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Regular Article |
From the Department of Anatomy II,*
Kumamoto University
School of Medicine, Kumamoto, Japan; and the Cardiovascular Research
Institute and Department of Anatomy,
University of California, San Francisco, California
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Abstract |
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3 times the pathogen-free value for at least 28 days. Remodeled
capillaries and venules were sites of focal plasma leakage and
extensive leukocyte adherence. Most systemic manifestations of the
infection occurred well after the peak of endothelial
proliferation, and the humoral immune response to M.
pulmonis was among the latest, increasing after 14
days. These data show that endothelial cell proliferation and
microvascular remodeling occur at an early stage of chronic airway
disease and suggest that the vascular changes precede widespread tissue
remodeling.
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Introduction |
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The properties and functional implications of angiogenic blood vessels have been examined in many animal models of cancer,5-8 but less is known about the vascular changes in chronic inflammation. One might expect the microvasculature to change gradually as protracted inflammatory diseases evolve, or it could change rapidly at the beginning and drive other aspects of the disease. Yet the time course of changes in the vasculature in chronic inflammation has not been addressed systematically, in part because of limited suitable animal models.
One animal model that has been useful for studying angiogenesis and vascular remodeling in chronic inflammation is the respiratory tract infection caused by Mycoplasma pulmonis in mice and rats. This infection causes severe, lifelong changes in the microvasculature of the airway mucosa,9-11 along with extensive tissue remodeling, leukocyte influx, epithelial and gland hyperplasia, and fibrosis in the airways and, in mice, pneumonitis.12-15 In rats infected with M. pulmonis, a familiar form of sprouting angiogenesis is the predominant change in the airway vasculature.9-11 In mice, two distinct types of changes occur in a strain-dependent manner.16 Sprouting angiogenesis occurs in C57BL/6 mice, but in C3H mice the airway microvasculature enlarges without increasing appreciably in length.16 This enlargement results from endothelial cell proliferation rather than vasodilatation.16,17 With the enlargement, endothelial cells of blood vessels in the anatomical position of capillaries acquire characteristics of venules, including leakiness, adherence of leukocytes, and expression of P-selectin and von Willebrand factor.18 These changes in endothelial cell phenotype could be key to the rapid influx of leukocytes that follows M. pulmonis infection and initiates the tissue remodeling.16,17
Like other chronic inflammatory conditions, M. pulmonis infection is accompanied by progressive tissue remodeling. Without therapeutic intervention, the disease is life-long. Interestingly, the enlargement of the microvasculature is evident as soon as 1 week after infection, before the full-blown disease develops.16 One possibility is that a sudden spurt of endothelial cell proliferation transforms the mucosal vasculature into a phenotype that supports the rapid influx of inflammatory cells and progression of the inflammatory response. One manifestation of such a phenotypic change would be the adherence of leukocytes to the endothelium. Alternatively, endothelial cells could proliferate at a uniform rate after the onset of the disease in parallel with the remodeling of other airway tissues. In either case, it is unclear how the rate of endothelial cell proliferation relates to the time course of vascular enlargement, disease progression, and overall severity. A further question is whether the proliferation of endothelial cells results in altered endothelial barrier function, consistent with the leakiness of angiogenic blood vessels in chronic inflammation and tumors.19-21
In addressing these questions, we sought to: 1) compare the time course of the microvascular enlargement with the time course of endothelial cell proliferation in the airway mucosa after M. pulmonis infection and identify the affected segments of the microvasculature; 2) determine whether the airway microvasculature becomes leaky at the peak of endothelial cell proliferation; 3) compare the time course of leukocyte adhesion with the pattern of endothelial cell proliferation; and (4) assess the temporal relationship between the vascular enlargement and overall disease severity.
Our strategy was to infect mice with M. pulmonis by intranasal inoculation and then to measure the time course of changes in blood vessel diameters, the number of 5-bromo-2'-deoxyuridine (BrdU)-labeled endothelial cells, and the amount of leukocyte adhesion in the microvasculature of the tracheal mucosa. We also examined the amount and location of blood vessel leakiness and assessed the time course and severity of the systemic immune response.
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Materials and Methods |
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Specific pathogen-free, 7- to 8-week-old male C3H/HeN mice (average body weight, 24 g) were purchased from Charles River Laboratories (Hollister, CA) and anesthetized intramuscularly with ketamine (100 mg/kg; Parke-Davis, Morris Plains, NJ) and xylazine (5 mg/kg; Ben Venue Laboratories, Bedford, OH). Mice were inoculated intranasally with 105 colony-forming units of M. pulmonis (strain UAB CT7) in a volume of 50 µl (25 µl per nostril). Infected and pathogen-free control mice were caged and housed separately under barrier conditions. All experiments were performed in accordance with the guidelines of the Committee for Animal Research of the University of California, San Francisco.
Staining of Airway Microvasculature
To make it easier to measure the size of blood vessels and to quantify intravascular leukocytes, the vasculature was stained by perfusion of a lectin that binds uniformly to the luminal surface of endothelial cells and leukocytes.22 Briefly, after anesthesia, mice were injected intravenously with biotinylated Lycopersicon esculentum lectin [100 µg lectin per 100 µl of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline; Vector Laboratories, Burlingame, CA]. After 2 to 3 minutes, the chest was opened and the vasculature was perfused with 1% paraformaldehyde and 0.5% glutaraldehyde in phosphate-buffered saline (PBS) for 3 minutes, followed by PBS for 2 minutes at a pressure of 120 mm Hg via a blunt 13-gauge needle inserted through the left ventricle into the ascending aorta. Tracheas were cut along the ventral midline, removed, and pinned luminal surface up on Petri dishes coated with Sylgard (Dow-Corning, Midland, MI). The specimens were permeabilized with 0.3% Triton X-100 in PBS overnight and then incubated in avidin-biotin-peroxidase complex (1:200; Vector) in 0.3% Triton X-100 in PBS overnight at room temperature. After washing with PBS for at least 2 hours, tracheas were then reacted for 10 to 15 minutes with 0.05% diaminobenzidine (Sigma Chemical Co., St. Louis, MO) and hydrogen peroxide in PBS. The tissues were washed thoroughly with deionized water, dehydrated with ethanol, removed from the Sylgard, flattened between two glass slides in absolute ethanol overnight, cleared in toluene, and mounted in Permount (Fisher Scientific, San Francisco, CA).
In other mice, the borders of endothelial cells were stained by vascular perfusion of silver nitrate.16,23 Briefly, the vasculature was perfused in succession with 1% paraformaldehyde and 0.5% glutaraldehyde in 75 mmol/L of cacodylate buffer (pH 7.4) at a pressure of 120 mm Hg for 3 minutes, followed by 0.9% NaCl for 2 minutes, 10 ml of 5% glucose delivered with a hand syringe at a rate of 1 ml per second, 7 ml of 0.2% silver nitrate for 7 seconds, 10 ml of 5% glucose for 10 seconds, and fixative again for 1 minute. The tracheas were incised along the midline, removed, and the silver halide was developed under a bright light for 15 minutes. Finally, the tracheas were dehydrated in alcohol, cleared in toluene, and mounted in Permount and observed as whole-mount preparations.
Measurement of Vessel Size
The diameter of arterioles, capillaries, and venules (10 vessels of each type, as determined by previously described criteria16 ) was measured in the mucosa of lectin-stained tracheal whole mounts from pathogen-free mice and mice infected for 4, 5, 7, and 14 days (n = 4 per group). Blood vessels in whole mounts of tracheas stained with lectin were measured with a Zeiss Axiophot microscope coupled to a color charge-coupled device video camera (model DXC-755; Sony, Tokyo, Japan), a real-time color video digitizing card (Video-Logic DVA-4000; Video-Logic, Cambridge, MA) in a personal computer, a digitizing tablet (model 1111A; GTCO Digipad, Rockville, MD), and image analysis software developed for this purpose in our laboratory.23
BrdU Labeling and Measurement of Endothelial Cell Proliferation
The thymidine analogue, BrdU (Sigma), was injected intravenously (1 mg in 100 µl of PBS per mouse) into pathogen-free mice and mice infected with M. pulmonis for 1, 3, 5, 7, 9, or 11 days or 2, 3, or 4 weeks (n = 3 to 6 per group). Three hours later, tissues were fixed by perfusion of 1% paraformaldehyde and 0.25% glutaraldehyde in PBS (pH 7.4; Sigma) for 3 minutes and with PBS for 2 minutes via the left ventricle at a pressure of 120 mm Hg. Preliminary experiments demonstrated that the combination of this dose of BrdU and the 3-hour interval between injection and vascular perfusion resulted in distinct labeling of proliferating endothelial cells in pathogen-free and infected tracheas. Tracheas and lungs were removed, washed with PBS, infiltrated with 30% sucrose in PBS at 4°C overnight, and embedded in OCT compound (Sakura Finetek USA Inc., Torrance, CA); 10-µm-thick sections were then cut on a cryostat.
BrdU-labeled cells were detected immunohistochemically in cryostat sections as described previously.24 Briefly, after air-drying, sections were digested with 0.005% pepsin (Sigma) in 0.01 N HCl at 37°C for 10 minutes and then 4 N HCl for 30 minutes at room temperature. Background staining was blocked by incubation in 5% normal goat serum for 30 minutes; then the sections were incubated with a mouse monoclonal antibody to BrdU (1: 200; DAKO, Carpinteria, CA) at room temperature for 2 hours or at 4°C overnight, followed by an alkaline phosphatase-conjugated goat anti-mouse IgG (1: 200; Jackson ImmunoResearch, West Grove, PA) at room temperature for 1 hour. The alkaline phosphatase-conjugated antibody was visualized as a red or dark blue reaction product using substrate kits I or IV (Vector Red or BCIP/NBT; Vector) in 0.1 mol/L of Tris-HCl buffer (pH 8.2 or 9.5, respectively) at room temperature. Sections were fixed again with 1% glutaraldehyde in PBS at room temperature for 5 minutes, washed in distilled water, counterstained with hematoxylin, and mounted in Aquatex (Merck, Darmstadt, Germany).
BrdU-labeled endothelial cell nuclei, identified as elongated oval regions of immunoreactivity in the lining of lectin-stained blood vessels, were counted in 10 to 12 sequential sections from the rostral trachea of each mouse. The percentage of BrdU-labeled blood vessels was expressed as the ratio of vessels having BrdU-labeled endothelial cells to the total number of vessel profiles per cross-section of trachea. In addition, the endothelial cell-labeling index was calculated as the ratio of number of BrdU-labeled endothelial nuclei to total number of endothelial nuclei stained with hematoxylin.
Some tracheal whole mounts were double-stained for lectin histochemistry and BrdU immunohistochemistry. After injection of L. esculentum lectin and perfusion fixation as above, tracheas were washed in PBS for at least 1 hour and then three times in deionized water for 5 minutes each. The tracheal whole mounts were digested with 0.003% pepsin (Sigma) in 0.01 N HCl (pH 2) at 37°C for 50 minutes, and then the luminal surface of the trachea was stroked gently with a fine camelhair brush to remove loosened epithelial cells, some of which were labeled with BrdU. The tissues were treated with 4 N HCl at room temperature for 50 minutes and then washed 7 times for 5 minutes each in deionized water and then 7 times in PBS. Background staining was blocked by incubation in 5% normal goat serum for 1 hour, then incubated with the mouse monoclonal antibody to BrdU (1:200, DAKO) at room temperature overnight, followed by an alkaline phosphatase-conjugated goat anti-mouse IgG (1:200, Jackson) at room temperature for 2 hours. The dark-blue alkaline-phosphatase reaction product was visualized with the BCIP kit for 5 to 10 minutes at room temperature as described above. The tracheas were incubated with avidin-biotin-peroxidase complex for visualizing the biotinylated lectin staining, dehydrated with ethanol, cleared with toluene, and mounted in Permount.
Quantification of Adherent Intravascular Leukocytes
Leukocytes adherent to the luminal surface of blood vessels were counted in lectin-stained tracheal whole mounts from pathogen-free mice and mice infected for 3, 4, 5, 7, or 14 days (n = 4 to 6 per group). In these preparations, adherent leukocytes were easily identified as lectin-stained, brown spheres within blood vessels. In each trachea, leukocytes were counted at a magnification of 400 in 20 regions of mucosa, each measuring 0.096 mm2 (total area 1.92 mm2 per trachea), located between cartilage rings. Values are expressed as number of adherent intravascular leukocytes per square millimeter of mucosal surface.
Localization and Measurement of Plasma Leakage
Sites of leakage were identified microscopically by using the particulate tracer Monastral blue pigment, which was injected intravenously (30 mg/kg in saline) 5 to 10 minutes before the vasculature was perfused with fixative.25 Tracheas were then removed and prepared as whole mounts.9,26 Sites of leakage were identified as patches of Monastral blue pigment trapped in vessel walls. For quantitative studies of leakage, Evans blue dye (30 mg/kg in saline; EM Sciences, Cherry Hill, NJ) was injected intravenously into anesthetized mice in a volume of 100 µl, as described previously.27 Thirty minutes later, the chest was opened, both atria were cut, and the vasculature was perfused with 1% paraformaldehyde in 0.05 mol/L citrate buffer (pH 3.5) for 2 minutes at a pressure of 120 to 140 mm Hg via a cannula in the left ventricle. Tracheas and bronchi were removed, gently blotted with filter paper, and weighed. Evans blue dye was extracted from the tissues using formamide and measured by spectrophotometry.27 Measurements were expressed as ng dye/mg wet weight of trachea. –
Assessment of Disease Severity
Blood (0.2 to 0.4 ml) was removed from anesthetized mice into heparinized syringes and centrifuged at 8000 rpm for 6 minutes for measurement of M. pulmonis antibody titers. Serum was diluted 1:5 with sterile saline, heat-inactivated at 56°C for 30 minutes, and then frozen until analyzed by indirect enzyme-linked immunosorbent assay (BioReliance Corporation, Rockville, MD). The enzyme-linked immunosorbent assay had a threshold sensitivity of 1 ng immunoglobulin per well. The lungs, bronchial lymph nodes, thymus, and spleen were removed, blotted dry, and weighed after vascular perfusion of fixative.
Statistics
Values are presented as means ± SE of data for three to six mice per group unless otherwise indicated. The significance of differences between groups was determined by Student’s t-test or by analysis of variance followed by the Dunn-Bonferroni test for multiple comparisons. Differences were considered significant when P < 0.05.
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Microvascular Enlargement
Major changes in the tracheal microvasculature were evident in
lectin-stained tracheas of M. pulmonis-infected mice
throughout the 28-day period of the study (Figure 1, A and B)
. All segments of the
microvasculature became significantly larger within a few days after
infection (Figure 1, C to H)
. The main arterioles in pathogen-free mice
were relatively straight tubes of uniform 13-µm diameter running
parallel to the cartilage rings (Figure 1C)
; after infection they had
the same general location but enlarged to a diameter of 31 µm
(Figure 1D)
. Capillaries, which included most of the mucosal vessels
overlying the cartilage rings, changed from 8-µm tubes without
adherent leukocytes (Figure 1E)
into 26-µm venule-like vessels
with abundant leukocytes adherent to their endothelium (Figure 1F)
.
These remodeled vessels had the normal anatomical location of
capillaries but had a venular phenotype. Similarly, venules, which were
normally the largest (average diameter, 36 µm) and most numerous
vessels in the region of mucosa between cartilage rings (Figure 1G)
,
increased in diameter to 62 µm (Figure 1H)
. The endothelium of
remodeled capillaries and venules was covered with leukocytes (Figure 1, F and H)
.
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). Capillary diameters (131% increase)
and venule diameters (48% increase) were significantly larger on day 5
(Figure 2)
. Vessels reached their maximal size at 7 days, when on
average the diameters of arterioles, capillaries, and venules were 148,
214, and 74% larger, respectively. The size of all types of vessels
tended to plateau after 7 days (Figure 2)
. The enlargement at 7 days
represented average diameter increases of 18 µm for arterioles and
capillaries and 26 µm for venules.
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Using BrdU to define the time course of endothelial cell
proliferation, we found that labeled nuclei were rare in the trachea
and bronchi of pathogen-free mice (Figure 3
; A, C, and E) but were common in
M. pulmonis-infected mice (Figure 3
; B, D, and F). After
infection, BrdU-labeled nuclei were most abundant in the airway
epithelium (Figure 3B)
, but labeled endothelial nuclei were easily
identified around the perimeter of the enlarged blood vessels in the
trachea (Figure 3D)
and bronchi (Figure 3F)
. Measurements showed that
1.1% of vessel profiles in sections of tracheas from pathogen-free
mice had BrdU-labeled endothelial cell nuclei, whereas after infection
the proportion was 2.4% at 1 day, 7.3% at 3 days, and 19.4% at 5
days (Figure 4)
. After the peak at 5
days, labeling declined to 12.2% at day 7, 5.9% at day 9, and
gradually decreased to 2.8% at 28 days (Figure 4)
.
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Examination of BrdU-labeled whole mounts revealed that labeled
endothelial cell nuclei were numerous in all segments of the tracheal
microvasculature at 3 to 7 days after infection but were uncommon there
in pathogen-free mice (Figure 3, G and H)
. After infection,
BrdU-labeled nuclei were abundant in the epithelium and were also found
in the lamina propria and smooth muscle.
Endothelial Cell Enlargement
Endothelial cells outlined by silver nitrate staining of tracheal
venules in pathogen-free mice had a polygonal shape and a uniform size
(Figure 5A)
. By comparison, during the
period of greatest endothelial cell proliferation (5 days after
infection), giant endothelial cells were scattered among endothelial
cells of normal size (Figure 5B)
. Although constituting only a small
minority of endothelial cells, giant cells were at least twice the size
of endothelial cells in normal venules and were very conspicuous. Sites
of leukocyte adherence and migration, appearing as tiny rings at
endothelial cell borders, were visible in venules of infected mice
(Figure 5B)
.
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Monastral blue did not leak from any blood vessel in the trachea
of pathogen-free mice (Figure 5C)
, but focal regions of extravasated
Monastral blue were in the wall of some tracheal vessels at the peak of
endothelial cell proliferation 5 days after infection (Figure 5D)
. The
distribution of leakage was not uniform. Some vessels had intense
Monastral blue labeling, but many had none. Most leaky sites were in
venules. Arterioles had none. Some sites of Monastral blue leakage were
near silver dots (ie, endothelial gaps23
) at endothelial
cell borders in silver nitrate-stained specimens (Figure 5D)
. However,
when the overall amount of plasma leakage in the trachea and bronchi
was measured with Evans blue, the values for infected mice were not
significantly different from those for pathogen-free mice at any time
point (Figure 6)
. Leakage expressed per
microgram of tissue was consistently greater in bronchi than in
tracheas and there was a trend toward higher values at 5 days, but the
large variability among animals obscured any differences because of the
infection.
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Adherent intravascular leukocytes, which were prominently stained
by the biotinylated L. esculentum lectin, were scarce in all
segments of the tracheal microvasculature in pathogen-free mice (Figure 1
; A, C, E, and G) but were abundant in infected mice (Figure 1
; B, F,
and H; and Figure 7
). Adherent leukocytes
were numerous in remodeled capillaries and venules (Figure 1, F and H)
but were rare in arterioles (Figure 1D)
. At 4 days after infection,
adherent leukocytes were 40 times as numerous as in pathogen-free
mice (1286 ± 388 versus 32 ± 8 intravascular
leukocytes per mm2
of mucosal surface area,
n = 4 mice per group, P < 0.0001) and
remained abundant at later time points (Figure 7)
. Extravascular
neutrophils, made visible by the reaction of myeloperoxidase in the
lectin histochemistry, were also abundant in the tracheal mucosa of
infected mice (Figure 1, B and H)
.
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The mice became lethargic and lost weight after inoculation with
M. pulmonis and from day 5 onward were significantly lighter
than the pathogen-free controls (Figure 8A)
. The weight of infected mice
gradually fell through day 21 and then stabilized, but pathogen-free
mice continued to gain weight during this period and at day 28 were
50% heavier than the infected mice.
|
. The weight of the bronchial lymph nodes reflected the
strong local immunological response to M. pulmonis infection
and was a sensitive indicator of disease progression. Bronchial lymph
nodes increased almost linearly in size from day 3 after infection: the
mean weight increased nearly 40-fold, from 2 mg at the outset to 77
mg at 28 days (Figure 8C)
. Lung weight gradually increased with the
influx of inflammatory cells and edema fluid and nearly doubled
throughout the first 28 days after infection (Figure 8D)
. The spleen
enlarged gradually and at 28 days was 70% heavier than in
pathogen-free mice (Figure 8E)
, mainly because of expansion of the
white pulp (data not shown). In contrast, thymus weight rapidly
decreased after infection. The thymus was significantly smaller on day
5 and by day 7 had lost 70% of its mass in comparison to corresponding
pathogen-free mice (Figure 8F)
. Overall, these measurements emphasize the slow, progressive infiltration of the lungs after M. pulmonis infection, accompanied by gradual but continuous enlargement of draining lymph nodes and spleen. The humoral immune response, as reflected by serological antibody titers to the infectious organisms, also occurred relatively late. By comparison, endothelial cell proliferation occurred rapidly after infection and the peak was transient, but the changes were long lasting. The rapid onset of vascular remodeling was accompanied by a reduction in body weight and shrinkage of the thymus.
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3 times baseline. Tracheal blood vessels doubled in
size during the first 7 days after infection and then vessel caliber
plateaued. All segments of the microvasculature were affected. In
addition, remodeled capillaries and venules, but not arterioles, were
sites of leukocyte adhesion. Sites of plasma leakage through
endothelial gaps were also present, but the overall amount of leakage
in the airways was not significantly increased, so the leaks seem to be
focal. Changes in the airway microvasculature were among the earliest
detected. Most systemic manifestations of the infection, including the
production of antibodies to M. pulmonis, followed these
changes. Endothelial Cell Proliferation
One of the aims of the present study was to determine the time
course of endothelial cell proliferation in C3H mice after M.
pulmonis infection. We were surprised to find that the peak in
endothelial cell division, as reflected by BrdU labeling, was brief and
occurred quite early, at only 5 days after the onset of the disease,
whereas tissue remodeling continued to evolve for weeks. At 5 days
11% of the endothelial cells took up BrdU during the 3-hour period
of labeling, compared to a baseline value of 1%. This labeling
index is within the range found in some other conditions accompanied by
microvascular remodeling and angiogenesis, such as wound healing,
delayed hypersensitivity reaction, and certain types of
tumors.28-30
After 5 days, the rate of endothelial cell
proliferation fell sharply but remained above baseline as the chronic
airway disease evolved.
Growth factors and other cytokines that stimulate endothelial cell proliferation after M. pulmonis infection may come from several sources. Mitogens from M. pulmonis organisms may directly affect endothelial cells as they do lymphocytes.31-33 Endothelial cell mitogens may also come from activated epithelial cells. M. pulmonis organisms adhere to the luminal surface of the airway epithelium, creating a polarized stimulus that produces phenotypic and functional changes in endothelial cells on the surface of blood vessels facing the epithelium.17 Endothelial cells on the opposite side of the same vessels are not similarly affected. M. pulmonis infection also caused the proliferation of epithelial cells, as it did endothelial cells, as indicated by increased BrdU labeling. In contrast to the polarized changes presumably mediated by activated epithelial cells, proliferating endothelial cells were uniformly distributed around the vessel circumference, suggesting that the mitogenic stimulus does not come from one direction.17 Therefore, M. pulmonis organisms attached to airway epithelial cells are unlikely to be the sole source of endothelial cell mitogens. Another obvious source of growth factors is the inflammatory cells that infiltrate the airway mucosa in abundance after M. pulmonis infection. Neutrophils, macrophages, and lymphocytes are well-documented sources of endothelial cell mitogens.11,34-36
Relation of Endothelial Cell Proliferation to Inflammatory Response
The present study provides clear evidence that the peak of the
endothelial cell proliferation at 5 days is a relatively early
component of the inflammatory response to M. pulmonis
infection, coinciding with the initial influx of leukocytes and
preceding the humoral immune response by more than a week. The
triggering stimulus for endothelial cell proliferation must be
activated even earlier. Biochemical changes, as indicated by increased
expression of tumor necrosis factor-
, interleukin-1, interleukin-6,
and interferon-
, are known to occur within a few hours after
infection.37
However, the identity and source of the
growth factors responsible for the proliferation of endothelial cells
are still under investigation.
The early onset of vascular remodeling after M. pulmonis infection is consistent with the time course of angiogenesis in the delayed hypersensitivity and graft-versus-host reactions, where blood vessel proliferation peaks at day 2 to 3 and lasts through day 6.29,38 Similarly, the peak of angiogenesis occurs at 3 to 5 days in fractured bones.30
Relation of Endothelial Cell Proliferation to Vascular Leakiness
The airway vasculature in rats infected with M. pulmonis for 4 weeks is abnormally leaky under baseline conditions.21 However, under the same conditions the overall amount of plasma leakage in the airways of mice infected with M. pulmonis for 4 weeks is within the range found in pathogen-free mice.16 In an earlier study in mice,16 we were unaware of the time course of endothelial cell proliferation and did not ask whether leakage occurs in the first few days after infection; we also did not look for focal regions of leakage that may be undetectable by Evans blue measurements of entire tracheas. In the present study, having learned about the peak of endothelial cell proliferation at 5 days, we measured Evans blue leakage at multiple times during the first 2 weeks after infection. Furthermore, we used the particulate tracer Monastral blue as a complementary test for focal sites of leakage.23,25 This approach revealed scattered sites of Monastral blue extravasation in remodeled capillaries and venules during the phase of rapid endothelial cell proliferation. Considering that the endothelial cells of arterioles also underwent proliferation, but no leakage was observed there, the leakage in remodeled vessels was not a necessary consequence of endothelial cell proliferation, although this may indicate that the media of arterioles opposes extravasation because of loss of endothelial barrier function. Although the experiments did not detect a significant increase in Evans blue accumulation after infection, even at the peak of endothelial cell proliferation, there was a trend toward more leakage around day 5, yet the interanimal variability precluded any meaningful interpretation.
The observations of Monastral blue extravasation are consistent with evidence that venules are the primary site of plasma leakage, as well as leukocyte migration, in a variety of pathological conditions, including the response to specific inflammatory mediators.39-46 Capillary-like vessels leak under some conditions, but the leaky vessels are typically newly formed by angiogenesis.47-49 Capillary-like vessels are the principal sites of leakage in the airways of M. pulmonis-infected rats, which have extensive angiogenesis,11,21 but these tiny new vessels do not form in C3H mice where microvascular enlargement is the dominant change.16
Change in Endothelial Cell Phenotype
Enlargement of the airway microvasculature in C3H mice after M. pulmonis infection is accompanied by distinctive changes in endothelial cells.16 In addition to undergoing proliferation, some endothelial cells enlarge to nearly twice normal size and resemble multinucleated giant endothelial cells located over atherosclerotic plaques.50,51 Giant endothelial cells in the airway microvasculature may be a manifestation of rapid proliferation, but they are unlikely to play a major role in increased vessel caliber because they were infrequent and their presence was limited to the period of most rapid endothelial cell proliferation.
M. pulmonis infection increases the expression of the leukocyte adhesion molecule P-selectin on remodeled capillaries and venules.18 P-selectin-dependent rolling is one of the earliest observable events in the recruitment of leukocytes to inflamed tissues.52 Our studies revealed that leukocyte adhesion to remodeled vessels occurred early in the disease in C3H mice, beginning during the period of rapid vascular remodeling and continuing thereafter. Sustained leukocyte migration is one of the most prominent features of M. pulmonis airway disease.12,13 Similar microvascular remodeling and leukocyte influx occur in many other inflammatory conditions.42,43 Rapid changes in endothelial cell phenotype may play a key role in the evolution of M. pulmonis disease by controlling leukocyte traffic into airway tissues. Thus, the early onset of microvascular remodeling may be a gatekeeper in the development of the chronic inflammatory airway disease.
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Acknowledgements |
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Footnotes |
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Supported in part by National Institutes of Health grants HL-24136 and HL-59157 from the National Heart, Lung, and Blood Institute (to D. M.).
Accepted for publication February 27, 2001.
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 D. M. McDonald and P. Baluk Imaging of Angiogenesis in Inflamed Airways and Tumors: Newly Formed Blood Vessels Are Not Alike and May Be Wildly Abnormal: Parker B. Francis Lecture Chest, December 1, 2005; 128(6_suppl): 602S - 608S. [Full Text] [PDF]
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 A. B. Aurora, P. Baluk, D. Zhang, S. S. Sidhu, G. M. Dolganov, C. Basbaum, D. M. McDonald, and N. Killeen Immune Complex-Dependent Remodeling of the Airway Vasculature in Response to a Chronic Bacterial Infection J. Immunol., November 15, 2005; 175(10): 6319 - 6326. [Abstract] [Full Text] [PDF]
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 T. R. Carlson, Y. Yan, X. Wu, M. T. Lam, G. L. Tang, L. J. Beverly, L. M. Messina, A. J. Capobianco, Z. Werb, and R. Wang Endothelial expression of constitutively active Notch4 elicits reversible arteriovenous malformations in adult mice PNAS, July 12, 2005; 102(28): 9884 - 9889. [Abstract] [Full Text] [PDF]
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 S. Frantz, K. A. Vincent, O. Feron, and R. A. Kelly Innate Immunity and Angiogenesis Circ. Res., January 7, 2005; 96(1): 15 - 26. [Abstract] [Full Text] [PDF]
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 P. Baluk, W. W. Raymond, E. Ator, L. M. Coussens, D. M. McDonald, and G. H. Caughey Matrix metalloproteinase-2 and -9 expression increases in Mycoplasma-infected airways but is not required for microvascular remodeling Am J Physiol Lung Cell Mol Physiol, August 1, 2004; 287(2): L307 - L317. [Abstract] [Full Text] [PDF]
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 M. Rusnati, M. Camozzi, E. Moroni, B. Bottazzi, G. Peri, S. Indraccolo, A. Amadori, A. Mantovani, and M. Presta Selective recognition of fibroblast growth factor-2 by the long pentraxin PTX3 inhibits angiogenesis Blood, July 1, 2004; 104(1): 92 - 99. [Abstract] [Full Text] [PDF] |
