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Potential Role for the Bone Morphogenetic Antagonist Gremlin in Pulmonary Hypertension

C. M. Costello

K. Meredith

E. Cahill

J. McBryan

M. Koenigshoff

O. Eickelberg

Sean Gaines

F. Martin

P. McLoughlin


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Conference: 2008 International PHA Conference and Scientific Sessions

Release Date: 06.20.2008

Presentation Type: Abstracts

Costello C.M.1, Howell K.1, Cahill E.1, McBryan J.2, Koenigshoff M.3, Eickelberg O.3, Gaine S.4, Martin F.2, McLoughlin P.1

1. School of Medicine and Medical Science, Conway Institute, University College Dublin, Ireland
2. School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Ireland
3. Dept. of Medicine II, University of Giessen Lung Center, Giessen, Germany
4. National Pulmonary Hypertension Unit, Mater Misericordiae University Hospital, Dublin, Ireland

BACKGROUND: Non-functioning mutations of the BMP receptor 2 (BMPR2) strongly predispose to the development of idiopathic pulmonary arterial hypertension (IPAH) but the precise pathways linking such mutations to the vascular disease remain unknown.  For example, we do not understand how this somatic mutation causes a pathological increase in vascular resistance in the pulmonary circulation but not in any other organ.  More recently it has been reported that inhibition of the BMP pathways plays an important role in mediating hypoxic pulmonary hypertension.  We therefore hypothesized that there are lung selective hypoxia-induced alterations in the expression of specific genes and that these lung selective pathways would contribute to the development of pulmonary hypertension in the presence of BMPR2 mutations. 

METHODSPrimary human microvascular endothelial cells from lung (HMVEC-L) and cardiac tissue (HMVEC-C) were cultured in normoxia or hypoxia (1% O2) for 3hr, 24hr or 48hrs. Affymetrix murine microarrays were used to examine changes in global gene expression in these cells and a subtractive strategy used to identify genes whose expression was altered in the pulmonary endothelium but not in the cardiac (systemic) endothelial cells.  Hypoxic murine tissues were generated by exposing mice to 10% oxygen for 48 hours.  Human lung samples from IPAH and control lungs were obtained at the time of lung transplantation.  Real-time PCR (TaqMan) was used to measure gene expression in cell culture, murine tissues and patient samples. Immunohistochemistry was used to determine gremlin protein in murine lung.

RESULTSGene array experiments suggested that the soluble BMP antagonist gremlin was upregulated in pulmonary but not cardiac endothelial cells in response to hypoxia.  Real time PCR analysis demonstrated that basal gremlin expression was more highly expressed in the lung than in the cardiac endothelium, and was significantly increased in response to hypoxia (48 hrs) in the pulmonary cells alone. Expression analysis in our in vivo murine lungs confirmed high basal expression of gremlin mRNA in control lungs compared to all other organs and a >3-fold up-regulation in the hypoxic lung without change in any systemic organ tested.  Immunohistochemical analysis demonstrated that gremlin protein was markedly increased in the hypoxic lung in vivo.  Functional studies showed that that gremlin antagonised BMP-stimulated HMVEC-L wound healing in vitro. Finally, real-time analysis indicated a > 8-fold increase in gremlin expression in lung tissue from IPAH patients compared to controls. 

COMMENTS AND CONCLUSIONS: This study shows, for the first time, that the soluble extracellular BMP antagonist gremlin is selectively up-regulated in both hypoxic and IPAH lungs. Given that BMP signaling is crucial for the maintenance of normal pulmonary vascular structure and function, this finding may give new insights into potential mechanisms mediating pulmonary hypertension. Furthermore, our novel finding that gremlin shows very high basal expression levels in the lung may offer an explanation for why the lung is the only organ that shows vascular abnormalities in the presence of a somatic BMPR2 mutation in patients with IPAH.  These findings identify a novel mechanism of BMP antagonism that may play an important role in the development of IPAH. 

This work was funded by the Health Research Board and the HEA (PRTLI).