Mark Pm Harms

Wouter Wieling

Willy Nmj Colier

Jacques Wm Lenders

Niels H Secher

M.D Johannes J Van Lieshout
email: j.j.vanlieshout@amc.uva.nl

Mark P M Harms

Willy N J M Colier

Jacques W M Lenders

Central and cerebrovascular effects of leg crossing in humans with sympathetic failure

Keywords: autonomic disease, blood flow velocity, cardiac output, near-infrared spectroscopy

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INTRODUCTION

In humans, the upright position challenges the cardiovascular system by gravitational displacement of blood to lower parts of the body with a decline in venous return and cardiac output (CO), and a reduction in cerebral perfusion pressure [START_REF] Rosner | Cerebral perfusion pressure, intracranial pressure, and head elevation[END_REF]. Adjustment to the postural decrease in central blood volume involves increased systemic vascular resistance through autonomic reflex activity, but patients with sympathetic dysfunction lack this ability to increase vasomotor tone resulting in a decrease in mean arterial pressure (MAP) [2;3]. In these patients the transcranial Doppler-determined middle cerebral artery flow velocity (MCA V mean ) and the frontal lobe oxygenation (O 2 Hb) determined by near-infrared spectroscopy decrease markedly and they develop symptoms of cerebral hypoperfusion when standing [START_REF] Harms | Orthostatic tolerance, cerebral oxygenation, and blood velocity in humans with sympathetic failure[END_REF].

Both patients with sympathetic dysfunction [START_REF] Van Lieshout | Physical manoeuvres for combating orthostatic dizziness in autonomic failure[END_REF] and recurrent vasovagal syncope [START_REF] Krediet | Leg crossing improves orthostatic tolerance in healthy subjects: a placebocontrolled crossover study[END_REF] can combat symptomatic orthostatic hypotension by crossing one leg against the other (leg crossing).

Leg crossing in the upright position in healthy subjects increases central venous pressure and CO with a reduction in low-frequency arterial pressure variability, signifying reduced sympathetic activity and suggesting that the central blood volume is restored [START_REF] Van Lieshout | Muscle tensing during standing: effects on cerebral tissue oxygenation and cerebral artery blood velocity[END_REF]. Although leg crossing has no effect on MAP in healthy subjects, both MCA V mean and O 2 Hb increase signifying enhanced cerebral perfusion [START_REF] Van Lieshout | Muscle tensing during standing: effects on cerebral tissue oxygenation and cerebral artery blood velocity[END_REF]. However, the effects of leg crossing on cerebral perfusion have not been evaluated in patients with orthostatic hypotension due to sympathetic failure. We therefore tested the hypothesis that in patients with autonomic failure, leg crossing enhances cerebral perfusion, by evaluating the cerebro-and central cardiovascular adaptation to orthostatic stress in patients with orthostatic hypotension due to sympathetic failure, and in age and gender matched control subjects.

METHODS

Eight patients (range 37-67 yr, 3 females) with severe orthostatic hypotension related to sympathetic failure classified as pure autonomic failure (PAF; n=7) and multiple system atrophy A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

(MSA; n=1) but with no symptoms or signs of heart disease participated in the study (Table 1).

Oral and written informed consent was obtained and the study was approved by Ethics Committee of the Academic Medical Center. Age and gender matched healthy subjects with intact autonomic circulatory control and normal orthostatic tolerance served as controls.

Protocol

At 8:00 AM after an overnight fast all subjects reported to the laboratory that was maintained at 22q C. The subjects were supine when instrumented at least 2 hours after a light breakfast without caffeine containing beverages. After instrumentation a test run was performed to familiarize the subjects to the protocol. After 10 minutes of supine rest, the subjects stood up and after 2 minutes upright they crossed their legs and maintained that position for 1 min, then they uncrossed the legs and maintained upright for another minute (Fig. 1). The duration of orthostatic stress was set so as to enable the patients to fulfill the protocol without symptoms of cerebral hypoperfusion and the protocol was repeated twice. A c c e p t e d M a n u s c r i p t (Oxymon, Artinis Medical Systems, Zetten, The Netherlands). The NIRS optodes were attached high on the forehead avoiding the temporalis muscle but sufficiently lateral to avoid the superior sagittal sinus with the transmitting and receiving optodes placed 5.5 cm apart [START_REF] Al-Rawi | Evaluation of a near-infrared spectrometer (NIRO 300) for the detection of intracranial oxygenation changes in the adult head[END_REF]. Changes in O 2 Hb and HHb (µmol.l -1 ) were reported with steady state standing values set at 0 µmol.l -1 .

Measurements

The MCA V mean was measured in the proximal segment and insonated (DWL Multidop X4, Sipplingen, Germany) through the posterior temporal ultrasound window. Once the optimal signal-to-noise ratio was obtained, the probe was secured with a head band. Both at rest and during exercise, determination of the MCA V mean has a coefficient of variation of ~5% [START_REF] Pott | Middle cerebral artery blood velocity and plasma catecholamines during exercise[END_REF]. The V mean was obtained from the maximal TCD frequency shifts over one beat divided by the corresponding beat interval and changes in MCA V mean were taken to represent those in cerebral blood flow (CBF).

End-tidal CO 2 (P ET CO 2 ) was measured by an infrared analyzer (Hewlett Packard 78345A, Roeblingen, Germany) as a reflection of partial arterial CO 2 pressure [START_REF] Immink | The postural reduction in middle cerebral artery blood velocity is not explained by PaCO 2[END_REF]. Thoracic electrical impedance (TI) [START_REF] Jonsson | Thoracic electrical impedance and fluid balance during aortic surgery[END_REF] (Kardio-Dynagraph, Diefenbach GmbH, Frankfurt/Main, Germany) was used as an index of changes in central blood volume whereas stroke volume (SV) was obtained from the blood pressure waveform using the Model flow method incorporating age, gender, height, and weight (BeatScope 1.0 software, FMS, Amsterdam, The Netherlands) [START_REF] Harms | Continuous stroke volume monitoring by modelling flow from non-invasive measurement of arterial pressure in humans under orthostatic stress[END_REF]. Model flow tracks fast changes in SV during postural stress as well as static exercise of relevance for leg crossing [START_REF] Van Dijk | Hemodynamic effects of legcrossing and skeletal muscle tensing during free standing in patients with vasovagal syncope[END_REF].

Data Acquisition and Analysis.

The signals of arterial pressure, the spectral envelope of MCA velocity, TI, P ET CO 2 , and a marker signal were A/D converted at 100 Hz with NIRS data sampled at 10 Hz. Signals were routed through an interface providing electrical isolation with offset and sensitivity adjustments when appropriate. Variables were also recorded on a thermo-writer (Graphtec WR7700 ™, Western Graphtec Inc., Irvine, CA) for on-line inspection. The MCA V mean was expressed as the A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. integral of the maximal frequency shifts over one beat divided by the corresponding beat interval. MAP was the integral of pressure over one beat divided by the beat interval and expressed both at heart and brain level (MAP mca ). Heart rate (HR) was the inverse of the interbeat pressure interval in beats.min -1 (bpm), CO was SV times HR, and total peripheral resistance (TPR) was the ratio of MAP to CO.

Beat-to-beat data were transformed to equidistantly re-sampled data at 2 Hz by polynomial interpolation and averages for both groups are shown in Fig 4 . Blood pressure, HR, V mean , P ET CO 2 , and TI were expressed in absolute values, whereas resting supine values for SV, CO and TPR were set at 100% (control) and changes were expressed as percentages. Within each subject, the average of all supine values measured over a period of 30 seconds prior to standing up was used as the baseline value and compared with the average value of 55 to 65 seconds of standing.

Similarly, the average of all values measured over a period of 30 seconds of quiet standing before leg crossing was used as the baseline value and compared with the average of the last 10 seconds of leg crossing. The average of the 30 seconds of quiet standing before leg crossing was then used as the baseline for evaluating the effect of leg crossing, the data of the three runs being averaged. The effect of leg crossing on cerebral and systemic circulatory variables were evaluated by the Wilcoxon signed rank test whereas differences between patients and controls were analyzed by the Mann-Whitney rank sum test. Correlation between variables were evaluated by nonlinear regression analysis. A P value <0.05 was considered to indicate a statistically significant difference. A c c e p t e d M a n u s c r i p t analysis. As a result, responses from 6 patients and their matched controls are reported of whom two patients (S7 and S8) had minor orthostatic complaints during standing which, however, disappeared with leg crossing.

RESULTS

In one patient (S2) the quality of the

Posture

In the supine position MAP and MCA V mean were high in the patients (Table 2). Standing induced a large fall in MAP in the patients (-46 (11-78)) but not in the controls (-4 (-8-8)) mmHg (P<0.05); accompanied by a larger fall in SV (46 (41-55) vs. 32 (15-46) %; P<0.05)) and CO (34 (21-42) vs. 7 (-9-25) %; P<0.05)) without the trend of an increase in TPR seen in the controls, such that TPR was significantly different in patients and controls during standing. Resting HR did not differ between the two groups of subjects when supine and during standing, HR increased to a comparable level in the two groups, whereas MCA V mean decreased only in the patients (from 77 (64-123) to 55 (38-77) cm.s -1 ) resulting in a comparable MCA V mean for patients and controls.

Upon standing, the decrease in O 2 Hb was also larger in the patients (-13.1 (-16.9 --4.0) vs. -4.9 (-14.6 --2.8) Pmol.l -1 ; P<0.05)), whereas the HHb increased in both groups of subjects, the larger increase being in the patients (7.5 (3.7-10.9) vs. 2.2 (1.7-9.1) Pmol.l -1 ; P<0.05)). The P ET CO 2 was comparable for patients and control subjects and independent of body position and resting TI and the trend for TI to increase on standing were also similar in the patients and control subjects Table 2).

Cardiovascular effects of leg crossing

At the onset of leg crossing, in each group CO and MAP increased ( In both patients and healthy subjects a marked transient decrease in TI (increase in central blood volume) in response to leg crossing was observed in 13 of 18 of each group (sign test p<0.05). After the initial drop, TI increased again and there was no significant change over one minute of leg crossing.

Cerebrovascular effects of leg crossing

In both patients and controls, leg crossing induced a similar increase in MCA V mean with a concomitant rise in O 2 Hb in the patients and a trend towards lower values for HHb (Figures 2345and Table 3). The P ET CO 2 was comparable for patients and controls and did not change during leg crossing. Within one minute after leg crossing, all values returned to baseline. The relationship between MCA V mean and MAP and CO during free standing, leg crossing and again free standing with three data points plotted for each subject is shown in figure 5. In the patients the regression coefficient for MAP and MCA V mean was 0.61 (p<0.02) and for CO and MCA V mean it was 0.52 (P<0.05). In the control subjects this value for MAP and MCA V mean was 0.34 (NS), whereas this was 0.71 for CO and MCA V mean (P<0.05).

DISCUSSION

In the present study, leg crossing consistently improved cerebral perfusion and oxygenation in patients with orthostatic hypotension related to sympathetic failure as in controls. However, the mechanisms of enhanced cerebral perfusion and oxygenation induced by leg crossing appeared different for healthy subjects in whom leg crossing increased CO without significantly affecting TPR or MAP. In the patients, MAP was increased and this was supported by an increase in TPR as well as in CO. The immediate drop in TI at the onset of leg crossing in both patients and healthy subjects suggests translocation of blood to the chest with less pooling due to sustained muscle tensing. However, given the defective vasomotor control in the patients, the increase in TPR induced by leg crossing is likely to be related to compression of venous and arterial vascular beds.

In healthy subjects the postural fall in venous return as a consequence of pooling of blood reduces SV and CO [START_REF] Harms | Orthostatic tolerance, cerebral oxygenation, and blood velocity in humans with sympathetic failure[END_REF]. However, an increase in vasomotor tone in response to cardiopulmonary and arterial baroreflex-mediated sympathoexcitation, local myogenic responses and venoarteriolar axon reflex activity, elevates mean and diastolic arterial pressure [3;16]. By contrast.

patients with sympathetic failure, however, cannot modulate vascular tone and thus develop orthostatic hypotension [2;3] because of an excessive fall in SV and CO, attributable to both increased pooling of venous blood and impaired inotropic and chronotropic cardiac responses [START_REF] Bevegård | Circulatory response to recumbent exercise and head-up tilting in patients with disturbed sympathetic cardiovascular control (postural hypotension). Observations on the effect of norepinephrine infusion and antigravity suit inflation in the head-up tilted position[END_REF]. The tendency for TI to be higher upon standing is consistent with a reduction in central blood volume in both groups (Table 2). In addition to the marked reduction in MAP and CO, a decrease in P ET CO 2 as an index of the arterial value might have contributed to the observed reduction in MCA V mean that occurred in the patients on standing. However, the contribution of P ET CO 2 to the postural reduction in MCA V mean is transient [START_REF] Immink | Transient influence of end-tidal carbon dioxide tension on the postural restraint in cerebral perfusion[END_REF] and in the present study, any such influence must have been small, if any. Also, leg crossing did not change P ET CO 2 rendering a contribution of partial arterial CO 2 pressure to the increase in cerebral perfusion during leg crossing unlikely. In healthy subjects, autonomic neural control of the cerebral circulation is tonically active [START_REF] Zhang | Autonomic neural control of dynamic cerebral autoregulation in humans[END_REF][START_REF] Ogoh | Autonomic neural control of the cerebral vasculature: acute hypotension[END_REF][START_REF] Van Lieshout | Point:Counterpoint: Sympathetic activity does/does not influence cerebral blood flow. Point: Sympathetic activity does influence cerebral blood flow[END_REF] but in patients with autonomic failure a reduced spillover of noradrenaline from the brain renders such an influence unlikely [START_REF] Mitchell | Jugular venous overflow of noradrenaline from the brain: a neurochemical indicator of cerebrovascular sympathetic nerve activity in humans[END_REF].

Under normal circumstances CBF is maintained across a range of MAP of ~ 60 to 150 mmHg by cerebral autoregulation (CA). Further, a reduction in MAP below the lower limit of CA challenges CBF, and a reduction in central blood volume and, therefore, in CO adversely influences the lower limit of CA [START_REF] Waldemar | Angiotensin-converting enzyme inhibition and regional cerebral blood flow in acute stroke[END_REF][START_REF] Jørgensen | Middle cerebral artery velocity during head-up tilt induced hypovolaemic shock in humans[END_REF][START_REF] Van Lieshout | Point:Counterpoint: Sympathetic activity does/does not influence cerebral blood flow. Point: Sympathetic activity does influence cerebral blood flow[END_REF]. The significant relationship between MAP and MCA V mean in the upright position in the patients (Fig 5) suggests that leg crossing in the patient raised MAP from below, to within the accepted autoregulatory range [START_REF] Panerai | Cerebral autoregulation: from models to clinical applications[END_REF]. Patients with autonomic failure are remarkably tolerant of postural hypotension [START_REF] Thomas | Preservation of autoregulation of cerebral blood flow in autonomic failure[END_REF] suggesting they have well maintained cerebral autoregulatory capacity. On the other hand, a parallel postural reduction in MAP and MCA V mean does occur in these patients suggesting a reduced autoregulatory capacity [START_REF] Lagi | Cerebral autoregulation in orthostatic hypotension. A transcranial Doppler study[END_REF]. Thus, whether CA is maintained in autonomic dysfunction remains debatable.

In the present study, MCA V mean was used as a measure of cerebral perfusion, assuming that in patients with sympathetic failure changes in MCA V mean are representative of those in CBF.

However, critical for the interpretation of the data is the extent to which MCA V mean reflects volume flow in cerebral circulation. Changes in resistance to flow occur mainly in the cerebral arteriolar bed mainly; the major cerebral conductance arteries are non-compliant and acting as a conduit for the pulsatile arterial flow. This is important because MCA V mean calculated from the frequency distribution of the Doppler shifts is related to CBF only if the insonated vessel diameter remains constant. This has been shown to be the case for the MCA over a ~30 mmHg range of blood pressure such that with MCA V mean do reflect changes in CBF [START_REF] Giller | Cerebral arterial diameters during changes in blood pressure and carbon dioxide during craniotomy[END_REF]. Indeed, direct observations made during craniotomy have revealed that lowering blood pressure by sodium nitroprusside does not affect MCA diameter [START_REF] Giller | Cerebral arterial diameters during changes in blood pressure and carbon dioxide during craniotomy[END_REF]. Moreover, orthostatic stress, as simulated by lower body negative pressure, does not alter the MCA diameter as assessed with MR imaging [START_REF] Serrador | MRI measures of middle cerebral artery diameter in conscious humans during simulated orthostasis[END_REF]. Nevertheless, the low blood pressure levels developed in the patients with sympathetic failure do not rule out the possibility that a considerable fall in blood pressure might have reduced the diameter of the MCA, such that the measurement of MCA V mean led to underestimation of the postural reduction in CBF [START_REF] Thoresen | Cerebral Doppler and misinterpretation of flow changes[END_REF]. of the capillaries, has been shown to reflect the brain capillary O 2 saturation over a two-fold variation in CBF [START_REF] Rasmussen | Capillary-oxygenation-level-dependent near-infrared spectrometry in frontal lobe of humans[END_REF]. Further, with postural stress, cerebrovascular oxygenation as determinated by NIRS has been shown to be directly related to cerebral perfusion as determined by the transcranial Doppler technique [9;33]. Thus, when MCA V mean and O 2 Hb increase in parallel, we believe it is reasonable to conclude that CBF changed in the same direction, as has been confirmed by clinical evaluation [9;34-36].

It must be acknowledged that there are potential complications when interpreting changes in cerebral perfusion in the upright position. Although the brain is elevated above heart, the contention that the cerebral perfusion pressure falls accordingly [START_REF] Hill | The influence of the force of gravity on the circulation[END_REF][START_REF] Rosner | Cerebral perfusion pressure, intracranial pressure, and head elevation[END_REF][START_REF] Gisolf | The siphon controversy: an integration of concepts and the brain as baffle[END_REF] is not generally accepted [START_REF] Hicks | The siphon controversy counterpoint: the brain need not be "baffling[END_REF]. For the brain, not only the inflow pressure but also the venous and cerebral spinal fluid pressures decline in proportion to the vertical distance above the heart [START_REF] Rosner | Cerebral perfusion pressure, intracranial pressure, and head elevation[END_REF][START_REF] Asgeirsson | Local vascular responses to elevation of an organ above the heart[END_REF][START_REF] Kongstad | The role of arterial and venous pressure for volume regulation of an organ enclosed in a rigid compartment with application to the injured brain[END_REF][START_REF] Dawson | Critical closing pressure explains cerebral hemodynamics during the Valsalva maneuver[END_REF] and veins above heart collapse because the surrounding tissue pressure is greater than the pressure inside the veins, so creating a Starling resistor [START_REF] Lopez-Muniz | Critical closure of pulmonary vessels analyzed in terms of Starling resistor model[END_REF]. Within the brain, a potential siphon exists at the level of the sinus sagittalis which, in contrast to the jugular veins, does not collapse upon standing up [START_REF] Mosso | Absence of gravity-dependent modulation of straight sinus flow velocity in healthy humans[END_REF]. However, because of the collapsible veins, gravitational pressure gradients are not matched on the arterial and venous sides of the circulation at all levels above the heart as would be necessary for a siphon to operate [37;47]. Indeed, the effects of posture on spinal fluid pressure [START_REF] Dawson | Standing up to the challenge of standing: a siphon does not support cerebral blood flow in humans[END_REF] and internal jugular vein jugular cross-sectional area [49;50] have been evaluated in healthy subjects and it has been shown that when humans are upright, the prevailing pressure for both the venous outflow and for the spinal fluid approaches zero at the base of the brain, so negating the idea that a siphon supports CBF. Thus, it seems that in upright humans, the magnitude of cerebrovascular resistance breaks the continuity requirement for a siphon, implying that the heart has to work against gravity to perfuse the brain. As far as the present study is concerned, we have no reason to suppose that these principles apply differently to patients with sympathetic failure.

In the present study in healthy subjects, both the MCA V mean and O 2 Hb increased in response to A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. leg crossing without significant changes in MAP confirming earlier findings [START_REF] Van Lieshout | Muscle tensing during standing: effects on cerebral tissue oxygenation and cerebral artery blood velocity[END_REF] and suggesting an increase in cerebral perfusion. There was a direct relationship between CO and MCA V mean that cannot be explained by changes in MAP (Fig 5) [51;52]. In the patients, the increase in cerebral perfusion as indicated by the increase in MCA V mean was comparable but, in contrast, MCA V mean was related to both CO and MAP. In healthy subjects the arterial baroreflex instantaneously maintains MAP by buffering changes in preload instantaneously. However, patients with sympathetic failure lack this buffering capacity and MAP is largely dependent on cardiac preload and CO. Thus, on this basis alone, it is not clear whether cerebral perfusion is determined by MAP as well as by CO (Fig 5). However, if we consider our results in more detail, it is clear that leg crossing increased MAP in each individual patient and there was an associated increase in MCA V mean indeed, it seems that leg crossing increased MAP in the patients from below, to within in the accepted cerebral autoregulatory range. In other words, the direct relationship between MCA V mean and MAP may be accounted for because standing allowed MAP to fall below the autoregulatory range. When MAP is within the autoregulatory range, there is no reason to suppose that the factors that regulate cerebral perfusion are different in healthy subjects and patients with sympathetic failure.

In conclusion, leg crossing consistently improves cerebral perfusion in patients with sympathetic failure as reflected by reflected in a rise of cerebral blood velocity and in cerebral oxygenation and this is associated with a relieve of symptoms. In healthy subjects, CO increases during leg crossing without significant changes in MAP whereas in the patients, there is an increase in MAP that is supported by an increase in TPR. There is no definite explanation for this increase in TPR in the patients with sympathetic failure, but we hypothesize that the positive mechanical effect of leg crossing on TPR overcomes the vasodilator effected caused by the absence of sympathetic vascular control. Patients with orthostatic intolerance should, therefore, be advised, in addition to increasing their salt intake and to use sleeping head-up tilted, also to apply leg crossing when

Legends to Figures

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Clinical

  Science Immediate Publication. Published on 16 Oct 2009 as manuscript CS20090038 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090038

  Arterial pressure was measured with a Finapres™ model 5 (Netherlands Organization for Applied Scientific Research, Biomedical Instrumentation, TNO-BMI, Amsterdam) from the middle finger of the non-dominant arm fixed in the anterior axillary line at heart level. Finger arterial pressure tracks beat-to-beat changes in arterial pressure during hypotension. Cerebral oxygenation was monitored using near infrared spectroscopy (NIRS) based on the transparency of tissue to light in the near-infrared region with the O 2 status dependent changes on absorption caused by chromophores dominated by oxy-and deoxy-haemoglobin (O 2 Hb and HHb) [8-10]. To estimate the concentration changes in O 2 Hb and HHb a differential path length factor of 6.0 was applied to account for the scattering of light in the tissue. A continuous wave NIRS instrument with three wavelengths (901, 848 and 770 nm) and 10 Hz sampling time was used Clinical Science Immediate Publication. Published on 16 Oct 2009 as manuscript CS20090038 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090038

Clinical

  Science Immediate Publication. Published on 16 Oct 2009 as manuscript CS20090038 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090038

  Doppler velocity spectrum in the upright posture was insufficient and in the standing position one patient (S5) developed serious orthostatic complaints and could, therefore, not complete three runs and these data were excluded from the Clinical Science Immediate Publication. Published on 16 Oct 2009 as manuscript CS20090038 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090038

  Fig 4) whereas TPR decreased. When analysed in the final 10 sec of leg crossing, in the controls, there was an increase in SV (16%) and CO (10%), a trend for a lower HR, but no significant change in TPR or MAP (Table 3, Fig 4). Pulse pressure increased from 48 (38-55) to 55 (50-58) mmHg (P<0.05). By contrast, in patients, leg crossing induced an increase in MAP and in pulse pressure Clinical Science Immediate Publication. Published on 16 Oct 2009 as manuscript CS20090038 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090038 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited (from 33 (23-48) to 40 (28-55) mmHg; P<0.05)). The underlying hemodynamic change was an increase in SV (8%), CO (6%) and TPR (11%). HR did not change (Table 3, Fig 4). Representative examples of the reproducibility of the responses to leg crossing manoeuvre in patients are provided in figures 2 and 3.

Clinical

  Science Immediate Publication. Published on 16 Oct 2009 as manuscript CS20090038 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090038 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Clinical

  Science Immediate Publication. Published on 16 Oct 2009 as manuscript CS20090038 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090038 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

  An attempt was therefore made to overcome this possibility by combining the use of the transcranial Doppler technique with the use of NIRS, which is based on different physical principles as an alternative means of assessing changes in CBF. The NIRS signal for the frontal lobe, which depends on oxygenation at the level Clinical Science Immediate Publication. Published on 16 Oct 2009 as manuscript CS20090038 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090038 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Clinical

  Science Immediate Publication. Published on 16 Oct 2009 as manuscript CS20090038 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090038

Clinical

  Science Immediate Publication. Published on 16 Oct 2009 as manuscript CS20090038 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090038 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited they stand up as a simple means of maintaining cerebral perfusion and avoiding postural syncope.

  crossing in a patient with pure autonomic failure Written informed consent of the patient was obtained for the photograph. Solid box, leg crossing and open boxes, standing legs uncrossed. Leg crossing induced an instantaneous increase in arterial pressure (BP), and cerebral blood velocity (CBV) and oxygenation (O 2 Hb) with a reduction in thoracic electrical impedance (TI) indicating an acute increase in thoracic blood volume.

Figure 2 Effects

 2 Figure 2

Figure 3 Reproducibility

 3 Figure 3

Figure 4 Cardiovascular

 4 Figure 4

Figure 5 Relationship

 5 Figure 5

Figure 1 BP

 1 Figure 1

Figure 2 Clinical

 2 Figure 2

Figure 3 Clinical

 3 Figure 3

Figure 4 Clinical

 4 Figure 4

Figure 5 Clinical

 5 Figure 5

Table 1 . Patient characteristics

 1

	Pure autonomic failure (PAF), multiple system atrophy (MSA), supine blood pressure (BPsup),
	standing blood pressure (BPstd), sleeping 12º head-up-tilt (HUT), dietary salt supplementation (NaCl),
	and Fludrocortisone (Flu)
	Patients Gender Disease	Treatment	BPsup	BPstd	Age	Weight	Height
					(mmHg)	(mmHg)	(yr)	(kg)	(cm)
	S1	m	PAF	none	156/68	97/51	65	78	172
	S2	m	PAF	HUT	175/101	76/47	51	83	176
	S3	f	PAF	none	175/99	85/48	40	65	176
	S4	f	PAF	NaCl/Flu	112/71	73/49	37	56	164
	S5	m	PAF	Flu	159/81	60/42	65	80	175
	S6	f	PAF	NaCl/Flu	163/98	75/55	61	65	160
	S7	m	MSA	Flu/HUT	135/84	76/45	54	96	196
	S8	m	PAF	NaCl/Flu/HUT	192/102	68/47	67	98	189

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Table 3 . Cardiovascular and cerebral responses to leg crossing in standing position

 3

	Mean arterial pressure at heart (MAP heart ) and brain level (MAP mca ), middle cerebral artery mean
	blood velocity (MCA V mean ), oxy-and deoxyhemoglobin (O 2 Hb and HHb) end-tidal CO 2
	(P ET CO 2 ), heart rate (HR), stroke volume (SV), cardiac output (CO), total peripheral resistance
	(TPR), and thoracic electrical impedance (TI). Data are median and range. * P<0.05 in comparison
	to standing before leg cross, † P<0.05 patients vs. controls.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

ACKNOWLEDGMENTS


MD Carmine Savoia

PhD Talin Ebrahimian

PhD Catherine A Lemarié

PhD Pierre Paradis

PhD Marc Iglarz

PhD Farhad Amiri

PhD Danesh Javeshghani

MD Ernesto L Schiffrin
email: ernesto.schiffrin@mcgill.ca

Countervailing vascular effects of rosiglitazone in

Keywords: hyperhomocysteinemia, rosiglitazone, atherosclerosis, endothelium, ADMA

Introduction

Peroxisome proliferator-activated receptor ! (PPAR!) belongs to a family of ligand-activated nuclear receptor and transcription factors which have been extensively studied in adipocytes [START_REF] Touyz | Peroxisome proliferator-activated receptors in vascular biology-molecular mechanisms and clinical implications[END_REF][START_REF] Tontonoz | Stimulation of adipogenesis in fibroblasts by PPAR!, a lipid-activated transcription factor[END_REF][START_REF] Duan | Peroxisome proliferators-activated receptor-!-mediated effects in the vasculature[END_REF], where they are involved in fat cell differentiation and lipid storage. PPAR! are present as well in liver and skeletal muscle, other targets of insulin action [START_REF] Touyz | Peroxisome proliferator-activated receptors in vascular biology-molecular mechanisms and clinical implications[END_REF][START_REF] Tontonoz | Stimulation of adipogenesis in fibroblasts by PPAR!, a lipid-activated transcription factor[END_REF][START_REF] Olefsky | PPAR gamma and the treatment of insulin resistance[END_REF], and modulate insulin sensitivity in all these tissues. Rosiglitazone is a high-affinity ligand for PPAR! belonging to the thiazolidinedione (TZD) or glitazone class of synthetic compounds clinically used in the treatment of type-2 diabetes mellitus [START_REF] Touyz | Peroxisome proliferator-activated receptors in vascular biology-molecular mechanisms and clinical implications[END_REF][START_REF] Duan | Peroxisome proliferators-activated receptor-!-mediated effects in the vasculature[END_REF]. TZDs promote fatty acid oxidation and enhance insulin sensitivity in liver and skeletal muscle in part through inhibition of agents from adipose tissue that promote insulin resistance [START_REF] Evans | PPARs and the complex journey to obesity[END_REF]. PPAR! is expressed in cardiovascular tissues, including heart, endothelium, vascular smooth muscle cells, and monocytes/macrophages [START_REF] Touyz | Peroxisome proliferator-activated receptors in vascular biology-molecular mechanisms and clinical implications[END_REF]. In vivo and in vitro studies have demonstrated that, independently of metabolic effects, PPAR! activators exert anti-proliferative, antioxidant and anti-inflammatory effects on the cardiovascular system, and that they may be anti-atherogenic [START_REF] Touyz | Peroxisome proliferator-activated receptors in vascular biology-molecular mechanisms and clinical implications[END_REF][START_REF] Duan | Peroxisome proliferators-activated receptor-!-mediated effects in the vasculature[END_REF][START_REF] Evans | PPARs and the complex journey to obesity[END_REF][START_REF] Tontonoz | PPAR! promotes monocyte/macrophage differentiation and uptake of oxidized LDL[END_REF][START_REF] Marx | Macrophages in human atheroma contain PPARgamma: Differentiation-dependent PPARgamma expression and reduction of MMP-9 activity through PPARgamma activation in mononuclear phagocytes in vitro[END_REF][START_REF] Ricote | Expression of the peroxisome proliferator-activated receptor ! (PPARgamma) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein[END_REF][START_REF] Staels | Activation of human aortic smooth-muscle cells is inhibited by PPAR" but not by PPAR! activators[END_REF][START_REF] Chinetti | CLA-1/SR-BI is expressed in atherosclerotic lesion macrophages and regulated by activators of peroxisome proliferator-activated receptors[END_REF]. Hence, this class of drugs may represent a therapeutic tool for vascular protection and prevention of progression of atherosclerosis and cardiovascular disease. However, experimental and clinical studies have provided controversial results on the role of TZDs in the prevention of cardiovascular events in diabetic patients, who are at high cardiovascular risk [START_REF] Robinson | Should we use PPAR agonists to reduce cardiovascular risk?[END_REF]. Hyperhomocysteinemia (H-Hcy) is an independent risk factor for coronary, cerebral, and peripheral atherosclerosis [START_REF] Welch | Homocysteine and atherothrombosis[END_REF][START_REF] Eikelboom | Homocysteine and cardiovascular disease: a critical review of the epidemiologic evidence[END_REF]. Cardiovascular risk associated with H-Hcy is particularly high when combined with other risk factors (i.e. diabetes mellitus, high cholesterol, hypertension). Methylene tetrahydrofolate reductase (MTHFR) is a key enzyme in the remethylation cycle of homocysteine, which is converted to methionine [START_REF] Welch | Homocysteine and atherothrombosis[END_REF]. A murine model of MTHFR deficiency, leading to increased plasma levels of homocysteine, develops atherosclerosis and vascular dysfunction when exposed to a high cholesterol diet (HCD) [START_REF] Chen | Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition[END_REF]. This model mimics a common cause of mild H-Hcy in humans [START_REF] Kang | Intermediate homocysteine: a thermolabile variant of methylenetetrahydrofolate reductase[END_REF][START_REF] Frosst | A candidate genetic risk factor for vascular diseas: a common mutation in methylenetetrahydrofolate reductase[END_REF]. We hypothesized that chronic administration of rosiglitazone would improve vascular structure and function in mthfr knockout mice fed HCD.

Methods

Animal Experiments

The study protocol was approved by the Animal Care Committee of the Lady Davis Institute and conducted in accordance with recommendations of the Canadian Council of Animal Care. Mice heterozygous for disruption of the mthfr gene presenting mild hyperhomocysteinemia were generated at the Montreal Children's Hospital Research Institute as already reported [START_REF] Chen | Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition[END_REF]. Heterozygous mthfr-deficient mice (mthfr +/-) and wild type controls were obtained by mating mthfr+/-mice with wild type BALB/cAnNCrlBR (Charles River, St-Constant, Qc, Canada). Old adult female mthfr +/-(n=24 divided in 4 groups as mentioned below) and wild type (n=6) littermate mice aged 11-12 months were studied. Homozygous mice have high mortality or delayed development and cerebellar abnormalities [START_REF] Chen | Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition[END_REF], and for this reason could not be studied. Heterozygous mice were divided into four groups (6 mice per group) fed or not HCD (6 mg/Kg/day; cholesterol 12.5 g/Kg; Research Diets #D12336, New Brunswick, NJ, USA), and treated or not with rosiglitazone (20 mg/Kg/day) for 90 days. Mice were sacrificed by CO 2 asphyxiation.

Measurement of plasma cholesterol

Blood samples from the mice were collected in tubes containing EDTA. Plasma was separated by centrifugation and stored at -80°C. Total plasma cholesterol was measured by enzymatic reaction.

Evaluation of PPAR! expression

Snap-frozen aorta at level of the coronary sinus was pulverized in lysis buffer (2 mM EDTA; 1 mM EGTA; with protease inhibitors: 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 1 mM PMSF, 1 mM Na 3 VO 4 ). Thereafter, samples were briefly sonicated and centrifuged (16000 g, 4°C, 30 min). Four µg of sample in a total incubation volume of 20 µµl as per manufacturer instructions was analyzed with an ELISA based kit (Active Motif, Carlsbad, CA, USA).

Study of internal carotid arteries

Internal carotid arteries (2-3 mm in length) were placed in cold physiological salt solution containing (mmol/L) NaCl 120, NaHCO 3 25, KCl 4.7, KH 2 PO 4 1.18, MgSO 4 1.18, CaCl 2 2.5, EDTA 0.026, and glucose 5.5. They were mounted on 2-glass microcannulae in a pressurized myograph. Endothelium-dependent relaxation was assessed by measuring the dilatory responses to acetylcholine (10 -9 to 10 -4 mol/L) in vessels pre-contracted with norepinephrine (5 x 10 -5 mol/L). Endothelium-independent relaxation was assessed by the dilatory response to sodium nitroprusside (10 -8 to 10 -4 mol/L). To evaluate nitric oxide (NO) availability, the concentration-response curve to acetylcholine was determined before and after 30-minute pre-incubation with the NO synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME, 10 -4 mol/L). Vessels were deactivated by perfusion with Ca ++ -free physiological salt solution containing 10 mmol/L EGTA for 30 minutes. Lumen and media were measured at an intraluminal pressure at 60 mm Hg as previously described [START_REF] Neves | Small artery mechanics in hyperhomocysteinemic mice: effects of angiotensin II[END_REF].

Plasma nitrite measurement

Plasma nitrite as an index of NO production was evaluated with a colorimetric assay as previously described [START_REF] Javeshghani | Increased blood pressure, vascular inflammation, and endothelial dysfunction in androgen-deficient follitropin receptor knockout male mice[END_REF]. Duplicate samples of plasma (50 µl) were incubated with cadmium overnight at 4°C with gentle shaking. Plasma was mixed with 0.01% N-(1-naphtyl) ethylenediamine followed by mixing with 0.1% p-aminobenzenesulfonamide and total nitrite was determined by measuring absorbance at 548 nm.

Plasma ADMA measurement

Blood samples were centrifuged at 3000 rpm for 10min at 4°C immediately after collection. Plasma samples were kept frozen at -80°C until analysis. Twenty µl of plasma as per manufacturer's instructions were analyzed by an ELISA-based kit (DLD Diagnostika GmBH, Hamburg, Germany).

Oxidative fluorescent microtopography

The oxidative fluorescent dye dihydroethidium (DHE) was used to evaluate in situ production of superoxide [START_REF] Rothe | Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2',7'-dichlorofluorescein[END_REF]. Unfixed frozen ring segments of aorta embedded in OCT were cut into 5-10 !m-thick sections and placed on a glass slide. DHE (2 x 10 -6 mol/L) was topically applied to each tissue section. Slides were incubated in a light-protected humidified chamber at 37°C for 30 minutes and then coverslipped [START_REF] Miller | Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis[END_REF]. Images were obtained with a Leica fluorescent microscope. The amount of DHE staining present in the vessel wall was quantified (Northern Eclipse program, EMPIX Imaging Inc., Missassauga, ON, Canada) and expressed as percentage of the DHE fluorescence per total surface area.

Western blot analysis for protein arginine methyltransferase (PRMT-1)

Protein was extracted from the coronary sinus area of snap frozen aorta as previously described [START_REF] Savoia | Angiotensin II/AT2 receptor-induced vasodilation in stroke-prone spontaneously hypertensive rats involves nitric oxide and cGMP-dependent protein kinase[END_REF]. Protein (100 !g) was separated by electrophoresis on 10% polyacrylamide gel and transferred onto a nitrocellulose membrane. Non-specific binding sites were blocked with 5% skimmed milk in Tris-buffered saline solution with Tween for 1 h at 24°C. Membranes were incubated overnight with anti-PRMT-1 polyclonal antibody (1:1000; Cell Signaling Technology, Danvers, MA). After incubation with the second antibody, the signal were revealed with chemiluminescence, visualized by autoradiography, and quantified densitometrically.

In silico analysis of the PRMT-1 promoter.

The mouse PRMT-1 gene was examined in silico using UCSC genome bioinformatics Web site [START_REF] Kent | The human genome browser at UCSC[END_REF]23]. The 52238857-52242238 bp region of chromosome 7 was analyzed in silico using rVista to determine conserved PPAR! and PPAR response elements between mouse and human [START_REF] Loots | rVISTA for comparative sequence-based discovery of functional transcription factor binding sites[END_REF]25].

Data analysis

Data are presented as mean ± SEM. Morphological data were compared by ANOVA followed by Student-Newman-Keuls post-hoc test. Statistical evaluation of mechanical parameters was performed by repeated-measures ANOVA. A value of P<0.05 was considered statistically significant.

Results

Mice with haploinsufficience in MTHFR presenting mild hyperhomocysteinemia [START_REF] Chen | Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition[END_REF], were treated or not with HCD with or without rosiglitazone. Wild type littermate was used as control. Plasma cholesterol (Figure 1a) was similar in all the animals fed the normal diet and increased as expected in mthfr +/-mice fed HCD compared to wild type mice (P<0.001), and was significantly reduced after rosiglitazone treatment (P<0.05 vs. mthfr +/-fed HCD). In mthfr +/-mice fed a normal diet, the expression level of PPAR! in aorta was similar to wild type mice (Figure 1b). In mthfr +/-mice fed HCD, PPAR! expression was significantly increased compared to wild type mice (P<0.05). Rosiglitazone treatment induced a further increase in PPAR! expression in mthfr +/-mice fed HCD compared to untreated mthfr +/-mice fed HCD (P<0.05). The M/L ratio of internal carotid arteries was similar in mthfr +/-mice fed normal diet compared to mthfr +/-mice fed HCD, and in both groups M/L was significantly greater than in wild type mice (P<0.001, Figure 2). M/L ratio and media cross-sectional area (CSA) were significantly smaller after rosiglitazone treatment only in mthfr +/-mice compared to untreated mthfr +/-mice when both groups were fed normal diet (P<0.01 and P<0.05 respectively). However, hypertrophic remodelling was not improved by rosiglitazone treatment of mthfr +/-mice fed HCD. Endothelium-dependent relaxation of carotid arteries was similar in mthfr +/-mice fed normal diet compared to wild type mice (Figure 3a), and was not different from mthfr +/-mice fed HCD or mthfr +/-mice fed the normal diet and treated with rosiglitazone. Vasodilation of carotid arteries in response to acetylcholine was significantly attenuated in mthfr +/-mice treated with rosiglitazone and fed HCD compared to HCD-fed mthfr +/-mice that did not receive rosiglitazone (P<0.05). Endothelium-independent relaxation was similar in all groups (Figure 3b). The NO synthase inhibitor L-NAME significantly reduced acetylcholine-induced dilation in all groups (Figure 3c). In carotid arteries from rosiglitazone-treated mthfr +/-fed HCD, acetylcholine-induced dilation was only slightly inhibited by L-NAME (Figure 3d), indicating poor NO synthesis or bioavailability. Superoxide generation in carotid arteries was higher in mthfr +/-mice fed normal diet compared to wild type mice (P<0.01, Figure 4). A further increase was observed in mthfr +/-mice fed HCD in comparison to mthfr +/-mice fed normal diet (P<0.01). Rosiglitazone did not affect superoxide production in carotid arteries of mthfr +/-mice fed normal or HCD. Plasma nitrite, an index of systemic NO production, was similar in mthfr +/-mice fed the normal or HCD compared to the wild type group. Rosiglitazone significantly reduced plasma nitrite concentration in both treatment groups (Figure 5a). Plasma levels of ADMA, an endogenous inhibitor of endothelial NO synthase (eNOS), were similar in mthfr +/-mice fed normal or HCD. Significantly elevated plasma ADMA levels were found in HCD fed mthfr +/-mice treated with rosiglitazone (P<0.05) (Figure 5b) The expression of PRMT-1, a key enzyme in the synthesis of ADMA, was similar in mthfr +/-mice compared to wild type mice independently of the diet. Rosiglitazone significantly enhanced PRMT-1 expression only in HCD-fed mthfr +/-(Figure 6). The analysis of prmt1 gene with the UCSC genome bioinformatics Web site revealed that it is contained on chromosome 7 and has four different alternative mRNA PRMT-1 splices (Figure 7b). The 52238857-52242238 bp region of chromosome 7 containing the two 5' exons and promoter region of the prmt1 gene was analyzed using the rVista software, and several PPAR! response elements conserved between mouse and human were found (Figure 7a,c). The UCSC genome browser revealed that several regions of the PRMT-1 promoter are highly conserved among several mammalian species (Figure 7d).

Discussion

Major findings from the present study of mild hyperhomocysteinemic mthfr +/ -mice demonstrate that: 1) rosiglitazone improved vascular remodeling of carotid arteries, in part by reducing plasma cholesterol levels in mice fed HCD; 2) PPAR! agonism was associated with impaired endothelial function in the carotid arteries of mthfr +/-mice fed HCD, associated with decreased plasma nitrite and enhanced PRMT-1 expression and plasma levels of ADMA, an endogenous eNOS inhibitor; 3) rosiglitazone failed to reduce increased levels of reactive oxygen species (ROS) in mthfr +/-mice. The association of several risk factors in a single individual is frequent in Western societies, and contributes to the development of atherosclerosis and cardiovascular dysfunction. Age, hypercholesterolemia and H-Hcy may participate in the progression of atherosclerosis, in part by inducing oxidative stress, endothelial dysfunction and inflammation [START_REF] Savoia | Inhibition of the renin angiotensin system: implications for the endothelium[END_REF][START_REF] Touyz | Reactive oxygen species in vascular biology: implications in hypertension[END_REF]. This is the case for the murine model of mild atherosclerosis studied here, which is characterized by advanced age, mild H-Hcy, and elevated plasma cholesterol secondary to HCD. PPAR! activators exert pleiotropic effects on a wide range of tissues beyond their actions on insulin-responsive organs and tissues. These actions include inhibition of mechanisms that contribute to the progression of atherosclerosis [START_REF] Touyz | Peroxisome proliferator-activated receptors in vascular biology-molecular mechanisms and clinical implications[END_REF][START_REF] Heikkinen | PPAR! in human and mouse physiology[END_REF]. TZDs may induce anti-atherogenic effects via different mechanisms, including the normalization of total plasma cholesterol, as shown in rats fed HCD [START_REF] Schaalan | Westernized-like-dietfed rats: effect on glucose homeostasis, lipid profile, and adipocyte hormones and their modulation by rosiglitazone and glimepiride[END_REF], or via direct anti-inflammatory, antioxidant, and anti-hypertrophic effects [START_REF] Touyz | Peroxisome proliferator-activated receptors in vascular biology-molecular mechanisms and clinical implications[END_REF][START_REF] Li | Peroxisome proliferator-activated receptor ! ligands inhibit development of atherosclerosis in LDL-receptor knock-out mice[END_REF][START_REF] Minamikawa | Potent inhibitory effect of troglitazone on carotid arterial wall thickness in type 2 diabetes[END_REF][START_REF] Chen | Troglitazone inhibits atherosclerosis in apolipoprotein E-20 knockout mice: pleiotropic effect on CD36 expresion and HDL[END_REF], which may be independent of their metabolic actions. As expected [START_REF] Chen | Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition[END_REF], rosiglitazone reduced the lipid deposition found in the aorta of mthfr +/- mice (data not shown), potentially via reduction of plasma cholesterol in the mice fed HCD. Inflammatory markers such as VCAM-1 and osteopontin, which were increased in mthfr +/-mice, were only slightly decreased by rosiglitazone (data not shown), suggesting that PPAR! agonism did not have a marked anti-inflammatory effect in this particular model. PPAR! agonists block cell proliferation and enhance apoptosis of vascular smooth muscle cells [START_REF] Marx | Peroxisome proliferator activated receptor ! activators inhibit gene expression and migration in human vascular smooth cells[END_REF][START_REF] Bruemmer | Regulation of the growth arrest and DNA damage inducible gene 45 (GADD45) by peroxisome proliferator-activated receptor ! in vascular smooth muscle cells[END_REF] suggesting a role of TZDs in the modulation of vascular remodeling. Growth and migration of vascular smooth muscle cells into the intima is a central step of atherosclerotic plaque formation [START_REF] Ross | Atherosclerosis-an inflammatory disease[END_REF]. Hence, rosiglitazone may reduce atherosclerotic lesions in part by improving the vascular remodeling [START_REF] Collins | Troglitazone inhibits formation of early atherosclerotic lesion in diabetic and non diabetic low density lipoprotein receptor deficient mice[END_REF]. Rosiglitazone attenuated intimal hyperplasia after arterial balloon catheter injury in a rat model of diet-induced H-Hcy, which presented a three-fold increase in proliferation of rat aortic vascular smooth muscle cells [START_REF] Murthy | Rosiglitazone reduces serum homocysteine levels, smooth muscle proliferation, and intimal hyperplasia in Sprague-Dawley rats fed high methionine diet[END_REF]. In persons with impaired glucose tolerance and no diabetes and cardiovascular disease, rosiglitazone modestly reduced carotid intima-media thickness [START_REF] Lonn | Effect of ramipril and of rosiglitazone on carotid intima-media thickness in people with impaired glucose tolerance or impaired fasting glucose: STARR (STudy of Atherosclerosis with Ramipril and Rosiglitazone)[END_REF], suggesting anti-atherogenic effects of PPAR! activation occurring particularly via remodeling of the vascular wall in humans. However, in type-2 diabetic patients, rosiglitazone had no significant effect on carotid atheroma as assessed by 3D carotid magnetic resonance imaging [START_REF] Varghese | Effect of rosiglitazone on progression of atherosclerosis: insights using 3D carotid cardiovascular magnetic resonance[END_REF]. Endothelial dysfunction contributes to the development of atherosclerotic lesions and vascular remodeling, and its presence and severity is predictive of adverse outcomes [START_REF] Schachinger | Prognostic impact of coronary vasodilator dysfunction on adverse long-term outcome of coronary heart disease[END_REF]. Several cardiovascular risk factors may contribute to endothelial dysfunction, including age and hypercholesterolemia, by promoting vascular oxidative stress and impaired NO generation or bioavailability [START_REF] Savoia | Inhibition of the renin angiotensin system: implications for the endothelium[END_REF][START_REF] Touyz | Reactive oxygen species in vascular biology: implications in hypertension[END_REF]. In particular, rabbits fed HCD had reduced NO levels and increased ADMA, an L-arginine analogue that acts as a competitive inhibitor of eNOS [START_REF] Bolayirli | Effects of atorvastatin therapy on hypercholesterolemic rabbits with respect to oxidative stress, nitric oxide pathway and homocysteine[END_REF]. We recently reported that mesenteric arteries of young mthfr +/-mice presented endothelial dysfunction due to increased oxidative stress and uncoupling of eNOS [START_REF] Virdis | Effect of hyperhomocysteinemia and hypertension on endothelial function in methylene tetrahydrofolate reductase-deficient mice[END_REF]. However, the precise mechanisms whereby H-Hcy produces endothelial dysfunction are incompletely defined. Besides decreased NO bioavailability due to increased ROS production, another potential mechanism for reduced NO effects in subjects with H-Hcy is decreased NO production due to increased levels of ADMA, [START_REF] Valance | Cardiovascular biology of the asymmetric dimethylarginine: dimethylarginine dimethylaminohyddrolase pathway[END_REF]. Homocysteine reduces the catabolism and induces the synthesis of ADMA, the latter partially dependent on PRMT-1 activation [START_REF] Lentz | Hyperhomocysteinemia, endothelial dysfunction, and cardiovascular risk: the potential role of ADMA[END_REF]. Acetylcholine-induced vasodilation of carotid arteries from old mthfr +/- was similar to that of age-matched wild type mice, and was 30% less then that observed in mesenteric arteries of younger mice [START_REF] Virdis | Effect of hyperhomocysteinemia and hypertension on endothelial function in methylene tetrahydrofolate reductase-deficient mice[END_REF]. It is possible that increased levels of homocysteine do not induce further impairment of endothelial function in addition to age-related endothelial dysfunction, despite the elevation of ROS levels in aorta of old mthfr +/-mice. Rosiglitazone, which reduced oxidative stress in young mthfr +/- [START_REF] Virdis | Effect of hyperhomocysteinemia and hypertension on endothelial function in methylene tetrahydrofolate reductase-deficient mice[END_REF], did not reduce increased ROS production in old mthfr +/-mice or improve endothelial function in these mice. Rosiglitazone treatment paradoxically resulted in greater impairment of endothelial function in mthfr +/-mice fed HCD. It has been recently reported that homocysteine competes for PPAR nuclear receptors with PPAR agonists and may impair the maximum activation of PPAR receptors by the latter. Thus, homocysteine elevation may have an important impact on PPAR agonist action [START_REF] Hayden | Homocysteine competes for the peroxisome proliferator-activated receptor nuclear receptors[END_REF]. Furthermore, reduced production of NO, plus increased scavenging by excess ROS, may contribute to explain these results. Indeed, mthfr +/-mice fed HCD presented the highest levels of ROS in aorta possibly due to the combined effect of age, high plasma cholesterol and H-Hcy. The endothelial dysfunction of HCD-fed mthfr +/-treated with rosiglitazone may also explain the absence of improvement of vascular remodeling in this group despite a favourable effect on lipid deposition in atherosclerotic lesions. This suggests a countervailing effect of TZDs on progression of atherosclerosis depending on how many risk factors are contemporaneously present. Interestingly, HCD-fed mthfr +/-mice treated with rosiglitazone presented increased expression of PPAR!, which has been recently reported to contribute to rosiglitazone-induced cardiotoxic effects [START_REF] Son | Cardiomyocyte expression of PPAR! leads to cardiac dysfunction in mice[END_REF]. An intriguing finding was that PRMT-1 was increased in rosiglitazone-treated HCD-fed mthfr +/- mice. PRMT-1 is the major source of ADMA in the vasculature [START_REF] Valance | Cardiovascular biology of the asymmetric dimethylarginine: dimethylarginine dimethylaminohyddrolase pathway[END_REF] and is partially activated by H-Hcy [START_REF] Virdis | Effect of hyperhomocysteinemia and hypertension on endothelial function in methylene tetrahydrofolate reductase-deficient mice[END_REF][START_REF] Son | Cardiomyocyte expression of PPAR! leads to cardiac dysfunction in mice[END_REF] as well as by LDL cholesterol [START_REF] Böger | LDL cholesterol upregulates synthesis of asymmetrical dimethylarginine in human endothelial cells: involvement of S-adenosyl methioninedependent methyltransferases[END_REF]. The promoter of prmt1 contains PPAR-! responsive elements which are highly conserved as shown in figure 7. Consistently with this finding, ADMA levels were increased approximately 2-fold in rosiglitazone-treated HCD-fed mthfr+/-mice. Thus, rosiglitazone via increased expression and therefore activity of PRMT-1 leading to production of ADMA may decrease NO production and promote endothelial dysfunction in HCD-fed old mthfr +/-mice, which already present high ROS generation in the vascular wall, thus amplifying this detrimental effect of H-Hcy and HCD. These observations may support and extend results of recent experimental and clinical studies that challenge the vascular protective role of PPAR! activators in cardiovascular disease. TZDs have adverse effects on advanced atherosclerosis by promoting plaque instability [START_REF] Thorp | Pioglitazone increases macrophage apoptosis and claque necrosis in advanced atherosclerotic lesions of non diabetic low-density lipoprotein receptor-null mice[END_REF], and PPAR! activation may contribute to monocytes differentiation into foam cells [START_REF] Tontonoz | PPAR gamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL[END_REF]. In humans, a recent meta-analysis of rosiglitazone treatment trials in diabetic patients has shown that rosiglitazone may be associated with increased risk of myocardial infarction and death from cardiovascular cause [START_REF] Nissen | Effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes[END_REF], although methodological limitations do not allow definitive conclusions and this conclusion remains highly controversial. As a result, there is a situation of equipoise regarding the safety of rosiglitazone until further data is obtained [START_REF] Psaty | Rosiglitazone and cardiovascular risk[END_REF].

Clinical perspectives

Important new findings from this study are that rosiglitazone may exert beneficial and detrimental effects on the cardiovascular system depending on pre-existing levels of cardiovascular risk. Rosiglitazone exerted vascular protective effects in old mice with H-Hcy by attenuating vascular remodeling as well as the atherosclerotic process. However, it was unable to reduce the increased oxidative stress in mthfr +/-mice fed or not HCD, and exerted minimal effect on inflammatory mediators in these mice. When these mice became hypercholesterolemic in addition to aged and H-HCy, the prior beneficial effect was counterbalanced by rosiglitazone-induced endothelial dysfunction, which may contribute to cardiovascular remodeling as well as to blunt any beneficial effect on atherosclerosis. These findings are consistent with current evidence for a role of PPAR! at the interface between the environment and control of metabolism [52]. Indeed, a common genetic variant of PPAR! in humans induces opposing effects on lipid and glucose metabolism depending on whether individuals are obese or sedentary, and depending on the fat composition of the diet [53]. In conclusion, the present study suggests mechanisms that may explain beneficial effects of PPAR! activation in lower risk subjects but detrimental actions in presence of high cardiovascular risk, which could explain some recent clinical observations in diabetic patients with advanced atherosclerosis.

Clinical

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Clinical

  Science Immediate Publication. Published on 01 Dec 2009 as manuscript CS20090289 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090289 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Clinical

  Science Immediate Publication. Published on 01 Dec 2009 as manuscript CS20090289 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090289 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

  52. Semple, R.K., Chatterjee, V.K., and O'Rahilly, S. (2006) PPAR gamma and human metabolic disease. J. Clin. Invest. 116,581-589 53. Luan, J., Brawne, P.O., Harding, A.H., Halsall, D.J., O'Rahilly, S., Chatterjee, V.K., and Wareham, N.J. (2001) Evidence of gene-nutrient interaction at the PPAR gamma locus. Diabetes. 50,686-689 Clinical Science Immediate Publication. Published on 01 Dec 2009 as manuscript CS20090289 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090289 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Figures legendsFigure 1 :

 1 Figures legendsFigure1: a) Plasma cholesterol levels in mthfr +/-and wild type mice. b) Expression of PPAR! at level of the coronary sinus of aorta in mthfr +/-and wild type mice. +/+: wild type control mice; +/-: mthfr +/-mice fed normal diet; +/-HCD: mthfr +/-mice fed high cholesterol diet; +/-R: mthfr +/ -mice fed normal diet and treated with rosiglitazone; +/-HCD/R: mthfr +/-mice fed high cholesterol diet and treated with rosiglitazone. *P<0.05.

Figure 2 :

 2 Figure 2: Media-to-lumen ratio and media cross-sectional area (CSA) of the carotid from mthfr +/- and wild type mice measured at 60 mm Hg intraluminal pressure. *P<0.05.

Figure 3 :

 3 Figure 3: a) Concentration-response curve to acetylcholine (Ach) and b) to sodium nitroprusside (SNP) of norepinephrine-pre-contracted mesenteric resistance arteries from mthfr +/-and wild type mice. c) and d) Concentration-response curve to acetylcholine ± the nitric oxide synthase (NOS) inhibitor L-NAME. *P<0.05.

Figure 4 :

 4 Figure 4: Dihydroethidium (DHE) staining (upper panel) of reactive oxygen species (ROS) in aorta of mthfr +/-and wild type mice. ROS production was increased in the aorta of mthfr +/-mice. *P<0.05; †P<0.05 vs. +/+, +/-, +/-R.

Figure 5 :

 5 Figure 5: a) Plasma nitrite (index of NO production) in mthfr +/-and wild type mice. b) Plasma concentration of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) in mthfr +/- mice. *P<0.05.

Figure 6 :

 6 Figure 6: Protein arginine methyltransferase-1 (PRMT-1) expression in the aorta of mthfr +/-mice. Results are presented as percentage of PRMT-1 expression in wild type control mice. *P<0.05.

Figure 7 :

 7 Figure 7: In silico analysis of prmt1 gene. The 52238857-52242238 bp region of chromosome 7 (a) containing the two 5' exons and the promoter region of the prmt1 gene responsible for the transcription of four different alternative mRNA PRMT-1 splices (b) are presented. Coding and non-coding exons are indicated by wide black and narrow gray rectangles, respectively. Direction of the transcription is indicated by arrowheads and is from right to left. c) Conserved PPAR and PPAR! response elements between mouse and human are indicated. d) Several regions of the prmt1 promoter that are highly conserved among mammals are displayed.

  Figure 1

6

 6 Figure 3

Figure 7 Clinical

 7 Figure 7

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Acknowledgments

The authors thank André Turgeon for his excellent technical work. This work was supported by grant 82790 from the Canadian Institutes of Health Research (CIHR), the Canada Research Chair (CRC) Program of CIHR/Government of Canada, and the Canada Fund for Innovation (CFI), all to ELS, and a fellowship from the Heart and Stroke Foundation to TE and CAL.

Disclosures

No conflicts to disclose. Clinical Science Immediate Publication. Published on 01 Dec 2009 as manuscript CS20090289 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20090289 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited