Abstract

Background

Acute respiratory distress syndrome (ARDS) is a leading cause of acute respiratory failure in critically ill medical and surgical patients. Despite significant advances in our understanding of the pathophysiology, management, and outcomes of ARDS, the morbidity and mortality remains unacceptably high, with a recent pooled estimate of mortality at 43%, ranging from 26 to 58% [1–4]. The pathogenesis of ARDS involves a complex network of cytokines and other pro-inflammatory compounds and the innate and adaptive immune system, leading to alveolar epithelial and endothelial damage [5]. Knowledge of the mechanisms underlying the pathogenesis of ARDS may identify novel therapeutic targets.

Increased extracellular matrix (ECM) turnover is a prominent feature of tissue injury in ARDS. Previous studies have shown that failure to remove ECM degradation products from the site of injury results in unremitting inflammation [6]. Hyaluronic acid (HA)—a nonsulfated glycosaminoglycan composed of repeating disaccharide units of D-glucuronic acid and N-acetyl glucosamine—is an important component of the ECM and is ubiquitously distributed in the lung parenchyma of humans [7]. Although HA has traditionally been regarded as a structural molecule, it has been shown to undergo dynamic regulation and possess bioactive properties [8].

The concentration of HA in the interstitium is a function of the equilibrium between synthesis and degradation, directly influencing circulating and tissue HA levels. The balance between these two processes is altered in many disease states. Increased HA deposition has been observed in the lung tissue of rats with bleomycin-induced lung injury [9] and in humans with ARDS [10, 11], whereas increased HA synthesis has been described in non-human primates with ARDS [12]. Hypoxemia alone has been shown to increase HA synthesis and hyaluronidase activity [13] and to upregulate the expression of the endogenous HA receptor, CD44 [14]. In bleomycin-induced alveolar injury in rats, an association between accumulation of HA in the lung interstitium and pulmonary edema has been demonstrated [9]. Furthermore, serum HA has been proposed as a prognostic factor in patients with ARDS undergoing extracorporeal CO₂ removal [15] and HA levels have been associated with other forms of organ injury particularly in sepsis [16, 17]. Urinary levels of HA have been shown to predict the eventual development of acute kidney injury and in-hospital mortality [18, 19]. Although these studies suggest a role for HA in the pathogenesis of lung injury and systemic inflammation, the mechanisms responsible for the observed associations between HA and patient outcomes are not well-understood.

Given the role of HA in pathologic inflammation in animal models of lung injury and clinical correlations in humans, we sought to determine the relationship between HA levels in the vascular and alveolar compartments and clinical outcomes. We hypothesized that increasing levels of HA would be associated with ARDS-related outcomes. We performed a secondary analysis of a prior randomized controlled trial in ARDS to test this hypothesis.

Methods

Results

Characteristics of enrolled patients are shown in Table 1. Eighty-six patients affected by ARDS were studied. The population consisted of predominately white (88%) men (63%) with a mean age of 50 years. In 47 (55%) of the patients, direct lung injury was the risk factor for ARDS (direct ARDS), whereas the other 39 (45%) had an extrapulmonary (indirect ARDS) risk factor for the syndrome (i.e., extrapulmonary sepsis or nonthoracic trauma). A majority of patients (57%) had moderate ARDS while 29% and 12% had mild and severe ARDS, respectively. The average highest SOFA score within 7 days was 9±4: 56 (65%) of the patients met criteria for sepsis at study enrollment. The majority of the patients (85%) were still alive after 28 days. Comparing subjects treated with placebo vs fish oil, there was no difference in day-0 BALF HA (median (IQR) 63 (23–216) ng/ml vs 56 (17–215) ng/mL; p=0.96) or day-0 serum HA concentration (median (IQR) 119 (53–245) ng/mL vs 148 (67–242) ng/mL; p=0.57]. Day-0 (study enrollment) serum HA levels (median 125.85 ng/mL, interquartile range (IQR) 55.81–241.58 ng/mL) and BALF HA levels (median 62.00 ng/mL, IQR 20.78–215.95 ng/mL) were positively correlated (r=0.26, p=0.02; Additional file3). However, this correlation was not observed in patients who survived and remained intubated on day 4 (p=0.47) or day 8 (p=0.64).

Table 1

Demographics and clinical characteristics of patients at study enrollment

Characteristic	Values in patients (n=86)
Age (years), mean±SD	50±17
Male sex, n (%)	54 (63)
Race, n (%)
White	76 (88)
African American	3 (4)
Othera	7 (8)
Co-morbidities, n (%)
Fish oil treatment, n (%)	38 (44)
Diabetes mellitus	18 (21)
Liver cirrhosis	6 (7)
Chronic kidney disease	2 (2)
ARDS risk factor, n (%)
Directb	47 (55)
Indirectc	39 (45)
ARDS classification of severityd,e, n (%)
Mild	25 (29)
Moderate	49 (57)
Severe	10 (12)
APACHE II score, mean±SD	22±6
Sepsis, n (%)	56 (65)
Day-0 serum HA (ng/mL), median (IQR)	125.85 (55.81–241.58)
Day-0 BALF HA (ng/mL), median (IQR)f	62.00 (20.78–215.95)
Lung injury score, mean±SD	3±1
Sequential organ failure assessment score, mean±SDg	9±4
28-Day mortality, n (%)	13 (15)

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ARDS acute respiratory distress syndrome, APACHE Acute Physiology and Chronic Health Evaluation, HA hyaluronic acid, BALF bronchoalveolar lavage fluid

^aIncludes American Indian, Asian, Pacific Islander, and unknown

^bDirect ARDS was defined as pneumonia, aspiration, inhalation injury, near drowning, or lung contusion as the risk factor for ARDS

^cIndirect ARDS was defined as extrapulmonary sepsis or nonthoracic trauma as the risk factor for ARDS

^dSeverity of ARDS was classified by the “Berlin” definition [22]

^eTwo patients did not have day-1 ratio of arterial oxygen partial pressure to fractional inspired oxyge (P/F) ratios recorded

^fTwo patients did not have day-0 BALF samples (n=84)

^gHighest sequential organ failure assessment score in first 7 days of study

HA levels are associated with measures of local organ injury

We tested for associations between HA levels in serum and BALF and measures of local lung injury severity. Considering the concentration of HA at study enrollment (day 0) and adjusting for age, sex, race, treatment group, and ARDS risk factor, a tenfold increase in day-0 serum HA concentration was associated with an increase of 0.30 in LIS (p=0.04; Fig. 1a and Table 2). A tenfold increase in BALF HA concentration was associated with a 0.27 increase in LIS (p=0.003; Fig. 1b and Table 2). Both serum and BALF HA levels were associated with severity of ARDS as defined by the Berlin criteria (p=0.001; Additional file4). Neither serum (p=0.14) nor BALF (p=0.47) HA levels were associated with quartile of VFDs (Table 2).

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Fig. 1

Compartmentalized hyaluronic acid levels are differentially associated with measures of local and systemic organ injury. Elevated alveolar levels of hyaluronic acid are associated with lung injury score (LIS) (a) but not sequential organ failure assessment (SOFA) score (b), while circulating levels are associated with both (c, d). [HA], concentration of hyaluronic acid. β values represent a change in units of either LIS (a, b) or SOFA (c, d) score per tenfold increase in [HA]. Solid lines represent regression lines determined via linear regression analyses, while hashed lines represent the 95% confidence interval of the regression line

Table 2

Association of early (day 0) hyaluronic acid levels with degree of end organ dysfunction

Outcome	Source	β (95% CI)a	p	β (95% CI)b	p
Lung injury score	Serum	0.35 (0.05–0.65)	0.02	0.30 (0.02–0.57)	0.04
	BALFc	0.31 (0.11–0.50)	0.002	0.27 (0.09–0.45)	0.003
Ventilator-free daysd	Serum	-0.59 (-1.21–0.03)	0.06	-0.47 (-1.10–0.15)	0.14
	BALFc	-0.09 (-0.51–0.33)	0.67	-0.15 (-0.58–0.27)	0.47
SOFAe	Serum	3.89 (2.18–5.61)	<0.001	4.02 (2.30–5.74)	<0.001
	BALFc	0.42 (-0.85–1.69)	0.52	0.72 (-0.56–2.00)	0.27

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SOFA sequential organ failure assessment score, BALF bronchoalveolar fluid,

^aLinear regression between log₁₀-transformed day-0 hyaluronic acid concentration and respective outcome measure (in units of lung injury score, quartiles of ventilator-free days, or units of SOFA)

^bMultiple linear regression adjusted for age, sex, race, treatment group, and acute respiratory distress syndrome etiology

^cTwo patients did not have day-0 BALF samples (n=84)

^dVentilator-free days were analyzed as quartiles, given the skewed distribution

In exploratory analyses, we sought to identify which component(s) of the LIS was responsible for the associations observed with the composite score. We found that both serum and BALF HA levels were strongly associated with the degree of hypoxemia (p=0.02 and p<0.001, respectively) and with the set positive end-expiratory pressure (PEEP) (p=0.01 and p=0.02, respectively; Additional file5 and Table 3). Neither serum nor BALF HA levels were associated with the number of affected quadrants on a chest radiograph (p=0.21 and p=0.53, respectively) or respiratory system compliance (p=0.89 and p=0.69, respectively; Table 3 and Additional file5). These data demonstrate that both circulating and alveolar HA levels are associated with severity of lung injury, primarily through associations with the degree of hypoxemia and the set PEEP.

Table 3

Association of early (day 0) hyaluronic acid levels with components of LIS

Lung injury score componenta	Source	β (95% CI)b	p	β (95% CI)c	p
Chest radiographd	Serum	-0.15 (-0.54–0.24)	0.44	-0.25 (-0.63–0.14)	0.21
	BALFe	-0.05 (-0.30–0.20)	0.69	-0.08 (-0.33–0.17)	0.53
Hypoxemiaf	Serum	0.66 (0.19–1.12)	0.006	0.57 (0.11–1.03)	0.02
	BALFe	0.57 (0.28–0.86)	<0.001	0.56 (0.28–0.85)	<0.001
Positive end-expiratory pressure	Serum	0.77 (0.16–1.38)	0.01	0.76 (0.16–1.35)	0.01
	BALFe	0.57 (0.17–0.96)	0.006	0.46 (0.07–0.85)	0.02
Respiratory system complianceg	Serum	0.005 (-0.35–0.36)	0.98	0.02 (-0.31–0.36)	0.89
	BALFe	0.09 (-0.14–0.32)	0.43	0.04 (-0.17–0.26)	0.69

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LIS lung injury score, BALF bronchoalveolar fluid

^aSee Additional file1: Table S1 for scoring of composite LIS via individual end-organ components

^bLinear regression between log₁₀-transformed day-0 hyaluronic acid concentration and respective outcome measure (in units of LIS)

^cMultiple linear regression adjusted for age, sex, race, treatment group, and acute respiratory distress syndrome etiology

^dOne patient did not have a chest radiograph available to interpret

^eTwo patients did not have day-0 BALF samples (n=84)

^fTwo patients did not have blood gas data available (n=84)

^gNine patients did not have compliance data available (n=77)

Discussion

Previous evidence supports a role for HA in lung injury and remodeling [9–15]. In this study, we built upon prior studies to show that increasing circulating and alveolar HA levels are associated with severity of lung injury, using simultaneously obtained serum and BALF samples from critically ill patients with ARDS. Alveolar HA appears to be primarily associated with respiratory organ dysfunction rather than systemic organ dysfunction, while circulating HA is associated with systemic organ dysfunction. These results suggest compartmentalized effects of HA in ARDS.

The mechanism by which HA contributes to or is a consequence of local lung injury remains to be determined; however, a few possible mechanistic models have been suggested. For example, low molecular weight forms of HA present during tissue injury can initiate inflammatory responses via Toll-like receptors [31]. Indeed, HA forms a network that maintains epithelial cell integrity during homeostasis [7, 8] and breakdown of this network could contribute to extravasation of protein-rich alveolar fluid. Furthermore, hypoxia induces both the production of HA and the activity of hyaluronidase in vitro, leading to its degradation into a low molecular-weight form [13]. We hypothesize that any one of these mechanisms could theoretically contribute to our observed association between alveolar HA concentration and lung injury severity.

In addition to the local effect in the lung, we hypothesize that HA likely has a systemic effect, as evidenced by our observation of an association between circulating HA levels and SOFA score. We found that in addition to respiratory dysfunction, liver, renal, and coagulation dysfunction drove the strong association observed with circulating HA levels and SOFA score. Previously, it has been shown that urinary HA levels predict the development of acute kidney injury [18, 19]. Furthermore, elevated concentrations of HA have been associated with hepatic dysfunction through decreased hepatic clearance [32]. To our knowledge, however, the association between circulating HA and coagulation dysfunction (i.e., thrombocytopenia) has not been reported. More studies are needed to further characterize these newly described relationships.

Although circulating and alveolar HA levels are strongly associated with SOFA score and LIS, respectively, they are not associated with 28-day mortality or VFDs. One potential reason for these findings may be due to the relatively low mortality rate observed in our cohort (15%) compared to previous population studies for all-comers with ARDS (43%), limiting our power to detect an effect and to generalize results to other populations [1–4]. Another is that VFDs may not be the best measure of lung injury severity, as other factors lead to prolonged mechanical ventilation such as neuromuscular weakness and encephalopathy. Additional work is needed to address these issues to better understand our findings.

This study has important limitations. First, our findings linking HA to severity of organ dysfunction were observed in a single population. In order to validate our findings, we would need to obtain serum and BALF from an independent population of patients with ARDS. Second, our findings do not identify the source of HA in the serum or BALF. HA may be released directly from the ECM of the lungs as a result of tissue degradation and/or remodeling or it may enter into the alveolar space because of increased vascular permeability. Our data showing weak positive correlation between circulating and alveolar HA levels at day 0 and the lack of correlation at days 4 and 8 suggest that HA in the two compartments may be somewhat independent. Third, the mortality in the ARDS patients enrolled in the fish oil study (15%) was lower than that observed in recent trials [33, 34]. This discrepancy limits the generalizability of our findings to a broader population of critically ill patients. In light of these limitations, additional studies will be required in order to generalize these results to patients with more severe ARDS, and to perform sampling of the alveolar fluid to see if HA levels increase prior to the development of lung injury in those at risk.

Conclusions

In conclusion, this study found that alveolar HA levels in patients recently diagnosed with ARDS are associated with measures of severity of lung injury—suggesting a local effect—while simultaneous circulating HA levels in these patients are associated with measures of other end-organ damage (SOFA score)—suggesting a systemic effect. These findings suggest a strong link between HA and local and systemic organ injury in ARDS. With further characterization of these conclusions, the measurement of HA concentrations may provide diagnostic or prognostic information for patients with ARDS.

Additional files

Acknowledgements

Abbreviations

Authors’ contributions

AJE assisted with development of the study design, contributed to the acquisition and interpretation of data, and drafted and edited the manuscript. PKB contributed to the interpretation of data and to drafting and editing the manuscript. RS contributed to the acquisition and cataloging of patient samples and assisted with drafting and editing the manuscript. MMW conceived the study, contributed to the acquisition and cataloging of patient samples, developed the study design, and contributed to the acquisition and interpretation of data. He also assisted with drafting and editing the manuscript. CRM conceived the study, developed the study design, and contributed to the acquisition and interpretation of data. She also assisted with drafting and editing of the manuscript. She takes responsibility that this study has been reported honestly, accurately, and transparently, and accepts accountability for the overall work by ensuring that questions pertaining to the accuracy or integrity of any portion of the work are appropriately investigated and resolved. All authors read and approved the final version of this manuscript.

Notes

Footnotes

Contributor Information

Anthony J. Esposito, Email: ude.notgnihsaw.u@99eja.

Pavan K. Bhatraju, Email: ude.notgnihsaw.u@ujartahb.

Renee D. Stapleton, Email: [email protected].

Mark M. Wurfel, Email: ude.notgnihsaw.u@lefruwm.

Carmen Mikacenic, Email: ude.notgnihsaw.u@necakimc.

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