This study analyzed the physiologic and molecular effects of Bmpr2 mutation in endothelium. We found that most Tie2-Bmpr2delx4+
mice developed elevated RVSP, although with large variability in degree. This was associated with increased muscularization of small arteries (Figure ), an increase in proliferating cells (Figure ), inflammatory cells(Figure ), thrombosis (Figure ), and apoptosis (Figure ). Molecular changes substantially matched the physiologic changes, with an increase in gene ontology groups related to proliferation (Figure ), apoptosis (Figure ), and thrombosis (Figure ). The increase in inflammatory cells seen is probably related both to the increase in cytokines (Figure , Additional File 5
) and the decrease in cell-cell adhesion genes (Figure ). BMPR2 has been previously shown to regulate endothelial barrier function in the context of leukocyte transmigration[40
]. We also saw large scale alteration in metabolic genes, including response to oxidative stress (Figure ); this matches our previous findings in smooth muscle and whole animals[27
]. Unlike expression of Bmpr2delx4+
mutation in smooth muscle, expression in PMVEC did not cause loss of differentiation markers. Overall, this is the first report of the molecular effects of Bmpr2 mutation in PMVEC, with results consistent with both our current physiology and our previous findings.
Physiologic results from this mouse were similar to results from an endothelial-specific BMPR2 knockout mouse described by Hong et al[41
], despite having very different methods of generation. The only difference was higher penetrance in our model (~80% as opposed to ~30%). Histologic details such as thromboses and perivascular inflammatory cells and levels of increased proliferating cells were all almost identical between the models, which strongly increases confidence that both models accurately represent the consequences of complete suppression of signaling through BMPR2 in the endothelium.
Both of these models are quite different in histologic detail than expression of a different Bmpr2 mutation, Bmpr2R899X
, in endothelium[15
]. Endothelial expression of Bmpr2R899X
, which retains intact Smad signaling[9
], results in vascular pruning and increased RVSP, but without the obvious increase in thromboses. This also matches our molecular data; increases in thrombotic pathways were not seen in PMVEC with Bmpr2R899X
mutation (Figure ). Our molecular results imply that neither increased thrombosis nor increased inflammation are required for the development of Bmpr2-mediated PAH, although obviously both could exacerbate disease.
Broadly, we believe that the sum of the data to date suggests that the normal role of BMPR2 in the adult pulmonary vasculature is as a master switch mediating injury response. After injury, suppression of BMPR2 signaling through its cytoplasmic tail results in a decrease in cell-cell adhesion to improve migration and leukocyte recruitment, a metabolic shift towards glycolysis to support production of proteins and lipids needed for proliferation, and an increase in propensity for both proliferation and apoptosis to support tissue remodeling and angiogenesis or angeogenic repair. Suppression of BMPR2 signaling through SMAD results in an increase in thrombosis and an increase in expression of cytokines, and in smooth muscle but not endothelium, dedifferentiation. In those with intact BMPR2, this naturally resolves as the injury is repaired. In the case of BMPR2 mutation, these injury responses can never be successfully resolved, leading to a pathologic continuation of the pro-thrombotic and pro-proliferative pathways which would have been adaptive in acute injury.
Decades ago, long before molecular origins of PAH were known, there was controversy over whether the initiating events in the disease involved inflammation, proliferation, thrombosis, or vasoconstriction through defective endothelial-smooth muscle communication[42
]. The discovery of BMPR2 as the familial PAH gene had promised a resolution to this controversy. However, the current study, as well as its predecessors in the literature, indicate that all of these are consequences of BMPR2 mutation alone, and our expression arrays suggest that they may all be relatively direct consequences of BMPR2 mutation, not an in vivo cascade. Our study does not address the issue of whether any are dispensable for the development of PAH. However, since each of these individually is capable of driving PAH in animal models[43
], it is possible that inflammation, proliferation, thrombosis, and vasoconstriction in the context of pulmonary vascular injury can not be easily separated; it is not that one is the cause and the other are bystanders, but rather they are inextricably linked.