S. Balaji1, M. Shah2, X. Wang1, M. Phillips2, M. Fahrenholtz1, C. M. Moles1, M. Rae1, S. G. Keswani1, S. E. Mclean2 1Texas Children’s Hospital And Baylor College Of Medicine,Division Of Pediatric Surgery,Houston, TX, USA 2University Of North Carolina At Chapel Hill School Of Medicine,Division Of Pediatric Surgery,Chapel Hill, NC, USA
Introduction: The management of pulmonary arterial hypertension(PAH) associated with congenital diaphragmatic hernia(CDH) is challenging. 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. Slit3-/- mice demonstrate CDH at birth and develop PAH over time with associated alterations in hyaluronan(HA) metabolism in perivascular matrix in the lungs. Inhaled IL-10 decreases or reverses the development of PAH in Slit3-/- mice with CDH. The role of IL-10 in regulating HA in pulmonary artery smooth muscle cells(PASMC) and pulmonary fibroblasts(PFB) has not been examined.
Methods: Primary PASMC and PFB isolated from C57BL/6J wildtype mice were cultured under static and mechanical tension and were treated with murine IL-10(200ng/ml). To determine the effect of IL-10 on regulation of pulmonary perivascular HA in CDH, 2-3m old Slit3-/- mice were treated twice with IL-10 mixed with HA-hydrogel, administered intranasally 7d apart. Lungs (n=3-4/group) were harvested and embedded. RNA was isolated from cell cultures and frozen lung samples, and hyaluronan synthases(HAS1-3) and hyaluronidases(Hyal1-2) were measured (qPCR). HA expression and localization(HA-binding protein) and leucocyte(CD45) and macrophage(CD206) infiltration was determined histologically in paraffin sections. Data represented as mean+/-SD; p<0.05 denotes significance; t-test
Results: PASMC expressed increased IL-6(2.8 fold; p<0.05), HAS1(31.4 fold; p<0.005) and MMP9(15.5 fold; p<0.05), but expressed lower collagen 1 and 3(1.75 fold; p<0.05) and MMP2(3.3 fold; p=0.005) under mechanical stress, which may partly explain the dysregulation of the HA in the perivascular matrix in CDH lungs. IL-10 over expression did not alter the HAS1, 2 or 3 expression of the PASMC under normal culture conditions. In PFB, however, IL-10 treatment significantly increased HAS1(2.5 fold; p<0.05) and decreased Hyal1(1.5 fold; p<0.05), suggesting a differential regulation of HA metabolism by IL-10 in the key cell types responsible for perivascular matrix turnover. Slit3-/- mice that developed PAH demonstrated significantly more HA deposition in the perivasculature, but exhibited a less dense, dysregulated matrix structure. Inhaled IL-10 hydrogel treatment for 2wk resulted in a more cohesive and dense distribution of HA. IL-10 treatment also significantly reduced inflammatory response in Slit3-/- murine lungs: CD45 infiltration(3.2 fold decrease; p<0.05) and macrophage infiltration(1.5 fold decerase; p<0.05)
Conclusion: These data provide evidence for a possible role for IL-10 in the regulation of altered HA metabolism, along with its anti-inflammatory role in the attenuation of PAH in CDH. Targeting the ECM of the pulmonary vasculature would represent a paradigm shift in treatment of PAH in CDH patients and the possible development of novel therapeutics.