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The Human Pulmonary Circulation: Historical Introduction.- Microcirculation of the Lung: Functional and Anatomic Aspects.- Pulmonary Vascular Development.- Pulmonary Vascular Function.- Pulmonary Vascular Mechanics.- Modeling of the Pulmonary Vasculature.- Metabolic and Clearance Function at the Pulmonary Microvascular Endothelial Surface in Pulmonary Hypertension.- The Pharmacology of the Major Vasoactive Mediators Relevant to the Pathogenesis of Pulmonary Hypertension.- The Normal Fetal and Neonatal Pulmonary Circulation.- Excitation-contraction Coupling and Regulation of Pulmonary Vascular Contractility.- Endothelial Regulation of Pulmonary Vascular Tone.- Acute Lung Injury: The Injured Lung Endothelium, Therapeutic Strategies for Barrier Protection and Vascular Biomarkers.- Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle.- Receptor-mediated Signal Transduction and Cell Signaling.- Role of Calcium as a Second Messenger in Signaling: A Focus on Endothelium.- Caveolae and Signaling in Pulmonary Vascular Endothelial and Smooth Muscle Cells.- The Chemistry of Biological Gases.- Role of Oxygen-derived Species in the Regulation of Pulmonary Vascular Tone.- Mitochondrial ROS and Redox State in Pulmonary Vascular O2 Sensing.- Cellular and Molecular Mechanisms of Pulmonary Vascular Smooth Muscle Cell Proliferation.- Role of Ca2+ in Vascular Smooth Muscle Gene Expression and Proliferation.- Biochemistry and Cellular Mechanisms of Apoptosis in Vascular Smooth Muscle and Endothelial Cells.- The Coagulation Cascade and its Regulation.- Platelets in Pulmonary Vascular Physiology and Pathology.- Lysis and Organization of Pulmonary Thromboemboli.- Interactions of Leukocytes and Coagulation Factors with the Vessel Wall.- Interaction of the Plasminogen System with the Vessel Wall.- Endothelial Apoptosis and Repair in Pulmonary Arterial Hypertension.- Bronchial Arterial Circulation in the Human.- Animal Models of Pulmonary Hypertension.- Transgenic and Gene Targeted Mouse Models for Pulmonary Hypertension.- Animal Models of Increased Lung Vascular Permeability.- Isolation and Culture of Pulmonary Vascular Smooth Muscle and Endothelial Cells.- Conventional Patch Clamp Techniques and High-throughput Patch Clamp Recordings on a Chip for Measuring Ion Channel Activity.- Measurement of Pulmonary Vascular Structure and Pulmonary Blood Distribution by MDCT and MR Imaging Techniques.- Quantification of DNA, RNA and Protein Expression.- Gene Cloning, Transfection and Mutagenesis.- Approaches for Manipulation of Gene Expression.- Bioinformatics, Genomics, and Functional Genomics: Overview.- Genomic Application to Study Pulmonary Hypertension.- Proteomics and Functional Proteomics.- Maintenance, Propagation and Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.- Identification of Adult Stem and Progenitor Cells in the Pulmonary Vasculature.- Differentiation of Embryonic Stem Cells to Vascular Cell Lineages.- Statistics and Clinical Data Analysis: A Reference Guide.- Hypoxic Pulmonary Vasoconstriction.- Pathogenic Roles of Ca2+ and Ion Channels in Hypoxia-mediated Pulmonary Hypertension.- Roles of Endothelium-derived Vasoactive and Mitogenic Factors in the Development of Chronic Hypoxia-mediated Pulmonary Hypertension.- Oxygen-sensitive Transcription Factors and Hypoxia-mediated Pulmonary Hypertension.- Developmental Regulation of Pulmonary Vascular Oxygen Sensing.- Pulmonary Vascular Remodeling by High Oxygen.- Pulmonary Vascular Remodeling: Cellular and Molecular Mechanisms.- Carbon Monoxide and Heme Oxygenase in the Regulation of Pulmonary Vascular Function and Structure.- Shear Stress, Cell Signaling and Pulmonary Vascular Remodeling.- Pulmonary Hypertension and the Extracellular Matrix.- Role of Progenitor Cells in Pulmonary Vascular Remodeling.- Receptor Signaling in Pulmonary Arterial Hypertension.- Role of Endothelium in the Development of Pulmonary Hypertension.- Coagulation and the Vessel Wall in Pulmonary Embolism.- Alveolar Epithelial Fluid Transport in Lung Injury.- High Altitude Pulmonary Edema .- Statins and Acute Lung Injury.- Genomics of Acute Lung Injury and Vascular Barrier Dysfunction.- Ventilator-induced Mechanical Stress and Lung Vascular Dysfunction.- Classification of Pulmonary Hypertension: History and Perspectives.- Epidemiology of Pulmonary Arterial Hypertension.- Air Pollution and the Pulmonary Vasculature.- Idiopathic Pulmonary Arterial Hypertension.- Genetics of Familial and Idiopathic Pulmonary Arterial Hypertension.- Pulmonary Arterial Hypertension Related to Scleroderma and Collagen Vascular Diseases.- Pathology and Management of Portopulmonary Hypertension.- Pathobiology and Treatment of Pulmonary Hypertension in HIV Disease.- Pulmonary Arterial Hypertension Secondary to Anorexigens and Other Drugs and Toxins.- Pulmonary Arterial Hypertension Related to Gauchers, Sarcoidosis and Other Disorders.- Pediatric Pulmonary Hypertension: An Integrated View from Pediatric Subspecialists.- Persistent Pulmonary Hypertension of the Newborn: Mechanisms and Treatment.- The Pulmonary Circulation in Congenital Heart Disease.- Pulmonary Hypertension Secondary to Congenital Systemic to Pulmonary (Left-to-Right) Shunts.- Surgical Evaluation of Congenital Heart Disease-associated Pulmonary Hypertension.- Pulmonary Veno-occlusive Disease.- Left Ventricular Diastolic Heart Function and Pulmonary Hypertension.- Pulmonary Hypertension Associated with Chronic Obstructive Pulmonary Diseases.- Pulmonary Hypertension Associated with Interstitial Lung Disease.- High Altitude Pulmonary Hypertension.- Pulmonary Hypertension and Congenital Heart Defects at High Altitude.- Pulmonary Embolism and Deep Vein Thrombosis.- Pulmonary Hypertension Due to Pulmonary Embolism and Thromboembolic Obstruction of Proximal and Distal Pulmonary Arteries.- Risk Factors for Chronic Thromboembolic Pulmonary Hypertension.- Evaluation of Small Vessel Arteriopathy in Chronic Thromboembolic Pulmonary Hypertension.- Hemolytic Anemia Associated Pulmonary Hypertension: Sickle Cell Disease and Thalassemia-associated Pulmonary Hypertension.- Pulmonary Hypertension due to Schistosomiasis.- Pulmonary Hypertension due to Capillary Hemangiomatosis.- Molecular Basis of Right Ventricular Hypertrophy and Failure in Pulmonary Vascular Disease.- Right Ventricular Dysfunction in Pulmonary Hypertension.- Large Vessel Pulmonary Arteritis.- Tumors of the Pulmonary Vascular Bed.- Cor Pulmonale.- Pregnancy and Contraception in Patients with Pulmonary Arterial Hypertension.- Cardiac Catheterization in the Patient with Pulmonary Hypertension.- Imaging of Pulmonary Vascular Diseases.- Histological and Pathological Diagnosis of Pulmonary Hypertension: Pathological Classification of Pulmonary Vascular Lesions.- Echocardiography in Pulmonary Vascular Disease.- Calcium Channel Blockers in the Treatment of Pulmonary Arterial Hypertension.- Prostacyclin and Prostaglandins.- Endothelin Receptor Antagonists for the Treatment of Pulmonary Arterial Hypertension.- Phosphodiesterase Inhibitors in the Treatment of Pulmonary Hypertension.- Nitric Oxide for Children.- The Serotonin System as a Therapeutic Target in Pulmonary Hypertension.- Combination Therapy for Pulmonary Arterial Hypertension.- Thrombolytic and Anti-coagulant Therapy for Pulmonary Embolism and Chronic Thromboembolic Pulmonary Hypertension.- Nursing Care of Patients with Pulmonary Arterial Hypertension.- Atrial Septostomy.- Evaluation of Patients with Chronic Pulmonary Thromboembolic Pulmonary Hypertension for Pulmonary Endarterectomy.- Pulmonary Endarterectomy.- Evaluation of Patients with Pulmonary Hypertension for Lung Transplantation.- Lung Transplantation for Pulmonary Hypertension.- Living-donor Lower Lobar Lung Transplantation for Pulmonary Arterial Hypertension.- Results of Lung Transplantation.- Index.

Experimental quality is directly proportional to cell quality. This might not be a valid mathematical equation, but it very succinctly and elegantly describes the work of biologists from all fields of research: molecular biology or cell biology, proliferation or apoptosis assays, patch clamp electrophysiology or flow cytometry. One element lies at the heart of the success of each of these procedures: viable and healthy cells. Many days and weeks of valuable experimentation time have been spent perfecting cell isolation techniques, simply to guarantee that we can gather reliable data. Although it is possible to purchase cells from various sources, most research groups have developed their own techniques for isolating cells from tissues, techniques which can be adapted relatively easily from one tissue type to another. In addition, although it is always desirable to use freshly isolated cells (less than 8 h after isolation), many investigators have turned to cell culture as a viable ­alternative to freshly dissociated cells, although this may present some scientific challenges and dilemmas. The current ­chapter addresses the isolation of pulmonary artery smooth ­muscle cells (PASMCs) and pulmonary artery endothelial (PAECs), particularly from humans, rats, and mice. Generally speaking, the methods we outline can also be applied to PASMCs and PAECs from other species, although some modifications may be required. Readers are advised to consult the extensive literature to identify the technique most applicable to their needs.

Advances in molecular biology and biochemistry have provided tools with which to explore and increase our understanding of mediators that regulate development and pathological changes in the pulmonary circulation. Combinations of techniques have allowed researchers to start to unravel complex biological pathways that have direct relevance to pulmonary research, such as those that contribute to cellular proliferation and vascular permeability. The aim of this chapter is to describe some of the established protocols to quantify DNA, RNA, and protein expression, discuss advances in these techniques, and illustrate how they can be applied. Expression levels of cell signaling components can be detected at the level of DNA, RNA, and protein. Southern blotting (also termed “Southern hybridization” and named for Edwin Southern, who developed the technique) allows for the detection of a specific DNA sequence in a preparation of DNA, typically from cellular nuclei. This method was modified and extended later for the detection of RNA and protein, named Northern and Western blots, respectively. All three of these protocols are based on similar principles and utilize similar methods. Alternatively, measurement of gene expression [using messenger RNA (mRNA) as a starting material for the generation of complementary DNA (cDNA)] can also be performed via reverse-transcription (RT) polymerase chain reaction (PCR), followed by agarose gel electrophoresis. A modification of this technique, real-time PCR (also termed “quantitative PCR”), allows for quantitative analysis of gene copy number. A number of other high-throughput techniques exist for the profiling of gene and protein expression, such as by use of DNA and protein microarrays; however, these will be discussed in more detail in subsequent sections. Many variations exist for the protocols outlined below and most buffers can be made using a variety of recipes or can be purchased from a number of suppliers. However, the principles remain the same; understanding the stages in each process and their purpose is key. This chapter outlines the methods that have proven to be versatile, reliable, and sensitive.

Mutagenesis is a change or alteration in the DNA sequences of a gene. Mutagenic events may occur spontaneously within the genome of an organism and many times do not lead to functional consequences or an altered phenotype. However, at times a change in the coding sequence of a gene manifests itself as a dysfunctional phenotypic trait or predisposes an individual to a particular disease. The human genome may contain variations or mutations in an individual or in a related group of individuals that predispose them to a particular disease. Identifying and understanding these mutations (or polymorphisms) in gene structure can aid in the understanding, diagnosis, and treatment of patients. Quite the opposite to the mutations that spontaneously occur and may be difficult to identify, purposefully introducing mutations into a candidate gene that is then expressed in a cell or mouse model systems can readily reveal important information. This chapter provides an overview of the strategy to clone a gene, express it in a cultured cell system, and elucidate the function of the expressed gene product using mutagenesis methods. The value of this type of experimental approach will be highlighted by reviewing two pulmonary genes that have been analyzed using in vitro mutagenesis methods, the potassium channel Kv1.5 and the bone morphogenetic protein receptor II.

Chronic hypoxia causes pulmonary hypertension (CHPH), which occurs in residents living in high altitude and patients with chronic obstructive pulmonary disease, interstitial lung disease, and sleep-disordered breathing. The pathogenic mechanisms of CHPH are unclear, but involve sustained vasoconstriction and excessive vascular remodeling. A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells is a major trigger for pulmonary vasoconstriction and an important stimulus for vascular smooth muscle proliferation. Multiple ion channels in the plasma membrane participate in the regulation of [Ca2+]cyt, while hypoxia upregulates Ca2+ channels and downregulates K+ channels in pulmonary vascular smooth muscle cells. This chapter discusses the potential mechanism by which hypoxia causes pulmonary hypertension via modulating Ca2+ and K+ channel expression and function, and regulating intracellular Ca2+ stores in the sarcoplasmic reticulum.

Adult stem cells retain some capacity for self-renewal, although it is limited in comparison with embryonic stem cells, and are more restricted in their differentiation capacity. Adult stem cells are also commonly referred to as tissue specific stem cells. Some adult stem cells are still able to give rise to several specialized cell types (multipotent stem cells), whereas others are limited to a single specialized cell type (unipotent stem cells). Progenitor cells, the progeny of adult stem cells, differ from stem cells in that the potential for long-term self-renewal is lost. Scientifically, progenitor cells are more differentiated than stem cells. Primary stem cell progeny progenitor cells, usually known as multipotent adult progenitor cells, have full lineage-specific potential, whereas next-generation progenitors (oligopotent progenitors) are more lineage-restricted. This adult stem cell and progenitor cell hierarchy system exists to preserve a homeostatic repair and maintenance of the body, replenishing specialized cells and sustaining the routine cellular turnover in regenerative organs. Furthermore, the properties of these cells make them good candidates for targeted drug/gene delivery to specific organs; for example, mesenchymal stem cells (MSCs) have recently been shown to preferentially home to the lung. Adult stem and progenitor cells may be either circulating or resident in a particular tissue/organ system, including the lung and pulmonary vasculature. This chapter briefly describes the adult stem and progenitor cells currently identified in the pulmonary vasculature and introduces methodological approaches to successfully identify stem and progenitor cells.

Spanning the plasmalemmal membrane of all cells are many ion channels, exchangers, and transporters, all interacting to control vascular tone via the regulation of cytosolic Ca2+ concentration. Among these channels are voltage-gated K+, Na+, and Ca2+ channels, voltage-independent Ca2+ and nonselective cation channels, Cl− channels, and ligand-gated ion (K+, Ca2+, and Na+) channels. Plasmalemmal Ca2+–Mg2+ -ATPase (or Ca2+ pump) and Na+–K+ -ATPase (or Na+ pump) and an array of exchangers – the Na+–Ca2+ exchanger (NCX), Na+–H+ exchanger (NHE), and Na+–Mg2+ exchanger – all coexist to maintain ionic homeostasis in the cell. Comprehensive studies of ion channel function under both normal and pathophysiological conditions have been made possible owing to the development of cell isolation and electrophysiological techniques. This chapter introduces the ion channels and transporters present in the pulmonary vasculature (focusing on their expression and function) and discusses the potential pathogenic role of ion channels and transporters in the development of pulmonary hypertension.

The term “channelopathy” refers to a disease whose primary underlying mechanism(s) involve dysfunctional ion channel activity. Long QT syndrome and cystic fibrosis are two prime examples of channelopathies, where altered transport of K+, Na+, and/or Cl− ions, respectively, leads to pathogenesis. Such findings have aimed a new spotlight at the overall physiological relevance of transmembrane ion flux in disease development and became of critical importance to industry with discoveries that many drugs can unintentionally induce various channelopathies such as epileptic seizures (often from inhibition of neuronal human NaV1.1 Na+ channels), or torsade de pointes (a lethal long QT syndrome produced most often by inhibition of cardiac human ERG K+ channels). As discussed extensively elsewhere in this monograph, ion channels with selective permeability to Na+, K+, and Ca2+ ions modulate pulmonary vasoreactivity, remodeling, and vascular permeability. Although biochemical and molecular biology techniques have contributed, a significant proportion of our knowledge of the role of ion channels in pulmonary physiology has been garnered by measuring transmembrane ionic currents in intact cells using patch clamp electrophysiology. The purpose of this chapter is to introduce the basics of conventional and high-throughput patch clamp technology and how this can be used to advance the research into the cause of pulmonary vascular diseases.

The maintenance of low resistance, pressure, and tone in the pulmonary circulation is dependent on the interaction of circulating and locally produced vasomodulatory regulators; many such vasoactive mediators act via receptor-mediated signaling pathways. Many types of receptors that regulate pulmonary vascular tone are expressed on the plasma membrane of cells, although vasomotor activity is also influenced by intracellular receptors, such as calcium-release receptors and receptors that regulate transcription (e.g., steroid hormone receptors). The pulmonary vasculature expresses a wide variety of receptor classes and subtypes that facilitate the interaction of cells of the pulmonary circulation with their extracellular environment (hormones, neurotransmitters, and other factors in the extracellular milieu play key roles in modifying blood flow under both physiological and patho-physiological conditions). One can identify such receptors by assessing the binding of radioligands, molecular cloning and expression studies, antisense approaches, and/or by conducting studies with transgenic or knockout animals. Receptor-mediated signaling in the pulmonary circulation changes with development and disease, is highly species specific, cell-type specific, and often depends on an intact endothelium. Moreover, the accessibility of plasma membrane receptors for neurotransmitters and hormones from the extracellular environment makes them excellent drug targets. The major classes of membrane receptors that regulate pulmonary vascular tone are G-protein-coupled receptors, ligand-gated ion channels, and receptor protein kinases [receptor tyrosine kinase and serine/threonine kinase receptors]. This chapter provides an overview of signaling by cell-surface receptors in the pulmonary circulation and highlights mediators whose activation regulates pulmonary vascular development, tone, and permeability.

Disease phenotypes are typically a result of interactions between both genetic variation and environmental conditions. More than 25,000 genes that make up the human genetic blueprint have been decoded. This extensive gene database along with the advent of DNA microarray technology and bioinformatics enable researchers to generate gene expression profiles in any given cell or tissue of interest. It is therefore possible to identify altered gene expression that occurs in a particular disease condition. These molecular breakthroughs open up new avenues for accurate diagnoses and genetic counseling, as well as opportunities for gene or protein therapy. Manipulating the expression of specific genes into their respective final ­protein products is an important tool for understanding the function of a specific gene that may have potential as a gene therapy. Traditional gene manipulation has focused on introducing a target gene into the cells and tissues through recombinant DNA and gene transfer techniques to express the gene of interest. To downregulate or inhibit the expression of a gene, antisense DNA and RNA interference (RNAi) fragments, which block RNA processing or translation of specific messenger RNAs (mRNAs), are methods that provide powerful and specific tools for studying gene regulation. This chapter discusses some of the most widely used molecular biology tools for downregulating gene expression: antisense-oligonucleotidemediated gene silencing and RNAi-mediated gene silencing.

Primary pulmonary hypertension (PPH) is a progressive, fatal disease that is characterized by a sustained elevation of pulmonary arterial pressure and pulmonary vascular resistance from an unknown cause. The incidence of PPH is about 1–2 per million in the general population, and the mean life expectancy after diagnosis is 2.5 years. Although PPH can occur in individuals of all ages and both genders, it predominantly affects women (Rubin, 1997).