Biomarkers Journal Open Access

Keywords

Coronavirus; SARS-CoV-2; Hyaluronan; Intensive care

Abbreviations

ALT: Alanine Transaminase; AST: Aspartate Transaminase; b- HABP: Biotinylated Hyaluronan Binding Protein; BMI: Body Mass Index; BSL2: Biosafety Level; CBC: Complete Blood Count; COVID-19: Coronavirus Disease 2019; CRP: C-Reactive Protein; CT: Computerized Tomography; ELISA: Enzyme Linked Immunosorbent Assay; HABP: Hyaluronan Binding Protein; HRP: Horseradish Peroxidase; ICU: Intensive Care Unit; IQR: Interquartile Range; LD: Lactate Dehydrogenase; PaO₂/FiO₂: Arterial Partial Pressure of Oxygen/Fractional Inspired Oxygen; PBS: Phosphate-Buffered Saline; ROS: Reactive Oxygen Species; RT-PCR: Reverse-Transcription Polymerase Chain Reaction; SAPS3: Simplified Acute Physiology Score 3; SOFA: Sequential Organ Failure Assessment; TE: Thromboembolism

Introduction

Coronavirus disease 2019 (COVID-19) has become a worldwide pandemic and a challenge for clinicians and researchers alike. Improvements in both treatment and knowledge of the pathophysiology has been made but much is still unknown. COVID-19 is characterized by inflammation of the lungs causing hypoxemia and respiratory failure. Autopsy studies have found increased lung weight and lungs filled with clear liquid jelly with the histologic appearance of hyaline membranes [1-5]. Hyaluronan has been found in exudate, plugs and the perialveolar interstitium of the lungs, thereby obstructing alveoli and impairing gas exchange [6]. Respiratory secretions of intubated patients with COVID-19 have a 20-folds higher lowmolecular hyaluronan content compared to that in samples from healthy controls [7]. A meta-analysis of RNA sequencing data of pulmonary cells obtained from bronchoalveolar lavage showed that genes encoding enzymes involved in hyaluronan metabolism are upregulated [8]. This growing evidence indicates a new pathogenic mechanism, where hyaluronan may contribute to hypoxemia and respiratory failure in COVID-19 [1,9]. Recently, it was demonstrated that high levels of circulating hyaluronan are correlated with disease severity of patients with COVID-19 [10]. However, the dynamics of plasma hyaluronan concentration throughout disease has not been studied, and no study has compared plasma hyaluronan levels between non-survivors and survivors. The aim of this study was to describe changes in plasma hyaluronan concentration in COVID-19 patients earlya fter Itnensive Care Unit (ICU) admission and later during ICU stay, and to compare hyaluronan concentrations between 30-day non-survivors and survivors.

Materials and Methods

Participants and data collection

We included COVID-19 patients who were treated in ICU at Uppsala University Hospital, Sweden, for at least 10 days between March 13th and April 16th 2020. Positive Reverse- Transcription Polymerase Chain Reaction (RT-PCR) was used to confirm COVID-19. Simplified Acute Physiology Score 3 (SAPS3) [11] was calculated on admission followed by daily recording of the Sequential Organ Failure Assessment (SOFA) score [12], respiratory (including PaO₂/FiO₂) and circulatory data. At discharge, vital status and time on invasive ventilation, vasopressor support and renal replacement therapy were noted. Information ons econdary infections were also collected. STROBE guidelines were followed for reporting.

Laboratory analysis

Complete Blood Count (CBC), kidney function, liver function, highly sensitive C-Reactive Protein (CRP) and ferritin were recorded on a daily basis as part of routine testing. CBC was analyzed on Sysmex XNTM (Sysmex, Kobe, Japan), while CRP, ferritin, kidney and liver markers were analyzed on Architect ci16200 (Abbott Laboratories, Abbott Park, IL, US). Sodium citrate tubes were collected early, within 2 days after ICU admission and at day 11-16 of ICU stay.

The ELISA-like assay for hyaluronan quantification is based on the specific andi rreversible binding of hyaluronan in the samples to immobilized link domain of aggrecan (also termed Hyaluronan Binding Protein, HABP) [13]. In short, COVID-19 patient´s blood samples after transportation to BSL2 laboratory were centrifuged at 3000 rpm for 10 min, at room temperature, and stored at -80°C. On the day of the assay proceeding, the samples were thawed out gradually on ice, and the virus infectivity was inactivated by heating to 56°C for 1 h. Hyaluronan standards (0 ng/ml-50 ng/ml) and patient samples, at appropriate dilutions inP BS containing 1% bovine serum albumin, were added on Maxisorb 96-well Nunc-Immuno plates precoated with 1 μg/ml HABP in 50 mM carbonate buffer, pH 9.5. After an incubation of 1 h, at 37°C, to allow the formation of HABP-hyaluronan complexes, followed by washing, biotinylated HABP (b-HABP) was added, and samples were incubated an additional 1 h. After washing away excess of b-HABP, the b-HABP bound to the hyaluronan trapped by the HABP-coated plates was detected with streptavidin-biotinylated HRP, followed by the substrate solution (3,3′,5,5′-Tetramethylbenzidine). The color development in the wells was terminated with 2 M H2SO4, and the absorbance at 450 nm was determined (Enspire Multimode Plate Reader, PerkinElmer).

Statistical analysis

All statistical analyses were performed using IBM® SPSS® statistics version 23 (SPSS, Inc., Chicago, IL, USA). Numerical variables were reported as median (Interquartile Range (IQR) or range) and categorical variables as number of observations (percent of total number of observations). The normality of distribution of continuous variables was tested by Kolmogorov- Smirnov test and visually assessed using histograms. Repeated measurements were compared using Wilcoxon signed-rank test. Kruskal-Wallis test was used when 3 groups were compared. Correlations and independent group analyses were carried out using Spearman Rank and Mann-Whitney U test, respectively. A p-value p<.05 was considered statistically significant.

Results

Nineteen patients with COVID-19 admitted to the ICU were included in this study. Median body mass index (BMI) was 30.8 (IQR 27.7-34.3), and all but one patient were men. Twelve patients (63%) had at least one comorbidity, with hypertension or diabetes being the most common. Patients were admitted to the ICU after a mediano f 9 days (IQR 7-11) of COVID-19 symptoms. SOFA scores were similar early and late during ICU stay (7 (IQR 5-7) vs. 7 (IQR 5-9)). Median length of ICU stay was 18 days (IQR 15-22), and the 30-day mortality after ICU admission was 21% (non-survivors n=4). Characteristics including comorbidities ofn on-survivors and survivors are presented in Table 1. Non-survivors and survivors had similar age and SAPS3 scores on admission. All patients at some point received invasive ventilation and vasopressor support (all were treated with noradrenalin), whereas 4 patients received renal replacement therapy (21%). Early blood samples were taken on average 1 day after ICU admission (range 0-2 days) and late samples were taken on average on day 12 in the ICU (range 11-16).

Table 1: Patient demographic characteristics and comorbidities, all patients, non-survivors (30 days) and survivors (30 days).

The median plasma hyaluronan concentrations of the healthy blood donors was 51 ng/ml (range 27-92). Plasma hyaluronan concentrations among patients with COVID-19 were higher early after admission compared with later during ICU stay (108 ng/ml (IQR 40-321) vs. 22 ng/ml (IQR 2-66), p=.001 (Figure 1).

Figure 1: Plasma hyaluronan concentrations (ng/ml) early after admission (n=19), late in the ICU stay (n=19) and healthy blood donors (n=3). Each line represents the dynamics for an individual patient (dashleinde s=non-survivors, solid lines=survivors).

There was no difference in plasma hyaluronan concentration between non-survivors and survivors early after admission, but the concentrations remained higher for non-survivors in late samples (Table 2).

Table 2: Hyaluronan concentration of early vs. late vs. healty blood donors.

Plasma hyaluronan concentrations in late samples from patients who acquired a secondary infection (n=15) were similar compared with those who did not. There was no correlation between early plasma hyaluronan concentration and SAPS3 or SOFA score for the corresponding day, whereas late plasma hyaluronan did not significantly correlate with same-day SOFA score in this small sample. There was no difference in either early or late plasma hyaluronan concentration in patients who received renal replacement therapy compared with those that did not. No correlation was found between either early or late plasma hyaluronan concentration and corresponding liver enzymes, bilirubin and creatinine plasma concentrations (Table 3).

Table 3: Correlation between hyaluronan (early/late) and clinical biochemistry.

The PaO₂/FiO₂ ratio increased over time from early after ICU admission to later during ICU stay (21 (IQR 16-24) vs. 23 (IQR 21-33), p=.034). There was no difference in PaO₂/FiO₂ ratio at admission between non-survivors and survivors, but later during ICU stay the PaO₂/FiO₂ ratio remained low for non-survivors compared with survivors (p=.001). No correlation was found between early and late plasma hyaluronan concentration and corresponding PaO₂/FiO₂ ratio. Furthermore, no difference was found between non-survivors and survivors during late ICU stay for liver enzymes, bilirubin and creatinine plasma concentrations (Table S1).'

Discussion

In this study of 19 critically ill COVID-19 patients, plasma hyaluronan levels decreased during ICU stay among survivors but remained higher among non-survivors. Plasma hyaluronan concentrations of the healthy blood donors in the present study were similar compared with previous studies [14-16]. In the present study, plasma hyaluronan concentration in COVID-19 patients early after ICU admission was high with a considerable variation. An earliers tudy that compared mildly, severely and critically ill patients with COVID-19 found an increase in plasma hyaluronan concentration with COVID-19 severity [10]. In that study, all patients in the critical group were admitted to the ICU and expressed the highest plasma hyaluronan concentration. The reported hyaluronan concentrations were higher compared to our results. However, they used another analytical method and had no healthy controls making direct comparison difficult.

Several mechanisms such as changes in production, transport or clearance can alter plasma hyaluronan concentrations. Hyaluronan is synthesized by three Hyaluronan Synthases (HAS 1-3) at the plasma membrane, and exported into the Extracellular Matrix (ECM). Inflammation increases the hyaluronan production leading to increased hyaluronan levels in the ECM [17,18] causing accumulation of hyaluronan in Acute Respiratory Distress Syndrome (ARDS) [19]. COVID-19 patients express high levels of pro inflammatory cytokines in the lungs [20], which might contribute to the high levels of hyaluronan found in autopsy studies [6] and respiratory secretions [7]. Secondary infections were not associated with increased plasma hyaluronan in our data and therefore we do not believe that additional bacterial or fungal infection in non-survivors is driving the difference.

Hyaluronan is degraded by Reactive Oxygen Species (ROS) and specific hyaluronidases in tissues and lymphatic pathways [21]. Normal lymphatic function is necessary to maintain a balance between production and clearance of hyaluronan. A substantial amount of hyaluronan is turned over in the lymphatic system itself, while the remaining hyaluronan is transported into the circulation [22]. Mediastinal lymph node enlargement is not considered a typical CT finding in COVID-19 patients, but lymphadenopathy may be underreported in radiological studies [23]. Autopsy reports of COVID-19 patients have shown pathological changes in hilar lymph nodes including congestion, which may be consistent with high hyaluronan concentrations. Changes in the lymphatic system may thus occur in COVID-19 and might contribute to stagnation of hyaluronan in the lungs [5,24]. The removal of hyaluronan from the circulation is very efficient and primarily takes place in the liver by sinusoidal endothelial cells. Plasma hyaluronan concentration is elevated in liver failure, and is a good marker for disease severity [25]. COVID-19 is associated with elevated liver enzymes and liver dysfunction, particularly in critically ill patients [26]. This study found no correlation between plasma hyaluronan concentration and liver enzymes, neither early after admission at the ICU nor at later stay, making it unlikely that liver dysfunction was affecting plasma hyaluronan concentration in this cohort. In addition, some hyaluronan is cleared in the kidneys but since no association was found with this probably is not an important factor in the present study.

The plasma hyaluronan concentrations reported are relatively low compared to other infectious such as influenza A, sepsis and dengue fever [14,27,28]. However, plasma concentration might not accurately reflect interstitial levels of hyaluronan. Accumulation of hyaluronan in tissues with limited lymph drainage, such as the alveolar space or changes in lymphatic flow, may result in stagnation of hyaluronan and diminished transport of hyaluronan into the circulation.

Our study had several limitations. The number of patients in this study is limited and this may increase the risk of type II error. Yet given the pilot nature of this study many of these findings are important to report. This study included patients that had been treated at the ICU for at least 13 days, thus excluding patients discharged from the ICU before day 13 and patients that died before day 13. Measuring plasma hyaluronan levels in these patients may yield additional insights.

Conclusion

This is the first study describing dynamics of plasma hyaluronan concentration in critically ill COVID-19 patients. The findings showing high plasma hyaluronan at ICU admission that do not decrease in patients who die, but normalize in survivors. Neither secondary infection, liver-nor renal failure were associated with differences in late plasma hyaluronan concentrations. Our finding strengthens the growing evidence that hyaluronan may play a role in the pathogenesis and severity of disease of respiratory failure in COVID-19. Further studies are warranted to confirm these findings and to determine which mechanisms contribute to the increased hyaluronan concentrations in plasma and the lungs, and determine if these may be amenable to pharmacological treatment.

Ethics Approval and Consent to Participate

The present study is a sub study of a prospective observational cohort study approved by the National Ethical Review Agency (COVID-19 patient samples; EPM; no. 2020-01623) and by the Regional Ethics Committee at Uppsala University (healthy blood donor samples; Ups-01-367). Informed consent was obtained from the patient or next of kin if the patient was unable to give consent. The declaration of Helsinki and its subsequent revisions were followed.

Consent for Publication

Not applicable

Availability of data and materials

The data that support the findings of this study are available on request from the corresponding author, JvdH. The data are not publicly available due to containing information that could compromise the privacy of research participants.

Competing interests

The authors declare that they have no competing interests

Funding

The study was funded by the SciLifeLab/KAW National COVID-19 research program project grant to MH (KAW 2020.0182); the Swedish Research Council to RF (2014-02569 and 2014-07606) and to MKM (2020-02258).

Authorship contributions

All authors participated in conception and design of the study. CK performed biochemical analyses. All authors had access to the data and participated in data collection and interpretation. JvdH drafted the manuscript, and all authors contributed to manuscript revision. All authors read and approved the final manuscript.

Acknowledgements

The authors thank research nurses Joanna Wessbergh and Elin Söderman, and the biobank assistants Erik Danielsson and Philip Karlsson for their expertise in compiling the study.

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