Sreedhar Kari

Harald Berger
email: harald.berger@mb.uni-magdeburg.de

Ulrich Gabbert

Raul Guinovart-Dıaz

Julian Bravo-Castillero

Reinaldo Rodrıguez-Ramos

Julian Bravo- Castillero

Evaluation of influence of interphase material parameters on effective material properties of three phase composites

Keywords: A. Fibers, A. Coating, B. Interphase, B. Modeling, B. Mechanical properties

In this paper, evaluation of effective material properties of different types of three phase composites (reinforcement, matrix and in between interphase) using numerical homogenization techniques (representative volume element (RVE) approach) based on finite element method is presented. The influence of interphase parameters like stiffness and volume

fraction of interphase on effective material properties of transversely randomly distributed uni-directional fiber composites and randomly distributed spherical particle composites is studied. The RVEs, required for transversely randomly distributed uni-directional fiber composites and randomly distributed spherical particle composites, are generated using a modified random sequential adsorption (RSA) algorithm with periodicity on opposite boundary surfaces. The numerical results are compared with analytical methods like

Introduction

Interphases, or interfacial zones, in fiber reinforced composite materials are the thin layers between the fiber and the matrix. These interphases are formed due to, for example, chemical reaction between the matrix and fiber materials or the use of protective coatings on the reinforcements during manufacturing to ensure good adhesion between reinforcement and matrix material. Although small in thickness, interphases can significantly affect the overall mechanical properties of the fiber or particle reinforced composites. The effective utilization of the fiber reinforced composites depends on efficient load transfer from the matrix to reinforcements through these interphases. It is the weakest link in the load path, and consequently most failures in fiber or particle reinforced composites, such as debonding and matrix cracking, occur in or near this region. Thus, it is crucial to fully understand the mechanism and effects of the interphase material parameters on the overall material properties of composites. The micro mechanical method provides the overall behavior of the three phase composites from the known properties of their constituents (fiber, matrix and interphase) through an analysis of a periodic representative volume element (RVE) or a unit cell model.

The effective material properties of two phase randomly distributed fiber and particle reinforced composites are extensively studied by using analytical and numerical analysis [START_REF] Berger | An analytical and numerical approach for calculating effective material coefficients of piezoelectric fiber composites[END_REF][START_REF] Berger | Unit cell models of piezoelectric fiber composites for numerical and analytical calculation of effective properties[END_REF][START_REF] Böhm | Multi-inclusion unit cell models for metal matrix composites with randomly oriented discontinuous reinforcements[END_REF][START_REF] Segurado | A numerical approximation to the elastic properties of spherereinforced composites[END_REF][START_REF] Gusev | Representative volume element size for elastic composites: a numerical study[END_REF][START_REF] Gusev | Fiber packing and elastic properties of transversely random unidirectional glass/epoxy composite[END_REF][START_REF] Hine | Numerical simulation of the effects of volume fraction, aspect ratio and fibre length distribution on the elastic and thermoelastic properties of short fibre composites[END_REF][START_REF] Guinovart | Closed-form expressions for the effective coefficients of a fiber-reinforced composite with transversely isotropic constituents-I Elastic and hexagonal symmetry[END_REF]. Pagano and Tandon [START_REF] Pagano | Elastic response of multidirectional coated fiber composites[END_REF][START_REF] Pagano | Thermo-elastic model for multidirectional coated-fiber composites: Traction formulation[END_REF] approximate the effective properties of thermoelastic composites with coated fibers using unit cell approach. They estimate the influence of coating of fiber on overall thermoelastic properties of composites. Dasgupta and Bhandarkar [START_REF] Dasgupta | A generalized self-consistent Mori-Tanaka scheme for fiber-composites with multiple interphases[END_REF] proposed a method to obtain the effective properties of transversely isotropic thermoelastic composites reinforced with coated cylindrical fibers. Yeh [START_REF] Yeh | The effect of interphase on the transverse properties of composites[END_REF] studied the effect of interphase on the transverse material properties of elastoplastic composites. Also Lagache et al. [START_REF] Lagache | Role of interphase on elastic behavior of composite materials: Theoretical and experimental analysis[END_REF] studied the effect of interphase on effective material properties of three phase composites numerically. Liu et al. [START_REF] Liu | Modeling of interphases in fiber-reinforced composites under transverse loading using boundary element method[END_REF] have modeled the interphases in fiber reinforced composites and studied the influence of interphases using boundary element method.

Guinovart et al. [START_REF] Guinovart | Closed-form thermoelastic moduli of a periodic three phase fiber reinforced composite[END_REF] developed closed form expressions to evaluate the effective material properties of thermoelastic three phase fiber composites. Most of the work, which deals with three phase composites, is restricted to regular arrangement of fiber composites. The aim of this paper is to point out the influence of material properties and volume fraction of interphases on the overall mechanical properties of three phase composites using RVE approach. In this work, the conventional numerical homogenization technique reported by Berger et al. [START_REF] Berger | An analytical and numerical approach for calculating effective material coefficients of piezoelectric fiber composites[END_REF][START_REF] Berger | Unit cell models of piezoelectric fiber composites for numerical and analytical calculation of effective properties[END_REF] and Kari et al. [START_REF] Kari | Computational evaluation of effective material properties of composites reinforced by randomly distributed spherical particles[END_REF] is extended to evaluate the effective material properties and influence of interphase of complex microstructured three phase composites like transversely randomly distributed fiber and randomly distributed spherical particle composites. The consideration of randomly distributed inclusions opens new possibilities to investigate more realistic composites which incorporates automatically such effects like the influence of different distances between the particles. All calculations are made under the assumption of small strains and elastic material behavior. In order to validate our developed numerical homogenization tools, comparisons are made between our numerical results and results reported in literature for uni-directional regular arrangement of fiber composites.

RVE models for numerical homogenization techniques

The RVE models and corresponding finite element modeling used to evaluate the effective material properties of regular arrangement of uni-directional three phase fiber composites are shown in Fig. 1.

The three phase uni-directional transversely randomly distributed fiber composite RVE models are generated using our developed user defined FORTRAN algorithms which are based on modified RSA algorithm reported by Wang [START_REF] Wang | Random sequential adsorption, series expansion and Monte Carlo simulation[END_REF]. The generation of microstructure for this case is based on two-dimensional X 1 -X 2 plane. First, random coordinates are generated for the center of one circular disc with prescribed radius (fiber radius plus interphase thickness corresponding to given interphase volume fraction) and is deposited on the X 1 -X 2 plane. The next randomly generated coordinates of centers of circular discs are checked for non-overlapping condition with previously deposited discs. If there is no overlap between the current disc and previously deposited discs, and the periodicity is satisfied, then the current disc will be deposited on the X 1 -X 2 plane. This process will be terminated when desired volume fraction is achieved or when no more discs can be added because of the jamming limit, which can occur at a certain volume fraction in RSA algorithm. Fig. 2 shows an example of RVE model and the corresponding finite element model of three phase transversely randomly distributed uni-directional fiber composite. Also the RVE models of randomly distributed spherical particle composites with interphase between the particles and matrix are generated using modified RSA algorithm. The center distance between spherical particle i and all other particles, which are previously accepted j=1…i-1 have to exceed a minimum value (2*r+s), where r is radius of the spherical particle plus interphase thickness and s is the minimum distance between any two spherical particles, imposed by the practical limitations for creating an adequate finite element mesh. If any surface of the spherical particle i cuts any of the cubic RVE surfaces, this condition has to be checked with the spherical volumes on the opposite surfaces because the microstructure of the composite is periodic. Fig. 3 shows the RVE model and the corresponding finite element modeling of the three phase spherical particle composites.

Results and discussion

The method of evaluation of effective material properties of three phase fiber and particle reinforced composites using numerical homogenization techniques is similar to the method explained in Berger et al. [START_REF] Berger | An analytical and numerical approach for calculating effective material coefficients of piezoelectric fiber composites[END_REF][START_REF] Berger | Unit cell models of piezoelectric fiber composites for numerical and analytical calculation of effective properties[END_REF] and Kari et al. [START_REF] Kari | Computational evaluation of effective material properties of composites reinforced by randomly distributed spherical particles[END_REF]. For the detailed description of the numerical homogenization techniques and corresponding periodic boundary conditions used to evaluate the effective stiffness coefficients for different types of composites one can refer to [START_REF] Berger | An analytical and numerical approach for calculating effective material coefficients of piezoelectric fiber composites[END_REF][START_REF] Berger | Unit cell models of piezoelectric fiber composites for numerical and analytical calculation of effective properties[END_REF][START_REF] Kari | Computational evaluation of effective material properties of composites reinforced by randomly distributed spherical particles[END_REF]. The material properties of constituents of three phase composites are taken from the literature given by Benveniste et al. [START_REF] Benveniste | Stress fields in composites with coated inclusions[END_REF]. Table 1 shows the material properties of the fiber, matrix and interphase used in this work.

Influence of volume fraction for square and hexagonal fiber arrangement

The effective material properties are obtained by increasing the volume fraction of fibers up to 70% by keeping the volume fraction of interphases constant at 0.075%. The effective material properties obtained using numerical homogenization technique are compared with analytical asymptotic homogenization method reported by Guinovart et al. [START_REF] Guinovart | Closed-form thermoelastic moduli of a periodic three phase fiber reinforced composite[END_REF][START_REF] Guinovart-Díaz | Modeling of three-phase fibrous composite using the asymptotic homogenization method[END_REF][START_REF] Guinovart-Díaz | A recursive asymptotic homogenization scheme for multi-phase fibrous elastic composites[END_REF]. Also a comparison is made between the square and hexagonal arrangements of fiber composites. Fig. 4 shows these comparisons. There is a good agreement between the analytical results and numerical predictions for all volume fractions considered in this study.

Along the longitudinal direction, the type of arrangement of fibers does not influence the effective material properties. But along the transverse direction, the effective Young's modulus eff t E is having higher value when compared with hexagonal arrangement. This is due the fact of tetrahedral symmetry in square arrangement. The transverse shear modulus is having higher value for hexagonal arrangement when compared with square arrangement. This is due to the fact that hexagonal arrangement is having transverse isotropy symmetry.

Influence of interphase material property on overall behavior of composites

In order to study the influence of interphase material properties on overall material behavior, the effective material properties are calculated by increasing the interphase Young's modulus from 10 to 250 GPa. The effective material properties are evaluated by keeping volume fraction of fiber, interphase and matrix materials and individual material properties of fiber and matrix as constant. Fig. 5 and6 show the variation of effective material properties with increase of interphase Young's modulus for transversely randomly distributed unidirectional fibers at 30% volume fraction and randomly distributed spherical particle composites at 15% volume fraction. In these cases the volume fraction of interfaces is 1% for the uni-directional fiber model and 3.78% for the spherical particle model. From Fig. 5, it can be observed that the transverse effective Young's modulus increases rapidly with the increase of interphase Young's modulus up to a certain value (say 100 GPa) and from that the influence is not so high. But the effective longitudinal Young's modulus is not much affected by increase of stiffness of interphase (5% difference between the interphase Young's modulus of 10 GPa and 250 GPa). Similarly the transverse shear modulus and Poisson's ratio are affected severely if the interphase material stiffness is too low. From Fig. 6, it can be observed that the overall material properties of randomly distributed spherical particle composites are also affected in all directions by interphase material properties especially if the interphase Young's modulus is too low.

Influence of interphase volume fraction

In this section, the influence of interphase volume fraction on effective material properties is studied by increasing the volume fraction of interphase from 1% to 10% for different interphase Young's moduli as shown in Fig 7 .   From Fig. 7, it can be observed that, if the interphase Young's modulus is low, by increasing the volume fraction of the interphase, the effective material properties are decreasing. This effect is more significant in case of transverse shear modulus than in longitudinal shear modulus. But by considering the interphase Young's modulus is equal to Young's modulus of fiber, then the effective material properties are increasing with the increase of interphase volume fraction. Also a decrease of effective material properties with increase of interphase volume fraction can be observed for randomly distributed spherical particle composites with an interphase Young's modulus of 34.48 GPa. Fig. 8 shows the variation of effective material properties with increase of interphase volume fraction for randomly distributed spherical particle composites.

In general, in case of uni-directional transversely isotropic composites, the material properties along transverse direction are much influenced by the interphase material parameters like, stiffness and thickness of interphase, than the material properties along longitudinal direction. For randomly distributed spherical particle composites, the effective material properties are sensitive to interphase material parameters in all directions.

Conclusions

The effective material properties of three phase composites having regular fiber distrubution like square and hexagonal arrangement are evaluated for different volume fractions and results are compared with analytical asymptotic homogenization method. The effects of interphase volume fraction and their material stiffness on the overall behavior of transversely randomly distributed fiber composites and randomly distributed spherical particle composites are studied. From these studies it is observed that, if the interphase material is not stiff enough to transmit the load between the phases, the resulting overall material properties are significantly affected especially across the transverse direction for transversely randomly distributed uni-directional fiber composites and for isotropic randomly distributed spherical particle composites. So it is necessary to choose appropriate materials at the interfaces to get an efficient load transfer between the phases.

Figure Captions

Fig. 1 .

 1 Fig. 1. FE models of RVEs for three phase regular arrangement of fiber composites. (a)

Fig. 2 .

 2 Fig. 2. Geometry and FE modelling of RVE of three phase transversely randomly distributed

Fig. 4 .Fig. 5 .

 45 Fig. 4. Variation of effective material properties with change in volume fraction for three

Fig. 6 .

 6 Fig. 6. Variation of effective material properties with increase of interphase stiffness for

Fig. 7 .Fig. 8 .

 78 Fig. 7. Variation of effective material properties with change in volume fraction of interphase.

  Fig.1

Fig. 4

 4 Fig.4

Fig. 5 ACCEPTED MANUSCRIPT 20 Fig. 6

 5206 Fig.5

Fig. 7 ACCEPTED MANUSCRIPT 22 Fig. 8

 7228 Fig. 7

Table 1

 1 Material properties used to analyze the three phase composites

	Material	Young's modulus (GPa) Poisson's ratio
	Matrix (Nickel)	214	0.31
	Fiber/particle (Tungsten)	345	0.28
	Interphase (Carbon coating)	34.48	0.20

Acknowledgments

This work was supported by Graduiertenkolleg 828 'Micro-Macro Interactions in Structured Media and Particle Systems'. The support of project PNCB IBMFQ 09-2006 is also gratefully acknowledged.


A Argüelles

A F Canteli

M A Castrillo

J Bonhomme

Interlaminar crack initiation and growth rate in a carbon-fibre epoxy composite under mode I fatigue loading

Keywords: Delamination, fatigue, fracture, composite material

In this paper, interlaminar crack initiation and propagation under mode-I loading of a composite material made of an epoxy matrix reinforced with unidirectional carbon fibres are experimentally assessed for different asymmetry ratios. In the experimental program, DCB specimens were tested and the number of cycles to failure necessary for the onset of crack growth was registered for the asymmetry ratios R=0.2 and R=0.5. The G-N curves under fatigue loading were determined, N being the number of cycles at which delamination commences for a given energy release rate. The G-da/dN curves associated with the previously generated crack growth were also determined for different G cr rates (90%, 80%, 70%, etc.) and two different asymmetry ratios.

For the asymmetry ratios, the fatigue limits show values in the order of 50% and 70%, respectively, of the critical fracture energy resulting from the static tests employing the Modified Beam Theory.

The fatigue limit is discretionally taken as the fatigue strength corresponding to 2.5 million cycles, i.e., the limit number of cycles considered for testing. The micrographs show that the morphology of the fracture toughness under mode-I loading are quantitatively but not qualitatively influenced by the stress ratios.

INTRODUCTION

Composites made of laminates exhibit a worrying susceptibility to the appearance and growth of cracks between the layers. This phenomenon, known as delamination, represents one of the most life-limiting failure modes of laminated composites. Delamination can be caused either by defects induced during the manufacturing process or generated in the structural elements during the service life of the material due to interlaminar stresses that can lead to the commencement of delamination.

Additionally, delamination growth generally results in a loss of resistance and stiffness in the structural members leading to failure of the structure. Although several decades have elapsed since the recognition of the importance of interlaminar failure [START_REF] O'brien | Characterization of delamination onset and growth in a composite laminate[END_REF][START_REF]Standard tests for toughened resin composites[END_REF], it still remains a determining factor limiting the use of structural elements made of laminated composites [START_REF] Paul | Evolution of U.S. military aircraft structures technology[END_REF]. In recent years, a number of research projects have been conducted that focus on the determination of the characteristics of interlaminar failure under static loading [START_REF] Hojo | Effect of stress ratio on nearthreshold propagation of delamination fatigue cracks in unidirectional CFRP[END_REF][START_REF] Hansen | Dependence of intralaminar fracture toughness on direction of crack propagation in unidirectional composites[END_REF][START_REF] Brunner | Experimental aspects of Mode I and Mode II fracture toughness testing of fibrereinforced polymer-matrix composites[END_REF]. However, new developments, predominantly in the aerospace field, compel us to complete the information on the behaviour of these materials with regard to delamination under varying loading conditions [START_REF] Ashcroft | Mode I Fracture of Epoxy Bonded Composite Joints: 1. Quasi-Static Loading[END_REF][START_REF] Hojo | Modes I and II interlaminar fracture toughness and fatigue delamination of CF/epoxy laminates with self-same epoxy interleaf[END_REF][START_REF] Shivakumar | A total fatigue life model for mode I delaminated composite laminates[END_REF][START_REF] Mahdi | A Comparison of Oven-cured and Induction-cured Adhesively Bonded Composite Joints[END_REF][START_REF] Matsubara | Mode II fatigue crack growth from delamination in unidirectional tape and satin-woven fabric laminates of high strength GFRP[END_REF][START_REF] Qiao | Mode-II Fatigue Fracture of Wood-Composite Bonded Interfaces[END_REF]. In this study, the initiation and propagation of interlaminar cracks under mode-I cycling loading is experimentally investigated using a composite made of unidirectional carbon fibre with an epoxy matrix, varying the asymmetry coefficient.

EXPERIMENTAL PROCEDURE.

Materials and specimens.

The composite material employed in this research program was manufactured from Hexcel AS4/8552 RC34 AW196 epoxy resin prepregs, reinforced with unidirectional carbon fibre AS4 sequentially piled and cured in an autoclave, resulting in a volume fraction of 60% . The laminate configuration consists of 16 plies with 0º orientation /insert/ 16 plies with 0º orientation, employing an RF-260-R Tygavac 20 micrometer thick insert film. Seven specimens were tested in accordance with ASTM D3039 for the static characterization of the material under tension and according to ASTM D3518 for static characterization under shear providing the following results:

Young moduli where sub-index 11 denotes fibre orientation and 22, the direction perpendicular to the fibres. The specimens were obtained from panels manufactured by cutting the laminates. A double cantilever beam (DCB) was used to characterize delamination under mode-I conditions for both static and cyclic loading. Figure 1 shows the geometry of the specimen used in accordance with ASTM in which a non-adhesive insert is placed midplane to provoke the initial delamination. Two hinges are glued, respectively, to the edge of both specimen legs, thus allowing the external load to be applied.

Figure 1.

Static and fatigue characterization.

All the tests performed to characterize the material both under static and cyclic loading were carried out in a servo-hydraulic MTS testing machine provided with a 5kN load cell controlled by a computer using the original software. For the calculation of the static fracture toughness of the material, three methods were applied in accordance with the ASTM D5528 Standard [START_REF] Astm D | Modo I Interlaminar fracture toughness of unidirectional fiber-reinforced polymer matrix composites[END_REF]: the Modified Beam Method (MBT), the Compliance Calibration Method (CC) and the Modified Compliance Calibration Method (MCC). A deformation rate of 0.5 mm/min was applied throughout the tests [START_REF] Hojo | Mode I delamination fatigue properties of interlayer-toughened CF/epoxy laminates[END_REF]. The experimental results obtained for the static fracture toughness G c according to the three methods are summarized in Table 1 showing an acceptable agreement:

Table 1.

The aim of the fatigue tests was to provide information on the material to be used in the fatigue life prediction of laminates. This objective was twofold:

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In the present study, delamination growth onset was decided by visual observation of the crack opening at the crack front. This procedure is in agreement with one of the three alternatives suggested by the ASTM D6115 standard [START_REF] Astm D | -97 Mode I fatigue delamination growth onset of unidirectional fiber-reinforced polymer matrix composites[END_REF] to determine the number of cycles until the onset of delamination though the method based on compliance increase is recommended as being more conservative under load control conditions. At this moment, the crack is ideally assumed to be initiated, though not having developed. This is repeated for different ∆G values, thus allowing the fatigue curve ∆G c (N) to be plotted. The procedure is similar to that applied for the conventional test to determine S-N curves. Two asymmetry coefficients R=0.2 and R=0.5 were here employed. In all cases, the maximum stress was maintained constant and the minimum stress was varied as a function of the adopted asymmetry coefficient [START_REF] Shivakumar | A total fatigue life model for mode I delaminated composite laminates[END_REF].

b) da/dN-∆G . crack growth rate curves

The crack growth rate, i.e., the da/dN-G curves, were determined for a fraction of the critical energy release rate G crit (80%) and for different R=δ min /δ max relations [START_REF] Hojo | Mode I delamination fatigue properties of interlayer-toughened CF/epoxy laminates[END_REF]. A frequency of 3 Hz was applied throughout the tests.

Although it is difficult and cumbersome to perform the test, it seems to be an effective way of obtaining valid information. The same asymmetry coefficients were used here as in the study of crack initiation, i.e., 0.2 and 0.5.

Since the aim of the tests is to gain knowledge about the onset of delamination and to determine the crack growth rate, they were all carried out under displacement control, contrary to usual fatigue tests, which are generally conducted under load control. This is due to the fact that, under load control, increasing stress arises at the crack front during its progression, thus causing crack instability of the specimen when the crack reaches a certain value, unless constant stress may be ensured at the crack tip via steady load variation. This means applying appropriate load control of the real crack length in the machine under consideration. The procedure employed consisted in controlling the displacement, i.e., the crack opening displacement, via the critical displacement obtained from the static characterization of the material.

By adjusting this displacement as the crack grows, it is possible to keep the stress state constant at the crack front throughout the test. The crack length values were read by means of a 100x portable microscope, located at one side of the specimen.

The test was stopped after every 10 4 cycles in order to determine the crack length and thus crack growth rate by optical observation without removing the specimen from the fixture. The mean load was applied while locating the crack front under the microscope and measuring crack growth. In this way, the crack growth rate da/dN was calculated. The energy release rate for the last 10 4 cycles was evaluated using the mean values of the crack length and the maximum and minimum loads.

EXPERIMENTALS RESULTS.

In the following, the experimental results obtained from the tests are presented. As can be observed, the growth rate slows down with increasing crack length. For better understanding of the process, Figure 5 shows both curves on the same graph for the same crack length. Although the overall behaviour is similar in both cases, significant differences arise. The crack growth rate becomes higher for crack lengths less than 4 mm. For crack lengths over this value, crack progression decreases, practically ceasing to grow for crack lengths over 10 mm and an asymmetry coefficient equal to 0.5 stops. It should be remembered that the maximum stress level was kept constant for both asymmetry coefficients, only the minimum stress being changed.

Beginning of delamination.

The crack growth rate increases for an asymmetry coefficient of 0.2, varying around 1.5⋅10 -4 mm/cycle, while for an asymmetry coefficient of 0.5, the growth rate varies around 1.64⋅10 -4 mm/cycle during the first 10 mm of crack length. In this same figure, a certain pattern of behaviour is observed, which is practically coincident for both cases, showing the same trend in crack growth, the highest crack growth rates corresponding to the increases in critical energy.

In Figure 7, the crack growth rate is represented as a function of critical delamination for an asymmetry coefficient energy of 0.5 and load levels of 80, 85 and 90% of the critical delamination energy of the material under static load.

Figure 7.

In this case, lower crack growth rates are obtained with decreasing load levels, presenting mean rates of 4.71⋅10 -5 mm/cycle, 6.99⋅10 -5 mm/cycle and 7.44⋅10 -5 mm/cycle for 80%, 85% and 90% ∆G, respectively, and crack lengths close to 20 mm. Figure 9 shows different fracture surface zones in another specimen tested under a stress ratio R= 0.5. Micrographs [a] and [d] depict the same zones as those referred to previously in Figure 8.

Fracture surfaces

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As regards the morphology of the fracture surface of the laminate under mode-I cyclic load, a certain difference between the generated fracture surfaces is observed when the stress ratio is changed. This becomes apparent when observing the higher degree of damage of the matrix-fibre interface occurring in the specimen tested at a higher stress level R = 0.2. Here, a certain separation between fibres and matrix is observed (see Figures 8 a) andc)) both in the initiation and in the growth zones. However, in the material subjected to a lower stress range, R=0.5, less separation and debonding of fibres is observed.

In the case of the higher stress level, the trajectories of the cracks generated were found to be mainly oriented along the interface fibre-matrix. Another important difference between the two fracture surfaces is found in the striation appearing in the resin. This effect is more noticeable in the case of the material subjected to a lower stress level, which could be explained by the presence of a lower propagation velocity at an overall level, since the fracture surface developed is broader.

CONCLUSIONS.

In this paper, certain parameters controlling the delamination process of a composite under mode-I fatigue loading were experimentally investigated. The results may be summarized as follows:

-As regards interlaminar crack initiation:

The determination of the fatigue curves was strongly influenced by the process of manufacturing the laminate, the accumulation of resin at the crack front and possibly by the film thickness, which led to pronounced variability in the data. As a consequence, the study and interpretation of the influence of these parameters requires the testing of a large number of specimens.

Fatigue fracture under mode-I loading may be characterized by carrying out the tests under displacement control, i.e., by regulating the opening of the crack lips as a function of the critical displacement obtained from the static characterization. The fatigue versus asymmetry coefficient curves show the expected behaviour, i.e., a higher fatigue limit with decreasing stress range.

-As regards crack growth:

Crack growth slows down with increasing crack lengths and can even stop progressing if the critical fracture energy supplied does not exceed the equivalent values calculated from the static characterization of the material. This could be due to the increasing compliance of the crack legs under cyclic loading caused by viscoelastic effects or to fibre bridging at the crack as evidenced by the R-curve of the material.

List of captions: Table 1. Fracture toughness obtained for the material used. Fig. 3 0,0E+00

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E 11 :

 11 144 GPa (C.V.: 7.8%) E 22 : 10.6 GPa (C.V.:5.9%) Ultimate stress: σ 11 : 1703 MPa (C.V.: 9.2%) σ 22 : 30.8 MPa (C.V.:11%) ACCEPTED MANUSCRIPT Shear modulus: G 12 : 5.36 GPa (C.V.:12%) Shear ultimate stress: τ máx : 67.7 MPa (C.V.:1.6%)

  a) To find out the number of cycles needed to initiate delamination for a given energy release rate, i.e., to determine the fatigue curves ∆G-N. b) To characterize the propagation phase by means of the crack growth curve da/dN-∆G . In order to reasonably extend the test duration without implying a pronounced increase in costs, tests were conducted up to 2.5 million instead of the 2 million cycles usually considered as a standard number of fatigue limit cycles . a) ∆G-N fatigue curves

Figure 2

 2 Figure 2 represents the curves characterizing the fatigue behaviour of the material (crack growth onset) for the two asymmetry coefficients considered in the tests.

Figure 2 .

 2 Figure 2.The values of the critical energy obtained from the tests are represented as a percentage. A reference fatigue limit, discretionally taken as the fatigue strength for 2.5 million cycles, i.e., the limit number of cycles, corresponds to approximately 50% of the critical fracture energy calculated from the static tests according to the Modified Beam Method for an asymmetry coefficient of 0.2, and to around 70% for an asymmetry coefficient of 0.5.The pronounced scatter observed by the results of the number of cycles elapsed until crack initiation could presumably be assigned to the strip of resin accumulated at the crack front as a result of the manufacturing process and to the 20 micrometer thickness of the insert film, which exceeds the 13 micrometer prescribed by ASTM D5528.3.2 Delamination crack rate.Figures3 and 4show the crack growth rate as a function of the crack length for the tested material for a loading level corresponding to 80% of the critical delamination energy and asymmetry coefficients of 0.5 and 0.2, respectively.

Figure 3 .

 3 Figure 3.

Figure 4 .

 4 Figure 4.

Figure 5 .

 5 Figure 5.

Figure 6

 6 Figure 6 represents the crack growth rate under fatigue loading as a function of the increase in the critical delamination energy, calculated using the Modified Beam Theory, for the two asymmetry coefficients considered in this study and a mean load level corresponding to 80% of the critical energy associated with static fracture of the material.

Figure 6 .

 6 Figure 6.

Figure 8

 8 Figure 8 depicts a number of fracture surfaces of one of the specimen tested under a stress ratio R=0.2. Micrograph [a] represents the transition zone between the insert initiating delamination and the crack front at a magnification of 1000x. In [b], the crack growth zone of the same specimen is shown under the same magnification as before. In [c], the fracture surface of the same specimen zone is represented at the same scale, this time for another crack, which is markedly longer. Finally, a detail of the fibre failure mode in the crack growth zone is shown at a magnification of 4000x.

Figure 8 .

 8 Figure 8.

Figure 1 .

 1 Figure 1. Specimen geometry, H mean =9.22mm.

Figure 2 .

 2 Figure 2. Fatigue curves for the material tested.

Figure 3 .

 3 Figure 3. Propagation rate versus the crack length for an asymmetry coefficient of 0,5.

Figure 4 .

 4 Figure 4. Propagation rate versus the crack length for an asymmetry coefficient 0,2.

Figure 5 .

 5 Figure 5. Propagation rate versus crack length for asymmetry coefficients of 0,2 and 0,5.

Figure 6 .

 6 Figure 6. Crack growth rate versus the increase in critical energy for different values of the asymmetry coefficient.

Figure 7 .

 7 Figure 7. Crack growth rates for different increases in the critical delamination energy and an asymmetry coefficient of 0,5.

Figure 8 .

 8 Figure 8. SEM micrographs of the material fracture surface for R = 0,2.

Figure 9 .

 9 Figure 9. SEM micrographs of the material fracture surface for R = 0,5.

  Fig.2

Fig. 6 1

 6 Fig.6

ACKNOWLEDGEMENTS

The authors are indebted to the Spanish Ministry of Science and Technology (Project MAT2003-09768-C03-03) for partial funding of this work.

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S Roussette

J C Michel
email: michel@lma.cnrs-mrs.fr

P Suquet

Nonuniform transformation field analysis of elastic-viscoplastic composites

Keywords: B. Mechanical properties, Modelling, Non-linear behaviour, Plastic deformation, Porosity

To exactly describe the macroscopic behaviour of composites with non linear dissipative constituents, it is necessary to take an infinitely large number of internal variables into account. Simplifying assumptions are usually adopted to reduce this number. A new method has been proposed [5], based on the transformation fields approach [1], but not taking transformation fields to be necessarily uniform. The interest of this new method has been shown in the case of composites with elasticplastic constituents. Here we deal with composites having elastic-viscoplastic and porous elastic-viscoplastic constituents. In the latter case, the viscoplastic strain has a dilatational part.

Introduction

To exactly describe the macroscopic behaviour of composites with non linear dissipative constituents, it is necessary to take an infinitely large number of internal variables into account. Simplifying assumptions are usually adopted to reduce this number. The transformation field analysis (TFA) method initially proposed by [START_REF] Dvorak | Transformation field analysis of inelastic composite materials[END_REF] for elastic-plastic composites and subsequently extended by other authors (e.g. [START_REF] Kattan | Overall damage and elastoplastic deformation in fibrous metal matrix composites[END_REF][START_REF] Fish | Finite deformation plasticity for composite structures: Computational models and adaptive strategies[END_REF][START_REF] Chaboche | Towards a micromechanics based inelastic and damage modeling of composites[END_REF]) to composites with more complex behaviour, including damage, provides an elegant means of reducing the number of internal variables by assuming uniform fields of internal variables in individual phases or sub-domains. Applications of this method to two-phase composites have shown, however, that it is necessary to subdivide the phases into many subdomains to obtain a satisfactory description of the overall behaviour. This subdivision is necessary because of the strong nonlinearity of the constitutive laws involved, which can generate highly heterogeneous mechanical fields within a single phase. In view of these findings, a new method was therefore proposed [START_REF] Michel | Nonuniform transformation field analysis[END_REF], which consists in decomposing the fields of internal variables into a small number of not necessarily uniform shape functions. Comparisons with the classical transformation field analysis approach have shown the interest of this method in the case of elastic-plastic composite structures [START_REF] Michel | Computational analysis of nonlinear composite structures using the nonuniform transformation field analysis[END_REF]. The present study focuses on the case of elastic-viscoplastic and porous elastic-viscoplastic composites. In the latter case, the viscoplastic strain has a dilatational part.

Constitutive relations for the individual constituents

The constituents on which we focus in the present study are of the elasticviscoplastic and porous elastic-viscoplastic type. These constituents show generalised standard behaviour, defined by the free energy w and the force potential ψ. In what follows, only the dissipative mechanisms described by the viscoplastic strain ε vp will be taken into account:

w(ε, ε vp ) = 1 2 (ε -ε vp ) : L : (ε -ε vp ), σ = ∂w ∂ε (ε, ε vp ), εvp = ∂ψ ∂σ (σ). (1)
In addition, we will assume the constituents to be isotropic. In this framework, the elastic tensor L is characterised by a bulk modulus k and a shear modulus G, and the force potential ψ depends on the stress σ only through its second invariant σ eq in the case of classical viscoplastic materials. In the case of porous viscoplastic materials, the force potential also depends on the first invariant σ m . Equation (1c) can therefore be written as follows:

εvp = 3 2 ∂ψ ∂σ eq (σ eq , σ m ) σ dev σ eq + 1 3 ∂ψ ∂σ m (σ eq , σ m ) δ, ( 2
)
where δ denotes Kronecker's symbol, σ dev the deviatoric part of σ, σ eq = ( 3 2 σ dev : σ dev ) 1/2 and σ m = tr(σ)/3. Let us now consider a representative volume element V of a composite material composed of N constituents. The generalised standard structure of the laws in question is known to be preserved in the change of scales. In other words, the composite itself shows generalised standard behaviour, but there is an infinitely large number of internal variables involved in its description. The state variables of the system are the macroscopic strain E and the viscoplastic strains ε vp (x) occurring at all the points x of V . Because of the linearity of the local problem (under prescribed state variables), the strain field ε(x) can be decomposed into the following sum:

ε(x) = A(x) : E + 1 |V | V D(x, x ) : ε vp (x )dx = A(x) : E +D * ε vp (x), (3)
where A(x) is the elastic strain-localisation tensor and D(x, x ) the non local operator giving the strain at point x resulting from a transformation strain ε vp (x ) at point x when the average strain is zero.

3 Incompressible viscoplasticity

Non uniform transformation fields

To reduce the number of internal variables and improve the classical transformation field analysis, Michel and Suquet [START_REF] Michel | Nonuniform transformation field analysis[END_REF] have described a method focusing on the non uniformity of the local fields of internal variables, taking the transformation fields to be non uniform. The viscoplastic strain field is decomposed into a set of fields called plastic modes, µ k :

ε vp (x, t) = M k=1 ε vp k (t) µ k (x). (4)
By contrast with what occurs with the classical transformation field analysis, the modes µ k are taken here to be non uniform and tensorial, which means that ε vp k is scalar. The total number of modes M can differ from the number N of constituents. Other assumptions are adopted to simplify the theory: (i) The support of each mode is entirely contained in a single phase. (ii) Under incompressible viscoplastic conditions, the modes are traceless tensor fields. (iii) The modes are orthogonal. This prerequisite is met when the modes have their support in different phases, but it has to be imposed when they involve a single phase. (iv) Lastly, to make the reduced internal variables ε vp k homogeneous with a strain, the modes are normalised:

µ k eq = 1, where µ k eq = ( 2 3 µ k : µ k ) 1/2 .

Reduced variables and influence factors

Upon multiplying (3) by µ k and averaging over V , given (4), we obtain:

e k = a k : E + M =1 D k ε vp , ( 5
)
where the reduced strain e k , the reduced strain-localisation tensor a k and the influence factors D k are defined by:

e k = µ k : ε , a k = µ k : A , D k = µ k : (D * µ ) . ( 6
)
By analogy with the equation for the reduced strain e k in ( 6), we define the reduced viscoplastic strain e vp k and the reduced stress τ k as follows:

e vp k = µ k : ε vp , τ k = µ k : σ . ( 7
)
Note that the whole set of ε vp k can be replaced by the whole set of e vp k , since:

e vp k = µ k : µ k ε vp k . ( 8
)

Constitutive relations for the reduced variables

Since the phase elasticity is isotropic and the modes are traceless fields supported in a single phase, the reduced stress τ k can be written:

τ k = 2G r (e k -e vp k ), (9)
where G r is the shear modulus of the phase r involved in mode k. The evolution of the reduced viscoplastic strain e vp k still remains to be described. Using (1c) and the definition (7a) of e vp k , we obtain:

ėvp k = µ k : εvp = 3 2 ∂ψ ∂σ eq (σ eq ) µ k : σ σ eq . ( 10
)
At this stage, an approximation is required to obtain a relation between the ėvp k 's and the τ k 's. This is done in [START_REF] Michel | Nonuniform transformation field analysis[END_REF] by substituting τ r eq = [ 10), taking M(r) to denote the number of modes involved in phase r:

M (r) k=1 (τ k ) 2 ] 1/2 for σ eq in (
ėvp k = 3 2 ∂ψ r ∂σ eq (τ r eq ) τ k τ r eq , τ r eq = ⎡ ⎣ M (r) k=1 (τ k ) 2 ⎤ ⎦ 1/2 . ( 11
)
The system of equations ( 5), ( 8), ( 9), (11) provides the set of reduced constitutive relations for the composite. This system is solved along a prescribed loading path, in either the space of macroscopic stresses or that of macroscopic strains [START_REF] Roussette | Analyse par champs de transformation de matériaux élastoviscoplastiques multiphases -Application aux combustibles MOX[END_REF], where the macroscopic stress is obtained by averaging the stress field which results from (1b), ( 3) and ( 4):

Σ = L : A : E + M k=1 ( L : D * µ k -L : µ k ) ε vp k . ( 12
)

Choosing the modes -the Karhunen-Loève decomposition

The accuracy of this method depends largely on the modes involved. In the present study, the modes are determined according to the following procedure. Let θ k (x), k = 1, ..., M T (r) be the viscoplastic strain fields in a given phase r. These fields are determined along certain specific loading paths in the space of macroscopic stresses and at different levels of the macroscopic strain by solving the complete non linear local problem numerically. In all the examples of section 5, a numerical method based on fast Fourier transforms [START_REF] Moulinec | A numerical method for computing the overall response of nonlinear composites with complex microstructure[END_REF][START_REF] Michel | Effective properties of composite materials with periodic microstructure: a computational approach[END_REF] was used to determine these viscoplastic strain fields, but any other numerical method as FEM could have been used. The Karhunen-Loève decomposition (also known in the literature as the proper orthogonal decomposition or as the principal component analysis) is then used to build a new set of modes µ k (x), k = 1, ..., M T (r):

µ k (x) = M T (r) =1 v k θ (x), (13)
where v k are the eigenvectors and λ k the eigenvalues of the correlation matrix:

M T (r) j=1 g ij v k j = λ k v k i , g ij = θ i : θ j . ( 14
)
It can be easily confirmed that the modes µ k thus obtained are orthogonal:

µ k : µ = λ k if k = , otherwise 0.
If we assume the eigenvalues λ k to be arranged in decreasing order of size, another advantage of using the Karhunen-Loève decomposition lies in the fact that only the first M(r) modes corresponding to the largest eigenvalues need to be taken into account. In the cases dealt with here, we chose the M(r) modes µ k making it possible to satisfy the following criterion:

⎛ ⎝ M (r) k=1 λ k ⎞ ⎠ ≥ 0.9999 ⎛ ⎝ M T (r) k=1 λ k ⎞ ⎠ . ( 15
)
In practice, this criterion is satisfied with M(r) = 2 or 3. As an example, Figure 1 shows the quick decrease in the eigenvalues calculated in the case of a composite with hexagonally distributed fibres, where the fibres and matrix both show power-law elastic-viscoplastic behaviour.

4 Compressible viscoplasticity

Non uniform transformation fields

When the viscoplasticity is compressible (as in the case of composites with porous viscoplastic constituents), the viscoplastic strain field can be decomposed as follows:

ε vp (x, t) = M k=1 ε vp k (t) µ k (x) + M =1 ε vp (t) µ (x) δ, ( 16
)
where the modes µ k are traceless tensor fields and the modes µ are scalar fields. With (16), (3) becomes:

ε(x) = A(x) : E + M k=1 (D * µ k )(x) ε vp k + M =1 (D * µ δ)(x) ε vp . ( 17
)

Reduced variables and influence factors

For further purposes, we now introduce the following reduced variables:

e k = µ k : ε , e vp k = µ k : ε vp , τ k = µ k : σ , ( 18
)
e k = µ k δ : ε , e vp k = µ k δ : ε vp , σ k = µ k δ : σ . ( 19
)
From ( 17) and the definitions (18a) and (19a) for the reduced strains e k and e k , the following equations can easily be deduced:

e k = a k : E + M =1 D k ε vp + M =1 F k ε vp , ( 20
)
e k = a k : E + M =1 F k ε vp + M =1 D k ε vp , ( 21
)
where

a k = µ k : A , D k = µ k : (D * µ ) , F k = µ k : (D * µ δ) , a k = µ k δ : A , F k = µ k δ : (D * µ ) and D k = µ k δ : (D * µ δ) .

Constitutive relations for the reduced variables

Since the elasticity of the phases is isotropic and the modes supported in a single phase, we have:

τ k = 2G r ( e k -e vp k ), σ k = 3k r ( e k -e vp k ), (22)
where k r and G r denote the bulk and shear moduli of the phase r with which the mode k is associated. Using (2) and the definitions (18b) and (19b) for the reduced viscoplastic strains e vp k and e vp k , we obtain:

˙ e vp k = 3 2 ∂ψ ∂σ eq (σ eq , σ m ) µ k : σ σ eq , ˙ e vp k = 1 3 ∂ψ ∂σ m (σ eq , σ m ) µ k δ : σ σ m . (23)
Here again, it is not possible in the non linear context to simply calculate the average of these products, and simplifying assumptions have to be used. In (23), σ eq is replaced by τ r eq = [

M (r) k=1 ( τ k ) 2 ] 1/2 and σ m is replaced by σ r m = [ M (r) k=1 ( σ k ) 2 ] 1/2 ,
so that the evolution of the reduced internal variables is given by:

˙ e vp k = 3 2 ∂ψ r ∂σ eq ( τ r eq , σ r m ) τ k τ r eq , ˙ e vp k = 1 3 ∂ψ r ∂σ m ( τ r eq , σ r m ) σ k σ r m , τ r eq = ⎡ ⎢ ⎣ M (r) k=1 ( τ k ) 2 ⎤ ⎥ ⎦ 1/2 , σ r m = ⎡ ⎢ ⎣ M (r) k=1 ( σ k ) 2 ⎤ ⎥ ⎦ 1/2 , ( 24
)
where M (r) (resp. M(r)) denotes the number of modes µ k (resp. µ k ) having their support in phase r. The macroscopic stress is obtained by averaging the stress field resulting from (1b), ( 16) and ( 17):

Σ = L : A : E + M k=1 ρ k ε vp k + M k=1 ρ k ε vp k , ρ k = (L : D * µ k ) -(L : µ k ) , ρ k = (L : D * µ k δ) -(L : µ k δ) . (25)

Choosing the modes -the Karhunen-Loève decomposition

In the case of compressible viscoplastic constituents, the viscoplastic strain fields θ k (x) are decomposed into a purely deviatoric part and a purely spherical part,

θ k (x) = θ k (x) + θ k (x)δ, (26)
and the Karhunen-Loève decomposition is applied separately to each of these parts.

Results and discussion

Configurations and material data

The case of incompressible viscoplasticity

The results in this case were obtained on a unidirectional fibre-matrix composite with circular fibres which are regularly distributed at the nodes of a hexagonal array. The volume fraction of the fibres is 0.25. The fibres and the matrix are assumed to have a power-law elastic-viscoplastic behaviour defined by a force potential of the form:

ψ(σ eq ) = σ 0 ε0 n + 1 σ eq σ 0 n+1 . ( 27
)
Two different viscosity exponents, corresponding to a linear viscosity and a strongly non-linear viscosity, are considered for the matrix: n = 1 and n = 8.

The other material properties used with each of these two viscosity exponents are shown in Table 1.

The case of compressible viscoplasticity

The results obtained in this case involved first the same hexagonal configuration as that studied above. The fibres are purely elastic and the matrix is a porous elastic-viscoplastic matrix defined by the elliptical force potential:

ψ(σ eq , σ m ) = σ 0 ε0 n + 1 9 4 A(f ) σ m σ 0 2 + B(f ) σ eq σ 0 2 n+1 2 , ( 28
)
where f denotes the porosity and where the expressions of A(f ) and B(f ) result from [START_REF] Michel | The constitutive law of nonlinear viscous and porous materials[END_REF] starting from an analytical self-consistent scheme and from a bounding method, respectively. Here again, the viscosity exponents n = 1 and n = 8 are considered for the matrix. The other material properties used here are shown in Table 2.

In a second step, the results obtained concern a square cell containing a large number of randomly distributed fibres (Figure 4a). The same force potential (28), the same fibre volume fraction (equal to 0.25) and the same material properties (Table 2) are used again here, but only the viscosity exponent n = 1 is considered for the matrix.

Loading

The composite is subjected to radial load paths in the space of the macroscopic stresses: Σ(t) = λ(t) Σ 0 , where Σ 0 is the prescribed stress direction. The following four stress directions are used: (29)

Σ (1) 0 = e 1 ⊗ e 1 , Σ (2)

Discussion

Figure 2 gives the predictions obtained with various models (TFA, NTFA with one mode per phase calculated at a fixed level of macroscopic strain E : Σ (1) 0 = 5%, and NTFA with two main eigen-modes per phase obtained using the Karhunen-Loève decomposition) when the fibres and the matrix have a power-law elastic-viscoplastic behaviour. The curves correspond to the loading path Σ [START_REF] Dvorak | Transformation field analysis of inelastic composite materials[END_REF] 0 . The predictions obtained with the classical TFA model are too rigid. Those obtained with NTFA model with one mode per phase are satisfactory, but the stress hardening estimate does not match the reference calculations. The NTFA model with two main eigen-modes per phase gives excellent results throughout the range of strains under consideration, which shows the usefulness of adopting the Karhunen-Loève decomposition, which means that the information will be optimally distributed at various loading times between the two eigen-modes having the highest eigenvalues.

Figures 3 and4b give the predictions obtained with the NTFA model in which the modes are determined using the Karhunen-Loève decomposition, in the case of elastic fibres and a porous elastic-viscoplastic matrix. The curves in Figures 3a and4b 0 . As in the context of incompressible viscoplasticity, the NTFA method again gives excellent results here.

Conclusion

The nonuniform transformation fields analysis method was extended here to the case of elastic-viscoplastic and porous elastic-viscoplastic constituents. The predictions obtained were accurate and the number of internal variables involved was reduced thanks to the use of the Karhunen-Loève decomposition.

0 = e 1 ⊗ e 2 + e 2 ⊗

 122 e 1 , Σ (3) 0 = e 1 ⊗ e 1 -e 2 ⊗ e 2 , Σ (4) 0 = e 1 ⊗ e 1 + e 2 ⊗ e 2 .

  Figures 3 and 4b give the predictions obtained with the NTFA model in which the modes are determined using the Karhunen-Loève decomposition, in the case of elastic fibres and a porous elastic-viscoplastic matrix. The curves in Figures3a and 4bcorrespond to the loading paths Σ (3) 0 and Σ (4) 0 , whereas those in Figure 3b correspond to the loading paths Σ (1) 0 and Σ (2)

Fig. 1 .Fig. 2 .Fig. 3 .

 123 Fig. 1. Example of eigenvalues.

Fig. 4 .

 4 Fig. 4. Randomly distributed fibres. Elastic fibres and porous elastic-viscoplastic matrix. (a) Microstructure. (b) n = 1. Loading along Σ (3) 0 and Σ (4) 0 (see (29) for the definition of the loading).

Table 1

 1 Material properties. Power-law elastic-viscoplastic fibres and matrix. E (GPa) ν σ 0 (MPa) ε0 (s -1 ) n

	Fibres	100	0.3	250	10 -5	1
	Matrix	180	0.3	50	10 -5	1
		180	0.3	57.825	10 -5	8

Table 2

 2 Material properties. Elastic fibres and porous elastic-viscoplastic matrix.

		E (GPa) ν	σ 0 (MPa) ε0 (s -1 )	n	Porosity
	Fibres	400	0.2
	Matrix	70	0.3	100	10 -2	1 or 8	0.07

Acknowledgements. The authors thank Rubens Sampaio for useful discussions about the Karhunen-Loève decomposition.


MD Tamás Radovits
email: radovitstamas@yahoo.com

Leila Seres

Domokos Gerő

Irina Berger

Csaba Szabó

Matthias Karck

Gábor Szabó

Single dose treatment with PARP-inhibitor INO-1001 improves aging-associated cardiac and vascular dysfunction

Keywords: aging, nitro-oxidative stress, poly(ADP-ribose) polymerase, cardiac function, endothelial function, immunohistochemistry

Overproduction of reactive oxygen species in aging tissues has been implicated in the pathogenesis of aging-associated cardiovascular dysfunction. Oxidant-induced DNA-damage activates the poly(ADP-ribose) polymerase (PARP) pathway, leading to tissue injury. In this study we investigated the acute effects of the PARP inhibitor INO-1001 on aging-associated cardiac and endothelial dysfunction. Using a pressure-volume conductance catheter, left ventricular pressure-volume analysis of young and aging rats was performed before and after a single injection of INO-1001. Endothelium-dependent and -independent vasorelaxation of isolated aortic rings were investigated by using acethylcholine and sodium nitroprusside. Aging animals showed a marked reduction of myocardial contractility and endotheliumdependent relaxant responsiveness of aortic rings. Single dose INO-1001-treatment resulted in acute improvement in their cardiac and endothelial function. Immunohistochemistry for nitrotyrosine and poly(ADP-ribose) confirmed enhanced nitro-oxidative stress and PARPactivation in aging animals. Acute treatment with INO-1001 decreased PARP-activation, but did not affect nitrotyrosine-immunoreactivity. Our results demonstrate that the agingassociated chronic cardiovascular dysfunction can be improved, at least, short term, by a single treatment course with a PARP-inhibitor, supporting the role of the nitro-oxidative stress-PARP-pathway in the age-related functional decline of the cardiovascular system. Pharmacological inhibition of PARP may represent a novel therapeutic utility to improve aging-associated cardiovascular dysfunction.

Introduction

There is emerging evidence that aging is an important risk factor in the development of ischemic heart disease. This may be due to an age-associated increase in coronary vascular resistance leading to reduced myocardial blood supply and flow reserve [START_REF] Hachamovitch | Alterations of coronary blood flow and reserve with aging in Fischer 344 rats[END_REF]. Numerous studies suggest that aging is associated with impaired function of endothelium in laboratory animals [START_REF] Tschudi | Effect of age on kinetics of nitric oxide release in rat aorta and pulmonary artery[END_REF] and humans [START_REF] Egashira | Effects of age on endothelium-dependent vasodilation of resistance coronary artery by acetylcholine in humans[END_REF] and this endothelial dysfunction predisposes the aging population to cardiovascular complications and micro-thrombus formation. Recent studies demonstrate that the cardiovascular dysfunction associated with advanced aging is related to the local formation of reactive oxygen and nitrogen species in the myocardium and coronary vasculature [START_REF] Bejma | Free radical generation and oxidative stress with aging and exercise: differential effects in the myocardium and liver[END_REF][START_REF] Van Der | Enhanced peroxynitrite formation is associated with vascular aging[END_REF][START_REF] Csiszar | Aging-induced phenotypic changes and oxidative stress impair coronary arteriolar function[END_REF].

Aging organisms are exposed to continuous oxidative injury, due to the higher rate of superoxide and other free radical production from the mitochondrial electron-transport chain [START_REF] Sohal | Hydrogen peroxide release by mitochondria increases during aging[END_REF]. Increases in reactive oxidant species (ROS) and other oxidants at old age can elicit oxidative modifications of various cell components, such as lipid, protein and particularly DNA (de la [START_REF] De La | Mitochondrial glutathione oxidation correlates with age-associated oxidative damage to mitochondrial DNA[END_REF].

Various oxygen and nitrogen species (peroxynitrite, hydrogen peroxide, hydroxyl radical and nitroxyl anion) have been established as pathophysiological relevant endogenous triggers of DNA single-strand breakage and activation of the poly(ADP-ribose) polymerase (PARP) enzyme (Virag & Szabo, 2002). When activated by DNA single strand breaks, PARP initiates an energy-consuming metabolic cycle by transferring ADP-ribose units from NAD + to nuclear proteins. This process results in rapid depletion of intracellular ATP-pools and impaired mitochondrial respiration, eventually leading to cellular energetic crisis, dysfunction and death via the necrotic route (Virag & Szabo, 2002).

Pharmacological attempts against nitro-oxidative stress using classic antioxidants, such as vitamin E (which works by scavenging toxic oxidation products), ascorbate or glutathione (which react with peroxynitrite, albeit at a relatively slow rate) resulted in conflicting results in experimental models of disease [START_REF] Ceriello | New insights on oxidative stress and diabetic complications may lead to a "causal" antioxidant therapy[END_REF]. Based on recent studies, pharmacological inhibition of PARP (Szabo C et al., 2004b;Soriano et al., 2001a;[START_REF] Szabo | Poly-ADP-ribose polymerase inhibition protects against myocardial and endothelial reperfusion injury after hypothermic cardiac arrest[END_REF][START_REF] Beller | Activation of the peroxynitrite-poly(adenosine diphosphate-ribose) polymerase pathway during neointima proliferation: a new target to prevent restenosis after endarterectomy[END_REF] or decomposition of peroxynitrite (Szabo et al., 2002;[START_REF] Pacher | Potent metalloporphyrin peroxynitrite decomposition catalyst protects against the development of doxorubicin-induced cardiac dysfunction[END_REF][START_REF] Radovits | The peroxynitrite decomposition catalyst FP15 improves aging-associated cardiac and vascular dysfunction[END_REF], which block the peroxynitrite -DNA injurypoly(ADP-ribose) polymerase pathway emerge as potent novel antioxidant therapeutic possibilities in multiple pathophysiological conditions. Chronic treatment with PARP-inhibitors PJ34 and INO-1001 for 2 months in a rodent model has been demonstrated to improve endothelial and cardiac dysfunction associated with aging (Pacher et al., 2002e, Pacher et al., 2004b) showing the involvement of the nitrooxidative stress -PARP -pathway in the pathophysiology of cardiac and vascular aging.

Considering the theoretical possibility of acutely interrupting this pathway by pharmacological inhibition of the PARP enzyme, thereby quickly restoring the ATP-pools and the normal energy supply of the cells, we investigated in this study whether cardiac and vascular dysfunction at old age may be beneficially affected even by a single treatment course with a potent pharmacological inhibitor of PARP.

Materials and Methods

Animals, treatment protocols. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). All procedures and handling of animals during the investigations were reviewed and approved by the Ethical Committee of the Land Baden-Württemberg for Animal Experimentation.

Young adult (3 month old, 200-250g) and aging (20 month old, 450-600g) male Lewis rats (Charles River, Sulzfeld, Germany) were housed in a room at a constant temperature of 22 ± 2 ºC with 12 hours light/dark cycles and fed a standard laboratory rat diet and water ad libitum.

Hemodynamic measurements. Young (n=7) and aging (n=7) rats were anesthetized with thiopentone sodium (60 mg kg -1 i.p.), tracheotomized, intubated and artificially ventilated.

Animals were placed on controlled heating pads and core temperature measured via a rectal probe was maintained at 37 ºC. The thoracic cavity was opened to permit access to the apex of the heart. All incisions were kept to a minimum to avoid major blood loss. The left ventricle was punctured by a 20 G plastic cannula, through which a 2F microtip pressure-volume catheter (SPR-838, Millar Instruments, Houston, TX, USA) was inserted into the left ventricular cavity. Mean arterial pressure was measured via the right femoral artery. After stabilization for 5 minutes, the signals were continuously recorded using a pressure-volume conductance system (Leycom, Zoetermeer, The Netherlands) coupled to an A/D converter (EMKA Technologies, Paris, France) at a sampling rate of 1000 s -1 , stored and displayed on a computer by the IOX Software System (EMKA Technologies, Paris, France). With the help of a special pressure-volume analysis program (EMKA Technologies, Paris, France) mean arterial pressure (MAP), maximal left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and developed pressure (DP) were computed and calculated.

Left ventricular pressure-volume relations were measured by transiently compressing the inferior vena cava. The slope (E es ) of the left ventricular end-systolic pressure-volume relationships (ESPVR), preload recruitable stroke work (PRSW) and maximal slope of systolic pressure increment -end-diastolic volume relation (+dP/dt-EDV) were calculated as load-independent indexes of left ventricular contractility. The above detailed left ventricular pressure-volume analysis was performed before and 60 minutes after a single dose iv. injection of the potent PARP-inhibitor INO-1001 (5mg kg -1 ). Myocardial INO-1001 concentrations as high as 100-400 nM can be achieved by dosing of the compound in doses similar to this one in rodents. These tissue concentrations are sufficient to provide a substantial inhibition of the cellular activity of PARP [START_REF] Xiao | Poly(ADP-Ribose) polymerase promotes cardiac remodeling, contractile failure, and translocation of apoptosis-inducing factor in a murine experimental model of aortic banding and heart failure[END_REF]. Myocardial sections of the rats used for the hemodynamic measurements were removed for immunohistochemical processing immediately after completing the left ventricular pressure-volume analysis.

In vitro organ bath experiments.

In additional experiments, young and aging rats received a single intraperitoneal injection of vehicle (control groups) or the PARP-inhibitor INO-1001 (5 mg kg -1 ) (treatment groups) intraperitoneally. 2 hours after the injection, animals were anesthetized with thiopentone sodium (60 mg kg -1 i.p.), the descending thoracic aorta was carefully removed and placed in cold (+4 ºC), oxigenized Krebs-Henseleit solution (118 mM NaCl, 4.7 mM KCl, 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 1.77 mM CaCl 2 , 25 mM NaHCO 3 , 11.4 mM glucose; pH=7.4). The aortae were prepared and cleaned from periadventitial fat and surrounding connective tissue and cut transversely into 4-mm width rings using an operation microscope. An additional thoracic aortic segment from each animal was prepared and separated for immunohistochemical processing.

Isolated aortic rings were mounted on stainless steel hooks in individual organ baths (Radnoti Glass Technology, Monrovia, CA, USA), containing 25 ml of Krebs-Henseleit solution at 37 ºC and aerated with 95% O 2 and 5% CO 2 . Special attention was paid during the preparation to avoid damaging the endothelium.

Isometric contractions were recorded using isometric force transducers (Radnoti Glass Technology, Monrovia, CA, USA), digitized, stored and displayed with the IOX Software System (EMKA Technologies, Paris, France).

The aortic rings were placed under a resting tension of 2 g and equilibrated for 60 minutes. During this period, tension was periodically adjusted to the desired level and the Krebs-Henseleit solution was changed every 30 minutes. Maximal contraction forces to potassium chloride (KCl, 100mM) were determined and aortic rings were washed until resting tension was again obtained. Phenylephrine (10 -6 M) was used to precontract the rings until a stable plateau was reached, and relaxation responses were examined by adding cumulative concentrations of endothelium-dependent dilator acethylcholine (ACh, 10 -9 -10 -4 M) and endothelium-independent dilator sodium nitroprusside (SNP, 10 -10 -10 -5 M). Contractile responses are expressed as grams of tension, relaxation is expressed as percent of contraction induced by phenylephrine (10 -6 M).

Immunohistochemical analysis. Myocardial sections and aortic segments separated for immunohistochemical processing were fixed immediately after excision in buffered paraformaldehyde solution (4%) for 1 day. Three adjacent sections were processed for both of the following types of immunohistochemical labelling. According to the methods previously described [START_REF] Liaudet | Protection against hemorrhagic shock in mice genetically deficient in poly(ADP-ribose)polymerase[END_REF], we performed immunohistochemical staining for nitrotyrosine (NT, a marker of nitrosative stress in general [START_REF] Halliwell | What nitrates tyrosine? Is nitrotyrosine specific as a biomarker of peroxynitrite formation in vivo?[END_REF]), and for poly(ADP-ribose) (PAR, the enzymatic product of PARP). Primary antibodies used for the stainings were polyclonal sheep anti-nitrotyrosine antibody (Upstate, Chicago, IL, USA) and mouse monoclonal anti-poly(ADP-ribose) antibody (Calbiochem, San Diego, CA, USA).

Statistical analysis.

All data are expressed as means ± s.e.mean. Intergroup comparisons were performed by using one-way analysis of variance followed by Student's unpaired t-test with Bonferroni´s correction for multiple comparisons. Differences were considered significant when p<0.05. When compared to the young group, aging in rats was associated with significantly decreased left ventricular contractility. The load independent, PV-loop derived contractility indexes (E max , PRSW, +dP/dt-EDV) showed a marked reduction in aging animals. After acute PARP-inhibition, we observed significantly increased E max and PRSW, indicating the rapid improvement in left ventricular contractility (Fig. 6.). Treatment with INO-1001 in young rats had no effect on any of the hemodynamic parameters studied (Fig. 56.).

Drugs

Discussion

In the current study we demonstrate that a single injection of PARP-inhibitor INO-1001 effectively decreases the age-related myocardial and vascular PARP-activation, resulting in acute improvement of left ventricular contractility and enhanced endotheliumdependent vasorelaxation in a rat model of aging-associated cardiovascular dysfunction.

Recent studies elucidated numerous cellular and molecular mechanisms responsible for the functional decline of the cardiovascular system at old age [START_REF] Csiszar | Role of oxidative and nitrosative stress, longevity genes and poly(ADP-ribose) polymerase in cardiovascular dysfunction associated with aging[END_REF]. The oxidative stress hypothesis (or free radical theory, as it was originally proposed) [START_REF] Harman | Aging: a theory based on free radical and radiation chemistry[END_REF]) is currently one of the most favored explanations for how aging leads to progressive cellular damage at the biochemical level. According to this theory, age-related loss of physiological function and aging is caused by the deleterious effects of progressive and irreversible accumulation of oxidative damage. Several previous studies have demonstrated that aging organisms have a higher rate of free radical production (O 2 -. and H 2 O 2 ) due to the incomplete terminal oxidation in the mitochondria at older age [START_REF] Sohal | Hydrogen peroxide release by mitochondria increases during aging[END_REF][START_REF] Nohl | Influence of mitochondrial radical formation on energy-linked respiration[END_REF][START_REF] Kim | Influence of age, exercise, and dietary restriction on oxidative stress in rats[END_REF]. Superoxide anions interact with the physiological mediator nitric oxide, forming the potent oxidant peroxynitrite. Hydroxyl radicals are formed mainly from hydrogen-peroxide in the Haber-Weiss reaction catalysed by iron. Nitro-oxidative stress accompanied by increased formation of hydrogen peroxide, hydroxyl radical, and peroxynitrite are endogenous inducers of DNA single strand breakage, that is the obligatory trigger of activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP), which mediates the cellular response to DNA injury (Virag & Szabo, 2002).

Depending on the severity of DNA damage, different cellular pathways can be triggered. In the case of mild DNA damage, PARP facilitates DNA repair and thus cell survival. Severe DNA injury causes excessive PARP activation that initiates an energyconsuming futile repair cycle by transferring ADP-ribose units from NAD + to nuclear proteins. The excessive nuclear poly(ADP-ribose) formation results in rapid depletion of intracellular NAD + and ATP-pools, slowing the rate of glycolysis and mitochondrial respiration, eventually leading first to cellular energetic crisis and dysfunction, then to cell necrosis (Soriano et al., 2001b). By this route, PARP activation in cardiomyocytes and endothelial cells causes functional impairment of contractile function at the cellular level and reduced ability of endothelial cells to produce nitric oxide when stimulated by an endothelium-dependent relaxant agonist, such as acetylcholine (Soriano et al., 2001b;[START_REF] Szabo | Endothelial dysfunction in a rat model of endotoxic shock. Importance of the activation of poly (ADP-ribose) synthetase by peroxynitrite[END_REF]Pacher et al., 2002a;Pacher et al., 2002b;Szabo et al., 2004a;Pacher et al., 2002d;Pacher et al., 2002e;[START_REF] Szabo | Role of nitrosative stress and poly(ADP-ribose) polymerase activation in myocardial reperfusion injury[END_REF]. Impairment of endothelial function in the coronary arteries may lead to regional or global myocardial ischaemia, which secondarily impairs cardiac performance. The increased ROS-formation and PARP activation together with the above discussed downstream molecular and intracellular mechanisms are considered to play an important role in the pathogenesis of various forms of chronic heart failure (Pacher et al., 2002d;Pacher et al., 2006).

Similar to other studies [START_REF] Radovits | The peroxynitrite decomposition catalyst FP15 improves aging-associated cardiac and vascular dysfunction[END_REF][START_REF] Van Der | Enhanced peroxynitrite formation is associated with vascular aging[END_REF][START_REF] Csiszar | Aging-induced phenotypic changes and oxidative stress impair coronary arteriolar function[END_REF][START_REF] Csiszar | Role of oxidative and nitrosative stress, longevity genes and poly(ADP-ribose) polymerase in cardiovascular dysfunction associated with aging[END_REF], we demonstrated increased immunoreactivity for nitrotyrosine and activation of PARP in the left ventricular myocardium and aortic wall of aging rats (Fig. 12.), which confirms the nitro-oxidative stress and the activation of the peroxynitrite-poly(ADPribose) polymerase pathway, and are consistent with the above discussed "free radical theory" of aging (Pacher et al., 2005;[START_REF] Ungvari | Role of oxidative-nitrosative stress and downstream pathways in various forms of cardiomyopathy and heart failure[END_REF].

INO-1001 effectively decreased PAR-formation in various models of disease, but there are conflicting data about the effect of PARP-inhibitors on tyrosine-nitration (Beller et al., 2006;[START_REF] Farivar | Poly (ADP) ribose polymerase inhibition improves rat cardiac allograft survival[END_REF]Pacher et al., 2002c). After single dose treatment with INO-1001 in aging rats we found decreased PAR staining (reflecting decreased PARP activity) both in the myocardium and the aortic intima, however, the immunoreactivity for protein-nitrotyrosine was unaffected. These findings are consistent with the hypothesis that short-term PARP inhibition is sufficient to affect the imbalance between the rapid and reversible polymerization and degradation of ADP-ribose units (by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG); [START_REF] Davidovic | Importance of poly(ADP-ribose) glycohydrolase in the control of poly(ADP-ribose) metabolism[END_REF], but it does not affect upstream processes such as peroxynitrite generation and action (a marker of which is nitrotyrosine), and/or it might exert feedback effects on the generation of oxidants, but this is not reflected in the staining for nitrotyrosine, as this is a rather stable product.

In aging vessels, the nitro-oxidative damage of the vascular smooth muscle layers were found to be less pronounced, when compared to the endothelium. (Fig. 1.) These immunohistochemical findings are in line with recent reports demonstrating that the vascular superoxide-overproduction at old age occurs mainly in the endothelial cells [START_REF] Van Der | Enhanced peroxynitrite formation is associated with vascular aging[END_REF], which are in addition more vulnerable to oxidative injury.

Endothelial dysfunction associated with advanced aging is a well-known phenomenon and can be explained by the reduced nitric oxide (NO) production of endothelial cells (Soriano et al., 2001b); or by the increased NO-inactivation by superoxide anions (peroxynitrite formation) resulting in altered NO bioavailibility [START_REF] Van Der | Enhanced peroxynitrite formation is associated with vascular aging[END_REF].

The underlying intracellular pathways and molecular mechanisms have been subject of intensive investigations recent years [START_REF] Van Der | Enhanced peroxynitrite formation is associated with vascular aging[END_REF]Pacher et al., 2004b;Pacher et al., 2002e;Pacher et al., 2002f). In accordance with these studies we report here impaired endothelium-dependent acetylcholine-induced relaxation of isolated aortic rings of aging rats.

The endothelium-independent relaxation induced by the exogenously administered NO-donor SNP was unaffected by aging, indicating the normal dilative capacity of the vascular smooth muscle. These functional data are consistent with our immunohistochemical findings showing signs of severe nitro-oxidative damage mainly in the endothelium of aging vessels. In contrast with a previous work using epinephrine for precontraction (Pacher et al., 2002e), we found a significant decrease in contraction forces induced by phenylephrine and potassium chloride in aging animals which was in line with the results of another study investigating vascular function of diabetic rats (Soriano et al., 2001a) and may be due to alterations in receptor density and/or receptor/effector coupling.

Previous studies report acute amelioration of endothelial dysfunction in chronic diseases and pathophysiological conditions: single doses of antioxidants (such as vitamin C), tetrahydrobiopterin and L-arginine were shown to improve endothelial dysfunction associated with hypertension, diabetes, chronic smoking or atherosclerosis (Pieper, 1997;[START_REF] Heitzer | Tetrahydrobiopterin improves endothelium-dependent vasodilation in chronic smokers : evidence for a dysfunctional nitric oxide synthase[END_REF]Taddei et al., 1998;Quyyumi, 1998). All of these therapeutical attempts focuses on the restoration of nitric oxide bioavailibility either by directly supplying the endothelial nitric oxide synthase with substrate/co-factor (and reversing eNOS-uncoupling) or by decreasing NO-removal by reducing nitro-oxidative stress. As previously reported, in vitro incubation of blood vessels with PARP-inhibitors resulted in rapid reversal of endothelial dysfunction in aortae from diabetic mice (Soriano et al., 2001a) and also in the early stage of atherosclerosis [START_REF] Benko | Restoration of the endothelial function in the aortic rings of apolipoprotein E deficient mice by pharmacological inhibition of the nuclear enzyme poly(ADP-ribose) polymerase[END_REF]. Similarly to the results of these studies we report here significantly enhanced endothelium-dependent vasorelaxation (improved endothelial function) in aortic rings of rats with advanced aging after a single dose injection of PARP-inhibitor INO-1001.

We propose that the aging-related overproduction of ROS represents a continuous trigger of DNA single strand breakage that, in turn keeps PARP in an activated state, which continuously depletes the ATP-pools of endothelial cells, thereby impairing the endotheliumdependent relaxant responsiveness. Under this continuing nitro-oxidative stress the endothelium is likely to exist in a state of chronic energy starvation (metabolic suppression) associated with dysfunction. Due to the reversibility of the rapid polymerization of poly(ADPribose) units, pharmacological interruption of this metabolic cycle by acute inhibition of PARP can lead to normalization of the energy balance of endothelial cells by rapid restoration of endothelial ATP-levels to normal levels [START_REF] Szabo | Poly(ADP-Ribose) polymerase inhibition reduces reperfusion injury after heart transplantation[END_REF], and according to some reports even to higher levels [START_REF] Csordas | Hydrogen peroxide-mediated necrosis induction in HUVECs is associated with an atypical pattern of caspase-3 cleavage[END_REF]. Considering the fact that -similarly to former studies -our results on young control rats show no direct vasodilatory effects of INO-1001, the found acute effect of PARP-inhibition on aging-associated endothelial dysfunction can be explained by this intracellular mechanism. However, most of the studies on ATP-levels supporting our hypothesis were conducted on cultured endothelial cells, obvious evidence for the proposed mechanism could be provided by directly measuring in vivo/ex vivo tissue ATPlevels, but this was not conducted in the present study, and therefore alternative explanations for the current findings are also theoretically possible.

Recent studies performing invasive hemodynamic measurements in aging rats report decreased cardiac performance and development of progressive heart failure after the age of 20 months [START_REF] Radovits | The peroxynitrite decomposition catalyst FP15 improves aging-associated cardiac and vascular dysfunction[END_REF]Pacher et al., 2004a;Pacher et al., 2004b;[START_REF] Anversa | Effects of age on mechanical and structural properties of myocardium of Fischer 344 rats[END_REF]. A recent study provided detailed echocardiographic evidence of a progressive decrement in multiple aspects of systolic and diastolic LV function in aging rats [START_REF] Boluyt | Echocardiographic assessment of age-associated changes in systolic and diastolic function of the female F344 rat heart[END_REF]. Consistent with these results, we demonstrated that advanced aging in rats is associated with impaired myocardial contractility, as reflected by the PV-loop-derived load-independent contractility-indexes E max , PRSW or +dP/dt-EDV. These indexes are widely used as sensitive cardiac contractile parameters, because they are independent from changes in loading conditions and therefore especially informative in assessing cardiac contractility in models, where preload and afterload are altered.

The possible molecular mechanism responsible for the rapid improvement of cardiac dysfunction are supposed to be the energy restoration of myocardial cells, similarly to that discussed detailed above. In other words, similar to the situation in blood vessels, it is possible (although remains to be directly confirmed) that PARP inhibition acutely affects cardiac contractility via improvement of cardiac high energy phosphate status. An acute improvement in the myocardial blood supply (due to improved endothelial function in the coronary microvasculature) may also be feasible.

In accordance with our previous work with other PARP-inhibitors [START_REF] Szabo | Poly(ADP-Ribose) polymerase inhibition reduces reperfusion injury after heart transplantation[END_REF] we found that INO-1001 did not affect cardiac function of young control rats. Thus the improved cardiac function seen in the aging treatment group is a specific phenomenon, reflecting a reversal of the aging-associated suppressed myocardial performance, rather than the consequence of some nonspecific direct cardiac effects of INO-1001.

This is the first study reporting rapid improvement of the chronic cardiac and vascular dysfunction associated with advanced aging by acute inhibition of the PARP enzyme. The current findings indicate the importance of the nitro-oxidative stress -PARP -pathway, especially its quickly reversible energy depleting aspects in the pathogenesis of myocardial and endothelial dysfunction at old age. The current work further supports the concept that PARP-inhibition may represent a potential therapy approach to improve cardiovascular dysfunction associated with aging. There are many studies and reviews about the clinical treatment perspectives of PARP-inhibitors (Jagtap and Szabo, 2005;[START_REF] Graziani | Clinical perspectives of PARP inhibitors[END_REF]. We probably already know enough about the safety of PARP inhibitors to consider them suitable for short-term treatment of acute situations where the inhibition of PARP may provide considerable benefit, e.g. in myocardial reperfusion injury. However, chronic use of PARP inhibitors may be more challenging because of the unknown potential long-term side effects. Whether or not chronic PARP inhibition is safe, there is no clear answer, as chronic safety studies have not yet been conducted with any of the orally bioavailable PARP inhibitors. Depending on the outcome of such studies, chronic PARP inhibition may or may not turn out to be clinically sustainable. It also remains to be tested as to how long is the period for which a single dose of the PARP inhibitor provides cardiovascular benefit. In chronic heart failure models there is evidence that the beneficial effect of PARP inhibition is sustained after discontinuation of the treatment (Pacher et al., 2002c), therefore the duration of the beneficial effect seen in the current study needs to be delineated.

  . Phenylephrine, acethylcholine and sodium nitroprusside (Sigma-Aldrich, Germany) were dissolved in normal saline, INO-1001, the potent indeno-isoquinolinone-based poly(ADP)-ribose polymerase inhibitor (Inotek Pharmaceuticals Corporation, Beverly, MA, USA;Jagtap et al., 2005) was dissolved in 5% glucose solution.ResultsImmunohistochemicalanalysis. Immunohistochemical staining showed increased immunoreactivity for nitrotyrosine and poly(ADP-ribose) -indicative of nitrosative stress and enhanced activation of PARP -in the left ventricular myocardium and in the aortic wall (mainly in the endothelium) of aging rats. (Fig. 1-2.) Single dose treatment with the potent PARP-inhibitor INO-1001 notably decreased PAR formation both in the myocardium and the aortic wall. Immunoreactivity for nitrotyrosine was not affected by acute PARP-inhibition. Figure 1. and 2. show representative stainings for NT and PAR in the young control, aging control and INO-1001 treatment groups. Vascular function. Similar to previous studies, the impairment of endothelial function in aging rats was demonstrated in our in vitro organ bath experiments. The aging-associated endothelial dysfunction was indicated by the reduced maximal relaxation of isolated aortic rings to ACh (61.2 ± 2.19% aging control vs. 80.8 ± 1.99% young control, p<0.05), and the rightward shift of the dose-response curve as compared with the young control group. (Fig. 3.A). Single dose treatment with PARP-inhibitor INO-1001 significantly improved the AChinduced, endothelium-dependent, nitric oxide mediated vasorelaxation in aging animals (maximal relaxation: 69.1 ± 2.25% aging treatment group vs. 61.2 ± 2.19% aging control, p<0.05). The same treatment had no effect in young rats. (Fig. 3.A) The endothelium-independent vascular smooth muscle function indicated by the vasorelaxation of aortic rings to SNP was not impaired in aging rats and was also unaffected by acute INO-1001 treatment. (Fig. 3.B) Maximal isometric forces produced by the isolated aortic rings precontracted by potassium chloride (100mM) and phenylephrine (10 -6 M) were significantly lower in the aging control group as compared with young animals, which was not influenced by acute PARP-inhibition. (Fig. 4.) Cardiac function. In the aging control group we found significantly decreased mean arterial pressure (MAP), maximal left ventricular systolic pressure (LVSP), developed pressure (DP) and increased left ventricular end-diastolic pressure (LVEDP). Single dose treatment with INO-1001 in aging rats significantly improved the hemodynamic parameters MAP, LVSP and DP (Fig. 5.).

  model of advanced aging-associated heart failure. Am. J.Physiol. Heart. Circ. Physiol., 287, H2132-7. PACHER, P., VASLIN, A., BENKO, R., MABLEY, J.G., LIAUDET, L., HASKO, G., MARTON, A., BATKAI, S., KOLLAI, M., SZABO, C., 2004b. A new, potent poly(ADPribose) polymerase inhibitor improves cardiac and vascular dysfunction associated with advanced aging. J. Pharmacol. Exp. Ther., 311, 485-91. PACHER, P., SCHULZ, R., LIAUDET, L., SZABO, C., 2005. Nitrosative stress and pharmacological modulation of heart failure. Trends Pharmacol. Sci., 26, 302-10. PACHER, P., LIAUDET, L., MABLEY, J.G., CZIRAKI, A., HASKO, G., SZABO, C., 2006.Beneficial effects of a novel ultrapotent poly(ADP-ribose) polymerase inhibitor in murine models of heart failure. Int. J.Mol. Med., 17, 369-75. PIEPER, G.M., 1997. Acute amelioration of diabetic endothelial dysfunction with a derivative of the nitric oxide synthase cofactor, tetrahydrobiopterin. J.Cardiovasc. Pharmacol., 29, 8- 15. QUYYUMI, A.A., 1998. Does acute improvement of endothelial dysfunction in coronary artery disease improve myocardial ischemia? A double-blind comparison of parenteral D-and L-arginine. J.Am. Coll. Cardiol., 32,

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Acknowledgements

This work was supported by a Grant from the German Research Foundation (SFB 414) to G.S. and by the Hungarian Research Fund (OTKA AT049488) and the National Institutes of Health (R01 GM060915) to C.S. The expert technical assistance of Anne Schuppe and Heike Ziebart are gratefully acknowledged.

Contraction forces induced by potassium chloride (KCl; 0.1M) (A) and phenylephrine (PE; 10 -6 M) (B). Each column represents mean ± s.e.mean of 12 experiments with thoracic aortic rings in all groups. *, P<0.05 versus young control.