- Email: [email protected]
Hypoxic Activation of Adventitial Fibroblasts*
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cell metaplasia in murine airways. Am J Respir Cell Mol Biol 2000; 22:253–260 Cohn L, Homer RJ, Marinov A, et al. Induction of airway mucus production By T helper 2 (Th2) cells: a critical role for interleukin 4 in cell recruitment but not mucus production. J Exp Med 1997; 186:1737–1747 Cohn L, Tepper JS, Bottomly K. IL-4-independent induction of airway hyperresponsivness by Th2, but not Th1, cells. J Immunol 1998; 161:3813–3816 Lee JJ, McGarry MP, Farmer SC, et al. Interleukin-5 expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma. J Exp Med 1997; 185:2143–2156 Grunig G, Warnock M, Wakil AE, et al. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 1998; 282:2261–2263 Wills-Karp M, Luyimbazi J, Xu X, et al. Interleukin-13: central mediator of allergic asthma. Science 1998; 282:2258 – 2261 Louahed J, Toda M, Jen J, et al. Interleukin-9 upregulates mucus expression in the airways. Am J Respir Cell Mol Biol 2000; 22:649 – 656 Temann UA, Geba GP, Rankin JA, et al. Expression of interleukin 9 in the lungs of transgenic mice causes airway inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness. J Exp Med 1998; 188:1307–1320 Temann UA, Prasad B, Gallup MW, et al. A novel role for murine IL-4 in vivo: induction of MUC5AC gene expression and mucin hypersecretion. Am J Respir Cell Mol Biol 1997; 16:471– 478 Haile´ S, Lefort J, Joseph D, et al. Mucous cell metaplasia and inflammatory cell recruitment are dissociated in allergic mice after antibody and drug-dependent cell depletion in a murine model of asthma. Am J Respir Cell Mol Biol 1999; 20:1–12 Cohn L, Homer RJ, MacLeod H, et al. Th2-induced airway mucus production is dependent on IL-4Ra, but not eosinophils. J Immunol 1997; 162:6178 – 6183 Singer M, Lefort J, Vargaftig BB. Granulocyte depletion and dexamethasone differentially modulate airways hyperreactivity, inflammation, mucus accumulation and secretion induced by rmIL-13 or antigen. Am J Respir Cell Mol Biol 2002; 26:74 – 84 Longpre M, Li D, Gallup M, et al. Allergen-induced IL-9 directly stimulates mucin transcription in respiratory epithelial cells. J Clin Invest 1999; 104:1375–1382 Nakanishi A, Morita S, Iwashita H, et al. Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma. Proc Natl Acad Sci U S A 2001; 98:5175–5180 Zhou Y, Dong Q, Louahed J, et al. Characterization of a calcium-activated chloride channel as a shared target of Th2 cytokine pathways and its potential involvement in asthma. Am J Respir Cell Mol Biol 2001; 25:486 – 491 Dolganov GM, Woodruff PW, Novikov A, et al. A novel method of gene transcript profiling in airway biopsy homogenates reveals increased expression of a Na⫹-K⫹-Cl- cotransporter (NKCC1) in asthmatic subjects. Genome Res 2001; 11:1473–1483 Shim JJ, Dabbagh K, Ueki IF, et al. IL-13 induces mucin production by stimulating epidermal growth factor receptors and by activating neutrophils. Am J Physiol 2001; 280:L134 – L140 Booth BW, Adler KB, Bonner JC, et al. Interleukin-13 induces proliferation of human airway epithelial cells in vitro via a mechanism mediated by transforming growth factoralpha. Am J Respir Cell Mol Biol 2001; 25:739 –743 Verdugo P. Goblet cell secretion and mucogenesis. Annu Rev Physiol 1990; 52:157–176
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43 Forstner G. Signal transduction, packaging, and secretion of mucins. Annu Rev Physiol 1995; 57:585– 605 44 Ohkawara Y, Lei XF, Stampfli MR, et al. Cytokine and eosinophil responses in the lung, peripheral blood, and bone marrow compartments in a murine model of allergen-induced airways inflammation. Am J Respir Cell Mol Biol 1997; 16:510 –520 45 Agusti C, Takeyama K, Cardell LO, et al. Goblet cell degranulation after antigen challenge in sensitized guinea pigs: role of neutrophils. Am J Respir Crit Care Med 1998; 158:1253–1258 46 Sommerhoff CP, Caughey GH, Finkbeiner WE, et al. Mast cell chymase: a potent secretagogue for airway gland serous cells. J Immunol 1989; 142:2450 –2456 47 Lundgren JD, Davey RT, Lundgren B, et al. Eosinophil cationic protein stimulates and major basic protein inhibits airway mucus secretion. J Allergy Clin Immunol 1991; 87: 689 – 698 48 Sommerhoff CP, Nadel JA, Basbaum CB, et al. Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells. J Clin Invest 1990; 85:682– 689 49 Hays SR, Woodruff PG, Khashayar R, et al. Allergen challenge causes inflammation but not goblet cell degranulation in asthmatic subjects. J Allergy Clin Immunol 2001; 108:753– 758 50 Li Y, Martin LD, Spizz G, et al. MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro. J Biol Chem 2001; 276:40982– 40990
Hypoxic Activation of Adventitial Fibroblasts* Role in Vascular Remodeling Kurt R. Stenmark, MD; Evgenia Gerasimovskaya, PhD; Raphael A. Nemenoff, PhD; and Mita Das, PhD
Substantial experimental evidence supports the idea that the fibroblast may play a significant role in the vascular response to injury, especially under hypoxic conditions. Fibroblasts have the ability to rapidly respond to hypoxic stress and to modulate their function to adapt rapidly to local vascular needs. Fibroblasts appear to be uniquely equipped to proliferate, transdifferentiate, and migrate under hypoxic conditions. Proliferative responses to hypoxia depend on the activation of G␣i and Gq kinase family members, and on the subsequent stimulation of protein kinase C and mitogen-activated protein kinase family members. Extracellular nucleotides (eg, adenosine triphosphate [ATP]) are likely to be increased in the hypoxic adventitial compartment and can act as autocrine/paracrine modifiers of the hypoxia-induced proliferative response. The proliferative effects of ATP appear to be mediated largely through G-protein-coupled P2Y receptors in fetal and neonatal fibroblasts. Hypoxia, acting through G␣-coupled pathways, also can directly up-regulate ␣-smooth muscle actin expression in fibroblast subpopulations, suggesting that hypoxia may play a Remodeling and Repair in Respiratory Diseases
direct role in mediating the “transdifferentiation” of fibroblasts into myofibroblasts in the vessel wall. In addition, chronic hypoxia causes stable (at least in vitro) phenotypic changes in fibroblasts that appear to be associated with changes in the signaling pathways used to elicit proliferation. However, it is also becoming clear that, similar to the heterogeneity described for vascular smooth muscle cells, numerous fibroblast subtypes exist in the vessel wall, and that each may respond in unique ways to hypoxia and other stimuli and thus serve special functions in response to injury. In fact, adventitia may be considered to be compartments in which cells with “stemcell-like” characteristics reside. Future work is needed to determine more precisely the role of the fibroblast in the wide variety of vascular complications observed in many humans diseases, and in the genes and gene products that confer unique properties to this important vascular cell. (CHEST 2002; 122:326S–334S)
markers. Indeed, it may be this apparent lack of differentiated characteristics that confers to the fibroblast a remarkable plasticity, thus allowing it a tremendous capacity for migration, rapid proliferation, synthesis of connective tissue components, contraction, cytokine production, and even transdifferentiation in response to the activation or stimulation by a variety of stimuli.1 Importantly, variations in oxygen concentrations have been shown to directly or indirectly affect nearly all of these functions in fibroblasts.2 The role of oxygen in modulating fibroblast gene expression and function has perhaps been the most welldocumented in the setting of wound healing, in which a hypoxic environment has been demonstrated to be a critical early component of many, but not all, of the cellular responses observed.3 As such, it is not surprising that fibroblasts may play an important role in the vascular response to hypoxia. We intend to briefly review the effects of decreased oxygen concentrations on vascular adventitial fibroblasts, especially as they may relate to pediatric lung injury.
Key words: adenosine triphosphate; extracellular nucleotides; fibroblast; growth; hypoxia; mitogen-activated protein kinases; myofibroblast; protein kinase C; transdifferentiation; vascular development
IN VIVO Response of the Adventitial Fibroblast to Hypoxia
Abbreviations: ATP ⫽ adenosine triphosphate; ERK ⫽ extracellular signal-regulated kinase; JNK ⫽ JUN N-terminal kinase; MAP ⫽ mitogen-activated protein; PA ⫽ pulmonary artery; PKC ⫽ protein kinase C; SM ⫽ smooth muscle; SMC ⫽ smooth muscle cell; TGF ⫽ transforming growth factor
and remodeling occur frequently in the vascuR epair lature, as evidenced by their occurrence in a wide
variety of cardiovascular abnormalities including pulmonary hypertension, systemic hypertension, atherosclerosis, vein graft remodeling, and restenosis following balloon dilatation of the coronary artery. The exposure of blood vessels to excessive hemodynamic stress (eg, hypertension), noxious blood-borne agents (eg, atherogenic lipids), locally released cytokines, or unusual environmental conditions (eg, hypoxia) requires readily available mechanisms to counteract these adverse stimuli and to preserve the structure and function of the vessel wall. The responses, which presumably developed evolutionarily to repair an injured tissue, often escape self-limiting control and can result, in the case of blood vessels, in lumen narrowing, decreased responsiveness to vasodilating stimuli, and obstruction to blood flow. Each cell (ie, endothelial, smooth muscle [SM], and fibroblast) in the vascular wall plays a specific role in the response to injury. However, while the roles of the endothelial cell and SM cell (SMC) in vascular remodeling have been studied extensively, until very recently comparatively little attention had been given to the adventitial fibroblast. Perhaps this is because the fibroblast is a relatively ill-defined cell that, at least compared to the SMC, exhibits few specific cellular
*From the Developmental Lung Biology Research Laboratory, University of Colorado Health Sciences Center, Denver, CO. Correspondence to: Kurt R. Stenmark, MD, Professor of Pediatrics, Head, Pediatric Critical Care Medicine, and Developmental Lung Biology Laboratory, University of Colorado Health Sciences Center, 4200 E Ninth Ave, Box B131, Denver, CO 80262; e-mail: [email protected] www.chestjournal.org
In animal models,4 the earliest and most dramatic structural changes following hypoxic exposure are found in the adventitial compartment of the vessel wall. Resident adventitial fibroblasts have been shown to exhibit early and sustained increases in proliferation that exceed those observed in endothelial or SMCs.4,5 In addition, early and dramatic changes in extracellular matrix protein synthesis occur.4 Early up-regulation of fibronectin messenger RNA expression and marked increases in type I collagen messenger RNA expression have been observed.3 Electron microscopic studies in the rat model of hypoxic pulmonary hypertension demonstrated the “transdifferentiation” of fibroblasts surrounding small arteries into myofibroblasts and ultimately into SMCs, which reside in a newly formed media.6 The increased expression of ␣-SM-actin-positive cells (eg, myofibroblasts) also has been observed in neonatal calves following acute hypoxic exposure.7 The fibroproliferative changes in the adventitia, particularly in resistance-size vessels, are associated ultimately with luminal narrowing and a progressive decrease in the ability of the vessel wall to respond to vasodilating stimuli.4
Hypoxia-Induced Proliferation Is Dependent on Mitogen-Activated Protein Kinases Much of the work done to date in defining the remodeling process has focused on the growth factors that are produced under hypoxic conditions and their potential effects on cell proliferation. Little work, however, has been done on the signaling pathways that might be induced by hypoxia itself and thus might affect vascular cell proliferation directly and/or markedly influence the response to locally produced growth factors. To better understand the intracellular pathways involved in hypoxiainduced signaling, we utilized a primary cell system generated from neonatal calves where marked increases in fibroblast proliferation in response to hypoxia have been CHEST / 122 / 6 / DECEMBER, 2002 SUPPLEMENT
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documented in vivo.4 Fibroblasts cultured from these calves were amenable to serum deprivation for 5 days, which allowed assessment of hypoxia-induced signaling in the absence of exogenous stimuli. Further, an approach in which the effects of serum and hypoxia were simultaneously evaluated in the same cells was utilized to determine whether there was a unique aspect of mitogen-activated protein (MAP) kinase signaling in hypoxia-induced proliferation. We found that hypoxia acts as a proliferative stimulus for pulmonary artery (PA) adventitial fibroblasts in the absence of exogenous mitogens (Fig 1), which is consistent with the observations of others.8 Hypoxia has been reported to activate MAP kinase signaling pathways in many cell types, although very few of those cells actually demonstrate a proliferative response under hypoxic conditions. The observations demonstrating that the mitogenic response to hypoxia of cultured human osteoblastic periodontal ligament cells is mediated by the
selective activation of extracellular signal-regulated kinase (ERK) 1/2,9 whereas that of adult bovine PA adventitial fibroblasts and renal mesangial cells is dependent on p38 MAP kinase activation,10,11 raise the possibility that MAP kinase signaling pathways utilized for hypoxia-induced proliferation are cell-type specific. We therefore chose to evaluate the effects of hypoxia on MAP kinase signaling in our system. Hypoxia caused a transient activation of ERK1/2 and JUN N-terminal kinase (JNK), and a biphasic activation of p38 MAP kinase in our cells.12 Using different antagonist strategies, ERK1/2, JNK1, and p38 MAP kinase, but not JNK2, were found to be necessary for hypoxia-induced proliferation. We also demonstrated that the hypoxia-induced proliferative responses appear to be mediated through different mechanisms than those activated during serum-stimulated growth.12 This was demonstrated by experiments showing the following: (1) that activation patterns of ERK, JNK, and especially p38 MAP
Figure 1. Hypoxia induces proliferation of neonatal bovine adventitial fibroblasts in vitro. Top left, A: hypoxic stimulation of DNA synthesis is greatest at 3 to 1% O2. Cells were plated at a density of 10 ⫻ 103 cells/cm2, were made quiescent by serum deprivation (0.1% fetal bovine serum) for 5 days, and then were exposed to 21%, 10%, and 3 to 1% O2 in the presence of [3H]thymidine for 24 h. Values are given as the mean ⫾ SE for this and all subsequent figures (four replicate wells). * ⫽ p ⬍ 0.05 compared to the 21% O2 results. Top left, B: chronic hypoxic exposure increases cell density above normoxic levels. Growth-arrested fibroblasts were exposed to normoxia (21% O2) and hypoxia (3 to 1% O2) for 3 days, were trypsinized, and were counted under a light microscope. * ⫽ p ⬍ 0.05 compared with cell counts under both normoxic (24 h and 72 h) and 24-h hypoxic exposure. Bottom left, C: hypoxia consistently induces an increase in proliferation in all fibroblast populations that are isolated from PA but only in selective fibroblast populations derived from the aorta. Adventitial fibroblasts were isolated from both the aorta and the PA of the same neonatal calf and were plated according to the above-mentioned protocol. DNA synthesis in response to normoxia and hypoxia was measured. * ⫽ p ⬍ 0.05 compared with corresponding normoxic value. Bottom right, D: hypoxia augments the serum-stimulated growth of fibroblasts. Fibroblasts were plated at a density of 250 cells/cm2 in media containing 10% fetal bovine serum and were allowed to attach overnight. On day 1, cells were exposed to normoxic and hypoxic gas for 30 min/d for up to 11 days. * ⫽ p ⬍ 0.05 compared to the corresponding normoxic value. 328S
Remodeling and Repair in Respiratory Diseases
kinases were different in response to hypoxia vs serum; (2) that blocking G␣i/o, ERK, and JNK1 nearly ablated the proliferative response induced by hypoxia yet had only moderate effects on serum-induced proliferation; (3) that blocking p38 MAP kinase with SB202190 attenuated hypoxia-induced proliferation yet led to an increase in serum-induced growth; and (4) that hypoxia led to synergistic augmentation of serum-induced growth. We were interested also in elucidating the upstream events induced by hypoxia that led to the activation of MAP kinase and protein kinase C (PKC) signaling. Based on observations suggesting that stimuli such as sheer stress, pH, and osmolality can activate G␣i-proteins with subsequent activation of MAP kinase signaling, we investigated the possibility that hypoxia in the absence of exogenous ligands directly activated G␣i/o-mediated signaling. Pertussis toxin, an antagonist of G␣i, markedly attenuated hypoxia-induced DNA synthesis and the activation of ERK and JNK but not p38 MAP kinase (Fig 2). These observations confirm that hypoxia itself can act as a growth-promoting stimulus for bovine neonatal adventitial fibroblasts through G␣i/o (and probably Gq)-mediated activation of a complex network of MAP kinases. A hypothetical schema illustrating our observations in this model system is shown in Figure 3.
Role of Extracellular Nucleotides in Fibroblast Proliferation Hypoxia appears to be capable of initiating DNA synthesis in at least some populations of fibroblasts in the absence of exogenous mitogens. It remains unclear, however, whether either the activation or augmentation of the hypoxia-induced growth response is due, at least in part, to autocrine/paracrine responses to factors secreted by fibroblasts during hypoxia, which act through G-proteincoupled pathways. One factor that could contribute to such an autocrine loop is adenosine triphosphate (ATP). Purines and pyrimidines (mainly ATP, adenosine diphosphate, adenosine, and uridine triphosphate) have widespread and specific extracellular signaling actions in the regulation of a variety of functions in many tissues and appear to have key roles in the development, proliferation, differentiation, and release of hormones, neurotransmitters, and cytokines.13 It is also becoming evident that alterations in the physiology of purinergic signaling may result in the development of a variety of pathologies including immune system, neurodegenerative and vascular disease.13 Importantly, in addition to nerves and circulating blood cells, vascular cells themselves appear to be a potent source of ATP and other adenine nucleotides. Figure 2. The inhibition of G␣i/o proteins by pretreatment with pertussis toxin attenuates hypoxia-induced stimulation of DNA synthesis and selectively inhibits MAP kinase activation. Top, A: hypoxia-induced increase in [3H]thymidine incorporation in PA fibroblasts is abolished in the presence of pertussis toxin. Pertussis toxin (100 ng/mL) was added to growth-arrested adventitial fibroblasts and incubated for 1 h, and then the cells were exposed to either normoxia or hypoxia in the presence of [3H]thymidine for 24 h. Middle, B: serum-stimulated growth of fibroblasts is attenuated by pertussis toxin. Quiescent fibroblasts were pretreated with pertussis toxin (100 ng/mL for 1 h) and stimulated www.chestjournal.org
with 10% fetal bovine serum, and growth was measured by DNA synthesis. * ⫽ p ⬍ 0.05 compared with normoxic or unstimulated results, ** ⫽ p ⬍ 0.05 compared with hypoxic or serum-stimulated results. Bottom, C: hypoxia-induced activation of ERK and JNK but not p38 MAP kinase is blocked by pertussis toxin. Representative blots for phospho-ERK, phospho-JNK, and phospho-p38 MAP kinase. CHEST / 122 / 6 / DECEMBER, 2002 SUPPLEMENT
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DNA synthesis, suggesting that the hypoxic activation of P2 receptors may be due to the release of endogenous ATP (16). These experiments suggest that hypoxia may create a purinergic network in which local ATP release, P2 receptor stimulation, and the altered degradation of ATP act to modulate hypoxia-induced fibroblast growth (16).
Hypoxia Induces Transdifferentiation of Fibroblasts to Myofibroblasts
Figure 3. Hypoxia-induced proliferation of fibroblasts is transduced through G-protein-mediated activation of MAP kinases.
These products are known to be released into the extracellular milieu in response to many vascular stress conditions including ischemia/oxidative stress, flow, and mechanical stretch.14 Since vascular cells have been shown to express metabotropic (P2Y) and/or ionotropic (P2X) subtypes of purinergic receptors, and since these receptors have been shown to be coupled to G-protein-dependent mitogenic signaling pathways,15 the released ATP can induce cell activation by an autocrine/paracrine mechanism. In fact, the extracellular concentration of ATP and the subsequent purinergic activation appear to play a critical role in determining the intracellular signaling set point of many key growth-regulating factors.15 Therefore, the release of ATP by environmental or chemical stimuli may contribute to activation of cellular signaling pathways, which raises the possibility that extracellular ATP is a critical modulator of signal transduction pathways operating to control proliferative responses. Using bovine PA adventitial fibroblasts and lung microvascular endothelial cells, we found that acute hypoxia (ie, 3% O2) induced the release of ATP from fibroblasts by 2.2-fold at 10 min, with persistent increases in extracellular ATP concentration observed at 24 h, and from endothelial cells by 60-fold, with a peak at 30 min.16 In addition, chronic hypoxia (ie, for 14 to 30 days) markedly attenuated the rate of extracellular ATP hydrolysis by ectonucleotidase(s), thus creating a situation in which a specific profile of nucleotide may have access to their respective receptors for a prolonged period. We next determined that ATP, uridine triphosphate, adenosine diphosphate-S and MeSATP increased [3H] thymidine incorporation (fourfold to fivefold [10⫺4 mol/L]) in fibroblasts, whereas adenosine was ineffective, indicating that P2Y metabatropic receptors and possibly P2X ionotropic receptors, rather than P1 receptors, are involved in the proliferative response to ATP.16 Hypoxia augmented the proliferative effect of ATP in an additive fashion, whereas ATP and/or hypoxia synergistically increased mitogeninduced DNA synthesis. Finally, we found that the P2 receptor antagonists suramin and cibacron blue 3GA (10⫺4 mol/L) attenuated both ATP and hypoxia-induced 330S
The changes in the proliferative and matrix-producing phenotype of the fibroblast under hypoxic conditions are accompanied by the appearance of ␣-SM-actin, in at least some of the cells in the adventitial compartment, indicating the transdifferentiation of some fibroblasts to myofibroblasts during the development of hypoxia-induced pulmonary hypertension.6,7 The presence of myofibroblasts has been documented extensively in active fibrotic lesions in many diseases including pulmonary fibrosis.17 Myofibroblasts appear to be ultrastructurally and metabolically distinct connective tissue cells acting as key participants in tissue remodeling, because of their own unique proliferative, migratory, and matrix-producing capabilities.18 The differentiation of fibroblasts into myofibroblasts appears to depend on a complex microenvironmental network in which growth factors, cytokines, adhesion molecules, and extracellular matrix components are involved. The stimulating activity of transforming growth factor (TGF)- on ␣-SM-actin and collagen production is well-accepted. However, other factors have also recently been demonstrated to be capable of stimulating ␣-SMactin expression in fibroblasts. Thrombin has been shown to differentiate normal lung fibroblasts to a myofibroblast phenotype via a G-protein-coupled receptor and PKC-⑀ dependent pathway.19 Since hypoxia also has been shown to be capable of activating G-protein-coupled receptor signaling pathways, we hypothesized that hypoxia itself might act as a stimulus to induce the differentiation of fibroblasts into myofibroblasts. We used clonal populations of neonatal bovine PA adventitial fibroblasts to test this possibility. Hypoxia induced a marked increase in ␣-SM-actin protein in fibroblast subpopulations.20 To evaluate the molecular mechanisms involved, fibroblasts were transiently transfected with an ␣-SM-actin promoter that was tagged with luciferase and then exposed to hypoxia. An increase in ␣-SM-actin promoter activity also was observed in response to hypoxia. Hypoxia-induced ␣-SM-actin promoter activity was largely independent of TGF- as the neutralizing antibody of TGF- blocked the hypoxia-induced promoter activity only by 40%, while it completely inhibited TGF--induced increases in promoter activity. To determine the role of G-proteins in hypoxia-induced ␣-SM-actin promoter activity, cells either were cotransfected with constitutively active constructs of Gi and Gq proteins or were treated with pertussis toxin. Augmentation (active Gi) and attenuation (pertussis toxin) of hypoxia-induced promoter activity supported the role for G␣i, but not Gq, in the differentiation of fibroblasts into myofibroblasts. This appears to differ in SMCs in which Gq-coupled pathways are critical for ␣-SM-actin regulaRemodeling and Repair in Respiratory Diseases
tion. PD98059, an inhibitor of ERK activation, potentiated the hypoxia-induced promoter activity. Therefore, we conclude that hypoxia induces the differentiation of fibroblasts into myofibroblasts. This supports our contention (see below) that numerous fibroblast subpopulations exist and that hypoxia (and certainly other stimuli) selectively activates specific fibroblast cell populations. Furthermore, our experiments provide preliminary evidence that the hypoxia-induced ␣-SM-actin expression in fibroblasts is mediated through G␣i.
Developmental Differences in the Response of Fibroblasts to Hypoxia Pathologic studies suggest that the PA adventitial response to chronic hypoxia is greater in infants and children than in adults.4 To explore the possibility of developmentally regulated differences in growth, fibroblasts were isolated from PA adventitia at different stages (ie, fetal, neonatal, and adult stages) during development and growth was assessed. We found that fetal and neonatal fibroblasts grew faster than adult cells in 10% serum.21 Rapidly growing fetal fibroblasts had increased PKC catalytic activity compared to adult cells.21 Using different PKC inhibitor strategies, we also demonstrated that high rates of growth in fetal cells were due in part to high levels of PKC activity.21 PKC signaling, however, is complex, with four Ca2⫹dependent and seven Ca2⫹-independent isozymes known to exist and to have different functions within the cell.22 Developmental changes in the expression of individual isozymes have been reported, and the increased expression of selected PKC isozymes has been linked to augmented growth capacity.23 Our data demonstrate that neonatal bovine PA adventitial fibroblasts express 7 of the 11 isozymes, three that are Ca2⫹-dependent (␣, I, and II) and four that are Ca2⫹-independent (␦, ⑀, , and ). The expression pattern of two isozymes, the Ca2⫹-dependent PKC-␣ and PKC-II, paralleled the developmental differences in growth, susceptibility to PKC inhibitors, and PKC catalytic activity.24 Antagonist strategies implicated these same Ca2⫹dependent isozymes in the enhanced growth of fetal and neonatal fibroblasts. Thus, it appears that the signaling pathways utilized to mediate hypoxia-induced proliferation are not only cell type-specific but are also age-specific or developmental-state specific.
Phenotypic Modulation of Fibroblasts in Response to Chronic Hypoxia In addition to the developmentally regulated changes in the proliferative and matrix-producing potentials of fibroblasts, an expanding body of experimental observations25,26 also supports the concept that significant changes in the phenotypic properties of resident fibroblasts can occur in the setting of fibroproliferative disease. We therefore tested the hypothesis that exposure to chronic hypoxia would induce specific alterations in resident fibroblasts, rendering them more sensitive to growth-promoting mitogens as well as hypoxia. Adventitial fibroblasts from www.chestjournal.org
age-matched control and chronically hypoxic calves were isolated simultaneously, and subsequent comparative experiments were performed in cells under identical conditions. We found, on a very consistent basis, that fibroblasts isolated from hypoxic hypertensive calves exhibited augmented growth responses to serum, hypoxia, and purified peptide mitogens compared to those obtained from agematched control animals.27 These proliferative attributes persisted through numerous cell passages in culture, suggesting acquired differences in fibroblast populations that were not simply due to short-term changes in the in vivo cellular milieu (ie, growth factor, cytokines, and matrix components). Recently, Welsh et al28 also reported that PA fibroblasts from chronically hypoxic rats appeared to have undergone a phenotypic switch. Thus, fibroblasts from both neonates and adults demonstrate a stable phenotypic change in response to hypoxia. Several possibilities must be considered in explaining how a stable phenotypic alteration of the adventitial fibroblast might come about. The first is that a large portion of resident fibroblasts is altered by changes that occur locally in the chronically hypoxic vascular wall. Hypoxia itself, a combination of hypoxia with subsequent hemodynamic changes, and/or a combination of hypoxia, hemodynamic changes, and changes in the local concentrations of cytokines, growth factors, and matrix proteins, must be considered as potential mediators of the phenotypic modulation of cells during the development of chronic hypoxia-induced pulmonary hypertension. Resident fibroblasts could be altered by these signals, conferring on them a new stable phenotype, which is manifested by intrinsically enhanced proliferative and matrix-producing capabilities. Another possibility is that selective expansion of a resident fibroblast population, with unusual or augmented growth properties, has occurred in vivo. This population could expand to the point where it is numerically the most important constituent of the activated or injured vessel wall. This population then might demonstrate selective advantage when cell cultures are performed and, thus, account for the apparent phenotypic change observed in fibroblast populations from the injured organ or vessel.
Fibroblast Heterogeneity in PA Adventitia Since lung, skin, and gingival tissue are composed of multiple fibroblast subpopulations, we investigated the hypothesis that the PA adventitia of neonatal calves is composed of several functionally distinct fibroblast subpopulations and that some subpopulations selectively expand during the development of hypoxic pulmonary hypertension. Fibroblast subpopulations were isolated from the PA adventitia of neonatal control calves using limiteddilution cloning techniques. These subpopulations exhibited marked and stable differences in morphology (Fig 4), cytoskeletal protein expression, and growth capabilities in response to serum.29 We investigated the possibility that fibroblast subpopulations also might exhibit markedly different proliferative responses to hypoxia. We found that proliferation under hypoxic conditions is highly variable CHEST / 122 / 6 / DECEMBER, 2002 SUPPLEMENT
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Figure 4. Fibroblast subpopulations isolated from PA adventitia by a limited-dilution cloning technique exhibited distinct morphologic characteristics. Top left, A, middle left, B, and bottom left, C: fibroblast subpopulations derived from PA adventitia demonstrated a rhomboidal appearance. Top right, D, middle right, E, and bottom right, F: subpopulations of adventitial fibroblasts appeared spindle-shaped (original magnification 10⫻).
among subpopulations with some showing greater than twofold increases in DNA synthesis in response to hypoxia (in the absence of growth factors), while others exhibit decreased DNA synthesis (Fig 5). Finally, to test the hypothesis that long-term hypoxic exposure was associated with a relative increase in the number of fibroblast subpopulations with hypoxia-induced proliferative ability, fibroblast subpopulations were isolated from neonatal calves exposed to hypoxia for 14 days. With regard to morphology, actin expression, and serumstimulated growth capabilities of subpopulations, there were no obvious differences between the hypoxic and the 332S
control calves. However, the number of fibroblast subpopulations with twofold or greater increases in DNA synthesis in response to hypoxia, was significantly greater in the adventitia of hypertensive calves (26%) compared to control calves (10%) [Fig 5]. These experiments provide further support to the hypothesis that the bovine PA adventitia is comprised of multiple phenotypically and functionally distinct fibroblast subpopulations and that chronic hypoxia is associated with the expansion of select fibroblast subpopulations.29 In addition, our data demonstrating numerous functionally distinct fibroblast subpopulations, some with remarkable growth potential, lend Remodeling and Repair in Respiratory Diseases
Figure 5. The proportion of fibroblast subpopulations with more than a twofold increase in DNA synthesis (hypoxia proliferative) was higher in the PA adventitia of hypertensive calves compared to control calves. Left, A: In vitro hypoxia-induced proliferative responses of 31 control (E) and 49 pulmonary hypertensive-derived (F) fibroblast subpopulations. Right, B: the percentage of hypoxiaproliferative fibroblast subpopulations was greater for the hypoxic hypertensive calves than for the control calves.
further support to the idea that the adventitial compartment may be where stem-like cells for the vasculature reside. Changes in the relative proportion of different fibroblast subsets may have profound effects not only on adventitial remodeling but on the whole vessel wall. It is possible that the alteration in the relative proportion of different fibroblast subsets with different capabilities for matrix protein and soluble growth factor production, might be expected to influence the behavior of adjacent SMCs or endothelial cells (ie, dynamic reciprocity). It is clear that there is a continuous feedback of information between the cell and the matrix, such that the extracellular matrix that is in contact with the cells is itself a product of cellular activity (by virtue of biosynthesis, degradation, and modification of matrix macromolecules) and that the extracellular matrix influences various fundamental aspects of cell behavior such as the deposition of matrix itself, cell proliferation, and the pattern of gene expression and cell migration. The activation of a fibroblast subset secreting unique matrix molecules and soluble factors thus may have an influence on neighboring SMCs or epithelial cells that is not exhibited under normal conditions.
Summary The fibroblast may play a significant role in the vascular response to injury, especially under hypoxic conditions, by www.chestjournal.org
modulating its function according to local vascular needs. Fibroblasts appear to be uniquely equipped to proliferate, transdifferentiate, and migrate under hypoxic conditions simultaneously. Hypoxia-induced proliferation of fibroblasts depends on the activation of G␣i and Gq, and on the subsequent stimulation of PKC and MAP kinase family members. Extracellular nucleotides (eg, ATP) acting through G␣i-coupled and Gq-coupled P2Y receptors, appear to act as autocrine/paracrine modifiers of the proliferative response of fibroblasts under hypoxic conditions. Hypoxia can also up-regulate ␣-SM-actin expression in fibroblasts, suggesting that hypoxia may contribute to the transdifferentiation of fibroblasts into myofibroblasts in the vessel wall. In addition, chronic hypoxia causes stable (at least in vitro) phenotypic changes in fibroblasts that appear to be associated with changes in the signaling pathways used to elicit proliferation. However, it is also becoming clear that, as with SMCs, numerous fibroblast subtypes exist in the vessel wall and that each may respond in unique ways to hypoxia and thus may serve special functions in response to injury. The adventitia of the vessel wall thus may be considered to be a compartment where cells with stem-cell-like characteristics reside. We believe, therefore, that the adventitial fibroblast may be a critical regulator of vascular function and structure under hypoxic conditions. This coordinated regulation of several functions by at least some adventitial fibroblast populations is CHEST / 122 / 6 / DECEMBER, 2002 SUPPLEMENT
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in contrast to the responses of mature SMCs, which, in general, dedifferentiate before they exhibit marked changes in proliferative or migratory behavior.
References 1 Sartore S, Chiavegato A, Faggin E, et al. Contribution of adventitial fibroblasts to neointima formation and vascular remodeling. Circ Res 2001; 89:1111–1121 2 Scheid A, Wenger RH, Christina H, et al. Hypoxia-regulated gene expression in fetal wound regeneration and adult wound repair. Pediatr Surg Int 2000; 16:232–236 3 Davidson JD, Mustoe TA. Oxygen in wound healing: more than a nutrient. Wound Repair Regen 2001; 9:175–177 4 Stenmark KR, Mecham RP. Cellular and molecular mechanisms of pulmonary vascular remodeling. Annu Rev Physiol 1997; 59:89 –144 5 Belknap JK, Orton EC, Ensley B, et al. Hypoxia increases bromodeoxyuridine labeling indices in bovine neonatal pulmonary arteries. Am J Respir Cell Mol Biol 1997; 16:366 –371 6 Sobin SS, Tremer HM, Hardy JD, et al. Changes in arteriole in acute and chronic hypoxic pulmonary hypertension and recovery in rat. J Appl Physiol 1983; 55:1445–1455 7 Stenmark KR, Durmowicz AG, Dempsey EC. Modulation of vascular wall cell phenotype in pulmonary hypertension. In: Bishop JE, Reeves JJ, Laurent GJ, eds. Pulmonary vascular remodeling. London, UK: Portland Press, 1995 8 Welsh D, Scott P, Plevin R, et al. Effects of hypoxia on IP3 generation and DNA synthesis in bovine pulmonary artery fibroblasts [abstract]. Am J Respir Crit Care Med 1996; 153:A576 9 Matsuda N, Morita N, Matsuda K, et al. Proliferation and differentiation of human osteoblastic cells associated with differential activation of MAP kinases in response to epidermal growth factor, hypoxia, and mechanical stress in vitro. Biochem Biophys Res Commun 1998; 249:350 –354 10 Sodhi CP, Batlle D, Sahai A. Osteopontin mediates hypoxiainduced proliferation of cultured mesangial cells: role of PKC and MAPK. Kidney Int 2000; 58:691–700 11 Seka Y, Takahashi N, Tobe K, et al. Hypoxia and hypoxia/ reoxygenation activate p65PAK, p38 mitogen-activated protein kinase (MAPK), and stress-activated protein kinase (SAPK) in cultured rat cardiac myocytes. Biochim Biophys Acta 1997; 239:840 – 844 12 Das M, Bouchey DM, Moore MJ, et al. Hypoxis-induced proliferative response of vascular adventitial fibroblasts is dependent on G protein-mediated activation of mitogenactivated protein kinases. J Biol Chem 2001; 276:15631– 15640 13 Abbracchio MP, Burnstock G. Purinergic signaling: pathophysiological roles. Jpn J Pharmacol 1998; 78:113–145 14 Vassort G. Adenosine 5⬘triphosphate: a p2-purinergic agonist in the myocardium. Physiol Rev 2001; 81:767– 806 15 Ostrom RS, Gregorian C, Insel PA. Cellular release of and response to ATP as key determinants of the set-point of signal transduction pathways. J Biol Chem 2000; 275:11735–11739 16 Gerasimovskaya EV, Ahmad S, White CW, et al. Extracellular ATP is an autocrine/paracrine regulator of hypoxia-induced fibroblast growth: signalling through extracellular signalregulated kinase 1/2 and the Egr-1 transcription factor J Biol Chem (in press) 17 Zhang K, Phan SH. Inhibition of myofibroblast apoptosis by transforming growth factor beta(1). Am J Respir Cell Mol Biol 1999; 21:658 – 665 18 Serini G, Gabbiani G. Mechanisms of myofibroblast activity and phenotypic modulation. Exp Cell Res 1999; 250:273–283 19 Bogatkevich GS, Tourkina E, Silver RM, et al. Thrombin 334S
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differentiates normal lung fibroblasts to a myofibroblasts phenotype via the proteolytically activated receptor-1 and a protein kinase C-dependent pathway. J Biol Chem 2001; 276:45184 – 45192 Das M, Short M, Nemenoff R, et al. Hypoxia induces transdifferentiation of selective vascular fibroblasts into myofibroblasts via G␣i and p38 MAP kinase pathways [abstract]. Am J Respir Crit Care Med 2002; 165:A361 Das M, Stenmark KR, Dempsey EC. Enhanced growth of fetal and neonatal pulmonary artery adventitial fibroblasts is dependent on protein kinase C. Am J Physiol 1995; 269: L660 –L667 Nishizuka Y. The protein C family and lipid mediators for transmembrane signaling and cell regulation. Alcohol Clin Exp Res 2001; 25:3S–7S Black JD. Protein kinase C-mediated regulation of cell cycle. Front Biosci 2000; 5:D406 –D423 Das M, Stenmark KR, Ruff LJ, et al. Selected isozymes of PKC contribute to augmented growth of fetal and neonatal bovine pulmonary artery fibroblasts. Am J Physiol 1997; 273:L1276 –L1284 Jordana M, Kirpalani H, Gauldie J. Heterogeneity of human lung fibroblast proliferation in relation to disease expression. In: Phipps RP, ed. Pulmonary fibroblast heterogeneity. Boca Raton, FL: CRC Press, 1992; 229 –249 Torry DJ, Richards CD, Podor TJ, et al. Anchorage-independent colony growth of pulmonary fibroblasts derived from fibrotic human lung tissue. J Clin Invest 1994; 93:1525–1532 Das M, Dempsey EC, Bouchey D, et al. Chronic hypoxia induces exaggerated growth responses in pulmonary artery adventitial fibroblasts. Am J Respir Cell Mol Biol 2000; 22:15–25 Welsh DJ, Peacock AJ, Maclean M, et al. Chronic hypoxia induces constitutive p38 mitogen activated protein kinase activity that correlates with enhanced cellular proliferation in fibroblasts from rat pulmonary but not systemic arteries. Am J Respir Crit Care Med 2001; 164:282–289 Das M, Dempsey EC, Reeves JT, et al. Phenotypically distinct fibroblast subpopulations in pulmonary artery adventitia: selective expansion in response to chronic hypoxia. Am J Physiol 2002; 282, L976 –L986
Overview of Pulmonary Fibrosis* Francis H.Y. Green, MD
Pulmonary fibrosis is a component of over 200 interstitial lung diseases. Some have known etiologies, however, for many diseases, the etiology remains unknown or obscure. This brief review examines the prevalence and classification of these diseases, the approach to be taken for the investigation of a patient suspected of having pulmonary fibrosis, the indications for the performance of lung biopsy, and *From the University of Calgary, Calgary, AB, Canada. This work was funded by the University of Calgary. Correspondence to: Francis H.Y. Green, MD, Professor, Pathology & Laboratory Medicine, Faculty of Medicine, University of Calgary, 3330 Hospital Dr NW, Calgary, AB, Canada T2N 4N1; e-mail: [email protected] Remodeling and Repair in Respiratory Diseases
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cell metaplasia in murine airways. Am J Respir Cell Mol Biol 2000; 22:253–260 Cohn L, Homer RJ, Marinov A, et al. Induction of airway mucus production By T helper 2 (Th2) cells: a critical role for interleukin 4 in cell recruitment but not mucus production. J Exp Med 1997; 186:1737–1747 Cohn L, Tepper JS, Bottomly K. IL-4-independent induction of airway hyperresponsivness by Th2, but not Th1, cells. J Immunol 1998; 161:3813–3816 Lee JJ, McGarry MP, Farmer SC, et al. Interleukin-5 expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma. J Exp Med 1997; 185:2143–2156 Grunig G, Warnock M, Wakil AE, et al. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 1998; 282:2261–2263 Wills-Karp M, Luyimbazi J, Xu X, et al. Interleukin-13: central mediator of allergic asthma. Science 1998; 282:2258 – 2261 Louahed J, Toda M, Jen J, et al. Interleukin-9 upregulates mucus expression in the airways. Am J Respir Cell Mol Biol 2000; 22:649 – 656 Temann UA, Geba GP, Rankin JA, et al. Expression of interleukin 9 in the lungs of transgenic mice causes airway inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness. J Exp Med 1998; 188:1307–1320 Temann UA, Prasad B, Gallup MW, et al. A novel role for murine IL-4 in vivo: induction of MUC5AC gene expression and mucin hypersecretion. Am J Respir Cell Mol Biol 1997; 16:471– 478 Haile´ S, Lefort J, Joseph D, et al. Mucous cell metaplasia and inflammatory cell recruitment are dissociated in allergic mice after antibody and drug-dependent cell depletion in a murine model of asthma. Am J Respir Cell Mol Biol 1999; 20:1–12 Cohn L, Homer RJ, MacLeod H, et al. Th2-induced airway mucus production is dependent on IL-4Ra, but not eosinophils. J Immunol 1997; 162:6178 – 6183 Singer M, Lefort J, Vargaftig BB. Granulocyte depletion and dexamethasone differentially modulate airways hyperreactivity, inflammation, mucus accumulation and secretion induced by rmIL-13 or antigen. Am J Respir Cell Mol Biol 2002; 26:74 – 84 Longpre M, Li D, Gallup M, et al. Allergen-induced IL-9 directly stimulates mucin transcription in respiratory epithelial cells. J Clin Invest 1999; 104:1375–1382 Nakanishi A, Morita S, Iwashita H, et al. Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma. Proc Natl Acad Sci U S A 2001; 98:5175–5180 Zhou Y, Dong Q, Louahed J, et al. Characterization of a calcium-activated chloride channel as a shared target of Th2 cytokine pathways and its potential involvement in asthma. Am J Respir Cell Mol Biol 2001; 25:486 – 491 Dolganov GM, Woodruff PW, Novikov A, et al. A novel method of gene transcript profiling in airway biopsy homogenates reveals increased expression of a Na⫹-K⫹-Cl- cotransporter (NKCC1) in asthmatic subjects. Genome Res 2001; 11:1473–1483 Shim JJ, Dabbagh K, Ueki IF, et al. IL-13 induces mucin production by stimulating epidermal growth factor receptors and by activating neutrophils. Am J Physiol 2001; 280:L134 – L140 Booth BW, Adler KB, Bonner JC, et al. Interleukin-13 induces proliferation of human airway epithelial cells in vitro via a mechanism mediated by transforming growth factoralpha. Am J Respir Cell Mol Biol 2001; 25:739 –743 Verdugo P. Goblet cell secretion and mucogenesis. Annu Rev Physiol 1990; 52:157–176
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43 Forstner G. Signal transduction, packaging, and secretion of mucins. Annu Rev Physiol 1995; 57:585– 605 44 Ohkawara Y, Lei XF, Stampfli MR, et al. Cytokine and eosinophil responses in the lung, peripheral blood, and bone marrow compartments in a murine model of allergen-induced airways inflammation. Am J Respir Cell Mol Biol 1997; 16:510 –520 45 Agusti C, Takeyama K, Cardell LO, et al. Goblet cell degranulation after antigen challenge in sensitized guinea pigs: role of neutrophils. Am J Respir Crit Care Med 1998; 158:1253–1258 46 Sommerhoff CP, Caughey GH, Finkbeiner WE, et al. Mast cell chymase: a potent secretagogue for airway gland serous cells. J Immunol 1989; 142:2450 –2456 47 Lundgren JD, Davey RT, Lundgren B, et al. Eosinophil cationic protein stimulates and major basic protein inhibits airway mucus secretion. J Allergy Clin Immunol 1991; 87: 689 – 698 48 Sommerhoff CP, Nadel JA, Basbaum CB, et al. Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells. J Clin Invest 1990; 85:682– 689 49 Hays SR, Woodruff PG, Khashayar R, et al. Allergen challenge causes inflammation but not goblet cell degranulation in asthmatic subjects. J Allergy Clin Immunol 2001; 108:753– 758 50 Li Y, Martin LD, Spizz G, et al. MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro. J Biol Chem 2001; 276:40982– 40990
Hypoxic Activation of Adventitial Fibroblasts* Role in Vascular Remodeling Kurt R. Stenmark, MD; Evgenia Gerasimovskaya, PhD; Raphael A. Nemenoff, PhD; and Mita Das, PhD
Substantial experimental evidence supports the idea that the fibroblast may play a significant role in the vascular response to injury, especially under hypoxic conditions. Fibroblasts have the ability to rapidly respond to hypoxic stress and to modulate their function to adapt rapidly to local vascular needs. Fibroblasts appear to be uniquely equipped to proliferate, transdifferentiate, and migrate under hypoxic conditions. Proliferative responses to hypoxia depend on the activation of G␣i and Gq kinase family members, and on the subsequent stimulation of protein kinase C and mitogen-activated protein kinase family members. Extracellular nucleotides (eg, adenosine triphosphate [ATP]) are likely to be increased in the hypoxic adventitial compartment and can act as autocrine/paracrine modifiers of the hypoxia-induced proliferative response. The proliferative effects of ATP appear to be mediated largely through G-protein-coupled P2Y receptors in fetal and neonatal fibroblasts. Hypoxia, acting through G␣-coupled pathways, also can directly up-regulate ␣-smooth muscle actin expression in fibroblast subpopulations, suggesting that hypoxia may play a Remodeling and Repair in Respiratory Diseases
direct role in mediating the “transdifferentiation” of fibroblasts into myofibroblasts in the vessel wall. In addition, chronic hypoxia causes stable (at least in vitro) phenotypic changes in fibroblasts that appear to be associated with changes in the signaling pathways used to elicit proliferation. However, it is also becoming clear that, similar to the heterogeneity described for vascular smooth muscle cells, numerous fibroblast subtypes exist in the vessel wall, and that each may respond in unique ways to hypoxia and other stimuli and thus serve special functions in response to injury. In fact, adventitia may be considered to be compartments in which cells with “stemcell-like” characteristics reside. Future work is needed to determine more precisely the role of the fibroblast in the wide variety of vascular complications observed in many humans diseases, and in the genes and gene products that confer unique properties to this important vascular cell. (CHEST 2002; 122:326S–334S)
markers. Indeed, it may be this apparent lack of differentiated characteristics that confers to the fibroblast a remarkable plasticity, thus allowing it a tremendous capacity for migration, rapid proliferation, synthesis of connective tissue components, contraction, cytokine production, and even transdifferentiation in response to the activation or stimulation by a variety of stimuli.1 Importantly, variations in oxygen concentrations have been shown to directly or indirectly affect nearly all of these functions in fibroblasts.2 The role of oxygen in modulating fibroblast gene expression and function has perhaps been the most welldocumented in the setting of wound healing, in which a hypoxic environment has been demonstrated to be a critical early component of many, but not all, of the cellular responses observed.3 As such, it is not surprising that fibroblasts may play an important role in the vascular response to hypoxia. We intend to briefly review the effects of decreased oxygen concentrations on vascular adventitial fibroblasts, especially as they may relate to pediatric lung injury.
Key words: adenosine triphosphate; extracellular nucleotides; fibroblast; growth; hypoxia; mitogen-activated protein kinases; myofibroblast; protein kinase C; transdifferentiation; vascular development
IN VIVO Response of the Adventitial Fibroblast to Hypoxia
Abbreviations: ATP ⫽ adenosine triphosphate; ERK ⫽ extracellular signal-regulated kinase; JNK ⫽ JUN N-terminal kinase; MAP ⫽ mitogen-activated protein; PA ⫽ pulmonary artery; PKC ⫽ protein kinase C; SM ⫽ smooth muscle; SMC ⫽ smooth muscle cell; TGF ⫽ transforming growth factor
and remodeling occur frequently in the vascuR epair lature, as evidenced by their occurrence in a wide
variety of cardiovascular abnormalities including pulmonary hypertension, systemic hypertension, atherosclerosis, vein graft remodeling, and restenosis following balloon dilatation of the coronary artery. The exposure of blood vessels to excessive hemodynamic stress (eg, hypertension), noxious blood-borne agents (eg, atherogenic lipids), locally released cytokines, or unusual environmental conditions (eg, hypoxia) requires readily available mechanisms to counteract these adverse stimuli and to preserve the structure and function of the vessel wall. The responses, which presumably developed evolutionarily to repair an injured tissue, often escape self-limiting control and can result, in the case of blood vessels, in lumen narrowing, decreased responsiveness to vasodilating stimuli, and obstruction to blood flow. Each cell (ie, endothelial, smooth muscle [SM], and fibroblast) in the vascular wall plays a specific role in the response to injury. However, while the roles of the endothelial cell and SM cell (SMC) in vascular remodeling have been studied extensively, until very recently comparatively little attention had been given to the adventitial fibroblast. Perhaps this is because the fibroblast is a relatively ill-defined cell that, at least compared to the SMC, exhibits few specific cellular
*From the Developmental Lung Biology Research Laboratory, University of Colorado Health Sciences Center, Denver, CO. Correspondence to: Kurt R. Stenmark, MD, Professor of Pediatrics, Head, Pediatric Critical Care Medicine, and Developmental Lung Biology Laboratory, University of Colorado Health Sciences Center, 4200 E Ninth Ave, Box B131, Denver, CO 80262; e-mail: [email protected] www.chestjournal.org
In animal models,4 the earliest and most dramatic structural changes following hypoxic exposure are found in the adventitial compartment of the vessel wall. Resident adventitial fibroblasts have been shown to exhibit early and sustained increases in proliferation that exceed those observed in endothelial or SMCs.4,5 In addition, early and dramatic changes in extracellular matrix protein synthesis occur.4 Early up-regulation of fibronectin messenger RNA expression and marked increases in type I collagen messenger RNA expression have been observed.3 Electron microscopic studies in the rat model of hypoxic pulmonary hypertension demonstrated the “transdifferentiation” of fibroblasts surrounding small arteries into myofibroblasts and ultimately into SMCs, which reside in a newly formed media.6 The increased expression of ␣-SM-actin-positive cells (eg, myofibroblasts) also has been observed in neonatal calves following acute hypoxic exposure.7 The fibroproliferative changes in the adventitia, particularly in resistance-size vessels, are associated ultimately with luminal narrowing and a progressive decrease in the ability of the vessel wall to respond to vasodilating stimuli.4
Hypoxia-Induced Proliferation Is Dependent on Mitogen-Activated Protein Kinases Much of the work done to date in defining the remodeling process has focused on the growth factors that are produced under hypoxic conditions and their potential effects on cell proliferation. Little work, however, has been done on the signaling pathways that might be induced by hypoxia itself and thus might affect vascular cell proliferation directly and/or markedly influence the response to locally produced growth factors. To better understand the intracellular pathways involved in hypoxiainduced signaling, we utilized a primary cell system generated from neonatal calves where marked increases in fibroblast proliferation in response to hypoxia have been CHEST / 122 / 6 / DECEMBER, 2002 SUPPLEMENT
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documented in vivo.4 Fibroblasts cultured from these calves were amenable to serum deprivation for 5 days, which allowed assessment of hypoxia-induced signaling in the absence of exogenous stimuli. Further, an approach in which the effects of serum and hypoxia were simultaneously evaluated in the same cells was utilized to determine whether there was a unique aspect of mitogen-activated protein (MAP) kinase signaling in hypoxia-induced proliferation. We found that hypoxia acts as a proliferative stimulus for pulmonary artery (PA) adventitial fibroblasts in the absence of exogenous mitogens (Fig 1), which is consistent with the observations of others.8 Hypoxia has been reported to activate MAP kinase signaling pathways in many cell types, although very few of those cells actually demonstrate a proliferative response under hypoxic conditions. The observations demonstrating that the mitogenic response to hypoxia of cultured human osteoblastic periodontal ligament cells is mediated by the
selective activation of extracellular signal-regulated kinase (ERK) 1/2,9 whereas that of adult bovine PA adventitial fibroblasts and renal mesangial cells is dependent on p38 MAP kinase activation,10,11 raise the possibility that MAP kinase signaling pathways utilized for hypoxia-induced proliferation are cell-type specific. We therefore chose to evaluate the effects of hypoxia on MAP kinase signaling in our system. Hypoxia caused a transient activation of ERK1/2 and JUN N-terminal kinase (JNK), and a biphasic activation of p38 MAP kinase in our cells.12 Using different antagonist strategies, ERK1/2, JNK1, and p38 MAP kinase, but not JNK2, were found to be necessary for hypoxia-induced proliferation. We also demonstrated that the hypoxia-induced proliferative responses appear to be mediated through different mechanisms than those activated during serum-stimulated growth.12 This was demonstrated by experiments showing the following: (1) that activation patterns of ERK, JNK, and especially p38 MAP
Figure 1. Hypoxia induces proliferation of neonatal bovine adventitial fibroblasts in vitro. Top left, A: hypoxic stimulation of DNA synthesis is greatest at 3 to 1% O2. Cells were plated at a density of 10 ⫻ 103 cells/cm2, were made quiescent by serum deprivation (0.1% fetal bovine serum) for 5 days, and then were exposed to 21%, 10%, and 3 to 1% O2 in the presence of [3H]thymidine for 24 h. Values are given as the mean ⫾ SE for this and all subsequent figures (four replicate wells). * ⫽ p ⬍ 0.05 compared to the 21% O2 results. Top left, B: chronic hypoxic exposure increases cell density above normoxic levels. Growth-arrested fibroblasts were exposed to normoxia (21% O2) and hypoxia (3 to 1% O2) for 3 days, were trypsinized, and were counted under a light microscope. * ⫽ p ⬍ 0.05 compared with cell counts under both normoxic (24 h and 72 h) and 24-h hypoxic exposure. Bottom left, C: hypoxia consistently induces an increase in proliferation in all fibroblast populations that are isolated from PA but only in selective fibroblast populations derived from the aorta. Adventitial fibroblasts were isolated from both the aorta and the PA of the same neonatal calf and were plated according to the above-mentioned protocol. DNA synthesis in response to normoxia and hypoxia was measured. * ⫽ p ⬍ 0.05 compared with corresponding normoxic value. Bottom right, D: hypoxia augments the serum-stimulated growth of fibroblasts. Fibroblasts were plated at a density of 250 cells/cm2 in media containing 10% fetal bovine serum and were allowed to attach overnight. On day 1, cells were exposed to normoxic and hypoxic gas for 30 min/d for up to 11 days. * ⫽ p ⬍ 0.05 compared to the corresponding normoxic value. 328S
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kinases were different in response to hypoxia vs serum; (2) that blocking G␣i/o, ERK, and JNK1 nearly ablated the proliferative response induced by hypoxia yet had only moderate effects on serum-induced proliferation; (3) that blocking p38 MAP kinase with SB202190 attenuated hypoxia-induced proliferation yet led to an increase in serum-induced growth; and (4) that hypoxia led to synergistic augmentation of serum-induced growth. We were interested also in elucidating the upstream events induced by hypoxia that led to the activation of MAP kinase and protein kinase C (PKC) signaling. Based on observations suggesting that stimuli such as sheer stress, pH, and osmolality can activate G␣i-proteins with subsequent activation of MAP kinase signaling, we investigated the possibility that hypoxia in the absence of exogenous ligands directly activated G␣i/o-mediated signaling. Pertussis toxin, an antagonist of G␣i, markedly attenuated hypoxia-induced DNA synthesis and the activation of ERK and JNK but not p38 MAP kinase (Fig 2). These observations confirm that hypoxia itself can act as a growth-promoting stimulus for bovine neonatal adventitial fibroblasts through G␣i/o (and probably Gq)-mediated activation of a complex network of MAP kinases. A hypothetical schema illustrating our observations in this model system is shown in Figure 3.
Role of Extracellular Nucleotides in Fibroblast Proliferation Hypoxia appears to be capable of initiating DNA synthesis in at least some populations of fibroblasts in the absence of exogenous mitogens. It remains unclear, however, whether either the activation or augmentation of the hypoxia-induced growth response is due, at least in part, to autocrine/paracrine responses to factors secreted by fibroblasts during hypoxia, which act through G-proteincoupled pathways. One factor that could contribute to such an autocrine loop is adenosine triphosphate (ATP). Purines and pyrimidines (mainly ATP, adenosine diphosphate, adenosine, and uridine triphosphate) have widespread and specific extracellular signaling actions in the regulation of a variety of functions in many tissues and appear to have key roles in the development, proliferation, differentiation, and release of hormones, neurotransmitters, and cytokines.13 It is also becoming evident that alterations in the physiology of purinergic signaling may result in the development of a variety of pathologies including immune system, neurodegenerative and vascular disease.13 Importantly, in addition to nerves and circulating blood cells, vascular cells themselves appear to be a potent source of ATP and other adenine nucleotides. Figure 2. The inhibition of G␣i/o proteins by pretreatment with pertussis toxin attenuates hypoxia-induced stimulation of DNA synthesis and selectively inhibits MAP kinase activation. Top, A: hypoxia-induced increase in [3H]thymidine incorporation in PA fibroblasts is abolished in the presence of pertussis toxin. Pertussis toxin (100 ng/mL) was added to growth-arrested adventitial fibroblasts and incubated for 1 h, and then the cells were exposed to either normoxia or hypoxia in the presence of [3H]thymidine for 24 h. Middle, B: serum-stimulated growth of fibroblasts is attenuated by pertussis toxin. Quiescent fibroblasts were pretreated with pertussis toxin (100 ng/mL for 1 h) and stimulated www.chestjournal.org
with 10% fetal bovine serum, and growth was measured by DNA synthesis. * ⫽ p ⬍ 0.05 compared with normoxic or unstimulated results, ** ⫽ p ⬍ 0.05 compared with hypoxic or serum-stimulated results. Bottom, C: hypoxia-induced activation of ERK and JNK but not p38 MAP kinase is blocked by pertussis toxin. Representative blots for phospho-ERK, phospho-JNK, and phospho-p38 MAP kinase. CHEST / 122 / 6 / DECEMBER, 2002 SUPPLEMENT
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DNA synthesis, suggesting that the hypoxic activation of P2 receptors may be due to the release of endogenous ATP (16). These experiments suggest that hypoxia may create a purinergic network in which local ATP release, P2 receptor stimulation, and the altered degradation of ATP act to modulate hypoxia-induced fibroblast growth (16).
Hypoxia Induces Transdifferentiation of Fibroblasts to Myofibroblasts
Figure 3. Hypoxia-induced proliferation of fibroblasts is transduced through G-protein-mediated activation of MAP kinases.
These products are known to be released into the extracellular milieu in response to many vascular stress conditions including ischemia/oxidative stress, flow, and mechanical stretch.14 Since vascular cells have been shown to express metabotropic (P2Y) and/or ionotropic (P2X) subtypes of purinergic receptors, and since these receptors have been shown to be coupled to G-protein-dependent mitogenic signaling pathways,15 the released ATP can induce cell activation by an autocrine/paracrine mechanism. In fact, the extracellular concentration of ATP and the subsequent purinergic activation appear to play a critical role in determining the intracellular signaling set point of many key growth-regulating factors.15 Therefore, the release of ATP by environmental or chemical stimuli may contribute to activation of cellular signaling pathways, which raises the possibility that extracellular ATP is a critical modulator of signal transduction pathways operating to control proliferative responses. Using bovine PA adventitial fibroblasts and lung microvascular endothelial cells, we found that acute hypoxia (ie, 3% O2) induced the release of ATP from fibroblasts by 2.2-fold at 10 min, with persistent increases in extracellular ATP concentration observed at 24 h, and from endothelial cells by 60-fold, with a peak at 30 min.16 In addition, chronic hypoxia (ie, for 14 to 30 days) markedly attenuated the rate of extracellular ATP hydrolysis by ectonucleotidase(s), thus creating a situation in which a specific profile of nucleotide may have access to their respective receptors for a prolonged period. We next determined that ATP, uridine triphosphate, adenosine diphosphate-S and MeSATP increased [3H] thymidine incorporation (fourfold to fivefold [10⫺4 mol/L]) in fibroblasts, whereas adenosine was ineffective, indicating that P2Y metabatropic receptors and possibly P2X ionotropic receptors, rather than P1 receptors, are involved in the proliferative response to ATP.16 Hypoxia augmented the proliferative effect of ATP in an additive fashion, whereas ATP and/or hypoxia synergistically increased mitogeninduced DNA synthesis. Finally, we found that the P2 receptor antagonists suramin and cibacron blue 3GA (10⫺4 mol/L) attenuated both ATP and hypoxia-induced 330S
The changes in the proliferative and matrix-producing phenotype of the fibroblast under hypoxic conditions are accompanied by the appearance of ␣-SM-actin, in at least some of the cells in the adventitial compartment, indicating the transdifferentiation of some fibroblasts to myofibroblasts during the development of hypoxia-induced pulmonary hypertension.6,7 The presence of myofibroblasts has been documented extensively in active fibrotic lesions in many diseases including pulmonary fibrosis.17 Myofibroblasts appear to be ultrastructurally and metabolically distinct connective tissue cells acting as key participants in tissue remodeling, because of their own unique proliferative, migratory, and matrix-producing capabilities.18 The differentiation of fibroblasts into myofibroblasts appears to depend on a complex microenvironmental network in which growth factors, cytokines, adhesion molecules, and extracellular matrix components are involved. The stimulating activity of transforming growth factor (TGF)- on ␣-SM-actin and collagen production is well-accepted. However, other factors have also recently been demonstrated to be capable of stimulating ␣-SMactin expression in fibroblasts. Thrombin has been shown to differentiate normal lung fibroblasts to a myofibroblast phenotype via a G-protein-coupled receptor and PKC-⑀ dependent pathway.19 Since hypoxia also has been shown to be capable of activating G-protein-coupled receptor signaling pathways, we hypothesized that hypoxia itself might act as a stimulus to induce the differentiation of fibroblasts into myofibroblasts. We used clonal populations of neonatal bovine PA adventitial fibroblasts to test this possibility. Hypoxia induced a marked increase in ␣-SM-actin protein in fibroblast subpopulations.20 To evaluate the molecular mechanisms involved, fibroblasts were transiently transfected with an ␣-SM-actin promoter that was tagged with luciferase and then exposed to hypoxia. An increase in ␣-SM-actin promoter activity also was observed in response to hypoxia. Hypoxia-induced ␣-SM-actin promoter activity was largely independent of TGF- as the neutralizing antibody of TGF- blocked the hypoxia-induced promoter activity only by 40%, while it completely inhibited TGF--induced increases in promoter activity. To determine the role of G-proteins in hypoxia-induced ␣-SM-actin promoter activity, cells either were cotransfected with constitutively active constructs of Gi and Gq proteins or were treated with pertussis toxin. Augmentation (active Gi) and attenuation (pertussis toxin) of hypoxia-induced promoter activity supported the role for G␣i, but not Gq, in the differentiation of fibroblasts into myofibroblasts. This appears to differ in SMCs in which Gq-coupled pathways are critical for ␣-SM-actin regulaRemodeling and Repair in Respiratory Diseases
tion. PD98059, an inhibitor of ERK activation, potentiated the hypoxia-induced promoter activity. Therefore, we conclude that hypoxia induces the differentiation of fibroblasts into myofibroblasts. This supports our contention (see below) that numerous fibroblast subpopulations exist and that hypoxia (and certainly other stimuli) selectively activates specific fibroblast cell populations. Furthermore, our experiments provide preliminary evidence that the hypoxia-induced ␣-SM-actin expression in fibroblasts is mediated through G␣i.
Developmental Differences in the Response of Fibroblasts to Hypoxia Pathologic studies suggest that the PA adventitial response to chronic hypoxia is greater in infants and children than in adults.4 To explore the possibility of developmentally regulated differences in growth, fibroblasts were isolated from PA adventitia at different stages (ie, fetal, neonatal, and adult stages) during development and growth was assessed. We found that fetal and neonatal fibroblasts grew faster than adult cells in 10% serum.21 Rapidly growing fetal fibroblasts had increased PKC catalytic activity compared to adult cells.21 Using different PKC inhibitor strategies, we also demonstrated that high rates of growth in fetal cells were due in part to high levels of PKC activity.21 PKC signaling, however, is complex, with four Ca2⫹dependent and seven Ca2⫹-independent isozymes known to exist and to have different functions within the cell.22 Developmental changes in the expression of individual isozymes have been reported, and the increased expression of selected PKC isozymes has been linked to augmented growth capacity.23 Our data demonstrate that neonatal bovine PA adventitial fibroblasts express 7 of the 11 isozymes, three that are Ca2⫹-dependent (␣, I, and II) and four that are Ca2⫹-independent (␦, ⑀, , and ). The expression pattern of two isozymes, the Ca2⫹-dependent PKC-␣ and PKC-II, paralleled the developmental differences in growth, susceptibility to PKC inhibitors, and PKC catalytic activity.24 Antagonist strategies implicated these same Ca2⫹dependent isozymes in the enhanced growth of fetal and neonatal fibroblasts. Thus, it appears that the signaling pathways utilized to mediate hypoxia-induced proliferation are not only cell type-specific but are also age-specific or developmental-state specific.
Phenotypic Modulation of Fibroblasts in Response to Chronic Hypoxia In addition to the developmentally regulated changes in the proliferative and matrix-producing potentials of fibroblasts, an expanding body of experimental observations25,26 also supports the concept that significant changes in the phenotypic properties of resident fibroblasts can occur in the setting of fibroproliferative disease. We therefore tested the hypothesis that exposure to chronic hypoxia would induce specific alterations in resident fibroblasts, rendering them more sensitive to growth-promoting mitogens as well as hypoxia. Adventitial fibroblasts from www.chestjournal.org
age-matched control and chronically hypoxic calves were isolated simultaneously, and subsequent comparative experiments were performed in cells under identical conditions. We found, on a very consistent basis, that fibroblasts isolated from hypoxic hypertensive calves exhibited augmented growth responses to serum, hypoxia, and purified peptide mitogens compared to those obtained from agematched control animals.27 These proliferative attributes persisted through numerous cell passages in culture, suggesting acquired differences in fibroblast populations that were not simply due to short-term changes in the in vivo cellular milieu (ie, growth factor, cytokines, and matrix components). Recently, Welsh et al28 also reported that PA fibroblasts from chronically hypoxic rats appeared to have undergone a phenotypic switch. Thus, fibroblasts from both neonates and adults demonstrate a stable phenotypic change in response to hypoxia. Several possibilities must be considered in explaining how a stable phenotypic alteration of the adventitial fibroblast might come about. The first is that a large portion of resident fibroblasts is altered by changes that occur locally in the chronically hypoxic vascular wall. Hypoxia itself, a combination of hypoxia with subsequent hemodynamic changes, and/or a combination of hypoxia, hemodynamic changes, and changes in the local concentrations of cytokines, growth factors, and matrix proteins, must be considered as potential mediators of the phenotypic modulation of cells during the development of chronic hypoxia-induced pulmonary hypertension. Resident fibroblasts could be altered by these signals, conferring on them a new stable phenotype, which is manifested by intrinsically enhanced proliferative and matrix-producing capabilities. Another possibility is that selective expansion of a resident fibroblast population, with unusual or augmented growth properties, has occurred in vivo. This population could expand to the point where it is numerically the most important constituent of the activated or injured vessel wall. This population then might demonstrate selective advantage when cell cultures are performed and, thus, account for the apparent phenotypic change observed in fibroblast populations from the injured organ or vessel.
Fibroblast Heterogeneity in PA Adventitia Since lung, skin, and gingival tissue are composed of multiple fibroblast subpopulations, we investigated the hypothesis that the PA adventitia of neonatal calves is composed of several functionally distinct fibroblast subpopulations and that some subpopulations selectively expand during the development of hypoxic pulmonary hypertension. Fibroblast subpopulations were isolated from the PA adventitia of neonatal control calves using limiteddilution cloning techniques. These subpopulations exhibited marked and stable differences in morphology (Fig 4), cytoskeletal protein expression, and growth capabilities in response to serum.29 We investigated the possibility that fibroblast subpopulations also might exhibit markedly different proliferative responses to hypoxia. We found that proliferation under hypoxic conditions is highly variable CHEST / 122 / 6 / DECEMBER, 2002 SUPPLEMENT
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Figure 4. Fibroblast subpopulations isolated from PA adventitia by a limited-dilution cloning technique exhibited distinct morphologic characteristics. Top left, A, middle left, B, and bottom left, C: fibroblast subpopulations derived from PA adventitia demonstrated a rhomboidal appearance. Top right, D, middle right, E, and bottom right, F: subpopulations of adventitial fibroblasts appeared spindle-shaped (original magnification 10⫻).
among subpopulations with some showing greater than twofold increases in DNA synthesis in response to hypoxia (in the absence of growth factors), while others exhibit decreased DNA synthesis (Fig 5). Finally, to test the hypothesis that long-term hypoxic exposure was associated with a relative increase in the number of fibroblast subpopulations with hypoxia-induced proliferative ability, fibroblast subpopulations were isolated from neonatal calves exposed to hypoxia for 14 days. With regard to morphology, actin expression, and serumstimulated growth capabilities of subpopulations, there were no obvious differences between the hypoxic and the 332S
control calves. However, the number of fibroblast subpopulations with twofold or greater increases in DNA synthesis in response to hypoxia, was significantly greater in the adventitia of hypertensive calves (26%) compared to control calves (10%) [Fig 5]. These experiments provide further support to the hypothesis that the bovine PA adventitia is comprised of multiple phenotypically and functionally distinct fibroblast subpopulations and that chronic hypoxia is associated with the expansion of select fibroblast subpopulations.29 In addition, our data demonstrating numerous functionally distinct fibroblast subpopulations, some with remarkable growth potential, lend Remodeling and Repair in Respiratory Diseases
Figure 5. The proportion of fibroblast subpopulations with more than a twofold increase in DNA synthesis (hypoxia proliferative) was higher in the PA adventitia of hypertensive calves compared to control calves. Left, A: In vitro hypoxia-induced proliferative responses of 31 control (E) and 49 pulmonary hypertensive-derived (F) fibroblast subpopulations. Right, B: the percentage of hypoxiaproliferative fibroblast subpopulations was greater for the hypoxic hypertensive calves than for the control calves.
further support to the idea that the adventitial compartment may be where stem-like cells for the vasculature reside. Changes in the relative proportion of different fibroblast subsets may have profound effects not only on adventitial remodeling but on the whole vessel wall. It is possible that the alteration in the relative proportion of different fibroblast subsets with different capabilities for matrix protein and soluble growth factor production, might be expected to influence the behavior of adjacent SMCs or endothelial cells (ie, dynamic reciprocity). It is clear that there is a continuous feedback of information between the cell and the matrix, such that the extracellular matrix that is in contact with the cells is itself a product of cellular activity (by virtue of biosynthesis, degradation, and modification of matrix macromolecules) and that the extracellular matrix influences various fundamental aspects of cell behavior such as the deposition of matrix itself, cell proliferation, and the pattern of gene expression and cell migration. The activation of a fibroblast subset secreting unique matrix molecules and soluble factors thus may have an influence on neighboring SMCs or epithelial cells that is not exhibited under normal conditions.
Summary The fibroblast may play a significant role in the vascular response to injury, especially under hypoxic conditions, by www.chestjournal.org
modulating its function according to local vascular needs. Fibroblasts appear to be uniquely equipped to proliferate, transdifferentiate, and migrate under hypoxic conditions simultaneously. Hypoxia-induced proliferation of fibroblasts depends on the activation of G␣i and Gq, and on the subsequent stimulation of PKC and MAP kinase family members. Extracellular nucleotides (eg, ATP) acting through G␣i-coupled and Gq-coupled P2Y receptors, appear to act as autocrine/paracrine modifiers of the proliferative response of fibroblasts under hypoxic conditions. Hypoxia can also up-regulate ␣-SM-actin expression in fibroblasts, suggesting that hypoxia may contribute to the transdifferentiation of fibroblasts into myofibroblasts in the vessel wall. In addition, chronic hypoxia causes stable (at least in vitro) phenotypic changes in fibroblasts that appear to be associated with changes in the signaling pathways used to elicit proliferation. However, it is also becoming clear that, as with SMCs, numerous fibroblast subtypes exist in the vessel wall and that each may respond in unique ways to hypoxia and thus may serve special functions in response to injury. The adventitia of the vessel wall thus may be considered to be a compartment where cells with stem-cell-like characteristics reside. We believe, therefore, that the adventitial fibroblast may be a critical regulator of vascular function and structure under hypoxic conditions. This coordinated regulation of several functions by at least some adventitial fibroblast populations is CHEST / 122 / 6 / DECEMBER, 2002 SUPPLEMENT
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in contrast to the responses of mature SMCs, which, in general, dedifferentiate before they exhibit marked changes in proliferative or migratory behavior.
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Overview of Pulmonary Fibrosis* Francis H.Y. Green, MD
Pulmonary fibrosis is a component of over 200 interstitial lung diseases. Some have known etiologies, however, for many diseases, the etiology remains unknown or obscure. This brief review examines the prevalence and classification of these diseases, the approach to be taken for the investigation of a patient suspected of having pulmonary fibrosis, the indications for the performance of lung biopsy, and *From the University of Calgary, Calgary, AB, Canada. This work was funded by the University of Calgary. Correspondence to: Francis H.Y. Green, MD, Professor, Pathology & Laboratory Medicine, Faculty of Medicine, University of Calgary, 3330 Hospital Dr NW, Calgary, AB, Canada T2N 4N1; e-mail: [email protected] Remodeling and Repair in Respiratory Diseases