Potential Role for the Bone Morphogenetic Antagonist Gremlin in Pulmonary Hypertension

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1 Potential Role for the Bone Morphogenetic Antagonist Gremlin in Pulmonary Hypertension Dr Sean Gaine National Pulmonary Hypertension Unit Mater Misericordiae University Hospital University College Dublin On behalf of Dr. Christine Costello

2 Background 1. The gene defect in BMPR2 responsible for familial PAH and many cases of idiopathic PAH doesn t cause disease in everyone with the defect. 2. Despite having this gene defect in every cell in the body, disease occurs only in the lung.

3 Hypothesis There must exist lung selective inducible genes that contribute to the development of PH in the presence of BMPR2 mutations. Hypoxia may selectively induce these gene in the lung rather than the systemic circulation These genes may explain the role of the BMP system in the development of PAH with or without the BMP mutation

4 Strategy adopted Primary human microvascular endothelial cells - LUNG Primary human microvascular endothelial cells CARDIAC Normoxia or hypoxia at different time points Human Affymetrix chips (22,283 genes) 0hr 3hr 24hr 48hr 0hr 3hr 24hr 48hr HMVEC-Lung HMVEC-Cardiac Identified genes differentially regulated in pulmonary endothelium

5 90 genes identified Identification of lung selective hypoxic responsive genes Fold over control values Lung cells Cardiac cells Costello et al. Am J Physiol Lung Cell Mol Physiol. 2008

6 Schematic of BMP signallingin familial PAH Mutations in the BMPR2 in familial PAH Vascular lesions in the pulmonary circulation Adapted from Balemans & Hull, Development Biology 2002

7 Schematic of modulation of BMP signalling Antagonists Modulation of BMP signalling: 1. I-Smad and Smurfs 2. Pseudoreceptors 3. Extracellular antagonists Endogenous BMP antagonists include: Gremlin Adapted from Balemans & Hull, Development Biology 2002

8 RNA values Relative mrna values Normalised Gremlin expression 1.0 significantly upregulated 0.5 only in lung 0.0 GREM 1 SLM 2 SPON1 ENPP2 PRKY M PEG1 LIM CH GREM 1 - Control * GREM 1 - Hypoxic Heart Kidney Liver Lung Spleen Thymus 4.5 * p < 0.001; control tissue compared to hypoxic tissue SLM 2 - Control SLM 2 - Hypoxic Costello et al. Am J Physiol Lung Cell Mol Physiol. 2008

9 Immunostaining of Gremlin in normoxic and hypoxic lungs Panels A & B: Normoxic murine lung tissue from 2 animals; Panels C & D: Hypoxic murine lung tissue from 2 animals Panels E: Omission of primary in normoxic animal; Panel F: Omission of primary in hypoxic animal Costello et al. Am J Physiol Lung Cell Mol Physiol. 2008

10 Gremlin inhibits BMP4 induced human endothelial cell wound healing % wound closure at 24hrs 80 # Vehicle BMP4 BMP4 + Gremlin BMP4 + Hypoxic CM Mean scratch width from 6 separate expts. Horizontal bars indicate median values. # indicates significant difference from all other groups (p<0.05). Costello et al. Am J Physiol Lung Cell Mol Physiol. 2008

11 Gremlin blocks BMP4-induced Smad phosphorylation in human lung cells PSmad/TSmad Vehicle Gremlin BMP4 Gremlin + BMP4 Phospho-Smad 1/5/8 Total Smad 1/5/8 GAPDH mins * n= mins Vehicle Gremlin BMP4 Gremlin + BMP4

12 Gremlin protein is highly expressed in human lung disease Slides A: Omission of primary in PH patient, Slides B-D: Gremlin staining (brown colour) in 3 PH patients

13 Relative Gremlin mrna values Gremlin gene expression is higher in humans with IPAH * * p< Control IPAH patients Costello et al. Am J Physiol Lung Cell Mol Physiol. 2008

14 Summary Identification of endogenous hypoxic responsive lung selective BMP antagonists - i.e. Gremlin and Follistatin Gremlin protein significantly increased in an in vivo murine model of hypoxia and highly expressed in human lung disease Human lung microvascular endothelial cells respond to BMP stimulation in vitro and Gremlin blocks these effects Results identify a novel mechanism of BMP antagonism that may play an important role in the development of PAH.

15 Background and Conclusion 1. The gene defect in BMPR2 responsible for familial PAH and many cases of idiopathic PAH doesn t cause disease in everyone with the defect. Perhaps a second hit may induce Gremlin which blocks any residual BMPR2 activity and precipitates the disease? 2. Despite having this gene defect in every cell in the body, disease occurs only in the lung. Could this be explained by the lung selective induction of BMP antagonists?

16 Acknowledgements Pulmonary Vascular Translational Medicine Group, UCD and Mater Hospital Collaborators: International Clinical Grant support Oliver Eickelberg (University of Giessen) Nick Morrell (Cambridge University) Staff of the National Pulmonary Hypertension Unit Sinead Doherty RN Patients from all the centres Health Research Board, Ireland Actelion Pharmaceuticals (unrestricted educational)

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18 Volume fraction of Gremlin Gremlin protein is significantly higher in hypoxic lung tissue 0.3 * * p < Normoxic lungs (n=4) Hypoxic Lungs (n=5) Costello et al. Am J Physiol Lung Cell Mol Physiol. 2008