Cardiovascular function depends on patent, continuous and stable blood vessel formation by endothelial cells (ECs). Blood vessel development initiates by vasculogenesis, as ECs coalesce into linear aggregates and organize to form central lumens that allow blood flow. Molecular mechanisms underlying in vivo vascular ‘tubulogenesis’ are only beginning to be unraveled. We previously showed that the GTPase-interacting protein called Rasip1 is required for the formation of continuous vascular lumens in the early embryo. Rasip1−/− ECs exhibit loss of proper cell polarity and cell shape, disrupted localization of EC–EC junctions and defects in adhesion of ECs to extracellular matrix. In vitro studies showed that Rasip1 depletion in cultured ECs blocked tubulogenesis. Whether Rasip1 is required in blood vessels after their initial formation remained unclear. Here, we show that Rasip1 is essential for vessel formation and maintenance in the embryo, but not in quiescent adult vessels. Rasip1 is also required for angiogenesis in three models of blood vessel growth: in vitro matrix invasion, retinal blood vessel growth and directed in vivo angiogenesis assays. Rasip1 is thus necessary in growing embryonic blood vessels, postnatal angiogenic sprouting and remodeling, but is dispensable for maintenance of established blood vessels, making it a potential anti-angiogenic therapeutic target.
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We thank the following for mouse lines: Ralf Adams for Cdh5(PAC)-CreERT2, Tom Sato for Tie2-Cre, Janet Rossant for Flk1-eGFP and Thomas Carroll for Sox2-Cre and CAG-CreERT2. We thank Dr. Hiromi Yanagisawa for use of cell culture equipment. The authors would like to thank Stephen Fu and Katherine Speichinger for excellent technical assistance. We thank the TIG group, and the Carroll, Olson, MacDonald and Cleaver labs for invaluable discussions and assistance. Finally, we are very grateful to Dr. Bob Hammer and the UTSW Transgenic Core for help with generating the Rasip1 conditional mice.
Most experiments were performed by Y.K. K.X. initiated experiments by crossing mouse lines and making initial observations. D.M.B. and S.F. carried out supportive experiments and D.M.B. finished key studies. K.T. and C.M. assisted with DIVAA implants. G.E.D. contributed to underlying ideas and analysis, contributed 3D in vitro data and read manuscript critically. O.C. supervised the overall project and contributed to the analysis. Y.K., D.M.B and O.C. wrote the manuscript.
This work was supported by NIH R01HL113498 to DMB, R01HL109604 to CM, R01HL105606 and R01HL108670 to GED, and CPRIT RP110405, R01HL113498 and R01DK079862 to OC.
Conflict of interest
The authors declare no competing or financial interests.
Yeon Koo and David M. Barry have contributed equally to this work.
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Generation of conditional Rasip1 knockout mouse. Schematic showing the wild-type allele of Rasip1 and the targeting vector (KOMP) used to generate the floxed allele. A LacZNeo cassette was used to interrupt the gene. Flippase recombinase (FLP) was used to remove the LacZ and Neo cassette which is flanked by flippase recognition target (FRT) sequences. The Rasip1 coding region is interrupted by removal of the third exon, which is flanked by loxP sites (floxed exon), in the presence of Cre recombinase (JPEG 435 kb)
Disrupted Rasip1 allele prevents lumen formation and disrupts junctional polarity. (A-F) Immunofluorescence staining for Flk1-eGFP and ZO1 in aortic ECs (at E8.25) showing that the LacZNeo cassette can disrupt Rasip1 function, thereby affecting lumen formation and adhesion polarity. Scale bars: A–F 15 μm (JPEG 954 kb)
Rasip1 is necessary for arterial fate. (A-B) Deletion of Rasip1 using Sox2-Cre disrupts maintenance of arterial fate by E9.0, as indicated by expression of the flow-responsive gene connexin 40 (CX40), as detected by RNA in situ hybridization. Scale bars: A–B 1 mm (JPEG 332 kb)
R26-YFP reporter shows that Cre recombinase is expressed in newly born angioblasts using Tie2-Cre. (A-B) Aortic ECs from 0-4 somite embryos were stained using a GFP antibody (red) and DAPI (blue) to mark Rosa26-YFP reporter expressing angioblasts in Tie2-Cre transgenic mice before and after lumen formation. M = mesoderm, EC = endothelial cell, End = endoderm. Scale bars: A–B 15 μm (JPEG 660 kb)
Rasip1 is necessary to polarize adhesion complexes away from the apical membrane but is not necessary for apical polarity. (A-F) Staining of ZO-1 and Rasip1 in aortae of E8.25 Rasip1f/f;Tie2-Cre and Rasip1f/+;Tie2-Cre embryos showing that junction polarity (junctions are abnormally localized apically, rather than their normal peripheral localization) is lost after deletion of Rasip1. Asterisks indicate the aortic lumen. (G-L) Staining of claudin5 and VEcad show that tight junction and adherens junction polarity, localization away from the apical membrane, are lost after deletion of Rasip1 using Tie2-Cre. (M-R) Staining of PECAM, endomucin and Podxl shows that apical polarity is normal after deletion of Rasip1 using Tie2-Cre. B, blood autofluorescence. Scale bars: A–R 15 μm (JPEG 1574 kb)
Rasip1 f/f ;Tie2-Cre embryos become hypoplastic at E9.0 and die from failed lumen formation. (A-B) Embryos become hypoplastic at E9.0 after Rasip1 deletion using Tie2-Cre and exhibit hemorrhages. (C-D) The dorsal aorta marked by PECAM and endomucin staining collapses after deletion of Rasip1 using Tie2-Cre at E9.0. Asterisks denote open lumens. Scale bars: A–B 1 mm, C-D 15 μm (JPEG 1950 kb)
CAG-Cre ERT2 (CAG) conditionally deletes Rasip1 in the retina. (A) In situ hybridization with anti-Rasip1 RNA Dig-labeled probe showing Rasip1 expression is vascular specific (throughout retinal vessels). (B) A diagram for tamoxifen treatment of neonatal mice for Rasip1 ablation. (C-E) Tamoxifen efficiency of recombination in blood vessels was verified by co-expression of isolectin B4 and the Rosa26-YFP reporter. Scale bars: A 500 μm, C–E 125 μm (JPEG 1496 kb)
Rasip1 is dispensable in adult vessels. (A) Rasip1 deletion confirmation in adult mice by detection of the deleted floxed allele using PCR. (B) Reverse transcriptase PCR of adult lung tissue showing that Rasip1 transcripts are ablated after treatment of Rasip1f/f;Cad5-CreERT2 adult mice with tamoxifen. (C) Western blot analysis showing Rasip1 protein levels are significantly reduced in adult Rasip1f/f;Cad5-CreERT2 mice treated with tamoxifen (lung). (D) The Rosa26-Tomato allele was used to visualize Rasip1-deficient ECs in the adult mouse ear vasculature. PECAM immunofluorescence shows that vessels are grossly normal. Scale bars: D 25 μm (JPEG 971 kb)
Rasip1 regulates blood vessel lumen formation in vitro. (A-D) 3D collagen vasculogenesis assay after reduction in Rasip1, Arhgap29 or both together with siRNA. Arrowheads denote open lumens and arrows denote failed lumen formation. (E) Quantification showing that lumen formation fails after reduction in Rasip1, Arhgap29 or both together. * = p < 0.01, n = 15. Scale bars: A–D 100 μm (JPEG 1442 kb)
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Koo, Y., Barry, D.M., Xu, K. et al. Rasip1 is essential to blood vessel stability and angiogenic blood vessel growth. Angiogenesis 19, 173–190 (2016). https://doi.org/10.1007/s10456-016-9498-5