Hernandez Maria, Romero-Vázquez Sara, Recalde Sergio, Bezunartea Jaione, Orduña Maite Moreno, Belza-Zuazu Idoia, García-Layana Alfredo, Adán Alfredo, Fernández-Robredo Patricia, Molins Blanca
Retinal Pathologies and New Therapies Group, Experimental Ophthalmology Laboratory, Department of Ophthalmology, Clínica Universidad de Navarra, Pamplona, Spain.
Navarra Institute for Health Research, IdiSNA, Pamplona, Spain.
Sci Rep. 2025 Aug 26;15(1):31408. doi: 10.1038/s41598-025-16631-z.
Choroidal neovascularization (CNV) and inflammation play an important role in retinal disease development and the acute phase reactant C-reactive protein (CRP) has been shown to contribute to Age-related macular degeneration (AMD) in vitro. Our aim was to evaluate whether monomeric and pentameric CRP (pCRP, mCRP) isoforms contribute to CNV in vivo and to characterize the mechanism of CRP dissociation in-vivo and in vitro. Both CRP isoforms were intravitreally (IVT) or intravenously (IV) injected in mice, CNV was laser-induced, retinography and fluorescein angiography were performed to evaluate edema. Lectin, mCRP, F4/80 and C5b9 localization were assessed by immunofluorescence and visualized under a confocal microscope. CNV, intensity of fluorescence of mCRP (IF mCRP) was also quantified. To confirm pCRP dissociation in RPE cells and mice, pCRP was coupled to a fluorochrome and IVT injected. A statistical increase in CNV areas was observed in pCRP IVT injected males (p < 0.05) while a statistical decrease was shown in females (p < 0.05). After IV injection, pCRP males showed an increase in CNV areas only vs. mCRP injected mice (p < 0.05) and in females the injection of pCRP injected mice showed higher CNV areas vs. vehicle (p < 0.05) and vs. mCRP injected mice (p < 0.05). Retinal edema after IVT CRP injection was observed mainly in mCRP injected mice. In females there was an IF mCRP statistical decrease in pCRP IVT injected mice vs. vehicle and a statistical increase in pCRP IV injected mice vs. vehicle (p < 0.05). Mice injected with IVT isoforms showed F4/80 positive cells and C5b-9 deposition around the CNV areas. mCRP labeling was observed in the intercellular space of the endothelial cells in the angiogenic area and detected in pCRP IVT injected animals, demonstrating the dissociation of pCRP into mCRP both in vitro and in vivo in proinflammatory microenvironments. In conclusion, CRP administration increased the area of CNV and the edema observed in the subretinal space, suggesting that CRP is activated in the CNV inflammatory environment. In addition, we demonstrated that pCRP dissociates in vivo into mCRP in damaged areas close to CNV, hypothesizing that the CNV process is exacerbated by mCRP.
脉络膜新生血管（CNV）和炎症在视网膜疾病的发展中起重要作用，并且急性期反应物C反应蛋白（CRP）已在体外实验中被证明与年龄相关性黄斑变性（AMD）有关。我们的目的是评估单体和五聚体CRP（pCRP，mCRP）异构体在体内是否对CNV有影响，并阐明CRP在体内和体外解离的机制。将两种CRP异构体分别通过玻璃体腔注射（IVT）或静脉注射（IV）到小鼠体内，通过激光诱导产生CNV，然后进行视网膜造影和荧光素血管造影以评估水肿情况。通过免疫荧光评估凝集素、mCRP、F4/80和C5b9的定位，并在共聚焦显微镜下观察。同时对CNV以及mCRP的荧光强度（IF mCRP）进行定量分析。为了证实pCRP在视网膜色素上皮（RPE）细胞和小鼠体内的解离情况，将pCRP与一种荧光染料偶联后进行玻璃体腔注射。观察到玻璃体腔注射pCRP的雄性小鼠CNV面积有统计学意义的增加（p < 0.05），而雌性小鼠则有统计学意义的减少（p < 0.05）。静脉注射后，pCRP雄性小鼠的CNV面积仅与注射mCRP的小鼠相比有增加（p < 0.05），在雌性小鼠中，注射pCRP的小鼠与注射赋形剂的小鼠相比以及与注射mCRP的小鼠相比，CNV面积均有更高的增加（p < 0.05）。玻璃体腔注射CRP后，视网膜水肿主要出现在注射mCRP的小鼠中。在雌性小鼠中，玻璃体腔注射pCRP的小鼠与注射赋形剂的小鼠相比，IF mCRP有统计学意义的降低，而静脉注射pCRP的小鼠与注射赋形剂的小鼠相比，IF mCRP有统计学意义的增加（p < 0.05）。注射IVT异构体的小鼠在CNV区域周围显示F4/80阳性细胞和C5b - 9沉积。在血管生成区域的内皮细胞胞间空间观察到mCRP标记，并在玻璃体腔注射pCRP的动物中检测到，这表明在促炎微环境中，pCRP在体内和体外均解离为mCRP。总之，给予CRP增加了CNV的面积以及视网膜下间隙中观察到的水肿，这表明CRP在CNV炎症环境中被激活。此外，我们证明pCRP在体内靠近CNV的受损区域解离为mCRP，推测CNV过程因mCRP而加剧。
