Olga Simó-Servat

Usefulness of the Vitreous Fluid Analysis in the Translational Research of Diabetic Retinopathy

Diabetic retinopathy (DR) is the major cause of acquired blindness in working-age adults. Current treatments for DR (laser photocoagulation, intravitreal corticosteroids, intravitreal antivascular endothelial growth factor (VEGF) agents, and vitreo-retinal surgery) are applicable only at advanced stages of the disease and are associated with significant adverse effects. Therefore, new pharmacological treatments for the early stages of the disease are needed. Vitreous fluid obtained from diabetic patients undergoing vitreoretinal surgery is currently used to explore the events that are taking place in the retina for clinical research. However, several confounding factors such as vitreous haemorrhage and concentration of vitreous proteins should be considered in the analysis of the results. In this paper we will focus on the vitreous fluid as a tool for exploring the mediators of DR and in particular the molecules related to inflammatory pathways. In addition, their role in the pathogenesis of DR will be discussed. The usefulness of new technologies such as flow cytometry and proteomics in identifying new candidates involved in the inflammatory process that occurs in DR will be overviewed. Finally, a more personalized treatment based on vitreous fluid analysis aiming to reduce the burden associated with DR is suggested.

Genetics in Diabetic Retinopathy: Current Concepts and New Insights

There is emerging evidence which indicates the essential role of genetic factors in the development of diabetic retinopathy (DR). In this regard it should be highlighted that genetic factors account for 25-50% of the risk of developing DR. Therefore, the use of genetic analysis to identify those diabetic patients most prone to developing DR might be useful in designing a more individualized treatment. In this regard, there are three main research strategies: candidate gene studies, linkage studies and Genome-Wide Association Studies (GWAS). In the candidate gene approach, several genes encoding proteins closely related to DR development have been analyzed. The linkage studies analyze shared alleles among family members with DR under the assumption that these predispose to a more aggressive development of DR. Finally, Genome-Wide Association Studies (GWAS) are a new tool involving a massive evaluation of single nucleotide polymorphisms (SNP) in large samples. In this review the available information using these three methodologies is critically analyzed. A genetic approach in order to identify new candidates in the pathogenesis of DR would permit us to design more targeted therapeutic strategies in order to decrease this devastating complication of diabetes. Basic researchers, ophthalmologists, diabetologists and geneticists should work together in order to gain new insights into this issue.

Circulating Biomarkers of Diabetic Retinopathy: An Overview Based on Physiopathology

Diabetic retinopathy (DR) is the main cause of working-age adult-onset blindness. The currently available treatments for DR are applicable only at advanced stages of the disease and are associated with significant adverse effects. In early stages of DR the only therapeutic strategy that physicians can offer is a tight control of the risk factors for DR. Therefore, new pharmacological treatments for these early stages of the disease are required. In order to develop therapeutic strategies for early stages of DR new diagnostic tools are urgently needed. In this regard, circulating biomarkers could be useful to detect early disease, to identify those diabetic patients most prone to progressive worsening who ought to be followed up more often and who could obtain the most benefit from these therapies, and to monitor the effectiveness of new drugs for DR before more advanced DR stages have been reached. Research of biomarkers for DR has been mainly based on the pathogenic mechanism involved in the development of DR (i.e., AGEs, oxidative stress, endothelial dysfunction, inflammation, and proangiogenic factors). This review focuses on circulating biomarkers at both early and advanced stages that could be relevant for the prediction or detection of DR.

Topical administration of DPP-IV inhibitors prevents retinal neurodegeneration in experimental diabetes

Aims/hypothesisThe main aims of the present study were: (1) to assess the expression and content of dipeptidyl peptidase IV (DPP-IV) in human and db/db mouse retinas, and in human vitreous fluid; and (2) to determine whether the topical administration of the DPP-IV inhibitors (DPP-IVi) would prevent retinal neurodegeneration and vascular leakage in db/db mice by reducing endogenous glucagon-like peptide 1 (GLP-1) degradation.MethodsTo assess the expression and content of DPP-IV, human samples of vitreous fluid and retinas were obtained from participants with type 2 diabetes (nxa0=xa08) and age-matched non-diabetic individuals (nxa0=xa08), as well as from db/db (nxa0=xa072) and db/+ (nxa0=xa028) mice. The interventional study, which included 72 db/db mice, consisted of the topical administration (eye drops) of saxagliptin, sitagliptin or vehicle for 14xa0days. DPP-IV mRNA levels were assessed by RT-PCR, and protein content was measured by ELISA or western blotting. GLP-1 was assessed by immunofluorescence, and its downstream effector exchange protein activated by cAMP-1 (EPAC-1) was used as a measure of GLP-1 receptor activation. Retinal analyses were performed in vivo by electroretinography and ex vivo by RT-PCR (Epac-1, Iba-1 [also known as Aif1]), western blotting (EPAC-1, glial fibrillar acidic protein [GFAP], glutamate−aspartate transporter [GLAST]) and immunofluorescence measurements (GLP-1, GFAP, ionised calcium binding adaptor molecule 1 [IBA-1], TUNEL, GLAST, albumin and collagen IV). Glutamate was quantified by HPLC. In addition, vascular leakage was examined by the Evans Blue method.ResultsDPP-IV was present in human vitreous fluid but in a range 100-fold less than in plasma. Both mRNA levels and protein content were much lower in the retina than in the liver or bowel, but were significantly higher in retinal pigment epithelium (RPE) from diabetic donors in comparison to non-diabetic donors (pxa0<xa00.05). Topical treatment with DPP-IVi prevented glial activation, apoptosis and vascular leakage induced by diabetes in db/db mice (pxa0<xa00.05). Moreover, it also significantly prevented diabetes-induced functional abnormalities in the electroretinogram. A significant increase of both GLP-1 and EPAC-1 was found after treatment with DPP-IVi (pxa0<xa00.05). Furthermore, GLAST downregulation induced by diabetes was prevented, resulting in a significant reduction of extracellular glutamate concentrations. All these effects were observed without any changes in blood glucose levels.Conclusions/interpretationThe topical administration of DPP-IVi is effective in preventing neurodegeneration and vascular leakage in the diabetic retina. These effects can be attributed to an enhancement of GLP-1, but other mechanisms unrelated to the prevention of GLP-1 degradation cannot be ruled out.

Proteomic Analysis of Early Diabetic Retinopathy Reveals Mediators of Neurodegenerative Brain Diseases

Purpose Current evidence suggests that retinal neurodegeneration is an early event in the pathogenesis of diabetic retinopathy. Our main goal was to examine whether, in the diabetic human retina, common proteins and pathways are shared with brain neurodegenerative diseases. Methods A proteomic analysis was performed on three groups of postmortem retinas matched by age: nondiabetic control retinas (n = 5), diabetic retinas without glial activation (n = 5), and diabetic retinas with glial activation (n = 5). Retinal lysates from each group were pooled and run on an SDS-PAGE gel. Bands were analyzed sequentially by liquid chromatography-mass spectrometry (LC/MS) using an Orbitrap Mass Spectrometer. Results A total of 2190 proteins were identified across all groups. To evaluate the association of the identified proteins with neurological signaling, significant signaling pathways belonging to the category “Neurotransmitters and Other Nervous System Signaling” were selected for analysis. Pathway analysis revealed that “Neuroprotective Role of THOP1 in Alzheimers Disease” and “Unfolded Protein Response” pathways were uniquely enriched in control retinas. By contrast, “Dopamine Degradation” and “Parkinsons Signaling” were enriched only in diabetic retinas with glial activation. The “Neuregulin Signaling,” “Synaptic Long Term Potentiation,” and “Amyloid Processing” pathways were enriched in diabetic retinas with no glial activation. Conclusions Diabetes-induced retinal neurodegeneration and brain neurodegenerative diseases, such as Alzheimers and Parkinsons diseases, share common pathogenic pathways. These findings suggest that the study of neurodegeneration in the diabetic retina could be useful to further understand the neurodegenerative processes that occur in the brain of persons with diabetes.

Mechanisms of retinal neuroprotection of calcium dobesilate: therapeutic implications

Current treatments for diabetic retinopathy (DR) are based on laser photocoagulation and intravitreal injections of corticosteroids or anti-vascular endothelial growth factor (VEGF) agents. These treatments are applicable only at advanced stages of the disease. In addition, they are expensive, require a vitreoretinal specialist and are associated with significant adverse effects. Therefore, new pharmacological treatments for the early stages of the disease are needed. Although DR is still often considered a microcirculatory disease of the retina, growing evidence suggests that retinal neurodegeneration is an early event in the pathogenesis of DR which participates in the microcirculatory abnormalities. For this reason, therapeutic strategies based on neuroprotection should be effective not only in preventing or arresting retinal neurodegeneration, but also in preventing the development and progression of the microvascular abnormalities that exist in the early stages of DR (Simó and Hernández, 2015). However, when the early stages of DR are the therapeutic target, it would be inconceivable to recommend an aggressive treatment such as intravitreal injections. Therefore, systemic treatments addressed to block the main pathways involved in the pathogenesis of DR are needed. However, most of these treatments can barely reach the retina at pharmacological concentrations and, in addition, could have serious adverse effects. Nevertheless, three classes of oral drugs have emerged as potential systemic treatments for DR: renin-angiotensin (RAS) system blockers, fenofibrate, and calcium dobesilate monohydrate (CaD). Regarding RAS, the current clinical evidence does not support the concept that they possess an extra value in preventing or arresting the progression of DR in hypertensive patients when compared with other anti-hypertensive agents. In the case of fenofibrate, several sub-studies have demonstrated its usefulness in arresting the progression of DR but not in preventing its development. Therefore, it seems reasonable to propose its use for patients with preexisting DR. However, fenofibrate for DR treatment has been approved only in Australia, Singapore, Philippines and Malaysia. By contrast CaD has been approved for the treatment of DR in numerous countries and clinical evidence supports its use in early stages of DR. Although CaD has been approved for the treatment of DR for many years, it has not been widely used in clinical practice. A poor understanding of its mechanisms of action has been one of the reasons for this. However, recent experimental evidence showing the neuroprotective action of CaD makes it a very attractive drug for early stages of DR. In fact, the American Diabetes Association in its more recent position statement defines DR as a “highly specific neurovascular complication” (Solomon et al., 2017). Therefore, drugs such as CaD which exert a multifaceted acction on neurovascular unit impairment can be contemplated as excellent candidates for targeting the early stages of DR. Systemic actions of CaD: The antioxidant and anti-inflammatory properties of CaD are among the systemic actions of CaD that could exert beneficial effects in DR. In addition, the beneficial hemorrheologic effects based on the evidence of CaD in reducing blood hyperviscosity and platelet aggregation facilitate capillary perfusion and reduce the inflammatory status.

Effects of Liposomal Formulation of Citicoline in Experimental Diabetes-Induced Retinal Neurodegeneration

Diabetic retinopathy (DR) has been classically considered a microcirculatory disease of the retina. However, there is growing evidence to suggest that retinal neurodegeneration is also an early event in the pathogenesis of DR. Citicoline has been successfully used as a neuroprotective agent in the treatment of glaucoma but their effects on DR remain to be elucidated. On this basis, the main aim of the present study was to evaluate the effect of topical administration of citicoline in liposomal formulation on retinal neurodegeneration in db/db mouse and to investigate the underlying mechanisms of action. The treatment (citicoline or vehicle) was topically administered twice daily for 15 days. Retinal analyses were performed in vivo by electroretinography and ex vivo by using Western blot and immunofluorescence measurements. We found that the liposomal formulation of citicoline prevented glial activation and neural apoptosis in the diabetic retina. The main mechanism implicated in these beneficial effects were the inhibition of the downregulation of synaptophysin and its anti-inflammatory properties by means of preventing the upregulation of NF-κB and TNF-α (Tumor Necrosis Factor α) induced by diabetes. Overall, these results suggest that topical administration of citicoline in liposomal formulation could be considered as a new strategy for treating the early stages of DR.

Assessment of advanced glycation end-products as a biomarker of diabetic outcomes

There are substantial differences in the onset and severity of diabetes complications that are not fully explained by HbA1c levels and other risk factors. HbA1c is the gold standard for assessing metabolic control, but has limited value to identify patients at risk of developing diabetic complications. The main disadvantage of HbA1c is that it does not provide information about glycemic variability and does not reflect long-term exposure to hyperglycemia. One of the main pathogenetic mechanisms of diabetic complications is the generation and accumulation of advanced glycation end-products (AGEs). Based on its fluorescence properties, AGEs may be measured in tissues such as the skin or lens. These non-invasive measurements of AGE accumulation may be considered as promising biomarkers of late diabetic complications, and our objective is to summarize the available evidence supporting this statement. However, further translational research and prospective clinical trials are needed before these new biomarkers may be incorporated into clinical practice.

Somatostatin and diabetic retinopathy: an evolving story

The story of how somatostatin (SST) has come to play an evolving role in the treatment of diabetic retinopathy (DR) has to be seen in the context of the growth of scientific knowledge regarding the pathophysiology of DR. The clinical observation published by Poulsen in 1953 [1] reporting that a patient with Sheehan’s syndrome presented a partial regression of proliferative diabetic retinopathy (PDR), and its post-mortem confirmation 13 years later [2] were essential preconditions for the theory that hypohysectomy could be a treatment for PDR. The studies published in 1970 by Merimee et al. [3] showing that GH (growth hormone) administration in diabetic patients with GH deficiency stimulated the development of DR led to the plausible mechanistic hypothesis that the effectiveness of hypophysectomy was mediated by decreased circulating levels of GH/insulin-like growth factor(IGF)-1. In fact, yttrium-90 pituitary implantation [4] and hypophysectomy [5] were proposed as treatments for PDR. However, it was unclear whether the observed benefit might have been related to improved blood glucose control after hypophysectomy rather than to the GH/IGF reduction itself. In addition, the results reported by Merimee et al. [3] could have been due to the worsening of metabolic control after GH administration. Furthermore, with the advances of treatment for PDR, such as laser photocoagulation, this type of aggressive approach became obsolete. The extensive investigation and clinical use of somatostatin analogues (SSA) over the past 30 years has provided a good opportunity to test again in a more appropriate and safer manner of the hypothesis that the inhibition of GH/IGF-1 could arrest the progression of DR. In the beginning of this century, pilot studies using SSA administered intramuscularly in patients with early PDR and severe non-proliferative DR (NPDR) revealed a decreased incidence of progression to PDR requiring panretinal laser treatment [6] or vitreo-retinal surgery [7]. However, a randomized double-blind placebo-controlled study with two arms (study 1 [NCT00131144] conducted in Europe using long-acting octreotide 20 and 30 mg, and another in North America and South America [NCT00130845] using longacting octreotide 30 mg) with an overall randomization of 899 patients completed in 2006 did not confirm these results [8]. This study revealed that long-acting octreotide given intramuscularly every 4 weeks in moderate-to-severe NPDR to low-risk PDR patients was unable to arrest DR progression significantly. The cohorts included in this clinical trial were recently used by Pivonello et al. [9] to evaluate the long-term safety of long-acting octreotide by means of a targeted post-hoc analysis. The authors confirmed the established cardiac, hepatic, and renal safety profile of longacting octreotide in diabetic patients with DR with a median duration of treatment of over 3.5 years. This is an important finding because DR is an independent predictor of diabetic nephropathy, cardiovascular disease, and stroke [10, 11], thus increasing even the risk of morbi-mortality of T2D subjects. Therefore, the lesson drawn from this paper regarding the safety of long-term administration of an SSA in a population with a high risk of suffering co-morbidities, is very relevant for the clinical practice. However, the reasons why long-acting octreotide failed to arrest the progression of DR remain to be elucidated. There are several important points which deserve to be commented on, and that could help us to shed light on this issue: 1) The role of circulating IGF-1 in the development and progression of DR is controversial, and this concept has not been supported by either large cross-sectional [12] or prospective clinical trials [13, 14]. In fact, acromegalic patients with diabetes do not present an increased incidence of DR, and when it appears it is not more aggressive [15]. * Rafael Simó rafael.simo@vhir.org

The ERM complex: a new player involved in diabetes-induced vascular leakage

BACKGROUNDnMicrovascular complications remain an important cause of morbidity in diabetic patients, and they are associated with a significant economic burden for healthcare systems. Vascular leakage is one of the earlier hallmarks in diabetic microvascular complications. Ezrin, radixin and moesin (ERM) proteins have recently been involved in vascular dysfunction under the effect of molecular mediators of diabetes complications. In this review we will present the available evidence regarding the role of these proteins in vascular leakage and their putative implication in diabetic microvascular complications.nnnMETHODS AND RESULTSnA comprehensive literature search of the electronic MEDLINE database was performed between November 2017 and January 2018. As a result, 36 articles have been reviewed and discussed.nnnDISCUSSIONnERM proteins are cytoskeleton-membrane linkers, and when activated in endothelial cells are able to induce cytoskeleton reorganization in stress fibers leading to the disassembly of focal adhesions and the formation of paracellular gaps which result in an increase of vascular permeability. The activation of these proteins is induced by mediators involved in diabetic complications such as PKC activation, TNF-α, AGEs and oxidative stress. In conclusion, ERMs play an essential role in endothelium homeostasis and can be envisaged as a new therapeutic molecular target for preventing or arresting diabetes-induced vascular leakage.