As noted, there is good evidence that adventitial fibroblasts in the pulmonary hypertensive vessel wall exhibit a hyper-proliferative, inflammatory, and invasive phenotype. Questions arise as to origins and mechanisms regulating this phenotype. Intriguingly, this phenotype resembles, in certain ways, the phenotypic characteristics of rheumatoid arthritis (RA) synovial fibroblasts (RASFs), cancer-associated fibroblasts and fibroblasts derived from the fibrotic lung, kidney and liver. It has been demonstrated that synovial fibroblasts (SF), perhaps more than other types of fibroblasts, acquire phenotypic characteristics commonly associated with transformed cells.[78
RASFs show “spontaneous” or “constitutive” activities associated with aggressive behavior and they differ from SFs of patients with osteoarthritis or normal SFs. For example, RASFs upregulate proto-oncogenes, matrix-specific degrading enzymes (MMPs), adhesion molecules, and cytokines, thus exhibiting a distinct “imprinted” phenotype which is stable over many passages in culture.[8
Similarly, primary fibroblasts isolated from fibrotic kidneys maintain their “activated” pro-fibrotic state even when cultured in vitro.[83
] Additionally, there are convincing data that demonstrate stable phenotypic differences in fibroblasts obtained from the lungs of patients with idiopathic pulmonary fibrosis (IPF). IPF fibroblasts are more resistant to apoptosis compared to fibroblasts isolated from nonfibrotic tissues.[84
] Fibroblasts isolated from the lungs of IPF patients also have documented increases in the expression of IL-13 receptor subunits.[85
] Another pathway in which phenotypic differences in receptor expression have been reported includes the CCL2:CCR2 pathway. Fibroblasts isolated from sites of scleroderma, including the lung, have increased CCR2 expression.[86
] It has also been demonstrated that IPF derived fibroblasts are hyper-responsive to cytokines, including TGFb, IL-13 and CCL2.[87
] Consistent with these observations is work from our laboratory, which demonstrates that hypoxia-induced pulmonary vascular remodeling is characterized by the emergence of a distinct adventitial fibroblast population that exhibits a constitutively activated, or “imprinted,” pro-inflammatory phenotype that is capable of inducing recruitment, retention and pro-inflammatory activation of monocytes/macrophages ().[41
] Importantly, in the absence of any exogenous stimulation, these constitutively-activated pro-inflammatory fibroblasts are equipped to generate a microenvironment characterized by high expression levels of pro-inflammatory cytokines such as IL-1beta and IL-6; macrophage chemo-attractant cytokines such as (CCL2/MCP1), CXCL12, SDF1, and CCL5 (RANTES), macrophage growth and activation factor GM-CSF, co-simulatory molecules capable of activating macrophages such as CD40L, as well as the adhesion molecule VCAM-1. Smooth muscle cells isolated from the same arteries of hypertensive animals exhibited either no or a far lesser degree of activation of all the aforementioned molecules and mediators.[41
Figure 5 Fibroblasts from the chronically hypoxic pulmonary artery express an “imprinted” pro-inflammatory phenotype. Fibroblasts derived from control and hypoxic pulmonary hypertensive calves were cultured and in the absence of any exogenous stimuli, (more ...)
The acquisition of stable, functional phenotypic changes in mesenchymal cells, such as the fibroblast described in the aforementioned conditions, probably requires epigenetic processes such as might occur in response to altered histone actelytation, DNA methylation and/or changes in micro-RNA expression profiles.[88
] Histone-dependent packaging of genomic DNA into chromatin is a central mechanism for gene regulation. Expression of inflammatory genes, DNA repair genes and proliferation genes is controlled by the degree of acetylation of histone and nonhistone proteins produced by histoneacetyltransferase (HAT) and histone deacetylase (HDACs).[90
] Several reports have documented such changes in HDAC activity in fibroblasts in rheumatoid arthritis (RA) and juvenile idiopathic arthritis, with recent reports demonstrating specific increases in HDAC-1 activity.[92
] Additional reports have demonstrated anti-inflammatory effects of small molecule HDAC inhibitors in animal models of inflammatory diseases, fibrotic vascular disease and in cancer.[94
] We recently reported that adventitial fibroblasts isolated from severely hypertensive, chronically hypoxic calves (described above) exhibited significantly elevated catalytic activity of HDACs, specifically Class 1 HDAC 1-3, which primarily localized to the nuclei and were linked to epigenetics through their ability to efficiently deacetylate nucleosomal histones.[41
] Most importantly, we found that specific catalytic inhibition of Class 1 HDACs was sufficient to suppress production of the constitutivelyexpressed pro-inflammatory mediators expressed by activated fibroblasts.[41
] These data 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 these adventitial fibroblasts. These findings are consistent with those of Kawabata et al. in RA with regard to specific increases in Class 1 HDAC activity and protein expression ().[92
The constitutively activated “imprinted” phenotype of fibroblasts is due, at least in part, to increased HDAC activity.
HDAC inhibitors have been shown to exert antiinflammatory effects both in vitro and in vivo in various inflammatory diseases, including RA, systemic lupus erythematosus, asthma, inflammatory lung disease, atherosclerosis, hemorrhage shock, diabetes, inflammatory bowel disease, osteoporosis, and macular degeneration.[96
] In vascular disease models, recent publications have demonstrated that HDAC inhibition can decrease neointima formation and decrease inflammation.[94
] Collectively, these results imply unforeseen potential for Class 1 HDAC-selective small molecule inhibitors for the treatment of pathologic vascular remodeling in the setting of some forms of PH. In this regard, numerous HDAC inhibitors are in preclinical and clinical development, including compounds that selectively inhibit Class 1 HDACs ().[95
HDAC inhibitors attenuate the pro-liferative and -inflammatory phenotype exhibited by the constitutively activated fibroblast from pulmonary hypertensive animals.
Another highly important mechanism through which cells become epigenetically altered is through DNA methylation changes. DNA methylation refers to the covalent attachment of a methyl group to the C5 position of cytosine residues in CpG dinucleotide sequences that are called CpG islands. DNA islands are often in the promoter or enhancer regions of genes and methylation of these sites can alter transcription. DNA methylation is involved in normal cellular control of gene expression and is dynamically regulated. However, changes in DNA methylation are also relevant to disease and may be of particular relevance to the changes in fibroblast phenotype that are observed in chronic fibrotic disorders. Five-methylcytosine DNA levels are reduced in RASF tissues and in cultured RASFs.[79
] Specifically, the promoter of an L1 element was partially demethylated, confirming a global genomic hypomethylation in RASFs. It was proposed that the hyper-aggressive and pro-inflammatory phenotype of RASFs was the result of a progressive loss of methylation marks and tissue-specific transcription factors, which are not normally expressed, are upregulated and are responsible for activation of many genes involved in the pathogenesis of rheumatoid arthritis. This concept was confirmed in experiments where 5-azaC (α DNA-hypomethylator) treatment of normal SFs lead to a phenotype identical to RASFs. Over 186 genes were upregulated in 5-azaC treated cells by greater than 2-fold, including growth factors and growth factor receptors, extracellular matrix proteins, adhesion molecules and matrix degrading enzymes. Furthermore, hypomethylation of certain receptors, specifically the death receptor, could explain the relative resistance to apoptosis, which has been reported in RASFs in certain patients.[79
] Additional data suggest that global genomic hypomethylation can be accompanied or followed by specific promoter hypermethylation.[100
There is growing evidence for abnormalities in DNA methylation in fibroblasts in other chronic fibrotic diseases, such as are observed in the lung and kidney.[101
] A recent study demonstrated epigenetic silencing of Thy- 1 by DNA hypermethylation specifically within fibroblast foci in patients with IPF, suggesting that this may be an important mechanism for pathogenetic fibroblast alterations since absence of Thy-1 correlates with a pro-fibrotic phenotype.[103
] Importantly, treatment with DNA methyltransferase restored Thy-1 expression in Thy-1-negative fibroblasts. Interestingly, the adventitial fibroblasts from hypertensive calves described above are Thy-1-negative while adventitial fibroblasts from healthy control calves are Thy-1-positive. Furthermore, a recent study showed that hypermethylation of RASALI, encoding an inhibitor or the Ras oncoprotein, is associated with the perpetuation of fibroblast activation and fibrogenesis in the kidney,[83
] and that kidney fibrosis is ameliorated in mice heterozygous for DNA (cytosine-5)-methyltransferase 1 (Dnmt1). Very intriguing data came from a recent study using lung fibroblasts that convincingly indicated that alterations of histone modifications alter DNA methylation.[104
] Sanders et al. showed that treatment with the HDAC inhibitor trichostatin (TSA) restored Thy-1 expression in Thy-1-negative cells in a time and concentration-dependent fashion and was associated with enrichment of histone acetylation. Bisulfite sequencing of the Thy-1 promoter region revealed demethylation of the previously hypermethylated CpG site in response to treatment with TSA.[104
] TSA treatment also upregulated total methyltransferase activity in these cells. The experimental observation that treatment with an HDAC inhibitor can restore Thy-1 (a proposed “fibrosis-suppressor” gene) expression in fibroblasts in fibrotic disease and change the phenotype (it decreased a-SM-actin expression) suggests that HDACs could be used as a therapeutic target for the treatment of fibrotic diseases such as IPF with or without pulmonary hypertension.
Because of the well-documented antiproliferative and anti-inflammatory properties of Class I HDACs in many cell types, including vascular wall and cardiac cells, we tested the effects of specific Class I HDAC inhibitors in a hypoxic model of PH. We found that two Class I HDAC inhibitors, MGCD0103 and MS-275, reduced hypoxia-mediated PH in rats in a manner that correlated with suppression of medial thickening of pulmonary arteries and inhibition of SMC proliferation in these vessels.[105
] Reduced SMC proliferation upon Class I HDAC inhibition was due, in part, to upregulation of the antiproliferative transcription factor, FoxO3a. Importantly, we also demonstrated that RV function was maintained in the face of Class I HDAC inhibition, and that indices of adverse ventricular remodeling (e.g., myocyte apoptosis and inflammation) were blunted by selective inhibition of Class I HDACs. This is in contrast to what was previously observed with the pan-HDAC inhibitor, trichostatin A (TSA) in a pulmonary artery banding model, and supports the hypothesis that isoform-selective HDAC inhibition will be safer than general HDAC inhibition in the setting of RV pressure overload. Both MGCD0103 (Mocetinostat) and MS-275 (Entinostat) are in clinical development for cancer and are well tolerated by humans, thus highlighting the translational potential of the present findings.
Two other reports have addressed effects of HDAC inhibitors in models of PH and RV remodeling. Valproic acid was shown to block RV cardiac hypertrophy in response to pulmonary artery banding (PAB) as well as in the setting of pulmonary hypertension caused by monocrotaline-induced lung injury.[106
] In contrast, TSA failed to block hypertrophy in response to PAB, and actually appeared to worsen RV function.[107
TSA is a potent, pan-HDAC inhibitor.[108
] The deleterious effects of this compound on the RV (e.g., decreased cardiac output, increased RV dilatation and apoptosis) could be a reflection of a protective role for HDAC(s) in this chamber of the heart. It is interesting to note that valproic acid, which exhibits selectivity for Class I HDACs, did not cause adverse effects in the PAB model.[107
] Our findings suggested that, with regard to the PH and RV, isoform-selective HDAC inhibition was safer than nonselective suppression of HDAC activity. This was evidenced by the ability of MGCD0103 to block RV apoptosis and inflammation and maintain RV contractile function in chronically hypoxic rats.[105
] Nonetheless, it should be noted that the model used for our studies (3 weeks of hypoxia in SD rats) was mild with regard to RV remodeling, and represented a model of PH caused by interstitial lung disease and/or hypoxemia (World Health Organization [WHO] Group III PH). It will be important to extend the current findings to more severe models of PH and RV dysfunction, such as the SUGEN plus hypoxia model, to determine whether the beneficial effects of Class I HDAC inhibitors are generalizable to other forms of PH, including WHO Group I iPAH.