S. Balaji1, M. Shah3, M. Phillips3, X. Wang1, C. Moles1, P. L. Bollyky2, S. E. Mclean3, S. G. Keswani1 1Baylor College Of Medicine & Texas Children’s Hospital,Pediatric Surgery,Houston, TX, USA 2Stanford Univeristy School Of Medicine,Division Of Infectious Diseases,Palo Alto, CA, USA 3University Of North Carolina,Division Of Pediatric Surgery,Chapel Hill, NC, USA
Introduction:
The management of pulmonary arterial hypertension (PAH) is challenging in patients with congenital diaphragmatic hernia (CDH) and is a significant source of morbidity and mortality in these patients. In CDH, the pulmonary arteries have thick walls due to smooth muscle cell hyperplasia, increased collagen deposition, and marked inflammation, leading to fibrotic remodeling of the perivasculature. Adult models of PAH and fibrosis demonstrate a dysregulation of hyaluronan (HA) metabolism in the pulmonary vasculature, which may contribute to increased inflammatory response. The role of HA has not been examined in neonatal PAH and CDH.
Methods:
The main challenge in studying CDH is the lack of a viable congenital murine model that develops PAH. We are characterizing the phenotype of the Slit3-/- mouse which shows a CDH at birth with a resultant increase in right ventricular pressures (surrogate for pulmonary arterial pressures) and evidence of vascular wall remodeling in the pulmonary arteries that mimics the human phenotype. To determine the dysregulation of HA metabolism in CDH, lungs from Slit3-/- or WT-control mice at 8-12 weeks (n=3-4/group) were harvested. RNA was isolated from frozen lung samples, and hyaluronan synthases (HAS1-3) and hyaluronidases (Hyal1-2) were measured (RT PCR). HA expression was determined in paraffin-embedded sections (HA-binding protein, staining). Data represented as mean+/-SD; p<0.5 denotes significance; t-test.
Results:
Compared to WT mice, the Slit3-/- mice progressively develop right ventricular remodeling, thickening of the pulmonary arteries, increased collagen, and TGFβ1 expression. Slit3-/- murine lungs expressed a significant (5-fold) increase in HAS1 gene expression (11.11+/-3.34 vs. 2.3+/-1.93, p<0.05) compared to WT-controls. There was no significant difference in the other 2 hyaluronan synthases, HAS2 (0.84+/-0.41 vs. 0.84+/-0.34) or HAS3 (0.69+/-0.24 vs. 1.15+/-0.8). Interestingly, the gene expression of hyaluronidases was also significantly higher in Slit3-/- murine lungs compared to WT-controls, including Hyal1 (3.24+/-0.68 vs. 1.02+/-0.11, p<0.01) and Hyal2 (2.58+/-0.57 vs. 0.84+/-0.15, p<0.01). HA staining demonstrated significantly more and dysregulated HA in the perivasculature in the Slit3-/- murine lungs.
Conclusion:
Using a Slit3-/- murine model, we demonstrated altered HA metabolism in CDH lungs. We also showed an increase in HA expression in the perivasculature in the lungs of Slit3-/- mice. These data provide evidence for a possible role of HA metabolism in the development of PAH in CDH, and HA may represent a novel therapeutic target. Targeting the ECM of the pulmonary vasculature would represent a paradigm shift in treatment of PAH in CDH patients.
