Removal of fatty acid-binding protein 4 positive stem cells affects the proliferation and self-renewal capability of clara cells in the lung

doi:10.3969/j.issn.2095-4344.1702

Abstract

Abstract:

BACKGROUND: In recent years, there have been many studies about fatty acid-binding protein 4 (FABP4); however, few studies focus on the expression and function of FABP4 in the lung.
OBJECTIVE: To systematically explore the expression and function of FABP4 in the lung.
METHODS: FABP4 Cre; Rosa26-tdTomato mice were selected to trace red fluorescent protein expression and immunofluorescence co-localization analysis was used to systematically detect the expression of FABP4 in the lung. We made a further study on function of FABP4 in lung by constructing FABP4 Cre; iDTR transgenic mice injected with diphtheria toxin to remove the FABP4⁺ cells from the lung.
RESULTS AND CONCLUSION: FABP4 could label macrophages, clara cells, type II alveolar cells, PDGFRα⁺ and vimentin⁺ mesenchymal cells in the lung of adult mice, and could not label smooth muscle cells and ciliated cells. In addition, percentages of clara cells and ciliated cells decreased when the FABP4⁺ cells were removed from the lung. We supposed that removal of FABP4⁺ cells with stem cell characteristics could influence the proliferation and self-renewal of clara cells in the lung. This study comprehensively revealed the expression pattern of FABP4 in lung of mice and preliminarily explored the FABP4 function in the lung, laying a foundation for the study of pulmonary immune response and homeostasis maintenance. Moreover, FABP4 may become a potential target for the treatment of lung diseases.

Key words: fatty acid binding protein 4, FABP4, lung, macrophage, interstitial cells, clara cells, National Natural Science Foundation of China

CLC Number:

Yang Ruichen, Liu Shuyue, Liu Huijuan. Removal of fatty acid-binding protein 4 positive stem cells affects the proliferation and self-renewal capability of clara cells in the lung[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(17): 2734-2738.

Figures/Tables 1

2.1 FABP4在成年小鼠肺中示踪使用成年FABP4 Cre; Rosa26-tdTomato小鼠，通过红色荧光蛋白来示踪FABP4在肺中的表达。结果显示，FABP4在小鼠肺泡区域可见红色荧光的表达，见图1A；在终末支气管周围也有表达，见图1B，尤其在肺泡区域表达较多。根据Tomato阳性细胞表达的位置可以判断，FABP4标记了肺中几种不同类型的细胞，提示其在肺中可能扮演了重要的角色。 2.2 FABP4能标记Ⅱ型肺泡细胞和clara细胞等上皮细胞实验研究了FABP4阳性细胞与肺中上皮细胞的共定位情况，图2中所示 PPC标记的Ⅱ型肺泡细胞在肺中分布比较均匀，排列在肺泡的边缘角落，细胞形态比较规则，FABP4阳性细胞与Ⅱ型肺泡细胞有部分共定位；同时检测了SCGB1A1标记的clara细胞与FABP4阳性细胞的共定位，观察到clara细胞特异性表达在支气管内部，排列整齐，FABP4只标记了一小部分clara细胞。作者推测肺支气管中的clara细胞来源于不同的谱系，而这群被标记的细胞与其他clara细胞不同。 2.3 FABP4能标记肺中间质细胞实验使用了两种间质细胞标记物PDGFRα和Vimentin来显示肺中的间质细胞，两种来源的间质细胞在肺中分布存在一些差异，其中PDGFRα阳性细胞分布在终末支气管和血管的周围，主要呈细丝状，Vimentin作为一个骨架蛋白在肺中呈现弥散性广泛分布。免疫荧光结果显示与Vimentin阳性间质细胞共标的FABP4阳性细胞比与PDGFRα阳性间质细胞共标的更多，见图3。 2.4 FABP4能标记肺中的巨噬细胞 F4/80可以特异性标记巨噬细胞，图4显示肺巨噬细胞主要分布于肺泡与肺间质中，偶见少量在支气管中游离存在。FABP4可以标记部分肺巨噬细胞，推测FABP4可能参与肺部的免疫应激反应。 2.5 FABP4不能标记肺平滑肌细胞和ciliated细胞经过与α-SMA和Tubulin免疫荧光染色分析发现，平滑肌表达在血管周围，呈现连续分布，在支气管周围也有间断存在的平滑肌层，但FABP4在这群细胞中无表达，见图5。FABP4来源细胞也没有标记支气管的纤毛细胞，说明FABP4这类细胞在肺的发育过程中可能没有参与肺中平滑肌细胞与支气管中纤毛细胞的形成。 2.6 FABP4阳性细胞去除的分析 FABP4 Cre;iDTR小鼠注射白喉毒素去除FABP4阳性细胞后，利用免疫荧光染色观察肺中上皮细胞和间质细胞的改变，结果显示：Vimentin阳性间质细胞略有下降，支气管中clara细胞和ciliated细胞均有不同程度的减少，见图6A，B，其对照iDTR小鼠肺中clara细胞比例为(60.73±2.82)%，FABP4 Cre;iDTR小鼠clara细胞比例下降至(53.62±3.10)%，P < 0.01，见图6C；对照iDTR小鼠ciliated细胞比例为(34.86±2.89)%，FABP4 Cre;iDTR小鼠ciliated细胞比例下降至(25.38±3.57)%，P < 0.01，见图6D，说明FABP阳性的这群clara细胞在肺中扮演极其重要的角色，对肺中clara细胞的再生与分化产生了影响。"

References

[1] Furuhashi M, Hotamisligil GS. Fatty acid-binding proteins: role in metabolic diseases and potential as drug targets. Nat Rev Drug Discov. 2008;7(6):489-503.

[2] Storch J, McDermott L. Structural and functional analysis of fatty acid-binding proteins. J Lipid Res. 2009;50 Suppl: S126-131.

[3] Mullassery D, Smith NP. Lung development. Semin Pediatr Surg. 2015; 24(4):152-155.

[4] Demircik F, Buch T, Waisman A. Efficient B cell depletion via diphtheria toxin in CD19-Cre/iDTR mice. PLoS One. 2013;8(3):e60643.

[5] Barkauskas CE, Cronce MJ, Rackley CR, et al. Type 2 alveolar cells are stem cells in adult lung. J Clin Invest. 2013;123(7):3025-3036.

[6] Zhang L, Whitsett JA, Stripp BR. Regulation of Clara cell secretory protein gene transcription by thyroid transcription factor-1. Biochim Biophys Acta. 1997;1350(3):359-367.

[7] Zepp JA, Zacharias WJ, Frank DB, et al. Distinct Mesenchymal Lineages and Niches Promote Epithelial Self-Renewal and Myofibrogenesis in the Lung. Cell. 2017;170(6):1134-1148.

[8] Hostettler KE, Gazdhar A, Khan P, et al. Multipotent mesenchymal stem cells in lung fibrosis. PLoS One. 2017;12(8):e0181946.

[9] Austyn JM, Gordon S. F4/80, a monoclonal antibody directed specifically against the mouse macrophage. Eur J Immunol. 1981;11(10):805-815.

[10] Mitchell J, Woodcock-Mitchell J, Reynolds S, et al. Alpha-smooth muscle actin in parenchymal cells of bleomycin-injured rat lung. Lab Invest. 1989; 60(5):643-650.

[11] Lee D, Wada K, Taniguchi Y, et al. Expression of fatty acid binding protein 4 is involved in the cell growth of oral squamous cell carcinoma. Oncol Rep. 2014;31(3):1116-1120.

[12] Uysal KT, Scheja L, Wiesbrock SM, et al. Improved glucose and lipid metabolism in genetically obese mice lacking aP2. Endocrinology. 2000;141(9):3388-3396.

[13] Agardh HE, Folkersen L, Ekstrand J, et al. Expression of fatty acid-binding protein 4/aP2 is correlated with plaque instability in carotid atherosclerosis. J Intern Med. 2011;269(2):200-210.

[14] Shum BO, Mackay CR, Gorgun CZ, et al. The adipocyte fatty acid-binding protein aP2 is required in allergic airway inflammation. J Clin Invest. 2006;116(8):2183-2192.

[15] von Eynatten M, Breitling LP, Roos M, et al. Circulating adipocyte fatty acid-binding protein levels and cardiovascular morbidity and mortality in patients with coronary heart disease: a 10-year prospective study. Arterioscler Thromb Vasc Biol. 2012;32(9):2327-2335.

[16] Wynn TA, Chawla A, Pollard JW. Macrophage biology in development, homeostasis and disease. Nature. 2013;496(7446):445-455.

[17] Lambrecht BN. Alveolar macrophage in the driver's seat. Immunity. 2006;24(4):366-368.

[18] Srikanth L, Venkatesh K, Sunitha MM, et al. In vitro generation of type-II pneumocytes can be initiated in human CD34(+) stem cells. Biotechnol Lett. 2016;38(2):237-242.

[19] Jiang Y, Ma S, Zhang H, et al. FABP4-mediated homocysteine-induced cholesterol accumulation in THP-1 monocyte-derived macrophages and the potential epigenetic mechanism. Mol Med Rep. 2016;14(1):969-976.

[20] Kuppe C, Kramann R. Role of mesenchymal stem cells in kidney injury and fibrosis. Curr Opin Nephrol Hypertens. 2016;25(4):372-377.

[21] Stone JS, Wisner SR, Bucks SA, et al. Characterization of Adult Vestibular Organs in 11 CreER Mouse Lines. J Assoc Res Otolaryngol. 2018;19(4):381-399.

[22] El Agha E, Kramann R, Schneider RK, et al. Mesenchymal Stem Cells in Fibrotic Disease. Cell Stem Cell. 2017;21(2):166-177.

[23] Dave JM, Bayless KJ. Vimentin as an integral regulator of cell adhesion and endothelial sprouting. Microcirculation. 2014;21(4):333-344.

[24] Li LF, Kao KC, Liu YY, et al. Nintedanib reduces ventilation-augmented bleomycin-induced epithelial-mesenchymal transition and lung fibrosis through suppression of the Src pathway. J Cell Mol Med. 2017;21(11): 2937-2949.

[25] Hamilton TG, Klinghoffer RA, Corrin PD, et al. Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms. Mol Cell Biol. 2003;23(11):4013-4025.

[26] Elizur A, Adair-Kirk TL, Kelley DG, et al. Clara cells impact the pulmonary innate immune response to LPS. Am J Physiol Lung Cell Mol Physiol. 2007;293(2):L383-392.

[27] Nkadi PO, Merritt TA, Pillers DA. An overview of pulmonary surfactant in the neonate: genetics, metabolism, and the role of surfactant in health and disease. Mol Genet Metab. 2009;97(2):95-101.

[28] Miyachi H, Reinhardt JW, Otsuru S, et al. Bone marrow-derived mononuclear cell seeded bioresorbable vascular graft improves acute graft patency by inhibiting thrombus formation via platelet adhesion. Int J Cardiol. 2018;266:61-66.

[29] Overholt KM, Otsuru S, Olson TS, et al. Identification of a murine CD45-F4/80lo HSC-derived marrow endosteal cell associated with donor stem cell engraftment. Blood Adv. 2017;1(27):2667-2678.

[30] Dyer EL. The lung: scientific foundations.Chest. 1992;101(2):24.