ORIGINAL PAPER

Effects of nintedanib on the microvascular architecture in a lungfibrosis model

Maximilian Ackermann1 • Yong Ook Kim2• Willi L. Wagner1 • Detlef Schuppan2,3 •

Cristian D. Valenzuela4 • Steven J. Mentzer4 • Sebastian Kreuz5 • Detlef Stiller5 •

Lutz Wollin5 • Moritz A. Konerding1

Received: 22 September 2016 / Accepted: 6 March 2017

� Springer Science+Business Media Dordrecht 2017

Abstract Nintedanib, a tyrosine kinase inhibitor approved

for the treatment of idiopathic pulmonary fibrosis, has anti-

fibrotic, anti-inflammatory, and anti-angiogenic activity.

We explored the impact of nintedanib on microvascular

architecture in a pulmonary fibrosis model. Lung fibrosis

was induced in C57Bl/6 mice by intratracheal bleomycin

(0.5 mg/kg). Nintedanib was started after the onset of lung

pathology (50 mg/kg twice daily, orally). Micro-computed

tomography was performed via volumetric assessment.

Static lung compliance and forced vital capacity were

determined by invasive measurements. Mice were sub-

jected to bronchoalveolar lavage and histologic analyses, or

perfused with a casting resin. Microvascular corrosion

casts were imaged by scanning electron microscopy and

synchrotron radiation tomographic microscopy, and quan-

tified morphometrically. Bleomycin administration resulted

in a significant increase in higher-density areas in the lungs

detected by micro-computed tomography, which was sig-

nificantly attenuated by nintedanib. Nintedanib signifi-

cantly reduced lung fibrosis and vascular proliferation,

normalized the distorted microvascular architecture, and

was associated with a trend toward improvement in lung

function and inflammation. Nintedanib resulted in a

prominent improvement in pulmonary microvascular

architecture, which outperformed the effect of nintedanib

on lung function and inflammation. These findings uncover

a potential new mode of action of nintedanib that may

contribute to its efficacy in idiopathic pulmonary fibrosis.

Keywords Idiopathic pulmonary fibrosis � Angiogenesisinhibitors � Intussusceptive angiogenesis � Microvascular

corrosion casting � Synchrotron radiation tomographic

microscopy

Introduction

Idiopathic pulmonary fibrosis (IPF) is a disabling, pro-

gressive, and ultimately fatal disease characterized by

fibrosis of the lung parenchyma and loss of pulmonary

function [1]. IPF is believed to be caused by repetitive

alveolar epithelial cell injury and dysregulated repair, with

uncontrolled proliferation of pulmonary fibroblasts and

their differentiation into myofibroblasts. These myofi-

broblasts deposit excessive amounts of extracellular matrix

components in the interstitial space [2]. A number of pro-

fibrotic mediators, including platelet-derived growth factor

(PDGF) [3], fibroblast growth factors (FGFs) [4], and

transforming growth factor (TGF)b [5], play important

roles in the pathogenesis of IPF. Pathologic angiogenesis

appears to be intrinsically associated with fibrogenic

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10456-017-9543-z) contains supplementarymaterial, which is available to authorized users.

& Maximilian Ackermann

maximilian.ackermann@uni-mainz.de

1 Institute of Functional and Clinical Anatomy, University

Medical Center of the Johannes Gutenberg University Mainz,

Johann-Joachim-Becher-Weg 13, 55128 Mainz, Germany

2 Institute of Translational Immunology and Research Center

for Immune Therapy (FZI), University Medical Center of the

Johannes Gutenberg University Mainz, Mainz, Germany

3 Division of Gastroenterology, Beth Israel Deaconess Medical

Center, Harvard Medical School, Boston, MA, USA

4 Laboratory of Adaptive and Regenerative Biology, Brigham

and Women’s Hospital, Harvard Medical School, Boston,

MA, USA

5 Immunology and Respiratory Diseases Research, Boehringer

Ingelheim Pharma GmbH & Co. KG, Biberach, Germany

123

Angiogenesis

DOI 10.1007/s10456-017-9543-z

progression in pulmonary fibrosis, but it is unclear how

morphological neovascularization correlates with the pro-

gression of fibrosis.

Nintedanib is a potent inhibitor of the receptor tyrosine

kinases FGF receptor-1, -2, -3, PDGF receptor a/b, vas-cular endothelial growth factor (VEGF) receptor-1, -2, -3,

and Flt-3 and of non-receptor tyrosine kinases like Src,

Lyn, and Lck, which occupy the intracellular ATP-binding

pocket [6]. In primary lung fibroblasts from patients with

IPF, nintedanib inhibited growth factor-stimulated migra-

tion and proliferation [7] and attenuated TGFb-inducedtransformation to myofibroblasts [8].

The in vivo efficacy of nintedanib was explored in sil-

ica-induced lung fibrosis in mice and bleomycin-induced

lung fibrosis in mice and rats [9]. Nintedanib exerted sig-

nificant anti-fibrotic and anti-inflammatory activity. Nin-

tedanib suppressed transcript levels of central fibrosis-

related genes (TGFb1, procollagen 1) and total collagen

levels and reduced the fibrotic score in histomorphometric

analyses of fibrotic lungs. Nintedanib reduced the levels of

lymphocytes in bronchoalveolar lavage fluid (BALF),

diminished levels of inflammatory mediators (IL-1b, IL-6,CXCL1/KC) and the percentage of myeloid dendritic cells

in lung tissue, and reduced pulmonary inflammation and

granuloma formation [9].

Preclinical studies have shown that inhibition of

VEGF, PDGF, and FGF signaling pathways reduces

tumor angiogenesis in the lung [10]. The potential impact

of anti-angiogenic activity in the treatment of IPF has not

been clarified [11]. Nintedanib inhibits proliferation of

endothelial cells, pericytes, and smooth muscle cells,

where it inhibits the proliferation of VEGF-stimulated

human umbilical vein endothelial cells, PDGF-BB-stim-

ulated human umbilical artery smooth muscle cells, and

PDGF-BB-stimulated bovine retinal pericytes [6]. Ninte-

danib also demonstrated anti-angiogenic efficacy in a

mouse model of xenograft tumors [6]. The objective of

this study was to explore the pulmonary vascular alter-

ations in the mouse model of bleomycin-induced lung

fibrosis and the impact of nintedanib on the remodeled

microvascular architecture.

Materials and methods

Animals

C57/BL6 mice (Charles River, Sulzfeld, Germany) aged

8–12 weeks, weighing 22–30 mg, were used in all exper-

iments. The care of the animals was consistent with

guidelines of the German Federal Association for

Accreditation of Laboratory Animal Care.

Bleomycin model and treatment

Induction of bleomycin-induced lung fibrosis, compound

treatment, and animal housing were carried out according

to standardized procedures.

Animals were short-term anesthetized with isoflurane

(3–5%). Bleomycin (Calbiochem, Darmstadt, Germany) at

a dose of 0.5 mg/kg (1.5–2 U/mg) in sterile isotonic saline

(50 lL per animal) was intratracheally instilled by means

of a 22 gauge plastic cannula (Vasofix, 0.5 9 25 mm, B.

Braun Melsungen, Germany) coupled to a 1-mL syringe.

Mice in the vehicle group were given the same volume of

sterile saline.

Treatment with nintedanib was started 7 days after

bleomycin instillation by twice daily oral (by gavage)

administration of 50 mg/kg (hydroxyethylcellulose [Na-

trosol�] suspension 0.5%) and continued until day 19 after

bleomycin application.

Experimental setup

On day 19, lung density was assessed by micro-computed

tomography (lCT) analysis. On day 20, final analyses were

conducted. Lung function was determined in all animals.

Half of the animals were subjected to bronchoalveolar

lavage, tissue sampling for histology/immunohistochem-

istry, and tissue sampling for hydroxyproline analysis and

mRNA analysis. The other half were subjected to vascular

corrosion casting and synchrotron radiation tomographic

microscopy (SRXTM).

Assessment of lung function

Mice were anesthetized with pentobarbital 48 mg/kg

combined with xylazine 2.32 mg/kg injected i.p. After

tracheostomy and intubation, the tracheal cannula was

connected to a FlexiVent system (SCIREQ, Montreal, PQ,

Canada) for pulmonary function testing. To prevent spon-

taneous breathing, animals received pancuronium bromide

0.64 mg/kg i.v. (Inresa Arzneimittel GmbH, Freiburg,

Germany). Ventilation was conducted with 150 breath/min,

a tidal volume of 10 mL/kg, and an end-expiratory pressure

of 3 cm H2O. After recruiting total lung capacity (30 cm

H2O), dynamic lung compliance and forced vital capacity

were measured. Static compliance was determined by

generating pressure volume loops with a maximal plateau

pressure of 30 cm H2O.

lCT

Animals were anesthetized with isoflurane 1.5% and fixed

in the prone position. l-CT images were acquired with a

Angiogenesis

123

Quantum FX lCT system (Perkin Elmer, Waltham, MA,

USA) with cardiac gating (without respiratory gating),

using the following parameters: 90 kV; 160 lA; FOV,

60 9 60 9 60 mm; spatial resolution 0.11 mm, resulting

in a total acquisition time of 4.5 min. Images were ana-

lyzed using MicroView 2.0 software (GE Healthcare,

Amersham, UK), which allows a semiautomatic segmen-

tation of the lungs. Hounsfield unit (HU) histograms were

obtained for the left and right lungs using bins of 10-HU

width. In the absence of a well-established standard to

quantify fibrotic changes in rodents with lCT, and to avoid

relying on an arbitrary threshold to identify fibrotic regions

[12, 13], the HU corresponding to the peak of the HU-

histogram for the segmented pixels was used as a measure

of fibrosis. Total lung volume was computed.

Bronchoalveolar lavage fluid (BALF)

Mice were killed by intraperitoneal injection of pentobar-

bital (400 mg/kg, Narcoren, Merial GmbH, Halbergmoos,

Germany). The left bronchus was sealed by a ligature, and

the right lung was lavaged twice with 400 lL PBS. Total

cell count of BAL cells and differential cell count were

determined by means of a Sysmex XT1800 iVet cell ana-

lyzer (Sysmex Europe GmbH, Norderstedt, Germany).

Histochemistry and immunohistochemistry

Analysis and quantitation of cellular proliferation (anti-

Ki67, Clone TEC-3, Dako, Hamburg, Germany) was per-

formed using immunohistochemistry. Cross sections of the

left lung lobe were harvested, formalin fixed, and embedded

in paraffin. Three microscopic fields at 409 magnification

were used. Picrosirius red staining was used to compare the

collagen content of fibrotic foci with normal tissue.

Ashcroft score

Fibrotic changes in each lung section were assessed as the

mean score of severity from observed microscopic fields

[14]. More than 25 fields within each lung section were

observed at a magnification of 1009. Scores from 0 (normal)

to 8 (total fibrosis) were assigned using a predetermined

scale. After examination of the whole section, the mean of

the scores from all fields was taken as the fibrotic score.

Hydroxyproline analysis

Whole collagen content of the right lung was quantified

colorimetrically using an assay of hydroxyproline. The

lung tissue was hydrolyzed with 1 mL 6 N HCl (Amresco,

OH, USA) at 110 �C for 16 h. Triplicates of 5 lL of the

supernatant were placed in a 96-well plate and mixed with

50 lL 0.1 M citrate buffer, pH 6.0, and 100 lL 150 mg

chloramine T (Sigma-Aldrich, Darmstadt, Germany) dis-

solved in citrate buffer (0.1 M, pH 6.0; Sigma-Aldrich) an

incubated at room temperature for 20 min. Next, 100 lL of

cooled Ehrlich’s (1.25 g distilled water-dissolved

dimethylbenzaldehyde; Sigma-Aldrich) was added to the

reaction mixture and incubated at 65 �C for 30 min.

Absorbance was measured at 550 nm in an Infinite

M200Pro spectrophotometer (TECAN, Wiesbaden, Ger-

many). Total hydroxyproline (lg/lung weight) was calcu-

lated on the basis of individual lung weights and the

corresponding relative hydroxyproline content.

Quantitative real-time polymerase chain reaction

Relative messenger RNA (mRNA) levels were quantified in

total lung RNA by reverse-transcriptase polymerase chain

reaction on a LightCycler 1.5 instrument (Roche, Man-

nheim, Germany) using the TaqMan methodology (Roche,

Indianapolis, IN, USA). Total RNA was isolated with

TRIzol Reagent (Sigma-Aldrich), and 1 lg total RNA was

reverse transcribed into cDNA using the iScript cDNA

Synthesis Kit (Bio-Rad, Hercules, CA, USA). TaqMan

reaction mixtures were purchased from Applied Biosystems.

Gapdh mRNA was used to normalize data and to control for

RNA integrity. TaqMan reactions were performed using a

Step One Plus sequence amplification system (Applied

Biosystems, Foster City, CA, USA). The results were

expressed as the ratio of the copy number of the target gene

divided by the number of copies of the housekeeping gene

(Gapdh) within the polymerase chain reaction run.

Quantitative reverse-transcriptase polymerase chain

reaction primers:

Acta2, forward 50-ACAGCCCTCGCACCCA-30, reverse50-CAAGATCATTGCCCCTCCAGAACGC-30, probe 50-GCCACCGATCCAGACAGAGT-30; Ifng, forward 50-CAGCAACAGCAAGGCGAAA-30, reverse 50-CTGGACCTGTGGGTTGTTGAC-30, probe 50-TCAAACTTGGCAATACTCATGAATGCATCCT-30;Mmp2, forward 50-CCGAGGACTATGACCGGGATAA-30,reverse 50-CTTGTTGCCCAGGAAAGTGAAG-30,probe 50-TCTGCCCCGAGACCGCTATGTCCA-30; Mmp3, forward

50-GATGAACGATGGACAGAGGATG-30, reverse 50-TGGTACCAACCTATTCCTGGTTGCTGC-30, probe 50-CAGAGAGTTAGATTTGGTGGGTACCA-30; Col1a1,

forward 50-TCCGGCTCCTGCTCCTCTTA-30, reverse 50-GTATGCAGCTGACTTCAGGGATGT-30, probe 50-TTCTTGGCCATGCGTCAGGAGGG-30; Tgfb1 forward

50-AGAGGTCACCCGCGTGCTAA-30, reverse 50-ACCGCAACAACGCCATCTATGAGAAAACCA-30,probe 50 -ACCGCAACAACGCCATCTATGA-

GAAAACCA-30; and Timp1, forward 50

TCCTCTTGTTGCTATCACTGATAGCTT-30, reverse 50-

Angiogenesis

123

CGCTGGTATAAGGTGGTCTCGTT-30, probe 50-TTCTGCAACTCGGACCTGGTCATAAGG-30; Tnfa, for-

ward 50-GGGCCACCACGCTCTTC-30, reverse 50-GGTCTGGGCCATAGAACTGATG-30, probe 50-ATGA-GAAGTTCCCAAATGGCCTCCCTC-30 were purchased

from Eurofins Genomics.

Quantitative vessel architecture analysis

by microvascular corrosion casting

After systemic heparinization with 2000 U/kg heparin i.p.,

mice were thoracotomized under deep anesthesia. The pul-

monary artery was cannulated through the right ventricle with

an olive-tipped cannula and perfused with 10 mL saline at

37 �C followed by 10 mL of a buffered 2.5% glutaraldehyde

solution (Sigma) at pH 7.4. After casting the microcirculation

with 10 mL of polyurethane-based casting resin PU4ii (vasQ-

tec, Zurich, Switzerland) and caustic digestion, the microvas-

cular corrosion casts were imaged after coating with gold in an

argon atmosphere with a Philips ESEM XL30 scanning elec-

tron microscope. From each lung series of stereopairs with tilt

angles of 6� were made for quantitative analyses. The stere-

opairs were color coded and reconstructed as anaglyphic ima-

ges.With the known tilt angle, calculations of individual points

marked interactively in both images of each stereopair were

carried out using macros defined for the Kontron KS 300

software (Zeiss, Oberkochen, Germany). For definition of the

dimensions of the microvascular unit, the intervascular dis-

tances as well as the vessel diameters and the variability of the

vessel diameters were assessed in the fibrotic areas only for

comparative purposes and depicted as cumulative percentage.

Qualitative vessel architecture analysis

by microvascular corrosion casting

Analysis of microvascular corrosion casts was used to

assess the effects of nintedanib on the vascular architecture.

Features of intussusceptive angiogenesis, a rapid mode of

vasculature expansion, were identified in the vascular cast

as tiny holes (hallmarks of intussusceptive pillars) and

small capillary loops with diameters between 2 and 5 lm.

The effect of nintedanib (?Bleo ?Nint) on fibrotic capsula

and foci was compared with a vehicle group (not treated

with bleomycin, -Bleo) and a positive control group

(treated with bleomycin but not nintedanib, ?Bleo).

Synchrotron radiation tomographic microscopy

(SRXTM)

The samples were scanned at an X-ray wavelength of 1 A

(corresponding to an energy of 12.398 keV) at the microto-

mography station of the Materials Science Beamline at the

Swiss Light Source of the Paul Scherrer Institut (Villigen,

Switzerland). The monochromatic X-ray beam (DE/E = 0.014%) was tailored by a slits system to a profile of

1.4 mm2. After penetration of the sample, X-rays were con-

verted into visible light by a thin Ce-doped YAG scintillator

screen (Crismatec Saint-Gobain, Nemours, France). Projection

images were further magnified by diffraction-limited micro-

scope optics and finally digitalized by a high-resolution CCD

camera (Photonic Science, East Sussex, UK). For the tissue

samples, the opticalmagnificationwas set to 209, andvascular

casts were scanned without binning with an optical magnifi-

cation, resulting in a voxel size 0.325 lm3. For each mea-

surement, 1001 projections were acquired along with dark and

periodic flat field images at an integration time of 4 s each

without binning. Data were postprocessed and rearranged into

flat field-corrected sinograms online. Reconstruction of the

volume of interestwas performed on a 16-nodeLinux PCFarm

(Pentium 4, 2.8 GHz, 512 megabytes RAM) using highly

optimized filtered back-projection. A global thresholding

approach was used for surface rendering. For 3D visualization

and surface rendering,Amira software (Burlington,MA,USA)

was installed on an Athlon 64 3500-based computer. The 3D

visualizations were analyzed with the program Avizo Fire 7.0.

Transmission electron microscopy

Fibrotic foci were analyzed by transmission electron

microscopy (TEM) to detect the potential role of alveolar

Table 1 The effect of

nintedanib on lung functionVehicle without bleomycin Bleomycin Bleomycin ? nintedanib

Mean SD (n = 15) Mean SD (n = 24) Mean SD (n = 24)

Cdyn (mL/cm H2O) 0.043 ±0.0022 0.034 ±0.0061* 0.034 ±0.0065

FVC (mL) 1.14 ±0.071 0.95 ±0.13* 0.99 ±0.13

Cstat (mL/cm H2O] 0.026 ±0.0016 0.020 ±0.0032* 0.022 ±0.0032

Lung function tests were conducted invasively at the end of the experiment (day 20). Pathology was

induced by intratracheal administration of bleomycin. Nintedanib treatment was conducted from day 7 to

day 19 (50 mg/kg b.i.d.)

Cdyn, Dynamic lung compliance; FVC, forced vital capacity; Cstat, static lung compliance, calculated at an

applied inflation pressure of 30 cm H2O

* p\ 0.0001 versus vehicle without bleomycin

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type II cells and alveolarmacrophages in the healing process.

Samples designated for TEMwere fixed with 2.5% buffered

glutaraldehyde and embedded in Epon (Serva, Heidelberg,

Germany). 700-A ultrathin sections were analyzed using a

Leo 906 digital transmission electron microscope (Leo,

Oberkochen, Germany).

Statistics

All data are presented as mean ± standard deviation of n

animals. Statistical differences between groups were ana-

lyzed by paired t test, one-way analysis of variance

(ANOVA) with subsequent Dunnett’s multiple comparison

test for all parametric data and Kruskal–Wallis test followed

by Dunn’s multiple comparison test for nonparametric data

(GraphPad Prism 6.0; GraphPad Software, Inc. La Jolla, CA,

USA), and p\ 0.05 was considered statistically significant.

Results

Nintedanib induces a trend toward improved lung

function

Twenty days after bleomycin administration, dynamic lung

compliance, forced vital capacity (FVC), and static lung

Pressure [cm H2O]

Lung

vol

ume

[mL]

0 10 20 300.0

0.2

0.4

0.6

0.8

25%

F/L

ratio

- Bleo

+ Bleo

+ Bleo

+ Nint

0.00

0.05

0.10

0.15

* * * *

* * *78%

Tissue density

***

***

- Bleo

+Bleo +Nint

+Bleo

(a) (b)

(c) (d)

Fig. 1 Functional lung testing showed that nintedanib was associated

with a trend toward improved lung function and a reduced prolifer-

ative activity. a Functional lung testing. Pressure volume loops were

conducted with an inflation pressure of up to 30 cm H2O. Data are

mean ± standard error of the mean. Vehicle group without bleomycin

stimulation (-Bleo), n = 13, positive control animals stimulated with

bleomycin 0.5 mg/kg (?Bleo), n = 24, bleomycin-stimulated ani-

mals treated with nintedanib 50 mg/kg twice daily from day 7 until

day 19 (?Bleo ?Nint), n = 23. b Tissue density assessed volumet-

rically by lCT. The ratio between the volume of the dense fibrotic

tissue and the total lung volume (F/L) is presented. Vehicle group

without bleomycin stimulation (-Bleo), n = 18, positive control

animals stimulated with bleomycin (?Bleo), n = 24, bleomycin-

stimulated animals treated with nintedanib 50 mg/kg twice daily

(?Bleo ?Nint), n = 24; ***p\ 0.001, ****p\ 0.0001. c Quantita-

tive assessment of interstitial pulmonary fibrosis was carried out

based on the standardized histopathologic quantification according to

Ashcroft. Treatment with nintedanib (?Bleo ?Nint) resulted in a

significant reduction of the fibrosis grade compared with bleomycin-

stimulated lungs (?Bleo); ***p\ 0.001. d Quantification of anti-Ki-

67-positive cells showed that treatment with nintedanib significantly

reduced proliferation rate. Data are mean ± standard error of the

mean (-Bleo), n = 12, (?Bleo), n = 12, (?Bleo ?Nint), n = 12;

***p\ 0.001

Angiogenesis

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compliance were significantly decreased compared with

mice treated with vehicle (Table 1). Nintedanib resulted in

a trend toward improved lung function with respect to FVC

(*20% improvement) (Table 1), static lung compliance

(*25% improvement) (Table 1; Fig. 1a) which did not

reach statistical significance. This trend was also recorded

in an experiment conducted in preparation for this study

(Supplementary Fig. 1).

Nintedanib reduces fibrotic density and bleomycin-

induced lung inflammation

Administration of bleomycin significantly increased lung

tissue density assessed by lCT, whereas treatment with

nintedanib significantly reduced the ratio of dense fibrotic

tissue to total lung volume by 78% (Fig. 1b, p\ 0.001).

These findings were in line with the histopathologic anal-

ysis of fibrotic tissue illustrated by Azan- and Sirius red-

positive areas (Fig. 2). The histopathologic quantification

of fibrosis grade using the Ashcroft score revealed a sig-

nificant reduction in fibrotic areas with nintedanib

(2.08 ± 1.99, p\ 0.001) compared with animals treated

with bleomycin only (positive controls) (4.89 ± 1.19),

while vehicle-treated animals had an Ashcroft score of

0.03 ± 0.16 (Fig. 1c). Remarkably, hydroxyproline con-

tent was not different between the positive controls and the

nintedanib-treated animals (not shown). Bleomycin-in-

stilled mice showed elevated levels of mRNA expression

of procollagen a1 (p\ 0.001), TGFb1 (p\ 0.01), TIMP-1

(p\ 0.001) and MMP-2 (p\ 0.001) (Fig. 3). Nintedanib

reduced the protein expression of TGFb1 (p\ 0.05),

MMP-2 (p\ 0.001), and TIMP-1 (p = ns). Surprisingly,

bleomycin treatment reduced the expression of alpha

smooth muscle actin (ACTA2; Supplemental Fig. 2). The

expression of MMP-3, IFNc, and TNFa was not signifi-

cantly different between the positive controls and ninte-

danib-treated mice (Supplemental Fig. 2).

Nintedanib reduces the amount of inflammatory

cells and suppresses proliferation

Cell proliferation was assessed by morphometric analysis

of anti-ki67-stained sections (Supplemental Fig. 3). In

animals not treated with bleomycin, 5.2 ± 5.6 positive

stained nuclei per field of view (FOV) were detected,

mainly attributed to the self-renewal of cells like alveolar

macrophages or alveolar epithelial type II cells. Bleomy-

cin-stimulated animals had an average proliferation rate of

96.4 ± 35.0 positive cells per FOV, which was nearly

20-fold higher than in the vehicle group. Treatment with

nintedanib resulted in a significant 63% reduction of this

proliferative activity (35.3 ± 20.4 positive stained cells per

FOV) (Fig. 1d). All differences reached statistical signifi-

cance (p\ 0.001).

Bleomycin caused a significant increase in total cells

(Fig. 4a), monocytes (Fig. 4b), and lymphocytes (Fig. 4c)

measured in the BALF, whereas neutrophil counts remained

low (Fig. 4d). Nintedanib decreased the total cell count in

- Bleo + Bleo + Bleo + Nint

Aza

nS

irius

red

Fig. 2 Increased tissue density and fibrotic foci was readily recognized in lower-resolution analysis of Azan- and Sirius-red stained sections in

positive controls (?Bleo). The grade of fibrosis in nintedanib-treated animals was reduced (?Bleo ?Nint). Bars 200 lm

Angiogenesis

123

BALF by 46% and reduced the monocyte and lymphocyte

cell counts by 57% (Fig. 4b) and 46% (Fig. 4c), respectively.

Nintedanib improves and largely normalizes

the microvascular architecture

Qualitatively, bleomycin stimulation reduced the integrity

of the alveolar morphology. Instead of perialveolar baskets,

large areas with densely packed vessel bulks in the fibrotic

foci, and a higher vascular density, especially in the foci,

were observed. There was no hierarchy of the vascularity,

but, instead, a chaotic tumor vessel-like arrangement with

tortuous vessel courses and numerous blind-ending vessels

(Fig. 5).

The mean intervessel distance in the positive controls

was 6.43 ± 2.58 lm, whereas nintedanib-treated animals

showed a mean intervessel distance of 10.89 ± 3.71 lm,

indicating significantly lower vessel densities (Fig. 6a,

p\ 0.001). The individual vessel diameters were assessed

in the vessels of fibrotic lesions. The vessels in bleomycin-

instilled lungs had a mean vessel diameter of

5.1 ± 1.38 lm in the foci, whereas the bleomycin-stimu-

lated, nintedanib-treated animals showed a mean vessel

diameter of 3.71 ± 1.0 lm (p\ 0.001) (Fig. 6b), indicat-

ing a normalizing effect of nintedanib on the microvascular

architecture in the fibrotic lesions.

Sprouting and intussusceptive angiogenesis

as a driver of fibrotic neovascularization

Altered vascular architecture occurred predominantly in the

subpleural and peribronchial regions in bleomycin-treated

Fig. 3 The expression of pro-fibrogenic, fibrolytic and pro-inflam-

matory transcripts in bleomycin-treated lungs (?Bleo). Relative lung

mRNA transcript levels of a procollagen a1(I), b TGFb1 (transform-

ing growth factor-b1), c TIMP-1 (tissue inhibitor of metallopro-

teinase-1), and d MMP-2 (matrix metalloproteinase isoform 2) were

determined by quantitative real-time polymerase chain reaction. Data

are mean ± standard deviation. The x-fold increase in expression

normalized to the expression in the vehicle group (-Bleo) is

presented. *p\ 0.05, **p\ 0.01, ***p\ 0.001

Angiogenesis

123

mice, whereas the vehicle group showed intact lung archi-

tecture (Fig. 7a, b). SRXTM qualitatively confirmed that the

lungs of bleomycin-treated animals had reduced integrity of

the alveolar morphology with densely packed vessel bulks

(Fig. 7c, d). Microvascular corrosion casting revealed the

appearance of both forms of angiogenesis, namely sprouting

angiogenesis depicted as small protrusions (Figs. 6c [blue

arrows], 7d [red squares]) and the fast adapting—shear

stress and flow dependent—intussusceptive angiogenesis

(Figs. 6c [red arrowheads], 7d [yellow circles]). The lungs

of mice treated with bleomycin and nintedanib also revealed

a lack of regular vascular hierarchy, but to a far lower extent

(Fig. 7e). The microvascular architecture in the lungs of

nintedanib-treated mice resembled normal, autochthonous

vascular lung architecture with alveolar plexus (Fig. 7f); the

diameters of the individual vessel segments showed little

variation, whereas the lungs of mice treated with bleomycin

alone were characterized by large caliber, sinusoidal vessel

networks with frequent vessel diameter changes (see sup-

plemental movies).

Nintedanib alleviates collagen accumulation

and restores alveolar structure

Semithin sections of alveolar tissue demonstrated an

accumulation of type II pneumocytes (Fig. 8a).

Ultrastructural analyses by TEM revealed striking mor-

phologic differences between nintedanib- and vehicle-

treated lungs (Fig. 8b–d). At first glance, huge masses of

mature-type striated collagen fibrils were found in bleo-

mycin- and vehicle-treated animals, especially in the

vicinity of the bronchial system (Fig. 8b). As an inflam-

matory, desmoplastic envelope, we observed inflammatory

cells (neutrophils, lymphocytes) framing the huge masses

of chaotically arranged collagen fibers (Fig. 8b). Occa-

sionally, marginalized fibroblasts revealed high-secreting

activity demonstrated by a high content of rough endo-

plasmic reticulum (Fig. 8b). The orientation of the collagen

fibers appeared highly divergent, and around the focal

accumulation of collagen fibers, high densities of vessels

with capillary wall building were seen (Fig. 8c). The lungs

(a) (b)

(c) (d)

0.2

0.4

0.6

-57%

* *

1

2

3

4

-46%

* *

1

2

3

4

-46%

* *

0.2

0.4

0.6

- Bleo

+ Bleo

+ Bleo

+ Nint

- Bleo

+ Bleo

+ Bleo

+ Nint

- Bleo

+ Bleo

+ Bleo

+ Nint

- Bleo

+ Bleo

+ Bleo

+ Nint

tota

l cel

l cou

nt x

105

mon

ocyt

ic c

ells

x 1

05

lym

phoc

ytes

x 1

05

neut

roph

ilic

cells

x 1

05

Fig. 4 Flow cytometric evaluation of bronchoalveolar lavage fluid.

Cytometry of BALF showed that treatment with nintedanib had

striking effects on the amount of inflammatory cells. Treatment with

nintedanib resulted in a trend toward reduced cell counts. Total cell

count: The total cell count (a) was reduced by 46%. Inflammatory

cells: The monocyte (b) and lymphocyte (c) counts were halved,

whereas neutrophils (d) were not affected by nintedanib. Data are

mean ± standard error of the mean. Vehicle group without bleomycin

stimulation (-Bleo), n = 9, positive control animals stimulated with

bleomycin (?Bleo), n = 12, bleomycin-stimulated animals treated

with nintedanib 50 mg/kg twice daily (?Bleo ?Nint), n = 12;

**p\ 0.01

Angiogenesis

123

of nintedanib-treated mice did not display the large bulks

of collagen fibers seen in the lungs of vehicle-treated,

bleomycin-stimulated animals (Fig. 8d), and the collagen

bundles appeared looser and more degraded (Fig. 8e).

Discussion

The present study investigated the effects of the tyrosine

kinase inhibitor nintedanib on three-dimensional morpho-

genetic evidence of angiogenesis in an animal model of

lung fibrosis, compared with conventional parameters of

angiogenesis and fibrosis.

First, treatment with nintedanib resulted in a trend

toward improved lung function and a reduction in inflam-

matory cells in the BALF. Second, lung tissue density,

assessed by lCT and histopathologic Ashcroft score, was

significantly improved by nintedanib, in line with a

reduction in proliferative activity. Third, nintedanib had

positive effects on microvascular architecture, increasing

intervascular distances and reducing vessel diameters. Data

obtained using SRXTM suggest a role for sprouting and

intussusceptive angiogenesis in alveolar tissue neoalveo-

larization on a sub-micron level. Finally, ultrastructural

analysis revealed a reduction in the bulk of collagen bun-

dles and an increase in activated macrophages and type 2

cells with nintedanib.

Functional testing of lung parameters revealed a trend

toward a better outcome with nintedanib. Clinical trials

have confirmed that nintedanib slows disease progression

in patients with IPF by significantly reducing the decline in

FVC [15, 16]. The limited effect of nintedanib observed in

this study might be explained by the study design, with a

relative short treatment duration and early tissue harvesting

limiting the time for alveolar restoration. The main

experimental limitation of the bleomycin animal model is

that bleomycin only causes an inflammatory response that

is triggered by the overproduction of free radicals and pro-

inflammatory cytokines [17], and the development of

fibrosis remains partially reversible [18].

Our analysis of BALF showed an increased number of

monocytes and lymphocytes in bleomycin-treated animals,

whereas nintedanib reduced the number of these cells.

Macrophages are critical drivers of fibrogenesis by pro-

moting inflammation and angiogenesis [19, 20], and recent

evidence suggests a role for activated macrophages in acute

exacerbations of IPF [21]. Our recent flow cytometry data

in compensatory lung regeneration confirm the active

contribution of macrophages and local alveolar type II cells

to alveolar growth in neoalveolarization and neovascular-

ization [22, 23].

Nintedanib is a potent inhibitor of the VEGF receptor,

FGF receptor, and PDGF receptor [6, 24], which trigger

pro-angiogenic signaling in lung fibrosis [25, 26]. In the

present study, treatment with nintedanib almost normal-

ized the vascular architecture, with restoration of alveolar

basket structures in fibrotic lungs. This finding of archi-

tectural restoration is in line with the improved lung

function in mice and in patients with IPF [15, 16, 27]. In

clinical trials, nintedanib reduced the decline in FVC

independent of the severity of lung function impairment at

baseline [27].

The reciprocal interaction between endothelial cells,

macrophages, and fibroblasts stimulates the fibrotic cas-

cade proceeding from the initial epithelial damage to the

excessive deposition of extracellular matrix [28, 29]. The

+ Bleo

+ Bleo+Nint

Fig. 5 Nintedanib restored a ‘‘normal’’ pulmonary vascular archi-

tecture. Scanning electron micrographs of microvascular corrosion

casts illustrate the striking architectural differences between the

positive control animals stimulated with bleomycin (?Bleo) and the

bleomycin-stimulated animals treated with nintedanib (?Bleo ?-

Nint). The microvascular architecture of the lungs of animals treated

with nintedanib was characterized by a recurrence of alveolar basket

structure, whereas the lungs of the positive controls showed huge

areas with densely packed vessel bulks constricting the airway

system. Bars 200 lm

Angiogenesis

123

loss of integrity of the basement membrane and alveolar–

capillary membrane enables the continuous destruction of

lung architecture by pro-angiogenic and pro-fibrotic stim-

uli. Thus, fibroblasts from patients with IPF express higher

levels of PDGF receptors and FGF receptors than controls

[7]. Nintedanib prevents the pro-proliferative and fibrotic

effects triggered by PDGF-BB, bFGF, and VEGF [7, 8].

Hilberg et al. [6] demonstrated that nintedanib inhibited the

proliferation of three cell types contributing to tumor

angiogenesis (endothelial cells, pericytes, and smooth

muscle cells) in epithelial lung carcinoma cell lines,

emphasizing the anti-proliferative and anti-angiogenic

potency of nintedanib.

Intussusceptive angiogenesis plays a pivotal role in

various proliferative diseases [30–32]. Our study is the

first, to our knowledge, to demonstrate a role for intus-

susceptive angiogenesis in fibrogenesis, as imaged by

scanning electron microscopy and SRXTM. These methods

allowed a high-definition display of vessel morphology,

providing 3-D detail down to the capillary level [33].

Mechanical stress and related changes in blood flow are

thought to play pivotal roles in the initiation of intussus-

ceptive remodeling. We have shown a rapid upregulation

of intussusceptive vessel expansion in a model of com-

pensatory lung growth after pneumonectomy [33].

Intussusceptive angiogenesis is distinct from sprouting

angiogenesis because it has no requirement for cell pro-

liferation, can rapidly expand an existing capillary net-

work, and can maintain organ function during replication

and remodeling. Thus, this pillar formation and branch

remodeling may represent an adaptive response to the

increasing blood flow and blood pressure during inflam-

mation and regeneration. The occurrence of intussusceptive

angiogenesis is related to the homing and mobilization of

endothelial precursor cells from the bone marrow or the

peripheral blood integrating and lining the endothelial wall

[30]. Interestingly, clinical findings have shown an imbal-

ance of circulating and proliferating endothelial progenitor

cells in patients with IPF, with enhanced endothelial pro-

genitor cell mobilization into the pulmonary vasculature

[34]. This homing may be associated with the recurrence of

intussusceptive angiogenesis. However, recent studies have

suggested that recruitment of circulating fibrocytes occurs

[29, 35] .These circulating fibrocytes are a subpopulation

of bone marrow hematopoietic-derived cells characterized

by the expression of procollagen type I and hematopoietic

markers, such as CD45 and CD34 [35]. The relative con-

tribution of circulating fibrocytes and endothelial progeni-

tor cells to the aggravation of pulmonary fibrosis remains

to be determined.

(a) (b) (c)

+ Bleo+ Bleo + Nint

+ Bleo+ Bleo + Nint

+ Bleo + Bleo + Nint + Bleo + Bleo + Nint

intervascular distances [µm] vessel diameter [µm]

Fig. 6 Nintedanib normalized microvascular architecture as assessed

by 3D-morphometry. Intervascular distances (a) and vessel diameters

(b) in fibrotic foci assessed tridimensionally in microvascular

corrosion casts shown as cumulative frequencies and box-whisker

plots with the median, 5th, 10th, 25th, 75th, 90th, and 95th

percentiles; mean (red). All p values were significant (p\ 0.001).

c Scanning electron micrographs of a fibrotic bleomycin lung

(?Bleo). Vascular casts showing a chaotic tumor-like vasculature

with sprouting angiogenesis (blue arrows). Adjacent to the bronchi

are intussusceptive pillars (red arrowheads), hallmarks of intussus-

ceptive angiogenesis. Lungs from positive controls stimulated with

bleomycin (?Bleo), n = 12, bleomycin-stimulated animals treated

with nintedanib 50 mg/kg twice daily (?Bleo ?Nint), n = 14.

Bars 20 lm. (Color figure online)

Angiogenesis

123

Notably, nintedanib treatment led to the development of

near-normal lung vasculature likely to restore the delicate

blood-gas exchange. Although excessive extracellular

matrix substantially accounts for the decline of lung

function in pulmonary fibrosis, microvascular remodeling

as irregularly shaped capillaries and/or the increase in

alveolar–capillary diameters further attenuates blood-gas

exchange [36]. In addition to vascular architectural chan-

ges, pulmonary capillary endothelial cells might serve as a

source of fibroblasts in pulmonary fibrosis via endothelial–

mesenchymal transition [37]. Taken together, the changes

in microvascularity may be considered a decisive mor-

phogenetic factor in the aggravation of pulmonary fibrosis.

In summary, this study provides the first evidence that the

anti-angiogenic activity of nintedanib restores the integrity

of lung architecture in pulmonary fibrosis. Our findings

suggest that pulmonary fibrogenesis and neoangiogenesis

interact in the progression of pulmonary fibrosis. Nintedanib

might interfere with these pivotal pathogenetic mechanisms

in pulmonary fibrosis, significantly improving lung function

and normalizing the microvascular architecture. Further

work is necessary to illuminate the dynamic capillary–

(a) (b)

(c) (d)

(e) (f)

Fig. 7 Sprouting and intussusceptive angiogenesis play a pivotal role

in fibrogenesis shown in SRXTM. a Three-dimensional evaluation of

microvascular corrosion casts by SRXTM illustrating the regular

alveolar duct structure accompanied by the limiting alveolar entrance

ring vessels. Bars 75 lm. b A typical example of vasculature in the

basket-shaped alveoli. Bars 30 lm. c Analysis of the fibrotic lungs ofbleomycin-stimulated animals revealed an increased, irregular vas-

cularity with loss of tissue integrity and double-layered vessels.

Bars 60 lm. d In fibrotic lungs intussusceptive holes (yellow circles)

were found, indicative of the occurrence of intussusceptive angio-

genesis around larger vessel structures. Several angiogenic sprouts are

highlighted with a red square. e In the lungs of animals treated with

nintedanib, the vascular density was decreased and regular alveolar

patterns were observed. Bars 50 lm. f After nintedanib treatment, the

alveolar entrance ring vessels (dashed red line) defining the alveolar

opening were predominantly found in the remodeled tissue re-

enabling the blood-gas exchange. Bars 30 lm (see movies in

supplemental material). (Color figure online)

Angiogenesis

123

alveolar interactions between structural adaptations of the

microvascular architecture and inflammation in pulmonary

fibrosis using SRXTM.

Acknowledgements This work is dedicated to the memory of the late

Prof. Moritz A. Konerding. The authors acknowledge the skillful

technical assistance of Kerstin Bahr (University Medical Center

Mainz), Janine Beier, Helene Lichius, and Andrea Vogtle (all

Boehringer Ingelheim, Biberach). Editorial assistance, funded by

Boehringer Ingelheim, was provided by Clare Ryles of Fleishman-

Hillard Fishburn. M.A., L.W., M.A.K., D.Sc., and S.J.M were

involved in the conception and design. M.A., Y.O.K., W.L.W.,

C.D.V., S.K., D.St., and L.W. were involved in the experimental

work, analysis, and interpretation. M.A., Y.O.K., W.L.W, C.D.V.,

D.Sc., S.J.M., S.K., D.St., and L.W. drafted the manuscript and

revised it for intellectual content. M.A. is the guarantor of this work

and, as such, had full access to all of the data in the study and takes

responsibility for the integrity of the data and the accuracy of the data

analysis.

Funding This study was funded in part by Boehringer Ingelheim,

Biberach, Germany

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* (a)

(b) (c)

(d) (e)

*

*

* *

* *

*

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c c

c c

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