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See detailEtude de la fibrose pulmonaire idiopathique canine: analyse du transcriptome, investigation des voies du TGF beta 1 et recherche de biomarqueurs
Krafft, Emilie ULg

Doctoral thesis (2014)

Canine idiopathic pulmonary fibrosis (cIPF) is a fibrotic disease of the pulmonary parenchyma, mainly seen in the West Highland white terrier. It is characterized by exercise intolerance and cough with a ... [more ▼]

Canine idiopathic pulmonary fibrosis (cIPF) is a fibrotic disease of the pulmonary parenchyma, mainly seen in the West Highland white terrier. It is characterized by exercise intolerance and cough with a progressive deterioration until death from respiratory insufficiency. Clinical, tomodensitometric and histological characteristics of cIPF have been described recently. However, this disease remains largely unknown and the clinicians are dealing with two major challenges: confirmation of the diagnosis, which requires many complementary exams, and absence of effective treatment. Identification of a targeted therapy is difficult without having a good understanding of the mechanisms leading to pulmonary parenchyma fibrosis. A similar disease, the idiopathic pulmonary fibrosis (IPF) is recognized in humans and cIPF might be interesting as a spontaneous model. This project in dogs was undertaken to answer, at least partly, to these challenges. The aims were to elucidate some mechanisms involved in cIPF pathogenesis and to identify biomarkers that could be used in the diagnosis process. The hypotheses were first that analysis of the transcriptome through microarray experiment would identify altered biological functions in cIPF, highlight specific molecules with an altered expression and identify potential biomarkers. Another hypothesis was the transforming growth factor beta 1 (TGFB1) pathways, considered central in the pathogenesis of IPF, would also be modified in cIPF. Finally, ET1, a known biomarker in human IPF, might also be an interesting biomarker in dogs. Gene expression analysis through microarray analysis, combined with the use of IPA, a data analysis program, identified altered biological functions in cIPF: cellular growth and proliferation, developmental processes, cellular movement, cell to cell signaling and interaction and antigen presentation. Some genes highlighted in the microarray experiment were then analyzed individually. Quantitative RT-PCR analysis confirmed an upregulation of the expression of CCL2, CCL7, CXCL14, IL8 and FAP (fibroblast activation protein) as well as a downregulation of the expression of PLUNC (palate, lung and nasal epithelium associated). We then complete the gene expression analysis with a search for potential biomarkers. Thirty-four potential biomarkers were identified with 32 biomarkers potentially measurable in blood (including CCL2, serum amyloid 1, IL8) and 2 biomarkers measurable only in the bronchoalveolar lavage fluid (BALF) (PLUNC and mesothelin). This approach was validated by measurement in serum of one of this biomarker: CCL2. CCL2 serum concentration was higher in affected WHWT compared to healthy WHWT and also higher in dogs with cIPF compared to dogs with chronic bronchitis (CB) or eosinophilic bronchopneumopathy (EBP). Based on serum CCL2 determination, cIPF was diagnosed with a sensibility of 92% and a specificity of 80%. We then studied TGFB1 and part of its storage, activation and signaling pathways. TGFB1 gene expression was not significantly different in the pulmonary parenchyma between affected and control dogs. However, in affected dogs, increased TGFB1 protein levels were seen by immunohistochemistry in fibrotic areas. High expression of bothTGFB1 type I receptor and phosphorylated Smad2/3, markers of an active intracellular TGFB1 signal, were seen in epithelial cells. No difference in expression for the storage proteins LTBP1 and LTBP2 was seen while expression of LTBP4 was significantly decreased in dogs with cIPF. Concerning the proteins involved in TGFB1 activation, gene expression was decreased for integrin subunit β8, increased for thrombospondin-1 and not modified for integrin subunit β6. Expression of Smad 7, involved in intracellular TGFB1 signal inhibition, was not modified. No difference for TGFB1 serum concentration was seen between WHWT with cIPF and healthy WHWT. A multivariate analysis performed on healthy dogs showed no age effect but a significant breed effect with higher levels in predisposed breeds. We evaluated part of the serotonin pathway, as one of its receptor (5HTR2B) was highlighted during the gene expression analysis. Serotonin has also been involved in the pathogenesis of human IPF and described to be of potential use as a biomarker in degenerative mitral valve disease in dogs. Expression of 2 serotonin receptors (5HTR2A and 5HTR2B), evaluated by quantitative RT-PCR in pulmonary tissue, was not different between dogs with cIPF and control dogs while expression of the serotonin transporter (5HTT) was significantly lower in affected dogs. No difference in serotonin serum level was seen between affected and healthy WHWT or between dogs with cIPF, CB or EBP. ET1 was evaluated as a biomarker in serum and BALF. ET1 serum concentration was not different between healthy WHWT and Beagles. Covariance analysis did not reveal any significant age effect. Serum levels were significantly higher in dogs with cIPF compared to dogs with CB or EBP. ROC curve analysis was then used to evaluate its diagnostic performances. The area under the curve was 0,818 with a sensitivity of 91.7% and a specificity of 87.5%. ET1 was also measured in the BALF in a small number of dogs. Its concentration was measurable in all dogs with cIPF while it was below the detection limit in all other dogs tested (healthy and with CB). Even though cIPF and human IPF are not completely identical from clinical, tomodensitometric or histological points of view, these results show that both canine and human diseases share molecular pathways, supporting the idea that cIPF might have an interest as spontaneous model. This work allowed a better understanding of cIPF pathogenesis. Gene expression analysis in the pulmonary parenchyma of affected dogs first identified several altered biological functions that should be analyzed in details in further studies. A more targeted analysis of some genes confirmed an upregulated expression of CCL2, CCL7, CXCL14, IL8 and FAP. Such a positive regulation of the expression of various inflammatory cytokines tends to suggest that inflammation might have a role in cIPF pathogenesis. Some of these cytokines also have profibrotic properties. PLUNC was one of the top down-regulated genes. Roles of the protein are still largely unknown; it might have a role in the inflammatory response and in the innate immunity. Developmental pathways were also altered in cIPF and quantitative RT-PCR confirmed an upregulation of FAP, a protein normally expressed in areas of tissue remodeling during fetal development and also positively regulated in human IPF. This study has shown that there is an active TGFB1 signal in the lungs of dogs with cIPF, especially at the level of the pathological epithelium. TGFB1 storage and activation pathways also seemed to be altered. Elevated TGFB1 circulating levels were found in predisposed breeds, which might explain at least partly their susceptibility for cIPF. Because of these results and its well-known profibrotic properties, we can suggest that TGFB1 is probably involved in cIPF pathogenesis and that modulation of its storage, activation or intracellular signaling might offer potential therapeutic targets. Our preliminary results are not in favor of a significant modification of the serotonin pathways in cIPF, although a decreased expression of 5HTT was seen in affected dogs and might have an impact on the amount of serotonin present locally. However, other studies are needed to conclude. Finally, several potential biomarkers have been identified and some of them were evaluated in details. While serum measurements performed for TGFB1 and serotonin indicated that these molecules have no interest as diagnostic biomarkers, ET1 and CCL2 were identified as interesting candidates with good diagnostic performances. However these results need to be confirmed in an independent validation cohort and the interest of combining both biomarkers should be evaluated. [less ▲]

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See detailCorrelation between thoracic ct-scan angiography findings and echocardiographic right pulmonary vein to pulmonary artery ratio in west highland white terriers with idiopathic pulmonary fibrosis.
Roels, Elodie ULg; Merveille, Anne-Christine ULg; Couvreur, T. et al

in Proceedings of the 24th Ecvim Meeting, Mainz, Germany - 4-6 September 2014 (2014, September)

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See detailVascular endothelial growth factor : a blood biomarker in canine pulmonary fibrosis.
Roels, Elodie ULg; Krafft, Emilie ULg; Heikkilä, H.P. et al

in Proceedings of the 24th Ecvim Meeting, Mainz, Germany - 4-6 September 2014 (2014, September)

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See detailLong-term outcome and use of 6-Minute Walk test in West Highland white Terriers with idiopathic pulmonary fibrosis
Lilja-Maula, LIO; Laurila, HP; Syrjä, P et al

in Journal of Veterinary Internal Medicine (2014), 28(2), 379-385

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See detailThe histopathology of idiopathic pulmonary fibrosis in West Highland white terriers shares features of both non-specific interstitial pneumonia and usual interstitial pneumonia in man
Syrjä, P; Heikkilä, HP; Lilja-Maula, L et al

in Journal of Comparative Pathology (2013), 149(2-3), 303-313

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See detailIs the CXC-Chemokine CXCL8 involved in the breed predisposition of west highland white terrier to canine idiopathic pulmonary fibrosis ?
Roels, Elodie ULg; Krafft, Emilie ULg; Laurila, HP et al

in Proceedings of the 23th ECVIM Meeting (2013, September)

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See detailIMMUNOHISTOCHEMICAL ANALYSIS OF TRANSFORMING GROWTH FACTOR BETA 1 AND ITS SIGNALLING PATHWAYS IN CANINE IDIOPATHIC PULMONARY FIBROSIS
Krafft, Emilie ULg; Laurila, HP; Rajamäki, MM et al

in Proceedings of the 23th ECVIM Meeting (2013, September)

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See detailIs 5-Hydroxytryptamine (serotonin) involved in the pathogenesis of idiopathic pulmonary fibrosis in dogs ?
Krafft, Emilie ULg; Roels, Elodie ULg; Laurila et al

in Proceedings of the 23th ECVIM Meeting (2013, September)

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See detailImmunohistochemical transforming growth factor Beta1 and its activation, storage and signalling pathways in canine idiopathic pulmonary fibrosis
Krafft, Emilie ULg; Laurila, HP; Rajamäki, MM et al

in Proceedings of the 23th ECVIM Meeting (2013, September)

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See detailAnalysis of gene expression in canine idiopathic pulmonary fibrosis
Krafft, Emilie ULg; Laurila, HP; peters, IR et al

in Veterinary Journal (2013), 198(2), 479-486

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See detailCCL2 as a serum biomarker of idiopathic pulmonary fibrosis in dogs
Krafft, Emilie ULg; Roels, Elodie ULg; Heikkilä, H.P. et al

Poster (2012, October 19)

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See detailGene expression profiles in canine idiopathic pulmonary fibrosis
Krafft, Emilie ULg; Heikkilä, H.P.; Peters, I. et al

in Proceedings of 17th International consortium on lung and airways fibrosis (2012, October 01)

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See detailCCL2 as a serum biomarker of idiopathic pulmonary fibrosis in dogs
Krafft, Emilie ULg; Roels, Elodie ULg; Heikkila, H.P. et al

in Proceedings of 22nd ECVIM Meeting - Masstricht, Netherlands (2012, September)

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See detailTransforming growth factor-beta 1 and its activating pathways in canine idiopathic pulmonary fibrosis
Krafft, Emilie ULg; Heikkila, H. P.; Day, M.J. et al

in Proceedings of the 22nd ECVIM-CA Congress (2012, September)

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See detailInvestigation of cytokine expression in canine idiopathic pulmonary fibrosis
Krafft, Emilie ULg; Heikkilä, HP; Vanherberghen, Morgane ULg et al

in Proceeding of 29th VCRS Symposium (2011, November 02)

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