Abstract
To investigate the role of vascular endothelial growth factor (VEGF) at different phases of diabetic retinopathy (DR), we assessed the retinal protein expression of VEGF-A164 (corresponding to the VEGF165 isoform present in humans, which is the predominant member implicated in vascular hyperpermeability and proliferation), HIF-1α and PKCβ/HuR pathway in Ins2 Akita (diabetic) mice at different ages. We used C57BL6J mice (WT) at different ages as control. Retina status, in terms of tissue morphology and neovascularization, was monitored in vivo at different time points by optical coherence tomography (OCT) and fluorescein angiography (FA), respectively. The results showed that VEGF-A164 protein expression increased along time to become significantly elevated (p < 0.05) at 9 and 46 weeks of age compared to WT mice. The HIF-1α protein level was significantly (p < 0.05) increased at 9 weeks of age, while PKCβII and HuR protein levels were increased at 46 weeks of age compared to WT mice. The thickness of retinal nerve fiber layer as measured by OCT was decreased in Ins2 Akita mice at 9 and 46 weeks of age, while no difference in the retinal vasculature were observed by FA. The present findings show that the retina of the diabetic Ins2 Akita mice, as expected for mice, does not develop proliferative retinopathy even after 46 weeks. However, diabetic Ins2 Akita mice recapitulate the same evolution of patients with DR in terms of both retinal neurodegeneration and pro-angiogenic shift, this latter indicated by the progressive protein expression of the pro-angiogenic isoform VEGF-A164, which can be sustained by the PKCβII/HuR pathway acting at post-transcriptional level. In agreement with this last concept, this rise in VEGF-A164 protein is not paralleled by an increment of the corresponding transcript. Nevertheless, the observed increase in HIF-1α at 9 weeks indicates that this transcription factor may favor, in the early phase of the disease, the transcription of other isoforms, possibly neuroprotective, in the attempt to counteract the neurodegenerative effects of VEGF-A164. The time-dependent VEGF-A164 expression in the retina of diabetic Ins2 Akita mice suggests that pharmacological intervention in DR might be chosen, among other reasons, on the basis of the specific stages of the pathology in order to pursue the best clinical outcome.
Highlights
Diabetic retinopathy (DR) is the major eye complication of diabetes mellitus and represents the leading cause of preventable blindness in the working age population, where roughly 90% of patients with type 1 diabetes and approximately 80% with type 2 diabetes for over 10 years will face the disease (Campbell and Doyle, 2019)
No agedependent changes in vascular endothelial growth factor (VEGF)-A164 protein basal levels, measured in an independent set of experiments, were observed among wild-type mice (Figure 3)
This altered milieu induces changes at microvascular level that result in the inability of capillaries to guarantee to the retina the proper blood supply, entailing the formation of non-perfused areas and the development of a hypoxic environment that promotes the production of VEGFA, a pivotal player in DR pathophysiology (Rigo et al, 2020)
Summary
Diabetic retinopathy (DR) is the major eye complication of diabetes mellitus and represents the leading cause of preventable blindness in the working age population, where roughly 90% of patients with type 1 diabetes and approximately 80% with type 2 diabetes for over 10 years will face the disease (Campbell and Doyle, 2019). Hallmarks of DR are the increasing thickness of the basement membrane, the hyper-permeability, and the formation of microaneurysms These functional alterations are followed by microvascular occlusions leading to a progressive retinal ischemia that induces the expression of the Vascular Endothelial Growth Factor-A (VEGF-A). VEGF-A165 is a potent mitogen for endothelial cells triggering their proliferation, migration and tube formation resulting in the growth of new blood vessels along the inside surface of the retina and in the vitreous that, in the diabetic retina are fragile and may break (Antonetti et al, 2012) These events may entail vitreous hemorrhage, subsequent fibrosis, and tractional retinal detachment with risk of permanent vision loss in the affected eye (Dulull et al, 2019). Analysis of VEGF-A interaction with binding domains of anti-angiogenic agents used in clinical practice is crucial in order to improve the design of new drugs (Platania et al, 2015)
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