Yuan Lab Research
The general interests of our research include vascular physiology, electrophysiology, and pathogenic mechanisms of pulmonary vascular disease. We are specifically interested in: 1)the regulation of excitation-contraction coupling and CA2+ signaling in vascular smooth muscle, 2) the transcriptional and functional regulation of ion channels (K+, Ca2+, and Cl- channels) in smooth muscle and endothelial cells, 3) the cellular and molecular mechanisms of hypoxic pulmonary vasoconstriction, 4) the pathogenic and therapeutic mechanisms of idiopathic and thromboembolic pulmonary hypertension, and 5) the functional role of on channels in stem cell proliferation and differentiation. We are using the combined techniques of patch clamping, digital imaging fluorescence microscopy, and molecular biology to investigate the roles of ion channels and intracellular Ca2+ in regulating vasomotor tone and pulmonary vascular smooth muscle proliferation and apoptosis. We are also studying how dysfunctional voltage-gated K+ (Kv) channels and upregulated transient receptor potential (TRP) channels in pulmonary arterial smooth muscle cells (PASMC) contribute to the development of pulmonary hypertension. The aims of our current research include:
- Understand the cellular and molecular mechanisms involved in hypoxia-mediated inhibition of Kv channels in PASMC
- Investigate the molecular mechanisms of PASMC proliferation and apoptosis
- Specify the pathogenic roles of K+ (e.g. Kv) and Ca2+ (e.g., TRP) channels in pulmonary arterial hypertension
- Search for new therapeutic approaches for pulmonary hypertension (e.g., K+ channel openers, pro-apoptotic/anti-proliferative agents)
- Determine the genetic variances associated with the susceptibility of developing idiopathic and thromboembolic pulmonary hypertension
- Identify the mechanisms that regulate membrane trafficking of ion channels in pathogenesis
Cellular Mechanisms of Pulmonary Arterial Hypertension
Pulmonary arterial hypertension (PAH) is caused by functional and structural changes in the pulmonary vasculature, which leads to increased pulmonary vascular resistance (PVR). Regardless of the initial pathogenic trigger, the major causes of elevated PVR in patients with PAH are chronic pulmonary vasoconstriction, pulmonary vascular remodeling, in situ thrombosis, and increased pulmonary vascular wall stiffness. Our current understanding of this mechanism suggests that multiple “hits” are involved in/required for the pulmonary vasculopathy observed in patients with PAH.
Figure 1. Schematic diagram depicting the potential mechanisms involved in the development of pulmonary arterial hypertension.
As diagramed in Figure 1, an increase in [Ca2+]cyt in PASMCs cab be caused by a variety of mechanisms, including but not limited to:
- Decreased Kv channel activity and subsequent depolarization (via opening of voltage-dependent Ca2+ channels)1
- Upregulated TRPC channels that participate in forming receptor-[ROC] and store-operated [SOC] Ca2+ channels2
- Upregulated membrane receptors (e.g., serotonin, endothelin, and/or leukotriene receptors) and their downstream cascades3
This increase in [Ca2+]cyt is known to cause pulmonary vasoconstriction, stimulate PASMC proliferation, and inhibit the BMP-signaling pathway that leads to antiproliferative and proapototic effects on PASMCs. Dysfunction of BMP signaling is due the following mechanisms:
- BMPR2 mutation and BMP-RII/BMP-RI downregulation4
- Kv channel function and expression inhibition
- PASMC apoptosis attenuation
- PASMC proliferation promotion
Increased angiopoietin-1 (Ang-1) synthesis and release5 from PASMCs enhance 5-HT production and downregulate BMP-RIa in pulmonary artery endothelial cells (PAEC), and further enhance PASMC contraction and proliferation. Inhibited nitric oxide (NO) and prostacyclin (PGI2) synthesis6 in PAECs, however, would attenuate the endothelium-derived relaxing effect on pulmonary arteries and promote sustained vasoconstriction and PASMC proliferation. Increased activity and expression of the 5-HT transporter (5-HTT)7 would serve as an additional pathway to stimulate PASMC growth via the mitogen-activated protein kinase (MAPK) pathway. Increased [Ca2+]cyt and Ca2+-sensitization also cause sustained PASMC contraction via the Rho kinase (ROC) pathway8. Furthermore, exogenous viral and bacterial infection and inflammation9 may contribute to vasoconstrcion and vascular medial hypertrophy in patients with mutations in multiple genes or with “susceptible predispositions” in these pathways. In addition, a variety of splicing factors, transcription factors, protein kinases, extracellular metalloproteinases, and circulating growth factors would serve as the so-called “hits” to mediate the phenotypical transition of normal cells to contractive or hypertrophied cells and to maintain the progression of PAH.
All superscript numbers correspond to mechanisms illustrated in Figure 1.
Ca2+ Signaling in Pulmonary Vasoconstriction and Vascular Remodeling
A rise in [Ca2+]cyt in PASMC is a major trigger for pulmonary vasoconstriction and an important stimulus for cell proliferation and migration. This subsequently causes pulmonary vascular remodeling characterized pathologically by intimal and medial hypertrophy, obliteration of small arteries due to excessive PAEC/PASMC proliferation and migration, and adventitial hypertrophy due to increased fibroblast (PAFB) proliferation and migration (Figure 2)
Figure 2. Schematic diagram showing the role of intracellular Ca2+ in pulmonary vasoconstriction and vascular remodeling.
Smooth muscle contraction is directly triggered by a rise in [Ca2+]cyt due to Ca2+ influx and release (from intracellular stores, e.g., sarcoplasmic reticulum, SR). The contractile proteins, actin and myosin, interact in a Ca2+-dependent pathway to result in contraction of PASMC. Calmodulin (CaM), an intracellular Ca2+-binding protein, binds to Ca2+ as [Ca2+]cyt rises. The Ca2+-CaM complex then activates myosin light chain kinase (MLCK) which, in turn, phosphorylates the myosin light chain (MLC). The phosphorylated MLC stimulates the activity of myosin ATPase, hydrolyzing ATP to release energy for the subsequent cycling of the myosin crossbridges with the actin filament. The formation of these crossbridges underlies PASMC contraction, prompting pulmonary vasoconstriction. A rise of [Ca2+]cyt also stimulates cell proliferation by activating cytoplasmic signal transduction proteins (e.g., CaMK, MAPK) and transcription factors (e.g., c-Fos/c-Jun, NFAT, CREB). Ca2+ also participates in regulating PASMC proliferation through its intimate involvement with the cell cycle. There are at least four Ca2+/CaM-sensitive steps in the cell cycle:
- transition from G0 (resting state) to G1 phase (the beginning of DNA synthesis)
- transition from G1 to S phase (an interphase during which replication of the nuclear DNA occurs)
- transition from G2 to M phase (mitosis)
- through M phase or mitosis.
An increase in [Ca2+]cyt in PASMC (and other cell types), which also rapidly increases nuclear [Ca2+] and gradually enhances stored [Ca2+] in the SR/ER, plays an important role in facilitating quiescent cells to get into the cell cycle and propelling cell passage through the cell cycle. Therefore, an increase in [Ca2+]cyt not only induces pulmonary vasoconstriction by triggering PASMC contraction, but also mediates pulmonary vascular remodeling by stimulating cell proliferation and migration (Figure 2).
Regulation of Intracellular Ca2+ in Pulmonary Artery Smooth Muscle Cells
The fundamental systems coordinating changes in [Ca2+]cyt are (Figure 3): a) Ca2+ entry via voltage-dependent Ca2+ channels (VDCC), receptor-operated Ca2+ channels (ROC), and/or store-operated Ca2+ channels (SOC); b) Ca2+ release and sequestration from and into the SR or ER; c) Ca2+ extrusion to the extracellular space by Ca2+-Mg2+ ATPase pumps; d) outward transportation of Ca2+ by the forward mode of Na+-Ca2+ exchangers (NCX) and inward transportation of Ca2+ by the reverse mode of NCX; e) mitochondrial Ca2+ release and sequestration; and f) release and sequestration from and into other intracellular Ca2+ stores (e.g., lysosomes).
K+ can be transported into virtually all mammalian cells against its electrochemical gradient. The mechanism responsible for this active transport is the ouabain-sensitive Na+/K+ pump (Na+/K+-ATPase) that expels 3 Na+ ions in exchange for 2 entering K+ ions. Hydrolysis of the terminal high-energy phosphate bond of ATP provides sufficient energy so that the Na+/K+ pump can concentrate K+ more than 20-fold in cells and thus can extrude Na+ against an equivalent concentration gradient. This transport is electrogenic because a single net positive charge is extruded during each cycle. The resulting current flow usually adds 1 to 2 mV to the resting membrane potential (Em) determined from ion gradients and permeabilities. The Na+/K+ pump compensates for the loss of K+ through the various types of K+-permeable channels (Figure 3).
Figure 3. Schematic diagram showing the mechanisms involved in regulating [Ca2+]cyt in pulmonary vascular smooth muscle cells.
There are three major causes of membrane depolarization: a) decrease in K+ currents (IK) due to downregulation and dysfunction of various K+ channels (e.g., voltage-gated, Ca2+-activated, and two-pore domain K+ channels); b) decrease in Na+/K+ ATPase activity (e.g., when ouabain is increased in the plasma and extracellular fluid); and c) increase in Cl- currents (ICl) due to upregulation and increased activity of Ca2+-activated Cl- (ClCa) channels. Membrane depolarization subsequently increases [Ca2+]cyt by inducing Ca2+ influx through VDCC and by eliciting Ca2+ release or mobilization from the SR or ER via ryanodine receptors (RyR) (Figure 4).
Figure 4. Schematic diagram showing the major causes for membrane depolarization and increases in [Ca2+]cyt due to membrane depolarization in pulmonary vascular smooth muscle cells.
The increased [Ca2+]cyt can be restored or recovered to the baseline level (approximately 100 nM) by i) Ca2+ extrusion via the Ca2+/Mg2+ ATPase in the plasma membrane, ii) outward Ca2+ transportation by the forward model of Na+/Ca2+ exchanger (NCX); and iii) Ca2+ sequestration or uptake into the SR/ER by the Ca2+/Mg2+ pump in the SR/ER membrane (SERCA). Inhibition of Na+/K+ ATPase (or Na+ pump) and/or activation of Na+-permeable channels in the plasma membrane would increase cytosolic Na+ concentration ([Na+]cyt). When [Na+]cyt is increased, the forward mode of NCX would change to the reverse mode and cause inward Ca2+ transportation and increase [Ca2+]cyt (Figure 3). In addition to causing pulmonary vasoconstriction, a rise in [Ca2+]cyt in PASMC is also an important stimulus for cell proliferation, migration, and for gene expression.
When membrane receptors, G protein-coupled receptors (GPCR) and tyrosine kinase receptors (TKR), in the plasma membrane are activated by extracellular ligands, phospholipace C (PLC) is activated to stimulate synthesis of IP3 and diacylglycerol (DAG), two important second messengers. DAG can open ROC, leading to Ca2+ influx (i.e., receptor-operated Ca2+ entry, ROCE) and an increase in [Ca2+]cyt. IP3 can activate IP3 receptors (IP3R) on the SR/ER membrane, stimulate Ca2+ release or mobilization from the SR/ER to the cytosol, and ultimately deplete Ca2+ in the SR/ER. Depletion of Ca2+ from the SR/ER triggers store-operated Ca2+ entry (SOCE), or capacitative Ca2+ entry (CCE), by activating SOC (Figure 5). The precise molecular entity of ROC and SOC has remained enigmatic. In the vasculature or vascular smooth muscle and endothelial cells, there is increasing evidence that the Orai1/2/3 and STIM1/2 (stromal interaction molecule) proteins, and transient receptor potential (TRP) channels are involved in forming functional ROC and SOC channels.
Figure 5. Schematic diagram showing the mechanisms involved in ROCE/SOCE and inward transportation of Ca2+ via the reverse mode of NCX in pulmonary vascular smooth muscle cells.
Pathogenic Roles of Ion Channels in Pulmonary Arterial Hypertension
In addition to the synthetic, structural, and functional abnormalities in the pulmonary vasculature, substantial and convincing evidence has recently emerged pointing to multiple derangements in complex membrane receptors, ion channels, and intracellular signaling pathways that contribute to the manifestation of pulmonary vascular disease, especially in idiopathic pulmonary arterial hypertension. It is now generally accepted that this disease involves a heterogeneous constellation of multiple genetic, molecular, and humoral abnormalities that all share a common end result, i.e., pulmonary vascular remodeling. Dysfunction of Kv channels, upregulation of TRPC/Orai channels and STIM proteins, upregulation of NCX, and abnormality of intracellular Ca2+ homeostasis are examples of cellular factors involved in sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in patients with idiopathic pulmonary arterial hypertension.