miR-27b controls venous specification and tip cell fate

Abstract

We discovered that miR-27b controls 2 critical vascular functions: it turns the angiogenic switch on by promoting endothelial tip cell fate and sprouting and it promotes venous differentiation. We have identified its targets, a Notch ligand Deltalike ligand 4 (Dll4) and Sprouty homologue 2 (Spry2). miR-27b knockdown in zebrafish and mouse tissues severely impaired vessel sprouting and filopodia formation. Moreover, miR-27b was necessary for the formation of the first embryonic vein in fish and controlled the expression of arterial and venous markers in human endothelium, including Ephrin B2 (EphB2), EphB4, FMS-related tyrosine kinase 1 (Flt1), and Flt4. In zebrafish, Dll4 inhibition caused increased sprouting and longer intersegmental vessels and exacerbated tip cell migration. Blocking Spry2 caused premature vessel branching. In contrast, Spry2 overexpression eliminated the tip cell branching in the intersegmental vessels. Blockade of Dll4 and Spry2 disrupted arterial specification and augmented the expression of venous markers. Blocking either Spry2 or Dll4 rescued the miR-27b knockdown phenotype in zebrafish and in mouse vascular explants, pointing to essential roles of these targets downstream of miR27b. Our study identifies critical role of miR-27b in the control of endothelial tip cell fate, branching, and venous specification and determines Spry2 and Dll4 as its essential targets. (Blood. 2012; 119(11):2679-2687) IntroductionAngiogenic balance and endothelial cell fate are determined by the extracellular signals generated by angiogenic growth factors (stimuli) and inhibitors. 1,2 Molecular mechanisms that determine angiogenic balance have been extensively studied; however, our understanding of the key intracellular events remains incomplete. Recent studies have shown that growing vasculature follows the gradients of VEGF, which are sensed by the nonproliferative endothelial tip cells that direct further expansion of the vascular sprout. The density and morphology of the growing vasculature is dictated by the frequency of tip cells. Following behind tip cells, proliferating stalk cells ensure sprout lengthening and lumen formation. Their fate is maintained by Delta like ligand 4 (Dll4), which is produced by the tip cells. Dll4 binds Notch on adjacent stalk cells, and the resulting signal represses tip fate and ensures proliferation and sprout lengthening toward the VEGF source. 3 Stalk cell proliferation and neovessel integrity depend on VEGF and other pro-angiogenic cytokines, such as basic fibroblast growth factor, which through cognate receptors activate mitogenic kinases converging on Erk1/2. 4 In normal tissues, VEGF release from the extracellular matrix is tightly controlled and improper VEGF gradients cause abnormally high numbers of tip cells and aberrant vascular patterns. 5,6 A large family of Sprouty (Spry) genes regulates secondary branching of the tubular structures in the kidney, lung, and ear. 7 This family encodes proteins Spry1 through 4 and sprouty-related domain 1 (SPRED1) and SPRED2. In...

Regulation of vascular leak and recovery from ischemic injury by general and VE-cadherin–restricted miRNA antagonists of miR-27

“…Inhibition of miR-27 has been described previously to inhibit angiogenesis in a choroidal neovascular model, and this correlated with an increase in expression of 2 anti-angiogenic molecules, Sprouty2 and Sema6A. 34 Inhibition of miR-27 can also inhibit angiogenesis through inhibition of sprout formation, achieved through Sema6A regulation 43,44 and D-like ligand 4. 44 Our results here show that angiogenesis can also be inhibited by overexpression of miR-27a with the in vitro reconstitution experiments, confirming the target as VE-cadherin.…”

“…34 Inhibition of miR-27 can also inhibit angiogenesis through inhibition of sprout formation, achieved through Sema6A regulation 43,44 and D-like ligand 4. 44 Our results here show that angiogenesis can also be inhibited by overexpression of miR-27a with the in vitro reconstitution experiments, confirming the target as VE-cadherin. We also demonstrated that inhibition of miR-27 activity promotes angiogenesis and, in a setting of a "controlled" angiogenic response as is seen in liver fibrosis, miR-27a levels are decreased.…”

miR-30a Regulates Endothelial Tip Cell Formation and Arteriolar Branching

“…Given its importance, it may not be surprising that in ECs Dll4 can be regulated or fine-tuned by multiple miRNAs, including the miR-30 and miR-27 family members. 13 Most likely, tuning interactions in which miR-30a and miR-27b act as a rheostat to dampen protein output to an optimal level may enable customized outputs in tip/stalk cells allowing adequate physiological control of angiogenesis and vessel branching. At present, information on the contribution of these miRNA in microvascular remodeling events associated with hypertension is lacking.…”

“…6 The exact regulatory mechanisms controlling Dll4 expression levels are unknown 7,11,12 but may involve microRNAs (miRNAs). 13 miRNAs are small (22 nucleotides) single-stranded noncoding RNAs that are produced in the nucleus, processed by the enzymes Drosha and Dicer, and incorporated into RNAinduced silencing complexes. miRNAs suppress gene expression at the posttranscriptional level by degrading or inhibiting of target mRNAs.…”

Cellular and physical mechanisms of branching morphogenesis

“…Together, these molecular and genetic studies have revealed that branching morphogenesis appears to be controlled by a conserved set of molecules, including fibroblast growth factors (FGFs), which signal through the mitogen-activated protein kinase (MAPK) cascade. Recent studies have also indicated a role for miRNAs in branching morphogenesis of the lung, kidney, salivary gland and vasculature (Biyashev et al, 2012;Chu et al, 2014;Hayashi et al, 2011;Jiang et al, 2013;Mujahid et al, 2013;Rebustini et al, 2012;Yu, 2014).…”