Figure 1.
Resting skin vessels contain high density HS moieties preferentially expressed at their basolateral compartments.
(A, C, E, F, G) Axial and longitudinal sections of vessels of (A, B) naïve frozen, or (C, D, E, F, G) paraffin embedded murine flank skin tissues. Tissues were stained for HS with monoclonal mouse IgM (10E4 epitope; red), goat anti mouse VE-cadherin (cyan), FITC-mouse monoclonal to α-SMA (green), and nuclei (blue). (H) HS and α-SMA profile of the fluorescence intensity along the purple arrow in the merged image. Double-headed black arrow denotes the vessel lumen as indicated by VE-cadherin. In C, D and G, small-headed arrows denote the endothelial apical side, large-headed arrows denote the endothelial basolateral side, and broken-line arrows denote the pericyte basolateral side. Images were taken at 100X magnification. Scale bar represents 5 µm.
Figure 2.
Low HS content within the apical glycocalyx of resting murine flank skin vessels.
(A) Paraffin embedded murine naïve dermal tissue immunostained for HS (red; left panel), glycocalyx (α-gal) with the lectin BS1-B4 (green; middle panel), and VE-cadherin (cyan). Images were taken at 100X magnification. Scale bar represents 5 µm. (B) Glycocalyx and HS fluorescence intensity along the purple arrow in the merged image (right panel). Double-headed black arrow denotes the vessel lumen as indicated by the apical glycocalyx. (C) Pixel ratio per cell (apical/basolateral as indicated by the areas marked with white lines) in naïve murine dermal tissue. Small-headed arrow denotes the endothelial apical side, large-headed arrow denotes the endothelial basolateral side, and broken-line arrow denotes the pericyte basolateral side. Data are the mean value and S.E.M. of 16 endothelial cells analyzed in sections derived from three independent experiments. P<0.0001 by paired t-test.
Figure 3.
H&E staining of naïve and inflamed flank skin.
(A) Naïve or (B) inflamed murine dermal tissue was stained and images were taken at 10X magnification. Scale bar represents 200 µm.
Figure 4.
HS and laminin are massively deposited in the basement membrane of inflamed skin vessels.
(A) Longitudinal and (B) axial paraffin sections of representative post capillary venules of CFA inflamed skin. (C) VE-cadherin and HS profile of the fluorescence intensity along the purple arrow in the axial section. Double-headed black arrow denotes the vessel lumen as indicated by VE-cadherin. In B, small-headed white arrow denotes the endothelial apical side. (D, E, F) Sections of naïve, and (G, H, I) inflamed murine dermal tissue, immunostained for HS (red), VE-cadherin (cyan), laminin with rabbit anti-mouse (green), and nucleus (blue). (F, I) merged images. Images were taken at 100X magnification. Scale bar represents 5 µm.
Figure 5.
HS deposition is not elevated in mice deficient in the main HS degrading enzyme, heparanase.
(A, C) Naïve, or (B, D) inflamed murine dermal tissue were removed from (A, B) wt or (C, D) heparanase KO mice, embedded in paraffin, and immunostained for HS (red), VE-cadherin (cyan), and nucleus (blue). Images were taken at 100X magnification. Scale bar represents 5 µm. In A the small-headed arrow denotes the endothelial apical side, the large-headed arrow denotes the endothelial basolateral side, and the broken-line arrow denotes the pericyte basolateral side. HS thickness (µm) in naïve (E), and inflamed (F) murine dermal vessels of either wt or heparanase KO mice. Data in each experimental group are mean value with SEM of 8 vessels in sections derived from three independent experiments.
Figure 6.
HS is localized mainly to the basement membrane in both resting and inflamed human dermal microvascular blood endothelial cells (HDBECs).
Immunostaining for HS (red), actin (green) and nuclei (blue) of resting or IL1-β- stimulated HDBEC monolayers grown for 16–18 hours on fibronectin. The mean fluorescence intensity values shown are the mean±SEM of ten fields of view from three independent experiments. Images were taken with a Delta-Vision microscope at 60X magnification. Scale bar represents 15 µm. N.S. not significant. ***P<0.0001 by Student's t test.
Figure 7.
HS is highly enriched in the basal lamina of lymph node HEVs.
(A, B) Cryosections of naïve lymph nodes derived from ROSAmT/mG × CD11c-Cre reporter mice immunostained for HS (grey). CD11c+ cells express the GFP protein (green), and endothelial cells express high levels of the tomato protein reporter (red). (C, D) Immunostaining of similar lymph node sections for HS (grey) and laminin (red). In B, small-headed arrow denotes the endothelial apical side as indicated by the tomato staining, and large-headed arrow denotes the endothelial basolateral side. Images were taken at (A) 10X magnification (scale bar represents 100 µm), and (B, C, D) 63X magnification; scale bars represent 10 µm.
Figure 8.
Differential distribution of HS and of PNAds in lymph node HEVs.
Consecutive sections of lymph node cryosections of the ROSAmT/mG × CD11c-Cre reporter mice stained for (B, C) HS (grey), or (E, F) PNAd (grey) using the 10E4 or MECA-79 IgMs, respectively. (A, C, D, F) Note the intense tomato protein label on the HEVs (red). (C, F) Merged images. (G) HS and MECA-79 profile of the fluorescence intensity along the purple arrow. Double-headed black arrow denotes the vessel lumen as indicated by the tomato staining. In C, small-headed arrow denotes the endothelial apical side, and large-headed arrow denotes the endothelial basolateral side. Images were taken at 63X magnification. Scale bars represent 10 µm.