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Spatiotemporal analysis of RhoA/B/C activation in primary human endothelial cells.

ABSTRACT: Endothelial cells line the vasculature and are important for the regulation of blood pressure, vascular permeability, clotting and transendothelial migration of leukocytes and tumor cells. A group of proteins that that control the endothelial barrier function are the RhoGTPases. This study focuses on three homologous (>88%) RhoGTPases: RhoA, RhoB, RhoC of which RhoB and RhoC have been poorly characterized. Using a RhoGTPase mRNA expression analysis we identified RhoC as the highest expressed in primary human endothelial cells. Based on an existing RhoA FRET sensor we developed new RhoB/C FRET sensors to characterize their spatiotemporal activation properties. We found all these RhoGTPase sensors to respond to physiologically relevant agonists (e.g. Thrombin), reaching transient, localized FRET ratio changes up to 200%. These RhoA/B/C FRET sensors show localized GEF and GAP activity and reveal spatial activation differences between RhoA/C and RhoB. Finally, we used these sensors to monitor GEF-specific differential activation of RhoA/B/C. In summary, this study adds high-contrast RhoB/C FRET sensors to the currently available FRET sensor toolkit and uncover new insights in endothelial and RhoGTPase cell biology. This allows us to study activation and signaling by these closely related RhoGTPases with high spatiotemporal resolution in primary human cells.

SUBMITTER: Reinhard NR

PROVIDER: S-EPMC4857094 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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Spatiotemporal analysis of RhoA/B/C activation in primary human endothelial cells.

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