Cellular and molecular mechanisms of pulmonary vascular remodeling: role in the development of pulmonary hypertension

Microvascular Research 68 (2004) 75 – 103 www.elsevier.com/locate/ymvre

Review

Cellular and molecular mechanisms of pulmonary vascular remodeling: role in the development of pulmonary hypertension Mehran Mandegar a, Yuan-Cheng B. Fung b, Wei Huang b, Carmelle V. Remillard a, Lewis J. Rubin a, Jason X.-J. Yuan a,* b

a Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA Department of Bioengineering, Irwin & Joan Jacob School of Engineering, University of California, San Diego, La Jolla, CA 92093, USA

Received 30 March 2004 Available online 20 July 2004

Abstract Pulmonary artery vasoconstriction and vascular remodeling greatly contribute to a sustained elevation of pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) in patients with pulmonary arterial hypertension (PAH). The development of PAH involves a complex and heterogeneous constellation of multiple genetic, molecular, and humoral abnormalities, which interact in a complicated manner, presenting a final manifestation of vascular remodeling in which fibroblasts, smooth muscle and endothelial cells, and platelets all play a role. Vascular remodeling is characterized largely by medial hypertrophy due to enhanced vascular smooth muscle cell proliferation or attenuated apoptosis and to endothelial cell over-proliferation, which can result in lumen obliteration. In addition to other factors, cytoplasmic Ca2+ in particular seems to play a central role as it is involved in both the generation of force through its effects on the contractile machinery, and the initiation and propagation of cell proliferation via its effects on transcription factors, mitogens, and cell cycle components. This review focuses on the role played by cellular factors, circulating factors, and genetic molecular signaling factors that promote a proliferative, antiapoptotic, and vasoconstrictive physiological milieu leading to vascular remodeling. D 2004 Elsevier Inc. All rights reserved. Keywords: Familial and idiopathic pulmonary arterial hypertension; Pulmonary hemodynamics; Primary pulmonary hypertension; Pulmonary vascular morphology; Pulmonary vascular resistance

Contents 1. 2.

Introduction: hemodynamics of pulmonary hypertension and the concept of feedback between pulmonary arterial pressure and tissue remodeling . . . . . . . . . . . . . . . . . . . . Anatomical foundation for pulmonary hypertension analysis . . . . . . . . . . . . . . . . . . . . 2.1. Pulmonary arteries and veins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: AP-1, activating protein-1; Ang-1, angiopoietin-1; AVD, apoptotic volume decrease; [Ca2+]cyt, cytosolic free Ca2+ concentration; [Ca2+]SR, intracellularly stored free Ca2+ concentration in the sarcoplasmic reticulum; [K+]i, intracellular K+ concentration; BMP, bone morphogenetic protein; BMP-R2, bone morphogenetic protein receptor-II; CCE, capacitative Ca2+ entry; CREB, cAMP response element binding protein; Em, membrane potential; ET-1, endothelin-1; FPAH, familial pulmonary arterial hypertension; 5-HT, 5-hydroxytryptamine, serotonin; 5-HTT, serotonin transport protein; 5-HTR, serotonin receptor protein; HPV, hypoxic pulmonary vasoconstriction; IK(V), K+ currents through voltage-gated K+ channel; IPAH, idiopathic pulmonary arterial hypertension; NO, nitric oxide; NOS, nitric oxide synthase; PAEC, pulmonary artery endothelial cell; PAH, pulmonary arterial hypertension; PAP, pulmonary arterial pressure; PASMC, pulmonary artery smooth muscle cell; PDGF, platelet-derived growth factor; PPH, primary pulmonary hypertension; PVR, pulmonary vascular resistance; ROC, receptor-operated Ca2+ channel; ROS, reactive oxygen species; SERCA, Ca2+-Mg2+ ATPase in the sarcoplasmic reticulum; Smad, ‘‘mothers against decapentaplegic’’ protein; SOC, store-operated Ca2+ channel; SPH, secondary pulmonary hypertension; SR, sarcoplasmic reticulum; TGF-h, transforming growth factor-h; TRP, transient receptor potential channel; TRPC, canonical transient receptor potential channel; TXA2, thromboxane A2; VDCC, voltage-dependent Ca2+ channel; VEGF, vascular endothelial cell growth factor. * Corresponding author. Division of Pulmonary and Critical Care Medicine, Department of Medicine, MC 0725, University of California, San Diego, 9500 Gilman Drive, San Diego, CA 92103-0725. Fax: +1-858-822-6533. E-mail address: [email protected] (J.X.-J. Yuan). 0026-2862/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2004.06.001

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2.2. Pulmonary capillaries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Pulmonary veins and pulmonary bronchi . . . . . . . . . . . . . . . . . . . . . . . . . . 3. The physics of the pulmonary circulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Mechanical properties of the pulmonary blood vessels . . . . . . . . . . . . . . . . . . . 3.2. Zero-stress state . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Blood pressure – flow relationship in the lung . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Distribution of pressure drop and resistance in pulmonary blood vessels . . . . . . . . . . 4. Fundamental molecular and pathological derangements in idiopathic pulmonary artery hypertension (IPAH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Classification of pulmonary hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Pathophysiology of IPAH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Pulmonary arterial vasoconstriction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Elevated cytoplasmic Ca2+ triggers vasoconstriction . . . . . . . . . . . . . . . . . . . . 5.2. Ca2+ sensitization in acute HPV. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Membrane depolarization leads to elevation of [Ca2+]cyt and causes contraction . . . . . . 5.4. Inhibition of voltage-gated K+ channels causes membrane depolarization . . . . . . . . . 5.5. The role of receptor-operated and store-operated Ca2+ channels in elevated [Ca2+]cyt . . . 6. Pulmonary arterial wall remodeling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Proliferation and hypertrophy of the pulmonary artery smooth muscle cells . . . . . . . . 6.1.1. Elevated [Ca2+]cyt propels cells to go through the cell cycle. . . . . . . . . . . . 6.1.2. Elevated [Ca2+]cyt in pulmonary artery endothelial cells increases AP-1 binding activity and growth factor synthesis . . . . . . . . . . . . . . . . . . . . . . . . 6.1.3. Sufficient Ca2+ in the SR is required for smooth muscle cell proliferation . . . . 6.2. Inhibition of apoptosis in the pulmonary artery smooth muscle cells . . . . . . . . . . . . 6.2.1. Dysfunction of K+ channels leads to inhibition of apoptosis. . . . . . . . . . . . 6.2.2. The antiapoptotic protein Bcl-2 inhibits K+ channels and enhances cell survival . 6.3. Transdifferentiation of adventitial fibroblasts into smooth muscle cells . . . . . . . . . . . 6.3.1. Hypoxia-induced proliferation and transdifferentiation of fibroblasts into pulmonary artery smooth muscle cells . . . . . . . . . . . . . . . . . . . . . . . 6.3.2. Fibroblast heterogeneity in pulmonary arterial adventitia . . . . . . . . . . . . . 7. Cellular and molecular mechanisms in the development of pulmonary hypertension. . . . . . . . 7.1. Role of downregulated voltage-gated K+ (KV) channels . . . . . . . . . . . . . . . . . . 7.1.1. Decreased KV channel activity leads to pulmonary vasoconstriction and pulmonary artery smooth proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.2. Decreased KV channel activity leads to inhibition of apoptotic volume decrease and apoptosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Role of upregulated canonical transient receptor potential (TRPC) channels and enhanced capacitative Ca2+ entry (CCE) . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.1. TRPC channels are involved in forming store-operated Ca2+ channels responsible for CCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.2. CCE is enhanced and TRPC channels are upregulated in proliferating PASMC. . 7.3.2. TRPC channels are upregulated in pulmonary artery smooth muscle cells from patients with IPAH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Role of bone morphogenetic protein receptor type II (BMP-R2) gene mutations . . . . . . 7.4. The role of 5-HT receptors and transporters. . . . . . . . . . . . . . . . . . . . . . . . . 7.4.1. 5-HT is a vasoconstrictor and a mitogen. . . . . . . . . . . . . . . . . . . . . . 7.4.2. Upregulated 5-HTT and increased 5-HT are associated with pulmonary hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4.3. 5-HTT gene polymorphism in patients with pulmonary hypertension . . . . . . . 7.5. Elevated angiopoietin-1 activity contributes to pulmonary vascular remodeling . . . . . . 7.6. Endothelial dysfunction in pulmonary hypertension. . . . . . . . . . . . . . . . . . . . . 7.7. Role of endothelium-derived factors and growth factors in pulmonary hypertension . . . . 7.7.1. Nitric oxide (NO) and NO synthase in pulmonary hypertension. . . . . . . . . . 7.7.2. Endothelin-1 (ET-1) and endothelin converting enzyme in pulmonary hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7.3. Angiogenic and growth factors in pulmonary hypertension . . . . . . . . . . . . 7.7.4. Thromboxane in pulmonary hypertension . . . . . . . . . . . . . . . . . . . . . 8. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction: hemodynamics of pulmonary hypertension and the concept of feedback between pulmonary arterial pressure and tissue remodeling Human lungs constitute the only organ in the body that receives the entire cardiac output at all times. Such a tremendous capacity can be demanding and places the pulmonary circulation system in a position that is vulnerable to injury as a result of developmental or acquired disorders affecting the heart or lungs, as well as conditions that may also affect the systemic vasculature. The pulmonary circulation is normally a high-flow, low-resistance, low-pressure system that carries blood into the pulmonary microcirculation where the blood takes up oxygen (O2) and unloads excess carbon dioxide (CO2). The most serious and potentially devastating chronic disorder of the pulmonary circulation is pulmonary hypertension, a hemodynamic abnormality of diverse etiology and pathogenesis. Pulmonary hypertension is an often fatal hemodynamic abnormality that is common to a variety of conditions. Pulmonary artery pressure (PAP) varies with age; however, from early childhood to the fifth decade of life, its upper limit is approximately 20 mm Hg (mean PAP) (Yuan and Rubin, 2001). Beyond the fifth decade, PAP varies depending on different physiological conditions and disease states. Variation of PAP can be encountered in healthy individuals living in higher altitude, divers, mountain climbers, athletes, and during exercise and rehabilitation. PAP is a product of cardiac output (CO) and pulmonary vascular resistance (PVR) as demonstrated by Eq. (1), where PVR is the vascular resistance of the whole lung, including the pulmonary arteries, capillaries, and veins.   PAP ¼ CO  PVRarteries þ PVRcapillaries þ PVRveins

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hypertension results when this positive feedback becomes too strong. The molecular, cellular, and pharmaceutical processes underlying tissue remodeling and PAP elevation are reviewed in great detail in later sections of this article. Eq. (1) further demonstrates the physical laws that govern the circulation of blood in the lungs. To make use of this equation, we must know its physical and anatomical foundations. The laws of physics predict that anytime a liquid (e.g., blood) flows through a cylindrical tubular structure (e.g., a blood vessel), the resistance (i.e., PVR) is inversely proportional to the fourth power of the radius of lumen of the tube. This is best demonstrated by the Poiseuille Eq. (2), where L is the length of the tube (or vessel), r is its inner radius, and g is the coefficient of viscosity of blood. Therefore, even small changes in the radius of the vessels can significantly change the PVR. 8Lg 1  4 ð2Þ p r When a circulatory bed is simulated by a system of tubes, Eq. (1) is exactly analogous to the Kirochoff equation of an electric circuit. From such an analogy, we

PVR ¼

ð1Þ

From this equation one concludes that PAP can be raised by in increase in (a) CO, or (b) arterial, capillary, or venous PVR. Therefore, one would expect a tremendous rise in PAP during periods of increased CO, such as during a progressively heavy exercise. However, in a healthy individual, the PAP is increased only slightly due to a compensatory increase in the cross-sectional area of the pulmonary vascular bed (which decreases PVR), as well as to the recruitment of previously unperfused vessels. Arterial distension is dependent upon the compliance of the blood vessel walls. Loss of this compliance due to vascular remodeling leads to progressively pronounced pulmonary hypertension. As demonstrated by Eq. (1), an increase in PVR of any of the three components of the pulmonary vasculature can lead to elevated PAP. The relative importance of the three terms (i.e., PVRarteries, PVRcapillaries, and PVRveins) in the lung is about equal. But if PAP is higher than normal, certain genes will be activated, causing tissue remodeling in the pulmonary arteries, so that PVRarteries will be increased even further in a vicious cycle. Thus, there exists a positive feedback between PVRarteries and PAP. Chronic pulmonary

Fig. 1. Typical cast of a small segment of arterial tree in human lung (A) showing the structure of the vascular tree. The proposed three schemes describing this complex structure are illustrated by the ‘Weibel Model’ (B), the ‘Strahler model’(C), and the ‘Diameter-defined Strahler’s system’ (D). Generation numbers are indicated on each branch. Reproduced with permission from Weibel (1963), Singhal et al. (1973), and Huang et al. (1996).

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know that when several tubes with resistances R1, R2,. . .,Rn form a parallel system of vessels, one can calculate a single overall resistance for the system (Rp) according to the following formula: 1 1 1 1 ¼ þ þ ... þ Rp R1 R2 Rn

ð3Þ

On the other hand, if the same tubes are networked in series, then a single overall resistance (Rs) for the network can be calculated by: Rs ¼ R1 þ R2 þ . . . þ Rn

ð4Þ

These equations imply that the overall resistance of a parallel network will always be less than that of any individual element within the network, while the overall resistance of a serial network will be cumulative of each individual element. Therefore, the greater the number of parallel elements in the network or the larger the radius of each element, the lower the overall resistance of the network, while the greater the number of elements in series in a network, the greater the overall resistance. A human lung has over 300 million arteries, several billion capillaries, and over 300 million veins. How many of these are in ‘‘parallel’’? How many are in ‘‘series’’? The answer to this question must be based on anatomy. Four decades of research on this anatomical problem has yielded a satisfactory answer which is reviewed in the Anatomical foundation for pulmonary hypertension analysis section below.

tree. The Strahler system (Fig. 1C) avoids the symmetric dichotomy assumption of the Weibel model. That is, the smallest noncapillary blood vessel is defined as an order 1 vessel. When two vessels of the same order meet, the order number of the confluent vessel is increased by one, and so on and so forth. The Strahler model applied to the study of human and cat lungs (Singhal et al., 1973; Yen et al., 1983, 1984) resulted in data showing very large overlaps of the diameters in successive-order vessels. Based on this data, calculations of vascular resistance became inaccurate. The Diameter-Defined Strahler’s system (Fig. 1D) solves this problem (Jiang et al., 1994). In this system, a new rule is added: when a vessel of order n with diameter Dn meets another vessel of order £n, the confluent vessel is called a vessel of order n + 1 if and only if its diameter is larger than Dn + (Sn + Sn + 1) / 2, where Sn and Sn + 1 are the standard deviations of the diameters of orders n and n + 1. All three of these structure schemes have been applied to describe the pulmonary arterial and/or venous trees in humans (Huang et al., 1996; Singhal et al., 1973), cats (Yen et al., 1983, 1984), dogs (Gan et al., 1993), and rats (Jiang et al., 1994). In the end, the answer to the parallel/series question posed at the end of the Introduction is now clear. Vessels of successive orders are connected in series and vessels in the same order are connected in parallel. Based on the Diameter-Defined Strahler’s system, Huang et al. (1996) iden-

Anatomical foundation for pulmonary hypertension analysis Pulmonary arteries and veins Pulmonary morphometry and modeling, that is, the study of the complex system of arteries, veins, and capillaries of the lung, has allowed for the extraction and extrapolation of hemodynamic data that is important in assessing pulmonary vascular disease. Fig. 1A shows a typical cast of a small segment of an arterial tree in the human lung. Three schemes have been proposed to describe the complex structure of the pulmonary circulation: the Weibel model, the Strahler model, and the Diameter-Defined Strahler’s system. The Weibel model (Fig. 1B) assumes a symmetrical dichotomy (Weibel, 1963). The largest vessel is designated as a vessel of generation one. After each bifurcation, the generation number of the offsprings is increased by one; all offsprings at a bifurcation are assumed to be equal in diameter and length and the average of the offsprings is used for morphological data. In human subjects, Weibel and Gomez (1962) reported a total of 28 generations in the pulmonary arterial tree and 23 generations in the bronchial

Fig. 2. Diameter (open circles) and length (open squares) of each segment of pulmonary arteries, and distribution of total cross-sectional area (closed circles) of all segments of each order of pulmonary arteries in human lungs (A and B). The diameter and length of each of the individual pulmonary arterial branches decline exponentially from order 1 to 15. The number of pulmonary arterial branches (B, solid squares) increases exponentially from order 1 to 15. Reproduced with permission from Huang et al. (1996).

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tified 15 orders of pulmonary arteries between the main pulmonary artery and the capillaries, and 15 orders of pulmonary veins between the capillaries and the left atrium. As shown in Fig. 2, the diameter and length of individual pulmonary arterial branches decline exponentially from order 1 to 15, whereas the number of pulmonary arterial branches increases exponentially from order 1 to 15. The increase in the cross-sectional area of pulmonary arteries, however, appears to be not exponential (Fig. 2A, closed circles). Based on the data from human lungs, approximately 25.5% of the total cross-sectional area in the pulmonary vasculature stem from large vessels (diameter >0.6 mm), 44.4% from medium-sized vessels (0.2 – 0.6 mm), and 30.2% from small vessels (diameter <0.2 mm). Given the relationship between PVR and vessel length and radius, total PVR can be significantly altered by changes in diameters (or radii) of all-calibre pulmonary arteries. Pulmonary capillaries The capillary blood vessels of the human lung are organized neither in series, nor in parallel, but in a network of sheet flow (Fig. 3) (Fung and Sobin, 1969,

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1972a,b).The system can be described as a sheet of blood flowing between two membranes which are joined together by ‘‘posts’’ of about 3 Am in diameter and a few micrometers long. Each capillary membrane has an outer side consisting of epithelial cells facing the alveolar space and an inner side consisting of the endothelial cells facing the blood. The size of the sheet can be described by thickness and by a solidity ratio, which is the ratio of the area of the blood space in plane view divided by the total area of the sheet. For capillary beds, sheet-flow resistance replaces PVR in the Poiseuille formula (Fung and Sobin, 1969). When connected, the capillary sheets form the alveoli, which make up the bulk of the lung. Based on microscopic studies and mathematical modeling, each alveolus, although similar is size and shape, is a nonregular 14-sided tetrakaidecahedron formed by cutting the six corners off a regular octahedron (Fig. 3C) (Fung and Sobin, 1969). This geometry allows for maximal space filling, thereby ensuring the maximal opportunity for effective gas exchange. Pulmonary veins and pulmonary bronchi The bifurcational systems of the pulmonary veins and pulmonary bronchi are similar to that of the pulmonary arteries. Because the venous pressure is lower, its tissue remodeling as a feedback from venous pressure increase has not be studied. Data on the morphometry of the airway are available based only on the Weibel (1963, 1991) model. Human pulmonary arteries always travel along the airways in ‘‘bronchovascular bundles’’ with similar irregular dichotomous branching model. The close relationship of the pulmonary arterial vessel and the airways start as early as 34 days into gestation (deMello et al., 1997). During early fetal development, it is believed that the airways act as a template for pulmonary blood vessel development, that is, the vessels form by vasculogenesis around the branching airways. Indeed, pulmonary arteries are invested with smooth muscle cells derived from the smooth muscle of adjacent bronchii immediately after their coalescence (Hall et al., 2000). These smooth muscle cells appear to migrate from the bronchii and to line up around the arteries, making up the innermost layers of the mature vessels. It is this layer of smooth muscle cells which can migrate into the intima and lead to the development of IPAH.

The physics of the pulmonary circulation

Fig. 3. A plan view of an interalveolar septum of cat lung (A) and a cross-sectional view of three interalveolar septa (B). A 14-sided tetrakaidecahedron model (C) of an alveolar duct formed by cutting the six corners off a regular octahedron. Reproduced with permission from Fung and Sobin, 1969.

The laws of physics relate pulmonary hypertension to the changes in morphology and composition of the system, the mechanical properties of the tissues, the pumping of the heart, and the boundary conditions. Theoretical biomechanics has advanced to a degree such that a precise and detailed prediction can be made of the blood pressure and flow

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anywhere in the lung when the morphological, constitutive, and boundary conditions are known. It is a useful tool for the investigation of the molecular mechanisms that underlie tissue remodeling. Mechanical properties of the pulmonary blood vessels Vascular remodeling induced by oxygen deficiency, changes in external load, or disease (diabetes, smoking) alters the stress – strain relationship within the vascular wall (Abe` et al., 1996; Liu and Fung, 1992a,b, 1993a,b). The mechanical properties of blood vessels are expressed by constitutive equations (Fung, 1993). Using these constitutive equations, only the elastic constants which change in the process of tissue remodeling remain undetermined. Huang et al. (2001) described the course of change of the mechanical (i.e., elastic) properties of pulmonary arteries from hypoxia-induced hypertensive rats. Over a 24-h hypoxic period, the changes in the incremental Young’s moduli in circumferential ( Yqq between 164 and 187 kN/ m2) and axial ( Yzz between 64 and 92 kN/m2) directions, and the Cross Young’s modulus ( Yqz, between 61 and 88 kN/m2) were statistically insignificant in 11th order main and third side-branch pulmonary arteries. Zero-stress state Fung (1983) and Vaishnav and Vossoughi (1983) independently showed that if a blood vessel is cut open or all loads are removed, the circular shape opens up and the wall becomes a sector; this is the blood vessel in its zero-stress state (Fig. 4). This illustrates that, compared to the in vivo or no-load (no transmural pressure or longitudinal stress) states, there are residual stresses and strains in blood vessels. The stress – strain relationship is very complex if residual strains exist; it is simplified in the zero-stress state. The opening angles at the zero-stress states varied in third- to ninth-order human pulmonary arteries (92 – 163j) and veins (89 – 128j) (Huang et al., 1998). The opening angle at the vascular zero-stress state changes during tissue remodeling. For example, the mean values of opening angle

Fig. 4. Definition of opening angle. (A) At no-load state, the internal pressure, external pressure, and longitudinal stress in a short ring-shaped segment are all zero. (B) Sector represents circumferential cross section of a blood vessel at zero-stress state. Opening angle is an angle subtended between two lines originating from midpoint to tips of inner wall (endothelium).

at the pulmonary trunk at times 0 (control), 2, 12, 48, 96, 144, 240, 480, and 720 h after exposure to hypoxia were, respectively, 294j, 378j, 385j, 374j, 246j, 267j, 193j, 195j, and 239j (Fung and Liu, 1991). Trends at other places on the arterial tree were similar, but magnitudes differed (Fung and Liu, 1991; Huang et al., 2001). Blood pressure– flow relationship in the lung The pulmonary pulsatile pressure – flow relation can be expressed in terms of vascular impedance, which is the ratio of the amplitude of oscillatory arterial pressure to the oscillatory inflow rate at a given frequency. Pulmonary vascular input impedance has been measured in many species (Engelberg and Dubois, 1959; Fung and Sobin, 1969; Huang et al., 1998; Zhuang et al., 1983). The question of how tissue remodeling affects the pressure – flow relationship has not yet been thoroughly worked out and will require more investigation. Distribution of pressure drop and resistance in pulmonary blood vessels The distribution of pressure drop is related to the resistance to blood flow, and the transport of nutrition in blood flow to organs and tissues. As stated earlier, the pulmonary circulation system is a low-pressure system compared to the systemic circulation. In the latter, the biggest pressure drop happens in the thick-walled and muscular arterioles. In the lungs, the pressure drop is evenly distributed in pulmonary arteries, capillary sheet, and pulmonary veins (Fig. 5). The pressure drop from the largest pulmonary artery to the smallest pulmonary arterioles generally is comparable to that in the capillaries (order 0) and veins in a normal lung. In disease state models (such as pulmonary arterial or venous hypertension), the changes in pressure drop and resistance distributions cannot be determined because insufficient data exist on the branching pattern, morphometry, morphology, and the elasticity of pulmonary blood vessels of the diseased human lungs as compared to normal lungs. Future studies need to focus on obtaining such critical data. Furthermore, while the pressure drop in pulmonary arteries and veins is similar, in some cases, the pressure drop in pulmonary veins may be larger than that in pulmonary arteries. Assuming that (a) the nature of the vascular smooth muscle cells, (b) their contractile machinery, (c) their chemistry, (d) the role of Ca2+ ions, and (e) the expressed and functional ion channels are similar, the pulmonary veins should be equally effective as the arteries in producing hypoxia-induced pulmonary arterial hypertension (PAH). Indeed, pulmonary veins exert a remote control which has a powerful effect on the arteries: the veins can reduce the resistance of the pulmonary capillaries to blood flow or, alternatively, contribute to an increase in the pressure in the pulmonary arteries. In the future, as much attention should be paid to

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in any kind of pulmonary hypertension, either due to obstructive or obliterative process, determines its effect on PVR. This is because the cross-sectional area of the pulmonary vascular system enlarges progressively from central pulmonary arteries to the capillaries. Therefore, the resistance to blood flow in the main pulmonary arteries is much greater than that of the pulmonary arterioles and capillary bed as a whole. This implies that the destruction or obliteration of millions of arterioles and capillaries would be required to equal the effect of occluding one lobar artery. That explains why the effect of an acute massive pulmonary embolus is so easily palpable in clinical settings. Obliterative pulmonary arterial hypertension, however, is a more chronic and progressive disease that mainly involves medium-sized and resistance arteries and arterioles (40 – 300 Am in lumen diameter). The predominant features of obliterative pulmonary arterial hypertension include pulmonary vascular remodeling, vasoconstriction, and in situ thrombosis (Rubin, 1997). Regardless of the cause, elevated PVR and the resulting pulmonary artery hypertension has a devastating effect on the heart. Pulmonary hypertension often puts excessive burden on the right ventricle due to the increased work load necessary to overcome the downstream pressure. Over time, this leads to right-sided heart failure, which is often the cause of demise in patients with pulmonary hypertension. Classification of pulmonary hypertension

Fig. 5. The longitudinal pressure distribution (A) in pulmonary blood vessels of the cat for the case in which the pressure in pulmonary arterial trunk, pa, is 20 cmH2O, the pressure in left atrium, pv, is 2 cmH2O, the alveolar gas pressure, pA, is zero, and the pleural pressure, pPL, is  7 cmH2O. Each tick mark on the horizontal axis represents the location of the exit end of each vessel in a given order (arteries from order 1 to order 11, veins from order  1 to order  11). The numerals 1 and 2 refer to symmetric and nonsymmetric branching patterns, respectively. (B) Distribution of the pressure drops in arteries, capillaries, and veins of the pulmonary vasculature (upper panel) and systemic (mesenteric and skeletal muscle) vasculature (lower panel). Reproduced with permission from Brody et al. (1968), Fronek and Zweifach (1974), Fung (1996), and Hakim et al. (1982).

the pulmonary venous smooth muscle as to the pulmonary arterial smooth muscle.

Fundamental molecular and pathological derangements in idiopathic pulmonary artery hypertension (IPAH) Pulmonary hypertension can occur in a variety of disease conditions including diseases in which pulmonary arteriopathy can be the primary disease (e.g., idiopathic pulmonary arterial hypertension) or diseases that can be present with pulmonary hypertension as a sequelae of other cardiopulmonary diseases, the so-called ‘‘secondary pulmonary hypertension (SPH).’’ The location of the vascular abnormality

The term ‘‘primary pulmonary hypertension’’ (PPH) was initially introduced 50 years ago to characterize a condition in which hypertensive vasculopathy existed exclusively in the pulmonary circulation without a demonstrable cause. Much research has been undertaken in the past five decades that has advanced our knowledge of this disease tremendously. The recent advances have been quite remarkable, ranging from the identification of a gene responsible for inherited forms of the disease and the application of molecular biologic techniques to explore its pathogenesis, to the development and commercialization of medical therapies and the refinement of surgical techniques for lung transplantation and pulmonary thromboendarterectomy. Recognizing these advancements and interest resulted in several international meetings of experts; two of them during the past 6 years. In the first of these recent meetings, held in Evian, France, in 1998, pulmonary hypertension was grossly classified into two broad categories: (a) the conditions that directly affect the pulmonary arterial tree, termed pulmonary arterial hypertension (PAH), and (b) the disorders that either predominantly affect the venous circulation or conditions that affect the pulmonary circulation by altering respiratory structure or function. Thus, PPH remained the term of choice to define familial or sporadic disease of undetermined cause. With further advancements in the elucidation of the pathogenesis and clinical pathophysiology of pulmo-

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nary hypertension, the Evian Classification needed a revision to account for the newfound knowledge. The most recent revision of this classification was, therefore, proposed at the 3rd World Conference on Pulmonary Hypertension in 2003 (Table 1). In this classification, PPH has been replaced with idiopathic PAH (IPAH) or, when supported by genetic basis, familial PAH (FPAH). This classification allows categorization by common clinical features and recognizes the similarity between IPAH and pulmonary hypertension of certain known etiologies. It should be noted that this classification is mostly a clinical classification and it does not accommodate the cellular and molecular perspectives and histological differences of the disease. From a physiologic point of view, pulmonary hypertension is divided into two broad categories: obliterative pulmonary hypertension and secondary pulmonary hypertension. Obliterative pulmonary arterial hypertension is an intrinsic disease of the pulmonary vascular smooth muscle and endothelial cells due to abnormalities at cellular and molecular levels (Rubin, 1997). These include mutations of membrane receptors, dysfunctional ion channels, abnormal Table 1 Revised nomenclature and classification of pulmonary hypertension (2003) Pulmonary arterial hypertension (PAH) Sporadic or idiopathic PAH (IPAH) Familial PAH (FPAH) PAH related to Collagen vascular disease Congenital systemic to pulmonary shunts (large, small, repaired or nonrepaired) Portal hypertension HIV infection Drugs and toxins Other (glycogen storage disease, Gaucher’s disease, hereditary hemorrhagic telangiectasia, hemoglobinopathies, myeloproliferative disorders, splenectomy) PAH associated with significant venous or capillary involvement Pulmonary venoocclusive disease Pulmonary capillary hemangiomatosis Pulmonary venous hypertension Left-sided atrial or ventricular heart disease Left-sided valvular heart disease Pulmonary hypertension associated with hypoxemia Chronic obstructive pulmonary disease Interstitial lung disease Sleep disordered breathing Alveolar hypoventilation disorders Chronic exposure to high altitude Pulmonary hypertension due to chronic thrombotic and/or embolic disease Thromboembolic obstruction of proximal pulmonary arteries Thromboembolic obstruction of distal pulmonary arteries Pulmonary embolism (tumor, parasites, foreign material) Miscellaneous Sarcoidosis, histiocytosis X, lymphangiomatosis, compression of pulmonary vessels (adenopathy, tumor, fibrosing mediastinitis)

membrane transporters, and effect of hormonal and agonist agents among other things which will be discussed in great detail later in this review. It is important to understand that the obliterative process impedes blood flow through small pulmonary vessels, leading to chronic and sustained pulmonary hypertension. Such processes can be seen in both familial and idiopathic (sporadic) PAH, pulmonary capillary hemangiomatosis, as well as in disorders that produce obliterative pulmonary artery disease as a complication of systemic processes such as in collagen vascular diseases (i.e., scleroderma, systemic lupus erythematosus, rheumatoid arthritis), HIV infection, drug/toxin ingestion (i.e., anorexigen use), persistent pulmonary arterial hypertension of the newborn, or hepatic failure. Secondary pulmonary hypertension (SPH), on the other hand, is a condition that develops because a physiological abnormality that initially arises independently and in the absence of any intrinsic pulmonary vascular disease. It can develop through several processes: (i) mechanical obstruction or paucity of the large pulmonary arteries (pulmonary thromboembolism, distal pulmonary arterial stenosis), (ii) parenchymal lung disease causing destruction of the pulmonary vascular bed and microcirculation (severe emphysema, interstitial pulmonary fibrosis, cystic fibrosis), (iii) chronic alveolar hypoxia-induced vasoconstriction (severe sleep apnea, chronic exposure to hypoxia in high altitude), (iv) hyperkinetic pulmonary hypertension due to excessive and chronic increase in the blood flow through the pulmonary vasculature [atrial or ventricular septal defect, congenital systemic-to-pulmonary shunts (e.g., Eisenmenger syndrome, portopulmonary hypertension)], (v) passive pulmonary hypertension due to impedance to the pulmonary venous drainage (mitral stenosis, pulmonary venoocclusive disease, chronic left ventricular dysfunction), and (vi) a combination of any two or more of the above mechanisms (destructive and hypoxic vasoconstrictive processes in chronic obstructive pulmonary disease). Although the pathophysiology of pulmonary hypertension can be distinctly classified into the obliterative and secondary groups, the histologic distinction between the groups is somewhat blurred, and it is often virtually impossible to distinguish the origin of the disease solely on the basis of histologic findings. For example, chronic hypoxia-induced pulmonary hypertension can lead to vascular wall remodeling whose characteristics are virtually indistinguishable from those of IPAH. The predominant features of obliterative pulmonary arterial hypertension include pulmonary vascular remodeling, vasoconstriction, and in situ thrombosis. Pathophysiology of IPAH Studies relating to the development of IPAH have revealed much about the molecular determinants of pulmonary vascular proliferation and tone. In IPAH, histologic findings are generally characterized by obliteration of the lumen of small- and medium-sized pulmonary arteries in

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association with medial hypertrophy, concentric laminar intimal fibrosis, fibrinoid degeneration, and formation of plexiform lesions and in situ thrombosis, both as pleiotropic manifestation of one disease. Evidence suggests that medial hypertrophy, which is the most consistent pathological finding in IPAH, is mainly due to the presence of intrinsic abnormalities in pulmonary artery smooth muscle cells (PASMC) function (Rubin, 1997). Cross-sectional vasoconstriction as a result of elevated arterial pressure leads to elastic stretch of the smooth muscle cells. Both elastic stretch of PASMC and elevated PAP have been shown to play a role in promoting pulmonary arterial cellular growth and synthetic activity (Hishikawa et al., 1994). Vasoconstriction and cellular proliferation may both involve signaling processes that result in parallel intracellular events involved in vascular remodeling and in the development of pulmonary hypertension. Furthermore, the clinical spectrum of severe pulmonary hypertension includes primary forms and secondary forms of the disease. Secondary forms of the disease can occur in association with and/or as a direct consequence of other ailments such as pulmonary embolism, congenital cardiac abnormalities, sarcoidosis, collagen vascular disorders, and infection with human immunodeficiency virus type 1 (HIV-1), all of which share the histologic features manifested by complex lumenoccluding vascular lesions (plexiform lesions) and in situ thrombosis (Tuder et al., 2001). The plexiform lesions, which occur in very small arteries and arterioles, are tufts of cellular capillary formations resembling a vascular plexus present within the lumen of dilated aneurysmal thin-walled arteries in about one in three lung biopsy specimen. Plexiform lesions also occur in patients with pulmonary hypertension secondary to other disorders; thus, it is not

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pathognomonic for IPAH (Cool et al., 1997; Smith et al., 1990). However, the origin of plexiform lesions appear to be somewhat different in these two disorders, although this has been the subject of debate. Some investigators believe smooth muscle proliferation and their transformation into myofibroblasts are responsible for the formation of these lesions (Cool et al., 1997; Smith et al., 1990; Yi et al., 2000). Others propose that, in IPAH, endothelial cells initiate the lesions in response to cytokines, growth factors, or vascular injury (Cool et al., 1997; Tuder et al., 1994; Yi et al., 2000). This theory is supported by the fact that the endothelial cells of the plexiform lesions isolated from the lungs of patients with IPAH proliferate in a monoclonal fashion, while the lesions from patients with secondary pulmonary hypertension arise from polyclonal cell populations (Lee et al., 1998a). Somatic mutations of the bone morphogenetic protein (BMP) receptor type II (BMP-R2) and BAX genes have been demonstrated in patients with IPAH, which may provide a growth advantage for these cells (Yeager et al., 1999). However, BMP-R2 mutations are present only in 40 – 50% of patients with familial PAH (FPAH) and not all plexiform lesions have somatic mutations (Deng et al., 2000; Yeager et al., 1999). Therefore, other molecular factors are likely involved in the acquisition of the selective growth advantage of endothelial cells in patients with severe pulmonary hypertension. More recently, Cool et al. (2003) have demonstrated the involvement of human herpes virus 8 (HHV-8) infection in lung tissues from 10 of 16 patients with IPAH. In this study, cells within the plexiform lesions as well as bronchoepithelial cells, inflammatory cells, and endothelial cells lining patent lung vessels tested positive for HHV-8 latency-associated nuclear antigen-1 (LANA-1) on immunohistochemi-

Fig. 6. Schematic illustration of pathophysiological components contributing to the development of sustained elevation of pulmonary vascular resistance (PVR) and artery pressure (PAP). Em, membrane potential; SMC, pulmonary artery smooth muscle cell; EC, pulmonary arterial endothelial cells.

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cal analysis, while cells from patients with secondary pulmonary hypertension lacked LANA-1. Smooth muscle cells were consistently negative for LANA-1. Additionally, the plexiform lesions from patients with IPAH had a histologic and immunohistochemical resemblance to cutaneous Kaposi sarcoma lesions, further supporting the possible involvement of HHV-8, since HHV-8 is a vasculotropic virus that is thought to be the cause of all clinical types of Kaposi sarcoma (Weiss et al., 1998). However, only one of three patients with HIV-1-associated severe pulmonary hypertension were positive of LANA-1 (HHV-8 infection), suggesting that HIV-1-associated pulmonary hypertension can occur independently of HHV8 infection (Cool et al., 2003). The third major characteristic pathophysiologic abnormality in obliterative pulmonary hypertension is in situ thrombosis. Endothelial cell dysfunction and its interaction with growth factors and platelets is believed to be in part responsible for this pathologic finding, by creating a procoagulant environment within the pulmonary vascular bed. Enhanced pro-coagulant activity due to elevated levels of plasma fibrinopeptide-A and prolonged half-life of fibrinogen has been demonstrated in patients with IPAH (Eisenberg et al., 1990). Furthermore, in as many as 70% of patients with IPAH, diminished fibrinolytic activity and the presence of elevated levels of plasminogen activator inhibitor have been reported (Welsh et al., 1996). Elevated levels of urinary metabolites of thromboxane, an indicator of platelet activation, are commonly detected in patients with IPAH as well as those with secondary pulmonary hypertension (Christman et al., 1992). The release of vasoconstrictors such as serotonin (5-hydroxytryptamine, or 5-HT) and thromboxane-A2 (TXA2), and stimulation of cell proliferation by platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) are associated with platelet activation. Furthermore, abnormal functioning of a von Willebrand factor has also been reported in patients with IPAH (Geggel et al., 1987). In patients with pulmonary hypertension secondary to congenital heart disease, the pulmonary vascular endothelium shows a significant increase in von Willebrand antigen immunostaining, implying a flow-induced change in the functional activity of these cells (Lopes and Maeda, 1998). It is therefore possible that elevated von Willebrand factor stimulates aggregation and adhesion of platelets to pulmonary vessel walls that are already damaged and stressed by pulmonary hypertension and/or intrinsic endothelial cell abnormalities. Aggregation and activation of platelets may lead to thrombus formation and may play a significant role in stimulating vasoconstriction and cellular proliferation by releasing vasoactive substances and mitogens. Defects in fibrinolysis pathways, on the other hand, may further encourage this process and exacerbate its obliterative results. While it is clear that platelets, fibroblasts, endothelial cells, and in situ thrombosis are all involved in pulmonary vascular remodeling, most experts now agree that PASMC prolifera-

tion and, to some extent, vasoconstriction lead to the vascular remodeling processes that underlie severe pulmonary hypertension. Fig. 6 illustrates the fundamental pathological players in the development of IPAH. This review focuses on molecular determinants that unite these two fundamental derangements (pulmonary vasoconstriction and vascular remodeling) and their role in the development and maintenance of severe pulmonary hypertension.

Pulmonary arterial vasoconstriction Pulmonary vasoconstriction in general can be interpreted in one of two forms: (a) longitudinal constriction (vessel shortening) or (b) cross-sectional constriction (vessel narrowing). In vivo, the length of blood vessels within the lungs is generally considered somewhat fixed, while their diameters constantly change to accommodate the everchanging physiological parameters and requirements. For the purposes of this article, vasoconstriction refers to an increase in tensile force which translates to the narrowing of the lumen of the vessel. Pulmonary vasoconstriction is a main contributing factor to PVR and, hence, elevated PAP. Also, as stated earlier, elevated PAP and the ensuing vasoconstriction can influence PASMC hypertrophy and hyperplasia (Hishikawa et al., 1994). In IPAH patients, acute application of vasodilators, such as inhalation of nitric oxide (NO) and infusion of prostacyclin (PGI2), adenosine or Ca2+ channel blockers (e.g., nifedipine and verapamil), reduces PVR and mean PAP (by 15– 20%) in 20– 25% patients (Tigno, Rubin, and Yuan, unpublished data). These observations indicate that sustained pulmonary vasoconstriction is an important contributor to the elevated PVR and PAP in some IPAH patients. Hypoxic pulmonary vasoconstriction (HPV), an adaptive mechanism unique to the lungs, is believed to be a major culprit in pulmonary hypertension secondary to hypoxic cardiopulmonary diseases [e.g., Eisenmenger’s syndrome, severe emphysema or chronic obstructive pulmonary disease (COPD)]. Hypoxia has been shown to induce vasoconstriction in isolated pulmonary arteries without endothelium (Yuan et al., 1990) and to cause contraction in isolated single PASMC (Murray et al., 1990), indicating that HPV is an intrinsic property of PASMC. HPV is important in that it redirects the blood flow away from the poorly ventilated area of the lungs and into the better-ventilated area to improve ventilation –perfusion matching and to maximize oxygenation of the pulmonary venous blood. Disease conditions such as COPD, obstructive sleep apnea, as well as living at high altitude cause chronic and sustained exposure to hypoxia. Sustained alveolar hypoxia results in sustained HPV, leading to vascular remodeling, pulmonary hypertension, right heart failure (cor pulmonale) and death. Although the precise mechanism by which hypoxia causes pulmonary vasoconstriction is still unclear, studies point to several possible pathways that may be responsible

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for these effects. Although somewhat different and complex in nature, the signaling pathways in response to acute or chronic hypoxia seem to relate, at least in part, to disturbed intracellular Ca2+ homeostasis. Elevated cytoplasmic Ca2+ triggers vasoconstriction Smooth muscle contraction is directly triggered by a rise in cytosolic free Ca2+ concentration ([Ca2+]cyt). 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 smooth muscle cell contraction, prompting vasoconstriction (Fig. 7) (Somlyo and Somlyo, 1994). Ca2+ sensitization in acute HPV The extent of pulmonary vasoconstriction is dependent on the level of [Ca2+]cyt and the sensitivity of the contractile apparatus to [Ca2+]cyt. As described in the previous section, actin – myosin crossbridging cycles require the phosphorylation of myosin light chain (MLC20) by MLCK, which itself is stimulated when [Ca2+]cyt increases. Vascular tone is determined by the phosphorylation/dephosphorylation ratio of MLC20, and therefore is dependent on the relative activities of MLCK and myosin phosphatase (Somlyo and Somlyo, 1994). However, the extent of the force generated by agonists is greater than that produced by depolarization (Morgan and Morgan, 1984). Therefore, there is an apparent increase in the sensitivity of the contractile apparatus to [Ca2+]cyt (i.e., Ca2+ sensitization) under such circumstances. Acute hypoxia results in a biphasic contraction, which coincides with a biphasic increase in [Ca2+]cyt (Robertson et al., 1995). Immediately after the initiation of hypoxia, both tension and [Ca2+]cyt simultaneously, yet transiently, increase. These transient changes are followed by a second phase where contraction is not only sustained, but it actually increases with time until hypoxia is ceased. [Ca2+]cyt, on the other hand, remains constantly elevated (relative to its baseline pre-hypoxia). Upon re-oxygenation, both tension and [Ca2+]cyt return to their pre-hypoxic levels. The dissociation between tension and [Ca2+]cyt during the sustained phase of HPV is believed to be due to sensitization of contractile apparatus to Ca2+. Many signaling pathways have been proposed to mediate Ca2+ sensitization during HPV, most of them dealing with increased kinase activity (Robertson and McMurtry, 2004). More particularly, protein kinase C, protein tyrosine kinases, Rho-kinase, p38-mitogen-activated (MAP) kinase have all been impli-

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cated. Because ATP is ultimately required for the phosphorylative action of intracellular kinases, it is not altogether surprising to find that glycolysis, a main generator of ATP, may also regulate Ca2+ sensitization during HPV. Finally, studies by Robertson et al. (1994, 2001) and Dipp et al. (2001) have suggested that an endotheliumderived constricting factor (EDCF) released during hypoxia (not ET-1!) preferentially causes vasoconstriction in the pulmonary vascular bed, but not in mesenteric vessels. Dipp et al. (2001) have shown that this elusive EDCF may cause Ca2+ sensitization via activation of Rho-kinase. In light of its role in HPV and as a key regulator of MLC phosphatase and contraction, Rho-kinase has gained much interest from those studying the physiological and molecular mechanisms underlying HPV. Membrane depolarization leads to elevation of [Ca2+]cyt and causes contraction Regulation of [Ca2+]cyt in PASMC is achieved mainly in two different ways: (a) trans-sarcolemmal influx of Ca2+ through Ca2+ channels or extrusion of Ca2+ through the plasma membrane Ca2+-Mg2+ATPase pump, and (b) the mobilization of Ca2+ from the sarcoplasmic reticulum (SR) through Ca2+ release channels or sequestration of Ca2+ into the SR by its Ca2+-Mg2+ ATPase pumps (SERCA) (Berridge, 1993; Blaustein, 1993). Accordingly, [Ca2+]cyt in PASMC can be increased by Ca2+ release from the intracellular stores (mainly, the SR) and Ca2+ influx through plasmalemmal Ca2+ channels (Fig. 7). Although mitochondria, lysosomes, and Golgi bodies can store Ca2+, the SR is the dominant intracellular store. Based on the sensitivity to inositol-1,4,5-trisphosphate (IP3) and ryanodine and/or distribution of their respective receptors, the vascular SR can be classified into two Ca2+ pools: (a) caffeine- and ryanodine-sensitive stores involved in Ca2+induced Ca2+ release, and (b) IP3-releasable stores that are sensitive to cyclopiazonic acid and thapsigargin (Berridge, 1993; Blaustein, 1993). SERCA is mainly responsible for the sequestration of cytosolic Ca2+ into the SR to keep a low [Ca2+]cyt and a high [Ca2+ ] in the SR ([Ca2+]SR) (Blaustein, 1993). Inhibition of SERCA by cyclopiazonic acid or thapsigargin prevents the reuptake of Ca2+ from the cytosol into the SR, thereby depleting the SR through Ca2+ leakage. In vascular smooth muscle cells (including PASMC), both IP3and ryanodine-sensitive SR stores have been identified and characterized (Blaustein and Lederer, 1999). The current data favor the idea that there are at least three types of SR stores in vascular smooth muscle cells based on the distribution and expression of IP3 and ryanodine receptors: the SR with only IP3 or ryanodine receptors and the SR with both IP3 and ryanodine receptors (Blaustein and Lederer, 1999). Ca2+ influx through the plasma membrane involves multiple Ca2+-permeable channels including: (a) voltagedependent Ca2+ channels (VDCC) regulated by changes in membrane potential (Em), (b) receptor-operated Ca2+ chan-

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Fig. 7. Proposed mechanism by which an increase in [Ca2+]cyt triggers pulmonary vasoconstriction and promotes pulmonary vasculature remodeling. Decreased activity of voltage-gated K+ (KV) channels and membrane depolarization can lead to a rise in [Ca2+]cyt by opening voltage-dependent Ca2+ channels (VDCC). In addition, activation of receptors (R), such as G protein-coupled receptors and receptor tyrosine kinases, leads to production of diacylglycerol (DAG) and inositol 1,4,5,-trisphosphate (IP3) and increases [Ca2+]cyt by opening receptor-operated Ca2+ channels (ROC) and inducing Ca2+ mobilization from the sarcoplasmic reticulum (SR). IP3 also opens store-operated Ca2+ channels (SOC) directly or indirectly by store depletion to further increase [Ca2+]cyt. Calmodulin (CaM) binds to Ca2+ as [Ca2+]cyt rises. The Ca2+/CaM complex binds to and activates myosin light chain kinase (MLCK). This leads to phosphorylation of the myosin light chain (MLC). MLC stimulates the activity of myosin ATPase, hydrolyzing ATP to generate energy for the cycling of the myosin crossbridges with the actin filament. The formation of these crossbridges underlies smooth muscle cell contraction, prompting vasoconstriction. Furthermore, elevated [Ca2+]cyt is responsible for propelling the quiescent cells into mitosis and cellular proliferation. The Ca2+/CaM complex activates at least four steps in the cell cycle (indicated by ‘‘+’’). Vasoconstriction and vascular remodeling (e.g., medial hypertrophy due to SMC proliferation) lead to chronic and sustained elevation of the pulmonary vascular resistance (PVR) and arterial pressure (PAP).

nels (ROC) activated by interaction of agonists with membrane receptors, and (c) store-operated Ca2+ channels (SOC) activated by depletion of Ca2+ from intracellular stores (Fig. 7) (Nelson et al., 1990; Parekh and Penner, 1997). The excitation –contraction coupling processes in pulmonary vascular smooth muscle is dependent on the function of all these channels. A change in Em is required for the electromechanical coupling that alters vascular tone by regulating the activity of VDCC, which are opened by

membrane depolarization and closed by membrane hyperpolarization (Nelson et al., 1990). In addition to elevating [Ca2+]cyt by opening VDCC, membrane depolarization also facilitates the production of IP3, which stimulates the release of SR Ca2+ into the cytoplasm (Ganitkevich and Isenberg, 1993), and promotes Ca2+ entry via reverse mode Na+/Ca2+ exchange (Blaustein and Lederer, 1999). Sustained elevation of [Ca2+]cyt induces a chronic state of vasoconstriction as described earlier and it contributes to smooth muscle cell

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hypertrophy and vascular remodeling, the hallmarks of IPAH, as will be discussed shortly. Inhibition of voltage-gated K+ channels causes membrane depolarization As we have just discussed, the Em in vascular smooth muscle cells is an important regulator of [Ca2+]cyt, hence of vascular tone. Resting Em in vascular smooth muscle cells normally ranges between 70 and 50 mV (Nelson and Quayle, 1995). At rest, the PASMC membrane is more permeable to K+ than to any other ion, therefore the Em is close to the equilibrium potential for K+ (EK); currents through K+ channels thus play a predominant role in modulation of the Em. Currents [IK(V)] through voltagegated K+ (KV) channels, in particular, have been demonstrated to be predominately responsible for the maintenance of the relatively negative Em in PASMC under resting conditions (Nelson and Quayle, 1995). According to Eq. (5), whole-cell IK(V) at any given time is determined by total number of functional KV channels expressed in the plasma membrane (N), the amount of current flowing through a single KV channel (i), and the steady-state open probability of a KV channel ( Popen). IKðVÞ ¼ N  i  Popen

ð5Þ

Em becomes more depolarized as a result of decreased IK(V) due to either a decrease in i, Popen, or N. In contrast, Em becomes more hyperpolarized as a result of increased IK(V) due to opening of KV channels (i.e., a rise in i or Popen) and/ or increasing expression of functional KV channels (i.e., N rises). Altered activity of K+ channels (e.g., KV channels) indirectly regulates the extent of Ca2+ influx through the VDCC by altering Em. Decreased expression and/or functioning of K+ channels lead to sustained membrane depolarization and contribute to sustained elevation of [Ca2+]cyt by (a) activating VDCC, (b) facilitating the production of inositol 1,4,5-trisphosphate (IP3) (Ganitkevich and Isenberg, 1993), and (c) promoting Ca2+ entry via the reverse mode of Na+/Ca2+ exchange (Blaustein and Lederer, 1999). Indeed, downregulated K+ channel expression and inhibited K+ channel function have been observed in PASMC from patients with IPAH; the resultant membrane depolarization increases [Ca2+]cyt by opening VDCC in PASMC, and causes pulmonary vasoconstriction and enhance pulmonary vascular medial hypertrophy [see Role of downregulated voltage-gated K+ (KV) channels section] (Yuan et al., 1998a,b). The role of receptor-operated and store-operated Ca2+ channels in elevated [Ca2+]cyt As discussed earlier, the sarcolemmal Ca2+ channels are regulated by changes in Em (e.g., VDCC), interactions of agonists with respective receptors (e.g., ROC), or depletion of

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Ca2+ from the intracellular stores (e.g., SOC). Neither ROC nor SOC activation require membrane depolarization. ROC, also called ligand-gated Ca2+ channels, are activated by the binding of ligands such as norepinephrine, serotonin (5-HT), and vasopressin (Nelson et al., 1990) to their specific membrane-bound receptors. SOC have been identified in many cell types, including PASMC, and are activated by the depletion of SR Ca2+ stores (Golovina et al., 2001). The influx of Ca2+ through SOC channels, called capacitative Ca2+ entry (CCE), is critical for refilling the empty SR with Ca2+ and maintaining a sustained increase in [Ca2+]cyt. Increased expression and function of SOC channels translates into enhanced CCE activity, which is directly responsible for a sustained elevation of [Ca2+]cyt and [Ca2+]SR (Fig. 7). The function of SOC and ROC is dependent on the expression of transient receptor potential (TRP) channel genes. Of the known TRP genes, some canonical TRP (TRPC) channel genes, such as TRPC1, TRPC2, TRPC4, TRPC5, and TRPC6 are expressed in human PASMC (Golovina et al., 2001; Yu and Yuan, unpublished observations). Additionally, TRPC1, TRPC3, and TRPC5 are expressed in pulmonary artery endothelial cells (PAEC) (Fantozzi et al., 2003). Upregulated TRP gene expression augments SOC activity and CCE, and has been shown to promote cell proliferation in human PASMC (Golovina et al., 2001). Recent data suggest that the increased expression of TRPC6 protein may play a role in the increased rate of growth and proliferation in PPHPASMC [see Role of upregulated canonical transient receptor potential (TRPC) channels and enhanced capacitative Ca2+ entry (CCE) section] (Yu et al., 2003a).

Pulmonary arterial wall remodeling Under normal conditions, the thickness and tissue mass of the pulmonary arterial walls are maintained at an optimal level by a fine balance between proliferation and apoptosis of fibroblasts, PASMC, and PAEC. If this balance is disturbed in favor of proliferation, the pulmonary arterial wall thickens, narrowing and eventually obliterating the vessel lumen, and leading to increased PVR (Fig. 8). This process also decreases pulmonary vascular compliance which accommodates for an increase in cardiac output (such as during a progressively heavy exercise) by allowing vasodilation and recruitment of previously unperfused vessels. Vascular remodeling refers to the structural changes that lead to hypertrophy and/or luminal occlusion. Loss of pulmonary vascular compliance and increased PVR due to pulmonary vascular remodeling leads to progressively pronounced pulmonary hypertension and has indeed been found to be the predominant pathological finding in IPAH (Rubin, 1997). Proliferation and hypertrophy of the pulmonary artery smooth muscle cells The molecular mechanisms responsible for pulmonary artery remodeling are incredibly complex. Vasoconstriction

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For example, MAP kinase II, an enzyme involved in the phosphorylation cascade that leads to a DNA synthesispromoting factor, is activated by a rise in [Ca2+]cyt (Berridge, 1993). Elevated [Ca2+]cyt also is involved in stimulating gene expression and cell proliferation as evidenced by the study of the activation and expression of the early responsive gene, c-fos. This gene contains two Ca2+-sensitive elements in its promoter: the serum response element (which binds with serum response factor, SRF, and ternary complex factor, TCF), and the cAMP response element that binds to cAMP response element binding protein (CREB). A rise in [Ca2+]cyt activates SRF and TCF, and a rise in nuclear [Ca2+] activates CREB, hence promoting activation and expression of c-fos (Hardingham et al., 1997). It has been shown that resting [Ca2+]cyt is significantly elevated in proliferating PASMC compared to that in growth-arrested cells supporting the contention that enhanced Ca2+ influx into the cytoplasmic space is required not only for smooth muscle contraction but also for cell growth and proliferation (Golovina et al., 2001; Platoshyn et al., 2000).

Fig. 8. The balance of cell proliferation and apoptosis in pulmonary artery smooth muscle cells (PASMC) maintains the thickness and tissue mass of the arterial walls at an optimal level. If this balance is disturbed such that there is more proliferation and/or less apoptosis, the arterial wall thickens, narrowing the lumen and ultimately leading to the obliteration of the vessel and to an overall increased PVR. The histological examination of pulmonary arteries in lung tissues isolated from normotensive patients (left) and IPAH patients (right) shows the severe degree of medial hypertrophy in the diseased artery.

and cellular proliferation may share a common pathway, involving signaling processes that result in parallel intracellular events in vascular remodeling and in the development of pulmonary hypertension. Increased proliferation and hypertrophy of PASMC have been implicated in the development of IPAH (Yuan and Rubin, 2001). Like vasoconstriction, this process seems to relate, at least in part, to disturbed [Ca2+]cyt homeostasis in PASMC. Elevated [Ca2+]cyt propels cells to go through the cell cycle [Ca2+]cyt has been shown to modulate smooth muscle cellular proliferation and growth. Elevated [Ca2+]cyt leads to a rapid increase in nuclear Ca2+ concentration (Allbritton et al., 1994) which is an essential component of proliferation of smooth muscle cells. Elevated [Ca2+]cyt propels the quiescent cells into the cell cycle where they undergo mitosis, promoting cellular proliferation (Hardingham et al., 1997). At least four steps in the cell cycle appear to be sensitive to Ca2+/calmodulin complex activation: (a) transition from G0 to G1 phase (from resting state to the beginning of DNA synthesis), (b) transition of G1 phase to S phase (DNA synthesis), (c) transition of G2 to M phase (mitosis), and (d) mitosis itself (Fig. 7). Some cytoplasmic signal transduction proteins that are involved in cellular proliferation are also Ca2+ dependent.

Elevated [Ca2+]cyt in pulmonary artery endothelial cells increases AP-1 binding activity and growth factor synthesis Activating protein-1 (AP-1) refers to a family of homoor heterodimeric transcription factors that control gene expression by directly regulating genes that contain AP-1 binding sites [5V-TGACTCA-3V (TRE) or 5V-TGACGTCA3V (CRE)] in their promoters, or indirectly by forming heterodimers with other types of transcription factors such as STAT (Michiels et al., 2001; Zhang et al., 1999). The family of AP-1 proteins is composed of Jun (v-Jun, c-Jun, JunB, and JunD), Fos (v-Fos, c-Fos, FosB, Fra1, and Fra2), or activating transcription factor (ATF2, ATF3/ LRF1, B-ATF) subunits (Michiels et al., 2001). The target genes for AP-1 (e.g., endothelin-1, VEGF, PDGF) are often involved in the regulation of cell proliferation, migration, and apoptosis and they are often called oncogenes (Semenza, 2000). Since it can target oncogenes, it is not surprising that overexpression of AP-1 has been shown to accelerate cellular proliferation and growth in tumor cells (Mathas et al., 2002). Genes encoding AP-1 are Ca2+ sensitive; sustained elevation of [Ca2+]cyt due to Ca2+ influx has been shown to upregulate the expression of c-fos and c-jun protooncogenes (Hardingham et al., 1998). However, different amplitude and duration in the elevation of [Ca2+]cyt in different compartments of the cell result in different transcriptional responses, for example, a different set of transcription factor genes are activated as a result of elevated [Ca2+]cyt due to influx via sarcolemmal Ca2+ channels as opposed to that due to Ca2+ release from the SR (Dolmetsch et al., 1998; Lipp et al., 1997). Chronic hypoxia-induced pulmonary hypertension is associated with increased synthesis or expression of ET-1, PDGF, and VEGF (Faller, 1999). In human PAEC, chronic hypoxia upregulates the mRNA and protein expression of

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TRPC4, a cation channel subunit involved in forming heterotetrameric SOC. This leads to an enhanced amplitude of SOC currents and CCE (Fantozzi et al., 2003). The resulting increase in [Ca2+]cyt augments AP-1 binding activity and may lead to an increased expression of AP-1 responsive genes (e.g., ET-1, PDGF, VEGF genes) in PAEC. The hypoxia-mediated increases in [Ca2+]cyt and AP-1 binding activity in PAEC may be partly responsible for hypoxia-induced pulmonary vascular remodeling. 2+

Sufficient Ca in the SR is required for smooth muscle cell proliferation Ca2+ stored within the SR plays an important role in the initiation of DNA synthesis and cell proliferation. Depletion of Ca2+ from the IP3-sensitive Ca2+ stores with the SR Ca2+Mg2+ ATPase inhibitors arrests cell growth, while its repletion via SERCA pumps allows continued sarcoplasmic/ endoplasmic reticular function (such as lipid synthesis and protein sorting and processing) and the resumption of the S phase of cell cycle (Mogami and Kojima, 1993). Therefore, increased [Ca2+]cyt and [Ca2+]SR are both required for PASMC mitosis and proliferation. Inhibition of apoptosis in the pulmonary artery smooth muscle cells The precise control of the balance between PASMC proliferation and apoptosis is important in maintaining the structural and functional integrity of the pulmonary vasculature. In IPAH, this balance seem to be disturbed such that there is increased PASMC proliferation and decreased apoptosis, leading to vessel wall thickening and vascular remodeling (Fig. 8) (Rabinovitch, 1998). Indeed, decreased apoptosis has been implicated in the development and maintenance of severe pulmonary hypertension (Zhang et al., 2003), whereas induction of apoptosis promotes the regression of hypertrophied pulmonary vascular wall in animal experiments (Rabinovitch, 1998). Dysfunction of K+ channels leads to inhibition of apoptosis Apoptotic volume decrease (AVD) and subsequent cell shrinkage is an early hallmark of apoptosis process (Remillard and Yuan, 2004). Maintenance of a high concentration of intercellular K+ ([K+]i) is required to maintain a normal cell volume (Maeno et al., 2000; Remillard and Yuan, 2004). Plasma membrane K+ channels play a role in regulating apoptosis in that their activation induces or accelerates AVD and apoptosis by enhancing cytoplasmic K+ loss. On the other hand, inhibition or decreased activity of K+ channels has been shown to cause accumulation of K+ in the cells, allowing the maintenance of a sufficiently high [K+]i to decelerate AVD and inhibit apoptosis (Fig. 9) (Bortner et al., 1997; Remillard and Yuan, 2004). In addition to its role in the control of cell volume, a high [K+]i is required for suppression of caspases and nucleases, which are believed to be the final mediators of apoptosis

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(Bortner and Cidlowski, 1999; Bortner et al., 1997; Thornberry and Lazebnik, 1998). Decreased expression and function of KV channels in PASMC, as witnessed in IPAH, attenuate the programmed cell death by decelerating AVD and inhibiting the activity of cytoplasmic caspases, which disrupts the balance between PASMC proliferation and apoptosis and promotes pulmonary vascular medial hypertrophy (Fig. 9) (Zhang et al., 2003). The antiapoptotic protein Bcl-2 inhibits K+ channels and enhances cell survival Bcl-2, an antiapoptotic membrane protein, attenuates apoptosis by (a) inhibiting cytochrome c release from the mitochondrial intermembrane space into the cytosol (Kluck et al., 1997), (b) inhibiting function of K+ channels (Ekhterae et al., 2001), (c) regulating influx of protons into the mitochondria (Shimizu et al., 1998), and (d) maintaining [Ca2+]SR (He et al., 1997). Overexpression of Bcl-2 in PASMC has been shown to downregulate the expression of KV channel a subunits. The resulting decrease in wholecell K+ currents or K+ loss inhibit AVD and apoptosis induced by apoptosis inducers (e.g., staurosporine) (Ekhterae et al., 2001; Krick et al., 2001). (Fig. 9). Expression of Bcl-2 mRNA has been shown to be upregulated in lung tissues from patients with sporadic and FPAH (Geraci et al., 2001). Furthermore, mutations in the BMP-R2 gene are known to be associated with familial and sporadic PAH (Deng et al., 2000). We have recently shown that BMP proteins downregulate Bcl-2 expression and induce apoptosis in normal human PASMC, whereas the BMP-mediated apoptosis is markedly inhibited in PASMC from IPAH patients (Zhang et al., 2003). It is, therefore, reasonable to hypothesize that the upregulated Bcl-2 gene expression in PASMC from IPAH patients may be in part related to the mutations in BMP-R2 gene and/or dysfunction of BMP mediated signaling. Transdifferentiation of adventitial fibroblasts into smooth muscle cells Repair of the injured tissue is an essential requirement for any living organism. Excessive hemodynamic stress (e.g., hypertension), noxious blood-borne agents (e.g., atherogenic lipids), locally released cytokines, or unusual environmental conditions (e.g., hypoxia) create adverse stimuli that require attention in order for the organism to be able to survive. Evolutionary pressures over long periods of time have perfected mechanisms that are readily available to counteract such stimuli and to preserve the structure and function of the vessel wall. These repair mechanisms, however, occasionally escape self-limiting control and can result, in case of blood vessels, in narrowing of the lumen, decreased responsiveness to vasodilators, and obstruction to blood flow. Smooth muscle cells, endothelial cells, and fibroblasts in the pulmonary vascular wall play specific roles in the response to injury. Fibroblasts are in a unique position for

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Fig. 9. Schematic diagram showing the roles of downregulated voltage-gated K+ (KV) channels and upregulated TRPC channels as well as increased Bcl-2 expression in the development of pulmonary hypertension by regulating [Ca2+]cyt and modulating cell proliferation and apoptosis. IP3, inositol 1,4,5trisphosphate; TRPC, canonical transient receptor potential channel gene; SOC, store-operated Ca2+ channels; ISOC, SOC currents; CCE, capacitative Ca2+ entry; [Ca2+]SR and [Ca2+]n, [Ca2+] in the sarcoplasmic reticulum (SR) and nucleus; AVD, apoptotic volume decrease; IK(V), K+ currents through KV channels; Cyt-c, cytochrome c; PVR, pulmonary vascular resistance; and PAP, pulmonary arterial pressure.

this role, since they are less differentiated, conferring a remarkable plasticity to these cells, allowing for a tremendous capacity for rapid migration, proliferation, synthesis of connective tissue, contraction, cytokine production, and, most importantly, transdifferentiation into other types of cells (e.g., PASMC) (Sartore et al., 2001). The role of oxygen in modulating fibroblast gene expression and, function has been well studied in the setting of wound healing where hypoxia can be a critical early component of many of the cellular responses (Davidson and Mustoe, 2001). Therefore, fibroblasts may play a role in vascular remodeling due to hypoxia. Indeed, in animal models, the adventitial compartment of the vessel walls has been found to undergo the earliest and most profound structural changes following exposure to hypoxia. Proliferation of fibroblasts has been shown to more sustained and exceed that of PAEC or PASMC in these models (Belknap et al., 1997). Hypoxia-induced proliferation and transdifferentiation of fibroblasts into pulmonary artery smooth muscle cells Hypoxia acts in vitro as a proliferative stimulus for pulmonary artery adventitial fibroblasts in the absence of exogenous mitogens (Stenmark et al., 2002). Hypoxia seems to act as a growth-promoting stimulus for adventitial fibroblasts through Ga i/o- and Gq-mediated activation of a complex network of kinases. Extracellular nucleotides such as ATP act as autocrine/paracrine modifiers through Gai-

and Gq-coupled P2Y receptors to promote proliferative response of fibroblasts under hypoxic conditions (Stenmark et al., 2002). Hypoxia-induced changes in fibroblasts’ proliferative and matrix-producing phenotypes are accompanied by the appearance of smooth muscle a-actin in tissues from pulmonary hypertensive subjects, suggesting that some of the fibroblasts are being transdifferentiated into myofibroblasts (Stenmark et al., 1995). This transdifferentiation involves a complex network of microenvironmental factors and pathways in which extracellular matrix components as well as growth factors, cytokines, and adhesion molecules may play a role. Although the precise mechanism of this effect is still not known, it appears that hypoxia stimulates expression of smooth muscle a-actin in fibroblasts, possibly through Gai, while it seems to be independent of TGFh signaling or Gq-coupled pathways (Stenmark et al., 2002). Fibroblast heterogeneity in pulmonary arterial adventitia The pulmonary vascular adventitia of neonatal calves has been found to contain multiple and functionally distinct subpopulations of fibroblasts (Stenmark et al., 2002). Proliferation under hypoxic conditions is highly variable among these subpopulations, with some exhibiting more than 2-fold increase in DNA synthesis, while others show a decrease in DNA synthesis. These observations suggest that

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hypoxia specifically selects certain phenotypically and functionally distinct subpopulations of fibroblasts to act as ‘stem cells’ for the vascular wall to expand and proliferate. Since each subpopulation of fibroblasts respond uniquely to hypoxia, they may serve special functions in response to injury. Thus, the adventitial fibroblasts residing in the vessel wall may be a critical regulator of vascular remodeling under hypoxic conditions.

Cellular and molecular mechanisms in the development of pulmonary hypertension In addition to the synthetic, structural, and functional abnormalities in the pulmonary vasculature discussed above, substantial and convincing evidence has recently emerged that point to multiple derangements in complex intracellular signaling pathways that can contribute to the manifestation of IPAH within and between individuals affected by this disease or condition. It is now generally accepted that this condition involves a heterogeneous constellation of multiple genetic, molecular, and humoral abnormalities that all share a common end result, that is, pulmonary vascular remodeling. Ion channel dysfunction and abnormal intracellular Ca2+ homeostasis are examples of cellular factors involved. Other factors include the circulating mediators and molecular signaling mechanisms that are involved in stimulation of gene transcription and promotion of the cell cycle (mitosis) and factors involved in creating a proliferative and vasoconstrictive milieu in the pulmonary arterial bed. Role of downregulated voltage-gated K+ (KV) channels As stated earlier, depolarization of Em, which is regulated by membrane K+ permeability, results in a sustained increase in [Ca2+]cyt via extracellular Ca2+ influx through VDCC and triggers the release of Ca2+ from the SR into the cytoplasm by facilitating the production of IP3. The transmembrane K+ current through voltage-gated K+ (KV) channels plays an important role in regulating the resting Em in PASMC (Yuan, 1995). Decreased KV channel activity leads to pulmonary vasoconstriction and pulmonary artery smooth proliferation In native cells, functional KV channels are heteromultimeric tetramers composed of pore-forming a and regulatory h subunits. In PASMC from IPAH patients, the amplitude of IK(V) and the mRNA/protein expression level of KV channel a subunits (e.g., KV1.2 and KV1.5) are both significantly decreased in comparison to PASMC from patients with non-pulmonary hypertension (NPH) cardiopulmonary diseases and SPH (Yuan et al., 1998a,b). These decreases translate to a more depolarized Em in IPAHPASMC. The membrane depolarization that occurs in PASMC from IPAH patients would cause a sustained

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increase in [Ca2+]cyt by promoting Ca2+ entry through opened VDCC. Indeed, in PASMC from IPAH patients, the resting [Ca2+]cyt is much higher than in PASMC from patients with NPH or SPH. The increased [Ca2+]cyt in IPAH-PASMC, as mentioned earlier, not only causes pulmonary vasoconstriction, but also stimulates PASMC proliferation, ultimately contributing to the development of pulmonary vascular remodeling (Fig. 9). Decreased KV channel activity leads to inhibition of apoptotic volume decrease and apoptosis As discussed earlier, activity of K+ channels regulates apoptosis and its consequent apoptotic volume decrease (AVD). In short, diminished [K+]i due to increased K+ efflux through plasmalemmal K+ channels results in cell shrinkage and activation of cytoplasmic caspases, while maintenance of sufficient K+ in the cytosol due to decreased activity of K+ channels inhibits apoptosis (Fig. 9). In PASMC from IPAH patients, apoptosis induced by BMP and staurosporine is inhibited in comparison to cells isolated from SPH patients (Zhang et al., 2003). Furthermore, overexpression of the KV1.5 gene (KCNA5) in PASMC increases IK(V), accelerates staurosporine-mediated apoptotic cell shrinkage, and enhances apoptosis (Brevnova et al., 2003). These data suggest that inhibited apoptosis in IPAHPASMC may be related to decreased KV channel activity. The downregulated KV channels in PASMC from IPAH patients may be a shared mechanism involved in mediating pulmonary vasoconstriction and in stimulating PASMC proliferation because of its dual role in increasing [Ca2+]cyt and decreasing K+ loss. Role of upregulated canonical transient receptor potential (TRPC) channels and enhanced capacitative Ca2+ entry (CCE) As discussed earlier, in addition to Ca2+ influx through VDCC, [Ca2+]cyt can also be increased by promoting Ca2+ influx through ROC and SOC (Fig. 7). Therefore, the elevated [Ca2+]cyt in IPAH-PASMC may be caused by multiple mechanisms; that is, enhanced Ca2+ entry through VDCC opened by membrane depolarization due to inhibited KV channels is only one of the important pathways for elevated [Ca2+]cyt and its resultant sequelae in the development of pulmonary vasoconstriction and vascular remodeling in IPAH. Activation of SOC by depletion of intracellular Ca2+ stores has been implicated in the elevation of [Ca2+]cyt in PASMC from IPAH patients. TRPC channels are involved in forming store-operated Ca2+ channels responsible for CCE The molecular composition of functional SOC, which are responsible for store depletion-mediated CCE, is still incompletely understood. However, numerous reports have indicated that the canonical TRP (TRPC) channels participate in forming functional SOC (and ROC) in many cells

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including PASMC. In PASMC, inhibition of TRPC1 and TRPC6 with antisense oligonucleotides attenuates CCE induced by passive depletion of the SR Ca2+ with cyclopiazonic acid (CPA), a blocker of the SERCA pump. Furthermore, inhibition of TRPC4 with small interfering RNA specifically targeting on TRPC4 mRNA also attenuates CPA-mediated CCE in PAEC (Fantozzi et al., 2003). These observations suggest that TRPC1, TRPC4, and TRPC6 are potentially involved in forming functional SOC in PASMC and PAEC. CCE is enhanced and TRPC channels are upregulated in proliferating PASMC In comparison to growth-arrested cells, proliferating human PASMC exhibit a high level of resting [Ca2+]cyt potentially maintained by a constant Ca2+ influx. Interestingly, PASMC proliferation is associated with a significant increase in mRNA and protein expression of TRPC channels such as TRPC1 and TRPC6 (Golovina et al., 2001; Yu et al., 2003a,b). Consistent with the upregulated TRPC channel expression, the amplitude of CCE is also significantly greater in proliferating PASMC than in growth-arrested cells. Inhibition of TRPC expression with antisense oligonucleotides markedly decrease the amplitude of CCE and significantly inhibits PASMC proliferation. Levels of various growth factors, such as PDGF, are noted to be higher in lung tissues of patients with severe pulmonary hypertension. Therefore, upregulation of TRPC channels appear to be a critical mechanism by which growth factors mediate PASMC proliferation. TRPC channels are upregulated in pulmonary artery smooth muscle cells from patients with IPAH Increased CCE due to increased formation of SOC by upregulated TRPC genes is also important in the development of pulmonary vascular remodeling in patients with PPH. Recently, our laboratory has compared the magnitude of CCE between PASMC isolated from patients undergoing lung transplant for IPAH and SPH. When matched for PAP and PVR, growth-arrested PASMC from IPAH patients demonstrate significantly higher resting [Ca2+]cyt when compared to cells from SPH patients. In addition, the magnitude of CCE is significantly greater in PASMC from IPAH patients than in cells from SPH patients. These data suggest that CCE is essential in maintaining the adequate cytoplasmic, nuclear, and SR Ca2+ required for PASMC proliferation. Enhanced CCE, possibly via upregulation of TRPC1, TRPC3, and TRPC6 channels (Yu et al., 2003a), may represent another critical downstream pathogenic event or mechanism involved in the development of severe pulmonary hypertension. Accordingly, interruption of CCE at any point, from agents that downregulate TRPC gene expression to specific blockers for SOC in PASMC, may prove beneficial in the development of therapeutic approaches for treatment of severe pulmonary hypertension.

Role of bone morphogenetic protein receptor type II (BMP-R2) gene mutations Mutations in the BMP-R2 gene have been associated with FPAH as well as in 15– 25% of patients with IPAH (Machado et al., 2001; Newman et al., 2001). BMPs are signaling molecules that belong to the transforming growth factor-h (TGF-h) superfamily and play an important role in regulating cell proliferation, differentiation, and apoptosis (Massague and Chen, 2000; Morrell et al., 2001; Yamamura et al., 2000). In humans, a variety of cell types including PASMC and PAEC synthesize and secrete BMPs. Similar to TGF-h, the signal transduction of BMP-mediated pathways involve two types of transmembrane serine-threonine kinase receptor proteins, BMP receptor type I (BMP-R1a and BMP-R1b) and type II (BMP-R2) (Heldin et al., 1997; Massague and Chen, 2000; Newman et al., 2001), which coassemble to form homo- or heteromeric proteins (Nohe et al., 2002). Binding of BMP ligand (e.g., BMP-2 and -7) to either of the receptors leads to hetero-oligomerization of BMP-R1 and BMP-R2 and formation of ligand– receptor complex, which in turn activates the downstream signaling elements such as the receptor-activated ‘‘mothers against decapentaplegic’’ (Smad) proteins. The activated BMP-R1 phosphorylates the R-Smad proteins (e.g., Smad-1, -5, and -8), which then dimerizes with co-Smad (e.g., Smad-4) to form a signaling complex that can translocate into the nucleus. The R-Smad/co-Smad complex is involved in regulation of transcription of certain ‘‘Smad-responsive’’ genes that contain the Smad binding sequence (5V-CAGAC-3V and 5VGTCTG-3V) in their promoter, some of which encode for proapoptotic or anti-proliferative proteins (Heldin et al., 1997; Massague and Chen, 2000; Newman et al., 2001). Fig. 10 illustrates the proposed mechanism for BMP signaling pathway in inducing apoptosis. In addition to R-Smad and co-Smads, humans also express antagonistic Smads such as Smad-6 and -7, which mediate negative feedback within TGF-h/BMP signaling pathways and regulatory inputs from other pathways (Hata et al., 2000; Imamura et al., 1997; Ishisaki et al., 1999). The antagonistic Smads compete with R-Smad for the activated tyrosine kinase and therefore inhibit activation of R-Smads (Massague and Chen, 2000). Smad-6 inhibits BMP signaling by blocking activation of Smad-1, -5, or -8 (Hata et al., 1998; Imamura et al., 1997; Ishisaki et al., 1999). Increased Smad in the nucleus can have three effects: (a) activation of gene transcription by binding onto the Smad-binding sequence in the promoter, (b) formation of heterogeneous polymers with other transcription factors to mediate apoptosis (Yamamura et al., 2000), and (c) formation of heterogeneous polymers with corepressors such as the homeodomain protein TGIF (Wotton et al., 1999) and two related proteins c-Ski and SnoN (Sun et al., 1999) to induce repression of target gene transcription (Fig. 10) (Massague and Chen, 2000). In mammalian cells, another inhibitor of Smads is the Smad-

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Fig. 10. Schematic diagram depicting the proposed role of bone morphogenic protein (BMP)-mediated apoptosis in human PASMC. There are two types of BMP receptors (BMP-RI and BMP-RII) which dimerize with one another and form a BMP ligand – receptor complex. The activated BMP-RI phosphorylates and activates the receptor-activated Smads (R-Smad) which then form dimerized complexes with Co-Smads and enter the nucleus. The R-Smad/co-Smad interact with DNA in the nucleus and regulate the transcription of various target genes whose primers contain the Smad binding sequence (5’-AGAC-3’). In the nucleus, Smad-1 (a R-Smad) and Smad-4 (a co-Smad) in association with different corepressors appear to be involved in downregulating the expression of Bcl2, an antiapoptotic protein that blocks the release of cytochrome c (Cyt-c) from the mitochondrial intermembrane space to the cytosol. Bcl-2 also downregulates the mRNA expression and inhibits the function of sarcolemmal K+ channels in PASMC. Downregulation of Bcl-2 via the BMP/BMP-R/Smad1 pathway thus leads to (a) an increase in K+ efflux and a decrease in cytoplasmic [K+] ([K+]cyt), which subsequently accelerate apoptotic volume decrease (AVD) and promote apoptosis; and (b) an increase in cytosolic Cyt-c ([Cyt-c]cyt), which induces cell apoptosis by activating caspases.

interacting protein-1, a zinc-finger/homeodomain protein that interacts with Smad-1 and -5 to inhibit BMP-mediated effects (Massague and Chen, 2000). Furthermore, activation of Smad proteins is inhibited by overexpression of calmodulin, a Ca2+-sensitive cytosolic protein. This attenuates the pro-apoptotic TGF-h/BMP signaling transduction pathway in human PASMC, suggesting that elevated [Ca2+]cyt can lead to activation of calmodulin and subsequent inhibition of Smad proteins (Fig. 10). Mutations in the BMP-R2 gene can negatively influence the binding of BMP ligand to either of the receptors due to decreased BMP-R2 protein expression or production of structurally dysfunctional proteins, therefore decreasing the formation of pro-apoptotic Smad complexes. The exact pathologic mechanisms by which mutations in the BMP-R2 gene mediate vascular remodeling in patients with IPAH is not clearly understood yet. Our previous study has sug-

gested that, in normal PASMC, BMPs induce apoptosis by downregulating Bcl-2 (Zhang et al., 2003). In PASMC from IPAH patients, the BMP-mediated inhibition of Bcl-2 and apoptosis are markedly attenuated because of dysfunctional BMP signaling. The interaction of BMPs with Bcl-2 proteins via BMP receptors provides a potential mechanism to explain why mutations of BMP-R2 causes pulmonary vascular medial hypertrophy: upregulation of Bcl-2 inhibits the release of cytochrome c from mitochondria, thereby attenuating PASMC apoptosis (Zhang et al., 2003). The role of 5-HT receptors and transporters The prolonged use of certain appetite suppressant medications (i.e., aminorex, fenfluramine, and dexfenfluramine) was determined to be a significant (30-fold increase vs. the general population) risk factor for the

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development of IPAH (Fishman, 1999). These drugs belong to a vast class of amphetamine- and epinephrine-like drugs that interact with the monoamine system in the brain, potently inhibiting neuronal 5-HT reuptake by inhibiting 5-HT transporters (5-HTT), and/or triggering indolamine release and a consequent increase in the amount of extracellular 5-HT (Eddahibi et al., 1999). This inhibition occurs not only in serotoninergic neurons, but also in other organ systems (including platelets, PAEC, and PASMC) that express the same 5-HTT protein (Lesch et al., 1994). Interestingly, the use of these anorexigenic drugs have not been associated with vascular remodeling in any other organ system but the lungs. These drugs have indeed been found to promote the development of plexogenic pulmonary arteriopathy mostly confined to the small arteries and arterioles in the lungs suggesting that they have a molecular target that is selectively present in resistance pulmonary arteries (Rothman et al., 1999). These observations sparked an interest in the ‘‘serotonin theory’’ of pulmonary hypertension. 5-HT is a vasoconstrictor and a mitogen Elevated levels of circulating 5-HT, a potent vasoconstrictor and mitogen (Fig. 11), have been reported under several conditions that lead to the development of pulmonary hypertension (Celada et al., 1994; MacLean et al., 2000). However, chronic treatment with phentermine and fenfluramine in combination has been shown to decrease plasma 5-HT levels in humans (Rothman et al., 2000). Additionally, some widely used antidepressants (e.g., selective 5-HT reuptake inhibitors that competitively inhibit 5-HTT) have not been shown to increase the risk of development of IPAH despite being used in a vast number of general population for years. On the other hand, the use of psychoanaleptic drugs may lower the odds ratio for IPAH and the use of selective inhibitors of 5-HTT may attenuate the development of PAH in experimental models, suggesting a possible role for 5-HTT (Louis, 1999). Taken all these observations into account, it appears that 5-HT is an important player in the pathogenesis of pulmonary hypertension most notably through its specific transporter. Concentration of serum 5-HT level does not necessarily reflect the 5-HT concentration in the local microenvironments surrounding PAEC and PASMC. As noted above, aminorex and fenfluramine-like drugs not only inhibit 5-HTT, but also elevate extracellular 5-HT levels and, possibly, alter the 5-HT turnover in the microenvironment. 5-HT is also released from a variety of pulmonary neuroendocrine cells and neuroepithelial bodies that are present throughout the airways and possibly from PASMC (Miyata et al., 2001). This increases the availability of free 5-HT in the local pulmonary microenvironment, making it more likely to have an effect on the pulmonary vasculature compared with systemic vessels. Vasoconstriction occurs when 5-HT binds to 5-HT receptors (5-HTR), such as 5-HT2 and 5-HT1B/1D receptors (Fig. 11). 5-HT can also be transported inside the cells

Fig. 11. Schematic diagram depicting the proposed role of 5-HT receptors (5-HTR) and transporters (5-HTT) in the development of pulmonary vasoconstriction and vascular medial hypertrophy. Serotonin (5-HT) may interact with either 5-HTR or 5-HTT on the cell membrane, which leads to activation of GTPase-activating protein (GAP) and/or Ras and/or Rac. The ensuing signals lead to activation of a still unknown mechanism involving NAD(P)H oxidase. This mechanism leads to the formation and release of reactive oxygen species (ROS), which in turn activates extracellular signalregulated kinase (ERK)-1 or ERK-2 and mitogen-activated protein (MAP) kinases. The result is upregulation of the expression of genes involved in both cellular hypertrophy and proliferation. 5-HTR activation also promotes the production of IP3 and diacylglycerol (DAG) which then induce Ca2+ influx by opening voltage-dependent (VDCC) and receptor-operated (ROC) Ca2+ channels and trigger Ca2+ release from the SR. The subsequent increase in [Ca2+]cyt causes smooth muscle cell contraction.

by 5-HTT. The 5-HTR and 5-HTT are abundantly expressed in the lung, where they are predominantly located in the PASMC (MacLean et al., 1996). Once internalized, 5-HT can exert its mitogenic and co-mitogenic effects on PASMC (Eddahibi et al., 1999; Lee et al., 1994). A clear distinction between the two classes of molecules, 5-HTT and 5-HTR, has so far been elusive. Either or both may participate in internalizing 5-HT and play a role in the PASMC response to 5-HT. Several subtypes of signal transducing 5-HTR have been characterized and cloned by pharmacological means. Some subtypes may activate phospholipase C or adenylate cyclase by acting on G proteins (Fanburg and Lee, 1997). It is postulated that these receptors operate at the cell surface, without necessarily mediating the uptake of 5-HT. Upregulated 5-HTT and increased 5-HT are associated with pulmonary hypertension Transport of 5-HT is enhanced by exposure of PASMC to hypoxia (Lee et al., 1989). 5-HTR predominates over transmembrane signaling, resulting in cell proliferation and hypertrophy (Lee et al., 1994). This proliferative response is augmented by monoamine oxidase inhibitors, which block the intercellular degradation of 5-HT. Since monoamine oxidase is unlikely to affect the 5-HT metabolism at the cell surface membrane, a rise in intercellular

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5-HT concentration may stimulate cell proliferation. 5-HTT is also involved in 5-HT signaling through a variety of other signaling molecules such as (a) tyrosine phosphorylation of GTPase-activating protein (GAP), (b) Ras and NAD(P)H oxidase activation to produce reactive oxygen species (ROS), (c) and activation of extracellular signal-regulated kinase (ERK1/ERK2) and MAP kinase to induce PASMC hyperplasia and hypertrophy (Fig. 11) (Lee et al., 1997, 1998b, 1999). Although the precise relationship between 5-HTR and 5-HTT has yet to be clearly defined, it appears that activation of either one by 5-HT (depending on cell type) can initiate a signaling process that activates cell proliferation and hyperplasia. Interestingly, IPAH develops in only a minority of the individuals who ingest appetite suppressants, suggesting that there may be a genetic predisposition in these patients that makes them vulnerable to the disease after a second ‘hit’ occurs, namely, due to the effect of these drugs. These medications may elevate 5-HT levels in the local milieu or may directly stimulate the overexpression of 5-HTT in PASMC in genetically predisposed patients, thereby causing pulmonary vasoconstriction, PASMC proliferation, and development of IPAH. Aminorex and fenfluramine derivatives interact with 5-HTT in a specific manner, further suggesting that the 5-HTT may be a critical target for appetite suppressants in initiating the development of IPAH (Rothman et al., 1999). Recent studies have demonstrated the increased expression of 5-HTT in lung tissues and pulmonary arteries isolated from patients with IPAH; there is marked enhancement of the proliferative growth response of cultured PASMC to 5-HT but not to other growth factors (Eddahibi et al., 2001a). Additionally, the increased expression of 5-HTT in patients with IPAH has been associated with polymorphism of the 5-HTT gene promoter (Eddahibi et al., 2001a). Mice with targeted 5-HTT gene disruption are less prone to develop severe hypoxic pulmonary hypertension than their wild-type controls (Eddahibi et al., 2000), while selective 5-HTT inhibitors attenuate hypoxic pulmonary hypertension. Conversely, increased 5-HTT expression has been shown to be associated with increased severity of hypoxic pulmonary hypertension (Eddahibi et al., 2001b). Therefore, it appears that the expression and/or function of 5-HTT in PASMC plays a critical role in the extent of hypoxia-induced pulmonary vascular remodeling. Taken together, these observations suggest that 5-HTT overexpression and/or activity in PASMC from patients with pulmonary hypertension may be responsible for the increased mitogenic responses to 5-HT. In addition to affecting the activity and expression of 5HTT, anorexigenic drugs (i.e., aminorex, fenfluramine, and chlorphentermine) have been found to be 5-HTT substrates. Because fenfluramine is mitogenic for rat PASMC and fibroblasts (Lee et al., 2001), it has been proposed that 5HTT substrates other than 5-HT may be mitogenic. According to this hypothesis, the drugs that behave as potent 5-

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HTT substrate are possibly translocated inside the PASMC where their level may reach a toxic level depending on the degree of drug retention. Individual patient susceptibility determines intrinsic drug toxicity, which may have effects similar to or greater than that of 5-HT (Rothman et al., 1999). Additionally, a high concentration of the drugs (e.g., fenfluramine) has been proposed to cause inhibition of K+ channels leading to elevated [Ca2+]cyt in PASMC (Miyata et al., 2001; Wang et al., 1998). Since patients with IPAH exhibit defects in K+ channel expression or function, it is plausible to hypothesize that in patients with impaired expression or function of K+ channels, even lower levels of intercellular drug concentrations that is achievable with the therapeutic dosing of these drugs may be sufficient to cause this effect. 5-HTT gene polymorphism in patients with pulmonary hypertension Genetic predisposition is one way to explain why anorexigenic drugs cause IPAH in only a minority of patients who ingest them. It has been well established that a polymorphism in the promoter region of the human 5HTT gene alters its transcriptional activity. Thus, the level of its expression is genetically predetermined based on its genotype. The polymorphism involves two alleles: the L allele is a 44-bp insertion (‘long 5-HTT gene promoter’) which has a 2- to 3-fold higher level of 5-HTT gene transcription as compared to the S allele which consists of the 44-bp deletion. It is believed that 60 – 70% of patients with IPAH have L/L genotype, while L/L genotype is present only in 20 – 30% of the control population of Caucasian subjects, suggesting that L/L genotype may confer genetic susceptibility to IPAH (Eddahibi et al., 1999). As mentioned earlier, mutations in BMP-R2 gene as well as mutations associated with impaired signaling through other members of the TGF-h receptor family have been strongly associated with familial as well as sporadic PAH (Newman et al., 2001; Trembath et al., 2001). It is not yet well established how the 5-HTT pathway is affected by a common BMP-R2 mutation. However, IPAH does not occur in all subjects with BMP-R2 mutations, suggesting that a second environmental or associated genetic factors may play a critical role. Since normal signaling from BMP-R2 is associated with suppression of PASMC proliferation and enhancement of PASMC apoptosis, it is plausible to hypothesize that it may antagonize the effects of 5-HT (Morrell et al., 2001, Zhang et al., 2003). The BMP-R2 mutation could therefore allow for a stronger response to the effects of 5-HT in the cells, particularly in patients with L/L genotype, allowing for heightened expression of the 5-HTT. It needs to be determined if patients carrying both BMP-R2 mutation and the 5-HTT polymorphism have heightened proliferative response to 5-HT in their PASMC. Whether or not aberrant BMP-R2 signal transduction and gene expression is complementary to increased 5-HTT signaling and gene expression is still unclear.

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In summary, anorexigens fenfluramine-like drugs may contribute to the development of IPAH in three ways: (a) by elevating the circulating 5-HT levels, (b) by acting as 5-HTT substrates that mimic 5-HT, and (c) by altering 5-HTT expression. Patients with the long 5-HTT gene promoter variant have a high 5-HT uptake at baseline, which can make them particularly susceptible to one or more of these mechanisms as a result of anorexigenic drug ingestion. Elevated angiopoietin-1 activity contributes to pulmonary vascular remodeling Angiopoietin-1 (Ang-1) is a 70-kDa protein that is secreted by PASMC during early stages of life as an angiogenic factor essential for lung vascular development and stabilization. Ang-1 binds its tyrosine kinase receptor (TIE2) which is only present on vascular endothelial cells. The ligand– receptor interaction between Ang-1 and TIE2 induces the recruitment, migration, and proliferation of smooth muscle cells around the endothelial vascular network during blood vessel formation (Papapetropoulos et al., 1999; Sullivan et al., 2003). After development is completed, Ang-1 is minimally expressed in human lung (Thistlethwaite et al., 2001). The exact role played by angiopoietin-1 in the development of pulmonary hypertension is controversial (Rudge et al., 2003), with two main groups leading the foray. Both points of view have their opponents and supporters which may help sway the readers’ opinions (Rudge et al., 2003). However, it is obvious that much work remains to be done before the role of Ang-1 in the development of pulmonary hypertension is elucidated. A summary of the ideologies follows. On the one hand, Du et al. (2003) recently demonstrated aberrant overexpression and high steady-state levels of Ang-1 in PASMC from adult patients with various forms of pulmonary hypertension (FPAH and IPAH). They have proposed that Ang-1 overexpression and tyrosine phosphorylation of the TIE2 receptor in pulmonary vascular endothelium directly correlate with the severity of their disease and can be used as molecular markers in patients with IPAH. Their data also suggest that Ang-1 overexpression in IPAH and FPAH may downregulate the expression of BMP-R1a in PAEC, which is required for BMP-R2 signaling (Yoshida et al., 2000) (Fig. 12). As has already been discussed, BMP-R2 mutations have been identified in FPAH patients [see Role of bone morphogenetic protein receptor type II (BMP-R2) gene mutations section]. Therefore, the Ang-1-mediated downregulation of BMP-1a (and, therefore, of BMP-R2) results in enhanced PASMC proliferation, thereby aggravating pulmonary hypertension. In addition to downregulating BMP-R1a expression, overexpression of Ang-1 in PASMC also mediates production and release of serotonin from PAEC, stimulating PASMC proliferation and migration via a paracrine mechanism. As shown in Fig. 12, Ang-1-induced, endotheliumderived serotonin acts as a mitogen by binding to 5-HTR or

5-HTT on the surface membrane of PASMC, inducing PASMC proliferation and migration. In this scenario, Ang1/TIE2/5-HT crosstalk between PAEC and PASMC is responsible for mediating pulmonary vascular medial hypertrophy, and ultimately the development of pulmonary hypertension. Six months after Thistlethwaite’s study was published, Zhao et al. (2003) published their findings on the protective role of angiopoietin-1 in experimental pulmonary hypertension. Rather than PASMC, they believe that pulmonary hypertension is due to endothelial cell dysfunction that causes disturbances in the endothelial vasodilator/vasoconstrictor balance. Using monocrotaline (MCT)-induced pulmonary hypertensive rats, they found that Ang-1 administration reversed the effects of MCT; that is, it (1) enhanced TIE2 receptor expression, (2) decreased right ventricular systolic pressure and right ventricular hypertrophy, (3) prevented apoptosis in the microvasculature, and (4) increased endothelial NO synthase expression, ultimately improving survival of the rats. Endothelial dysfunction in pulmonary hypertension One of the complex and multifactorial processes that contribute to the development of pulmonary hypertension involves endothelial cell dysfunction. Endothelial cells play an integral role in the maintenance of normal vascular structure and function. Excessive endothelial cell proliferation in conjunction with neoangiogenesis results in the formation of plexiform lesions, which are commonly found in pathological examination of the pulmonary arteries of patients with PAH. Endothelial cells are also responsible for producing a wide array of growth factors and vasoactive mediators which regulate the physical and biochemical properties of the pulmonary vessels. Most of these mediators affect PASMC growth and contractility. Altered production of various endothelial vasoactive mediators, such as endothelin-1, NO, serotonin, thromboxane, and prostacyclin, has been associated with the development of PAH by facilitating vasoconstriction and PASMC hypertrophy, leading to vascular remodeling (see the following section). Endothelial cells also serve as a shield that protects the underlying vascular tissue layers from exposure to diverse blood-borne factors. This is generally called endothelial barrier function. Injury to the endothelium or dysfunction of the endothelial cells can lead to exposure of the smooth muscle cells and fibroblasts to these factors, promoting pathological changes as well as thrombosis that can lead to obliteration of the vascular lumen. Loss of endothelial barrier function may result from the actions of several mediators and factors. Endothelial cell permeability may be altered by excessive production of VEGF by alveolar epithelium in response to hypoxia (Christou et al., 1998), and inflammatory mediators, cytokines, oxidants (McQuaid and Keenan, 1997). The endothelium can also become ‘‘leaky’’ due to widening of the

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Fig. 12. Proposed mechanisms involved in angiopoietin-1 (Ang-1)-mediated pulmonary vascular remodeling. Ang-1 released from pulmonary artery smooth muscle cells (SMC) binds toTIE2 on the surface membrane of pulmonary arterial endothelial cells (EC) and leads to an increase in 5-HT production and downregulation of BMP-RI gene in EC. 5-HT released from EC then interacts with 5-HT receptors (5-HTR) and transporters (5-HTT) causing SMC contraction by increasing [Ca2+]cyt and stimulating SMC proliferation by activating ERK/MAPK pathway. Downregulated BMP-RI gene in EC would lead to inhibition of BMP-BMP-R-Smad signaling pathway, which induces EC proliferation and inhibits SMC apoptosis. Therefore, overexpression of Ang-1 in SMC triggers pulmonary vasoconstriction and stimulates SMC/EC proliferation by both autocrine and paracrine mechanisms.

intercellular tight junctions due to mechanical stress or the action of mediators such as thrombin (Bogatceva et al., 2002). As a result of the loss of endothelial barrier integrity, proliferative mediators can leak into and come in contact with the underlying vascular tissue cells such as smooth muscle cells and fibroblasts, enhancing cellular proliferation and causing medial and adventitial hypertrophy, both prominent features of IPAH. The endothelium maintains normal coagulation through the interaction of various substances and factors such as tissue-type plasminogen activator, urokinase-type plasminogen activator, von Willebrand factor, thrombomodulin, humoral factors, platelets, and heparan sulfates (Vane et al., 1990). Dysfunctional or injured endothelium, therefore, can enhance intraluminal thrombosis, which leads to further narrowing or obstruction of the pulmonary vessel lumen and worsened pulmonary hypertension. Another factor is the slowing of the blood flow in the pulmonary circulation of patients with PAH, which occurs due to luminal narrowing secondary to vascular remodeling. This event, accompanied by a relative deficiency of the antithrombotic molecules such as prostacyclin and NO, further enhances thrombogenicity (Cool et al., 1999). Additionally, exposure of platelets to the subendothelial tissue activates the platelets (Ruf and Morgenstern, 1995), resulting in thrombosis and the release of vasomediators and growth factors which may in turn contribute to progression of PAH. Circulating endothelial cells may be involved in the reparative response to vascular injury and tumorigenesis. Although the extent of the role that these cells play in the development of IPAH has yet to be determined, it has been

shown that patients with IPAH have elevated number of circulating endothelial cells (Bull et al., 2003; Tuder et al., 2001). Whether these cells are sloughed off due to injury, actively shed in progressive IPAH, or derived from the endothelial progenitor cells obtained from mobilization of bone marrow by growth factors such as VEGF is not clear. Role of endothelium-derived factors and growth factors in pulmonary hypertension Under normal conditions, the pulmonary vasculature is a highly compliant system that may accommodate up to a 6-fold rise in cardiac output without any significant rise in PAP via vasodilation and recruitment of previously unperfused vessels. This mechanism is maintained by a variety of active mediators produced by PAEC. Endothelium-derived relaxing factors such as nitric oxide (NO) and prostacyclin usually act in coordination with endothelium-derived constricting factors such as ET-1, thromboxane, and serotonin to accommodate changes in the cardiac output and to keep the PAP relatively constant. An imbalance of these endotheliumderived factors can elevate vasomotor tone, promote PAEC and PASMC proliferation, induce pulmonary vascular remodeling, and incite thrombosis. Nitric oxide (NO) and NO synthase in pulmonary hypertension NO is a biologically reactive molecule with several physiological and pathophysiological effects within the vascular, immune, and nervous systems. The roles of NO in inhibiting smooth muscle cell growth and constriction,

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and in inhibiting platelet aggregation are well established (Singh and Evans, 1997). Generation of NO from L-arginine is catalyzed by NO synthase (NOS), which has three distinct isoforms (Barnes and Belvisi, 1993). NOS-III (i.e., endothelial NOS, eNOS), which is a Ca2+-dependent constitutive isoform, is expressed basally in vascular endothelial cells, while NOS-I (i.e., neuronal NOS, nNOS) is expressed in neuronal cells. NO generated by NOS-I and III are responsible for maintaining normal vascular tone and neuronal signal transduction. NOS-II (i.e., inducible NOS, iNOS), which is expressed in a variety of cells within the body, is a Ca2+-independent isoform, that is induced in response to certain cytokines and endotoxins. NOS-II can generate NO in great quantities over long periods of time, and is the predominant isoform during inflammatory processes (Nathan and Xie, 1994). Pulmonary arteries of patients with pulmonary hypertension with severe morphologic abnormalities exhibit diminished expression of NOS-III (Giaid and Saleh, 1995). Furthermore, NOS-III mutant mice exhibit increased PAP and impaired relaxation to acetylcholine, suggesting that NOS-III may play an important role in maintenance of normal vascular tone (Steudel et al., 1997). Conversely, however, PAP and pulmonary vascular structure are unaffected in NOS-I-knockout mice, suggesting that NOS-I may not be involved in pulmonary hypertension, although NOS-I produced by nerve endings in the vessel wall may affect vascular tone in pulmonary arteries. Endothelin-1 (ET-1) and endothelin converting enzyme in pulmonary hypertension ET-1 is a potent vasoconstrictor peptide that is produced by the endothelial cells. As a mitogen that promotes inflammation and smooth muscle cell proliferation, ET-1 has been found to play a crucial role in vascular remodeling and development of pulmonary hypertension (Kirchengast and Munter, 1999). ET-1 is initially produced as a 38amino-acid active peptide, which is then converted into a 21-amino-acid peptide by the action of endothelin converting enzyme (ECE) (Yanagisawa, 1994). Expression of ECE1 has been demonstrated in the airway epithelium, PASMC, macrophages, and PAEC from lungs of normal and pulmonary hypertensive patients, and is particularly abundant in PAEC from mildly to severely pulmonary hypertensive patients (Giaid, 1998). The vasoconstrictor effect of ET-1 is mediated through ETA receptors on PASMC plasma membranes. ETB receptors on PAEC membranes mediate the vasodilatory effects of ET-1 (via NO production), modulate the synthesis of ET-1, and are responsible for clearance of circulating ET-1 (Goldie, 1998). The effects of ET-1 on the vascular system are diverse and complex. Blockade of both ETA and ETB receptors are required to blunt the effect of ET-1 on the pulmonary circulation. In animal studies, bosentan, an orally active nonpeptide which is a competitive antagonist of both ETA and ETB receptors, has been demonstrated to effectively

arrest pulmonary vascular remodeling associated with pulmonary hypertension (Ono and Matsumori, 2002). Human trials of this drug have been very promising in causing reversal of IPAH and regression of its associated histologic changes (Rubin et al., 2002). Interactions between ET-1 and NO has been demonstrated in several studies. In patients with pulmonary hypertension, ET-1 and NO production are increased and decreased, respectively (Giaid and Saleh, 1995). In fact, the ETB receptor has been demonstrated to mediate vasorelaxation through the release of NO (Yanagisawa, 1994) and hypoxia inhibits ETB receptor-mediated NO synthesis by NOS (Sato et al., 1999). During chronic hypoxia, impaired NOS and vasodilation have been associated with impaired cyclic guanosine monophosphate (cGMP)-dependent mechanisms (Berkenbosch et al., 2000). Phosphodiesterase inhibitors (e.g., sildenafil) that increase the intracellular concentration of cGMP and cyclic adenosine monophosphate (cAMP) cause pulmonary vasodilation. Since inhibition of phosphodiesterases is influenced by naturiuretic peptide activity (Zhao et al., 2003), drugs that induce activity of naturiuretic peptides may protect against the structural changes associated with chronic hypoxia. In this regard, the beneficial effects of sildenafil have been reported in published case reports (Prasad et al., 2000). Angiogenic and growth factors in pulmonary hypertension Angiogenic factors play a crucial role in lung development and exert a protective effect by modulating adaptation to various abnormal conditions. Defects in the function of TGF-h and VEGF, an angiogenic factor, have been implicated in the pathophysiology of pulmonary hypertension. VEGF-A expression, abundant in the adult lung (Monacci et al., 1993), is regulated in part by hypoxia. Chronic hypoxia has been shown to increase the expression of VEGF-A and its receptors VEGFR-1 and -2 in rat lung (Christou et al., 1998; Tuder et al., 1995). Targeted knockout of a single VEGF-A gene allele in mice causes lethal impairment of angiogenesis (Carmeliet et al., 1996). The expression of VEGF-B, a more recently discovered member of VEGF family which is also expressed in lower levels in the walls of pulmonary arteries, does not appear to be regulated by hypoxia or cytokines (Olofsson et al., 1999). The role of VEGF-B in pulmonary hypertension is still unclear since VEGF-B knockout mice are healthy and fertile, except for an abnormally small heart and coronary artery dysfunction and impairment of recovery from cardiac ischemia (Bellomo et al., 2000). Endogenous VEGF-B does not significantly counteract the development of chronic hypoxic pulmonary hypertension, although its overexpression in the lung by means of adenoviral gene transfer has been shown to be as potent as VEGF-A in attenuating the development of pulmonary hypertension and vascular remodeling (Louzier et al., 2003). Finally, blockade of VEGF receptors by tyrosine kinase inhibitors leads to pulmonary vascular remodeling and mild

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pulmonary hypertension in normoxic rats, and causes severe irreversible pulmonary hypertension in chronically hypoxic rats (Taraseviciene-Stewart et al., 2001). This is further supported by the finding that prostaglandins, which are used in the treatment of IPAH in humans, promote production of VEGF, thereby preventing the progression of the disease (Machado et al., 2001). Thromboxane in pulmonary hypertension The arachidonic acid metabolite, thromboxane A 2 (TXA2), which is a vasoconstrictor, a smooth muscle mitogen, and an agonist for platelet aggregation, contributes to vascular remodeling and histopathologic changes associated with IPAH. Patients with IPAH have elevated levels of urinary 11-dehydro-TXB2, a major urinary metabolite of TXA2 (Christman et al., 1992), as well as elevated total body synthesis of TXA2, suggesting that TXA2 may play a role in pathogenesis of IPAH (Robbins et al., 2001). Although it is not precisely determined where TXA2 is produced in patients with IPAH, however, activated alveolar macrophages and platelets are one possibility (Robbins et al., 2001).

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hypertrophy in small to medium-sized pulmonary arteries, narrowing of the vessel lumen, increased PVR, and pulmonary hypertension. As research continues, one would hope to develop means of preventing or effectively treating this debilitating and ultimately fatal disease. From what we have learned so far, it appears that a vast number of diverse, and often clinically hard to detect, abnormalities are involved in the development and maintenance of this condition. This obviously makes it unappealing, and likely ineffective, to develop numerous therapeutic modalities to reverse each unique pathogenic derangements in any one individual. However, since all of these abnormalities seem to share one final pathway, that is, vascular remodeling, we suggest that any effective future therapeutic modality should strive to involve prevention and/ or reversal of vascular remodeling by inhibiting proliferation and promoting apoptosis in PASMC and possibly by preventing vasoconstriction at a cellular level. A combined modality to tackle these three fronts may provide synergistic effects and ultimately improve the prognosis for IPAH.

Acknowledgments Summary To unravel the etiology of IPAH, numerous studies in the recent past have tackled its pathophysiology from many different angles. It is now clear that this is a disease entity that defies a single predominating pathophysiological cascade theory, but rather it involves a heterogeneous constellation of multiple genetic, molecular, and humoral abnormalities. Although each abnormality is likely important in itself, none appears to be sufficient to cause the disease by itself. Interestingly, some of the derangements, such as the BMP-R2 gene mutations, occur in all vascular smooth muscle cells, yet manifest as disease only in the pulmonary vascular bed. This supports the notion of multiple hit theory in which some of the hits confer pulmonary specificity. For instance, inheritance of BMP-R2 gene mutation, followed by acquiring mutations in KV channels, can trigger the development of severe IPAH. Overall, abnormalities that are responsible for the development and maintenance of IPAH can be classified in one of three broad categories: (a) cellular factors that create a proliferative, antiapoptotic, and vasoconstrictive physiological milieu, (b) circulating factors that promote a proliferative, antiapoptotic, and vasoconstrictive physiological milieu, and (c) genetic molecular signaling factors that augment gene transcription and the cell cycle thereby promoting a proliferative, antiapoptotic, and vasoconstrictive physiological milieu. All these diverse abnormalities seem to share a final manifestation, vascular remodeling in which fibroblasts, PASMC, PAEC, and platelets all play a role. The resulting disturbance in the balance between proliferation and apoptosis, as well as derangements in the vascular tone due to vasoconstriction, leads to medial

This work is supported in part by grants from the National Institutes of Health (HL 64945, HL 54043, HL 66012, HL 69758, HL 66941 and HL 43026). The authors would like to thank O. Platoshyn, S. Zhang, Y. Yu, I. Fantozzi, E.E. Brevnova, D. Ekhterae, S. Krick, B.R. Lapp, S.S. McDaniel, H. Kim, C.L. Bailey, M.A. Sweeney, J. Kriett, and P.A. Thistlethwaite for their contribution and assistance to this work.

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