Figure 8: DHHC21 deficiency prevents inflammation-mediated vascular barrier dysfunction.
(a,b) Following LPS or thermal injury, Zdhhc21dep/dep mice displayed decreased plasma protein extravasation in the lungs. (a) Images of four replicates used to qualitatively demonstrate SIRS-induced Evans Blue extravasation in whole left lung lobes (signal intensity low=blue, high=red). (b) Quantitative results of Evans Blue extravasation assays. Values were normalized to Zdhhc21+/+mice receiving sham operation. Results represent mean±s.e.m. from four independent experiments. *P<0.05 versus Zdhhc21+/++sham, #P<0.05 versus Zdhhc21+/++SIRS. (c,d) Histamine-induced transvascular flux of FITC-albumin from mesenteric microvessels. (c) Representative images at 0, 5, 10 and 15 min after histamine application. Scale bars, 100 μm. Arrowheads indicate the observed microvessel. (d) Quantification of plasma transvascular flux. For each genotype, value was normalized to its own t=0 min. Results represent mean±s.e.m. from six mice. *P<0.05 versus Zdhhc21+/++histamine. (e) The effects of Zdhhc21 knockdown or loss-of-function on endothelial permeability to FITC-albumin with or without 2-BP (100 μM). Thrombin (10 U ml−1) was applied to MLMVEC monolayers and their permeability was calculated based on albumin transendothelial diffusion rate. Bar graph represents mean±s.e.m. at n≥3. *P<0.05 versus Zdhhc21+/+ EC+vehicle, #P<0.05 versus Zdhhc21+/+ EC+thrombin. (f) Dynamic changes of endothelial barrier resistance to thrombin (10 U ml−1) in MLMVECs isolated from Zdhhc21+/+ and Zdhhc21dep/dep mice. TER value of each time point is normalized to baseline. Solid line tracing represents the mean resistance, and shadow represents standard error. Embedded graph shows maximum TER changes. Bar graph represents mean±s.e.m. *P<0.05 versus Zdhhc21+/+ EC+vehicle , #P<0.05 versus Zdhhc21+/+ EC+thrombin. (g) Blunted TER response to thrombin (10 U ml−1) was partially rescued in cells overexpressing wild-type DHHC21. Zdhhc21 (DDK tagged) or empty vector was overexpressed in Zdhhc21dep/dep ECs. Results represent mean resistance±s.e.m. The efficiency of Zdhhc21 overexpression was verified by Western blotting using anti-DDK tag and anti-DHHC21 antibody (Embedded graph).
