The Orphan Receptor Tie1 Controls Angiogenesis and Vascular Remodeling by Differentially Regulating Tie2 in Tip and Stalk Cells

SUMMARY Tie1 is a mechanistically poorly characterized endothelial cell (EC)-specific orphan receptor. Yet, Tie1 deletion is embryonic lethal and Tie1 has been implicated in critical vascular pathologies, including atherosclerosis and tumor angiogenesis. Here, we show that Tie1 does not function independently but exerts context-dependent effects on the related receptor Tie2. Tie1 was identified as an EC activation marker that is expressed during angiogenesis by a subset of angiogenic tip and remodeling stalk cells and downregulated in the adult quiescent vasculature. Functionally, Tie1 expression by angiogenic EC contributes to shaping the tip cell phenotype by negatively regulating Tie2 surface presentation. In contrast, Tie1 acts in remodeling stalk cells cooperatively to sustain Tie2 signaling. Collectively, our data support an interactive model of Tie1 and Tie2 function, in which dynamically regulated Tie1 versus Tie2 expression determines the net positive or negative effect of Tie1 on Tie2 signaling.


Supplemental Experimental Procedures Generation of Tie1 MiCreM knockin mice
For the knockin approach, a gene targeting vector was constructed which upon homologous recombination would insert a MeriCreMer cDNA (Srinivas et al, 2001;Zhang et al, 1996) into the first exon of the Tie1 locus. The targeting construct consisted, from 5' to 3', of a short homologous arm upstream of the ATG start codon of the Tie1 gene, a splice donor site, an intron and a splice acceptor site (intron), all taken from the rabbit β--globin gene (Kouskoff et al, 1993), the coding sequence of codon--improved Cre (iCre) (Shimshek et al, 2002), flanked by two mutated estrogen receptor sites (Mer) (Zhang et al, 1996), a poly--adenylation signal (pA) from the rabbit β--globin gene (Kouskoff et al, 1993), a FRT--flanked Neomycin (Neo) resistance gene, a long homologous arm, and finally the diphtheria toxin subunit A gene (DT--A) to select against random integration events ( Figure S1). Gene targeting experiments were performed in embryonic day (E) 14 embryonic stem cells. Correct homologous recombination of the targeting vector results in replacement of the start codon in the first exon of the Tie1 locus with the inducible MeriCreMer cassette. Correctly targeted Tie1 alleles were identified by PCR after screening a total of 240 Neomycin--resistant colonies.

Retina image analyses
Retina analyses were performed using the Fiji software (ImageJ). Vessel outgrowth was measured by analyzing the distance of the vascular front from the central optic nerve.
Relative vessel area was calculated as IB--4 + area per retina area. For analysis of the tips, number of tips at the total retina front were counted and depicted as number of tips per mm vessel front. Branches in the vasculature were analyzed by skeletonizing the IB--4 stained images and counting the total number of branches. Regression analysis was performed by counting ColIV + IB--4 -structures and correlating them to the vessel area. For the analysis of EC apoptosis, cleaved caspase3 + structures that co--localized with IB--4 staining were counted and correlated to the vessel area. ERG1 + nuclei co--localized with CD31 were counted.
Relative pericyte coverage was analyzed by measuring desmin--positive area and correlating it to the vessel area. If relative values (ColIV and cleaved caspase3 analysis) are plotted then single values per mouse were normalized to the average of the WT littermates.

In situ hybridization (ISH) on whole--mounted retina
A plasmid containing mouse Tie1 cDNA region spanning the fibronectin domains 2 and 3 (FN2 and FN3) was used for synthesizing the ISH probes. Digoxigenin (DIG)--labeled sense and antisense RNA probes were synthesized using T7 and T3 polymerases, respectively. ISH was Savant et. al performed with minor modifications as previously described (Powner et al, 2012). Briefly, eyes of P6 mouse pups were removed and fixed for 20 min in 4% PFA on ice. After isolation and dissection, retinas were stored in 100% methanol at --20° C overnight. Hybridization was performed at 66°C. RNA labeled probes were detected by an alkaline--phosphatase--coupled antibody (Roche Applied Sciences) and nitroblue tetrazolium/5--bromo--4--chloro--3--indolyl phosphate (NBT/BCIP) was used as a chromogenic substrate for alkaline phosphatase (Promega). After color development, retinas were extensively washed with PBS.
Subsequently, retinas were incubated with IB--4 conjugated to Alexa--568 (1:200) for blood vessel staining. Bright field and fluorescent images were acquired simultaneously using a Zeiss LSM 880 equipped with a 20x objective. Images were processed and background corrected with a rolling ball algorithm in the Fiji software (ImageJ). To better visualize colocalization, a pseudo color (green) was applied to the bright field image.

HUVEC stimulation assay
To study angiopoietin induced signaling, HUVEC were starved overnight in EC basal medium (Endopan3) containing 0.5% FCS or for 4 hrs without FCS. Cells were stimulated with 400 ng/ml of human, recombinant Ang1 or Ang2 (R&D) in EC basal medium for indicated time.
Cells were then washed with cold 1x PBS and lysed for protein extraction.
Corresponding isotype controls, goat IgG--PE and mouse IgG--APC (R&D), were used. Sample acquisition was performed with a BD FACS Canto II flow cytometer and subsequent analysis was done using FlowJo software.

Spheroid sprouting assay
The mosaic spheroid sprouting assays were performed with Tie1--GFP and wild--type (WT) HUVEC. For this, 1:1 mixture of WT and Tie1--GFP cells were resuspended in EC growth medium containing 20% methocel (Sigma). 25 μl cell suspension drops were pipetted on non--adherent plastic plates. Subsequently, plates were turned upside--down to form the so called hanging drops in which HUVEC spheroids are formed. Plates were incubated for 24 hrs at 37°C and spheroids were harvested by washing the plates with 10% FCS/PBS. Next, spheroids were centrifuged at 200 g for 5 min and resuspended in 20% FCS and 80% methocel. The collagen matrix was prepared on ice using Collagen type I (isolated from rat tail tendons), Medium 199 and NaOH (1 M) in 8:1:1 ratio. Additionally, 1x HEPES buffer was added to the mix for adjusting the pH. The Collagen solution was mixed with the spheroid solution in 1:1 ratio and transferred to a 24--well plate. For polymerization, gels were incubated for 30 min at 37°C and then stimulated with 75 ng/ml VEGF (R&D) in EC basal medium. Sprouting spheroids were imaged using the cell observer (Zeiss) and analyzed using the Fiji software (ImageJ).

Scratch wound assay
For a 2D migration assay, WT, Tie1--GFP or a 1:1 mixture of WT and Tie1--GFP HUVEC were seeded on 0.2% gelatin--coated cover slips. A scratch was made using the 100 µl pipette tip and cells were allowed to migrate for 5 hrs. Cells were then fixed with 4% PFA/PBS for 20 min at RT followed by staining with Alexa--546 conjugated Phalloidin (Invitrogen, A22283) to visualize the actin cytoskeleton of migrating cells. Nuclei were counterstained with Hoechst dye (1:3000). Cells in the wound area were imaged using the cell observer (Zeiss) and analyzed using the Fiji software (ImageJ).

Shear stress experiments
Shear stress experiments were performed using the ibidi flow chambers (ibidi cells in focus, 15 µ--Slide I 0.4 Luer, catalog no. 80176) connected to an IPS high precision peristaltic pump with a flow rate of 11 ml/min (IDEX corporation, ISM935). Laminar shear at 10 dynes/cm 2 was obtained by pumping EC media at 1.6 ml/min flow rate on Tie1--silenced and control cells seeded in the ibidi chambers. Shear stress experiments were performed for 48 hrs in a sterile, 5% CO2 incubator at 37°C.