Figure 1.
Isolation of lymphatic endothelial cell population.
FACS was performed on cells isolated from the back skin of wild type mice. (A) Events are first gated on FSC and SSC for debris. The gated events were then plotted to determine Calcein Am incorporation, which will occur only in live cells. We observed a variation in levels of Calcein incorporation in different cell type as shown by the multiple populations observed. We then gated the positive events for caicein Am as shown in the left plot. The right plot shows the viable cells plotted for expression of CD31 and podoplanin, which we identified to be LEC population. (B) Western blotting for PROX-1 and LYVE-1 on cell lysate from isolated CD45-/CD31+/ podoplanin+ population and cultured human microvascular lymphatic endothelial cell (HMVEC) shows comparable expression of these lymphatic endothelial markers in both cell types. (C) Isolated CD45-/CD31+/ podoplanin+ cells were cultured and stained for podoplanin and PROX-1. Immunofluorecscent image of the cells is shown. Scale bar 100µm. (D) Isolated CD45-/CD31+/ podoplanin+ cells and CD45-/CD31-/ podoplanin- were seeded on Matrigel for 24hours and only CD45-/CD31+/ podoplanin+ cells were able to form tube like structures. Scale bar 100µm.
Figure 2.
Quantification of LECs in vivo.
(A-C) Single cell suspension was isolated from non-treated wild type mice, 3, or 7 days post CHS (N>5). Viable/CD45- , viable/CD45+, viable/CD45-/ CD31+/podoplanin+, and viable/CD45-/ CD31+ cells were quantified using flow cytometry analysis and the FLOWJo software. We observed that the percentage of viable CD45+ cells within the viable cell population in the skin of mice was elevated at days 3, and 7 post-CHS compared to non-treated animals (p<0.05). The percentage of the CD45- cell population within the viable population decreased at days 3 and day7 post-CHS compared to non-treated. Animals (p<0.05) We observed that the percentage of viable CD45-/CD31+/ podoplanin+ cells within the CD45- population increased significantly (p<0.05) at day 3 post CHS as compared to non-treated, and 7 days post CHS. (E) Immunofluorescence analysis was performed for podoplanin (green) and DAPI (blue) to identify lymphatic vessels in the skin. Arrows indicate location of lymphatic vessel. Scale bar 200µm. (F) Quantification of lymphatic vessels was performed using imageJ. Vessels were counted if a lumen was observed and if at least one nucleus was within the podoplanin signal. No significant change in the lymphatic density was observed. G) Quantification of the lymphatic vessel cross sectional area in pixel2. We found a significant difference in the size of the vessels at day 3 post-CHS as compared to the other time points. Significance (p<0.05) is denoted with a * and a bracket is used to identified the significantly different groups.
Figure 3.
Identification of proliferating LECs.
(A) EdU was injected every 24 hours I.P into mice either without treatment or after CHS. Single cell suspensions were made and viable CD45-/CD31+/ podoplanin+ cells were isolated using FACS. Cells were then fixed with 2% PFA, and stained using click it chemistry with an Alexa647 dye and DAPI. Flow cytometry analysis was performed on the sample to identify DAPI + /EdU + cell population. Sample flow cytometry plot of the FCS-A to EdU fluorescence for all three groups, non-treated, injected from day 0 to day3, and from day 4 to day 7. (B) Quantification of LECs or total CD31+ cells that stained for DAPI + /EdU + in non-treated mice, in mice injected from day 0 to day 3 post CHS, and in mice injected at day 4 to day 7. We observed a significant increase in the number of LECs and total CD31+ cells that intercalated EdU from day 0 to 3, the treated anmials with injection from day 4 to 7, and the non-treated groups (N=3) (p<0.001).
Figure 4.
(A-D) Mice were injected I.D at the base of the tail with IGC to highlight lymphatic architecture at days 0, 3, 5, and 7 post-CHS treatment. We show images from one representative mouse. Oxazolone was applied on the right thigh of the mouse and the left side was left untreated. The yellow dotted line is an outline of the mouse body. We observed extravasation of the ICG dye after the onset of CHS. (E-I) We show images of a representative mouse at day 3 post-CHS to compare the changes occurring in the CHS treated side and non-treated side. The thigh area was increase in size to help visualize the leakiness of the dye in the interstitial area.
Figure 5.
Lymphatic flow measurement using near infrared dye ICG.
A-D Mice were injected I.D. with IGC at the base of the tail to quantify the contractility and qualify the ejection fraction of the lymph at days 0(A), 3(B), and 7(C) of post-CHS in the contralateral view at day 3(D) (N=3). Selected frames are showed from the same sample mice to illustrate the changes that occur in the lymphatic flow. Spatio-temporal fluorescent intensity maps are shown to visualize the ejection fraction of the ICG dye. We observed that mice pre-treatment (A), at day 7 post-CHS (B) or the contralateral side at day 3post-CHS (D) have forward flow, but that at day 3 (C) on the side of inflammation the lymph flow is perturbed and shows backflow.
Figure 6.
Identification of pro-angiogenic.- lymphangiogenic factors significantly changes in CHS at day 3 post-CHS compared to non-treated and day 7.
Multiplex assay was performed on skin homogenate on non-treated, day 3, and day 7 mice post-CHS challenge using the manufacturer’s protocol. We report the mean ratio ±std of the concentration normalized to non-treated values for the analytes that were significantly different. * p<0.05 (FDR<0.1) day3 versus non-treated. # p<0.05 (FDR<0.1) day 3 versus day 7.