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Crim1 maintains retinal vascular stability during development by regulating endothelial cell Vegfa autocrine signaling.

ABSTRACT: Angiogenesis defines the process in which new vessels grow from existing vessels. Using the mouse retina as a model system, we show that cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is highly expressed in angiogenic endothelial cells. Conditional deletion of the Crim1 gene in vascular endothelial cells (VECs) causes delayed vessel expansion and reduced vessel density. Based on known Vegfa binding by Crim1 and Crim1 expression in retinal vasculature, where angiogenesis is known to be Vegfa dependent, we tested the hypothesis that Crim1 is involved in the regulation of Vegfa signaling. Consistent with this hypothesis, we showed that VEC-specific conditional compound heterozygotes for Crim1 and Vegfa exhibit a phenotype that is more severe than each single heterozygote and indistinguishable from that of the conditional homozygotes. We further showed that human CRIM1 knockdown in cultured VECs results in diminished phosphorylation of VEGFR2, but only when VECs are required to rely on an autocrine source of VEGFA. The effect of CRIM1 knockdown on reducing VEGFR2 phosphorylation was enhanced when VEGFA was also knocked down. Finally, an anti-VEGFA antibody did not enhance the effect of CRIM1 knockdown in reducing VEGFR2 phosphorylation caused by autocrine signaling, but VEGFR2 phosphorylation was completely suppressed by SU5416, a small-molecule VEGFR2 kinase inhibitor. These data are consistent with a model in which Crim1 enhances the autocrine signaling activity of Vegfa in VECs at least in part via Vegfr2.

SUBMITTER: Fan J

PROVIDER: S-EPMC3879820 | biostudies-other | 2014 Jan

REPOSITORIES: biostudies-other

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Crim1 maintains retinal vascular stability during development by regulating endothelial cell Vegfa autocrine signaling.

Fan Jieqing J Ponferrada Virgilio G VG Sato Tomohito T Vemaraju Shruti S Fruttiger Marcus M Gerhardt Holger H Ferrara Napoleone N Lang Richard A RA

Development (Cambridge, England) 20131218 2

Angiogenesis defines the process in which new vessels grow from existing vessels. Using the mouse retina as a model system, we show that cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is highly expressed in angiogenic endothelial cells. Conditional deletion of the Crim1 gene in vascular endothelial cells (VECs) causes delayed vessel expansion and reduced vessel density. Based on known Vegfa binding by Crim1 and Crim1 expression in retinal vasculature, where angiogenesis is ...[more]

PMID: 24353059

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Project description:PurposeThere are reports that a b-isoform of vascular endothelial growth factor-A 165 (VEGFA165b) is predominant in normal human vitreous, switching to the a-isoform (VEGFA165a) in the vitreous of some diseased eyes. Although these isoforms appear to have a different ability to activate the VEGF receptor 2 (VEGFR2) in various endothelial cells, the nature of their ability to activate intracellular signaling pathways is not fully characterized, especially in retinal endothelial cells. We determined their activation potential for two key intracellular signaling pathways (MAPK, AKT) over complete dose-response curves and compared potential effects on the expression of several VEGFA165 target genes in primary human retinal microvascular endothelial cells (HRMECs).MethodsTo determine full dose-response curves for the activation of MAPK (ERK1/2), AKT, and VEGFR2, direct in-cell western assays were developed using primary HRMECs. Potential differences in dose-response effects on gene expression markers related to endothelial cell and leukocyte adhesion (ICAM1, VCAM1, and SELE) and tight junctions (CLDN5 and OCLN) were tested with quantitative PCR.ResultsActivation dose-response analysis revealed much stronger activation of MAPK, AKT, and VEGFR2 by the a-isoform at lower doses. MAPK activation in primary HRMECs displayed a sigmoidal dose-response to a range of VEGFA 165 a concentrations spanning 10-250 pM, which shifted higher into the 100-5,000 pM range with VEGFA 165 b. Similar maximum activation of MAPK was achieved by both isoforms at high concentrations. Maximum activation of AKT by VEGFA 165 b was only half of the maximum activation from VEGFA 165 a. At a lower intermediate dose, where VEGFA 165 a activated intracellular signaling stronger than VEGFA 165 b, the changes in VEGFA target gene expression were generally greater with VEGFA 165 a.ConclusionsIn primary HRMECs, VEGFA 165 a could maximally activate MAPK and AKT at lower concentrations where VEGFA 165 b had relatively little effect. The timing for maximum activation of MAPK was similar for the isoforms, which is different from that reported for non-retinal endothelial cells. Although differences in VEGFA 165 a and VEGFA 165 b are limited to the sequence of their six C-terminal six amino acids, this results in a large difference in their ability to activate at least two key intracellular signaling pathways and VEGF-target gene expression in primary human retinal endothelial cells.

Dataset Information

Crim1 maintains retinal vascular stability during development by regulating endothelial cell Vegfa autocrine signaling.

Publications

Crim1 maintains retinal vascular stability during development by regulating endothelial cell Vegfa autocrine signaling.

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