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New drug targets for pulmonary hypertension: Rho GTPases in pulmonary vascular remodelling
  1. B Wojciak-Stothard
  1. Dr B Wojciak-Stothard, BHF Laboratories, Department of Medicine, University College London, 5 University Street, WC1E 6JJ, UK; b.wojciak-stothard{at}ucl.ac.uk

Abstract

Rho GTPases, key regulators of actin dynamics, play a major role in vascular processes such as endothelial permeability, cell motility, angiogenesis, nitric oxide production, smooth muscle contractility, cell proliferation and differentiation. In the lung, Rho GTPases control pulmonary vascular tone and remodelling. Their basal activity is important in fetal lung development and vascular adaptation to changes in oxygen levels, but their continuous activation in neonatal or adult lung leads to the development of pulmonary hypertension (PH), a condition characterised by excessive remodelling and hyperconstriction of pulmonary arteries. This review, based on recent molecular, cellular and animal studies, focuses on the current understanding of Rho GTPases signalling in pulmonary vascular physiology and pathophysiology. It also discusses the existing and prospective treatments targeting Rho GTPases in the management of PH.

  • Rho GTPases, therapy, pulmonary hypertension, vascular remodelling
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Pulmonary arterial hypertension (PH) is a condition of multifactorial origin. The World Health Organization aetiology based classification helps physicians to evaluate the patient and establish a therapeutic plan.1 2 PH is a disease characterised by abnormal remodelling of small pulmonary arteries, leading to increased pulmonary vascular resistance and right heart failure. Endothelial dysfunction is believed to be an early component of the pulmonary hypertensive process and involves a decrease in the production of vasorelaxants, such as nitric oxide and prostacyclin, and an increase in the production of vasoconstrictors, such as endothelin 1.3 This leads to an increase in vascular smooth muscle proliferation, extracellular matrix deposition, and hypercontractility. Alveolar hypoxia is believed to be an important contributor to the chronic PH associated with residence at high altitude and chronic lung diseases such as chronic obstructive pulmonary disease, cystic fibrosis, asthma and sleep apnoea, as well as persistent pulmonary hypertension of the newborn (PPHN).

In spite of clear clinical benefits, current drug treatments for PH involving antithrombotic agents, prostanoids, endothelin receptor antagonists and phosphodiesterase type 5 inhibitors are not entirely effective and raise concerns about routes of administration, side effects and insufficient long term prognosis.4

Recent studies have identified the Rho family of proteins as new drug targets for PH. Rho GTPases are affected by changes in oxygen levels and regulate nitric oxide production, endothelial permeability and smooth muscle contractility, which are important in the pathogenesis of PH. New evidence from animal studies shows that some of the currently used drugs for PH such as statins or the inhibitors of 5 phosphodiesterase may, at least in part, act through modification of the RhoA/Rho kinase pathway (fig 1).

Figure 1 Pathways affecting RhoA activity in pulmonary vasculature (see text for details). RhoA oscillates between an active guanosine triphosphate (GTP) bound state, and an inactive guanosine diphosphate (GDP) bound state. To exert its effects, RhoA has to be anchored to the cell membrane by an isoprenoid tail. Isoprenylation of Rho is inhibited by statins. Phosphorylation of RhoA by protein kinase A (PKA) or protein kinase G (PKG) promotes the association of RhoA with guanine dissociation inhibitor (GDI), that renders the protein inactive. Nitric oxide, phosphodiesterase 5 inhibitors (PDE5 inhibitors) or prostanoids increase the levels of cyclic guanosine monophosphate (cGMP) or cyclic adenosine monophosphate (cAMP), thus inactivating RhoA. The inhibitors of Rho kinase prevent endothelial dysfunction, smooth muscle cell (SMC) contractility, growth and differentiation. ADMA, asymmetric methylarginine; DDAH, dimethylarginine dimethylaminohydrolases; GAPs, GTPase activating proteins; GEFs, guanosine nucleotide exchange factors; VSMC, vascular smooth muscle cells.

The main objectives of this review are: (1) to present an overview of current research into Rho GTPases; (2) to describe how they function in the regulation of pulmonary vascular tone; and (3) to provide evidence of their possible therapeutic potential.

RHO GTPASES: AN OVERVIEW

Rho GTPases are regulatory molecules that link surface receptors to the organisation of the actin cytoskeleton.5 In addition to their role in the regulation of actin dynamics, Rho family members regulate diverse cellular functions including cell motility, cell cycle progression, cell survival, transcriptional regulation, membrane trafficking, and cytokinesis.611 In humans, 22 Rho genes encode more than 25 proteins, that have been divided into six groups of which RhoA, Rac and Cdc42 are best characterised.12 These proteins show approximately 30% homology with Ras (hence the name “Ras homologous”) and 80–90% homology with each other.5

Rho GTPases are activated by vasoactive substances, cytokines and growth factors (for example, thrombin, histamine, angiotensin II, endothelin 1’ PDGF, VEGF, prostaglandin E2) via heterotrimeric G protein coupled receptors, tyrosine kinase receptors, or integrin clustering.1315 They are also activated by mechanical and physical stimuli such as shear stress, stretch, pressure and hypoxia.1618

Rho GTPases activity cycle

The Rho GTPases act as molecular switches that cycle between an active guanosine 5c-triphosphate (GTP) bound and an inactive guanosine 5c diphosphate (GDP) bound conformation19 (fig 1). In their GTP bound state, they interact with downstream targets (effectors) to elicit cellular responses. The activity cycle is regulated by several proteins. The guanosine nucleotide exchange factors (GEFs) facilitate the exchange of GDP for GTP, while GTPase activating proteins (GAPs) attenuate GTPase signalling by increasing the rate of GTP hydrolysis. Guanine dissociation inhibitors (GDIs) sequester GDP bound Rho GTPases in the cytoplasm and inhibit their spontaneous GDP–GTP exchange activity. To exert their biological effects, Rho GTPases have to be anchored to the cell membrane by an isoprenoid tail. Isoprenoids are intermediates of the cholesterol de novo synthesis and their production is inhibited by statins.20 Recent evidence also shows that the activity of Rho GTPases can be controlled by phosphorylation/dephosphorylation cycle, dependent upon the level of nitric oxide. In vascular smooth muscle cells nitric oxide increases the levels of cyclic GMP that activate protein kinase G (PKG). PKG phosphorylates RhoA on Ser188, allowing GDI to extract RhoA from membranes and prevent its activation.21 RhoA can also be phosphorylated at the same residue by cyclic AMP dependent protein kinase A (PKA).22

Tools used in studies on Rho GTPases activation

The use of inhibitors helped to elucidate the physiological importance of Rho GTPases. Statins inhibit cholesterol synthesis by inhibiting the rate limiting enzyme in its pathway, HMG CoA reductase. They also inhibit synthesis of isoprenoids, the intermediates of the cholesterol synthesis, important for the formation of lipid attachments required for the activation and membrane localisation of Rho GTPases.20 23

More specific inhibition or activation of Rho GTPases can be achieved with mutant proteins, introduced into cells by microinjection or expressed following plasmid transfection or infection with viral constructs. Created by a single amino acid substitution, dominant negative mutants compete with endogenous GTPases for binding to cellular exchange factors (GEFs), while constitutively activated forms remain predominantly in the GTP bound, active form.24

A variety of bacterial toxins have been used to modify the activity of Rho GTPases.25 The C3 family of exoenzymes, C3 ADP-ribosyl transferase from Clostridium botulinum, Clostridium limosum transferase, B cereus transferase and epidermal differentiation inhibitor (EDIN) from Staphylococcus aureus, specifically inactivate Rho by ADP ribosylation that impairs its activation by GEFs. Clostridium difficile toxins A and B inactivate all Rho family proteins by glucosylating the nucleotide binding site. Salmonella typhimurium SptP acts as GAP protein for Rho family members. Other toxins, such as cytotoxic necrotising factor from Escherichia coli and the dermonecrotising toxin of Pertussis bacteria, activate Rho GTPases.25 Bacterial toxins and other proteins of interest can be introduced into the cells by cell penetrating peptides (CPPs) derived from the TAT protein from HIV virus (TAT peptide) or Drosophila antennapedia peptide,26 with less damage than the conventional techniques.27 28

The Rho kinase inhibitors include the pyridine derivative Y-27632,29 the closely related compound Y-3288530 and fasudil with its derivatives: HA-1077 and H-1152.31 32 Y-27632 was originally developed as a smooth muscle relaxant and was shown to inhibit stress fibre formation in fibroblasts, endothelial contraction and thrombin induced DNA synthesis in rat aortic smooth muscle cells (SMC).33

Rho GTPases in vascular endothelial cells

Two Rho GTPases, RhoA and Rac1, are thought to play opposing roles in the regulation of endothelial barrier function. RhoA was first shown to promote formation of stress fibres—long, contractile bundles of actomyosin filaments in cells—whereas Rac1 was shown to promote formation of lamellipodia.6 34 Activation of RhoA in vascular endothelial cells increases contractile forces that pull the intercellular junctions apart, resulting in increased permeability.15 Rac1 counteracts the actions of RhoA by increasing cell-to-cell adhesion.13 35

Apart from its effects on endothelial permeability, RhoA inhibits production of vasodilator nitric oxide (NO) by decreasing expression of nitric oxide synthase (NOS) in endothelial cells.36 37 In a reciprocal manner, NO may decrease RhoA expression and activity by reducing RhoA protein stability or inhibiting membrane translocation and activation of RhoA.38 39 Shortage of NO activates RhoA in cultured endothelial and vascular smooth muscle cells,38 40 and leads to vascular remodelling and hypertension.41 These data indicate that a fine balance exists between NO and RhoA pathways in normal physiological conditions, and malfunction of either pathway has severe pathophysiological implications.

Rho GTPases in vascular smooth muscle cells

RhoA is abundantly expressed in vascular smooth muscle and its activation increases vascular tone. Acting via its effector, Rho kinase (ROCK), RhoA helps to maintain a sustained contraction by increasing the levels of myosin light chain phosphorylation and allowing contraction to proceed even in the presence of relatively low levels of calcium—an effect known as calcium sensitisation.42 Rho kinase can also facilitate SMC contractility by increasing the levels of intracellular calcium, following inhibition of voltage gated K+ channels (KV).43 RhoA and Rho kinase are important for differentiation and survival of vascular SMC (VSMC) and their prolonged inhibition induces SMC apoptosis in vitro.4446 Rho proteins can also regulate extracellular matrix production in various cell types47 48 and their inhibition reduces neointima formation in PH animals.49

RHO GTPASES IN THE PULMONARY VASCULATURE

The most important function of Rho GTPases in pulmonary vasculature is the regulation of vascular tone. Pulmonary vessels have to respond quickly to changes in oxygenative conditions in order to regulate oxygen supply for the whole body. Hypoxia activates RhoA and Rho in pulmonary vasculature, and the implications of this activation strongly depend on the developmental stage of the lung and the duration of hypoxia.

Fetal and neonatal lung

In fetal life hypoxia notably augments branching morphogenesis in the lung, the effect associated with the activation of RhoA.50 High activity of RhoA in pulmonary vasculature in utero helps to maintain a high level of vascular resistance.51 52 Pulmonary vascular resistance falls at birth, when pulmonary arteries dilate and blood flow increases. Postnatal dilatation is characterised by extensive remodelling of pulmonary arteries. Endothelial cells become thinner and lose actomyosin filaments.53 VSMC also show reduction in F-actin content and relax, possibly due to a decrease in the circulating vasoconstrictor leukotrienes and increased release of bradykinin.54 Failure to adapt to extrauterine life causes persistent pulmonary hypertension of the newborn (PPHN), a condition often caused by perinatal exposure to chronic hypoxia and manifested by increased pulmonary vascular resistance.52 Recent data from animal models of the disease suggest that abnormal pulmonary arterial remodelling may result from sustained changes in the activities of RhoA and Rho kinase in pulmonary arterial endothelial and smooth muscle cells.55 56 Cultured pulmonary artery endothelial cells from piglets with chronic hypoxia induced PPHN show chronic activation of RhoA and inhibition of Rac1, changes that contribute to the abnormal phenotype and the breakdown of the endothelial barrier function.55 Loss of endothelial barrier function allows proliferative mediators to leak into the underlying vascular tissue cells such as SMC and fibroblasts, enhancing cellular proliferation and causing medial and adventitial hypertrophy characteristic of PH. Normal phenotype and permeability can be restored by inhibiting RhoA/Rho kinase pathway with dominant negative RhoA (N19RhoA], C3 transferase, Y-27632 or activating Rac1 by over-expressing constitutively activated Rac1 or treating cells with sphingosine-1-phosphate (S1P). S1P is a natural, platelet derived lipid of considerable clinical and therapeutic interest.57 58

Adult lung

Acute hypoxia induces hypoxic pulmonary vasoconstriction (HPV), a transient reaction that contributes to ventilation–perfusion matching in the lung by diverting blood flow to oxygen-rich areas. HPV can be fully inhibited by the Rho kinase inhibitor Y-27632 in rat lungs, which indicates the importance of the RhoA/Rho kinase pathway in this response.59 60

Chronic hypoxia causes a sustained activation of RhoA and Rho kinase in pulmonary arteries leading to endothelial dysfunction and medial smooth muscle proliferation and hypercontractility, which is characteristic of PH. The importance of RhoA in pulmonary arterial remodelling was demonstrated in patients and animals with PH.2 61 RhoA may contribute to this condition by mediating the effects of vasoconstrictors such as angiotensin II, endothelin 1 (ET 1) and acetylcholine and/or down regulating eNOS expression and NO production in endothelial cells. Our recent data show that both oxygen and nitric oxide deprivation activate RhoA and inhibit Rac1 in cultured primary pulmonary artery endothelial cells.40 55 Interestingly, cardiovascular risk factor and NOS inhibitor, asymmetric methylarginine (ADMA), activates RhoA40 and its levels are increased in the lungs of patients and animals with PH.62 63 Pharmacological targeting of the enzymes metabolising ADMA, dimethylarginine dimethylaminohydrolases (DDAH), may be important in new strategies for treatment of PH.

THERAPEUTIC POTENTIAL OF RHO INHIBITORS

Recent studies suggest that Rho GTPases may, at least in part, mediate the effects of drugs used in the treatment of PH, such as statins, 5 phosphodiesterase inhibitors and endothelin receptor antagonists. Specific inhibitors of Rho kinase, fasudil and Y-27632, show great therapeutic potential in animal models of PH, and the acute administration of fasudil reduces vascular resistance in patients with severe PH. The long term effects of Rho kinase inhibitors are currently assessed in clinical trials.

Statins

Statins have been clinically used for primary and secondary prevention of atherothrombosis. They have beneficial, “pleiotropic” effects on the pulmonary vasculature, that include augmentation of endothelial function, increase of eNOS expression and NO mediated vasodilation, as well as inhibition of proliferation of endothelial and vascular smooth muscle cells.20 23 Evidence that this occurs via inhibition of Rho/Rho kinase dependent pathways has been provided both in vitro and in vivo. Simvastatin decreased neointimal VSMC proliferation and upregulated cell cycle inhibitors, endothelial NO synthase and bone morphogenetic protein receptor type 1α in monocrotaline induced PH in rats.49 Other authors found that simvastatin slowed down the progression of PH, although it did not reduce mortality in the same rat model of PH.64 Simvastatin also prevented chronic hypoxia induced PH in rats by reducing signals from vascular endothelial growth factor and increasing endothelial cell apoptosis, the effects associated with RhoA inhibition and increased expression of Rac1.65

Recent clinical studies helped to evaluate the suitability of statins in the treatment of PH. An open label observational study on 16 patients with PH has shown beneficial effects of simvastatin (20–80 mg/day) on 6 min walk performance and haemodynamics without complications.66 A clinical trial, based at Imperial College London, is currently randomising patients with PH to simvastatin or placebo (ClinicalTrials.gov Identifier: NCT00180713). Another clinical trial in PH combines simvastatin and aspirin in a randomised, double blind, placebo controlled study (NCT 00384865).64

While the results from the animal models of PH are encouraging, some studies have shown adverse effects of statins on endothelial function. Prolonged treatment of mice with atorvastatin downregulated endothelial NO production, after the treatment was withdrawn. This was caused by a massive membrane translocation and activation of Rho, when the availability of isoprenoids was restored upon the removal of atorvastatin.37

Main messages

  • Rho GTPases are important for pulmonary vascular homeostasis in neonatal and adult lung, but their continuous activation causes pulmonary arterial remodelling and pulmonary hypertension (PH).

  • The outcome of currently used therapies for the treatment of PH is unsatisfactory. The Rho kinase inhibitors, Y-2762 and fasudil, demonstrate a dramatic therapeutic potential in treatment of PH, in particular in cases refractory to nitric oxide.

  • Combinational therapy involving Rho and Rho kinase inhibitors and conventional drugs, such as prostacyclin, shows most promise. Activation of Rac1 in pulmonary endothelium may prove beneficial in prevention of endothelial dysfunction in PH.

Phosphodiesterase type 5 inhibitors

The type 5 phosphodiesterase (PDE5) is the major cGMP degrading enzyme in the lung and its activity and protein levels are upregulated in PH.6769 Cyclic GMP is required for the vasodilatory signals of prostaglandin PGI2, NO and atrial natriuretic peptide (ANP), likely to result from RhoA inhibition in vascular smooth muscle.38 Sildenafil, an orally active, selective inhibitor of PDE5 was shown to inhibit RhoA/Rho kinase dependent remodelling of the pulmonary artery in chronically hypoxic PH rats through enhanced RhoA phosphorylation and cytosolic sequestration by GDI.70 Its suitability for PH treatment was revealed in a clinical trial involving 278 patients with PH.71

Main research questions

  • Further work is required to establish the effects of chronic administration of Rho and Rho kinase inhibitors.

  • More integrated translational research is needed to evaluate the suitability of drug targets established in animal models for use in clinical practice.

  • More studies on new Rho kinase inhibitors and selective routes of drug delivery are also needed, in particular those involving viral vectors and cell penetrating fusion proteins.

Endothelin 1 receptor antagonists and prostanoids

Endothelin 1 was shown to activate RhoA and Rho kinase in the pulmonary circulation of chronically hypoxic hypertensive rats, the effect prevented by ETA receptor antagonists.72 It is therefore conceivable that ETA/B receptor antagonists used in clinical practice, such as bosentan, may exert their effects via the RhoA/Rho kinase pathway.

Although prostacyclin lacks a direct inhibitory effect on Rho kinase,2 it may affect RhoA activity and its downstream effectors by increasing the levels of intracellular cAMP.22

Rho kinase inhibitors

Rho kinase inhibitors show great therapeutic promise. Intravenous administration of fasudil or Y-27632 attenuated PH and pulmonary artery remodelling and enhanced eNOS expression in monocrotalline and chronic hypoxia induced PH in rodents.73 74 Rho kinase inhibitors were found to have a synergistic effect with sildenafil in the treatment of PH induced by chronic hypoxia70 and prevent pulmonary arterial remodelling in high flow induced PH in rats.75

Inhaled fasudil selectively reduces PA pressure, as opposed to inhaled NO, which reduces both pulmonary and systemic pressures in rat models of PH.76

In a clinical trial involving nine patients with severe PH, intravenous fasudil (30 mg for 30 min) reduced pulmonary vascular resistance without producing systemic hypotension or any other side effects.61 Clinical trials with long term oral administration of fasudil in patients with PH are underway in Japan.2 Recently, inhibitors of Rho kinase were shown to have acute vasodilatory effects in pulmonary hypertensive neonatal rats unresponsive to NO, which shows their potential as drug targets in neonates with PH that is refractory to NO.77 Combination therapy of fasudil and prostacyclin in monocrotalline induced PH in rats was significantly more beneficial in the treatment of the disease, when compared with each monotherapy.2 While intravenous administration of fasudil proved successful in reducing pulmonary vascular resistance in PH, the majority of animal models suggest that inhaled administration of Y-27632 and fasudil will provide a more selective route for treating those diseases. At present intravenous fasudil is the only clinically available Rho kinase inhibitor, but its oral forms and other compounds are undergoing investigation.

In spite of the evidence of positive effects of Rho kinase inhibitors in PH, a prolonged treatment may result in an inhibition of beneficial adaptive responses to hypoxia. For example, inhibition of Rho kinase in chronic hypoxia induced PH in rats attenuated angiogenesis in the pulmonary circulation.78

Viral gene transfer

The use of recombinant adenoviruses to inhibit Rho pathway had beneficial effects on endothelial function in cultured cells from PH animals.55 More studies will be needed to verify suitability of viral gene transfer in the treatment of PH. However, the data from animal models are encouraging. Adenoviral gene therapy was successfully used in intratracheal transduction of the eNOS gene which ameliorated hypoxia induced PH in mice.79 E-NOS transduced bone marrow endothelial progenitor cells reversed the establishment of monocrotalline induced PH in rats.80 Recombinant adenoviruses can also be delivered via in vivo inhalation in mice.81 Many hurdles still remain in the successful gene therapy of PH, including site specific gene delivery, sustained gene expression, and immune response to vectors.4

Bacterial toxins

Bacterial toxins can be successfully used in vitro to modify the activity of Rho GTPases; however, their suitability for in vivo use requires further studies. Subcutaneous administration of C3 transferase in mice was shown to reduce cerebral ischaemia.37

REFERENCES

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Footnotes

  • Competing interests: None.

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