@article{apparao_newman_zhang_khosla_randell_sannes_2010, title={Temporal Changes in Expression of FoxA1 and Wnt7A in Isolated Adult Human Alveolar Epithelial Cells Enhanced by Heparin}, volume={293}, ISSN={["1932-8494"]}, DOI={10.1002/ar.20805}, abstractNote={AbstractPre‐ and postnatal developmental studies of the lung have provided compelling evidence demonstrating multiple factors that orchestrate alveolar epithelial cell differentiation. The extent to which reactivation of certain developmental pathways in the adult might influence the course of differentiation of alveolar type 2 cells (AT2) into AT1 cells is not known. In this study, we examined selected members of the forkhead (Fox) family of transcription factors and the Wnt (wingless) family of signaling proteins for expression during human alveolar cell differentiation in vitro and determined their potential responses to sulfated components of extracellular matrix (ECM), like those shed from cell surfaces or found in basement membrane and modeled by heparin. Isolated adult human AT2 cells cultured over a 9‐day period were used to define the temporal profile of expression of targeted factors during spontaneous differentiation to AT1‐like cells. FoxA1 protein was upregulated at early to intermediate time points, where it was strongly elevated by heparin. Gene expression of wnt7A increased dramatically beginning on day 3 and was enhanced even further on days 7 and 9 by heparin, whereas protein expression appeared at days 7 and 9. These temporal changes of expression suggest that sulfated ECMs may act to enhance the increase in FoxA1 at the critical juncture when AT2 cells commence the differentiation process to AT1 cells, in addition to enhancing the increase in wnt7A when the AT1 cell phenotype stabilizes. Collectively, these factors may act to modulate differentiation in the adult human pulmonary alveolus. Anat Rec, 293:938–946, 2010. © 2010 Wiley‐Liss, Inc.}, number={6}, journal={ANATOMICAL RECORD-ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY}, author={Apparao, K. B. C. and Newman, Donna R. and Zhang, Huiying and Khosla, Jody and Randell, Scott H. and Sannes, Philip L.}, year={2010}, month={Jun}, pages={938–946} } @article{gabr_reed_newman_pohl_khosla_sannes_2007, title={Alterations in cytoskeletal and immune function-related proteome profiles in whole rat lung following intratracheal instillation of heparin}, volume={8}, ISSN={["1465-993X"]}, DOI={10.1186/1465-9921-8-36}, abstractNote={AbstractBackgroundHeparin has been shown to modify fundamental biologic processes ranging from blood coagulation and cell proliferation to fibrogenesis and asthma. The goal of this study was to identify specific or broad biologic responses of the rat lung to intratracheal instillation of heparin by targeted proteomic analysis.MethodsRats were given either aerosolized 500 μg heparin in 250 μl saline or saline alone. Lungs were harvested at 0, 24, or 96 hours post-treatment and isolated proteins analyzed by two-dimensional gel electrophoresis. Proteins which increased and decreased significantly in treated groups above controls were then selected for identification by mass spectrometry.ResultsAlthough heparin treatments resulted in a general reduction in cytosolic protein expression, there were significant increases within members of discrete groups of proteins. At 24 hours, proteins which function in cytoskeletal organization and in calcium signaling were up-regulated between 2- and 27-fold above baseline and untreated controls. Increased proteins include annexins V and VI, septin 2, capping G protein, actin-related protein 3, moesin, RhoGDP dissociation inhibitor, and calcyclin. A group of proteins relating to immune response and tumor suppressor function were either up-regulated (tumor suppressor p30/hyaluronic acid binding protein-1, Parkinson disease protein 7, proteosome 28 subunit/interferon-γ inducible protein, and proteosome subunit macropain α-1) or strongly down-regulated (transgelin). At 96 hours, most proteins that had increased at 24 hours remained elevated but to a much lesser degree.ConclusionThese cumulative observations demonstrate that whole lung heparin treatment results in significant up-regulation of selected groups of proteins, primarily those related to cytoskeletal reorganization and immune function, which may prove to be relevant biomarkers useful in analysis of lung exposures/treatments as well as in system biology studies.}, journal={RESPIRATORY RESEARCH}, author={Gabr, Amir A. and Reed, Mathew and Newman, Donna R. and Pohl, Jan and Khosla, Jody and Sannes, Philip L.}, year={2007}, month={May} } @article{leiner_newman_li_walsh_khosla_sannes_2006, title={Heparin and fibroblast growth factors affect surfactant protein gene expression in type II cells}, volume={35}, DOI={10.1165/rcmb.2006-01590C}, number={5}, journal={American Journal of Respiratory Cell and Molecular Biology}, author={Leiner, K. A. and Newman, D. and Li, C. M. and Walsh, E. and Khosla, J. and Sannes, P. L.}, year={2006}, pages={611–618} } @article{newman_li_simmons_khosla_sannes_2004, title={Heparin affects signaling pathways stimulated by fibroblast growth factor-1 and-2 in type II cells}, volume={287}, ISSN={["1522-1504"]}, DOI={10.1152/ajplung.00284.2003}, abstractNote={Undersulfation of the basement membrane matrix of alveolar type II (AT2) cells compared with that of neighboring type I cells is believed to account for some of the known morphological and functional differences between these pneumocytes. Heparin, a model for sulfated components of basement membrane matrices, is known to inhibit fibroblast growth factor (FGF)-2-stimulated DNA synthesis as well as gene expression of FGF-2 and its receptor in AT2 cells. To determine whether these end points result from specific effects of heparin on FGF-related signaling pathways, isolated rat AT2 cells were treated with 100 ng/ml FGF-1 or FGF-2 in the presence of up to 500 μg/ml heparin. In addition, experiments were done on cells grown in the presence of 20 mM sodium chlorate (sulfation inhibitor). High-dose heparin reduced FGF-1- or FGF-2-stimulated phosphorylation of mitogen-activated protein kinase kinases (MEK1/2), p44/42 mitogen-activated protein kinases (MAPK/ERK1/2), stress-activated protein kinase/c-Jun NH2-terminal kinase, Akt/protein kinase B, and p90RSK. FGF-2-stimulated signaling was more sensitive to heparin's effects than was signaling stimulated by FGF-1. Heparin had an additive effect on the reduced [3H]thymidine incorporation in FGF-2-treated AT2 cells caused by inhibition of the MEK/ERK pathway by the MEK inhibitor PD-98059. The data suggest that heparin's known capacity to alter DNA synthesis and, possibly, other biological end points is realized via cross talk between multiple signaling pathways.}, number={1}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY}, author={Newman, DR and Li, CM and Simmons, R and Khosla, J and Sannes, PL}, year={2004}, month={Jul}, pages={L191–L200} } @article{li_newman_cesta_tompkins_khosla_sannes_2003, title={Modulation of fibroblast growth factor expression and signal transduction in type II cells}, volume={123}, ISSN={["0012-3692"]}, DOI={10.1378/chest.123.3_suppl.429S}, abstractNote={repair and wound healing. PDGFs are synthesized and secreted by most inflammatory cell types present within the milieu of the asthmatic airway. We have previously reported that airway fibroblasts from severe asthmatics produce more type I procollagen in response to PDGF stimulation as compared to patients with mild asthma and normal control subjects; therefore, we hypothesized that the enhanced responsiveness to PDGFs in patients with severe asthma is linked to an increased expression of PDGF receptors. In an ongoing study, 5 subjects with severe asthma, 10 subjects with mild-to-moderate asthma, and 6 normal control subjects underwent bronchoscopy with endobronchial biopsy. Biopsies were placed in Dulbecco’s modified Eagle’s serum supplemented with fetal bovine serum (10%), streptomycin (100 g/mL), penicillin (10,000 U/mL), and gentamicin (100 g/mL), and cultured until fibroblast growth was established at 50% confluency (approximately 8 to 20 days). Immunostaining with vimentin (Dako; Carpenteria, CA), Ab-1 (Calbiochem; San Diego, CA) and -smooth muscle actin (Dako) confirmed fibroblast identity. To determine baseline fibroblast expression of PDGF receptors (PDGFRs) [PDGFRand PDGFR], we developed a sandwich enzyme-linked immunosorbent assay for these receptors that quantifies receptor protein levels in fibroblast cell lysates. Receptor protein levels were expressed in nanograms per 100 g of total cell protein. There were no significant differences in baseline expression of PDGFRbetween the groups (severe, 7.6 ng/100 g protein; mild to moderate, 12.50 ng/100 g protein; normal control, 11.33 ng/100 g protein; p 0.35). However, there was a significantly greater baseline expression of PDGFRin the severe asthmatic group, as compared to both the mild/moderate asthmatic and normal control groups (severe, 15.20 ng/100 g protein; mild-to-moderate, 13.30 ng/100 g protein; normal control, 3.67 ng/100 g protein; p 0.0024). Our data suggests that airway fibroblasts from severe asthmatics may be of a synthetic phenotype, with altered capabilities in collagen production, as compared to those from patients with mild-to-moderate asthma and normal control subjects, and this may be driven by an increased expression of PDGFR. Modulation of Fibroblast Growth Factor Expression and Signal Transduction in Type II Cells*}, number={3}, journal={CHEST}, author={Li, CM and Newman, D and Cesta, M and Tompkins, L and Khosla, J and Sannes, PL}, year={2003}, month={Mar}, pages={429S–429S} } @article{pagan_khosla_li_sannes_2002, title={Effect of growth factor-fibronectin matrix interaction on rat type II cell adhesion and DNA synthesis}, volume={28}, ISSN={["0190-2148"]}, DOI={10.1080/019021402753462013}, abstractNote={Type II cells attach, migrate, and proliferate on a provisional fibronectin-rich matrix during alveolar wall repair after lung injury. The combination of cell-substratum interactions via integrin receptors and exposure to local growth factors are likely to initiate the signals required for cell proliferation, differentiation, reepithelialization, and ultimate restoration of the alveolar wall structure. Accordingly, primary cultured type II cells have been shown to bind fibronectin, in part through the α 5 β 1 integrin, and to respond to growth factors that induce type II cell proliferation, such as fibroblast growth factor 1 (FGF-1). The purpose of this study was to determine whether or not FGF-1 modifies type II cell attachment to fibronectin, and if together they affect DNA synthesis. Attachment assays showed that FGF-1 treatment enhanced type II cell adhesion to fibronectin. This effect correlated with an increase in β 1 integrin cell surface expression, and with the formation of cytoskeletal stabilizing structures such as lamellipodial extensions and stress fibers. FGF-1 also induced an increase in thymidine in corporation into DNA. Together FGF-1 and fibronectin appear to promote adhesion, cytoskeletal organization, and in creased DNA synthesis, and in this way influence cell-substratum interactions and signaling during alveolar repair.}, number={2}, journal={EXPERIMENTAL LUNG RESEARCH}, author={Pagan, I and Khosla, J and Li, CM and Sannes, PL}, year={2002}, month={Mar}, pages={69–84} } @article{li_newman_khosla_sannes_2002, title={Heparin inhibits DNA synthesis and gene expression in alveolar type II cells}, volume={27}, ISSN={["1535-4989"]}, DOI={10.1165/rcmb.2002-0002OC}, abstractNote={Responses of isolated type II alveolar cells to fibroblast growth factors (FGF) have been shown to be sensitive to the level of sulfation in extracellular matrix (ECM) substrata. These observations may reflect the specific in situ distribution and level of sulfation of ECM within the alveolar basement membranes (ABM) associated with type II cells. The goal of this study was to determine if the model sulfated ECM heparin modified DNA synthesis and gene expression by type II cells in a concentration dependent-manner. Isolated rat type II cells were exposed to different concentrations of heparin (0.005-500 micro g/ml) in serum-free medium for 1-3 d with or without FGF-1 or FGF-2. The effects of heparin were examined by [(3)H]thymidine incorporation into DNA, total cell protein, cell number, and selected gene expression. Results indicated that heparin inhibited [(3)H]thymidine uptake in a concentration-dependent manner. Total protein, cell number, and FGF-2 protein expression and mRNA of FGF-1, -2, and FGF receptor-2 detected by reverse transcriptase-polymerase chain reaction were decreased by heparin. These results demonstrate that sulfated molecules in the ABM may play important regulatory role(s) in selected type II cell activities during normal cell homeostasis, turnover, and repair after lung injury.}, number={3}, journal={AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY}, author={Li, CM and Newman, D and Khosla, J and Sannes, PL}, year={2002}, month={Sep}, pages={345–352} } @article{wang_sakamoto_khosla_sannes_2002, title={Pre- and postnatal lung development, maturation, and plasticity: Detection of chondroitin sulfates and decorin in developing fetal and neonatal rat lung}, volume={282}, ISSN={["1522-1504"]}, DOI={10.1152/ajplung.00160.2001}, abstractNote={Chondroitin sulfates and their related proteoglycans are components of extracellular matrix that act as key determinants of growth and differentiation characteristics of developing lungs. Changes in their immunohistochemical distribution during progressive organ maturation were examined with monospecific antibodies to chondroitin sulfate, a nonbasement membrane chondroitin sulfate proteoglycan, and the specific chondroitin sulfate-containing proteoglycan decorin in whole fetuses and lungs from newborn and adult rats. Alveolar and airway extracellular matrix immunostained heavily in the prenatal rat for both chondroitin sulfate and chondroitin sulfate proteoglycan, whereas decorin was confined to developing airways and vessels. These sites retained their respective levels of reactivity with all antibodies through 1–10 days postnatal but thereafter became progressively more diminished and focal in alveolar regions. The heavy staining seen early in development was interpreted to reflect a significant and wide distribution of chondroitin sulfates, chondroitin sulfate proteoglycans, and decorin in rapidly growing tissues, whereas the reduced and more focal reactivity observed at later time points coincided with known focal patterns of localization of fibrillar elements of the extracellular matrix and a more differentiated state.}, number={3}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY}, publisher={American Physiological Society}, author={Wang, YQ and Sakamoto, K and Khosla, J and Sannes, PL}, year={2002}, month={Mar}, pages={L484–L490} } @article{li_khosla_hoyle_sannes_2001, title={Transforming growth factor-beta(1) modifies fibroblast growth factor-2 production in type II cells}, volume={120}, ISSN={["0012-3692"]}, DOI={10.1378/chest.120.1_suppl.S60}, abstractNote={Transforming growth factor (TGF)-β 1 is an inflammatory cytokine that plays multiple roles in pulmonary fibrosis. In vascular epithelium, it has been shown to regulate production and activity of fibroblast growth factor (FGF)-2, a potent type II cell mitogen in the lung. Such a relationship could have important consequences in prefibrotic change in the lung alveolus, where reepithelialization of alveolar surfaces is crucial. The goal of this study was to determine if FGF-2 production by alveolar type II cells is modulated by TGF-β 1 or FGF-1, another type II cell mitogen. Isolated rat type II cells were exposed to 0 to 40 ng/mL of TGF-β 1 or 0 to 500 ng/mL of FGF-1 in serum-free medium for 1 to 3 days. Using a specific immunoassay, significant increases in FGF-2 protein in type II cell lysates were achieved after 1 day of exposure to 100 ng/mL of FGF-1 and after 3 days of treatment with 8 ng/mL of TGF-β 1 . Similarly, transcripts for FGF-2 were dramatically increased with TGF-β 1 or FGF-1, as were those for FGF receptor (FGFR)-1. These interactions were dramatically effected by the addition of heparin, a model sulfated extracellular matrix (ECM). Heparin as low as 0.01 mg/mL significantly downregulated expression of TGF-β 1 and FGF-1–stimulated FGF-2 and FGFR-1. These results demonstrate important regulatory links between FGF-2, sulfated ECMs, and both TGF-β 1 and FGF-1, which could contribute to the modulation of normal cell turnover, development, and repair processes attendant to fibrosis in the lung.}, number={1}, journal={CHEST}, author={Li, CM and Khosla, J and Hoyle, P and Sannes, PL}, year={2001}, month={Jul}, pages={60S–61S} } @article{li_khosla_pagan_hoyle_sannes_2000, title={TGF-beta 1 and fibroblast growth factor-1 modify fibroblast growth factor-2 production in type II cells}, volume={279}, ISSN={["1040-0605"]}, DOI={10.1152/ajplung.2000.279.6.l1038}, abstractNote={ Fibroblast growth factor (FGF)-2, which stimulates DNA synthesis by type II cells in the lung, has been shown to be regulated by transforming growth factor (TGF)-β1, an important inflammatory cytokine, in vascular epithelium. The goal of this study was to determine if FGF-2 production by alveolar type II cells is modulated by TGF-β1 or FGF-1, which also stimulates DNA synthesis by type II cells. Isolated rat type II cells were exposed to 0–40 ng/ml of TGF-β1 or 0–500 ng/ml of FGF-1 in serum-free medium for 1–5 days. With a specific immunoassay, significant increases of FGF-2 protein in type II cell lysates to levels above those in control cells were achieved after 1 day of exposure to 100 ng/ml of FGF-1 and after 3 days of treatment with 8 ng/ml of TGF-β1. Similarly, transcripts for FGF-2 were dramatically increased above those in control cells with TGF-β1 or FGF-1, as were those for FGF receptor-1. These results demonstrate important regulatory links between FGF-2 and both TGF-β1 and FGF-1 in the alveolar epithelium that could contribute to the regulation of normal cell turnover, development, and the repair processes after injury in the lung. }, number={6}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY}, author={Li, CM and Khosla, J and Pagan, I and Hoyle, P and Sannes, PL}, year={2000}, month={Dec}, pages={L1038–L1046} } @article{sannes_khosla_li_pagan_1998, title={Sulfation of extracellular matrices modifies growth factor effects on type II cells on laminin substrata}, volume={275}, ISSN={["1040-0605"]}, DOI={10.1152/ajplung.1998.275.4.l701}, abstractNote={The alveolar basement membrane contains a variety of extracellular matrix (ECM) molecules, including laminin and sulfated glycosaminoglycans of proteoglycans. These mixtures exist within microdomains of differing levels of sulfate, which may specifically interact to be key determinants of the known capacity of the type II cell to respond to certain growth factors. Isolated type II cells were exposed to either acidic fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), or keratinocyte growth factor (KGF; FGF-7) on culture wells precoated with laminin alone or in combination with chondroitin sulfate (CS), high-molecular-weight heparin, or their desulfated forms. Desulfated heparin significantly elevated FGF-1- and FGF-2-stimulated DNA synthesis, whereas desulfated CS and N-desulfated heparin elevated FGF-7-stimulated DNA synthesis by type II cells on laminin substrata. When FGF-1 was mixed into the various test matrix substrata, DNA synthesis was significantly increased in all cases. These results demonstrated that decreased levels of sulfate in ECM substrata act to upregulate responses to heparin-binding growth factors by alveolar epithelial cells on laminin substrata.}, number={4}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY}, author={Sannes, PL and Khosla, J and Li, CM and Pagan, I}, year={1998}, month={Oct}, pages={L701–L708} } @article{sannes_khosla_peters_1997, title={Biosynthesis of sulfated extracellular matrices by alveolar type II cells increases with time in culture}, volume={273}, ISSN={["1040-0605"]}, DOI={10.1152/ajplung.1997.273.4.l840}, abstractNote={The aim of this study was to determine the extent to which sulfate incorporated into biosynthesized basement membrane (BM) components increased as isolated type II cells progress toward a more type I cell-like phenotype from 7 to 21 days in culture. Specific sulfate cytochemistry, using high iron diamine, showed that type I-like cells in 21-day cultures deposited a more highly sulfated extracellular matrix. Biosynthetic labeling experiments using [35S]cysteine or [35S]sulfate as precursors confirmed the increased capacity of 21-day type I-like cells to biosynthesize sulfated BM components compared with type II-like cells in 7-day cultures, including a novel sulfated laminin. These biochemical changes in sulfation of BM components coincide with the established phenotypic transition from type II to type I cells during prolonged culture. More importantly, the data suggest that regulation of sulfation constitutes a potential mechanism by which type I and type II cells alter their environment in such a manner as to stabilize phenotype and modulate responses to growth factors.}, number={4}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY}, author={Sannes, PL and Khosla, J and Peters, BP}, year={1997}, month={Oct}, pages={L840–L847} } @article{adler_khosla_kim_1988, title={Morphometric and biochemical characterization of a chamber system for maintaining differentiated guinea pig respiratory mucosal cells between air and liquid phases.}, volume={137}, journal={American Review of Respiratory Disease}, author={Adler, K. B. and Khosla, J. and Kim, K. C.}, year={1988}, pages={14} }