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.

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.

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.

The adaptor protein Shc integrates growth factor and ECM signaling during postnatal angiogenesis.

Angiogenesis requires integration of cues from growth factors, extracellular matrix (ECM) proteins, and their receptors in endothelial cells. In the present study, we show that the adaptor protein Shc is required for angiogenesis in zebrafish, mice, and cell-culture models. Shc knockdown zebrafish embryos show defects in intersegmental vessel sprouting in the trunk. Shc flox/flox; Tie2-Cre mice display reduced angiogenesis in the retinal neovascularization model and in response to VEGF in the Matrigel plug assay in vivo. Functional studies reveal a model in which Shc is required for integrin-mediated spreading and migration specifically on fibronectin, as well as endothelial cell survival in response to VEGF. Mechanistically, Shc is required for activation of the Akt pathway downstream of both integrin and VEGF signaling, as well as for integration of signals from these 2 receptors when cells are grown on fibronectin. Therefore, we have identified a unique mechanism in which signals from 2 critical angiogenic signaling axes, integrins and VEGFR-2, converge at Shc to regulate postnatal angiogenesis.

Bmper inhibits endothelial expression of inflammatory adhesion molecules and protects against atherosclerosis.

OBJECTIVE: Bone morphogenetic proteins (Bmps) are important mediators of inflammation and atherosclerosis, though their mechanism of action is not fully understood. To better understand the contribution of the Bmp signaling pathway in vascular inflammation, we investigated the role of Bmper (Bmp endothelial cell precursor-derived regulator), an extracellular Bmp modulator, in an induced in vivo model of inflammation and atherosclerosis. METHODS AND RESULTS: We crossed apolipoprotein E-deficient (ApoE(-/-)) mice with mice missing 1 allele of Bmper (Bmper(+/-) mice used in the place of Bmper(-/-) mice that die at birth) and measured the development of atherosclerosis in mice fed a high-fat diet. Bmper haploinsufficiency in ApoE(-/-) mice (Bmper(+/-);ApoE(-/-) mice) led to a more severe phenotype compared with Bmper(+/+);ApoE(-/-) mice. Bmper(+/-);ApoE(-/-) mice also exhibited increased Bmp activity in the endothelial cells in both the greater and lesser curvatures of the aortic arch, suggesting a role for Bmper in regulating Bmp-mediated inflammation associated with laminar and oscillatory shear stress. Small interfering RNA knockdown of Bmper in human umbilical vein endothelial cells caused a dramatic increase in the inflammatory markers intracellular adhesion molecule 1 and vascular cell adhesion molecule 1 at rest and after exposure to oscillatory and laminar shear stress. CONCLUSIONS: We conclude that Bmper is a critical regulator of Bmp-mediated vascular inflammation and that the fine-tuning of Bmp and Bmper levels is essential in the maintenance of normal vascular homeostasis.

Role of PECAM-1 in arteriogenesis and specification of preexisting collaterals.

RATIONALE: Hemodynamic forces caused by the altered blood flow in response to an occlusion lead to the induction of collateral remodeling and arteriogenesis. Previous work showed that platelet endothelial cell adhesion molecule (PECAM)-1 is a component of a mechanosensory complex that mediates endothelial cell responses to shear stress. OBJECTIVE: We hypothesized that PECAM-1 plays an important role in arteriogenesis and collateral remodeling. METHODS AND RESULTS: PECAM-1 knockout (KO) and wild-type littermates underwent femoral artery ligation. Surprisingly, tissue perfusion and collateral-dependent blood flow were significantly increased in the KO mice immediately after surgery. Histology confirmed larger caliber of preexisting collaterals in the KO mice. Additionally, KO mice showed blunted recovery of perfusion from hindlimb ischemia and reduced collateral remodeling, because of deficits in shear stress-induced signaling, including activation of the nuclear factor κB pathway and inflammatory cell accumulation. Partial recovery was associated with normal responses to circumferential wall tension in the absence of PECAM-1, as evidenced by the upregulation of ephrin B2 and monocyte chemoattractant protein-1, which are 2 stretch-induced regulators of arteriogenesis, both in vitro and in vivo. CONCLUSIONS: Our findings suggest a novel role for PECAM-1 in arteriogenesis and collateral remodeling. Furthermore, we identify PECAM-1 as the first molecule that determines preexisting collateral diameter.

Platelet-endothelial cell adhesion molecule-1 regulates endothelial NO synthase activity and localization through signal transducers and activators of transcription 3-dependent NOSTRIN expression.

BACKGROUND: NO produced by the endothelial NO synthase (eNOS) is an important regulator of cardiovascular physiological and pathological features. eNOS is activated by numerous stimuli, and its activity is tightly regulated. Platelet-endothelial cell adhesion molecule-1 (PECAM-1) has been implicated in regulating eNOS activity in response to shear stress. The current study was conducted to determine the role of PECAM-1 in the regulation of basal eNOS activity. METHODS AND RESULTS: We demonstrate that PECAM-1-knockout ECs have increased basal eNOS activity and NO production. Mechanistically, increased eNOS activity is associated with a decrease in the inhibitory interaction of eNOS with caveolin-1, impaired subcellular localization of eNOS, and decreased eNOS traffic inducer (NOSTRIN) expression in the absence of PECAM-1. Furthermore, we demonstrate that activation of blunted signal transducers and activators of transcription 3 (STAT3) in the absence of PECAM-1 results in decreased NOSTRIN expression via direct binding of the signal transducers and activators of transcription 3 to the NOSTRIN promoter. CONCLUSIONS: Our results reveal an elegant mechanism of eNOS regulation by PECAM-1 through signal transducers and activators of transcription 3-mediated transcriptional control of NOSTRIN.

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.

Inhibition of tumor angiogenesis by a natural fragment of a tRNA synthetase.

Human tyrosyl- and tryptophanyl-tRNA synthetases (TyrRS and TrpRS, respectively) link protein synthesis to signal-transduction pathways, including angiogenesis. Fragments of TyrRS stimulate angiogenesis, whereas those of TrpRS (T2-TrpRS) inhibit angiogenesis. Thus, these two synthetases acquired opposing activities during evolution, possibly as a coordinated mechanism for regulating angiogenesis. The recent identification of the cellular target of T2-TrpRS sheds light into the mechanism of angiogenesis inhibition. This mechanism provides a molecular basis for the lack of effect of T2-TrpRS on the normal vasculature. With these features, we suggest that this fragment of a tRNA synthetase might safely be used to arrest neovascularization of tumors. In particular, an anti-angiogenesis agent that stops the growth of tumor vessels without affecting normal vessels might serve as an adjunct to cytotoxic therapy.

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.

PECAM-1 is necessary for flow-induced vascular remodeling.

OBJECTIVE: Vascular remodeling is a physiological process that occurs in response to long-term changes in hemodynamic conditions, but may also contribute to the pathophysiology of intima-media thickening (IMT) and vascular disease. Shear stress detection by the endothelium is thought to be an important determinant of vascular remodeling. Previous work showed that platelet endothelial cell adhesion molecule-1 (PECAM-1) is a component of a mechanosensory complex that mediates endothelial cell (EC) responses to shear stress. METHODS AND RESULTS: We tested the hypothesis that PECAM-1 contributes to vascular remodeling by analyzing the response to partial carotid artery ligation in PECAM-1 knockout mice and wild-type littermates. PECAM-1 deficiency resulted in impaired vascular remodeling and significantly reduced IMT in areas of low flow. Inward remodeling was associated with PECAM-1-dependent NFkappaB activation, surface adhesion molecule expression, and leukocyte infiltration as well as Akt activation and vascular cell proliferation. CONCLUSIONS: PECAM-1 plays a crucial role in the activation of the NFkappaB and Akt pathways and inflammatory cell accumulation during vascular remodeling and IMT. Elucidation of some of the signals that drive vascular remodeling represent pharmacologically tractable targets for the treatment of restenosis after balloon angioplasty or stent placement.

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.

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.

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.