Louise Moller
email: louisem@dadlnet.dk

Hans S Jorgensen

Taagehoej Finn

Jens Ol Jensen

Jorgensen

Hans Stodkilde-Jorgensen

Jensen

Jens Otto Lunde Jorgensen

Fasting in healthy subjects is associated with intrahepatic accumulation of lipids as assessed by 1 H-Magnetic Resonance Spectroscopy

Keywords: Fasting, Fatty liver, 1 H-Magnetic Resonance Spectroscopy, Lipids, 3-hydroxybutyrate acids

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Introduction

Non-alcoholic fatty liver disease (NAFLD) is prevalent in obese subjects and is by many considered an important feature of the metabolic syndrome. Although the majority of such cases are asymptomatic, several reports underscore an associations between NAFLD and dyslipidemia, hypertension, diabetes and cardiovascular disease [START_REF] Cortez-Pinto | Non-alcoholic fatty liver: another feature of the metabolic syndrome?[END_REF]. The correlation between NAFLD and insulin resistance is close, and is independent of body mass index (BMI), gender and age [START_REF] Marchesini | Association of nonalcoholic fatty liver disease with insulin resistance[END_REF][START_REF] Westerbacka | Women and men have similar amounts of liver and intra-abdominal fat, despite more subcutaneous fat in women: implications for sex differences in markers of cardiovascular risk[END_REF][START_REF] Roberts | Pediatric nonalcoholic fatty liver disease (NAFLD): a "growing" problem?[END_REF][START_REF] Marchesini | Nonalcoholic fatty liver disease: a feature of the metabolic syndrome[END_REF].

Furthermore the morbidity and mortality in patients with NAFLD is increased [START_REF] Angulo | Obesity and nonalcoholic fatty liver disease[END_REF], which is attributed to cardiovascular disease, diabetes, and liver cirrhosis [START_REF] Schindhelm | Alanine aminotransferase as a marker of non-alcoholic fatty liver disease in relation to type 2 diabetes mellitus and cardiovascular disease[END_REF][START_REF] Schindhelm | Nonalcoholic fatty liver disease and cardiovascular disease risk[END_REF][START_REF] Bugianesi | NASH and the risk of cirrhosis and hepatocellular carcinoma in type 2 diabetes[END_REF].

Elevation of free fatty acids (FFAs) seems to be pivotal for the development of NAFLD, but the amount of intrahepatic lipids (IHL) is also determined by factors such as the capacity of the hepatocytes for uptake and turnover of FFAs, the release of triglyceride (TG) in very-low-densitylipoprotein particles, and TG storage in spherical droplets [START_REF] Bradbury | Lipid metabolism and liver inflammation. I. Hepatic fatty acid uptake: possible role in steatosis[END_REF]. Donnelly et al. have calculated that in patients with NAFLD 59% of the TG in the liver derives from FFAs, 26.2% from de novo lipogenesis and 14.9% from the diet [START_REF] Donnelly | Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease[END_REF].

Intervention studies reveal that the pool of IHL may change rapidly, especially in response to dietary changes. In patients with type 2 diabetes, two weeks of hypocaloric diet or an 8% weight reduction reduce IHL by 27% and 81%, respectively [12;13]. Furthermore two weeks of low fat diet have been shown to decrease IHL by 20% in overweight non-diabetic subjects [START_REF] Westerbacka | Dietary fat content modifies liver fat in overweight nondiabetic subjects[END_REF]. In rats as little as three days of high fat feeding causes a threefold increase in IHL [START_REF] Samuel | Mechanism of hepatic insulin resistance in nonalcoholic fatty liver disease[END_REF].

It is well described that fasting induces an increase in circulating levels of FFAs and a decrease in both hepatic and peripheral insulin sensitivity. This is associated with an increase in both intramyocellular lipid content [16;17] and hepatic production of ketone bodies [START_REF] George | Fuel metabolism in starvation[END_REF], both of which reflect mobilisation and oxidation of lipid intermediates. Therefore fasting is likely to be associated with significant changes in IHL in human subjects, but this remains to be experimentally verified.

The aim of the present study therefore was to measure changes in IHL during fasting in normal subjects by 1 H-MR Spectroscopy ( 1 H-MRS) and to correlate this to circulating levels of ketone bodies.

Materials and Methods

Subjects

The study population comprised eight healthy, non-obese young males, age 23.6±0.7 yrs.

[mean±SE], BMI 22.8±0.4 kg/m 2 , fasting plasma glucose 4.3±0.1 mmol/l, glycosylated haemoglobin A (HbA1c) 5.5±0.1%, alanine aminotransferase (ALT) 23.4±2.8 U/l, TG 0.73±0.08 mmol/l, total cholesterol 4.2±0.2 mmol/l, high-density lipoproteins (HDL) 1.5±0.1 mmol/l, lowdensity lipoproteins (LDL) 2.4±0.2 mmol/l. All subjects were students from the local university.

The exclusion criteria included: a family history of type 2 diabetes, use of any medications, alcohol consumption above 21 units per week, known liver disease, claustrophobia and carriage of magnetic implant.

All subjects were instructed to consume a diet with no major deviations from the national recommendations (max 30% of the energy from fat, 50-60% from carbohydrates and 10-20% from protein), to avoid high fat meals and to abstain from alcohol 3 days before each study period.

During the fasting period they were allowed to drink tap or mineral water and to perform normal ambulatory activities, excluding any kind of exercise.

Design

The subjects were randomly assigned to one of two groups: 1) 1 H-MRS examination at baseline and after 36 hours of fasting (36h-fast) (n=6); 2) 1 H-MRS examination at baseline and after 12 hours of fasting (12h-fast) (n=3). Blood samples were drawn in both groups immediately after the fast. One of the subjects participated in both groups separated by two months.

The protocol was approved by the regional Ethics Committee, the nature and potential risks were explained, before participants gave written informed consent. The study was conducted according to the declaration of Helsinki (2000) of the World Medical Association.

H-MRS:

1 H-MRS was performed using a 1.5 T scanner (Signa Excite 1.5 Tesla, Twin Speed, GE medical systems, Milwaukee, USA). The subjects were allowed to eat and drink until the beginning of the study period. The baseline examination was performed at 20.00h, group 1 was fasted for 36 hours, group 2 overnight (12 hours), before re-examination at 08.00h.

During each visit, three 1 H-MRS measurements (spectra 1-3) were made to enable calculations of the inter-individual variation in baseline IHL and the intra-day coefficient of variation (CV). First, spectra 1 and 2 were obtained (with the same shimming and spatial position), then the subject was taken out of the magnet, allowed to move around a few minutes, and repositioned, before spectra 3 was made. Subjects were positioned feet first in the supine position.

A belt was strapped around the lower part of the thorax and upper part of abdomen to minimize respiratory movement of the diaphragm and liver. To generate the spectra, a standard whole-body coil was used for radio frequency transmission and signal reception. The exact orientation of the liver was verified in a 3-plane T2 Localizer pulse sequence.

Oblique plane T1 Gradient Echo sequence using single-breath holding technique with TR 140 msec, TE 2.2/4.4.were made to enable identification of the area of interest.

The volume of interest (VOI 2 * 2 * 3 cm 3 ) was carefully positioned in the lower posterior part of the liver (area 6), avoiding inclusion of costae, visible vessels and bile ducts. Autoshim was performed to optimize the magnetic field homogeneity. Water suppressed point resolved spectroscopy sequence during free breathing (TE: 30 msec, TR: 2000 msec, number of acquisitions: 128) was applied, using water as autocenter frequency. The full width at half maximum (FWHM) of the unsuppressed water peak was 10.2±1.6 Hz.

Each session lasted approximately 40 min.

Analytical Procedures and calculations

Relative lipid content

The spectra were analysed using SAGE (GE medical systems, version 7). The height of the suppressed water signal intensity peak (S water ) was measured at ~4.8 ppm, the lipids/ (-CH2-)n signal intensity (S lipid ) peak at ~1.4 ppm, spectra from subject nr 4, before and after the 36h-fast are provided in Figure 1. We used peak height rather than the area under the curve (AUC), as our FWHM was relative low and constant (10.2±1.6); moreover using AUC caused a higher CV.

The relative lipid content (RLC) was calculated using the following formula:

Validation of data

The CV was calculated by dividing the standard deviation with the mean (of the three RLC), and presented in terms of percentage. To demonstrate the intra-day CV the median CV of RLC was determined.

Blood analysis

A blood sample was drawn at the end of the fasting period. Plasma glucose was measured in duplicate immediately after sampling on a Beckman Glucoanalyzer (Beckman Instruments, Palo Alto, CA, USA). Serum samples were frozen immediately and stored at -20 °C. Insulin, growth hormone and cortisol were analysed using time-resolved flouroimmunoassay (AutoDELFIA, PerkinElmer, Wallac, Turku, Finland), C-peptide by ELISA (DakoCytomation, Cambridgeshire, United Kingdom), FFAs were analysed by a commercial kit (Wako Chemicals, Neuss, Germany).

ALT was determined by a commercial method (Pyridoxal Phosphate Activated Cobas Integra 800, Roche, Mannheim, Germany). Glycerol, lactate, alanine and 3-hydroxybutyrates (3-OHB) were measured using a COBAS biocentrifugal analyzer with flurometric attachment (Roche Diagnostics, Welwyn Garden City, UK) [START_REF] Harrison | Blood glucose, lactate, pyruvate, glycerol, 3-hydroxybutyrate and acetoacetate measurements in man using a centrifugal analyser with a fluorimetric attachment[END_REF].

Statistical Analysis

The windows software Intercooled Stata 9.0 was used for the statistical analysis. To analyse the changes in IHL, the mean of the three RLC, at baseline and after the fasting period, were used as the best estimates of the "true" values. Student's paired t-test was used. For estimation of the different components of variation in the RLC measurements two Bland-Altman analysis were made followed by a 2 component analysis of variance on the two standard deviations. For statistical significant results, a p-value < 0.05 was required. Since the distribution of the RLC data were skewed, data were log transformed before applying relevant statistical tests, and only medians are presented. Unless otherwise stated, data are presented as mean±SE.

Results

Baseline variation in RLC

The inter-individual variation in RLC before the fasting periods ranged from 0.25% to 0.72% (5th vs. 95th percentile) with a median of 0.51%. Since one subjects participated in both the 12 and 36h-fast, a mean of his baseline RLC was used.

Validation of RLC measured by 1 H-MRS

The median CV for RLC measured by 1 H-MRS was 11.6%. None of the spectra were significantly different, as seen in Figure 3. After performing a 2 component analysis we found that 25-30% of the variation was due to differences between spectra 1 and 2, attributed to elements of uncertainty in the analysis of data, equipment errors and respiration movement, the remaining 70-75% was attributed to repositioning before the third spectra was made. This underlines the importance of precision when the voxel is placed.

Changes in IHL during the fasting periods

The IHL increased significantly during the 36h-fast. Median RLC increased from 0.42% to 0.74%, p=0.009 (CI: 1.34 -3.54). The median percentage change in RLC during the 36h-fast was 156% (range: 4-252%, p<0.05). (Table 1 and Figure 2) No significant change in the IHL was observed after the 12h-fast [RLC: 0.72% (before) vs. 0.42% (after), p=0.45].

Analytes in blood

Mean±SE levels of the measured analytes at the end of each fasting period are provided in Table 2. Due to the low sample size a statistical comparison between the levels after the 2 fasting studies was not performed, but, as expected, the concentrations of all lipid intermediates increased with more prolonged fasting. There was a significant positive correlation between the increase in RLC in terms of percentage (RLC %) and the 3-OHB concentration after the 36h-fast (r=0.85, p=0.03), as illustrated in Figure 4. The RLC% as well as RLC after the 36h-fast did not correlate to either levels of FFAs (p=0.31, p=0.60) or ALT levels (p=0.68, p=0.20) after the 36h-fast.

Discussion

This study demonstrates for the first time that fasting is associated with an increase in IHL in human subjects. Apart from extending our knowledge about substrate metabolism in humans, the data also demonstrate that food intake must be taken into consideration when interpreting IHL results.

Fasting is associated with distinct and dynamic changes in substrate metabolism, and Cahill reports that approximately 40% of FFAs are metabolised via hepatic ketogenesis after long term fasting, reflecting that ketone bodies becomes increasingly important as fuel for the brain [START_REF] George | Fuel metabolism in starvation[END_REF]. Our study showed that the increase in IHL after 36 hours of fasting was positively correlated to the hepatic 3-OHB production. Whereas high levels of IHL in steady state conditions such as the metabolic syndrome seem to be predictive of disease activity, the observed increase during fasting is more likely to reflect a physiological adaptation to alteration in substrate metabolism in general and the increase in hepatic ketogenesis in particular.

In several publications ALT levels have been used as a surrogate marker of IHL, but in our study ALT did not correlate to any indices of IHL. This may reflect the short duration of our study, but it is also plausible that the mechanisms underlying the increased IHL during fasting differ from those of NAFLD.

Since our study population consisted of lean and healthy adult males, without any evidence of hepatic disease or steatosis, one would anticipate IHL to be low. We found a baseline median RLC of 0.51%, with inter-individual variation ranging from 0.25% to 0.72% (5th vs. 95th percentile). Compared to the median of 1.9 recorded by Szczepainak et al. in a group with low risk of steatosis [START_REF] Szczepaniak | Magnetic resonance spectroscopy to measure hepatic triglyceride content: prevalence of hepatic steatosis in the general population[END_REF], IHL in our subjects is indeed low. Several factors, including dietary habits, physical fitness, age and gender are known to influence IHL in otherwise healthy subjects.

Westerbacka et al, who measured IHL by 1 H-MRS in obese non-diabetic women before and after 2 weeks of isocaloric high-fat or low-fat diet, recorded a 20% decrease in IHL in the low-fat diet group, and a 35% increase in the high-fat diet group [START_REF] Westerbacka | Dietary fat content modifies liver fat in overweight nondiabetic subjects[END_REF], yet a single high fat meal does not seem to influence IHL [START_REF] Szczepaniak | Magnetic resonance spectroscopy to measure hepatic triglyceride content: prevalence of hepatic steatosis in the general population[END_REF]. A high level of habitual physical activity is associated with low IHL [START_REF] Perseghin | Habitual physical activity is associated with intrahepatic fat content in humans[END_REF].

Cross-sectional studies indicate that age is inversely related to IHL [23;24], whereas the impact of gender remains controversial.

In conclusion, this study shows for the first time that IHL increases during fasting in healthy human subjects and that this increase correlates positively with circulating levels of 3-OHB. This contributes to our knowledge about the regulation of substrate metabolism during alterations in nutritional supply. It also emphasises that nutritional status should be standardised when assessing IHL in patients. Spectra from subject nr 4, before and after the 36h-fast. A: suppressed water peak, B: lipid peak.

Figure 2 RLC at baseline ( ) and after 36h-fast ( ) from each subject in group 1(n=6). Median RLC at baseline ( ) and after 36h-fast ( ), (p=0.009).

Reproducibility of the three measurements of RLC obtained at the 18 MR The dotted line depict the optimal correlation (slope=1). A: The correlation between RLC 1 and 2, r=0.97 (p<0.01).

B: correlation between RLC 1 and 3, r=0.95 (p<0.01). C: correlation between 2 and 3, r=0.93 (p<0.01).

Scatter plot illustrating the correlation between the change in RLC during the 36h-fast in terms of percentage and the 3-OHB immediately after the 36h-fast (r=0.85, p=0.03).

  of water suppression.

Figure 1 Figure 2 13S

 12 Figure 1

Figure 3 Figure 4 14S

 34 Figure 3

TABLE 1

 1 Change in RLC (%) during 12 and 36 hours of fasting.

	Subject	12h-fast	36h-fast
	1		151.7
	2		160.1
	3		167.9
	4		260.8
	5		76.6
	6	-8.7	-4.2
	7	10.1
	8	-4 1 . 1

TABLE 2

 2

	Blood samples drawn after 12 and 36 hours of fasting,
	respectively.
	Values are given as mean±SE.

Acknowledgments

Thanks to Lisa Erika Dalman for assistance during the MR sessions.

The study was sponsored by Pfizer Inc. with an unrestricted research grant.


Hans-Joachim Kabitz

Anja Schwoerer

; Hinrich-Cordt Bremer

Florian Sonntag

Stephan Walterspacher

David Walker

Vanessa Schaefer

Nicola Ehlken

Gerd Staehler

Michael Halank

Hans Klose

Hossein A Ghofrani

Marius M Hoeper

Ekkehard Gruenig

MD Wolfram Windisch
email: wolfram.windisch@uniklinik-freiburg.de

Impairment of respiratory muscle function in pulmonary hypertension

Keywords: Exercise, Pulmonary heart disease, Pulmonary hypertension, Respiratory muscle strength, Twitch pressure

It has been suggested that impaired respiratory muscle function occurs in patients with pulmonary hypertension (PH). However, comprehensive investigation of respiratory muscle function -including the application of non-volitional tests -needed to verify impairment of respiratory muscle strength in PH-patients has yet not been performed.

Respiratory muscle function was assessed in 31 PH-patients (20 female, mean pulmonary artery pressure 51±20 mmHg, median WHO-class 3.0±0.5, pulmonary arterial hypertension n=25, chronic-thrombembolic PH n=6) and in 31 control subjects (20 female) well-matched for gender, age and body-mass index. A six-minute walking test was performed to rate exercise capacity.

Volitionally assessed maximal inspiratory (7.5±2.1 vs 6.2±2.8 kPa; p=0.04) and expiratory (13.3±4.2 vs 9.9±3.4 kPa; p<0.001) mouth pressures, sniff nasal (8.3±1.9 vs 6.6±2.2 kPa; p=0.002) and transdiaphragmatic (11.3±2.5 vs 8.7±2.5 kPa; p<0.001) pressures, non-volitionally assessed twitch mouth (1.46±0.43 vs 0.97±0.41 kPa; p<0.001) and transdiaphragmatic (2.08±0.55 vs 1.47±0.72 kPa; p=0.001) pressures during bilateral anterior magnetic phrenic nerve stimulation were markedly lower in PH-patients compared to control subjects. Maximal inspiratory mouth (r=0.58; p<0.001) and sniff transdiaphragmatic (r=0.43; p=0.02) pressures were correlated to six-minute walking distance in PH-patients.

The present study provides strong evidence that inspiratory muscle strength is reduced in PHpatients compared to well-matched control subjects. Furthermore, six-minute walking distance is significantly linked to parameters assessing inspiratory muscle strength.

Introduction

Daily activities of patients suffering from pulmonary hypertension (PH) are limited by dyspnea on exertion and fatigue. These symptoms have been mainly attributed to a mismatch in systemic oxygen delivery and oxygen demand due to the overloaded right ventricle [START_REF] Naeije | Breathing more with weaker respiratory muscles in pulmonary arterial hypertension[END_REF]. However, impairment of respiratory muscle function might partly contribute to these symptoms as has been previously suggested in patients with chronic left heart failure [START_REF] Mancini | Respiratory muscle function and dyspnea in patients with chronic congestive heart failure[END_REF][START_REF] Mcparland | Inspiratory muscle weakness and dyspnea in chronic heart failure[END_REF][START_REF] Hughes | Diaphragm strength in chronic heart failure[END_REF][START_REF] Meyer | Inefficient ventilation and reduced respiratory muscle capacity in congestive heart failure[END_REF]. Moreover, reduced inspiratory muscle strength is known to be an independent predictor of prognosis in patients with chronic left heart failure [START_REF] Meyer | Respiratory muscle dysfunction in congestive heart failure: clinical correlation and prognostic significance[END_REF]. In contrast, the role of impaired inspiratory muscle strength in PHpatients remains unclear.

The only pre-existing study on respiratory muscle strength which indicated possible respiratory muscle dysfunction in patients with PH has used volitional tests on respiratory muscle strength only [START_REF] Meyer | Respiratory muscle dysfunction in idiopathic pulmonary arterial hypertension[END_REF]. However, volitional tests are highly dependent on the patient's co-operation and motivation and are thus fraught with variability [START_REF] Polkey | Measurement of respiratory muscle strength[END_REF][START_REF] Ats/Ers | ATS/ERS Statement on respiratory muscle testing[END_REF]. Therefore, additional measurements for assessment of inspiratory muscle function have been demanded recently to be investigated in patients with PH [START_REF] Naeije | Breathing more with weaker respiratory muscles in pulmonary arterial hypertension[END_REF].

It has been clearly outlined that the definitive diagnosis of inspiratory muscle weakness requires non-volitional tests, such as measurement of twitch pressures during magnetic phrenic nerve stimulation [START_REF] Polkey | Measurement of respiratory muscle strength[END_REF]. For this reason, the present study was aimed at prospectively evaluating respiratory muscle function in PH-patients.

Materials and Methods

The study was performed in agreement with the ethical standards laid down in the Declaration of Helsinki. The study protocol was approved by the institutional review board for human studies at the Albert-Ludwigs University Freiburg, Germany. All participants gave their written informed consent.

Patients and control subjects

Thirty-one patients (20 women) who were treated in experienced PH-centers and who suffered from PH (selected forms of pulmonary arterial hypertension, n=25; chronic-thrombembolic PH, n=6) according to the 2003 Venice classification [START_REF] Simonneau | Clinical classification of pulmonary hypertension[END_REF] were enrolled in this prospective study. PH was diagnosed in an experienced PH-center (including right heart catheterization and echocardiography in all patients). All patients were clinically stable with no evidence of syncope within two months prior to the study. Appropriate medical therapy was provided in all patients.

Criteria for inclusion of patients were WHO functional classes II-IV [START_REF] Mcgoon | Screening, early detection, and diagnosis of pulmonary arterial hypertension: ACCP evidence-based clinical practice guidelines[END_REF]. Exclusion criteria covered primary lung diseases, rib cage abnormalities, neuromuscular disorders, oral steroids (>10 mg Prednisolone-equivalent/day) and malnutrition.

In addition, 31 healthy subjects (20 women and 11 men), well-matched for gender, age and body-mass index served as controls. All patients and controls were naive to magnetic stimulation and the experimental setting.

Lung function parameters, exercise testing and serological markers

(ITGV) assessed during body-plethysmography was used as the best available measure of functional residual capacity. A six-minute walking test with assessment of the six-minute walking distance (6MWD), dyspnea using the modified Borg Dyspnea Scale (BDS) and blood gas analysis (BGA) was performed [START_REF] Ats | ATS statement: guidelines for the six-minute walk test[END_REF]. In addition, a venous blood sample was drawn from sitting patients to provide data on NT-proBNP and uric acid.

Pressure and airflow recordings

The pneumotachograph ZAN-100-Flowhandy-II ® and the pressure transducer ZAN-400 ® (ZAN ® , Oberthulba, Germany) were used for airflow and pressure recordings. Ventilatory drive (P 0.1 ) and effective inspiratory impedance (P 0.1 /V T /t i ), used as a marker of the load imposed on the inspiratory muscles, were assessed according to recent recommendations [START_REF] Ats/Ers | ATS/ERS Statement on respiratory muscle testing[END_REF][START_REF] Whitelaw | Occlusion pressure as a measure of respiratory center output in conscious man[END_REF]. Maximal inspiratory (PImax, measured from residual volume) and expiratory (PEmax, assessed from total lung capacity) mouth pressure, and respiratory capacity (P 0.1 /PImax) were measured in line with previous recommendations [START_REF] Ats/Ers | ATS/ERS Statement on respiratory muscle testing[END_REF][START_REF] Windisch | Peak or plateau maximal inspiratory mouth pressure: which is best?[END_REF][START_REF] Leech | Respiratory pressures and function in young adults[END_REF][START_REF] Fernandez | P 0.1/PIMax: an index for assessing respiratory capacity in acute respiratory failure[END_REF]. Sniff pressures were obtained at functional residual capacity as outlined previously [START_REF] Heritier | Sniff nasal inspiratory pressure. A noninvasive assessment of inspiratory muscle strength[END_REF]. Twitch mouth (TwPmo) and transdiaphragmatic (TwPdi) pressures including inspiratory flow (F trig ) and pressure (P trig ) during inspiratory pressure triggering were assessed by bilateral anterior magnetic phrenic nerve stimulation (BAMPS) (Magstim ® 200 2 , Magstim ® Inc., Wales, United Kingdom) [START_REF] Mills | Bilateral magnetic stimulation of the phrenic nerves from an anterolateral approach[END_REF][START_REF] Kabitz | Controlled twitch mouth pressure reliably predicts twitch esophageal pressure[END_REF]. Further details on the methods for making these measurements have been published previously [START_REF] Kabitz | Controlled twitch mouth pressure reliably predicts twitch esophageal pressure[END_REF][START_REF] Windisch | Influence of different trigger techniques on twitch mouth pressure during bilateral anterior magnetic phrenic nerve stimulation[END_REF].

Predetermination of study endpoints

The primary endpoint of the current study was defined as the difference in inspiratory muscle strength between PH-patients and control subjects assessed by TwPmo due to its superior non-

Statistical analysis

Sigma-Stat ® Software (Version 3.1, Systat Software, Inc., Point Richmond, California, USA) was used for statistical analysis. All data are presented as mean ± standard deviation (SD). The null-hypothesis (H 0 ) was determined as there is no difference in mean TwPmo between PHpatients and controls. For H 0 rejection 29 subjects are needed in each group following sample size determination (unpaired t-test, power 0.9, two-sided type I error 0.05) with an estimated SD of 0.4 kPa for mean TwPmo and a difference of at least 0.35 kPa between the two groups.

Comparison between two groups was performed using the unpaired t-test/Mann-Whitney ranksum test for normally/non-normally distributed data. Due to the fact that TwPmo was designated as primary endpoint no data adjustment for multiple comparisons has been performed.

Correlation analysis was performed using Pearson product-moment correlation. Linear regression analysis was performed where appropriate [START_REF] Bland | Regression analysis[END_REF]. A p-value <0.05 was considered statistically significant. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Results

Anthropometric and lung function characteristics for all participants are presented in Table 1.

Hemodynamic and serological parameters for PH-patients are given in Table 2. There is no difference in gender composition, age and BMI when comparing patients with PH and controls.

Lung function parameters revealed mildly reduced values for forced expiratory volume in 1 second (FEV 1 ), forced vital capacity (FVC) and a trend towards reduced maximal expiratory flow at 50 percent of FVC (MEF 50 ) in PH-patients compared to controls (Table 1).

Exercise testing, ventilatory characteristics and volitional tests on respiratory muscle function

Results of the six-minute walking test with 6MWD, BDS-scores and BGA-data are given in Table 1. All PH-patients stated that their exercise capacity was limited by dyspnea and not by skeletal muscle exhaustion or weakness of the legs. The 6MWD was significantly shorter (p<0.001) and BDS was higher (p<0.001) in PH-patients compared to controls (Table 1). In addition, oxygen and carbon dioxide partial pressures were lower in PH-patients compared to controls both at rest and following exercise (p<0.001 in all instances). Data for ventilatory characteristics and volitional tests on respiratory muscle function are presented in Table 3.

Breathing frequency (18±5 vs 17±5 /min; p=0.30), tidal volume (0.8±0.3 vs 0.7±0.2 l; p=0.23), ventilatory drive and respiratory capacity (Table 3) showed trends towards higher values in PHpatients compared to controls. However, this did not reach statistical significance. Importantly, the load imposed on the inspiratory muscles is equal between PH-patients and controls (Table 3).

In contrast, all volitional tests on respiratory muscle strength revealed significantly reduced values for PH-patients compared to controls (Figure 1).

Non-volitional tests on inspiratory muscle strength

Results for non-volitional assessment of inspiratory muscle strength, represented by twitch pressures and corresponding data are given in Table 3. TwPmo was sufficiently recorded in all of the 31 patients and in 29 controls. The remaining two control subjects declined magnetic stimulation. Catheter placement for measurement of transdiaphragmatic pressures was declined by five controls and by five PH-patients. Group comparison revealed highly significant reduction of TwPmo (p<0.001) and TwPdi (p=0.001) in PH-patients compared to controls (Table 3).

Values for TwPmo and TwPdi in PH-patients and controls are illustrated in Figure 2. Adequacy of twitch pressures assessment was assured in both PH-patients and controls, as can be deduced by the lack of difference in P trig and F trig values between the two groups (Table 3).

Relationship between respiratory muscle function and exercise capacity in patients with pulmonary hypertension

PImax (r=0.58; p<0.001) and SnPdi (r=0.43; p=0.02), but not twitch pressures (p>0.05 in both instances), were significantly correlated to 6MWD in PH-patients. The relationship between PImax and 6MWD following linear regression analysis is illustrated in Figure 3. In contrast, hemodynamic characteristics assessed during right heart catheterization -mean pulmonary artery pressure, pulmonary vascular resistance, central venous pressure, and cardiac index -were not correlated to 6MWD (p>0.05 in all instances). Following stepwise multiple linear regression analysis, 6MWD can only be predicted from PImax (p<0.01) but neither from TwPmo nor mean pulmonary artery pressure (p>0.8 in both instances), considering PImax, TwPmo and mean pulmonary artery pressure.

Discussion

The present study establishes evidence of substantially impaired respiratory muscle function in a representative collective of patients with PH by using both volitional and non-volitional tests on respiratory muscle function. The main finding of the present study is that respiratory muscle strength is markedly reduced in patients suffering from PH when compared to controls. These differences are more distinct considering non-volitional tests on inspiratory muscle strength as reflected by TwPmo and TwPdi in comparison to volitional tests.

There is solely one pre-existing study on respiratory muscle dysfunction in patients with PH, applying volitional tests on respiratory muscle function only [START_REF] Meyer | Respiratory muscle dysfunction in idiopathic pulmonary arterial hypertension[END_REF]. However, the definitive diagnosis of respiratory muscle weakness in PH-patients can only be established by the use of non-volitional tests on inspiratory muscle function [START_REF] Naeije | Breathing more with weaker respiratory muscles in pulmonary arterial hypertension[END_REF]. since volitional tests are highly variable due to their dependency on the participant making a truly maximal effort [START_REF] Polkey | Measurement of respiratory muscle strength[END_REF][START_REF] Ats/Ers | ATS/ERS Statement on respiratory muscle testing[END_REF].

The finding of impaired respiratory muscle function is suggested to have major clinical impact on patients suffering from PH. The application of combined respiratory and exercise training to PH-patients resulted in an improvement each in quality of life, WHO functional class, peak oxygen consumption and achieved workload [START_REF] Mereles | Exercise and respiratory training improve exercise capacity and quality of life in patients with severe chronic pulmonary hypertension[END_REF]. The findings of the present study might serve as a pathophysiological background for these reported improvements. This is supported by the observation that respiratory muscle training in patients with chronic left heart failure resulted in improvement of both inspiratory muscle strength and important clinical parameters such as dyspnea on exertion and quality of life [START_REF] Mancini | Benefit of selective respiratory muscle training on exercise capacity in patients with chronic congestive heart failure[END_REF][START_REF] Dall'ago | Inspiratory muscle training in patients with heart failure and inspiratory muscle weakness: a randomized trial[END_REF]. Therefore, future studies are needed to Several studies have suggested that respiratory muscle strength is reduced in patients with chronic left heart failure [START_REF] Mcparland | Inspiratory muscle weakness and dyspnea in chronic heart failure[END_REF][START_REF] Hughes | Diaphragm strength in chronic heart failure[END_REF][START_REF] Meyer | Respiratory muscle dysfunction in congestive heart failure: clinical correlation and prognostic significance[END_REF][START_REF] Chua | Inspiratory muscle strength is a determinant of maximum oxygen consumption in chronic heart failure[END_REF][START_REF] Witt | Respiratory muscle weakness and normal ventilatory drive in dilative cardiomyopathy[END_REF], but, again, solely volitional tests on respiratory muscle function have been applied, thus reducing the reliability of these findings [START_REF] Polkey | Measurement of respiratory muscle strength[END_REF][START_REF] Ats/Ers | ATS/ERS Statement on respiratory muscle testing[END_REF]. However, when using non-volitional tests with assessment of twitch pressures, the reduction in inspiratory muscle strength in patients with chronic left heart failure was reported to be less evident and hence not to be of clinical importance since inspiratory muscle strength remains well preserved [START_REF] Hughes | Diaphragm strength in chronic heart failure[END_REF][START_REF] Carmo | Diaphragmatic function in patients with chronic left ventricular failure[END_REF]. This is in clear contrast to findings of the present study in PH-patients in whom substantially reduced inspiratory muscle strength has been clearly established by a 34% difference of non-volitionally assessed TwPmo compared to controls. Interestingly, patients with CTEPH presented with even more impaired respiratory muscle strength compared to patients with PAH. However, due to the low and unequal sample sizes between the two groups, data are purely descriptive without any further statistical analyses performed. The reasons for the observed differences remain unclear and future studies are needed to address this issue.

The underlying pathophysiological mechanisms responsible for the impairment of respiratory muscle strength in PH-patients have not yet been conclusively investigated. Based on observations mainly derived from chronic left heart failure, several mechanisms accounting for a reduction in respiratory muscle strength have been suggested. Structural skeletal muscle abnormalities and abnormal expression of myosin isoforms highly suggestive of fiber type transformation predominantly pronounced in the diaphragm of patients with chronic left heart failure have been reported [START_REF] Lindsay | Histological abnormalities of muscle from limb, thorax and diaphragm in chronic heart failure[END_REF]. Furthermore, fiber type changes associated with a myopathic pattern [START_REF] Stassijns | Effects of dilated cardiomyopathy on the diaphragm in the Syrian hamster[END_REF], cross-sectional muscle fiber reduction [START_REF] Howell | Congestive heart failure: differential adaptation of the diaphragm and latissimus dorsi[END_REF] and fast-to-slow transformations for myosin and regulatory proteins [START_REF] Tikunov | Changes in myofibrillar protein composition of human diaphragm elicited by congestive heart failure[END_REF][START_REF] Tikunov | Chronic congestive heart failure elicits adaptations of endurance exercise in diaphragmatic muscle[END_REF] failure [START_REF] Macfarlane | Cellular basis for contractile dysfunction in the diaphragm from a rabbit infarct model of heart failure[END_REF] the occurrence of a depressed oxidative capacity of the working muscles [START_REF] Drexler | Alterations of skeletal muscle in chronic heart failure[END_REF], linked to underperfusion of respiratory muscles and deoxygenation [START_REF] Mancini | Respiratory muscle deoxygenation during exercise in patients with heart failure demonstrated with near-infrared spectroscopy[END_REF], has been described in chronic left heart failure. However, in contrast to patients with chronic left heart failure in previous studies, PH-patients in the current study presented without relevant functional restrictions of the left heart, as can be seen by normal values for left ventricular ejection fraction and pulmonary artery wedge pressure. In addition, by taking into account the more reliable non-volitional tests on inspiratory muscle strength, the PH-patients of the present study are suggested to have more severely impaired inspiratory muscle strength compared to chronic left heart failure patients of previous studies. Therefore, even if there are several similarities between PH and chronic left heart failure pathophysiological findings in these patients cannot easily be transferred to PHpatients and further investigations are needed to confirm these suggestions. Importantly, none of the PH-patients in the present study suffered from malnutrition or wasting since these factors are known to be potential confounders with regard to reduced respiratory muscle strength in chronic left heart failure [START_REF] Anker | Wasting as independent risk factor for mortality in chronic heart failure[END_REF]. Finally, PH-patients in the current study presented with hypocapnia both at rest and following exercise, indicating higher demands on the inspiratory muscles. This overstraining might in part account for reduced inspiratory muscle strength in PH-patients.

Interestingly, hypocapnia has recently been recognized as an independent marker of mortality in PH-patients [START_REF] Hoeper | Prognostic value of blood gas analyses in patients with idiopathic pulmonary arterial hypertension[END_REF].

The present study indicates that maximal inspiratory mouth pressure and sniff transdiaphragmatic pressure are significantly linked to exercise capacity in PH-patients.

Interestingly, reduced inspiratory muscle strength, as assessed by maximal inspiratory mouth pressure, is known to be an important determinant of maximal oxygen consumption and exercise tolerance [START_REF] Chua | Inspiratory muscle strength is a determinant of maximum oxygen consumption in chronic heart failure[END_REF] and, moreover, an independent predictor of prognosis [START_REF] Meyer | Respiratory muscle dysfunction in congestive heart failure: clinical correlation and prognostic significance[END_REF] Minute ventilation is known to be increased in patients with PH both at rest and during exercise [START_REF] Sun | Exercise pathophysiology in patients with primary pulmonary hypertension[END_REF]. Accordingly, in both the present and a previous study [START_REF] Meyer | Respiratory muscle dysfunction in idiopathic pulmonary arterial hypertension[END_REF], ventilatory drive revealed trends towards higher values while respiratory capacity tended to be reduced in PH-patients compared to controls. Importantly, no difference could be observed in the effective inspiratory impedance between PH-patients and controls, indicating that the load imposed on the inspiratory muscles is not increased in PH-patients [START_REF] Whitelaw | Occlusion pressure as a measure of respiratory center output in conscious man[END_REF]. Therefore, impairment of respiratory muscle function in PHpatients cannot be explained by known changes in breathing pattern. Since changes in lung volume are known to have substantial impact on respiratory muscle strength care was taken to apply all tests on respiratory muscle function at comparable lung volumes both in PH-patients and control subjects. This was assured by the computer device as outlined above. Importantly, pre-existing differences in ITGV as the best available measure of functional residual capacity between PH-patients and control subjects have been ruled out as confounding factor. Both PHpatients and normal subjects presented with ITGV values within the predicted norm. However, there was a non-significant difference of approximately 0.3 l in ITGV between PH-patients and control subjects. Since it is known that TwPdi decreases by 0.34 kPa/l changes in ITGV [START_REF] Polkey | Diaphragm strength in chronic obstructive pulmonary disease[END_REF] this difference might account for a reduction in TwPdi of 0.1 kPa for PH-patients compared to control subjects. However, this leaves the major component of 0.5 kPa of difference unexplained and, therefore, the observed marked differences in respiratory muscle function between PHpatients and control subjects cannot be attributed to differences in lung volume. Since lung volume correction has been established for TwPdi only and effects are suggested to be at best small no further parameter of inspiratory muscle strength was corrected for lung volume. The present study has certain limitations which need to be addressed. Firstly, the assessment of muscle function focused on respiratory muscles only. A generalized systemic muscle weakness process in PH-patients is likely to accompany the demonstrated respiratory muscle weakness in PH-patients as has been demonstrated in patients with chronic left heart failure [START_REF] Lindsay | Histological abnormalities of muscle from limb, thorax and diaphragm in chronic heart failure[END_REF]. However, since peripheral muscle function was not investigated in the present study this remains speculative and clearly needs further investigations. Secondly, patients with PH showed mild airway obstruction compared to controls. However, it is well known that mild peripheral airway obstruction is common in patients suffering from PH, and values presented in the current study are comparable to those published previously [START_REF] Meyer | Peripheral airway obstruction in primary pulmonary hypertension[END_REF]. Therefore, inclusion of PH-patients with mild airway obstruction could not be avoided. Nevertheless, impairment of lung function in PHpatients was at best slight and is, therefore, suggested to be irrelevant with regard to the observed marked differences in respiratory muscle strength between PH-patients and controls.

In conclusion, this study provides strong evidence that inspiratory muscle strength is reduced in PH-patients compared to well-matched controls. Furthermore, six-minute walking distance in PH-patients is significantly linked to parameters of inspiratory muscle strength.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 3 Sep 2007 as manuscript CS20070238 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070238 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  volitional and at the same time non-invasive characteristics. Secondary endpoints were differences in the non-volitional but invasive TwPdi and volitionally assessed respiratory muscle function between PH-patients and controls, and associations between parameters assessing respiratory muscle function and 6MWD in PH-patients.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 3 Sep 2007 as manuscript CS20070238 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070238

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 3 Sep 2007 as manuscript CS20070238 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070238 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  investigate possible improvements of respiratory muscle strength in PH-patients undergoing respiratory muscle training. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 3 Sep 2007 as manuscript CS20070238 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070238 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  in the diaphragm have been reported. In addition, besides an intracellular calcium regulation disorder in the diaphragms of patients with chronic left heart S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 3 Sep 2007 as manuscript CS20070238 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070238 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  in patients with chronic S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 3 Sep 2007 as manuscript CS20070238 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070238 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society left heart failure. Whether this compares to PH-patients remains unclear and requires additional investigations.

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  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 3 Sep 2007 as manuscript CS20070238 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070238 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  F trig = inspiratory flow at triggering twitch pressures; P 0.1 = mouth occlusion pressure after 0.1 seconds of inspiration; P 0.1 /PImax = respiratory capacity (higher values indicate reduced capacity); P 0.1 /V T /t i = effective inspiratory impedance; PEmax = maximal expiratory mouth pressure; PImax = maximal inspiratory mouth pressure; P trig = inspiratory pressure at triggering twitch pressures; SnPdi = Sniff transdiaphragmatic pressure; SnPna = Sniff nasal pressure; TwPdi = twitch transdiaphragmatic pressure; TwPmo = twitch mouth pressure. *n=26; † n=29. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 3 Sep 2007 as manuscript CS20070238 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070238 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

FiguresFigure 1 .

 1 Figures

Figure 2 .

 2 Figure 2. Values for non-volitional assessed twitch mouth (TwPmo) and transdiaphragmatic (TwPdi) pressures in patients with pulmonary hypertension as compared to control subjects. Solid bars represent mean values for control subjects. Mean values for patients with pulmonary hypertension are represented by the hatched bars. T-bars represent 95 % confidence interval of the mean. S t a g e 2 ( a ) P O S T -P R I N T

Figure 3 .

 3 Figure 3. Relationship between maximal inspiratory mouth pressure (PImax) and six-minute walking distance (6MWD) in patients with pulmonary hypertension following linear regression analysis. The regression line between PImax and 6MWD is represented by the solid line. Dashed line reflects 95 percent confidence interval for the regression. Dotted line represents 95 percent confidence interval for the population. S t a g e 2 ( a ) P O S T -P R I N T

Table 1 .

 1 Anthropometric characteristics, lung function and exercise parameters in patients with pulmonary hypertension (n=31) and control subjects (n=31). Clinical Science Immediate Publication. Published on 3 Sep 2007 as manuscript CS20070238 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070238Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

			Tables
			Control subjects	PH-patients	p-value
	Gender (f/m)	[n]	20/11	20/11
	Age	[a]	55.0 ± 12.2	56.5 ± 14.3	0.66
	BMI	[kg/m²]	26.6 ± 4.9	26.0 ± 5.5	0.67
	ITGV	[l]	2.9 ± 0.6	3.2 ± 1.0 †	0.21
	ITGV	[% pred]	88 ± 17	99 ± 26 †	0.08
	FEV 1	[% pred]	98 ± 13	84 ± 19	0.001
	FVC	[% pred]	110 ± 13	97 ± 18	0.001
	FEV 1 /FVC	[%]	78 ± 6	74 ± 9	0.054
	MEF 50	[% pred]	70 ± 25	60 ± 28	0.16
	6MWD	[m]	619 ± 79	407 ± 118*	<0.001
	BDS		0.2 ± 0.5	6.4 ± 2.4*	<0.001
	PaO 2 rest	[mmHg]	77 ± 5	68 ± 9 †	<0.001
	PaCO 2 rest	[mmHg]	39 ± 3	35 ± 3 †	<0.001
	PaO 2 exercise	[mmHg]	87 ± 8	64 ± 16 †	<0.001
	PaCO 2 exercise	[mmHg]	38 ± 3	34 ± 4 †	<0.001
	Values are expressed as mean (± SD). BDS = Borg dyspnea scale; BMI = body-mass index; FEV 1 = forced
	expiratory volume in 1 second; FVC = forced vital capacity; ITGV = intrathoracic gas volume; MEF 50 = maximal
	expiratory flow at 50 percent of FVC; % pred = percent predicted; PaCO 2 rest/exercise = carbon dioxide partial pressure
	at rest / following exercise; PaO 2 rest/exercise = oxygen partial pressure at rest / following exercise; 6MWD = distance
	walked in 6 minutes. *n=30; † n=18.

S t a g e 2 ( a ) P O S T -P R I N T © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 2 .

 2 Hemodynamic and serological parameters in patients with pulmonary hypertension (n=31).

			PH-patients
	WHO II/III/IV	[n]	2/23/6
	MPAP	[mmHg]	51 ± 20
	PVR	[dyn*s*cm -5 ]	759 ± 425
	PAWP	[mmHg]	9 ± 5
	CVP	[mmHg]	6 ± 3
	CI	[l/min/m²]	2.6 ± 0.5
	EF	[%]	56 ± 8
	NT-proBNP	[pg/ml]	1117 ± 1656
	Uric acid	[mg/dl]	6.8 ± 2.6
	Values are expressed as mean (± SD). PAWP = pulmonary artery wedge pressure; CI = cardiac index; CVP =
	central venous pressure; EF = left ventricular ejection fraction (assessed by echocardiography); NT-proBNP = N-
	terminal pro brain-derived natriuretic peptide; MPAP = mean pulmonary artery pressure; PVR = pulmonary vascular
	resistance; WHO II/III/IV = World Health Organization functional classes.

S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 3 Sep 2007 as manuscript CS20070238 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070238 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 3 .

 3 Ventilatory characteristics, tests on respiratory muscle strength and corresponding data in patients with pulmonary hypertension (n=31) and control subjects (n=31).

			Control subjects	PH-patients	p-value
	P 0.1	[kPa]	0.23 ± 0.10	0.28 ± 0.15	0.12
	P 0.1 /V T /t i	[kPa/l/s]	0.5 ± 0.2	0.5 ± 0.2	0.68
	P 0.1 /PImax	[%]	3 ± 2	6 ± 7	0.13
	PImax	[kPa]	7.5 ± 2.1	6.2 ± 2.8	0.04
	PEmax	[kPa]	13.3 ± 4.2	9.9 ± 3.4	<0.001
	SnPna	[kPa]	8.3 ± 1.9	6.6 ± 2.2	0.002
	SnPdi	[kPa]	11.3 ± 2.5*	8.7 ± 2.5*	<0.001
	TwPmo	[kPa]	1.46 ± 0.43 †	0.97 ± 0.41	<0.001
	TwPdi	[kPa]	2.08 ± 0.55*	1.47 ± 0.72*	0.001
	P trig	[kPa]	0.5 ± 0.1 †	0.5 ± 0.1	0.74
	F trig	[ml/s]	32 ± 14 †	32 ± 12	0.88
	Values are expressed as mean (± SD).

Acknowledgements

We are grateful to all participants for their effort devoted to this study, to Bruno Kopp on behalf of PH e.V. for their support, to Roland Merklein (ZAN ® GmbH, Oberthulba, Germany) for software assistance, to Dr. Sandra Dieni for proofreading the manuscript and to Stephanie Rubenbauer for statistical consulting.

Founding sources: Support for this project was provided by a grant of the German Pulmonary Hypertension Group (Pulmonale Hypertonie e.V.), Rheinstetten, Germany. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 3 Sep 2007 as manuscript CS20070238 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070238 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society


Flavio Ribichini
email: flavio.ribichini@univr.it

Francesco Pugno

Valeria Ferrero

William Wijns

Giovanni Vacca

Corrado Vassanelli

Renu Virmani

Prof Flavio Ribichini

Long-Term Histologic and Immunohistochemic Findings in Human Venous Aorto-Coronary Bypass Grafts

Keywords: atherosclerosis, pathology, saphenous vein graft, angioplasty, stents percent diameter stenosis, EL: elastic lamina, M: media, AP: atheromatous plaque, FP: fibrous plaque, NI: neo-intima, SMC: smooth-muscle cells, UN: underlying plaque. * asterisks denotes disrupted media

come

Introduction.

The implantation of endovascular stents in saphenous vein grafts (SVG) used as bypass conduits to revascularise the native coronary circulation is common practice in interventional cardiology. The percutaneous treatment of narrowed veins offers an excellent alternative to a second operation that in many cases can be avoided or at least delayed. Indeed, repeat bypass surgery is generally burdened by technical difficulties, older age, and a higher rate of graft occlusion [START_REF] Foster | Comparison of operative mortality for initial and repeat coronary artery bypass grafting: the Coronary Artery Surgical Study (CASS) Registry Experience[END_REF].

Recent advances in stent technology and anti-platelet therapy, and the development of aspiration and protective devices to avoid distal embolisation during the procedure have improved the safety and short-mid term results of the transcatheter treatment of SVG [START_REF] Baim | Randomized trial of a distal embolic protection device during percutaneous intervention of saphenous vein aorto-coronary bypass grafts[END_REF]. Nevertheless, the progressive atherosclerotic degeneration of the venous conduit and the high rate of restenosis observed after stented angioplasty in this setting reduce the long-term efficacy of this approach that is burdened by a 40% rate of vessel failure compared to 15 to 20% in native coronary arteries [START_REF] Choussat | Long-term clinical outcome after endoluminal reconstruction of diffusely degenerated saphenous vein grafts with less-shortening wallstents[END_REF].

The pathologic analysis of SVG treated with coronary stents is performed from surgical or autopsy specimens which are not easily available. Indeed, studies in this field are scarce and the description of pathologic changes of stented grafts available from previous reports is limited to relatively short periods.

Previous studies performed in human venous bypass grafts have described the morphologic characteristics of restenosis after implantation of two different stent types [START_REF] Anderson | Vascular pathology of balloon-expandable flexible coil stents in humans[END_REF][START_REF] Van Beusekom | Histology after stenting of human saphenous vein bypass grafts: observations from surgically excised grafts 3 to 320 days after stent implantation[END_REF][START_REF] Depre | Pathology of restenosis in saphenous bypass grafts after long term stent implantation[END_REF]; other two studies have analysed the composition of in-stent restenotic plaques retrieved by means of directional atherectomy [START_REF] Strauss | Directional atherectomy for tretament of restenosis within coronary stents: clinical, angiographic and histologic results[END_REF][START_REF] Nikol | Restenosis in human vein bypass grafts[END_REF].

In the present study we analyse a series of SVG 11 6 years old (range 5-18 years) containing metallic stents that had been in place for as much as 5 years (range 5-61 months). The histology and the immunohistochemistry of the plaque composition and the cellular infiltration around the metallic stents are described.

The report is focused on the description of the salient features observed in this specific and rare histopathologic setting. Such information may be of help to evaluate possible differences of efficacy between drug-eluting stents (DES) and conventional metallic stents implanted in degenerated SVG.

Methods

Our study describes the pathology of 7 SVG containing 12 coronary stents, retrieved from 6 patients (table 1). All patients gave their informed consent to the analysis of the surgical specimens and angiographic images, and the approval of the Ethical Committee of the institution where the study material was analysed (Ospedale Maggiore di Novara, Italy) was obtained.

All patients were treated with balloon dilatation and/or stent implantation because of recurrence of angina secondary to the stenosis of one graft. At a mean time of 11 6 years the patients were re-operated because of recurrent angina secondary to the angiographic documentation of a total occlusion of the graft in 3 cases, and to stent restenosis and or progression of the disease in other grafts in the remaining patients. At the time of the repeat surgical intervention the stented grafts were harvested, rinsed with saline an fixed in 9% neutral buffered formalin. The proximal edge of the graft was marked with a suture stitch to identify the anatomic orientation and allow comparison with the angiographic images.

Pathologic examination.

Using the suture stitch as a landmark, the localisation of the stents within the SVG was assessed by a radiographic examination that allows the precise identification of each stent length, the detection of overlapped or under-expanded segments and the comparison with the last digital angiographic image of the graft before surgical retrieval. The SVG segments were placed into processing vials and dehydrated in a graded series of alcohols. Samples were then infiltrated with methylmethacrylate plastic and placed in airtight vials in a 39°C waterbath for polymerisation. Afterwards, blocks were cut at 2-3mm intervals along the stented and non stented vein segments. Plastic sections (4-6 m thick) were then cut, adhered to glass and allowed to dry. The plastic was then removed, and the sections selected for analysis (nearly 100 per segment) were rehydrated and stained. In segments selected for immunohistochemistry the stent wires were carefully removed under a dissecting microscope taking care not to alter the macroscopic wall structure before paraffin embedding. Plastic and paraffin sections were stained with hematoxylin and eosin and Movat pentachrome.

The SVG segments showing different degrees of lumen stenoses were selected for analysis i.e, total occlusion, severe, moderate or mild stenosis of the vein that correspond to diameter stenosis 100%, <100 50%, <50 30% or <30 respectively, and cross-sectional area stenosis of 100%, >75%, 75-50% and <50% respectively. The following area measurements were made on each section: stent area, underlying plaque, lipid cores, calcified cores, vessel lumen, and in-stent neointima. Fibrocellular tissue was graded as paucicellular or fibrotic [<30 spindled cells in high power fields (HPF)], moderately cellular (30-100 spindled cells HPF) and hypercellular (>100 spindled cells HPF). Sections were further magnified (x200) and 4 fields containing stent struts (12, 3,6 and 9 o'clock) were selected for analysis of neointimal details [neointimal thickness (mm) and area (mm 2 ), neovessels and inflammatory cell density around the stent struts in terms of cells/mm 2 that was classified as being slight: 1-10 inflammatory cells/struts, moderate: 11 to 20 cells/struts, or severe: >20 cells/struts]. The stent strut penetration was categorised as being associated with Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. the fibrous plaque (or an intact fibrous cap or intact media), medial injury, or lipid core or atheroma penetration [START_REF] Farb | Pathology of acute and chronic coronary stenting in humans[END_REF].

Immunohistochemistry.

Immunostaining for the detection of smooth muscle cells (SMC), macrophages, cells containing angiotensin1-converting enzyme (ACE), proliferation marker Ki-67 and estrogen receptors (ER), were performed in selected samples of non-stented and stented segments of all veins using previously described techniques [10].

Cellular ACE activity was investigated because ACE has been observed in the atherosclerotic plaque as a mediator of inflammation and in the process of wound healing after percutaneous injury of the vessel wall, and to identify endothelial cells [START_REF] Diet | Increased accumulation of tissue ACE in human atherosclerotic coronary artery disease[END_REF][START_REF] Ohishi | Upregulation of angiotensin-converting enzyme during the healing process after injury at the site of percutaneous transluminal coronary angioplasty in humans[END_REF]. Estrogenic activity was investigated because of the protective mechanisms exerted by estrogenes in the vascular wall [START_REF] Dai-Do | 17 beta-estradiol inhibits proliferation and migration of human vascular smooth muscle cells: similar effects in cells from postmenopausal females and in males[END_REF][START_REF] Chandrasekar | Coronary artery endothelial protection after local delivery of 17beta-estradiol during balloon angioplasty in a porcine model: a potential new pharmacologic approach to improve endothelial function[END_REF][START_REF] Geraldes | Specific contribution of estrogen receptors on mitogenactivated protein kinase pathways and vascular cell activation[END_REF]. The total vessel area of positively stained segments for each immunologic reaction was quantified by computer-aided planimetry of each specimen using an acquisition program Image Pro Plus (Media Cybernetics, software version 4.0, Silver Spring, MD, USA) in order to determine a percentage of tissue surface containing different cellular types.

Statistical analysis.

Continuous data are expressed as means and standard deviations; discrete variables are given as absolute values and percentages. Two-tailed Student's t test was used for comparison of parametric variables and the chi-square or exact test for discrete variables. A linear regression analysis was used to correlate the number of inflammatory cells around the stent struts and neointimal thickness. A probability value <5% was considered significant.

Results.

Histology.

Patients clinical characteristics, procedural details of stent implantation, timing and the reason for surgical re-intervention are shown in Table 1.

The angiographic examination obtained before repeat bypass surgery showed 3 totally occluded SVG (each one containing one, two and three stents respectively, and variable degrees of neointimal growth within the stents). In the remaining cases, a mild stenosis within the stent was analysed in 3 stents in 3 different patients and a moderate-severe stenosis in other 3 stents implanted in 3 different patients. Segments of SVG with mild, moderate and severe stenosis not covered by the stents were also analysed.

Stents showing medial fracture were associated with a significantly greater mean neointimal thickness (1.37 0.68mm 2 versus 0.81 0.47mm 2 , p<0.02). In one case, the stent struts of two sequential stents were partially apposed to a large aneurysm, leaving a space between the struts and the SVG wall filled with thrombus, and atheroma (Figure 1a and1b). The degree of stent wall indentation, the luminal SVG % Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

diameter stenosis and area narrowing, as well as the main histologic characteristics, are reported in Table 2, that briefly describes the classification scheme proposed by Virmani et al [START_REF] Virmani | Lessons from sudden coronary death. A comprehensive morphological classification scheme for atherosclerotic lesions[END_REF].

Histology of the stented SVG segments: in totally occluded SVG, red thrombus was observed in all cases as well as large amount of organised thrombus containing neovessels (Figure 2). The presence of neovessels has been well described in native coronary arteries with total occlusion [START_REF] Depre | Neovascularization in human coronary atherosclerotic lesions[END_REF]. In 2 of the 3 totally occluded cases a stenosis of the graft was present proximally to the stented segment and in one case the stenotic plaque was in the mid portion of the under-expanded stent, two mechanisms previously related to SVG stent occlusion [START_REF] Depre | Pathology of restenosis in saphenous bypass grafts after long term stent implantation[END_REF]. Histology of the underlying stenotic lesions showed predominance of smooth muscle cells that extended from the metallic wires towards the vessel lumen within an extensive extracellular proteoglycan/collagen matrix (Figure 2). The lumen side of the stent showed a slight to moderate degree of inflammation (i.e. >1 to <20 cells adjacent to each stent strut), and giants cells were frequently observed. The vessel wall showed a thin smooth muscular layer of the vein media (average 900 300 m) and of the collagenous adventitia (average 650 400 m). When the stent struts caused laceration or rupture of the vein medial wall, a thickened adventitia (>1mm) was observed in correspondence of a thickened neointima toward the luminal side. Large calcium deposits (range 2 to 8.7mm 2 ) were observed in all cases, always surrounded by inflammatory infiltrate of histiocytes and lymphocytes.

In patent stents, plaques causing different degrees of luminal stenoses were characterised by a similar histologic pattern, mainly composed by SMC and extracellular matrix. A moderate to severe infiltration of inflammatory cells (10-20 or >20 cells/strut) was observed when the stent strut was adjacent to injured media or a lipid core rather than a fibrous plaque, confirming in the veins a mechanism previously described in native coronary arteries [START_REF] Farb | Pathology of acute and chronic coronary stenting in humans[END_REF]. Furthermore, when the stent struts were in contact with an underlying plaque mainly composed of a necrotic core, no healing was present (Figure 3a) and plaque and thrombus protruding into the vein lumen were observed (Figure 3b). Different degrees of inflammation around the stent struts correlated with neointimal thickness in patent vessels, indeed, by linear regression analysis, a significant correlation was found between the areas occupied by inflammatory cells and the intimal thickness around the stent struts of plaques analysed in patent conduits (r 2 =0.43, p<0.001).

The degree of penetration in the vessel wall was not different for self-expanding or balloon expanding stents. This may correspond to aggressive post-stent high-pressure balloon dilatation or to the injury caused by the self-expanding stent type that exerts a permanent radial force on the vessel wall. An interesting pathologic feature of plaques causing only a mild narrowing of the vein lumen is the presence of a circular layer of calcium around the stent. In these cases, organised thrombus was rarely observed and neointimal growth was only mild (Figure 4).

Histology of the non-stented SVG segments: vein plaques were fibrous with calcified areas and chronic inflammatory infiltrates. Stenoses causing SVG lumen narrowing were mainly composed of organised thrombus with neovessels. Histiocytes and less frequently lymphocytes were observed beneath the endothelial layer that covers the intima. In the lumen of the SVG, red thrombus was observed in as many of 70% of analysed segments (Figure 5). Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Restenotic or primary lesions: primary lesions were composed mostly by moderately cellular plaques (30-100 spindled cells HPF) while restenotic plaques showed a trend to be predominantly paucicellular (<30 spindled cells/HPF). No significant differences in extracellular matrix formation or cell proliferation were observed between primary and restenotic SVG tissue, in contrast to previously described findings in arterial tissue [START_REF] Riessen | Regional differences in the distribution of the proteoglycans biglycan and decorin in the extracellular matrix of atherosclerotic and restenotic human coronary arteries[END_REF]. The lack of a diagnostic pattern that differentiate primary from restenotic plaques in SVG has been reported previously by other authors [START_REF] Van Beusekom | Histology after stenting of human saphenous vein bypass grafts: observations from surgically excised grafts 3 to 320 days after stent implantation[END_REF][START_REF] Nikol | Restenosis in human vein bypass grafts[END_REF].

Stents in place for less than 6 months: pathology of stent analysed within 6 months of implantation revealed moderate-severe inflammation of the media around the stent wires (10-20 or >20 cells/strut respectively), neointimal proliferation and SMC; the endothelial layer was not always observed and when present, the endothelial cells showed large nuclei and polygonal cellular shape.

Stents in place from 6 months to 2 years: neoendothelium showed a thin layer of cells with a normal aspect covering the neointima located within the stented segments. Large calcified semicircular areas with moderate inflammation near the calcified core and the metallic wires were observed. In some areas, macrophages included iron pigments.

Stents in place from 2 to 5 years: appeared basically as described above, with more intense infiltration of lymphocytes under the endothelial layer, and giant cells close to the stent struts. Calcified areas of the vessel wall were larger the older the SVG (mean calcified surface in veins >10 years 6.8 3.4mm 2 versus 3.4 3.0mm 2 in veins 10 years, p<0.01).

Immunohistochemistry

Immunostaining for alpha actin identified SMC's as the cellular component of 40 to 80% of the media layer in all cases, and rare elements in the adventitia. In the lumen side, cellular components of the neointima within the stent revealed SMC's accounting for 55% of the neointimal area, and a rich extracellular fibrous matrix. There were more SMC's close to the neoendothelium than close to the stent struts (53% versus 12%, p<0.01), but the percentage area of stained cells did not differ among the different types of tissue analysed (Table 3). KP-1 staining was observed in areas of cluster of macrophages in all cases. In the underlying plaque KP-1 stained macrophages situated close to the necrotic or the calcified cores and the external side of the metallic stent struts. Some inflammatory cells were also observed in the adventitia. In the neointima, KP-1 positive cells were in contact with the metallic struts (Figure 6a), and beneath the neo-endothelial layers when this was present. In plaques causing severe in-stent restenosis KP-1 positive cells were significantly more frequent than in plaques causing only mild in-stent stenosis (Table 3). In non-stented segments KP-1 stained cells were also present close to necrotic, atheromatous or calcified cores.

ACE activity was detected mostly in cells that co-localised for KP-1 in identical sections, suggesting their inflammatory nature. These cells were most commonly observed close to the stent wires (Figure 6b), and in some cases close to the adventita. A second type of ACE positive cells that did not stain for KP-1 were identified as endothelial cells of neovessels of the plaque and cells of the neo-endothelium that faced the vessel lumen. A third group of ACE positive cells that co-localised for alpha actin in identical sections was Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. observed in the media and the sub-endothelial layer of the neointima with a spindle-shape morphology. In plaques causing in-stent restenosis there was significantly more ACE activity in macrophages compared with plaques of non-stented vein segments (Table 3).

Immunostaining for Ki-67 showed few positive cells, suggesting the poor proliferative state of the old veins.

There was no difference as to the amount of Ki-67 positive cells between restenotic or "de-novo" lesions and between plaques causing severe or mild in-stent obstructions (Table 3).

Immunostaining for ER was completely negative in all the sections examined irrespective of the patient's gender and of the presence of a stent.

Discussion

Despite the improvement of the immediate results of transcatheter treatment of diseased SVG [START_REF] Baim | Randomized trial of a distal embolic protection device during percutaneous intervention of saphenous vein aorto-coronary bypass grafts[END_REF], the longterm outcomes remain sub-optimal [START_REF] Choussat | Long-term clinical outcome after endoluminal reconstruction of diffusely degenerated saphenous vein grafts with less-shortening wallstents[END_REF]. The reasons for such unfavorable results are probably related to the nature of the venous wall and the changes that follow the implantation of the vein in the systemic circulation [START_REF] Kalan | Comparison of morphologic changes and luminal sizes of saphenous vein and internal mammary artery after simultaneous implantation for coronary arterial bypass grafting[END_REF]. Metallic stents may interact with the venous wall in a different manner compared to the arterial wall, the more fragile media layer and the friable atherosclerotic type of plaque of the SVG can be easily damaged by percutaneous devices inducing a subsequent exaggerated reparative response.

Our study extends the available knowledge about the long-term effects of metallic stents in SVG up to 5 years after implantation, and about the evolution of the atherosclerotic disease of the SVG wall up to 18 years. The main findings of our research can be summarised as follows.

1. Histology shows, on the one hand, that the media layer of the SVG, being thinner than that of coronary arteries, is likely more susceptible to the mechanical damage caused by the stents and the balloon pressure, and that the easier media fracture ensues aggressive neointimal proliferation. On the other hand, very longterm chronic inflammation close to the metallic wires is a relevant phenomenon, and similarly to what has also been reported in coronary arteries, this finding correlates with thicker neointima within the stents [START_REF] Farb | Pathology of acute and chronic coronary stenting in humans[END_REF][START_REF] Farb | Morphological predictors of restenosis after coronary stenting in humans[END_REF].

However, organised thrombus with neovessels represent the main component of the lumen-narrowing material (particularly in totally occluded conduits), while substantial amounts of plaque easily protrude into the SVG lumen after stent placement. The larger diameter and the lower elasticity of the vein conduit may cause a less brisk flow in the SVG that fosters platelet apposition, thrombus formation and subsequent lumen narrowing. Thus, the atherosclerotic material of SVG lesions appears highly friable because it lacks excess collagen and is richer in lipids, necrotic cores and macrophages facilitating plaque prolapse through the stent struts.

Unlike native coronary arteries, in which calcification is basically seen in the intima, embedded in the collagen matrix, and focally precipitated in necrotic cores, atherosclerosis of the degenerated SVG is characterized by large areas of calcification of the vessel wall itself. In particular, in segments of veins with only mild in-stent proliferation calcium deposition may assume a circular pattern embedded in a thin Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. muscular wall, and these annular calcified areas are more extensive the longer the time of the implant in the arterial circulation.

2. Immunohistochemic assays show a long-lasting presence of macrophages staining positively for KP-1 and ACE, confirming a preliminary report from our group in which ACE activity in cells close to the stent wires was observed after 6 months of stent implantation in an artery [START_REF] Ribichini | Angiotensin-converting enzyme tissue activity in the diffuse in-stent restenotic plaque[END_REF]. Such observation extends the concept proposed by Komatsu et al that demonstrated ACE activity as a mediator of inflammation but being limited to short time after balloon arterial wall injury [START_REF] Komatsu | Neointimal tissue response at sites of coronary stenting in humans[END_REF]. This phenomenon was observed predominantly in sections of veins that received a coronary stent. The presence of ACE was not only referred to inflammatory cells; indeed, ACE immunostaining was observed in endothelial cells, as expected, but also in groups of neointimal spindle-shaped cells that stained also positively alpha actin. This finding suggests a possible role of the enzyme in the transformation of SMC-like cells from a dedifferentiated to a differentiated phenotypic state or from other type of cells such as infiltrating macrophages or fibroblasts as it has been previously proposed in native coronary arteries [10,[START_REF] Ohishi | Upregulation of angiotensin-converting enzyme during the healing process after injury at the site of percutaneous transluminal coronary angioplasty in humans[END_REF][START_REF] Komatsu | Neointimal tissue response at sites of coronary stenting in humans[END_REF]. A predominant role of ACE in the repairing process of wall damage in SVG is supported by the threefold greater ACE activity observed in human SVG compared to the internal thoracic artery of patients re-operated 20 years after a first coronary bypass surgery [START_REF] Borland | Differential action of angiotensin II and activity of angiotensin-converting enzyme in human bypass grafts[END_REF].

Immunostaining for the detection of ER was totally negative in these samples. The absence of ER in the saphenous vein is in agreement with previous observations [START_REF] Bergqvist | Estrogen and progesterone receptors in vessel walls. Biochemical and immunochemical asays[END_REF][25]; some rare ER may be found in veins of fertile women, but not in men or post-menopausal women [START_REF] Bergqvist | Estrogen and progesterone receptors in vessel walls. Biochemical and immunochemical asays[END_REF]. The lack of the anti-atherosclerotic effects of estrogens in the structure of the vein wall may contribute another possible reason for the aggressive atherosclerotic degeneration of the veins used as by-pass grafts [START_REF] Geraldes | Specific contribution of estrogen receptors on mitogenactivated protein kinase pathways and vascular cell activation[END_REF].

The rare presence of cells staining with the proliferation marker Ki-67, put in evidence the poor proliferative state of this type of tissue, and suggests that organised thrombus and migrated cells rather than cell proliferation could be the principal reasons of SVG lumen narrowing.

The recent availability of DES has dramatically improved the efficacy of percutaneous coronary interventions in native vessels but very scarce information is available regarding the efficacy of DES implanted in old degenerated SVG [START_REF] Ge | Treatment of saphenous vein graft lesions with drugeluting stents: immediate and midterm outcome[END_REF][START_REF] Vermeersch | Randomized double-blind comparison of sirolimuseluting stent versus bare-metal stent implantation in diseased saphenous vein grafts: six-month angiographic, intravascular ultrasound, and clinical follow-up of the RRISC Trial[END_REF][START_REF] Rocca | Targeted stent use in clinical practice based on evidence from the Basel Stent Cost Effectiveness Trial (BASKET)[END_REF]. Furthermore, data about the histopathology of such kind of stents implanted in old SVG is not available so far. Commercially available DES inhibit restenosis on coronary arteries exerting mainly anti-blastic and anti-proliferative effects [START_REF] Costa | Molecular basis of restenosis and drug-eluting stents[END_REF]; therefore, from a practical standpoint, DES may exert different levels of effectiveness (either higher or lower) in old, degenerated atherosclerotic SVG according to the predominant histopathologic characteristics observed in these plaques, as described in the present study. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Sep 2007 as manuscript CS20070243 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070243

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Sep 2007 as manuscript CS20070243 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070243

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Sep 2007 as manuscript CS20070243 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070243

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Sep 2007 as manuscript CS20070243 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070243

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Sep 2007 as manuscript CS20070243 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070243

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Sep 2007 as manuscript CS20070243 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070243

Figure 1 .

 1 Figure 1. (a): X-ray photography of a SVG containing 2 overlapped metallic stents at the site of a large aneurysm (arrow) and a third stent beyond. (b): hematoxylin-eosin cross-section of the same SVG (x2)showing the patent lumen of the stents and the aneurysm lumen filled with fresh thrombus (arrows).

Figure 2 .

 2 Figure 2. Movat Pentachrome staining (x4) of a totally occluded SVG: the lumen of the conduit is occupied by a thick neointima within the stent and a totally occlusive organized thrombus (arrows).

Figure 3 .Figure 4 .

 34 Figure 3. (a): Cross-section of a SVG implanted a self-expanded metallic stent and developing late in-stent restenosis. The stent struts show deep apposition close to the media layer on the left side of the SVG section and a thick neointima developed within the stent lumen close to the struts (asterisk). The wires on the right side (arrow) are close to a necrotic core and only a thin layer of healing neointima is observed in the lumen side of the stent struts. (Movat Pentachrome section x2). (b): Atherosclerotic plaque and large thrombus protruding into the SVG lumen through the stent struts (asterisk). (Movat Pentachrome section x4)

Figure 5 .

 5 Figure 5. Movat Pentachrome stained cross-section (x4) of a non-stented segment of SVG previously treated with balloon angioplasty and showing late luminal narrowing (asterisk), thickened neointima (1), organized thrombus (2), red thrombus (3) and neovessels of the thickened adventita (4) are evident.

Figure 6 .

 6 Figure 6. (a): Immunohistochemistry staining showing brown-colored cells positive for human panmacrophagic antibody KP-1 close to the stent wires removed for paraffin embedding (asterisk). The KP-1 positive staining demonstrate the inflammatory nature of the cells around the metallic struts (x200). (b): ACE immunostaining showing positive reaction (brown-colored elements) in inflammatory cells close to the metallic strent wire removed for paraffin embedding (asterisk). S t a g e 2 ( a ) P O S T -P R I N T

S t a g e 2

 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Sep 2007 as manuscript CS20070243 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070243 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Sep 2007 as manuscript CS20070243 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070243 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Sep 2007 as manuscript CS20070243 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070243 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Sep 2007 as manuscript CS20070243 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070243 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Sep 2007 as manuscript CS20070243 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070243 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Sep 2007 as manuscript CS20070243 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070243 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 3 . Quantitative immunohistochemic analysis. Type of SVG sample alpha actin * p value

 3

	KP1*	p value	ACE*	p value	Ki-67*	p value	ER

Abbreviations: SVG: saphenous vein graft, ACE: angiotensin-converting enzyme. (*) indicates % of plaque area.

© 2007 The Authors Journal compilation © 2007 Biochemical Society

S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 12 Sep 2007 as manuscript CS20070243 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070243 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

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