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PEGylated Serp-1 Markedly Reduces Pristane-Induced Experimental Diffuse Alveolar Hemorrhage, Altering uPAR Distribution, and Macrophage Invasion

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Diffuse alveolar hemorrhage (DAH) is one of the most serious clinical complications of systemic lupus erythematosus (SLE). The prevalence of DAH is reported to range from 1 to 5%, but while DAH is considered a rare complication there is a reported 50-80% mortality. There is at present no proven effective treatment for DAH and the therapeutics that have been tested have significant side effects. There is a clear necessity to discover new drugs to improve outcomes in DAH. Serine protease inhibitors, serpins, regulate thrombotic and thrombolytic protease cascades. We are investigating a Myxomavirus derived immune modulating serpin, Serp-1, as a new class of immune modulating therapeutics for vasculopathy and lung hemorrhage. Serp-1 has proven efficacy in models of herpes virus-induced arterial inflammation (vasculitis) and lung hemorrhage and has also proved safe in a clinical trial in patients with unstable coronary syndromes and stent implant. Here, we examine Serp-1, both as a native secreted protein expressed by CHO cells and as a polyethylene glycol modified (PEGylated) variant (Serp-1m5), for potential therapy in DAH. DAH was induced by intraperitoneal (IP) injection of pristane in C57BL/6J (B6) mice. Mice were treated with 100 ng/g bodyweight of either Serp-1 as native 55 kDa secreted glycoprotein, or as Serp-1m5, or saline controls after inducing DAH. Treatments were repeated daily for 14 days (6 mice/group). Serp-1 partially and Serp-1m5 significantly reduced pristane-induced DAH when compared with saline as assessed by gross pathology and H&E staining (Serp-1, p = 0.2172; Serp-1m5, p = 0.0252). Both Serp-1m5 and Serp-1 treatment reduced perivascular inflammation and reduced M1 macrophage (Serp-1, p = 0.0350; Serp-1m5, p = 0.0053), hemosiderin-laden macrophage (Serp-1, p = 0.0370; Serp-1m5, p = 0.0424) invasion, and complement C5b/9 staining. Extracellular urokinase-type plasminogen activator Guo et al. Serpin Reduces Lupus Lung Hemorrhage receptor positive (uPAR+) clusters were significantly reduced (Serp-1, p = 0.0172; Serp-1m5, p = 0.0025). Serp-1m5 also increased intact uPAR+ alveoli in the lung (p = 0.0091). In conclusion, Serp-1m5 significantly reduces lung damage and hemorrhage in a pristane model of SLE DAH, providing a new potential therapeutic approach.
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ORIGINAL RESEARCH
published: 16 February 2021
doi: 10.3389/fcvm.2021.633212
Frontiers in Cardiovascular Medicine | www.frontiersin.org 1February 2021 | Volume 8 | Article 633212
Edited by:
Klaus T. Preissner,
University of Giessen, Germany
Reviewed by:
Margarethe Geiger,
Medical University of Vienna, Austria
Djuro Kosanovic,
I.M. Sechenov First Moscow State
Medical University, Russia
*Correspondence:
Liqiang Zhang
liqiang.zhang@asu.edu
Alexandra R. Lucas
arlucas5@asu.edu
Specialty section:
This article was submitted to
Atherosclerosis and Vascular
Medicine,
a section of the journal
Frontiers in Cardiovascular Medicine
Received: 24 November 2020
Accepted: 20 January 2021
Published: 16 February 2021
Citation:
Guo Q, Yaron JR, Wallen JW III,
Browder KF, Boyd R, Olson TL,
Burgin M, Ulrich P, Aliskevich E,
Schutz LN, Fromme P, Zhang L and
Lucas AR (2021) PEGylated Serp-1
Markedly Reduces Pristane-Induced
Experimental Diffuse Alveolar
Hemorrhage, Altering uPAR
Distribution, and Macrophage
Invasion.
Front. Cardiovasc. Med. 8:633212.
doi: 10.3389/fcvm.2021.633212
PEGylated Serp-1 Markedly Reduces
Pristane-Induced Experimental
Diffuse Alveolar Hemorrhage,
Altering uPAR Distribution, and
Macrophage Invasion
Qiuyun Guo 1,2 , Jordan R. Yaron1, John W. Wallen III 3, Kyle F. Browder 1, R yan Boyd 4,
Tien L. Olson 4, Michelle Burgin 1, Peaches Ulrich 1, Emily Aliskevich 1, Lauren N. Schutz 1,
Petra Fromme 4, Liqiang Zhang 1
*and Alexandra R. Lucas 1
*
1Center for Personalized Diagnostics and Center for Immunotherapy, Vaccines and Virotherapy, The Biodesign Institute,
Arizona State University, Tempe, AZ, United States, 2Department of Oncology, Tongji Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan, China, 3Exalt Therapeutics LLC, Las Vegas, NV, United States,
4Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, AZ, United States
Diffuse alveolar hemorrhage (DAH) is one of the most serious clinical complications of
systemic lupus erythematosus (SLE). The prevalence of DAH is reported to range from
1 to 5%, but while DAH is considered a rare complication there is a reported 50–80%
mortality. There is at present no proven effective treatment for DAH and the therapeutics
that have been tested have significant side effects. There is a clear necessity to discover
new drugs to improve outcomes in DAH. Serine protease inhibitors, serpins, regulate
thrombotic and thrombolytic protease cascades. We are investigating a Myxomavirus
derived immune modulating serpin, Serp-1, as a new class of immune modulating
therapeutics for vasculopathy and lung hemorrhage. Serp-1 has proven efficacy in
models of herpes virus-induced arterial inflammation (vasculitis) and lung hemorrhage
and has also proved safe in a clinical trial in patients with unstable coronary syndromes
and stent implant. Here, we examine Serp-1, both as a native secreted protein expressed
by CHO cells and as a polyethylene glycol modified (PEGylated) variant (Serp-1m5),
for potential therapy in DAH. DAH was induced by intraperitoneal (IP) injection of
pristane in C57BL/6J (B6) mice. Mice were treated with 100 ng/g bodyweight of either
Serp-1 as native 55 kDa secreted glycoprotein, or as Serp-1m5, or saline controls
after inducing DAH. Treatments were repeated daily for 14 days (6 mice/group). Serp-1
partially and Serp-1m5 significantly reduced pristane-induced DAH when compared
with saline as assessed by gross pathology and H&E staining (Serp-1, p=0.2172;
Serp-1m5, p=0.0252). Both Serp-1m5 and Serp-1 treatment reduced perivascular
inflammation and reduced M1 macrophage (Serp-1, p=0.0350; Serp-1m5, p=0.0053),
hemosiderin-laden macrophage (Serp-1, p=0.0370; Serp-1m5, p=0.0424) invasion,
and complement C5b/9 staining. Extracellular urokinase-type plasminogen activator
Guo et al. Serpin Reduces Lupus Lung Hemorrhage
receptor positive (uPAR+) clusters were significantly reduced (Serp-1, p=0.0172;
Serp-1m5, p=0.0025). Serp-1m5 also increased intact uPAR+alveoli in the
lung (p=0.0091). In conclusion, Serp-1m5 significantly reduces lung damage
and hemorrhage in a pristane model of SLE DAH, providing a new potential
therapeutic approach.
Keywords: lupus, diffuse alveolar hemorrhage, immune modulator, Serp-1, inflammation, recombinant protein
therapeutic, vasculitis
INTRODUCTION
Systemic lupus erythematosus (SLE), or lupus, is an autoimmune
disease characterized by immune cell hyperactivity, production
of antibodies against self-antigens, such as double-stranded (ds)
DNA, histones, and ribonucleoprotein (RNP). The etiology of
SLE is only partially defined and has been linked to abnormal
genetic, hormonal, and environmental responses (14). The
incidence of this disease is 20–70 per 100,000 people, and the
incidence in women is 6–10 times that of men. Patients with
SLE have a wide range of clinical symptoms, including skin
rash, nephritis, non-erosive arthritis, serositis, cardiovascular
involvement, hematological, and respiratory disorders (with
pulmonary fibrosis and hypertension). In some cases (1) The
most serious clinical manifestation of SLE is diffuse alveolar
hemorrhage (DAH), with prevalence ranging from 1 to 5%, but
causing >50–80% mortality in affected SLE patients (24). Lupus
DAH is characterized by neutrophilic capillaritis with destruction
of alveolar septae and infiltration of hemosiderin-laden
macrophages (58). The current treatment options for DAH
include steroids, cyclophosphamide, rituximab, methotrexate,
azathioprine, respiratory support, and among others. The efficacy
of these treatments is limited and there are many significant side
effects, that include hypertension, diabetes, osteoporosis, adrenal
suppression, infertility, pulmonary fibrosis, hepatotoxicity, and
risk for future malignancies. Therefore, there is an urgent, unmet
need for new drugs to improve treatment for DAH.
Pristane (2, 6, 10, and 14 tetramethylpentadecane, TMPD)
is an isoprenoid alkane found at high concentration in mineral
oil, and in low concentration in vegetables (9). It is also found
in the liver of some sharks (10). Intraperitoneal (IP) injections
of pristane can induce in mice a wide range of autoantibodies
specific to, or associated with, SLE (11,12), making pristane
an accepted method to establish mouse models of SLE. IP
injection of pristane in C57BL/6J (B6) mice causes severe
alveolar hemorrhage within 2 weeks, manifested by alveolar and
perivascular inflammation (capillaritis, small vessel vasculitis),
endothelial injury and hemorrhage (7,1315). Many previous
studies have proven that this model can closely simulate the
pathological process of DAH.
Serp-1 is a purified 55 kDa secreted glycoprotein originally
derived from MYXV, belonging to the SERPIN superfamily.
Our previous research has demonstrated that purified Serp-
1 protein treatment is beneficial in a wide range of immune
mediated disorders, from arthritis to vasculitis to transplant (16
21). Serp-1 reduces macrophage cell infiltration into transplanted
hearts, kidneys and aorta in rodent models, with improved
histopathological evidence of acute and chronic rejection (16,17,
22). In a mouse model of inflammatory vasculitis induced by
mouse gamma herpesvirus-68 (MHV-68) infection in interferon
gamma receptor deficient mice (IFNγR/) and also in an
aortic transplant model, Serp-1 significantly reduced arterial
inflammation and plaque growth. Additionally, Serp-1 treatment
reduced lung hemorrhage and consolidation and improved
survival in mouse gamma herpesvirus-68 (MHV68) infected
mice, a model for inflammatory vasculitis and lethal lung
hemorrhage (20,21). In clinical trials, Serp-1 treatment proved
safe and significantly reduced markers for myocardial damage
after coronary stent implant in phase I and IIa clinical trials
in patients with unstable angina pectoris or non-ST elevation
myocardial infarction (NSTEMI), with no significant major
adverse reactions (MACE =0) and no neutralizing antibody
detected (23).
Urokinase type plasminogen activator (uPA) binds to the uPA
receptor (uPAR). The uPA/uPAR complex sits at the leading, or
invading, edge of inflammatory macrophage cells. In addition
to a role in thrombolysis, the uPA/uPAR complex also activates
plasmin which in turn activates matrix metalloproteinases
(MMPs). MMPs break down connective tissue (collagen and
elastin), to allow immune cells to infiltrate tissues. The uPA/uPAR
complex thus functions both in fibrinolysis and in inflammatory
cell activation and invasion, the latter being considered the
predominant function. Serp-1 binds and inhibits thrombolytic
protease, tissue- and urokinase-type plasminogen activators (tPA
and uPA, respectively) as well as thrombotic proteases, thrombin
and factor Xa. Serp-1 binds to the uPA/ uPAR complex on
the macrophage plasma membrane surface (24,25). Serp-1
inhibition of macrophage migration is dependent upon uPAR
expression in vitro in monocytes and in vivo in the aortic
transplant model. Serp-1 efficacy was previously found to be
dependent on uPAR expression in the donor aorta in aortic
transplant models in mice (18,24,26). Serp-1 treatment is thus
projected to either reduce excess thrombolysis, or to rebalance
an imbalance in both thrombotic and thrombolytic cascades,
and to reduce inflammation in this SLE lung hemorrhage model.
In previous studies another member of the Serpin superfamily,
Alpha-1-antitrypsin (AAT), has shown anti-inflammatory and
immunomodulatory functions, inhibiting the activation and
recruitment of inflammatory cells when given for 1 week prior
to pristane injection. Human AAT (hAAT) reduced the severity
of DAH in B6 mice; hAAT transgenic mice completely prevented
DAH induced by pristane (27).
The half-life for Serp-1 in circulating blood was 20 min in
clinical trial up to 1.36 days in mouse and rabbit models, and
Frontiers in Cardiovascular Medicine | www.frontiersin.org 2February 2021 | Volume 8 | Article 633212
Guo et al. Serpin Reduces Lupus Lung Hemorrhage
is dependent upon the model examined (23,28). PEGylation
has been demonstrated to improve the half-life and reduce
antigenicity in prior work with other proteins (29). In this
study, we examined treatment with either PEGylated Serp-1, here
termed Serp-1m5, or with the native non-PEGylated secreted
Serp-1 in the SLE DAH mouse model for efficacy and compared
to Saline control alone. Based on prior studies, we have postulated
that Serp-1 will prove effective and safe for the treatment of DAH.
MATERIALS AND METHODS
Proteins and Chemicals
Serp-1 (m008.1L; NCBI Gene ID# 932146) was expressed in a
Chinese hamster ovary (CHO) cell line (Viron Therapeutics Inc.,
London, ON, CA). The Serp-1 protein used in this research is
GMP-compliant and purified by continuous chromatographic
separation. The purity of Serp-1 is >95%, as determined by
Coomassie stained SDS-PAGE and reverse-phase HPLC. Serp-
1 was endotoxin-free by LAL (limulus amebocyte lysate) assay.
Serp-1 was incubated with mPEG-NHS (5 K) (Nanocs Inc.,
#PG1-SC-5k-1, NY) in PBS buffer (pH 7.8) at 4C overnight
to modify the protein according to standard PEGylation
protocols. PEGylated Serp-1 (Serp-1m5) was purified by
FPLC using an ÄKTA pure protein purification system
with Superdex-200.
Hematoxylin and eosin for H&E staining and trichrome
reagents were from Sigma-Aldrich. Information about each
antibody used for immunohistochemical staining in this study is
provided below when first mentioned.
Animals
All animal procedures in this study were approved by the
Institutional Animal Care and Use Committee of Arizona State
University under protocol #20-1761R and conform to national
and international guidelines for animal care. Eighteen wild-type
female C57BL6/J mice aged 6–8 weeks old were treated with
pristane. Female mice are reported in prior studies to be preferred
for the development of DAH model (13,27). Each mouse was
injected with 0.5 ml of pristane (Sigma-Aldrich) intraperitoneally
(IP) at day 0. These mice were then randomly divided into three
groups, i.e., saline, Serp-1 or Serp-1m5 treatment groups (6 mice
each group, n=6). Six normal mice were also examined, without
pristane or Serp-1, and six mice had Serp-1 treatment without
pristane. No adverse effects were seen [Toxicity for Serp-1 has
been extensively tested and proven to be minimal in preclinical
and clinical trials, as previously reported (1621,2326)]. Each
mouse was given one IP injection of 100 µL saline or 100 ng/g
bodyweight of clinical grade Serp-1 or Serp-1m5 in 100 µL of
Saline after pristane induction. The treatments were repeated
every day until the 14th day. The mice were euthanized by CO2
asphyxiation on the 15th day and lung tissues were divided; one
half was frozen at 80C for later protein analysis, and one half
fixed in 10% neutral-buffered formalin for at least 3 days before
processing and paraffin embedding. There were no early deaths
or complications in any treatment group.
Lung Pathological Evaluation
DAH in lung specimens was initially assessed by gross
observation of excised lungs, prior to either fixation or freezing.
Lung tissues were fixed in 10% neutral-buffered formalin after
collection and then processed in a Leica TP1050 tissue processor
and embedded in paraffin with a Leica EG1160 embedding
station, as previously described (2126). Tissue blocks were cut
into 5 µm sections using a Leica RM2165 microtome.
Sections were stained with hematoxylin and eosin (H&E) and
by Masson’s trichrome using standard procedures, as previously
described (2127,30). DAH was classified into three degrees
of severity according to the percentage of hemorrhage on
H&E stained sections as assessed by a blinded histological
analysis of DAH score as follows: (1) No hemorrhage (0%);
(2) Partial hemorrhage (25–75%); (3) Complete hemorrhage
(75–100%). Prussian blue staining (Electron Microscopy Science
company) was performed with standard protocol to analyze the
hemorrhage status.
Sections were additionally stained for immunohistochemical
analysis (IHC) for CD3 (Abcam, ab6590, 1:100), CD4 (Abcam,
ab183685, 1:1,000), Ly6G (Invitrogen, 14-5931-82, 1:100),
arginase-1 (Cell Signaling, 93668, 1:200), iNOS (Abcam, ab15323,
1:100), C5b/9 antibody (Abcam, ab 55811), and uPAR (R&D
Systems, AF534,1:100). HRP-conjugated secondary antibodies
against rabbit or goat IgG were applied at a dilution of 1:500 for
1 h at room temperature. HRP-conjugated secondary antibody
given alone without primary antibody was used as negative
control for each stain. Antigens were revealed with ImmPACT
DAB (Vector Labs, USA), counterstained with Gil’s formula #3
Hematoxylin and mounted with Cytoseal XYL.
Slides were examined and images collected as objective-
calibrated TIFFs on an Olympus BX51 upright microscope
equipped with an Olympus DP74 color CMOS high-resolution
camera operated by cellSens Dimensions v1.16. Images
(Olympus, Waltham, MA, USA). Images were subsequently
analyzed live and processed in ImageJ/FIJI. Positively stained
cells were counted per high power field for each group; three
high power fields examined per mouse and lung section.
Lung Tissue Protein Extraction and
Analysis
For each mouse, lungs were collected after euthanasia at 15
days post-pristane injection. One lung was fixed in neutral
buffered formalin for later histological analysis. The other lung
was frozen at 80C for later biochemical assays. Half of the
collected frozen tissue was homogenized as whole lung tissue
with a blade homogenizer into 400 µL RIPA with EDTA buffer
containing 1×protease inhibitor cocktail (Bimake, Houston,
TX, USA, #B14001) and 1 mM phenylmethanesulfonyl fluoride
(PMSF) on ice. Homogenized samples were rotated at 4C for
1 h and centrifuged at 13,000×g for 15 min at 4C. Supernatant
containing total protein was transferred to a new tube for
ELISA assays.
Half of the frozen lung tissue was homogenized into 400 µL
PBS buffer containing protease inhibitors cocktail, PMSF, and
1 mM EDTA. Homogenized samples were processed by two
Frontiers in Cardiovascular Medicine | www.frontiersin.org 3February 2021 | Volume 8 | Article 633212
Guo et al. Serpin Reduces Lupus Lung Hemorrhage
cycles freeze-thaw, followed by centrifugation at 13,000×g at
4C for 15 min. Supernatant without membrane proteins was
then collected for cell membrane free soluble uPAR (csuPAR)
protein analysis.
Elisa Assays
uPAR (R&D Systems, DY531) levels in lung tissues were
quantified with ELISA kits following manufacturer’s instructions.
Quantified protein level of lung tissue was normalized to total
protein, which was determined with the BCA (bicinchoninic
acid) protein assay kit (Thermo Fisher Scientific, #23227).
Mass Spectrometry Analysis of C3 Binding
by Serp-1
For Serp-1 interactome analysis, 100 µL of streptavidin magnetic
beads (Thermo Scientific, #88816) were washed, resuspended in
1 mL EBC buffer (50 mM Tris-HCl, 120 mM NaCl, 0.5% NP-40,
pH 8.0) with 1.0% BSA. Ten microliters of biotin-labeled Serp-
1 antibody (AxB7.9-biotin) was added and the mixture rotated
at RT (20C) for 2 h. Beads were washed in EBC, resuspended
in 500 µL EBC buffer with 2% BSA and 20 µg Serp-1 added,
and incubated with 250 µL plasma at 4C overnight followed
by buffer wash. Total binding proteins were collected by boiling
the beads with 40 µL of 6 ×SDS reducing dye. SDS-PAGE was
performed, and bands cut from gel for interactome analysis by
mass spectrometry (MS) at the ASU/Biodesign MS center.
Flow Cytometry Analysis of Splenocytes
Spleens were isolated from mice and cells dissociated in
ice-cold RPMI-1640 containing 20% FBS using a 70 µm cell
strainer for immediate Flow cytometry analysis. Red blood cells
were lysed using RB C lysis buffer (155 mM NH4Cl, 12 mM
NaHCO3, 0.1 mM EDTA) for 10 min at room temperature and
pelleted splenocytes were washed with RPMI-1640 containing
20% FBS. Splenocytes were deposited into 96-well round-
bottom polystyrene plates (106splenocytes per well). Cells
were either directly stained as follows or stimulated with
Cell Activation Cocktail (Biolegend) in the presence of 1X
Brefeldin A (Biolegend) for 90 min prior to staining. Cells
were blocked on ice with TruStain FcX anti-mouse CD16/32
Fc receptor blocker (Biolegend) for 10 min and stained with
eBioscience Fixable Viability Dye eFluor780 (Thermo Fisher),
according to manufacturer’s procedure. Surface markers were
stained for 30 min at 4C at manufacturer’s recommended
dilution in 3% BSA/PBS. Cells were fixed and permeabilized
using the eBioscience Foxp3 Transcription Factor staining
buffer kit (Thermo Fisher) for 1 h according to manufacturer’s
procedure. Intracellular markers were stained for 30 min
at 4C at manufacturer’s recommended dilution in 3%
BSA/PBS. Antibodies used were: CD4-PE/Cy7 (clone RM4-
5, Biolegend), CD8-BV480 (clone 53-6.7, BD Biosciences),
NK1.1-SB600 (clone PK136, Thermo Fisher), FoxP3-eFluor450
(clone FJK-16s, Thermo Fisher), IFNγ-APC (clone XMG1.2,
Biolegend), GATA3-BV711 (clone L50-823, BD Biosciences),
RoRγt-BV650 (clone Q31-378, BD Biosciences), CD11c
(clone BV480, BD Biosciences), CD11b (clone M1170, BD
Biosciences), F4/80(clone BV711, Biolegend), and CD163 (clone
PercCP/eF710, Thermo).
Cells were analyzed on an Attune NxT with autosampler
(Thermo Fisher) by the ASU Knowledge Enterprise Core
Research Flow Cytometry facility and data were processed
with FlowJo v10.
Statistical Analysis
Graphing and statistical analysis were performed using
GraphPad Prism v8.4.3 (GraphPad Software, San Diego,
CA, USA). Mean values were calculated for each analysis
and are presented as mean ±SEM. Differences between
groups were compared using analysis of variance (ANOVA),
Fishers LSD (least significant difference) secondary analysis
and unpaired Student’s T-test. P<0.05 were considered
significant, represented in the figures as p<0.05, ∗∗p<0.01,
and ∗∗∗p<0.001.
RESULTS
Native Serp-1 and PEGylated Serp-1
(Serp-1m5) Treatment Reduce
Pristane-Induced DAH in C57/BL/6 SLE
Mouse Model
When each mouse was euthanized by CO2inhalation, the lungs
were immediately collected and imaged by a digital camera,
before further processing (Figure 1A). All mice survived to 15
days post-pristane injections. According to the severity of the
hemorrhage observed on gross pathology specimens, the lungs
were divided into four grades: severe, moderate, mild, and no
bleeding. As shown in Figure 1B, the whole lung pathology
specimens demonstrate severe DAH in the saline treatment
group, 5/6 severe DAH (5/6, N=6) and 1 moderate DAH
(1/6; N=6). The Serp-1 treatment group contained five severe
DAH (5/6; N=6) and one non-DAH (1/6; N=6). In the
Serp-1m5 treatment group, three cases were severe (3/6; N=
6), two cases were mild (2/6; N=6), and one case had no
DAH (1/6; N=6) (Figure 1;p=0.2677). No hemorrhage
was detected in normal healthy mice with or without Serp-1
treatment after euthanasia (Data not shown). Further evaluation
of the hemorrhage was based on H&E staining and Prussian blue
staining. Four 20 ×fields were examined in randomly selected
areas on the H&E sections for each mouse and scored according
to the degree of bleeding as follows: 0, no hemorrhage; 1, 0–25%
hemorrhage; 2, 25–50% hemorrhage; 3, 50–75% hemorrhage;
4, 75–100% hemorrhage. Representative histology images are
presented in Figure 2A. The average DAH score for each
mouse was calculated. Measurements were performed by two
independent experimenters blinded to the treatments given to
pristane injected mice. On histological examination, the DAH
score of the Serp-1 treatment group indicates a trend toward a
reduction and the DAH score for the Serp-1m5 treatment group
is significantly lower than that of the saline group (Figure 2B;
Serp-1, p=0.2172; Serp-1m5, p=0.0252). Prussian blue
staining was used to detect hemosiderin laden macrophages.
Serp-1 and Serp-1m5 treatments both significantly reduced
Frontiers in Cardiovascular Medicine | www.frontiersin.org 4February 2021 | Volume 8 | Article 633212
Guo et al. Serpin Reduces Lupus Lung Hemorrhage
FIGURE 1 | Serp-1 and its reengineered derivative Serp-1m5 reduce the prevalence of hemorrhage in pristane induced DAH mouse model. All mice induced by
pristane were euthanized by CO2on the 15th day of treatments with saline (6 mice), Serp-1 (6 mice), or Serp-1m5 (6 mice). Whole lungs from mice in each group
were collected (A) and examined based on four grades of hemorrhage: severe, moderate, mild, or none (B).
FIGURE 2 | Histological analysis of DAH during treatment with saline control (N=6 mice), GMP Serp-1 (N=6 mice), or Serp-1m5 (N=6 mice) proteins given daily.
(A) Representative micrograph images of H&E stained lung sections at 14 days follow up. (B) DAH scores were evaluated by taking four 20×field of each section,
and calculating the average score as standardized: 0, no hemorrhage; 1, 0–25% hemorrhage; 2, 25–50% hemorrhage; 3, 50–75% hemorrhage; 4, 75–100%
hemorrhage. The DAH score of Serp-1m5 is significantly lower than that of the saline group (p=0.0252). (C) Representative micrographs of Prussian blue staining at
14 days for each treatment group. (D) Both Serp-1 (p=0.0370) and Serp-1m5 (p=0.0424) significantly reduced detected hemosiderin laden macrophage counts in
lungs form the pristane induced DAH model. *p<0.05.
detected hemosiderin laden macrophage cells when they were
compared to the saline treatment group (Figures 2C,D; Serp-1,
p=0.0370; Serp-1m5, p=0.0424). On H&E stained sections,
there was a significant reduction in perivascular mononuclear
cell infiltrates with Serp-1m5 treatments and a trend for Serp-
1 treatment (Figure 3) (ANOVA, P=0.0104; Serp-1m5, p=
0.0026, Serp-1, p=0.1215). Trichrome staining revealed a trend
toward a reduction in collagen or fibrous tissue staining around
areas of excess hemorrhage, with both Serp-1m5 and Serp-1
treatments (Figure 4).
Serp-1 and Serp-1m5 Treatment Reduced
M1 Macrophages and Neutrophils in
Pristane-Induced DAH Model
The DAH in the pristane induced mouse model is reported
to be macrophage dependent (13). We characterized the
proinflammatory M1 macrophage polarization in the mouse
lung tissue sections by IHC staining for iNOS (iNOS+). As
shown in Figure 5, the lungs of mice treated by Serp-1 and
Serp-1m5 treatments had significantly lower numbers of iNOS+
Frontiers in Cardiovascular Medicine | www.frontiersin.org 5February 2021 | Volume 8 | Article 633212
Guo et al. Serpin Reduces Lupus Lung Hemorrhage
FIGURE 3 | Saline treated mice had marked perivascular mononuclear cell infiltrates after pristane induction DAH (A). Serp-1 treatment produced a non significant
reduction in perivascular inflammatory cell counts (B). Serp-1m5 treatment significantly reduced perivascular inflammatory cell counts when compared to the saline
treated controls (C). Perivascular mononuclear cell counts ±SEM are illustrated in panel (D). *p<0.05.
FIGURE 4 | Trichrome staining indicated a nonsignificant increase in collagen
staining in areas of hemorrhage in the saline treated controls. Serp-1 and
Serp-1m5 treatment produced a non-significant decrease in fibrous tissue
staining, here illustrated as measured thickness of fibrous tissue.
M1 macrophages than that of saline control (Figures 5A,B;
Serp-1, p=0.0350; Serp-1m5, p=0.0053). Additionally, lung
tissues treated with Serp-1 and Serp-1m5 also have significantly
less detected numbers of Ly6G+neutrophils than the saline
treatment group (Figure 5C; Serp-1, p=0.0371; Serp-1m5,
p=0.004). We also performed IHC staining for arginase-1
(Arg-1) to characterize the anti-inflammatory M2 macrophages
(Figure 5D), but no statistical differences among these three
groups were observed for Arg-1+M2 macrophage. Arg-
1+M2 macrophage staining detected a nonsignificant trend
toward increased numbers. Serp-1 and Serp-1m5 treatment
groups have a trend to increased CD3+T cells (Figure 5E)
when compared to saline treated mice but this does not
achieve significance (p=0.3595). There were no identified
changes in different in CD4+T helper cell staining either
(Figure 5F;p=0.1015).
Serp-1m5 Treatment Reduced Macrophage
Counts on Flow Cytometry Analysis in Cell
Isolates From the Spleen of Mice After 15
Days of Induction With Pristane
In order to examine the potential systemic immune cell responses
to Serp-1 and Serp-1m5 treatments on mice after pristane
induction of DAH, we examined splenocyte isolates from each
mouse at 14 days follow up. We did not observe consistent
changes in spleen size among these three groups when we
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Guo et al. Serpin Reduces Lupus Lung Hemorrhage
FIGURE 5 | DAH Serp-1 and Serp-1m5 treatments reduced M1 macrophage and neutrophil numbers in lung sections after pristane-induced DAH, but did not
significantly affect M2 macrophage counts, CD3+cell, or CD4+cell infiltration. (A) Representative micrographs (40×) of iNOS +IHC staining in lungs at 14 days
post-pristine injection. (B) Serp-1 and Serp-1m5 group had significantly lower numbers of M1 macrophage counts marked by positive staining for iNOS IHC staining
(Serp-1, p=0.0350; Serp-1m5, p=0.0053) when compared to the saline treatment group. (C) The Serp-1m5 treated group had significantly lower numbers of
Ly6G+neutrophils (Serp-1, p=0.0371; Serp-1m5, p=0.004). Serp-1 treatment had a strong trend toward reduced neutrophil counts, but did not reach
significance. The number of Arg-1 +M2 macrophages (D), CD3+T cells (E), or CD4+T cells (F) were not statistically different among the three groups, although a
non-significant trend toward increased Arg+cells was seen. *p<0.05, **p<0.01.
collected mouse tissues after 15 days of induction with pristane
(17 mice in total–6 with saline, 6 with Serp-1, and 5 with
Serp-1m5 treatment; the spleen from one mouse in the Serp-
1m5 group was not collected and was not tested by flow
cytometry). Flow cytometry analysis of splenocytes demonstrated
significantly decreased F4/80+macrophages (Figure 6;p=
0.0173) in live splenocytes from Serp-1m5 treated mice. No
change in detected CD163+M2 in F4/80+macrophages,
nor in CD11b or CD11c cells in live splenocytes was seen
(Figure 6B). A significant increase in CD4+T cells in
live splenocytes (p=0.0268) and the TH1/Th2 ratio (p
=0.0287) was detected (Figure 7); A significant decrease
in Tregs (p=0.0285) and GATA3+CD4+Th2 cells (p
=0.0097) in CD4+T cells were detected with Serp-1m5
but not the unmodified Serp-1 (Figure 7); There was no
significant change in the frequency of NK cells, CD8+T cells,
CD11b+cells, CD11c+cells in living spleen cells, IFNg+CD4+
Th1 cells, Th17 cells in CD4+T cells, and CD163+F480+M2
macrophages in F4/80+macrophages among the three groups
(Figures 6,7).
Serp-1 and Serp-m5 Reduce C5b/9
Complement Positive Cell Counts in DAH
Model Lung Sections
Serp-1 pulled down human plasma C3 and vitronectin as
determined by mass spectrometry. C5b/9 is a final stage in
complement activation, the membrane attack complex. We have
demonstrated a significant reduction in cells staining positively
in both bronchial (ANOVA p=0.0008) and parenchymal tissues
(ANOVA p=0.0101) in lung sections from mice treated. Both
Serp-1 and Serp-1m5 produced significant reduction; Serp-1,
bronchus p=0.0146; parenchyma p=0.0077 and Serp-1m5,
brochus p=0.0002, parenchyma p=0.0074; Figure 8).
Serp-1 and Serp-1m5 Treatment Reduced
Cell Membrane Free Soluble uPAR in the
Lung Tissue of Pristane-Induced DAH
Mouse Model
The uPA-uPAR interaction plays an important role during
inflammatory cell invasion and activation. It has been reported
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Guo et al. Serpin Reduces Lupus Lung Hemorrhage
FIGURE 6 | Serp-1m5 treatment reduces macrophages in the spleen of mice after 15 days of induction with pristane. (A) Representative images of flow cytometry for
F4/80+macrophages in live splenocytes demonstrating reduced F4/80+cells with Serp-1m5 treatment. (B) Flow cytometry of macrophages demosntrated
decreased frequency of F4/80+macrophages (p=0.0173) in live splenocytes in Serp-1m5 treated mice compared with saline treated mice while no significant
differences in the frequency of CD11b+cells, CD11c+cells in living spleen cells and CD163+F480+M2 macrophages in F480+macrophages among the three
groups. *p<0.05.
earlier this year that cell free uPAR, i.e., soluble uPAR (suPAR),
is related to organ damage in SLE patients (31). Our research has
previously demonstrated that the immune modulation produced
by Serp-1 in aortic allografts as well as Serp-1 inhibition
of macrophage activation and diapedesis in tissue culture is
dependent on the uPAR (25). In prior work, Serp-1 lost its ability
to reduce inflammation and to reduce plaque growth in uPAR
knock out aortic allograft transplants in mouse models (18).
The depletion of uPAR also abolished the function of Serp-1
to promote wound healing (30). We therefor performed IHC
staining for uPAR to characterize uPAR expression after pristine
induction, with or without serpin treatments. It can be seen from
the IHC staining of lung tissue that Serp-1 and Serp-1m5 reduce
non-specific uPAR+clusters (Figures 9A–C; Serp-1, p=0.0172;
Serp-1m5, p=0.0025), detected as non-cell associated clumps
of positive staining in the lungs, when compared to the saline
treatment group. In contrast, there was increased detection of
intact uPAR+stained alveoli along the inner rim (Figures 9A–C;
Serp-1m5 vs. saline, p=0.0091). Control experiments without
primary antibody did not detect nonspecific staining. To further
quantitatively analyze the dissociation of uPAR, we compared
the uPAR extracted from tissue without detergent (PBS only) to
the total uPAR extracted with RIPA buffer containing 0.1% SDS.
ELISA was performed to determine the concentration of uPAR
in each extraction. We set the average ratio of lung tissue cell
membrane free uPAR (csuPAR) to total uPAR in each lung treated
with saline as one and normalized all the ratios of csuPAR/total
uPAR (Figure 9D). The ratio of csuPAR to total uPAR of Serp-
1 and Serp-1m5 groups was significantly lower than that of the
saline treated group (Serp-1, p=0.0004; Serp-1m5, p=0.0002).
This data confirmed our observation in the IHC staining to
uPAR (Figures 9A–C).
DISCUSSION
The pathogenesis of DAH is reported to be caused by defects in
macrophage phagocytic function and reduction in the removal
of apoptotic cells. Apoptotic fragments activate autoreactive B
cells and T cells, leading to the production of autoantibodies
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Guo et al. Serpin Reduces Lupus Lung Hemorrhage
FIGURE 7 | Flow cytometry of lymphocytes indicate a decreased frequency of GATA3+CD4+Th2 cells (p=0.0097), Tregs cells (p=0.0285) in CD4+T cells and
increased frequency of CD4+T cell in live splenocytes (p=0.0268), ratio of Th1/Th2 (p=0.0287) in Serp-1m5 treated mice compared with saline treated mice while
no significant differences in the frequency of NK cells, CD8+T cells, IFNg+CD4+Th1 cells, Th17 cells in CD4+T cells among the three groups. *p<0.05, **p<0.01.
and the formation of circulating immune complexes (ICs). ICs
are then believed to activate the classical complement pathway,
thereby causing pulmonary capillary vasculitis, damage to the
basement membrane, and capillary leakage with extravasation of
red blood cells (RBC) and bleeding into the alveolar cavity. This
continuous auto-Ab-mediated enhancement of the complement
system also causes complement depletion and reduces the
ability of phagocytes to remove dead cell debris, thus initiating
a vicious circle (32). Intraperitoneal injection of pristane in
B6 mice is an accepted model for lupus that simulates the
SLE DAH pathological process (14,33,34). After injection,
pristane migrates to the lungs, resulting in increased cell death,
activation of inflammatory cells such as macrophages, activation
of the classical pathway of complement and ICs formation and
additionally alveolar hemorrhage similar to human DAH occurs.
Studies have demonstrated that this process is macrophage
dependent in the mouse DAH model. Neutrophil depletion
is however reported to not be protective in prior mouse
model studies when using antibody to neutrophil elastase.
In contrast treatment with clodronate liposomes (CloLip) to
reduce macrophages was able to prevent DAH (13). Cell debris
depends on the opsonization of natural IgM, C3, and CR3/CR4
on the surface of macrophage cells to activate downstream
inflammatory pathways. CD11b/mice are protected against
the development of pristane-induced DAH (5), and mice with C3
deficiency and CD18 deficiency are also resistant. Reduction of
complement in wild-type mice by cobra venom factor (CVF) can
prevent DAH (13), while antibody to suppress neutrophils was
not effective.
From prior research, it is understood that macrophages play a
decisive role in the SLE DAH pathological process. Our research
has demonstrated that the administration of Serp-1 or Serp-
1m5 after intraperitoneal injection of pristane can significantly
reduce the occurrence and severity of DAH (Figures 1,2).
We performed immunohistochemistry using typical markers of
M1(iNOS), M2 (ARG1) macrophages and neutrophils (Ly6G).
Quantitation of stained cells demonstrated that Serp-1 and
Serp-1m5 treatments significantly reduced M1 macrophage
polarization and neutrophil infiltration in the lungs of the DAH
mouse model. Flow cytometry analysis of spleen cells was also
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Guo et al. Serpin Reduces Lupus Lung Hemorrhage
FIGURE 8 | C5b/9 staining is markedly increased in the bronchial epithelium
and in the surrounding lung parenchyma in saline treated mice with pristane
induced DAH (A). Serp-1 (B) and Serp-1m5 (C) significantly detected reduced
bronchial staining. Bar graphs demonstrate significant changes in the mean
cell counts ±SEM for cells positively stained for C5b/9 in the bronchial
epithelium (D) and in the lung parenchyma (E); Bronchus cell counts—p=
0.0008 ANOVA; Parenchyma ANOVA—p=0.0101. *p<0.05, **p<0.01,
***p<0.001.
consistent with the findings on immunostaining in the lungs.
Flow cytometry analysis demonstrated that the macrophage
isolates from the spleen of Serp-1m5 treated mice after pristane
induction were significantly reduced when compared to saline
treated pristane injected control mice. In contrast, the number
of M2 macrophages did not change significantly. Therefore, it
is deduced that the efficacy of Serp-1 and Serp1m5 treatment
is associated with a significant reduction in M1 macrophage.
Variable changes in lymphocytes counts on IHC with some
significant differences in CD4, Treg, and Th2 counts on flow
cytometry were observed and on flow cytometry. These T cell
changes were limited to Serp-1m5 treatments. These findings are
consistent with previous studies with Serp-1 suggesting a more
significant effect on macrophage responses. However, the more
pronounced effects of Serp-1m5 both on lung hemorrhage, M1
macrophage and Ly6G counts on IHC and also greater effects
of Serp-1m5 on splenocyte analyses might suggested that some
of the enhanced activity of the Serp-1m5 protein is due to a
larger effect overall on immune cell responses in the DAH model
in mice.
M1 macrophages are involved in the pathogenesis of various
autoimmune inflammatory diseases, including multiple sclerosis,
rheumatoid arthritis, inflammatory bowel diseases, asthma, and
SLE (1,3539), we have proposed that targeting M1 cells
with Serp-1 treatment will provide a potential treatment for
autoimmune diseases, suggesting that Serp-1 can inhibit the
activation of macrophage M1 to protect SLE patients against
the development of DAH. Serp-1 is a proven inhibitor of
activated serine proteases, functioning to bind, and inhibit
both tPA and uPA as well as thrombin and fXa as noted in
the introduction. The thrombotic and thrombolytic cascades
also activate immune and inflammatory cell responses and
conversely platelets and thrombosis are activated on the surfaces
of dysfunctional endothelial cells and/or activated macrophages
in the arterial wall. uPA and tPA are plasminogen activators,
serine proteases, in the thrombolytic, clot dissolving cascade, and
can initiate matrix metalloproteinase activation and connective
tissue degradation allowing immune cell invasion. Complement
is also serine protease central to immune cell responses.
We have posited that Serp-1 inhibits the migration of M1
macrophages to the lung via blockade of the uPA/uPAR complex
on the surface of activated inflammatory phagocytes. Activated
macrophage expression of uPA and tPA leads to the activation of
MMPs allowing cell invasion into damaged tissues by breaking
down surrounding connective tissue or the arterial endothelial
glycocalyx. It is, however, not known whether Serp-1 may
directly promote macrophage polarization to a M2 phenotype.
The uPAR is part of a large lipid raft that interacts with
many cell surface integrins and low density lipoprotein related
protein receptor (LRPR) as well as chemokine receptors. We
had previously demonstrated that Serp-1 alters macrophage
migration in vitro via uPAR and filamin B (an actin binding
protein) dependent mechanisms. Our previous studies have
demonstrated that Serp-1 can reduce monocyte/ macrophage
adhesion and migration across endothelial monolayers in vitro
and into mouse ascites in vivo. Serp-1 applied to monocytes
alters the expression of filamin B and CD18, increasing filamin
B and decreasing CD-18 expression. These alterations in gene
expression are uPAR dependent and application of siRNA
to filamin blocked Serp-1 mediated inhibition of monocyte
migration in vitro. Filamin b and uPAR are co-localized and
co-immunoprecipitated with Serp-1 (25). Therefore, we would
suggest that Serp-1 exerts its anti-inflammatory effects by
modifying uPAR-CD18 and filamin b in monocytes to mediate
the decrease of M1 macrophages in lung tissue. We would like
to further note that in prior reported studies of the mechanisms
of DAH development, DAH was resistant to CD18-deficient
mice (5).
Serp-1 treatment may also have the added benefit of reducing
activation of fibrinolysis and thus may directly mediate a
reduction in bleeding. The uPA/uPAR complex is traditionally
considered to act predominantly on cellular activation and
immune cell responses rather than as a primary regulator of
thrombolysis. tPA, the fibrinolytic serine protease that Serp-
1 also inhibits, is considered the central mediator of the
regulation of clot lysis in the blood stream. In this study,
our immunohistochemical study of uPAR in lung tissues
demonstrated that when compared with saline treated DAH
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Guo et al. Serpin Reduces Lupus Lung Hemorrhage
FIGURE 9 | Serp-1 and Serp-1m5 treatment reduce soluble uPAR in the pristane-induced DAH model. (A) The representative pictures of uPAR IHC staining. (B)
Serp-1 and Serp-1m5 reduced cell-free uPAR+clusters in IHC staining when compared to the saline treatment group (Serp-1, p=0.0172; Serp-1m5, p=0.0025).
(C) Serp-1m5 increased intact uPAR+stained alveoli along the inner rim when compared to the saline treatment group (p=0.0091; Serp-1m5 vs. saline). (D) The
ratio of csuPAR to total uPAR of Serp-1 and Serp-1m5 tested by ELISA of lung tissue was significantly lower than that of the saline group (Serp-1, p=0.0004;
Serp-1m5, 0.0002). *p<0.05, **p<0.01, and ***p<0.001.
mice, the soluble uPAR in the Serp-1 treatment group was
significantly reduced, while uPAR on the intact alveolar cells
was preserved significantly. This is consistent with the uPAR
ELISA analysis of lung tissue that similarly detected Serp-
1 reductions in soluble uPAR. uPAR is composed of three
homologous domains and is connected to the cell surface
through glycosylphosphatidylinositol (GPI) anchors. The three
domains of uPAR include the main uPA (40), and the
extracellular matrix protein vitronectin binding site (41). uPAR
is easily cleaved by several proteases, including physiologically
related enzymes such as neutrophil elastase, plasmin and uPA
itself (4244).
Soluble uPAR is currently considered to be a biomarker
of inflammation and immune system activation but has not
to date been examined nor associated with DAH in SLE
patients. Elevated suPAR levels are associated with a variety of
inflammatory diseases, such as systemic inflammatory response
syndrome (SIRS), cancer, local segmental glomerulosclerosis,
cardiovascular disease, type 2 diabetes, asthma, liver failure,
COVID, and SLE (31,4550). After DAH occurs, the alveolar
basement membrane tissue is destroyed by immune complex
(IC) deposition and inflammatory cell adhesion, red blood cells
then extravasate (leak out of damaged vessels), and abnormal
blood coagulation pathways are initiated. Cleavage of uPAR
by plasmin and uPA causes the release of soluble uPAR from
the cell surface membrane into surrounding tissues, weakening
of the anchoring of cells to the extracellular matrix, reducing
endothelial cell connections and, at the same time, triggers a
series of proteolytic cascade reactions to degrade the components
of the extracellular matrix leading to further destruction of
alveolar tissue structure and increased bleeding. Therefore, Serp-
1 can reduce the cleavage of uPAR, maintain cell adhesion
to the extracellular matrix as well as reducing inflammatory
cell invasion via inhibiting uPA and plasmin and activation of
matrix metalloproteinases. As we have seen in our experiments,
soluble uPAR is significantly reduced, and uPAR in intact
alveolar tissue cells increased. Meanwhile, Serp-1 reduces the
downstream protease cascade by modulating the coagulation
and fibrinolysis process, thereby preventing further destruction
of lung tissue structure and pulmonary hemorrhage. Activation
of complement is also central to immune responses. We
have demonstrated Serp-1 binding to C3 in human plasma
by MS analysis. We also report here a reduction in C5b/9
IHC complement staining with serpin treatments, which may
be secondary to efficacy in reducing uPA/uPAR activity or
conversely complement. Prior work would suggest that uPAR
is a principal target. However, binding and modulation of a
second pathway via C3 may also contribute to the benefits
seen with serpin treatments in this lupus DAH model. These
findings support a reduction in overall immune cell activation
with serpin treatment but do not provide a final proof of
mechanism. The studies do suggest a close correlation between
Frontiers in Cardiovascular Medicine | www.frontiersin.org 11 February 2021 | Volume 8 | Article 633212
Guo et al. Serpin Reduces Lupus Lung Hemorrhage
uPAR expression, as well as complement activation, both serine
protease pathways, and serpin efficacy in this mouse model of
pristane induced SLE DAH.
In summary, Serp-1 and PEGylated Serp-1 (Serp-1m5)
effectively reduce the frequency and severity of pristane-
induced diffuse pulmonary hemorrhage in mice through multiple
factors, while the modified Serp-1 protein has greater efficacy
than its wild type does in this experimental DAH model.
This research proves that Serp-1 and its derivates are very
promising candidates of therapeutics for DAH, which has no
proven effective treatment available now. In this direction,
the molecular mechanism under the therapeutic effectiveness
to DAH and the technologies for the modification will be
further studied.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included
in the article/supplementary material, further inquiries can be
directed to the corresponding author/s.
ETHICS STATEMENT
The animal study was reviewed and approved by Biodesign
Institute, ASU Institutional Animal Care and Use
Committee Animal Protocol Review ASU Protocol Number:
20-1761R RFC 1.
AUTHOR CONTRIBUTIONS
QG, JRY, LZ, and ARL designed the study, LZ and QG developed
materials. LZ, QG, and JRY performed the animal studies and
histology assays, RB and TO assisted in the protein purification,
KB, EA, MB, and LNS performed histology, JW and PF provided
scientific feedback and discussion, QG, LZ, JRY, and SL analyzed
the data, QG, ARL, and LZ wrote the manuscript. All authors
reviewed the manuscript.
FUNDING
This work was supported by prior grants from NIH (Grant
numbers 1 R01 AI100987-01A1; RO1 DE020820-01A1;
1RC1HL100202-01), AHA (GRNT33460327), and startup
funding from ASU, Biodesign Institute as well as the ASU
Skysong Challenge fund to the Lucas lab.
ACKNOWLEDGMENTS
We would like to acknowledge Dr. Westley H. Reeves for his
initial observations and discussion on the SLE DAH model.
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177-x
Conflict of Interest: The method of Serp-1 modification has been submitted
for a provisional patent application with title of “New composition of
immunomodulating Serpin, Serp-1” (PCT Application No. 63/017,598). ARL,
LZ, QG, JRY, and JW are inventors of this patent. JW is employed by Exalt
Therapeutics but has received no funding for his work on this project nor the
research presented in this manuscript.
The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential
conflict of interest.
Copyright © 2021 Guo, Yaron, Wallen, Browder, Boyd, Olson, Burgin, Ulrich,
Aliskevich, Schutz, Fromme, Zhang and Lucas. This is an open-access article
distributed under the terms of the Creative Commons Attribution License (CC BY).
The use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice.
No use, distribution or reproduction is permitted which does not comply with these
terms.
Frontiers in Cardiovascular Medicine | www.frontiersin.org 13 February 2021 | Volume 8 | Article 633212
... Treatment with purified native Serp-1 has demonstrated both acute and long-term efficacy in modulating inflammation in a wide range of inflammatory disorders and injuries, including atherosclerosis, transplant, wound healing, and spinal cord injury [39][40][41][42]. More recently, a modified Serp-1 protein, PEGSerp-1, with a longer halflife (~8 h), was shown to reduce inflammation and fibrosis in healing corneal wounds, and reduced macrophage invasion of alveoli in a mouse model of diffuse alveolar hemorrhage [43][44][45]. Based on these previous studies, we examined whether the pegylated version of the viral Serp-1 protein, PEGSerp-1, would ameliorate the chronic inflammatory pathology of DMD. ...
... Serp-1 was endotoxin-free, as detected by limulus amebocyte lysate (LAL) assay. Serp-1 was incubated with mPEG-NHS (5 K) (Nanocs Inc., #PG1-SC-5k-1, New York, NY, USA) in PBS (pH 7.8) at 4 • C overnight to modify the protein according to standard PEGylation protocols [43]. PEGylated-Serp-1 was purified by FPLC using an ÄKTA pure protein purification system with Superdex-200 [43]. ...
... Serp-1 was incubated with mPEG-NHS (5 K) (Nanocs Inc., #PG1-SC-5k-1, New York, NY, USA) in PBS (pH 7.8) at 4 • C overnight to modify the protein according to standard PEGylation protocols [43]. PEGylated-Serp-1 was purified by FPLC using an ÄKTA pure protein purification system with Superdex-200 [43]. ...
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