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Vascular Microarchitecture of Murine Colitis-Associated Lymphoid Angiogenesis.

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THE ANATOMICAL RECORD 292:621–632 (2009)
Vascular Microarchitecture of Murine
Colitis-Associated Lymphoid Angiogenesis
Laboratory of Adaptive and Regenerative Biology, Brigham and Women’s Hospital,
Harvard Medical School, Boston, Massachusetts
Molecular and Integrative Physiological Sciences, Harvard School of Public Health,
Boston, Massachusetts
Department of Anatomy, Johannes Gutenberg University, Mainz, Germany
In permissive tissues, such as the gut and synovium, chronic inflammation can result in the ectopic development of anatomic structures that
resemble lymph nodes. These inflammation-induced structures, termed
lymphoid neogenesis or tertiary lymphoid organs, may reflect differential
stromal responsiveness to the process of lymphoid neogenesis. To investigate the structural reorganization of the microcirculation involved in colonic lymphoid neogenesis, we studied a murine model of dextran sodium
sulfate (DSS)-induced colitis. Standard 2-dimensional histology demonstrated both submucosal and intramucosal lymphoid structures in DSSinduced colitis. A spatial frequency analysis of serial histologic sections
suggested that most intramucosal lymphoid aggregates developed de
novo. Intravital microscopy of intravascular tracers confirmed that the
developing intramucosal aggregates were supplied by capillaries arising
from the quasi-polygonal mucosal plexus. Confocal optical sections and
whole mount morphometry demonstrated capillary networks (185 46
lm diameter) involving six to ten capillaries with a luminal diameter of
6.8 1.1 lm. Microdissection and angiogenesis PCR array analysis demonstrated enhanced expression of multiple angiogenic genes including
CCL2, CXCL2, CXCL5, Il-1b, MMP9, and TNF within the mucosal
plexus. Intravital microscopy of tracer particle flow velocities demonstrated a marked decrease in flow velocity from 808 901 lm/sec within
the feeding mucosal plexus to 491 155 lm/sec within the capillary
structures. We conclude that the development of ectopic lymphoid tissue
requires significant structural remodeling of the stromal microcirculation.
A feature of permissive tissues may be the capacity for lymphoid angioC 2009 Wiley-Liss, Inc.
genesis. Anat Rec, 292:621–632, 2009. V
Key words: microcirculation;
genesis; colitis
Abbreviations used: CFSE ¼ 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester; 2D ¼ 2-dimensional; 3D ¼ 3-dimensional; DiI ¼ 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate; DiO ¼ 3,30 -dioctadecyloxacarbocyanine perchlorate; DMSO ¼ dimethyl sulfoxide; FITC ¼ fluoroscein isothiocyanate; H and E ¼ hematoxylin and eosin; HEV ¼ high
endothelial venule; Mab ¼ monoclonal antibodies; PBS ¼ phosphate buffered saline; TLO ¼ tertiary lymphoid organs.
Grant sponsor: NIH; Grant numbers: HL47078 and HL75426.
*Correspondence to: Steven J. Mentzer, Room 259, Brigham
and Women’s Hospital, 75 Francis Street, Boston, MA 02115.
E-mail: [email protected]
Received 11 September 2008; Accepted 21 January 2009
DOI 10.1002/ar.20902
Published online in Wiley InterScience (www.interscience.wiley.
In normal circumstances, the peripheral immune system is organized into secondary lymphoid organs such
as regional lymph nodes and Peyer’s patches (Picker and
Butcher, 1992). In some chronic inflammatory diseases,
anatomic structures that resemble lymph nodes with B
cell follicles and T cell zones form de novo. These inflammation-induced ectopic lymphoid structures have been
termed lymphoid neogenesis or tertiary lymphoid organs
(Ruddle, 1999; Hjelmstrom, 2001). Some tissues, including the gut, have been associated with lymphoid neogenesis in inflammatory disease states, whereas other
tissues, such as the skin, are rarely associated with ectopic lymphoid aggregates (Aloisi and Pujol-Borrell,
2006). These tissue-specific differences suggest the importance of stromal responsiveness to lymphoid
Attempts to investigate stromal adaptation to chronic
inflammation have largely focused on endothelial cells.
Endothelial cell acquisition of adhesive and chemoattractant properties has been proposed as a mechanism for
regulating lymphoid traffic to inflamed tissues (von
Andrian and Mempel, 2003). High endothelial venules
(HEV), endothelial cells characterized by a cuboidal morphology, and a distinctive molecular phenotype (e.g.,
PNAdþ and CCL21þ), are a variable finding in chronically inflamed tissues (Armengol et al., 2001; Weninger
et al., 2003; Barone et al., 2005; Manzo et al., 2005).
Much of the variability in endothelial adhesive function
and chemokine expression may reflect the underlying
microvascular architecture. Stromal reorganization,
including the adaptive structural change in the microvasculature, may provide an explanation for variability
in both endothelial phenotype and tissue responsiveness.
To investigate the structural reorganization of the
microcirculation involved in lymphoid neogenesis, we
studied a murine model of dextran sodium sulfate
(DSS)-induced colitis. Both submucosal and intramucosal lymphoid aggregates were identified in the mouse
colon. A frequency analysis suggested that most intramucosal lymphoid aggregates developed de novo. The
developing intramucosal lymphoid aggregates were supplied by capillaries arising from the quasi-polygonal mucosal plexus. The time course of aggregate development
and the gene expression within the mucosa suggest that
the structural changes were the result of inflammationinduced sprouting angiogenesis.
changed to water for the remainder of the experimental
Clinical Colitis Score
Using RFID tagging of each mouse (AVID,, body weights and clinical scores were
recorded daily. A modification of a previously described
method (Waidmann et al., 2002), the colitis score incorporated posture (0 normal; 1 abnormal), activity level (0
normal; 1 abnormal), ruffled fur (0 absent; 1 present),
rectal prolapse (0 absent; 1 present), feces (0 normal; 2
liquid; 4 bloody), weight loss (0 10%; 2 ¼ 10%–20%; 4
20%). A score of less than 3 was considered minimal
or no colitis, 3–5 was moderate colitis, and a score
greater than 5 was severe colitis.
Microvideo Endoscopy
As previously described (Ravnic et al., 2007a), the
microvideo endoscopy was performed using a multi-purpose rigid endoscope (KSVEA Rigid; 64018 BSA) (Karl
Storz, Germany) with a 2.7 mm diameter and 18 cm
length. The rigid optical system included a 30 degree
wide angle forward oblique telescope. The KSVEA rigid
endoscope used a 175 Watt Xenon light source. The analog video images were digitized for archiving and
Stereologic Sampling
Small mucosal tissue blocks (10 15 mm) were
freshly excised from DSS-treated mice, embedded in
OCT compound (Miles Labs, Elkhart, IN), and prepared
for cryosectioning. Vertical cryosections were prepared
in 7–10 lm thickness slides, stained with hematoxylin
and eosin (H and E), and evaluated for lymphoid aggregates. Preliminary microscopic evaluation of each block
was performed to ensure an acceptable tissue preparation. In adequately prepared specimens, the number of
lymphoid aggregates was assessed based on H and E
staining (Carlsen et al., 2002). Multiple sections were
obtained from two parallel regions a minimum of 750
lm apart; more sections were obtained in regions of
apparent confluence so that discrete aggregates could be
judged. The histology sections were evaluated with a
500 lm 750 lm grid projection by at least two independent observers. The mean aggregate number of these
two regions was recorded for each time point.
C57B/6 mice (Jackson Laboratory, Bar Harbor, ME),
25–33 g, were used in all experiments. The care of the
animals was consistent with guidelines of the American
Association for Accreditation of Laboratory Animal Care
(Bethesda, MD).
Dextran Sulfate Administration
In C57/B6 mice, the DSS (TdB Consultancy AB, Uppsala, Sweden) model of colitis was similar to that
described previously (Okayasu et al., 1990). Briefly, DSS
was freshly prepared and added daily to the drinking
water at a final concentration of 3%. The mice were
assessed daily for clinical signs and total body weight.
The DSS treatment was continued for 6 days then
Immunohistochemistry was performed with commercially available primary antibodies used at a 1:50 concentration. The anti-CD4 (GK1.5, Abcam, Cambridge,
UK), anti-CD19 (1D3, BD Pharmigen), anti-CD11b (M1/
70, BD Pharmigen, San Jose, CA), F4/80 (CI:A3-1, BD
Pharmigen) antibodies were used with a goat anti-rat biotinylated second antibody and developed with neutralite avidin-texas red conjugate (Southern Biotechnology,
(MEC7.46, Cell Sciences, Canton, MA) was developed
with the neutralite avidin-texas red conjugate (Southern
Biotechnology). The Flk-1/KDR/VEGFR2 antibody (ThermoFisher Scientific) and the anti-CD20 antibody
(EP459Y, Abcam) were detected with Qdot 525 goat
F(ab’)2 anti-rabbit IgG conjugate (Invitrogen, Eugene,
Immunofluorescence Staining
Cryostat sections were obtained from colon specimens
were treated with O.C.T. compound and snap frozen. After warming the slide to 27 C, the sections were fixed
for 10 min (2% paraformaldehyde and PBS at pH 7.43).
The slides were washed with buffer (PBS, 5% sheep serum, 0.1% azide, 1 mM MgCl2, 1 mM CaCl2) and blocked
with 20% sheep serum, 20% goat serum, 0.1% azide in
PBS. The slides were treated with monoclonal antibodies
(Mab) at 10–20 lg/mL. The slides were incubated for 1
hr at 27 C and washed twice. The detection antibody
was added and incubated for 20 min at 27 C. The slides
were washed twice and examined by fluorescence
Optical System
The exteriorized tissue was imaged using a Nikon
Eclipse TE2000 inverted epifluorescence microscope
using Nikon objectives of 10, 20, and 40 linear magnification with infinity correction. An X-Cite (Exfo;
Vanier, Canada) 120 watt metal halide light source and
a liquid light guide was used to illuminate the tissue
samples. Excitation and emission filters (Chroma, Rockingham, VT) in separate LEP motorized filter wheels
were controlled by a MAC5000 controller (Ludl, Hawthorne, NY) and MetaMorph software 7.5 (MDS Analytical Technologies, Downingtown, PA). The CFSE tracer
(ex 480 nm, em 520 nm) was imaged with 25 nm band
pass filters (Omega, Brattleboro, VT). The intravital videomicroscopy 14-bit fluorescent images were digitally
recorded on a C9100-02 camera (Hamamatsu, Japan).
The C9100-02 camera has a hermetic vacuum-sealed aircooled head and on-chip electron gain multiplication
(2000). Images with 1000 1000 pixel resolution were
routinely obtained at 50 fps; frame rates exceeding 50
fps were obtained with binning and subarrays. The
images were recorded in image stacks comprising 30
sec–10 min video sequences.
3-Dimensional Tissue Mounts
The structure of the colon microcirculation was characterized by fluorescent vessel painting (Ravnic et al.,
2005). After systemic heparinization, the aorta was cannulated and perfused with 15 mL of 37 C phosphate buffered saline (PBS) followed by perfusion with a buffered
2.5% glutaraldehyde solution (Sigma). The systemic circulation was perfused with 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) or 3,30 dioctadecyloxacarbocyanine perchlorate (DiO) (10–25
mL) as described previously (Ravnic et al., 2005). Immediately following tracer infusion, the organs were harvested, prepared in a 25 C PBS bath, and fixed
overnight between glass slides in 4% formalin. After a
brief rinse with distilled water, the specimens were
stained with DAPI (Vector, Burlingame, CA) and permanently mounted with Vectashield mounting medium
(Vector). The fluorescently labeled microvessels were
imaged using a Nikon Eclipse TE2000 inverted epifluorescence microscope. Structured illumination confocal
microscopy was performed with an Optigrid system
(Qioptiq, Rochester, NY) (Lee et al., 2008). The Optigrid
uses a one-dimensional optical grid in the form of a Ronchi grating mounted on a piezo-electrically driven actuator. The pattern is moved perpendicular to the grid lines
three times producing three separate images that are
digitally recombined using a proprietary software algorithm (Volocity 4.4; Improvision, Natick, MA).
Mucosal Microdissection
After euthanasia, subtotal colectomy (ascending to
transverse) was performed. The lumen was flushed and
opened along the mesenteric border (McDonald and
Newberry, 2007). The mucosa was copiously irrigated
with cold PBS (4 C) until all debris was removed as
determined by stereomicroscopy. The colon wall was immobilized on a standard microscope slide and the mucosa, superficial to the lamina propria, was removed
using gentle dissection with a second microscope slide.
Limited dissection of the intact superficial (50–100 lm
thick) mucosa was confirmed by light microscopy.
Tissue Processing
Standard RNA isolation procedures were used, including separate laboratory space for tissue harvesting, RNA
isolation, and PCR processing. Pipets and consumables
were regularly treated by UV irradiation; work surfaces
were routinely cleaned with DNA Exitus Plus (Applichem, Cheshire, CT), standard disinfectants and UV
treatment. Routine wipe tests of work areas were performed to screen for nucleic acid contamination.
RNA Isolation
Total RNA was isolated using Qiagen RNeasy Midi Kit
(Qiagen, Valencia, CA). Briefly, the fresh tissue was triturated using a 20-G needle until uniformly homogeneous. The tissue lysate was centrifuged at 3000g for 10
min and the supernatant (lysate) was removed by pipetting. An equal volume of 70% ethanol was added to
lysate and gently mixed. The sample was placed in an
RNeasy midi column, centrifuged for 5 min at 3000g and
the flow-trough was discarded. After additional RPE
buffer was added to the column, the tube was again centrifuged for 5 min at 3000g to dry the RNeasy silica-gel
membrane. The RNeasy column was transferred to a collection tube and elution was performed using RNase-free
water and centrifugation for 3 min at 3000g. Generally,
a second elution step was not performed. Genomic DNA
contamination was eliminated by RNase-Free DNase Set
(Qiagen). Briefly, 1–2 lg of potentially contaminated
RNA was treated with DNase buffer, RNase inhibitor,
and DNase I. In all RNA isolations, the total RNA quality was assessed by using an Agilent 2100 Bioanalyzer
(Agilent Technologies, Palo Alto, CA). RNA integrity
numbers (RIN) (Schroeder et al., 2006) of the RNA samples were uniformally greater than 7.3 (mean, 8.5;
range, 7.3–9.8).
Angiogenesis PCR Arrays
First Strand cDNA Synthesis used RT2 First Strand
Kit from SuperArray Bioscience Corporation. Mouse
Angiogenesis RT2 Profiler PCR Array and RT2 Real-
Timer SyBR Green/ROX PCR Mix were purchased from
SuperArray Bioscience Corporation (Frederick, MD).
Angiogenesis Genes
The angiogenesis genes examined in our study include
the following (abbreviation, gene name): Angiopoietin 1
(Angpt1, 1110046O21Rik/Ang-1), Angiopoietin 2 (Angpt2,
Agpt2/Ang- 2), Chemokine (C-C motif) ligand 2 (CCL2,
AI323594/HC11), Collagen, type IV, alpha 3 (Col4a3,
[a]3(IV)/alpha3(IV)), Colony Stimulating Factor 3 (granulocyte) (CSF3, Csfg/G-CSF), Chemokine (C-X-C motif)
ligand 1 (CXCL1, Fsp/Gro1)), Chemokine (C-X-C motif)
ligand 2 (CXCL2, CINC-2a/GROb), Chemokine (C-X-C
motif) ligand 5 (CXCL5, AMCF-II/ENA-78), Fibroblast
growth factor 1 (Fgf1, Dffrx/Fam), Fibroblast growth factor 2 (Fgf2, Fgf-2/Fgfb), Fibroblast growth factor 6 (Fgf6,
Fgf-6), Fibroblast growth factor receptor 3 (Fgfr3, Fgfr-3/
HBGFR), Heart and neural crest derivatives transcript 2
(Hand2, AI225906/AI661148), Interferon gamma (Ifng,
IFN-g/IFN-gamma), Interleukin 1 beta (Il1b, IL-1beta/Il1b), Interleukin 6 (Il6, Il-6), Leptin (Lep, ob/obese), Matrix metallopeptidase 9 (MMP9, AW743869/B), T-box 4
(Tbx4, 3930401C23), Transforming growth factor alpha
(Tgfa, wa-1/wa1), Transforming growth factor, beta 1
(Tgfb1, TGF-beta1/TGFbeta1), Transforming growth factor, beta 2 (Tgfb2, BB105277/Tgf-beta2), Transforming
growth factor, beta 3 (Tgfb3, Tgfb-3), Tumor necrosis factor (TNF, DIF/TNF-alpha), Thymidine phosphorylase
(Tymp, 2900072D10Rik/Ecgf1), Vascular endothelial
growth factor A(Vegfa, VEGF- A/VEGF120), Vascular endothelial growth factor B (Vegfb, VEGF-B/Vrf), Vascular
endothelial growth factor C (Vegfc, AW228853/VEGF-C).
Quantitative PCR
Real-time PCR was performed with SYBR green qPCR
master mixes that include a chemically-modified hot
start Taq DNA polymerase (SABioscience). PCR was performed on ABI 7300 Real-Time PCR System (Applied
Biosystems). For all reactions, the thermal cycling conditions were an initial 50 C for 2 min and 95 C for 10 min
followed by 40 cycles of denaturation at 95 C for 15 sec
and simultaneous annealing and extension at 60 C for
1 min.
via the tail vein with 150 lL of the prepared solution.
The CFSE and FITC-dextran tracers (ex 480 nm, em
520 nm) were imaged with 25 nm band pass filters
Multi-Frame Particle Tracking
Tracking of the green and infra-red particles was performed on digitally recorded and distance calibrated
multi-image ‘‘stacks’’ (Ravnic et al., 2006). The image
stacks produced a sequential time history of velocity and
direction as the acquired images were time stamped
based on the 100 mHz system bus clock of the Xeon
processor (Intel, Santa Clara, CA). The movement of
individual particles was tracked using the MetaMorph
(MDS Analytical Technologies) object tracking applications. The intensity centroids of the particles were identified and their displacements tracked through planes in
the source image stack. For displacement reference, the
algorithm used the location of the particle at its first
position in the stack. Each particle was imaged as a
high contrast fluorescent disk and its position was determined with sub pixel accuracy. The image of the particle
was tracked using a cross correlation centroid-finding
algorithm to determine the best match of the particle/
cell position in successive images. With routine distance
calibration, the overlay of the image stack provided a
quantitative assessment of the particle/cell path. From
the XY coordinates, velocity, mean displacement, and
mean vector length were calculated.
Time-Series Flow Visualization
The stream acquired images were stacked to create a
time-series of 500 or 1000 consecutive frames. The
stacks were systematically analyzed to ensure the absence of motion artifact. The stack ‘‘maximum’’ operation
selected the highest intensity value for each pixel location throughout the time-series. The resultant image
produced a time-series reconstruction of particle locations during the time interval of the image stack.
Statistical Analysis
The nanoparticles were developed by Molecular Probes
(Invitrogen, Eugene, OR) for intravascular particle
tracking (Ravnic et al., 2007b). Characteristics of the
particles included superior fluorescence intensity, small
size (500 nm), and low surface charge content (6.2 lEq/
g). The nanoparticles used in this study were green (ex
488; em 510) and infra-red (ex 655 nm; em 710 nm).
Gene expression was calculated using the comparative
cycle threshold (Ct) method (Livak and Schmittgen,
2001). Although the data was monitored for nonideal
efficiencies, comparable amplification of the target genes
and reference genes was assumed. Every effort to optimize the reaction efficiency was made. Validation assays
using serial dilutions of the target and reference genes
were not routinely performed. The DSS-induced colitis
and control data were plotted as a scattergram and a
linear regression was calculated with 95% prediction
bands after the data was imported into Origin 8.0 (OriginLab, North Hampton, MA). Linear regression was
uniformly P < 0.0001. In nanoparticle velocity analyses,
the unpaired Student’s t-test for samples of unequal variances was used to calculate statistical significance. The
data was expressed as mean one standard deviation.
The significance level for the sample distribution was
defined as P < 0.01.
Plasma-Marker Fluorescence Labeling
A 5-(and-6)-carboxyfluorescein diacetate, succinimidyl
ester (CFSE) (Invitrogen, Eugene, OR) labeling solution
was prepared in dimethyl sulfoxide (DMSO) as described
(Becker et al., 2004; Ravnic et al., 2006). The freshly
prepared CFSE (400 lL) was injected into the tail vein
of an anesthetized mouse. In some mice, a 10% 250,000
kD fluorescein isothiocyanate (FITC)-dextran (Sigma) solution in normal saline was prepared. Mice were injected
Fig. 1. DSS-induced colitis in adult mice. After DSS exposure for 5
days, the mice demonstrated persistent inflammatory changes in the
colonic mucosa. Microendoscopy of the mouse descending colon 5
to 60 days after the initial DSS exposure showed gross mucosal
changes, including erythema and ulceration, reminiscent of human colitis (A, control; B, DSS colitis). (C–F) Hematoxylin and eosin histology
of submucosal and intramucosal mononuclear aggregates (white
ovals) in mice 28–30 days after the onset of DSS colitis. The mononuclear infiltration in many regions of the colon was nearly transmural (C
and D). Other areas demonstrated intramucosal aggregates superficial
to the lamina propria (E and F)(Bar A,B ¼ 200 lm; C,D ¼ 160 lm).
Colon Lymphoid Neogenesis
Consistent with previous reports (Neurath et al.,
2000), the mice in this study (N ¼ 92 mice) initially
developed weight loss and clinical signs of colitis: the
mean weight dropped to 76% 6% of baseline on day 7
and gradually recovered to 100% 4% of baseline
weight on day 28. Similarly, the clinical colitis scores,
including ruffled fur, inactivity, and diarrhea, peaked on
day 8 (score 10 4) and returned to baseline on day 19
(score 0.3 2). Despite the clinical improvement over
the first 2–3 weeks, microendoscopy demonstrated
ongoing colonic inflammation (Fig. 1). Serial histologic
sections demonstrated submucosal aggregates that frequently involved both the submucosa and mucosal
crypts; these aggregates appeared to span the lamina
propria (Fig. 1C,D). Relatively smaller intramucosal
aggregates were identified within the mucosa; that is,
superficial to the lamina propria (Fig. 1E,F).
Lymphoid Membrane Markers
Fig. 2. Histologic studies of lymphoid aggregates in both acute and
chronic DSS-induced colitis. Random and systematic sections were
obtained from DSS colitis mice at various time points after the induction of colitis (refer Methods). Histologic sections demonstrated an
increasing frequency of submucosal (N ¼ 39 mice; closed circles) and
intramucosal (N ¼ 27 mice; open circles) mononuclear aggregates.
Linear curve fit for the submucosal (solid line) and intramucosal aggregates (dashed line) are shown with 95% confidence bands (dotted
Fig. 3. Immunohistochemical phenotyping of the submucosal and
intramucosal aggregates. Immunophenotyping demonstrated T-cell
(CD4) and B-cell (CD19) expression within both the submucosal and
intramucosal aggregates (day 30 shown). The B-cell prevalence, as
documented by CD19 and CD20 expression, increased in frequency
between 30 and 60 days and was associated with an increase in the
Spatial frequency analysis of the mononuclear aggregates within the submucosa demonstrated an increase in
size and prevalence during the 60 day study period (Fig.
2, close circles; R ¼ 0.79; F ¼ 61.8; P < 0.0001). Similarly, the smaller intramucosal aggregates also increased
in size and prevalence (Fig. 2, open circles; R ¼ 0.62; F
¼ 16.4; P < 0.0001). As expected, immunophenotyping
demonstrated T-cell (CD4) and B-cell (CD19) expression
within the submucosal aggregates (Fig. 3A–C). The prevalence of B cells within the aggregates, as demonstrated
by anti-CD19 and anti-CD20 staining, increased in frequency between 30 and 60 days. The increase in B-cell
frequency, demonstrated by immunofluorescence staining, was associated with an increasing frequency of
lymphoid follicles. Although most intramucosal and
development of lymphoid follicles (not shown). Intramucosal aggregates occasionally demonstrated a notable density of monocytes with
a prominent CD11b and F4/80 expression (Bar ¼ 100 lm) (day 30
shown). Other intramucosal nodules demonstrated more balanced distribution of lymphocytes and monocytes.
Fig. 4. Fluorescent vessel painting of the mucosal plexus microcirculation 30 days after the onset of DSS-induced colitis. The colitis
mice had the microcirculation flushed, fixed, and labeled with the
intravascular lipophilic tracer (red ¼ DiI). The colon was then prepared
as a whole mount and examined by wide field (A–D) and structured
illumination confocal microscopy. Intramucosal structures, consistent
with the distribution of intramucosal aggregates, were visible on lower
power (A,C). Higher magnification (B,D) demonstrated a small vessel
network in the plane of, and contiguous with, the mucosal plexus (B,D
Bar ¼ 80 lm).
submucosal aggregates were phenotypically similar, 6%–
10% of the intramucosal mononuclear aggregates demonstrated a predominance of monocytoid markers
(CD11b and F4/80) (Fig. 3D–F).
vascular microarchitecture, the mucosal plexus was
examined using intravital videomicroscopy and fluorescent vessel painting. In control mice, the mucosal
plexus—a quasi-polygonal network of vessels surrounding the mucosal crypts—was a continuous plexus without a specialized vascular supply to lymphoid tissue. In
contrast, 21 to 60 days after the onset of inflammation,
intravital microscopy studies of the mucosal plexus demonstrated distinctive microcirculatory structures composed of a small network of capillaries. The structures
Mucosal Plexus Angiogenesis
The development of intramucosal lymphoid tissue suggested the possibility of structural changes in the mucosal microcirculation. To investigate changes in the
Fig. 5. Angiogenic gene expression in chemically-induced colitis (A)
7 days, (B) 14 days, (C) 31 days, and (D) 65 days after the onset of
DSS exposure. In the SuperArray assay, angiogenic inflammatory
mediators including CCL2, CXCL2, CXCL5, MMP9, IL-1b, and TNF
are shown as solid squares (n). The angiogenic factors Angpt1,
Angpt2, Vegfa, Vegfb, and Vegfc are shown as solid triangles (s). The
remainder of the SuperArray angiogenesis PCR array genes are shown
as open circles (O). A linear regression was performed on the data
from each time point: (A) 0.975, (B) 0.857, (C) 0.916, and (D) 0.993 (P
< 0.0001); 95% prediction bands are shown.
were 185 46 lm (N ¼ 12) in diameter and appeared to
be contiguous with the mucosal plexus (Fig. 4). Morphometry based on fluorescent vessel painting and 3D
tissue mounts of the capillaries indicated a microvessel
diameter of 6.8 1.1 lm (N ¼ 6). Consistent with the
histologic analysis, the rarity of similar structures in
control mice suggested that the capillary structures
developed de novo. To explore the gene expression potentially involved in sprouting angiogenesis, mRNA was
isolated from microdissected mucosal plexus in DSSinduced colitis and control mice. The expression of genes
implicated in angiogenesis was explored using the angiogenesis pathway PCR arrays at 4 timepoints after the
induction of DSS colitis: 7, 14, 31, and 65 days (Fig. 5).
The expression of CXCL2, Il-1b, CXCL5, CCL2, TNF,
and MMP9 peaked at 14 days after the onset of DSSinduced colitis (Fig. 5B). In this bulk RNA, angiogenic
factors with a less notable inflammatory association,
such as Angpt1, Angpt2, Vegfa, Vegfb, and Vegfc, were
not significantly elevated relative to controls (Fig. 6).
Intravital Microscopy of the
Lymphoid Aggregate
The functional implications of the lymphoid angiogenesis was investigated by fluorescent intravital videomicroscopy. In the chronic phase, 30–60 days after the
onset of chemically-induced colitis, intravenously
injected fluorescent nanoparticles were tracked through
the mucosal capillary structures (Fig. 7A,B). Nanoparticle flow demonstrated that the particles passed directly
from the mucosal plexus into the capillary structures,
confirming both structural and functional continuity.
Frequently, the particles exited the capillary structures
and passed into deeper collecting veins. The flow
through the mucosal plexus structures was notable for a
Fig. 6. Angiogenic gene expression in chemically-induced colitis after the onset of DSS exposure.
Microdissected colon mucosa was obtained from colitis and control mice at 7, 14, 31, and 65 days after
the onset of colitis. Gene expression was determined by real-time qPCR. Gene expression is presented
relative to control values.
significant decrease in flow velocity when compared to
the feeding vessels within the mucosal plexus (Fig. 7C–
E). The analysis of intravital microscopy recordings (N ¼
6 mice) showed that particles passing into these de novo
vessels demonstrated a mean velocity of 491 155 lm/
sec. In contrast, the feeding vessels of the mucosal
plexus demonstrated a mean velocity of 808 901 lm/
sec (Fig. 7F; P < 0.01).
In this report, we studied the microvascular adaptations associated with prolonged inflammation in DSSinduced murine colitis. Although both submucosal and
intramucosal lymphoid aggregates were identified, the
development of lymphoid neogenesis within the superficial mucosa was associated with structural reorganization of the microcirculation. A frequency analysis
suggested that most intramucosal lymphoid aggregates
developed de novo. The intramucosal aggregates were
supplied by capillaries arising from the quasi-polygonal
mucosal plexus. The expression of genes associated with
both inflammation and angiogenesis suggested that the
structural changes were the result of inflammationinduced sprouting angiogenesis.
The structural changes observed in this study highlight the importance of the mucosal stroma in sustaining
a peripheral immune response. Previous work has
focused on the plasticity of vascular endothelial cells in
adapting to peripheral inflammation. Endothelial cells
can undergo dramatic inflammation-induced changes in
morphology—from flat conduit lining cells to cuboidal
HEV cells (Freemont, 1988; Sasaki et al., 1994; Peng,
1996). On a molecular level, the stromal-endothelial
interactions in ulcerative colitis and rheumatoid arthritis can stimulate the expression of PNAd, CCL21, and
CXCL13 proteins (Takemura et al., 2001; Salomonsson
et al., 2003; Carlsen et al., 2004; Manzo et al., 2005).
Consistent with other studies of inflammation (Weninger
et al., 2003), PNAd expression in our model was
Fig. 7. Intravital microscopy of DSS-induced intramucosal aggregates after intravenous injection of fluorescent plasma markers and
infra-red nanoparticle tracers. (A) A plasma-marker angiogram of an
intramucosal capillary structure in DSS colitis. A large submucosal
vein draining some of the capillaries is seen (arrow). (B) The same
mouse with digital recombination of 500 consecutive infra-red images
obtained at 20 msec intervals (Bar ¼ 100 lm). Fluorescence reflects
the infra-red nanoparticles within the capillary structure during the 500
image time series. (C–E) Instantaneous velocities of three nanoparticles as they were tracked through intramucosal structures (N ¼ 3
mice); arrows delineate the anatomic extent of the capillary structures.
(F) Combined data of 600 particles tracked in six mice; the mean velocity of particles in vessels feeding the structure and passing through
the structure are shown. The data was plotted with the box defining
the 25th and 75th velocity precentiles with the whiskers defining the
fifth and 95th percentile. The median value was plotted as a square.
inconsistent (not shown) suggesting that the variability
in HEV morphology and PNAd expression may reflect
different stages of ectopic lymphoid aggregate development. Regardless, the significant structural remodeling—including the apparent sprouting growth of a
complex arrangement of capillaries—suggests that stromal adaptations play an important role in the pathophysiology of prolonged DSS-induced colitis.
Our exploratory analysis of mRNA expression within
the mucosal plexus demonstrated several mediators previously associated with angiogenesis. Three members of
the CXC chemokine family, known to promote angiogenesis (Strieter et al., 2005), were expressed at high levels
during the peak of the inflammation. mRNA From the
CXCL1, CXCL2, and CXCL5 genes were expressed at
levels 90-to 8000-fold greater than controls. These CXC
family chemokines signal through the CXCR2 receptor—
a receptor that has been implicated in vivo in models of
corneal neovascularization (Addison et al., 2000) and
wound repair (Devalaraja et al., 2000). The association
of MMP9 with tissue remodeling (Page-McCaw et al.,
2007) suggests a functional role for extracellular proteases in the remodeling necessary for the development
of both lymphoid aggregates and lymphoid angiogenesis.
Furthermore, the inflammatory mediators IL-1b and
TNF also have been implicated in angiogenesis (Maruotti et al., 2006). In contrast, the expression of angiogenic factors such as Angpt1 and Vegfa were not
elevated relative to controls. This finding may reflect the
bulk sampling of mRNA. Although the mucosal plexus
was microdissected from the remainder of the colon wall,
the samples included bulk mRNA from the perivascular
inflammatory cells as well as the mucosal plexus vessels.
Discrete spatial sampling, enabled by laser capture
microdissection, may be necessary to elucidate the participation of these factors.
Gut-associated lymphoid tissue has been separated
into effector sites, which consist of lymphocytes scattered
throughout the superficial mucosal tissue and the induction sites present in organized lymphoid tissues (Mowat,
2003; Spahn and Kucharzik, 2004). The inductive sites
include Peyer’s patches, mesenteric lymph nodes, and
isolated lymphoid follicles. The contemporary understanding of immunologic function is that antigen presentation and the generation of antigen-specific effector
cells occurs in inductive tissues, and that effector cells
migrate into superficial mucosal tissues. Our observation
of progressive organization of the superficial mucosal
compartment suggests that this initial functional distinction may evolve during the subacute phase of inflammation leading to the presence of both inductive and
effector elements with the chronically inflamed colonic
mucosa. This functional evolution within mucosal tissue
is suggested by human studies of secondary and ectopic
lymphoid tissue (Manzo et al., 2007). Distorted crypt
architecture, intramucosal inflammatory cells, and
lymphoid aggregates were features present in 79% of
ongoing inflammatory bowel disease and were highly
predictive of chronic colitis (Surawicz and Belic, 1984).
The development of inductive sites may also provide
an explanation for the spatial distribution of the intramucosal aggregates. The sporadic distribution of intramucosal lymphoid aggregates does not reflect any
vascular microarchitectural feature that might predispose to capillary sprouting and lymphoid angiogenesis.
Rather than a structural predisposition to lymphoidassociated angiogenesis, we suspect that the spatial distribution of lymphoid aggregates may reflect the location
of antigen presenting cells, such as dendritic cells, in the
initiation of lymphoid neogenesis (Carragher et al.,
2008). The importance of antigen presenting cells in
lymphoid neogenesis may also help explain the occasional concentration of monocytoid cells within the intramucosal aggregates.
An advantage of our study was the use of intravascular tracers to demonstrate structural continuity between
the capillary structures and the mucosal plexus. Because
the particles were inert and charge-neutral, they could
be tracked through the microcirculation without the concern of unanticipated biomolecular interactions with
vascular lining cells. The particles provided a useful
measure of both flow velocity and network flow fields
within the sprouting capillary structures. An interesting
observation was the diminished flow velocity within the
capillary structures; velocities were sufficiently diminished to be within the physiologic range of rolling velocities in secondary lymphoid tissue (Stein et al., 1999).
Thus, even if some of the typical secondary lymphoid
organ receptor-ligand interactions were not present, the
microhemodynamic conditions were nonetheless suitable
for lymphoid adhesion and transmigration (Li et al.,
Finally, an assessment of capillary structure and
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blood flow within the mucosal aggregates. Assuming an
average of 10 capillaries with cylindrical geometry, and
a cardiac output of 20 mL/min (Janssen et al., 2002), the
perfusion of an average capillary structure would be
0.0015% of cardiac output; that is, 10% of the perfu-
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(Hay and Hobbs, 1977). This finding indicates that even
small and developing lymphoid structures possess the
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microarchitecture, associates, colitis, angiogenesis, vascular, murine, lymphoid
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