Figure 2.

Inhibition of HIF-1α impairs lymphangiogenesis and delays lymphatic repair. A) Representative microlymphangiography 3 wk following surgery (boxed); arrow indicates direction of lymphatic flow. Lymphatic tracer is visualized crossing the wound (white arrow) in controls. B) Immunofluorescent photomicrograph for LYVE-1 demonstrating lymphatic vessels traversing wound (arrow) in controls. C) Number of podoplanin⁺ vessels per high-power field (left panel) and representative photomicrographs (right panel) in YC-1, 2ME, and respective controls. Arrowheads indicate lymphatics; boxed region in gross photograph represents region used for analysis. D) Number of LYVE-1⁺ vessels per high-power field and representative photomicrographs (red, LYVE-1; blue, nuclear stain) demonstrating reduced numbers of lymphatic vessels following YC-1 treatment. E) Western blot of protein harvested from tissue centered at the wound (boxed) 3 wk after surgery in animals treated with YC-1, 2ME, or respective vehicle control (fold change relative to control). F) Number of VWF⁺ vessels per high-power field. G–I) Cross-sectional lymph node area (μm²; G) and lymphatic vessel density (H) in popliteal nodes following YC-1 (G–I), 2ME (G–I), or vehicle control treatment in PBS- or CFA-OVA-treated animals, demonstrating reduced lymph node size and lymphatic density following HIF-1α inhibition. I) Immunofluorescent images of LYVE-1 (red)- and DAPI (blue)-stained lymph nodes. J) Number of podoplanin⁺ lymphatic vessels in DFO- or PBS-supplemented Matrigel plugs. Representative low-power (×10; left) and high-power (×40; right; boxed) photomicrographs of podoplanin-stained Matrigel plugs are shown. K) Low- and high-power images of podoplanin stains showing specificity for lymphatic vessels (arrow) with lack of staining of blood vessel endothelium (arrowhead). Scale bars = 100 μm (B, I, J, K); 75 μm (C); 50 μm (D). n.s., not significant. *P < 0.05, **P < 0.01.