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ABSTRACT |
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INTRODUCTION
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Pulmonary hypertension occurs commonly in patients with chronic hypoxic lung disease and is characterized by the remodeling of the pulmonary artery walls. The molecular mechanisms underlying such remodeling are unknown but we have recently shown that the stress-activated (Jnk and p38) mitogen-activated protein (MAP) kinases are activated in pulmonary artery fibroblasts following acute hypoxia. We now show that Erk and p38 MAP kinases are constitutively activated in fibroblasts derived from the remodeled pulmonary, but not the systemic circulation from rats exposed to chronically hypoxic conditions. Moreover, we find that such fibroblasts show sustained enhanced proliferative capacities relative to pulmonary artery fibroblasts derived from normoxic rats or to aortic fibroblasts from either normoxic or hypoxic rats. Finally, abrogation of p38, but not Erk MAP kinase activity by use of specific inhibitors, prevents the enhanced proliferative capacity exhibited by pulmonary artery fibroblasts. Taken together, these data suggest that enhanced p38 MAP kinase activity provides a molecular mechanism to explain the proliferation of pulmonary artery fibroblasts required for remodeling of the pulmonary vasculature.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Pulmonary hypertension occurs commonly in patients with
chronic hypoxic lung disease and is characterized by the remodeling of the pulmonary artery walls by processes including hypertrophy and hyperplasia (1). The three major cell types comprising the pulmonary artery walls
fibroblasts, smooth muscle,
and endothelial cellsall proliferate at a higher rate when
maintained under conditions of hypoxia than when cultured under normoxic conditions (2). Such structural remodeling of the
pulmonary arteries, together with hypoxia-induced vasoconstriction and polycythemia (3), leads to the rise of pulmonary
artery pressure (PAP) observed in pulmonary hypertension.
Although hypoxia induces structural remodeling and vasoconstriction of the pulmonary arteries, the effects of hypoxia on systemic vascular cells are quite different (4). Indeed, because hypoxia does not result in vasoconstriction but rather induces vasodilation of systemic vessels, we have proposed that there are fundamental differences in oxygen sensing and cell signaling between systemic and pulmonary artery cells (5). We have focused on the role of vascular fibroblasts (4) because it is the matrix proteins generated by these cells in the media and adventitia in response to hypoxia that render the vessels indistensible by vasodilators, the consequence being a fixed state of pulmonary hypertension. Our previous studies have suggested that there may be a link between vasoconstriction and remodeling because, whereas acute hypoxia increases the rate of replication of bovine pulmonary artery fibroblasts, fibroblasts from the bovine systemic circulation do not show this enhanced cell proliferation (4).
The molecular mechanisms by which hypoxia stimulates proliferation of pulmonary artery fibroblasts, but not fibroblasts from the systemic circulation, are unknown. There is, however, considerable evidence in the literature that the mitogen-activated protein (MAP) kinases, Erk1 and Erk2, and the related stress-activated kinases, Jnk and p38 MAP kinase, which have been implicated as key regulators of cell proliferation, can be activated in response to hypoxic stress (6, 7). These MAP kinases, which are all activated by a common threonine-X-tyrosine regulatory motif by their distinct upstream dual specificity (thr/tyr) MAP kinase kinases (6), are an important group of serine/threonine signaling kinases. These modulate the phosphorylation, and hence, activation, status of transcription factors, and link transmembrane signaling with gene induction events in the nucleus (6). We have shown in our model of acute bovine pulmonary artery hypoxia that the stress-activated MAP kinase, p38, is activated in response to hypoxia and appears to contribute to the enhanced levels of proliferation exhibited by fibroblasts exposed to acute hypoxia (20).
In this current study we have investigated whether fibroblasts derived from the pulmonary and systemic circulations of chronically hypoxic rats in which irreversible pulmonary vascular remodeling has taken place (8) exhibit similar differential replicative properties. In addition, we hypothesized that pulmonary artery cells from chronically hypoxic rats would exhibit growth and signaling characteristics different from fibroblasts derived from control rats kept under normoxic but otherwise matched conditions. To test these hypotheses, we obtained pulmonary and aortic fibroblasts from normoxic and chronically hypoxic rats and carried out proliferation and cell-signaling studies under conditions of normoxia or acute hypoxia. In particular, we investigated the roles of Erk and stress-activated MAP kinases in hypoxia-induced pulmonary artery fibroblast proliferation and pulmonary vascular remodeling following chronic exposure to hypoxia.
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INTRODUCTION
METHODS
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DISCUSSION
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Materials
All reagents were of Analar grade and were obtained from Sigma (Poole, Dorset, UK) unless otherwise specified. [3H]Thymidine was purchased from DuPont (Stevenage, Hertfordshire, UK). All tissue culture flasks and media were obtained from Gibco (Paisley, Renfrewshire, UK). Fetal calf serum was obtained from Imperial Laboratories (Andover, Hants, UK). Rabbit polyclonal antibodies specific for the activated dual phosphorylated forms of the three MAP kinase family members (Erk1/Erk2, Jnk, and p38) and their appropriate control antibodies for MAP kinase expression were obtained from New England Biolabs (Hertfordshire, UK).
Chronic Hypoxic Rat Model of Pulmonary Hypertension
Pulmonary hypertensive rats were prepared (in the laboratory of M. MacLean) using the technique of MacLean and coworkers (9). Briefly, male Wistar rats (specific pathogen free, Harlan UK Ltd, 28- 30 d old at the start of the experiment, ~ 65 g) were placed in a hypobaric chamber. This was depressurized to 550 mbar (oxygen concentration reduced to 10%) over 2 d. The temperature in the chamber was maintained at 21-22° C and the chamber was ventilated with air at approximately 45 L/min. Rats were maintained in these hypoxic, hypobaric conditions for 2 wk and studied immediately after removal from the chamber. Age-matched control rats were maintained in room air. Pulmonary hypertension (PHT) was assessed by measuring the ratio of right ventricular (RV)/total ventricular (TV) weight. The right ventricle was carefully dissected free from the septum and left ventricle and both were blotted lightly and weighed. This is a reliable index of the degree of PHT in rats (8, 10). The right ventricular-to- total ventricular ratio was greater in the chronic hypoxic (CH) rats (0.352 ± 0.007 versus 0.247 ± 0.005, n = 12; p < 0.001) indicating a significant degree of pulmonary hypertension.
Primary Culture of Rat Pulmonary Artery Fibroblasts (RPAF) and Rat Aortic Fibroblasts (RAF)
Freshly excised rat lung and heart tissue was obtained as described above. The pulmonary artery was dissected free from the heart into the lungs and the whole section was cut longitudinally and opened into a flat sheet. Similarly, the aorta was dissected free from the heart and lungs. Fibroblasts were prepared using the technique of Freshney (11), with some modifications. Briefly, muscular tissue and endothelial cell layers were removed by gentle abrasion of the vessel. The remaining tissue (adventitia) was then dissected into 1-mm3 portions. Approximately 25 portions of tissue were evenly distributed over the base of a 25-cm2 culture flask containing 2 ml of Dulbecco's modified Eagle medium DMEM with 20% fetal calf serum (FCS), supplemented with penicillin/streptomycin (400 IU/ml and 400 µg/ml) and amphotericin B (5 µg/ml). The explants were incubated in a humidified atmosphere of 5% CO2 in air at 37° C. We have previously shown by staining for actin (12) that this technique provides a pure culture of fibroblasts (13). Cells were maintained in DMEM containing 10% FCS, supplemented with penicillin/streptomycin (200 IU/ml and 200 µg/ml) and L-glutamine (27 mg/ml) and used between passage 3 and 10.
Growth of Cells in a Hypoxic Environment
A humidified temperature-controlled incubator (LEEC model GA156; Colwick, Nottingham, UK) was used as a hypoxic chamber. This incubator allows control of internal oxygen levels between 0 and 21% while CO2 level is simultaneously controlled at 5%. For these experiments, primary cultures of RPAF or RAF cells derived from normoxic or chronically hypoxic rats were transferred to 24-well plates. From our previous work (14) we elected to use a tissue culture supernatant PO2 of 35 mm Hg, which could be obtained by maintaining an atmosphere of 2% oxygen. Our previous studies (4) have established that a 6-h preincubation under these conditions of hypoxia is required to achieve the desired PO2 levels (35 mm Hg) in the culture medium. Once established, we found the PO2 levels to remain constant at 35 mm Hg (4).
Proliferation Assay: DNA Synthesis as Measured by [3H]Thymidine Incorporation
To measure DNA replication as a measure of cellular proliferation, rat pulmonary and aortic fibroblast cells were grown to approximately 60% confluency in 24-well plates at 37° C and then serum starved for 24 h before culturing (under conditions of normoxia or acute hypoxia) in the presence or absence of 5% serum for a further 24 h. The fibroblasts were pulsed with [3H]thymidine (0.1 µCi/well) 4 h before the end of the 24 h of stimulation to allow estimation of DNA synthesis by incorporation of [3H]thymidine. At 24 h the medium was removed and the cells were washed twice with 0.5 ml phosphate-buffered saline (PBS). Washing with 5% trichloroacetic acid (TCA) precipitated cellular proteins and the lipid fractions were extracted by washing with 100% ethanol. The remaining cell contents were dissolved in 0.3 M NaOH. The contents of each well were transferred to scintillation vials, to which was added 5 ml of Ecosint A scintillation fluid. Vials were vortexed thoroughly before radioactive counts were measured by scintillation counter. Counts were measured in DPMs (disintegrations per minute).
Western Blot Analysis
Sample preparation. RPAF and RAF cells were grown to 90% confluency in 6-well plates and stimulated with 5% serum for the times indicated. Reactions were terminated by the addition of 2× ice-cold RIPA buffer (50 mM Tris [pH 7.4], 150 mM sodium chloride, 2% [vol/ vol] NP 40, 0.25% [wt/vol] sodium deoxycholate, 1 mM ethyleneglycoltetraacetic acid [EGTA], 10 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], chymostatin [10 µg/ml], leupeptin [10 µg/ml], antipain [10 µg/ml], and pepstatin A [10 µg/ml]) and kept on ice for 30 min to facilitate the extraction of cellular proteins. The samples were centrifuged at 12,000 × g for 15 min and the resulting supernatants containing the solubilized proteins were used for Western blot analysis. Protein content of the samples was determined by the Micro BCA Protein Reagent Kit (Pierce, Rockford, IL).
Western blotting. Samples (50 µg) were boiled in reducing loading buffer and electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolving gels under reducing conditions. The resolved proteins were transferred to PVDF (Millipore, Watford, UK) and blocked at room temperature with 10% nonfat dried milk in PBS/Tween 20 (0.1%) under constant agitation. The primary rabbit antidual phosphorylated, activated MAP kinase antibody was incubated with the blot for at least 1 h at room temperature. The blots were washed in PBS/Tween before incubating with sheep anti-rabbit immunoglobulin horseradish peroxidase (Ig HRP) (New England Biolabs, Beverly, MA) in 5% nonfat dried milk for 1 h with constant agitation. For each member of the MAP kinase family studied, activation was assessed by reactivity with the antiactive, dually phosphorylated antibody while expression was determined by an antibody that recognized both the active and inactive forms of the enzyme. The blots were thoroughly washed and then incubated with ECL reagent (Amersham Life Sciences, Buckinghamshire, UK) and exposed to film. Prestained molecular weight markers were used to calibrate the molecular weight range of the resolved proteins. Even protein loading/sample recovery from SDS-PAGE gels was corroborated by Ponceau Red staining (15).
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RESULTS |
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Differential Effects of Acute Hypoxia on DNA Synthesis of Pulmonary and Aortic Fibroblasts from Normoxic and Chronically Hypoxic Rats
Fibroblasts explanted from rat pulmonary arteries (RPAF) undergo DNA synthesis when cultured in the presence of 5% serum following serum starvation for 24 h (Figure 1A; p < 0.05). When such cells are simultaneously exposed to acute hypoxia, there is a substantial increase in the resultant level of DNA synthesis observed (Figure 1A). In contrast, fibroblasts explanted from the systemic rat aorta (RAF) do not exhibit an enhanced level of DNA synthesis in response to acute hypoxia (Figure 1B). Consistent with these differential effects of hypoxia on RPAF and RAF cells, RPAF cells from chronically hypoxic rats exhibited increased levels of DNA synthesis relative to control rats even under subsequent conditions of normoxia (Figures 1A and 1B). RAF cells showed similar levels of replication regardless of whether the explants were derived from normoxic or chronically hypoxic rats (Figure 1D). No further increase above the enhanced level of DNA synthesis observed in RPAF cells from chronically hypoxic rats could be achieved by exposing these cells to acutely hypoxic conditions (Figure 1C).
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Differential p38 and Erk MAP Kinase Activity in RPAF and RAF Cells from Normoxic and Chronically Hypoxic Rats
We have previously shown that exposure of bovine pulmonary artery fibroblasts to acute hypoxia induces the activation of the stress-activated protein kinases, Jnk and p38 MAP kinase. We therefore investigated whether these signal transducers played a role in the enhanced proliferation and remodeling of fibroblasts observed after prolonged (14 d) exposure to chronic hypoxia. To do this, we have examined the activation of the three major classes of MAP kinases by detecting their dually phosphorylated (tyr/thr) forms by Western blotting using specific antiactive phosphokinase antibodies (16). Serum stimulation of RPAF from control rats incubated under normoxic conditions showed a weak multiphasic activation of p38 MAP kinase as indicated by its dual phosphorylation of its regulatory thr/tyr motif (Figure 2A, upper panel ). In contrast, p38 MAP kinase was found to be strongly constitutively active in RPAF cells derived from rats subjected to chronically hypoxic conditions, and this strong activity could be further enhanced by serum stimulation, particularly in the period 16-48 h poststimulation (Figure 2A, upper panel ). This increase in p38 MAP kinase activity was not due to an upregulation of p38 expression, as Western blot analysis revealed comparable levels of p38 expression in RPAF cells from chronically hypoxic and normoxic rats (Figure 2A, lower panel ). RAF cells, however, despite expressing substantial levels of p38, did not show significant activation of p38 MAP kinase in response to serum, nor was this activity modulated following chronic exposure of the rats to hypoxia (Figure 2B).
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A similar pattern was observed for Erk MAP kinase in pulmonary fibroblasts: whereas serum induced Erk (particularly p42 Erk2) activity in normoxic RPAF cells in a multiphasic manner (peaks at 1 h and 24 h), RPAF from chronically hypoxic rats showed strong constitutive activity of Erk MAP kinase (Figure 3A). Although there appeared to be a slight upregulation in Erk expression in these cells, this upregulation was not sufficient to explain the very high levels of kinase activity observed (Figure 3A). As with p38 MAP kinase, chronic exposure to hypoxia did not modulate Erk activation in RAF cells, which already appeared to exhibit a considerable level of constitutive Erk activity that was not significantly modulated by addition of serum (Figure 3B). Although Jnk MAP kinase could be weakly activated by the addition of serum to RPAF cells, there was no significant difference in the protein levels of Jnk expressed, nor in the kinetics or extent of Jnk activation following serum stimulation of hypoxic RPAF relative to that observed in RPAF from control normoxic rats (Figure 4A). Moreover, no significant activation of Jnk in response to serum could be detected in RAF cells derived from either normoxic or hypoxic rats (Figure 4B).
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Differential Roles for p38 and Erk MAP Kinase in RPAF and RAF Cell DNA Synthesis from Normal and Chronically Hypoxic Rat Cells
As p38 and Erk MAP kinase activity was constitutively active in RPAF cells derived from hypoxic but not normoxic rats, we decided to investigate, using specific pharmacological inhibitors, the role of these kinases in the hypoxia-enhanced proliferation of RPAF cells. A selective and potent inhibitor of the Erk MAP kinase cascade, U0126, mediates its effects by binding to and inactivating the Erk-specific MAP kinase kinase, MEK, whereas it has no effect on any of the components of the Jnk or p38 MAP kinase cascades (16). Similarly, the compound SB203580 is a selective and potent inhibitor of p38 MAP kinase that does not affect either Erk or Jnk MAP kinases (17). These reagents therefore are useful pharmacological tools in identifying the functional activities mediated by p38 and Erk MAP kinases. Although various doses of the inhibitors were used (SB203580 and U0126: 0.1 µM-20 µM), only one dose is presented, as increasing the dose of these inhibitors gave rise to similar results. As expected, although preincubation for 1 h with SB203580 (5 µM) inhibited p38 activity (Figure 5A), in RPAF and PAF cells, preincubation for 1 h with U0126 (1 µM) profoundly inhibited both the hypoxia- and serum-stimulated Erk MAP kinase activation in these cells (Figure 5B).
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1] or
2 [THaving demonstrated the activity of these kinase inhibitors, we tested their effect in DNA synthesis assays (Figure 6) to probe the roles of the constitutive Erk and p38 MAP kinase activity observed in RPAF, but not RAF cells derived from chronically hypoxic rats. These results showed that SB203580 and U0126 had no effect on the DNA synthesis resulting from basal (results not shown) or serum stimulation of RPAF or PAF cells derived from normoxic rats (Figures 6A and 6B). In contrast, the p38 inhibitor SB203580 completely abrogated the enhanced serum-stimulated DNA synthesis observed in RPAF, but not PAF cells from chronically hypoxic rats (Figures 6A and 6B), such that the response was now equivalent to that observed in cells derived from control normoxic rats. The Erk MAP kinase cascade inhibitor U0126 had no effect on this hypoxia-induced enhancement of DNA synthesis.
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DISCUSSION |
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In this study we have shown that pulmonary artery fibroblasts from chronically hypoxic rats appear to have undergone a phenotypic switch whereby they exhibit enhanced proliferative responses relative to those observed with RPAF derived from normoxic animals even when the cells from the hypoxic animals are grown under normoxic conditions. This phenotypic switch appears to be irreversible in that hypoxia-enhanced proliferation is maintained following multiple passages of these primary cultures under normoxic conditions. This mirrors the changes observed in vivo in patients with hypoxic pulmonary hypertension, where remodeling of the pulmonary arteries due to increased cellular proliferation remains even after oxygen therapy (18). Sustained enhanced proliferative capacities have recently also been reported for fibroblasts isolated from the pulmonary artery of neonatal calves with the severe fibroproliferative changes characteristic of hypoxic neonatal pulmonary hypertension (19). This study showed that the proliferative responses of such fibroblasts could be further enhanced following subsequent exposure to acute hypoxia. However, in our system, the augmented responses to serum observed in RPAF could not be further enhanced by reexposure to acute hypoxia. This suggests that the mechanisms regulating pulmonary vascular remodeling in neonates and adults may be quite different and may provide a rationale for the well-established finding that the effects of hypoxia are more marked in neonates relative to adults (19). Interestingly, no remodeling of the systemic circulation is observed in the patients with hypoxic pulmonary hypertension (5) and this is reflected in our rat model where fibroblasts from rat aorta did not show enhanced proliferative capacities following chronic exposure to hypoxia.
The molecular mechanisms by which hypoxia stimulates proliferation of pulmonary artery fibroblasts, but not fibroblasts from the systemic circulation, are unknown. There is considerable evidence in the literature, however, that the mitogen-activated protein kinases Erk1 and Erk2 and the related stress-activated kinases Jnk and p38 MAP kinase can be activated in response to hypoxic stress (6, 7). Indeed, we have shown in our model of acute bovine pulmonary artery hypoxia that the stress-activated MAP kinases Jnk and p38, but not the classical Erk1 and Erk2 MAP kinases, are activated in response to acute hypoxia (20). We therefore addressed the role of all three classes of these MAP kinases by investigating whether there was differential activation of these kinases in response to serum in fibroblasts derived from the pulmonary and systemic circulation of normoxic versus chronically hypoxic rats.
Although Jnk was strongly activated by hypoxia in the acute model (20), we could find no evidence of Jnk activation in RPAF derived from rats subjected to chronic hypoxia (Figure 4). Moreover, we could not detect any differential activation of Jnk in response to serum in RPAF derived from chronically hypoxic versus normoxic rats (Figure 4). Furthermore, only the p54 isoform of Jnk could be detected in these cells. However, as the Jnk activation observed in response to hypoxia in the acute model was transient, peaking between 3 and 6 h following hypoxic challenge, these results may indicate that whereas a Jnk kinase signal may be required for the initiation of hypoxia-mediated events, it is not involved in the remodeling process. In contrast, we found strong and constitutive activation of Erk and p38 MAP kinases in RPAF from chronically hypoxic rats but not normoxic rats (Figures 2 and 3). In addition, this induction of constitutive Erk and p38 MAP kinase activity was not observed in RAF from either chronically hypoxic rats or normoxic rats. Taken together, these results suggested that Erk and/or p38 MAP kinase played a role in the enhanced proliferative responses observed in remodeled fibroblasts from chronically hypoxic rats.
To address the relative roles of Erk and p38 MAP kinases in serum and/or hypoxia-induced proliferation of RPAF and RAF cells, we performed DNA synthesis assays in the presence of specific inhibitors of the Erk (U0126) and p38 (SB203580) MAP kinase cascades. Given the widely established roles for Erk MAP kinases in promoting cell proliferation (21), and the strong constitutive activation of this MAP kinase in RPAF cells by chronic hypoxia (Figure 3), it could be considered surprising that the Erk cascade inhibitor U0126 had no effect on the enhancement of serum-stimulated proliferation observed in RPAF from chronically hypoxic rats. However, examination of the levels of Erk MAP kinase activity in RAF cells showed that these cells also exhibited considerable levels of constitutive Erk MAP kinase activity despite an inability to display enhanced proliferative capacities following exposure to chronic hypoxia. Indeed, Erk activity was found to be strongly and constitutively activated in RAF cells regardless of the oxygen status of the rats from which they were derived. Taken together with the finding that Erk activity could be stimulated in RPAF cells from normoxic rats following stimulation with serum, these results could suggest that Erk activity serves to render cells permissive for growth factor-mediated stimulation of proliferation. Thus, the enhanced Erk MAP kinase signals observed in hypoxic RPAF cells could contribute to the augmented growth factor-stimulated responses observed in response to chronic hypoxia reported in the bovine neonatal study (19).
In contrast, and consistent with the differential patterns of p38 MAP kinase activity observed in RPAF and RAF from chronically hypoxic versus normoxic rats (Figure 2), we found that although p38 activity appeared to be essential for the enhanced proliferative response observed in RPAF from chronically hypoxic rats, it did not appear to play an important role in transducing serum-induced proliferation in either RPAF or RAF cells. This important role for p38 in transducing proliferative signals in response to hypoxia reflects our earlier studies on the bovine acute hypoxia model, which showed that p38 activation was dependent on the presence of a maintained hypoxic environment as sustained p38 activity was blocked by reoxygenation. Moreover although p38 MAP kinase has generally been considered to play a role in the transduction of apoptotic (7) rather than proliferative signals as evidenced by negative regulation of cyclin D expression (24) and its activation under conditions of cellular stress (25, 26) including ischemic damage and reperfusion of cardiac myocytes (27, 28), there is some evidence not only that crosstalk between Jnk and p38 may be required for apoptosis (29, 30) but also that activation of p38 MAP kinase can promote cellular activation, proliferation, and differentiation (31). Pertinently, whereas most of the physiological and chemical stresses that can induce apoptosis generally activate both JNK and p38 (29, 30), we find that under these stress conditions of chronic hypoxia, Jnk does not appear to be activated and p38 acts to promote cellular proliferation.
Hypoxia inducible factor (HIF-1) appears to play a universal role in the regulation of hypoxic gene expression (32), and, indeed, a number of genes including erythropoietin (EPO) (33) have been found to be regulated by HIF-1. It is therefore possible that p38 could be involved in gene expression, which may be responsible for the differential responses of fibroblasts from pulmonary and systemic arteries as there is compelling evidence in the literature that HIF-1 is regulated through p38 and other members of the MAP kinase family (34).
Finally, there is an urgent need for drugs that could reverse or prevent the pulmonary vascular remodeling that occurs in nearly all forms of cardiac and respiratory disease. Currently most vasodilating drugs used to counteract pulmonary hypertension result in systemic hypotension. However, because systemic fibroblasts do not replicate in the face of hypoxia and p38 MAP kinase does not appear to be necessary for the normal replication of systemic vascular fibroblasts, it is possible that therapies targeting RPAF p38 MAP kinase could lead to the control of pulmonary vascular remodeling without affecting the systemic circulation.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr. A. J. Peacock, Scottish Pulmonary Vascular Unit, Level 8, Western Infirmary, Glasgow G11 6NT, Scotland, UK.
(Received in original form August 10, 2000 and in revised form February 16, 2001).
This research was funded by the British Lung Foundation and the Chest Heart and Stroke Association, Scotland.| |
References |
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METHODS
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DISCUSSION
REFERENCES
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