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(Circulation Research. 2009;104:236.)
© 2009 American Heart Association, Inc.

Integrative Physiology

Interleukin-6 Overexpression Induces Pulmonary Hypertension

M. Kathryn Steiner, Olga L. Syrkina, Narasaish Kolliputi, Eugene J. Mark, Charles A. Hales, Aaron B. Waxman

From the Pulmonary Critical Care Unit (M.K.S., O.L.S., N.K., C.A.H., A.B.W.), Department of Medicine; and Department of Pathology (E.J.M.), Massachusetts General Hospital, Harvard Medical School, Boston.

Correspondence to M. Kathryn Steiner, Division of Pulmonary Critical Care Medicine, University of Massachusetts Memorial Medical Center, 55 Lake Ave North, Worcester, MA 01655. E-mail marciakathryn.steiner{at}umassmemorial.org

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inflammatory cytokine interleukin (IL)-6 is elevated in the serum and lungs of patients with pulmonary artery hypertension (PAH). Several animal models of PAH cite the potential role of inflammatory mediators. We investigated role of IL-6 in the pathogenesis of pulmonary vascular disease. Indices of pulmonary vascular remodeling were measured in lung-specific IL-6–overexpressing transgenic mice (Tg⁺) and compared to wild-type (Tg^–) controls in both normoxic and chronic hypoxic conditions. The Tg⁺ mice exhibited elevated right ventricular systolic pressures and right ventricular hypertrophy with corresponding pulmonary vasculopathic changes, all of which were exacerbated by chronic hypoxia. IL-6 overexpression increased muscularization of the proximal arterial tree, and hypoxia enhanced this effect. It also reproduced the muscularization and proliferative arteriopathy seen in the distal arteriolar vessels of PAH patients. The latter was characterized by the formation of occlusive neointimal angioproliferative lesions that worsened with hypoxia and were composed of endothelial cells and T-lymphocytes. IL-6–induced arteriopathic changes were accompanied by activation of proangiogenic factor, vascular endothelial growth factor, the proproliferative kinase extracellular signal-regulated kinase, proproliferative transcription factors c-MYC and MAX, and the antiapoptotic proteins survivin and Bcl-2 and downregulation of the growth inhibitor transforming growth factor-β and proapoptotic kinases JNK and p38. These findings suggest that IL-6 promotes the development and progression of pulmonary vascular remodeling and PAH through proproliferative antiapoptotic mechanisms.

Key Words: interleukin-6 • pulmonary artery hypertension • proliferation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Pulmonary vascular remodeling is associated with increased pulmonary vascular resistance, pulmonary artery hypertension (PAH), and right heart failure. Advanced PAH is characterized by arteriopathy, which includes muscularization of distal pulmonary arterioles, concentric intimal thickening, and obstruction of the vascular lumen by proliferating endothelial cells to form plexiform lesions.¹ Evidence suggests that PAH is associated with genetic perturbations favoring cellular growth, proliferation, and angiogenesis² and inhibitors of apoptosis, previously thought to be only expressed in cancer cells, promoting a proliferative cellular phenotype, resulting in pulmonary vascular remodeling in PAH.^3,4 The histopathologic features and known genetic susceptibilities of this condition have led to the hypothesis that PAH arises from hyperproliferation of pulmonary artery smooth muscle cells (PASMCs) and endothelial cells (PAECs).

In addition to the formation of proliferative neointimal lesions and muscularization of the pulmonary vascular bed, perivascular inflammatory cell infiltrates are also present in advanced human cases of PAH. These infiltrates consist of T cells, B cells, and macrophages, suggesting that cytokines and growth factors associated with these inflammatory cells may be promoting PAEC and PASMC hyperproliferation.⁵ The proinflammatory cytokine interleukin (IL)-6 is consistently increased in the serum and lungs^6–8 of patients with idiopathic PAH and in inflammatory diseases^6,9–11 that are associated with PAH. In addition, Kaposi sarcoma–associated herpes virus, which may cause PAH in human immunodeficiency virus–negative Castleman’s disease, encodes a constitutively active form of IL-6,¹² resulting in unregulated cell growth and escape from host antitumor defenses. Furthermore, unchecked production of IL-6 in tissues leading to chronic inflammation has exhibited a strong association with many cancers.¹³ Given mounting evidence for the role of inflammation and cancer-like mechanisms in the pathogenesis of PAH, we investigated whether IL-6 promotes the development of pulmonary arteriopathy and consequent PAH. We show that lung-specific overexpression of IL-6 in mice replicates the pathological lesions observed in advanced PAH, including both distal arteriolar muscularization and plexogenic arteriopathy, and leads to increased pulmonary vascular resistance (PVR) and PAH. At the cellular and molecular level, these vasculopathic changes are associated with the activation of vascular endothelial growth factor (VEGF) and the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK), with subsequent increases in protooncogene c-MYC/MAX transcription factor complex and the antiapoptotic proteins survivin and Bcl-2, with downregulation of the growth inhibitor transforming growth factor (TGF)-β and proapoptotic kinases JNK and p38. This suggests that IL-6 participates in the development of distal pulmonary proliferative arteriopathy and consequent elevation in PVR and development of PAH.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
For details, see the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org.

Indices of pulmonary vascular remodeling were measured in lung-specific IL-6–overexpressing transgenic mice (Tg⁺) and compared to wild-type (Tg^–) controls in both normoxic and chronic hypoxic conditions.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
IL-6 Overexpression Increases Pulmonary Artery Pressure and Ventricular Hypertrophy
To test the hypothesis that increased IL-6 may cause increased PVR and PAH, we measured the right ventricular systolic pressure (RVSP) in IL-6 Tg⁺ and Tg^– mice. Under normoxic conditions, Tg⁺ mice had elevated RVSP compared to Tg^– mice (Figure 1a). In Tg⁺ mice, 3 weeks of exposure to 10% oxygen almost doubled RVSP compared to baseline, and this value was almost 2.6 times higher than the RVSP in hypoxic Tg^– mice (Figure 1a).

View larger version (27K): [in this window] [in a new window]   Figure 1. IL-6 Tg+ mice have PAH at baseline that worsens with hypoxia. a, RVSP is higher in Tg+ mice (P<0.05 vs normoxic Tg–, P<0.05 vs hypoxic Tg–). b, RV/LV+S are higher in IL-6 Tg+ mice (P<0.05 vs normoxic Tg–, P<0.05 vs hypoxic Tg–). c, RV weight is higher in IL-6 Tg+ mice (P<0.05 vs normoxic Tg–, P<0.05 vs hypoxic Tg–) and increases further in hypoxia (P<0.05 vs normoxic Tg+). d, Representative photomicrographs of IL-6 Tg+ and Tg– mouse hearts in normoxic and hypoxic conditions. IL-6 Tg+ right ventricles are hypertrophied at baseline, and they hypertrophy further with hypoxia (hematoxylin/eosin staining; magnification, x25; scale bar=0.01 mm).

Ventricular wall thickness increased in response to chronic pressure overload, which is a consequence of elevated resistance in the pulmonary artery. Right ventricular hypertrophy (RVH), as measured by right ventricle weight/(left ventricle weight+septum weight [RV/LV+S]) and absolute RV weight, were greater in Tg⁺ mice than in Tg^– mice under normoxic conditions (Figure 1b and 1c). Hypoxia produced even greater RVH in Tg⁺ mice, whereas there was no change in ventricular wall thickness in Tg^– mice (Figure 1b and 1c). The histological appearance of the hearts was consistent with RVH measurements, showing that right ventricular wall mass was greater in Tg⁺ mice in both normoxic and hypoxic conditions (Figure 1d). See the online data supplement for additional data.

IL-6 Overexpression Induced Muscularization Throughout the Entire Pulmonary Vascular Bed
To determine the cause of increased PVR, we examined specific regions of the pulmonary vascular tree for remodeling. Examination of the proximal branches of the main PA revealed that the elastic lamina was increased in normoxic Tg⁺ mice compared to their Tg^– counterparts (Figure 2c versus 2a) and was quantitatively confirmed by counting the number of elastic lamina (Figure 2q). Following hypoxia, the number of elastic lamina in Tg⁺ mice more than tripled compared to baseline and exceeded the number in hypoxic Tg^– littermates by a factor of 5. Main PA branches in Tg⁺ mice exhibited an increase in not only the number of elastic lamina but also the percentage vessel medial wall thickness, relative to the Tg^– control, under both normoxic and hypoxic conditions (Figure 2r). The medial wall of the main PA branches in Tg⁺ mice more than doubled in thickness in response to hypoxia compared to their Tg⁺ normoxic controls, whereas PA medial wall thickness did not change in hypoxic Tg^– mice compared to normoxic controls.

View larger version (37K): [in this window] [in a new window]   Figure 2. Pulmonary artery (PA) tree of IL-6 Tg+ mice has increased muscularization that worsens with hypoxia. a through l, Representative photomicrographs of the elastic lamina of the PA vasculature of IL-6 Tg+ and Tg– mice in normoxic and hypoxic conditions. Main PA branches (Tg– [a and b] vs Tg+ [c and d]), PA at the level of the TBs (Tg– [e and f] vs Tg+ [g and h]), PA distal to TB (acinar) (Tg– [i and j] vs Tg+ [k and l]). Elastic tissue stain; magnification, x400; scale bar=0.001 mm. m through p, Representative photomicrographs of smooth muscle hypertrophy of distal acinar arterioles of the PA vasculature of IL-6 Tg+ in normoxic and hypoxic conditions (Tg– [m and n] vs Tg+ [o and p]). Immunohistochemistry with -smooth muscle actin; magnification, x400; scale bar=0.001 mm. q through t, Thickness of the medial wall is increased at all levels of the PA tree of IL-6 Tg+ mice compared to Tg– mice at baseline and worsens with hypoxia. q, Number of elastic lamina of main PA branches (P<0.05 vs normoxic Tg–, P<0.05 vs normoxic Tg+, P<0.05 vs hypoxic Tg–). r, Percentage wall thickness of the main PA branches (*P<0.05 vs hypoxic Tg–, *P<0.05 vs normoxic Tg+, P<0.05 vs normoxic Tg–). s, Percentage wall thickness of the TB PA vessels (*P<0.05 vs hypoxic Tg–, *P<0.05 vs normoxic Tg+, P<0.05 vs normoxic Tg–, P<0.05 vs normoxic Tg–). t, Percentage wall thickness of the acinar pulmonary arteriolar vessels (P<0.05 vs normoxic Tg–, P<0.05 vs normoxic Tg–, *P<0.05 vs hypoxic Tg–, *P<0.05 vs normoxic Tg+).

The terminal bronchioles (TBs) and distal acinar arterioles were examined for evidence of muscularization. The most notable findings were that the distal acinar arterioles of Tg⁺ mice were muscularized at baseline and became more thickly muscularized in hypoxia unlike the Tg^– mice arterioles, as shown by elastic staining (Figure 2k and 2l [Tg⁺] versus 2i and 2j [Tg^–]) and by immunohistochemistry with -smooth muscle actin (Figure 2o and 2p [Tg⁺] versus 2m and 2n [Tg^–]). See the online data supplement for detailed results, together with the quantitative results of the medial wall thickness of both the TBs and acinar vessels.

IL-6 Overexpression Induced Arteriolar Neointimal Occlusive Lesions
We examined the vascular bed for occlusive neointimal lesions that, like arteriole muscularization, may contribute to increased PVR. We found that the arterioles in Tg⁺ mice had thick intimal walls, with many of the arteriolar lumens being partially (27±5%) or completely occluded (4±2%). Hypoxia increased the number of partially (55±3%) or completely occluded arterioles (14±1%, Figure 3b, 3d, and 3e). In contrast, all arterioles in the lungs of Tg^– normoxic mice were patent, and the intima was not thickened. Only after exposure to hypoxia did the arteriolar lumens become partially occluded, with the number of these vessels reaching 3±2% (Figure 3a, 3c, and 3e).

View larger version (61K): [in this window] [in a new window]   Figure 3. Arterioles of IL-6 Tg+ mice have neointimal occlusive lesions. a through d, Representative photomicrographs of the lung parenchyma of IL-6 Tg+ and Tg– mice showing neointimal hyperplasia of acinar arterioles in IL-6 Tg+ mice in normoxic conditions (b) and occlusive arteriopathy in hypoxic conditions (d). No neointimal hyperplastic or occlusive lesions are seen in Tg– mice (a and c) (hematoxylin/eosin staining; magnification, x400; scale bar=0.001 mm). Arrows indicate arterioles. e, Pie chart demonstrates that IL-6 Tg+ mice have a higher percentage (%) of partially (P.) occluded and closed luminal acinar arterioles at baseline that worsens with hypoxia compared with Tg– mice. Hematoxylin/eosin stain included in key to demonstrate representative examples of an open (blue), partially occluded (red), and closed acinar arteriole (yellow); magnification, x40; scale bar=0.001 mm.

Hypoxia Induced Loss of Pulmonary Arterioles in IL-6–Overexpressing Mice
See the online data supplement and supplemental Figure I for results.

IL-6–Induced Distal Pulmonary Vascular Wall Endothelial Cellular Growth and Proliferation
To determine whether PAEC types contribute to intimal wall thickening in arterioles of Tg⁺ mice, we performed immunohistochemical analysis of factor VIII, an endothelial cell marker. We found that the arteriole walls were thickened, in part, by multiple layers of PAECs. Under both normoxic and hypoxic conditions, the PAEC layers either formed smooth and thick concentric occlusive lesions or plexogenic-like occlusive lesions caused by a piling up of PAECs in a nonuniform fashion (Figure 4c and 4d), whereas arteriolar walls in Tg^– mice had a normal appearance under both normoxic and hypoxic conditions (Figure 4a and 4b). At baseline and in hypoxic conditions, PAECs lining the arterioles of Tg^– mice exhibited minimal expression of VEGF receptor (VEGFR)2 (Figure 4e and 4f), whereas PAECs had elevated VEGFR2 expression in Tg⁺ mice (Figure 4g and 4h), suggesting that the growth potential of these cells was increased and confirming that the plexogenic-like lesions in Tg⁺ mice consisted predominately of PAECs. In Tg⁺ mice, increases in PAECs and VEGFR2 expression were associated with increased proliferation within the intimal wall of arterioles at baseline and in hypoxic conditions, as assessed by staining for the proliferation marker proliferating-cell nuclear antigen (PCNA) (Figure 4k and 4l). No change in PCNA levels were detected in the Tg^– lungs (Figure 4i and 4j).

View larger version (60K): [in this window] [in a new window]   Figure 4. Angioproliferative lesions are present in Tg+ mice distal arterioles. a through l, Representative photomicrographs showing the formation of thick occlusive neointimal lesions. Endothelial cells (factor VIII) are forming thick layers in the distal acinar arterioles of Tg+ mice (c and d) and have increased expression of VEGFR2 (g and h), which was not seen in Tg– mice (a and b or e and f). There is increased cellular proliferation in the walls of the distal arterioles of IL-6 Tg+ mice (PCNA) (k and l) in normoxic and hypoxic conditions, which was not seen in Tg– mice (i and j). Immunohistochemistry staining; magnification, x400; scale bar=0.001 mm.

Characterization of Pulmonary Vasculopathy: Progrowth/Proproliferative Factors and Prosurvival/Antiapoptotic Mediators Are Activated in IL-6 Tg⁺ Mice
We determined whether factors that stimulate growth, proliferation, and survival and inhibit apoptosis of PAECs and PASMCs were underlying the mechanism through which IL-6 promotes the characteristic pathophysiologic phenotype of PAH. This was preformed by investigating a number of key modulators that may be involved, at the level of the protein in immunoblot analysis of whole lung lysates (supplemental Figure II, a through e). See the online data supplement for the results.

Inflammatory Cells Contribute to IL-6–Induced Neointimal Lesions in Arterioles
Given that lymphocytes form conglomerates near major airways in this mouse model,¹⁴ we examined the pulmonary vascular bed for evidence of a similar cellular inflammatory response at sites of arteriolar occlusive lesions. Under normoxic conditions, the number of periarteriolar lymphocytes (determined by the high nuclear to cytoplasmic ratio) was greater in Tg⁺ mice than in Tg^– mice (Figure 5c), with T cells, determined by immunohistochemistry (Figure 5g), but not B cells (Figure 5k) being increased within the pulmonary vascular bed of Tg⁺ animals. Other inflammatory cells were not seen (Figure 5a through 5d). Following hypoxia, these T cells (Figure 5d) contributed to the obstruction of the arteriolar lumen. This was confirmed by immunoblot analysis of whole lung lysates (supplemental Figure III). Taken together, IL-6 mainly recruits lymphocytes, and the lymphocytes that are recruited to the pulmonary vascular bed are predominately T cells, not B cells. Further characterization of lymphocyte recruitment and function was determined in IL-6 Tg⁺ mice. The data can be seen in the online data supplement, together with supplemental Figure III.

View larger version (72K): [in this window] [in a new window]   Figure 5. Inflammatory cells present in the neointimal lesions of the arterioles of Tg+ mice. a through l, Representative photomicrographs showing an increase in periarteriolar lymphocytes (Giemsa stain: c vs. a), specifically T cells (CD3: g vs. e), compared to B cells (B220: k vs. i) in IL-6 Tg+ mice with little change in Tg– mice at baseline. In hypoxic conditions, CD3+ T cells are obliterating the arteriolar lumen of IL-6 Tg+ mice (d and h) vs. (b and f). Magnification, x400; scale bar=0.001 mm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Patients with severe PAH and animal models of PAH^15–21 exhibit increases in inflammatory cells, growth factors, and cytokines. IL-6, a pleiotropic cytokine, is frequently elevated, suggesting that PAH development is associated with IL-6–induced inflammation. Our results demonstrate that IL-6 lung-specific overexpression produces distal arteriolar-occlusive plexogenic lesions and arteriolar wall muscularization. These changes in the distal vascular bed are associated with and may lead to proximal pulmonary artery wall hypertrophy and RVH, as well as increased RVSP and PVR. Injection of recombinant human IL-6 (rhIL-6) also produces RVH in rats²² and mice.¹⁸ However, they lack the associated distal obliterative muscularized vascular lesions observed in the transgenic mice that constitutively overexpress IL-6. Importantly, however, IL-6 knockout mice exposed to hypoxia are resistant to the development of increased RVSP.²³ The lack of correlation between pulmonary vascular remodeling and the presence of elevated pulmonary artery pressures in other murine models^24–27 has slowed our understanding of the pathobiology of PAH. IL-6 Tg⁺ mice, in which the pathological and physiological changes observed in the pulmonary artery bed correlate with the severity of PAH, may enable a better understanding of PAH pathobiology, including the role of increased IL-6 in the development of PAH in humans.

A major finding in this study is that distal vascular remodeling in Tg⁺ mice is similar to that seen in patients with severe PAH,⁵ with (1) concentric intimal wall thickening, (2) plexogenic lesions, (3) recruitment of inflammatory cells, and (4) distal arteriolar wall muscularization. These features occurred de novo under normoxic conditions and worsened with hypoxia. In other rodent models,^15–21 vascular remodeling is limited to either dysregulated PAECs or PASMCs, but not both. The Tg⁺ mouse, which exhibits all 4 main pathological features of PAH, is, to our knowledge, the only in vivo model that recapitulates the pathological features of PAH in humans. Therefore, this model may reveal how interactions between hyperproliferative PASMCs and PAECs and inflammatory cells, as well as the pathobiological phenotypes of these cells contribute to PAH development.

Disorganized PAEC proliferation leading to the formation of neointimal obliterative lesions is described in many cases of idiopathic PAH⁵ or associated PAH and may be why the human form of severe PAH is difficult to treat with the present available drugs.^28–30 This has led to the search for newer models of PAH in which a neointima is formed and occludes the vascular lumen. A number of 2-hit injurious murine models have been able to reproduce neointimal occlusive lesions,^15,31–33 as well as a genetically altered model³⁴; however, less than 5% of these mice developed these lesions. Our study is of interest because we show that by solely overexpressing IL-6 without an additional stress, PAECs are stimulated to either form smooth concentric multilayers, leading to thickening of the intimal wall, or to pile up on top of one another, narrowing the distal arteriolar lumen and forming a plexiform lesion. Both features were present in all mice under normoxic conditions, when PAH is mild, and increased under hypoxic conditions, when RVSP is maximal and the disease is severe. This suggests that aberrant PAEC proliferation and lesion formation are pathologically relevant and useful markers of disease progression and that overexpression of IL-6, a single genetic perturbation, is able to reproduce the characteristic obliterative lesions seen in the aforementioned models and replicate that of human disease.

Distal extension of smooth muscle into small peripheral, normally nonmuscular, pulmonary arteries within the respiratory acinous is notable in all forms of PAH. The cellular processes underlying muscularization of this distal part of the pulmonary vascular bed are incompletely understood but are thought to result from the abnormal growth of PASMCs, which have impaired responses to antiproliferative proapoptotic stimuli such as bone morphogenic protein (BMP) and TGF-β.^2,35–37 We show that lung-specific overexpression of IL-6 induces 3 forms of muscularization. First, IL-6 results in distal extension of smooth muscle into the small peripheral pulmonary arteries at the level of the acinous, and, with the added insult of hypoxia, the medial wall further hypertrophies. Secondly, IL-6 results in an increase in the medial wall thickness of the main and bronchial level pulmonary arteries, and, thirdly, there is an increase in the number of layers of elastic lamella, both of which increase further in hypoxic conditions. The combination of these striking changes in muscularization have not been observed in other PAH murine models. However, in the spontaneously hypertensive rat,³⁸ increased arterial medial wall thickness is associated with increased number of lamina in major blood vessels, as well as increased wall thickness, although less striking than in hypoxic IL-6 Tg⁺ mice. Furthermore, the hypertrophic changes observed are augmented under increased pressure, suggesting that secondary structural adaptations become superimposed on primary genetic ones. In IL-6 Tg⁺ mice, where growth development is altered,¹⁴ as in the fawn-hooded rat,³⁹ early genetic abnormalities in pulmonary vascular development may contribute to the progression of PAH in the adult Tg⁺ mice with and without a hypoxic injurious stimulus.

It is unclear what triggers PAECs and PASMCs to have a proproliferative phenotype while maintaining an insensitivity toward growth inhibitory stimuli in patients with PAH. In humans, plexiform lesions express angiogenic factors including VEGF and its receptor VEGFR2,⁴⁰ suggesting that VEGF may play a proangioproliferative role in the development of plexiform lesions, a growth factor shared by the plexiform lesions observed in IL-6 Tg⁺ mice, as well as other animal models with angioproliferative lesions.³³ VEGF may also be an important survival factor for PASMCs in the presence of IL-6. IL-6 triggers cultured smooth muscle cell proliferation both directly, through upregulation of VEGFR2 expression and phosphorylation, and indirectly, through upregulation of matrix metalloproteinase-9.⁴¹ IL-6–induced VEGF expression may also indirectly increase the number of PASMCs by transforming PAECs into smooth muscle–like cells, as observed in cultured human PAECs.⁴² These findings, taken with our results, suggest that the presence of abnormal levels of IL-6 may activate, amplify, and maintain the growth and proliferation of PAECs and PASMCs by upregulating VEGF expression.

TGF-β/BMP signaling, a network of proteins that control cell growth, is impaired and the TGF-β receptor is absent in the PAECs in the core of plexiform lesions in PAH.^43,44 This suggests that PAECs in plexiform lesions have lost their check-and-balance system to control PAEC growth, giving rise to a hyperproliferative PAEC phenotype. In addition, PASMCs from patients with PAH are resistant to the antiproliferative effects of TGF-β,² suggesting that the failure of TGF-β to suppress PASMC growth in PAH may, in part, underlie the increased muscularization of normally nonmuscularized distal pulmonary arteries of patients with PAH. IL-6 has recently been found to negatively regulate the TGF-β/BMP signaling cascade.⁴⁵ In the IL-6 Tg⁺ mouse model, in which both muscularization and angioproliferative lesions are abundant and PAH is present, we show that the expression of TGF-β is reduced, in a rich milieu of angioproliferative growth factor VEGF and its receptor. Taken together, the IL-6 Tg⁺ mouse model shares similar growth factor characteristics to that of patients with PAH, and thus this model may enable investigators to delineate the trigger behind the molecular imbalance that favors the increased expression of proliferative growth factors.

IL-6 overexpression may predispose to proliferative cellular phenotypes and exaggerated PAH as a result of unopposed MAPK intracellular signaling, normally countered by antiproliferative TGF-β–mediated signaling. Both p38^MAPK and ERK are noted to be unopposed in PASMCs from patients with mutations in the TGF-β/BMP pathway, resulting in a proliferative apoptotic resistant phenotype.⁴⁶ The IL-6 Tg⁺ mice share a similar biology to PASMCs of the patient, whereby ERK activity also is upregulated in an unopposed environment, which is, in part, attributable to the lack of TGF-β and, in part, attributable to the lack of proapoptotic MAPKs p38 and pJNK. These findings are also consistent with in vitro work, in which IL-6–stimulated human endothelial cells⁴⁷ also have reduced p38 and pJNK. In other cell systems, IL-6 activates the MAPK signaling pathway via ERK and, in turn, blocks the TGF-β/BMP pathway by preventing the nuclear translocation of Smad, a downstream BMP signaling protein,^48,49 resulting in cellular proliferation. Given that this mouse model and the vasculature of PAH patients are deficient in growth controlling TGF-β/BMP proteins⁴⁶ in the setting of elevated IL-6 levels and that both share ERK activation, further investigation of unopposed IL-6/ERK signaling may uncover the mechanism by which vascular cells switch from a balanced growth controlled state to an excessively proproliferative one.

IL-6 overexpression may predispose to exaggerated PAH as a result of coordinating a number of downstream proproliferative, prosurvival, and antiapoptotic signaling pathways (Figure 6). We found that c-myc and its obligatory binding partner, MAX, may be key in the downstream proliferative signal cascade of IL-6, promoting cellular proliferative phenotypes in PAH. However, it is also clear from our work that IL-6 selectively blocks apoptosis within the pulmonary vascular bed by downregulating TGF-β and proapoptotic MAPKs, pJNK, and p38, while upregulating prosurvival factors survivin and Bcl-2. Taken together, these complex obliterative vascular lesions are likely forming because IL-6 is favoring a proproliferative antiapoptotic cell state, as observed in angioproliferative lesions of patients with PAH.

View larger version (21K): [in this window] [in a new window]   Figure 6. Hypothetical mechanisms leading to hyperproliferative apoptotic-resistant PASMC and PAEC phenotypes, angioproliferative occlusive lesions and muscularization of distal vessels in PAH. IL-6 induces angioproliferative growth factor VEGF and intracellular MAPK ERK to activate proproliferative transcription factor complex c-MYC and MAX, resulting in an increase in cell cycle genes to promote PASMC and PAEC proliferation. Concurrently, IL-6 downregulates growth inhibitor TGF-β and other MAPKs that are proapoptotic (p38 and JNK1), while upregulating apoptotic inhibitors survivin and Bcl-2, resulting in apoptotic resistant PASMC and PAEC phenotypes. Inflammatory cells, specifically T cells, may have an important role in maintaining the secretion of the proproliferative cytokine IL-6.

See the online data supplement for an expanded discussion regarding IL-6 and its role in regulating proliferative and antiapoptotic pathways.

Elevation of IL-6 in the serum and inflammatory cellular infiltrate in plexiform lesions in PAH patients and now IL-6 Tg⁺ mice with replicative pathophysiological changes of PAH suggest that cellular immunity may play an active role in the dysregulation of PAECs and PASMCs and the development of PAH. Further discussion regarding our findings and controversies of the role inflammation may play in this disease is highlighted in the online data supplement.

The importance of the loss of small pulmonary arteriolar vessels, which may contribute to the development of PVR, is controversial; for further discussion regarding our data, see the online data supplement.

In summary, our work supports the hypothesis that IL-6 directly promotes a proproliferative apoptotic resistant milieu within the PA wall, resulting in a vasculopathy mirroring that seen in patients with severe PAH. Unlike other murine models, this transgenic mouse model replicates the pathophysiology of human PAH, with muscularization and arteriolar-occlusive changes occurring in the distal vascular bed, leading to elevated PVR and secondary chronic pressure overload occurring in the main PA and right ventricle. Our work demonstrates that, in the pulmonary vascular bed, regulation of inflammatory cytokine IL-6 is tightly linked with cellular growth and proliferation through a number of proliferative apoptotic resistant downstream pathways. Future work to establish the underlying mechanism of these complicated signaling systems in the lung-specific IL-6 Tg⁺ mice, and to determine several of the key molecules necessary to inhibit the excessive proliferative cellular state, will improve our understanding of the disease and thereby help in developing new therapies that target the angioproliferative lesions in patients with refractory PAH. The development of PAH in mice overexpressing IL-6 in the lung, together with the presence of increased IL-6 in PAH patients, suggests that IL-6 is integral to the development and progression of pulmonary vascular remodeling, PVR, and PAH.

	Acknowledgments

 
We acknowledge David A. Schoenfeld, PhD (Biostatistical Center, Harvard Medical School).

Sources of Funding

This work was supported by National Heart, Lung, and Blood Institute grants HL074859 (to A.B.W.) and HL007874 and HL039150 (to C.A.H.).

Disclosures

None.

	Footnotes

 
Original received June 23, 2008; revision received November 27, 2008; accepted December 2, 2008.

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