betageri-et-al-2023-the-matricellular-protein-ccn3-supports-lung-endothelial-homeostasis-and-function-html.html

). Impaired

vascular regeneration affects organ physiology (

), and has

been associated with poor outcomes in aging-associated disor-
ders, including idiopathic pulmonary

ﬁ

brosis (IPF) (

IPF is a chronic, progressive, and fatal lung disease char-

acterized by aberrant wound healing, exaggerated deposi-
tion of extracellular matrix (ECM) by persistently activated

ﬁ

broblasts (myo

ﬁ

broblasts) (

), and several vascular

abnormalities. Among the latter, studies reported capillary
rarefaction, vascular leakage, and endothelial transcrip-
tional alterations (

), including limited expres-

sion of gene markers of lung endothelial progenitor cells
(also known as general capillary ECs, gCap ECs) (

). Current

therapeutic options are only able to slow disease progres-
sion, thus the prognosis is still poor, and the median sur-
vival is only 3

–

5 yr after diagnosis (

). Although most

studies have focused on nonvascular cell types as central
players of the disease, as well as preferred therapeutic tar-
gets for IPF, recent evidence shed light on the important
contribution of the pulmonary vasculature in orchestrating
wound-healing responses after lung injury (

10.1152/ajplung.00248.2022

). This

highlights an opportunity for new endothelial-focused ther-
apeutic strategies.

Using a mouse model of bleomycin-induced lung

ﬁ

brosis,

we have recently demonstrated that the pulmonary vascula-
ture of young and aged mice displays divergent behaviors
upon bleomycin injury, with aged animals exhibiting abnor-
mal epigenetic and transcriptional responses, resulting in
aberrant lung

ﬁ

brosis resolution (

To untangle the mechanistic underpinnings behind this

divergent vascular response, we have recently performed
RNA sequencing (RNA-seq) analysis on freshly sorted lung
ECs from the lungs of young and aged mice following the
bleomycin challenge (

). This analysis led to the identi

ﬁ

ca-

tion of

Ccn3

, a gene encoding for the matricellular protein

Cellular Communication Network Factor 3, also known as
nephroblastoma overexpressed (Nov).

Ccn3

was upregulated

in young lung ECs in response to bleomycin injury, but not in
aged lung ECs under the same experimental conditions (

CCN3 is a secreted matricellular protein of the CCN fam-

ily that also includes CCN1 (CYR61), CCN2 (encoding con-
nective tissue growth factor, CTGF), CCN4 (WISP-1), CCN5
(WISP-2), and CCN6 (WISP-3) (

). These proteins are a

subset of ECM proteins that serve regulatory roles in
response to extracellular stimuli rather than structural roles

Correspondence: N. Caporarello (caporarello.nunzia@mayo.edu).
Submitted 4 August 2022 / Revised 23 November 2022 / Accepted 19 December 2022

L154

1040-0605/23 Copyright

2023 the American Physiological Society.

http://www.ajplung.org

Am J Physiol Lung Cell Mol Physiol

324: L154

–

L168, 2023.

First published December 27, 2022; doi:

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(

). Among these proteins, CCN2 (CTGF) plays an impor-

tant role in

ﬁ

brotic disorders in multiple tissues and organs

such as periodontal tissues, kidney, liver, pancreas, skin,
and lung (

). CCN2 (CTGF) is a pro

ﬁ

brotic marker, acti-

vated by TGF-

(

) and involved in facilitating lung

ﬁ

bro-

blast activation, proliferation, and ECM synthesis (

Interestingly, different studies showed that CCN3 overex-
pression counteracts pro

ﬁ

brotic CCN2 expression in murine

skin

ﬁ

broblasts (

) as well as in rat mesangial cells (

). In

addition, in a mouse model of diabetic nephropathy, treat-
ment with recombinant human CCN3 completely blocks
renal

ﬁ

brosis (

). This suggests a role for CCN3 as an en-

dothelial-derived negative regulator of

ﬁ

brosis and a pos-

sible therapeutic target in

ﬁ

brotic diseases.

Although the role of CCN3 has been studied in nonvas-

cular cell types, its role and function in vascular biology
remain relatively understudied. Here, we investigated the
role of CCN3 in promoting lung vascular endothelial ho-
meostasis and function to uncover its importance as a
potential endothelial-targeted therapeutic strategy to pro-
mote vascular repair in lung

ﬁ

brosis.

METHODS

Bleomycin Mouse Studies

All animal studies were performed under protocols

approved by the Mayo Clinic Institutional Animal Care and
Use Committee (IACUC) and conforming to the Animal
Research Reporting

In Vivo

Experiments (ARRIVE) guide-

lines. Col1

1-GFP transgenic mice (FVB strain) were gener-

ated as previously described (University of California San
Diego, La Jolla, CA) (

) and kindly provided by Dr. Derek

Radisky. Mice had access to food and water ad libitum and
were on a 12/12 light/dark cycle. Two- or 18-mo-old Col1

GFP mice (female and male) were intratracheally injected
with bleomycin (1.2 U/kg) or PBS, as previously described (

). Thirty or 75 days after bleomycin delivery, lungs were

harvested and processed for FACS isolation of lung ECs using
a strategy detailed in our previous study (

). The RNA seq

analysis of sorted lung ECs shown in this work is extracted
from a previously generated experiment, performed using
freshly FACS-isolated lung ECs from young and aged mice
injected with PBS or bleomycin (end point: 30 days) (

Cell Culture

Human lung microvascular endothelial cells (HLMECs)

were purchased from Lonza or Cell Applications and main-
tained in endothelial cell growth basal medium (EBM2)
supplemented with microvascular endothelial cell growth

medium SingleQuots (Lonza, Walkersville, MD) or micro-
vascular endothelial cell growth media (Cell Applications,
CA) containing 5% FBS. All experiments were performed
with endothelial cells within fourth passage. In experi-
ments involving siRNA or stimulating cells with recombinant
CCN3 protein (Cat. No. 12026, PeproTech, Cranbury, NJ, resus-
pended in nuclease-free water), serum was reduced to 0.1%.
Normal human lung

ﬁ

broblasts (NHLFs) were purchased from

Lonza (Walkersville, MD) and maintained in EMEM (ATCC,
Manassas, VA) containing 10% FBS. In experiments involving
stimulating cells with TGF

(Cat. No. 14-8348-62, Thermo

Fisher, Waltham, MA), serum was reduced to 0.1%. All
experiments were performed with

ﬁ

broblasts between pas-

sages 3 and 7.

RNA Interference

Transient RNA interference was performed with siGENOME

Non-Targeting Control siRNA Pool #1 (D-001206-13-05,
Horizon Discovery Biosciences) or siGENOME Human NOV
siRNA (L-010527-02-0005, Horizon Discovery Biosciences)
by using Lipofectamine RNAiMAX reagent (13778150.
Thermo Fisher Scienti

ﬁ

c, Waltham, MA). On the day of

transfection, the medium was switched to 0.1% FBS. Cells
were harvested 72 h after the time of transfection.

RNA Isolation and qRT-PCR Analysis

Total mRNA was isolated using RNeasy micro kit (Qiagen,

Valencia, CA), followed by Nanodrop concentration and pu-
rity analysis. cDNA was synthesized with SuperScript VILO
(Thermo Fisher Scienti

ﬁ

c, Waltham, MA); RT-PCR was per-

formed using FastStart Essential DNA Green Master (Roche
Diagnostics, Mannheim, Germany) and analyzed using a
LightCycler 96 (Roche Diagnostics, Mannheim, Germany).
RT-PCR primers used in this study (Integrated Technologies,
Coralville, IA) are listed in

Table 1

. qPCR array was performed

by using RT

Pro

ﬁ

ler PCR Array Human Endothelial Cell

Biology (Qiagen, Valencia, CA). For qPCR array, data repre-
sent fold changes relative to the control siRNA or control
untreated cells and normalized to the housekeeping gene

Actb

. All signi

ﬁ

cant genes from these analyses are included

in the supplement (Supplemental Tables S1 and S2).

Protein Extraction and Western Blot Analysis

Cell proteins were extracted using RIPA lysis buffer

(Thermo Fisher Scienti

ﬁ

c, Waltham, MA) supplemented with

protease and phosphatase inhibitor cocktail (Thermo Fisher
Scienti

ﬁ

c, Waltham, MA). Protein concentration was deter-

mined using Pierce BCA Protein Assay Kit (Thermo Fisher
Scienti

ﬁ

c, Waltham, MA). Ten micrograms of protein were

Table 1.

Human and mouse primer sequences for qPCR analysis

Primer

Forward

Reverse

CCN3

AACTGCATTGAACAGACCACA

ATTGACGGTTCCTATTGGTGAC

GAPDH

GGAAGGGCTCATGACCACAG

ACAGTCTTCTGGGTGGCAGTG

ACTA2

GTGAAGAAGAGGACAGCACTG

CCCATTCCCACCATCACC

COL1A1

AAGGGACACAGAGGTTTCAGTGG

CAGCACCAGTAGCACCATCATTTC

CCN2

AAAAGTGCATCCGTACTCCCA

CCGTCGGTACATACTCCACAG

Ccn3

AGTGCCCCAGTATATCACCGA

TGCGGTCACAGTAGAGACCA

Gapdh

GTGGAGTCATACTGGAACATGTAG

AATGGTGAAGGTCGGTGTG

CCN3 SUPPORTS LUNG ENDOTHELIAL FUNCTION

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resolved by 7.5% SDS-PAGE, transferred to polyvinylidene

ﬂ

uoride membranes, and probed overnight at 4

C with

the following primary antibodies: GAPDH (Cell Signaling
Technology Cat. No. 2118, 1:1,000 dilution, Danvers, MA,
RRID:AB_561053) and CCN3 (Cell Signaling Technology Cat.
No. 8767, 1:1,000 dilution, Danvers, MA, RRID:AB_2797661).
Blots were then washed and incubated with appropriate IgG-
HRP-conjugated secondary antibodies for 1 h at room tem-
perature. The bands were then visualized using Super Signal
West Pico Plus (Thermo Fisher Scienti

ﬁ

c, Waltham, MA) and

ChemiDoc (Bio-Rad, Hercules, CA), according to the manu-
facturer

’

s protocol. All antibodies were commercially avail-

able and were validated by the respective manufacturers.

VE-Cadherin and Phalloidin Staining

To visualize cell-cell junctions and cell morphology,

HLMECs were plated 80,000 cells/well on a 12-well plate and
transfected with control siRNA or CCN3 siRNA. After 24 h,
cells were lifted and plated (60,000 cells/well) to visualize
a con

ﬂ

uent monolayer on a 24-well glass bottom plate.

Culture media was then removed, and cells were washed
with PBS at 37

C. Cells were then

ﬁ

xed in 10% formalin so-

lution for 15 min at room temperature, washed with PBS,
and permeabilized in 0.1% Triton X-100 for 5 min at room
temperature. Cells were then blocked with 5% normal goat
serum and 2% BSA for 1 h and subsequently primary rabbit
antibody solution of vascular endothelial (VE)-cadherin
(Cell Signaling Technology Cat. No. 2500, 1:400 dilution,
Danvers, MA, RRID:AB_10839118) was added for an over-
night incubation at 4

C. Cells were then washed with PBS and

secondary antibody solution of goat anti-rabbit (Thermo Fisher
Scienti

ﬁ

c Cat. No. A-11034, 1:500 dilution, Waltham, MA,

RRID:AB_2576217), DAPI (Biolegend, Cat. No. 422801, 1:1,000
dilution, San Diego, CA), and phalloidin (Cytoskeleton, Inc.,
Cat. No. PHDR1, 1:40 dilution, Denver, CO) was added for 1 h
incubation at room temperature. After washing with PBS, the
cells were imaged, and intercellular gaps were quanti

ﬁ

ed with

Fiji (

) by thresholding and measuring both total gap area and

size of gap area (Fiji, RRID:SCR_002285). All antibodies were
commercially available and were validated by the respective
manufacturers.

Ki67 Staining

Human lung endothelial cells that were either control or

CCN3 silenced, or treated with recombinant CCN3 were
washed,

ﬁ

xed, and permeabilized in 0.1% Triton X-100 for

5 min at room temperature. Cells were then blocked with 4%
normal goat serum for 1 h and subsequently primary mouse
antibody solution of Ki67 (Santa Cruz Biotechnology Cat. No.
sc-23900, 1:100 dilution, Dallas, TX, RRID:AB_627859) was
added for an overnight incubation at 4

C. Cells were then

washed with PBS and secondary antibody solution of Alexa
Fluor 488 goat anti-mouse (Thermo Fisher Scienti

ﬁ

c Cat. No.

A-11001, 1:500 dilution, Waltham, MA, RRID:AB_2534069)
and counterstained with DAPI (Biolegend, Cat. No. 422801,
1:1,000 dilution, San Diego, CA) was added for 2 h at room
temperature. Cells positive for Ki67 were determined in each
well from 25

ﬁ

elds at

10 magni

ﬁ

cation using a Bio-Tek

Cytation5, analyzed using a visual intensity-based threshold
by Bio-Tek Gen5 Image software (Gen5 RRID:SCR_017317),

and expressed as percentage of total cell number (determined
with DAPI count), as previously described (

). All antibodies

were commercially available and were validated by the re-
spective manufacturers.

Wound-Healing Assay

Human lung endothelial cells were plated at a density of

80,000 cells/well in 12-well plates. After 24 h, cells were
transfected with either control or

CCN3

siRNA and 24 h later,

a single scratch was made with sterile 200

L pipette tip to

obtain a cell-free area, as previously described (

) and

the media was changed to endothelial media with 5% FBS to
stimulate cell growth. For experiments involving recombi-
nant CCN3, cells were pretreated with endothelial media
containing 0.1% FBS or 0.1% FBS and 100 ng/mL human
recombinant CCN3 protein (Cat. No. 12026, PeproTech,
Cranbury, NJ) for 2 h. Subsequently, a single scratch was per-
formed as done for the RNAi experiments, and the respective
media concentrations were replaced. Wound closure was
monitored at 12-, 18-, and 24-h time points for knockdown
experiments in full serum medium, and a longer period of
12-, 24- and 48-h time points for experiments performed
with recombinant CCN3 and low serum conditions. Images
were taken with an Olympus CKX53 inverted microscope
equipped with a digital camera. The wound area was then
measured using ImageJ (NIH) (

) software (ImageJ, RRID:

SCIR_003070). Data are represented by the percent wound
area open, calculated as follows, adopted from Ref.

% Wound area closed

open

ð Þ

open

ð Þ

open

ð Þ

100

% Wound area open

100

% Wound area closed

where

open

(0) represents the area of the wound that is open

time 0

and

open

(

) represents the area of the wound that

is open at

time t

In Vitro Angiogenesis Assay

Control or CCN3-silenced cells (10,000 cells/well), trans-

fected via RNA interference strategy as described above,
were seeded onto

-slide chambers slides (Cat. No. 81506,

Ibidi, Fitchburg, WI) coated with 10

L of polymerized

Matrigel matrix (Cat. No. CB40234A, Fisher Scienti

ﬁ

Waltham, MA) in low serum media, as previously described
(

). After a 2-h serum starvation period, cells were

stimulated with 5% (full) serum media containing 50 ng/
mL of VEGF-A (Cat. No. 100-20A, PeproTech, Cranbury,
NJ) and 1 mM sphingosine-1-phosphate (Cat. No. 1370,
Tocris Bioscience, Bristol, UK), as previously described (

After 6 h, the well area (

4 objective) was photographed to

quantify the ability of cells to organize into capillary-like
structures and

10 objective was used for representative

images. Quanti

ﬁ

cation was performed with regards to total

number of junctions and total vessel length via AngioTool
0.6 software (

) [AngioTool (RRID:SCR_016393)]. For

experiments using recombinant CCN3, control cells or cells
treated with 100 ng/mL of recombinant CCN3 for 2 h were
seeded onto

-slide chambers slides coated with 10

L of

polymerized Matrigel matrix in low media containing ei-
ther 0 ng/mL or 100 ng/mL human recombinant CCN3

CCN3 SUPPORTS LUNG ENDOTHELIAL FUNCTION

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protein. After 2 h, the cells were stimulated with low serum
media containing 50 ng/mL of VEGF-A and either 0 ng/mL
or 100 ng/mL recombinant human CCN3 protein in the ab-
sence of sphingosine-1-phosphate to create an environment
with reduced angiogenic stimuli. The well area was then
imaged using

4 and

10 objectives as above for both

quanti

ﬁ

cation and representative images.

Isolation and Testing of Endothelial-Derived
Conditioned Media

HLMECs were transfected with control or

CCN3

siRNA.

The media was replaced 6 h after transfections and cells
were incubated for 72 h. Conditioned media (CM) from con-
trol- and CCN3-silenced HLMECs was then collected, cen-
trifuged at 1,100 rpm for 5 min to remove cellular debris,
and concentrated 3X with Amicon Ultra-4 Centrifugal Filter
Unit with Ultracell-3 membrane (Cat. No. UFC800396,
Millipore Sigma, Burlington, MA) before being applied to
recipient NHLFs, previously primed with 2 ng/mL of TGF

(Cat. No. 14-8348-62, Thermo Fisher, Waltham, MA) for 24
h. After 48 h, RNA isolation and qRT-PCR analysis on recip-
ient NHLFs were performed as described in

RNA Isolation

and qRT-PCR Analysis

. Recipient cells were then

ﬁ

xed, per-

meabilized, and stained with phalloidin to visualize cell
morphology, and counterstained with DAPI.

Statistical Analysis

The individual data points shown in all plots represent

data from independent mice or biological replicates from
cell culture experiments. Variables were summarized by
mean and standard deviation with statistical comparisons
between groups performed using Student

’

test or one-way

analysis of variance (followed by Tukey

’

s post hoc test). All

analyses and plots were generated with GraphPad Prism
9.3.0 (La Jolla, CA) with statistical signi

ﬁ

cance de

ﬁ

ned as

0.05 (GraphPad Prism, RRID:SCR_002798).

RESULTS

Ccn3

Expression Is Upregulated in Young But Not Aged

Lung ECs After Bleomycin-Induced Lung Fibrosis

Analysis of RNA-seq performed on freshly sorted lung ECs

(

) reveals that

Ccn3

expression in lung ECs is upregulated in

young mice 30 days after bleomycin injury (

Fig. 1

), a time

point associated with early resolution phase of bleomycin-
induced lung

ﬁ

brosis in young mice (

). In contrast, no differ-

ences were observed in

Ccn3

expression in aged lung ECs at

the same time point (

Fig. 1

). To investigate the dynamic alter-

ations in

Ccn3

expression during bleomycin injury, we meas-

ured

Ccn3

transcripts in sorted lung ECs in a larger cohort of

young and aged mice, at 30 and 75 days after bleomycin injury
(this latter time point associated with lung

ﬁ

brosis resolution

in young mice) (

). We observed that in lung ECs isolated

from young mice,

Ccn3

transcript levels signi

ﬁ

cantly increase

at 30 days after injury and return to baseline levels at 75 days
after injury. In contrast, no elevation of

Ccn3

levels was

observed in lung ECs isolated from aged animals at the same
time points (

Fig. 1

). Previous studies focused on human um-

bilical vein endothelial cells (HUVECs) and human dermal mi-
crovascular endothelial cells (HDMECs) highlighted the role of
CCN3 in supporting vascular homeostasis by reducing in

ﬂ

am-

mation (

) and promoting angiogenesis (

). These observa-

tions align with the transient expression of

Ccn3

observed in

young mice, correlating with the time course of

ﬁ

brosis resolu-

tion and regeneration of lung vascular network. Thus, our data
suggest that the role of CCN3 in restoring endothelial homeo-
stasis and promoting vascular repair may be extended to the
lung endothelium.

CCN3 Loss and Gain of Function Leads to
Transcriptional Changes in Lung ECs

Since CCN3 has been found as a negative regulator of pro-

ﬁ

brotic CCN2 (

), we

ﬁ

rst assessed whether this

Figure 1.

CCN3 is transiently upregulated in lung ECs from young but not aged mice after bleomycin injury.

: histograms of RNA expression RPKM val-

ues showing upregulation of

Ccn3

in lung ECs isolated from young mice 30 days after bleomycin but no elevation in lung ECs isolated from aged mice

at the same time point (young sham 2-mo-old,

= 5; young 2-mo-old

day 30

after bleomycin,

= 4; aged sham 18-mo-old,

= 3; aged 18-mo-old after

bleomycin,

= 4). Values are summarized as means and SD.

: qPCR analysis of

Ccn3

transcripts in FACS-isolated lung endothelial cells (CD45

CD326

/GFP

/CD31

population) isolated from young and aged mice either sham or challenged with bleomycin (young sham 2-mo-old,

= 6; young

2-mo-old,

day 30

after bleomycin,

= 9; young 2-mo-old,

day 75

after bleomycin,

= 8; aged sham 18-mo-old,

day 0

= 7; aged 18-mo-old,

day 30

after

bleomycin,

= 9; aged 18-mo-old,

day 75

after bleomycin,

= 7). Data are expressed as means ± SD and analyzed using one-way analysis of variance

(followed by Tukey

’

s post hoc test). Aged lung ECs failed to upregulate

Ccn3

at any of the time points analyzed. EC, endothelial cell; RPKM, reads per kil-

obase of exon per million reads mapped.

0.01;

0.001; ns, not signi

ﬁ

cant.

CCN3 SUPPORTS LUNG ENDOTHELIAL FUNCTION

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effect could be extended to human lung ECs. We found that
silencing of CCN3 in HLMECs led to a signi

ﬁ

cant increase in

transcripts for

CCN2

(

), con

ﬁ

rming that CCN3 coun-

teracts CCN2 in HLMECs.

Then, to evaluate the effects of CCN3 loss and gain of func-

tion broadly on endothelial transcriptional state, we used a
qPCR array and measured a panel of endothelial-related genes
in control- or CCN3-silenced HLMECs or in HLMECs either
untreated or treated with human recombinant CCN3 (

Fig. 2,

and Supplemental Tables S1 and S2). We found that loss

of CCN3 leads to signi

ﬁ

cant reduction in genes associated

with endothelial homeostasis, including the proangiogenic
gene

KIT

(receptor tyrosine kinase), which is also a marker of

gCap ECs (

) (

). In addition, we found a

reduction in levels of

NOS3

(nitric oxide synthase 3),

whose loss we previously linked with endothelial dysfunc-
tion and lung

ﬁ

brosis (

ACE

(angiotensin-converting

enzyme), and trending decrease of the pan-endothelial marker

PECAM1

(platelet and endothelial cell adhesion molecule 1)

(

). In addition,

HMOX1

(heme oxygenase 1) (

), an anti-

oxidant enzyme, was found signi

ﬁ

cantly reduced (

Furthermore, upon CCN3 silencing, we found reduced tran-
scripts for the antiapoptotic

BCL2

(B-cell lymphoma 2) (

whereas upon recombinant CCN3 treatment, we found
reduced transcripts for proapoptotic genes, including

FAS

(Fas

cell surface death receptor) and

TNFSF10

(TNF Superfamily

10), the latter also shown to stimulate collagen production by
NHLFs as well as mouse

ﬁ

broblasts (

), and found

increased in serum of patients with IPF (

) (

). In addi-

tion, upon silencing of CCN3, we found increased levels of the
proin

ﬂ

ammatory gene

CCL5

(c-c motif chemokine ligand 5),

also known as RANTES (

), which has been associated

with accumulation of in

ﬂ

ammatory cells within the lung in

interstitial lung disease (

), whereas upon treatment with

Figure 2.

CCN3 loss and gain of function leads to transcriptional changes.

: gene expression analysis by qPCR demonstrates signi

ﬁ

cant increase of

CCN2

transcript in CCN3-silenced HLMECs. Data are expressed as means ± SD from

= 4 independent experiments and analyzed using Student

’

test

(

0.0001).

: Western blot analysis and relative quanti

ﬁ

cation con

ﬁ

rm reduction of CCN3 protein expression in CCN3-silenced HLMECs. Data are

expressed as means ± SD from

= 3 independent experiments and analyzed using Student

’

test (

0.05).

: schematic of the experimental work-

ﬂ

ow.

–

: transcriptional changes in control- and CCN3-silenced HLMECs for 72 h (

= 5 independent experiments) or control- and HLMECs treated

with 100 ng/mL recombinant CCN3 for 9 h (

= 4 independent experiments) were analyzed by using an endothelial cell biology pro

ﬁ

ler PCR array. Data

are expressed as means ± SD and analyzed using Student

’

test (

0.05,

0.01,

0.001,

0.0001). HLMEC, human lung microvas-

cular endothelial cell; PCR, polymerase chain reaction. [Image created with BioRender.com and published with permission.]

CCN3 SUPPORTS LUNG ENDOTHELIAL FUNCTION

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recombinant CCN3, we found reduced levels of proin

ﬂ

amma-

tory genes, such as

IL1

(interleukin-1 beta), and

IL11

(interleu-

kin 11), both highly abundant in

ﬁ

brotic lungs (

) (

Finally, in CCN3-silenced cells, we found reduced transcripts
for matrix metalloprotease-2 (

MMP2

) (

), important for

neovascularization (

) and also shown to have an anti

ﬁ

brotic

role in bleomycin-induced lung

ﬁ

brosis (

). Interestingly, sev-

eral pro

ﬁ

brotic genes, including

ADAM17

(a disintegrin and

metalloprotease 17), associated with vascular remodeling and
hypertension (

PDGFR

, a marker of endothelial to mesen-

chymal transition (EndMT), and

FGF1

(

ﬁ

broblast growth factor

1), a cytokine highly abundant in

ﬁ

brotic lungs (

), were

downregulated after treatment with recombinant CCN3 (

Fig.

). We also found genes that were unchanged in response to

either loss or gain of function of CCN3. Of note, among these
unchanged genes, we found important regulators of in

ﬂ

am-

mation and EndMT such as

VEGF

(vascular endothelial

growth factor),

IL6

(interleukin-6), and

TGFB

(transforming

growth factor beta).

Altogether, these data show that CCN3 is associated with

transcriptional signatures important for endothelial function,
including endothelial homeostasis, cell survival, and in

ﬂ

am-

mation/

ﬁ

brosis.

Loss of CCN3 in Human Lung ECs Enhances Lung
Fibroblast Activation via Paracrine Signals

We have previously demonstrated that in the setting of

lung

ﬁ

brosis, normal lung ECs facilitate the deactivation of

lung

ﬁ

broblasts via soluble paracrine signals, and that aging-

associated endothelial dysfunction impairs this effect (

Our data showing increased transcripts for proin

ﬂ

ammatory

genes and reduced transcripts for anti

ﬁ

brotic genes in

CCN3-silenced HLMECs led us to investigate whether these
endothelial alterations could in

ﬂ

uence the activation of nor-

mal lung

ﬁ

broblasts through the secretion of soluble factors.

To identify putative pro

ﬁ

brotic factors that could be secreted

from CCN3-silenced HLMECs and subsequently activating

ﬁ

brotic programs in neighboring

ﬁ

broblasts, results from the

PCR array were further analyzed for transcripts encoding
secreted factors. We found a signi

ﬁ

cant increase of

TIMP1

(tissue inhibitor of metalloproteinase), involved in regulat-
ing the production and degradation of ECM proteins (

In addition, we found a signi

ﬁ

cant reduction in

PLAU

(uroki-

nase plasminogen activator) (

Fig. 3

), whose reduction has

been related to the development of lung

ﬁ

brosis (

) and

IL7

(Interleukin-7), recognized to inhibit TGF

production

Figure 3.

Loss of CCN3 in human lung ECs

enhances lung

ﬁ

broblast activation via para-

crine signals.

: loss of CCN3 in HLMECs

leads to alterations in genes encoding for
secreted factors

IL7, TIMP1,

and

PLAU.

Data

are expressed as means ± SD and analyzed
using Student

’

test (

= 0.08,

= 0.12,

0.05).

: schematic of the experimen-

tal work

ﬂ

ow. Conditioned media was col-

lected and concentrated from HLMECs
transfected with either nontargeting or

CCN3

siRNA (72 h). The conditioned media

was then applied to NHLFs primed with 2
ng/mL TGF-

and RT-PCR was performed

to evaluate pro

ﬁ

brotic gene expression

on recipient

ﬁ

broblasts.

: primed

ﬁ

bro-

blasts cultured in conditioned medium
collected from CCN3-silenced HLMECs
displayed elevated levels of pro

ﬁ

brotic

markers

COL1A1

and

ACTA2

compared

with

ﬁ

broblasts exposed to conditioned

medium generated from control silenced
HLMECs. Data are expressed as means ±
SD from

= 3 independent experiments

and analyzed using Student

’

test (

0.1 and

= 0.054, respectively).

ﬁ

bro-

blasts were primed with TGF

and then

exposed for 3 days to conditioned media
collected from control or CCN3-silenced
HLMECs. At the end of the incubation,
cells were stained with phalloidin to visu-
alize cell morphology and counterstained
with DAPI. Cells exposed to conditioned
medium generated from CCN3-silenced
HLMECs exhibit a more elongated pheno-
type. EC, endothelial cell; HLMEC, human
lung microvascular endothelial cell; NHLF,
normal human lung

ﬁ

broblast. [Image cre-

ated with BioRender.com and published
with permission.]

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exogeneous human recombinant CCN3 protein to HLMECs
and observed increased cell migration in comparison with
untreated cells (

Fig. 4,

and

). Thus, these data show that

upon knockdown of

CCN3

levels, endothelial migratory

function is impaired, whereas the addition of exogeneous
CCN3 stimulates endothelial migration. Through further
analysis of the PCR array results from CCN3-silenced cells,
we found a signi

ﬁ

cant reduction of promigratory genes,

including

FN1

(

ﬁ

bronectin 1) (

ITGAV

(integrin alpha v

subunit) (

), and

MMP1

(

) (

Fig. 4

). However, treatment

with exogeneous recombinant CCN3 protein also led to a sig-
ni

ﬁ

cant reduction in

MMP1

, thus suggesting that migratory

function was determined by a combination of transcrip-
tional alterations (

Fig. 4

). Altogether, our data support the

role of CCN3 in promoting endothelial cell migration.

CCN3 Supports Lung Endothelial Barrier Integrity

In the normal lung, the endothelial barrier is tightly regu-

lated to form a con

ﬂ

uent vascular monolayer, regulating the

paracellular transport of plasma proteins and solutes (

Upon

ﬁ

brotic injury, disruption of the endothelial monolayer

results in the formation of paracellular gaps and increased
vascular permeability (

). Clinically, increases in vascular

permeability have been observed in patients with IPF where
the degree of leakiness is associated with the severity of the
disease (

To evaluate whether CCN3 could be involved in maintaining

the con

ﬂ

uent endothelial monolayer by regulating intercellular

gaps, we transfected a con

ﬂ

uent monolayer of HLMECs with

control siRNA or

CCN3

siRNA. Immuno

ﬂ

uorescence was per-

formed to stain for VE-cadherin, a key intercellular junction
molecule. We observed that control HLMECs display continu-
ous and ordered cell-cell junctions along with a few intercellu-
lar gaps, while the loss of

CCN3

resulted in increased presence

of intercellular gaps and lack of continuous cell-cell junctions
(

Fig. 5,

and

Quanti

ﬁ

cation of total gap area (

Fig. 5

) and size and per-

cent of gap area (Supplemental Fig. S1) con

ﬁ

rmed this obser-

vation. We also observed that upon CCN3 silencing, HLMECs
lose the continuous band along cell peripheries, which were
evident in control cells and instead form a more serrated or
zig-zag pattern, consistent with a phenotype of disrupted

Figure 5.

Loss of CCN3 impairs endothelial

barrier integrity.

: representative immuno

ﬂ

orescence images (

10 objective magni

ﬁ

ca-

tion) of HLMECs transfected with control or

CCN3

siRNA (72 h), stained for VE-cadherin

(green) and Phalloidin (red) (F-actin). Nuclei
were counterstained for DAPI. Arrows high-
light the inter-cellular gaps.

: arrows highlight

the intercellular gaps.

: three frames per well

were analyzed (

40 cells) and total gap

area was quanti

ﬁ

ed via ImageJ and normalized

for cell count (obtained through quanti

ﬁ

cation of

nuclei visualized by DAPI). Data distribution is visual-
ized via violin plot from control siRNA (

= 15 images)

and

CCN3

siRNA (

= 16 images) expressing

the median as the dotted line. Data are ana-
lyzed using Student

’

test,

= 3 independent

experiments (

0.0001).

: loss of CCN3

in HLMECs leads to reduction in genes associ-
ated with endothelial barrier integrity. Data are
expressed as means ± SD and analyzed using
Student

’

test (

= 0.11,

0.01). DAPI,

,6-diamidino-2-phenylindole; HLMEC, human

lung microvascular endothelial cell.

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endothelial barrier, as previously described (

). These obser-

vations are consistent with transcriptional

ﬁ

ndings as further

analysis of the PCR array results from CCN3-silenced cells
highlighted a reduction in

SPHK1

(sphingosine kinase 1), a

protein involved in maintaining endothelial barrier integrity
via phosphorylation of sphingosine to sphingosine-1 phos-
phate (

), and

COL18A1,

a proteoglycan whose reduc-

tion is associated with increased vascular permeability
(

) (

Fig. 5

). These

ﬁ

ndings demonstrate that

CCN3

plays

an important and complex role in the maintenance of lung
vascular barrier integrity.

CCN3 Stimulates in Vitro Angiogenesis in Human Lung
ECs

Angiogenesis is de

ﬁ

ned as the growth of new blood ves-

sels from preexisting ones (

). This process is critical and

necessary to support tissue repair after injury, and its fail-
ure has been linked to impaired wound healing (

). To

evaluate whether silencing of

CCN3

impacts lung endothe-

lial angiogenic capacity, we performed an in vitro angio-
genesis assay. Equal number of control and CCN3-silenced
HLMECs were placed on Matrigel (Supplemental Fig. S2

)

and stimulated with VEGFA and sphingosine-1-phosphate
(S1P) as proangiogenic cocktail (

), where VEGFA serves

as a proangiogenic factor (

) and S1P promotes cell

migration and capillary-like network formation in vitro
(

). Quanti

ﬁ

cation of total number of junctions and

total vessel length indicated that loss of CCN3 signi

ﬁ

cantly

impairs vascular network formation (

Fig. 6,

and

). This

observation was supported by the transcriptional reduc-
tion of proangiogenic genes:

PGF

(placental growth factor)

(

) and

EDN1

(endothelin-1) (

) (

), and by the ele-

vation of

TYMP

(thymidine phosphorylase), this latter

involved in aberrant angiogenesis (

) (

). To test the

effect of exogenous recombinant CCN3 on in vitro angio-
genesis, we performed the in vitro angiogenesis assay
stimulating the cells with VEGF in the absence of S1P,
allowing us to assess whether CCN3 could still support in
vitro angiogenesis in an environment where promigratory
cues are lacking. We found that the addition of exogeneous
human recombinant CCN3 protein to VEGFA-stimulated-
HLMECs improved endothelial network formation (

Fig. 6,

and

) in comparison to HLMECs treated with VEGF-A

alone. In addition, we observed a signi

ﬁ

cant increase in

the number of junctions and the total vessels length in the
presence of both recombinant CCN3 and VEGFA in com-
parison to VEGFA alone (

). Interestingly, we also

found a signi

ﬁ

cant reduction of

TYMP,

involved in aber-

rant angiogenesis, in our PCR array (

Then, we assessed whether this effect could be mediated

by differences in cell proliferation of HLMECs in response
to CCN3 loss or gain of function. Immuno

ﬂ

uorescence

staining and analysis of the proliferation marker Ki67 (

Fig.

and

) showed no signi

ﬁ

cant differences in prolifera-

tion of HLMECs following manipulation of CCN3 levels.
These

ﬁ

ndings, together with our previous migration

experiments, suggest that the effect of CCN3 in supporting
in vitro angiogenesis is not dependent on increased cell
proliferation, but rather mediated by the promotion of en-
dothelial cell migration.

DISCUSSION

Mounting evidence has revealed the involvement of endo-

thelial dysfunction in the progression of various lung-related
diseases (

). Recent

ﬁ

ndings from our group and others

have highlighted the importance of the pulmonary vascula-
ture in orchestrating wound-healing responses after lung
injury. These responses are mediated by endothelial-derived
paracrine factors with proreparative and anti

ﬁ

brotic effects

on neighboring cells, including epithelial cells (

), inter-

stitial macrophages (

), and interstitial

ﬁ

broblasts (

). In

addition, pulmonary ECs secrete basement membrane pro-
teins (

), that play an active role in epithelial-endothelial

association and alveolarization during development (

but that also promote vascular and epithelial regeneration in
response to injury (

). Hence, studies investigating the con-

tribution of the pulmonary vasculature in the progression of
chronic lung diseases with underlying vascular abnormal-
ities, such as IPF (

), may hold translational potential.

Here, we found that the matricellular protein CCN3

plays an important role in regulating homeostasis and
function of lung ECs. Transcriptional analysis of freshly
sorted lung ECs showed a transient upregulation of

Ccn3

during the resolution phase of bleomycin-induced lung

ﬁ

brosis in young, but not aged mice, suggesting an impor-
tant role for

Ccn3

in promoting endothelial function

during resolution of lung

ﬁ

brosis (

In vitro studies showed that the loss of CCN3 in HLMECs

leads to endothelial transcriptional alterations and impairs
endothelial migratory and angiogenic capabilities, along
with barrier integrity. Our data showing increased intercellu-
lar gaps after CCN3 silencing in HLMECs suggest disruption
of the endothelial barrier. This phenotype is relevant for
human disease as it is widely recognized that patients with
IPF have increased pulmonary vascular permeability, which
correlates with worse outcomes (

). These data suggest

that in aged mice, there is a correlation between the lack of
upregulation of

Ccn3

in lung ECs with poor endothelial func-

tion and failure of resolution. Interestingly, addition of
recombinant CCN3 protein improves endothelial function as
assessed by migration and in vitro angiogenesis assays in
HLMECs. Notably, a recent work investigated the role of
CCN3 in the vasculature of patients affected by systemic
sclerosis (SSc), a connective tissue disease with underlying
vasculopathy and autoimmune features, leading to organ

ﬁ

brosis, including lung

ﬁ

brosis (

). This study reported

reduced CCN3 expression in dermal blood vessels of patients
with SSc and showed that recombinant CCN3 promotes both
migration and angiogenesis of human dermal microvascular
ECs in culture (

). Our

ﬁ

ndings highlighting the importance

of CCN3 in the maintenance of vascular homeostasis and
function are consistent with these previous observations.
Interestingly, our data demonstrate that manipulation of
CCN3 levels has no signi

ﬁ

cant impact on lung endothelial

proliferation, in agreement with previous studies showing
no effect of CCN3 on endothelial proliferation of HUVEC (

)

and HDMECs (

). Thus, improved angiogenic function

upon augmentation of CCN3 levels is attributed to the pro-
motion of endothelial migration and organization of the vas-
cular network, rather than cell proliferation. In addition, our

ﬁ

ndings revealing altered expression of in

ﬂ

ammatory and

CCN3 SUPPORTS LUNG ENDOTHELIAL FUNCTION

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ﬁ

brotic genes upon loss and gain of function of CCN3 in lung

ECs are in agreement with a previous study demonstrating
that CCN3 reduces nuclear factor kappa B (NF-

B) nuclear

translocation, regulating endothelial in

ﬂ

ammatory activa-

tion in cultured HUVEC (

One potential mechanism through which CCN3 can exert

its bene

ﬁ

cial effect on vascular ECs is through binding of

surface integrin receptors, particularly integrins

and

(

). Using in vitro cultures of HUVECs, it has been

demonstrated that CCN3 mediates endothelial survival
through integrin

, endothelial adhesion through integ-

rins

, and

and endothelial migration through

integrins

and

(

). These processes mediated by

integrins are critical for angiogenesis and vascular remod-
eling during development and wound healing as well as
pathological tissue remodeling (

100

). Of particular inter-

est, integrin

is prominently expressed throughout the

vasculature, at low levels in quiescent vasculature but

Figure 6.

Loss of CCN3 impairs in vitro angiogenic function while exogeneous CCN3 recovers this function.

: schematic depicting the experimental

work

ﬂ

ow. Control- and CCN3-silenced HLMECs were plated on polymerized Matrigel basement membrane for 6 h and analyzed for the ability to form

tube-like structures.

: representative images (

10 objective magni

ﬁ

cation) of HLMECs transfected with control or

CCN3

siRNA (72 h) indicate that loss

of CCN3 resulted in a more disordered, less continuous network.

: quanti

ﬁ

cation of total number of junctions and total vessel length, parameters of in

vitro angiogenesis, via AngioTool 0.6 software shows reduction of both parameters in CCN3-silenced cells compared with control cells.

: schematic

depicting the experimental work

ﬂ

ow. Control HLMECs cells or those treated with 100 ng/mL recombinant CCN3 were plated on Matrigel basement

membrane for 6 h and analyzed for the ability to form tube-like structures.

: representative images (

10 objective magni

ﬁ

cation) of HLMECs treated

with 0 ng/mL or 100 ng/mL human recombinant CCN3 protein indicate that exogeneous CCN3 promoted a more continuous, ordered network.

: quanti-

ﬁ

cation via AngioTool 0.6 software shows increases in total number of junctions and total vessels length in HLMECs treated with recombinant CCN3 in

comparison with untreated control cells de

ﬁ

cient in S1P. Data are expressed as means ± SD from

= 3 independent experiments and analyzed using

Student

’

test (

0.05,

0.01).

: loss and gain of function of CCN3 in HLMECs leads to changes in genes associated with angiogenesis. Data

are expressed as means ± SD and analyzed using Student

’

test (

= 0.057,

0.05,

0.01,

0.0001). Representative immuno

ﬂ

uores-

cence images (

10 objective magni

ﬁ

cation) of HLMECs transfected with control or

CCN3

siRNA (72 h) (

) and HLMECs treated with 0 ng/mL or 100 ng/

mL human recombinant CCN3 protein (

), stained for Ki67 (green). Nuclei were counterstained with DAPI. Quanti

ﬁ

cation of percent of Ki67 positive, DAPI

positive cells, via Bio-Tek Gen5 Image software shows no signi

ﬁ

cant change in CCN3-silenced cells (

) and recombinant CCN3 protein treated cells

compared with respective controls (

). Data are expressed as means ± SD from

= 3 independent experiments and analyzed using Student

’

test (

0.05,

0.01). DAPI, 4

,6-diamidino-2-phenylindole; HLMEC, human lung microvascular endothelial cells. [Image created with BioRender.com and

published with permission.]

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induced in response to cytokines and growth factors dur-
ing vascular remodeling (

100

) and has been previously

established as critical for angiogenic function both in
vitro and in vivo (

100

101

). Consistent with these studies,

we found that integrin

, implicated in endothelial

migration (

), is downregulated in CCN3-silenced cells,

further highlighting the involvement of integrin signal-
ing in mediating CCN3 function. Altogether, these
studies strongly support a role of CCN3 in promoting en-
dothelial homeostasis, migration, and in vitro angiogene-
sis. Further investigation will be needed to de

ﬁ

ne the link

between CCN3 expression, integrins involved in pulmo-
nary vascular remodeling, and experimental models of
persistent lung

ﬁ

brosis.

Recent evidence has shown that endothelial-derived

soluble signals to neighboring cells are important to pro-
mote alveolar epithelial repair (

102

103

) and can in

ﬂ

uence

ﬁ

broblast activation (

). With regards to endothelial-

mesenchymal cross talk, previous work from our labora-
tory has demonstrated that normal lung ECs possess the
ability to promote lung

ﬁ

broblast deactivation (

). In addi-

tion, our group and others have demonstrated that dys-
functional lung ECs lose this ability, and instead promote

ﬁ

broblast activation through the release of pro

ﬁ

brotic

soluble factors (

). In line with these studies, we

demonstrated that upon loss of

CCN3

, conditioned media

from these altered lung ECs activated pro

ﬁ

brotic programs

in recipient normal lung

ﬁ

broblasts. Interestingly, we

observed downregulation of the anti

ﬁ

brotic genes

PLAU

and

IL17,

and upregulation of the pro

ﬁ

brotic gene

TIMP1

Thus, indicating that modulation of CCN3 expression
altered the expression and secretion of soluble factors
involved in paracrine communication between the lung
vasculature and

ﬁ

broblasts. Further work will be required

to reveal the contributions of individual secreted soluble
factors to the endothelial paracrine effect on lung

ﬁ

bro-

blasts. These

ﬁ

ndings suggest that perturbations in CCN3

levels in lung ECs, as seen in the pulmonary vasculature of
the aged animals, promote

ﬁ

broblast activation, contribut-

ing to the development of a pro

ﬁ

brotic environment that

facilitates persistent lung

ﬁ

brosis.

Another potential mechanism through which altered

endothelial CCN3 expression promotes a pro

ﬁ

brotic envi-

ronment could be related to the modi

ﬁ

cation of mechani-

cal properties of the microenvironment. It has been
shown that CCN3 counteracts the level of the known pro

ﬁ

brotic marker CCN2 (CTGF) in dermal and mesangial

ﬁ

broblasts (

), and our present work con

ﬁ

rms that

this effect can be extended to the pulmonary endothe-
lium. In addition, CTGF is overexpressed in

ﬁ

brotic dis-

eases in multiple organs, including lungs (

104

–

106

where it promotes lung

ﬁ

broblast activation, abundant

ECM synthesis, and increased local tissue stiffness (

107

https://doi.org/10.6084/m9.

110

). Since increased rigidity of the microenvironment

behaves as a positive feedback loop, further promoting

ﬁ

broblast activation (

), an important extension of

this work will be to de

ﬁ

ne the ability of lung ECs to modify

the surrounding ECM composition and stiffness through
secretion of CCN3 and whether altered endothelial CCN3
levels leads to pathological mechanical changes that exac-
erbate lung

ﬁ

brosis.

An implication of this study is the need to identify thera-

peutic strategies to increase CCN3 levels and function in the
pulmonary vasculature. Our in vivo data are consistent with
transient elevations in CCN3 promoting vascular repair and

ﬁ

brosis resolution. Thus, an important extension of this

work will include the development of therapeutic strategies
to augment endogenous levels of lung endothelial CCN3. To
avoid systemic exposure to a proangiogenic and migratory
stimulus, tools for localized

CCN3

mRNA delivery such as

lung endothelial-speci

ﬁ

c lipid nanoparticles may be imple-

mented. Future studies aimed at restoring endothelial CCN3
levels during experimentally induced lung

ﬁ

brosis in aged

mice, where the unresolved injury that better recapitulates
the persistent

ﬁ

brosis of IPF patients, will be needed to

address the translational potential of our

ﬁ

ndings. In terms

of clinical relevance, additional work to assess the levels of
CCN3 in lung specimens from patients with IPF is warranted
to corroborate CCN3 as a potential therapeutic target to pro-
mote vascular repair in lung

ﬁ

brosis. In conclusion, our work

suggests that endothelial CCN3 regulates lung endothelial
homeostasis and function and plays an important role in
promoting lung vascular regeneration, thereby supporting
lung repair after injury. Altogether, our study suggests that
targeting endothelial CCN3 could represent a potential new
strategy to ameliorate the vascular abnormalities that
accompany the progression of IPF.

DATA AVAILABILITY

Data will be made available upon reasonable request.

SUPPLEMENTAL DATA

Supplemental Tables S1 and S2 and Supplemental Figs. S1

–

S3:

ﬁ

gshare.21583092.v2

ACKNOWLEDGMENTS

Graphical abstract image created with BioRender.com and

published with permission.

GRANTS

This work was supported by National Institutes of Health (NIH)

grant HL092961 (to D.J.T.), HL158733 (to G.L.), HL159704 (to
P.A.L), HL158018 (to P.A.L), HL153026 (to P.A.L), NIH T32 HL
105355 Training Program in Lung Physiology and Biomedical
Engineering (to P.A.L and K.R.B.), the American Lung Association
Dalsemer Award (to N.C.) and the Boehringer Ingelheim Discovery
Award IPF/ILD (to N.C.).

DISCLOSURES

No con

ﬂ

icts of interest,

ﬁ

nancial or otherwise, are declared by

the authors.

AUTHOR CONTRIBUTIONS

K.R.B., D.J.T., and N.C. conceived and designed research; K.R.B.,

P.A.L., and A.J.H. performed experiments; K.R.B., P.A.L., and N.C.
analyzed data; K.R.B. and N.C. interpreted results of experiments;
K.R.B. prepared

ﬁ

gures; K.R.B. drafted manuscript; G.L., D.J.T.,

and N.C. edited and revised manuscript; K.R.B., P.A.L., A.J.H.,
G.L., D.J.T., and N.C. approved

ﬁ

nal version of manuscript.

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