Depleting hypothalamic somatostatinergic neurons recapitulates diabetic phenotypes in mouse brain, bone marrow, adipose and retina.

AIMS/HYPOTHESIS: Hypothalamic inflammation and sympathetic nervous system hyperactivity are hallmark features of the metabolic syndrome and type 2 diabetes. Hypothalamic inflammation may aggravate metabolic and immunological pathologies due to extensive sympathetic activation of peripheral tissues. Loss of somatostatinergic (SST) neurons may contribute to enhanced hypothalamic inflammation. METHODS: The present data show that leptin receptor-deficient (db/db) mice exhibit reduced hypothalamic SST neurons, particularly in the periventricular nucleus. We model this finding, using adeno-associated virus delivery of diphtheria toxin subunit A (DTA) driven by an SST-cre system to deplete these neurons in Sstcre/gfp mice (SST-DTA). RESULTS: SST-DTA mice exhibit enhanced hypothalamic c-Fos expression and brain inflammation as demonstrated by microglial and astrocytic activation. Bone marrow from SST-DTA mice undergoes skewed haematopoiesis, generating excess granulocyte-monocyte progenitors and increased proinflammatory (C-C chemokine receptor type 2; CCR2hi) monocytes. SST-DTA mice exhibited a 'diabetic retinopathy-like' phenotype: reduced visual function by optokinetic response (0.4 vs 0.25 cycles/degree; SST-DTA vs control mice); delayed electroretinogram oscillatory potentials; and increased percentages of retinal monocytes. Finally, mesenteric visceral adipose tissue from SST-DTA mice was resistant to catecholamine-induced lipolysis, displaying 50% reduction in isoprenaline (isoproterenol)-induced lipolysis compared with control littermates. Importantly, hyperglycaemia was not observed in SST-DTA mice. CONCLUSIONS/INTERPRETATION: The isolated reduction in hypothalamic SST neurons was able to recapitulate several hallmark features of type 2 diabetes in disease-relevant tissues.

Fasting and fasting-mimicking treatment activate SIRT1/LXRα and alleviate diabetes-induced systemic and microvascular dysfunction.

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.

Novel Methods to Mobilize, Isolate, and Expand Mesenchymal Stem Cells.

Numerous studies demonstrate the essential role of mesenchymal stem cells (MSCs) in the treatment of metabolic and inflammatory diseases, as these cells are known to modulate humoral and cellular immune responses. In this manuscript, we efficiently present two novel approaches to obtain MSCs from equine or human sources. In our first approach, we used electro-acupuncture as previously described by our group to mobilize MSCs into the peripheral blood of horses. For equine MSC collection, culture, and expansion, we used the Miltenyi Biotec CliniMACS Prodigy system of automated cell manufacturing. Using this system, we were able to generate appoximately 100 MSC colonies that exhibit surface marker expression of CD105 (92%), CD90 (85%), and CD73 (88%) within seven days of blood collection. Our second approach utilized the iPSC embryoid bodies from healthy or diabetic subjects where the iPSCs were cultured in standard media (endothelial + mesoderm basal media). After 21 days, the cells were FACS sorted and exhibited surface marker expression of CD105, CD90, and CD73. Both the equine cells and the human iPSC-derived MSCs were able to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Both methods described simple and highly efficient methods to produce cells with surface markers phenotypically considered as MSCs and may, in the future, facilitate rapid production of MSCs with therapeutic potential.

Intravitreal Administration of AAV2-SIRT1 Reverses Diabetic Retinopathy in a Mouse Model of Type 2 Diabetes.

Purpose: The expression of silent information regulator (SIRT) 1 is reduced in diabetic retinopathy (DR). Previous studies showed that alterations in SIRT1 messenger RNA (mRNA) and protein expression are implicated in progressive inflammation and formation of retinal acellular capillaries. Treatment with the SIRT1 agonist, SRT1720, improved visual response by restoration of a- and b-wave responses on electroretinogram scotopic measurements in diabetic (db/db) mice. In this study, we investigated the effects of intravitreal SIRT1 delivery on diabetic retinal pathology. Methods: Nine-month-old db/db mice received one intravitreal injection of either AAV2-SIRT1 or AAV2-GFP control virus, and after 3 months, electroretinography and optomotor responses were measured. Their eyes were then removed and analyzed by immunohistochemistry and flow cytometry. Results: SIRT1 mRNA and protein levels were increased following AAV2-SIRT1 administration compared to control virus AAV2-GFP injected mice. IBA1+ and caspase 3 expression were decreased in retinas of db/db mice injected with AAV2-SIRT1, and reductions in scotopic a- and b-waves and high spatial frequency in optokinetic response were prevented. Retinal hypoxia inducible factor 1α (HIF-1α) protein levels were reduced in the AAV2-SIRT1-injected mice compared to control-injected mice. Using flow cytometry to assess changes in intracellular HIF-1α levels, endothelial cells (CD31+) from AAV-2 SIRT1 injected mice demonstrated reduced HIF-1α expression compared to db/db mice injected with the control virus. Conclusions: Intravitreal AAV2-SIRT1 delivery increased retina SIRT1 and transduced neural and endothelial cells, thus reversing functional damage and improving overall visual function. Translational Relevance: AAV2-SIRT1 gene therapy represents a beneficial approach for the treatment of chronic retinal conditions such as DR.

Circulating SARS-CoV-2+ megakaryocytes are associated with severe viral infection in COVID-19.

Several independent lines of evidence suggest that megakaryocytes are dysfunctional in severe COVID-19. Herein, we characterized peripheral circulating megakaryocytes in a large cohort of inpatients with COVID-19 and correlated the subpopulation frequencies with clinical outcomes. Using peripheral blood, we show that megakaryocytes are increased in the systemic circulation in COVID-19, and we identify and validate S100A8/A9 as a defining marker of megakaryocyte dysfunction. We further reveal a subpopulation of S100A8/A9+ megakaryocytes that contain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein and RNA. Using flow cytometry of peripheral blood and in vitro studies on SARS-CoV-2-infected primary human megakaryocytes, we demonstrate that megakaryocytes can transfer viral antigens to emerging platelets. Mechanistically, we show that SARS-CoV-2-containing megakaryocytes are nuclear factor κB (NF-κB)-activated, via p65 and p52; express the NF-κB-mediated cytokines interleukin-6 (IL-6) and IL-1ß; and display high surface expression of Toll-like receptor 2 (TLR2) and TLR4, canonical drivers of NF-κB. In a cohort of 218 inpatients with COVID-19, we correlate frequencies of megakaryocyte subpopulations with clinical outcomes and show that SARS-CoV-2-containing megakaryocytes are a strong risk factor for mortality and multiorgan injury, including respiratory failure, mechanical ventilation, acute kidney injury, thrombotic events, and intensive care unit admission. Furthermore, we show that SARS-CoV-2+ megakaryocytes are present in lung and brain autopsy tissues from deceased donors who had COVID-19. To our knowledge, this study offers the first evidence implicating SARS-CoV-2+ peripheral megakaryocytes in severe disease and suggests that circulating megakaryocytes warrant investigation in inflammatory disorders beyond COVID-19.

Specific mesoderm subset derived from human pluripotent stem cells ameliorates microvascular pathology in type 2 diabetic mice.

Human induced pluripotent stem cells (hiPSCs) were differentiated into a specific mesoderm subset characterized by KDR+CD56+APLNR+ (KNA+) expression. KNA+ cells had high clonal proliferative potential and specification into endothelial colony-forming cell (ECFCs) phenotype. KNA+ cells differentiated into perfused blood vessels when implanted subcutaneously into the flank of nonobese diabetic/severe combined immunodeficient mice and when injected into the vitreous of type 2 diabetic mice (db/db mice). Transcriptomic analysis showed that differentiation of hiPSCs derived from diabetics into KNA+ cells was sufficient to change baseline differences in gene expression caused by the diabetic status and reprogram diabetic cells to a pattern similar to KNA+ cells derived from nondiabetic hiPSCs. Proteomic array studies performed on retinas of db/db mice injected with either control or diabetic donor-derived KNA+ cells showed correction of aberrant signaling in db/db retinas toward normal healthy retina. These data provide "proof of principle" that KNA+ cells restore perfusion and correct vascular dysfunction in db/db mice.

Characterizing the Retinal Phenotype in the High-Fat Diet and Western Diet Mouse Models of Prediabetes.

We sought to delineate the retinal features associated with the high-fat diet (HFD) mouse, a widely used model of obesity. C57BL/6 mice were fed either a high-fat (60% fat; HFD) or low-fat (10% fat; LFD) diet for up to 12 months. The effect of HFD on body weight and insulin resistance were measured. The retina was assessed by electroretinogram (ERG), fundus photography, permeability studies, and trypsin digests for enumeration of acellular capillaries. The HFD cohort experienced hypercholesterolemia when compared to the LFD cohort, but not hyperglycemia. HFD mice developed a higher body weight (60.33 g vs. 30.17g, p < 0.0001) as well as a reduced insulin sensitivity index (9.418 vs. 62.01, p = 0.0002) compared to LFD controls. At 6 months, retinal functional testing demonstrated a reduction in a-wave and b-wave amplitudes. At 12 months, mice on HFD showed evidence of increased retinal nerve infarcts and vascular leakage, reduced vascular density, but no increase in number of acellular capillaries compared to LFD mice. In conclusion, the HFD mouse is a useful model for examining the effect of prediabetes and hypercholesterolemia on the retina. The HFD-induced changes appear to occur slower than those observed in type 2 diabetes (T2D) models but are consistent with other retinopathy models, showing neural damage prior to vascular changes.

Selective LXR agonist DMHCA corrects retinal and bone marrow dysfunction in type 2 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) make up the main cellular mechanism by which intracellular cholesterol is regulated and play important roles in inflammation and disease pathogenesis. N, N-dimethyl-3ß-hydroxy-cholenamide (DMHCA), a selective LXR agonist, specifically activates the cholesterol efflux arm of the LXR pathway without stimulating triglyceride synthesis. In this study, we use a multisystem approach to understand the effects and molecular mechanisms of DMHCA treatment in type 2 diabetic (db/db) mice and human circulating angiogenic cells (CACs), which are hematopoietic progenitor cells with vascular reparative capacity. We found that DMHCA is sufficient to correct retinal and BM dysfunction in diabetes, thereby restoring retinal structure, function, and cholesterol homeostasis; rejuvenating membrane fluidity in CACs; hampering systemic inflammation; and correcting BM pathology. Using single-cell RNA sequencing on lineage-sca1+c-Kit+ (LSK) hematopoietic stem cells (HSCs) from untreated and DMHCA-treated diabetic mice, we provide potentially 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 beneficial effects of DMHCA treatment on diabetes-induced retinal and BM pathology.

Effect of Polyphenols From Campomanesia adamantium on Platelet Aggregation and Inhibition of Cyclooxygenases: Molecular Docking and in Vitro Analysis.

Campomanesia adamantium is a medicinal plant of the Brazilian Cerrado. Different parts of its fruits are used in popular medicine to treat gastrointestinal disorders, rheumatism, urinary tract infections and inflammations. Despite its widespread use by the local population, the mechanisms involving platelet aggregation and the inhibition of cyclooxygenase by C. adamantium are unknown. This study evaluated the chemical composition, antioxidant activities and potential benefits of the C. adamantium peel extract (CAPE) and its components in the platelet aggregation induced by arachidonic acid in platelet-rich plasma. Aspects of the pharmacological mechanism were investigated as follows: platelet viability, calcium mobilization, levels of the cyclic nucleotides cAMP and cGMP, thromboxane B2 levels, and the inhibitory effects on COX-1 and COX-2 were studied in vitro and using molecular docking in the catalytic domain of these proteins. The major CAPE constituents standing out from the chemical analysis are the flavonoids, namely those of the flavones and chalcones class. The results showed that CAPE, quercetin and myricetin significantly decreased arachidonic acid-induced platelet aggregation; the assays showed that CAPE and quercetin decreased the mobilization of calcium and thromboxane B2 levels in platelets and increased cAMP and cGMP levels. Moreover, CAPE inhibited the activity of COX-1 and COX-2, highlighting that quercetin could potentially prevent the access of arachidonic acid more to the catalytic site of COX-1 than COX-2. These results highlight CAPE's potential as a promising therapeutic candidate for the prevention and treatment of diseases associated with platelet aggregation.

Restructuring of the Gut Microbiome by Intermittent Fasting Prevents Retinopathy and Prolongs Survival in db/db Mice.

Intermittent fasting (IF) protects against the development of metabolic diseases and cancer, but whether it can prevent diabetic microvascular complications is not known. In db/db mice, we examined the impact of long-term IF on diabetic retinopathy (DR). Despite no change in glycated hemoglobin, db/db mice on the IF regimen displayed significantly longer survival and a reduction in DR end points, including acellular capillaries and leukocyte infiltration. We hypothesized that IF-mediated changes in the gut microbiota would produce beneficial metabolites and prevent the development of DR. Microbiome analysis revealed increased levels of Firmicutes and decreased Bacteroidetes and Verrucomicrobia. Compared with db/db mice on ad libitum feeding, changes in the microbiome of the db/db mice on IF were associated with increases in gut mucin, goblet cell number, villi length, and reductions in plasma peptidoglycan. Consistent with the known modulatory effects of Firmicutes on bile acid (BA) metabolism, measurement of BAs demonstrated a significant increase of tauroursodeoxycholate (TUDCA), a neuroprotective BA, in db/db on IF but not in db/db on AL feeding. TGR5, the TUDCA receptor, was found in the retinal primary ganglion cells. Expression of TGR5 did not change with IF or diabetes. However, IF reduced retinal TNF-α mRNA, which is a downstream target of TGR5 activation. Pharmacological activation of TGR5 using INT-767 prevented DR in a second diabetic mouse model. These findings support the concept that IF prevents DR by restructuring the microbiota toward species producing TUDCA and subsequent retinal protection by TGR5 activation.