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. 2023 Apr 20;12:e80900. doi: 10.7554/eLife.80900

Figure 1. Establishment of the diphtheria toxin (DT)-induced endothelial cell (EC) ablation model.

(A) Transgenic mice harboring Cre recombinase cDNA downstream from a 2.5 kb fragment of the VE-Cadherin (Cdh5) mouse promoter (B6.FVB-Tg(Cdhn5-cre)7Milia) were crossed with mice with Cre-inducible expression of DTR (C57BL/6-Gt(ROSA)26Sortm1(HBEGF)Awai/J) giving rise to Cdh5-DTR binary transgenic mice. (B) Immunostaining for human diphtheria toxin receptor (DTR) (red) and ECs (anti-CD144, green) and merged image showing co-localization (yellow) in lung sections from Cdh5-DTR binary transgenic mice 3 days post intra-tracheal (IT) delivery of saline or DT (10 ng). Scale bar is 50μm. (C) Immunostaining for activated caspase 3 (aCasp-3, red) in lung sections from binary transgenic mice 3 or 7 days after treatment DT or saline (DAPI nuclear staining in blue). Scale bar is 100μm. (D) Representative plots of lung EC numbers assessed by flow cytometry (CD144) in binary transgenic mice 3 days after IT delivery of saline or DT. (E) Summary flow cytometric data showing the percent of total lung cells staining positive using CD144, CD31, or CD34 antibodies in DT-treated binary transgenic mice 3 or 7 days post-treatment (dpt) compared with saline. Data represented as mean ± SEM. n=5 for 3d timepoint, n=3 for 7d timepoint, multiple unpaired t-tests were performed with multiple comparisons using Holm-Sidak correction. More details are in Figure 1—figure supplement 1.

Figure 1.

Figure 1—figure supplement 1. Sublethal dose of diphtheria toxin (DT) induces pulmonary hypertension and selective increase in lung permeability.

Figure 1—figure supplement 1.

(A) Single intra-tracheal (IT) administration of DT at doses below 20 ng was compatible with survival to 7 days post-treatment. (B) Doses above 1 ng resulted in a dose-dependent increase in right ventricular systolic pressure (RVSP). (C) Vascular permeability as measured by Evans blue uptake remained unaltered following DT administration at 3 and 7 days in the liver, spleen, kidney, and brain. Data represented as mean ± SEM. An unpaired multiple t-tests with Holm-Sidak multiple comparisons method with alpha (0.05) was used. n = 3 biological replicates per group.