T Cell-Derived IL-17A Induces Vascular Dysfunction via Perivascular Fibrosis Formation and Dysregulation of⋅NO/cGMP Signaling

Figures

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  Figure 1. Overexpression of IL-17A in T cells results in vascular dysfunction per se. (a) Cytokine profile of CD4-IL-17Aind/+ mice in plasma measured with a Bio-Plex assay kit. IL-17Aind/+ control mice were normalized to 100% (indicated with the dashed line); percentual change compared to control is shown for CD4-IL-17Aind/+ mice; n = 3–6; either Student's unpaired t-test or Mann–Whitney t-test. (b) Cumulative concentration relaxation curves of aortic rings of CD4-IL-17Aind/+ and IL-17Aind/+ control mice in response to ACh (b1) and GTN (b2); n = 12–15; two-way ANOVA with Bonferroni post hoc test. (c) Maximal aortic constriction in response to a phenylephrine bolus in organ chamber from CD4-IL-17Aind/+ and IL-17Aind/+ control mice; n = 13; Student's unpaired t-test. (d) Cardiac function measured via echocardiography; n = 5; Student's unpaired t-test. (e) Systolic blood pressure detection via carotid artery telemetry catheter implantation of mice over 48 hours; n = 3–5. Data (a–d) are presented as mean ± SEM.

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  Figure 2. T cell-specific IL-17A overexpression does not result in an infiltration of inflammatory cells into the aortic wall; nevertheless, CD11b+ cells are more reactive. (a) Flow cytometric analysis of aortas from CD4-IL-17Aind/+ compared to IL-17Aind/+ control mice. Dot plot shows gating strategy of aortic samples. Cells were pregated on living cells and gated on CD45.2+, CD90.2−, CD11b+, F4/80+, and either Ly6G+Ly6C+ neutrophils or Ly6Csingle+ monocytes. Quantitative analysis of aortic flow cytometric analysis; n = 3–7 mice per group; either Student's unpaired t-test or Mann–Whitney t-test. (b) ROS/RNS measurement in whole peripheral blood was performed after stimulation with PDBu for 20 minutes; repeated measurements of pooled samples; n = 11–12 mice per group; unpaired Student's t-test. (c) ROS/RNS detection in isolated CD11b+ splenocytes after 20 min stimulation with PDBu; n = 5; Student's unpaired t-test. Data are presented as mean ± SEM.

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  Figure 3. No eNOS uncoupling in CD4-IL-17Aind/+ mice. (a) NO production in aortic tissue was measured via electron paramagnetic resonance- (EPR-) based spin trapping. a2 shows the quantification of the delta intensity normalized to dry weight of samples; n = 9–10; Student's unpaired t-test. (b) Oxidative fluorescence microtopography of aortic sections of CD4-IL-17Aind/+ and IL-17Aind/+ mice. b1: representative picture of isolated aortic sections stained with dihydroethidium (DHE, 1 μM). Autofluorescence of the laminae is visible in green, superoxide formation in red fluorescence; scale bar = 50 μm. b2: quantification of superoxide formation in the aortas is shown as a percentage of control; n = 6–7; unpaired Student's t-test. (c) ROS formation in aortic tissue (endothelial scan) of CD4-IL-17Aind/+ and IL-17Aind/+ mice, incubated with L-NAME (eNOS inhibitor) or buffer. c1: representative DHE photomicrotopographs of aortic cryosections with focus on the endothelial layer; ROS formation appears in red. c2: quantification; n = 3; comparison of IL-17Aind/+ control mice with CD4-IL-17Aind/+ mice or comparison of IL-17Aind/++L-NAME mice with CD4-IL-17Aind/++L-NAME mice; Mann–Whitney t-test. (d)…

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  Figure 4. IL-17A induced perivascular fibrosis in CD4-IL-17Aind/+ mice and increased fibroblast migration and proliferation. (a) Combined Masson's trichrome–Verhoeff's elastica (MTC-VES) staining (a1) and Sirius red staining (a2) of aortic rings of CD4-IL-17Aind/+ and control mice. Representative pictures are shown; scale bar for both stainings = 100 μm. a3: quantification of aortic wall thickness and thickness of collagen-positive area in adventitia are shown. Thickness was measured at 10 different points per section with ImageJ software; n = 6; unpaired Student's t-test. (b) Aortic sprouting assay of CD4-IL-17Aind/+ and control mice. b1: representative image of aortic rings on Matrigel covered plates at 5x magnification. Quantification of sprout length (b2) and number of sprouts (b3); n = 2–3 mice. (c) Aortic ring staining after sprouting assay of CD4-IL-17Aind/+ and control mice. Aortic rings were stained for DAPI (blue), α-smooth muscle cell actin (αSMa, yellow), fibroblast-specific protein 1 (FSP1, green), and CD31 (red); scale bar = 60 μm. A representative image per group is shown; n = 2–3 mice per group. (d) MTT analysis of fibroblasts and smooth muscle cells (SMCs) cultivated with…

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  Figure 5. T cell-specific IL-17A overexpression results in a downregulation of sGC/VASP signaling in the aorta, with a concomitant upregulation of Vcam1 and Mmp2 mRNA. (a, b) Western blot of aortic protein (a) and PVAT protein (b) of CD4-IL-17Aind/+ and control mice of p-eNOS (S1177), eNOS, PYK2, sGCα1, sGCβ1, pVASP (S239), and GCH-1. a1, b1: original Western blot; a2, b2: quantification of the signal, aorta: n = 4–16 (partially, 3-4 aortas were pooled per sample), Mann–Whitney t-test or unpaired Student's t-test. PVAT: n = 5–11, Student's unpaired t-test. (c) Quantitative real-time PCR of aortic mRNA of Vcam1, Cybb, Nos3, Mmp2, Mmp9, and Tgfb, n = 4–8, Mann–Whitney t-test or unpaired Student's t-test. Data are shown as mean ± SEM.