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Background: We examined components of systemic and intestinal renin-angiotensin system on gut barrier permeability, glucose homeostasis, systemic inflammation, and progression of diabetic retinopathy (DR) in human subjects and mice with type 1 diabetes (T1D). Methods: T1D individual with (n=18) and without (n=20) DR and controls (n=34) were examined for changes in gut-regulated components of the immune system, gut leakage markers (FABP2 [fatty acid binding protein 2] and peptidoglycan), and Ang II (angiotensin II); Akita mice were orally administered a Lactobacillus paracasei (LP) probiotic expressing humanized ACE2 (angiotensin-converting enzyme 2) protein (LP-ACE2) as either a prevention or an intervention. Akita mice with genetic overexpression of humanAce2 by small intestine epithelial cells ( Vil-Cre.hAce2KI-Akita ) were similarly examined. After 9 months of T1D, circulatory, enteral, and ocular end points were assessed. Results: T1D subjects exhibit elevations in gut-derived circulating immune cells (ILC1 cells) and higher gut leakage markers, which were positively correlated with plasma Ang II and DR severity. The LP-ACE2 prevention cohort and genetic overexpression of intestinal ACE2 preserved barrier integrity, reduced inflammatory response, improved hyperglycemia, and delayed development of DR. Improvements in glucose homeostasis were due to intestinal MasR activation, resulting in a GSK-3β (glycogen synthase kinase-3 beta)/c-Myc (cellular myelocytomatosis oncogene)-mediated decrease in intestinal glucose transporter expression. In the LP-ACE2 intervention cohort, gut barrier integrity was improved and DR reversed, but no improvement in hyperglycemia was observed. These data support that the beneficial effects of LP-ACE2 on DR are due to the action of ACE2, not improved glucose homeostasis. Conclusions: Dysregulated systemic and intestinal renin-angiotensin system was associated with worsening gut barrier permeability, gut-derived immune cell activation, systemic inflammation, and progression of DR in human subjects. In Akita mice, maintaining intestinal ACE2 expression prevented and reversed DR, emphasizing the multifaceted role of the intestinal renin-angiotensin system in diabetes and DR.

The molecular signaling cascades that regulate angiogenesis and microvascular remodeling are fundamental to normal development, healthy physiology, and pathologies such as inflammation and cancer. Yet quantifying such complex, fractally branching vascular patterns remains difficult. We review application of NASA’s globally available, freely downloadable VESsel GENeration (VESGEN) Analysis software to numerous examples of 2D vascular trees, networks, and tree-network composites. Upon input of a binary vascular image, automated output includes informative vascular maps and quantification of parameters such as tortuosity, fractal dimension, vessel diameter, area, length, number, and branch point. Previous research has demonstrated that cytokines and therapeutics such as vascular endothelial growth factor, basic fibroblast growth factor (fibroblast growth factor-2), transforming growth factor-beta-1, and steroid triamcinolone acetonide specify unique “fingerprint” or “biomarker” vascular patterns that integrate dominant signaling with physiological response. In vivo experimental examples described here include vascular response to keratinocyte growth factor, a novel vessel tortuosity factor; angiogenic inhibition in humanized tumor xenografts by the anti-angiogenesis drug leronlimab; intestinal vascular inflammation with probiotic protection by Saccharomyces boulardii, and a workflow programming of vascular architecture for 3D bioprinting of regenerative tissues from 2D images. Microvascular remodeling in the human retina is described for astronaut risks in microgravity, vessel tortuosity in diabetic retinopathy, and venous occlusive disease.

Rationale: There is incomplete knowledge of the impact of bone marrow cells on the gut microbiome and gut barrier function. Objective: We postulated that diabetes mellitus and systemic ACE2 (angiotensin-converting enzyme 2) deficiency would synergize to adversely impact both the microbiome and gut barrier function. Methods and Results: Bacterial 16S rRNA sequencing and metatranscriptomic analysis were performed on fecal samples from wild-type, ACE2 −/y , Akita (type 1 diabetes mellitus), and ACE2 −/y -Akita mice. Gut barrier integrity was assessed by immunofluorescence, and bone marrow cell extravasation into the small intestine was evaluated by flow cytometry. In the ACE2 −/y -Akita or Akita mice, the disrupted barrier was associated with reduced levels of myeloid angiogenic cells, but no increase in inflammatory monocytes was observed within the gut parenchyma. Genomic and metatranscriptomic analysis of the microbiome of ACE2 −/y -Akita mice demonstrated a marked increase in peptidoglycan-producing bacteria. When compared with control cohorts treated with saline, intraperitoneal administration of myeloid angiogenic cells significantly decreased the microbiome gene expression associated with peptidoglycan biosynthesis and restored epithelial and endothelial gut barrier integrity. Also indicative of diabetic gut barrier dysfunction, increased levels of peptidoglycan and FABP-2 (intestinal fatty acid-binding protein 2) were observed in plasma of human subjects with type 1 diabetes mellitus (n=21) and type 2 diabetes mellitus (n=23) compared with nondiabetic controls (n=23). Using human retinal endothelial cells, we determined that peptidoglycan activates a noncanonical TLR-2 (Toll-like receptor 2) associated MyD88 (myeloid differentiation primary response protein 88)-ARNO (ADP-ribosylation factor nucleotide-binding site opener)-ARF6 (ADP-ribosylation factor 6) signaling cascade, resulting in destabilization of p120-catenin and internalization of VE-cadherin as a mechanism of deleterious impact of peptidoglycan on the endothelium. Conclusions: We demonstrate for the first time that the defect in gut barrier function and dysbiosis in ACE2 −/y -Akita mice can be favorably impacted by exogenous administration of myeloid angiogenic cells.

The gut is a well-established route of infection and target for viral damage by SARS-CoV-2. This is supported by the clinical observation that about half of COVID-19 patients exhibit gastrointestinal (GI) symptoms. We asked whether the analysis of plasma could provide insight into gut barrier dysfunction in patients with COVID-19 infection. Plasma samples of COVID-19 patients (n=30) and healthy control (n=16) were collected during hospitalization. Plasma microbiome was analyzed using 16S rRNA sequencing, metatranscriptomic analysis, and gut permeability markers including FABP-2, PGN and LPS in both patient cohorts. Almost 65% (9 out 14) COVID-19 patients showed abnormal presence of gut microbes in their bloodstream. Plasma samples contained predominately Proteobacteria, Firmicutes, and Actinobacteria. The abundance of gram-negative bacteria (Acinetobacter, Nitrospirillum, Cupriavidus, Pseudomonas, Aquabacterium, Burkholderia, Caballeronia, Parabhurkholderia, Bravibacterium, and Sphingomonas) was higher than the gram-positive bacteria (Staphylococcus and Lactobacillus) in COVID-19 subjects. The levels of plasma gut permeability markers FABP2 (1282±199.6 vs 838.1±91.33; p=0.0757), PGN (34.64±3.178 vs 17.53±2.12; pvs 249.6±17.06; p=0.0049) were higher in COVID-19 patients compared to healthy subjects. These findings support that the intestine may represent a source for bacteremia and may contribute to worsening COVID-19 outcomes. Therapies targeting the gut and prevention of gut barrier defects may represent a strategy to improve outcomes in COVID-19 patients.

AIMS/HYPOTHESIS: Homo Sapiens evolved under conditions of intermittent food availability and prolonged fasting between meals. Periods of fasting are important for recovery from meal-induced oxidative and metabolic stress, and tissue repair. Constant high energy-density food availability in present-day society contributes to the pathogenesis of chronic diseases, including diabetes and its complications, with intermittent fasting (IF) and energy restriction shown to improve metabolic health. We have previously demonstrated that IF prevents the development of diabetic retinopathy in a mouse model of type 2 diabetes (db/db); however the mechanisms of fasting-induced health benefits and fasting-induced risks for individuals with diabetes remain largely unknown. Sirtuin 1 (SIRT1), a nutrient-sensing deacetylase, is downregulated in diabetes. In this study, the effect of SIRT1 stimulation by IF, fasting mimicking cell culture conditions (FMC) or pharmacological treatment using SRT1720 was evaluated on systemic and retinal metabolism, systemic and retinal inflammation and vascular and bone marrow damage. METHODS: The effects of IF were modelled in vivo using db/db mice and in vitro using bovine retinal endothelial cells or rat retinal neuroglial/precursor R28 cell line serum starved for 24 h. mRNA expression was analysed by quantitative PCR (qPCR). SIRT1 activity was measured via histone deacetylase activity assay. NR1H3 (also known as Liver X receptor alpha (LXRα)) acetylation was measured via western blot analysis. RESULTS: IF increased Sirt1 mRNA expression in mouse liver and retina when compared with non-fasted animals. IF also increased SIRT1 activity eightfold in mouse retina while FMC increased SIRT1 activity and expression in retinal endothelial cells when compared with control. Sirt1 expression was also increased twofold in neuronal retina progenitor cells (R28) after FMC treatment. Moreover, FMC led to SIRT1-mediated LXRα deacetylation and subsequent 2.4-fold increase in activity, as measured by increased mRNA expression of the genes encoding ATP-binding cassette transporter (Abca1 and Abcg1). These changes were reduced when retinal endothelial cells expressing a constitutively acetylated LXRα mutant were tested. Increased SIRT1/LXR/ABC-mediated cholesterol export resulted in decreased retinal endothelial cell cholesterol levels. Direct activation of SIRT1 by SRT1720 in db/db mice led to a twofold reduction of diabetes-induced inflammation in the retina and improved diabetes-induced visual function impairment, as measured by electroretinogram and optokinetic response. In the bone marrow, there was prevention of diabetes-induced myeloidosis and decreased inflammatory cytokine expression. CONCLUSIONS/INTERPRETATION: Taken together, activation of SIRT1 signalling by IF or through pharmacological activation represents an effective therapeutic strategy that provides a mechanistic link between the advantageous effects associated with fasting regimens and prevention of microvascular and bone marrow dysfunction in diabetes.

In diabetic dyslipidemia, cholesterol accumulates in the plasma membrane, decreasing fluidity and thereby suppressing the ability of cells to transduce ligand-activated signaling pathways. Liver X receptors (LXRs) are the main cellular mechanism by which intracellular cholesterol is regulated and play important roles in inflammation and disease pathogenesis. N,N-dimethyl-3β-hydroxy-cholenamide (DMHCA), a selective LXR agonist, specifically activates the cholesterol efflux arm of the LXR pathway without stimulating triglyceride synthesis. Thus, DMHCA possesses superior clinical potential as a cholesterol lowering agent than current LXR pan-agonist. In this study, we use a multi-systems approach to understand the effects and molecular mechanisms of DMHCA treatment in type 2 diabetic db/db mice and human -derived circulating angiogenic cells (CACs), which are vascular reparative cells. We find that DMHCA is sufficient to correct the retina-bone marrow (BM) axis in diabetes, thereby restoring retinal structure, function, and cholesterol homeostasis, rejuvenating membrane fluidity in circulating vascular reparative cells, hampering systemic inflammation, and correcting BM dysfunction. Using single-cell RNA-seq on lineage-sca1+cKit+(LSK) hematopoietic stem cells (HSCs) from untreated and DMHCA-treated diabetic mice, we provide novel insights into hematopoiesis and reveal DMHCA’s mechanism of action in correcting diabetic HSCs by reducing myeloidosis and increasing CACs and erythrocyte progenitors. Taken together, these findings demonstrate the broad and pleiotropic effects of DMHCA treatment, which has exciting potential to correct the retina-BM axis in diabetic subjects.

Human induced pluripotent stem cells (hiPSC) differentiated into a specific mesoderm subset expressing vascular endothelial growth factor receptor 2, neural cell adhesion molecule 1, and apelin G protein-coupled receptor APJ (called KNA+), possessed all of the phenotypic and functional endothelial colony forming cell potential that resides in differentiating hiPSC. Thus, we postulated KNA+ mesoderm treatment would correct diabetic retinal capillary vasodegeneration. KNA+ cells derived from diabetic (D) and non-diabetic (N) patient-derived hiPSC displayed comparable phenotypic and functional properties. N-KNA+ or D-KNA+ intravitreal injections into type II diabetic ( db/db ) murine eyes led to retinal vascular engraftment, showed no toxicity, visual acuity was significantly improved, and diabetes-induced scotopic and photopic ERG defects were corrected. Proteomic arrays revealed that hiPSC-derived KNA+ cell administration restored several aberrant cell signaling pathways in the neural retina. These results support the efficacy of both hiPSC-derived D-KNA+ and N-KNA+ cells in correcting vasodegeneration in the diabetic murine retina.

SummaryIn diabetes, the retina, a tissue with unique metabolic needs, demonstrates dysregulation of the intricate balance between nutrient availability and utilization. This results in cholesterol accumulation, pro-inflammatory and pro-apoptotic changes, and consequently neurovascular damage. Sirtuin 1 (SIRT1), a nutrient sensing deacetylase, is downregulated in the diabetic retina. In this study, the effect of SIRT1 stimulation by fasting or by pharmacological activation using SRT1720, was evaluated on retinal cholesterol metabolism, inflammation and neurovascular damage. SIRT1 activation, in retinal endothelial cells (REC) and neuronal retinal progenitor cells (R28), led to Liver X Receptor alpha (LXRα) deacetylation and subsequent increased activity, as measured by increased ATP-binding cassette transporter (ABC) A1 and G1 mRNA expression. In turn, increased cholesterol export resulted in decreased REC cholesterol levels. SIRT1 activation also led to decreased inflammation. SIRT1 activation, in vivo, prevented diabetes-induced inflammation and vascular and neural degeneration. Diabetes-induced visual function impairment, as measured by electroretinogram and optokinetic response, was significantly improved as a result of SIRT1 activation. Taken together, activation of SIRT1 signaling is an effective therapeutic strategy that provides a mechanistic link between the advantageous effects associated with fasting regimes and prevention of diabetic retinopathy (DR).

SIRT1 is critical to vascular health and regulates glucose and lipid metabolism through its deacetylase activity. Type 2 diabetes-associated decreases in SIRT1 levels result in increased levels of acetylated LXR leading to reduced expression of reverse cholesterol transport genes and increased expression of NFκB-controlled pro-inflammatory genes. Diabetic circulating angiogenic cells (CAC) which are critical in vascular repair also demonstrate reduced SIRT1 and increasing its levels corrects CAC dysfunction (1). db/db mice were given SRT-1720 hydrochloride (100 mg/kg body weigh/day for 2 or 6 mo.) in their chow or fed normal chow beginning at diabetes onset. Retinal tissue, blood and bone marrow (BM) were collected. Electroretinograms (ERG) were performed at 2 and 6 mo. The BM supernatant was analyzed for cytokines. BM hematopoietic stem cells and CACs were assessed by flow cytometry. Retinal lipids were assessed by Mass Spectroscopy. At 2 mo, the retina of SRT1720-treated db/db mice demonstrated an increase in the ratio of total cholesterol/demosterol/lanosterol (197±85) compared to db/db (65±12. p Disclosure C.P. Vieira: None. A. Longhini: None. S. Li Calzi: None. M.D. DuPont: None. J.V. Busik: None. M.B. Grant: None. Funding National Institutes of Health (EY025383)

Increasing evidence points to inflammation as a key component in diabetes-associated retinal perturbations, however, the underlying molecular mechanisms are not yet fully understood. The aims of this study were to characterize peripheral blood mononuclear cells (PBMC) in diabetics with and without DR and to elucidate the biologic role of NLRP3 inflammasome in these cells. PBMC from 33 diabetics (T1D n=9 and T2D n=24) and 18 controls were assessed by flow cytometry. To examine the inflammasome activation, monocytes and neutrophils were treated with the TLR-2 agonist, peptidoglycan (PGN) (10µg/ml) for 3 hours followed by stimulation with a potassium ionophore, nigericin. NLRP3, IL-1β, Caspase-1, IL-18 levels were assessed. Neutrophil counts were significantly higher (1.6 folds, p Disclosure M.D. DuPont: None. A. Longhini: None. G. Sreejit: None. R. Prasad: None. P. Nagareddy: None. M.B. Grant: None. Funding National Institutes of Health (EY028037)

Retinitis pigmentosa (RP) is an inherited retinal disease with a prevalence of 1/4,000. RP is highly genetically heterogeneous and there are over 80 genes associated with RP to date. One particular USH2A variant, p.C759F, has long been reported in RP cases but its pathogenicity was questioned by a recent study. Here, by leveraging large scale next-generation sequencing data from 982 non-Asian RP probands, we used binomial tests to evaluate the enrichment of this allele in RP cohort. We observed significant enrichment of this allele both in homozygous state and in compound heterozygous state with another USH2A protein-truncating allele. The results highlighted the clinical significance of the USH2A p.C759F allele in RP cases, which is important for accurate molecular diagnosis.