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
). 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
). 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
). 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
). 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
). 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
). 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
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
). 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
). 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
). 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
). 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.