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. Author manuscript; available in PMC: 2023 May 27.
Published in final edited form as: Circ Res. 2022 Apr 20;130(11):1662–1681. doi: 10.1161/CIRCRESAHA.121.320420

Figure 3. KLF10 deficient CD4+ T cells release IL-9 that mediates perivascular fibrosis.

Figure 3.

A, IL-9 levels were measured in plasma in PBS or Ang II treated Cre and TKO mice (n=4 in PBS groups, and n=8 in Ang II-treated groups). B, mRNA expression level of Il9 in the PBMCs, heart, kidney, and aorta in Ang II-treated Cre and TKO mice (n=6 in aortic groups, and n=8 in others). C-D, Representative flow cytometry plots and the percentage of CD4+IL9+ T cells gated in CD3+ T cells in the aorta (C, n=5) and spleen (D, n=5). E, Representative immunofluorescence staining, and the number of CD4+IL9+ T cells in the aorta in Ang II treated Cre and TKO mice (n=5, scale bars= 50μm). F-G, Schematic diagram of the experimental setup for CD4+T cell isolation from Ang II-treated Cre or TKO mice (F), and quantification of Il9 mRNA in isolated CD4+ T cells, and IL-9 protein released into the supernatants of CD4+ T cells (G, n=6). H-I, Schematic diagram of the experimental setup of CD4+ T cell isolation from Cre control mice for in vitro treatment (H), quantification of Il9 mRNA in the treated CD4+ T cells, and IL-9 protein released into the supernatants of treated CD4+ T cells (I). J, Schematic diagram of the experimental setup for recombinant mIL-9 treatment in Cre mice. K, GLS in mIL-9 or PBS treated Cre mice after Ang II infusion (n=6). L, the value of PWV and circumferential strain in mIL-9 or PBS treated Cre mice after 28 days Ang II treatment (n=6). M, the ratio of albumin and creatinine, and the level of KIM-1 in urine in mIL-9 or IgG treated Cre mice after 28 days Ang II treatment (n=6). N-O, Representative images of Masson trichrome staining and Sirius red staining, and the area of perivascular fibrosis and adventitial collagen in the aorta (n=6, scale bars= 200μm). P values correspond to two-way ANOVA with Tukey's multiple comparisons tests (A, and I), or unpaired two-tailed t tests (B, G, and K-O). C-E, P values correspond to unpaired two-tailed Mann-Whitney U-tests. PBMC indicates peripheral blood mononuclear cells; Ang II, angiotensin II; GLS, global longitudinal strain; PWV, pulse wave velocity; Circ Strain, circumferential strain; KIM-1, kidney injury molecule-1.