Cédric Filipe

Laetitia Lam

Shang Leen

Laurent Brouchet

Audrey Billon

Vincent Benouaich

Vincent Fontaine

Pierre Gourdy

Françoise Lenfant

Jean-François Arnal

Alain-Pierre Gadeau

Henrik Laurell
email: laurell@toulouse.inserm.fr

Estradiol accelerates endothelial healing through the retrograde commitment of uninjured endothelium

Keywords: confocal microscopy, BrdU, 5-Bromo-2deoxyuridine, REGEN, Regenerative area, RE, ReEndothelialized area, RetroP, Retrograde Proliferating zone Endothelial regeneration, arterial injury, estrogen, carotid injury model, "en face" confocal microscopy

Although the accelerative effect of 17ß-estradiol (E2) on endothelial regrowth has been clearly demonstrated, the local cellular events accounting for this beneficial vascular action are still uncertain. In the present work, we compared the kinetics of endothelial healing of mouse carotid arteries after endovascular and perivascular injury. Both basal reendothelialization as well as the accelerative effect of E2 were similar in the two models. Three days after endothelial denudation, a "regenerative area" was observed in both models, characterized by similar changes in gene expression after injury, visualized by "en face" confocal microscopy (EFCM). A precise definition of the injury limits was only possible with the perivascular model, since it causes a complete and lasting decellularization of the media. Using this model, we demonstrated that migration of uninjured endothelial cells precedes proliferation (BrdU incorporation) and that these events occur at earlier time points with E2 treatment. We have also identified an uninjured retrograde zone as an intimate component of the endothelial regeneration process. Thus, in the perivascular model the "regenerative area" can be subdivided into a retrograde zone and a reendothelialized area. Importantly, both areas are significantly enlarged by E2.. In conclusion, the combination of the electric perivascular injury model and EFCM is well adapted to the visualization of the endothelial monolayer and to investigate cellular events involved in reendothelialization. This process is accelerated by E2 as a consequence of the retrograde commitment of an uninjured endothelial zone to migrate and proliferate contributing to an enlargement of the "regenerative area".

Introduction

The integrity and functionality of the arterial endothelium, the very thin cell monolayer positioned at the interface between the blood and the vessel wall, plays a crucial role in the physiology of circulation.

Deendothelialization is the consequence of the treatment of coronary atherosclerosis disease by percutaneous transluminal coronary angioplasty, most often associated with intracoronary stents. One major drawback of this therapeutical approach is the residual in-stent restenosis, due to smooth muscle cell proliferation and intimal extracellular matrix accumulation. Drug-eluting stents releasing antimitotics (sirolimus, paclitaxel) efficiently prevent smooth muscle cell proliferation and thereby in-stent restenosis, but increase the risk of late stent thrombosis. This very serious event, commonly associated with sudden death or acute myocardial infarction, is at least partly the consequence of the inhibition of the reendothelialization of the stent struts by the antimitotics [START_REF] Farb | Stent Thrombosis redux--the FDA Perspective[END_REF][START_REF] Luscher | Drugeluting stent and coronary thrombosis: biological mechanisms and clinical implications[END_REF].

The endothelial healing after injury process has previously mostly been studied in larger animals.

More than 20 years ago, a series of pioneer studies unraveled several cellular mechanisms involved in endothelial regrowth after injury in rats and rabbits [START_REF] Haudenschild | Endothelial regeneration. II. Restitution of endothelial continuity[END_REF][START_REF] Reidy | Endothelial regeneration. III. Time course of intimal changes after small defined injury to rat aortic endothelium[END_REF][START_REF] Schwartz | Endothelial regeneration. I. Quantitative analysis of initial stages of endothelial regeneration in rat aortic intima[END_REF], refs in [START_REF] Van Belle | Endothelial regrowth after arterial injury: from vascular repair to therapeutics[END_REF]. To further gain insight in the molecular mechanisms, the possibility to use transgenic mice provides its obvious advantage. However, the endovascular injury model in mice, proposed by Lindner et al. [START_REF] Lindner | Mouse model of arterial injury[END_REF] is laborious due to the very small size of the mouse carotid artery. As an alternative method, we more recently proposed a model of perivascular electric injury of the carotid artery [START_REF] Brouchet | Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta[END_REF], based on a femoral artery injury model developed by Carmeliet and colleagues [START_REF] Carmeliet | Vascular wound healing and neointima formation induced by perivascular electric injury in mice[END_REF]. Even though the perivascular carotid model has been used in several published studies [START_REF] Billon | The estrogen effects on endothelial repair and mitogen-activated protein kinase activation are abolished in endothelial NO synthase knockout mice, but not by NO synthase inhibition by N-nitro-L-arginine methyl ester (L-NAME)[END_REF][START_REF] Fontaine | Essential role of bone marrow fibroblast growth factor-2 in the effect of estradiol on reendothelialization and endothelial progenitor cell mobilization[END_REF][START_REF] Umetani | 27-Hydroxycholesterol is an endogenous SERM that inhibits the cardiovascular effects of estrogen[END_REF][START_REF] Zhu | The Scavenger Receptor Class B Type I Adaptor Protein PDZK1 Maintains Endothelial Monolayer Integrity[END_REF], no systematic comparison with the endovascular injury has yet been reported, although both models behave similarly in terms of accelerated endothelial healing by 17βestradiol (E2) estimated three days post injury by Evans blue staining [START_REF] Billon | The estrogen effects on endothelial repair and mitogen-activated protein kinase activation are abolished in endothelial NO synthase knockout mice, but not by NO synthase inhibition by N-nitro-L-arginine methyl ester (L-NAME)[END_REF]. Indeed, it is now clear that the physiological and pathophysiological role of E2 is not limited to reproductive organs, but involves many other tissues, in particular those belonging to the cardiovascular system. In particular, E2 exerts several beneficial effects at the level of the endothelium: it promotes endothelial NO production, prevents VCAM-1 expression and endothelial apoptosis (refs in [START_REF] Arnal | Estrogens in vascular biology and disease: where do we stand today?[END_REF][START_REF] Mendelsohn | Molecular and cellular basis of cardiovascular gender differences[END_REF]). In addition, E2 stimulates endothelial regrowth after endothelial denudation in rats [START_REF] Krasinski | Estradiol accelerates functional endothelial recovery after arterial injury[END_REF] and in mice [START_REF] Brouchet | Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta[END_REF][START_REF] Iwakura | Estrogen-mediated, endothelial nitric oxide synthase-dependent mobilization of bone marrow-derived endothelial progenitor cells contributes to reendothelialization after arterial injury[END_REF][START_REF] Strehlow | Estrogen increases bone marrow-derived endothelial progenitor cell production and diminishes neointima formation[END_REF]. Interestingly, inhibition of neointimal proliferation and acceleration of reendothelialization with the local administration of E2 following balloon angioplasty in a pig model has been recently reported [START_REF] Chandrasekar | Local delivery of 17-beta-estradiol decreases neointimal hyperplasia after coronary angioplasty in a porcine model[END_REF][START_REF] New | Estrogen-eluting, phosphorylcholine-coated stent implantation is associated with reduced neointimal formation but no delay in vascular repair in a porcine coronary model[END_REF]. Moreover, implantation of E2-eluting stents appears feasible and safe in humans [START_REF] Abizaid | First human experience with the 17-beta-estradiol-eluting stent: the Estrogen And Stents To Eliminate Restenosis (EASTER) trial[END_REF].

E2 effects can be mediated by estrogen receptor alpha (ERα) and beta (ERβ), also named NR3A1 and NR3A2, referring to their classification within the nuclear receptor superfamily [START_REF]A unified nomenclature system for the nuclear receptor superfamily[END_REF]. The accelerative effect of E2 on reendothelialization is mediated by ERα [START_REF] Brouchet | Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta[END_REF] and endothelial NO synthase appears absolutely required for this effect [START_REF] Billon | The estrogen effects on endothelial repair and mitogen-activated protein kinase activation are abolished in endothelial NO synthase knockout mice, but not by NO synthase inhibition by N-nitro-L-arginine methyl ester (L-NAME)[END_REF][START_REF] Iwakura | Estrogen-mediated, endothelial nitric oxide synthase-dependent mobilization of bone marrow-derived endothelial progenitor cells contributes to reendothelialization after arterial injury[END_REF]. Interestingly, the effect of E2 on endothelial healing remains unaltered upon inhibition of NO synthase activity by L-NAME [START_REF] Billon | The estrogen effects on endothelial repair and mitogen-activated protein kinase activation are abolished in endothelial NO synthase knockout mice, but not by NO synthase inhibition by N-nitro-L-arginine methyl ester (L-NAME)[END_REF]. In addition, ERα also mediates the beneficial effect of E2 on medial hyperplasia [START_REF] Pare | Estrogen receptor-alpha mediates the protective effects of estrogen against vascular injury[END_REF]. However, at least in mice, ERβ is not necessary for both these E2 effects on arterial healing [START_REF] Brouchet | Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta[END_REF][START_REF] Karas | Estrogen inhibits the vascular injury response in estrogen receptor beta-deficient female mice[END_REF].

To further study the cellular mechanisms accounting for the accelerative effect of E2 on reendothelialization, we have compared the kinetics of reendothelialization in the conventional endovascular and a perivascular electric injury model using Evans blue staining. Since the endothelial cell monolayer is rather poorly visualized by immunohistochemical analyses on frozen transversal sections, we also applied « en face» confocal microscopy (EFCM) to more precisely visualize the regenerated endothelium, as well as the expression of several molecular key actors. We confirmed, in agreement with previous work in rat [START_REF] Schwartz | Endothelial regeneration. I. Quantitative analysis of initial stages of endothelial regeneration in rat aortic intima[END_REF], the appearance of the "REGENerative area" (REGEN) characterized by endothelial cell proliferation and gene expression changes. At variance with the endovascular model, the injury limit can be precisely defined in the perivascular model by EFCM analysis. This feature revealed the implication of retrograde zone in the adjacent uninjured endothelium in the regenerative process. The commitment of this zone, named RetroP, could be of profound importance in the beneficial action of E2, as its size is increased upon E2 treatment. C57Bl/6-TgN(ACTbEGFP)1Osb transgenic mice (GFP-Tg, Jackson Laboratories, Bar Harbor, Maine) [START_REF] Okabe | Green mice' as a source of ubiquitous green cells[END_REF] with at least eight backcrosses with C57BL/6J mice, were used in bone marrow grafting experiments. Two weeks after ovariectomy, recipient C57Bl/6 mice were lethally irradiated (9Gy, γsource) and received an intravenous injection of 4x10 6 donor BM cells 24 h later. One month later, mice were implanted with either a placebo or an E2 pellet. Chimeric mice were sacrificed 3 days post-injury.

Materials and methods

Mice

Mice were exposed to placebo or E2 for 2 weeks before artery injury and until sacrifice as previously described [START_REF] Brouchet | Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta[END_REF]. We systematically checked that placebo-treated ovariectomized mice had an atrophied uterus (<20 mg) and non-detectable (<5pg/ml i.e. 20 x 10 -12 M) circulating levels of E2, whereas those implanted with an E2 releasing pellet had a significant increase in uterine weight and serum E2 concentrations (100 to 150 pg/ml, not shown).

Mouse carotid injury

Endovascular injury was performed mechanically by a modification of the protocol described by Lindner and co-workers [START_REF] Lindner | Mouse model of arterial injury[END_REF]. Briefly, animals were anaesthetized by intraperitoneal injection of a mix of ketamine and xylasine (1% and 0.1% (w/v), respectively). The right common carotid artery was exposed from bifurcation, via an anterior incision of the neck. The left external carotid was ligated distally and looped proximally with 7-0 silk suture. Silk loops were placed around internal and common carotids to temporally restrict blood flow in the area of surgical manipulation. The external carotid was incised. The injury was performed using a 0.3 mm diameter swab built by loosely winding and sticking an 8-0 silk suture around 0.16 mm diameter blunted guide wire. The swab was introduced, advanced through the common left carotid artery and withdrawn 3 times. A 4 mm length denudation from bifurcation of the common carotid artery was performed using the silk suture on the common carotid as injury. The device was removed and the external carotid ligated proximally. Blood flow was then restored in internal and common carotids. The skin incision was closed and animals were allowed to wake up under warm conditions.

The carotid electric injury was performed essentially as previously described [START_REF] Brouchet | Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta[END_REF]. Briefly, the left common carotid artery was exposed as described above. The electric injury was applied to the distal part (4 mm precisely) of the common carotid artery with a bipolar microregulator.

One to 7 days later, the endothelial regeneration process was evaluated by staining the denuded areas with Evans blue dye as previously described [START_REF] Brouchet | Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta[END_REF]. Briefly, 50 µL of solution containing 5% Evans blue diluted in saline was injected into the tail vein 10 minutes before euthanasia, followed by fixation with a perfusion of 4% phosphate-buffered formalin (pH 7.0) for 5 minutes. The left common carotid artery was dissected from the aortic arch to the carotid bifurcation. The artery was then opened longitudinally. The total and stained carotid artery areas were planimetered after image digitalization. The percentage of reendothelialization was calculated by 1-(A dayS /A day0 )* 100, where A dayS represents the remaining deendothelialized area at the day of sacrifice, and A day0 the initially deendothelialized area.

En face confocal microscopy

Intravascular blood was first removed with an intra cardiac injection of PBS 1X. The carotids were carefully dissected and fixed for 20 min in PBS containing 4% paraformaldehyde. Carotids were longitudinally opened and fixation was quenched with 100 mM glycine (pH 7.4), permeabilized for 10 min in 0.1% Triton X-100, and washed in PBS. Fixed tissues were blocked with a solution I (75 mM NaCl, 18mM Na 3 citrate, 2% goat serum, 1% BSA, 0.05% Triton X-100, and 0.02% NaN 3 ) during 2h at room temperature. Primary antibody are diluted in solution I and incubated with carotids during 48h at 4°C. Then, tissues were washed with solution II (75 mM NaCl, 18 mM Na 3 citrate, 0.05% Triton X-100) for 2h and rinsed in PBS. Carotids were incubated, 1h at room temperature, with fluorophore-conjugated antibodies (Molecular probes) diluted in solution I. Finally tissues were washed in solution I for 1h at room temperature. Primary antibodies used were: CD31 (01951D, Pharmingen), eNOS (sc654, Santa Cruz Biotechnologies), von Willebrand Factor (A0082, Dako). Secondary antibodies (Invitrogen) were all conjugated with Alexa Fluor 633. To label the nuclei, propidium iodide was used (Sigma-Aldrich). All preparations were mounted with Kaiser's glycerol gelatin (Merck). Microscopy imagery was performed on a LEICA SP2 confocal microscopy and quantification was performed with ZEISS LSM 510 software.

Lengths of the reendothelialized area (RE) and of the retrograde proliferating zone (RetroP) are means of at least 10 measures spanning the carotid obtained with the software Zeiss LSM Image Browser v.3.1 (Carl Zeiss Jena Gmbh, Jena, Germany). The assembly of micrographs was achieved with PhotoStich v.3.1 (Canon, Courbevoie, France)

BrdU incorporation and staining

To label proliferating cells 3 or 5 days post-injury, the mice received an intraperitoneal injection of 100 µL 5-Bromo-2-deoxyuridine solution (33 mg/mL, Roche Diagnostic) 15 hours before sacrifice. At earlier time points [START_REF] Lindner | Mouse model of arterial injury[END_REF][START_REF] Reidy | Endothelial regeneration. V. Inhibition of endothelial regrowth in arteries of rat and rabbit[END_REF] or 50 h post-injury), the mice received an injection at time 0 and then approximately every 16 h. After tissue recovery, samples were fixed in 100% methanol during 30 min and permeabilized with 2N HCl during 7 min at 37°C. After 3 washes in PBS, samples were treated with a Borate solution (24.6 g/L Na 2 B 4 O 7, 4.36 g/L KH 2 PO 4 ) during 15 min. Carotid arteries were subsequently incubated with solution I (previously described) for 2 hours at RT. Finally, tissues were incubated with anti-BrdU antibody conjugated with Alexa fluor 633 (Invitrogen) during 1 hour at RT and rinsed in solution II (described above). A mean length of each zone (REGEN, RE, RetroP) was measured was obtained as described above.

Statistics

one-factor ANOVA was performed. To test the respective role of E2 treatment and type of injury or time on reendothelialization, a two-factor ANOVA was performed. When an interaction was observed between the two factors, the four groups were compared by a one-factor ANOVA. P<0.05 was considered as statistically significant.

Results

Endovascular and perivascular electric injury models exhibit similar kinetics of both basal and E2 stimulated reendothelialization

In order to compare the reendothelialization process after endovascular and perivascular electric carotid injury, we performed an injury of equal length (4 mm) in both models in ovariectomized mice treated with either placebo or E2. Under these conditions the reendothelialization is completed seven days after both types of injury (data not shown), as previously observed [START_REF] Brouchet | Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta[END_REF]. Therefore, the kinetics of reendothelialization was analyzed at 3 and 5 days post-injury. Interestingly, no difference in the kinetics of the endothelial healing was observed between both models (Figure 1). To test the respective role of injury type and E2 treatment on reendothelialization at each time point, a two-factor ANOVA was performed and revealed a significant effect of E2 on acceleration of reendothelialization at day 3 and day 5 (both P<0.05), but no effect of the type of injury and no interaction between the two parameters. Thus, no difference in the rate of the endothelial healing could be detected between both models.

"En face" confocal immunohistochemical analysis of endovascular and perivascular electric carotid injury reveals similarities between the two models in terms of reendothelialization

We next wanted to gain further insight in molecular and cellular events during reendothelialization in both injury models at 3 days post injury. Since the visualization of the endothelial cell monolayer by transversal sections is limited, we employed EFCM on longitudinally opened and flattened mouse carotid arteries. As the endothelial regeneration in both models progresses mainly from the proximal edge of the lesion, in the direction of the blood flow, we focused on this region for further studies. Staining by propidium iodide confirmed that the endothelial and smooth muscle cell nuclei could be distinguished by their orientation, size and shape. Indeed, in contrast to smooth muscle, endothelial cell nuclei were oriented in the direction of the blood flow, and were larger and rounder in shape than smooth muscle cell nuclei. Compared to the intact endothelium, EFCM analysis revealed a decreased expression of von irrespective of the injury model (Figures 2A-C, 2A'-C').

We then determined the proliferative index of the endothelial cells following a peritoneal injection of BrdU, 15 hours prior to sacrifice. The fraction of BrdU positive cells after endovascular injury (38.6 ± 4.6%) and perivascular injury (44.3 ± 3.9%) did not differ significantly (P=0.35) (Figures 2D and2D').

In agreement with previous reports [START_REF] Lindner | Mouse model of arterial injury[END_REF], endovascular carotid injury using a thin swab preserves most of the vascular smooth muscle cells (Figure 2E). In contrast, perivascular electric carotid injury provokes a complete decellularization of both the intima and the media (Figure 2E'), without any cellular recolonization of the latter one week after injury. Thus, at variance with the endovascular model, the electric model permits the precise definition of the injury limit using EFCM.

Electric injury model analyzed by EFCM also allowed to determine two distinct areas: the "ReEndothelialized area" (RE) corresponding to the part of the injured intima that has been recolonized with endothelial cells and the "REGENerative area" (REGEN), corresponding to the zone of proliferating cells. In these ovariectomized mice, the REGEN was only slightly larger than the RE. Interestingly, the overexpression of eNOS and the lowered expression of vWF and CD31 were overlapping with the REGEN both in the endovascular (Figures 2A-D) and in the perivascular (Figures 2A'-D') model.

Taken together, we show here that even though the media exhibits major differences in the two injury models, the endothelial cells of the REGEN had similar characteristics in both models. Since both injury models exhibited similar kinetics of reendothelialization (Figure 1), but only the electric model in combination with EFCM allowed a precise localization of the injury limit due to the absence of cellular recolonization of the media (Figure 2E'), this latter model was chosen for most of the subsequent experiments.

"En face" confocal immunohistochemical analysis of perivascular electric carotid injury reveals the absence of medial smooth muscle regeneration within in the first week post-injury.

One of the surprising features of the model of perivascular electric carotid injury was the total absence of regeneration of the media at day 7, at a time when the reendothelialization was completed. We therefore decided to explore the regeneration of the medial layer at day 1, 3 and 5 post-injury. We first observed that, at these different time point, the limit of injury remained abrupt, with no visible evidence of migrating cells and with no change in smooth muscle cell density (Fig 2E'). Second, we determined the proliferative index of the smooth muscle cells following a peritoneal injection of BrdU, 15 hours prior to sacrifice. The fraction of BrdU positive cells was always < 1% of the cells examined at the edge of the injury, i.e. less than one medial cell per carotid artery examined (not shown). Third, the "en face" technique also offers the opportunity to precisely determine the abundance of bone-marrow derived cells in hematopoietic chimeric GFP mice (bone marrow from GFP transgenic mice grafted to irradiated wild type mice). However, whereas the number of GFP positive cells in the REGEN was about 4% and were very abundant in the adventitia, no GFP positive cells could be detected in the media (( 14) and not shown).

Analysis by EFCM of E2-stimulated reendothelialization at day 3 after perivascular injury reveals the commitment of a retrograde zone.

E2 treatment elicited two main modifications at day 3 post-injury. First, EFCM confirmed that E2 accelerates the reendothelialization process (Figures 3 and4), in agreement with data obtained through Evans blue staining (Figure 1). Second, E2 induces the enlargement of REGEN in the adjacent uninjured area (Figure 3B). BrdU labeling enabled the visualization of this "Retrograde Proliferating zone" (RetroP), defined as the area with a density ≥10% of BrdU positive cells. RetroP was present in placebo-treated mice, but was impressively increased by E2 (Figure 3B and4). Changes in expression levels, such as increased eNOS, decreased CD31 and vWF, observed in RE also overlapped with RetroP (Figures 3B andC).

The quantification showed that E2 increased RE by 32.9%, and REGEN by 43.5%, both P<0.05 (Figure 4A). Moreover, the combination of EFCM and BrdU labeling allowed us to define the proliferation index as well as the total number of endothelial cells. Compared to placebo, E2 increased in the RE the total number of BrdU-positive cells by 93.4 % and the total cell number by 86.1 %, both P<0.05 (Figure 4B and4C). As the density of BrdU-positive cells in RE was not significantly increased by E2, the increase in total number of BrdU-positive cells was mainly due to the increased size of RE. The proliferative effect of E2 was even more spectacular in the RetroP. Indeed, E2 not only increased the size of RetroP (from 75±7 µm to 163±29 µm, P<0.05), but also the percentage of BrdU-positive cells in this zone (from 16.0 ± 1.3 to 42.4 ± 1.2%, P<0.05), this latter value being similar to that found in the RE.

Altogether, E2 drastically (10-fold) increased the total number of BrdU-positive cells in the RetroP (Figure 4C).

In the endovascular injury model 3 days post injury, where only REGEN could be defined due to the lack of a precise determination of the injury limit, E2 increased the REGEN (+37.4%, P<0.05) to a similar extent as in the perivascular injury model (not shown). Indeed, a two-way ANOVA did not reveal any interaction between the E2 effect and the model used. Furthermore, the percentage of BrdU-positive cells in REGEN was very similar in E2 and placebo-treated mice (35.7 ± 3.2% and 36.0 ± 2.1% respectively, P=0.87, not shown), which is very close to the observations made in the perivascular injury model.

Early events in reendothelialization: endothelial migration precedes proliferation -effect of E2

To characterize the initial cellular responses in endothelial regrowth with respect to cell migration and proliferation, we used the perivascular model followed by repeated intraperitoneal administrations of BrdU on average every 16h. At 24 h post-injury, neither endothelial cell migration nor cell proliferation was observed in both E2-and placebo-treated mice (data not shown).

At 36 h after injury, the front of endothelial cells had clearly started to colonize the injured area, and the reendothelialized surface was significantly increased by E2 (+50.4%, P<0.05) (Figure 5A and5B).

However, as for the observation at 24 h, no BrdU labeled cells were detected in any condition at 36 h post injury (Figure 5A). When the same analysis was performed 50 h post injury, cellular proliferation was observed in half (3/6) of the E2-treated mice, but never (0/6) in placebo-treated mice (Figure 5C). Of note, in BrdU positive E2 treated carotids, the percentage of labeled cells (39.2±5.0%) was already close to that observed at day 3 or day 5, indicating that the onset of proliferation quickly reached its maximal level.

The results shown in figures 5A and 5B clearly showed that migration of endothelial cells precedes the proliferative response, and that both of these processes appeared earlier under E2 treatment.

The fact that the RE at 36h was larger in E2-treated mice than in control mice in the absence of proliferation could be explained by several mechanisms. One possibility is that an equal number of cells had migrated into the injured area in both placebo and E2 treated mice, but that the cells in the latter group had an increased size and therefore covered a larger surface. To test this hypothesis, the cellular density in RE was measured by counting of propidium iodide stained nuclei in the intima of carotids 36h and 50h post-injury. Since we did not observe any significant difference in cellular density in the two conditions, this would imply that more cells are recruited in the healing process under E2 treatment. These cells could either derive from circulating cells and/or from increased migration of surrounding uninjured endothelial cells. In order to determine whether the recruitment and incorporation of bone-marrow derived cells could account for the observed difference, we performed transplantation of GFP-positive bone-marrow into wild type mice. The "en face" confocal microscopy technique offers the opportunity to precisely determine the percentage of bone-marrow derived cells at different levels in the artery wall. Unexpectedly, E2 did not increase the density of GFP positive cells in the RE (3.4±1.2%,) compared to placebo (4.2±0.9%), NS (data not shown), although it increased the total number of GFP positive cells in the REGEN in proportion to the increase in its size, as previously reported [START_REF] Iwakura | Estrogen-mediated, endothelial nitric oxide synthase-dependent mobilization of bone marrow-derived endothelial progenitor cells contributes to reendothelialization after arterial injury[END_REF][START_REF] Strehlow | Estrogen increases bone marrow-derived endothelial progenitor cell production and diminishes neointima formation[END_REF]. However, we found that E2 engages the migration of more endothelial cells from the uninjured area into the injured area. Indeed, as shown in figure 5C, depicting data from carotids 50 hours post injury, E2 decreased the cell density further upstream the injury limit (in the retrograde uninjured endothelium) than did the placebo. Noteworthy, the decrease in the cell number in this region coincided with the increase in cells observed downstream the injury limit (in the RE) in E2-treated mice. Thus, E2 mobilized a larger retrograde area and thereby a larger number of endothelial cells to migrate.

Taken together, these results demonstrate that endothelial cell migration constitutes an early cellular response to injury which precedes endothelial proliferation, and that E2 generated an earlier and enhanced action of both of these processes through the recruitment of a larger area of uninjured endothelium. As the surface of uninjured "committed" endothelium, with decreased cellular density, was considerably larger than the retrograde proliferating zone (RetroP) at both day 2 (figure 5C) and at day 3 (not shown), we next sought to examine the evolution of RetroP at a later time point.

Accentuation of the retrograde proliferation at day 5 post injury in Placebo-and E2-treated mice.

The size of RetroP at day 5 was significantly larger than at day 3 both in placebo and E2 treated mice (Figure 6) and corresponded well with the size of the "committed zone", exhibiting a decreased cell density at day 3 (not shown). The size of both RE and RetroP in both placebo and E2 treated mice increased progressively and significantly from day 2 to day 3 and from day 3 to day 5 (P<0.05)(Figure 6B). Moreover, both RE and RetroP were significantly larger under E2 treatment compared to placebo at all time points (Figure 6B). These analyses by EFCM confirmed the effect of E2 on reendothelialization obtained by Evans blue staining (Figure 1). At Day 30, when reendothelialization was completed for about 3 weeks, no proliferation was observed in both placebo-and E2-treated mice (not shown).

In summary, these data suggest that the decreased endothelial cell density observed in the retrograde uninjured endothelium at earlier time points give rises to a subsequent proliferative response covering a similar surface. E2 not only increased the RE corresponding to the previously reported acceleration of reendothelialization, but it clearly promoted the progressive recruitment of a retrograde zone in the adjacent uninjured endothelium (RetroP) which substantially contributed to the regenerative capacity of the REGEN.

E2 does not alter medial smooth muscle regeneration

We also explored the regeneration of the medial layer at day 1, 3 and 5 post-injury in response to E2. As in placebo-treated mice, the limit of injury remained abrupt, with no visible migrating cells and with no change in smooth muscle cell density, < 1% of proliferating smooth muscle cells, and no GFP positive cells could be detected in the injured media at variance with the intima and adventitia (not shown). Altogether, these data confirm that the limit of injury can be reliably defined by the front of the smooth muscle cells. artery associated to the ligature of external carotid artery, rapidity and reliability (thrombosis rate 5-10% versus 30% in the endovascular). Moreover, perivascular electric injury can be easily transferred from one experimenter to another and requires less operator skill than the endovascular injury. Taken together, all these similarities allowed us to conclude that perivascular electric injury represents a valid and alternative model to endovascular injury.

(ii)

More than two decades ago several researchers, as Reidy, Clowes, Schwartz, and others [START_REF] Clowes | Kinetics of cellular proliferation after arterial injury. III. Endothelial and smooth muscle growth in chronically denuded vessels[END_REF][START_REF] Haudenschild | Endothelial regeneration. II. Restitution of endothelial continuity[END_REF][START_REF] Reidy | Endothelial regeneration. III. Time course of intimal changes after small defined injury to rat aortic endothelium[END_REF][START_REF] Schwartz | Endothelial regeneration. I. Quantitative analysis of initial stages of endothelial regeneration in rat aortic intima[END_REF][START_REF] Spagnoli | Morphometric analysis of gap junctions in regenerating arterial endothelium[END_REF] beautifully described different cellular events involved in reendothelialization models of endovascular artery injury in the rat or the rabbit. In concordance with our data, Schwartz and colleagues showed that proliferation (using 3H-thymidine incorporation) of endothelial cells from each side of the adjacent uninjured area was not restricted to the front edge, but involved up to 100 rows of endothelial cells, contributing to a "regeneration zone" [START_REF] Schwartz | Endothelial regeneration. I. Quantitative analysis of initial stages of endothelial regeneration in rat aortic intima[END_REF]. Using BrdU incorporation, we observed here in mouse a similar "regenerative area" (REGEN). At variance with the study by Schwartz et al., we did not observe any proliferating cells 24h or even 36 h post-injury. It is possible that this divergence could in part be due to differences between blood vessels (carotid versus aorta) or species (mouse versus rat). Speciesdependent differences in regenerative capacity have indeed been reported. The endothelium of the rabbit carotid is, for instance, less prone to heal than that of rats [START_REF] Reidy | Endothelial regeneration. V. Inhibition of endothelial regrowth in arteries of rat and rabbit[END_REF] Not surprisingly, we observed that the regenerative process was also characterized by marked alterations of gene expression. It is of interest to note that these changes appear over the entire REGEN including both injured and uninjured areas. Increased eNOS expression was reported 10 years ago by Berk and coworkers in the regenerative area after rat carotid injury [START_REF] Poppa | Endothelial NO synthase is increased in regenerating endothelium after denuding injury of the rat aorta[END_REF]. The expression of eNOS is also increased in proliferating cultured endothelial cells [START_REF] Arnal | Regulation of endothelial nitric oxide synthase mRNA, protein, and activity during cell growth[END_REF]. More recently, Losordo and colleagues demonstrated that the eNOS is also indispensable in the accelerative effect of E2 on reendothelialization [START_REF] Iwakura | Estrogen-mediated, endothelial nitric oxide synthase-dependent mobilization of bone marrow-derived endothelial progenitor cells contributes to reendothelialization after arterial injury[END_REF] even though it is independent of the NO producing activity of eNOS [START_REF] Billon | The estrogen effects on endothelial repair and mitogen-activated protein kinase activation are abolished in endothelial NO synthase knockout mice, but not by NO synthase inhibition by N-nitro-L-arginine methyl ester (L-NAME)[END_REF].

(iii) We also explored the regeneration of the smooth muscle cells in the injured media in the perivascular electric model. Surprisingly, at variance to the rapid endothelial healing, we could not detect any significant features of migration or proliferation of the adjacent uninjured smooth muscle cells during the first week, neither infiltration of bone marrow-derived cells in the media. We can not provide an explanation for the absence of rapid regeneration of the media. The high expression of eNOS in the regenerated endothelium could be one contributing factor, as elevated eNOS levels are known to inhibit smooth muscle cell proliferation and migration [START_REF] Janssens | Human endothelial nitric oxide synthase gene transfer inhibits vascular smooth muscle cell proliferation and neointima formation after balloon injury in rats[END_REF][START_REF] Der Leyen | Gene therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene[END_REF]. The absence of the underlying smooth muscle cells and the lack of their regeneration (at least during the first week) have two major consequences. First, only the electric injury model allows to precisely define the limit of the injury, and thereby to further divide REGEN into a reendothelialized area (RE) and a "retrograde proliferating zone" (RetroP). Second, it suggests, given the similarities between the results observed in the two models of reendothelialization, that the underlying smooth muscle cells in the injured area do not appear to be of importance in endothelial healing. However, it does not rule out that those in the RetroP could still play an important role. The suggested lack of role of the smooth muscle cells in the injured zone for both basal and estradiolinduced reendothelialization (Figure 1) should not be interpreted as a general conclusion concerning the mechanisms involved in other treatments that influence the kinetics of endothelial healing.

(iv) At day 3 post-injury, the enlargement of REGEN by E2 (Figure 4A) was the outcome of an increased surface of both the RE and the RetroP, a zone expanding the REGEN into the adjacent uninjured area. E2 treatment did not alter the percentage of the BrdU-positive cells in the RE, but increased this percentage in the RetroP to a level similar to that observed in the RE (Figure 4B). Thus, E2 recruited cells to proliferate mainly by increasing in the size of the proliferative cell area (REGEN = RE + RetroP).

These phenomena were magnified at day 5 post injury, in particular the effect of E2 on RetroP (Figure 6).

Earlier time points revealed that E2 initially accelerated cell migration, leading to a decrease in cellular density in the upstream uninjured endothelium over a distance that was 3-fold longer under E2 (Figure 5C). This is likely to be directly contributing factor to the increased size of RetroP, defined by proliferating cells, observed after E2 treatment (Figure 6). Thus, the spatio-temporal "commitment" of the adjacent uninjured endothelium occured earlier under E2, leading to a larger and precocious RetroP, which could represent a key event in reendothelialization and the emergence of REGEN.

Whereas E2 is known to promote endothelial cell migration and proliferation in vitro [START_REF] Geraldes | Estrogen regulation of endothelial and smooth muscle cell migration and proliferation: role of p38 and p42/44 mitogen-activated protein kinase[END_REF][START_REF] Geraldes | Specific contribution of estrogen receptors on mitogen-activated protein kinase pathways and vascular cell activation[END_REF][START_REF] Morales | Estrogen promotes angiogenic activity in human umbilical vein endothelial cells in vitro and in a murine model[END_REF], the present study is, to our knowledge, the first demonstration of a clear distinction between E2-stimulated migration and proliferation of endothelial cells in vivo. It was initially believed that E2 accelerated reendothelialization mainly through a local effect on adjacent residing uninjured endothelium. However, several studies have shown that arterial healing can be significantly modulated by circulating bone marrow (BM)-derived cells as monocyte-macrophages [START_REF] Fujiyama | Bone marrow monocyte lineage cells adhere on injured endothelium in a monocyte chemoattractant protein-1-dependent manner and accelerate reendothelialization as endothelial progenitor cells[END_REF], platelets [START_REF] Massberg | Platelets secrete stromal cell-derived factor 1alpha and recruit bone marrow-derived progenitor cells to arterial thrombi in vivo[END_REF] or endothelial progenitor cells (EPCs) [START_REF] Aicher | Mobilizing endothelial progenitor cells[END_REF][START_REF] Asahara | Endothelial progenitor cells for postnatal vasculogenesis[END_REF][START_REF] Rafii | Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration[END_REF]. E2 increases the number of circulating EPCs as well as the incorporation of BM-derived cells in the reendothelialized area [START_REF] Iwakura | Estrogen-mediated, endothelial nitric oxide synthase-dependent mobilization of bone marrow-derived endothelial progenitor cells contributes to reendothelialization after arterial injury[END_REF][START_REF] Strehlow | Estrogen increases bone marrow-derived endothelial progenitor cell production and diminishes neointima formation[END_REF]. However, the present work does not suggest that the incorporation of these BM-derived cells substantially contribute in the endothelial regeneration process, as the percentage of GFP positive cells was low (about 4%) and did not differ in E2 or placebo treated mice.

We can not exclude a potential role of non-BM derived progenitor cells, since homing of these cells seems important in neovascularization after hind leg ischemia [START_REF] Aicher | Nonbone marrow-derived circulating progenitor cells contribute to postnatal neovascularization following tissue ischemia[END_REF]. Third, platelets are abundant in the deendothelialized area [START_REF] Brouchet | Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta[END_REF][START_REF] Massberg | A crucial role of glycoprotein VI for platelet recruitment to the injured arterial wall in vivo[END_REF] and release factors that can directly [START_REF] Pakala | Mitogenic effect of serotonin on vascular endothelial cells[END_REF] or indirectly [START_REF] Massberg | Platelets secrete stromal cell-derived factor 1alpha and recruit bone marrow-derived progenitor cells to arterial thrombi in vivo[END_REF] contribute to accelerate reendothelialization. Indeed, we previously reported that BM FGF2 is absolutely required in the accelerative effect of E2 in reendothelialization, whereas extra-BM FGF2 is dispensable [START_REF] Fontaine | Essential role of medullar FGF2 in the effect of estradiol on reendothelialization and EPCs mobilization[END_REF], suggesting that FGF2 is a key mediator of the E2 action at the level of the BM. The "en face" analysis by EFCM, does not suggest that BM-derived cells contribute substantially as "cobble stones" of the regenerated endothelial "pavement", at least not in the repair process in the models used in the present study, in which the injury is limited to 4 mm and reendothelialization completed after one week. This does not exclude that the incorporation of BM-derived progenitor cells in the intima could be of importance in the repair of arterial injuries spanning larger surfaces for which healing takes several weeks or even stays incomplete.

Nonetheless, despite the scarcity of these cells, they could play an important role through a paracrine action. The identification of the specific BM-derived cell populations responsible for the reendothelialization process will require the development of the cell-specific inactivation of ERα in these BM-derived populations, tools that are not yet available.

endothelial zone to proliferate (RetroP), contributing to an enlargement of the "REGENerative area" and to an acceleration of reendothelialization. A similar scenario ought henceforth to be considered in other situations of accelerated reendothelialization (eg. by statins) [START_REF] Walter | Statin therapy accelerates reendothelialization: a novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells[END_REF]. We believe that the perivascular electric injury coupled with "en face" confocal microscopy (EFCM) will represent a powerful model to further investigate the cellular mechanisms underlying the reendothelialization process. A better understanding of the mechanisms of artery healing will help to propose new strategies to optimize post-angioplasty and instent reendothelialization. An enlargement of the "regenerative area" and the "commitment" of surrounding uninjured tissue, endothelial or not, ought also to be considered in other healing processes. C: Intima tunica with immunostaining against vWF or CD31.

Nuclei appear in blue. As in Figure 2, the sharp limit allow to perfectly define the initial site of the injury, and subsequently to precisely define the "reendothelialized area" (RE), injured intimal area colonized by new endothelium, indicated by the full arrowheads. BrdU incorporation allowed to define a "Regenerative endothelial area" (REGEN), indicated by the empty arrowheads. REGEN was enlarged by E2 as a consequence of an increase in both the RE and the "retrograde proliferating zone" (RetroP). C: Intimal cellular density around the site of injury, 50 h post-injury. The number of propidium iodidestained nuclei were counted in each 75µm portion, upstream (-) of and downstream (+) from the injury limit. E2 decreased the cell density further in the retrograde uninjured endothelium than did the placebo.

Thus, the larger retrograde mobilization of endothelial cells to migrate by E2 accounted for the main increase of cells downstream the injury limit and thus a larger reendothelialized area.

  All procedures involving experimental animals were performed in accordance with the principles and guidelines established by the National Institute of Medical Research (INSERM). They were housed in cages in groups of 5, kept in a temperature-controlled facility on a 12-hour light-dark cycle, and fed normal laboratory mouse chow diet. At 4 weeks of age, C57BL/6J mice (Charles Rivers, L'arbresle, France) were ovariectomized, and pellets releasing either placebo or E2 (17β-estradiol 0.1mg, 60 days release, i.e. 80 µg/kg/d, Innovative Research of America, Sarasota, FL) were implanted subcutaneously in the back of the animals.

Figure 1 .

 1 Figure 1. Effect of estradiol (E2) on the reendothelialization process evaluated by Evans blue staining 3 and 5 days after endovascular or perivascular injury.

Figure 2 .

 2 Figure 2. Visualization of the intima of carotid arteries 3 days after endovascular (left) or perivascular (right) injury in ovariectomized mouse. Top: Representative Evans blue staining of the remaining deendothelialized area in carotids harvested 3

Figure 3 .

 3 Figure 3. Effect of E2 on reendothelialization 3 days after perivascular electric injury in ovariectomized mice treated with E2 or placebo (Pl).A: Representative carotids harvested 3 days after injury and Evans blue staining of the remaining

Figure 4 .Figure 5 .

 45 Figure 4. Quantification of the effect of E2 on reendothelialization evaluated after perivascular electric injury. "En face" confocal immunohistochemical analysis of the intima tunica allowed the definition and measurement of:A: the respective length of the "reendothelialized area" (RE), "retrograde proliferating zone" (RetroP) and

Figure 6 .A:

 6 Figure 6. Accentuation of the retrograde proliferation 5 days post-injury in placebo-and E2-treated mice.A: Representative image of intima tunica 5 days after injury. In order to visualize cells in proliferation,

Acknowledgments:

The work was supported in part by Université Toulouse III and Faculté de Médecine de Toulouse-Rangueil, INSERM, the European Vascular Genomics Network No. 503254, Agence Nationale de la Recherche ANR-05-PCOD-011-01 "E2 and artery", the Fondation de France, the Fondation de l'Avenir, and the Conseil Régional Midi-Pyrénées and Aquitaine in France. We thank Cochard P., Jauneau A., Martinez Y and. Knibiehler B for the support and analysis of confocal data, Barreira Y. at the I2MR animal facility, Bayard F. for fruitful discussions and Fouque M.J. for excellent technical assistance. C.F. was supported by a grant from the Groupe de Réflexion et de Recherche Cardiovasculaire and V.F. was supported by a grant from the Nouvelle Société Française d'Athérosclérose.

The work was supported in part by

Discussion

The main achievements of the present study are:

The conventional endovascular and the perivascular electric injury model were found to be undistinguishable in terms of basal and E2-elicited reendothelialization.

(ii) A "REGENerative area" characterized by endothelial cell proliferation and gene expression changes is similar in both of the mouse models, and E2 elicit an enlargement of this REGEN.

(iii)

In contrast to the endovascular model, the underlying smooth muscle cells are destroyed in the injured area of the perivascular electric model, and do not regenerate during the first week, allowing to precisely define the injury limit. This permitted to subdivide REGEN into a reendothelialized area (RE, in the uninjured area) and a retrograde proliferating zone (RetroP, in the adjacent uninjured endothelium).

(iv) E2 strongly increased the retrograde commitment of adjacent uninjured endothelium, which is first distinguished by the mobilization of endothelial cells and a decrease in cellular density, followed by a proliferative response. This increase of RetroP, in addition to the increase of RE, contributed to the enlargement of the "regenerative area", which are likely to represent key events in the acceleration of reendothelialization.

(i) Endovascular and electric perivascular injuries can be similar in their efficiency to destroy the endothelium, but they differ significantly in their effects on the underlying cell populations in the vessel wall. Endovascular injury preserves most of the adventitial cells as well as most of the medial smooth muscle cells [START_REF] Lindner | Mouse model of arterial injury[END_REF]. In both models the intima is regenerated after one week, however in the perivascular model, the media does not show any sign of regeneration during the time of reendothelialization. As both models had similar characteristics with respect to basal and E2-elicited reendothelialization (evaluated by Evans Blue staining), changes in gene expression and proliferative response (see below), this would suggest a minor, if any, role of the smooth muscle cells in the injured area in the reendothelialization process. However, smooth muscle cells in the RetroP area might play an important role (see below). The perivascular model offers various major advantages, such as no modification of the flow in the carotid


Annie Britton
email: <a.britton@ucl.ac.uk>

Martin J Shipley

Archana Singh-Manoux

Michael G Marmot

Britton Annie

Shipley Martin

Singh-Manoux Archana

Successful aging: the contribution of early-life and midlife risk factors

Keywords: Adult, Aging, psychology, Female, Health Behavior, Health Promotion, Health Status, Humans, Life Style, Logistic Models, London, epidemiology, Male, Middle Aged, Risk Factors, Social Support, Socioeconomic Factors Aging, cohort studies, health behaviors, inequalities INTRODUTION

published or not. The documents may come

Successful aging: the contribution of early-life and midlife risk factors.

1
but it is also a challenge in terms of provisions for health care and continued healthy functioning for this growing group of individuals. Thus, it is important to ensure that these extra years are not only free from major disease but that there is maintenance of mental and physical functioning. This will reduce the enormous economic and social responsibility faced by the population. It is estimated that nearly half of lifetime health care expenditure is realized during the post-65 years ( ) and the total US per capita costs due to aging are projected 2 to increase by 18 percent between 2000 and 2050 ( ). 3

One of the most commonly used terms to describe a good old age is successful aging , often attributed to R. J. Havighurst in the " "

1960s, who defined it as adding life to the years ( ). Interest in successful aging remained high though the successive decades and there " " 4 was a general realization among biomedical researchers that of life may be as important as of years added to life ( ). quality quantity

Whilst a precise definition of successful aging has not been agreed, there is general consensus that it includes freedom from chronic disease and the ability to continue to function effectively, both physically and mentally, in old age ( , ). 6 7 Key research and policy areas include investigating the factors that permit individuals to continue to function effectively, both physically and mentally, into older age and determining whether these factors are modifiable. Prospective epidemiological studies with substantial numbers of participants with phenotypic information useful for gerontololgical research are rare but essential to identify risk factors for health and survival at older ages ( ). Previous research has shown income ( ), education ( , , ), ethnicity ( , ), exercise ( 8 9 8 10 11 9 10 , , , ), diet ( ), smoking habits ( , , , , , ) and social networks ( , ) all to be related to successful aging. The 10 12 13 14 14 8 9 14 15 16 17 10 12 relative importance of these factors remains unclear and our primary objective here was to compare early and mid-life predictors of successful aging.

METHODS

The For the analysis of successful aging we included the 5,823 men and women with no prevalent disease at phase 1, who had measures of functioning at phase 7 and who had attended five or more phases of follow-up. The latter exclusion was to reduce potential reporting bias since those who attend most phases have the greatest opportunity for declaring the presence of major diseases. Any residual confounding due to this was removed by adjusting for the number of phases attended in all analyses. Prevalent disease at Phase 1 was defined as a self-report in the questionnaire on coronary heart disease (CHD), cancer, diabetes or depression. The incidence of disease (CHD, stroke, cancer, diabetes, depression and the ATPIII metabolic syndrome) was determined from all relevant data collected between Phase 1 and Phase 7 from self-reports in questionnaires, medication use and from clinical examinations (metabolic syndrome) together with supporting evidence from general practitioners and hospitals (CHD). Good functioning at Phase 7 was defined as being in the best third of the sex-specific distribution for three or four of the four measures: Walking speed, lung function, Alice Heim 4-I (AH4-I) cognitive test and Physical Component Score of Short Form36 General Health Survey ( ) (or two or three in the top third for the 22 3 10 marked eight foot walking course. Participants were asked to walk to the other end of the course at your usual walking pace, just as if you

"
were walking down the street to go the shops. Walk all the way past the other end of the tape before you stop . Times were recorded in " seconds to two decimal places. Three tests were conducted and the mean walk time was used in the analysis. Lung function was assessed using forced expiratory volume (FEV) which is the volume of air expelled in the first second of a forced expiration starting from full inspiration ( ) The AH4-I is composed of a series of 65 verbal and mathematical reasoning items of increasing difficulty. This is a test of 23 inductive reasoning that measures the ability to identify patterns and infer principles and rules ( ). 24

Statistical analysis

Logistic regression analyses were performed, separately in men and women, to determine the association between phase 1 factors and successful aging at phase 7. All analyses were adjusted for age at Phase 1 and the number of phases attended (5, 6 or 7) to remove potential reporting bias since participants who came to more phases had more opportunities to report adverse health outcomes. The overall effect of early life factors, midlife health behaviors and psychosocial factors were summarized by creating a score for each from the individual factors (see ). A high score on each summary measure indicates a more favourable level. The effect of each of these on appendix successful aging was expressed per 1 standard deviation change in each score.

Sensitivity analyses

We undertook additional sets of sensitivity analyses using slight variations on the definition of successful aging in order to assess whether it influenced the role of risk factors for successful aging. First, successful aging was defined using only the disease criteria and then only the functioning criteria. Second, the occurrence of metabolic syndrome was excluded from the definition of disease in recognition that it measures a constellation of risk factors not a clinical disease. Finally, cognitive functioning was excluded from the definition of successful aging.

RESULTS

Of the 10,308 participants in the study, 402 (6 percent) men and 238 (7 percent) women had prevalent disease at Phase 1. Of the remaining subjects, 77 percent (5179 men and 2231 women) attended five or more of the seven phases of the study. A further 1039 men and 548 women were excluded because they either did not attend Phase 7 or had missing data on functioning at Phase 7, or had unknown metabolic syndrome status at all phases. The remaining 5823 subjects (4140 men and 1683 women) form the sample for this analysis. The 4485 subjects excluded from the analyses tended to be older (mean age at Phase 1, 45.1 years v 43.9), more often women (39 percent v 29 percent) and from the lowest SEP group (33 percent v 15 percent). Of the 5823 subjects in the analysis, 548 (12.8 percent) men and 246 (14.6 percent) women were successfully aging at phase 7. During follow up 2286 participants (38.9 percent) developed at least one of the specified major diseases (CHD, stroke, cancer, diabetes, depression, metabolic syndrome). ( ) Table 1 As we would expect, participants who were younger at the start of the study are more likely to be disease free and functioning well at follow-up (average age at Phase 7, of those who were successfully aging compared to those who were not was 57.5 vs. 60.8 for men and 57.3 vs. 61.3 for women). Therefore, all analyses are adjusted for age. SEP in midlife (employment grade) was strongly related to successful aging for both men and women. Those in the highest employment grades were much more likely to be successfully aging than those in the lowest grades (age adjusted odds ratio 7.06, 95 percent confidence interval: 3.4, 14.6 for men). As this effect is so strong and is related to many other risk factors, we present both the age and age and SEP adjusted odds ratios in . With age adjustment alone, table 2 education, height, smoking, diet, physical activity, decision latitude and work support were associated with successful aging for men. After additional adjustment for SEP, decision latitude was no longer statistically significant. For women, the affects of SEP adjustment were more pronounced. Height, smoking, alcohol, and physical activity remain associated with successful aging after adjustment for age and SEP.

The relative importance of early life factors, midlife SEP, and midlife behavioural and psychosocial factors are shown in . A table 3 one standard deviation improvement in grouped early-life factors is associated with an increased likelihood of successful aging (OR 1.41, 95 percent confidence interval: 1.27, 1.57 for men). When all the factors are adjusted for each other, the strongest effects are seen for SEP for both men and women, but independent, significant effects are seen for all other groupings except psychosocial factors in women. When stratified into two age categories (data not shown), early life and behavioural factors were more important for young men (35 44 at baseline) then older men (45 55 at baseline). There were no interactions with age among the women.

-

Sensitivity analysis

The sensitivity analysis (tables 2A, 2B, 2C, 2D), undertaken with different definitions of successful aging, do not substantially change our original conclusions, although there were changes in magnitudes of effects. For example, we found much stronger effects of social position when successful aging was defined as high functioning (table 2B) compared to when successful aging was defined as avoidance of disease, i.e. ignoring functioning. (table 2A). Exclusion of the metabolic syndrome made very little impact (table 2C). Excluding cognitive functioning from the definition reduced the effects of education and social position, but all other effects were broadly similar (table 2D).

Further analyses removing the adjustment for the number of phases attended made only a very small difference to the magnitude of the estimates presented. When the participants with less than five visits were included in the analyses, the odds ratio estimates of SEP in Table

were increased by at most 4 and the age and SEP adjusted odds ratios for all other factors showed only small changes. Analysis of the 2 % 535 deaths that occurred between Phase 1 and Phase 7 showed that low SEP was associated with increased mortality with the hazard ratio (95 CI) for low versus high SEP being 2.33 (1.71, 3.18) in men (p<0.001) and 1.67 (1.02, 2.72) in women (p 0.04).

% =

DISCUSSION

Position in the social hierarchy in midlife is strongly associated with the chances of entering early old age free from major disease and with good functioning. Independent of SEP, there are contributions from early life factors, midlife health behaviors and midlife psychosocial factors. Therefore, strategies aimed at attenuating the modifiable risk factors would lessen the inequalities dealt out in childhood and experienced throughout the life course and thus increase everyone s chances of living to a successful old age. This finding is ' supported by previous analyses of cohorts in which successful aging was predicted by variables assessed before the age of 50 and by several factors that were under some personal control and therefore potentially modifiable ( ). 11

Although a precise definition of successful aging does not yet exist, most researchers agree that it needs to include freedom from chronic disease and high function ( , ). In the analysis shown in this paper we have chosen to interpret this broad definition to include 6 7

high physical and cognitive functioning. The decision to include good cognition in our definition of successful aging was based on the fact that increasing lifespan makes cognition an increasingly important health measure for older adults. In developed countries, dementia prevalence is around 1.5 at age 65 years, and doubles every 4 years to reach 30 at 80 years of age. ( ) Thus, it is important to consider % %

25

cognitive function in research on successful aging, particularly one that is subject to age related decline. ( ) However, in order to ensure 26 that our results were not driven by one aspect of our definition of successful aging, we undertook sensitivity analysis using different definitions of successful aging. These results suggest no major differences in the importance of the predictors of successful aging. For instance, removing cognition from the definition reduces the importance of social position somewhat but it still remains a strong predictor of successful aging.

Examination of the sensitivity of our results to the effect of removing and controlling for those participants who did not attend all phases of the study showed that the effects of social position may have been marginally underestimated but had little effect on the other factors. In addition, attrition of the cohort due to mortality is greater among those of lower social position and reinforces our observations that those of higher social position age more successfully. Hence, the gradient of successful aging with socio-economic position would be increased if the definition of successful aging also included death.

There were some differences between the importance of predictors in men and women ( ). In the early life factors, education was table 2 important for men and height, reflecting childhood factors, was more important in women. Among the health behaviours, diet was important for men and alcohol for women. Finally, among the psychosocial factors, work support did not play a role among women. These gender differences in the importance of predictors for successful aging warrant further investigation. It is important to note that all predictors were assessed very early in midlife (average age for men was 43.8 and for women 44.3 years) and their impact on successful aging might vary later in the lifecourse. This is likely to be particularly true of measures of social support and social network, which are likely to become important for older adults.

There are a number of limitations to this study. Caution is needed when extrapolating results to the whole population as the study consisted of London-based office workers. In particular, we did not have sufficient numbers to look at the effects of ethnicity. Secondly, we did not look at the probability of having good physical and mental functioning in later life among those with chronic disease as we excluded prevalent disease at baseline. It would be interesting to explore the factors that enable those with co-morbidities and disabilities to continue to function and maintain independence. This is still considered to be successful aging by some ( ). A strength of this study is " " 5 that disease incidence was determined by ongoing surveillance and exposure variables measured before disease diagnosis. Therefore the sequence of events is clear, and we have avoided the problem of reverse causation whereby health behaviors change due to disease labeling.

The importance of socioeconomic factors for health through the life-course is well documented ( , ). However, the role of 18 27 socioeconomic factors in predicting successful aging remains little explored. One exception to this general rule is the investigation into predictors of dementia in old age. Education is widely believed to have a protective effect on dementia ( , ). Data from a 28 29 population-based study were recently used to develop an equation for the prediction of the risk of late-life dementia in people of middle age ( ). Age, low education and vascular risk factors like hypertension, hypercholesterolemia, and obesity in mid life were found to be 30 highly predictive of dementia during the 20-year follow-up period.

Results from the Berlin Aging Study showed successful aging to be relatively free from the effects of social class ( ). However, as 31

the analysis was based on those over 70 years of age it was necessarily restricted to those who had survived up to this age. It is possible that the effects of socioeconomic position are diluted when examined amongst the oldest old. Factors believed to protect against dementia at earlier times have been found to have little effect at the end of life ( ). The focus of our analysis is on successful aging in early old age 32 and results show SEP to be an important correlate. Our measure of SEP is a global measure and is linked to education, income, cognitive complexity of occupation and perhaps other lifestyle factors. We show SEP to be associated with successful aging after adjustments for early life factors, health behaviors and some psychosocial factors. The precise mechanisms through which SEP influences disease and functioning outcomes needs further investigation. Certainly, the influence of factors associated with socioeconomic position is large.

In this study, we have shown that even with a disadvantaged start to life (including little education and poor parents), the likelihood of entering old age with enhanced physical and mental capabilities is influenced by midlife health behaviors and exposure to psychosocial stressors. From a public health perspective, our results provide a reflection both on the definition of successful aging and the predictors of " " successful aging. In terms of definition, it is vital to emphasize the importance of both a disease free and a high functioning status. The analysis on the predictors of successful aging suggests that targeting modifications to health behaviors in early midlife might be beneficial for successful aging. A focus on health behaviors might also have the consequence of reducing social inequalities in health at older ages.

  Whitehall II study was established in 1985 as a longitudinal population-based study to examine the socioeconomic gradient in health and disease among 10,308 civil servants (6,895 men and 3,413 women).( ) All civil servants aged 35 55 years in 20 London based 18 departments were invited to participate by letter. In total, 73 percent of those invited agreed to take part in Phase 1. Baseline examination (Phase 1) took place during 1985 1988, and involved a clinical examination and a self-administered questionnaire containing sections on

	£	£
	Early life factors (father s social class, age when left education and adult height) were ascertained from self-completed questionnaire '
	and clinic data. Adult height was used in this way as it has been shown to be associated with prenatal and childhood exposures. ( ) 19
	Measures of health behaviors at Phase 1: Smoking, exercise, diet, and alcohol were assessed by questionnaire. Smoking was grouped
	as current, ex-smoker and never-smokers. Exercise was derived from questions on frequency and number of hours per week taken in
	activities that were mildly energetic, moderately energetic or vigorous. These were grouped as vigorous or moderate if they did one or
	more hours per week of these, or as none/mild. A summary index of poor diet was defined if 2 or all of the following applied: most
	frequently used bread was white, usually used milk was whole, and fruit or vegetables were eaten less often than daily. Alcohol
	consumption in the last week was expressed in units of alcohol where 1 unit 8 grams ethanol. =
	Measures of psychosocial factors at Phase 1: The first set of factors were work based and consisted of the central components of the
	job strain model, that is, psychological job demands, decision latitude, and social support at work ( ). Four items dealt with psychological 20
	job demands, and 15 items dealt with decision authority and skill discretion, which were combined into an index of decision latitude (or
	job control). Social support at work is the sum of two subscales: support from co-workers and support from supervisor. Social network was
	assessed using an adapted version of the Berkman & Syme scale (1979). This scale assesses the frequency and the number of friends,
	relatives and work colleagues seen; and participation in social and religious groups; high scores indicating a bigger network ( ). Sample 21
	selection: Participants were classed as aging successfully if they were free from major disease up to Phase 7 and had good physical and
	mental functioning at Phase 7.

demographic characteristics, health, lifestyle factors, work characteristics, social support and life events. Clinical examination included measures of blood pressure, anthropometry, biochemical measurements, neuroendocrine function, and sub-clinical markers of cardiovascular disease. Subsequent phases of data collection have alternated between postal questionnaire alone (phases 2, 4, 6) and postal questionnaire accompanied by a clinical examination (phases 1, 3, 5 and 7). The median length of follow up from Phase 1 to Phase 7 was 17 years, with 535 individuals dying during this period. The clinical examination where physical functioning and cognitive tests were administered, phase 7, was attended by 6944 participants. The University College London Ethics Committee approved the study.

Measures of socioeconomic position (SEP) at phase 1: Based on salary and work role, the civil service defines a hierarchy of employment grades ranging from senior executive officers to clerical and support staff. This is a three level variable representing high (administrative grades), intermediate (professional or executive grades) and low (clerical or support grades) SEP. People in the three SEP groups differ with respect to salary, social status and level of responsibility. Although mostly white collar, respondents covered a wide range of SEP, with annual salaries in 1995 ranging from 4,995 to 150,000.

TABLE 3

 3 All odds ratios are adjusted for number of phases attended in addition to adjustments given Odds ratio of successful aging associated with a one standard deviation improvement in socio-economic position, early life factors, health behaviors and psychosocial factors. Adjusted for age and all the other factors in the table + All odds ratios are adjusted for number of phases attended in addition to adjustments given

	(

* *

[START_REF] Strawbridge | Successful aging: predictors and associated activities[END_REF]

[START_REF] Strawbridge | Successful aging: predictors and associated activities[END_REF]

[START_REF] Strawbridge | Successful aging: predictors and associated activities[END_REF]

[START_REF] Strawbridge | Successful aging: predictors and associated activities[END_REF]

[START_REF] Strawbridge | Successful aging: predictors and associated activities[END_REF]

Ackowledgements:

The Whitehall II study has been supported by grants from the British Medical Research Council; British Economic and Social Research

Footnotes:

All authors designed the study, were involved in data acquisition and prepared the text. Dr. Britton conducted the Author Contributions: literature review and wrote the first draft. Mr Shipley was primarily responsible for statistical analyses. Professor Marmot is responsible for quality assurance and control.

None.

Sponsor s Role: % * Among those who had no prevalent disease at Phase 1, who attended 5 or more Phases of follow-up and had measures of functioning Phase 7.

† Good functioning is defined as being in the best third of the sex-specific distribution for three or four of the four measures (or two or three in the top third for the 26 of subjects who only had three of % the four measures).