Background
In patients with congenital cardiac communications, pulmonary vascular abnormalities pose a risk for immediate postoperative hemodynamic disturbances which are sometimes severe enough to be considered as life-threatening medical emergencies. Besides, persistent pulmonary hypertension late after operation may affect treatment success [
1‐
3]. The severity of postoperative pulmonary vascular reactivity depends on the degree of preoperative pulmonary vascular remodeling which is generally related to patient age, type of cardiac anomaly and presence of extracardiac syndromes, such as Down syndrome (trisomy 21) [
4]. Intraoperative factors play a role: metabolic and electrolyte abnormalities, imbalance between endogenous vasoconstrictors and vasodilators, and systemic inflammatory response to cardiopulmonary bypass (CPB) [
5]. Postoperatively, hypoxia and acidosis have been considered as major triggers for pulmonary vasoconstriction [
6,
7].
Large (unrestrictive) cardiac communications may be associated with pulmonary overcirculation with variable degrees of pulmonary congestion. Patients may present with congestive heart failure and failure to thrive and are predisposed to recurrent upper and lower respiratory tract infections. Viral infections have been extensively investigated in pediatric patients undergoing cardiac surgery. They were shown to impact on major outcomes, such as duration of postoperative mechanical ventilation, length of intensive care unit and hospital stays, and mortality [
8‐
10]. However, the impact of preoperative exposure to respiratory viruses on the postoperative behavior of the pulmonary circulation has not been examined so far.
Respiratory viral infections may affect pulmonary microcirculation in at least two ways. First, they may cause local alveolar hypoxia, a major stimulus for pulmonary vasoconstriction. Second, infected airway epithelial cells cause cascade expression of proinflammatory cytokines, chemokines and growth factors, thus inducing local inflammation, extracellular matrix reorganization and cell proliferation [
11‐
17]. Because small airways and arteries share the same microenvironment in the lungs [
18], the biological events that take place as a result of viral infections may in theory, contribute to both airway and vascular remodeling.
In this study, we investigated the possible role of preoperative exposure to respiratory viruses and post-CPB inflammatory reaction in determining the behavior of the pulmonary circulation following elimination of left-to-right cardiac shunts. We examined that in a prospective pediatric cohort taking into consideration classical risk factors for postoperative pulmonary hypertension as well. We also looked for a possible relationship between the presence/absence of viral genomes in the airways prior to surgery and the level of pulmonary arterial pressure after discharge from the hospital.
Methods
Study design, setting and patients
This was a prospective cohort study comprising patients who were admitted to the Heart Institute (InCor), University of São Paulo School of Medicine, São Paulo Brazil, from November 2016 to September 2021, for surgical repair of congenital cardiac communications. Patients were consecutively enrolled based on the following criteria: age 1 month to 3 years; heart with biventricular physiology; presence of unrestrictive cardiac communications, i.e., with a diameter of the post-tricuspid communication greater than 50% of the aortic annulus diameter on transthoracic echocardiography; absence of pulmonary stenosis; clinical features suggestive of at least moderately elevated pulmonary arterial pressure: a loud second heart sound at pulmonic region, increased pulmonary/systemic blood flow ratio with absence of significant pressure gradients across the septal defects (echocardiography) and increased pulmonary vascular markings on chest radiographs; absence of extracardiac syndromes other than Down syndrome; and absence of any signs of ongoing or recent inflammatory or infectious diseases. The study was based essentially on patients who were eligible for surgical treatment with no need for cardiac catheterization. Patients with significant right-to-left shunting and persistently low peripheral oxygen saturation (< 85%), suggesting the presence of advanced pulmonary vasculopathy, were not included. An informed consent signed by family members was necessary for patient inclusion. The study protocol was approved by the Institutional Scientific and Ethics Committee, CAPPesq no. 3.735.675.
General diagnostic data
Data recorded at baseline included details of the clinical history and physical examination, such as signs and symptoms of congestive heart failure and failure to thrive, presence or absence of Down syndrome, occurrence of acute respiratory infections and eventual hospitalization prior to referral to the Heart Institute. The diagnosis of Down syndrome was confirmed by genetic testing. Chest radiographs, ECG and complete echocardiographic data were available for all patients. Transthoracic echocardiography was used to assess cardiovascular anatomy and blood flow parameters. The pulmonary/systemic blood flow ratio was calculated based on systolic flow parameters in the right and left ventricular outflow tracts. Pulmonary venous flow was estimated by measuring the velocity–time integral of blood flow in pulmonary veins. Patients with a pulmonary blood flow ratio greater than 2.50 and a velocity–time integral of pulmonary venous flow > 24.0 cm were considered to have pulmonary over circulation which was generally associated with normal (> 93%) peripheral oxygen saturation. Subjects with a pulmonary/systemic blood flow ratio < 2.00, a velocity–time integral of blood flow in pulmonary veins < 20.0 cm and peripheral oxygen saturation < 93% were presumed to have heightened pulmonary vascular resistance, thus requiring special attention postoperatively. The tricuspid annular plane systolic excursion (TAPSE) was used to estimate right ventricular systolic function.
Perioperative management
Patients underwent open cardiac surgery for repair of cardiac lesions. Intraoperatively, they had pulmonary arterial pressure and systemic arterial pressure levels recorded before and after cardiopulmonary bypass (CPB). In our institution, patients who are at risk for postoperative pulmonary hemodynamic disturbances, i.e., those with unrestrictive cardiac communications and preoperative signs of pulmonary hypertension are routinely weaned from CPB on 20 ppm inhaled nitric oxide. A pulmonary arterial catheter was inserted by the surgeon to facilitate hemodynamic monitoring in the intensive care unit. Postoperative analgosedation was performed using fentanyl, midazolan and ketamine singly or in combination. Alternatively, morphine and dexmetomidine were used in combination in subjects with a very stable clinical course. Milrinone, epinephrine and norepinephrine were used as inotropic/vasoactive agents. Postoperatively, patients were kept on inhaled nitric oxide during the entire period of mechanical ventilation, except those in whom ventilation was prolonged for reasons other than pulmonary hypertension.
Primary outcome: postoperative hemodynamics
Invasive assessment of pulmonary and systemic arterial pressures was carried out for at least 2.5 days postoperatively (readings taken at 2-h intervals). Patients with unstable clinical course required longer periods of hemodynamic monitoring. To analyse differences between the study groups (i.e., patients with versus without viral genomes in the respiratory tract) pressure curves were constructed based on data obtained during the first 12 h of intensive care unit stay. In that period, patients were still deeply sedated, stable on mechanical ventilation receiving 20 ppm inhaled nitric oxide, and already free from major post-CPB hemodynamic instabilities. We also computed pulmonary/systemic mean arterial pressure ratio (PAP/SAP) and calculated the mean of first 4 values corresponding to the first 6 h of postoperative monitoring. In the study, this parameter was referred to as early postoperative PAP/SAP. It was also used to compare groups.
Other assessments during and after hospitalization
Postoperative clinical events related to pulmonary vascular tone instability and acute right/left ventricular dysfunction were recorded. These included: (1) typical pulmonary hypertensive crisis defined as a sustained elevation of pulmonary arterial pressure (mean pulmonary arterial pressure > 75% of systemic arterial pressure level) with a decline in systemic pressure (≥ 20%) and oxygen desaturation (< 90%); (2) systemic hypotension with a pulmonary/systemic mean arterial pressure ratio in the range of 50–75% requiring frequent changes in the doses of vasoactive-inotropic drugs; (3) prolonged and/or recurrent hemodynamic and respiratory disturbances not promptly responsive to sedation and manual ventilation; and (4) all hemodynamic and respiratory critical instabilities requiring cardiorespiratory resuscitation. Transient elevations of pulmonary arterial pressure, even to suprasystemic levels, that were rapidly reversed by sedation and manual ventilation were not characterized as clinical events. Interpretations were made independently by 3 physicians (AAL, AMT, and on-duty intensivist).
After discharge from the hospital patients were followed up for 6 months, and then pulmonary hemodynamics was re-evaluated noninvasively by means of transthoracic echocardiography. An adequate tricuspid regurgitation jet Doppler signal was required to estimate systolic pulmonary arterial pressure. That was not possible in subjects with negligible tricuspid regurgitation. All factors and covariates that were recorded perioperatively were tested for a possible role in predicting post-hospitalization pulmonary arterial pressure.
Respiratory viruses
Nasopharyngeal and tracheal aspirates were obtained from all patients for detection and identification of respiratory viruses. Nasopharyngeal aspirates were obtained 2–3 days before surgery in the complete absence of respiratory symptoms. Tracheal aspirates were collected in the operating room just after orotracheal intubation. Nucleic acid extraction was performed using the EASYMAG system (bioMérieux, France) according to the manufacturer’s instructions. Samples were subjected to real-time polymerase chain reaction (RT-PCR, one step) using the ABI 7500 system (Applied Biosystems, CA, USA). A Multiplex Kit (XGen, Pinhais, PR, Brazil) was used for detection of 19 respiratory pathogens including 18 viruses and Mycoplasma pneumoniae.
Pre- and postoperative inflammatory profile
Peripheral venous blood was collected 2–3 days before surgery and 4 h after CPB termination for analysis of serum levels of 36 inflammatory mediators. Proteins were analyzed by immunoblotting using a human cytokine array (R&D Systems, Minneapolis, MN, USA). Samples were processed in duplicate and proteins were semiquantified by chemiluminescence. The results were obtained taking the average signal of each pair of duplicate spots and expressed as units of pixel intensity (upi). Preoperative and postoperative samples were always run in the same assay.
Data obtainment
Preoperative and postoperative clinical and hemodynamic assessments and laboratory analyses (respiratory viruses and inflammatory mediators) were carried out in a blinded fashion.
Statistical analysis
Unless otherwise specified, numerical data are presented as medians with interquartile ranges. Categorical data are presented as number of cases and percentages. Descriptive statistics was performing using the Mann–Whitney test and the Chi-square family of tests for comparisons between groups. The Wilcoxon test, Friedman’s test and linear regression analysis were used to test for differences and associations within subjects. Inferential statistics was carried out using the General Linear model (one-way GLM analysis and two-way GLM analysis for repeated measures) to test for differences between groups regarding the behavior of hemodynamic parameters and curves. In this case, the distribution of the dependent variables was tested for closeness to the normal (Gaussian) distribution and when necessary, variables were analyzed after Box–Cox transformation. Predictors of hemodynamic abnormalities and clinical events were identified using univariate, bivariate and multivariate Logistic regression analysis. Once a predictor was identified, a second variable was characterized as a confounder or enhancer if its inclusion in the statistical model resulted in a > 10% change in the original odds ratio. In all tests, 0.05 was set as the significant level. Statistical analysis was performed using the SPSS statistical software, version 28 (IBM, Armonk, NY, USA).
Discussion
Viruses are the leading cause of respiratory disorders and lung cell injury. This is particularly so in the pediatric population [
25‐
27]. In the setting of cardiac surgery, respiratory abnormalities initiated preoperatively in the course of viral diseases may be further exacerbated by the systemic inflammatory reaction elicted by CPB [
28] placing patients at greater risk for poor postoperative outcomes [
8‐
10,
29,
30]. To the best of our knowledge, the present study represents the first initiative to investigate the relationship between respiratory viruses and the behavior of the pulmonary circulation after surgery for congenital cardiac shunts. Our findings suggest that respiratory viruses might have somehow contributed to the development of pulmonary vascular changes preoperatively, thus increasing the predisposition to postoperative pulmonary hypertension. Post-CPB inflammatory reaction seemed to play a role as a triggering factor. Altered postoperative hemodynamics accounted for clinical events in the intensive care unit with longer times of mechanical ventilation. Finally, respiratory viruses and early postoperative hemodynamics played an interactive role in determining the level of pulmonary artery pressure after discharge from the hospital. Obtainment of tracheal aspirates was crucial for demonstrating the aforementioned associations. The presence of genetic material for respiratory viruses in nasopharynx was by no means predictive of any hemodynamic or clinical events.
Some of our patients had respiratory viral infections in their clinical history, others did not. The question might be raised if respiratory viruses remain active in asymptomatic individuals. In addition, because different pathogens were detected in the study population, it would be worth discovering which ones are capable of infecting the distal lung as to cause relevant changes in small airways and eventually vessels. Although these are difficult points to address in the context of the present study, preliminary thoughts can be formulated with regard to rhinovirus, the most prevalent agent in our cohort. Rhinoviruses are known for their chronic behavior in many instances and their ability to alter the biology of peripheral lung cells. Although infections are usually mild and limited to the upper respiratory tract, there is growing evidence that they can induce lower respiratory tract pathology with severe pulmonary and extrapulmonary complications in adults and children [
31,
32]. In the pediatric population, rhinovirus infections have been linked clinically with serious lower airway illnesses including asthma exacerbation, cystic fibrosis, bronchitis, bronchiolitis, pneumonia and croup [
33‐
36]. Although most rhinovirus serotypes replicate optimally at 33 °C, some strains can actually replicate well at 37 °C and infect lower airways in humans [
37]. Of special interest in terms of pulmonary vascular biology, rhinovirus-infected lung vascular endothelial cells generate several inflammatory and cytopathic responses [
38]. Rhinovirus-infected bronchial epithelial cells express inflammatory mediators including IP-10 which was shown to be involved in vascular smooth muscle cell migration and proliferation [
11,
15,
39]. Furthermore, type 2 immune response which is induced during rhinovirus infections [
40,
41] was shown to play a central role in pulmonary vascular remodeling [
13,
42]. In the present study, we were able to demonstrate the hypertensive behavior of the pulmonary circulation for the specific subgroup of rhinovirus carriers. Although the study was not sufficiently powered to demonstrate the individual role of other viruses, we speculate that some might be involved. For example, human bocavirus, the second most prevalent agent in our cohort was shown to persist in the infected host, induce the expression of cytokines and growth factors and elict Th1 and Th2 immune responses [
43,
44].
In our patients, the inflammatory response to surgery under CPB was characterized by an increase in the number of circulating monocytes, a marked increase in neutrophil to lymphocyte ratio, a decrease in platelet count and changes in serum levels of several cytokines and related proteins. The inflammatory reaction seemed to explain the hypertensive postoperative behavior of the pulmonary circulation at least in part. While it is known that multiple mechanisms are involved in pulmonary vasoconstriction and hypertension in acute conditions [
45], it is worth commenting on the role of some inflammatory mediators. IL-1, IL-6 and TNF-α protein levels were shown to be closely associated with PI3K/Akt signaling pathway activation in early stages of monocrotaline-induced pulmonary hypertension in rats [
46]. The chemokine-like cytokine MIF, a central element in innate and adaptative immunity was shown to enhance pulmonary vasoconstriction induced by hypoxia and potentiate constriction pre-evoked by agonists in isolated pulmonary artery rings [
47]. Although the chemokine MCP-1 has been shown to play an important pathophysiological role in pulmonary vascular remodeling [
48‐
50], its involvement in acute pulmonary vasoconstriction is probably indirect. MCP-1 is expressed during endothelial cell activation, attracts inflammatory cells and enhances the release of several other mediators of inflammation [
51]. Interestingly, MCP-1 was shown to play a role in the recovery of monocyte function after pediatric cardiac surgery along with IL-1RA and IL-10 [
52]. In the present study, the increase in IL-1 activity following CPB was inferred from the ~ tenfold increase in serum level of IL-1RA. Because of the spare receptor phenomenon, large quantities of IL-1RA are required to functionally inhibit the biological effects of negligible amounts of IL-1 [
53]. Our results showed that preoperative exposure to respiratory viruses and postoperative overexpression of IL-1/IL-1RA, MIF and MCP-1 acted as combined risk factors for postoperative pulmonary hypertension. Other cytokines (e.g., IL-6 and IL-21) probably played a role as judged by the data presented in Table
5. Furthermore, because the level of several inflammatory proteins changed significantly following surgery, as shown in Table
4, the spectrum of mediators with relevant effects on the pulmonary and systemic circulation and the heart may be even wider.
We also investigated factors that have been classically linked to postoperative pulmonary hypertension in this population. While patient’s age (maximum of 35 months in the study) did not seem to influence postoperative pulmonary hemodynamics, the presence of Down syndrome may have played an indirect role via the complexity of cardiac anomaly (atrioventricular septal defect in 61.5% of cases) with longer surgical times (CPB duration) and presumably, more pronounced inflammatory response. Peripheral oxygen saturation was found to be an important predictor of postoperative hemodynamics. This was probably due to the fact that oxygen saturation reflected the status of the pulmonary circulation at baseline. In fact, bedside oxygen saturation was negatively correlated with pre-CPB pulmonary artery pressure measured directly in the operating room. Although oxygen saturation did not correlate with the presence/absence of viral genomes in the airways, we cannot totally exclude chronic airway abnormalities as a cause for lower-than-normal oxygen saturation levels in some individuals. Upper airway obstructions may have contributed to imbalances between ventilation and perfusion especially in patients with Down syndrome. Bidirectional shunting within the cardiac chambers as a result of heightened pulmonary vascular resistance may also have contributed to systemic oxygen desaturation in some cases.
Acknowledgements
We gratefully acknowledge the assistance of Mrs. Roseli Polo in all phases of the study. We also acknowledge Dr. Ana Maria Thomaz, Dr. Leína Zorzanelli and Dr. Rilvani C. Gonçalves for helping with patient care management and Dr. Juliano Penha for specific study-related surgical actions. We appreciate the collaborative participation of the pediatric nurses Mrs. Angela M. L. Marques and Renata S. Tito and the collaborative work of pediatric cardiologists and cardiac surgeons, intensivists and all colleagues of the multiprofessional team involved in assisting patients at the Heart Institute (InCor), São Paulo, Brazil. Part of a doctoral thesis by Kelly Cristina de Oliveira Abud to the Program in Cardiology, University of São Paulo School of Medicine, São Paulo, Brazil.
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