ß1 integrin is a sensor of blood flow direction.

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 ß1 integrin is a key sensor of force direction because it is activated by unidirectional, but not bidirectional, shearing forces. ß1 integrin activation by unidirectional force was amplified in ECs that were pre-sheared in the same direction, indicating that alignment and ß1 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 ß1 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, ß1 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.

Pressure and stiffness sensing together regulate vascular smooth muscle cell phenotype switching.

Vascular smooth muscle cells (VSMCs) play a central role in the progression of atherosclerosis, where they switch from a contractile to a synthetic phenotype. Because of their role as risk factors for atherosclerosis, we sought here to systematically study the impact of matrix stiffness and (hemodynamic) pressure on VSMCs. Thereby, we find that pressure and stiffness individually affect the VSMC phenotype. However, only the combination of hypertensive pressure and matrix compliance, and as such mechanical stimuli that are prevalent during atherosclerosis, leads to a full phenotypic switch including the formation of matrix-degrading podosomes. We further analyze the molecular mechanism in stiffness and pressure sensing and identify a regulation through different but overlapping pathways culminating in the regulation of the actin cytoskeleton through cofilin. Together, our data show how different pathological mechanical signals combined but through distinct pathways accelerate a phenotypic switch that will ultimately contribute to atherosclerotic disease progression.

Lamin-A Mechano-Protects the Heart.

In this issue of Developmental Cell, Cho et al. (2019) find that lamin-A levels in the nuclear envelope are regulated in response to mechanical stimuli to prevent the nucleus from rupture, keep DNA repair factors in the nucleus, and consequentially "mechano-protect the genome."

Atheroprone flow activates inflammation via endothelial ATP-dependent P2X7-p38 signalling.

Objective: Atherosclerosis is a focal disease occurring at arterial sites of disturbed blood flow that generates low oscillating shear stress. Endothelial inflammatory signalling is enhanced at sites of disturbed flow via mechanisms that are incompletely understood. The influence of disturbed flow on endothelial adenosine triphosphate (ATP) receptors and downstream signalling was assessed. Methods and results: Cultured human endothelial cells were exposed to atheroprotective (high uniform) or atheroprone (low oscillatory) shear stress for 72 h prior to assessment of ATP responses. Imaging of cells loaded with a calcium-sensitive fluorescent dye revealed that atheroprone flow enhanced extracellular calcium influx in response to 300 µM 2'(3')-O-(4-Benzoylbenzoyl) adenosine-5'-triphosphate. Pre-treatment with pharmacological inhibitors demonstrated that this process required purinergic P2X7 receptors. The mechanism involved altered expression of P2X7, which was induced by atheroprone flow conditions in cultured cells. Similarly, en face staining of the murine aorta revealed enriched P2X7 expression at an atheroprone site. Functional studies in cultured endothelial cells showed that atheroprone flow induced p38 phosphorylation and up-regulation of E-selectin and IL-8 secretion via a P2X7-dependent mechanism. Moreover, genetic deletion of P2X7 significantly reduced E-selectin at atheroprone regions of the murine aorta. Conclusions: These findings reveal that P2X7 is regulated by shear forces leading to its accumulation at atheroprone sites that are exposed to disturbed patterns of blood flow. P2X7 promotes endothelial inflammation at atheroprone sites by transducing ATP signals into p38 activation. Thus P2X7 integrates vascular mechanical responses with purinergic signalling to promote endothelial dysfunction and may provide an attractive potential therapeutic target to prevent or reduce atherosclerosis.

Experimental Approaches to Study Endothelial Responses to Shear Stress.

SIGNIFICANCE: Shear stress controls multiple physiological processes in endothelial cells (ECs). RECENT ADVANCES: The response of ECs to shear has been studied using a range of in vitro and in vivo models. CRITICAL ISSUES: This article describes some of the experimental techniques that can be used to study endothelial responses to shear stress. It includes an appraisal of large animal, rodent, and zebrafish models of vascular mechanoresponsiveness. It also describes several bioreactors to apply flow to cells and physical methods to separate mechanoresponses from mass transport mechanisms. FUTURE DIRECTIONS: We conclude that combining in vitro and in vivo approaches can provide a detailed mechanistic view of vascular responses to force and that high-throughput systems are required for unbiased assessment of the function of shear-induced molecules. Antioxid. Redox Signal. 25, 389-400.

Endothelial repair in stented arteries is accelerated by inhibition of Rho-associated protein kinase.

AIMS: Stent deployment causes endothelial cells (EC) denudation, which promotes in-stent restenosis and thrombosis. Thus endothelial regrowth in stented arteries is an important therapeutic goal. Stent struts modify local hemodynamics, however the effects of flow perturbation on EC injury and repair are incompletely understood. By studying the effects of stent struts on flow and EC migration, we identified an intervention that promotes endothelial repair in stented arteries. METHODS AND RESULTS: In vitro and in vivo models were developed to monitor endothelialization under flow and the influence of stent struts. A 2D parallel-plate flow chamber with 100 µm ridges arranged perpendicular to the flow was used. Live cell imaging coupled to computational fluid dynamic simulations revealed that EC migrate in the direction of flow upstream from the ridges but subsequently accumulate downstream from ridges at sites of bidirectional flow. The mechanism of EC trapping by bidirectional flow involved reduced migratory polarity associated with altered actin dynamics. Inhibition of Rho-associated protein kinase (ROCK) enhanced endothelialization of ridged surfaces by promoting migratory polarity under bidirectional flow (P < 0.01). To more closely mimic the in vivo situation, we cultured EC on the inner surface of polydimethylsiloxane tubing containing Coroflex Blue stents (65 µm struts) and monitored migration. ROCK inhibition significantly enhanced EC accumulation downstream from struts under flow (P < 0.05). We investigated the effects of ROCK inhibition on re-endothelialization in vivo using a porcine model of EC denudation and stent placement. En face staining and confocal microscopy revealed that inhibition of ROCK using fasudil (30 mg/day via osmotic minipump) significantly increased re-endothelialization of stented carotid arteries (P < 0.05). CONCLUSIONS: Stent struts delay endothelial repair by generating localized bidirectional flow which traps migrating EC. ROCK inhibitors accelerate endothelial repair of stented arteries by enhancing EC polarity and migration through regions of bidirectional flow.