To investigate the association between mid-peripheral capillary free zones (CFZs) and retinal structural and functional metrics in diabetics without diabetic retinopathy (DR). This cross-sectional study included 45 eyes from 28 diabetics without DR and 46 eyes from 31 controls (mean age in both groups, 59 years). Macular optical coherence tomography (OCT) scans were acquired for retinal nerve fibre layer (RNFL) and ganglion cell layer thickness measurements. Thickness measurements were obtained using the Early Treatment of Diabetic Retinopathy Study grid. Retinal mid-peripheral CFZs were computed from OCT angiography images using custom MATLAB software. Retinal function was evaluated as a pilot exploratory objective using full-field flash electroretinography. Correlations between mid-peripheral CFZs and retinal structure and function were assessed using linear mixed-effect models, accounting for the association between eyes, while receiver operating characteristic curves were used to compare the multimodal models. Larger periarteriole CFZs were associated with thinner inner inferior RNFL thickness (β = −0.48, p = 0.03) in diabetics without DR. Functionally, there was no significant association between the mid-peripheral CFZs and ERG parameters (p > 0.05) in the no DR group; these findings should be interpreted with caution given the pilot nature of the functional data. The multimodal model of vascular and structural parameters had a modestly improved area under the curve (AUC) and specificity compared to the model of vascular parameters alone (AUC = 0.85 versus 0.83, specificity = 0.65 versus 0.54, respectively). These findings demonstrate that enlarged mid-peripheral periarteriole CFZs are associated with thinner RNFL in diabetics without clinical retinopathy. The multimodal model of vascular and structural metrics showed modestly improved diagnostic ability. This study shows early novel retinal vascular and neural associations in diabetics without clinical retinopathy and demonstrates the potential utility of a multimodal model for discriminating this group from healthy controls.

This review examines the protective angiotensin-converting enzyme 2/angiotensin-(1–7)/Mas receptor axis of the renin–angiotensin system. We sought to integrate evidence from preclinical and early human studies on how activation of this pathway influences vascular, cardiac, renal, hepatic and metabolic health, and to identify methodological lessons for developing future therapies. Animal models show that augmenting this axis improves endothelial function, limits cardiac remodelling, enhances insulin sensitivity, reduces renal injury and mitigates hepatic steatosis associate with MASH/NAFLD. Early-phase trials of recombinant angiotensin-converting enzyme 2 and angiotensin-(1–7) analogues demonstrate favourable safety profiles and clear pharmacodynamic target engagement but inconsistent immediate clinical effects. Emerging work highlights the value of biomarker ratios, gene and lifestyle based modulation and patient stratification strategies. Activation of the angiotensin-converting enzyme 2/angiotensin-(1–7)/Mas receptor axis offers a promising route to address cardiometabolic disease. Filling gaps in long-term outcomes, receptor-specific mechanisms and precision patient selection could transform this counter-regulatory pathway into a cornerstone of next-generation therapies.

Using micro-computed tomography, we identified a network of skull channels in the calvarium of type 2 diabetic (T2D) mice that remained structurally intact and numerically stable despite long-standing disease. The retention of calvaria bone marrow structural integrity was associated with preserved hematopoietic capacity under chronic diabetic conditions, which was not observed in the bone marrow of long bones. A distinctive feature of the calvarial bone marrow compartment was its direct exposure to cerebrospinal fluid (CSF), a property not shared by tibial bone marrow. To characterize the biochemical environment of the murine calvarium, we profiled oxysterols in CSF using mass spectrometry. The CSF exhibited elevated levels of neurotrophic and anti-inflammatory oxysterols, including 22-hydroxycholesterol (22-OHC) and 27-hydroxycholesterol (27-OHC). To assess whether this protective oxysterol signature was conserved in humans, we analyzed CSF samples from diabetic and non-diabetic individuals with obesity-associated idiopathic intracranial hypertension (IIH). Human CSF contained 7α-hydroxy-3-oxo-4-cholestenoic acid (7-HOCA), a metabolite of 27-OHC, supporting the conservation of this neuroprotective profile across species. Given the anatomical proximity of the calvarium to the eye, we hypothesized that calvaria bone marrow may serve as a reservoir for immune cells recruited to the injured or infected retina. The calvaria bone marrow was the predominant source of myeloid angiogenic cells (MACs) and neutrophils, mobilizing these cells at levels approximately 20-fold higher than long bones. These findings demonstrate that calvarial bone marrow plays a critical role in retinal immune defense, while maintaining both structural integrity and functional capacity despite chronic T2D.

Diabetic retinopathy (DR) is a leading cause of vision loss and is associated with reduced nuclear retinoid X receptor (RXR) activity. Retinal explants exposed to high glucose showed downregulation of RXR, peroxisome proliferator-activated receptor (PPAR), and liver X receptor-β and their lipid metabolism targets (Abca1, Scd1, and Acox1). Supplementation with the RXR agonist UAB126 restored nuclear receptor signaling and preferentially activated RXR/PPAR heterodimers. We evaluated UAB126 microparticles (UAB126-MP), a sustained-release RXR agonist formulation, for pharmacokinetic, molecular, and functional effects in db/db mouse retinas. A single intravitreal injection of UAB126-MP provided extended drug exposure for up to 6 months and increased retinal RXRα expression. Functionally, UAB126-MP preserved scotopic and photopic electroretinographic responses at 2 months and sustained cone photoreceptor function at 6 months. However, it did not reduce acellular capillaries. At 6 months, immunostaining and flow cytometry revealed decreased retinal macrophage/microglial infiltration and reduced CD45-positive myeloid cells and monocytes. These findings indicate that RXR agonism supports neuronal protection and immune modulation but does not prevent vasodegeneration, suggesting primarily neuroprotective and anti-inflammatory actions. Collectively, UAB126-MP confers durable retinal protection through nuclear receptor-mediated pathways, underscoring its potential as a long-acting therapeutic strategy for DR. ARTICLE HIGHLIGHTS:High glucose exposure of retinal explants reduces expression of nuclear receptors retinoid X receptor (RXR), peroxisome proliferator-activated receptor, and liver X receptor-β, and treatment with UAB126 increases the formation of RXR-peroxisome proliferator-activated receptor heterodimers. Sustained release of UAB126-loaded microparticles enables intravitreal drug delivery for up to 6 months. Intravitreal UAB126 microparticles injection in diabetic mice confers long-term neuroprotection and anti-inflammatory benefits. RXR-targeted therapy represents a promising approach strategy for managing diabetic retinopathy.

Purpose:To compare the width of the mid-peripheral capillary free zones (CFZs; periarteriole and perivenule) between diabetics with no diabetic retinopathy (DR) versus controls. Methods:The 20° × 20° optical coherence tomography angiography images of paired arterioles, venules, and their adjacent capillaries within the macular and inferomacular regions of the superficial vascular plexus were obtained from 46 eyes of 28 diabetics with no DR (mean age, 59 years; range, 40-71 years) and 46 eyes of 31 controls (mean age, 59 years; range, 46-78 years). There was no significant difference in age between groups (P = 0.77). The macular and inferomacular images were montaged to generate a wider field of view, followed by the application of a vesselness filter and Otsu thresholding. The mid-peripheral CFZ width was calculated using previously established MATLAB algorithms. Generalized linear mixed models were used to compare the mid-peripheral CFZs between groups, accounting for correlation between eyes. Results:The periarteriole CFZ width was greater in diabetics with no DR (73.3 ± 6.49 µm) compared to controls (67.3 ± 7.08 µm; P < 0.001, Cohen's d = 0.88). Similarly, the perivenule CFZ width was larger in diabetics with no DR (60.8 ± 6.40 µm) compared to controls (54.8 ± 4.58 µm; P < 0.001, Cohens' d = 1.08). Conclusions:Our results demonstrate larger mid-peripheral CFZ width in diabetics with no DR. The mid-peripheral CFZs show promise as a potential novel retinal vascular biomarker for early DR detection. Translational Relevance:Our study shows the potential clinical utility of the mid-peripheral CFZs for early DR detection.

Purpose: We previously reported that the systemic administration of preprogrammed mouse hematopoietic bone marrow-derived progenitor cells (HSPCs) improved visual function and restored a functional retinal pigment epithelial (RPE) layer. Here, we investigated the potential impact of donor vs. host age on systemic cellular therapy in a murine model of retinal degeneration. Methods: HSPCs from young (8 weeks) and old (15 months) mice were programmed ex vivo with a lentiviral vector expressing the RPE65 gene (LV-RPE65) and systemically administering into young or old SOD2 KD mice. Visual loss and pathological changes were evaluated by electroretinogram (ERG), optical coherence tomography (OCT), histology, and immunohistochemistry. Results: Old donor HSPCs administered to old manganese superoxide dismutase (SOD2) knockdown (KD) recipient mice offered the least benefit. This was exemplified by the reduced recruitment and incorporation of LV-RPE65 HSPC into the RPE layer, as well as decreased improvement in visual function, retinal thinning, and limited reduction in oxidative damage and microglial activation. LV-RPE65 HSPC from young mice incorporated into the RPE layer of old SOD2 KD mice, though to a lesser extent than young cells administered to young hosts, offered some level of protection. By contrast, LV-RPE65 HSPCs from old mice, located to the subretinal space of young host mice, reduced visual loss, although some retinal pathology was observed. Conclusions: The administration of LV-RPE65 HSPC from old donors to old SOD2 KD mice offered the least improvement. Translational Relevance: Our findings highlight how both donor and recipient age impact the success of HSPC-based retinal therapy and using cells from aged donors for AMD treatment may have some limitations.

Diabetes mellitus (DM) is a chronic metabolic disorder that results in hyperglycemia, leading to multiple microvascular and macrovascular complications, including significant ocular damage resulting in the development of diabetic retinopathy (DR) and diabetic macular edema (DME). Many factors contribute to the pathogenesis of DR and DME, including hyperglycemia-mediated vascular and neuronal abnormalities and local and systemic inflammation. Growth hormone (GH) and insulin-like growth factor-1 (IGF-1) have been implicated in the initiation and progression of DR and DME through a variety of mechanistic processes. In this review, we provide a comprehensive synopsis of the diverse roles and molecular pathways supporting IGF-1 in the pathogenesis of DR and DME, elucidating its range of effects from detrimental to protective, depending on the context and stage of disease. We further investigate the underlying inflammatory processes regulated by IGF-1 and examine how the interaction of IGF-1 with key signaling molecules influences these inflammatory mechanisms. Additionally, the potential of serum IGF-1 as a biomarker for the progression of DR and DME in clinical practice is discussed. Finally, we consider current therapeutic approaches for DR and DME in relation to IGF-1 and explore novel therapeutic targets and innovative delivery methods. By providing an in-depth understanding of IGF-1’s role in the pathogenesis and progression of DR and DME, this review underscores the diagnostic utility of serum IGF-1 and puts forth new treatment strategies to improve the management of DR and DME.

Strategies to improve metabolic health include calorie restriction, time restricted eating and fasting several days per week or month. These approaches have demonstrated benefits for individuals experiencing obesity, metabolic syndrome, and prediabetes. However, their impact on established diabetes remains incompletely studied. The chronicity of type 2 diabetes (T2D) requires that interventions must be undertaken for extended periods of time, typically the entire lifetime of the individual. In this study, we examined the impact of intermittent fasting (IF), with an every-other-day protocol for a duration of 6 months in a murine model of T2D, the db/db (D) mouse on metabolism and liver steatosis. We compared D-IF mice with diabetic ad-libitum (AL; D-AL), control-IF (C-IF) and control-AL (C-AL) cohorts. We demonstrated using lipidomic, microbiome, metabolomic and liver transcriptomic studies that chronic IF improved carbohydrate utilization and glucose homeostasis without weight loss and reduced white adipose tissue inflammation and significantly impacted lipid metabolism in the liver. Microbiome studies and predicted functional analysis of gut microbiota showed that IF increased beneficial bacteria involved in sphingolipid (SL) metabolism. The metabolomic studies showed that oxidation of lipid species and ceramide levels were reduced in D-IF compared to D-AL. The liver lipidomic analysis and liver microarray confirmed a reduction in overall lipid content in D-IF mice compared to D-AL mice, especially in the feeding state as well as an overall reduction in oxidized lipids and ceramides. These studies support that long-term IF can improve glucose homeostasis and dramatically altered lipid metabolism in the absence of weight loss.

Introduction and Objective: Tryptophan (Trp) is absorbed via angiotensin converting enzyme 2 (ACE2) dependent and independent mechanisms. In chronic diabetes a reduction of ACE2 and of its dimerization partner the Trp transporter BOAT1 limits Trp absorption. Loss of ACE2 and BOAT1 supports the need for alternative mechanisms for Trp absorption. We sought to bypass ACE2 by using the dipeptide isoleucine-tryptophan (IW). Di- and tripeptides are absorbed via SLC15A1, an alternative amino acid transporter that is minimally affected by diabetes. This study examined IW’s therapeutic potential in prevention of gut dysbiosis and diabetic retinopathy in the model of type 2 diabetes, db/db mice. Methods: ACE2 dependent Trp absorption was achieved using oral gavage of genetically modified Lactobacillus paracasei expressing soluble ACE2 (LP-ACE2) and was compared to ACE2 independent absorption using oral administration of IW (5 mg/kg/day. Acellular capillaries, a marker of retinal vasodegeneration, were the diabetic retinopathy endpoint. Gut barrier integrity and dysbiosis were assessed at 2 and 6 months of diabetes. Results: Metatranscriptomic analysis revealed that both IW and LP-ACE2 corrected gut dysbiosis. IW and LP-ACE2 treatments restored gut barrier integrity, reduced plasma gut microbial antigens, and corrected diabetes-induced dysbiosis. Both treatments decreased gut inflammation (IL-1β, IL-2, IL-6, and IFN-γ), with reductions in acellular capillaries by 40.9% (LP-ACE2) and 43.18% (IW). IW increased levels of the beneficial Trp derived bacterial metabolite, indole proprionic acid (IPA) which enhances AhR/PXR signaling in intestinal epithelial cells, improving gut barrier function. Both treatments doubled intestinal GLP-1 and GIP levels compared to untreated db/db mice. Conclusion: IW and microbiota-derived metabolites address diabetes-induced dysbiosis, inflammation, and vascular damage. By activating indole/AhR/PXR signaling pathways, they preserve gut and retinal health. M.B. Grant: None. R. Prasad: None. NEI (033620, 012601)

This study investigated the therapeutic potential of the nuclear retinoid X receptor (RXR) in mitigating the progression of alpha-synucleinopathies (αSNPs), particularly in Parkinson’s disease (PD). PD-like pathology in mice was successfully induced through the co-delivery of AAV expressing human α-synuclein (αS) and αS preformed fibrils (PFFs) into the substantia nigra pars compacta (SNpc). Significant increases in Lewy body (LB)-like inclusions, loss of tyrosine hydroxylase-positive (TH+) neurons, and reductions in dopamine (DA) levels in the striatum were observed. Additionally, diminished levels of PPARα and NURR1—proteins essential for neuronal survival—along with elevated expression of IBA1 and GFAP, markers of microglial activation and astrocytic gliosis, respectively, are associated with the pathogenesis of Parkinson’s disease. AAV-mediated overexpression of human RXRα demonstrated preservation of TH+ neurons, prevention of DA decline, and attenuation of αS accumulation. Furthermore, RXR-treated PD brains showed a reduced number of GFAP+ and Iba1+ cells, decreased GFAP+ and IBA1+ immunoreactivity, and fewer and less widespread LB-like aggregates. RXR overexpression also enhanced the production of PPARα and NURR1. These findings suggest that RXRα upregulation promotes neuroprotection by mitigating αSNPs and chronic neuroinflammation, a major contributor to PD progression. This research underscores the therapeutic potential of targeting nuclear receptors, such as RXR, in neurodegenerative diseases like PD.

Purpose: To investigate the therapeutic potential of inducible pluripotent stem cell (hiPSC)-based vascular repair, we evaluated two vascular reparative cell populations, CD34+ cells derived from hiPSC (hiPSC-CD34+) and endothelial colony forming cells (ECFCs) derived from hiPSC (iPS-ECFCs), alone and in combination, in a type 2 diabetic (db/db) mouse model of DR. Methods: hiPSC-CD34+ cells (1 × 104) or iPSC- ECFCs (1 × 105) alone or in combination (1.1 × 105) were injected into the vitreous of immunosuppressed db/db mice with six months of established diabetes. One month post-injection, mice underwent electroretinography (ERG) and optical coherence tomography (OCT) to evaluate functional and structural retinal recovery with iPSC administration. Immunohistochemistry (IHC) was used to assess recruitment and incorporation of cells into the retinal vasculature. Retinas from the experimental groups were analyzed using Functional Proteomics via Reverse Phase Protein Array (RPPA). Results: Functional assessment via ERG demonstrated significant improvements in retinal response in the diabetic cohorts treated with either hiPSC-derived CD34+ cells or hiPSC-ECFCs. Retinal thickness, assessed by OCT, was restored to near-nondiabetic levels in mice treated with hiPSC-CD34+ cells alone and the combination group, whereas hiPSC-ECFCs alone did not significantly affect retinal thickness. One month following intravitreal injection, hiPSC-CD34+ cells were localized to perivascular regions, whereas hiPSC-ECFCs were observed to integrate directly into the retinal vasculature. RPPA analysis revealed interaction-significant changes, and this was interpreted as a combination-specific, non-additive host responses (m6A, PI3K–AKT–mTOR, glycolysis, endothelial junction pathways). Conclusions: The studies support that injection of hiPSC-CD34+ cells and hiPSC-ECFCs, both individually and in combination, showed benefit; however, iPSC combination-specific effects were identified by measurement of retinal thickness and by RPPA.

Purpose:We evaluated the safety and bioactivity of carboxyamidotriazole (CAI) using two approaches, a polymeric CAI-PLGA nanoemulsion in the mouse model of choroidal neovascularization (CNV) and CAI-loaded bioresorbable intravitreal implant in a rabbit model of vascular leakage. Methods:Mice underwent laser rupture of Bruch's membrane to induce CNV followed by a single (2 µL volume) intravitreal injection of either vehicle (n = 11); CAI nanoparticles (0.5 µg, 1 µg, 2 µg, 400 nM, 800 nM, and 1.6 µM, respectively); aflibercept 10 µg; or CAI nanoparticles (1 µg) + aflibercept 10 µg. New Zealand white rabbits underwent either sham intravitreal injection, aflibercept 500 µg injection, or CAI-PLGA intravitreal implant. Vascular leakage was induced with injections of VEGF on days 23 and 53. On days 30 and 60, all groups underwent vitreous fluorophotometry and fundus imaging. On day 60, the rabbits were euthanized, and their eyes were enucleated. Results:Intravitreal injection of the CAI-Nano at the dose of 1 µg significantly decreased choroidal neovascular volume, to 25% of saline on day 7 and 30% on day 14, which was comparable to aflibercept. Vitreous fluorophotometry revealed significantly lower levels of fluorescein in the aflibercept and CAI implant groups compared to the sham group on day 30. On day 60, the CAI implant group showed significantly reduced neovascularization as compared with the aflibercept groups. No toxicity was observed in any group. Conclusions:CAI in nanoparticle formulation or as a sustained release bioresorbable implant showed potent efficacy and caused no retinal toxicity in murine and rabbit models. Translational Significance:CAI demonstrates strong potential as a sustained release anti-angiogenic therapy with effective long-term durability.

Hallmark findings in age-related macular degeneration (AMD) include the accumulation of extracellular lipid and vasodegeneration of the choriocapillaris. Choroidal inflammation has long been associated with AMD, but little is known about the immune landscape of the human choroid. Using 3D multiplex immunofluorescence, single-cell RNA sequencing, and flow cytometry, we unravel the cellular composition and spatial organization of the human choroid and the immune cells within it. We identify two populations of choroidal macrophages with distinct FOLR2 expression that account for the majority of myeloid cells. FOLR2+ macrophages predominate in the nondiseased eye, express lipid-handling machinery, uptake lipoprotein particles, and contain high amounts of lipid. In AMD, FOLR2+ macrophages are decreased in number and exhibit dysfunctional lipoprotein metabolism. In mice, FOLR2+ macrophages are negative for the postnatal fate-reporter Ms4a3, and their depletion causes an accelerated AMD-like phenotype. Our results show that prenatally derived resident macrophages decline in AMD and are implicated in multiple hallmark functions known to be compromised in the disease.

BACKGROUND:Most organs are maintained lifelong by resident stem/progenitor cells. During development and regeneration, lineage-specific stem/progenitor cells can contribute to the growth or maintenance of different organs, whereas fully differentiated mature cells have less regenerative potential. However, it is unclear whether vascular endothelial cells (ECs) are also replenished by stem/progenitor cells with EC-repopulating potential residing in blood vessels. It has been reported recently that some EC populations possess higher clonal proliferative potential and vessel-forming capacity compared with mature ECs. Nevertheless, a marker to identify vascular clonal repopulating ECs (CRECs) in murine and human individuals is lacking, and, hence, the mechanism for the proliferative, self-renewal, and vessel-forming potential of CRECs is elusive.METHODS:We analyzed colony-forming, self-renewal, and vessel-forming potential of ABCG2 (ATP binding cassette subfamily G member 2)-expressing ECs in human umbilical vessels. To study the contribution of Abcg2-expressing ECs to vessel development and regeneration, we developed Abcg2CreErt2;ROSA TdTomato mice and performed lineage tracing during mouse development and during tissue regeneration after myocardial infarction injury. RNA sequencing and chromatin methylation chromatin immunoprecipitation followed by sequencing were conducted to study the gene regulation in Abcg2-expressing ECs.RESULTS:In human and mouse vessels, ECs with higher ABCG2 expression (ABCECs) possess higher clonal proliferative potential and in vivo vessel-forming potential compared with mature ECs. These cells could clonally contribute to vessel formation in primary and secondary recipients after transplantation. These features of ABCECs meet the criteria of CRECs. Results from lineage tracing experiments confirm that Abcg2-expressing CRECs (AbcCRECs) contribute to arteries, veins, and capillaries in cardiac tissue development and vascular tissue regeneration after myocardial infarction. Transcriptome and epigenetic analyses reveal that a gene expression signature involved in angiogenesis and vessel development is enriched in AbcCRECs. In addition, various angiogenic genes, such as Notch2 and Hey2, are bivalently modified by trimethylation at the 4th and 27th lysine residue of histone H3 (H3K4me3 and H3K27me3) in AbcCRECs.CONCLUSIONS:These results are the first to establish that a single prospective marker identifies CRECs in mice and human individuals, which holds promise to provide new cell therapies for repair of damaged vessels in patients with endothelial dysfunction.

The accessibility of the retina with the use of non-invasive and relatively low-cost ophthalmic imaging techniques and analytics provides a unique opportunity to improve the detection, diagnosis and monitoring of systemic diseases. The National Heart, Lung, and Blood Institute conducted a workshop in October 2022 to examine this concept. On the basis of the discussions at that workshop, this Roadmap describes current knowledge gaps and new research opportunities to evaluate the relationships between the eye (in particular, retinal biomarkers) and the risk of cardiovascular diseases, including coronary artery disease, heart failure, stroke, hypertension and vascular dementia. Identified gaps include the need to simplify and standardize the capture of high-quality images of the eye by non-ophthalmic health workers and to conduct longitudinal studies using multidisciplinary networks of diverse at-risk populations with improved implementation and methods to protect participant and dataset privacy. Other gaps include improving the measurement of structural and functional retinal biomarkers, determining the relationship between microvascular and macrovascular risk factors, improving multimodal imaging 'pipelines', and integrating advanced imaging with 'omics', lifestyle factors, primary care data and radiological reports, by using artificial intelligence technology to improve the identification of individual-level risk. Future research on retinal microvascular disease and retinal biomarkers might additionally provide insights into the temporal development of microvascular disease across other systemic vascular beds. In this Roadmap arising from an NHLBI workshop, Chew and colleagues explore the use of retinal imaging biomarkers for the prediction, diagnosis and monitoring of systemic cardiovascular diseases. The authors identify knowledge gaps and research opportunities to translate retinal imaging biomarkers into clinical practice.

Introduction: Diabetic retinopathy (DR) is characterized by retinal endothelial cell (REC) death and inadequate vascular repair by dysfunctional circulating angiogenic cells (CACs). We have previously demonstrated an increase in ASM-mediated C16 ceramide and ceramide-rich platform (CRP) formation as a major pathogenic mechanism in the development of REC damage and CAC dysfunction in DR. Using anti-ceramide Abs, we aimed to prevent REC damage and restore CAC-mediated repair by dispersing CRPs, thus restoring retinal microvascular health and preventing DR. Methods: Anti-ceramide Abs were administered in vitro or ex vivo at 10 (short chain fragment antibody; scFv) /100 (monoclonal antibody; mAb) μg/mL and in vivo (subcutaneously) at 60μg/g (mAb) bodyweight in STZ induced diabetic mouse model. Migration of CACs was assessed using trans-well assays ex vivo. In vivo vascular degeneration was captured using FITC-albumin technique. In vivo migration of GFP+ CACs (JAX:006567) was examined via confocal imaging. Results: In vitro studies in BRECs show generation of vast ceramide rich platforms followed by apoptosis upon acute stimulation with 20ng/mL TNFa or 10ng/mL IL-1β. This phenotype was fully ameliorated when treated with anti-ceramide scFv. STZ mice showed a significant increase (42% CRP pos. cells) in CRP formation on day 3, with gradual increases on day 7, 14, 42, and 180 (70% pos.) post-hyperglycemia (n=9-12, p<0.0001. Anti-ceramide treatment reduced CRP formation on CACs and protected from retinal vascular permeability 6-weeks post hyperglycemia in vivo (n=10-12; p<0.01. In vivo migration and vascular repair of CACs was impaired in 6-month diabetic STZ mice compared to their age matched controls and rescued following anti-ceramide Abs intervention (scFv and mAb) in vivo (n=4-7; p<0.08). Conclusion: Anti-ceramide Abs restore microvascular health via normalization of both REC and CACs homeostasis to halt or reverse DR. Disclosure J.V. Busik: Consultant; Ceramedix, Inc. T.F. Dorweiler: None. A. Singh: None. M.B. Grant: None. R. Kolesnick: Stock/Shareholder; Ceramedix Holding LLC. Funding R01EY030766R01EY025383R01EY016077

Bone marrow (BM) derived vascular reparative cells, myeloid angiogenic cells (MACs), work with vascular wall derived cells, endothelial colony forming cells (ECFCs), to orchestrate vessel repair. Both populations of cells are found in the circulation making their identification at times problematic. Importantly, the circulating levels of both ECFCs and MACs reflect vascular injury and disease. In humans, MACs are often identified by the surface marker CD34. Circulating levels of CD34+ cells are reduced following coronary angioplasty, acute coronary syndromes, unstable angina, and in individuals with diabetic complications including diabetic retinopathy (DR). The aim of this chapter is to summarize the literature pertinent to the role of MACs and ECFCs in vascular repair specifically in the eye with the goal of understanding the potential of these cells as treatment for retinal degenerative diseases.