Fig 1.
Detection of epifascial lymphatic collectors in patients.
A) Intradermal injection of Patent Blue into the first two interdigital spaces of a patient’s foot. Note the centripetal flow of the marker in the lymphatics. B) Isolation of a lymphatic collector, which is marked blue (arrow), in the hypodermis of the thigh. C-G) Staining with antibodies against CD31 (green) and Prox1 (red) (C-F), and negative control with only secondary antibodies (G). Lymphatic endothelial cell express both of the markers (as seen at higher magnification in D, F). Vasa vasorum in the adventitia (a) and media (m) express only CD31. Bars = 150 μm in C; 25 μm in D; 15 μm in F, and 200 μm in E,G.
Fig 2.
Semi- and ultra-thin sections of human epifascial lymphatic collectors.
A) Semithin cross-section showing the collapsed lumen (L) of the collector. The media (M) is densely packed with smooth muscle cells (SMCs). Note the thin circular layer of SMCs (arrow) at the border to the adventitia. B-F) Ultrathin sections. B) TEM picture showing a capillary in the media of a collector, consisting of endothelial cells and pericytes. E, erythrocyte. C) TEM picture showing the lumen (L) of a lymphatic collector. The lymphatic endothelial cell possesses slender processes directed towards the SMCs in the media. Note the existence of dark (arrow) and light (asterisk) SMCs. D) TEM picture of the collector shown in A). The collapsed lumen is lined by lymphatic endothelial cells (LEC). Note the rivet-like junction (arrow) between a SMC and a LEC. E) TEM picture of the media showing SMCs. The majority of the SMCs has a dark cytoplasm, some are light (asterisk). F) TEM picture of a dark SMC with ‘starfish morphology’. The lumen (L) of the collector is lined by LECs. Bars = 150 μm in A, 2μm in B,C,E,F, and 1μm in D.
Fig 3.
Immunofluorescence studies of initial foreskin lymphatics and epifascial lymphatic collectors.
A-C) Initial lymphatics. Staining with the antibodies D2-40 (A, B) and CCBE1 (A, C). D2-40 marks LECs, while CCBE1 demarcates both LECs and blood vascular endothelium. Bar = 50μm. D-F) Lymphatic collector. Staining with the antibodies D2-40 (D, E) and CCBE1 (D, F). D2-40 is not expressed, while CCBE1 demarcates the LECs of collectors. Bar = 200μm. G) LYVE-1 (red) marks initial lymphatics in foreskin. L, lumen of lymphatics; Ep, epidermis. Bar = 75μm. H) No LYVE-1 signal is detectable in lymphatic collectors. L, lumen of the collector. Dapi (blue) marks all nuclei. Bar = 150μm.
Fig 4.
Immunofluorescence studies of initial lymphatics and epifascial lymphatic collectors.
A-C) Staining of initial lymphatics with the antibodies Prox-1 (A, B) and vimentin (A, C). Vimentin is expressed in initial lymphatics. L, lumen of the lymphatics. Arrowheads point to the nuclei of LECs. Bar = 25μm. D) Staining of a lymphatic collector with antibodies against vimentin (green). Note expression in LECs, in numerous cells of the adventitia and some scattered cells in the media. Bar = 200μm. E) Focal expression of β-catenin in lymphatic collectors (L) and in larger vessels of the adventitia. Bar = 100μm. F) Focal expression of ESAM-1 in initial lymphatics (L) and strong expression in dermal capillaries. Dapi (blue) marks all nuclei. Bar = 35μm.
Fig 5.
TEM studies of ICLCs in the media of lymphatic collectors.
A) ICLC (arrow) with slender cell body and several cytoplasmic processes surrounded by SMCs. Bar = 5μm. B) Long process of an ICLC (arrows) in close contact to SMCs. Bar = 2μm. C) Higher magnification of B), showing accumulations of caveolae (arrows) in SMCs adjacent to the ICLC. Bar = 0.8μm. D) Peg-like protrusions of SMCs with caveolae (arrows) separated by dense plaques underlying the plasma membrane, adjacent to an ICLC process. Bar = 0.2μm.
Fig 6.
Immunofluorescence studies of ICLC in lymphatic collectors.
Staining with antibodies against vimentin (green) (A, B, D) and PDGFRα (red) (A, C). Note double-positive ramified ICLC (arrow in A-C) in the media of the collector. Bar = 50μm. D) Higher magnification showing vimentin-positive cells with long processes (arrows), which possess varicose-like swellings. Dapi (blue) marks all nuclei. Bar = 35μm.
Fig 7.
Immunofluorescence studies of lymphatic collectors and dermis.
A, B) Staining with anti-cKIT (CD117) antibodies of lymphatic collectors. Granulated, round cells that express c-KIT (arrows) are located in the adventitia of the collectors. Bar = 250μm in A, and 25μm in B. C-E) Staining of a lymphatic collectors with anti-CD34 (red) and anti-αSMA (green) antibodies. C,D) In large collectors, CD34+ cells (arrow) are found in the adventitia, and in the outer parts of the media between the αSMA-positive SMCs. Bar = 150μm. D) Higher magnification of C showing nucleated CD34+ cells (arrow). No double-positive cells are visible. Bar = 60μm. E) Smaller caliber collector stained with anti-CD34 (red) and anti-αSMA (green) antibodies. CD34+ cells are found in all parts of the media. Bar = 200μm. F) Staining of foreskin with anti-CD34 (green) and anti-Lyve-1 (red) antibodies. Blood capillaries beneath the epidermis express CD34. The Lyve-1-positive lymphatics are CD34-negative. Dapi (blue) marks all nuclei. Bar = 150μm.