Pulmonary hypertension contributes to the morbidity and

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1 Reviews Hypoxia-Induced Pulmonary Vascular Remodeling Cellular and Molecular Mechanisms Kurt R. Stenmark, Karen A. Fagan, Maria G. Frid Downloaded from by guest on July 21, 2018 Abstract Chronic hypoxic exposure induces changes in the structure of pulmonary arteries, as well as in the biochemical and functional phenotypes of each of the vascular cell types, from the hilum of the lung to the most peripheral vessels in the alveolar wall. The magnitude and the specific profile of the changes depend on the species, sex, and the developmental stage at which the exposure to hypoxia occurred. Further, hypoxia-induced changes are site specific, such that the remodeling process in the large vessels differs from that in the smallest vessels. The cellular and molecular mechanisms vary and depend on the cellular composition of vessels at particular sites along the longitudinal axis of the pulmonary vasculature, as well as on local environmental factors. Each of the resident vascular cell types (ie, endothelial, smooth muscle, adventitial fibroblast) undergo site- and time-dependent alterations in proliferation, matrix protein production, expression of growth factors, cytokines, and receptors, and each resident cell type plays a specific role in the overall remodeling response. In addition, hypoxic exposure induces an inflammatory response within the vessel wall, and the recruited circulating progenitor cells contribute significantly to the structural remodeling and persistent vasoconstriction of the pulmonary circulation. The possibility exists that the lung or lung vessels also contain resident progenitor cells that participate in the remodeling process. Thus the hypoxia-induced remodeling of the pulmonary circulation is a highly complex process where numerous interactive events must be taken into account as we search for newer, more effective therapeutic interventions. This review provides perspectives on each of the aforementioned areas. (Circ Res. 2006;99: ) Key Words: pulmonary hypertension pulmonary vasoconstriction fibrocyte inflammation progenitor cell adventitia Pulmonary hypertension contributes to the morbidity and mortality of adult and pediatric patients with various lung and heart diseases. 1 8 Importantly, many of these diseases or conditions are associated with persistent or intermittent hypoxia, either globally or regionally, within confined areas of the lung. These diseases include those associated with upper airway obstruction, such as obstructive sleep apnea, Pickwickian syndrome, obesity hypoventilation syndrome, and damage or injury to the respiratory center either as a primary disorder (Ondine s curse) or secondary to trauma or other neurologic disease. A number of primary lung diseases are also associated with the presence of chronic hypoxia, including chronic obstructive pulmonary disease (COPD), cystic fibrosis, diffuse interstitial fibrosis, bronchopulmonary dysplasia, radiation fibrosis, infiltrative lung tumors, and collagen vascular disease. 1 7 Neuromuscular and skeletal disorders affecting the chest wall such as scoliosis, Duchenne muscular dystrophy, and poliomyelitis may also impair ventilation and be associated with hypoxia-induced pulmonary hypertension. 1,4,8 In many of these conditions, parenchymal lung injury and inflammation contribute directly to the pulmonary hypertension, but there is little question that hypoxia also contributes to the vascular changes and ultimately the pulmonary hypertension observed in each of these conditions. The idea that hypoxia alone can cause pulmonary hypertension and significant structural remodeling of pulmonary arteries (PAs) in humans is supported by observations that in persons living at high altitude there is chronic elevation of pulmonary artery (PA) pressure only, a small portion of which may be reversible with administration of oxygen. 9 Furthermore, in high altitude residents, a far greater increase in PA pressure in response to exercise is observed than in sea-level dwellers. 9 In the lungs of these persons, increased expression of -smooth muscle actin ( -SM-actin) is observed in the walls of small PAs, which normally have little if any smooth muscle, and larger, more proximal vessels exhibit a thickened media and adventitia, findings collectively considered as hallmarks of hypoxia-induced pulmonary vascular remodeling and hypertension. In further support of the idea that chronic hypoxia alone can cause rapid and significant changes in human PAs are the results of a simulated climb of Mount Everest in studies termed Oper- Original received May 26, 2006; revision received July 24, 2006; accepted August 21, From the Department of Pediatrics (K.R.S., M.G.F.), Developmental Lung Biology Laboratory; and Department of Medicine (K.A.F.), Cardiovascular Pulmonary Research Laboratory, University of Colorado at Denver and Health Sciences Center. Correspondence to Kurt R. Stenmark, MD, Professor of Pediatrics, Head, Pediatric Critical Care Medicine, Developmental Lung Biology Laboratory, 4200 E 9th Ave, Box B131, Denver, CO Kurt.Stenmark@UCHSC.edu 2006 American Heart Association, Inc. Circulation Research is available at DOI: /01.RES f 675

2 676 Circulation Research September 29, 2006 ation Everest II. 10,11 In this study, volunteers were exposed to decreasing levels of hypobaric hypoxia over a 6-week period. Catheterization at rest and on exercise was performed after exposure to progressively higher altitudes. After 40 days, pressures were significantly higher than those observed in response to acute hypoxia before the climb, and in addition, there was a lack of vasodilator response to the acute administration to 100% oxygen, findings considered as evidence that there had been induction of significant structural remodeling in the pulmonary vascular bed over this relatively short period of time. 11 The importance of chronic hypoxia as a contributing factor to the development of pulmonary hypertension thus seems relevant to a better understanding of a wide variety of human clinical conditions. The purpose of this review is to examine the cellular and molecular changes that occur in the PAs in response to chronic hypoxic exposure and to review our current understanding of the mechanisms whereby hypoxia initiates changes in the phenotype of various vascular wall cells. The work discussed is derived largely from studies in a variety of animal models. These models, despite their shortcomings, appear to have direct relevance to a better understanding of hypoxia-induced or -associated pulmonary hypertension in humans. Potential interventions, when removal of the hypoxic stimulus is not possible, that may ameliorate or reverse hypoxia-induced vascular changes, will also be discussed. Hypoxia-Induced Vascular Remodeling: Observations in Animal Models Chronic hypoxic exposure induces changes in the structure of PAs, as well as in biochemical and functional phenotypes of each of the cell types composing the artery, from the hilum of the lung to the most peripheral vessels in the alveolar wall. Morphological Changes of the Pulmonary Arteries in Response to Chronic Hypoxia A number of features are observed nearly universally in the PAs of mammals who develop pulmonary hypertension on chronic exposure to hypoxia. (Importantly, there are species that have adapted to residing in chronically hypoxic conditions in which vascular remodeling is not observed, eg, pika, yak, snow pig, llama ) The magnitude of the changes induced depends on the species studied, the sex of the animal, and the developmental stage at which the animal is exposed to hypoxia Structural changes include the appearance of SM-like cells (as defined by the expression of -SM-actin) in previously nonmuscularized vessels of the alveolar wall, so called distal extension of smooth muscle, an apparent sine qua non of hypoxia-induced pulmonary hypertension. In addition, there is medial and adventitial thickening of the muscular and elastic vessels. The medial thickening is believed to be attributable to hypertrophy and increased accumulation of smooth muscle cells as well as increased deposition of extracellular matrix proteins, predominantly collagen and elastin. The adventitial thickening is assumed to be caused by accumulation of fibroblasts and myofibroblasts and an often marked increase in extracellular matrix accumulation (collagens, elastin, fibronectin, tenascin). 13,15,20 Intimal changes have been consistently observed in hypoxic rat models of pulmonary hypertension, yet they are usually minimal, at least from a morphological point of view. 13,15,21 However, in hypoxic neonatal calves with extreme elevations of pulmonary arterial pressure (mean, 100 mm Hg), intimal thickening is more pronounced and perhaps more similar to the changes observed in humans. 22,23 Lastly, many studies have also reported a reduction in the cross-sectional area of the pulmonary vascular bed resulting from loss of small blood vessels (sometimes termed rarefaction or pruning) following chronic hypoxic exposure. 13,24 27 This, along with the remodeling described above, is thought to be responsible for the structural or nonvasoconstrictive component of chronic hypoxic pulmonary hypertension. However, this concept of vascular rarefaction has recently been challenged by reports demonstrating angiogenic responses in the pulmonary capillaries in response to hypoxia. 28,29 This angiogenesis is thought to counteract pulmonary hypertension by lowering perfusion resistance in the pulmonary vascular bed. In support of this idea is the fact that lung overexpression of angiostatin, an antiangiogenic cleavage product of plasminogen, aggravates pulmonary hypertension in chronically hypoxic mice. 30 In addition, overexpression of vascular endothelial growth factor (VEGF) in the lung has been shown to protect against hypoxic pulmonary hypertension. 27 Site-Specific Changes in Pulmonary Vascular Structure: Cellular Mechanisms The cellular and molecular mechanisms used to effect cellular and structural changes at specific locations within the pulmonary circulation are different. This is because the cellular composition of the artery changes along the longitudinal axis of the pulmonary circulation. Although it has been said that pulmonary hypertension is a disease of the distal lung circulation, it is increasingly appreciated that structural change in larger vessels may contribute directly to right ventricular work and failure and to changing flow dynamics and thus distal vascular remodeling. An understanding of the vascular remodeling induced by chronic hypoxia thus requires an evaluation of the cells participating and the potential mechanisms through which these cellular changes are effected at different vascular sites. Vascular Remodeling in Large Proximal Pulmonary Arteries In the large PAs (conducting or elastic), the media and adventitia both increase in thickness in response to chronic hypoxic exposure. In the rat, adventitial thickening is early and dramatic, whereas thickening of the media lags behind. 13,15 Adventitial fibroblasts demonstrate earlier and more significant increases in DNA synthesis compared with smooth muscle cells (SMCs). In fact, medial SMCs were not observed to demonstrate an increase in DNA synthesis until at least day 3, and, at this point in time, the labeling index increased from less than 0.5 to only approximately 1 to 1.5. The findings in the mouse are similar, with a brief rise in the medial cell labeling index in the main PA at days 4 to 6, followed by a rapid decline in medial SMC labeling and total SMC number compared with controls at 3 weeks. This

3 Stenmark et al Hypoxic Pulmonary Remodeling 677 Figure 1. Schematic representation of the potential cellular mechanisms involved in hypoxia-induced remodeling of a pulmonary artery composed of phenotypically heterogeneous cell populations. In this model, cells (designated Immature or Nonmuscle ) exist within the media that exhibit heightened proliferative, migratory, and synthetic capabilities. In response to hypoxia itself, or hemodynamic stresses associated with hypoxia, these cells undergo rapid expansion and contribute to vessel wall thickening, therefore allowing the differentiated SMCs to maintain contractile/homeostatic functions. Thus in this model, media itself contains the cells necessary for structural adaptation to stress or injury. suggests that the medial thickening is largely attributable to increased matrix (elastin and collagen) deposition and cell hypertrophy. The mouse adventitia also undergoes thickening with an accumulation of cells and matrix proteins (predominately collagen). 31,32 In large animals (eg, calf and pig), the responses are different, with early and dramatic medial thickening predominating. 33,34 The differences may be explained by the fact that the cellular composition of the proximal pulmonary and systemic vessels of larger mammalian species (including the cow, lamb, pig, and human) is more complex than that of the rodent species (Figure 1). 14,35 39 The media of large conducting pulmonary and systemic vessels in various large mammalian species, including the human, is composed of multiple phenotypically distinct SMC populations. 14,35 38 These different SMC populations appear to serve different functions based on observations of distinct ion channel expression and proliferative and matrix-producing capabilities in response to many stimuli including hypoxia There is evidence to support the argument that these cells are derived from distinct lineages and are not simply a common cell exhibiting different states of differentiation ,47 Although it is evident that these phenotypically distinct cell populations serve different functions in health and disease, at present, the embryonic origin of distinct cell populations, the ratio of different cells at a given vascular site in a given species, and the mechanisms directing the developmental assembly of these cells types into a functioning, mature blood vessel wall remain to be determined. It has been shown in larger mammalian species that the medial thickening of proximal PAs in response to chronic hypoxia can be accounted for, in large part, by the proliferation of a distinct SM-like subpopulation residing within the media. 37,48 This SM-like population exists in a relatively undifferentiated state (as assessed by SM-specific cytoskeletal and contractile protein marker expression) in comparison with other SMCs within the vessel wall. The in situ observations are consistent with cell culture studies demonstrating that only less differentiated SM-like cells demonstrated increases in proliferation in response to hypoxic exposure. 42,43,49 SMCs that maintain a more differentiated phenotype in culture exhibited growth inhibition under hypoxic conditions. 42 The adventitia of the large PA of calves underwent less thickening than in rats or mice, perhaps because of the fact that the PA media in larger mammals contains cells capable of rapidly responding to hypoxia and increases in wall tension by proliferating and secreting matrix proteins (Figure 1). Because thickening serves to decrease wall stress, the existence of such a cell population within the media of large vessels may provide the vascular wall with a mechanism to adapt rapidly to increased wall tension. Thus, highly differentiated SMCs are spared from dedifferentiating and proliferating and may maintain their normal homeostatic functions, such as contraction in these vessels. Little is known regarding the mechanisms that confer unique proliferative characteristics to specific cell populations that exist in the large PAs. It has been demonstrated that less differentiated and more proliferation-prone medial cells are characterized by exuberant responses to G protein coupled receptor (GPCR) agonists, compared with the differentiated medial SMCs that do not exhibit proliferative responses to hypoxia. 37,42,43,49 These observations suggest that there may be differences in receptor expression and/or intracellular signaling among various medial cell types. Differences in endothelin production, receptor expression, and responses have been described in distinct cell populations derived from the ovine PA. 50,51 Medial SM-like cells, that respond with increased proliferation to hypoxia both in vivo and in vitro, have been demonstrated to exhibit augmented responses to stimulation of the protein kinase C (PKC) pathway. 17,52 Hypoxia-induced activation of GPRCs, with subsequent signaling through G i - and G q -mediated activation of extracellular signal-regulated kinase 1/2 (ERK1/2) has also been shown to be necessary for hypoxia-induced proliferation. 53 Differences in camp response-element binding protein (CREB) expression have also been shown to function as molecular determinants of SMC proliferative capability under hypoxic conditions. 54 Whether hypoxic inducible factor (HIF)-1, the prototypic hypoxia-inducible transcription factor, is directly involved in

4 678 Circulation Research September 29, 2006 hypoxia-induced proliferation of specific medial SMC populations remains unclear at present. 55 Collectively, these observations support the existence of distinct medial cell populations with membrane-bound receptors sensitive to hypoxic activation that engage specific intracellular signaling pathways conferring unique hypoxic proliferative responses. As mentioned above, marked increases in collagen and elastin accumulation are observed in proximal vessels in response to hypoxic exposure and contribute to medial and adventitial thickening. Less is known of the pathways stimulating matrix protein production under hypoxic conditions than of those responsible for cell proliferation. There is reason to believe the former are distinctly different and perhaps even more complex than those regulating proliferation. For instance, hypoxia decreases elastin production in cultured bovine main PA SMCs, whereas in vivo, in chronically hypoxic animals of all examined species, marked increases in elastin accumulation in the PA media are observed. 56,57 On the other hand, hypoxia appears to directly increase collagen production and expression of transforming growth factor (TGF)-, an important regulator of collagen synthesis in fibroblasts, both in vivo and in vitro. 32,34,57 59 The importance of TGF- in hypoxia-induced collagen production and vascular remodeling is demonstrated in recent studies in which a dominant negative mutation in the TGF- receptor blocks chronic hypoxia-induced pulmonary hypertension in rats. 60 In addition, administration of rh relaxin, a hormone with direct effects on collagen and fibronectin production by fibroblasts, and other antifibrotic agents reduce hypoxia-induced pulmonary hypertension, 61 findings that again emphasize the importance of matrix accumulation even in large vessels in hypoxia-induced pulmonary hypertension. More work is needed to determine the mechanisms regulating proliferation and matrix protein production by the various cells comprising the vascular wall of the proximal large PAs under hypoxic conditions, as these changes obviously have significant effects on vascular wall compliance and thus on pulmonary blood flow and right heart function. Many studies now support the concept that the changes observed in large blood vessels in response to chronic hypoxia and other stimuli are important and have significant effects on right heart function. 31,32,62 Increases in wall stiffness in large vessels contribute to the overall increases in impedance and contribute as much as 30% to 40% of the increase in load to which the right ventricle is exposed. 62,63 In addition, this increase in stiffness, associated with vascular remodeling in proximal vessels, leads to an inability of conductance vessels to store and deliver entire stroke volume of the right ventricle and ultimately results in a loss of pulmonary flow during diastole. This disturbance of flow not only increases the load on the right ventricle but also may contribute to decrease in NO production in distal PA endothelial cells, which require steady pulsatile flow for optimal NO production. Vascular Remodeling in Distal Muscular Pulmonary Arteries The distal pulmonary circulation, which is the primary site of hypoxic pulmonary vasoconstriction, also undergoes significant structural change in response to chronic hypoxic exposure. Therefore, an important question is whether the cellular mechanisms that act to cause structural changes in the distal vessels are the same as those used in the proximal arteries. Studies in various species raise the possibility that they are different. Significant differences in the electrophysiologic properties between proximal and distal SMCs exist and contribute to the unique nature of the distal circulation. 40,41,64 Distal PA SMCs cultured from normal calves, demonstrate a resistance to traditional growth-promoting stimuli, including platelet-derived growth factor (PDGF)-BB, angiotensin II, and TGF- 1, as well as to serotonin and endothelin. Importantly, hypoxia also consistently inhibited growth of distal PA cells in the presence or absence of serum. 65 These responses to growth factors and cytokines differ significantly from those described in the far more commonly used bovine vascular SMCs (VSMCs) derived from the media of the main PA, where PDGF, serotonin, endothelin, and angiotensin stimulate cell proliferation rather than cell hypertrophy The responses are also different from those observed in SMCs derived from the rat or mouse PA, where, again, proliferative responses to these factors as well as to hypoxia have been reported. Thus, it appears that the medial SMCs in the distal pulmonary circulation, at least in some species, comprise a uniform population of well-differentiated and relatively growth-resistant cells. Studies in the human pulmonary circulation also suggest differences in proliferative capacity between SMCs derived from proximal and distal vessels. 69 In some reports, cells described as SM like and derived from the distal vessels exhibit greater proliferative responses to mitogens than cells from the proximal vessels. However, it is unclear whether the cells obtained were only from normotensive patients or whether pulmonary hypertensive patients were also included. Furthermore, cells were characterized as SMCs based only on expression of -SMactin (but no other SM-specific marker), raising the possibility that nonmedial cells were cultured and studied, as might occur with myofibroblasts (also -SM-actin cells). Observations of a uniform population of growth-resistant SMCs in the distal circulation raise a question as to what, besides hyperplasia of the resident medial SMCs, could explain the marked medial thickening in response to chronic hypoxic exposure. Importantly, in every study assessing cell proliferation in animals exposed to hypoxic conditions, early and significant increases in fibroblast proliferation were observed. 15,33,70 In addition, hypoxia has been demonstrated to induce differentiation of a fibroblast into a myofibroblast phenotype and to increase myofibroblast numbers in the PA adventitia. 71 In the systemic circulation, early activation, differentiation, and subsequent migration of fibroblasts/myofibroblasts into the media have been suggested as a mechanism used to contribute to thickening. 72 The possibility that this or a similar mechanism is operating to affect medial thickening in small PAs was suggested more than 20 years ago by Sobin et al and, more recently, by Jones and colleagues. 13,73,74 Both investigators have presented evidence in support of the idea that either an adventitial fibroblast or an interstitial lung fibroblast is activated by hypoxia, recruited,

5 Stenmark et al Hypoxic Pulmonary Remodeling 679 Figure 2. Schematic representation of the potential cellular mechanisms involved in hypoxia-induced remodeling of a pulmonary artery composed of a uniform population of differentiated SMCs. In this model, SMCs within the medial compartment are, by and large, growth resistant. Thus, alternative cellular mechanisms are used to thicken a vessel wall in response to hypoxia and associated hemodynamic stresses. These include proliferation and differentiation of fibroblasts into myofibroblasts in the adventitia, as well as their migration into the medial and even intimal compartments. Circulating progenitor cells may also be recruited to the adventitial, medial, or intimal compartments, where they assume mesenchymal or even SMC-like characteristics. Endothelial cells have also been shown to be capable of transitioning into mesenchymal cells, thus raising the possibility that they can also contribute to vascular thickening. Lastly, tissue progenitor cells residing in the adventitia could also participate in the remodeling process. and differentiates into a SM-like cell in the newly muscularized vessel. It seems logical that cells with a larger repertoire of functional capabilities, such as adventitial fibroblasts or even recently described adventitial stem-like cells, may be more capable of proliferating, migrating, and secreting matrix proteins than highly differentiated contractile medial SMCs, the function of which is to control vascular tone and blood flow within the distal circulation. 20,75 Recent in vivo and in vitro studies evaluated the cellular composition of the distal PA media of normoxic and chronically hypoxic neonatal calves. The media of control vessels was comprised of a phenotypically uniform population of SMCs, whereas at least 2 distinct cell populations were detected in chronically hypoxic vessels. One was comprised of highly differentiated and significantly hypertrophied SMCs, and another was an undifferentiated cell population with high proliferative and migratory capabilities. Experimental data suggested that this latter cell population was of a nonresident, nonmedial origin (be it adventitial, interstitial, or circulating) and that these cells have migrated into the media. This possibility was supported by recent studies showing that hypoxia induces recruitment of mesenchymal precursors, including fibrocytes, into the vessel wall. 76,77 Fibrocytes comprise a subpopulation of circulating leukocytes (CD45, CD11b ) that, at the site of tissue injury, can assume a mesenchymal phenotype ( -SMactin, type-i collagen ) and the functions of resident fibroblasts or myofibroblasts. Fibrocytes have been described as contributing to fibrosis in a number of other fibroproliferative conditions in the lung In summary, there is experimental data to suggest that the cellular mechanisms contributing to remodeling of the distal vasculature (as schematically depicted in Figure 2) are different from those of the proximal circulation (Figure 1), especially in larger mammalian species, in which proximal vessels are comprised of heterogeneous cell populations with distinct growth and differentiation properties. In vessels in which the media is comprised of a uniform population of differentiated SMCs, adventitial cells as well as circulating progenitors may provide important cellular sources of the medial thickening (Figure 2), which has traditionally been ascribed to resident SMC proliferation. More studies are needed in this area to determine the mechanisms controlling medial thickening of the distal circulation, because it constitutes a critical part of the hypoxic pulmonary vascular remodeling response. Structural Changes in Nonmuscular Alveolar Wall Vessels Perhaps the most characteristic, yet least understood, change in the pulmonary vasculature that occurs in response to chronic hypoxic exposure is the muscularization of the vascular segments that normally lack a muscular coat converting the precapillary segment into a resistance structure. It is the first cellular event to occur in response to chronic hypoxic exposure, as well as the first to disappear on withdrawal of the hypoxic stimulus. 13 Mural cells are reported to increase in size and number, and a media and adventitia are formed to produce a new structural vessel profile. Several mechanisms have been invoked to explain the distal muscularization process. Certain vessels, termed partially muscular, contain pericytes and/or intermediate cells. These cells exhibit SM-like characteristics, although they can be distinguished morphologically and biochemically from a differentiated SMC. 13,15,81 In vessels where either pericytes or intermediate cells are present, it has been suggested that hypoxia induces differentiation and proliferation of these cells such that they may directly contribute to the observed muscularization. Other mechanisms, including those discussed below may also operate to contribute to distal muscularization. In the smallest alveolar wall vessels, which normally lack any elastic lamina and possess a capillary-like wall structure,

6 680 Circulation Research September 29, 2006 Figure 3. Schematic representation of the potential cellular mechanisms involved in hypoxia-induced remodeling of a nonmuscular pulmonary artery. At least 2 possibilities have been implicated in this process. Hypoxia can induce phenotypic modulation of a resident interstitial lung fibroblast into a myofibroblast, and its recruitment to the pulmonary vessel wall, where it ultimately assumes a SMC-like phenotype. Another possibility is that a subset of circulating leukocytes with the potential to express mesenchymal characteristics (termed fibrocytes) is exuded from the vessel, incorporate themselves into the vascular wall, and assume mesenchymal characteristics. the mechanisms of muscularization may be different and are probably more complex than simply migration of pericytes down the vessel. One suggested mechanism is the recruitment of interstitial fibroblasts through a process of migration, alignment, and incorporation into the vessel wall (Figure 3). 74,82 It is also possible that in these vessels, especially so-called corner vessels in the alveolus, there is extravasation of inflammatory and mesenchymal precursor cells. 77,83 Similar to their role in muscularized vessels discussed above, these cells could differentiate into SM-like cells and contribute to hypoxia-induced distal muscularization (Figure 3). The possibility that endothelial cells transdifferentiate into a SM-like cell and contribute to distal muscularization also needs to be considered. Endothelial/mesenchymal transdifferentiation plays an important role in vascular development of the pulmonary and systemic circulations. 84 Microvascular endothelial cells have been reported to have the capability to transdifferentiate into mesenchymal-like cells. 85 Another intriguing possibility, although never explored to our knowledge in hypoxic pulmonary disease, is that lung epithelial cells may transdifferentiate into mesenchymal cells. Epithelial/mesenchymal transition (EMT) is critical during normal embryonic development and is increasingly appreciated to contribute to fibrosis and cancer development and progression. 86,87 TGF- and Wnt signaling are important transducers of the EMT process. These systems are known to be regulated by hypoxia, thus supporting the idea that EMT could also contribute to the distal PA muscularization process. A better understanding of distal muscularization could provide new opportunities for therapy. Cell-Specific Changes Induced by Hypoxia Changes in Endothelial Cell Phenotype in Chronic Hypoxic Pulmonary Hypertension As stated above, chronic hypoxic exposure increases intimal thickness in the pulmonary circulation by causing hypertrophy and hyperplasia of the endothelial cells and thickening of the subendothelial space. 15,21,33,34,70 There can be focal disruption of the pulmonary endothelial cell basement membrane, creating a patchy appearance of microfibrillar material and a thickened subendothelial space, much like that reported in the aorta of hypertensive rats, observations consistent with experiments showing that hypoxic exposure increases lung vascular permeability measured by direct protein extravasation. 88,89 Influx of plasma proteins into the vessel wall may activate vascular wall proteases, initiating a series of remodeling events in a series of events described by Rabinovitch. 90 Endothelial cells, in response to hypoxia, have been shown to produce more laminin, fibronectin, and elastin, all of which affect endothelial as well as SMC function. 91 On the other hand, chronically hypoxic endothelial cells produce and secrete less heparan sulfate proteoglycans than do normoxic cells, suggesting that chronic hypoxia impairs the ability of the endothelium to produce growth-restrictive molecules for the SMCs. 92 Importantly, the endothelial changes observed differ at various sites along the longitudinal axis of the PA. 13,15 It is also interesting that significant differences are noted between PA endothelial cells of the yak, a species resistant to the development of pulmonary hypertension, and the closely related bovine species, the most susceptible to hypoxia-induced pulmonary hypertension of all species examined, observations also supporting the idea that the endothelium plays a critical role in mediating many of the changes observed in the hypoxia-induced hypertensive process. 12,22 These changes in pulmonary endothelial cell structure and local environmental milieu are accompanied by alterations in their physiological and metabolic functions. Hypoxic exposure decreases antithrombotic potential, increases permeability and expression of inflammatory cell markers, and interferes with a variety of cell plasma membrane-dependent receptors, metabolic and transport functions of the endothelial cell. 91,93 95 Hypoxia exerts profound effects on the physical state and composition of endothelial cell plasma membrane lipids, membrane fluidity, and plasma membrane phospholipid and fatty acid composition. 93,96,97 The mechanisms through which hypoxia alters plasma membranes in the pulmonary circulation are not clear. Increased contents of

7 Stenmark et al Hypoxic Pulmonary Remodeling 681 Figure 4. Molecules secreted by the chronically hypoxic endothelial cells. Factors listed in parenthesis are downregulated. malondialdehyde and conjugated dienes suggest that lipid peroxidation occurs and plays a role. 93 Another potential mechanism is activation of membrane phospholipases, which would account for the reduction in the content of plasma membrane phospholipids and the concomitant increase in the release of fatty acids. 93 Increases in phospholipase A and C and diacyl glycerol (DAG) lipase activity have been demonstrated. 98 Membrane changes observed in hypoxic endothelial cells contribute directly to the alterations in growth factor receptor and function, as well as in the transport and processing of biogenic amines, extracellular nucleotides, and adenosine, and thus could have significant effects on pulmonary vascular tone and structure. 94,97,99 Hypoxia alters surface coagulant properties of endothelial cells (Figure 4), suppressing normal thrombomodulin production and inducing procoagulant activity, largely through the increased expression of tissue factor. 100,101 Hypoxia also increases interleukin (IL)-1 and IL-6 mrna levels and IL-1 production in cultured endothelial cells, probably through nuclear factor B (NF- B) or NF-IL Hypoxia (and/or IL-1) can also increase expression of endothelial leukocyte molecule (ELAM)-1, intracellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 on endothelial cells. These observations suggest that hypoxia induces changes in the endothelial cell, leading to prothrombotic and proinflammatory interactions with circulating cells that collectively participate in the pulmonary hypertensive response. Indeed, increased adherence of both leukocytes and platelets have been observed in chronically hypoxic rats. This proinflammatory response could also facilitate recruitment and migration of progenitor cells into the vessel wall. 103,104 Chronic hypoxic exposure is associated with a number of changes in the production and release of potent vasoactive substances by the endothelium which can exert significant effects not only on the contractile state of SMCs but on their proliferative and synthetic state as well 105,106 (Figure 4). Decreased production and/or activity of prostacyclin and NO have been reported Increases in the production of platelet-activating factor (PAF), leukotrienes, HETEs, endothelin, serotonin, and PDGF have been observed In addition, recent reports have suggested that there is a release of extracellular ATP by PA endothelial cells under hypoxic conditions. 114 This is interesting because ATP is also released under conditions such as osmotic swelling, which, as discussed above, has been described in the setting of hypoxic exposure. 115 Extracellular ATP has direct effects not only on the endothelial cell, where, acting through purinergic receptors and AMP-activated protein kinase (AMPK), it participates in controlling NO release, but also on other endothelial cell functions. 116 Released nucleotides also directly activate SMCs and fibroblast proliferation and migration. 114,117,118 The direct relevance of the release (or lack thereof) of many of these molecules to the hypoxia-induced hypertensive process has been confirmed in whole animal studies. Rats treated with PAF inhibitors and 5-lipoxygenase inhibitors, as well as mice lacking 5-lipoxygenase, demonstrate less pulmonary hypertension and vascular remodeling in response to hypoxia. 119,120 Endothelin receptor antagonists inhibit hypoxia-induced pulmonary hypertension. 112 Germline deletions in NO synthase 3 (NOS3) are associated with increased right ventricular pressure and distal muscularization in both normoxia and hypoxia. 121,122 However, 1 group of investigators found a decrease in pulmonary arterial muscularization early in the course of hypoxic exposure. 123 Increased production of the NOS3 cofactor tetrahydrobiopterin is associated with attenuated development of hypoxic pulmonary hypertension and vascular remodeling, whereas NOS3 deficiency results in enhanced pulmonary hypertension and remodeling. 124 Deficiency in other NOS isoforms has not been clearly associated with increased pulmonary vascular responses to hypoxia. 125 Decreases in other vasodilator genes, such as atrial natruretic peptide adrenomedullin, and heme oxygenase-1, and prostacyclin, are all also associated with enhanced hypoxia-induced pulmonary hypertension and/or remodeling Conversely, increased expression of the serotonin (5-hydroxytryptamine [5-HT]) transporter (5-HTT) gene is associated with enhanced hypoxic pulmonary hypertension. 130 Decreased expression of the 5-HT-1B receptor is associated with decreased hypoxia responses. 131 Deficiency in the endothelin-1b receptor in rats is associated with increased hypoxic pulmonary hypertension and remodeling. Interestingly, germline overexpression of endothelin (ET)-1 in a mouse did not result in significant pulmonary hypertension

8 682 Circulation Research September 29, 2006 Figure 5. Chronic hypoxia exhibits significant effects on Ca 2 handling and thus on the vasoconstrictive and proliferative (not shown) status of the PA SMCs. Hypoxia downregulates K v channel expression, leading to membrane depolarization. Activation of voltagedependent Ca 2 channels (VDCCs), as well as store- and receptor-operated channels (SOC and ROC, respectively) leads to Ca 2 entry. There can also be RhoA/Rho kinase mediated Ca 2 sensitization of VSMC contraction. Numerous GPCR-dependent vasoconstrictors induced by hypoxia, including ET-1, 5-HT, thromboxane (TXA 2 ), thrombin, and sphingosine-1 phosphate (S1P), lead to stimulation of guanine nucleotide exchange factors (GEFs) and activation of the small GTPase RhoA, which binds to and activates Rho kinase. Activated Rho kinase, in turn, phosphorylates the myosin-binding subunit (MBS) of MLCP and/or the MLCP inhibitory protein CPI-17. Inhibition of MLCP increases phosphorylation of MLC and augments contraction at a given level of cytosolic [Ca 2 ] and Ca 2 /calmodulin (CaM)-mediated activation of MLCK. PLC indicates phospholipase C- ; RyR, ryanodine receptor; SR, sarcoplasmic reticulum. but was associated with pulmonary inflammation and fibrosis. 132 The collective body of evidence described above demonstrates that the endothelial cell is a direct target of hypoxia. Marked changes in its permeability, coagulant, inflammatory, and protein synthetic capabilities are observed in response to hypoxic exposure. These changes can affect directly or indirectly underlying SMCs and adventitial fibroblasts, thus contributing to chronic abnormalities in vascular tone and structure. Changes in SMC Phenotype in Chronic Hypoxic Pulmonary Hypertension: Specific Contribution to Vascular Tone and Structure The severity of chronic hypoxic pulmonary hypertension is determined, at least in part, by the extent of structural changes in the medial compartment of the pulmonary arterial wall. These changes include SMC proliferation, hypertrophy, matrix protein production, and recruitment of adventitial or circulating cells. Hypoxia, blood-borne and locally produced cytokines, and mechanical stress collectively act to drive the cellular responses within the media. These stimuli activate a cascade of intracellular signaling mechanisms, including tyrosine kinases, mitogen-activated protein kinases (MAPKs), PKC, phosphoinositidyl 3-kinase (PI3K), SMAD phosphorylation, calcium (Ca 2 ) entry and Rho kinases, which collectively act to control SMC contractility, growth, differentiation, and matrix protein synthesis (Figure 5). Synergy between different stimuli and the resulting cross-talk between signal transduction pathways augment the extent of vascular changes within the media. Susceptibility to these stimuli is enhanced when inhibiting mechanisms, such as endothelial barrier function, local production of heparan sulfates and prostaglandin I 2 (PGI 2 ) and NO-induced increases in cyclic nucleotides, are impaired. Intrinsic (genetic, developmental, acquired, or epigenetic) differences in growth and matrix synthetic capacity, as well as local and regional phenotypic heterogeneity of pulmonary SMCs also regulate the pattern of remodeling of the tunica media in response to chronic hypoxia. Changes in receptor expression or function

9 Stenmark et al Hypoxic Pulmonary Remodeling 683 as well as in Ca 2 handling have been observed in SMCs from hypertensive pulmonary arteries. Examples of these changes are discussed. Hypoxia-Induced Changes in Serotonin Transporter and Receptor Function in SMCs Several studies have shown a role for 5-HT in the regulation of acute and chronic hypoxic responses, particularly as they relate to contraction and growth of SMCs. 130, HT is a vasoactive and, in some SMCs, a mitogenic molecule released from platelets, pulmonary neuroendocrine cells, and endothelial cells. Moreover, increases in plasma serotonin levels have been observed under hypoxic conditions. 136 Exposure of PA SMCs to elevated levels of 5-HT results in increased SMC contraction and proliferation. 134 These observations have generated great interest in elucidating the mechanisms through which serotonin exerts its effects and contributes to the remodeling process. An active transporter that internalizes serotonin (5-HTT) and multiple cell surface serotonin receptors (5-HT-2A, 5-HT-1B/1D, and 5-HT-2B) have been described on SMCs, and it appears that a balance of transporter and receptor expression and function determines the cellular responses to serotonin. 135 Overexpression of the 5-HTT gene in rats exposed to chronic hypoxia augmented vascular remodeling, as did continuous infusion of serotonin. 130 Administration of the 5-HTT inhibitors citalopram and fluoxetine partially reduced increased pulmonary vascular resistance in chronically hypoxic mice, a finding supported by another experiment, in which hypoxic pulmonary hypertension was attenuated in knockout mice with disruption of 5-HTT. 137,138 It has also been demonstrated that the increased contractile response to 5-HT in PAs isolated from chronically hypoxic wild-type mice is mediated through the 5-HT-1B receptor and that 5-HT-1BR knockout mice develop less pulmonary vascular remodeling and hypertension than do wild-type mice. 131 However, Marcos et al reported that chronic hypoxia-induced pulmonary hypertension and increased vessel muscularization were not reduced by the 5-HT-1B/1D receptor antagonist GR The 5-HT-2B receptor has also been implicated in hypoxic pulmonary hypertension, as it has been shown to regulate 5-HTT activity HT-2BR null mice fail to develop hypoxic pulmonary hypertension despite maintaining an acute response to hypoxia. 139 Moreover, 5-HT-1BR, 5-HT-2BR, and 5-HTT are colocalized in PA SMCs, and the 5-HT-2BR functionally interacts with the 5-HT-1BR. This cross-talk between the G q -coupled 5-HT-2BR and the G i -coupled 5-HT- 1BR has been suggested to facilitate the development of pulmonary hypertension. 135 It is possible that serotonin receptors act by producing reactive oxygen species via the NADPH oxidase-like enzyme to induce SMC proliferation. 140 Collectively, these observations suggest that the mechanisms of action of 5-HT, its transporter, and receptors are rather complex and may be species and cell type dependent. Future studies will be needed to delineate how hypoxia effects serotonin-induced changes in SMC phenotype and the relation of these changes to hypoxia-induced vascular remodeling. Chronic Hypoxia-Induced Changes in Ionic Balance and Calcium Homeostasis in SMCs: Effects on Tone and Proliferation Accumulating evidence indicates that both subacute and chronic hypoxia cause intrinsic changes in the ionic balance and calcium homeostasis of PA SMCs, resulting in membrane depolarization, elevation in resting intracellular calcium [Ca 2 ] i, persistent vasoconstriction, and changes in electrophysiological and calcium responses to vasoconstrictors and vasodilators McMurtry et al proposed almost 2 decades ago that chronic hypoxia might have some overlap with the cellular and molecular mechanisms involved in mediating acute hypoxic pulmonary vasoconstriction. 144 Furthermore, because chronic hypoxia is also associated with variable degrees of medial SMC hypertrophy and increases in numbers of SMCs or SM-like cells, a common hypothesis is that pulmonary vasoconstriction and PA SMC proliferation use overlapping signaling processes that contribute to vasoconstrictive and structural changes. For example, hypoxia-induced increases in [Ca 2 ] i cause pulmonary vasoconstriction and also act as an important stimulus for SMC proliferation. 145 These observations led to the hypothesis that hypoxia-induced decreases in K v channel currents, membrane depolarization, and increased [Ca 2 ] i in SMCs may serve as a shared mechanism to cause pulmonary vasoconstriction and stimulate SMC proliferation. Indeed, chronic hypoxia reduces K channel activity and also mediates changes in the transcriptional and translational control of K channel genes in PA SMCs. 141,143 Chronic hypoxia downregulates the mrna and protein expression of several pore-forming -subunits of K v channels in rat PA SMCs (K v 1.1, 1.5, 1.6, 2.1, and 4.3), whereas no appreciable expression changes are seen in mesenteric artery SMCs. 146 In addition to effects on SMC proliferation, decreases in K v channel expression may actually protect the cell from apoptosis. Inhibition of K channel activity can attenuate proapoptotic volume decreases by maintaining sufficient potassium within the cytoplasm to inhibit apoptosis. 143 It is not known exactly what controls chronic hypoxia-induced changes in K v expression. However, many vasoactive agonists and growth factors, which are known to be released by endothelial cells (including ET-1) in response to hypoxia as described above, can have effects on K v channel expression. 143,145 Concordant with the observations of decreases in K v channel expression, sustained membrane depolarization, activation of L-type calcium channels, and increases in intracellular calcium, are observations that calcium channel antagonists nifedipine and verapamil attenuate hypoxia-induced pulmonary hypertension. 147 However, other studies showed that the effects of calcium channel blockers are often only partial or temporary and are sometimes complicated by changes in cardiac output. 148 Interestingly, in chronically hypoxic rats, nifedipine was ineffective in reducing pulmonary hypertension, whereas another vasodilator, NIP121, caused significant reduction in PA pressure and vascular resistance. 149 More dramatically, the elevated resting [Ca 2 ] i in PA SMCs of chronically hypoxic rats was unaffected by nifedipine but was reduced instantaneously to the level of control PA SMCs by the removal of extracellular Ca

10 684 Circulation Research September 29, 2006 These observations suggest that Ca 2 influx pathways, other than voltage-dependent calcium channels (VDCCs), are involved in chronic hypoxic pulmonary vasoconstriction and remodeling. Nonselective cation channels encoded by the canonical transient receptor potential (TRPC) gene family constitute alternative pathways of Ca 2 entry into VSMCs. Multiple TRPC subtypes have been identified in PA SMCs, with the existence of functionally distinct receptor-operated and storeoperated cation entry pathways The store-operated Ca 2 channel TRPC-1 and the receptor-operated Ca 2 channel TRPC-6 are coexpressed in PA SMCs. Chronic hypoxic exposure upregulated TRPC expression and enhanced both store-operated and receptor-operated calcium entry into PA SMCs. 152 Upregulation of TRPC by hypoxia appears subtype specific, such that TRPC-1 and TRPC-6 expression is doubled or tripled, whereas other TRPCs are unaltered. The mechanisms regulating hypoxia-induced upregulation of these Ca 2 channels are currently unclear. However, recent studies show that, during serum-induced proliferation, TRPC-1 mrna is upregulated, resting Ca 2 is elevated, and store-operated Ca 2 entry is enhanced and that TRPC-6 expression is increased by PDGF in PA SMCs. 151,155 PA SMC proliferation can be blocked by antisense oligonucleotides against TRPC, supporting the idea that their upregulation is a required step in certain SMC growth processes. Increases in store- and receptor-operated calcium entries in chronically hypoxic PA SMCs may also contribute to alterations in pulmonary vasoreactivity. Increases in TRPC expression could contribute to the increase in vasoreactivity to vasoconstrictors such as endothelin, angiotensin II, and serotonin observed in PAs from chronically hypoxic animals. This is attributable to the fact that these vasoconstrictors activate GPCRs, which stimulate phospholipase C, to generate inositol-1,4,5-trisphosphate (IP 3 ) and DAG, which in turn may work synergistically to promote Ca 2 entry through TRPC channels. Thus, upregulation of TRPC channels, in conjunction with elevated circulating levels of vasoconstrictors and increased agonist receptors (ie, ET-A receptor and 5-HT-2BR) in PA SMCs may provide a powerful mechanism for increasing and maintaining vascular tone in chronic hypoxic pulmonary hypertension (Figure 5). Intracellular signaling, via GTP-RhoA and its downstream effector Rho kinase (Rho/Rho kinase signaling), is also increasingly appreciated as an important signaling pathway in the pathogenesis of chronic hypoxic pulmonary hypertension because of its potential to cause sustained vasoconstriction and its effects on the proliferative and differentiation state of vascular wall cells. 156,157 The contractile state of VSMCs is regulated by phosphorylation (causing contraction) and dephosphorylation (causing relaxation) of the 20-kDa regulatory myosin light chain (MLC). Phosphorylation of MLC is catalyzed by Ca 2 /calmodulin-dependent MLC kinase (MLCK) and dephosphorylation by calcium-independent MLC phosphatase (MLCP), which is targeted to myosin by its regulatory myosin binding subunit (MBS). Thus, the balance and activity of MLCK and MLCP regulate contraction. At a given level of cytosolic Ca 2, the activity of both enzymes can be modulated by second messenger-mediated pathways to change MLC phosphorylation and thus force of contraction; in other words, the activity of these pathways will change the calcium sensitivity of contraction. 156,157 There is indirect evidence that hypoxia activates RhoA in VSMCs. 157,158 Furthermore, several vasoconstrictors speculated to participate in hypoxia-induced sustained vasoconstriction (including ET-1, 5-HT, and thromboxane) activate RhoA in VSMCs through GPCR-mediated signaling pathways. 156,157 Interestingly, GPCR-independent signaling to RhoA also occurs and can be mediated by receptor and nonreceptor tyrosine kinases and by recruitment of other GTPases. 156,157 The importance of Rho kinase signaling in chronic hypoxic pulmonary hypertension is highlighted by the experiments of McMurtry et al, in which the Rho kinase inhibitor Y27632, but not nifedipine, caused marked vasodilation in the hypertensive animals. 159 The severity of chronic hypoxic pulmonary hypertension has also been shown to be reduced by chronic blockade of the Rho/Rho kinase signaling. 29,159 Collectively, these observations suggest that chronic hypoxia induces changes in SMC Ca 2 handling and intracellular Ca 2 concentrations through numerous mechanisms, as well as changes in Rho/Rho kinase signaling, which act in concert to have profound effects on the contractile and proliferative state of SMCs (Figure 5). Changes in Adventitial Fibroblast Phenotype and Function in Chronic Hypoxic Pulmonary Hypertension A rapidly emerging concept is that the vascular adventitia acts as a biological processing center for the retrieval, integration, storage, and release of key regulators of vessel wall function. In fact, under certain conditions, the adventitial compartment may be considered the principle injury-sensing tissue of the vessel wall. 20 In all species studied, hypoxic exposure induces early and often dramatic adventitial remodeling. 15,20,70 Accumulating experimental data suggest that, in response to vascular stresses including hypoxia and overdistention, the adventitial fibroblast is the first cell to be activated, to proliferate, to upregulate contractile and extracellular matrix proteins, and to release factors that can directly affect medial SMC tone and growth, as well as stimulate the recruitment of inflammatory and progenitor cells. 15,20,33,70,72,77 Each of these hypoxia-induced changes in fibroblast phenotype modulates either directly or indirectly overall vascular function and structure. In vitro experimental studies support the notion that adventitial fibroblasts have greater propensity to proliferate under hypoxic conditions than do resident PA SMCs It has even been suggested that hypoxia-induced proliferation is a unique property of the PA fibroblast. 161 Current evidence implies that activation of G i and G q family members, perhaps in a ligand-independent fashion, with subsequent stimulation of PKC and mitogen-activated PKC family members, are important regulators of hypoxia-induced fibroblast proliferation. 114,118,160 Activation of PI3K and synergistic interaction with Akt, mammalian target of rapamycin (mtor), and p70 ribosomal protein S6 kinase have also recently been demonstrated to be essential for the proliferative response of PA

11 Stenmark et al Hypoxic Pulmonary Remodeling 685 adventitial fibroblast to hypoxia. 114,118 Transcriptional targets of hypoxia and/or the aforementioned signaling pathways include Egr-1, HIF-1, and HIF-2. Knockdown experiments have shown that both Egr-1 and HIF-2 participate in the proliferative response. 163,164 It has also been shown that downstream targets of HIF-1, angiotensin converting enzyme (ACE), and the angiotensin II receptor type 1 (AT 1 ) are upregulated by hypoxia and that inhibitors of ACE and AT 1 can block hypoxia-induced, HIF-dependent, adventitial fibroblast proliferation In addition, mice partially deficient in either HIF-1 or HIF-2 have attenuated hypoxia-induced pulmonary hypertension and remodeling. 168,169 It should be stressed that the arterial adventitia, like the lung interstitium, is composed of heterogeneous fibroblast populations and that only specific subsets of fibroblasts appear capable of proliferating in response to hypoxia. 20,170 Experimental evidence suggests that a selective expansion of these hypoxia-sensitive fibroblast subpopulations accounts for the marked accumulation of fibroblasts in the adventitia of chronically hypoxic animals, also consistent with observations that fibrogenic foci in the lung are comprised of selective fibroblast populations. 20,170 PA adventitial fibroblasts derived from chronically hypoxic animals exhibit heightened growth responses to a number of growth promoting stimuli including hypoxia as well as alterations of proliferation associated signaling pathways, compared with fibroblasts derived from normoxic animals. 161,162,171,172 For instance, the atypical PKC isozyme acts as a proproliferative kinase in fibroblasts from chronically hypoxic animals, whereas it exhibits antiproliferative actions in fibroblasts from normoxic animals. 172 These observations raise the possibility that chronic hypoxia leads to the emergence of fibroblasts in the adventitia that have lost their ability to limit stimulus-induced proliferation. Hypoxia-activated PA adventitial fibroblasts may exert significant effects on other cell types within the vessel wall through the production of paracrine factors (by HIF-1 dependent mechanisms), which have potent stimulatory effects on SMC proliferation, or production of reactive oxygen species (ROS), generated in large part through a distinct NADPH oxidase system. 173,174 Fibroblast-produced ROS can exert potent effects on vascular tone either directly by stimulating contraction of SMCs and/or by acting as a sink and destroying endothelial derived NO before its optimal effects on SMC relaxation have been achieved. 174 In support of the important role of NADPH-derived radicals in hypoxia-induced pulmonary hypertension and vascular remodeling is the fact that both responses are completely abolished in NADPH (GP91Fox) knockout mice. 175 Hypoxia-induced pulmonary hypertension and numerous other vasculopathies are all associated with early and dramatic increases in the differentiation of fibroblasts into a myofibroblast ( -SM-actin ) phenotype. 71,74,77, Activated fibroblasts and myofibroblasts contribute to the structural remodeling of the vessel through increased production of collagen and other extracellular matrix proteins including fibronectin, tenascin, and elastin. 20,34,77,176,177 Myofibroblast accumulation can also contribute to the abnormalities of tone observed in chronic hypoxic pulmonary hypertension. The contractile properties of myofibroblasts appear to be different from those of SMCs with a slower onset of contraction in response to vasoactive stimuli and a failure to relax in response to vasodilating stimuli reported. 176 Hypoxia can induce significant changes in the production of chemokines and cytokines by the adventitial fibroblast including MCP-1, SDF-1, thrombin, and VEGF. 20,76 These chemokines can serve to recruit inflammatory cells and circulating progenitor cells (discussed below). In addition, activated adventitial fibroblasts are known to secrete a variety of proangiogenic factors. 20,72,76 In this context, it has been reported that hypoxia induces a rapid expansion of the PA adventitial vasa vasorum. 76,77 Expansion of the vasa vasorum appears important in contributing to the progression of many vascular diseases, possibly by providing a conduit for delivery of inflammatory and progenitor cells to the vessel wall. 20,180 Transcriptional Mechanisms Contributing to Phenotypic Changes in Pulmonary Vascular Wall Cells Under Hypoxic Conditions See the online data supplement, under Section 1, available at Role of Inflammation, Progenitor Cells, and Vasa Vasorum in Hypoxia-Induced Vascular Remodeling Relatively little attention has been given to the possibility that pulmonary inflammation and/or a noninflammatory accumulation of circulating monocytes/macrophages might contribute to the abnormalities of structure and function seen in the PAs following chronic hypoxic exposure. However, there is accumulating evidence to support the idea that both acute and chronic exposure of animals to even moderate hypoxia results in increased expression of lung inflammatory cytokines, chemokines, and adhesion molecules and in the accumulation of leukocytes within the lungs and in and around lung blood vessels. It is increasingly appreciated that factors produced by leukocytes have significant effects on the phenotype of local vascular wall cells, including increases in proliferation and matrix protein production and changes in the responses to vasoconstricting or vasodilating substances. Additionally, these inflammatory cells induce recruitment of circulating mesenchymal precursors that could directly contribute to the remodeling process (Figure 6). Several recent animal studies document the enhanced expression of inflammatory mediators (monocyte chemoattractant protein [MCP]-1, macrophage inflammatory protein [MIP]-2, IL-1, IL-6) and increased numbers of macrophages and neutrophils in the lungs of mice and rats exposed to acute hypoxia. 128,181,182 At early time points, the increase in macrophage and neutrophil numbers was associated with an increase in albumin extravasation consistent with the development of mild vascular leak. This finding is similar to acute hypoxia-induced changes in lung vascular permeability reported by others and is also observed in response to acute hypoxia in the mesenteric circulation. 183,184 Human studies also suggest that acute hypoxic exposure is associated with leukocyte recruitment and cytokine production in the lung. In

12 686 Circulation Research September 29, 2006 Figure 6. Schematic representation of the potential roles of resident adventitial fibroblasts and circulating monocytes/ fibrocytes in the hypoxia-induced structural vascular remodeling process. (1) Hypoxia activates resident adventitial fibroblasts (or medial SMCs) to secrete chemokines/cytokines that (2) induce recruitment of circulating monocytes/ fibrocytes through the vasa vasorum and pulmonary capillaries to the pulmonary perivascular space, where (3) these recruited cells exhibit both direct and indirect effects on the remodeling process, such as differentiation into mesenchymal cells/myofibroblasts, production of collagens, and stimulation of resident vessel wall cell contraction and proliferation, and stimulate the process of neovascularization. patients with high-altitude pulmonary edema (HAPE), increased numbers of neutrophils and macrophages, in bronchoalveolar lavage fluid, as compared with healthy individuals, have been observed. 185,186 In addition, increased levels of tumor necrosis factor (TNF)-, IL-1-, IL-6, and IL-8 have been noted, levels that quickly reversed to normal following return to normoxic conditions. 186 Chronic hypoxic exposure results in the robust and persistent appearance of mononuclear cells in the PA adventitia and media of both weanling rats and neonatal calves. 77 Interestingly, at least in these 2 animal models, hypoxia-induced accumulation of mononuclear cells appeared specific to the pulmonary circulation because no macrophage recruitment was noted in systemic vessels (aorta, femoral, carotid). Further, no neutrophils were identified in the pulmonary perivascular areas at any of the time points ( 24 hours) evaluated. 77 A substantial proportion of the mononuclear cells that accumulated around PAs was comprised by fibrocytes (cells characterized by dual expression of leukocytic and mesenchymal markers). As mentioned earlier, fibrocytes may play a particularly important role in the hypoxia- or injuryinduced vascular remodeling processes (Figure 6). They have been described as important contributors to the fibrosis observed in lung injury, wound healing, and asthma , The recruited monocytes and fibrocytes release a variety of factors, which can act to increase the proliferation and/or differentiation of resident fibroblasts and SMCs. In addition, fibrocytes are potent producers of extracellular matrix proteins, especially collagen, raising the possibility that nonresident, recruited mesenchymal cells contribute to vascular fibrosis Fibrocytes also produce angiogenic factors and could contribute to the neovascularization of the vasa vasorum described above. 78,80 Other investigators have also suggested a role for circulating bone marrow derived cells in chronic hypoxia induced remodeling. 190 The contribution of circulating mononuclear cells/fibrocytes to the hypoxia-induced vascular remodeling process was confirmed by depletion studies, in which the number of these cells was reduced in the circulation of hypoxic rats using intravenous injections of liposome-encapsulated clodronate or gadolinium chloride. 77 A striking reduction in adventitial thickening as well as a near complete inhibition of the accumulation of collagen, fibronectin, and tenascin-c were documented. 77 Questions arise as to how leukocytes and precursor cells traffic into the vascular wall, particularly the adventitia, under conditions of chronic hypoxia. In the systemic circulation, the adventitial vasa vasorum microcirculation undergoes marked neovascularization in a number of vasculopathies and is thought to serve as a conduit for continued delivery of inflammatory and progenitor cells to the vessel wall. 180,191,192 Similar changes have been documented in the pulmonary arteries of chronically hypoxic calves. 76 Furthermore, experiments have shown that inhibition of plaque neovascularization reduced macrophage accumulation and progression of advanced atherosclerosis. 180 Other experiments have shown that hypoxic fibroblasts support the growth of vasa endothelial cells. 193 Thus, local fibroblasts and recruited inflammatory progenitor cells play a role in driving the neovascularization of the vasa and contribute to a feed-forward loop of vascular remodeling. Future experiments need to be directed at determining the factors that recruit and then retain inflammatory and progenitor cells in the vessel wall so as to ultimately design specific therapies to turn the process off. Chronic Intermittent Hypoxia and Pulmonary Vascular Remodeling See the online data supplement, under Section 2. Sources of Funding This work was supported by NIH grant SCOR HL ; NIH Program Project grant HL ; American Heart Association Grants PM Z (to K.R.S. and M.G.F.) and EIA N (to K.A.F.); and NIH grant R01 HL (to K.A.F.). None. Disclosures

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18 Hypoxia-Induced Pulmonary Vascular Remodeling: Cellular and Molecular Mechanisms Kurt R. Stenmark, Karen A. Fagan and Maria G. Frid Circ Res. 2006;99: doi: /01.RES f Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX Copyright 2006 American Heart Association, Inc. All rights reserved. Print ISSN: Online ISSN: The online version of this article, along with updated information and services, is located on the World Wide Web at: Data Supplement (unedited) at: Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: Subscriptions: Information about subscribing to Circulation Research is online at: