Exploring epigenetic and microRNA approaches for γ-globin gene regulation.

Therapeutic interventions aimed at inducing fetal hemoglobin and reducing the concentration of sickle hemoglobin is an effective approach to ameliorating acute and chronic complications of sickle cell disease, exemplified by the long-term use of hydroxyurea. However, there remains an unmet need for the development of additional safe and effective drugs for single agent or combination therapy for individuals with ß-hemoglobinopathies. Regulation of the γ-globin to ß-globin switch is achieved by chromatin remodeling at the HBB locus on chromosome 11 and interactions of major DNA binding proteins, such as KLF1 and BCL11A in the proximal promoters of the globin genes. Experimental evidence also supports a role of epigenetic modifications including DNA methylation, histone acetylation/methylation, and microRNA expression in γ-globin gene silencing during development. In this review, we will critically evaluate the role of epigenetic mechanisms in γ-globin gene regulation and discuss data generated in tissue culture, pre-clinical animal models, and clinical trials to support drug development to date. The question remains whether modulation of epigenetic pathways will produce sufficient efficacy and specificity for fetal hemoglobin induction and to what extent targeting these pathways form the basis of prospects for clinical therapy.

The Flavor Enhancer Maltol Increases Pigment Aggregation in Dermal and Neural Melanophores in Xenopus laevis Tadpoles.

Melanophores are pigmented cells that change the distribution of melanosomes, enabling animals to appear lighter or darker for camouflage, thermoregulation, and protection from ultraviolet radiation. A complex series of hormonal and neural mechanisms regulates melanophore pigment distribution, making these dynamic cells a valuable tool to screen toxicants as they rapidly respond to changes in the environment. We found that maltol, a naturally occurring flavor enhancer and fragrance agent, induces melanophore pigment aggregation in a dose-dependent manner in Xenopus laevis tadpoles. To determine if maltol affects camouflage adaptation, we placed tadpoles into maltol baths situated over either a white or a black background. Maltol induced pigment aggregation in a similar dose-dependent pattern regardless of background color. We also tested how maltol treatment compares to melatonin treatment and found that the degree of pigment aggregation induced by maltol is similar to treatment with melatonin but that maltol induces over a much longer time course. Last, maltol had no effect on mRNA expression in the brain of genes that regulate camouflage-related pigment aggregation. The present results suggest that maltol does not exert its effects via the camouflage adaptation mechanism or via melatonin-related mechanisms. These results are the first to identify a putative toxicological effect of maltol exposure in vivo and rule out several mechanisms by which maltol may exert its effects on pigment aggregation. Environ Toxicol Chem 2020;39:381-395. © 2019 SETAC.

Role of GluN2A NMDA receptor in homocysteine-induced prostaglandin E2 release from neurons.

Hyperhomocysteinemia or systemic elevation of homocysteine is a metabolic condition that has been linked to multiple neurological disorders where inflammation plays an important role in the progression of the disease. However, it is unclear whether hyperhomocysteinemia contributes to disease pathology by inducing an inflammatory response. The current study investigates whether exposure of primary cultures from rat and mice cortical neurons to high levels of homocysteine induces the expression and release of the proinflammatory prostanoid, Prostaglandin E2 (PGE2). Using enzymatic assays and immunoblot analysis we show concurrent increase in the activity of cytosolic phospholipase A2 (cPLA2) and level of cyclooxygenase-2 (COX2), two enzymes involved in PGE2 biosynthesis. The findings also show an increase in PGE2 release from neurons. Pharmacological inhibition of GluN2A-containing NMDAR (GluN2A-NMDAR) with NVP-AAM077 significantly reduces homocysteine-induced cPLA2 activity, COX2 expression, and subsequent PGE2 release. Whereas, inhibition of GluN2B-containing NMDAR (GluN2A-NMDAR) with Ro 25-6981 has no effect. Complementary studies in neuron cultures obtained from wild type and GluN2A knockout mice show that genetic deletion of GluN2A subunit of NMDAR attenuates homocysteine-induced neuronal increase in cPLA2 activity, COX2 expression, and PGE2 release. Pharmacological studies further establish the role of both extracellular-regulated kinase/mitogen-activated protein kinase and p38 MAPK in homocysteine-GluN2A NMDAR-dependent activation of cPLA2-COX2-PGE2 pathway. Collectively, these findings reveal a novel role of GluN2A-NMDAR in facilitating homocysteine-induced proinflammatory response in neurons.

Perspective: Sistas In Science - Cracking the Glass Ceiling.

In this perspective, we describe our experience as women of color scientists from diverse backgrounds and similar struggles embarking upon the National Heart, Lung and Blood Institute-funded program called PRIDE (Programs to Increase Diversity among Underrepresented Minorities Engaged in Health-Related Research). Under the leadership of our mentor and friend, Betty Pace, MD, a renowned and successful African American physician-scientist, the PRIDE Program was designed to address the difficulties experienced by junior-level minority investigators in establishing independent research programs and negotiating tenure and full professor status at academic institutions. The strength of PRIDE's innovative formula was pairing us with external senior mentors and, importantly, allowing us to serve as peer mentors to each other. We believe this "Sister's Keeper" paradigm is one solution for women to overcome their limitations and extend understandings and best practices worldwide for science, medicine, and global health.

Human trabecular meshwork cells express BMP antagonist mRNAs and proteins.

Glaucoma patients have elevated aqueous humor and trabecular meshwork (TM) levels of transforming growth factor-beta2 (TGF-ß2). TGF-ß2 has been associated with increased extracellular matrix (ECM) deposition (i.e. fibronectin), which is attributed to the increased resistance of aqueous humor outflow through the TM. We have previously demonstrated that bone morphogenetic protein (BMP) 4 selectively counteracts the profibrotic effect of TGF-ß2 with respect to ECM synthesis in the TM, and this action is reversed by the BMP antagonist gremlin. Thus, the BMP and TGF-ß signaling pathways antagonize each other's antifibrotic and profibrotic roles. The purpose of this study was to determine whether cultured human TM cells: (a) express other BMP antagonists including noggin, chordin, BMPER, BAMBI, Smurf1 and 2, and (b) whether expression of these proteins is regulated by exogenous TGF-ß2 treatment. Primary human trabecular meshwork (TM) cells were grown to confluency and treated with TGF-ß2 (5 ng/ml) for 24 or 48 h in serum-free medium. Untreated cell served as controls. qPCR and Western immunoblots (WB) determined that human TM cells expressed mRNAs and proteins for the BMP antagonist proteins: noggin, chordin, BMPER, BAMBI, and Smurf1/2. Exogenous TGF-ß2 decreased chordin, BMPER, BAMBI, and Smurf1 mRNA and protein expression. In contrast, TGF-ß2 increased secreted noggin and Smurf2 mRNA and protein levels. BMP antagonist members are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of BMP antagonist members may lead to functional changes in the human TM.

Transforming growth factor-ß2 induces expression of biologically active bone morphogenetic protein-1 in human trabecular meshwork cells.

PURPOSE: There are limited studies on the factors that regulate the processing of TGF-ß2 and extracellular matrix (ECM) proteins into their mature form. Bone morphogenic protein 1 (BMP1) is an enzyme responsible for the cleavage and maturation of growth factors and ECM proteins. The purpose of our study was to determine whether cultured human trabecular meshwork (TM) cells express BMP1, BMP1 expression is regulated by TGF-ß2, BMP1 is biologically active, and BMP1 regulates LOX activity. METHODS: Primary human TM cells were isolated and subjected to quantitative PCR (qPCR) and Western immunoblotting (WB) for BMP1. BMP1 immunolocalization was performed in TM tissues. qPCR was used to determine BMP1 mRNA expression and WB results were used to determine BMP1 protein expression. BMP1 activity was measured in TM cells treated with TGF-ß2 or with a combination of TGF-ß2/UK383367. Lysyl oxidase (LOX) enzyme activity was evaluated by WB in TM cells treated with BMP1 or with a combination of BMP1/ß-aminoprorionitrile (BAPN). RESULTS: Human TM cells expressed mRNA and protein for BMP1. Exogenous TGF-ß2 increased mRNA expression compared to their controls (P<0.05). An ELISA showed TGF-ß2-induced BMP1 secretion compared to their controls in all cell strains (P<0.05). Secreted BMP1 stimulated LOX enzymatic activity in TM cells. CONCLUSIONS: BMP1 is expressed in the human TM. TGF-ß2 induction of BMP1 may be responsible for increased processing of growth factors and ECM proteins into their mature forms, resulting in TM stiffness and resistance to ECM degradation.

The effects of transforming growth factor-ß2 on the expression of follistatin and activin A in normal and glaucomatous human trabecular meshwork cells and tissues.

PURPOSE: To compare follistatin (FST) and activin (Act) expression in normal and glaucomatous trabecular meshwork (TM) cells and tissues and determine if exogenous TGF-ß2 regulates the expression of FST and Act in TM cells. METHODS: Total RNA was isolated from TM cell strains, and mRNA expression for FST 317/344 isoforms and Act was determined via RT-PCR and quantitative PCR (qPCR). Western immunoblotting and immunocytochemistry determined FST and Act A protein levels in normal TM (NTM) and glaucomatous TM (GTM) cells. Cells were treated with recombinant human TGF-ß2 protein at 0 to 10 ng/mL for 0 to 72 hours. qPCR, Western immunoblotting, immunocytochemistry, and ELISA immunoassay were utilized to determine changes in FST and Act A mRNA and protein levels. In addition, NTM and GTM tissue samples were examined by immunohistochemistry for expression of FST, FST 315, FST 288, and Act A. RESULTS: Both FST mRNA and protein levels were significantly elevated in GTM cells. FST mRNA transcripts FST 317/344 were also significantly elevated in GTM cells. Immunohistochemistry showed FST levels were significantly elevated in GTM tissues. Exogenous TGF-ß2 significantly induced FST mRNA and protein expression. Immunohistochemistry demonstrated that Act A protein levels were significantly higher in NTM tissues compared to GTM tissues. CONCLUSIONS: FST is elevated in GTM cells and tissues. FST is known to be an inhibitor of bone morphogenetic proteins (BMPs), which, coupled with the ability of TGF-ß2 to upregulate FST levels, may indicate a possible role of FST in the pathogenesis of glaucoma. These results suggest that additional endogenous molecules in human TM may regulate TGF-ß2 signaling via inhibition of BMP family members.

The evolving landscape of quality measurement for heart failure.

Heart failure (HF) is a major cause of mortality and morbidity, representing a leading cause of death and hospitalization among U.S. Medicare beneficiaries. Advances in science have generated effective interventions to reduce adverse outcomes in HF, particularly in patients with reduced left ventricular ejection fraction. Unfortunately, effective therapies for heart failure are often not utilized in an effective, safe, timely, equitable, patient-centered, and efficient manner. Further, the risk of adverse outcomes for HF remains high. The last decades have witnessed the growth of efforts to measure and improve the care and outcomes of patients with HF. This paper will review the evolution of quality measurement for HF, including a brief history of quality measurement in medicine; the measures that have been employed to characterize quality in heart failure; how the measures are obtained; how measures are employed; and present and future challenges surrounding quality measurement in heart failure.

Impact of medication nonadherence on hospitalizations and mortality in heart failure.

BACKGROUND: Limited literature exists on the association between medication adherence and outcomes among patients with heart failure. METHODS AND RESULTS: We conducted a retrospective longitudinal cohort study of 557 patients with heart failure with reduced ejection fraction (HFrEF) (defined by EF <50%) in a large health maintenance organization. We used multivariable Cox proportional hazards models to assess the relationship between adherence (with angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, ß-blockers, and aldosterone antagonists) and the primary outcome of all-cause mortality plus cardiovascular hospitalizations. Mean follow-up time was 1.1 years. Nonadherence (defined as <80% adherence) was associated with a statistically significant increase in the primary outcome in the cohort overall (hazard ratio 2.07, 95% confidence interval 1.62-2.64; P < .0001). This association remained significant when all 3 classes of heart failure medications and the components of the composite end point were considered separately and when the adherence threshold was varied to 70% or 90%. CONCLUSIONS: Medication nonadherence was associated with an increased risk of all-cause mortality and cardiovascular hospitalizations in a community heart failure population. Further research is needed to define systems of care that optimize adherence among patients with heart failure.

Association of longitudinal measures of hemoglobin and outcomes after hospitalization for heart failure.

BACKGROUND: Cross-sectional assessments of hemoglobin (Hb) are associated with mortality in patients with heart failure (HF). Our objectives were to characterize patterns of change in Hb over time in patients with HF and to evaluate the relationship between longitudinal measures of Hb and adverse outcomes. METHODS: The study included 2,478 patients with a primary discharge diagnosis of HF from January 2001 to December 2006. Outcomes included time to death and time to death or HF hospitalization. The association between baseline Hb and outcomes was evaluated using multivariable Cox regression. The longitudinal association was evaluated using a time-dependent Hb predictor variable and using anemia trajectory groups. RESULTS: For a median of 475 days, baseline Hb was associated with a trend toward increased mortality (hazard ratio [HR] 1.02, 95% CI 0.99-1.06 per g/dL decline). With a time-dependent approach, the magnitude of the association was greater (HR 1.35, 95% CI 1.30-1.39 per g/dL decline). In trajectory analysis, 35% of the cohort had variable patterns of anemia. Persistently low Hb (HR 1.65, 95% CI 1.27-2.14) and a progressive decline in Hb (HR 1.54, 95% CI 1.16-2.05) were associated with increased mortality risk. Patients with recovery of anemia had similar outcomes as those patients who are persistently nonanemic. Results were similar for the composite of death or HF hospitalization. CONCLUSIONS: Variability in Hb over time is common in patients with HF, and declining Hb is associated with a poor prognosis. Longitudinal characterization of Hb levels has greater prognostic significance than a single measurement. Systematic surveillance of Hb levels may help identify high-risk patients with heart failure.