Impact of The Protective Renin-Angiotensin System (RAS) on The Vasoreparative Function of CD34+ CACs in Diabetic Retinopathy

Purpose: In diabetes, the impaired vasoreparative function of Circulating Angiogenic Cells (CACs) is believed to contribute to the progression of diabetic retinopathy (DR). Accumulating evidence suggests that the protective arm of renin-angiotensin system (RAS) ACE2 Angiotensin-(1-7) Mas plays an important role in restoring the function of diabetic CACs. We examined the protective RAS in CACs in diabetic individuals with different stages of retinopathy. Methods: Study subjects (n43) were recruited as controls or diabetics with either no DR, mild non-proliferative DR (NPDR), moderate NPDR, severe NPDR or proliferative DR (PDR). Fundus photography and fluorescein angiograms were analyzed using Vessel Generation Analysis (VESGEN) software in a cohort of subjects. CD34+ CACs were isolated from peripheral blood of diabetics and control subjects. RAS gene expressions in CACs were measured by qPCR. The vasoreparative function of CACs was assessed by migration ability toward CXCL12 using the QCM 5M 96-well chemotaxis cell migration assay. Results: ACE2 gene is a key enzyme converting the deleterious Angiotensin II to the beneficial Angiotensin-(1-7). ACE2 expression in CACs from diabetic subjects without DR was increased compared to controls, suggestive of compensation (p0.0437). The expression of Mas (Angiotensin-(1-7) receptor) in CACs was also increased in diabetics without DR, while was reduced in NPDR compared to controls (p0.0002), indicating a possible loss of compensation of the protective RAS at this stage of DR. The presence of even mild NPDR was associated with CD34+ CAC migratory dysfunction. When pretreating CACs of DR subjects with Angiotensin-(1-7), migratory ability to a chemoattractant CXCL12 was restored (p0.0008). By VESGEN analysis, an increase in small vessel density was observed in NPDR subjects when compared with the controls. Conclusions: These data suggest the protective RAS axis within diabetic CACs may help maintain their vasoreparative potential. The VESGEN analysis supports the presence of retinal repair in small vessels. The loss of the protective arm of RAS may predict the progression of DR

miTuner - a kit for microRNA based gene expression tuning in mammalian cells

The purpose of this RFC is to introduce a modular expression tuning kit for use in mammalian cells. The kit enables the regulation of the gene expression of any gene of interest (GOI) based on synthetic microRNAs, endogenous microRNAs or a combination of both

Loss of Angiotensin-Converting Enzyme 2 Exacerbates Diabetic Retinopathy by Promoting Bone Marrow Dysfunction

Angiotensin-converting enzyme 2 (ACE2) is the primary enzyme of the vasoprotective axis of the renin angiotensin system (RAS). We tested the hypothesis that loss of ACE2 would exacerbate diabetic retinopathy by promoting bone marrow dysfunction. ACE2-/y were crossed with Akita mice, a model of type 1 diabetes. When comparing the bone marrow of the ACE2-/y-Akita mice to that of Akita mice, we observed a reduction of both short-term and long-term repopulating hematopoietic stem cells, a shift of hematopoiesis towards myelopoiesis, and an impairment of lineage-c-kit+ hematopoietic stem/progenitor cell (HS/PC) migration and proliferation. Migratory and proliferative dysfunction of these cells was corrected by exposure to angiotensin-1–7 (Ang-1–7), the protective peptide generated by ACE2. Over the duration of diabetes examined, ACE2 deficiency led to progressive reduction in electrical responses assessed by electroretinography and to increases in neural infarcts observed by fundus photography. Compared to Akita mice, ACE2-/y-Akita at 9-months of diabetes showed an increased number of acellular capillaries indicative of more severe diabetic retinopathy. In diabetic and control human subjects, CD34+ cells, a key bone marrow HS/PC population, were assessed for changes in mRNA levels for MAS, the receptor for Ang-1–7. Levels were highest in CD34+ cells from diabetics without retinopathy. Higher serum Ang-1–7 levels predicted protection from development of retinopathy in diabetics. Treatment with Ang-1–7 or alamandine restored the impaired migration function of CD34+ cells from subjects with retinopathy. These data support that activation of the protective RAS within HS/PCs may represent a therapeutic strategy for prevention of diabetic retinopathy

Renin-Angiotensin System (RAS) in Hematopoietic Stem/Progenitor Cells (HS/PC) Predicts Vaso-Reparative Dysfunction and Progression of Diabetic Retinopathy (DR)

Purpose: We tested the hypothesis that loss of angiotensin converting enzyme 2 (ACE2) within diabetic HS/PCs (Hematopoietic Stem/Progenitor Cells) would be detrimental to HS/PC reparative function, and alter their ability to contribute to vascular remodeling in human subjects and rodent models of DR (Diabetic Retinopathy). Methods: Subjects (n52) were recruited as controls (n13) or diabetics (n39) with either no DR, mild non-proliferative DR (NPDR), moderate NPDR, severe NPDR or proliferative DR (PDR). Fluorescein angiograms were analyzed using Vessel Generation Analysis (VESGEN) software in a cohort of subjects. CD34+ HS/PCs were isolated from peripheral blood. RAS (Renin-Angiotensin System) gene expression and migration was measured. Diabetic ACE2 knockout (KO)C57BL6-Ins2 (Akita) mice at 3, 6 and 9 months of diabetes were compared to age-matched controls. Bone marrow HS/PC populations were analyzed by flow cytometry and migration and proliferation studies performed. Results: ACE2 gene expression in human CD34+ cells from diabetics without DR was increased compared to controls (p0.0437). Mas receptor mRNA was also increased in diabetics without DR, but reduced with the onset of NPDR (p0.0002), suggesting a loss of compensation. DR was associated with CD34+ cell migratory dysfunction. By VESGEN analysis, vessel density measured by several confirming parameters in early NPDR (n3) was greater than in normal retina (n6) in both arteries and veins, which suggests active retinal remodeling. ACE2KO-Akita and Akita cohorts showed reduced retinal thickness by OCT (Optical Coherence Tomography) at 9 months of diabetes. Absence of ACE2 in 9-month Akita mice led to an accelerated increase in acellular capillaries compared to diabetic alone. Electroretinogram (ERG) in ACE2KO-Akita mice resulted in persistent deterioration of the neural retina. Reparative function studies showed that ACE2KO exacerbated diabetes-induced impairment of LK (Low Potassium) cell migration and proliferative functions as early as 3-month of diabetes (p0.0019). Conclusions: Retinopathy and adverse vascular remodeling in subjects with diabetes was associated with a loss of the protective arm of RAS in HS/PCs. Loss of ACE2 exacerbated vascular dysfunction in diabetic mice

miMeasure – a standard for miRNA binding site characterization in mammalian cells

This RFC proposes a standard for the quantitative characterization of miRNA binding sites (miRNA-BS) in mammalian cells. The miMeasure standard introduces a ready-to-use standard measurement plasmid (pSMB_miMeasure, BBa_K337049) enabling rapid experimental characterization of any miRNA-BS of choice. We recommend a new standard unit, RKDU (relative knock-down unit) to describe the knock-down efficiency of a miRNA-BS in a specific cell type. pSMB_miMeasure allows for an easy and fast measurement of RKDU while providing effective normalization against variance stemming from differences in transfection efficiency and from other sources

The global burden of cancer attributable to risk factors, 2010-19 : a systematic analysis for the Global Burden of Disease Study 2019

Background Understanding the magnitude of cancer burden attributable to potentially modifiable risk factors is crucial for development of effective prevention and mitigation strategies. We analysed results from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019 to inform cancer control planning efforts globally. Methods The GBD 2019 comparative risk assessment framework was used to estimate cancer burden attributable to behavioural, environmental and occupational, and metabolic risk factors. A total of 82 risk-outcome pairs were included on the basis of the World Cancer Research Fund criteria. Estimated cancer deaths and disability-adjusted life-years (DALYs) in 2019 and change in these measures between 2010 and 2019 are presented. Findings Globally, in 2019, the risk factors included in this analysis accounted for 4.45 million (95% uncertainty interval 4.01-4.94) deaths and 105 million (95.0-116) DALYs for both sexes combined, representing 44.4% (41.3-48.4) of all cancer deaths and 42.0% (39.1-45.6) of all DALYs. There were 2.88 million (2.60-3.18) risk-attributable cancer deaths in males (50.6% [47.8-54.1] of all male cancer deaths) and 1.58 million (1.36-1.84) risk-attributable cancer deaths in females (36.3% [32.5-41.3] of all female cancer deaths). The leading risk factors at the most detailed level globally for risk-attributable cancer deaths and DALYs in 2019 for both sexes combined were smoking, followed by alcohol use and high BMI. Risk-attributable cancer burden varied by world region and Socio-demographic Index (SDI), with smoking, unsafe sex, and alcohol use being the three leading risk factors for risk-attributable cancer DALYs in low SDI locations in 2019, whereas DALYs in high SDI locations mirrored the top three global risk factor rankings. From 2010 to 2019, global risk-attributable cancer deaths increased by 20.4% (12.6-28.4) and DALYs by 16.8% (8.8-25.0), with the greatest percentage increase in metabolic risks (34.7% [27.9-42.8] and 33.3% [25.8-42.0]). Interpretation The leading risk factors contributing to global cancer burden in 2019 were behavioural, whereas metabolic risk factors saw the largest increases between 2010 and 2019. Reducing exposure to these modifiable risk factors would decrease cancer mortality and DALY rates worldwide, and policies should be tailored appropriately to local cancer risk factor burden. Copyright (C) 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.Peer reviewe

Per2-Mediated Vascular Dysfunction Is Caused by the Upregulation of the Connective Tissue Growth Factor (CTGF).

Period 2-mutant mice (Per2m/m), which possess a circadian dysfunction, recapitulate the retinal vascular phenotype similar to diabetic retinopathy (DR). The vascular dysfunction in Per2m/m is associated with an increase in connective tissue growth factor (CTGF/CCN2). At the molecular level, CTGF gene expression is dependent on the canonical Wnt/β-catenin pathway. The nuclear binding of β-catenin to a transcription factor, lymphoid enhancer binding protein (Lef)/ T-cell factor (TCF/LEF), leads to downstream activation of CTGF. For this study, we hypothesized that the silencing of Per2 results in nuclear translocation and subsequent transactivation of the CTGF gene. To test this hypothesis, we performed immunofluorescence labeling for CTGF in retinal sections from wild-type (WT) and Per2m/m mice. Human retinal endothelial cells (HRECs) were transfected with siRNA for Per2, and the protein expression of CTGF and β-catenin was evaluated. The TCF/LEF luciferase reporter (TOPflash) assay was performed to validate the involvement of β-catenin in the activation of CTGF. Per2m/m retinas exhibited an increased CTGF immunostaining in ganglion cell layer and retinal endothelium. Silencing of Per2 using siRNA resulted in an upregulation of CTGF and β-catenin. The TOPflash assay revealed an increase in luminescence for HRECs transfected with Per2 siRNA. Our studies show that loss of Per2 results in an activation of CTGF via nuclear entry of β-catenin. Our study provides novel insight into the understanding of microvascular dysfunction in Per2m/m mice

Combined single-cell and spatial transcriptomics reveal the molecular, cellular and spatial bone marrow niche organization

The bone marrow constitutes the primary site for life-long blood production and skeletal regeneration. However, its cellular and spatial organization remains controversial. Here, we combine single-cell and spatially resolved transcriptomics to systematically map the molecular, cellular and spatial composition of distinct bone marrow niches. This allowed us to transcriptionally profile all major bone-marrow-resident cell types, determine their localization and clarify sources of pro-haematopoietic factors. Our data demonstrate that Cxcl12-abundant-reticular (CAR) cell subsets (Adipo-CAR and Osteo-CAR) differentially localize to sinusoidal and arteriolar surfaces, act locally as 'professional cytokine-secreting cells' and thereby establish peri-vascular micro-niches. Importantly, the three-dimensional bone-marrow organization can be accurately inferred from single-cell transcriptome data using the RNA-Magnet algorithm described here. Together, our study reveals the cellular and spatial organization of bone marrow niches and offers a systematic approach to dissect the complex organization of whole organs

The increase in CTGF staining in <i>Per2</i>^<i>m/m</i> retinas.

<p>(A) Retinal sections were stained with CTGF antibodies. The photomicrographs show an increase in staining for CTGF (green) in the GCL (white arrows) and OPL (white arrows) in <i>Per2</i>^m/m mice; the bar-graph shows quantification of mean fluorescence intensity for (B) GCL and (C) OPL. (D) Retinal endothelial cells were stained with BS-isolectin (red) prior to euthanasia. The staining reveals an increase in CTGF expression in vessels co-stained with BS-1 isolectin. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, Inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PR, photoreceptors. n = 3.</p

<i>Per2</i>^<i>m/m</i> mice show a decrease in retinal thickness.

<p>(A) The OCT scans of the whole retina showing representative images from the WT and <i>Per2</i>^<i>m/m</i> mice. RNFL, retinal nerve fiber layer; GCL/IPL, Ganglion cell layer/inner plexiform layer; INL, Inner nuclear layer; OPL, Outer plexiform layer; ONL, Outer nuclear layer; ELM/IS/OS, external limiting membrane/inner segment of photoreceptors/outer segment of photoreceptors; RPE, Retinal pigment epithelial layer. (B) The measurements of retinal thickness were performed by placing the 2 calipers (C01 & C02) near the optic nerve; the bar chart shows a decrease in retinal thickness of <i>Per2</i>^<i>m/m</i>. n = 6, *p<0.05.</p