We provide evidence that APN can mitigate pulmonary arterial remodeling in vivo
. Furthermore, data from in vitro
studies confirm a direct suppressive effect of APN on the proliferation of PASMCs, and suggest that the effect may be mediated in part by a downregulation of the SRF-SRE pathway. These data complement findings from our previous study, which demonstrated that APN−/−
mice in this model of pulmonary hypertension had increased arterial remodeling and elevated pulmonary artery pressures (17
). Overall, these studies add to the growing evidence linking metabolism, inflammation, and pulmonary vascular disease (3
), and suggest a potential therapeutic role for the manipulation of adipokine activity in pulmonary hypertension.
The discovery and characterization of multiple bioactive mediators derived from adipose tissues that can influence immunity and tissue repair clearly establish a link between metabolism, vascular inflammation, and remodeling (42
). Although most of the data are derived from studies of systemic vascular processes, an appreciation of the effects on pulmonary vascular disease has been increasing. Studies of human samples and animal models support a mechanistic role for insulin resistance, apolipoprotein E deficiency, and peroxisome proliferator–activated receptor-γ (PPAR-γ) activity in the pathogenesis of pulmonary hypertension (4
). Furthermore, treatment with PPAR-γ agonists, such as rosiglitazone, was shown to mitigate pulmonary hypertension and pulmonary arterial remodeling in animal models, similar to the effects on systemic vascular remodeling (45
). An increased incidence of pulmonary hypertension and pulmonary vascular remodeling in obesity is also evident (48
). In light of the obesity-associated downregulation of APN expression along with its anti-remodeling activity, these data suggest that APN may play a mechanistic role, connecting obesity and metabolism with increased pulmonary arterial remodeling. In fact, Hansman and colleagues suggested that the elevation of APN by PPAR-γ agonists could explain the beneficial effects of this therapy on pulmonary hypertension (25
). Our initial study was among the first to demonstrate a direct effect of APN on pulmonary hypertension (17
). Two subsequent studies have reported data consistent with our experiments (22
). Together, these data strongly suggest that APN has a protective role in pulmonary vascular disease (4
In our murine models of OVA-induced pulmonary hypertension (3
), allergic pulmonary inflammation was induced to stimulate pulmonary vascular remodeling and pulmonary hypertension. There is increasing evidence suggesting that perivascular inflammation may contribute to the pathogenesis of the obstructive arterial lesions seen in pulmonary hypertension (5
). This may be most relevant in pulmonary hypertension related to infections, such as schistosomiasis (the most common form of pulmonary hypertension worldwide) (52
) and autoimmunity, but may also be important in other forms of pulmonary hypertension (51
). Inflammation likely provides a direct stimulus for vascular remodeling, possibly via the release of growth factors and other mediators, or via metabolic changes such as focal hypoxia (53
Eosinophils in particular are known as potent sources of growth factors that are mitogenic for SMCs (54
), and were shown to be necessary for airway remodeling in models of chronic allergic airway inflammation (55
). Given that APN−/−
mice exhibited increased eosinophil recruitment into the lung in our model of pulmonary hypertension, some of the increased remodeling seen in these mice may result from increased inflammation. However, ΔGly-APN mice, which exhibit reduced pulmonary arterial remodeling, did not show reduced inflammation, suggesting that APN directly suppresses the growth of PASMCs, independent of its effects on inflammation. We speculate that the very strong stimulus for inflammation in this model of pulmonary hypertension overwhelmed any anti-inflammatory effects of APN.
The anti-remodeling activities of APN are well-documented in the systemic vasculature, liver, lung, and heart (57
). In vitro
, APN suppresses the proliferation and migration of vascular SMCs (61
), and in vivo
mice exhibit an increased accumulation of SMCs after vascular mechanical injury (62
). These data suggest that APN could also inhibit pulmonary arterial remodeling in pulmonary hypertension (specifically, the accumulation of SMCs in the pulmonary vasculature). Consistent with this idea, APN was shown to bind to the pulmonary vascular endothelium, and adenovirus-mediated overexpression of APN mitigated pulmonary vascular remodeling in a hypoxic model of pulmonary hypertension (22
). Thus, based on these data and the data presented here, APN is likely to suppress pulmonary vascular remodeling in pulmonary hypertension via its direct effects on PASMCs. However, the molecular mechanisms for this suppression remain unclear.
One of the most prominent features of vascular remodeling is an increased mass of SMCs. Unlike other muscle cells, SMCs remain plastic, and can alternate between a contractile state and a proliferative state in response to pathophysiologic stress. Thus, the increased numbers of muscle cells seen in pulmonary vascular remodeling are likely derived from existing SMCs or myofibrolasts that change into a highly proliferative and synthetic phenotype before forming new muscle (63
). We reasoned that one mechanism by which APN could affect the proliferation of PASMCs involves modulating the phenotype of PASMCs. Because the SRF-SRE pathway is one of the major regulators of the SMC phenotype, we hypothesized that this pathway could be a potential target of the antiproliferative activity of APN. Consistent with this, gene-expression profiling of laser-microdissected intrapulmonary arteries in mice with hypoxia-induced pulmonary hypertension demonstrated an upregulation of SRF, suggesting a role for this pathway in the pathogenesis of pulmonary hypertension (65
SRF is a phylogenetically conserved, MADs-box transcription factor that mediates the rapid transcriptional response to growth and differentiation signals in SMCs. SRF-SRE controls the expression of more than 200 genes, including immediate early genes such as c-fos
, which are involved in cellular proliferation (30
). The knockdown of SRF in primary vascular smooth muscle cells leads to cell-cycle arrest in G1 with impaired proliferation (31
), and the inducible deletion of SRF in the SMCs of adult mice leads to a thinning of smooth muscle layers in the gut and bladder, with an associated dilation of the intestinal tract and the death of mice within 2–3 weeks of SRF deletion (66
). Thus, factors that affect the SRF-SRE pathway will likely exert a profound effect on the proliferative capacity of SMCs, and could influence vascular remodeling. Although our data clearly demonstrate an effect of APN on SRF activity, the exact molecular mechanisms behind this interaction remain unknown.
In addition to its effects on SRF, APN may modulate the growth and proliferation of PASMCs in pulmonary hypertension via other mechanisms. For example, APN was shown to inhibit the growth factor–mediated activation of the mammalian target of rapamycin (mTOR) via adenosine monophosphate-activated kinase, which increases the tuberous sclerosis complex–mediated suppression of mTOR (68
). APN also directly binds growth factors such as platelet-derived growth factor isoform B homodimer, which could work to limit proliferation by sequestering it from its receptor in PASMCs (4
). Furthermore, APN inhibits Rho (Ras homologue) kinase signaling, which was shown to play a role in the development of pulmonary hypertension (70
In conclusion, our study adds to the growing body of evidence suggesting that metabolism may influence the pathogenesis of pulmonary hypertension. Specifically, we demonstrated that APN inhibits the development of pulmonary arterial remodeling and pulmonary hypertension in this model. Furthermore, we demonstrated that APN can inhibit the SRF-SRE pathway, which suggests a novel mechanism for our findings. These data have direct relevance to the pathogenesis of pulmonary hypertension, and may identify potential therapeutic targets in this important disorder.