Composition of the extracellular matrix of lymphatic novel threadlike structures: Is it keratin?

Background. The lumen of novel threadlike structures (NTSs) is enclosed by a single layer of endothelial cells surrounded by extracellular matrix (ECM). We hypothesized that collagen may be a component of the ECM associated with lymphatic NTSs. Methods. Six female New Zealand white rabbits were anesthetized, and the NTS structures within lymphatic vessels were identified by contrast-enhanced stereomicroscopy or alcian blue staining. Isolated NTS specimens were stained with acridine orange, YOYO-1, and 1,1′-dioctadecyl- 3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). The structural and molecular composition of the ECM was investigated using transmission electron microscopy (TEM), electrospray ionization-mass spectrometry, and proteomic analysis. Results. The lymph vessel wall was stained red by DiI, and rod-shaped nuclei were stained green by YOYO-1. The area surrounding the NTS was also stained red and contained green rod-shaped nuclei. TEM images showed that the NTS consisted of many ECM fibers and the ECM fibers appeared to be 100 nm in diameter and had narrowly spaced striated bands. Proteomic analysis of the lymphatic NTS-associated ECM identified 4 proteins: keratin 10, cytokeratin 3, cytokeratin 12, and soluble adenylyl cyclase. Conclusion. The TEM study suggested that the lymphatic NTS-associated ECM did not contain collagen. This was confirmed by proteomic analysis, which showed that keratin was the major component of the ECM.