Material

Monocytes/macrophages are critical in orchestrating the tissue-repair response. However, the mechanisms that govern macrophage regenerative activities during the sequential phases of repair are largely unknown. In the present study, we examined the dynamics and functions of diverse monocyte/macrophage phenotypes during the sequential stages of skin repair. By combining the analysis of a new CCR2-eGFP reporter mouse model with conditional mouse mutants defective in myeloid cell–restricted CCR2 signaling or VEGF-A synthesis, we show herein that among the large number of inflammatory CCR2+Ly6C+ macrophages that dominate the early stage of repair, only a small fraction strongly expresses VEGF-A that has nonredundant functions for the induction of vascular sprouts. The switch of macrophage-derived VEGF-A during the early stage of tissue growth toward epidermal-derived VEGF-A during the late stage of tissue maturation was critical to achieving physiologic tissue vascularization and healing progression. The results of the present study provide new mechanistic insights into CCR2-mediated recruitment of blood monocyte subsets into damaged tissue, the dynamics and functional consequences of macrophage plasticity during the sequential repair phases, and the complementary role of macrophage-derived VEGF-A in coordinating effective tissue growth and vascularization in the context of tissue-resident wound cells. Our findings may be relevant for novel monocyte-based therapies to promote tissue vascularization.

E ndothelial cells form the inner lining of all blood vessels and not only regulate transport of nutrients to the underlying tissue but also coordinate the formation of new blood vessels, a process termed angiogenesis. Therefore, endothelial cells are highly plastic cells that are capable of switching from a resting quiescent state in normal conduit blood vessels to a highly proliferative and migratory state when angiogenesis takes place. Resting quiescent endothelial cells are termed phalanx cells, 1 whereas migratory angiogenic endothelial cells are referred to as tip cells, which are followed by proliferating so-called stalk cells.2 Although the mechanisms regulating tip and stalk cell behavior have been extensively studied, relatively little is known about the control of the phalanx state.Shear stress, the force that laminar blood flow exerts on endothelial cells, is thought to be one of the factors that determine the quiescent state of endothelial cells.3 This biomechanical stimulus induces the expression of the transcription factor Krüppel-like factor 2 (KLF2), which orchestrates a network of genes that elicit a quiescent endothelial cell phenotype. 4,5 Among the factors that are upregulated by KLF2 are antiinflammatory and antithrombotic proteins, whereas proinflammatory and prothrombotic factors are downregulated by KLF2. 4 Although not all effects of shear stress on endothelial cells are mediated by KLF2, KLF2 coordinates approximately half of the gene expression programs evoked by shear stress. 5,6See accompanying editorial on page 13Recent studies have highlighted the importance of cellular metabolism for the control of endothelial cell phenotype. 7,8 Particularly, it was shown that angiogenic endothelial cells rely heavily on glycolysis for migration and proliferation. The enzyme PFKFB3 is a key regulator of glycolysis in endothelial cells that has been shown to promote angiogenic sprouting.9-11 However, how resting endothelial cells control © 2014 American Heart Association, Inc. Objective-Cellular metabolism was recently shown to regulate endothelial cell phenotype profoundly. Whether the atheroprotective biomechanical stimulus elicited by laminar shear stress modulates endothelial cell metabolism is not known. Approach and Results-Here, we show that laminar flow exposure reduced glucose uptake and mitochondrial content in endothelium. Shear stress-mediated reduction of endothelial metabolism was reversed by silencing the flow-sensitive transcription factor Krüppel-like factor 2 (KLF2). Endothelial-specific deletion of KLF2 in mice induced glucose uptake in endothelial cells of perfused hearts. KLF2 overexpression recapitulates the inhibitory effects on endothelial glycolysis elicited by laminar flow, as measured by Seahorse flux analysis and glucose uptake measurements. RNA sequencing showed that shear stress reduced the expression of key glycolytic enzymes, such as 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3 (PFKFB3), phosphofructokinase-1, and hexokinase 2 in a KLF2-dependent ma...

MicroRNAs (miRs) are small non-coding RNAs that post-transcriptionally control gene expression. Inhibition of miRs by antisense RNAs (antimiRs) might be a therapeutic option for many diseases, but systemic inhibition can have adverse effects. Here we show that light-activatable antimiRs efficiently and locally restricted target miR activity in vivo. We use an antimiR-92a and establish a therapeutic benefit in diabetic wound healing. AntimiR-92a is modified with photolabile protecting groups, so called ‘cages'. Irradiation activates intradermally injected caged antimiR-92a without substantially affecting miR-92a expression in other organs. Light activation of caged antimiR-92a improves healing in diabetic mice to a similar extent as conventional antimiRs and derepresses the miR-92a targets Itga5 and Sirt1, thereby regulating wound cell proliferation and angiogenesis. These data show that light can be used to locally activate therapeutically active antimiRs in vivo.

Noncoding RNAs have been shown to exert important physiological and pathophysiological functions. Various studies suggest that modulating noncoding RNAs may provide a therapeutic option. Noncoding RNAs comprise small RNAs, mainly microRNAs, and long noncoding RNAs. MicroRNAs postranscriptionally regulate gene expression pattern by binding to the 3'untranslated region of a given target mRNA, thereby blocking protein translation or inducing its degradation. Long noncoding RNAs on the contrary have more diverse functions acting as epigenetic regulators, molecular scaffolds, or decoys. In this article, we summarize examples of microRNAs and long noncoding RNAs, which might be promising novel targets for treatment of cardiovascular diseases, such as heart failure, acute myocardial infarction, fibrosis, as well as atherosclerosis. Furthermore, we give insights into the available tools to inhibit or overexpress noncoding RNAs and discuss the challenges for translation. Strategies for improving RNA therapeutics and reducing toxicity, for example, by augmenting tissue specificity or cellular uptake will be discussed.

Rationale: Vascularization is critical to maintain organ function. Although many molecular pathways were shown to control vessel growth, the genuine process of capillary formation under different conditions is unclear. Objective: Here, we elucidated whether clonal expansion contributes to vessel growth by using Confetti mice for genetic tracing of clonally expanding endothelial cells (ECs). Methods and Results: In postnatal retina angiogenesis, we predominantly observed random distribution of fluorescence labeled ECs indicative of random integration or cell mixing. However, in models of pathophysiological angiogenesis (retinopathy of prematurity), as well as ischemia-induced angiogenesis in limbs and hearts, clonally expanded ECs were significantly more abundant (≤69%). Inhibition of VEGFR2 (vascular endothelial growth factor receptor 2) reduced clonal expansion after ischemia. To determine the mechanism underlying clonal expansion in vivo, we assessed gene expression specifically in clonally expanded ECs selected by laser capture microscopy. Clonally expanded ECs showed an enrichment of genes involved in endothelial-to-mesenchymal transition. Moreover, hypoxia-induced clonal expansion and endothelial-to-mesenchymal transition in ECs in vitro suggesting that hypoxia-enhanced endothelial-to-mesenchymal transition might contribute to vessel growth under ischemia. Conclusions: Our data suggest that neovascularization after ischemia is partially mediated by clonal expansion of ECs. Identification of the pathways that control clonal expansion may provide novel tools to augment therapeutic neovascularization or treat pathological angiogenesis.

The vascular bone marrow niche influences outcome in chronic myeloid leukemia via the E-selectin-SCL/TAL1-CD44 axis

Objective-Jumonji C (JmjC) domain-containing proteins modify histone and nonhistone proteins thereby controlling cellular functions. However, the role of JmjC proteins in angiogenesis is largely unknown.