Hypoxia-induced Inhibition of Tropoelastin Synthesis by Neonatal Calf Pulmonary Artery Smooth Muscle Cells Anthony G. Durmowicz, David B. Badesch, William C. Parks, Robert P. Mecham, and Kurt R. Stenmark Cardiovascular-Pulmonary Research Laboratory and Webb-Waring Lung Institute, University of Colorado Health Sciences Center, Denver, Colorado and Department of Medicine, Jewish Hospital at Washington University Medical Center, S1. Louis, Missouri
Animals chronically exposed to hypoxia develop characteristic structural changes in the pulmonary arterial vasculature including cell hypertrophy, hyperplasia, and increased deposition of extracellular matrix proteins. The medial smooth muscle cells' (SMC) increase in tropoelastin mRNA expression and elastin deposition as determined by in situ hybridization and histologic examination appears to contribute significantly to this increase in matrix protein accumulation. The primary stimulus for the increased tropoelastin production, which persists in vitro, is unknown but mechanical forces and hypoxia seem to play a role. In order to determine the direct effects of hypoxia on tropoelastin production by pulmonary artery SMC, cultured neonatal bovine pulmonary artery SMC were exposed to 3 %, 10%, and 21% O2 concentrations for 48, 72, and 120 h and soluble tropoelastin was measured by direct immunoassay. Tropoelastin mRNA levels were also determined by Northern and slot blot analysis after 48 h of incubation under hypoxic conditions. SMC cultured in 3 % and 10% O2 for 120 h showed dose-dependent decreases (11-fold and 2-fold, respectively) in measured tropoelastin levels compared with SMC cultured in 21% O2 conditions. This decrease was not due to cell damage or accumulation of toxic metabolites while under hypoxic conditions nor to a change in tropoelastin partitioning between the cell and media. Tropoelastin mRNA levels were also decreased under hypoxic conditions. Secreted, cell layer, and total protein synthesis determined by L-(3H]leucine incorporation again showed a dose-dependent decrease under hypoxic conditions but not to the same extent as tropoelastin production. Total protein synthesis after 120 h in hypoxia was decreased by 2-fold in 3 % conditions and by 22 % in 10% O 2 conditions compared with control SMC in 21% O2 conditions. These data suggest that the direct effects of hypoxia alone are not responsible for the increased tropoelastin production seen by SMC in the pulmonary vasculature upon chronic exposure to hypoxia.
Hypoxic pulmonary hypertension is associated with morphologic and structural alterations in the pulmonary arterial wall including cellular hypertrophy, hyperplasia, and increased deposition of extracellular matrix proteins such as elastin and collagen (1-4). Increased synthesis of tropoelastin and procollagen by pulmonary artery (PA) tissue from pulmonary hypertensive neonatal calves has been previously documented (5, 6). Recent studies utilizing in situ hybridization techniques have demonstrated that the increase (Received in original form January 22, 1991 and in revised form April 5, 1991) Address correspondence to: Anthony G. Durmowicz, M.D., Box B-133, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. Abbreviations: calf serum, CS; Dulbecco's modified Eagle's media, DMEM; lactate dehydrogenase, LHD; pulmonary artery, PA; smooth muscle cell(s), SMC. Am. J. Respir. Cell Mol. BioI. Vol. 5. pp. 464-469, 1991
in elastin seen in pulmonary hypertensive vessels may be due to recruitment of contractile vascular smooth muscle cells (SMC) into an elastin-producing synthetic cell (7). This is confirmed by studies showing that the increase in PA SMC tropoelastin synthesis appears to be the result of stable phenotypic changes in the SMC because the upregulation of tropoelastin synthesis is maintained by SMC cultured from pulmonary hypertensive arteries even under normoxic conditions (5). However, the primary stimulus leading to upregulated elastin production in the setting of hypoxic pulmonary hypertension is unknown. Many in vivo and in vitro studies suggest that hemodynamic forces alone, particularly increased vascular wall tension, are capable of inducing matrix protein synthesis in vascular SMC. Hypoxia alone has also been shown to influence matrix protein synthesis as evidenced by both increased fibronectin, thrombospondin, and "hypoxia-associated" protein levels (8-10) and decreased collagen and proteoglycan synthesis (11-14). Thus, hypoxia is capable of causing a redirection of cell protein synthesis
Durmowicz, Badesch, Parks et al.: Hypoxic Inhibition of Tropoelastin Synthesis
as well as affecting production of SMC mitogens and inhibitors (15-18). The direct effect of hypoxia on SMC tropoelastin synthesis has not been studied. We therefore assessed the role of decreased oxygen tension in regulating tropoelastin production in cultured neonatal calf PA SMC in an effort to gain a greater understanding of the stimuli responsible for the phenotypic switch of the SMC in hypoxic pulmonary hypertension. The data demonstrate a marked decrease in tropoelastin levels and gene expression under hypoxic conditions. These findings suggest that the direct effects of hypoxia do not account for the increased SMC tropoelastin production seen in hypoxic pulmonary hypertension.
Materials and Methods Tissue Culture Neonatal (15-day) bovine PA SMC were isolated by explant culture as previously described (19). Unless otherwise specified, cultures were maintained in Dulbecco's modified Eagle's media (DMEM) supplemented with 5 % calf serum (CS), 20 mM Hepes (pH 7.4), 10 mM nonessential amino acid solution, 10 U/ml penicillin, 0.1 mg/ml streptomycin, 10 J.tg/ml gentamicin, and 3.7 g/liter sodium bicarbonate (all reagents from Sigma Chemical Co., St. Louis, MO). Cultures were incubated in a humidified air, 5 % CO z atmosphere at 37 0 C. Early-passage (2 to 7) cells were used for all experiments.
In Vitro Exposure to Hypoxia SMC were plated at an equal density into 24-well tissue culture plates or 75-cm z flasks and grown to confluence. Culture plates were then placed in airtight Plexiglas" controlled-atmosphere chambers (Bellco Biotechnology, Vineland, NJ) and exposed to O, at 3%, 10%, or 21% concentrations plus 5% COz and the balance N, at a continuous flow of 3 to 4liters/min for 20 min daily. After the specified treatment period, cells were harvested for the quantitative determination described below. To assess if equilibration between gas and liquid phases of the cultured cells had occurred and to determine the level of hypoxia to which the cells were actually exposed, O, concentration was measured in the media after 10 to 12 h of gas exposure with a Clark's electrode. The corresponding partial pressure of O, was estimated by calculating the ratio of Paz (mm Hg) and current (nanoamps) at 21% O, concentration and applying it to currents conducted at lower Oz concentrations. Media incubated at 3 % O, conducted a current of 2.0 nanoamps, which correlated with a Paz of 25 mm Hg. Medium incubated at 10% O, conducted a current of 5.0 nanoamps correlating with a Po, of 63 mm Hg, and medium at 21% O, conducted a current of 10.2 nanoamps, which correlated with a Paz of 128 mm Hg. Cell media lactate dehydrogenase (LD H) levels were measured from cells exposed to hypoxic conditions to assess possible hypoxia-associated cell damage. There were no differences in media LD H levels from cells cultured in either 3 %, 10%, or 21% O, conditions (data not shown). Tropoelastin Determinations Confluent calfPA SMC cultured in DMEM and 5 % CS were placed in the appropriate O2 concentration (3 %, 10% or
465
21%). 48 h before measuring tropoelastin levels, the culture medium was replaced with DMEM containing 5 % CS and the crosslinking inhibitors IS-aminoproprionitrile (100 J.tg/ml) and penicillamine (50 J.tg/ml) (Sigma). After the specified time of exposure to hypoxia, media were removed from the wells for tropoelastin level determination. Cells were removed from the wells by trypsinization, and cell numbers were determined with a hemocytometer. Experiments in each O, concentration at each time point were performed at least twice with six separate samples (culture wells) per condition (3 %, 10%, or 21% Oz). Data were presented either as the result of a single experiment (Figure 1) or as the compilation of seven experiments with tropoelastin levels measured in 3 % and 10% O, conditions expressed relative to those of SMC cultured under 21% conditions from the same individual experiment (Figure 2). Soluble tropoelastin was measured by an enzyme-linked protein-binding immunoassay. Bovine tropoelastin used for the standards and secreted tropoelastin protein were detected with a rabbit polyc1onal antiserum to bovine tropoelastin (20). A goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (Sigma) and the substrates 2.2-azino-di[3-etbyl-benzthiozoline sulfonate] (ATBS) and hydrogen peroxide (Kirkegaard and Perry, Gaithersburg, MD) were used for detection. A standard curve was prepared with each assay, and duplicates of each sample were processed. Tropoelastin levels were normalized to cell number. Cell-associated tropoelastin levels were measured by removing the media from cells previously exposed to the appropriate experimental conditions and washing the cell layer twice with buffered saline. Cells were scraped from the bottom of each culture dish well, and 0.5 ml of 0.1 N acetic acid was added. The plates were then gently agitated overnight at 4 0 C, the cell debris spun down by centrifugation, and the supernatant evaporated using a Speed Vac Concentrator (Savant Instruments, Farmingdale, NY). The pellet was dissolved in 0.3 ml DMEM, 5% CS, and tropoelastin levels were determined as above. The ratio of cell-associated to soluble tropoelastin in each well was then calculated. Tropoelastin Synthesis following Recovery from Hypoxia Confluent PA SMC were cultured under 3 %, 10%, or 21% O, conditions for 72 h in DMEM, 5% CS with 100 J.tg/ml IS-aminoproprionitrile and 50 J.tg/ml penicillamine added then returned to 21% Oz conditions. After an additional 48 h, the media were removed and soluble tropoelastin levels measured and normalized to cell number. RNA Isolation and mRNA Detection Confluent SMC layers from 75-cm z flasks exposed to 3 %, 10%, or 21% o, 5 % COz, and the balance N z conditions for 48 h were trypsinized, pelleted, and washed twice with buffered saline. Total RNA was isolated by homogenization in guanidine thiocyanate and differential ethanol precipitation (21). Quantification was determined by absorbance at 260 nm ultraviolet light absorbance and equal loading of Northern blot lanes assessed by ethidium bromide staining. For slot blots, total RNA (1,000 ng) was denatured in 1 M formaldehyde-50% formamide, serially diluted, and adsorbed to nitrocellulose using a slot blot manifold (Bio-Rad,
466
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991
Richmond, CA). For Northern hybridizations, total RNA (5 j.tg/lane) was denatured, resolved by formaldehyde-l % agarose electrophoresis, and blotted to nitrocellulose. Tropoelastin mRNA was hybridized to nick-translated (Multiprime DNA labeling system; Amersham International, Amersham, UK) T66, an exon-specific bovine cDNA. After washing and exposure for 12 h (Northern) or 6 h (slot blot) to XAR-5 film (Eastman Kodak, Rochester, NY) with intensifying screens, relative mRNA levels were determined by densitometric scanning using a Hoeffer GS-300 scanning densitometer (22). Total Protein Synthesis To determine total protein synthesis, media maintained for 120 h at the determined O, concentration were replaced with leucine-free Eagle's minimal essential medium (GIBCO, Grand Island, NY) for 90 min, and L-(3H]leucine (New England Nuclear, Boston, MA) was added to each well at a final concentration of 10 j.tCi/mi. Incubation was continued for 8 h in the appropriate oxygen chamber. A 100-j.t1 aliquot of medium was mixed with 900 j.tl of 0 0 C, 10% trichloroacetic acid (Sigma) and incubated for 1 h at 4 C. Precipitates were collected on Whatman GF/C filters, rinsed twice with 2 ml of95% ethanol, and air-dried. Cell layers were washed with buffered saline, solubilized in 1 N NaOH, neutralized with 1 N HCI, and precipitated with 10% trichloroacetic acid as above. Precipitated (3H]leucine was quantified by scintillation spectrophotometry. Medium and cell-associated counts were summated to approximate total protein synthesis. 0
Statistical Methods Tropoelastin and acid-precipitable radioactivity (protein synthesis determination) levels in 3 % and 10% O, culture conditions were compared with 21% Oz conditions with an unpaired Student's t test. P values < 0.05 were considered significant. Tropelastin time course data in 3 % and 10% Oz conditions were analyzed using an ANOVA with comparisons made by Fischer's protected least significance test at the 95 % significance level.
70
limits of
detection
10
21
Oxygen concentration (%)
Figure 1. Tropoelastin production by calf pulmonary artery (PA) smooth muscle cells (SMC) grown under hypoxic conditions for 120 h. SMC exposed to different o, concentrations (3 %, 10%, or 21%) for 120 h had tropoelastin levels measured over the final 48 h of incubation. Tropoelastin production was normalized per cell number. There was an approximate l l-fold reduction in tropoelastin levels by SMC cultured in 3% Oi, while SMC cultured in 10% O, caused about a 2-fold reduction in tropoelastin levels compared with control SMC grown in 21% Oz. * P < 0.05.
To determine whether possible cell damage or toxic metabolite accumulation, which may occur during culture under hypoxic conditions, could be the cause of the decreased tropoelastin levels measured after hypoxic exposure, 120
100 !!!~
GIGI
>
:::l
~~ eN
80
:;:0
=~ gN a.-
00
60
~E
GI GI
~
Results Exposure of neonatal PA SMC to hypoxia resulted in O, concentration and time-dependent decreases in soluble tropoelastin levels (Figures 1 and 2). This decrease was greatest in 3% o, (Po z , 25 mm Hg) at 120 h (Figure 1). At this time point, levels decreased approximately ll-fold compared with controls (21% Oz; Po., 128 mm Hg). During the same period, tropoelastin levels were reduced approximately 2-fold in 10% Oz, (Po z, 63 mm Hg). In addition, the decrease in measured tropoelastin in 3 % O, conditions was more rapid and more pronounced than that under 10% at earlier time points (Figure 2). To determine if the decreased tropoelastin levels measured after culture in hypoxic conditions were secondary to a change in partitioning of soluble tropoelastin between the cell and media, levels of cell-associated and secreted tropoelastin were measured after culture in hypoxia. There was no difference in the ratio of cell-associated to secreted tropoelastin under any O, condition (Figure 3).
..
-GI GI-
~a.
40
a:
20
----
--0--
3%02 10%02
O+----r------,------,----...---------, o 24 48 72 96 120 Time (hours)
Figure 2. Time plot of relative tropoelastin synthesis in 3 % and 10% O, culture conditions (compared with control 21% Oz conditions). SMC were cultured in differing Oz concentrations (3 %, 10%, or 21%) for either 48, 72, or 120 h, and tropoelastin levels were measured over the final 48 h of incubation in test conditions. Points shown are pooled data from seven experiments and are expressed relative to tropoelastin levels measured in 21% O, culture conditions from each individual experiment. There were significant dose- and time-dependent decreases in tropoelastin levels by calf PASMC cultured under hypoxic conditions at all time points except 48 h in 10% Oz. Incubation for longer periods in lower Oz concentrations resulted in the greatest decrease in tropoelastin levels. * P < 0.05 compared to 21% level.
Durmowicz, Badesch, Parks et al.: Hypoxic Inhibition of Tropoelastin Synthesis
467
100
95
.~ . 0
0.
0
~
90
}
1
85
w
~
80
Total RNA (n g)
Figure 5. Slot blot hybridization of SMC tropoelastin RNA. Total RNA from calf PA SMC cultured in 3%, 10%, or 21% O2 conditions for 48 h was denatured in formaldehyde, serially diluted, adsorbed to nitrocellulose using a slot blot manifold (Bio-Rad), and hybridized with 32P-labeled 1'66. The hybridization signals were quantified by densitometric scanning of slots on X-ray film and are presented as the slope of densitometry values (arbitrary units) plotted against ng RNA.
75 10
21
Oxygen
concentration
(~'- )
Figure 3. Effectof hypoxic exposure on partitioning of tropoelastin between PA SMC and media. SMC were cultured in 3 %, 10%, or 21% O2 conditions for 72 h, and both soluble and cell-associated tropoelastin levels were determined . Values are expressed for extracellular tropoelastin as a percentage of total tropoelastin production. There was no difference in tropoelastin partitioning between SMC and media at any O2 concentration .
(3H]leucine incorporation, was determined (Figure 6). Cell-associated and secreted SMC protein synthesis both decreased under lowered oxygen tensions. At 3 % 01, labeled protein in the cell layer was decreased approximately 50 %, whereas culture in 10% O 2 caused approximately a 20 % reduction in cell-associated protein.
tropoelastin levels were measured from SMC that had been exposed to hypoxic conditions for 72 h and then returned to 21% O 2 for 48 h. Preexposure to either 3% or 10% hypoxic conditions did not affect subsequent tropoelastin production under normoxic conditions (data not shown) . To assess the mechanism of hypoxia-induced reduction of tropoelastin production, total RNA samples isolated from SMC grown under hypoxic conditions for 48 h were probed for tropoelastin mRNA expression using both Northern and slot blotting techniques. These results indicated that tropoelastin-specific mRNA levels decreased in a manner similar to tropoelastin protein levels, with lower steady-state mRNA amounts seen in both 3 % and 10% O 2 conditions (Figures 4 and 5) . These observations support the thought that hypoxia-mediated decreases in tropoelastin levels could at least partially be mediated at a pretranslational level although post-translational modulation may also likely be present. To assess the effect of hypoxia on total protein synthesis in cultured SMC, total protein synthesis, as measured by
Figure 4. Northern hybridization of RNA from calf PA SMC cultured in differing O2 concentrations . Left panel: Total RNA (5 ~g/lane) was isolated from calf PASMC cultured in 3%, JO%, or 21% O2 conditions, denatured, resolved by formaldehyde-l % agarose electrophoresis, and blotted to nitrocellulose. Tropoelastin mRNA was hybridized to 32P-labeled 1'66, an exon-specific bovine cDNA . Equivalent loadings of RNA were determined by sample absorbance at 260 nm of ultraviolet light. After washing, blots were exposed to XAR-5 film (Kodak) for 12 h with intensifying screens. Right panel : Graph depicting the relative signal strength of tropoelastin gene-specific bands as quantified by densitometry (compared with 21% O2 conditions as control).
Discussion Neonatal bovine PA SMC tropoelastin levels decreased under hypoxic conditions in a time- and O 2 concentrationdependent fashion. It was shown that this decrease was not the result of a change in partitioning between the cell and the media nor as a result of cell damage when assessed by media LDH levels and the ability of the cells to recover from hypoxic conditions. The decrease in tropoelastin levels was associated with a decrease in tropoelastin steady-state mRNA levels in both 3 % and 10% O 2 conditions, suggesting pretranslational modulation of tropoelastin synthesis. However, the large differences between tropoelastin protein levels measured in 3 % and 10 % O 2 conditions (Figures 1 and 2) taken in conjunction with the similar decreases in tropoelastin steady-state mRNA levels in 3 % and 10% O 2
Oxygen Concentra tion J%
10% 21%
,.
O.yg e" Cone.n lt.lIon (%1
21
468
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991
20
•
....
.!! E
med ium
10
Do.
~U
~
a.
,;
u
'"
10
21
Oxygen concentration ('%o)
Figure 6. SMC protein synthesis after 120 h of incubation in differing O, concentrations. L-[3H]leucine incorporation was used to measure secreted, cell layer, and total protein synthesis by calf PA SMC after incubation in 3%, 10%, or 21% Oz conditions for 120 h (see MATERIALS AND METHODS). Incubation in 3% O. resulted in an approximate 2-fold decrease in secreted, cell layer, and total protein synthesis compared with 21% O, control. Culture in 10% O, showed approximately a 20% decrease in secreted, cell layer, and total protein synthesis. * P < 0.05 compared to 21% value; # P = 0.088; $ P = 0.056.
(Figures 4 and 5) suggest both pre- and post-translational hypoxia-mediated mechanisms may be involved in decreasing measured tropoelastin levels. Total protein synthesis was also reduced by hypoxia but it did not appear to be reduced to the same extent as was observed for tropoelastin. These results demonstrate a marked reduction of tropoelastin protein levels and gene expression under hypoxic conditions, suggesting hypoxia alone is not sufficient to cause either the increase in tropoelastin production observed in pulmonary hypertensive vessels in vivo or the stable increase in tropoelastin production observed in SMC cultured from chronically hypoxic pulmonary hypertensive calves (5, 7). Animals exposed to chronic hypoxia develop severe pulmonary hypertension and changes in the pulmonary vessel wall that include significant increases in production and deposition of elastin and collagen (1-4). Possible stimuli for the increased matrix protein synthesis in pulmonary vascular remodeling have previously been thought to include hypoxia, mechanical forces, or other factors. Experimental evidence suggests mechanical forces such as pressure and increased wall tension play an important role. For example, pulsatile stretch rapidly induced collagen synthesis in cultured vascular SMC (23), and mechanical stretch of isolated PA rings rapidly induces elastin and collagen mRNA expression (24). Furthermore, when pressure and flow are reduced in vivo by coarctation of the left PA, hypoxia-mediated structural remodeling is markedly reduced (25). In the neonatal calf model of hypoxic pulmonary hypertension in which the arterial POz is roughly the equivalent of our culture conditions in 3 % Oz (27 mm Hg), we have demonstrated that tropoelastin mRNA levels are markedly increased in lobar pulmonary arteries from hypoxic pulmonary hypertensive calves, whereas the tropoelastin mRNA levels in the thoracic
aorta do not change (5) . Interestingly, the upregulation of tropoelastin synthesis by SMC in the pulmonary vascular media in vivo occurs at a tissue Po, that is likely even less than in 3 % O, culture conditions in vitro, where a great reduction in tropoelastin levels are seen . In addition, in this in vivo model there is a marked increase in pressure in the pulmonary circulation, with mean PA pressures increasing from 25 mm Hg to 120 mm Hg, while no change in pressure is noted in the systemic circulation. We have shown that lobar pulmonary veins from the hypoxic pulmonary hypertensive calves demonstrate marked decreases in tropoelastin mRNA levels compared with normoxic controls (26). Because right to left shunting of blood across the foramen ovale and ductus arteriosus occurs in the presence of suprasystemic pulmonary hypertension, the pulmonary venous blood in the pulmonary hypertensive animals maintains a low oxygen tension yet with decreased pressure and flow. Thus, because veins, and thus venous smooth muscle, can respond to stretch with increased matrix production (27), pressure and flow may be necessary for tropoelastin gene expression. The direct effects of hypoxia, in the absence of these mechanical factors, may downregulate tropoelastin synthesis. Interaction among vascular wall cells may be necessary for the increased tropoelastin production and accumulation observed in vivo. In support of this theory is the observation that conditioned media from hypoxic endothelial cells induced proliferation of SMC in vitro (17). In addition, it has been shown that an intact endothelium was necessary for the induction of increased elastin and collagen synthesis by isolated pulmonary arteries in response to stretch (24). Furthermore, tropoelastin production by SMC is stimulated by growth factors including transforming growth factor-S and insulin-like growth factor-I (19, 28, 29). Since hypoxia has been shown to increase both endothelial cell permeability in vitro (8) and capillary permeability in vivo (30), it may allow circulating growth factors access to vascular wall cells. Decreases in the production of matrix proteins by SMC, fibroblasts, and endothelial cells under hypoxic conditions have been shown in several other in vitro studies (11-14). The mechanism of this hypoxia-induced inhibition is not known. Cell metabolism may be downregulated in response to hypoxia, and, in fact, our data demonstrated a reduction in total protein synthesis. However, not all cell protein production is inhibited by hypoxia or other oxidative stresses. Hypoxic or anoxic cultured endothelial cells produced increased fibronectin, laminin, and thrombospondin protein and mRNA (8, 9) . Also, incubation of cells under hypoxic conditions or in the presence of hydrogen peroxide has been shown to cause induction of heat shock protein synthesis (15, 16), and certain "hypoxia-associated proteins" have recently been described as increasing under hypoxic conditions in culture (to). The apparent reduction in tropoelastin relative to total protein synthesis in hypoxic SMC seen in this study may be due to inhibition of tropoelastin production in the face of increases in other types of proteins such as fibronectin, heat shock proteins, or hypoxia-associated proteins. Alternatively, increased tropoelastin turnover under hypoxic conditions could explain the greater decrease in measured tropoelastin levels relative to total protein synthesis but not the concomitant decrease in tropoelastin mRNA levels seen.
Durmowicz, Badesch, Parks et al.: Hypoxic Inhibition of Tropoelastin Synthesis
Nevertheless, the importance of the findings reported here (that the increased elastin deposition seen in chronic hypoxia pulmonary hypertension is probably not due to the direct effects of hypoxia alone on SMC) does not depend upon the decrease being entirely specific for tropoelastin, as, in fact, it almost certainly is not. The regulation of PA SMC tropoelastin synthesis and proliferation in the development of chronic pulmonary hypertension is likely complex. The direct effectsof hypoxia, by themselves, are not likely to account for the increased tropoelastin production seen in chronic hypoxic pulmonary hypertension. Further work is needed to define the role of hypoxia in redirecting cell protein production and metabolism, in induction of cell mitogens, and of its interaction with mechanical forces to produce vascular remodeling in chronic hypoxia-induced pulmonary hypertension. Acknowledgments: Dr. Durmowicz was supported by National Institutes of Health (NIH) Grant HL-07670; Dr. Badesch was supported by NIH Clinical Investigator Award HL-02408-0l, Pfizer Scholars Award in Cardiovascular Medicine, and a grant from RJR Nabisco; Dr. Mecham was supported by NIH Grants HL-29594 and HL-41040; and Dr. Stenmark was supported by NIH Grants HL14985 and HL-01503 and an American Lung Association Career Investigator Award.
References 1. Meyrick, 8., and L. Reid. 1980. Hypoxia induced structural changes in the media and adventitia of the rat hilar pulmonary artery and their regression. Am. J. Pathol. 100:151-178. 2. Stenmark, K. R., J. Fasules, D. M. Hyde et a11987. Severe pulmonary hypertension and arterial adventitial changes in newborn calves at 4300m. J. Appl. Physiol. 62:821-830. 3. Meyrick, B., and L. Reid. 1979. Hypoxia and incorporation of 3H thymidine by cells of the rat pulmonary arteries and alveolar wall. Am. J. Pathol. 96:51-70. 4. Sobin, S. S., H. M. Tremer, J. D. Hardy, and H. P. Chiodo. 1983. Changes in arteriole in acute and chronic hypoxic pulmonary hypertension and recovery in rat. J. Appl. Physiol. 55:1445-1455. 5. Mecham, R. P., L. A. Whitehouse, D. S. Wrennetal. 1987. Smooth muscle-mediated connective tissue remodeling in pulmonary hypertension.
Science 237:423-426. 6. Crouch, E. C., W. C. Parks, J. L. Rosenbaum et al.1989. Regulation of collagen production by medial smooth muscle cells in hypoxic pulmonary hypertension. Am. Rev. Respir. Dis. 140:1045-1051. 7. Prosser, I. W., K. R. Stenmark, M. Suthar, E. C. Crouch, R. P. Mecham, and W. C. Parks. 1989. Regional heterogeneity of elastin and collagen gene expression in intralobar arteries in response to hypoxic pulmonary hypertension as demonstrated by in situ hybridization. Am. J. Pathol. 165:1073-1088. 8. Ogawa, S., H. Gerlach, C. Esposito, A. Pasagian-Macaulay, J. Brett, and D. Stem. 1990. Hypoxia modulates barrier and coagulant function of cultured bovine endothelium. J. Clin. Invest. 85: 1090-1098. 9. Lynch, D. C., P. L. Ansell, and R. B. Levene. 1988. Effects of anoxia on gene expression in human endothelial cells. J. Cell Bioi. 107:58Ia. (Abstr.) 10. Zimmerman, L. H., R. A. Levine, and H. W. Farber. 1990. Transcriptional regulation of hypoxins in cultured endothelial cells. Am. Rev. Respir. Dis. 141(Suppl.):A351. (Abstr.)
469
II. Stavenow, L., P. Falke, and A. Berglund. 1983. Effects of different injurious stimuli on cell death, proliferation, and collagen secretion by rabbit aortic smooth muscle cells and human umbilical vein endothelial cells in culture. Med. Bio/. 61:214-218. 12. Humphries, D. E., S. L. Lee, B. L. Fanburg, and J. E. Silbert. 1986. Effects of hypoxia and hyperoxia on proteoglycan production by bovine pulmonary artery endothelial cells. J. Cell. Physiol. 126:249-253. 13. Levene, C. I., C. P. Bartlet, C. Fomieri, and G. Heale. 1985. Effect of hypoxia and carbon monoxide on collagen synthesis in cultured porcine and bovine aortic endothelium. Br. J. Exp. Pathol. 66:399-408. 14. Webster, D. F., and H. C. Burry. 1982. The effects of hypoxia on human skin, lung, and tendon cells in vitro. Br. J. Exp. Pathol. 63:50-55. 15. Li, G. C., and D. C. Shrieve. 1982. Thermal tolerance and specific protein synthesis in Chinese hamster fibroblasts exposed to prolonged hypoxia.
Exp. Cell Res. 142:464-468. 16. Courgeon, A. M., E. Rollet, J. Becker, C. Maisonhaute, and M. BestBelpomme. 1988. Hydrogen peroxide induces actin and some heat shock proteins in Drosophila cells. Eur. J. Biochem. 171:163-170. 17. Vender, R. L., D. R. Clemmons, L. Kwock, and M. Friedman. 1987. Reduced oxygen tension induces pulmonary endothelium to release a pulmonary smooth muscle mitogen(s). Am. Rev. Respir. Dis. 135:622-627. 18. Pasricha, P., P. Hassoun, E. Teufel, and B. Fanburg. 1989. Further characterization of a unique inhibitor of pulmonary artery smooth muscle cell growth produced by hypoxic pulmonary artery cells. Am. Rev. Respir. Dis. 139(Suppl.):AI71. (Abstr.) 19. Badesch, D. B., P. K. Lee, W. C. Parks, and K. R. Stenmark. 1989. Insulin-like growth factor-I stimulates elastin synthesis by bovine pulmonary arterial smooth muscle cells. Biochem. Biophys. Res. Commun. 160:382-387. 20. Prosser, I. W., L. A. Whitehouse, W. C. Parks et al. Polyclonal antibodies to tropoelastin and the specific detection and measurement oftropoelastin in vitro. Biochemistry. Submitted. 21. MacDonald, R. J., G. H. Swift, A. E. Przybyla, andJ. M. Chirgwin. 1987. Isolation of RNA using guanidinium salts. Methods Enzymol. 152:219227. 22. Parks, W. c., H. Secrist, L. C. WU, and R. P. Mecham. 1988. Developmental regulation of tropoelastin isoforms. J. Bioi. Chem. 263:44164423. 23. Leung, D. Y. M., S. Glagov, and M. B. Mathews. 1977. A new in vitro system for studying cell response to mechanical stimulation. Exp. Cell
Res. 109:285-298. 24. Tozzi, C. A., G. J. Poiani, A. M. Harangozo, C. D. Boyd, and D. J. Riley. 1989. Pressure-induced connective tissue synthesis in pulmonary artery segments is dependent on intact endothelium. J. Clin. Invest. 84: 10051012. 25. Rabinovitch, M., M. A. Konstam, W. J. Gamble et al. 1983. Changes in pulmonary blood flow affect vascular response to chronic hypoxia in rats.
Circ. Res. 54:432-441. 26. Parks, W. C., R. P. Mecham, E. C. Crouch, E. C. Orton, and K. R. Stenmark. 1989. Response of lobar vessels to hypoxic pulmonary hypertension. Am. Rev. Respir. Dis. 140:1455-1457. 27. Tozzi, C. A., J. Sumka, S. Hayes, G. J. Poiani, and D. J. Riley. 1988. Differential responsiveness of pulmonary artery, aorta, and vein to stretch-induced increases in collagen mRNA levels. Am. Rev. Respir. Dis. 137(Suppl.):532. (Abstr.) 28. Foster, J., C. B. Rich, and J. R. Fiorini. 1987. Insulin-like growth factor I, somatomedin C, induces the synthesis of tropoelastin in aortic tissue. Collagen and Related Research 7: 161-169. 29. Liu, J., and J. M. Davidson. 1988. The elastogenic effect of recombinant transforming growth factor beta on porcine aortic smooth muscle cells.
Biochem. Biophys. Res. Commun. 154:859-901. 30. Stelzner, T. J., S. W. Chang, R. F. O'Brien, and J. V. Weil. 1988. Subacute hypoxic exposure increases lung trans vascular protein escape in rats. Chest 93: 157s-158s.