Identification of Dhx15 as a Major Regulator of Liver Development, Regeneration, and Tumor Growth in Zebrafish and Mice.

RNA helicase DHX15 plays a significant role in vasculature development and lung metastasis in vertebrates. In addition, several studies have demonstrated the overexpression of DHX15 in the context of hepatocellular carcinoma. Therefore, we hypothesized that this helicase may play a significant role in liver regeneration, physiology, and pathology. Dhx15 gene deficiency was generated by CRISPR/Cas9 in zebrafish and by TALEN-RNA in mice. AUM Antisense-Oligonucleotides were used to silence Dhx15 in wild-type mice. The hepatocellular carcinoma tumor induction model was generated by subcutaneous injection of Hepa 1-6 cells. Homozygous Dhx15 gene deficiency was lethal in zebrafish and mouse embryos. Dhx15 gene deficiency impaired liver organogenesis in zebrafish embryos and liver regeneration after partial hepatectomy in mice. Also, heterozygous mice presented decreased number and size of liver metastasis after Hepa 1-6 cells injection compared to wild-type mice. Dhx15 gene silencing with AUM Antisense-Oligonucleotides in wild-type mice resulted in 80% reduced expression in the liver and a significant reduction in other major organs. In addition, Dhx15 gene silencing significantly hindered primary tumor growth in the hepatocellular carcinoma experimental model. Regarding the potential use of DHX15 as a diagnostic marker for liver disease, patients with hepatocellular carcinoma showed increased levels of DHX15 in blood samples compared with subjects without hepatic affectation. In conclusion, Dhx15 is a key regulator of liver physiology and organogenesis, is increased in the blood of cirrhotic and hepatocellular carcinoma patients, and plays a key role in controlling hepatocellular carcinoma tumor growth and expansion in experimental models.

Tcf20 deficiency is associated with increased liver fibrogenesis and alterations in mitochondrial metabolism in mice and humans.

BACKGROUND & AIMS: Transcription co-activator factor 20 (TCF20) is a regulator of transcription factors involved in extracellular matrix remodelling. In addition, TCF20 genomic variants in humans have been associated with impaired intellectual disability. Therefore, we hypothesized that TCF20 has several functions beyond those described in neurogenesis, including the regulation of fibrogenesis. METHODS: Tcf20 knock-out (Tcf20-/- ) and Tcf20 heterozygous mice were generated by homologous recombination. TCF20 gene genotyping and expression was assessed in patients with pathogenic variants in the TCF20 gene. Neural development was investigated by immufluorescense. Mitochondrial metabolic activity was evaluated with the Seahorse analyser. The proteome analysis was carried out by gas chromatography mass-spectrometry. RESULTS: Characterization of Tcf20-/- newborn mice showed impaired neural development and death after birth. In contrast, heterozygous mice were viable but showed higher CCl4 -induced liver fibrosis and a differential expression of genes involved in extracellular matrix homeostasis compared to wild-type mice, along with abnormal behavioural patterns compatible with autism-like phenotypes. Tcf20-/- embryonic livers and mouse embryonic fibroblast (MEF) cells revealed differential expression of structural proteins involved in the mitochondrial oxidative phosphorylation chain, increased rates of mitochondrial metabolic activity and alterations in metabolites of the citric acid cycle. These results parallel to those found in patients with TCF20 pathogenic variants, including alterations of the fibrosis scores (ELF and APRI) and the elevation of succinate concentration in plasma. CONCLUSIONS: We demonstrated a new role of Tcf20 in fibrogenesis and mitochondria metabolism in mice and showed the association of TCF20 deficiency with fibrosis and metabolic biomarkers in humans.

The pituitary tumour-transforming gene 1/delta-like homologue 1 pathway plays a key role in liver fibrogenesis.

BACKGROUND AND AIMS: PTTG1 is almost undetectable in adult livers but is highly expressed in hepatocarcinoma. While little is known about its involvement in liver fibrosis, PTTG1 expression is associated with DLK1. We assessed the role of the PTTG1/DLK1 pathway in fibrosis progression and the potential therapeutic effect of PTTG1 silencing in fibrosis. METHODS: Pttg1 and Dlk1 were studied in liver and isolated cell populations of control and fibrotic rats and in human liver biopsies. The fibrotic molecular signature was analysed in Pttg1-/- and Pttg1+/+ fibrotic mice. Finally, Pttg1 silencing was evaluated in rats as a novel antifibrotic therapy. RESULTS: Pttg1 and Dlk1 mRNA selectively increased in fibrotic rats paralleling fibrosis progression. Serum DLK1 concentrations correlated with hepatic collagen content and systemic and portal haemodynamics. Human cirrhotic livers showed greater PTTG1 and DLK1 transcript abundance than non-cirrhotic, and reduced collagen was observed in Pttg1 Pttg1-/- mice. The liver fibrotic molecular signature revealed lower expression of genes related to extracellular matrix remodelling including Mmp8 and 9 and Timp4 and greater eotaxin and Mmp13 than fibrotic Pttg1+/+ mice. Finally, interfering Pttg1 resulted in reduced liver fibrotic area, lower α-Sma and decreased portal pressure than fibrotic animals. Furthermore, Pttg1 silencing decreased the transcription of Dlk1, collagens I and III, Pdgfrß, Tgfrß, Timp1, Timp2 and Mmp2. CONCLUSIONS: Pttg1/Dlk1 are selectively overexpressed in the cirrhotic liver and participate in ECM turnover regulation. Pttg1 disruption decreases Dlk1 transcription and attenuates collagen deposition. PTTG1/DLK1 signalling is a novel pathway for targeting the progression of liver fibrosis.

Graphene-Dendrimer Nanostars for Targeted Macrophage Overexpression of Metalloproteinase 9 and Hepatic Fibrosis Precision Therapy.

Fibrosis contributes to ∼45% of all deaths in industrialized nations, but no direct antifibrotic therapeutic interventions exist to date. Graphene-based nanomaterials exhibit excellent versatility in electronics, and emerging trends exploit their properties for biomedical applications, especially for drug and gene delivery. We designed constructs of graphene nanostars linked to PAMAM-G5 dendrimer for the selective targeting and delivery of a plasmid expressing the collagenase metalloproteinase 9 under the CD11b promoter into inflammatory macrophages in cirrhotic livers. Graphene nanostars preferentially accumulated in inflammatory macrophages M1 in less than 3 h in a manner unaffected by covalent linkage to dendrimers. Dendrimer-graphene nanostars efficiently delivered the plasmid encoding for metalloproteinase 9 into macrophages, allowing the synthesis and secretion of the metalloproteinase to digest adjacent collagen fibers. In turn, metalloproteinase 9 overexpression promoted the macrophage switch from inflammatory M1 to pro-regenerative M2 in 3 days. This targeted gene therapy reduced selectively and locally the presence of collagen fibers in fibrotic tracts where inflammatory macrophages accumulated in cirrhotic mice without affecting the activation state of hepatic stellate cells. Overall, this treatment significantly reduced hepatic injury and improved liver restoration in mice with liver cirrhosis treated for 10 days. Graphene-dendrimer nanostars targeted the macrophage overexpression of metalloproteinase 9, selectively reducing hepatic fibrosis, and might be a good treatment for diseases associated with fibrosis and inflammatory macrophage accumulation.

Cerium Oxide Nanoparticles Protect against Oxidant Injury and Interfere with Oxidative Mediated Kinase Signaling in Human-Derived Hepatocytes.

Cerium oxide nanoparticles (CeO2NPs) possess powerful antioxidant properties, thus emerging as a potential therapeutic tool in non-alcoholic fatty liver disease (NAFLD) progression, which is characterized by a high presence of reactive oxygen species (ROS). The aim of this study was to elucidate whether CeO2NPs can prevent or attenuate oxidant injury in the hepatic human cell line HepG2 and to investigate the mechanisms involved in this phenomenon. The effect of CeO2NPs on cell viability and ROS scavenging was determined, the differential expression of pro-inflammatory and oxidative stress-related genes was analyzed, and a proteomic analysis was performed to assess the impact of CeO2NPs on cell phosphorylation in human hepatic cells under oxidative stress conditions. CeO2NPs did not modify HepG2 cell viability in basal conditions but reduced H2O2- and lipopolysaccharide (LPS)-induced cell death and prevented H2O2-induced overexpression of MPO, PTGS1 and iNOS. Phosphoproteomic analysis showed that CeO2NPs reverted the H2O2-mediated increase in the phosphorylation of peptides related to cellular proliferation, stress response, and gene transcription regulation, and interfered with H2O2 effects on mTOR, MAPK/ERK, CK2A1 and PKACA signaling pathways. In conclusion, CeO2NPs protect HepG2 cells from cell-induced oxidative damage, reducing ROS generation and inflammatory gene expression as well as regulation of kinase-driven cell survival pathways.

Optimizing Glutaraldehyde-Fixed Tissue Heart Valves with Chondroitin Sulfate Hydrogel for Endothelialization and Shielding against Deterioration.

Porcine glutaraldehyde-fixed pericardium is widely used to replace human heart valves. Despite the stabilizing effects of glutaraldehyde fixation, the lack of endothelialization and the occurrence of immune reactions contribute to calcification and structural valve deterioration, which is particularly significant in young patients, in whom valve longevity is crucial. This report shows an optimization system with which to enhance endothelialization of fixed pericardium to mimic the biological function of a native heart valve. The glutaraldehyde detoxification, together with the application of a biodegradable methacrylated chondroitin sulfate hydrogel, reduces aldehydes cytotoxicity, increases the migration and proliferation of endothelial cells and the recruitment of endothelial cell progenitors, and confers thromboresistance in fixed pericardium. The combination of glutaraldehyde detoxification and a coating with chondroitin sulfate hydrogel promotes in situ mechanisms of endothelialization in fixed pericardium. We offer a new solution for improving the long life of bioprosthetic valves and exploring the means of making valves suitable to endothelialization.

Implantation of healthy matrix-embedded endothelial cells rescues dysfunctional endothelium and ischaemic tissue in liver engraftment.

OBJECTIVE: Liver transplantation is limited by ischaemic injury which promotes endothelial cell and hepatocyte dysfunction and eventually organ failure. We sought to understand how endothelial state determines liver recovery after hepatectomy and engraftment. DESIGN: Matrix-embedded endothelial cells (MEECs) with retained healthy phenotype or control acellular matrices were implanted in direct contact with the remaining median lobe of donor mice undergoing partial hepatectomy (70%), or in the interface between the remaining median lobe and an autograft or isograft from the left lobe in hepatectomised recipient mice. Hepatic vascular architecture, DNA fragmentation and apoptosis in the median lobe and grafts, serum markers of liver damage and phenotype of macrophage and lymphocyte subsets in the liver after engraftment were analysed 7 days post-op. RESULTS: Healthy MEECs create a functional vascular splice in donor and recipient liver after 70% hepatectomy in mouse protecting these livers from ischaemic injury, hepatic congestion and inflammation. Macrophages recruited adjacent to the vascular nodes into the implants switched to an anti-inflammatory and regenerative profile M2. MEECs improved liver function and the rate of liver regeneration and prevented apoptosis in donor liver lobes, autologous grafts and syngeneic engraftment. CONCLUSIONS: Implants with healthy endothelial cells rescue liver donor and recipient endothelium and parenchyma from ischaemic injury after major hepatectomy and engraftment. This study highlights endothelial-hepatocyte crosstalk in hepatic repair and provides a promising new approach to improve regenerative medicine outcomes and liver transplantation.

A small population of liver endothelial cells undergoes endothelial-to-mesenchymal transition in response to chronic liver injury.

Rising evidence points to endothelial-to-mesenchymal transition (EndMT) as a significant source of the mesenchymal cell population in fibrotic diseases. In this context, we hypothesized that liver endothelial cells undergo EndMT during fibrosis progression. Cirrhosis in mice was induced by CCl4 A transgenic mouse expressing a red fluorescent protein reporter under the control of Tie2 promoter (Tie2-tdTomato) was used to trace the acquisition of EndMT. Sinusoidal vascular connectivity was evaluated by intravital microscopy and high-resolution three-dimensional confocal microscopy. A modest but significant fraction of liver endothelial cells from both cirrhotic patients and CCl4-treated Tie2-tdTomato mice acquired an EndMT phenotype characterized by the coexpression of CD31 and α-smooth muscle actin, compared with noncirrhotic livers. Bone morphogenetic protein-7 (BMP-7) inhibited the acquisition of EndMT induced by transforming growth factor-ß1 (TGF-ß1) treatment in cultured primary mouse liver endothelial cells from control mice. EndMT was also reduced significantly in vivo in cirrhotic Tie2-tdTomato mice treated intraperitoneally with BMP-7 compared with untreated mice (1.9 ± 0.2 vs. 3.8 ± 0.3%, respectively; P < 0.05). The decrease of EndMT in cirrhotic livers correlated with a significant decrease in liver fibrosis (P < 0.05) and an improvement in the vascular disorganization rate (P < 0.05). We demonstrated the acquisition of the EndMT phenotype by a subpopulation of endothelial cells from cirrhotic livers in both animal models and patients. BMP-7 treatment decreases the occurrence of the EndMT phenotype and has a positive impact on the severity of disease by reducing fibrosis and sinusoidal vascular disorganization.NEW & NOTEWORTHY A subpopulation of liver endothelial cells from cirrhotic patients and mice with liver fibrosis undergoes endothelial-to-mesenchymal transition. Liver endothelial cells from healthy mice could transition into a mesenchymal phenotype in culture in response to TGF-ß1 treatment. Fibrotic livers treated chronically with BMP-7 showed lower EndMT acquisition, reduced fibrosis, and improved vascular organization.

Monocyte-endothelial cell interactions in the regulation of vascular sprouting and liver regeneration in mouse.

BACKGROUND & AIMS: Regeneration of the hepatic mass is crucial to liver repair. Proliferation of hepatic parenchyma is intimately dependent on angiogenesis and resident macrophage-derived cytokines. However the role of circulating monocyte interactions in vascular and hepatic regeneration is not well-defined. We investigated the role of these interactions in regeneration in the presence and absence of intact monocyte adhesion. METHODS: Partial hepatectomy was performed in wild-type mice and those lacking the monocyte adhesion molecule CD11b. Vascular architecture, angiogenesis and macrophage location were analyzed in the whole livers using simultaneous angiography and macrophage staining with fluorescent multiphoton microscopy. Monocyte adhesion molecule expression and sprouting-related pathways were evaluated. RESULTS: Resident macrophages (Kupffer cells) did not migrate to interact with vessels whereas infiltrating monocytes were found adjacent to sprouting points. Infiltrated monocytes colocalized with Wnt5a, angiopoietin 1 and Notch-1 in contact points and commensurate with phosphorylation and disruption of VE-cadherin. Mice deficient in CD11b showed a severe reduction in angiogenesis, liver mass regeneration and survival following partial hepatectomy, and developed unstable and leaky vessels that eventually produced an aberrant hepatic vascular network and Kupffer cell distribution. CONCLUSIONS: Direct vascular interactions of infiltrating monocytes are required for an ordered vascular growth and liver regeneration. These outcomes provide insight into hepatic repair and new strategies for hepatic regeneration.

Overexpression of angiopoietin-2 in rats and patients with liver fibrosis. Therapeutic consequences of its inhibition.

BACKGROUND & AIMS: Studies in experimental models of cirrhosis showed that anti-angiogenic treatments may be effective for the treatment of liver fibrosis. In this context, angiopoietins are potential therapeutic targets as they are involved in the maintenance and stabilization of newly formed blood vessels. In addition, angiopoietin-2 is expressed in fibrotic livers and its inhibition in tumours results in vessel stability. Therefore, our study was aimed to assess the therapeutic utility of inhibiting angiopoietin-2. METHODS: Circulating levels of angiopoietin-1 and angiopoietin-2 were quantified by ELISA in CCl4 -treated rats and in patients with cirrhosis. In vivo blockade of angiopoietin-2 in rats with liver fibrosis was performed with a chemically programmed antibody, CVX-060. RESULTS: High levels of angiopoietin-2 were found in the systemic and suprahepatic circulation of cirrhotic patients and the ratio angiopoietin-1/angiopoietin-2 inversely correlated with prognostic models for alcoholic liver disease. Chronic treatment of CCl4 -treated rats with CVX-060 was associated with a significant decrease in inflammatory infiltrate, normalization of the hepatic microvasculature and reduction in VCAM-1 vascular expression. The anti-angiopoietin-2 treatment was also associated with less liver fibrosis and with lower levels of circulating transaminases. CVX-060 treatment was not associated with either vascular pruning in healthy tissue or compensatory overexpression of VEGF. CONCLUSIONS: Inhibition of angiopoietin-2 is an effective and safe treatment for liver fibrosis in CCl4 -treated rats, acting mainly through the induction of vessel normalization and the attenuation of hepatic inflammatory infiltrate. Therefore, inhibition of angiopoietin-2 offers a therapeutic alternative for liver fibrosis.

Increased nitric oxide production in lymphatic endothelial cells causes impairment of lymphatic drainage in cirrhotic rats.

BACKGROUND AND AIM: The lymphatic network plays a major role in maintaining tissue fluid homoeostasis. Therefore several pathological conditions associated with oedema formation result in deficient lymphatic function. However, the role of the lymphatic system in the pathogenesis of ascites and oedema formation in cirrhosis has not been fully clarified. The aim of this study was to investigate whether the inability of the lymphatic system to drain tissue exudate contributes to the oedema observed in cirrhosis. METHODS: Cirrhosis was induced in rats by CCl(4) inhalation. Lymphatic drainage was evaluated using fluorescent lymphangiography. Expression of endothelial nitric oxide synthase (eNOS) was measured in primary lymphatic endothelial cells (LyECs). Inhibition of eNOS activity in cirrhotic rats with ascites (CH) was carried out by L-N(G)-methyl-L-arginine (L-NMMA) treatment (0.5 mg/kg/day). RESULTS: The (CH) rats had impaired lymphatic drainage in the splanchnic and peripheral regions compared with the control (CT) rats. LyECs isolated from the CH rats showed a significant increase in eNOS and nitric oxide (NO) production. In addition, the lymphatic vessels of the CH rats showed a significant reduction in smooth muscle cell (SMC) coverage compared with the CT rats. CH rats treated with L-NMMA for 7 days showed a significant improvement in lymphatic drainage and a significant reduction in ascites volume, which were associated with increased plasma volume. This beneficial effect of L-NMMA inhibition was also associated with a significant increase in lymphatic SMC coverage. CONCLUSIONS: The upregulation of eNOS in the LyECs of CH rats causes long-term lymphatic remodelling, which is characterised by a loss of SMC lymphatic coverage. The amelioration of this lymphatic abnormality by chronic eNOS inhibition results in improved lymphatic drainage and reduced ascites.

Liver-targeted nanoparticles delivering nitric oxide reduce portal hypertension in cirrhotic rats.

Nitric oxide (NO) is a small vasodilator playing a key role in the pathogenesis of portal hypertension. Here, we assessed the potential therapeutic effect of a NO donor targeted to the liver by poly(beta-amino ester) nanoparticles (pBAE NPs) in experimental cirrhosis. Retinol-functionalized NO donor pBAE NPs (Ret pBAE NPs) were synthetized with the aim of actively targeting the liver. Administration of Ret pBAE NPs resulted in uptake and transfection by the liver and spleen. NPs were not found in other organs or the systemic circulation. Treatment with NO donor Ret pBAE NPs (30 mg/ kg body weight) significantly decreased aspartate aminotransferase, lactate dehydrogenase and portal pressure (9.75 ± 0.64 mmHg) compared to control NPs (13.4 ± 0.53 mmHg) in cirrhotic rats. There were no effects on mean arterial pressure and cardiac output. Liver-targeted NO donor NPs reduced collagen fibers and steatosis, activation of hepatic stellate cells and mRNA expression of profibrogenic and proinflammatory genes. Finally, Ret pBAE NPs displayed efficient transfection in human liver slices. Overall, liver-specific NO donor NPs effectively target the liver and mitigated inflammation and portal hypertension in cirrhotic rats. The use of Ret pBAE may prove to be an effective therapeutic strategy to treat advanced liver disease.

Factors involved in extracellular matrix turnover in human derived cardiomyocytes.

BACKGROUND: The molecular mechanisms by which myocardial ischemia translates into ventricular remodeling remain unclear. METHODS: We investigated whether hypoxia and proinflammatory cytokines are specific inducers of remodeling signals in an in vitro model of cultured adult human ventricular myocytes (AC16 cells). RESULTS: Hypoxia modified the ratio of matrix remodeling factors by increasing the aminoterminal propeptide of type III procollagen (PIIINP) and reducing tissue inhibitor of matrix metalloproteinase type 1 (TIMP-1) secretion in AC16 cells. These effects, however, were not associated with either modifications in expression of matrix metalloproteinase type 2, collagen-I or metalloproteinase activity. Hypoxia does, actually increase the production of the cardiac antifibrogenic growth factors, Apelin and VEGF, through an Hypoxia Inducible Factor type 1-dependent mechanism. Concerning proinflammatory signaling pathways, IL1ß emerged as a powerful inducer of matrix turnover, since it significantly enhanced PIIINP, TIMP-1 and hyaluronic acid production and increased metalloproteinase activity. In contrast, TNFα did not modify matrix turnover but markedly induced the production of Apelin and VEGF. CONCLUSION: Hypoxia and increased TNFα activity likely exert cardioprotective actions by activating the cardiac antifibrogenic factors Apelin and VEGF. In contrast, IL1ß is a strong promoter of interstitial collagen remodeling that may contribute to ventricular dilation and heart failure in the ischemic myocardium.

Monocyte-endothelial cell interactions in vascular and tissue remodeling.

Monocytes are circulating leukocytes of innate immunity derived from the bone marrow that interact with endothelial cells under physiological or pathophysiological conditions to orchestrate inflammation, angiogenesis, or tissue remodeling. Monocytes are attracted by chemokines and specific receptors to precise areas in vessels or tissues and transdifferentiate into macrophages with tissue damage or infection. Adherent monocytes and infiltrated monocyte-derived macrophages locally release a myriad of cytokines, vasoactive agents, matrix metalloproteinases, and growth factors to induce vascular and tissue remodeling or for propagation of inflammatory responses. Infiltrated macrophages cooperate with tissue-resident macrophages during all the phases of tissue injury, repair, and regeneration. Substances released by infiltrated and resident macrophages serve not only to coordinate vessel and tissue growth but cellular interactions as well by attracting more circulating monocytes (e.g. MCP-1) and stimulating nearby endothelial cells (e.g. TNF-α) to expose monocyte adhesion molecules. Prolonged tissue accumulation and activation of infiltrated monocytes may result in alterations in extracellular matrix turnover, tissue functions, and vascular leakage. In this review, we highlight the link between interactions of infiltrating monocytes and endothelial cells to regulate vascular and tissue remodeling with a special focus on how these interactions contribute to pathophysiological conditions such as cardiovascular and chronic liver diseases.

PPAR-γ Agonist GW1929 Targeted to Macrophages with Dendrimer-Graphene Nanostars Reduces Liver Fibrosis and Inflammation.

Macrophages play essential roles during the progression of chronic liver disease. They actively participate in the response to liver damage and in the balance between fibrogenesis and regression. The activation of the PPARγ nuclear receptor in macrophages has traditionally been associated with an anti-inflammatory phenotype. However, there are no PPARγ agonists with high selectivity for macrophages, and the use of full agonists is generally discouraged due to severe side effects. We designed dendrimer-graphene nanostars linked to a low dose of the GW1929 PPARγ agonist (DGNS-GW) for the selective activation of PPARγ in macrophages in fibrotic livers. DGNS-GW preferentially accumulated in inflammatory macrophages in vitro and attenuated macrophage pro-inflammatory phenotype. The treatment with DGNS-GW in fibrotic mice efficiently activated liver PPARγ signaling and promoted a macrophage switch from pro-inflammatory M1 to anti-inflammatory M2 phenotype. The reduction of hepatic inflammation was associated with a significant reduction in hepatic fibrosis but did not alter liver function or hepatic stellate cell activation. The therapeutic antifibrotic utility of DGNS-GW was attributed to an increased expression of hepatic metalloproteinases that allowed extracellular matrix remodeling. In conclusion, the selective activation of PPARγ in hepatic macrophages with DGNS-GW significantly reduced hepatic inflammation and stimulated extracellular matrix remodeling in experimental liver fibrosis.

RNF41 orchestrates macrophage-driven fibrosis resolution and hepatic regeneration.

Hepatic inflammation is a common trigger of chronic liver disease. Macrophage activation is a predictive parameter for survival in patients with cirrhosis. Ring finger protein 41 (RNF41) negatively regulates proinflammatory cytokines and receptors; however, the precise involvement of macrophage RNF41 in liver cirrhosis remains unknown. Here, we sought to understand how RNF41 dictates macrophage fate in hepatic fibrosis and repair within the inflammatory milieu. We found that RNF41 expression is down-regulated in CD11b+ macrophages recruited to mouse fibrotic liver and to patient cirrhotic liver regardless of cirrhosis etiology. Prolonged inflammation with TNF-α progressively reduced macrophage RNF41 expression. We designed a macrophage-selective gene therapy with dendrimer-graphite nanoparticles (DGNPs) to explore the influence of macrophage RNF41 restoration and depletion in liver fibrosis and regeneration. RNF41 expression induced in CD11b+ macrophages by DGNP-conjugated plasmids ameliorated liver fibrosis, reduced liver injury, and stimulated hepatic regeneration in fibrotic mice with or without hepatectomy. This therapeutic effect was mainly mediated by the induction of insulin-like growth factor 1. Conversely, depletion of macrophage RNF41 worsened inflammation, fibrosis, hepatic damage, and survival. Our data reveal implications of macrophage RNF41 in the control of hepatic inflammation, fibrosis, and regeneration and provide a rationale for therapeutic strategies in chronic liver disease and potentially other diseases characterized by inflammation and fibrosis.

Optimization of 3D autologous chondrocyte-seeded polyglycolic acid scaffolds to mimic human ear cartilage.

Auricular reconstruction in children with microtia is one of the more complex procedures in plastic surgery. Obtaining sufficient native material to build an ear requires harvesting large fragments of rib cartilage in children. Herein, we investigated how to optimize autologous chondrocyte isolation, expansion and re-implantation using polyglycolic acid (PGA) scaffolds for generating enough cartilage to recapitulate a whole ear starting from a small ear biopsy. Ear chondrocytes isolated from human microtia subjects grew slower than microtia rib or healthy ear chondrocytes and displayed a phenotypic shift due to the passage number. Rabbit ear chondrocytes co-cultured with mesenchymal stem cells (MSC) at a 50 : 50 ratio recapitulated the cartilage biological properties in vitro. However, PGA scaffolds with different proportions of rabbit chondrocytes and MSC did not grow substantially in two months when subcutaneously implanted in immunosuppressed mice. In contrast, rabbit chondrocyte-seeded PGA scaffolds implanted in immunocompetent rabbits formed a cartilage 10 times larger than the original PGA scaffold. This cartilage mimicked the biofunctional and mechanical properties of an ear cartilage. These results indicate that autologous chondrocyte-seeded PGA scaffolds fabricated following our optimized procedure have immense potential as a solution for obtaining enough cartilage for auricular reconstruction and opens new avenues to redefine autologous cartilage replacement.

Atherosclerotic plaque development in mice is enhanced by myeloid ZEB1 downregulation.

Accumulation of lipid-laden macrophages within the arterial neointima is a critical step in atherosclerotic plaque formation. Here, we show that reduced levels of the cellular plasticity factor ZEB1 in macrophages increase atherosclerotic plaque formation and the chance of cardiovascular events. Compared to control counterparts (Zeb1WT/ApoeKO), male mice with Zeb1 ablation in their myeloid cells (Zeb1∆M/ApoeKO) have larger atherosclerotic plaques and higher lipid accumulation in their macrophages due to delayed lipid traffic and deficient cholesterol efflux. Zeb1∆M/ApoeKO mice display more pronounced systemic metabolic alterations than Zeb1WT/ApoeKO mice, with higher serum levels of low-density lipoproteins and inflammatory cytokines and larger ectopic fat deposits. Higher lipid accumulation in Zeb1∆M macrophages is reverted by the exogenous expression of Zeb1 through macrophage-targeted nanoparticles. In vivo administration of these nanoparticles reduces atherosclerotic plaque formation in Zeb1∆M/ApoeKO mice. Finally, low ZEB1 expression in human endarterectomies is associated with plaque rupture and cardiovascular events. These results set ZEB1 in macrophages as a potential target in the treatment of atherosclerosis.

Prevention of fibrosis progression in CCl4-treated rats: role of the hepatic endocannabinoid and apelin systems.

Endocannabinoids behave as antifibrogenic agents by interacting with cannabinoid CB2 receptors, whereas the apelin (AP) system acts as a proangiogenic and profibrogenic mediator in the liver. This study assessed the effect of long-term stimulation of CB2 receptors or AP receptor (APJ) blockade on fibrosis progression in rats under a non-discontinued fibrosis induction program. The study was performed in control and CCl(4)-treated rats for 13 weeks. Fibrosis-induced rats received a CB2 receptor agonist (R,S)-3-(2-iodo-5-nitrobenzoyl)-1-(1-methyl-2-piperidinylmethyl)-1H-indole (AM1241) (1 mg/kg b.wt.), an APJ antagonist [Ala(13)]-apelin-13 sequence: Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Ala (F13A) (75 µg/kg b.wt.), or vehicle daily during the last 5 weeks of the CCl(4) inhalation program. Mean arterial pressure (MAP), portal pressure (PP), hepatic collagen content, angiogenesis, cell infiltrate, and mRNA expression of a panel of fibrosis-related genes were measured in all animals. Fibrosis-induced rats showed increased hepatic collagen content, reduced MAP, portal hypertension, and increased expression of the assessed messengers in comparison with control rats. However, fibrotic rats treated with either AM1241 or F13A had reduced hepatic collagen content, improved MAP and PP, ameliorated cell viability, and reduced angiogenesis and cell infiltrate compared with untreated fibrotic rats. These results were associated with attenuated induction of platelet-derived growth factor receptor ß, α-smooth muscle actin, matrix metalloproteinases, and tissue inhibitors of matrix metalloproteinase. CB2 receptor stimulation or APJ blockade prevents fibrosis progression in CCl(4)-treated rats. The mechanisms underlying these phenomena are coincident despite the marked dissimilarities between the CB2 and APJ signaling pathways, thus opening new avenues for preventing fibrosis progression in liver diseases.

Hypoxia and proinflammatory factors upregulate apelin receptor expression in human stellate cells and hepatocytes.

BACKGROUND: The activation of the apelin receptor (APJ) plays a major role in both angiogenic and fibrogenic response to chronic liver injury. However, the mechanisms that govern the induction of APJ expression have not been clarified so far. METHODS: The regulation and the role of APJ in cultured human liver cells were investigated. Tissular expression of APJ and α-smooth muscle actin was analysed by immunocolocalisation in human cirrhotic liver and in control samples. mRNA and protein expression of APJ were analysed in two cell lines, LX-2 (as hepatic stellate cells, HSCs) and HepG2 (as hepatocytes), under hypoxic conditions or after exposure to proinflammatory or profibrogenic factors. Additionally, both hepatic cell lines were stimulated with apelin to assess cell survival and the expression of angiogenic factors. RESULTS: The APJ-positive signal was negligible in control livers. In contrast, APJ was highly expressed in HSCs and slightly expressed in hepatocytes of human cirrhotic liver. Sustained hypoxia and lipopolysaccharide stimulated the expression of APJ in LX-2 cells. Moreover, hypoxia, tumour necrosis factor α and angiotensin II induced the expression of APJ in HepG2 cells. Activation of APJ stimulated angiopoietin-1 expression and cell survival in LX-2 cells and, in turn, triggered the synthesis of vascular endothelial growth factor type A and platelet-derived growth factor-BB in HepG2 cells. CONCLUSIONS: These results suggest that hypoxia and inflammatory factors could play a major role in the activation of the hepatic apelin system leading to angiogenic and fibroproliferative response occurring in chronic liver disease.