Knowing that a mutation in the bone morphogenetic protein receptor type 2 (BMPR2) gene is present in most patients with a hereditary form of pulmonary hypertension (PH) has allowed us to propose therapies that might be of value in patients with all forms of PH.1,2 The premise is based on evidence from cultured cells and experimental animals. Taken together, these studies indicate that dysfunction of BMPR2 and related genes leads to an adverse response of the pulmonary circulation to injury, and that this can cause the progressive elevation in pulmonary arterial (PA) pressure and right heart failure in patients with PA hypertension (PAH).
The adverse response to injury is manifest as rarefaction of the pulmonary arteries seen on the angiogram (Figure 1). This is caused by loss of the most distal arteries, attributed at least in part to programmed cell death of the endothelial cell lining.3 As a consequence, at least in part because of dysfunction of the remaining endothelial cells in small and larger arteries, there is an expansive proliferation of cells that have features of smooth muscle, and this culminates in progressive occlusion of the lumen of the vessel. The origin of these smooth muscle-like cells can be endothelial cells,4 fibroblasts,5 or even inflammatory cells.6 Another feature related to dysfunction of the endothelial and other cells of the vessel wall is the abnormal recruitment of inflammatory cells that contribute further to the adverse response to injury. They do so both by releasing factors that amplify proliferation of resident cells, and also by releasing an enzyme (elastase) that causes elastic fibers to degrade. The degradation and ultimate loss of elastic fibers cause vessel stiffening7; the breakdown products of elastin also promote cell proliferation8 and inflammation.9
Figure 1: The adverse response to injury is manifest as rarefaction of the pulmonary arteries seen on the angiogram, and this results from loss of the most distal arteries by programmed cell death of the endothelial cell lining. As a consequence of dysfunction of the endothelial cells in larger arteries, there is an expansive proliferation of cells that have features of smooth muscle that culminate in progressive occlusion of the lumen of the vessel. Another feature related to dysfunction of the endothelium of the vessel wall is the abnormal recruitment of inflammatory cells that contribute further to the adverse response to injury. They do so both by releasing factors that amplify proliferation, and also by contributing to factors that cause elastic fibers to degrade.
While BMPR2 mutations are present in >70% of familial and 25% of sporadic cases of idiopathic PAH,1,10,11 dysfunction of the BMPR2 receptor alone is insufficient to cause PAH, but may be necessary to allow it to develop in response to one or more injurious perturbations of the pulmonary circulation. Moreover, patients without a mutation and PAH and experimental models of PAH show low expression levels of BMPR2.12 The focus on BMPR2 signaling is important because of the protective nature of this pathway. BMPR2 signaling has been shown to be critical in preventing adverse remodeling by promoting survival of pulmonary arterial endothelial cells, and doing so can prevent damage or facilitate regeneration of damaged microvessels.13 BMPR2 signaling also inhibits pulmonary arterial proliferation in response to growth factors (Figure 2).14 In addition, BMPR2 signaling promotes motility of pulmonary arterial smooth muscle cells15 while repressing growth,16 and BMPR2 signaling is anti-inflammatory.17
Figure 2: This is a schema proposing that BMPR2 signaling was critical in preventing the adverse remodeling in response to a variety of injuries that culminate in the development of PAH. BMPR2 signaling inhibits pulmonary artery smooth muscle cell proliferation in response to growth factors that are liberated by elastases in response to injury and inflammation, and conversely BMP signaling has the opposite effect on endothelial cells in sustaining the survival of endothelial cells and the regeneration of the microvessels in response to injury. Both processes involve PPAR_, but in smooth muscle cells a target is apolipoprotein E, and in endothelial cells a target is apelin. Apelin also inhibits smooth muscle cell proliferation, as do nitro-fatty acids that regulate PPAR_ in both cell types.
The BMPR2 receptor has been found to direct transcription factors to turn on target genes that protect the vessel wall, and reasoning suggests that knowing what those factors and target genes are will ultimately allow us to replace them, and therefore, to rescue dysfunction of the BMPR2 receptor and to either arrest or reverse the disease process.
It has been shown that when there is injury to the vessel wall, an enzyme that degrades elastin, an elastase, releases growth factors that are normally stored in the matrix surrounding the cells of the vessel wall.18 One of the most otent of these growth factors is platelet-derived growth factor (PDGF). It binds to its receptor on the surface of smooth muscle cells and activates a signaling messenger called pERK19 to induce expression of genes that make cells proliferate. But when BMPR2 is functional, it counteracts this pathway by directing a molecule called PPARγ to the nucleus. PPARγ can block the pERK signal, and it induces expression of genes that prevent cell division (cell cycle inhibitors called p27 and p21).20 Another gene that responds toPPARγ is apolipoprotein E, and the apolipoprotein E protein blocks the PDGF receptor.21 In adipocytes (fat cells) PPAR_ produces a molecule called adiponectin that sequesters PDGF (Figure3).22 So now the growth factor is incapacitated, the receptor is incapacitated; the signal is blocked and replaced by signals that inhibit the cell from dividing. In cultured cells PPAR_ has been shown to be pivotal in directing BMPR2’s ability to prevent proliferation.14,23 When smooth muscle cells are stimulated to divide by the growth factor PDGF, they stop dividing when you activate BMP signaling. However, when PPAR_ is incapacitated by an inhibitor, this protective advantage is lost.
Figure 3: This is a schema showing that when there is injury to the vessel wall, there is elastase activity and release of PDGF, inducing a signaling messenger called pERK. This is a factor that facilitates cell proliferation. To counteract this process, BMPR2 directs PPAR_ to the nucleus. PPAR_ can block the pERK, and it also induces genes that prevent cell division (cell cycle inhibitors p27 and p21). PPAR_ also induces production of a gene product, apolipoprotein E. This protein blocks the PDGF receptor. In adipocytes PPAR_ induces production of adiponectin, a protein that sequesters the growth factor, PDGF.
It follows that a mouse genetically engineered not to produce PPARγ in smooth muscle cells will spontaneously develop PAH. The pressure in the right ventricle is elevated, the right side of the heart hypertrophies, and the small vessels become abnormally thick walled, and there is right ventricular hypertrophy.14 Mice that lack a protective target of PPARγ namely apolipoprotein E, also develop spontaneous PAH, but only as they age. However, if these mice are fed a high-fat diet they develop PAH earlier.23 This PAH can be reversed by treating the mice with an activator of PPARγ, a drug that is used to treat metabolic syndrome.
Because PPARγ activation is used to treat metabolic syndrome (also known as insulin resistance or Type 2 diabetes), our studies suggested that there may be a high incidence of this complication in patients with PAH and dysfunctional PPARγ activation. Indeed, further studies by our group showed that women with PAH are twice as likely to have metabolic syndrome than the general population of women.24 In addition, if you divide the women with PAH into the group that has insulin resistance vs. the group that doesn’t have this condition, those with insulin resistance are more likely over a 6-month period to have adverse events that include worsening heart failure, need for hospitalization, or transplantation. This information, however, suggested that we might also be able to rescue dysfunction of the BMPR2 receptor by activating PPARγ.
This was tested in pulmonary arterial smooth muscle cells from mice that were engineered to only express half the amount of BMPR2, and from a patient with a mutation in BMPR2. In both cases, the profound increase in pulmonary arterial smooth muscle cell number in response to a growth factor could not be prevented by activating the receptor because it was deficient. However, when we activated PPARγ, we could rescue the dysfunction of the receptor and inhibit the proliferative response to a growth factor.14
We also showed that the BMPR2 counteracts endothelial vulnerability to injury, allowing these cells to recover and regenerate. 13 In endothelial cells, BMPR2 also activates PPARγ, but PPARγ now forms a complex with another transcription factor called beta-catenin and together they induce expression of genes that regulate endothelial cell survival and angiogenesis. 25 Prominent among these genes is apelin, a small protein that others have shown can promote endothelial health (Figure 4). It was interesting that synthetic drugs used to treat metabolic syndrome were not effective in activating PPARγ in endothelial cells because they disrupted its interaction with beta-catenin. However endogenous activators of PPARγ (those that the cells produce) such as nitro-fatty acids26 were highly effective in inducing PPAR_ activity and inducing the production of apelin by pulmonary arterial endothelial cells.25 In fact, we were able to show that in mice that develop PAH because they lack PPARγ in endothelial cells, administration of apelin could reverse the pathology. Apelin not only promotes endothelial cell survival, but it is a protein that is released by endothelial cells and has beneficial effects in preventing smooth muscle cell proliferation in response to growth factors (Figure 5). Thus, it can rescue BMPR2 dysfunction in endothelial or smooth muscle cells.
Figure 4: This is a schema showing that in pulmonary arterial endothelial cells, BMPR2 signaling induces a complex between PPARγ and beta-catenin. Nitro-fatty acids that are released from mitochondrial membranes by reactive oxygen species can also promote the interaction between PPARγand beta-catenin and rescue dysfunction of BMPR2 by inducing the target endothelial cell survival gene, apelin.
Figure 5: This schema shows that apelin is produced as a consequence of the PPARγ– beta-catenin interaction and gene regulation. Apelin has autocrine function in protecting endothelial cells, possibly through the APJ receptor, and also paracrine
function in preventing pulmonary arterial smooth muscle cell proliferation. Thus apelin should be able to help regenerate normal microvessels and reverse occlusive neointimal formation by inducing apoptosis of abnormal smooth muscle-like cells, and may therefore be a promising therapy for PAH.
In addition, loss of BMPR2 activity is proinflammatory in smooth muscle cells17 and some data show that the same is true in endothelial cells. Agents that rescue BMPR2 dysfunction, such as apelin or endogenous nitro-fatty acids, are also known for their antiinflammatory properties, thus further protecting the vessel wall.27
Finally, we have embarked on a strategy that searches drug libraries for an agent that will have as an “off-target” effect the added benefit of activatingBMPR2, and have found one such agent with very promising results in both our experiments with cultured human vascular cells as well as rats with PH.
Key Words—BMPR2, platelet-derived growth factor, pulmonary arterial hypertension, pulmonary arterial smooth muscle cells