1 Cardiovascular Pulmonary and Developmental Lung Biology Research Laboratories, University of Colorado Health Sciences Center, Denver 80262; and 2 Denver Veterans Administration Medical Center, Denver, Colorado 80220
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ABSTRACT |
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Proliferation of fibroblasts contributes to the adventitial thickening observed during the development of hypoxia-induced pulmonary hypertension. However, whether all or only specific subpopulations of fibroblasts proliferate during this process is unknown. Because lung, skin, and gingiva contain multiple fibroblast subpopulations, we hypothesized that the pulmonary artery (PA) adventitia of neonatal calves is composed of multiple fibroblast subpopulations and that only selective subpopulations expand under chronic hypoxic conditions. Fibroblast subpopulations were isolated from PA adventitia of control calves using limited dilution cloning techniques. These subpopulations exhibited marked differences in morphology, actin expression, and serum-stimulated growth. Only select fibroblast subpopulations demonstrated the ability to proliferate in response to hypoxia. Fibroblast subpopulations were similarly isolated from calves exposed to hypoxia (14 days). With regard to morphology, actin expression, and serum-stimulated growth of subpopulations, there were no obvious differences in fibroblast subpopulations between the hypoxic and the control calves. However, the number of fibroblast subpopulations with about a twofold increase in hypoxia-induced DNA synthesis was significantly greater in the hypoxic calves (26%) compared with control calves (10%). We conclude that the bovine PA adventitia comprises numerous phenotypically and biochemically distinct fibroblast subpopulations and that select subpopulations expand in response to chronic hypoxia.
fibrosis; vascular disease; vascular remodeling; fibroblast growth; hypoxia-induced proliferation
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INTRODUCTION |
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HYPOXIA-INDUCED pulmonary hypertension complicates the clinical course of many important lung diseases in both children and adults (23, 42, 46). The pulmonary hypertension and accompanying structural remodeling is particularly severe in infants, and often the earliest and most dramatic structural changes are found in the adventitial compartment of the vessel wall (31, 46). In animal models, the resident adventitial fibroblasts have been shown to exhibit early and sustained increases in proliferation and matrix protein synthesis under hypoxic conditions, and these changes have been associated with luminal narrowing and a progressive decrease in the ability of the vessel wall to respond to vasodilating stimuli (3, 23, 30, 45, 46). Furthermore, in response to different stimuli, adventitial fibroblasts isolated from hypoxia-induced pulmonary hypertensive neonatal calves demonstrate greater growth capabilities than those from control animals (8), which could be the result of a generalized or polyclonal activation of resident adventitial fibroblasts. Alternatively, it could be the result of the presence of increased numbers of specific fibroblast subpopulations with distinct growth characteristics as has been suggested in the setting of lung fibrosis and scleroderma (15, 20, 21, 24, 32). However, it is not known whether the pulmonary artery (PA) adventitia is composed of heterogeneous fibroblast subpopulations with unique functional characteristics and, if so, whether these fibroblast subpopulations contribute selectively to the fibroproliferative vascular changes observed in cases of severe hypoxia-induced pulmonary hypertension.
The existence of phenotypic and functional heterogeneity among the resident fibroblast populations of different organs has been well described (6, 19, 26, 34, 36, 37, 39, 43). Evidence of fibroblast heterogeneity within the same anatomical site or organ system has also been well documented (2, 11, 15, 16, 18, 20, 34). Fluorescence-activated cell sorting (FACS) on the basis of cell-surface antigen expression and limited dilution cloning techniques have both been utilized to demonstrate the existence of heterogeneous fibroblast populations within the lung with regard to morphology, synthesis of prostaglandin (PG) E2, synthesis of collagen, and growth capability (11, 24, 34, 35, 48). Importantly, utilization of either technique results in the generation of stable subpopulations or clones that maintain unique and distinct functional characteristics for prolonged periods of time in culture. Accumulating evidence also supports the concept that persistent local pathophysiological stimuli may provide a natural selective drive, as a result of which certain fibroblast clones or subpopulations with distinct proliferative or matrix-producing capabilities will emerge and contribute specifically to the disease process in fibroblast-enriched organ systems (15, 20, 32, 49). However, it is unknown whether fibroblast subpopulations that are uniquely susceptible to activation and proliferation in response to hypoxia exist within the pulmonary circulation or any other organ system. Furthermore, it is not known whether chronic hypoxia would provide an environment such that fibroblasts with unique hypoxia-induced growth advantages would emerge and increase in number and thus contribute in select ways to the adventitial thickening and fibrosis during the development of hypoxic pulmonary hypertension.
We hypothesized that the normal PA adventitia is composed of heterogeneous subpopulations of fibroblasts and that only select subpopulations possess the capability of proliferating under hypoxic conditions in the absence of exogenous mitogens. Furthermore, we hypothesized that chronic in vivo hypoxia would create a growth advantage for these fibroblast subpopulations and would result in a selective increase in their number in the thickened PA adventitia of chronically hypoxic animals. A positive answer to this question would lend support to the idea that certain fibroblast subpopulations are activated in response to a given pathophysiological condition and may thus selectively contribute to specific disease processes. Because no markers have been developed that allow FACS sorting of bovine fibroblasts and because we did not want to "preselect" fibroblast subpopulations based on any one marker of fibroblast phenotype, our approach was to isolate fibroblast subpopulations using limited dilution cloning techniques and then to evaluate these subpopulations for differences in morphology, contractile protein expression, and growth capabilities. In addition, we specifically evaluated the subpopulations for their ability to proliferate in response to hypoxia in the absence of any exogenous mitogens. We also exposed a group of neonatal calves to chronic hypoxia for 14 days to promote the development of severe pulmonary hypertension and adventitial thickening and fibrosis. Adventitial fibroblast subpopulations from these vessels were then generated using the same techniques and also evaluated for changes in morphology, contractile protein expression, and growth in response to hypoxia. Our findings support the idea that the PA adventitia is composed of multiple functionally heterogeneous fibroblast subpopulations. Only specific fibroblast subpopulations exhibit an ability to proliferate under hypoxic conditions. However, these fibroblast subpopulations appear to undergo selective expansion in response to chronic hypoxia and therefore may contribute to the adventitial thickening and fibrosis observed during the development of chronic hypoxia-induced pulmonary hypertension.
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MATERIALS AND METHODS |
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Materials.
Human platelet-derived growth factor PDGF-BB and basic
fibroblast growth factor (bFGF) were purchased from Bachem Fine
Chemicals (Torrance, CA) and suspended in Eagle's minimal essential
medium (MEM) with 2% fatty acid-free bovine serum albumin
(BSA). MEM, trypsin-EDTA 10× suspension, penicillin,
streptomycin, amphotericin B, Hanks' balanced salt solution (HBSS)
with HEPES (with and without CaCl2), and monoclonal
antibody specific for -smooth muscle (SM) actin (A-2547 from clone
1A4) were from Sigma (St. Louis, MO). Elastase was purchased
from Boehringer Mannheim (Indianapolis, IN). Collagenase II and soybean
trypsin inhibitor were from Worthington (Lakewood, NJ). Fetal bovine
serum (FBS) was purchased from Gemini Bio-Products (Calabasas, CA).
[3H]Thymidine was from ICN Biochemicals (Irvine, CA).
Rabbit antibodies against bovine aortic SM myosin were kindly provided
by Dr. R. S. Adelstein (National Heart, Lung, and Blood Institute,
Bethesda, MD) (25). Affinity-purified rabbit antibody
against von Willebrand factor was purchased from DAKO. Conditioned
media were prepared for cloning experiments by removing medium from
rapidly growing culture of bovine fetal fibroblasts and filtering it
through a 0.22-µM filter to remove any cell debris.
Isolation and growth of neonatal bovine main PA adventitial
fibroblast subpopulations.
We harvested adventitia from the main PA of 15-day-old normoxic and
hypoxic neonatal calves. Normoxic calves were born and remained at Fort
Collins, CO [1,524 m altitude, barometric pressure (PB) = 650 Torr]. Hypoxic calves were born at Fort Collins altitude, but after they were 1 day old, they were placed into an altitude chamber at simulated altitude (4,570 m, PB = 445 Torr),
where they remained for 2 wk to induce severe pulmonary hypertension as
previously described (45). At postmortem examination,
adventitial tissue was isolated, carefully dissected free of blood
vessels and fat under a dissecting microscope, and cut into small
pieces. The tissue pieces were incubated with Ca2+-free
HBSS for 30 min at 37°C and then with HBSS containing
Ca2+, elastase, collagenase, albumin, and soybean trypsin
inhibitor for 90 min at 37°C on a rotator. The tissue was gently
triturated with a sterile Pasteur pipette after each 30 min of
incubation. The dispersed cells were passed through a 100-µm nylon
cell strainer (Falcon) to remove any undigested tissue pieces, diluted
in MEM containing 10% FBS to inactivate the enzymes, and centrifuged at 900 rpm for 10 min. Using a light microscope and hemocytometer, we
counted the cells and serially diluted this cell suspension with the
media containing 30% fetal conditioned media and 10% FBS. Cells were
plated at a density of 0.5 cells · well1 · 0.2 ml
1 in
96-well plates. These cultures were examined at frequent intervals using a light microscope and maintained at 37°C and 5%
CO2 with a biweekly change of media containing 30% fetal
conditioned media and 10% FBS. The cells were maintained in 96-well
plates for 2 wk. Wells with cells were scored to determine cloning
efficiency. Once cells reached confluence in the microtiter wells, they
were trypsinized and transferred to 24-well culture dishes in MEM
containing 10% FBS. When the cells had grown again to confluence, they
were transferred serially into 12-well, 6-well culture dishes and
25-mm, 75-mm culture flasks, respectively. In all experiments,
fibroblasts were studied between the third and the 15th passages.
Characterization of fibroblast subpopulations by morphology and
immunofluorescence staining for -SM actin, myosin, and factor VIII.
Because our goal was to obtain pure subpopulations of fibroblasts, we
first analyzed individual cell populations for their morphological
appearance and then examined expression of SM-specific markers in each
isolated cell population. Cells were grown to confluence on Tissue-Tek
chamber slides in 10% FBS-MEM, fixed with cold methanol for 10-15
min at 4°C, and processed for indirect immunostaining as follows. For
double immunofluorescence staining of
-SM actin and SM myosin, fixed
cells were incubated with a cocktail of monoclonal
-SM actin and
polyclonal anti-SM myosin antibodies (diluted 1:100 and 1:1,000,
respectively) for 1 h at room temperature. After three washes in
PBS, cells were incubated with a cocktail of biotinylated anti-mouse
IgG and FITC-conjugated anti-rabbit IgG (both diluted at 1:100 and both
purchased from Sigma) for 1 h at room temperature. The staining
for
-SM actin was accomplished by incubation with streptavidin-Texas
red (1:50, Amersham). All stained cells were examined with a Nikon
Optiphot epifluorescence photomicroscope.
Growth of PA fibroblast subpopulations in the presence of serum. To examine the in vitro growth characteristics of fibroblast subpopulations, serum-stimulated growth was measured as previously described (9, 10). Cells were sparsely seeded (1.0 × 104 cells/well) in MEM containing 10% FBS in 24-well plates, and cell counts were performed on alternate days between day 0 and day 10. Media were supplemented, but not replaced, with fresh media containing 10% FBS on day 4 and day 8 to avoid blunting growth of the more rapidly proliferating cells. To assess changes in cell number, the cells were trypsinized for 10 min, gently triturated after addition of an equal volume of 10% FBS-MEM, and counted with a standard hemocytometer under light microscope. Data were expressed as cell number × 104/well. Population doubling time was calculated in exponentially growing cells according to a previously described method (33).
Assessment of hypoxia-induced DNA synthesis. To test whether fibroblast subpopulations isolated from the PA adventitia of neonatal calves had the ability to replicate in response to low oxygen concentration, cells were seeded at a density of 7.5 × 103/cm2 in media containing 10% FBS, allowed to attach overnight, and then growth arrested for 72 h with 0.1% FBS-MEM. At the end of 72 h, the medium was replaced with fresh MEM only. Quiescent fibroblasts were exposed to normoxia and hypoxia in the presence of [3H]thymidine for 24 h in sealed humidified gas chambers as previously described (7, 8). At the end of 24 h, cells were harvested for measurement of thymidine incorporation. Briefly, the assay medium was removed, and cells were rinsed with PBS and fixed with 0.2% perchloric acid. After an additional rinse with PBS, the acid-precipitated cellular material was solubilized with 0.01 N sodium hydroxide-0.1% sodium dodecyl sulfate. The contents of each well were then added to 4 ml of Ecoscint H (National Diagnostics, Atlanta, GA), and radioactivity was measured with a Beckman LS 7500 beta-scintillation counter (Irvine, CA). Cell counts were obtained at the end of the 24-h incubation period. Incorporation of [3H]thymidine into DNA was expressed as counts per million per cell.
[3H]Thymidine incorporation in response to growth factors. DNA synthesis in response to purified mitogens was measured under serum-free conditions according to the previously described method (8, 9). Cells were seeded at 7.5 × 103/cm2 in MEM-10% serum, allowed to attach for 24 h, and then growth arrested for 72 h with MEM-0.1% serum. At time 0 of the test period, medium was replaced with MEM alone, and purified mitogens (PDGF, 30 ng/ml; bFGF, 40 ng/ml) and [3H]thymidine (0.5 µCi/well) were added to each well and exposed to normoxia and hypoxia for 24 h in sealed humidified gas chambers. At the end of the incubation, cells were processed for the measurement of [3H]thymidine incorporation as mentioned in Assessment of hypoxia-induced DNA synthesis.
Data analyses.
All data are presented as arithmetic means ± SE. Each observation
was reproduced in cells isolated from at least three different animals.
One-way analysis of variance followed by Student-Newman-Keuls multiple
comparison test was used for individual comparisons within and between
groups of data points. Distribution relationship between the animals
and the percentage of "hypoxia-proliferative" fibroblast subpopulations was evaluated by the 2 method. Data were
considered significantly different at P < 0.05.
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RESULTS |
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Isolation of purified fibroblast clones or subpopulations from the PA adventitia of neonatal control calves. To determine whether clones, or at least highly purified primary fibroblast subpopulations, could be generated from the PA adventitia, we utilized the limited dilution cloning technique. Aggregate fibroblast populations were enzymatically dispersed from cleaned adventitia and suspended in media. Serial dilutions of this cell suspension were made such that ~0.5 cells were placed into each well of a 96-well plate. Thus some wells likely received no cells, whereas others received one or potentially more cells. For each of eight neonatal control calves, we prepared three 96-well plates. We then determined the number of wells with cells that had grown to confluence at the end of 2 wk. We found in the calf with the highest "cloning" efficiency an average (for the three plates) of 17 ± 0.6 confluent wells, whereas in the calf with the poorest cloning efficiency there were only 6 ± 2.8 such wells. On average for all eight calves, 13% of wells (12.8 ± 1.4 wells in 96-well plates) showed growth of fibroblasts to confluence. Nearly 100% of the wells that demonstrated growth in the 96-well plates survived serial passaging for expansion into quantities sufficient for biochemical and functional characterization. Thus our limited dilution cloning technique could be used to establish purified primary fibroblast subpopulations from the main PA adventitia.
Heterogeneity of morphology and contractile protein expression in
fibroblast subpopulations.
We sought to determine whether PA adventitia, like other
fibroblast-enriched tissues (2, 11, 12, 15, 16, 18, 19, 35, 37,
40), was composed of multiple morphologically, biochemically,
and functionally distinct populations by analyzing the fibroblast
subpopulations generated from each animal. We found that, when examined
by light microscopy, the cells in the >120 subpopulations that were
evaluated could be described as having two general morphologies, cells
that appeared rounded or rhomboidal in shape (Fig.
1, A-C) and cells that
were more elongated or spindle shaped (Fig. 1, E-G).
Within these two broad classifications, there were differences among
subpopulations in characteristics such as cell size, pattern of
organization (i.e., whorls vs. linear), and shape (Fig. 1). These
morphological findings supported the idea that in neonatal calves, the
PA adventitia is composed of a mixed population of fibroblasts.
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Heterogeneity in growth potential of fibroblast subpopulations.
To determine if the isolated fibroblast subpopulations exhibited
differences in growth capabilities, we generated growth curves for 42 different fibroblast subpopulations isolated from PA adventitia of
neonatal control calves. Marked differences in growth capacity in the
presence of 10% serum-containing media were noted between the
subpopulations. An example demonstrating the range of growth differences in three subpopulations is shown in Fig.
3A. Using doubling time as a
measurement of growth rate, we found that the variability in growth
among the subpopulations was great, as indicated by the histogram (Fig.
3B). Doubling times varied from as short as 11 h to as
long as 80 h and in some cases over 100 h. The average slope
(ratio of cell count/day) of the growth curve of the clones also
demonstrated marked heterogeneity of growth between cell populations (Table 1). Finally,
the saturation density at confluence also varied remarkably among the
fibroblast clones (Table 1) consistent with possible differences in
cell size, contact inhibition or lack thereof, or both.
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Heterogeneity among fibroblast subpopulations in their growth
response to hypoxia.
We considered the possibility that, in addition to differences in
morphology, actin staining, and growth capability in serum, fibroblast
subpopulations might differ in their proliferative response to hypoxia.
In a preliminary experiment, we selected two different fibroblast
subpopulations from the same neonatal calf, plated them at 10 × 103 cells/well, and grew them for 7 days in the presence of
serum under either normoxic or hypoxic conditions. At the end of 7 days, subpopulation A had cell counts of 79 ± 9 × 103/well in normoxia and 80 ± 11 × 103 in hypoxia, whereas subpopulation B had
counts of 58 ± 4 × 103 in normoxia and 90 ± 5 × 103 in hypoxia. These findings suggested that
despite slower growth in normoxia, subpopulation B
demonstrated a significantly higher growth rate under hypoxic
conditions, whereas subpopulation A did not. We then growth
arrested these same two subpopulations for 72 h with 0.1% FBS
containing media and exposed them to hypoxia in absence of any
exogeneous mitogens for 24 h and measured DNA synthesis. Hypoxia
selectively increased thymidine incorporation in subpopulation
B but not in A (Fig. 4),
suggesting that hypoxia acts as growth-promoting stimulus for only
selected fibroblast subpopulations. The fibroblast subpopulations that
had a more than twofold increase in hypoxia-induced proliferation were
termed as hypoxia proliferative, and the subpopulations that did not exhibit any increase in DNA synthesis in response to
hypoxia were designated as hypoxia nonproliferative.
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Adventitial fibroblast subpopulations from chronically hypoxic
calves.
Because hypoxic pulmonary hypertension is associated with dramatic
fibroproliferative changes in the adventitia (45, 46), we
considered the possibility that the adventitia of hypoxia-induced pulmonary hypertensive calves might have fibroblast subpopulations that
differed from control calves in regard to morphology, -SM actin
expression, and growth capabilities either under normoxic or hypoxic
conditions. First, we found that the average cloning efficiency of
fibroblasts from hypoxic hypertensive calves (11.3 ± 1.0) was
very similar to that of the control calves (12.8 ± 1.4). In 32 fibroblast subpopulations from hypertensive calves that were examined
with regard to morphology and
-SM actin expression, 41% exhibited a
rounded morphology and 59% of the subpopulations were elongated (Table
2), which was not different from the 32 and 68% distribution observed, respectively, in control calves. The
microscopic appearances of the round and elongated cells in the
subpopulations from hypertensive calves were similar to those from
control calves. In hypoxic hypertensive calves, 61% of subpopulations stained positively for
-SM actin expression and 11% did not, which
was not different from the values in control calves (68 and 14%,
respectively). As in control calves, even for subpopulations that
positively expressed actin, the pattern of expression could differ,
where, for example, some fibroblasts showed actin expression in
stress fibers, whereas in others, it was expressed diffusely within the cytoplasm. Rounded cells could be either actin positive, negative, or mixed, and elongated cells could either be actin positive
or mixed (Table 2).
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DISCUSSION |
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The main findings in the present study were that: 1) the adventitia of the normal neonatal bovine PA is composed of numerous phenotypically distinct subpopulations of fibroblasts and 2) chronic hypoxia-induced pulmonary hypertension is associated with a selective increase in the number of resident fibroblast subpopulations with enhanced growth capability under hypoxic conditions. Given the extensive literature showing fibroblast heterogeneity in other tissues (11, 15, 24, 34, 35, 48), it was perhaps not surprising that we would find fibroblast heterogeneity in the PA adventitia, even though this has not been previously reported. Remarkable, however, was the nature, magnitude, and frequency of the differences between the subpopulations. The characteristics which differed between subpopulations included: 1) morphology, 2) the presence or absence of actin staining, 3) actin expression pattern, i.e., cytoplasmic vs. stress fiber distribution, 4) the serum-stimulated growth rate under normoxia and hypoxia, 5) the DNA synthesis (in the absence of serum) in normoxia and hypoxia, and 6) growth factor (PDGF and bFGF)-induced proliferative responses under normoxic and hypoxic conditions. Differences in these characteristics were observed among subpopulations generated from several animals. Importantly, the growth rate pattern of the heterogeneic subpopulations obtained from one animal had a striking resemblance to subpopulations generated from other animals, indicating that the observed heterogeneity was a consistent and true characteristic of fibroblast subpopulations of the bovine PA adventitia. Our results are consistent with reports demonstrating heterogeneity of cells composing the endothelium and media (4, 14, 27, 38) and, as discussed below, extend the heterogeneity concept to vascular fibroblasts by showing in a single study that numerous functionally distinct subpopulations exist within the adventitia.
The present study examined only a few of many known fibroblast characteristics. From lung tissue, for example, fibroblasts have been shown to be heterogeneous with regard to morphology (15, 34), proliferative capacity (6, 20, 24, 34, 48), surface marker (Thy-1, MHC class II, CD4) expression (11, 34), type and amount of collagen production (11, 16), intracellular metabolic pathways (15), and cytokine production (15). In addition, fibroblasts from nonpulmonary tissues have been shown to be heterogeneous with regard to proliferation (2, 22), size and shape (18, 19), presence of lipid droplets (29), collagen synthesis (13, 16, 21, 36, 40, 49, 51), expression of growth factors (16), response to and synthesis of PGE2, and expression of C1q surface receptors, among others. One wonders about heterogeneity in some of the other key functions of fibroblasts, such as capacity for transition to other cell types (i.e., myofibroblast and/or SM cell), migration, protease expression, and propensity to apoptosis. Previous studies have usually focused on a single characteristic or group of characteristics of fibroblast heterogeneity that might account for a particular fibroblast function, response to injury, or disease process. The present study on heterogeneity itself has expanded the prior concepts by showing within a mixed fibroblast population from a single normal neonatal tissue (i.e., the PA adventitia) the marked and frequent variability in numerous characteristics of fibroblast subpopulations.
One important aspect of fibroblast function, the ability to proliferate under hypoxic conditions, has received only limited attention in the literature. We and others have documented that aggregate populations of fibroblasts from the PA demonstrate increased DNA synthesis under hypoxic conditions (7, 8, 44, 50). Similar findings have been reported in aggregate populations of fibroblasts from skin and gingiva (1, 5, 32, 44, 47). Our findings suggest that proliferation under hypoxic conditions is not a response that characterizes all fibroblast subpopulations but, rather, that it is a response limited to only select subpopulations of fibroblasts. It is possible, for instance, that some vascular beds or tissues have a higher proportion of hypoxia-proliferative subpopulations than others. For example, the PA could have a higher proportion of hypoxia-proliferative fibroblast subpopulations than certain systemic vascular beds. This might explain our previously reported findings wherein aggregate fibroblast populations from the pulmonary circulation demonstrate more consistent proliferative responses to hypoxia than aggregate fibroblast populations from the aorta (7). It could also explain the marked variability in hypoxic responses in aggregate fibroblast populations derived from one animal versus another, since mixed populations could vary considerably in the numbers of hypoxia-proliferative cells they contain. Furthermore, the fact that purified populations of fibroblasts with unique hypoxic responses can be generated and maintained in culture will allow investigation into the basic mechanisms that contribute to hypoxia-induced cell proliferation.
It should be noted that we were not able to identify a characteristic that allowed us to predict whether a fibroblast would proliferate under hypoxic conditions or not. This is unlike our findings for hypoxia-responsive and nonresponsive cells within the media of the vessel wall (14). Furthermore, our findings suggest a wide variability of the proliferative responses to hypoxia among fibroblast subpopulations, which is different from our findings of nearly an "all or none" phenomenon in medial smooth muscle cell. It will be important in the future to identify proteins that either confer or associate with hypoxic responsiveness so that more accurate evaluations of select cell expansion can be evaluated in vivo.
Although this study was designed simply to demonstrate that different characteristics exist among the subpopulations of fibroblasts, some comment can be made about the interrelationship of characteristics within a given subpopulation. For example, cells that were smaller and rounded usually (but not always) grew faster in serum than cells that were larger and spindle shaped. But the relationship between morphology and growth rate was weak, because either round-shaped cells or spindle-shaped cells could grow rapidly, and either shape could grow slowly. Nor could morphology be related to the actin expression or the expression pattern. Also, the expression or the pattern of expression of actin did not relate to proliferation. Furthermore, subpopulations that replicated rapidly in normoxia did not necessarily do so in hypoxia. These findings, together with the great spectrum among the subpopulations of growth rates in normoxia and hypoxia, suggest an enormous heterogeneity in aggregate fibroblast populations.
Given the inherent heterogeneity of fibroblasts within the vascular
adventitia, we wondered how these subpopulations would respond to a
specific in vivo stimulus, namely chronic hypoxia, which, in the
newborn calf, causes a thickened and hypercellular PA adventitia
(45). There were at least three possibilities: 1) all or nearly all of the fibroblast subpopulations could
increase their replicative capacity in response to chronic hypoxia,
2) those subpopulations that normally reside in the
adventitia and that have the potential for a replicative response to
hypoxia might show a heightened response, and 3)
subpopulations of fibroblasts that are particularly responsive to
hypoxia might become more numerous. Comparing subpopulations from
control versus hypoxic calves indicates no statistically significant
shift in the spectrum of proliferative responses and a similar range of
hypoxia-induced growth responses in normoxic and hypoxic calves. These
findings oppose, respectively, the first and second possibilities
listed. However, the finding of more subpopulations with greater than twofold increase in hypoxia-induced proliferation in hypoxic calves than in control calves supports the third possibility, namely, an
increase in the number of hypoxia-proliferative subpopulations. Furthermore, similar morphologies and -SM actin expression patterns in fibroblast subpopulations isolated from control and hypoxic calves
at least raise the possibility that the hypoxic exposure promoted
expansion of these selective subpopulations. Our findings of selective
expansion of hypoxia-proliferative fibroblast subpopulations during
hypoxia-induced pulmonary hypertension is consistent with the recent
report of Panchenko et al. (32) demonstrating selective proliferation of a subset of hypoxia-adapted fibroblasts during skin fibrosis.
In view of the important role of cell-cell interactions in the coordinate control of cell activity, any alteration in the relative proportion of different fibroblast subpopulations within a tissue might be expected to significantly influence the behavior of the entire organ. Expansion of hypoxia-proliferative fibroblast subpopulations under hypoxic conditions could lead, through alterations in the production of paracrine factors as well as extracellular matrix proteins, to changes in SM as well as endothelial function. Fibroblasts are known to significantly influence endothelial and epithelial function (39). For example, fibroblasts from patients with psoriasis induce normal keratinocytes to display many of the aberrations in cell proliferation and differentiation associated with psoriasis (17). Expansion of fibroblasts with unique characteristics could thus have potentially significant and as yet unexplored effects on endothelial and SMC function and could thus contribute in many ways to the functional abnormalities described for hypertensive PA.
On the basis of the results of the present study we propose that the adventitia of the normal PA contains an array of fibroblast subpopulations, differing markedly in numerous biochemical and functional characteristics. One wonders what biological advantage might accrue to such great heterogeneity. A speculative answer for vascular fibroblasts might refer to the large number of normal functions required of these cells, such as providing elasticity for a threefold pressure change within the lumen during each cardiac cycle, structural integrity for a three- to fourfold change in mean pressure during exercise, responding to the hormonal changes during pregnancy, as well as participating in the developmental changes within the arterial wall throughout fetal, neonatal, and adult life. Furthermore, in response to injury, fibroblasts have been shown to play an important role in the structural remodeling of the vascular wall (28, 41). Specific fibroblast subpopulations with certain functions may increase in number in response to specific stimuli and play unique roles in the structural alterations occurring during injury. Although we have suggested that this may occur with chronic hypoxia in the PA adventitia, it needs both confirmation and examination of mechanism through further research.
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ACKNOWLEDGEMENTS |
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We thank Steve Hofmeister and Sandi Walchak for harvesting bovine PA tissue, preparing the final figures, and immunofluorescence staining of the cells.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants HL-64917-01A1, HL-14985, and SCOR-56481. M. Das was supported by a postdoctoral fellowship from the American Heart Association, Arizona, Colorado & Wyoming Affiliate, a Giles Filley Research Award from the American Physiological Society, and a research grant from the American Lung Association.
Preliminary results were presented at Experimental Biology Meetings in New Orleans, LA (8c), and San Francisco, CA (8a); the 10th International Vascular Biology Meeting in Cairns, Australia, 1998; and the American Thoracic Society Annual Meeting in San Diego, CA (8b).
Address for reprint requests and other correspondence: M. Das, Developmental Lung Biology Research Labs, B-131, Univ. of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262 (E-mail: Mita.Das{at}UCHSC.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajplung.00382.2001
Received 27 September 2001; accepted in final form 21 November 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alaluf, S,
Muir-Howie H,
Hu HL,
Evans A,
and
Green MR.
Atmospheric oxygen accelerates the induction of a postmitotic phenotype in human dermal fibroblasts: the key protective role of glutathione.
Differentiation
66:
147-155,
2000[ISI][Medline].
2.
Azzarone, B,
and
Macieira-Coelho A.
Heterogeneity of the kinetics of proliferation within human skin fibroblastic cell populations.
J Cell Sci
57:
177-187,
1982[ISI][Medline].
3.
Belknap, JK,
Orton EC,
Ensley B,
and
Stenmark KR.
Hypoxia increases bromodeoxyuridine labeling indices in bovine neonatal pulmonary arteries.
Am J Respir Cell Mol Biol
16:
366-371,
1997[Abstract].
4.
Benzakour, O,
Kanthou C,
Kanse SM,
Scully MF,
Kakkar VV,
and
Cooper DN.
Evidence for cultured human vascular smooth muscle cell heterogeneity: isolation of clonal cells and study of their growth characteristics.
Thromb Haemost
75:
854-858,
1996[ISI][Medline].
5.
Bonakdar, MP,
Barber PM,
and
Newman HN.
The vasculature in chronic adult periodontitis: a qualitative and quantitative study.
J Periodontol
68:
50-58,
1997[ISI][Medline].
6.
Bordin, S,
Page RC,
and
Narayanan AS.
Heterogeneity of normal human diploid fibroblasts: isolation and characterization of one phenotype.
Science
223:
171-173,
1984[ISI][Medline].
7.
Das, M,
Bouchey DM,
Moore MJ,
Hopkins DC,
Nemenoff RA,
and
Stenmark KR.
Hypoxia-induced proliferative response of vascular adventitial fibroblasts is dependent on G-protein-mediated activation of mitogen-activated protein kinases.
J Biol Chem
276:
15631-15640,
2001
8.
Das, M,
Dempsey EC,
Bouchey D,
Reyland MR,
and
Stenmark KR.
Chronic hypoxia induces exaggerated growth responses in pulmonary artery adventitial fibroblasts: potential contribution of specific protein kinase C isozymes.
Am J Respir Cell Mol Biol
22:
15-25,
2000
8a.
Das, M,
Dempsey EC,
and
Stenmark KR.
Selective subpopulations of pulmonary artery adventitial fibroblasts exhibit unique proliferative and apoptotic capabilities (Abstract).
FASEB J
12:
A339,
1998[ISI].
8b.
Das, M,
Dempsey EC,
and
Stenmark KR.
Proliferation and apoptosis contribute to adventitial changes during hypoxia-induced pulmonary hypertension (Abstract).
Am J Respir Crit Care Med
159:
A194,
1999.
8c.
Das, M,
Stenmark KR,
and
Dempsey EC.
Hypoxia stimulates growth of selective subsets of pulmonary artery adventitial fibroblasts (Abstract).
FASEB J
11:
A557,
1997.
9.
Das, M,
Stenmark KR,
and
Dempsey EC.
Enhanced growth of fetal and neonatal pulmonary artery adventitial fibroblasts is dependent on protein kinase C.
Am J Physiol Lung Cell Mol Physiol
269:
L660-L667,
1995
10.
Das, M,
Stenmark KR,
Ruff LJ,
and
Dempsey EC.
Selected isozymes of PKC contribute to augmented growth of fetal and neonatal bovine PA adventitial fibroblasts.
Am J Physiol Lung Cell Mol Physiol
273:
L1276-L1284,
1997
11.
Derdak, S,
Penney DP,
Keng P,
Felch ME,
Brown D,
and
Phipps RP.
Differential collagen and fibronectin production by Thy 1+ and Thy 1 lung fibroblast subpopulations.
Am J Physiol Lung Cell Mol Physiol
263:
L283-L290,
1992
12.
Desmouliere, A,
Rubbia-Brandt L,
Abdiu A,
Walz T,
Macieira-Coelho A,
and
Gabbiani G.
-Smooth muscle actin is expressed in a subpopulation of cultured and cloned fibroblasts and is modulated by
-interferon.
Exp Cell Res
201:
64-73,
1992[ISI][Medline].
13.
Falanga, V,
Zhou LH,
Takagi H,
Murata H,
Ochoa S,
Martin TA,
and
Telfman T.
Human dermal fibroblast clones derived from single cells are heterogeneous in the production of mRNAs for alpha 1 (I) procollagen and transforming growth factor-beta 1.
J Invest Dermatol
105:
27-31,
1995[Abstract].
14.
Frid, MG,
Aldashev AA,
Dempsey EC,
and
Stenmark KR.
Smooth muscle cells isolated from discrete compartments of the mature vascular media exhibit unique phenotypes and distinct growth capabilities.
Circ Res
81:
940-952,
1997
15.
Fries, KM,
Blieden T,
Looney RJ,
Sempowski GD,
Silvera MR,
Willis RA,
and
Phipps RP.
Evidence of fibroblast heterogeneity and the role of fibroblast subpopulations in fibrosis.
Clin Immunol Immunopathol
72:
283-292,
1994[ISI][Medline].
16.
Goldring, SR,
Stephenson ML,
Downie E,
Krane SM,
and
Korn JH.
Heterogeneity in hormone responses and patterns of collagen synthesis in cloned dermal fibroblasts.
J Clin Invest
85:
798-803,
1990[ISI][Medline].
17.
Haase, I,
Hobbs RM,
Romero MR,
Broad S,
and
Watt FM.
A role for mitogen-activated protein kinase activation by integrins in the pathogenesis of psoriasis.
J Clin Invest
108:
527-536,
2001
18.
Hakkinen, L,
and
Larjava H.
Characterization of fibroblast clones from periodontal granulation tissue in vitro.
J Dent Res
71:
1901-1907,
1992[Abstract].
19.
Hou, LT,
and
Yaeger JA.
Cloning and characterization of human gingival and periodontal ligament fibroblasts.
J Periodontol
64:
1209-1218,
1993[ISI][Medline].
20.
Irwin, CR,
Picardo M,
Ellis I,
Sloan P,
Grey AM,
McGurk M,
and
Schor SL.
Inter- and intra-site heterogeneity in the expression of fetal-like phenotypic characteristics by gingival fibroblasts: potential significance for wound healing.
J Cell Sci
107:
1333-1346,
1994
21.
Jelaska, A,
Arakawa M,
Broketa G,
and
Korn JH.
Heterogeneity of collagen synthesis in normal and systemic sclerosis skin fibroblasts.
Arthritis Rheum
39:
1338-1346,
1996[ISI][Medline].
22.
Jelaska, A,
and
Korn JH.
Anti-Fas induces apoptosis and proliferation in human dermal fibroblasts: differences between foreskin and adult fibroblasts.
J Cell Physiol
175:
19-29,
1998[ISI][Medline].
23.
Jones, R,
and
Reid L.
Vascular remodelling in clinical and experimental pulmonary hypertensions.
In: Pulmonary Vascular Remodelling, edited by Bishop JE,
Reeves JT,
and Laurent GJ.. London: Portland, 1995, p. 47-115.
24.
Jordana, M,
Schulman J,
McSharry C,
Irving LB,
Newhouse MT,
Jordana G,
and
Gauldie J.
Heterogeneous proliferative characteristics of human adult lung fibroblast lines and clonally derived fibroblasts from control and fibrotic tissue.
Am Rev Respir Dis
137:
579-584,
1988[ISI][Medline].
25.
Kawamoto, S,
and
Adelstein RS.
Characterization of myosin heavy chains in cultured aorta smooth muscle cells.
J Biol Chem
262:
7282-7288,
1987
26.
Koumas, L,
King AE,
Critchley HOD,
Kelly RW,
and
Phipps RP.
Existence of functionally distinct Thy 1+ and Thy 1 human female reproductive tract fibroblasts.
Am J Pathol
159:
925-935,
2001
27.
Lemire, JM,
Covin CW,
White S,
Giachelli CM,
and
Schwartz SM.
Characterization of cloned aortic smooth muscle cells from young rats.
Am J Pathol
144:
1068-1081,
1994[Abstract].
28.
Li, G,
Chen S-J,
Oparil S,
Chen Y-F,
and
Thompson JA.
Direct in vivo evidence demonstrating neointimal migration of adventitial fibroblasts after balloon injury of rat carotid arteries.
Circulation
101:
1362-1365,
2000
29.
Maksvytis, HJ,
Niles RM,
Simanovsky L,
Minassian IA,
Richardson LL,
Hamosh M,
Hamosh P,
and
Brody JS.
In vitro characteristics of the lipid-filled interstitial cell associated with postnatal lung growth: evidence for fibroblast heterogeneity.
J Cell Physiol
118:
113-123,
1984[ISI][Medline].
30.
Meyrick, B,
and
Reid L.
Hypoxia and incorporation of [3H]-thymidine by cells of the rat pulmonary arteries and alveolar wall.
Am J Pathol
96:
51-70,
1979[Abstract].
31.
Murphy, JD,
Rabinovitch M,
Goldstein JD,
and
Reid LM.
The structural basis of persistent pulmonary hypertension of the newborn infant.
J Pediatr
98:
962-967,
1981[ISI][Medline].
32.
Panchenko, MV,
Farber HW,
and
Korn JH.
Induction of heme oxygenase-1 by hypoxia and free radicals in human dermal fibroblasts.
Am J Physiol Cell Physiol
278:
C92-C101,
2000
33.
Patterson, MK, Jr.
Measurement of growth and viability of cells in culture.
In: Methods in Enzymology, edited by Jakoby WB,
and Pastan IH.. New York: Academic, 1979, vol. 58, p. 141-152.
34.
Phipps, RP,
Penney DP,
Keng P,
Quill H,
Paxhia A,
Derdak S,
and
Felch ME.
Characterization of two major populations of lung fibroblasts: distinguishing morphology and discordant display of Thy 1 and class II MHC.
Am J Respir Cell Mol Biol
1:
65-74,
1989[ISI][Medline].
35.
Raghu, G,
Chen Y,
Rusch V,
and
Rabinovitch PS.
Differential proliferation of fibroblasts cultured from normal and fibrotic human lungs.
Am Rev Respir Dis
138:
703-708,
1988[ISI][Medline].
36.
Rodemann, HP,
Bayreuther K,
Francz PI,
Dittmann K,
and
Albiez M.
Selective enrichment and biochemical characterization of seven human skin fibroblast cell types in vitro.
Exp Cell Res
180:
84-93,
1989[ISI][Medline].
37.
Rodemann, HP,
Muller GA,
Knecht A,
Norman JT,
and
Fine LG.
Fibroblasts of rabbit kidney in culture. I. Characterization and identification of cell-specific markers.
Am J Physiol Renal Fluid Electrolyte Physiol
261:
F283-F291,
1991
38.
Rosenberg, RD.
Vascular-bed-specific hemostasis and hypercoagulable states: clinical utility of activation peptide assays in predicting thrombotic events in different clinical populations.
Thromb Haemost
86:
41-50,
2001[ISI][Medline].
39.
Schor, SL,
and
Schor AM.
Clonal heterogeneity in fibroblast phenotype: implications for the control of epithelial-mesenchymal interactions.
Bioessays
7:
200-204,
1987[ISI][Medline].
40.
Sempowski, GD,
Borrello MA,
Blieden TM,
Barth RK,
and
Phipps RP.
Fibroblast heterogeneity in the healing wound.
Wound Repair Regen
3:
120-131,
1995.
41.
Shi, Y,
O'Brien J,
Fard A,
Mannion JD,
Wang D,
and
Zalewski A.
Adventitial myofibroblasts contribute to neointimal formation in injured porcine coronary arteries.
Circulation
94:
1655-1664,
1996
42.
Siu, GJ,
Liu YH,
Chang XS,
Anand IS,
Harris E,
Harris P,
and
Heath D.
Subacute infantile mountain sickness.
J Pathol
155:
61-70,
1988[ISI][Medline].
43.
Smith, TJ,
Sempowski GD,
Wang HS,
Vecchio PJD,
Lippe SD,
and
Phipps RP.
Evidence for cellular heterogeneity in primary cultures of human orbital fibroblasts.
J Clin Endocrinol Metab
80:
2620-2625,
1995[Abstract].
44.
Steinbrech, DS,
Longaker MT,
Mehrara BJ,
Saadeh PB,
Chin GS,
Gerrets RP,
Chau DC,
Rowe NM,
and
Gittes GK.
Fibroblast response to hypoxia: the relationship between angiogenesis and matrix regulation.
J Surg Res
84:
127-133,
1999[ISI][Medline].
45.
Stenmark, KR,
Fasules J,
Voelkel NF,
Henson J,
Tucker A,
Wilson H,
and
Reeves JT.
Severe pulmonary hypertension and arterial adventitial changes in newborn calves at 4300 m.
J Appl Physiol
62:
821-830,
1987
46.
Stenmark, KR,
and
Mecham RP.
Cellular and molecular mechanisms of pulmonary vascular remodeling.
Annu Rev Physiol
59:
89-144,
1997[ISI][Medline].
47.
Tokuda, Y,
Crane S,
Yamaguchi Y,
Zhou L,
and
Falanga V.
The levels and kinetics of oxygen tension detectable at the surface of human dermal fibroblast cultures.
J Cell Physiol
182:
414-420,
2000[ISI][Medline].
48.
Torry, DJ,
Richards CD,
Podor TJ,
and
Gauldie J.
Anchorage-independent colony growth of pulmonary fibroblasts derived from fibrotic human lung tissue.
J Clin Invest
93:
1525-1532,
1994[ISI][Medline].
49.
Trojanowska, M,
LeRoy EC,
Eckes B,
and
Krieg T.
Pathogenesis of fibrosis: type I collagen and the skin.
J Mol Med
76:
266-274,
1998[ISI][Medline].
50.
Welsh, DJ,
Scott P,
Plevin R,
Wadsworth R,
and
Peacock AJ.
Hypoxia enhances cellular proliferation and inositol 1, 4, 5-triphosphate generation in fibroblasts from bovine pulmonary artery but not from mesenteric artery.
Am J Respir Crit Care Med
158:
1757-1762,
1998
51.
Whiteside, TL,
Ferrarini M,
Hebda P,
and
Buckingham RB.
Heterogeneous synthetic phenotype of cloned scleroderma fibroblasts may be due to aberrant regulation in the synthesis of connective tissues.
Arthritis Rheum
31:
1221-1229,
1988[ISI][Medline].