Antiproliferative heparan sulfate inhibiting hyaluronan and transforming growth factor-β expression in human lung fibroblast cells

Abstract

The objective of this study was to examine the effects of heparan sulfate (HS) on factors involved in the remodeling of connective tissue observed in patients with fibrotic respiratory disorders such as asthma. A suitable working model is to stimulate human fetal lung fibroblasts in vitro with structurally different forms of HS. Highly sulfated and iduronic acid (IdoUA)-rich HS specifically decreased cell proliferation, production of hyaluronan (HA), transforming growth factor (TGF)-␤ 1 , and TGF-␤-induced ␣-smooth muscle actin but did not affect the overall proteoglycan production in the cells. These repressed factors are suggested to play a critical role in the early stages of remodeling and myofibroblast activation. Low sulfated and IdoUA-poor HS did not display any effects on these factors. Furthermore, analysis of the protein expression pattern by twodimensional gel electrophoresis revealed a 70% increased expression of annexin II, which has previously been shown to have a high affinity for both heparin and HS. Heat-shock protein 27 and arsenite translocating factor, both involved in actin organization and polymerization, were also increased in the HS-stimulated cells. Thus, the reduced expression of HA and TGF-␤ 1 , both important in the development of fibrosis, seems to be mediated by specific changes in protein expression of the fibroblast. The observed inhibition of cell proliferation, HA, and TGF-␤ 1 allows speculation of highly sulfated HS as an antifibrotic candidate in the early stage of remodeling.

Specific Haptoglobin Expression in Bronchoalveolar Lavage during Differentiation of Circulating Fibroblast Progenitor Cells in Mild Asthma

“…Twodimensional gel electrophoresis was performed according to a previously described protocol. 17 Gels were stained either with Comassie G250 (Pierce, Rockford, IL) according to instructions from the manufacturer, or silver nitrate according to a previously described protocol. 18 Gels were scanned using a GS-710 gel scanner (Bio-Rad, Hercules, CA) and image analysis was performed using PDQuest v7.01 computer software (Bio-Rad, Hercules, CA).…”

“…Equal amounts of protein samples were loaded for all western blotting which was performed according to a previous described protocol. 17 Primary polyclonal sheep anti-human haptoglobin antibody was used, diluted 1:1000 (Abcam Ltd, Cambridge, UK). The secondary antibody used was a polyclonal rabbit anti-sheep antibody, conjugated with horseradish peroxidase (DAKO, Glostrup, Denmark).…”

“…Two-Dimensional Gel Electrophoresis (2-DE), Image Analysis, and Identification of Peptides Using MALDI-TOF. Two-dimensional gel electrophoresis was performed according to a previously described protocol . Gels were stained either with Comassie G250 (Pierce, Rockford, IL) according to instructions from the manufacturer, or silver nitrate according to a previously described protocol …”

“…Western Blot. Equal amounts of protein samples were loaded for all western blotting which was performed according to a previous described protocol . Primary polyclonal sheep anti-human haptoglobin antibody was used, diluted 1:1000 (Abcam Ltd, Cambridge, UK).…”

Alterations in cytoskeletal and immune function-related proteome profiles in whole rat lung following intratracheal instillation of heparin

“…Heparin, a model heparan sulfate proteoglycan (HSPG), has been previously shown to reduce or inhibit proliferation and gene and protein expression in isolated rat lung type II alveolar cells [4-6]. Similarly, heparin and/or heparan sulfate have been shown to reduce proliferation in lung fibroblasts [7-9] and lung smooth muscle cells [10,11]. The current proteomic study was undertaken to expand investigation into the effects of heparin-like molecules on whole lung, in vivo.…”