Levels of circulating endothelial cells are low in idiopathic pulmonary fibrosis and are further reduced by anti-fibrotic treatments

BMC Medicine

Andrea Cossarizza⁶  Luca Richeldi³  Giacomo Sgalla³  Carlo Vancheri⁷  Alessandro Sanduzzi Zamparelli⁵  Marialuisa Bocchino⁵  Luigi Zucchi²  Cristiano Matteo Carbonelli²  Fabrizio Luppi⁸  Gloria Montanari⁸  Elisa Persiani⁸  Lara Gibellini⁴  Elena Bianchini¹  Stefania Cerri⁸  Sara De Biasi⁴ 

[1] Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy;Pulmonology Unit, Department of Cardiology, Thoracic and Vascular Surgery and Critical Care Medicine, IRCCS - Arcispedale Santa Maria Nuova, Reggio Emilia, Italy;Department of Respiratory Medicine, University of Southampton, Southampton, UK;Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia School of Medicine, via Campi, 287-41125, Modena, Italy;Respiratory Medicine Section, Department of Clinical Medicine and Surgery, “Federico II” University of Naples, Naples, Italy;Dipartimento Sperimentale Interaziendale, Campus San Lazzaro, University of Modena and Reggio Emilia, Reggio Emilia, 42122, Italy;Regional Centre for Rare Lung Diseases, Department of Clinical and Experimental Medicine, University of Catania, Catania, Italy;Department of Medical and Surgical Sciences for Children and Adults, University of Modena and Reggio Emilia, Modena, Italy

关键词: Pirfenidone;    Nintedanib;    Idiopathic pulmonary fibrosis;    Endothelial cells;    Circulating fibrocytes;   

Others  :  1232406
DOI  :  10.1186/s12916-015-0515-0

 received in 2015-07-14, accepted in 2015-10-16,  发布年份 2015

【 摘 要 】

Background

It has been suggested that circulating fibrocytes and endothelial cells actively participate in the intense remodelling of the pulmonary vasculature in patients with idiopathic pulmonary fibrosis (IPF). Indeed, fibrotic areas exist that have fewer blood vessels, whereas adjacent non-fibrotic tissue is highly vascularized. The number of circulating endothelial cells (CEC) and endothelial progenitor cells (EPC) might reflect the balance between vascular injury and repair. Thus, fibrocytes as well as endothelial cells could potentially be used as biomarkers of disease progression and treatment outcome.

Methods

Peripheral blood samples were collected from 67 patients with a multidisciplinary diagnosis of IPF and from 45 age-matched and sex-matched healthy volunteers. Buffy coat was isolated according to standard procedures and at least 20 million cells were stained with different monoclonal antibodies for the detection of CEC, EPC and circulating fibrocytes. For the detection of CEC and EPC, cells were stained with anti-CD45, anti-CD34, anti-CD133, anti-CD14, anti-CD309 and with the viability probe Far-Red LIVE/DEAD. For the detection of circulating fibrocytes, cells were first stained with LIVE/DEAD and the following monoclonal antibodies: anti-CD3, anti-CD19, anti-CD45, anti-CD34 and anti-CD14, then cells were fixed, permeabilized and stained with fluorochrome-conjugated anti-collagen I monoclonal antibodies.

Results

Patients with IPF displayed almost undetectable levels of circulating fibrocytes, low levels of CEC, and normal levels of EPC. Patients treated with nintedanib displayed higher levels of CEC, but lower levels of endothelial cells expressing CD309 (the type II receptor for vascular endothelial growth factor). Treatment with both nintedanib and pirfenidone reduced the percentage of CEC and circulating fibrocytes.

Conclusions

Levels of CEC were reduced in patients with IPF as compared to healthy individuals. The anti-fibrotic treatments nintedanib and pirfenidone further reduced CEC levels. These findings might help explain the mechanism of action of these drugs and should be explored as predictive biomarkers in IPF.

【 授权许可】

   
2015 De Biasi et al.

【 图 表 】

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

【 参考文献 】

• [1]Collard HR, Bradford WZ, Cottin V, Flaherty KR, King TE, Koch GG et al.. A new era in idiopathic pulmonary fibrosis: considerations for future clinical trials. Eur Respir J. 2015; 46(1):243-9.
• [2]Richeldi L. Efficacy endpoints for idiopathic pulmonary fibrosis trials. Lancet Respir Med. 2015; 3(5):335-7.
• [3]Vij R, Noth I. Peripheral blood biomarkers in idiopathic pulmonary fibrosis. Transl Res. 2012; 159(4):218-27.
• [4]King TE, Pardo A, Selman M. Idiopathic pulmonary fibrosis. Lancet. 2011; 378(9807):1949-61.
• [5]Andersson-Sjoland A, de Alba CG, Nihlberg K, Becerril C, Ramirez R, Pardo A et al.. Fibrocytes are a potential source of lung fibroblasts in idiopathic pulmonary fibrosis. Int J Biochem Cell Biol. 2008; 40(10):2129-40.
• [6]Bucala R, Spiegel LA, Chesney J, Hogan M, Cerami A. Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol Med. 1994; 1(1):71-81.
• [7]Strieter RM, Keeley EC, Burdick MD, Mehrad B. The role of circulating mesenchymal progenitor cells, fibrocytes, in promoting pulmonary fibrosis. Trans Am Clin Climatol Assoc. 2009; 120:49-59.
• [8]Metz CN. Fibrocytes: a unique cell population implicated in wound healing. Cell Mol Life Sci. 2003; 60(7):1342-50.
• [9]Quan TE, Cowper S, Wu SP, Bockenstedt LK, Bucala R. Circulating fibrocytes: collagen-secreting cells of the peripheral blood. Int J Biochem Cell Biol. 2004; 36(4):598-606.
• [10]Phillips RJ, Burdick MD, Hong K, Lutz MA, Murray LA, Xue YY et al.. Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest. 2004; 114(3):438-46.
• [11]Mehrad B, Burdick MD, Zisman DA, Keane MP, Belperio JA, Strieter RM. Circulating peripheral blood fibrocytes in human fibrotic interstitial lung disease. Biochem Biophys Res Commun. 2007; 353(1):104-8.
• [12]Abe R, Donnelly SC, Peng T, Bucala R, Metz CN. Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol. 2001; 166(12):7556-62.
• [13]Gomperts BN, Strieter RM. Fibrocytes in lung disease. J Leukoc Biol. 2007; 82(3):449-56.
• [14]Maharaj S, Shimbori C, Kolb M. Fibrocytes in pulmonary fibrosis: a brief synopsis. Eur Respir Rev. 2013; 22(130):552-7.
• [15]Smadja DM, Dorfmuller P, Guerin CL, Bieche I, Badoual C, Boscolo E et al.. Cooperation between human fibrocytes and endothelial colony-forming cells increases angiogenesis via the CXCR4 pathway. Thromb Haemost. 2014; 112(5):1002-13.
• [16]Malli F, Koutsokera A, Paraskeva E, Zakynthinos E, Papagianni M, Makris D et al.. Endothelial progenitor cells in the pathogenesis of idiopathic pulmonary fibrosis: an evolving concept. PLoS One. 2013; 8(1):e53658.
• [17]Smadja DM, Mauge L, Nunes H, d’Audigier C, Juvin K, Borie R et al.. Imbalance of circulating endothelial cells and progenitors in idiopathic pulmonary fibrosis. Angiogenesis. 2013; 16(1):147-57.
• [18]Strieter RM, Mehrad B. New mechanisms of pulmonary fibrosis. Chest. 2009; 136(5):1364-70.
• [19]Ebina M, Shimizukawa M, Shibata N, Kimura Y, Suzuki T, Endo M et al.. Heterogeneous increase in CD34-positive alveolar capillaries in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2004; 169(11):1203-8.
• [20]Cosgrove GP, Brown KK, Schiemann WP, Serls AE, Parr JE, Geraci MW et al.. Pigment epithelium-derived factor in idiopathic pulmonary fibrosis: a role in aberrant angiogenesis. Am J Respir Crit Care Med. 2004; 170(3):242-51.
• [21]Raghu G, Collard HR, Egan JJ, Martinez FJ, Behr J, Brown KK et al.. An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management. Am J Respir Crit Care. 2011; 183(6):788-824.
• [22]Cossarizza A, Bertoncelli L, Nemes E, Lugli E, Pinti M, Nasi M et al.. T cell activation but not polyfunctionality after primary HIV infection predicts control of viral load and length of the time without therapy. PLoS One. 2012; 7(12):e50728.
• [23]Cossarizza A, Cousin D. Overcoming challenges in cellular analysis: multiparameter analysis of rare cells. Science. 2015; 347(6220):443.
• [24]Guaraldi GZS, Cossarizza A, Vernacotola L, Carli F, Lattanzi A, Beghetto B et al.. Randomized trial to evaluate cardiometabolic and endothelial function in patients with plasma HIV-1 RNA suppression switching to darunavir/ritonavir with or without nucleoside analogues. HIV Clin Trials. 2013; 14(4):140-8.
• [25]Moeller A, Gilpin SE, Ask K, Cox G, Cook D, Gauldie J et al.. Circulating fibrocytes are an indicator of poor prognosis in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2009; 179(7):588-94.
• [26]Bournazos S, Fahim A, Hart SP. Identification of fibrocytes in peripheral blood. Am J Respir Crit Care Med. 2009; 180(12):1279.
• [27]Simler NR, Brenchley PE, Horrocks AW, Greaves SM, Hasleton PS, Egan JJ. Angiogenic cytokines in patients with idiopathic interstitial pneumonia. Thorax. 2004; 59(7):581-5.
• [28]Wollin L, Maillet I, Quesniaux V, Holweg A, Ryffel B. Antifibrotic and anti-inflammatory activity of the tyrosine kinase inhibitor nintedanib in experimental models of lung fibrosis. J Pharmacol Exp Ther. 2014; 349(2):209-20.
• [29]Richeldi L. Treatments for idiopathic pulmonary fibrosis. N Engl J Med. 2014; 371(8):783.
• [30]Inomata M, Kamio K, Azuma A, Matsuda K, Kokuho N, Miura Y et al.. Pirfenidone inhibits fibrocyte accumulation in the lungs in bleomycin-induced murine pulmonary fibrosis. Respir Res. 2014; 15:16. BioMed Central Full Text
• [31]Raghu G, Johnson WC, Lockhart D, Mageto Y. Treatment of idiopathic pulmonary fibrosis with a new antifibrotic agent, pirfenidone: results of a prospective, open-label phase II study. Am J Respir Crit Care Med. 1999; 159(4 Pt 1):1061-9.
• [32]Richeldi L, Cottin V, Flaherty KR, Kolb M, Inoue Y, Raghu G et al.. Design of the INPULSIS trials: two phase 3 trials of nintedanib in patients with idiopathic pulmonary fibrosis. Respir Med. 2014; 108(7):1023-30.
• [33]Richeldi L, du Bois RM, Raghu G, Azuma A, Brown KK, Costabel U et al.. Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis. N Engl J Med. 2014; 370(22):2071-82.
• [34]Spagnolo P, Maher TM, Richeldi L. Idiopathic pulmonary fibrosis: recent advances on pharmacological therapy. Pharmacol Ther. 2015; 152:18-27.
• [35]Tzouvelekis A, Bonella F, Spagnolo P. Update on therapeutic management of idiopathic pulmonary fibrosis. Ther Clin Risk Manag. 2015; 11:359-70.
• [36]Chaudhary NI, Roth GJ, Hilberg F, Muller-Quernheim J, Prasse A, Zissel G et al.. Inhibition of PDGF, VEGF and FGF signalling attenuates fibrosis. Eur Respir J. 2007; 29(5):976-85.
• [37]Hostettler KE, Zhong J, Papakonstantinou E, Karakiulakis G, Tamm M, Seidel P et al.. Anti-fibrotic effects of nintedanib in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis. Respir Res. 2014; 15(1):157. BioMed Central Full Text
• [38]Spagnolo P, Sverzellati N, Rossi G, Cavazza A, Tzouvelekis A, Crestani B et al.. Idiopathic pulmonary fibrosis: an update. Ann Med. 2015; 47(1):15-27.
• [39]Rangarajan S, Kurundkar A, Kurundkar D, Bernard K, Sanders YY, Ding Q, et al. Novel mechanisms for the anti-fibrotic action of nintedanib. Am J Respir Cell Mol. Biol. 2015, doi:10.1165/rcmb.2014-0445OC.