Figure 4
Pressure overload had induced the remodeling of cardiac lymphatic vessels and the numbers of lymphatic endothelial cells was correlated with L+ macrophages in hypertrophic and failing hearts. (A) Quantification by immunofluorescence of lymphatic vessels found in cardiac tissue after 1 week and 6 weeks of TAC or SHAM. *P < 0.05 **P < 0.01 vs corresponding SHAM, by t test. (B) Percent of CD45-CD31+PODO+ lymphatic cells expressing LYVE-1 in cardiac tissue after 1 and 6 weeks of SHAM or TAC (flow gating strategy detailed in supplementary 3c). *P < 0.05; **P < 0.01 vs corresponding SHAM by t test. (C) The number of endothelial lymphatic cells (CD45-CD31+LYVE-1+, present in cardiac tissue of each individual mouse was positively correlated with the number of L+ subsets identified after 6 weeks of TAC . rs = 0.6; P = 0.034 by Spearman. The line comes from a linear regression model. (D) Representative image of lymphatic vessels in SHAM and chronic failing heart after 6 weeks of TAC (scale bar, 500 µm) and (E) quantification of open myocardial and subepicardial lymphatic vessels. Data are presented in mean ± SEM. **P < 0.01 by Mann–Whitney U test. (F) Representative staining of lymphatic collectors in heart of mice following 6 weeks of TAC or SHAM, 10 min after the injection of 15 µl of 10 mg/mL FITC—2000 KDa dextran in the apex of heart and quantification of FITC dextran fluorescence in cardiac draining lymph node of mice following injection. Data are presented in mean ± SEM. **P < 0.01; by Mann–Whitney test. (G) Representative flow cytometry dot plots of heart draining MLN of mice with SHAM or TAC after intra-myocardial injection of fluorescent beads and their quantification (expressed as number of beads found per 200 000 events) (n = 4–6). *P < 0.05; by Mann–Whitney test. (H,I) RT-PCR analysis of the CCL19, CXCL12 and CCL21 chemokine expression (H) and the atypical chemokine scavenging receptors (ACKR)3 and 4 (I) performed on CD45-CD31+LYVE−1+ isolated from heart after 1 week of SHAM or TAC. Data were obtained using the comparative 2−ΔCt method to housekeeping gene expression normalization. Data are presented in mean ± SEM of cells isolated from pooled 4 to 6 mouse hearts. Significance *P < 0.05; **P < 0.01 by Mann–Whitney test.
