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. 2016 Jul;55(1):105-16.
doi: 10.1165/rcmb.2015-0152OC.

Lysophosphatidic Acid Signaling through the Lysophosphatidic Acid-1 Receptor Is Required for Alveolarization

Manuela Funke  1   2   3 , Lars Knudsen  4 , David Lagares  3 , Simone Ebener  1   2 , Clemens K Probst  3 , Benjamin A Fontaine  3 , Alicia Franklin  3 , Manuela Kellner  4 , Mark Kühnel  4 , Stephanie Matthieu  4 , Roman Grothausmann  4 , Jerold Chun  5 , Jesse D Roberts Jr  6 , Matthias Ochs  4 , Andrew M Tager  3
Affiliations

Lysophosphatidic Acid Signaling through the Lysophosphatidic Acid-1 Receptor Is Required for Alveolarization

Manuela Funke et al. Am J Respir Cell Mol Biol. 2016 Jul.

Abstract

Lysophosphatidic acid (LPA) signaling through one of its receptors, LPA1, contributes to both the development and the pathological remodeling after injury of many organs. Because we found previously that LPA-LPA1 signaling contributes to pulmonary fibrosis, here we investigated whether this pathway is also involved in lung development. Quantitative assessment of lung architecture of LPA1-deficient knock-out (KO) and wild-type (WT) mice at 3, 12, and 24 weeks of age using design-based stereology suggested the presence of an alveolarization defect in LPA1 KO mice at 3 weeks, which persisted as alveolar numbers increased in WT mice into adulthood. Across the ages examined, the lungs of LPA1 KO mice exhibited decreased alveolar numbers, septal tissue volumes, and surface areas, and increased volumes of the distal airspaces. Elastic fibers, critical to the development of alveolar septa, appeared less organized and condensed and more discontinuous in KO alveoli starting at P4. Tropoelastin messenger RNA expression was decreased in KO lungs, whereas expression of matrix metalloproteinases degrading elastic fibers was either decreased or unchanged. These results are consistent with the abnormal lung phenotype of LPA1 KO mice, being attributable to reduced alveolar septal formation during development, rather than to increased septal destruction as occurs in the emphysema of chronic obstructive pulmonary disease. Peripheral septal fibroblasts and myofibroblasts, which direct septation in late alveolarization, demonstrated reduced production of tropoelastin and matrix metalloproteinases, and diminished LPA-induced migration, when isolated from LPA1 KO mice. Taken together, our data suggest that LPA-LPA1 signaling is critically required for septation during alveolarization.

Keywords: LPA1; alveolarization; elastin; lung development; matrix metalloproteinase.

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Figures

Figure 1.
Figure 1.
Airspace enlargement in lysophosphatidic acid receptor 1 (LPA1) knock-out (KO) mice. LPA1 KO mice exhibit enlarged and simplified distal airspaces throughout the alveolar stage of lung development. Representative toluidine blue–stained sections of fixed wild type (WT) and LPA1 KO mouse lungs (A and B) at 4 days and (C and D) at 3 weeks, (E and F) 12 weeks, and (G and H) 24 weeks of age. Original magnification  × 20. Scale bars represent 50 μm (A and B) or 100 μm (C–H).
Figure 2.
Figure 2.
Quantitative assessment of airspace enlargement in LPA1 KO mice by design-based stereology. (A) Total V(sep,lung) (cm3) was significantly reduced in LPA1 KO compared with WT mice at 12 weeks (*P = 0.02) and 24 weeks of age (†P = 0.009). (B) Vν (alv) (μm3) was significantly increased in LPA1 KO compared with WT mice at 3 weeks (*P = 0.02), 12 weeks (*P = 0.02), and 24 weeks of age (†P = 0.03). (C) Total N(alv,lung) were significantly decreased in LPA1 KO compared with WT mice at 3, 12, and 24 weeks of age (*P < 0.001). Total numbers of alveoli in WT mice at 12 and 24 weeks were greater than in WT mice at 3 weeks of age by 38% and 44%, respectively (†P = 0.00017 and ‡P = 0.00044). (D) Total S(alvepi,lung) (cm2) was significantly decreased in LPA1 KO compared with WT mice at 3 (*P = 0.01), 12 (†P < 0.001) and 24 (†P < 0.001) weeks of age. (E) Correlation between NV(alv,par) (1/cm3) and total Sv(alvepi/par) in WT mice, considering mice assessed at 3, 12, and 24 weeks together. (F) Total V(lung) (cm3) were significantly different between LPA1 KO and WT mice only at 12 weeks of age, when they were lower in LPA1 KO mice. *P < 0.001. For all comparisons, the following numbers of mice were examined: for mice at 3 weeks of age, seven WT and seven LPA1 KO mice were examined; for mice at 12 weeks of age, five WT and five LPA1 KO mice were examined; and for mice at 24 weeks of age, eight WT and seven LPA1 KO mice were examined. In A, B, D, and F, data are presented as mean values ± standard deviation. In C, data are presented as individual animal values with means indicated by horizontal lines. N(alv,lung), numbers of alveoli per lung; NV(alv,par), density of alveolar number within lung parenchyma; S(alvepi,lung), alveolar surface per lung; Sv(alvepi/par), density of alveolar surface area within lung parenchyma; V(sep,lung), septal tissue volume per lung; Vν(alv), volume-weighted mean volume of alveoli.
Figure 3.
Figure 3.
Early-phase alveolarization is completed by 3 weeks of age in WT and LPA1 KO mice. Representative electron microscopy images of (A) LPA1 KO and (B) WT mice indicate that mature single-layer capillary networks (arrowheads) are present in both genotypes. Four lungs per group. Original magnification × 2,200. Scale bar represents 10 μm. AE2, type 2 alveolar epithelial cells (arrows).
Figure 4.
Figure 4.
Branching morphogenesis is similar in WT and LPA1 KO mice. Three-dimensional reconstructions of airways from scanning laser optical tomography of (A) WT and (B) LPA1 KO mice at 3 weeks of age show similar branching morphology. Four lungs per group.
Figure 5.
Figure 5.
Disorganized and reduced septal elastic fibers in the lungs of LPA1 KO mice. Hart’s resorcin-fuchsin staining of lung sections without (A–H) and with (I–L) prior tissue oxidation, and tartrazine counterstaining. Original magnification × 20; scale bars are 100 μm. Comparison of stained nonoxidized sections of WT (A, inset C) and LPA1 KO (B, inset D) mouse pup lungs at P4 demonstrates less organized, less condensed elastic fibers in the alveolar septa of the KO animals. Nonoxidized lung sections from 3-week-old WT (E, inset G) and LPA1 KO (F, inset H) mice also demonstrate less organized, less condensed elastic fibers in the KOs, with areas in KO lungs in which the elastic fibers appear not to extend from the alveolar walls into the tips of the septa. Comparison of stained sections from 3-week-old WT (I, inset K) and LPA1 KO (J, inset L) mice that were oxidized before staining to visualize all components of the elastic fiber system demonstrate overall decreased elastic fibers in the KO animals.
Figure 6.
Figure 6.
Reduced elastin and matrix metalloproteinase (MMP) production in the lungs of LPA1 KO mice. (A) Tropoelastin mRNA expression was significantly reduced in total lung homogenates from LPA1 KO compared with WT mice at 3 weeks of age (*P < 0.03); (B) MMP-2 and (C) MMP-7 mRNA expression was not significantly different between LPA1 KO and WT mice at 3 weeks of age; (D) MMP-9 and (E) MMP-12 mRNA expression was significantly reduced in total lung homogenates from LPA1 KO mice (*P < 0.03 and †P = 0.04); and (F) TIMP-3 mRNA expression was not significantly different. Data in A to F are presented as the mean expression of the gene of interest relative to GAPDH, ± SEM and have been combined from three independent experiments with three to six WT and three to four LPA1 KO mice/experiment. (G) Western blotting suggests that levels of α-elastin protein are reduced in the lungs of LPA1 KO compared with WT mice at 3 weeks of age. Differences in MMP-9 and MMP-12 protein levels were less apparent, but their expression was not increased in the KO animals. GAPDH, glyceraldehyde phosphate dehydrogenase; TIMP, tissue inhibitor of metalloproteinase.
Figure 7.
Figure 7.
Impaired tropoelastin production and migration of LPA1–deficient peripheral septal fibroblasts (PSFBs). (A) PSFBs isolated from LPA1 KO mice at 3 weeks of age demonstrated significantly reduced tropoelastin expression compared with PSFBs isolated from WT mice of the same age (*P = 0.02). Collagen and fibronectin (FN) expression were not significantly different between LPA1 KO and WT PSFBs. Data are presented as mean expression of the gene of interest relative to GAPDH, ± SEM. (B) PSFBs isolated from LPA1 KO mice at 3 weeks of age demonstrated significantly reduced migration induced by LPA (10 μM) compared with PSFBs isolated from WT mice of the same age (*P = 0.00006) but had similar migration induced by platelet-derived growth factor (PDGF)-BB (10 ng/ml). Data are presented as mean fold-increase of migration induced by the chemoattractant compared with migration induced by media alone, ± SEM. (C) PSFBs isolated from LPA1 KO mice at 3 weeks of age demonstrated significantly reduced MMP-2 (*P = 0.02), MMP-7 (*P = 0.02), and MMP-9 (†P = 0.0002) expression and significantly increased TIMP-3 expression (‡P = 0.00025) compared with PSFBs isolated from WT mice of the same age. Data are presented as mean expression of the gene of interest relative to GAPDH, ± SEM.
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