J Richard
email: julien.richard@cerfacs.fr

F Nicoud

Presqu'île de Giens (Var) Towards the Effect of the Fluid Ftructure Coupling on the Aeroacoustic Instabilities of Solid Rocket Motors

Keywords: Fluid-structure interaction, unstationary, LES

The industrial optimization needs nowadays the treatment of coupled phenomena such as aero-thermal or aero-elasticity. The following paper presents the construction and validation of a coupled software assembly, through the realization of simple test cases with a known analytical solution, with the aim to subsequently use it to solve an industrial configuration coming from the solid propulsion.

Introduction

Large solid propellant rocket motors may suffer aero-acoustic instabilities arising from a coupling between the burnt gas flow and the acoustic eigenmodes of the combustion chamber [START_REF] Culick | Remarks on Acoustic Oscillations in a Solid Propellant Rocket[END_REF]. During the firing, these instabilities generate pressure oscillations leading to thrust oscillations that could endanger the launcher integrity. Given the size and cost of any single firing test or launch, being able to predict and avoid these instabilities at the design level is of the utmost industrial importance. The full scale calculations of the Ariane 5 Solid Rocket Motors (P230) have allowed to highlight the impact of fluidstructure coupling on some elements of the structure such as the frontal thermal inhibitors. Inhibitors are designed with elastomers which burn at a lower speed than propergol, creating diaphragms with small rigidity across the flow. The large deformation and the flapping of these thermal inhibitors have already been calculated in a fully coupled way in the region of the second segment [START_REF] Wasistho | Numerical Study of Turbulent Flow in SRM with Protruding Inhibitors[END_REF] . It was shown that they have a strong influence on the amplitude of the thrust oscillations [START_REF] Ribereau | Numerical Simulation of Thrust Oscillations of Ariane 5 Solid Rocket Boosters[END_REF] [START_REF] Della Pieta | Couplage Fluide/Structure applique en propulsion spatiale[END_REF] . However, interaction between the structural response of some parts of the launcher, like the head end or the propergol, with the instabilities have not been investigated. The objective of this paper is to evaluate and validate the articulation of the numerical tools needed for these investigations, through the realization of simple test cases for which analytical solution can be obtained. This will lead, in a latter paper to an application on a subscaled version of the Ariane 5 SRM.

Presentation of the numerical software

Classically, the fluid-structure interaction problem consits in solving simultaneously both the fluid (1) and the structural (2) equations where some variables of one work as a source term for the other.

d dt (AW ) + F c (W, x, ˙ x) = R(W, x) (1)
M d 2 dt ( U) + D d dt ( U) + K U = f ext (W ( x,t), x) (2)
In this semidiscrete formulation already presented by Lesoinne in [START_REF] Lesoinne | Stability Analysis of Dynamic Meshes for Transient Aeroelastic Computations[END_REF], dots stand for time-derivatives, x is the displacement or position vector of the moving fluid grid points, W is the fluid state vector, A results from the discretization of the fluid equations, F c = F -ẋW is the vector of Arbitrary Lagrangian Eulerian (ALE) convective fluxes, F is the vector of convective fluxes, R the vector of diffusive fluxes, U the structural displacement vector, and finally M, D and K are respectively the mass, damping and stiffness matrices of the structural system. In order to solve the problem of fluid-structure interactions, we have chosen to use a Conventional Serial Staggered (CSS) method with subcycling, which has already been well studied and advocated [START_REF] Farhat | On the Accuracy, Stability, and Performance of the Solution of Three-Dimensional Nonlinear Transient Aeroelastic Problems by Partitioned Procedures[END_REF][13] [START_REF] Piperno | Partitioned procedures for the transient solution of coupled aeroelastic problems-Part II : Energy transfer analysis and three-dimensional applications[END_REF]. Used software are quicly presented in this part, then the CSS method is reminded.

AVBP

Two different modeling tools are used for this work. First of all, aero-acoustics calculations are performed with the Large Eddy Simulation (LES) solver AVBP [START_REF] Schönfeld | Steady and unsteady flows simulations using the hybrid flow solver AVBP[END_REF] developed at CERFACS and IFP, which has already been validated for aero-acoustic applications [START_REF] Schmitt | Large-eddy simulation and experimental study of heat transfer, nitric oxide emissions and combustion instability in a swirled turbulent high-pressure burner[END_REF] . AVBP is designed to solve the Navier-Stokes equations for three-dimensional compressible flows over unstructured meshes. The numerical discretization can be based on a Lax-Wendroff scheme which is 2nd order in both space and time. The sub-grid scale terms are modelled thanks to the Smagorinsky or the WALE model [START_REF] Nicoud | Subgrid-scale stress modelling based on the square of the velocity gradient tensor[END_REF].

MARC

The solids deformations are computed thanks to the structural analysis software MARC, using a finite element method (FEM) and developed by MSC-SOFTWARE. It is specialized in the treatment of non-linear materials, commonly encountered in solid propulsion. It allows static, dynamic and modal computations [20]. The numerical scheme used in our computations is the trapezoidal Newmark method [START_REF] Huges | The Finite element method. Linear static and dynamic finite element analysis[END_REF] which has already been advocated for coupling applications [START_REF] Giordano | Shock wave impacts on deforming panel, an application of fluid-structure interaction[END_REF] [START_REF] Piperno | Partitioned procedures for the transient solution of coupled aeroelastic problems-Part II : Energy transfer analysis and three-dimensional applications[END_REF]. It consists of writing the structure's equilibrium at the time t n+1 , knowing its state at t n , and the external force f at t n+1 :

4 ∆t 2 s M + 2 ∆t s D + K u n+1 = f n+1 + 4 ∆t 2 s M + 2 ∆t s D u n + 4 ∆t s M + D un + M ün (3)

PALM

The PALM coupler developed at CERFACS is used to synchronize these two solvers [START_REF] Buis | PALM : A Computational Framework for assembling High Performance Computing Applications[END_REF]. It also provides data transmission between the different tools at the fluid-structure interface without large intrusion in the different solvers. As a first step, the interpolation between the unmatching meshes is achieved with a nearest-point method.

Coupling architecture

In most cases the time step ∆t f for the fluid is smaller than the structural one ∆t s . We consider that the coupling time step ∆t c is equal to the structural one ∆t s . The following steps are done for each coupling step of the CSS method in order to go from t n to t n+1 = t n + ∆t s :

-1 : Predict the structural u n+1 p displacement at time t n+1 . A common predictor is :

u n+1 p = u n + α 0 ∆t s un + α 1 ∆t s ( un -un-1 ) (4)
It is worth to notice that the choice of α 0 = 1 and α 1 = 0 raises to a first order predictor, while α 0 = 1 and α 1 = 0.5 defines a second order predictor. -2 : Advance the fluid system to t n + ∆t s while updating the position of the fluid grid in order to match the position u n+1 p at the end of the coupling steps. Because ∆t f < ∆t s this step is achieve by subcycling the fluid solver. -3 : Transfer the fluid pressure P n+1 S to the structure. It's worth to notice that P n+1 S is not necessary the fluid pressure at the interface at time t n+1 . Many choices for P n+1 S can be found in the literature and we have retained three of them which are explained in details later. -4 : Finally integrate the structure to t n+1 . In order to ensure the Geometric Conservation Law (GCL) [START_REF] Lesoinne | Geometric conservation laws for flow problems with moving boundaries and deformable meshes, and their impact on aeroelastic computations[END_REF] the velocity of the fluid/structure interface is kept constant during the subcycling and equal to u n+1 p -u n p ∆t s . If no thermal inhibitors are considered, the assumption of small deformations is well justified in the context of solid propulsion described in the introduction. This approximation allows to model the wall motion in the LES solver by a transpiration boundary condition, which reproduces correctly the coupled solid surfaces motion speed [13][18]. This approach was preferred as a first step since it avoids considering mesh deformation, so it simplifies the overall chain complexity.

0D test configuration 3.1 Device description

The coupled system is composed for its fluid portion of an adiabatic room, closed at its left side by a fixed wall and at its right by a movable piston. It has a section S 0 and a length L 0 when the piston is at x = 0. The piston has a mass m and is attached to an external fixed point through a spring of rigidity k. Displacement, velocity, and acceleration of the piston at time t are respectively x(t), ẋ(t), and ẍ(t).

P 0 K X X=0 L 0 FIGURE 1 -0D coupling system
The following assumptions are first made for the system : -The gas is perfect with an adiabatic coefficient γ -The gravity effect is neglected -The spring mass is neglected -The spring is considered with no dissipation

0D Modelling

Lefrançois and Boufflet have shown in [START_REF] Lefrançois | An Introduction to Fluid-Structure Interaction : Application to the Piston Problem[END_REF] that applying Newton's second law to the piston and considering that its position at rest corresponds to x(t) = ẋ(t) = ẍ(t) = 0 and P = P 0 leads to :

m ẍ(t) = -k x(t) + S 0 (P(x(t),t) -P 0 ) (5)
All fluid variables are supposed to be to be uniform in the chamber (0D assumption), which means that P(x(t),t) = P(t). Due to adiabatic walls, there is no thermal flux between the gas and the chamber. Then, supposing that the compression is done in reversible way, allows to apply Laplace's law which states that :

P(x(t),t) = P(t) = P 0 L γ 0 (L 0 + x(t)) γ (6)
By re-injecting (6) in ( 5) and linearizing for small displacements around x = 0, we get :

m * ẍ(t) = -k x(t) + S 0 -γ P 0 L 0 x(t) (7)
Solving [START_REF] Giordano | Shock wave impacts on deforming panel, an application of fluid-structure interaction[END_REF] gives us both the temporal evolution of the piston position and the chamber's pressure. This corresponds respectively to equations ( 8) and [START_REF] Lefrançois | An Introduction to Fluid-Structure Interaction : Application to the Piston Problem[END_REF].

x(t) = x 0 cos k + γP 0 S 0 /L 0 m t (8)
P(t) = P 0 + γP 0 L 0 x 0 cos k + γP 0 S 0 /L 0 m t + π = P 0 + P 1 cos k + γP 0 S 0 /L 0 m t + π (9)

Energy consideration

By using a simple switch of variable, it is possible to write that pressure and structure are vibrating at the same circular frequency ω = k+γP 0 S 0 /L 0 m , but with a phase difference φ = π. Two scalars are introduced as follow : k e = x 0 P 1 cos(φ), and h = ω∆t c . Piperno and Fahrat [START_REF] Piperno | Partitioned procedures for the transient solution of coupled aeroelastic problems-Part II : Energy transfer analysis and three-dimensional applications[END_REF] have calculated the amount of energy imbalance created at the fluid structure interface ∆E N after N oscillations for a general case including structural dissipation and for different choices of P n+1 S . According to their results ∆E N ∼ Nπ(δE F + δE S ) and in our particular case with subcycling we have :

δE F = k e (α 0 -1)h + 1 4 - 7α 0 12 + 3α 1 2 h 3 + O(h 4 ) ( 10
)
δE S is depending on the choice of P n+1 S . For the first choice, P n+1 S = P n+1 :

δE S = O(h 4 ) (11)
For the second choice,

P n+1 S = 1 ∆t c R t n+1
t n P(t)dt :

δE S = k e - h 2 + h 3 8 + O(h 4 ) (12)
For the third choice,

P n+1 S = 2 ∆t c R t n+1
t n P(t)dt -P n S :

δE S = O(h 4 ) (13)
Using a first order predictor (which means α 0 = 1 and α 1 = 0) and noticing that in this case k e < 0, the first and third choice of P n+1 S are attended to produce energy at the third order, which is sufficient with regard to the accuracy of the fluid and structural solvers. On the other hand, the second choice of P n+1 S will create energy at the first order, which must be seen in our simulations as a linear growth of the oscillations's amplitudes

Numerical Results

The structure is modeled in two dimensions for this test case, the spring being represented in MARC by a bloc of material of Young's Modulus E M . It has a section S M0 = S 0 and an unstretched lenght of L M0 . Assuming that the poisson coefficient is set to zero, and that the displacements are small, this is equivalent to a spring of rigidity k = E M * S 0 L M0 . Considering the 0D assumption, AVBP is replaced in the coupled software assembly by a simple 0D program which uses the information from MARC to compute P n+1 S according to Laplace's law. Figure [START_REF] Buis | PALM : A Computational Framework for assembling High Performance Computing Applications[END_REF] shows that the three choices of P n+1 S give a good agreement with theoretical results, the piston vibrating at the predicted frequency. In the same way figure [START_REF] Culick | Remarks on Acoustic Oscillations in a Solid Propellant Rocket[END_REF], shows that the first and third choices of P n+1 S seem to well conserve the energy of the system, whereas as predicted previously, the second choice leads to a linear growth of the amplitude, thus of the energy. This one dimensional test case is designed in order to account for the acoustics in a fluid-solid coupled system. Starting from rest, the fluid part will be disturbed with a pressure Dirac impulsion. After some iterations, coupled eigenfrequencies should appear while looking at the pressure signal in the chamber.

Device description

The set-up is composed of an adiabatic chamber filled with gas, closed at its left by a fixed wall and at the right by a deformable block of rubber fixed on its right side, see figure [START_REF] Della Pieta | Couplage Fluide/Structure applique en propulsion spatiale[END_REF]. The fluid chamber has a section S 0 and length l 0 when the block of rubber is at rest at x = l 0 . The rubber of density ρ S , has a Young modulus E and a Poisson ratio ν, and is fixed at x = L 0 . Displacement (or position, depending the contest), velocity, and acceleration of a point U of the solid at time t are respectively U(t), ˙ U(t), and ¨ U(t). The following assumptions are first made for the system : -The gas is perfect with an adiabatic coefficient γ -There is no external volume forces nor on the solid, neither on the fluid -Both fluid and solid are considered with no dissipation -The deformations of the solid are small, which allows to approximate the interface location by l 0

Acoustic Modelisation

Considering an eigen circular frequency ω c of the coupled system, we have :

For the Fluid :

C G = √ γRT and k G = ω c C G
For the Solid :

C S = (1-ν)E ρ S (1+ν)(1-2ν) and k S = ω c C S
The reader can find a general method to evaluate the speed of sound in a solid in [START_REF] Bacon | Mécanique Des Solides Déformables[END_REF]. P being the oscillating pressure and U the oscillating velocity, the wave propagation for both fluid and solid, can be written :

P G,S = A + G,S e jk G,S x + A - G,S e -jk G,S x (14)
UG,S = 1 ρ G,S C G,S (A + G,S e jk G,S x -A - G,S e -jk G,S x ) (15)
Using ( 14) and [START_REF] Ribereau | Numerical Simulation of Thrust Oscillations of Ariane 5 Solid Rocket Boosters[END_REF] with the boundary conditions which are :

-UG (0) = US (0) = 0 (wall no-slip condition for the fluid and embedding for the solid) -UG (l) = US (l) (velocity continuity at the fluid structure interface located at l 0 ) -P G (l) = P S (l) (Stress continuity at the fluid structure interface located at l 0 ) one can find that ω c has to be the solution of [START_REF] Schmitt | Large-eddy simulation and experimental study of heat transfer, nitric oxide emissions and combustion instability in a swirled turbulent high-pressure burner[END_REF] which can be solved numerically.

tan( ω c C S (l 0 -L 0 )) tan( ω c C G (l 0 )) = ρ S C S ρ G C G (16)

Numerical Results

Both fluid and structure are modeled in two dimensions for this test case. In order to avoid any acoustic interference, a symmetry boundary condition has been used for the fixed bottom and top walls. This trick still ensures the absence of flow and thermal flux normal to the walls, and suppresses the impact of a noslip condition on the one dimensional assumption. The solid is modelled in MARC by a bloc of material of Young's Modulus E = 4.274 10 5 Pa and Poisson's coefficient ν = 0. It has a section S M0 = S 0 and an unstretched length of L M0 . The calculation with the first and second choice of P n+1 S have been achieved using coupling time step ten times bigger than the fluid one (ie ∆t coupling = ∆t MARC = 10∆t AV BP ), and frequencies results are represented on figure [START_REF] Fabignon | Instabilities and pressure oscillations in solid rocket motors[END_REF]. Unfortunately the third choice for P n+1 S appeared to be quickly numerically unstable. We don't have any valuable explanation to this behavior for the moment. t n P(t)dt), one can notice that some modes are damped while others seem to be fed in comparison with the spectra obtained with P n+1 S = P n+1 . This results is explainable with the same energy consideration than in the 0D part. Indeed, writing pressure and displacement at the fluid structure interface for one of the coupled eigen-modes leads to [START_REF] Schönfeld | Steady and unsteady flows simulations using the hybrid flow solver AVBP[END_REF] which shows that the pressure and the structure are vibrating at the same circular frequency ω c but with a phase depending of k G and l 0 . The reader can check that the phase φ is π if 1 2 sin( 4π f c l 0 C G ) > 0 where f c is en eigenfrequency of the coupled system, and φ = 0 in other cases. Using the same scalar k e and h as in the 0D part, it comes out that the second choice of P n+1 S will feed in energy some modes and damp the others. This can be visualized on Fig. [START_REF] Farhat | On the Accuracy, Stability, and Performance of the Solution of Three-Dimensional Nonlinear Transient Aeroelastic Problems by Partitioned Procedures[END_REF].

Conclusion

As predicted in the literature, it follows from these two test cases that the choice of P n+1 S is an important factor of the CSS method. As expected analytically, the first choice has shown a good energy conservation and a good behavior when dealing with acoustics. That is the reason why it appears to be adapted for our next applications. Indeed, including the fluid-structure interaction in solid propulsion simulation implies to be able to deal correctly with self-exited acoustic modes (see figure [START_REF] Huges | The Finite element method. Linear static and dynamic finite element analysis[END_REF]) rising from a coupling between the hydrodynamics and the acoustic eigenmodes of the propellant's chamber (see figure [START_REF] Giordano | Shock wave impacts on deforming panel, an application of fluid-structure interaction[END_REF] and figure [START_REF] Lefrançois | An Introduction to Fluid-Structure Interaction : Application to the Piston Problem[END_REF]). The second choice must be avoided due to its poor energy conservation and it has shown unphysical feeding or damping for some modes. The third one gives good results in 0D, but it has shown itself numerically unstable in the 1D case.

FIGURE 2 -

 2 FIGURE 2 -Fourrier's Transform of the displacement FIGURE 3 -Displacement's amplitude

FIGURE 4 -

 4 FIGURE 4 -Acoustic coupling system

FIGURE 5 -

 5 FIGURE 5 -Fourrier's Transform of the pressure at the middle of the chamber for the first choice of P n+1 S

FIGURE 6 -

 6 FIGURE 6 -Feeded and Damped modes

FIGURE 7 -FIGURE 9 -

 79 FIGURE 7 -Instabilities mechanism FIGURE 8 -Example of self-exited frequencies in a solid rocket motor


Silvia Ferrè

Erika Baldoli

Marzia Leidi

Jeanette A M Maier
email: jeanette.maier@unimi.it

Jeanette Am Maier

Magnesium deficiency promotes a proatherogenic phenotype in cultured human endothelial cells via activation of NFkB

Keywords: endothelial cells, magnesium, NFkB, cytokines, MMP Magnesium Mg, matrix metalloprotease MMP, inhibitor of metalloprotease TIMP, interleukin IL, granulocyte macrophage-colony stimulating factor GM-CSF, platelet derived growth factor PDGF, tumor necrosis factor TNF, coronary artery disease CAD, Vascular Cell Adhesion Molecule VCAM, plasminogen activator inhibitor PAI, human umbilical vein endothelial cells HUVEC, lipopolysaccaride LPS, standard deviation SD

Phenotypic modulation of endothelium to a dysfunctional state contributes to the pathogenesis of atherosclerosis, partly through the activation of the transcription factor NFkB. Several data indicate that magnesium deficiency caused by prolonged insufficient intake and/or defects in its homeostasis may be a missing link between diverse cardiovascular risk factors and atherosclerosis. Here we report that endothelial cells cultured in low magnesium rapidly activate NFkB, an event which is prevented by exposure to the anti-oxidant trolox. It is well known that NFkB activation correlates with marked alterations of the cytokine network. In the present study, we show that exposure of endothelial cells to low magnesium increases the secretion of RANTES, interleukin 8 and platelet derived growth factor BB, all important players in atherogenesis. Moreover, we describe the increased secretion of matrix metalloprotease-2 and -9 and of their inhibitor TIMP-2. Interestingly, by zymography we show that metalloprotease activity predominated over the inhibitory effect of TIMP-2.

These results indicate that low magnesium promotes endothelial dysfunction by inducing proinflammatory and pro-atherogenic events.

A C C E P T E D

M A N U S C R I P T

INTRODUCTION

Vascular endothelium, which comprises a monolayer of more than ten trillion endothelial cells, represents a dynamic interface between the blood and the surrounding tissues [START_REF] Galley | Physiology of the endothelium[END_REF]. Apart from forming a selective semi-permeable barrier, the endothelial cells actively and reactively control vascular tone and hemostasis as well as the ingress and egress of leukocytes. Consequently, it is not surprising that endothelial dysfunction is the common denominator that links cardiovascular risk factors and some diseases (diabetes, hyperlipemia, hypertension) to atherosclerotic burden [START_REF] Vanhoutte | Endothelial dysfunction: the first step toward coronary arteriosclerosis[END_REF].

Indeed, endothelial dysfunction contributes to the initiation and progression of atherosclerotic disease and has been suggested to be an independent vascular risk factor [START_REF] Esper | Endothelial dysfunction: a comprehensive appraisal[END_REF].

Also magnesium (Mg) deficiency has been reported to be involved in the pathogenesis of cardiovascular diseases in general and of atherosclerosis in particular [START_REF] Maier | Low magnesium and atherosclerosis: an evidence-based link[END_REF]. Indeed, Mg fortification of drinking water succeeded in the inhibition of atherogenesis in APO-E-deficient mice fed with a high cholesterol diet [START_REF] Ravn | Oral magnesium supplementation induces favorable antiatherogenic changes in ApoE-deficient mice[END_REF] and oral Mg therapy has been associated with significant improvement of endothelial function in patients with coronary artery disease (CAD) [START_REF] Shechter | Oral magnesium therapy improves endothelial function in patients with coronary artery disease[END_REF]. In addition, the Atherosclerosis Risk in Communities Study concluded that the highest risk for CAD occurred in subjects with the lowest serum Mg, even after controlling for the traditional CAD risk factors [START_REF] Liao | Is low magnesium concentration a risk factor for coronary heart disease? The Atherosclerosis Risk in Communities (ARIC) Study[END_REF].

Evidence suggests that the occidental diet is relatively deficient in Mg because of preference for calorie-rich, micronutrient-poor foods, low Mg content in water and soil, and processing of many food items [START_REF] Ford | Dietary magnesium intake in a national sample of US adults[END_REF]. Moreover, it should be recalled that Mg deficiency is a significant clinical complication arising in patients with diabetes mellitus, in nephropathics, in individuals treated with some classes of diuretics or anticancer drugs, as well as in alcoholists. Hypomagnesemia is also common in hospitalized patients, in particular in the elderly [START_REF] Ford | Dietary magnesium intake in a national sample of US adults[END_REF].

In cultured endothelial cells, low Mg increases the adhesion of monocytes to cultured endothelial cells via the upregulation of Vascular Cell Adhesion Molecule (VCAM), induces plasminogen activator inhibitor (PAI) 1, augments the levels of the pro-inflammatory cytokine interleukin (IL)-1 alpha, impairs endothelial proliferation and promotes cellular senescence [START_REF] Maier | Low magnesium promotes endothelial cell dysfunction: implications for atherosclerosis, inflammation and thrombosis[END_REF][START_REF] Killilea | A connection between magnesium deficiency and aging: new insights from cellular studies[END_REF]. Recently, endothelial function has been shown significantly impaired in a model of inherited hypomagnesemia in mice (MgL mice) [START_REF] Paravicini | Dysregulation of vascular TRPM7 and annexin-1 is associated with endothelial dysfunction in inherited hypomagnesemia[END_REF]. In agreement with the results obtained in cultured endothelial cells, MgL aortas had higher expression of VCAM and PAI-1 than their controls [START_REF] Paravicini | Dysregulation of vascular TRPM7 and annexin-1 is associated with endothelial dysfunction in inherited hypomagnesemia[END_REF].

In addition, in Mg deficient rodents an inflammatory response has been described [START_REF] Mazur | Magnesium and the inflammatory response: potential physiopathological implications[END_REF]. All these results point to the fact that low Mg promotes the acquisition of an inflammatory phenotype in the endothelium.

The family of transcription factors named NFkB controls the global inflammatory response and

A C C E P T E D M A N U S C R I P T

regulates the proatherogenic program in endothelial cells [START_REF] Kempe | NFkB controls the global pro-inflammatory response in endothelial cells: evidence for the regulation of a pro-atherogenic program[END_REF]. In resting cells, NFkB subunits are held in the cytosol because of their association with inhibitory proteins denominated IkB [START_REF] Kempe | NFkB controls the global pro-inflammatory response in endothelial cells: evidence for the regulation of a pro-atherogenic program[END_REF].

Upon cytokine signalling or environmental stress, NFkB activation is initiated [START_REF] Denk | Activation of NF-kappa B via the Ikappa B kinase complex is both essential and sufficient for proinflammatory gene expression in primary endothelial cells[END_REF]. The canonical pathway of NFkB activation involves the rapid degradation of IkB by the proteasome, the release of NFkB subunits, their translocation to the nucleus and binding to the cognate DNA motifs in target genes.

Because i) endothelial dysfunction, early event in atherogenesis, is driven by NFkB [START_REF] Denk | Activation of NF-kappa B via the Ikappa B kinase complex is both essential and sufficient for proinflammatory gene expression in primary endothelial cells[END_REF], and ii) Mg deficiency affects endothelial behaviour and contributes to atherosclerosis [START_REF] Maier | Low magnesium and atherosclerosis: an evidence-based link[END_REF][START_REF] Maier | Low magnesium promotes endothelial cell dysfunction: implications for atherosclerosis, inflammation and thrombosis[END_REF], we investigated NFkB in human umbilical vein endothelial cells (HUVEC) cultured in low Mg. We show that Mg deficiency activates NFkB and this event associates with the overexpression of the chemokines IL-8

and RANTES, of platelet derived growth factor (PDGF)-BB, Tissue Inhibitor of Matrix Metalloproteases (TIMP)-2 and metalloproteases (MMP) 2 and 9, all implicated in inflammation and atherogenesis.

A C C E P T E D M A N U S C R I P T

MATERIALS AND METHODS

2.1.

Cell culture and reporter gene assay. Human umbilical vein endothelial cells (HUVEC) were cultured in M199 containing 10% fetal bovine serum, 1 mM glutamine, 1 mM penicillin and streptomycin, Endothelial Cell Growth Factor (ECGF, 150 µg/ml), 1 mM sodium pyruvate and heparin (5 units/ml) on 2% gelatin-coated dishes [START_REF] Maier | Low magnesium promotes endothelial cell dysfunction: implications for atherosclerosis, inflammation and thrombosis[END_REF]. A magnesium free medium was purchased by Invitrogen (San Giuliano M.se, Italy) and utilized to vary the concentrations of magnesium by the addition of MgSO 4 [START_REF] Maier | Low magnesium promotes endothelial cell dysfunction: implications for atherosclerosis, inflammation and thrombosis[END_REF]. On the basis of previous reports [START_REF] Maier | Low magnesium promotes endothelial cell dysfunction: implications for atherosclerosis, inflammation and thrombosis[END_REF], we cultured HUVEC in medium containing 0.1 or 1.0 mM. In all the experiments the cells were seeded in growth medium; after 24 h, the medium was changed to expose the cells to either 0.

A C C E P T E D M A N U S C R I P T mixtures.
For supershift experiments, nuclear extracts were incubated with antibodies (1 µg) against p65 and p50 (Santa Cruz Biotechnology-Tebu Bio, Magenta, Italy) for 60 min on ice followed by 25 min of incubation with oligonucleotide.

Protein-DNA complexes were resolved on 5% acrylamide gels. Gels were dried and radiolabelled bands detected by autoradiography. The experiments were repeated three times with similar results.

2.3. Protein Array. HUVEC were cultured in medium containing 0.1 or 1.0 mM Mg for 2 days. 5 ml of conditioned media were centrifuged and utilized to incubate the membranes on which 40 antibodies against cytokines were spotted in duplicate (RayBiotech-Tebu Bio, Milano, Italy). The assay was performed according to the manufacturer's instructions. Densitometry was performed by the ImageJ software. Data are expressed as the mean ± SD of two separate experiments in duplicate.

TransAM assay.

TransAM assays were performed according to the manufacturer's instructions (Active Motif, Carlsbad, CA, USA). TransAM NFkB family kits are ELISA-based kits designed specifically to quantify NFkB activation. In brief, 5 µg of nuclear protein samples were incubated for 1 h in a 96-well plate coated with an oligonucleotide that contains the NFkB consensus site (5'-GGGACTTTCC-3') to which activated NFkB factors contained in nuclear extracts specifically bind.

By using an antibody directed against an epitope on p50 or p65 that is accessible only when NFkB is activated and bound to its target DNA, the NFkB complex bound to the oligonucleotide is detected. After incubation for 1 h with a secondary horseradish peroxidase-conjugated antibody, specific binding was detected by colorimetric estimation on a spectrophotometer at 450 nm. Results are expressed as arbitrary units and represent the mean ± SD of two separate experiments in triplicate.

Immunoblot analysis and ELISA.

Culture media were resolved by SDS-PAGE, transferred to nitrocellulose sheets at 200 mA for 16 h, and probed with anti-PDGF-BB, anti-RANTES, anti-IL-6, anti-MMP-9 and anti-MMP-2 antibodies (Santa Cruz Biotechnology-Tebu Bio). Secondary antibodies were labeled with horseradish peroxidase (GE Healthcare). The SuperSignal chemiluminescence kit (Pierce, Rockford, IL) was used to detect immunoreactive proteins. In some experiments, nuclear extracts were obtained using the Nuclear Extract kit (Active Motif) and processed by western blot using antibodies against NFkB subunits p50 or p65 (Tebu Bio). All the experiments were repeated at least three times with comparable results.

The amounts of TIMP-2 and IL-8 were measured in 1:2 diluted medium using a double-antibody sandwich ELISA (GE Healthcare) according to the manufacturer's instructions. The concentrations

A C C E P T E D M A N U S C

A C C E P T E D M A N U S C R I P T

RESULTS

Activation of NFkB in Mg deficient HUVEC

Because i) Mg deficiency promotes inflammation and atherogenesis [START_REF] Maier | Low magnesium and atherosclerosis: an evidence-based link[END_REF][START_REF] Mazur | Magnesium and the inflammatory response: potential physiopathological implications[END_REF] and ii) NFkB regulates many genes involved in inflammation and atherosclerosis [START_REF] Kempe | NFkB controls the global pro-inflammatory response in endothelial cells: evidence for the regulation of a pro-atherogenic program[END_REF][START_REF] Denk | Activation of NF-kappa B via the Ikappa B kinase complex is both essential and sufficient for proinflammatory gene expression in primary endothelial cells[END_REF], we examined the effects of low The localization of p50 and p65, which are known to translocate to the nucleus upon activation, was then assessed by western blot. We found that both p50 and p65 accumulate in the nuclei within 1 h of culture in 0.1 mM Mg (fig. 1C) and that LPS markedly induced the nuclear translocations of the two proteins in HUVEC in 0.1 and in 1.0 mM Mg.

The increase in NFkB binding activity by Mg deficiency correlated with the functional induction of NFkB as demonstrated by reporter gene assays using a vector expressing luciferase under the control of multiple copies of the NFkB responsive consensus. The construct was transiently transfected into HUVEC and the activity of luciferase was monitored as described in the methods.

An increase of luciferase activity was observed in cells exposed to 0.1 mM Mg both under basal conditions and after exposure to LPS, indicating that the NFkB complexes are transcriptionally active (fig. 1D).

Inhibition of NFkB activation by trolox in Mg deficient HUVEC.

Mg deficiency has been associated with oxidative stress [START_REF] Wolf | Magnesium deficiency and endothelial dysfunction: is oxidative stress involved?[END_REF] which is known to activate NFkB [START_REF] Denk | Activation of NF-kappa B via the Ikappa B kinase complex is both essential and sufficient for proinflammatory gene expression in primary endothelial cells[END_REF].

We therefore evaluated whether an anti-oxidant might prevent NFkB activation in HUVEC cultured in low Mg. Among many anti-oxidants available, for these studies we selected trolox, a watersoluble analog of α-tocopherol which has been demonstrated to exert a more potent cytoprotective effect than α-tocopherol [START_REF] Mabile | Alpha-Tocopherol and trolox block the early intracellular events (TBARS and calcium rises) elicited by oxidized low density lipoproteins in cultured endothelial cells, Free Radic[END_REF]. HUVEC were pretreated with trolox (40 µM) for 60 min before being exposed to 0.1 mM Mg containing medium for 1 h.

A C C E P T E D M A N U S C R I P T

We first evaluated nuclear translocation of p65 and p50 in HUVEC cultured in 0.1 or 1.0 mM Mg in the presence or in the absence of trolox. We found that trolox inhibited p65 and p50 nuclear accumulation (fig. 2A). Quantification through the TransAM assay confirmed this finding for both p50 and p65 (fig. 2B).

These results strongly suggest that NFkB activation by Mg deficiency is mediated by oxidant species.

Modulation of molecules involved in atherogenesis and inflammation in Mg deficient HUVEC.

We utilized a protein array tailored for proteins involved in inflammation and atherogenesis to study how culture of HUVEC in low Mg modulated the levels of these molecules. To this purpose, HUVEC were cultured for 48 h in 0.1 or 1.0 mM Mg. The medium was then collected and utilized for the proteomic analysis. The results were quantified by densitometry. Figure 3 shows a significant increase of the secretion of PDGF-BB, TIMP-2 and IL-8, and to a lower extent, of granulocyte macrophage-colony stimulating factor (GM-CSF) and RANTES in HUVEC in 0.1 mM Mg. No modulation of secreted transforming growth factor β1, IL-6 and tumor necrosis factor (TNF) α was detected. On the basis of their relevance in atherosclerosis, some of these proteins were further investigated by western blot or ELISA. Western blot confirms that, while IL-6 is not altered, secreted PDGF-BB and RANTES are increased in Mg deficient HUVEC (fig. 4, left panel).

Figure 4 (right panel) also shows the results of the densitometric analysis. We then measured the levels of IL-8 and TIMP-2 by ELISA. Figures 5A andB show the increase of the two secreted proteins in Mg deficient HUVEC vs controls.

MMP activity in Mg deficient HUVEC.

Because TIMP-2 antagonizes the action of MMP-2 and -9 [START_REF] Baker | Metalloprotease inhibitors: biological actions and therapeutic opportunities[END_REF], we evaluated the effect of Mg deficiency on the expression and on the activity of these proteases. By western blot we found that HUVEC in 0.1 mM Mg secreted higher amounts of the gelatinases MMP-9 and MMP-2 in respect to the controls (fig. 6A).We then analyzed medium collected from HUVEC cultured for 48 h in 0.1 or 1.0 mM Mg by zymography. Two clear bands of gelatinolytic activity, sized to 88 and 68 kDa, corresponding to activated MMP-9 and -2 respectively, were detected. Figure 6B shows the higher gelatinolytic activity of MMP-2 and -9 in the medium from cell cultured in 0.1 mM vs 1.0 mM Mg.

A C C E P T E D M A N U S C R I P T

DISCUSSION

Mg is the fourth most abundant cation in the human body. Approximately 60-65% of the total body Mg is mineralized in bone, whereas the remaining part is mainly localized in the intracellular compartments and only 1% is circulating in the blood. Current dietary guidelines recommend adequate intake of Mg (310-420 mg daily) to maintain a proper serum Mg concentration without drastically depleting the Mg stores. Hypomagnesaemia (serum Mg concentration below 0.74 mmol/L, or 1.8 mg/dL) can be the result of some therapeutic regimens and of various pathological conditions, including hereditary diseases [START_REF] Ford | Dietary magnesium intake in a national sample of US adults[END_REF][START_REF] Wolf | Magneium and tumors: ally or foes?[END_REF][START_REF] Verive | Evaluating the frequency rate of hypomagnesemia in critically ill pediatric patients by using multiple regression analysis and a computer-based neural network[END_REF][START_REF] Glaudemans | A missense mutation in the Kv1.1 voltage-gated potassium channel-encoding gene KCNA1 is linked to human autosomal dominant hypomagnesemia[END_REF]. In rodents, serum Mg concentration drops to 0.1 mM after 8 days on a Mg deficient diet. This is the concentration of extracellular Mg we used in our experimental model because it allows to detect significant effects in vitro. It should be pointed out, however, that we previously observed endothelial dysfunction also after exposure to a 0.5 mM Mg containing medium [START_REF] Maier | Low magnesium promotes endothelial cell dysfunction: implications for atherosclerosis, inflammation and thrombosis[END_REF]. On the other hand, it is possible that locally restricted dramatic changes in Mg concentration occur and influence endothelial cells in a specific district.

Mg is essential to maintain health and lower the risk of cardiovascular disease. Recent evidence from animal and clinical studies suggests that low Mg is associated with inflammatory processes and atherogenesis [START_REF] Laires | Role of cellular magnesium in health and human disease[END_REF]. Being endothelial dysfunction a critical step in atherogenesis, it is noteworthy that low Mg induces IL-1, VCAM and PAI-1 and promotes a senescent phenotype in cultured endothelial cells [START_REF] Maier | Low magnesium promotes endothelial cell dysfunction: implications for atherosclerosis, inflammation and thrombosis[END_REF][START_REF] Killilea | A connection between magnesium deficiency and aging: new insights from cellular studies[END_REF]. However, the molecular mechanisms involved have not been been elucidated. We anticipated a role of the transcription factor NFkB, crucial regulator of inflammation and atherosclerosis [START_REF] Kempe | NFkB controls the global pro-inflammatory response in endothelial cells: evidence for the regulation of a pro-atherogenic program[END_REF][START_REF] Denk | Activation of NF-kappa B via the Ikappa B kinase complex is both essential and sufficient for proinflammatory gene expression in primary endothelial cells[END_REF], which is activated in experimental and human atherosclerosis [START_REF] Monaco | Canonical pathway of NFkB activation selectively regulates proinflammatory and prothrombotic responses in human atherosclerosis[END_REF]. In Mg deficient HUVEC NFkB is rapidly activated. In particular, we have analyzed the prototypical NFkB complex demonstrating the involvement of p50 and p65 in low Mg dependent NFkB activation. Our results are in agreement with data obtained in primary cultured cerebral vascular smooth muscle cells demonstrating that low extracellular Mg activates NFkB [START_REF] Altura | Expression of NFkB and proto-oncogenes c-fos and c-jun are induced by low magnesium in aortic and cerebral vascular smooth muscle cells: possible link to hypertension, atherogenesis and stroke[END_REF]. In addition, Mg supplementation markedly attenuates NFkB nuclear translocation and protects IkB from degradation in LPS-treated endothelial cells [START_REF] Rochelson | Magnesium sulphate suppresses inflammatory responses by human umbilical endothelial cells through the NFkB pathway[END_REF]. Interestingly, in a model of cyclosporine-induced nephrotoxicity in rats, Mg supplementation showed marked beneficial effects by preventing NFkB activation which is responsible for renal damage [START_REF] Asai | Activation of transcription factors AP-1 and NFkB in chronic cyclosporine A nephrotoxicity: role in beneficial effects of magnesium supplementation[END_REF]. Therefore, Mg levels seem to participate to the complex regulation of NFkB activity in various systems. Mg deficiency has been shown to induce oxidative stress in endothelial cells [START_REF] Wolf | Magnesium deficiency and endothelial dysfunction: is oxidative stress involved?[END_REF] and oxidative stress is implicated in NFkB activation [START_REF] Monaco | Canonical pathway of NFkB activation selectively regulates proinflammatory and prothrombotic responses in human atherosclerosis[END_REF]. In atherogenesis, oxidative stress mediates signalling pathways underlying vascular inflammation, starting from the initiation of fatty streak development, through lesion progression, to plaque rupture [START_REF] Monaco | Canonical pathway of NFkB activation selectively regulates proinflammatory and prothrombotic responses in human atherosclerosis[END_REF]. Our results suggest that oxidant species are implicated in NFkB activation, at least early after exposure to low Mg containing medium. Indeed, in Mg deficient HUVEC, the anti-

A C C E P T E D M A N U S C R I P T
oxidant trolox, a synthetic cell-permeable analogue of α-tocopherol, prevents p50 and p65 nuclear translocation and activation. We therefore propose that in Mg deficient HUVEC oxidative stress might activate NFkB and, therefore, trigger NFkB signalling pathway. Accordingly, in HUVEC grown in 0.1 mM Mg we have shown the upregulation of IL-1α, a pro-inflammatory cytokine target of NFkB, which mediates some of low Mg effects on these cells [START_REF] Maier | Low magnesium promotes endothelial cell dysfunction: implications for atherosclerosis, inflammation and thrombosis[END_REF]. In the present study we utilized protein array to obtain a broad profile of the cytokines and growth factors released by Mg deficient HUVEC. Indeed, atherosclerotic plaque development involves several synchronized events such as adhesion, migration and proliferation of cells, all mediated by growth factors and cytokines, some of which resulted significantly increased in Mg-deficient HUVEC. In particular, in cells cultured in low Mg we describe the induction of RANTES, IL-8 and PDGF-BB, all targets of NFkB [START_REF] Monaco | Canonical pathway of NFkB activation selectively regulates proinflammatory and prothrombotic responses in human atherosclerosis[END_REF][START_REF] Khachigian | Nuclear factor-kappa B interacts functionally with the platelet-derived growth factor B-chain shear-stress response element in vascular endothelial cells exposed to fluid shear stress[END_REF]. A great attention has been focused on PDGF-BB in the origin of atherosclerosis, because of its mitogenic and chemotactic effects on vascular smooth muscle cells [START_REF] Ross | The pathogenesis of atherosclerosis: a perspective for the 1990s[END_REF]. Indeed, the abnormal proliferation and migration of these cells are major processes in the development of vascular disease, not only in atherosclerosis but also in restenosis after angioplasty. PDGF-BB is upregulated by pro-inflammatory stimuli from the early stages of atherogenesis and PDGF-BB mRNA expression is increased in atherosclerotic lesions [START_REF] Ross | Localization of PDGF-B protein in macrophages in all phases of atherogenesis[END_REF][START_REF] Barrett | Sis (platelet-derived growth factor B chain) gene transcript levels are elevated in human atherosclerotic lesions compared to normal artery[END_REF].

We also show an increase of the secretion of the chemokines RANTES and IL-8 in Mg-deficient HUVEC. Interestingly, chemokines are important actors in the progression of atherosclerosis as well as in plaque destabilization [START_REF] Zernecke | Chemokines in atherosclerosis. An update[END_REF]. In particular, the enhanced expression of IL-8 and RANTES within human atherosclerotic lesions has been reported [START_REF] Monaco | Canonical pathway of NFkB activation selectively regulates proinflammatory and prothrombotic responses in human atherosclerosis[END_REF][START_REF] Zernecke | Chemokines in atherosclerosis. An update[END_REF]. IL-8 is critical for chemotaxis and adhesion of monocytes to the endothelial cells, pivotal steps in atherogenesis, and also promotes vascular smooth muscle cell proliferation and migration, crucial events for the progression of the plaque [START_REF] Zernecke | Chemokines in atherosclerosis. An update[END_REF]. Similarly, RANTES directs leukocyte attraction and is also involved in neointima formation in atherosclerosis-prone mice. Hence, it is not surprising that antagonizing RANTES reduces atherosclerotic lesion formation and stabilizes the plaque [START_REF] Zernecke | Chemokines in atherosclerosis. An update[END_REF]. We therefore conclude that low Mg contributes to atherosclerosis by stimulating endothelial secretion of IL-8, RANTES and PDGF-BB. In our experimental conditions, however, we did not observe any modulation of TNFα and IL-6 which have been shown increased in animal model fed with a Mg-restricted diet [START_REF] Mazur | Magnesium and the inflammatory response: potential physiopathological implications[END_REF]. We hypothesize that different cell types, and not endothelial cells, might contribute to secrete these cytokines in vivo.

Protein array also revealed the increase of GM-CSF, another cytokine which is transcriptionally dependent upon NFkB activation. This finding is puzzling because GM-CSF regulates intimal cell proliferation in the early step of atherosclerosis [START_REF] Zhu | GM-CSF regulates intimal cell proliferation in nascent atherosclerotic lesions[END_REF]. More studies are necessary to confirm the regulation of GM-CSF levels in HUVEC cultured in low Mg.

A C C E P T E D M A N U S C R I P T

In atherosclerotic plaques, also the expression of MMPs, which degrade collagen fibrils leading to the loss of fibrous cap integrity, is increased [START_REF] Monaco | Canonical pathway of NFkB activation selectively regulates proinflammatory and prothrombotic responses in human atherosclerosis[END_REF]. Dysregulated extracellular matrix metabolism has a role in vascular remodelling during the development and complication of human atherosclerotic lesions. The expression of these proteases is normally tightly regulated at the transcriptional level.

A second level of control is represented by the requirement of extracellular activation of the latent pro-enzyme. A further level of control of MMP activity involves the irreversible binding of MMPs to the specific tissue inhibitors TIMPs. We found that Mg deficient HUVEC secreted increased amounts of MMP-2 and -9 and of their inhibitor TIMP-2. We anticipated that the increased amounts of TIMP-2 might represent a mechanism to counterbalance MMP activity. However, by zymography we show that MMP-2 and -9 activity overrides the inhibitory effect of TIMP-2. We therefore conclude that MMP/TIMP balance is shifted toward greater MMP activity in Mg-deficient HUVEC. This result further highlights how Mg deficiency might contribute to atherosclerosis.

Indeed, an increased expression of MMPs in human atherosclerotic plaques and, in particular, a marked upregulation of MMP-9 in unstable carotid plaque have been demonstrated [START_REF] Galis | Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques[END_REF]. Recently, genetic variation within the MMP-2 promoter region was associated with cap thickness and therefore it may influence the role of MMP-2 in plaque vulnerability [START_REF] Volcik | MMP-2 genetic variation is associated with measures of fibrous cap thickness: The Atherosclerosis Risk in Communities Carotid MRI Study[END_REF].

CONCLUSION

Our data indicate that low

  Mg on NFkB activation. We first performed EMSA on nuclear extracts prepared from HUVEC cultured in 1.0 or 0.1 mM Mg for 1 h. In nuclear extracts of HUVEC in 1.0 mM Mg, no retarded band was detected whereas two distinct bands could be clearly shown in the samples from cells exposed to 0.1 mM Mg (fig.1A, left panel). It is noteworthy that these bands were markedly induced by treatment with LPS (1 µg/ml), which is used as a positive control, in HUVEC in 0.1 and in 1.0 mM Mg. On the basis of the supershift analysis (fig.1B), these two bands were identified as the p50/p50 homodimer and p50/p65 heterodimer. Interestingly, in the presence of LPS a third shifted band -by supershift identified as the homodimer p65/65 -was detected. All the bands were specific as assessed by competition assays showing their suppression by incubation with a 50-fold excess of unlabelled NFkB probe (fig 1A, left panel). The bands revealed by EMSA were quantified by densitometry (fig. 1A, right panel).

Figure 1 .

 1 Figure 1. Induction of NFkB activity in HUVEC cultured in low Mg. (A) EMSA of the NFkB DNA binding activity was performed on nuclear extracts from HUVEC cultured in 0.1 or 1.0 mM Mg for 1 h, in the presence or in the absence of LPS (left panel). SP and UN are the specific and unspecific competition, respectively, of the LPS-treated HUVEC in 1.0 mM Mg. The binding activity of the constitutively expressed transcription factor oct-1 was used to assess equal loading. Right panel: each band detected by EMSA was quantified by densitometry and expressed on an arbitrary scale. (B) HUVEC were exposed to LPS for 1 h. Nuclear extracts were incubated with 1 µg of anti-p65 or anti-p50 antibodies and then electrophoresed. The shifting of the different NF-kB subunits is indicated by white arrowheads. (C) Nuclear extracts from HUVEC cultured in medium containing 0.1 or 1.0 mM Mg in the presence or in the absence of LPS were analyzed by western blot using antibodies against p65 or p50. TBP is used as a nuclear marker and GAPDH as an indicator of contamination by cytosol. (D) Luciferase assay was performed in HUVEC cultured in 0.1 or 1.0 mM Mg as described in the methods. Luciferase activity was detected by fluorimetry. Results are shown as the mean ± SD of three separate experiments..

Figure 2 .

 2 Figure 2. Inhibition of low Mg-induced NFkB activation by trolox. (A) Nuclear extracts of HUVEC in 0.1 or 1.0 mM Mg in the presence or in the absence of trolox (40 µM) for 1 h were obtained. Western blot was performed with antibodies against p65 or p50. TBP is shown as loading and fraction purity control. GAPDH shows that cytosolic contamination is very low. (B) p65 and p50 activity was quantified by TransAM NFkB analysis on nuclear extracts of HUVEC cultured in 0.1 or 1.0 mM Mg with or without trolox. Results are expressed as arbitrary units and represent the mean ± SD of 3 separate experiments.

Figure 3 .

 3 Figure 3. Protein arrays on media collected from HUVEC cultured in 0.1 or 1.0 mM Mg for 48 h.Densitometric analysis on array spots was performed and data are expressed as the mean ± SD.

Figure 4 .

 4 Figure 4. Amounts of secreted RANTES, PDGF-BB and IL-6 in Mg deficient HUVEC. A representative western blot on conditioned media from HUVEC cultured in 0.1 or 1.0 mM Mg is

Figure 5 .

 5 Figure 5. Increased secretion of IL-8 and TIMP-2 in Mg deficient HUVEC. (A) IL-8 and (B) TIMP-2 were detected by ELISA in media collected from HUVEC cultured in 0.1 or 1.0 mM Mg. Data are expressed as the mean of 3 different experiments in triplicate ± SD.

Figure 6 .

 6 Figure 6. Increased secretion of MMP-2 and -9 in Mg deficient HUVEC. Representative western blot (A) and zymography (B) on media collected from cells cultured in 0.1 or 1.0 mM Mg are shown.

  R I P T of TIMP-2 and IL-8 were determined by interpolation from a standard curve and shown as the mean ± SD of four separate experiments in triplicates.2.6. Gelatin zymography. Conditioned media were incubated at 4 °C overnight with gelatin-

	Sepharose. Zymography was performed using 8% polyacrylamide gels co-polymerized with 1
	mg/ml gelatin type B (Sigma Aldrich) under non-reducing conditions without heating. Gels were
	then washed twice for 30 minutes in 2.5% Triton X-100 at room temperature and incubated
	overnight in collagenase buffer (50 mmol/L Tris-HCl, pH 7.5, 10 mmol/L CaCl 2 , 150 mmol/L
	NaCl) at 37 °C. Gels were stained in Coomassie Blue R 250 (Bio-Rad, Milano, Italy) in a mixture of
	methanol:acetic acid:water (4:1:5) for 1 hour and destained in the same solution without the dye.
	Gelatinase activities were visualized as distinct bands.

2.7. Statistical Analysis. Statistical significance was determined using the Student's T test and set at p values less than 0.05. In the figures *: p< 0.05 **: p< 0.01; ***: p< 0.001.

ACKNOWLEDGEMENTS

The authors thank prof. Lorenza Tacchini for helpful suggestions and support. This work is supported by MIUR-PRIN 2007 grant number 2007ZT39FN.


Teresa Rampino

Marilena Gregorini

Giulia Bedino

Giovanni Piotti

Elisa Gabanti

Adalberto Ibatici

Nadia Sessarego

Cristina Piacenza

Teresa Chiara

Pasquale Balenzano

Esposito

Chiara Teresa Balenzano

Pasquale Esposito

Francesca Bosio

Grazia Soccio

Francesco Frassoni

Antonio Dal Canton
email: dalcanton@smatteo.pv.it

Prof Antonio Dal

MESENCHYMAL STROMAL CELLS IMPROVE RENAL INJURY IN ANTI-THY 1 NEPHRITIS BY MODULATING INFLAMMATORY CYTOKINES AND SCATTER FACTORS

Keywords: Mesenchymal stromal cells, Mesangioproliferative nephritis, Interleukins, Hepatocyte growth factor, Anti Thy-1 nephritis, Scatter factors smooth muscle actin BSA: bovin serum albumin DMEM: Dulbecco's Modified Eagle Medium EDTA: ethylenediaminetetraacetic acid EGFP

INTRODUCTION

Mesenchymal stromal cells (MSC) are multipotent cells that can be isolated from bone marrow, and fetal tissues (1) Because of their differentiation into mature cells of various types, and their easy expansion in vitro (2) MSC are a promising cell therapy that has been tried to regenerate injured organs in experimental and human disease [START_REF] Bi | Stromal cells protect against acute tubular injury via a endocrine effect[END_REF][START_REF] Morigi | Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renal failure[END_REF]. In addition,

MATERIALS AND METHODS

Animals.

Experiments were performed according to guidelines of our ethical committee for animal studies. Wild-type Sprague Dawley rats (Charles River, Calco, Italy) and transgenic Sprague-Dawley rats that express EGFP (Japan Slc, Hamamatsu, Japan) were used. The rats carrying the EGFP transgenic were established with the same construct and technique used to produce EGFP transgenic mice [START_REF] Okabe | Green mice' as a source of ubiquitous green cells[END_REF].

Isolation and Culture of MSC and Mesangial Cells.

Sprague Dawley EGFP transgenic rats were sacrificed by CO 2 inhalation. The total marrow cell content of both femurs and tibias was flushed from bones and EGFP positive cells were plated at a concentration of 10 6 cells/ml with Murine MesenCult medium (Stemcell Technologies, Vancouver, Canada) and incubated at 37°C with 5% humidified CO 2 atmosphere. After 48 h of culture, non-adherent cells were discarded; fresh medium was added and replaced twice a week. When cultures reached sub confluence, adherent cells were detached with 0.05% trypsin (Sigma-Aldrich, St Louis). Collected cells were replanted at a concentration ranging between 0.05-0.15x10 5 /ml of MesenCult medium for several passages. Rat mesangial line (CRL-2573) was obtained by ATCC (American Type Culture Collection, Manassas, USA). The cells were at passage 8, expressed normal genes of wild type mesangial cells, and were positive for desmin and vimentin and negative for cytokeratin 8. The cells were seeded at a concentration of 64,000/cm 2 on a culture flask and cultured in Dulbecco's Modified Eagle Medium (DMEM) high glucose medium (Gibco, Invitrogen, Paisley, UK), containing Penicillin/Streptomycin 1%, Amphotericin B, G418 (0.4 mg/mL) (Gibco) and Fetal Bovine Serum 15% (Euroclone, Torquay, UK), at 37°C in 5% humidified CO 2 atmosphere. After 48 h of culture, non-adherent cells were discarded; fresh medium was added and replaced twice a week. When cultures reached sub confluence, adherent cells were detached with 0.25% trypsin-ethylenediaminetetraacetic Acid (EDTA). Collected cells were replanted for several passages at a concentration ranging between 12,000 and 20,000/cm 2 .

Immunocytochemical Characterization and Osteogenic and Adipogenic Differentiation of MSC.

Cultured EGFP MSC were evaluated by flow cytometry starting from the 3 rd passage for CD90, CD73, and CD45, CD11b (Becton Dickinson, San Josè; CA) using FacsCalibur flow cytometer (BD Biosciences, San Josè, CA). The differentiation into osteocytes and adipocytes was evaluated as previously described [START_REF] Pittenger | Multilineage potential of adult human mesenchymal stem cells[END_REF]. Histochemical analysis of the cell layer was performed evaluating calcium deposition (Alizarin red staining, Sigma-Aldrich, St Louis MO). For adipogenic induction, 2x10 4 MSC/cm 2 were seeded in chamber slides with Mesencult. At confluence cells were stimulated with adipogenic medium (Cambrex, Walkersville, MD USA) for 3 weeks replacing the medium every 3 days. Cells were stained with Oil-Red O (Bioptica, Milan Italy).

In vivo experiments.

We studied a total of 5 groups of rats. Group A: 20 rats were injected at day 0 via tail vein (vtv) with 400 µl of saline that contained 400 µg of anti-Thy 1.1 IgG mAb (Cedarlane Laboratories, Toronto, Canada), as previously described [START_REF] Rampino | Neutralization of macrophage-stimulating protein ameliorates renal injury in anti-Thy 1 glomerulonephritis[END_REF]. Group B: 25 rats were injected vtv at day 0 with 400 µg of anti-Thy 1.1 IgG mAb, followed after 3 days by injection vtv of 1 ml of phosphate-bufferd saline (PBS) containing 3x10 6 MSC isolated A c c e p t e d M a n u s c r i p t from EGFP rats. Group C: 20 rats were injected vtv at day 0 with 400 µg of anti-Thy 1.1 IgG mAb, followed after 3 days by injection vtv of 3x10 6 rat mesangial cells. Group D: 20 rats were injected vtv at day 0 with 400 µl of saline, followed at day 3 by injection vtv of 1 ml of PBS containing 3x10 6 MSC isolated from EGFP rats. Group E: 20 rats were injected vtv with 400 µl of saline. Five rats of each group were sacrificed at day 1, 7, 10, 14 and five rats of group B were sacrificed 12 hours after MSC infusion. Soon after sacrifice kidneys, lung and spleen were removed and half fixed in 10% neutral-buffered formalin, half frozen in liquid nitrogen. All rats were male, weighed 180-200 g and were housed at constant temperature (20 C°) and humidity (75%) under controlled light cycle, with free access to water and standard chow diet. 24-hour urine was obtained by housing individual rats in metabolic cages for up to 24 hours on days 0, 7, 10, 14; urine volume was measured and samples were stored at -20 C°. Blood was drawn on day 0 from the caudal vein, and on days 7, 10, 14 by cardiac exsanguination and soon centrifuged, and serum was stored at -20 C°.

In vivo tracking of circulating anti-Thy 1.1 Ab.

MSC (1x10 5 ) cultured in chamber slides were fixed with formalin-acetate 10% for 10 minutes, washed in PBS and incubated in dark-humid chamber at room temperature for 60 minutes with rat serum (100 µl) sampled immediately before and 1, 24, 48, 72 h after anti-Thy1.1 Ab injection. Moreover, we used anti-Th1.1 Ab at concentration of 1 µg/µl adding 20 µl of Ab in a total volume of 100 µl of normal rat serum, as positive control. After three washings in PBS the cells were treated with the secondary antibody (Universal Biotinylated Link, Dako, Glostrup, Denmark) and the streptavidin-biotin-peroxidase complex (Dako). Visualization was in 3, 3-diaminobenzidine (Dako). Harris hematoxylin was used to counter stain the nuclei lightly. Finally the cells were dehydrated in increasing alcohol scale (95°C to 100°C, xylol) and the cover slip was mounted with synthetic non aqueous mounting media (Dako) for analysis with microscope Olympus IX8 connected with a computer system for cell imaging and CCD camera (Magnification X200) (Olympus Optical Co, Tokyo, Japan). All experiments were duplicated.

Deposition of anti-Thy1.1 Ab.

Anti-Thy 1.1 Ab deposition was detected by immunohistochemistry in formalin fixed kidney of rats of all groups sacrificed at day 1. Three-µm thick sections were collected on poly-L-lysine-coated slides (Dako), were dewaxed in xylol, passed in a decreasing series of alcohol, and finally rehydrated with distilled water. Endogenous peroxidase was blocked with H 2 O 2 3.7% vol/vol followed by H 2 O for 15 min. After 3 washings in 150 mM PBS the sections underwent microwave antigen retrieval. Subsequently in order to visualize anti-Thy 1.1 Ab deposition the sections were incubated with ready-to-use anti-mouse secondary antibody EnVision Plus (Dako) and colour reaction was developed with 3, 3diaminobenzidine (Dako) as the chromogen. The slides were then counterstained with Harris hematoxylin. The extent of anti-Thy 1.1 Ab deposited was evaluated in blinded manner in 40 random glomeruli in each kidney section by image analysis, using Olympus IX8 microscope connected with CCD camera and Cell-R software on image processing computer. We measured the ratio between the glomerular anti-Thy 1.1 Ab positive area and glomerular tuft area. Glomerular deposition of C3 was detected in frozen tissue of rats of all groups sacrificed at day 1. Five-µm thick sections were collected on poly-L-lysine-coated slides (Dako), were fixed in acetone for 5 minutes at room temperature, and then passed in distilled water. Subsequently the sections were exposed to polyclonal sheep anti-human C3 FITC Ab, 1:600 cross-reacting with rat C3 (ICN, Segrate, Italy) v/v PBS/Bovine Serum Albumin (BSA) 1% for one hour at room temperature. After 3 washings in PBS the cover slips were mounted with aqueous mounting media for analysis with fluorescence microscope. Negative controls included both omission of the primary Ab and substitution of IgG for primary antibodies. The extent of C3 deposition was evaluated in blinded manner in 40 random glomeruli in each kidney section as described above.

Deposition of C3 complement fraction.

Biochemical measurements.

Serum creatinine and urinary protein concentration were measured at days 0, 7, 10, 14 by an automated method (Abbott Laboratories, Abbott Park, IL), and the Bio-Rad Protein Assay (Bio-Rad Laboratories, Richmond, CA). Interleukin-6 (IL-6) and transforming growth factor-β (TGF-β) were quantified in rat serum at days 7, 10, 14 by ELISA (R&D Systems, Minneapolis, USA). IL-6 was measured in 24 h urine at days 7, 10, 14 by ELISA. All experiments were quadruplicated.

Renal Histopathology

Renal Morphology.

3-µm thick sections cut along the maximal sagittal plane of formalin-fixed kidney were stained with hematoxylin-eosin and were examined by two investigators in double blind fashion, using an Olympus IX8 microscope connected with a CCD camera and software imaging analysis Cell-R. A thorough analysis of glomerular lesions was performed in all glomeruli present in three sections of all kidneys of groups A, B and C. The following types of lesions were distinguished: (i) mesangiolysis, (ii) ballooning, (iii) microaneurysm, (iv) matrix or cellular crescent, (v) necrosis. The damage score was established using the following criteria: glomeruli without obvious changes were factored with 0, glomeruli with mesangiolysis and/or ballooning involving <25% of the glomerular area were factored with 1, glomeruli with mesangiolysis and/or ballooning involving 26 to 50% of the glomerular area were graded with 2, glomeruli with mesangiolysis and/or ballooning involving 51 to 75% of the glomerular area were graded with 3, glomeruli with mesangiolysis and/or ballooning involving >75% of the glomerular area and/or microaneurysm and/or crescents and/or glomerular necrosis were graded with 4. The results were expressed as the percentage of glomeruli in each grade score. Furthermore, we evaluated the extent of ballooning and necrosis as the ratio between the glomerular area replaced by ballooning and necrosis and total glomerular area.

Expression of ED-1 Ag, PCNA, α-SMA, PDGF-β, HGF, Met, MSP and Ron.

The glomerular expression of ED-1 Ag, proliferating cell nuclear antigen (PCNA), αsmooth muscle actin (α-SMA), plateled derived growth factor-β (PDGF-β), HGF, Met, MSP, and Ron were studied by immunohistochemistry in formalin fixed tissue. Three-µm thick sections of paraffin embedded tissue were collected on poly-L-lysinecoated slides (Dako), they were dewaxed in xylol, passed in a decreasing series of alcohol, and finally rehydrated with distilled water. Endogenous peroxidase was blocked with H 2 O 2 3.7% vol/vol followed by H 2 O for 15 min. After 3 washings in 150 mM of PBS the sections underwent microwave antigen retrieval. Subsequently the sections were exposed overnight at 4 C° to the following antibodies: 1) monoclonal mouse anti-rat ED-1 (Serotec Ltd, Oxford, UK), 1:300; 2) monoclonal mouse anti-PCNA, mAb (Santa Cruz Biotechnology, CA), 1:200; 3) monoclonal mouse anti-human α-SMA), 1:100, (Dako); 4) polyclonal rabbit anti-human PDGF-β (Santa Cruz Biotechnology), 1:200; 5) polyclonal rabbit anti-human HGF (Santa Cruz Biotechnology, 1:50; 6) monoclonal mouse antihuman Met (Novocastra LaboratoriesLtd, UK), 1:20; 7) polyclonal goat anti-mouse MSP (Santa Cruz Biotechnology), 1:600; 8) monoclonal mouse anti human RON (Transduction Laboratories, Lexington, KY), 1:750. After 3 washings in PBS the immunocomplex was visualized with the biotin-streptavidin-peroxidase complex and 3,3-diaminobenzidine (DAKO). Sections were faintly counterstained with Harris hematoxylin. Negative controls included both omission of the primary Ab and substitution of IgG for primary antibodies. In all glomeruli of three sections of each kidney we counted the cells positive for ED-1 Ag and PCNA and we measured the extent of PDGF-β, α-SMA, HGF, MSP and Ron expression as the ratio between the glomerular PDGF-β, α-SMA, HGF, MSP, Ron positive area and the whole glomerular area. Glomerular capillary Met expression was measured as the ratio between the number of Met stained glomerular capillaries and total number glomerular capillaries.

Tracking of EGFP positive cells.

We tracked by immunohistochemistry EGFP positive cells in kidney, spleen, lung excised 12 hours and at days 7, 10, 14 after injection of MSC. Three-µm thick sections of paraffin embedded tissue were collected on poly-L-lysine-coated slides (Dako), they were dewaxed in xylol, passed in a decreasing series of alcohol, and finally rehydrated with distilled water. Endogenous peroxidase was blocked with H 2 O 2 3.7% vol/vol followed by H 2 O for 15 min. After 3 washings in 150 mM of PBS the sections underwent microwave antigen retrieval. Subsequently the sections were exposed overnight at 4 C° to monoclonal mouse anti-green fluorescent protein (GFP) antibody IgG1, (Chemicon, Temecula, CA), diluted 1:1000 in PBS/BSA 1%. EGFP positive cells were visualized with the biotinstreptavidin-peroxidase complex and coloured in 3,3-diaminobenzidine (DAKO). Sections were faintly counterstained with Harris hematoxylin. Negative controls included omission of the primary Ab and substitution of IgG for primary antibodies. Positive controls were sections of kidney of EGFP rat. Ten sections of each organ were analyzed.

Statistical Analysis.

ANOVA followed by the Newman-Keuls test or Student t test were used for comparison of the means. Differences in renal injury scores were evaluated with Mann-Whitney and Kruskal Wallis tests. MSC isolated from bone marrow of Sprague Dawley EGFP rats differentiated into osteogenic and adipogenic cells when cultured in appropriate conditions (figure 1 suppl) Flow cytometric analysis showed that MSC were positive for CD90 (≥95%), CD73 (≥95%) and were negative for CD45 (<5%), CD11b (<5%).

Tracking of circulating anti-Thy 1.1 Ab.

In preliminary experiments we incubated MSC in vitro with rat serum sampled 1, 24, 48 and 72 hours after i.v. injection of anti-Thy 1.1 Ab and we detected MSC-binding antibody present in serum by immunocytochemistry. Figure 2 suppl shows no serum binding activity before antibody injection and intense staining of cell-bound antibody 1 hour after its injection. Progressively attenuated staining is visible after 24 and 48 hours and no residual antibody is detectable after 72 hours. These results exclude that residual circulating antibody can interfere with MSC infused after 72 hours.

In vivo experiments.

Throughout the study, no significant differences were observed in body weight, water and food intake among the five groups of rats.

Glomerular deposition of anti-Thy1.1 Ab and C3 complement fraction.

In order to demonstrate that the pathogenic steps that start anti-Thy 1 disease occurred similarly in untreated rats as in rats treated with MSC, we evaluated the glomerular deposition of anti-Thy 1.1 and C3 complement fraction within 24 hours of antibody injection. As expected, no deposition of anti-Thy 1.1 Ab nor C3 was observed in rats of groups D and E. 24 hours after the injection of anti-Thy 1.1 both the antibody and C3 were deposited in glomeruli similarly in groups A, B and C (figure3 suppl).

Renal function.

Baseline (day 0) serum creatinine was similar in the five groups of rats. In groups A, B e C creatinine increased significantly on day 7 compared with day 0; group A from 0.41±0.10 (mean±SD) to 0.62±0.14 mg/dl (P <0.05); group B from 0.51±0.20 to 0.74±0.20 mg/dl (P <0.05); group C from 0.44±0.30 to 0.64±0.05 mg/dl (P<0.05). At day 10, creatinine remained higher than baseline in group A (0.59±0.01mg/dl) and group C (0.58±0.03mg/dl), but not in group B (0.45±0.01mg/dl); indeed at day 10 creatinine was significantly lower in B than in A and C (P<0.001 B vs A and C). At day 14 creatinine decreased to levels no longer different from baseline in all three groups: group A (0.51±0.07mg/dl), group B (0.44±0.14mg/dl), group C (0.57±0.01 mg/dl). No change of creatinine occurred in rats not treated with anti-Thy 1.1 IgG (group E), even in those receiving MSC (groupD).

Baseline urinary protein excretion was similar in the five groups. Proteinuria rose similarly in groups A, B and C on day 3, then proteinuria increased further in groups A and C, but not in group B. In fact, proteinuria was significantly higher in groups A and C than in group B at days 7, 10 and 14. Furthermore, on day 14 proteinuria returned to levels not different from baseline in group B, while it remained higher than baseline in groups A and C. No change in proteinuria occurred in rats not treated with anti-Thy 1.1 IgG (groupE), even in those receiving MSC (group D) (figure 2). A damage score was established ranging from 0 (no obvious change) to 4 (mesangiolysis and /or ballooning involving >75% of the glomerular area and /or microaneurysm and /or crescents and/ or glomerular necrosis). A representative picture of renal histology is shown in figure 3. No histological changes were observed in groups D and E. The damage scores of glomeruli in groups A and B are summarized in figure 4. In group A, a high percentage of glomeruli were factored 3-4, while a low percentage of glomeruli was normal. The proportion of normal vs severely damaged glomeruli was inverted in group B; a high percentage of glomeruli scored 0 and a minority scored 3-4. In group C, the injury scores were similar as in group A: a high percentage of glomeruli were factored 3-4 (68%, 60% and 47% on days 7, 10 and 14 respectively), while a low percentage of glomeruli was normal (10%, 19%, and 20% on days 7, 10 and 14 respectively). Moreover, we quantified by image analysis the glomerular area occupied by ballooning and/or necrosis and we found that the extension of injured area was reduced in nephritic rats treated with MSC on days 7, 10 and 14, compared with untreated rats and rats receiving mesangial cells. MSC did not induce histological changes in healthy rats (group D) (figure 4 suppl).

Renal Histopathology

Glomerular monocytes infiltration and Ron expression

As shown in figure 5, the number of ED-1 positive cells was significantly lower in group B than in groups A and C at day 7 and day 10. As expected from the spontaneous course of the disease ( 9), the number of glomerular monocytes declined in all three groups at day 14. Mesangial cells did not affect monocyte infiltration in nephritic rats, whereas no inflammatory infiltration was found in healthy rats receiving MSC (group D) and in the control group E. Since in anti-Thy 1 disease the MSP/Ron system attracts monocytes into the glomeruli and stimulates mesangial cell proliferation (15-16), we investigated the effects of MSC on glomerular expression of Ron. MSC decreased glomerular expression of Ron on days 7 and 10 in nephritic rats (group B), but did not modify Ron expression in healthy rats (group D). Ron expression did not also change in nephritic rats receiving mesangial cells (group C).

Glomerular expression of α-SMA, PCNA, MSP and PDGF-β

Anti-Thy1 disease is a mesangioproliferative nephritis in which the proliferation of activated mesangial cells is stimulated by MSP released locally by mesangial cells themselves [START_REF] Gaertner | Glomerular oxidative and antioxidative systems in experimental mesangioproliferative glomerulonephritis[END_REF][START_REF] Rampino | Macrophage-stimulating protein is produced by tubular cells and activates mesangial cells[END_REF] as well as by PDGF-β released by mesangial cells and by monocytes [START_REF] Floege | Novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis by aptamers[END_REF]. The injection of MSC modulated glomerular endocapillary proliferation (PCNA positive cells), mesangial cell activation (α-SMA positive cells) and the release of MSP and PDGF-β release (figure 6). In fact, in nephritic rats injected with MSC a steady rise of endocapillary proliferation occurred at days 7, 10 and 14, while in rats not treated with MSC and rats treated with mesangial cells (A and C) a peak increase in proliferation occurred at day 7 followed by a decline at day 10 and by a further rise at day 14. Similary glomerular expression of α-SMA was steady in nephritic treated rats while it peaked at day 7 and fell at day 10 in rats not treated with MSC. It is noteworthy that the suppression of glomerular proliferation in nephritic rats not treated with MSC was synchronous at day 10 with the maximum extent of severe glomerular injury as indicated by ballooning and necrosis. Moreover, in nephritic rats MSP and PDGF-β expression was also significantly suppressed by MSC at day 7 and at day 10, while, in non nephritic rats (groups D) MSC did not affect a-SMA, PCNA, MSP and PDGF-β glomerular expression. There was no change in group E. In non-nephritic rats HGF (figure 5 suppl) and Met (figure 6 suppl) were diffusely expressed in tubules, while they were almost absent in glomeruli. De novo expression of HGF in glomeruli occurred in all rats with anti-Thy 1 disease, but the level of HGF was steadily high in MSC treated rats, while it declined on day 14 in untreated rats and rats treated with mesangial cells. It is notable that MSC injected in healthy rats (group D) did not upregulate HGF. Glomerular expression of Met increases slightly in rats with anti-Thy 1 disease that were not treated with MSC (group A and C). In contrast, treatment with MSC induced a striking increase in glomerular Met expression at day 10 and at day 14 (figure 7). Interestingly, MSC induced overexpression of Met in endothelial cells of glomerular capillaries, but only in nephritic rats (figure 6 suppl panel a and b).

MSC localization.

We examined ten kidney sections and we detected very few EGFP positive cells (0-5 for total kidney/section) in the kidney of each group of rats at each time point. EGFP positive cells were located in tubule, in the interstitium or in the glomerular tuft. We found 3-4 cells/field in spleen sections at 12 h after MSC infusion and occasionally we detected MSC in lung sections at the same time (Figure 7 suppl).

IL-6 and TGF-β levels.

As shown in,figure 8 serum levels of IL-6 increased in all groups with anti-Thy 1 disease, but the rise was striking in groups not treated with MSC in comparison with the modest rise occurring in rats treated with MSC. The suppression of IL-6 induced by MSC was especially evident at day 10 when serum IL-6 peaked in untreated rats. The infusion of MSC induced modest, but significant increase in serum IL-6 in rats without nephritis (D P<0.001 vs E). Also urinary IL-6 increased significantly in all groups with nephritis and also the rise in urinary IL-6 was significantly attenuated by treatment with MSC throughout the study period. TGF-β serum levels rose significantly in all groups with anti-Thy 1 nephritis and the rise was significantly attenuated by treatment with MSC at days 10 and 14. MSC did not induce an increase of TGF β serum levels in non nephritic rats (group D) (figure 8).

DISCUSSION

The purpose of our study was to investigate the effects of treatment with MSC on the course of anti-Thy 1 nephritis. We used MSC expressing CD 90 in accordance with the minimal criteria for defining MSC provided by the ISCT [START_REF] Dominici | Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement[END_REF]. CD 90 is targeted by the anti-Thy 1.1 Ab that is injected to induce the disease, therefore residual circulating antibody may react with CD90 positive MSC and interfere with their viability and their physiological functions. Conversely, MSC may bind circulating antibody and prevent it from reaching and injuring mesangial cells. In order to obviate such interferences, we made preliminary experiments to determine when the injected antibody is completely cleared from the circulation. We found no residual MSC-binding antibody in serum three days after its injection and consequently we infused MSC three days after the induction of the disease. The main finding of our study is that MSC infused intravenously mitigate anti-Thy 1 disease. In fact, treatment with MSC anticipated the recovery of serum creatinine to baseline levels at day 10 and reduced proteinuria in each of the days studied, returning it to baseline at day 14. Furthermore, MSC and not mesangial cells infusion had striking effects on glomerular pathology. In rats treated with MSC about half of the glomeruli were spared from any lesion in each of the days studied and only a minority scored 4, i.e. had severe lesions. In contrast, in rats not treated with MSC glomeruli scoring 4 were the prevalent population while normal glomeruli were a minority. MSC offered the most impressive protection at day 10, when MSC reduced the percentage of glomeruli scoring 4 from 60% (group A) and 56% (group C) to 18% (group B), while raising the percentage of intact glomeruli from 14% (group A) and 19 % (group C) to 48% (group B). The less aggressive course of the disease in rats infused with MSC did not depend on a decrease in anti-Thy 1.1 Ab bound to mesangial cells, or on suppression of complement activation. In fact, the amount of antibody and of C3 complement fraction deposited in glomeruli was the same in rats treated with MSC as in control nephritic rats. In anti-Thy 1 nephritis monocytes invade the glomerulus and cause glomerular injury by releasing reactive oxygen species and inflammatory cytokines [START_REF] Gaertner | Glomerular oxidative and antioxidative systems in experimental mesangioproliferative glomerulonephritis[END_REF]. The activated monocytes that infiltrate the glomerulus express Ron and are recruited into the tuft by the powerful chemotactic effect of MSP [START_REF] Rampino | Macrophage-stimulating protein is produced by tubular cells and activates mesangial cells[END_REF][START_REF] Floege | Novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis by aptamers[END_REF][START_REF] Rampino | Neutralization of macrophage-stimulating protein ameliorates renal injury in anti-Thy 1 glomerulonephritis[END_REF]. PDGF-β and TGF-β are other powerful chemoattractants for monocytes, which are released in the glomerulus in anti-Thy 1 disease [START_REF] Floege | Factors involved in the regulation of mesangial cell proliferation in vitro and in vivo[END_REF]. In this study we show that MSC suppress glomerular MSP and Ron expression. Furthermore, MSC in nephritic rats decrease the local levels of PDGF-β and circulating levels of TGF-β. Such inhibition of different chemotactic systems is a straightforward explanation for the reduction of infiltrating monocytes and for the attenuation of glomerular injury. Anti-Thy 1 disease represents a nominal model of mesangioproliferative glomerulonephritis [START_REF] Bagchus | Glomerulonephritis induced by monoclonal anti-Thy 1.1 antibodies. A sequential histological and ultrastructural study in the rat[END_REF][START_REF] Floege | Factors involved in the regulation of mesangial cell proliferation in vitro and in vivo[END_REF]. In fact, the disease is characterized by an initial mesangiolysis that is followed by reparative overgrowth of mesangial cells [START_REF] Bagchus | Glomerulonephritis induced by monoclonal anti-Thy 1.1 antibodies. A sequential histological and ultrastructural study in the rat[END_REF][START_REF] Kriz | Pathways to recovery and loss of nephrons in anti-thy 1 nephritis[END_REF]. Excessive mesangial proliferation is driven by growth factors that include MSP, PDGF-β, and IL-6 (16, 17, 18, 21, 22,). Activated proliferating mesangial cells express α-SMA and PCNA [START_REF] Rampino | Macrophage-stimulating protein is produced by tubular cells and activates mesangial cells[END_REF][START_REF] Floege | Novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis by aptamers[END_REF][START_REF] Rampino | Neutralization of macrophage-stimulating protein ameliorates renal injury in anti-Thy 1 glomerulonephritis[END_REF][START_REF] Bessho | Counteractive effects of HGF on PDGF-induced mesangial cell proliferation in a rat model of glomerulonephritis[END_REF]. Accordingly, in untreated nephritic rats, at day 7 we found overexpressed markers of mesangial cell activation (α-SMA) and proliferation (PCNA) and high levels of growth factors (local PDGF-β, MSP and serum and urinary IL-6). At day 10, in spite of further rise in growth factors, mesangial cell proliferation transiently fell in coincidence with the occurrence of peak necrosis, that reduced the number of alive cells responding to growth factors. Mesangial growth resumed at day 14, still sustained by growth factors, in particular by MSP and IL-6. Mesangial cell therapy did not modify the course of nephritis. In contrast treatment with MSC reduced the number of PCNA positive cells at day 7, and maintained growth steady at days 10 and 14. Such stabilization was associated with levels of PDGF-β, MSP and IL-6 lower than in untreated rats. Altogether, these results suggest that MSC regulate the repair response driven by PDGF-β, MSP and IL-6, making glomerular proliferation less aggressive and more steady. We speculate that by preventing mesangial overgrowth MSC reduce the risk of shift to the fibrogenic form of the disease. Accordingly, MSC inhibit the release of PDGF-β, one major inducer of collagen accumulation and glomerular scarring [START_REF] Floege | Novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis by aptamers[END_REF][START_REF] Floege | Factors involved in the regulation of mesangial cell proliferation in vitro and in vivo[END_REF][START_REF] Ostendorf | Specific antagonism of PDGF prevents renal scarring in experimental glomerulonephritis[END_REF]. It is notable that MSC work in nephritic rats and not in healthy rats, in fact the number of PCNA and α-SMA positive cells, as well as the local levels of PDGF-β and MSP, did not change in non nephritic rats. These findings confirm the hypothesis suggested by others (Krampera et al) i.e. that MSC need an inflammatory environment to be committed to induce a reparative effect. We also found a little increase of IL-6 in healthy rats treated with MSC compared with untreated rats, but the range of IL-6 remained normal [START_REF] Chen | Flk-1+ mesenchymal stem cells aggavate collagen-induced arthritis by up-regulating interleukin-6[END_REF]. In anti-Thy 1 disease, the lysis of mesangial stalk causes loss of glomerular capillaries, rarefaction of the capillary network and formation of microaneurysms [START_REF] Bagchus | Glomerulonephritis induced by monoclonal anti-Thy 1.1 antibodies. A sequential histological and ultrastructural study in the rat[END_REF], therefore the reconstruction of new glomerular capillaries is part of the healing process [START_REF] Kriz | Pathways to recovery and loss of nephrons in anti-thy 1 nephritis[END_REF]. The HGF/Met system is a powerful inducer of neoangiogenesis and exogenous administration of HGF to rats with anti-Thy 1 disease has been shown to accelerate glomerular repair through proliferation of endothelial cells and regeneration of glomerular capillaries [START_REF] Mori | Hepatocyte growth factor-stimulating endothelial cell growth and accelerating glomerular capillary repair in experimental progressive glomerulonephritis[END_REF]. In our study, treatment with MSC induced a striking expression of Met receptor in glomerular capillaries only in nephritic rats at days 10 and 14. We suggest that MSC-induced Met expression made local HGF operative and that the HGF/Met system mediated the reconstruction of glomerular capillaries. Studies in disease models have shown that intravenously injected MSC home to injured areas and participate in tissue regeneration by differentiating into mature tissue cells (4) and/or inducing the release of soluble factors [START_REF] Bi | Stromal cells protect against acute tubular injury via a endocrine effect[END_REF][START_REF] Togel | Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms[END_REF][START_REF] Gnecchi | Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement[END_REF][START_REF] Gnecchi | Paracrine mechanism in adult stem cell signaling and therapy[END_REF]. We have tracked MSC 12 hours, 3, 7 and 14 days after their injection and we have found very rare MSC in the glomerular tuft, in the interstitium and in the tubules. These results indicate that MSC did not participate in glomerular repair by replacing injured glomerular cells, but they improved the course of anti-Thy 1 disease by modulating the principal cytokines and growth factors involved in the pathogenesis of antiThy 1 nephritis. In particular, MSC reduced glomerular mononuclear infiltration by suppressing MSP and attenuated mesangial activation and glomerular endocapillary proliferation by down-regulating inflammatory and fibrogenic cytokines, such as TGF-β, PDGF-β and IL-6. In this way, MSC promoted glomerular repair and prevented glomerular scarring. Furthermore, MSC contributed to the regeneration of glomerular capillaries by inducing Met expression. Thus, MSC injection resulted into the reset of the two Scatter Factor systems, potentiating HGF/Met and disactivating MSP/Ron. Our study does not clarify the mechanisms by which MSC operated such complex regulation. A renal paracrine action is suggested by the local changes induced by MSC in the glomerular expression of HGF/Met, MSP/Ron systems and PDGF-b. However, others have already shown that the protective effect of MSC is independent of their engraftment in the injured tissue. In fact, elegant studies have demonstrated in experimental models of acute tubular necrosis and myocardial infarction that the protective effect of MSC could be reproduced by injecting conditioned medium from cultured MSC [START_REF] Bi | Stromal cells protect against acute tubular injury via a endocrine effect[END_REF][START_REF] Gnecchi | Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement[END_REF]. Thus, the effect of MSC is mediated by soluble factors, that can be released by MSC wherever they are in the body and reach the site of injury through the circulation [START_REF] Bussolati | Contribution of stem cells to kidney repair[END_REF][START_REF] Bussolati | Contribution of stem cells to kidney repair[END_REF][START_REF] Morigi | Human bone marrow mesenchymal stem cells accelerate recovery of acute renal injury and[END_REF]. Furthermore, recent studies have demonstrated that MSC release soluble We studied 5 groups of rats. Group A: rats in which anti-Thy 1 nephritis was induced by injecting anti-Thy 1.1 Ab intravenously on day 0. These rats were studied to define the spontaneous course of the disease. Group B: rats in which anti-Thy 1 nephritis was induced as in group A, that were treated with MSC infused intravenously on day 3. These rats were studied to understand whether MSC modify the course of the disease and the mechanisms there of. Group C: rats in which anti-Thy 1 nephritis was induced as in groups A and B, that were treated with mesangial cells on day 3. These rats were studied to investigate the systemic and renal effects of MSC-sized cells endowed with potential tropism to the kidney. Group D: rats injected with saline on day 0, followed by injection of MSC on day 3. These rats were studied to investigate the effects of MSC on normal kidney. Group E: control rats injected with saline on day 0. Slashes indicate days in which rats were sacrificed. Five rats for each group were sacrificed at day 1, 7, 10, 14. Five rats of group B were sacrificed 12 hours after MSC infusion.

Clinical

  Science Immediate Publication. Published on 28 Jun 2010 as manuscript CS20100147 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100147

Clinical

  Science Immediate Publication. Published on 28 Jun 2010 as manuscript CS20100147 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100147 A c c e p t e d M a n u s c r i p t

  Clinical Science Immediate Publication. Published on 28 Jun 2010 as manuscript CS20100147 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100147 A c c e p t e d M a n u s c r i p t RESULTS Characterization of rat MSC.

Clinical

  Science Immediate Publication. Published on 28 Jun 2010 as manuscript CS20100147 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100147 A c c e p t e d M a n u s c r i p t Glomerular injury.

Clinical

  Science Immediate Publication. Published on 28 Jun 2010 as manuscript CS20100147 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100147 A c c e p t e d M a n u s c r i p t HGF/Met system.

Clinical

  Science Immediate Publication. Published on 28 Jun 2010 as manuscript CS20100147 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100147 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 28 Jun 2010 as manuscript CS20100147 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100147 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 28 Jun 2010 as manuscript CS20100147 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100147 A c c e p t e d M a n u s c r i p t microvescicles (MV). MV are a novel mechanism of cell-to-cell communication which can transfer to target cells mRNA and /microRNA sequences. In fact, Bruno et al. have shown that injection of MSC-derived MV protected against acute tubular injury by shuttling m-RNA into tubular cells(32, 33). The transfer of transcripts can modify cell behaviour and may be bidirectional from stem cells to injured cells or vice versa. This hypothesis may explain why we found MSC effect only in nephritic rats and not in non nephritic rats and also the inefficacy of mesangial cell infusion.In conclusion, we have shown that MSC ameliorate the course of anti-Thy 1 nephritis by suppressing local and systemic release of inflammatory cytokines and growth factors and for the first time by modulating the activity of the scatter factor systems. We injected MSC intravenously after the disease had been established, thus reproducing conditions that make treatment with MSC easily feasible in the clinical setting.

1

 1 s c r i p t prolong survival in mice. Stem Cells 26, 2075-2082. 32. Bruno, S., Grange, C., Deregibus, M.C., Calogero, R.A., Saviozzi, S., Collino, F., Morando, L., Busca, A., Falda, M., Bussolati, B., Tetta, C., and Camussi, G. (2009) Mesenchymal stem cell-derived microvesicles protect against acute tubular injury. J. Am. Soc. Nephrol. 20, 1053-1067. 33. Camussi, G., Deregibus, M.C., and Tetta, C. (2010) Paracrine/endocrine mechanism of stem cells on kidney repair: role of microvesicle-mediated transfer of genetic information. Curr. Opin. Nephrol. Hypertens. 19, 7-12. Clinical Science Immediate Publication. Published on 28 Jun 2010 as manuscript CS20100147 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100147 A c c e p t e d M a n u s c r i p t TITLES AND LEGENDS TO FIGURES Figure Experimental design.

Figure 2

 2 Figure 2Urinary protein excretion rate. Groups are defined in figure1. Symbols are mean daily (24 h) urinary protein excretion, bars are SD. # day 0 P <0.001 vs day 7 and day 10 in groups A and C, °day 0 P <0.005 vs day 7 and day 10 in group B, *P <0.0001 vs B at days 7, 10, 14.

Figure 3

 3 Figure 3 Renal morphology at day 10. Groups are defined in figure 1. Representative renal histology of groups A, B, C, D and E at day 10. Hematotoxylin-eosin. (Magnification X200).

Figure 4

 4 Figure 4Glomerular damage score. Groups are defined in figure1. Numbers are the average percentage of glomeruli scoring 0, 1, 2, 3 and 4 at days 7, 10, and 14 (Kruskal Wallis test group A, P <0.0001 vs group B).

Figure 5

 5 Figure 5 Glomerular monocytes and Ron expression. Groups are defined in figure 1. Panel a. Columns are the mean number of monocytes/glomerulus. Bars are SD. °P <0.0001 vs A and C; *P <0.05 vs A and C; §P <0.0001 vs A, B and C. Panel b. Columns are the mean percent glomerular area staining for Ron: *P <0.001 vs A and C at days 7 and 10; °P <0.05 vs A, B and C at days 7,10.

Figure 6

 6 Figure 6Glomerular expression of α-SMA, PCNA, PDGF-β, MSP. Groups are defined in figure1.

Figure 7

 7 Figure 7 Glomerular expression of HGF and Met Groups are defined in figure 1. Panel a. Columns are the mean percent glomerular area staining for HGF: *P <0.05 vs A and C at day 7; °P<0.01 vs A, B and C at days 7, 10, and 14; #P <0.05 group B day 7 vs group B days 10 and 14; §P <0.05 groups A and C day 10 vs groups A and C day 14. Panel b. Columns show percent of glomerular capillaries stained for Met: *P <0.001vs A, C, D and E at days 10 and 14; and A, B, C, D, E at day 7; °P <0.05 vs A, and C at days 10, and 14.

Figure 8

 8 Figure 8 IL-6 and TGF-β levels Groups are defined in figure 1. Panel A: serum IL-6 levels. Columns are means, bars are SD: *P <0.05 vs A and C. °P <0.005 vs E, #P <0.001 vs E Panel B: urine IL-6/urine protein concentration (PTU). Columns are means, bars are SD: *P <0.05 vs A and C; °P <0.05 vs D and E. Panel C: Columns are means, bars SD: *P <0.05 vs A and C; °P <0.001 vs D and E.

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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

All authors state no conflict of interest. The manuscript has not been published previously and is not being considered concurrently by another journal.