Elisabetta Poluzzi

Carlo Piccinni

Paolo Carta

Aurora Puccini

Monica Lanzoni

Domenico Motola

Alberto Vaccheri

Fabrizio Ponti

Nicola Montanaro
email: nicola.montanaro@unibo.it

Fabrizio De Ponti

Cardiovascular events in statin recipients: impact of adherence to treatment in a 3-year record linkage study

Keywords: statins, medication adherence, general practice, cardiovascular disease, medical recordlinkage

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Randomised controlled trials have clearly shown the benefits of lipid-lowering drug treatment for cardiovascular prevention, but how much these translate into actual benefit in the general population is not as well established. Whereas there is consensus on the benefits in secondary prevention [1,2] recommendations to use statins in primary prevention are currently a matter of debate [3][4][5][6] and in fact statins are used mostly in the latter setting (75% of statin recipients [7]). Some authors cast doubt on the benefit of statins in patients with low cardiovascular risk [START_REF] Atomson | Should statins be prescribed for primary prevention of cardiovascular disease in patients with chronic kidney disease?[END_REF] whereas others suggested the use of statins (alone or even in the so-called polypill) even in normolipidemic subjects [4,9,[START_REF] Brugts | The benefits of statins in people without established cardiovascular disease but with cardiovascular risk factors: metaanalysis of randomised controlled trials[END_REF].

Lack of adherence is probably the most relevant factor differencing randomised controlled trials from clinical practice.

In the general population, lack of adherence to statin therapy remains a widespread phenomenon and, consequently, an important matter of concern both in terms of cost-effectiveness and riskbenefit profile. We have previously shown that, among statin recipients, less than 50% were prescribed an amount of drug consistent with daily treatment and this coverage was only slightly higher in patients in secondary prevention, in those aged 50-69 years and among males. Moreover, adherence significantly increased with the complexity of other cardiovascular treatments [11]. On the contrary, prescription of a highly active statin regimen (which can be used as a proxy of the severity of hypercholesterolemia) did not correlate with coverage.

The role of adherence to statins in preventing cardiovascular events in general practice is currently under investigation by different points of view (i.e., risk-benefit and cost-benefit profile) and some investigators have already published useful experiences in different countries [START_REF] Perreault | Effect of statin adherence on cerebrovascular disease in primary prevention[END_REF][START_REF] Dragomir | Relationship between Adherence Level to Statins, Clinical Issues and Health-Care Costs in Real-Life Clinical Setting[END_REF][START_REF] Perreault | Impact of better adherence to statin agents in the primary prevention of coronary artery disease[END_REF][15][16]. However, the scenario is far from complete and the evidence is still partial and insufficient to support Health Policy strategies [17]. The aim of this study was to evaluate the occurrence of cardiovascular events in all patients treated with statins in Emilia Romagna (about 4,000,000 inhabitants), focusing on the relationship between the occurrence of events and the degree of adherence to therapy.

Methods

Sources of data

Drug prescription data were retrieved from the Emilia Romagna Regional Health Authority Database, which provides the following information for each reimbursed prescription: identification code of the drug, ATC (Anatomical Therapeutic Chemical) code, number of packages dispensed, patient code, date of prescription. The patient code allows retrieval of his/her drug history without individual identification.

We collected data of statins and other drugs used for cerebro-or cardiovascular risk prevention (i.e., antiplatelet agents, nitrates, antihypertensives, antidiabetics), prescribed by general practitioners (GPs) and reimbursed between January 2003 and February 2008 by the Health Authority of Emilia Romagna (with both urban and rural area, 4,027,275 inhabitants). The following ATC codes were considered: A10 -drugs used in diabetes; B01A -antithrombotic agents; C01DA -antianginal drugs (nitrates); C02, C03, C07, C08, C09 -drugs used in the treatment of hypertension; C10lipid modifying agents.

All statins are reimbursed by Italian Health Service for (i) familial hypercholesterolaemia, (ii) patients with previous cardiovascular or cerebrovascular events, or (iii) patients with a 10-year cardiovascular risk higher than 20%. Data of hospital admissions for cardiovascular or cerebrovascular events, occurred from January 2003 to February 2008, were retrieved from the hospital discharge registry which provides the following information: ICD-9 code (410-414: ischemic heart disease; 430-438: cerebrovascular Adherence was evaluated by analysing the amount of statins received during the 3 year-period:

• for each year, patients were considered "covered" if they received at least 300 tablets (allowing a tolerance of ~20% over the 365 day period) [11] and

• patients who met the above criterion throughout the 3-year follow-up were considered as "adherent".

The amount of statins was evaluated as number of tablets purchased. In fact, the availability of different dosage strengths for each active substance allow patients to take their daily medication as one tablet. To evaluate the association between cardiovascular events and adherence to statin treatment, a logistic multivariate regression analysis was performed, by grouping non-adherent patients into 3 different categories: "strongly non adherent", "slightly non adherent" and "highly variable" patients (see table 2 for definitions). Age, sex, comorbidity (diabetes, hypertension-angina-heart failure and thrombosis in the first year of the follow-up) were included as covariates in the models.

Statistical analyses

Only patients surviving the 3-year follow-up period were included in the logistic analysis.

To better describe the role of the adherence in cardiovascular prevention, two different stratified models for primary and secondary prevention cohort were estimated. Patients in secondary prevention were those who experienced a cardiovascular event before the recruitment. The effect of adherence was assessed by a generalization of Mantel-Haenszel method in different subgroups samples. The differences in subgroups were tested with Pearson χ 2 test. A p-value <0.05 was considered significant.

Results

Adherence to statin regimen

The cohort of statin recipients consisted of 137,217 subjects, with a mean age of 67 ±10 years and 1:1 female/male ratio (Table 1). During the three years of follow up, 6,491 subjects (5%) died.

Seventy-seven percent of subjects (106,167) received prescriptions of the same statin and simvastatin was the most prescribed (32% of patients), followed by atorvastatin (24%). The remaining 23% of patients were switched to another statin, especially rosuvastatin (23.6%) or atorvastatin (12.6%). Moreover, 72% received only highly active statin regimens (i.e., at dosages expected to reduce LDL-cholesterol by more than 30% [11]).

Eighty-five percent of patients received additional cardiovascular treatments on the first year of follow-up: in particular, 77% (105,341) of subjects concomitantly received drugs for hypertension 1). Along the remaining two years of follow-up, a further 2.5% of patients per year received antihypertensive prescriptions, a further 5% received antithrombotics and a further 2.5% received antidiabetics.

In our cohort, 35% did not receive statin prescriptions in the 2 previous years (new users).

The proportions of patients receiving more than 300 tablets/year of statins and that of patients receiving 201-300 tablets/year were stable along the three years of follow-up (45% and 24% respectively), whereas that of patients receiving 101-200 tablets decreased from 24% to 19% and that of patients taking less than 101 tablets showed an appreciable increase, from 6% in the first year to 13% in the third one.

Sixty-two percent (81,666) of patients were covered for at least 1 year, 44% (60,894) for at least 2 years and 24% (32,355) for the whole 3-year period and only this last figure represents the adherent population according to our criteria. (Figure 1). Among the 98,371 patients who did not meet the criteria to be considered "adherent", 28% (38,328) were "slightly non-adherent", 30% (41,678) "strongly non-adherent", and 13% (18,382) "highly variable" (Table 2).

Cardiovascular events

Within the whole cohort of 137,217 patients, 29% (39,708) experienced cerebro-or cardiovascular events during the 3 year-follow-up (annual rate: 13%): 54% of events were represented by coronary heart disease, followed by cerebrovascular accidents (18%).

Cerebro-and cardiovascular events occurred more frequently in non-adherent patients rather than adherent group (reference), with the following ranking: strongly non-adherent (adjOR=1. 19 3). By considering separately new users and already in treatment patients, the analysis confirmed the significant variables, with minor differences between the two groups. The following variables weighed on the occurrence of CV events more in new users rather than in those already in treatment: strongly non adherent or highly variable regimens, previous CV events and concomitant anti-thrombotic drugs.

Subgroup analysis showed a higher risk of events among non-adherent patients irrespective of risk factors: adjOR 1.40 (95% CI 1.35-1.45) in patients using statins for primary prevention and adjOR=1.59 (95% CI 1.51-1.68) in those using statins for secondary prevention (Figure 2). The only exception was represented by the younger age group (<50 years), for whom the confidence interval includes 1. Moreover, for subjects older than 80 years both in primary and secondary prevention, and in patients with diabetes in primary prevention, the association between lack of adherence and CV events was statistically stronger than in unstratified analysis.

Considering patients' prescriptions twelve months before and after the occurrence of an event, 53% of patients increased their number of tablets after the event; so that a fraction (15%) of the noncovered patients became covered in the year after the event. On the other hand, 26% of patients did not change their number of tablets and the remaining 21% even decreased it.

Discussion

In our cohort of 137,217 patients, more than three quarters of statin recipients did not adhere to drug therapy throughout the 3-year follow-up and, these patients showed more than 40% increase of cardiovascular events when compared with adherent statin recipients.

Although the issue of lack of adherence to statin treatment has been already reported [11,18], in our opinion, a population-based cohort study is important because it attempts to translate adherence to statin regimen into actual cardiovascular benefit in unselected patients in a community setting. Some previous studies provided useful findings to clarify this topic. A study on a large Israeli [19] found a strong association between intensity of statin therapy (in terms of number of tablets/year) and improved survival both in primary and secondary prevention, even higher than that found in clinical trials (up to 45% of risk reduction). Also a Canadian study showed the important role of adherence to statins in the prevention of both cerebrovascular events (reduction of risk: 26%) and coronary disease (18%) [START_REF] Perreault | Effect of statin adherence on cerebrovascular disease in primary prevention[END_REF][START_REF] Dragomir | Relationship between Adherence Level to Statins, Clinical Issues and Health-Care Costs in Real-Life Clinical Setting[END_REF][START_REF] Perreault | Impact of better adherence to statin agents in the primary prevention of coronary artery disease[END_REF]. Two recent Italian studies focused on incident statin recipients for primary prevention. On one hand, Deambrosis et al. found a better cholesterol outcome in adherent patients, but a paradoxical association between adherence and hospitalisation for coronary events [15]. This finding was probably affected by a confounding factor, represented by the basal cardiovascular risk, which conditioned independently both adherence and hospital admissions. On the other hand, Corrao et al. found a protective role of adherence on nonfatal ischemic heart disease (reduction of risk: 15-20%) [16].

Our results (although not directly comparable to those of the above studies because of different parameters in outcomes and in observed population) substantially agree on the clinical importance of continued statin use and provide a wider picture on cardiovascular outcomes in all users (both incident users and already in treatment at recruitment).

The approach used in this study has strengths and limitations. In our opinion, one strength was represented by performing our analysis in a large community setting, including all statin recipients, no matter what the length of therapy before the recruitment was, and including all meaningful cardiovascular outcomes. On the one hand, the findings provided by this approach refer to prevalent statin users, whereas other studies frequently selected only incident users, which represent only a minority of overall statin recipients. On the other hand, our method intrinsically lacks in completeness on prior morbidity and drug history, and could be affected by the immeasurable time bias [20] because of the lack of information on statin exposure during hospitalisations. In the worst case scenario, this bias could have generated a misclassification of 586 non-adherent subjects (0.4% of the cohort), who would pass to adherent status by considering all hospitalisation days as covered days. On the basis of these data, we found a possible overestimation of the risk of events among 11 non-adherent patients by 6 and 8%, respectively in case of primary and secondary prevention. A further limitation was the lack of information on lipidemia, which was an obstacle in the correct association between adherence and cardiovascular outcomes. However information on lipidemia would be very difficult to collect even by using physician's records. In addition, since our prescription data were obtained only from reimbursement database, we probably excluded patients not meeting the Italian reimbursement criteria, because at a lower CV risk. This lack of data could have caused a slight overestimation of the role of adherence in the prevention of CV events. Moreover, a positive attitude of patients towards their own health could be a confounding factor, influencing independently both statin adherence and event occurrence, but also this information is not available in administrative databases, which are the source of our study. Only studies based on questionnaire or primary care database could collect this data and could quantify the magnitude of the healthy adherer effect. This phenomenon is usually indicated as "healthy adherer effect", as already acknowledged by Dragomir [START_REF] Dragomir | Relationship between Adherence Level to Statins, Clinical Issues and Health-Care Costs in Real-Life Clinical Setting[END_REF], Rasmussen [21] and Anderson [22], who stated the difficulties in its quantification.

Nonetheless a generic "healthy adherer behaviour" could have generated a tolerable overestimation of the protective effect of adherence.

Finally, because of the low frequency and the delayed onset of cardiovascular events in low-risk patients, a longer follow up could provide further evidence on this population.

When addressing the health policy implications of our results showing the close relationship between adherence to statins and their effectiveness, the fact that 3 out of 4 of statin recipients were not adherent suggests that statin use in our community setting produces both scarce gain in terms of health and waste of economic resources.

For efficient resource allocation, quantification of the non-adherent population to optimise overall health gain in the community is a priority [17]. Each Health Authority faces two different scenarios:

(a) encouraging efforts to cover a large population entails inherent problems in ensuring adherence, increased overall burden of side effects, in an attempt to reduce as many as possible cardiovascular (which implies less effort to ensure adherence, decreased overall burden of side effects, lower drug cost, but incomplete population protection from cardiovascular risk) [17].

In the light of these potential benefit and limitations, there is debate on which strategy is better, but certainly the specific risk profile of the resident population and the availability of economic resources should guide decision makers.

In our community setting, we interpret the paradoxically lower odds of cardiovascular events in strongly non-adherent patients as an indication that this group was at lower risk than the other nonadherent patients. Indeed, the strongly non-adherent group probably included a number of subjects who actually did not require pharmacological lipid-lowering treatment. Therefore, we would not recommend measures to improve adherence in this group. Probably, efforts to improve adherence should focus on the 28% that was not strictly adherent, but received a yearly amount of drug able to cover at least 6 months (slightly non adherent patients), and on the 13% that alternated covered years with years at very low intensity of treatment (highly variable patients). Notably, the last group showed the highest odds of cardiovascular events (about 70% higher than adherent patients).

Conclusions

This study shows the key role of adherence to statins in the prevention of cardiovascular events at any level of risk in a community setting. Appropriate use of statins needs both strict selection of patients before starting drug treatment and patient-physician cooperation for continued drug use whenever the treatment was appropriately started.

F o r P e e r R e v i e w 19

Abstract

Purpose

In the general population, lack of adherence to statin therapy remains a widespread phenomenon and an important matter of concern both in terms of cost-effectiveness and risk-benefit profile. This study aimed to evaluate the occurrence of cardiovascular events in Italian statin recipients, focusing on the relationship between degree of adherence to therapy and occurrence of events in a 3-year follow-up.

Methods

Our cohort consisted of all patients from Emilia Romagna (4,027,275 inhabitants) who received statin prescriptions in January-February 2005 and was followed up to 36 months for cardiovascular hospital admission (ie. coronary disease, cerebrovascular accidents, peripheral artheropathy), adherence to statin treatment (proportion of days covered: ≥80%) and use of other cardiovascular drugs. The relationship between adherence and cardiovascular events was analysed by multivariate logistic regression; age, sex, other cardiovascular drugs and previous events were covariates of the model.

Results

Patients non-adherent to statin regimen over the 3-year period (76% of the cohort) had higher odds of events, irrespective of risk factors, by more than 40% when compared with adherent patients.

Odds of events were in particular: strongly non-adherent adjOR=1.19 (CI95% 1.15-1.23), slightly non-adherent adjOR=1.25 (1.21-1.30), highly variable in the amount of statins received adjOR=1.69 (1.62-1.77).

Conclusions

This study shows the key role of adherence to statins in the cardiovascular prevention at any level of risk. Appropriateness of statin use needs not only careful selection of patients to be treated, but also cooperation between patient and physician to ensure continued drug use whenever treatment is appropriate.

Introduction

Randomised controlled trials have clearly shown the benefits of lipid-lowering drug treatment for cardiovascular prevention, but how much these translate into actual benefit in the general population is not as well established. Whereas there is consensus on the benefits in secondary prevention [1,2] recommendations to use statins in primary prevention are currently a matter of debate [3][4][5][6] and in fact statins are used mostly in the latter setting (75% of statin recipients [7]). Some authors cast doubt on the benefit of statins in patients with low cardiovascular risk [START_REF] Atomson | Should statins be prescribed for primary prevention of cardiovascular disease in patients with chronic kidney disease?[END_REF] whereas others suggested the use of statins (alone or even in the so-called polypill) even in normolipidemic subjects [4,9,[START_REF] Brugts | The benefits of statins in people without established cardiovascular disease but with cardiovascular risk factors: metaanalysis of randomised controlled trials[END_REF].

Lack of adherence is probably the most relevant factor differencing randomised controlled trials from clinical practice.

In the general population, lack of adherence to statin therapy remains a widespread phenomenon and, consequently, an important matter of concern both in terms of cost-effectiveness and riskbenefit profile. We have previously shown that, among statin recipients, less than 50% were prescribed an amount of drug consistent with daily treatment and this coverage was only slightly higher in patients in secondary prevention, in those aged 50-69 years and among males. Moreover, adherence significantly increased with the complexity of other cardiovascular treatments [11]. On the contrary, prescription of a highly active statin regimen (which can be used as a proxy of the severity of hypercholesterolemia) did not correlate with coverage.

The role of adherence to statins in preventing cardiovascular events in general practice is currently under investigation by different points of view (i.e., risk-benefit and cost-benefit profile) and some investigators have already published useful experiences in different countries [START_REF] Perreault | Effect of statin adherence on cerebrovascular disease in primary prevention[END_REF][START_REF] Dragomir | Relationship between Adherence Level to Statins, Clinical Issues and Health-Care Costs in Real-Life Clinical Setting[END_REF][START_REF] Perreault | Impact of better adherence to statin agents in the primary prevention of coronary artery disease[END_REF][15][16]. However, the scenario is far from complete and the evidence is still partial and insufficient to support Health Policy strategies [17]. The aim of this study was to evaluate the occurrence of cardiovascular events in all patients treated with statins in Emilia Romagna (about 4,000,000 inhabitants), focusing on the relationship between the occurrence of events and the degree of adherence to therapy.

Methods

Sources of data

Drug prescription data were retrieved from the Emilia Romagna Regional Health Authority Database, which provides the following information for each reimbursed prescription: identification code of the drug, ATC (Anatomical Therapeutic Chemical) code, number of packages dispensed, patient code, date of prescription. The patient code allows retrieval of his/her drug history without individual identification.

We collected data of statins and other drugs used for cerebro-or cardiovascular risk prevention (i.e., antiplatelet agents, nitrates, antihypertensives, antidiabetics), prescribed by general practitioners (GPs) and reimbursed between January 2003 and February 2008 by the Health Authority of Emilia Romagna (with both urban and rural area, 4,027,275 inhabitants). The following ATC codes were considered: A10 -drugs used in diabetes; B01A -antithrombotic agents; C01DA -antianginal drugs (nitrates); C02, C03, C07, C08, C09 -drugs used in the treatment of hypertension; C10lipid modifying agents.

All statins are reimbursed by Italian Health Service for (i) familial hypercholesterolaemia, (ii) patients with previous cardiovascular or cerebrovascular events, or (iii) patients with a 10-year cardiovascular risk higher than 20%. Data of hospital admissions for cardiovascular or cerebrovascular events, occurred from January Adherence was evaluated by analysing the amount of statins received during the 3 year-period:

• for each year, patients were considered "covered" if they received at least 300 tablets (allowing a tolerance of ~20% over the 365 day period) [11] and

• patients who met the above criterion throughout the 3-year follow-up were considered as "adherent".

The amount of statins was evaluated as number of tablets purchased. In fact, the availability of different dosage strengths for each active substance allow patients to take their daily medication as one tablet. To evaluate the association between cardiovascular events and adherence to statin treatment, a logistic multivariate regression analysis was performed, by grouping non-adherent patients into 3 different categories: "strongly non adherent", "slightly non adherent" and "highly variable" patients (see table 2 for definitions). Age, sex, comorbidity (diabetes, hypertension-angina-heart failure and thrombosis in the first year of the follow-up) were included as covariates in the models.

Statistical analyses

Only patients surviving the 3-year follow-up period were included in the logistic analysis.

To better describe the role of the adherence in cardiovascular prevention, two different stratified models for primary and secondary prevention cohort were estimated. Patients in secondary prevention were those who experienced a cardiovascular event before the recruitment. The effect of adherence was assessed by a generalization of Mantel-Haenszel method in different subgroups samples. The differences in subgroups were tested with Pearson χ 2 test. A p-value <0.05 was considered significant.

Results

Adherence to statin regimen

The cohort of statin recipients consisted of 137,217 subjects, with a mean age of 67 ±10 years and 1:1 female/male ratio (Table 1). During the three years of follow up, 6,491 subjects (5%) died.

Seventy-seven percent of subjects (106,167) received prescriptions of the same statin and simvastatin was the most prescribed (32% of patients), followed by atorvastatin (24%). The remaining 23% of patients were switched to another statin, especially rosuvastatin (23.6%) or atorvastatin (12.6%). Moreover, 72% received only highly active statin regimens (i.e., at dosages expected to reduce LDL-cholesterol by more than 30% [11]).

Eighty-five percent of patients received additional cardiovascular treatments on the first year of follow-up: in particular, 77% (105,341) of subjects concomitantly received drugs for hypertension 1). Along the remaining two years of follow-up, a further 2.5% of patients per year received antihypertensive prescriptions, a further 5% received antithrombotics and a further 2.5% received antidiabetics.

In our cohort, 35% did not receive statin prescriptions in the 2 previous years (new users).

The proportions of patients receiving more than 300 tablets/year of statins and that of patients receiving 201-300 tablets/year were stable along the three years of follow-up (45% and 24% respectively), whereas that of patients receiving 101-200 tablets decreased from 24% to 19% and that of patients taking less than 101 tablets showed an appreciable increase, from 6% in the first year to 13% in the third one.

Sixty-two percent (81,666) of patients were covered for at least 1 year, 44% (60,894) for at least 2 years and 24% (32,355) for the whole 3-year period and only this last figure represents the adherent population according to our criteria. (Figure 1). Among the 98,371 patients who did not meet the criteria to be considered "adherent", 28% (38,328) were "slightly non-adherent", 30% (41,678) "strongly non-adherent", and 13% (18,382) "highly variable" (Table 2).

Cardiovascular events

Within the whole cohort of 137,217 patients, 29% (39,708) experienced cerebro-or cardiovascular events during the 3 year-follow-up (annual rate: 13%): 54% of events were represented by coronary heart disease, followed by cerebrovascular accidents (18%).

Cerebro-and cardiovascular events occurred more frequently in non-adherent patients rather than adherent group (reference), with the following ranking: strongly non-adherent (adjOR=1.19; 95% CI 1.15-1.23), slightly non-adherent (1.25; 1.21-1.30), highly variable (1.69;1.62-1.77). These events increased with age (e.g., patients older than 80: adjOR=2.78; 95% CI 2.56-3.03) and were statistically more frequent in men (adjOR=1.39; 95% CI 3). By considering separately new users and already in treatment patients, the analysis confirmed the significant variables, with minor differences between the two groups. The following variables weighed on the occurrence of CV events more in new users rather than in those already in treatment: strongly non adherent or highly variable regimens, previous CV events and concomitant anti-thrombotic drugs.

Subgroup analysis showed a higher risk of events among non-adherent patients irrespective of risk factors: adjOR 1.40 (95% CI 1.35-1.45) in patients using statins for primary prevention and adjOR=1.59 (95% CI 1.51-1.68) in those using statins for secondary prevention (Figure 2). The only exception was represented by the younger age group (<50 years), for whom the confidence interval includes 1. Moreover, for subjects older than 80 years both in primary and secondary prevention, and in patients with diabetes in primary prevention, the association between lack of adherence and CV events was statistically stronger than in unstratified analysis.

Considering patients' prescriptions twelve months before and after the occurrence of an event, 53% of patients increased their number of tablets after the event; so that a fraction (15%) of the noncovered patients became covered in the year after the event. On the other hand, 26% of patients did not change their number of tablets and the remaining 21% even decreased it.

Discussion

In our cohort of 137,217 patients, more than three quarters of statin recipients did not adhere to drug therapy throughout the 3-year follow-up and, these patients showed more than 40% increase of cardiovascular events when compared with adherent statin recipients.

Although the issue of lack of adherence to statin treatment has been already reported [11,18], in our opinion, a population-based cohort study is important because it attempts to translate adherence to statin regimen into actual cardiovascular benefit in unselected patients in a community setting. [19] found a strong association between intensity of statin therapy (in terms of number of tablets/year) and improved survival both in primary and secondary prevention, even higher than that found in clinical trials (up to 45% of risk reduction). Also a Canadian study showed the important role of adherence to statins in the prevention of both cerebrovascular events (reduction of risk: 26%) and coronary disease (18%) [START_REF] Perreault | Effect of statin adherence on cerebrovascular disease in primary prevention[END_REF][START_REF] Dragomir | Relationship between Adherence Level to Statins, Clinical Issues and Health-Care Costs in Real-Life Clinical Setting[END_REF][START_REF] Perreault | Impact of better adherence to statin agents in the primary prevention of coronary artery disease[END_REF]. Two recent Italian studies focused on incident statin recipients for primary prevention. On one hand, Deambrosis et al. found a better cholesterol outcome in adherent patients, but a paradoxical association between adherence and hospitalisation for coronary events [15]. This finding was probably affected by a confounding factor, represented by the basal cardiovascular risk, which conditioned independently both adherence and hospital admissions. On the other hand, Corrao et al. found a protective role of adherence on nonfatal ischemic heart disease (reduction of risk: 15-20%) [16].

Some previous studies provided useful findings to clarify this topic. A study on a large Israeli

Our results (although not directly comparable to those of the above studies because of different parameters in outcomes and in observed population) substantially agree on the clinical importance of continued statin use and provide a wider picture on cardiovascular outcomes in all users (both incident users and already in treatment at recruitment).

The approach used in this study has strengths and limitations. In our opinion, one strength was represented by performing our analysis in a large community setting, including all statin recipients, no matter what the length of therapy before the recruitment was, and including all meaningful cardiovascular outcomes. On the one hand, the findings provided by this approach refer to prevalent statin users, whereas other studies frequently selected only incident users, which represent only a minority of overall statin recipients. On the other hand, our method intrinsically lacks in completeness on prior morbidity and drug history, and could be affected by the immeasurable time bias [20] because of the lack of information on statin exposure during hospitalisations. In the worst case scenario, this bias could have generated a misclassification of 586 non-adherent subjects (0.4% of the cohort), who would pass to adherent status by considering all hospitalisation days as covered days. On the basis of these data, we found a possible overestimation of the risk of events among 11 non-adherent patients by 6 and 8%, respectively in case of primary and secondary prevention. A further limitation was the lack of information on lipidemia, which was an obstacle in the correct association between adherence and cardiovascular outcomes. However information on lipidemia would be very difficult to collect even by using physician's records. In addition, since our prescription data were obtained only from reimbursement database, we probably excluded patients not meeting the Italian reimbursement criteria, because at a lower CV risk. This lack of data could have caused a slight overestimation of the role of adherence in the prevention of CV events. Moreover, a positive attitude of patients towards their own health could be a confounding factor, influencing independently both statin adherence and event occurrence, but also this information is not available in administrative databases, which are the source of our study. Only studies based on questionnaire or primary care database could collect this data and could quantify the magnitude of the healthy adherer effect. This phenomenon is usually indicated as "healthy adherer effect", as already acknowledged by Dragomir [START_REF] Dragomir | Relationship between Adherence Level to Statins, Clinical Issues and Health-Care Costs in Real-Life Clinical Setting[END_REF], Rasmussen [21] and Anderson [22], who stated the difficulties in its quantification.

Nonetheless a generic "healthy adherer behaviour" could have generated a tolerable overestimation of the protective effect of adherence.

Finally, because of the low frequency and the delayed onset of cardiovascular events in low-risk patients, a longer follow up could provide further evidence on this population.

When addressing the health policy implications of our results showing the close relationship between adherence to statins and their effectiveness, the fact that 3 out of 4 of statin recipients were not adherent suggests that statin use in our community setting produces both scarce gain in terms of health and waste of economic resources.

For efficient resource allocation, quantification of the non-adherent population to optimise overall health gain in the community is a priority [17]. Each Health Authority faces two different scenarios:

(a) encouraging efforts to cover a large population entails inherent problems in ensuring adherence, increased overall burden of side effects, in an attempt to reduce as many as possible cardiovascular Deleted: difficult to collect. (which implies less effort to ensure adherence, decreased overall burden of side effects, lower drug cost, but incomplete population protection from cardiovascular risk) [17].

In the light of these potential benefit and limitations, there is debate on which strategy is better, but certainly the specific risk profile of the resident population and the availability of economic resources should guide decision makers.

In our community setting, we interpret the paradoxically lower odds of cardiovascular events in strongly non-adherent patients as an indication that this group was at lower risk than the other nonadherent patients. Indeed, the strongly non-adherent group probably included a number of subjects who actually did not require pharmacological lipid-lowering treatment. Therefore, we would not recommend measures to improve adherence in this group. Probably, efforts to improve adherence should focus on the 28% that was not strictly adherent, but received a yearly amount of drug able to cover at least 6 months (slightly non adherent patients), and on the 13% that alternated covered years with years at very low intensity of treatment (highly variable patients). Notably, the last group showed the highest odds of cardiovascular events (about 70% higher than adherent patients).

Conclusions

This study shows the key role of adherence to statins in the prevention of cardiovascular events at any level of risk in a community setting. Appropriate use of statins needs both strict selection of patients before starting drug treatment and patient-physician cooperation for continued drug use whenever the treatment was appropriately started.

F

F o r P e e r R e v i e w 19

  : atherosclerosis; 443: other peripheral vascular disease; 444: arterial embolism and thrombosis;), type of the hospital and ward, duration of the hospital stay and patient code. Data regarding age, sex and deaths were anonymously retrieved from vital statistics. All the databases are managed by the Drug Policy Service of the Health Department of the Emilia-Romagna Region, which provides anonymous data by attributing an anonymous code to each patient. This code allows the record linkage of individual patient data among different databases according to the provisions of privacy rules. The protocol was approved by the institutional ethics committee. Study design Our cohort consisted of all patients who received at least one statin prescription between January and February 2005 (recruitment period) and at least another prescription of statins during the first year of follow-up. Patient drug prescriptions and cardiovascular hospital admissions were analysed for 36 months following recruitment (March 2005 -February 2008, follow-up). Co-prescriptions of cardiovascular drugs other than statins (recorded in the first year of follow-up) were used as a component of patient risk profile, as well as hospital discharge records for cardiovascular events occurred before recruitment (January 2003-December 2004).

  All analyses were performed using STATA version 10 (StataCorp. 2001. College Station, TX: Stata Corporation) and SAS version 9.1 (SAS Institute Inc., Cary, NC, USA).

  b) envisaging a strategy more stringently identifying the population with a likely benefit

  ATomson C (2009) Should statins be prescribed for primary prevention of cardiovascular disease in patients with chronic kidney disease? BMJ 339:b2949. doi: 10.1136/bmj.b2949.:b2949-9. Hlatky MA (2008) Expanding the orbit of primary prevention--moving beyond JUPITER. N Engl J Med 20;359:2280-2282 10. Brugts JJ, Yetgin T, Hoeks SE, Gotto AM, Shepherd J, Westendorp RG, de Craen AJ, Knopp RH, Nakamura H, Ridker P, van Domburg R, Deckers JW (2009) The benefits of statins in people without established cardiovascular disease but with cardiovascular risk factors: metaanalysis of randomised controlled trials. BMJ 338:b2376. doi: 10.1136/bmj.b2376.:b2376-11. Poluzzi E, Strahinja P, Lanzoni M, Vargiu A, Silvani MC, Motola D, Gaddi A, Vaccheri A, Montanaro N (2008) Adherence to statin therapy and patients' cardiovascular risk: a pharmacoepidemiological study in Italy. Eur J Clin Pharmacol 64:425-432 12. Perreault S, Ellia L, Dragomir A, Cote R, Blais L, Berard A, Lalonde L (2009) Effect of statin adherence on cerebrovascular disease in primary prevention. Am J Med 122:647-655 13. Dragomir A, Cote R, White M, Lalonde L, Blais L, Berard A, Perreault S (2009) Relationship between Adherence Level to Statins, Clinical Issues and Health-Care Costs in Real-Life Clinical Setting. Value Health 13:87-94 14. Perreault S, Dragomir A, Blais L, Berard A, Lalonde L, White M, Pilon D (2009) Impact of better adherence to statin agents in the primary prevention of coronary artery disease. Eur J Clin Pharmacol 65:1013-1024

3 Keywords:

 3 statins, medication adherence, general practice, cardiovascular disease, medical record-

  2003 to February 2008, were retrieved from the hospital discharge registry which provides the following information: ICD-9 code (410-414: ischemic heart disease; 430-438: cerebrovascular : atherosclerosis; 443: other peripheral vascular disease; 444: arterial embolism and thrombosis;), type of the hospital and ward, duration of the hospital stay and patient code. Data regarding age, sex and deaths were anonymously retrieved from vital statistics. All the databases are managed by the Drug Policy Service of the Health Department of the Emilia-Romagna Region, which provides anonymous data by attributing an anonymous code to each patient. This code allows the record linkage of individual patient data among different databases according to the provisions of privacy rules. The protocol was approved by the institutional ethics committee. Study design Our cohort consisted of all patients who received at least one statin prescription between January and February 2005 (recruitment period) and at least another prescription of statins during the first year of follow-up. Patient drug prescriptions and cardiovascular hospital admissions were analysed for 36 months following recruitment (March 2005 -February 2008, follow-up). Co-prescriptions of cardiovascular drugs other than statins (recorded in the first year of follow-up) were used as a component of patient risk profile, as well as hospital discharge records for cardiovascular events occurred before recruitment (January 2003-December 2004).

  All analyses were performed using STATA version 10 (StataCorp. 2001. College Station, TX: Stata Corporation) and SAS version 9.1 (SAS Institute Inc., Cary, NC, USA).

  heart failure, 54% (73,963) used antithrombotics and 16% (21,530) used antidiabetics (Table

Formatted

  In other words, a statin adherent regimen may avoid about 1 out of 3 cardiovascular events.

Formatted:

  Font: (Default) Times New Roman, 12 pt, Font color: Auto, Complex Script Font: Times New Roman

  b) envisaging a strategy more stringently identifying the population with a likely benefit

Fig. 1 Fig. 2

 12 Figure captions

  Fig.1

Table 1 . Descriptive analysis of patients' characteristics and events

 1

				Number of patients	%	Events	% in the class
	Gender a	F	67,238	49%	16,210	24%
			M	69,858	51%	23,498	34%
			<50	6,750	5%	943	14%
	Age class	50-65	41,914	31%	8,573	20%
			65-80	73,763	54%	23,707	32%
			>80	14,790	11%	6,519	44%
	Previous CV events b	Yes	29,144	21%	15,504	53%
			No	108,073	79%	24,238	22%
	Diabetes	c	Yes	21,530	16%	8,007	37%
			No	115,687	84%	31,735	27%
	Angina, hypertension, heart failure c	Yes	105,341	77%	36,480	35%
			No	31,876	23%	3,262	10%
	Thrombosis c	Yes	73,963	54%	29,106	39%
			No	63,254	46%	10,636	17%
	Three year adherence d	Yes	32,355	24%	8,803	27%
			No	104,862	76%	30,939	30%
	New users	Yes	48,386	35%	13,681	28%
			No	88,831	65%	26,061	29%

a indication of gender was missing in 121 patients; b previous CV events were observed for the period 2003-2004; c concomitant CV disorders were evaluated by the prescription of the relevant drugs in the first year of follow-up. The following ATC codes were considered: A10 -drugs used in diabetes; C01DA -antianginal drugs (nitrates); C02, C03, C07, C08, C09 -drugs used in the treatment of hypertension and B01A -antithrombotic agents; d patients were considered adherent when they were prescribed at least 300 doses of statins for each of the 3-year follow-up; patients who died during follow-up were included in the non-adherent group.

Table 2 . Distribution of patients according to adherence to statins

 2

	N.	%

Table 3 . Variables influencing occurrence of cardiovascular events

 3

	New users	Already in treatment patients		Total
	N = 48,386	N = 88,831	N = 137,217
	OR (CI95%)	adjOR (CI95%)	OR (CI95%)	adjOR (CI95%)	OR (CI95%)	adjOR (CI95%)

a reference: adherent subjects; b reference: female gender; c reference: <50 years;; d reference: subjects without previous cardiovascular events; e subjects without antidiabetic drugs;

f reference: subjects without prescription of drugs for angina, hypertension or heart failure; g reference: subjects without prescriptions of antithrombotic agen

Year-by-year coverage of patients with statin therapy.

  The size of each circle is proportional to the corresponding percentages of patients (value inside); and the density of grey of the circle to the cumulative level of coverage, from black (always covered) to white (never covered). The analysis includes only patients alive at the end of the relevant year: the total alive patients were 136,013, 133,513, 130,727 in 2005, 2006 and 2007,

respectively Fig.

2 Influence of lack of adherence to statins on cardiovascular events, stratified by sex, age, diabetes and other cardiovascular risk factors

			N° events		A
	total		24238
	sex	F	11201		1st year			2nd year			3rd year
		M	13021
		<50	532
	age class	50-65	5264							yes
		65-80	14582									24%
		>80	3860
		No	19448
	diabetes	Yes	4790				yes
	angina, hypertension or heart failure	No Yes	2561 yes 21677					31%				7%
	thrombosis	Yes No	15851 8387		46%					yes		6%
	total Were patients covered in the year? sex age class diabetes	F M <50 50-65 65-80 >80 No	N° events 15504 5009 10477 411 3306 9125 2659 12287	0.5	F o r P e e r 53% 15% yes F o r 0.7 0.9 1.1 1.3 1.40 B F o r P P 12% e e r e e r	1.5	yes 1.7 yes	1.9	8% 7% 4%	2.1	2.3	2.5
	angina, hypertension or heart failure thrombosis Fig.2	Yes No Yes No Yes	3217 701 14803 2249 13255	0.5	0.7	0.9	1.1	R e v i e 39% R e v i e R e 1.3 1.5 1.7 1.59 v i e	1.9	6% 33%	2.1	2.3	2.5
					deaths 1%			deaths 3%	w deaths 5% w w
	Fig. 1
								21 22

; Points in the middle of lines indicate odds ratios; horizontal lines, 95% confidence intervals. (A) primary prevention: patients without hospital admission for CV event in two years before the recruitment (B) secondary prevention: patients with at last one hospital admission for CV event in two years before recruitment . 1 Cardiovascular

events in statin recipients: impact of adherence to treatment in a 3-year record-linkage study

  Elisabetta Poluzzi 1 , Carlo Piccinni 1 , Paolo Carta 1 , Aurora Puccini 2 , Monica Lanzoni 2 , Domenico Motola 1 , Alberto Vaccheri 1 , Fabrizio De Ponti 1 , and Nicola Montanaro 1

	1 Department of Pharmacology, University of Bologna, Via Irnerio 48, I-40126 Bologna, Italy
	2 Drug Policy Service, Emilia Romagna Regional Health Authority, Viale Aldo Moro 21, I-40127
	Bologna, Italy
	Corresponding author
	Nicola Montanaro Department of Pharmacology, University of Bologna Via Irnerio 48 40126 Bologna, Italy e-mail: nicola.montanaro@unibo.it F o r
	P
	e
	e r
	R
	e
	v i e
	w
	2

  1.35-1.43), in patients using statins for secondary prevention (2.54; 2.46-2.62), those co-medicated for hypertension, angina or heart failure

	(2.77; 2.65-2.89), using antithrombotics (1.87; 1.81-1.93), and with diabetes (1.36; 1.31-1.40; see
	table
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	9

Table 1 . Descriptive analysis of patients' characteristics and events

 1

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

a indication of gender was missing in 121 patients; b previous CV events were observed for the period 2003-2004; c concomitant CV disorders were evaluated by the prescription of the relevant drugs in the first year of follow-up. The d patients were considered adherent when they were prescribed at least 300 doses of statins for each of the 3-year follow-up; patients who died during follow-up were included in the non-adherent group.

Table 2 . Distribution of patients according to adherence to statins

 2 never covered and at least 1 year with less than 200 tablets; d large differences among the number of tablets per year (ie. Patients with differences of at least 200 pills year by year).

	N.	%

c

Table 3 . Variables influencing occurrence of cardiovascular events

 3

	New users	Already in treatment patients		Total
	N = 48,386	N = 88,831	N = 137,217
	OR (CI95%)	adjOR (CI95%)	OR (CI95%)	adjOR (CI95%)	OR (CI95%)	adjOR (CI95%)

a reference: adherent subjects;

b reference: female gender; c reference: <50 years;; d reference: subjects without previous cardiovascular events; e subjects without antidiabetic drugs;

f reference: subjects without prescription of drugs for angina, hypertension or heart failure; g reference: subjects without prescriptions of antithrombotic agen Formatted: Underline, Font color: Blue Formatted: Underline, Font color: Blue Formatted: Underline, Font color: Blue Formatted: Underline, Font color: Blue Formatted: Underline, Font color: Blue

European Journal of Clinical Pharmacology

Acknowledgments

The study was supported by funds from the Emilia Romagna Region and the University of Bologna.

The opinions expressed herein by A.P. and M.L. do not necessarily reflect those of the Emilia Romagna Health Authority.

Acknowledgments

The study was supported by funds from the Emilia Romagna Region and the University of Bologna.

The opinions expressed herein by A.P. and M.L. do not necessarily reflect those of the Emilia Romagna Health Authority.


Hanna Hlawaty

Nadine Suffee

Angela Sutton

Olivier Oudar

Oualid Haddad

Veronique Olivier

Christelle Laguillier-Morizot

Liliane Gattegno

Didier Letourneur

Nathalie Charnaux
email: nathalie.charnaux@jvr.aphp.fr

Low molecular weight fucoidan prevents intimal hyperplasia in rat injured thoracic aorta through the modulation of matrix metalloproteinase-2 expression

Keywords: Fucoidan, intimal hyperplasia, smooth muscle cell, endothelial cell, MMP-2 LMWF, low molecular weight fucoidan, MMP, matrix metalloproteinase, SDF-1, stromal cell-derived factor-1, VEGF, vascular endothelial growth factor, VSMC, vascular smooth muscle cell

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Cardiovascular disease, due essentially to atherosclerosis, causes a major part of deaths in western countries. The arterial wall consists of three layers: the (inner) intima, the media and the (outer) adventitia. Intimal hyperplasia is the principal mechanism of restenosis after balloon angioplasty, with intimal damage and media overstretch leading to vascular smooth muscle cell (VSMC) proliferation, platelet deposition and leukocyte recruitment [1]. It is currently believed that medial VSMC proliferation and migration, as well as transmigration and retention of blood borne inflammatory cells play roles in the formation of obstructive atherosclerotic lesions. As some attempts to modify the response to arterial injury failed [2,3], an understanding of the patho-physiological process of neo-intima formation, extracellular matrix (ECM) remodeling, and the identification of appropriate cellular or molecular targets for therapeutic treatment are still of interest.

The first response to vascular injury in the rat is a dramatic increase of VSMC proliferation in the media, which occurs 1 to 3 days after injury [4,5]. The second phase of lesion development, beginning at day 3, involves the migration of VSMCs into the intima [6], resulting in neo-intima formation, where VSMCs are normally not found in the rat. During the third phase of lesion development, VSMCs proliferate within the neo-intima [6]. Proliferation and migration of VSMCs lead to intimal hyperplasia, fibrous cap formation and atherosclerosis development [7].

Matrix metalloproteinases (MMPs) play an important role in the pathogenesis of restenosis after arterial angioplasty. They orchestrate ECM degradation, facilitate migration and proliferation of VSMCs leading to neo-intimal development [8]. MMP-2 (gelatinase A, type IV collagenase) is one of the MMP members that has been extensively studied and has been found to A c c e p t e d M a n u s c r i p t 4 play an important role in increasing restenosis after either inflammatory or mechanical injury to the vascular wall [9,10]. MMP-2 is expressed abundantly in atherosclerotic and restenotic lesions, and the inhibition of its activity by synthetic compounds greatly reduces neo-intima formation [11].

Vascular endothelial cells play an important role in the control of vascular function and present a large surface area for the exchange of materials between blood and tissues. They participate in vascular homeostasis, interacting with circulating cells, on the one hand, and cells present in the vascular wall, VSMCs, on the other hand. In pathological processes, the balance between vascular endothelial cells and VSMCs is disturbed by endothelial activation and dysfunction leading to local inflammation, thrombosis and vasoconstriction [12].

Fucoidan is a sulfated polysaccharide extracted from brown seaweed that reduces rat smooth muscle cell proliferation in vitro in a more intensive manner than heparin [13]. We have previously shown the therapeutic potential of low molecular weight fucoidan (LMWF) in reduction of in-stent restenosis in a rabbit model, vascular tissue repair [14] and in critical hindlimb ischemia in a rat model [15]. Interestingly, LMWF has been shown to release the glycosaminoglycan-bound stromal cell-derived factor-1 (SDF-1)/CXCL12, which mobilizes progenitor cells [16][17][18] and may participate in angiogenesis with vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) [19].

The aim of this work was to evaluate the ability of LMWF to inhibit VSMC mobility and stimulate HUV-EC-C migration in vitro and to assess its in vivo therapeutic effect using a rat model of intimal hyperplasia after balloon injury.

A c c e p t e d M a n u s c r i p t 5

Materials and methods

Polysaccharide

LMWF was isolated and hydrolyzed by a radical depolymerization process [20] from high molecular weight (HMW) extracts of brown marine algae. The characteristics of LMWF according to previously reported analytical methods [21] are as follows: weight average molecular mass 8 ± 1 kDa; fucose content 35% (wt/wt); uronic acid content 3% (wt/wt); and sulfate content 34% (wt/wt). The anticoagulant activity in vitro of the LMWF has been established by an activated partial thromboplastin time (APTT), and the amount of LMWF required to obtain an APTT of 80 sec (control 40 sec) was 25 g/ml [22].

Rat model of intimal hyperplasia in thoracic aorta

The animal protocol was approved by the Bichat University Institutional Animal Care and Use Committee. Adult male Sprague-Dawley rats (n = 12, 280-300 g, purchased from Janvier, CERJ, Laval, France) were anesthetized with intraperitoneal pentobarbital injection (50 mg/kg, CEVA Santé Animale, Libourne, France) [15]. A 2F Fogerty balloon catheter (Baxter Healthcare Corporation, Irvine, CA, USA) was inserted through an incision made in the external carotid artery and advanced along the length of the common carotid artery to the thoracic aorta [23]. The balloon was then inflated and passed three times along the length of the aorta. The balloon catheter was removed, the external carotid artery was permanently ligated and the skin wound was repaired. Then, the animals were divided into two groups: the first one received the LMWF solution (5 mg/kg/day, n = 6) and the second one received the saline solution (control animals, n = 6) via intramuscularly injection (IM) in the right leg for 14 days. The LMWF injections were A c c e p t e d M a n u s c r i p t 6 performed once a day in the morning and the LMWF concentration in plasma was stable at 10 µg/ml up to 6 hours as described [14]. The rats were sacrificed by an overdose of pentobarbital sodium. The uninjured arteries from control rats were divided into segments used for morphological analysis. Blood samples (5 ml) were withdrawn by heart puncture at the time of sacrifice. Finally, organs (lung, kidney, heart, liver and spleen) were weighted.

Blood analysis and white blood cell count

Two weeks after balloon injury, the blood samples were harvested and were either centrifuged for ELISA assays, or assayed for determination of blood cell counts (white blood cells, monocytes, eosinophils, basophils, red blood cells, platelets, haemoglobin, hematocrit) or biochemical analysis (creatinine, urea, glucose, aspartate and alanine aminotransferase, triglycerides, total and HDL cholesterol) by an automated Coulter XE 2100 (Beckman Coulter France, Roissy, France) or COBAS 6000 (Roche, Meylan, France).

Tissue harvest and histology processing

Two weeks after balloon injury, rats were killed by pentobarbital overdose. The thoracic aortas were harvested, flushed with saline solution, cleaned of adipose tissue and cut into 4-cm-long tubes. The aortic tubes were divided into two groups. The first one (thoracic, n = 6) was fixed in 4% para-formaldehyde (Sigma-Aldrich, Lyon, France), embedded in paraffin, and cut in 9-µmthick cross sections for histology study. The second one (thoracic, n = 6) was frozen in liquid nitrogen-cooled isopentane, cut in 9-µm-thick cross sections with a Leica CM1900 cryostat (Leica Microsystems, Rueil-Malmaison, France) for immunohistochemistry study. Cell density in the neo-intima was observed under a microscope and analyzed for 10 different sections per group.

A c c e p t e d M a n u s c r i p t 7

Morphological studies

After fixation in 4% paraformaldehyde, rat aortas were stained with hematoxylin and eosin solution (Sigma-Aldrich). The intimal and medial areas were quantified using a light microscope (Leica DMRXA microscope, Leica Microsystems) coupled to HistoLab image analysis software (HistoLab Software, Microvision Instruments, Evry, France). Digital planimetry with the use of a video camera mounted on a microscope analyzed the borders of the external elastic lamina, internal elastic lamina, and vessel lumen. Specific software (HistoLab Software) allowed quantification of the neo-intimal, medial, and adventitial areas. Neo-intimal growth was estimated by using the neo-intimal area, medial area and the ratio of intimal to medial areas. At least 3 sections of each stained samples were used for quantification, representing different levels of the arterial segment.

Immunofluorescence of carotid artery

Adjacent 9-µm-thick fresh arterial cross sections were immunostained with mouse anti-human endothelium CD31 (rat cross-reactive, clone RECA-1, dilution 1/20; Abcam, Paris, France) and mouse anti-human smooth-muscle α-actin (α-SMA) (rat cross-reactive, clone 1A4, dilution 1/100; DAKO Corporation, Carpinteria, CA, USA) as previously described [1]. Afterwards, slides were co-incubated with the appropriate secondary antibodies (5 µg/ml; Invitrogen, Cergy Pontoise, France). Negative control sections were incubated only with the secondary antibodies.

Representative immunofluorescence photomicrographs were taken using a Leica DMRXA microscope (Leica Microsystems).

For proliferating cell nuclear antigen (PCNA) immunostaining, cryostat sections were incubated with a mouse monoclonal anti-human PCNA antibody (rat cross-reactive, clone PC10, dilution

Gel in situ zymography

The frozen tissue disposed above was sliced into 10 μm with a cryostat. Then a polyethylene terephthalate-base film coated with 7 μM crosslinked gelatin (Wako Pure Chemical industries, Osaka, Japan) was placed on and was incubated for 24 h at 37 °C in a humidified chamber. After incubation, the film was stained with 1% amido black 10B (Wako Pure Chemical industries) in 70% (v/v) methanol (VWR, Strasbourg, France) and 10% (v/v) acetic acid (VWR) for 15 min.

The film was destained with distilled water, 70% (v/v) methanol and 10% (v/v) acetic acid solution. Lysis of the substrate was assessed by examination under a fluorescent microscope (Zeiss Axiophot microscope, Carl Zeiss France, Le Pecq, France). The specificity of the proteolysis was examined on adjacent serial sections incubated on a film containing the protease inhibitor 1,10-phenanthroline, indicating that the activity was caused by MMPs.

Cell culture

Human Vascular Endothelial Cells (HUV-EC-Cs, N° CRL-1730, ATCC, LGC Molsheim, France) were cultured in Endothelial Cell Basal Medium 2 (ECBM2, PromoCell, Heidelberg, Germany) supplemented with 10% fetal bovine serum, EGF (Epidermal Growth Factor, 5.0 ng/ml), hydrocortisone (0.2 μg/ml), VEGF (0.5 ng/ml), bFF (basic Fibroblast Factor, 10 ng/ml), R3 IGF-1 (Insulin like Growth Factor, 20 ng/ml), Ascorbic Acid (1μg/ml), heparin (22.5 μg/ml), antibiotics (penicillin-streptomycin, 1%, Invitrogen) and L-Glutamine (1%, Invitrogen) at 37 °C in 5% CO 2 . (human Epidermal Growth Factor, 5.0 ng/ml), hbFF (human basic Fibroblast Factor, 2 ng/ml), insulin (5 ng/ml), antibiotics (penicillin-streptomycin, 1%, Invitrogen) and L-Glutamine (1%, Invitrogen) at 37 °C in 5% CO 2 .

The media was changed twice a week. Twenty-four hours before migration, HUV-EC-Cs and VSMCs were plated in 24-well plates at a density of 5 x 10 4 cells per well and cultured at 37 °C in 5% CO 2 .

As demonstrated [24,25], the presence of growth factors such as VEGF, EGF, bFGF and IGF-1 in the culture medium of both HUV-EC-Cs and VSMCs, mimics the atherogenic conditions in in vitro cultures. For migration, spreading, proliferation of HUV-EC-Cs, heparin was not added to the supplemented basal cell culture medium.

Spreading assay

Spreading of HUV-EC-C or VSMC cells were carried out in respective supplemented basal cell culture media, as previously described [26]. Briefly, serum-deprived cells incubated with LMWF (0.1, 1 and 10 µg/ml) were allowed to spread on fibronectin (BD Bioscience Pharmingen, Le Pont de Claix, France) for 2 h. Then the cells were permeabilized in 0.05% Triton X-100 (Sigma-Aldrich) and were stained with Alexa Fluor 546 phalloidin (dilution 1/100, Invitrogen) and observed with a fluorescence microscope (Zeiss Axiophot, Carl Zeiss France). Cell areas were evaluated on 30 cells by analysis with the NIH (release Beta 3b) software (National Institutes of Health). Alternatively, HUV-EC-C cells were transfected either with MMP-2 siRNA-1 or MMP-2 siRNA-2 or SNC RNA (Eurogentec, Seraing, Belgium) for 6 h, then washed and supplemented A c c e p t e d M a n u s c r i p t 10 endothelial cell medium was added for 24 h. Then, HUV-EC-C cells were allowed to spread on fibronectin for 2 h.

Wound migration assay

The in vitro migratory activity of HUV-EC-Cs and VSMCs was measured using a wound migration assay and performed in respective supplemented cell basal culture media. HUV-EC-Cs and VSMCs (50-70% confluence) were wounded uniformly using a sterile 1.15 mm diameter pipette tip. Then, the cells were treated with LMWF (0.1, 1 and 10 µg/ml) during 24 h, then washed and incubated with respective supplemented cell basal culture media. Standard photographs of the wounded areas were taken using a phase contrast microscopy (Olympus CK40 microscope, X10 objective, Olympus France, Rungis, France) immediately after wounding and then 48 h after migration (15 images at each time point). Distance between cells at both sides of the wound was measured for 5 pairs of cells per image. The cell migration distance was calculated by subtracting the distance at the lesion edge at 48 h from the distance measured at 0 h. Then, HUV-EC-Cs were wounded uniformly using a sterile 1.15 mm diameter pipette tip and photographs were taken 48 h later.

Alternatively, HUV-EC-Cs were transfected either with MMP-2 siRNA-1 or MMP-2 siRNA-2 or SNC RNA (Eurogentec) for 6 h, then washed and supplemented endothelial cell basal culture medium was added for 24 h. The percentage of inhibition was [(D1-D2)/D1] x 100.

D1 was the difference between the number of SNC RNA-transfected cells treated with LMWF and that of untreated SNC RNA-transfected cells. D2 was the difference between the number of MMP-2 siRNA-transfected cells treated with LMWF and that of untreated MMP-2 siRNAtransfected cells.

Cell migration and invasion assays in Boyden chambers

HUV-EC-C and VSMC migration and invasion assays were performed in respective supplemented cell basal culture media using Bio-coat cell migration chambers (BD Bioscience Pharmingen) [27]. Briefly, inserts were coated with fibronectin (100 µg/ml, BD Bioscience Pharmingen) for migration or Matrigel (320 µg/ml, BD Bioscience Pharmingen) for invasion assays, respectively. Untreated control cells or cells treated with 10 µg/ml LMWF were placed in the upper chamber and allowed to migrate or invade. After twenty-four hours, cells migrated through the porous membrane were stained with Mayer's hemalum (Sigma-Aldrich) and counted manually by two different observers who performed the blind data acquisition.

In order to analyze the influence of conditioned medium from HUV-EC-Cs pre-incubated or not with LMWF on VSMC migration, VSMCs were placed in the upper part of the Boyden chamber and allowed to migrate through fibronectin towards (placed in the lower part of the Boyden chamber): 1-unconditioned endothelial cell supplemented culture medium or 2unconditioned endothelial cell supplemented culture medium in the presence of 10 µg/ml LMWF, 3-conditioned HUV-EC-C supplemented culture medium or 4-conditioned HUV-EC-C supplemented culture medium from HUV-EC-Cs pre-incubated with 10 µg/ml LMWF for 24 h. The percentage of inhibition was calculated as described [28].

Gelatin zymographic assay

HUV-EC-Cs and VSMCs were treated with 10 µg/ml LMWF. Gelatinolytic activities of pro-MMP-2 and active MMP-2 were measured in cell conditioned media as previously described Total MMP activity was defined as the sum of the pro-form and the active form of MMP-2.

Assessment of Viability and Cell Proliferation

Viable adherent HUV-EC-Cs and VSMCs were quantified after LMWF treatment, using a MTT test (Sigma-Aldrich). Untreated cells (3x10 5 ) or cells pre-treated with 10 µg/ml LMWF for 24 h, were incubated with 10 µM bromo-2-deoxyuridine (BrdU, Invitrogen) for 4 h for HUV-EC-Cs and 16 h for VSMCs before cell collection. After BrdU labelling, the cells were washed in PBS, fixed in 1% paraformaldehyde (Sigma-Aldrich) and permeabilized with 0.05% Triton X100 (Sigma-Aldrich) for 3 min. The cells were then treated with DNaseI (Sigma-Aldrich) for 30 min at 37 °C, to expose incorporated BrdU, washed with PBS and stained with Alexa Fluor 647 conjugated anti-BrdU antibody (dilution 1/100, Invitrogen), for 30 min at room temperature.

Flow cytometry analysis was performed and the percentage of BrdU-positive cells was calculated.

ELISA assay

ELISA assay of total MMP-2 level was performed in the conditioned medium from HUV-EC-C or VSMC cells pre-treated with 10 µg/ml LMWF for 24 h. MMP2 Elisa kit (Quantikine Human/Mouse/Rat MMP-2 (total) Immunoassay, DMP200) was purchased from R&D Systems (Lyon, France).

Western blotting

HUV-EC-Cs and VSMCs were incubated with 10 µg/ml LMWF for 24 h and assayed for Western blot as previously described [30]. The supernatant was collected and protein A c c e p t e d M a n u s c r i p t 13 concentration was determined by bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, USA). Total MMP-2 protein was probed using anti-MMP-2 rabbit polyclonal IgG antibodies (dilution 1/500; Santa Cruz Biotechnology, Heidelberg, Germany) and revealed with horseradish peroxidase (HRP) anti-rabbit immunoglobulin G (dilution 1/2000; Santa Cruz Biotechnology). The gels were then washed and proteins were detected using Enhanced chemiluminescence detection reagents (GE Healthcare, Orsay, France).

Real-time RT-PCR and siRNA transfection

Real-time RT-PCR assays were performed using a Step-One with a MMP-2 TaqMan kit (Applied Biosystem, Courtaboeuf, France). MMP-2 mRNA levels were normalized with two housekeeping gene levels as described in the manufacturer's instructions (TaqMan Inventoried Assay; GAPDH: Hs02758991_g1, β-actin: Hs00357333_g1, MMP-2: Hs1548724_m1).

Two MMP-2 gene-specific sense and antisense 21-nt single stranded RNAs with symmetric 2 nt 3'(2'-deoxy) thymidine overhangs were designed, chemically synthesized and HPLC purified (Eurogentec). Small-interfering RNA (siRNA) sequences corresponding to MMP-2 were: 5'-CAG-GUG-AUC-UUG-ACC-ACC-AGA-AdTdT-3', (reverse) 5'-UUC-UGG-UCA-AGA-UCA-CCU-GdTdT-3' for MMP-2 siRNA-1 and (forward) 5'-GAC-AAA-UUC-UGG-AGA-UAC-AdTdT-3', (reverse) 5'-UGU-AUC-UCC-AGA-AUU-UGU-CdTdT-3' for MMP-2 siRNA-2.

HUV-EC-Cs or VSMCs were transfected with 100 nM siRNA in serum-free medium using jetSI™-ENDO transfectant reagent (Eurogentec) following the manufacturer's instructions.

Mock cells were cultured in parallel and transfected with jetSI™-ENDO lacking siRNA. In each experiment, a SNC RNA (Eurogentec) was used as a negative control. Cells transfected with MMP-2 siRNA-1 or MMP-2 siRNA-2 or SNC RNA or mock-transfected were used 3 days posttransfection for further analysis.

Statistical Analysis

For the determination of statistical significance, an ANOVA test was performed with the Statview software (StatView 4.5 Abacus Concepts, Berkeley, USA). A P value of < 0.05 was used as the criterion of statistical significance.

Results

LMWF prevents intimal hyperplasia in balloon-injured rat thoracic aorta.

Rats were submitted to balloon angioplasty followed by 2 weeks of treatment with saline or LMWF injections. No rat died during the procedures or the 14-day treatment with LMWF (5 mg/kg/day IM). Biochemical and haematological blood parameters and organ weight were not different in samples obtained from LMWF-treated versus control animals (data not shown).

Histological sections of thoracic aortas that were uninjured (Fig. 1a, left panel) or balloon-injured and vehicle-treated (Fig. 1a, middle panel), or balloon-injured and LMWF-treated (Fig. 1a, right panel) are presented. Morphological analysis indicated that the balloon angioplasty of rat thoracic aortas increased significantly the neo-intimal area of these aortas compared with aortas in uninjured animals (5 ± 0.30 versus 0.2 ± 0.02 mm 2 respectively, P < 0.01, n = 6), whereas the medial area was not different (Table 1). The neo-intimal area in the aortas was significantly reduced by 40 ± 5% in LMWF-treated animals as compared with vehicle-treated ones (3 ± 0.50 versus 5 ± 0.30 mm 2 respectively, P < 0.01, n = 6). The intima/media ratio was also significantly decreased by 46 ± 9% in LMWF-treated animals as compared with vehicle-treated animals (0.27 ± 0.04 versus 0.50 ± 0.08, P < 0.01, n = 6). Cell density was decreased by 30 ± 5% in LMWFtreated animals as compared with vehicle-treated animals (28 ± 4 versus 40 ± 3 cell/mm 2 , respectively, P < 0.01, n = 6) (Table 1). The decrease of VSMC proliferation was confirmed by To determine the presence of an endothelial cell lining in the vascular graft two weeks after balloon injury and LMWF treatment, we performed immunohistochemistry analysis of arterial cross sections using fluorescent antibodies against the endothelial cell marker CD31. The immunohistological staining revealed the presence of CD31 positive cells located in the luminal part of the neo-intima layer (Fig. 1c). Furthermore, treatment of rats with LMWF increased the intensity levels of CD31 immunostaining in the neo-intima layer as compared to vehicle-treated animals (Fig. 1c). To determine the localization of VSMCs, -SMA immunostaining was performed. As expected, aorta from vehicle-treated rat exhibited high levels of -SMA staining in the neo-intima (Fig. 1c). In contrast, aorta from LMWF-treated rats showed fewer -SMA positive cells in the neo-intimas.

Antagonistic effects of LMWF on migration and proliferation of human vascular endothelial and smooth muscle cells

In order to determine the cellular and molecular mechanisms underlying the effects of LMWF in reducing the intimal hyperplasia, we explored its influence on cell growth, spreading and migration of both, human endothelial and vascular smooth muscle cells in vitro.

The proliferative activity of HUV-EC-Cs and VSMCs was quantified after 24 h of cell incubation with LMWF, using MTT assay. No cell toxicity was observed even at high LMWF dose (100 µg/ml) in both cell types (data not shown). LMWF increased significantly the number of viable HUV-EC-Cs in a dose-dependent manner reaching a maximum at 10 µg/ml, compared with untreated cells (40 ± 4% increase, P < 0.05, n = 3) (Fig. 2a). Similarly, the number of BrdU- LMWF had no significant effect on the number of viable VSMCs, whatever the concentration used (Fig. 3a) using MTT assay. However, the number of BrdU-positive VSMCs, treated with 10 µg/ml LMWF for 24 h, decreased by 35 ± 9.9% compared to untreated cells after BrdU incorporation (P < 0.05) (data not shown).

We then addressed the effects of LMWF on spreading and migration of both cell types in vitro. Cells were allowed to spread on fibronectin and then observed by fluorescent microscopy 2 h after cell incubation with LMWF at increasing concentrations (0.1, 1, 10 µg/ml). As shown in Fig. 2b, HUV-EC-C spreading was significantly increased by 22.5 ± 4% when cells were incubated with 10 µg/ml LMWF as compared with untreated cells (P < 0.05, n = 3). However, LMWF was devoid of effect on VSMC spreading in the same experimental conditions whatever the concentrations used (Fig. 3b).

Two experimental approaches were used to explore the influence of LMWF treatment on cell mobility. In the wound migration assay, LMWF significantly increased HUV-EC-C migration in a dose-dependent manner up to 45 ± 5% (at 10 µg/ml LMWF) as compared with untreated cells (P < 0.01, n = 3) (Fig. 2c). In contrast, LMWF decreased significantly VSMC migration in a dose-dependent manner up to 40 ± 3% (at 10 µg/ml LMWF) as compared to untreated cells (P < 0.01, n = 3) (Fig. 3c). Furthermore, the data described above on cell mobility were confirmed by the use of a Boyden chamber approach. Indeed, when upper chambers were coated with Matrigel (referred to as "invasion assay" in Fig. 2d), the ability of HUV-EC-Cs incubated with LMWF to invade through the reconstituted extracellular matrix is 7.5-fold higher than that of untreated cells at 10 A c c e p t e d M a n u s c r i p t 17 µg/ml LMWF (P < 0.01, n = 3). In these experimental conditions, 10 µg/ml LMWF significantly decreased VSMC invasion by 65 ± 7% as compared to untreated cells (P < 0.001, n = 3) (Fig. 3d).

It was previously demonstrated that endothelial cells could influence vascular smooth muscle cell activities [22]. Therefore, we addressed the question whether the conditioned medium of HUV-EC-Cs pre-incubated with LMWF could affect VSMC migration. As shown in Fig. 3e, the conditioned medium from HUV-EC-Cs significantly induced the migration of VSMCs across fibronectin (P < 0.001, n = 3) as compared to unconditioned endothelial cell medium.

Interestingly, the ability of VSMCs to migrate towards conditioned medium from HUV-EC-Cs pre-incubated with 10 µg/ml LMWF was reduced by 58.3 ± 4% as compared with conditioned medium from untreated HUV-EC-Cs (P < 0.01, n = 3). It is to note that the presence of LMWF in unconditioned endothelial cell medium, placed in the lower chamber, slightly but significantly increased VSMC migration (P < 0.05) as compared to unconditioned medium (Fig. 3e).

LMWF modulates MMP-2 activity in HUV-EC-Cs and VSMCs

To analyze the molecular mechanisms underlying the effects of LMWF on HUV-EC-C and VSMC migration, we investigated whether this sulfated polysaccharide could have an influence on MMP-2 level and activity. Fucoidan cell treatment induced an increase in the level of mRNA encoding for MMP-2 in HUV-EC-Cs up to 40 ± 3% at 10 µg/ml LMWF (P < 0.05) (Fig. 4a). As shown in Fig. 4b, we also demonstrated using an ELISA assay that the level of total MMP-2 in the conditioned medium from HUV-EC-Cs pre-incubated with 10 µg/ml LMWF for 24 h was increased by 5.4-fold as compared with untreated cells (P < 0.01, n = 3). Furthermore, pro-MMP-2 expression detected by gelatin zymography was also significantly increased by 3.2fold in the conditioned medium from cells treated with 10 µg/ml LMWF as compared to untreated cells (Fig. 4c) (P < 0.01, n = 3). This result was confirmed by Western blot (up to 2.5fold increase at 10 µg/ml LMWF) (P < 0.01) (Fig. 4d). These data suggest that LMWF increased MMP-2 expression in HUV-EC-Cs at both transcriptional and translational levels.

In contrast, 10 µg/ml LMWF significantly decreased MMP-2 mRNA level in VSMCs by 20 ± 5% as compared to untreated cells (P < 0.05) (Fig. 5a). Furthermore, the incubation of VSMCs with 10 µg/ml LMWF resulted in a 6-fold decrease in total MMP-2 level in the conditioned medium (P < 0.01, n = 3) (Fig. 5b). This result was strengthened by gelatin zymography where pro-MMP-2 expression in LMWF-incubated VSMCs was reduced by about 3-fold at 10 µg/ml LMWF (P < 0.01, n = 3) (Fig. 5c). This result was also confirmed by Western blot (up to 40 ± 8% decrease for 10 µg/ml LMWF, P < 0.05, n = 3) (Fig. 5d).

MMPs have been demonstrated to be involved in cell migration. Therefore, in order to demonstrate a causal link between LMWF effects on HUV-EC-C and VSMC migration and MMP-2, MMP-2 RNA interference assays were carried out. When HUV-EC-Cs were transfected with MMP-2 siRNA-1 or MMP-2 siRNA-2, MMP-2 mRNA expression was inhibited by 60 ± 5% or 70 ± 7% respectively as compared to control SNC RNA-transfected cells (data not shown).

The decrease in MMP-2 mRNA expression was similar in siRNA-2 MMP-2-transfected VSMCs while no significant effect was observed with siRNA-1 MMP-2 (data not shown). As assessed by the wound healing assay, migration of cells transfected with MMP-2 siRNA-1 or MMP-2 siRNA-2 was decreased by 50 ± 5% or 60 ± 5% respectively as compared to SNC RNA-transfected cells (P < 0.01, n = 3) (Fig. 6a). The migration of MMP-2 siRNA-1 or MMP-2 siRNA-2-transfected cells induced by LMWF was inhibited by 57.9 ± 4% or 60.8 ± 7% when compared with SNC RNA-transfected cells (Fig. 6a). In addition, migration of VSMCs transfected with MMP-2 siRNA-2 was decreased by 45 ± 5% as compared to SNC RNA-transfected cells (P < 0.01, n = 3) (Fig. 6b). In this experimental condition, the migration of LMWF-treated MMP-2 siRNA-2- This data suggest that LMWF affect both HUV-EC-C and VSMC migration at least partly through MMP-2 expression.

LMWF treatment decreases MMP expression and activity in vivo

To verify whether LMWF could decrease the MMP activities in vivo, in situ zymography was carried out on the cross section of injured thoracic rat aortas. Gelatinolytic activity was markedly detected as green light in the whole neo-intima layer due to gelatin digestion (the presence of MMP-gelatinolytic activities), but not in the surrounding media layer (Fig. 6c). Treatment with LMWF during 14 days decreased the MMP activities in the neo-intima layer compared to vehicle treatment (Fig. 6c, lower versus upper panel of the middle column). These gelatinolytic activities were specific since they were inhibited by 1,10-phenanthroline, a broad MMP inhibitor (Fig. 6c, right column).

Discussion

Restenosis of the treated vessel is a common limitation of endovascular procedures and is one of the causes of recurrence of clinical symptoms after revascularization. Restenosis is closely related to neo-intima formation which is a complex process, classically believed to be the consequence of vascular smooth muscle cell proliferation and migration and the synthesis of extracellular matrix [31]. Recently, it has been proposed that the neo-intimal lesion also consists of progenitor cells attracted to the site of injury [32]. In this report, we provide evidences that LMWF treatment prevents the formation of intimal hyperplasia in rat injured thoracic aorta, since A c c e p t e d M a n u s c r i p t 20 the thoracic aorta intima/media ratio was largely decreased (46%) in LMWF-treated rats as compared to vehicle-treated rats. Fucoidan is a sulfated polysaccharide present in brown algae belonging mainly to Fucales and Laminariales orders. Although much effort has been made during the past years to elucidate the structures of sulfated polysaccharides such as heparin and fucoidan, the structural complexity with respect to glycosidic linkage and sulfate position are still limitations to correlate the structures and biological activities of these polysaccharides. It is however established that fucoidan biological activities rely on the sulfate groups, and it is assumed that the recognition of a particular pattern of sulfation may allow specific interaction with targeted proteins. Nevertheless, reduced into low molecular weight fractions, fucoidans have been used for in vivo animal studies. Fucoidan is often presented as a heparin-like molecule due to its ability to interact with several cytokines (growth factors, chemokines) and to exert direct or indirect modulating effect upon their biological activity. Fucoidan is of a particular pharmacological interest because, in addition to its non-animal origin, it exhibits various biological effects, among them anti-inflammatory activities and low anti-coagulant and antithrombotic activities [33,34]. Fucoidans are known to bind growth factors, such as FGFs, and protect them from proteolysis [33]. Fucoidans can release the glycosaminoglycan-bound SDF-1/CXCL12 from its tissue storage sites and can also bind to this chemokine [17,34].

Our results obtained from an in vivo rat model are in accordance with previous studies which demonstrated that fucoidans could reduce intimal hyperplasia. Indeed, it was previously reported that LMWF prevents neo-intimal formation in a rabbit model of in-stent restenosis in iliac artery [14] or after aortic allograft in rats [32]. LMWF promotes therapeutic revascularization in a rat model of critical hindlimb ischemia [15,35]. Our data also suggest that LMWF increases vascular endothelial cell proliferation, spreading and migration, but decreases vascular smooth muscle cell migration. These data agree with previous in vitro and in vivo studies [14,36,37]. The molecular A c c e p t e d M a n u s c r i p t 21 mechanisms by which fucoidans may inhibit the neo-intima formation are multiple: On the one hand, fucoidans, especially LMWF, promote growth factor effects, such as 33] or VEGF [38] leading to neovascularization. The proposed mechanism is therefore that fucoidans may act in vivo by trapping and protecting endogenously released growth factors from deactivation and proteolytic cleavage or may also displace endogenous growth factors from their tissue heparan sulfate storage sites thus increasing their bioavailability. However, LMWF was also demonstrated to have therapeutic effects in angiogenesis on its own, since a beneficial effect in revascularization was reported with LMWF alone in vivo [39]. On the other hand, another reported effect of fucoidans is the ability to promote progenitor stem cell mobilization via the release of the chemokine SDF-1/CXCL12 from heparan sulfate storage sites [34]. SDF-1/CXCL12 is involved in angiogenesis [40] Interestingly, this induction is largely reduced when using conditioned medium from vascular endothelial cells pre-incubated with LMWF. Strikingly, LMWF in unconditioned HUV-EC-C culture medium, placed in the lower part of the Boyden chamber, induced slightly VSMC migration as compared to control unconditioned HUV-EC-C medium. This data suggests that in these experimental conditions, LMWF potentializes the migratory effects of growth factors present in the unconditioned culture medium, such as VEGF, as described [38]. In contrast, conditioned medium of HUV-EC-Cs pre-treated with LMWF reduced the migration of VSMCs as compared to conditioned medium of untreated HUV-EC-Cs, suggesting that LMWF may reduce the level of vascular endothelial cell-secreted molecules able to induce VSMC migration or may interfere with them and inhibit their chemotactic effects on VSMCs.

In the present study, in situ zymography revealed that LMWF significantly decreases MMP-2 activity in the neo-intima. According to the in vitro study, LMWF increases MMP-2 protein expression and activity in HUV-EC-Cs. In contrast, a decrease in MMP-2 protein expression and activity is observed in VSMCs. Furthermore, the level of total MMP-2 in the conditioned medium from cells incubated with 10 µg/ml LMWF was 5.4-fold higher in HUV-EC-Cs, but 6-fold lower in VSMCs than in untreated controls cells. The remodelling of the vascular wall in response to injury critically depends on the action of extracellular proteases, such as the MMPs, and is accompanied by the migration of VSMCs [29]. This enzyme family consists of a number of structurally related proteinases capable of degrading ECM proteins, including basement membrane collagen, denatured collagen (gelatin), laminin, various proteoglycans and elastin [44]. Previous studies have shown that the MMP expression or activation is upregulated in human atherosclerotic lesions and in rat arteries after balloon catheter injury [31]. Increased pro- HUV-EC-Cs were either untreated or incubated with 1 or 10 µg/ml LMWF for 24 h.

Table 1. LMWF significantly reduced intimal hyperplasia

Treatment with LMWF significantly reduced the neo-intimal and media areas, the intima/media ratio, cell number in neo-intima or media and cell density. The data are expressed as mean + SEM.

Page 31 of 38 A c c e p t e d M a n u s c r i p t

  ; DAKO Corporation), revealed by incubation with rhodamine-stained goat anti-mouse IgG (dilution 1/100; Invitrogen).

  Muscle Cells (VSMC, PromoCell) were cultured in Smooth Muscle Cell Basal Medium 2 (SMCBM2, PromoCell) supplemented with 5% fetal bovine serum, hEGF

[

  29]. The MMP activities were estimated from the 72 and 66 kDa gelatinolytic bands corresponding to pro-MMP-2 and active MMP-2, as previously described[29]. Densitometric

  A c c e p t e d M a n u s c r i p t analysis of scanned gelatinolytic bands were performed with NIH (release Beta 3b) software.

  PCNA immunostaining intensity in neointima layer in aorta from LMWF-treated animals as compared to vehicle ones (Fig.1b, right versus middle panel).

  EC-Cs, treated with 10 µg/ml LMWF for 24 h, increased by 25 ± 8.1% compared to untreated cells after BrdU incorporation (P < 0.05) (data not shown).

  not significantly different to that of LMWF-treated SNC-transfected cells (Fig.6b).

  and regulates endothelial cell branching morphogenesis [41]. Although most therapeutic strategies to prevent restenosis are designed to inhibit VSMC proliferation directly, VSMC proliferation might be indirectly inhibited by re-endothelialization, as endothelial cells secrete antiproliferative and antithrombotic substances [42]. Recently, a strategy using in vivo gene transfer of human Hepatocyte Growth Factor that exclusively stimulated endothelial cells without replication of VSMC growth into injured vessels, results in the successful inhibition of neo-intima formation in rat balloon injured carotid artery [42]. It was also demonstrated in post-angioplasty intimal hyperplasia that active vascular endothelial cells participate in the secretion of growth factors for vascular smooth muscle cells [43] and contribute to the pro-inflammatory response through the secretion of cytokines. In addition, active vascular endothelial cells increase VSMC migration by elaborating factors like platelet-derived growth factor and alter VSMC adhesion molecule expression. Thereafter, the interrelations between vascular endothelial and smooth muscle cells may play a major role in the neo-intima A c c e p t e d M a n u s c r i p t 22 development. In that context, our data demonstrated that conditioned medium from vascular endothelial cells induces the migration across fibronectin of vascular smooth muscle cells.

  MMP-2 and pro-MMP-2 activities were detected at 1-st, 3-rd and 7-th days after arterial injury, respectively in rabbit model of intimal hyperplasia[31].Sweeney et al. [17] also indicated that plasma MMP-9 significantly increased after intravenous injection of HMW fucoidan. In contrast, LMWF did not induce an increase in MMP-9 level in vivo. It was also demonstrated that fucoidan significantly inhibited UVB-induced MMP-1 mRNA expression in a dosedependent manner[45]. In addition, peptide-based inhibitors of MMP activity have been shown to block VSMC migration[46]. Our data demonstrate that LMWF increase MMP-2 expression at both transcriptional and translational levels, as well as MMP-2 activity. Furthermore, our results suggest a direct linkage between LMWF and MMP-2, since MMP-2 downregulation by RNA interference affects LMWF-induced effects on cell migration in vitro. The precise molecular mechanisms of action of LMWF in various cells are in most cases unknown. It was demonstrated that fucoidan can enter into cells through a endocytosis pathway [14] and regulate various signalling pathways [45,47]. Our data hypothesize that LMWF could reduce intimal hyperplasia through the modulation of MMP-2 expression in various cell types involved in the neo-intima development. In vascular endothelial cells, LMWF would increase MMP-2 expression while decreasing it in VSMCs and leading to antagonist effect on cell migration, enhanced for endothelial and reduced for VSMCs. In summary, our data highlight the protective effects of fucoidans on the undesired formation of neo-intima after balloon injury and propose a new underlying mechanism besides the known effect of fucoidans on growth factor activities and the promotion of progenitor stem cell mobilization via the release of the chemokine SDF-1/CXCL12. Other LMW heparin preparations have already shown properties similar to cellular effects induced by LMWF [14,37]. Nevertheless, LMWF exhibit lower anticoagulant activity [13] and a rapid degradation of internalized heparin in VSMCs has been observed conversely to LMWF [37].

  Figure legends

Fig. 2 .

 2 Fig. 2. LMWF increased HUV-EC-C proliferation, spreading, migration and invasion.

  . For viability MTT assay, the results for three independent experiments were expressed as percentage of control cells without LMWF treatment. The number of viable untreated cells was arbitrary set to 100%. *, P < 0.05 versus untreated cells.(b). For spreading assay, the cells were incubated with LMWF at the indicated concentrations (0.1, 1 or 10 µg/ml), allowed to spread on fibronectin for 2 h, and stained with Alexa Fluor 546 phalloidin. They were observed under a fluorescence microscope (lower panel), photographed and theirs areas were evaluated by NIH (release Beta 3b) software (upper panel, histograms). The results for three independent experiments were expressed as percentage of control cells without LMWF treatment. The spreading of untreated cells was arbitrary set to 100%. Bar=5 µm. *, P < 0.05 versus untreated cells. (c). Wound migration assay was performed with a monolayer of HUV-EC-Cs, either untreated or incubated with LMWF at the indicated concentrations. Lower panel: Representative phase contrast photographs were taken 48 h after treatment. Bar = 200 µm. Upper panel: Histograms showing HUV-EC-C wound migration in each condition. The results were expressed in mm of cell displacement. The data are expressed as mean ± SEM. *, P < 0.05 versus untreated cells; **, P < 0.01 versus untreated cells. (d). In the cell invasion assay (Boyden chamber), cells, incubated with 0.1, 1 or 10 µg/ml LMWF and migrated through the Matrigel-coated membrane, were stained with Mayer's hemalum and counted. The results are expressed as cell number per field. The datas are expressed as mean + SEM for three independent experiments. *, P < 0.05 versus untreated cells; **, P < 0.01 versus untreated cells.

Fig. 3 .

 3 Fig. 3. LMWF decreased VSMC migration and invasion.

  Photographs were taken under a fluorescence microscope (lower panel) and the cell areas were

Fig. 4 .

 4 Fig. 4. LMWF increased MMP-2 expression in HUV-EC-Cs.

  (a). LMWF increased the level of mRNA encoding for MMP-2 in HUV-EC-Cs as assessed by real-time PCR. Results are expressed in arbitrary unit (AU). Data are expressed as mean + SEM. *, P < 0.05 versus untreated cells. MMP-2 mRNA level was arbitrary set to 1 in untreated cells. (b). Total MMP-2 expression was detected by ELISA assay in the conditioned medium from HUV-EC-Cs either untreated or incubated with 10 µg/ml LMWF. All data are expressed as mean + SEM. *, P < 0.05 versus untreated cells; **, P < 0.01 versus untreated cells. (c) Lower panel: MMP-2 activity in the cell conditioned medium was assessed by gelatin zymography. Upper panel: Representative histograms of gelatin zymography. Results are expressed in arbitrary unit (AU). All data are expressed as mean + SEM. **, P < 0.01 versus untreated cells. (d). Total MMP-2 protein was detected by Western blot in the conditioned medium from HUV-EC-Cs either untreated or incubated with 1 or 10 µg/ml LMWF. Results are expressed in arbitrary unit (AU). All data are expressed as mean + SEM. **, P < 0.01 versus untreated cells.

Fig. 5 .

 5 Fig. 5. LMWF decreased MMP-2 expression in VSMCs.

  . The migration of VSMCs induced by 10 µg/ml LMWF was unchanged when cells were transfected with MMP-2 siRNA-2 as compared to control SNC RNA-transfected cells. The results were expressed in mm of cell displacement. The data are expressed as mean ± SEM. § , P < 0.01 versus LMWF-untreated SNC RNA-transfected cells; *, P < 0.05 versus LMWF-untreated SNC RNA-transfected cells. (c). MMP activities were observed in rat injured thoracic aortas by in situ zymography. Representative examples of cross-sections were observed using fluorescence microscopy (blue fluorescence of DAPI nuclear staining, left column; green fluorescence of in situ gelatin lysis by MMPs after in situ zymography, middle column; green fluorescence of in situ gelatin lysis by MMPs after in situ zymography in presence of MMP inhibitor, right column. All pictures are taken with X4 magnification. n, neo-intima; m, media. The arrows indicate the in situ MMP activities in the neo-intima peripheral layer.

  Figure 1

  Figure 2

  Figure 3

  Figure 4

  Figure 5

Figure 6

 6 Figure 6

Table 1

 1

Acknowledgments

This work was supported by the Direction de la Recherche et des Enseignements Doctoraux (Ministère de l'Enseignement Supérieur et de la Recherche), the University Paris 13 and INSERM. N. Suffee was supported by a fellowship from Ministère de l'Enseignement Supérieur et de la Recherche. The authors would like to thank Liliane Louedec (INSERM U698) for her technical assistance with the animal experiments.

Nevertheless, knowledge of underlying mechanisms of fucoidan effects and the establishment of structure-activity relationship are essential steps for this new therapeutic approach.

Conflict of interest