Endothelial Cell Reactive Oxygen Species and Ca2+ Signaling in Pulmonary Hypertension

Abstract

Pulmonary hypertension (PH) refers to a disorder characterized by elevated pulmonary arterial pressure, leading to right ventricular overload and eventually right ventricular failure, which results in high morbidity and mortality. PH is associated with heterogeneous etiologies and distinct molecular mechanisms, including abnormal migration and proliferation of endothelial and smooth muscle cells. Although the exact details are not fully elucidated, reactive oxygen species (ROS) have been shown to play a key role in promoting abnormal function in pulmonary arterial smooth muscle and endothelial cells in PH. In endothelial cells, ROS can be generated from sources such as NADPH oxidase and mitochondria, which in turn can serve as signaling molecules in a wide variety of processes including posttranslational modification of proteins involved in Ca²⁺ homeostasis. In this chapter, we discuss the role of ROS in promoting abnormal vasoreactivity and endothelial migration and proliferation in various models of PH. Furthermore, we draw particular attention to the role of ROS-induced increases in intracellular Ca²⁺ concentration in the pathobiology of PH.

Keywords: Calcium homeostasis; Endothelial cell; Hypoxia; Mitochondria; NADPH oxidase; Pulmonary hypertension; Reactive oxygen species.

Fig. 1

Reactive oxygen species (ROS) and endothelial nitric oxide synthase (eNOS) uncoupling. Under normal circumstances (left panel), eNOS utilizes various cofactors including l-arginine, molecular O₂ and (6R-)5,6,7,8-tetrahydrobiopterin (BH₄) to catalyze nitric oxide (NO) formation (at the heme site). The eNOS enzyme includes various regulatory subunits including a calmodulin (CaM) binding site. In situations of oxidative stress (right panel), local increases in superoxide (O₂^•) lead to: (1) formation of peroxinitrites (ONOO⁻) due to reactions with NO, causing oxidation of BH₄ (to BH₃) and inactivation of this critical cofactor and (2) oxidative modification of the Zn-thiolate cluster. Together, these events lead to decreased catalytic efficiency of eNOS and generation of O₂^• instead of NO. Increased O₂^• in turn can augment further uncoupling of eNOS via a feed-forward mechanism

Fig. 2

Mechanisms of reactive oxygen species (ROS)-induced Ca²⁺ entry in endothelial cells (ECs) in pulmonary arterial hypertension (PAH). Increases in ROS from cytosolic (primarily NADPH oxidase) and mitochondrial sources can lead to both release of Ca²⁺ from internal endoplasmic reticulum (ER) stores as well as influx through receptor-operated (ROC) and store-operated (SOC) channels on the cell membrane. Release of Ca²⁺ from the ER can activate SOCs in order to replete ER Ca²⁺ stores via store-operated Ca²⁺ entry (SOCE). The sources of increased ROS in ECs include: NADPH oxidase, mitochondria and physical stimuli like shear stress. Based on data from both ECs and smooth muscle cells (SMCs) in PAH, mitochondrial ROS generation may be linked to abnormal HIF-1α stabilization and glycolytic shift

Fig. 3

Functional consequences of elevated reactive oxygen species (ROS) and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in pulmonary endothelial cells (ECs). Elevations of both ROS and [Ca²⁺]_i promote normal physiologic responses, such as hypoxic vasoconstriction, maladaptive responses such as EC adaptation to shear stress and stretch and lastly, pathologic responses such as increased migration and proliferation