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.National Institutes of Health (U.S.) (Grant R01 GM 49039

Single-Walled Carbon Nanohorns as Promising Nanotube-Derived Delivery Systems to Treat Cancer

Cancer has become one of the most prevalent diseases worldwide, with increasing incidence in recent years. Current pharmacological strategies are not tissue-specific therapies, which hampers their efficacy and results in toxicity in healthy organs. Carbon-based nanomaterials have emerged as promising nanoplatforms for the development of targeted delivery systems to treat diseased cells. Single-walled carbon nanohorns (SWCNH) are graphene-based horn-shaped nanostructure aggregates with a multitude of versatile features to be considered as suitable nanosystems for targeted drug delivery. They can be easily synthetized and functionalized to acquire the desired physicochemical characteristics, and no toxicological effects have been reported in vivo followed by their administration. This review focuses on the use of SWCNH as drug delivery systems for cancer therapy. Their main applications include their capacity to act as anticancer agents, their use as drug delivery systems for chemotherapeutics, photothermal and photodynamic therapy, gene therapy, and immunosensing. The structure, synthesis, and covalent and non-covalent functionalization of these nanoparticles is also discussed. Although SWCNH are in early preclinical research yet, these nanotube-derived nanostructures demonstrate an interesting versatility pointing them out as promising forthcoming drug delivery systems to target and treat cancer cells

Nanoparticles to Target and Treat Macrophages:The Ockham's Concept?

Nanoparticles are nanomaterials with three external nanoscale dimensions and an average size ranging from 1 to 1000 nm. Nanoparticles have gained notoriety in technological advances due to their tunable physical, chemical, and biological characteristics. However, the administration of functionalized nanoparticles to living beings is still challenging due to the rapid detection and blood and tissue clearance by the mononuclear phagocytic system. The major exponent of this system is the macrophage. Regardless the nanomaterial composition, macrophages can detect and incorporate foreign bodies by phagocytosis. Therefore, the simplest explanation is that any injected nanoparticle will be probably taken up by macrophages. This explains, in part, the natural accumulation of most nanoparticles in the spleen, lymph nodes, and liver (the main organs of the mononuclear phagocytic system). For this reason, recent investigations are devoted to design nanoparticles for specific macrophage targeting in diseased tissues. The aim of this review is to describe current strategies for the design of nanoparticles to target macrophages and to modulate their immunological function involved in different diseases with special emphasis on chronic inflammation, tissue regeneration, and cancer

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

Biotinylated polyurethane-urea nanoparticles for targeted theranostics in human hepatocellular carcinoma

Over the past years, significant efforts have been devoted to explore novel drug delivery and detection strategies for simultaneous therapy and diagnostics. The development of biotinylated polyurethane-urea nanoparticles as theranostic nanocarriers for targeted drug and plasmid delivery, for fluorescence detection of human hepatocellular carcinoma cells, is described herein. These targeted nanoparticles are specifically designed to incorporate biotin into the polymeric matrix, since many tumor types overexpress receptors for biotin as a mechanism to boost uncontrolled cell growth. The obtained nanoparticles were spherical, exhibited an average diameter ranging 110–145 nm, and showed no cytotoxicity in healthy endothelial cells. Biotinylated nanoparticles are selectively incorporated into the perinuclear and nuclear area of the human hepatocellular carcinoma cell line, HepG2, in division, but not into growing, healthy, human endothelial cells. Indeed, the simultaneous incorporation of the anticancer drugs, phenoxodiol or sunitinib, together with plasmid DNA encoding green fluorescent protein, into these nanoparticles allows a targeted pharmacological antitumor effect and furthermore, selective transfection of a reporter gene, to detect these cancer cells. The combined targeted therapy and detection strategy described here could be exploited for liver cancer therapy and diagnostics, with a moderate safety profile, and may also be a potential tool for other types of cancer.Spain. Ministerio de Educación y CienciaDGI (Spain) (CTQ 2011-29336-C03/PPQ)Catalonia (Spain). Departament d'Universitats, Recerca i Societat de la Informació (DURSI) (Grant 2009 SGR-961)CIBER-BB

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

Treatment with chondroitin sulfate to modulate inflammation and atherogenesis in obesity

Background and aims: Osteoarthritic patients treated with high doses of chondroitin sulfate (CS) have a lower incidence of coronary heart disease - but the mechanistic aspects of these beneficial effects of CS remain undefined. We examined how CS treatment affects the formation of atheroma via interaction with endothelial cells and monocytes. Methods: We characterized arterial atheromatous plaques by multiphoton microscopy and serum pro-inflammatory cytokines by immunoenzymatic techniques in obese mice receiving CS (1 g/kg/day, i.p.) or vehicle for 6 days. Effects of CS on signaling pathways, cytokine secretion and macrophage migration were evaluated in cultures of human coronary endothelial cells and in a monocyte cell line stimulated with TNF-α by Western blot, immunoenzymatic techniques and transwell migration assays. Results: Treatment of obese mice with CS reduced the extension of foam cell coverage in atheromatous plaques of arterial bifurcations by 62.5%, the serum concentration of IL1β by 70%, TNF-α by 82% and selected chemokines by 25-35%. Cultures of coronary endothelial cells and monocytes stimulated with TNF-α secreted less pro-inflammatory cytokines in the presence of CS (P < 0.01). CS reduced the activation of the TNF-α signaling pathway in endothelial cells (pErk 36% of reduction, and NFκB 33% of reduction), and the migration of activated monocytes to inflamed endothelial cells in transwells (81 ± 6 vs. 13 ± 2, P < 0.001). Conclusions: CS interferes with the pro-inflammatory activation of monocytes and endothelial cells driven by TNF-α thus reducing the propagation of inflammation and preventing the formation of atherosclerotic plaques

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.S

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

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.We acknowledge the support from grants PDI2019-105502RB-100 and PID2022-138243OB-I00 to MM-R, PID2019-111669RB, BES-2017-080823 to IP "ESF Investing in your future", and RTI2018-094734-B-C21 to WJ and PM-L, from MCIN/AEI/10.13039/501100011033. RedFibro (RED2022-134485-T) of the 2022 caLL for aid to «RESEARCH NETWORKS», within the framework of the Programa Estatal del Plan Estatal de Investigación Científica, Técnica y de Innovación 2021–2023. Consolidated Research Group of the Generalitat de Catalunya AGAUR (2021 SGR 00881 to MM-R) and PM-L financed ESF Investing in your future, support from AGAUR of the Generalitat de Catalunya. Fundación Mutua Madrileña AP171442019, European Cooperation in Science & Technology (COST) ACTION CA17112 Prospective European Drug-Induced Liver Injury Network, and the Project 201 916/31 Contribution of mitochondrialoxysterol and bile acid metabolism to liver carcinogenesis 2019 by Fundació Marató TV3. PM-L was additionally supported by a fellowship from the Ramon y Cajal Program (RYC2018-0Z23971-I) and a grant (PID2021-123426OB-I00) from the Spanish Ministerio de Ciencia, Innovación y Universidades. CIBERehd is financed by the Instituto de Salud Carlos III.Peer reviewe

Fisiopatología del edema y de la formación de ascitis en la cirrosis hepática experimental

[spa] DE LA TESIS: INTRODUCCIÓN: La formación de ascitis es el trastorno más importante que presentan los pacientes cirróticos. Dicho proceso constituye un fenómeno extraordinariamente complejo en el que se ha implicado a diversos sistemas neurohormonales y sustancias endógenas capaces de regular el metabolismo renal de sodio y agua, o la reactividad vascular. Actualmente, se considera que la disfunción renal y el desarrollo de ascitis es el resultado de la vasodilatación arterial en el territorio esplácnico, promovida por la hipertensión portal. Esto da lugar a dos secuencias de acontecimientos bien diferenciados. En primer lugar, la vasodilatación esplácnica aumenta el aflujo sanguíneo en este territorio produciendo un incremento de la presión capilar y de la permeabilidad vascular, así como una mayor formación de linfa. En segundo lugar, la disminución de la presión arterial, como respuesta homeostática compensatoria, activa los sistemas vasoconstrictores endógenos. Estos sistemas, sin embargo, tienen propiedades antidiuréticas y antinatriuréticas, por lo que la activación de los mismos también produce retención renal de sodio y agua. La concurrencia simultánea de estas dos perturbaciones estimula el acumulo de líquido intraperitoneal. Sin embargo, todavía se desconocen los territorios vasculares implicados en la formación de ascitis ni los mecanismos moleculares que regulan la fisiopatología de ese desorden. OBJETIVOS: La presente tesis doctoral tuvo como objetivos investigar cuáles son las áreas vasculares que presentan una permeabilidad vascular alterada en las ratas cirróticas con ascitis y, por otra parte, analizar el impacto de vías de señalización proangiogéncias en los procesos que conducen al aumento de la permeabilidad vascular que origina la ascitis en la cirrosis experimental. RESULTADOS: El hígado y el mesenterio son los tejidos que presentan una permeabilidad vascular aumentada en las ratas cirróticas con ascitis. Los factores vascular endothelial growth factor A (VEGF-A) y la angiopoyetina-2 se encuentran sobreexpresados en el hígado y el mesenterio de las ratas cirróticas con ascitis. El bloqueo agudo del receptor 2 de VEGF reduce drásticamente la permeabilidad vascular en el hígado y el mesenterio de las ratas cirróticas con ascitis. Por otra parte, la apelina, un péptido proangiogénico de reciente descubrimiento, se encuentra aumentada en el suero de los pacientes y las ratas con cirrosis. La expresión de dicho péptido y de su receptor se encuentra hiperactivada específicamente en el hígado de las ratas cirróticas con ascitis. Más concretamente, la apelina se sobreexpresa en las células estrelladas hepáticas (CEH), y su receptor, en dichas células y en el parénquima del hígado cirrótico. El bloqueo crónico del receptor de la apelina reduce la angiogénesis y la fibrosis hepática, mejora la hemodinámica sistémica y la función renal en las ratas cirróticas con ascitis. Esos efectos beneficiosos en la hemodinámica y la angiogénesis hepática se vieron asociados con la disminución drástica de la producción de ascitis o incluso la desaparación total de la misma. Para averiguar los agentes que regulan la expresión hepática de este péptido se utilizó una línea de CEH (LX-2). La angiotensina-II y la endotelina-1 (dos factores profibrogénicos) son los principales agentes que estimulan la expresión de apelina en las células LX-2. Precisamente, la apelina actúa de mediador en la estimulación de colágeno-I y de receptor beta de PDGF que realizan angiotensina-II y endotelina-1 en las CEHs. CONCLUSIÓN: El intenso proceso de angiogénesis hepática y mesentérica contribuye de manera significativa al desarrollo de la ascitis en la cirrosis hepática. Por tanto, estos datos apoyan la hipótesis de que el bloqueo de dianas terapéuticas implicadas en angiogénesis puede constituir un avance en el tratamiento de las alteraciones de la permeabilidad vascular que dan lugar a la formación de ascitis en esta enfermedad