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Regular Articles |
From the Department of Anatomy and Cardiovascular Research
Institute,*
University of California, San Francisco,
California, and the Department of Comparative
Medicine,
University of Alabama,
Birmingham, Alabama
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Chronic inflammation may also be accompanied by changes in the phenotype of endothelial cells.11 In the chronic airway disease produced in rats by Mycoplasma pulmonis infection, for example, substance P induces plasma leakage not only from venules but also from newly formed capillary-like vessels.15 In comparison, in the airway mucosa of normal rats, substance P causes plasma leakage from venules but not capillaries.16 The leakage from capillary-like vessels in infected rats is due to a change in the phenotype of capillary endothelial cells, one feature of which is increased expression of neurokinin receptors.15
It is unclear whether new vessel growth and vessel enlargement are distinct processes, different stages of a single process, or processes typical of particular tissues or particular inflammatory stimuli. Furthermore, it is unclear whether changes of endothelial cell phenotype are a consistent feature of chronic inflammation.
We have sought to examine these issues by developing an animal model to study the stages and types of microvascular remodeling associated with chronic airway inflammation. For this purpose we have used M. pulmonis infection of the respiratory tract of rats and mice. Our previous studies have shown that the growth of new blood vessels is a prominent feature of the chronically inflamed airway mucosa of infected rats.15,17 Although less is known about vascular remodeling in the airways of infected mice, studies have shown that the severity of M. pulmonis-induced respiratory disease in mice is strain dependent.18-20 Such studies can take advantage of the wealth of information on immune and inflammatory responses in mice and the advent of genetically targeted mutants, which could eventually be used to address molecular mechanisms of vascular remodeling.
The overall goal of the present study was to develop a better understanding of the distinction between new vessel growth and vessel enlargement in a model of chronic inflammation in mice. In particular, we sought to compare the stages of vascular remodeling in mouse strains with known differences in severity of respiratory disease after infection with M. pulmonis. We used C57BL/6 mice, which develop mild disease, and C3H mice, which develop severe disease.18-20 Previous studies have shown that, compared with C57BL/6 mice, C3H mice have greater airway exudate, epithelial hyperplasia, lymphoid hyperplasia, and inflammatory cell infiltrate in the alveoli and have higher mortality.18
Our specific aims were to 1) determine whether the vascular remodeling in C57BL/6 and C3H mice infected with M. pulmonis involves the growth of new blood vessels, changes in existing vessels, or both, 2) determine whether the enlargement of vessels after infection is accompanied by an increase in number or size of endothelial cells, 3) characterize changes in endothelial cell phenotype associated with vascular remodeling in chronic airway disease, and 4) determine whether the remodeled vessels are abnormally leaky under baseline conditions or are abnormally sensitive to inflammatory mediators.
Our strategy was to induce M. pulmonis infection in pathogen-free C57BL/6 and C3H mice by intranasal inoculation. At 1, 2, 4, or 8 weeks after inoculation the tracheal microvasculature was stained by perfusion of the lectin Lycopersicon esculentum,21 and the number, size, and length of vessels were measured morphometrically. Alternatively, endothelial cell borders were stained by vascular perfusion of silver nitrate,16 and the number and size of endothelial cells were measured. The phenotype of the endothelial cells was characterized by the binding of lectins,21 expression of von Willebrand factor (vWF),22 and adhesion of leukocytes. In all cases, the three-dimensional architecture of the vessels was analyzed in tracheal whole mounts. In addition, endothelial permeability was assessed in vivo under baseline conditions and after substance P, by measuring the leakage of intravascular Evans blue dye.23
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Pathogen-free, 8-week-old, male and female C3H mice (Frederick Cancer Research Facility, Frederick, MD (C3H/HeNCr) or Charles River Laboratories, Hollister, CA (C3H/HeNCrlBR)) and C57BL/6 mice (Charles River (C57BL/6NCrlBR)) were used in these experiments. Mice were anesthetized (87 mg/kg ketamine and 13 mg/kg xylazine intraperitoneally) and inoculated intranasally with ~105 colony-forming units of M. pulmonis (strain UAB CT7) in a volume of 50 µl. Infected and pathogen-free control mice were caged and housed separately under barrier conditions and handled in accordance with the procedures of the Committee on Animal Research, University of California, San Francisco.
Vascular Perfusions and Staining
At 1, 2, 4, or 8 weeks after inoculation, mice were anesthetized with Nembutal (30 to 50 mg/kg intraperitoneally) and supplemented as necessary. Infected mice tended to be more sensitive to anesthetic and were given less for the initial injection. Titers of antibody to M. pulmonis were measured in serum from pathogen-free control and infected mice at the time of the experiment. Blood (0.2 ml) was withdrawn from the jugular vein into a heparinized syringe and centrifuged for 6 minutes at 8000 rpm. Plasma was withdrawn and re-centrifuged, and 100 µl of the plasma was added to 400 µl of sterile saline, heated to 56°C for 30 minutes, and then frozen until sent for analysis (MA Bioservices, Rockville, MD).
To perfuse the mice, the chest cavity was opened, the atria were removed to allow outflow of blood and perfusate, and a cut was made in the left ventricle. A cannula was inserted into the ascending aorta and clamped. Fixative or other solutions were perfused at 120 mm Hg. The luminal surface of the vasculature of some mice was stained by perfusion of biotinylated L. esculentum lectin21 (Vector Laboratories, Burlingame, CA). The vasculature was perfusion fixed for 3 minutes with 1% paraformaldehyde and 0.5% glutaraldehyde in phosphate-buffered saline (PBS), pH 7.4, and the vasculature was then perfused with PBS for 60 seconds, 25 ml of PBS plus 1% bovine serum albumin (BSA) for 80 seconds, 25 ml of lectin (5 or 10 µg/ml in PBS/BSA) for 80 seconds, 25 ml of PBS/BSA for 60 seconds, and PBS for 60 seconds. The tracheas were removed, opened flat, and stained using avidin-biotin complex/diaminobenzidine (ABC/DAB) histochemistry.21 Tracheas were dehydrated through alcohols and mounted whole in Permount embedding medium. The rostral portions of some tracheas were embedded in glycol methacrylate, sectioned (3 to 4 µm thickness), stained with toluidine blue, and mounted in Permount embedding medium.
The endothelial cell borders were stained in the vasculature of some mice by perfusion of silver nitrate.16 The vasculature was perfusion fixed for 3 minutes with 1% paraformaldehyde and 0.5% glutaraldehyde in 75 mmol/L cacodylate, pH 7.4, and the the vasculature was then perfused with 0.9% NaCl for 1 minute, 10 ml of 5% glucose for 10 seconds, 7 ml of 0.2% silver nitrate for 7 seconds, 10 ml of 5% glucose for 10 seconds, and fixative for 60 seconds. The tracheas were removed and opened flat, exposed to light for 15 minutes to develop the silver, and then dehydrated through alcohols and mounted whole in Permount. Tracheas stained with biotinylated lectins or silver nitrate were examined with an upright microscope (Axiophot, Zeiss) equipped with Nomarski differential interference contrast optics.
Morphometric Measurements of Vessels and Endothelial Cells
Morphometric measurements of blood vessels were made in whole mounts of tracheas stained with lectin or silver nitrate using a Zeiss Axiophot microscope coupled to a color charge-coupled device (CCD) video camera (Sony, model DXC-750), a digitizing card (VideoLogic DVA-4000, Cambridge, MA), monitor, and software written for this purpose in our laboratory.16 Vessel area density, which represents the fraction of the total tissue area occupied by the wall or lumen and reflects the overall number, length, and size of vessels, was measured in lectin-stained tracheas by overlaying the live video microscopic image with a computer-generated square lattice. The number of points of the lattice that intersected vessels was scored. Vessel area density was expressed as the fraction (percentage) of lattice points that intersected vessels. Vessel length density, which represents the length of blood vessels in a given area of tissue and reflects the total length of vessels independent of vessel size and number, was measured by overlaying an array of sinusoidal lines, and the number of intersections of vessels with the lines was scored. Length density was computed from the total line length and number of intersections. Measurements of vessel area density and vessel length density were made on 10 fields per trachea and four tracheas per group (n = 4). The diameter of different types of tracheal microvessels was measured in 10 vessels of each type per trachea and four tracheas per group (n = 4). We used the number of vessels that traverse the cartilage as an index of the number of vessels in the trachea. This index was determined by placing a line of known length on the video screen parallel to the axis of the cartilage and counting the number of vessels that intersected the line. Results were expressed as the number of vessels per unit length of the intersection line (number per millimeter). Measurements were made on 10 cartilage segments per trachea and four tracheas per group (n = 4). Luminal endothelial cell surface area was measured in silver-stained venules by tracing the cell border of 10 endothelial cells per trachea and four tracheas per group (n = 4).
Characterization of Endothelial Cell Phenotype
Binding of Wheat Germ Agglutinin Lectin
Previous studies in the airways of rats have shown that the luminal surface of endothelial cells in venules does not bind the lectin wheat germ agglutinin (WGA), whereas that of endothelial cells in arterioles and capillaries does.21 The vessels in the airways of mice bind WGA in a similar pattern; thus, the pattern of WGA binding was used as one feature of the phenotype of the endothelial cells in remodeled vessels of infected mice. Biotinylated WGA (Vector) was perfused through the vasculature of pathogen-free and infected C57BL/6 and C3H mice, and the tracheas were processed using a protocol similar to that used with L. esculentum lectin (see above).
Staining for von Willebrand Factor
Previous studies have shown that vWF immunoreactivity is greater on the venous side of the circulation.22,24-26 We characterized the distribution of vWF in tracheal whole mounts in which the vessels were stained by perfusion of fluorescein-L. esculentum lectin. vWF was stained by immersing the intact trachea in antibodies to vWF. The vasculature was perfusion fixed for 3 minutes with 1% paraformaldehyde in PBS, pH 7.4, and then perfused with PBS for 60 seconds, 25 ml of PBS plus 1% BSA for 80 seconds, 25 ml of fluorescein L. esculentum lectin (20 µg/ml in PBS/BSA) for 80 seconds, 25 ml of PBS/BSA for 60 seconds, and PBS for 60 seconds. The tracheas were removed, opened flat, and pinned on slabs of Sylgard (Dow Corning, Midland, MI). The tracheas were permeabilized in PBS plus 0.3% Triton X-100 (Sigma Chemical Co., St. Louis, MO) for 3 hours, incubated overnight with rabbit polyclonal primary antibody to vWF (diluted 1:400; Dako, Carpinteria, CA), washed, incubated for 4 to 6 hours with Cy3-conjugated goat anti-rabbit secondary antibody (diluted 1:200; Jackson ImmunoResearch Laboratories, West Grove, PA), washed, and mounted intact in Vectashield (Vector). Fluorescently stained tracheas were examined with a confocal microscope (Zeiss LSM 410).
Adhesion of Leukocytes
Adherent leukocytes were readily visible in tracheal vessels perfused with biotinylated L. esculentum lectin and stained by the ABC/DAB peroxidase reaction (see above). Using video microscopy (see above), the number of adherent leukocytes was determined in segments of vessels in the position of capillaries overlying the tracheal cartilage. The length and diameter of each vessel segment were also measured, and the number of leukocytes expressed as the number per luminal vessel surface area. Measurements were made on 10 vessels per trachea and four tracheas per group (n = 4).
Measurement of Relative Abundance of Segments of the Microvasculature
The abundance of arterioles, capillaries, and venules was determined morphometrically in L. esculentum-stained tracheal whole mounts. Using video projections and a digitizing tablet as described above, the length of each type of microvessel segment was traced in an entire video microscopic field. The tracheal vasculature in mice was organized in a pattern similar to that in rats,16 although there were fewer branches between feeding arterioles and collecting venules. The microscope fields measured approximately 360 x 580 µm. The length of blood vessels per unit area of tracheal tissue (vessel length density, mm/mm2) and the fraction (percentage) of the total vessel length were calculated for each type of microvessel in each of four microscope fields per trachea and four tracheas per group (n = 4).
Measurement of Plasma Leakage
The amount of plasma leakage was compared in the tracheas of pathogen-free mice and mice infected with M. pulmonis for 4 weeks. Leakage was measured in the baseline state and after intravenous injection of the inflammatory mediator substance P. Plasma leakage was measured using the tracer dye Evans blue as described previously.23 Briefly, Evans blue (30 mg/kg; EM Sciences, Cherry Hill, NJ) was injected into one femoral vein of anesthetized mice, and 30 seconds later substance P (5 µg/kg in 100 µl) or vehicle was injected into the other femoral vein. Five minutes later the vasculature was perfusion fixed (1% paraformaldehyde in 50 mmol/L citrate buffer, pH 3.5) for 1 minute. Tracheas were removed, blotted dry, and weighed. Evans blue was extracted from the tracheas with formamide and measured with a spectrophotometer. Measurements were made in eight vehicle-treated (baseline) mice (n = 8) and six substance-P-stimulated mice (n = 6).
Statistics
Data are presented as means ± SE of data from four tracheas per group except where otherwise noted. Significant differences between means were evaluated using analysis of variance followed by Dunn-Bonferroni's test or unpaired Student's t-test. P < 0.05 was considered statistically significant.
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The architecture of the tracheal vessels in pathogen-free C57BL/6
mice and C3H mice resembled that in pathogen-free rats.16
The vessels in lectin-stained tracheal whole mounts formed repeating,
orderly networks: feeding arterioles were in most of the
intercartilaginous regions parallel to the cartilaginous rings,
terminal arterioles branched from the feeding arterioles toward the
cartilaginous rings, capillaries crossed the cartilaginous rings,
postcapillary venules were near the edge of the rings, and collecting
venules were in the intercartilaginous region parallel to the rings
(Figure 1, A and E)
. Most of the vessels
were in a plane of the mucosa beneath the epithelial basement membrane.
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, were enlarged and slightly increased in number at 4 weeks (Figure 1C)
, and were much more numerous at 8 weeks (Figure 1D)
. This
new vessel network was no longer confined to a single plane, and some
vessels were perpendicular to the epithelium (Figure 1D)
. The venules
were enlarged at all time points (Figure 1, B–D)
. Vascularity of the
tracheal mucosa of C57BL/6 mice, as measured by the area density of
vessels, increased from a pathogen-free baseline value of 34% to 49%
at 1 week after infection to 64% at 8 weeks (Figure 2A)
. The total length of tracheal vessels
in C57BL/6 mice, estimated by length density measurements, was 40%
higher than baseline at 8 weeks after infection (Figure 3A)
, and the number of vessels crossing
each cartilaginous ring was 120% higher than baseline at 8 weeks
(Figure 3B)
.
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. The vessel area density in tracheas of C3H
mice increased from a baseline value of 41% in pathogen-free mice to
65% at 1 week after infection and further to 64% at 8 weeks (Figure 2B)
. The vessel length density did not change in C3H mice (Figure 3A)
nor did the number of vessels (Figure 3B)
. The remodeled vessels in the
infected tracheas of C3H mice remained in a plane beneath the
epithelium.
When viewed in tissue sections, tracheal vessels in pathogen-free mice
were found in the loose connective tissue of the mucosa, and the
endothelial cells were flattened (Figure 4A)
. In comparison, tracheal vessels in
infected C57BL/6 (Figure 4B)
and C3H (Figure 4C)
mice were
conspicuously larger in diameter and the endothelium remained
flattened. The mucosa of infected mice contained numerous inflammatory
cells.
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Increased Number of Endothelial Cells in Enlarged Vessels
Because of the conspicuous vessel enlargement in the tracheas of
M. pulmonis-infected C3H mice, we determined whether there
was an increase in the number of endothelial cells. To do so, we
measured the luminal vessel surface area and the area of endothelial
cells in pathogen-free and infected C3H mice. The vessel diameters in
all segments of the tracheal microvasculature at 1 week and 4 weeks
after infection were increased compared with pathogen-free controls
(Figure 5)
. At 4 weeks after infection,
the arteriole diameters were 1.9-fold that of pathogen-free mice, the
capillaries 2.6-fold, the postcapillary venules 2.3-fold, and the
collecting venules 1.7-fold. Based on measurements of vessel length and
diameter (Figure 5)
, the surface area of the microvasculature in
infected C3H mice was 2.1-fold that of pathogen-free controls.
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. Interestingly, the
endothelial cell surface area was 32% greater at 1 week after
infection but at 2 weeks and 4 weeks was not significantly different
from the baseline values (Figure 7)
.
Comparing the time course of the increases in vessel diameter and the
changes in endothelial area suggests that the vessels were dilated at 1
week (vessel enlargement with larger endothelial cells) and then the
number of endothelial cells increased at 2 and 4 weeks (vessel
enlargement with normal-sized endothelial cells). However, even at 1
week, the enlargement of endothelial cells was not sufficient to
account for the increased surface area of the vessels; the endothelial
cells in collecting venules were 32% larger whereas the luminal
surface area was 66% larger, indicating 26% more endothelial cells.
At 4 weeks, the vessels remained enlarged whereas the average
endothelial cell area returned to normal; thus, an estimated 71% more
endothelial cells accompanied the increased surface area in collecting
venules. These results indicate that the infection increased the number
of endothelial cells in the tracheal vasculature of C3H mice.
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In addition to the different changes in the architecture of
vessels in C57BL/6 and C3H mice after infection, some of the functional
properties of the remodeled vessels were very different in the two
strains. In C57BL/6 mice, new capillary-sized vessels connected to
other capillary-sized vessels or to venules (Figure 6C)
. Numerous
blind-ending vessels, which appeared to be vascular sprouts, were also
present (Figure 6D)
. Such sprouts were common in tracheas of infected
C57BL/6 mice but not in pathogen-free mice or infected C3H mice. In C3H
mice, abrupt transitions in the characteristics of vessels occurred, in
which arterioles joined venules with little or no intervening
capillaries (Figure 6, E and F)
.
In addition to these changes in morphology, the remodeled vessels exhibited changes in lectin binding, vWF expression, and leukocyte adhesion.
Binding of Wheat Germ Agglutinin Lectin
One phenotypic characteristic of venular endothelial cells, unlike
capillary endothelial cells, is that the lectin WGA does not bind to
their luminal surface.21
In tracheal vessels of
pathogen-free C3H and C57BL/6 mice, WGA bound to the luminal surface of
endothelial cells in arterioles and capillaries but bound weakly or not
at all in venules (Figure 8A)
. In C57BL/6
mice infected with M. pulmonis, endothelial cells in the new
capillary-sized vessels bound WGA (Figure 8C)
, indicating a similarity
to capillary endothelial cells, whereas endothelial cells in enlarged
venules did not bind WGA. In infected C3H mice, endothelial cells in
the enlarged vessels in the anatomical position of capillaries bound
WGA weakly or not at all (Figure 8E)
, indicating their conversion to a
venule-like phenotype.
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vWF immunoreactivity is another marker that distinguishes
endothelial cells of different segments of the
vasculature.22,24-26
In tracheal vessels of pathogen-free
mice, vWF immunoreactivity was moderate in endothelial cells in
arterioles, high in venules, and low in capillaries (Figure 8B)
. In
tracheal vessels of infected C57BL/6 mice, vWF immunoreactivity was
very low in endothelial cells of the new capillary-sized vessels and
high in enlarged venules (Figure 8D)
. In infected C3H mice, vWF
immunoreactivity was high in endothelial cells in the enlarged,
venule-like vessels in the anatomical position of capillaries (Figure 8F)
.
Adhesion of Leukocytes
Tracheal venules in pathogen-free rats contain occasional adherent
leukocytes,27
and the number is increased by inflammatory
stimuli.16,27
Similarly, tracheal venules of pathogen-free
mice contain occasional leukocytes, whereas capillaries contained
almost none (Figure 1, A and E)
. In infected C3H mice, leukocytes were
numerous in venules and in remodeled vessels in the position of
capillaries (Figures 1F and 6, E and F
). The number of adherent
leukocytes in remodeled vessels overlaying the cartilage in the
position of capillaries was more than 100-fold higher in infected C3H
mice (1266 ± 100 per mm2
of vessel surface area at 4
weeks) than in pathogen-free mice (12 ± 11 per mm2).
Relative Proportion of Different Vessel Types
The abundance of the different types of microvessels was measured
in the tracheal mucosa of C57BL/6 and C3H mice (Table 1)
. In C57BL/6 mice, the length density
of arterioles did not change significantly at 8 weeks after infection,
but the length density of capillaries increased by 73%, and the length
density of venules increased by 175% compared with pathogen-free mice
(Table 1)
. As a result, the proportion of arterioles decreased in
infected C57BL/6 mice, the proportion of capillaries did not change,
and the proportion of venules increased from 18% to 28%.
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. As
a result, the proportion of venules increased from 26% in
pathogen-free C3H mice to 55% in infected mice, whereas the proportion
of capillaries declined from 57% to 31%. Plasma Leakage in Remodeled Vessels
The amount of Evans blue leakage per milligram of trachea in C3H
or C57BL/6 mice infected for 4 weeks was similar to that in
pathogen-free controls (Figure 9, A and B)
. However, substance P (5 µg/kg intravenously) caused significantly
more leakage in infected mice of both strains than in pathogen-free
controls (Figure 9, A and B)
. Thus, the remodeled vessels in M.
pulmonis-infected mice of both strains were abnormally leaky in
response to substance P.
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Time Course of Vascular Remodeling
In both C57BL/6 and C3H strains of mice, the vessel area density
was increased at 1 week after infection and remained above baseline
values at 2, 4, and 8 weeks. At 1 and 2 weeks, this increased vessel
density reflected enlargement of the vessels. Some of this increased
vessel size may be due to vasodilatation (ie, increased vessel diameter
without increased numbers of endothelial cells), because the size of
endothelial cells was increased at 1 week in C3H mice. This stage of
the infection initiates production of several cytokines, including
tumor necrosis factor-
, interleukin-1, and
interleukin-6,19
but precedes the appearance of significant
titers of circulating antibodies to M.
pulmonis.18
Subsequently, the number of vessels in the tracheal mucosa of infected C57BL/6 mice was increased in some focal regions at 4 weeks and was increased throughout the length of the trachea at 8 weeks. In comparison, the vessel diameters in C3H mice at 4 weeks and 8 weeks remained approximately twice their normal value, and no new vessel growth was detected. The size of the endothelial cells in tracheal vessels of C3H mice declined to control values at 2 and 4 weeks, despite unchanged vessel size, indicating increased numbers of endothelial cells. This process of remodeling appears to be an initial vasodilatation and endothelial cell enlargement, followed by an increase in the number of endothelial cells. The number of endothelial cells may have increased by proliferation of resident cells, although recruitment of circulating endothelial precursor cells to the vessel lumen may also occur.28
The initial stage of vascular remodeling (ie, vessel enlargement) was similar in the two strains, whereas new vessels grew only in C57BL/6 mice. The process of new vessel growth may be either a stage of vascular remodeling, driven by the severity or duration of the host inflammatory/immune response, or a distinct pathway, driven by a qualitatively different host response. By several criteria, C3H mice have a greater inflammatory/immune response to M. pulmonis infection,18,19 yet no significant new vessel growth occurs. Thus, the new vessel growth in C57BL/6 mice and the vessel enlargement in C3H mice may not reflect differences in the severity of infection but instead may indicate qualitative differences in the inflammatory/immunological responses of the two strains. One scenario is that an innate immune response to M. pulmonis infection induces vessel dilatation, which, if prolonged for more than 1 to 2 weeks, can induce endothelial cell proliferation but not new vessel growth. Subsequently, an acquired immune response can induce new vessel growth depending on the nature of that response. Further characterization of the inflammatory/immune responses in the two strains is needed to examine this question.
Angiogenesis in Chronic Inflammation
A potential source of confusion in describing changes in the microvasculature in chronic inflammation is usage of the term angiogenesis. Although often defined as the growth of new vessels from existing ones,1,29,30 angiogenesis has also been used to describe vascular changes in chronic inflammation that involve endothelial cell proliferation without new vessel growth.31,32 New vessel growth is readily identified in avascular tissues or in rapidly growing tissues such as tumors, but it may be difficult to identify in well vascularized tissues such as the airway mucosa. In the present study, we identified new vessel growth in the airways of chronically inflamed C57BL/6 mice that clearly corresponds to the usual definition of angiogenesis. However, in C3H mice, we identified vessel enlargement with increased numbers of endothelial cells but without new vessel growth. Should this type of microvascular remodeling also be considered as a form of angiogenesis? How prevalent is it in chronic inflammation? Refinement of the terminology used to describe these different microvascular responses would facilitate the communication of studies of chronic inflammation.
Endothelial Cell Phenotype
Endothelial cells in different segments of the microvasculature have specialized phenotypes.22,24,33,34 For example, endothelial cells in venules are distinct from those in capillaries by virtue of their expression of receptors for certain inflammatory mediators,35 lack of cell surface binding of the lectin WGA,21 and adhesion of leukocytes and decreased barrier function after inflammatory stimuli.16,27,34 It is unclear how these specialized phenotypic properties are regulated under normal or pathological conditions.
Although the biochemical basis and functional significance of the differential binding of WGA to the surface of endothelial cells is not known, it can be used as a phenotypic marker to help distinguish venules from capillaries. In the present study, we found that remodeled vessels in chronic inflammation in the two strains of mice differentially regulated this phenotypic marker. The luminal endothelial surface of new capillary-sized vessels in M. pulmonis-infected C57BL/6 mice bound WGA, indicating a similarity to capillaries. In contrast, the luminal endothelial surface of enlarged vessels in the location of capillaries in C3H mice did not bind WGA, indicating their conversion to a venule-like phenotype.
An additional feature that appears to distinguish different segments of the microvasculature is the amount of vWF immunoreactivity. Although vWF has been used as a marker for endothelial cells in culture and in tissue sections for many years, 22,24-26,36,37 most studies have focused on endothelial cells from large vessels. The amount of vWF immunoreactivity is high in endothelial cells on the venous side of the circulation22,25 and at sites of arterial branching37 but low in capillaries.24 However, the differential expression of vWF in different segments of the microvasculature has not been fully documented. In the present study, examination of the intact microvascular network in whole mounts of normal tracheas readily revealed that vWF immunoreactivity was moderate in arterioles, low in capillaries, and high in venules. Again, we found that remodeled vessels in chronic inflammation differentially regulated the amount of vWF. Endothelial cells of new capillary-sized vessels in M. pulmonis-infected C57BL/6 mice had very little vWF immunoreactivity, indicating their similarity to capillary endothelial cells, whereas endothelial cells of enlarged vessels in the location of capillaries in C3H mice had much more, indicating their conversion to a venule-like phenotype.
Adherent leukocytes were a prominent feature of chronically inflamed tracheas, particularly in C3H mice. Adherent leukocytes were not found in the new capillary-like vessels in infected C57BL/6 mice but were found in the enlarged venule-like vessels in C3H mice. Although some studies of chronically inflamed tissues have documented conversion of venules to lymph-node-like high endothelial venules specialized for lymphocyte migration,38 the enlarged venules in tracheas of C3H mice infected with M. pulmonis did not show markers of high endothelial venules.39
Thus, in addition to the size, shape, and location of the vessels, we were able to use three phenotypic markers to assess whether endothelial cells had features of venules, namely, lack of binding of WGA, strong vWF immunoreactivity, and the ability to support adhesion of leukocytes. These markers were part of the phenotype of endothelial cells in normal venules and changed in a coordinated fashion in the remodeled vessels in chronically inflamed tracheas. vWF immunoreactivity was not strictly correlated with leukocyte adhesion or lack of WGA binding, as arterioles contained moderate amounts of vWF but bound WGA and did not support leukocyte adhesion. Therefore, this set of features appears to represent at least two independent markers of vessel phenotype. Examining changes in endothelial cell phenotype during vascular remodeling, and comparing the changes in the two types of remodeling in C57BL/6 and C3H mice, may help shed light on the regulation of specialized endothelial cell phenotype.
Although we have described the changes in endothelial cell phenotype in
terms of features that can normally be used to distinguish capillaries
from venules, additional phenotypic features may be distinctive to
endothelial cells in remodeling or angiogenic vessels, and not found in
the normal microvasculature. Some such features have recently been
reported. For example, the integrin
vß340
and the tie-family of
tyrosine kinase receptors41
may be selectively expressed by
angiogenic endothelial cells. The relation between these distinctly
angiogenic markers and the expression of capillary-like or venule-like
phenotypes is unknown.
Plasma Leakage in Chronically Inflamed Tracheas
Previous studies have provided evidence for increased leakage of plasma from angiogenic blood vessels.42 However, in the present study, we found that the baseline amount of plasma leakage was similar in pathogen-free mice and mice infected for 4 weeks, consistent with previous findings in F344 rats.17 The method we used for assessing leakage, namely, measuring extravasated Evans blue dye in the whole trachea after a 5-minute circulation period, may not detect very localized leakage or brief episodes of leakage. Another possibility is that plasma leakage occurs earlier than 4 weeks and then ceases. Nevertheless, our findings indicate that despite the extensive vascular remodeling at 4 weeks, which is a period of rapid vessel growth in C57BL/6 mice, there is limited leakage under baseline conditions.
Unlike the baseline level of plasma leakage, the amount of leakage induced by substance P was much higher in infected mice than in pathogen-free mice. In rats, the abnormal sensitivity to substance P after M. pulmonis infection appears to be due, at least in part, to up-regulation of receptors for substance P.15 The role of the increased expression of substance P receptors in the pathophysiology of M. pulmonis infection is unknown, but the recent production of mice deficient in these receptors43 may help answer the question.
Characterization of Vascular Remodeling in Tissue Whole Mounts
The different patterns of vascular architecture in infected C57BL/6 and C3H mice could be readily distinguished in lectin-stained tracheal whole mounts. In addition, as the location of individual vessels within the microvasculature could be identified, abnormalities in endothelial cell phenotype were also apparent. These features were not readily apparent in tracheas cut into thin sections. Although the whole-mount approach is not feasible with most pathological specimens, our findings indicate that different forms of vascular remodeling could be distinguished by morphometry of tissue sections combined with staining for specific markers of endothelial cell phenotype. For example, vascular remodeling similar to that observed in C3H mice could be detected in tissue sections by an increase in the diameter of the vessels without an increase in vessel number and by an increase in the proportion of vessels with strong vWF immunoreactivity.
Comparison with Other Studies of Vascular Remodeling in Chronic Inflammation
Although vascular remodeling is a common feature of chronic inflammation, few studies have documented the type of remodeling or the phenotype of endothelial cells in the remodeled vessels. Our previous studies of M. pulmonis infection of the airways of rats indicate that new capillary-sized vessels form in the tracheal mucosa of some strains of rat.15 In infected F344 rats, new capillary-sized vessels form across the cartilage,15 similar to the new vessel growth in C57BL/6 mice, but enlargement and proliferation of venules was not assessed in rats. Although different strains of rat also show different susceptibilities to M. pulmonis, we have not seen a vascular response similar to that of infected C3H mice in the strains that we have examined to date.
The type of vascular remodeling seen in C3H mice has been previously described in a model of aseptic chronic inflammation in adult rats.32 In this model, a piece of sterile tissue was implanted next to the cremaster muscle, and the muscle capillaries enlarged and changed functional properties without the growth of new vessels. An increase in endothelial cell number was also reported, which was postulated to be a result of chronic hyperemia.44
Psoriasis is a chronic inflammatory condition in which the microvessels enlarge, become more tortuous, change their structural properties,11-14 and contain proliferating endothelial cells.11 The growth of new vessels via sprouting has not been documented in psoriasis. Dermal microvessels in the position of capillaries assume phenotypic properties of venules, as assessed by the ultrastructure of the basement membrane and the presence of endothelial fenestrae.12 These particular phenotypic properties, which are visible only by transmission electron microscopy, may be specific to venules of the dermis, whereas other properties, such as the lack of binding of WGA lectin and high vWF immunoreactivity, have not been examined. Recently developed animal models of psoriasis45 may help to characterize further the type of microvascular remodeling.
Morphometric studies of tissue sections from airway biopsies suggest that both the number and the size of vessels can increase in human asthma.6,7,46 Characterization of the phenotypic properties of the vessels may further aid in defining the nature of the microvascular remodeling and in determining the amounts of new vessel growth and vessel enlargement in chronic airway inflammation.
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Supported in part by NIH grants HL-24136 and HL-59157.
Accepted for publication July 18, 1998.
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, interleukin 1, interleukin 6, and 
interferon in C3H/HeN and C57BL/6N mice in acute Mycoplasma pulmonis disease. Infect Immun 1995, 63:4084-4090[Abstract]
vß3 for angiogenesis. Science 1994, 264:569-571This article has been cited by other articles:
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