Age-Related Maculopathy Susceptibility 2 and Complement Factor H Polymorphism and Intraocular Complement Activation in Neovascular Age-Related Macular Degeneration.

Purpose: To investigate the association of risk alleles in complement factor H (CFH) and age-related maculopathy susceptibility 2 (ARMS2) with complement activation products in the aqueous humor in eyes with neovascular age-related macular degeneration (nAMD) including polypoidal choroidal vasculopathy (PCV), retinal angiomatous proliferation (RAP), and pachychoroid neovasculopathy (PNV). Design: Prospective, comparative, observational study. Participants: Treatment-naïve patients with nAMD and cataract patients as controls. Methods: The study included 236 eyes of 236 patients with nAMD and 49 control eyes of 49 patients. Aqueous humor samples were collected from 67 eyes with drusen-associated nAMD, 72 eyes with PCV, 26 eyes with RAP, and 71 eyes with PNV before intravitreal anti-VEGF injection and cataract surgery in the 49 control eyes. Clinical samples were measured for complement component 3a (C3a), C4a, and C5a using a bead-based immunoassay. Genotyping of the ARMS2 A69S (rs10490924), CFH I62V (rs800292), and CFH Y402H (rs1061170) was performed using TaqMan genotyping. Main Outcome Measures: The levels of complement activation products (C3a, C4a, and C5a) in the aqueous humor in each genotype of ARMS2 and CFH. Results: The C3a level in the aqueous humor was significantly elevated (P = 0.006) in patients with nAMD and the ARMS2 A69S risk allele, whereas the levels of the complement activation products were not associated with CFH I62V and Y402H genotypes. Among the control eyes, no significant differences were seen in any complement activation products for all genetic polymorphisms. The levels of the complement activation products in the aqueous humor of eyes with the nAMD subtypes for each genetic polymorphism did not show significant differences. Conclusions: The C3a concentration in the aqueous humor was significantly higher in Japanese nAMD patients with the ARMS2 A69S risk allele, whereas it was not elevated in the patients with CFH I62V. Age-related maculopathy susceptibility 2 A69S polymorphism is strongly associated with local complement activation in nAMD patients.

The complex formation of MASP-3 with pattern recognition molecules of the lectin complement pathway retains MASP-3 in the circulation.

The complement system plays an important role in host defense and is activated via three different activation pathways. We have previously reported that mannose-binding lectin-associated serine protease (MASP)-3, unlike its splicing variant MASP-1, circulates in an active form and is essential for the activation of the alternative pathway (AP) via the activation of complement factor D (FD). On the other hand, like MASP-1 and MASP-2 of the lectin pathway (LP), MASP-3 forms a complex with the pattern recognition molecules (PRMs) of the LP (LP-PRMs). Both MASP-1 and MASP-2 can be activated efficiently when the LP-PRMs complexed with them bind to their ligands. On the other hand, it remains unclear how MASP-3 is activated, or whether complex formation of MASP-3 with LP-PRMs is involved in activation of MASP-3 or its efficiency in the circulation. To address these issues, we generated wild-type (WT) and four mutant recombinant mouse MASP-3 proteins fused with PA (human podoplanin dodecapeptide)-tag (rmMASP-3-PAs), the latter of which have single amino acid substitution for alanine in the CUB1 or CUB2 domain responsible for binding to LP-PRMs. The mutant rmMASP-3-PAs showed significantly reduced in-vivo complex formation with LP-PRMs when compared with WT rmMASP-3-PA. In the in-vivo kinetic analysis of MASP-3 activation, both WT and mutant rmMASP-3-PAs were cleaved into the active forms as early as 30 minutes in the circulation of mice, and no significant difference in the efficiency of MASP-3 cleavage was observed throughout an observation period of 48 hours after intravenous administration. All sera collected 3 hours after administration of each rmMASP-3-PA showed full restoration of the active FD and AP activity in MASP-3-deficient mouse sera at the same levels as WT mouse sera. Unexpectedly, all mutant rmMASP-3-PAs showed faster clearance from the circulation than the WT rmMASP-3-PA. To our knowledge, the current study is the first to show in-vivo kinetics of MASP-3 demonstrating rapid activation and clearance in the circulation. In conclusion, our results demonstrated that the complex formation of MASP-3 with LP-PRMs is not required for in-vivo activation of MASP-3 or its efficiency, but may contribute to the long-term retention of MASP-3 in the circulation.

Enhanced Delivery of Thermoresponsive Polymer-Based Medicine into Tumors by Using Heat Produced from Gold Nanorods Irradiated with Near-Infrared Light.

The aim of this study was to establish a drug delivery system (DDS) for marked therapy of tumors using a thermoresponsive polymer, polyoxazoline (POZ). The effectiveness of the following was investigated: (i) the delivery of gold nanorods (GNRs) to tumor tissues, (ii) heat production of GNR upon irradiation with near-infrared (NIR) light, and (iii) high accumulation of an intravenously injected radiolabeled POZ as a drug carrier in tumors by sensing heat produced by GNRs. When the GNR solution was irradiated with NIR light (808 nm), the solution temperature was increased both in a GNR-concentration-dependent manner and in a light-dose-dependent manner. POZ, with a lower critical solution temperature of 38 °C, was aggregated depending on the heat produced by the GNR irradiated by NIR light. When it was intratumorally pre-injected into colon26-tumor-bearing mice, followed by NIR light irradiation (GNR+/Light+ group), the tumor surface temperature increased to approximately 42 °C within 5 min. Fifteen minutes after irradiation with NIR light, indium-111 (111In)-labeled POZ was intravenously injected into tumor-bearing mice, and the radioactivity distribution was evaluated. The accumulation of POZ in the tumor was significantly (approximately 4-fold) higher than that in the control groups (GNR+/without NIR light irradiation (Light-), without injection of GNR (GNR-)/Light+, and GNR-/Light- groups). Furthermore, an in vivo confocal fluorescence microscopy study, using fluorescence-labeled POZ, revealed that uptake of POZ by the tumor could be attributed to the heat produced by GNR. In conclusion, we successfully established a novel DDS in which POZ could be efficiently delivered into tumors by using the heat produced by GNR irradiated with NIR light.

Changes in complement activation products after anti-VEGF injection for choroidal neovascularization in age-related macular degeneration and pachychoroid disease.

We evaluated changes in the complement system resulting from anti-vascular endothelial growth factor (VEGF) in eyes with age-related choroidal neovascularization (CNV) including neovascular age-related macular degeneration, pachychoroid neovasculopathy, and polypoidal choroidal neovasculopathy. We measured the concentrations of the complement activation products (C3a, C4a), VEGF, and monocyte chemotactic protein-1 in the aqueous humor during intravitreal anti-VEGF injections for CNV. The VEGF level decreased significantly (P < 0.001), while the C3a and C4a levels increased significantly (P < 0.001 for both comparisons) 1 month after two monthly anti-VEGF injections. The VEGF level was correlated with the C3a (R = 0.328, P = 0.007) and C4a (R = - 0.237, P = 0.055) levels at baseline, but the correlation between the VEGF and C3a levels (R = - 0.148, P = 0.242) changed significantly (P = 0.028 by analysis of covariance) after anti-VEGF treatment. The C3a increase after anti-VEGF therapy did not change the visual outcomes in eyes with CNV for 1 year. Dysregulation of the complement system can be induced after anti-VEGF therapy.

A novel complement inhibitor sMAP-FH targeting both the lectin and alternative complement pathways.

Inhibition of the complement activation has emerged as an option for treatment of a range of diseases. Activation of the lectin and alternative pathways (LP and AP, respectively) contribute to the deterioration of conditions in certain diseases such as ischemia-reperfusion injuries and age-related macular degeneration (AMD). In the current study, we generated dual complement inhibitors of the pathways MAp44-FH and sMAP-FH by fusing full-length MAp44 or small mannose-binding lectin-associated protein (sMAP), LP regulators, with the N-terminal five short consensus repeat (SCR) domains of complement factor H (SCR1/5-FH), an AP regulator. The murine forms of both fusion proteins formed a complex with endogenous mannose-binding lectin (MBL) or ficolin A in the circulation when administered in mice intraperitoneally. Multiple complement activation assays revealed that sMAP-FH had significantly higher inhibitory effects on activation of the LP and AP in vivo as well as in vitro compared to MAp44-FH. Human form of sMAP-FH also showed dual inhibitory effects on LP and AP activation in human sera. Our results indicate that the novel fusion protein sMAP-FH inhibits both the LP and AP activation in mice and in human sera, and could be an effective therapeutic agent for diseases in which both the LP and AP activation are significantly involved.

Evidence for Activation of Lectin and Classical Pathway Complement Components in Aqueous Humor of Neovascular Age-Related Macular Degeneration.

PURPOSE: The complement system is activated via 3 different pathways; the lectin pathway (LP), classical pathway (CP), and alternative pathway. To investigate the possible roles for the LP or CP in the development of neovascular age-related macular degeneration (nAMD), we compared aqueous humor levels of complement proteins of the LP and CP between eyes with nAMD and those with cataract as controls. METHODS: Seventeen eyes from 17 patients with treatment-naïve nAMD and 9 eyes from 9 patients with cataract were studied. Aqueous humor samples were collected before intravitreal aflibercept or ranibizumab injection for the nAMD patients and before cataract surgery for the cataract patients. Aqueous humor levels of complement C4 of the LP and CP, complement C3 of all 3 complement pathways, and mannose-binding lectin-associated serine protease (MASP)-2 of the LP were measured by enzyme-linked immunosorbent assay. Aqueous humor levels of C4a and C3a, the activation products of C4 and C3, respectively, were measured by a bead-based immunoassay. The ratios of C4a to C4 and C3a to C3, representing the degree of C4 and C3 activation, respectively, were calculated in individual patients. RESULTS: The aqueous humor levels of C4, C3, and MASP-2 were significantly lower in the nAMD eyes compared to the controls (p = 0.008, p = 0.011, and p = 0.018, respectively). In contrast, the aqueous humor levels of C4a and C3a, as well as the C4a/C4 and C3a/C3 ratios, were significantly higher in the nAMD eyes compared to the controls (p = 0.039, p = 0.003, p < 0.001, and p < 0.001, respectively). CONCLUSIONS: This study provides evidence for significant intraocular activation of either or both of the LP and CP in nAMD eyes that might be involved in the development of nAMD. The significantly lower levels of MASP-2 in the aqueous humor of the nAMD eyes were likely due to MASP-2 consumption by activation of the LP.

Cutting Edge: Role of MASP-3 in the Physiological Activation of Factor D of the Alternative Complement Pathway.

The complement system, a part of the innate immune system, can be activated via three different pathways. In the alternative pathway, a factor D (FD) plays essential roles in both the initiation and the amplification loop and circulates as an active form. Mannose-binding lectin-associated serine proteases (MASPs) are key enzymes of the lectin pathway, and MASP-1 and/or MASP-3 are reported to be involved in the activation of FD. In the current study, we generated mice monospecifically deficient for MASP-1 or MASP-3 and found that the sera of the MASP-1-deficient mice lacked lectin pathway activity, but those of the MASP-3-deficient mice lacked alternative pathway activity with a zymogen FD. Furthermore, the results indicate that MASP-3 but not MASP-1 activates the zymogen FD under physiological conditions and MASP-3 circulates predominantly as an active form. Therefore, our study illustrates that, in mice, MASP-3 orchestrates the overall complement reaction through the activation of FD.

Essential Roles for Mannose-Binding Lectin-Associated Serine Protease-1/3 in the Development of Lupus-Like Glomerulonephritis in MRL/lpr Mice.

The complement system, composed of the three activation pathways, has both protective and pathogenic roles in the development of systemic lupus erythematosus (or lupus), a prototypic autoimmune disease. The classical pathway contributes to the clearance of immune complexes (ICs) and apoptotic cells, whereas the alternative pathway (AP) exacerbates renal inflammation. The role of the lectin pathway (LP) in lupus has remained largely unknown. Mannose-binding lectin (MBL)-associated serine proteases (MASPs), which are associated with humoral pattern recognition molecules (MBL or ficolins), are the enzymatic constituents of the LP and AP. MASP-1 encoded by the Masp1 gene significantly contributes to the activation of the LP. After the binding of MBL/ficolins to pathogens or self-altered cells, MASP-1 autoactivates first, then activates MASP-2, and both participate in the formation of the LP C3 convertase C4b2a, whereas, MASP-3, the splice variant of the Masp1 gene, is required for the activation of the zymogen of factor D (FD), and finally participates in the formation of the AP C3 convertase C3bBb. To investigate the roles of MASP-1 and MASP-3 in lupus, we generated Masp1 gene knockout lupus-prone MRL/lpr mice (Masp1/3-/- MRL/lpr mice), lacking both MASP-1 and MASP-3, and analyzed their renal disease. As expected, sera from Masp1/3-/- MRL/lpr mice had no or markedly reduced activation of the LP and AP with zymogen forms of complement FD. Compared to their wild-type littermates, the Masp1/3-/- MRL/lpr mice had maintained serum C3 levels, little-to-no albuminuria, as well as significantly reduced glomerular C3 deposition levels and glomerular pathological score. On the other hand, there were no significant differences in the levels of serum anti-dsDNA antibody, circulating ICs, glomerular IgG and MBL/ficolins deposition, renal interstitial pathological score, urea nitrogen, and mortality between the wild-type and Masp1/3-/- MRL/lpr mice. Our data indicate that MASP-1/3 plays essential roles in the development of lupus-like glomerulonephritis in MRL/lpr mice, most likely via activation of the LP and/or AP.

Genotoxicity-suppressing effect of aqueous extract of Connarus ruber cortex on cigarette smoke-induced micronuclei in mouse peripheral erythrocytes.

INTRODUCTION: According to published information, it has not been determined whether the inhalation of cigarette smoke can induce chromosome aberrations and/or point mutations in mice, though cigarette smoke is clearly carcinogenic to mice. We tested clastogenicity of inhaled cigarette smoke in mouse by a micronucleus test using peripheral erythrocytes. Since it is important to determine the in vivo anti-genotoxic effect against inhaled cigarette smoke to reduce the risk of tobacco carcinogenesis, we also tested in vivo anti-gnotoxic effect against inhaled cigarette smoke of a Connarus extract whose in vitro anti-genotoxic effect was shown. RESULTS: Male ICR mice were exposed for 1 min to a 6-fold dilution of the smoke once a day for up to 14 consecutive days. Although the frequencies of reticulocytes with micronucleus (MNRETs) and erythrocytes with micronuclei (MN erythrocytes) did not increase within 72 h after a single inhalation of cigarette smoke, the frequency of MN erythrocytes increased significantly upon inhalation for 7 and 14 days. When the Connarus extract was fed to mice at >23.7 ppm during the inhalation period of 14 days, frequency of MN erythrocytes was significantly lower than that at 0 ppm. In vitro antioxidant activity of Connarus extract was almost same to that of vitamin C. The antioxidant activity of the Connarus extract might play an important role in its anti-genotoxic effect against cigarette smoke in vivo, like vitamins C. CONCLUSIONS: Consecutive inhalation of cigarette smoke is clastogenic to mouse bone marrow as shown by the increased frequency of MN erythrocytes. Also, it was shown the possibility that the Connarus extract reduces the risk of tobacco carcinogenesis.

Role of complement in a murine model of peanut-induced anaphylaxis.

Peanut allergy is severe and persisting from childhood to adulthood. However, there is no effective prophylaxis or treatment for peanut allergy. Little is known to about the molecular process in the pathogenesis of peanuts allergy, especially in innate immunity. Thus we investigated the role of complement activation in murine peanut anaphylaxis. Complement component C3 deposition on peanut extract (PE) was evaluated using sera from wild-type (WT), mannose-binding lectin associated serine protease (MASP)-1/3 deficient, MASP-2 deficient, and C4 deficient mice. Sera from interferon regulatory factor-4 (IRF-4) deficient mice, which lack serum immunoglobulin, were also used. In anaphylaxis study, mice were pretreated with propranolol and a long-acting form of IL-4, and injected with PE. Mice were then assessed for plasma C3a levels and hypothermia shock by ELISA and rectal temperature measurement, respectively. C3 deposition on PE was abolished in immunoglobulin- and C4-deficient sera. No difference in C3 deposition levels were observed among WT, MASP-1/3 deficient and MASP-2 deficient sera. IgM, IgG2b, IgG3, C1q, and ficolin-A deposits were detected on PE. In anaphylaxis study, MASP-1/3 deficient mice showed elevation of plasma C3a levels similar to WT mice. However, they were significantly reduced in C4- and MASP-2-deficient mice compared to WT mice. Consistently, PE-induced anaphylactic shock was prevented in C4 deficient mice and partially in MASP-2 deficient mice. In conclusion, PE activates complement via both the lectin and classical pathways in vivo, and the complement activation contributes to hypothermia shock in mice.

Mice deficient in ficolin, a lectin complement pathway recognition molecule, are susceptible to Streptococcus pneumoniae infection.

Mannose-binding lectin (MBL) and ficolin are complexed with MBL-associated serine proteases, key enzymes of complement activation via the lectin pathway, and act as soluble pattern recognition molecules in the innate immune system. Although numerous reports have revealed the importance of MBL in infectious diseases and autoimmune disorders, the role of ficolin is still unclear. To define the specific role of ficolin in vivo, we generated model mice deficient in ficolins. The ficolin A (FcnA)-deficient (Fcna(-/-)) and FcnA/ficolin B double-deficient (Fcna(-/-)b(-/-)) mice lacked FcnA-mediated complement activation in the sera, because of the absence of complexes comprising FcnA and MBL-associated serine proteases. When the host defense was evaluated by transnasal infection with a Streptococcus pneumoniae strain, which was recognized by ficolins, but not by MBLs, the survival rate was significantly reduced in all three ficolin-deficient (Fcna(-/-), Fcnb(-/-), and Fcna(-/-)b(-/-)) mice compared with wild-type mice. Reconstitution of the FcnA-mediated lectin pathway in vivo improved survival rate in Fcna(-/-) but not in Fcna(-/-)b(-/-) mice, suggesting that both FcnA and ficolin B are essential in defense against S. pneumoniae. These results suggest that ficolins play a crucial role in innate immunity against pneumococcal infection through the lectin complement pathway.

Mouse ficolin B has an ability to form complexes with mannose-binding lectin-associated serine proteases and activate complement through the lectin pathway.

Ficolins are thought to be pathogen-associated-molecular-pattern-(PAMP-) recognition molecules that function to support innate immunity. Like mannose-binding lectins (MBLs), most mammalian ficolins form complexes with MBL-associated serine proteases (MASPs), leading to complement activation via the lectin pathway. However, the ability of murine ficolin B, a homologue of human M-ficolin, to perform this function is still controversial. The results of the present study show that ficolin B in mouse bone marrow is an oligomeric protein. Ficolin B, pulled down using GlcNAc-agarose, contained very low, but detectable, amounts of MASP-2 and small MBL-associated protein (sMAP) and showed detectable C4-deposition activity on immobilized N-acetylglucosamine. These biochemical features of ficolin B were confirmed using recombinant mouse ficolin B produced in CHO cells. Taken together, these results suggest that like other mammalian homologues, murine ficolin B has an ability to exert its function via the lectin pathway.

Mannose-binding lectin and its associated proteases (MASPs) mediate coagulation and its deficiency is a risk factor in developing complications from infection, including disseminated intravascular coagulation.

The first line of host defense is the innate immune system that includes coagulation factors and pattern recognition molecules, one of which is mannose-binding lectin (MBL). Previous studies have demonstrated that MBL deficiency increases susceptibility to infection. Several mechanisms are associated with increased susceptibility to infection, including reduced opsonophagocytic killing and reduced lectin complement pathway activation. In this study, we demonstrate that MBL and MBL-associated serine protease (MASP)-1/3 together mediate coagulation factor-like activities, including thrombin-like activity. MBL and/or MASP-1/3 deficient hosts demonstrate in vivo evidence that MBL and MASP-1/3 are involved with hemostasis following injury. Staphylococcus aureus infected MBL null mice developed disseminated intravascular coagulation (DIC), which was associated with elevated blood IL-6 levels (but not TNF-α and multi-organ inflammatory responses). Infected MBL null mice also develop liver injury. These findings suggest that MBL deficiency may manifest into DIC and organ failure during infectious diseases.

Essential role of mannose-binding lectin-associated serine protease-1 in activation of the complement factor D.

The complement system is an essential component of innate immunity, participating in the pathogenesis of inflammatory diseases and in host defense. In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as recognition molecules, and MBL-associated serine protease (MASP) is a key enzyme; MASP-2 is responsible for the lectin pathway activation. The function of other serine proteases (MASP-1 and MASP-3) is still obscure. In this study, we generated a MASP-1- and MASP-3-deficient mouse model (Masp1/3-/-) and found that no activation of the alternative pathway was observed in Masp1/3-/- serum. Mass spectrometric analysis revealed that circulating complement factor D (Df) in Masp1/3-/- mice is a zymogen (pro-Df) with the activation peptide QPRGR at its N terminus. These results suggested that Masp1/3-/- mice failed to convert pro-Df to its active form, whereas it was generally accepted that the activation peptide of pro-Df is removed during its secretion and factor D constitutively exists in an active form in the circulation. Furthermore, recombinant MASP-1 converted pro-Df to the active form in vitro, although the activation mechanism of pro-Df by MASP-1 is still unclear. Thus, it is clear that MASP-1 is an essential protease of both the lectin and alternative complement pathways.

Mannose-binding lectin (MBL)-associated serine protease (MASP)-1 contributes to activation of the lectin complement pathway.

The complement system plays an important role in innate immunity. In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as recognition molecules, and MBL-associated serine protease (MASP) is a key enzyme. It has been suggested that MASP-2 is responsible for the activation of C4. Other serine proteases (MASP-1 and MASP-3) are also associated with MBL or ficolins; however, their functions are still controversial. In this study, a MASP-1- and MASP-3-deficient mouse model (MASP1/3(-/-)) was generated by a gene targeting strategy to investigate the roles of MASP-1 and MASP-3 in the lectin pathway. Serum derived from MASP1/3(-/-) mice showed significantly lower activity of both C4 and C3 deposition on mannan-agarose, and this low activity was restored by the addition of recombinant MASP-1. MASP-1/3-deficient serum showed a significant delay for activation of MASP-2 compared with normal serum. Reconstitution of recombinant MASP-1 in MASP-1/3-deficient serum was able to promote the activation of MASP-2. From these results, we propose that MASP-1 contributes to the activation of the lectin pathway, probably through the activation of MASP-2.

Eyelid lengthening combined with penetrating keratoplasty for exposure keratopathy in Graves' ophthalmopathy--a case report.

Upper eyelid retraction is a well-known component of Graves' disease. With greater degrees of retraction, corneal exposure is usually increased. We report here on a patient with corneal perforation following exposure keratopathy due to upper eyelid retraction. The patient was treated with penetrating keratoplasty and an upper eyelid lengthening procedure using Goretex dura substitute as an interpositional graft material. The exposure keratopathy resolved postoperatively and this condition has been maintained for 45 months since the operation, with a good cosmetic outcome and symmetry of the palpebral fissures.