Eleni Tzima

A mechanosensory complex that mediates the endothelial cell response to fluid shear stress.

Shear stress is a fundamental determinant of vascular homeostasis, regulating vascular remodelling, cardiac development and atherogenesis, but the mechanisms of transduction are poorly understood. Previous work showed that the conversion of integrins to a high-affinity state mediates a subset of shear responses, including cell alignment and gene expression. Here we investigate the pathway upstream of integrin activation. PECAM-1 (which directly transmits mechanical force), vascular endothelial cell cadherin (which functions as an adaptor) and VEGFR2 (which activates phosphatidylinositol-3-OH kinase) comprise a mechanosensory complex. Together, these receptors are sufficient to confer responsiveness to flow in heterologous cells. In support of the relevance of this pathway in vivo, PECAM-1-knockout mice do not activate NF-κB and downstream inflammatory genes in regions of disturbed flow. Therefore, this mechanosensing pathway is required for the earliest-known events in atherogenesis.

Integrins in Mechanotransduction

Mechanical forces are crucial to the regulation of cell and tissue morphology and function. At the cellular level, forces influence cytoskeletal organization, gene expression, proliferation, and survival. Integrin-mediated adhesions are intrinsically mechanosensitive and a large body of data implicates integrins in sensing mechanical forces. We review the relationship between integrins and mechanical forces, the role of integrins in cellular responses to stretch and fluid flow, and propose that some of these events are mechanistically related.

Activation of integrins in endothelial cells by fluid shear stress mediates Rho-dependent cytoskeletal alignment

Fluid shear stress is a critical determinant of vascular remodeling and atherogenesis. Both integrins and the small GTPase Rho are implicated in endothelial cell responses to shear but the mechanisms are poorly understood. We now show that shear stress rapidly stimulates conformational activation of integrin αvβ3 in bovine aortic endothelial cells, followed by an increase in its binding to extracellular cell matrix (ECM) proteins. The shear‐induced new integrin binding to ECM induces a transient inactivation of Rho similar to that seen when suspended cells are plated on ECM proteins. This transient inhibition is necessary for cytoskeletal alignment in the direction of flow. The results therefore define the role of integrins and Rho in a pathway leading to endothelial cell adaptation to flow.

Activation of Rac1 by shear stress in endothelial cells mediates both cytoskeletal reorganization and effects on gene expression

Hemodynamic shear stress is a fundamental determinant of vascular remodeling and atherogenesis. Changes in focal adhesions, cytoskeletal organization and gene expression are major responses of endothelial cells to shear stress. Here, we show that activation of the small GTPase Rac is essential for gene expression and for providing spatial information for shear stress‐induced cell alignment. Fluorescence resonance energy transfer (FRET) localizes activated Rac1 in the direction of flow. This directional Rac1 activation is downstream of shear‐induced new integrin binding to extracellular matrix. Additionally, Rac1 mediates flow‐induced stimulation of nuclear factor κB (NF‐κB) and the subsequent expression of intercellular cell adhesion molecule 1 (ICAM‐1), an adhesion receptor involved in the recruitment of leukocytes to atherosclerotic plaque. These studies provide a unifying model linking three of the main responses to shear stress that mediate both normal adaptation to hemodynamic forces and inflammatory dysfunction of endothelial cells in atherosclerosis.

Role of Small GTPases in Endothelial Cytoskeletal Dynamics and the Shear Stress Response

Fluid shear stress caused by blood flow is a major determinant of vascular remodeling and arterial tone and can lead to development of atherosclerosis. The endothelial monolayer in vivo acts as a signal transduction interface for hemodynamic forces; these forces determine the shape, cytoskeletal organization, and function of endothelial cells, allowing the vessels to cope with physiological or pathological conditions. The Ras superfamily of GTPases have been revealed to be master regulators of many cellular activities. In particular, the GTPases RhoA, Rac1, and Cdc42 are known to regulate cell shape changes through effects on the cytoskeleton, but their ability to influence polarity, microtubule dynamics, and transcription factor activity is just as significant. Shear stress modulates the activity of small GTPases, which are critical for both cytoskeletal reorganization and changes in gene expression in response to shear stress. The goal of this article is to review what is known about Ras and more so about Rho GTPases in mechanotransduction and the responses of cells to fluid flow. Several distinct signaling pathways can be coordinately activated by flow, and small GTPases are strongly implicated in some of them; thus possible connections will be explored and a unifying hypothesis offered.

The novel fragment of tyrosyl tRNA synthetase, mini-TyrRS, is secreted to induce an angiogenic response in endothelial cells

Aminoacyl tRNA synthetases—enzymes that catalyze the first step of protein synthesis—in mammalian cells are now known to have expanded functions, including activities in signal transduction pathways, such as those for angiogenesis and inflammation. The native synthetases themselves are procytokines, having no signal transduction activities. After alternative splicing or natural proteolysis, specific fragments that are potent cytokines and that interact with specific receptors on cell surfaces are released. In this manner, a natural fragment of human tyrosyl tRNA synthetase (TyrRS), mini‐TyrRS, has been shown to act as a proangiogenic cytokine. The mechanistic basis for the action of mini‐TyrRS in angiogenesis has yet to be established. Here, we show that mini‐TyrRS is exported from endothelial cells when they are treated with tumor necrosis factor‐α. Mini‐TyrRS binds to vascular endothelial cells and activates an array of angiogenic signal transduction pathways. Mini‐TyrRS‐induced angiogenesis requires the activation of vascular endothelial growth factor receptor‐2 (VEGFR2/Flk‐1/KDR). Mini‐ TyrRS stimulates VEGFR2 phosphorylation in a VEGF‐ independent manner, suggesting VEGFR2 transactivation. Transactivation of VEGFR2 and downstream angiogenesis require an intact Glu‐Leu‐Arg (ELR) motif in mini‐TyrRS, which is important for its cytokine activity. These studies therefore suggest a mechanism by which mini‐TyrRS induces angiogenesis in endothelial cells and provide further insight into the role of mini‐TyrRS as a link between translation and angiogenesis.—Greenberg, Y., King, M., Kiosses, W. B., Ewalt, K., Yang, X., Schimmel, P., Reader, J. S., and Tzima, E. The novel fragment of tyrosyl tRNA synthetase, mini‐ TyrRS, is secreted to induce an angiogenic response in endothelial cells. FASEB J. 22, 1597–1605 (2008)

Pericytes Regulate Vascular Basement Membrane Remodeling and Govern Neutrophil Extravasation during Inflammation

During inflammation polymorphonuclear neutrophils (PMNs) traverse venular walls, composed of the endothelium, pericyte sheath and vascular basement membrane. Compared to PMN transendothelial migration, little is known about how PMNs penetrate the latter barriers. Using mouse models and intravital microscopy, we show that migrating PMNs expand and use the low expression regions (LERs) of matrix proteins in the vascular basement membrane (BM) for their transmigration. Importantly, we demonstrate that this remodeling of LERs is accompanied by the opening of gaps between pericytes, a response that depends on PMN engagement with pericytes. Exploring how PMNs modulate pericyte behavior, we discovered that direct PMN-pericyte contacts induce relaxation rather than contraction of pericyte cytoskeletons, an unexpected response that is mediated by inhibition of the RhoA/ROCK signaling pathway in pericytes. Taking our in vitro results back into mouse models, we present evidence that pericyte relaxation contributes to the opening of the gaps between pericytes and to the enlargement of the LERs in the vascular BM, facilitating PMN extravasation. Our study demonstrates that pericytes can regulate PMN extravasation by controlling the size of pericyte gaps and thickness of LERs in venular walls. This raises the possibility that pericytes may be targeted in therapies aimed at regulating inflammation.

Biologically active fragment of a human tRNA synthetase inhibits fluid shear stress-activated responses of endothelial cells

Human tryptophanyl-tRNA synthetase (TrpRS) is active in translation and angiogenesis. In particular, an N-terminally truncated fragment, T2-TrpRS, that is closely related to a natural splice variant is a potent antagonist of vascular endothelial growth factor-induced angiogenesis in several in vivo models. In contrast, full-length native TrpRS is inactive in the same models. However, vascular endothelial growth factor stimulation is only one of many physiological and pathophysiological stimuli to which the vascular endothelium responds. To investigate more broadly the role of T2-TrpRS in vascular homeostasis and pathophysiology, the effect of T2-TrpRS on well characterized endothelial cell (EC) responses to flow-induced fluid shear stress was studied. T2-TrpRS inhibited activation by flow of protein kinase B (Akt), extracellular signal-regulated kinase 1/2, and EC NO synthase and prevented transcription of several shear stress-responsive genes. In addition, T2-TrpRS interfered with the unique ability of ECs to align in the direction of fluid flow. In all of these assays, native TrpRS was inactive, demonstrating that angiogenesis-related activity requires fragment production. These results demonstrate that T2-TrpRS can regulate extracellular signal-activated protein kinase, Akt, and EC NO synthase activation pathways that are associated with angiogenesis, cytoskeletal reorganization, and shear stress-responsive gene expression. Thus, this biological fragment of TrpRS may have a role in the maintenance of vascular homeostasis.

Localized α4 Integrin Phosphorylation Directs Shear Stress–Induced Endothelial Cell Alignment

Vascular endothelial cells respond to laminar shear stress by aligning in the direction of flow, a process which may contribute to atheroprotection. Here we report that localized &agr;4 integrin phosphorylation is a mechanism for establishing the directionality of shear stress–induced alignment in microvascular endothelial cells. Within 5 minutes of exposure to a physiological level of shear stress, endothelial &agr;4 integrins became phosphorylated on Ser988. In wounded monolayers, phosphorylation was enhanced at the downstream edges of cells relative to the source of flow. The shear-induced &agr;4 integrin phosphorylation was blocked by inhibitors of cAMP-dependent protein kinase A (PKA), an enzyme involved in the alignment of endothelial cells under prolonged shear. Moreover, shear-induced localized activation of the small GTPase Rac1, which specifies the directionality of endothelial alignment, was similarly blocked by PKA inhibitors. Furthermore, endothelial cells bearing a nonphosphorylatable &agr;4(S988A) mutation failed to align in response to shear stress, thus establishing &agr;4 as a relevant PKA substrate. We thereby show that shear-induced PKA-dependent &agr;4 integrin phosphorylation at the downstream edge of endothelial cells promotes localized Rac1 activation, which in turn directs cytoskeletal alignment in response to shear stress.

Evidence for Annexin II-S100A10 Complex and Plasmin in Mobilization of Cytokine Activity of Human TrpRS

In mammalian cells, specific aminoacyl-transfer RNA (tRNA) synthetases have cytokine functions that require interactions with partners outside of the translation apparatus. Little is known about these interactions and how they facilitate expanded functions that link protein translation to other cellular pathways. For example, an alternative splice fragment of tryptophanyl-tRNA synthetase (TrpRS) and a similar natural proteolytic fragment are potent angiostatic factors that act through the vascular endothelial-cadherin receptor and Akt signaling pathway. Here we demonstrate mobilization of TrpRS for exocytosis from endothelial cells and the potential for plasmin to activate the cytokine function of the extracellular synthetase. Direct physical evidence showed that the annexin II-S100A10 complex, which regulates exocytosis, forms a ternary complex with TrpRS. Functional studies demonstrate that both annexin II and S100A10 regulate trafficking of TrpRS. Thus, complexes of mammalian tRNA synthetases with seemingly disparate proteins may in general be relevant to understanding how their expanded functions are implemented.