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.