Ruby A. Fernandez

Enhanced Ca 2+ -Sensing Receptor Function in Idiopathic Pulmonary Arterial Hypertension

Rationale: A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. Increased resting [Ca2+]cyt and enhanced Ca2+ influx have been implicated in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH). Objective: We examined whether the extracellular Ca2+-sensing receptor (CaSR) is involved in the enhanced Ca2+ influx and proliferation in IPAH-PASMC and whether blockade of CaSR inhibits experimental pulmonary hypertension. Methods and Results: In normal PASMC superfused with Ca2+-free solution, addition of 2.2 mmol/L Ca2+ to the perfusate had little effect on [Ca2+]cyt. In IPAH-PASMC, however, restoration of extracellular Ca2+ induced a significant increase in [Ca2+]cyt. Extracellular application of spermine also markedly raised [Ca2+]cyt in IPAH-PASMC but not in normal PASMC. The calcimimetic R568 enhanced, whereas the calcilytic NPS 2143 attenuated, the extracellular Ca2+-induced [Ca2+]cyt rise in IPAH-PASMC. Furthermore, the protein expression level of CaSR in IPAH-PASMC was greater than in normal PASMC; knockdown of CaSR in IPAH-PASMC with siRNA attenuated the extracellular Ca2+-mediated [Ca2+]cyt increase and inhibited IPAH-PASMC proliferation. Using animal models of pulmonary hypertension, our data showed that CaSR expression and function were both enhanced in PASMC, whereas intraperitoneal injection of the calcilytic NPS 2143 prevented the development of pulmonary hypertension and right ventricular hypertrophy in rats injected with monocrotaline and mice exposed to hypoxia. Conclusions: The extracellular Ca2+-induced increase in [Ca2+]cyt due to upregulated CaSR is a novel pathogenic mechanism contributing to the augmented Ca2+ influx and excessive PASMC proliferation in patients and animals with pulmonary arterial hypertension.

Upregulated expression of STIM2, TRPC6, and Orai2 contributes to the transition of pulmonary arterial smooth muscle cells from a contractile to proliferative phenotype

Pulmonary arterial hypertension (PAH) is a progressive disease that, if left untreated, eventually leads to right heart failure and death. Elevated pulmonary arterial pressure (PAP) in patients with PAH is mainly caused by an increase in pulmonary vascular resistance (PVR). Sustained vasoconstriction and excessive pulmonary vascular remodeling are two major causes for elevated PVR in patients with PAH. Excessive pulmonary vascular remodeling is mediated by increased proliferation of pulmonary arterial smooth muscle cells (PASMC) due to PASMC dedifferentiation from a contractile or quiescent phenotype to a proliferative or synthetic phenotype. Increased cytosolic Ca(2+) concentration ([Ca(2+)]cyt) in PASMC is a key stimulus for cell proliferation and this phenotypic transition. Voltage-dependent Ca(2+) entry (VDCE) and store-operated Ca(2+) entry (SOCE) are important mechanisms for controlling [Ca(2+)]cyt. Stromal interacting molecule proteins (e.g., STIM2) and Orai2 both contribute to SOCE and we have previously shown that STIM2 and Orai2, specifically, are upregulated in PASMC from patients with idiopathic PAH and from animals with experimental pulmonary hypertension in comparison to normal controls. In this study, we show that STIM2 and Orai2 are upregulated in proliferating PASMC compared with contractile phenotype of PASMC. Additionally, a switch in Ca(2+) regulation is observed in correlation with a phenotypic transition from contractile PASMC to proliferative PASMC. PASMC in a contractile phenotype or state have increased VDCE, while in the proliferative phenotype or state PASMC have increased SOCE. The data from this study indicate that upregulation of STIM2 and Orai2 is involved in the phenotypic transition of PASMC from a contractile state to a proliferative state; the enhanced SOCE due to upregulation of STIM2 and Orai2 plays an important role in PASMC proliferation.

Inhibition of the Ca2+-sensing receptor rescues pulmonary hypertension in rats and mice

A recent study from our group demonstrated that the Ca2+-sensing receptor (CaSR) was upregulated, and the extracellular Ca2+-induced increase in cytosolic Ca2+ concentration ([Ca2+]cyt) was enhanced in pulmonary arterial smooth muscle cells from patients with idiopathic pulmonary arterial hypertension and animals with experimental pulmonary hypertension (PH). However, it is unclear whether CaSR antagonists (for example, NPS2143) rescue the development of experimental PH. We tested the rescue effects of NPS2143 in rats with monocrotaline (MCT)-induced PH and mice with chronic hypoxia-induced PH. For the NPS2143 treatment group, rats and mice were i.p. injected with NPS2143 once per day from days 14 to 24. Four weeks after MCT injection or exposure to normobaric hypoxia, the right ventricular (RV) systolic pressure, right heart hypertrophy (RV/LV+S ratio) and RV myocardial fibrosis were rescued or nearly restored to normal levels by NPS2143 treatment. The rescue effects of NPS2143 on experimental PH further support a critical role for the CaSR in the PH mechanism. Therefore, NPS2143 may be a promising potential treatment for pulmonary arterial hypertension.

Pathogenic Role of Store-Operated and Receptor-Operated Channels in Pulmonary Arterial Hypertension

Pulmonary circulation is an important circulatory system in which the body brings in oxygen. Pulmonary arterial hypertension (PAH) is a progressive and fatal disease that predominantly affects women. Sustained pulmonary vasoconstriction, excessive pulmonary vascular remodeling, in situ thrombosis, and increased pulmonary vascular stiffness are the major causes for the elevated pulmonary vascular resistance (PVR) in patients with PAH. The elevated PVR causes an increase in afterload in the right ventricle, leading to right ventricular hypertrophy, right heart failure, and eventually death. Understanding the pathogenic mechanisms of PAH is important for developing more effective therapeutic approach for the disease. An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC migration and proliferation which lead to pulmonary vascular wall thickening and remodeling. It is thus pertinent to define the pathogenic role of Ca2+ signaling in pulmonary vasoconstriction and PASMC proliferation to develop new therapies for PAH. [Ca2+]cyt in PASMC is increased by Ca2+ influx through Ca2+ channels in the plasma membrane and by Ca2+ release or mobilization from the intracellular stores, such as sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER). There are two Ca2+ entry pathways, voltage-dependent Ca2+ influx through voltage-dependent Ca2+ channels (VDCC) and voltage-independent Ca2+ influx through store-operated Ca2+ channels (SOC) and receptor-operated Ca2+ channels (ROC). This paper will focus on the potential role of VDCC, SOC, and ROC in the development and progression of sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in PAH.

miRNA208/Mef2 and TNF-α in Right Ventricular Dysfunction: The Transition From Hypertrophy to Failure

Pulmonary arterial hypertension (PAH) is a rare but progressive and deadly disease caused by functional and structural changes in the pulmonary vasculature, which lead to an increase in pulmonary vascular resistance. Regardless of the initial pathogenic trigger, the major causes of increased pulmonary vascular resistance in patients with PAH are sustained pulmonary vasoconstriction, pulmonary vascular remodeling, in situ thrombosis, and increased pulmonary vascular wall stiffness. Despite expanding research into the diagnosis and treatment of pulmonary hypertension, death rates from pulmonary hypertension have continued to increase 2.5% per year for women and 0.9% per year for men during the past decade.1 Patients with PAH, if untreated, die mainly because of progressive right heart failure, and the response of the right ventricle (RV) to the increased afterload is an important determinant of outcome in patients.2 During the development of pulmonary hypertension, an initial adaptive response of the RV to the increased afterload is to increase its wall thickness and contractility with varying degrees of RV hypertrophy.3 However, with disease progression, sustained long-term pressure overload of the RV can lead to progressive contractile dysfunction and eventually cause RV failure with further RV dilation. Little is known about the molecular and cellular mechanisms, which underlie the development of RV failure. The mechanism that determines the transition of RV function from compensated hypertrophy to decompensated failure is also uncertain. Article, see p 56 MicroRNAs (miRNAs), as crucial regulators of cardiovascular development and cardiac remodeling, have attracted increasing interest in recent years. Drake et al4 compared the gene expression patterns between RV hypertrophy in hypoxia-induced …

Mutant hERG channel traffic jam. Focus on “Pharmacological correction of long QT-linked mutations in KCNH2 (hERG) increases the trafficking of Kv11.1 channels stored in the transitional endoplasmic reticulum”

ION CHANNELS ARE MACROMOLECULAR pore-forming structures that regulate the selective distribution of ions across the cell membrane or between intracellular compartments. They are fundamental in several physiological processes including cell volume regulation, neurotransmission, and muscle contraction. Therefore, it is not surprising that disruption of ion channel function by genetic mutation can be associated with cardiovascular disorders. KCNH2, also known as the human ether-a-go-go related gene (hERG), encodes the pore-forming subunit of a delayed rectifier voltage-dependent K channel (9). These channels are expressed in numerous cell types, but are most notable for their role in the heart. Normal hERG K channel activity is important for maintaining cardiac electrical activity. The gating properties of hERG K channels makes their contribution ideal for prolonging the duration of the plateau phase (slow activation and rapid inactivation) and for initiating the terminal repolarization phase (rapid recovery from inactivation and slow deactivation) of the action potential. Controlling action potential duration is critical for ensuring sufficient Ca 2 entry to enable cardiac contraction, while the delayed repolarization phase provides some protection against premature contractions (9). Loss-of-function (LOF) mutations can attenuate hERG K channel activity, thus increasing the propensity for the development of life-threatening arrhythmias. For example, LOF mutations in KCNH2 can cause chromosome 7-linked long QT (type 2; LQT2) syndrome, a disorder characterized by abnormal cardiac electrical activity, due to prolongation of the QT interval (2). Although this LOF can be attributed to decreased expression, dominant-negative (DN) effects, or changes in

Deacetylation of MicroRNA-124 in Fibroblasts Role in Pulmonary Hypertension

The molecular mechanisms involved in the development of pulmonary hypertension (PH) remain unclear, although many investigators have demonstrated that abnormalities in gene expression in pulmonary vascular fibroblasts, smooth muscle cells, and endothelial cells are involved in the pathogenesis of PH. The control of gene expression is a complicated process, involving multiple layers of regulation. There are 3 distinct mechanisms of epigenetic regulation, DNA methylation, histone modifications, and gene silencing mediated by microRNAs (miRNAs). DNA methylation occurs on cytosine residues in CpG regions and is regulated by DNA methyltransferases (DNMTs). DNA methylation is essential for normal development, and 60% to 80% of the human genome CpGs are methylated. Methylation of most CpGs is constant, changing only in response to different cellular processes. In cancers and other diseases, hypermethylation of so-called CpG islands, which are CG-dense regions close to transcription start sites, found in tumor suppressor genes has been reported, leading to gene silencing. These data demonstrate that DNA methylation status is a frequently altered epigenetic modification in human diseases. In addition to DNA methylation, histone modifications represent another layer of regulation of gene expression. For the transcription machinery to be recruited to their target genes, the DNA needs to be accessible. The ability of the transcription machinery to reach the DNA is mainly controlled by histone acetyltransferases and histone deacetylases (HDACs). Histone acetyltransferases acetylate lysine residues and relax the chromatin structure, allowing for transcription factors to bind to the DNA and activate transcription. HDACs remove acetyl residues from histones, resulting in a condensed chromatin structure and transcriptional repression. The last layer of gene expression regulation is controlled by miRNAs, which are small noncoding RNAs that bind to their complementary sequence in the 3′ untranslated regions of their target mRNAs, resulting in gene silencing. Article, see p 67 These pathways of gene …

Smooth Muscle Cell Ion Channels in Pulmonary Arterial Hypertension: Pathogenic Role in Pulmonary Vasoconstriction and Vascular Remodeling

Pulmonary arterial hypertension (PAH) is a progressive hemodynamic disease that impacts right heart function ultimately resulting in mortality due to right heart failure. Increased pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) observed in PAH patients can be attributed in part to sustained vasoconstriction and excessive remodeling of the distal pulmonary arteries. Pulmonary vasoconstriction is a result of pulmonary artery smooth muscle cell (PASMC) contraction while pulmonary vascular remodeling is associated with increased cell proliferation and decreased apoptosis. Spanning the plasma membrane of PASMC are macromolecular pore-forming proteins known as ion channels that allow for passive distribution of ion across the membrane when open. Ion channel activity is crucial for driving critical physiological functions. Posttranscriptional regulation of ion channel expression and ion channel dysfunction in PASMCs have been linked to changes in vascular tone and the initiation of vascular remodeling. This chapter will introduce smooth muscle cell ion channels that regulate physiological function in the pulmonary vasculature and summarize their potential pathogenic contribution to the development of pulmonary vascular diseases such as PAH.

Adult Lung Stem Cells

Over the past decade a wealth of information has been divulged on stem cells present in the lung both in the pulmonary vasculature and the respiratory tract. Cells have been identified with the capability of repopulating the lung and others that contribute to the pathogenesis or, conversely, have therapeutic benefit in pulmonary vascular disease. The isolation of a single-resident lung stem cell capable of repopulating any lung epithelium still remains elusive. What is currently known about stem and progenitor cells in the lung suggests that a non-classical stem cell hierarchy exists with a novel array of cellular mechanisms controlling proliferation and differentiation of such cells. This chapter serves to provide an up-to-date review of what is currently known about stem and progenitor cells within the lung.