After injection of the VEGF receptor antagonist in the fully formed granuloma, the preexisting blood vessels in the middle layer regressed and underwent apopto-sis, accompanied by expans
Trang 1results in microvessel attrition and disorganization of wound tissue
KRISHNAMURTHY P GUDEHITHLU, NAILA AHMED, HENRY WU, NATALIA O LITBARG,
SANDRA L GARBER, JOSE A L ARRUDA, GEORGE DUNEA, and ASHOK K SINGH
CHICAGO, ILLINOIS
Vascular endothelial growth factor (VEGF) is a potent growth factor that is indispens-able for the development of blood vessels in the fetus and for wound healing in adults VEGF likely plays a role in maintaining the blood vessels once they have been formed.
It is not clear, however, whether a low tissue VEGF (caused either by disease or by systemic administration of VEGF antagonists) can cause abnormalities in preexist-ing blood vessels, especially of wound tissue that requires high local levels of VEGF for healing The present study investigated the effect of VEGF antagonism on blood vessels of foreign-body granulomas (a model of wound-healing tissue) Granulo-mas were induced by implanting perforated polyvinyl tubes into the subcutaneous tissue of rats and allowed to develop for 14 days, at which time the implanted tubes were completely encapsulated by the subcutaneous tissue The encapsulated granulomas consisted of 3 distinct histological layers, of which the middle layer was well perfused by a rich supply of microvessels Morphologically, the granuloma remained “stable” after developing for 14 days At 1 week, VEGF levels in the granuloma fluid, which is in equilibrium with the interstitial fluid, were 25 times higher than in the plasma VEGF levels in the granuloma fluid continued to increase for up
to 3 weeks, reflecting the high dependence of the wound tissue on ambient VEGF levels After injection of the VEGF receptor antagonist in the fully formed granuloma, the preexisting blood vessels in the middle layer regressed and underwent apopto-sis, accompanied by expansion of the extracellular matrix (predominately collagen I) into areas normally devoid of matrix We conclude that wound tissue is sensitive
to ambient VEGF levels, and that a low VEGF condition resulting from VEGF receptor antagonism can disrupt the healing of wound tissue (J Lab Clin Med 2005;145:
194 –203)
Abbreviations: ED 50 ⫽ median effective dose; ELISA ⫽ enzyme-linked immunosorbent assay; FITC
⫽ fluorescein isothiocyanate; PAS ⫽ periodic acid-Schiff; PCNA ⫽ proliferating cell nuclear anti-gen; TUNEL ⫽ terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling; VEGF ⫽ vascular nick-endothelial growth factor; vWF ⫽ von Willebrand factor
V ascular endothelial growth factor (VEGF), a
powerful endothelial cell growth factor,
pro-motes the development of new blood vessels
during fetal development, as shown by its critical
pres-ence in fetal tissues and by experiments in which its deletion proved to be lethal.1–3VEGF is also critical in inducing new blood vessels in wound-healing tissue.4,5 That even blood vessels in the normal adult tissue From the Division of Nephrology, Cook County Hospital, Chicago,
IL; Section of Nephrology, University of Illinois and the Chicago
Veterans Administration Medical Center, Chicago, IL; and the
Hek-toen Institute for Medical Research, Chicago, IL.
Supported by the National Kidney Foundation of Illinois, the Juvenile
Diabetes Foundation International (grant JDA 1-2000-241), and the
U.S Department of Veteran Affairs Merit Review Program (J.A.L.A.
and R.B.R.).
Submitted for publication September 9, 2004; accepted for publica-tion February 15, 2005.
Reprint requests: Ashok K Singh, PhD, Hektoen Institute for Medical Research, 2100 West Harrison St., Chicago, IL 60612; e-mail: singhashok@comcast.net.
0022-2143/$ – see front matter
© 2005 Mosby, Inc All rights reserved.
doi:10.1016/j.lab.2005.02.007
194
Trang 2continue to be responsive to VEGF is indicated by
experiments in which VEGF injection into the cornea
or skeletal muscle of adult mice resulted in sprouting of
new blood vessels.6,7It is less clear, however, whether
low tissue VEGF, caused either by disease or by
sys-temic administration of VEGF antagonists for
treat-ment of solid cancers,8,9 can cause abnormalities in
preexisting blood vessels, especially of wound tissue
that depends on high local levels of VEGF for growth
and sustenance
To address this issue, we induced a foreign-body
granuloma by implanting a perforated polyvinyl tube in
the subcutaneous tissue of rat By 2 weeks, the
im-planted tube was completely covered by new tissue and
blood vessels (granuloma) This tissue growth was
de-pendent on high VEGF levels, as suggested by high
levels of VEGF found in the granuloma fluid that
accumulated inside the tube Thus, although induced by
an external stimulus, a foreign-body granuloma
repre-sents new tissue growth, having similarities to wound
healing, and thus is a suitable model for examining the
effects of VEGF antagonism on a healing tissue
We first measured VEGF level in the granuloma fluid
during the development of the granuloma, to ascertain
that VEGF level was associated with the formation of
new blood vessels and associated tissues To study the
role of low VEGF on granuloma blood vessels, we
injected an antagonist against the VEGF receptor in the
granuloma soon after tube implantation (to study the
effects on new blood vessel formation) and, more
im-portant, after its complete encapsulation (to study the
effects on preexisting blood vessels) The antagonist
was injected at a dose that blocked VEGF binding to its
receptor locally in the granuloma but was too low to
affect systemic VEGF reactivity
MATERIALS AND METHODS
Construction of the perforated polyvinyl tube for
subcu-taneous implantation A piece of polyvinyl chloride tubing
(length, 20 mm; diameter, 7 mm) (PVC 180; Nalge Nunc,
Rochester, NY) was sealed at both open ends by heat
appli-cation to create an enclosed chamber (inside volume, 0.5 mL).
Eight holes (diameter, 0.5 mm) were drilled around the
cham-ber to allow steady diffusion between the tube contents and
the surrounding tissue The tubes were stored in 70% alcohol
for sterility Before implantation in the rats, the tubes were
washed vigorously with sterile saline solution and air-dried.
Surgical implantation of the polyvinyl tube in rats
Ani-mal experiments were conducted under the protocol approved
by the institutional animal care and use committee
Sprague-Dawley rats (males, 225–250 g) were anesthetized with a 1:5
mixture of ketamine 50 mg/mL and acepromazine 10 mg/mL
at a dose of 0.1 mL/100 g The animals’ backs were shaved
and cleaned with alcohol and povidone In each animal, two
1-cm incisions were made on either side of the lumbar region.
Using blunt dissection, a subcutaneous pocket was made around the incisions, into which the polyvinyl tubes were inserted The incisions were closed with silk sutures, and the animal was allowed to recover and form a granuloma around the tube.
Injection of VEGF receptor antagonist in the granu-loma VEGF receptor antagonist was purchased from R & D Systems, Minneapolis, MN (cat # 471-F-1) The VEGF re-ceptor antagonist is a soluble recombinant mouse VEGF R1 (Flt-1)/Fc chimera that is active in inhibiting the VEGF-dependent proliferation of cultured human umbilical vein endothelial cells According to the manufacturer, the median effective dose (ED50) of this activity is 10 –30 ng/mL The VEGF receptor antagonist treatment was initiated fol-lowing 2 protocols The first protocol was designed to study the effect of the VEGF receptor antagonist on new blood vessel formation in the granuloma; the second protocol was designed to study its effects on preexisting blood vessels In the first protocol, administration of the VEGF receptor antag-onist was initiated 3 days after implantation of the plastic tube, and the injections were continued for 2 weeks, after which the granulomas were harvested In the second protocol, administration of the VEGF receptor antagonist was initiated
2 weeks after tube implantation (after complete encapsulation
of the tube had occurred), and the injections were continued for 2 weeks, after which the granulomas were harvested Animals were classified as either control or experimental in each protocol Each experimental animal received 0.5 mL sterile saline solution containing 100 ng of the antagonist/ granuloma on the first day and 0.1 mL containing 20 ng of the antagonist/granuloma on subsequent alternate days Controls received similar volumes of saline solution in the granuloma
at the same times as the experimental animals The animals were injected under restraint by passing the 25-gauge syringe needle through the skin, the granuloma, and the polyvinyl wall of the tube into the cavity of the granuloma ( Fig 1 ).
Estimation of the intragranuloma and blood levels of VEGF receptor antagonist Because of the unavailability of
an assay for VEGF receptor antagonist, we estimated the antagonist level by determining the clearance rate of the granuloma fluid This was carried out in a separate group of
Fig 1. Method of injecting test substances and aspirating granuloma fluid from the polyvinyl tube granuloma in live rats (under re-straint).
Trang 3rats (n ⫽ 5) implanted with polyvinyl tubes Two weeks after
tube implantation, a trace amount of radioactive ( 125 I) human
albumin (prepared by the chloramine-T method 10 ) was
in-jected in the granuloma Samples (25 L) of granuloma fluid
and serum were collected at time 0 and every 30 minutes
thereafter for 3 hours The samples were precipitated with
10% trichloroacetic acid, and the radioactivity in the
precip-itates was measured in a gamma counter The data were
plotted to obtain a clearance curve for injected ( 125 I) human
albumin.
Determination of VEGF levels in the granuloma fluid.
VEGF165levels in the granuloma fluid and serum were
de-termined by a sandwich enzyme-linked immunosorbent assay
(ELISA) kit (murine; R&D Systems) The interference
be-cause of the presence of VEGF receptor antagonist (present in
the granuloma fluid of experimental rats) in the VEGF ELISA
was determined by adding increasing amounts of the
antag-onist to standard solutions of VEGF and comparing the
con-sequent VEGF levels with the VEGF levels in samples not
containing the antagonist ELISA found no difference in
measured VEGF levels in the free and
antagonist-containing solutions, confirming that the copresence of the
VEGF receptor antagonist in the samples did not interfere
with the VEGF measurements.
Processing of granulomas Each granuloma was
surgi-cally resected and its wet weight recorded About 1/4 of the
granuloma was placed in a nonformalin fixative (Histochoice;
Amresco, Solon, OH) for histological studies, and the
remain-ing 3/4 was processed for hemoglobin determination.
Hemoglobin determination in the granulomas for
deter-mination of blood content in the granulomas Hemoglobin
concentration was determined spectrometrically as a
surro-gate index of the blood content of the granuloma Each
granuloma was placed in ice-cold water containing heparin to
promote red cell lysis and avoid blood clotting The tissue
was homogenized in a polytron homogenizer The
homoge-nate was centrifuged at 15,000 g for 10 minutes The
super-natant was filtered through 0.45- filters to obtain a clear
filtrate The filtrate was scanned in a spectrophotometer in the
400 – 800 nm range to measure the 2 typical absorption peaks
of hemoglobin at 540 and 575 nm Hemoglobin concentration
was determined from the absorption at 575 nm using a
stan-dard curve constructed from fresh diluted human blood with
known hemoglobin concentration, also read at 575 nm
As-suming the hemoglobin content in normal blood to be 15
g/dL, the hemoglobin concentration was converted to L
blood/granuloma and L blood/g of tissue.
Histology and immunocytochemistry Tissues fixed in
Histochoice were embedded in paraffin, and 4--thick
sec-tions were cut in a microtome Secsec-tions were deparaffinized
and stained with trichrome and periodic acid-Schiff (PAS)
stains for visualizing extracellular matrix and general tissue
morphology.
The nature of the extracellular matrix was further examined
by immunostaining, by first incubating with goat anti–type I
collagen, goat anti–type III collagen (Southern Biotechnology
Associates, Birmingham, AL) and rabbit anti-human
fi-bronectin (Chemicon, Temecula, CA) antibodies, followed by
washing and reincubating with the appropriate secondary anti-goat IgG-alkaline phosphatase conjugate (or anti-rabbit IgG-alkaline phosphatase antibodies) (Sigma, St Louis, MO) The slides were washed, and the bound alkaline phosphatase was developed with fast-red naphthol (Sigma).
Slides were similarly immunostained for VEGF, VEGF-R1 and -R2, and proliferating cell nuclear antigen (PCNA) by first incubating with the primary antibody [rabbit anti-mouse VEGF (NeoMarkers, Fremont, CA), goat anti-mouse VEGF-R1 (Flt-1) and R2 (Flk-1) (R&D Systems), or anti-mouse PCNA (clone 19A2; Biogenex, San Ramon, CA)], followed by an appropriate second antibody, fluorescein iso-thiocyanate (FITC)-labeled in cases of VEGF, VEGF-R1, and VEGF-R2 and peroxidase-labeled in cases of PCNA FITC-labeled slides were examined and photographed under epiflu-orescence (Nikon, New York, NY), and peroxidase labeled slides were developed with diaminobenzidine-H2O2(brown color) and examined under a light microscope (Nikon) Microvessel density was studied by staining sections for type IV collagen Sections were first incubated with primary goat anti–type IV collagen (Southern Biotechnology Associ-ates), followed by washing and reincubation with the second-ary anti-goat IgG-alkaline phosphatase conjugate (Sigma) The slides were washed, and the bound alkaline phosphatase was developed with fast-red naphthol (Sigma).
The organization of the microvessels (only those with ⬎ 3– 4 endothelial cells) in the granuloma tissue was examined
by double immunostaining for ␣-smooth muscle actin, to highlight pericytes, and von Willebrand factor (vWF; also known as factor VIII), to highlight endothelial cells (Smaller microvessels containing 1–3 endothelial cells could not be analyzed in this manner, because they were not stainable by factor VIII antibody.) The tissue was permeabilized by pre-treatment with trypsin (10 g/mL; Sigma) at room tempera-ture for 10 minutes, then reacted with monoclonal anti–␣-smooth muscle actin (Sigma) antibody, followed by anti-mouse IgG conjugated to alkaline phosphatase The enzyme reaction was developed with fast-red naphthol (red color) Subsequently, the tissue was reacted with rabbit anti-vWF antibody (Sigma) and anti-rabbit IgG antibody conjugated to horseradish peroxidase and developed with diaminobenzi-dine-H2O2(brown color) The tissue was counterstained with hematoxylin (blue color).
Apoptosis in the nuclei of microvessels was visualized by double-staining for type IV collagen (to highlight microves-sels) and DNA fragments (to highlight apoptotic nuclei in microvessels) The sections were first immunostained for type
IV collagen (red color), as described earlier Subsequently, apoptosis was visualized by staining for DNA fragments by the reaction of tissue with dT terminal transferase in the presence of biotinylated DNA bases, followed by streptavi-din-peroxidase and diaminobenzidine-H2O2reaction (brown color) using reagents supplied by Travegen (Gaithersberg, MD) The sections were finally counterstained with hematox-ylin (blue color).
Quantification of immunocytochemical changes Rep-resentative areas of type I collagen, type III collagen, fi-bronectin, and VEGF-stained slides from several granulomas
Trang 4were photographed (n ⬎ 10) and analyzed for intensity of
appropriate color (red for fibronectin and types I and III
collagens, green for VEGF) using the Image J software
(JAVA imaging software inspired by the National Institutes
of Health and available free at http://rsb.info.nih.gov ) The
percentage changes between the control and VEGF-receptor
antagonist groups reported in the results and figure legends
were derived from the arbitrary intensity units on a scale of 0
(zero intensity)–255 (maximum intensity) P values of
changes (Student t test) are indicated in the text.
Microvessels stained with type IV collagen were quantified
by counting the number of vessels per high-power field.
Apoptosis was quantified by randomly examining 20
mi-crovessels in the medial layer of each granuloma (n ⫽ 3 from
each group), and approximately 300 healthy and apoptotic
endothelial nuclei were scored (brown-stained) to arrive at a
statistical evaluation of the extent of apoptosis in the
mi-crovessels PCNA-positive cells were counted from several (n
⫽ 20) high-power fields of control and antagonist-treated
granulomas (n ⫽ 4 of each group).
All quantitative data are expressed as mean ⫾ standard
error, and statistical comparisons were made using the
Stu-dent t test.
RESULTS
Induction of the subcutaneous foreign body
granu-loma. In preliminary experiments, we studied the
for-mation of granuloma after the surgical implantation of
a polyvinyl tube in the subcutaneous tissue of rat After
implantation, animals were killed at 1 week and 2, 3,
and 4 weeks, and granulomas were extracted The
gran-ulomas were examined, wet-weighed, homogenized to
determine hemoglobin (or blood) content, and
pro-cessed for histology At 1 week, the polyvinyl tube was
completely encapsulated with a thin layer of tissue,
which was supplied by at least 2 large blood vessels
extending from the surrounding tissue By 2 weeks, the
granuloma appeared thicker than at 1 week Also by 2
weeks (not seen at 1 week), the holes around the plastic
tube were plugged by tissue, which appeared to be
highly vascular The granulomas increased in weight
until 2 weeks, after which the weights remained
con-stant for up to 4 weeks
The granuloma was organized in 3 histologically
demarcated layers (Fig 2) The inner layer in contact
with the polyvinyl tube was approximately 200 thick
and consisted of mononuclear epithelioid cells and
fi-broblasts, had little extracellular matrix, and was
de-void of blood vessels The medial layer was thicker and
consisted of tightly packed fibroblastic cells and
extra-cellular matrix fibers (stained blue by trichrome)
ar-ranged parallel to the length of the granuloma This
layer was also richly endowed with fine blood vessels
The outer layer was more amorphous, with loose
con-nective tissue and large blood vessels One-week-old
granulomas were as histologically organized as the older granulomas, except that each of the 3 histological layers was thinner than in the older granulomas The organization of the microvessels was examined immunohistochemically by double-staining with anti–
␣-smooth muscle actin to visualize the pericytes and with anti-vWF to highlight the endothelial lining of the microvessels It was observed that 100% of the mi-crovessels containing at least 3– 4 endothelial cells also had pericytes surrounding the endothelial layer, indi-cating that the microvasculature in the granuloma was similar to that seen in normal adult state (not shown) The histological pattern including microvessels ob-served 2 weeks after implantation of the polyvinyl tube remained unchanged during the next 2 weeks, suggest-ing stabilization of the healsuggest-ing process
Granuloma fluid. Within days after implantation of the polyvinyl tube, an accumulation of a clear fluid inside the tube was seen, which was in a dynamic equilibrium with the interstitial fluid/plasma, being re-placed by 50% within 90 minutes, as determined by radiotracer studies (Fig 3) It resembled plasma (or serum) This was confirmed by the similarity of its electrophoretic profile (sodium dodecyl sulfate–polyac-rylamide gel electrophoresis) to that of rat plasma and serum (not shown).Fig 4shows the VEGF concentra-tion in the granuloma fluid of control animals between
Fig 2. Histology of the foreign-body granuloma formed 2 weeks after the subcutaneous implantation of a polyvinyl tube in the rat The granuloma appeared to be organized in 3 histological demarcated layers The inner layer (⬃ 200 thick) contained mostly mononu-clear epithelioid cells and fibroblasts The thicker medial layer con-sisted of tightly packed fibroblastic cells, extracellular matrix (blue stain), and microvessels (arrows) The outer layer was made up of loose connective tissue and large blood vessels (Trichrome stain; original magnification ⫻ 50.)
Trang 5weeks 1 and 4 VEGF levels in the serum of control
animals remained below 0.1 ng/mL at all times (data
not shown) The VEGF levels in the granuloma fluid at
1 week were 25 times higher than that in the serum and
continued to increase to 50 times higher than that in the
serum by the third week, after which they remained
unchanged
Effect of injecting VEGF receptor antagonist during
granuloma formation. Initiating the VEGF-receptor
an-tagonist injections in the granuloma soon after
polyvi-nyl tube implantation (first protocol) resulted in incom-plete encapsulation of the tube In contrast, control granulomas exhibited well-formed, complete encapsu-lation These results were reflected in significantly lower wet weights and blood content of the experimen-tal granulomas, confirming the antiangiogenic activity
of the VEGF receptor antagonist on new blood vessel formation (Table I) Note that even though the blood content/granuloma decreased in the antagonist-injected granulomas, the blood content/g of tissue remained the same (Table I)
In the next set of experiments, we tested the effect of the VEGF receptor antagonist in formed granulomas by injecting the antagonist starting 2 weeks after implan-tation of the polyvinyl tube (second protocol) Exami-nation of these granulomas revealed well-formed gran-ulomas that were more fibrous, less elastic, and more leathery to the touch than the control tissues Despite these qualitative differences, the wet weights and blood content of the VEGF receptor antagonist–injected gran-ulomas were similar to those of the control grangran-ulomas (Table I) However, important histological changes were seen in the microvessels of the granulomas (see below) Because we were interested primarily in exam-ining the effects of the VEGF receptor antagonist in preexisting blood vessels, we carried out further studies only in the granulomas from the second protocol experiment
Estimation of the intragranuloma and blood levels of VEGF receptor antagonist. We found the half-life of the granuloma fluid to be 90 minutes (Fig 3) Given the initial concentration of the antagonist injected in the granuloma of 200 ng/mL (10 ⫻ ED50) on the first day and 40 ng/mL on subsequent days (2 ⫻ ED50) (see Materials and Methods), one can see that these concen-trations will decline to below ED50within a matter of a few hours after injection as the antagonist diffuses out
of the granuloma into the blood Assuming rapid clear-ance of the antagonist from the plasma, one could then estimate that the drug would be at its highest concen-tration in the blood soon after the intragranuloma in-jection The blood concentration could be estimated to
be 300-fold less than the intragranuloma concentration, because the whole body fluid content of the animal (150
mL for a 250-g rat) is 300 times that of the intragranu-loma volume (0.5 mL) Therefore, over the period of 2 weeks of antagonist injections, the antagonist must have reached the 10-fold ED50level only once for a few hours after the first injection and, subsequently, the 2-fold ED50 for a few hours every other day in the granuloma For most of the remaining time, the antag-onist must have been less than the ED50 level in the granuloma The blood levels must be less than the ED50 levels at all times
Fig 4. Comparison of VEGF levels in the granuloma fluid from
control and VEGF receptor antagonist–injected animals at different
times before and after injection of the antagonist Each bar represents
a mean of 4 samples *Denotes statistical difference at P ⬍ 05
compared with control sample at the same time VEGF levels were
comparable between controls and experimental until 2 weeks (ie,
before injection of VEGF receptor antagonist) After injection of the
receptor antagonist, VEGF levels decreased in the experimental
gran-ulomas compared with controls, suggesting inhibition of the VEGF
production in the granuloma by the antagonist VEGF levels in serum
were ⬍ 0.1 ng/mL at all times (not shown).
Fig 3. Clearance of 125 I-albumin from the polyvinyl tube granuloma
fluid As indicated, a half-life of approximately 90 minutes was
calculated for the clearance of 125 I-albumin.
Trang 6VEGF in the granuloma fluid and tissue after VEGF
re-ceptor antagonist injection. Fig 4shows VEGF
concen-trations in the granuloma fluid from weeks 1– 4 in the
experiment where the VEGF receptor antagonist was
injected after 2 weeks of tube implantation (second
protocol) For the first 2 weeks of granuloma formation
and before VEGF receptor antagonist injection, VEGF
levels were similar in the control and experimental
granulomas, as expected After the start of the VEGF
receptor antagonist injection, VEGF levels in the
ex-perimental granulomas became significantly lower than
in the control granulomas injected with saline solution
The lower VEGF level was not because of interference
in the immunoassay due to the copresence of the VEGF
receptor antagonist in the granuloma fluid, because the
antagonist per se did not interfere in the assay for
VEGF (see Materials and Methods) By
immunocyto-chemical staining, VEGF was localized predominantly
to the inner layer of the granuloma, and consistent with
the decreased fluid levels of VEGF, a significant
de-crease in tissue-associated VEGF was also observed in
this area (Fig 5)
Extracellular matrix changes in the granuloma after
treatment with VEGF receptor antagonist. Granulomas
from VEGF receptor antagonist–injected animals (sec-ond protocol) were histologically distinguishable from the control granulomas by the expansion of the extra-cellular matrix (PAS-positive material) from the medial layer to the inner layer, resulting in disorganization of the inner layer Moreover, the extracellular matrix in these granulomas appeared to lose its orderly orienta-tion compared with that in controls (Fig 6) The nature
of the PAS-positive material was further examined by immunostaining of the granuloma sections for collagen
I, collagen III, and fibronectin The medial and outer layers of the control granulomas were filled predomi-nately with collagen I, which increased in amount and expanded into the inner layer after injection of the VEGF receptor antagonist (Fig 7A, B) (control ⫽ 35.2
⫾ 3.3 vs antagonist treated ⫽ 55.1 ⫾ 2.8 red intensity
units/unit area; n ⫽ 12; P ⬍ 05) Small amounts of
fibronectin were diffusely distributed in the inner and medial layers of the control granulomas, and these appeared to decrease slightly after VEGF receptor an-tagonist injection (data not shown) Collagen III was present in minor amounts throughout the granuloma and did not appear to change after the antagonist treat-ment (data not shown)
Fig 5. Immunofluorescent localization of VEGF in control and experimental (treated with the VEGF receptor
antagonist) granulomas In the control granuloma, VEGF was localized predominantly to the inner layer, which
decreased significantly after treatment with the VEGF receptor antagonist (Control VEGF intensity ⫽ 91.1 ⫾
6.3 versus antagonist-treated ⫽ 51.0 ⫾ 4.6 green intensity units/unit area; n ⫽ 12; P ⬍ 05; original
magnification ⫻ 100.)
Table I Wet weights and blood content of granulomas injected with VEGF receptor antagonist
Wet weight (g)
Blood content (L/granuloma)
Blood content (L/g of tissue) VEGF receptor antagonist injected 3 days after tube implantation (first protocol)
VEGF receptor antagonist injected 14 days after tube implantation (second protocol)
Granulomas were harvested for wet weights and blood content (by hemoglobin determination) at the end of the VEGF receptor antagonist injections (17 days after tube implantation in the first protocol and 28 days after tube implantation in the second protocol).
*P ⬍ 05 compared with its respective control.
Trang 7Microvascular changes in granuloma injected with
VEGF receptor antagonist. The microvasculature of the
granulomas was examined by immunostaining for type
IV collagen, which highlighted the endothelial
base-ment membrane of the microvessels (Fig 7C and D; low
power) Whereas the control tissue exhibited a high
density of microvessels in the medial layer (53 ⫾ 6
capillaries/high-power field; n ⫽ 12), the experimental
tissue demonstrated significantly fewer and weaker
mi-crovessels in this layer (23 ⫾ 4 capillaries/high-power
field; n ⫽ 12; P ⬍ 05), suggestive of degenerating
vessels (Fig 7E and F; high power)
Apoptosis in the microvessels of the granuloma. The
granuloma sections were double-stained for DNA
frag-mentation and type IV collagen to examine the
endo-thelial cells for signs of apoptosis (Fig 8) There was a
statistically significant 8-fold increase in the degree of
apoptosis in the microvessels of the granulomas treated
with VEGF receptor antagonist compared with
controls
Cell proliferation in the microvessels of the
granu-loma. To examine cell proliferation in the granuloma,
tissues were immunostained for PCNA, a marker of cell
proliferation Most of the proliferative activity was
present in the medial layer, which is rich in
microves-sels Compared with controls, the VEGF receptor
an-tagonist–injected granulomas demonstrated 70% fewer
PCNA positive cells in the medial layer, suggesting that
the antagonist exerted a significant antiproliferative
ef-fect on the microvessels (Fig 9)
DISCUSSION
When a perforated polyvinyl tube was implanted
subcutaneously into rats, it was surrounded by a
gran-uloma amply sustained by a new vascular network derived from surrounding blood vessels The histolog-ical pattern observed in this granuloma appeared to be very similar to other foreign-body granulomas de-scribed in the literature.4,10 –13The polyvinyl tube gran-uloma model was preferred because it created an en-closed wound tissue that allowed us to inject the drug (VEGF receptor antagonist) directly into the confined wound tissue and to conveniently harvest the solid wound tissue for histological evaluation After encap-sulation (1 week after implantation), the implanted tube collected a plasma-like fluid that could be sampled for measuring VEGF levels
The origin of the granuloma fluid is not completely understood Based on the immunocytochemical studies,
it appears that VEGF was secreted primarily by the inner layer of the granuloma, then diffused and accu-mulated in the polyvinyl tube Being derived from a rapidly growing wound tissue, this fluid is rich in VEGF and other growth factors The fluid is in equi-librium with the interstitial fluid (fluid bathing the gran-uloma tissue), and as such reflects the fluid environ-ment of the granuloma tissue Clearance experienviron-ments with radioactive albumin showed that the fluid was replaced by 50% in 90 minutes (half-life)
Injecting the VEGF receptor antagonist into the granuloma soon after implantation of the polyvinyl tube (after 3 days) prevented the formation of a complete granuloma, as expected based on the crit-ical role of VEGF in new blood vessel formation in wound healing.4,5This resulted in decreased weight and lower blood content of the granulomas This experiment confirmed the bioactivity of the VEGF receptor antagonist used in this study All subsequent
Fig 6. Histological changes in control and experimental (rats treated with the VEGF receptor antagonist)
granulomas Experimental granulomas were distinguishable from the control granulomas by the expansion of the
extracellular matrix (pink) from the medial layer to the inner layer Furthermore, the extracellular matrix in the
experimental granulomas appeared disorganized compared with that of the control granulomas (PAS stain;
original magnification ⫻ 150.)
Trang 8studies were performed in pre-formed granulomas to
allow us to study the effect of VEGF receptor
antag-onist on preexisting blood vessels
Injecting the VEGF receptor antagonist after
com-plete formation of the granuloma had a dramatic effect
on the already-formed blood vessels and matrix The
blood vessels in the middle layer appeared to have
atrophied, and there was a concomitant expansion of
the extracellular matrix, consisting of predominantly
type I collagen (interstitial collagen), into the inner
layer that was in contact with the polyvinyl tube This
resulted in disruption of the inner layer, which now
looked craggy and disorganized The extracellular
ma-trix itself, originally organized in parallel to the length
of the granuloma, lost its directional arrangement so that the tissue was less elastic and more leathery to the touch Neither the weight of the granuloma nor its blood content (as measured by tissue hemoglobin) changed, possibly because the changes were limited only to the fine capillaries of the middle layer In these capillaries, the blockade of VEGF caused inhibition and apoptosis of the endothelial cells, as detected by decreased PCNA staining and increased terminal de-oxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) reaction
It should be noted that although in a disease state (like cancer), any VEGF antagonist would have to be delivered systemically, we preferred to apply the an-tagonist directly in the wound, to avoid indirect and confounding effects of the antagonist on the systemic vascular tissue and other vital organs
The VEGF-receptor antagonist that we used in the study is a soluble receptor that blocks VEGF action by preventing VEGF from binding to its natural receptor
on the endothelial cell Unexpectedly, the antagonist treatment reduced VEGF levels in the granuloma fluid The reason for this decrease in VEGF is not known, but could be related to accelerated catabolism of VEGF or excessive turnover of the granuloma fluid Further stud-ies are needed to investigate these possibilitstud-ies Because an assay for VEGF receptor antagonist was not available, we estimated the antagonist level in gran-uloma and blood by determining the clearance rate of
Fig 7. Distribution of extracellular matrix (type I collagen) and
microvessels (type IV collagen) by immunostaining (red) in control
and experimental (rats treated with the VEGF receptor antagonist)
granulomas A and B, Control granulomas showed well-organized
type I collagen mostly in the extracellular regions of the medial layer
(A) In granulomas injected with the VEGF-receptor antagonist, type
I collagen appeared less well organized, increased in the medial layer,
and extended into the inner layer (B) (Uncounterstained; original
magnification ⫻ 50.) C and D, Architecture of granuloma blood
vessels visualized by type IV collagen immunostaining (red) at low
power Well-formed robust blood vessels and microvessels appeared
in the medial layer of control granulomas (C) In granulomas treated
with the VEGF receptor antagonist, the blood vessels were fewer in
number and appeared to be degenerating (D) (Uncounterstained;
original magnification ⫻ 50.) E and F, Microvascular changes in the
medial layer of granulomas visualized by type IV collagen
immuno-staining (red) at high power Whereas well-formed capillaries are
visible in the control granulomas (E), there appear to be fewer and
weaker microvessels in the medial layer of granulomas injected with
the VEGF receptor antagonist, suggestive of degenerating vessels
(F) [Uncounterstained (background color adjusted to highlight red);
original magnification ⫻ 200.]
Fig 8. Representative photomicrographs showing endothelial nuclei
of a microvessel undergoing apoptosis (stained for TUNEL) in the
medial layer of control (A) and experimental (rats treated with the VEGF receptor antagonist) (B) granulomas Normal nuclei are
stained blue, and apoptotic nuclei are stained brown (Blue
counter-stain; original magnification ⫻ 600.) (C) Quantitative comparison of
apoptotic nuclei found in the microvessels of control and VEGF receptor antagonist–injected granulomas There was an 8-fold in-crease in the number of apoptotic endothelial nuclei in the VEGF
receptor antagonist group compared with controls *P ⬍ 05
com-pared with controls (n ⫽ 300 nuclei).
Trang 9the granuloma fluid using radioactive albumin as a
tracer Based on the experimentally determined
clear-ance rate of the granuloma fluid, we showed that the
intragranuloma antagonist dose used produced
antago-nist levels in the pharmacological range in the
granu-lomas and only trace levels in blood
Low tissue VEGF levels have been found in diabetic
nephropathy,14,15 other chronic glomerular diseases,16
experimental models of reduced renal mass,17
mesan-giolysis (induced by anti-Thy 1 antibody),18 diabetic
peripheral vascular damage,19,20 and
hyperoxia-in-duced lung endothelial damage.21 The harmful effects
of such low VEGF levels has been reversed by the
systemic administration of VEGF or by gene transfer,
an effect due largely to new blood vessel formation but
not necessarily exerted on the preexisting blood vessels
and the surrounding matrix.19,20,22
Although a strong association has been documented
between disease and low VEGF, the specific
patholog-ical effects exerted on the preexisting blood vessels by
a low VEGF environment have not yet been clarified
Our results suggest that in a healing tissue (and possibly
even in the normal adult tissue), whereas the major
blood vessels seem to be refractory to low VEGF, the
microvessels and the surrounding matrix are sensitive
to the ambient VEGF levels Low VEGF levels caused
attrition and disorganization of the pre-existing
mi-crovessels We also showed that the vessel regression
after administration of the VEGF receptor antagonist was due to an antiproliferative effect on the endothelial cells, resulting in cell death by apoptosis
So far, a direct effect of low VEGF levels on preex-isting blood vessels has been reported only in experi-ments conducted in tumors In such experiexperi-ments, de-creasing VEGF caused selective endothelial cell death
in blood vessels that lacked pericytes but spared those blood vessels that had pericytic support.23,24Recently, however, Maynard et al25found that in pregnant mice,
a soluble VEGF receptor (sFlt1) released from the uterus reduced systemic VEGF levels and caused glo-merular endothelial cell changes similar to those seen in the renal endotheliosis of preeclampsia These results would also indicate that VEGF continues to be an important endothelial growth factor in the adult life and that its reduction can result in endothelial cell death in existing blood vessels
We conclude that VEGF, a classical angiogenic fac-tor critical during fetal development, also regulates the maturation of wound tissue Low VEGF levels induced
by disease or therapeutic manipulation may cause dam-aging effects in a healing tissue, and possibly also in normal adult vasculature
The authors thank Linda Wanna and P Sethupathi for help with the animal surgery and Bhakti Patel, Sreya Patri, Valentina Svoren, and Lev Rappaport for help with the immunocytochemistry.
Fig 9 A and B, Representative photomicrographs showing cells immunostained (brown) for PCNA in the
medial layer of control and experimental (rats treated with the VEGF receptor antagonist) granulomas (Blue
counterstain blue; original magnification ⫻ 200.) C, Compared with controls, VEGF receptor antagonist treated
granulomas displayed 70% fewer PCNA-positive cells in the medial layer, suggesting that the antagonist exerted
a significant antiproliferative effect on the microvessels *P ⬍ 05 compared with controls.
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