The Role of Resolvins, Protectins and Marensins in Non-Alcoholic Fatty Liver Disease (NAFLD).

Increased triacylglycerols' (TAG) synthesis, insulin resistance, and prolonged liver lipid storage might lead to the development of non-alcoholic fatty liver disease (NAFLD). Global prevalence of NAFLD has been estimated to be around 25%, with gradual elevation of this ratio along with the increased content of adipose tissue in a body. The initial stages of NAFLD may be reversible, but the exposition to pathological factors should be limited. As dietary factors greatly influence various disease development, scientists try to find dietary components, helping to alleviate the steatosis. These components include n-3 polyunsaturated (PUFA) fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acids (DHA). This review focused on the role of resolvins, protectins and merensins in NAFLD.

New Insights on Complement Inhibitor CD59 in Mouse Laser-Induced Choroidal Neovascularization: Mislocalization After Injury and Targeted Delivery for Protein Replacement.

PURPOSE: The membrane attack complex (MAC) in choriocapillaris (CC) and retinal pigment epithelium (RPE) increase with age and disease (age-related macular degeneration). MAC assembly can be inhibited by CD59, a membrane-bound regulator. Here we further investigated the role of CD59 in murine choroidal neovascularization (CNV), a model involving both CC and RPE, and tested whether CR2-CD59, a soluble targeted form of CD59, provides protection. METHODS: Laser-induced CNV was generated in wild type and CD59a-deficient mice (CD59-/-). CNV size was measured by optical coherence tomography, and CR2-CD59 was injected intraperitoneally. Endogenous CD59 localization and MAC deposition were identified by immunohistochemistry and quantified by confocal microscopy. Cell-type-specific responses to MAC were examined in retinal pigment epithelial cells (ARPE-19) and microvascular endothelial cells (HMEC-1). RESULTS: CD59 levels were severely reduced and protein was mislocalized in the RPE surrounding the lesion. CNV lesion size and subretinal fluid accumulation were exacerbated in CD59-/- when compared with those in WT mice, and an increase in MAC deposition was noted. In contrast, CR2-CD59 significantly reduced both structural features of CNV severity. In vitro, MAC inhibition in ARPE-19 cells prevented barrier function loss and accelerated wound healing and cell adhesion, whereas in HMEC-1 cells, CR2-CD59 decelerated wound healing and cell adhesion. CONCLUSION: These data further support the importance of CD59 in controlling ocular injury responses and indicate that pharmacological inhibition of the MAC with CR2-CD59 may be a viable therapeutic approach for reducing complement-mediated ocular pathology.

Bioactive products formed in humans from fish oils.

Resolvins, maresins, and protectins can be formed from fish oils. These specialized pro-resolving mediators (SPMs) have been implicated in the resolution of inflammation. Synthetic versions of such SPMs exert anti-inflammatory effects in vitro and when administered to animal models. However, their importance as endogenous products formed in sufficient amounts to exert anti-inflammatory actions in vivo remains speculative. We biased our ability to detect SPMs formed in healthy volunteers by supplementing fish oil in doses shown previously to influence blood pressure and platelet aggregation under placebo-controlled conditions. Additionally, we sought to determine the relative formation of SPMs during an acute inflammatory response and its resolution, evoked in healthy volunteers by bacterial lipopolysaccharide (LPS). Bioactive lipids, enzymatic epoxyeicosatrienoic acids (EETs), and free radical-catalyzed prostanoids [isoprostanes (iPs)] formed from arachidonic acid and the fish oils, served as comparators. Despite the clear shift from ω-6 to ω-3 EETs and iPs, we failed to detect a consistent signal, in most cases, of SPM formation in urine or plasma in response to fish oil, and in all cases in response to LPS on a background of fish oil. Our results question the relevance of these SPMs to the putative anti-inflammatory effects of fish oils in humans.

Dissecting the complement pathway in hepatic injury and regeneration with a novel protective strategy.

Liver resection is commonly performed under ischemic conditions, resulting in two types of insult to the remnant liver: ischemia reperfusion injury (IRI) and loss of liver mass. Complement inhibition is recognized as a potential therapeutic modality for IRI, but early complement activation products are also essential for liver regeneration. We describe a novel site-targeted murine complement inhibitor, CR2-CD59, which specifically inhibits the terminal membrane attack complex (MAC), and we use this protein to investigate the complement-dependent balance between liver injury and regeneration in a clinical setting of pharmacological inhibition. CR2-CD59 did not impact in vivo generation of C3 and C5 activation products but was as effective as the C3 activation inhibitor CR2-Crry at ameliorating hepatic IRI, indicating that the MAC is the principle mediator of hepatic IRI. Furthermore, unlike C3 or C5 inhibition, CR2-CD59 was not only protective but significantly enhanced hepatocyte proliferation after partial hepatectomy, including when combined with ischemia and reperfusion. Remarkably, CR2-CD59 also enhanced regeneration after 90% hepatectomy and improved long-term survival from 0 to 70%. CR2-CD59 functioned by increasing hepatic TNF and IL-6 levels with associated STAT3 and Akt activation, and by preventing mitochondrial depolarization and allowing recovery of ATP stores.

Evaluation of adenovirus-delivered human CD59 as a potential therapy for AMD in a model of human membrane attack complex formation on murine RPE.

PURPOSE: Complement-mediated damage to the retinal pigment epithelium (RPE), Bruch membrane, and choroid has been associated with pathogenesis in age-related macular degeneration (AMD). The terminal step of complement activation involves lysis of cells by the insertion of the membrane attack complex (MAC) in the plasma membrane. The hypothesis that local overexpression of human CD59 (hCD59) delivered by an adenovirus (Ad) vector to primary murine RPE cells in vitro, RPE in vivo, or cornea ex vivo protects those cells from human MAC deposition and lysis was tested. METHODS: A humanized model of MAC deposition on murine cells and murine ocular tissues including RPE and cornea was developed to permit testing of human complement regulators in mice. A recombinant adenovirus-expressing hCD59 was generated, and this virus was injected into the subretinal space of adult mice. Subsequently, eyecups from these mice were exposed to human serum, and the levels of MAC deposition on the RPE were quantified. hCD59 was also expressed on murine cornea ex vivo and in murine hepatocytes, and primary RPE cells in vitro and levels of human MAC deposition and cell lysis were measured. RESULTS: Adenovirus-mediated delivery of hCD59 to the RPE, cornea, or cells in culture protects those cells from human MAC deposition and MAC-mediated damage and vesiculation. CONCLUSIONS: The humanized model of MAC deposition on murine ocular tissues allows testing of human complement regulators that may have potential in the treatment of AMD or other diseases associated with complement activation.

Recent developments in the understanding and management of paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinuria (PNH) has been recognised as a discrete disease entity since 1882. Approximately a half of patients will eventually die as a result of having PNH. Many of the symptoms of PNH, including recurrent abdominal pain, dysphagia, severe lethargy and erectile dysfunction, result from intravascular haemolysis with absorption of nitric oxide by free haemoglobin from the plasma. These symptoms, as well as the occurrence of thrombosis and aplasia, significantly affect patients' quality of life; thrombosis is the leading cause of premature mortality. The syndrome of haemolytic-anaemia-associated pulmonary hypertension has been further identified in PNH patients. There is currently an air of excitement surrounding therapies for PNH as recent therapeutic developments, particularly the use of the complement inhibitor eculizumab, promise to radically alter the symptomatology and natural history of haemolytic PNH.

Complement inhibitors targeted to the proximal tubule prevent injury in experimental nephrotic syndrome and demonstrate a key role for C5b-9.

In glomerular diseases of diverse etiologies, dysfunction of the glomerular barrier to protein passage results in proteinuria, and proteinuria is considered an independent risk factor that plays a direct role in inflammation, interstitial fibrosis, and renal failure. The mechanism by which proteinuria leads to nephrotoxic injury is unclear, but a role for complement in mediating interstitial damage appears likely. We describe a strategy for Ag-specific targeting of complement inhibitors using a single chain Ab fragment and show that complement inhibitors targeted to the tubular epithelium protect against tubulointerstitial injury and renal dysfunction in a rat model of puromycin-induced nephrosis. The targeting of systemically administered complement inhibitors markedly enhanced their efficacy and obviated the need to systemically inhibit complement, thus reducing the risk of compromising host defense and immune homeostasis. Targeted inhibition of complement activation by Crry, and of membrane attack complex (MAC) formation by CD59 was equally therapeutic, demonstrating that the MAC plays a key role in proteinuria-induced tubulointerstitial injury. CD59 activity was dependent on its being targeted to the site of complement activation, and this is the first report of specific inhibition of the MAC in vivo after systemic administration of inhibitor. The data establish the MAC is a valid target for pharmaceutical intervention in proteinuric disorders and provide an approach to investigate the role of the MAC in complement-dependent disease under clinically relevant conditions.

Correction of the PNH defect by GPI-anchored protein transfer.

Hemolytic anemia is a major feature of paroxysmal nocturnal hemoglobinuria (PNH). Intravascular red blood cell (RBC) destruction is caused by increased sensitivity of the abnormal erythrocyte to complement-mediated lysis, due to the GPI absence of a membrane-bound glycosylphosphatidylinositol (GPI)-linked protein, which functions as an inhibitor of reactive lysis (CD59). Both in vivo and in vitro models have suggested the feasibility of cell-to-cell transfer of GPI proteins, and patients with hemolysis could potentially benefit from transfer of CD59 to their deficient erythrocytes. We studied the ability of RBC components prepared from outdated packed RBC collections, as well as high-density lipoprotein (HDL) preparations, rich in CD55 and CD59, to promote protein transfer, as assessed by flow cytometry, immunoblotting, and susceptibility to complement-mediated lysis. By flow cytometry, CD55 and CD59 were present on RBC-derived microvesicles that stained with an antiglycophorin antibody Ab; in addition, soluble CD59 and CD55 were detected by immunoblot in soluble fractions eluated from RBC units stored for more than 35 days, but not in fresh blood. Both commercial HDL preparations and those prepared in our laboratory contained CD55 and CD59, as assayed by immunoblot. When RBC that were deficient (GPI)-anchored protein, obtained from five patients, with PNH were incubated with HDL preparations for 2 to 4 hours, there was significant transfer of both proteins to the cell surface, as demonstrated by flow cytometry. Washed RBC microvesicles, prepared by ultrasonification, also mediated transfer of GPI-linked proteins to deficient RBC. Pretreatment of microvesicles, RBC eluate preparations, and HDL with phosphatidylinositol-specific, phospholipase C, abrogated protein transfer to deficient cells, indicating that increased cell-associated CD55 and CD59 levels were related to insertion of the intact GPI moiety, rather than to simple adhesion. PNH RBC that were exposed to HDL, RBC eluate preparations, or microvesicles demonstrated decreased in vitro complement-mediated hemolysis in the Ham test. Transfer of GPI-linked proteins from soluble preparations containing CD55 and CD59 to PNH erythrocytes is feasible and may have clinical utility.