Abstract

Vascular remodeling in chronic hypoxic pulmonary hypertension includes marked fibroproliferative changes in the pulmonary artery (PA) adventitia. Although resident PA fibroblasts have long been considered the primary contributors to these processes, we tested the hypothesis that hypoxia-induced pulmonary vascular remodeling requires recruitment of circulating mesenchymal precursors of a monocyte/macrophage lineage, termed fibrocytes. Using two neonatal animal models (rats and calves) of chronic hypoxic pulmonary hypertension, we demonstrated a dramatic perivascular accumulation of mononuclear cells of a monocyte/macrophage lineage (expressing CD45, CD11b, CD14, CD68, ED1, ED2). Many of these cells produced type I collagen, expressed α-smooth muscle actin, and proliferated, thus exhibiting mesenchymal cell characteristics attributed to fibrocytes. The blood-borne origin of these cells was confirmed in experiments wherein circulating monocytes/macrophages of chronically hypoxic rats were in vivo-labeled with DiI fluorochrome via liposome delivery and subsequently identified in the remodeled pulmonary, but not systemic, arterial adventitia. The DiI-labeled cells that appeared in the vessel wall expressed monocyte/macrophage markers and procollagen. Selective depletion of this monocytic cell population, using either clodronate-liposomes or gadolinium chloride, prevented pulmonary adventitial remodeling (ie, production of collagen, fibronectin, and tenascin-C and accumulation of myofibroblasts). We conclude that circulating mesenchymal precursors of a monocyte/macrophage lineage, including fibrocytes, are essential contributors to hypoxia-induced pulmonary vascular remodeling. Vascular remodeling in chronic hypoxic pulmonary hypertension includes marked fibroproliferative changes in the pulmonary artery (PA) adventitia. Although resident PA fibroblasts have long been considered the primary contributors to these processes, we tested the hypothesis that hypoxia-induced pulmonary vascular remodeling requires recruitment of circulating mesenchymal precursors of a monocyte/macrophage lineage, termed fibrocytes. Using two neonatal animal models (rats and calves) of chronic hypoxic pulmonary hypertension, we demonstrated a dramatic perivascular accumulation of mononuclear cells of a monocyte/macrophage lineage (expressing CD45, CD11b, CD14, CD68, ED1, ED2). Many of these cells produced type I collagen, expressed α-smooth muscle actin, and proliferated, thus exhibiting mesenchymal cell characteristics attributed to fibrocytes. The blood-borne origin of these cells was confirmed in experiments wherein circulating monocytes/macrophages of chronically hypoxic rats were in vivo-labeled with DiI fluorochrome via liposome delivery and subsequently identified in the remodeled pulmonary, but not systemic, arterial adventitia. The DiI-labeled cells that appeared in the vessel wall expressed monocyte/macrophage markers and procollagen. Selective depletion of this monocytic cell population, using either clodronate-liposomes or gadolinium chloride, prevented pulmonary adventitial remodeling (ie, production of collagen, fibronectin, and tenascin-C and accumulation of myofibroblasts). We conclude that circulating mesenchymal precursors of a monocyte/macrophage lineage, including fibrocytes, are essential contributors to hypoxia-induced pulmonary vascular remodeling. Significant fibroproliferative changes in the pulmonary artery (PA) adventitia are prominent characteristics of chronic pulmonary hypertension of many causes, including hypoxia. These processes occur in both large and small PAs as a result of adventitial cell proliferation, production/deposition of extracellular matrix proteins, especially collagens, and accumulation of myofibroblasts [α-smooth muscle actin (α-SMA)-expressing fibroblasts].1Stenmark KR Mecham R Cellular and molecular mechanisms of pulmonary vascular remodeling.Annu Rev Physiol. 1997; 59: 89-144Crossref PubMed Scopus (290) Google Scholar, 2Jeffery T Morrell NW Molecular and cellular basis of pulmonary vascular remodeling in pulmonary hypertension.Prog Cardiovasc Dis. 2002; 45: 173-202Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 3Rabinovitch M Pulmonary vascular remodeling in hypoxic pulmonary hypertension.in: Yuan JX-J Hypoxic Pulmonary Vasoconstriction: Cellular and Molecular Mechanisms. Kluwer Academic Publishers, Dordrecht2004: 403-418Crossref Google Scholar, 4Meyrick B The pathology of pulmonary artery hypertension.Clin Chest Med. 2001; 22: 393-404Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 5Humbert M Morrell NW Archer SL Stenmark KR MacLean MR Lang IM Christman BW Weir EK Eickelberg O Voelkel NF Rabinovitch M Cellular and molecular pathobiology of pulmonary arterial hypertension.J Am Coll Cardiol. 2004; 43: 13S-24SAbstract Full Text Full Text PDF PubMed Scopus (1278) Google Scholar These fibroproliferative changes have long been assumed to be due to activation, proliferation, and differentiation of resident adventitial fibroblasts. However, the possibility that nonresident cells may contribute directly to the hypoxia-induced pulmonary remodeling process emerges from recent reports in various disease models (including those of the lung) that demonstrate recruitment of circulating, bone marrow-derived cells capable of assuming a mesenchymal/fibroblast phenotype at the site of tissue injury.6Pereira R Halford K O'Hara M Leeper D Sokolov B Pollard M Bagasra O Prockop D Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice.Proc Natl Acad Sci USA. 1995; 92: 4857-4861Crossref PubMed Scopus (823) Google Scholar, 7Davie NJ Crossno JT Frid MG Hofmeister SE Reeves JT Hyde DM Carpenter TC Brunetti JA McNiece IK Stenmark KR Hypoxia-induced pulmonary artery adventitial remodeling and neovascularization: contribution of progenitor cells.Am J Physiol. 2004; 286: L668-L678Google Scholar, 8Hashimoto N Jin H Liu T Chensue S Phan SH Bone marrow-derived progenitor cells in pulmonary fibrosis.J Clin Invest. 2004; 113: 243-252Crossref PubMed Scopus (645) Google Scholar, 9Hayashida K Fujita J Miyake Y Kawada H Ando K Ogava S Fukuda K Bone marrow-derived cells contribute to pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension.Chest. 2005; 127: 1793-1798Crossref PubMed Scopus (93) Google Scholar Among different types of circulating mesenchymal progenitors, mononuclear cells (MNCs) of a monocyte/macrophage lineage, and specifically a subpopulation of MNCs termed “fibrocytes,” have recently received significant attention with regard to their potential role in various fibroproliferative diseases.10Quan TE Cowper S Wu SP Bockenstedt LK Bucala R Circulating fibrocytes: collagen-secreting cells of the peripheral blood.Int J Biochem Cell Biol. 2004; 36: 598-606Crossref PubMed Scopus (488) Google Scholar, 11Metz CN Fibrocytes: a unique cell population implicated in wound healing.Cell Mol Life Sci. 2003; 60: 1342-1350Crossref PubMed Scopus (201) Google Scholar, 12Phillips RJ Burdick MD Hong K Lutz MA Murray LA Xue YY Belperio JA Keane MP Strieter RM Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis.J Clin Invest. 2004; 114: 438-446Crossref PubMed Scopus (916) Google Scholar, 13Moore B Kolodsick J Thannickal V Cooke K Moore T Hogaboam C Wilke C Toews G CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury.Am J Pathol. 2005; 166: 675-684Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar Fibrocytes comprise a subpopulation of circulating MNCs of a monocyte/macrophage lineage (CD11b+, CD13+, CD14+) that can exhibit fibroblast properties at the site of tissue injury (collagen production, differentiation into myofibroblasts).10Quan TE Cowper S Wu SP Bockenstedt LK Bucala R Circulating fibrocytes: collagen-secreting cells of the peripheral blood.Int J Biochem Cell Biol. 2004; 36: 598-606Crossref PubMed Scopus (488) Google Scholar, 11Metz CN Fibrocytes: a unique cell population implicated in wound healing.Cell Mol Life Sci. 2003; 60: 1342-1350Crossref PubMed Scopus (201) Google Scholar, 12Phillips RJ Burdick MD Hong K Lutz MA Murray LA Xue YY Belperio JA Keane MP Strieter RM Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis.J Clin Invest. 2004; 114: 438-446Crossref PubMed Scopus (916) Google Scholar, 13Moore B Kolodsick J Thannickal V Cooke K Moore T Hogaboam C Wilke C Toews G CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury.Am J Pathol. 2005; 166: 675-684Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 14Pilling D Buckley CD Salmon M Gomer RH Inhibition of fibrocyte differentiation by serum amyloid P.J Immunol. 2003; 171: 5537-5546PubMed Google Scholar, 15Yang L Scott PG Giuffre J Shankowsky HA Ghahary A Tredget EE Peripheral blood fibrocytes from burn patients: identification and quantification of fibrocytes in adherent cells cultured from peripheral blood mononuclear cells.Lab Invest. 2002; 82: 1183-1192Crossref PubMed Scopus (214) Google Scholar Fibrocytes have been shown to be an important source of tissue fibroblasts in asthma and lung fibrosis.12Phillips RJ Burdick MD Hong K Lutz MA Murray LA Xue YY Belperio JA Keane MP Strieter RM Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis.J Clin Invest. 2004; 114: 438-446Crossref PubMed Scopus (916) Google Scholar, 13Moore B Kolodsick J Thannickal V Cooke K Moore T Hogaboam C Wilke C Toews G CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury.Am J Pathol. 2005; 166: 675-684Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 16Schmidt M Sun G Stacey MA Mori L Mattoli S Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma.J Immunol. 2003; 170: 380-389Google Scholar However, to our knowledge, there are no studies demonstrating the contribution of circulating fibrocytes or other types of mesenchymal precursors of a mononuclear origin to pulmonary vascular remodeling in the setting of hypoxic pulmonary hypertension. We thus tested the hypothesis that chronic hypoxic exposure induces recruitment of circulating fibrocytes to the pulmonary circulation, and that these cells contribute directly and substantially to pulmonary vascular remodeling. Two neonatal animal models (rat and calf) of chronic hypoxia-induced pulmonary hypertension were used to test this hypothesis in an effort to determine how generalized and consistent the response was across species. In these models we sought to 1) identify the contribution of MNCs and fibrocytes to PA adventitial thickening using immunofluorescence analysis with a panel of cell type-specific antibodies; 2) delineate the direct contribution of these cells to fibroproliferative processes (collagen production, proliferation, accumulation of myofibroblasts) in hypoxic pulmonary vascular remodeling using double-label immunofluorescent staining for leukocyte antigens, markers of fibrosis (procollagen), cell replication [5-bromo-2′-deoxyuridine (BrdU)], and myofibroblasts (α-SMA); 3) assess the blood-borne origin of these cells in the remodeled pulmonary adventitia of chronically hypoxic animals by in vivo labeling of these cells in the circulation and subsequent determination of their localization in the lung and systemic vasculature; and 4) evaluate the role of these cells in hypoxic pulmonary vascular remodeling process by selectively depleting them and subsequently assessing the impact on pulmonary adventitial fibrosis and myofibroblast accumulation. Two animal species were used as models of hypoxia-induced neonatal pulmonary hypertension: weanling rats and, to confirm the findings in a large mammalian species, calves. The advantages of using the hypoxic calf model have been described before1Stenmark KR Mecham R Cellular and molecular mechanisms of pulmonary vascular remodeling.Annu Rev Physiol. 1997; 59: 89-144Crossref PubMed Scopus (290) Google Scholar and include remarkable thickening of PA adventitia, which resembles the pathological picture in human neonatal pulmonary hypertension.2Jeffery T Morrell NW Molecular and cellular basis of pulmonary vascular remodeling in pulmonary hypertension.Prog Cardiovasc Dis. 2002; 45: 173-202Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 3Rabinovitch M Pulmonary vascular remodeling in hypoxic pulmonary hypertension.in: Yuan JX-J Hypoxic Pulmonary Vasoconstriction: Cellular and Molecular Mechanisms. Kluwer Academic Publishers, Dordrecht2004: 403-418Crossref Google Scholar, 4Meyrick B The pathology of pulmonary artery hypertension.Clin Chest Med. 2001; 22: 393-404Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar Rats were used in this study as an animal model of hypoxic pulmonary hypertension because these rodents, in contrast to mice, develop marked PA adventitial thickening17Sobin SS Chen PC Ultrastructural changes in the pulmonary arterioles in acute hypoxic pulmonary hypertension in the rat.High Alt Med Biol. 2000; 1: 311-322Crossref PubMed Scopus (11) Google Scholar and because certain experimental manipulations can be performed in rats but are not economically feasible in calves. The Wistar-Kyoto rat strain was chosen because it develops more severe hypoxic pulmonary hypertension than the Sprague-Dawley strain.18Bochnowicz S Osborn RR Luttmann MA Louden C Hart T Hay DW Underwood DC Differences in time-related cardiopulmonary responses to hypoxia in three rat strains.Clin Exp Hypertens. 2000; 22: 471-492Crossref PubMed Scopus (7) Google Scholar Weanling (4 weeks old) male Wistar-Kyoto rats were purchased from Charles River Laboratories (Wilmington, MA). Experimental hypoxic groups of rats (n = 4 to 6, each time point) were exposed to hypobaric hypoxia (PB = 380 mmHg) for 24 hours to 4 weeks. Age-matched controls were kept at ambient altitude. One-day-old male Holstein calves were purchased from Laluna dairy farm (Fort Collins, CO). The experimental hypoxic group (n = 7) was exposed for 2 weeks to hypobaric hypoxia (PB = 445 mmHg), while age-matched controls (n = 6) were kept at ambient altitude (Denver, CO; PB = 640 mmHg).1Stenmark KR Mecham R Cellular and molecular mechanisms of pulmonary vascular remodeling.Annu Rev Physiol. 1997; 59: 89-144Crossref PubMed Scopus (290) Google Scholar Animals of both species were euthanized by overdose of sodium pentobarbital (160 mg/kg body weight). Standard veterinary care was used following institutional guidelines: for rats, at the University of Colorado Health Sciences Center for Laboratory Animal Care (Denver, CO) in compliance with Institutional Animal Care and Use Committee-approved protocols; for calves, at the Department of Physiology, School of Veterinary Medicine, Colorado State University (Fort Collins, CO). Freshly obtained tissue samples were embedded in O.C.T. (Sakura Finetek, Torrance, CA) and frozen at −70°C until use. The following antibodies against leukocyte antigens were used in the study: bovine-specific monoclonal antibodies (mAbs) against CD45, CD11b, CD14 (15 μg/ml; VMRD Inc., Pullman, WA), CD68 (EBM11, 1:100; DakoCytomation Corp., Carpinteria, CA); rat-specific mAbs against CD45, CD11b (OX-1, OX-42, 1:100; Chemicon Int., Temecula, CA), mAbs against ED1, ED2 (1:10, 1:50; BD Pharmingen, San Diego, CA), and anti-granulocyte mAbs (RK-4, 1:50; Cedarline Laboratories, Ontario, Canada). The following antibodies against markers of fibrosis were used: mAbs against type I procollagen (SP1.D8, 1:10; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), mAbs against collagen-prolyl-4-hydroxylase-α (6–6H2, 1:50; Medicorp, Montreal, Canada), mAbs against ED-A-FN (ist-9, 1:10; Oxford Biotechnology, Oxfordshire, UK), and rabbit polyclonal anti-tenascin-C Abs (1:100, Chemicon Int.). To define the expression of a smooth muscle marker α-SMA, mAb 1A4 was used (1:100; Sigma Chemical Co., St. Louis, MO). Proliferating cells were identified using fluorescein isothiocyanate-conjugated anti-BrdU mAbs (1:3, BD Pharmingen). Tissue cryosections (5 μm) were fixed in methanol:acetone (1:1). Nonspecific binding was blocked with fetal bovine serum:phosphate-buffered saline (PBS) (1:1), and sections were incubated overnight with primary Abs at 4°C. Secondary biotinylated Abs (Vector Laboratories, Burlingame, CA), streptavidin-Alexa-594 (red) and streptavidin-Alexa-488 (green) (Molecular Probes, Eugene, OR) were used at dilutions recommended by the manufacturers. Immunolabeled sections were mounted in VectaShield/DAPI (Vector Laboratories) and examined under a Zeiss fluorescent microscope with an AxioVision digital imaging system (Carl Zeiss MicroImaging, Inc., Thornwood, NY). For double-label confocal microscopy, labeling with antibodies against CD45, CD11b, CD14, CD68, and ED1 was accomplished first. Next, anti-procollagen or α-SMA mAbs, directly labeled with Zenon-Alexa-fluor kit (Molecular Probes), were applied for 1 to 2 hours. Images were analyzed on DeltaVision digital deconvolution confocal microscope (Olympus America Inc., Melville, NY). Photographs were taken with a Photometrics Quantix cooled digital charge-coupled device camera (Olympus). Quantitative analysis of the percentage of monocytic cells co-expressing procollagen, BrdU, and α-SMA was performed on 12 to 18 stained lung sections that were obtained from four to six animals (rats and calves, respectively). Rats were exposed to hypoxia for 24 hours, 48 hours, 72 hours, 96 hours, and 1, 2, 3, and 4 weeks. Lung cryosections were immunolabeled for a monocyte/macrophage marker CD11b, and the perimeter of the free PA wall (contiguous to alveoli) was examined within the radial distance of ≥150 μm outward from the external elastic lamina. CD11b+ cell number was assessed per unit volume (10-μm radial distance by 500-μm perimeter length). Rats (72-hour hypoxic or normoxic) were injected with BrdU (100 μg/kg body weight) at 24, 18, and 2 hours before euthanization. Liposomes were prepared as previously described.19Van Rooijen N Sanders A Liposome mediated depletion of macrophages: mechanisms of action, preparation of liposomes and applications.J Immunol Methods. 1994; 174: 83-93Crossref PubMed Scopus (1483) Google Scholar For liposome-mediated in vivo DiI-labeling of circulating monocytes/macrophages, liposomes containing red fluorochrome, DiI, were introduced into the circulation via a series of intravenous injections (see below). These liposomes are readily taken up via phagocytosis by circu-lating phagocytic MNCs of a monocyte/macrophage lineage (but not by other subsets of MNCs), and DiI-fluorochrome is incorporated into the cell membrane, resulting in selective labeling of circulating monocytes/macrophages.19Van Rooijen N Sanders A Liposome mediated depletion of macrophages: mechanisms of action, preparation of liposomes and applications.J Immunol Methods. 1994; 174: 83-93Crossref PubMed Scopus (1483) Google Scholar, 20Danenberg HD Fishbein I Gao J Mönkkönen J Reich R Gati I Moerman E Golomb G Macrophage depletion by clodronate-containing liposomes reduces neointimal formation after balloon injury in rats and rabbits.Circulation. 2002; 106: 599-605Crossref PubMed Scopus (206) Google Scholar DiI liposomes were intravenously injected (0.1 ml/10 g body weight) into anesthetized rats (n = 5) every third day during the 3-week hypoxic (or normoxic) exposure, as well as on 3 consecutive days before the end of the experiment before euthanasia. Tissues were frozen in O.C.T., and unfixed cryosections were examined for the red DiI fluorescence. After capturing the image demonstrating DiI fluorescence, the cryosection was fixed in acetone:methanol, which completely bleached DiI fluorescence, and then labeled with antibodies against CD11b, and/or ED1, and/or ED2. To deplete circulating MNCs of a monocyte/macrophage lineage, we used two separate approaches: serial intravenous injections of liposomes containing clodronate (Cl2MBP; gift of Roche Diagnostics GmbH, Mannheim, Germany) or intravenous injections of gadolinium chloride (GdCl3, Sigma Chemical Co.). These methods are commonly used to target and deplete phagocytic cells. Clodronate has an extremely short half-life in the circulation and therefore is not toxic.19Van Rooijen N Sanders A Liposome mediated depletion of macrophages: mechanisms of action, preparation of liposomes and applications.J Immunol Methods. 1994; 174: 83-93Crossref PubMed Scopus (1483) Google Scholar However, when delivered into phagocytic cells using liposomes as vehicles, clodronate accumulates in the cell and induces apoptosis after exceeding a threshold concentration.19Van Rooijen N Sanders A Liposome mediated depletion of macrophages: mechanisms of action, preparation of liposomes and applications.J Immunol Methods. 1994; 174: 83-93Crossref PubMed Scopus (1483) Google Scholar Importantly, Cl2MBP-liposomes do not cross vascular endothelial barriers and thus would not be taken up by resident tissue phagocytic cells. The selective action of GdCl3 on monocytes/macrophages is based on the fact that this compound is readily dissolved in normal saline; however, when injected into the bloodstream, it rapidly aggregates into relatively large colloidal particles at neutral pH. Similar to liposomes, the particles of GdCl3 are taken up exclusively by circulating phagocytic MNCs but not by other cells. Once inside the phagocytic cell and after exceeding the threshold concentration, GdCl3 causes cell apoptosis.21Singh B Pearce JW Gamage LN Janardhan K Caldwell S Depletion of pulmonary intravascular macrophages inhibits acute lung inflammation.Am J Physiol. 2004; 286: L363-L372Google Scholar Experimental hypoxic and normoxic rats (n = 8, each group) were injected intravenously with Cl2MBP-liposomes bi-weekly for 4 weeks (first injection was at 0.1 ml/10 g body weight, subsequent injections were at 0.05 ml/10 g body weight). Control hypoxic and normoxic rats were intravenously injected with PBS liposomes (n = 6) on the same schedule. GdCl3 (2 mg/ml, 10 mg/kg body weight) was administered as intravenous injections bi-weekly for 4 weeks of hypoxic or normoxic exposure (n = 6, each), and control rats were injected with PBS (n = 8). Data are presented as mean ± SEM. Statistical analysis used Student's t-test and one-way analysis of variance; significance accepted at P < 0.05. In normoxic animals of both species (rats and calves), the PA adventitia was thin [Figure 1, A (rat) and C (calf)], whereas in animals exposed to chronic hypobaric hypoxia (rats for 4 weeks and calves for 2 weeks), the PA adventitia was remarkably thickened [Figure 1, B (rat) and D (calf)]. We first examined whether MNCs of a monocyte/macrophage lineage were recruited to the PA in response to chronic hypoxic exposure using a panel of antibodies to monocyte/macrophage antigens (in rats, CD45, CD11b, ED1, ED2; in calves, CD45, CD11b, CD14, CD68). In normoxic animals (both rats and calves), the adventitia contained very few cells expressing these antigens [Figure 2, normoxia columns, A and C (rat) and E (calf)]. However, in chronically hypoxic animals of both species, numerous cells expressing monocyte/macrophage antigens were observed in the thickened adventitia of both large and small PAs [Figure 2, hypoxia columns, B and D (rat) and F (calf)]. Accumulation of these cells was specific to the lung vasculature, because the aorta, carotid, and femoral arteries of hypoxic animals were devoid of such cells (data not shown). Moreover, neutrophils were not identified in the PA adventitia of either hypoxic calves (at 2 weeks of hypoxic exposure) or hypoxic rats (starting from 24 hours and up to 4 weeks of hypoxic exposure) (data not shown).Figure 2Hypoxia induces a robust appearance of MNCs in the PA adventitia. Cryosections of PAs from rats (A–D) and calves (E, F) were labeled with antibodies against MNC/macrophage antigens (red) and cell nuclei (DAPI, blue). Few MNCs are present in the adventitia of normoxic rats and calves (single large arrowheads in the normoxia columns). In the adventitia of chronically hypoxic rats and calves, abundant MNCs of a monocyte/macrophage phenotype are present (triple large arrowheads in the hypoxia columns). For rats, micrographs A and B show main PAs, whereas C and D show distal PAs (80 to 100 μm in diameter). Green autofluorescence of the elastic lamellae in A--D defines the boundaries of the PA tunica media. The PA lumens are marked with small green arrows. M, media; A, adventitia. Scale bars: 100 μm (A, B); 20 μm (C–F).View Large Image Figure ViewerDownload Hi-res image Download (PPT) A kinetic analysis demonstrated a progressive accumulation of CD11b+ monocytes/macrophages in the pulmonary perivascular space of hypoxic (24 hours to 4 weeks) rats with a peak accumulation index of 11 at 4 weeks of hypoxic exposure compared to 2.5 in normoxic rats (Figure 3). Furthermore, a progressive increase in the distance from the external elastic lamellae (x axis) of the CD11b+ cell peak was observed, which correlated with the progressive increase in the thickening of PA adventitia in response to chronic exposure to hypoxia. Extensive collagen deposition, accumulation of myofibroblasts, and cell proliferation in the PA adventitia are prominent features of hypoxia-induced pulmonary vascular remodeling.1Stenmark KR Mecham R Cellular and molecular mechanisms of pulmonary vascular remodeling.Annu Rev Physiol. 1997; 59: 89-144Crossref PubMed Scopus (290) Google Scholar, 2Jeffery T Morrell NW Molecular and cellular basis of pulmonary vascular remodeling in pulmonary hypertension.Prog Cardiovasc Dis. 2002; 45: 173-202Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 3Rabinovitch M Pulmonary vascular remodeling in hypoxic pulmonary hypertension.in: Yuan JX-J Hypoxic Pulmonary Vasoconstriction: Cellular and Molecular Mechanisms. Kluwer Academic Publishers, Dordrecht2004: 403-418Crossref Google Scholar, 4Meyrick B The pathology of pulmonary artery hypertension.Clin Chest Med. 2001; 22: 393-404Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 5Humbert M Morrell NW Archer SL Stenmark KR MacLean MR Lang IM Christman BW Weir EK Eickelberg O Voelkel NF Rabinovitch M Cellular and molecular pathobiology of pulmonary arterial hypertension.J Am Coll Cardiol. 2004; 43: 13S-24SAbstract Full Text Full Text PDF PubMed Scopus (1278) Google Scholar, 22Short M Nemenoff RA Zawada WM Stenmark KR Das M Hypoxia induces differentiation of pulmonary artery adventitial fibroblasts into myofibroblasts.Am J Physiol. 2004; 286: C416-C425Crossref Scopus (87) Google Scholar We thus evaluated whether any of the MNCs of a monocyte/macrophage lineage accumulating in the pulmonary perivascular space in response to chronic hypoxia exhibited a phenotype characteristic of a fibrocyte (ie, produced collagen, expressed α-SMA, proliferated).10Quan TE Cowper S Wu SP Bockenstedt LK Bucala R Circulating fibrocytes: collagen-secreting cells of the peripheral blood.Int J Biochem Cell Biol. 2004; 36: 598-606Crossref PubMed Scopus (488) Google Scholar, 11Metz CN Fibrocytes: a unique cell population implicated in wound healing.Cell Mol Life Sci. 2003; 60: 1342-1350Crossref PubMed Scopus (201) Google Scholar To identify fibrocytes in the PA adventitia, double-label immunostaining was performed for MNC-macrophage antigens (in rat, CD45, CD11b, ED1, ED2; in calf, CD45, CD11b, CD14, CD68) and intracellular collagen precursor molecules [in rat, collagen-prolyl-4-hydroxylase-α (cP4Hα); in calf, α-I procollagen (pColl)]. Routine and confocal fluorescence microscopy demonstrated that a substantial proportion of MNCs, accumulating in the remodeled PA adventitia of hypoxic animals, was comprised of collagen-expressing fibrocytes (64.3 + 4.3% in rats, and 38.1 + 3.6% in calves) [Figure 4, A–C (rat) and D–E (calf)]. No fibrocytes were identified in the PA adventitia of normoxic calves and/or rats (not shown) We determined if any MNCs co-expressed α-SMA and thus contributed directly to the accumulation of myofibroblasts (α-SMA-expressing fibroblasts) in the PA adventitia of chronically hypoxic animals. Analysis of double-label immunostaining demonstrated that, in hypoxic animals of both animal species, numerous adventitial cells co-expressed macrophage antigens (CD11b in rats and CD68 in calves) and α-SMA [Figure 5, A and B (rat) and C and D (calf)]. Deconvolution confocal microscopy showed that, among all MNCs within the remodeled adventitia, 19.2 ± 4.3% MNCs (in rats) and 7.8 ± 3.2% MNCs (in calves) co-expressed α-SMA. No α-SMA-expressing MNCs were identified in the PA adventitia of normoxic calves and rats (not shown). The PA adventitia of hypoxic rats demonstrated marked increases in nuclear BrdU incorporation (Figure 6B), whereas no proliferating adventitial cells were observed in normoxic rats (Figure 6A). Analysis of double-label immunofluorescent staining of lung tissues from chronically hypoxic rats demonstrated that a significant number (15.4 ± 2.3%) of MNCs expressing CD45 and/or ED2 had incorporated BrdU (Figure 6B, arrows; CD45 is shown). Cells other than MNCs, potentially resident fibroblasts, were also observed to have incorporated BrdU (Figure 6B, arrowheads). The relative contribution of cells expressing MNC markers to the overall BrdU labeling index in the PA adventitia at 72 hours of hypoxic exposure was 28.6 ± 4.1%. Thus, the MNCs, accumulating within the remodeled PA adventitia, contributed significantly to the overall increases in cell proliferation observed in the