ARHGEF18 is a flow-responsive exchange factor controlling endothelial tight junctions and vascular leakage

Surya Prakash Rao Batta, Marc Rio, Corentin Lebot, Céline Baron-Menguy, Maxence Bodet, Reda Moutaoukil, Robin Le Ruz, Ibtissam Babahnini, Gervaise Loirand, Anne-Clémence Vion

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To cite this version:

Surya Prakash Rao Batta, Marc Rio, Corentin Lebot, Céline Baron-Menguy, Maxence Bodet, et al. ARHGEF18 is a flow-responsive exchange factor controlling endothelial tight junctions and vascular leakage. Cell Reports, 2025, 44 (3), pp.115288. 10.1016/j CELREP.2025.115288. hal-05241273

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Cell Reports

# ARHGEF18 is a flow-responsive exchange factor controlling endothelial tight junctions and vascular leakage

## Graphical abstract

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## Authors

Surya Prakash Rao Batta, Marc Rio, Corentin Lebot, ..., Ibtissam Babahnini, Gervaise Loirand, Anne-Clémence Vion

## Correspondence

gervaise.loirand@univ-nantes.fr (G.L.),
anne-clemence.vion@univ-nantes.fr
(A.-C.V.)

## In brief

Batta et al. identified ARHGEF18 as a flow-responsive RhoGEF with high phosphorylation-dependent activity under physiological shear stress and as necessary to maintain tight junctions by recruiting ZO-1 and Claudin5 via RhoAP38 signaling pathway. *In vivo*, ARHGEF18 loss results in compromised tight junctions and arterial bleeding.

## Highlights

* Physiological shear stress promotes ARHGEF18 phosphorylation and activity

* Active ARHGEF18 helps in tight junction maintenance and basal adhesion

* *In vivo*, ARHGEF18 promotes vascular barrier and prevents bleeding in arterial territories

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## Article

# ARHGEF18 is a flow-responsive exchange factor controlling endothelial tight junctions and vascular leakage

Surya Prakash Rao Batta,<fnref n="1" /> Marc Rio,<fnref n="1" /> Corentin Lebot,<fnref n="1" /> Céline Baron-Menguy,<fnref n="1" /> Maxence Bodet,<fnref n="1" /> Reda Moutaoukil,<fnref n="1" /> Robin Le Ruz,<fnref n="1" /> Ibtissam Babahnini,<fnref n="1" /> Gervaise Loirand,<fnref n="1" />,\* and Anne-Clémence Vion<fnref n="1" />,2,\*

## SUMMARY

The shear stress resulting from blood flow is a major regulator of endothelial cell (EC) biology and morphology. Rho protein-mediated cytoskeleton remodeling is an early and essential step of EC responses to flow. However, how Rho protein signaling is controlled by shear stress remains unclear. Here we demonstrate that phosphorylation, activity, and expression of the Rho nucleotide exchange factor (RhoGEF) ARHGEF18 in ECs are modulated by the magnitude of shear stress. When phosphorylated, ARHGEF18 interacts with tight junctions; participates in EC elongation, alignment, and migration; and allows the maintenance of the endothelial barrier under physiological flow conditions. In mice, ARHGEF18 is involved in tight junction formation, flow response of ECs, and the control of vascular permeability. Together, our results identified ARHGEF18 as the first flow-sensitive RhoGEF in ECs, whose activity is essential for the maintenance of intercellular junctions and the control of vascular permeability in vivo.

## INTRODUCTION

Mechano-sensitivity and mechano-transduction play crucial roles in shaping life. Cells, individually or collectively, need to respond appropriately to mechanical cues coming from their environment to adapt, survive, and to ensure healthy tissue development and homeostasis.<fnref n="1" /> In the vasculature, endothelial cells (ECs) are constantly exposed to the mechanical stress generated by blood flow.<fnref n="2" />,³ ECs are therefore able to sense small variations in the direction, magnitude, and regularity of blood flow-induced shear stress (SS)⁴,⁵ and consequently modulate their functions to generate adapted responses.⁶ This leads, for example, to arterio-genesis,⁷ vascular patterning,⁸,⁹ and acute regulation of vessel tone,¹⁰ which are critical for both the development and maintenance of a healthy cardiovascular system. Conversely, failure of ECs to adapt to changes in blood flow or chronic exposure to abnormal flow leads to vascular diseases such as hypertension, atherosclerosis,<fnref n="1" />,¹¹ or intracranial aneurysm.¹²

The shear stress generated by laminar flow as observed in linear parts of the vascular tree is essential for the alignment of ECs in the direction of flow and the control of their functions,¹³,¹⁴ thanks to several mechanosensing mechanisms. Well-studied examples include ion channels,¹⁵ primary cilia expressed at the apical surface of cells,¹⁶,¹⁷ but also include mechano-sensing complexes at the cell-cell interface such as the PECAM-1/ VEGFRs/VE-cadherin complex⁶ and at cell membrane-extracellular matrix attachment such as focal adhesions and integrins.¹⁸ These sensors and complexes play an essential role in translating

flow-induced physiological forces into intracellular signals, resulting in various cellular responses such as chemokine release, nitric oxide production, cell proliferation, and junctional reorganization. Rho protein-mediated actin cytoskeleton reorganization is one of the first steps in these processes.¹⁹⁻²³ Exposure of EC to physiological SS rapidly induces the translocation of Rho proteins from the cytosol to the plasma membrane associated with their activation.²⁴ Indeed, the overexpression of dominant-negative mutants of RhoA or Rac1 in ECs prevents their proper elongation and alignment in response to flow.²⁵ Both transient¹³,¹⁸,²⁵,²⁶ and sustained²⁴ activation of Rho proteins upon SS are required for the establishment and maintenance of SS-induced EC alignment. Activation of Rho proteins consists of their transition from an inactive state (GDP-bound) to an active state (GTP-bound).²⁷ Rho guanine nucleotide exchange factors (RhoGEFs) induce the activation of Rho proteins by promoting the release of GDP and subsequent GTP loading.²⁸ In contrast to the ubiquity of Rho proteins, RhoGEF expression is tissue specific and their activation is signal/time specific,²⁹ placing them at the center of the control of the multiple effects of Rho proteins on cell functions. Some RhoGEFs, such as FGD5,³⁰ have been described to be expressed exclusively in ECs, while others, although not specific to ECs, have been characterized for their role in mechanosensitivity of ECs. The RhoGEFs Tiam1 and Vav2 are involved in the rapid activation of Rac1 in ECs exposed to SS.³¹ The RhoGEF Trio keeps Rac1 active at the downstream side of the EC and is essential for long-term flow-induced cell alignment³² as well as barrier integrity through the formation of

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<footnote n="1">¹Université de Nantes, CHU Nantes, CNRS, INSERM, l'Institut du thorax, 44000 Nantes, France</footnote>

<footnote n="2">²Lead contact</footnote>

<footnote>\*Correspondence: gervaise.loirand@univ-nantes.fr (G.L.), anne-clemence.vion@univ-nantes.fr (A.-C.V.)</footnote>

<footnote>https://doi.org/10.1016/j.celrep.2025.115288</footnote>
a NOTCH1/VE-Cadh/LAR/Trio complex.33 Finally, ARHGEF18 has been identified as the RhoGEF responsible for RhoA activation in response to force sensing by ICAM in ECs, thereby promoting leukocyte transmigration.34

Here, using unbiased RNA screening in ECs, we identified ARHGEF18 as a novel flow-sensitive RhoGEF. We demonstrated that ARHGEF18 is a RhoA-specific RhoGEF activated under physiologic flow that interacts with ZO1 and Claudin5 in ECs. We showed that ARHGEF18 is involved in EC alignment in response to flow by controlling p38 activity, tight junction, and focal adhesion formation through its guanine nucleotide exchange activity. In vivo, endothelial-specific deletion of ARHGEF18 in mice resulted in retinal and brain hemorrhages, as well as vascular leakage in the kidney and mesenteric arteries, consistent with a physiological role endothelial junction stability.

## RESULTS

### Shear stress regulates ARHGEF18 expression and activity

To identify RhoGEFs whose expression is modulated by SS, we performed a 3'RNA profiling on ECs exposed to different SS levels: no flow (static, 0 dyn/cm²), pathological low SS (LSS, 3.6 dyn/cm²), physiological SS (PSS, 16 dyn/cm²), and pathological high SS (HSS, 36 dyn/cm²). The observed increased expression of the flow-dependent gene *KLF2* with increased level of SS validated our experimental conditions (Figures 1A and S1A). Among the genes with differential expression between static and SS, we identified several RhoGEFs whose expression was dependent on the level of SS (Figures 1A and S1B). Among these, only *ARHGEF18* showed an expression that was inversely correlated with the level of SS (Figure S1B). This inverse correlation between ARHGEF18 mRNA expression and SS levels was further confirmed by qRT-PCR analysis (Figure 1B). However, at the protein level, ARHGEF18 expression increased under pathological SS conditions (LSS and HSS) compared with physiological condition (PSS) (Figure 1C). This difference in the expression pattern between ARHGEF18 mRNA and protein suggests that additional post-transcriptional effects of SS could affect ARHGEF18 protein levels. We next assessed whether the nucleotide exchange activity of ARHGEF18 on RhoA and Rac1 was also modulated by SS by performing pull-down assay using nucleotide-free RhoA<sup>G17A</sup> and Rac1<sup>G15A</sup> mutants to capture active RhoGEF.35 Positive controls using vascular endothelial growth factor (VEGF) and thrombin stimulation, have validated our pull-down assay, showing, as expected, the capture of GEF-H1 and p115 by RhoA<sup>G17A</sup>, respectively (Figure S1C). Under static condition, we were able to detect ARHGEF18 among the proteins coprecipitated with RhoA<sup>G17A</sup>, indicating a basal nucleotide exchange activity of ARHGEF18 on RhoA in EC in the absence of flow (Figure 1D). Under flow conditions, the maximal association between ARHGEF18 and RhoA<sup>G17A</sup> was detected in ECs subjected to PSS, indicating a high exchange activity of ARHGEF18 on RhoA under physiological flow conditions, which was reduced in pathological SS situations (Figure 1D). In contrast, we were unable to capture ARHGEF18 with Rac1<sup>G15A</sup>, regardless of SS conditions (Figure 1E), while Vav, a known RacGEF,36 was found among the proteins copre-

cipitated with Rac<sup>G15A</sup> (Figure S1D). Taken together, these data demonstrate that ARHGEF18 is a RhoGEF specific for RhoA in ECs whose expression and activity are modulated by flow, with a maximal activity under physiological SS conditions.

### ARHGEF18 is expressed in arterial EC and interacts with tight junction proteins

ARHGEF18 has been shown to be involved in the formation of tight junctions in epithelial cells.37 We therefore investigated the possible interaction of endogenous ARHGEF18 with endothelial cell-cell junction proteins by co-immunoprecipitation experiments. No or little non-specific interactions were observed in our experimental setup (isotype control, Figure S1E) and the immunoprecipitated fractions showed a good enrichment of the target proteins (Figure S1E). ARHGEF18 was not detected in the protein fraction co-immunoprecipitated with VE-cadherin but was present in the immunoprecipitated fraction of the ZO-1 complex (Figure 1F). We confirmed the association of ARHGEF18 and ZO-1 in the same protein complex by detecting ZO-1 in the proteins co-immunoprecipitated with an anti-flag antibody from the lysates of ECs expressing a flag-tagged ARHGEF18 (Figure 1G). We also detected a large amount of the major EC tight junction protein claudin 5 in this immunoprecipitate, whereas VE-cadherin was not found (Figure 1G). Taking advantage of ECs with a low expression of the flag-tagged ARHGEF18, we also observed areas where ARHGEF8 is concentrated at the cell-cell junction or in its near proximity (Figure 1H). These results are consistent with the localization of ARHGEF18 within the tight junction complex. We next used immunofluorescence staining of mouse retinas to characterize the expression of ARHGEF18 in the vasculature. Using isolectin B4 staining to label ECs, we observed ARHGEF18 expression in the arteries of the retina at postnatal day 6, and in some capillary areas but not in the veins (Figure 1I). In 4-week-old mouse retina, this arterial expression was maintained but the capillary cluster were lost (Figure S1F). Interestingly the staining was stronger in arteries close to the optic nerve (Figure S1F) and became fainter as the artery caliber decreased and as the network ramified to become capillary (Figure S1F). Altogether, these *in vivo* and *in vitro* data show that ARHGEF18 is expressed in arterial ECs and integrates into a complex with ZO-1 and claudin 5 to localize to the tight junctions.

### ARHGEF18 participates in EC adhesion and migration

We used short hairpin RNA (shRNA) and small interfering RNA (siRNA) silencing strategies to knock down the expression of ARHGEF18 (shRNA Figure S2A, siRNA, data not shown) and analyzed its role in ECs. We first evaluated the consequence of ARHGEF18 depletion on RhoA activity by pull-down assay. Under PSS condition, the activity of RhoA was decreased by 30% in ARHGEF18-depleted EC compared with controls (Figure S2B). Surprisingly, although ARHGEF18 was not characterized as GEF for Rac1,38 the activity of Rac1 was also decreased by 30% in ARHGEF18-depleted EC compared with controls (Figure S2C). We next assessed the functional role of ARHGEF18 in ECs in basic cell processes that depend on RhoA activity and the dynamic organization of the actin cytoskeleton such as adhesion and migration under static conditions. Real-time impedance measurement after EC seeding showed that
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<figure><img src="image_16.png" /><figcaption>Figure 1. ARHGEF18 expression and activity is SS dependent</figcaption></figure>

(A) MA plot comparing gene expression in ECs subjected for 24 h to static condition and high shear stress (HSS). Green dots: up-regulated genes in HSS condition; red dots: down-regulated genes in HSS condition. n = 4.

(B) ARHGEF18 mRNA expression level in EC exposed to static, low shear stress (LSS), physiological shear stress (PSS) or HSS for 24 h. Data are shown as mean ± SEM. \*p < 0.05; \*\*p < 0.01; one-way ANOVA.

(C) ARHGEF18 protein expression level in ECs exposed to static, LSS, PSS, or HSS condition for 24 h. Data are shown as mean ± SEM. \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; one-way ANOVA.

(D) ARHGEF18 activity determined from the amount of ARHGEF18 captured by GST-RhoA<sup>G17A</sup> pull-down assay in EC exposed to static, LSS, PSS, or HSS condition for 24 h. Data are shown as mean ± SEM. \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; one-way ANOVA.

(E) ARHGEF18 captured by GST-Rac1<sup>G15A</sup> pull-down assay in ECs subjected to static, LSS, PSS, or HSS condition for 24 h (representative of three independent experiments).

(F) Co-immunoprecipitation of ARHGEF18 with VE-cadherin (left) or ZO-1 (right). Co-immunoprecipitation efficiency was assessed by the presence of β-catenin and claudin 5 in the immunoprecipitated fraction of VE-cadherin complex and ZO-1 complex respectively (upper lanes) (representative of three independent experiments).

(G) Co-immunoprecipitation of CLD5, ZO1, and VE-cadherin with FLAG-tagged ARHGEF18 (WT) (representative of three independent experiments).

(H) FLAG staining in HUVECs expression of a flag-tagged ARHGEF18 (WT). Upper panel: green, FLAG; magenta: VE-cadherin. Middle panel: FLAG; lower panel: ZO-1.

(I) Immunofluorescent staining of ARHGEF18 in mouse retina from P6 animals. Green: Arhgef18, Magenta: IsolectinB4 (representative of seven independent animals). Scale bar, 50 µm. See also Figures S1 and S2.

ARHGEF18 depletion reduced both the slope of impedance rise and the plateau, indicating that ARHGEF18 regulates early adhesion and spreading of ECs (Figures S2D–S2F). To further characterize this effect at the single-cell level, we plated ECs onto L-shaped micro-patterned fibronectin to overcome variability in cell morphology by forcing cells to individually adhere and adopt a normalized geometry, allowing a better comparison and quantification of the number and size of focal adhesions. Silencing of ARHGEF18 led to a significant decrease in the number of long focal adhesions (>5 µm) compared with control conditions suggesting a defect in focal adhesion maturation in ARHGEF18-depleted ECs (Figures S2G and S2H). To assess the role of ARHGEF18 in EC migration, we used an *in vitro* wound healing assay that revealed that the closure of the wound was slowed down by ARHGEF18 knockdown (Figures S2I and

S2J). Finally, since we have demonstrated an interaction among ARHGEF18, ZO-1, and claudin 5, we then assessed whether ARHGEF18 deletion may affect endothelial permeability under static condition. Surprisingly, no difference in the leakage of fluorescent-labeled dextran tracers (3 kD and 70 kD) was observed between ARHGEF18-depleted and control EC monolayers under static condition (Figure S2K). These results suggest that ARHGEF18 promotes adhesion, focal adhesion site formation, and migration of ECs without affecting their barrier properties when not exposed to flow.

## ARHGEF18 participates in the flow-induced response of ECs

We next sought to determine whether the high ARHGEF18 activity in ECs under PSS condition contributes to the physiological
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(J) Maximum displacement (µm) of HUVECs expressing a non-targeting shRNA (shNT) or an Arhgef18 shRNA (shA18.1) over 18 h, exposed to PSS. n = 3 (400–700 track analyzed). Dotted red line represents the mean of maximum displacement under static condition. Data are shown as mean ± SEM. ***p < 0.001, unpaired t test.

(K) Linearity of progression of HUVECs expressing a non-targeting shRNA (shNT) or an Arhgef18 shRNA (shA18.1) over 18 h, exposed to PSS. n = 3 (400–700 track analyzed). Dotted red line represents the mean of linearity of progression under static condition. Data are shown as mean ± SEM. ***p < 0.001, unpaired t test. See also Figures S3 and S4 and Videos S1, S2, S5, and S6.

response of ECs to flow. As expected, control ECs (shNT) were aligned and elongated in the direction of flow. They exhibited a continuous VE-cadherin junction with junction-associated lamellipodia formation at the front and rear of the cells, continuous ZO-1 staining, and cortical actin cables (Figure 2A, upper panel). In contrast, ARHGEF18-depleted ECs exposed to PSS failed to elongate and align in the direction of the flow as evidenced by the significant decrease in the flow response index (Figure 2A, lower panel; Figures 2B, S3A, and S3B). In addition, ARHGEF18

knockdown did not affect the proliferation rate of ECs under PSS, ruling out a possible effect of EC density/packing on the cell shape change observed in ARHGEF18-depleted ECs subjected to PSS (Figure S3C). Despite the presence of VE-cadherin at junctions, the shape of adheren junctions appeared to be altered in ARHGEF18-deficient ECs compared with the control (Figure 2A). Indeed, the use of a classification method to assess junctional activity³⁹ revealed that ARHGEF18-deficient ECs had more active adherens junctions than control ECs (Figure S3D).

<caption>**Figure 2. ARHGEF18 participates in EC response to SS and tight junction formation under physiological SS**</caption>

(A) HUVECs expressing a non-targeting shRNA (shNT) or an Arhgef18 shRNA (shA18.1), exposed to physiological SS (PSS) for 24 h. Flow direction is indicated by the arrow. Scale bar, 50 µm.

(B) Cell orientation and elongation with the flow in HUVECs expressing a non-targeting shRNA (shNT) or an Arhgef18 shRNA (shA18.1), exposed to PSS for 24 h. Each dot represents a cell. n = 5 with 200–600 cells analyzed by experiment. Data are shown as median. ***p < 0.001; Mann-Whitney t test.

(C) ZO-1 localized at junction in HUVECs expressing a non-targeting shRNA (shNT) or an Arhgef18 shRNA (shA18.1), exposed to physiological SS for 24 h. n = 3, 9 areas analyzed by experiment. Data are shown as mean ± SEM. ***p < 0.001; unpaired t test.

(D) Actin localization at junction in HUVECs expressing a non-targeting shRNA (shNT) or an Arhgef18 shRNA (shA18.1), exposed to physiological SS for 24 h. n = 3, 9 areas analyzed by experiment. Data are shown as mean ± SEM. ***p < 0.001; unpaired t test.

(E) Claudin5 staining in a mix population of WT cells (GFP negative, indicated as “WT” on images) and cells expressing an Arhgef18 shRNA (shA18.1, GFP positive, green, indicated as “Sh” on images), exposed to physiological SS. Green, GFP; red, Claudin5; magenta: VE-cadherin.

(F) Quantification of the Claudin5 staining at junction. n = 3, 12 couple of cells analyzed per experiment. Data are shown as mean ± SEM.

(G) Number of holes in-between cells in HUVECs expressing a non-targeting shRNA (shNT) or an Arhgef18 shRNA (shA18.1), exposed to physiological SS for 24 h. n = 3. 10 areas analyzed by experiment. Data are shown as mean ± SEM. ***p < 0.001; unpaired t test.

(H) Duration of the holes in HUVECs expressing a non-targeting shRNA (shNT) or an Arhgef18 shRNA (shA18.1), exposed to physiological SS for 24 h. Data are shown as mean ± SEM. ***p < 0.001; unpaired t test.

(I) Representative view of the cells tracking for 18 h under PSS (around 100 cells presented, green: shNT, red: shA18.1).
Moreover, the localization of ZO-1 at junction appeared disrupted and was significantly decreased by ARHGEF18 knockdown (Figures 2A, 2C, and S3A). This was accompanied by a decrease in the cortical actin in ARHGEF18-deficient ECs compared with control (Figures 2A, 2D, and S3A). To further determine the role of ARHGEF18 on EC junction under PSS condition, we analyzed claudin 5 labeling at the cell-cell junction, taking advantage of the mosaicism of ECs expressing ARHGEF18 shRNA, detectable by GFP labeling in the EC monolayer. We observed that the maximal intensity of claudin 5 labeling at junction (colocalizing with VE-cadherin) between two ECs decreased when one of the two ECs expressed the ARHGEF18-shRNA. This junctional localization of claudin 5 was even more strongly reduced if the two ECs on both sides of the junction expressed the ARHGEF18-shRNA (Figures 2E, 2F, and S3E). This effect is reflected by an overall decrease in claudin 5 and ZO-1 expression analyzed by western blot in ARHGEF18-deficient ECs subjected to PSS compared with control cells (Figures S3F and S3G). The reduced ZO-1 expression was only observed when ECs were exposed to PSS, indicating a PSS-specific action of ARHGEF18 on tight junctions. These data suggest that the loss of ARHGEF18 expression leads to tight junction destabilization under PSS attested by the exclusion of claudin 5 and ZO-1 from junction, which could be linked to an effect on claudin 5 and ZO-1 protein expression or stability. As a result, we observed an increase in the number of gaps between ECs in ARHGEF18-deficient ECs compared with control (Figures 2A-2G and S3A). To assess the dynamics of these holes, we performed live imaging experiments under PSS and quantified their duration. The holes in-between ECs were not larger (data not shown) but remained open for a longer time in ARHGEF18-deficient ECs compared with control (Figure 2H, Videos S1 and S2). In these experiments, we also quantified the motility of ECs under static and PSS conditions. Under static condition, ARHGEF18-deficient ECs were more motile than controls (Figures S3H and S3I, Videos S3 and S4), but their direction was as random as in the control ECs (Figures S3H and S3J, Videos S3 and S4). We also observed no holes in the layer of both control and ARHGEF18-deficient ECs (Videos S3 and S4), reinforcing the idea that hole formation and permeability in ARHGEF18-deficient ECs is dependent on flow input. As expected, exposure of ECs to PSS increased the speed of control ECs and the directionality of their trajectories (Figures 2I-2K, Video S5), but these flow-induced effects were compromised in ARHGEF18-deficient ECs, leading to a significant decrease in the distance, and direction of ARHGEF18-deficient ECs migration compared with controls under PSS condition (Figures 2I-2K; Videos S5 and S6). To then analyze focal adhesions, which are known to be involved in EC response to flow,²¹ we performed immunofluorescence staining for paxillin on ECs exposed to PSS. Paxillin staining showed that focal adhesions, which appeared elongated and collectively oriented in control ECs, became more punctuate and disorganized in ARHGEF18-deficient ECs (Figures S4A and S4B). Surprisingly, we observed no change in the overall level of paxillin phosphorylation assessed by western blot in ARHGEF18-deficient ECs compared with controls (Figures S4C and S4D). Collectively, these data suggest that ARHGEF18 contributes to EC response to physiological

flow by promoting tight junction formation/stabilization, actin recruitment at the cell-cell junction, and by participating in EC migration with the flow.

## ARHGEF18 activity-dependent p38 activation is essential for EC response to flow

We wanted to investigate the possible involvement of the AKT, p38 MAPK, and ERK1/2 signaling pathways, which are known to be flow sensitive and play a central role in focal adhesion formation and actin remodeling in ECs,⁴⁰⁻⁴⁶ in ARHGEF18-mediated effects. Western blot analysis showed that ERK1/2 and AKT phosphorylation levels were unaffected in ARHGEF18-deficient ECs compared with control ECs (Figures S5A and S5B). In contrast, PSS-induced p38 phosphorylation was significantly reduced by ARHGEF18 silencing (Figures 3A and S5C) suggesting that ARHGEF18 participates in flow-induced activation of p38 and that p38 may be involved in the ARHGEF18-dependent effect on tight junction and focal adhesion in ECs under physiological flow condition. Indeed, pharmacological inhibition of p38 by SB239063 (100 nM) reproduced the effects of ARHGEF18 depletion by inducing a loss of cell alignment and elongation (Figures S5D and S5E). Inhibition of p38 also induced delocalization of ZO-1 and actin fibers from junctions in ECs under PSS (Figures S5D, S5F, and S5G). This loss of junctional ZO-1 was not accompanied by an increase in the number of gaps between ECs, suggesting an additional role for ARHGEF18 on cell contact maintenance, which would be independent of p38. Finally, inhibition of p38 in ECs under PSS resulted in a loss of focal adhesion organization compared with control as observed with ARHGEF18 depletion (Figures S5H and S5I).

The phosphorylation of the tyrosine residue Y606 in the Dbl domain of ARHGEF18 has been shown to be necessary for its GEF activity³⁷ and predicted to be under the control of an Src family kinase (SFK). By immuno-precipitation of phosphorylated proteins, we found that ARHGEF18 was phosphorylated on tyrosine residues and that this phosphorylation was increased under PSS conditions (Figure 3B, isotype control in Figure S6A). To identify the kinase upstream of ARHGEF18 phosphorylation, we first evaluated the effect of two known inhibitors of SFK with different affinity for their members (SRC inhibitor I: half maximal inhibitory concentration [IC₅₀] = 44 nM and 88 nM, for Src and Lck, respectively; SU6656: IC₅₀ = 20 nM, 130 nM, and 170 nM for YES, LYN and FYN, respectively⁴⁷) on ARHGEF18 phosphorylation in ECs under static conditions. Interestingly, SU6656 (50 nM) abolished tyrosine phosphorylation of ARHGEF18, whereas SRC inhibitor I (100 nM) had no effect (Figure S6B). Moreover, SU6656 strongly reduced the level of tyrosine phosphorylation of ARHGEF18 under PSS (Figure S6C). According to the differential inhibitory properties on SFK of SU6656 and SRC inhibitor I, these data suggested that YES could be the upstream SFK mediating ARHGEF18 phosphorylation. To assess this hypothesis, we silenced YES or FYN in ECs and analyzed tyrosine phosphorylation of ARHGEF18 under PSS. Only YES depletion decreased the level of tyrosine phosphorylation of ARHGEF18, consistent with a direct or indirect control of ARHGEF18 phosphorylation by YES (Figure 3C).
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<figure><img src="image_25.png" /><figcaption>Figure 3. YES mediates ARHGEF18 phosphorylation and its GEF activity is essential for flow-dependent p38 signaling</figcaption></figure>

(A) p38 phosphorylation by western blot in HUVECs expressing a non-targeting shRNA (shNT) or an Arhgef18 shRNA (sha18.1). 24 h of flow. Static: n = 3; PSS: n = 8. Data are shown as mean ± SEM.
**p < 0.01; **p < 0.001 two-way ANOVA.

(B) ARHGEF18 western blot from phosphotyrosine immunoprecipitation from HUVECs exposed to static, low SS (LSS), physiological SS (PSS), or high SS condition (HSS), 24 h of flow. Data are shown as mean ± SEM.

(C) Representative images of blot for ARHGEF18, YES, and FYN with the associated quantification of ARHGEF18 from phosphor-tyrosine immunoprecipitation from Control HUVECs or HUVECs silenced for FYN and YES, exposed to physiological SS (PSS) for 24 h. Data are shown as mean ± SEM.

(D) Representative images and quantification of p38 phosphorylation by western blot in HUVECs expressing a WT form (A18WT) or a mutant form (A18Y606A) of ARHGEF18, exposed to PSS for 24 h. Data are shown as mean ± SEM. Wilcoxon t test.

(E) Representative images of HUVECs exposed to physiological SS for 24 h, expressing a WT form (A18WT) or a mutant form (A18Y606A) of ARHGEF18 and stained for VE-cadherin. Flow direction is indicated by the arrow. Scale bar, 50 µm.

(F) Cell orientation and elongation with the flow in HUVECs exposed to PSS for 24 h, expressing a WT form (A18WT) or a mutant form (A18Y606A) of ARHGEF18. Each dot represents a cell. Between 1,000 and 1,350 cells analyzed. n = 3. Data are shown as mean ± SEM. **p < 0.01; unpaired t test.

(G) Representative images of ZO-1 staining in a mix population of WT cells (FLAG negative, non-red) and cells expressing the mutant form (A18Y606A) of ARHGEF18 (FLAG positive, red), exposed to physiological SS for 24 h. Green: ZO-1; red: FLAG; magenta: VE-cadherin. Scale bar, 50 µm.

(H) Quantification of the ZO-1 staining at junction. n = 3, 25 couples of cells analyzed per experiment. Data are shown as mean ± SEM. See also Figures S5 and S6.

To further characterize the link among ARHGEF18, RhoA, and p38, we sought to prevent the flow-induced activation of ARHGEF18. As we found that flow-induced ARHGEF18 activation might depend on YES-mediated phosphorylation of Y606 of ARGHEF18, we substituted this tyrosine with an alanine residue and expressed this phosphoresistant ARHGEF18 mutant (ARHGEF18Y606A) in ECs silenced for endogenous ARHGEF18. The pull-down assay with RhoAG17A showed that, in contrast to WT-ARHGEF18, ARHGEF18Y606A did not associate with RhoA under PSS condition, indicating a key role for Tyr606 phosphorylation in the GEF activity of ARHGEF18 under physiological flow conditions (Figure S6D). This loss of activity in ARHGEF18Y606A-expressing ECs was associated with a decrease in p38 phosphorylation compared with the one of ARHGEF18-WT-expressing EC (Figure 3D). Morphologically,

the expression of ARHGEF18Y606A rendered ECs unable to align in response to physiological flow, contrary to ECs expressing WT-ARHGEF18 (Figures 3E and 3F). To assess the consequences of ARHGEF18Y606A expression on tight junction, we analyzed ZO-1 labeling in mosaic cells and again took advantage of the fact that not all ECs expressed the phosphoresistant ARHGEF18Y606A mutant, detectable by FLAG labeling. We observed that the intensity of ZO-1 labeling at the junction between two ECs (colocalized with VE-cadherin) decreased when one of the two neighboring ECs expressed ARHGEF18Y606A compared with the intensity between two non-transfected ECs, and was further reduced when both ECs on either side of the junction expressed ARHGEF18Y606A (Figures 3G and 3H). In ECs with local overexpression of the ARHGEF18Y606A mutant, we observed a focal loss of ZO-1 staining at the membrane of
<figure><img src="image_26.png" /><figcaption>Figure 4. ARHGEF18 participates in vascular patterning and response to flow in vivo</figcaption></figure>

(A) Western blot from aortas from *Arhgef18-iEC-KO* mice (A18 iEC-KO) and littermate controls.

(B) Quantification of ARHGEF18 in *Arhgef18-iEC-KO* aortas (A18 iEC-KO) and controls (median ± IQ; Mann-Whitney test). \*\*p < 0.01.

(C) Quantification of Claudin5 in *Arhgef18-iEC-KO* aortas (A18 iEC-KO) and controls (median ± IQ; Mann-Whitney test). \*\*p < 0.01.

(D) Quantification of ZO-1 in *Arhgef18-iEC-KO* aortas (A18 iEC-KO) and controls (median ± IQ; Mann-Whitney test). \*p < 0.05.

(E) Representative images of retinas from P6 pup whole-mounts stained with isolectin B4 (IB4, black). Scale bar, 500 µm.

(F) Radial expansion of the vascular plexus in *Arhgef18-iEC-KO* mice (A18 iEC-KO) and littermate controls (median ± IQ; Mann-Whitney test). \*\*p < 0.01.

(G) Density of the vascular plexus in *Arhgef18-iEC-KO* mice and littermate controls (median ± IQ; unpaired t test). \*\*\*p < 0.001.

(H) Representative images of retinal artery stained for isolectin (IB4, magenta) and ZO-1 (green) of P6 pups (observed in three littermate control and four *Arhgef18-iEC-KO*).

(I) Representative images of aortas mounted “en face” and stained for isolectin (IB4, magenta) and ZO-1 (green) from P6 pups, imaged using super-resolution structured illumination microscopy.

(J) Quantification of the ZO-1 staining at junction using plot profile analysis. n = 3, 10 to 21 couples of cells analyzed per experiment. Data are shown as mean ± SEM.

(K) Representative images of aortas mounted “en face” and stained for VE-cadherin (gray) from 4-week-old mice. Scale bar, 50 µm.

(L) Cell elongation (aspect ratio) with the flow in aortas. WT, n = 6; *Arhgef18-iEC-KO* mice, n = 7. Each dot represents a cell with 200–300 cells analyzed by experiment. Data are shown as mean ± SEM. \*\*\*p < 0.001; unpaired t test.

(M) Junction status based on their morphology (WT, n = 3; *Arhgef18-iEC-KO* mice, n = 6, 300 patches per *n*, mean ± SEM, \*p < 0.05, two-way ANOVA). See also Figure S7.

the area where ARHGEF18Y606A accumulated, suggesting that ARHGEF18, through its GEF activity, could participate in the addressing of ZO-1 to the membrane (Figure 3G, lower panel). Overall, these data show that ARHGEF18 is activated by physiological flow through a mechanism involving Tyr606 phosphorylation by YES, and that ARHGEF18 activation is required for p38 phosphorylation, tight junction organization, and EC alignment under physiological flow condition.

### ARHGEF18 loss in vivo hampers vascular development and induces vascular leakage

To assess the function of ARHGEF18 *in vivo*, we generated tamoxifen-inducible endothelial-specific ARHGEF18 knockout mice (*Arhgef18 fl/fl*; CDH5-iCre<sup>ERT2+/wt</sup> mice; referred to as *Arhgef18-iEC-KO*). The efficiency of tamoxifen treatment to induce

ARHGEF18 deletion in ECs expressing CDH5-iCre and floxed *Arhgef18* alleles *in vitro* was confirmed by western blot (Figure S7A). In mice, analysis of *Arhgef18* expression in the aortas (Figures 4A and 4B) and in the lungs (Figures S7B and S7C) showed a marked decrease in *Arhgef18-iEC-KO* compared with control littermates (*Arhgef18 fl/fl*; mice treated with tamoxifen). Interestingly, *Arhgef18-iEC-KO* aortas also displayed a reduced expression of ZO-1 and claudin 5 compared with control mice (Figures 4C and 4D). The reduction of claudin 5 expression was also visible in the lung (Figure S7C). Six days postnatally (P6), the retinal vasculature of *Arhgef18-iEC-KO* mice showed a reduced radial expansion and a decreased vascular density compared with littermate controls (Figures 4E–4G). This phenotype was transient since no more difference was observed between the retinal vasculature of 4-week-old *Arhgef18-iEC-KO*
<figure><img src="image_27.png" /></figure>

and littermate control mice (data not shown). The reduced vascular density observed in the *Arhgef18*-iEC-KO retina (P6) was unlikely due to a reduced proliferation as the density of ECs present in the retinal vasculature was unchanged between *Arhgef18*-iEC-KO and littermate controls (Figures S7D and S7E). We then assessed the consequence of *Arhgef18* deletion on tight junction appearance *in vivo* by the analysis of ZO-1 localization. At P6, ZO-1 fluorescent labeling at junction was reduced in ECs of both retinal arteries and aorta of *Arhgef18*-iEC-KO mice compared with littermate control (Figures 4H–4J). Interestingly, the loss of ZO-1 fluorescent labeling at junction was less visible in the veins and capillaries of the retina (Figure S7F), in which ARHGEF18 is not detectable by immunofluorescence (Figure 1H), confirming a link between ARHGEF18 expression and ZO-1 localization *in vivo*. Shape analysis of ECs showed a defect

<caption>**Figure 5.** *Arhgef18* prevents general vascular leakage and hemorrhages in retina and brain</caption>

(A) Representative retinal whole-mount images from P6 pups stained with isolectin (IB4, magenta) and TER119 (green). Scale bars, upper images, 500 µm and lower images, 50 µm.

(B) Quantification of erythrocytes outside the vasculature (control, *n* = 7; *Arhgef18*-iEC-KO, *n* = 9; median ± IQ; Mann-Whitney *t* test).

(C) Quantification of leaking spot per retina (control, *n* = 7; *Arhgef18*-iEC-KO, *n* = 9; median ± IQ; Mann-Whitney *t* test).

(D) Representative retinal whole-mount images from P12 pups stained with isolectin (IB4, green), SM22 (red) and TER119 (violet). Scale bar, 200 µm.

(E) Quantification of erythrocytes outside the vasculature (control, *n* = 3; A18 iEC-KO, *n* = 11; median ± IQ; Mann-Whitney *t* test).

(F) Representative photographs of brain from P6 animals.

(G) Number of animals with micro-hemorrhages (control, *n* = 23; KO, *n* = 24).

(H) Representative images of thick brain slices from P6 pups stained with isolectin (IB4, green), SM22 (red) and TER119 (violet). Red arrow indicates leaking spots/Representative of three WT and four KO.

(I) Representative images of mesenteric arteries from *Arhgef18*-iEC-KO mice (A18 iEC-KO) and littermate controls (P7), after dextran dye circulation (green, 3 kD; red, 70 kD).

(J) Number of animals with positive cells in the media (*n* = 4).

in blood flow-induced EC elongation, quantified by the aspect ratio, in the aorta of *Arhgef18*-iEC-KO mice compared with littermate controls, although the overall cell orientation remained in the direction of the flow (Figures 4K, 4L, and S7G). Moreover, ECs of *Arhgef18*-iEC-KO mouse aorta displayed slightly but significantly more active adherens junctions compared with control (Figures 4K and

4M). As tight junctions are associated with vascular barrier maintenance,⁴⁸ we assessed vascular leakage in the retinal vasculature by looking at erythrocyte extravasation. *Arhgef18*-iEC-KO mouse retinas presented twice as many erythrocytes outside the vasculature than littermate control mouse retinas, both as diffused leakage with erythrocytes spread within the vascular space and as focal hemorrhages (Figures 5A–5C). At P12, this leakage was still present but only as a diffuse leakage, without focal hemorrhages (Figures 5D and 5E) and was no longer observed in 4-week-old *Arhgef18*-iEC-KO mice (data not shown). This bleeding was not a side effect of the expression of the recombinase as retinas from CDH5-iCre<sup>ERT2+/wt</sup> mice without any flox allele but injected with tamoxifen did not display increased erythrocytes leakage nor decreased radial expansion compared with their littermate controls (Figures S7H–S7J). The
retinal vasculature being an extension of the brain vasculature with common developmental origins,⁴⁹ we also assessed potential vascular leakage in the brain of the *Arhgef18*-iEC-KO mice (P6). Interestingly, we observed small red spots at the surface of the brain in 50% of the *Arhgef18*-iEC-KO mice, but only in 10% of the littermate controls (Figure 5F). To confirm this finding, we stained thick sections of the brain for erythrocytes and while we did not see any hemorrhage in control mice, we regularly found erythrocytes leakage in *Arhgef18*-iEC-KO mice (Figure 5G). Most of them were localized in the cortex in the immediate vicinity of penetrating arterioles. Interestingly we observed different features of bleeding at P6, with fresh bleeding visualized by accumulation of intact erythrocytes outside the vasculature (Figure 5H, *Arhgef18*-iEC-KO #1, red arrow) and resolving bleedings with dead and phagocytized erythrocytes (Figure 5H, *Arhgef18*-iEC-KO #2, red arrow). Finally, we evaluated the possible specificity of these leakages to the vasculature of the central nervous system. Evans blue extravasation was significantly increased in the kidneys of *Arhgef18*-iEC-KO mice (P7), compared with littermate controls but not in the liver or the lung (Figure S7K). Additionally, we assessed the vascular permeability of mesenteric arteries *ex vivo* under flow. After 30 min of perfusion, positive cells for the 3-kD dextran were visible in most of the arteries from A18-iEC-KO mice while none of the arteries from control animal had positive cells (Figures 5I and 5J). No vascular leakage was observed for the 70-kD dextran (Figure 5I). Together these data exclude a specific action of ARHGEF18 on the vasculature of the central nervous system and confirmed our *in vitro* results, showing the involvement of ARHGEF18 in flow response of ECs and in the in the control of vascular permeability *in vivo* in mice.

DISCUSSION

Our work identifies ARHGEF18 as a new SS-sensitive RhoGEF in ECs that is involved in EC elongation and alignment, and in the maintenance of the endothelial barrier under physiological flow conditions. ARHGEF18, also known as p114-RhoGEF, is a RhoA-specific RhoGEF, but has been shown to be able to trigger RAC1 activation in HEK and fibroblasts, probably through an indirect mechanism.³⁸,⁵⁰ In ECs, we showed that silencing ARHGEF18 was able to reduce RAC1 activity without directly interacting with it, suggesting an indirect effect of ARHGEF18 as in other cell types. ARHGEF18 has been described mainly in epithelial cells where it controls tight junction maturation and the establishment of apicobasal polarity³⁷,⁵¹ via RhoA and RAC1 activation.³⁸,⁵⁰,⁵² In these cells, ARHGEF18 is shown to activate RhoA at tight junctions, interacting directly with myosin IIA and regulating tight junction assembly in a spatiotemporal manner, thus controlling long-range intercellular communication in epithelia and collective behavior of epithelial cells.³⁷,⁵³ Here we add a new role for ARHGEF18 by demonstrating its involvement in the mechanosensing machinery of ECs and identifying the underlying molecular mechanisms: ARHGEF18 expression, phosphorylation, and activity are modulated by the level of SS applied to ECs. ARHGEF18 activity is at its highest when ECs are exposed to physiological SS while its expression is at its lowest level. These observations are consistent with the existence of a negative reg-

ulatory loop between ARHGEF18 activity and expression. Indeed, activation of RhoA/Rho kinase signaling has been shown to generate a negative feedback loop on RhoA-GEF expression, including ARHGEF18.⁵⁴ This regulatory mechanism could therefore restrict ARHGEF18-induced RhoA activation by physiological flow, in agreement with the need of a moderate RhoA activity under physiological laminar flow to maintain EC alignment.²⁶

It is well admitted that EC response to laminar flow is mediated by a rise in cytoskeletal tension that increases traction forces and intercellular tension, and promotes the assembly of adherens and tight junctions.⁵⁵,⁵⁶ As a major regulator of cytoskeletal reorganization, the dynamic regulation of RhoA is critical for SS-induced morphological reshaping of ECs.²⁵,²⁶ Our results show that depletion of ARHGEF18 or expression of a non-phosphorylatable catalytically inactive ARHGEF18 mutant impaired EC elongation and alignment with the flow in physiological conditions, thus indicating that ARHGEF18 is an essential component of the regulatory mechanisms that control RhoA activity and collective EC behavior of under flow. However, ARHGEF18 depletion only partially inhibits RhoA activity and flow-induced EC alignment suggesting the participation of other RhoGEFs in flow-induced activation of RhoA. Two other RhoGEFs, Tiam1 and Trio, have been shown to be involved in the flow response of ECs.³¹,³²,⁵⁷ But unlike ARHGEF18, which appears to act on tight junctions through RhoA, Trio and Tiam1 regulate Rac1 activity, thereby stabilizing adherens junctions (Trio)³² and favoring planar polarity (Tiam1).³¹ The concomitant regulation of Trio, Tiam1, and ARHGEF18 in response to shear stress may therefore help to refine the balance between RhoA and Rac1 activity required to fine-tune the EC response to flow. Interestingly ARHGEF18 have been shown to participate in planar polarity in epithelial cells as well and might also act on flow-induced planar polarity.

We showed that activation of ARHGEF18 by physiological flow depends on the SFK YES. This role of YES in ARHGEF18 activation is consistent with the impairment of PSS-induced EC alignment observed when SFK proteins are inhibited⁵⁸ and the recent description of YES as a flow-sensitive protein.⁵⁹ We propose that YES could be the kinase responsible for the phosphorylation and activation of ARHGEF18 in ECs, thereby contributing to the maintenance of endothelial tight junctions and prevention of vascular leakage under physiological condition. This effect of ARHGEF18 on barrier maintenance appears to be specific to its activation by physiological SS, since neither holes nor permeability defects were observed in ARHGEF18-depleted EC monolayers under static conditions. In addition, the loss of the dynamics of hole opening and closing induced by ARHGEF18 depletion under physiological flow highlights a clear role of ARHGEF18 in EC motility and their ability to close opened holes under physiological SS.

Additionally, our data also identified p38 MAPK as a downstream effector of ARHGEF18 on tight junction and EC alignment under physiological SS. ARHGEF18 would act on p38 through the activation of RhoA, as the RhoA/Rho kinase pathway is known to induce p38 phosphorylation.⁶⁰ In addition to RhoA/p38 signaling, other pathways likely contribute to the effect of ARHGEF18 since the intercellular gaps induced by ARHGEF18 depletion were not reproduced by expression of the inactive ARHGEF18 mutant or by p38 inhibition in EC under flow. This
result supports the concept that RhoGEFs could regulate cell
functions through scaffolding properties independent of their
guanine nucleotide exchange activity on RHO proteins.⁶¹,⁶²

In our models, the destabilization of tight junctions and junc-
tional actin, and the associated increase in adherent junction ac-
tivity in ARHGEF18-deficient ECs, points toward a role of
ARHGEF18 in mediating cell-cell tension arising from flow. This
is consistent with the observed co-localization of ARHGEF18
with the tight junction proteins ZO-1 and claudin-5. Interestingly,
ZO-1 has been recently shown to regulate tension on endothelial
cell-cell contacts, in part via the recruitment of ARHGEF18 to a
junctional protein complex.⁶³ Here we show that depletion of
ARHGEF18 both *in vitro* and *in vivo* or expression of an inactive
ARHGEF18 mutant compromised junctional recruitment of
ZO-1, providing evidence that ARHGEF18 activity is mandatory
to bring ZO-1 at tight junction and that ARHGEF18 and ZO-1
reciprocally need each other to localize at the junctional protein
complex. Although ARHGEF18 does not co-localize with VE-
cadherin, ARHGEF18 depletion also affects adherens junctions,
without altering VE-cadherin localization. This last result con-
firms that ARHGEF18 is a key component of the molecular com-
plex through which tight junction can indirectly regulate adhe-
rens junctions, as reported in ECs under static conditions.⁶³
Interestingly, loss of ARHGEF18 not only disrupts tight junction
under physiological SS but also impairs focal adhesion formation
and organization, supporting the existence of a cross-talk be-
tween tight junction and focal adhesion. Cell-cell junction and
matrix adhesion are two processes highly interrelated and
depend on the activation of multiple RhoGEFs and RhoGT-
Pases.⁶⁴,⁶⁵ We reported ARHGEF18 as one of the contributors.
the first described to be able to act on both basal adhesion
and cell-cell junctions in ECs.

Since the generation and the transmission of force at cell-cell
junctions and focal adhesions are important for the assembly
and the distribution of adhesive complexes and junctions, this
observation provides further evidence that the maintenance of
junctional tension is an important function of ARHGEF18. This ef-
fect is likely to be the cause of the significant alteration of EC
migration under flow, both in speed and directionality, induced
by ARHGEF18 depletion *in vitro*. In *Arhgef18*-iEC-KO mice
*in vivo*, this defect in migration could be partly responsible for
the reduction in radial expansion and vessel density of the retinal
vasculature.

In mice, complete deletion of ARHGEF18 resulted in embryonic
lethality.⁶⁶ *Arhgef18* null embryos showed reduced vasculature,
increased hemorrhage and edema, which is in line with our obser-
vation. Our mouse model, harboring EC-specific deletion of
ARHGEF18 after birth, presents bleedings in the retina and the
brain associated with a loss of ZO-1 at endothelial junctions. In
the aortas, we also observed a decrease in ZO-1 expression asso-
ciated with its exclusion from junction. We did not observe spon-
taneous micro-hemorrhages outside the central nervous system
but we found vascular leakage in the kidney and in the mesenteric
arteries, two territories exposed to high physiological flow. This
vascular phenotype of *Arhgef18*-iEC-KO confirms *in vivo* the
role of ARHGEF18 in the maintenance of tight junctions and EC
alignment under physiological laminar flow. While not specific to
the cerebral vasculature, the effect of ARHGEF18 deletion is

mainly visible in this territory, potentially due to the high level of
claudin 5 expression in cerebral ECs and crucial role of tight junc-
tions in the maintenance of blood brain barrier.

In humans, a variant of the *ARHGEF18* gene has been shown
to be associated with systemic capillary leak syndrome,⁶⁷ a rare
disorder characterized by repeated, transient, unprovoked epi-
sodes of hypotensive shock and peripheral edema due to a tran-
sient endothelial hyperpermeability, consistent with the role of
ARHGEF18 identified in this study. Mutations in *ARHGEF18*
have been also reported in inherited retinal dystrophy, character-
ized by retina degeneration affecting photoreceptors, but pa-
tients carrying ARHGEF18 mutations also displayed attenuated
retinal vessel density, which could be in line with a primary effect
of those mutations on vascular ECs.⁶⁸

In summary, ARHGEF18 is a novel mechanosensitive RhoGEF
activated by physiological flow that plays a central role in the
regulation of tight junction, thereby driving the physiological
alignment of ECs and the endothelial permeability.

## Limitations of the study

This work described the role of ARHGEF18 in ECs. However,
staining of endogenous ARGHEF18 in EC *in vitro* was not ob-
tained due to technical limitations that we could not overcome.
We have circumvented this by expressing a tagged version of
ARHGEF18, but having the dynamics of ARHGEF18 localization
under flow would be critical in understanding exactly how it pro-
motes tight junction maintenance.

While the effect of flow on the expression, phosphorylation
and activity of ARHGEF18 is clear in our study, the direct kinase
activity of YES on ARHGEF18 needs to be confirmed as well as
how physiological SS control YES kinase activity, although this
kinase has been described as flow-sensitive by others.⁵⁹

We have shown in this study that ARHGEF18 directly controls
RhoA activity and indirectly controls Rac1 activity. We did not
investigate the possible role of ARHGEF18 on other Rho proteins
such as RhoG, also known to participate in migration and tight
junctions.⁶⁹,⁷⁰ In addition, although we observed a strong effect
of ARHGEF18 deletion on focal adhesion formation, we did not
identify the mechanism by which ARHGEF18 can modulate
both cell-cell and cell-matrix interactions.

Finally, since this study shows the role of ARHGEF18 in con-
trolling physiological function, i.e., the maintenance of tight junc-
tions in arterial territories, it would be interesting to take this work
further and evaluate the regulation of ARHGEF18 in vascular dis-
eases and its contribution to endothelial dysfunction in hyperten-
sion, atherosclerosis, or intracranial aneurysm.

## RESOURCE AVAILABILITY

**Lead contact**

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Dr Anne-Clémence Vion, <anne.clemence.vion@univ-nantes.fr>.

**Materials availability**

* This study did not generate new unique reagents.

* All unique/stable reagents and mouse lines generated in this study are available from the lead contact with a completed materials transfer agreement.
## Data and code availability

* Data: 3'SRP data have been deposited at the European Nucleotide Archive portal and are publicly available as of the date of publication. Accession numbers are listed in the **key resources table**.

* Code: 3'SRP pipeline codes are publicly available at https://bio.tools/3SRP as of the date of publication. Accession numbers are listed in the **key resources table**.

* Other items: Any additional information required to reanalyze the data reported in this paper is available from the **lead contact** upon request.

## ACKNOWLEDGMENTS

We thank Dr. Sauzeau and Dr. Quillard for helpful comments on the manuscript. We are most grateful to the genomics (GenoA), bioinformatics (BiRD), and microcoscopy (MicroPICell) core facilities (SFR Bonamy, Nantes, France), members of Biogenouest and IBISA, for their expert services. We also thank the Institut Français de Bioinformatique funded by the French National Agency (ANR) (ANR-11-INBS-0013) and the national infrastructure France-Bioimaging (ANR-10-INBS-04), as well as the animal house facility (UTE) of Nantes Université. This work was supported by ANR grants Programme d'Investissements d'Avenir ANR-16-IDEX-0007 (NeXT Initiative), ANR-21-CE14-0016 (to A.-C.V.) and the local fund Genavie (to S.P.R.B.).

## AUTHOR CONTRIBUTIONS

Conceptualization, A.-C.V.; methodology, A.-C.V.; formal analysis, A.-C.V. and S.P.R.B.; investigation, M.R., C.L., C.B.-M., R.M., A.-C.V., S.P.R.B., I.B., and M.B.; data curation, A.-C.V.; writing – original draft, A.-C.V. and S.P.R.B.; writing – review & editing, A.-C.V. and G.L.; visualization, A.-C.V. and S.P.R.B.; supervision, A.-C.V.; funding acquisition, A.-C.V.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* **KEY RESOURCES TABLE**

* **EXPERIMENTAL MODEL AND SUBJECT DETAILS**

* Mouse strains

* Mammalian cell lines

* **METHOD DETAILS**

* *In vivo* permeability assay

* *Ex vivo* permeability assay

* Microfluidic chamber experiments

* Mice EC isolation and tamoxifen treatment *in vitro*

* 3'Sequencing RNA profiling

* Generation of lentiviral vectors

* Lentivirus production, transduction and induction of gene of interest vectors

* Pulldown assay

* Immunoprecipitation and Co-immunoprecipitation assay

* Micropatterning assay

* Western blotting

* *In vitro* immunofluorescence staining

* *In vivo* immunofluorescence staining

* Impedance-based assay

* *In vitro* permeability assay

* Wound healing assay

* Proliferation assay

* Live imaging experiments

* Microscope image acquisition

* **QUANTIFICATION AND STATISTICAL ANALYSIS**

* Flow-induced orientation analysis

* Cell junction activity analysis

* Basal adhesion analysis

* Live imaging analysis

* Statistical analysis

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2025.115288.

Received: October 26, 2023

Revised: November 20, 2024

Accepted: January 17, 2025

Published: February 19, 2025

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>anti-ARHGEF18</td><td>Genetex</td><td>Cat# GTX102223, RRID:AB_1949683</td></tr><tr><td>anti-ARHGEF18</td><td>HPA</td><td>Cat# HPA071867, RRID:AB_2686466</td></tr><tr><td>anti-Akt</td><td>CST</td><td>Cat# 9272, RRID:AN_329827</td></tr><tr><td>anti-phospho-Akt</td><td>CST</td><td>Cat# 9271, RRID:AB_329825</td></tr><tr><td>anti-Claudin5</td><td>Invitrogen</td><td>Cat# 34-1600, RRID:AB_2533157</td></tr><tr><td>anti-Claudin5</td><td>Abnova</td><td>Cat# MAB19903, RRID:AB_3675832</td></tr><tr><td>anti-ERG</td><td>abcam</td><td>Cat# ab92513, RRID:AB2630401</td></tr><tr><td>anti-ERK1/2</td><td>CST</td><td>Cat# 4695, RRID:AB_390779</td></tr><tr><td>anti-phospho-ERK1/2</td><td>CST</td><td>Cat# 9101, RRID:AB_331646</td></tr><tr><td>anti-FLAG</td><td>Merck</td><td>Cat# F1804, RRID:AB_262044</td></tr><tr><td>anti-GAPDH</td><td>MerckMillipore</td><td>Cat# MAB374, RRID:AB_2107445</td></tr><tr><td>anti-GEF-H1</td><td>CST</td><td>Cat# 4076, RRID:AB_2060032</td></tr><tr><td>anti-GST</td><td>Millipore</td><td>Cat# PC53-100UG, RRID:AB_213827</td></tr><tr><td>anti-paxillin</td><td>abcam</td><td>Cat# ab32084, RRID:AB_779033</td></tr><tr><td>anti-paxillin (phospho Y118)</td><td>abcam</td><td>Cat# ab194738, RRID:AB_3675833</td></tr><tr><td>anti-p38</td><td>CST</td><td>Cat# 9212, RRID:AB_330713</td></tr><tr><td>anti-p38 (phospho Thr180/Tyr182)</td><td>CST</td><td>Cat# 9211, RRID:AB_331641</td></tr><tr><td>anti-p115-RhoGEF</td><td>CST</td><td>Cat# 3669, RRID:AB_2059739</td></tr><tr><td>anti-Rac1</td><td>CST</td><td>Cat# 2465, RRID:AB_2176152</td></tr><tr><td>anti-RhoA</td><td>CST</td><td>Cat# 2117, RRID:AB_10693922</td></tr><tr><td>anti-SM22</td><td>abcam</td><td>Cat# ab14106, RRID:AB_443021</td></tr><tr><td>anti-TER119</td><td>R&amp;D</td><td>Cat# MAB1125, RRID:AB_2297123</td></tr><tr><td>anti-phospho-Tyr</td><td>CST</td><td>Cat# 9411, RRID:AB_331228</td></tr><tr><td>anti-Vav</td><td>abcam</td><td>Cat# ab92890, RRID:AB_10562279</td></tr><tr><td>anti-VE-cadherin</td><td>R&amp;D</td><td>Cat# AF938, RRID:AB_355726</td></tr><tr><td>anti-VE-cadherin</td><td>R&amp;D</td><td>Cat# AF1002, RRID:AB_2077789</td></tr><tr><td>anti-ZO-1</td><td>Thermo Fisher Scientific</td><td>Cat# 61-7300, RRID:AB_2533938</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>p38 specific inhibitor (SB239063)</td><td>TOCRIS</td><td>Cat. No. 1962/10</td></tr><tr><td>SRC inhibitor I</td><td>TOCRIS</td><td>Cat. No. 3642</td></tr><tr><td>SU6656</td><td>TOCRIS</td><td>Cat. No. 6475/5</td></tr><tr><td>polybrene</td><td>Sigma-Aldrich</td><td>H9268</td></tr><tr><td>puromycin</td><td>Sigma-Aldrich</td><td>P8833</td></tr><tr><td>doxycycline</td><td>Sigma-Aldrich</td><td>D9891</td></tr><tr><td>gelatin</td><td>Sigma-Aldrich</td><td>G1393</td></tr><tr><td>Isolectin B4 488</td><td>life technologies</td><td>I21411</td></tr><tr><td>Phalloidin Alexa 488</td><td>life technologies</td><td>A12379</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>10236276001</td></tr><tr><td>fluorescent dextran dyes 3 kDa</td><td>Thermofisher</td><td>D3329</td></tr><tr><td>fluorescent dextran dyes 70 kDa</td><td>Thermofisher</td><td>D1540</td></tr><tr><td colspan="3"><strong>Critical commercial assays</strong></td></tr><tr><td>Edu-click647</td><td>Base Click</td><td>BCK-EdU647</td></tr><tr><td>Q5 site directed mutagenesis kit</td><td>NEB</td><td>E0554</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Deposited data</strong></td></tr><tr><td>3'SRP data</td><td>Accession numbers: PRJEB83681</td><td>N/A</td></tr><tr><td>codes</td><td>https://bio.tools/3SRP</td><td>N/A</td></tr><tr><td>Hiseq Rapid SBS kit v2 50 cycles</td><td>illumina</td><td>#FC-402-4022</td></tr><tr><td colspan="3"><strong>Experimental models: Cell lines</strong></td></tr><tr><td>HUVEC</td><td>Promocell</td><td>Cat.No.C-12203</td></tr><tr><td colspan="3"><strong>Experimental models: Organisms/strains</strong></td></tr><tr><td>Mouse: C57/BL6J, Arhgef18<sup>lox/lox</sup></td><td>derivated from Arhgef18<sup>tm1a(KOMP)Mbp</sup>from the MRC</td><td>N/A</td></tr><tr><td>Mouse: CDH5(PAC)-CreERT2</td><td>provided by Pr R.Adams</td><td>N/A</td></tr><tr><td>Mouse: CDH5(PAC)-CreERT2; Arhgef18<sup>lox/lox</sup></td><td>in house</td><td>N/A</td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td>Arhgef18_Fw</td><td>ACAGCTCTGCGATCGCCACCATGACGG TCTCTCAGAAAGGG</td><td>N/A</td></tr><tr><td>Arhgef18_Rev</td><td>GCTGTCTCGAGAATTAAGAAGAAGATGA CGTCTTCTTTGC</td><td>N/A</td></tr><tr><td>ORF18-Oligo1</td><td>ATGACGGTCTCTCAGAAAGGG</td><td>N/A</td></tr><tr><td>ORF18-Oligo2</td><td>GTACGTCGGTCAGCAGGATAG</td><td>N/A</td></tr><tr><td>ORF18-Oligo3</td><td>CTCACCTTCCGCAAGGAAGAC</td><td>N/A</td></tr><tr><td>ORF18-Oligo4</td><td>GAAGTTGCGTTGCCGCTCCTG</td><td>N/A</td></tr><tr><td>ORF18-Oligo5</td><td>CCCACCAGGACAGCTATGTG</td><td>N/A</td></tr><tr><td>ORF18-Oligo6</td><td>GAAGAAGATGACGTCTTCTTTGCTGG</td><td>N/A</td></tr><tr><td>shNT sens</td><td>CAAATCACAGAATCGTCGTAT</td><td>N/A</td></tr><tr><td>shNT Antisense</td><td>ATACGACGATTCTGTGATTTG</td><td>N/A</td></tr><tr><td>shArhgef18.1 sens</td><td>GGCCACAATGAAGCTGTTAGT</td><td>N/A</td></tr><tr><td>shArhgef18.1 Antisense</td><td>ACTAACAGCTTCATTGTGGCC</td><td>N/A</td></tr><tr><td>shArhgef18.2 sens</td><td>CAAAACTTGATCAAGAAAATT</td><td>N/A</td></tr><tr><td>shArhgef18.2 Antisense</td><td>AATTTTCTTGATCAAGTTTTG</td><td>N/A</td></tr><tr><td>shArhgef18.3 sens</td><td>CGGGCTACGACTGCACAAACA</td><td>N/A</td></tr><tr><td>shArhgef18.3 Antisense</td><td>TGTTTGTGCAGTCGTAGCCCG</td><td>N/A</td></tr><tr><td>shArhgef18.4 sens</td><td>AAGACAAGATGTCCTTTATGA</td><td>N/A</td></tr><tr><td>shArhgef18.4 Antisense</td><td>TCATAAAGGACATCTTGTCTT</td><td>N/A</td></tr><tr><td>shArhgef18.5 sens</td><td>CGATTTTATTTGTAAAGTTGA</td><td>N/A</td></tr><tr><td>shArhgef18.5 Antisense</td><td>TCAACTTTACAAATAAAATCG</td><td>N/A</td></tr><tr><td>shArhgef18.6 sens</td><td>GAGCAAATGTTCCTATTTTCG</td><td>N/A</td></tr><tr><td>shArhgef18.6 Antisense</td><td>CGAAAATAGGAACATTTGCTC</td><td>N/A</td></tr><tr><td colspan="3"><strong>Software and algorithms</strong></td></tr><tr><td>Fiji (ImageJ)</td><td>GraphPad</td><td>N/A</td></tr><tr><td>Prism 10</td><td>GraphPad</td><td>N/A</td></tr></tbody></table>

## EXPERIMENTAL MODEL AND SUBJECT DETAILS

### Mouse strains

Mice were maintained at the “unité thérapeutique experimental” of Nantes university under standard husbandry conditions. The following mouse strains were used: C57/BL6J, Arhgef18<sup>lox/lox</sup> (generated from mice carrying the conditional knockout first Arhgef18<sup>tm1a(KOMP)Mbp</sup> from the MRC), CDH5(PAC)-CreERT2 (Kindly provided by Pr R.Adams). To generate endothelium specific Arhgef18-knock-out (KO) mice, we mated Arhgef18<sup>lox/lox</sup> to CDH5(PAC)-CreERT2 transgenic mice to generate mice containing both the CDH5(PAC)-CreERT2 allele and Arhgef18 floxed alleles (CDH5(PAC)-CreERT2; Arhgef18<sup>lox/lox</sup> mice: Arhgef18-iEC-floxed mice). Cre-mediated deletion in EC (Arhgef18-iEC-KO mice) was induced by tamoxifen (T5648, Sigma) treatment (intraperitoneal injection of 20 µg/g animal at postnatal days 1, 2 and 3). Littermates tamoxifen-treated Arhgef18<sup>lox/lox</sup> mice were used as controls.
Validation of non-effect of the tamoxifen alone was done on CDH5(PAC)-CreERT2 mice for key experiments (bleeding in the retinas). Retinas, brain, lungs, and aortas were collected at postnatal day 6 (P6), 12 (P12) or at 4 week-old (4WO) onwards. Animal sex was not determined as the animals were used before weaning. The investigators were blinded to allocation during experiments. Animal procedures were performed in accordance with the animal license APAFIS #33434-2021101214003205.

## Mammalian cell lines

Human Umbilical Vein Endothelial Cells (HUVECs) (passage 2 to 6; PromoCell), chosen for their accessibility and well characterized response to flow, were routinely cultured in EBM media supplemented with the provided growth factors kit (Promocell) at 37°C, 5% CO₂. Sex of the cells was mixed as we used vial from pooled donors.

## METHOD DETAILS

### In vivo permeability assay

Arhgef18-iEC-KO pups (P6) and their littermate controls were injected intraperitoneally with Evans Blue (1%). Twenty minutes after injection, pups were sacrificed, and an intracardiac perfusion was performed with heparinated PBS to remove intravascular Evans Blue. Organs (liver, lung, kidney) were collected and weighed. Evans Blue was then eluted in formamide (2h at 55°C, ON at RT). After centrifugation, the absorbance was measured at 620 nm using a spectrophotometer and normalized to the organ weight (g).

### Ex vivo permeability assay

Mesenteric arteries of Arhgef18-iEC-KO pups (P7) and their littermate controls were dissected and mounted in a video-monitored perfusion system (Living Systems Instrumentation) as previously described Briefly, arterial segment was cannulated on 2 glass micropipettes, using nylon monofilaments to secure both ends and bathed in a 5-mL chamber containing a physiological salt solution maintained at a pH of 7.4, a $pO_2$ of 160 mmHg, and a $pCO_2$ of 37 mmHg. Flow was controlled by a servo-perfusion system. After 5min of PSS perfusion, a solution of dextran dyes (70kD and 3kD, 1/2000 in physiological salt solution) were perfused intraluminal for 30min, followed by a wash with physiological salt solution only for 30min. arteries were then fixed in PFA 4% and directly observed on a Nikon microscope.

### Microfluidic chamber experiments

For flow experiments dedicated to pull-down, co-immunoprecipitation, or WB lysate collection, cells were cultured on 0.2% gelatin-coated slides (Menzel Glazer) and submitted to unidirectional laminar SS using peristaltic pumps (Gilson) connected to a glass reservoir (ELLIPSE) and the chamber containing the slide for 24h. For flow experiments dedicated immunofluorescence staining and live imaging, cells were cultured on 0.2% gelatin-coated 0.4 ibidi slides (IBIDI) and unidirectional laminar SS was applied using the pumping system and control unit form IBIDI. Local SS was calculated using Poiseuille's law and averaged to 3.6 dyn/cm² (pathological low SS: LSS) 16 dyn/cm² (physiological SS: PSS) or 36 dyn/cm² (pathological high SS: HSS).⁷¹⁻⁷³ For inhibition studies, confluent HUVEC were treated with either the p38 specific inhibitor for 1h prior to physiological SS, or the SFK inhibitors for 3h prior to physiological SS. Treatment was maintained during the flow stimulation.

### Mice EC isolation and tamoxifen treatment in vitro

Brain endothelial cells, isolated from *arhgef18f/f* (Control) or *arhgef18f/f*; Cdh5-CreERT2 mice cortical gray matter, were obtain through collagenase dispase digestion and BSA and Percoll density gradient centrifugation. Purified vessels were seeded onto collagen IV/fibronectin-coated dishes at high density (3 brain for 4cm²). Cells were then grown in EGM2-MV (promocell) with 5 µg/mL puromycin during the first 5 days.⁷⁴ Brain ECs were then treated with hydroxy-tamoxifen (1ug/mL) for 3 days to induce recombination.

## 3'Sequencing RNA profiling

**Experimental protocol:** The protocol was performed according to the 3'-digital gene expression (3'-DGE) approach developed by the Broad institute as described in https://doi.org/10.21203/rs.3.pex-1336/v1. The libraries were prepared from 10 ng of total RNA in 4µL. The mRNA poly(A) tails were tagged with universal adapters, well-specific barcodes and unique molecular identifiers (UMIs) during template-switching reverse transcription. Barcoded cDNAs from multiple samples were then pooled, amplified and tagmented using a transposon-fragmentation approach which enriches for 3'ends of cDNA: 200ng of full-length cDNAs were used as input to the Nextera DNA Flex Library Prep kit (ref #20018704, Illumina) and Nextera DNA CD Indexes (24 Indexes, 96 Samples) (ref #20018707, Illumina) according to the manufacturer's protocol (Nextera DNA Flex Library Document, ref #1000000025416 v04, Illumina). Size library was controlled on 2200 Tape Station Sytem (Agilent Technologies). A library of 350-800 bp length was run on a HiSeq 2500 using Hiseq Rapid SBS kit v2 50 cycles (ref #FC-402-4022).

**Bioinformatics protocol:** Raw fastq pairs match the following criteria: the 16 bases of the first read correspond to 6 bases for a designed sample-specific barcode and 10 bases for a unique molecular identifier (UMI). The second read (104 bases) corresponded to the captured poly(A) RNAs sequence.
Bioinformatics steps were performed using a snakemake pipeline (https://bio.tools/3SRP). Samples demultiplexing was performed with a python script. Raw paired-end fastq files were transformed into a single-end fastq file for each sample. Alignment on refseq reference transcriptome, available from the UCSC download site, was performed using bwa. Aligned reads were parsed and UMs were counted for each gene in each sample to create an expression matrix containing the absolute abundance of mRNAs in all samples. Reads aligned on multiple genes or containing more than 3 mismatches with the reference were discarded. The expression matrix was normalized and differentially expressed genes (DEG) were searched using the R package deseq2.⁷⁵

## Generation of lentiviral vectors

For RNA interference, the sequence for non-target shRNA was previously described (see key resource tables). For Arhgef18, specific sense and antisense DNA sequences were identified using free online services (http://sidirect2.rnai.jp/; http://web.stanford.edu/group/markkaylab/cgi-bin/).⁷⁶ The sequences that met the optimal criteria were selected and synthesized as oligos in the form of miR-E backbone. The oligos were then amplified, cloned into LT3GEPIR and sequenced as previously described. The resultant lentiviral vector drives the expression of shRNA along with EGFP under TRE3G promoter, a doxycycline inducible promoter. For Arhgef18 overexpression constructs, Arhgef18 was amplified from HUVEC cDNA by overlap-extension PCR using the oligos and primers in key resource tables. The resultant amplicon was digested and clone into LT3(AsiSI)N1-GPIR (modified LT3GEPIR) vector in-between AsiSI and EcoRI restriction sites. The resultant vector LT3N1-GPIR-Arhgef18 encodes Arhgef18-EGFP fusion protein under TRE3G promoter (WT-ARHGEF18). GEF mutant version of Arhgef18 was generated by site directed mutagenesis of tyrosine to alanine at amino acid position 606 of the canonical sequence (Q6ZSZ5-4 isoform) using primers A18-Y606A-SDM-Fr (5'-CATAAC CAAAGCCCCAGTGCTGGTG-3'), A18-Y606A-SDM-Rev (5'-CGTTGTGTAACCAGGAGAATG-3') and Q5 site directed mutagenesis kit (NEB E0554). This mutation corresponds to the Y260A mutation described by Terry and colleague³⁷ and Acharya and colleague⁷⁷ based on the Q6ZSZ5-1 isoform sequence. This LT3N1-GPIR-Arhgef18 vectors were further modified to substitute EGFP with 3XHA-FLAG tag, shArhgef18.6 to target endogenous Arhgef18 and P2A sequence to replace IRES using standard molecular cloning techniques. The resultant vector, Arhgef18-HA/FLAG_shArhgef18.6, is designated as ARHGEF18Y606A in the main text.

## Lentivirus production, transduction and induction of gene of interest vectors

Lentivirus vector carrying either the shRNA or Arhgef18 ORF was transfected into HEK293T cells along with packaging vectors psPAX2 and pVSVG2 (provided by Prof. Dr.Utz Fischer's lab) using polyethylenimine (764965; Sigma-Aldrich) transfection agent. After overnight incubation, the medium was replaced with fresh medium (DMEM+10%serum+1%PenStrep). 48h and 72h after transfection, viral supernatant was collected, filtered through 0.22 µm filter and used to infect HUVECs (passage 2) in the presence of polybrene (8 µg/mL). The transduced HUVECs were selected with puromycin (8 µg/ml) for 48h and maintained in the presence of puromycin (1µg/ml). The induction of either shRNA or full length ARHGEF18 was performed at least 2 days before the experiment by supplementing the medium with doxycycline (1 µg/ml) and maintained throughout the experimental procedure.

## Pulldown assay

Pulldown of active small GTPases (RHOA/RAC) or active ARHGEF18 was done as previously described.⁷⁸ Briefly, HUVECs (WT, shRNA or A18-Y606A) subjected to various SS conditions for indicated time, were lysed in either RBD lysis buffer (50 mM Tris-HCl (pH 7.6), 500 mM NaCl, 1% (vol/vol) Triton X-100, 0.1% (wt/vol) SDS, 0.5% (wt/vol) deoxycholate and 10 mM MgCl2) or GEF lysis buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% (vol/vol) Triton X-100, 1 mM DTT) supplemented with protease and phosphatase inhibitor cocktail. Cell debris was removed by centrifugation and equal volumes of cell lysate was incubated with 25-50 µg of either GST-RBD/PBD for active small-GTPases; or GST tagged RHOA<sup>G17A</sup>/RAC1<sup>G15A</sup> for active GEF pulldown for 45 min. After incubation, beads were washed three time in respective wash buffers. Captured proteins were detected using Western Blot.

## Immunoprecipitation and Co-immunoprecipitation assay

HUVECs subjected to various SS conditions for indicated time, were lysed in IP lysis buffer (1% Nonidet P-40, 25 mM Tris-HCl pH 7.4, 100 mM NaCl, 10 mM MgCl2, 10% glycerol) supplemented with protease and phosphatase inhibitor cocktail. Lysis buffer was homogenized passing through a 32-gauge syringe needle and cellular debris were removed by centrifugation (5 min, 10000 rpm). A small amount of the lysate was collected in order to assess the presence of the protein in the total cell lysate. Beads were first equilibrated in IP buffer and then coated with 0.5-1 µg of the corresponding antibodies. Cell lysates were then incubated with the coated beads, supernatants and immunoprecipitants (washed 3 times) were stored to proceed for Western blot.

## Micropatterning assay

Pattern shape was designed with FIJI software to have an L shaped pattern 50 × 50 µm long and 6 µm large. PDMS stencils were placed in IBIDI wells and cleaned in a Plasma cleaner for 5 min. A hydrophobic treatment was done using PLL PEG (Poly-lysine Polyethylene glycol 100µg/ml) for 30min. Patterns were then prepared using the Primo device (Alveole). Briefly, the wells with PLPP (photoinitiator, Alveole) were placed under a microscope with the PRIMO device. Focus was adjusted to UV reflection and the patterning sequence was launched with 1200 mJ/mm² UV exposure. Once patterned, wells were profusely washed with PBS and then coated with fibronectin (10µg/ml). Six hundred cells were seeded per well for 1 h, then washed and the remaining cells were left to adhere for 12 h.
## Western blotting

HUVECs were washed with cold PBS and scraped off in NETF buffer supplemented with protease and phosphatase inhibitors. Lysates were centrifuged and protein supernatant was quantified using the Lowry protein assay (Bio-Rad). Lysates were reduced in lamelli buffer and electrophoresis was performed using precast SDS-PAGE gels (Biorad) and transferred on to PVDF membranes (Biorad). Equal loading was checked using Ponceau red solution. Membranes were then blocked in 5% milk and probed with primary antibodies over-night (see below) and respective secondary antibodies (1:3000). The membranes were developed using ECL (Biorad) using the Chemidoc imaging system (Biorad). After initial immunodetection, membranes were re-probed with anti-GAPDH antibody. The protein bands were quantified using fiji and expressed as a relative value to either GAPDH or as the ratio of phosphorylated to total protein.

## In vitro immunofluorescence staining

HUVECs were fixed with 4% PFA in PBS for 10 min at room temperature (RT). Blocking/permeabilization was performed using blocking buffer consisting of 5% BSA (Sigma-Aldrich), 0.5% Triton X-100 (Sigma-Aldrich), 0.01% sodium deoxycholate (Sigma-Aldrich), and 0.02% sodium azide (Sigma-Aldrich) in PBS at pH 7.4 for 45 min at RT. Primary antibodies were incubated at the desired concentration in 1:1 Blocking buffer/PBS at RT for 2 h and secondary antibodies were incubated at the desired concentration in 1:1 blocking buffer/PBS for 1 h at RT (Donkey anti-Rabbit (life tech, 1/500), Donkey anti-Rat (life tech, 1/500), Donkey anti-Goat (life tech, 1/500), Donkey anti-Mouse (life tech, 1/500)). DAPI (Sigma-Aldrich, 1/10000, 5 min) was used for nuclear labeling and phalloidin 488 (life technologies, 1/500, 45min) was used to labeled polymerized actin.

## In vivo immunofluorescence staining

Retinas and Aortas were fixed with 4% PFA in PBS overnight at 4°C. Aortas were open "en face" before staining. Brains were sliced using a vibratome (100μM section). Blocking/permeabilization was performed using blocking buffer consisting of 1% FBS, 3% BSA (Sigma-Aldrich), 0.5% Triton X-100 (Sigma-Aldrich), 0.01% sodium deoxycholate (Sigma-Aldrich), and 0.02% sodium azide (Sigma-Aldrich) in PBS at pH 7.4 for 1 h at RT. Primary antibodies were used at the desired concentration in 1:1 Blocking buffer/PBS overnight at 4°C and secondary antibody were incubated at the desired concentration in 1:1 blocking buffer/PBS for 2 h at RT (Donkey anti-Rabbit (life tech, 1/500), Donkey anti-Rat (life tech, 1/500), Donkey anti-Goat (life tech, 1/500)). DAPI was used for nuclear labeling and Isolectin B4 488 was used to labeled endothelial cells. Retinas were mounted in Mowiol.

## Impedance-based assay

Cell adhesion assay was performed using xCELLigence RTCA instrument (Roche). Briefly, HUVECs (P4-P6) expressing either shNT (non-target), sh18.1 or sh18.4 were plated in E-plate 96 well (5232368001; Agilent) at a density of 20,000cells/well. Cell adhesion was measured as electrical impedance between electrodes and represented as cell index value over a period of time.

## In vitro permeability assay

HUVECs were plated onto gelatin (0.2%, G1393; Sigma-Aldrich) coated 0.4mm trans-well inserts (353095; Corning). Once the cells formed a confluent monolayer, fluorescent dextran dyes (3 kDa, and 70 kDa) were added to the top chamber (Conc. 1 mg/ml) and medium from lower chamber was collected at different time points. Fluorescence was measured using Varioskan LUX (Thermofisher; Excitation at 595nm and emission at 615nm for 3kDA dextran; Excitation at 570nm and emission at 590nm for 70kDa dextran).

## Wound healing assay

Confluent monolayer of HUVECs were scratched using a pipette tip. Cell debris were washed and the medium was replaced with fresh complete medium. Cell migration was video-recorded under optimal conditions (37°C and 5%CO₂) using an inverted Ti Eclipse microscope equipped with a 10x/0.25 phase contrast objective and a DS-QI2 CMOS camera (Nikon France, Champigny Sur Marne). Images were recorded every 5 min during 24h. Wound area was manually measured at different time points using Fiji and rate of wound closure was calculated by area relative to time.

## Proliferation assay

Proliferation assay was performed using the Edu-click647 kit (BCK-EdU647; Base Click). HUVECs expressing the shRNAs were plated in µ-Slide VI 0.4 (80606; IBID) at a density of 30,000cells per channel in starving medium (EBM+0.5%serum+doxy+puro). After 16h, cells were stimulated with complete medium (EBM+2%serum+doxy+puro) in the presence of EdU (5 µM) under static or flow for 8h. Cells were fixed in 4%formaldehyde for 10 min, stained for EdU and nuclei were labeled with DAPI (62248; Thermofisher) according to the kit manual. DAPI+ and EdU+ cells were counted using Fiji.

## Live imaging experiments

Ibidi slides containing ECs exposed to 6h of flow were transferred onto the microscope (Nikon Eclipse Ti inverted equipped a temperature and CO2 control chamber) together with the ibidi pumping system.

Physiological flow (PSS) was maintained for 16h and phase contrast images were taken every 3min.
Microscope image acquisition

Images from mesenteric arteries ex vivo were acquired using a Eclipse Ti inverted (Nikon) equipped with a ELWD Plan-fluor 40x/0.6 objective. Images from fluorescently labeled HUVECs were acquired using a confocal A1 SIM microscope (Nikon) equipped with a Plan-Apochromat 20x/0.8 NA Ph2 objective or with a 60x/1.4 plan apochromat objective; or using an Eclipse Ti2 inverted microscope (Nikon). Images were taken at room temperature using NIS Element software. Images of Retinas and Aortas were taken using an LFOV FLIM inverted confocal microscope (Nikon) equipped with a Plan-Apochromat 20x/0.8 NA Ph2 objective or with a Plan-Apochromat 60x/1.4 NA DIC objective. The microscope was equipped with a photon multiplier tube detector. Images were taken at room temperature using NIS element software (Nikon).

QUANTIFICATION AND STATISTICAL ANALYSIS

Flow-induced orientation analysis

Cell shape was quantified using a dedicated FIJI macro. Basically, VE-Cadherin staining was detected in order to segment the border of each endothelial cell. Based on this segmentation a skeleton was built and aspect ratio (major axis lenght by minor axis lenght, AR) as well as the angle between the flow direction and the main axis was determined for all entire ECs visible on the image. Flow index corresponding to the ratio of AR and the angle was calculated for each EC. Flow index thus integrates both the elongation and the alignment of endothelia cells: The higher the flow index is, the better the cell response to the flow. Based on the VE-cadherin staining, gaps in the endothelial monolayer were quantified manually, only gaps with an area above 100um were taken in account.

Cell junction activity analysis

ZO-1 intensity at the junctions was quantified using FIJI. Briefly, an enlarged mask from the VE-Cadherin segmentation previously described was generated and applied on the ZO-1 staining image, percentage of the ZO-1 staining in this area was extracted for each image. Actin cable formation was measured by the intensity of the phalloidin staining at the junctions using the same enlarged mask applied on the Actin staining image. Both analyses were made automatically by FIJI with identical parameter for mask enlargement and threshold values. Claudin5 localization at junction was quantified using a mix of Wildtype ECs and ECs expressing the shRNA. Using FIJI, intensity plots for both Claudin5 and VE-cadherin staining were obtained by drawing a line of approximately 15 µm in between two cells.

Adherent junction morphology analysis was done in HUVECs or Aortas stained for VE-cadherin using the patch and classified MATLAB code previously developed by Bentley and colleague³⁹ adapted for 2D images. Two status were defined: activated vs. stabilized, and divided into 3 level: from low to high. Activated junctions were defined as serrated or reticular and stabilized as straight think junctions. Each image taken was divided into small pieces of images allowing to visualize only a portion of the junction and these small images were presented blindly and randomly to the user who then classified the junction accordingly. This technique ensured an unbiased analysis of the junctions Irrespective of the treatment and entire shape of the cell.

Basal adhesion analysis

For micropatterning images, paxillin channel was thresholded and the focal adhesion length was quantified for each focal adhesion detected. The focal adhesions were then classified by size within each cell. Quantification was done blindly. For SS exposed ECs, focal adhesion type was assessed qualitatively by classifying blindly (by 2 operators) the overall aspect of Paxillin staining in the cell. Cells with long, numerous and parallel focal adhesions were classified 1 and cells with short, few and disorganized focal adhesions were classified 0.

Live imaging analysis

Endothelial cells were individualized using thresholding of the phase contrast images. Each cell was then tracked using TrackMate plugging in FIJI. Only cells tracked over the entire duration of the movie were used. Cell speed and linearity of its path were extracted from the TrackMate analysis.

Statistical analysis

Statistical analysis was performed using GraphPad Prism software. For in vitro and in vivo experiments, when only 2 conditions were compared, a paired T test or a Wilcoxon T test were used depending on the distribution of the data. For multiple comparison an ordinary one-way ANOVA (data distribution was assumed to be normal) was used, followed by a Tukey test. Details of the statistical test used for each experiment can be found in the figure legends.