Tight junction protein Claudin-5 and lung fibrosis

Am J Physiol Lung Cell Mol Physiol. 2011 Oct 14. [Epub ahead of print]
Altered expression of tight junction molecules in alveolar septa in lung injury and fibrosis.
Ohta H, Chiba S, Ebina M, Furuse M, Nukiwa T.
Source
1Tohku University Graduate school of Medicine.
Abstract
The dysfunction of alveolar barriers is a critical factor in the development of lung injury and subsequent fibrosis, but the underlying molecular mechanisms remain poorly understood. To clarify the pathogenic roles of tight junctions in lung injury and fibrosis, we examined the altered expression of claudins, the major components of tight junctions, in the lungs of disease models with pulmonary fibrosis. Among the 24 known claudins, claudin-1, claudin-3, claudin-4, claudin-7, and claudin-10 were identified as components of airway tight junctions. Claudin-5 and claudin-18 were identified as components of alveolar tight junctions and were expressed in endothelial and alveolar epithelial cells, respectively. In experimental bleomycin-induced lung injury, the levels of mRNA encoding tight junction proteins were reduced, particularly that of claudin-18. The integrity of the epithelial tight junctions was disturbed in the fibrotic lesions 14 days after the intraperitoneal instillation of bleomycin. These results suggest that bleomycin mainly injured alveolar epithelial cells and impaired alveolar barrier function. In addition, we analyzed the influence of transforming growth factor-β (TGF-β), a critical mediator of pulmonary fibrosis that is upregulated after bleomycin-induced lung injury, on tight junctions in vitro. The addition of TGF-β decreased the expression of claudin-5 in human umbilical vein endothelial cells (HUVECs) and disrupted the tight junctions of epithelial cells (A549). These results suggest that bleomycin-induced lung injury causes pathogenic alterations in tight junctions and that such alterations seem to be induced by TGF-β.

PMID: 22003091 [PubMed - as supplied by publisher]

SPARC in lung fibrosis

A recent study with bone marrow chimera mice suggested that, bleomycin induces inflammation and fibrosis in wild type through TNF synthesis that triggers TGF-β release; TGF-β promotes fibroblasts deposition and regulates TNF synthesis from macrophages.

B: In WT>SPARC KO chimeras, despite a normal parenchyma inflammation, collagen deposition by fibroblasts is greatly reduced and results in milder fibrosis.

C: In SPARC KO>WT chimeras, the inability of SPARC KO macrophages to down-modulate TNF production in response to TGF-β results in exaggerated and persistent inflammation and severe fibrosis.


Reference
Am J Pathol. 2011 Oct 11. [Epub ahead of print]
SPARC Oppositely Regulates Inflammation and Fibrosis in Bleomycin-Induced Lung Damage.
Sangaletti S, Tripodo C, Cappetti B, Casalini P, Chiodoni C, Piconese S, Santangelo A, Parenza M, Arioli I, Miotti S, Colombo MP.
Source
Molecular Immunology Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy.
Abstract
Fibrosis results from inflammatory tissue damage and impaired regeneration. In the context of bleomycin-induced pulmonary fibrosis, we demonstrated that the matricellular protein termed secreted protein acidic and rich in cysteine (SPARC) distinctly regulates inflammation and collagen deposition, depending on its cellular origin. Reciprocal Sparc(-/-) and wild-type (WT) bone marrow chimeras revealed that SPARC expression in host fibroblasts is required and sufficient to induce collagen fibrosis in a proper inflammatory environment. Accordingly, Sparc(-/-) WT chimeras showed exacerbated inflammation and fibrosis due to the inability of Sparc(-/-) macrophages to down-regulate tumor necrosis factor production because of impaired responses to tumor growth factor-β. Hence, the use of bone marrow cells expressing a dominant-negative form of tumor growth factor-β receptor type II under the monocyte-specific CD68 promoter, as a decoy, phenocopied Sparc(-/-) donor chimeras. Our results point to an unexpected dual role of SPARC in oppositely influencing the outcome of fibrosis.
Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
PMID: 22001347 [PubMed - as supplied by publisher]

CD69 and lung fibrosis

CD69-/- mice exhibited reduced inflammation and fibrosis.

Reference:
Respir Res. 2011 Oct 5;12(1):131. [Epub ahead of print]
Attenuation of lung inflammation and fibrosis in CD69-deficient mice after intratracheal bleomycin.
Yamauchi K, Kasuya Y, Kuroda F, Tanaka K, Tsuyusaki J, Ishizaki S, Matsunaga H, Iwamura C, Nakayama T, Tatsumi K.
Abstract
ABSTRACT:

BACKGROUND:
Cluster of differentiation 69 (CD69), an early activation marker antigen on T and B cells, is also expressed on activated macrophages and neutrophils, suggesting that CD69 may play a role in inflammatory diseases. To determine the effect of CD69 deficiency on bleomycin(BLM)-induced lung injury, we evaluated the inflammatory response following intratracheal BLM administration and the subsequent fibrotic changes in wild type (WT) and CD69-deficient (CD69-/-) mice.

METHODS:
The mice received a single dose of 3 mg/kg body weight of BLM and were sacrificed at 7 or 14 days post-instillation (dpi). Lung inflammation in the acute phase (7 dpi) was investigated by differential cell counts and cytokine array analyses of bronchoalveolar lavage fluid. In addition, lung fibrotic changes were evaluated at 14 dpi by histopathology and collagen assays. We also used reverse transcription polymerase chain reaction to measure the mRNA expression level of transforming growth factor beta1 (TGF-beta1) in the lungs of BLM-treated mice.

RESULTS:
CD69-/- mice exhibited less lung damage than WT mice, as shown by reductions in the following indices: (1) loss of body weight, (2) wet/dry ratio of lung, (3) cytokine levels in BALF, (4) histological evidence of lung injury, (5) lung collagen deposition, and (6) TGF-beta1 mRNA expression in the lung.

CONCLUSION:
The present study clearly demonstrates that CD69 plays an important role in the progression of lung injury induced by BLM.

PMID: 21970554 [PubMed - as supplied by publisher] Free

Immunoglobulin free light chains are increased in hypersensitivity pneumonitis and IPF

from PLoSOne
PLoS One. 2011;6(9):e25392. Epub 2011 Sep 28.
Immunoglobulin free light chains are increased in hypersensitivity pneumonitis and idiopathic pulmonary fibrosis.
Groot Kormelink T, Pardo A, Knipping K, Buendía-Roldán I, García-de-Alba C, Blokhuis BR, Selman M, Redegeld FA.
Source
Division of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
Abstract
BACKGROUND:
Idiopathic pulmonary fibrosis (IPF), a devastating lung disorder of unknown aetiology, and chronic hypersensitivity pneumonitis (HP), a disease provoked by an immunopathologic reaction to inhaled antigens, are two common interstitial lung diseases with uncertain pathogenic mechanisms. Previously, we have shown in other upper and lower airway diseases that immunoglobulin free light chains (FLCs) are increased and may be involved in initiating a local inflammation. In this study we explored if such a mechanism may also apply to HP and IPF.

METHODS:
In this study we examined the presence of FLC in serum and BAL fluid from 21 IPF and 22 HP patients and controls. IgG, IgE and tryptase concentrations were measured in BAL fluid only. The presence of FLCs, plasma cells, B cells and mast cells in lung tissue of 3 HP and 3 IPF patients and 1 control was analyzed using immunohistochemistry.

RESULTS:
FLC concentrations in serum and BAL fluid were increased in IPF and HP patients as compared to control subjects. IgG concentrations were only increased in HP patients, whereas IgE concentrations were comparable to controls in both patient groups. FLC-positive cells, B cells, plasma cells, and large numbers of activated mast cells were all detected in the lungs of HP and IPF patients, not in control lung.

CONCLUSION:
These results show that FLC concentrations are increased in serum and BAL fluid of IPF and HP patients and that FLCs are present within affected lung tissue. This suggests that FLCs may be involved in mediating pathology in both diseases.

PMID: 21980441 [PubMed - in process] PMCID: PMC3182208

BIBF 1120 for IPF

BIBF 1120 is developed  by Boehringer Ingelheim. It is a tyrosine kinase inhibitor targeting multiple tyrosine kinase receptors including platelet-derived growth factor receptors (PDGFR) α and β, vascular endothelial growth factor receptors
(VEGFR) 1, 2, and 3, and fibroblast growth factor receptors (FGFR) 1, 2, and 3.

A recent 12-month, randomized, double-blind, placebo-controlled, phase 2 trial (To Improve Pulmonary Fibrosis with BIBF 1120 [TOMORROW]) with 4 doses (50 mg once a day, 50 mg twice a day, 100 mg twice a day, or 150 mg twice a day) may have some positive effect (N = 428 patients). Although highest dose was associated with significant side effects, 150 mg twice a day was associated with a trend toward a reduction in the decline in lung function, with fewer acute exacerbations and preserved quality of life. Although the differences in primary end point among the study groups were not statistically significant, the benefits observed in the study were indeed clinically relevant.

It is encouraging that the trial was, at least, not another failed IPF trial, compared to others (pirfenidone, systemic glucocorticoids, azathioprine and cyclophosphamide, interferon-γ-1b, phosphodiesterase-5 inhibitors, or endothelin receptor antagonists).

I do hope that phase III trial gives more positive news. Since lower doses did not show much, I would guess that next  trial uses highest dose, 150 mg b.i.d.

References:
Richeldi L, et al. Efficacy of a tyrosine kinase inhibitor in idiopathic pulmonary fibrosis. N Engl J Med. 2011 Sep 22;365(12):1079-87. PubMed PMID: 21992121.
NEJM link: http://www.nejm.org/doi/full/10.1056/NEJMoa1103690.


Downey GP. Resolving the scar of pulmonary fibrosis. N Engl J Med. 2011 Sep
22;365(12):1140-1. PubMed PMID: 21992127.
NEJM link: http://www.nejm.org/doi/full/10.1056/NEJMe1108558.

FoxO3a and Lung Fibrosis

Dr. Henke and associates recent suggested that FoxO3a is largely inactive in IPF fibroblasts to promote IPF fibroblast proliferation. They previously demonstrated that lower Pten activity in IPF fibroblasts, higher PI3K-AKT activity on collagen matrix. In this study, the authors showed that phosphorylated, inactive, FoxO3 is higher, whereas total FoxO3 is lower, in IPF fibroblasts on collagen. They went on to show that p27 was lower in IPF.  They proposed model: 

Reference:
Nho RS, Hergert P, Kahm J, Jessurun J, Henke C. Pathological Alteration of
FoxO3a Activity Promotes Idiopathic Pulmonary Fibrosis Fibroblast Proliferation
on Type I Collagen Matrix. Am J Pathol. 2011 Sep 3. [Epub ahead of print] PubMed PMID: 21893017.

Human Lung Stem Cells?

Recently, Dr. Piero Anversa of Brigham and Women’s reported identification of human lung stem cells, appeared in May 12, 2011, issue of New England Journal of Medicine. The flow cytometric identification was based on c-kit antigen. The authors showed that the stem cells isolated were clonal and pluripotent. c-kit+ cells expressed KLF4, nanog,, oct4, and sox2. In a cryo injury mouse model, the authors showed that c-kit+  cells were, amazingly, able to promote lung regeneration!

In an editorial, Dr. Chapman of UCSF stated that "Kajstura et al. resets the starting point for clinical investigation into the role of stem cells and progenitor cells in lung disease". 

However, several scientists in the field were more skeptical. In Nature Medicine Community Corner, Dr. Stripp and Dr. Hogan of Duke questioned the experimental methodology (eg, RT-PCR and antibody staining) and proper controls.  They urged that more vigorous validation is needed. More importantly, the c-kit-CreER mice is needed to trace if the c-kit+ cells in the LUNG are pluripotent, hierarchical differentiation. Dr. Thannickal of UAB also suggested “The phenotype and fate of endogenous lung stem cells and associated alterations of their tissue niche in disease states will need to be characterized.” He also questioned if there is any deficiency in c-kit+ cells in COPD or IPF. In a reply, Dr. Anversa sounded very angry implying that the comments by Drs. Hogan and Stripp were "border on personal attacks".

Dr. Anversa's early work "Bone marrow cells regenerate infarcted myocardium" published in Nature 2001, stated that Lin- c-kit+ bone marrow cells were able to generate myocardium de novo. Dr. Weissman demonstrated that BM hematopoietic stem cells (KTLS) robustly reconstituted peripheral blood leukocytes, but did not contribute appreciably to any nonhematopoietic tissues, indicating that "transdifferentiation" of circulating HSCs and/or their progeny is an extremely rare event, if it occurs at all. 

References

Kajstura J, Rota M, Hall SR, Hosoda T, D'Amario D, Sanada F, Zheng H, Ogórek B, Rondon-Clavo C, Ferreira-Martins J, Matsuda A, Arranto C, Goichberg P, Giordano G, Haley KJ, Bardelli S, Rayatzadeh H, Liu X, Quaini F, Liao R, Leri A, Perrella MA, Loscalzo J, Anversa P. Evidence for human lung stem cells. N Engl J Med. 2011 May 12;364(19):1795-806. PubMed PMID: 21561345.

Chapman HA. Toward lung regeneration. N Engl J Med. 2011 May
12;364(19):1867-8. PubMed PMID: 21561353.

Hogan, B., Stripp, B. & Thannickal, V.J. Lung stem cells: looking beyond the hype. Nat Med. 2011 Jul 7;17(7):788-9. doi: 10.1038/nm0711-788. PubMed PMID: 21738157.

Anversa P, Kajstura J, Leri A, Loscalzo J. Tissue-specific adult stem cells in the human lung. Nat Med. 2011 Sep 7;17(9):1038-9. doi: 10.1038/nm.2463. PubMed PMID: 21900912.

Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, Pickel J,
McKay R, Nadal-Ginard B, Bodine DM, Leri A, Anversa P. Bone marrow cells regenerate infarcted myocardium. Nature. 2001 Apr 5;410(6829):701-5. PubMed PMID:11287958. 

Wagers AJ, Sherwood RI, Christensen JL, Weissman IL. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science. 2002 Sep 27;297(5590):2256-9. Epub 2002 Sep 5. PubMed PMID: 12215650.