Ιntroduction
The pulmonary microvascular endothelium possesses numerous metabolic properties, many of which are catalyzed by ectoenzymes expressed on the luminal pulmonary endothelial surface [
1]. Angiotensin-converting enzyme (ACE) is the ectoenzyme that catalyzes the conversion of angiotensin I to angiotensin II, as well as the degradation of bradykinin, thus regulating the vascular tone. Given the strategic location of the lungs, and the extent of the pulmonary capillary endothelial surface, the pulmonary vasculature is the main site of ACE activity [
1].
Pulmonary capillary endothelium-bound (PCEB) ACE activity has been widely studied in animals and humans by indicator-dilution techniques and has been shown to provide a quantifiable, direct and sensitive measurement of endothelial function under normal conditions and pulmonary diseases [
1,
2].
In acute respiratory distress syndrome (ARDS), most commonly occurring during sepsis, pulmonary capillary endothelial cells appear to be among the first lung cells to be insulted, leading to impaired metabolic functionality [
3,
4]. In this respect, PCEB-ACE activity has been shown to decrease early during ARDS and to correlate with its severity [
5].
Activated protein C (APC) system dysregulation plays an important role in the pathogenesis of sepsis-associated organ dysfunction [
6]. In 2004, the use of recombinant human APC (rhAPC;
drotrecogin alfa (activated); Xigris; Eli Lilly, Indianapolis, IN, USA) in patients with sepsis-induced organ failure was recommended based on the results of the PROWESS study [
7,
8]. We hypothesized that rhAPC administration in septic patients with ARDS would improve pulmonary endothelial function, as assessed by PCEB-ACE activity indices.
It should be noted that the drug was withdrawn from the market after the negative results of the PROWESS-SHOCK study [
9]. However, our study on the effect of rhAPC on pulmonary endothelial function of septic patients with ARDS was performed during the period that the drug was in use in patients with sepsis-induced organ dysfunction.
Methods
Compliance with Ethics Guidelines
The study was approved by the Evangelismos Hospital Research Ethics Committee (94/9-9-2003). All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964, also as revised in 2013. Ιnformed written consent was obtained from all patients’ next-of-kin prior to any study procedure. Our study was conducted and completed in 2004, before the International Committee of Medical Journal Editors initiated the policy requiring investigators to deposit information about trial designs. Consequently, our study was not registered.
Study Population and Measurements
In 2004, 19 critically-ill septic patients with ARDS treated with rhAPC were enrolled in the study. All subjects were hospitalized in a general intensive care unit (ICU), were mechanically ventilated, and had at least two organ dysfunctions consequent to infection, one of which was sepsis-associated respiratory dysfunction. All patients suffered from severe sepsis or septic shock and, furthermore, all patients had ARDS except three who had acute lung injury, according to the respective definitions at the time of the study [
10,
11]; based on the current definitions, they are all considered to have suffered from sepsis or septic shock and ARDS [
12,
13]. The decision to treat with rhAPC was taken by the patients’ attending physicians, in compliance with the administration criteria valid at that time [
8]. They all received rhAPC at 24 μg/kg/h for 96 h in a continuous IV drip. All the precautions were taken regarding the risk of bleeding before and after major or minor surgical procedures. Major bleeding events were not noticed. After the completion of the study, patients were followed up and complete clinical and laboratory data were recorded for 28 days or until their discharge from the ICU. At the end of the study, the subjects were grouped according to their outcome (28 days survival or prior discharge from the ICU) into survivors and non-survivors.
PCEB-ACE activity was measured prior to initiation of rhAPC treatment (measurement #1 at baseline), during the 96-h infusion (measurement #2 at 24 h; measurement #3 at 48 h; measurement #4 at 96 h) and 3 days after the end of the rhAPC infusion (measurement #5, day 7). No patient was under ACE inhibitors. Each patient’s baseline measurement served as control of him/herself.
A detailed description of the methodology used is given elsewhere [
2]. Trace amounts of the radiolabelled synthetic ACE substrate
3[H] benzoyl-Phe-Ala-Pro (BPAP) were injected as a rapid bolus into a (jugular or subclavian) central vein, and arterial blood was withdrawn into a fraction collector for analysis.
Single transpulmonary pass substrate utilization is expressed as percent metabolism (%
M) and transpulmonary hydrolysis (
v = [
E] ×
tc ×
kcat/
Km), where [
E] equals the enzyme concentration available for reaction,
tc the capillary transit time,
kcat the catalytic rate constant, and
Km the Michaelis–Menten constant. Functional capillary surface area (FCSA) was additionally calculated as hydrolysis × pulmonary plasma flow and depends on the total enzyme mass that is available for reaction, and the constants
kcat and
Km. Both
v and %
M are indices that reflect ACE activity per capillary, while FCSA is a reflection of ACE activity per vascular bed [
1,
2,
14].
Statistical Analysis
Data are presented as mean ± SE, or median (interquartile range) when data distribution was skewed. Comparisons between groups were made by Student’s t test or Mann–Whitney test, when appropriate, for continuous data, and Chi square test for qualitative data. Differences within time were examined by the Friedman test. Mixed effect models were fitted to examine the effect of time, outcome and their interaction. The Tukey–Kramer test was used for multiple comparisons adjustment. All p values are two-sided. Differences were considered significant at p < 0.05.
Discussion
In sepsis, the dysregulated interaction between inflammation and coagulation underlies organ dysfunction [
6]. On this basis, it was suggested that natural anticoagulants, that are down-regulated in sepsis, could be used as a salvage therapy in septic organ failure. Recombinant human APC was the only anticoagulant initially shown to be effective in decreasing mortality in severely septic patients [
7]. Furthermore, several experimental studies have documented the beneficial effects of the treatment with rhAPC (intravenous or inhaled) on inflammation-induced lung injury [
15‐
19].
Pulmonary capillary ACE activity has been proved to be a reliable index of endothelial dysfunction in variable pulmonary diseases [
1,
5,
20]. Contrary to other surrogate markers of endothelial injury, such as circulating adhesion molecules and other soluble endothelial-derived proteins, its measurement by means of indicator-dilution type techniques provides a direct and sensitive quantification of pulmonary endothelial function at the patients’ bedside. Both substrate transpulmonary hydrolysis (
v) and FCSA have been shown to decrease early during the ARDS continuum and to be inversely related to LIS [
5].
In this study we evaluated baseline PCEB-ACE activity in a homogenous, septic population with ARDS, in the sense that they all fulfilled the criteria for rhAPC administration. We rationalized that a beneficial effect of rhAPC on pulmonary microcirculation would be depicted as an amelioration of the PCEB-ACE activity indices. Our hypothesis was not confirmed: rhAPC treatment had no significant effect on pulmonary endothelial function as estimated by this method. Our results coincided with the uncertainty that emerged about the efficacy and the concerns regarding the use of rhAPC in the treatment of sepsis. The PROWESS-SHOCK study that was conducted for the re-examination of the risk/benefit profile of rhAPC failed to document a mortality benefit [
9], leading to the withdrawal of the product from the market. Despite rhAPC withdrawal, related research has been recently published, driving us to present these previously obtained data. In this context, a study using the pulmonary leak index of 67Gallium transferrin technique to measure alveolo-capillary permeability in patients with ARDS treated with rhAPC also failed to reveal a beneficial effect of the treatment [
21].
In our study population, the estimated BPAP percent metabolism and hydrolysis were considerably decreased compared to the values of normal volunteers with no lung pathologies [
1,
2,
22]. Further analysis of our results attempted to examine a possible relation of PCEB-ACE activity with patient outcome. Indeed, poor outcome (defined as mortality) was associated with more prominent pulmonary capillary endothelial dysfunction, as expressed by the significantly lower values of PCEB-ACE activity indices (i.e. %
M and
v). These results were in accordance with the estimations of disease and respiratory severity indices and scores: when mixed effect models were fitted, non-survivors were found to express worse PO
2/FiO
2, as well as LIS and SOFA scores. However, it is worth mentioning that the values of %
M and hydrolysis (
v) were statistically lower in the non-survivors group from the very first measurement (baseline measurement) compared to survivors, in contrast to all other measured disease severity indices, which showed no significant difference between the two groups at baseline (data not shown). This may denote a superiority of the PCEB-ACE activity indices in providing early information on outcome.
Τhe complex pathophysiology of sepsis and the heterogeneity of septic patients are perhaps reasons why clinicians are still lacking specific treatment options for this syndrome. Our study population consisted of septic patients with ARDS and similar clinical severity, with the limitation of the small size. Nevertheless, our results support a potential implementation of direct markers of endothelial dysfunction in everyday clinical practice. Such markers accompanied by clinical severity scores and other surrogate indices might serve the need for better risk stratification and personalized treatment in sepsis.
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