Nicotine dose-dependent epigenomic-wide DNA methylation changes in the mice with long-term electronic cigarette exposure.

Epigenomic-wide DNA methylation profiling holds the potential to reflect both electronic cigarette exposure-associated risks and individual poor health outcomes. However, a systemic study in animals or humans is still lacking. Using the Infinium Mouse Methylation BeadChip, we examined the DNA methylation status of white blood cells in male ApoE-/- mice after 14 weeks of electronic cigarette exposure with the InExpose system (2 hr/day, 5 days/week, 50% PG and 50% VG) with low (6 mg/ml) and high (36 mg/ml) nicotine concentrations. Our results indicate that electronic cigarette aerosol inhalation induces significant alteration of 8,985 CpGs in a dose-dependent manner (FDR<0.05); 7,389 (82.2%) of the CpG sites are annotated with known genes. Among the top 6 significant CpG sites (P-value<1e-8), 4 CpG sites are located in the known genes, and most (3/5) of these genes have been related to cigarette smoking. The other two CpGs are close to/associated with the Phc2 gene that was recently linked to smoking in a transcriptome-wide associations study. Furthermore, the gene set enrichment analysis highlights the activation of MAPK and 4 cardiomyocyte/cardiomyopathy-related signaling pathways (including adrenergic signaling in cardiomyocytes and arrhythmogenic right ventricular cardiomyopathy) following repeated electronic cigarette use. The MAPK pathway activation correlates well with our finding of increased cytokine mRNA expression after electronic cigarette exposure in the same batch of mice. Interestingly, two pathways related to mitochondrial activities, namely mitochondrial gene expression and mitochondrial translation, are also activated after electronic cigarette exposure. Elucidating the relationship between these pathways and the increased circulating mitochondrial DNA observed here will provide further insight into the cell-damaging effects of prolonged inhalation of e-cigarette aerosols.

A mitochondrial contribution to anti-inflammatory shear stress signaling in vascular endothelial cells.

Atherosclerosis, the major cause of myocardial infarction and stroke, results from converging inflammatory, metabolic, and biomechanical factors. Arterial lesions form at sites of low and disturbed blood flow but are suppressed by high laminar shear stress (LSS) mainly via transcriptional induction of the anti-inflammatory transcription factor, Kruppel-like factor 2 (Klf2). We therefore performed a whole genome CRISPR-Cas9 screen to identify genes required for LSS induction of Klf2. Subsequent mechanistic investigation revealed that LSS induces Klf2 via activation of both a MEKK2/3-MEK5-ERK5 kinase module and mitochondrial metabolism. Mitochondrial calcium and ROS signaling regulate assembly of a mitophagy- and p62-dependent scaffolding complex that amplifies MEKK-MEK5-ERK5 signaling. Blocking the mitochondrial pathway in vivo reduces expression of KLF2-dependent genes such as eNOS and inhibits vascular remodeling. Failure to activate the mitochondrial pathway limits Klf2 expression in regions of disturbed flow. This work thus defines a connection between metabolism and vascular inflammation that provides a new framework for understanding and developing treatments for vascular disease.

MEKK3-TGFß crosstalk regulates inward arterial remodeling.

Arterial remodeling is an important adaptive mechanism that maintains normal fluid shear stress in a variety of physiologic and pathologic conditions. Inward remodeling, a process that leads to reduction in arterial diameter, plays a critical role in progression of such common diseases as hypertension and atherosclerosis. Yet, despite its pathogenic importance, molecular mechanisms controlling inward remodeling remain undefined. Mitogen-activated protein kinases (MAPKs) perform a number of functions ranging from control of proliferation to migration and cell-fate transitions. While the MAPK ERK1/2 signaling pathway has been extensively examined in the endothelium, less is known about the role of the MEKK3/ERK5 pathway in vascular remodeling. To better define the role played by this signaling cascade, we studied the effect of endothelial-specific deletion of its key upstream MAP3K, MEKK3, in adult mice. The gene's deletion resulted in a gradual inward remodeling of both pulmonary and systematic arteries, leading to spontaneous hypertension in both vascular circuits and accelerated progression of atherosclerosis in hyperlipidemic mice. Molecular analysis revealed activation of TGFß-signaling both in vitro and in vivo. Endothelial-specific TGFßR1 knockout prevented inward arterial remodeling in MEKK3 endothelial knockout mice. These data point to the unexpected participation of endothelial MEKK3 in regulation of TGFßR1-Smad2/3 signaling and inward arterial remodeling in artery diseases.

Activation of Smad2/3 signaling by low fluid shear stress mediates artery inward remodeling.

Endothelial cell (EC) sensing of wall fluid shear stress (FSS) from blood flow governs vessel remodeling to maintain FSS at a specific magnitude or set point in healthy vessels. Low FSS triggers inward remodeling to restore normal FSS but the regulatory mechanisms are unknown. In this paper, we describe the signaling network that governs inward artery remodeling. FSS induces Smad2/3 phosphorylation through the type I transforming growth factor (TGF)-ß family receptor Alk5 and the transmembrane protein Neuropilin-1, which together increase sensitivity to circulating bone morphogenetic protein (BMP)-9. Smad2/3 nuclear translocation and target gene expression but not phosphorylation are maximal at low FSS and suppressed at physiological high shear. Reducing flow by carotid ligation in rodents increases Smad2/3 nuclear localization, while the resultant inward remodeling is blocked by the EC-specific deletion of Alk5. The flow-activated MEKK3/Klf2 pathway mediates the suppression of Smad2/3 nuclear translocation at high FSS, mainly through the cyclin-dependent kinase (CDK)-2-dependent phosphosphorylation of the Smad linker region. Thus, low FSS activates Smad2/3, while higher FSS blocks nuclear translocation to induce inward artery remodeling, specifically at low FSS. These results are likely relevant to inward remodeling in atherosclerotic vessels, in which Smad2/3 is activated through TGF-ß signaling.

Smooth Muscle Cell Reprogramming in Aortic Aneurysms.

The etiology of aortic aneurysms is poorly understood, but it is associated with atherosclerosis, hypercholesterolemia, and abnormal transforming growth factor ß (TGF-ß) signaling in smooth muscle. Here, we investigated the interactions between these different factors in aortic aneurysm development and identified a key role for smooth muscle cell (SMC) reprogramming into a mesenchymal stem cell (MSC)-like state. SMC-specific ablation of TGF-ß signaling in Apoe-/- mice on a hypercholesterolemic diet led to development of aortic aneurysms exhibiting all the features of human disease, which was associated with transdifferentiation of a subset of contractile SMCs into an MSC-like intermediate state that generated osteoblasts, chondrocytes, adipocytes, and macrophages. This combination of medial SMC loss with marked increases in non-SMC aortic cell mass induced exuberant growth and dilation of the aorta, calcification and ossification of the aortic wall, and inflammation, resulting in aneurysm development.

Isoform-Specific Roles of ERK1 and ERK2 in Arteriogenesis.

Despite the clinical importance of arteriogenesis, this biological process is poorly understood. ERK1 and ERK2 are key components of a major intracellular signaling pathway activated by vascular endothelial growth (VEGF) and FGF2, growth factors critical to arteriogenesis. To investigate the specific role of each ERK isoform in arteriogenesis, we used mice with a global Erk1 knockout as well as Erk1 and Erk2 floxed mice to delete Erk1 or Erk2 in endothelial cells, macrophages, and smooth muscle cells. We found that ERK1 controls macrophage infiltration following an ischemic event. Loss of ERK1 in endothelial cells and macrophages induced an excessive macrophage infiltration leading to an increased but poorly functional arteriogenesis. Loss of ERK2 in endothelial cells leads to a decreased arteriogenesis due to decreased endothelial cell proliferation and a reduced eNOS expression. These findings show for the first time that isoform-specific roles of ERK1 and ERK2 in the control of arteriogenesis.

Author Correction: SUMOylation of VEGFR2 regulates its intracellular trafficking and pathological angiogenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Noninvasive In Vivo Quantification of Adeno-Associated Virus Serotype 9-Mediated Expression of the Sodium/Iodide Symporter Under Hindlimb Ischemia and Neuraminidase Desialylation in Skeletal Muscle Using Single-Photon Emission Computed Tomography/Computed Tomography.

BACKGROUND: We propose micro single-photon emission computed tomography/computed tomography imaging of the hNIS (human sodium/iodide symporter) to noninvasively quantify adeno-associated virus 9 (AAV9)-mediated gene expression in a murine model of peripheral artery disease. METHODS: AAV9-hNIS (2×1011 viral genome particles) was injected into nonischemic or ischemic gastrocnemius muscles of C57Bl/6J mice following unilateral hindlimb ischemia ± the α-sialidase NA (neuraminidase). Control nonischemic limbs were injected with phosphate buffered saline or remained noninjected. Twelve mice underwent micro single-photon emission computed tomography/computed tomography imaging after serial injection of pertechnetate (99mTcO4-), a NIS substrate, up to 28 days after AAV9-hNIS injection. Twenty four animals were euthanized at selected times over 1 month for ex vivo validation. Forty-two animals were imaged with 99mTcO4- ± the selective NIS inhibitor perchlorate on day 10, to ascertain specificity of radiotracer uptake. Tissue was harvested for ex vivo validation. A modified version of the U-Net deep learning algorithm was used for image quantification. RESULTS: As quantitated by standardized uptake value, there was a gradual temporal increase in 99mTcO4- uptake in muscles treated with AAV9-hNIS. Hindlimb ischemia, NA, and hindlimb ischemia plus NA increased the magnitude of 99mTcO4- uptake by 4- to 5-fold compared with nonischemic muscle treated with only AAV9-hNIS. Perchlorate treatment significantly reduced 99mTcO4- uptake in AAV9-hNIS-treated muscles, demonstrating uptake specificity. The imaging results correlated well with ex vivo well counting (r2=0.9375; P<0.0001) and immunoblot analysis of NIS protein (r2=0.65; P<0.0001). CONCLUSIONS: Micro single-photon emission computed tomography/computed tomography imaging of hNIS-mediated 99mTcO4- uptake allows for accurate in vivo quantification of AAV9-driven gene expression, which increases under ischemic conditions or neuraminidase desialylation in skeletal muscle.

Publisher Correction: N-terminal syndecan-2 domain selectively enhances 6-O heparan sulfate chains sulfation and promotes VEGFA165-dependent neovascularization.

The original version of this Article contained errors in Figures 1, 3 and 4. In panels b and d of Figure 1, the labels 'Sdc4-/-' were inadvertently replaced by 'Sdc4+/+'. In panels c and d of Figure 3, the labels 'Sdc4-/-' were replaced by 'Sdc2-/-'. In panel f of Figure 3, the labels 'FGF2' were replaced by 'VEGFA165'. In panel e of Figure 6, a 'Sdc2-/-' label was inadvertently included. This has now been corrected in the PDF and HTML versions of the Article.

N-terminal syndecan-2 domain selectively enhances 6-O heparan sulfate chains sulfation and promotes VEGFA165-dependent neovascularization.

The proteoglycan Syndecan-2 (Sdc2) has been implicated in regulation of cytoskeleton organization, integrin signaling and developmental angiogenesis in zebrafish. Here we report that mice with global and inducible endothelial-specific deletion of Sdc2 display marked angiogenic and arteriogenic defects and impaired VEGFA165 signaling. No such abnormalities are observed in mice with deletion of the closely related Syndecan-4 (Sdc4) gene. These differences are due to a significantly higher 6-O sulfation level in Sdc2 versus Sdc4 heparan sulfate (HS) chains, leading to an increase in VEGFA165 binding sites and formation of a ternary Sdc2-VEGFA165-VEGFR2 complex which enhances VEGFR2 activation. The increased Sdc2 HS chains 6-O sulfation is driven by a specific N-terminal domain sequence; the insertion of this sequence in Sdc4 N-terminal domain increases 6-O sulfation of its HS chains and promotes Sdc2-VEGFA165-VEGFR2 complex formation. This demonstrates the existence of core protein-determined HS sulfation patterns that regulate specific biological activities.

Restoration of brain circulation and cellular functions hours post-mortem.

The brains of humans and other mammals are highly vulnerable to interruptions in blood flow and decreases in oxygen levels. Here we describe the restoration and maintenance of microcirculation and molecular and cellular functions of the intact pig brain under ex vivo normothermic conditions up to four hours post-mortem. We have developed an extracorporeal pulsatile-perfusion system and a haemoglobin-based, acellular, non-coagulative, echogenic, and cytoprotective perfusate that promotes recovery from anoxia, reduces reperfusion injury, prevents oedema, and metabolically supports the energy requirements of the brain. With this system, we observed preservation of cytoarchitecture; attenuation of cell death; and restoration of vascular dilatory and glial inflammatory responses, spontaneous synaptic activity, and active cerebral metabolism in the absence of global electrocorticographic activity. These findings demonstrate that under appropriate conditions the isolated, intact large mammalian brain possesses an underappreciated capacity for restoration of microcirculation and molecular and cellular activity after a prolonged post-mortem interval.

Molecular Imaging of Factor XIII Activity for the Early Detection of Mouse Coronary Microvascular Disease.

Coronary microvascular disease (MVD) remains a major clinical problem due to limited mechanistic understanding and a challenging diagnosis. In the present study we evaluated the utility of targeted imaging of active factor XIII (FXIII) for detection of coronary MVD associated with thrombus. We hypothesized that a high specificity and sensitivity FXIII targeted radiolabeled probe can serve as a biomarker for cross-linked thrombi in the microvasculature, and thus an indicator for underlying coronary MVD. To evaluate this approach, a coronary MVD model was established for local induction of singlet oxygen and reactive oxygen species (ROS) via a photochemical reaction (PCR). Methods: PCR was used to induce endothelial injury and microthrombi via focal over-production of ROS only in the coronary microvasculature. Oxidative stress was initially evaluated in primary coronary endothelial cells to optimize parameters of PCR, which were then translated to in vivo experiments. To develop the coronary MVD model, 64 mice were assigned to one of four groups after thoracotomy: 1) sham control; 2) rose bengal; 3) green light; or 4) their combination. Following interventions, the mice underwent transmission electron microscopy, fluorescent myocardial perfusion, coronary angiography, and immunohistochemical staining. Echocardiography (n = 12) and gene expression (n = 10) studies were also performed after MVD induction to monitor serial changes in cardiac function and explore possible mechanisms. To diagnose early onset MVD, FXIII radioactivity was assessed in 104 mice using ex vivo gamma well counting (GWC) and in 14 mice using in vivo serial single photon emission computed tomography / computed tomography (SPECT/CT) imaging of a FXIII targeted technetium-labeled probe (99mTc-NC100668). Results:In vitro experiments demonstrated that photosensitizer concentration and light illumination time were critical parameters for PCR. In vivo experiments demonstrated manifestations of clinical MVD, including endothelial damage, a "no flow zone," arteriole rarefaction with patent epicardial coronary arteries, infiltration of inflammatory cells in the PCR-treated region, and preserved cardiac function. Gene expression also demonstrated a pro-thrombotic and impaired fibrinolytic status. In the early stages of MVD, enhanced FXIII activity was confirmed within the MVD region using GWC and in vivo SPECT/CT imaging. Conclusion: Our results demonstrate that molecular imaging of FXIII activity may allow for early detection of coronary MVD associated with thrombus, in a novel pre-clinical model.

Sex-dependent differences in central artery haemodynamics in normal and fibulin-5 deficient mice: implications for ageing.

Mouse models provide unique opportunities to study vascular disease, but they demand increased experimental and computational resolution. We describe a workflow for combining in vivo and in vitro biomechanical data to build mouse-specific computational models of the central vasculature including regional variations in biaxial wall stiffness, thickness and perivascular support. These fluid-solid interaction models are informed by micro-computed tomography imaging and in vivo ultrasound and pressure measurements, and include mouse-specific inflow and outflow boundary conditions. Hence, the model can capture three-dimensional unsteady flows and pulse wave characteristics. The utility of this experimental-computational approach is illustrated by comparing central artery biomechanics in adult wild-type and fibulin-5 deficient mice, a model of early vascular ageing. Findings are also examined as a function of sex. Computational results compare well with measurements and data available in the literature and suggest that pulse wave velocity, a spatially integrated measure of arterial stiffness, does not reflect well the presence of regional differences in stiffening, particularly those manifested in male versus female mice. Modelling results are also useful for comparing quantities that are difficult to measure or infer experimentally, including local pulse pressures at the renal arteries and characteristics of the peripheral vascular bed that may differ with disease.

SUMOylation of VEGFR2 regulates its intracellular trafficking and pathological angiogenesis.

Regulation of VEGFR2 represents an important mechanism for the control of angiogenesis. VEGFR2 activity can be regulated by post-translational modifications such as ubiquitination and acetylation. However, whether VEGFR2 can be regulated by SUMOylation has not been investigated. Here we show that endothelial-specific deletion of the SUMO endopeptidase SENP1 reduces pathological angiogenesis and tissue repair during hindlimb ischemia, and VEGF-induced angiogenesis in the cornea, retina, and ear. SENP1-deficient endothelial cells show increased SUMOylation of VEGFR2 and impaired VEGFR2 signalling. SUMOylation at lysine 1270 retains VEGFR2 in the Golgi and reduces its surface expression, attenuating VEGFR2-dependent signalling. Moreover, we find that SENP1 is downregulated and VEGFR2 hyper-SUMOylated in diabetic settings and that expression of a non-SUMOylated form of VEGFR2 rescues angiogenic defects in diabetic mice. These results show that VEGFR2 is regulated by deSUMOylation during pathological angiogenesis, and propose SENP1 as a potential therapeutic target for the treatment of diabetes-associated angiogenesis.

Inhibiting Integrin α5 Cytoplasmic Domain Signaling Reduces Atherosclerosis and Promotes Arteriogenesis.

BACKGROUND: Fibronectin in endothelial basement membranes promotes endothelial inflammatory activation and atherosclerosis but also promotes plaque stability and vascular remodeling. The fibronectin receptor α5 subunit is proinflammatory through binding to and activating phosphodiesterase 4D5, which inhibits anti-inflammatory cyclic adenosine monophosphate and protein kinase A. Replacing the α5 cytoplasmic domain with that of α2 resulted in smaller atherosclerotic plaques. Here, we further assessed plaque phenotype and compensatory vascular remodeling in this model. METHODS AND RESULTS: α5/2 mice in the hyperlipidemic apolipoprotein E null background had smaller plaques in the aortic root, with reduced endothelial NF-κB activation and inflammatory gene expression, reduced leukocyte content, and much lower metalloproteinase expression. However, smooth muscle cell content, fibrous cap thickness, and fibrillar collagen were unchanged, indicating no shift toward vulnerability. In vivo knockdown of phosphodiesterase 4D5 also decreased endothelial inflammatory activation and atherosclerotic plaque size. α5/2 mice showed improved recovery from hindlimb ischemia after femoral artery ligation. CONCLUSIONS: Blocking the fibronectin-Integrin α5 pathway reduces atherosclerotic plaque size, maintains plaque stability, and improves compensatory remodeling. This pathway is therefore a potential therapeutic target for treatment of atherosclerosis.

Quantification of intramyocardial blood volume with 99mTc-RBC SPECT-CT imaging: A preclinical study.

BACKGROUND: Currently, there is no established non-invasive imaging approach to directly evaluate myocardial microcirculatory function in order to diagnose microvascular disease independent of co-existing epicardial disease. In this work, we developed a methodological framework for quantification of intramyocardial blood volume (IMBV) as a novel index of microcirculatory function with SPECT/CT imaging of 99mTc-labeled red blood cells (RBCs). METHODS: Dual-gated myocardial SPECT/CT equilibrium imaging of 99mTc-RBCs was performed on twelve canines under resting conditions. Five correction schemes were studied: cardiac gating with no other corrections (CG), CG with attenuation correction (CG + AC), CG + AC with scatter correction (CG + AC + SC), dual cardiorespiratory gating with AC + SC (DG + AC + SC), and DG + AC + SC with partial volume correction (DG + AC + SC + PVC). Quantification of IMBV using each approach was evaluated in comparison to those obtained from all corrections. The in vivo SPECT estimates of IMBV values were validated against those obtained from ex vivo microCT imaging of the casted hearts. RESULTS: The estimated IMBV with all corrections was 0.15 ± 0.03 for the end-diastolic phase and 0.11 ± 0.03 for the end-systolic phase. The cycle-dependent change in IMBV (ΔIMBV) with all corrections was 23.9 ± 8.6%. Schemes that applied no correction or partial correction resulted in significant over-estimation of IMBV and significant under-underestimation of ΔIMBV. Estimates of IMBV and ΔIMBV using all corrections were consistent with values reported in the literature using invasive techniques. In vivo SPECT estimates of IMBV strongly correlated (R2 ≥ 0.70) with ex vivo measures for the various correction schemes, while the fully corrected scheme yielded the smallest bias. CONCLUSIONS: Non-invasive quantification of IMBV is feasible using 99mTc-RBCs SPECT/CT imaging, however, requires full compensation of physical degradation factors.

Novel application and serial evaluation of tissue-engineered portal vein grafts in a murine model.

AIM: Surgical management of pediatric extrahepatic portal vein obstruction requires meso-Rex bypass using autologous or synthetic grafts. Tissue-engineered vascular grafts (TEVGs) provide an alternative, but no validated animal models using portal TEVGs exist. Herein, we preclinically assess TEVGs as portal vein bypass grafts. MATERIALS & METHODS: TEVGs were implanted as portal vein interposition conduits in SCID-beige mice, monitored by ultrasound and micro-computed tomography, and histologically assessed postmortem at 12 months. RESULTS: TEVGs remained patent for 12 months. Histologic analysis demonstrated formation of neovessels that resembled native portal veins, with similar content of smooth muscle cells, collagen type III and elastin. CONCLUSION: TEVGs are feasible portal vein conduits in a murine model. Further preclinical evaluation of TEVGs may facilitate pediatric clinical translation.

Hyperoxia causes miR-34a-mediated injury via angiopoietin-1 in neonatal lungs.

Hyperoxia-induced acute lung injury (HALI) is a key contributor to the pathogenesis of bronchopulmonary dysplasia (BPD) in neonates, for which no specific preventive or therapeutic agent is available. Here we show that lung micro-RNA (miR)-34a levels are significantly increased in lungs of neonatal mice exposed to hyperoxia. Deletion or inhibition of miR-34a improves the pulmonary phenotype and BPD-associated pulmonary arterial hypertension (PAH) in BPD mouse models, which, conversely, is worsened by miR-34a overexpression. Administration of angiopoietin-1, which is one of the downstream targets of miR34a, is able to ameliorate the BPD pulmonary and PAH phenotypes. Using three independent cohorts of human samples, we show that miR-34a expression is increased in type 2 alveolar epithelial cells in neonates with respiratory distress syndrome and BPD. Our data suggest that pharmacologic miR-34a inhibition may be a therapeutic option to prevent or ameliorate HALI/BPD in neonates.

MicroCT angiography detects vascular formation and regression in skin wound healing.

Properly regulated angiogenesis and arteriogenesis are essential for effective wound healing. Tissue injury induces robust new vessel formation and subsequent vessel maturation, which involves vessel regression and remodeling. Although formation of functional vasculature is essential for healing, alterations in vascular structure over the time course of skin wound healing are not well understood. Here, using high-resolution ex vivo X-ray micro-computed tomography (microCT), we describe the vascular network during healing of skin excisional wounds with highly detailed three-dimensional (3D) reconstructed images and associated quantitative analysis. We found that relative vessel volume, surface area and branching number are significantly decreased in wounds from day 7 to days 14 and 21. Segmentation and skeletonization analysis of selected branches from high-resolution images as small as 2.5µm voxel size show that branching orders are decreased in the wound vessels during healing. In histological analysis, we found that the contrast agent fills mainly arterioles, but not small capillaries nor large veins. In summary, high-resolution microCT revealed dynamic alterations of vessel structures during wound healing. This technique may be useful as a key tool in the study of the formation and regression of wound vessels.

Endothelial miR-17∼92 cluster negatively regulates arteriogenesis via miRNA-19 repression of WNT signaling.

The contribution of endothelial-derived miR-17∼92 to ischemia-induced arteriogenesis has not been investigated in an in vivo model. In the present study, we demonstrate a critical role for the endothelial-derived miR-17∼92 cluster in shaping physiological and ischemia-triggered arteriogenesis. Endothelial-specific deletion of miR-17∼92 results in an increase in collateral density limbs and hearts and in ischemic limbs compared with control mice, and consequently improves blood flow recovery. Individual cluster components positively or negatively regulate endothelial cell (EC) functions in vitro, and, remarkably, ECs lacking the cluster spontaneously form cords in a manner rescued by miR-17a, -18a, and -19a. Using both in vitro and in vivo analyses, we identified FZD4 and LRP6 as targets of miR-19a/b. Both of these targets were up-regulated in 17∼92 KO ECs compared with control ECs, and both were shown to be targeted by miR-19 using luciferase assays. We demonstrate that miR-19a negatively regulates FZD4, its coreceptor LRP6, and WNT signaling, and that antagonism of miR-19a/b in aged mice improves blood flow recovery after ischemia and reduces repression of these targets. Collectively, these data provide insights into miRNA regulation of arterialization and highlight the importance of vascular WNT signaling in maintaining arterial blood flow.