Consolidation and maintenance of long-term memory involve dual functions of the developmental regulator Apterous in clock neurons and mushroom bodies in the Drosophila brain.

Memory is initially labile but can be consolidated into stable long-term memory (LTM) that is stored in the brain for extended periods. Despite recent progress, the molecular and cellular mechanisms underlying the intriguing neurobiological processes of LTM remain incompletely understood. Using the Drosophila courtship conditioning assay as a memory paradigm, here, we show that the LIM homeodomain (LIM-HD) transcription factor Apterous (Ap), which is known to regulate various developmental events, is required for both the consolidation and maintenance of LTM. Interestingly, Ap is involved in these 2 memory processes through distinct mechanisms in different neuronal subsets in the adult brain. Ap and its cofactor Chip (Chi) are indispensable for LTM maintenance in the Drosophila memory center, the mushroom bodies (MBs). On the other hand, Ap plays a crucial role in memory consolidation in a Chi-independent manner in pigment dispersing factor (Pdf)-containing large ventral-lateral clock neurons (l-LNvs) that modulate behavioral arousal and sleep. Since disrupted neurotransmission and electrical silencing in clock neurons impair memory consolidation, Ap is suggested to contribute to the stabilization of memory by ensuring the excitability of l-LNvs. Indeed, ex vivo imaging revealed that a reduced function of Ap, but not Chi, results in exaggerated Cl- responses to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in l-LNvs, indicating that wild-type (WT) Ap maintains high l-LNv excitability by suppressing the GABA response. Consistently, enhancing the excitability of l-LNvs by knocking down GABAA receptors compensates for the impaired memory consolidation in ap null mutants. Overall, our results revealed unique dual functions of the developmental regulator Ap for LTM consolidation in clock neurons and LTM maintenance in MBs.

Elucidating Gender-Specific Distribution of Imipramine, Chloroquine, and Their Metabolites in Mice Kidney Tissues through AP-MALDI-MSI.

Knowledge of gender-specific drug distributions in different organs are of great importance for personalized medicine and reducing toxicity. However, such drug distributions have not been well studied. In this study, we investigated potential differences in the distribution of imipramine and chloroquine, as well as their metabolites, between male and female kidneys. Kidneys were collected from mice treated with imipramine or chloroquine and then subjected to atmospheric pressure matrix-assisted laser desorption ionization-mass spectrometry imaging (AP-MALDI-MSI). We observed differential distributions of the drugs and their metabolites between male and female kidneys. Imipramine showed prominent distributions in the cortex and medulla in male and female kidneys, respectively. Desipramine, one of the metabolites of imipramine, showed significantly higher (*** p < 0.001) distributions in the medulla of the male kidney compared to that of the female kidney. Chloroquine and its metabolites were accumulated in the pelvis of both male and female kidneys. Interestingly, they showed a characteristic distribution in the medulla of the female kidney, while almost no distributions were observed in the same areas of the male kidney. For the first time, our study revealed that the distributions of imipramine, chloroquine, and their metabolites were different in male and female kidneys.

Increase in the number of new cases of ANCA-associated vasculitis in the COVID-19 vaccine era.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is an autoimmune vasculitis characterized by the production of antibodies against ANCA, with unclear pathogenesis. With the ongoing COVID-19 pandemic, COVID-19 mRNA vaccination has been available in Japan since February 2021. Although autoimmune symptoms have been reported after COVID-19 vaccinations, there have been no clinical investigations regarding the relationship between COVID-19 mRNA vaccines and the pathogenesis of AAV. Thus, the present study aimed to investigate whether the administration of COVID-19 mRNA vaccines affects the development of AAV. The study identified patients with new-onset AAV who were MPO-ANCA or PR3-ANCA positive and met the entry criteria of the AAV EMA classification algorithm. The study compared the number of new AAV cases per year before and after the start of the COVID-19 mRNA vaccine program in Japan. The study found that the annual number of new cases of AAV in Japan's Nagasaki Prefecture increased by approximately 1.5-fold since the COVID-19 vaccine program was initiated, suggesting a possible link between the COVID-19 mRNA vaccines and the development of AAV. Although the study provides insight into the clinical evaluation and management of autoimmune symptoms following COVID-19 vaccination, further investigation of the possible association between COVID-19 mRNA vaccines and the pathogenesis of AAV is required.

Inhibition of calcium/calmodulin-dependent protein kinase IV in arthritis: dual effect on Th17 cell activation and osteoclastogenesis.

OBJECTIVE: To investigate the role of calcium/calmodulin-dependent protein kinase IV (CaMK4) in the development of joint injury in a mouse model of arthritis and patients with RA. METHODS: Camk4-deficient, Camk4flox/floxLck-Cre, and mice treated with CaMK4 inhibitor KN-93 or KN-93 encapsulated in nanoparticles tagged with CD4 or CD8 antibodies were subjected to collagen-induced arthritis (CIA). Inflammatory cytokine levels, humoral immune response, synovitis, and T-cell activation were recorded. CAMK4 gene expression was measured in CD4+ T cells from healthy participants and patients with active RA. Micro-CT and histology were used to assess joint pathology. CD4+ and CD14+ cells in patients with RA were subjected to Th17 or osteoclast differentiation, respectively. RESULTS: CaMK4-deficient mice subjected to CIA displayed improved clinical scores and decreased numbers of Th17 cells. KN-93 treatment significantly reduced joint destruction by decreasing the production of inflammatory cytokines. Furthermore, Camk4flox/floxLck-Cre mice and mice treated with KN93-loaded CD4 antibody-tagged nanoparticles developed fewer Th17 cells and less severe arthritis. CaMK4 inhibition mitigated IL-17 production by CD4+ cells in patients with RA. The number of in vitro differentiated osteoclasts from CD14+ cells in patients with RA was significantly decreased with CaMK4 inhibitors. CONCLUSION: Using global and CD4-cell-targeted pharmacologic approaches and conditionally deficient mice, we demonstrate that CaMK4 is important in the development of arthritis. Using ex vivo cell cultures from patients with RA, CaMK4 is important for both Th17 generation and osteoclastogenesis. We propose that CaMK4 inhibition represents a new approach to control the development of arthritis.

NAD+ Levels Are Augmented in Aortic Tissue of ApoE-/- Mice by Dietary Omega-3 Fatty Acids.

BACKGROUND: Maintaining bioenergetic homeostasis provides a means to reduce the risk of cardiovascular events during chronological aging. Nicotinamide adenine dinucleotide (NAD+) acts as a signaling molecule, and its levels were used to govern several biological pathways, for example, promoting angiogenesis by SIRT1 (sirtuin 1)-mediated inhibition of Notch signaling to rejuvenate capillary density of old-aged mice. NAD+ modulation shows promise in the vascular remodeling of endothelial cells. However, NAD+ distribution in atherosclerotic regions remains uncharacterized. Omega-3 polyunsaturated fatty acids consumption, such as docosahexaenoic acid and eicosapentaenoic acid, might increase the abundance of cofactors in blood vessels due to omega-3 polyunsaturated fatty acids metabolism. METHODS: Apolipoprotein E-deficient (ApoE-/-) mice were fed a Western diet, and the omega-3 polyunsaturated fatty acids-treated groups were supplemented with docosahexaenoic acid (1%, w/w) or eicosapentaenoic acid (1%, w/w) for 3 weeks. Desorption electrospray ionization mass spectrometry imaging was exploited to detect exogenous and endogenous NAD+ imaging. RESULTS: NAD+, NADH, NADP+, NADPH, FAD+, FADH, and nicotinic acid adenine dinucleotide of the aortic arches were detected higher in the omega-3 polyunsaturated fatty acids-treated mice than the nontreated control. Comparing the distribution in the outer and inner layers of the arterial walls, only NADPH was detected slightly higher in the outer part in eicosapentaenoic acid-treated mice. CONCLUSIONS: Supplementation of adding docosahexaenoic acid or eicosapentaenoic acid to the Western diet led to a higher NAD+, FAD+, and their metabolites in the aortic arch. Considering the pleiotropic roles of NAD+ in biology, this result serves as a beneficial therapeutic strategy in the animal model counter to pathological conditions.

Lipid biomarkers that reflect postoperative recurrence risk in lung cancer patients who smoke: a case-control study.

BACKGROUND: The risk of postoperative recurrence is higher in lung cancer patients who smoke than non-smokers. However, objective evaluation of the postoperative recurrence risk is difficult using conventional pathological prognostic factors because of their lack of reproducibility. Consequently, novel objective biomarkers that reflect postoperative risk in lung cancer patients who smoke must be identified. Because cigarette smoking and oncogenesis alter lipid metabolism in lung tissue, we hypothesized that the lipid profiles in lung cancer tissues are influenced by cigarette smoking and can reflect the postoperative recurrence risk in smoking lung cancer patients. This study aimed to identify lipid biomarkers that reflect the smoking status and the postoperative recurrence risk. METHODS: Primary tumor tissues of lung adenocarcinoma (ADC) (n = 26) and squamous cell carcinoma (SQCC) (n = 18) obtained from surgery were assigned to subgroups according to the patient's smoking status. The ADC cohort was divided into never smoker and smoker groups, while the SQCC cohort was divided into moderate smoker and heavy smoker groups. Extracted lipids from the tumor tissues were subjected to liquid chromatography-tandem mass spectrometry analysis. Lipids that were influenced by smoking status and reflected postoperative recurrence and pathological prognostic factors were screened. RESULTS: Two and 12 lipid peaks in the ADC and SQCC cohorts showed a significant positive correlation with the Brinkman index, respectively. Among them, in the ADC cohort, a higher lipid level consisted of three phosphatidylcholine (PC) isomers, PC (14:0_18:2), PC (16:1_16:1), and PC (16:0_16:2), was associated with a shorter recurrence free period (RFP) and a greater likelihoods of progressed T-factor (≥ pT2) and pleural invasion. In the SQCC cohort, a lower m/z 736.5276 level was associated with shorter RFP and greater likelihood of recurrence. CONCLUSIONS: From our data, we propose three PC isomers, PC (14:0_18:2), PC (16:1_16:1), and PC (16:0_16:2), and a lipid peak of m/z 736.5276 as novel candidate biomarkers for postoperative recurrence risk in lung ADC and SQCC patients who are smokers.

Lipid Polyunsaturated Fatty Acid Chains in Mouse Kidneys Were Increased within 5 min of a Single High Dose Whole Body Irradiation.

To understand the ultra-early reaction of normal organ lipids during irradiation, we investigated the response of lipids, including polyunsaturated fatty acid (PUFA) chains, which are particularly susceptible to damage by ROS, in mice's kidneys, lungs, brains, and livers within 5 min of single high-dose irradiation. In this study, we set up three groups of C56BL/6 male mice and conducted whole-body irradiation with 0 Gy, 10 Gy, and 20 Gy single doses. Kidney, lung, brain, and liver tissues were collected within 5 min of irradiation. PUFA-targeted and whole lipidomic analyses were conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results showed that PUFA chains of kidney phosphatidylcholine (PC), phosphatidylethanolamine (PE), and triacylglycerol (TG) significantly increased within 5 min of 10 Gy and 20 Gy irradiation. The main components of increased PUFA chains in PC and PE were C18:2, C20:4, and C22:6, and in TG the main component was C18:2. The kidney lipidomes also showed significant changes from the perspective of lipid species, mainly dominated by an increase in PC, PE, TG, and signal lipids, while lipidomes of the lung, brain, and liver were slightly changed. Our results revealed that acute PUFA chains increase and other lipidomic changes in the kidney upon whole-body irradiation within 5 min of irradiation. The significantly increased lipids also showed a consistent preference for possessing PUFA chains. The lipidomic changes varied from organ to organ, which indicates that the response upon irradiation within a short time is tissue-specific.

Osteopontin-induced vascular hyperpermeability through tight junction disruption in diabetic retina.

Diabetic retinopathy is a major cause of blindness in developed countries, and is characterized by deterioration of barrier function causing vascular hyperpermeability and retinal edema. Vascular endothelial growth factor (VEGF) is a major mediator of diabetic macular edema. Although anti-VEGF drugs are the first-line treatment for diabetic macular edema, some cases are refractory to anti-VEGF therapy. Osteopontin (OPN) is a phosphoglycoprotein with diverse functions and expressed in various cells and tissues. Elevated OPN level has been implicated in diabetic retinopathy, but whether OPN is involved in hyperpermeability remains unclear. Using streptozotocin-induced diabetic mice (STZ mice) and human retinal endothelial cells (HRECs), we tested the hypothesis that up-regulated OPN causes tight junction disruption, leading to vascular hyperpermeability. The serum and retinal OPN concentrations were elevated in STZ mice compared to controls. Intravitreal injection of anti-OPN neutralizing antibody (anti-OPN Ab) suppressed vascular hyperpermeability and prevented decreases in claudin-5 and ZO-1 gene expression levels in the retina of STZ mice. Immunohistochemical staining of retinal vessels in STZ mice revealed claudin-5 immunoreactivity with punctate distribution and attenuated ZO-1 immunoreactivity, and these changes were prevented by anti-OPN Ab. Intravitreal injection of anti-OPN Ab did not change VEGF gene expression or protein concentration in retina of STZ mice. In an in vitro study, HRECs were exposed to normal glucose or high glucose with or without OPN for 48 h, and barrier function was evaluated by transendothelial electrical resistance and Evans blue permeation. Barrier function deteriorated under high glucose condition, and was further exacerbated by the addition of OPN. Immunofluorescence localization of claudin-5 and ZO-1 demonstrated punctate appearance with discontinuous junction in HRECs exposed to high glucose and OPN. There were no changes in VEGF and VEGF receptor-2 expression levels in HRECs by exposure to OPN. Our results suggest that OPN induces tight junction disruption and vascular hyperpermeability under diabetic conditions. Targeting OPN may be an effective approach to manage diabetic retinopathy.

STING, a cytosolic DNA sensor, plays a critical role in atherogenesis: a link between innate immunity and chronic inflammation caused by lifestyle-related diseases.

AIMS: Lifestyle-related diseases promote atherosclerosis, a chronic inflammatory disease; however, the molecular mechanism remains largely unknown. Endogenous DNA fragments released under over-nutrient condition provoke sterile inflammation through the recognition by DNA sensors. Here, we investigated the role of stimulator of interferon genes (STING), a cytosolic DNA sensor, in atherogenesis. METHODS AND RESULTS: Apolipoprotein E-deficient (Apoe-/-) mice fed a western-type diet (WTD), a hypercholesterolaemic mouse model, showed higher STING expression and markers for DNA damage such as γH2AX, p53, and single-stranded DNA (ssDNA) accumulation in macrophages in the aorta compared with wild-type (WT) mice. The level of cGAMP, a STING agonist, in the aorta was higher in Apoe-/- mice. Genetic deletion of Sting in Apoe-/- mice reduced atherosclerotic lesions in the aortic arch, lipid, and macrophage accumulation in plaques, and inflammatory molecule expression in the aorta compared with the control. Pharmacological blockade of STING using a specific inhibitor, C-176, ameliorated atherogenesis in Apoe-/- mice. In contrast, bone marrow-specific STING expression in Apoe-/- mice stimulated atherogenesis. Expression or deletion of STING did not affect metabolic parameters and blood pressure. In vitro studies revealed that STING activation by cGAMP or mitochondrial DNA accelerated inflammatory molecule expression (e.g. TNF-α or IFN-ß) in mouse and human macrophages. Activation of nuclear factor-κB and TANK binding kinase 1 was involved in STING-associated vascular inflammation and macrophage activation. Furthermore, human atherosclerotic lesions in the carotid arteries expressed STING and cGAMP. CONCLUSION: Stimulator of interferon genes stimulates pro-inflammatory activation of macrophages, leading to the development of atherosclerosis. Stimulator of interferon genes signalling may serve as a potential therapeutic target for atherosclerosis.

High-fat diet induces a predisposition to follicular hyperkeratosis and neutrophilic folliculitis in mice.

BACKGROUND: Neutrophilic folliculitis is an inflammatory condition of hair follicles. In some neutrophilic folliculitis, such as in patients with acne and hidradenitis suppurativa, follicular hyperkeratosis is also observed. Neutrophilic folliculitis is often induced and/or exacerbated by a high-fat diet (HFD). However, the molecular mechanisms by which an HFD affects neutrophilic folliculitis are not fully understood. OBJECTIVE: Our aim was to elucidate how an HFD promotes the development of neutrophilic folliculitis. METHODS: Mice were fed an HFD, and their skin was subjected to histologic, RNA sequencing, and imaging mass spectrometry analyses. To examine the effect of an HFD on neutrophil accumulation around the hair follicles, phorbol 12-myristate 13-acetate (PMA) was used as an irritant to the skin. RESULTS: Histologic analysis revealed follicular hyperkeratosis in the skin of HFD-fed mice. RNA sequencing analysis showed that genes related to keratinization, especially in upper hair follicular keratinocytes, were significantly upregulated in HFD-fed mice. Application of PMA to the skin induced neutrophilic folliculitis in HFD-fed mice but not in mice fed a normal diet. Accumulation of neutrophils in the skin and around hair follicles was dependent on CXCR2 signaling, and CXCL1 (a CXCR2 ligand) was produced mainly by hair follicular keratinocytes. Imaging mass spectrometry analysis revealed an increase in fatty acids in the skin of HFD-fed mice. Application of these fatty acids to the skin induced follicular hyperkeratosis and caused PMA-induced neutrophilic folliculitis even in mice fed a normal diet. CONCLUSION: An HFD can facilitate the development of neutrophilic folliculitis with the induction of hyperkeratosis of hair follicles and increased neutrophil infiltration around the hair follicles via CXCR2 signaling.

Circulating Vitreous microRNA as Possible Biomarker in High Myopic Eyes with Macular Hole.

High myopia is a major cause of irreversible visual impairment globally. In the present study, we investigated the microRNA (miRNA) profile in the vitreous of macular hole (MH) and high myopic MH. We performed miRNA analysis using TaqMan® Low Density Arrays (Thermo Fisher Scientific, Waltham, MA, USA) to investigate the circulating vitreous miRNA profile from patients with MH (axial length < 26.5 mm, n = 11) and high myopic MH (axial length ≥ 26.5 mm, n = 11) who underwent pars plana vitrectomy. The vitreous inflammatory cytokine signature was examined in high myopic MH eyes using a multiplex assay. A miRNA-Array analysis revealed that let-7c was significantly up-regulated and miR-200a was significantly down-regulated in high myopic MH eyes compared to those in MH eyes. The bioinformatics analysis for up-regulated miRNA targeted gene identified 23 pathways including mitogen-activated protein kinase (MAPK) and several inflammatory signaling pathways, whereas the bioinformatics analysis for down-regulated miRNA targeted genes showed 32 enriched pathways including phosphoinositide 3-kinase/protein kinase B (PI3K/AKT). The levels of inflammatory cytokines including IP-10, IFN-γ, and MCP-1 were significantly higher in the vitreous of high myopic MH eyes. These results suggest that specific miRNAs expressed in the vitreous may be associated with the pathological condition of high myopic MH and the above mentioned miRNAs may contribute to the development of inflammatory status in the vitreous of high myopic eyes.

Mass spectrometry in the lipid study of cancer.

Introduction: Cancer is a heterogeneous disease that exploits various metabolic pathways to meet the demand for increased energy and structural components. Lipids are biomolecules that play essential roles as high energy sources, mediators, and structural components of biological membranes. Accumulating evidence has established that altered lipid metabolism is a hallmark of cancer.Areas covered: Mass spectrometry (MS) is a label-free analytical tool that can simultaneously identify and quantify hundreds of analytes. To date, comprehensive lipid studies exclusively rely on this technique. Here, we reviewed the use of MS in the study of lipids in various cancers and discuss its instrumental limitations and challenges.Expert opinion: MS and MS imaging have significantly contributed to revealing altered lipid metabolism in a variety of cancers. Currently, a single MS approach cannot profile the entire lipidome because of its lack of sensitivity and specificity for all lipid classes. For the metabolic pathway investigation, lipid study requires the integration of MS with other molecular approaches. Future developments regarding the high spatial resolution, mass resolution, and sensitivity of MS instruments are warranted.

Sub internal limiting membrane hemorrhage followed by bilateral optic disc hemorrhage in Kikuchi-Fujimoto disease: a case report.

BACKGROUND: Kikuchi-Fujimoto disease (KFD) is a necrotizing lymphadenitis, and presents fever of unknown origin and cervical lymphadenopathy. Ocular complications are unusual in KFD. Here we report a case of sub internal limiting membrane (ILM) hemorrhage followed by bilateral optic disc hemorrhage in KFD. CASE PRESENTATION: A 16-year-old Japanese man perceived a sudden decrease of right vision 3 days after onset of fever with unknown origin and left cervical lymphadenopathy. At presentation, visual acuity (VA) of right eye was 0.05 in decimal chart (1.30: converted to logarithm of minimum angle of resolution: logMAR). Fundus photograph showed extensive sub-ILM hemorrhage in right eye, and optic disc hemorrhages in both eyes. Fluorescein angiography presented hypo- and hyperfluorescences in optic disc of right eye, and hyperfluorescence in the disc of left eye. To make a definitive diagnosis, cervical lymph node biopsy was performed, and KFD was diagnosed pathologically. Thereafter, fever, headache and the cervical lymphadenopathy disappeared spontaneously. The sub-ILM hemorrhage was drained into the vitreous cavity by neodymium:yttrium-aluminum-garnet laser (Nd: YAG) hyaloidotomy. VA recovered to 1.5 (- 0.18: logMAR VA) in right eye. CONCLUSION: Sub-ILM hemorrhage and optic disc hemorrhage are a KFD-related ocular complication.

FABP5 Is a Sensitive Marker for Lipid-Rich Macrophages in the Luminal Side of Atherosclerotic Lesions.

Lipid-rich macrophages in atherosclerotic lesions are thought to be derived from myeloid and vascular smooth muscle cells. A series of studies with genetic and pharmacological inhibition of fatty acid binding protein 4 (FABP4) and FABP5 and bone marrow transplant experiments with FABP4/5 deficient cells in mice have demonstrated that these play an important role in the development of atherosclerosis. However, it is still uncertain about the differential cell-type specificity and distribution between FABP4- and FABP5-expressing cells in early- and late-stage atherosclerotic lesions. In this study, we first explored spatial distribution of FABP4/5 in atherosclerotic lesions in apolipoprotein E deficient (ApoE-/-) mice. FABP4 was only marginally detected in early and advanced lesions, whereas FABP5 was abundantly expressed in these lesions. In advanced lesions, the FABP5-positive area was mostly restricted to the foam cell layer adjacent to the lumen above collagen and elastic fibers with a high signal/noise ratio. Oil red O (ORO) staining revealed that FABP5-positive cells were lipid-rich in early and advanced lesions. Together, most of lipid-rich FABP5-positive cells reside adjacent to the lumen above collagen and elastic fibers. We next studied involvement of FABP5 in lesion formation of atherosclerosis using ApoE-/- FABP5-/- mice. However, deletion of FABP5 did not affect the development of atherosclerosis. These findings, along with previous reports, suggest a novel notion that FABP5 is a sensitive marker for bone marrow-derived lipid-rich macrophages in the luminal side of atherosclerotic lesions, although its functional significance remains elusive.

Mass spectrometry-based phospholipid imaging: methods and findings.

Introduction: Imaging is a technique used for direct visualization of the internal structure or distribution of biomolecules of a living system in a two-dimensional or three-dimensional fashion. Phospholipids are important structural components of biological membranes and have been reported to be associated with various human diseases. Therefore, the visualization of phospholipids is crucial to understand the underlying mechanism of cellular and molecular processes in normal and diseased conditions. Areas covered: Mass spectrometry imaging (MSI) has enabled the label-free imaging of individual phospholipids in biological tissues and cells. The commonly used MSI techniques include matrix-assisted laser desorption ionization-MSI (MALDI-MSI), desorption electrospray ionization-MSI (DESI-MSI), and secondary ion mass spectrometry (SIMS) imaging. This special report described those methods, summarized the findings, and discussed the future development for the imaging of phospholipids. Expert opinion: Phospholipids imaging in complex biological samples has been significantly benefited from the development of MSI methods. In MALDI-MSI, novel matrix that produces homogenous crystals exclusively with polar lipids is important for phospholipids imaging with greater efficiency and higher spatial resolution. DESI-MSI has the potential of live imaging of the biological surface while SIMS is expected to image at the subcellular level in the near future.

Preferential Incorporation of Administered Eicosapentaenoic Acid Into Thin-Cap Atherosclerotic Plaques.

OBJECTIVE: n-3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have beneficial effects on atherosclerosis. Although specific salutary actions have been reported, the detailed distribution of n-3 polyunsaturated fatty acids in plaque and their relevance in disease progression are unclear. Our aim was to assess the pharmacodynamics of EPA and DHA and their metabolites in atherosclerotic plaques. Approach and Results: Apolipoprotein E-deficient (Apoe-/-) mice were fed a Western diet supplemented with EPA (1%, w/w) or DHA (1%, w/w) for 3 weeks. Imaging mass spectrometry analyses were performed in the aortic root and arch of the Apoe-/- mice to evaluate the distribution of EPA, DHA, their metabolites and the lipids containing EPA or DHA in the plaques. Liquid chromatography-mass spectrometry and histological analysis were also performed. The intima-media thickness of atherosclerotic plaque decreased in plaques containing free EPA and EPAs attached with several lipids. EPA was distributed more densely in the thin-cap plaques than in the thick-cap plaques, while DHA was more evenly distributed. In the aortic root, the distribution of total EPA level and cholesteryl esters containing EPA followed a concentration gradient from the vascular endothelium to the media. In the aortic arch, free EPA and 12-hydroxy-EPA colocalized with M2 macrophage. CONCLUSIONS: Administered EPA tends to be incorporated from the vascular lumen side and preferentially taken into the thin-cap plaque.

Higher Accumulation of Docosahexaenoic Acid in the Vermilion of the Human Lip than in the Skin.

The vermilion of the human lip is a unique facial area because of certain distinguishing features from the adjacent tissues such as the white lip (skin) and oral mucosa. However, the distinction in terms of molecular distribution between the vermilion and skin has remained unexplored. Therefore, we aimed to map the human lip by mass spectrometry imaging to gain understanding of the free fatty acid distribution in the vermilion. The lip specimens trimmed off during cheiloplasty were analyzed using desorption electrospray ionization-mass spectrometry imaging. Distributions of two monounsaturated fatty acids and three polyunsaturated fatty acids were observed in the human lip tissue: palmitoleic acid (POA) and oleic acid (OA) and linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA), respectively. Although POA, OA, LA, and AA were differentially distributed across the vermilion and skin, DHA showed a higher accumulation in the epithelium of the vermilion compared to that in the skin. Our results clearly demonstrated the difference in fatty acid distributions between the vermilion and skin. The highly abundant DHA in the epithelium of the vermilion may have an antioxidant role and may thus protect the lip from aging. Our findings can provide a novel strategy for treating lip disorders.

CD4+ CD52lo T-cell expression contributes to the development of systemic lupus erythematosus.

The cell-surface glycoprotein CD52 is widely expressed in lymphocytes. CD4+CD52hi T cells are functioning suppressor CD4+T cells. We investigated the role of the immune regulation of CD4+CD52 T cells in systemic lupus erythematosus (SLE). CD4+CD52lo T cells were increased in SLE patients, in positive correlation with SLEDAI, anti-ds-DNA antibody, and IgG concentration. Circulating follicular helper-like T cells (Tfh-like cells) were also increased in SLE, in positive correlation with CD4+CD52lo T cells. Chemokine receptor 8 (CCR8) expression in CD4+CD52lo T cells was increased. In vitro experiments using CD4 T cells of SLE patients showed that thymus and activation-regulated chemokine (TARC), a ligand of CCR8, contributed to the development of CD4+CD52hi T cells into CD4+CD52lo T cells. Our findings suggest that CD4+CD52lo T-cell upregulation is involved in the production of pathogens by autoantibodies, and TARC may contribute to the development of SLE through an aberrant induction of CD4+CD52lo T cells.

MicroRNA-204-3p inhibits lipopolysaccharide-induced cytokines in familial Mediterranean fever via the phosphoinositide 3-kinase γ pathway.

Objective: We sought to identify the microRNA (miRNA) profile and potential biomarkers in FMF and to clarify their gene targets to elucidate the pathogenesis of FMF. Methods: We performed an miRNA microarray using serum from FMF patients in attack and in remission. We then examined the expression of miRNAs in macrophages derived from THP-1 cells stimulated with toll-like receptor (TLR) ligands. Macrophages derived from THP-1 cells transfected with pre-miRNA were stimulated with lipopolysaccharides (LPSs) for the quantification of inflammatory cytokine production. To identify the target genes, we overexpressed their miRNA and performed a complementary DNA microarray. Transfection with reporter construct and the precursor miRNA was performed to confirm the suppression of target mRNA. Results: We found that miR-204-3p was greatly decreased in the serum from FMF patients in attack. The expression of miR-204-3p was suppressed by LPS stimulation in the macrophages derived from THP-1 cells and the inhibition of miR-204-3p significantly induced the production of TLR4-related cytokines. The bioinformatic analysis showed that miR-204-3p is predicted to target genes implicated in the TLR pathway through the regulation of PI3Kγ signalling. The reporter assay revealed that miR-204-3p directly suppressed the luciferase activity of 3'-UTR of PIK3CG reporter construct. The inhibition of PI3Kγ resulted in decreased amounts of IL-6 and IL-12p40 in monocytes from FMF patients. Conclusion: These data suggest that serum miR-204-3p has potential as a useful biomarker in FMF patients and that miR-204-3p serves as a suppressor of inflammatory cytokine production in FMF by targeting the PI3Kγ pathway.

Crim1 maintains retinal vascular stability during development by regulating endothelial cell Vegfa autocrine signaling.

Angiogenesis defines the process in which new vessels grow from existing vessels. Using the mouse retina as a model system, we show that cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is highly expressed in angiogenic endothelial cells. Conditional deletion of the Crim1 gene in vascular endothelial cells (VECs) causes delayed vessel expansion and reduced vessel density. Based on known Vegfa binding by Crim1 and Crim1 expression in retinal vasculature, where angiogenesis is known to be Vegfa dependent, we tested the hypothesis that Crim1 is involved in the regulation of Vegfa signaling. Consistent with this hypothesis, we showed that VEC-specific conditional compound heterozygotes for Crim1 and Vegfa exhibit a phenotype that is more severe than each single heterozygote and indistinguishable from that of the conditional homozygotes. We further showed that human CRIM1 knockdown in cultured VECs results in diminished phosphorylation of VEGFR2, but only when VECs are required to rely on an autocrine source of VEGFA. The effect of CRIM1 knockdown on reducing VEGFR2 phosphorylation was enhanced when VEGFA was also knocked down. Finally, an anti-VEGFA antibody did not enhance the effect of CRIM1 knockdown in reducing VEGFR2 phosphorylation caused by autocrine signaling, but VEGFR2 phosphorylation was completely suppressed by SU5416, a small-molecule VEGFR2 kinase inhibitor. These data are consistent with a model in which Crim1 enhances the autocrine signaling activity of Vegfa in VECs at least in part via Vegfr2.