Perivascular fibrosis is mediated by a KLF10-IL-9 signaling axis in CD4+ T cells

Perivascular fibrosis, characterized by increased amount of connective tissue around vessels, is a hallmark for vascular disease. Angiotensin II (Ang II) contributes to vascular disease and end-organ damage via promoting T-cell activation. Despite recent data suggesting the role of T cells in the progression of perivascular fibrosis, the underlying mechanisms are poorly understood. Kruppel-like Factor 10 is a transcription factor expressed in T cell subsets. We sought to investigate the role of KLF10 in CD4+ T cells in regulating vascular damage in an AngII mouse model. CD4-targeted KLF10 deficient (TKO) and CD4-Cre (WT) mice were generated and subjected to 28 days of Ang II infusion. Differential gene expression profiling of non-stripped aorta, stripped aorta, isolated aortic fibroblasts from CD4-Cre (Cre) or CD4-Cre KLF10-fl/fl mice(TKO) mice after 28 days Angiotensin II (Ang II) treatment were performed to ascertain the underlying global transcriptomic changes involved in perivascular fibrosis. Since increased IL-9 is a profibrotic cytokine, we hypothesized that perivascular fibrosis might be rescued by administration of anti-IL-9 neutralizing monoclonal antibodies (mAbs) in Ang II-treated TKO mice. Then differential gene expression profiling of non-stripped aorta from TKO mice with or without anti-IL9-antibody treatment after 28 days Ang II infusion. To further understand the differences in phenotypic heterogeneity in fibroblasts during the progression of perivascular fibrosis between TKO and control mice in response to Ang II treatment, we performed single-cell RNA-sequencing (scRNA-seq) of the descending aorta. Collectively, comparative RNA-seq transcript profiling identified several pathways associated with periadventitial remodeling and that the calcium signaling pathway is dominantly upregulated in non-stripped aortas of TKO mice after Ang II treatment. Indeed, the profibrotic genes Col8a1, Mmp2, Fmod, and Angptl1, which were identified as significantly increased in TKO aortas and in isolated fibroblasts by both bulk RNA seq and scRNA seq, were all decreased after treatment with anti-IL-9 mAbs. These results indicate that the KLF10-IL-9 signaling axis in CD4+ T cells tightly regulate the processes of Ang II-induced pathological perivascular fibrosis and provide novel therapeutic opportunities for the treatment of hypertensive-associated diseases. 1) Differential gene expression profiling of non-stripped aotra, stripped aorta, aortic fibroblasts from CD4-Cre(Cre) or CD4-Cre KLF10-fl/fl mice(TKO) mice after 28 days Angiotensin II (Ang II) treatment. 2) Differential gene expression profiling of non-stripped aotra from TKO mice with or without anti-IL9-antibody treatment after 28 days Ang II infusion. 3) Single cell RNA-sequencing was perform on descending aorta from Cre or TKO after Ang II treatment.