Drebrin is Required for Myosin-facilitated Actin Cytoskeletal Remodeling during Pulmonary Alveolar Development.

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.

Discoidin domain receptor-2 enhances secondary alveolar septation in mice by activating integrins and modifying focal adhesions.

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 ß1-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 ß1-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.

Neuropilin-1 directs PDGFRα-entry into lung fibroblasts and signaling from very early endosomes.

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.

Platelet-derived Growth Factor-α and Neuropilin-1 Mediate Lung Fibroblast Response to Rigid Collagen Fibers.

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 ß1-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.

Neuropilin-1 and platelet-derived growth factor receptors cooperatively regulate intermediate filaments and mesenchymal cell migration during alveolar septation.

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 PDGFRß. 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.

Platelet-derived growth factor receptor-α and Ras-related C3 botulinum toxin substrate-1 regulate mechano-responsiveness of lung fibroblasts.

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-ß (ßPIX). 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.

Glucocorticoids Retain Bipotent Fibroblast Progenitors during Alveolar Septation in Mice.

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.

Fibroblast growth factor signaling in myofibroblasts differs from lipofibroblasts during alveolar septation in mice.

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.

In search of the elusive lipofibroblast in human lungs.

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?

Regulation of fibroblast lipid storage and myofibroblast phenotypes during alveolar septation in mice.

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.

Paracrine cellular and extracellular matrix interactions with mesenchymal progenitors during pulmonary alveolar septation.

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.

Platelet-derived growth factor-A regulates lung fibroblast S-phase entry through p27(kip1) and FoxO3a.

BACKGROUND: Secondary pulmonary alveolar septal formation requires platelet derived growth factor (PDGF-A) and platelet derived growth factor receptor-alpha (PDGFRα), and their regulation influences alveolar septal areal density and thickness. Insufficient PDGFRα expression in lung fibroblasts (LF) results in failed septation. METHODS: Mice in which the endogenous PDGFRα-gene regulates expression of the green fluorescent protein were used to temporally and spatially track PDGFRα-signaling. Transition from the G1/G0 to the S-phase of the cell cycle was compared in PDGFRα-expressing and non-expressing LF using flow cytometry. Laser scanning confocal microscopy was used to quantify p27(kip1) and forkhead box "other" 3a (FoxO3a) in the nuclei of alveolar cells from mice bearing the PDGFRα-GFP knock-in, and p27(kip1) in mice with a conditional deletion of PDGFRα-gene function. The effects of PDGF-A on the phosphorylation and the intracellular location of FoxO3a were examined using Western immuoblotting and immunocytochemistry. RESULTS: In neonatal mouse lungs, entry of the PDGFRα-expressing LF subpopulation into the S-phase of the cell cycle diminished sooner than in their non-expressing LF counterparts. This preferential diminution was influenced by PDGFRα-mediated signaling, which phosphorylates and promotes cytoplasmic localization of FoxO3a. Comparative observations of LF at different ages during secondary septation and in mice that lack PDGFRα in alveolar LF demonstrated that nuclear localization of the G1 cyclin-dependent kinase inhibitor p27(kip1) correlated with reduced LF entry into S-phase. CONCLUSIONS: Nuclear localization of FoxO3a, an important regulator of p27(kip1) gene-expression, correlates with diminished proliferation of the PDGFRα-expressing LF subpopulation. These mechanisms for diminishing the effects of PDGFRα-mediated signaling likely regulate secondary septal formation and their derangement may contribute to imbalanced fibroblast cell kinetics in parenchymal lung diseases.

Platelet-derived growth factor-A and sonic hedgehog signaling direct lung fibroblast precursors during alveolar septal formation.

Alveolar septal formation is required to support the respiration of growing mammals; in humans effacement of the alveolar surface and impaired gas exchange are critical features of emphysema and pulmonary fibrosis. Platelet-derived growth factor-A (PDGF-A) and its receptor PDGF-receptor-α (PDGFRα) are required for secondary septal elongation in mice during postnatal days 4 through 12 and they regulate the proliferation and septal location of interstitial fibroblasts. We examined lung fibroblasts (LF) to learn whether PDGFRα expression distinguished a population of precursor cells, with enhanced proliferative and migratory capabilities. We identified a subpopulation of LF that expresses sonic hedgehog (Shh) and stem cell antigen-1 (Sca1). PDGF-A and Shh both increased cytokinesis and chemotaxis in vitro, but through different mechanisms. In primary LF cultures, Shh signaled exclusively through a noncanonical pathway involving generation of Rac1-GTP, whereas both the canonical and noncanonical pathways were used by the Mlg neonatal mouse LF cell line. LF preferentially oriented their primary cilia toward their anterior pole during migration. Furthermore, a larger proportion of PDGFRα-expressing LF, which are more abundant at the septal tips, bore primary cilia compared with other alveolar cells. In pulmonary emphysema, destroyed alveolar septa do not regenerate, in part because cells fail to assume a configuration that allows efficient gas exchange. Better understanding how LF are positioned during alveolar development could identify signaling pathways, which promote alveolar septal regeneration.

Vitamin A deficiency alters airway resistance in children with acute upper respiratory infection.

OBJECTIVE: To assess whether vitamin A deficiency alters the recovery of total respiratory resistance (TRR) following acute upper respiratory tract infection (URI). METHODS: This is a case control study of children, age 4-6 years and grouped as: URI, (n = 74), URI and wheezing, (URI-wheezing, n = 52), and healthy controls (n = 51). Vitamin A and total respiratory resistance (TRR) were assessed using the modified relative dose response (MRDR) and forced oscillometry, respectively. RESULTS: Children with URI and URI-wheezing had lower retinol, 32.4 ± 13.12 and 18.3 ± 6.83 µg/dl respectively, compared to controls, 56.9 ± 29.82 µg/dl (ANOVA, P < 0.001). The MRDR was elevated in children in the URI or URI-wheezing groups 0.066 ± 0.045 and 0.021 ± 0.021, respectively, compared to controls 0.007 ± 0.006 (ANOVA, P < 0.0001). The TRR in the URI and URI-wheezing groups differed from controls. During convalescence, the TRR failed to decline in the URI-group only when the MRDR was >0.06. In the URI-wheezing group, TRR declined independently of retinol and MRDR. CONCLUSION: Vitamin A contributes to preservation of airway function during and in recovery after upper respiratory infection in children.

Fibroblasts expressing PDGF-receptor-alpha diminish during alveolar septal thinning in mice.

In mice, secondary alveolar septal formation primarily occurs during a brief postnatal period and is accompanied by transient expansion of the interstitial lung fibroblast (LF) population. PDGF-A, which solely signals through PDGF-receptor-alpha (PDGF-Rα), is required for expansion, but the receptor's relevant downstream targets remain incompletely defined. We have evaluated the proliferation, apoptosis, and differential response to the selective protein tyrosine kinase inhibitor, imatinib, by pdgfrα-expressing LF (pdgfrα-LF) and compared them with their nonexpressing LF counterparts. Our objective was to determine whether diminished signaling through PDGF-Rα-mediated pathways regulates the decline in myofibroblasts, which accompanies septal thinning and ensures more efficient alveolar gas exchange. Using quantitative stereology and flow cytometry at postnatal d 12 and 14, we observed that imatinib caused a selective suppression of proliferation and an increase in apoptosis. The number of the alpha smooth muscle actin (αSMA) producing pdgfrα-LF was also reduced. Using cultures of neonatal mouse LF, we showed that imatinib did not suppress PDGF-Rα gene expression but reduced PDGF-A-mediated Akt phosphorylation, potentially explaining the increase in apoptosis. Our findings are relevant to bronchopulmonary dysplasia in which positive pressure ventilation interferes with myofibroblast depletion, septal thinning, and capillary maturation.

PDGF-Ralpha gene expression predicts proliferation, but PDGF-A suppresses transdifferentiation of neonatal mouse lung myofibroblasts.

BACKGROUND: Platelet-derived growth factor A (PDGF-A) signals solely through PDGF-Ralpha, and is required for fibroblast proliferation and transdifferentiation (fibroblast to myofibroblast conversion) during alveolar development, because pdgfa-null mice lack both myofibroblasts and alveoli. However, these PDGF-A-mediated mechanisms remain incompletely defined. At postnatal days 4 and 12 (P4 and P12), using mouse lung fibroblasts, we examined (a) how PDGF-Ralpha correlates with ki67 (proliferation marker) or alpha-smooth muscle actin (alphaSMA, myofibroblast marker) expression, and (b) whether PDGF-A directly affects alphaSMA or modifies stimulation by transforming growth factor beta (TGFbeta). METHODS: Using flow cytometry we examined PDGF-Ralpha, alphaSMA and Ki67 in mice which express green fluorescent protein (GFP) as a marker for PDGF-Ralpha expression. Using real-time RT-PCR we quantified alphaSMA mRNA in cultured Mlg neonatal mouse lung fibroblasts after treatment with PDGF-A, and/or TGFbeta. RESULTS: The intensity of GFP-fluorescence enabled us to distinguish three groups of fibroblasts which exhibited absent, lower, or higher levels of PDGF-Ralpha. At P4, more of the higher than lower PDGF-Ralpha + fibroblasts contained Ki67 (Ki67+), and Ki67+ fibroblasts predominated in the alphaSMA + but not the alphaSMA- population. By P12, Ki67+ fibroblasts comprised a minority in both the PDGF-Ralpha + and alphaSMA+ populations. At P4, most Ki67+ fibroblasts were PDGF-Ralpha + and alphaSMA- whereas at P12, most Ki67+ fibroblasts were PDGF-Ralpha- and alphaSMA-. More of the PDGF-Ralpha + than - fibroblasts contained alphaSMA at both P4 and P12. In the lung, proximate alphaSMA was more abundant around nuclei in cells expressing high than low levels of PDGF-Ralpha at both P4 and P12. Nuclear SMAD 2/3 declined from P4 to P12 in PDGF-Ralpha-, but not in PDGF-Ralpha + cells. In Mlg fibroblasts, alphaSMA mRNA increased after exposure to TGFbeta, but declined after treatment with PDGF-A. CONCLUSION: During both septal eruption (P4) and elongation (P12), alveolar PDGF-Ralpha may enhance the propensity of fibroblasts to transdifferentiate rather than directly stimulate alphaSMA, which preferentially localizes to non-proliferating fibroblasts. In accordance, PDGF-Ralpha more dominantly influences fibroblast proliferation at P4 than at P12. In the lung, TGFbeta may overshadow the antagonistic effects of PDGF-A/PDGF-Ralpha signaling, enhancing alphaSMA-abundance in PDGF-Ralpha-expressing fibroblasts.

Association of nutritional status with the response to infection with Leishmania chagasi.

Outcomes of infection with Leishmania chagasi range from self-resolving infection to visceral leishmaniasis (VL). Risk factors determining development of disease are not totally understood, but probably include environmental influences and host genetics. We assessed whether nutrition influenced the outcome of Leishmania infection by comparing relatives of children with VL with either self-resolving Leishmania spp. infection or apparently uninfected households. We observed a decrease in body mass index (P < 0.0005) and mid-upper arm circumference for age (P = 0.022) z-scores for children with VL. Levels of vitamin A were lower in active children with VL as measured by serum retinol (P = 0.035) and the modified-relative-dose-response test (P = 0.009). Higher birth weight (P = 0.047) and albumin concentrations (P = 0.040) protected against disease. Increased breastfeeding time (P = 0.036) was associated with asymptomatic infection. The results indicate that modifiable nutritional aspects are associated with the outcome of Leishmania spp. infection in humans.

Platelet-derived growth factor receptor-alpha-expressing cells localize to the alveolar entry ring and have characteristics of myofibroblasts during pulmonary alveolar septal formation.

Platelet-derived growth factor-A and its receptor, platelet-derived growth factor receptor-alpha (PDGF-Ralpha), are required for formation of the secondary pulmonary alveolar septa in mice. However, it remains unclear how these molecules direct the secondary septation process. We have examined the abundance, location, and the accumulation of alpha-smooth muscle actin (alphaSMA), neutral lipid droplets, and elastin in the proximity of PDGF-Ralpha-expressing alveolar cells during postnatal days 4 through 12 in the mouse. PDGF-Ralpha-expressing cells preferentially have characteristics of myofibroblasts and were more likely to contain alphaSMA than are alveolar cells that do not express PDGF-Ralpha. PDGF-Ralpha expressing cells were preferentially located in the alveolar entry ring (AER) where alphaSMA and elastic fibers accumulate. In contrast, PDGF-Ralpha expression inversely correlated with neutral lipid accumulation, which was more prominent at the alveolar base, distant from the AER. PDGF-Ralpha-expressing alveolar cells accumulate in the AER where they may promote mechanical stability during respiration. In addition to defining how alveolar septa form, these findings may have implications for the treatment of diseases which involve alveolar effacement such as emphysema and pulmonary fibrosis.

Arg-Gly-Asp-containing domains of fibrillins-1 and -2 distinctly regulate lung fibroblast migration.

Development of the extracellular matrix is a critical feature of alveolar formation and actively involves pulmonary interstitial fibroblasts. The elastic fiber network is an interconnected system of load-bearing fibers that also influences the behavior of adjacent cells, particularly the interstitial lung fibroblasts (LF). We hypothesized that discrete domains of fibrillins-1 and -2 interact with LF integrins and direct their migration in the presence of platelet-derived growth factor (PDGF)-A. Surfaces coated with recombinant peptides lacking or including an arginine-glycine-aspartic acid (RGD) motif were used to study LF migration across porous filters and on protein-coated glass. Exon 24 of fibrillin-2 (Fib2 24), which encodes for an RGD-containing transforming growth factor-beta-binding (TB) domain, stimulated migration with greater directional persistence and more effectively stimulated trans-filter migration at low concentrations. Exons 36-44 of fibrillin-1 (Fib1 36-44), which include epidermal growth factor-like domains and an RGD-containing TB domain, induce more lamlellipodia and more widespread remodeling of the leading edge, resulting in greater migration velocity than did Fib2 24. Distinct structural features in regions that surround the RGD motifs may differentially regulate how the PDGF receptor-alpha promotes integrin distribution and actin filament remodeling at the cell's leading edge. Understanding how fibrillins regulate LF migration may help elucidate how the elastic fiber system could be restored as an interconnected unit, which fails to occur in emphysematous lungs.