochemistry, RNA-Seq, and macrophage depletion. The liver was successfully regenerated after PHx when rats were transfused with fresh RBCs (F-RBCs). However, in rats resuscitated with stored RBCs (S-RBCs), the regeneration process was disrupted, as detected by delayed hepatocyte proliferation and lack of hypertrophy. The delayed regeneration was associated with elevated numbers of hemorrhage-activated liver macrophages (Mhem) secreting HO-1. Depletion of macrophages prior to PHx and transfusion improved the regeneration process. Gene expression profiling revealed alterations in numerous genes belonging to critical pathways, including cell cycle and DNA replication, and genes associated with immune cell activation, such as chemokine signaling and platelet activation and adhesion. Our results implicate activated macrophages in delayed liver regeneration following S-RBC transfusion via HO-1 and PAI-1 overexpression.

Opposite physiological and pathological mTORC1-mediated roles of the CB1 receptor in regulating renal tubular function.

Activation of the cannabinoid-1 receptor (CB1R) and the mammalian target of rapamycin complex 1 (mTORC1) in the renal proximal tubular cells (RPTCs) contributes to the development of diabetic kidney disease (DKD). However, the CB1R/mTORC1 signaling axis in the kidney has not been described yet. We show here that hyperglycemia-induced endocannabinoid/CB1R stimulation increased mTORC1 activity, enhancing the transcription of the facilitative glucose transporter 2 (GLUT2) and leading to the development of DKD in mice; this effect was ameliorated by specific RPTCs ablation of GLUT2. Conversely, CB1R maintained the normal activity of mTORC1 by preventing the cellular excess of amino acids during normoglycemia. Our findings highlight a novel molecular mechanism by which the activation of mTORC1 in RPTCs is tightly controlled by CB1R, either by enhancing the reabsorption of glucose and inducing kidney dysfunction in diabetes or by preventing amino acid uptake and maintaining normal kidney function in healthy conditions.

Cell-autonomous and non-cell-autonomous functions of caspase-8.

Cells in vivo do not act in isolation. Therefore, when attempting to predict the results of pharmaceutical modulation of the function of a protein, we must also take into account the non-cell-autonomous consequences of such modulation. Studies of caspase-8 initially indicated that it serves as the proximal enzyme in cellular self-destruction dictated through the extrinsic cell-death pathway. Later studies revealed that it also participates in mechanisms affecting cell growth and survival. This essay presents a brief account of a study indicating that, apart from functional changes that are cell autonomous, tissue-specific deletion of caspase-8 in mice also has non-cell-autonomous effects with consequences that might even be the opposite of the cell-autonomous ones.

Chronic hypoxia in pregnant mice impairs the placental and fetal vascular response to acute hypercapnia in BOLD-MRI hemodynamic response imaging.

INTRODUCTION: Brief hypercapnic challenge causes acute placental hypoperfusion with fetal brain sparing on BOLD-MRI. We hypothesize that this non-invasive imaging strategy can distinguish between normal pregnancy and chronic placental hypoperfusion (using the maternal hypoxia model). METHODS: Eighteen pregnant female ICR mice were randomized to three groups: normoxia, late-onset hypoxia (12%O2;E13.5-17.5) and early-onset hypoxia (12%O2;E10.5-17.5). On E17.5, animals were imaged in a 4.7-T Bruker-Biospec MRI scanner. Fast coronal True-FISP was performed to identify organs of interest (placenta and fetal heart, liver and brain). BOLD-MRI was performed at baseline and during a 4-min hypercapnic challenge (5%CO2). %-change in placental and fetal signal was analyzed from T2*-weighted gradient echo MR images. Following MRI, fetuses and placentas were harvested, weighed and immuno-stained. RESULTS: In normoxic mice, hypercapnia caused reduction in BOLD-MRI signal in placenta (-44% ± 7%; p < 0.0001), fetal liver (-32% ± 7%; p < 0.0001) and fetal heart (-54% ± 12%; p < 0.002), with relative fetal brain sparing (-12% ± 5%; p < 0.0001). These changes were markedly attenuated in both hypoxia groups. Baseline fetal brain/placenta SI ratio was highest in normoxic mice (1.14 ± 0.017) and reduced with increasing duration of hypoxia (late-onset hypoxia: 1.00 ± 0.026; early-onset hypoxia: 0.91 ± 0.016; p = 0.02). Both hypoxic groups exhibited fetal growth restriction with prominent placental glycogen-containing cells, particularly in early-onset hypoxia. There was increased fetal neuro- and intestinal-apoptosis in early-onset hypoxia only. CONCLUSIONS: BOLD-MRI with brief hypercapnic challenge distinguished between normoxia and both hypoxia groups, while fetal neuroapoptosis was only observed after early-onset hypoxia. This suggests that BOLD-MRI with hypercapnic challenge can identify chronic fetal asphyxia before the onset of irreversible brain injury.