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SummaryCleaved high-molecular-weight kininogen (HKa) or its peptide domain 5 (D5) alone exert anti-adhesive properties in vitro related to impeding integrin-mediated cellular interactions. However, the anti-adhesive effects of HKa in vivo remain elusive. In this study, we investigated the effects of HKa on leukocyte recruitment and neointima formation following wire-induced injury of the femoral artery in C57BL/6 mice. Local application of HKa significantly reduced the accumulation of monocytes and also reduced neointimal lesion size 14 days after injury. Moreover, C57BL/6 mice transplanted with bone marrow from transgenic mice expressing enhanced green fluorescence protein (eGFP) showed a significantly reduced accumulation of eGFP+-cells at the arterial injury site and decreased neointimal lesion size after local application of HKa or the polypeptide D5 alone. A differentiation of accumulating eGFP+-cells into highly specific smooth muscle cells (SMC) was not detected in any group. In contrast, application of HKa significantly reduced the proliferation of locally derived neointimal cells. In vitro, HKa and D5 potently inhibited the adhesion of SMC to vitronectin, thus impairing their proliferation, migration, and survival rates. In conclusion, application of HKa or D5 decreases the inflammatory response to vascular injury and exerts direct effects on SMC by impeding the binding of integrins to extracellular matrix components. Therefore, HKa and D5 may hold promise as novel therapeutic substances to prevent neointima formation.

Pulmonary hypertension (PH) is characterized by increased proliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs). Forkhead box O (FoxO) transcription factors are key regulators of cellular proliferation. Here we show that in pulmonary vessels and PASMCs of human and experimental PH lungs, FoxO1 expression is downregulated and FoxO1 is inactivated via phosphorylation and nuclear exclusion. These findings could be reproduced using ex vivo exposure of PASMCs to growth factors and inflammatory cytokines. Pharmacological inhibition and genetic ablation of FoxO1 in smooth muscle cells reproduced PH features in vitro and in vivo. Either pharmacological reconstitution of FoxO1 activity using intravenous or inhaled paclitaxel, or reconstitution of the transcriptional activity of FoxO1 by gene therapy, restored the physiologically quiescent PASMC phenotype in vitro, linked to changes in cell cycle control and bone morphogenic protein receptor type 2 (BMPR2) signaling, and reversed vascular remodeling and right-heart hypertrophy in vivo. Thus, PASMC FoxO1 is a critical integrator of multiple signaling pathways driving PH, and reconstitution of FoxO1 activity offers a potential therapeutic option for PH.

Aims MicroRNA (miR)-92a is an important regulator of endothelial proliferation and angiogenesis after ischaemia, but the effects of miR-92a on re-endothelialization and neointimal lesion formation after vascular injury remain elusive. We tested the effects of lowering miR-92a levels using specific locked nucleic acid (LNA)-based antimiRs as well as endothelial- specific knock out of miR-92a on re-endothelialization and neointimal formation after wire-induced injury of the femoral artery in mice. Methods and results MiR-92a was significantly up-regulated in neointimal lesions following wire-induced injury. Pre-miR-92a overexpression resulted in repression of the direct miR-92a target genes integrin a5 and sirtuin1, and reduced eNOS expression in vitro. MiR-92a impaired proliferation and migration of endothelial cells but not smooth muscle cells. In vivo, systemic inhibition of miR-92a expression with LNA-modified antisense molecules resulted in a significant acceleration of re-endothelialization of the denuded vessel area. Genetic deletion of miR-92a in Tie2-expressing cells, representing mainly endothelial cells, enhanced re-endothelialization, whereas no phenotypewas observed in mice lacking miR-92a expression in haematopoietic cells. The enhanced endothelial recovery was associated with reduced accumulation of leucocytes and inhibition of neointimal formation 21 days after injury and led to the de-repression of the miR-92a targets integrin a5 and sirtuin1. Conclusion Our data indicate that inhibition of endothelial miR-92a attenuates neointimal lesion formation by accelerating re-endothelialization and thus represents a putative novel mechanism to enhance the functional recovery following vascular injury.