Metabolic adaptation to tyrosine kinase inhibition in leukemia stem cells.

Tyrosine kinase inhibitors (TKIs) are very effective in treating chronic myelogenous leukemia (CML), but primitive, quiescent leukemia stem cells persist as a barrier to the cure. We performed a comprehensive evaluation of metabolic adaptation to TKI treatment and its role in CML hematopoietic stem and progenitor cell persistence. Using a CML mouse model, we found that glycolysis, glutaminolysis, the tricarboxylic acid cycle, and oxidative phosphorylation (OXPHOS) were initially inhibited by TKI treatment in CML-committed progenitors but were restored with continued treatment, reflecting both selection and metabolic reprogramming of specific subpopulations. TKI treatment selectively enriched primitive CML stem cells with reduced metabolic gene expression. Persistent CML stem cells also showed metabolic adaptation to TKI treatment through altered substrate use and mitochondrial respiration maintenance. Evaluation of transcription factors underlying these changes helped detect increased HIF-1 protein levels and activity in TKI-treated stem cells. Treatment with an HIF-1 inhibitor in combination with TKI treatment depleted murine and human CML stem cells. HIF-1 inhibition increased mitochondrial activity and reactive oxygen species (ROS) levels, reduced quiescence, increased cycling, and reduced the self-renewal and regenerating potential of dormant CML stem cells. We, therefore, identified the HIF-1-mediated inhibition of OXPHOS and ROS and maintenance of CML stem cell dormancy and repopulating potential as a key mechanism of CML stem cell adaptation to TKI treatment. Our results identify a key metabolic dependency in CML stem cells persisting after TKI treatment that can be targeted to enhance their elimination.

Cholesterol crystal formation is a unifying pathogenic mechanism in the development of diabetic retinopathy.

AIMS/HYPOTHESIS: Hyper-reflective crystalline deposits found in retinal lesions have been suggested to predict the progression of diabetic retinopathy, but the nature of these structures remains unknown. METHODS: Scanning electron microscopy and immunohistochemistry were used to identify cholesterol crystals (CCs) in human donor, pig and mouse tissue. The effects of CCs were analysed in bovine retinal endothelial cells in vitro and in db/db mice in vivo using quantitative RT-PCR, bulk RNA sequencing, and cell death and permeability assays. Cholesterol homeostasis was determined using 2H2O and 2H7-cholesterol. RESULTS: We identified hyper-reflective crystalline deposits in human diabetic retina as CCs. Similarly, CCs were found in the retina of a diabetic mouse model and a high-cholesterol diet-fed pig model. Cell culture studies demonstrated that treatment of retinal cells with CCs can recapitulate all major pathogenic mechanisms leading to diabetic retinopathy, including inflammation, cell death and breakdown of the blood-retinal barrier. Fibrates, statins and α-cyclodextrin effectively dissolved CCs present in in vitro models of diabetic retinopathy, and prevented CC-induced endothelial pathology. Treatment of a diabetic mouse model with α-cyclodextrin reduced cholesterol levels and CC formation in the retina, and prevented diabetic retinopathy. CONCLUSIONS/INTERPRETATION: We established that cholesterol accumulation and CC formation are a unifying pathogenic mechanism in the development of diabetic retinopathy.

Transthyretin proteoforms of intraocular origin in human subretinal fluid.

Understanding the molecular composition of ocular tissues and fluids could inform new approaches to prevalent causes of blindness. Subretinal fluid accumulating between the photoreceptor outer segments and retinal pigment epithelium (RPE) is potentially a rich source of proteins and lipids normally cycling among outer retinal cells and choroid. Herein, intact post-translationally modified proteins (proteoforms) were extracted from subretinal fluids of five patients with rhegmatogenous retinal detachment (RRD), analyzed by tandem mass spectrometry, and compared to published data on these same proteins as synthesized by other organs. Single-nuclei transcriptomic data from non-diseased human retina/RPE were used to identify whether proteins in subretinal fluid were of potential ocular origin. Two human donor eyes with normal maculas were immunoprobed for transthyretin (TTR) with appropriate controls. The three most abundant proteins detected in subretinal fluid were albumin, TTR, and apolipoprotein A-I. Remarkably, TTR relative to the other proteins was more abundant than its serum counterpart, suggestive of TTR being synthesized predominantly locally. Six proteoforms of TTR were detected, with the relative amount of glutathionylated TTR being much higher in the subretinal fluid (12-43%) than values reported for serum (<5%) and cerebrospinal fluid (0.4-13%). Moreover, a putative glycosylated TTR dimer of 32,428 Da was detected as the fourth most abundant protein. The high abundance of TTR and putative TTR dimer in subretinal fluid was supported by analysis of available single-nuclei transcriptomic data, which showed strong and specific signal for TTR in RPE. Immunohistochemistry further showed strong diffuse TTR immunoreactivity in choroidal stroma that contrasted with vertically aligned signal in the outer segment zone of the subretinal space and negligible signal in RPE cell bodies. These results suggest that TTR in the retina is synthesized intraocularly, and glutathionylation is crucial for its normal function. Further studies on the composition, function, and quantities of TTR and other proteoforms in subretinal fluid could inform mechanisms, diagnostic methods, and treatment strategies for age-related macular degeneration, familial amyloidosis, and other retinal diseases involving dysregulation of physiologic lipid transfer and oxidative stress.

Plasma Microbiome in COVID-19 Subjects: An Indicator of Gut Barrier Defects and Dysbiosis.

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) complications. We aimed to investigate 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 = 146) and healthy individuals (n = 47) were collected during hospitalization and routine visits. Plasma microbiome was analyzed using 16S rRNA sequencing and gut permeability markers including fatty acid binding protein 2 (FABP2), peptidoglycan (PGN), and lipopolysaccharide (LPS) in both patient cohorts. Plasma samples of both cohorts contained predominately Proteobacteria, Firmicutes, Bacteroides, and Actinobacteria. COVID-19 subjects exhibit significant dysbiosis (p = 0.001) of the plasma microbiome with increased abundance of Actinobacteria spp. (p = 0.0332), decreased abundance of Bacteroides spp. (p = 0.0003), and an increased Firmicutes:Bacteroidetes ratio (p = 0.0003) compared to healthy subjects. The concentration of the plasma gut permeability marker FABP2 (p = 0.0013) and the gut microbial antigens PGN (p < 0.0001) and LPS (p = 0.0049) were significantly elevated in COVID-19 patients compared to healthy subjects. These findings support the notion that the intestine may represent a source for bacteremia and 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.

AAV8-antiVEGFfab Ocular Gene Transfer for Neovascular Age-Related Macular Degeneration.

Sustained suppression of VEGF is needed in many patients with neovascular age-related macular degeneration (NVAMD), and gene transfer of a VEGF-neutralizing protein is a promising approach to achieve it. Initial clinical trials testing this approach have shown encouraging signals, but evidence of robust transgene expression and consistent antiangiogenic and antipermeability activity has been lacking. In this study, we demonstrate expression of an anti-human VEGF antibody fragment (antiVEGFfab) after subretinal injection of AAV8-antiVEGFfab. In transgenic mice expressing human VEGF in retina (rho/VEGF mice), a model of type 3 choroidal neovascularization (NV), eyes injected with ≥1 × 107 gene copies (GC) of AAV8-antiVEGFfab had significantly less mean area of NV than null vector-injected eyes. A dose-dependent response was observed with modest reduction of NV with ≤3 × 107, >50% reduction with ≥1 × 108 GC and almost complete elimination of NV with 3 × 109 or 1 × 1010 GC. In Tet/opsin/VEGF mice, in which doxycycline-induced high expression of VEGF leads to severe vascular leakage and exudative retinal detachment (RD), reduction of total RD by 70%-80% occurred with 3 × 109 or 1 × 1010 GC of AAV8-antiVEGFfab, an effect that was sustained for at least a month. These data strongly support initiating clinical trials testing subretinal injection of AAV8-antiVEGFfab in patients with NVAMD.

Vorapaxar and Amyotrophic Lateral Sclerosis: Coincidence or Adverse Association?

BACKGROUND: Vorapaxar, a novel antiplatelet thrombin PAR-1 inhibitor, is currently approved for post myocardial infarction and peripheral artery disease indications with concomitant use of clopidogrel and/or aspirin. The vorapaxar safety profile was acceptable. However, aside from heightened bleeding risks, excesses of solid cancers and diplopia, there were more amyotrophic lateral sclerosis (ALS) diagnoses after vorapaxar. STUDY QUESTION: To assess the Food and Drug Administration (FDA) reviews on the potential association of vorapaxar with ALS. STUDY DESIGN: The review the public FDA records on reported adverse events after vorapaxar. MEASURES AND OUTCOMES: Incidence of ALS after vorapaxar and placebo. RESULTS: The ALS risk appears very small, about 1 case per 10,000 treated subjects, but quite probable. Indeed, there were overall 2 placebo and 4 vorapaxar ALS incidences in the Phase III clinical trials. CONCLUSIONS: Potential adverse association of vorapaxar with ALS risks may be related to off-target neuronal PAR receptor(s) blockade beyond platelet inhibition.

Should the Dose of Antiplatelet Drugs Be Adjusted for Body Weight? The Example of Vorapaxar.

BACKGROUND: In contrast to the vast majority of pharmaceuticals on the market, antiplatelet agents are widely prescribed in a uniform, 'one size fits all' manner, without conventional dose adjustments. However, strong evidence yielded from clinical trials repeatedly suggests that patients with a low body weight (LBW), the elderly and those with renal or hepatic impairment may benefit from reduced doses, while younger, heavier patients, males and diabetics may benefit from a dose escalation. Vorapaxar, a thrombin protease-activated receptor-1 inhibitor, has been tested in the TRA2P and TRACER clinical trials, but its efficacy and safety in patients with a LBW is unclear. OBJECTIVE: To determine the impact of LBW on primary end point rates (PER) and bleeding risk after vorapaxar, as yielded from the TRA2P and TRACER secondary FDA review. RESULTS: The LBW (<60 kg) groups in TRA2P (n = 1,852; 7%) and TRACER (n = 1,046; 8%) were small. However, the PER repeatedly suggested inferiority of vorapaxar over placebo in both the successful TRA2P study (10.6 vs. 8.4%; p = 0.012) and the failed TRACER study (19.3 vs. 18.2%; p = not significant). In TRA2P, the PER monotonically escalated with increasing weight for placebo, while those in the vorapaxar arm formed a flat U- or J-shaped distribution across the weight quintiles. In TRACER, the PER by weight quintile appear much higher, but also more random than in TRA2P. The bleeding rates in TRA2P were higher for the 2 lowest-weight quintiles with both placebo and vorapaxar. In TRACER, bleeding rates were more than doubled when compared to TRA2P, and they varied little by weight quintile, with a slight decrease for the heaviest patients in the placebo population and being the highest in the 2 lowest-weight quintiles after vorapaxar. CONCLUSION: The FDA analyses revealed no definite proof that LBW is associated with reduced efficacy of vorapaxar. While these data are striking, they can be explained by better outcomes in LBW placebo patients already sufficiently treated with dual-antiplatelet therapy. In contrast to efficacy, both TRA2P and TRACER definitely suggest that bleeding rates after vorapaxar are higher in patients with LBW. Dose adjustment for antiplatelet agents may soon become a reality.

Independent Predictors of Major Adverse Events following Coronary Stenting over 28 Months of Follow-Up.

BACKGROUND: Despite recent advances in stent design and constantly improving protective pharmacological strategies, complications and adverse events following percutaneous coronary interventions (PCI) are still major factors influencing morbidity and mortality. Therefore, predicting secondary vascular occlusions represents an unmet medical need. OBJECTIVE: The aim of our study was to triage clinical and laboratory predictors of major adverse clinical events (MACE) following coronary stenting. METHODS: This was a prospective, case-controlled, single-center study, which included 94 consecutive patients with documented coronary disease who underwent PCI with drug-eluting stent (DES) implantation. All patients received dual antiplatelet therapy with aspirin and clopidogrel. Numerous clinical characteristics and laboratory biomarkers were assessed before stenting and were correlated with poststenting MACE over the mean follow-up of 28 months. MACE included death, nonfatal myocardial infarction, target vessel revascularisation, stroke, stent thrombosis, angina recurrence and instent restenosis. RESULTS: Twenty-three patients experienced MACE. Independent MACE predictors after PCI with DES implantation were antecedent diabetes mellitus (RR = 0.45; 95% CI 0.20-0.97; p = 0.045), prior thrombolytic therapy (RR = 0.42; 95% CI 0.27-0.83; p = 0.039), baseline plasminogen activator inhibitor -1 (PAI-1; p = 0.008) and plasma von Willebrand factor (vWF) activity (p = 0.007). Other clinical characteristics and laboratory indices showed no correlation with MACE. CONCLUSIONS: Background diabetes mellitus, prior thrombolytic therapy, PAI-1 and vWF prestenting activity may be useful for MACE prediction over 28 months of follow-up.

Thrombin generation and platelet reactivity at hospital discharge and 6-month outcome after the acute coronary syndrome in diabetic and nondiabetic patients.

OBJECTIVES: Increased plasma thrombogenesis and blood platelet reactivity are associated with a worse outcome in patients with the acute coronary syndrome (ACS). The aim of this study was to test the clinical utility of combining a thrombin generation test and platelet aggregation in predicting future ischemic events after ACS. METHODS: The study included patients hospitalized due to ST-elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention with stent implantation. Blood for platelet aggregation and thrombin generation was collected at hospital discharge. We performed whole-blood platelet aggregation with arachidonic acid (AA), collagen, adenosine diphosphate and thrombin receptor-activating peptide (TRAP) as agonists and the thrombin generation test using a fluorescence method. Patients were followed for up to 6 months. The combined end point of the study consisted of death, stroke, myocardial infarction or repeated target vessel revascularization. RESULTS: The study enrolled 161 patients. The end point occurred in 30 patients (18.6%). Thrombin generation showed a significantly prolonged lag time, time to thrombogram peak and start of the tail of the thrombogram in diabetic patients who reached the study end point but not in nondiabetics. End point occurrence was not connected with platelet reactivity at hospital discharge in the whole group. In the diabetic subgroup, increased platelet aggregation induced with AA and TRAP at hospital discharge was connected with a more frequent occurrence of the study end point. CONCLUSIONS: In diabetic patients after STEMI, thrombin generation measures as well as TRAP- and AA-induced platelet aggregation at hospital discharge are associated with an ensuing ischemic event during the 6-month follow-up.

Interferon-dependent signaling is critical for viral clearance in airway neutrophils.

Neutrophilic inflammation characterizes several respiratory viral infections, including COVID-19-related acute respiratory distress syndrome, although its contribution to disease pathogenesis remains poorly understood. Blood and airway immune cells from 52 patients with severe COVID-19 were phenotyped by flow cytometry. Samples and clinical data were collected at 2 separate time points to assess changes during ICU stay. Blockade of type I interferon and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) signaling was performed in vitro to determine their contribution to viral clearance in A2 neutrophils. We identified 2 neutrophil subpopulations (A1 and A2) in the airway compartment, where loss of the A2 subset correlated with increased viral burden and reduced 30-day survival. A2 neutrophils exhibited a discrete antiviral response with an increased interferon signature. Blockade of type I interferon attenuated viral clearance in A2 neutrophils and downregulated IFIT3 and key catabolic genes, demonstrating direct antiviral neutrophil function. Knockdown of IFIT3 in A2 neutrophils led to loss of IRF3 phosphorylation, with consequent reduced viral catabolism, providing the first discrete mechanism to our knowledge of type I interferon signaling in neutrophils. The identification of this neutrophil phenotype and its association with severe COVID-19 outcomes emphasizes its likely importance in other respiratory viral infections and potential for new therapeutic approaches in viral illness.

Type I interferon-dependent IFIT3 signaling is critical for viral clearance in airway neutrophils.

Neutrophilic inflammation characterizes several respiratory viral infections including COVID-19-related ARDS, although its contribution to disease pathogenesis remains poorly understood. Here, we identified two neutrophil subpopulations (A1 and A2) in the airway compartment of 52 severe COVID-19 subjects, where loss of the A2 subset correlated with increased viral burden and reduced 30-days survival. A2 neutrophils showcased a discrete antiviral response with an increased interferon signature. Blockade of type I interferon attenuated viral clearance in A2 neutrophils and downregulated IFIT3 and key catabolic genes, demonstrating direct antiviral neutrophil function. Knockdown of IFIT3 in A2 neutrophils led to loss of IRF3 phosphorylation with consequent reduced viral catabolism, providing the first discrete mechanism of type I interferon signaling in neutrophils. The identification of this novel neutrophil phenotype and its association with severe COVID-19 outcomes emphasizes its likely importance in other respiratory viral infections and potential for new therapeutic approaches in viral illness.

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.

Plasma microbiome in COVID-19 subjects: an indicator of gut barrier defects and dysbiosis.

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; p<0.0001), and LPS (405.5±48.37 vs 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.

Hepatocyte growth factor is upregulated in ischemic retina and contributes to retinal vascular leakage and neovascularization.

In patients with macular edema due to ischemic retinopathy, aqueous levels of hepatocyte growth factor (HGF) correlate with edema severity. We tested whether HGF expression and activity in mice with oxygen-induced ischemic retinopathy supports a role in macular edema. In ischemic retina, HGF was increased in endogenous cells and macrophages associated with retinal neovascularization (NV). HGF activator was increased in and around retinal vessels potentially providing vascular targeting. One day after intravitreous injection of HGF, VE-cadherin was reduced and albumin levels in retina and vitreous were significantly increased indicating vascular leakage. Injection of VEGF caused higher levels of vitreous albumin than HGF, and co-injection of both growth factors caused significantly higher levels than either alone. HGF increased the number of macrophages on the retinal surface, which was blocked by anti-c-Met and abrogated in chemokine (C-C motif) ligand 2 (CCL2)-/- mice. Injection of anti-c-Met significantly decreased leakage within 24 hours and after 5 days it reduced retinal NV in mice with ischemic retinopathy, but had no effect on choroidal NV. These data indicate that HGF is a pro-permeability, pro-inflammatory, and pro-angiogenic factor and along with its activator is increased in ischemic retina providing support for a potential role of HGF in macular edema in ischemic retinopathies.

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.

Molecular mechanisms of retinal ischemia.

Each day, the retina converts an immense number of photons into chemical signals that are then transported to higher order neural centers for interpretation. This process of photo transduction requires large quantities of cellular energy and anabolic precursors, making the retina one of the most metabolically active tissues in the body. With such a large metabolic demand, the retina is understandably sensitive to perturbations in perfusion and hypoxia. Indeed, retinal ischemia underlies many prevalent retinal disorders including diabetic retinopathy (DR), retinal vein occlusion (RVO), and retinopathy of prematurity (ROP). Retinal ischemia leads to the expression of growth factors, cytokines, and other cellular mediators which promote inflammation, vascular dysfunction, and ultimately, vision loss. This review aims to highlight the most recent and compelling findings that have advanced our understanding of the molecular mechanisms underlying retinal ischemias.

Progenitor cell combination normalizes retinal vascular development in the oxygen-induced retinopathy (OIR) model.

Retinopathy of prematurity (ROP) is a disorder of the developing retina of preterm infants. ROP can lead to blindness because of abnormal angiogenesis that is the result of suspended vascular development and vaso-obliteration leading to severe retinal stress and hypoxia. We tested the hypothesis that the use of the human progenitor cell combination, bone marrow-derived CD34+ cells and vascular wall-derived endothelial colony-forming cells (ECFCs), would synergistically protect the developing retinal vasculature in a mouse model of ROP, called oxygen-induced retinopathy (OIR). CD34+ cells alone, ECFCs alone, or the combination thereof were injected intravitreally at either P5 or P12 and pups were euthanized at P17. Retinas from OIR mice injected with ECFCs or the combined treatment revealed formation of the deep vascular plexus (DVP) while still in hyperoxia, with normal-appearing connections between the superficial vascular plexus (SVP) and the DVP. In addition, the combination of cells completely prevented aberrant retinal neovascularization and was more effective anatomically and functionally at rescuing the ischemia phenotype than either cell type alone. We show that the beneficial effects of the cell combination are the result of their ability to orchestrate an acceleration of vascular development and more rapid ensheathment of pericytes on the developing vessels. Lastly, our proteomic and transcriptomic data sets reveal pathways altered by the dual cell therapy, including many involved in neuroretinal maintenance, and principal component analysis (PCA) showed that cell therapy restored OIR retinas to a state that was closely associated with age-matched normal retinas. Together, these data herein support the use of dual cell therapy as a promising preventive treatment for the development of ROP in premature infants.

Mousetap, a Novel Technique to Collect Uncontaminated Vitreous or Aqueous and Expand Usefulness of Mouse Models.

Vitreous or aqueous humour taps are widely used in patients or large animals with retinal diseases to monitor disease biomarkers, search for novel biomarkers, assess the integrity of the blood-retinal barrier, or perform pharmacokinetic or pharmacodynamics studies. Although there are many useful mouse models of retinal diseases, the small size of mouse eyes has precluded vitreous or aqueous taps. Herein we describe a novel technique, mousetap, which allows collection of vitreous or aqueous humour uncontaminated by blood or tissue surrounding the vitreous cavity. Mousetap was used to obtain vitreous samples from several mouse models of retinal vascular diseases and vitreous albumin measured by ELISA was highly reproducible among mice of the same model. The mean vitreous albumin concentration differed widely among control mice and mice of different models and correlated with fluorescein angiographic assessment of vascular leakage severity. Protein arrays showed increases in levels of several vasoactive proteins in the vitreous from mice with oxygen-induced ischemic retinopathy compared with age-matched controls; almost all of these proteins are increased in the vitreous of patients with the most common human ischemic retinopathy, proliferative diabetic retinopathy. Thus, mousetap facilitates the use of mice for studies previously reserved for large animal models and patients.

Mortality and oral anticoagulants in the Food and Drug Administration Adverse Event Reporting System.

OBJECTIVE: The comparative crude death rates (CDR) among non-vitamin K antagonist oral anticoagulants (NOACs) are unknown. Further, whether NOACs improve survival when compared with warfarin is also unclear. We compared CDR co-reported for four NOACs combined or separately versus warfarin within the US Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS) database. METHODS: We selected CDR from the FAERS database linked to four NOACs and warfarin. The primary endpoints were differences in proportional reporting ratios (PRRs), and Chi-Square (χ2)for dabigatran, rivaroxaban, apixaban and edoxaban when compared with warfarin. RESULTS: The FAERS database contains significantly less death reports associated with all NOACs combined (14 917 out of 128 267 reports (11.63%); PRR=1.089; χ2=70.0; p=6.05e-17) than for warfarin (19 493 out of 153 911 reports (12.67%)). The numbers for rivaroxaban (6318 out of 64 512 reports or (9.79%); PRR=1.293; χ2=359.4; p=3.72e-80), apixaban (1693 out of 17 789 reports (9.52%); PRR=1.331; χ2=145.8; p=1.43e-33) and edoxaban (53 out of 755 reports (7.02%); PRR=1.804; χ2=21.18; p=4.18e-06) were favourable as compared with warfarin, while the numbers of fatalities co-reported with dabigatran (6989 out of 46 250 reports (15.11%); PRR=0.838; χ2=185.2; p=3.61e-42) were higher than for warfarin. CONCLUSION: Overall, based on these CDR, NOACs appear to be associated with a mortality benefit over warfarin. Among NOACs, we observed remarkably similar for factor Xa inhibitors (rivaroxiban, apixaban and edoxaban) but unfavourable signal for the direct thrombin inhibitor (dabigatran). However, these data are clearly not sufficient to change the prescription patterns.