Introduction

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Hypoxia-induced pulmonary hypertension complicates the clinical course of many important pulmonary diseases in children and adults (1). The pulmonary hypertension and accompanying structural remodeling is particularly severe in infants (2). In this regard, it is important to note that the earliest and most dramatic structural changes after hypoxic exposure are found in the adventitial compartment of the vessel wall (5, 6). In animal models, the resident adventitial fibroblasts have been shown to exhibit early (24 h) and sustained increases in proliferation and to exhibit dramatic increases in extracellular matrix protein synthesis (7). These fibroproliferative changes are ultimately associated with luminal narrowing and a progressive decrease in the ability of the vessel wall to respond to vasodilating stimuli (3). However, the mechanisms contributing to the excessive fibroproliferative response in the pulmonary artery adventitia under conditions of chronic hypoxia remain poorly understood.

An expanding body of experimental observations supports the concept that significant changes in the phenotypic properties of resident fibroblasts can occur in fibroproliferative states and might contribute significantly to the generation and maintainence of the fibroproliferative response. For example, stable increases in the proliferative capacity of mesenchymal cells have been observed in the affected organs of patients with atherosclerosis, progressive systemic sclerosis, idiopathic pulmonary fibrosis, interstitial renal fibrosis, and hepatic fibrosis (10). In animal models of lung and renal fibrosis, increased proliferative capacity of fibroblasts isolated from fibrotic tissue has also been demonstrated (10, 15). The phenotype has been observed to be stable for many population doublings, suggesting significant and persistent changes that become intrinsic to the cell itself (i.e., not dependent on changes in the local environment from which the cell was isolated). Little work has been done to evaluate changes in the signaling pathways that confer enhanced proliferative capabilities on fibroblasts from fibrotic organs.

We have recently shown that significant changes in protein kinase C (PKC) isozyme expression occurs in fibroblasts during normal vascular development and that specific isozymes contribute to the augmented proliferative capacity of fibroblasts from immature animals (17). Further, unique and stable changes in the proliferative potential of pulmonary artery (PA) smooth-muscle cells isolated from the hypertensive vessel wall have been shown to be associated with significant changes in PKC activity and isozyme expression (18, 19). Unique tyrosine phosphorylation patterns in response to acidic fibroblast growth factor (FGF) have been observed in fibroblasts isolated from humans with autosomal dominant polycystic kidney disease (20). Thus, changes in signaling pathways could contribute to "acquired" changes in growth potential. We therefore sought to investigate the possibility that fibroblasts isolated from the adventitia of neonatal bovine animals with severe hypoxia-induced pulmonary hypertension would demonstrate enhanced proliferative capabilities to both peptide mitogens as well as to hypoxia. Additionally, we sought to investigate a potential signaling pathway that might contribute to augmented proliferative capabilities of the cells. On the basis of our previous studies demonstrating a role for PKC isozymes as important contributors to the enhanced growth of mesenchymal cells during development as well as in response to hypoxia (17, 19), we elected to examine the role of PKC isozymes in mediating augmented growth of fibroblasts. We demonstrated that fibroblasts isolated from animals with severe fibroproliferative changes exhibit augmented growth potential. Further, a unique and synergistic interaction between the growth-promoting effects of hypoxia and peptide mitogens was documented in these fibroblasts in vitro. The augmented proliferative characteristics of fibroblasts isolated from the PA of calves chronically exposed to hypoxia for 2 wk beginning on Day 1 of life (Neo-Hyp) were associated with activation of PKC-I and - isozymes, suggesting that changes in the activity of specific PKC isozymes may contribute to the enhanced growth properties of fibroblasts isolated from the diseased vessel wall.

	Materials and Methods

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Materials

Human platelet-derived growth factor (PDGF)-BB, basic FGF (bFGF), and insulin-like growth factor (IGF)-I were purchased from Bachem California, Inc. (Torrance, CA) and suspended in Eagle's minimum essential medium (MEM) with 2% fatty acid-free bovine serum albumin (BSA). MEM, BSA, trypsin-ethylenediaminetetraacetic acid (EDTA) 10× suspension, penicillin, streptomycin, and amphotericin B were from Sigma Chemical (St. Louis, MO). Fetal bovine serum (FBS) was purchased from Gemini Bio-Products, Inc. (Calabasas, CA). Leupeptin, aprotinin, phenylmethylsulfonyl fluoride, sodium fluoride, calpain inhibitor I, calpastatin, pepstatin, protein A sepharose, histone HI, and mercaptoethanol were also from Sigma. [³²P]-adenosine triphosphate (ATP) was purchased from New England Nuclear (Boston, MA). Anti-PKC-I and PKC-µ antibodies and horseradish peroxidase (HRP)-conjugated goat antirabbit immunoglobulin G were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PKC- antibody was purchased from both GIBCO BRL (Gaithersburg, MD) and Santa Cruz Biotechnology. Molecular weight markers and nitrocellulose membranes were from GIBCO BRL and Bio-Rad Laboratories (Richmond, CA), respectively. Enhanced chemiluminescence detection kits were from Amersham Corp. (Arlington Heights, IL). Reagents for protein determination were purchased from Bio-Rad Laboratories. Phorbol 12-myristate 13-acetate (PMA), Ro31-8220, and GF109203X were obtained from LC Services (Waltham, MA). All were dissolved in dimethylsulfoxide (DMSO) and diluted to working concentrations in phosphate-buffered saline (PBS). [³H]thymidine was from ICN Biochemicals (Irvine, CA).

Isolation and Growth of PA Adventitial Fibroblasts

PA adventitia were harvested from neonatal calves. The calves were exposed to either normoxia (Neo-C) or hypoxia (Neo-Hyp) (simulated altitude of 4,570 m in a hypobaric chamber) for 14 d beginning 1 d after birth as previously described (6). Fibroblasts were isolated, grown, and characterized according to the previously described method (21). All cells were maintained in MEM, pH 7.4, supplemented with 10% serum, 100 U/ml penicillin, and 0.1 mg/ ml streptomycin, and incubated in a humidified atmosphere with 5% CO₂ at 37°C. Medium was changed twice weekly and cells were harvested with trypsin (0.2 g/liter)- EDTA (0.5 g/liter). Early passage (passages 1-6) cells were used. The growth characteristics and light microscopic appearance of the cells were unchanged up to passage 6.

Proliferation in the Presence and Absence of Serum

Serum-stimulated growth of PA adventitial fibroblasts was measured as previously described (21). For these measurements, PA adventitial fibroblasts from Neo-C and Neo-Hyp calves were seeded at the same density (5 × 10³ cells/ cm²) in MEM-10% serum in 24-well plates, and cell counts were performed on alternate days between Days 0 and 10. Media were supplemented, but not replaced, with fresh 10% serum-containing media on Days 4 and 8 to avoid blunting the growth of the more rapidly proliferating cells (21). To detect changes in cell number, the cells were trypsinized for 10 min, gently triturated after addition of an equal volume of MEM-10% serum, and counted with a standard hemocytometer. Data are expressed as cell numbers × 10³/well.

To test whether there is a difference in the growth rate of PA adventitial fibroblasts isolated from Neo-C and Neo-Hyp calves in the absence of serum, cells (7.5 × 10³/ cm²) were seeded in MEM-10% serum and allowed to attach overnight. The medium was changed with MEM- 0.1% serum after 24 h and [³H]thymidine incorporation and cell numbers were measured after every 24 h up to Day 7.

Growth of Quiescent Cells after Restimulation with Serum

To test whether fibroblasts isolated from Neo-C and Neo-Hyp calves retained the differences in proliferative capabilities after several days of growth arrest, cells were seeded in 10% serum/MEM. On Day 1, the cells were growth-arrested with medium containing 0.1% serum. At the end of 7 d of quiescence, the cells were restimulated with 10% serum. At 24 and 48 h after the addition of the serum, the cell counts were performed.

Response to Hypoxia

To test whether cells from Neo-C and Neo-Hyp calves have the ability to replicate in response to low oxygen concentration, cells were seeded sparsely (250 cells/cm²) in 10% FBS/MEM and allowed to attach overnight. The effects of different oxygen concentrations on the proliferation of mammalian fibroblasts have been shown to be dependent on cell density (22). Cultures plated between 50 and 250 cells/cm² had a reproducible growth pattern that is consistently sensitive to different oxygen concentrations. From Day 1, sparsely seeded fibroblasts were exposed to normoxia (20% oxygen) or hypoxia (3% oxygen) in the presence of serum as previously described (23). The hypoxic chambers were re-purged with these gas mixtures every 24 h for up to 11 d. At the end of the experiment, cells were trypsinized and counted. Results are expressed as cell counts × 10³/well.

Responses to Peptide Mitogens

Neo-C and Neo-Hyp PA adventitial fibroblasts were seeded in MEM-10% serum at 7.5 × 10³/cm², grown for 1 d, and then growth-arrested for 72 h with MEM-0.1% serum. DNA synthesis in response to purified peptide mitogens, 30 ng/ml PDGF-BB, 100 ng/ml IGF-I, and 40 ng/ml bFGF, was measured under serum-free conditions as previously described (21).

To test whether there are differences in the synergestic effects of hypoxia and mitogens, PDGF-BB, IGF-I, bFGF, and [³H]thymidine were added to the quiescent Neo-C and Neo-Hyp cells under serum-free conditions and incubated at 37°C for 30 min. Cells were then treated with normoxia (20% O₂) and hypoxia (3% O₂) for 24 h. At the end of 24 h, cells were harvested for counting thymidine incorporation according to a previously described method (21).

Application of PKC Antagonist Strategies

Normoxic growth. Adventitial fibroblasts from Neo-C and Neo-Hyp calves were seeded at a density of 5 × 10³/cm² in MEM-10% serum as previously described. After 24 h, PKC antagonists Ro31-8220 (3 µm), PMA (1 µm), and GF109203X (3 µm) were applied to the cells. Cell counts were obtained at 0 h of addition of test conditions to confirm equal cell numbers in each group, and at 4 d after addition of these compounds. Ro31-8220 and GF109203X were re-added to the cells on Day 3. PMA was left in for the duration of the growth assay and re-added after 2 d to prevent a reversal of the downregulated state of PKC as previously described (17).

Hypoxic growth. To detect the role of PKC in the hypoxia-induced augmented growth of cells, Neo-C and Neo-Hyp fibroblasts were seeded sparsely (250 cells/cm²) in 10% FBS/MEM. On Day 1, Ro31-8220 (2 µm) and PMA (1 µm) were added to the culture media. Cells were incubated at 37°C for 30 min and then exposed to 20% or 3% oxygen for 6 d. The Belcro chambers were refilled with fresh gas mixtures every 24 h. At the end of the experiment, cells were trypsinized and counted with a hemocytometer. Results are expressed as cell counts × 10³/well.

Immunoblot Analysis of PKC Isozymes

To detect differences in protein levels of PKC isozymes between the two cell types, Neo-C and Neo-Hyp PA adventitial fibroblasts (6.6 to 26.6 × 10³/cm²) were plated in 15 ml of medium containing 10% serum in T-75 flasks. Cells were grown for 4 to 5 d in the presence of serum. Total cell lysates were prepared according to a previously described method (17). For analysis of PKC isozyme expression, 20 µg of protein were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane, stained with ponceau S to confirm equal protein loading for both cell types, and blocked in 5% dry milk/PBS/0.05% Tween 20 for 2 h at room temperature. Nitrocellulose membranes were incubated with PKC isozyme-specific antibodies directed against I, , and µ PKC. Immune complexes were detected using HRP-conjugated secondary antibody and enhanced chemiluminescence as described previously (17). Protein levels were quantitated by densitometry (NIH Image Software; NIH, Bethesda, MD). Data are expressed as percentages of the Neo-C value.

Immunokinase Assay of PKC Isozymes

To test whether hypoxia can stimulate PKC isozyme activity, PA adventitial fibroblasts from both Neo-C and Neo-Hyp calves were plated at the density of 12.5 × 10³/cm² in 10 ml of 10% FBS/MEM in 100 mm petri dishes. After 24 h, the medium was changed to 0.1% FBS/MEM and the cells were growth-arrested for 72 h. At the end of 72 h, the medium was changed again to fresh MEM sufficient to cover the monolayer. The cells were then exposed to hypoxia as described earlier and lysed after different lengths of time according to the previously described method (17). Cell lysates (250 to 500 µg protein) were incubated with 2 µg PKC-I or - antibodies at 4°C overnight. Immune complexes were collected by incubation with protein A-Sepharose beads for 1 h at 4°C. The beads were harvested by centrifugation and washed three times with kinase assay buffer (40 mM Tris [pH 7.4], 20 mM MgCl₂, 20 µm cold ATP, and 2.5 mM CaCl₂) to remove nonspecifically bound proteins. Immunoprecipitated proteins were resuspended in kinase assay buffer and incubated with 50 µg/ml phosphatidylserine, 4 µm dioleoylglycerol, 5 µCi [³²P] -ATP, and 400 µg/ml histone HI for 10 min at 30°C. The reaction was terminated by adding SDS sample buffer and samples were boiled for 4 min at 95°C. The samples were analyzed by SDS-PAGE and visualized by autoradiography after drying.

Data Analysis

All data are expressed as arithmetic means ± standard error (SE); n equals the number of replicate wells or flasks per test condition in representative experiments. One-way and two-way analyses of variance followed by the Student-Newman-Keuls multiple comparison test were used for individual comparisons within and between groups of data points. Data were considered significantly different if P < 0.05.

	Results

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Growth Responses of Neo-C and Neo-Hyp Cells under Serum-Stimulated and -Depleted Conditions

Dramatic fibroproliferative changes occur in the vessel wall of neonatal calves exposed to hypoxia for 14 d (6). Quantitative analysis of proliferating cells in the adventitia of the main pulmonary artery, assessed with antibodies against Ki-67, showed increases in fibroblast DNA synthesis, data consistent with our previous observations in large intralobar pulmonary arteries (24). To determine whether adventitial fibroblasts isolated from the main PA of calves with fibroproliferative changes exhibited augmented growth potential in vitro, we compared the proliferative response to a maximal serum stimulus in adventitial fibroblasts from 15-d-old neonatal control calves (Neo-C) with that in fibroblasts from 15-d-old neonatal calves exposed to hypobaric hypoxia for 14 d (Neo-Hyp). Fibroblasts isolated from Neo-C calves showed an increase in cell count by Day 3, and the count increased to a plateau by Day 7. When grown under similar conditions, fibroblasts isolated from Neo-Hyp calves had higher cell counts than did the cells from the control calves (Figure 1A). The higher growth rate of Neo-Hyp over Neo-C cells was apparent by Day 4 and the difference was maintained throughout the 10 d of observation. A significantly higher plateau density was observed in Neo-Hyp than in Neo-C fibroblasts (Neo-Hyp versus Neo-C: 417 ± 16 versus 206 ± 9.3 × 10³).

View larger version (15K): [in this window] [in a new window]   Figure 1.   Proliferative responses of PA adventitial fibroblasts from pulmonary hypertensive (Neo-Hyp) and control (Neo-C) calves. (A) Serum-stimulated Neo-Hyp fibroblasts proliferate faster and achieve higher plateau densities than do Neo-C fibroblasts. Cells were seeded at a low density (5 × 103 cells/cm2) and grown in MEM-10% serum. (Values are means ± SE for this and all subsequent figures; n = 4 replicate wells, * P < 0.05 compared with Day 0 result and matched Neo-C result at same time.) Similar results in both direction and degree were obtained in separate experiments with different cell populations from at least three separate animals. (B) Neo-Hyp cells have greater [3H]thymidine incorporation after serum withdrawal. Cells were seeded at a density of 7.5 × 103/cm2 in MEM-10% serum and allowed to grow overnight, and the medium was changed with 0.1% serum on Day 1 (* P < 0.05 compared with Neo-C fibroblasts). (C) Restimulation of quiescent fibroblasts with serum. After 7 d of serum withdrawal, growth-arrested cells were again stimulated with medium containing 10% serum. * P < 0.05 compared with Neo-C cells.

When placed in 0.1% serum, fibroblasts from the Neo-C calves exhibited relatively low rates of DNA synthesis after 1 d of serum deprivation, which then decreased by Day 2 to an extremely low nadir that was maintained for 7 d (Figure 1B). Under similar serum-deprived conditions, DNA synthesis (cpm/cell × 10³) in fibroblasts from Neo-Hyp calves was higher than that from the control calves on Day 1 (408 ± 26 versus 92 ± 9.5, respectively), Day 2 (145 ± 4.7 versus 12 ± 3.3), and Day 3 (68 ± 6.4 versus 5.5 ± 0.3). By Day 4 and thereafter, there was no difference between the two groups (Figure 1B). When cells that had been serum-deprived for 7 d were again placed in medium containing 10% serum, enhanced proliferative responses were observed in Neo-Hyp compared with Neo-C fibroblasts (Figure 1C). Therefore, in both the presence and absence of serum, adventitial fibroblasts isolated from Neo-Hyp calves exhibit a significantly higher growth potential than do fibroblasts isolated from Neo-C animals.

Proliferative Response of Neo-C and Neo-Hyp Fibroblasts to Hypoxia

To determine the in vitro effects of hypoxia on the proliferative capabilities of these two cell types, the growth rates under hypoxic conditions (3% O₂) in the presence and absence of serum were compared. When Neo-C fibroblasts were cultured in medium containing 10% serum at a low density (250 cells/cm²), we observed an increase in cell counts over a period of 11 d in 3% O₂ relative to 20% O₂ (Figure 2A). Neo-Hyp fibroblasts plated under same conditions showed a greater increase in cell growth in response to hypoxia (Figure 2A). The numbers of Neo-Hyp and Neo-C fibroblasts after 11 d of hypoxic exposure were 70.5 ± 15 × 10³ and 30.5 ± 4 × 10³, respectively.

View larger version (19K): [in this window] [in a new window]   Figure 2.   Proliferative response of PA fibroblasts under hypoxic conditions. (A) Neo-Hyp fibroblasts demonstrate augmented growth in response to 3% O2. Matched cell populations were seeded under very sparse conditions (250 cells/cm2). Cells were exposed to either normoxic (20% O2) or hypoxic (3% O2) gas for 30 min/d for up to 11 d. Dotted line and open squares represent Neo-C normoxia; dotted line and solid squares, Neo-C hypoxia; solid line and open circles, Neo-Hyp normoxia; solid line and solid circles, Neo-Hyp hypoxia. * P < 0.05 compared with Neo-C hypoxic results. (B) Hypoxia induces increased DNA synthesis in Neo-Hyp fibroblasts. A total of 7.5 × 103 cells/cm2 were plated, growth-arrested for 72 h with medium containing 0.1% serum, and then exposed to normoxia or hypoxia for 24 h (n = 4 replicate wells; * P < 0.05 compared with Neo-C hypoxic results). Similar results were reproduced with at least two other groups of matched cell populations.

We also wanted to determine whether hypoxia could stimulate adventitial fibroblast DNA synthesis in the absence of serum. We found that fibroblasts from Neo-Hyp calves had higher DNA synthesis not only under normoxic conditions but also under hypoxic conditions compared with the Neo-C fibroblasts (0.1% serum for 72 h and then exposure to 20% or 3% O₂ for 24 h) (Figure 2B). Thus, fibroblasts isolated from both Neo-C and Neo-Hyp calves exhibit a unique ability to grow in response to reduced oxygen tensions. However, the hypoxia-induced proliferative responses are greater for Neo-Hyp fibroblasts than for Neo-C fibroblasts, in both the presence and absence of serum.

Response to Peptide Mitogens in Neo-C and Neo-Hyp Fibroblasts

Changes in the local concentration of peptide growth factors such as PDGF, bFGF, and IGFs have been demonstrated in the setting of hypoxic pulmonary hypertension (1). To determine whether the Neo-Hyp fibroblasts exhibited increased responsiveness to locally important peptide mitogens, [³H]thymidine incorporation was measured in both Neo-C and Neo-Hyp fibroblasts after stimulation with PDGF-BB, IGF-I, and bFGF. For these experiments, the cells were growth-arrested before the addition of mitogens. When fibroblasts from Neo-Hyp calves were stimulated with purified mitogens under normoxic conditions, DNA synthesis was greater in response to each of these mitogens than in Neo-C cells (Figure 3). When Neo-C cells were exposed to the mitogens and then grown in hypoxic conditions (3% O₂), we observed an increase in DNA synthesis that was not statistically significant compared with the normoxic response. In contrast, we found that when quiescent Neo-Hyp cells were stimulated with the previously mentioned mitogens and then exposed to hypoxia for 24 h, marked increases in thymidine incorporation in response to all three peptide mitogens were observed compared with the responses seen under normoxic conditions (Figure 3). Therefore, Neo-Hyp fibroblasts exhibit an augmented growth response to peptide mitogens under hypoxic conditions that is not seen in Neo-C cells.

View larger version (14K): [in this window] [in a new window]   Figure 3.   Hypoxic pulmonary hypertension-induced change in responsiveness of bovine adventitial fibroblasts to peptide mitogens. (A) Neo-Hyp fibroblasts have higher DNA synthesis in response to PDGF-BB under both normoxic and hypoxic conditions. PDGF-BB was applied at 30 ng/ml. (B) [3H]thymidine incorporation after stimulation with IGF-I under both 20% and 3% O2 pressure is greater in Neo-Hyp than Neo-C fibroblasts. IGF-I was applied at 100 ng/ml. (C) bFGF (40 ng/ml) induces greater increase in DNA synthesis under both normoxic and hypoxic conditions in Neo-Hyp than Neo-C fibroblasts. For all three panels, the cells were seeded at the density of 7.5 × 103/cm2 and growth-arrested for 72 h (n = 4 replicate wells; * P < 0.05 compared with Neo-C result, ** P < 0.05 compared with Neo-C and Neo-Hyp under normoxic condition results). Similar results were reproduced in at least two independent groups of matched cell populations.

PKC Antagonists Attenuate Serum-Stimulated Growth of Neo-C and Neo-Hyp Fibroblasts

Because developmental differences in growth of PA adventitial fibroblasts and smooth-muscle cells (SMCs) have been correlated with differences in susceptibility to different PKC antagonists and differences in PKC activity (17, 21), we sought to determine whether the enhanced growth properties of Neo-Hyp fibroblasts are mediated through the PKC signaling pathway. We began by comparing the susceptibility of the two cell types to growth inhibition by a PKC inhibitor (Ro31-8220) which is thought to inhibit all PKC isozymes. Because similar patterns in the growth differences between Neo-C and Neo-Hyp fibroblasts in normoxia and hypoxia were detected in both the presence and absence of serum, the studies with PKC antagonists were performed only in the presence of serum. Further, because apoptotic effects of PKC inhibitors on cells in serum- deprived conditions have been reported (25), this experimental design ensured that we were evaluating antiproliferative and not apoptotic effects (18). We found that the growth of both Neo-C and Neo-Hyp fibroblasts was inhibited by Ro31-8220 (Figure 4A). The cell counts for Neo-C and Neo-Hyp cells after 4 d of treatment with 3 µm Ro31-8220 were decreased by 87 and 67%, respectively. Another PKC antagonist, GF109203X, a structural analogue of Ro31-8220, was also used to examine the role of PKC in the augmented growth of Neo-Hyp cells (Figure 4B). GF109203X (3 µm) has been shown to inhibit all the PKC isozymes with relative specificity for the classical isozymes (26). The proliferation of both cell types in response to maximum serum stimulus was blocked by this PKC inhibitor.

View larger version (20K): [in this window] [in a new window]   View larger version (22K): [in this window] [in a new window]   Figure 4.   Hypoxia-induced proliferative response of bovine PA fibroblasts is dependent on PKC. (A) The normoxic serum-stimulated growth of both Neo-C and Neo-Hyp cells is susceptible to the inhibitory effects of Ro31-8220. Cells were seeded at the density of 5 × 103/cm2. Cell counts were performed 4 d after initial application (and 2 d after reapplication) of Ro31-8220 (3 µm). DMSO was used as vehicle for all test conditions. (B) Inhibition of normoxic serum-stimulated growth of Neo-Hyp fibroblasts by GF109203X also blocks the proliferation of fibroblasts isolated from both Neo-C and Neo-Hyp calves in response to serum and normoxic oxygen pressure. Cells were seeded at 5 × 103/cm2 in medium containing 10% serum. On Day 1, GF109203X (3 µm) was added to the media. The inhibitor was re-added after 2 d. Cell counts were performed 5 d after initial plating. (C) The hypoxia-induced augmented growth of both Neo-C and Neo-Hyp fibroblasts is also blocked by Ro31-8220. Cells were plated under very sparse conditions (250/cm2). Ro31-8220 (2 µm) was added to the cultures and cells were exposed to 20% and 3% O2 pressure for up to 6 d. The cells were exposed to a fresh gas mixture every 24 h. The normoxic results were consistent with the previous data under 20% O2 for both cell types with higher initial seeding densities (n = 4 replicate wells for panels A and B; * P < 0.05 compared with control data for each cell population). Similar results were reproduced in a minimum of two other groups of matched cell populations.

Because the growth-inhibitory effects of both PKC antagonists on Neo-Hyp and Neo-C cells followed a similar trend under normoxic conditions, only Ro31-8220 was used to evaluate the role of PKC in the hypoxia-induced growth of the cells. The enhanced growth capability of Neo-C and Neo-Hyp fibroblasts in the presence of 10% serum and hypoxia was also sensitive to the inhibitory effects of 2 µm Ro31-8220 (Figure 4C). Therefore, PKC signaling pathways appeared to be important in both serum-stimulated normoxic and hypoxic growth of Neo-C and Neo-Hyp fibroblasts.

Hypoxia-Induced Growth Is Associated with Activation of Specific PKC Isozymes

The PKC enzyme family consists of 11 distinct isozymes with different regulatory and biochemical properties (26). Because developmental differences in expression levels of Ca²⁺-dependent PKC- and -II have been linked to developmental differences in growth of PA adventitial fibroblasts (17), we sought to determine which PKC isozymes might have a role in the augmented growth properties of Neo-Hyp fibroblasts. We used a PKC downregulation strategy (prolonged treatment with 1 µm PMA), a method we used previously to implicate individual isozymes in growth responses of fibroblasts during development and one that has also been utilized by others (17, 27, 28). Our previous studies indicated that prolonged PMA treatment induced degradation of the four PKC isozymes , II, , and  , but not I, , and µ isozymes in PA fibroblasts (17). Therefore, if PMA downregulation-susceptible isozymes were involved in the enhanced proliferative responses, this strategy should decrease the proliferative response of the cell. If the effect of downregulation were to mimic the effects of Ro31-8220 and GF109203X, it would suggest that downregulation-sensitive PKC isozymes are required for the growth response. On the other hand, if the effect of downregulation did not mimic the effects of Ro31-8220 and GF109203X, downregulation-resistant PKC isozymes would be implicated in the growth response. This comparative strategy has been previously validated (17, 18).

We found that serum-stimulated normoxic growth of Neo-C fibroblasts was blocked by 50% after PKC downregulation with PMA (Figure 5A). In contrast, the growth of Neo-Hyp fibroblasts under normoxic conditions was not inhibited by PKC downregulation (Figure 5A). PKC downregulation also exerted inhibitory effects on enhanced growth of Neo-C cells in response to hypoxia and 10% serum. However, PKC downregulation by prolonged PMA treatment had no effect on hypoxia-induced augmented growth of Neo-Hyp cells in the presence of 10% serum (Figure 5B). Therefore, it appears that the downregulation-resistant classical PKC-I and atypical PKC- and -µ isozymes might be important in the augmented growth of Neo-Hyp fibroblasts under both normoxic and hypoxic conditions.

View larger version (34K): [in this window] [in a new window]   View larger version (29K): [in this window] [in a new window]   Figure 5.   Differential effects of phorbol ester-induced PKC downregulation on the growth of PA adventitial fibroblasts. (A) PKC downregulation induced by prolonged treatment with 1 µm PMA inhibits the serum-stimulated normoxic growth of Neo-C fibroblasts but not of Neo-Hyp fibroblasts. Cells were plated at a density of 5 × 103/cm2 in medium containing 10% serum and treated with 1 µm PMA. Changes in growth were then assessed over a period of 4 d. PMA was left in the media during this interval and re-added on Day 3 to maintain the downregulated state. (B) Under hypoxic conditions, PKC downregulation blocks the hypoxic growth only of Neo-C fibroblasts. Cells were seeded at 250/cm2 in 10% serum/MEM. The normoxic results for both cell types were consistent with the data under 20% O2 for the previously described experiment with higher initial seeding densities. For both panels, n = 4 replicate wells; * P < 0.05 compared with the control result of each cell population. Similar results were reproduced in a minimum of two other groups of matched cell populations.

PKC-I and - Protein Levels Are Higher in' Fibroblasts from Neo-Hyp Calves

To determine whether differences in PKC isozyme expression parallelled the differences in growth as predicted by the antagonist and downregulation strategies described earlier, downregulation-resistant PKC isozyme (I, , and µ) levels in matched Neo-C and Neo-Hyp cell lysates were compared. Because all experiments using PKC inhibitors were carried out in the presence of 10% serum, the lysates were prepared from serum-stimulated fibroblasts. We found a significantly higher expression level of the downregulation-resistant I isozyme of PKC in Neo-Hyp fibroblasts than in Neo-C fibroblasts in response to serum stimulation (Figure 6). Neo-Hyp cells also possessed higher levels of the PKC- isozyme than did Neo-C fibroblasts. No significant differences in the expression of the Ca²⁺-independent PKC-µ isozyme were observed between the two cell types (Figure 6). Therefore, higher levels of the downregulation-resistant PKC-I and - isozymes were observed in rapidly proliferating Neo-Hyp compared with Neo-C fibroblasts.

View larger version (22K): [in this window] [in a new window]   Figure 6.   Expression of PKC-I and - is higher in Neo-Hyp fibroblasts than in Neo-C cells. (A) Representative immunoblots for downregulation-resistant isozymes. A total of 20 µg of protein from Neo-C and Neo-Hyp PA adventitial fibroblasts was resolved by SDS-PAGE, transferred to nitrocellulose, then probed with anti-PKC-I, -, and -µ antibodies. (B) Quantitative analysis of expression pattern for each downregulation-resistant isozyme. Results are pooled from three separate experiments (n = 3). For each experiment, cells from different animals at each treatment were used. Values are expressed as percentages of the Neo-C value. * P < 0.05 compared with Neo-C result.

Increases in PKC-I and - Activity in Response to Hypoxia

To determine whether hypoxia can activate PKC-I and - isozymes in Neo-C and Neo-Hyp fibroblasts, and whether differences in the hypoxia-induced activation pattern correlate with the hypoxia-induced growth differences between the two cell types, we compared PKC-I and - isozyme-specific activity in hypoxia-exposed matched Neo-C and Neo-Hyp cell lysates. To eliminate the effects of exogenous growth factors, the cells were growth-arrested for 72 h with medium containing 0.1% serum and then exposed to either 20% or 3% O₂ for different lengths of time. Hypoxia induced an increase in PKC-I activity in both cell types after 1 h of hypoxic exposure (Figure 7A), however there were no differences in the activation pattern between the two cell types. Likewise, no differences in the basal activity of PKC-I was observed between quiescent Neo-C and Neo-Hyp fibroblasts.

View larger version (54K): [in this window] [in a new window]   Figure 7.   PKC-I- and --specific activities are enhanced in both Neo-C and Neo-Hyp fibroblasts in response to hypoxia. (A) Representative blots for PKC-I activity and PKC-I protein level in Neo-C and Neo-Hyp fibroblasts in response to hypoxia. Growth- arrested cells were exposed to hypoxia, cell lysates were prepared, and PKC isozyme was immunoprecipitated with a PKC-I-specific antibody. The immunoprecipitated proteins were incubated with histone HI substrate and [32P]-ATP. Phosphorylated histones were separated by SDS-gel electrophoresis. (B) Representative blots for PKC- activity and level in Neo-C and Neo-Hyp cells upon hypoxic exposure at different times after exposure to hypoxia. Similar results were obtained in six independent experiments.

In contrast to PKC-I, growth-arrested Neo-Hyp fibroblasts had 2- to 4-fold higher basal PKC- activity than did Neo-C cells (results from a total of six independent experiments) (Figure 7B). PKC- activity was increased in both cell types upon exposure to acute hypoxia. In the Neo-C fibroblasts, PKC- activity was induced by 2- to 4-fold at 10 min, 2-fold at 60 min, and 4-fold at 24 h (Figure 7B). The high basal PKC- activity of Neo-Hyp fibroblasts was further increased within 1 h of hypoxic exposure (Figure 7B). Therefore, hypoxia activates PKC-I and - isozymes in both Neo-C and Neo-Hyp fibroblasts, whereas the higher basal activity of PKC- correlates with a greater proliferative rate of Neo-Hyp cells compared with the fibroblasts isolated from control calves.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study we addressed the hypothesis that fibroblasts isolated from the pulmonary artery of neonatal animals with severe fibroproliferative changes would exhibit augmented proliferative capabilities compared with fibroblasts isolated from control animals. We found, on a very consistent basis (i.e., from three matched pairs of animals), that fibroblasts isolated from Neo-Hyp calves exhibited enhanced growth responses to serum, hypoxia, and purified peptide mitogens compared with those obtained from age-matched control animals (Neo-C). Importantly, we also observed a synergistic interaction between hypoxia and purified peptide mitogens on growth in fibroblasts isolated from the hypertensive animals that was not observed in fibroblasts from the control animals. These proliferative attributes persisted through numerous cell passages in culture, suggesting acquired differences in fibroblast populations which were not simply due to changes in the in vivo cellular milieu (i.e., growth factors, cytokines, or matrix components). In further support of this idea is the fact that in nearly all experiments paired sets of fibroblasts, which were obtained from control and hypertensive animals on the same day and which were subsequently handled identically, were used. We thus conclude that the fibroproliferative changes characteristic of hypoxic neonatal pulmonary hypertension are associated with significant changes in the proliferative phenotype of resident adventitial fibroblasts.

The finding of enhanced proliferative capability of fibroblasts isolated from the pulmonary artery of calves with hypoxia-induced fibroproliferative changes is consistent with previous studies which evaluated the growth potential of fibroblasts isolated from other organs exhibiting fibroproliferative changes (10, 21). Human lung fibroblasts from early and late stages of fibrosis have been demonstrated to exhibit different proliferative potential. Fibroblasts from early fibrotic lesions have a greater proliferative potential than do fibroblasts from normal lung, and both have greater proliferative potential than fibroblasts from tissue with longer-standing and dense fibrosis (10). Also, at a relatively early stage of the disease, fibroblasts from humans with active pulmonary fibrosis proliferate significantly faster than fibroblasts from those without fibrosis (11, 13). This observation holds for both parent cells and clonal populations from fibrotic and nonfibrotic tissues (11). Other studies have demonstrated that collagen synthesis of cells from fibrotic lung is elevated when compared with that from control cells (29). In the setting of acute lung injury, Chen and colleagues have demonstrated the existence of fibroblasts not only with enhanced proliferative potential but also with the ability to proliferate in the absence of exogenous peptide growth factors (12). This proliferative phenotype was shown to be stable for at least five to seven passages and did not appear to depend on autocrine release of trophic factors. Similar ex vivo stability of altered fibroblast phenotypic properties has been observed in fibroblasts from patients with progressive systemic sclerosis, renal interstitial fibrosis, autosomal polycystic kidney disease, hepatic fibrosis, and gingivitis (14, 20). In all cases, these attributes persist through numerous cell passages in culture. Thus, the results of the current study are consistent with the concept raised by us and others that in fibroproliferative diseases, stable alteration of mesenchymal cell phenotype can and does occur.

The augmented growth properties of Neo-Hyp fibroblasts in response to serum, purified mitogens, and hypoxia compared with Neo-C cells strongly suggest that there might be differences in the signaling pathways contributing to these responses. Indeed, in SMCs obtained from hypertensive animals, acquisition of new growth properties that include increased sensitivity to the effects of hypoxia have been documented (18). At least some of these effects are mediated through the PKC signaling pathway. Therefore, we were particularly interested in the possibility that members of the PKC enzyme family were playing an important role in conferring heightened growth potential on Neo-Hyp fibroblasts. In previous developmental studies, we reported that enhanced growth responses of immature compared with adult PA adventitial fibroblasts were dependent on the PKC signaling pathway (21). Specifically, the PKC- and -II isozymes were shown to participate in conferring augmented proliferative responses on immature PA adventitial fibroblast populations (17). Experiments in the present study using PKC inhibitors and downregulation strategies were consistent with a role for PKC- and -II in serum-stimulated growth of fibroblasts isolated from neonatal control animals. In addition, we found that the fibroblasts from the PA of chronically hypoxic animals, which demonstrated even greater proliferative capabilities than did normal neonatal fibroblasts, expressed higher levels of PKC-I (an alternative splice product of the same gene as PKC-II) and PKC-. These results suggest that there may be changes in the PKC isozyme profile that occur not only during development but also in response to chronic hypoxia. Interestingly, among the PKC genes, the PKC- gene and its gene products PKC-I and -II are subject to the most extensive regulation, with both tissue-specific and developmentally regulated expression as well as evolutionarily conserved mechanisms for transcriptional and post-transcriptional regulation (30, 31). Overexpression of PKC-I in vascular SMCs has been shown to increase the rate of cell proliferation and to accelerate the entry into the S phase of these cells (32). PKC- has also been implicated in the mitogenic response of nonvascular and vascular cells (33). PKC- is particularly abundant in fetal tissues and adult rat liver, an organ which retains rejuvenative capacity, consistent with the notion that PKC- plays a key role in cell proliferation (34). Thus, our observations are consistent with the ideas that changes in PKC isozyme distribution can occur in specific cell populations in response to chronic stimuli and that these changes can participate in conferring unique functional properties on the cells.

Previous studies have demonstrated that SMCs isolated from the pulmonary arteries of chronically hypoxic neonatal calves also demonstrate enhanced growth capabilities compared with SMCs from control animals. Questions therefore arose as to whether chronic hypoxia elicited similar changes in PKC isozyme profiles in all vascular wall cells exhibiting augmented growth capabilities. Our experiments demonstrate that the PKC isozymes used by adventitial fibroblasts from chronically hypoxic calves which contribute to serum and hypoxic growth are different than those utilized by the adjacent SMC population from the same animal. In SMCs the PKC- isozyme, rather than PKC-I or -, was found to be the major signaling mediator for the heightened growth properties (18). Thus, although chronic exposure to hypoxia induces increases in the growth capabilities of cells in both the medial and adventitial compartments, the mechanisms through which this is accomplished, at least with regard to utilization of specific PKC isozymes, is different.

The high basal activity of PKC- in serum-deprived "quiescent" fibroblasts from chronically hypoxic calves could contribute to the greater growth capabilities exhibited by these fibroblasts compared with those from control animals. High basal activities of PKC- in fibroblasts with high proliferative capabilities are consistent with recent observations in tumor cells where high expression of PKC- in tumor compared with nontumor cells has recently been shown to be associated with a higher proliferative capacity (35). The greater basal activity of PKC- of Neo-Hyp fibroblasts compared with Neo-C cells may also explain the lack of effect that PKC downregulation by prolonged treatment with phorbol esters had on the growth of Neo-Hyp fibroblasts. The PKC- isozyme is not stimulated by the usual activators of PKC (such as calcium, phorbol esters, or diacylglycerol) and thus is not susceptible to downregulation by phorbol ester. PKC- has been shown to be activated by phosphoinositol 3-kinase (36) and to play a pivotal role in cytokine-induced activation of nuclear factor (NF)-B, an inducible transcriptional activator that participates in the control of cell proliferation (37). Interestingly, hypoxia has been shown to activate NF-B and also to stimulate phosphoinositol 3-kinase (38, 39), observations consistent with our demonstration that hypoxia activates PKC- and stimulates proliferation. Therefore, adventitial fibroblasts in response to chronic hypoxia may acquire a unique proliferative phenotype through the alteration of PKC isozyme signaling intermediates.

Several possibilities must be considered in explaining how a stable phenotypic alteration of the adventitial fibroblast population might come about. First, a large proportion of resident fibroblasts are altered by changes that occur locally in the chronically hypoxic vascular wall. Hypoxia itself, a combination of hypoxia with the subsequent hemodynamic changes, and ultimately the combination of hypoxia, hemodynamics, and changes in the local concentrations of cytokines, growth factors, and matrix proteins must be considered. Resident fibroblasts could then be altered by these signals, conferring upon them a new, stable differentiated state that is manifested by intrinsically enhanced proliferative capacity. Another possibility is that selective expansion of a normally resident fibroblast population occurs in vivo in response to chronic stimulus, making it numerically the most abundant constituent of the activated or injured vessel wall. Thus, this population might demonstrate selective advantage when cell cultures are done. Distinguishing between these two possibilities in the current study is clearly impossible. Future work should be directed at examining the possibility that even seemingly morphologically homogenous populations of adventitial fibroblasts are actually composed of subpopulations that differ with respect to certain phenotypes.

Our findings that fibroblasts from the normal pulmonary arterial wall also exhibit a moderate increase in growth under hypoxic conditions are consistent with the observations of Peacock and colleagues (40) in bovine adventitial fibroblasts, and with other observations in skin fibroblasts (41). The finding that the effects of hypoxia were more marked in low-density cultures than in confluent cultures for both cell populations (Neo-C and Neo-Hyp) is also supported by observations that have been reported for cultured human skin, lung, and tendon fibroblasts (42). The mechanism underlying the cell density-dependent hypoxic response of the cells is not well understood. In contrast to fibroblasts, SMCs do not tolerate this low plating density. We also found consistently that hypoxia, in the absence of any exogenous mitogens, stimulates the proliferation of "quiescent" fibroblasts. However, we found that SMCs, whether from control or hypertensive animals, need priming or the presence of mitogens to exihibit proliferative responses under hypoxic conditions (23). Therefore, PA adventitial fibroblasts, whether from control or chronically hypoxic animals, compared with the SMCs possess unique signaling mechanisms that allow them to replicate in response to hypoxia in the absence of exogenous mitogens.

In conclusion, our findings support the idea that severe hypoxia-induced pulmonary hypertension is associated with a significant change in the proliferative phenotype of the resident fibroblast populations in the adventitia. Fibroblasts with enhanced growth responses to hypoxia and peptide mitogens, in this setting, provide a mechanism whereby excessive fibroproliferative changes can occur. Our findings suggest that the growth of fibroblasts from hypertensive animals is mediated at least in part through specific PKC isozymes. Controlling fibroproliferative responses must be aimed at first discovering the causes for the switch in phenotypic properties of fibroblasts; therapies must then be directed at cells whose growth responses to mitogenic stimuli are uniquely different from those observed in cells from uninjured vessel walls.