The microRNA-30 family targets DLL4 to modulate endothelial cell behavior during angiogenesis

Abstract

IntroductionDLL4 plays a fundamental role in vascular development and angiogenesis. 1,2 DLL4 haploinsufficiency results in extensive arterial defects and embryonic lethality, 3 indicating that the developing vasculature is sensitive to minor alterations in DLL4 dosage. DLL4 expression is mainly restricted to the endothelium of nascent vessels, particularly the tip cells, where it maintains stalk cell identity in neighboring cells, thereby regulating vessel sprouting and branching in response to angiogenic stimuli. 4 The importance of optimal DLL4 expression in physiologic angiogenesis is illustrated through its regulation of intersegmental vessel (ISV) development in zebrafish. Morpholino (MO) knockdown of dll4 in zebrafish results in an increased number of endothelial cells within the ISVs and ectopic ISV branching from the dorsal aorta (DA) because of overactivation of Vegfa signaling. 5,6 DLL4 is relevant in pathologic angiogenesis and is overexpressed in human tumors, often in association with markers of inflammation, hypoxia and angiogenesis. [7][8][9] Inhibition of DLL4 suppresses experimental tumor growth by inducing nonproductive, deregulated angiogenesis. 10,11 We and others have shown that DLL4 expression is up-regulated in lymphatic endothelial cells (LECs) after infection by Kaposi sarcoma herpesvirus (KSHV), 12,13 an oncogenic ␥-herpesvirus that is the etiologic agent of Kaposi sarcoma (KS). KS is an angioproliferative neoplasm composed of cells of endothelial origin. 14 Although accurate regulation of DLL4 levels is a hallmark of angiogenesis, the mechanisms that finely regulate DLL4 expression are not completely defined. Therefore we hypothesized that, in addition to well-known transcriptional mechanisms that affect DLL4 expression, DLL4 is regulated at the posttranscriptional level.MicroRNAs (miRNAs) are small, noncoding RNAs that influence target gene expression through mRNA degradation and translation inhibition. 15 Implicated in key cellular processes, miRNAs play a role in angiogenesis and cancer. 16,17 miR-27b is the only miRNA thus far implicated in DLL4 regulation 18 ; however, this miRNA also regulates sprouty homologue 2 (SPRY2) and semaphorin 6A (SEMA6A), and it is unclear whether its proposed suppression of DLL4 specifically leads to vascular defects. 18,19 We previously described the miRNA signature in KSHV-infected LECs (KLECs). 20 These data indicated significant down-regulation of members of the miR-30 miRNA family postinfection (PI). Encoded by 6 genes and expressed from 4 distinct transcripts across the human genome, the members of the miR-30 family share an identical seed sequence and hence have common predicted targets. 21 Here we show that miR-30b and miR-30c target DLL4 in vitro and in vivo, and that the miR-30 family regulates angiogenesis. Methods Cell cultureLECs were purchased from Promocell and grown in endothelial growth medium MV (Promocell) supplemented with 10 ng/mL VEGF-C (R&D Systems). HUVECs were purchased from Promocell and grown in endothelial growth medium MV2 ...

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

“…Similar effects were reported for miR-30b and miR-30c overexpression. 25 These data suggest that because all miR-30 family members share an identical seed, they all have the potential of targeting dll4 in gain-of-function experiments.…”

Latency-Associated Nuclear Antigen of Kaposi Sarcoma–Associated Herpesvirus Promotes Angiogenesis through Targeting Notch Signaling Effector Hey1

“…Moreover, the KSHV genome itself encodes viral cytokine-and chemokine-like factors that can activate endothelial cells and stimulate angiogenesis (4,(8)(9)(10)(11)(12). However, although it is a welldefined angiogenesis-associated pathway, whether the Notch signaling pathway can be manipulated by KSHV in Kaposi sarcoma angiogenesis, is largely unknown (13,14).…”

Regulation of NF-κB signaling by oxidized glycerophospholipid and IL-1β induced miRs-21-3p and -27a-5p in human aortic endothelial cells

“…Seven of the 11 previously expressed miRNAs have been implicated as regulators of angiogenesis. miRs-126-5p, -126-3p, -27a-3p, -103a-3p, -30a-5p, and -let-7f are proangiogenic and target known inhibitors of angiogenesis, including the genes SPRED1 , DLK1 , SEMA6 , TSP-1 , and DLL4 to promote vessel development ( 11,(44)(45)(46)(47). miR-24-3p, through regulation of the transcription factor of GATA2 and PAK4 , acts to inhibit angiogenesis ( 48 ).…”