Minh-Quyen Q Le
email: minh-quyen.le@insa-lyon.fr

Minh Tu Pham

Mahdi Tavakoli

Richard Moreau

Sliding Mode Control of a Pneumatic Haptic Teleoperation System with On/off Solenoid Valves

Keywords: Pneumatic actuator, on/off solenoid valve, sliding mode control, haptic teleoperation, stability

This paper presents a novel bilateral control scheme for pneumatic teleoperation systems that are actuated by lowcost solenoid valves. A sliding mode control is incorporated into a two-channel, bilateral teleoperation architecture involving position-position, force-force, or force-position schemes. An analysis of stability and transparency of the closed-loop teleoperation system is carried out. The proposed control design is verified on a single-degree-of-freedom pneumatic teleoperation system with four on/off solenoid valves. Moreover, simulation results demonstrate high accuracies in terms of position and force tracking in the teleoperation system.

I. INTRODUCTION

Teleoperators are designed to enable humans to manipulate dangerous, remote, or delicate tasks with enhanced safety, at lower cost, or even with better accuracy than direct manipulation. Teleoperation has found applications in many areas including space and underwater explorations, nuclear waste handling, surgery, rehabilitation, training, education, and entertainment [START_REF] Hokayem | Bilateral teleoperation: An historical survey[END_REF], [START_REF] Hannaford | Scaling, impedance, and power flows in force reflecting teleoperation[END_REF].

In a teleoperation system, the human operator imposes a force on the master manipulator which results in a displacement that is transmitted to the slave [START_REF] Shimoga | A survey of perceptual feedback issues in dexterous telemanipulation: Part I. Finger force feedback[END_REF]. When the slave follows the master's motion and the force interaction with the environment is driven to the operator, the teleoperation system is said to be bilateral. The goal of bilateral teleoperation is to achieve "transparency", meaning that through the master manipulator, the operator feels as if he/she is directly operating on the remote environment [START_REF] Ferre | Advances in Telerobotics[END_REF].

In this study, we investigate the use of electro-pneumatic actuators in a teleoperation system. Compared to the electrical actuators, which are most used today in teleoperation, pneumatic actuators have higher power-tomass ratio and can generate larger force without any reduction mechanism [START_REF] Le | Development of a hybrid control for a pneumatic teleoperation system using on/off solenoid valves[END_REF]. Moreover, they are inert to magnetic fields, which is crucial in certain applications such as robot-assisted surgery under MRI guidance [START_REF] Yu | An fMRI compatible haptic interface with pneumatic actuation[END_REF].

In most recent work involving pneumatic actuation, servo-valves, rather than solenoid (on/off) valves, have been used to achieve high performances in various position or force control tasks. Servo-valves, however, are typically expensive due to requiring high-precision manufacturing. Therefore, in this paper, fast-switching on/off valves are used due to their advantages in terms of low cost and small size. One of the objectives of this paper is to show that good transparency can be obtained with these inexpensive components as actuators of the teleoperation system.

The traditional control approach for systems with solenoid valves involves using Pulse Width Modulation (PWM) to control the output mass flow rate of the valve [START_REF] Noritsugu | Pulse-width modulated feedback force control of a pneumatically powered robot hand[END_REF][START_REF] Shih | Position control of a pneumatic rodless cylinder using fuzzy PWM control method[END_REF][START_REF] Taghizadeh | Improving dynamic performances of PWM-driven servo-pneumatic systems via a novel pneumatic circuit[END_REF]. A main disadvantage of the PWM control is the chattering phenomenon caused by the high-frequency switching of the valve in steady state [START_REF] Le | Comparison of a PWM and a Hybrid Force Control for a Pneumatic Actuator Using On/off Solenoid Valves[END_REF], [START_REF] Le | Transparency of a pneumatic teleoperation system using on/off solenoid valves[END_REF].

To overcome the drawbacks of PWM-based control of solenoid valves, this paper presents a nonlinear sliding mode control inspired by Nguyen et al. [START_REF] Nguyen | Accurate sliding-mode control of pneumatic systems using low-cost solenoid valves[END_REF]. The sliding control law is used in a two-channel (2CH) bilateral teleoperation architecture with three different schemes: position-position, force-force, and force-position. These architectures are chosen due to their simplicity in terms of implementation of the control law. We provide in this paper both a tracking performance analysis and a stability analysis for the closedloop system using a Lyapunov candidate function.

For the sake of simplicity, the master and slave actuators are considered to be identical in this study -indeed, the master and the slave are one degree-of-freedom (DOF) pneumatic manipulators. It should be noted that this paper does not deal with the problem of compensating for time delays in a teleoperation system's communication channel.

The structure of this paper is described as follows. The modeling of the pneumatic manipulator is presented in Section II. Section III describes the sliding controller design for a 2CH master-slave telemanipulator. Section IV presents simulation results that validate the proposed control laws. Finally, concluding remarks and perspectives are given in Section V.

II. MODEL OF THE PNEUMATIC SYSTEM

As mentioned above, the master and the slave manipulators are identical, thus only one pneumatic robot is presented in this section. To describe the air flow dynamics in a cylinder, we assume that air is a perfect gas, the mass flow rate leakages are negligible, and the temperature variation in chambers is negligible with respect to the supply temperature. A schematic of the one-DOF pneumatic actuation system is shown in Fig. 1. The device consists of a pneumatic cylinder, four solenoid valves, a force sensor, and a position sensor. The behavior of the pressure inside each chamber of the cylinder can be expressed as [

γ γ   = -         = +     (1)
where U 1 , U 2 , U 3 and U 4 are the control voltages of valve 1, valve 2, valve 3, and valve 4; y and y & are the position (m) and velocity (m/s) of the piston; P p and P n are the pressures inside chambers p and n (Pa); V p and V n are the volumes of chambers p and n (m 3 ); S p and S n are the piston cylinder area in the chambers p and n (m 2 ); q p and q n are the mass flow rates in the chambers p and n (kg/s); T a is the temperature of the supply air (K); r is the perfect gas constant (J/(kg.K)) and γ is the polytropic constant. The mass flow rate characteristics of the on/off valves can be expressed as q P P U U q U U P U U q P P U U q P P U U q U U P U U q P P U U

 = =  = = =   - = =  = =   = = =   - = =  (2)
where P sa and P atm are the pressures of the supply air and atmosphere. The '0' state of the input voltage corresponds to a closed valve and the '1' state corresponds to an open valve. All the states where U 1 = U 2 = 1 and U 3 = U 4 = 1 are prohibited to avoid a bypass of the valves. The functions in (2) are given by a standard expression in which the mass flow rate of the valve is regulated by the air passage through an orifice [START_REF] Mccloy | Discharge characteristics of servo valve orifices[END_REF]:

2 if 0.433 (sonic) ( , ) 1
C P T C P T C  ≤       =  -      -    -        (3)
In the above, C val is the valve flow rate coefficient; P up and P down are respectively the absolute upstream and downstream stagnation pressures of the valve (Pa); T atm is the atmosphere temperature, and T up is the upstream stagnation temperature.

The dynamics of the piston and the load are

p p n n st ext

My S P S P by F F

= - --+ && & ( 4
)
where b is the viscous friction coefficient (N.s/m); M is the moving load (kg); F st is the stiction force; and F ext is the external force (N). For the sake of simplicity, the stiction force is supposed to be negligible.

III. TELEOPERATION BASED ON SLIDING CONTROL

In order to facilitate the control law design, a switching scheme for the four solenoid valves in Fig. 1 is defined so that each of the master and slave robots has the three modes of operation shown in Table I Here, U (the 4 th row) is defined as the input vector of the valves, and u (the 3 rd row) is the discrete (switching) control can be chosen either as u = sign(s) or u = -sign(s) where s is the sliding surface.

In order to bring the system to the sliding surface s = 0 at steady state, we define a neighbourhood of radius ε << 1 around zero. When |s| is within this neighbourhood, the third mode (u = 0) is used to save energy. In summary, we get the control law as

/ ( ) if 0 if sign s s u s ε ε + - >  =  ≤   (5)

A. Open-loop models of master and slave

Ignoring the stiction force in (4), the dynamics of the master and slave robots can be written as

= - - + = - - - && & && & (6)
where f h and f e are the operator force exerted on the master and the environment force exerted on the slave; y m and y s are the master and slave positions. Differentiating (6) and using (1)-( 3), the dynamics of the master and slave manipulators are obtained after some manipulations as [START_REF] Nguyen | Accurate sliding-mode control of pneumatic systems using low-cost solenoid valves[END_REF] ,

m m h m s s e s m m m h m s s s e s m h m s e s f M u f M u y f M u y f M u f M u f M u α β α β α β α β α α + + - -   + + = + - =     = - + = - = -- = -     + = - =     1 , 1 , 1, , 1 , 0 , 0
where i = m or s (for master or slave, respectively), and

U = [U1 U2 U3 U4 ] [1 0 1 0] [0 1 0 1] [0 0 0 0] 875 2 2 , , ( ) ( )
p p i n n i i i i p i n i S P S P b y y M M V y V y γ α   = - - +       && & (8) , , ( , ) ( , ) ( ) ( )
p sa p i n i atm n i a p i n i S q P P q P P S rT M V y M V y β γ +   = +       (9) , , ( , ) ( , ) ( ) ( )
p p i atm sa n i n i a p i n i S q P P q P P S rT M V y M V y β γ -   = +       (10)

B. Closed-loop teleoperation system 1) Position error based (PEB)

A position-error-based, also called position-position, system involves the simplest bilateral controller in which no force sensors are required. This architecture involves two types of data transmission between the master and the slave: position from the master to the slave and vice versa. The pneumatic-actuated PEB teleoperation system with a sliding mode control is shown in Fig. 2.

Sliding mode control

Cylinder (1), ( 4)

Valves (2), (3)
Fig. 2. Position-error-based approach with sliding mode control

In Fig. 2, s is the sliding surface; u m and u s are the discrete control signals for the master and the slave manipulators; and e = y s -y m is the position tracking error. Also, f h * and f e * are the operator's and the environment's exogenous input forces, respectively.

In the PEB scheme, the sliding surface s is defined as

where e = y s -y m is the position error between the master and the slave and ξ and ω are constant and positive parameters.

The control laws u m and u s are defined as u s = -u m =sign(s).

In the following, we analyze the position error convergence and the stability of the closed-loop system.

Consider the following Lyapunov candidate function

2 1 2 V s = (12)
The sliding surface s = 0 is reached within a finite time if the following condition is satisfied:

V ss s η = < - & & (13)
for some constant η > 0. Thus, from [START_REF] Le | Transparency of a pneumatic teleoperation system using on/off solenoid valves[END_REF]

M M y S P S P b B y K y f M M y S P S P b B y K y f

+ = - -+ - + + = - -+ - - && & && (23)
To establish the boundedness of λ, we consider the following points:

• Since we are dealing with a physical system, the piston positions y m and y s are bounded at the beginning (i.e., at t = 0). Also, the interaction forces f h and f e are bounded at the beginning. • In practice, the chamber pressures P p,i and P n,i are lowerbounded by the atmospheric pressure (P atm ) and upperbounded by the supply pressure (P sa ). This means that the first-order differential equations [START_REF] Yu | An fMRI compatible haptic interface with pneumatic actuation[END_REF] f & are also bounded. Eventually, it is found that λ, which is the sum of several bounded functions, is bounded. Therefore, the sliding condition in ( 13) is ensured, which implies that the position tracking error tends to zero (and that the overall system is stable). In fact, from ( 13), s will be bounded and converges to zero. Note that a drawback of the PEB method is that it does not guarantee a good transparency in term of force tracking. In order to improve the tracking performance, other schemes are proposed in the next sections.

2) Force error based (FEB)

A force-error-based, also called force-force, system is rarely used in the 2CH bilateral teleoperation system since two force sensors are required with no significant performance improvement. However, compared to the PEB architecture, this architecture can improve the force tracking performance. Fig. 3 shows the pneumatic-actuated FEB teleoperation system with a sliding mode control.

Consider the control law u s = u m = sign(s) where the sliding surface is defined as

h e s e f f = = - ( 24
)
where e is the force tracking error. Using the same Lyapunov function as in [START_REF] Nguyen | Accurate sliding-mode control of pneumatic systems using low-cost solenoid valves[END_REF], we need to show that the sliding condition ( 13) is satisfied.

q p,m q n,m Electro-pneumatic system Valves (2), (3)
Cylinder ( 1), [START_REF] Ferre | Advances in Telerobotics[END_REF] q p,m q n,m Electro-pneumatic system q p,s q n,s

Electro-pneumatic system

Cylinder (1), ( 4)

Valves (2), (3)
q p,s q n,s

Electro-pneumatic system

Cylinder (1), ( 4)

Valves (2), (3)
Fig. 3. Force-error-based approach with sliding mode control From (

m m m s m s s h m m m m e s m s s m m m s m s M y u M y u f M y u f M y u M y u M y u α β α β α β α β α α + + - -   - - = -+ + =   = - + = - = -+ - = -     - = -+ =   &&& &&& & & &&& &&& &&& &&& 7), we get ( ), 1 ( ), 1 ( ), 1, ( ), 1 ( ) , 0 ( ) , 0 m
Case 1: s > 0. In this case u m = u s = 1. From (13), we need ( )

h e m m m s s s s f f M y y α β α β η + + = -= - - + -- < - & & & &&& &&& (26)
As it was demonstrated above, m y &&& , s y &&& , m α and s α are bounded. Thus, by choosing a valve with large orifice, m β + and s β + can be made sufficiently large to satisfy (26). Case 2: s < 0. In this case, u m = u s = -1. We have ( )

h e m m m s s s s f f M y y α β α β η - - = -= - + + -+ > & & & &&& &&& (27)
Similar to Case 1, the stability of the system can be guaranteed by choosing a large enough value of m βand s β -. Consequently, the force tracking error converges to zero and the overall system is stable. However, the FEB method does not guarantee a good position tracking performance. In order to overcome the PEB and FEB architecture drawbacks, we use the scheme described in the following section.

3) Direct force reflection (DFR)

A direct-force-reflection, also called force-position, system has advantages over the position-position and forceforce architectures. The pneumatic-actuated DFR system with a sliding mode control is illustrated in Fig. 4.

Sliding mode control

Sliding mode control q p,s q n,s

Electro-pneumatic system

Cylinder (1), ( 4)

Valves (2), (3) Valves (2), (3)
Cylinder (1), [START_REF] Ferre | Advances in Telerobotics[END_REF] q n,m Electro-pneumatic system Fig. 4. Direct-force-reflection approach with sliding mode control

Here, s m and s s are the sliding surfaces for the master and slave systems, respectively; e m is the force tracking error of the master side; and e s is the position tracking error of the slave. This architecture involves two types of data transmission between the master and the slave: force from the slave to master and position from the master to the slave. Hence, the transparency is improved in terms of force and position tracking, compared to these previous methods. This statement will be justified later in the simulation.

In this section, we use a Lyapunov function to prove the stability of the DFR system. First, we will show the stability of the force-controlled master manipulator. Afterwards, we will show the stability of the position-controlled slave manipulator. However, the stability of the overall system is difficult to show due to the different sliding surfaces for the master and the slave. Indeed, the following proves the stability of each manipulator, but not the stability of the overall teleoperation system.

a)

Force convergence in the closed-loop master system

Since the desired force for the master robot, i.e., the slave/environment contact force f e is assumed to be bounded at the beginning, we need to show that f h converges to f e in a finite time. The sliding surface s m and the Lyapunov function V m are defined as in ( 24) and ( 12), respectively. The controller u m is chosen to be similar to the FEB system in section III.B. Consequently, the sliding surface (the force tracking error) tends to zero, i.e., f h tends towards f e .

b)

Position convergence in the closed-loop slave system

The sliding surface s s and the Lyapunov function V s are defined as in [START_REF] Le | Transparency of a pneumatic teleoperation system using on/off solenoid valves[END_REF] and [START_REF] Nguyen | Accurate sliding-mode control of pneumatic systems using low-cost solenoid valves[END_REF], respectively. The controllers u s is chosen to be similar to the PEB system in section III.B. The straightforward reasoning described in section III.B.1 allows us to infer that φ is bounded. Therefore, there exists a high value of s β -such as (31) is satisfied. Case 2: s s < 0. In this case, u s = 1. Thus, we need

s ϕ β η + + > (33)
This condition is achieved by choosing a large enough s β -. Note that for both cases, the convergence of the sliding surface (and thus the position tracking errors) to zero is proved, so x s tends towards x m .

As it will be confirmed in the next section, the DFR architecture provides a good transparency in terms of both position and force tracking.

IV. SIMULATIONS

In this section, we verify the efficiency and performance of a teleoperation system using pneumatic actuators with on/off solenoid valves. The pneumatic and mechanical models of the master and the slave actuators are given by ( 1)-( 3

h f t t = +
is chosen as the input signal of the teleoperation system. For the PEB and DFR systems, the sliding surface of the position-controlled slave is defined in [START_REF] Le | Transparency of a pneumatic teleoperation system using on/off solenoid valves[END_REF]. To place the master and the slave closed-loop poles for fast responses without overshoot, the parameters ξ = 1 and ω= 100 rad/s are chosen.

Fig. 5 shows the master and the slave force and position tracking profiles for the PEB teleoperation system, where the slave is in contact with an soft environment whose dynamic is Z e = s 2 + 10s + 500. Figures 6 and7 illustrate the same profiles for the FEB and the DFR systems, respectively.

As illustrated in Fig. 5, the PEB system provides a good position tracking responses. However, the force response is deteriorated because no force sensor is used. On the other hand, the force tracking performance of the FEB system is improved, thanks to the knowledge of the force information. Nonetheless, the transparency is not perfect in term of position tracking. As it can be seen in Fig. 6, the slave's movement does not accurately track the master's movement.

The DFR system in Fig. 7 provides an improvement in position tracking response with respect to the FEB system. Moreover, it displays a superior force tracking performance compared to the PEB. Since the DFR uses the measurement of slave/environment contact forces, the feeling of contact motion is much more realistic. Therefore the performance of the teleoperation system is improved significantly by feeding the operator with the slave contact force. This result agrees with the previous theoretical work.

2 . 1 :Case 2 :

 212 Case s m > 0. In this case, u m = 1. Using the expression of h f & in (25) and the definition of s m as in (24) we have it was demonstrated in section III.B.1, e f & , m y &&& and m α can be shown to be bounded. Thus, the stability condition (29) is satisfied by choosing a large value of m β + . s m < 0. In this case, u m = -1. From (24) and (Similar to Case 1, it is possible to choose a large enough value of m β -to ensure the stability of the master robot.

  ), and[START_REF] Yu | An fMRI compatible haptic interface with pneumatic actuation[END_REF]. The dynamic model of the operator's hand and the environment are presented in(22).The 2CH bilateral teleoperation systems with three architectures PEB, FEB and DFR are implemented in Matlab/Simulink.The initial values of the position and the velocity are set as (pressures in the chambers are supposed to be equal to the atmospheric pressure. The environment is assumed to be passive (f e * = 0) and the operator's exogenous force * 15sin( ) 10 sin[START_REF] Le | Development of a hybrid control for a pneumatic teleoperation system using on/off solenoid valves[END_REF]

Sliding Mode Control of a Pneumatic Haptic Teleoperation System with on/off Solenoid Valves

	M.Q. Le, M.T. Pham, M. Tavakoli, R. Moreau

2011 IEEE International Conference on Robotics and Automation Shanghai International Conference Center May 9-13, 2011, Shanghai, China 978-1-61284-380-3/11/$26.00 ©2011 IEEE 874

	Chamber p	Chamber n	Position sensor
	P p , V p , T p	P n , V n , T n	Force sensor
	q p		q n
	Valve 1	Valve 2	Valve 3	Valve 4
	P sa	P atm	P atm	P sa
	U 1	U 2	U 3	U 4
		DSP controller
				F
	Desired force or motion		y
	Fig. 1. Electro-pneumatic system with four valves

TABLE I :

 I THREE POSSIBLE CONTROL MODES

		Mode 1	Mode 2	Mode 3
	Chamber p	filled	exhausted	closes
	Chamber n	exhausted	filled	closes
	Control u	1	-1	0

& & &

& & & &

& &

Among the three architectures, the DFR scheme seems to be a better choice to obtain a good transparency. Although the various teleoperation controllers have previously been compared from a performance perspective in the literature, this is the first study to show that it is possible to achieve stability and satisfactory performance using manipulators actuated by low-cost switching on/off valves.

V. CONCLUSION

In this paper, pneumatic actuators with inexpensive solenoid valves are chosen for the development of a masterslave teleoperation system. To efficiently control the switching on/off valves for haptic teleoperation system, sliding mode controllers are proposed and implemented in a realistic simulation. A comparison of the transparency and stability has been investigated between three control architectures (PEB, FEB and DFR) in a two-channel bilateral teleoperation system. The results show that better force and position tracking is obtained in the DFR architecture than in the PEB and FEB architectures. The simulation results are encouraging for future work aimed at implementing the proposed sliding bilateral control on an experimental testbed. Another aspect of this work is to incorporate sliding mode control in more complex teleoperation architectures (e.g., three or four-channel methods) in order to improve the performance of the teleoperation system.


Belen Torondel
email: belen.torondel@lshtm.ac.uk

Manasi Nandi

Peter Kelly

Beata Wojciak-Stothard

Ingrid Fleming

James Leiper

Adenoviral mediated overexpression of DDAH improves vascular tone regulation

Keywords: Carotid Arteries, Endothelial Cells, Endothelium, Vascular, Gene Therapy, Mice, Transgenic, Microscopy, Fluorescence, Nitric Oxide Ddah 1 +/-mice, ADMA, Adenovirus, Gene transfer, Vascular Reactivity

Dimethylarginine dimethylaminohydrolase(DDAH) degrades asymmetric dimethylarginine(ADMA), an endogenously produced nitric oxide (NO) synthase inhibitor.In mammals two isoforms of DDAH, DDAH1 and DDAH2,are expressed in the cardiovascular system,suggesting that ADMA concentrations are actively regulated in blood vessels,raising the possibility that cardiovascular metabolism of ADMA constitutes a novel mechanism for the regulation of NO production.The purpose of this study was to determine the role of DDAH-catalyzed asymmetric methylarginine metabolism in the regulation of vascular function. Overexpression of DDAH1 or 2 in HUVEC increases DDAH activity, reduces ADMA concentrations and increases NO production.Similarly, overexpression of DDAH 1 or 2 in DDAH1+/+ mice carotid vessels increases NO production and attenuates the response to phenylephrine,enhances acetylcholine relaxation and attenuates the effect of exogenously applied ADMA.Finally,overexpression of either DDAH1 or 2 completely reversed the vascular dysfunction seen in DDAH1+/-mice.These data indicate that basal concentrations of ADMA in blood vessels are sufficient to regulate NO production,that increases in the level of either DDAH1 or 2 improves vascular function and that overexpression of either DDAH1 or 2 is sufficient to compensate for life long exposure to elevated ADMA.Thus therapeutic manipulation of DDAH expression or activity may

Nitric oxide (NO) is a key physiological mediator in the cardiovascular, neuronal and immune systems.

Numerous reports have detailed the effects of reduced NO production on signaling in these systems and reduced NO generation has been associated with many pathological conditions [START_REF] Ignarro | Nitric oxide donors and cardiovascular agents modulating the bioactivity of nitric oxide: an overview[END_REF] . In the cardiovascular system genetic or pharmacological inhibition of NO production leads to attenuation of vascular relaxation, increased vascular resistance and increased blood pressure. Therefore endogenous mechanisms that regulate NO production are of considerable interest [START_REF] Vallance | Blocking NO synthesis: how, where and why?[END_REF] . In the cardiovascular system, NO is synthesized by the endothelial isoform of nitric oxide synthase (eNOS) and the activity of this enzyme has been reported to be regulated at many levels including transcription, translation, post-translational modification and co-factor availability [START_REF] Vallance | Blocking NO synthesis: how, where and why?[END_REF] . In addition to these mechanisms, eNOS activity can be regulated by the level of endogenously produced asymmetrically methylated arginines which are competitive enzyme inhibitors [START_REF] Vallance | Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure[END_REF] . In humans, two endogenously produced asymmetric methylarginines are present; asymmetric dimethylarginine (ADMA) and Nmonomethylarginine (L-NMMA). However ADMA is found at approximately 10 fold the concentration of L-NMMA and is therefore thought to be the most functionally significant asymmetric methylarginine [START_REF] Vallance | Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure[END_REF] .

Methylarginines are generated by the post-translational methylation of certain arginine residues in proteins catalysed by a family of protein arginine methyltransferases (PRMT's) [START_REF] Clarke | Protein methylation[END_REF][START_REF] Mcbride | State of the arg: protein methylation at arginine comes of age[END_REF] . Proteolysis of arginine methylated proteins leads to the liberation of free methylarginines which have the potential to compete with arginine at the active site of NOS and thereby inhibit enzyme activity. An increasing volume of literature documents the association between raised plasma ADMA concentrations and a range of cardiovascular disease states [START_REF] Vallance | Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure[END_REF][START_REF] Inoue | Decrease of 3-methylhistidine and increase of NG,NG-dimethylarginine in the urine of patients with muscular dystrophy[END_REF][START_REF] Lluch | Plasma concentrations of nitric oxide and asymmetric dimethylarginine in human alcoholic cirrhosis[END_REF][START_REF] Zoccali | Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study[END_REF] . In some of these reports decreased NO production has been implicated in disease pathology and it has been suggested that inhibition of eNOS activity by ADMA is a mechanism of disease. However until recently direct evidence confirming this causal relationship between ADMA and NO in vivo has been lacking. In addition to endogenous production, asymmetric methylarginines are also metabolized in vivo. The enzyme dimethylarginine dimethylaminohydrolase (DDAH) hydrolyses both ADMA and L-NMMA to citrulline and di-or mono-methylamine respectively. In humans two isoforms of DDAH (DDAH1 & 2) are found with distinct but overlapping tissue distributions [START_REF] Leiper | Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distributions and homology with microbial arginine deiminases[END_REF] . In the cardiovascular system both DDAH1 and 2 are expressed in both endothelial and smooth muscle cells suggesting that in blood vessels asymmetric methylarginine concentrations are actively regulated and that this pathway constitutes a novel mechanism for the regulation of NO and vascular function.

Consistent with this suggestion, global heterozygous deletion of DDAH1 in mice attenuates vascular NO signaling resulting in endothelial dysfunction and increased systemic vascular resistance and blood pressure in vivo 10 . Conversely, global overexpression of DDAH1 in mice increases the concentration of nitrogen oxides (NOx) in the plasma and is associated with reduced systolic blood pressure 11 . In transgenic mice globally overexpressing DDAH2 increased cardiac NOx was apparent however no change in systolic blood pressure was observed 12 . However, the role of DDAH in the regulation of vascular NO signaling has only been studied in DDAH1 transgenic mice 13 and therefore the precise roles of DDAH1 and 2 remains unclear.

In the present study we aimed to study the effect of DDAH catalyzed asymmetric methylarginine metabolism in blood vessels. We developed adenoviral vectors for the expression of human DDAH1 and

Construction of recombinant adenovirus

Recombinant DDAH I and DDAH II adenoviruses (AdDDAH I and AdDDAH II) were generated using the protocol from Stratagene AdEasy System (http://www.coloncancer.org/adeasy/protocol2.htm). 14 Adenoviral vectors expressing green fluorescent protein (GFP) were used as controls.

Cell culture:

Human umbilical vein endothelial cells (HUVECs) and all cell culture reagents were purchased from PROMOCELL and cultured as described previously 15 and used prior to passage 6. HUVECs were grown in 6-well plates until confluent and infected with adenoviruses at an MOI that we have previously determined to result in optimal infection (MOI=1000) in medium containing 2% fetal calf serum (FCS) for 1 hour, following which the medium was replaced with Phenol Red-free culture medium containing 10% FCS. The media was collected for nitrite and ADMA determination 24 hours after infection and the cells processed for fluorescence microscopy or western blotting.

Nitrite determination

To measure nitrite, a stable end product of NO degradation, an aliquot of medium (100µl) from each culture well was mixed with 100 µl of the Griess reagent (1% sulfanilamide and 0.1% naphthylenediamine dihydrochloride in 2% phosphoric acid). The mixture was incubated for 10 minutes at room temperature to allow the colour to develop, and the absorbance at 540nm was measured in a microplate reader. Concentrations were determined by comparison with a sodium nitrite standard curve.

ADMA/SDMA measurements

Levels of asymmetric dimethylarginine (ADMA) and its biologically inactive isomer symmetric dimethylarginine (SDMA) were quantitated by HPLC in conditioned medium as previously described 10 .

Western blotting

Cells or tissues were lysed in cell lysis buffer (125mM HEPES, pH 7.5, 750mM NaCl, 5% Igepal CA-630, 50mM MgCl2, 5mM EDTA and 10% glycerol, Upstate Biotechnology), the protein concentration determined using a Bio-Rad protein assay kit with bovine serum albumin standards. Protein samples (50µg) were separated on a 12% SDS-polyacrilamide gels, transferred to nitrocellulose membranes, and probed with affinity-purified polyclonal antibodies specific for DDAH1 or 2 14 , α-tubulin or GFP.

Inmunoreactive bands were visualized with a horseradish peroxidase conjugated secondary antibody using an ECL detection kit (Amersham).

DDAH Activity assay.

HUVECs were grown to confluence in 75 cm 2 flasks and infected as described above. 24 hours post infection, cells were scraped from the flask in ice cold lysis buffer (sodium phosphate, pH 6.6 (10mM), containing 0.1% Triton X-100) and incubated on ice for 20 minutes. Cell debris was removed by centrifugation and supernatants assayed for DDAH activity using a colorimetric assay for the determination of enzymatically formed citrulline as previously described 16 .

Infection of Carotid arteries.

All the experiments were carried out under a UK Home Office License and conducted according to the Animal Scientific Procedures Act 1986. Male mice 10-12 weeks-old (DDAH1+/-or DDAH1+/+ on C57Bl6 background) were sacrificed with an overdose of Isofluorane, carotid arteries exposed by dissection in situ and the distal ends ligated. A mixture of adenovirus and cell culture medium (5µl of 1x10 9 plaque-forming units/ml virus plus 20µl of Dulbecco's modified Eagle's medium (DMEM) containing 2% fetal calf serum (FCS) was introduced into the proximal end of each carotid artery (one side with AdDDAH1 or 2 and the contralateral artery with AdGFP) following which the proximal end was ligated. The ligated segment was removed and incubated in tissue culture medium (DMEM containing 10% FCS) for 4h after which the ligatures were removed, the vessel cut into four ~4mm segments and then incubated in fresh tissue culture medium for a further 24 h. Following incubation vessels were either processed for microscopy, mounted in myographs for functional studies or homogenized for western blotting. Tissue culture medium was collected for nitrite determination.

Fluorescence microscopy.

Fluorescence microscopy was used to monitor viral infection of cultured endothelial cells and carotid arteries. For endothelial cells cultures, cells were stained with DAPI to identify nuclei and the transfection efficiency determined. At the MOI used >90% infection of endothelial cells was observed and no difference in the transfection efficiency between GFP, GFP-DDAH1 and GFP-DDAH2 viruses was apparent. For analysis of infected arteries, rings were opened and fixed in 4% formaldehyde dissolved in PBS for 15 min and permeabilized for 3 min in 0.1% Triton X-100 (Sigma). F-actin was labeled with 1 µg/ml TRITC-phalloidin (Sigma) for 1 h. Single plain images of the cells were obtained by confocal laser scanning microscopy (Bio-Rad Radiance 2100) with Bio-Rad software (LaserSharp 2000 version 5.1) 14 .

Determination of Carotid Arterial Vascular Reactivity.

Carotid arteries from male 10-12 week-old DDAH 1+/-and wild-type littermates on a pure C57Bl6 background were used for functional studies. Rings were mounted in myograph (Danish Myotechnology) and bathed in PSS (37 0 C gassed with 5% CO 2 in O 2 ) at a resting tension determined by the diameter of the vessel. After equilibration, vessels were stimulated with thromboxane A2 mimetic U-46619 (10 -6 mol/L) until responses were reproducible. Functional integrity of the endothelium was confirmed routinely by the presence of relaxation induced by acetylcholine (ACh) 10 -6 mol/L during contraction obtained with phenylephrine (PE) 10 -6 mol/L. For wild type animals only arteries which presented more that 60% relaxation, were considered as endothelial intact and used for the experiments. However as we hypothesized that DDAH 1 deletion will cause endothelial dysfunction, this value was reduced to 30% for vessels from DDAH 1+/-mice. Concentration-response curves to phenylephrine (10 -8 to 10 -4 mol/L) and acetylcholine (10 -9 to 3x10 -6 mol/L) were constructed. In order to study ADMA contraction, vessels were precontracted with an effective concentration of PE to achieve 20% of maximal response (PE EC 20 ) and a cumulative concentration response curve to ADMA (10 -8 to 10 -3 mol/L) was determined. After myography, fluorescence microscopy was used to determine transfection efficiency as described above.

Statistical analysis

Statistical analysis was performed with GraphPad Prism version 3.0. All data are given as mean± standard error of the mean (SEM). Tests of significance were conducted using t student test for differences between data groups, where p<0.05 were considered significant. Phenylephrine contraction in carotid arteries was expressed as absolute tension in grams (g). Relaxation was expressed as a percentage of the phenylephrine-induced contraction. ADMA contractile effect was expressed as a percentage of the maximal response to U46619 (10 -6 mol/L). The concentrations of agonist producing half-maximum effect

Incubation of HUVEC with viruses expressing GFP or GFP + DDAH1 or 2 resulted in >90 infection

efficiency as judged by GFP fluorescence (Figure 1a). No differences in the infection efficiency of GFP, GFP-DDAH1 and GFP-DDAH2 viruses were apparent. 24 h following infection, cells expressing either GFP alone of GFP + DDAH1 or 2 were lysed and soluble fractions were subjected to western blotting using anti-DDAH 1 or 2 and anti-α tubulin antibodies (Figure 1b). In GFP infected cells, a band corresponding to endogenous DDAH1 and 2 was detectable. Following infection with viruses expressing DDAH1 or 2 increased immunoreactivity was observed confirming overexpression of the proteins.

Aliquots of cell lysates were also assayed for DDAH activity using a colorimetric assay to detect the citrulline reaction product (Material and Methods). Infection of HUVEC with viruses encoding either DDAH1 or 2 increased DDAH activity compared to cells infected with a virus expressing GFP alone (Figure 1c, GFP = 100%, DDAH1 =140.6±10.1 %, DDAH2 = 150.8±8.9 % n = 5, p<0.05).

Conditioned medium from infected cells was also collected 24h post infection and analyzed to determine methylarginine and nitrite concentrations. Conditioned medium from cells overexpressing either DDAH 1 or 2 had significantly lower concentrations of ADMA compared to cells expressing GFP alone (Figure 1d, GFP = 8.11±0.09 ng/ml, DDAH1 = 6.32±0.04 ng/ml, DDAH2 = 5.71±0.02 ng/ml n = 8, p<0.05). Levels of SDMA, which is not a substrate for DDAH, were unchanged (data not shown). The reduction in ADMA elaboration was accompanied by increased accumulation of nitrite, a stable breakdown product of NO (Figure 1e Carotid arteries from C57Bl6 mice were incubated with virus in situ and maintained in static culture as described (Materials and Methods). One carotid artery was infected with viruses expressing GFP and the contralateral carotid artery was infected either with GFP-DDAH1 or GFP-DDAH2. 24h following incubation, conditioned medium was removed from the vessels for determination of GFP protein levels and nitrite concentrations. Infection of vessels with adenoviruses was verified by GFP fluorescence (Figure 2a). No difference in the level of GFP protein in vessels infected with either DDAH1 or 2 expressing viruses was apparent indicating that the infection efficiency of the two viruses was similar (Figure 2b). Increased nitrite accumulation in the medium of DDAH1 or 2 infected vessels compared to vessels expressing GFP alone was observed (Figure 2c, GFP = 2.94±0.92 µmol/L, DDAH1 = 8.67 ±1.23 µmol/L, DDAH2 = 6.77±1.39µmol/L n=5, p<0.05). Infection of vessels with adenoviruses encoding GFP alone did not alter nitrite accumulation compared to uninfected vessels (GFP = 2.94±0.92 µmol/L, control= 2.77±0.89 µmol/L n=5, p>0.05) and there was no significant difference nitrite accumulation between DDAH1 and DDAH2 infected vessels. Infection was confirmed by GFP fluorescence For measurement of vascular reactivity, carotid arteries were incubated with viruses in situ, removed and cultured ex vivo (material and methods). Infection was confirmed by GFP fluorescence, no difference in infection efficiency of the viruses used was apparent (data not shown). Segments were mounted under isometric tension in myographs. Endothelial integrity was confirmed as described and then dose response curves to phenylepherine, acetylcholine and ADMA were constructed (Figure 3 and Table 1). Initially, we established that viral infection of blood vessels did not alter responses to vasoconstrictors and vasodilators (supplement figure). Infection with either DDAH1 or DDAH2 significantly reduced the maximal contraction to phenylepherine (Figure 3a), increased the maximal relaxation to acetylcholine (Figure 3b) and decreased the contractile response to exogenous ADMA (Figure 3c) compared to control GFP-infected vessels. Only infection with DDAH1 caused a significant shift in the EC 50 of these agents however infection with DDAH2 showed a similar trend that did not reach statistical significance in these experiments (Table 1).

Infection of carotid arteries from DDAH1+/-mice.

In order to determine if the vascular dysfunction previously observed in DDAH1+/-mice could be reversed by adenoviral delivery of either DDAH1 or 2 we studied carotid arteries from DDAH1+/-mice.

Initially we confirmed that vascular responses in the carotid arteries from DDAH1+/-mice showed a similar pattern of endothelial dysfunction, increased phenylepherine and ADMA contraction and reduced acetylcholine relaxation, to that which we had observed in aorta and pulmonary arteries (Figure 4).

Infection with either DDAH1 or DDAH2 significantly reduced the maximal contraction to phenylepherine (Figure 5a), increased the maximal relaxation to acetylcholine (Figure 5b) and decreased the contractile response to exogenous ADMA (Figure 5c) such that the endothelial dysfunction caused by DDAH1 haploinsufficiency was completely reversed. In contrast to wild type vessels, infection of DDAH1+/-carotid arteries with either DDAH1 or 2 significantly shifted both the maximal response and the EC 50 values (Table 1). In this study we first used primary human umbilical vein endothelial cells (HUVECs) as they are known to constitutively express eNOS, DDAH1 and DDAH2 and to release measurable quantities of both ADMA and NO (detected in the medium as nitrite) 15,17,18 . We chose to overexpress DDAH isoforms using the adenovirus system as this has previously been shown to result in highly efficient infection of vascular cells in culture and tissues ex vivo. In our studies we observed >90% infection of HUVEC in culture that resulted in high level expression of DDAH isoforms. Despite very substantial overexpression at the protein level we observed a relatively modest increase in total DDAH activity (~30%) that was associated with a similar reduction in ADMA elaboration (~30%). This discrepancy between protein levels and enzymatic activity is also apparent in several other studies where DDAH transfection has been used to modulate ADMA levels 11,12,15,19,20 and might indicate that either 1) overexpression of DDAH produces predominantly misfolded and inactive protein or 2) that DDAH proteins are subject to posttranslation regulation. We have fractionated DDAH proteins produced by overexpression and found them to be soluble proteins residing in the cytosol suggesting that they are not grossly misfolded and perhaps supporting the notion that DDAH enzymes can be regulated post-translationally.

To determine the effect of DDAH overexpression on NO synthesis we measured the accumulation of nitrite, a stable breakdown product of NO, in the conditioned medium of transfected HUVEC. We observed a very large increase in 24h nitrite accumulation following transfection with either DDAH1 or 2.

These data indicate that basal ADMA is sufficient to exert significant inhibition of eNOS activity in primary human endothelial cells and that relatively modest increases in total DDAH activity and associated reduction in ADMA are sufficient to cause a very substantial increase in endothelial NO production. In our experiments there was no significant difference in nitrite production between DDAH1 and DDAH2 transfected cells indicating that, at least at the level of overexpression, there is functional redundancy between DDAH isoforms in endothelial cells.

To extend our observation of endothelial cells in culture into blood vessels we studied mouse carotid arteries that had been infected with adenoviruses in situ. We determined the efficiency of infection by measuring GFP fluorescence and observed that intraluminal infection of blood vessels resulted in almost exclusively endothelial expression of GFP. Consistent with our observations from cell culture, vascular infection with adenoviruses expressing either DDAH1 or DDAH2 resulted in a large increase in nitrite in the conditioned medium. When we determined the vascular reactivity of infected carotid arteries, consistent with our observation of increased NO production, we observed a significant reduction in the maximal response to phenylepherine and a significant increase in the maximal acetylcholine response in vessels overexpressing either DDAH1 or 2 when compared to GFP infected vessels. These observations demonstrate for the first time that in intact blood vessels basal ADMA concentrations regulate eNOS activity and that DDAH activity is insufficient to maintain ADMA concentrations below inhibitory levels. These findings are consistent with studies of genetically modified mice that globally overexpress either DDAH1 or 2 11,12 . In these mice substantial overexpression of DDAH resulted in increased DDAH activity (~3 fold for DDAH1 and ~0.5 fold for DDAH2), reduced circulating ADMA (-60% for DDAH1 and -25% for DDAH2) and increased nitrite concentrations in the urine of DDAH1 transgenic mice or in the cardiac tissue of DDAH2 transgenic mice. When cardiovascular function was studied in these mice significant changes were only observed in DDAH1 overexpressing mice where a significant increase in heart rate and a trend towards lower mean arterial pressure and systemic vascular resistance was observed. In contrast, no difference in systolic blood pressure was observed in DDAH2 overexpressing mice 11,12 . These differences in haemodynamic phenotype might result from differences in the magnitude of DDAH overexpression. However, the effect of DDAH overexpression on vascular function has only been studied in cerebral vessels from DDAH1 transgenic mice which overexpress DDAH1 in both endothelial and smooth muscle cells 13 . In these vessels enhanced relaxation in response to acetylcholine was observed however it was not possible to determine the relative contribution of endothelial and smooth muscle cell derived ADMA to this effect. Our data indicate that endothelial overexpression of either DDAH1 or 2 is sufficient to enhance responses to vasodilators and reduce responses to vasoconstrictors in systemic blood vessels which would lower vascular resistance and blood pressure in vivo.

In our final set of experiments we determined the ability of adenovirus-mediated DDAH gene delivery to reverse the endothelial dysfunction seen in DDAH1+/-mice. These mice have life-long exposure to elevated ADMA concentrations that are similar to those seen in many human cardiovascular disease states. Whilst it is clear that raised ADMA causes endothelial dysfunction it is not known whether this phenotype is reversible by acute elevation of DDAH activity. Previously we have demonstrated that endothelial dysfunction in aorta from DDAH1+/-mice could be reversed with high concentrations of exogenous arginine ex vivo 10 . However recent trials of high dose arginine supplementation have produced negative results indicating that this is not a viable mechanism to reverse the effects of ADMA Alternatively, post-translational activation of DDAH proteins would achieve the same result. However, to date, no post-translational modifications of DDAH that result in increased activity have been reported.

Our observation that following DDAH overexpression there is a poor correlation between DDAH protein level and activity, might indicate that post-translational mechanisms exist to modulate DDAH activity.

These observations merit further investigation.

It is well established that reduced NO production following surgical procedures such as venous grafting and angioplasty contributes to vascular smooth muscle cell proliferation and restenosis. A number of therapeutic approaches to reduce smooth muscle cell proliferation in these situations, including drug eluting stents and VEGF gene therapy have been investigated 23,24 . It has previously been reported that endothelial cells repopulating injured vessels have higher intracellular ADMA concentrations that would be predicted to attenuate NO production 25,[START_REF] Goto | Possible involvement of facilitated polyol pathway in augmentation of intimal hyperplasia in rabbits with alloxan-induced hyperglycemia[END_REF] . Our data suggest that local adenovirus-mediated delivery of DDAH genes would be sufficient to reduce endothelial ADMA concentrations and promote NO production in these situations. Several studies have reported that vascular delivery of NOS genes improves endothelial function in models of cardiovascular disease [START_REF] Alexander | Gene transfer of endothelial nitric oxide synthase improves nitric oxide-dependent endothelial function in a hypertensive rat model[END_REF][START_REF] Khurana | A direct mechanical method for accurate and efficient adenoviral vector delivery to tissues[END_REF][START_REF] Miller | Targeting endothelial cells with adenovirus expressing nitric oxide synthase prevents elevation of blood pressure in stroke-prone spontaneously hypertensive rats[END_REF] and our current findings indicate that manipulation of DDAH levels could achieve similar results.

F o r P e e r R e v i e w

2NO production 2 )

 2 in vascular cells and tissues. Overexpression of DDAH1 or 2 in HUVECs increased DDAH activity, reduced ADMA concentrations and increased NO production. Similarly, adenoviral mediated overexpression of DDAH1 or 2 in mice carotid vessels increased NO production in static culture. When carotid vessels were studied using myography, overexpression of DDAH1 or 2 attenuated the response to PE, enhanced ACh relaxation and attenuated the effect of exogenously applied ADMA. Finally, overexpression of either DDAH1 or 2 completely reversed the vascular dysfunction seen in DDAH1+/mice. These data indicate that 1) basal concentrations of ADMA in blood vessels are sufficient to regulate increases in the level of either DDAH1 or 2 improves vascular function 3) overexpression of either DDAH1 or 2 is sufficient to compensate for DDAH1 haploinsufficiency.

(

  EC 50 values) was determined from the individual concentration-response curves by nonlinear regression analysis and expressed as negative logM. For comparisons of concentration response curves, two-way ANOVA was used. The n values indicate the number of experiments and animals used.

  , GFP = 1.45±0.23 µmol/L, DDAH1 = 3.71±0.61 µmol/L, DDAH2 = 4.11±1.06 µmol/L n=8, p<0.05).

  Infection of murine carotid arteries.

  This study has several major findings. Overexpression of either DDAH1 or 2 in human endothelial cells is sufficient to reduce ADMA elaboration and increase basal NO generation. In murine blood vessels similar effects are observed with increased NO production resulting in enhanced responses to vasodilators and reduced contractile responses. Finally the profound endothelial dysfunction that results from life long DDAH1 haploinsufficiency in mice is completely reversed by adenovirus mediated delivery of either DDAH1 or DDAH2 genes. Taken together these data indicate that in primary vascular cells and tissues basal ADMA concentrations are sufficient to exert tonic inhibition of eNOS and that DDAH activity is insufficient to maintain ADMA concentrations below an inhibitory threshold. Furthermore, functional redundancy exists between DDAH isoforms with increased expression of either DDAH1 or 2 being sufficient to increase vascular function and reverse the effect of DDAH1 haploinsufficiency.

21.

  Our data presented here indicate that adenovirus-mediated delivery of either DDAH1 or DDAH2 is sufficient to substantially reverse the endothelial dysfunction of DDAH1+/-mice. These observations suggest that in human cardiovascular diseases where reduced DDAH activity and increased ADMA play a role in pathology, interventions resulting in increased expression and/or activity of either DDAH1 or 2 useful. We have previously reported that retinoic acid-induced upregulation of DDAH2 expression in endothelial cells is associated with reduced ADMA elaboration and increased NO production demonstrating that, in principle, transcriptional upregulation of DDAH can result in beneficial increases in endothelial NO production22 . Numerous recent studies have indicated that various treatments can regulate DDAH gene expression and that these changes in DDAH level are associated with altered ADMA elaboration and NO synthesis.

  Leiper J, Nandi M, Torondel B et al. Disruption of methylarginine metabolism impairs vascular homeostasis. Nat Med 2007 February;13(2):198-203. (11) Dayoub H, Achan V, Adimoolam S et al. Dimethylarginine dimethylaminohydrolase regulates nitric oxide synthesis: genetic and physiological evidence. Circulation 2003 December 16;108(24):3042-7. (12) Hasegawa K, Wakino S, Tatematsu S et al. Role of asymmetric dimethylarginine in vascular injury in transgenic mice overexpressing dimethylarginie dimethylaminohydrolase 2. Circ Res 2007 July 20;101(2):e2-10. (13) Dayoub H, Rodionov R, Lynch C et al. Overexpression of dimethylarginine dimethylaminohydrolase inhibits asymmetric dimethylarginine-induced endothelial dysfunction in the cerebral circulation. Stroke 2008 January;39(1):180-4. (14) Wojciak-Stothard B, Torondel B, Tsang LY et al. The ADMA/DDAH pathway is a critical regulator of endothelial cell motility. J Cell Sci 2007 March 15;120(Pt 6):929-42. (15) Smith CL, Birdsey GM, Anthony S, Arrigoni FI, Leiper JM, Vallance P. Dimethylarginine dimethylaminohydrolase activity modulates ADMA levels, VEGF expression, and cell phenotype. Biochem Biophys Res Commun 2003 September 5;308(4):984-9. (16) Knipp M, Vasak M. A colorimetric 96-well microtiter plate assay for the determination of enzymatically formed citrulline. Anal Biochem 2000 November 15;286(2):257-64. (17) Pollock JS, Forstermann U, Mitchell JA et al. Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells. Proc Natl Acad Sci U S A 1991 December 1;88(23):10480-4.

  Li H, Oehrlein SA, Wallerath T et al. Activation of protein kinase C alpha and/or epsilon enhances transcription of the human endothelial nitric oxide synthase gene. Mol Pharmacol 1998 April;53(4):630-7. (19) Kostourou V, Troy H, Murray JF et al. Overexpression of dimethylarginine dimethylaminohydrolase enhances tumor hypoxia: an insight into the relationship of hypoxia and angiogenesis in vivo. Neoplasia 2004 July;6(4):401-11. (20) Hasegawa K, Wakino S, Tanaka T et al. Dimethylarginine dimethylaminohydrolase 2 increases vascular endothelial growth factor expression through Sp1 transcription factor in endothelial cells. Arterioscler Thromb Vasc Biol 2006 July;26(7):1488-94. (21) Wilson AM, Harada R, Nair N, Balasubramanian N, Cooke JP. L-arginine supplementation in peripheral arterial disease: no benefit and possible harm. Circulation 2007 July 10;116(2):188-95. (22) Achan V, Tran CT, Arrigoni F, Whitley GS, Leiper JM, Vallance P. all-trans-Retinoic acid increases nitric oxide synthesis by endothelial cells: a role for the induction of dimethylarginine dimethylaminohydrolase. Circ Res 2002 April 19;90(7):764-9. (23) Khurana R, Shafi S, Martin J, Zachary I. Vascular endothelial growth factor gene transfer inhibits neointimal macrophage accumulation in hypercholesterolemic rabbits. Arterioscler Thromb Vasc Biol 2004 June;24(6):1074-80. (24) Windecker S, Meier B. Late coronary stent thrombosis. Circulation 2007 October 23;116(17):1952-65. (25) Masuda H, Goto M, Tamaoki S, Azuma H. Accelerated intimal hyperplasia and increased endogenous inhibitors for NO synthesis in rabbits with alloxan-induced hyperglycaemia. Br J Pharmacol 1999 January;126(1):211-8.

  Figures legends:

Fig 2 .

 2 Fig 2. Wild type mice carotid arteries infected with AdDDAH1 a. En face confocal image of an infected

Fig 3 .

 3 Fig 3. Concentration response curves to a. Phenylephrine, b. Acetylcholine, c. ADMA in carotid arteries

Fig 4 .

 4 Fig 4. Concentration response curves to a. Phenylephrine, b. Acetylcholine, c. ADMA in carotid arteries

Fig 5 .

 5 Fig 5. Concentration response curves to a. Phenylephrine, b. Acetylcholine, c. ADMA in carotid arteries

  Figure 1. a.b.

  Fig.4

Force

(

  73.30±3.97) % (83.29±5.71) %* (78.36±5.10) %* 2.65x10 -5 (1.63-4.33)x10 -5 5.83x10 -5 (2.85-8.92)x10 -5* 3.94x10 -5 (1.87-8.30)x10 -5* (82.53± 4.97) % (72.60± 11.61) %* (69.34± 0.12) %* Wild Type DDAH 1 +/-*p<0.001, AdDDAH versus AdGFP (EC 50 = concentration producing half-maximal effect, CI=confidence interval; Emax=maximal effect, S.E.M=standard error of the mean).

Table 1 . EC 50 and Emax values from functional studies on isolated mouse carotid arteries from wild type and DDAH 1+/-mice.

 1

		EC 50	(95% CI)	Emax ± ± ± ±S.E.M
	Phenylephrine
	AdGFP	1.51x10 -6 (0.86-2.65)x10 -6	(1.93±0.18) g
	AdDDAH 1	2.77x10 -6 (1.05-4.42)x10 -6*	(1.48±0.16) g*
	AdDDAH 2	2.16x10 -6 (0.68-9.91)x10 -6	(1.72±0.21) g*
	Acetylcholine
	AdGFP	2.22x10 -8 (1.56-3.17)x10 -8	(81.13±2.74) %
	AdDDAH 1	1.77x10 -8 (1.26-2.51)x10 -8*	(92.47±2.16) %*
	AdDDAH 2	2.08x10 -8 (1.32-3.25)x10 -8	(87.60±2.91) %*
	ADMA AdGFP	1.84x10 -5 (0.87-3.87)x10 -5	(62.89±5.84) %
	AdDDAH 1	6.13x10 -5 (3.10-9.96)x10 -5*	(50.51±5.30) %*
	AdDDAH 2	2.82x10 -5 (1.41-5.65)x10 -5	(54.34±4.80) %*
		EC 50	(95% CI)	Emax ± ± ± ±S.E.M
	Phenylephrine
	AdGFP	2.27x10 -7 (0.91-5.67)x10 -7	(2.40±0.34) g
	AdDDAH 1	3.17x10 -7 (0.81-9.97)x10 -7*	(1.48±0.31) g*
	Acetylcholine AdDDAH 2	1.50x10 -7 (0.41-5.41)x10 -7*	(1.90±0.36) g*
	AdGFP	1.54x10 -8 (0.88-2.71)x10 -8
	AdDDAH 1	1.16x10 -8 (0.57-2.35)x10 -8*
	AdDDAH 2	1.01x10 -8 (0.53-1.92)x10 -8*
	ADMA
	AdGFP
	AdDDAH 1
	AdDDAH 2

Table 1 . EC 50 and Emax values from functional studies on isolated mouse carotid arteries from wild type and DDAH 1+/-mice.

 1 -8 (0.88-2.71)x10 -8 1.16x10 -8 (0.57-2.35)x10 -8* 1.01x10 -8 (0.53-1.92)x10-8*

		EC 50	(95% CI)	Emax ± ± ± ±S.E.M
	Phenylephrine
	AdGFP	1.51x10 -6 (0.86-2.65)x10 -6	(1.93±0.18) g
	AdDDAH 1	2.77x10 -6 (1.05-4.42)x10 -6*	(1.48±0.16) g*
	AdDDAH 2	2.16x10 -6 (0.68-9.91)x10 -6	(1.72±0.21) g*
	Acetylcholine
	AdGFP	2.22x10 -8 (1.56-3.17)x10 -8	(81.13±2.74) %
	AdDDAH 1	1.77x10 -8 (1.26-2.51)x10 -8*	(92.47±2.16) %*
	AdDDAH 2	2.08x10 -8 (1.32-3.25)x10 -8	(87.60±2.91) %*
	ADMA AdGFP	1.84x10 -5 (0.87-3.87)x10 -5	(62.89±5.84) %
	AdDDAH 1	6.13x10 -5 (3.10-9.96)x10 -5*	(50.51±5.30) %*
	AdDDAH 2	2.82x10 -5 (1.41-5.65)x10 -5	(54.34±4.80) %*
		EC 50	(95% CI)	Emax ± ± ± ±S.E.M
	Phenylephrine
	AdGFP	2.27x10 -7 (0.91-5.67)x10 -7	(2.40±0.34) g
	AdDDAH 1	3.17x10 -7 (0.81-9.97)x10 -7*	(1.48±0.31) g*
	Acetylcholine AdDDAH 2	1.50x10 -7 (0.41-5.41)x10 -7*	(1.90±0.36) g*
	AdGFP	1.54x10
	AdDDAH 1
	AdDDAH 2
	ADMA
	AdGFP
	AdDDAH 1
	AdDDAH 2

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Acknowledgments

The authors wish to thank Tiziana Bacheti and Herpreet Gill for all their technical support.

Sources of funding:

This work was funded by grants from the British Heart Foundation (RG/05/002) and from the European Vascular Genomics Network funded by the European Union (contract number LSHM-CT-2003-503254).