Role of cGMP signalling in pericytes during lung fibrosis

Andreas Friebe, Würzburg

Mice lacking NO-sensitive guanylyl cyclase (NO-GC) globally or in specific cell types have been proven to be excellent models to study NO/cGMP-mediated signalling in many cellular functions including contraction, growth and survival. Based on our work in the previous three years, we will now concentrate on lung physiology and pathophysiology with a special focus on fibrosis. Although NO-GC has been first isolated from rat and bovine lung, the cell type that expresses high levels of the enzyme has not been identified to date. In the last funding period, we have found NO-GC to be highly expressed in lung pericytes. Pericytes are mural cells found in precapillary arterioles, capillaries and postcapillary venules. The identification of NO-GC in pericytes is clinically intriguing as these cells are found in virtually all organs and tissues. The exact function of pericytes is still under investigation. Based on their ability to contract and relax, pericytes have been postulated to regulate capillary diameter and thus possibly contribute to blood pressure regulation. Pericytes are also thought to be involved in the development of lung fibrosis. Interestingly, a role of NO-GC in fibrotic processes has been demonstrated recently using our global KO mice. We have acquired a mouse strain that expresses Cre recombinase in pericyte precursor cells under the control of the Foxd1 promotor. In lineage tracing experiments, Foxd1-expressing cells have been shown to develop into lung pericytes which contribute significantly to extracellular matrix deposition and scarring during bleomycin-induced lung fibrosis. Foxd1-Cre mice are currently being crossed with our floxed NO-GC mice. Using these animals, we will elucidate the role of NO-GC in the development of bleomycin-induced lung fibrosis. This will include 1) the isolation and culture of pericytes for identification of members of the NO/cGMP cascade as well as contraction/relaxation studies, 2) bleomycin induction of lung fibrosis in our cell-specific KO strains, including the clinically relevant evaluation of novel NO-GC activators and stimulators as agents to prevent/relieve fibrotic processes and 3) the effect of NO-GC on cigarette smoke-exposed animals in conjunction with lung fibrosis. In addition, we will try to dissect which of the NO-GC isoforms (NO-GC1 or NO-GC2) has the major impact in the disease. In collaboration with other groups of this research unit, these data will be complemented using mice lacking cGKI and BK channel. Furthermore, we will use transgenic mice expressing a cGMP biosensor in Foxd1-derived pericytes to monitor cGMP kinetics in culture and in whole lung during disease development. In sum, our project will provide valuable information on the function of cGMP in a so far neglected cell type, the pericyte, and will shed light on the role of NO-GC during lung fibrosis.

Fig. 1: PDE3A expression in mice lacking NO-GC. (A) PDE3A expression in platelets and aorta. PDE3A expression was detected in platelets from WT and NO-GC KO mice using Western blotting (n=6 for each genotype; loading controls are omitted for clarity). (B) PDE3A in aorta is downregulated in NO-GC SMKO mice. Mice carrying homozygous floxed alleles for NO-GC and the CreERT2 recombinase gene under the control of the SM-MHC promoter were injected with tamoxifen to induce smooth muscle-specific knockout of NO-GC. At the indicated time points, NO-GC expression in aortic rings as well as the systolic blood pressure were determined (n=6 for each genotype). PDE3A expression was found to be reduced by 50% in mice 50 days after tamoxifen injection. Whether the reduction in PDE3A is a consequence of the decrease in NO-GC (and cGMP) or of the increase in blood pressure will be determined in this project. (C) Abolished cGMP/cAMP crosstalk in platelets from mice lacking NO-GC. For the measurement of cGMP/cAMP crosstalk, platelets were preincubated for 5 min with increasing concentrations of the NO donor GSNO and then cAMP synthesis was stimulated with 100 nM PGE1 (n=6 for each genotype). (C) Andreas Friebe
Fig. 2: NO-GC expression in lung pericytes. Both immunohistological pictures show murine lung tissue stained with antibodies against NO-GC (red) and αSMA (green) (A) or NO-GC (red) and CD31 (green), a specific marker for endothelium (B and C). The antibody against NO-GC was home-made and is very specific based on negative results in GCKO tissue. NO-GC-positive cells are wrapped around small vessels/capillaries which is typical for pericytes. (C) Enlarged area showing the close contact of pericytes wrapping around endothelial cells. In addition to the localization and specific morphology, we have identified these cells as pericytes using desmin staining (data not shown). Note the expected expression of NO-GC in a larger blood vessel in (A; arrow) which co-stains for αSMA. (C) Andreas Friebe

Overview of the NO-GC knockout models generated in our laboratory.

GC Knockout Abbreviation Cre line Specificity Area of Investigation
Guanylyl Cyclase KO NO-GC KO Ella-Cre ubiquitous
Smooth Muscle GCKO SMKO SMMHC-CreERT2 cell-specific vascular relaxation, blood pressure, gastrointestinal motility
Endothelial Cell GCKO ECKO Tie2-Cre cell-specific angiogenesis, permeability
Interstitial Cells of Cajal GCKO ICCKO cKit-CreERT2 cell-specific gastrointestinal motility
Fibroblast-like Cell GCKO FLCKO PDGFRα-CreERT2 cell-specific gastrointestinal motility
Neuron-specific GCKO NestinKO Nestin-Cre cell-specific baroreceptor, general neuronal functions
Cardiomyocyte GCKO CMKO αMHC-CreERT2 cell-specific heart function, baroreceptor
Platelet-Specific GCKO PSKO Pf4-Cre cell-specific platelet function

Double or triple mutants are also available (e.g. SMC/CM, SMC/ICC, SMC/FLC and SMC/ICC/FLC).

Project-related publications

PI of this project; PIs of other FOR 2060 projects are in bold.

1.    Straubinger J, Schottle V, Bork N, Subramanian H, Dunnes S, Russwurm M, Gawaz M, Friebe A, Nemer M, Nikolaev VO, Lukowski R. Sildenafil does not prevent heart hypertrophy and fibrosis induced by cardiomyocyte AT1R signaling. J Pharmacol Exp Ther. 2015;354:406-16. [Project 3Project 4, and Project 5] [pubmed]

2.    Hoffmann LS, Etzrodt J, Willkomm L, Sanyal A, Scheja L, Fischer AW, Stasch JP, Bloch W, Friebe A, Heeren J, Pfeifer A. Stimulation of soluble guanylyl cyclase protects against obesity by recruiting brown adipose tissue. Nat Commun. 2015;6:7235. [Project 3] [pubmed]

3.    Bettaga N, Jäger R, Dünnes S, Groneberg D, Friebe A. Cell-specific impact of nitric oxide-dependent guanylyl cyclase on arteriogenesis and angiogenesis in mice. Angiogenesis. 2015;18:245-54. [Project 3] [pubmed]

4.    Groneberg D, Zizer E, Lies B, Seidler B, Saur D, Wagner M, Friebe A. Dominant role of interstitial cells of Cajal in nitrergic relaxation of murine lower oesophageal sphincter. J Physiol. 2015;593:403-14. [Project 3] [pubmed]

5.    Beyer C, Zenzmaier C, Palumbo-Zerr K, Mancuso R, Distler A, Dees C, Zerr P, Huang J, Maier C, Pachowsky ML, Friebe A, Sandner P, Distler O, Schett G, Berger P, Distler JH. Stimulation of the soluble guanylate cyclase (sGC) inhibits fibrosis by blocking non-canonical TGFβ signalling. Ann Rheum Dis. 2015;74:1408-16. [Project 3] [pubmed]