Perioperative outcome of laparoscopic left lateral liver resection is improved by using a bioabsorbable staple line reinforcement material in a porcine model

Hypothesis Laparoscopic liver surgery is significantly limited by the technical difficulty encountered during transection of substantial liver parenchyma, with intraoperative bleeding and bile leaks. This study tested whether the use of a bioabsorble staple line reinforcement material would improve outcome during stapled laparoscopic left lateral liver resection in a porcine model.
The success of laparoscopic cholecystectomy has driven the application of minimally invasive techniques to other disease processes, and led to the use of laparoscopy in solid organ surgery [1–9]. Since the report of the first laparoscopic liver resection for a 6-cm focal nodular hyperplasia, laparoscopic surgery for the treatment of liver diseases has become more popular [28–30]. In 1995, excision of a segment 4 hepatic tumor was reported. The first successful laparoscopic left lateral hepatectomy (segments 2 and 3) in a patient with a benign adenoma was performed in 1996 [8].
Technological advances and the development of new equipment have facilitated the growing trend of laparoscopic liver resections [2]. However, laparoscopic liver surgery remains technically challenging. Compared to open surgery, the laparoscopic surgeon is limited by the lack of an effective means to divide substantial liver parenchyma. Transection of the liver can be performed laparoscopically in several different ways, however bile leakage rates of 1–16% and bleeding complication rates of 6–55% have been reported [9, 30–32].
We hypothesized that the addition of a bioabsorbable membrane to reinforce standard laparoscopic stapling devices would reduce the bleeding and biliary complications of stapled laparoscopic liver resections. We tested this hypothesis in a prospective survival study of 20 pigs undergoing laparoscopic left lateral liver resections.
Methods
Study design
A total of 20 female pigs each weighing approximately 40 kg were used for the study. All animals were maintained in accordance with the recommendations of the institutional animal care and use committee at the Mount Sinai School of Medicine. Animals were randomly assigned at the time of surgery to either group A (n = 10) in which the stapling devices used for laparoscopic left lateral segmentectomy were reinforced with a bioabsorbable membrane or group B (n = 10), in which standard stapling devices were used. Animals were followed prospectively for a 6-week period after which they were sacrificed.
Surgery
All animals were premedicated with ketamine (22 mg/kg) and atropine (0.04 mg/kg) and anesthetized with thiopentothal (15 mg/kg) prior to intubation. Animals were then mechanically ventilated with an initial tidal volume of 10 ml/kg and a respiratory rate of 15 breaths per minute. The tidal volume was adjusted to maintain arterial PaCO2 of 35–40 mmHg during the experiment. Anesthesia was maintained with inhaled isofluorane (1.5%).
The pig was placed in a supine position. A standard open technique was used to gain access to the abdomen and a total of five trocars (Karl Storz Endoscopy America, Culver City, CA, USA) were used for the procedure (Fig. 1).
Fig. 1Schematic illustration of trocars placement in the porcine model
Pneumoperitoneum was established to a pressure of 15 mmHg. After inspection of the abdominal cavity with a 10-mm 30° laparoscope, the remaining trocars were placed under direct vision. Two 10-mm trocars were used in the left and right upper quadrants. One 12-mm trocar was positioned in the left anterior axillary line for liver retraction. The liver capsule and parenchyma were inspected superiorly and inferiorly and the line of transection was visualized between the liver hilum, along the surface of the entire left liver lobes towards the diaphragm. Subsequently, the left lateral segmentectomy was demarcated (segments 2 and 3 of the liver) within 1 cm along the falciform ligament. If necessary, the diaphragmatic and minor omental attachments to the liver were divided to mobilize the left lobe. Once mobilized, attention was turned to transection of the liver parenchyma. This was accomplished with sequential firings of a 3.5-mm endo GIA stapler (US Surgical, Norwalk, CT). In group A, the stapler cartridges were buttressed with a bioabsorble reinforcement material (Seamguard®, W.L. Gore, Flagstaff, Arizona) (Fig. 2). In group B the standard stapler cartridges were used. As the liver parenchyma was divided, no attempt was made to isolate the major feeding vessels (major branches of the left hepatic artery and vein) to segments 2 and 3; rather, they were divided en masse with the liver parenchyma. Upon completion of the transection, hemostasis was achieved with the ultrasonic scalpel (Ethicon Endo-Surgery, Cincinatti, Ohio, USA), which works by means of a vibrating blade or scissors and can effectively seal small vessels and bile ducts, and/or electrocautery. The resected specimen was placed into an endobag and retrieved through the umbilical trocar site that was extended approximately 3.5–5.0 cm to accommodate the specimen. The abdomen was irrigated and inspected prior to closure of the trocar sites. No intra-abdominal drains were used.
Fig. 2An absorbable polymer membrane (Bioabsorbable Seamguard, W.L. Gore, Flagstaff, Arizona, USA) is constructed as a buttress mat integrated in the stapler systems
Data collection
Operative data recorded at the time of surgery included operative time, specimen size, and blood loss as estimated by amount of fluid collected in a suction container minus irrigation fluid used. Animals were prospectively followed with regard to clinical outcome for 6 weeks. Clinical status (temperature, blood pressure = BP, heart rate = HR) was examined routinely on a daily basis unless clinical condition mandated additional assessment. Routine blood tests (bilirubin, ALT, AST, alkaline phosphatase, GGT, and complete blood count) were obtained 2 days and 6 weeks postoperatively.
Animals were sacrificed at 6 weeks and examined for intra-abdominal abnormalities such as abscess or bile leaks. Standard methylene blue was injected into the biliary tree to examine for active biliary leaks. A standardized pressure-controlled injector using a green sized syringe injected 5–10 ml methylene blue into the ligated common bile duct. Subsequently, the cut edge of the liver was examined both macroscopically and microscopically for evidence of biliary leak. The cut edge of the liver was then sent for histopathological analysis.
Statistical analysis
To obtain a 50% reduction rate in postoperative outcome after laparoscopic liver resection an 80% power analysis showed a series of 20 consecutive animals to be operated. Moreover, a 95% confidence interval was obtained to achieve statistical significance. Data were compared between groups with Student’s t-test and chi-square tests.
Results
All animals tolerated the procedure well and were healthy during the entire follow up. Clinical status (temperature, blood pressure, heart rate) examined routinely on a daily basis were not different. All animals resumed normal diet within 1 day and had their first bowel movement within 2–3 days. Routine blood tests (bilirubin, ALT, AST, alkaline phosphatase, GGT, white blood cell, hemoglobin, hematocrit) obtained 2 days and 6 weeks postoperatively showed no abnormalities in either group.
Mean operative time was not different between groups (group A 64 ± 11 min versus group B 68 ± 9 min, p = ns). Intraoperative blood loss was significantly higher in group B (25 ± 5 mL versus 185 ± 9 mL, p < 0.05) (Fig. 3). An average of six 60-mm stapler cartridge firings per animal were used in each group to transect the left lateral liver lobe (p = ns). Resected specimen size was similar in both groups (average size 10 × 8 × 4 cm; average weight 0.41 kg). There was no morbidity or mortality in either group. However, two animals in group B were found to have bile collections in the previous operating field at the time of necropsy. When the livers were subjected to methylene blue injection via the common bile duct (CBD), two additional animals in group B were found to have evidence of biliary leak (a total of four of ten animals in group B versus zero of ten in group A, p < 0.05).
Fig. 3Intraoperative blood loss in groups A and B
Histopathological examination of the cut surface of the liver in group A revealed no evidence of the staple line reinforcement material, indicating that it had been totally reabsorbed. There was no bile duct damage seen and only mild mononuclear inflammation was seen in the portal areas. Subcapsular sinusoid dilation and congestion was seen with sharp demarcation between areas of dilation and absence of dilation in a pattern suggestive of fixation artifact although ischemic or anoxic damage cannot be ruled out. In group B marked fibrotic changes and damaged vascular and biliary endothelium were seen (Fig. 4 and 5).
Fig. 4Macroscopic illustration of bile duct damage at the liver’s transection site of group B in which conventional stapling technique was performedFig. 5Microscopic examination (20×) of resection line of group A (above) and group B (below)
Discussion
While new technology is rapidly expanding the realm of laparoscopic surgery to include major hepatic resections, the majority of laparoscopic hepatic operations currently performed are for diagnostic purposes [3–5]. One of the major limitations to laparoscopic liver resections is the difficulty encountered in division of substantial liver parenchyma. Commonly employed devices for standard open liver resections including the Cavitron ultrasonic surgical aspirator (CUSA) (Valleylab, Boulder, CO), and the Argon Beam coagulator (Valleylab, Boulder, CO) are available for use in laparoscopy [34–36].
Transecting the liver can be performed in laparoscopically several ways (varying from gasless procedures to hand-assisted or strict laparoscopic liver resections), each technique with its own risk of bleeding and bile leaks. In the past, the percentage of these complications has not changed considerably, remaining between 10% and 50% [9, 31]. Commonly used laparoscopic devices such as the ultrasonic scalpel or the Ligasure (Valleylab, Boulder, CO) are limited in their ability to divide liver parenchyma because of the small size of the active heating area.
The addition of various buttressing materials to stapling devices has been tried as a means to improve results and reduce complications rates in both pulmonary and gastrointestinal surgery [17–23]. We hypothesized that the addition of a buttressing material to standard laparoscopic stapling devices wouldprovide a novel technique for liver transection. One buttressing material (Seamguard®, W.L. Gore, Flagstaff, Arizona) is a porous fibrous structure composed solely of synthetic bioabsorbable poly (glycolide:trimethylene carbonate) copolymer. Degraded via a combination of hydrolytic and enzymatic pathways, the copolymer has been found to be both biocompatible and nonantigenic, with a history of use as bioabsorbable sutures, membranes, and other implantable devices. When used for staple line reinforcement, it can be expected to retain mechanical strength for 4–5 weeks and be completely absorbed by the end of 6 months [11]. The fact that this material is completely bioabsorbable should reduce concerns over possible long-term complications such as migration, erosion, calcification, and infection. In addition, this synthetic copolymer does not carry the risk of animal source contamination. The histopathological examination of the resection line of both groups showed fibrotic changes in group B in contrast to group A. This may be caused by excess mechanical stress which was avoided in group A due to addition of a staple line reinforcement.
Our results show that transaction using conventional stapling devices is inadequate but that the addition of a bioabsorbable staple line reinforcement material to standard laparoscopic stapling devices can reduce intraoperative blood loss during transection of liver parenchyma. In addition, there was a reduction in postoperative bile leaks, although in this study these leaks were not clinically significant. Bioabsorbable reinforcement improves on complications and is a viable addition to laparoscopic liver resection.

liver resections
staple line reinforcement
laparoscopic surgery
complications
bile duct leak
hemorrhage.


Sodium and potassium intake and risk of cardiovascular events and all-cause mortality: the Rotterdam Study

Background Dietary electrolytes influence blood pressure, but their effect on clinical outcomes remains to be established. We examined sodium and potassium intake in relation to cardiovascular disease (CVD) and mortality in an unselected older population. Methods A case–cohort analysis was performed in the Rotterdam Study among subjects aged 55 years and over, who were followed for 5 years. Baseline urinary samples were analyzed for sodium and potassium in 795 subjects who died, 206 with an incident myocardial infarction and 181 subjects with an incident stroke, and in 1,448 randomly selected subjects. For potassium, dietary data were additionally obtained by food-frequency questionnaire for 78% of the cohort. Results There was no consistent association of urinary sodium, potassium, or sodium/potassium ratio with CVD and all-cause mortality over the range of intakes observed in this population. Dietary potassium estimated by food frequency questionnaire, however, was associated with a lower risk of all-cause mortality in subjects initially free of CVD and hypertension (RR = 0.71 per standard deviation increase; 95% confidence interval: 0.51–1.00). We observed a significant positive association between urinary sodium/potassium ratio and all-cause mortality, but only in overweight subjects who were initially free of CVD and hypertension (RR = 1.19 (1.02–1.39) per unit). Conclusion The effect of sodium and potassium intake on CVD morbidity and mortality in Western societies remains to be established.
Introduction
Observational and experimental data support an independent, positive relationship between sodium intake and blood pressure, most clearly in hypertensive populations [1–3]. Potassium intake, on the other hand, has been inversely related to blood pressure [3, 4]. Since hypertension is a strong predictor of cardiovascular disease (CVD), especially stroke, inadequate intake of sodium and potassium is likely to be associated with increased cardiovascular morbidity and mortality [1]. Only recently, population-based studies on dietary salt intake in relation to CVD and non-cardiovascular events have received priority [5]. Alderman et al. were among the first to report an increased risk of myocardial infarction with low urinary sodium in treated hypertensive men [6]. In a subsequent analysis of NHANES I data, an inverse association of sodium intake with all-cause and cardiovascular mortality was found [7]. Estimation of salt intake by 24-h dietary recall and other methodological aspects of this analysis, however, have been criticized [8–10]. Salt intake was not significantly related to coronary or all-cause mortality in the large cohorts of the Scottish Heart Health Study [11] and the MRFIT trial [12]. A recent systematic review of 11 randomized trials showed no effect of long-term sodium reduction on overall mortality, but this meta-analysis included only 17 fatal events and should be interpreted with caution [13]. He et al. showed that high sodium intake was a strong risk factor for congestive heart failure in overweight participants of the NHANES I follow-up study [14], and also predictive for CVD and all-cause mortality in this group [15]. Similarly, in a Finnish cohort, 24-h urinary sodium excretion predicted mortality and risk of coronary heart disease only in the presence of overweight [16]. With regard to incidence of stroke, the Finnish study showed no association with urinary sodium [16]. Stroke mortality was neither predicted by dietary sodium intake in MRFIT [12]. In the WHO Cardiovascular Diseases and Alimentary Comparison (CARDIAC) Study in 24 countries, however, sodium intake appeared to be a risk factor for stroke in men [17]. As a consequence of these inconsistent findings, there is currently no consensus as to the cardiovascular risks of salt intake.
Tobian et al. demonstrated a lower risk of hemorrhagic stroke and mortality in hypertensive rats that had been given potassium supplements, an effect that was not mediated by blood pressure reduction [18]. Khaw and Barrett-Connor confirmed this independent protective effect of dietary potassium against stroke in humans [19]. Also in the CARDIAC study [17], the Cardiovascular Health Study [20] and the Nurses Health Study [21] the intake of potassium was inversely related to risk of stroke. Data on dietary potassium in relation to coronary and all-cause mortality in humans are scanty. We examined the relationship of sodium and potassium intake with cardiovascular events and all-cause mortality in the older cohort of the population-based Rotterdam Study.
Methods
The Rotterdam Study
This case–cohort analysis formed part of the Rotterdam Study, a population-based prospective study among 7,983 men and women aged 55 years and older in the Netherlands [22]. The Medical Ethics Committee of the Erasmus Medical Centre Rotterdam approved the study, and written informed consent was obtained from all participants. From August 1990 until June 1993, a trained research assistant collected data on health, medication use, lifestyle, and risk indicators for chronic diseases during a home interview. Subjects were subsequently invited at the study centre for clinical examination and assessment of diet.
Assessment of diet
Subjects were interviewed at the study centre by a trained dietician, who used a validated, semi-quantitative food frequency questionnaire [23]. The intake of total energy, alcohol, macronutrients, and a large number of micronutrients was computed using Dutch food composition tables [24]. No information on salt use was obtained and therefore data on dietary sodium were considered unreliable for this analysis.
Clinical examination
Height and body weight were measured with the subject wearing indoor clothing without shoes. The body mass index was computed as weight divided by height squared. A trained research assistant measured sitting systolic and diastolic blood pressure twice with a random-zero sphygmomanometer after a 5-min rest, and values were averaged. Hypertension was defined as a systolic blood pressure ≥160 mmHg or diastolic blood pressure ≥95 mmHg or use of antihypertensive medication. Diabetes mellitus was considered present when the subject reported antidiabetic treatment, or when random or post-load plasma glucose levels were 11.1 mmol/l or higher. CVD was considered present in case of a verified history of myocardial infarction, stroke, coronary bypass grafting, or percutaneous transluminal coronary angioplasty. Serum total and HDL cholesterol level (mmol/l) were determined by standard laboratory methods [25].
Assessment of sodium and potassium excretion
Participants collected an overnight urine sample before visiting the research centre and recorded collection times on the jar. They were not aware that samples would be used for estimation of electrolyte intake. At the research centre, volumes were recorded, urines were swirled and 100 ml samples were taken. Samples were stored in plastic tubes at −20°C for future laboratory determinations. Urinary sodium, potassium and creatinine determinations were performed by Vitros® 250 (formerly Ektachem 250) Chemistry System (Johnson & Johnson, Ortho-Clinical Diagnostics Inc., Rochester, New York). Determination of electrolytes and creatinine were based on potentiometry and enzymatic conversion, respectively. Urinary sodium and potassium concentrations (mmol/l) were standardized to 24-h values using recorded collection times and urinary volumes (ml). In addition, urinary sodium/potassium ratio was computed.
Follow-up procedures
The present analysis is based on follow-up data collected from baseline (1990–1993) until 1 January 1998. Informed consent for collection of follow-up data was obtained from 7,802 participants (98%). Information on vital status was obtained at regular intervals from municipal population registries. General practitioners (GPs) used a computerized information system to record fatal and non-fatal events in the research area (covering 85% of the cohort). In the Netherlands, the GP forms the link to all specialized medical care and clinical events are unlikely to be missed by this follow-up procedure. Research physicians verified all information on incident events using GP records and hospital discharge letters. Events were coded independently by two physicians according to the International Classification of Diseases, 10th revision (ICD-10) [26]. Coded events were reviewed by a medical expert in the field, whose judgment was considered definite in case of discrepancies.
Myocardial infarction comprised ICD-10 code I21 and stroke comprised ICD-10 codes I60-I67. Both fatal and non-fatal incident events were recorded. For the present study, only first events were considered. Events followed by death within 28 days were classified as fatal. CVD mortality comprised fatal myocardial infarction, fatal stroke, sudden cardiac death and other forms of fatal CVD (ICD-10 codes I20-I25, I46, I49, I50, I60-I67, I70-I74, and R96).
Study population
Of 7,129 subjects who visited the research centre, 6,605 adequately performed a timed overnight urine collection for which collection times were recorded and volumes exceeded 150 ml. Of those, 5,531 had blood pressure readings and these subjects were eligible for the present analysis. We followed a case–cohort approach for efficiency reasons. Assessment of urinary sodium, potassium and creatinine excretion was performed in all subjects who died (n = 795, including 217 cardiovascular deaths), and in those who experienced a myocardial infarction (n = 206) or stroke (n = 181) during follow-up. A random sample of 1,500 control subjects was taken from the eligible cohort for assessment of electrolyte excretions. Urine samples could not be retrieved for 52 of these subjects, and data on urinary sodium, potassium and creatinine were thus obtained in 1,448 subjects. Dietary data were available for 1,205 subjects (83%) of the random sample, 518 subjects (65%) who died during follow-up, 157 subjects (72%) who died from CVD, 170 subjects (83%) with an incident myocardial infarction and 147 subjects (81%) with an incident stroke. Reasons for missing dietary data were participation in the pilot phase of the Rotterdam Study, low cognitive function, and logistic reasons, as described in more detail elsewhere [23]. Of the random sub-cohort (n = 1,448), 783 subjects (54%) were free of CVD and hypertension at baseline.
Data analysis
Pearson correlations were computed to examine inter-relationships between urinary and dietary measures of electrolyte intake and associations with total energy intake.
The association of urinary and dietary electrolytes with incident myocardial infarction, incident stroke, cardiovascular mortality and all-cause mortality was evaluated in a case–cohort design with standard Cox proportional-hazards models with modification of the standard errors based on robust variance estimates [27, 28]. We used the method according to Barlow in which the random cohort is weighted by the inverse of the sampling fraction from the source population. Members of the random cohort are included from baseline until failure or censoring, whereas cases outside the cohort are included at the time of their event. For the Cox models we used Proc MI and Proc MIanalyze, in conjunction with Proc Phreg (SAS 8.2).
Relative risks (RR) with 95% confidence intervals (95%-CI) were computed per 1 standard deviation increase in urinary sodium (mmol/24 h), urinary potassium (mmol/24 h) and dietary potassium intake (mg/day), and per 1 unit increase in urinary sodium/potassium ratio. Two-sided P-values below 0.05 were considered statistically significant. Adjustment was made for age, sex and, in urinary analyses, for 24-h urinary creatinine excretion (model 1). In a second analysis (model 2), additional adjustment was made for body mass index (kg/m2), smoking status (current, past, or never), diabetes mellitus (yes/no), use of diuretics (yes/no), and highest completed education (three categories). In a third analysis (model 3), dietary confounders were additionally adjusted for, i.e. daily intake of total energy (kJ), alcohol (g), calcium (g), and saturated fat (g). In the analysis for urinary sodium we additionally included urinary potassium in this model, and vice versa.
Analyses were repeated after exclusion of subjects with a history of CVD or hypertension to avoid biased risk estimates due to intentional dietary changes. Within this sub-cohort, a predefined stratified analysis of urinary sodium and urinary sodium/potassium ratio with cardiovascular and all-cause mortality was performed in subjects with a high body mass index (i.e., ≥25 kg/m2), using model 3.
Also in the sub-cohort free of CVD and hypertension, the distribution of 24-h urinary sodium excretion was divided into quartiles to be able to examine the relationship with all-cause mortality at extreme intakes. Quartiles of urinary sodium (cut-off levels: 66, 105 and 151 mmol/24 h) were entered categorically into the fully adjusted model (model 3), using the lower quartile as the reference.
Results
The study had a median follow-up of 5.5 years. Baseline characteristics of the study population are shown in Table 1. Randomly selected controls (n = 1,448) were expectedly healthier at baseline than cases, as indicated by a lower prevalence of hypertension, diabetes, and CVD.
Table 1Baseline characteristics of the study populationRandom sampleCasesIncident MIIncident strokeCVD mortalityAll-cause mortalityNo. of subjects1,448206181217795In random sample (%)31312829Age (year)69.2 (8.7)71.0 (8.0)74.0 (8.5)76.8 (8.4)76.9 (8.9)Men (%)4162455149Body mass index (kg/m2)26.4 (3.8)26.3 (3.4)26.0 (3.3)26.2 (3.8)25.7 (3.8)Smoking status (%)a    Current2329282326    Former4148424740    Never3623292935Alcohol use (%)8174807173Educational level (%)a,b    Low5861606566    Intermediate3231343028    High108656Serum cholesterol (mmol/l)    Total6.6 (1.2)6.3 (1.3)6.5 (1.2)6.6 (1.4)6.3 (1.3)    HDL1.4 (0.4)1.3 (0.4)1.3 (0.4)1.2 (0.4)1.3 (0.4)Blood pressure (mmHg)    Systolic140 (22)145 (23)149 (24)146 (25)145 (25)    Diastolic74 (11)74 (12)75 (13)73 (13)73 (14)Hypertension (%)c3744535547Diabetes mellitus (%)d1021222621History of CVD (%)e1735173928Values are means with standard deviations, or percentages; CVD, cardiovascular disease; MI, myocardial infarctionaValues not always add up to 100% due to roundingbHighest achieved level of education; low, primary education, or less; intermediate, secondary general or vocational education; high, higher vocational education, universitycSystolic blood pressure ≥160 mmHg or diastolic blood pressure ≥95 mmHg or use of antihypertensive medicationdPlasma glucose ≥11.1 mmol/l or treated with oral antidiabetes medication or insulineVerified history of cardiovascular disease, i.e. myocardial infarction, stroke, coronary bypass-grafting, or percutaneous transluminal coronary angioplasty
Baseline urinary excretions and dietary intakes are presented in Table 2. In the random sample, 24-h urinary sodium excretion estimated from overnight urine collection was 117 mmol (i.e., 2.7 g/day, which corresponds to a NaCl intake of 6.8 g/day). Urinary potassium excretion was 45 mmol/24 h (1.8 g/day), which was half the amount estimated by food frequency questionnaire (3.6 g/day). The correlation between urinary and dietary potassium was 0.21 (P < 0.001).
Table 2Baseline urinary excretions and dietary intakes of Dutch men and women aged 55 years and over: The Rotterdam StudyRandom subcohortCasesIncident MIIncident strokeCVD mortalityAll-cause mortalityUrinary excretionaVolume (l/24 h)1.4 (0.6)1.4 (0.6)1.4 (0.6)1.3 (0.6)1.3 (0.6)Sodium (mmol/24 h)117 (69)124 (68)115 (72)99 (61)107 (66)Potassium (mmol/24 h)45 (22)47 (22)45 (23)44 (24)44 (22)Sodium/potassium2.8 (1.5)2.7 (1.3)2.7 (1.3)2.5 (1.4)2.6 (1.6)Creatinine (mmol/24 h)9.2 (4.9)9.8 (4.7)8.4 (4.4)8.1 (4.7)8.1 (4.4)Sodium/creatinine13.8 (6.6)13.6 (6.1)14.6 (7.1)14.0 (8.0)14.8 (7.9)Potassium/creatinine5.4 (2.2)5.3 (2.1)5.8 (2.1)6.1 (2.6)6.1 (2.5)Dietary intakebTotal energy (mJ/day)8.3 (2.1)8.6 (2.2)8.4 (2.2)8.3 (2.0)8.5 (2.2)Saturated fat (g/day)32 (12)34 (13)34 (13)33 (13)34 (12)Calcium (g/day)1.1 (0.4)1.1 (0.4)1.1 (0.4)1.1 (0.5)1.1 (0.4)Sodium (g/day)c2.2 (0.7)2.3 (0.6)2.2 (0.6)2.2 (0.7)2.2 (0.7)Potassium (g/day)3.6 (0.8)3.7 (0.8)3.6 (0.8)3.6 (0.9)3.6 (0.9)Values are means with standard deviations; CVD, cardiovascular disease; MI, myocardial infarctionaBased on one timed overnight urine samplebDietary data were available for 1,205 subjects of the random sample (83%), 170 MI cases (83%), 147 stroke cases (81%), 157 CVD deaths (72%), and 518 deaths from any cause (65%)cOnly from foods, discretionary sources not included
RR for cardiovascular events and all-cause mortality per 1-SD increase in 24-h urinary sodium are presented in Table 3. Urinary sodium was not significantly associated with incident myocardial infarction, incident stroke, or overall mortality. For CVD mortality, however, a borderline significant inverse association was observed (RR = 0.77 (0.60–1.01) per 1-SD, model 3) but the relationship was attenuated after excluding subjects with a history of CVD or hypertension (RR = 0.83 (0.47–1.44) per 1-SD, model 3). In subjects initially free of CVD, the risk of all-cause mortality was also examined across quartiles of 24-h urinary sodium (median values: 45, 87, 125 and 190 mmol, respectively). RR in consecutive quartiles, using the lower quartile as the reference, were 0.80 (0.43–1.49), 0.66 (0.34–1.27) and 0.98 (0.54–1.78), respectively (model 3). In a subgroup analysis of CVD free subjects with a body mass index ≥25 kg/m2, the association of urinary sodium with CVD mortality or all-cause mortality was neither statistically significant (RR = 0.91 (0.44–1.89) and RR = 1.19 (0.86–1.66) per 1-SD, respectively; model 3).
Table 3Relative risk of urinary sodium with cardiovascular events and all-cause mortality in Dutch men and women aged 55 years and overAll subjectsaSubjects initially free of CVD and hypertensionaIncident MIRR, model 1b1.13 (0.95–1.34)1.04 (0.75–1.43)RR, model 2c1.16 (0.98–1.39)1.07 (0.77–1.50)RR, model 3d1.19 (0.97–1.46)1.14 (0.77–1.69)Incident strokeRR, model 11.09 (0.89–1.33)1.16 (0.84–1.61)RR, model 21.09 (0.87–1.35)1.15 (0.81–1.62)RR, model 31.08 (0.80–1.46)1.02 (0.66–1.58)CVD mortalityeRR, model 10.74 (0.60–0.91)0.84 (0.59–1.22)RR, model 20.83 (0.68–1.02)0.95 (0.66–1.39)RR, model 30.77 (0.60–1.01)0.83 (0.47–1.44)All-cause mortalityRR, model 10.90 (0.81–1.02)1.00 (0.83–1.20)RR, model 20.96 (0.84–1.09)1.10 (0.91–1.34)RR, model 30.95 (0.81–1.12)1.12 (0.86–1.46)RR, Relative risk with 95% confidence interval per standard deviation increase in urinary sodium (mmol/24 h), obtained by Cox proportional hazard analysisaNumber of cases and subjects in random sample given in Table 1bAjusted for age, sex and (for urinary sodium) 24-h urinary creatinine excretioncAs model 1, with additional adjustment for body mass index, smoking status, diabetes, use of diuretics, highest completed educationdAs model 2, with additional adjustment for daily intake of total energy, alcohol, calcium, saturated fat and 24-h urinary potassium excretioneCardiovascular mortality comprises fatal myocardial infarction, fatal stroke, sudden cardiac death and other forms of fatal CVD
Findings for potassium are presented in Table 4. Urinary potassium tended to be positively associated with incident CVD events or mortality, especially in subjects who were initially free of CVD and hypertension. After full adjustment for confounders (model 3), however, none of these associations were statistically significant. Urinary potassium did neither predict all-cause mortality. For dietary potassium, similar results were obtained except for risk of all-cause mortality that was significantly reduced both in the entire cohort (RR = 0.78 (0.65–0.94 per 1-SD) and in subjects initially free of CVD and hypertension (RR = 0.71 (0.51–1.00), model 3).
Table 4Relationship of urinary and dietary potassium with cardiovascular events and all-cause mortality in Dutch men and women aged 55 years and overAll subjectsaSubjects initially free of CVD and hypertensionaUrinary excretion (mmol/24 h)Dietary intake (mg/day)Urinary excretion (mmol/24 h)Dietary intake (mg/day)Incident MIRR, model 1b1.10 (0.89–1.35)0.98 (0.85–1.13)1.15 (0.84–1.59)1.14 (0.85–1.54)RR, model 2c1.16 (0.94–1.43)0.94 (0.81–1.09)1.25 (0.94–1.74)1.07 (0.78–1.46)RR, model 3d1.11 (0.87–1.43)0.90 (0.65–1.24)1.22 (0.79–1.87)1.32 (0.65–2.67)Incident strokeRR, model 11.09 (0.87–1.36)0.99 (0.84–1.17)1.12 (0.79–1.60)1.07 (0.79–1.43)RR, model 21.12 (0.89–1.42)0.99 (0.84–1.16)1.15 (0.77–1.71)1.20 (0.86–1.68)RR, model 31.17 (0.86–1.58)1.02 (0.71–1.46)1.11 (0.61–2.04)1.06 (0.50–2.29)CVD mortalityeRR, model 11.13 (0.90–1.41)0.97 (0.82–1.14)1.63 (1.14–2.33)1.23 (0.83–1.84)RR, model 21.14 (0.92–1.42)0.95 (0.81–1.12)1.66 (1.08–2.56)1.19 (0.78–1.83)RR, model 31.23 (0.94–1.60)0.97 (0.72–1.31)1.45 (0.84–2.54)1.43 (0.67–3.03)All-cause mortalityRR, model 11.04 (0.91–1.18)0.91 (0.82–1.01)1.06 (0.88–1.28)0.95 (0.78–1.17)RR, model 21.06 (0.86–1.31)0.89 (0.80–0.99)1.06 (0.86–1.31)0.90 (0.73–1.12)RR, model 31.08 (0.91–1.28)0.78 (0.65–0.94)0.95 (0.71–1.26)0.71 (0.51–1.00)RR, Relative risk with 95% confidence interval per standard deviation increase in urinary or dietary potassium, obtained by Cox proportional hazard analysisaNumber of cases and subjects in random sample given in Table 1bAjusted for age, sex and (for urinary potassium) 24-h urinary creatinine excretioncAs model 1, with additional adjustment for body mass index, smoking status, diabetes, use of diuretics and highest completed educationdAs model 2, with additional adjustment for daily intake of total energy, alcohol, calcium, saturated fat and 24-h urinary sodium excretioneCardiovascular mortality comprises fatal myocardial infarction, fatal stroke, sudden cardiac death and other forms of fatal CVD
Data for urinary sodium/potassium ratio (Table 5) showed no relationship with CVD events and mortality. When restricting this analysis to CVD free subjects with a body mass index ≥25 kg/m2, urinary sodium/potassium ratio was significantly associated with all-cause mortality (RR = 1.19 (1.02–1.39) per unit, model 3), but not with CVD mortality (RR = 0.86 (0.60–1.25)).
Table 5Relationship of urinary sodium/potassium ratio with cardiovascular events and all-cause mortality in Dutch men and women aged 55 years and overAll subjectsaSubjects initially free of CVD and hypertensionaIncident MIRR, model 1b1.03 (0.93–1.14)0.92 (0.76–1.13)RR, model 2c1.02 (0.92–1.13)0.90 (0.73–1.10)RR, model 3d1.04 (0.93–1.17)0.91 (0.72–1.16)Incident strokeRR, model 11.01 (0.89–1.13)1.01 (0.83–1.23)RR, model 20.99 (0.86–1.13)0.99 (0.77–1.20)RR, model 30.99 (0.83–1.18)0.90 (0.66–1.22)CVD mortalityeRR, model 10.88 (0.77–1.01)0.85 (0.65–1.11)RR, model 20.93 (0.81–1.06)0.86 (0.66–1.13)RR, model 30.92 (0.80–1.07)0.91 (0.65–1.27)All-cause mortalityRR, model 10.99 (0.91–1.06)1.04 (0.91–1.18)RR, model 20.99 (0.92–1.08)1.06 (0.93–1.22)RR, model 31.01 (0.91–1.12)1.13 (0.93–1.36)RR, Relative risk with 95% confidence interval per 1 unit increase in urinary sodium/potassium ratio, obtained by Cox proportional hazard analysisaNumber of cases and subjects in random sample given in Table 1bAdjusted for age, sex and 24-h urinary creatinine excretioncAs model 1, with additional adjustment for body mass index, smoking status, diabetes, use of diuretics and highest completed educationdAs model 2, with additional adjustment for daily intake of total energy, alcohol, calcium, and saturated fateCardiovascular mortality comprises fatal myocardial infarction, fatal stroke, sudden cardiac death and other forms of fatal CVD
Discussion
In an unselected population of older Dutch subjects we found no consistent association of urinary sodium and potassium with CVD events or mortality. Dietary potassium estimated by food frequency questionnaire, however, was associated with a lower risk of all-cause mortality. Urinary sodium/potassium ratio was positively associated with mortality risk, but only in overweight subjects without CVD and hypertension at baseline.
Electrolyte intake was assessed from one overnight urine collection, which provides a crude estimate of short-term intake [29, 30]. Luft et al. examined the utility of nocturnal sodium excretion under controlled intake conditions, in which daily sodium intake was randomly varied [31]. In that study, on a randomly selected day, both 24-h and nocturnal sodium excretion estimated the daily intake reasonably well. Nevertheless, it is likely that misclassification has attenuated the relationships with CVD events and mortality in our study. Incomplete urine collection was partly adjusted for by adding urinary creatinine excretion to the multivariate models. In addition, we examined the urinary sodium/potassium ratio, which is less influenced by incomplete urine collection. To exclude bias due to dietary changes, we repeated all analyses in a subgroup without CVD or hypertension at baseline.
Salt intake was not consistently related to CVD or mortality in our study. An explanation for the absence of a positive relationship, apart from regression dilution bias, may be the relatively narrow range of salt intake in the Netherlands and the lack of contrast in exposure within a single population. An increased risk of mortality was observed for high salt intake in overweight Finnish subjects with 24-h urinary excretions close to 200 mmol (RR = 1.56 per 100 mmol) [16]. However, this could not be confirmed in our analysis of quartiles of sodium intake in relation to overall mortality (RR = 0.98 in CVD free subjects with median sodium excretion of 190 mmol/24 h). The absence of a relationship between salt intake and mortality in our study corroborates the findings from the large Scottish Heart Health Study among almost 12,000 middle-aged subjects with 24-h urine samples [11]. Follow-up data of the MRFIT trial neither showed a relationship between dietary sodium intake estimated by 24-h recall and cardiovascular events or mortality [12]. However, other prospective epidemiological studies do suggest that sodium intake is related to morbidity and mortality [6, 7, 15, 16], although this may be confined to specific subgroups with overweight, hypertension or high salt intake. In overweight subjects, we did find a positive relationship between urinary sodium/potassium ratio and overall mortality (19% increase in risk per unit change in sodium/potassium ratio).
A protective effect of potassium intake against stroke, as previously reported [19–21], could not be confirmed by our data. We neither observed an association between potassium intake and coronary events. Long-term rather than short-term intake may be relevant and therefore we estimated habitual potassium intake during the preceding year by food frequency questionnaire. Mortality risk was reduced by 29% per 1-SD increase in dietary potassium, although only in subjects initially free of CVD and hypertension. Except for misclassification, although less likely than for sodium, we have no explanation for the absent relationship between potassium intake and CVD events. In the Scottish Heart Health Study, 24-h urinary potassium excretion was inversely related to all-cause mortality and coronary events [11]. Data on potassium intake in relation to mortality, however, are sparse and more prospective population-based studies are needed to draw conclusions. Preferably, the effect of dietary potassium on CVD should be examined in a randomized trial.
Prolonged differences in blood pressure of 5 mmHg may result in a one-third reduction in stroke and one-fifth reduction in coronary events [32]. Meta-analysis of randomized controlled trials showed that sodium reduction around 2 g per day could lower blood pressure by 2–3 mmHg, with the effect being twice as large in hypertensives [33]. The World Health Organization recommends that people should consume less than 5 g of salt (i.e. 2 g of sodium) per day in order to prevent CVD [34]. From this and other epidemiological studies we conclude that effect of dietary salt on clinical cardiovascular endpoints and overall mortality within the range of intake commonly observed in Western countries has not yet been established. More research is needed to settle the discussion regarding this major public health issue.

sodium
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Diagnosis of acute myeloid leukemia according to the WHO classification in the Japan Adult Leukemia Study Group AML-97 protocol

We reviewed and categorized 638 of 809 patients who were registered in the Japan Adult Leukemia Study Group acute myeloid leukemia (AML)-97 protocol using morphological means. Patients with the M3 subtype were excluded from the study group. According to the WHO classification, 171 patients (26.8%) had AML with recurrent genetic abnormalities, 133 (20.8%) had AML with multilineage dysplasia (MLD), 331 (51.9%) had AML not otherwise categorized, and 3 (0.5%) had acute leukemia of ambiguous lineage. The platelet count was higher and the rate of myeloperoxidase (MPO)-positive blasts was lower in AML with MLD than in the other WHO categories. The outcome was significantly better in patients with high (≥50%) than with low (<50%) ratios of MPO-positive blasts (P < 0.01). The 5-year survival rates for patients with favorable, intermediate, and adverse karyotypes were 63.4, 39.1, and 0.0%, respectively, and 35.5% for those with 11q23 abnormalities (P < 0.0001). Overall survival (OS) did not significantly differ between nine patients with t(9;11) and 23 with other 11q23 abnormalities (P = 0.22). Our results confirmed that the cytogenetic profile, MLD phenotype, and MPO-positivity of blasts are associated with survival in patients with AML, and showed that each category had the characteristics of the WHO classification such as incidence, clinical features, and OS.
Introduction
The French-American-British (FAB) classification of acute myeloid leukemia (AML), based on morphological and cytochemical findings, was established in 1976 and has since become the standard classification [1, 2]. However, specific chromosomal and genetic abnormalities that have been extracted from analyses of prognostic factors for AML are recognized as important in selecting treatment strategies and are reflected in the AML classification as factors that are required to establish the disease entity [3]. The 1999 World Health Organization (WHO) classification includes morphological, immunological, cytogenetic, genetic, and clinical features [4–6]. The WHO and FAB classifications differ in several aspects. The blast threshold required for a diagnosis of AML was reduced from 30 to 20%, and new AML categories have been added for cytogenetic abnormalities, the presence of multilineage dysplasia (MLD), as well as a history of chemotherapy and subtypes for acute basophilic leukemia, acute panmyelosis with myelofibrosis, and myeloid sarcoma. The WHO classification comprises more subtypes and is more comprehensive than the FAB classification.
Cytogenetic features are important prognostic factors in AML [3, 7–12]. However, 11q23 abnormalities have not yet been established as a cytogenetic risk classification. Over 30 partner genes with 11q23 abnormalities have been described, and some reports indicate that patients with t(9;11) have a relatively more favorable prognosis than those with other partner chromosomes/partner genes [13–16].
In the present study, we reviewed stained smears of blood and bone marrow from patients who were registered in the Japan Adult Leukemia Study Group (JALSG) AML-97 trial, and classified them into FAB subtypes and WHO categories. We also evaluated their survival on the basis of the WHO classification, the myeloperoxidase (MPO)-positivity of blasts, and cytogenetic findings including 11q23 abnormalities.
Patients and methods
Patients
Between December 1997 and July 2001, 809 patients aged from 15 to 66 years with untreated AML (excluding M3) were registered from 103 institutions in the AML-97 trial of the JALSG. The patients were diagnosed with AML according to the FAB criteria at each institution. Patients with a history of MDS, hematological abnormalities before the diagnosis of AML, or a history of chemotherapy were not eligible for the AML-97 trial.
Treatment strategies
Details of the JALSG AML-97 treatment protocol are described elsewhere [17]. In brief, all patients underwent induction therapy consisting of idarubicin (3 days) and Ara-C (7 days). Patients who achieved complete remission were randomized into one of two arms of consolidation chemotherapy alone or in combination with maintenance chemotherapy. Patients who were placed into intermediate/poor risk groups according to the JALSG scoring system [17] and who had an HLA-identical sibling (≤50 years old) were simultaneously assigned to receive allogeneic hematopoietic stem cell transplantation during their first remission.
Morphologic and cytochemical analyses
Peripheral blood and bone marrow smears from registered patients were sent to Nagasaki University for staining with May-Giemsa, MPO, and esterase, and the diagnosis was then reevaluated by the Central Review Committee for Morphological Diagnosis. Patients were subsequently categorized according to the FAB and WHO classifications. Dyserythropoietic features were defined as >50% dysplastic features in at least 25 erythroblasts and dysgranulopoietic features including ≥3 neutrophils with hyposegmented nuclei (pseudo-Pelger–Heut anomaly), and hypogranular or agranular neutrophils (>50% of ≥10 neutrophils). Dysmegakaryopoietic features were defined as ≥3 megakaryocytes that were micronuclear, multiseparate nuclear, or large mononuclear [18].
We assessed the ratios (%) of MPO-positive blasts on MPO-stained bone marrow smears using the diaminobenzidine method [19].
Cytogenetic analysis
Cytogenetic analysis was performed at either laboratories in participating hospitals or authorized commercial laboratories. The karyotypes of leukemic cells were collected through the JALSG AML-97 case report forms and reviewed by the Central Review Committee for Karyotyping. The patients were classified into favorable, intermediate, or adverse risk groups based on karyotypes according to results of the Medical Research Council (MRC) AML 10 trial [3]. The favorable risk group included patients with t(8;21) and inv(16), whether alone or in combination with other abnormalities. The intermediate risk group included those with a normal karyotype and other abnormalities that were not classified as either favorable or adverse. The adverse risk group included patients with a complex karyotype with four or more numerical or structural aberrations, −5, deletion (5q), and −7, whether alone or in combination with intermediate risk or other adverse risk abnormalities.
Statistical analysis
The overall survival (OS) for all patients was defined as the interval from the date of diagnosis to that of death. We applied the Kaplan–Meier method to estimate OS and 5-year survival. We compared survival rates between groups using the log-rank test (Stat View J 5.0). Differences were examined by the Chi-square test using Excel software. All P-values are two-sided, and values <0.05 were considered significant.
Results
Patient characteristics
Of the 809 registered patients, 638 were consistent with the WHO classification. Data were incomplete for 10 of the 638 patients. Table 1 lists the characteristics of the patients. The median age of all 638 patients (390 males and 248 females) was 45 years (range 15–66 years). The median values of WBC, hemoglobin (Hb), platelets, and the ratio of blasts in the bone marrow were 13.7 × 109/l, 8.3 g/dl, 52.0 × 109/l, and 56.0%, respectively.
Table 1Patient characteristicsAge (year)45 (15–66)Male/female390/248WBC count (×109/l)13.7 (0.4–709)Hemoglobin (g/dl)8.3 (3.8–17.2)Platelet count (×109/l)52 (0–890)Bone marrow blasts (%)56 (6–99)Values are presented as the median (range)
WBC white blood cell
FAB classification
Table 2 shows the FAB classification of the 638 patients. Most were classified as M2 (n = 261; 40.9%), followed by M4 (n = 148; 23.2%), and M1 (n = 109; 17.1%) with M0, M4Eo, M5a, M5b, M6, M7, and acute leukemia of ambiguous lineage comprising the remainder in that order.
Table 2Number of patients according to the FAB classificationSubtypeDescriptionNo. of patients%M0Minimally differentiated acute myeloid leukemia (AML)304.7M1AML without maturation10917.1M2AML with maturation26140.9M4Acute myelomonocytic leukemia (AMMoL)14823.2M4EoAMMoL with eosinophils233.6M5aAcute monoblastic leukemia193.0M5bAcute monocytic leukemia243.8M6Acute erythroleukemia162.5M7Acute megakaryoblastic leukemia50.8Acute leukemia of ambiguous lineage30.5Total638100
WHO classification and clinical characteristics
Table 3 shows the patients categorized according to the WHO classification. The first category of AML with recurrent genetic abnormalities accounted for 171 patients (26.8%), 133 (20.8%) were in the second category of AML with MLD, 331 (51.9%) were in the fourth category of AML not otherwise categorized, and 3 (0.5%) were categorized as having acute leukemia of ambiguous lineage. Most patients in the second category were identical to those with a de novo MLD phenotype. We found that 144 patients diagnosed with the MLD phenotype comprised 133 (92.4%) in the second category, 10 (7.0%) with 11q23 abnormalities, and 1 (0.7%) with acute leukemia of ambiguous lineage. Figure 1 shows the OS of each category. The 5-year survival rates of the first, second, and fourth categories were 58.2, 22.5, and 40.9% (P < 0.0001), respectively.
Table 3Number of patients according to the WHO classificationCategory and subtypeNo. of patients%I. AML with recurrent genetic abnormalities17126.8
t(8;21)(q22;q22);(AML1/ETO)11317.7 inv(16)(p13;q22) or t(16;16)(p13;q22);(CBFβ/MYH11)264.1
t(15;17)(q22;q12)(PML/RARα)–– 11q23(MLL)abnormalities325.0II. AML with multilineage dysplasia13320.8 Following MDS–– Without antecedent MDS13320.8III. AML and MDS, therapy-related–– Alkylating agent-related–– Topoisomerase type II inhibitor-related–– Other types––IV. AML not otherwise categorized33151.9 AML, minimally differentiated253.9 AML without maturation9915.5 AML with maturation10816.9 Acute myelomonocytic leukemia (AMMoL)639.9 AMMoL with eosinophilia50.8 Acute monoblastic leukemia81.3 Acute monocytic leukemia162.5 Acute erythroid leukemia60.9 Acute megakaryoblastic leukemia10.2Acute leukemia of ambiguous lineage30.5Total638100
Fig.1Overall survival of patients categorized according to the WHO classification
Table 4 compares the clinical features among the WHO categories. The mean values of platelets, WBC, Hb, and the ratio (%) of blasts in bone marrow and of MPO-positive blasts significantly differed, whereas age did not significantly differ. Patients in the second category had a higher platelet count (111.0 × 109/l), whereas those with 11q23 abnormalities had a lower count (38.3 × 109/l) compared with those of other subtypes.
Table 4Comparison of clinical findings of patients diagnosed according to the WHO classificationCategoryPlatelets (×109/l ± SE)WBC (×109/l ± SE)Hb (g/dl ± SE)Age (year ± SE)Blasts in bone marrow (%±SE)MPO positivity of blasts (%±SE)I
t(8;21)76.7 ± 56.43 (113)a
1.4 ± 0.6 (113)7.8 ± 0.2 (113)41.6 ± 1.3 (113)49.9 ± 2.0 (113)93.3 ± 3.3 (108)inv(16)57.8 ± 52.03 (26)6.6 ± 1.2 (26)9.2 ± 0.5 (26)44.5 ± 2.6 (26)50.5 ± 4.1 (26)66.9 ± 6.7 (26)11q2338.3 ± 30.8 (32)4.3 ± 1.1 (32)8.9 ± 0.4 (32)41.6 ± 2.4 (32)56.3 ± 3.7 (32)43.6 ± 6.1 (32)II111.0 ± 121.5 (133)3.0 ± 0.5 (133)8.3 ± 0.2 (133)44.2 ± 1.2 (133)48.0 ± 1.8 (133)34.0 ± 3.1 (126)IV72.8 ± 91.7 (330)5.1 ± 0.3 (331)8.8 ± 0.1 (330)43.8 ± 0.7 (331)65.7 ± 1.2 (328)53.7 ± 1.9 (312)
P < 0.0001
P < 0.0001
P = 0.0004
P = 0.4077
P < 0.0001
P < 0.0001
SE standard error, WBC white blood cell, MPO myeloperoxidase, Hb hemoglobin
aNumber of patients
The WBC count of patients with t(8;21) was 1.4 × 109/l and lower than in other subtypes. The MPO-positive rate of blasts among patients with t(8;21) was higher (93.3%) and that of patients in the second category was lower (34.0%), than in other subtypes. All patients were grouped as high- or low-MPO according to ≥50% or <50% of MPO-positive blasts, respectively. A total of 339 patients (53.1%) were classified as high-MPO, 268 (42.0%) as low-MPO, and the MPO status of blasts could not be assessed in 31 (4.9%). Figure 2 shows the OS of patients with high- or low-MPO. The 5-year survival rate for patients with high or low-MPO was 50.7 and 29.6%, respectively (P < 0.0001).
Fig. 2Overall survival of patients with high or low MPO-positive blasts
Cytogenetics
All 638 patients were classified into favorable (n = 139; 21.8%), intermediate (n = 413; 64.7%), and adverse (n = 54; 8.5%) cytogenetic risk groups (Table 5). Figure 3 shows the OS according to this stratification. The 5-year survival rates were 63.4, 39.3, and 0.0% in the favorable, intermediate (except for those with 11q23 abnormalities), and adverse risk groups, respectively, and 35.5% in the group with 11q23 abnormalities (P < 0.0001).
Table 5Distribution of patients classified by cytogenetic riskCytogenetic risk groupNo. of patients%Favorable13921.8
t(8;21)11317.7 inv(16)264.1Intermediate41364.7 Normal karyotype26741.8 11q23325.0 Ph(+)71.1
t(7;11)(p15;p15)40.6
t(6;9)40.6 Other13120.5Adverse548.5 Complex416.4 −720.3 abn350.8 del5q20.3 −510.2 Other30.5Total638100.0
Fig. 3Overall survival of patients stratified according to cytogenetic risk groups. Significant differences were observed between patients with a favorable, intermediate (except 11q23), and adverse karyotype (P < 0.0001)
The numbers of patients with or without MLD and high- or low-MPO in each cytogenetic risk group are listed in Table 6. None of those with the MLD phenotype were classified into the favorable risk group, while 129 (89.6%) and 15 (10.4%) of 144 patients with MLD were classified into intermediate or adverse risk groups, respectively. Only 15 patients (4.4%) in the high-MPO group were classified as having an adverse risk, while 11 (4.1%) in the low-MPO group were included in the favorable risk group.
Table 6Relationship between cytogenetic risk groups and MLD phenotype or MPO-positive rates of blastsFavorable n = 139Intermediate n = 445Adverse n = 54TotalMLD +0129 (89.5%)15 (10.4%)144 −138 (28.2%)292 (59.6%)38 (7.8%)490 Unknown1214MPO High123 (36.3%)201 (59.3%)15 (4.4%)339 Low11 (4.1%)221 (82.5%)36 (13.4%)268 Unknown523331High- and low-MPO indicates a percentage of myeloperoxidase positive blasts ≥50 or <50%, respectively
MLD multilineage dysplasia
The 32 patients with 11q23 abnormalities comprised 11 (34.4%) with t(11;19), 9 (28.1%) with t(9;11), 5 (15.6%) with del(11)(q23), 4 (12.5%) with t(6;11), and 3 (9.4%) with t(11;17). Figure 4 shows the OS of the intermediate risk group. The 5-year survival rate was 44.0% in patients with a normal karyotype, 35.5% in those with 11q23 abnormalities, and 30.6% in other patients including those with t(7;11), t(6;9), and Ph(+) abnormalities, respectively (P = 0.033).
Fig. 4Overall survival of patients with subtypes of intermediate cytogenetic risk. Significant differences were observed between patients with a normal karyotype and those with 11q23 abnormalities (P = 0.033)
Table 7 shows the relationship between t(9;11) (n = 9) and other 11q23 abnormalities (n = 23). More patients with low-MPO, without MLD, or with the FAB M5 subtype were found in the group with t(9;11) than with other 11q23 abnormalities. The survival rates between the two groups did not significantly differ (P = 0.22, data not shown).
Table 7Comparison of t(9;11) and other 11q23 abnormalitiesNo. of patientsAuerMPO*MLD*FABMedian age (year)Median survival (day)+−HighLow+−M1M2M4M4EoM5a**M5b
t(9;11)9091809003060391031.00Other 11q232351813101013131312348520.00Total3252714181022131618344.5531.5High- and low-MPO indicates a percentage of myeloperoxidase-positive blasts ≥50 or <50%, respectively
MLD multilineage dysplasia* P < 0.05, ** P < 0.01
Discussion
We attempted to classify selected patients who were reviewed morphologically and had available chromosomal data according to the WHO system. However, our series had some limitations in terms of analysis and patient selection. Although we obtained chromosomal data, genetic data were not available. Patients who were diagnosed with AML M3 or who had t(15;17), a history of MDS, or preceding hematological abnormalities, or who had previously undergone chemotherapy, were not eligible for the present study. However, multicenter trials might have some advantages in diagnosing AML according to the WHO classification, because morphological diagnoses and karyotypes are reviewed by the corresponding institutional committees.
The incidence of each category of the WHO classification was similar to those in several reports when patients with t(15;17) and therapy-related AML were excluded [20–22]. We and several others have shown that approximately 30% of patients have recurrent genetic abnormalities. Multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) assays have recently been applied to analyze cytogenetic abnormalities [21, 23, 24]. This method might cause the frequency of the first WHO category to increase. Thus, the multiplex RT-PCR assay might have to be incorporated into the WHO system. The JALSG has started a cohort study in which all AML patients in participating hospitals are registered and analyzed according to the WHO classification. That study should clarify the real ratios of the AML subtypes in the WHO classification.
Few reports have included clinical data with the WHO classification. We found that the platelet count was higher among patients in the second category than in other categories. This supports our previous finding that the platelet count is higher in patients with AML accompanied by the MLD phenotype [25]. Among patients with MLD, none were in the favorable risk group, whereas the intermediate or adverse risk ratios among these patients were 89.6 and 10.4%, respectively. These differences might influence the finding that OS was better among patients without than with MLD (P = 0.0002, data not shown). Previous studies have also associated the MLD phenotype with a poorer outcome, although MLD is not significantly prognostic on multivariate analysis [18, 26], and a German group showed that dysplastic features correlate with adverse karyotypes [26]. Furthermore, patients in the second category had a lower MPO-positive rate of blasts, whereas those with t(8;21) had a higher rate. Patients with high- and low-MPO were more frequently observed in the favorable and adverse risk groups, respectively. Multivariate analysis has shown that MPO is a significant factor affecting OS [19]. We did not assess prognostic factors by multivariate analysis here because the main theme of this study was to categorize patients according to the WHO classification, and we have already examined these in a previous series [18, 19].
Several studies have demonstrated the impact of specific cytogenetic abnormalities on survival in AML [3, 7–12, 20–22]. The cytogenetic risk groups stratified the AML patients in the present study according to the MRC system, as in these reports [3]. Therefore, we confirmed the clinical usefulness of cytogenetics as the first category of the WHO classification. We found that 32 patients had 11q23 abnormalities. The MRC system revealed that de novo and secondary AML patients with 11q23 abnormalities had an intermediate outcome with an OS rate of 45% at 5 years (n = 60; median age, 17 years) in a younger cohort [3] and an OS rate of 0% at 5 years (n = 11; median age 64 years) in an elderly cohort [7]. In contrast, SWOG/ECOG trials including adult de novo AML patients (age, 16–55 years) assigned those with 11q abnormalities to the unfavorable cytogenetic subgroup [8]. Our data showed that patients with 11q23 abnormalities have an intermediate rather than adverse outcome. The prognostic effect of 11q23 abnormalities might depend on the partner gene. Several studies have shown that 11q23 abnormalities with t(6;11) and t(10;11) are associated with a poor prognosis, whereas t(9;11) is associated with a superior OS and such patients might respond well to intensive treatment, especially when the chemotherapy regimen includes high-dose cytarabine [15, 27–30]. The CALGB study has shown that the median OS of 13.2 months among 23 patients with t(9;11) was significantly longer than the 7.7 months among 24 patients with other 11q23 rearrangements (P = 0.009) [30]. In a recent CALGB series of 54 patients with 11q23 abnormalities, 27 patients with t(9;11) had an intermediate outcome and a median OS of 13.2 months, whereas those with t(6;11) or t(11;19) had a poor outcome of 7.2 or 8.4 months [15]. Conversely, Schoch et al. showed that 14 patients with t(9;11) had a median OS of 10.0 months compared with the 12.8 months of 26 patients with other MLL rearrangements, and that the two cytogenetic groups did not significantly differ [13]. Our data showed that nine patients with t(9;11) were more frequently involved in M5. The MPO and MLD features significantly differed between patients with t(9;11) and those with other 11q23 abnormalities. However, the CALGB study found no significant differences in myelodysplastic features between the two cytogenetic groups [30]. In terms of OS, our results showed no significant differences between patients with t(9;11) and those with other 11q23 abnormalities (P = 0.22). Some problems are associated with the analyses of 11q23 abnormalities. We had few patients with these abnormalities, particularly individual translocations, and genetic analysis was not performed. Thus, the prognostic risk of 11q23 abnormalities cannot be concluded from the present study. Nonetheless, these abnormalities were never associated with a favorable risk. To classify 11q23 abnormalities into each prognostic risk group, further investigations and genetic analyses of a large number of patients with 11q23 abnormalities are required.
The fourth WHO category, which is not otherwise categorized, accounted for 52% of patients in the present study. Most of them were classified into the intermediate risk group, and no prognostic subdivisions were valuable. Using cytogenetic features as a prognostic factor in groups with a normal karyotype has limitations, and such patients accounted for 64.6% of the intermediate risk group (data not shown). Additional factors are required to stratify these patients. We and several others suggested that differences could be based on molecular genetic analysis [22, 31–35]. For example, FLT3 mutations are important biomarkers of a normal karyotype and might be valuable for stratifying the intermediate risk group. Further follow-up studies might also shed light on the roles of FLT3 ITD mutations in the development of AML and aid their use as novel molecular targeting agents against AML [22, 32]. Bienz et al. identified CEBPA mutations, FLT3-ITD, and differing levels of BAALC expression as having independent prognostic significance in patients with a normal karyotype [33]. If these genetic markers can be confirmed as being of clinical significance, genetic analyses will probably be incorporated into the WHO classification.
In summary, our results confirmed those of previous studies showing the prognostic significance of cytogenetics, MLD, and MPO-positivity of blasts in AML. Furthermore, we categorized patients with de novo AML according to the WHO classification and showed the clinical characteristics and OS of each category.

who classification
aml
myeloperoxidase
11q23 abnormalities
multilineage dyplasia


P2 receptors in atherosclerosis and postangioplasty restenosis

Atherosclerosis is an immunoinflammatory process that involves complex interactions between the vessel wall and blood components and is thought to be initiated by endothelial dysfunction [Ross (Nature 362:801–09, 1993); Fuster et al. (N Engl J Med 326:242–50, 1992); Davies and Woolf (Br Heart J 69:S3–S11, 1993)]. Extracellular nucleotides that are released from a variety of arterial and blood cells [Di Virgilio and Solini (Br J Pharmacol 135:831–42, 2002)] can bind to P2 receptors and modulate proliferation and migration of smooth muscle cells (SMC), which are known to be involved in intimal hyperplasia that accompanies atherosclerosis and postangioplasty restenosis [Lafont et al. (Circ Res 76:996–002, 1995)]. In addition, P2 receptors mediate many other functions including platelet aggregation, leukocyte adherence, and arterial vasomotricity. A direct pathological role of P2 receptors is reinforced by recent evidence showing that upregulation and activation of P2Y2 receptors in rabbit arteries mediates intimal hyperplasia [Seye et al. (Circulation 106:2720–726, 2002)]. In addition, upregulation of functional P2Y receptors also has been demonstrated in the basilar artery of the rat double-hemorrhage model [Carpenter et al. (Stroke 32:516–22, 2001)] and in coronary artery of diabetic dyslipidemic pigs [Hill et al. (J Vasc Res 38:432–43, 2001)]. It has been proposed that upregulation of P2Y receptors may be a potential diagnostic indicator for the early stages of atherosclerosis [Elmaleh et al. (Proc Natl Acad Sci U S A 95:691–95, 1998)]. Therefore, particular effort must be made to understand the consequences of nucleotide release from cells in the cardiovascular system and the subsequent effects of P2 nucleotide receptor activation in blood vessels, which may reveal novel therapeutic strategies for atherosclerosis and restenosis after angioplasty.
Introduction
Atherosclerosis is a pathological phenomenon primarily affecting the large conduit arteries, for example the aorta, coronary, carotid iliac, and femoral arteries. The development of atherosclerotic lesions in arteries involves the intimal recruitment of smooth muscle cells (SMC) within the blood vessel wall, and also the infiltration of blood-derived cells [1]. This process necessitates the proliferation and migration of SMC from the underlying media and the endothelial adhesion of leukocytes and their infiltration into the subendothelium. A similar intimal accumulation of SMC also takes place during the postangioplasty restenotic process. Although the factors involved in intimal cell recruitment are not clearly identified, it is becoming evident that endothelial dysfunction is a key factor in the development of vascular disease. Experimental evidence suggests that an intact endothelium plays a central role in maintaining a low proliferative state of SMC under normal conditions [10]. In arterial injury, endothelial cells, SMC, and various blood cells can release chemotactic factors and mitogens, including ATP and other nucleotides (see [4] for review). Activation of P2 nucleotide receptors has been shown to induce not only the proliferation and migration of vascular SMC but also apoptosis, a process involved in the evolution of the atherosclerotic plaque [11]. In addition, P2 receptors mediate both vasorelaxation and vasoconstriction of arteries that may be involved in the vascular remodeling accompanying atherosclerosis and postangioplasty restenosis [5]. A better understanding of the causative agents and mechanisms of proliferation and migration of vascular SMC as well as recruitment of blood-derived cells by the endothelium could lead to prevention, attenuation, or even reversal of intimal thickening, which may dramatically reduce morbidity and mortality from vascular diseases such as atherosclerosis and restenosis after angioplasty. In this respect, a better understanding of the physiological role of P2 receptors in both normal and pathological blood vessels could potentially lead to a breakthrough in the fight against vascular disease.
P2 receptors in the cardiovascular system
Extracellular nucleotides bind to cell surface receptors known as P2 receptors, which are present in many tissues. To date, these receptors have been classified into two main families: the P2X receptors that are ligand-gated ion channels comprised of homo- or hetero-oligomers [12] and P2Y receptors that are seven membrane spanning receptors coupled via G proteins (Gq/11 or Gi/o) to phospholipase C (PLC) and/or adenylate cyclase [12–14]. In turn, PLC activation generates inositol 1,4,5-triphosphate (IP3), a mediator of Ca2+ release from intracellular stores, and diacylglycerol, an activator of protein kinase C (PKC), whereas adenylate cyclase generates cyclic adenosine monophosphate (cAMP), an activator of protein kinase A (PKA). The cloning of seven P2X (P2X1, P2X2, P2X3, P2X4, P2X5, P2X6, P2X7) and eight P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14) receptor subtypes has made it possible to use molecular and pharmacological approaches to study the distribution and functional properties of specific P2 receptor subtypes at the tissue and cellular level.
P2 receptors in vascular cells
The normal arterial wall consists of three layers: the intima, media, and adventitia. The single layer of endothelial cells facing the vessel lumen is a very important component of the vascular wall in terms of releasing both vasodilators such as nitric oxide (NO) and prostacyclin (PGI2) and vasoconstrictors like thromboxane A2 and endothelin. The principal P2Y receptor subtypes that have been functionally characterized in endothelial cells are P2Y1 and P2Y2, but mRNAs for P2Y4 and P2Y6 receptors have also been detected [4]. Endothelium-dependent vasorelaxation has been attributed to the release of NO and PGI2 after binding of nucleotides to P2Y1 and P2Y2 receptors in endothelial cells [15], whereas vasoconstrictor effects in SMC result from the action of nucleotides on P2Y2 and P2X receptors [16, 17]. In most blood vessels, P2Y1 receptors for ADP are present on the endothelium and regulate vasodilatation by Ca2+-dependent (PLC-mediated) activation of nitric oxide synthase (NOS) and generation of endothelial-dependent relaxing factor (EDRF) (see [4] for review). Endothelial prostacyclin production is also stimulated by P2Y1 and P2Y2 receptors, but this seems to play a minimal role in vasodilatation, at least under physiological conditions (see [18] for review). Recent studies have indicated that in the aorta of P2Y2-null mice the endothelium-dependent relaxation by ATP and ATPγS was inhibited, demonstrating the role of the P2Y2 receptors, but that a relaxation by UTP and UDP was maintained, suggesting the additional involvement of P2Y6 receptors [19]. The majority of the cells in intact blood vessels are SMC, which occupy most of the media, and are involved in vasoconstriction and vasorelaxation of the vessel. P2Y2Rs in SMC mediate the induction of immediate-early and delayed-early cell cycle-dependent genes, consistent with a role for P2Y2Rs in vascular proliferation of SMC [20, 21]. A recent study demonstrated that P2Y2 is the predominant functional receptor that responds to ATP and UTP in rat aortic SMC [22]. In human cerebral arteries, P2Y6 seems to be the predominant subtype and induces vasoconstriction when activated by UDP/UTP [23, 24]. This is consistent with findings from rat pulmonary and mesenteric arteries [25, 26]. In addition, a recent study on P2X1 knockout mice further supports the prominent contractile effect of the P2Y6 subtype in mesenteric arterial trees [27]. Taken together, it seems that the principal receptor mediating UTP/UDP-induced contractile responses in blood vessels might be the P2Y6 subtype. Other studies have reported the presence of both P2Y4 and P2Y6 receptors in rat aortic SMC [21, 28, 29]. P2Y1 receptors are expressed in SMC of a number of blood vessel types and like their EC counterparts mediate vasodilatation most likely through the activation of K+ channels (see [18] for review). The presence of several P2X receptor subtypes also has been reported in human saphenous vein SMC, including P2X1, P2X2, P2X4, and P2X7 receptors [30]. The outermost layer of the blood vessel consists of connective tissue and fibroblasts, which have not been appreciated in the regulation of vascular tone. A recent study showed that fibroblasts can migrate into the neointima, suggesting their possible involvement in the development of vascular diseases such as atherosclerosis and restenosis after angioplasty [31]. Since human and rat fibroblasts are known to express P2Y1, P2Y2, P2Y4, P2Y6, P2X3, P2X4, and P2X7 receptors [32], further investigation is needed to establish the role of fibroblast P2 receptors in either physiological or pathophysiological conditions.
P2 receptors in blood cells
G protein-coupled P2Y receptors whose activation leads to intracellular calcium mobilization have been observed in neutrophils [4, 33, 34], and turkey erythrocytes express both P2Y1 and P2X7 receptors [35, 36]. ATP and UTP act as secretagogues by binding to P2Y2Rs to enhance exocytosis of primary granules in neutrophils [37]. In macrophages, ATP activates a P2X7 receptor that differs from other ligand-gated ion channel P2X receptors by its ability to generate plasma membrane pores when activated [38]. Human T lymphocytes also have been shown to express P2X7 receptors [39]. Human monocytes and macrophages coexpress P2X1, P2X4, and P2X7 receptors, whereas granulocytes only express P2X7 receptors [40]. P2Y1, P2Y2, and P2Y6 receptors also are expressed in monocytes, B lymphocytes, and polymorphonuclear granulocytes [41]. Human platelets express P2Y1, P2Y12, and P2X1 receptors [42–44]. Thus, the diversity of P2 receptor expression in blood cells, as well as endothelial cells, SMC, and fibroblasts, suggests that this receptor superfamily plays a significant role in the regulation of cardiovascular functions.
P2 receptors regulate nucleotide-induced vascular smooth muscle cell proliferation
Proliferation of SMC is a hallmark of vascular diseases such as atherosclerosis and restenosis following angioplasty. ATP and other nucleotides are released by aggregating platelets and damaged vascular cells, such as endothelial cells and SMC in pathological or stress conditions [45]. Extracellular nucleotides can act via P2X and P2Y receptors to induce acute responses such as the regulation of vascular tone [46]. However, it is generally believed that the ionotropic P2X receptors do not mediate the chronic responses of nucleotides such as cell proliferation. The mitogenic effect of extracellular nucleotides on vascular SMC (VSMC) has been known for years [47]. However, a potent antiproliferative effect of UTP on VSMC also has been reported in human arterial and venous SMC [48]. In either case, the P2 receptor subtype(s) responsible for these effects on the proliferation of VSMC has not been determined. Earlier studies have shown that ATP or UTP increased DNA and protein synthesis in subcultured rat aortic VSMC [49, 50]. In the same cell culture model, however, Malam-Souley et al. [51] were unable to detect increases in DNA synthesis after ATP/UTP stimulation, although ATP or UTP upregulated the expression of mRNA to several cell cycle progression-related genes. Because P2X receptor agonists were essentially inactive, it was concluded that a P2U-like receptor (now termed P2Y2) was responsible for the mitogenic effects of ATP/UTP. However, the role of a P2Y4 receptor cannot be excluded, because the nucleotide agonist profile between rat P2Y2 and P2Y4 receptors is essentially indistinguishable [52]. Indeed, Harper et al. [53] suggested that the P2Y4 receptor mediated ATP/UTP-induced proliferation of rat aortic VSMC. Recent studies indicated that ATP, UTP, or ITP, three agonists of the cloned porcine P2Y2 receptor, increased DNA and protein synthesis and cell number in coronary artery SMC [54]. Indeed, treatment of pig coronary artery SMC with UTP, ATP, or ITP caused a concentration-dependent increase in DNA and protein synthesis and cell number, whereas UDP only caused a small increase in protein synthesis. Intriguingly, ATP was much more potent and efficacious than UTP, ITP, and UDP in increasing DNA synthesis and expression of PCNA, a protein marker of cell proliferation, suggesting that another receptor may contribute to the proliferative response [54]. In vivo experiments have shown that intimal thickening of collared rabbit carotid arteries was greatly enhanced by in situ UTP application and was closely associated with osteopontin expression in medial SMC [6]. Osteopontin is chemotactic for SMC and is associated with arterial smooth muscle cell proliferation [55]. Moreover, both UTP and ATP increased osteopontin expression in cultured SMC, whereas ADP, UDP, and 2-MeSATP were ineffective, which suggests a role for the P2Y2R in which ATP and UTP are equipotent. Direct evidence for involvement of the P2Y2R was provided by inhibition of UTP-induced osteopontin expression in cultured SMC by P2Y2 antisense oligonucleotides [6]. P2Y6 receptors also have been shown to mediate proliferation of SMC in rat aorta [56].
Role of P2 receptors in the migration of vascular SMC
Recent studies indicate that the extracellular nucleotides ATP, ADP, UTP, and UDP serve as directional cues for the migration of rat aortic SMC [57]. At identical concentrations, the most powerful migratory response induced by these nucleotides was elicited by UTP. Nucleotide-induced migration of SMC is the consequence of both chemotaxis and chemokinesis and may result either from the activation of one particular P2 nucleotide receptor subtype or from activation of several P2 receptor subtypes. The ability of UTP at submicromolar levels to stimulate migration of SMC supports the hypothesis that this response could have physiological consequences and is essentially mediated by P2Y2 receptor activation without excluding participation of other P2Y receptor subtypes. The difference in the capacity of UTP and ATP to elicit migration of SMC could be due to the inhibition of nucleotide-induced cell migration by adenosine generated from ATP catabolism by cell surface ectonucleotidases. Indeed, ATP and UTP were equally effective in causing migration of SMC when ATP was prevented from degradation by addition of the ectonucleotidase inhibitor α,β-methylene-ATP [57]. Several P2Y receptor subtypes could be involved in nucleotide-induced migration of SMC. It has been shown in rat aortic SMC that the P2Y2 receptor is the predominant P2Y receptor subtype [28, 58], whereas lower levels of P2Y4 and P2Y1 receptor mRNA were detected [58]. The very low level of P2Y1 receptor mRNA expression was consistent with the absence of ADP-induced migration of cultured rat aortic SMC, demonstrating that P2Y1 is not involved in this process [58]. In addition, the same study showed that a commercially available solution of hexokinase-treated UDP (UTP-free) induced cell migration equally well as untreated UDP, thereby demonstrating that UDP is chemotactic for aortic SMC by activation of the P2Y6 receptor. Conversely, migration of rat aortic SMC induced by UTP occurred even when UTP degradation by nucleoside diphosphate kinase was inhibited, demonstrating the involvement of P2Y2 and/or P2Y4 receptor(s) [58].
The increased migration of SMC in response to extracellular nucleotides could be related to increases in extracellular matrix (ECM) protein expression. Indeed, previous studies have shown that UTP induces osteopontin expression in rat and rabbit aortic SMC [51, 6]. Increased expression of osteopontin, an RGD-containing ECM protein, is associated with the activation of rat arterial SMC in vitro and in vivo [57]. The increase in osteopontin expression plays a key role in UTP-induced migration of rat aortic SMC, since a monoclonal antibody against osteopontin fully abolished UTP-induced migration [57], whereas an antibody against vitronectin, another ECM protein also involved in migration of human SMC [59], had no effect on the migration of rat aortic SMC [57]. UTP induces increases in osteopontin mRNA expression by increasing both osteopontin mRNA stabilization and osteopontin promoter activity [60]. Recent studies have shown that activation of an AP-1 binding site located 76 bp upstream of the transcription start in the rat osteopontin promoter is involved in UTP-induced osteopontin expression. Using a luciferase promoter deletion assay, Renault et al. [61] identified a new region of the rat osteopontin promoter (-1837 to -1757) that is responsive to UTP. This region contains an NFκB site located at -1800 and an Ebox located at -1768. Supershift electrophoretic mobility shift and chromatin immunoprecipitation assays identified NFκB and USF-1/USF-2 as DNA binding proteins induced by UTP. Using dominant negative mutants of IκB kinase and USF transcription factors, it was confirmed that NFκB and USF-1/USF-2 are involved in the UTP-induced expression of osteopontin.
This ability of nucleotides to act as chemoattractants for rat arterial SMC in a concentration range potentially found in pathological vessels [62] and the findings of previous studies demonstrating the mitogenic activity of extracellular nucleotides for these cells suggest that nucleotides released from mechanically stretched vascular or damaged cells during the angioplasty process may participate in arterial wall remodeling.
Role of P2 receptors in nucleotide-induced vascular inflammation
In addition to their mitogenic effects, extracellular nucleotides may also cause cell recruitment by inducing lymphocyte and macrophage adhesion to human pulmonary artery endothelial cells as demonstrated in vitro [63]. Nucleotides can also modulate rat aortic smooth muscle cell adhesion and migration by increasing the expression of osteopontin [51, 57], a protein involved in both processes. Moreover, extracellular nucleotides may play a role in intra-arterial attraction of monocytes by inducing an increased expression of monocyte-chemoattractant protein-1 by arterial SMC [21]. Stimulation of P2 receptors is coupled to the release of the proinflammatory cytokines interleukin (IL)-1β, IL-1α, IL-8, and tumor necrosis factor (TNF)-α (see [4] for review) that are of obvious relevance to inflammation in atherosclerosis. Activation of P2X7 receptors on monocytes/macrophages enhances release of proinflammatory cytokines that modulate NO production and expression of inducible nitric oxide synthase (iNOS) (see [64] for review), mediators of immune cell activation that is an early step in atherosclerotic lesion development.
Monocyte recruitment into the vessel wall is a complex process that includes cell rolling, firm attachment, and directed migration. It is now becoming evident that adhesion molecules such as VCAM-1 play an important role in leukocyte adherence to vascular endothelial cells [65, 66]. VCAM-1 expression is induced or upregulated by proinflammatory cytokines such as TNF-α and IL-1β in cellular components of the arterial wall including endothelial cells, smooth muscle cells, and fibroblasts [67-69]. ATP and UTP have been shown to induce cell-cell adhesion in a human monocyte/macrophage lineage and neutrophil adherence to human endothelial cell monolayers [63, 70].
Recent studies have shown that local UTP delivery via an osmotic pump to collared rabbit carotid arteries induced intimal accumulation of macrophages, similar to oxidized low-density lipoprotein (LDL), a response that was mediated by activation of P2Y2 receptors [6]. Leukocyte migration depends on the activities of adhesion proteins (e.g., selectins and integrins) on leukocytes and vascular endothelial cells. We have demonstrated that activation of P2Y2 receptors in endothelial cells causes the expression of VCAM-1 that mediates the adherence of monocytes to vascular endothelium [71], leading to their penetration into the vessel wall to promote arterial inflammation associated with atherosclerosis.
Recent studies have revealed that a Src homology-3 (SH3) binding domain in the C-terminal tail of the P2Y2 receptor promotes the nucleotide-induced association of Src with the P2Y2 receptor, leading to the transactivation of growth factor receptors, such as the epithelial growth factor (EGF) and vascular endothelial growth factor (VEGF) receptors, and nucleotide-induced upregulation of VCAM-1 [72, 73]. Since leukocyte infiltration and migration are key processes involved in atherosclerosis, these findings suggest that P2Y2 receptors represent a novel target for reducing arterial inflammation associated with cardiovascular disease.
P2 receptors in vascular apoptosis
Apoptosis (programmed cell death) has been reported to occur in various vascular diseases such as atherosclerosis, restenosis, and hypertension [74, 75]. The major cell types undergoing apoptosis in human atherosclerotic lesions are arterial SMC [11, 76–78] and macrophages [79]. In restenosis following balloon angioplasty, there is a peak in the proliferation and apoptosis of rat vascular SMC 14 days postangioplasty [76]. Furthermore, apoptosis of arterial SMC has been described in animal models of intimal thickenings [80] and probably takes part in the normal process involved in the control of hyperplasia. In contrast, apoptosis of SMC in advanced human atherosclerotic plaques may destabilize the fibrous lesion to promote plaque rupture and its clinical consequences.
As a mediator of cell-to-cell communication, ATP can trigger a variety of biological responses after being rapidly released in large amounts from various sources, including activated platelets, endothelial cells, nerve terminals, antigen-stimulated T cells, and other cell types following hypoxia, stress, and tissue damage. For example, in human umbilical cord vein endothelial cells (HUVEC), a substantial release of ATP (and UTP) is induced by shear stress [81], which may lead to alterations in the balance between proliferation and apoptosis regulated by P1 adenosine and P2 (particularly P2X7) nucleotide receptors [82]. P2X7 and P1 receptors have been previously linked to apoptosis in other cell types, including immune cells, astrocytes, and thymocytes [83–85]. The P2X7R also has been shown to mediate ATP-induced cell death in human embryonic kidney cells [86], human cervical epithelial cells [87], and primary rat cortical neurons [88]. In human arterial SMC, adenosine-induced apoptosis is essentially mediated via the A2b-adenosine receptor subtype and involves a cAMP-dependent pathway [89].
As an important constituent of atherosclerotic plaques, fibroblasts share several features with smooth muscle cells. In human fibroblasts, P2X7 was identified as the main nucleotide receptor involved in the high glucose concentration-dependent responses modulated by ATP, including morphological changes, enhanced apoptosis, caspase-3 activation, and IL-6 release [90]. In the immune system, ATP also plays important roles through nucleotide receptors in leukocyte functions. P2X7 receptor-mediated apoptosis has been demonstrated in various types of leukocytes, including a lymphocytic cell line, murine thymocytes, murine peritoneal macrophages, human macrophages, mesangial cells, dendritic cells, and microglial cells [83, 91–96]. Extracellular ATP acting via the P2X7 receptor activates the transcription factor NFκB by selectively targeting NFκB p65 (Rel A) in the N9 mouse microglial cell line [97]. It also has been reported that the P2X7 receptor modulates macrophage production of TNF-α, IL-1β, and nitric oxide (NO) following lipopolysaccharide (LPS) exposure [98], consistent with a role for the P2X7 receptor in inflammation. In HUVEC, TNF-α markedly increases apoptotic cell death via the activation of caspase-3 [74]. Recent reports indicate that ATP/ADP activate NFκB and induce apoptosis probably through P2X7 receptors in porcine aortic endothelial cells [99]. These studies have provided compelling evidence suggesting a role for P2 and P1 receptor-mediated apoptosis in vascular diseases; however, further studies are needed to determine the precise pathways involved and to accumulate direct evidence that these pathways contribute significantly to the development of atherosclerosis, hypertension, and restenosis.
Modulation of P2 receptors in vascular injury
Experimental arterial intimal hyperplasia can be induced by balloon angioplasty or by the perivascular placement of a silicone collar around an artery. An influx of leukocytes precedes the migration and proliferation of vascular SMC into the intima in both these models [100]. In normal adult rat aorta, P2Y2 mRNA was found in the endothelial cell lining, while a sustained expression of P2Y2 mRNA was detected in few medial SMC [20]. In contrast, P2Y2 mRNA was detected in all medial SMC of rat fetal aortas and in most of the aortic SMC of intimal lesions after balloon angioplasty, with an overexpression in cells lining the lumen both 1 and 3 weeks after injury.
In the collar model, neointimal formation appears to be triphasic [100]. The first phase is characterized by vascular infiltration of leukocytes beginning 2 h after collar placement around a rabbit carotid artery. The second phase begins within 12 h of collar placement and is characterized by medial replication of SMC. The third phase is characterized by the appearance, beginning at day 3 after collar placement, of subendothelial SMC. In situ hybridization with sham-operated rabbit carotid arteries indicated that P2Y2 mRNA expression was localized to CD31-positive aortic endothelial cells and not medial SMC [6]. High levels of P2Y2 mRNA were detected in medial SMC 3 days after collar placement, before appearance of neointima. At day 14, all intimal and medial SMC were P2Y2 positive. Fura-2 digital imaging of single SMC, used to measure changes in myoplasmic calcium concentration in response to P2Y receptor agonists, confirmed an increase in P2Y2 receptor activity. However, the same study showed that P2Y4 mRNA was equivalently expressed in sham-operated and collared arteries or cultured rabbit carotid SMC, whereas P2Y6 mRNA was not detected in carotid arteries or cultured SMC. In a more recent study, it was shown that P2Y2 receptor upregulation occurs in stented porcine coronary artery, a clinically relevant model of arterial injury [54]. P2Y2 receptor mRNA levels were significantly increased in coronary SMC dispersed from stented segments of coronary arteries 3 weeks after stent angioplasty compared with SMC from unstented segments. There was no significant difference observed in levels of P2Y6 mRNA in the stented and unstented artery segments, whereas P2Y4 receptor mRNA was undetectable.
Upregulation of functional P2Y receptors also occurs in the basilar artery of the rat double-hemorrhage model [7], in the coronary artery of diabetic dyslipidemic pigs [8], and in human atherosclerotic lesions (Seye and Desgranges, unpublished data). It has been proposed that upregulation of P2Y receptors could be a potential diagnostic indicator for the early stages of atherosclerosis [9]. Interestingly, a more recent study showed that high shear stress, associated with vascular diseases, can selectively upregulate P2Y2 and P2Y6 receptors in perfused arterial SMC [101]. P2X1 and P2X4 receptors have been shown to be upregulated in rabbit intimal thickenings [102]. Taken together, these findings strongly suggest that at least some P2 receptor subtypes (most notably P2Y2) are implicated in the development of vascular disease.
Pathophysiological significance of P2 receptor modulation in vascular injury
Migrating and proliferating SMC in the arterial media, together with infiltrating macrophages and T lymphocytes, are the main cell types that comprise atherosclerotic and restenotic lesions [1]. In the early stages of intimal hyperplasia, SMC are modified from a differentiated, contractile phenotype to an immature, synthetic phenotype, and this enables them to migrate into the intima, to proliferate, and to secrete extracellular matrix components. In many respects, this shift in phenotype is a reversal of the normal differentiation pattern of vascular SMC during fetal and early postnatal life [103, 104]. However, there is still a lack of knowledge concerning the phenotypic regulation of SMC during vasculogenesis and vascular disease. A high level of P2 receptor expression could be related to the altered phenotype of SMC in intimal thickenings. Partially dedifferentiated SMC are found in rat arterial intimal lesions after balloon angioplasty [105, 106], and their phenotype has been compared with that of newborn rat aortic SMC [107]. Interestingly, it has been reported that medial SMC of rat embryonic aorta also exhibit high P2Y2 expression, similar to intimal thickenings [20]. Other studies have shown that P2Y1 and P2Y2 receptor transcripts are strongly upregulated with phenotypic changes in rat SMC, whereas P2X1 mRNA is completely downregulated, and P2Y4 and P2Y6 mRNA levels are unchanged [58, 28]. Taken together, these and other results suggest that P2Y2 receptor expression is upregulated in the entire cell population of intimal thickenings and is closely associated with a poorly differentiated phenotype of SMC. The dramatic increase in P2Y2 mRNA expression observed in balloon angioplasty-induced intimal lesions would suggest increased activity of extracellular nucleotides with consequent enhancement of cell proliferation and vasoreactivity. Indeed, extracellular nucleotides, particularly ATP and UTP, have been shown to induce cell cycle progression and proliferation of cultured arterial SMC [21, 49, 50, 51] and vasoconstriction in the absence of endothelial cells [17, 108, 109]. Since both neointimal hyperplasia and vasoconstrictive remodeling have been found to be involved in postangioplasty restenosis [1, 5, 107, 110], these findings suggest that extracellular nucleotides may play a significant role in this process, at least as long as functional endothelial cells, which regulate intimal thickening [111, 112] and nucleotide-induced vasorelaxation [113, 114], are not regenerated.
Increased P2Y2 receptor expression in the neointima may by itself be sufficient to enhance the local effects of extracellular nucleotides on the proliferation of SMC. Although the expression of other P2 receptors has been described in arterial SMC [21, 29, 115], the P2Y2 receptor seems to be more specifically involved in the response of SMC to ATP and UTP [116], particularly in the potentiation of proliferation [21, 50]. The effects of extracellular nucleotides are not only dependent on the nature and number of P2 receptors present on target cells, but also on the local concentrations of nucleotide agonists. Although in vivo concentrations of extracellular nucleotides are difficult to measure, various in vitro experiments suggest that extracellular nucleotides are released from blood and vascular cells exposed to various physicochemical conditions, e.g., stress, hypoxia, and other factors [117, 118, 119] associated with the angioplasty process.
P2Y2 receptors in SMC are involved in nucleotide-induced constriction of normal arteries [17, 108, 109]. Long lasting alterations in vasomotricity after endothelial cell denudation, resulting in increased sensitivity to vasoconstrictive substances, have previously been demonstrated [113, 114]. It appears that like other receptors for vasoconstrictive factors such as angiotensin II [120], endothelin [121], and platelet-derived growth factor (PDGF) [122], which are overexpressed in neointima, P2Y2 receptors may play an important role in controlling the vasoactive properties of pathological arteries, particularly in chronic constriction at the lesion site that is postulated to be one of the processes leading to postangioplasty restenosis [5, 110].
Conclusion
P2 receptor subtypes, including P2Y2, P2X1, and P2X4, appear to play a role in responses to endothelial injury that are thought to be key events in the initiation of atherosclerosis and restenosis after angioplasty. P2Y2R upregulation and activation in endothelial cells and SMC promote leukocyte transmigration and intimal thickening in arteries of animal models of vascular injury, suggesting a possible regulatory role for this receptor in the mechanisms leading to neointimal hyperplasia after angioplasty. Although there are no selective P2Y2 receptor antagonists yet available, recent progress in siRNA technology has made it possible to design small RNA interference molecules that can selectively inhibit P2 receptor subtype expression. Such molecules can be efficiently delivered into the vessel wall using adenoviral vectors. In addition, P2 receptor transgenic mice (i.e., mice in which the relevant receptor subtype has been deleted or overexpressed) will be valuable tools to substantiate the role that nucleotides and P2 receptors play in the etiology of cardiovascular disease. Further delineation of the signaling pathways involved in these P2 receptor-mediated processes may help limit or prevent vascular diseases such as atherosclerosis and restenosis after angioplasty.

atherosclerosis
restenosis
inflammation
proliferation
migration
smooth muscle cell
nucleotide receptors