Alveolar septation increases gas-exchange surface area and requires coordinated cytoskeletal rearrangement in lung fibroblasts (LFs) to balance the demands of contraction and cell migration. We hypothesized that DBN (drebrin), a modulator of the actin cytoskeleton in neuronal dendrites, regulates the remodeling of the LF cytoskeleton. Using mice bearing a transgelin-Cre-targeted deletion of Dbn in pulmonary fibroblasts and pericytes, we examined alterations in alveolar septal outgrowth, LF spreading and migration, and actomyosin function. The alveolar surface area and number of alveoli were reduced, whereas alveolar ducts were enlarged, in mice bearing the dbn deletion (DBNΔ) compared with their littermates bearing only one dbn-Flox allele (control). Cultured DBNΔ LFs were deficient in their responses to substrate rigidity and migrated more slowly. Drebrin was abundant in the actin cortex and lamella, and the actin fiber orientation was less uniform in lamella of DBNΔ LFs, which limited the development of traction forces and altered focal adhesion dynamics. Actin fiber orientation is regulated by contractile NM2 (nonmuscle myosin-2) motors, which help arrange actin stress fibers into thick ventral actin stress fibers. Using fluorescence anisotropy, we observed regional intracellular differences in myosin regulatory light chain phosphorylation in control LFs that were altered by dbn deletion. Using perturbations to induce and then release stalling of NM2 on actin in LFs from both genotypes, we made predictions explaining how DBN interacts with actin and NM2. These studies provide new insight for diseases such as emphysema and pulmonary fibrosis, in which fibroblasts inappropriately respond to mechanical cues in their environment.

The extracellular matrix (ECM) of the pulmonary parenchyma must maintain the structural relationships among resident cells during the constant distortion imposed by respiration. This dictates that both the ECM and cells adapt to changes in shape, while retaining their attachment. Membrane-associated integrins and discoidin domain receptors (DDR) bind collagen and transmit signals to the cellular cytoskeleton. Although the contributions of DDR2 to collagen deposition and remodeling during osseous development are evident, it is unclear how DDR2 contributes to lung development. Using mice ( smallie, Slie/Slie, DDR2Δ) bearing a spontaneous inactivating deletion within the DDR2 coding region, we observed a decrease in gas-exchange surface area and enlargement of alveolar ducts. Compared with fibroblasts isolated from littermate controls, DDR2Δ fibroblasts, spread more slowly, developed fewer lamellipodia, and were less responsive to the rigidity of neighboring collagen fibers. Activated β1-integrin (CD29) was reduced in focal adhesions (FA) of DDR2Δ fibroblasts, less phospho-zyxin localized to and fewer FA developed over ventral actin stress fibers, and the adhesions had a lower aspect ratio compared with controls. However, DDR2 deletion did not reduce cellular displacement of the ECM. Our findings indicate that DDR2, in concert with collagen-binding β1-integrins, regulates the timing and location of focal adhesion formation and how lung fibroblasts respond to ECM rigidity. Reduced rigidity sensing and mechano-responsiveness may contribute to the distortion of alveolar ducts, where the fiber cable-network is enriched and tensile forces are concentrated. Strategies targeting DDR2 could help guide fibroblasts to locations where tensile forces organize parenchymal repair.

Platelet-derived growth factor receptor-α (PDGFRα) is absolutely required for the development of secondary pulmonary alveolar septa. Our earlier observations indicated that PDGFRα resides intracellularly as well as on the plasma membrane of murine lung fibroblasts (LF). We have examined how neuropilin-1 (Nrp1), a surface receptor without kinase activity, regulates the intracellular trafficking of PDGFRα in LF obtained from mice, some bearing a targeted deletion of Nrp1 in myofibroblasts. Using the proximity ligation assay, we observed that PDGFRα and Nrp1 colocalized in both early antigen-1 (EEA1) containing sorting endosomes and with adaptor protein containing a pleckstrin homology domain and a phosphotyrosine-binding domain-1 (APPL1) in very early endosomes (VEE). These findings were confirmed using live-cell imaging, which demonstrated that recently internalized PDGFRα was observed in Rab5-containing vesicles residing within 100 nm of the plasma membrane. Nrp1 deletion reduced the phosphorylation of Akt (protein kinase B), the major downstream target of PDGFRα, and limited accumulation of inositol-3 phosphates in APPL1-containing endosomes after exposure to PDGFA. PDGFRα co-immunoprecipitated with APPL1, indicating that PDGFRα enters VEE. Targeted deletion of Nrp1 or APPL1-depletion in control LF reduced the activity of an Akt1 biosensor following stimulation with PDGFA. Our findings demonstrate that Nrp1 enhances the entry of PDGFRα into APPL1 containing VEE and that APPL1 enhances PDGFRα signaling. Therefore, Nrp1 promotes endosomal signaling by PDGFRα offering a potential mechanism to explain our prior observation that Nrp1 supports the formation of alveolar ducts and alveoli during secondary septation in mice.

During pulmonary secondary alveolar septation, the rudimentary distal saccule subdivides by extending tissue sheets into the saccular air space, creating alveoli, which open into the alveolar duct. The sheets originate from saccular mesenchymal cells, which contain α-SMA (αSMA [ACTA2]) and abut elastic fibers (myofibroblasts [MF]), characteristics that are shared by cells that subsequently occupy the secondary septal tips. During elongation, collagen fibers are positioned to provide a scaffold for translocating septal mesenchymal cells. We hypothesized that collagen fibers direct the migration, orientation, and location of MFs during septal elongation. To address this hypothesis, we examined how electrospun collagen fibers direct the migration of fibroblasts bearing targeted deletions of PDGFRα (platelet-derived growth factor receptor-α) or Nrp1 (neuropilin-1), after their isolation from lungs that exhibit reduced secondary septation. We observed that deletion of either gene reduced Rac1 activation and the speed of migration of lung fibroblasts (LF) along electrospun fibers. The deletions did not reduce the proportion of LF that displayed collagen-binding integrins and increased the proportion of LF bearing activated β1-integrin. LF bearing the PDGFRα deletion failed to localize focal adhesions over electrospun fibers, suggesting that they may not appropriately sense and respond to regionally increased stiffness near the fibers. In lungs of mice bearing the PDGFRα deletion, collagen fibers are delocalized from ACTA2-containing MF, and their orientation deviated from the plane of the alveolar walls. Diminished PDGFRα or Nrp1 reduces LF localization to stiffer regions of fibrillar collagen substrates, suggesting that signaling through these receptors enables responsiveness to regional differences in extracellular matrix rigidity.

Editorial FocusThe lipofibroblast: more than a lipid-storage depotStephen E. McGowanStephen E. McGowanDepartment of Veterans Affairs Research Service and Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, IowaPublished Online:01 May 2019https://doi.org/10.1152/ajplung.00109.2019This is the final version - click for previous versionMoreSectionsPDF (101 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Lipid-laden cells (initially named lipid-interstitial cells, LIC, and later termed lipofibrobasts, LiF) were first observed in the pulmonary alveolar interstitium of rats and mice during the saccular and alveolar stages of development (7, 10). Their location and neutral rather than phospholipid storage granules distinguished them from alveolar type 2 (AT2) epithelial cells and macrophages, and their proliferation kinetics distinguished them from interstitial cells that lacked lipid granules (3). The LiF population peaked during the first two postnatal weeks, when plasma chylomicrons and very-low-density lipoproteins were elevated (3, 15). When cultured, the lipid granules dissipated and the cells contained ACTA2 and produced elastin, but lipid storage could be enhanced by exposure to neonatal rat serum or peroxisome proliferator-activated receptor agonists (14, 17). When fibroblasts, which had been isolated in the saccular stage and loaded with [3H]triolein, were cocultured with AT2 cells, the triolein was observed in surfactant lipoproteins, leading to the hypothesis that LiF provide surfactant lipids when alveoli rapidly increase in number (26). Comparable to hepatic stellate cells, LiF store retinyl esters, an endogenous precursor for retinoic acid, which supports elastin synthesis and alveolar formation (16). However, an understanding of the function of LiFs was limited by the transient presence of lipid granules and because the cells lost the granules when cultured.With the development of more sensitive tools to analyze gene expression and proteins in small populations or individual cells, the unique phenotype of LiFs has been defined at a molecular level during alveolarization and during compensatory lung growth after pneumonectomy (1, 6). Signaling by PDGF-A through platelet-derived growth factor receptor-α (PDGFRα) is required for alveolarization, and LiF express PDGFRα, but their lineage is not directly defined by PDGF-A signaling (25). Lineage-tracing established that sonic hedgehog signaling differentiates fibroblast progenitors to a myofibroblast (MF), α-smooth muscle actin (αSMA)-containing phenotype before postnatal day 2 (P2) when Lif progenitors begin to express the differentiated adipocyte protein perilipin 2 (adipose differentiation-related protein, ADRP), the protein that encapsulates neutral lipid droplets (11, 12, 21). Myofibroblast differentiation depends on inactivation of a Wnt inhibitory pathway and on sustained sonic hedgehog signaling. Lipofibroblasts are most abundant from P4 to P8 and exhibit other markers of white adipocyte-like differentiation [transcription factor-21 (Tcf21)] and G0/G1 switch protein-2 (GOS2) (18). Using transgelin (tagln)-Cre to delete PDGFRα, increased adipocyte progenitor markers Tcf21, peroxisome proliferator activated receptor-γ (PPARγ), and delta-like ligand-1 (Dlk1-C) and reduced the proportion of fibroblasts that contained α-smooth muscle actin, ACTA2 (αSMA) (18). Thus, whereas PDGFRα gene expression does not drive the lineage distinction between LiF and MF, it influences the characteristics of an incompletely committed progenitor population (Fig. 1).Fig. 1.Divergence and differentiation of alveolar fibroblast subtypes. Lung fibroblast progenitors express platelet-derived growth factor receptor-α (PDGFRα), and PDGFRα and its ligand PDGF-A are required for secondary alveolar septation. These progenitors differentiate along divergent pathways towards lipofibroblasts (LiF) or myofibroblasts (MF). (┤), suppresses; (→), activates. ACTA2, α-smooth muscle actin (αSMA); CDCP1, Cub-domain containing protein-1; Dlk1, delta-like noncanonical Notch ligand-1; FGF10, fibroblast growth factor 10; Fra-2, Fos-related antigen-2; Gli1, glioma-associated oncogene homolog 1; PPARγ, peroxisome proliferator-activated receptor-γ; Smad, mammalian homolog of Drosophila mothers against decapentaplegic; Tcf21, transcription factor-21; TGF-β, transforming growth factor- β. Italicized text or dashed lines are effects involving TCF21 in the heart, whereas no data are available for the lung.Download figureDownload PowerPointPark and associates (22) studied lung cell populations that lineage-labeled for Tcf21, which is a specific marker of white adipose tissue and defines progenitor populations in the heart and kidney (2, 8). Lineage labeling at embryonic day (E)11.5, shortly after outpouching of the laryngeo-tracheal groove, which heralds airway branching, identified both peribronchial and vascular smooth muscle cells. Labeling during the canalicular stage (E14.5 or E15.5) identified cells that at E18.5 (the early saccular stage) were also observed more diffusely in the distal parenchyma. Using 10 μm sections for immunochemistry they observed that 20–30% of Tcf21 lineage-labeled cells also stained for PDGFRα, and that colocalization occurred in a majority of cells when adults were lineage labeled. Although, their approach was less precise than others using three-dimensional reconstruction and stereology, they showed that PDGFRα+ cells that were Tcf21+ also contained neutral lipid droplets. An important and unique observation by Park and associates is that the Tcf21 is expressed in the adult lung, in PDGFRα-expressing cells that contain perilipin 2. Prior studies using electron microscopy, histochemical staining for lipids, or fluorescent markers with a lower quantum yield did not identify lipid droplets in adults. These earlier studies raised doubts about the existence of potential relevance of LiF to lung disease in adults. Using adult mouse lungs, Park and associates also observed that lineage-labeled perilipin-2 positive cells colocalized with Col1a1 (collagen I), expressed fibroblast growth factor-10 (FGF10) and the membrane-tethered Col13a1 (collagen XIII). Using adenovirus to exogenously express Tcf21 in primary lung fibroblasts, which were isolated at P7 from wild-type or Tcf21 lineage-labeled mice, they observed more intense lipid-droplet staining in individual cells, although viral transduction of Tcf21 did not increase the number of lipid-droplet containing cells. Because only some of the lung fibroblasts would endogenously express Tcf21, and the cells were not cultured under conditions which enhance droplet accumulation, this indicates that Tcf21 enables fibroblasts to assume a lipid storage phenotype. This suggests that manipulating Tcf21 expression alters lipid acquisition by lung fibroblasts that may not have expressed Tcf21 in the embryonic lung.Others have studied the adipocytic properties of Tcf21-expresssing cardiac fibroblast precursors (CFP) of which many arise from the epicardium (23). The use of Tcf21CreER to conditionally delete prokineticin receptor-1 (PKR1), and unmask a dTomato Tcf21 lineage label in adult mice, enabled CFP cells to accumulate more lipid, which deposited near coronary arteries. Signaling through PKR1 dampened adipogenic signals and promoted development of a perivascular, αSMA-containing dTomato+ population. After consuming a high-fat diet, Tcf21-traced CFP-accumulated lipid droplets, and the diet augmented accumulation when PKR1 had been deleted. There were obverse effects on αSMA and platelet and endothelial adhesion molecule-1 (PECAM1), and traced cells, which on the normal diet only contained αSMA, now also contained perilipin. These findings suggest that Tcf21 sustains fibroblast progenitors in a less-differentiated state. This is supported by a subsequent observation that the PKR1-ligand, prokineticin-2, promotes asymmetric cell division, whereby one daughter cell retains plasticity, a characteristic of progenitor cells (24). A similar hypothesis arose from studies addressing interactions between Tcf21 and transforming growth factor-β (TGF-β).The TGF-β signaling intermediate SMAD3, which drives the fibroblast to myofibroblast transdifferentiation, is antagonized by Tcf21 in human coronary artery smooth muscle cells (9). Binding of Tcf21 to its cognate DNA binding element (which can be shared by SMAD3) antagonizes SMAD3-mediated differentiation of smooth muscle cells (SMCs), which pathologically increases in atherosclerotic coronary disease. The authors hypothesized that Tcf21 retains SMCs in a dedifferentiated progenitor state by antagonizing SMAD3, which, if unchecked, leads to differentiation towards an αSMA- and Col1a1-expressing atherogenic phenotype. Myofibroblast differentiation and function are also regulated by molecular contacts between the cell surface and extracellular matrix (20, 27). Administering dexamethasone to newborn mice before isolating lung fibroblasts at P8 (close to P7, when Park and associates showed that Tcf21 is most abundantly expressed) enlarges the population of fibroblasts that contain lipid droplets, αSMA, and bare markers of preadipocytes [more Dlk1 and less sterol regulatory element-binding protein 1 (SREBP1)] and more phosphorylated SMAD1/5 (19). It would have been enlightening if Park and coworkers had assessed αSMA as well as lipid accumulation in cultured lung fibroblasts after introducing the Ad5-Tcf21 construct. In the future, lineage tracing could be used to study potential antagonism between Tcf21 and SMAD2/3 signaling and how this is altered by glucocorticoids. This may help develop strategies to allay transdifferentiation into the myofibroblasts and ameliorate lung fibrosis. Others have shown that LiFs transdifferentiate to myofibroblasts during the development and revert to LiFs during the resolution of bleomycin-induced pulmonary fibrosis in mice (5).If Tcf21 is required to maintain a balance between myofibroblastic and lipid-storage characteristics, then, like myofibroblast profusion, excessive lipofibroblasts may be detrimental. This occurs in a rare neonatal interstitial lung disease, pulmonary interstitial glycogenosis (PIG) (13). These infants develop hypoxemia and pulmonary hypertension because alveolar walls are fewer and thickened. The thickened walls are overpopulated by glycogen-rich, vimentin-staining mesenchymal cells, which also contain abundant neutral lipid droplets (4). The first characterization of lipid-laden mesenchymal cells in neonatal rats demonstrated that LiFs also contained abundant glycogen at and immediately after birth (7). This not only confirms that LiFs are found in humans, but that they are deregulated in disease.Nearly 50 years after they were first recognized morphologically, LiFs are being recognized as more than epiphenomena or purveyors of surfactant lipids. The study by Park and coworkers (22) is casting a signature role for LiFs as a pivotal alveolar fibroblast subtype that may ultimately lead to new approaches to lung repair and regeneration.DISCLOSURESNo conflicts of interest, financial or otherwise, are declared by the author.AUTHOR CONTRIBUTIONSS.E.M. drafted manuscript; edited and revised manuscript; approved final version of manuscript.REFERENCES1. Ardini-Poleske ME, Clark RF, Ansong C, Carson JP, Corley RA, Deutsch GH, Hagood JS, Kaminski N, Mariani TJ, Potter SS, Pryhuber GS, Warburton D, Whitsett JA, Palmer SM, Ambalavanan N; LungMAP Consortium. LungMAP: The Molecular Atlas of Lung Development Program. Am J Physiol Lung Cell Mol Physiol 313: L733–L740, 2017. doi:10.1152/ajplung.00139.2017. Link | ISI | Google Scholar2. Braitsch CM, Kanisicak O, van Berlo JH, Molkentin JD, Yutzey KE. Differential expression of embryonic epicardial progenitor markers and localization of cardiac fibrosis in adult ischemic injury and hypertensive heart disease. J Mol Cell Cardiol 65: 108–119, 2013. doi:10.1016/j.yjmcc.2013.10.005. Crossref | PubMed | ISI | Google Scholar3. Brody JS, Kaplan NB. Proliferation of alveolar interstitial cells during postnatal lung growth. Evidence for two distinct populations of pulmonary fibroblasts. Am Rev Respir Dis 127: 763–770, 1983. PubMed | ISI | Google Scholar4. Deutsch GH, Young LR. Lipofibroblast Phenotype in pulmonary interstitial glycogenosis. Am J Respir Crit Care Med 193: 694–696, 2016. doi:10.1164/rccm.201509-1809LE. Crossref | PubMed | ISI | Google Scholar5. El Agha E, Moiseenko A, Kheirollahi V, De Langhe S, Crnkovic S, Kwapiszewska G, Szibor M, Kosanovic D, Schwind F, Schermuly RT, Henneke I, MacKenzie BA, Quantius J, Herold S, Ntokou A, Ahlbrecht K, Braun T, Morty RE, Günther A, Seeger W, Bellusci S. Two-way conversion between lipogenic and myogenic fibroblastic phenotypes marks the progression and resolution of lung fibrosis. Cell Stem Cell 20: 1–13, 2017. doi:10.1016/j.stem.2017.03.011.Crossref | PubMed | ISI | Google Scholar6. Green J, Endale M, Auer H, Perl AK. Diversity of interstitial lung fibroblasts is regulated by platelet-derived growth factor receptor α kinase activity. Am J Respir Cell Mol Biol 54: 532–545, 2016. doi:10.1165/rcmb.2015-0095OC. Crossref | PubMed | ISI | Google Scholar7. O'Hare KH, Sheridan MN. Electron microscopic observations on the morphogenesis of the albino rat lung, with special reference to pulmonary epithelial cells. Am J Anat 127: 181–205, 1970. doi:10.1002/aja.1001270205. Crossref | PubMed | Google Scholar8. Ide S, Finer G, Maezawa Y, Onay T, Souma T, Scott R, Ide K, Akimoto Y, Li C, Ye M, Zhao X, Baba Y, Minamizuka T, Jin J, Takemoto M, Yokote K, Quaggin SE. Transcription factor 21 is required for branching morphogenesis and regulates the Gdnf-axis in kidney development. J Am Soc Nephrol 29: 2795–2808, 2018. doi:10.1681/ASN.2017121278. Crossref | PubMed | ISI | Google Scholar9. Iyer D, Zhao Q, Wirka R, Naravane A, Nguyen T, Liu B, Nagao M, Cheng P, Miller CL, Kim JB, Pjanic M, Quertermous T. Coronary artery disease genes SMAD3 and TCF21 promote opposing interactive genetic programs that regulate smooth muscle cell differentiation and disease risk. PLoS Genet 14: e1007681, 2018. doi:10.1371/journal.pgen.1007681. Crossref | PubMed | ISI | Google Scholar10. Kaplan NB, Grant MM, Brody JS. The lipid interstitial cell of the pulmonary alveolus. Age and species differences. Am Rev Respir Dis 132: 1307–1312, 1985. PubMed | ISI | Google Scholar11. Kugler MC, Loomis CA, Zhao Z, Cushman JC, Liu L Munger JS. Sonic hedgehog signaling regulates myofibroblast function during alveolar septum formation in murine postnatal lung. Am J Respir Cell Mol Biol 57: 280–293, 2017. doi:10.1165/rcmb.2016-0268OC. Crossref | PubMed | ISI | Google Scholar12. Li C, Li M, Li S, Xing Y, Yang CY, Li A, Borok Z, De Langhe S, Minoo P. Progenitors of secondary crest myofibroblasts are developmentally committed in early lung mesoderm. Stem Cells 33: 999–1012, 2015. doi:10.1002/stem.1911. Crossref | PubMed | ISI | Google Scholar13. Liptzin DR, Baker CD, Darst JR, Weinman JP, Dishop MK, Galambos C, Brinton JT, Deterding RR. Pulmonary interstitial glycogenosis: diagnostic evaluation and clinical course. Pediatr Pulmonol 53: 1651–1658, 2018. doi:10.1002/ppul.24123. Crossref | PubMed | ISI | Google Scholar14. Maksvytis HJ, Niles RM, Simanovsky L, Minassian IA, Richardson LL, Hamosh M, Hamosh P, Brody JS. In vitro characteristics of the lipid-filled interstitial cell associated with postnatal lung growth: evidence for fibroblast heterogeneity. J Cell Physiol 118: 113–123, 1984. doi:10.1002/jcp.1041180203. Crossref | PubMed | ISI | Google Scholar15. McGowan SE, Doro MM, Jackson S. Expression of lipoprotein receptor and apolipoprotein E genes by perinatal rat lipid-laden pulmonary fibroblasts. Exp Lung Res 27: 47–63, 2001. doi:10.1080/019021401459761. Crossref | PubMed | ISI | Google Scholar16. McGowan SE, Doro MM, Jackson SK. Endogenous retinoids increase perinatal elastin gene expression in rat lung fibroblasts and fetal explants. Am J Physiol Lung Mol Cell Physiol 273: L410–L416, 1997. doi:10.1152/ajplung.1997.273.2.L410. Link | ISI | Google Scholar17. McGowan SE, Jackson SK, Doro MM, Olson PJ. Peroxisome proliferators alter lipid acquisition and elastin gene expression in neonatal rat lung fibroblasts Am J Physiol Lung Mol Cell Physiol 273: L1249–L1257, 1997. doi:10.1152/ajplung.1997.273.6.L1249. Link | ISI | Google Scholar18. McGowan SE, McCoy DM. Regulation of fibroblast lipid storage and myofibroblast phenotypes during alveolar septation in mice. Am J Physiol Lung Cell Mol Physiol 307: L618–L631, 2014. doi:10.1152/ajplung.00144.2014. Link | ISI | Google Scholar19. McGowan SE, McCoy DM. Glucocorticoids retain bipotent fibroblast progenitors during alveolar septation in mice. Am J Respir Cell Mol Biol 57: 111–120, 2017. doi:10.1165/rcmb.2016-0376OC. Crossref | PubMed | ISI | Google Scholar20. Noskovičová N, Heinzelmann K, Burgstaller G, Behr J, Eickelberg O. Cub domain-containing protein 1 negatively regulates TGF-β signaling and myofibroblast differentiation. Am J Physiol Lung Cell Mol Physiol 314: L695–L707, 2018. doi:10.1152/ajplung.00205.2017. Link | ISI | Google Scholar21. Ntokou A, Klein F, Dontireddy D, Becker S, Bellusci S, Richardson WD, Szibor M, Braun T, Morty RE, Seeger W, Voswinckel R, Ahlbrecht K. Characterization of the platelet-derived growth factor receptor-α-positive cell lineage during murine late lung development. Am J Physiol Lung Cell Mol Physiol 309: L942–L958, 2015. doi:10.1152/ajplung.00272.2014. Link | ISI | Google Scholar22. Park J, Ivey MJ, Deana Y, Riggsbee KL, Sörensen E, Schwabl V, Sjöberg C, Hjertberg T, Park GY, Swonger JM, Rosengreen T, Morty RE, Ahlbrecht K, Tallquist MD. The Tcf21 lineage constitutes the lung lipofibroblast population. Am J Physiol Lung Cell Mol Physiol. In press. 10.1152/ajplung.00254.2018. Link | ISI | Google Scholar23. Qureshi R, Kindo M, Arora H, Boulberdaa M, Steenman M, Nebigil CG. Prokineticin receptor-1-dependent paracrine and autocrine pathways control cardiac tcf21+ fibroblast progenitor cell transformation into adipocytes and vascular cells. Sci Rep 7: 12804, 2017. doi:10.1038/s41598-017-13198-2. Crossref | PubMed | ISI | Google Scholar24. Qureshi R, Kindo M, Boulberdaa M, von Hunolstein JJ, Steenman M, Nebigil CG. A prokineticin-driven epigenetic switch regulates human epicardial cell stemness and fate. Stem Cells 36: 1589–1602, 2018. doi:10.1002/stem.2866. Crossref | PubMed | ISI | Google Scholar25. Surate Solaligue DE, Rodríguez-Castillo JA, Ahlbrecht K, Morty RE. Recent advances in our understanding of the mechanisms of late lung development and bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol 313: L1101–L1153, 2017. doi:10.1152/ajplung.00343.2017. Link | ISI | Google Scholar26. Torday J, Hua J, Slavin R. Metabolism and fate of neutral lipids of fetal lung fibroblast origin. Biochim Biophys Acta 1254: 198–206, 1995. doi:10.1016/0005-2760(94)00184-Z. Crossref | PubMed | ISI | Google Scholar27. Tsujino K, Li JT, Tsukui T, Ren X, Bakiri L, Wagner E, Sheppard D. Fra-2 negatively regulates postnatal alveolar septation by modulating myofibroblast function. Am J Physiol Lung Cell Mol Physiol 313: L878–L888, 2017. doi:10.1152/ajplung.00062.2017. Link | ISI | Google ScholarAUTHOR NOTESAddress for reprint requests and other correspondence: S. E. McGowan, Division of Pulmonary, Critical Care, and Occupational Medicine, C33B GH, Department of Internal Medicine, University of Iowa Hospitals and Clinics, 200 Hawkins Dr., Iowa City, IA 52242 (e-mail: [email protected]edu). Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation Related ArticlesThe Tcf21 lineage constitutes the lung lipofibroblast population 01 May 2019American Journal of Physiology-Lung Cellular and Molecular PhysiologyCited ByRetinoids stored locally in the lung are required to attenuate the severity of acute lung injury in male mice15 February 2023 | Nature Communications, Vol. 14, No. 1The molecular consequences of androgen activity in the human breastCell Genomics, Vol. 3, No. 3Comparison of the Behavior of Perivascular Cells (Pericytes and CD34+ Stromal Cell/Telocytes) in Sprouting and Intussusceptive Angiogenesis12 August 2022 | International Journal of Molecular Sciences, Vol. 23, No. 16Adipose tissue and adipose secretome in systemic sclerosis15 September 2021 | Current Opinion in Rheumatology, Vol. 33, No. 6Origin and functional heterogeneity of fibroblasts10 February 2020 | The FASEB Journal, Vol. 34, No. 3 More from this issue > Volume 316Issue 5May 2019Pages L869-L871 https://doi.org/10.1152/ajplung.00109.2019PubMed30840482History Received 5 March 2019 Accepted 5 March 2019 Published online 1 May 2019 Published in print 1 May 2019 Metrics

Generation of secondary alveolar septa occurs primarily after birth in humans and is complete in mice postnatally, when mechanical stresses vary as air space pressure oscillates. Alveolar mesenchymal cells deposit elastic fibers, which limit cell strain; although when the elastic fiber network is incomplete, this function is also served by the intracellular cytoskeleton. Intermediate filament proteins support deformation during cell division and migration, which occur during septal elongation. Because platelet-derived growth factor receptor-α (PDGFRα) signaling is essential for alveolar septation, we hypothesized that neuropilin-1 (NRP1) may link PDGFRα to cytoskeletal deformation. During cell migration, NRP1 links receptor tyrosine kinase signaling to cytoskeletal and focal adhesion remodeling. Therefore, we examined the consequences of nrp1 gene deletion in alveolar mesenchymal cells (myofibroblasts and pericytes). NRP1 depletion reduced the proportion of mesenchymal cells that contain nestin and desmin within the subpopulation that lacked PDGFRα but contained PDGFRβ. Desmin was reduced at alveolar entry rings, air spaces were enlarged, and surface area was reduced after NRP1 depletion. PDGFRα and NRP1 colocalized to membrane lipid rafts, which are known to contain Src kinase. NRP1 depletion reduced alveolar mesenchymal cell migration and PDGF-A-mediated activation of Src kinase, which may limit accumulation of desmin at septal tips (alveolar entry rings). Cooperation between NRP1 and PDGF signaling is required for secondary septation, and manipulation of NRP1 could promote alveolar regeneration without producing fibrosis.

AIMA virus- and oncogene-free induced pluripotent stem cell (iPSC) reprogramming method was developed with cord blood-derived mononuclear cells (CBDMNC) and peripheral blood mononuclear cells (PBMNC) from patients with genetic lung diseases.METHODiPSC reprogramming used small molecules, hematopoietic stem cell (HSC) expansion media and episomal vectors that lacked Myc and Lin28.RESULTSAll iPSC colonies were fully reprogrammed based on SSEA4 expression. A total of 300,000 CBDMNC was the optimal cell number for cell reprogramming, which was associated with a 13-fold increase in CD34+ cells upon exposure to HSC media. Cell reprogramming was not observed in the absence of HSC expansion media. The method also reprogrammed PBMNC in patients with cystic fibrosis or α-1 antitrypsin deficiency. Oncogene-free iPSC cell lines differentiated into all three germ cell lineages.CONCLUSIONThis iPSC reprogramming approach satisfies an important regulatory requirement for iPSC-based cell therapies with lower clinical risk from CBDMNC and PBMNC.

Although pulmonary alveolar interstitial fibroblasts are less specialized than their epithelial and endothelial neighbors, they play essential roles during development and in response to lung injury. At birth, they must adapt to the sudden mechanical changes imposed by the onset of respiration and to a higher ambient oxygen concentration. In diseases such as bronchopulmonary dysplasia and interstitial fibrosis, their adaptive responses are overwhelmed leading to compromised gas-exchange function. Thus, although fibroblasts do not directly participate in gas-exchange, they are essential for creating and maintaining an optimal environment at the alveolar epithelial-endothelial interface. This review summarizes new information and concepts about the ontogeny differentiation, and function of alveolar fibroblasts. Alveolar development will be emphasized, because the development of strategies to evoke alveolar repair and regeneration hinges on thoroughly understanding the way that resident fibroblasts populate specific locations in which extracellular matrix must be produced and remodeled. Other recent reviews have described the disruption that diseases cause to the fibroblast niche and so my objective is to illustrate how the unique developmental origins and differentiation pathways could be harnessed favorably to augment certain fibroblast subpopulations and to optimize the conditions for alveolar regeneration.

Glucocorticoids have been widely used and exert pleiotropic effects on alveolar structure and function, but do not improve the long-term clinical outcomes for patients with bronchopulmonary dysplasia, emphysema, or interstitial lung diseases. Treatments that foster alveolar regeneration could substantially improve the long-term outcomes for such patients. One approach to alveolar regeneration is to stimulate and guide intrinsic alveolar progenitors along developmental pathways used during secondary septation. Other investigators and we have identified platelet-derived growth factor receptor-α-expressing fibroblast subpopulations that are alternatively skewed toward myofibroblast or lipofibroblast phenotypes. In this study, we administered either the glucocorticoid receptor agonist dexamethasone (Dex) or the antagonist mifepristone to mice during the first postnatal week and evaluated their effects on cellular proliferation and adoption of α-smooth muscle actin and lipid droplets (markers of the myofibroblast and lipofibroblast phenotypes, respectively). We observed that Dex increased the relative abundance of fibroblasts with progenitor characteristics, i.e., containing both α-smooth muscle actin and lipid droplets, uncoupling protein-1 (a marker of brown and beige adipocytes), delta-like ligand-1, and stem cell antigen-1. Dex enhanced signaling through the Smad1/5 pathway, which increased uncoupling protein-1 in a lung fibroblast progenitor cell line. We conclude that glucocorticoid receptor manipulation can sustain fibroblast plasticity, and posit that targeting downstream glucocorticoid responsive pathways could steer fibroblast progenitors along more desirable regenerative pathways.

Aim: Nonviral induced pluripotent stem cell (IPSC) reprogramming is not efficient without the oncogenes, Myc and Lin28. We describe a robust Myc and Lin28-free IPSC reprogramming approach using reprogramming molecules. Methods: IPSC colony formation was compared in the presence and absence of Myc and Lin28 by the mixture of reprogramming molecules and episomal vectors. Results: While more colonies were observed in cultures transfected with the aforementioned oncogenes, the Myc and Lin28-free method achieved the same reprogramming efficiency as reports that used these oncogenes. Further, all colonies were fully reprogrammed based on expression of SSEA4, even in the absence of Myc and Lin28. Conclusion: This approach satisfies an important regulatory pathway for developing IPSC cell therapies with lower clinical risk.

Platelet-derived growth factor (PDGF)-A, which only signals through PDGF-receptor-α (PDGFR-α), is required for secondary alveolar septal formation. Although PDGFR-α distinguishes mesenchymal progenitor cells during the saccular stage, PDGFR-α-expressing alveolar cells persist through adulthood. PDGF-A sustains proliferation, limits apoptosis, and maintains α-smooth muscle actin (α-SMA)-containing alveolar cells, which congregate at the alveolar entry ring at postnatal day (P)12. PDGFR-α-expressing, α-SMA-containing alveolar cells redistribute in the elongating septum, suggesting that they migrate to the alveolar entry rings, where mechanical tension is higher. We hypothesized that PDGFR-α and Ras-related C3 botulinum toxin substrate 1(Rac1) are required for mechanosensitive myofibroblast migration. Spreading of PDGFR-α-deficient lung fibroblasts was insensitive to increased rigidity, and their migration was not reduced by Rac1-guanine exchange factor (GEF)-inhibition. PDGFR-α-expressing fibroblasts migrated toward stiffer regions within two-dimensional substrates by increasing migrational persistence (durotaxis). Using a Förster resonance energy transfer (FRET) biosensor for Rac1-GTP, we observed that PDGFR-α was required for fibroblast Rac1 responsiveness to stiffness within a three-dimensional collagen substrate, which by itself increased Rac1-FRET. Rho-GTPase stabilized, whereas Rac1-GTPase increased the turnover of focal adhesions. Under conditions that increased Rac1-GTP, PDGFR-α signaled through both phosphoinositide-3-kinase (PIK) or Src to engage the Rac1 GEF dedicator of cytokinesis-1 (Dock180) and p21-activated-kinase interacting exchange factor-β (βPIX). In cooperation with collagen fibers, these signaling pathways may guide fibroblasts toward the more rigid alveolar entry ring during secondary septation. Because emphysema and interstitial fibrosis disrupt the parenchymal mechanical continuum, understanding how mechanical factors regulate fibroblast migration could elicit strategies for alveolar repair and regeneration.

Pulmonary alveolar fibroblasts produce extracellular matrix in a temporally and spatially regulated pattern to yield a durable yet pliable gas-exchange surface. Proliferation ensures a sufficient complement of cells, but they must differentiate into functionally distinct subtypes: contractile myofibroblasts (MF), which generate elastin and regulate air-flow at the alveolar ducts, and, in mice and rats, lipofibroblasts (LF), which store neutral lipids. PDGF-A is required but acts in conjunction with other differentiation factors arising from adjacent epithelia or within fibroblasts. We hypothesized that FGF receptor (FGFR) expression and function vary for MF and LF and contributes to their divergent differentiation. Whereas approximately half of the FGFR3 was extracellular in MF, FGFR2 and FGFR4 were primarily intracellular. Intracellular FGFR3 localized to the multivesicular body, and its abundance may be modified by Sprouty and interaction with heat shock protein-90. FGF18 mRNA is more abundant in MF, whereas FGF10 mRNA predominated in LF, which also express FGFR1 IIIb, a receptor for FGF10. FGF18 diminished fibroblast proliferation and was chemotactic for cultured fibroblasts. Although PDGF receptor-α (PDGFR-α) primarily signals through phosphoinositide 3-kinase and Akt, p42/p44 MAP kinase (Erk1/2), a major signaling pathway for FGFRs, influenced the abundance of cell-surface PDGFR-α. Observing different FGFR and ligand profiles in MF and LF is consistent with their divergent differentiation although both subpopulations express PDGFR-α. These studies also emphasize the importance of particular cellular locations of FGFR3 and PDGFR-α, which may modify their effects during alveolar development or repair.

Although protease-antiprotease imbalance is widely thought to contribute to the genesis of emphysema, the involvement of the alveolar macrophage in this process is poorly defined. We have quantified the uptake of radioiodinated human neutrophil elastase by human alveolar macrophages in monolayer culture and assessed the enzyme's fate during periods as long as 48 h after uptake using molecular sieve chromatography. Approximately half of the radiolabel eluted with enzymatically inactive material of molecular sizes corresponding to either degraded or alpha1-protease-inhibitor-bound elastase. The remainder of the radiolabel eluted at 29,000 daltons, in fractions containing enzyme that solubilized particulate elastin, and likely represented intact neutrophil elastase. Macrophages from smokers and nonsmokers showed similar characteristics of incorporation and disposition of neutrophil elastase. Lysates of uncultured alveolar macrophages from smokers and nonsmokers contained (mean +/- SE, n = 4) 7.4 +/- 0.8 and 3.3 +/- 1.4 ng of neutrophil elastase activity per 10(6) cells, respectively (not significant). However, as smokers have 11-fold more cells obtainable by lavage, the total lavaged elastase loads in alveolar macrophages were 561.7 +/- 72.3 and 21.3 +/- 6.8 (p less than 0.01) in smokers and nonsmokers, respectively. We conclude that the alveolar macrophage may clear and inactivate neutrophil elastase in the lung. In addition, by conserving some ingested elastase in an enzymatically active form, the macrophage may serve as an elastase reservoir. In areas of high macrophage density, as around the respiratory bronchioles of smokers, the macrophage could release a portion of the incorporated elastase and damage lung elastin.

Alveolar development in humans primarily occurs postnatally and requires a carefully orchestrated expansion of distal epithelial and mesenchymal progenitor populations and coordinated differentiation, to create a highly segmented gas-exchange surface. The regulation of alveolarization normally assimilates cues from paracrine cell-cell, cell-extracellular matrix, and mechanical interactions which are superimposed on cells and the extracellular matrix through phasic respiratory movement. In bronchopulmonary dysplasia, the entire process is precociously initiated when cellular and extracellular components are adapted to the saccular stage where movement and circulation are much more limited. This review focuses on mesenchymal cells (fibroblasts, endothelial cells, and pericytes), and epithelial cells are primarily discussed as sources of growth factor ligands or recipients of ligands produced by mesenchymal cells. Some interstitial fibroblasts differentiate to contractile myofibroblasts, containing a smooth muscle-actin rich cytoskeleton, which connects with tensile and elastic elements in the extracellular matrix, and together comprise a load-bearing network that diffuses mechanical forces during respiration. Other interstitial fibroblasts assimilate neutral lipid droplets, which regulate the differentiation of distal epithelial progenitors and surfactant production by alveolar type 2 cells. Pericytes organize and reinforce the capillary network as it expands to match the coverage of type 1 epithelial cells. Hyperoxia and the mechanical load imposed by positive pressure mechanical ventilation disrupt these paracrine interactions, leaving thickened alveolar walls, airways and arterioles, thereby diminishing gas-exchange surface area. Better understanding of these mechanisms of alveolar septation will lead to more effective treatments to preserve and perhaps augment the surface usual sequence of events that drive alveolarization.

Although the pulmonary interstitial lipofibroblast (LF) has been widely recognized in rat and mouse lungs, their presence in human lungs remains controversial. In a recent issue of the Journal, Tahedl and associates (Tahedl D, Wirkes A, Tschanz SA, Ochs M, Mühlfeld C. Am J Physiol Lung Cell Mol Physiol 307: L386-L394, 2014) address this controversy and provide the most detailed stereological analysis of LFs in mammals other than rodents. Strikingly, their observations demonstrate that LFs were only observed in rodents, which contrasts with earlier reports. This editorial reviews the anatomical, physiological, and biochemical characteristics of the LF to better understand the significance of LFs for lung development and disease. Although lipid droplets are a signature of the LF cell type, it remains unclear whether lipid storage is the defining characteristic of LFs, or whether other less overt properties determine the importance of LFs. Are lipid droplets an adaptation to the neonatal environment, or are LFs a surrogate for other properties that promote alveolar development, and do lipid droplets modify physiology or disease in adults?

Signaling through platelet-derived growth factor receptor-α (PDGFRα) is required for alveolar septation and participates in alveolar regeneration after pneumonectomy. In both adipose tissue and skeletal muscle, bipotent pdgfrα-expressing progenitors expressing delta-like ligand-1 or sex-determining region Y box 9 (Sox9) may differentiate into either lipid storage cells or myofibroblasts. We analyzed markers of mesenchymal progenitors and differentiation in lung fibroblasts (LF) with different levels (absent, low, or high) of pdgfrα gene expression. A larger proportion of pdgfrα-expressing than nonexpressing LF contained Sox9. Neutral lipids, CD166, and Tcf21 were more abundant in LF with a lower compared with a higher level of pdgfrα gene expression. PDGF-A increased Sox9 in primary LF cultures, suggesting that active signaling through PDGFRα is required to maintain Sox9. As alveolar septation progresses from postnatal day (P) 8 to P12, fewer pdgfrα-expressing LF contain Sox9, whereas more of these LF contain myocardin-like transcription factor-A, showing that Sox9 diminishes as LF become myofibroblasts. At P8, neutral lipid droplets predominate in LF with the lower level of pdgfrα gene expression, whereas transgelin (tagln) was predominantly expressed in LF with higher pdgfrα gene expression. Targeted deletion of pdgfrα in LF, which expressed tagln, reduced Sox9 in α-actin (α-SMA, ACTA2)-containing LF, whereas it increased the abundance of cell surface delta-like protein-1 (as well as peroxisome proliferator-activated receptor-γ and tcf21 mRNA in LF, which also expressed stem cell antigen-1). Thus pdgfrα deletion differentially alters delta-like protein-1 and Sox9, suggesting that targeting different downstream pathways in PDGF-A-responsive LF could identify strategies that promote lung regeneration without initiating fibrosis.