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Impact of surgical and pharmacological interventions on fetal lung growth in pulmonary hypoplasia

Final Report Summary - INCREASELUNGGROWTH08 (Impact of surgical and pharmacological interventions on fetal lung growth in pulmonary hypoplasia)

Project context and objectives

Pulmonary hypoplasia is a significant cause of perinatal death with an incidence of 15 %. Defects that result in pulmonary hypoplasia usually arise in the early stages of lung development. A common example (1 every 2 500 newborns) of such malformation is congenital diaphragmatic hernia (CDH). Lungs affected by CDH have fewer alveoli, thickened alveolar walls, increased interstitial tissue, markedly diminished alveolar air space and gas-exchange surface area. There are further a reduced number of vessels, adventitial thickening, medial hyperplasia and peripheral extension of the muscle layer into the smaller intra-acinary arterioles. Lungs are also less compliant, which relates to interstitial changes. This leads in essence to respiratory deficiency and pulmonary hypertension in the neonatal period with a mortality of 40 % and frequent requirement of prolonged mechanical ventilation or even extra corporeal membrane oxygenation (ECMO).

Prenatal interventions that trigger lung development in order to increase postnatal survival have been attempted. The concept of percutaneous, single port foetal endoscopic tracheal occlusion (FETO) using an inflatable balloon was first evaluated in sheep and is meanwhile applied clinically. However, the procedure remains invasive with the main risk of preterm premature rupture of the membranes and still 30 to 40 % succumb, the majority due to persistent hypoplasia.

Therefore, pharmacological agents to trigger lung development have been investigated experimentally by using different administration routes and dosages. These include vitamins, hyperoncotic substances and growth factors.

Our goal was to conduct an interdisciplinary study, including experimental, molecular and clinical medicine. In an experimental section, we aimed to explore additional (non-surgical) treatment options to accelerate foetal lung development and proliferation and thereby ultimately increase postnatal survival of affected babies. However, such basic animal research has to be justified by potential future clinical applications. Therefore it is also necessary to be able to predict the outcome for affected baby as accurate as possible, to allow stratification of foetuses to different prognostic groups and eventually propose experimental therapies for babies with fatal prognosis. The evaluation of these babies was part of the clinical section.

Work performed and preliminary results

During the study period 35 pregnant rats carrying 425 foetuses were operated. Preliminary results of the respective projects were presented at various national and international meetings:

1. Haader, M.; Hiden, U.; Malvasio, V.; Kuess, A.; Ruttenstock, E.; Desoye, G.; Lang, U.; Saxena, A.; Klaritsch, P. 'Impact of intra-amniotic administration of insulin-like growth factor (IGF-1) on fetal rat lungs with nitrofen-induced diaphragmatic hernia'. Book of Abstracts. 2012; 12-12.- ISPO 2012 Scientific Meeting; 3 and 4 March 2012; Graz, Austria. (ISBN: 978-3-200-02541-7).
2. Mang, M.; Haader, M.; Hiden, U.; Desoye, G.; Kuess, A.; Saxena, A.; Lang, U.; Klaritsch, P. 'Effect of intraperitoneal insulin like growth factor-2 (IGF-2) injection on lung development of fetal rats'. Book of Abstracts. 2012; 16-16. - ISPO 2012 Scientific Meeting; 3 and 4 March 2012; Graz, Austria. (ISBN: 978-3-200-02541-7).
3. Haader, M.; Hiden, U.; Ruttenstock, E; Lang, U; Saxena, A; Klaritsch, P. 'Der Einfluss von intraamniotisch appliziertem IGF-1 auf die fetale Rattenlunge mit Nitrofen-induzierter Zwerchfellhernie. Geburtshilfe und Frauenheilkunde'. 2012; 72(5):427-427.-Jahrestagung der Österreichischen Gesellschaft für Gynäkologie und Geburtshilfe (OEGGG); 6 to 9 June 2012; Graz, Austria.
4. Ruttenstock, E.; Haader, M.; Hiden, U.; Lang, U.; Saxena, A.; Klaritsch, P. 'Intraamniotische Applikation von IGF-2 bei Rattenfeten mit Nitrofen-induzierter kongenitaler Zwerchfellhernie. Geburtshilfe und Frauenheilkunde'. 2012; 72(5):433-433.-Jahrestagung der OEGGG; 6 to 9 June 2012; Graz, Austria.
5. Ruttenstock, E. M.; Haader, M.; Desoye, G.; Lang, U.; Saxena, A. K.; Klaritsch, P. 'Intra-amniotic IGF-2 treatment improves development of hypoplastic lungs in a rat model of congenital diaphragmatic hernia'. 13th European Paediatric Surgeons' Association (EUPSA) and 59th British Association of Paediatric Surgeons (BAPS) Congress of Paediatric Surgery; 13 to 16 June 2012; Rome, Italy.

Scientific publications of these studies are currently under preparation.

Summarised results:

Intra-amniotically administered recombinant human IGF-1 (rhIGF-1) reduced foetal body weights, lung weights and lung-to-body-weight-ratios in comparison to the control group, but compared to placebo only body weights were significantly decreased (p less than 0.05). Protein expression of IGF-1 was also decreased (p less than 0.01) but there was an increase (p less than 0.01) of messenger ribonucleic acid (mRNA) expression of IGF-2r in the IGF-1 group compared to the placebo group.

Intra-amniotic IGF-1 administration did not lead to improved lung development in fetal rats with CDH, most likely due to suppressive effects of the applied agent.

Mean body and lung weights were decreased in the female IGF-1 group as compared to placebo (p less than 0.05). The female placenta-to-body weight ratio was elevated (p less than 0.01). The mRNA expression levels of podoplanin, IGF-2r and Vegfr-2 were up-regulated in male IGF-1 treated foetuses compared to male placebo group (p less than 0.05). IGF-1 protein was decreased in female IGF-1 treated foetuses (p less than 0.05).

Whilst intra-amniotic injection of IGF-1 seems beneficial for lung maturation and vasculogenesis in male foetuses, a rather suppressive effect was observed in the female foetuses. This gender specific outcome is of crucial importance for the development of further treatment strategies.

Administration of IGF-2 lead to upregulation of FGF10 and Ki67 gene expression levels compared to placebo. VEGFr-2 gene expression levels were significantly down-regulated in IGF-2 treated foetuses compared to placebo group. VEGFr-1 gene expression levels were similar in both groups (0.88 ± 0.14 versus 1.00 ± 0.29; p=0.137).

IGF-2 treatment seems to have a positive impact on lung branching (FGF10) and alveolar proliferation (Ki67) of hypoplastic lungs in the nitrofen-induced CDH model. Intra-amniotic IGF-2 treatment could potentially be beneficial to lung development of hypoplastic lungs of babies with CDH.

Intraperitoneal application of IGF-2 increased mRNA expression of Ki-67 and tPA. VEGF only showed a trend for increased expression. The other markers did not reveal any significant differences. However, there was a trend for an increased VEGF-mRNA expression and BMP-4 and FGF10 protein expression.

Foetal intraperitoneal administration of IGF-2 seems to advance general proliferation of lung tissue and formation of small lung vessels and thus may promote branching of airway structures.

Following intra-amniotic injection of either recombinant VEGF-A or saline (placebo) mRNA expression levels of Ttf-1, IGF-2r, FGF10 and Plat were significantly downregulated in VEGF-A treatment group compared to placebo group.

Intra-amniotic administration of VEGF-A appears to have a suppressing effect on several aspects of development of hypoplastic lungs in the nitrofen-induced CDH model. Further studies are needed to explore whether other members of the vascular endothelial growth factor family, known to exert a positive effect on lung maturation, could be used in the treatment of hypoplastic lungs.

Clinically:

In close collaboration with the former host institution, advanced clinical evaluations of pregnancies affected by foetal CDH in the setting of a specialised outpatient clinic at the unit for maternal-foetal medicine at the Department of Obstetrics and Gynaecology that meanwhile acts as national referral centre with direct link to the foetal surgery centre in Leuven for transfer of patients and data. Affected patients were evaluated according to their prognosis and treatment options, as well as data gathering and management. All necessary data, amongst others foetal organ scans by two-dimensional (2D) and three-dimensional (3D) ultrasound or foetal magnetic resonance imaging (MRI) were assessed. If eligible, patients were offered to participate in one of the two ongoing European trials on FETO in severe and moderate cases of CDH (total trial available at http://www.totaltrial.eu online).

In collaboration with the former host several scientific contributions were published:

1. Mayer S., Klaritsch P., Petersen S., Done E., Sandaite I., Till H., Claus F., Deprest J. A. 'The correlation between lung volume and liver herniation measurements by fetal MRI in isolated congenital diaphragmatic hernia: a systematic review and meta-analysis of observational studies'. Prenat Diagn 2011; 31(11):1086-96.
2. Beck V., Davey M. G., Mayer S., Froyen G., Deckx S., Klaritsch P., Roubliova X. I., Petersen S. G., Deprest J. A. 'A longer tracheal occlusion period results in increased lung growth in the nitrofen rat model'. Prenat Diagn 2012; 32(1):39-44.

Additionally, we successfully spread that knowledge within the professional community of the country by organising events and information material:

1. Klaritsch, P. 'Prenatal diagnosis' of CDH. ISPO 2012 Scientific Meeting; 3 and 4 March 2012; Graz, Austria, 2012.
2. Klaritsch, P. Fetale Trachealokklusion. Jahrestagung der Österreichischen Gesellschaft für Gynäkologie und Geburtshilfe (OEGGG); 6 to 9 June 2012; Graz, Austria, 2012.
3. Klaritsch, P. 'Ultrasound diagnosis of congenital diaphragmatic hernia', 13th Congress of the World Federation for Ultrasound in Medicine and Biology; 26 to 29 August 2011; Vienna, Austria.
4. Santner, A.; Häusler, M.; Csapo, B.; Lang, U.; Höllwarth, M.; Klaritsch, P. 'Incidence and outcome of congenital diaphragmatic hernia in Styria', 13th Congress of the World Federation for Ultrasound in Medicine and Biology ; 26 to 29 August 2011; Vienna, Austria.
5. Klaritsch, P. 'CDH - differential diagnosis, antenatal ultrasound assessment and determination of prognosis'. Austrian Total-Trial Startup Meeting - Randomised controlled trial of Fetoscopic Endoluminal Tracheal Occlusion (FETO) versus expectant management in foetuses with left sided Congenital Diaphragmatic Hernia (CDH) and moderate pulmonary hypoplasia; 28 February 2009; Graz, Austria.
6. Klaritsch, P. 'Ultraschallkriterien und sonographische Differentialdiagnosen der CDH'. 27 February 2009; Graz, Austria.

Potential impact of final results (including the socio-economic impact and the wider societal implications)

Clinically

Lung growth could be partially accelerated by prenatal administration of growth factors, especially by IGF-2. The next logic steps would include studies of postnatal survival and ventilatory mechanics. This could be achieved using ventilation models in rabbits. If the proposed therapeutic approaches turn out to be beneficial for postnatal survival and lung function, further investigation to test adverse effects (in large animal models, e.g. sheep) may be useful. The design of a sufficient pharmacological treatment modality that could be applied by minimal invasive interventions (e.g. fetoscopic intra-tracheal administration) as an adjunct to the FETO or by ultrasound (US) guided needle procedures alone could eventually increase the number of surviving babies.

We believe that the conducted research may open the door for pharmacological therapy for this condition. As such it could lead to a wider application of foetal therapy for CDH and from a socio-economic point of view, reduce the financial burden of prolonged intensive care.