To understand the relationship between BMPR2 haploinsufficiency and pulmonary hypertension, this study examined BMPR2+/− mice and explored three issues: 1) whether BMPR2 haploinsufficiency affects the development of the mice; 2) whether haploinsufficiency alone leads to pulmonary hypertension; and 3) whether haploinsufficiency increases the susceptibility to pulmonary hypertension in the setting of inflammation. The results showed that BMPR2 heterozygosity caused approximately 20% fatality in mice during embryonic development, but did not lead to pulmonary hypertension under unstressed conditions. The study also showed that BMPR2+/− mice were more sensitive to inflammation-induced pulmonary hypertension than wildtype mice.
BMP signaling plays an important role in embryonic development, and is involved in gastrulation, mesoderm formation, neural patterning, skeletal development, and organogenesis (for reviews, see 5,30
). Homozygous knockout mice deficient in BMP signaling proteins, such as BMP-2, BMP-4, BMPR2, BMPR-IA (Alk-3), ActR-I (Alk-2), Smad1, or Smad5, all die during embryonic development 30
. By contrast, animals with heterozygous mutations in these genes are viable and reported to be grossly phenotypically normal. In this study, we found that approx. 20% of BMPR2+/− mice died in utero
and/or during weaning, which suggests that haploinsufficiency of BMPR2 affects early development. Although we observed no apparent abnormality in the surviving BMPR2+/− mice under unstressed conditions in this study, some of these mice may have had minor developmental defects, and these defects could affect the phenotype of the mice under stressed conditions.
Inflammation has been suggested to be an important mechanism in the development of pulmonary arterial hypertension. Evidence supporting the hypothesis include: 1) inflammatory cells are found in the vicinity of remodeled pulmonary vessels with plexiform lesion; 2) proinflammatory cytokines and chemokines are increased in patients with pulmonary arterial hypertension; 3) pulmonary arterial hypertension is a common complication of autoimmune diseases involving systemic inflammation, such as scleroderma and systemic lupus erythematosus; and 4) in a rat model of pulmonary arterial hypertension, monocrotaline administration causes pulmonary endothelial injury and inflammation, which is followed by pulmonary vascular remodeling (for reviews, see 17–20
). In the present study, we examined the effect of inflammation on the development of pulmonary hypertension in BMPR2+/− mice. Adenovirus-mediated overexpression of 5LO in the lung was used as the inflammatory stress because 5LO expression is increased in the human disease. The results showed that BMPR2+/− mice responded to the inflammation with an immediate marked increase of RVSP (peaking at day 7), and delayed muscularization of distal pulmonary arterioles (28 days after Ad5LO treatment). The early increase of RVSP is related to enhanced pulmonary vasoconstriction, due, at least in part, to increased thromboxane A2
production in the mice. The later muscularization of distal arterioles could be caused by the initial vasoconstriction, increased release of growth factors by inflammatory cells, and/or endothelial cell activation/injury caused by the inflammation. Further study is required to identify the specific mechanism. The overall degree of the muscularization found in the Ad5LO-treated BMPR2+/− mice is mild. This could be due to the transgene expression, as well, as the inflammation is transient. Further studies are required to demonstrate whether increased pulmonary vascular injury by sustained inflammation leads to extensive pulmonary vascular remodeling and persistent pulmonary hypertension in BMPR2+/− mice. BMPR2+/− mice were found to produce significantly higher amounts of thromboxane A2
than wildtype mice during 5LO overexpression. This observation is consistent with previous reports that thromboxane A2
production is significantly enhanced in patients with pulmonary arterial hypertension 31–33
. Mechanistically, however, this finding raised the question of how BMPR2 deficiency leads to increased thromboxane A2
production. No report in the literature has linked BMP signaling to thromboxane A2
production. As the thromboxane A2
levels in the wildtype and BMPR2+/− mice were the same before Ad5LO delivery, the difference in thromboxane A2
production in these mice resides in the different responses to 5LO overexpression or inflammation. We, therefore, examined platelet activation in vivo
and pulmonary epithelial cell activation in cell culture, as thromboxane synthase is most abundantly expressed in platelets (2,187 ng/mg protein) 34
, and lung has the highest content of the enzyme, 765 ng/mg, among solid organ tissues 34
. The cells in lung that express thromboxane synthase are mainly bronchial epithelial cells and alveolar macrophages in humans, and also small pulmonary artery smooth muscle cells in rats 29
. Examining plasma sP-selectin and urinary serotonin, two platelet activation markers, showed that both of the markers tended to be higher in BMPR2+/− than wildtype mice, but neither difference reached statistical significance. Thus, platelet activation may not be the primary or the only source of the enhanced thromboxane A2
production in BMPR2+/− mice during 5LO expression. The pulmonary epithelial cell line, A549 cells, was found to produce a very small amount of thromboxane A2
under basal conditions, but the production of this prostanoid was stimulated markedly by IL-1β. BMP did not affect the basal level of thromboxane A2
production, but reduced IL-1β stimulation significantly by 20%. These effects suggest that BMP signaling does not regulate thromboxane A2
production directly, but interferes with IL-1β signaling.
IL-1β signaling activates two pathways, one leading to NFκB activation and the other, MAP kinase JNK and, subsequently, AP-1. A previous study has shown that BMP-7 inhibits IL-1β-induced JNK and AP-1 activation, but does not affect IL-1β-induced NFκB activation in human mesangial cells 35
. If a similar selective inhibition occurs in pulmonary epithelial cells, it could explain the partial inhibitory effect of BMPs on the IL-1β-stimulated thromboxane A2
production in A549 cells. Further studies are required to understand the specific interaction between BMP and IL-1 signaling in these cells.
is a potent vasoconstrictor and platelet activator 36,37
. It also inhibits voltage-gated-potassium channels 38
, and has synergistic effects with serotonin in causing vascular smooth muscle cell proliferation 39
. These effects, when persistently produced by multiple inflammatory insults or stresses, could contribute to pulmonary vascular remodeling and sustained pulmonary hypertension. Understanding the relationship between BMPR2 haploinsufficiency and thromboxane A2
production under inflammatory stress could shed light on the mechanism of heterozygous BMPR2 mutation-mediated IPAH.