Role of the Aryl Hydrocarbon Receptor in Su5416/Hypoxia-induced Pulmonary Hypertension: A New Mechanism for an “Old” Model
Pulmonary arterial hypertension (PAH) remains a disease with a poor prognosis, despite recent advances in treatment. A better understanding of the mechanisms underlying the pathogenesis of PAH in animal models could facilitate the development of new therapeutic strategies. Previous studies showed that a single
injection of the compound SU5416, a potent vascular endothelial growth factor receptor (VEGFR)-1 and -2 inhibitor, caused pulmonary artery endothelial cell (PAEC) apoptosis, emphysema, and a mild increase in pulmonary arterial pressure in rats (1, 2). Surprisingly, exposure of the injected animals to chronic hypoxia triggered severe pulmonary hypertension (PH). Unlike in other models of PH (such as application of monocrotaline or exposure to chronic hypoxia), proliferative vascular remodeling in SU5416/hypoxia-induced PH was accompanied by the formation of obstructive intimal lesions in the peripheral pulmonary arteries that resembled plexiform lesions in humans (2). For this reason, the SU5416/hypoxia rat model has become a preferred animal model of PH. Paradoxically, the induction of PAEC apoptosis by SU5416 appears to be a key event in the development of PH in this model, as this process is prevented by the administration of pan-
caspase inhibitor or knockout of programmed cell death 4 (2, 3). According to one concept, SU5416 may induce so-called “escape angiogenesis,” a compensatory (rebound) response that occurs after antiangiogenic drug therapy (4, 5). Alternatively, it has been suggested that massive apoptosis of PAECs resulting from the loss of survival signaling leads to the emergence of apoptosis- resistant cells with increased proliferative potential (6).
However, the precise mechanism underlying the synergistic action of SU5416 and hypoxia in inducing angio-obliterative PH is still elusive.
In this issue of the Journal, Dean and colleagues (pp. 320–330) demonstrate that the proproliferative effect of SU5416 in the pulmonary circulation may result, at least partly, from activation of the aryl hydrocarbon receptor (AhR) and thus possibly from a VEGFR-independent pathway (Figure 1) (7). The study is based on the group’s previous finding of an upregulation of AhR in the SU5416/hypoxia rat model (8). AhR is a transcription factor that can be activated in vivo by a variety of endogenous and exogenous aromatic hydrocarbons. In the present study, the AhR antagonist CH223191 reversed the rise in right ventricular systolic pressure
VEGFR
Testosterone
17-Estradiol
16-OHE1
CYP1A1 CYP1B1
Apoptosis
2-OHE2
Pulmonary hypertension with
proliferating endothelial cells
Figure 1. Putative mechanisms of pulmonary vascular remodeling in the SU5416/hypoxia rat model of pulmonary hypertension. Lines ending with arrows indicate a stimulatory effect, whereas lines ending with a crossbar indicate an inhibitory effect. Solid lines indicate pathways reported for the SU5416/hypoxia rat model. Dashed lines indicate hypothetical pathways. See text for details. 2-OHE2 = 2-hydroxyestradiol; 16a-OHE1 =
16a-hydroxyestrone; AhR = aryl hydrocarbon receptor; ARNT = aryl hydrocarbon receptor nuclear translocator; HIF = hypoxia-inducible factor; VEGFR = vascular endothelial growth factor receptor.
Supported by the German Research Foundation, Excellence Cluster Cardiopulmonary System and CRC1213, project B06.
and pulmonary vascular remodeling in SU5416/hypoxia-treated female rats. SU5416 increased the proliferation of human blood outgrowth endothelial cells and pulmonary artery smooth muscle cells (PASMCs) in an AhR-dependent manner. The authors found that SU5416-induced AhR activation in the rat lung upregulated the expression of aromatase and CYP1A1, enzymes that are responsible for estrogen synthesis and hydroxylation. Interestingly, this group previously showed that inhibition of aromatase had a therapeutic effect in female mice and rats with experimental PH, whereas the estrogen metabolite 16a-hydroxyestrone enhanced the proliferation of human PASMCs and induced PH in mice (9, 10). In addition, urinary 16a-hydroxyestrone levels were found to be increased in female mice with hypoxia-induced PH and female patients with hereditary PAH carrying a BMPR2 mutation (9, 11). Collectively, these findings suggest that SU5416 activates AhR, which results in the increased formation of estrogen
metabolites, promoting pulmonary vascular remodeling in females. Thus, the study by Dean and colleagues (7) offers a novel explanation for the development of PH in the SU5416/hypoxia rat model (Figure 1).
Because the extent to which the suggested pathway is involved in the pathogenesis of PAH in humans is not completely clear, it is important to address this newly proposed mechanism in further studies. Interestingly, the expression of AhR was found to be increased in PASMCs from female patients with PAH (8). Furthermore, despite the fact that aromatase expression is not altered in these cells (10), the aromatase inhibitor anastrozole significantly increased the 6-minute walk distance in both female and male patients with PAH compared with placebo in a recent small-scale clinical trial (12). Still, reports regarding the role
hypoxia, which acts as a “second hit” in the SU5416/hypoxia model (Figure 1).
Although SU5416 promoted the expression of ARNT/HIF-1b, it also caused a significant downregulation of HIF-1a. Because a synergism between ARNT and HIF-1a in the SU5416/hypoxia rat model is thus rather unlikely, further investigations should take into account HIF-2a regulation. Recently, two independent groups demonstrated that HIF-2a stabilization in the endothelium resulted in severe PH in mice (15, 16). Against this background, it is tempting to speculate that a second hit of chronic hypoxia along with increased expression of ARNT/HIF-1b may be associated with the upregulation of HIF-2a.
The description by Dean and colleagues (7) of a novel AhR-dependent mechanism underlying PH development in the SU5416/hypoxia rat model adds an exciting new aspect to PH
pathogenesis in this model. It would be interesting to see whether the proposed mechanism is independent of the effect of SU5416 as a VEGFR antagonist. At least some components of this mechanism also appear to be present in human PAH. Thus, in future studies, it will be important to decipher the relevance of the suggested pathway in human PAH in greater detail to evaluate its potential as a treatment target for this disease. ■
Author disclosures are available with the text of this article at www.atsjournals.org.
Ievgen Strielkov, Ph.D. Norbert Weissmann, Ph.D.
Excellence Cluster Cardiopulmonary System Justus Liebig University Giessen
Giessen, Germany
of estrogen metabolism in PH are rather contradictory. For
instance, anastrozole reverses SU5416/hypoxia-induced PH in
female, but not male, rats, whereas knockout of estrogen- hydroxylating CYP1B1 in this model prevents PH only in male mice (9, 10). In this context, it is noteworthy that the main part of the present study was conducted in female animals. Considering that treatment with SU5416/hypoxia induces PH of a similar magnitude in both genders, it would be of interest for future studies to evaluate the contribution of AhR activation in males.
Surprisingly, although SU5416 plus hypoxia was shown to increase CYP1A1 expression in rat lungs, hypoxia alone exerted the opposite effect in mouse lungs and isolated human PAECs (7, 13).
At least in human PAECs, this inhibitory response appears to be dependent on hypoxia-inducible factor (HIF)-2a (13).
Unfortunately, to the best of our knowledge, no data regarding the expression of CYP1A1 in patients with PAH are currently available. Because CYP1A1-derived estrogen metabolites possess both proproliferative (16a-hydroxyestrone) and antiproliferative
(2-hydroxyestradiol) properties (14), the role of CYP1A1 in the pathogenesis of PAH merits further investigation.
The authors propose a cross-talk between the AhR pathway and HIF signaling, which could explain the exaggerated pulmonary vascular remodeling observed after exposure to chronic hypoxia in SU5416-treated rats. They found that SU5416 enhanced the expression of aryl hydrocarbon receptor nuclear translocator (ARNT), which is required for AhR DNA binding. Because ARNT (also known as HIF-1b) is involved in hypoxic signaling, its upregulation may potentiate the proproliferative effect of chronic
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EDITORIALS
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