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Local guidance of emerging vessel sprouts requires soluble Flt-1.

ABSTRACT: Blood vessel networks form via sprouting of endothelial cells from parent vessels. Extrinsic cues guide sprouts after they leave the initiation site, but these cues are likely insufficient to regulate initial outward movement, and many embryonic vessel networks form in the absence of a strong extrinsic gradient. We hypothesized that nascent sprouts are guided by spatial cues produced along their own vessels, and that soluble Flt-1 (sFlt-1) participates in this guidance. Analysis of developing vessels with perturbed flt-1 function revealed misguided emerging sprouts, and transgenic sFlt-1 rescued sprout guidance parameters. sflt-1 activity in endothelial cells immediately adjacent to the emerging sprout significantly improved local sprout guidance. Thus, we propose that a vessel-intrinsic system initially guides emerging sprouts away from the parent vessel, utilizing spatially regulated expression of sFlt-1 in conjunction with exogenous VEGF-A. Local sprout guidance defects are predicted to contribute to vessel dysmorphogenesis during perturbed development and disease.

SUBMITTER: Chappell JC

PROVIDER: S-EPMC2747120 | biostudies-other | 2009 Sep

REPOSITORIES: biostudies-other

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Local guidance of emerging vessel sprouts requires soluble Flt-1.

Chappell John C JC Taylor Sarah M SM Ferrara Napoleone N Bautch Victoria L VL

Developmental cell 20090901 3

Blood vessel networks form via sprouting of endothelial cells from parent vessels. Extrinsic cues guide sprouts after they leave the initiation site, but these cues are likely insufficient to regulate initial outward movement, and many embryonic vessel networks form in the absence of a strong extrinsic gradient. We hypothesized that nascent sprouts are guided by spatial cues produced along their own vessels, and that soluble Flt-1 (sFlt-1) participates in this guidance. Analysis of developing ve ...[more]

PMID: 19758562

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Project description:Angiogenesis is a key process in several ocular disorders and cancers. Soluble Flt-1 is an alternatively spliced form of the Flt-1 gene that retains the ligand-binding domain, but lacks the membrane-spanning and intracellular kinase domains of the full-length membrane bound Flt-1 (mbFlt-1) protein. Thus, sFlt-1 is an endogenous inhibitor of VEGF-A mediated angiogenesis. Synthetic mopholino oligomers directed against splice site targets can modulate splice variant expression. We hypothesize that morpholino-induced upregulation of sFlt-1 will suppress angiogenesis in clinically relevant models of macular degeneration and breast cancer.In vivo morpholino constructs were designed to target murine exon/intron 13 junction of the Flt-1 transcript denoted VEGFR1_MOe13; standard nonspecific morpholino was used as control. After nucleofection of endothelial and breast adenocarcinoma cell lines, total RNA was extracted and real-time RT-PCR performed for sFlt-1 and mbFlt-1. Intravitreal injections of VEGFR1_MOe13 or control were done in a model of laser-induced choroidal neovascularization and intratumoral injections were performed in MBA-MD-231 xenografts in nude mice. VEGFR1_MOe13 elevated sFlt-1 mRNA expression and suppressed mbFlt-1 mRNA expression in vitro in multiple cellular backgrounds (p<0.001). VEGFR1_MOe13 also elevated sFlt/mbFlt-1 ratio in vivo after laser choroidal injury 5.5 fold (p<0.001) and suppressed laser-induced CNV by 50% (p = 0.0179). This latter effect was reversed by RNAi of sFlt-1, confirming specificity of morpholino activity through up-regulation of sFlt-1. In the xenograft model, VEGFR1_MOe13 regressed tumor volume by 88.9%, increased sFlt-1 mRNA expression, and reduced vascular density by 50% relative to control morpholino treatment (p<0.05).Morpholino oligomers targeting the VEGFR1 mRNA exon/intron 13 junction promote production of soluble FLT-1 over membrane bound FLT-1, resulting in suppression of lesional volume in laser induced CNV and breast adenocarcinoma. Thus, morpholino manipulation of alternative splicing offers translational potential for therapy of angiogenic disorders.

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Local guidance of emerging vessel sprouts requires soluble Flt-1.

Publications

Local guidance of emerging vessel sprouts requires soluble Flt-1.

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