Vasodilation is a defence mechanism during inflammation and infection that is regulated by nitric oxide produced by endothelial nitric oxide synthase (eNOS) and the generation of endothelium-dependent hyperpolarization. Several viral infections have been shown to alter vascular biology, but effects of bacterial pathogens are unclear. Here using an ex vivo blood vessel model and human primary endothelial cells, we show that Klebsiella pneumoniae, a prevalent bloodstream pathogen, inhibits vasodilation pathways. The type VI secretion system (T6SS) effector VgrG4 activates the mitochondrial receptor NLRX1, which leads to increased mitochondrial reactive oxygen species and phosphorylation of the eNOS inhibitory site by the kinase PKCβ, which in turn reduces eNOS activity. K. pneumoniae capsule polysaccharide also activates the phosphatase PP2Ac, which reduces phosphorylation of the eNOS activation site. VgrG4-induced mitochondrial reactive oxygen species attenuate endothelium-dependent hyperpolarization by impairing signalling of the Ca2+-activated K+ channel axis. This work reveals that T6SS activity can modulate host vascular biology by targeting eNOS post-translational modifications.

ABSTRACT Mitochondrial quality control (MQC) is essential for retinal homeostasis, yet how distinct mitophagy pathways are coordinated within specialized retinal cell types remains poorly understood. Here, we show that Müller glia engage distinct mitophagy programmes that are differentially activated across physiological, metabolic stress, and differentiation contexts. Using pathway-resolved analyses supported by mouse and human single-cell transcriptomic datasets, we demonstrate that PINK1-dependent and receptor-mediated mitophagy pathways coexist within Müller glia and exhibit distinct functional and spatial regulation. To enable precise, time-resolved interrogation of these processes, we developed MQ-MG2, a spontaneously immortalised Muller glial model stably expressing the Mito-QC reporter while preserving endogenous mitophagy adaptors and metabolic features of primary Muller cells. Using this system, we identify context-dependent activation of mitophagy pathways with spatial relevance in vivo and reveal transient coordination of PINK1-dependent and receptor-associated mitophagy during Muller glial neurogenic differentiation. Suppression of fission-dependent mitophagy impaired the acquisition of complex neurite features in MQ-MG2, with a comparable phenotype observed following targeted PINK1 deletion in human neurogenic cells. Together, these findings position Muller glia as active integrators of mitochondrial quality control, capable of engaging distinct mitophagy programmes according to cellular context.

mRNA localisation is a critical posttranscriptional mechanism that confers a spatiotemporal dimension to the control of gene expression. Among diverse outcomes, this process can result in compartmentalised protein synthesis and consequently, elicit localised cellular responses. Targeting mRNAs to their destination is often determined by localisation elements (LEs) contained in untranslated regions within targeted transcripts. Although mRNA localisation has been widely explored in the context of subcellular biology, its roles in tissue function are only just beginning to emerge. A defined set of transcripts accumulate at the leading edge of endothelial tip cells that guide emerging vessels during sprouting angiogenesis. This includes RAB13 and NET1 mRNAs, which encode proteins implicated in cytoskeletal remodelling processes underpinning cell motility. In this study, we tested the anti-angiogenic potential of antisense oligonucleotide (ASO)-based strategies designed to perturb RAB13 and NET1 localisation. Upon confirming that ASOs targeting LEs mislocalise these mRNAs without altering steady-state levels of the encoded proteins, we applied them to a series of in vitro, ex vivo and in vivo angiogenesis assays. Remarkably, the mislocalisation of RAB13 and NET1 inhibits chemotaxis and vessel sprouting in response to pro-angiogenic stimuli. Furthermore, vessel sprouting from mouse choroidal explants and retinal angiogenesis are also hindered by mRNA mislocalisation. Altogether, our strategy for disrupting spatial control of gene expression in endothelial cells opens new mechanistic avenues for the manipulation of vessel formation.

This study used serial block-face scanning electron microscopy (SBF-SEM), a nanoscale imaging technique in x-y-z planes, to investigate 3D ultrastructural changes in the retinal neurovascular unit (NVU) associated with diabetes. We hypothesised that this approach would reveal previously uncharacterised pathological alterations that contribute to the development of diabetic retinal disease (DRD). Retinas from male diabetic and non-diabetic mice, as well as from human male donors with and without diabetes, were prepared for SBF-SEM imaging. Retinal tissue was microdissected, fixed and embedded for serial sectioning and 3D reconstruction. Ultrastructural analysis of the NVU was performed in capillary regions exclusively within the superficial vascular plexus of both mouse and human retinas. Image stacks were processed using Microscopy Image Browser for contrast normalisation and segmentation, with 3D visualisation performed in Amira software. Quantitative analyses were conducted on pericyte–endothelial cell peg-and-socket formations, cell–basement membrane (BM) interactions, endothelial tubule formation and vascular BM thickness. SBF-SEM revealed novel 3D ultrastructural changes in the retinal NVU of diabetic mice and humans, including: (1) partial detachment and reduced frequency of pericyte–endothelium peg-and-socket formations (p<0.05–0.001); (2) localised detachment of endothelial cells and pericytes from the vascular BM (p<0.05–0.01), along with macroglial cell retraction from the outer vascular BM; and (3) increased formation of endothelial tubules (p<0.01–0.001). These changes were observed in the absence of any obvious vascular BM thickening, as no significant differences in mean or maximum BM thickness were found between diabetic and non-diabetic retinal capillaries analysed in this study. This study provides new insights into the early ultrastructural changes in the retinal NVU in DRD, offering a basis for a better understanding of the pathological processes that contribute to the development of this disease. Links to all raw image stacks analysed in this article are available at https://doi.org/10.5281/zenodo.15210333 . The MATLAB vascular BM thickness measurement script is available at the GitHub link https://github.com/Curtis-WWIEM/BM_thickness .

Diabetic retinal disease (DRD) is characterised by progressive neurovascular unit (NVU) dysfunction, often occurring before visible microvascular damage. Our previous studies suggested that the accumulation of acrolein (ACR)-derived protein adducts on retinal Müller cells and neuronal proteins may contribute to NVU dysfunction in diabetes, although this has yet to be directly tested. In this study, we evaluated the effects of the novel ACR-scavenging drug 2-hydrazino-4,6-dimethylpyrimidine (2-HDP) on retinal NVU dysfunction in experimental diabetes and explored its potential for systemic delivery in humans. Sprague Dawley rats were divided into three groups: non-diabetic rats; streptozocin (STZ)-induced diabetic rats; and STZ-induced diabetic rats treated with 2-HDP in their drinking water throughout the duration of diabetes. Endpoint measures were taken at varying time points, ranging from 1 to 6 months post-diabetes induction. Retinal function and structure were evaluated using electroretinography (ERG) and spectral-domain optical coherence tomography (SD-OCT). Retinal vessel calibre, BP and vasopermeability (assessed by Evans Blue leakage) were also monitored. Immunohistochemistry was employed to assess retinal neurodegenerative and vasodegenerative changes, while cytokine arrays were used to investigate the effect of 2-HDP on diabetes-induced retinal inflammation. The accumulation of the ACR–protein adduct Nε-(3-formyl-3,4-dehydropiperidino)lysine (FDP-Lys) in human diabetic retinas was analysed. Computational chemistry simulations were performed to predict 2-HDP’s passive permeability properties and its potential for systemic delivery. 2-HDP treatment had no effect on blood glucose, body weight, water intake, HbA1c levels or BP in diabetic rats (p>0.05). However, it protected against retinal FDP-Lys accumulation (p<0.05) and neurophysiological dysfunction, preserving ERG waveforms at 3 and 6 months post-diabetes induction (p<0.05 to p<0.001 for scotopic for a-wave, b-wave and summed oscillatory potentials). SD-OCT imaging revealed that 2-HDP prevented retinal thinning at 3 months (p<0.01) and protected against synaptic dysfunction, as evidenced by preserved synaptophysin expression (p<0.01 and p<0.001 for inner and outer plexiform layers, respectively). It also prevented neurodegeneration by maintaining retinal ganglion cells, amacrine cells, bipolar cells, and photoreceptors (p<0.05 to p<0.01). In addition, 2-HDP prevented retinal arteriolar dilation (p<0.01), reduced microvascular permeability (p<0.05) and attenuated microvascular damage, as indicated by preserved pericyte numbers and reduced acellular capillary formation (p<0.05). Mechanistically, 2-HDP inhibited microglial activation (p<0.05), suppressed the upregulation of proinflammatory molecules associated with NVU dysfunction in the diabetic retina (p<0.05 to p<0.001) and preserved the expression of the Müller cell glutamate-handling proteins, glutamate aspartate transporter 1 and glutamine synthetase (p<0.05 to p<0.01). FDP-Lys accumulation was observed in post-mortem human retinas from individuals with type 2 diabetes (p<0.05), in a pattern that was similar to that in the rat model of diabetes. Molecular dynamics simulations showed that the neutral form of 2-HDP readily crosses cell membranes, with enhanced permeation in the presence of ACR, highlighting its potential for systemic delivery. 2-HDP protects against retinal NVU dysfunction in diabetic rats by reducing FDP-Lys accumulation, preserving neuroretinal function and preventing microvascular damage, independent of glycaemic control. These results, combined with evidence from human diabetic retinas and molecular dynamics simulations, support 2-HDP’s potential as a promising therapeutic agent for DRD, warranting further preclinical and clinical investigation.

Diabetes, a pervasive and enduring health challenge, imposes significant global implications on health, financial healthcare systems, and societal well-being. This study undertakes a comprehensive exploration of various structural learning algorithms to discern causal pathways amongst potential risk factors influencing diabetes progression. The methodology involves the application of these algorithms to relevant diabetes data, followed by the conversion of their output graphs into Causal Bayesian Networks (CBNs), enabling predictive analysis and the evaluation of discrepancies in the effect of hypothetical interventions within our context-specific case study. This study highlights the substantial impact of algorithm selection on intervention outcomes. To consolidate insights from diverse algorithms, we employ a model-averaging technique that helps us obtain a unique causal model for diabetes derived from a varied set of structural learning algorithms. We also investigate how each of those individual graphs, as well as the average graph, compare to the structures elicited by a domain expert who categorised graph edges into high confidence, moderate, and low confidence types, leading into three individual graphs corresponding to the three levels of confidence. The resulting causal model and data are made available online, and serve as a valuable resource and a guide for informed decision-making by healthcare practitioners, offering a comprehensive understanding of the interactions between relevant risk factors and the effect of hypothetical interventions. Therefore, this research not only contributes to the academic discussion on diabetes, but also provides practical guidance for healthcare professionals in developing efficient intervention and risk management strategies.

In many retinopathies, hypoxia stimulates pathogenic neovascularization. The precise impact of hypoxia and how it may drive a switch in retinal microvascular endothelial cell metabolism during active angiogenesis remains ill-defined. This study has sought to understand dynamic shifts in the metabolic profile of retinal microvascular endothelial cells exposed to hypoxia and during ischemia-induced neovascularization. The impact of manipulating glycolytic metabolism by intravitreal injection of the glycolytic inhibitor 3-(3-Pyridinyl)-1-(4pyridinyl)-2-propen-1-one) (3PO) on pre-retinal neovascularization was investigated using the oxygen-induced retinopathy (OIR) model. Metabolic pathways generating adenosine triphosphate (ATP) in human retinal microvascular endothelial cells (HRMECs) was evaluated using qPCR and the Seahorse XFe96 analyzer under normal culture conditions and hypoxia ± 3PO. The role of glycolysis in HRMEC angiogenesis related processes such as tubulogenesis, proliferation and migration were assessed when glycolysis was blocked by 3PO. Our study showed that intravitreal injection of 3PO in the OIR model inhibited pre-retinal neovascularization compared to vehicle injected controls (P < 0.0001). While hypoxia increased glycolysis in HRMECs, treatment with 3PO reduced their glycolytic activity under normoxia and hypoxia culture conditions. Treatment with 3PO, reduced glycolytic mRNA expression of GLUT1, HK1, PFKFB3, ENO2 and VEGFA. Finally, glycolytic inhibition reduced tubulogenesis (p < 0.05), migratory capacity (p < 0.001) and proliferation (p < 0.01) of HRMECs in vitro. This data suggests that retinal angiogenesis can be modulated by manipulating the glycolytic pathway using 3PO in vivo and that 3PO treatment in vitro can diminish the angiogenic potential of HRMECs.

Vasodilation is a crucial protective response to inflammation and infection. Endothelial cells control vasodilation through the bioavailability of eNOS-produced nitric oxide (NO), and the generation of endothelium-dependent hyperpolarization (EDH). Here, we demonstrate that Klebsiella pneumoniae, one of the most prevalent blood stream infection pathogens, inhibits agonist-induced vasodilation by blunting the NO-dependent pathway and attenuating the EDH pathway. The type VI secretion system (T6SS) effector VgrG4 licences the kinase PKCβ in an NLRX1-controlled mitochondria reactive oxygen species (mtROS)-dependent manner to phosphorylate the eNOS inhibitory site Thr495, effectively dampening eNOS activity. The capsule polysaccharide, on the other hand, limits the phosphorylation of the eNOS activation site Ser1177 by inducing the phosphatase PP2Ac upon activation of an EGF receptor-dependent pathway. VgrG4-induced mtROS attenuates the EDH pathway. Overall, this work reveals a new anti-host activity of the T6SS and illustrates how pathogens can control vascular biology by targeting eNOS post translational modifications. ### Competing Interest Statement J.A.B. declares consultancy fees from VaxDyn and GSK. The other authors have declared that no conflict of interest exists.

Impaired function of the retinal neurovascular unit (NVU) is an early event in diabetic retinopathy (DR). It has been previously shown that topical delivery of the dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin can protect against diabetes-mediated dysfunction of the retinal NVU in the db/db mouse. The aim of the present study was to examine whether sitagliptin could prevent the DR-like lesions within the NVU of the new non-diabetic model of DR, the Trpv2 knockout rat (Trpv2+/-). For that purpose, at 3 months of age, Trpv2+/- rats were topically treated twice daily for two weeks with sitagliptin or PBS-vehicle eyedrops. Trpv2+/+ rats treated with vehicle served as the control group. Body weight and glycemia were monitored. Optical coherence tomography recordings, fundus images and retinal samples were obtained to evaluate sitagliptin effects. The results revealed that sitagliptin eye drops had no effect on body weight or glycemia. Vehicle-treated Trpv2+/- rats exhibited retinal thinning and larger diameters of major retinal blood vessels, upregulation of inflammatory factors and oxidative markers, glial activation and formation of acellular capillaries. However, topical administration of sitagliptin significantly prevented all these abnormalities. In conclusion, sitagliptin eye drops exert a protective effect against DR-like lesions in Trpv2+/- rats. Our results suggest that sitagliptin eye drops carry significant potential to treat not only early-stages of DR but also other diseases with impairment of the NVU unrelated to diabetes.

The recovery of mitochondrial quality control (MQC) may bring innovative solutions for neuroprotection, while imposing a significant challenge given the need of holistic approaches to restore mitochondrial dynamics (fusion/fission) and turnover (mitophagy and biogenesis). In diabetic retinopathy, this is compounded by our lack of understanding of human retinal neurodegeneration, but also how MQC processes interact during disease progression. Here, we show that mitochondria hyperfusion is characteristic of retinal neurodegeneration in human and murine diabetes, blunting the homeostatic turnover of mitochondria and causing metabolic and neuro-inflammatory stress. By mimicking this mitochondrial remodelling in vitro, we ascertain that N6-furfuryladenosine enhances mitochondrial turnover and bioenergetics by relaxing hyperfusion in a controlled fashion. Oral administration of N6-furfuryladenosine enhances mitochondrial turnover in the diabetic mouse retina (Ins2 Akita males), improving clinical correlates and conferring neuroprotection regardless of glycaemic status. Our findings provide translational insights for neuroprotection in the diabetic retina through the holistic recovery of MQC.

AimsDental pulp stem cells (DPSCs) contain a population of stem cells with a broad range of differentiation potentials, as well as more lineage-committed progenitors. Such heterogeneity is a significant obstacle to experimental and clinical applications. The aim of this study is to isolate and characterize a homogenous neuronal progenitor cell population from human DPSCs.MethodologyPolysialylated-neural cell adhesion molecule (PSA-NCAM+) neural progenitors were isolated from the dental pulp of three independent donors using magnetic-activated cell sorting (MACS) technology. Immunofluorescent staining with a panel of neural and non-neural markers was used to characterize the magnetically isolated PSA-NCAM+ fraction. PSA-NCAM+ cells were then cultured in Neurobasal A supplemented with neurotrophic factors: dibutyryl cyclic-AMP, neurotrophin-3, B27 and N2 supplements to induce neuronal differentiation. Both PSA-NCAM+ and differentiated PSA-NCAM+ cells were used in Ca2+ imaging studies to assess the functionality of P2X3 receptors as well as membrane depolarization.ResultsPSA-NCAM+ neural progenitors were isolated from a heterogeneous population of hDPSCs using magnetic-activated cell sorting and anti-PSA-NCAM MicroBeads. Flow cytometry analysis demonstrated that immunomagnetic sorting significantly increased the purity of PSA-NCAM+ cells. Immunofluorescent staining revealed expression of pan-neuronal and mature neuronal markers, PGP9.5 and MAP2, respectively, as well as weak expression of the mature sensory markers, peripherin and islet1. ATP-induced response was mediated predominately by P2X3 receptors in both undifferentiated and differentiated cells, with a greater magnitude observed in the latter. In addition, membrane depolarizations were also detected in cells before and after differentiation when loaded with fast-voltage-responding fluorescent molecule, FluoVolt (TM) in response to potassium chloride. Interestingly, only differentiated PSA-NCAM+ cells were capable of spontaneous membrane oscillations.ConclusionsIn summary, DPSCs contain a population of neuronal progenitors with enhanced neural differentiation and functional neural-like properties that can be effectively isolated with magnetic-activated cell sorting (MACS).

Aims/Purpose: We hypothesize that exacerbated mitochondrial fusion abrogates mitophagy in diabetic retinopathy [DR]. Our aim was to rescue this process pharmacologically to prevent vision loss in a mouse model of type‐1 diabetes. Methods: We generated a drug screening platform mimicking mitochondrial hyperfusion in DR. To this end, retinal Müller cells (mouse primary and MIO‐M1 cell line) were antagonized for mitochondrial fission using a Drp1 inhibitor peptide under elevated glucose (30.5 mM) conditions. Pharmacological ‘hits’ rescuing mitophagy in conditions of diabetes‐induced hyperfusion were assessed for their capability to improve mitochondrial health (membrane potential [ΔΨm]) and bioenergetics (Seahorse). The bio‐activity of lead compounds were corroborated in vivo using diabetic mitophagy reporter mice (Mito‐QC Ins2 Akita ), while effectiveness to alleviate retinal degeneration was evaluated via electroretinography (ERG), retinal thickness (SD‐OCT) and morphometric analysis of retinal neurons. Results: Among all drugs screened, we identified PA‐01 (a PINK1‐mitophagy activator) as capable of safely rescuing mitophagy under conditions of diabetes‐induced mitochondrial hyperfusion. Accordingly, PA‐01 rescued mitochondrial health (ΔΨm) and bioenergetics in Müller glia cultures, by optimizing metabolic flux (basal‐ and ATP‐linked respiration). The bio‐activity of PA‐01 was demonstrated in vivo, where long‐term oral administration rescued mitochondrial hyperfusion and mitophagy in the diabetic retina, while further preventing neurodegeneration. This was reflected in i) improved scotopic ERG responses (increased a‐wave and b‐wave amplitudes), ii) improved SD‐OCT retinal thickness and iii) neuroprotection to photoreceptors, synaptic terminals and amacrine cells. Conclusions: Exacerbated mitochondrial fusion contributes to DR pathology by inhibiting mitophagy in Müller glia. Rescuing this process pharmacologically holds promising therapeutic potential to alleviate vision loss in DR.

BACKGROUND:Preeclampsia is prevalent in women with diabetes, but the mechanism is unclear. We previously found that oxidized, glycated lipoproteins robustly upregulated soluble fms-like tyrosine kinase-1 (sFlt1), a key mediator of preeclampsia. Here, we determined the role of protein kinase C (PKC) and its subtypes in sFlt1 regulation in placental trophoblasts, and whether this mechanism might mediate the effect of modified lipoproteins. METHODS:Cultured human HTR8/SVneo and BeWo trophoblasts were treated with the PKC activator phorbol-12-myristate-13-acetate (PMA) for 24h, ± PKC inhibitors GF109203X (general), Ro31-8220 (PKCα-selective), LY333531 (PKCβ-selective) and rottlerin (PKCδ-selective). The effect of 'heavily oxidized, glycated' low-density lipoproteins (HOG-LDL) vs. native LDL (N-LDL), ± high glucose (30 mM), was evaluated in HTR8/SVneo cells. sFlt1 secretion (ELISA), mRNA expression (RT-qPCR), and cellular PKC activity were measured. RESULTS:PMA stimulated robust sFlt1 release and mRNA expression in both cell lines; these effects were inhibited by GF109203X, Ro31-8220 and LY333531 in a concentration-dependent manner. Rottlerin inhibited sFlt1 in BeWo, but modestly enhanced it in HTR8/SVneo cells. HOG-LDL enhanced PKC activity vs. N-LDL in HTR8/SVneo cells. Also, HOG-LDL, but not high glucose, significantly increased sFlt1 secretion and mRNA expression; this response was inhibited by GF109203X, Ro31-8220 and LY333531 at concentrations comparable to those that blocked PMA induction of sFlt1. CONCLUSION:Modified lipoproteins upregulate sFlt1 in trophoblasts via a PKC-mediated mechanism, involving at least α and β isoforms. The data suggest potential therapeutic targets to reduce the risk of preeclampsia in women with diabetes.