12-HETE/GPR31 induces endothelial dysfunction in diabetic retinopathy.

12-hydroxyeicosatetraenoic acid (12-HETE), a major metabolite of arachidonic acid, is converted by 12/15-lipoxygenase and implicated in diabetic retinopathy (DR). Our previous study demonstrated a positive correlation between 12-HETE and the prevalence of DR. However, reasons for the increased production of 12-HETE are unclear, and the underlying mechanisms through which 12-HETE promotes DR are unknown. This study aimed to elucidate the correlation between 12-HETE and DR onset, investigate potential mechanisms through which 12-HETE promotes DR, and seek explanations for the increased production of 12-HETE in diabetes. We conducted a prospective cohort study, which revealed that higher serum 12-HETE levels could induce DR. Additionally, G protein-coupled receptor 31 (GPR31), a high-affinity receptor for 12-HETE, was expressed in human retinal microvascular endothelial cells (HRMECs). 12-HETE/GPR31-mediated HRMEC inflammation occurred via the p38 MAPK pathway. 12-HETE levels were significantly higher in the retina of mice with high-fat diet (HFD)- and streptozotocin (STZ)-induced diabetes than in those with only STZ-induced diabetes and healthy controls. They were positively correlated with the levels of inflammatory cytokines in the retina, indicating that HFD could induce increased 12-HETE synthesis in patients with diabetes in addition to hyperglycemia. Conclusively, 12-HETE is a potential risk factor for DR. The 12-HETE/GPR31 axis plays a crucial role in HRMEC dysfunction and could be a novel target for DR prevention and control. Nevertheless, further research is warranted to provide comprehensive insights into the complex underlying mechanisms of 12-HETE in DR.

Knockdown of ChREBP ameliorates retinal microvascular endothelial cell injury and angiogenic responses in diabetic retinopathy.

PURPOSE: To examine whether and how carbohydrate response element-binding protein (ChREBP) plays a role in diabetic retinopathy. METHODS: Western blotting was used to detect ChREBP expression and location following high glucose stimulation of Human Retinal Microvascular Endothelial Cells (HRMECs). Flow cytometry, TUNEL staining, and western blotting were used to evaluate apoptosis following ChREBP siRNA silencing. Cell scratch, transwell migration, and tube formation assays were used to determine cell migration and angiogenesis. Diabetic models for wild-type (WT) and ChREBP knockout (ChKO) mice were developed. Retinas of WT and ChKO animals were cultivated in vitro with vascular endothelial growth factor + high glucose to assess neovascular development. RESULTS: ChREBP gene knockdown inhibited thioredoxin-interacting protein and NOD-like receptor family pyrin domain containing protein 3 expression in HRMECs, which was caused by high glucose stimulation, reduced apoptosis, hindered migration, and tube formation, and repressed AKT/mTOR signaling pathway activation. Compared with WT mice, ChKO mice showed suppressed high glucose-induced alterations in retinal structure, alleviated retinal vascular leakage, and reduced retinal neovascularization. CONCLUSIONS: ChREBP deficiency decreased high glucose-induced apoptosis, migration, and tube formation in HRMECs as well as structural and angiogenic responses in the mouse retina; thus, it is a potential therapeutic target for diabetic retinopathy.

Histologic correlates of "Choroidal abnormalities" in Neurofibromatosis type 1 (NF1).

Neurofibromatosis type 1 (NF1) is a rare autosomal dominant disorder characterized by proliferation of cells from neural crest origin. The most common manifestations are cutaneous, neurologic, skeletal and ocular. The distinction of NF1 from other syndromes with multiple café-au-lait macules may be difficult in the pediatric age group, and ocular findings, especially Lisch nodules (i.e., melanocytic hamartomas on the irides), are a useful, early diagnostic tool. In recent years, novel ocular manifestations descriptively referred to as "choroidal abnormalities", choroidal "hyperpigmented spots" and "retinal vascular abnormalities" have been recognized in NF1. Choroidal abnormalities (CA) appear as bright patchy nodules that can be best detected with near-infrared ocular coherence tomography imaging (NIR-OCT). Because of their high specificity and sensitivity for NF1, CA have been added as an ocular diagnostic criterion of NF1 as an alternative to Lisch nodules. Although CA are important ocular diagnostic criteria for NF1, the histologic correlates are controversial. We present the postmortem ocular pathology findings of an NF1 patient for whom clinical notes and ocular imaging were available. Findings in this patient included choroidal hyperpigmented spots on funduscopy and retinal vascular abnormalities, both of which have been reported to be closely associated with CA. Histologic examination of the eyes showed multiple clusters of melanocytes of varying sizes in the choroid. Pathologic review of 12 additional postmortem eyes from 6 NF1 patients showed multiple, bilateral choroidal melanocytic aggregates in all eyes. These findings suggest that the CA seen on NIR-OCT and the hyperpigmented spots seen clinically in NF1 patients are manifestations of multifocal choroidal melanocytic clusters, consistent with choroidal melanocytic hamartomas. Lisch nodules, often multiple, were present in all eyes with morphology that differed from the choroidal hamartomas. As such, although CA and Lisch nodules are melanocytic hamartomas, there are clear phenotypical differences in their morphologies.

Decreased retinal capillary density as a beneficial response to 24-week high-speed circuit resistant training in healthy older adults.

PURPOSE: To determine the changes in retinal microvascular density after a 24-week high-speed circuit resistance training program (HSCT) in healthy older adults. METHODS: Thirty healthy older adults were recruited and randomly assigned to either a training group (HSCT) or a non-training (CON) group. Fifteen subjects (age 73.3 ± 7.76 yrs) in the HSCT group exercised three times per week on non-consecutive days for 24 weeks. Fifteen subjects in the CON group (age 72.2 ± 6.04 yrs) did not have formal physical training. Both eyes of each subject were imaged using optical coherence tomography angiography (OCTA) at baseline and at the 24-week follow-up. The vessel densities of the retinal vascular network (RVN), superficial vascular plexus (SVP), and deep vascular plexus (DVP) were measured. RESULTS: There were no demographic differences between the study groups. There were significant decreases in the retinal vessel densities of RVN, SVP and DVP in the HSCT group (P < 0.05). However, there were no significant changes in all three vascular measurements in the CON group (P > 0.05), although the changes showed a decreasing trend. The decreased vessel densities were doubled in the HSCT group in comparison to the CON group. However, the differences between groups did not reach a significant level (P > 0.05). CONCLUSIONS: This is the first study to reveal the decreased retinal vessel densities as a possible imaging marker for the beneficial effects of the 24-week HSCT program in older adults.

High Vasohibin-2 expression correlated with autophagy in proliferative diabetic retinopathy.

Vasohibin-2 (VASH2) is confirmed to be associated with angiogenesis. To investigate the vitreous levels of VASH2 and how VASH2 induces angiogenesis in proliferative diabetic retinopathy (PDR), a total of 120 eyes were enrolled in this prospective and randomized controlled study and the vitreous level of VASH2 was quantified by Luminex liquid suspension chip. Vector systems were applied in human retinal microvascular endothelial cells (HRMECs) for VASH2 gene overexpression, along with interfering lentiviral vectors (VASH2-shRNA) for VASH2 gene silencing. Cell migration, autophagic flux, as well as the expression of α-tubulin, detyrosinated ⍺-tubulin, LC3 II/LC3 I, P62 were detected under normal, VASH2 overexpression, or interference conditions. The level of VASH2 in PDR patients was significantly higher (218.61 ± 30.14 pg/ml) than that in ERM/MH patients (80.78 ± 2.05 pg/ml) (P = 0.001). The migration ability of HRMECs was significantly increased in VASH2 overexpression group, while in the interfering group, the migration ability decreased. VASH2 increased the detyrosination of ⍺-tubulin. The high fluorescence intensity of autophagic flux showed an activation of autophagy in VASH2 overexpression group, which was also confirmed by the increase of LC3 II/LC3 I ratio and the decrease of P62. Collectively, the present study shows in PDR, vitreous level of VASH2 is higher. VASH2 promotes neovascularization by inducing autophagy, suggesting VASH2 could be a new anti-angiogenic drug target for PDR.

Research on the role of exosomes secreted by immortalized adipose-derived mesenchymal stem cells differentiated into pericytes in the repair of high glucose-induced retinal vascular endothelial cell damage.

Diabetic retinopathy, a leading cause of vision impairment, is marked by microvascular complications in the retina, including pericyte loss, a key indicator of early-stage disease. This study explores the therapeutic potential of exosomes derived from immortalized adipose-mesenchymal stem cells differentiated into pericyte-like cells in restoring the function of mouse retinal microvascular endothelial cells damaged by high glucose conditions, thereby contributing to the understanding of early diabetic retinopathy intervention strategies. To induce immortalized adipose-mesenchymal stem cells differentiation into pericyte-like cells, the study employed pericyte growth supplement. And confirmed the success of cell differentiation through the detection of α-smooth muscle actin and neural/glial antigen 2 expression by Western blot and immunofluorescence. Exosomes were isolated from the culture supernatant of immortalized adipose-mesenchymal stem cells using ultracentrifugation and characterized through Western blot for exosomal markers (CD9, CD81, and TSG101), transmission electron microscopy, and nanoparticle tracking analysis. Their influence on mouse retinal microvascular endothelial cells under high glucose stress was assessed through various functional assays. Findings revealed that exosomes, especially those from pericyte-like immortalized adipose-mesenchymal stem cells, were efficiently internalized by retinal microvascular endothelial cells and effectively counteracted high glucose-induced apoptosis. These exosomes also mitigated the rise in reactive oxygen species levels and suppressed the migratory and angiogenic properties of retinal microvascular endothelial cells, as demonstrated by Transwell and tube formation assays, respectively. Furthermore, they preserved endothelial barrier function, reducing hyperglycemia-induced permeability. At the molecular level, qRT-PCR analysis showed that exosome treatment modulated the expression of critical genes involved in angiogenesis (VEGF-A, ANG2, MMP9), inflammation (IL-1ß, TNF-α), gap junction communication (CX43), and cytoskeletal regulation (ROCK1), with the most prominent effects seen with exosomes from pericyte-like immortalized adipose-mesenchymal stem cells. High glucose increased the expression of pro-angiogenic and pro-inflammatory markers, which were effectively normalized post-exosome treatment. In conclusion, this research highlights the reparative capacity of exosomes secreted by pericyte-like differentiated immortalized adipose-mesenchymal stem cells in reversing the detrimental effects of high glucose on retinal microvascular endothelial cells. By reducing apoptosis, oxidative stress, inflammation, and abnormal angiogenic behavior, these exosomes present a promising avenue for therapeutic intervention in early diabetic retinopathy. Future studies can focus on elucidating the precise molecular mechanisms and exploring their translational potential in vivo.

Retinal microvascular function and incidence and trajectories of clinically relevant depressive symptoms: the Maastricht Study.

BACKGROUND: Cerebral microvascular dysfunction may contribute to depression via disruption of brain structures involved in mood regulation, but evidence is limited. We investigated the association of retinal microvascular function, a proxy for microvascular function in the brain, with incidence and trajectories of clinically relevant depressive symptoms. METHODS: Longitudinal data are from The Maastricht Study of 5952 participants (59.9 ± 8.5 years/49.7% women) without clinically relevant depressive symptoms at baseline (2010-2017). Central retinal arteriolar equivalent and central retinal venular equivalent (CRAE and CRVE) and a composite score of flicker light-induced retinal arteriolar and venular dilation were assessed at baseline. We assessed incidence and trajectories of clinically relevant depressive symptoms (9-item Patient Health Questionnaire score ⩾10). Trajectories included continuously low prevalence (low, n = 5225 [87.8%]); early increasing, then chronic high prevalence (early-chronic, n = 157 [2.6%]); low, then increasing prevalence (late-increasing, n = 247 [4.2%]); and remitting prevalence (remitting, n = 323 [5.4%]). RESULTS: After a median follow-up of 7.0 years (range 1.0-11.0), 806 (13.5%) individuals had incident clinically relevant depressive symptoms. After full adjustment, a larger CRAE and CRVE were each associated with a lower risk of clinically relevant depressive symptoms (hazard ratios [HRs] per standard deviation [s.d.]: 0.89 [95% confidence interval (CI) 0.83-0.96] and 0.93 [0.86-0.99], respectively), while a lower flicker light-induced retinal dilation was associated with a higher risk of clinically relevant depressive symptoms (HR per s.d.: 1.10 [1.01-1.20]). Compared to the low trajectory, a larger CRAE was associated with lower odds of belonging to the early-chronic trajectory (OR: 0.83 [0.69-0.99]) and a lower flicker light-induced retinal dilation was associated with higher odds of belonging to the remitting trajectory (OR: 1.23 [1.07-1.43]). CONCLUSIONS: These findings support the hypothesis that cerebral microvascular dysfunction contributes to the development of depressive symptoms.

tRNA-derived fragment tRF-30 propels diabetes-induced retinal microvascular complications by regulating STAT3 signaling.

Transfer RNA-derived fragments (tRFs) represent a novel class of non-coding RNA transcripts that possess specific biological functions. However, the involvement of tRFs in retinal microvascular diseases remains poorly understood. In this study, we aimed to reveal whether modulation of tRF-30 expression could attenuate pathological retinal neovascular diseases. Our findings demonstrate a significant upregulation of tRF-30 expression levels in both in vivo models of diabetic retinopathy (DR) and in vitro endothelial sprouting models. Conversely, inhibition of tRF-30 expression suppressed the formation of abnormal neovascularization in the retina in vivo, while reducing the proliferation and migration activity of retinal vascular endothelial cells in vitro. We also found that tRF-30 modulates retinal neovascularization through the tRF-30/TRIB3/signal transducer and activated transcription 3 signaling pathway. Furthermore, we validated a significant upregulation of tRF-30 expression levels in the vitreous humor of DR patients, with high levels of both validity and specificity in diagnostic testing. Collectively, our findings highlight a pro-angiogenic role for tRF-30 in DR. Intervening in the tRF-30 signaling pathway may represent a promising prevention and treatment strategy for retinal angiogenesis.

circZNF532 promotes endothelial-to-mesenchymal transition in diabetic retinopathy by recruiting TAF15 to stabilize PIK3CD.

Endothelial-to-mesenchymal transition (EndMT) is a pivotal event in diabetic retinopathy (DR). This study explored the role of circRNA zinc finger protein 532 (circZNF532) in regulating EndMT in DR progression. Human retinal microvascular endothelial cells (HRMECs) were exposed to high glucose (HG) to induce the DR cell model. Actinomycin D-treated HRMECs were used to confirm the mRNA stability of phosphoinositide-3 kinase catalytic subunit δ (PIK3CD). The interaction between TATA-box-binding protein-associated factor 15 (TAF15) and circZNF532/PIK3CD was subsequently analyzed using RNA immunoprecipitation (RIP), RNA pull-down. It was found that HG treatment accelerated EndMT process, facilitated cell migration and angiogenesis, and enhanced PIK3CD and p-AKT levels in HRMECs, whereas si-circZNF532 transfection neutralized these effects. Further data showed that circZNF532 recruited TAF15 to stabilize PIK3CD, thus elevating PIK3CD expression. Following rescue experiments suggested that PIK3CD overexpression partially negated the inhibitory effect of circZNF532 silencing on EndMT, migration, and angiogenesis of HG-treated HRMECs. In conclusion, our results suggest that circZNF532 recruits TAF15 to stabilize PIK3CD, thereby facilitating EndMT in DR.

Ocular biomarkers of cognitive decline based on deep-learning retinal vessel segmentation.

BACKGROUND: The current literature shows a strong relationship between retinal neuronal and vascular alterations in dementia. The purpose of the study was to use NFN+ deep learning models to analyze retinal vessel characteristics for cognitive impairment (CI) recognition. METHODS: We included 908 participants from a community-based cohort followed for over 15 years (the prospective KaiLuan Study) who underwent brain magnetic resonance imaging (MRI) and fundus photography between 2021 and 2022. The cohort consisted of both cognitively healthy individuals (N = 417) and those with cognitive impairment (N = 491). We employed the NFN+ deep learning framework for retinal vessel segmentation and measurement. Associations between Retinal microvascular parameters (RMPs: central retinal arteriolar / venular equivalents, arteriole to venular ratio, fractal dimension) and CI were assessed by Pearson correlation. P < 0.05 was considered statistically significant. The correlation between the CI and RMPs were explored, then the correlation coefficients between CI and RMPs were analyzed. Random Forest nonlinear classification model was used to predict whether one having cognitive decline or not. The assessment criterion was the AUC value derived from the working characteristic curve. RESULTS: The fractal dimension (FD) and global vein width were significantly correlated with the CI (P < 0.05). Age (0.193), BMI (0.154), global vein width (0.106), retinal vessel FD (0.099), and CRAE (0.098) were the variables in this model that were ranked in order of feature importance. The AUC values of the model were 0.799. CONCLUSIONS: Establishment of a predictive model based on the extraction of vascular features from fundus images has a high recognizability and predictive power for cognitive function and can be used as a screening method for CI.

Alcohol consumption and microvascular dysfunction: a J-shaped association: The Maastricht Study.

BACKGROUND: Microvascular dysfunction (MVD) is an important contributor to major clinical disease such as stroke, dementia, depression, retinopathy, and chronic kidney disease. Alcohol consumption may be a determinant of MVD. OBJECTIVE: Main objectives were (1) to study whether alcohol consumption was associated with MVD as assessed in the brain, retina, skin, kidney and in the blood; and (2) to investigate whether associations differed by history of cardiovascular disease or sex. DESIGN: We used cross-sectional data from The Maastricht Study (N = 3,120 participants, 50.9% men, mean age 60 years, and 27.5% with type 2 diabetes [the latter oversampled by design]). We used regression analyses to study the association between total alcohol (per unit and in the categories, i.e. none, light, moderate, high) and MVD, where all measures of MVD were combined into a total MVD composite score (expressed in SD). We adjusted all associations for potential confounders; and tested for interaction by sex, and history of cardiovascular disease. Additionally we tested for interaction with glucose metabolism status. RESULTS: The association between total alcohol consumption and MVD was non-linear, i.e. J-shaped. Moderate versus light total alcohol consumption was significantly associated with less MVD, after full adjustment (beta [95% confidence interval], -0.10 [-0.19; -0.01]). The shape of the curve differed with sex (Pinteraction = 0.03), history of cardiovascular disease (Pinteraction < 0.001), and glucose metabolism status (Pinteraction = 0.02). CONCLUSIONS: The present cross-sectional, population-based study found evidence that alcohol consumption may have an effect on MVD. Hence, although increasing alcohol consumption cannot be recommended as a policy, this study suggests that prevention of MVD may be possible through dietary interventions.

Aberrant SUMO2/3 modification of RUNX1 upon SENP1 inhibition is linked to the development of diabetic retinopathy in mice.

Our previous report established that RUNX family transcription factor 1 (RUNX1) promotes proliferation of mouse retinal microvascular endothelial cells (mRMECs) and exacerbates diabetic retinopathy (DR). However, the mechanism behind the upregulation of RUNX1 remains unclear. This study aims to investigate the possible correlation between histone SUMOylation and RUNX1 in DR, as well as the involved molecules. A mouse model of diabetes was induced by streptozotocin (STZ). These mice had increased retinal thickness and elevated production of inflammatory cytokines. Additionally, they showed elevated levels of SUMO1 and SUMO2/3, but reduced levels of SUMO specific peptidase 1 (SENP1) in retinal tissues. Co-immunoprecipitation and Western blot assays revealed that the RUNX1 protein was primarily modified by SUMO2/3, and SENP1 inhibited SUMO2/3 modification, thereby reducing RUNX1 expression. Overexpression of SENP1 alleviated symptoms in mice and alleviated inflammation. In vitro experiments demonstrated that the SENP1 overexpression suppressed the proliferation, migration, and angiogenesis of high-glucose-induced mRMECs. However, further overexpression of RUNX1 counteracted the alleviating effects of SENP1 both in vivo and in vitro. In conclusion, this study demonstrates that the downregulation of SENP1 in DR leads to SUMO2/3-dependent activation of RUNX1. This activation promotes proliferation of mRMECs and exacerbates DR symptoms in mice.

Inhibition of diabetes-induced Drp1 deSUMOylation prevents retinal vascular lesions associated with diabetic retinopathy.

Retinal microvascular endothelial cell (RMEC) injury plays an important role in the pathophysiology diabetic retinopathy (DR). The GTPase dynamin-related protein 1 (Drp1), crucial to mitochondrial dynamics, has been implicated in hyperglycaemia-induced microvascular damage. Moreover, Drp1 can be deSUMOylated by the enzyme sentrin/SUMO-specific protease 3 (SENP3). Whether SENP3/deSUMOylated Drp1 can aggravate DR is unclear. Therefore, we designed this experiment to investigate the role of SENP3/desumoylated Drp1 in DR in vitro and in vivo. Murine RMECs (mRMECs) were classified into a control (CON), high-glucose (HG) and high-glucose + SENP3-siRNA (HG-siRNA) groups. The SENP3 and SUMOylated/deSUMOylated drp1 levels, mitochondrial morphology, mitochondrial membrane potential (MMP) and apoptosis rate were evaluated. In vivo, mice were assigned to a normal, type 2 diabetic or type 2 diabetic SENP3-siRNA mouse groups. Then, blood-retinal barrier function and retinal tissue structure were evaluated. As compared to those in the control group, the SENP3 and Drp1 levels, degree of mitochondrial fragmentation, extent of MMP loss and apoptosis rate of mRMECs were significantly increased in the HG group. However, inhibited SENP3 expression increased the level of SUMOylated Drp1 in the mRMECs and reduced the hyperglycaemia-induced mitochondrial damage and apoptosis rate. These experimental results were confirmed by diabetic animal experiments showing that inhibited SENP3 expression attenuated the increase in retinal permeability and diabetic retinopathy, suggesting that SENP3/deSUMOylated Drp1 activation aggravated DR by disrupting mitochondrial dynamics and apoptosis. Furthermore, blocking SENP3 expression significantly attenuated RMEC damage and DR.

The interface targeting hnRNPA2B1 regulates the repression of transthyretin against human retinal microvascular endothelial cells in high-glucose environment.

BACKGROUND: The interaction between transthyretin (TTR) and heterogeneous nuclear ribonucleoprotein (hnRNP)A2B1 is involved in the neovascularization of human retinal microvascular endothelial cells (hRECs) under hyperglycemic conditions. However, whether the TTR-hnRNPA2B1 interface can be altered and how this protein-protein interaction and associated downstream pathways are regulated is unclear. METHODS: We performed homologous sequential analysis and binding energy assays using Discovery Studio and designed substitution targeting three fragments of the interface (fragment 1: aa 34-39, -RKAADD-; fragment 2, aa 61-68, -EEEFVEGI-; and fragment 3, aa 96-102, -TANDSGP-) to disrupt or stabilize the TTR-hnRNPA2B1 complex and were subjected to Co-immunoprecipitation analysis. To investigate the effect of TTR-hnRNPA2B1 interface alterations on the physiological properties of hRECs, we performed CCK-8, EdU, migration, wound healing and tube formation assays. To study the downstream genes, we performed qRT-PCR and western blot. RESULTS: Nineteen TTR substitutions were recombinantly expressed in soluble form, results indicated that reducing the binding energy stabilized the TTR-hnRNPA2B1, while increasing the binding energy had the opposite effect. The native TTR significantly prohibited the proliferation, DNA synthesis, migration and tube formation capacities of hRECs, while fragment 1 always reduced these effects. However, the I68R and D99R substitutions in fragments 2 and 3, respectively, increased the inhibitory effect of TTR. Furthermore, our qRT-PCR and western blot results showed that the expression and protein levels of STAT-4, miR-223-3p and FBXW7 were also regulated by the alteration of the TTR-hnRNPA2B1 interface. CONCLUSION: This work suggests that the formation of the TTR-hnRNPA2B1 complex plays vital role in hyperglycemia, and modification of this interface regulates the TTR-mediated inhibition of hREC neovascularization via the STAT-4/miR-223-3p/FBXW7 pathway. This mechanism could have important implications for diabetic retinopathy treatment.

Retinal microvascular complexity as a putative biomarker of biological age: a pilot study.

Physiological changes associated with aging increase the risk for the development of age-related diseases. This increase is non-specific to the type of age-related disease, although each disease develops through a unique pathophysiologic mechanism. People who age at a faster rate develop age-related diseases earlier in their life. They have an older "biological age" compared to their "chronological age". Early detection of individuals with accelerated aging would allow timely intervention to postpone the onset of age-related diseases. This would increase their life expectancy and their length of good quality life. The goal of this study was to investigate whether retinal microvascular complexity could be used as a biomarker of biological age. Retinal images of 68 participants ages ranging from 19 to 82 years were collected in an observational cross-sectional study. Twenty of the old participants had age-related diseases such as hypertension, type 2 diabetes, and/or Alzheimer's dementia. The rest of the participants were healthy. Retinal images were captured by a hand-held, non-mydriatic fundus camera and quantification of the microvascular complexity was performed by using Sholl's, box-counting fractal, and lacunarity analysis. In the healthy subjects, increasing chronological age was associated with lower retinal microvascular complexity measured by Sholl's analysis. Decreased box-counting fractal dimension was present in old patients, and this decrease was 2.1 times faster in participants who had age-related diseases (p = 0.047). Retinal microvascular complexity could be a promising new biomarker of biological age. The data from this study is the first of this kind collected in Montenegro. It is freely available for use.

25-hydroxyvitamin D3 inhibits oxidative stress and ferroptosis in retinal microvascular endothelial cells induced by high glucose through down-regulation of miR-93.

BACKGROUND: The decrease of vitamin D plays a critical role in diabetes mellitus (DM)-induced oxidative stress and vascular endothelial injury. Therefore, we investigated the effect and mechanism of 25-hydroxyvitamin D3 (25 (OH) D3) on oxidative stress and ferroptosis induced by high glucose in human retinal microvascular endothelial cells (hRMVECs). And the objective of this paper was to propose a new strategy for the prevention and treatment of diabetic retinopathy (DR). METHODS: First, hRMVECs were transfected with mimics NC or miR-93. After that, cells were treated with 100 nM / 500 nM 25 (OH) D3 and then cultured in a high glucose (30 mM) environment. Subsequently, qRT-PCR was employed to detect the expression level of miR-93; CCK-8 for the proliferation of cells in each group; biochemical tests for the level of intracellular reactive oxygen species (ROS), malondialdehyde (MDA), reduced glutathione (GSH) and ferrous ion (Fe2+); and Western blot for the expression of ferroptosis-related proteins glutathione peroxidase 4 (GPX4) and SLC7A11). RESULTS: Under a high glucose environment, 25 (OH) D3 at 100 nM/500 nM could significantly promote the proliferation of hRMVECs, remarkably decrease the level of intracellular ROS/MDA, and up-regulate the level of GSH. Besides, 25 (OH) D3 greatly reduced Fe2+ level in the cells while increased protein level of GPX4 and SLC7A11. Subsequently, we found that high glucose induced miR-93 expression, while 25 (OH) D3 markedly decreased high glucose-induced miR-93 overexpression. Furthermore, overexpression of miR-93 inhibited the functions of 25 (OH) D3 by activating ROS (ROS and MDA were up-regulated while GSH was down-regulated) and inducing Fe2+ (Fe2+ level was up-regulated while GPX4 and SLC7A11 level was down-regulated) in cells. CONCLUSION: 25 (OH) D3 may inhibit oxidative stress and ferroptosis in hRMVECs induced by high glucose via down-regulation of miR-93.

Retinopathy is associated with impaired cognition in patients undergoing peritoneal dialysis.

Objective: Previous studies have shown a relationship between retinopathy and cognition including population with and without chronic kidney disease (CKD) but data regarding peritoneal dialysis (PD) are limited. This study aims to investigate the relationship between retinopathy and cognitive impairment in patients undergoing peritoneal dialysis (PD). Methods: In this observational study, we recruited a total of 107 participants undergoing PD, consisting of 48 men and 59 women, ages ranging from 21 to 78 years. The study followed a cross-sectional design. Retinal microvascular characteristics, such as geometric changes in retinal vascular including tortuosity, fractal dimension (FD), and calibers, were assessed. Retinopathy (such as retinal hemorrhage or microaneurysms) was evaluated using digitized photographs. The Modified Mini-Mental State Examination (3MS) was performed to assess global cognitive function. Results: The prevalence rates of retinal hemorrhage, microaneurysms, and retinopathy were 25%, 30%, and 43%, respectively. The mean arteriolar and venular calibers were 63.2 and 78.5 µm, respectively, and the corresponding mean tortuosity was 37.7 ± 3.6 and 37.2 ± 3.0 mm-1. The mean FD was 1.49. After adjusting for age, sex, education, mean arterial pressure, and Charlson index, a negative association was revealed between retinopathy and 3MS scores (regression coefficient: -3.71, 95% confidence interval: -7.09 to -0.33, p = 0.03). Conclusions: Retinopathy, a condition common in patients undergoing PD, was associated with global cognitive impairment. These findings highlight retinopathy, can serve as a valuable primary screening tool for assessing the risk of cognitive decline.

tRNA-derived fragment tRF-1020 ameliorates diabetes-induced retinal microvascular complications.

Transfer RNA (tRNA)-derived fragments are the non-coding single-stranded RNAs involved in several physiological and pathological processes. Herein, we investigated the role of tRF-1020, a tRNA fragment, in diabetes-induced retinal microvascular complications. The results showed that the levels of tRF-1020 expression were down-regulated in diabetic retinal vessels and retinal endothelial cells following high glucose or H2 O2 stress. Overexpressing tRF-1020 led to decreased endothelial cell viability, proliferation, migration, and tube formation and alleviated retinal vascular dysfunction as shown by decreased retinal acellular capillaries, vascular leakage, and inflammation. By contrast, tRF-1020 silencing displayed the opposite effects. tRF-1020 regulated endothelial angiogenic functions and retinal vascular dysfunction by targeting Wnt signalling. Moreover, the levels of tRF-1020 expression were reduced in aqueous humour and vitreous samples of the patients with diabetic retinopathy. Collectively, tRF-1020 is a potential target for the diagnosis and treatment of diabetic retinopathy.

High-sugar high-fat treatment induces autophagy of retinal microvascular endothelial cells.

OBJECTIVE: To investigate the role of high-sugar high-fat treatment in inducing autophagy of rat retinal microvascular endothelial cells. METHODS: The optimal concentrations and time points of glucose and oxidized low-density lipoprotein (ox-LDL) in inducing rat retinal microvascular endothelial cells were determined by examining the proliferate rate by CCK-8 assay. They were divided into control group (blank control), model group (treatment of 50 mM glucose and 10 µg/ml ox-LDL for 24 h), chloroquine group (treatment of 20 µM chloroquine, 50 mM glucose and 10 µg/ml ox-LDL for 24 h), resveratrol group (treatment of 50 µM resveratrol, 50 mM glucose and 10 µg/ml ox-LDL for 24 h) and MITO-Tempol group (treatment of 20 µM MITO-Tempol, 50 mM glucose and 10 µg/ml ox-LDL for 24 h). Reactive oxygen species (ROS) level in rat retinal microvascular endothelial cells induced with high sugar high-fat treatment was measured by flow cytometry. In addition, protein levels of cathepsin B and cathepsin D in rat retinal microvascular endothelial cells induced with high sugar high-fat treatment were examined by immunofluorescence, and protein levels of LC3 A/B and the autophagy substrate P62 were detected by Western blot. RESULTS: Primary retinal microvascular endothelial cells were isolated from neonatal Sprague-Dawley (SD) rats. ROS level was significantly higher in model group than that of control group (P < 0.05). Compared with that of model group, ROS level was significantly reduced in chloroquine group and MITO-Tempol group, which was significantly elevated in resveratrol group (P < 0.05). Positive expressions of cathepsin B and cathepsin D were significantly reduced in model group than those of control group (P < 0.05). They were significantly elevated in chloroquine group and MITO-Tempol group, and reduced in resveratrol group than those of model group (P < 0.05). LC3 A/B and P62 were significantly upregulated in model group than those of control group (P < 0.05). Compared with those of model group, LC3 A/B and P62 were significantly downregulated in chloroquine group and MITO-Tempol group, and upregulated in resveratrol group (P < 0.05). CONCLUSION: High-sugar high-fat treatment induces autophagy of rat retinal microvascular endothelial cells, which can be intervened to a certain extent by chloroquine and MITO-Tempol.

Microvascular endothelial dysfunction in heart failure patients: An indication for exercise treatment?

BACKGROUND: Endothelial dysfunction represents a diagnostic marker to differentiate disease severity in chronic heart failure (CHF) patients. Retinal vessel phenotyping was applied in CHF patients as it has been acknowledged as a sensitive diagnostic tool to quantify microvascular health and overall cardiovascular risk. METHODS: The central retinal arteriolar (CRAE) and venular diameter equivalents (CRVE) as well as the retinal microvascular function, quantified by arteriolar (aFID) and venular flicker-light induced dilatation (vFID), were analyzed in 26 CHF patients. These data were compared with 26 age- and sex-matched healthy peers. The effects of an exercise intervention on retinal microvascular health in one CHF patient were investigated to demonstrate potentially beneficial effects of exercise treatment in a case report format as proof of concept. RESULTS: CHF patients showed narrower CRAE (170 ± 16 µm vs. 176 ± 16 µm, p = 0.237) and wider CRVE (217 ± 20 µm vs. 210 ± 17 µm, p = 0.152), resulting in a significantly lower arteriolar-to-venular diameter ratio (AVR, 0.79 ± 0.07 vs. 0.84 ± 0.06, p = 0.004) compared to controls. More strikingly, CHF patients showed significantly lower mean aFID (1.24 ± 1.14% vs. 3.78 ± 1.85%, p < 0.001) and vFID (2.89 ± 1.33% vs. 3.88 ± 1.83%, p = 0.033). Twelve weeks of exercise therapy induced wider CRAE (143 ± 1.0 µm vs. 153 ± 0.9 µm), narrower CRVE (183 ± 3.1 µm vs. 180 ± 2.4 µm) and improved aFID (0.67% vs. 1.25%) in a male 78 years old CHF patient. CONCLUSIONS: aFID is a sensitive diagnostic tool to quantify microvascular impairments in CHF patients. Exercise treatment in CHF patients has high potential to improve retinal microvascular health as a marker for vascular regeneration and overall risk reduction, which warrants further examination by randomized controlled trials.