O2068: Integrin a3b1 Regulates Alveolar Basement Membrane Formation during Distal Lung Development through Epithelial Differentiation

Background: Epithelial-extracellular matrix (ECM) interactions are essential for lung development. Alveolar epithelial cells (AECs) connect to the lung ECM through integrins, transmembrane heterodimeric ab ECM receptors. In many branched organs, epithelial-laminin (LM) interactions have been shown to regulate early organogenesis, with integrin a3b1 as the dominant epithelial LM-binding integrin receptor, but the specific role of a3b1 integrin in lung development is unknown. LM is a principal component of the alveolar basement membrane (BM), a specialized ECM structure in the distal lung. During the development of the distal lung, AECs rapidly differentiate into cuboidal type 2 (AT2) and flat, matrix-producing type 1 (AT1) AECs, which are essential for gas exchange. Since a3b1 integrin regulates epithelial differentiation in other contexts, we hypothesize that integrin a3b1 is required for distal lung development through the regulation of epithelial differentiation.

Methods: We generated mice with epithelial-specific deletion of the a3 integrin subunit from embryonic day 9.5 (E9.5) using Shh-Cre, (a3Shh mice), thereby directly targeting a3b1 integrin. Lung morphometry was quantified by alveolar duct volume density using ImageJ. FlexiVent and mouse ox measurements assessed alveolar function. Immunostained lung sections assessed AEC differentiation and ECM localization. Quantitative PCR detected differences in BM proteins. Transmission electron microscopy visualized alveolar BM structure at postnatal day 28 (P28).

Results: By histological examination, P28 a3Shh mice exhibited impaired distal lung development, characterized by thickened, hypercellular alveolar septa and alveolar duct expansion. Immunostained lungs for AT2 and AT1 markers revealed impaired AEC differentiation in P28 a3Shh, where overabundant AT2 filled the septum, and there was paucity of AT1, an abnormal finding quantified by increased AT2 to AT1 ratio (3.8+/-0.3 in a3Shh versus 2.0+/-0.3 in a3f/f, p <.05). By immunostaining, increased collagen IV was mislocalized in the interstitial space in P28 a3Shh.Cre lungs, rather than co-localizing with laminin and BM proteins. AECs deficient in a3b1 produced abnormal laminin isoforms, with persistence of embryonic laminin-111 and failure to produce AT1-derived mature laminin-332 isoforms at P28, indicating failed developmental programming of laminin isoform switching due to loss of AT1. Since the acquisition of mature laminin isoforms is required for assembly of the similar fused glomerular BM, we examined alveolar BM structure in a3Shh and control lungs by electron microscopy. Epithelial and endothelial matrix components failed to fuse in a3Shh, generating a wide, fractured BM. Disruption in BM structure resulted in restrictive lung function, as measured by Flexivent pulmonary function testing (resistance 1.11 vs. 0.58 cmH2O/ml/s in a3Shh vs. control mice; compliance 0.02 vs. 0.04 ml/cmH2O in a3Shh vs. control, p <.05), and adult a3Shh mice were mildly hypoxemic compared to a3f/f by MouseOx measurement.

Conclusion: Epithelial a3b1 is required for distal lung development through regulation of AEC differentiation, which directs proper laminin isoforms switching essential for alveolar BM formation.