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Published as doi: 10.1096/fj.06-6477com.
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Binding of elastin peptides to S-Gal protects the heart against ischemia/reperfusion injury by triggering the RISK pathway

Arnaud Robinet*, Hervé Millart*, Floriane Oszust*, William Hornebeck{dagger},1 and Georges Bellon{dagger}

* Laboratoire de Pharmacologie Médicale, Université de Reims-Champagne-Ardenne, Faculté de Médecine, Reims, France; and

{dagger} Laboratoire de Biochimie et Biologie Moléculaire, Université de Reims-Champagne-Ardenne, Faculté de Médecine, UMR 6198, Centre National de la Recherche Scientifique, Reims, France

1Correspondence: Laboratoire de Biochimie, Université de Reims-Champagne-Ardenne, Faculté de Médecine, CNRSUMR 6–19, 51, Rue Cognacq Jay, 51095 Reims cedex, France. E-mail: william.hornebeck{at}univ-reims.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Elastin peptides (EPs) generated by hydrolysis of elastic fibers by elastinolytic enzymes display a wide spectrum of biological activities. Here, we investigated their influence on rat heart ischemia-mediated injury using the Langendorff ex vivo model. EPs, i.e., kappa elastin, at 1.32- and 660-nM concentrations, when administered before the ischemia period, elicited a beneficial influence against ischemia by accelerating the recovery rate of heart contractile parameters and by decreasing significantly creatine kinase release and heart necrosis area when measured at the onset of the reperfusion. All effects were S-Gal-dependent, as being reproduced by (VGVAPG)3 and as being inhibited by receptor antagonists, such as lactose and V14 peptide (VVGSPSAQDEASPL). EPs interaction with S-Gal triggered NO release and activation of PI3-kinase/Akt and ERK1/2 in human coronary endothelial cells (HCAECs) and rat neonatal cardiomyocytes (RCs). This signaling pathway, as designated as RISK, for reperfusion injury salvage kinase pathway, was shown to be responsible for the beneficial influence of EPs on ischemia/reperfusion injury on the basis of its inhibition by specific pharmacological inhibitors. EPs survival activity was attained at a concentration averaging that present into the blood circulation, supporting the contention that these matrikines might offer a natural protection against cardiac injury in young and adult individuals. Such protective effect might be lost with aging, since we found that hearts from 24-month-old rats did not respond to EPs.—Robinet, A., Millart, H., Oszust, F., Hornebeck, W., Bellon, G. Binding of elastin peptides to S-Gal protects the heart against ischemia/reperfusion injury by triggering the RISK pathway.


Key Words: preconditioning • postconditioning • cardioprotection • aging


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE FRAGMENTATION OF ELASTIN, ONE OF THE longest-lived protein in humans, by elastinolytic enzymes is a hallmark of aging tissues and is deeply increased in cardiovascular diseases as athero-arteriosclerosis and aortic aneurism (1 2 3) . Such elastolysis was also observed in myocardial tissue during ischemia, where release of potent elastolytic proteinases i.e., elastase, cathepsin G, proteinase 3 and gelatinase B from neutrophils was found to disrupt human heart elastic lamina (4) . Locally produced elastin peptides (EPs) exhibit a wide panel of biological effects, acting as regulators of cell migration, differentiation, or proliferation (5 6 7) . Most effects are mediated through the interaction of EPs with a 67-kDa elastin binding protein, identified as an enzymatically inactive spliced variant of ß-Galactosidase (S-Gal) (7 8 9) . EPs were considered to display a detrimental influence on the cardiovascular system as exhibiting chemotactic activity for neutrophils, as inducing a T-helper-type 1 polarization of human blood lymphocytes, or as triggering the expression of neutral proteinases from fibroblasts and aorta smooth muscle cells (5 , 10 11 12) .

However, EPs were recently described to induce an endothelium- and NO-dependent vasorelaxation and to possess potent proangiogenic activity, both properties being mediated through S-Gal occupancy by these peptides (13 , 14) .

The crucial cardioprotective function of NO in myocardial ischemia (MI) and reperfusion injury is now well documented (15 16 17 18 19) . For instance, NO donors attenuate ischemia/reperfusion (I/R), arrhythmias, and myocardial infarct size, and improve postischemic coronary blood flow and contractile function (18 , 19) . Also, NO when endogenously produced through adenovirus-mediated endothelial NOS expression or exogenously administered, protects hearts from myocardial infarct-mediated left ventricular remodeling and apoptosis and limits the extent of myocardial infarction (16 , 17 , 20) . NO is generated through the PI3-kinase-Akt (PKB)-eNOS activation cascade which, together with ERK1/2 activation, constitutes a universal prosurvival signaling pathway mediating myocardial protection at reperfusion (21 , 22) . We recently reported that up-regulation of collagenase-1 following treatment of fibroblasts with EPs involved PI3-kinase and ERK signaling pathways (23) . Also, ERK1/2 activation was observed following EPs interaction with S-Gal in lymphocytes, aorta smooth muscle cells, and melanoma cells (10 , 24 , 25) .

We hypothesized that EPs might exert cardioprotection against myocardial ischemia reperfusion injury by inducing NO release and by targeting the RISK pathway. To verify this hypothesis, we used the Langendorff-perfused rat heart system as an ex vivo ischemia model. Our data indicated that EPs, at a concentration reported to be present in the blood circulation, induced a physiological protection of the myocardium against I/R injury when administered at the onset of reperfusion or throughout the experimental protocol. A much higher amount was necessary to obtain a significant preconditioning effect. EP-mediated beneficial influence on heart function and survival appeared to be S-Gal-, NO-, RISK- and age-dependent.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Insoluble elastin and soluble kappa-elastin (KE) peptides (average molecular weight=75kDa) were obtained as described previously (26) . (VGVAPG)3, a peptide reproducing the sequence of elastin binding to S-Gal and V14 peptide (VVGSPSAQDEASPL), a peptide reproducing the sequence of S-Gal interacting with EPs, were obtained from Neosystem (Strasbourg, France), and scrambled peptide: (VVGPGA)3 came from Sequentia (Evry, France). Their purity (>97%) was confirmed by HPLC and by fast atom bombardment mass spectrometry. All peptides were solubilized in Krebs-Henseleit Buffer (KHB) prior to use. N-w-nitro-L-arginine methyl ester (L-NAME) was from Sigma (Saint-Quentin Fallavier, France) and total nitric oxide assay from R&D Systems Europe (Lille, France). U0126, an inhibitor of MEK, LY294002, an inhibitor of PI3-kinase, mouse anti-human Akt monoclonal antibody, rabbit anti-human p-Ser473–Akt polyclonal antibodies, mouse anti-human p-Thr202 and p-Tyr204-Erk1,2 monoclonal antibodies were obtained from Cell Signaling (Montigny le Bretonneux, France). Horseradish peroxidase (HRP)-conjugated sheep anti-rabbit or anti-mouse IgG antibodies were from EUROMEDEX (Soufflewehersheim, France). To raise S-Gal specific antibodies, V14 was injected intraperitoneally into rabbits. Antibodies were purified from antisera on a sepharose-activated affinity column (Amersham, Orsay, France) coupled with V14. Specific antibodies were eluted with 3M MgCl2 and dialyzed overnight with phosphate-buffered saline (Life Technologies, Cergy-Pontoise, France).

PI3-kinase p85 and p-PI3-kinase p85 (Tyr 508) antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Cell cultures
Human coronary artery endothelial cells (HCAECs) were purchased from PromoCell (Heidelberg, Germany) and were used between the second and eight passages. HCAECs were cultured in endothelial cell growth medium (ECGM) MV (PromoCell, Heidelberg, Germany) supplemented with 0.4% (wt/vol) ECGS/H, 2% (vol/vol) fetal calf serum (FCS), 10 ng/ml epidermal growth factor (EGF), 1 µg/ml hydrocortisone, 50 ng/ml amphoterecin B and 50 µg/ml gentamicin. Neonatal rat ventricular myocytes (RCs) were isolated from neonatal rat hearts using the Worthington Neonatal Cardiomyocyte Isolation System kit. Briefly, ventricular tissues are first rinsed in sterile calcium- and magnesium-free Hank’s balanced salt solution (CMF HBSS) solution lacking Ca2+ and Mg2+ and incubated overnight with 50 ng/ml trypsin, then with a 5 M excess of soybean trypsin inhibitor. After 3 successive washings in HBSS, tissues were then treated with 300 U/ml of collagenase in HBSS for 45 min under constant stirring. The cell suspension was then filtered (70-µm pores) and centrifuged for 5 min at 100 g. To achieve myocyte enrichment, dissociated cells were preplated twice in T75 flasks for 90 min, a time period during which nonmyocyte cells attached to the bottom of the cultured flask. Cardiomyocytes (125x103 cells/cm2) were then cultured in L-15 Leibovitz medium (Worthington, Lakewood, NJ) supplemented with 5% (vol/vol) fetal calf serum and 1% (vol/vol) penicillin and streptomycin solution (Life Technologies; Invitrogen, Cergy-Pontoise, France).

Isolated heart preparation and experimental protocol
All experimentation procedures on animals adhered to the "Principles of Laboratory Animal Care" (National Institutes of Health, publication N23, rev. 1985). Two, six, and twenty-four month-old male Wistar rats were fed a standard diet and acclimatized in a quiet quarantine room for 5 days before the experiments. They were anesthetized with 50 mg/kg of sodium pentobarbital injected intraperitoneally and heparinized with a 500 IU injection. After deep anesthesia was reached, the chest was quickly opened. The hearts were promptly excised, placed first into a beaker of ice-cold perfusion solution to isolate an adequate length of aorta artery for cannulation. The hearts were perfused with oxygenated KHB at 37°C in accordance with the nonworking Langendorff mode. We used the balloon method for recording of isovolumetric pressure in the isolated perfused hearts. Retrograde perfusion was started immediately after the aortic cannulation at a constant pressure (67 mmHg) and a latex balloon (Hugo Sachs Elektronic) was inserted into the left ventricle through the left atrium and connected to a pressure transducer (Spectramed model P10EZ transducer, Gould 8000S recorder; Gould Electronics, Ballainvilliers, France). The balloon was filled with distilled water, such that the initial diastolic pressure was kept constant at 10 mmHg.

All experiments lasted a total of 120 min (Fig. 1 ). All hearts were allowed to stabilize for 20 min. with KHB (NaCl: 118 mM, KCl: 4.7 mM, CaCl2: 1.25 mM, MgSO4: 1.2 mM, KH2PO4 1.2 mM, glucose: 11 mM, NaHCO3: 22.6 mM, EDTA: 0.027 mM). The temperature of the perfusion buffer was adjusted to 37°C by means of a Harvard thermocirculator and a water thermostat type VTS 13, and the buffer was continuously bubbled with 95% O2 and 5% CO2. Measurement recorded after a 20-min stabilization period was considered as the baseline values. The 40-min global ischemia period was produced by closing the inflow of the physiological solution. The hearts were maintained at 37°C in a thermocontrolled chamber containing 0.9% (vol/vol) physiological serum in normal saline solution. This ischemia period was followed by a 40-min reperfusion period. In "postconditioning" experimental conditions, isolated rat hearts were perfused with KHB during the stabilization and reperfusion periods (control: Ctrl) or for 10 min at the onset of the reperfusion period with 1.32 or 600 mM EPs, either KE or (VGVAPG)3. [(VVGPGA)3 peptide was used as negative control]. Antagonists, i.e., lactose (10–4 M), V14 peptide (1.3 µM), L-NAME (10 mM), U0126 (1 µM), LY294002 (10 µM) were injected 5 min before EPs administration and for a period of 20 min. In a second set of experiments, EPs were administered 20 min before the ischemia period and for 10 min, that is, "early preconditioning" condition. Antagonists were added 5 min. before EPs treatment and for a period of 20 min. (Fig. 1) . Finally, in some experiments, hearts, after a 20-min stabilization period with KHB, were constantly perfused in the presence of EPs, except during the ischemia period.


Figure 1
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  		Figure 1. Schematic representation of the experimental protocol. Rat hearts were stabilized during 20 min with KHB. and subjected to a 40-min ischemia period followed by a further 40-min reperfusion period. At indicated times, hearts were treated with EPs or insulin in the absence or presence of antagonists either under "postconditioning" (1) or "early preconditioning" (2) conditions.

Heart parameters measurement
The contractile parameters: left ventricular diastolic pressure (LVEDP) and rate-pressure product (RPP) were quantified during the whole experiments (120 min). LVEDP data (mmHg) were determined during the postischemia period. The rate-pressure product (RPP; mmHg · beat–1 · min–1) was calculated by multiplying the developed pressure and the heart rate. Reperfusion arrhythmias were not diagnosed. The mean coronary flow (MCF; ml/min) was measured using the perfusate draining out of the right atrium for 1 min.

Effluents from the hearts were collected and creatine kinase (CK/mIU · min–1 · g–1 of wet weight), a marker of myocardial cell cytolysis, was determined at t95 min of experiments, a time where enzymatic CK activity reached a maximal value in all experimental conditions. Measurements were based on an enzymatic method using a commercially available reagent pack (Roche-Boehringer, Meylan, France) according to the manufacturer instructions.

Myocardial infarct size was determined after 30 min of postischemia reperfusion by quantitative image analysis. Briefly, hearts were perfused for 8 min at a flow rate of 2 ml/min with a 1% 2,3,5-triphenyltetrazolium chloride (TTC) dissolved in Krebs buffer for staining of area necrosis. Hearts were removed from the cannula and incubated in 1% (vol/vol) TTC in saline solution at 37°C for 10 min. The hearts were then fixed in formalin. Area of necrosis was measured from cross-sectional slices through the ventricles, which were then photographed. Photomicrographs were quantified by measuring the area of stained vs. unstained tissue as black pixels relative to total pixels with the use of the Adobe Photoshop software.

NO production by coronary artery endothelial cells and cardiomyocytes
HCAECs (104 cells) or/and RCs (104 cells) were seeded in 24-well culture plates and cultured for various periods in FCS-free ECGM MV in the absence (control) or presence of varying concentrations of either KE or (VGVAPG)3. Antagonists of S-Gal such as lactose (10–4 M), L-NAME (10 µM), and V-14 (1.3 µM) were added in the culture medium in the presence of EPs. At the end of incubation, the medium was collected and NO was measured using a total nitric oxide assay that involves the conversion of nitrate by the enzyme nitrate reductase. The detection of total nitrite was then determined as a colored azo dye product of the Griess reaction by recording the absorbance at 540–570 nm.

Western blot analysis
At the end of EPs treatment, hearts were snap frozen for PI3-kinase, Akt, and ERK1/2 analyses. Frozen hearts were powdered in a prechilled mortar and pestled with liquid nitrogen. Ice-cold lysis buffer (Tris-HCl buffer 50 mM, pH 7.4, NaCl 150 mM, Nonidet-P40 1% (vol/vol), sodium deoxycholate 1% (wt/vol), SDS 0.1% (wt/vol), iodoacetamide 5 mM, PMSF 1 mM) was added to powdered tissue. The homogenate was centrifuged at 15,000 g at 4°C, for 15 min. Protein concentration was measured using the Bradford method (Interchim, Montluçon, France). Aliquots of supernatant containing equal amounts of protein (20 µg) were analyzed by Western blot analysis. Nonspecific binding sites on Immobilon P membranes (Millipore, Saint-Quentin en Yvelines, France) were blocked with 5% (wt/vol) nonfat milk in Tris-buffered saline-containing Tween-20 (TBTS). Membranes were then incubated overnight at 4°C in 5% (wt/vol) nonfat milk in TBTS containing PI3-kinase, phospho-PI3-kinase, phospho-Akt, Akt, phospho-ERK1/2 antibodies (all at 1:1000 dilution), and then incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibodies in TBTS (1:3000 dilution) for 1 hour at 20°C. Immunocomplexes were visualized by chemiluminescence using the ECL+ system, according to the manufacturer’s instructions (Amersham, Orsay, France).

Expression of results and statistical analysis
Statistical analyses were performed using SPSS8.0 software for Windows. Values were reported as mean ± SEM of 5 experiments. Data were analyzed using simple one-way ANOVA. If the F ratios were significant, post hoc tests (Dunnett’s multiple comparison test) were applied to assess significance (P <0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The cardioprotective effect of EPs following ischemia-reperfusion is S-Gal-dependent
Forty minutes of normothermic global ischemia were chosen as an insult in our experimental model since untreated reperfused rat hearts recovered 42 ± 5% of their baseline function at the end of the experiments. As documented in previous studies, early reperfusion intervention represents an approach more appropriate to the clinical scenario (27) . In keeping, EPs were first administered 1 min after the reperfusion period. Kinetics of heart parameters, each of them measured by intervals of 5 min, as resting tension (LVEDP), mean coronary flow (MCF), rate pressure product (RPP), and creatine kinase (CK) release, were determined and the extent of myocardial infarct was finally appreciated by quantifying heart necrotic areas.

Results reported in Fig. 2 A indicate that EPs, that is, kappa elastin (KE) originating from organoalkaline hydrolysis of insoluble elastin (28) maintained LVEDP to normal levels (<50 mm Hg) during the whole reperfusion period and values were highly significant vs. control at any time point analyzed from 80 to 120 min of reperfusion. Similarly, recovery of RPP following reperfusion was improved by KE (Fig. 2B ). As earlier as following 5 min of reperfusion, RPP reaches 85 ± 9% and 89 ± 10% recovery using the highest concentration (660 nM) of KE, as compared to 25 ± 9% recovery in control. Importantly, EPs displayed a beneficial influence, in a significant statistical manner, at low concentration (1.32 nM: 0.1 µg/ml). Similar beneficial effects of EPs were obtained on MCF heart parameter. For sake of comparison, insulin (10 mU/ml) was administered under similar experimental conditions. Data from Fig. 2A and B indicate that it shows a significant lower beneficial influence on contractile parameters and MCF as compared to KE


Figure 2
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  		Figure 2. Influence of EPs on contractile parameters and coronary flow on rat heart following ischemia-reperfusion. LVEDP (mmHg), RPP (%) and MCF (%), as determined relative to their basal values at the beginning of the experiment, were measured in Langendorff-perfused rat hearts. Hearts were subjected to a 40-min ischemia period followed by either a 40-min reperfusion period with KHB (control, Ctrl) or for 10 min with 1.32 or 660 nM kappa elastin (KE) or 10 mU/ml insulin. After EPs or insulin injection, hearts were perfused with KHB.

Parallelly, myocardial infarct size decreased significantly following KE administration with, for instance, a 2- fold decrease at the highest concentration (660 nM) of KE used; that correlated with concomitant diminution of CK release (Fig. 3) . Here again, insulin had a much lower effect on myocardial infarct size and CK release.


Figure 3
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  		Figure 3. Beneficial effects of EPs on myocardial infarct size and creatine kinase (CK) release following ischemia-reperfusion. Necrosis area and CK release were determined in rat hearts subjected to a 40-min ischemia period followed by either a 40-min reperfusion period with KHB (Ctrl) or for 10 min with 1.32 or 660 nM kappa elastin (KE) or 10 mU/ml insulin. After KE or insulin injection, hearts were perfused with KHB. A) Photomicrographs of corresponding necrosis area. B) Variation of necrosis area and CK release following insulin or KE treatments. Area of necrosis was measured from cross-sectional slices through the ventricles. Photomicrographs were quantified and necrosis area was expressed as percentage of black pixels relative to total pixels. CK activity was determined in perfusion effluents from hearts at t 95 min, i.e., 5 min after the end of KE or insulin injection. CK release was expressed as percentage relative to the basal value obtained in control (Ctrl).

Most EP-directed biological effects are mediated through S-Gal (7 , 9) . To ascertain its involvement in EP-induced cardioprotection, experiments were reproduced using (VGVAPG)3, a tropoelastin sequence known to interact specifically with S-Gal (7) . As shown in Table 1 , this peptide reproduced KE effects on LVEDP, RPP, and CK release. On the contrary, a scrambled peptide (VVGPGA)3, unable to adopt a type VIII ß-turn conformation that was previously shown to be a prerequisite for EPs to interact with S-Gal (29) was inactive. Furthermore, S-Gal participation in the observed effects was examined by coadministration of lactose (10–4 M), which is known to decrease the affinity of elastin to S-Gal, or V14 (1.3 µM), a peptide reproducing the S-Gal sequence known to interact with GXXPG motif. As shown in Table 1 , both agents totally prevented (VGVAPG)3-mediated effects.


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  		Table 1. S-Gal’s effects on LVEDP, RPP, and CK release

The cardioprotective effect of EPs on ischemia-reperfusion injury is NO-mediated
Previous investigations indicated that EP-mediated endothelium-dependent vasorelaxation could be inhibited by L-NAME, an eNOS inhibitor (13) . We thus hypothesized that NO could be involved in EP-induced cardioprotection. We first analyzed the influence of EPs on NO release by human coronary endothelial cells (HCAECs) and rat cardiomyocytes (RCs), both cells expressing S-Gal (Fig. 4 A). Whatever cell type, either KE or (VGVAPG)3 (660 nM) induced an average two-fold increase in NO production (Fig. 4B, C ). Maximal effect occurred following 15 min incubation of EPs with cells. Then NO secretion decreased to basal levels. For both cell types, EP-mediated NO release could be totally prevented by lactose and V14. The scrambled peptide was ineffective, indicating that EPs interaction with S-Gal was specific and entirely responsible of NO generation. To demonstrate the involvement of NO in EPs effects in the Langendorff heart ischemia model, L-NAME (10 µM) was coadministered with EPs at the onset of reperfusion. As shown in Fig. 4D , the eNOS inhibitor suppressed the beneficial contribution of EPs on LVEDP and necrosis area.


Figure 4
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  		Figure 4. The cardioprotective effect of EPs is NO dependent. A) Identification of S-Gal (67 KDa) in cell extracts from rat cardiomyocytes (RCs), human coronary artery endothelial cells (HCAECs) and human fibrosarcoma cells (HT-1080) by Western-blot. EPs mediate the release of NO from HCAECs (B) and RCs (C). Cells were seeded in 24-well culture plates and cultured for various periods in FCS-free ECGM MV in the absence (control) or presence of 660 nM KE, (VGVAPG)3 (P), or (VVGPGA)3 (SP). In some experiments, antagonists such as lactose (10–4 M), L-NAME (L-N) (10 µM), or V14 (1.3 µM) were injected in the presence of EPs. NO release was determined using Griess reaction and expressed as µmole/l. C) NO synthase inhibitor (L-NAME) impedes the beneficial effect of EPs on LVEDP and necrosis area. LVEDP (mmHg) was measured in Langendorff-perfused rat hearts under the following conditions: Ctrl: hearts were subjected to a 40-min ischemia period followed by a further 40-min reperfusion period with KHB. P: hearts were subjected to a 40-min ischemia period and then reperfused for a further 10-min period with 660 nM (VGVAPG)3 in the absence or presence of L-NAME (10µM) followed by a further 30-min reperfusion period with KHB Necrosis area was measured in Langendorff-perfused rat hearts, as described in Fig. 3 .

RISK pathway is involved in EP-mediated cardioprotection
Activation of the PI3-kinase/Akt/ERK1/2 pathway at the time of reperfusion also designated as the reperfusion injury salvage kinase (RISK) pathway, was largely described as an important target for ischemic postconditioning (20) . We first analyzed whether EPs could trigger such signaling pathway in HCAECs and RCs in culture. Figure 5 A shows that p85 PI3-kinase subunit was activated as early as 5 min following incubation of EPs with either HCAECs or RCs. Activation was only transient and returned to nearly basal level at 60 min. Akt activation followed a similar kinetics as RCs, but phosphorylation was delayed in HCAECs (maximal activation at 15 min), although a more sustained effect could be observed for this cell type (Fig. 5B ). Finally, 15-min incubation between cells and EPs was necessary to achieve maximal ERK1/2 activation (Fig. 5C ). Strikingly, EPs activated mainly ERK 1 in HCAECs, while ERK 2 was majorly affected in RCs. In keeping with S-Gal-mediated effect on NO production, lactose totally prevented ERK1/2 activation by EPs (Fig. 5C ).


Figure 5
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  		Figure 5. EPs activate the RISK pathway in HCAECs and RCs. HCAECs (104 cells) and RCs (104 cells) were seeded in 24-well culture plates and cultured for various periods from t0 to t60 min. in FCS-free ECGM MV in the presence of 660 nM (VGVAPG)3, i.e., P. Phosphorylation of PI3-kinase (A), Akt (B), and ERK1/2 (C), as well as total PI3-kinase, Akt and ERK1/2 were analyzed from cell extracts by Western blot analysis. Lactose, i.e., L; (10–4 M) was used as an antagonist to ascertain that EPs effect on ERK1/2 activation following a 15-min incubation period with 660 nM P was S-Gal-mediated (C). The respective bands were quantified by densitometric analysis and the phosphorylated forms were expressed as arbitrary units (AU) relative to total PI3-kinase, Akt, or ERK1/2 protein level, respectively, as a function of time.

We further examined the extent of Akt activation from extracts of rat hearts following 40 min of reperfusion by Western blot analysis. Results reported in Fig. 6 A demonstrate that KE (660 nM) as well as (VGVAPG)3 peptide (660 nM) treatments lead to a substantial increase in pSer473-Akt relative to total Akt. We also demonstrated that EPs could also significantly activate ERK1/2 in our ex vivo ischemia model by Western blot analysis (Fig. 6B ). Such activation was totally abolished in the presence of U0126, a specific MEK inhibitor (data not shown). In addition, U0126 and LY294002, a PI3-kinase inhibitor, suppressed the beneficial influence of EPs on necrosis area (Fig. 6C ). Parallelly, U0126 or LY294002 when concomitantly administered with (VGVAPG)3 peptide (660 nM), abolished the positive effect of EPs on LVEDP and RPP (Fig. 6D ). Altogether, these data indicate that EPs exerted their protective effect on cardiac function through triggering the RISK-pathway.


Figure 6
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  		Figure 6. Involvement of the RISK pathway in EPs-mediated effect. Influence of EPs (KE or P) in Akt and Erk 1/2 activation. Akt (A) and ERK1,2 (B) were analyzed by Western blot analysis, from rat hearts subjected to a 40-min ischemia period followed by either a 40-min reperfusion period with KHB (ctrl) or for 10 min with either 660 nM (VGVAPG)3 peptide (P) or kappa elastin (KE) followed by 30 min reperfusion with KHB. Rat heart extracts were subjected to Western blot analysis using mouse anti-human Akt monoclonal antibody and rabbit anti-human pSer473-Akt polyclonal antibodies (A), or mouse anti-human ERK1/2 monoclonal antibody and mouse anti-human phospho-ERK1/2 monoclonal antibody antibodies (B). Horseradish peroxidase (HRP)-conjugated sheep anti-rabbit or anti-mouse IgG antibodies were used as secondary antibodies at 1/3,000 dilution. Immunocomplexes were visualized by chemiluminescence using the ECL+ system and bands were quantified by densitometric analysis. Results were expressed as arbitrary units (AU) and as mean ± SEM (n=5, *P < 0.05). Influence of PI3-kinase and MEK inhibition on EPs-mediated effects on necrosis area (C), LVEDP and RPP (D). Necrosis area was measured as described as in Fig. 3 , except that, after a 40-min ischemia period, hearts were reperfused with 10 µM LY294002 (LY) or 1 µM U0126 (U) in the presence or absence of 660 nM (VGVAPG)3 peptide (P). Necrosis area was expressed as a percentage of black pixels relative to total pixels. *P < 0.05, NS = not significant. D) LVEDP (mmHg) and RPP (%), as determined relative to the basal value determined at the beginning of the experiment, were measured as follows: hearts were subjected to a 40-min ischemia period followed by either a 40-min reperfusion period with KHB (ctrl) or for 10 min with either 1 µM U0126 (U), 10 µM LY294002 ((LY), or 600 nM (VGVAPG)3 peptide (P). In some experiments, hearts were perfused concomitantly with (VGVAPG)3 and U0126 or LY294002. Then, hearts were perfused with KHB for a further 30-min period. LVEDP was measured 10 min after the end of EPs injection and RPP at the onset of the 30-min. period of KHB reperfusion. *P < 0.05, NS = not significant.

EP-preconditioned hearts are protected from ischemia-reperfusion injury
We initiated experiments to evaluate whether EPs could still protect against ischemia when delivered prior to ischemia insult. For that purpose, isolated rat hearts were stabilized with KHB for 20 min, then exposed for 10 min to 660 nM KE or 660 nM (VGVAPG)3 peptide, and then to a 10-min drug-free perfusion period. Results reported in Fig. 7 A show that EPs still improve RPP to nearly total recovery, such beneficial influence being also observed on LVEDP and myocardial infarct size, as well as on CK release (not shown). However, contrary to data obtained when EPs were used in ischemic postconditioning, statistical significance was only observed at a high EP concentration, i.e., 660 nM. Indeed, no significant effect was obtained with 1.32 and 13.2 nM EPs. The 10-min lag phase of the drug-free reperfusion period just before ischemia may account for such differential sensitivity. To approach physiological conditions, hearts were perfused with EPs from t20 to t40 min and from t80 to t120 min of the experiment at a concentration close to that present in the blood circulation, i.e., 1.32 nM. Results from Fig. 7A (P-C) demonstrate that, under these conditions, EPs exhibited a beneficial effect on RPP, similar to that obtained in postconditioning experiment.


Figure 7
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  		Figure 7. Preconditioning beneficial effect of EPs on ischemia-reperfusion injury. RPP (A and B) was measured in Langendorff-perfused rat hearts under early preconditioning conditions: after a 20-min stabilization period with KHB, hearts were subjected to a 20-min perfusion period with either KHB (ctrl), 1.32, or 660 nM kappa elastin (KE) or 1.32 or 660 nM (VGVAPG)3 peptide (P) followed by a 40-min ischemia period and then by a 40-min reperfusion period with KHB. In some experiments, hearts were perfused both before the ischemia period and at the onset of the reperfusion period with 1.32 nM (VGVAPG)3 (P-C). Results for RPP are expressed as a percentage relative to the basal value determined at the beginning of the experiment. RPP values measured during the reperfusion period from hearts perfused with 660 nM KE or P under preconditioning conditions and with 1.32 nM P under both pre- and postconditioning conditions (P-C) are all significant with P < 0.05. B) RPP (%) and MCF (%) were measured under early preconditioning conditions as follows: hearts were subjected to a 40-min stabilization period with KHB (ctrl) or to a 20-min stabilization period followed by a 10-min perfusion period with 660 nM kappa elastin (KE) or (VGVAPG)3 peptide (P) and then by a 10-min perfusion period with KHB. Antagonists of S-Gal, i.e., 10–4 M lactose (L) and 1 µM V14, were injected 15 min after the start of the experiment over a 20-min total period of perfusion in the presence of KE or P (L+KE, L+V14, L+P, L+V14) similarly as reported in Fig. 1 . Hearts were then subjected to a 40-min ischemia period followed by a 40-min reperfusion period with KHB. RPP and MCF were measured during the reperfusion period at t90 min. *P < 0.05.

Here again, the effects of EPs on RPP and MCF appeared S-Gal- and NO-dependent as being inhibited by S-Gal antagonists, i.e., lactose (10–4 M) or V14 (1.30 µM) and NOS inhibitor, i.e., L-NAME (10 µM) (Fig. 7B ).

Cardioprotective effect of EPs is serum-independent but age-dependent
We initially hypothesized that under physiological conditions, interaction of EPs with serum constituents might hide their S-Gal reacting motif. Therefore, reperfusion medium was supplemented with 5% (vol/vol) bovine serum and EPs were administered, as described before, at the onset of reperfusion. No significant difference could be observed in EPs-mediated effect on cardiac contractile parameters and CK release, whether experiments were performed in the absence or presence of serum (not shown). We then turned our attention to aging since EPs vasorelaxing effects were previously demonstrated to be age-dependent (30) . Thus, postconditioning experiments were reproduced using hearts from young (12 wk), adult (6 mo), and old (24 mo) animals. Data from Fig. 8 A indicated that hearts from young and adult animals responded similarly to EPs, as demonstrated by LVEDP measurements. On the contrary, contractile parameters of old rat hearts were unmodified by EPs administration. That was concomitant with lower EPs-induced Akt activation. (Fig. 8B ).


Figure 8
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  		Figure 8. The protective effect of EPs is age-dependent. A) LVEDP (mmHg) was measured under postconditioning conditions as follows: young (3 mo), adult (6 mo), and aged (24 mo) rat hearts were subjected to a 40-min. ischemia period followed by a further 10-min reperfusion period in the absence (C) or presence of 660 nM (VGVAPG)3 (P) and then, by a 30-min reperfusion period with KHB. *P < 0.05. B) Representative Western blots from tissue extracts showing a decrease of phospho-Akt level in old vs. young rats.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A recent epidemiological study performed on 1389 individuals between 59 and 70 yr revealed that the concentration of elastin peptides in blood circulation averaged 10 µg/ml (31) . Therefore, in addition to local elastolysis, most cells of the organism, including endocardial, endothelial cells, and cardiomyocytes could be in contact with these matrikines. Data from this study pinpoint that elastin peptides behave as potent survival factors in the ex vivo Langendorff rat ischemia model. Importantly, cardioprotection was evidenced when peptide concentration, close or below the level present in the blood circulation, was delivered at the onset of reperfusion, that is, "ischemia postconditioning effect" or when administered throughout the experiment. These findings are in keeping with recent data showing that expressing recombinant elastin or fragments within myocardial scar reduced its expansion and prevented left ventricular enlargement after a myocardial infection (32 , 33) . Here, we demonstrated that the protective function of elastin peptides on ischemia-reperfusion injury could be entirely attributed to their interaction with S-Gal, also designated as elastin binding protein (EBP).

Its involvement in directing the biological effects of elastin peptides was recently questioned since mice lacking ß-galactosidase transcript did not present vascular defects and continued to develop normally (28) . However, the beneficial influence of EPs on rat ischemia was here found to be totally suppressed by lactose, in keeping with lectin-like property of S-Gal and by a QDEA-containing 14-mer peptide corresponding to a specific S-Gal sequence following ß-Gal splicing. Also, a scrambled peptide, i.e., (VVGPGA)3 not containing either a VGV sequence, reported to be the sequence inducing dose-dependent vasorelaxation of aortic rings (13 , 34) or a GXXPG type VIII forming ß-turn structure leading to MMP overexpression in normal or transformed cells was inactive (34) . Thus, probably, S-Gal displays only minimal influence on development but might act as a critical element in tissue repair and survival following elastase-driven elastolysis. Besides, it needs to be emphasized that several matrix proteins, apart from elastin, including fibrillins, fibronectin, tenascins, and several collagens do contain multiple GXXPG motifs, which interact with S-Gal (35) . Several of these fragments produced by proteolysis (14 , 35 , 36) of matrix proteins were recently found to be as potent as VGVAPG in promoting an angiogenic phenotype in a matrigel assay (37) , thus supporting the pivotal importance of S-Gal in cardiac and vascular functions.

As earlier suggested by G. Faury and coworkers (13) , here, we confirmed that elastin peptides could trigger NO release from human coronary endothelium cells and rat cardiomyocytes in culture. The up-regulation of NO production observed by supplementation of cardiomyocytes culture medium by EPs suggested that these elastin fragments, similarly as documented with endothelial cells, might prevent myocyte death during lethal reperfusion-induced injury. Of note, however, nitric oxide release could be higher in rat cardiac microvessel endothelial cells as compared to human coronary counterparts (38) .

Such EP-mediated NO overproduction appeared entirely responsible of the major improvement of the heart function following ischemia as documented by L-NAME inhibition of such beneficial influence. It is well known that NO might represent a valuable therapeutic target in myocardial ischemia and heart failure (27 , 39 , 40) . Thus EPs as other NO donors, in the absence of ischemia, can mimick the molecular and functional aspects of ischemia-induced late PC (41) .

The enhancing effect of elastin peptides on NO production by both endothelial cells and cardiomyocytes is fast, thus involving eNOS, whose activation is mediated by calcium and calmodulin pathways. It needs to be emphasized that the most reported biological activities of elastin fragments were also found to be similarly calcium- and calmodulin-dependent (26) . eNOS activation necessitates the upstream activation of PI3-kinase/Akt signaling cascade, which together with ERK1/2 constitutes the RISK pathway. Akt appears to play a pivotal "pharmacological postcond" function against ischemia and adenovirally transfected rat hearts, which constitutively express active Akt were shown to confer significant protection against ischemia-reperfusion injury (42) . The prosurvival PI3-kinase/Akt survival pathway together with its downstream targets appears therefore as one main heart survival pathway that can be induced by several agents, including those activating G protein-coupled receptors when delivered during the first minutes of the reperfusion period (43 , 44) . Of significance, the "elastin receptor system" comprising S-Gal, cathepsin A, and Neu-1 was described to belong to G protein-coupled receptor family (25) .

For many cell types, a specific ERK1/2 activation pathway is induced following binding of elastin peptides to their cognate receptor. In fibroblasts, S-Gal occupancy by EPs was found to trigger ERK1/2 activation by a combined mechanism in which IB p110{gamma} PI3-kinase/RAF-1/MEK and PKA/B-RAF/MEK pathways cooperate to induce sustained ERK1/2 activation (23) . In aorta smooth muscle cells, ERK activation following interaction of EPs with S-Gal was found to be instead Ras-dependent (25) . We here evidenced that components of the RISK pathway, together with NO production, could be activated by EPs using either endothelium cells, cardiomyocytes in culture or the ex vivo Langendorff rat heart model.

Interfering with the activity of any component of this signaling system, i.e., PI3-kinase, eNOS and ERK led to entire inhibition of EP-mediated cardioprotection. It suggested that a "linear" signaling axis was triggered by EPs. A general scheme was recently proposed where PI3-kinase-Akt pathway results in phosphoregulation of eNOS, which then activates guanylate cyclase via NO-guanylate cyclase further activating PKG via cGMP, which, in turn, opens the mitochondrial potassium (K) ATP channel leading to reactive oxygen species (ROS) production and ERK activation (45) . It needs to be delineated that we recently demonstrated that PKG was involved in ERK-mediated MT1-MMP and proangiogenic phenotype induced by EPs (unpublished observations); also, opening of K channels and ROS release by EPs were described for other cell types (46 , 47) .

EPs-mediated cardioprotection following ischemia/reperfusion injury somewhat constitutes a paradox since active concentration of peptides is naturally found at higher concentration into blood circulation, whatever age or pathological status, meaning that we ought to be protected against such cardiac complication. However, aging appeared to be the limiting factor, with whole beneficial influence being lost when hearts from old rats were treated by EPs. That is in keeping with previous investigations by Faury et al. who demonstrated that aorta rings of 30-month-old rats did not respond to EPs (47 , 48) . It is well documented that aging is associated with decreased eNOS expression and NO production, which contribute to impaired angiogenesis and endothelium-dependent vasodilatation (49 , 50) . That might be attributed to receptor(s)-uncoupling as here shown for "elastin-receptor system," which similarly as described in lymphocytes, exhibited age-dependent modifications (51) . The mechanism involved in such elastin receptor-impaired activity with aging is unknown but might be linked to age-dependent reduction in sialidase activity, as observed in mouse synaptic plasma membranes (52) .

Overall, our data suggest that elastin peptides, but also other matrix fragments containing GXXPG sequences, might be considered as cardioprotective agents against ischemia-reperfusion injury in young and adult individuals. The pivotal function of matrikines in directing tumor progression has been extensively investigated. Recently, endostatin, a circulating fragment from type XVIII collagen, was described to relaxed small and large arteries through activation of PI3-kinase, Akt and eNOS (35) . It suggested that not only EPs but also other matrix fragments could be key regulators of injury following ischemia/reperfusion.


   ACKNOWLEDGMENTS
 
This work has been supported by the French Ministery of Research, and the University of Reims Champagne-Ardenne ("Bonus Qualité Recherche" 2005 and 2006). The authors thank Mrs. F. Moreau and Mr. J. Pisani for their excellent technical assistance.

Received for publication October 5, 2006. Accepted for publication February 1, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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