CXC Chemokines Function as a Rheostat for Hepatocyte Proliferation and Liver Regeneration

Background

Our previous in vitro studies have demonstrated dose-dependent effects of CXCR2 ligands on hepatocyte cell death and proliferation. In the current study, we sought to determine if CXCR2 ligand concentration is responsible for the divergent effects of these mediators on liver regeneration after ischemia/reperfusion injury and partial hepatectomy.

Methods

Murine models of partial ischemia/reperfusion injury and hepatectomy were used to study the effect of CXCR2 ligands on liver regeneration.

Results

We found that hepatic expression of the CXCR2 ligands, macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC), was significantly increased after both I/R injury and partial hepatectomy. However, expression of these ligands after I/R injury was 30-100-fold greater than after hepatectomy. Interestingly, the same pattern of expression was found in ischemic versus non-ischemic liver lobes following I/R injury with expression significantly greater in the ischemic liver lobes. In both systems, lower ligand expression was associated with increased hepatocyte proliferation and liver regeneration in a CXCR2-dependent fashion. To confirm that these effects were related to ligand concentration, we administered exogenous MIP-2 and KC to mice undergoing partial hepatectomy. Mice received a “high” dose that replicated serum levels found after I/R injury and a “low” dose that was similar to that found after hepatectomy. Mice receiving the “high” dose had reduced levels of hepatocyte proliferation and regeneration whereas the “low” dose promoted hepatocyte proliferation and regeneration.

Conclusions

Together, these data demonstrate that concentrations of CXC chemokines regulate the hepatic proliferative response and subsequent liver regeneration.     

FTY720 reduces post-ischemic brain lymphocyte influx but does not improve outcome in permanent murine cerebral ischemia

Background

The contribution of neuroinflammation and specifically brain lymphocyte invasion is increasingly recognised as a substantial pathophysiological mechanism after stroke. FTY720 is a potent treatment for primary neuroinflammatory diseases by inhibiting lymphocyte circulation and brain immigration. Previous studies using transient focal ischemia models showed a protective effect of FTY720 but did only partially characterize the involved pathways. We tested the neuroprotective properties of FTY720 in permanent and transient cortical ischemia and analyzed the underlying neuroimmunological mechanisms.

Methodology/Principal Findings

FTY720 treatment resulted in substantial reduction of circulating lymphocytes while blood monocyte counts were significantly increased. The number of histologically and flow cytometrically analyzed brain invading T- and B lymphocytes was significantly reduced in FTY720 treated mice. However, despite testing a variety of treatment protocols, infarct volume and behavioural dysfunction were not reduced 7d after permanent occlusion of the distal middle cerebral artery (MCAO). Additionally, we did not measure a significant reduction in infarct volume at 24h after 60 min filament-induced MCAO, and did not see differences in brain edema between PBS and FTY720 treatment. Analysis of brain cytokine expression revealed complex effects of FTY720 on postischemic neuroinflammation comprising a substantial reduction of delayed proinflammatory cytokine expression at 3d but an early increase of IL-1β and IFN-γ at 24 h after MCAO. Also, serum cytokine levels of IL-6 and TNF-α were increased in FTY720 treated animals compared to controls.

Conclusions/Significance

In the present study we were able to detect a reduction of lymphocyte brain invasion by FTY720 but could not achieve a significant reduction of infarct volumes and behavioural dysfunction. This lack of neuroprotection despite effective lymphopenia might be attributed to a divergent impact of FTY720 on cytokine expression and possible activation of innate immune cells after brain ischemia.     

Characterization of Microparticles after Hepatic Ischemia-Reperfusion Injury

Background

Hepatic ischemia-reperfusion (I/R) is a well-studied model of liver injury and has demonstrated a biphasic injury followed by recovery and regeneration. Microparticles (MPs) are a developing field of study and these small membrane bound vesicles have been shown to have effector function in other physiologic and pathologic states. This study was designed to quantify the levels of MPs from various cell origins–platelets, neutrophils, and endolethial cells–following hepatic ischemia-reperfusion injury.

Methods

A murine model was used with mice undergoing 90 minutes of partial hepatic ischemia followed by various times of reperfusion. Following reperfusion, plasma samples were taken and MPs of various cell origins were labeled and levels were measured using flow cytometry. Additionally, cell specific MPs were further assessed by Annexin V, which stains for the presence of phosphatidylserine, a cell surface marker linked to apoptosis. Statistical analysis was performed using one-way analysis of variance with subsequent Student-Newman-Keuls test with data presented as the mean and standard error of the mean.

Results

MPs from varying sources show an increase in circulating levels following hepatic I/R injury. However, the timing of the appearance of different MP subtypes differs for each cell type. Platelet and neutrophil-derived MP levels demonstrated an acute elevation following injury whereas endothelial-derived MP levels demonstrated a delayed elevation.

Conclusion

This is the first study to characterize circulating levels of cell-specific MPs after hepatic I/R injury and suggests that MPs derived from platelets and neutrophils serve as markers of inflammatory injury and may be active participants in this process. In contrast, MPs derived from endothelial cells increase after the injury response during the reparative phase and may be important in angiogenesis that occurs in the regenerating liver.     

Cilostazol activates function of bone marrow-derived endothelial progenitor cell for re-endothelialization in a carotid balloon injury model

Background

Cilostazol(CLZ) has been used as a vasodilating anti-platelet drug clinically and demonstrated to inhibit proliferation of smooth muscle cells and effect on endothelial cells. However, the effect of CLZ on re-endothelialization including bone marrow (BM)-derived endothelial progenitor cell (EPC) contribution is unclear. We have investigated the hypothesis that CLZ might accelerate re-endothelialization with EPCs.

Methodology/Principal Findings

Balloon carotid denudation was performed in male Sprague-Dawley rats. CLZ group was given CLZ mixed feed from 2weeks before carotid injury. Control group was fed normal diet. CLZ accelerated re-endothelialization at 2 weeks after surgery and resulted in a significant reduction of neointima formation 4 weeks after surgery compared with that in control group. CLZ also increased the number of circulating EPCs throughout the time course. We examined the contribution of BM-derived EPCs to re-endothelialization by BM transplantation from Tie2/lacZ mice to nude rats. The number of Tie2-regulated X-gal positive cells on injured arterial luminal surface was increased at 2 weeks after surgery in CLZ group compared with that in control group. In vitro, CLZ enhanced proliferation, adhesion and migration activity, and differentiation with mRNA upregulation of adhesion molecule integrin αvβ3, chemokine receptor CXCR4 and growth factor VEGF assessed by real-time RT-PCR in rat BM-derived cultured EPCs. In addition, CLZ markedly increased the expression of SDF-1α that is a ligand of CXCR4 receptor in EPCs, in the media following vascular injury.

Conclusions/Significance

CLZ promotes EPC mobilization from BM and EPC recruitment to sites of arterial injury, and thereby inhibited neointima formation with acceleration of re-endothelialization with EPCs as well as pre-existing endothelial cells in a rat carotid balloon injury model. CLZ could be not only an anti-platelet agent but also a promising tool for endothelial regeneration, which is a key event for preventing atherosclerosis or restenosis after vascular intervention.     

The Suppressive Effect of Resveratrol on HIF-1α and VEGF Expression after Warm Ischemia and Reperfusion in Rat Liver

Background

Hypoxia-inducible factor-1α (HIF-1α) is overexpressed in many human tumors and their metastases, and is closely associated with a more aggressive tumor phenotype. The aim of the present study was to investigate the effect of resveratrol (RES) on the expression of ischemic-induced HIF-1α and vascular endothelial growth factor (VEGF) in rat liver.

Methods

Twenty-four rats were randomized into Sham, ischemia/reperfusion (I/R), and RES preconditioning groups. I/R was induced by portal pedicle clamping for 60 minutes followed by reperfusion for 60 minutes. The rats in RES group underwent the same surgical procedure as I/R group, and received 20 mg/kg resveratrol intravenously 30 min prior to ischemia. Blood and liver tissue samples were collected and subjected to biochemical assays, RT-PCR, and Western blot assays.

Results

I/R resulted in a significant (P<0.05) increase in liver HIF-1α and VEGF at both mRNA and protein levels 60 minutes after reperfusion. The mRNA and protein expressions of HIF-1α and VEGF decreased significantly in RES group when compared to I/R group (P<0.05).

Conclusion

The inhibiting effect of RES on the expressions of HIF-1α and VEGF induced by I/R in rat liver suggested that HIF-1α/VEGF could be a promising drug target for RES in the development of an effective anticancer therapy for the prevention of hepatic tumor growth and metastasis.     

Carvacrol Alleviates Ischemia Reperfusion Injury by Regulating the PI3K-Akt Pathway in Rats

Background

Liver ischemia reperfusion (I/R) injury is a common pathophysiological process in many clinical settings. Carvacrol, a food additive commonly used in essential oils, has displayed antimicrobials, antitumor and antidepressant-like activities. In the present study, we investigated the protective effects of carvacrol on I/R injury in the Wistar rat livers and an in vitro hypoxia/restoration (H/R) model.

Methods

The hepatoportal vein, hepatic arterial and hepatic duct of Wistar rats were isolated and clamped for 30 min, followed by a 2 h reperfusion. Buffalo rat liver (BRL) cells were incubated under hypoxia for 4 h, followed normoxic conditions for 10 h to establish the H/R model in vitro. Liver injury was evaluated by measuring serum levels of alanine aminotransferase (ALT) and aspatate aminotransferase (AST), and hepatic levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and malondiadehyde (MDA), and hepatic histology and TUNEL staining. MTT assay, flow cytometric analysis and Hoechst 33258 staining were used to evaluate the proliferation and apoptosis of BRL cells in vitro. Protein expression was examined by Western Blot analysis.

Results

Carvacrol protected against I/R-induced liver damage, evidenced by significantly reducing the serum levels of ALT and AST, histological alterations and apoptosis of liver cells in I/R rats. Carvacrol exhibited anti-oxidative activity in the I/R rats, reflected by significantly reducing the activity of SOD and the content of MDA, and restoring the activity of CAT and the content of GSH, in I/R rats. In the in vitro assays, carvacrol restored the viability and inhibited the apoptosis of BRL cells, which were subjected to a mimic I/R injury induced by hypoxia. In the investigation on molecular mechanisms, carvacrol downregulated the expression of Bax and upregulated the expression of Bcl-2, thus inhibited the activation of caspase-3. Carvacrol was also shown to enhance the phosphorylation of Akt.

Conclusion

The results suggest that carvacrol could alleviate I/R-induced liver injury by its anti-oxidative and anti-apoptotic activities, and warrant a further investigation for using carvacrol to protect I/R injury in clinic.     

Bone marrow-derived mesenchymal stem cells ameliorate hepatic ischemia reperfusion injury in a rat model

Background

Ischemia-reperfusion (I/R) injury associated with living donor liver transplantation impairs liver graft regeneration. Mesenchymal stem cells (MSCs) are potential cell therapeutic targets for liver disease. In this study, we demonstrate the impact of MSCs against hepatic I/R injury and hepatectomy.

Methodology/Principal Findings

We used a new rat model in which major hepatectomy with I/R injury was performed. Male Lewis rats were separated into two groups: an MSC group given MSCs after reperfusion as treatment, and a Control group given phosphate-buffered saline after reperfusion as placebo. The results of liver function tests, pathologic changes in the liver, and the remnant liver regeneration rate were assessed. The fate of transplanted MSCs in the luciferase-expressing rats was examined by in vivo luminescent imaging. The MSC group showed peak luciferase activity of transplanted MSCs in the remnant liver 24 h after reperfusion, after which luciferase activity gradually declined. The elevation of serum alanine transaminase levels was significantly reduced by MSC injection. Histopathological findings showed that vacuolar change was lower in the MSC group compared to the Control group. In addition, a significantly lower percentage of TUNEL-positive cells was observed in the MSC group compared with the controls. Remnant liver regeneration rate was accelerated in the MSC group.

Conclusions/Significance

These data suggest that MSC transplantation provides trophic support to the I/R-injured liver by inhibiting hepatocellular apoptosis and by stimulating regeneration.     

FTY720 Treatment in the Convalescence Period Improves Functional Recovery and Reduces Reactive Astrogliosis in Photothrombotic Stroke

Background

The Sphingosine-1-phosphate (S1P) signaling pathway is known to influence pathophysiological processes within the brain and the synthetic S1P analog FTY720 has been shown to provide neuroprotection in experimental models of acute stroke. However, the effects of a manipulation of S1P signaling at later time points after experimental stroke have not yet been investigated. We examined whether a relatively late initiation of a FTY720 treatment has a positive effect on long-term neurological outcome with a focus on reactive astrogliosis, synapses and neurotrophic factors.

Methods

We induced photothrombotic stroke (PT) in adult C57BL/6J mice and allowed them to recover for three days. Starting on post-stroke day 3, mice were treated with FTY720 (1 mg/kg b.i.d.) for 5 days. Behavioral outcome was observed until day 31 after photothrombosis and periinfarct cortical tissue was analyzed using tandem mass-spectrometry, TaqMan®analysis and immunofluorescence.

Results

FTY720 treatment results in a significantly better functional outcome persisting up to day 31 after PT. This is accompanied by a significant decrease in reactive astrogliosis and larger post-synaptic densities as well as changes in the expression of vascular endothelial growth factor α (VEGF α). Within the periinfarct cortex, S1P is significantly increased compared to healthy brain tissue.

Conclusion

Besides its known neuroprotective effects in the acute phase of experimental stroke, the initiation of FTY720 treatment in the convalescence period has a positive impact on long-term functional outcome, probably mediated through reduced astrogliosis, a modulation in synaptic morphology and an increased expression of neurotrophic factors.     

Recombinant Adiponectin Ameliorates Liver Ischemia Reperfusion Injury via Activating the AMPK/eNOS Pathway

Background

It is of importance to minimize ischemia reperfusion (I/R) injury during liver operations. Reducing the inflammatory reaction is an effective way to achieve this goal. Notably, adiponectin (APN) was found to have anti-inflammatory activity in heart and renal I/R injury. Herein, we investigated the role of APN in liver I/R injury.

Methods

Wistar rats were randomized to four groups: (1) sham group; (2) I/R control group; (3) I/R+APN group; and (4) I/R+APN+AMPK inhibitor group. Liver and blood samples were collected 6h and 24h after reperfusion. Liver function and histopathologic changes were assessed. Macrophage and neutrophil infiltration was detected by immunohistochemistry staining, while pro-inflammatory cytokines and chemokines released in the liver were measured using ELISA and RT-PCR, respectively. Apoptosis was analyzed by TUNEL staining and caspase-3 expression in the liver. Downstream molecules of APN were investigated by Western blotting.

Results

Circulatory APN was down-regulated during liver I/R. When exogenous APN treatment was administered during liver I/R, alanine transaminase (ALT) and aspartate aminotransferase (AST) were decreased, and less hepatocyte necrosis was observed. Less inflammatory cell infiltration and pro-inflammatory cytokines/chemokines release were also observed in the I/R+APN group when compared with the I/R control group. APN treatment also reduced hepatocyte apoptosis, evidenced by reduced TUNEL positive cells and less caspase-3 expression in the reperfused liver. Finally, the AMPK/eNOS pathway was found to be activated by APN, and administration of an AMPK inhibitor reversed the beneficial effects of APN.

Conclusion

APN can protect the liver from I/R injury by reducing the inflammatory response and hepatocyte apoptosis, a process that likely involves the AMPK/eNOS pathway. The current study provides a potential pharmacologic target for liver I/R injury.     

The Role of N-Acetyltransferase 8 in Mesenchymal Stem Cell-Based Therapy for Liver Ischemia/Reperfusion Injury in Rats

Objective

To evaluate the impact of mesenchymal stem cells (MSCs) against hepatic I/R injury and explore the role of N-acetyltransferase 8 (NAT8) in the process.

Methods

We investigated the potential of injected MSCs systemically via the tail vein in healing injuried liver of the SD rat model of 70% hepatic I/R injury by measuring the biochemical and pathologic alterations. Subsequently, we evaluated the expression levels of NAT8 by western blotting in vivo. Concurrently, hydrogen peroxide (H₂O₂)-induced apoptosis in the human normal liver cell line L02 was performed in vitro to evaluate the protective effects of MSC conditioned medium (MSC-CM) on L02 cells. In addition, we downregulated and upregulated NAT8 expression in L02 cells and induced apoptosis by using H₂O₂ to study the protective role of NAT8.

Results

MSCs implantation led to a significant reduced liver enzyme levels, an advanced protection in the histopathological findings of the acutely injured liver and a significantly lower percentage of TUNEL-positive cells, which were increased after I/R injury. In vitro assays, MSC-CM inhibited hepatocyte apoptosis induced by H₂O_2. Moreover, overexpression or downregulation of NAT8 prevented or aggravated hepatocyte apoptosis induced by H₂O₂, respectively.

Conclusions

MSC transplantation provides support to the I/R-injured liver by inhibiting hepatocellular apoptosis and stimulating NAT8 regeneration.     

Crosstalk between chemokine receptor CXCR4 and cannabinoid receptor CB2 in modulating breast cancer growth and invasion

Background

Cannabinoids bind to cannabinoid receptors CB₁ and CB₂ and have been reported to possess anti-tumorigenic activity in various cancers. However, the mechanisms through which cannabinoids modulate tumor growth are not well known. In this study, we report that a synthetic non-psychoactive cannabinoid that specifically binds to cannabinoid receptor CB₂ may modulate breast tumor growth and metastasis by inhibiting signaling of the chemokine receptor CXCR4 and its ligand CXCL12. This signaling pathway has been shown to play an important role in regulating breast cancer progression and metastasis.

Methodology/Principal Findings

We observed high expression of both CB₂ and CXCR4 receptors in breast cancer patient tissues by immunohistochemical analysis. We further found that CB₂-specific agonist JWH-015 inhibits the CXCL12-induced chemotaxis and wound healing of MCF7 overexpressing CXCR4 (MCF7/CXCR4), highly metastatic clone of MDA-MB-231 (SCP2) and NT 2.5 cells (derived from MMTV-neu) by using chemotactic and wound healing assays. Elucidation of the molecular mechanisms using various biochemical techniques and confocal microscopy revealed that JWH-015 treatment inhibited CXCL12-induced P44/P42 ERK activation, cytoskeletal focal adhesion and stress fiber formation, which play a critical role in breast cancer invasion and metastasis. In addition, we have shown that JWH-015 significantly inhibits orthotopic tumor growth in syngenic mice in vivo using NT 2.5 cells. Furthermore, our studies have revealed that JWH-015 significantly inhibits phosphorylation of CXCR4 and its downstream signaling in vivo in orthotopic and spontaneous breast cancer MMTV-PyMT mouse model systems.

Conclusions/Significance

This study provides novel insights into the crosstalk between CB₂ and CXCR4/CXCL12-signaling pathways in the modulation of breast tumor growth and metastasis. Furthermore, these studies indicate that CB₂ receptors could be used for developing innovative therapeutic strategies against breast cancer.     

Microfluidic Endothelium for Studying the Intravascular Adhesion of Metastatic Breast Cancer Cells

Background

The ability to properly model intravascular steps in metastasis is essential in identifying key physical, cellular, and molecular determinants that can be targeted therapeutically to prevent metastatic disease. Research on the vascular microenvironment has been hindered by challenges in studying this compartment in metastasis under conditions that reproduce in vivo physiology while allowing facile experimental manipulation.

Methodology/Principal Findings

We present a microfluidic vasculature system to model interactions between circulating breast cancer cells with microvascular endothelium at potential sites of metastasis. The microfluidic vasculature produces spatially-restricted stimulation from the basal side of the endothelium that models both organ-specific localization and polarization of chemokines and many other signaling molecules under variable flow conditions. We used this microfluidic system to produce site-specific stimulation of microvascular endothelium with CXCL12, a chemokine strongly implicated in metastasis.

Conclusions/Significance

When added from the basal side, CXCL12 acts through receptor CXCR4 on endothelium to promote adhesion of circulating breast cancer cells, independent of CXCL12 receptors CXCR4 or CXCR7 on tumor cells. These studies suggest that targeting CXCL12-CXCR4 signaling in endothelium may limit metastases in breast and other cancers and highlight the unique capabilities of our microfluidic device to advance studies of the intravascular microenvironment in metastasis.     

Antisense Oligonucleotide against hTERT (Cantide) Inhibits Tumor Growth in an Orthotopic Primary Hepatic Lymphoma Mouse Model

Background

Human xenograft models, resulting from orthotopic transplantation (implantation into the anatomically correct site) of histologically intact tissue into animals, are important for investigating local tumor growth, vascular and lymphatic invasion at the primary tumor site and metastasis.

Methodology/Principal Findings

We used surgical orthotopic transplantation to establish a nude mouse model of primary hepatic lymphoma (PHL), HLBL-0102. We performed orthotopic transfer of the HLBL-0102 tumor for 42 generations and characterized the tumor cells. The maintenance of PHL characteristics were supported by immunohistochemical and cytogenetic analysis. We also report the antitumor effect of Cantide, an antisense phosphorothioate oligonucleotide against hTERT, on the growth of HLBL-0102 tumors. We showed a significant, dose-dependent inhibition of tumor weight and serum LDH activity in the orthotopically transplanted animals by Cantide. Importantly, survival was prolonged in Cantide-treated HLBL-0102 tumor-bearing mice when compared to mock-treated mice.

Conclusions/Significance

Our study provided the basis for the development of a clinical trial protocol to treat PHL.     

Salvage liver transplantation for patients with recurrent hepatocellular carcinoma after curative resection

Objective

To summarize the experience with salvage liver transplantation (SLT) for patients with recurrent hepatocellular carcinoma (HCC) after primary hepatic resection in a single center.

Methods

A total of 376 adult patients with HCC underwent orthotopic liver transplantation (OLT) at Organ Transplantation Center, the First Affiliated Hospital of Sun Yat-sen University, between 2004 and 2008. Among these patients, 36 underwent SLT after primary liver curative resection due to intrahepatic recurrence. During the same period, one hundred and forty-seven patients with HCC within Milan criteria underwent primary OLT (PLTW group), the intra-operative and post-operative parameters were compared between these two groups. Furthermore, we compared tumor recurrence and patient survival of patients with SLT to 156 patients with HCC beyond Milan criteria (PLTB group). Cox Hazard regression was made to identify the risk factors for tumor recurrence.

Results

The median interval between initial liver resection and SLT was 35 months (1–63 months). The intraoperative blood loss (P<0.05) and transfusion volume (P<0.05) were larger in the SLT group than in the PLTW group. The operation time was longer in the SLT group (P<0.05). The post-operative complications incidence, tumor recurrence rate, patients'' survival rate, and tumor-free survival rate were comparable between these two groups (all P>0.05). When compared to those patients with HCC beyond Milan criteria undergoing primary OLT, patients undergoing SLT achieved a better survival and a lower tumor recurrence. Cox Proportional Hazards model showed that vascular invasion, including macrovascular and microvascular invasion, as well as AFP level >400 IU/L were risk factors for tumor recurrence after LT.

Conclusions

In comparison with primary OLT, although SLT is associated with increased operation difficulties, it provides a good option for patients with HCC recurrence after curative resection.     

S1P1 Receptor Modulation Preserves Vascular Function in Mesenteric and Coronary Arteries after CPB in the Rat Independent of Depletion of Lymphocytes

Background

Cardiopulmonary bypass (CPB) may induce systemic inflammation and vascular dysfunction. Sphingosine 1-phosphate (S1P) modulates various vascular and immune responses. Here we explored whether agonists of the S1P receptors, FTY720 and SEW2871 improve vascular reactivity after CPB in the rat.

Methods

Experiments were done in male Wistar rats (total n = 127). Anesthesia was induced by isoflurane (2.5–3%) and maintained by fentanyl and midazolam during CPB. After catheterization of the left femoral artery, carotid artery and the right atrium, normothermic extracorporeal circulation was instituted for 60 minutes. In the first part of the study animals were euthanized after either 1 hour, 1 day, 2 or 5 days of the recovery period. In second part of the study animals were euthanized after 1 day of postoperative period. We evaluated the contractile response to phenylephrine (mesenteric arteries) or to serotonin (coronary artery) and vasodilatory response to acethylcholine (both arteries).

Results

Contractile responses to phenylephrine were reduced at 1 day recovery after CPB and Sham as compared to healthy control animals (Emax, mN: 7.9±1.9, 6.5±1.5, and 11.3±1.3, respectively). Mainly FTY720, but not SEW2871, caused lymphopenia in both Sham and CPB groups. In coronary and mesenteric arteries, both FTY720 and SEW2871 normalized serotonin and phenylephrine-mediated vascular reactivity after CPB (p<0.05) and FTY720 increased relaxation to acetylcholine as compared with untreated rats that underwent CPB.

Conclusion

Pretreatment with FTY720 or SEW2871 preserves vascular function in mesenteric and coronary artery after CPB. Therefore, pharmacological activation of S1P₁ receptors may provide a promising therapeutic intervention to prevent CPB-related vascular dysfunction in patients.     

Total intermittent Pringle maneuver during liver resection can induce intestinal epithelial cell damage and endotoxemia

Objectives

The intermittent Pringle maneuver (IPM) is frequently applied to minimize blood loss during liver transection. Clamping the hepatoduodenal ligament blocks the hepatic inflow, which leads to a non circulating (hepato)splanchnic outflow. Also, IPM blocks the mesenteric venous drainage (as well as the splenic drainage) with raising pressure in the microvascular network of the intestinal structures. It is unknown whether the IPM is harmful to the gut. The aim was to investigate intestinal epithelial cell damage reflected by circulating intestinal fatty acid binding protein levels (I-FABP) in patients undergoing liver resection with IPM.

Methods

Patients who underwent liver surgery received total IPM (total-IPM) or selective IPM (sel-IPM). A selective IPM was performed by selectively clamping the right portal pedicle. Patients without IPM served as controls (no-IPM). Arterial blood samples were taken immediately after incision, ischemia and reperfusion of the liver, transection, 8 hours after start of surgery and on the first post-operative day.

Results

24 patients (13 males) were included. 7 patients received cycles of 15 minutes and 5 patients received cycles of 30 minutes of hepatic inflow occlusion. 6 patients received cycles of 15 minutes selective hepatic occlusion and 6 patients underwent surgery without inflow occlusion. Application of total-IPM resulted in a significant increase in I-FABP 8 hours after start of surgery compared to baseline (p<0.005). In the no-IPM group and sel-IPM group no significant increase in I-FABP at any time point compared to baseline was observed.

Conclusion

Total-IPM in patients undergoing liver resection is associated with a substantial increase in arterial I-FABP, pointing to intestinal epithelial injury during liver surgery.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01099475","term_id":"NCT01099475"}}NCT01099475     

Non-invasive Imaging of Endothelial Progenitor Cells in Tumor Neovascularization Using a Novel Dual-modality Paramagnetic/Near-Infrared Fluorescence Probe

Objective

Bone-marrow derived endothelial progenitor cells (EPCs) play an important role in tumor neovasculature. Due to their tumor homing property, EPCs are regarded as promising targeted vectors for delivering therapeutic agents in cancer treatment. Consequently, non-invasive confirmation of targeted delivery via imaging is urgently needed. This study shows the development and application of a novel dual-modality probe for in vivo non-invasively tracking of the migration, homing and differentiation of EPCs.

Methods

The paramagnetic/near-infrared fluorescence probe Conjugate 1 labeled EPCs were systemically transplanted into mice bearing human breast MDA-MB-231 tumor xenografts. Magnetic resonance imaging (MRI) and near-infrared (NIR) fluorescence optical imaging were performed at different stages of tumor development. The homing of EPCs and the tumor neovascularization were further evaluated by immunofluorescence.

Results

Conjugate 1 labeled EPCs can be monitored in vivo by MRI and NIR fluorescence optical imaging without altering tumor growth for up to three weeks after the systemic transplantation. Histopathological examination confirmed that EPCs were recruited into the tumor bed and then incorporated into new vessels two weeks after the transplantation. Tumor size and microvessel density was not influenced by EPCs transplantation in the first three weeks.

Conclusions

This preclinical study shows the feasibility of using a MRI and NIR fluorescence optical imaging detectable probe to non-invasively monitor transplanted EPCs and also provides strong evidence that EPCs are involved in the development of endothelial cells during the tumor neovascularization.     

Interleukin-8 is activated in patients with chronic liver diseases and associated with hepatic macrophage accumulation in human liver fibrosis

Background

Interleukin-8 (IL-8, CXCL8) is a potent chemoattractant for neutrophils and contributes to acute liver inflammation. Much less is known about IL-8 in chronic liver diseases (CLD), but elevated levels were reported from alcoholic and hepatitis C-related CLD. We investigated the regulation of IL-8, its receptors CXCR1 and CXCR2 and possible IL-8 responding cells in CLD patients.

Methodology

Serum IL-8 levels were measured in CLD patients (n = 200) and healthy controls (n = 141). Intrahepatic IL-8, CXCR1 and CXCR2 gene expression was quantified from liver samples (n = 41), alongside immunohistochemical neutrophil (MPO) and macrophage (CD68) stainings. CXCR1 and CXCR2 expression was analyzed on purified monocytes from patients (n = 111) and controls (n = 31). In vitro analyses explored IL-8 secretion by different leukocyte subsets.

Principal Findings

IL-8 serum levels were significantly increased in CLD patients, especially in end-stage cirrhosis. Interestingly, patients with cholestatic diseases exhibited highest IL-8 serum concentrations. IL-8 correlated with liver function, inflammatory cytokines and non-invasive fibrosis markers. Intrahepatically, IL-8 and CXCR1 expression were strongly up-regulated. However, intrahepatic IL-8 could only be associated to neutrophil infiltration in patients with primary biliary cirrhosis (PBC). In non-cholestatic cirrhosis, increased IL-8 and CXCR1 levels were associated with hepatic macrophage accumulation. In line, CXCR1, but not CXCR2 or CXCR3, expression was increased on circulating monocytes from cirrhotic patients. Moreover, monocyte-derived macrophages from CLD patients, especially the non-classical CD16⁺ subtype, displayed enhanced IL-8 secretion in vitro.

Conclusions

IL-8 is strongly activated in CLD, thus likely contributing to hepatic inflammation. Our study suggests a novel role of IL-8 for recruitment and activation of hepatic macrophages via CXCR1 in human liver cirrhosis.     

Prophylactic activated recombinant factor VII in liver resection and liver transplantation: systematic review and meta-analysis

Background and Aim

Intraoperative blood loss is a frequent complication of hepatic resection and orthotopic liver transplantation. Recombinant activated coagulation factor VII (rFVIIa) is a coagulation protein that induces hemostasis by directly activating factor X. There is no clear information about the prophylactic value of rFVIIa in hepatobiliary surgery, specifically in liver resection and orthotopic liver transplantation. The aim of this study was to assess the effect of rFVIIa prophylaxis to prevent mortality and bleeding resulting from hepatobiliary surgery.

Methods

Relevant randomized trials were identified by searching The Cochrane Central Register of Controlled Trials in The Cochrane Library, MEDLINE, EMBASE, and Science Citation Index. Randomized clinical trials comparing different rFVIIa prophylactic schemas against placebo or no intervention to prevent bleeding in hepatobiliary surgery were included. Adults undergoing liver resection, partial hepatectomy, or orthotopic liver transplantation were included. Dichotomous data were analyzed calculating odds ratios (ORs) and 95% confidence intervals (CIs). Continuous data were analyzed calculating mean differences (MD) and 95% CIs.

Results

Four randomized controlled trials were included. There were no significant differences between rFVIIa and placebo for mortality (OR 0.96; 95% CI 0.35–2.62), red blood cell units (MD 0.32; 95% CI −0.08–0.72) or adverse events (OR 1.55; 95% CI 0.97–2.49).

Conclusions

The available information is limited, precluding the ability to draw conclusions regarding bleeding prophylaxis in hepatobiliary surgery using rFVIIa. Although an apparent lack of effect was observed in all outcomes studied, further research is needed.