Shear-induced endothelial cell-cell junction inclination

Abstract

Atheroprone regions of the arterial circulation are characterized by time-varying, reversing, and oscillatory wall shear stress. Several in vivo and in vitro studies have demonstrated that flow reversal (retrograde flow) is atherogenic and proinflammatory. The molecular and structural basis for the sensitivity of the endothelium to flow direction, however, has yet to be determined. It has been hypothesized that the ability to sense flow direction is dependent on the direction of inclination of the interendothelial junction. Immunostaining of the mouse aorta revealed an inclination of the cell-cell junction by 13 degrees in direction of flow in the descending aorta where flow is unidirectional. In contrast, polygonal cells of the inner curvature where flow is disturbed did not have any preferential inclination. Using a membrane specific dye, the angle of inclination of the junction was dynamically monitored using live cell confocal microscopy in confluent human endothelial cell monolayers. Upon application of shear the junctions began inclining within minutes to a final angle of 10 degrees in direction of flow. Retrograde flow led to a reversal of junctional inclination. Flow-induced junctional inclination was shown to be independent of the cytoskeleton or glycocalyx. Additionally, within seconds, retrograde flow led to significantly higher intracellular calcium responses than orthograde flow. Together, these results show for the first time that the endothelial intercellular junction inclination is dynamically responsive to flow direction and confers the ability to endothelial cells to rapidly sense and adapt to flow direction.

Fig. 1.

Schematic of the flow-induced inclination hypothesis of the endothelial cell (EC)-cell junction in the direction of flow. Liu et al. (25) measured an increase in the slope of the trailing-edge membrane (θ₁) and decrease in the slope of the leading-edge membrane (θ₂) at the top of the junction after initiation of flow (gray vs. versus black: flow-unadapted condition). However, their study was limited to surface topology. Here, it was hypothesized that ECs also adapt to steady flow by inclination of their cell-cell junction (θ_3, or the complementary angle θ₄) to reduce the propagation of tension from cell to cell.

Fig. 2.

EC-cell junctions inclined uniformly in direction of flow in regions of laminar steady flow. A: en face tissue sections of the mouse aorta stained with platelet endothelial cell adhesion molecule 1 (PECAM-1) antibody show alignment of ECs with a fusiform shape in the descending aorta. B: a more disordered morphology (polygonal cells) was observed at the inner curvature of the aorta. Scale bars are 20 μm. C: XZ cross sections of the aortic endothelial layer cut longitudinally along the alignment trajectory showed an inclination of the tip of the EC-cell junction in direction of flow (sections I) as compared with transversal sections (perpendicular to flow, sections II) and arbitrary sections of the inner curvature (sections III). D: bar graph depicts EC-cell junction inclination angle mean values for each group. E: polar plots illustrate the distribution of the frequency of the inclination angles (by increment of 5° angles) for each group.

Fig. 3.

EC-cell junctions inclined after onset of flow in confluent human umbilical vein endothelial cells (HUVECs). A: PECAM-1 staining of HUVECs in unsheared condition (static, left) and after 30 min of a 16 dyn/cm² steady shear stress (flow, right). EC-cell junction inclinations are shown on XZ cross sections (bottom). Scale bar is 20 μm. B: bar graph depicts EC-cell junction inclination angle mean values for various magnitudes of shear stress in the range of 8–32 dyn/cm², which did not alter the equilibrium angle of inclination. NS, no significant difference. *P < 0.05. C: polar plots illustrate the distribution of the inclination angles as explained in Fig. 2.

Fig. 4.

Glycocalyx degradation did not alter flow-induced EC-cell junction inclination. HUVECs were subjected to a 16 dyn/cm² steady shear stress for 30 min after 2-h pretreatment with 50 μg/ml heparinase III (Hep). A and B: PECAM-1 immunostaining with XZ cross sections showing inclination of the EC-cell junction in Hep-pretreated cells and control (Ctrl)-untreated experiment. Scale bars are 20 μm. C: removal of heparan sulfate by Hep treatment was confirmed by Western blot. D: EC-cell junction inclination angles for each condition described above.

Fig. 5.

Flow-induced EC-cell junction inclination was independent of cytoskeletal structure. The cytoskeletal structure was disrupted by pretreatment of HUVEC confluent monolayers with 0.1 μM cytochalasin D (CD), 50 μM blebbistatin (Bleb), or 5 μM Taxol for 2 min, 30 min, and 2 h, respectively. Cytoskeleton disruption was verified by staining for phalloidin (A and B) and myosin IIA (C and D) and Taxol-induced inhibition of depolymerization of microtubules by acetylated-tubulin (E and F). Scale bars are 20 μm. Differences in myosin IIA staining were more detectable when using a lower cell seeding density where cytoplasmic retraction was observed (D). However, no cell retraction was observed on confluent monolayers. A wider PECAM-1 membrane staining including increased number of protusions after blebbistatin pretreatment was noticed (insets). G: steady shear stress of 30 min at 16 dyn/cm² did not show any significant difference in EC-cell inclination after cytoskeletal disruption.

Fig. 6.

Dynamic monitoring of the EC-cell junction inclination. A: representation of the inclination angle θ₄ of a range of 12 EC-cell junctions initially inclined in opposite direction of flow (θ₄ < 0), in upright position, or in direction of flow (θ₄ > 0) followed over a time course of 30 min of a 16 dyn/cm² orthograde flow. After 25 min of shear stress, all junctions were considered as flow adapted because they all inclined in the direction of flow. Step retrograde flow immediately after the 30-min time point led to a uniformly rapid reversal of the junctional inclination. XZ sections were acquired every 2 to 5 min over the 45-min period. B: XZ scans illustrating two EC-cell junctions gradually inclining in direction of flow in the first 30 min then suddenly readapting their position to the new flow direction. A representative video of dynamic inclination is provided in the Supplemental Material (Video S2).

Fig. 7.

Step changes of flow direction were associated with higher intracellular calcium (Ca²⁺_i) responses. A: after an initial 30-min period of slowly ramped-up and -down flow, flow-adapted HUVECs were subjected to a 1-min step flow in either forward or reverse direction. Unadapted cells were not subjected to the initial slow pre-shear but were left mounted on the chamber for 30 min with a reduced flow (0.5 dyn/cm² for 1 min) every 10 min to avoid hypoxia. Sudden step shear of 5 dyn/cm² minimally stimulated Ca²⁺_i in both unadapted (gray line, n = 24 cells) and preinclined cells (“orthograde,” dashed line, n = 20 cells). In contrast, dramatic Ca²⁺_i increases were observed when flow was reversed (“retrograde,” solid line, n = 24 cells). Values are means ± SD. *Significant differences of Ca²⁺_i responses in retrograde flow condition as compared with both orthograde flow and unadapted (P < 0.05). For all conditions, experiments were replicated five times with different HUVECs. Data were acquired every 5 s over a 4-min period. B: junctional tension in static (unadapted), orthograde, and retrograde flow. A potential explanation for the sudden increase in Ca²⁺_i responses in retrograde flow can be given by the theory of tension force propagation at the membrane first proposed by Fung and Liu (13). Inclination of the cell-cell junction in flow-adapted cells reduces transmission of tension forces, while a sudden retrograde flow dramatically increases membrane tension from cell to cell. This is likely the basis for the increased permeability of the endothelium in regions of oscillatory flow in the vasculature.