Experimental eye research最新文献_第6页

Antagonizing NK-1R modulates pain perception following corneal injury

IF 3 2区医学 Q1 OPHTHALMOLOGY

Experimental eye research

Pub Date : 2025-02-01 DOI: 10.1016/j.exer.2025.110230

Pier Luigi Surico , Amirreza Naderi , Rohan Bir Singh, Francesca Kahale, Yeganeh Farsi, Seokjoo Lee, Aytan Musayeva, Yihe Chen, Reza Dana

Substance P (SP) expressed by corneal nerves, is an 11-amino acid long neuropeptide from the tachykinin family, encoded by the Tac1 gene, and binds to neurokinin receptors. SP overexpression is associated with various pathological responses in the cornea including vasodilation, pain, inflammation, and angiogenesis in the normally avascular tissue. This study investigates the role of neurokinin-1 receptor (NK-1R) mediated signaling in nociception, nerve regeneration, and neuronal activation following mechanical corneal injury in mice. Corneal injuries were induced in age- and sex-matched C57BL/6 mice by removing corneal epithelium and partial anterior stroma. Following injury, mice were treated with either L-733,060, an NK-1R antagonist, or vehicle, administered topically twice daily for 21 days. Corneal SP levels were measured using ELISA, and nerve regeneration was assessed by quantifying corneal nerve fiber density (CNFD) via β-Tubulin III staining. Gene expression of neuronal markers (ATF3, GFAP, cFos, TRPV1, and TRPM8) in the trigeminal ganglia was measured using qPCR. Pain responses were evaluated using the eye-wiping test (EWT) and palpebral ratio (PR). Results indicated a persistent increase in corneal SP post-injury, significantly reduced by NK-1R antagonism. At 21 days, NK-1R antagonist-treated mice showed higher CNFD, reduced expression of neuronal activation markers, and lower pain perception compared to controls. These findings suggest that SP/NK-1R signaling is critical in corneal nociception post-injury, and its inhibition reduces pain, prevents neuronal hyperactivation, and supports nerve regeneration.

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引用次数: 0

Role of m6A methyltransferase METTL3 in keratoconus pathogenesis m6A甲基转移酶METTL3在圆锥角膜发病中的作用。

IF 3 2区医学 Q1 OPHTHALMOLOGY

Experimental eye research

Pub Date : 2025-02-01 DOI: 10.1016/j.exer.2024.110207

Huimin Yu , Shengqian Dou , Huijin Wang , Yaru Sun , Junpeng Qu , Ting Liu , Xiaoxue Liu , Chao Wei , Hua Gao

Keratoconus (KC) is the most common ectatic corneal disease with unknown pathogenesis. This study aimed to investigate the role of methyltransferase-like enzyme 3 (METTL3) in KC pathogenesis. In the present study, we examined the levels of METTL3 and other N6-methyladenosine (m⁶A) modification-related proteins in KC samples and human stromal keratocyte (HTK) cells stimulated by mechanical stretch (MS) using Western blotting and immunohistochemistry. The level of m⁶A RNA methylation was quantified using the m⁶A RNA methylation assay kit. Genetic (Mettl3 knockdown mice) and pharmacological (STM2457) approaches were employed to investigate the effect of METTL3 on the expression of metalloproteinases (MMPs) in MS-treated corneal stromal cells (CSCs) via Western blotting and real-time polymerase chain reaction. Moreover, YAP signaling activity was assessed to explore the relationship between METTL3 and MMPs in MS-treated CSCs.

Increased expression of METTL3 and decreased expression of METTL14, WTAP, and YTHDF2 were detected in KC samples and MS-stimulated HTK cells. Correspondingly, the m⁶A levels in KC specimens and MS-stimulated CSCs were significantly higher than those in controls but were significantly reduced when METTL3 activity was genetically and pharmacologically blocked. Inhibition of METTL3 significantly reduced the expression of MMP1 and MMP3 in mechanically stretched CSCs and reduced YAP activity. Furthermore, pharmacologically inhibiting YAP signaling in MS-stimulated HTK cells significantly reduced MMP1 and MMP3 expression. Our findings highlight the pathogenic role of METTL3 in KC. Further investigation is required to investigate the underlying mechanism.

圆锥角膜（KC）是最常见的扩张性角膜疾病，其发病机制尚不清楚。本研究旨在探讨甲基转移酶样酶3 （METTL3）在KC发病机制中的作用。在本研究中，我们使用Western blotting和免疫组织化学检测了机械拉伸（MS）刺激的KC样品和人间质角质细胞（HTK）细胞中METTL3和其他n6 -甲基腺苷（m6A）修饰相关蛋白的水平。使用m6A RNA甲基化检测试剂盒定量m6A RNA甲基化水平。采用基因（Mettl3敲除小鼠）和药理学（STM2457）方法，通过Western blotting和实时聚合酶链反应（real-time polymerase chain reaction）研究Mettl3对ms处理的角膜基质细胞（CSCs）金属蛋白酶（MMPs）表达的影响。此外，我们还评估了YAP信号活性，以探索ms处理的CSCs中METTL3和MMPs之间的关系。在KC样品和ms刺激的HTK细胞中检测到METTL3的表达增加，METTL14、WTAP和YTHDF2的表达减少。相应地，KC标本和ms刺激的CSCs中的m6A水平显著高于对照组，但当METTL3活性被遗传和药理学阻断时，m6A水平显著降低。抑制METTL3可显著降低机械拉伸CSCs中MMP1和MMP3的表达，并降低YAP活性。此外，在ms刺激的HTK细胞中，药理学抑制YAP信号显著降低MMP1和MMP3的表达。我们的研究结果强调了METTL3在KC中的致病作用，需要进一步研究其潜在机制。

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引用次数: 0

PI3K signaling and lysyl oxidase is critical to corneal stroma fibrosis following mustard gas injury PI3K信号和赖氨酸氧化酶对芥子气损伤后角膜间质纤维化至关重要。

IF 3 2区医学 Q1 OPHTHALMOLOGY

Experimental eye research

Pub Date : 2025-02-01 DOI: 10.1016/j.exer.2024.110213

Nishant R. Sinha , Alexandria C. Hofmann , Laila A. Suleiman , Riley Laub , Ratnakar Tripathi , Shyam S. Chaurasia , Rajiv R. Mohan

Sulfur mustard gas (SM), an alkylating and vesicating agent, has been used frequently in many wars and conflicts. SM exposure to the eye results in several corneal abnormalities including scar/fibrosis formation. However, molecular mechanism for SM induced corneal fibrosis development is poorly understood. After SM insult to the eye, excessive synthesis/secretion of extracellular matrix components (ECM) including collagen (COL) I, COL III, and lysyl oxidase (LOX) by corneal myofibroblasts causes corneal fibrosis, however, precise mechanism remains elusive. This study tested the hypothesis that Phosphoinositide 3-kinase (PI3K) signaling alterations post SM in cornea enhances stromal ECM synthesis and corneal fibrosis. New Zealand White Rabbits were used. The right eyes were exposed to SM (200 mg-min/m³) and left eye to the air for 8min at MRI Global. Rabbit corneas were collected on day-3, day-7, and day-14 for molecular analysis. SM exposure caused a significant increase in mRNA expression of PI3K, AKT, COL I, COL III, and LOX and protein levels of LOX in a time-dependent manner in rabbit corneas. The in vitro studies were performed with human corneal stromal fibroblasts (hCSFs) by growing cultures in −/+ nitrogen mustard (NM) and LY294002, a PI3K specific inhibitor, for 30min, 8h, 24h, 48h, and 72h. NM significantly increased mRNA and protein levels of PI3K, AKT, COL I, COL III, and LOX. On the contrary, LY294002 in NM hCSFs significantly reduced PI3K, AKT, COL I, COL III, and LOX protein expression. We concluded that PI3K signaling mediates stromal collagen synthesis and LOX production following SM injury.

硫磺芥子气（SM）是一种烷基化和起泡剂，在许多战争和冲突中被频繁使用。SM暴露于眼睛会导致几种角膜异常，包括瘢痕/纤维化形成。然而，SM诱导角膜纤维化发生的分子机制尚不清楚。SM损伤眼睛后，角膜肌成纤维细胞过度合成/分泌细胞外基质成分（ECM），包括胶原（COL） I、COL III和赖氨酸氧化酶（LOX），导致角膜纤维化，但其确切机制尚不清楚。本研究验证了SM后角膜中PI3K信号通路改变可促进间质ECM合成和角膜纤维化的假说。实验使用新西兰大白兔。右眼暴露于SM （200mg-min/m3）中，左眼暴露于空气中8min。分别于第3天、第7天和第14天采集兔角膜进行分子分析。SM暴露导致兔角膜PI3K、AKT、COL I、COL III和LOX mRNA表达和LOX蛋白水平呈时间依赖性显著升高。人角膜间质成纤维细胞（hCSFs）在-/+氮芥（NM）和LY294002 （PI3K特异性抑制剂）中培养30min、8h、24h、48h和72h。NM显著提高PI3K、AKT、COL I、COL III和LOX的mRNA和蛋白水平。相反，LY294002在NM hCSFs中显著降低PI3K、AKT、COL I、COL III和LOX蛋白的表达。我们得出结论，PI3K信号介导SM损伤后间质胶原合成和LOX产生。

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引用次数: 0

How crosstalk between mitochondria, lysosomes, and other organelles can prevent or promote dry age-related macular degeneration 线粒体、溶酶体和其他细胞器之间的相互作用如何预防或促进干性老年性黄斑变性。

IF 3 2区医学 Q1 OPHTHALMOLOGY

Experimental eye research

Pub Date : 2025-02-01 DOI: 10.1016/j.exer.2024.110219

Aparna Lakkaraju , Patricia Boya , Marie Csete , Deborah A. Ferrington , James B. Hurley , Alfredo A. Sadun , Peng Shang , Ruchi Sharma , Debasish Sinha , Marius Ueffing , Susan E. Brockerhoff

Organelles such as mitochondria, lysosomes, peroxisomes, and the endoplasmic reticulum form highly dynamic cellular networks and exchange information through sites of physical contact. While each organelle performs unique functions, this inter-organelle crosstalk helps maintain cell homeostasis. Age-related macular degeneration (AMD) is a devastating blinding disease strongly associated with mitochondrial dysfunction, oxidative stress, and decreased clearance of cellular debris in the retinal pigment epithelium (RPE). However, how these occur, and how they relate to organelle function both with the RPE and potentially the photoreceptors are fundamental, unresolved questions in AMD biology. Here, we report the discussions of the “Mitochondria, Lysosomes, and other Organelle Interactions” task group of the 2024 Ryan Initiative for Macular Research (RIMR). Our group focused on understanding the interplay between cellular organelles in maintaining homeostasis in the RPE and photoreceptors, how this could be derailed to promote AMD, and identifying where these pathways could potentially be targeted therapeutically.

细胞器如线粒体、溶酶体、过氧化物酶体和内质网形成高度动态的细胞网络，并通过物理接触部位交换信息。虽然每个细胞器都有独特的功能，但这种细胞器间的相互作用有助于维持细胞的稳态。年龄相关性黄斑变性（AMD）是一种毁灭性致盲疾病，与线粒体功能障碍、氧化应激和视网膜色素上皮（RPE）细胞碎片清除减少密切相关。然而，这些是如何发生的，以及它们如何与RPE和潜在的光感受器的细胞器功能相关，是AMD生物学中根本的，未解决的问题。在这里，我们报告了2024年瑞安黄斑研究倡议（RIMR）的“线粒体，溶酶体和其他细胞器相互作用”任务组的讨论。我们的研究小组专注于了解细胞器在维持RPE和光感受器内稳态方面的相互作用，这是如何促进AMD的，并确定这些途径在哪里可能成为治疗的靶点。

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引用次数: 0

Senescent retinal pigment epithelial cells promote angiogenesis in choroidal neovascularization via the TAK1/p38 MAPK pathway 衰老的视网膜色素上皮细胞通过TAK1/p38 MAPK通路促进脉络膜新生血管的血管生成。

IF 3 2区医学 Q1 OPHTHALMOLOGY

Experimental eye research

Pub Date : 2025-02-01 DOI: 10.1016/j.exer.2025.110232

Yinhao Wang , Huiling Ma , Qianjie Yang , Kuangqi Chen , Hui Ye, Xinglin Wang, Jianhua Xia, Xiaodan Chen, Xiawei Wang, Ye Shen, Hongguang Cui

Senescent retinal pigment epithelial cells play a key role in neovascular age-related macular degeneration (nAMD); however, the mechanisms underlying the angiogenic ability of these cells remain unclear. Herein, we investigated the effects of the senescent adult retinal pigment epithelial cell line-19 (ARPE-19) on wound healing, cell migration and survival, and tube formation abilities of human umbilical vein endothelial cells (HUVECs). Additionally, we used Brown Norway rats to establish a laser-induced choroidal neovascularization (CNV) model for further nAMD-related studies. We found that the wound healing, cell migration, and tube formation abilities of HUVECs were significantly enhanced following culture in conditioned media from senescent ARPE-19 cells; this was attributed to the activation of the transforming growth factor β-activated kinase 1 (TAK1)/p38 MAPK pathway. Consistently, we found that the TAK1 inhibitors 5Z-7-oxozeaenol and takinib reversed the effects of conditioned media from senescent ARPE-19 cells on the wound healing, migration, survival, and tube formation abilities of HUVECs. We further investigated the therapeutic effects of 5Z-7-oxozeaenol on the laser-induced CNV rat model. We found that TAK1 was activated in IB4+ areas in laser-induced CNV lesions; inhibiting the activity of TAK1 using 5Z-7-oxozeaenol significantly alleviated CNV lesion formation and fluorescein leakage in fundus fluorescein angiography and greatly improved a-waves, b-waves, and OP values, as recorded by electroretinography. Thus, senescent RPE cells may promote angiogenesis via the TAK1/p38 MAPK pathway. Further, inhibiting TAK1 expression alleviates pathological neovascularization and improves retinal function in a laser-induced CNV rat model, highlighting the therapeutic potential of this approach for treating nAMD.

衰老视网膜色素上皮细胞在新生血管性年龄相关性黄斑变性（nAMD）中起关键作用然而，这些细胞血管生成能力的机制尚不清楚。在此，我们研究了衰老成人视网膜色素上皮细胞系19 （ARPE-19）对人脐静脉内皮细胞（HUVECs）伤口愈合、细胞迁移和存活以及成管能力的影响。此外，我们用布朗挪威大鼠建立了激光诱导脉络膜新生血管（CNV）模型，用于进一步的namd相关研究。我们发现，衰老的ARPE-19细胞在条件培养基中培养后，HUVECs的伤口愈合、细胞迁移和成管能力显著增强；这归因于转化生长因子β活化激酶1 (TAK1)/p38 MAPK通路的激活。同样，我们发现TAK1抑制剂5z -7-氧zeaenol和takinib逆转了衰老ARPE-19细胞条件培养基对huvec伤口愈合、迁移、存活和管形成能力的影响。我们进一步研究了5z -7-氧玉米烯醇对激光诱导CNV大鼠模型的治疗作用。我们发现，在激光诱导的CNV病变中，TAK1在IB4+区被激活；5z -7-氧zeaenol抑制TAK1活性可显著减轻眼底荧光素血管造影中CNV病变形成和荧光素渗漏，并可显著改善视网膜电图记录的a波、b波和OP值。因此，衰老的RPE细胞可能通过TAK1/p38 MAPK途径促进血管生成。此外，在激光诱导的CNV大鼠模型中，抑制TAK1表达可缓解病理性新生血管并改善视网膜功能，突出了该方法治疗nAMD的治疗潜力。

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引用次数: 0

UPLC-MS/MS-based serum metabolomics analysis for comprehensive pathological myopia profiling 基于 UPLC-MS/MS 的血清代谢组学分析，用于病理近视综合分析。

IF 3 2区医学 Q1 OPHTHALMOLOGY

Experimental eye research

Pub Date : 2025-02-01 DOI: 10.1016/j.exer.2024.110152

Xin Liu , Yue Wu , Yuying Liu , Wenzhe Qian , Liandi Huang , Yixiang Wu , Bilian Ke

Pathological myopia (PM) is associated with ocular morbidities that cause blindness. PM often occurs in eyes with high myopia (HM) while they are distinctly different. Identifying the differences in metabolites and metabolic pathways between patients with PM and HM may provide information about the pathogenesis of PM, which is currently unknown. This study aimed to reveal the comprehensive metabolic alterations associated with PM. Thirty patients with PM, 27 with simple HM and 27 with low myopia (LM) were enrolled in this study. Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) was performed, and a Venn diagram was generated to explore the overlapping differential metabolites and enriched pathways between each set of two groups. The area under the receiver operating characteristic curve (AUC) was computed to assess the discrimination capacity of each metabolite marker. A total of 134, 125 and 81 differential metabolites were identified in each comparison. Thirty-two differential metabolites were overlapped between the PM vs HM comparison and the PM vs LM comparison. Of these 32 metabolites, 16 were common to all three comparisons; among these metabolites, high levels of 4-hydroxy-l-glutamic acid and low levels of succinic semialdehyde and 2,3-dinor-8-iso prostaglandin F2α appeared to be risk factors for PM. The remaining 16 metabolites were shared only between the PM versus HM and PM versus LM comparisons, most of which are lipid molecules. Pathway analysis revealed that alanine, aspartate and glutamate metabolism was the key metabolic pathway altered in PM patients. Overall, significant differences in the metabolites and metabolic pathways were observed in patients with PM. The metabolic differences identified in this study included differential factors between PM and HM patients, addressing current gaps in PM research. These findings provide a novel perspective of the molecular mechanism of PM.

病理性近视（PM）与导致失明的眼部疾病有关。病理性近视通常发生在高度近视（HM）患者的眼睛中，但两者却截然不同。确定高度近视患者和高度近视患者之间代谢物和代谢途径的差异，可为目前尚不清楚的高度近视发病机制提供信息。本研究旨在揭示与 PM 相关的全面代谢改变。本研究共纳入了 30 名 PM 患者、27 名单纯 HM 患者和 27 名低度近视（LM）患者。研究人员采用超高效液相色谱/串联质谱法（UPLC-MS/MS）对两组患者的代谢物进行了分析，并绘制了维恩图（Venn diagram），以探讨两组患者之间重叠的差异代谢物和富集途径。计算接收者操作特征曲线下面积（AUC）以评估每种代谢物标记物的鉴别能力。在每组比较中，分别发现了 134、125 和 81 个差异代谢物。在 PM 与 HM 的比较和 PM 与 LM 的比较中，有 32 个差异代谢物重叠。在这 32 个代谢物中，有 16 个代谢物在所有三项比较中都有共性；在这些代谢物中，高水平的 4-hydroxy-l-glutamic acid 和低水平的琥珀酸半醛及 2,3-二去甲-8-异前列腺素 F2α 似乎是 PM 的风险因素。其余 16 种代谢物仅在 PM 与 HM 和 PM 与 LM 的比较中共享，其中大部分是脂质分子。通路分析显示，丙氨酸、天冬氨酸和谷氨酸代谢是 PM 患者发生改变的关键代谢通路。总体而言，在 PM 患者体内观察到了代谢物和代谢途径的明显差异。本研究发现的代谢差异包括 PM 和 HM 患者之间的不同因素，填补了目前 PM 研究的空白。这些发现为 PM 的分子机制提供了一个新的视角。

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引用次数: 0

Role of sulfated GAGs in shear mechanical properties of human and porcine cornea 硫酸化GAGs对人和猪角膜剪切力学性能的影响。

IF 3 2区医学 Q1 OPHTHALMOLOGY

Experimental eye research

Pub Date : 2025-02-01 DOI: 10.1016/j.exer.2024.110181

Hamed Hatami-Marbini, Md Esharuzzaman Emu

The corneal extracellular matrix is mainly composed of collagen fibers, proteoglycans (PGs), and glycosaminoglycans (GAGs). The present work was done to investigate the effect of GAGs on linear viscoelastic shear properties of human and porcine cornea. A clear understanding of structural functions of GAGs could result in the development of new intervention methods for diseased conditions that involve changes to the expression of GAGs/PGs. Here, we used keratanase II enzyme to deplete sulfated GAGs from porcine and human donor corneal disks. After quantifying the GAG content, collagen fiber diameter, and interfibrillar spacings of control and GAG-depleted specimens using the Blyscan assay and transmission electron microscopy, we performed torsional rheometry to determine their shear properties at different levels of axial strain. We found that the GAG content of control human (52.35 ± 3.40 μg/mg dry tissue) and porcine cornea (48.59 ± 7.79 μg/mg dry tissue) significantly reduced following keratanase II enzyme treatment. Moreover, we observed that the diameter of collagen fibers (28.78 ± 2.33 nm) and interfibrillar spacing (45.93 ± 2.33 nm) of human specimens were significantly smaller than the collagen fiber diameter (34.77 ± 21.90 nm) and interfibrillar spacing (54.28 ± 3.99 nm) of porcine corneal samples. Although GAG depletion did not have any significant effect on the collagen fiber diameter, it significantly increased the interfibrillar spacing in both porcine and human samples. Within the range of linear viscoelastic behavior, the shear stiffness of human and porcine corneal samples did not depend on the shear strain but significantly increased with increasing the applied axial strain. The average complex shear modulus was found to be between 1.0 KPa and 6.5 KPa and between 8.5 KPa and 31 KPa for control porcine and human corneal discs, respectively. The GAG removal caused significant reduction of shear stiffness in both human and porcine corneal samples. Based on these findings, we conclude that sulfated GAGs are important in defining shear properties of porcine and human corneas and significant GAG content variation adversely affects corneal shear modulus.

角膜细胞外基质主要由胶原纤维、蛋白聚糖和糖胺聚糖（GAGs）组成。本文研究了聚乙二醇酯对人和猪角膜线粘弹性剪切性能的影响。清楚地了解GAGs的结构功能可能会导致开发新的疾病干预方法，这些方法涉及改变GAGs/ pg的表达。在这里，我们使用角化酶II酶来消耗来自猪和人供体角膜盘的硫酸化GAGs。在使用Blyscan测定法和透射电镜对对照和无GAG样品的GAG含量、胶原纤维直径和纤维间距进行量化后，我们进行了扭转流变测定，以确定它们在不同水平轴向应变下的剪切性能。我们发现，角化酶II酶处理后，对照组人（52.35±3.40 μg/mg干组织）和猪角膜（48.59±7.79 μg/mg干组织）的GAG含量显著降低。人角膜的胶原纤维直径（28.78±2.33 nm）和纤维间距（45.93±2.33 nm）明显小于猪角膜的胶原纤维直径（34.77±21.90 nm）和纤维间距（54.28±3.99 nm）。虽然GAG缺失对胶原纤维直径没有显著影响，但在猪和人的样品中，它显著增加了纤维间间距。在线性粘弹性范围内，人和猪角膜试样的剪切刚度不依赖于剪切应变，而是随着施加轴向应变的增加而显著增加。猪和人椎间盘的平均复合剪切模量分别在1.0 ~ 6.5 KPa和8.5 ~ 31 KPa之间。在人类和猪角膜样品中，GAG的移除导致剪切刚度的显著降低。基于这些发现，我们得出结论，硫酸酸化的GAG在定义猪和人角膜的剪切特性方面很重要，而显著的GAG含量变化会对角膜剪切模量产生不利影响。

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引用次数: 0

Clodronate liposome-mediated macrophage depletion ameliorates iron overload-induced dry eye disease 氯膦酸脂质体介导的巨噬细胞耗竭可改善铁超载引起的干眼病。

IF 3 2区医学 Q1 OPHTHALMOLOGY

Experimental eye research

Pub Date : 2025-02-01 DOI: 10.1016/j.exer.2024.110204

Jing Lu , Fangfang Lu , Zhengwu Peng , Zihe Zhang , Weijie Jiang , Xia Meng , Xin Yi , Tuo Chen , Zhigang Fei , Yu Wang , Jiahuan Yi , Xujie Deng , Jia Zhang , Zhi Wang , Qiguo Xiao

Dry eye disease (DED) is a prevalent ophthalmic disease that affects millions of people worldwide. Iron overload and macrophage inflammation have been implicated in the development of murine DED, though the specific role of macrophages under iron overload conditions remains unclear. This study aimed to establish a novel iron overload-induced mouse model of DED and investigate macrophage involvement. The model was induced via intraperitoneal injection of D-glucoside iron. Results showed that macrophage depletion via clodronate liposomes (CL) significantly mitigated iron deposit, decreased ocular surface inflammation, improved tear production and restored the structure of ocular surface tissues. Furthermore, CL specifically targeted pro-inflammatory M1 macrophages and reduced levels of the inflammatory cytokines IL-1β, IL-6, and TNF-α, effectively alleviating symptoms of DED. In conclusion, this study characterized a novel iron overload-induced DED mouse model and demenstrated that macrophage depletion mitigated the pathological changes in ocular surface and lacrimal gland tissues caused by iron overload, suggesting potential therapeutic strategies for further investigation in the treatment of DED.

干眼病（DED）是一种流行的眼部疾病，影响着全世界数百万人。铁超载和巨噬细胞炎症与小鼠DED的发展有关，尽管巨噬细胞在铁超载条件下的具体作用尚不清楚。本研究旨在建立一种新的铁负荷诱导的小鼠DED模型，并探讨巨噬细胞的参与。采用d -葡萄糖苷铁腹腔注射法制备大鼠模型。结果表明，氯膦酸脂质体（CL）对巨噬细胞的消耗可显著减轻铁沉积，减少眼表炎症，改善泪液分泌，恢复眼表组织结构。此外，CL特异性靶向促炎M1巨噬细胞，降低炎症细胞因子IL-1β、IL-6和TNF-α水平，有效缓解DED症状。总之，本研究建立了一种新的铁超载诱导的DED小鼠模型，并证明巨噬细胞消耗减轻了铁超载引起的眼表和泪腺组织的病理变化，为进一步研究治疗DED提供了潜在的治疗策略。

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引用次数: 0

Mass spectrometric detection of keratins in tear fluid 泪液中角蛋白的质谱检测。

IF 3 2区医学 Q1 OPHTHALMOLOGY

Experimental eye research

Pub Date : 2025-02-01 DOI: 10.1016/j.exer.2025.110231

Saleh Ahmed , Jeremy Altman , Garrett Jones , Tae Jin Lee , Danielle M. Robertson , Wenbo Zhi , Shruti Sharma , Ashok Sharma

Purpose

Keratin contamination is a common problem in mass spectrometry proteomic analyses, particularly in bottom-up mass spectrometry. The purpose of this study was to determine the protein contaminants introduced during the proteomic analysis of tear fluid.

Methods

Human tear fluid samples were collected using Schirmer strips. Proteomic analyses were performed using liquid chromatography–tandem mass spectrometry (LC-MS/MS) on blank Schirmer strips and tear fluid samples, with empty vials serving as controls for assessing environmental contaminant proteins.

Results

We detected 26 contaminant proteins (18 keratins and 8 non-keratins). 98.2% of the total protein contamination can be attributed to the 9 keratins, including KRT10 (23.6%), KRT1 (23.5%), KRT2 (15.7%), KRT14 (7.6%), KRT16 (7.0%), KRT5 (6.1%), KRT9 (5.9%), KRT6B (4.6%), and KRT6A (4.3%). A comparison to the proteomic profile of blank Schirmer strips and controls (empty vials) found a strong correlation (R² = 0.9753), indicating that these proteins were not from the blank Schirmer strips but are environmental contaminants. On the other hand, several keratins including KRT19, KRT13, KRT4, KRT7, KRT15, KRT8 and KRT18 were present in tear fluid, but either not detected or were negligible in blank strips. Another set of keratins, including KRT5, KRT6A, KRT14, KRT16, and KRT17, were identified as components of tear fluid as well as environmental contaminants.

Conclusions

This study revealed nine major contaminant keratins in the mass spectrometry analysis. Several other keratins were identified as constituents of tear fluid. Background subtraction is necessary for the accurate analysis of tear fluid using mass spectrometry.

目的：角蛋白污染是质谱分析中常见的问题，特别是在自下而上的质谱分析中。本研究的目的是确定泪液蛋白质组学分析过程中引入的蛋白质污染物。方法：采用Schirmer试纸采集人泪液。蛋白质组学分析采用液相色谱-串联质谱（LC-MS/MS）对空白席尔默条带和泪液样品进行分析，空瓶作为评估环境污染物蛋白质的对照。结果：共检出26种污染蛋白（角蛋白18种，非角蛋白8种）。总蛋白污染的98.2%可归因于9种角蛋白，包括KRT10（23.6%）、KRT1（23.5%）、KRT2（15.7%）、KRT14（7.6%）、KRT16（7.0%）、KRT5（6.1%）、KRT9（5.9%）、KRT6B（4.6%）和KRT6A（4.3%）。空白Schirmer条带与空白对照（空瓶）的蛋白质组学分析结果显示，两者具有很强的相关性（R2 = 0.9753），表明这些蛋白质不是来自空白Schirmer条带，而是环境污染物。另一方面，泪液中存在KRT19、KRT13、KRT4、KRT7、KRT15、KRT8和KRT18等几种角蛋白，但在空白条中未检测到或可忽略不计。另一组角蛋白，包括KRT5、KRT6A、KRT14、KRT16和KRT17，被确定为泪液成分以及环境污染物。结论：本研究在质谱分析中揭示了9种主要的污染物角蛋白。其他几个角蛋白被确定为泪液的成分。背景减法是使用质谱法准确分析泪液的必要条件。

{"title":"Mass spectrometric detection of keratins in tear fluid","authors":"Saleh Ahmed , Jeremy Altman , Garrett Jones , Tae Jin Lee , Danielle M. Robertson , Wenbo Zhi , Shruti Sharma , Ashok Sharma","doi":"10.1016/j.exer.2025.110231","DOIUrl":"10.1016/j.exer.2025.110231","url":null,"abstract":"<div><h3>Purpose</h3><div>Keratin contamination is a common problem in mass spectrometry proteomic analyses, particularly in bottom-up mass spectrometry. The purpose of this study was to determine the protein contaminants introduced during the proteomic analysis of tear fluid.</div></div><div><h3>Methods</h3><div>Human tear fluid samples were collected using Schirmer strips. Proteomic analyses were performed using liquid chromatography–tandem mass spectrometry (LC-MS/MS) on blank Schirmer strips and tear fluid samples, with empty vials serving as controls for assessing environmental contaminant proteins.</div></div><div><h3>Results</h3><div>We detected 26 contaminant proteins (18 keratins and 8 non-keratins). 98.2% of the total protein contamination can be attributed to the 9 keratins, including KRT10 (23.6%), KRT1 (23.5%), KRT2 (15.7%), KRT14 (7.6%), KRT16 (7.0%), KRT5 (6.1%), KRT9 (5.9%), KRT6B (4.6%), and KRT6A (4.3%). A comparison to the proteomic profile of blank Schirmer strips and controls (empty vials) found a strong correlation (R<sup>2</sup> = 0.9753), indicating that these proteins were not from the blank Schirmer strips but are environmental contaminants. On the other hand, several keratins including KRT19, KRT13, KRT4, KRT7, KRT15, KRT8 and KRT18 were present in tear fluid, but either not detected or were negligible in blank strips. Another set of keratins, including KRT5, KRT6A, KRT14, KRT16, and KRT17, were identified as components of tear fluid as well as environmental contaminants.</div></div><div><h3>Conclusions</h3><div>This study revealed nine major contaminant keratins in the mass spectrometry analysis. Several other keratins were identified as constituents of tear fluid. Background subtraction is necessary for the accurate analysis of tear fluid using mass spectrometry.</div></div>","PeriodicalId":12177,"journal":{"name":"Experimental eye research","volume":"251 ","pages":"Article 110231"},"PeriodicalIF":3.0,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142947124","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Screening of a retinal-targeting Adeno-Associated Virus (AAV) via DNA shuffling

IF 3 2区医学 Q1 OPHTHALMOLOGY

Experimental eye research

Pub Date : 2025-02-01 DOI: 10.1016/j.exer.2025.110245

Yixin Yu , Xiangwei Zhou , Wei Peng , Yuan Wang , Mingzhu Li , Ying Zhu , Zicheng Song , Fei Wu , Chunsheng Dong

Due to its unique physiological structure and functions, the eye has received considerable attention in the field of adeno-associated virus (AAV) gene therapy. Inherited retinal degenerative diseases, which arise from pathogenic mutations in mRNA transcripts expressed in the eye's photoreceptor cells or retinal pigment epithelium (RPE), are the most common cause of vision loss. However, current retinal gene therapy mostly involves subretinal injection of therapeutic genes, which treats a limited area, entails retinal detachment, and requires sophisticated techniques. Intravitreal (IVT) injection provides an alternative method with less invasion and more convenience for retinal gene therapy. In the present study, we performed a directed evolution via DNA shuffling in RHO-GFP mice and identified a novel recombinant AAV vector (AAV-M04) suitable for IVT injection in the gene delivery of retinal tissue. Compared with AAV2, AAV9, and AAV2.7m8, AAV-M04 vector exhibited higher transduction efficiency in retinal ganglion cell line-5 (RGC-5) cells as well as in human embryonic stem cell derived retinal organoids. Importantly, when delivered via IVT injection in mice, the AAV-M04 vector also showed better delivery efficiency of transgene as indicated by the red fluorescence protein mScarlet. The red fluorescence was distributed in a wider retinal area of AAV-M04 injected mice, suggesting the potent retinal targeting of AAV-M04 vector. In addition, AAV-M04 qualities including the packaging efficiency, the thermal stability, and the capsid integrity were superior to controls, which were important in drug manufacture. In summary, we screened a novel AAV-M04 vector with great retinal-targeting via IVT injection, which provides the potential of AAV-M04 for effective gene therapy of retinal diseases.

{"title":"Screening of a retinal-targeting Adeno-Associated Virus (AAV) via DNA shuffling","authors":"Yixin Yu , Xiangwei Zhou , Wei Peng , Yuan Wang , Mingzhu Li , Ying Zhu , Zicheng Song , Fei Wu , Chunsheng Dong","doi":"10.1016/j.exer.2025.110245","DOIUrl":"10.1016/j.exer.2025.110245","url":null,"abstract":"<div><div>Due to its unique physiological structure and functions, the eye has received considerable attention in the field of adeno-associated virus (AAV) gene therapy. Inherited retinal degenerative diseases, which arise from pathogenic mutations in mRNA transcripts expressed in the eye's photoreceptor cells or retinal pigment epithelium (RPE), are the most common cause of vision loss. However, current retinal gene therapy mostly involves subretinal injection of therapeutic genes, which treats a limited area, entails retinal detachment, and requires sophisticated techniques. Intravitreal (IVT) injection provides an alternative method with less invasion and more convenience for retinal gene therapy. In the present study, we performed a directed evolution via DNA shuffling in RHO-GFP mice and identified a novel recombinant AAV vector (AAV-M04) suitable for IVT injection in the gene delivery of retinal tissue. Compared with AAV2, AAV9, and AAV2.7m8, AAV-M04 vector exhibited higher transduction efficiency in retinal ganglion cell line-5 (RGC-5) cells as well as in human embryonic stem cell derived retinal organoids. Importantly, when delivered via IVT injection in mice, the AAV-M04 vector also showed better delivery efficiency of transgene as indicated by the red fluorescence protein mScarlet. The red fluorescence was distributed in a wider retinal area of AAV-M04 injected mice, suggesting the potent retinal targeting of AAV-M04 vector. In addition, AAV-M04 qualities including the packaging efficiency, the thermal stability, and the capsid integrity were superior to controls, which were important in drug manufacture. In summary, we screened a novel AAV-M04 vector with great retinal-targeting via IVT injection, which provides the potential of AAV-M04 for effective gene therapy of retinal diseases.</div></div>","PeriodicalId":12177,"journal":{"name":"Experimental eye research","volume":"251 ","pages":"Article 110245"},"PeriodicalIF":3.0,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143028473","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0