Eukaryotic initiation factor 6 regulates mechanical responses in endothelial cells.

The repertoire of extratranslational functions of components of the protein synthesis apparatus is expanding to include control of key cell signaling networks. However, very little is known about noncanonical functions of members of the protein synthesis machinery in regulating cellular mechanics. We demonstrate that the eukaryotic initiation factor 6 (eIF6) modulates cellular mechanobiology. eIF6-depleted endothelial cells, under basal conditions, exhibit unchanged nascent protein synthesis, polysome profiles, and cytoskeleton protein expression, with minimal effects on ribosomal biogenesis. In contrast, using traction force and atomic force microscopy, we show that loss of eIF6 leads to reduced stiffness and force generation accompanied by cytoskeletal and focal adhesion defects. Mechanistically, we show that eIF6 is required for the correct spatial mechanoactivation of ERK1/2 via stabilization of an eIF6-RACK1-ERK1/2-FAK mechanocomplex, which is necessary for force-induced remodeling. These results reveal an extratranslational function for eIF6 and a novel paradigm for how mechanotransduction, the cellular cytoskeleton, and protein translation constituents are linked.

Mechanical forces regulate endothelial-to-mesenchymal transition and atherosclerosis via an Alk5-Shc mechanotransduction pathway.

The response of endothelial cells to mechanical forces is a critical determinant of vascular health. Vascular pathologies, such as atherosclerosis, characterized by abnormal mechanical forces are frequently accompanied by endothelial-to-mesenchymal transition (EndMT). However, how forces affect the mechanotransduction pathways controlling cellular plasticity, inflammation, and, ultimately, vessel pathology is poorly understood. Here, we identify a mechanoreceptor that is sui generis for EndMT and unveil a molecular Alk5-Shc pathway that leads to EndMT and atherosclerosis. Depletion of Alk5 abrogates shear stress-induced EndMT responses, and genetic targeting of endothelial Shc reduces EndMT and atherosclerosis in areas of disturbed flow. Tensional force and reconstitution experiments reveal a mechanosensory function for Alk5 in EndMT signaling that is unique and independent of other mechanosensors. Our findings are of fundamental importance for understanding how mechanical forces regulate biochemical signaling, cell plasticity, and vascular disease.

Mechanical Forces and Their Effect on the Ribosome and Protein Translation Machinery.

Mechanical forces acting on biological systems, at both the macroscopic and microscopic levels, play an important part in shaping cellular phenotypes. There is a growing realization that biomolecules that respond to force directly applied to them, or via mechano-sensitive signalling pathways, can produce profound changes to not only transcriptional pathways, but also in protein translation. Forces naturally occurring at the molecular level can impact the rate at which the bacterial ribosome translates messenger RNA (mRNA) transcripts and influence processes such as co-translational folding of a nascent protein as it exits the ribosome. In eukaryotes, force can also be transduced at the cellular level by the cytoskeleton, the cell's internal filamentous network. The cytoskeleton closely associates with components of the translational machinery such as ribosomes and elongation factors and, as such, is a crucial determinant of localized protein translation. In this review we will give (1) a brief overview of protein translation in bacteria and eukaryotes and then discuss (2) how mechanical forces are directly involved with ribosomes during active protein synthesis and (3) how eukaryotic ribosomes and other protein translation machinery intimately associates with the mechanosensitive cytoskeleton network.

Mechanical Regulation of Protein Translation in the Cardiovascular System.

The cardiovascular system can sense and adapt to changes in mechanical stimuli by remodeling the physical properties of the heart and blood vessels in order to maintain homeostasis. Imbalances in mechanical forces and/or impaired sensing are now not only implicated but are, in some cases, considered to be drivers for the development and progression of cardiovascular disease. There is now growing evidence to highlight the role of mechanical forces in the regulation of protein translation pathways. The canonical mechanism of protein synthesis typically involves transcription and translation. Protein translation occurs globally throughout the cell to maintain general function but localized protein synthesis allows for precise spatiotemporal control of protein translation. This Review will cover studies on the role of biomechanical stress -induced translational control in the heart (often in the context of physiological and pathological hypertrophy). We will also discuss the much less studied effects of mechanical forces in regulating protein translation in the vasculature. Understanding how the mechanical environment influences protein translational mechanisms in the cardiovascular system, will help to inform disease pathogenesis and potential areas of therapeutic intervention.

The guidance receptor plexin D1 is a mechanosensor in endothelial cells.

Shear stress on arteries produced by blood flow is important for vascular development and homeostasis but can also initiate atherosclerosis1. Endothelial cells that line the vasculature use molecular mechanosensors to directly detect shear stress profiles that will ultimately lead to atheroprotective or atherogenic responses2. Plexins are key cell-surface receptors of the semaphorin family of cell-guidance signalling proteins and can regulate cellular patterning by modulating the cytoskeleton and focal adhesion structures3-5. However, a role for plexin proteins in mechanotransduction has not been examined. Here we show that plexin D1 (PLXND1) has a role in mechanosensation and mechanically induced disease pathogenesis. PLXND1 is required for the response of endothelial cells to shear stress in vitro and in vivo and regulates the site-specific distribution of atherosclerotic lesions. In endothelial cells, PLXND1 is a direct force sensor and forms a mechanocomplex with neuropilin-1 and VEGFR2 that is necessary and sufficient for conferring mechanosensitivity upstream of the junctional complex and integrins. PLXND1 achieves its binary functions as either a ligand or a force receptor by adopting two distinct molecular conformations. Our results establish a previously undescribed mechanosensor in endothelial cells that regulates cardiovascular pathophysiology, and provide a mechanism by which a single receptor can exhibit a binary biochemical nature.

A key role for the novel coronary artery disease gene JCAD in atherosclerosis via shear stress mechanotransduction.

AIMS: Genome-wide association studies (GWAS) have consistently identified an association between coronary artery disease (CAD) and a locus on chromosome 10 containing a single gene, JCAD (formerly KIAA1462). However, little is known about the mechanism by which JCAD could influence the development of atherosclerosis. METHODS AND RESULTS: Vascular function was quantified in subjects with CAD by flow-mediated dilatation (FMD) and vasorelaxation responses in isolated blood vessel segments. The JCAD risk allele identified by GWAS was associated with reduced FMD and reduced endothelial-dependent relaxations. To study the impact of loss of Jcad on atherosclerosis, Jcad-/- mice were crossed to an ApoE-/- background and fed a high-fat diet from 6 to16 weeks of age. Loss of Jcad did not affect blood pressure or heart rate. However, Jcad-/-ApoE-/- mice developed significantly less atherosclerosis in the aortic root and the inner curvature of the aortic arch. En face analysis revealed a striking reduction in pro-inflammatory adhesion molecules at sites of disturbed flow on the endothelial cell layer of Jcad-/- mice. Loss of Jcad lead to a reduced recovery perfusion in response to hind limb ischaemia, a model of altered in vivo flow. Knock down of JCAD using siRNA in primary human aortic endothelial cells significantly reduced the response to acute onset of flow, as evidenced by reduced phosphorylation of NF-КB, eNOS, and Akt. CONCLUSION: The novel CAD gene JCAD promotes atherosclerotic plaque formation via a role in the endothelial cell shear stress mechanotransduction pathway.

Haemodynamics Regulate Fibronectin Assembly via PECAM.

Fibronectin (FN) assembly and fibrillogenesis are critically important in both development and the adult organism, but their importance in vascular functions is not fully understood. Here we identify a novel pathway by which haemodynamic forces regulate FN assembly and fibrillogenesis during vascular remodelling. Induction of disturbed shear stress in vivo and in vitro resulted in complex FN fibril assembly that was dependent on the mechanosensor PECAM. Loss of PECAM also inhibited the cell-intrinsic ability to remodel FN. Gain- and loss-of-function experiments revealed that PECAM-dependent RhoA activation is required for FN assembly. Furthermore, PECAM-/- mice exhibited reduced levels of active ß1 integrin that were responsible for reduced RhoA activation and downstream FN assembly. These data identify a new pathway by which endothelial mechanotransduction regulates FN assembly and flow-mediated vascular remodelling.

Endothelial Mechanosignaling: Does One Sensor Fit All?

SIGNIFICANCE: Forces are important in the cardiovascular system, acting as regulators of vascular physiology and pathology. Residing at the blood vessel interface, cells (endothelial cell, EC) are constantly exposed to vascular forces, including shear stress. Shear stress is the frictional force exerted by blood flow, and its patterns differ based on vessel geometry and type. These patterns range from uniform laminar flow to nonuniform disturbed flow. Although ECs sense and differentially respond to flow patterns unique to their microenvironment, the mechanisms underlying endothelial mechanosensing remain incompletely understood. RECENT ADVANCES: A large body of work suggests that ECs possess many mechanosensors that decorate their apical, junctional, and basal surfaces. These potential mechanosensors sense blood flow, translating physical force into biochemical signaling events. CRITICAL ISSUES: Understanding the mechanisms by which proposed mechanosensors sense and respond to shear stress requires an integrative approach. It is also critical to understand the role of these mechanosensors not only during embryonic development but also in the different vascular beds in the adult. Possible cross talk and integration of mechanosensing via the various mechanosensors remain a challenge. FUTURE DIRECTIONS: Determination of the hierarchy of endothelial mechanosensors is critical for future work, as is determination of the extent to which mechanosensors work together to achieve force-dependent signaling. The role and primary sensors of shear stress during development also remain an open question. Finally, integrative approaches must be used to determine absolute mechanosensory function of potential mechanosensors. Antioxid. Redox Signal. 25, 373-388.

Pulling on my heartstrings: mechanotransduction in cardiac development and function.

PURPOSE OF REVIEW: Endothelial cells line the surface of the cardiovascular system and display a large degree of heterogeneity due to developmental origin and location. Despite this heterogeneity, all endothelial cells are exposed to wall shear stress (WSS) imparted by the frictional force of flowing blood, which plays an important role in determining the endothelial cell phenotype. Although the effects of WSS have been greatly studied in vascular endothelial cells, less is known about the role of WSS in regulating cardiac function and cardiac endothelial cells. RECENT FINDINGS: Recent advances in genetic and imaging technologies have enabled a more thorough investigation of cardiac hemodynamics. Using developmental models, shear stress sensing by endocardial endothelial cells has been shown to play an integral role in proper cardiac development including morphogenesis and formation of the conduction system. In the adult, less is known about hemodynamics and endocardial endothelial cells, but a clear role for WSS in the development of coronary and valvular disease is increasingly appreciated. SUMMARY: Future research will further elucidate a role for WSS in the developing and adult heart, and understanding this dynamic relationship may represent a potential therapeutic target for the treatment of cardiomyopathies.

Platelet endothelial cell adhesion molecule-1 mediates endothelial-cardiomyocyte communication and regulates cardiac function.

BACKGROUND: Dilated cardiomyopathy is characterized by impaired contractility of cardiomyocytes, ventricular chamber dilatation, and systolic dysfunction. Although mutations in genes expressed in the cardiomyocyte are the best described causes of reduced contractility, the importance of endothelial-cardiomyocyte communication for proper cardiac function is increasingly appreciated. In the present study, we investigate the role of the endothelial adhesion molecule platelet endothelial cell adhesion molecule (PECAM-1) in the regulation of cardiac function. METHODS AND RESULTS: Using cell culture and animal models, we show that PECAM-1 expressed in endothelial cells (ECs) regulates cardiomyocyte contractility and cardiac function via the neuregulin-ErbB signaling pathway. Conscious echocardiography revealed left ventricular (LV) chamber dilation and systolic dysfunction in PECAM-1(-/-) mice in the absence of histological abnormalities or defects in cardiac capillary density. Despite deficits in global cardiac function, cardiomyocytes isolated from PECAM-1(-/-) hearts displayed normal baseline and isoproterenol-stimulated contractility. Mechanistically, absence of PECAM-1 resulted in elevated NO/ROS signaling and NRG-1 release from ECs, which resulted in augmented phosphorylation of its receptor ErbB2. Treatment of cardiomyocytes with conditioned media from PECAM-1(-/-) ECs resulted in enhanced ErbB2 activation, which was normalized by pre-treatment with an NRG-1 blocking antibody. To determine whether normalization of increased NRG-1 levels could correct cardiac function, PECAM-1(-/-) mice were treated with the NRG-1 blocking antibody. Echocardiography showed that treatment significantly improved cardiac function of PECAM-1(-/-) mice, as revealed by increased ejection fraction and fractional shortening. CONCLUSIONS: We identify a novel role for PECAM-1 in regulating cardiac function via a paracrine NRG1-ErbB pathway. These data highlight the importance of tightly regulated cellular communication for proper cardiac function.

Natural aminoacyl tRNA synthetase fragment enhances cardiac function after myocardial infarction.

A naturally-occurring fragment of tyrosyl-tRNA synthetase (TyrRS) has been shown in higher eukaryotes to 'moonlight' as a pro-angiogenic cytokine in addition to its primary role in protein translation. Pro-angiogenic cytokines have previously been proposed to be promising therapeutic mechanisms for the treatment of myocardial infarction. Here, we show that systemic delivery of the natural fragment of TyRS, mini-TyrRS, improves heart function in mice after myocardial infarction. This improvement is associated with reduced formation of scar tissue, increased angiogenesis of cardiac capillaries, recruitment of c-kitpos cells and proliferation of myocardial fibroblasts. This work demonstrates that mini-TyrRS has beneficial effects on cardiac repair and regeneration and offers support for the notion that elucidation of the ever expanding repertoire of noncanonical functions of aminoacyl tRNA synthetases offers unique opportunities for development of novel therapeutics.

Rac[e] to the pole: setting up polarity in endothelial cells.

Mechanical forces influence many biological processes via activation of signaling molecules, including the family of Rho GTPases. Within the endothelium, the mechanical force of fluid shear stress regulates the spatiotemporal activation of Rho GTPases, including Rac1. Shear stress-induced Rac1 activation is required for numerous essential biological processes, including changes in permeability, alignment of the actin cytoskeleton, redox signaling, and changes in gene expression. Thus, identifying mechanisms of Rac1 activation and the spatial cues that direct proper localization of the GTPase is essential in order to gain a comprehensive understanding the role of Rac1 in shear stress responses. This commentary will highlight our current understanding of how Rac1 activity is regulated in response to shear stress, as well as the downstream consequences of Rac1 activation.

Haemodynamic and extracellular matrix cues regulate the mechanical phenotype and stiffness of aortic endothelial cells.

Endothelial cells (ECs) lining blood vessels express many mechanosensors, including platelet endothelial cell adhesion molecule-1 (PECAM-1), that convert mechanical force into biochemical signals. While it is accepted that mechanical stresses and the mechanical properties of ECs regulate vessel health, the relationship between force and biological response remains elusive. Here we show that ECs integrate mechanical forces and extracellular matrix (ECM) cues to modulate their own mechanical properties. We demonstrate that the ECM influences EC response to tension on PECAM-1. ECs adherent on collagen display divergent stiffening and focal adhesion growth compared with ECs on fibronectin. This is because of protein kinase A (PKA)-dependent serine phosphorylation and inactivation of RhoA. PKA signalling regulates focal adhesion dynamics and EC compliance in response to shear stress in vitro and in vivo. Our study identifies an ECM-specific, mechanosensitive signalling pathway that regulates EC compliance and may serve as an atheroprotective mechanism that maintains blood vessel integrity in vivo.

A novel pathway spatiotemporally activates Rac1 and redox signaling in response to fluid shear stress.

Hemodynamic forces regulate embryonic organ development, hematopoiesis, vascular remodeling, and atherogenesis. The mechanosensory stimulus of blood flow initiates a complex network of intracellular pathways, including activation of Rac1 GTPase, establishment of endothelial cell (EC) polarity, and redox signaling. The activity of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can be modulated by the GTP/GDP state of Rac1; however, the molecular mechanisms of Rac1 activation by flow are poorly understood. Here, we identify a novel polarity complex that directs localized Rac1 activation required for downstream reactive oxygen species (ROS) production. Vav2 is required for Rac1 GTP loading, whereas, surprisingly, Tiam1 functions as an adaptor in a VE-cadherin-p67phox-Par3 polarity complex that directs localized activation of Rac1. Furthermore, loss of Tiam1 led to the disruption of redox signaling both in vitro and in vivo. Our results describe a novel molecular cascade that regulates redox signaling by the coordinated regulation of Rac1 and by linking components of the polarity complex to the NADPH oxidase.

Endothelial Shc regulates arteriogenesis through dual control of arterial specification and inflammation via the notch and nuclear factor-κ-light-chain-enhancer of activated B-cell pathways.

RATIONALE: Arteriogenesis, the shear stress-driven remodeling of collateral arteries, is critical in restoring blood flow to ischemic tissue after a vascular occlusion. Our previous work has shown that the adaptor protein Shc mediates endothelial responses to shear stress in vitro. OBJECTIVE: To examine the role of the adaptor protein Shc in arteriogenesis and endothelial-dependent responses to shear stress in vivo. METHODS AND RESULTS: Conditional knockout mice in which Shc is deleted from endothelial cells were subjected to femoral artery ligation. Hindlimb perfusion recovery was attenuated in Shc conditional knockout mice compared with littermate controls. Reduced perfusion was associated with blunted collateral remodeling and reduced capillary density. Bone marrow transplantation experiments revealed that endothelial Shc is required for perfusion recovery because loss of Shc in bone marrow-derived hematopoietic cells had no effect on recovery. Mechanistically, Shc deficiency resulted in impaired activation of the nuclear factor κ-light-chain-enhancer of activated B-cell-dependent inflammatory pathway and reduced CD45⁺ cell infiltration. Unexpectedly, Shc was required for arterial specification of the remodeling arteriole by mediating upregulation of the arterial endothelial cell marker ephrinB2 and activation of the Notch pathway. In vitro experiments confirmed that Shc was required for shear stress-induced activation of the Notch pathway and downstream arterial specification through a mechanism that involves upregulation of Notch ligands delta-like 1 and delta-like 4. CONCLUSIONS: Shc mediates activation of 2 key signaling pathways that are critical for inflammation and arterial specification; collectively, these pathways contribute to arteriogenesis and the recovery of blood perfusion.

RhoA goes GLOBAL.

Mechanical forces influence nearly all aspects of biology. Cells are equipped with numerous mechanosensitive proteins that activate various signaling cascades in response to mechanical cues from their environment. Much interest lies in understanding how cells respond to external stresses. A number of studies have highlighted the coordination of mechanical and chemical signaling cascades downstream of integrins. In recent years, the study of mechanotransduction has expanded to other mechanosensitive adhesion receptors, such as platelet endothelial cell adhesion molecule-1 (PECAM-1). This commentary will highlight our current understanding of integrin and PECAM-1-mediated mechanotransduction and expand on the observation that a localized mechanical stress can elicit a global mechanosignaling response.