During the previous funding period we determined that the BMPR2-PPAR? axis protects against the development of pulmonary arterial hypertension (PAH) by regulating different genes in pulmonary arterial (PA) endothelial (EC) and smooth muscle cells (SMC). We hypothesized that this cell-specific gene regulation resulted from differences in nuclear proteins that interacted with PPAR?. To identify PPAR? interacting proteins by affinity purification and mass spectrometry, we transfected HEK293 cells with FLAG-tagged PPAR?. As expected, we found proteins known to associate with PPAR?, but, surprisingly, there were multiple novel interacting proteins, related to DNA damage sensing and repair. Moreover, in PA EC exposed to the DNA damaging effects of doxorubicin (Dox), the interaction between PPAR?and these proteins was particularly prominent. We now propose to pursue the functional significance of two of these interactions: the one between PPAR? and the DNA damage sensing complex MRN (MRE11, Rad50 and NBS1) and the one between PPAR? and p53, that is important in cell cycle arrest, DNA repair and mitochondrial metabolism.
In Specific Aim I, we confirm our studies using PPAR? siRNA, to show that reduced formation of the PPAR?-MRN complex decreases activation of ATM, ?H2AX and pCHK1/2 and results in unrepaired DNA. We then determine whether impaired sensing and repair of DNA damage is also observed when PPAR? is reduced as a consequence of loss of BMPR2. In addition, we investigate whether other DNA damaging stimuli, such as hypoxia and reoxygenation and TNF-?, result in unrepaired DNA when PPAR? or BMPR2 are reduced. Studies showing an increase in unrepaired DNA in PA EC of PAH patients are pursued, as we determine whether DNA can be repaired by FK506, an agent that 'rescues' BMPR2 signaling when the receptor is deficient, or by NO2-FA, an endogenous PPAR? agonist.
In Specific Aim II, we determine whether reduced BMPR2 (via decreased PPAR?) impairs the interaction between PPAR? and p53 in response to DNA damaging stimuli. We also investigate whether the PPAR?-p53 complex is deficient in PA EC, from PAH patients including those with a BMPR2 mutation. The functional significance of the PPAR?-p53 interaction is pursued, based upon preliminary data indicating that (i) PPAR? influences the transcriptional activity of p53 and (ii) that the PPAR?-p53 complex regulates genes that influence PA EC survival, cell cycle, DNA repair, and mitochondrial biogenesis and DNA synthesis. An unbiased ChIPSeq approach is applied to find novel targets of PPAR?-p53 mediated gene regulation.
In Specific Aim III, we study mice with postnatal deletion of BMPR2 or of the DNA damage sensor MRE11 in EC, with two goals: (i) to relate the development of pulmonary hypertension (PH) to unrepaired DNA and (ii) to determine if improving BMPR2 signaling with FK506 or inducing a PPAR?-p53 interaction with nutlin- 3a salvages DNA repair and prevents or reverses PH.

Public Health Relevance

Our proposed studies link a genetic predisposition to pulmonary arterial hypertension (PAH) with impaired resolution of DNA damage. We will show how a known PAH mutation in bone morphogenetic protein receptor (BMPR)2 impairs the function of a nuclear protein, peroxisome proliferator activated receptor (PPAR)?, in sensing DNA damage in the cells of the vessel wall, in repairing the DNA, and in controlling cell growth and metabolism until the repair is accomplished. Experiments follow in which we try to rescue the function of the BMPR2-PPAR? axis to prevent the DNA damage and the alterations in metabolism that promote loss and obliteration of pulmonary arteries in PAH.

National Institute of Health (NIH)
Research Project (R01)
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Vascular Cell and Molecular Biology Study Section (VCMB)
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Xiao, Lei
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Stanford University
Schools of Medicine
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Hopper, Rachel K; Moonen, Jan-Renier A J; Diebold, Isabel et al. (2016) In Pulmonary Arterial Hypertension, Reduced BMPR2 Promotes Endothelial-to-Mesenchymal Transition via HMGA1 and Its Target Slug. Circulation 133:1783-94
Vattulainen-Collanus, Sanna; Akinrinade, Oyediran; Li, Molong et al. (2016) Loss of PPARγ in endothelial cells leads to impaired angiogenesis. J Cell Sci 129:693-705
Spiekerkoetter, Edda; Sung, Yon K; Sudheendra, Deepti et al. (2015) Low-Dose FK506 (Tacrolimus) in End-Stage Pulmonary Arterial Hypertension. Am J Respir Crit Care Med 192:254-7
Diebold, Isabel; Hennigs, Jan K; Miyagawa, Kazuya et al. (2015) BMPR2 preserves mitochondrial function and DNA during reoxygenation to promote endothelial cell survival and reverse pulmonary hypertension. Cell Metab 21:596-608
de Jesus Perez, Vinicio; Yuan, Ke; Alastalo, Tero-Pekka et al. (2014) Targeting the Wnt signaling pathways in pulmonary arterial hypertension. Drug Discov Today 19:1270-6
Sawada, Hirofumi; Saito, Toshie; Nickel, Nils P et al. (2014) Reduced BMPR2 expression induces GM-CSF translation and macrophage recruitment in humans and mice to exacerbate pulmonary hypertension. J Exp Med 211:263-80
Morrell, Nicholas W; Archer, Stephen L; Defelice, Albert et al. (2013) Anticipated classes of new medications and molecular targets for pulmonary arterial hypertension. Pulm Circ 3:226-44
Perez, Vinicio A de Jesus; Ali, Ziad; Alastalo, Tero-Pekka et al. (2011) BMP promotes motility and represses growth of smooth muscle cells by activation of tandem Wnt pathways. J Cell Biol 192:171-88
Alastalo, Tero-Pekka; Li, Molong; Perez, Vinicio de Jesus et al. (2011) Disruption of PPARγ/β-catenin-mediated regulation of apelin impairs BMP-induced mouse and human pulmonary arterial EC survival. J Clin Invest 121:3735-46
Rabinovitch, Marlene (2010) PPARgamma and the pathobiology of pulmonary arterial hypertension. Adv Exp Med Biol 661:447-58

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