Rebecca Schuster

The Duffy antigen/receptor for chemokines (DARC) regulates prostate tumor growth

The Duffy antigen/receptor for chemokines (DARC) is a promiscuous chemokine receptor that binds to members of the CXC chemokine family possessing angiogenic properties. The DARC is expressed on erythrocytes and endothelial cells and is required for Plasmodium vivax infection of erythrocytes. Approximately 70% of African‐Americans lack erythrocyte expression of the DARC as a genetic mechanism of protection against malaria infection. African‐American men have a 60% greater incidence of prostate cancer and a 2‐fold higher mortality rate than Caucasian men. Using a transgenic model of prostate cancer with DARC‐deficient mice, we tested the hypothesis that lack of DARC expression on erythrocytes contributes to enhanced prostate tumor growth. In vitro, erythrocytes from wild‐type mice but not DARC‐deficient mice cleared angiogenic chemokines produced by prostate cancer cells and reduced endothelial cell chemotaxis. In vivo, tumors from DARC‐deficient mice had higher intra‐tumor concentrations of angiogenic chemokines, increased tumor vessel density, and greatly augmented prostate tumor growth. The data suggest that the DARC functions to clear angiogenic CXC chemokines from the prostate tumor microcirculation and that the lack of erythroid DARC, as occurs in the majority of African‐Americans, may be a contributing factor to the increased mortality to prostate cancer in this popula‐tion.—Shen, H., Schuster, R., Stringer, K. F., Waltz, S. E., Lentsch, A. B. The Duffy antigen/receptor for chemokines (DARC) regulates prostate tumor growth. FASEB J. 20, 59–64 (2006)

Ischemic and non-ischemic acute kidney injury cause hepatic damage

Recent studies have documented that remote organs are affected by ischemic injury to the kidney. Here we studied whether the liver also suffers damage during induction of renal ischemia-reperfusion in rats and compared this to bilateral nephrectomy. Hepatic levels of tumor necrosis factor-alpha increased significantly after 6 and 24 h of renal ischemia or nephrectomy. Malondialdehyde, an index of lipid peroxidation, increased while total glutathione was decreased in the liver in both the renal ischemia and nephrectomy groups, suggesting activation of oxidative stress. Expression of liver spermine-spermidine acetyl transferase, an enzyme upregulated in early phases of hepatic injury was significantly increased 6 h after either kidney ischemia or nephrectomy. Apoptosis was increased in hepatocytes 24 h after nephrectomy. We also found histological evidence of hepatocyte injury following both ischemia and bilateral nephrectomy. Infusion of reduced glutathione, before the induction of renal ischemia, significantly improved liver architecture and was associated with a reduction in hepatic malondialdehyde and serum alanine transaminase levels. Our study shows that acute kidney ischemia or renal failure activates oxidative stress and promotes inflammation, apoptosis, and tissue damage in hepatocytes.

Hepatocyte signaling through CXC chemokine receptor‐2 is detrimental to liver recovery after ischemia/reperfusion in mice

CXC chemokines and their receptor, CXC chemokine receptor‐2 (CXCR2), are important components of the hepatic inflammatory response to ischemia/reperfusion (I/R). However, direct effects of CXC chemokines on hepatocytes during this response have not been studied. Wild‐type and CXCR2−/− mice were subjected to 90 minutes of partial hepatic ischemia followed by up to 96 hours of reperfusion. CXCR2−/− mice had significantly less liver injury at all reperfusion times compared with wild‐type mice. Early neutrophil recruitment (12 hours) was diminished in CXCR2−/− mice, but within 24 hours it was the same as that of wild‐type mice. Hepatocyte proliferation and regeneration was accelerated in CXCR2−/− mice compared with wild‐type mice. These effects were associated with increased activation of nuclear factor κB and signal transducers and activators of transcription‐3, despite there being no difference in the expression of proliferative factors such as tumor necrosis factor α, interleukin‐6, and hepatocyte growth factor. To establish whether the accelerated proliferation and regeneration observed in CXCR2−/− mice was due to effects on hepatocytes rather than just a generalized decrease in acute inflammatory injury, mice were treated with the CXCR2 antagonist, SB225002, after neutrophil recruitment and injury were maximal (24 hours after reperfusion). SB225002 treatment increased hepatocyte proliferation and regeneration in a manner identical to that observed in CXCR2−/− mice. Treatment of primary wild‐type hepatocytes with macrophage inflammatory protein‐2 revealed that low concentrations protected against cell death, whereas high concentrations induced cell death. These effects were absent in hepatocytes from CXCR2−/− mice. Conclusion: Our data suggest that hepatocyte CXCR2 regulates proliferation and regeneration after I/R injury and reveal important differences in the role of this receptor in liver regeneration and repair induced under different conditions that may be related to ligand concentration. (HEPATOLOGY 2008.)

Age-dependent responses to hepatic ischemia/reperfusion injury

The current study explored the concept that adult and pediatric populations differ in their response to major injury. Male C57BL/6 mice of a “young adult” (8-12 weeks) or “mature adult” (12-13 months) age were subjected to partial hepatic ischemia and reperfusion. Mature adult mice displayed significantly more liver injury than young adult mice as assessed histologically and by serum levels of alanine aminotransferase. Interestingly, there was far less neutrophil accumulation in the livers of mature adult mice. However, liver-recruited neutrophils from mature adult mice had a higher activation state than those from young adult mice. Activation of the inflammatory transcription factor, NF-κB, was suppressed in whole livers from mature adult mice. In isolated liver cells, Kupffer cells showed no difference in NF-κB activation, but hepatocytes from mature adult mice had delayed NF-κB activation in response to TNF. Furthermore, isolated hepatocytes from young adult mice produced abundant amounts of the chemokine, macrophage inflammatory protein-2, whereas hepatocytes from mature adult mice produced little, if any macrophage inflammatory protein-2. Mature adult mice had much lower hepatic expression of the cytoprotective protein, heat shock protein 70, than did young adult mice. In contrast, serum heat shock protein 70 levels, which has been linked to subsequent tissue injury, were higher in mature adult mice than in young adult mice. These data suggest that there are multiple alterations at the cellular and molecular levels that contribute to enhanced postischemic liver injury in mature adult mice.

Interleukin-37 reduces liver inflammatory injury via effects on hepatocytes and non-parenchymal cells.

Background and Aim:  The purpose of the present study was to determine the effects of interleukin‐37 (IL‐37) on liver cells and on liver inflammation induced by hepatic ischemia/reperfusion (I/R).

Interleukin-33 is hepatoprotective during liver ischemia/reperfusion in mice.

Interleukin (IL)‐33 is a recently identified member of the IL‐1 family that binds to the receptor, ST2L. In the current study, we sought to determine whether IL‐33 is an important regulator in the hepatic response to ischemia/reperfusion (I/R). Male C57BL/6 mice were subjected to 90 minutes of partial hepatic ischemia, followed by up to 8 hours of reperfusion. Some mice received recombinant IL‐33 (IL‐33) intraperitoneally (IP) before surgery or anti‐ST2 antibody IP at the time of reperfusion. Primary hepatocytes and Kupffer cells were isolated and treated with IL‐33 to assess the effects of IL‐33 on inflammatory cytokine production. Primary hepatocytes were treated with IL‐33 to assess the effects of IL‐33 on mediators of cell survival in hepatocytes. IL‐33 protein expression increased within 4 hours after reperfusion and remained elevated for up to 8 hours. ST2L protein expression was detected in healthy liver and was up‐regulated within 1 hour and peaked at 4 hours after I/R. ST2L was primarily expressed by hepatocytes, with little to no expression by Kupffer cells. IL‐33 significantly reduced hepatocellular injury and liver neutrophil accumulation at 1 and 8 hours after reperfusion. In addition, IL‐33 treatment increased liver activation of nuclear factor kappa light‐chain enhancer of activated B cells (NF‐κB), p38 mitogen‐activated protein kinase (MAPK), cyclin D1, and B‐cell lymphoma 2 (Bcl‐2), but reduced serum levels of CXC chemokines. In vitro experiments demonstrated that IL‐33 significantly reduced hepatocyte cell death as a result of increased NF‐κB activation and Bcl‐2 expression in hepatocytes. Conclusion: The data suggest that IL‐33 is an important endogenous regulator of hepatic I/R injury. It appears that IL‐33 has direct protective effects on hepatocytes, associated with the activation of NF‐κB, p38 MAPK, cyclin D1, and Bcl‐2 that limits liver injury and reduces the stimulus for inflammation. (HEPATOLOGY 2012)

Hepatocyte exosomes mediate liver repair and regeneration via sphingosine-1-phosphate

BACKGROUND & AIMS Exosomes are small membrane vesicles involved in intercellular communication. Hepatocytes are known to release exosomes, but little is known about their biological function. We sought to determine if exosomes derived from hepatocytes contribute to liver repair and regeneration after injury. METHODS Exosomes derived from primary murine hepatocytes were isolated and characterized biochemically and biophysically. Using cultures of primary hepatocytes, we tested whether hepatocyte exosomes induced proliferation of hepatocytes in vitro. Using models of ischemia/reperfusion injury and partial hepatectomy, we evaluated whether hepatocyte exosomes promote hepatocyte proliferation and liver regeneration in vivo. RESULTS Hepatocyte exosomes, but not exosomes from other liver cell types, induce dose-dependent hepatocyte proliferation in vitro and in vivo. Mechanistically, hepatocyte exosomes directly fuse with target hepatocytes and transfer neutral ceramidase and sphingosine kinase 2 (SK2) causing increased synthesis of sphingosine-1-phosphate (S1P) within target hepatocytes. Ablation of exosomal SK prevents the proliferative effect of exosomes. After ischemia/reperfusion injury, the number of circulating exosomes with proliferative effects increases. CONCLUSIONS Our data shows that hepatocyte-derived exosomes deliver the synthetic machinery to form S1P in target hepatocytes resulting in cell proliferation and liver regeneration after ischemia/reperfusion injury or partial hepatectomy. These findings represent a potentially novel new contributing mechanism of liver regeneration and have important implications for new therapeutic approaches to acute and chronic liver disease.

Activation of hepatocytes by extracellular heat shock protein 72

Heat shock protein (HSP) 72 is released by cells during stress and injury. HSP-72 also stimulates the release of cytokines in macrophages by binding to Toll-like receptors (TLR) 2 and 4. Circulating levels of HSP-72 increase during hepatic ischemia-reperfusion injury. The role of extracellular HSP-72 (eHSP-72) in the injury response to ischemia-reperfusion is unknown. Therefore, the objective of the present study was to determine whether eHSP-72 has any direct effects on hepatocytes. Primary mouse hepatocytes were treated with purified human recombinant HSP-72. Conditioned media were evaluated by ELISA for the cytokines, TNF-alpha, IL-6, and macrophage inflammatory protein 2 (MIP-2). Stimulation of hepatocytes with eHSP-72 did not induce production of TNFalpha or IL-6 but resulted in dose-dependent increases in MIP-2 production. To evaluate the pathway responsible for this response, expression of TLR2 and TLR4 was confirmed on hepatocytes by immunohistochemistry. Hepatocyte production of MIP-2 was significantly decreased in hepatocytes obtained from TLR2 or TLR4 knockout mice. MIP-2 production was found to be partially dependent on NF-kappaB because inhibition of NF-kappaB with Bay 11-7085 significantly decreased eHSP-72-induced MIP-2 production. Inhibitors of p38 mitogen-activated protein kinase or c-Jun NH(2)-terminal kinase had no effect on production of MIP-2 induced by eHSP-72. The data suggest that eHSP-72 binds to TLR2 and TLR4 on hepatocytes and signals through NF-kappaB to increase MIP-2 production. The fact that eHSP-72 did not increase TNF-alpha or IL-6 production may be indicative of a highly regulated signaling pathway downstream from TLR.

A murine model of mild traumatic brain injury exhibiting cognitive and motor deficits

BACKGROUND Mild traumatic brain injury (TBI) is a serious public health concern affecting more than 1.7 million people in the United States annually. Mild TBI is difficult to diagnose and is clinically associated with impaired motor coordination and cognition. METHODS We subjected mice to a mild TBI (mTBI-1 or mTBI-2) induced by a weight drop model. We assessed brain injury histologically and biochemically, the latter by serum neuron-specific enolase and glial fibrillary acidic protein. Systemic and brain inflammation were measured by cytokine array. We determined blood-brain barrier integrity by cerebral vascular leakage of micromolecular and macromolecular fluorescent molecules. We evaluated mice using a rotarod device and novel object recognition to measure motor coordination and cognition, respectively. RESULTS Mice undergoing mTBI-1 or mTBI-2 had significant deficits in motor coordination and cognition for several days after injury compared with controls. Furthermore, both mTBI-1 and mTBI-2 caused micromolecular leakage in the blood-brain barrier, whereas only mTBI-2 caused macromolecular leakage. Serum neuron-specific enolase and glial fibrillary acidic protein were elevated acutely and corresponded to the degree of injury, but returned to baseline within 24 h. Serum cytokines interleukin-6 and keratinocyte-derived chemokine were significantly increased within 90 min of TBI. Interleukin-6 levels correlated with the degree of injury. CONCLUSIONS The current study provides a reproducible model of mild TBI in mice that exhibits pathologic features of mild TBI in humans. Furthermore, our data suggest that serum cytokines, such as IL-6, may be effective biomarkers for severity of head injury.

Peroxisome proliferator‐activated receptor‐γ protects against hepatic ischemia/reperfusion injury in mice

The function of peroxisome proliferator‐activated receptor‐γ (PPARγ) in hepatic inflammation and injury is unclear. In this study, we sought to determine the role of PPARγ in hepatic ischemia/reperfusion injury in mice. Male mice were subjected to 90 minutes of partial hepatic ischemia followed by up to 8 hours of reperfusion. PPARγ was found to be constitutively activated in hepatocytes but not in nonparenchymal cells. Upon induction of ischemia, hepatic PPARγ activation rapidly decreased and remained suppressed throughout the 8‐hour reperfusion period. This reduced activation was not a result of decreased protein availability as hepatic nuclear PPARγ, retinoid X receptor‐α (RXRα), and PPARγ/RXRα heterodimer expression was maintained. Accompanying the decrease in PPARγ activation was a decrease in the expression of the natural ligand 15‐deoxy‐Delta12,14‐prostaglandin J2. This was associated with reduced interaction of PPARγ and the coactivator, p300. To determine whether PPARγ activation is hepatoprotective during hepatic ischemia/reperfusion injury, mice were treated with the PPARγ agonists, rosiglitazone and connecting peptide. These treatments increased PPARγ activation and reduced liver injury compared to untreated mice. Furthermore, PPARγ‐deficient mice had more liver injury after ischemia/reperfusion than their wild‐type counterparts. Conclusion: These data suggest that PPARγ is an important endogenous regulator of, and potential therapeutic target for, ischemic liver injury. (HEPATOLOGY 2007.)