A MST1–FOXO1 cascade establishes endothelial tip cell polarity and facilitates sprouting angiogenesis

Hypoxia is a main driver of sprouting angiogenesis, but how tip endothelial cells are directed to hypoxic regions remains poorly understood. Here, we show that an endothelial MST1–FOXO1 cascade is essential for directional migration of tip cells towards hypoxic regions. In mice, endothelial‐specific deletion of either MST1 or FOXO1 leads to the loss of tip cell polarity and subsequent impairment of sprouting angiogenesis. Mechanistically, MST1 is activated by reactive oxygen species (ROS) produced in mitochondria in response to hypoxia, and activated MST1 promotes the nuclear import of FOXO1, thus augmenting its transcriptional regulation of polarity and migration‐associated genes. Furthermore, endothelial MST1‐FOXO1 cascade is required for revascularization and neovascularization in the oxygen-induced retinopathy model. Together, the results of our study delineate a crucial coupling between extracellular hypoxia and an intracellular ROS‐MST1‐FOXO1 cascade in establishing endothelial tip cell polarity during sprouting angiogenesis.

T he vascular system expands its network from pre-existing vessels by sprouting angiogenesis for supplying oxygen and nutrients to avascular and hypoxic tissues. In response to numerous angiogenic cues from oxygen-and nutrientdeprived tissues, endothelial cells (ECs), the main components of the vascular lumen, adopt a series of morphogenic behaviors, such as tip ECs and stalk ECs, for coordinating sprouting angiogenesis [1][2][3] . Tip ECs are championed cells and highly migratory, leading the sprouts in the direction of a guidance cue, while stalk ECs are proliferative, supplying building blocks for sprout elongation 1,2,4 . Haemodynamic forces drive lumen expansion into newly formed sprouts to deliver oxygen-and nutrient-rich blood flow 5,6 . These overall processes are finely regulated by various extrinsic cues and corresponding intrinsic signaling in the ECs. Lately, significant advances have been made in the understanding of intrinsic transcriptional and metabolic changes in tip ECs 7-11; however, how they are directed-the EC polarization at the vascular front-into the avascular, hypoxic area is poorly understood.
Mammalian sterile 20-like kinases 1 and 2 (MST1/2) have been identified as mediators of oxidative stress 12,13 and lately characterized as the major component of the Hippo pathway 14,15 . The mammalian core Hippo pathway components encompass MST1/ 2, large tumor suppressor homolog 1 and 2 (LATS1/2), and Yesassociated protein (YAP) or its paralog transcriptional coactivator with PDZ-binding motif (TAZ). YAP/TAZ are transcription coactivators that mainly interact with the TEAD/TEF family of transcription factors and play crucial roles in regulating cellular proliferation, differentiation and migration, tissue growth, and organ morphogenesis 14,15 . We and others lately have found that YAP/TAZ play critical roles in the morphogenesis of tip ECs and proliferation of stalk ECs by regulating cytoskeletal rearrangement and metabolic activity during sprouting angiogenesis 10,[16][17][18] . LATS1/2 are direct upstream regulators of YAP/TAZ, limiting their activities through phosphorylation-dependent cytoplasmic retention and destabilization 14,15 . Indeed, endothelial deletion of LATS1/2 enhances activities of YAP/TAZ, leading to a dense and hyperplastic network, uncoordinated outgrowth, numerous filopodia bursts in tip ECs, and increased proliferating ECs in growing retinal vessels 10 . Overall, this LATS1/2-YAP/TAZ cascade responds to vascular endothelial growth factor-A (i.e., VEGF) and regulates angiogenesis 10,16 . MST1/2 are serine/threonine kinases that are expressed ubiquitously in most tissues and cell types [12][13][14]19 . MST1/2 phosphorylate and activate LATS1/2, and thereby inactivate YAP/TAZ in the canonical Hippo pathway. However, these kinase-substrate relationships are highly cell typeand context-dependent [19][20][21][22][23][24][25] . Specifically, MST1 is activated by cellular stress such as ultraviolet radiation, serum starvation, hydrogen peroxide, and reactive oxygen species (ROS) 26 , followed by phosphorylation of its cellular substrates including Forkhead box (FOXO) proteins 13,19,21,22 . In fact, MST1 mediates oxidative stress-induced neuronal cell death through phosphorylation of FOXO1 at serine 212, which leads to disruption of the association between FOXO1 and 14-3-3 proteins, subsequently enhancing nuclear import of FOXO1 19 . Of importance in ECs, FOXO1 is a crucial gatekeeper for EC quiescence mediated through reducing glycolysis, mitochondrial respiration, and proliferation by suppressing MYC during sprouting angiogenesis 11 .
Here, we unveil that MST1 acts as an upstream regulator of FOXO1 rather than of LATS1/2 and plays key roles in sprouting angiogenesis by establishing endothelial polarity at tip ECs. Moreover, hypoxia rather than VEGF monopolizes the MST1-FOXO1 cascade in this context. Our results demonstrate a crucial coupling between extracellular hypoxia and an intracellular MST1-FOXO1 cascade, which facilitates sprouting angiogenesis.

MST1 is involved in establishing endothelial polarization.
Considering that MST1/2 are upstream regulators of LATS1/2 in the Hippo pathway, we hypothesized that the roles of MST1/2 could be similar to those of LATS1/2 but opposite to those of YAP/TAZ in sprouting angiogenesis, based on our previous report 10 . To investigate the role of MST1 in sprouting angiogenesis, we generated Mst1 iΔEC mice by crossing Mst1 flox/flox mice 27 with VE-cadherin Cre-ER T2 mice 28 (Fig. 1a), confirmed EC-specific deletion of MST1 in these animals ( Supplementary  Fig. 1a-f), and examined retinal angiogenesis during the postnatal period. Cre-ER T2 -positive but flox/flox-negative mice among the littermates for each experiment were defined as wild-type (WT) mice. EC-specific deletion of MST1 in Mst1 iΔEC mice from P1 led to impaired retinal angiogenesis at P6. Compared with WT mice, Mst1 iΔEC mice exhibited respectively 29 and 60% reduced vascular branching and density, and 72% reduced 5-ethynyl-2′deoxyuridine (EdU) incorporation (proliferation), but no difference in cleaved caspase3 (apoptosis) in the ECs at the vascular front (Fig. 1b, c and Supplementary Fig. 2a-c). Moreover, tip ECs at the vascular front of Mst1 iΔEC mice exhibited reduced both sprout number and length, but no difference in filopodia formation per sprout compared with WT mice (Fig. 1d, f). Of special interest, Mst1 iΔEC mice showed abnormally aligned VE-cadherin (marking endothelial junctions) and ETS-related gene (ERG, marking endothelial nuclei) in the vascular front of the growing retinal vessels (Fig. 1e).
These findings led us to ask whether MST1 regulates endothelial polarization during sprout elongation. To address this question, we further analyzed shapes and locations of EC nuclei in the vascular front by visualizing ERG; these nuclei are elliptical in actively migrating cells and spherical in static cells 29 . Consistent with previous reports 7 , the nuclei of tip ECs were largely elliptical in WT mice. In contrast, they were spherical and not directed into the avascular area although filopodia properly extended in Mst1 iΔEC mice (Fig. 1d, g). Moreover, ERG + nuclei of tip ECs overlapped each other in Mst1 iΔEC mice but positioned in a planar fashion in WT mice (Fig. 1g).
We additionally assessed the orientation of Golgi apparatus in tip ECs to analyze EC polarity according to previous reports 29,30 . Of note, the Golgi apparatus were polarized towards the anterior or posterior of the nuclei in tip ECs of WT mice, whereas such polarization was lost in tip ECs of Mst1 iΔEC mice ( Fig. 1h and Supplementary Fig. 2d). These data indicate that MST1 plays a critical role in endothelial polarization during sprouting angiogenesis.
MST1 is crucial for perpendicular vascular branching. Because retinal angiogenesis analyzed at P6 represents sprouting angiogenesis in two-dimensional space 31 , we determined the role of MST1 in angiogenesis in three-dimensional space by analyzing retinal deep vascular plexus formation and brain angiogenesis at P12 32 (Fig. 2a). Of importance, perpendicular vascular sprouting was not extended into the retinal deep layer in Mst1 iΔEC mice; instead, sprouts passed each other, displaying aberrant vessel crossings (Fig. 2b-e). As a result, vascular network formation in the retinal deep layer was severely impaired in Mst1 iΔEC mice at P12. Nevertheless, no apparent impairments in lumen formation (outlined by ICAM2 distribution), collagen type IV deposition (basement membrane), or barrier integrity [shown by TER119 + red blood cells (RBCs) inside vascular lumen] were shown in Mst1 iΔEC mice ( Fig. 2f-h). Similarly, brain angiogenesis in cerebellum but not in cerebrum was also impaired with accompanying reduced vascular density without affecting barrier integrity (no change in GLUT1 intensity) compared with WT mice at P12 (Fig. 2i-l). These results indicate that endothelial MST1 is critical for perpendicular vascular branching.

MST1 does not rely on the Hippo pathway in angiogenesis.
Because MST2 is closest to MST1 among the five members of the MST kinase family 33 , we determined whether Mst2 null mice 34 exhibited similar phenotypes in retinal angiogenesis during the postnatal period ( Supplementary Fig. 3a). Although no apparent differences were detected in retinal angiogenesis between WT and Mst2 null mice, the defective phenotypes of double Mst1 iΔEC -Mst2 null mice were similar to those of Mst1 iΔEC mice (Supplementary Fig. 3b, c), indicating that endothelial MST1 but not MST2 is critical in sprouting angiogenesis.
To see whether LATS1/2 are downstream regulators of MST1 in sprouting angiogenesis, we characterized the vascular phenotypes in growing retinal vessels of Lats1/2 iΔEC mice 35 19,21,22 . Of note, FOXO1 was highly and specifically expressed in most ECs but showed differential subcellular localizations in different regions of growing retinal blood vessels (Fig. 3a, b). Consistent with a previous report 11 , a diffuse nucleocytoplasmic localization of FOXO1 at the vascular front but relatively intense nuclear localization at the vascular plexus was shown. Beyond those findings, we identified relatively strong nuclear localization of FOXO1 at tip ECs of WT mice (Fig. 3b). However, of special note, Mst1 iΔEC mice exhibited a nucleocytoplasmic localization of FOXO1 at tip ECs. Moreover, angiopoietin-2 (Angpt2), which is expressed at tip ECs under control of FOXO1, was markedly reduced in Mst1 iΔEC mice ( Fig. 3c-e). These data imply that MST1 may promote nuclear import of FOXO1 in tip ECs during sprouting angiogenesis.
FOXO1 is required for establishing endothelial polarization.
To address the role of nuclear FOXO1 in tip ECs during sprouting elongation, we generated Foxo1 iΔEC mice by crossing Foxo1 flox/flox mice 38 with VE-cadherin Cre-ER T2 mice ( Fig. 4a and Supplementary Fig. 5a, b). When FOXO1 was specifically deleted in ECs of Foxo1 iΔEC mice from P1, the mice at P6 showed retarded radial vessel growth (Fig. 4b, c). Foxo1 iΔEC mice exhibited three major defective subtypes of vascular sprouts depending on the regionally variant subcellular localization of FOXO1 in growing retinal blood vessels: flat type, hill type at the peri-arterial ECs, and glomeruloid type at the peri-venous ECs ( Supplementary Fig. 5c). However, regardless of these subtypes, abnormally aligned VE-cadherin and ERG + nuclei were detected in all tip ECs of Foxo1 iΔEC mice (Fig. 4d). In fact, tip ECs were stacked with vascular front ECs and the ERG + nuclei of tip ECs were spherical and randomly positioned, but the filopodia extension of tip ECs was directed to the avascular region in Foxo1 iΔEC mice (Fig. 4e, h). Of interest, the ERG + nuclei of tip ECs strikingly overlapped each other in Foxo1 iΔEC mice, but arranged in a planar distribution in WT mice (Fig. 4f). Moreover, the polarization of Golgi apparatus was lost in tip ECs of Fox-o1 iΔEC mice ( Fig. 4g and Supplementary Fig. 5d). Thus, the vascular phenotypes of Foxo1 iΔEC mice are similar to those of Mst1 iΔEC mice, implying that FOXO1 could be a downstream substrate of MST1 for endothelial polarization during sprouting angiogenesis.
Hypoxia activates MST1-FOXO1 cascade in primary cultured ECs. To reveal the factors that activate MST1 during sprouting angiogenesis, we analyzed the publicly available microarray data (GSE19284) that identified enriched genes in tip ECs 39 . Of note, the genes related to ROS, including ROS biosynthesis and response to ROS, and those related to hypoxia were enriched in tip ECs compared to non-ECs (Fig. 5a). These findings led us to hypothesize that a hypoxic microenvironment in the vascular front constantly induces intracellular ROS synthesis, which activates the MST1-FOXO1 cascade at tip ECs. To address this hypothesis, primary cultured human umbilical vascular endothelial cells (HUVECs) were exposed to hypoxia (1% O 2 ). After hypoxia exposure, increased MST1 phosphorylation at Thr183 (pMST1) was detected already at 30 min, gradually increased without alteration of the activity of canonical Hippo pathway, peaked at 6 h, decreased thereafter, and returned to basal level at 36 h. Under the same condition, increased changes in HIF1α protein level and FOXO1 phosphorylation at Ser212 (pFOXO1) were positively correlated with increases in pMST1 (Fig. 5b, c; Supplementary Figs. 6 and 7a-e). These data imply that hypoxia activates MST1 and FOXO1 in ECs, leading us to ask whether ROS directly contributes to activate the MST1-FOXO1 cascade and where ROS is produced in ECs under hypoxia. The process of ROS biosynthesis by NADPH oxidase, xanthine oxidase and mitochondrial electron transport chain requires oxygen as a substrate. However, even though the exact mechanism remains elusive, ROS can be produced in hypoxia, which is mainly dependent on mitochondrial electron transport chain 40,41 . To delineate the relationship among hypoxiaintracellular ROS-MST1-FOXO1 in ECs, we treated Trolox (a ROS scavenger) and rotenone (a mitochondrial electron transport chain I inhibitor) to the HUVECs. Both Trolox and rotenone suppressed the hypoxia-induced stabilization of HIF1α protein level by 31 and 48% and pMST1 by 35 and 41%, respectively (Fig. 5d, e), indicating that hypoxia-induced MST1 activation is primarily mediated through intracellular ROS biosynthesis at the mitochondria in ECs. We then asked whether pFOXO1 depends on pMST1, according to previous report 19,22 . Of note, Fig. 1 MST1 plays a critical role in establishing endothelial polarization. a Diagram depicting the experimental schedule for endothelial cell (EC)-specific deletion of MST1 in retinal vessels from P1 and their analyses at P6 using Mst1 iΔEC mice. b, c Images of CD31 + retinal vessels and comparisons of indicated parameters in WT (n = 6) and Mst1 iΔEC (n = 6) mice. Scale bars, 200 μm. d Magnified images of CD31 + vessels and ERG + nuclei of ECs in WT and Mst1 iΔEC mice. The insets (white dashed-line boxes) are 3D reconstructed and magnified in g. Scale bars, 50 μm. e Images showing abnormally aligned VEcadherin (VECAD) and ERG + nuclei of ECs in the vascular front of Mst1 iΔEC mice compared with those of WT mice. Middle and bottom panels show VECAD and ERG signals of insets (white dashed-line boxes) in top panels. Scale bars, 100 μm. f Comparisons of indicated parameters in WT (n = 5) and Mst1 iΔEC (n = 5) mice. g 3D reconstructed images of ERG + nuclei of ECs from the front and a 45°angle showing that the ERG + nuclei of ECs overlap each other in Mst1 iΔEC mice. h Images of CD31 + vessels, ERG + nuclei of ECs, and GM130 + Golgi apparatus at tip ECs of WT and Mst1 iΔEC mice. The yellow dashed line outlines CD31 + vessels. Note that GM130 + Golgi apparatus are polarized towards the anterior or posterior of the nuclei in tip ECs of WT mice (yellow arrowheads), while such polarization is lost in tip ECs of Mst1 iΔEC mice (yellow arrows). Scale bars, 20 μm. Data represent mean (bar) ± s.d. (error bars). P values, versus WT by two-tailed unpaired t-test. NS not significant. Source data are provided as a Source Data file immunoprecipitation analysis using the HUVECs revealed that endogenous MST1 physically interacts with endogenous FOXO1 (Fig. 5f). Moreover, depletion of MST1 using its corresponding small interfering RNA (siRNA) abrogated the hypoxia-induced pFOXO1 in parallel (Fig. 5g). These results imply that MST1 directly mediates pFOXO1 through hypoxia-induced intracellular ROS biosynthesis (Fig. 5h), which is consistent with previous findings in other cell types 19 .

MST1 governs the nuclear import of FOXO1 under hypoxia.
Considering activation of phosphatidylinositol-3-OH kinase (PI3K)/AKT signaling by VEGF promotes nuclear export of FOXO1 through phosphorylation at Ser256, we postulated that activated MST1 may predominantly promote nuclear import of FOXO1 through phosphorylation at Ser212 against the export stimulated by VEGF-induced PI3K/AKT activation. To decipher this postulation, we confirmed that hypoxia-MST1-FOXO1 cascade operates independently of VEGF/VEGFR2-PI3K-AKT-FOXO1 cascade (Supplementary Fig. 7   In contrast, no apparent differences were detected in the localizations of FOXO1 in the ECs of the vascular front and plexus. These results imply that MST1 activation plays a dominant role in nuclear-cytoplasmic shuttling of FOXO1 in tip ECs that are exposed to extreme hypoxia (Fig. 6f).
MST1-FOXO1 cascade establishes cell polarity in EC migration. Given that Mst1 iΔEC and Foxo1 iΔEC mice failed to induce tip EC polarization, we postulated that the MST1-FOXO1 cascade is critical for inducing tip EC polarization into the avascular area. To evaluate the role of the MST1-FOXO1 cascade in establishing cell polarity during directional EC migration into the avascular area, we employed a wound scratch assay in confluent siCont-ECs, siMST1-ECs, and siFOXO1-ECs (HUVECs transfected with siRNA targeting the FOXO1 gene) and examined the EC behaviors at the wound margin ( Supplementary Fig. 8a, b). While the gap was 95% closed in siCont-ECs at 15 h after scratching, it was only 35 and 43% closed in siMST1-ECs and siFOXO1-ECs, respectively. Of importance, siMST1-ECs and siFOXO1-ECs lost polarization toward the gap area in the leading edge, while siCont-ECs were polarized toward the gap. (Fig. 7a-c and Supplementary Movies 1-3).
We further tracked each EC using a CellTracker 42 and analyzed speed and directionality of the ECs. siCont-ECs showed a linear cell morphology and migrated toward the gap. siMST1-ECs showed a mixed cell morphology of linear and cobblestonelike appearance and migrated randomly regardless of the direction of the gap, and siFOXO1-ECs exhibited a cobblestone-like appearance with small displacement between imaging intervals (Supplementary Fig. 8c and Movies 4-6). Although the average and instantaneous speeds of each EC among siCont-, siMST1-, and siFOXO1-ECs were similar for 15 h, ∼90% of siCont-ECs were displaced into the gap, while 57 and 61% of siMST1-and siFOXO1-ECs, respectively, were randomly displaced (Fig. 7d, e). Moreover, while siCont-ECs exhibited persistent and directional migration, siMST1-and siFOXO1-ECs randomly migrated without directional persistence (defined by the net displacement divided by the total migrating path of each cell) (Fig. 7f). Furthermore, at 9 h after scratching, in the leading edge of siMST1-and siFOXO1-ECs compared with those of siCont-ECs, there were randomly positioned Golgi apparatus, microtubule organizing centers including microtubules (shown by tubulin) and centrosome (shown by pericentrin), and caveolin; fewer incorporated vinculin into the focal adhesion site; and smaller lamellipodia (Fig. 7g, h and Supplementary Fig. 8d-g).
These results indicate that MST1 and FOXO1 are crucial for establishing endothelial polarity in the direction of cell migration. MST1-FOXO1 commonly regulates genes related to EC migration. To elucidate how MST1 and FOXO1 regulate directional EC migration at the transcriptional level, we performed RNA sequencing analysis in siCont-, siMST1-, and siFOXO1-ECs. Ingenuity Pathway Analysis was used to achieve Gene Ontology (GO) analysis in comparisons of siCont-ECs versus siMST1-ECs and siCont-ECs versus siFOXO1-ECs. The GO term Cellular Movements was ranked first and second in the comparison of siMST1-ECs and siFOXO1-ECs to siCont-ECs, respectively ( Supplementary Fig. 9a), implying that MST1 and FOXO1 commonly regulate genes related to Cellular Movements. To decipher how much MST1 and FOXO1 cooperate to regulate genes related to cellular movements, we employed the EdgeR method in R and found 659 and 3,680 differential expressed genes (DEGs) in the comparisons of siCont-ECs versus siMST1-ECs and siCont-ECs versus siFOXO1-ECs, respectively. Only 369 genes among DEGs were commonly regulated by MST1 and FOXO1 ( Supplementary Fig. 9b). Furthermore, we clustered the commonly regulated 369 genes and found that 286 genes were commonly up-or down-regulated by MST1 and FOXO1 (Supplementary Fig. 9c), which were assumed to be the genes regulated by the MST1-FOXO1 cascade. Of interest, GO biological process analysis with the genes regulated by MST1-FOXO1 cascade revealed that GO terms including EC migration, sprouting angiogenesis, and cell-substrate junction assembly were ranked in the top 10 enrichment candidates, and GO terms related to angiogenesis showed significantly high Q scores [-log 10 (p-value)] ( Supplementary Fig. 9d, e). Collectively, these results delineate that MST1-FOXO1 cascade establishes endothelial cell polarity through transcriptional control of cell migration and adhesion which affects cell polarity and vice versa 43 (Fig. 7i).
MST1-FOXO1 cascade is required for pathological angiogenesis. To examine whether endothelial MST1 is required to maintain retinal vessel integrity, we deleted MST1 in ECs of Mst1 iΔEC mice from age 8 weeks and analyzed them 4 weeks later ( Supplementary Fig. 11a). No definite differences were found between WT and Mst1 iΔEC mice in radial length, vascular density, RBC leakage, and distributions of VE-cadherin and ERG + nuclei of the retinal vessels ( Supplementary Fig. 11b-e). These results indicate that endothelial MST1 is dispensable for maintaining integrity of retinal vessels. To see whether endothelial MST1 and FOXO1 has a significant role in pathologic angiogenesis, we employed an oxygen-induced retinopathy (OIR) model (Supplementary Fig. 11f). Mst1 iΔEC -OIR and Foxo1 iΔEC -OIR mice exhibited impaired revascularization (196 and 167% increased avascular area, respectively) and neovascularization [60 and 85% reduced neovascular tuft (NVT) area, respectively] compared with WT-OIR mice (Fig. 8a, c). FOXO1 was enriched in the nuclei of ECs and Angpt2 was highly expressed in tip ECs and NVT ECs, where aberrant sprouting occurs, compared with adjacent ECs in WT-OIR mice. However, Mst1 iΔEC -OIR mice showed 70 and 71% reduced FOXO1 intensity and 64 and 71% reduced Angpt2 expression in tip ECs and NVT ECs, respectively, compared with those of WT-OIR mice. Furthermore, Angpt2 expression was nearly absent in tip ECs and NVT ECs of Foxo1 iΔEC -OIR mice compared with those of WT-OIR mice Fig. 4 FOXO1 is required for establishing endothelial polarization. a Diagram depicting the experiment schedule for EC-specific deletion of FOXO1 in retinal vessels from P1 and their analyses at P6. b, c Images of CD31 + vessels and comparisons of indicated parameters in WT (n = 5) and Foxo1 iΔEC (n = 5) mice. Scale bar, 500 μm. d Images showing VECAD and ERG + nuclei of ECs at the vascular front of WT and Foxo1 iΔEC mice. Middle and bottom panels show VEcadherin (VECAD) and ERG signals of insets (dashed-line boxes) in top panels. Scale bars, 100 μm. e Magnified images of CD31 + vessels and ERG + nuclei of ECs. Scale bars, 50 μm. f 3D reconstructed images of CD31 + vessels and ERG + nuclei of ECs in WT and Foxo1 iΔEC mice. g Images of CD31 + vessels, ERG + nuclei of ECs and GM130 + Golgi apparatus at tip ECs in WT and Foxo1 iΔEC mice. The yellow dashed line outlines CD31 + vessels. Note that GM130 + Golgi apparatus are polarized towards the anterior or posterior of the nuclei in tip ECs of WT mice (yellow arrowheads), while such polarization is lost in tip ECs of Foxo1 iΔEC mice (yellow arrows). Scale bars, 20 μm. h Comparisons of indicated parameters in WT (n = 5) and Foxo1 ΔEC (n = 5) mice. Data Supplementary Fig. 11g, h). In addition, the Golgi apparatus were polarized towards the anterior or posterior of the nuclei in tip ECs of WT-OIR mice. On the contrary, such polarizations were lost in tip ECs of Mst1 iΔEC -OIR and Fox-o1 iΔEC -OIR mice (Fig. 8d, e). Thus, these results imply that MST1 facilitates the nuclear import of FOXO1 in tip ECs as well as in NVT ECs during OIR progression. Moreover, endothelial MST1-FOXO1 cascade is required for revascularization and neovascularization and contributes to establishing endothelial polarity in revascularization.

Discussion
Rapidly growing tissues during development and solid tumors swiftly expanding into surrounding parenchymal tissues constantly face hypoxia due to inadequate angiogenesis and blood  supply 44,45 . In turn, hypoxia is a leading driver for sprouting and intussusceptive angiogenesis, but how it guides tip ECs toward the hypoxic area has been an enigma. An ample number of previous studies 1, [46][47][48][49] have suggested that the VEGF gradient could be a major determinant for tip EC polarization during sprouting angiogenesis. However, there is devoid of sprouting angiogenesis with accompanying tip EC polarization in VEGFrich adult organs such as endocrine glands, liver, kidney, and muscles 50 , raising the possibility that hypoxia could activate an alternative pathway for guiding tip ECs in sprouting angiogenesis into the avascular region. In this study, we uncovered a coupling between extracellular hypoxia and intracellular ROS-MST1-FOXO1 pathway, which establishes the polarity of tip EC during sprouting angiogenesis. Among diverse roles of MST1, we have defined the roles in the behavior of tip ECs of growing blood vessels. Of note, our findings indicate that hypoxia-induced ROS biosynthesis from mitochondria could be a major upstream regulator of MST1 activation in ECs. Activated MST1 promotes planar and perpendicular vascular branching for regulating tip EC polarity and sprouting angiogenesis. In general, MST1 can be activated in two ways: cleavage and phosphorylation 26 . Cleaved MST1 under apoptotic stimuli translocates from cytosol into the nucleus, promoting chromatin condensation during cellular apoptosis 26 . On the other hand, a full-length MST1 can be autophosphorylated at Thr183 under cellular stresses by disrupting the interaction between thioredoxin-1 and SARAH (Salvador/ RASSF1/Hippo) domain 51 , and activated MST1 phosphorylates cytosolic substrates such as SAV, LATS1/2, and FOXOs 26 . Not surprisingly, the physiologic activity of MST1 and its cellular substrates are cell type-and context-dependent 20,52,53 . Our analyses using the genetically modified mice revealed that endothelial MST1 does not undertake a canonical Hippo pathway. Instead, endothelial MST1 phosphorylates FOXO1 under hypoxia in HUVECs, and this kinase-substrate interaction drives nuclear import of FOXO1 in tip ECs. Of importance, this hypoxia-MST1-FOXO1 cascade operates independently of VEGF/VEGFR2 signaling in regulating sprouting angiogenesis. Thus, endothelial MST1 is involved in the regulatory cascade for nuclear transport of FOXO1 rather than its cleavage and in regulation of apoptosis and chromatin condensation or its phosphorylation to LATS1/2 for the canonical Hippo pathway for establishing tip EC polarity during sprouting angiogenesis.
Then, how does nuclear FOXO1 regulates polarity of tip ECs? Beyond the traditional roles of FOXOs as transcriptional factors for maintaining cellular homeostasis 54 , the newly identified roles of FOXOs in neuronal polarization and positioning have been highlighted 55,56 . They control axon-dendrite polarity and morphogenesis in developing granular neurons through regulating polarity-associated gene expression 56 . Thus, these novel roles of FOXOs in neurons could be shared with the roles described here for FOXO1 in tip EC polarization in terms of cell behaviors and regulating polarity-associated gene expression. Nevertheless, this involvement is apart from the deciphered role of FOXO1 in metabolic gatekeeping to maintain EC homeostasis 11 during establishment of the vascular plexus of growing vessels. In this regard, determining specific FOXO1 transcriptional output will be of interest.
Several models have been proposed to explain the specific transcriptional regulation of FOXO1 54 . FOXO1 can bind numerous sites with members of transcriptional machinery because the length of its DNA binding motif is short 57 , on the other hand, FOXO1 interacts with other transcriptional factors or cofactors and can initiate transcriptional activity with or even without direct binding to DNA 58 . Of particular note, FOXO1 that is phosphorylated by MST1 could not bind to DNA, even if it translocates to the nucleus 59 . Together, our finding suggest that MST1-FOXO1 cascade may regulate polarity/migration-associated genes by interacting with other transcriptional machineries rather than direct binding to DNA, which needs further investigations. In addition, although we identified the hypoxiaintracellular ROS-MST1 as the upstream signal of FOXO1 at tip ECs, a limitation of this study is the inability to analyze the regulatory roles of FOXO1 in gene expression and PTMs of FOXO1 in ECs in different regions of growing retinal vessels. Defined analysis at the single cell level at the different regions of ECs is warranted for further study to understand a contextdependent role of FOXO1 during angiogenesis.
In conclusion, this study demarcates a crucial coupling between extracellular hypoxia and intracellular ROS-MST1-FOXO1 pathway, which establishes the polarity of tip ECs during developmental and pathologic angiogenesis.

Methods
Mice and treatment. Specific pathogen-free (SPF) C57BL/6 J mice and R26-tdTomato were purchased from the Jackson Laboratory. VE-cadherin-Cre-ER T2 28 , Mst1 flox/flox27 , Mst2 null 34 , and Foxo1 flox/flox38 mice were transferred, established and bred in SPF animal facilities at KAIST and fed with free access to a standard diet (PMI Lab diet) and water. In order to induce Cre activity in the Cre-ER T2 mice, tamoxifen (Sigma-Aldrich, T5648) was given with following dosages and schedules: for neonatal mice, 100 μg of tamoxifen dissolved in corn oil (Sigma-Aldrich, C8267) was injected into the stomach daily from P1 to P3; for OIR mice, 200 μg of tamoxifen was injected intraperitoneally (i.p.) daily from P12 to P14; for adult mice aged over 8 weeks, 2 mg of tamoxifen was injected i.p. for 5 consecutive days from the indicated time point. For anesthesia, mice were injected i.p. with the anesthetic solution (ketamine 40 mg/kg and xylazine 5 mg/kg). We complied with all ethical regulations for animal testing and research and performed all animal experiments under the approval from the Institute Animal Care and Use Committee (No. KA2017-31) of KAIST.
Histological analyses. Briefly, eyeballs were enucleated and fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature (RT). After preparing the retinas from eyeballs, they were additionally fixed in 1% PFA for 1 h at RT. Samples were blocked with 5% donkey (or goat) serum in PBST (0.3% Triton X-100 in PBS) for 30 min, incubated in primary antibodies (diluted at a ratio of 1:200 in blocking solution) at 4°C overnight, washed in PBST, and incubated in secondary antibodies in blocking solution at RT for 2 h. The retinas were washed in PBST and mounted on microscope glass slides with Vectashield (Vector Laboratories, H-1200). IF Fig. 6 MST1 activation governs to promote nuclear import of FOXO1 under hypoxia. a-c Images and comparisons of the nuclear enrichment of FOXO1 in siCont-ECs and siMST1-ECs exposed to normoxia or hypoxia (1% O 2 ) in the absence (−) or presence (+) of VEGF (200 ng/ml) for 30 min (n = 3, each group). Scale bars, 20 μm. Data represent mean (bar) ± s.d. (error bars). P values, normoxia with VEGF versus hypoxia with VEGF by one-way ANOVA with Tukey's post hoc test. NS not significant. d Immunoblot analyses of indicated proteins in nuclear and cytoplasmic fractions of HUVECs exposed to normoxia (−) or hypoxia (1% O 2 ) (+) without (−) or with (+ ) VEGF stimulation (200 ng/ml, 30 min). e Images and comparisons of the nuclear enrichment of GFP in HEK293T cells transfected with gene constructs encoding either GFP-tagged FOXO1 (FOXO1-WT or WT) or non-phosphorylatable staining of brain were performed as follows. Mice were anesthetized and perfusionfixed with 4% PFA. After dissecting the brain from skull, the brain was additionally fixed in 4% PFA at 4°C for 6 h. The samples were cut into 150 μm sections by a vibratome (Leica, VT1200 S), immunostained, and mounted. Primary cultured HUVECs were fixed in 2% PFA for 10 min at RT. After blocking, the cells were stained with primary and secondary antibodies using the same procedure as tissue staining, and mounted with DAKO mounting medium. Primary antibodies and reagent used for IF were as follows: hamster anti-CD31 monoclonal (Millipore, MAB1398Z); isolectin B4 (IB4), Alexa Fluor 594-conjugated (Thermo Fisher Scientific, I21413); rat anti-VE-cadherin monoclonal (BD Pharmingen, 555289); mouse anti-VE-cadherin monoclonal (BD Biosciences, #555661); rabbit anti-ERG monoclonal (Abcam, ab92513); rabbit anti-cleaved-caspase-3 (Cl-CASP3) polyclonal (CST, #9664); rabbit anti-pHH3 polyclonal (Millipore, 05-806); rabbit anticollagen IV polyclonal (Bio-Rad, 2150-1470); rat anti-ICAM2 monoclonal (BD EdU incorporation assay for proliferating ECs. Six milligrams of 5-ethynyl-2′deoxyuridine (EdU) (Invitrogen, A10044) was dissolved in 1 ml of Milli-Q water as a stock solution. Then, 5 μl of the stock solution per gram of body weight was injected i.p. 3 h before killing. Retinas were isolated and processed as described above. EdU-incorporated cells were detected with the Click-iT EdU Alexa Fluor-555 Imaging Kit (Invitrogen, C10338).
RNA interference. HUVECs were transfected with a pool of siRNAs using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocols.
Retroviral infection. The GFP-tagged human FOXO1 cDNA was cloned into the pMSCV-puro vector (GFP-FOXO1-WT). Non-phosphorylatable FOXO1 (GFP-FOXO1-S212A) was generated by overlap extension PCR. The FLAG-tagged human MST1 cDNA was cloned into the pMSCV-puro vector (FLAG-MST1). The retroviruses were produced in HEK 293 T cells with each indicated gene constructs using Lipofectamine P3000 (Invitrogen) and HUVECs were infected with each indicated retrovirus using Hexadimethrine bromide (Sigma-Aldrich). pMSCVpuro vector was used as a control vector.
Wound scratch assay. Briefly, wound scratch assay were performed on confluent layers of transfected HUVECs. 48 h after siRNA transfection, cells were seeded into 24-well cell culture plate confluently and were incubated for 24 h to allow them to adhere. Migration was initiated by scratching wound with the 200 μl pipet tip followed by two washes with EGM2 media. Phase images of migration were taken every 10 min for 15 h on a Cell observer (Zeiss). For IF analyses of cell migration, cells were maintained on 8-well glass slide (Lab-Tek, 154534) and fixed with 2% PFA for 10 min at 9 h after initiating cell migration by scratch. Cells were maintained in an incubation chamber at 37°C, 5% CO 2 and 95% relative humidity during the experiment.
Morphometric analyses. Morphometric measurements of retinas and brains were performed by using the ImageJ software (NIH). Radial length of retinal vessel was measured as the distance from the optic disc to the peripheral vascular front in each leaflet of the retina and averaged. Retinal vascular density was measured as CD31 + or IB4 + retinal vessel area divided by total measured area of the retina and presented as a percentage. The number of branching points was measured manually in four 500 µm × 500 µm fields located between an artery and a vein in each retina and averaged. The number of ERG + nuclei of ECs was counted in five 200 µm × 200 µm fields and averaged per sample. The number of EdU incorporated ECs, pHH3 + labeled proliferating ECs and CASP3 + apoptotic ECs were measured in four 500 µm × 500 µm fields and averaged per sample. Perpendicular growth of retinal vessels was measured as the length of the vessels reaching out from the superficial layer to the vascular front ahead to the deep layer. The total number of sprouts and filopodia was first measured in a 500 µm × 500 µm field and then normalized to a length of 100 µm along the angiogenic front, which was measured four times per sample and averaged. The number of filopodia per sprout was calculated by dividing the number of filopodia by the number of sprouts, which was measured in five sprouts per sample and averaged. Sprout length was examined in six 100 µm × 100 µm areas of vascular front in each retina and averaged. The angle of ERG + nuclei of ECs was measured as the angle of the nuclear long axis, which was projected in the first quadrant with 90°being considered as the direction of filopodia extension. Nuclear ellipticity was calculated by the distance of nuclear long axis (Height) divided by the maximum vertical distance to the nuclear long axis (Width). RBC leakage was measured in 200 µm × 200 µm fields as RBC-stained area outside of the vessels divided by the measured area and averaged. NVT area in the OIR model was measured using the Lasso tool of Adobe Photoshop software and divided by total CD31 + vessel area. Staining intensities were measured in ten regions of interest (ROIs) of vessels or, if indicated, other vascular regions in each retina and averaged. For comparison, the values were subtracted by the background signals in non-vascularized areas and averaged, which were then normalized by the average of those of control and presented as fold change or percentage. The stained area of specific molecules in retinal vessels was measured using the same threshold values in ImageJ software and divided by the total area of CD31 + or IB4 + retinal vessels. High-resolution confocal images were taken using ×40 lens and then analyzed using the IMARIS software (Bitplane). Analyses of the 3D structures of ERG + nuclei of ECs and CD31 + vessels were performed using the surface module of IMARIS software (Bitplane). Vascular density in brains was measured as CD31 + vessel area divided by total measured area of brain with Z-projected images of 150 μm-thickness and presented as a percentage. Identical measurements of those performed with retina were employed for other morphometric analyses for brain. Nuclear FOXO1 expression in HUVECs was measured using ImageJ software as the co-localized FOXO1 + and DAPI + area divided by total FOXO1 + area. For cell migration and polarity analyses, the width of the wounds was analyzed using ImageJ software and wound closure rate (μm/min) was calculated by dividing the difference between the width at 0 h and 15 h by 900 (15 h × 60 min) in six measurements per sample. The Golgi apparatus, MTOC and pericentrin polarization were measured in 15-20 cells per sample using ImageJ software as the angle obtained from the centre of the nucleus to the organelles. Average speed and net displacement were compared among each groups from the extracted data. Distance from origin and instantaneous speed were calculated and compared from the data measured every 30 min. Directional persistence was calculated as the net displacement divided by the total migrating path of each cell and compared among each groups.
Generation of the anti-phospho-FOXO1 (Ser212) polyclonal antibody. The rabbit antibody against phospho-FOXO1 (Ser212) was generated as follows 13,19 . Briefly, the phosphopeptide antigen C-SAGWKNpSIRHNLS was synthesized, HPLC purified, conjugated to keyhole limpet hemocyanine, and injected into two rabbits over an 8-week period (one primary injection and three boosting injections) (Abclon). The antiserum was obtained for affinity purification to remove nonspecific antibodies, increase sensitivity and reduce background.
Immunoblotting and immunoprecipitation assays. The cells were lysed on ice in RIPA lysis buffer supplemented with protease and phosphatase inhibitors (CST, ). Cell lysates were centrifuged for 10 min at 4°C, 15,000 g. Protein concentrations of the supernatants were quantitated using the detergent-insensitive Pierce BCA protein assay kit (Thermo Scientific, 23227). Lamni buffer was added to total protein lysates and samples were denatured at 95°C for 5 min. Aliquots of each protein lysate (10-20 μg) were subjected to SDS-polyacrylamide gel electrophoresis.
Fractionation of nuclear and cytoplasmic proteins. Cells were washed with icecold PBS and lysed with cytoplasmic extraction reagent (10 mM HEPES (pH 7.8), 10 mM KCL, 1.5 mM MgCl2, 0.5 mM 1,4-dithiothretiol, and protease inhibitor). Cells were scrape-loaded into pre-chilling tubes, incubated for 10 min on ice and spun at 15,000 g at 4°C with 10% NP-40 solution. Then, supernatants were collected as the cytosolic fraction. Cell pellets were further washed with ice-cold PBS 2 times to remove cytosolic proteins. Washed cell pellets were suspended in lamni buffer as the nuclear fraction. Nuclear and cytosolic fraction were confirmed by immunoblotting for LAMIN B1 and GAPDH as nuclear and cytoplasmic markers, respectively.
RNA-sequencing analysis. HUVECs were cultured in 60 mm dish and transfected with GL2 (siCont-ECs), MST1 (siMST1-ECs) and FOXO1 (siFOXO1-ECs) siRNA. At 48 h after siRNA transfection, each siCont-, siMST1-, and siFOXO1-ECs were incubated under hypoxia for 3 h. After extracting total RNA from normoxic-ECs, hypoxic-ECs, siCont-ECs, siMST1-ECs, and siFOXO1-ECs using Trizol reagent (Invitrogen), 1 μg of total RNA was used to construct cDNA libraries with the TruSeq RNA library kit. The protocol consisted of poly A-selected RNA extraction, RNA fragmentation, random hexamer primed reverse transcription, and 100 nt paired-end sequencing by Illumina HiSeq 2500. The libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide and qualified using an Agilent Technologies 2100 Bioanalyzer. The sequencing adapters were removed by Trimmomatic v0.26 61 . Then, the trimmed reads were mapped to the human Ensembl reference genome (GRCh38.91) using HISAT2 aligner 62 so as to acquire the BAM file. Raw read counts were achieved from the following using the HT-Seq, then used to analyze the differentially expressed genes (DEG) between samples by applying EdgeR method. Hierarchically clustered heatmaps of DEG among siCont-ECs, siMST1-ECs, and siFOXO1-ECs were generated in R using the heatmap function. To analyze Gene Ontology biological process and get the Fold enrichment score and Q-score, the ingenuity pathway analysis (QIAGEN) was used and all DEGs were mapped to Gene Ontology database Released 2018-04-04 (http:// www.geneontology.org/) 63,64 .
RNA extraction and quantitative RT-PCR. Total RNA was extracted from samples using RNeasy mini kit (Qiagen) according the manufacturer's protocols. A total of 1 μg of extracted RNA was transcribed into cDNA using GoScript TM Reverse Transcription System (Promega). cDNA was mixed with primers and FastStart SYBR Green Master (Roche), and mRNA expression levels were measured by real-time PCR QuantStudio3 (Thermo Fisher Scientific). The primers were designed using Primer-BLAST. The list of qRT-PCR primers used in this study is described in Supplementary Table 1.
Transcriptional profile analysis of microarray data. Gene set enrichment analysis (GSEA) was performed with v6.1 of the Molecular Signature Database (http:// www.broadinstitute.org/gsea/msigdb), and the gene sets which were <0.05 nominal P-value were stated.
OIR mouse model. Briefly, P7 mice were exposed to 75% oxygen in a hyperoxic chamber (COY laboratories, O2 Control InVivo Cabinet, Grass Lake, Michigan) for 5 days with their nursing mother and then returned to room air for 5 days.
Code availability. R scripts used in the present work are available from the authors upon request.
Statistics and reproducibility. No methods were not used to predetermine sample size. Reproducibility was ensured by performing more than five and three independent in vivo and in vitro experiments, respectively. Animals or samples were not randomized and the investigators were not blinded during experiments. Both male and female neonatal mice were analyzed at P6 and P12 and only male mice were used at 12 weeks of age. No animals were excluded from analysis. All values are presented as mean ± standard deviation (s.d.). Statistical significance was determined by the two-tailed unpaired t-test between 2 groups or the one-way ANOVA followed by Tukey's honest significant difference (HSD) test with ranks for multiple-group comparison. Statistical analysis was performed using GraphPad Prism 7.0 (GraphPad Software). Statistical significance was set to P-value < 0.05.
Reporting summary. Further information on experimental design is available in the Nature Research Reporting Summary linked to this article. Fig. 8 MST1-FOXO1 cascade is required for pathologic angiogenesis. a Images of CD31 + vessels in the superficial layer of retinas and avascular area (red) in WT-OIR, Mst1 iΔEC -OIR, and Foxo1 iΔEC -OIR mice. Scale bars, 500 μm. b Images of subcellular localization of FOXO1 in CD31 + vessels at vascular front (revascularization) and vascular plexus (neovascularization) in WT-OIR, Mst1 iΔEC -OIR, and Foxo1 iΔEC -OIR mice. Scale bars, 100 μm. Note that WT-OIR mice exhibited a nuclear localization of FOXO1 (yellow arrowheads), while Mst1 iΔEC -OIR mice showed a diffuse nucleocytoplasmic localization of FOXO1 (yellow arrows) in tip ECs and NVT ECs. c Comparisons of indicated parameters in WT-OIR (n = 5), Mst1 iΔEC -OIR (n = 5) and Foxo1 iΔEC -OIR (n = 5) mice. Data represent mean (bar) ± s.d. (error bars). P values, versus WT by two-tailed unpaired t-test. d Images of CD31 + vessels, ERG + nuclei of ECs and GM130 + Golgi apparatus at tip ECs in WT-OIR, Mst1 iΔEC -OIR and Foxo1 iΔEC -OIR mice. The images of the inset (white dashed-line boxed) are magnified in e. The yellow dashed line outlines CD31 + vessels. Scale bars, 50 μm. e Images of ERG + nuclei of ECs and GM130 + Golgi apparatus at tip ECs in WT-OIR, Mst1 iΔEC -OIR, and Foxo1 iΔEC -OIR mice. The yellow dashed line outlines CD31 + vessels. Note that GM130 + Golgi apparatus are polarized towards the anterior or posterior of the nuclei in tip ECs of WT-OIR mice (yellow arrowheads), while such polarization is lost in tip ECs of Mst1 iΔEC -OIR and Foxo1 iΔEC -OIR mice (yellow arrows). Scale bars, 100 μm. Source data are provided as a Source Data file

Data availability
The RNA-sequencing data generated with this study have been deposited in Gene Expression Omnibus under the accession number GSE116033. The source data underlying all Figs and Supplementary Figs are provided as a Source Data file. A Reporting Summary for this article is available as a Supplementary Information file. All other data that support the findings of this study are available from the corresponding author upon reasonable request. Expression data from the published studies were obtained from the accession number GSE19284 in the Gene Expression Omnibus 39 .