Basic Science Studies

Funder - American Heart Association Title - Role of endothelium-derived microvesicles in pulmonary hypertension Goals -  To investigate the role of pulmonary endothelium-derived microvesicles, and their miRNA cargos, in the pathogenesis of pulmonary vascular remodeling and pulmonary hypertension. Description In the pulmonary vasculature, the pulmonary vascular endothelial cells (PVEC) and pulmonary artery smooth muscle cells (PASMC) are two key cell types that play a major role in the pathobiology of pulmonary hypertension (PH). As the “signal initiator” in the pulmonary vasculature, PVEC affect the function of PASMC by secreting bioactive agents, myoendothelial injunctions (MEJ) and/or releasing extracellular vesicles (EV) that contain various cargo components including proteins and RNAs etc., under both normal and pathological conditions. So far, our knowledge about the role of endothelial-derived EV and its cargo in the PASMC function and pathogenesis of PH is very limited. In this proposal, we will investigate the role of PVEC-derived microvesicles (MV), especially that of the miRNA cargo in these MV, in the pathogenesis of pulmonary vascular remodeling and PH. In preliminary studies we found that in hypoxia, MV released by mouse PVEC in culture (H-MV) induced mouse PASMC proliferation in vitro and pulmonary vascular remodeling and PH in mice in room air (vs MV released by mouse PVEC in normoxia, H-MV vs N-MV). Using miRNA deep sequencing, we identified 9 miRNAs that were significantly induced in H-MV (v.s. N-MV), including miR-210, a miRNA that is known to induce SMC proliferation and PH. Interestingly, all the other identified miRNAs either suppressed or did not alter SMC proliferation in vitro, including a novel miRNA, miR-212-5p, that had the strongest inhibitory effect on SMC proliferation. We also found that miR-212-5p played a protective role in the pathogenesis of hypoxia-induced PH in mice. Based on these findings, we hypothesize that the endothelium, via MV, promotes vascular remodeling during hypoxia but also provides a break against continued progression of the disease via releasing anti-proliferative miRNAs, such as miR-212, packaged in the MV.
Funder - NIH: NHLBI Title - Role of MicroRNA-17-92 and PDLIM5 Signaling in Pulmonary Arterial Hypertension Description This project aims to elucidate the molecular mechanisms underlying the microRNA-17~92/PDLIM5 signaling-mediated pulmonary artery smooth muscle cell phenotype change and its functional impact in the genesis of pulmonary hypertension. We are continuing to investigate the role of PDLIM5 in the metabolic shift and to carry out a PDLIM5-targeted drug development process for the treatment of pulmonary hypertension.
Title -  Endothelium-derived Extracellular Vesicles, MicroRNAs and Pulmonary Hypertension Description In this grant, the role of microRNAs and the endothelium derived extracellular vesicles in the pathogenesis of Pulmonary Hypertension is being studied. Another grant with this same title is developing a novel method of engineering the extracellular vesicles for treatment of pulmonary hypertension.
Funder - NIH/NIGMS Title - Fra-1 - A20 Signaling and Resolution of Pneumonia-Induced Sepsis Description The counter balancing of pro- and anti-inflammatory gene transcription is crucial for normal homeostasis after septic tissue injury. Aberrant regulation of this transcriptional balance culminates in an unchecked systemic inflammation, leading to lung tissue damage and edema, respiratory failure and ultimately death. However, the exact mechanisms underlying pathological inflammation in sepsis are poorly understood, and thus the strategies to accelerate the resolution of sepsis are very limited. Studies in the proposal will test the novel hypothesis that pathogenic signaling caused by sepsis is the result of a macrophage-specific Fra-1/AP-1 restricted expression of anti- inflammatory A20, a crucial ubiquitin-editing enzyme that terminates uncontrolled activation of NF- κB and MAP kinase signaling. The proposed studies will offer novel insights and targets for therapies to accelerate lung injury repair in patients with pseudomonas pneumonia and sepsis.
Funder - NIH: NHLBI Title - Role of Nrf2 in Alveolar Epithelial Cell Regeneration During Lung Repair
Funder - NIH: NHLBI Title - Structure and Function of organelles in red blood cells Description The higher number of mitochondria rich reticulocytes in circulating blood, could potentially promote the elevated levels of reactive oxygen species, changes in oxygen metabolism and the cell lysis seen in the SCD. The molecular mechanism of increased mitochondria in red blood cells associated with SCD is not clear. This study investigates to examine the link between the aberrant mitophagy and sickle cell pathology in order to develop new strategies for the treatment of SCD using mitophagy restoration drugs.
Title - The role of erythrocyte mitochondrial retention in sickle cell disease Description Sickle cell disease (SCD) is an inherited blood disorder that affects millions of people worldwide and results in health care costs of at least $2.4 billion per year in the United States alone.1 It is caused by a mutation in the β -globin gene which leads to the formation of hemoglobin S (HbS). HbS is able to polymerize and leads to RBC sickling, hemolysis, acute and chronic pain, chronic hemolytic anemia, multisystem organ damage, and a much-shortened life expectancy. Novel targeted therapeutic approaches are essential to overcome the cascade of the events that begin with HbS polymerization. Recently many investigators have demonstrated that SCD organ pathology is associated with oxidative stress. Oxidative stress occurs when there is an increase in oxidants without a similar increase in antioxidants. Excessive ROS accumulation triggers a cascade of oxidative reactions that damage lipids, proteins of red blood cells ultimately leading to hemolysis or early destruction. Although much progress has been made to ROS mediated complications in SCD patients, further studies are essential in an attempt to understand the source of ROS and factors involved in HbS polymerization and hemolytic process. We have demonstrated in our laboratory that SCD RBCs retain mitochondria. In addition, we have shown that these retained mitochondria create excessive intracellular ROS generation and are associated with hemolysis. Our preliminary data also show that these mitochondria cause an increased oxygen consumption in the red blood cells. We hypothesize that erythrocyte mitochondrial retention causes exacerbation of SCD pathogenesis by two non-mutually exclusive mechanisms 1) Mitochondria generate excessive ROS leading to hemolysis and 2) Mitochondria increased oxygen consumption leading to a hypoxic intracellular environment that causes Hb S polymerization. An understanding of mitochondrial oxygen consumption and consequential oxidative stress in the pathogenesis of SCD represents a novel opportunity for the development of targeted therapeutic agents. The possibility of mitochondria- derived ROS generation and oxygen consumption in RBCs are novel targets that have not been investigated before. Our long-term goal is to translate the novel finding of mitochondria-retaining SCD RBCs into new pharmaceutical therapies for sickle cell disease.
Title - Advancing fetal hemoglobin to the clinical using highly predictive in-vivo strategies Description Elevated fetal hemoglobin levels lessen the severity of sickle cell disease (SCD) and increase the lifespan of patients. Effective treatment of the large numbers of SCD patients projected in the U.S. and worldwide in the coming years would be best accomplished with an affordable, easily-administered, orally-available drug therapy designed to increase Fetal Hemoglobin (HbF) levels to a target >30% distributed throughout a large percentage of erythrocytes. Hydroxyurea (HU), currently the sole FDA-approved drug for SCD, is only effective in approximately 50% of patients and the HbF remains heterogeneously distributed among erythrocytes resulting in a large fraction lacking the protective effects of HbF. A logical approach to increase HbF that has been successfully pursued by our laboratory is to intervene with the epigenetic mechanism executing the switch from HbF to HbA expression in adults using pharmacological inhibitors of enzymes that catalyze repressive epigenetic modifications associated with γ-globin gene silencing. Our laboratory has developed and utilized an in vivo baboon model for over thirty years to investigate globin gene regulation and the in vivo activity of HbF-inducing drugs. Simian primates such as the baboon are widely acknowledged as the best animal models for testing the ability of new drugs to increase γ-globin expression because the activity of HbF-inducing agents is predictive of effects in man due to conservation of the structure and developmental stage-specific regulation of the β-like globin genes in simian primates. Initial studies from our laboratory demonstrating that DNA methyltransferase (DNMT) inhibitors increased HbF in baboons were followed by a number of clinical trials that confirmed their effectiveness in SCD patients and validated use of the baboon model. Recently we have shown that the LSD1 inhibitor RN-1 increased γ-globin expression in the sickle cell disease mouse model and in baboons and that long term treatment of baboons was well tolerated. In this proposal we will 1) investigate a combinatorial drug regimen targeting both DNMT1 and LSD1 for effects on HbF induction and reduction of potentially adverse hematological side-effects, 2) continue to advance new highly specific “third generation” LSD1 inhibitors with reduced ability to cross the blood brain barrier with the goal of increasing HbF to therapeutic levels, maximizing F cell numbers, and raising the therapeutic index, and 3) investigate new compounds that expand the BFU-E subpopulation permissive for γ-globin expression, alone and in combination with inhibitors of epigenetic-modifying enzymes, for additional stimulation of HbF induction. We envision that the results of these studies will directly impinge in the design of new clinical trials to increase HbF for the therapy of sickle cell disease.
Title - The impact of selenium and selenoprotein levels on hemolysis and pain in sickle cell disease Description Although the cause of SCD, a mutation in the gene for hemoglobin, has been known for many years, there is little understanding of why the spectrum of symptoms varies so much among afflicted individuals. Our lab first linked hemolysis in sickle cell patients to abnormal mitochondrial ROS. In this study, we propose that the differences in disease severity that SCD patients experience is associated with selenium deficiency and reduced GPX1 activity, which is linked to mitochondrial RBC OCR and mitochondrial ROS generation. These proposed studies will address the gap in the research regarding the varying severity of anemia in SCD by examining the association between selenium levels and GPX1 activity and their impact on hemoglobin levels and increases the number of sickle cell associated events (SCE) in children and adults with SCD. Establishing an association between the expression and or activity of selenoproteins and mitochondrial RBC bioenergetics and mitochondrial ROS generation would likely lead to the development of a new intervention strategy for mitigating the burden of SCD, and decreasing the profound morbidity and early mortality. The potential of providing a safe dietary supplement to the SCD community could be a novel strategy that could be implemented in developed as well as developing nations and could profoundly alter people’s lives by effecting the course of the disease.