Transcriptional and Epigenetic Controls of Vascular Homeostasis458Implication of microRNA 199a3p and 199a5p in vascular function : modulation of the eNOS/NO pathway459Role of endothelial cell adenosine deaminase acting on RNA-2 in ischemic/inflammatory disease in vivo460Adventitial activation by sonic hedgehog signaling is critical for vascular remodeling
Authors: V Joris, A Gatsiou, J-M Daniel, L Menchi, I Lobysheva, GL Condorelli, JL Balligand, D Catalucci, C Dessy, FF Lunella, C Amrhein, S Gunther, C Garcia Gonzalez, A Schneider, T Braun, A Zeiher, S Dimmeler, K Stellos, J Dutzmann, L Korte, A Koch, J Bauersachs, D Sedding
Journal: Cardiovascular Research (2016)

# 458   Implication of microRNA 199a3p and 199a5p in vascular function : modulation of the eNOS/NO pathway {#article-title-2}

Introduction: MiR 199a3p and 199a5p have been mainly implicated in proliferation, cell survival and remodeling in context of cancerogenesis but more recent data proposed a role in cardiovascular functions.

Objectives: We seek to assess how miR199a3p and miR199a5p modulate endothelial function by identifying their molecular targets in endothelial cells.

Methods: Mice were treated with miR199a3p/5p inhibitors and after 30 days, the contractile profile and endothelial function were evaluated ex vivo in the aorta. Cultured endothelial cells were transfected with miR199a3p/5p inhibitors or a scramble sequence. NO production, expression and/or activity of eNOS and its major regulators were measured by EPR and Western blot respectively. Implication of Akt pathway was analysed using LY294002 treatment. Angiogenesis was investigated in 2D-culture in matrigel support.

Results: Vessels from mice treated with antagomirs against miR199a3p or 5p showed a larger NO-dependent relaxation compared to controls. Circulating HbNO measured by EPR in venous blood collected from these mice was significantly increased compared to controls suggesting a role of both miRs in the control of the NOS/ NO pathway.

Repression of miR 199a3p/5p improved eNOS activity through an increased eNOS phosphorylation at serine 1177 and a decreased phosphorylation status of threonine 495. The eNOS allosteric regulator, cav-1, was not modulated by LNA treatments. However, both treatments promoted Akt activation and calcineurin expression. Furthermore, addition of LY294002 inhibited the increase of eNOS phosphorylation at serine 1177 induced by LNA treatment. Interestingly, repression of miR199a5p also promoted SOD expression, suggesting that miR199a5p also improved NO bioavailability. In addition, inhibition of miR199a5p upregulated VEGF production and promoted tubes formation in endothelial cells.

Conclusion: Our results demonstrate that miR199a3p/5p modulate the NOS/NO pathway in the endothelium by promoting NO production and repressing NO degradation. These results also suggest a strong implication of the Akt pathway in this modulation. Interestingly, miR199a5p shows additional control of angiogenesis.

# 459   Role of endothelial cell adenosine deaminase acting on RNA-2 in ischemic/inflammatory disease in vivo {#article-title-3}

Background: RNA binding proteins (RBPs) are key players in posttranscriptional regulation of mRNAs. Adenosine deaminase acting on RNA-2 (ADAR2) enzyme binds to double-stranded RNAs and controls brain development. The role of ADAR2 in endothelial cell function has not been described so far.

Methods and Results: ADAR2 is expressed in human and murine endothelial cells and is 2-fold induced by hypoxia or hind limb ischemia in mice (P<0.05 for all). ADAR2 deficiency resulted in 73±12% impairment of leukocyte infiltration, in 53±4% reduced neovascularization, and a 40±6% decreased blood-flow recovery of ischemic muscle tissues in a hindlimb ischemia mouse model (P<0.001 for all). Mechanistically, ADAR2 knockdown causes a 2-fold downregulation of 522 transcripts, whereas 113 transcripts are found to be upregulated in RNA-seq experiments. Among the endothelial cell enriched, highly ADAR2-regulated transcripts was interleukin-6 signal transducer (IL6ST also known as glycoprotein-130, gp130), the receptor of interleukin-6 (IL-6). Silencing of ADAR2 resulted in a downregulation of gp130 mRNA and protein expression in endothelial cells by 65±5% and 50±5%, respectively (P<0.001 for both). Similarly, the expression of gp130 mRNA was decreased by 50±25% (P<0.05) in murine lung endothelial cells, which derived from transgenic ADAR2-null mice. Silencing of ADAR2 reduced by at least 2-fold the IL-6-mediated STAT3 phosphorylation, the mRNA expression of the IL-6-induced downstream genes MCP-1, VCAM-1 and E-selectin, and the IL-6-induced platelet and leukocyte adhesion on endothelial cells and endothelial cell network formation (P<0.05 for all). Of interest, ADAR2 regulated gp130 mRNA stability. Co-silencing of ADAR2 and Drosha completely restored the ADAR2-regulated gp130 mRNA expression indicating that ADAR2 regulates the maturation of specific microRNAs (miRs). Indeed, ADAR2 regulates by at least 2-fold the expression of 20 microRNAs, including of miR-579 and miR-320e, which are predicted to bind to the 3’-untranslated region of IL6ST mRNA.

Conclusions: This study shows for the first time that ADAR2 controls miR biogenesis and thus IL-6/STAT3 signalling in endothelial cells.

# 460   Adventitial activation by sonic hedgehog signaling is critical for vascular remodeling {#article-title-4}

Background: Adventitial cells have been suggested to contribute to vascular remodeling processes, but the signaling pathways are largely unknown. Sonic hedgehog (Shh) is a regulator of vasculogenesis and promotes angiogenesis in the adult. Shh induces activation of its membrane-bound downstream protein Smoothened (Smo) and also other signaling pathways.

Purpose: The aim of this study was to analyze the impact of the adventitial layer on vascular remodeling processes and to define the underlying cellular mechanisms.

Methods and Results: Following wire-induced injury of the femoral artery of C57BL/6J mice, the formation of a neointimal lesion at 21 days was preceded by high proliferation rates of adventitial fibroblasts and massive adventitial thickening at 7 and 14 days.

In vitro, Shh and platelet-derived growth factor (PDGF)-BB strongly induced proliferation and migration of human adventitial fibroblasts (AoAF) compared with Shh or PDGF-BB alone. Importantly, the specific Smo inhibitor GDC-0449 (Vismodegib) nearly completely reversed the complementary effects of Shh and PDGF-BB. Mechanistically, we found that PDGF-BB selectively induced trafficking of Smo to the plasma membrane of AoAF but not human coronary artery smooth muscle cells (SMC) or mesenchymal stem cells via activation of protein kinase A (PKA). The activation of Smo resulted in downstream signalling via RhoA but not the Gli family of transcription factors. Following stimulation with Shh and PDGF-BB, the supernatant of AoAF but not leukocytes strongly induced proliferation and migration of SMC and this effect was also blocked by GDC-0449.

In vivo, perivascular application of GDC-0449 via a thermo-sensitive gel significantly reduced neointima formation and proliferation of adventitial and neointimal cells following wire-induced injury. Following adventitial transplantation of ubiquitously GFP expressing mice, only very view GFP+ cells could be detected within the neointimal lesion. To investigate the potential paracrine effect of the activated adventitial layer, aortic adventitial grafts to the injured femoral artery were explanted at 14 days and incubated in serum-free media. Proliferation and migration rates of SMC in response to the supernatant of these grafts were significantly increased compared with the supernatant of native aortic adventitia or neointimal/medial samples. Further protein array analyses of these supernatants identified the up regulation of the same pattern of growth factors/cytokines as in the supernatant of Shh and PDGF-BB stimulated AoAF (especially interleukin-6 and -8).

Conclusion: Dilation of the murine femoral artery is immediately followed by adventitial enrichment of cytokine-producing fibroblasts, whose paracrine function is essential for the sustained inflammatory response and the subsequent proliferation and migration of local SMC. Mechanistically, AoAF cytokine production is under the predominant control of non-canonical Shh signaling.