Retinal Endothelial Dysfunction Research Articles

Possible Role of Endothelial-Derived Cellular and Exosomal-miRNAs in Lipid-Mediated Diabetic Retinopathy: Microarray Studies

Diabetic retinopathy (DR) is a salient cause of blindness worldwide. There is still an immense need to understand the pathophysiology of DR to discover better diagnostic and therapeutic modalities. Human retinal endothelial cells (HRECs) were treated with 15-HETE or D-glucose, then miRNAs were isolated, and a microarray was performed. MirWALK 2 and Ingenuity Pathway Analysis (IPA) were used to analyze the microarray results. Exosomal miRNAs from 15-HETE-treated HRECs were isolated, microarrayed, and then imported into IPA for further analysis. The microarray results showed that 15-HETE downregulated 343 miRNAs and upregulated 297 miRNAs in HRECs. High glucose treatment induced a differential expression of HREC-miRNAs where 185 miRNAs were downregulated and 244 were upregulated. Comparing the impact of 15-HETE versus DG or diabetic mouse retina elaborated commonly changing miRNAs. Pathway and target analysis for miRNAs changed in 15-HETE-treated HRECs revealed multiple targets and pathways that may be involved in 15-HETE-induced retinal endothelial dysfunction. The HREC-exosomal miRNAs were differentially expressed after 15-HETE treatment, with 34 miRNAs downregulated and 45 miRNAs upregulated, impacting different cellular pathways. Here, we show that 15-HETE induces various changes in the cellular and exosomal miRNA profile of HRECs, highlighting the importance of targeting the 12/15 lipoxygenase pathway in DR.

Sulodeksyd w leczeniu retinopatii cukrzycowej

Diabetes mellitus is causing dysfunction of the vascular endothelial cells, which results in various pathologies within the microvascular and macrovascular system including retinopathy, neuropathy, nephropathy, and angiopathy. Endothelial dysfunction is caused by hyperglycemia via different mechanisms, such as damage to the glycocalyx, activation of the inflammation, oxidative stress, stimulation of cellular senescence, induction of imbalance between coagulation and fibrinolysis. Many studies suggests that inhibition of retinal endothelial dysfunction may be an effective way of slowing the progress of diabetic retinopathy. Sulodexide (glycosaminoglycans composed from heparin-like and dermatan fractions)is associated with a protective and regenerating action on the glycocalyx, slows down senescence of human venous and arterial endothelial cells. Moreover its mechanism of action involves an anti-inflammatory, anti-proteolytic, and antioxidant effect, including interference with metabolic and non-metabolic stress. In present study sulodexide was chosen to evaluate its therapeutic properties for the treatment of diabetic retinopathy.

Effects of tumor necrosis factor-α and interleukin-1β on human retinal endothelial cells

Uveitis, or intraocular inflammation, is a potentially blinding condition that mostly affects the working-age population. The cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β, play a role in the pathogenesis of non-infectious uveitis and have been linked to the breakdown of the inner blood-retinal barrier, composed mainly of retinal endothelial cells, leading to macular oedema and vascular leakage. However, the effects of TNF-α and IL-1β on human retinal endothelial function are not fully understood. In this work, we investigated the impact of TNF-α and IL-1β on several aspects of human retinal endothelial cell biology. Through a real-time biosensor, the impact of TNF-α and IL-1β on formation of a retinal endothelial cell barrier was analyzed. Changes in junctional components were assessed via RT-qPCR and immunolabelling. Cell survival, necrosis and apoptosis were appraised via cell proliferation and flow cytometric studies. Tumor necrosis factor-α and IL-1β impaired the electrical resistance of the retinal endothelial cell barrier, while the addition of a potentially barrier-impairing cytokine, IL-6, did not enhance the effect of TNF-α and IL-1β. Level of the gene transcript encoding zonula occludens (ZO)-1 was diminished, while ZO-1 protein configuration was changed by TNF-α and IL-1β. Both cytokines affected human retinal endothelial cell proliferation and viability, while only TNF-α increased rates of necrosis. These results indicate that TNF-α and IL-1β are important drivers of retinal endothelial dysfunction in non-infectious uveitis, suggesting that targeting these cytokines is critical when treating complications of uveitis, such as macular oedema and vascular leakage.

Intraocular Pressure-Induced Endothelial Dysfunction of Retinal Blood Vessels Is Persistent, but Does Not Trigger Retinal Ganglion Cell Loss.

Research has been conducted into vascular abnormalities in the pathogenesis of glaucoma, but conclusions remain controversial. Our aim was to test the hypothesis that retinal endothelial dysfunction induced by elevated intraocular pressure (IOP) persists after IOP normalization, further triggering retinal ganglion cell (RGC) loss. High intraocular pressure (HP) was induced in mice by episcleral vein occlusion (EVO). Retinal vascular function was measured via video microscopy in vitro. The IOP, RGC and their axons survival, levels of oxidative stress and inflammation as well as vascular pericytes coverage, were determined. EVO caused HP for two weeks, which returned to baseline afterwards. Mice with HP exhibited endothelial dysfunction in retinal arterioles, reduced density of RGC and their axons, and loss of pericytes in retinal arterioles. Notably, these values were similar to those of mice with recovered IOP (RP). Levels of oxidative stress and inflammation were increased in HP mice but went back to normal in the RP mice. Our data demonstrate that HP induces persistent endothelial dysfunction in retinal arterioles, which persists one month after RP. Oxidative stress, inflammation, and loss of pericytes appear to be involved in triggering vascular functional deficits. Our data also suggest that retinal endothelial dysfunction does not affect RGC and their axon survival.

High Glucose Aggravates Retinal Endothelial Cell Dysfunction by Activating the RhoA/ROCK1/pMLC/Connexin43 Signaling Pathway.

PurposeThis research aims to explore the mechanism underlying the relationship between RhoA/ROCK signaling and Connexin43 (Cx43) in retinal endothelial cell dysfunction and to evaluate the protective effect of ROCK inhibitors against retinal endothelial cell dysfunction in diabetic retinopathy (DR) models.MethodsTUNEL staining, hematoxylin and eosin staining, a retinal digestion assay, and Evans blue assay were conducted to explore the effect of fasudil in alleviating retinal dysfunction induced by DR. ELISA, the CCK-8 assay, and flow cytometry were conducted to study inflammation, viability, and apoptosis of mouse retinal microvascular endothelial cells treated with high glucose and ROCK inhibitors. The qRT–PCR and Western blotting were used to evaluate the expression of RhoA, ROCK1, ROCK2, MLC, pMLC, and Cx43. Co-immunoprecipitation was used to verify the interaction between pMLC and Cx43. Immunofluorescence and scrape-loading and dye transfer were used to evaluate the expression and function of Cx43.ResultsMarked endothelial cell dysfunction resulting from the activation of RhoA/ROCK1 signaling was found in in vivo and in vitro models of DR. Via interaction with pMLC, which is downstream of RhoA/ROCK1, a significant downregulation of Cx43 was observed in retinal endothelial cells. Treatment with ROCK inhibitors ameliorated retinal endothelial dysfunction in vitro. The ROCK inhibitor, fasudil, significantly alleviated retinal dysfunction as shown by a decrease of retinal acellular capillaries, an improvement of vascular permeability, and a reduction of cell apoptosis in vivo.ConclusionsOur study highlights a novel mechanism that high glucose could activate RhoA/ROCK1/pMLC signaling, which targets the expression and localization of Cx43 and is responsible for cell viability, apoptosis, and inflammation, resulting in retinal endothelial cell injury. ROCK inhibitors markedly ameliorate endothelial cell dysfunction, suggesting their therapeutic potential for diabetic retinopathy.

Adiponectin Ameliorates Hyperglycemia-Induced Retinal Endothelial Dysfunction, Highlighting Pathways, Regulators, and Networks.

BackgroundThe pathophysiology of diabetic retinopathy (DR) is multifaced. A low level of circulating adiponectin (APN) in type 2 diabetes is associated with microvasculature complications, and its role in the evolution of DR is complex.AimThis study is designed to explore the potential impact of APN in the pathogenesis of DR, linking the changes in cellular and biological processes with the pathways, networks, and regulators involved in its actions.MethodsHuman microvascular retinal endothelial cells (HMRECs) were exposed to 30mM glucose (HG) and treated with globular adiponectin (30μg/mL) for 24 hours. The cells were evaluated for reactive oxidative stress (ROS) and apoptosis. RT-PCR profile arrays were utilized to evaluate the profile of genes involved in endothelial functions, angiogenesis, extracellular matrix, and adhesion molecules for hyperglycemic HMRECs treated with adiponectin. In addition, the barrier function, leukocyte migration, and angiogenesis were evaluated. The differential expressed genes (DEGs) were outlined, and bioinformatic analysis was applied.ResultsAdiponectin suppresses ROS production and apoptosis in HMRECs under HG conditions. Adiponectin improved migration and barrier functions in hyperglycemic cells. The bioinformatic analysis highlighted that the signaling pathways of integrin, HMGB1, and p38 AMPK, are mainly involved in the actions of APN on HMRECs. APN significantly affects molecular functions, including the adhesion of cells, chemotaxis, migration of WBCs, and angiogenesis. STAT3, NFKB, IKBKB, and mir-8 are the top upstream regulators, which affect the expressions of the genes of the data set, while TNF and TGFB1 are the top regulators.ConclusionAdiponectin significantly counteracts hyperglycemia at various cellular and molecular levels, reducing its impact on the pathophysiological progression towards DR in vitro using HMRECs. Adiponectin ameliorates inflammatory response, oxidative stress, and endothelial barrier dysfunction using a causal network of NFBk complex, TNF, and HMGB1 and integrin pathways.

Trimetazidine mitigates high glucose-induced retinal endothelial dysfunction by inhibiting PI3K/Akt/mTOR pathway-mediated autophagy

ABSTRACT Trimetazidine (TMZ), as a metabolic regulator, has been widely testified to exhibit positive therapeutic effects on various disease models, but its role in diabetic retinopathy has not been reported. Therefore, this study was designed with the purpose of exploring the effects of TMZ on high-glucose (HG)-induced retinal endothelial dysfunction and its underlying mechanism. To establish DR model in vitro, 30 mM glucose was applied to induce human retinal endothelial cells (HRECs). Cell proliferation, invasion, and migration were examined by means of Cell Counting Kit-8, transwell, and wound healing assays, respectively. The tubule formation experiment was used to test the tubulogenesis ability and fluorescein isothiocyanate (FITC)-albumin was utilized to measure the permeability of monolayer HRECs. In addition, immunofluorescence and Western blot were employed to detect protein expression. Compared with the HG-induced group, TMZ concentration dependently inhibited the proliferation, migration, and angiogenesis of HG-induced HRECs, decreased the permeability of monolayer HRECs, and increased the protein expression levels of Claudin-5 and VE-cadherin. In addition, TMZ intervention increased the expression of p-PI3K, p-AKT, and p-mTOR but decreased the expression of LC3I, LC3II, and Beclin 1, which were then partially reversed by P13 K inhibitor (LY294002). Moreover, the autophagy agonist rapamycin (RAPA) was also testified to reverse the inhibitory effects of TMZ on the proliferation, migration, and angiogenesis of HG-induced HRECs. In summary, TMZ inhibited excessive autophagy by activating PI3K/Akt/mTOR pathway, thereby improving retinal endothelial dysfunction induced by HG.

Endothelial Dysfunction in Diabetic Retinopathy.

Diabetic retinopathy (DR) is a diabetic complication which affects retinal function and results in severe loss of vision and relevant retinal diseases. Retinal vascular dysfunction caused by multifactors, such as advanced glycosylation end products and receptors, pro-inflammatory cytokines and chemokines, proliferator-activated receptor-γ disruption, growth factors, oxidative stress, and microRNA. These factors promote retinal endothelial dysfunction, which results in the development of DR. In this review, we summarize the contributors in the pathophysiology of DR for a better understanding of the molecular and cellular mechanism in the development of DR with a special emphasis on retinal endothelial dysfunction.

Adipokines and endothelial dysfunction in obesity WHO°III

Obesity is closely associated with the metabolic syndrome (MetS) and subsequent low-grade inflammation links to endothelial dysfunction (ED) and cardiovascular disease. The impact of adipokines on retinal ED is not fully understood, in particular not in severe obesity. The aim of the study was to identify the association of the MetS and prespecified adipokines on retinal ED in obesity WHO°III. 92 obese patients (obesity WHO°III) were assessed for the MetS (IDF), neck circumference, adipokines and inflammatory markers (hsCRP, TNFα, Il-6, MCP-1, sICAM, sVCAM, IGF-BP3, RBP 4 and adiponectin). Retinal ED as determined by the arterio-venous-ratio (AVR) and retinal vessel diameters (CRAE, CRVE) was measured using retinal photographs. Obese subjects with MetS (MetS+ group) differed from the MetS− by neck circumference, fasting plasma glucose, insulin, HOMA-IR, triglycerides and HDL-C. Importantly, IL-6, sICAM and adiponectin were significantly different between groups, while measures of retinal ED showed no differences. Univariate linear regression revealed a significant association between neck circumference and ED for patients with MetS, and a significant association between adiponectin and CRAE for patients without MetS. This study shows that ED in obesity WHO°III is independent of MetS or inflammation and that neck circumference has an impact on ED in obesity WHO°III.

High prevalence of retinal endothelial dysfunction in obesity WHO class III

BackgroundThe extent of retinal endothelial dysfunction (ED) in patients with obesity is unknown. We evaluated markers of endothelial dysfunction to assess cardiovascular risk in patients with obesity WHO III° and their interrelation with classical cardiovascular risk factors. Methods120 patients (mean age 42.7±10.6years, 87 women) were prospectively evaluated for metabolic and cardiovascular risk using anthropometry, cardiovascular risk factors, lipid and glucose profiles. Intima media thickness (IMT) as marker of subclinical atherosclerosis, ED of retinal vessels, and the arteriole-to-venule ratio (AVR) of retinal vessels were assessed. ResultsThe mean BMI in our cohort was 48.7kg/m2. We diagnosed an overall prevalence of impaired glucose metabolism of 69.2%. 71.6% and 65.2% presented with arterial hypertension or dyslipidemia, respectively. Prevalences of retinal ED, pathologically reduced AVR, and enlarged IMT were 62.7%, 56.6% and 30%, respectively. Markers of endothelial function demonstrated correlation of neck to height ratio with dilatation of arteries (r=−0.333, p=0.01) and HDL cholesterol with dilatation of veins (r=−0.393, p=0.002). AVR was significantly related to neck circumference (r=−0.269, p=0.004). ConclusionRetinal ED, AVR, and IMT as direct noninvasive surrogate measures of cardiovascular risk showed a high prevalence in patients with obesity WHO III°. We found no association of classical parameters for metabolic or cardiovascular risk with markers of endothelial dysfunction. Therefore, we have to hypothesize that other factors also play a pivotal role in the development of vascular pathology in patients with obesity.

Association Between Diabetic Retinopathy and Flow-Mediated Vasodilation in Type 2 DM

Objective: Retinal endothelial dysfunction is a key in the etiogenesis of diabetic retinopathy (DR), in patients with type 2 diabetes mellitus (DM). Brachial artery flow-mediated vasodilation (FMD) is a marker of endothelial function associated with production of endogenous nitric oxide. Using FMD, we investigated the relationship between macrovascular function and DR.Methods: We studied 74 patients with type 2 DM, including non-DR (NDR) (n = 30); mild nonproliferative DR (NPDR) (n = 16); moderate NPDR (n = 10); severe NPDR (n = 10); and proliferative DR (PDR) (n = 8); and 21 age-matched controls. We measured FMD in each group. Retinal blood flow and pulsatility ratios were measured using laser Doppler velocimetry.Results: FMD decreased significantly in patients with DM compared with healthy control subjects. No significant differences were found in FMD among the NDR, mild NPDR, and moderate NPDR groups. FMD decreased significantly in the severe NPDR and PDR groups compared with the NDR group. FMD was significantly and negatively correlated with duration of DM and pulsatility ratio.Conclusion: Systemic endothelial dysfunction appears to be associated with DR and vascular abnormalities in patients with type 2 DM.

Hyperglycemia Impairs Acetylcholine-Induced Vasodilation of Retinal Arterioles Through Polyol Pathway–Independent Mechanisms in Rats

We previously reported that acetylcholine (ACh)–induced vasodilation of retinal arterioles is diminished in diabetic rats; however, the underlying mechanism(s) of this phenomenon has not been fully elucidated. To determine the role of the polyol pathway in the diabetes-induced retinal vascular dysfunction, we investigated the effect of GP-1447, an inhibitor of aldose reductase, on the attenuation of ACh-induced vasodilation of retinal arterioles seen in diabetic rats. Male Wistar rats were treated with streptozotocin (STZ) and experiments were performed 2 weeks later. The STZ-treated animals were given drinking water containing 5% d-glucose to shorten the term for the development of retinal vascular dysfunction. Treatment with GP-1447 was initiated immediately after STZ treatment and continued throughout the 2-week experimental period. The attenuation of retinal vascular responses to ACh were not modified by treatment with GP-1447, whereas the aldose reductase inhibitor completely prevented diabetes-induced thinning of the retina and sorbitol accumulation in the retina and the lens. These results suggest that mechanisms that are independent of the polyol pathway may contribute to the onset of retinal endothelial dysfunction, although the pathway plays an important role in morphological changes of retina and formation of cataracts in diabetic rats.

Acute Hyperglycemia-Induced Endothelial Dysfunction in Retinal Arterioles in Cats

To investigate the effects of acute hyperglycemia on retinal microcirculation and endothelial function in cats and removal of superoxide to prevent retinal endothelial dysfunction from hyperglycemia. Hyperglycemia was induced by intravenous injection of 25% glucose to maintain the plasma glucose concentration at 30 mM. Laser Doppler velocimetry was used to measure the vessel diameter (D) and blood velocity (V) simultaneously and calculated retinal blood flow (RBF) in second-order retinal arterioles in cats. Intravitreous, endothelial-dependent vasodilator bradykinin (BK) and endothelium-independent vasodilator sodium nitroprusside (SNP) were administered into the vitreous cavity to evaluate endothelial function in the retinal arterioles. To control osmolality, 25% mannitol was administered the same way. Systemic hyperoxia was induced to noninvasively examine endothelial function during hyperglycemia. To determine the effect of the superoxide on the hyperglycemia-induced changes in the retinal circulation, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) was administered in drinking water for 14 days before the experiment. The D, V, and RBF increased with acute hyperglycemia and mannitol compared with baseline. BK-induced increases in D, V, and RBF significantly declined, whereas SNP-induced increases were unattenuated during acute hyperglycemia. Return of the decreased RBF to baseline after cessation of systemic hyperoxia was significantly (P < 0.05) inhibited by acute hyperglycemia. TEMPOL significantly (P < 0.05) prevented a decrease in the BK-induced increase in RBF during hyperglycemia. The results suggest that acute hyperglycemia increases RBF via increased osmolality and may cause retinal endothelial dysfunction partially via increased oxidative stress. Systemic hyperoxia can be used to noninvasively evaluate retinal endothelial function during hyperglycemia.

Hyperglycemia Accelerates Impairment of Vasodilator Responses to Acetylcholine of Retinal Blood Vessels in Rats

We previously reported that vascular endothelial functions in both retinal and systemic circulation are impaired 6-8 weeks after induction of hyperglycemia with streptozotocin (STZ) in rats. However, it remains to be elucidated whether the period required for the onset of endothelial dysfunction is different, depending on vascular beds and severity of hyperglycemia. In this study, we examined the effects of several vasodilators on the diameter of retinal blood vessel and blood pressure in Control, STZ (STZ treatment alone), and STZ+Glc (STZ treatment plus D-glucose feeding) rats. The overall structures of the retina and the retinal capillary network were also evaluated. The vasodilator effects of acetylcholine on retinal arterioles were significantly reduced in the STZ+Glc group, but not in the STZ group, 2 weeks after induction of hyperglycemia. There were no significant differences in acetylcholine-induced decreases in blood pressure among the three experimental groups. The responses to NOR3, forskolin, and adenosine were unaffected by hyperglycemia. The retinal thickness was significantly reduced in the STZ+Glc group. No significant changes were observed in the morphology and the density of retinal capillary network by immunohistochemical techniques. These results suggest that endothelium-dependent vasodilatory mechanisms of retinal arterioles are more vulnerable than those of peripheral resistance vessels to the effects of hyperglycemia. Hyperglycemia shortens the period required for onset of retinal endothelial dysfunction, depending on its severity.