Endothelial Cell Shape Change Research Articles

Study of corneal endothelial cell layer 3 months after Descemet membrane endothelial keratoplasty

Background To study the endothelial cell layer 3 months after Descemet membrane endothelial keratoplasty (DMEK) using specular microscopy to provide data about the endothelial cell density (ECD), endothelial cell loss (ECL) percentage, and changes in endothelial cell shape (hexagonality) and size [coefficient of variation (CV)] and to study the factors causing these changes in endothelial cell layer after DMEK. Patients and methods This study included 20 patients investigated by specular microscopy 3 months after DMEK. We discuss the postoperative ECD, factors that affect the ECD, factors that affect hexagonality and CV, visual outcome, and advantages of DMEK. The study was approved by the ethics committee of Ain Shams University. Results This study showed that the mean ECD postoperative was 1664.80 cells/mm2, with mean ECL of 34.67%. This cell loss increased in cases of difficult graft unfolding. Donor age and preservation time had no effect on postoperative ECD. A percentage decrease in hexagonal cells and a percentage increase in CV were noticed in cases that received grafts from donors with history of diabetes mellitus. Postoperative ECD and hexagonal cell percentage had no correlation with central corneal thickness in this study. Conclusion DMEK results in significant improvement of postoperatively visual acuity (VA). Advantages of DMEK like no corneal sutures, short recovery time, less cylindrical aberration, decreased incidence of graft rejection, and good visual outcome make it the best choice to treat eyes with corneal endothelial dysfunction.

Dynamically regulated focal adhesions coordinate endothelial cell remodelling in developing vasculature.

The assembly of a mature vascular network involves coordinated endothelial cell (EC) shape changes, including the process of EC elongation. How EC elongation is dynamically regulated in vivo is not fully understood. Here, we have generated a zebrafish mutant that is deficient for the integrin adaptor protein Talin 1 (Tln1). Using a new focal adhesion (FA) marker line expressing endothelial Vinculinb-eGFP, we demonstrate that EC FAs function dynamically and are lost in our tln1 mutants, allowing us to uncouple the primary roles of FAs in EC morphogenesis from the secondary effects that occur due to systemic vessel failure or loss of blood flow. Tln1 loss led to compromised F-actin rearrangements, perturbed EC elongation and disrupted cell-cell junction linearisation in vessel remodelling. Finally, chemical induction of actin polymerisation restored actin dynamics and EC elongation during vascular morphogenesis. Together, we identify that FAs are essential for EC elongation and junction linearisation in flow-pressured vessels and that they influence actin polymerisation in cellular morphogenesis. These observations can explain the severely compromised vessel beds and vascular leakage observed in mutant models that lack integrin signalling. This article has an associated 'The people behind the papers' interview.

4D diSPIM Myography Reveals Changes in Endothelial Cell Shape and Ca2+ Signaling in Response to Intravascular Pressure in ex vivo Resistance‐sized Arteries

The vascular endothelium is critical in promoting vasodilation of resistance‐sized arteries through Ca2+dependent pathways. The endothelium is exposed to mechanical stimulation through changing intravascular pressure and flow. Understanding how the endothelium integrates this information into a change in vascular function requires the ability to simultaneously measure changes vessel diameter and EC Ca2+ signaling with high spatial and temporal resolution. We hypothesized that endothelial cell shape and Ca2+ signaling is pressure dependent. To test this, we combined 4D Dual Inverted Selective Plane of Illumination Microscopy (diSPIM; 3i, CO) with pressure myography. This system allows for imaging of three‐dimensional tissue preparations over time with very low photobleaching and phototoxicity, millisecond temporal resolution, and isometric 320 nm spatial resolution over a large imaging volume (330 x 330 x 150 mm). We designed and fabricated pressure myography chambers that position ex vivo pressurized arteries in the correct orientation for acquisition of high‐resolution fluorescent data via diSPIM while using an inverted microscope base to continuously capture vessel diameter measurements via transmitted IR light. Resistance‐sized mesenteric arteries from mice expressing an endothelium specific GECI are positioned between the diSPIM objectives and maintained at physiologic temperature and pH. We captured fluorescent images of whole pressurized arteries at 80, 60, 40, and 20 mmHg before and after the generation of myogenic tone and in the absence of extracellular Ca2+. As static intravascular pressure is increased, ECs increase in width and decrease in thickness. Myogenic tone minimizes changes in EC shape with changing pressure, however increasing static intravascular pressure decreases local EC Ca2+ signaling. The data demonstrate a relationship between intravascular pressure and EC shape and Ca2+ signaling in intact resistance arteries. This technique provides a method of studying the effects of physiologically relevant mechanical stimuli and the roles of various mechanosensitive mechanisms on vascular function.

Marcksl1 modulates endothelial cell mechanoresponse to haemodynamic forces to control blood vessel shape and size

The formation of vascular tubes is driven by extensive changes in endothelial cell (EC) shape. Here, we have identified a role of the actin-binding protein, Marcksl1, in modulating the mechanical properties of EC cortex to regulate cell shape and vessel structure during angiogenesis. Increasing and depleting Marcksl1 expression level in vivo results in an increase and decrease, respectively, in EC size and the diameter of microvessels. Furthermore, endothelial overexpression of Marcksl1 induces ectopic blebbing on both apical and basal membranes, during and after lumen formation, that is suppressed by reduced blood flow. High resolution imaging reveals that Marcksl1 promotes the formation of linear actin bundles and decreases actin density at the EC cortex. Our findings demonstrate that a balanced network of linear and branched actin at the EC cortex is essential in conferring cortical integrity to resist the deforming forces of blood flow to regulate vessel structure.

Endothelial cells on the move: dynamics in vascular morphogenesis and disease.

The vascular system is a hierarchically organized network of blood vessels that play crucial roles in embryogenesis, homeostasis and disease. Blood vessels are built by endothelial cells – the cells lining the interior of blood vessels – through a process named vascular morphogenesis. Endothelial cells react to different biomechanical signals in their environment by adjusting their behavior to: (1) invade, proliferate and fuse to form new vessels (angiogenesis); (2) remodel, regress and establish a hierarchy in the network (patterning); and (3) maintain network stability (quiescence). Each step involves the coordination of endothelial cell differentiation, proliferation, polarity, migration, rearrangements and shape changes to ensure network integrity and an efficient barrier between blood and tissues. In this review, we highlighted the relevance and the mechanisms involving endothelial cell migration during different steps of vascular morphogenesis. We further present evidence on how impaired endothelial cell dynamics can contribute to pathology.

Changes in corneal endothelial cell shape after treatment with one drop of ROCK inhibitor

We performed an investigation of the temporal impact on corneal endothelial cell (CEC) shape after ophthalmic instillation of one drop of the ROCK inhibitor, ripasudil. Subjects comprised 32 healthy adult volunteers, each of whom instilled ripasudil in the right eye. CEC shape [defined as CEC density (CECD), coefficient of variation (CV), and hexagonal cell rate (6A)], central corneal thickness (CCT), intraocular pressure (IOP), and conjunctival hyperemia were evaluated before instillation (baseline) and at 15min, 1h, 2h, and 4-6h after instillation by non-contact specular microscopy and slit-lamp microscopy. The incidence of pseudo gutta was also assessed. The CECD (cells/mm2) significantly decreased after instillation as follows: 2661 cells/mm2 at baseline, 2578 cells/mm2 at 15min, 2527 cells/mm2 at 1h, 2575 cells/mm2 at 2h, and 2613 cells/mm2 at 4-6h. After instillation, CV significantly increased and 6A significantly decreased. Pseudo gutta appeared in 18 subjects (56.3%) after instillation. There was no difference in CCT before and after instillation. IOP significantly decreased after instillation. The most severe conjunctival hyperemia was observed at 15min after instillation, but had nearly disappeared within 2h. Pseudo gutta appeared in more than half of the subjects, and the CEC shape changed upon instillation of one drop of ripasudil; however, it recovered at 4-6h after instillation. Therefore, these changes would not greatly impact clinical treatment. Similar to the presence of conjunctival hyperemia, changes in CEC shape occur at early stages after instillation of ripasudil.

Cortical Actin Dynamics in Endothelial Permeability.

The pulmonary endothelial cell forms a critical semi-permeable barrier between the vascular and interstitial space. As part of the blood-gas barrier in the lung, the endothelium plays a key role in normal physiologic function and pathologic disease. Changes in endothelial cell shape, defined by its plasma membrane, determine barrier integrity. A number of key cytoskeletal regulatory and effector proteins including non-muscle myosin light chain kinase, cortactin, and Arp 2/3 mediate actin rearrangements to form cortical and membrane associated structures in response to barrier enhancing stimuli. These actin formations support and interact with junctional complexes and exert forces to protrude the lipid membrane to and close gaps between individual cells. The current knowledge of these cytoskeletal processes and regulatory proteins are the subject of this review. In addition, we explore novel advancements in cellular imaging that are poised to shed light on the complex nature of pulmonary endothelial permeability.

Endoglin moves and shapes endothelial cells.

Vascular malformations result from improper blood vessel responses to molecular and mechanical signals. Two studies now show that endothelial cell migration and cell shape changes are perturbed in mutants lacking the TGFβ/BMP co-receptor endoglin, leading to arteriovenous shunts. Endoglin coordinates endothelial cell responses to ligand-receptor signalling and flow-mediated mechanical cues.

Endoglin controls blood vessel diameter through endothelial cell shape changes in response to haemodynamic cues.

The hierarchical organization of properly sized blood vessels ensures the correct distribution of blood to all organs of the body, and is controlled via haemodynamic cues. In current concepts, an endothelium-dependent shear stress set point causes blood vessel enlargement in response to higher flow rates, while lower flow would lead to blood vessel narrowing, thereby establishing homeostasis. We show that during zebrafish embryonic development increases in flow, after an initial expansion of blood vessel diameters, eventually lead to vessel contraction. This is mediated via endothelial cell shape changes. We identify the transforming growth factor beta co-receptor endoglin as an important player in this process. Endoglin mutant cells and blood vessels continue to enlarge in response to flow increases, thus exacerbating pre-existing embryonic arterial-venous shunts. Together, our data suggest that cell shape changes in response to biophysical cues act as an underlying principle allowing for the ordered patterning of tubular organs.

Rac-1 as a new therapeutic target in cerebro- and cardio-vascular diseases.

Growing evidence indicates that overproduction of reactive oxygen species (ROS) plays a prominent role in the development of cardio- and cerebro-vascular diseases. Among the mechanisms identified to produce oxidative stress in the vascular wall, those mediated by membrane-bound NAD(P)H oxidases represent a major one. NAD(P)H oxidases are a family of enzymes that generate ROS both in phagocytic and non-phagocytic cell types. Vascular NAD(P)H oxidase contains the membrane-bound subunits Nox1, Nox2 (gp91phox), Nox4 and p22phox, the catalytic site of the oxidase, and the cytosolic components p47phox and p67phox. Rac1 (Ras-related C3 botulinum toxin substrate1) is a small GTPase essential for the assembly and activation of NADPH oxidase. Several molecular and cellular studies have reported the involvement of Rac1 in different cardiovascular pathologies, such as vascular smooth muscle proliferation, cardiomyocyte hypertrophy, endothelial cell shape change, atherosclerosis and endothelial dysfunction in hypertension. In addition, increased activation of NADPH oxidase by Rac1 has been reported in animals and humans after myocardial infarction and heart failure. The Rac1/NADPH pathway has also been found involved in different pathologies of the cerebral district, such as ischemic stroke, cognitive impairment, subaracnoid hemorrhage and neuronal oxidative damage typical of several neurodegenerative disorders. In addition, thrombotic events are an important step in the onset of cardio- and cerebrovascular diseases. Rac1 has been found involved also in platelet activation, inducing actin polymerization and lamellipodia formation, which are necessary steps for platelet aggregation. Taken together, the evidence candidates Rac1 as a new pharmacological target of cardiovascular and cerebrovascular diseases. Although the involvement of Rac1 in the beneficial pleiotropic effects of drugs such as statins is well known, and the onset of numerous side effects has raised concern for the management of some patient groups. Interestingly, a novel selective Rac1 inhibitor, NSC23766, has recently been introduced; its use has been reported mainly in the oncology field. Future studies are needed to extend its application to cardio- and cerebro-vascular diseases, and translate its use to humans.

Neural Wiskott-Aldrich Syndrome Protein (N-WASP)-mediated p120-Catenin Interaction with Arp2-Actin Complex Stabilizes Endothelial Adherens Junctions

Stable adherens junctions (AJs) are required for formation of restrictive endothelial barrier. Vascular endothelial cadherin from contiguous endothelial cells forms AJs, which are stabilized intracellularly by binding of p120-catenin and cortical actin. Mechanisms inducing cortical actin formation and enabling its linkage with p120-catenin remain enigmatic. We altered the function of neural Wiskott-Aldrich syndrome protein (N-WASP), which induces actin polymerization through actin-related protein 2/3 complex (Arp2/3), to address the role of N-WASP in regulating AJ stability and thereby endothelial permeability. We show that depletion of N-WASP in endothelial cells impaired AJ adhesion and favored the organization of actin from cortical actin to stress fibers, resulting thereby in formation of leaky endothelial barrier. Exposure of the N-WASP-depleted endothelial cell monolayer to the permeability-increasing mediator, thrombin, exaggerated AJ disruption and stress fiber formation, leading to an irreversible increase in endothelial permeability. We show that N-WASP binds p120-catenin through its verprolin cofilin acid (VCA) domain, induces cortical actin formation through Arp2, and links p120-catenin with cortical actin. The interaction of N-WASP with p120-catenin, actin, and Arp2 requires phosphorylation of N-WASP at the Tyr-256 residue by focal adhesion kinase. Expression of the VCA domain of N-WASP or phosphomimicking (Y256D)-N-WASP mutant in endothelial cells stabilizes AJs and facilitates barrier recovery after thrombin stimulation. Our study demonstrates that N-WASP, by mediating p120-catenin interaction with actin-polymerizing machinery, maintains AJs and mitigates disruption of endothelial barrier function by edemagenic agents, therefore representing a novel target for preventing leaky endothelial barrier syndrome.

Vascular Lumen Formation

The vascular system developed early in evolution. It is required in large multicellular organisms for the transport of nutrients, oxygen, and waste products to and from tissues. The vascular system is composed of hollow tubes, which have a high level of complexity in vertebrates. Vasculogenesis describes the de novo formation of blood vessels, e.g., aorta formation in vertebrate embryogenesis. In contrast, angiogenesis is the formation of blood vessels from preexisting ones, e.g., sprouting of intersomitic blood vessels from the aorta. Importantly, the lumen of all blood vessels in vertebrates is lined and formed by endothelial cells. In both vasculogenesis and angiogenesis, lumen formation takes place in a cord of endothelial cells. It involves a complex molecular mechanism composed of endothelial cell repulsion at the cell-cell contacts within the endothelial cell cords, junctional rearrangement, and endothelial cell shape change. As the vascular system also participates in the course of many diseases, such as cancer, stroke, and myocardial infarction, it is important to understand and make use of the molecular mechanisms of blood vessel formation to better understand and manipulate the pathomechanisms involved.

Microtubule‐associated protein EB3 regulates calcium signaling and facilitates increase in endothelial permeability

End binding proteins (EBs) are highly conserved microtubule plus‐end tracking accessory factors that bind to growing microtubule ends and suppress microtubule catastrophe events. We investigated the roles of EB1 and EB3 in regulating endothelial cytoskeletal dynamics and cell shape change, the primary determinants of the permeability of endothelial barrier. Protein depletion experiments showed that EB3, but not EB1, facilitates thrombin‐induced release of calcium from endoplasmic reticulum (ER), and thereby activates calcium entry from extracellular stores. We observed that EB3 interacts with inositol 1,4,5‐thriphosphate receptor type 3 (IP3R3) in lung microvasculature. Cell permeant variant of IP3R3 derivative peptide containing EB3 consensus motif reduced the interaction between EB3 and IP3R3 and mitigated increased endothelial permeability response associated with the cell shape changes. Furthermore, infusion of IP3R3 derivative peptide in ex vivo murine lung preparation attenuates the increase in lung vascular liquid permeability (Kf,c) in response to activation of the Protease‐Activated Receptor ‐1. Intravenous injection of this peptide prevents lung injury and reduces the lethality of polymicrobial sepsis in mice. These data indicate that EB3 regulates calcium signaling in endothelium and facilitates pulmonary vascular leak in disease states.

Synthesis and Antitumor Activity of 1,5-Disubstituted 1,2,4-Triazoles as Cis-Restricted Combretastatin Analogues

A series of 1-aryl-5-(3',4',5'-trimethoxyphenyl) derivatives and their related 1-(3',4',5'-trimethoxyphenyl)-5-aryl-1,2,4-triazoles, designed as cis-restricted combretastatin analogues, were synthesized and evaluated for antiproliferative activity, inhibitory effects on tubulin polymerization, cell cycle effects, and apoptosis induction. Their activity was greater than, or comparable with, that of the reference compound CA-4. Flow cytometry studies showed that HeLa and Jurkat cells treated with the most active compounds 4l and 4o were arrested in the G2/M phase of the cell cycle in a concentration dependent manner. This effect was accompanied by apoptosis of the cells, mitochondrial depolarization, generation of reactive oxygen species, activation of caspase-3, and PARP cleavage. Compound 4l was also shown to have potential antivascular activity, since it induced endothelial cell shape change in vitro and disrupted the sprouting of endothelial cells in the chick aortic ring assay.

Novel aspects of the roles of Rac1 GTPase in the cardiovascular system

Rac1 GTPase is an established master regulator of cell motility through cortical actin re-organization and of reactive oxygen species generation through regulation of NADPH oxidase activity. Numerous molecular and cellular studies have implicated Rac1 in various cardiovascular pathologies: vascular smooth muscle proliferation, cardiomyocyte hypertrophy, and endothelial cell shape change. The physiological relevance of these in vitro findings, however, is just beginning to be reassessed with the newly developed, conditional mouse mutagenesis technology. Conditional gene targeting has also revealed unexpected, cell type-specific roles of Rac1. The aim of this review is to summarize the recent advance made in Rac1 research in the cardiovascular system, with special focus on its novel roles in the regulation of endothelial function, angiogenesis, and endothelium-mediated neuroprotection.

Endothelial Cells Form a Phalanx to Block Tumor Metastasis

Tumor blood vessels deliver oxygen poorly, thereby contributing to tumor hypoxia and upregulation of proangiogenic cytokines in an escalating feedback loop. Mazzone et al. (2009) now show that reducing the amount of a protein involved in endothelial oxygen sensing leads to changes in endothelial cell shape that interrupt this feedback loop and reduce tumor metastasis.

Role of calcineurin in thrombin‐mediated endothelial cell contraction

Barrier function and shape changes of endothelial cells (EC) are regulated by phosphorylation/dephosphorylation of key signaling and contractile elements. EC contraction results in intercellular gap formation and loss of the selective vascular barrier to circulating macromolecules. EC dysfunction elicited by thrombin was found to correlate with actin microfilament redistribution. It is known that calcineurin (Cn) is involved in thrombin-induced EC dysfunction because inhibition of Cn potentiates PKC activity and the phosphorylation state of EC myosin light chain is also affected by Cn activity. Immunofluorescent detection of Cn catalytic subunit (CnA) isoforms coexpressed with GFP was visualized on paraformaldehyde (PFA) fixed bovine pulmonary artery endothelial cells (BPAEC). Actin microfilaments were stained with Texas Red-phalloidin. Cytotoxic effects of transfections or treatments and the efficiency of transfections were assessed by flow cytometry. Treatment of BPAEC with Cn inhibitors (cyclosporin A and FK506) hindered recovery of the cells from thrombin-induced EC dysfunction. Inhibition of Cn in the absence of thrombin had no effect on cytoskeletal actin filaments. We detected attenuated thrombin-induced stress fiber formation and changes in cell shape only when cells were transfected with constitutively active CnA and not with various CnA isoforms. Flow cytometry (FCM) analysis has proved that cytotoxic effect of treatments is negligible. We observed that Cn is involved in the recovery from thrombin-induced EC dysfunction. Inhibition of Cn caused prolonged contractile effect, while overexpression of constitutively active CnA resulted in reduced thrombin-induced stress fiber formation.

Microtubule-Targeted Drugs Inhibit VEGF Receptor-2 Expression by both Transcriptional and Post-Transcriptional Mechanisms

Cytoskeletal polymers control a wide range of cellular functions, including proliferation, migration, and gene expression. As changes in endothelial cell shape and motility are required to form vascular networks, we hypothesized that disassembly of actin filaments or microtubules may impact endothelial vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) expression as a critical determinant of angiogenesis. We therefore investigated the effect of actin filament- and microtubule-disrupting agents on VEGFR1 and VEGFR2 expression by endothelial cells. Microtubule (MT) disassembly greatly inhibited endothelial VEGFR2 expression, whereas VEGFR1 expression levels remained largely unchanged. These suppressive effects were neither conveyed by increased VEGFR2 shedding nor by shortened protein half-life, suggesting that transcriptional mechanisms account for the observed effects. In line with this conclusion, MT disruption significantly suppressed endothelial VEGFR2 mRNA accumulation. The treatment considerably decreased transcriptional activity of 5'-deletional VEGFR2 promoter gene constructs. MT disruption-mediated repression was conveyed by a GC-rich region harboring two consensus Sp1-binding sites. Electrophoretic mobility-shift assay analysis demonstrated that constitutive Sp1-dependent DNA binding is decreased by MT disassembly. In addition, we provide evidence for additional post-transcriptional regulatory mechanisms, as the VEGFR2 mRNA half-life is significantly reduced by MT-disrupting agents. Hence, both inhibition of the rate of gene transcription and increased mRNA turnover represent critical molecular mechanisms by which MT disruption inhibits VEGFR2 expression.

Gαq-TRPC6-mediated Ca2+ Entry Induces RhoA Activation and Resultant Endothelial Cell Shape Change in Response to Thrombin

RhoA activation and increased intracellular Ca(2+) concentration mediated by the activation of transient receptor potential channels (TRPC) both contribute to the thrombin-induced increase in endothelial cell contraction, cell shape change, and consequently to the mechanism of increased endothelial permeability. Herein, we addressed the possibility that TRPC signals RhoA activation and thereby contributes in actinomyosin-mediated endothelial cell contraction and increased endothelial permeability. Transduction of a constitutively active Galphaq mutant in human pulmonary arterial endothelial cells induced RhoA activity. Preventing the increase in intracellular Ca2+ concentration by the inhibitor of Galphaq or phospholipase C and the Ca2+ chelator, BAPTA-AM, abrogated thrombin-induced RhoA activation. Depletion of extracellular Ca2+ also inhibited RhoA activation, indicating the requirement of Ca2+ entry in the response. RhoA activation could not be ascribed to storeoperated Ca2+ (SOC) entry because SOC entry induced with thapsigargin or small interfering RNA-mediated inhibition of TRPC1 expression, the predominant SOC channel in these endothelial cells, failed to alter RhoA activity. However, activation of receptor-operated Ca2+ entry by oleoyl-2-acetyl-sn-glycerol, the membrane permeable analogue of the Galphaq-phospholipase C product diacylglycerol, induced RhoA activity. Receptor-operated Ca2+ activation was mediated by TRPC6 because small interfering RNA-induced TRPC6 knockdown significantly reduced Ca2+ entry. TRPC6 knockdown also prevented RhoA activation, myosin light chain phosphorylation, and actin stress fiber formation as well as inter-endothelial junctional gap formation in response to either oleoyl-2-acetyl-sn-glycerol or thrombin. TRPC6-mediated RhoA activity was shown to be dependent on PKCalpha activation. Our results demonstrate that Galphaq activation of TRPC6 signals the activation of PKCalpha, and thereby induces RhoA activity and endothelial cell contraction.

Regulation of Endothelial Nitric Oxide Synthase by Thrombin

Endothelial cells play a pivotal role in the regulation of vascular function. Not only do endothelial cells modulate vascular tone and control the initiation and progression of vascular inflammation through regulated secretion of vasoactive agents and by surface expression of adhesion molecules, but they also provide a hemocompatible vessel lining by molecular control of platelet aggregation, coagulation, and fibrinolysis.1,2 One of the many vasoactive agents important in maintaining both vascular integrity and hemostasis is thrombin, the main effector serine protease of the coagulation cascade.3,4 Physiologically, thrombin is short lived in the circulation, and in the context of a normal endothelium, thrombin activates the protein C system to terminate its own production.2,4 However, in pathological conditions, at sites of vascular injury, thrombin has a host of direct actions. It is a potent activator of platelets, it induces shape and permeability changes of endothelial cells, it mobilizes adhesion molecules to the endothelial surface, it stimulates autocoid and proinflammatory cytokine production, it regulates blood vessel diameter by modulating endothelium-dependent vasodilation and endothelium-independent vascular smooth muscle vasoconstriction, and it stimulates vascular cell growth.1–7 Endothelial effects of thrombin have been attributed to NO production through activation of endothelial NO synthase (eNOS), which requires phosphorylation of Ser1179.8,9 Exact mechanisms whereby thrombin regulates eNOS phosphorylation remain unclear, although Ca2+/calmodulin and Akt have been implicated to be critical. In the current issue of Hypertension , Motley et …