Beata Wojciak-Stothard

Regulation of TNF-alpha-induced reorganization of the actin cytoskeleton and cell-cell junctions by Rho, Rac, and Cdc42 in human endothelial cells.

We have investigated the role of the small guanosine‐trisphosphate (GTP)‐binding proteins, Rho, Rac, and Cdc42, in the early responses of human umbilical vein endothelial cells (HUVECs) to TNF‐α (tumor necrosis factor‐α). Quiescent confluent HUVECs incubated with TNF‐α for 5–30 min showed an increased formation of membrane ruffles, filopodia, and actin stress fibres followed by cell retraction and formation of intercellular gaps. This process was accompanied by the dispersion of cadherin‐5 from intercellular junctions. TNF‐α also induced a transient increase in polymerized F‐actin, as determined both by measuring G‐actin content and by quantifying fluorescent emission from fluorescein isothiocyanate (FITC)‐phalloidin‐labelled F‐actin. Microinjection of cells with activated RhoA protein led to an increase in polymerized actin, formation of stress fibres, cell retraction as well as dispersion of cadherin‐5. The proteins Cdc42 and Rac induced qualitatively similar effects to Rho, although not as dramatic and in addition induced formation of filopodia and lamellipodia. Microinjection of cells with a Rho inhibitor, C3 transferase, prevented gap formation caused by TNF‐α. Similar effects were observed in cells microinjected with the dominant inhibitory proteins N17Cdc42 and N17Rac1. Cell retraction and gap formation were also prevented by inhibitors of myosin light chain kinase (MLCK). Our data suggest that Cdc42, Rac, and Rho are activated in a hierarchical cascade following stimulation with TNF‐α leading to actomyosin‐mediated cell retraction and formation of intercellular gaps. J. Cell. Physiol. 176:150–165, 1998.

Disruption of methylarginine metabolism impairs vascular homeostasis.

Asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA) are endogenously produced amino acids that inhibit all three isoforms of nitric oxide synthase (NOS). ADMA accumulates in various disease states, including renal failure, diabetes and pulmonary hypertension, and its concentration in plasma is strongly predictive of premature cardiovascular disease and death. Both L-NMMA and ADMA are eliminated largely through active metabolism by dimethylarginine dimethylaminohydrolase (DDAH) and thus DDAH dysfunction may be a crucial unifying feature of increased cardiovascular risk. However, despite considerable interest in this pathway and in the role of ADMA as a cardiovascular risk factor, there is little evidence to support a causal role of ADMA in pathophysiology. Here we reveal the structure of human DDAH-1 and probe the function of DDAH-1 both by deleting the DDAH1 gene in mice and by using DDAH-specific inhibitors which, as we demonstrate by crystallography, bind to the active site of human DDAH-1. We show that loss of DDAH-1 activity leads to accumulation of ADMA and reduction in NO signaling. This in turn causes vascular pathophysiology, including endothelial dysfunction, increased systemic vascular resistance and elevated systemic and pulmonary blood pressure. Our results also suggest that DDAH inhibition could be harnessed therapeutically to reduce the vascular collapse associated with sepsis.

Shear stress–induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases

Shear stress induces endothelial polarization and migration in the direction of flow accompanied by extensive remodeling of the actin cytoskeleton. The GTPases RhoA, Rac1, and Cdc42 are known to regulate cell shape changes through effects on the cytoskeleton and cell adhesion. We show here that all three GTPases become rapidly activated by shear stress, and that each is important for different aspects of the endothelial response. RhoA was activated within 5 min after stimulation with shear stress and led to cell rounding via Rho-kinase. Subsequently, the cells respread and elongated within the direction of shear stress as RhoA activity returned to baseline and Rac1 and Cdc42 reached peak activation. Cell elongation required Rac1 and Cdc42 but not phosphatidylinositide 3-kinases. Cdc42 and PI3Ks were not required to establish shear stress–induced polarity although they contributed to optimal migration speed. Instead, Rho and Rac1 regulated directionality of cell movement. Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement. Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow.

Microtubules Regulate Migratory Polarity through Rho/ROCK Signaling in T Cells

Background Migrating leukocytes normally have a polarized morphology with an actin-rich lamellipodium at the front and a uropod at the rear. Microtubules (MTs) are required for persistent migration and chemotaxis, but how they affect cell polarity is not known. Methodology/Principal Findings Here we report that T cells treated with nocodazole to disrupt MTs are unable to form a stable uropod or lamellipodium, and instead often move by membrane blebbing with reduced migratory persistence. However, uropod-localized receptors and ezrin/radixin/moesin proteins still cluster in nocodazole-treated cells, indicating that MTs are required specifically for uropod stability. Nocodazole stimulates RhoA activity, and inhibition of the RhoA target ROCK allows nocodazole-treated cells to re-establish lamellipodia and uropods and persistent migratory polarity. ROCK inhibition decreases nocodazole-induced membrane blebbing and stabilizes MTs. The myosin inhibitor blebbistatin also stabilizes MTs, indicating that RhoA/ROCK act through myosin II to destabilize MTs. Conclusions/Significance Our results indicate that RhoA/ROCK signaling normally contributes to migration by affecting both actomyosin contractility and MT stability. We propose that regulation of MT stability and RhoA/ROCK activity is a mechanism to alter T-cell migratory behavior from lamellipodium-based persistent migration to bleb-based migration with frequent turning.

Reduced MicroRNA-150 Is Associated with Poor Survival in Pulmonary Arterial Hypertension

RATIONALE MicroRNAs (miRNAs or miRs) are implicated in the pathogenesis of various cardiovascular diseases, including pulmonary arterial hypertension (PAH). OBJECTIVES We sought to measure changes in plasma levels of miRNAs in patients with PAH and relate them to the severity of the disease. METHODS A microarray screen was performed on total plasma RNA from eight patients with PAH and eight healthy control subjects. Quantitative polymerase chain reaction confirmed reduced miR-150 concentrations and was then used to measure miR-150 levels in (1) two separate cohorts of patients with PAH, from London (n = 145) and Sheffield (n = 30), respectively; (2) circulating microvesicles and blood cells; and (3) lungs from a monocrotaline rat model. MEASUREMENTS AND MAIN RESULTS Fifty-eight miRNAs showed differences in plasma concentration and miR-150 the largest down-regulation in PAH. Receiver-operator-characteristic analysis showed both raw and normalized plasma miR-150 levels correlated with 2-year survival (P < 0.01) in patients with PAH. Cox regression analysis confirmed miR-150 levels as a significant predictor of survival. Age, baseline cardiac index, World Health Organization functional class, 6-minute walk distance, disease duration, and red cell distribution width also predicted survival. Entering these covariates in a multivariable model verified plasma miR-150 levels as an independent predictor of survival in PAH (hazard ratio, 0.533; P = 0.010). miR-150 levels also predicted survival in a second, independent PAH cohort. miR-150 levels were significantly reduced in circulating microvesicles from patients with PAH and the lungs of the monocrotaline rat. CONCLUSIONS Reduced circulating miR-150 levels are associated with poor survival in PAH.

The ADMA/DDAH pathway is a critical regulator of endothelial cell motility

Asymmetric dimethylarginine (ADMA) is an inhibitor of nitric oxide production associated with abnormal blood vessel growth and repair, however, the mechanism of action of ADMA is not well understood. We studied the role of exogenous and endogenous ADMA in the regulation of cell motility and actin cytoskeleton in porcine pulmonary endothelial cells (PAECs) and pulmonary microvascular endothelial cells (PMECs) from knockout mice that lack one of the enzyme metabolising ADMA, dimethylarginine dimethylaminohydrolase I (DDAHI) as well as endothelial cells overexpressing DDAH in vitro. We show that ADMA induced stress fibre and focal adhesion formation and inhibited cell motility in primary pulmonary endothelial cells. The effects of ADMA depended on the activity of RhoA and Rho kinase and were reversed by overexpression of DDAH, nitric oxide donors and protein kinase G activator, 8-bromo-cGMP. ADMA also inhibited the activities of Rac1 and Cdc42 in cells but these changes had a minor effect on cell motility. Endogenous ADMA increased RhoA activity and inhibited cell motility in PMECs from DDAHI knockout mice and inhibited angiogenesis in vitro. These results are the first demonstration that metabolism of cardiovascular risk factor ADMA regulates endothelial cell motility, an important factor in angiogenesis and vascular repair.

Modulation of Rac1 Activity by ADMA/DDAH Regulates Pulmonary Endothelial Barrier Function

Endogenously produced nitric oxide synthase inhibitor, asymmetric methylarginine (ADMA) is associated with vascular dysfunction and endothelial leakage. We studied the role of ADMA, and the enzymes metabolizing it, dimethylarginine dimethylaminohydrolases (DDAH) in the regulation of endothelial barrier function in pulmonary macrovascular and microvascular cells in vitro and in lungs of genetically modified heterozygous DDAHI knockout mice in vivo. We show that ADMA increases pulmonary endothelial permeability in vitro and in in vivo and that this effect is mediated by nitric oxide (NO) acting via protein kinase G (PKG) and independent of reactive oxygen species formation. ADMA-induced remodeling of actin cytoskeleton and intercellular adherens junctions results from a decrease in PKG-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and a subsequent down-regulation of Rac1 activity. The effects of ADMA on endothelial permeability, Rac1 activation and VASP phosphorylation are prevented by overexpression of active DDAHI and DDAHII, whereas inactive DDAH mutants have no effect. These findings demonstrate for the first time that ADMA metabolism critically determines pulmonary endothelial barrier function by modulating Rac1-mediated remodeling of the actin cytoskeleton and intercellular junctions.

Role of RhoB in the regulation of pulmonary endothelial and smooth muscle cell responses to hypoxia.

Rationale: RhoA and Rho kinase contribute to pulmonary vasoconstriction and vascular remodeling in pulmonary hypertension. RhoB, a protein homologous to RhoA and activated by hypoxia, regulates neoplastic growth and vasoconstriction but its role in the regulation of pulmonary vascular function is not known. Objective: To determine the role of RhoB in pulmonary endothelial and smooth muscle cell responses to hypoxia and in pulmonary vascular remodeling in chronic hypoxia-induced pulmonary hypertension. Methods and Results: Hypoxia increased expression and activity of RhoB in human pulmonary artery endothelial and smooth muscle cells, coincidental with activation of RhoA. Hypoxia or adenoviral overexpression of constitutively activated RhoB increased actomyosin contractility, induced endothelial permeability, and promoted cell growth; dominant negative RhoB or manumycin, a farnesyltransferase inhibitor that targets the vascular function of RhoB, inhibited the effects of hypoxia. Coordinated activation of RhoA and RhoB maximized the hypoxia-induced stress fiber formation caused by RhoB/mammalian homolog of Drosophila diaphanous-induced actin polymerization and RhoA/Rho kinase-induced phosphorylation of myosin light chain on Ser19. Notably, RhoB was specifically required for hypoxia-induced factor-1&agr; stabilization and for hypoxia- and platelet-derived growth factor-induced cell proliferation and migration. RhoB deficiency in mice markedly attenuated development of chronic hypoxia-induced pulmonary hypertension, despite compensatory expression of RhoA in the lung. Conclusions: RhoB mediates adaptational changes to acute hypoxia in the vasculature, but its continual activation by chronic hypoxia can accentuate vascular remodeling to promote development of pulmonary hypertension. RhoB is a potential target for novel approaches (eg, farnesyltransferase inhibitors) aimed at regulating pulmonary vascular tone and structure.

The ADMA/DDAH Pathway Regulates VEGF-Mediated Angiogenesis

Objectives—Asymmetrical dimethylarginine (ADMA) is a nitric oxide synthase (NOS) inhibitor and cardiovascular risk factor associated with angiogenic disorders. Enzymes metabolising ADMA, dimethylarginine dimethylaminohydrolases (DDAH) promote angiogenesis, but the mechanisms are not clear. We hypothesized that ADMA/DDAH modifies endothelial responses to vascular endothelial growth factor (VEGF) by affecting activity of Rho GTPases, regulators of actin polymerization, and focal adhesion dynamics. Methods and Results—The effects of ADMA on VEGF-induced endothelial cell motility, focal adhesion turnover, and angiogenesis were studied in human umbilical vein endothelial cells (HUVECs) and DDAH I heterozygous knockout mice. ADMA inhibited VEGF-induced chemotaxis in vitro and angiogenesis in vitro and in vivo in an NO-dependent way. ADMA effects were prevented by overexpression of DDAH but were not associated with decreased proliferation, increased apoptosis, or changes in VEGFR-2 activity or expression. ADMA inhibited endothelial cell polarization, protrusion formation, and decreased focal adhesion dynamics, resulting from Rac1 inhibition after decrease in phosphorylation of vasodilator stimulated phosphoprotein (VASP). Constitutively active Rac1, and to a lesser extent dominant negative RhoA, abrogated ADMA effects in vitro and in vivo. Conclusion—The ADMA/DDAH pathway regulates VEGF-induced angiogenesis in an NO- and Rac1-dependent manner.

Simvastatin and sildenafil combine to attenuate pulmonary hypertension

Statins have been proposed to be a potential treatment for pulmonary arterial hypertension. If introduced into clinical practice, the statin would have to be used in conjunction with established therapy. We investigated the effects of combining simvastatin with a phosphodiesterase type-5 inhibitor, sildenafil, in the rat model of hypoxia-induced pulmonary hypertension. Rats were allocated to either: 1) a prevention protocol, to receive simvastatin 20 mg·kg−1·day−1 by intraperitoneal injection or sildenafil 75 mg·kg−1·day−1 orally or the combination (or vehicle) for 2 weeks beginning at the start of exposure to hypoxia (10% inspired oxygen); or 2) a treatment protocol, where the same agents were administered in the last 2 weeks of a 4-week period of hypoxia. In both protocols, the combination of sildenafil and simvastatin lowered pulmonary artery pressure and produced a significantly greater reduction in right ventricular hypertrophy and pulmonary vascular muscularisation than either drug alone. Moreover, the combination augmented significantly endothelial nitric oxide synthase expression and cGMP levels in the lung and right ventricle above that produced by either drug independently and resulted in greater inhibition of RhoA activity. These data suggest that simvastatin can be usefully combined with sildenafil in the treatment of pulmonary arterial hypertension to achieve greater therapeutic benefit.