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Antagomir-mediated silencing of endothelial cell specific microRNA-126 impairs ischemia-induced angiogenesis.

ABSTRACT: MicroRNAs are negative regulators of gene expression that play a key role in cell-type specific differentiation and modulation of cell function and have been proposed to be involved in neovascularization. Previously, using an extensive cloning and sequencing approach, we identified miR-126 to be specifically and highly expressed in human endothelial cells (EC). Here, we demonstrate EC-specific expression of miR-126 in capillaries and the larger vessels in vivo. We therefore explored the potential role of miR-126 in arteriogenesis and angiogenesis. Using miR-reporter constructs, we show that miR-126 is functionally active in EC in vitro and that it could be specifically repressed using antagomirs specifically targeting miR-126. To study the consequences of miR-126 silencing on vascular regeneration, mice were injected with a single dose of antagomir-126 or a control 'scramblemir' and exposed to ischemia of the left hindlimb by ligation of the femoral artery. Although miR-126 was effectively silenced in mice treated with a single, high dose (HD) of antagomir-126, laser Doppler perfusion imaging did not show effects on blood flow recovery. In contrast, quantification of the capillary density in the gastrocnemius muscle revealed that mice treated with a HD of antagomir-126 had a markedly reduced angiogenic response. Aortic explant cultures of the mice confirmed the role of miR-126 in angiogenesis. Our data demonstrate a facilitary function for miR-126 in ischemia-induced angiogenesis and show the efficacy and specificity of antagomir-induced silencing of EC-specific microRNAs in vivo.

SUBMITTER: van Solingen C

PROVIDER: S-EPMC3828868 | biostudies-other | 2009 Aug

REPOSITORIES: biostudies-other

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Antagomir-mediated silencing of endothelial cell specific microRNA-126 impairs ischemia-induced angiogenesis.

van Solingen Coen C Seghers Leonard L Bijkerk Roel R Duijs Jacques M G J JM Roeten Marko K MK van Oeveren-Rietdijk Annemarie M AM Baelde Hans J HJ Monge Matthieu M Vos Joost B JB de Boer Hetty C HC Quax Paul H A PH Rabelink Ton J TJ van Zonneveld Anton Jan AJ

Journal of cellular and molecular medicine 20090801 8A

MicroRNAs are negative regulators of gene expression that play a key role in cell-type specific differentiation and modulation of cell function and have been proposed to be involved in neovascularization. Previously, using an extensive cloning and sequencing approach, we identified miR-126 to be specifically and highly expressed in human endothelial cells (EC). Here, we demonstrate EC-specific expression of miR-126 in capillaries and the larger vessels in vivo. We therefore explored the potentia ...[more]

PMID: 19120690

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Project description:Thioredoxin-2 (Trx2), a major antioxidant protein in mitochondria, enhances nitric oxide bioavailability and inhibits ASK1-dependent apoptosis in endothelial cells (ECs). However, the in vivo role of Trx2 in angiogenesis has not been defined. Here we used EC-specific transgenesis of Trx2 (Trx2-TG) in mice to determine the in vivo function of Trx2 in arteriogenesis and angiogenesis.In a femoral artery ligation model, Trx2-TG mice had enhanced capacity in limb perfusion recovery and ischemic reserve capacity compared to the nontransgenic littermates. Ischemia-initiated arteriogenesis in the upper limb was augmented in Trx2-TG mice. Trx2-TG mice also showed significantly enhanced capillary formation and maturation in the lower limb. In nontransgenic limb, ischemia specifically induced a downregulation of Trx2 protein, leading to increased oxidative stress, ASK1 activation, and EC apoptosis. In contrast, Trx2-TG maintained a constitutive level of Trx2, reducing the ischemia-induced deleterious responses. We then defined the mechanism by which Trx2 increases angiogenesis using ECs isolated from Trx2-TG mice. Trx2-TG ECs showed increased NO and NO-dependent migration. In addition, these cells were more resistant to oxidative stress-induced activation of ASK1 signaling and apoptosis. Moreover, Trx2-augmented EC survival is NO-independent. To define the relative contributions of Trx2-increased NO and Trx2-reduced ASK1 apoptotic activity to angiogenesis in vivo, we examined Trx2 effects on ischemia-induced angiogenesis in eNOS-deficient mice. The eNOS deletion caused severe impairment in the functional flow recovery in response to ischemia. Trx2 expression in eNOS-KO mice still dramatically inhibited ischemia-induced ASK1 and EC apoptosis, leading to an enhanced functional flow recovery.These in vivo and in vitro data support that Trx2 maintains EC function by two parallel pathways-scavenging ROS to increase NO bioavailability and inhibiting ASK1 activity to enhance EC survival, facilitating ischemia-mediated arteriogenesis and angiogenesis.

Project description:RATIONALE:Notch signaling regulates vascular development. However, the implication of the Notch ligand Delta-like 4 (Dll4) in postischemic angiogenesis remains unclear. OBJECTIVE:We investigated the role of Dll4/Notch signaling in reparative angiogenesis using a mouse model of ischemia. METHODS AND RESULTS:We found Dll4 weakly expressed in microvascular endothelial cells of normoperfused muscles. Conversely, Dll4 is upregulated following ischemia and localized at the forefront of sprouting capillaries. We analyzed the effect of inhibiting endogenous Dll4 by intramuscular injection of an adenovirus encoding the soluble form of Dll4 extracellular domain (Ad-sDll4). Dll4 inhibition caused the formation of a disorganized, low-perfused capillary network in ischemic muscles. This structural abnormality was associated to delayed blood flow recovery and muscle hypoxia and degeneration. Analysis of microvasculature at early stages of repair revealed that Dll4 inhibition enhances capillary sprouting in a chaotic fashion and causes excessive leukocyte infiltration of ischemic muscles. Furthermore, Dll4 inhibition potentiated the elevation of the leukocyte chemoattractant CXCL1 (chemokine [C-X-C motif] ligand 1) following ischemia, without altering peripheral blood levels of stromal cell-derived factor-1 and monocyte chemoattractant protein-1. In cultured human monocytes, Dll4 induces the transcription of Notch target gene Hes-1 and inhibits the basal and tumor necrosis factor-alpha-stimulated production of interleukin-8, the human functional homolog of murine CXCL1. The inhibitory effect of Dll4 on interleukin-8 was abolished by DAPT, a Notch inhibitor, or by coculturing activated human monocytes with Ad-sDll4-infected endothelial cells. CONCLUSIONS:Dll4/Notch interaction is essential for proper reparative angiogenesis. Moreover, Dll4/Notch signaling regulates sprouting angiogenesis and coordinates the interaction between inflammation and angiogenesis under ischemic conditions.

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Antagomir-mediated silencing of endothelial cell specific microRNA-126 impairs ischemia-induced angiogenesis.

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Antagomir-mediated silencing of endothelial cell specific microRNA-126 impairs ischemia-induced angiogenesis.

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