Fig 1.
(A) Right ventricular systolic pressures are significantly elevated in BMPR2 mutant mice with six weeks of transgene activation using doxycycline at 1g/kg in western diet; this elevation was prevented through administration of SB2014741 in pumps for the final four weeks. Circles represent individual mice; columns are averages of log2-transformed values; error bars are SEM. SB204741 did not affect control mice; both vehicle and treated mice are included in the control column as left and right groups of circles, respectively. (B) Cardiac Index does not change between groups, measured as cardiac output in ml/minute as determined by echocardiography divided by body surface area in square meters. (C) Immunoflourescence staining for CD45 in a 10x field of distal alveoli in agarose-inflated lungs. An increase in CD45+ cells is immediately apparent with BMPR2 mutation and with SB204741 treatment in controls. (D) BMPR2 mutant mice have ~2x the inflammatory cells per field at baseline, but SB204741 treatment has divergent effects on inflammatory cells in control and BMPR2 mutant mice, causing significant increases and decreases respectively (* = p<0.05, § = p<0.01). (E) BMPR2 mutant mice have roughly twice the numbers of partially and fully muscularized vessels per field for small and medium sized vessels; this is substantially normalized by SB204741 (* = p<0.01).
Fig 2.
SB204741 prevents arteriole wall stiffening in BMRP2 mutant animals.
(A) In mutant animals treated for 4 weeks with SB204741, the average elastic modulus is significantly lower than their untreated counterparts. The histogram shows representative vessels with a Gaussian fit of elastic modulus distributions and calculated median values. Inset bar graph shows median stiffness values. (B) Fluorescent images (green = CD31; red = αSMA), with a topographical map of vessel height overlaid with a colorimetric representation of the elastic modulus. Values in the graph in (A) are expressed as mean ± standard error. n = 3 per group, *p<0.05 compared to WT, #p<0.05 compared to vehicle treated.
Fig 3.
SB204741 reduces SRC phosphorylation and downstream activation through reduction of motility of SRC.
(A) Western blots from BMPR2 mutant or WT mice whole lung treated with SB204741 or vehicle. BMPR2 mutants show increased phosphorylation of SRC target CAS; SRC activity and phosphorylation is reduced with SB204741 treatment. (B) Densitometry for pSRC, pCAS, and pCAV1 phosphorylation. Values are normalized to total protein and β-actin (i.e. pSRC/SRC/β-actin). n = 3, *p<0.05 compared to WT, #p<0.05 compared to vehicle treated.
Fig 4.
SB204741 restricts motion of pSRC.
(A) SB204741 reduces tubulin and perinuclear SRC motility, both of which are increased in mutant microvascular endothelial cells. Eularian analysis of fluorescently labeled tubulin and SRC in endothelial cells shows elevated motility in vehicle treated mutant cells, as well as a significant decrease in motility in mutants cells treated with SB204741. Values are expressed as mean ± standard error. n = 5–10, *p<0.05 compared to WT, #p<0.05 compared to vehicle treated.
Fig 5.
(A): Principal components analysis found a strong difference between BMPR2 mutants and controls along Principal Component 1 (PC1). Treatment with SB204741 caused nearly opposite changes in PC vector in control and mutant mouse lungs (large arrows). Circles and diamonds refer to individual arrays for control and BMPR2 mutants respectively: open and filled shading are for vehicle and SB204741 treatment respectively. (B): Heat map of normalized gene expression for 100 genes most affected by SB204741 treatment. Each column is a gene, with rows treatment/genotype groups. Red corresponds to high expression and blue to low. In general, SB204741 eliminates differences between control and BMPR2 mutant mice, by moving gene expression in opposite directions (BMPR2 mutants become more like controls, but controls become more like BMPR2 mutants). (C): Representative examples of significantly overrepresented gene ontology groups. Angular width of each wedge is proportional to the number of genes altered by SB204741 in the group as a fraction of the 234 with a 95% confidence of change of over 20%. Radius is proportional to–log of the p-value (so longer is more significant). Circles correspond to multiple comparisons adjusted p = 0.05 and p = 0.01. Overlap is approximate, and demonstrates that most genes belong to more than one ontology group (lower level ontology groups not shown).
Fig 6.
(A) SB204741 causes convergence of expression of most specific genes in the cytoskeletal component ontology group from Fig 5B between control and BMPR2 mutant lungs. (B) SB204741 results in reduced expression of most genes in the muscle contractility gene ontology group, which was the most statistically significant group in Fig 4B. This brings expression levels of BMPR2 mutant mice to control levels. Error bars are standard deviation. Grey lines are from control mice; black lines are from BMPR2 mutant mice in both A and B.
Fig 7.
(A) SB204741 inhibits the elevated contractility of mutant microvascular cells in response to TGF-β1. Mutant microvascular smooth muscle cells exhibit a nearly five-fold increase in TGF-β1 induced contractility compared to their WT counterparts after 72 hours of treatment. TGF-β1 induced contractility in mutant microvascular smooth muscle cells is prevented when cells are treated concurrently with SB204741. Mutant cells synthesize (B) and activate (C) higher amounts of TGF-β1 than WT, and neither TGF-β1 synthesis nor activation is changed in mutant cells by SB204741. Values are expressed as mean ± standard error. n = 3–12 per group, *p<0.05 compared to WT, #p<0.05 compared to no treatment. Significance determined by a two-way ANOVA followed by a Holm-Sidak post hoc test.
Fig 8.
A proposed molecular mechanism for HTR2B antagonism to prevent heritable PAH.
Mutations in the tail domain of BMPR2 result in increased SRC transport and signaling. Antagonism of HTR2B inhibits the translocation of SRC and decreases SRC signaling, causing a decrease in expression of SRC regulated genes. Functionally, this results in increased small vessel compliance, reduced inflammatory infiltrate, and decreased vascular smooth muscle contractility which together contribute to a restoration in mean pulmonary arterial pressures.