Abstract
Ocular vascular dysfunction is a major contributing factor to the pathogenesis of glaucoma. In recent years, there has been a renewed interest in the role of angiotensin II (Ang II) in mediating the disease progression. Despite its (patho)physiological importance, the molecular mechanisms underlying Ang II-mediated oxidative stress remain largely unexplored in the ocular vasculature. Here, we provide the first direct evidence of the alterations of proteome and signalling pathways underlying Ang II-elicited oxidative insult independent of arterial pressure changes in the ophthalmic artery (OA) and retina (R) employing an in vitro experimental model. Both R and OA were isolated from male C57Bl/6J mice (n = 15/group; n = 5/biological replicate) and incubated overnight in medium containing either vehicle or Ang II (0.1 μM) at physiological conditions. Label-free quantitative mass spectrometry (MS)-based proteomics analysis identified a differential expression of 107 and 34 proteins in the R and OA, respectively. Statistical and bioinformatics analyses revealed that protein clusters involved in actin cytoskeleton and integrin-linked kinase signalling were significantly activated in the OA. Conversely, a large majority of differentially expressed retinal proteins were involved in dysregulation of numerous energy-producing and metabolic signalling pathways, hinting to a possible shift in retinal cell bioenergetics. Particularly, Ang II-mediated downregulation of septin-7 (Sept7; p < 0.01) and superoxide dismutase [Cu-Zn] (Sod1; p < 0.05), and upregulation of troponin T, fast skeletal muscle (Tnnt3; p < 0.05) and tropomyosin alpha-3 chain (Tpm3; p < 0.01) in the OA, and significant decreased expressions of two crystallin proteins (Cryab; p < 0.05 and Crybb2; p < 0.0001) in the R were verified at the mRNA level, corroborating our proteomics findings. In summary, these results demonstrated that exogenous application of Ang II over an acute time period caused impairment of retinal bioenergetics and cellular demise, and actin cytoskeleton-mediated vascular remodelling in the OA.
Highlights
The ocular system is an exceptionally metabolically active organ and despite its relatively small size, it ranks among the highest energy consumers, far exceeding the metabolic rate of the brain [1]
Combined bottom-up LC-mass spectrometry (MS)/MS proteomics analysis of triplicates of both groups identified a total of 592 and 985 proteins from the ophthalmic artery and retina, respectively, at less than 1% (< 1%) false discovery rate (FDR)
There have been considerable efforts in the past decades directed towards the study of angiotensin II (Ang II)-induced oxidative stress and the underlying alterations in the functionality of various blood vessels, which have steadily expanded our understanding of the role of Ang II in different tissues and cells [30,31,32,33,34]
Summary
The ocular system is an exceptionally metabolically active organ and despite its relatively small size, it ranks among the highest energy consumers, far exceeding the metabolic rate of the brain [1]. A salient feature in energy homeostasis involves the complex ocular vasculature. Blood vessels are highly dynamic components of the circulatory system that constantly undergo functional adaptation to various stimuli via vascular remodelling in order to supply blood to the different components of the eye with maximum efficiency. It has been elegantly demonstrated that any imbalance and/or deficiencies in the vasculature of the visual system culminate in disease conditions [7,8,9]
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