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Recent studies described a protection from atherosclerosis by targeting of the Th1 key transcription factor T box expressed in T cells (T-bet). It remains to be established, whether T-bet is involved in mediating angiotensin II (ATII) induced vascular dysfunction and oxidative stress. ATII (1mg/kg/d) was administered by osmotic minipumps for one week and radiotelemetry was performed in T-bet-/- vs. control C57/BL6(WT) mice. Vascular dysfunction was assessed by isometric tension studies and reactive oxygen species (ROS) were detected by using DHE staining of aortic cryosections, aortic rings Lucigenin enhanced chemiluminescence (ECL) and L-012 enhanced blood oxidative burst. Protein expression was determined by western blot and mRNA expression by realtime PCR. CD4+ cytokines were measured by ELISA and myelomonocytic cells were analyzed by FACS and IHC. Aortic T-bet protein expression was increased in response to ATII in WT mice. In T-bet deficient mice AT II caused a similar increase of blood pressure as compared to WT. In contrast to WT, T-bet-/- showed an attenuated vascular dysfunction, ROS production and expression of NADPH oxidase subunits in response to AT II. Furthermore, iNOS mRNA expression and protein tyrosine nitration in the aorta was markedly reduced in T-bet-/- mice which showed a significant decrease of macrophages in the vascular wall caused by a reduced presence of pro-inflammatory cells after AT II treatment compared to WT. In a rescue experiment we transferred WT CD4+ T cells in T-bet deficient mice which resulted in increased vascular oxidative stress and dysfunction. We conclude, that T-bet mediates, at least in part, ATII induced vascular damage and might represent a novel target to treat vascular dysfunction and inflammation in arterial hypertension.

As reported by Mach and co-workers CD40 ligand is involved in the vascular infiltration of immune cells and development of atherosclerotic lesions. Accordingly, it was found that the pathogenesis of atherosclerosis was attenuated in CD40L-/- mice compared to the wild-type mice based on a reduction of macrophage and CD4+ T cells migration into the atherosclerotic plaque. Furthermore, T cell mediated CD40L release, in turn, led to thrombocyte and macrophage activation in thrombosis. Here, we show that CD40L plays an important role in the intravascular Angiotensin-II-induced inflammatory process and analyzed the function of CD40 ligand during Angiotensin-II-induced oxidative stress and vascular dysfunction in arterial hypertension.In an established murine model we used osmotic minipumps containing angiotensin II (1mg/kg/d). Vascular function was recorded by isometric tension studies, reactive oxygen species (ROS) were monitored in blood and heart of CD40L-/- mice by using enhanced chemiluminescence. ELISA of CD4+ T cell supernatants as well as Western blot analysis and realtime RT-PCR have been used to analyze the expression of pro-inflammatory cytokines, NAPDH oxidase subunits and T cell transcription factors.CD40L-/- mice showed an ameliorated endothelial function and decreased oxidative stress in the heart and blood after angiotensin II treatment compared to their wild-type littermates. Moreover, CD40L-/- mice displayed significantly decreased levels of Th1 cytokines released by splenic CD4+ T cells. This observation was accompanied by lower expression levels of NOX-2 in aorta and splenic T-bet as well as P-Selectin ligand from CD40L-/- mice. In summary, our results demonstrate that CD40L-/- deficiency improves angiotensin II induced vascular dysfunction and oxidative stress further supporting our previous observations [Wenzel et al. Circulation 2011] that immune cells and inflammatory pathways significantly contribute to angiotensin II induced arterial hypertension.