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Persistent accumulation of monocytes/macrophages in the pulmonary artery adventitial/perivascular areas of animals and humans with pulmonary hypertension has been documented. The cellular mechanisms contributing to chronic inflammatory responses remain unclear. We hypothesized that perivascular inflammation is perpetuated by activated adventitial fibroblasts, which, through sustained production of pro-inflammatory cytokines/chemokines and adhesion molecules, induce accumulation, retention, and activation of monocytes/macrophages. We further hypothesized that this pro-inflammatory phenotype is the result of abnormal activity of histone-modifying enzymes, specifically, class I histone deacetylases (HDACs).
Pulmonary adventitial fibroblasts from chronically hypoxic hypertensive calves (termed PH-Fibs) expressed a constitutive and persistent pro-inflammatory phenotype defined by high expression of IL-1β, IL-6, CCL2(MCP-1), CXCL12(SDF-1), CCL5(RANTES), CCR7, CXCR4, GM-CSF, CD40, CD40L, VCAM-1. The pro-inflammatory phenotype of PH-Fibs was associated with epigenetic alterations as evidenced by increased activity of HDACs, and the findings that class I HDAC inhibitors markedly decreased cytokine/chemokine mRNA expression levels in these cells. PH-Fibs induced increased adhesion of THP-1 monocytes, and produced soluble factors that induced increased migration of THP-1 and murine bone marrow-derived macrophages (BMDMs), as well as activated monocytes/macrophages to express pro-inflammatory cytokines and pro-fibrogenic mediators (TIMP1 and COL1) at the transcriptional level. Class I HDAC inhibitors markedly reduced the ability of PH-Fibs to induce monocyte/migration and pro-inflammatory activation.
The emergence of a distinct adventitial fibroblast population with an epigenetically-altered pro-inflammatory phenotype capable of recruiting, retaining and activating monocytes/macrophages characterizes pulmonary hypertension-associated vascular remodeling, and thus could contribute significantly to chronic inflammatory processes in the pulmonary artery wall.
Several studies have documented that pulmonary hypertension (PH)-associated vascular remodeling is characterized by the early and persistent accumulation of mononuclear cells in the perivascular adventitia in animal models of PH including chronically hypoxic calves, rats, mice, and monocrotaline-treated rodents (1–3). Further, in most chronic forms of human PH, perivascular accumulation of monocytes/macrophages is a common feature (4–6) and elevated levels of inflammatory cytokines, including IL-1β and IL-6, have been shown to predict survival in idiopathic and familial pulmonary arterial hypertension (7). Thus, pulmonary vascular remodeling is potentially perpetuated by a local/ adventitial chronic inflammatory response.
Traditionally, vascular inflammation has been considered an “inside-out” response centered on monocyte/macrophage recruitment to the intima of blood vessels. However, growing evidence supports a new paradigm of an “outside-in” hypothesis, in which vascular inflammation is initiated and perpetuated by adventitial fibroblasts (8–11). The participation of fibroblasts in the regulation/orchestration of immune responses has traditionally been regarded as insignificant while monocytes, dendritic cells and T-lymphocytes are the established key players. However, recent experimental evidence suggests that mesenchymal cells, specifically fibroblasts, are responsive to non-antigen specific danger signals, which activate TLR-NFκB signaling pathways. Thus innate activation of fibroblasts may play a central role in the initiation and perpetuation of inflammatory tissue responses (8, 11–13). Whether pulmonary adventitial fibroblasts, in the setting of PH, exhibit features of a phenotype compatible with innate immune activation characterized by generation of canonical pro-inflammatory cytokines/chemokines, as well as adhesion molecules is unknown. Moreover, in the pulmonary circulation, it is currently unknown if adventitial fibroblasts are capable of recruiting and retaining monocytes/macrophages to the pulmonary artery wall and with subsequent activation of monocytes/macrophages toward a pro-inflammatory phenotype.
Acquired and stable changes in the phenotype of mesenchymal cells may require epigenetic processes such as might occur in response to altered histone acetylation (14). Histone-dependent packaging of genomic DNA into chromatin is a central mechanism for gene regulation. Expression of inflammatory genes, DNA repair, and proliferation have been shown to be controlled by the degree of acetylation of histone and non-histone proteins produced by histone acetyltransferases (HATs) and histone deacetylases (HDACs) (15–18). Several reports have documented changes in HDAC activity in fibroblasts in rheumatoid arthritis and juvenile idiopathic arthritis, with recent reports demonstrating specific increases in HDAC-1 activity (19, 20). Additional reports have demonstrated anti-inflammatory effects of small molecule HDAC inhibitors in animal models of inflammatory diseases, in fibrotic vascular disease, and in cancer (16, 21, 22). However, to our knowledge, no previous studies have addressed the possible role of HDACs in the control of inflammatory gene expression in the setting of PH.
The goal of this study was to test the hypothesis that, in the setting of chronic hypoxic PH, adventitial fibroblasts exhibit a “persistently activated” pro-inflammatory phenotype capable of inducing recruitment, retention and pro-inflammatory activation of monocytes/macrophages. Our approach was to determine whether, in the setting of chronic hypoxic PH, pulmonary adventitial fibroblasts: 1) exhibit an activated pro-inflammatory phenotype (express elevated levels of cytokines, chemokines, adhesion molecules); 2) promote migration, adhesion and pro-inflammatory activation of monocytes/macrophages; and 3) whether epigenetic modifications due to abnormal activity of histone-modifying enzymes contribute to this activated phenotype.
The neonatal calf and rat models of severe hypoxia-induced PH have been described previously (1). Briefly, one day-old male Holstein calves were exposed to hypobaric hypoxia (PB=445 mmHg) for 2 weeks (n=7), while age-matched controls (n=7) were kept at ambient altitude (PB=640 mmHg). Wistar-Kyoto rats were exposed to hypobaric hypoxia (PB = 380 mmHg) for 4 weeks (n = 9). Age-matched controls (n = 9) were kept at ambient altitude.
Monocrotaline treatment of rats (experimental group, n = 12, controls, n=12) were performed as previously described (23).
Standard veterinary care was used following institutional guidelines: for rats, at University of Colorado Denver Center for Laboratory Animal Care 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). Animals of both species were euthanized by overdose of sodium pentobarbital (160 mg/kg body weight).
Frozen sections of lung tissue from human subjects with idiopathic pulmonary arterial hypertension (iPAH) (n = 5) and controls (n = 4) were used for immunocytochemical analyses (see Acknowledgements).
Isolation of adventitial fibroblasts from distal pulmonary arteries was performed as previously described (24). Experiments were performed on cells at passages 4–10. Conditioned media was collected from confluent fibroblast cultures and used in migration and/or activation studies with monocytes (24 hrs, serum-free media) or murine macrophages (72 hrs, complete media). THP-1 monocytes (a human leukemic monocytic cell line routinely used in monocyte assays) were purchased from American Type Culture Collection (ATCC, Manassas, VA). Murine bone marrow-derived macrophages (BMDMs) were isolated from C57/Bl6 mice as previously described (25).
Mouse monoclonal Ab (mAb) or rabbit polyclonal Ab (pAb) against the following antigens were used: bovine CD14 (mAb, 15 µg/ml; VMRD, Pullman, WA), human CD68 (cross-reacts with bovine and rat antigens) (mAb, 1:100 dilution; DAKO, Carpinteria; CA), rat CD11b (mAb, 1:50 dilution; Chemicon, Temecula, CA), human VCAM-1 (cross-reacts with bovine) (goat Ab, 1:50; Santa Cruz), bovine IL-1β (mAb, 15mg/ml; VMRD, Pullman, WA), vimentin (chicken Ab, 1:500 dilution; Millipore, Temecula, CA), heat shock protein-47 (Hsp47) (mAb, 1:400 dilution; Calbiochem, San Diego, CA), α-SM-actin (mAb, 1:200 dilution; Sigma-Aldridge, Saint Louis, MI), SM-myosin heavy chains (SM-MHC, pAb, 1:2000, generously provided by Dr. R. Adelstein, NIH, Bethesda, MD). Immuno-labeled sections were mounted in VectaShield with DAPI (Vector Labs) and examined under a Ziess fluorescent microscope, and images were acquired using AxioVision digital imaging system.
Total RNA isolation from cultured cells, first-strand cDNA synthesis, and real-time RT-PCR were performed as described previously (24). Specific TaqMan probes used with mouse BMDM RNA, as well as bovine-specific IL-10 probe, were the commercially available TaqMan Gene Expression Assays obtained from Applied Biosystems. The sequences for all other primers (bovine and human) are listed in Table 1 and were designed as previously published (26). Results are presented as relative expression to HPRT using the delta CT method or as fold change using delta-delta CT method (27).
Western Blotting was performed as per manufacturer’s suggestions (BioRad, Hercules, CA).
HDAC activity in fibroblasts was measured using a fluorescent substrate that is selectively deacetylated by class I HDACs -1, -2 and -3, which was synthesized (Genscript) as previously described (28). Immunoblotting was performed with HDAC-specific antibodies, HDAC1 , HDAC2 , HDAC3  (Cell Signaling Technology, Danvers, MA). Effects of HDAC inhibitors on fibroblast production of cytokines were assessed by incubating serum-starved fibroblasts with the pan HDAC inhibitor Suberoylanilide Hydroxamic Acid (SAHA, 10 µM, ChemieTek, Indianapolis, IN) or class I HDAC inhibitor apicidin (3 µM) (Enzo, New York, NY) and collecting Fib-CM from treated fibroblasts for further assays. The concentrations of HDAC inhibitors, SAHA and apicidin, used in this study were chosen based on extensive previous titration assays, performed in our laboratories, on various fibroblast types (cardiac, pulmonary, lung interstitial) as the effective, yet not toxic. Cell viability was checked by the Trypan Blue assay.
THP-1 monocytes (2.0 × 106) and/or murine BMDMs (1×106) were incubated with Fib-CM (1ml) for 24 hours. THP-1 and BMDM mRNA was used for real-time RT-PCR analysis.
Fibroblast conditioned medium (Fib-CM) was placed on the bottom of a transwell plate. THP-1 monocytes (Calcien AM-labeled; Invitrogen, Carlsbad; CA) and/or murine BMDMs were placed on the top of a 3 µm Transwell insert (THP-1 monocytes: Fluroblock, BD Biosciences, San Jose, CA; BMDMs: Costar, Lowell, MA). The relative number of cells that migrated through the insert pores toward Fib-CM were assessed by fluorescence measurement (Omega PolarStar fluorometer, BMG Labtech, Cary, NC) or by counting the cells (Calcein AM- or DAPI-stained) using MetaMorph software (Molecular Devices, Sunnyvale, CA).
Confluent fibroblast cultures, grown in 24-well plates, were serum-deprived for 24 hrs. THP-1 cells were labeled with Calcein AM (Invitrogen), stimulated with 10 ng/ml CXCL12(SDF-1) to stimulate integrin conformation, and 500K were added to each well of fibroblasts for 30 min. at 37C. Non-adherent THP-1 cells were removed, followed by 4 washes with Ca++, Mg++ containing HBSS. The relative number of remaining adherent THP-1 was determined by fluorometric measurement.
Values are expressed as mean ± SEM. Student t-test and one-way ANOVA were used for statistical analysis. Differences with P values less than 0.05 were considered statistically significant.
Marked accumulation of monocytes/macrophages (defined by expression of CD14, CD11b, CD68 antigens) was observed in the pulmonary perivascular adventitia of chronically hypoxic calves and rats, monocrotaline-treated rats with PH, as well as in human subjects with idiopathic PAH (Fig. 1). These observations emphasize the adventitial/perivascular accumulation of monocytes/macrophages in several forms of PH.
Consistent with the previous data on fibroblasts from the large elastic PAs (29), adventitial fibroblasts isolated from distal pulmonary arteries of chronically hypoxic hypertensive calves (hereafter termed “PH-Fibs”) were significantly smaller in size than those of control calves (termed “CO-Fibs”) and proliferated at markedly higher rates (data not shown). The phenotype of PH-Fibs and CO-Fibs was characterized by immunofluorescence analysis using mesenchymal, smooth muscle, hematopoietic/progenitor, and monocyte/macrophage markers. Both PH-Fibs and CO-Fibs expressed mesenchymal antigens: a myofibroblast marker α-smooth muscle(SM)-actin (Fig. 2A,B), as well as heat-shock protein-47 (Hsp47, a molecular chaperone for type I pro-collagen), and vimentin (Fig. 2E,F, only PH-Fibs shown). Both PH-Fibs and CO-Fibs populations lacked expression of smooth-muscle-specific marker SM-myosin heavy chain (Fig. 2C, only PH-Fibs shown), and lacked expression of CD34, CD14, and CD68 (not shown). However, a marked difference in the phenotype of PH-Fibs and CO-Fibs was observed in the fact that PH-Fibs were almost completely deficient in THY1 mRNA expression, whereas CO-Fibs expressed THY1 at high levels (Fig. 2G).
Under serum-free conditions, PH-Fibs, as compared to CO-Fibs, exhibited constitutively augmented mRNA expression of canonical pro-inflammatory cytokines (IL-1β, IL-6, GM-CSF), chemokines/cognate receptors (CCL2(MCP-1), CXCL12(SDF-1), CCL5(RANTES), CCR7, CXCR4), co-stimulatory molecules (CD40L, CD40), as well as vascular cell adhesion molecule-1, VCAM-1 (Fig. 3, Table 2). These phenotypical differences between PH-Fibs and CO-Fibs were maintained in culture through multiple passages (tested up to passage 10), thus representing a stable pro-inflammatory phenotype of PH-Fibs. Notably, the pro-inflammatory phenotype of PH-fibs was characterized by the lack of expression of pro-inflammatory TNFα, IL-12A (P35), IL-12B (P40), Th2 cytokines IL-4, IL-13, or Th17 cytokine IL-17A. Little-to-no expression of IL-10, a canonical anti-inflammatory cytokine, was noted in both CO- and PH-Fibs.
RT-PCR results were confirmed at the protein level by immunofluorescent cytochemistry, ELISA, and Western blot analyses. As shown in Fig. 4, in vivo immunocytochemical analysis of lung tissues demonstrated augmented expression of IL-1β, CCL2(MCP-1), CXCL12(SDF-1), and VCAM-1 in the pulmonary adventitia of chronically hypertensive calves. In vitro analysis of cultured cells confirmed these observations by demonstrating augmented expression of these proteins by PH-Fibs compared to CO-Fibs. A 5.4-fold increase in CCL2(MCP-1) production in PH-Fibs (58.5 ± 8.1 pg/ml versus 10.8 ± 7.1 pg/ml in CO-Fibs) was shown by ELISA analysis, and a 1.8-fold increase in VCAM-1 expression by PH-Fibs compared to CO-Fibs was shown by Western blot analysis. Limitations on availability of bovine-specific antibodies prevented evaluation, at the protein level, of other molecules that were significantly increased at the mRNA level.
In contrast, little expression of a pro-inflammatory phenotype was observed in smooth muscle cells (SMC), isolated from the pulmonary arteries of hypertensive animals (Fig. 5A). In fact, PH-Fibs exhibited markedly higher mRNA expression levels than those of SMC isolated from the same pulmonary arteries of hypertensive animals (PH-SMC) for IL-1β, IL-6, CCL2(MCP-1), CXCL12(SDF-1), CCL5(RANTES), CXCR4, CD40, and VCAM-1 (Fig. 5B and Table 3). No significant difference was observed between PH-Fibs and PH-SMC for mRNA expression levels of TNFα, GM-CSF, CCR7, and CD40L (Fig. 5B).
To address the possible role of HDACs in the distinct phenotype of PH-Fibs, HDAC catalytic activity was quantified. As shown in Fig. 6A-a, class I HDAC catalytic activity was significantly increased in PH-Fibs compared to CO-Fibs. The activity was completely blocked by the selective class I HDAC inhibitor, apicidin, confirming that deacetylation of the substrate was mediated by members of HDAC class I. Elevated class I HDAC catalytic activity correlated with increased abundance of HDACs -1, -2 and -3 as determined by Western blotting (Fig. 6A-b,-c).
To determine whether HDAC activity contributed to the pro-inflammatory phenotype of PH-Fibs, pharmacological HDAC inhibitors were used. Incubation of PH-Fibs with the pan-HDAC inhibitor, SAHA, or with the selective class I HDAC inhibitor, apicidin, resulted in attenuation of a pro-inflammatory phenotype as defined by dramatically reduced mRNA expression levels of IL-6, CCL2(MCP-1), CXCL12(SDF-1), GM-CSF, and VCAM-1 (Fig. 6B). Notably, changes in mRNA expression levels of CCL5(RANTES) (Fig. 6B), CXCR4 and CCR2 (not shown) were statistically insignificant, indicating specific targeting of HDAC inhibitors. HDAC inhibition did not result in increased mRNA expression of the anti-inflammatory cytokine IL-10. HDAC inhibitors did not affect, at the concentrations tested, cell viability as tested by Trypan Blue assay.
We next tested whether PH-Fibs induced increased migration of THP-1 monocytes and primary murine BMDMs. Serum-free culture medium, conditioned by PH-Fibs (PH-Fib-CM), as compared to culture media conditioned by CO-Fibs (CO-Fib-CM), induced (1.84 ± 0.11)-fold higher transwell migration of THP-1 monocytes (Fig. 7A, left panel). PH-Fib-CM also increased migration of primary murine BMDMs (2.10 ± 0.06)-fold (Fig. 7A, right panel). We also sought to determine if PH-Fibs exhibited increased adhesion for THP-1 monocytes and show that PH-Fibs induced (1.6 ± 0.04)-fold higher adhesion of monocytes, compared to CO-Fibs (Fig. 7B). We also evaluated whether pro-inflammatory PH-fibs could promote activation of monocytes and macrophages towards a pro-inflammatory phenotype. Incubation of both human THP-1 monocytes and murine BMDMs with PH-Fib-CM, but not with CO-Fib-CM, resulted in markedly upregulated mRNA expression of canonical pro-inflammatory mediators and pro-fibrotic molecules (Figs. 7C,D). Specifically, in response to PH-Fib-CM, THP-1 monocytes showed increased mRNA expression of IL-1β (8.04 ± 2.1-fold increase compared to CO-Fib-CM), IL-6 (2.32 ± 0.23-fold increase), CCL2(MCP-1) (7.44 ± 1.27-fold increase), and type-1 collagen (COL1A1) (3.95 ± 0.65-fold increase) (Fig. 7C). In murine BMDMs incubated with PH-Fib-CM, mRNA expression of IL-1β increased (6.56 ± 0.35)-fold compared to CO-Fib-CM, IL-6 increased (12.0 ± 0.81)-fold, CCL2(MCP-1) increased (2.7 ± 0.11)-fold, and Col1a1 increased (1.32 ± 0.06)-fold (Fig. 7D), and TIMP-1 increased 2-fold (not shown).
We next asked if the attenuation of the pro-inflammatory phenotype in PH-Fibs observed after application of HDAC inhibitors was associated with reduced functional abilities of PH-Fibs to recruit and activate monocytes/macrophages. Indeed, treatment of PH-Fibs with SAHA and apicidin decreased the chemotactic activity of PH-Fib-CM for the monocytes (Fig. 8A) and significantly attenuated their ability to activate pro-inflammatory cytokine production in THP-1 monocytes (Fig. 8B). Induction of IL-6 or COL1A1 expression by PH-Fib-CM was not affected by class I HDAC inhibitors at the time point tested (not shown).
The present study demonstrates that hypoxia-induced pulmonary vascular remodeling is characterized by the emergence of a distinct adventitial fibroblast population (termed here PH-Fibs) that exhibits a constitutively activated “imprinted” pro-inflammatory phenotype, capable of inducing recruitment, retention, and pro-inflammatory activation of monocytes and macrophages. Remarkably, this pro-inflammatory phenotype of PH-Fibs was characterized by high expression levels of canonical pro-inflammatory cytokines (IL-1β, IL-6), macrophage chemo-attractant cytokines [CCL2(MCP-1), CXCL12(SDF-1), CCL5(RANTES)], macrophage growth factor (GM-CSF), a co-stimulatory molecule capable of activating macrophages (CD40L), as well as by increased expression of the adhesion protein VCAM-1 in the absence of any exogenous stimulation. In contrast, smooth muscle cells, isolated from the same arteries of hypertensive animals, did not exhibit a pro-inflammatory phenotype. Our study supports the hypothesis that, mechanistically, the phenotype of PH-Fibs was due to epigenetic alterations, as demonstrated by increased catalytic activity and protein expression of class I histone deacetylases (HDACs). This hypothesis was further supported by our observations that apicidin, a specific class I HDAC inhibitor, preferentially and dramatically decreased expression of a specific subset of pro-inflammatory mediators and caused a marked reduction in the ability of PH-Fibs to induce monocyte migration and activation.
Pulmonary hypertension (PH) is characterized by dramatic changes in the structure of pulmonary arteries and the phenotype of vascular wall cells. In several forms of PH, including the calf model of severe hypoxia-induced PH presented here, the adventitia displays dramatic thickening, which was originally assumed to be exclusively caused by excessive accumulation of fibroblasts and myofibroblasts. New experimental data, however, have expanded this concept by demonstrating dramatic perivascular accumulation of inflammatory cells, suggesting that inflammation correlates with and constitutes an essential part of vascular remodeling in many diseases, including experimental PH and PAH in humans (1, 4, 6, 30–32). In the current study, using inter-species (rat, calf, human) analysis of pulmonary perivascular cellular composition in several forms of PH (hypoxia-induced and monocrotaline-induced experimental PH in animal models, as well as idiophathic PAH in humans), we documented and confirmed consistent accumulation of monocytes/macrophages in the pulmonary adventitia. These observations raise questions as to what specific vascular cell type is responsible for inducing inflammatory cell accumulation and activation in the adventitia. The presented data suggest that a specific population of pro-inflammatory pulmonary adventitial fibroblasts is the candidate cell type.
Functionally, pro-inflammatory PH-Fibs produced soluble factors that were capable of recruiting, retaining and activating monocytes (i.e. THP-1) and macrophages (BMDMs). Candidate factors produced by PH-Fibs responsible for recruitment of monocytes/macrophages both in vivo and in vitro are CCL2(MCP-1), a canonical monocyte attractant cytokine, and CCL12(SDF-1). CCL2-dependent recruitment of monocytes/macrophages has recently been implicated in the pathogenesis of a number of chronic inflammatory conditions, characterized by vascular remodeling (32, 33). CCL12(SDF-1) has been found to be upregulated in hypoxic lung and to be critical for recruitment of CXCR4-positive macrophages, which play critical roles in vascular remodeling (34, 35). Importantly, the persistence of inflammatory cells in pulmonary adventitia observed under sustained hypoxic exposure requires up-regulation of specific molecules capable of retaining circulating cells in the local microenvironment. PH-Fibs generated GM-CSF, a macrophage growth and survival factor that has been shown to control retention of macrophages in the tissue and has been suggested to be a critical cytokine in regulating a variety of tissue inflammatory responses including lung inflammation (36, 37). Moreover, PH-Fibs exhibited increased expression of both CXCL12(SDF-1) and VCAM-1, which have been shown as crucial in the retention of hematopoietic progenitor cells within the tissue, e.g. bone marrow (38). The current study also demonstrates that PH-Fibs produced soluble factors that induced a phenotypic alteration in monocytes/macrophages that was compatible with innate immune activation. Monocytes and macrophages exposed to culture supernatant from PH-Fibs exhibited a pro-inflammatory [IL-1β, IL-6, and CCL2(MCP-1)] gene transcription profile indicative of activated TLR-NFκB signaling pathways. These findings are of particular interest, since in patients with idiopathic and familial PAH, increased expression levels of IL-1β, IL-6, CCL2(MCP-1) have been proposed to predict survival (7, 39–41). Pro-inflammatory IL-6 has recently been implicated in the pathogenesis of hypoxia-induced lung inflammation and pulmonary vascular remodeling using experimental animal models (42, 43). Additionally, generation of IL-6 and IL-1β by monocytes/macrophages is compatible with activation of TLR- and inflammasome signaling pathways activated by danger associated molecular patterns (DAMPs) (44). Such DAMPs likely degenerately activate TLR/Inflammasome pathways across species, which would account for the observation that soluble factors derived from bovine PH-Fibs activated human (THP-1) and murine (BMDMs) monocytes/macrophages. Alternatively, IL-6 and IL-1β secreted by PH-Fibs could activate STAT-dependent and NFkB dependent signaling pathways that lead to activation of macrophages (25, 45).
Intriguingly, PH-Fibs induced expression of NFκB- and AP1-target pro-fibrogenic genes, pro-collagen type I (COL1A1) and TIMP-1, in THP-1 monocytes and BMDM macrophages, which is consistent with a previously described pro-fibrogenic macrophage phenotype, characteristic of chronic inflammation and tissue remodeling in pulmonary hypertension and other diseases (46–48). In pulmonary circulation, in adult atherosclerotic pulmonary arteries, Liptay et al. proposed that extracellular matrix gene expression (fibronectin and type I pro-collagen) was intimately associated with non-foamy neointimal macrophages (47). Expression of virtually all types of collagens, as well as of TIMP-1, at both mRNA and protein levels, has recently been reported in macrophages, where the authors propose that collagen synthesis in macrophages may represent a specific feature of a hitherto unrecognized pro-fibrogenic macrophage phenotype that adds to a spectrum of macrophage functional heterogeneity (49). Thus, our data is consistent with previous reports and suggest a phenotypic switch of monocytes/macrophages, exposed to soluble factors from PH-Fibs, toward not only a pro-inflammatory, but also a potentially pro-fibrogenic phenotype.
We have made the novel observation that, in the setting of severe hypoxic PH, pulmonary adventitial fibroblasts express a distinct pro-inflammatory phenotype, stable in culture for numerous passages. Similar findings of persistently activated fibroblasts were reported for diseased tissues in other organs, including synovial fibroblasts from patients with rheumatoid arthritis, tumor-associated fibroblasts, fibroblasts from systemic sclerosis patients, and lung fibroblasts from patients with idiopathic pulmonary fibrosis (50–52). Although, the molecular basis for such a distinct and stable phenotype remains unclear, a potential role of epigenetic modulation has been suggested. In the present study, PH-Fibs, isolated from severely hypertensive animals, were found to exhibit significantly elevated catalytic activity of HDACs, a family of enzymes that are known to play critical roles in the control of epigenetics (53). Specifically, class I HDACs (HDAC1, HDAC2 and HDAC3), which primarily localize to nuclei, are linked to epigenetics through their ability to efficiently deacetylate nucleosomal histones. Class I HDAC catalytic activity was increased in PH-Fibs, and specific catalytic inhibition of class I HDACs was sufficient to suppress production of pro-inflammatory mediators by PH-Fibs. The data thus suggest that transcriptional changes due to epigenetics, which are mitotically heritable and occur in the absence of underlying changes in DNA sequence, could mechanistically explain the stable, pro-inflammatory phenotype of PH-Fibs. Although some studies have reported decreased HDAC activity in lung diseases such as COPD and bronchial asthma (54), as well as in rheumatoid arthritis (55), recent reports have demonstrated that both the total HDAC activity and specifically the expression of HDAC-1 are significantly increased in rheumatoid arthritis, both whole tissues and synovial fibroblasts (19, 20). Importantly, the data of Kawabata et al. (19) in rheumatoid arthritis are very similar to our results with regard to increases in class I HDAC activity and protein expression. Kawabata et al. discuss in detail the possible explanations for the discrepancies of their results from those reported earlier (55). Similar results (i.e. increased HDAC-1 synovial fibroblasts from RA patients) have also been shown by Horiuchi et al., who suggested that increased HDAC-1 activity might be involved in rheumatoid arthritis pathogenesis by regulating cell cycle and survival in synovial tissues (20). Furthermore, anti-inflammatory effects of HDAC inhibitors (consistent with our results) have been shown both in vitro, as well as in vivo in various inflammatory diseases, including that of rheumatoid arthritis, systemic lupus erythematosus, asthma, inflammatory lung diseases, atherosclerosis, hemorrhagic shock, diabetes, inflammatory bowel diseases, osteoporosis, macular degeneration, neurodegenerative and CNS diseases (16). In vascular disease models, recent publications have demonstrated that HDAC inhibition can decrease neointima formation (56), and decrease inflammation (21). The possibility that specific class I HDAC inhibitors may be beneficial in cardiovascular disease has been suggested by us previously (17). Collectively, these results imply unforeseen potential for class I HDAC-selective small molecule inhibitors for the treatment of pathological vascular remodeling in the setting of some forms of PH. In this regard, numerous HDAC inhibitors are in pre-clinical and clinical development, including compounds that selectively inhibit class I HDACs (22).
Vascular inflammation has traditionally been considered an “inside-out” response centered on monocyte/macrophage recruitment to the intima of blood vessels, wherein injured vascular endothelial cells produce inflammatory mediators and express surface adhesion molecules that participate in monocyte homing to the luminal surface and their transmigration into the intima and/or media. However, growing experimental evidence supports a new paradigm of an “outside-in” hypothesis, in which the adventitial compartment is viewed as a critical regulator of vessel wall function, with vascular inflammation being initiated in the adventitia and then progressing inward, toward the media and intima (8). In support of this “outside-in” hypothesis is the observation, in a wide variety of systemic vascular injuries, of an almost immediate influx of monocytes/macrophages into the adventitial compartment (8, 11, 57–60). The findings of the current study support the “outside-in” hypothesis and provide novel information demonstrating that severe hypoxia-induced PH is associated with the emergence of adventitial fibroblasts with a pro-inflammatory phenotype that may orchestrate recruitment, retention and activation of circulating inflammatory cells. Thus, even though vascular endothelial cells are well known as producers of cytokines and chemokines essential in the recruitment of inflammatory cells (33), it is becoming increasingly clear that the adventitial fibroblast is capable of a wide array of responses, including production of mediators controlling inflammatory responses, and activation and differentiation of the recruited leukocytes.
Our findings of a uniquely distinct adventitial fibroblast in PH raise questions regarding the potential origin of this cell. Our previous work, using animal models of chronic hypoxic PH, demonstrated a dramatic perivascular accumulation of circulating fibrocytes (mesenchymal cells of a monocyte/macrophage lineage) that produced collagen, expressed α-SM-actin, and actively proliferated in the pulmonary adventitia, i.e. a phenotype closely related to that described for PH-Fibs in the present study. In contrast to tissue fibroblasts, fibrocytes lack THY1 expression (61), an observation similar to that described here for PH-Fibs. Thus, the possibility that the PH-Fibs population arose from a circulating, hematopoietic rather than the resident origin cannot be excluded. However, it should be noted that the cells used for the study did not express hematopoietic/progenitor markers, at least at the time point in culture analyzed. Other potential origins of these cells may include endothelial-mesenchymal transition from the adventitial vasa vasorum endothelial cells (62) or even from resident vascular progenitor cells (63).
In conclusion, our study has identified a novel adventitial fibroblast population with a stable pro-inflammatory phenotype that likely results from epigenetic modifications brought about by HDACs. Our data support the “outside-in” hypothesis for the pathogenesis of PH and suggest that adventitial pro-inflammatory fibroblasts orchestrate recruitment, retention and activation of monocytes/macrophages toward a pro-inflammatory phenotype. These findings therefore begin to explain the very common observations of pulmonary perivascular inflammation in humans with PAH and in animal models of PH, and suggest a pivotal role for fibroblast/ macrophage interactions in the PH disease process. Our study also highlights synthetic HDAC inhibitors or tailored immune-modulatory agents that target innate signaling pathways in fibroblasts and/or macrophages as promising candidates for therapy and prevention of PH.
The authors are thankful to Marcia McGowan and Stephen Hofmeister for outstanding help in preparing the manuscript, Aimee L. Anderson for technical assistance with BMDM experiments, Dr. Kendall Hunter, Dr. Dale Brown, Stephen Hofmeister and Sandi Walchak for hemodynamic measurements in calves, Kelley Colvin for technical assistance with the rat model, and Drs. Mary Weiser-Evans and Raphael Nemenoff and respective members of their laboratory, for assistance with the BMDM migration studies.
Lung tissues from idiopathic PAH patients and control subjects were provided by the Pulmonary Hypertension Breakthrough Initiative (PHBI), which is funded by the Cardiovascular Medical Research Education Fund (CMREF). The tissues were procured at the Transplant Procurement Centers at Stanford University, University of California, San Diego, Vanderbilt University and Allegheny General Hospital.
This manuscript was supported by National Institutes of Health grants HL084923-04 and HL014985-37.