Role of Adrenomedullin 2/Intermedin in the Pathogenesis of Neovascular Age-Related Macular Degeneration.

Adrenomedullin 2 (AM2; also known as intermedin) is a member of the adrenomedullin (AM) peptide family. Similarly to AM, AM2 partakes in a variety of physiological activities. AM2 has been reported to exert protective effects on various organ disorders; however, its significance in the eye is unknown. We investigated the role of AM2 in ocular diseases. The receptor system of AM2 was expressed more abundantly in the choroid than in the retina. In an oxygen-induced retinopathy model, physiological and pathologic retinal angiogenesis did not differ between AM2-knockout (AM2-/-) and wild-type mice. In contrast, in laser-induced choroidal neovascularization, a model of neovascular age-related macular degeneration, AM2-/- mice had enlarged and leakier choroidal neovascularization lesions, with exacerbated subretinal fibrosis and macrophage infiltration. Contrary to this, exogenous administration of AM2 ameliorated the laser-induced choroidal neovascularization-associated pathology and suppressed gene expression associated with inflammation, fibrosis, and oxidative stress, including that of VEGF-A, VEGFR-2, CD68, CTGF, and p22-phox. The stimulation of human adult retinal pigment epithelial (ARPE) cell line 19 cells with TGF-ß2 and TNF-α induced epithelial-to-mesenchymal transition (EMT), whereas AM2 expression was also elevated. The induction of EMT was suppressed when the ARPE-19 cells were pretreated with AM2. A transcriptome analysis identified 15 genes, including mesenchyme homeobox 2 (Meox2), whose expression was significantly altered in the AM2-treated group compared with that in the control group. The expression of Meox2, a transcription factor that inhibits inflammation and fibrosis, was enhanced by AM2 treatment and attenuated by endogenous AM2 knockout in the early phase after laser irradiation. The AM2 treatment of endothelial cells inhibited endothelial to mesenchymal transition and NF-κB activation; however, this effect tended to be canceled following Meox2 gene knockdown. These results indicate that AM2 suppresses the neovascular age-related macular degeneration-related pathologies partially via the upregulation of Meox2. Thus, AM2 may be a promising therapeutic target for ocular vascular diseases.

Adrenomedullin-Receptor Activity-Modifying Protein 2 System Ameliorates Subretinal Fibrosis by Suppressing Epithelial-Mesenchymal Transition in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a leading cause of visual impairment. Anti-vascular endothelial growth factor drugs used to treat AMD carry the risk of inducing subretinal fibrosis. We investigated the use of adrenomedullin (AM), a vasoactive peptide, and its receptor activity-modifying protein 2, RAMP2, which regulate vascular homeostasis and suppress fibrosis. The therapeutic potential of the AM-RAMP2 system was evaluated after laser-induced choroidal neovascularization (LI-CNV), a mouse model of AMD. Neovascular formation, subretinal fibrosis, and macrophage invasion were all enhanced in both AM and RAMP2 knockout mice compared with those in wild-type mice. These pathologic changes were suppressed by intravitreal injection of AM. Comprehensive gene expression analysis of the choroid after LI-CNV with or without AM administration revealed that fibrosis-related molecules, including Tgfb, Cxcr4, Ccn2, and Thbs1, were all down-regulated by AM. In retinal pigment epithelial cells, co-administration of transforming growth factor-ß and tumor necrosis factor-α induced epithelial-mesenchymal transition, which was also prevented by AM. Finally, transforming growth factor-ß and C-X-C chemokine receptor type 4 (CXCR4) inhibitors eliminated the difference in subretinal fibrosis between RAMP2 knockout and wild-type mice. These findings suggest the AM-RAMP2 system suppresses subretinal fibrosis in LI-CNV by suppressing epithelial-mesenchymal transition.

Studies on safety and efficacy of particles containing a mixture of hydroxyapatite-argentum-titanium oxide (HAT) and sheets coated with HAT particles to be used in masks to improve nasal allergy: II. Cellular, in vivo, and clinical studies.

PURPOSE: We report the manufacture of particles containing a mixture of hydroxyapatite-argentum-titanium oxide (HAT), followed by attachment to nonwoven polyester fabrics to produce HAT-coated sheets (HATS) for use in masks. The purpose of the present study was to perform cellular, in vivo, and clinical studies to further examine the safety of HATS for use in masks to improve nasal allergy. METHODS: Reverse mutation tests for HAT were performed using five bacterial strains. A cellular toxicity test was performed using a Chinese hamster cell line incubated with the HATS extracts. Skin reactions after intradermal administration were examined in rabbits. Skin sensitization tests in guinea pigs were performed using the HATS extracts. HAT was administered to the nasal cavity and conjunctival sac of the rabbits. An oral administration study was performed in rats. Finally, a human skin patch test was performed using the HATS. RESULTS: Reverse mutation tests showed negative results. The cellular toxicity test showed that the HATS extract had moderate cytotoxicity. The intradermal skin reaction and skin sensitization tests were all negative. The administration of HAT to the nasal cavity and intraocular administration showed negative results. No toxicity was observed after oral administration of HAT powder up to a dose of 2000 mg/kg. Finally, the skin patch test result was negative. CONCLUSION: Although HAT showed moderate cytotoxicity, in vivo results indicated that HAT is safe because it does not come in direct contact with cells in normal usage, and HATS is safe when used in masks.

Endogenous Calcitonin Gene-Related Peptide Deficiency Exacerbates Postoperative Lymphedema by Suppressing Lymphatic Capillary Formation and M2 Macrophage Accumulation.

Lymphedema is a chronic condition caused by disruption of lymphatic vessels, which often occurs after invasive surgery. Calcitonin gene-related peptide (CGRP) is a 37-amino acid peptide produced by alternative splicing of the primary transcript of the calcitonin/CGRP gene (Calca). CGRP was initially identified as a neuropeptide released primarily from sensory nerves and involved in regulating pathophysiological nociceptive pain. However, recent studies have shown CGRP is also released from a variety of other cells and possesses multiple functions. In this study, CGRP knockout (-/-) mice were used to show the actions of endogenous CGRP in postoperative lymphedema. After generating a mouse postoperative tail lymphedema model, the edema was observed to be more severe in CGRP-/- mice than in wild-type mice. Numbers of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1)-positive lymphatic capillaries were decreased and lymphatic capillary formation-related factors were down-regulated in CGRP-/- mice. In addition, accumulation of M2 but not M1 macrophages was selectively reduced in the edematous tissue of CGRP-/- mice. Selective depletion of M2 macrophages decreased lymphatic capillary formation and worsened lymphedema in wild-type mice but not CGRP-/- mice, where numbers of M2 macrophages were already diminished. These findings suggest that endogenous CGRP acts to ameliorate postoperative lymphedema by enhancing lymphatic capillary formation and that M2 macrophages play critical roles. CGRP may be a useful therapeutic target for the treatment of postoperative lymphedema.

Development of a Novel Model of Central Retinal Vascular Occlusion and the Therapeutic Potential of the Adrenomedullin-Receptor Activity-Modifying Protein 2 System.

Central retinal vein occlusion (CRVO) is an intractable disease that causes visual acuity loss with retinal ischemia, hemorrhage, and edema. In this study, we developed an experimental CRVO model in mice and evaluated the therapeutic potential of the pleiotropic peptide adrenomedullin (ADM) and its receptor activity-modifying protein 2 (RAMP2). The CRVO model, which had phenotypes resembling those seen in the clinic, was produced by combining i.p. injection of Rose bengal, a photoactivator dye enhancing thrombus formation, with laser photocoagulation. Retinal vascular area, analyzed using fluorescein angiography and fluorescein isothiocyanate-perfused retinal flat mounts, was decreased after induction of CRVO but gradually recovered from day 1 to 7. Measurements of retinal thickness using optical coherence tomography and histology revealed prominent edema early after CRVO, followed by gradual atrophy. Reperfusion after CRVO was diminished in Adm and Ramp2 knockout (KO) mice but was increased by exogenous ADM administration. CRVO also increased expression of a coagulation factor, oxidative stress markers, and a leukocyte adhesion molecule in both wild-type and Adm KO mice, and the effect was more pronounced in Adm KO mice. Using retinal capillary endothelial cells, ADM was found to directly suppress retinal endothelial injury. The retinoprotective effects of the Adm-Ramp2 system make it a novel therapeutic target for the treatment of CRVO.

Investigation of the therapeutic mechanism of subthreshold micropulse laser irradiation in retina.

PURPOSE: Subthreshold micropulse laser irradiation has been used for the treatment of retinal edema; however, there are few reports about the mechanism of its therapeutic effect. In this study, we compared threshold short pulse and subthreshold micropulse laser irradiation in mice and investigated their mechanism. METHODS: Nine to 12-week-old male C57BL/6J mice were used in this study. After general anesthesia, threshold short pulse or subthreshold micropulse laser irradiation was performed on the right eye using IQ577. Enucleation was performed 24 h after the laser irradiation, and histological and gene expression analyses were carried out. RESULTS: Coagulation spots and atrophy of the retinal pigment epithelium were observed after threshold short pulse laser irradiation but not after subthreshold micropulse laser irradiation. Twenty-four hours after laser, aquaporin (AQP) 1, 2, 7, and 11 levels were significantly elevated by 1.7- to 3-fold in the threshold short pulse laser group compared with non-treated control group. AQP 3 was increased significantly and prominently by 100-fold. VEGF-A and VEGFR2 were upregulated 1.5- and 2.3-fold, respectively. In the subthreshold micropulse laser group, AQP 3 was increased by 6-fold compared with the non-treated control group. Angiopoietin-1 and the adrenomedullin (AM) receptor CLR were decreased by 0.6-fold and 0.5-fold, respectively. CONCLUSION: Threshold short pulse laser irradiation caused retinal damage and prominent changes in the expression of various genes. Contrarily, subthreshold micropulse laser irradiation did not induce retinal damage; it upregulated AQP 3, which might have improved retinal edema by drainage of subretinal fluid.

Screen of Non-annotated Small Secreted Proteins of Pseudomonas syringae Reveals a Virulence Factor That Inhibits Tomato Immune Proteases.

Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) is an extracellular model plant pathogen, yet its potential to produce secreted effectors that manipulate the apoplast has been under investigated. Here we identified 131 candidate small, secreted, non-annotated proteins from the PtoDC3000 genome, most of which are common to Pseudomonas species and potentially expressed during apoplastic colonization. We produced 43 of these proteins through a custom-made gateway-compatible expression system for extracellular bacterial proteins, and screened them for their ability to inhibit the secreted immune protease C14 of tomato using competitive activity-based protein profiling. This screen revealed C14-inhibiting protein-1 (Cip1), which contains motifs of the chagasin-like protease inhibitors. Cip1 mutants are less virulent on tomato, demonstrating the importance of this effector in apoplastic immunity. Cip1 also inhibits immune protease Pip1, which is known to suppress PtoDC3000 infection, but has a lower affinity for its close homolog Rcr3, explaining why this protein is not recognized in tomato plants carrying the Cf-2 resistance gene, which uses Rcr3 as a co-receptor to detect pathogen-derived protease inhibitors. Thus, this approach uncovered a protease inhibitor of P. syringae, indicating that also P. syringae secretes effectors that selectively target apoplastic host proteases of tomato, similar to tomato pathogenic fungi, oomycetes and nematodes.

Adrenomedullin Suppresses Vascular Endothelial Growth Factor-Induced Vascular Hyperpermeability and Inflammation in Retinopathy.

Diabetic macular edema (DME) is caused by blood-retinal barrier breakdown associated with retinal vascular hyperpermeability and inflammation, and it is the major cause of visual dysfunction in diabetic retinopathy. Adrenomedullin (ADM) is an endogenous peptide first identified as a strong vasodilator. ADM is expressed in the eyes and is up-regulated in various eye diseases, although the pathophysiological significance is largely unknown. We investigated the effect of ADM on DME. In Kimba mice, which overexpress human vascular endothelial growth factor in their retinas, the capillary dropout, vascular leakage, and vascular fragility characteristic of diabetic retinopathy were observed. Intravitreal or systemic administration of ADM to Kimba mice ameliorated both the capillary dropout and vascular leakage. Evaluation of the transendothelial electrical resistance and fluorescein isothiocyanate-dextran permeability of an endothelial cell monolayer using TR-iBRB retinal capillary endothelial cells revealed that vascular endothelial growth factor enhanced vascular permeability but that co-administration of ADM suppressed the effect, in part by enhancing tight junction formation between endothelial cells. In addition, a comprehensive PCR array analysis showed that ADM administration suppressed various molecules related to inflammation and NF-κB signaling within retinas. From these results, we suggest that by exerting inhibitory effects on retinal inflammation, vascular permeability, and blood-retinal barrier breakdown, ADM could serve as a novel therapeutic agent for the treatment of DME.

Major Cys protease activities are not essential for senescence in individually darkened Arabidopsis leaves.

BACKGROUND: Papain-like Cys Proteases (PLCPs) and Vacuolar Processing Enzymes (VPEs) are amongst the most highly expressed proteases during leaf senescence in Arabidopsis. Using activity-based protein profiling (ABPP), a method that enables detection of active enzymes within a complex sample using chemical probes, the activities of PLCPs and VPEs were investigated in individually darkened leaves of Arabidopsis, and their role in senescence was tested in null mutants. RESULTS: ABPP and mass spectrometry revealed an increased activity of several PLCPs, particularly RD21A and AALP. By contrast, despite increased VPE transcript levels, active VPE decreased in individually darkened leaves. Eight protease knock-out lines and two protease over expressing lines were subjected to senescence phenotype analysis to determine the importance of individual protease activities to senescence. Unexpectedly, despite the absence of dominating PLCP activities in these plants, the rubisco and chlorophyll decline in individually darkened leaves and the onset of whole plant senescence were unaltered. However, a significant delay in progression of whole plant senescence was observed in aalp-1 and rd21A-1/aalp-1 mutants, visible in the reduced number of senescent leaves. CONCLUSIONS: Major Cys protease activities are not essential for dark-induced and developmental senescence and only a knock out line lacking AALP shows a slight but significant delay in plant senescence.

Pathophysiological function of endogenous calcitonin gene-related peptide in ocular vascular diseases.

Calcitonin gene-related peptide (CGRP; official name CALCA) has a variety of functions and exhibits both angiogenic and anti-inflammatory properties. We previously reported the angiogenic effects of the CGRP family peptide adrenomedullin in oxygen-induced retinopathy; however, the effects of CGRP on ocular angiogenesis remain unknown. Herein, we used CGRP knockout (CGRP(-/-)) mice to investigate the roles of CGRP in ocular vascular disease. Observation of pathological retinal angiogenesis in the oxygen-induced retinopathy model revealed no difference between CGRP(-/-) and wild-type mice. However, much higher levels of the CGRP receptor were present in the choroid than the retina. Laser-induced choroidal neovascularization (CNV), a model of exudative age-related macular degeneration, revealed more severe CNV lesions in CGRP(-/-) than wild-type mice, and fluorescein angiography showed greater leakage from CNV in CGRP(-/-). In addition, macrophage infiltration and tumor necrosis factor (TNF)-α production were enhanced within the CNV lesions in CGRP(-/-) mice, and the TNF-α, in turn, suppressed the barrier formation of retinal pigment epithelial cells. In vivo, CGRP administration suppressed CNV formation, and CGRP also dose dependently suppressed TNF-α production by isolated macrophages. From these data, we conclude that CGRP suppresses the development of leaky CNV through negative regulation of inflammation. CGRP may thus be a promising therapeutic agent for the treatment of ocular vascular diseases associated with inflammation.

Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination.

Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins.

Effects of Adrenomedullin on Doxorubicin-Induced Cardiac Damage in Mice.

Doxorubicin (DOX) is one of the best known anticancer drugs, and is used in the treatment of lymphoma, lung cancer, stomach cancer, and a number of other cancers. However, DOX has some serious side effects, the worst being lethal heart failure. Occasionally, its side effects result in the cessation of the anticancer treatment, thus having a serious adverse influence on prognosis. Agents that can be administered as alternative prophylactics or to ameliorate the side effects of DOX will be useful in increasing the safety and efficacy of anticancer therapy. Adrenomedullin (AM) is a peptide hormone secreted by many organs, including the heart; it has an organ-protective effect, including antiapoptotic, anti-inflammatory, and antioxidative stress. Blood AM levels increase with heart failure; endogenic AM has been suggested in order to protect the heart. Furthermore, exogenous AM administration has shown therapeutic effects for heart failure in patients. However, it is unclear whether AM can protect the heart against drug-induced cardiac injury in vivo. The present study was performed in order to investigate the effects of AM on DOX-induced cardiac damage. Male BALB/c mice were treated with DOX and/or AM. Exogenous AM improved the survival ratio of DOX-treated mice. In addition, AM reduced serum lactate dehydrogenase (LDH) levels following DOX treatment. On pathological examination, AM was shown to inhibit DOX-induced cardiac tissue damage, mitochondrial abnormality, and cell death. These findings suggest that AM has a protective effect against DOX-induced cardiac damage.

Pseudomonas syringae pv. syringae uses proteasome inhibitor syringolin A to colonize from wound infection sites.

Infection of plants by bacterial leaf pathogens at wound sites is common in nature. Plants defend wound sites to prevent pathogen invasion, but several pathogens can overcome spatial restriction and enter leaf tissues. The molecular mechanisms used by pathogens to suppress containment at wound infection sites are poorly understood. Here, we studied Pseudomonas syringae strains causing brown spot on bean and blossom blight on pear. These strains exist as epiphytes that can cause disease upon wounding caused by hail, sand storms and frost. We demonstrate that these strains overcome spatial restriction at wound sites by producing syringolin A (SylA), a small molecule proteasome inhibitor. Consequently, SylA-producing strains are able to escape from primary infection sites and colonize adjacent tissues along the vasculature. We found that SylA diffuses from the primary infection site and suppresses acquired resistance in adjacent tissues by blocking signaling by the stress hormone salicylic acid (SA). Thus, SylA diffusion creates a zone of SA-insensitive tissue that is prepared for subsequent colonization. In addition, SylA promotes bacterial motility and suppresses immune responses at the primary infection site. These local immune responses do not affect bacterial growth and were weak compared to effector-triggered immunity. Thus, SylA facilitates colonization from wounding sites by increasing bacterial motility and suppressing SA signaling in adjacent tissues.

Functional differentiation of RAMP2 and RAMP3 in their regulation of the vascular system.

Adrenomedullin (AM) is a vasoactive peptide that possesses various bioactivities. AM receptors are dimers consisting of CLR with one of two accessory proteins, RAMP2 or RAMP3. The functional difference between CLR/RAMP2 and CLR/RAMP3 and the relationship between the two receptors remain unclear. To address these issues, we generated RAMP2 and RAMP3 knockout (-/-) mice and have been studying their physiological activities in the vascular system. AM-/- and RAMP2-/- mice die in utero due to blood vessel abnormalities, which is indicative of their essential roles in vascular development. In contrast, RAMP3-/- mice were born normally without any major abnormalities. In adult RAMP3-/- mice, postnatal angiogenesis was normal, but lymphangiography using indocyanine green (ICG) showed delayed drainage of subcutaneous lymphatic vessels. Moreover, chyle transport by intestinal lymphatics was delayed in RAMP3-/- mice, which also showed more severe interstitial edema than wild-type mice in a tail lymphedema model, with characteristic dilatation of lymphatic capillaries and accumulation of inflammatory cells. In scratch-wound assays, migration of isolated RAMP3-/- lymphatic endothelial cells was delayed as compared to wild-type cells, and AM administration failed to enhance the re-endothelialization. The delay in re-endothelialization was due to a primary migration defect rather than a decrease in proliferation. These results suggest that RAMP3 regulates drainage through lymphatic vessels, and that the AM-RAMP3 system could be a novel therapeutic target for controlling postoperative lymphedema.

TIME FOR COFFEE is an essential component in the maintenance of metabolic homeostasis in Arabidopsis thaliana.

Plants often respond to environmental changes by reprogramming metabolic and stress-associated pathways. Homeostatic integration of signaling is a central requirement for ensuring metabolic stability in living organisms. Under diurnal conditions, properly timed rhythmic metabolism provides fitness benefits to plants. TIME FOR COFFEE (TIC) is a circadian regulator known to be involved in clock resetting at dawn. Here we explored the mechanism of influence of TIC in plant growth and development, as initiated by a microarray analysis. This global profiling showed that a loss of TIC function causes a major reprogramming of gene expression that predicts numerous developmental, metabolic, and stress-related phenotypes. This led us to demonstrate that this mutant exhibits late flowering, a plastochron defect, and diverse anatomical phenotypes. We further observed a starch-excess phenotype and altered soluble carbohydrate levels. tic exhibited hypersensitivity to oxidative stress and abscisic acid, and this was associated with a striking resistance to drought. These phenotypes were connected to an increase in total glutathione levels that correlated with a readjustment of amino acids and polyamine pools. By comparatively analyzing our transcriptomic and metabolomic data, we concluded that TIC is a central element in plant homeostasis that integrates and coordinates developmental, metabolic, and environmental signals.

Vascular endothelial adrenomedullin-RAMP2 system is essential for vascular integrity and organ homeostasis.

BACKGROUND: Revealing the mechanisms underlying the functional integrity of the vascular system could make available novel therapeutic approaches. We previously showed that knocking out the widely expressed peptide adrenomedullin (AM) or receptor activity-modifying protein 2 (RAMP2), an AM-receptor accessory protein, causes vascular abnormalities and is embryonically lethal. Our aim was to investigate the function of the vascular AM-RAMP2 system directly. METHODS AND RESULTS: We generated endothelial cell-specific RAMP2 and AM knockout mice (E-RAMP2(-/-) and E-AM(-/-)). Most E-RAMP2(-/-) mice died perinatally. In surviving adults, vasculitis occurred spontaneously. With aging, E-RAMP2(-/-) mice showed severe organ fibrosis with marked oxidative stress and accelerated vascular senescence. Later, liver cirrhosis, cardiac fibrosis, and hydronephrosis developed. We next used a line of drug-inducible E-RAMP2(-/-) mice (DI-E-RAMP2(-/-)) to induce RAMP2 deletion in adults, which enabled us to analyze the initial causes of the aforementioned vascular and organ damage. Early after the induction, pronounced edema with enhanced vascular leakage occurred. In vitro analysis revealed the vascular leakage to be caused by actin disarrangement and detachment of endothelial cells. We found that the AM-RAMP2 system regulates the Rac1-GTP/RhoA-GTP ratio and cortical actin formation and that a defect in this system causes the disruption of actin formation, leading to vascular and organ damage at the chronic stage after the gene deletion. CONCLUSIONS: Our findings show that the AM-RAMP2 system is a key determinant of vascular integrity and homeostasis from prenatal stages through adulthood. Furthermore, our models demonstrate how endothelial cells regulate vascular integrity and how their dysregulation leads to organ damage.

A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice.

BACKGROUND: Microinjection of clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-related RNA and DNA into fertilized eggs is a novel approach for creating gene-modified mice. Blastocysts obtained just before implantation may be appropriate for testing the fidelity of CRIPSR/Cas9-mediated genome editing because they can be individually handled in vitro and obtained 3days after microinjection, thus allowing researchers to check mutations rapidly. However, it is not known whether indel mutations caused by the CRISPR/Cas9 system can be reproducibly detected in embryos. In this study, we assessed the detection of CRISPR/Cas9-induced mutations in embryos. RESULTS: T7 endonuclease I was more effective than Surveyor nuclease for detecting mutations in annealed fragments derived from 2 plasmids, which contained nearly identical sequences. Mouse fertilized eggs were microinjected with CRISPR/Cas9-related RNA/DNA to examine whether non-homologous end joining-mediated knockout and homologous recombination-mediated knockin occurred in the endogenous receptor (G protein-coupled) activity modifying protein 2 (Ramp2) gene. Individual blastocysts were lysed to obtain crude DNA solutions, which were used for polymerase chain reaction (PCR) assays. T7 endonuclease I-based PCR and sequencing analysis demonstrated that 25-100% of the embryos were knockout embryos and 7-57% of the embryos were knockin embryos. Our results also established that crude DNA from a single blastocyst was an appropriate template for Whole genome amplification and subsequent assessment by PCR and the T7 endonuclease I-based assay. CONCLUSIONS: The single blastocyst-based assay was useful for determining whether CRISPR/Cas9-mediated genome editing worked in murine embryos.

Adrenomedullin-RAMP2 system is crucially involved in retinal angiogenesis.

Adrenomedullin (ADM) is an endogenous peptide first identified as a strong vasodilating molecule. We previously showed that in mice, homozygous knockout of ADM (ADM(-/-)) or its receptor regulating protein, RAMP2 (RAMP2(-/-)), is embryonically lethal due to abnormal vascular development, thereby demonstrating the importance of ADM and its receptor signaling to vascular development. ADM expression in the retina is strongly induced by ischemia; however, its role in retinal pathophysiology remains unknown. Here, we analyzed oxygen-induced retinopathy (OIR) using heterozygous ADM and RAMP2 knockout mice models (ADM(+/-) or RAMP2(+/-), respectively). In addition, we analyzed the role of the ADM-RAMP2 system during earlier stages of retinal angiogenesis using an inducible endothelial cell-specific RAMP2 knockout mouse line (DI-E-RAMP2(-/-)). Finally, we assessed the ability of antibody-induced ADM blockade to control pathological retinal angiogenesis in OIR. In OIR, neovascular tufts, avascular zones, and hypoxic areas were all smaller in ADM(+/-) retinas compared with wild-type mice. ADM(+/-) retinas also exhibited reduced levels of VEGF and eNOS expression. DI-E-RAMP2(-/-) showed abnormal retinal vascular patterns in the early stages of development. However, ADM enhanced the proliferation and migration of retinal endothelial cells. Finally, we found intravitreal injection of anti-ADM antibody reduced pathological retinal angiogenesis. In conclusion, the ADM-RAMP2 system is crucially involved in retinal angiogenesis. ADM and its receptor system are potential therapeutic targets for controlling pathological retinal angiogenesis.

Calcitonin gene-related peptide is involved in inflammatory pain but not in postoperative pain.

BACKGROUND: The aim of this study was to clarify the roles of calcitonin gene-related peptide (CGRP) in postoperative pain and inflammatory pain. METHODS: αCGRP knockout mice that the authors have developed and wild-type mice were used. Pain behaviors were assessed after incision and complete Freund's adjuvant (CFA) injection. Changes in CGRP and c-Fos expression in the dorsal horn were also examined. RESULTS: Guarding pain scores in αCGRP knockout mice were lower than those in wild-type mice at 24 h (3.8 ± 1.6 vs. 6.8 ± 1.5, P = 0.044) and 48 h (1.8 ± 1.7 vs. 6.0 ± 1.5, P = 0.001) after CFA injection (n = 8 to 9). Withdrawal latencies to heat stimulation in αCGRP knockout mice were higher than those in wild-type mice at 24 to 72 h after CFA injection (4.9 ± 1.0 vs. 3.4 ± 0.8 at 24 h, P = 0.04; 5.1 ± 0.3 vs. 3.2 ± 0.9 at 48 h, P = 0.047; and 5.4 ± 1.6 vs. 3.5 ± 0.5 s at 72 h, P = 0.045) (n = 11 to 13), but withdrawal thresholds to mechanical stimulation were comparable. CGRP expression was increased at 24 h after CFA injection in wild-type mice, and the c-Fos-positive profile was increased at 4 h after CFA injection (ipsilateral vs. contralateral: 12.3 ± 4.6 vs. 1.3 ± 1.9, P < 0.0001) and maintained at 24 h (10.0 ± 4.1 vs. 0.8 ± 1.3, P < 0.0001) (n = 4 to 6). CONCLUSION: These results suggest that contribution of the αCGRP system depends on the modality of pain and the stage of inflammation.

Phytophthora infestans effector AVRblb2 prevents secretion of a plant immune protease at the haustorial interface.

In response to pathogen attack, plant cells secrete antimicrobial molecules at the site of infection. However, how plant pathogens interfere with defense-related focal secretion remains poorly known. Here we show that the host-translocated RXLR-type effector protein AVRblb2 of the Irish potato famine pathogen Phytophthora infestans focally accumulates around haustoria, specialized infection structures that form inside plant cells, and promotes virulence by interfering with the execution of host defenses. AVRblb2 significantly enhances susceptibility of host plants to P. infestans by targeting the host papain-like cysteine protease C14 and specifically preventing its secretion into the apoplast. Plants altered in C14 expression were significantly affected in susceptibility to P. infestans in a manner consistent with a positive role of C14 in plant immunity. Our findings point to a unique counterdefense strategy that plant pathogens use to neutralize secreted host defense proteases. Effectors, such as AVRblb2, can be used as molecular probes to dissect focal immune responses at pathogen penetration sites.