Abdelrahman Y. Fouda

Background: Efferocytosis, the phagocytic clearance of apoptotic and damaged cells, promotes inflammation resolution and tissue repair following ischemic stroke. This study investigated temporal changes in efferocytosis and phagocytosis-related transcriptional programs during acute experimental stroke, examined the effects of aging on these responses, and assessed whether similar immune signatures are present in human ischemic stroke. Methods: Publicly available transcriptomic datasets from murine transient middle cerebral artery occlusion (tMCAO; GSE104036 and GSE112348), permanent middle cerebral artery occlusion (pMCAO; GSE137482), and human peripheral blood after ischemic stroke (GSE16561) were analyzed using OmicSoft/Ingenuity-style pathway analysis. Functional validation included in vivo assessment of efferocytosis after tMCAO and in vitro phagocytosis assays using bone marrow-derived macrophages from young and aged mice. Results: Both acute tMCAO models exhibited robust inflammatory activation together with sustained activation of phagocyte-related pathways during the first 24 hours after stroke. Human peripheral blood demonstrated similar inflammatory and phagocytic signatures, supporting translational relevance. Increased efferocytosis at 24 hours after tMCAO was associated with neuroprotection. Although both young and aged mice activated phagocytosis-related pathways after pMCAO, aged mice showed reduced phagosome formation. Consistent with these findings, macrophages from aged mice exhibited enhanced inflammatory responses and impaired uptake of apoptotic cells. Conclusions: A conserved post-stroke immune response characterized by inflammatory activation and phagocyte-mediated clearance was identified across murine and human datasets. Efficient efferocytosis was associated with neuroprotection, whereas aging impaired apoptotic cell clearance and promoted a pro-inflammatory macrophage phenotype, highlighting efferocytosis as a potential therapeutic target for ischemic stroke.

Ischemia-induced retinopathy is a defining feature of prevalent ocular conditions, including diabetic retinopathy and central retinal artery or vein occlusion. Therapeutic interventions for ischemic retinopathies show limited efficacy and adverse effects, highlighting the need to thoroughly investigate the underlying mechanisms. Histone deacetylase 3 (HDAC3), a member of the histone deacetylase family, plays a central role in regulating gene expression in myeloid cells (microglia and macrophages). We recently showed that myeloid HDAC3 deletion promotes tissue repair and functional recovery after retinal ischemia-reperfusion (IR) injury via efferocytosis, a process by which myeloid cells engulf and clear apoptotic cells. Here, we investigated the mechanism by which myeloid HDAC3 deletion enhances efferocytosis. Employing an in vitro efferocytosis assay coupled with RNA sequencing on HDAC3 KO macrophages revealed that the secreted protein, CD5 molecule-like (CD5L), was the most upregulated among other pro-efferocytic genes. In vivo, we found that CD5L levels markedly increased in the retinas of myeloid HDAC3 KO mice subjected to IR injury, and its expression colocalized with myeloid cells. Co-immunoprecipitation experiments showed that HDAC3 represses CD5L expression in a liver X receptor (LXRα)-dependent manner. Additionally, we found that CD36, a receptor for CD5L that facilitates the clearance of apoptotic cells, was upregulated in retinal myeloid cells after IR. In vitro, CD5L treatment enhanced efferocytosis via CD36. We then evaluated the role of CD5L in retinal IR injury using in vivo neuronal, vascular, structural, and functional endpoints. CD5L KO mice showed worsened outcomes after IR, whereas treatment with recombinant CD5L was protective against retinal ischemic injury. Collectively, our findings suggest that deleting HDAC3 enhances macrophage efferocytosis by upregulating the CD5L/CD36 axis. CD5L may serve as a promising therapeutic target to improve outcomes in ischemic retinopathy.

Traumatic optic neuropathy (TON) occurs due to trauma to the optic nerve, resulting in blindness. Current management focuses primarily on supportive care, highlighting an urgent need to identify novel treatment targets. Neuronal expression of the enzyme histone deacetylase 3 (HDAC3) has been previously implicated in retinal ganglion cell (RGC) degeneration after optic nerve crush (ONC), a model of TON. Here we investigated the role of myeloid HDAC3 (i.e., HDAC3 expressed in microglia and macrophages) in RGC loss, axonal degeneration, and efferocytosis, a reparative process by which phagocytic myeloid cells engulf apoptotic cells. ONC injury was performed on myeloid-specific HDAC3 knockout (KO) and floxed control mice. Neurodegeneration and efferocytosis assays were assessed using retina flatmount immunolabeling and confocal imaging. RGC function was evaluated using pattern electroretinography (PERG). Axonal sprouting was quantified by anterograde transport of cholera toxin B injected intravitreally. Myelin debris clearance was assessed in optic nerves in vivo and in vitro using bone-marrow-derived macrophages isolated from myeloid HDAC3 KO and control mice. Myeloid HDAC3 deletion preserved RGC and improved axonal regeneration after ONC, together with improved retinal function assessed by PERG. Furthermore, the deletion of HDAC3 enhanced the phagocytic function of myeloid cells to effectively remove apoptotic cells and myelin debris, both in vivo and in vitro. These protective effects were associated with the deletion of HDAC3 specifically in macrophages, since microglial-only deletion of HDAC3 did not preserve RGC count or function. The enhanced efferocytosis function of HDAC3 KO macrophages was at least partly dependent on increasing the expression of the phagocytic tyrosine kinase receptor, MerTK. The deletion of myeloid HDAC3 enhances efferocytosis, leading to neuroprotection, regeneration, and functional recovery following ONC. Targeting myeloid-HDAC3 presents a novel therapeutic strategy for TON.

Objective: Diabetic retinopathy (DR) is a leading cause of blindness, and understanding its progression from non-proliferative (NPDR) to sight-threatening proliferative diabetic retinopathy (PDR) is crucial. Systemic lactate dehydrogenase (LDH) has been implicated in various disease processes. We investigated the association between systemic LDH levels at the time of NPDR diagnosis and the 1-year risk of progression to PDR and its complications. Methods: We conducted a retrospective, propensity-matched cohort study using the TriNetX US Collaborative Network. Patients with type 2 diabetes and a new diagnosis of NPDR were stratified into three groups based on a single LDH measurement taken within 6 months of the index date: low (<200 U/L), moderate (201–280 U/L), and high (≥281 U/L). Two separate analyses were performed: one comparing the low-LDH group to the moderate-LDH group, and another comparing the low-LDH group to the high-LDH group. The primary outcomes were the 1-year absolute risks and risk ratios (relative risk, RR) for PDR, tractional retinal detachment (TRD), and vitreous hemorrhage (VH). Results: Comparing the low-LDH cohort to the moderate-LDH cohort, the moderate-LDH group had a higher 1-year absolute risk of PDR (3.93% vs. 2.96%), TRD (1.35% vs. 0.99%), and VH (4.38% vs. 3.51%). Comparing the low-LDH group to the high-LDH group, the high-LDH cohort showed an increased risk for PDR (3.66% vs. 3.00%), TRD (1.27% vs. 0.96%), and VH (1.27% vs. 0.96%). Conclusions: Our findings demonstrate a consistent, dose-dependent relationship between higher systemic LDH levels and an increased risk of progression to PDR and its complications.

Pathological retinal neovascularization (NV) contributes to vision loss in diabetic retinopathy (DR) and retinopathy of prematurity, which are the leading causes of blindness in working-age adults and children, respectively. Retinal hypoxia is a key driver of pathological neovascularization that results in uncontrolled vessel sprouting and the formation of immature and leaky blood vessels. Anti-vascular endothelial growth factor and laser therapies are the standard of care to mitigate vision loss, but their limited effectiveness underlies the need to identify new therapeutic targets. The goal of the current study was to define the role of the enzyme histone deacetylase 3 (HDAC3) in the pathogenesis of experimental NV. Pathological neovascularization was induced by subjecting C57BL/6 J mouse pups to oxygen-induced retinopathy (OIR). Retinal tissues were analyzed by Western blotting and immunofluorescent labeling was conducted on mouse retinal flatmounts and human retinal sections from patients with DR. In vitro studies used cultured bovine retinal endothelial cells (REC) subjected to oxygen-glucose deprivation (OGD) followed by reoxygenation (R). Cells were treated with the HDAC3 inhibitor, RGFP966, the mitochondrial fission inhibitor, Mdivi-1 or DMSO as a control. Endpoints included assays of cell migration, untargeted proteomic analysis, Seahorse analysis of glycolysis, and mitochondrial morphology using MitoTracker dye. Using the methods described above, we found that HDAC3 expression was increased in retinal vessels of OIR mice and human DR retinal samples. HDAC3 also was upregulated in REC following OGD/R. Treatment with RGFP966 (2, 8 μM) attenuated OGD/R-induced angiogenesis as determined by cell migration. In confirmation, siRNA-mediated HDAC3 knockdown attenuated REC migration whereas HDAC3 overexpression increased it. OGD/R induced a strong upregulation of the rate-limiting glycolysis enzyme, hexokinase 2 (HK2), as determined by untargeted proteomic analysis, which correlated with increased glycolysis and mitochondrial fission. Treatment with RGFP966 or Mdivi-1 (5 μM), blocked HK2 upregulation, suppressed glycolytic flux, and reduced mitochondrial fission. Our findings indicate that HDAC3 plays a crucial role in pathological neovascularization by driving endothelial cell metabolic reprogramming toward glycolysis via the induction of mitochondrial fission and HK2 signaling. Targeting HDAC3 or its downstream metabolic pathways may offer a promising therapeutic strategy for mitigating pathological NV. Retinal endothelial cells (REC) respond to oxygen glucose deprivation/reperfusion (OGD/R) injury by increasing the expression of HDAC3 which, in turn, upregulates hexokinase 2 (HK2) and mitochondrial fission. These then go on to metabolically reprogram the REC toward a more glycolytic phenotype and promote the process of pathological angiogenesis of the retina. Inhibiting HDAC3 by RGFP966 protects against the OGD/R-induced metabolic changes. In a similar fashion, the inhibition of mitochondrial fission with Mdivi-1 mitigates the glycolytic shift and HK2 expression. These findings suggest a working model in which HDAC3-induced mitochondrial fission upregulates HK2, induces glycolysis and promotes REC pathological angiogenesis.

Background: Diabetic retinopathy (DR) is a leading cause of vision loss, and its progression from non-proliferative (NPDR) to sight-threatening proliferative diabetic retinopathy (PDR) is a critical event. Systemic lactate dehydrogenase (LDH), a marker of cellular hypoxia and turnover, has been implicated in various disease processes. We investigated the association between systemic LDH levels at the time of NPDR diagnosis and the 1-year risk of progression to PDR and other sight-threatening complications. Methods: We conducted a retrospective, propensity-matched cohort study using the TriNetX US Collaborative Network. Patients with a new NPDR diagnosis were stratified into three groups based on a single LDH measurement taken within six months of the index date: low (< 200 U/L), moderate (201–280 U/L), and high (≥281 U/L). Two separate 1:1 nearest-neighbor propensity-matched analyses were performed: one comparing the low-LDH group to the moderate-LDH group, and a second comparing the low-LDH group to the high-LDH group. Matching was performed on a comprehensive set of demographic, metabolic, vascular, and ocular covariates. The primary outcomes were the 1-year absolute risks and risk ratios (RR) for PDR, tractional retinal detachment (TRD), and vitreous hemorrhage (VH). Results: In the first analysis, the low-LDH cohort was compared to the moderate-LDH cohort. The moderate-LDH group had a higher 1-year absolute risk of PDR (3.93 % vs. 2.96 %, RR 1.33, 95 % CI 1.14–1.54), TRD (1.35 % vs. 0.99 %, RR 1.36, 95 % CI 1.07–1.73), and VH (4.38 % vs. 3.51 %, RR 1.25, 95 % CI 1.10–1.42). In the second analysis, comparing the low-LDH group to the high-LDH group, the high-LDH cohort showed an increased risk for PDR (3.66 % vs. 3.00 %, RR 1.22, 95 % CI 1.05–1.42), TRD (1.27 % vs. 0.96 %, RR 1.32, 95 % CI 1.04–1.67), and VH (1.27 % vs. 0.96 %, RR 1.22, 95 % CI 1.08–1.40). Conclusion: Our findings demonstrate a consistent, dose-dependent relationship between higher systemic LDH levels and an increased risk of progression to PDR and its complications. This suggests that systemic LDH levels may be an independent and readily available biomarker for identifying patients with NPDR who are at higher risk for sight-threatening outcomes.

Extracellular vesicles (EVs) are bilayer lipid membrane particles that are released by every cell type. These secretions are further classified as exosomes, ectosomes, and microvesicles. They contain biomolecules (RNAs, proteins, metabolites, and lipids) with the ability to modulate various biological processes and have been shown to play a role in intercellular communication and cellular rejuvenation. Various studies suggest exosomes and/or microvesicles as a potential platform for drug delivery. EVs may deliver lipids and nucleotides directly to an injury site in an axon, promoting growth cone stabilization and membrane expansion as well as repair, thus positively modulating adult axon regeneration. In this review, we will provide a perspective on the metabolite composition of EVs in adult axonal regeneration relevant to the central nervous system (CNS), specifically that pertaining to the optic nerve. We will present an overview of the methods for isolation, enrichment, omics data analysis and quantification of extracellular vesicles with the goal of providing direction for future studies relevant to axon regeneration. We will also include current resources for multi-omics data integration relevant to extracellular vesicles from diverse cell types.

Traumatic optic neuropathy (TON) is a serious injury to the optic nerve that can lead to vision impairment or blindness. Current treatment options include corticosteroids or surgical decompression. There is no consensus on the treatment of TON due to insufficient evidence supporting the effectiveness of current options. Cluster of differentiation 47 (CD47) is a transmembrane protein that can be upregulated on apoptotic cells. It prevents their phagocytic clearance or efferocytosis by communicating a “don’t eat me” signal when it binds to signal regulatory protein α (SIRPα) on myeloid cells. We found that CD47 is upregulated in the retina after the optic nerve crush (ONC) injury model of TON, along with the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Antibody neutralization of TNF-α after ONC resulted in blunted CD47 levels whereas treating ex-vivo retinal explants with recombinant TNF-α resulted in increased levels of CD47, indicating that TNF-α stimulates CD47 expression. To investigate the therapeutic potential of blocking the CD47/SIRPα axis after ONC, we intravitreally treated WT mice with anti-CD47, anti-SIRPα, or anti-IgG (control) post-ONC injury. Anti-CD47 treatments resulted in improved efferocytosis, attenuated neurodegeneration, improved retinal thickness, and enhanced retinal function as compared to anti-IgG control treatment post-ONC. Conversely, anti-SIRPα did not show statistically significant neuronal preservation after ONC. Myeloid-specific SIRPα deletion reduced retinal thinning and only showed a neuroprotective trend for retinal ganglion cell count and function after ONC. Collectively, our data indicate that the CD47-SIRPα axis is a promising therapeutic target to improve retinal outcomes following ONC injury, with CD47 neutralization proving more effective than targeting SIRPα.

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