β1 integrin is a sensor of blood flow direction

ABSTRACT Endothelial cell (EC) sensing of fluid shear stress direction is a critical determinant of vascular health and disease. Unidirectional flow induces EC alignment and vascular homeostasis, whereas bidirectional flow has pathophysiological effects. ECs express several mechanoreceptors that respond to flow, but the mechanism for sensing shear stress direction is poorly understood. We determined, by using in vitro flow systems and magnetic tweezers, that β1 integrin is a key sensor of force direction because it is activated by unidirectional, but not bidirectional, shearing forces. β1 integrin activation by unidirectional force was amplified in ECs that were pre-sheared in the same direction, indicating that alignment and β1 integrin activity has a feedforward interaction, which is a hallmark of system stability. En face staining and EC-specific genetic deletion studies in the murine aorta revealed that β1 integrin is activated and is essential for EC alignment at sites of unidirectional flow but is not activated at sites of bidirectional flow. In summary, β1 integrin sensing of unidirectional force is a key mechanism for decoding blood flow mechanics to promote vascular homeostasis. This article has an associated First Person interview with the first author of the paper.

. Controls for magnetic tweezers experiments. (A) Beads coated with antibodies targeting b1 integrins remained attached to HUVEC in the presence of magnetic force. HUVECs were loaded with the calcium fluorescent dye Cal-520 and then incubated with beads coated with 8E3, 9EG7, HUTS4 or K20 antibodies. The position of the beads was recorded by fluorescent microscopy before and after the application of unidirectional force (~16 pN) for 2 min. (B) Force application to poly-D lysine coated beads did not activate calcium signalling. HUVECs were loaded with Cal-520 and then incubated with beads coated with poly-D lysine. Beads were exposed to unidirectional force (~16 pN), bidirectional force (1Hz ~16 pN) or no force. Calcium responses were recorded for 3 min using fluorescence microscopy. Representative images are shown. Data were pooled from 5 independent experiments. The median amplitude of the first peak of the calcium response was calculated. Values are shown as means ± SEM.  Figure S3. b3 integrins induce Ca 2+ accumulation in response to unidirectional but not bidirectional shearing force. HUVECs were loaded with Cal-520 and then incubated with beads coated with antibodies targeting β3 integrins (7E3). Beads were exposed to unidirectional force (~16 pN), bidirectional force (1Hz ~16 pN) or no force. Calcium responses were recorded for 3 min using fluorescence microscopy. Representative images are shown. Data were pooled from 5 independent experiments. The median amplitude of the first peak of the calcium response was calculated. Values are shown as means ± SEM and differences were tested using a one-way ANOVA test, with Tukey's test for multiple comparisons. **p<0.01. Figure S4. β1 integrin activation by unidirectional flow is not regulated by TRPV4 or Piezo1. (A) qRT-PCR validation of silencing of Piezo1 and TRPV4. HUVECs were transfected with siRNA targeting Piezo1, TRPV4 or with scrambled (scr) sequences as a control. After 72 h, the expression levels of Piezo1 and TRPV4 mRNA were assessed by qRT-PCR. Mean expression levels +/-SEM is shown. Data were pooled from three independent experiments. Differences between means were analysed using a paired t-test. **P<0.01. (B) HUVECs were transfected with siRNA targeting Piezo1, TRPV4 or with scrambled sequences as a control. After 72 h, cells were exposed to unidirectional flow for 3 min or remained under static conditions. Cells were stained with antibodies targeting active β1 integrins (12G10; green) and DAPI (nuclei; blue). Representative images and 12G10 mean fluorescence values ± SEM are shown. Data were pooled from 3 experiments. Differences were analysed using a one-way ANOVA with Tukey's test for multiple comparisons. **p<0.01. HUVECs were loaded with the calcium fluorescent dye (Cal-520). Some cultures were treated with EGTA to remove calcium from the extracellular media or were left untreated as a control. HUVECs were then exposed to unidirectional or 1 Hz bidirectional flow as indicated and calcium responses were recorded for 3 min using fluorescence microscopy. Data were pooled from 4 independent experiments. The median amplitude of the first peak of the calcium response was calculated. Values are shown as means ± SEM and differences were tested using a two-way ANOVA. *p<0.05, **p<0.01. Figure S7. Examples of calcium response quantitation in single cells. (A) HUVECs were loaded with Cal-520 and exposed to unidirectional flow. Calcium responses were recorded for 3 min using fluorescence microscopy. Using ImageJ software, the mean fluorescent intensity (y axis) for individual cells was measured each second (x axis). Representative images (left panel) and fluorescence data from two cells (right panel) are shown. (B) The peak amplitude for each cell was calculated by deducting the minimum intensity value from the maximum intensity value and then dividing by the minimum intensity value (peak amplitude= (max-min)/min).

Movie 1. Magnetic tweezers displaced paramagnetic beads in suspension.
The ability of the magnetic tweezers system to generate magnetic force was tested by monitoring displacement of suspended superparamagnetic beads. Beads were recorded prior and after the application of unidirectional force (at 2 s).
Movie 2. Molecular dynamic simulations of the aVβ3 integrin exposed to unidirectional force. The ectodomain of the aVβ3 integrin (αV is shown in blue and β3 integrin is shown in red) in which a 200 kJ mol -1 nm -1 force parallel to the membrane was applied on the βA domain of the head region of the inactive form. Water and ions are omitted for clarity. The integrin Calf-2 and β-tail domains were restrained to mimic the cell membrane (see Methods for more details).
Movie 3. Example of calcium response to unidirectional flow. HUVECs were loaded with Cal-520 and exposed to unidirectional flow for 3 min. Calcium responses were recorded for 3 min using fluorescence microscopy. Data shown are representative of 15 independent experiments that gave closely similar results.
Movie 4. EC at the outer curvature of the aortic arch express active β1 integrins at the apical surface. Mouse aortic arches were stained en face with antibodies targeting active β1 integrins (9EG7; red). EC were co-stained using anti-PECAM-1 antibodies (green) and nuclei were counterstained using DAPI (nuclei; blue). Fluorescence was measured at the outer curvature region using super-resolution confocal microscopy. A zseries stack of images was generated and represented in 3D using Imaris software. Note the presence of red signal (active β1 integrins) on the apical (upper) and basal (lower) surfaces.