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Background and objective Involvement of pulmonary vascular remodelling is a characteristic sign in COPD. Vascular mediators such as vascular endothelial growth factor (VEGF) and prostacyclin may regulate fibroblast activity. The objective was to study the synthesis of VEGF and interactions with prostacyclin and transforming growth factor (TGF)-β1 in lung fibroblasts from patients with COPD and healthy control subjects. To further explore the autocrine role of synthesized VEGF on fibroblast activity, studies were performed in human lung fibroblasts (HFL-1). Methods Primary distal lung fibroblast cultures were established from healthy individuals and from COPD patients (GOLD stage IV). Lung fibroblasts were stimulated with the prostacyclin analogue iloprost and the profibrotic stimuli TGF-β1. VEGF synthesis was measured in the cell culture medium. Changes in proliferation rate, migration and synthesis of the extracellular matrix (ECM) proteins proteoglycans were analysed after stimulations with VEGF-A isoform 165 (VEGF165; 1–10 000 pg/mL) in HFL-1. Results Iloprost and TGF-β1 significantly increased VEGF synthesis in both fibroblasts from COPD patients and control subjects. TGF-β1-induced VEGF synthesis was significantly reduced by the cyclooxygenase inhibitor indomethacin in fibroblasts from COPD patients. VEGF significantly increased proliferation rate and migration capacity in HFL-1. VEGF also significantly increased synthesis of the ECM proteins biglycan and perlecan. The VEGF receptors (VEGFR), VEGFR1, VEGFR2 and VEGFR3, were all expressed in primary lung fibroblasts and HFL-1. Conclusion VEGF is synthesized in high amounts by distal lung fibroblasts and may have a crucial role in ongoing vascular remodelling processes in the distal lung compartments.

Chronic lung allograft dysfunction including Bronchiolitis obliterans syndrome (BOS) is common after lung transplantation. Histologically, BOS is recognized as fibrotic lesions with accumulated extracellular matrix (ECM) in small airways. Lung fibroblasts are major producers of ECM and vascular endothelial growth factor (VEGF). In this study we hypothesize that VEGF is involved in BOS development after lung transplantation.We investigated the effect of profibrotic transforming growth factor (TGF-β) on VEGF synthesis in lung fibroblasts isolated from distal lung tissue biopsies taken from patients at 3, 6 and 12 months after lung transplantation (n = 14). Co-expression of VEGF receptor (VEGFR) 2 and collagen marker prolyl4-hydroxylase (p4OH) were analyzed in lung tissue from patients with BOS (n = 11).VEGF synthesis from distal derived lung fibroblasts were significantly lower 3 months after lung transplantation (168.6 ± 133.7; n = 7) compared to non-transplanted subjects (451.8 ± 185.9; n = 9; p = 0.0033) and increased over time at 6 months (584.1 ± 264.9; n = 9; p = 0.0033) and 12 months (451.1 ± 207.5; n = 8; p = 0.0065) post transplantation. TGF-β significantly induced VEGF synthesis at all time points. At 12 months post transplantation there was significantly less VEGF synthesis after TGF-β stimulation in fibroblasts obtained from BOS patients (1170 ± 450.2; n = 4) compared to patients without any chronic rejection process (1980 ± 417.9; n = 4; p 0.039). The numbers of cells expressing VEGFR2/p4OH were increased in patients with BOS (33.2 ± 10.9; n = 11) compared to control subjects (10.1 ± 9.9; n = 11; p 0.001).Our results support that changes in VEGF/VEGFR2 axis could be involved in BOS development and marker of poor outcome.

Pulmonary vascular remodelling is a characteristic sign in different lung disorders. Vascular endothelial growth factor (VEGF) is a key mediator in vascular remodelling. The objective was to study the role of VEGF on fibroblast activity, such as extracellular matrix (ECM) synthesis, migration and proliferative capacity and to study if VEGF is affected in different lung disorders. Studies were performed in human lung fibroblasts (HFL-1) and primary distal fibroblasts cultures from control subjects, patients with COPD (GOLD IV) and patients with bronchiolitis obliterans syndrome (BOS). Lung fibroblasts were stimulated with TGF-b1. VEGF165 synthesis was measured in the cell culture medium. Changes in synthesis of the ECM proteins proteoglycans and procollagen I, proliferation rate and migration were analysed after stimulations with VEGF165 (1-10000 pg/ml). TGF-β1 significantly enhanced VEGF synthesis in all cell types. There were no differences in VEGF synthesis between fibroblasts from control subjects and COPD patients, whereas patients with BOS had a higher VEGF synthesis (p In conclusion, VEGF is promoting specific ECM synthesis and fibroblast activation. VEGF may have a crucial role in the interplay between fibroblasts and other cells in vascular remodelling processes in the distal lung.

Involvement of pulmonary vascular remodelling is a characteristic sign in COPD. Vascular mediators such as vascular endothelial growth factor (VEGF) and prostacyclin may regulate fibroblast activity. The objective was to study the synthesis of VEGF and interactions with prostacyclin and transforming growth factor (TGF)-βPrimary distal lung fibroblast cultures were established from healthy individuals and from COPD patients (GOLD stage IV). Lung fibroblasts were stimulated with the prostacyclin analogue iloprost and the profibrotic stimuli TGF-βIloprost and TGF-βVEGF is synthesized in high amounts by distal lung fibroblasts and may have a crucial role in ongoing vascular remodelling processes in the distal lung compartments.

Background: Nanoparticles (1-100 nm) possess unusual properties related to size, shape, and chemical composition and can be used for specific targeting of cells and potential drug delivery. However, the use of nanoparticles for drug delivery into the lung requires careful evaluations, in vitro and ex vivo . In this study, we investigated the use of human lung fibroblasts (HFL-1) and precision-cut lung slices (PCLS) for testing nanoparticles. Methods: Lung fibroblasts and murine PCLS were stimulated with different sizes and concentrations of SiO2, CuO and Ag nanoparticles. Changes in cell viability were measured as amount of released lactate dehydrogenase (LDH) or proliferative capacity. Synthesis of extracellular matrix proteoglycans and procollagen I were analysed in fibroblasts after stimulation with Ag nanoparticles. Results: Silica-based nanoparticles (20 nm; 10 and 100 μg/ml) and CuO nanoparticles (20 nm; 1 μg/ml) did not affect cell viability from either fibroblasts or PCLS, whereas SiO2 (1000 μg/ml) and CuO (10 μg/ml) induced LDH release compared to untreated controls in both test systems. Ag nanoparticles (10 nm; 2 μg/ml) and (75 nm; 2 μg/ml) reduced proliferation rate in HFL-1 after 48 h (p Discussion: Our data indicate that depending on chemical composition, size and concentration of the nanoparticles different physiological responses are obtained. Careful characterization of nanoparticles is of importance for toxicity and therapeutic applications in nanomedicine.

Many lung-transplanted (LTx) patients develop obliterative bronchiolitis (OB) with deposition of extracellular matrix, accompanied by fibrocyte infiltration and increased lumen of vessels. In this study we hypothesize that fibrocytes, VEGF and prostacyclin (PGI2) are involved in remodelling processes after LTx. We investigated TGF-β activated peripheral fibroblast phenotypes regarding production of VEGF 165 /PGI2. Fibrocytes and fibroblast phenotypes were identified by prolyl4-hydroxylase (p4OH), CD45, VEGFR2 or PGIR. VEGF production from fibroblasts after LTx significantly decreased 3mo after LTx compared to healthy controls (p

Pulmonary fibrosis is characterized by excessive accumulation of connective tissue, along with activated extracellular matrix (ECM)‐producing cells, myofibroblasts. The pathological mechanisms are not well known, however serotonin (5‐HT) and 5‐HT class 2 (5‐HT 2) receptors have been associated with fibrosis. The aim of the present study was to investigate the role of 5‐HT 2B receptors in fibrosis, using small molecular 5‐HT 2B receptor antagonists EXT5 and EXT9, with slightly different receptor affinity. Myofibroblast differentiation [production of alpha‐smooth muscle actin (α‐SMA)] and ECM synthesis were quantified in vitro, and the effects of the receptor antagonists were evaluated. Pulmonary fibrosis was also modeled in mice by subcutaneous bleomycin administrations (under light isoflurane anesthesia), and the effects of receptor antagonists on tissue density, collagen‐producing cells, myofibroblasts and decorin expression were investigated. In addition, cytokine expression was analyzed in serum. Lung fibroblasts displayed an increased α‐SMA (P

Polyamines are organic cations, which are considered essential for normal cell cycle progression. This view is based on results from numerous studies using a variety of enzyme inhibitors or polyamine analogues interfering with either the metabolism or the physiological functions of the polyamines. However, the presence of non-specific effects may be hard to rule out in such studies. In the present study, we have for the first time used a transgenic cell system to analyze the importance of polyamines in cell growth. We have earlier shown that expression of trypanosomal ODC in an ODC-deficient variant of CHO cells (C55.7) supported growth of these otherwise polyamine auxotrophic cells. However, one of the transgenic cell lines grew much slower than the others. As shown in the present study, the level of ODC activity was much lower in these cells, and that was reflected in a reduction of cellular polyamine levels. Analysis of cell cycle kinetics revealed that reduction of growth was correlated to prolongation of the G1, S, and G2+M phases in the cells. Providing exogenous putrescine to the cells resulted in a normalization of polyamine levels as well as cell cycle kinetics indicating a causal relationship.

BACKGROUND: Lung transplantation (LTx) is established as a life-saving treatment in end-stage lung disease. However, long-term survival is hampered by the development of chronic rejection, almost synonymous with bronchiolitis obliterans syndrome (BOS). The rejection is characterized by deposition of extracellular matrix in small airways. Fibroblasts/myofibroblasts are the main producers of extracellular matrix molecules such as proteoglycans. This study compared fibroblast phenotype and activity in the wound healing process at different points after LTx in patients who later did, or did not, develop BOS. METHODS: Distally derived fibroblasts from patients 6 and 12 months after LTx and from healthy controls were analyzed for production of the proteoglycans versican, perlecan, biglycan, and decorin, with and without transforming growth factor (TGF)-I²(1). Fibroblast migration and proliferation were also studied. RESULTS: At 6 and 12 months after LTx, versican production was higher in fibroblasts from LTx patients (p < 0.01 p < 0.01) than from controls. Fibroblasts from patients who later developed BOS were more responsive to TGF-I²(1)-induced synthesis of versican and biglycan than patients without signs of rejection (p < 0.05). Production of perlecan and decorin was negatively correlated with fibroblast proliferation in fibroblasts at 6 months after LTx. In a more detailed case study of 2 patients, one with and one without BOS, the altered proteoglycan profile was associated with impaired lung function. CONCLUSIONS: LTx changes the phenotype of fibroblasts to a non-proliferative but extracellular matrix-producing cell due to wound healing involving TGF-I²(1). If not controlled, this may lead to development of BOS. (Less)

Ornithine decarboxylase (ODC) has a very fast turnover in mammalian cells, but is a stable enzyme in T. brucei and other trypanosmatid parasites like Leishmania donovani. However, Crithidia fasciculata, which is a phylogenetically closely related trypanosomatid to L. donovani, has an ODC with a rapid turnover. Interestingly, C. fasciculata ODC, but not L. donovani ODC, is rapidly degraded also in mammalian systems. In order to obtain information on what sequences are important for the rapid degradation of C. fasciculata ODC, we produced a variety of C. fasciculata/L. donovani ODC hybrid proteins and characterized their turnover using two different mammalian expression systems. The results obtained indicate that C. fasciculata ODC contains several sequence elements essential for the rapid turnover of the protein and that these regions are mainly located in the central part of the enzyme.

Background Prostacyclin analogs are potent vasodilators and possess anti-inflammatory properties. However, the effect of prostacyclin on extracellular matrix (ECM) in COPD is not well known. Collagen fibrils and proteoglycans are essential ECM components in the lung and fibroblasts are key players in regulating the homeostasis of ECM proteins. The aim was to study the synthesis of prostacyclin and its effect on fibroblast activity and ECM production, and in particular collagen I and the collagen-associated proteoglycans biglycan and decorin. Methods Parenchymal lung fibroblasts were isolated from lungs from COPD patients (GOLD stage IV) and from lungs and transbronchial biopsies from control subjects. The prostacyclin analog iloprost was used to study the effect of prostacyclin on ECM protein synthesis, migration, proliferation and contractile capacity of fibroblasts. Results TGF-β1 stimulation significantly increased prostacyclin synthesis in fibroblasts from COPD patients (p Conclusions Iloprost reduced collagen I synthesis and fibroblast contractility but did not affect the collagen-associated proteoglycans or proliferation rate in fibroblasts from COPD patients. Enhanced prostacyclin production could lead to improper collagen network fibrillogenesis and a more emphysematous lung structure in severe COPD patients.