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The Effects of Exogenous Surfactant Treatment in a Murine Model of Two-Hit Lung Injury

Zambelli, Vanessa PhD*; Bellani, Giacomo MD, PhD*†; Amigoni, Maria MD; Grassi, Alice MD*; Scanziani, Margherita MD; Farina, Francesca PhD*; Latini, Roberto MD; Pesenti, Antonio MD*†

doi: 10.1213/ANE.0000000000000549
Critical Care, Trauma, and Resuscitation: Research Report

BACKGROUND: Because pulmonary endogenous surfactant is altered during acute respiratory distress syndrome, surfactant replacement may improve clinical outcomes. However, trials of surfactant use have had mixed results. We designed this animal model of unilateral (right) lung injury to explore the effect of exogenous surfactant administered to the injured lung on inflammation in the injured and noninjured lung.

METHODS: Mice underwent hydrochloric acid instillation (1.5 mL/kg) into the right bronchus and prolonged (7 hours) mechanical ventilation (25 mL/kg). After 3 hours, mice were treated with 1 mL/kg exogenous surfactant (Curosurf®) (surf group) or sterile saline (NaCl 0.9%) (vehicle group) in the injured (right) lung or did not receive any treatment (hydrochloric acid, ventilator-induced lung injury). Gas exchange, lung compliance, and bronchoalveolar inflammation (cells, albumin, and cytokines) were evaluated. After a significant analysis of variance (ANOVA) test, Tukey post hoc test was used for statistical analysis.

RESULTS: At least 8 to 10 mice in each group were analyzed for each evaluated variable. Surfactant treatment significantly increased both the arterial oxygen tension to fraction of inspired oxygen ratio and respiratory system static compliance (P = 0.027 and P = 0.007, respectively, for surf group versus vehicle). Surfactant therapy increased indices of inflammation in the acid-injured lung compared with vehicle: inflammatory cells (685 [602–773] and 216 [125–305] × 1000/mL, respectively; P < 0.001) and albumin in bronchoalveolar lavage (BAL) (1442 ± 588 and 743 ± 647 μg/mL, respectively; P = 0.027). These differences were not found (P = 0.96 and P = 0.54) in the contralateral (uninjured) lung (inflammatory cells 131 [78–195] and 119 [87–149] × 1000/mL and albumin 135 ± 100 and 173 ± 115 μg/mL).

CONCLUSIONS: Exogenous surfactant administration to an acid-injured right lung improved gas exchange and whole respiratory system compliance. However, markers of inflammation increased in the right (injured) lung, although this result was not found in the left (uninjured) lung. These data suggest that the mechanism by which surfactant improves lung function may involve both uninjured and injured alveoli.

Published ahead of print December 11, 2014.

From the *Department of Health Science, University of Milano-Bicocca, Monza, Italy; Department of Emergency, San Gerardo Hospital, Monza, Italy; and Department of Cardiovascular Research, Istituto di Ricerche Farmacologiche, Milano, Italy.

Accepted for publication September 25, 2014.

Published ahead of print December 11, 2014.

Funding: The present study was partially supported by an unrestricted grant from Chiesi Farmaceutici (Parma, Italy), which also provided the exogenous surfactant for free. The funding sources had no involvement in study design or data collection, analysis or interpretation, or in the decisions concerning the drafting or the submission of this manuscript.

The authors declare no conflicts of interest.

This report was presented, in part, at the 22nd SMART Congress (2011).

Reprints will not be available from the authors.

Address correspondence to Giacomo Bellani, MD, PhD, Department of Health Science, University of Milano-Bicocca, Via Cadore 48, Monza, Italy. Address e-mail to giacomo.bellani1@unimib.it.

Acute respiratory distress syndrome (ARDS) is characterized by an in-hospital mortality rate as high as 40%.1 The acute onset of hypoxemia, diffuse inflammatory infiltrates,2,3 and decreased pulmonary mechanics4 are typical of these syndromes.5 Despite promising preliminary studies,6 no pharmacological approach has improved ARDS patient outcomes. Although low tidal volume mechanical ventilation (MV) improves outcomes, MV can induce lung injury (ventilator-induced lung injury [VILI]) by causing overdistension of pulmonary units that may be already damaged by a primary insult, generating repetitive opening and closing of atelectatic regions and exacerbating lung injury and inflammation.7–9 The mechanical stress upregulates cytokine production and neutrophil recruitment in the lung, which contribute to tissue injury.10–12

Pulmonary surfactant is a complex mixture of lipids (90%) and proteins (10%) secreted by type II pneumocytes into the alveolar spaces. Its main function is to prevent alveolar collapse by reducing the surface tension at the alveolar air–liquid interface, and thus reduce the work of breathing. The first evidence on the effect of surfactant on ARDS pathophysiology was demonstrated in 1982, when Hallman et al.13 discovered that lung surfactant is altered during ARDS. The function of endogenous surfactant may deteriorate because of plasma proteins or reactive oxygen species and proteases released during the pulmonary inflammatory process,14–16 with a subsequent increase in alveolar surface tension, decreased lung compliance,17 and edema formation.18 The exogenous surfactant treatment in ARDS patients thus has a physiological basis and evidence of benefit when used to treat the respiratory distress syndrome of premature infants.19 Several preclinical and clinical studies have evaluated the efficacy of exogenous surfactant replacement in ARDS, but the results are mixed.20 One possibility for this inconsistency is that different studies have used different types of treatment. The timing, dose, and specific composition and activity of the exogenous surfactant are fundamental variables that can affect the effectiveness of treatment.20 We have demonstrated21 that a single bolus of exogenous porcine-derived surfactant (Curosurf®; Chiesi Farmaceutici, Parma, Italy) improved lung function in a murine model of acid-induced lung injury. Interestingly, this beneficial effect persisted 2 weeks after lung injury. To better mimic the clinical situation, we recently22 characterized a double-hit murine model of ARDS because the evolution of lung injury may also be exacerbated by positive-pressure prolonged ventilation. The key feature of this model is the concurrent presence of a unilateral lung (right) subjected to a double insult (acid aspiration and MV) and the other lung (left) that is only mechanically ventilated. This model mimics well the inhomogeneity of ARDS in humans because there are nonaerated areas (the right lung) and overdistended areas (the left lung). The aim of this study was to evaluate the effect of natural surfactant administration in this murine model of ARDS. We hypothesized that by delivering the exogenous surfactant (Curosurf) selectively in the acid-injured lung, a partial recruitment of collapsed alveoli and increased compliance would occur. By these mechanisms, tidal volume may be redistributed throughout the lung, reducing overdistension, and possibly reducing inflammation.

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METHODS

Animals

Male C57BL/6J mice (22–25 g) were obtained from Harlan Laboratories (Udine, Italy) and maintained under standard laboratory conditions in the University of Milano-Bicocca in Monza (Italy). Procedures involving animals and their care were conducted in conformity with the institutional guidelines complying with national (D.L. n. 116, G.U., suppl. 40, 18 Febbraio 1992, Circolare n. 8, G.U., 14 Luglio 1994) and international laws and policies (EEC Council Directive 86/609, OJ L 358, 1, December 12, 1987; the US National Research Council Guide for the Care and Use of Laboratory Animals, 2011). The experimental protocol was submitted to the Italian Ministry of Health and approved by the Animal Care Unit of the University of Milan-Bicocca (Monza, Italy).

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General Experimental Protocol

The 2-hit lung injury model was performed as described previously.22,23 Animals were anesthetized with ketamine (120 mg/kg), xylazine (0.8 mg/kg), and fentanyl (90 μg/kg) by intraperitoneal injection and connected to a rodent ventilator (Inspira ASV, Harvard Apparatus, Holliston, MA). The variables set up for MV were the following: tidal volume 25 mL/kg, respiratory rate 100/min, positive end-expiratory pressure 2 cmH2O, fraction of inspired oxygen (FiO2) 0.5, and inspiratory on expiratory ratio 35%. Immediately after tracheal intubation, muscle paralysis was obtained by an intraperitoneal injection of pancuronium bromide (2 mg/kg). Hydrochloric acid (HCl) 1.5 mL/kg 0.1 M was instilled into the right lung. After 3 hours, mice underwent intratracheal instillation into the right lung with 1 mL/kg exogenous surfactant (80 mg phospholipids/mL, surf group) or sterile saline 0.9% (vehicle group). The exogenous surfactant used was porcine-derived surfactant (Curosurf). A group of mice did not receive intratracheal instillation of either vehicle or surfactant but of HCl (HCl VILI) only. For the entire duration of the experiment (7 hours), the animals were placed on a heating pad so that body temperature was maintained constant at about 36.5 to 37 degrees.

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Anesthesia Maintenance

Adequate levels of sedation and hydration were maintained by continuous infusion into the carotid artery of fentanyl 57 μg/kg/h, ketamine 100 mg/kg/h, and pancuronium bromide 2 mg/kg/h in Ringer’s acetate solution.

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Hemodynamic Monitoring

During the experiment, hemodynamic variables (invasive arterial blood pressure and heart rate) and airway pressure were monitored using a pressure transducer, which was interfaced to a PowerLab (AD Instruments, Colorado Springs, CO) signal transduction unit. A recruitment maneuver (RM; 30 cm H2O for 10 seconds) was performed immediately after intubation and then every 60 minutes, being the plateau pressure and respiratory system static compliance (Cstat) measured before and after an RM.

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Assessment of the Injury

The mice were killed after 7 hours of MV by exsanguination under deep anesthesia. However, all animals that died between 6 and 7 hours of MV were considered for analysis for hemodynamics, BAL fluid, and blood collection for leukocyte count and cytokine concentration measurement (not for arterial blood gas analysis). The animals that died before 6 hours of MV were excluded from the analysis.

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Pressure–Volume Curve Assessment

At the seventh hour from the beginning of the experiment, a last RM was performed, and compliance before and after RM was measured. Next, a pressure–volume curve was constructed by delivering 3 steps of 200 μL of inspiratory volume from functional residual capacity by the ventilator and measuring plateau airway pressure for each step. Three values of compliance were obtained by calculating the ratio between the insufflated volume and the plateau pressure. A mean value was then calculated.

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Arterial Blood Gas Measurement

After a period of oxygen administration with FiO2 of 1, a blood sample was withdrawn from the catheter in the carotid artery and analyzed (0.1 mL) with an I-STAT 1 portable analyzer (Oxford Instruments S.M., Burke e Burke, Menfis Biomedica, Milan, Italy). The cartridge used for the analysis allowed the measurement of oxygen partial pressure, carbon dioxide partial pressure, and pH, and calculated the values of HCO3, base excess (BE), saturation of oxygen, and total CO2.

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Peripheral Total Leukocyte Count

The same blood sample was used for the peripheral total leukocyte count. Twenty microliters of blood was resuspended in 200 μL of Turk (a nuclear dyer), and leukocytes were counted in a Burker chamber.

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Selective Bronchoalveolar Lavage

After animal exsanguination, the thorax was opened and a macroscopic observation of the lungs allowed identification of the localization of the acid injury, as a hemorrhagic and nonrecruitable zone. BAL was then performed separately for each lung by alternatively clamping the left and right bronchus. Lavage was performed 3 times for each lung, with 600 or 400 μL of lavage solution (0.9% saline solution and protease inhibitor), respectively, for the right and left lungs. The BAL samples obtained were then centrifuged for 10 minutes, 1500 rpm, 4°C; the supernatant was then stocked at −80°C. The cell pellet was resuspended in 500 μL PBS (Dulbecco’s phosphate-buffered saline, GIBCO). One-hundred microliters was placed in 200 μL of Turk for total leukocyte count in a Burker chamber, while 100 μL was centrifuged by a Cytospin (StatSpin Cytofuge 2, Bio-Optica, Milan) and then colored with a Diff-Quick kit (Medion Diagnostics, Düdingen, Switzerland), which differently stains the nucleus and cytoplasm, thus allowing us to obtain the differential cell count.

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Protein Concentration

Protein concentration in BAL fluid was measured by the bicinchoninic acid method. Briefly, 200 μL of reagent (composed by bicinchoninic acid and CuSO4, 1:50) was added to samples. BAL samples from right and left lungs were analyzed separately. A standard curve was constructed with increasing concentrations of bovine serum albumin. A spectrophotometer reading was then performed at 570 nm.

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Albumin and Cytokine Concentration

Albumin, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were assayed by enzyme-linked immunosorbent assays according to manufacturer instructions (Abnova Corporation, Taiwan; for Albumin and R&D systems, Minneapolis, MN).

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Statistical Analysis

Sample size was in line with previous literature data: for each group, 8 to 10 mice were analyzed.21,22,24,25 Data are expressed as mean ± SD for normally distributed data and as median (interquartile range) when nonnormally distributed. Kaplan-Meier estimates of survival curves were based on the results of the log-rank test, and 95% confidence intervals were calculated.26 Differences between group means were assessed using 1-way ANOVA and, when this was significant, Tukey post hoc test was used to assess differences between groups. We used Shapiro-Wilk test and Levene test to assess normality and the equality of variance, respectively, to all of the ANOVA residuals. Because of nonnormal distribution, total cell counts in BAL and cytokine concentration were transformed by the natural logarithm before analysis of variance. P values of <0.05 were considered statistically significant. Statistical analysis was performed by SPSS 19.0 (IBM, Chicago, IL), and by Sigmaplot 11.0 (Systat Software Inc., Chicago, IL).

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RESULTS

We used 58 mice (22 in HCl VILI group, 18 in both surf and vehicle group); of these, 41 survived >6 hours. Acid-induced damage in the right lung was confirmed by macroscopic examination after euthanasia. Using macroscopic analysis, when we identified that HCl instillation involved the contralateral lung, we excluded that animal from analysis (approximately 3%). For each group, 8 to 10 mice were analyzed. Some analysis could not be performed in some animals because of technical problems (e.g., tracheal rupture preventing the BAL execution or arterial blood gases machine malfunctioning); however, the total number of analyses performed in each group is reported in the tables.

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Survival

Some animals with HCl instillation became hypotensive. Those for which arterial blood pressure reached 40 mm Hg or died slowly were eliminated from the study. The survival rate was 83% (confidence interval, 100%–66%) in the group of animals treated with surfactant, 72% (confidence interval, 93%–51%) in the vehicle group, and 59% (confidence interval, 80%–38%) in the untreated (HCl VILI) group (log rank, P = 0.224) (Fig. 1); although the lack of significance could have been attributable to the wide confidence intervals of the proportion of survival rate.

Figure 1

Figure 1

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Physiological Parameters

No differences in terms of hemodynamics and airway pressure were observed in the examined groups throughout the experiment (Table 1), nor was the fluid infusion rate different between groups (HCl VILI: 16.8 ± 4, surf: 15.4 ± 5, vehicle: 16.7 ± 2 mL/kg/h).

Table 1

Table 1

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Gas Exchange

After 7 hours of MV from acid instillation, there was a statistically significant (P = 0.019 versus HCl VILI and P = 0.027 versus vehicle) improvement in arterial oxygen tension in the surf group compared with the other groups (Fig. 2). The 3 groups did not differ with regard to arterial carbon dioxide tension (Table 2), whereas, the pH and BE values were significantly higher in surfactant-treated mice compared with the untreated and vehicle groups (Table 2).

Figure 2

Figure 2

Table 2

Table 2

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Mechanical Properties

Respiratory system Cstat measured during the experiment did not differ among groups, but from the sixth hour, the surf group showed higher Cstat than the other 2 groups (Fig. 3). These data probably indicated better elastic properties and were confirmed by the measurement of the Cstat using the pressure–volume curve constructed at the end of the experiment (Fig. 4).

Figure 3

Figure 3

Figure 4

Figure 4

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Leukocytes Count in Blood and BAL

Peripheral white blood cell counts were significantly lower (P = 0.01) in the surf group (1033 ± 314 per mm3) compared with the HCl VILI group (1976 ± 830 per mm3) and with the vehicle group (2036 ± 691 per mm3). On the contrary, in right lung BAL, the surf group showed an important cellular recruitment in the alveolar space because total cell counts were significantly higher compared with the other groups (Table 3).

Table 3

Table 3

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Microvascular Permeability

The total protein concentration in BAL was higher in the right lung of surf group compared with the other 2 groups, but there was an inverse trend in the left lung (surf group trending toward lower protein levels) (Fig. 5). When focusing on the albumin concentration, we found significantly higher levels in the right (acid-injured) lung of surf group mice, but no difference was found in the contralateral lung.

Figure 5

Figure 5

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Cytokine Concentration

BAL levels of inflammatory mediator release in the right lung tended to be higher in the surfactant group (Fig. 6). Indeed, there was an increase in the right lung of IL-1β and TNF-α levels in the surf group compared with the HCl VILI group.

Figure 6

Figure 6

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DISCUSSION

Surfactant alterations in lung injury contribute to the pathophysiology of ARDS. This complex substance allows collapsed alveoli to also open during low inspiratory pressure and to keep alveolar size during inspiration.17 Gregory et al.27 demonstrated a significant decrease in total surfactant pool in ARDS patients and showed that these alterations occur early in both the at-risk and ARDS patients. In a rat model of VILI,28 alterations of surfactant system precede lung dysfunction, and the primary cause of surfactant impairment is attributable to serum proteins. Based on its modifications during ARDS and its encouraging results in neonatal patients, surfactant therapy has been tested in the treatment of adult ARDS. Some clinical trials demonstrated beneficial effects in terms of oxygenation,29–32but several other studies demonstrated no improvement or severe side effects.33 This variable response to exogenous surfactant could be because of the origin of lung injury. The more favorable response was found in direct-injury animal models more so than in indirect-injury animals,20 and these observations resemble the response pattern seen in humans.32,34

In the present study, we evaluated the effects of porcine-derived surfactant treatment (Curosurf) in a 2-hit murine model of ARDS. This particular experimental model mimics well the dishomogeneity typical of clinical disease. Indeed, the right lung received the acid instillation before prolonged MV, leading to the subsequent reduction of available areas to ventilate causing the contralateral (left lung) to overdistend. As demonstrated recently, 22 mice that undergo 2-hit lung injury show, in addition to severe defects of oxygenation and mechanical properties, inflammatory cell recruitment, and increased albumin levels in bilateral BAL samples, the addition of prolonged MV aggravates the preexisting lung injury (induced by acid), and because of tidal volume redistribution, it may affect the contralateral, noninjured lung.22

In this experimental setting, we evaluated whether local (only in the right lung) exogenous surfactant administration could induce beneficial effects by redistributing the air volume in the whole lung. In our model, death occurred predominantly toward the end of the experiment, almost after the fifth hour of MV. In our small study, survival was not significantly different in surfactant-treated mice compared with the vehicle group and untreated mice. At the end of the experiment, Curosurf-treated animals showed an improvement in gas exchange: oxygen partial pressure (PaO2) was higher (≤25%) than both vehicle and HCl VILI groups. No difference was found in carbon dioxide tension, whereas pH and BE were significantly better in the surf group than in the other 2 groups. These gas exchange results are in line with those obtained in our previous study, performed on unilateral acid-induced lung injury.21 Improvements in lung function were also seen via pulmonary mechanical properties. Respiratory system Cstat improved significantly during the experiment. In particular, all 3 groups had similar compliance until the fifth hour; then 3 hours after the treatment, it begun to differ among groups; and the surf group showed better pulmonary elastic properties compared with the other groups. This beneficial effect on lung mechanical function was expected, considering the surfactant physiological role in reduction of surface tension, which could positively affect oxygenation. On the other hand, we found that surfactant treatment increased inflammation only in the injured lung. In our previous study,21 we demonstrated reduced neutrophil fraction in BAL 24 hours after lung injury. On the contrary, in this experiment, we found an intense inflammatory process 7 hours after lung injury (earlier time), with more cellular infiltrates and cytokine levels in alveoli of the right lung of surfactant-treated mice than in vehicle or untreated animals. Our results do not allow us to elucidate the mechanism by which surfactant promotes inflammation: potential mechanisms include immune reaction to exogenous proteins and a direct mechanical action. However, these results are in line with other published studies. In vitro and in vivo studies demonstrated that exogenous surfactant treatment increases the infiltration of leukocytes in the lungs.35–37 In particular, an in vitro study35 performed on polymorphonuclear cells revealed important chemotactic effects of exogenous surfactant (Curosurf) on this type of cell. Moreover, in 2002, Stamme et al.24 used an isolated and ventilated murine lung model to show how exogenous surfactant treatment enhances inflammatory cytokines release by inducing an overventilation and stretching of alveolar units. Nieman et al.36 hypothesized that the effect of the exogenous surfactant on the inflammation could depend on the mechanical effects of the instillation process as well as to surfactant preparation properties. Surfactant instillation may stimulate the release of cytokines (TNF, IL-1, or platelet-activating factor) and cause adhesion of leukocytes to the vascular endothelium.38 Finally, in a recent report, the administration of a bovine-derived exogenous surfactant did not decrease lung inflammation, whereas it led to an increase of several inflammatory cytokines.39

Results on the left lung partially confirmed our hypothesis: we reasoned that by delivering the exogenous surfactant selectively in the right lung, the contralateral lung could be protected from injury induced by overdistension. Indeed, mice treated with surfactant tended to have a reduced inflammatory process when compared with untreated and vehicle-treated mice; although markers of inflammation increased in the right lung, they did not increase in the left lung, although because of the lack of an a priori calculation of the sample size, this result should be interpreted with caution.

In conclusion, in this study, we found that administration of porcine exogenous surfactant (Curosurf) improved lung function in a 2-hit model of ARDS, characterized by acid instillation and prolonged MV. In addition, we found that surfactant administration increased inflammation in the lung directly injured by acid but had no effect on the inflammation of the contralateral lung; the increased inflammation caused by surfactant administration (for which we did not provide a mechanistic explanation) might be involved in the controversial findings deriving from clinical trials. Although we cannot exclude that surfactant had a direct action on the contralateral, uninjured lung, our data suggest that surfactant may improve lung function by promoting redistribution of tidal volume to both lungs, preventing overdistension of ventilated regions and decreasing VILI.

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DISCLOSURES

Name: Vanessa Zambelli, BiolD, PhD.

Contribution: This author helped conduct the study, analyze the data, and write the manuscript.

Attestation: Vanessa Zambelli has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Giacomo Bellani, MD, PhD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Giacomo Bellani has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Name: Maria Amigoni, MD.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Maria Amigoni has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Alice Grassi, MD.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Alice Grassi has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Margherita Scanziani, MD.

Contribution: This author helped conduct the study and analyze the data.

Attestation: Margherita Scanziani has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Francesca Farina, BiolD, PhD.

Contribution: This author helped conduct the study.

Attestation: Francesca Farina has seen the original study data and approved the final manuscript.

Name: Roberto Latini, MD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Roberto Latini has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

Name: Antonio Pesenti, MD.

Contribution: This author helped design the study, analyze the data, and write the manuscript.

Attestation: Antonio Pesenti has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.

This manuscript was handled by: Avery Tung, MD.

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ACKNOWLEDGMENTS

We thank Valentina Milani, PhD (Medical Statistician, Laboratory of Medical Statistics, Department of Cardiovascular Research, IRCCS—Istituto di Ricerche Farmacologiche Mario Negri, Milan) for the statistical advice.

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