Therapeutic angiogenesis is likely to require the administration of factors that complement each other. Activation of the receptor tyrosine kinase (RTK) Flk1 by vascular endothelial growth factor (VEGF) is crucial, but molecular interactions of other factors with VEGF and Flk1 have been studied to a limited extent. Here we report that placental growth factor (PGF, also known as PlGF) regulates inter- and intramolecular cross talk between the VEGF RTKs Flt1 and Flk1. Activation of Flt1 by PGF resulted in intermolecular transphosphorylation of Flk1, thereby amplifying VEGF-driven angiogenesis through Flk1. Even though VEGF and PGF both bind Flt1, PGF uniquely stimulated the phosphorylation of specific Flt1 tyrosine residues and the expression of distinct downstream target genes. Furthermore, the VEGF/PGF heterodimer activated intramolecular VEGF receptor cross talk through formation of Flk1/Flt1 heterodimers. The inter- and intramolecular VEGF receptor cross talk is likely to have therapeutic implications, as treatment with VEGF/PGF heterodimer or a combination of VEGF plus PGF increased ischemic myocardial angiogenesis in a mouse model that was refractory to VEGF alone.

Vascular endothelial growth factor A (here referred to as VEGF) is an endothelium-specific growth factor that binds to two distinct receptor tyrosine kinases, designated Flt-1 and KDR/Flk-1. VEGF stimulates autophosphorylation of both receptors, but little is known about their signal transduction properties. In this study, we used porcine aortic endothelial (

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IntroductionMicroRNAs (miRNAs) are highly conserved, single-stranded noncoding short RNA molecules (18-24 nucleotides) that regulate gene expression at the posttranscriptional level. miRNAs silence gene expression by inhibiting the translation of proteins from mRNAs or by promoting the degradation of mRNAs. After transcription of the primary miRNA transcripts from the genome, their maturation is mediated by the 2 RNase III endonucleases Dicer and Drosha. Then, mature miRNAs are incorporated into the RNA-induced silencing complex, 1 which mediates the binding of the miRNA to the 3Ј-untranslated region (3Ј-UTR) of the target mRNA leading either to translational repression or degradation of the target mRNA. 2 Because miRNAs control specific expression patterns of target genes, miRNAs represent attractive candidates to interfere with neovascularization.Increasing evidence indicates that miRNAs are important regulators of vascular development and angiogenesis. 3,4 In this context, first studies addressed the function of the miRNAprocessing enzymes Dicer and/or Drosha to explore the general role of miRNAs for angiogenesis. Depletion of Dicer in zebrafish or mice revealed an aberrant vessel growth, and silencing of Dicer in endothelial cells reduced in vitro angiogenesis. [5][6][7] To date, several miRs that regulate endothelial cell function and angiogenesis have been identified, 8 including the pro-angiogenic miRs miR-130a, 9 miR-210, 5,10,11 and miR-378. 12 In addition, miR-126 was shown to regulate vascular integrity and angiogenesis during development and in ischemia-induced angiogenesis. [13][14][15] In contrast, miR-221 and miR-222, 7,16 miR-15 and miR-16, 17,18 and members of the miR-17-92 cluster 19,20 inhibit angiogenesis.In our previous study, we found that the members of the miR-23ϳ27ϳ24 cluster, miR-27a and miR-27b, were highly expressed in endothelial cells. 6 In addition, miR-27b was downregulated after Dicer and Drosha silencing, and inhibition of miR-27b significantly reduced endothelial cell sprouting in vitro, 6 indicating that miR-27b exerts pro-angiogenic effects. Recently, Zhou et al demonstrated that the miR-23ϳ27ϳ24 cluster regulates angiogenesis. 21 In muscle stem cells, miR-27b down-regulates Pax3 expression during myogenic differentiation. 22 Moreover, miR-27 down-regulates Runx1 expression during granulocyte differentiation 23 and the nuclear receptor peroxisome proliferatoractivated receptor-␥ (PPAR-␥) in adipocytes. 24 The myocyte enhancer factor 2C (MEF2C) is another important target of miR-27b during heart development. 25 However, the specific functions and targets of miR-27 in endothelial cells are largely unexplored. As the family members miR-27a and miR-27b differ in only one nucleotide and share the same seed sequence, we investigated the specific role of both family members for the angiogenic activity of endothelial cells and determined the effects on neovascularization. Here we identified the angiogenesis inhibitor semaphorin 6A as a The online version of this article contains a ...

Histone deacetylases (HDACs) deacetylate histones and nonhistone proteins, thereby affecting protein activity and gene expression. The regulation and function of the cytoplasmic class IIb HDAC6 in endothelial cells (ECs) is largely unexplored. Here, we demonstrate that HDAC6 is upregulated by hypoxia and is essential for angiogenesis. Silencing of HDAC6 in ECs decreases sprouting and migration in vitro and formation of functional vascular networks in matrigel plugs in vivo. HDAC6 regulates zebrafish vessel formation, and HDAC6-deficient mice showed a reduced formation of perfused vessels in matrigel plugs. Consistently, overexpression of wild-type HDAC6 increases sprouting from spheroids. HDAC6 function requires the catalytic activity but is independent of ubiquitin binding and deacetylation of a-tubulin. Instead, we found that HDAC6 interacts with and deacetylates the actin-remodelling protein cortactin in ECs, which is essential for zebrafish vessel formation and which mediates the angiogenic effect of HDAC6. In summary, we show that HDAC6 is necessary for angiogenesis in vivo and in vitro, involving the interaction and deacetylation of cortactin that regulates EC migration and sprouting.

Vascular endothelial growth factor-A (VEGF-A) plays a major role in tumor angiogenesis and raises the concentration of intracellular free calcium ([Ca2+]i). Carboxyamidotriazole (CAI), an inhibitor of calcium influx and of angiogenesis, is under investigation as a tumoristatic agent. We studied the effect of CAI and the role of [Ca2+]i in VEGF-A signaling in human endothelial cells. VEGF-A induced a biphasic [Ca2+]i signal. VEGF-A increased the level of intracellular inositol 1,4,5-trisphosphate (IP3), which suggests that VEGF-A releases Ca2+ from IP3-sensitive stores and induces store-operated calcium influx. Reduction of either extracellular or intracellular free Ca2+ inhibited VEGF-A-induced proliferation. CAI inhibited IP3 formation, both phases of the calcium signal, nitric oxide (NO) release, and proliferation induced by VEGF-A. CAI prevented neither activation of VEGF receptor-2 (VEGFR-2) (KDR/Flk-1), phospholipase C-g, or mitogen-activated protein kinase (MAP kinase) nor translocation of nuclear factor of activated T cells (NFAT). We conclude that calcium signaling is necessary for VEGF-A-induced proliferation. MAP kinase activation occurs independently of [Ca2+]i but is not sufficient to induce proliferation in the absence of calcium signaling. Inhibition of the VEGF-A-induced [Ca2+]i signal and proliferation by CAI can be explained by inhibition of IP3 formation and may contribute to the antiangiogenic action of CAI. Calcium-dependent NO formation may represent a link between calcium signaling and proliferation.

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