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Remodeling of the pulmonary arteries is a common feature among the heterogeneous disorders that cause pulmonary hypertension. In these disorders, the remodeled pulmonary arteries often demonstrate inflammation and an accumulation of pulmonary artery smooth muscle cells (PASMCs) within the vessels. Adipose tissue secretes multiple bioactive mediators (adipokines) that can influence both inflammation and remodeling, suggesting that adipokines may contribute to the development of pulmonary hypertension. We recently reported on a model of pulmonary hypertension induced by vascular inflammation, in which a deficiency of the adipokine adiponectin (APN) was associated with the extensive proliferation of PASMCs and increased pulmonary artery pressures. Based on these data, we hypothesize that APN can suppress pulmonary hypertension by directly inhibiting the proliferation of PASMCs. Here, we tested the effects of APN overexpression on pulmonary arterial remodeling by using APN-overexpressing mice in a model of pulmonary hypertension induced by inflammation. Consistent with our hypothesis, mice that overexpressed APN manfiested reduced pulmonary hypertension and remodeling compared with wild-type mice, despite developing similar levels of pulmonary vascular inflammation in the model. The overexpression of APN was also protective in a hypoxic model of pulmonary hypertension. Furthermore, APN suppressed the proliferation of PASMCs, and reduced the activity of the serum response factor–serum response element pathway, which is a critical signaling pathway for smooth muscle cell proliferation. Overall, these data suggest that APN can regulate pulmonary hypertension and pulmonary arterial remodeling through its direct effects on PASMCs. Hence, the activation of APN-like activity in the pulmonary vasculature may be beneficial in pulmonary hypertension.
Our research provides mechanistic insights into the beneficial effects of adiponectin on pulmonary arterial remodeling in pulmonary hypertension. These data establish that adiponectin can directly suppress remodeling via effects on pulmonary artery smooth muscle cells. In addition, the data provide insights into how metabolism and obesity may affect pulmonary vascular disease.
Pulmonary hypertension is an important pulmonary disorder that leads to significant morbidity and mortality and can occur in association with multiple diseases, many of which share a common pathologic appearance characterized by pulmonary arterial inflammation associated with an abnormal accumulation of pulmonary artery smooth muscle cells (PASMCs) in the pulmonary vasculature (1, 2). Accumulating evidence suggests that pulmonary vascular inflammation is an important stimulus for the pathologic changes seen in various types of pulmonary hypertension in both human and animal models (1–5). A role for inflammation in the pathogenesis of pulmonary hypertension was suggested by studies demonstrating the presence of increased concentrations of cytokines in patients with pulmonary hypertension (6, 7) and the presence of leukocytes in and around the remodeled vasculature of the lung (8–10). Furthermore, in animal models, pulmonary vascular inflammation induces arterial remodeling and pulmonary hypertension (3, 11–14). It has been suggested that inflammatory cells release mediators that stimulate remodeling of the vessel wall, in part by directly promoting the proliferation of PASMCs (3, 5, 15–17).
Recent experimental evidence suggests that adipose tissue may contribute to the pathogenesis of inflammatory vascular diseases such as atherosclerosis through the secretion of multiple bioactive mediators (adipokines) that influence energy homeostasis, inflammation, and tissue remodeling (18–20). One of the most important adipokines is adiponectin (APN), which has a wide range of metabolic, anti-inflammatory, and antiproliferative activities (21). Interestingly, persons with obesity have lower amounts of circulating APN compared with lean individuals, suggesting that decreased concentrations of APN may contribute to the increased incidence of vascular diseases associated with obesity. Links between APN and pulmonary vascular disease are not fully defined. However, recent data from murine models of pulmonary hypertension suggest that APN deficiency can increase the severity of pulmonary vascular inflammation, pulmonary arterial remodeling, and pulmonary hypertension (4, 17, 22, 23). In our previous study, APN-deficient (APN−/−) mice developed increased eosinophil recruitment into the lungs and increased pulmonary vascular remodeling after the induction of allergic vascular inflammation (17). This increased remodeling was largely secondary to the proliferation of PASMCs within the pulmonary arteries. Although APN deficiency may have exacerbated the pulmonary vascular disease in this model indirectly via its effects on vascular inflammation, other data suggest that APN may also directly inhibit pulmonary arterial remodeling, independent of its effects on inflammation (4, 24, 25). Based on these data, we hypothesize that APN could suppress pulmonary arterial remodeling via direct suppressive effects on PASMC proliferation (25). In the data presented here, we demonstrate that APN can suppress pulmonary arterial remodeling and pulmonary hypertension independently of its effects on pulmonary inflammation. Additional in vitro data show that APN can directly suppress the proliferation of PASMCs, and that APN decreases the activity of the serum response factor–serum response element (SRF-SRE) pathway, a critical signaling pathway for smooth muscle cell (SMC) proliferation (26–31). These data support a potential role for APN in the pathogenesis of pulmonary hypertension by modulating pulmonary arterial remodeling. Some of the results of this study were previously reported as an abstract (32).
APN−/− mice and ΔGly-APN mice were backcrossed more than seven generations onto a C57BL/6 background (17, 33). Wild-type C57BL/6 control mice were obtained from the National Cancer Institute (Bethesda, MD). Male mice were used at age 6–8 weeks. All protocols were approved by the Institutional Animal Care and Use Committee of Massachusetts General Hospital.
The high-dose ovalbumin (OVA) model of pulmonary hypertension was performed as described (3). PBS was used to challenge control mice. Mice were analyzed 24 hours after the last challenge. The hypoxic model of pulmonary hypertension was performed as previously described (17, 34). We also developed an additional high-dose OVA model that induced pulmonary hypertension at normoxia in wild-type mice. Additional information is available in the online supplement.
Histopathologic and quantitative measurements of pulmonary artery wall thickness were performed as previously described (17). Additional information is available in the online supplement.
Right-ventricular systolic pressure (RVSP) was measured as previously described (17). Additional information is available in the online supplement.
RNA was purified from the lung and analyzed by quantitative RT-PCR (QPCR), as previously described (35). Additional information is available in the online supplement. Lung extracts and serum were collected, diluted 1:1,000 and 1:10,000, respectively, and used in a commercial ELISA kit to measure protein concentrations of mouse adiponectin (B-Bridge International, Mountain View, CA).
Information regarding the APN binding assay is available in the online supplement.
Lungs were homogenized in PBS containing protease inhibitors (Roche, Indianapolis, IN). Crude lysates were centrifuged at 900 × g to remove debris, and the supernatant was filtered and collected. Additional information is available in the online supplement.
PASMCs were isolated from the main pulmonary arteries of male C57BL/6 mice, as previously described (36). Cells were used in experiments after passages 3–6. For proliferation assays, PASMCs were seeded in 96-well plates at 5,000 cells per well. PASMCs were starved in 0.1% BSA medium overnight. Purified APN (ALEXIS-Enzo Life Sciences, Farmingdale, NY) or 18 μg of lung protein extracts were added, and cells were incubated for 72 hours. Proliferation was assayed using the CyQUANT NF Cell Proliferation Assay Kit (Invitrogen, Carlsbad, CA).
For the determination of SRE activity, PASMCs were transfected with SRE–luciferase and Renilla–luciferase (10:1), using Lipofectamine 2000 (Invitrogen) (38). The next day, cells were serum-starved for 2 hours and then stimulated with serum, serum plus APN, or lung protein extracts for 6 hours. SRE activity was assessed using a dual-luciferase reporter assay system (Promega, Madison, WI), as previously described (37).
Results are shown as mean ± SEM. Groups were compared using a Student's t test. Between-group comparisons of means were performed by 2-way ANOVA. P < 0.05 was regarded as significant.
We hypothesized that APN can suppress pulmonary arterial remodeling via its direct effects on the proliferation of PASMCs. Thus we sought to determine if elevations in the level of APN would exert effects on pulmonary vascular remodeling, independent of its effects on inflammation. For these experiments, we used a transgenic strain of mice reported to have 2- to 3-fold higher APN concentrations in serum (ΔGly-APN mice) compared with wild-type mice (33). These mice were produced by the transgenic insertion of a mutant form of APN. Unexpectedly, these mice exhibited increased expression and secretion of native APN, without secretion of the mutant form. These mice were protected from high fat diet–induced insulin resistance and had a modified profile of fat deposition, but were otherwise normal and of equal size and weight to age-matched and sex-matched wild-type mice (the ΔGly-APN mice were backcrossed nine generations onto a C57BL/6 background). We confirmed that the ΔGly-APN mice had increased concentrations of APN in plasma compared with wild-type mice, as demonstrated previously (data not shown) (33). Next we used male ΔGly-APN and wild-type control mice in the high-dose OVA model of pulmonary hypertension (3). Only male mice were used for these experiments, because of their more dramatic phenotype in the OVA model of lung inflammation. As seen in previous studies, PBS challenges of wild-type mice did not result in detectable inflammation or pulmonary arterial remodeling (3, 17). Moreover, no inflammation or pulmonary arterial remodeling was detectable in the PBS-challenged ΔGly-APN mice (data not shown). OVA-challenged wild-type mice developed prominent eosinophilic vascular inflammation, associated with pulmonary arterial remodeling (Figures 1Ai and 1Aiii). However, OVA-challenged ΔGly-APN mice exhibited less pulmonary arterial remodeling than wild-type mice, despite the development of eosinophilic vascular inflammation (Figures 1Aii and 1Aiv). Staining for α-SMC actin confirmed that cells within the vasculature were SMCs (Figure 1B), and image analysis indicated a significant reduction in pulmonary arterial wall thickness in ΔGly-APN mice compared with vessels in wild-type mice (Figure 1C). Although this high-dose OVA model produces extensive pulmonary artery remodeling, it does not produce elevated pulmonary artery pressures in wild-type mice unless the mice are rendered hypoxic during measurements (3). Consequently, we developed a modified model with more frequent dosing of OVA for a longer period. Wild-type mice in this model developed similar extensive pulmonary arterial remodeling, but also demonstrated elevations in pulmonary artery pressure, as assessed by RVSP, while breathing room air (Figure 2A). Consistent with our remodeling data, ΔGly-APN mice had reduced right ventricular pressures compared with wild-type mice, with concentrations similar to those in PBS-challenged mice (Figure 2A). Other hemodynamic measurements, including systemic blood pressure, heart rate, and right ventricular diastolic pressure, were not different between the two genotypes after either PBS or OVA challenges (data not shown).
To provide further evidence for the effects of APN on the pulmonary vasculature and to investigate whether the effects of adiponectin were dependent on allergic inflammation, we used ΔGly-APN mice in the hypoxic model of pulmonary hypertension. After 3 weeks of continuous exposure to 10% oxygen, ΔGly-APN mice manifested lower RVSP than wild-type mice (Figure 2B). Control mice maintained in normoxia did not have elevated RVSPs, and the systemic hemodynamics were not different between the two genotypes with either normoxia or hypoxia (data not shown). These data demonstrate that APN can modulate pulmonary hypertension in two different models of disease.
We also quantified the inflammatory response in ΔGly-APN and wild-type mice in the high-dose OVA model of pulmonary hypertension. As already stated, we did not observe significant inflammation in the PBS-challenged wild-type and ΔGly-APN mice, as expected (data not shown). Surprisingly, the numbers of inflammatory cells around the pulmonary vessels (Figure 1A) and in BAL fluid were not different between OVA-challenged ΔGly-APN mice and wild-type mice (Figures 3A and 3B). Lymphocyte recruitment and activation were also unaffected by the overexpression of APN (Figures 3C and 3D). In addition, we saw no effects of increased APN concentrations on the lung RNA levels of a panel of chemokines that had been upregulated in APN−/− mice in a model of pulmonary hypertension (17), or of a panel of growth factors that could regulate the proliferation of PASMCs in response to inflammation in the lungs (Figures 3E and 3F). As shown previously (17), PBS-challenged mice did not develop increased concentrations of chemokines or growth factors (data not shown). Thus, despite the prominent effects of APN on pulmonary arterial remodeling, we saw no inhibition of inflammation because of increased concentrations of APN. These data suggest that APN exerts direct effects on the remodeling response, independent of its effects on inflammation.
Our in vivo data suggest a direct suppressive effect of APN on pulmonary arterial remodeling. Therefore, we reasoned that APN can directly influence the proliferation of PASMCs. To address this question, we isolated and cultured PASMCs from wild-type mice (36), and used QPCR to measure the expression of the known APN receptors AdipoR1, AdipoR2, T-cadherin, and calreticulin. Both AdipoR1 and AdipoR2 were detected in RNA isolated from cultured PASMCs, but not the other receptors (data not shown). To demonstrate that APN binds to PASMCs, we incubated PASMCs with purified APN on ice for 30 minutes, washed the cells with cold PBS, and isolated the cellular proteins. Western blotting of the protein extracts with an antibody to APN demonstrated the presence of APN, consistent with the binding of APN to PASMCs (Figure 4A). We then stimulated cultured PASMCs with serum and increasing concentrations of APN, and measured proliferation after 72 hours. As shown by others (25), APN suppressed the proliferation of PASMCs in a dose-dependent manner (Figure 4B).
To provide a more relevant test of the situation in vivo, we also used protein isolated from the lungs of wild-type, ΔGly-APN, and APN−/− mice after high-dose OVA immunization and challenge in a proliferation assay. APN protein was detected in the lung extracts of wild-type and ΔGly-APN mice, but not in those of APN−/− mice, and lung APN concentrations were higher in ΔGly-APN mice than in wild-type mice, as measured by Western blotting and ELISA (Figures 4C and 4D). We incubated these lung extracts with cultured wild-type PASMCs for 72 hours. Compared with protein extracted from wild-type lungs, protein from the lungs of APN−/− mice led to an increased proliferation of PASMCs, whereas protein from the lungs of ΔGly-APN mice stimulated less proliferation of PASMCs (Figure 4D). These data suggest that the APN−/− mice in our model had a lung protein milieu that enhanced the proliferation of PASMCs relative to wild-type mice, whereas the overexpression of APN produced a lung protein profile that inhibited the proliferation of PASMCs. In addition, these data are consistent with our hypothesis that APN can directly regulate pulmonary arterial remodeling by suppressing the proliferation of PASMCs.
In response to various stimuli, SMCs can change their phenotype from contractile to highly proliferative and synthetic (38). This process is critical in the response of SMCs to physiologic stress, and is mediated in part via the SRF-SRE pathway. This pathway was demonstrated to play a central role in regulating many SMC-specific genes, and is essential for the development and proliferation of SMCs (28, 29, 31, 39–41). We hypothesized that APN may modulate PASMC proliferation in part via changes in SRF-SRE activity. To explore this possibility, we transfected PASMCs with an SRE–luciferase construct (37), and treated the cells with 20% serum and increasing concentrations of APN. SRE activity was then measured with a dual-luciferase reporter assay. As expected, SRE activity was increased in PASMCs with 20% serum treatment, but APN suppressed the SRE response to serum in a dose-dependent manner (Figure 5A). We also tested the effects of lung protein extracts taken from wild-type and ΔGly-APN mice after high-dose OVA immunization and challenge. Consistent with the proliferation data, protein from the lungs of ΔGly-APN mice induced less SRE activity than protein from the lungs of wild-type mice (Figure 5B). These data suggest that the antiproliferative effect of APN on PASMCs could be mediated in part via the suppression of SRF-SRE activity in these cells.
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 (3–5, 16, 24, 25), 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, 24, 25, 43, 44). 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 (45–47). An increased incidence of pulmonary hypertension and pulmonary vascular remodeling in obesity is also evident (48–50). 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, 23). 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, 17), 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, 51). 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, 56). 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 (57–60). In vitro, APN suppresses the proliferation and migration of vascular SMCs (61), and in vivo, APN−/− 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, 23). 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, 64). 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 and Egr1, 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, 67). 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, 69). Furthermore, APN inhibits Rho (Ras homologue) kinase signaling, which was shown to play a role in the development of pulmonary hypertension (70, 71).
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.
The authors thank Barry Sandall and Carol Leary for their technical support.
This work was supported by National Institutes of Health grants HL088297 (B.D.M.), T32 HL07874 (B.D.M. and M.W.), DK55758 (P.E.S.), and HL074352 (K.D.B.).
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1165/rcmb.2010-0316OC on November 12, 2010
Author Disclosure: K.D.B. received sponsored grants from Ikaria LLC and the National Institutes of Health for more than $100,001 each. B.M. received sponsored grants from the Roche Organ Transplant Research Foundation for $50,001–$100,000 and the National Institutes of Health for more than $100,001. P.E.S. received sponsored grants from the National Institutes of Health and the Juvenile Diabetes Research Foundation for more than $100,001 each. None of the other authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.