Identification of potential molecular targets associated with proliferative diabetic retinopathy

Diabetic retinopathy (DR) is one of the most common microvascular complications in diabetes mellitus, and a primary cause of blindness and visual disturbance globally [1]. DR can be divided into non-proliferative and proliferative forms based on the type of microvascular lesions and related ischemic injury [2]. It has been predicted that the global prevalence of diabetes will continue to grow significantly for the next several decades, as a result of increasing incidence of type 1 diabetes rather than increasing type 2 diabetes which has already reached an epidemic level [3]. The incidence of DR increases with diabetic progression and results from a complex process with various molecular and biochemical participants, including oxidative stress, endoplasmic reticulum stress and mitochondrial damage amongst others [4]. The basic mechanisms underlying DR have been extensively studied, however, effective prevention and therapeutic interventions for this disease are still not available.

At present, DR treatments mainly focus on laser photocoagulation, intravitreal pharmacotherapy, anti-VEGF, and glycemic control [5]. Intravitreal anti-inflammatory agents have been effective in treating DR [6], and intravitreal triamcinolone acetonide shows an antiangiogenic effect toward proliferative DR [7]. Vascular endothelial growth factor (VEGF) plays a critical role in promoting vascular permeability, cell migration as well as proliferation of vascular endothelial cells, vasculogenesis, and angiogenesis [8]. VEGF is intimately involved with the progression of proliferative DR and diabetic macular edema facilitating changes in retinal capillary permeability, and advances in anti-VEGF therapies, in age-related macular degeneration, have accelerated the application of anti-VEGF therapies in DR [9]. Oxidative stress is a critical factor in the etiology of DR. Metabolic abnormalities, caused by the increase of glucose concentration in diabetes, can lead to the overproduction of the superoxide radical involved in the uncoupling of mitochondrial electron transport chains, ultimately resulting in oxidative stress [10, 11]. Antioxidants, including Vitamins and polyphenols, are considered a beneficial therapeutic strategy during the treatment of DR. These compounds inhibit the reactive oxygen species, free radicals and enhance the antioxidant defense system [12]. Despite some progress, DR remains a prevalent vision-threatening disease.

In this study, we used the microarray dataset “GSE60436” generated by Ishikawa K et al., [13]. In that study, they identified a subset of differentially expressed genes (DEGs) from active fibrovascular membranes (FVMs) and normal retinas or inactive FVMs and normal retinas, these DEGs were then evaluated by functional analysis. In our study, we aim to identify pivotal genes associated with the pathogenesis of proliferative DR and explore their function and upstream and downstream targets. The common DEGs from active/ inactive FVMs and normal retinas were identified, and then subjected to functional enrichment, protein-protein interaction (PPI) network and module analyses. In addition, the miRNA-target and transcription factors (TFs)-target were predicted to explore the potential regulatory relationships.

In this study, we identified 1475 co-DEGs from active/inactive-FVMs samples. The upregulated co-DEGs were found to participate in angiogenesis [Hypoxia Inducible Factor 1 Subunit Alpha (HIF1A) and Placental Growth Factor (PGF)] and mitosis-related processes (the genes in upregulated module-A, including KIF11 and BUB1). The downregulated co-DEGs were enriched in G − protein coupled receptor signaling and other pathways (The genes in downregulated module-A, including GNB3 and GNG8).

The important pathological features of proliferative DR including abnormal growth of retinal blood vessels and angiogenesis have all been linked to VEGF signaling. High blood sugar can trigger hypoxia in retinal tissues, and this hypoxia acts as a crucial factor in the regulation of VEGF-induced angiogenesis through the production of HIF [17]. HIF1A is an hypoxia induced TF and the lack of HIF1A suppresses the formation of the retinal intermediate vascular plexus in mouse models [18]. PGF, a homologue of VEGF, plays an important role in placental development, and has been shown to be involved in pathological angiogenesis both in ocular and non-ocular cancers [19]. In addition, increased expression of PGF has also been observed in the vitreous of DR patients [19]. Bender et al., revealed that PGF and VEGFA are both essential for follicular angiogenesis in primates, and neither can function alone and result in normal follicular angiogenesis [20]. Huang et al. showed that PGF negatively effects retinal endothelial cell barrier function by activating VEGFR1 and VEGFR2 and inhibiting glucose-6-phosphate dehydrogenase and the antioxidant pathways [21]. Ishikawa K et al., reported that the genes that were significantly upregulated in active FVMs were predominantly part of the angiogenesis process [13]. Therefore, we speculate that PGF and VEGF are important in the progression of FVM to proliferative DR.

MicroRNAs (miRNAs) are small non-coding RNAs of around 22 nucleotides (nt) in length, which mediate the expression of protein-coding genes, and are becoming increasingly popular biomarkers used in both prognostic and diagnostic assays [22, 23]. Targets for miR-136 and miR-374 were significantly enriched in the co-DEGs identified in this study. However, the relationship between miR-136 or miR-374 and DR is not well reported, and this is the first publication linking them. miR-136 is highly expressed in pre-eclampsia and inhibits the formation of HUVEC capillaries via dysregulation of VEGF. In addition, the expression levels of miR-136 inversely correlates with VEGF [24]. In paclitaxel-resistant ovarian cancer cells, pre-miR-136 transfection significantly decreased angiogenesis and induced apoptosis, and regulation of miR-136 expression could re-sensitize paclitaxel-resistant cells [25]. C-C Motif Chemokine Ligand 3 represses the expression of miR-374b, which subsequently accelerates VEGF-A level and angiogenesis in osteosarcoma cells [26]. Thus, we speculate that miR-136 and miR-374 may participate in the regulation of angiogenesis in proliferative DR.

KIF11 is a member of the kinesin-like protein family. Mutations in KIF11 have been shown to cause familial exudative vitreoretinopathy associated with retinal detachment [27]. Birtel et al., revealed that KIF11 is important in both ocular development and the maintenance of retinal morphology and function and defects in this protein have been associated with retinal ciliopathy [28]. KIFs are motor proteins involved in the activities of the centrosome and spindle during mitosis. In our study, KIFs (KIF11, KIF15, KIF20A) and other mitosis-associated genes, like BUB1, were significantly enriched in one module involved in cell division and mitotic nuclear division. The proliferation of retinal endothelial cells is a significant event in the progression of DR [29]. In addition, a previous study has shown that high glucose significantly increases the proliferation of retinal endothelial cells and the expression levels of VEGF, also, it was more effective under intermittent high glucose, than constant high glucose conditions [30]. Therefore, we suggest that the genes in this module play a key role in proliferative DR through the regulation of the proliferation of retinal endothelial cells.

G proteins are a kind of heterotrimeric guanine nucleotide-binding protein consisting of three subunits (alpha, beta and gamma subunits), which play an important role in intracellular signal transduction, including insulin, epinephrine, dopamine signaling molecule transduction [31]. GNB3 encodes G Protein Subunit Beta 3. The GNB3 gene polymorphisms, including C825T, have been linked to type 2 diabetes mellitus and its complications [32]. GNB3 knockout lead to the malfunction of cone photoreceptors and ON-bipolar cells in murine retinas [33]. In addition, it was revealed that a naturally occurring mutation in GNB3 results in retinal degeneration in chickens [34]. A homozygous missense variant in GNB3 results in a unique stationary retinal disorder with dual anomalies in visual processing, night blindness and photophobia [35]. G protein subunits (GNB3, GNB5, GNG3, GNG7, GNG8, GNG13) are downregulated in proliferative DR, and significantly enriched in module A which is associated with cellular responses to glucagon stimuli including G − protein coupled receptor signaling pathways and other biological processes. Therefore, we conclude that G proteins may play a crucial role in visual signal transduction in proliferative DR.

Although this study has made several novel observations, all the results were based on bioinformatics analysis, and further experimental verification is required. In addition, in an ideal situation, healthy retinas, non-proliferative diabetic retinas and autologous retinas should be used as controls for a more comprehensive comparison with retinas from subjects with FVM-associated proliferative DR. However, such microarray studies are limited by challenges in obtaining samples. Additionally, the small sample size in this study may also limit the application of our observations. Genes that are important for proliferative DR might be excluded as a result of the small sample size and inter-sample variation.

In brief, we investigated differentially expressed gene profiles in proliferative DR. Several genes, including HIF1A, PGF, KIF11, G protein subunits, and miR-136, miR-374 were found to be involved in angiogenesis, retinal endothelial cell proliferation, and visual signal transduction in proliferative DR. These findings could provide valuable insight and foundations for the study of proliferative DR’s pathomechanism and may provide an idea of potential therapeutic targets for its treatment.