Intestinal Intervention Strategy Targeting Myeloid Cells to Improve Hepatic Immunity during Hepatocarcinoma Development

Innate immunity in the tumor microenvironment plays a pivotal role in hepatocarcinoma (HCC) progression. Plant seeds provide serine-type protease inhibitors (SETIs), which can have a significant influence on liver inflammation and macrophage function. To elucidate the influence of SETIs to counter pro-tumorigenic conditions, at the early stages of HCC development, it was used as an established model of diethylnitrosamine/thioacetamide-injured liver fed with a standard diet (STD) or high-fat diet (42%) (HFD). The administration of SETIs improved survival and ameliorated tumor burden via modulation of monocyte-derived macrophages as key effectors involved in diet-induced HCC development. RT-qPCR analyses of hepatic tissue evidenced a diet-independent downregulatory effect of SETIs on the transcripts of CD36, FASN, ALOX15, and SREBP1c; however, animals fed with an STD showed opposing effects for PPAR and NRLP3 levels. These effects were accompanied by a decreased production of IL-6 and IL-17 but increased that of TNF in animals receiving SETIs. Moreover, only animals fed an HFD displayed increased concentrations of the stem cell factor. Overall, SETIs administration decreased the hepatic contents of lysophosphatydilcholine, phosphatidylinositol, phosphatidylcholine, and phosphatidyl ethanolamine. Notably, animals that received SETIs exhibited increased hepatic proportions of CD68+CX3CR1+CD74+ cells and at a higher rate in those animals fed an HFD. Altogether, the data evidence that oral administration of SETIs modulates the tumor microenvironment, improving hepatic innate immune response(s) and favoring a better antitumoral environment. It represents a path forward in developing coadjutant strategies to pharmacological therapies, with either a preventive or therapeutic character, to counter physiopathological conditions at early stages of HCC development.

Hepatic Ischemia-Reperfusion Impairs Blood-Brain Barrier Partly Due to Release of Arginase From Injured Liver

Aim: Hepatic ischemia-reperfusion (HIR) induces remote organs injury, including the brain. The homeostasis of the brain is maintained by the blood-brain barrier (BBB); thus, we aimed to investigate whether HIR impaired BBB and attempted to elucidate its underlying mechanism.Methods: Cell viability of human cerebral microvascular endothelial cells (hCMEC/D3) was measured following 24 h incubation with a serum of HIR rat undergoing 1 h ischemia and 4 h reperfusion, liver homogenate, or lysate of primary hepatocytes of the rat. The liver homogenate was precipitated using (NH4)2SO4 followed by separation on three columns and electrophoresis to identify the toxic molecule. Cell activity, apoptosis, proliferation, cell cycle, and expressions of proteins related to cell cycle were measured in hCMEC/D3 cells incubated with identified toxic molecules. HIR rats undergoing 1 h ischemia and 24 h reperfusion were developed to determine the release of an identified toxic molecule. BBB function was indexed as permeability to fluorescein and brain water. Endothelial cell proliferation and expressions of proteins related to the cell cycle in cerebral microvessels were measured by immunofluorescence and western blot.Results: Toxic molecule to BBB in the liver was identified to be arginase. Arginase inhibitor nor-NOHA efficiently attenuated hCMEC/D3 damage caused by liver homogenate and serum of HIR rats. Both arginase and serum of HIR rats significantly lowered arginine (Arg) in the culture medium. Arg addition efficiently attenuated the impairment of hCMEC/D3 caused by arginase or Arg deficiency, demonstrating that arginase impaired hCMEC/D3 via depriving Arg. Both arginase and Arg deficiency damaged hCMEC/D3 cells by inhibiting cell proliferation, retarding the cell cycle to G1 phase, and downregulating expressions of cyclin A, cyclin D, CDK2, and CDK4. HIR notably increased plasma arginase activity and lowered Arg level, increased the BBB permeability accompanied with enhanced brain water, and decreased the proliferative cells (marked by Ki67) in cerebral microvessels (marked by CD31) and protein expressions of cyclin A, cyclin D, CDK2 and CDK4 in isolated brain microvessels. Oral supplement of Arg remarkably attenuated these HIR-induced alterations.Conclusion: HIR leads to substantial release of arginase from the injured liver and then deprives systemic Arg. The Arg deficiency further impairs BBB via inhibiting the proliferation of brain microvascular endothelial cells by cell cycle arrest.

Role of circulating angiogenin levels in portal hypertension and TIPS

Background Pathogenesis of portal hypertension is multifactorial and includes pathologic intrahepatic angiogenesis, whereby TIPS insertion is an effective therapy of portal hypertension associated complications. While angiogenin is a potent contributor to angiogenesis in general, little is known about its impact on TIPS function over time. Methods In a total of 118 samples from 47 patients, angiogenin concentrations were measured in portal and inferior caval vein plasma at TIPS insertion (each blood compartment n = 23) or angiographic intervention after TIPS (each blood compartment n = 36) and its relationship with patient outcome was investigated. Results Angiogenin levels in the inferior caval vein were significantly higher compared to the portal vein (P = 0.048). Ten to 14 days after TIPS, inferior caval vein angiogenin level correlated inversely with the portal systemic pressure gradient (P<0.001), measured invasively during control angiography. Moreover, patients with TIPS revision during this angiography, showed significantly lower angiogenin level in the inferior caval vein compared to patients without TIPS dysfunction (P = 0.01). Conclusion In cirrhosis patients with complications of severe portal hypertension, circulating levels of angiogenin are derived from the injured liver. Moreover, angiogenin levels in the inferior caval vein after TIPS may predict TIPS dysfunction.

Therapeutic Effects of CXCL9-overexpressing Human Umbilical Cord Mesenchymal Stem Cells on Liver Fibrosis in Rats

Background: Umbilical cord mesenchymal stem cells (UC-MSCs) transplantation have become a promising treatment for liver fibrosis. However, UC-MSCs have limited anti-fibrosis ability, and their homing ability of UC-MSCs to the injured liver sites appears to be poor. In this study, we aimed to determining if overexpression of CXCL9 could have the synergistic anti-fibrosis effect with UC-MSCs, and whether it can promote the homing ability of UC-MSCs.Methods: Overexpression of CXCL9 in UC-MSCs (CXCL9-UC-MSCs) was attained by transfection of naive UC-MSCs with the lenti-CXCL9-mCherry. The impact of transplanted CXCL9-UC-MSCs on both repairing of liver fibrosis and homing was evaluated and compared with lenti-mCherry empty vector transfected UC-MSCs (control UC-MSCs).Results: After puromycin screening, UC-MSCs could stably express CXCL9 without affecting the stem and differentiation ability of UC-MSCs. In addition, biochemical analysis showed that the liver function of CXCL9-UC-MSCs was significantly increased in comparison with that of control UC-MSCs (P < 0.05). Futhermore, histopathology after 4 weeks of cell therapy demonstrated that the content of collagen fibers decreased obviously, the pseudo-lobules almost disappeared, and the morphology of hepatic lobules was basically normal. Frozen sections were performed 24 hours and 4 weeks after the cell injection. It can be seen that the fluorescence expression of the CXCL9-UC-MSCs group was significantly higher than that of the control UC-MSCs group, which proved that CXCL9-UC-MSCs have a stronger chemotactic ability, and can stay longer than control UC-MSCs in the injured liver.Conclusion: Overexpression of CXCL9 improves the efficacy of UC-MSC therapy for liver fibrosis repair, thereby promoting the homing and staying of UC-MSCs to injured hepatic sites in a rat model of liver fibrosis.

Liver Injury and the Macrophage Issue: Molecular and Mechanistic Facts and Their Clinical Relevance

The liver is an essential immunological organ due to its gatekeeper position to bypassing antigens from the intestinal blood flow and microbial products from the intestinal commensals. The tissue-resident liver macrophages, termed Kupffer cells, represent key phagocytes that closely interact with local parenchymal, interstitial and other immunological cells in the liver to maintain homeostasis and tolerance against harmless antigens. Upon liver injury, the pool of hepatic macrophages expands dramatically by infiltrating bone marrow-/monocyte-derived macrophages. The interplay of the injured microenvironment and altered macrophage pool skews the subsequent course of liver injuries. It may range from complete recovery to chronic inflammation, fibrosis, cirrhosis and eventually hepatocellular cancer. This review summarizes current knowledge on the classification and role of hepatic macrophages in the healthy and injured liver.

Snake extract–laden hemostatic bioadhesive gel cross-linked by visible light

Bioadhesives reduce operation time and surgical complications. However, in the presence of blood, adhesion strength is often compromised. Inspired by the blood clotting activity of snake venom, we report a visible light–induced blood-resistant hemostatic adhesive (HAD) containing gelatin methacryloyl and reptilase, which is a hemocoagulase (HC) extracted from Bothrops atrox. HAD leads to the activation and aggregation of platelets and efficiently transforms fibrinogen into fibrin to achieve rapid hemostasis and seal the tissue. Blood clotting time with HAD was about 45 s compared with 5 to 6 min without HAD. HAD instantaneously achieved hemostasis on liver incision (~45 s) and cut rat tail (~34 s) and reduced blood loss by 79 and 78%, respectively. HAD is also efficient in sealing severely injured liver and abdominal aorta. HAD has great potential to bridge injured tissues by combing hemostasis with adhesives.

Discovery of chemerin as the new chemoattractant of human mesenchymal stem cells

Background The homing capacity of human mesenchymal stem cells (hMSCs) to the injured sites enables systemic administration of hMSCs in clinical practice. In reality, only a small proportion of MSCs are detected in the target tissue, which is a major bottleneck for MSC-based therapies. We still don’t know the mechanism how MSCs are chemo-attracted to certain target organ and engrafted through trans-endothelial migration. In this study, we aimed to determine the mechanism how the circulating hMSCs home to the injured liver. Methods and results When we compare the cytokine array between normal and injured mouse liver at 1-day thioacetamide (TAA)-treatment, we found that chemerin, CXCL2, and CXCL10 were higher in the injured liver than normal one. Among three, only chemerin was the chemoattractant of hMSCs in 2D- and 3D-migration assay. Analysis of the signal transduction pathways in hMSCs showed that chemerin activated the phosphorylation of JNK1/2, ERK1/2 and p38, and finally upregulated CD44, ITGA4, and MMP-2 that are involved in the transendothelial migration and extravasation of MSCs. Upstream transcription regulators of CD44, ITGA4, and MMP-2 after chemerin treatment were MZF1, GATA3, STAT3, and STAT5A. To develop chemerin as a chemoattractant tool, we cloned gene encoding the active chemerin under the CMV promoter (CMV-aChemerin). We analyzed the migration of hMSCs in the 3D model for space of the Disse, which mimics transmigration of hMSCs in the liver. CMV-aChemerin-transfected hepatocytes were more effective to attract hMSC than control hepatocytes, leading to the enhanced transendothelial migration and homing of hMSCs to liver. The homing efficiency of the intravascularly-delivered hMSCs to liver was evaluated after systemic introduction of the CMV-aChemerin plasmid packed in liposome-vitamin A conjugates which target liver. CMV-aChemerin plasmid targeting liver significantly enhanced homing efficiency of hMSCs to liver compared with control plasmid vector. Conclusions Chemerin is the newly found chemoattractant of hMSCs and may be a useful tool to manipulate the homing of the intravascularly-administered hMSC to the specific target organ.