Segundo método de validación: Comprobación de los puntos

Previous studies have shown that hemodynamic shear stress plays a crucial role in the maintaining endothelial barrier function; a reduction in the shear force in several pathological situations lead to increased junction failure (161, 162). Here, we cultured confluent monolayer of human umbilical vein endothelial cells (HUVECs) on collagen gels and studied the cell-cell junction evolution without shear flow. We find that gaps nucleate in the endothelial junctions, subsequently, grow and stabilize, but heal eventually (Figure 5-2a). As shown in Figure 5-2b, the junctions disrupt more frequently at the vertices

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(tricellular junctions) rather than the borders (bicellular junctions). The intercellular gap formed persists over 30 mins (Figure 5-2c). Our quantitative measurement shows that the possibility of observing a disruption (gap) at vertices is approximately 9 times higher than that at borders (Figure 5-2d). Taken together, our data suggest that endothelial cell junctions experience disruptions and subsequent healing without shear flow, and the disruptions occur more frequently at the vertices compared to the borders.

5.3.2 Intercellular cellular force profile determines the sites of initiation of junction disruption

The intercellular force profile of a representative cell in the network obtained using our computational approach is shown in Figure 5-2e. Clearly, the VE-cadherin bonds at the vertices are subject to larger forces compared to the borders. To understand the underlying reason for the presence of large forces at vertices, we examined principle stress induced by cell contraction at the border and the vertex. Since cortical actin has a quasisarcomeric structure along the bicellular junction (border), the contractile stresses generated by the cortical actin act along the borders as shown in Figure 5-2f, left panel. When two neighboring cells have the same levels of contraction, the deformations induced by the two cells are identical, and no forces are transmitted through the VE-cadherin bonds. For a tricellular junction, tensile forces (arrows) generated by cortical actin contraction lead to a ‘tug of war’ at the vertices, resulting in high levels of force (Figure 5-2f, right panel). In a

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levels, the force profile deviates from the ideal case, but even in this case, the tricellular junctions sustain larger forces as shown in Figure 5-2e.

Previous work (163) suggests that catch-bond behavior is responsible for the maturation and reinforcement of junctions, while the slip-bond behavior is the reason for the failure of junctions. Recent experiments (144, 145, 158) on individual cadherin bond have shown that the kinetics of VE-cadherin bonds can be described by a catch-slip model as shown in

Figure 5-1d. This model naturally gives a catch-slip transition force, 𝑓_𝑐𝑠, at which lifetime of the bond reaches its maximum. To understand how disruptions are initiated, we studied the binding probability of under three conditions: low, intermediate and high contraction levels, corresponding to different RhoA levels (represented by the parameter, 𝛼_𝑐𝑝) respectively. The initiation sites of disruption are defined as the locations with low binding probability (<0.25) when all VE-cadherins are connected. When the contraction level is low, the forces on VE-cadherin bonds are much smaller than 𝑓_𝑐𝑠, the VE-cadherins are in the catch regime where smaller force gives lower binding probability (𝑃_𝑏). Since the intercellular forces are lowest at the middle of bicellular junction (Figure 5-2e), the initiation sites of disruptions are found at these positions as shown in Figure 5-2g. When the contraction level is in the intermediate range, the forces on VE-cadherin bonds are close to 𝑓𝑐𝑠, the lifetime and binding probability (𝑃𝑏) reach their maximum levels. Therefore, no disruptions are observed as shown in Figure 5-2g. When the contraction level is high, the forces on VE-cadherin bonds are much higher than 𝑓_𝑐𝑠, the unbinding kinetics fall into the

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slip regime where larger force gives lower binding probability (𝑃_𝑏). Therefore, the disruptions are found to initiate at the tricellular junctions as shown in Figure 5-2g. Our experiments show that the disruptions occur more frequently (Figure 5-2b) and are more likely (9 times higher) at the vertex than the border (Figure 5-2d). Based on our model, the endothelial network studied in the experiments is in the high contractility range (large 𝛼𝑐𝑝) and VE-cadherins are in the slip regime. Hence, we applied the chemomechanical

feedback parameters (𝛼_𝑐𝑝) corresponding to a high level of contraction for the VE- cadherins in the rest of the study.

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Figure 5-2 Gap formation in endothelial cells and estimation of intercellular forces. (a) A snapshot of HUVECs cultured as a confluent monolayer on a thin collagen gel. Three states of a typical triple cell junction (vertex): nucleation, growth and stabilization and healing. (b) The frequency of rupture at vertices is higher compared to borders; (c) the duration of disruption at the vertex and the border (right panel) are similar; mean ± s.d., 𝑛 ≥ 15, ∗

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𝑃 < 0.05. (d) Probability of observing gaps at vertices or at borders. (e) Intercellular force map along the cell-cell junctions of a representative cell showing the triple cell junctions (vertex) bear significantly larger forces compared to two cell junctions (border). (f) Schematics of VE-cadherin bonds experiencing forces generated by identical levels of contraction in all cells. At the triple cell junctions, significant intercellular forces are generated due to the “tug of war” between the boundaries that intersect at the junctions. (g) Initiation sites of disruption for different levels of cytoplasmic contractile states. At low contractility, the cadherins forces are in the catch regime (𝑓 < fcs), and junction disruption initiates at the borders. At intermediate contractility (𝑓~ fcs), no junction disruptions are nucleated. When contractility is high, the cadherin forces are in the slip regime (𝑓 > fcs) and the junction disruption initiates at the vertices.

5.3.3 Junction disruptions nucleate from the vertex and stall when the driving force