METABOLIC MECHANISMS IN PHYSIOLOGIC AND PATHOLOGIC OXYGEN SENSING By OLIVIA ROSE STEPHENS Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular Medicine CASE WESTERN RESERVE UNIVERSITY August 2019 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of OLIVIA ROSE STEPHENS candidate for the degree of Doctor of Philosophy*. Committee Chair Prasad Sathyamangla, PhD Thesis Advisor Serpil Erzurum, MD Clinical Mentor Kristin Highland, MD Committee Member Bela Anand-Apte, MBBS, PhD Committee Member Satish Kalhan, MD Date of Defense June 26, 2019 *We also certify that written approval has been obtained for any proprietary material contained therein. i Table of Contents List of tables……………………………………………………………………………iv List of figures…………………………………………………………..………………v Abstract…………………………………………………………………..……….……vii 1. General Introduction I. Hypoxia and HIF-1 Physiological response to hypoxia………………………………………...1 HIF- 1…………………………………...…………………………………….1 Other regulators of HIF-1……………………………………………………2 HIF-1 target genes…………………………………………………………..3 HIF- 2……………...………………………………………………………….3 II. β-adrenergic receptors β-adrenergic receptors (βAR)………………………………………………4 βAR signaling ………………………………………………………………..5 βAR pathways ……………………………………………………………….6 βAR regulation of HIF-1……………………………………………………..7 βAR under hypoxia…………………………………………………………..9 III. Pulmonary Arterial Hypertension Pulmonary Arterial Hypertension (PAH)…………………………………10 Molecular pathology of PAH………………………………………………11 HIF in PAH ………………………………………………………………….12 βAR in PAH…………………………………………………………………12 β-blockers in PAH ………………………………………………………….13 IV. Microparticles and Mitochondria Microparticles…...………………………………………………………….14 Microparticle contents……………………………………………………..16 Basic mitochondrial structure and function………………………………17 Release of intact mitochondria…………………………………………....18 Mitochondrial DAMPs……………………………………………...………19 Mitochondrial transfer……………………………………………...………19 2. Interdependence of hypoxia and β-adrenergic receptor signaling in Pulmonary Arterial Hypertension Abstract………………………………………..……………………………21 Introduction…………………………………………………………………22 Materials and methods.………………...…………………………………25 ii Results……………………………………………………………………...30 Discussion………………………………………………………………….39 Tables……………………………………………………….……………...47 Figures…………………………………………………….………………..48 3. Flow cytometric detection and characterization of cell-free mitochondria in murine and human circulation Abstract……………………………………………………………………..61 Introduction...………………………………………………………...…….62 Materials and methods……………………………………………...........64 Results ………………………………………………………..……...........67 Discussion...………………………………………………………………..71 Figures……………………………………………………………..……….75 Supplemental figures………………………………………………...……82 4. Discussion and future directions Expanding the β-adrenergic signaling model……………………….…..83 Predicting treatment response in Pulmonary Arterial Hypertension.…84 What is the mechanistic link between βAR and HIF-1?.......................85 Developing therapeutics based on ligand bias……………………….…87 Characterization of circulating mitochondria …………………….….…..89 Release of whole mitochondria in pathological conditions………...…..89 References…………………………………………………………………..........…..91 iii List of Tables Table 1: Characteristics of PAH patients with phenotype of high or low RV Glucose uptake……………………………………………………………………...…47 iv List of Figures Chapter 2 Figure 1: βAR ligands differentially regulate HIF-1 and cAMP in vitro………...….48 Figure 2: βAR ligands differentially affect hypoxia-induced HIF-1 activity in vitro……………………………………...…………………………………...50 Figure 3: Isoproterenol and salbutamol have opposing effects on erythropoietic response in vivo………………………………………………………..51 Figure 4: Overexpression of β2AR in HEK293 cells increases basal HIF-1 activity and downstream effects under normoxia……………………………………53 Figure 5: Hypoxia blunts cAMP response to isoproterenol and salbutamol in vitro……………………………………………………………………...55 Figure 6: PAH patients with the phenotype of high RV glucose uptake have more severe disease………………………………….…………………………56 Figure 7: Mononuclear cells from PAH patients with the phenotype of high RV glucose uptake do not produce cAMP in response to isoproterenol……..57 Figure 8: PAH patients with the high RV glucose uptake phenotype do not respond to carvedilol…………………………………….…………………………….59 Chapter 3 Figure 1: Optimization of MP isolation via centrifugation…………………….……75 Figure 2: Murine MPs stain positive for MitoTracker Green………………………76 Figure 3: MPs from GFP-mito mice are GFP positive and MitoTracker Red positive…………………………..………………………………….78 v Figure 4: Murine circulating mitochondria stain positive for CD41 and CD144 but not CD45…………………………………………………………………..79 Figure 5: Human circulating mitochondria stain positive for CD41, CD144, and CD45……………..……………………………………………………….80 Supplemental Figure 1: Illustrated method for optimizing isolation of plasma MP…………………………………………..………………………………….82 vi Metabolic Mechanisms in Physiologic and Pathologic Oxygen Sensing Abstract by OLIVIA ROSE STEPHENS The β-adrenergic receptor (βAR) exists in an equilibrium of inactive and active conformational states, which is modulated by ligands resulting in downstream signaling. In addition to cAMP, βAR regulates hypoxia-inducible factor 1 (HIF-1). We hypothesized that HIF-1 signaling occurs via a unique, independent βAR conformation and that Pulmonary Arterial Hypertension (PAH) patients with HIF- biased conformations would have blunted cAMP response. We found isoproterenol and salbutamol, both cAMP agonists, had opposing effects on HIF- 1 in cells and mice. Additionally, hypoxia blunted agonist-stimulated cAMP in vitro, consistent with receptor equilibrium shifting towards HIF-activating conformations. βAR overexpression in cells increased HIF-1 activity and glycolysis which was blunted by HIF-1 inhibitors, suggesting increased βAR increases basal HIF-1 signaling. Because PAH is also characterized by HIF- related glycolytic shift, we dichotomized PAH patients in the PAHTCH trial (NCT01586156) based on right ventricular glucose uptake to evaluate βAR signaling. Patients with high glucose uptake had more severe disease than those with low uptake and had no response to βAR ligands. The findings expand the paradigm of βAR regulation and uncover a novel PAH subtype that might benefit from β-blockers. vii Circulating cell-free mitochondrial components are well characterized as mediators of inflammation. Recent studies show cells also release microparticles (MPs) containing intact mitochondria under conditions of stress or injury. However, detection of cell-free mitochondria and their cellular origin has not been studied in non-pathological conditions. Thus, we hypothesize that intact mitochondria are detectable in the circulation under physiological conditions. To test this, plasma MPs were analyzed via flow cytometry. Murine platelet-depleted plasma showed a small cluster of MPs which was 65% positive for the mitochondrial marker MitoTracker Green (MT Green). Additionally, transgenic mice expressing mitochondrial GFP had GFP positive MPs in their plasma. Human plasma also contained cell-free mitochondria, with approximately 11% of the total MPs staining MT green positive. Platelets and endothelial cells were sources of MT green positive MPs in mice and humans, based on cell-specific surface markers. Leukocytes were also a source of mitochondria in humans but not mice. Together these data show multiple cell types release intact mitochondria into the circulation in healthy individuals. viii Chapter 1: Introduction I. Hypoxia and HIF Physiologic responses to hypoxia Exposure to hypoxia, or low oxygen concentrations, leads to a number of acute physiological effects. Low blood oxygen level is detected through peripheral chemoreceptors. Peripheral chemoreceptors detect minute changes in not only blood oxygen, but also carbon dioxide and pH. When low blood oxygen is detected, peripheral chemoreceptors stimulate vasoconstriction and bradycardia. However, peripheral chemoreceptors also stimulate increased ventilation, which leads to tachycardia. This overcomes the initial depression of heart rate. Thus, the acute physiologic response to hypoxia is tachycardia. HIF-1 Prolonged exposure to hypoxia results in a number of cellular and molecular adaptations that are primarily regulated by hypoxia-inducible factor-1 (HIF-1). HIF- 1 is a transcription factor that consists of an α and a β subunit. Both subunits are constitutively expressed. However, the α subunit is degraded in an oxygen dependent manner (163). Specifically, HIF-1α is hydroxylated by prolyl hydroxylase domain proteins (PHD) using oxygen as a substrate. This hydroxylation provides a binding site for von Hippel Lindau (VHL) which interacts with an E3 ligase that ubiquitinates HIF-1α. Ubiquitination targets HIF-1α for proteasomal degradation (60, 69). Thus, when there is not enough oxygen to serve as a substrate for hydroxylation (i.e. hypoxia), HIF-1α accumulates and 1 translocates to the nucleus to dimerize with HIF-1β. As a heterodimer, HIF-1 binds hypoxia response elements (HRE) to stimulate transcription of many genes. Other regulators of HIF-1 In addition to oxygen, PHD also utilize α-ketoglutarate, an intermediate of the tricarboxylic acid (TCA) cycle, as a substrate for hydroxylation of HIF-1α. Thus HIF-1α levels are sensitive to changes α-ketoglutarate levels. PHD is inhibited by pyruvate and tricarboxylic