Cytokine 131 (2020) 155086

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Cytokine
journal homepage: www.elsevier.com/locate/cytokine

Short communication

Patients with acute respiratory distress syndrome exhibit increased T

stromelysin1 activity in the blood samples
⁎
Sandeep Arthama,1, Arti Vermaa,1, Andrea Sikora Newsomea, Payaningal R. Somanatha,b,
a
Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia and Charlie Norwood VA Medical Center, Augusta, GA 30912, United States
b
Georgia Cancer Center, Vascular Biology Center and Department of Medicine, Augusta University, Augusta, GA 30912, United States

A R T I C LE I N FO A B S T R A C T

Keywords: Enzyme activity analyses in the blood are expected to be reliable, non-invasive diagnostic as well as prognostic
Stromelysin1 markers to reflect disease progression in acute lung injury (ALI). The objective of the current study was to
MMP3 evaluate the enzymatic activity of stromelysin1 (matrix metalloprotease-3) in the plasma/serum samples col-
ARDS lected from ALI patients compared to the samples collected from healthy controls. Gene expression omnibus
Acute lung injury
(GEO) database analysis indicated a correlation between increased stromelysin1 gene expression and the in-
Biomarker
cidence of ALI in various animal models. Our analysis of patient plasma/serum samples from healthy controls
and ALI patients revealed a significant, 3-fold increase in stromelysin1 activity in ALI plasma/serum compared to
healthy subjects with no difference in stromelysin1 activity between the serum and plasma samples.
Interestingly, no significant difference in stromelysin1 activity between non-smoking and smoking subjects was
observed. These findings provide fundamental information on the potential reliability of stromelysin1 activity
analysis, combined with other biomarkers in development, in blood samples for the early detection of ALI.

1. Introduction ARDS went unrecognized by the clinician. [2]. The probability of

More than five decades have passed since the first formal descrip-
tion of acute lung injury (ALI) and its more severe form, acute re-
spiratory distress syndrome (ARDS). Therapeutic advances are most
centered around supportive care with new drug therapies to halt the
disease progression and promote resolution of injury severely lacking
[1]. Even despite these advances in supportive care, recent studies in-
dicate that ARDS continues to have high mortality with an overall
average of 40% mortality in ARDS patients and significantly increased
mortality with each increase in the ARDS severity category [2,3].
Further, the possibility exists that new pharmacological therapies
would not work in all ARDS cases because of the heterogeneity of the
disease physiology and response limiting the universal application of
certain treatment strategies.
Early diagnosis of ALI is crucial for the initiation of an effective
therapy that enhances patient survival. Even though previous studies
tested several markers for endothelial and epithelial injury, none of
those validated diagnostic biomarkers are currently approved, thus the
ARDS diagnosis heavily relies on the clinical features and chest imaging
[4,5]. According to a recent clinical study, 39.8% of all patients with
clinician recognition of ARDS increased with an increase in the severity
of the disease, higher nurse-to-patient ratios, higher physician-to-pa-
tient ratios, younger patient age, lower PaO2/FIO2 ratio, and the pre-
sence of pneumonia or pancreatitis [2]. This high rate of under-
diagnoses indicates the need for a reliable and readily available
diagnostic marker that can be used by clinicians to recognize ARDS at
an early stage thus prompting timely use of known strategies and re-
ducing mortality.
In our recent study in pursuit of potential therapeutic targets for
ARDS, we made an astute observation that stromelysin1 (matrix me-
talloprotease-3; MMP3) activity correlated with the extent of lung in-
jury in mice, and stromelysin1 inhibition significantly reduced en-
dotoxin-induced ALI [6]. Since the stromelysin1 activity can be
measured in body fluids, we compared stromelysin1 activity in serum/
plasma samples of ARDS patients with that of the healthy volunteer
subjects. In our analysis, a significant 3-fold increase in stromelysin1
activity was observed in the ARDS patient samples. In accordance with
these results, we provide the first evidence that stromelysin1 activity in
patient plasma/serum may be a suitable, non-invasive diagnostic
marker for ALI/ARDS.

⁎
Corresponding author at: Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, HM102 – Augusta University, Augusta, GA 30912,
United States.
E-mail address: sshenoy@augusta.edu (P.R. Somanath).
1
Equal Contribution.

https://doi.org/10.1016/j.cyto.2020.155086
Received 16 April 2019; Received in revised form 28 March 2020; Accepted 30 March 2020
1043-4666/ © 2020 Elsevier Ltd. All rights reserved.
S. Artham, et al. Cytokine 131 (2020) 155086

2. Materials and methods

P-value

0.1990
0.1972
0.6657
0.6123
0.0229
0.8419
0.0147
0.0034
0.0048
0.5363

0.0008
0.0018
0.0009
0.0420
0.0187
Sample size (n) 2.1. Stromelysin1 gene expression analysis from preclinical studies

Gene Expression Omnibus (GEO) is a public data repository of high

throughput gene sequencing and microarrays that allows users to
2–6

analyze the expression profiles of the gene(s) of interest from previously

2
2
6
6
6
3
6
8

3
3
3
6
3
3
performed pre-clinical studies [7]. We searched GEO datasets using the
Lung injured (Mean ± SEM)

keywords “stromelysin1/MMP3 and acute lung injury” or “strome-

lysin1/MMP3 and inflammation”. These searches matched several
studies spanning over a decade from the years 2002–2018. The studies
were arranged chronologically, and the gene expression levels of stro-
0.1592

0.2236
0.1558
0.1652
0.3903
0.3414
238.0
98.75
2.200
69.37
18.94
100.0
65.92
14.77

1.889 melysin1 were downloaded for different groups in all studies. The gene
Stromelysin1 - Gene expression values

expression was compared between control and injury groups using the
±
±
±
±
±
±
±
±
±

±
±
±
±
±
±
student t-test. One-way ANOVA was used in studies with more than two
815.8
206.8
22.80
482.4
296.0
589.2
747.7
145.1
4.004

13.27
2.794
1.679
7.720
8.779
8.623
arms indicating a dose-dependent effect. The mean and standard de-
viation (SD) values are presented for each study.
Control (Mean ± SEM)

−0.04522 ± 0.2201
Chronologically ordered studies over a decade from GEO datasets showing increased stromelysin1 gene expression values with various modes of lung injury.

0.3702 ± 0.08693

2.2. Gene expression analysis based on the model of lung injury and
6.337 ± 0.09207

7.265 ± 0.09699
0.1587

7.416 ± 0.2470
85.47
34.20
1.050
16.88
16.88
16.88
38.99
8.674

7.867 ± 2.961

pathway analysis
±
±
±
±
±
±
±
±
±

All the studies were grouped based on the type of lung injury and
219.9
149.4
24.25
290.8
290.8
290.8
269.8
83.35
4.146

the expression level of stromelysin1 and subsequently compared with

the respective control group. Only pre-clinical studies in mice, rats, or
cell lines were included. The expression of stromelysin1 within 24 h of
MMP3 - Septic neutrophil response to lipopolysaccharide and high mobility group box 1

Intratracheal lipopolysaccharide (IT LPS)-induced lung injury in Rag-1 null mice (day 1)

injury was included for studies that had information and data for var-
ious time points after injury. Pathways, annotations, and biological
Intratracheal lipopolysaccharide (IT LPS)-induced lung injury in WT mice (day 1)

systems (BioSystems) that cite genes for the current profiles were
downloaded from GEO profile data and were arranged based on the
Leukemia inhibitory factor neutralization effect on E. coli pneumonia lung

source and organisms. The sources included KEGG, REACTOME,

Fra-1 deficient lung response to fibrotic agent bleomycin (Fra-1 null)

Wikipathways, and GEO pathway interactions.

Fra-1 deficient lung response to fibrotic agent bleomycin (WT)

2.3. Patient plasma and serum samples

Ventilator-associated lung induced injury sensitive strains
Lipopolysaccharide lung injury + mechanical ventilation

Serum and plasma samples collected from healthy subjects (smoking

and non-smoking) and ARDS patients were obtained from the Discovery
Life Sciences (Los Osos, CA). Experiments were performed based on the
protocol exemption approved by Augusta University’s Institutional
Review Board for de-identified patient samples.
Phosgene effect on lungs (24 hrs.)
Lipopolysaccharide lung injury

2.4. Stromelysin1 activity assay

Ventilator-induced lung injury
Ventilator-induced lung injury

Inflammatory lung injury

The enzymatic activity of stromelysin1 was determined using a

Mechanical Ventilation
Hyperoxic lung injury

fluorescence resonance energy transfer peptide and immunocapture

assay as previously described [6]. Briefly, 50 μg total protein of patient
Mode of Injury

serum/plasma samples were incubated at 4 °C for 2 h with rabbit

polyclonal anti-stromelysin1 antibody (Cat no. sc-6839; Santa Cruz
protein

Biotechnology, Dallas, TX). A/G agarose beads were then added and
allowed to incubate overnight at 4 °C. The beads were then washed and
samples were transferred to a black 96well plate and 100 μL of 2 mmol/
Human
Species

Mouse
Mouse

Mouse
Mouse
Mouse
Mouse
Mouse

Mouse
Mouse
Mouse
Mouse
Mouse

L 5-FAM/QXL 520 fluorescence resonance energy transfer peptide

Rat

Rat

(FRET peptide) (cat no. 60580–01; AnaSpec, San Jose, CA) in assay
2002
2005
2005
2005
2005
2005
2005
2005
2007

2007
2010
2010
2012
2013
2013
Year

buffer was added per well. Negative control wells were included that
contained the FRET peptide without any sample to account for any
degradation and background fluorescence. Plates were incubated for
Altemeier WA et al.
Altemeier WA et al.
Altemeier WA et al.

Rajasekaran S et al.
Rajasekaran S et al.
Aggarwal NR et al.
Aggarwal NR et al.
Jacobson JR et al.

Quinton LJ et al.

15 h at 37 °C, and the relative fluorescence units were read and mon-
Sciuto AM et al.

Nonas SA et al.
Cho HY et al.

Silva E et al.
Ma SF et al.
Ma SF et al.

itored at excitation/emission wavelengths of 485/528 nm in a Synergy

HT multimode microplate fluorescence reader (BioTek, Winooski, VT)
Study

running Gen5 data analysis software.

111849110
111849110

3. Results
10883390
10892593
11471376
11471376
11471376
10828576
10897676
28178955

38520806
66691092
66674612
99875778
1245690
GEO ID

3.1. Stromelysin1 gene expression is enhanced in different types of lung

injury.
Table 1

10.1
10.2
No.

2.1
2.2
3.1
3.2
3.3

8.1
8.2
1

4
5
6

GEO datasets retrieved with the search terms ‘Acute lung injury and

2
S. Artham, et al.
Fig. 1. GEO database analysis on the effect of (A) lipopolysaccharide, (B) mechanical, (C) inflammation, and (D) bleomycin-induced lung injury on stromelysin1
gene expression. The data is presented as Mean ± SD.
stromelysin1/MMP3′ spanned over a decade (2002–2013) were ordered
chronologically (Table 1). These studies were subsequently analyzed for
stromelysin1 gene expression in lung injury animals. Most of the studies
that presented stromelysin1 expression within the first 24 h after injury
showed a significant increase in the stromelysin1 gene expression cor-
relating with ALI. We further grouped these preclinical studies based on
the different modes of ALI including mechanical ventilation, endotoxin/
lipopolysaccharide (LPS), inflammation, and bleomycin-induced ALI.
Studies with various modes of lung injury indicated greater strome-
lysin1 gene expression in injured animals as compared to control in
most of the studies except for mechanical ventilation-induced lung in-
jury. While gene expression might be relevant, it is also important to
assess the enzymatic activity of stromelysin1 in these animal models.
(Fig. 1).
3.2. Stromelysin1 gene expression correlates with inflammation and growth
factor-regulated pathways
Pathways, annotations, and biological systems (BioSystems) that
cite genes for the current profiles were obtained from the GEO datasets
(Table 2). Intriguingly, we not only observed changes in the in-
flammation associated signaling pathways but also changes in several
other pathways including signaling by G-protein coupled receptors
(GPCR), signaling associated with growth factor transactivation, and
many others were involved in stromelysin1 gene expression associated
with lung injury.
3.3. Human serum and plasma samples from the ARDS patients exhibit
significantly higher levels of stromelysin1 activity compared to healthy
subjects
Next, we determined the stromelysin1 activity level in plasma and
serum samples collected from ARDS patients compared to healthy
smoking and non-smoking subjects. Human subject characteristics are
shown in Table 3. Comparison of stromelysin1 activity in blood samples
3
Cytokine 131 (2020) 155086
from healthy human subjects with ARDS patients showed a 3 to 6-fold
increase in stromelysin1 activity in ARDS subjects (Fig. 2A). The stro-
melysin1 activity within the serum and plasma components of blood
samples showed no difference in activity indicating the flexibility of
using either the serum or plasma samples from patients (Fig. 2B).
Smoking is an important confounding factor in the etiology of lung
injury. Analysis of blood stromelysin1 activity in healthy smokers
versus healthy non-smokers showed no difference in the basal strome-
lysin1 activity in the blood of healthy subjects (Fig. 2C).
3.4. Stromelysin1 gene expression is enhanced in inflammatory conditions
in general
GEO datasets retrieved with the search terms ‘inflammation and
stromelysin1/MMP3′ spanned over a decade (2002–2013) were ana-
lyzed for stromelysin1 gene expression in non-pulmonary tissues with
severe inflammation. The majority of these studies indicated elevated
stromelysin1 gene expression associated with inflammatory conditions
such as rheumatoid arthritis, colitis and muscle damage in sepsis.
(Fig. 3). These results indicated that stromelysin1 levels are elevated in
inflammatory conditions in general and hence its specificity as a bio-
marker may only depend on its specific measurements in biological
samples such as the bronchoalveolar lavage.
4. Discussion
The current diagnostic criteria provided by the Berlin definition of
ARDS lack a sensitive biomarker that can be used to diagnose ARDS [8].
Previous studies for biomarkers in ALI/ARDS have utilized multiplex
ELISA to study stromelysin1 plasma expression and have not studied
the utility of stromelysin1 activity as a diagnostic marker [9–13].
Confirmation of the role of stromelysin1 in the pathogenesis of ALI/
ARDS must also be demonstrated by its increased activity and not just
presence. Detailed analysis of several preclinical research data from the
GEO repository database studying lung injury indicated a significant
S. Artham, et al. Cytokine 131 (2020) 155086

Table 2
Linked signaling pathways for stromelysin1 expression during lung injury. Data obtained from GEO profile datasets.
Inflammation associated and extracellular matrix related pathways

BSID Name Source Organism

1473615 IL-17 signaling pathway KEGG Conserved

Biosystem
813210 TNF signaling pathway KEGG Conserved
Biosystem
200269 Rheumatoid arthritis KEGG Conserved
Biosystem
1474480 IL-17 signaling pathway KEGG Mus musculus
1474301 IL-17 signaling pathway KEGG Homo sapiens
1470923 Interleukin-4 and 13 signaling REACTOME Homo sapiens
1458260 Photodynamic therapy-induced WikiPathways
NF-kB survival signaling
1367346 Degradation of the extracellular REACTOME Mus musculus
matrix
1367333 Extracellular matrix organization REACTOME Mus musculus
1270258 Activation of Matrix REACTOME Homo sapiens
Metalloproteinases
1270257 Degradation of the extracellular REACTOME Homo sapiens
matrix
1270244 Extracellular matrix organization REACTOME Homo sapiens
1269318 Signaling by Interleukins REACTOME Homo sapiens
1269310 Cytokine Signaling in Immune REACTOME Homo sapiens
system
1269170 Immune System REACTOME Homo sapiens
812263 TNF signaling pathway KEGG Mus musculus
812256 TNF signaling pathway KEGG Homo sapiens
200310 Rheumatoid arthritis KEGG Mus musculus
200309 Rheumatoid arthritis KEGG Homo sapiens
198900 Matrix Metalloproteinases WikiPathways
198370 Matrix Metalloproteinases WikiPathways

Non-Inflammatory pathways

BSID Name Source Organism

522987 Transcriptional misregulation in cancer KEGG Conserved Biosystem

1367106 EGFR Transactivation by Gastrin REACTOME Mus musculus
1269543 Signaling by GPCR REACTOME Homo sapiens
1269379 Signal Transduction REACTOME Homo sapiens
1367105 Gastrin-CREB signalling pathway via PKC and MAPK REACTOME Mus musculus
1367053 Signaling by GPCR REACTOME Mus musculus
1270259 Collagen degradation REACTOME Homo sapiens
1270247 Assembly of collagen fibrils and other multimeric structures REACTOME Homo sapiens
1270245 Collagen formation REACTOME Homo sapiens
1269593 EGFR Transactivation by Gastrin REACTOME Homo sapiens
711361 Oncostatin M Signaling Pathway WikiPathways
523020 Transcriptional misregulation in cancer KEGG Mus musculus
523016 Transcriptional misregulation in cancer KEGG Homo sapiens
138019 p75(NTR)-mediated signaling Pathway Interaction Homo sapiens
1269592 Gastrin-CREB signalling pathway via PKC and MAPK REACTOME Homo sapiens

increase in the gene expression of stromelysin1 and its correlation with
increased inflammation, GPCR, and growth factor signaling. While the
association of increased stromelysin1 gene expression with pathways
other than inflammation is intriguing, this association could be because
of the different modes of injury and multiple signaling pathways
eventually culminating in a single effect. In agreement with this hy-
pothesis, we have recently shown lung endothelial cells, in addition to
inflammatory cells, to be a major source of stromelysin1 expression
with LPS-induced injury through a novel Akt1-FoxO1/3a signaling
pathway [6]. Interestingly, Akt signaling is also involved in resolution
of lung injury [14]. Therefore, role of stromelysin1 in different stages of
lung injury could be explored in future.
In addition to our demonstration of the clinical utility of strome-
lysin1 as a target for ARDS treatment [6], we also show for the first time
that stromelysin1 activity in the blood samples of ARDS patients as
compared to healthy controls can be utilized as a screening method. We
observed a 2–3-fold increase in stromelysin1 activity in ARDS patients,
and this increase was not observed in healthy smokers. From our
analysis, measurement of stromelysin1 expression and its activity in
human biological fluids such as blood samples may serve as a very
important tool for the early diagnosis of ALI/ARDS. Our study sets the
stage for a large scale analysis of stromelysin1 activity in the body fluids
of ALI/ARDS patients such as the plasma/serum and bronchoalveolar
lavage that will prove whether stromelysin1 activity measurement
could be used as a reliable early screening marker as well as a prog-
nostic marker. Interestingly, Ware et al. previously studied various
biomarkers of several pathways associated with ARDS. These include a
marker of epithelial injury, a marker for endothelial injury as well as
markers for inflammation among others. While this study found that a
two-biomarker panel (RAGE & Ang-2) performed in moderate range
even though outperformed clinician recognition of ARDS. This study
did not include stromelysin1 and hence it would be interesting to ob-
serve a three-biomarker panel in future studies for further improving
the diagnostic range of ARDS [15].
Our study is a pilot study and has several limitations. Firstly, we had
a small cohort and hence we couldn’t obtain diversity within the sample

4
S. Artham, et al. Cytokine 131 (2020) 155086

Table 3
(A) Table showing patient characteristics of age matched healthy subjects used as controls. (B) Table showing patient characteristics of ARDS subjects (whose
samples were used in study) classified under international disease-10 code J80 (ARDS).
A

# Sample ID Sample Date Gender Age Ethnicity Smoke Matrix

1 R313816 4/3/2017 Male 26 Black Yes Lithium Heparin

2 R313824 4/3/2017 Male 21 Black Yes Lithium Heparin
3 N124401 4/6/2017 Male 40 Black No Lithium Heparin
4 R314046 4/6/2017 Male 36 Black No Lithium Heparin
5 R314059 4/6/2017 Female 35 Black No Lithium Heparin
6 R313897 4/4/2017 Male 33 Black Yes Na Heparin
7 R313904 4/4/2017 Male 33 Black Yes Na Heparin
8 DLS17-025697 11/23/2016 Male 37 Black Yes Serum
9 DLS17-025776 11/29/2016 Male 44 Black Yes Serum
10 DLS17-025695 11/29/2016 Male 63 Black No Serum

# Sample ID Sample Date Gender Age Ethnicity Matrix

1 DLS14-32067 6/14/2016 M 33 Black Na Heparin

2 DLS15-18601 3/6/2016 F 38 White Lithium Heparin
3 DLS16-69301 11/16/2016 M 46 Black Lithium Heparin
4 DLS15-18166 1/22/2016 M 27 White Lithium Heparin
5 DLS15-18537 3/2/2016 M 20 White Lithium Heparin
6 DLS14-31892 6/10/2016 M 73 White Serum
7 DLS16-31851 8/4/2016 M 37 White Serum
8 DLS16-32224 9/11/2016 M 44 White Serum
9 DLS15-18413 2/29/2016 F 43 White Lithium Heparin
10 DLS15-17563 11/28/2015 M 41 White Na Heparin

Fig. 2. Stromelysin1 activity in patients’ samples. (A) Plasma and serum samples from ARDS human patients were compared with age-matched controls for stro-
melysin1 activity. Additional wells containing the FRET peptide devoid of any samples was included as negative controls to account for any background fluorescence.
(B) Both serum and plasma of ARDS patients had a significant increase in stromelysin1 activity compared to healthy subjects. (C) Stromelysin1 activity was
comparable between healthy smokers and non-smokers. ‘ns’ - not significant. The data is presented as Mean ± SD.

for greater specificity. Second, we limited this study only to a single stromelysin1 activity analysis cannot be considered a specific marker
biomarker due to encouraging preclinical observation but in the future, for ALI as stromelysin1 gene expression is enhanced in inflammatory
this will be extended to include a few other biomarkers. Third, conditions and tissue injury in general. Finally, our study was limited to

5
S. Artham, et al.
Fig. 3. GEO database analysis indicating changes in stromelysin1 gene expression associated with (A) murine rheumatoid arthritis, (B) human rheumatoid arthritis,
(C) human ulcerative colitis, (D) dextran sodium sulfate-induced murine ulcerative colitis, and (E) the effect of sepsis in mice on skeletal muscle. The data is
presented as Mean ± SD.
a single cohort and included ARDS patients irrespective of the source of
lung injury. In conclusion, this study is first of its kind to study activity
and not just the presence of stromelysin1 in plasma/serum samples of
ARDS patients as a potential diagnostic marker and found a significant
3-fold difference between ARDS and control groups. In spite of these
limitations, the specificity of increased stromelysin1 activity in ARDS
patients could be improved if the same is determined in the bronch-
oalveolar lavage samples collected from these patients. These en-
couraging results recommend larger clinical studies for further scrutiny
of stromelysin1 activity as a potential early screening biomarker in
ARDS patients along with other previously studied biomarkers such as
RAGE, Ang-2 and IL6.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
Funds were provided by the NHLBI grant R01HL103952, NCATS
grant UL1TR002378, Wilson Pharmacy Foundation (intramural) and
Translational Research Initiative grant (intramural). This work has been
6
Cytokine 131 (2020) 155086
accomplished using the resources and facilities at the VA Medical
Center in Augusta, GA. The funders had no role in the study design, data
collection, analysis, and decision to publish the data. The contents of
the manuscript do not represent the views of the Department of Veteran
Affairs or the United States Government.
Author contributions
Conception and design: SA, AV, AN, and PRS; Data production,
analysis, and interpretation: SA, AV, AN, and PRS; writing the manu-
script: SA, AV, AN, and PRS. All authors reviewed the manuscript.
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