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Choroidal neovascularization (CNV) is the primary pathogenic process underlying wet age-related macular degeneration, leading to severe vision loss. Despite current anti-vascular endothelial growth factor (VEGF) therapies, several limitations persist. Crocetin, a major bioactive constituent of saffron, exhibits multiple pharmacological activities, yet its role and mechanism in CNV remain unclear. Here, we investigated the potential effects of crocetin on CNV using in vitro and in vivo models. In human umbilical vein endothelial cells, crocetin demonstrated inhibition of VEGF-induced cell proliferation, migration, and tube formation in vitro, as assessed by CCK-8 and EdU assays, transwell and scratch assays, and tube formation analysis. Additionally, crocetin suppressed choroidal sprouting in ex vivo experiments. In the human retinal pigment epithelium (RPE) cell line ARPE-19, crocetin attenuated cobalt chloride-induced hypoxic cell injury, as evidenced by CCK-8 assay. As evaluated by quantitative PCR and Western blot assay, it also reduced hypoxia-induced expression of VEGF and hypoxia-inducible factor 1α (HIF-1α), while enhancing zonula occludens-1 expression. In a laser-induced CNV mouse model, intravitreal administration of crocetin significantly reduced CNV size and suppressed elevated expressions of VEGF, HIF-1α, TNFα, IL-1β, and IL-6. Moreover, crocetin treatment attenuated the elevation of phospho-S6 in laser-induced CNV and hypoxia-induced RPE cells, suggesting its potential anti-angiogenic effects through antagonizing the mechanistic target of rapamycin complex 1 (mTORC1) signaling. Our findings indicate that crocetin may hold promise as an effective drug for the prevention and treatment of CNV. • Crocetin inhibits cell proliferation, migration, and tube formation of HUVECs. • Crocetin attenuates hypoxic injury in RPE cells in vitro. • Intravitreal injection of crocetin suppresses laser-induced CNV formation in mice. • Crocetin is a potential therapeutic drug for CNV. [ABSTRACT FROM AUTHOR]

Extracellular ATP is an important damage-associated molecular pattern molecule that plays key roles in innate immunity. In fish, however, the mechanism for extracellular ATP release remains largely undefined. Pannexin1 (Panx1) is a newly discovered extracellular ATP release channel with a wide tissue distribution and diverse biological functions in mammals. In the present study, we identified and characterized a Panx1 homolog cDNA, termed poPanx1 , from Japanese flounder Paralichthys olivaceus , which is one of the most important economic mariculture fish species in China. PoPanx1 is a membrane protein that is composed of 437 amino acids with an estimated molecular mass of 48.7 kDa and an isoelectric point of 6.46. The poPanx1 mRNA ubiquitously expresses in all examined tissues but with predominant expression in hepatopancreas in unstimulated healthy adult Japanese flounder. In Japanese flounder head kidney primary cells, poPanx1 gene expression could be significantly induced by pathogen-associated molecular patterns (PAMPs; polyinosinic–polycytidylic acid and bacterial endotoxin LPS) stimulations. In vivo experiments revealed that poPanx1 mRNA expression was significantly up-regulated upon immune challenges with Edwardsiella tarda and Vibrio anguillarum . Furthermore, we showed that poPanx1 is an important channel protein for PAMP-induced extracellular ATP release that is required for activation of purinergic signaling in fish innate immunity. Taken together, our findings suggest that the ATP release channel, poPanx1 , is a novel immune response gene in purinergic signaling of Japanese flounder P . olivaceus . [ABSTRACT FROM AUTHOR]

Abstract: Deposition of amyloid β (Aβ) containing plaques in the brain is one of the neuropathological hallmarks of Alzheimer''s disease (AD). It has been suggested that modulation of neuronal activity may alter Aβ production in the brain. We postulate that these changes in Aβ production are due to changes in the rate-limiting step of Aβ generation, APP cleavage by γ-secretase. By combining biochemical approaches with fluorescence lifetime imaging microscopy, we found that neuronal inhibition decreases endogenous APP and PS1 interactions, which correlates with reduced Aβ production. By contrast, neuronal activation had a two-phase effect: it initially enhanced APP–PS1 interaction leading to increased Aβ production, which followed by a decrease in the APP and PS1 proximity/interaction. Accordingly, treatment of neurons with naturally secreted Aβ isolated from AD brain or with synthetic Aβ resulted in reduced APP and PS1 proximity. Moreover, applying low concentration of Aβ42 to cultured neurons inhibited de novo Aβ synthesis. These data provide evidence that neuronal activity regulates endogenous APP–PS1 interactions, and suggest a model of a product-enzyme negative feedback. Thus, under normal physiological conditions Aβ may impact its own production by modifying γ-secretase cleavage of APP. Disruption of this negative modulation may cause Aβ overproduction leading to neurotoxicity. [Copyright &y& Elsevier]

P2X4 receptor (P2X4R) is a member of trimeric ATP-gated receptor channel family. Despite the importance of P2X4R in innate immunity has been addressed in mammals, the immunological significance of P2X4R has not been characterized in fish. In the present study we identified a full-length P2X4R cDNA sequence from Japanese flounder Paralichthys olivaceus (termed poP2X4R ) by RT-PCR and RACE approaches and analyzed its gene expression patterns under normal and immune challenge conditions. Qualitative RT-PCR analyses revealed that poP2X4R has a widespread distribution in all examined tissues but dominantly expressed in hepatopancreas. In Japanese flounder head kidney macrophages and peripheral blood lymphocytes, poP2X4R was rapidly and significantly up-regulated by the immune challenges of LPS, poly(I:C) and zymosan. In addition, poP2X4R was up-regulated in spleen, head kidney and gill tissues by Edwardsiella tarda infections. Furthermore, we showed that poP2X4R is a membrane glycoprotein which could interact with ATP release channel Pannexin1, an important component in extracellular ATP-activated purinergic signaling pathways involved in Japanese flounder innate immune response. From a comparative immunological point of view, our results have provided new evidence for the involvement of extracellular ATP-gated P2XRs in fish innate immunity. [ABSTRACT FROM AUTHOR]

Calmodulin (CaM) is a multifunctional calcium sensor protein that participates in various cellular processes under normal, stress and pathological conditions. In crabs, however, the involvement of CaM in response to environmental stress and immune challenges has not been revealed yet. In the present study, a CaM cDNA ( EsCaM ) was identified from Chinese mitten crab Eriocheir sinensis and its mRNA expression patterns in response to ambient (salinity and pH) stress and immune challenges was examined. EsCaM encodes a 149-amino-acid protein with a calculated molecular mass of 16.8 kDa and an isoelectric point of 4.09. In unstimulated healthy E . sinensis , EsCaM mRNA transcript was detected in all tested tissues with predominant expression in hepatopancreas and the lowest expression in haemocytes. Ambient salinity (15‰ and 30‰ salinities) and pH (pH 6 and 8.5) stress significantly altered EsCaM mRNA expression in gill, hepatopancreas, haemocytes, intestine and muscle in Chinese mitten crab. In addition, EsCaM gene expression was significantly and rapidly induced as early as 2 h after LPS and Poly(I:C) immune stimulations in haemocytes in vitro . Furthermore, EsCaM expression was significantly up-regulated in E . sinensis haemocytes, gill, hepatopancreas, intestine and muscle in response to Edwardsiella tarda and Vibrio anguillarum challenges. Collectively, our findings suggest that EsCaM is an important stress and immune response gene in Chinese mitten crab. [ABSTRACT FROM AUTHOR]

Abstract: Lipopolysaccharide-induced TNF-alpha factor (LITAF) is an important transcription factor in transcriptional regulation of TNF-alpha and other cytokines. Here we identified a full-length LITAF homolog cDNA, termed EsLITAF, that contains a 71 bp 5′-untranslated sequence, an open reading frame consisting of 381 bp, and a 208 bp 3′-untranslated sequence in Chinese mitten crab (Eriocheir sinensis), an economically important freshwater crustacean species in China. EsLITAF protein possesses a conserved C-terminal LITAF-like domain with two CXXC motifs and is comprised of 126 amino acids with a theoretical molecular mass of 13.1 kDa and an isoelectric point of 6.36. Blast search against GenBank database revealed that EsLITAF protein shared the highest sequence identity (84%) with the counterpart of Pacific white shrimp (Litopenaeus vannamei). EsLITAF mRNA transcript expresses ubiquitously in all examined tissues with the highest expression in hepatopancreas and lowest expression in haemocytes. Furthermore, EsLITAF mRNA expression could be significantly and rapidly induced in haemocytes by LPS and Poly(I:C) stimulations in vitro. Moreover, EsLITAF gene expression was up-regulated in haemocytes, gill and hepatopancreas after Edwardsiella tarda and Vibrio anguillarum challenges. Taken together, we have identified and characterized a new crustacean LITAF homolog from the Chinese mitten crab. [Copyright &y& Elsevier]