Discussion
The results of this study demonstrate that the immunity cluster predominates in cataracts, with a higher abundance of macrophage-derived Ti-EVs in high myopic cataracts, which strongly correlates with the AQP1 cluster. It was also observed that the eye morphogenesis cluster may also work in concert with AQP1, potentially driving the progression of high myopic cataracts through this pathway.
These findings were for the first time elucidate the mechanism of cataract formation from the perspective of Ti-EVs derived from the lens capsule. Ti-EVs constitute a specific class of extracellular vesicles that originate from and reside within the interstitial spaces of tissues. These vesicles are secreted by various cell types within tissues and play pivotal roles in intercellular communication and the regulation of tissue microenvironments. Notably, Ti-EVs derived from lens capsules exhibit remarkable tissue specificity, maintaining their identity despite the complex ocular environment. This specificity enables them to accurately reflect the pathophysiological characteristics and behaviors of the cells within their original tissues. Consequently, this tissue-specific nature of these Ti-EVs allows for a precise analysis of their components, which will facilitate a deeper understanding of the pathological processes underlying high myopic cataract.
In the realm of cataract research, such as high myopic cataract, the lens capsules hold a paramount significance, serving as a cornerstone in unraveling the complexities of the disease. However, the quantity of EVs that can be obtained from these tissues is minuscule due to their delicate thinness and minute size, making the efficient extraction and in-depth analysis of their contents an essential endeavor. To address this challenge, our research team innovatively adopted a cutting-edge methodology that integrates microfluidic technology with PBA. This combination allowed us to meticulously extract and meticulously analyze Ti-EVs. Leveraging the precision and sensitivity of these advanced techniques, we were able to achieve a remarkable breakthrough: successfully isolating a median of 6.24*10^7 particles/mL EVs from a mere single capsular membrane. Furthermore, utilizing the powerful PBA platform, we were able to detect and identify a comprehensive list of 264 proteins, providing us with invaluable insights into the composition and function of these EVs.
In our findings, the immunity cluster holds a dominant position in the development of cataracts. The interplay between immune response and cataract formation has been increasingly acknowledged. Immune responses involving cytokines and inflammatory mediators have been implicated in the process of cataract formation [20]. Recent research has underscored the significance of the immune disfunction caused by systemic inflammation, notably from conditions such as periodontitis, in intensifying cataract development [20-22]. In a large-scale national survey involving 11,205 participants, Huang et al. investigated the systemic immune-inflammation index (SII), which was determined by neutrophils, lymphocytes, and platelets. They observed that a high SII level exceeding 500*109/L was positively associated with cataract development among women, contributing valuable insights into the relationship between high SII levels and risk of cataract in adults in the United States [20]. Yeh et al. revealed that periodontitis can provoke systemic inflammation and oxidative stress, both of which are linked to the onset of various eye diseases, including cataracts [21]. This implies that immune disruptions triggered by oral microbiome from periodontitis may play a role in the oro-optic-network and promote the development of cataract.
In this study, it was observed that the lens capsule of patients with high myopic cataract contained a significantly higher abundance of macrophage-derived Ti-EVs. This finding is consistent with earlier research, which suggested that macrophage activation, recruitment, and the subsequent macrophage-mediated inflammation play pivotal roles in the development of lens opacification [23-25]. Previous studies have demonstrated that, in the aqueous humor of myopic eyes, there is an elevated presence of proinflammatory cytokines [24], such as interleukin-6 (IL-6) and matrix metalloproteinases (MMPs), which could create an environment that favors macrophage activation and recruitment. Similarly, the increased level of oxidative stress due to higher oxygen tension around the lens could also activate macrophages and other immune cells [25], leading to an influx of macrophages into the lens capsule. The activated macrophages, in turn, release EVs, which interacted with lens epithelial cells, inducing changes in their behavior, and contributing to the opacity of the lens. The precise mechanisms by which Ti-EVs mediate lens opacification are still being elucidated, but it is clear that the presence of these vesicles in the lens capsule is closely associated with the development of high myopic cataract.
Aquaporin 1 (AQP1) is a membrane-embedded water channel protein that play a crucial role in facilitating the passive transport of water molecules across cellular membranes along osmotic gradients. Studies have shown that AQP1 is involved in the maintenance of osmotic balance and the regulation of cellular hydration, making it an essential component for proper cellular function [26-28]. In the lens, AQP1 also performs a vital function by enabling the efficient movement of water. This facilitates the precise control of water content within lens fibers, which is critical for maintaining the lens’ transparency and refractive properties. The delicate balance of water movement regulated by AQP1 ensures that the lens remains clear and functions optimally, allowing for clear vision [29]. When the normal function of AQP1 is compromised, it can lead to alterations in the hydration state of lens fibers, resulting in the opacity of the lens [30]. An increased level of AQP1 protein expression was noted in the lens epithelial cells of cataract patients [31].
AQP1 is also known to play a crucial role in the regulation of macrophage function, particularly in modulating inflammatory responses. Studies have shown that AQP1 can attenuate macrophage-mediated inflammation by inhibiting the activation of p38 mitogen-activated protein kinase (MAPK) pathways, which are critical in the inflammatory response [32]. This suggests that AQP1 may help maintain a balance in macrophage polarization, potentially preventing excessive inflammation that could exacerbate conditions like high myopia and its associated cataracts [33]. In addition, the polarization of macrophages towards an M2 phenotype, which is associated with tissue repair and anti-inflammatory responses, can also be influenced by AQP1 levels [34], which would be beneficial for initial repair but may also lead to excessive inflammation and fibrosis if not properly regulated, contributing to cataract formation. We further analyzed AQP1 expression within macrophage subclusters and discovered a notable increase in the Eye morphogenesis cluster, which was characterized by high expression of HAVCR1, CPM, and MRC1, indicating potential collaboration between this cluster and AQP1.
Based on our findings, we postulate that the development of high myopic cataract could be attributed to a complex interplay of multiple factors, such as change in the ocular morphology, that disrupt the normal functioning of macrophages during the progression of high myopia. As a result, the altered macrophage function could lead to dysregulation in the expression of AQP1, which disrupt the lens homeostasis, promoting the onset and progression of cataract. Therefore, our hypothesis suggests a potential causal link between macrophage dysfunction, altered AQP1 expression, and the development of high myopic cataract, highlighting the need for further research to elucidate the underlying mechanisms and explore potential therapeutic interventions.
A key limitation of this study is the small sample size, which may restrict the generalizability of our findings. A larger sample size would be necessary to confirm the statistical significance and robustness of our results, allowing for more accurate and reliable conclusions to be drawn. In addition, the number of proteins that can be detected by PBA are limited, which prevented us from identifying all relevant proteins involved in the processes we investigated. Future research could explore the utilization of more advanced techniques or platforms, could provide a more comprehensive understanding of the biological mechanisms at play. Moreover, the potential mechanisms proposed in our study require further biological validation to confirm their validity. The markers identified need to undergo clinical validation to assess their therapeutic potential in real-world settings. This validation process is crucial for translating our research findings into practical applications that can benefit patients and improve clinical outcomes.
In summary, this study investigated the alteration in subpopulation patterns of single Ti-EVs derived from the lens capsules of patients with high myopic cataract and age-related cataract by PBA. By analyzing their protein profiling, we observed a significantly higher abundance of macrophage-derived Ti-EVs in high myopic cataracts, which strongly correlates with the AQP1 cluster. The interaction between macrophage and AQP1 may provide new insights into the underlying mechanisms driving the progression of high myopic cataracts.