Serum Inter–a-Trypsin Inhibitor and Matrix Hyaluronan
Promote Angiogenesis in Fibrotic Lung Injury
Stavros Garantziotis1, Enrique Zudaire2, Carol S. Trempus1, John W. Hollingsworth3, Dianhua Jiang3,
Lisa H. Lancaster4, Elizabeth Richardson1, Lisheng Zhuo5, Frank Cuttitta2, Kevin K. Brown6, Paul W. Noble3,
Koji Kimata5, and David A. Schwartz1,7
1National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina;2Angiogenesis Core Facility, National Cancer Institute,
Gaithersburg, Maryland;3Duke University Medical Center, Durham, North Carolina;4Vanderbilt University Medical Center, Nashville, Tennessee;
5Institute for Molecular Science of Medicine, Aichi Medical University, Aichi, Japan;6National Jewish Medical and Research Center, Denver,
Colorado; and7National Heart, Lung, and Blood Institute, Bethesda, Maryland
Rationale: The etiology and pathogenesis of angiogenesis in idio-
pathic pulmonary fibrosis (IPF) is poorly understood. Inter-a-trypsin
may contribute to the angiogenic response to tissue injury.
Objectives: To determine whether IaI promotes HA-mediated angio-
genesis in tissue injury.
Methods: An examination was undertaken of angiogenesis in IaI-
sufficient and -deficient mice in the bleomycin model of pulmonary
fibrosis and in angiogenesis assays in vivo and in vitro. IaI and HA in
patients with IPF were examined.
Measurements and Main Results: IaI significantly enhances the angio-
genic response toshort-fragmentHAinvivoand in vitro.laldeficiency
lung angiogenesis after bleomycin exposure in mice. IaI is found in
is particularly strong in vascular areas around fibroblastic foci. Serum
levels of IaI and HA are significantly elevated in patients with IPF
compared with control subjects. High serum IaI and HA levels are
associated with decreased lung diffusing capacity, but not FVC.
Conclusions: Our findings indicate that serum IaI interacts with HA,
the vascular response to lung injury and may lead to aberrant
Clinical trial registered with www.clinicaltrials.gov (NCT00016627).
Keywords: inter–a-trypsin inhibitor; hyaluronan; angiogenesis; pulmo-
Idiopathic pulmonary fibrosis (IPF) is a progressive, lethal
interstitial lung disease, characterized by unremitting scarring
of alveolar tissue and respiratory failure. The histological
correlate of IPF is usual interstitial pneumonitis (UIP). The
biological mechanism of UIP pathogenesis is unknown, but may
involve an aberrant healing response to repetitive tissue injury
(1). Angiogenesis is an essential component of wound healing,
and is likely to be involved in lung repair and the pathogenesis
of UIP. Both human and animal research has investigated
angiogenesis in the development of fibrosis. A seminal article
by Turner-Warwick more than 40 years ago demonstrated
extensive angiogenesis in human IPF lungs (2). However, the
existing literature offers a mixed picture, with some reports
suggesting that a net-angiogenic imbalance exists in UIP lungs
(3), and others showing that angiostatic factors are increased in
fibroblastic foci of UIP lungs (4). Ultimately, these contrasting
results could be explained by an overall temporal and spatial
heterogeneity in angiogenic activity in pulmonary fibrosis (5, 6).
Several factors are likely to affect angiogenesis in IPF. Extra-
cellular matrix deposition is significantly increased in IPF (7).
Matrix components, such as fibronectin, vitronectin, versican, and
hyaluronan, have known angiogenic properties (8–11). Hyalur-
properties (13), whereas short-fragment HA (sHA), which is
produced in inflammation and tissue injury, is highly angiogenic
(14), and mediates its effects via cell receptors, such as CD44 and
RHAMM (10). HA binds to cells most efficiently in complex with
inter–a-trypsin inhibitor (IaI) (15).
IaI is a composite protein containing a light chain (called
urinary trypsin inhibitor [UTI] or bikunin), and two heavy
chains that can bind HA (16). IaI is synthesized in the liver and
secreted into the circulation, where it can be found in high
concentrations of up to 0.5 mg/ml. From the serum, IaI reaches
sites of injury via extravasation. The exact role of IaI is unclear.
However, it is known that IaI has antiinflammatory properties
(17, 18), and that it can bind HA, and thus stabilize extracellular
matrix (19). Furthermore, it is known that HA can promote
inflammation after lung injury (20). We therefore hypothesized
that IaI could reach injured areas in the lung, bind HA, and
have antiinflammatory activity there. We also hypothesized that
AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject
Pulmonary fibrosis leads to respiratory failure and death.
The pathogenesis of pulmonary fibrosis is still unclear, but
angiogenesis is believed to be a significant component.
Extracellular matrix plays an important role in angiogenesis.
What This Study Adds to the Field
Serum inter–a-trypsin inhibitor (IaI) and matrix hyalur-
onan are necessary for the angiogenic response to lung
injury. IaI serum levels are inversely associated with gas
exchange in fibrosis patients, indicating that IaI may be
linked to aberrant angiogenesis.
(Received in original form March 7, 2008; accepted in final form August 12, 2008)
Supported in part by grants from National Institute of Environmental Health
Services (ES11961) and National Heart, Lung, and Blood Institute (HL91335),
and by the Intramural Research Program of the National Institutes of Health, the
National Institute of Environmental Health Sciences, the National Cancer In-
stitute, and the National Heart, Lung, and Blood lnstitute.
Correspondence and requests for reprints should be addressed to Stavros
Garantziotis, M.D., Staff Clinician Medical Director, Clinical Research Unit,
National Institute of Environmental Health Sciences, Research Triangle Park,
NC 27709. E-mail: email@example.com
This article has an online supplement, which is available from the issue’s table of
contents at www.atsjournals.org
Am J Respir Crit Care Med
Originally Published in Press as DOI: 10.1164/rccm.200803-386OC on August 14, 2008
Internet address: www.atsjournals.org
Vol 178. pp 939–947, 2008
IaI may interact with HA and promote angiogenesis in response
to fibrotic lung injury.
In this study, we demonstrate that IaI significantly enhances
the angiogenic effect of sHA in vitro. IaI is necessary in vivo
for angiogenesis in a matrigel model of angiogenesis and the
absence of IaI leads to enhanced inflammation and decreased
angiogenesis in bleomycin-induced lung injury. In patients with
IPF, serum concentrations of IaI and HA are elevated com-
pared with control subjects, and are inversely correlated with
lung diffusion capacity for carbon monoxide (DLCO), but not
FVC measurements. IaI is found in fibroblastic foci in IPF,
where it colocalizes with HA. The colocalization is particularly
strong in vascular areas around fibroblastic foci. In aggregate,
our findings indicate that serum IaI interacts with HA, and
promotes angiogenesis in tissue injury and pulmonary fibrosis.
Some of the results of these studies have been submitted in the
form of an abstract at the International American Thoracic
Society Conference, Toronto, Canada, 2008 (21).
In Vitro Vascular Tube Formation Assay
Human microvascular endothelial cells type 1 (22) were seeded on
grown to 50–70% confluence. Endotoxin-free long-fragment HA (Hea-
lon; Advanced Medical Optics, Santa Ana, CA) was sonicated to
produce sHA (see Figure E1 in the online supplement), and added to
some wells at 0.5 mg/ml, with or without IaI (30 mg/ml). Tubular
structures were photographed after 10 hours’ incubation, and branching
points per node were measured for quantification of angiogenesis.
IaI-deficient mice have been backcrossed onto C57BL/6 for more than
10 generations (19). Littermates were used as control animals. C57BL/6
mice from Jackson Laboratories (Bar Harbor, ME) were used for some
experiments examining expression of IaI heavy chains (ITIH). Mice were
kept in a specific pathogen-free facility. Experiments were approved by
the Duke and National Institute of Environmental Health Sciences
Animal Care and Use Committee.
In Vivo Matrigel Angiogenesis Assay
Matrigel (BD BioSciences, San Jose, CA) mixed with 20 U/ml heparin
and 100 ng bFGF (R&D Systems, Minneapolis, MN) was instilled
subcutaneously (0.5 ml) into isoflurane-anesthetized mice. One plug was
injected per mouse. After 7 days, mice were killed via CO2asphyxiation,
the matrigel plugs were removed and fixed in 10% buffered formalin,
embedded in paraffin, sectioned, and stained. Some plugs were weighed,
homogenized in Dulbecco’s phosphate-buffered saline (DPBS), the
hemoglobin content was measured with Drabkin’s solution (Sigma, St.
Louis, MO) according to the manufacturer’s instructions, and the results
were normalized to 100 mg of matrigel plug (23).
Fibrotic Lung Injury
Mice received 2 U/kg intratracheal bleomycin (Sigma), and were killed
by CO2asphyxiation at different time points after instillation. Lung
lavage was performed with 3 ml cold saline. Cell counts were performed
using the Shandon Cytospin 3 setup (Thermo-Fisher, Waltham, MA)
after cell staining with hematoxylin. The right lungs and liver snips were
snap-frozen and stored at 2808C until used, and the left lungs were
inflated with 10% buffered formalin and paraffin embedded.
Histology and Immunohistochemistry
Formalin-fixed human lungs (from surgical biopsies or autopsies) and
blue, hematoxylin and eosin, or with rabbit anti-IaI (Dako, Carpinteria,
CA), biotinylated HA binding protein (Seikagaku America, East Fal-
mouth, MA), CD31 (Dako), or Factor 8 (Abcam, Cambridge, MA).
Matrigel vessels were stained with CD34 (eBioscience, San Diego, CA),
as described elsewhere (24).
mRNA Expression Analysis
mRNA was isolated from frozen lungs and livers by the Trizol method.
Real-time reverse transcriptase–polymerase chain reaction was per-
formed for the IaI heavy chain 1 (forward GGTCTTTGGCTCTAAA
GTGCAATC and reverse GGTGGCTTCCTTGAGCTTTGT) and
heavy chain 2 (forward GCCATCCACATCTTCAATGAGAG and
reverse CGCTTGAGAAAGCTGTAGAGCTG) using SYBR-Green
assay and the 7900HT sequence detection system (Applied Biosystems,
Foster City, CA). The housekeeping protein 18s RNA was used as the
reference mRNA. Fluorescence values for each gene were normalized
to those of 18s, and expressed as fold change over control groups using
the comparative Ctmethod (25).
Patient recruitment and selection was described in detail elsewhere (26).
Briefly, patients and unaffected control subjects were recruited at
Vanderbilt University (Nashville, TN) and National Jewish Medical
and Research Center (Denver, CO), after approval by the institutional
review boards and issue of a certificate of confidentiality from the
National Institutes of Health. Standard criteria were used to establish
the diagnosis of IPF (27, 28). After informed consent, all subjects
underwent blood draw and FVC and DLCOmeasurements.
Serum IaI Measurement
Serum IaI levels were determined by using an inhibitory ELISA for the
IaI light-chain UTI/bikunin. ELISA plates were coated with purified
UTI (Mochida Pharmaceutical, Tokyo, Japan) at 48C overnight, and
blocked with 3% bovine serum albumin. Totals of 50 ml each of sample
or standard (0–25 mg/ml) and 50 ml each of diluted rabbit anti-human
UTI antiserum were added to the well, and the plates were incubated
at 378C for 1 hour. Detection was performed with horseradish
peroxidase–conjugated goat anti-rabbit IgG. Color development was
performed with TMB solution (Kirkegaard and Perry, Gaithersburg,
MA), and the absorbance at 450 nm was measured on a Molecular
Devices VersaMAX spectrophotometer (Sunnyvale, CA). Assays were
performed in triplicate.
Analysis of variance, Student’s t test, and the chi-square test were
performed using SPSS (SPSS, Inc., Chicago, IL) and GraphPad (Graph-
statistically significant. Results are represented as mean (6SEM).
IaI Enhances the Angiogenic Effect of sHA In Vitro
It is known that IaI binds to HA in the extracellular matrix and
potentiates CD44–HA binding (29). We therefore hypothesized
that IaI promotes angiogenesis through enhanced sHA binding.
We first examined the effect of IaI on vascular tube formation
in vitro. We found that human microvascular endothelial cells
type 1 have more robust angiogenic response to sHA when IaI
is added to the medium (Figure 1A). We quantified the effect
by counting the number of vascular tubes per ‘‘node’’ (i.e.,
branching point), a measure of network complexity. At a con-
centration of 0.5/ml, sHA had no significant effect on vascular
network formation compared with control. Addition of IaI to
sHA significantly enhanced vascular tube formation and com-
plexity (Figure 1B). Long-fragment HA and IaI alone had no
effect on angiogenesis in these experiments. Addition of IaI to
the long-fragment HA medium did not change the long-
fragment HA response (data not shown), indicating that IaI is
important for the angiogenic effect of sHA binding. Interest-
ingly, although the IaI-sHA addition produced similar branch
points per node as the positive control (fetal bovine serum), the
940 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 1782008
vascular tubes had a less mature appearance, suggesting that
other factors besides HA are important for the development of
IaI Is Necessary for In Vivo Angiogenesis in the Murine
To further examine whether IaI was necessary for angiogenesis
in the whole animal, we used an in vivo matrigel angiogenesis
model. We found that IaI-deficient mice (Figure 2A, top row)
had minimal angiogenesis into the matrigel plug as compared
with IaI-sufficient mice (Figure 2A, bottom row). When we
measured the hemoglobin content of the plugs as a means of
quantification of angiogenesis, we saw a statistically significant
difference between IaI-sufficient and -deficient mice. This was
wholly attributable to the absence of hemoglobin and angio-
genesis in plugs from IaI-deficient mice (Figures 2B–2C).
IaI Is Expressed in the Liver in Response to Lung Injury, but
Reaches the Lung during Inflammation and Fibrosis
Angiogenesis is considered important in the pathogenesis of
pulmonary fibrosis (4–6). We therefore evaluated the effect of
IaI in a murine model of fibrotic lung injury. First, we de-
termined the origin of IaI in injured lung by examining IaI
heavy-chain expression after intratracheal bleomycin exposure.
We examined lung and liver mRNA expression of IaI heavy
chains ITIH1 and ITIH2, because they are components of
circulating IaI in the mouse. At baseline, there was a roughly
50-fold higher mRNA expression of IaI heavy chains ITIH1 and
Figure 1. In vitro vascular tube
formation by human microvas-
cular endothelial cells type 1.
(A) Short-fragment hyaluronan
(sHA) induces vascular tube
formation, an effect that is
significantly enhanced by in-
Negative control was 0% fetal
bovine serum (FBS). Positive
control was 5% FBS. (B) Quan-
tification of vascular tube for-
comparison testing; n 5 6
wells/group). Arrowheads indi-
cate vascular nodes. Error bars
assay. (A) Photographs of matrigel plugs
explanted from inter–a-trypsin inhibitor
(IaI)–deficient mice (top panels) or IaI-
sufficient mice (bottom panels) 7 days
after injection. There is a virtual absence
of vascularization in plugs from IaI-
deficient mice. (B) Quantification of plug
hemoglobin content. No hemoglobin
was detected in plugs from IaI-deficient
mice. The difference from IaI-sufficient
littermates is statistically significant. Error
bars represent SEM. (C) Histologic exam-
ination of matrigel plugs. There were no
visible cells or angiogenesis in plugs re-
moved from IaI-deficient mice (top panel)
compared with IaI-sufficient mice (bot-
tom panel, arrows) (CD34 staining; orig-
inal magnification 5 3200; n 5 7 mice/
In vivo matrigel angiogenesis
Garantziotis, Zudaire, Trempus, et al.: IaI/Hyaluronan Mediate Angiogenesis in Injury 941
ITIH2 in the liver compared with the lung (Figures 3A and 3B).
We noted a significant up-regulation of heavy-chain mRNA
expression in the liver in response to bleomycin, peaking
between 7 and 14 days after bleomycin lung injury (Figure
3C). In contrast, the initially low mRNA expression of heavy
chains in the lung declined even further, possibly due to influx
of inflammatory cells and fibroblasts that do not express IaI
mRNA (Figure 3D). In nonfibrotic lungs, IaI and HA staining is
evident only in the subbronchial and subendothelial space (30).
However, fibrotic lungs stained strongly for IaI after bleomycin
exposure (Figures 3E–3F), and IaI colocalized with HA in
fibrotic areas of the lung (Figure 3G). Similar results were
obtained at 7 and 14 days after bleomycin exposure (data not
shown). This was in contrast to naive lungs, where there is little
HA–IaI colocalization (Figure 3H). These results suggest that,
after bleomycin lung injury, IaI message is primarily expressed
in the liver, and IaI protein is reaching the lung via the circu-
lation rather than through local lung production.
IaI Ameliorates Inflammation after Bleomycin Exposure and
Promotes Angiogenesis in Response to Lung Injury
We then examined the effect of IaI on lung inflammation after
bleomycin exposure. IaI-deficient mice demonstrated signifi-
cantly increased cellular inflammation after bleomycin exposure
compared with IaI-sufficient mice (Figures E2A and E2B). This
effect persisted throughout 21 days of observation (Figure
E2C). Whole-lung lavage HA levels were significantly higher
in IaI-deficient mice compared with IaI-sufficient mice (Figure
E2D). There was no difference in the degree of fibrosis by
histology and lung collagen quantification at 21, 28, and 42 days
after bleomycin exposure (data not shown). We examined
angiogenesis in IaI-deficient and -sufficient mice at baseline, 7,
inhibitor (IaI) heavy chains in liver and
lung at baseline and after intratracheal
bleomycin exposure in C57BL/6 mice.
(A) inter–a-trypsin inhibitor heavy chain
(ITIH1) and (B) ITIH2 are expressed in
liver (arbitrarily set at 100% reference)
more than 50-fold than in lung at base-
line. (C) After bleomycin exposure, ITIH1
expression in the liver (open squares) is
up-regulated twofold and ITIH2 expres-
sion (open triangles) is up-regulated more
than sixfold. The highest up-regulation is
seen between Days 7 and 14 after bleo-
mycin (*P , 0.001). (D) Lung ITIH1 and
ITIH2 expression decreases after bleomy-
cin exposure. Error bars represent SEM.
(E) IaI is detectable through immunohis-
tochemistry (brown staining) in fibrotic
lungs 3 weeks after bleomycin exposure
(original magnification 5 340). (F) Iso-
type control for (E). (G) Staining of
bleomycin-exposed lung for hyaluronan
(left panel), and IaI (middle panel). There
is hyaluronan–IaI colocalization evident
in yellow (right panel) (original magnifi-
cation 5 3200). (H) Staining of unex-
posed lung for hyaluronan (left panel)
and IaI (middle panel). Hyaluronan is
mainly in the subepithelial space, and
IaI mainly in bronchial epithelia, as well
as in vessels (a result of serum IaI) with
little colocalization (original magnifca-
Expression of inter–a-trypsin
942 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 1782008
14, and 21 days after bleomycin lung injury through factor VIII
staining. No difference was observed at baseline (Figures 4A
and 4B). IaI-sufficient mice (Figure 4C) had higher vascular
density in fibrotic areas than IaI-deficient mice (Figure 4D),
a difference that was statistically significant after quantification
(Figure 4E) at Days 14 and 21 after bleomycin exposure.
IaI and HA Colocalize in UIP Lungs
We examined whether IaI is found in the lung tissue of patients
with IPF. We stained lungs for IaI and HA in patients with
histologically verified UIP. We found that fibroblastic foci in
UIP lungs (Figure 5A) stained positive for IaI (Figure 5B). HA
staining was also strongly positive in fibroblastic foci, evidenced
difference was observed in baseline vessels between (A)
inter–a-trypsin inhibitor (IaI)-sufficient and (B) IaI-deficient
IaI-sufficient mice show more vascularity in fibrotic areas
than (D) IaI-deficient mice (factor VIII immunohistochem-
istry; original magnification 5 3100). (E) The difference in
vascularity is statistically significant when quantified. Two
independent observers wereinvolvedin thismeasurement.
One observer took photographs of all fibrotic areas in
a longitudinal section of lung, and a second, blinded
14 per group). Error bars represent SEM.
Angiogenesis in bleomycin-exposed mice. No
localize in fibroblastic foci in usual interstitial pneumonitis
(UIP). (A) Representative photomicrograph of a fibroblastic
focus in a patient with UIP (hematoxylin and eosin;
original magnification 5 3400). (B) Same fibroblastic
focus stained for IaI (original magnification 5 3400). IaI
is present in the fibroblastic focus, staining most strongly
in the margins. (C and D). Alcian blue staining of a lung
with UIP fibroblastic foci (arrows) with (C) or without (D)
hyaluronidase pretreatment (original magnification 5
3200). Hyaluronidase treatment significantly diminishes
the blue–green staining within the fibroblastic focus.
Inter–a-trypsin inhibitor (IaI) and hyaluronan
Garantziotis, Zudaire, Trempus, et al.: IaI/Hyaluronan Mediate Angiogenesis in Injury 943
by positive Alcian blue staining, which was abolished by
hyaluronidase pretreatment (Figures 5C and 5D). Confocal
immunohistochemistry showed that IaI and HA colocalized in
fibroblastic foci (Figure6).
appeared particularly strong in the periphery of fibroblastic
foci, where blood vessels are found (Figures 7A–7D). Further-
more, colocalization of IaI and HA was most prominent in the
subendothelial space of blood vessels adjacent to fibroblastic
foci (Figures 7E–7G). We then stained lungs with different
types of interstitial fibrotic lung diseases for IaI (Figure E3).
Healthy lungs and lungs with hypersensitivity pneumonitis,
acute interstitial pneumonia, and nonspecific interstitial pneu-
monia stained faintly for IaI, and the staining was found mostly
in vessels and in the subendothelial basal membrane, as has
been previously described (Figures E3A–E3C). Sarcoidosis
lungs stained positive for IaI in the margins of sarcoidosis
nodules (Figure E3D). UIP lungs stained positive in fibroblastic
foci (Figure E3E). These results suggest that fibroblastic foci in
UIP constitute a unique type of tissue injury among other
interstitial lung diseases, where IaI can be found.
colocalize in fibroblastic foci in usual interstitial pneumo-
nitis (UIP). (A) Hematoxylin–eosin stain of a fibroblastic
focus (original magnification 5 3400). (B) Immunohisto-
chemistry for IaI (red; original magnification 5 3400). (C)
Immunohistochemistry for hyaluronan (green; original mag-
nification 5 3400). (D) Merged image for IaI and hyalur-
onan colocalization (yellow; original magnification 5 3400).
Inter–a-trypsin inhibitor (IaI) and hyaluronan
and hyaluronan colocalize in the suben-
dothelial space. (A) Fibroblastic focus
stained for hematoxylin and eosin. The
same fibroblastic focus stains positive for
(B) hyaluronan and (C) IaI, particularly in
the periphery (arrows), where vessels are
marked with CD31 (D) (3200 original
magnification). The subendothelial area
and basal membrane of a small-sized
blood vessel stain positive for (E) IaI and
(F) hyaluronan, which colocalize in this
location (G) (original magnification 5
Inter–a-trypsin inhibitor (IaI)
944 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 1782008
IaI Serum Concentrations Are Increased in Sera of
Patients with IPF
We measured the serum concentrations of IaI in 44 patients
with IPF and 43 control subjects (Figure 8). We found that
patients with IPF had significantly higher serum IaI concen-
trations compared with control subjects (Figure 8A). We then
investigated the association of serum IaI concentration with
parameters of lung function in 30 of the 44 patients, for whom
complete lung function data were available. Serum IaI concen-
trations did not correlate with FVC (Figure 8B), but showed
a significant inverse correlation with the DLCOmeasurements in
patients with IPF (Figure 8C). To ensure that our correlations
were not skewed by outliers, we compared patients with IPF in
the lower 50% range of IaI levels to patients in the upper 50%
range of IaI levels. There were no significant differences
between groups with regard to male:female ratio, smoker:
nonsmoker ratio, and age (Table 1). Patients with low or high
IaI serum concentration did not differ in regard to FVC,
indicating that serum IaI is not a specific marker of fibrosis
severity (Figure 8D). However, patients with low serum IaI
concentration had significantly higher DLCOlevels than patients
with high serum IaI (Figure 8E). In aggregate, these results
indicate that serum IaI concentrations are associated with
severity of deficits in gas exchange in patients with IPF.
The development of lung fibrosis is believed to be associated
with aberrant injury and repair. Angiogenesis is required for the
normal healing response to tissue injury. Newly formed vessels
deliver nutrients and inflammatory cells to the injury site, thus
promoting wound healing. In chronic diseases like IPF, this
balance may be disrupted, leading to aberrant angiogenesis with
detrimental rather than salutary effects. Our findings indicate
that serum IaI interacts with HA, and promotes angiogenesis in
lung injury. This conclusion is supported by several comple-
mentary approaches. First, we show that, in vitro, IaI promotes
the angiogenic effects of sHA. Second, we show that, in lung
tissue injury, IaI interacts with HA to establish the angiogenic
response. This underscores the synergistic roles of systemic and
local factors in the course of chronic lung injury, and suggests
that IaI may play a role in IPF. Third, we demonstrate that,
among patients with IPF, serum IaI concentrations correlate
with a disproportional decrease in DLCO, independent of re-
striction (FVC), and colocalize with HA in the fibroblastic foci.
Our findings suggest that IaI contributes to the vascular re-
sponse to lung injury, and may lead to aberrant angiogenesis.
This article is the first to document that IaI is important for
HA-mediated angiogenesis in relevant models of tissue injury
and repair. Our results suggest that circulating IaI is the source
of the IaI found in injured tissue, and that extravasation of
serum IaI in regions of tissue injury can bind local HA and
facilitate neovascularization as a component of tissue repair. In
this way, IaI may actually provide a positive angiogenic
feedback loop in tissue injury–vessels deliver IaI in the area
of injury, and IaI enhances local HA binding to promote further
angiogenesis. It is interesting that IaI deficiency completely
impairs angiogenesis in the matrigel model, whereas, in the
bleomycin model, IaI-deficient mice still display some angio-
genesis. We speculate that the explanation lies with HA levels.
The enhancing effect of IaI in HA binding is particularly
prominent at low and intermediate HA levels (29). Therefore,
the high tissue HA levels after bleomycin injury (20) may
counterbalance the effect of IaI deficiency. Indeed, we found
Figure 8. Inter–a-trypsin inhibitor (IaI) serum concentra-
tions. (A) Significantly higher serum IaI concentrations in
patients with idiopathic pulmonary fibrosis (IPF; gray bars)
compared with control subjects (white bars). (B) No
correlation exists between serum IaI concentrations and
FVC. (C) Correlation between serum IaI concentrations
and diffusion capacity for carbon monoxide (DLCO). (D)
No difference in FVC between patients with high (white
bar) and low (gray bar) serum IaI concentrations. (E) DLCO
is significantly higher in patients with low serum IaI
concentrations (white bar) than in patients with high
serum IaI concentrations (gray bar). Error bars represent
Garantziotis, Zudaire, Trempus, et al.: IaI/Hyaluronan Mediate Angiogenesis in Injury 945
that high concentrations of sHA had significant angiogenic
effect in vitro, even in the absence of IaI (data not shown). In
that sense, IaI may be particularly useful as a homeostatic
agent, stimulating HA effects during low-level or chronic injury,
such as in IPF. HA levels are elevated in IPF lung lavage fluid
(31). HA can be degraded to lower molecular weight forms
through reactive oxygen species or hyaluronidase activity, and
de novo sHA is synthesized by fibroblasts and smooth muscle
cells after cytokine or growth factor stimulation (32). Thus,
although we did not have the opportunity to measure HA levels
or sizes in lungs of patients with IPF, there is some evidence
that sHA is the active HA component in IPF lungs (32). We
speculate, therefore, that IaI likely binds to sHA and poten-
tiates its angiogenic actions in IPF.
The results presented here suggest that IaI plays a role in the
pathogenesis of IPF. Based on our presented animal data, we
believe that,inthesettingofIPF,IaI reaches thefibroblasticfocus
via the surrounding vessels, which explains the strong IaI staining
in the perivascular area of fibroblastic foci. Our findings suggest
matrigel model, IaI is essential for angiogenesis. Second, in
bleomycin lung fibrosis, IaI deficiency decreases angiogenesis in
the fibrotic area. Third, IaI and HA colocalize specifically in the
inversely with DLCOin patients with IPF.
Our findings indicate that the interaction between IaI and
HA may play a role in aberrant angiogenesis in the pathogen-
esis of IPF. Angiogenesis is a part of the healing response after
tissue injury; however, aberrant angiogenesis may instead
support fibroproliferation and inhibit tissue repair (33). The
presence of neovascularization in fibrotic lungs was pointed out
decades ago (2). Newly formed vessels in IPF are fully in-
tegrated into the fibrotic tissue lattice (5, 34) and form a vascular
network, which extends from the pleura to the parenchyma
(35). The factors triggering IPF-related angiogenesis are in-
completely understood. Studies have demonstrated the pres-
ence of both angiogenic factors, such as IL-8, macrophage
inflammatory protein-2, epithelial neutrophil–activating protein
(ENA)-78 and vascular endothelial growth factor (3, 36–38),
and angiostatic factors, such as pigment epithelium–derived
factor (PEDF) and CXCL11 (4, 39), in fibrotic lungs. Thus,
a debate exists whether there is too much, too little, or simply
heterogeneous angiogenesis in IPF (6). We observed that the
presence of IaI led to significantly more angiogenesis in a murine
model of fibrotic lung injury, and IaI colocalized with HA in
vascular areas in UIP lungs. However, in patients with IPF, high
IaI concentrations were associated not with higher, but with
significantly lower gas exchange capacity (DLCO), indepen-
dently of lung restriction (FVC). This suggests the presence of
ongoing lung injury and repair in IPF lungs. We speculate that,
in fibrotic areas, angiogenic and angiostatic factors are compet-
ing. Epithelia in fibrotic lungs may play an important role in this
aspect. Epithelia overlying the fibroblastic foci are a rich source
of chemokines and cytokines, which, in aggregate, promote
a fibrotic microenvironment (40). However, it is possible that
distressed epithelia send ‘‘conflicting signals’’ to the underlying
stroma. On one hand, epithelial cells express proangiogenic
signals, such as vascular endothelial growth factor and ENA-78
(36, 38), which stimulate angiogenesis. On the other hand,
epithelial cells also release antiangiogenic factors, such as
PEDF (4), thereby inhibiting angiogenesis. The leading edge
of the fibroblastic focus may therefore be the edge of the
angiostatic environment, with compensatory angiogenic activity
promoted by IaI and HA. The net effect is the formation of
vessels in the leading edge of fibroblastic foci. These vessels are
not connected to ventilated parts of the lung, thus increasing
shunt and ventilation–perfusion mismatch in patients with IPF
(41, 42). The physiologic correlate is decreased DLCO, dispro-
portionate to the degree of restriction (FVC).
DLCO and gas exchange are independent predictors of
mortality in IPF (43–45). The degree of gas exchange impair-
ment in IPF is likely a result of several coexisting processes (i.e.,
alveolar obliteration, pulmonary arterial hypertension, and
vascular shunting). These processes are related, but not always
interdependent: for example, pulmonary arterial pressures do
not always correlate with FVC, and they are only loosely related
to DLCO(44, 46). Furthermore, DLCOcorrelates poorly with
histological abnormalities in IPF (47, 48). Our findings suggest
that, in chronic lung injury, the ongoing angiogenic response to
persistent lung injury may not be directly associated with the
degree of restriction. In our study, serum concentrations of IaI
were associated with decreased DLCO, but not FVC (Figure 8).
Shunt flow is a major contributor to hypoxemia in IPF (41, 42).
Taken together, our results imply that aberrant angiogenesis
may independently contribute to impaired gas exchange in IPF.
In summary, we have shown that IaI is a novel mediator of
angiogenesis, and that this action is predicated on binding to HA
in the extracellular matrix. IaI is a systemic factor, and reaches
areas of injury via the circulation. Overall, serum IaI concen-
trations are elevated in patients with IPF, indicating that IaI may
exchange (DLCO), independent of the degree of lung restriction
(FVC). Because gas exchange deficits predict mortality in IPF,
ourfindings suggestthat aberrant angiogenesis,mediatedat least
partly by matrix HA and serum IaI, may be an independent
contributor of morbidity and mortality in IPF.
Conflict of Interest Statement: None of the authors has a financial relationship
with a commercial entity that has an interest in the subject of this manuscript.
1. Garantziotis S, Steele MP, Schwartz DA. Pulmonary fibrosis: thinking
outside of the lung. J Clin Invest 2004;114:319–321.
2. Turner-Warwick M. Precapillary systemic-pulmonary anastomoses.
3. Keane MP, Arenberg DA, Lynch JP III, Whyte RI, Iannettoni MD,
Burdick MD, Wilke CA, Morris SB, Glass MC, DiGiovine B, et al.
The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in
idiopathic pulmonary fibrosis. J Immunol 1997;159:1437–1443.
Schwarz MI, Cool CD, Worthen GS. Pigment epithelium–derived
factor in idiopathic pulmonary fibrosis: a role in aberrant angiogenesis.
Am J Respir Crit Care Med 2004;170:242–251.
TABLE 1. PATIENT DEMOGRAPHICS BY SERUM INTER–a-TRYPSIN
INHIBITOR LEVELS: LOWER 50% TO UPPER 50%
(n 5 16)
(n 5 15) CharacteristicsChi-Squaret Test
Systemic steroid use,
use*, yes/no, %
Other medication (bosentan),
Mean age 6 SEM, yr
64 6 4 67 6 40.58
Definition of abbreviation: IaI 5 inter–a-trypsin inhibitor.
* Cyclophosphamide, azathioprine, methotrexate, IFN-g, etanercept, colchicine.
946AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 1782008
5. Ebina M, Shimizukawa M, Shibata N, Kimura Y, Suzuki T, Endo M,
Sasano H, Kondo T, Nukiwa T. Heterogeneous increase in CD34-
positive alveolar capillaries in idiopathic pulmonary fibrosis. Am
J Respir Crit Care Med 2004;169:1203–1208.
6. Keane MP. Angiogenesis and pulmonary fibrosis: feast or famine? Am
J Respir Crit Care Med 2004;170:207–209.
in pulmonary fibrosis. Am J Respir Crit Care Med 1996;154:1819–1828.
8. George EL, Georges-Labouesse EN, Patel-King RS, Rayburn H, Hynes
RO. Defects in mesoderm, neural tube and vascular development in
mouse embryos lacking fibronectin. Development 1993;119:1079–1091.
9. Jang YC, Tsou R, Gibran NS, Isik FF. Vitronectin deficiency is
associated with increased wound fibrinolysis and decreased microvas-
cular angiogenesis in mice. Surgery 2000;127:696–704.
10. Slevin M, Krupinski J, Gaffney J, Matou S, West D, Delisser H, Savani
RC, Kumar S. Hyaluronan-mediated angiogenesis in vascular disease:
uncovering RHAMM and CD44 receptor signaling pathways. Matrix
11. Zheng PS, Wen J, Ang LC, Sheng W, Viloria-Petit A, Wang Y, Wu Y,
Kerbel RS, Yang BB. Versican/PG-M G3 domain promotes tumor
growth and angiogenesis. FASEB J 2004;18:754–756.
12. Fraser JR, Laurent TC, Laurent UB. Hyaluronan: its nature, distribu-
tion, functions and turnover. J Intern Med 1997;242:27–33.
Early-response gene signalling is induced by angiogenic oligosacchar-
ides of hyaluronan in endothelial cells: inhibition by non-angiogenic,
high-molecular-weight hyaluronan. Int J Cancer 1997;71:251–256.
14. West DC, Hampson IN, Arnold F, Kumar S. Angiogenesis induced by
degradation products of hyaluronic acid. Science 1985;228:1324–1326.
15. Majors AK, Austin RC, de la Motte CA, Pyeritz RE, Hascall VC, Kessler
SP, Sen G, Strong SA. Endoplasmic reticulum stress induces hyalur-
onan deposition and leukocyte adhesion. J Biol Chem 2003;278:47223–
16. Zhuo L, Hascall VC, Kimata K. Inter-alpha-trypsin inhibitor, a covalent
protein–glycosaminoglycan–protein complex. J Biol Chem 2004;279:
17. Garantziotis S, Hollingsworth JW, Ghanayem RB, Timberlake S, Zhuo L,
Kimata K, Schwartz DA. Inter-alpha-trypsin inhibitor attenuates com-
plement activation and complement-induced lung injury. J Immunol
18. Wu R, Cui X, Lim YP, Bendelja K, Zhou M, Simms HH, Wang P.
Delayed administration of human inter-alpha inhibitor proteins
reduces mortality in sepsis. Crit Care Med 2004;32:1747–1752.
19. Zhuo L, Yoneda M, Zhao M, Yingsung W, Yoshida N, Kitagawa Y,
Kawamura K, Suzuki T, Kimata K. Defect in SHAP-hyaluronan
complex causes severe female infertility: a study by inactivation of the
bikunin gene in mice. J Biol Chem 2001;276:7693–7696.
20. Teder P, Vandivier RW, Jiang D, Liang J, Cohn L, Pure E, Henson PM,
Noble PW. Resolution of lung inflammation by CD44. Science 2002;
21. Garantziotis S, Zudaire E, Hollingsworth JW, Jiang D, Loyd JE,
Richardson E, Zhuo L, Noble PW, Kimata K, Schwartz DA. Serum
inter-alpha-trypsin inhibitor and matrix hyaluronan mediate aberrant
angiogenesis in idiopathic pulmonary fibrosis. AM J Respir Crit Care
22. Ades EW, Candal FJ, Swerlick RA, George VG, Summers S, Bosse DC,
Lawley TJ. HMEC-1: establishment of an immortalized human
microvascular endothelial cell line. J Invest Dermatol 1992;99:683–690.
23. Zhang B, Cao H, Rao GN. Fibroblast growth factor-2 is a downstream
mediator of phosphatidylinositol 3-kinase-Akt signaling in 14,15-epox-
24. Trempus CS, Morris RJ, Bortner CD, Cotsarelis G, Faircloth RS, Reece
JM, Tennant RW. Enrichment for living murine keratinocytes from
the hair follicle bulge with the cell surface marker CD34. J Invest
25. Livak KJ, Schmittgen TD. Analysis of relative gene expression data
using real-time quantitative PCR and the 22DDCTmethod. Methods
26. Steele MP, Speer MC, Loyd JE, Brown KK, Herron A, Slifer SH, Burch
LH, Wahidi MM, Phillips JA III, Sporn TA, et al. Clinical and
pathologic features of familial interstitial pneumonia. Am J Respir
Crit Care Med 2005;172:1146–1152.
27. American Thoracic Society. Idiopathic pulmonary fibrosis: diagnosis and
treatment: international consensus statement. American Thoracic
Society (ATS), and the European Respiratory Society (ERS). Am J
Respir Crit Care Med 2000;161:646–664.
28. American Thoracic Society; European Respiratory Society. American
Thoracic Society/European Respiratory Society international multi-
disciplinary consensus classification of the idiopathic interstitial
pneumonias. Am J Respir Crit Care Med 2002;165:277–304.
29. Zhuo L, Kanamori A, Kannagi R, Itano N, Wu J, Hamaguchi M,
Ishiguro N, Kimata K. SHAP potentiates the CD44-mediated leuko-
cyte adhesion to the hyaluronan substratum. J Biol Chem 2006;281:
30. Kobayashi H, Sun GW, Terao T. Immunolocalization of hyaluronic acid
and inter-alpha-trypsin inhibitor in mice. Cell Tissue Res 1999;296:
31. Bjermer L, Lundgren R, Hallgren R. Hyaluronan and type III procolla-
gen peptide concentrations in bronchoalveolar lavage fluid in idio-
pathic pulmonary fibrosis. Thorax 1989;44:126–131.
32. McKee CM, Penno MB, Cowman M, Burdick MD, Strieter RM, Bao C,
Noble PW. Hyaluronan (HA) fragments induce chemokine gene
expression in alveolar macrophages: the role of HA size and CD44.
J Clin Invest 1996;98:2403–2413.
33. Tzouvelekis A, Anevlavis S, Bouros D. Angiogenesis in interstitial lung
diases: a pathogenetic hallmark or a bystander? Respir Res 2006;7:82.
34. Renzoni EA, Walsh DA, Salmon M, Wells AU, Sestini P, Nicholson
AG, Veeraraghavan S, Bishop AE, Romanska HM, Pantelidis P, et al.
Interstitial vascularity in fibrosing alveolitis. Am J Respir Crit Care
35. Cool CD, Groshong SD, Rai PR, Henson PM, Stewart JS, Brown KK.
Fibroblast foci are not discrete sites of lung injury or repair: the
fibroblast reticulum. Am J Respir Crit Care Med 2006;174:654–658.
36. Keane MP, Belperio JA, Burdick MD, Lynch JP, Fishbein MC, Strieter
RM. ENA-78 is an important angiogenic factor in idiopathic pulmo-
nary fibrosis. Am J Respir Crit Care Med 2001;164:2239–2242.
37. Keane MP, Belperio JA, Moore TA, Moore BB, Arenberg DA, Smith
RE, Burdick MD, Kunkel SL, Strieter RM. Neutralization of the CXC
chemokine, macrophage inflammatory protein-2, attenuates bleomy-
cin-induced pulmonary fibrosis. J Immunol 1999;162:5511–5518.
38. Simler NR, Brenchley PE, Horrocks AW, Greaves SM, Hasleton PS,
Egan JJ. Angiogenic cytokines in patients with idiopathic interstitial
pneumonia. Thorax 2004;59:581–585.
39. Strieter RM, Starko KM, Enelow RI, Noth I, Valentine VG. Effects of
interferon-g 1b on biomarker expression in patients with idiopathic
pulmonary fibrosis. Am J Respir Crit Care Med 2004;170:133–140.
40. Selman M, Pardo A. Role of epithelial cells in idiopathic pulmonary
fibrosis: from innocent targets to serial killers. Proc Am Thorac Soc
41. Agusti AG, Roca J, Gea J, Wagner PD, Xaubet A, Rodriguez-Roisin R.
Mechanisms of gas-exchange impairment in idiopathic pulmonary
fibrosis. Am Rev Respir Dis 1991;143:219–225.
42. Gunther A, Enke B, Markart P, Hammerl P, Morr H, Behr J, Stahler G,
Seeger W, Grimminger F, Leconte I, et al. Safety and tolerability of
bosentan in idiopathic pulmonary fibrosis: an open label study. Eur
Respir J 2007;29:713–719.
43. Flaherty KR, Andrei AC, Murray S, Fraley C, Colby TV, Travis WD,
Lama V, Kazerooni EA, Gross BH, Toews GB, et al. Idiopathic
pulmonary fibrosis: prognostic value of changes in physiology and six-
minute-walk test. Am J Respir Crit Care Med 2006;174:803–809.
44. Hamada K, Nagai S, Tanaka S, Handa T, Shigematsu M, Nagao T,
Mishima M, Kitaichi M, Izumi T. Significance of pulmonary arterial
pressure and diffusion capacity of the lung as prognosticator in
patients with idiopathic pulmonary fibrosis. Chest 2007;131:650–656.
45. Schwartz DA, Helmers RA, Galvin JR, Van Fossen DS, Frees KL,
Dayton CS, Burmeister LF, Hunninghake GW. Determinants of
survival in idiopathic pulmonary fibrosis. Am J Respir Crit Care
46. Lettieri CJ, Nathan SD, Barnett SD, Ahmad S, Shorr AF. Prevalence
and outcomes of pulmonary arterial hypertension in advanced
idiopathic pulmonary fibrosis. Chest 2006;129:746–752.
47. Cherniack RM, Colby TV, Flint A, Thurlbeck WM, Waldron JA Jr,
Ackerson L, Schwarz MI, King TE Jr. Correlation of structure and
function in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med
48. Fulmer JD, Roberts WC, von Gal ER, Crystal RG. Morphologic–
physiologic correlates of the severity of fibrosis and degree of cellularity
in idiopathic pulmonary fibrosis. J Clin Invest 1979;63:665–676.
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