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(Circulation. 2004;109:2917-2923.)
© 2004 American Heart Association, Inc.

Basic Science Reports

p66^ShcA Modulates Tissue Response to Hindlimb Ischemia

Germana Zaccagnini, PhD^*; Fabio Martelli, PhD^*; Pasquale Fasanaro, MSc; Alessandra Magenta, PhD; Carlo Gaetano, MD; Anna Di Carlo, PhD; Paolo Biglioli, MD; Marco Giorgio, PhD; Ines Martin-Padura, PhD; Pier Giuseppe Pelicci, MD; Maurizio C. Capogrossi, MD

From Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’Immacolata–IRCCS, Rome (F.M, P.F., A.M., M.C.C.); Laboratorio di Biologia Vascolare e Terapia Genica, Centro Cardiologico Monzino–IRCCS, Milan (G.Z., A.D.); Dipartimento di Chirurgia Cardiovascolare, Centro Cardiologico Monzino–IRCCS, Milan (P.B.); and Istituto Europeo di Oncologia, Milan (I.M.-P., M.G., P.G.P.), Italy.

Correspondence to Fabio Martelli, PhD, Laboratorio Patologia Vascolare, Istituto Dermopatico dell’Immacolata–IRCCS, Via dei Monti di Creta 104, 00167 Rome, Italy. E-mail f.martelli{at}idi.it

Received July 31, 2003; de novo received January 28, 2004; revision received March 2, 2004; accepted March 4, 2004.

	Abstract

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Background— Oxidative stress plays a pivotal role in ischemia and ischemia/reperfusion injury. Because p66^ShcA-null (p66^ShcA–/–) mice exhibit both lower levels of intracellular reactive oxygen species and increased resistance to cell death induced by oxidative stress, we investigated whether tissue damage that follows acute ischemia or ischemia/reperfusion was altered in p66^ShcA–/– mice.

Methods and Results— Unilateral hindlimb ischemia was induced by femoral artery dissection, and ischemia/reperfusion was induced with an elastic tourniquet. Both procedures caused similar changes in blood perfusion in p66^ShcA wild-type (p66^ShcAwt) and p66^ShcA–/– mice. However, significant differences in tissue damage were found: p66^ShcAwt mice displayed marked capillary density decrease and muscle fiber necrosis. In contrast, in p66^ShcA–/– mice, minimal capillary density decrease and myofiber death were present. When apoptosis after ischemia was assayed, significantly lower levels of apoptotic endothelial cells and myofibers were found in p66^ShcA–/– mice. In agreement with these data, both satellite muscle cells and endothelial cells isolated from p66^ShcA–/– mice were resistant to apoptosis induced by simulated ischemia in vitro. Lower apoptosis levels after ischemia in p66^ShcA–/– cells correlated with decreased levels of oxidative stress both in vivo and in vitro.

Conclusions— p66^ShcA plays a crucial role in the cell death pathways activated by acute ischemia and ischemia/reperfusion, indicating p66^ShcA as a potential therapeutic target for prevention and treatment of ischemic tissue damage.

Key Words: ischemia • reperfusion • free radicals • apoptosis • muscles

	Introduction

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Reactive oxygen species (ROS) formation plays a causal role in tissue injury that follows both ischemia and ischemia/reperfusion (I/R).^1–8 ROS production increases during simulated ischemia of cultured cardiomyocytes,^3,4 and in vivo studies have demonstrated ROS formation in cardiac tissue during ischemia without reperfusion.^5,7,8 Furthermore, oxidative damage has been observed in focal cerebral ischemia lacking reperfusion, and treatment with antioxidants reduced infarct volume.²

I/R is a complex set of events with severe pathological consequences. Reperfusion initiates both local and systemic damage in part through rapid ROS generation, and numerous studies show that ROS scavengers improved I/R injury occurring to hindlimb skeletal muscles.^1,9–11

Nevertheless, the molecular mechanisms responsible for cell death induced by ischemia remain obscure.

The mammalian adapter protein ShcA has 3 isoforms: p46, p52, and p66. All ShcA isoforms contain a common structure, but the p66^ShcA isoform has a unique domain at the N terminus.^12,13 p66^ShcA protein is phosphorylated at serine/threonine residues in response to various stimuli, including UV and H₂O₂ treatment.¹⁴ Specifically, in response to UV, p66^ShcA is phosphorylated mainly at serine 36.¹⁴ p66^ShcA-null (p66^ShcA–/–) mouse fibroblast cells display lower levels of intracellular ROS and increased resistance to oxidative damage. Wild-type p66^ShcA (p66^ShcAwt), but not a phosphorylation-defective mutant, can restore the normal stress response.^14–16 Moreover, p66^ShcA–/– mice are less susceptible to chemical-induced oxidative damage, have lower levels of oxidative stress, are less susceptible to atherosclerosis, and have an extended life span.^14,16,17

In this study the role of p66^ShcA in muscle damage that follows acute hindlimb ischemia was investigated. Results show that p66^ShcA–/– mice were resistant to tissue damage induced by ischemia and I/R, demonstrating that p66^ShcA plays a crucial role in the cell death pathways activated by acute ischemia.

	Methods

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Animal Model and Surgical Procedures
All experimental procedures complied with the Guidelines of the Italian National Institutes of Health and with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, Md) and were approved by the institutional Animal Care and Use Committee. One hundred twenty-nine Sv-Ev p66^ShcAwt and p66^ShcA–/– mice were previously described.¹⁴ Their genetic background was identical except for the p66^ShcA locus. For each experiment, 6 to 8 male 2- to 3-month-old mice were used. Surgical procedures are described in the Data Supplement.

Histology and Morphometric Analysis
Hematoxylin-eosin section preparation,¹⁸ capillary density measurement,¹⁹ and arteriolar length density assessment¹⁸ were previously described. Necrotic muscle fibers were identified by morphology, differential eosin staining, and presence of infiltrating cells near the degenerating fibers on the whole section at x400 magnification. Apoptosis mediated by DNAse I and II was identified by terminal deoxynucleotidyl transferase assay (Roche) according to the manufacturer’s instructions. The experimental procedure for lectin staining is described in the Data Supplement. Histological and immunohistochemical analysis was performed by 2 blinded readers with comparable results.

Western Blotting
Western blotting was performed as previously described²⁰ (see Data Supplement).

Cell Cultures
Endothelial and satellite cell isolation and culture conditions are described in the Data Supplement. Apoptosis (n=3 to 6) was assessed by measuring the amount of nucleosomes generated during the apoptotic fragmentation of cellular DNA by cell death detection enzyme-linked immunosorbent assay (Roche) according to the manufacturer’s instructions.

Oxidative Stress Measurement
Oxidative stress levels of in vitro cultured cells were measured with the use of the DCFH-DA oxidant-sensitive fluorescent probe²¹ as described in the Data Supplement.

To measure oxidative damage levels in adductor muscles, protein carbonylation and thiobarbituric acid reactive substances (TBARS) were measured (see Data Supplement). Although both methods have some caveats, these markers are widely accepted to measure in vivo oxidative stress.^22,23

Statistical Analysis
Unless differently stated, variables were analyzed by Student t test. A value of P0.05 was deemed statistically significant. Results are reported as mean±SEM values.

	Results

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Similar Severity of Hindlimb Ischemia in p66^ShcAwt and p66^ShcA–/– Mice
To evaluate the role of p66^ShcA in the in vivo response to ischemia, the femoral artery of both p66^ShcAwt and p66^ShcA–/– mice was removed to induce unilateral hindlimb ischemia.²⁴ Ischemia of comparable severity was induced at 2 days after surgery in mice of the 2 genotypes as assessed by laser-Doppler perfusion imaging (LDPI) (LDPI ischemic/contralateral ratio: wt=0.152±0.02; –/–=0.153±0.02; P=NS).

To better characterize how femoral artery dissection affects perfusion of different components of the vasculature, the number of blood vessels that remained perfused after surgery was analyzed. The vasculature of p66^ShcAwt and p66^ShcA–/– mice was stained by intra-aortic injection of biotinylated Lycopersicon esculentum lectin, which binds to the N-acetyl glucosamine residues on the endothelial cell surface and stains perfused vessels.²⁵ By this approach, the number of vessels that are still perfused due to the residual flow after ischemia is analyzed. Although the number of lectin-positive capillaries was >3-fold lower in the ischemic muscle compared with the contralateral limb (lectin-positive capillaries: wt=32.3±10.8; –/–=39.0±10.8, expressed as percentage of the contralateral), almost all the arterioles and venules were stained in both the ischemic and the contralateral normoperfused limb (percentage of lectin-positive arterioles and venules: wt=97.0±1.9; –/–=95.7±1.6). No significant difference between genotypes was observed in the staining of capillaries, arterioles, or venules.

Vascular Endothelial Growth Factor Is Upregulated by Ischemia in p66^ShcA–/– Mice
Vascular endothelial growth factor (VEGF) is an angiogenic growth factor expressed in response to tissue ischemia and, at least in certain cell types, after ROS exposure.²⁶ Thus, it was tested whether VEGF expression was comparable in p66^ShcAwt and p66^ShcA–/– mice in both normoperfused and ischemic adductor muscles. VEGF was expressed similarly in normoperfused mice of both genotypes; moreover, VEGF expression was comparably upregulated in p66^ShcAwt and p66^ShcA–/– mice as soon as 4 hours after ischemia and remained elevated for the following 44 hours (Figure 1).

View larger version (38K): [in this window] [in a new window]   Figure 1. Similar VEGF induction in p66ShcAwt and p66ShcA–/– mice after ischemia. Adductor muscle extracts were derived from normoperfused (norm) or ischemic (isch) mice at 4, 24, and 48 hours after femoral artery dissection. VEGF was detected with the use of a specific antibody. A, Representative Western blotting. Arrowheads indicate different VEGF isoforms. Bottom panel shows red Ponceau staining of relevant nitrocellulose membranes, indicating similar protein loading in each lane. B, Amount of VEGF in cell lysates was quantified by scanning densitometry. Differences between p66ShcAwt and p66ShcA–/– mice at each time point were not statistically significant. OD indicates optical density.

Ischemia Induces an Electrophoretic Mobility Shift of p66^ShcA
p66^ShcA phosphorylation at serine 36 is necessary for the cell death response to oxidative stress.¹⁴ After 4 hours of ischemia, an alteration of p66^ShcA electrophoretic mobility compatible with serine 36 phosphorylation was observed (Figure 2), indicating that p66^ShcA may play a role in the response to ischemia.

View larger version (64K): [in this window] [in a new window]   Figure 2. Representative Western blotting shows the electrophoretic mobility upshift of p66ShcA induced by ischemia. Adductor muscle extracts were derived from normoperfused (norm) or ischemic (isch) mice 4 hours after femoral artery dissection of p66ShcAwt and p66ShcA–/– mice. p66ShcA and tubulin (loading control) were detected with the use of specific antibodies. Arrowheads indicate different p66ShcA isoforms.

p66^ShcA–/– Mice Are Resistant to Tissue Damage Induced by Ischemia
ROS have been implicated in cell death, by both apoptosis and necrosis, that follows ischemia.¹ Thus, tissue damage after ischemia was evaluated in adductor muscles of p66^ShcAwt and p66^ShcA–/– mice. To this aim, muscle fibers, capillaries, and arterioles of p66^ShcAwt and p66^ShcA–/– mice were studied in adductor muscle sections stained with hematoxylin-eosin. Morphometric analysis of normoperfused muscles did not show any significant difference between p66^ShcAwt and p66^ShcA–/– mice in arteriole length density (mm/mm³: wt=15.8±1.8; –/–=13.3±3.0) and in muscle fiber density (fibers per square millimeter: wt=688±53; –/–=647±68). Conversely, capillary density was significantly lower in p66^ShcA–/– than in p66^ShcAwt mice (capillaries per square millimeter: wt=729±41; –/–=568±19; P<0.002), suggesting that p66^ShcA may be involved in angiogenesis in the absence of ischemia.

On femoral artery dissection, significant differences in the damage to both vascular and muscle tissue were found. As expected, in p66^ShcAwt mice, femoral artery dissection induced extensive necrotic damage to muscle fibers that was concentrated in discrete areas of the adductor muscle (Figure 3A); necrotic fibers were evaluated by morphological criteria, differential eosin staining, and presence of infiltrating cells near the degenerating fibers. In contrast, minimal tissue damage was observed in p66^ShcA–/– mice. At 2 days after surgery, the number of necrotic fibers was almost 4-fold lower in p66^ShcA–/– mice (Figure 3B). Similarly, capillary density decreased in p66^ShcAwt mice after ischemia, but no decrease was observed in p66^ShcA–/– mice (Figure 3C).

View larger version (84K): [in this window] [in a new window]   Figure 3. Increased resistance to ischemia in p66ShcA–/– mice. A, Representative hematoxylin-eosin–stained sections of p66ShcAwt and p66ShcA–/– mice adductor muscles. Magnification x400; bar=60 µm. Black arrows indicate infiltrating cells; white arrows, necrotic myofibers; and white asterisk, non-necrotic myofiber. B, Lower number of necrotic muscle fibers in p66ShcA–/– mice 2 days after femoral artery dissection. Bars are necrotic fibers per square millimeter; P<0.009. C, No decrease in capillary density in p66ShcA–/– mice after ischemia. Values are expressed as percent capillary density of the relevant normoperfused (norm.) mouse (*P<0.008).

When arteriolar length density after femoral artery dissection was examined, no significant difference with normoperfused mice was found in mice of both genotypes (not shown). These data suggest that the residual perfusion was sufficient to preserve arterioles and venules from ischemia-induced vascular degeneration.

p66^ShcA-Null Cells Are Resistant to Ischemia-Induced Apoptosis
Ischemia has been shown to induce cell death by both apoptosis and necrosis.^1,2,27 Thus, apoptosis induced by ischemia was measured in mice of both genotypes at 8 hours after ischemia, when overt necrosis was not present. Terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick-end labeling (TUNEL)–positive nuclei were readily detectable in both muscle fibers and endothelium (Figure 4A), and the number of apoptotic nuclei was >5-fold lower in p66^ShcA–/– than in p66^ShcAwt mice (Figure 4B). Qualitatively similar data were obtained at 24 hours after ischemia, but quantification was difficult because of progressing tissue degeneration (not shown).

View larger version (37K): [in this window] [in a new window]   Figure 4. Targeted mutation of p66ShcA induces resistance to ischemia-induced apoptosis. A, Representative sections of adductor muscle of p66ShcAwt and p66ShcA–/– mice after 8 hours of ischemia. Magnification x400; bar=60 µm. TUNEL assay shows lower apoptosis levels in p66ShcA–/– mice. Inset shows a x3 enlargement of TUNEL-positive nuclei, indicated by black arrows. B, Mean TUNEL-positive nuclei number per square millimeter; *P<0.021, wt vs –/–. C and D, Higher viability and lower apoptosis rates of p66ShcA–/– satellite myoblasts and endothelial cells after simulated ischemia. p66ShcAwt and p66ShcA–/– cells were grown in simulated ischemia conditions for the indicated time. C, Cell viability was determined by trypan blue exclusion. Results are expressed as percentage of treated vs untreated controls (*P<0.001; #P<0.002; P<0.011). D, Apoptosis rates were determined by measuring the cytoplasmic histone-associated DNA fragments. Results are expressed as fold apoptosis induction vs untreated wt cells (*P<0.03; #P<0.003; P<0.001).

To assess whether increased resistance to ischemia was also observed in isolated cells, satellite myoblasts and endothelial cells were derived from p66^ShcAwt and p66^ShcA–/– mice and cultured in either normoxic conditions or conditions that simulated in vitro certain aspects of ischemia (1% O₂ for myoblasts, 1% O₂ and serum deprivation for endothelial cells). Both myoblasts and endothelial cells derived from p66^ShcA–/– mice displayed increased survival (Figure 4C) and lower apoptosis rates after exposure to simulated ischemia (Figure 4D).

Lower Oxidative Stress in p66^ShcA–/– Cells After Ischemia
Lower basal levels of intracellular oxidative stress were observed in p66^ShcA–/– fibroblasts and mice.^14–16 Thus, it was tested whether the same was true in adductor muscles after ischemia. To this aim, tissue levels of TBARS and protein carbonylation were measured in adductor muscle extracts at 2 and 4 hours after ischemia. Although in p66^ShcAwt mice both TBARS and protein carbonylation increased, no increase was observed in p66^ShcA–/– mice (Figure 5A and 5B).

View larger version (21K): [in this window] [in a new window]   Figure 5. Lower oxidative stress in p66ShcA-null cells and mice during ischemia. A, TBARS were determined in adductor muscle extracts 2 and 4 hours after ischemia. Values are expressed as nanomoles per gram of dry tissue (wt versus –/–; *P<0.002; #P<0.01). B, Protein carbonylation was determined in adductor muscle extracts 2 and 4 hours after ischemia. Protein carbonyl groups were derivatized with 2,4-dinitrophenyl hydrazine and then detected by Western blotting with an antibody to the DNP-protein conjugates. Amount of conjugates in cell lysates was quantified by scanning densitometry. Results are expressed as percentage of normoperfused nonischemic mice (wt versus –/–; *P<0.03; #P<0.04). C, Endothelial cells were exposed to simulated ischemia (isch.) for 2 or 4 hours, and intracellular oxidative stress was determined by measuring fluorescence associated with DCFH-DA oxidant-sensitive fluorescent probe. Values are expressed as percentage of fluorescence of wt cells grown in control conditions. Statistical significance was calculated by ANOVA (*P<0.03; #P<0.01; P<0.03).

Next, oxidative stress was measured in cultured endothelial cells exposed to simulated ischemia, measuring fluorescence associated with a DCFH-DA oxidant-sensitive probe. In the absence of ischemia, p66^ShcA–/– endothelial cells had lower levels of oxidative stress. After exposure to simulated ischemia for 2 or 4 hours, increase of oxidative stress levels was much lower in p66^ShcA–/– than in p66^ShcAwt cells (Figure 5C).

p66^ShcA–/– Mice Are Resistant to Tissue Damage Induced by I/R
It has been shown that ROS formation plays a causal role in tissue injury that follows I/R.^1–8 To confirm the role of p66^ShcA in tissue damage response to oxidative stress, both p66^ShcAwt and p66^ShcA–/– mice were subjected to 1 hour of ischemia with an elastic tourniquet, followed by 24 hours of reperfusion. LDPI documented a reduction in hindlimb blood flow consequent to ischemia (LDPI ischemic/contralateral ratio: wt=0.29±0.05; –/–=0.27±0.04) and the following reperfusion 10 minutes after tourniquet removal (LDPI ischemic/contralateral ratio: wt=0.87±0.06; –/–=0.92±0.09); in both circumstances, perfusion was similar in p66^ShcAwt and p66^ShcA–/– mice.

Histological damage was assessed by evaluating capillary density and muscle fiber degeneration in hematoxylin-eosin–stained sections of gastrocnemius, a muscle not compressed and possibly damaged by the tourniquet. As observed in adductor muscles, capillary density of normoperfused gastrocnemius muscles was significantly lower in p66^ShcA–/– than in p66^ShcAwt mice (capillaries per square millimeter: wt=813±45; –/–=685±59; P<0.04). On I/R, significant differences in the damage to both vascular and muscle tissues were found. After 1 day of reperfusion, the number of necrotic fibers was 5-fold lower in p66^ShcA–/– than in p66^ShcAwt mice (Figure 6A). Similarly, capillary density decreased to a much lower extent in p66^ShcA–/– than in p66^ShcAwt mice (Figure 6B).

View larger version (22K): [in this window] [in a new window]   Figure 6. p66ShcA–/– mice are resistant to tissue damage induced by I/R. p66ShcAwt and p66ShcA–/– mice were subjected to tourniquet ischemia for 1 hour, followed by 24 hours of reperfusion. A, Lower number of necrotic muscle fibers in gastrocnemius of p66ShcA–/– mice at 1 day after reperfusion. Bars show necrotic fibers per square millimeter; *P<0.003. B, Lower decrease in capillary density in gastrocnemius of p66ShcA–/– mice after I/R. Values are expressed as percentage of capillary density of the relevant normoperfused (norm.) mouse (*P<0.001).

	Discussion

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ROS have been implicated in cell death, by both apoptosis and necrosis, that follows ischemia and I/R.¹ Moreover, protection by antioxidant species of various ischemic and I/R organs, including hindlimb skeletal muscles, has been shown.^2,6,9–11 However, the molecular mechanisms responsible for cell death in ischemia remain poorly understood. The present study provides evidence demonstrating that targeted mutation of p66^ShcA induces resistance to ischemia, both in vivo and in vitro. In agreement with this finding, tolerance to oxidative stress induced by preconditioning is associated with p66^ShcA downmodulation.²⁸ Moreover, in dilated cardiomyopathy, which is associated with increased levels of oxidative stress–mediated cell death, p66^ShcA expression is increased.²⁹ Although the role of p66^ShcA in the response to ischemia of the heart, brain, and other organs is still unknown, one may speculate that p66^ShcA–/– mice may live longer,¹⁴ at least in part, because they are less sensitive to ischemia.

Although several models can be configured to explain the resistance of p66^ShcA–/– mice to ischemia, it is remarkable that p66^ShcA regulates intracellular ROS levels that, in turn, directly affect both apoptosis and necrosis. In agreement with previous reports,^15–17 lower levels of intracellular oxidative stress were measured in p66^ShcA–/– cells and mice after ischemia. Furthermore, several authors reported that skeletal muscle damage induced by I/R is largely dependent on ROS generation.^10,11 In agreement with data obtained in ischemic mice, it was found that tissue damage after hindlimb I/R was much lower in p66^ShcA–/– mice.

Results obtained may be due to both decreased production and increased scavenging of ROS. In agreement with the latter hypothesis, in p66^ShcA–/– cells the activity of the mammalian forkhead homolog FKHRL1 is increased and redox-dependent forkhead inactivation is reduced.¹⁵ In addition, expression of FKHRL1 results in an increase in both catalase and Mn–superoxide dismutase.^15,30 However, similar levels of Mn–superoxide dismutase protein were expressed in p66^ShcAwt and p66^ShcA–/– mice both before and after ischemia (F. Martelli, PhD, and M.C. Capogrossi, MD, unpublished data, 2004).

p66^ShcA phosphorylation at serine 36 is necessary for the cell death response to oxidative stress.¹⁴ After ischemia, an alteration of p66^ShcA electrophoretic mobility compatible with serine 36 phosphorylation was observed, further implicating p66^ShcA in the response to ischemia.

Previous reports have shown that p53, at least in certain tissues, is necessary for cell death induction by ischemia.³¹ However, different levels of p53 expression do not seem to be involved in the resistance to ischemia of p66^ShcA–/– mice, because p53 was upregulated to a comparable extent in both p66^ShcAwt and p66^ShcA–/– mice (F. Martelli, PhD, and M.C. Capogrossi, MD, unpublished data, 2004). Although p53 role in the skeletal muscle response to ischemia is unknown, p66^ShcA may act as a downstream effector of p53 in ischemia-induced apoptosis.¹⁶

Finally, it was found that p66^ShcA–/– mice have lower capillary density in both adductor and gastrocnemius muscles, suggesting that p66^ShcA may be involved in angiogenesis. Indeed, during embryonic development, ShcA is predominantly expressed in the cardiovascular system, and mice that express none of the ShcA isoforms have defective embryonic angiogenesis.³² Because p66^ShcA–/– cells have lower ROS levels^15,16 (this report), it is possible that a ROS-dependent signal transduction pathway of 1 or more growth factors may be impaired in p66^ShcA–/– mice.³³

In conclusion, the present work demonstrates that p66^ShcA plays a crucial role in the cell death pathways activated by ischemia. Although the mechanisms of p66^ShcA-dependent signaling require further investigation, p66^ShcA may represent a suitable therapeutic target for the prevention of tissue damage occurring during arterial occlusion and organ transplantation.

	Acknowledgments

 
This work was supported in part by Ministero della Salute (ICS-030.6/RF00-49, ICS-120.4/RF00-90, RF01/Conv 188, RF02/Conv 228), AIRC (grant 266/01), and MURST (PNR-T12 66084).

	Footnotes

 
*The first 2 authors contributed equally to this work.

The Data Supplement, which contains additional information about Methods, is available online at http://www.circulationaha.org.

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