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. 2012 Jun 11;197(6):837-49.
doi: 10.1083/jcb.201110132. Epub 2012 Jun 4.

Smooth Muscle-Endothelial Cell Communication Activates Reelin Signaling and Regulates Lymphatic Vessel Formation

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Free PMC article

Smooth Muscle-Endothelial Cell Communication Activates Reelin Signaling and Regulates Lymphatic Vessel Formation

Sophie Lutter et al. J Cell Biol. .
Free PMC article

Abstract

Active lymph transport relies on smooth muscle cell (SMC) contractions around collecting lymphatic vessels, yet regulation of lymphatic vessel wall assembly and lymphatic pumping are poorly understood. Here, we identify Reelin, an extracellular matrix glycoprotein previously implicated in central nervous system development, as an important regulator of lymphatic vascular development. Reelin-deficient mice showed abnormal collecting lymphatic vessels, characterized by a reduced number of SMCs, abnormal expression of lymphatic capillary marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and impaired function. Furthermore, we show that SMC recruitment to lymphatic vessels stimulated release and proteolytic processing of endothelium-derived Reelin. Lymphatic endothelial cells in turn responded to Reelin by up-regulating monocyte chemotactic protein 1 (MCP1) expression, which suggests an autocrine mechanism for Reelin-mediated control of endothelial factor expression upstream of SMC recruitment. These results uncover a mechanism by which Reelin signaling is activated by communication between the two cell types of the collecting lymphatic vessels--smooth muscle and endothelial cells--and highlight a hitherto unrecognized and important function for SMCs in lymphatic vessel morphogenesis and function.

Figures

Figure 1.
Figure 1.
SMC recruitment and ECM deposition during collecting vessel formation. (A–H) Immunofluorescence staining of wild-type ear skin at the indicated stages (P12, P14, P16, or P21) with antibodies against LYVE-1 (green, A–C and E–G), α-SMA (red, A–D and H), desmin (red, E–G; green, H), PECAM-1 (blue, A–C and E–G), and Podoplanin (blue, D). Arrows in A indicate areas of LYVE-1 down-regulation before SMC recruitment. The arrow in D indicates an NG2-positive blood vessel, and the arrowhead in D indicates an NG2-negative collecting vessel. P, postnatal day. (I–L) Immunofluorescence staining of wild-type ear skin at P14 (I, I′, K, and K′) and P16 (J, J′, L, and L′) with antibodies against Collagen IV (green I–J′), Laminin-α5 (green K–L′), α-SMA (red), and LYVE-1 (blue). Arrows show Laminin-α5 expression in luminal valves and arrowheads indicate expression of Laminin-α5 by SMC. The broken line in K outlines the LYVE-1 collecting vessel. The asterisks in K and L show LYVE-1+ lymphatic capillaries. Bars, 50 µm. (M) Schematic timeline of dermal collecting lymphatic vessel differentiation.
Figure 2.
Figure 2.
Reelin is a lymphatic endothelial-specific matrix molecule that shows differential distribution in lymphatic capillaries and collecting vessels. (A and A′) Immunofluorescence of E16 dorsal skin for Reelin (green), LYVE-1 (red), and PECAM-1 (blue). Arrowheads and arrows indicates Reelin-positive and -negative lymphatic and blood vessels, respectively. (B–G′) Immunofluorescence staining of P6 mesentery (B–C′) and P21 ear-skin (D–G′), with antibodies against Reelin (green) and α-SMA (red). Blue staining (in B) is for PECAM-1 and (in D–E′) for LYVE-1. Boxed areas in D and E are magnified in D′ and E′, showing Reelin expression only. Vessel borders are highlighted by broken lines in B′, C′, D′, E′, and F′). Tissue was unpermeabilized in F–G′ to detect extracellular proteins, and the fluorescent signal was amplified using TSA technology. The arrowhead in F indicates weak Reelin immunoreactivity outside capillary LECs. Bars: (A, D, E, F and G) 50 µm; (B and C) 500 µm; (D’ and E’) 20 µm.
Figure 3.
Figure 3.
Reelin secretion and processing is stimulated by SMC contact. (A) qPCR analysis showing relative RELN expression in three independent samples of LEC, BEC, and HUVSMC and two samples of HAoSMC. Mean ± SEM is plotted (error bars). (B) Schematic of Reelin structure, showing antibody binding sites (asterisks), cleavage sites, and products. The arrows and arrowheads indicate polypeptides that are visualized in Western blotting: arrow, full-length protein; arrowhead, C-terminally cleaved polypeptide; open arrowhead, N- and C-terminally cleaved polypeptide. Adapted from Jossin et al. (2007). (C and D) Analysis of whole-cell lysate (C) and medium (D) of LEC and SMC cultured either alone or together by immunoprecipitation and Western blotting with antibodies to Reelin. The arrows indicates full-length Reelin, arrowheads indicate cleavage products. Analysis of whole cell lysate in C by Western blotting with antibodies to CD31/PECAM-1 (for LEC) and α-SMA (for SMC) show that equivalent numbers of cells were used. (E) Quantification of Reelin protein levels, normalized to CD31/PECAM-1, in whole cell lysate (black bars) and culture medium (gray bars). Data represent mean (n = 3) ± SEM (error bars). (F) Analysis of LEC-conditioned medium alone or after incubation with SMC. Note the cleavage product of 180 kD only in the presence of SMC (open arrowhead).
Figure 4.
Figure 4.
Reduced SMC coverage and defective collecting lymphatic vessel differentiation in Reln-deficient mice. (A, B, and D–I) Immunofluorescence of ear skin from wild-type (A, D, F, and H) and Reln−/− (B, E, G, and I) mice for the indicated proteins. For A and B, individual images (projections of confocal z stacks) were aligned to trace the entire collecting vessel from the proximal (Q1) to distal (Q4) end, and the overlapping blood vasculature was blacked out for clarity (original images are shown in Fig. S2). Broken lines outline the collecting vessels. (C) LYVE-1 immunoreactivity in wild-type and Reln−/− vessels (mean ± SEM [error bars]; n = 6 vessels). (J) SMC coverage in vessel quarters (mean ± SEM; n ≥ 12 vessels). (K) Quantification of widest and narrowest vessel points and the difference between the two (n ≥ 12 vessels). The boxes represent interquartile range, lines indicate mean, and whiskers indicate minimum and maximum values. *, P < 0.05; **, P < 0.01; ***, P < 0.005. Bars: (A and B) 200 µm; (D–I) 50 µm.
Figure 5.
Figure 5.
Functional impairment in Reln−/− collecting lymphatic vessels. (A–D) P21 wild-type (A and B) and Reln−/− (C and D) ear skin, injected with FITC-dextran (green) and costained with antibodies against α-SMA (red; A and C) and LYVE-1 (blue, A and C; and red, B and D). The broken line in A and C indicates the injection site, and arrows point to adjacent lymphatic vessels. Arrows in B and D point to LYVE-1–positive capillaries, and the arrowhead in B indicates an LYVE-1–negative collecting vessel filled with FITC-dextran. The broken line in B outlines the collecting vessel and its connection to LYVE-1+ capillaries (not outlined). (E) Quantification of Evans blue dye content in inguinal lymph nodes of control and Reln−/− mice after 5 min of injection into the hind limb footpad. Values from individual lymph nodes and mean values are plotted (error bars; n = 11 [Ctrl] and 6 [Reln−/−] mice). (F) Efficiency of fluorescent signal, representing efficiency of lymphatic flow, in a defined region of a vessel after ICG injection into the hind limb footpad over time. (G) Representative images of control and Reln−/− hind limbs after ICG dye injections at three time points (excluding a 6–8-s delay before recording was started). In control but not in Reln−/− mice, the dye reached the inguinal lymph node (asterisks). Bars: (A–D) 100 µm; (G) 0.5 cm.
Figure 6.
Figure 6.
LECs respond to Reelin via noncanonical signaling pathway. (A and B) Fold change in expression of SMC recruitment genes after Reelin stimulation. Note the strong increase in MCP1 expression at 4 and 8 h in cells stimulated with full-length Reelin containing supernatant from HEK 293 cells only. The mean ± SEM of three experiments is plotted (error bars). *, P < 0.05; **, P < 0.001. (C–E) Immunofluorescence staining of P21 ear skin from wild-type (C), Apoer2−/−;Vldlr−/− (D), and Dab1Scm (E) mice using LYVE-1 (green), α-SMA (red), and Podoplanin (blue) antibodies. Individual images were aligned to trace the vessel from the proximal to the distal end, and overlapping blood vasculature was blacked out for clarity. A segment from the middle section of each vessel is displayed (original images are shown in Fig. S5). (F) SMC coverage in vessel quarters (mean ± SEM [error bars]; n ≥ 6 vessels per genotype). *, P = 0.04. (G) Quantification of the widest and narrowest vessel points and the difference between the two. Boxes represents interquartile range, lines indicates mean, and whiskers indicate minimum and maximum values. (H) LYVE-1 immunoreactivity in collecting vessels (mean ± SEM [error bars]; n ≥ 6 vessels). All differences in F–H were nonsignificant, except where indicated. Bars, 200 µm.
Figure 7.
Figure 7.
Model of Reelin signaling during collecting lymphatic vessel formation. Reelin (red stars) is produced by LEC (gray cells). Upon SMC (blue cell) contact, Reelin is secreted from LEC (red cells) and cleaved to release diffusible fragments (red gradient). Reelin can stimulate expression of MCP1 in LECs to enhance further SMC recruitment.

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