Cell-specific differences in ET-1 system in adjacent layers of main pulmonary artery. A new source of ET-1

Elena Tchekneva1, Mayme L. Lawrence1, and Barbara Meyrick1,2

Departments of 1 Pathology and 2 Medicine, Center for Lung Research, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2650


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Endothelin-1 (ET-1) is a potent vasoconstrictor that causes sustained constriction of the pulmonary artery and modulates normal vascular tone. Endothelial cells were thought to be the major source of ET-1, but recent studies show that vascular smooth muscle cells (SMCs) are also capable of its synthesis. We examined the ET-1 and endothelin-converting enzyme-1 (ECE-1) system in cells cultured from two adjacent layers, subendothelial (L1) and inner medial (L2), of normal sheep main pulmonary artery and the response of this system to exogenous ET-1 and transforming growth factor-beta 1 (TGF-beta 1). End points include assessment of preproET-1 (ppET-1) and ECE-1 gene coexpression, measurement of intracellular and released ET-1, and ECE-1 activity. RT-PCR analysis revealed that ppET-1 and ECE-1 transcripts were greater in L1 than in L2 cells. The L1 cells also synthesized (L1, 3.2 ± 0.1; L2, 1.2 ± 0.1 fmol/106 cells) and released (L1, 9.2 ± 0.5; L2, 2.3 ±0.1 fmol/ml) greater amounts of ET-1 than L2 cells. The L2 cells internalized exogenous ET-1 in a dose-dependent manner (EC50 8 nmol/l) and were more responsive to exogenous ET-1 than L1 cells, showing upregulation of both the ppET-1 and ECE genes. TGF-beta 1 downregulated ET-1-stimulated ppET-1 and ECE-1 transcripts but only in L2 cells. In addition, L1 cells showed greater ECE-1 activity than L2 cells, and in both, the activity was sensitive to the metalloprotease inhibitor phosphoramidon. We conclude that the ET-1 system in L1 and L2 cells is distinct. The data suggest that the two cell types have diverse functions in the arterial wall; the L1 cells, like endothelial cells, provide a local source of ET-1; and since the L2 cells are more responsive to exogenous ET-1, they are likely to affect normal pulmonary vascular tone.

endothelin-converting enzyme; reverse transcriptase-polymerase chain reaction; smooth muscle cells; cell heterogeneity; endothelin-1; smooth muscle cell


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE VASCULAR WALL IS COMPOSED of a heterogeneous population of smooth muscle (SM) cells (SMCs). Frid et al. (12, 13) demonstrated at least four cell populations (smooth muscle and non-muscle-like) in the wall of normal bovine main pulmonary artery. The four cell types were found in three distinct layers of the arterial wall, one in the subendothelial layer (L1), one in the inner medial layer (L2), and two in the outer medial layer (L3S and L3R). These distinctions were made on the basis of morphology, their complement of smooth muscle markers, and proliferative activity in culture. The L1 cells were found to be less differentiated than other SMC types, containing only small amounts of alpha -SM actin. Morphologically and functionally distinct SMCs also have been described for systemic vessels, e.g., normal rat aorta (26, 40) and canine carotid artery (17).

Endothelin (ET) is a potent vasoconstrictor that contributes to regulation of vascular tone in both normal and diseased states. Three isoforms of ET (ET-1, ET-2, and ET-3) have been described, and each isopeptide has been shown to be encoded by distinct genes (18). The production of ET-1 involves expression of a 212-amino acid precursor, preproendothelin-1 (ppET-1), that is cleaved proteolytically to a 38-amino acid precursor peptide, Big ET-1. Cleavage of Big ET-1 by the metalloprotease endothelin-converting enzyme-1 (ECE-1) results in the biologically active (21- amino acid) ET-1. ET-1 was originally isolated from porcine endothelial cells (42), and, consequently, the endothelium was postulated to be the predominant source of this peptide. It is now clear that SMCs produce ET-1 (15, 37, 43) and that ppET-1 gene expression is regulated by exogenous ET-1, but whether adjacent but distinct types of vascular SMCs synthesize and release similar amounts of ET-1 and whether ET-1 synthesis is regulated in a similar manner in each cell type are not known.

In a previous study, we showed regional variability in ET-1 synthesis in SMCs of sheep pulmonary artery (37). Cells cultured from the inner medial layer (L2) of the main artery showed greater levels of ppET-1 gene expression and intracellular Big and mature ET-1 than those from the midregion (9-10th generation) artery. The present study examines the hypothesis that the synthesis and autoregulation of ET-1 are distinctive in cells from adjacent layers of the main pulmonary artery. Cells were cultured from the L1 and L2 layers of sheep main pulmonary artery, and ppET-1 and ECE-1 gene coexpression and intracellular levels of mature ET-1 and ECE-1 activity were examined and compared in these two cell types. In addition, their response to exogenous ET-1 was examined as was the effect of transforming growth factor-beta 1 (TGF-beta 1), a growth factor that is abundant in the media of normal sheep pulmonary artery (27), on ET-1-stimulated ppET-1 and ECE-1 gene expression. Our data demonstrate that subendothelial cells express, synthesize, and release greater amounts of ET-1 than L2 cells and that the L2 cells internalize and are more responsive to exogenous ET-1 than subendothelial cells. Basal ECE-1 activity is greater in L1 than in L2 cells and in both cells is metalloprotease sensitive. Furthermore, ET-1-induced upregulation of ppET-1 and ECE-1 gene expression is modulated by TGF-beta 1 only in L2 cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Cell culture. Cells were isolated from two adjacent layers of the main pulmonary artery from mixed-breed yearling sheep (n = 4). Subendothelial (L1) cells were isolated by deep scraping of the luminal surface following gentle removal of the endothelial layer. Additional L1 cells were obtained by subsequent removal and dissection of the visible remaining subendothelial layer. The L1 cells were seeded at low density. Colonies of L1 cells (50-100 cells) were identified by their morphology under phase-contrast microscopy, isolated in cloning rings, trypsinized, and transferred into culture dishes. SMCs from the inner medial layer (L2) were isolated using either an explant technique (38) or enzymatic dissociation (6). The L1 and L2 cells were cultured in RPMI 1640 medium containing 100 U/ml penicillin, 100 µg/ml streptomycin, 40 µg/ml gentamicin, 2 mmol/l L-glutamine (GIBCO BRL; Grand Island, NY) and 15% heat-inactivated calf serum (Atlanta Biologicals; Norcross, GA). For all studies, the cells were used in log phase and experiments were carried out in RPMI 1640 medium containing 0.1% BSA (Sigma; St. Louis, MO). Cells were used in between passages three and six.

Characterization of L1 and L2 cells. The smooth muscle nature of the cultured cells was assessed by morphology, and the presence of alpha -SM actin and SM myosin (12) using immunocytochemical techniques and confocal microscopy. Cells were double reacted with a monoclonal antibody against alpha -SM actin (Sigma) and a rabbit antibody to SM myosin (Dr. R. S. Adelstein). Briefly, semiconfluent layers of both L1 and L2 cells were washed three times with PBS and fixed in absolute alcohol at -20°C for 10 min. Each cell type was incubated with either primary anti-alpha -SM actin (1:150) followed by incubation with biotinylated anti-mouse IgG and visualized using streptavidin-Texas Red (1:100; Amersham; Arlington Heights, IL) or anti-SM myosin (1:1,500) followed by FITC-conjugated anti-rabbit IgG diluted 1:100 (Sigma). Negative controls included cells treated with only the secondary antibodies.

Both SMC types were also tested for the presence of endothelial cell markers, factor VIII activity and uptake of acetylated low-density lipoprotein (AcLDL) labeled with the BODIPY FL fluorophore according to manufacturer's protocols (Molecular Probes; Eugene, OR). Cultured pulmonary artery endothelial cells were used as controls.

Experimental protocols. In all experiments, cells that were at early stages of superconfluence were used. For assessment of ppET-1 and ECE-1 gene expression, L1 and L2 cells were stimulated with exogenous ET-1 (10 nmol/l) for 90 min in RPMI 1640 medium containing 0.1% BSA. For measurement of intracellular and released ET-1, both cell types were incubated with and without ET-1 for 18 h. Untreated L1 and L2 cells served as controls. ET-1 was selected for use in these experiments because it is known to act as an autoregulator of ppET-1 mRNA in SMCs (15). Cultured pulmonary artery endothelial cells served as controls for the ppET-1 and ECE-1 gene expression experiments. In other experiments, L1 and L2 cells were preincubated with 1-100 ng/ml recombinant TGF-beta 1 (R&D Systems; Minneapolis, MN) for 18 h and then treated with exogenous ET-1 (10 nmol/l) for 90 min. Smooth muscle cell numbers and viability were determined following trypsinization and trypan blue staining. ECE-1 activity was measured in crude cell membrane fractions from unstimulated L1 and L2 cells. To examine whether the ECE-1 activity was metalloprotease inhibitor sensitive, conversion of Big ET-1 by ECE-1 was detected in crude membrane fractions preincubated with 10-9 to 10-4 mol/l phosphoramidon (Sigma) at 37°C for 15 min.

Analysis of ppET-1 and ECE-1 mRNA by RT-PCR. Total RNA was isolated using the RNA STAT-60 reagent (TEL-TEST "B;" Friendswood, TX). The sense and antisense primers for ppET-1 were generated from the cDNA sequence (37) and were as follows: 5'-TTG TGG CTT TCC AAG GAG CTC CAG-3' (bases 298-321) and 5'-CGG TTG TCC CAG GCT TTC ATG-3' (bases 694-674). The sense and antisense primers for ECE-1 were generated from the human cDNA sequence (30) and were 5'-CCG GCC GGG ATC CTG CAG GCA CCA-3' (bases 1720-1743) and 5'-GAG GCC TTC GTG GGA GCT CTC AGG-3' (bases 2166-2142) (30). Optimal expression of the ppET-1 and ECE-1 genes was obtained using 33 cycles and an annealing temperature of 60°C. These conditions resulted in a single band of 396 bases for ppET-1 and 446 bases for ECE-1.

Our previously described RT-PCR protocol for visualization of ppET-1 transcripts (37) was found to work well for ECE-1. Briefly, 4 µg of total RNA were reverse transcribed in a total volume containing 50 U/µl human placenta RNase inhibitor, 1 mmol/l each deoxynucleotide triphosphate 10, 0.04 A260 units of oligo-p(dT)15 primer, 40 U of avian myeloblastosis virus RT in buffer, 50 mmol/l Tris · HCl, 8 mmol/l MgCl2, 30 mmol/l KCl, 1 mmol/l dithiothreitol (pH 8.5 at 20°C). The RT reaction was carried out for 1 h at 41°C and stopped by heating the sample to 99°C for 5 min. The excess RNA was removed by treatment with 1 U of RNase H at 37°C for 20 min followed by heating at 99°C for 5 min. The resultant first-strand cDNA was amplified by PCR using a sample mixture containing 1 µl of RT sample, 42 µl of distilled water, 0.5 µl of 5' and 3' primers (20 µmol/l each), 5 µl of 10× PCR buffer (10 mmol/l Tris · HCl, 15 mmol/l MgCl2, and 500 mmol/l KCl, pH 8.3) and 1.25 U of Taq DNA polymerase (Boehringer Mannheim; Indianapolis, IL). Each sample of the amplified cDNA was visualized after ethidium bromide staining and separated electrophoretically on an 1.8% agarose gel. Three types of negative control included samples without mRNA, RT, and first-strand cDNA. Optical density of the cDNA bands was determined by a computerized image-analysis system (Molecular Analyst Alias; GS-700 Imaging Densitometer; Bio-Rad; Hercules, CA) and normalized to RT-PCR products of hG3PDH (CLONTECH Laboratories, Palo Alto, CA) generated from each sample.

Measurement of ET-1. Intracellular and released levels of mature ET-1 were determined in homogenates of L1 and L2 cells and in conditioned medium. After 18-h incubation, the conditioned medium was collected from each sample and centrifuged at 1,000 rpm for 10 min, the supernatant was passed through a 0.45-µm filter, and the cell-free supernatant was used to determine released mature ET-1.

To assess intracellular levels of mature ET-1, the cells from these same experiments were washed three times in serum-free medium, trypsinized, and centrifuged at 1,000 rpm for 10 min. The cell pellet was then homogenized by a Teflon glass pestle in 400 µl of PBS with 0.1% Triton X-100, the homogenates were centrifuged at 3,000 rpm for 15 min, and the supernatant was collected. Both cell supernatants and conditioned medium were acidified by 2 mol/l HCl and loaded onto Sep-Pak C18 cartridges, which had been prewashed with 100% methanol, acetonitrile-5 mmol/l trifluoroacetic acid (TFA), and distilled H2O with 5 mmol/l TFA. The cartridges were washed in distilled H2O containing 5 mmol/l TFA and eluted with 2 ml of 80% methanol containing 0.1% TFA. The samples were dried and reconstituted in assay buffer. Mature ET-1 was measured using an ELISA (Biotrak ELISA system, Amersham International). The assay is sensitive to values of >1 fmol and cross-reacts with 100% of ET-1 and ET-2 (ET-2 is not generally found in the walls of the pulmonary vasculature) but not with ET-3 (<0.001% cross-reactivity). The concentration of ET-1 in cellular extracts is expressed in femtomoles per 106 cells and in conditioned medium in femtomoles per milliliter. Each sample was run in duplicate.

Isolation of crude cell membrane fractions and measurement of ECE-1 activity. At the end of the experiment, crude cell membrane fractions were separated by a modification of the technique described by Xu and colleagues (41). All procedures were carried out at 4°C. Confluent layers of SMCs were rinsed twice in ice-cold PBS, the cells were removed by scraping, and they were collected in 5 ml of PBS. After centrifugation at 1,000 rpm for 10 min, the pellets were homogenized by a Teflon-glass pestle in 400 µl of distilled water for 2 min followed by immediate addition of 4 ml of buffer B containing 20 mmol/l Tris · HCl, 20 µmol/l pepstatin A, 1 mmol/l phenylmethylsulfonyl fluoride, 1 mmol/l p-chloromercuriphenylsulfonic acid, and 250 mmol/l sucrose (Sigma) at pH 7.4. The homogenates were centrifuged at 1,000 g for 10 min, and the resultant supernatant was recentrifuged at 100,000 g for 2 h. The resultant pellets (crude membrane fractions) were resuspended in buffer B to give 1 mg protein/ml.

ECE-1 activity was determined in the crude membrane fractions by measurement of the amount of mature ET-1 generated from exogenous Big ET-1 (Peptides International) (41). Because ECE-1 activity is both substrate and protein dependent, the assay was run using concentrations of 0-10 µmol/l of Big ET-1 as the substrate and 0-15 µg of crude membrane fractions. We found that for L1 cells values within the linear range were achieved when 0.25 µmol/l of Big ET-1 was incubated with 3 µg of crude membrane fractions per 50- µl reaction mixture [0.5 mol/l NaCl in 0.1 mol/l sodium phosphate buffer (pH 6.8)] at 37°C for 30 min and terminated by addition of 50 µl of 5 mmol/l EDTA. For L2 cells, 0.1 µmol/l of Big ET-1 was incubated with 10 µg of crude membrane fractions. The reaction mixture was directly assayed for mature ET-1. Two negative controls were run for each assay, samples without substrate (Big ET-1) and samples without the cell membrane fractions. Membrane fractions from cultured endothelial cells were run as a positive control. Each sample was run in duplicate. ECE-1 activity is expressed in fmol ET-1 · 40 µg protein-1 · 130 min-1.

Statistics. Data are presented as means ± SD of either duplicate or triplicate determinations from each of two or more different cell lines from two or three different animals. The number of experiments is given in the figure legends and represents individual experiments because for each group of animals, the data collected from individual cell lines showed the same trend. Data were compared using Student's t-test and were considered significant when P <=  0.01.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of L1 and L2 cells. Phase-contrast microscopy revealed that the cells from the L1 and L2 layers of the normal sheep pulmonary artery (Fig. 1) have a different morphology, confirming previous findings by Frid and colleagues (12). The L1 cells were "rhomboidal" in shape, initially formed a monolayer (Fig. 1), and at superconfluence formed a dense multilayer with appearance of larger irregularly shaped cells in the most superficial layer. L2 cells showed the morphology of typical SMCs in that they were spindle shaped and had a typical "hill-and-valley" pattern (Fig. 1).


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Fig. 1.   Light micrograph of toluidine blue-stained 1-µm section shows subendothelial layer (L1) and inner medial layer (L2) of normal sheep pulmonary artery (A). ×70. Phase-contrast micrographs show subconfluent rhomboidal shaped L1 cells (B) and spindle-shaped L2 cells (C) cultured from normal sheep main pulmonary artery. ×280.

Figure 2 shows representative confocal micrographs of the combination of alpha -SM actin and SM myosin staining. Localization of alpha -SM actin in cytoplasmic and cell membrane-bound epitopes is depicted in red, whereas SM myosin localizes mainly to microfilaments and is depicted in green; yellow represents sites of congruence of alpha -SM actin with SM myosin. A little alpha -SM actin was demonstrated in the perinuclear region in subconfluent L1 cells (Fig. 2A), but at superconfluence both markers were apparent in the irregularly shaped cells of the superficial layer (Fig. 2B). L2 cells showed colocalization of both markers (Fig. 2C). Control cells that were not treated with antibody showed no fluorescence (e.g., Fig. 2D).


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Fig. 2.   Confocal micrographs of L1 and L2 cells from sheep pulmonary artery after incubation with antibodies to alpha -smooth muscle (SM) actin (red) and SM myosin (green). Yellow represents sites of congruence of alpha -SM actin and SM myosin. A: subconfluent L1 cells show little expression of alpha -SM actin and SM myosin. B: at superconfluence, large stellate-shaped superficial L1 cells contain both alpha -SM actin and SM myosin alone and in conjunction. C: L2 cells show colocalization of both alpha -SM actin and SM myosin. D: control cells that were not treated with antibodies showed no fluorescence. ×280.

By fluorescence microscopy, control endothelial cells showed both factor VIII reactivity and uptake of AcLDL (Fig. 3, A and B). L1 cells also showed factor VIII activity (Fig. 3C), although uptake of AcLDL was not a feature (Fig. 3D). L2 cells showed minimal uptake of AcLDL and were negative for factor VIII.


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Fig. 3.   Fluorescence micrographs of L1 and L2 cells from normal sheep main pulmonary artery. Endothelial cells (A) and L1 cells (C) show factor VIII reactivity. Endothelial cells (B) but not L1 cells (D) show uptake of acetylated low-density lipoprotein. ×280.

ppET-1 and ECE-1 gene expression. RT-PCR demonstrated that expression of the ppET-1 gene was significantly higher in L1 than in L2 cells (n = 5, P < 0.01). Similarly, ECE-1 gene expression was significantly higher in L1 than in L2 cells (n = 4, P < 0.01). A representative RT-PCR analysis for ppET-1 and ECE-1 gene transcripts is shown in Fig. 4A. Normalized to human glyceraldehyde-3-phosphate dehydrogenase (hG3PDH), the densitometry readings show that the level of ppET-1 transcripts in the L1 cells was twice that in the L2 cells and ECE-1 transcripts were 30% greater than in L2 cells (Fig. 4B).



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Fig. 4.   A: RT-PCR analysis demonstrating preproendothelin-1 (ppET-1) and endothelin-converting enzyme-1 (ECE-1) gene expression in L1 and L2 cells cultured from sheep pulmonary artery. S, Hae III DNA size marker. B: densitometric readings of ppET-1 and ECE-1 gene expression normalized to human glyceraldehyde-3-phosphate dehydrogenase (hG3PDH; loading marker). Open bars, L1 cells. Solid bars, L2 cells. Data are expressed as means ± SD; n = 4 cell lines from 3 animals. * P < 0.01 compared with L1 cells.

Intracellular and released ET-1 and response to exogenous ET-1. In agreement with our findings for ppET-1 gene expression, intracellular ET-1 levels were 2.6 times higher in L1 than in L2 cells (means ± SD: L1, 3.2 ± 0.1; L2, 1.2 ± 0.1 fmol/106 cells; n = 3) and levels of released ET-1 were four times greater from L1 than from L2 cells (L1, 9.2 ± 0.5; L2, 2.3 ± 0.1 fmol/ml; n = 3). Incubation with ET-1 caused a dose-dependent (EC50 8 nmol/l) increase in intracellular ET-1 in L2 cells but not in L1 cells (Fig. 5).


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Fig. 5.   Representative dose-response curve for endothelin-1 (ET-1; 0.01-1000 nmol/l) shows levels of intracellular ET-1 in L1 and L2 cells after exposure for 18 h. After treatment, cells were washed 3 times in serum-free medium before cell pellet was prepared for analysis. Intracellular levels of ET-1 in L1 cells () remained at baseline with each concentration of ET-1. Intracellular ET-1 levels in L2 cells () showed dose-dependent increase (EC50 8 nmol/l) and followed concentrations of exogenous ET-1. Data are means ± SD of duplicate measurements in 3 cell lines from 2 animals.

Effect of exogenous ET-1 on ppET-1 and ECE-1 gene expression and regulation by TGF-beta 1. Exogenous ET-1 alone caused striking increases in ppET-1 (70%) and ECE-1 (30%) gene expression in L2 cells (Fig. 6A). Pretreatment with TGF-beta 1 followed by ET-1 resulted in a dose-dependent decrease in ppET-1 and ECE-1 transcripts (Fig. 6A). Densitometric readings for ppET-1 and ECE-1 normalized to hG3PDH following 1 ng/ml TGF-beta 1 showed a 20% decrease for ppET-1 and ECE-1 gene transcription compared with ET-1 stimulation alone (Fig. 6B). Further increases in concentration of TGF-beta 1 (10 and 100 ng/ml) resulted in greater suppression of ET-1-induced ppET-1 and ECE-1 gene expression to levels that were ~70% less than with ET-1 alone. L1 cells, on the other hand, showed higher expression of the ppET-1 and ECE-1 genes at baseline than L2 cells but responded little to either exogenous ET-1 or TGF-beta 1 and ET-1 (Fig. 7).



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Fig. 6.   A: representative (n = 3 cell lines from 2 animals) RT-PCR analysis of ppET-1 and ECE-1 gene expression in control (lane 2) L2 cells and after exposure to 10 nmol/l ET-1 for 90 min (lane 3). Pretreatment of L2 cells with 1, 10 and 100 ng/ml transforming growth factor-beta 1 (TGF-beta 1) for 18 h followed by 90-min incubation with 10 nmol/l ET-1 resulted in a dose-dependent decrease in both ppET-1 and ECE-1 gene expression (lanes 4, 5, and 6, respectively). B: densitometric readings for ppET-1 and ECE-1 transcripts in L2 normalized to hG3PDH (loading control). Open bars, ECE-1. Solid bars, ppET. Data are expressed as means ± SD of duplicate density measurements.




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Fig. 7.   A: representative RT-PCR analysis (n = 3 animals) of ppET-1 and ECE-1 gene coexpression in L1 cells (lane 2). L1 cells were unresponsive to either 10 nmol/l ET-1 alone (lane 3) or TGF-beta 1 (1, 10 and 100 ng/ml) followed by ET-1 (lanes 4, 5, and 6, respectively). B: densitometric readings for ppET-1 and ECE-1 transcripts in L1 cells normalized to hG3PDH (loading control). Open bars, ECE-1. Solid bars, ppET-1. Data are expressed as means ± SD of 2 independent density measurements.

ECE-1 activity. Basal ECE-1 activity in crude membrane fractions of L1 cells was twice that in L2 cells (L1 cells, 495.8 ± 187.4; L2 cells, 239.8 ± 80.6 ECE activity as fmol ET-1 · 40 µg-1 · 130 min-1; n = 4, P = 0.01), reflecting the difference in ECE-1 gene expression (Fig. 4). Phosphoramidon inhibited ECE-1 activity in a dose-dependent manner in both L1 and L2 cells (IC50, 0.03 µmol/l).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In a previous study, we demonstrated that normal sheep pulmonary artery shows regional variability in ppET-1 gene expression, the main pulmonary artery expressing greater levels of the ppET-1 gene than the midregion artery (9-10th generation) (37). We also showed that these differences could be recapitulated in SMCs cultured from the L2 layer. The present study extends those findings and shows that cells cultured from the adjacent L1 and L2 layers of the main pulmonary artery show phenotypic differences in the ppET-1-ECE-1 system in that ppET-1 and ECE-1 gene expression, released ET-1, intracellular ET-1 concentrations, and ECE-1 activity are greater in L1 than in L2 cells. Furthermore, L2 cells are more responsive to exogenous ET-1 than L1 cells; only L2 cells take up exogenous ET-1; and TGF-beta 1 modulates ET-1-stimulated ppET-1 and ECE-1 gene expression in L2 but not in L1 cells. These studies demonstrate phenotypic differences in the ET-1-ECE-1 system in cells from adjacent layers of sheep pulmonary artery and add to our understanding of the complex pattern of vasoactive mediator release that contributes to the regulation of vascular tone.

Heterogeneity of SMCs in main pulmonary artery. Heterogeneity of vascular SMCs is now well established in mammalian species (1-3, 17, 40), and the recent studies of Frid and colleagues (12, 13) have shown SMC heterogeneity in bovine main pulmonary artery. Our study demonstrates that cells cultured from the L1 and L2 layers of normal sheep pulmonary artery contain similar populations. In addition, at superconfluence, large superficial cells with both alpha -SM actin and SM myosin emerged from the underlying rhomboidal cells. These studies demonstrate at least two distinct types of SMCs in sheep main pulmonary artery and suggest that the L1 cells under certain conditions such as the intimal thickening in patients with chronic and primary pulmonary hypertension may give rise to a more well-differentiated SMC.

Source and function of L1 and L2 cells. The source and function of the L1 cell are not certain. Few studies have examined cells from normal animals, so our current understanding is based mainly on cells and tissues from pathological states and from embryos. For example, under pathological conditions, migrating medial and even adventitial SMCs have been suggested as a source of subendothelial SMCs (2, 17, 22, 33). Endothelial cells also have been suggested as a source. In the quail embryo, endothelial cells have been reported to differentiate into mesenchymal cells. The embryonic endothelial cells lose their endothelial markers and express alpha -SM actin (10). The present study perhaps confirms the latter notion in that the L1 cells show factor VIII activity at subconfluence and at superconfluence exhibit SMC markers.

Functionally, it is also possible that the L1 cells act as stem cells. For example, cells from the subendothelial or luminal layer have been reported to replace large areas of damaged endothelial cells in rat carotid artery following balloon injury (5), and stellate subendothelial cells containing cytoplasmic filaments have been suggested to replace damaged endothelial cells in intimal explants from bovine pulmonary artery (25). The position of the L1 cells, their close contact to the endothelial cells as well as to inner medial SMCs, and their high level of ET-1 synthesis and release (see below) confirm the notion that L1 cells may represent a cell intermediate between endothelial and mature SMCs, rather like a pericyte (31).

ET-1 synthesis and release. Vascular SMCs from aorta, coronary arteries, umbilical vein, and pulmonary artery have been shown previously to express the ppET-1 (7, 36, 37, 43) and ECE-1 (7, 36) genes. The present study demonstrates that cells cultured from the L1 and L2 layers of the pulmonary artery have a different ET-1-ECE-1 system.

ET-1 is known to upregulate ppET-1 transcripts in SMCs as well as in endothelial cells through an autocrine-positive feedback mechanism involving activation of protein kinase C (15, 29). Our study confirms a positive feedback loop for L2, but not for L1, cells of the main pulmonary artery and demonstrates coexpression of ECE-1 and ppET-1 in response to exogenous ET-1. The difference in behavior between the two cell types demonstrates that they are functionally and metabolically different. The disparate responses in the ET-1-ECE-1 system is likely to include distinctions in their ET-1 receptor populations. In rat endothelial cells, ETB has been demonstrated to regulate expression of ppET-1 mRNA (29), and in mesangial cells, ETB has been shown to modulate stimulation of ET-1 mRNA and to extend the half-life of ppET-1 transcripts (19). Human SMCs have been shown to express both ETA and ETB receptors (9, 16). Our preliminary studies suggest that the ET-1 receptor populations on L1 and L2 cells are different. The ETA and ETB transcripts are present on both cell types but are less abundant in L1 than L2 cells, and the ETB receptor shows the greatest level of expression in L2 cells (Balyakina Y and Meyrick B, unpublished data). The differences in ET-1 receptor populations on the two cell types is likely to explain, at least in part, their different responses to exogenous ET-1.

As previously described for SMCs (28), our study demonstrates that L2 cells from sheep main pulmonary artery have the ability to internalize exogenous ET-1. Internalization and subsequent externalization have been shown to occur by way of ETA and represent pathways of intracellular signaling as well as prolongation of the ET-1 vasoconstrictor effect (14, 23). In contrast to that finding, a study in which medial SMCs from normal rat aorta were grown in collagen gels failed to show contraction in response to ET-1 (40). Further studies are needed to clarify these findings.

ECE-1. ECE-1 is an important activation protease in the biosynthesis of ETs. It is a novel membrane-bound neutral metalloprotease structurally related to neutral endopeptidase 24.11 and Kell blood group protein (30, 41). Another form, ECE-2, has been described recently (11). Although that form is structurally and functionally similar to ECE-1, its pH optimum is in the acidic range. At least three isoforms of ECE-1 have been described (32, 34, 39). Immunocytochemical techniques applied to various human tissues, including the lung, have revealed that two isoforms are membrane bound and the other is cytosolic (32). All three isoforms are the result of alternative splicing of the ECE-1 gene. Our data demonstrate cell-specific differences in ECE-1 mRNA and activity in L1 and L2 cells. Basal ECE-1 activity, like ECE-1 transcripts, was higher in the L1 than in the L2 cells and parallels the synthesis of ET-1 by those cells. ECE-1 activity has been shown to be of two types depending on its sensitivity to the metalloprotease inhibitor phosphoramidon (24). Vascular SMCs have been demonstrated to have phosphoramidin-sensitive ECE-1 (24), and our data confirm this finding for both L1 and L2 cells.

Response of L1 and L2 cells to TGF-beta 1. In a previous study, we demonstrated that high levels of TGF-beta 1 gene expression and protein are present in the wall of normal sheep main pulmonary artery and are localized mainly over the medial SMCs and the medial-adventitial border; little expression is found over the intima (27). The potent growth factor TGF-beta 1 has been reported to enhance ppET-1 mRNA and ET-1 synthesis in various cultured cells, including pulmonary artery endothelium and vascular SMCs (15, 21). Whether TGF-beta 1 enhances transcription or prolongs the half-life of ppET-1 mRNA is unclear. Regulation of ET-1 by TGF-beta 1 has been suggested to involve a nuclear factor-1-binding element in the ppET-1 gene (35). Our data are the first to demonstrate that ET-1-stimulated ppET-1 and ECE-1 gene coexpression is regulated by TGF-beta 1 in L2 but not in L1 cells. The finding for L2 cells confirms our earlier report showing that TGF-beta 1 caused a 50% reduction in ET-1-stimulated intracellular ET-1 (37). The mechanism for this modulation is not certain. Cristiani and colleagues (8) reported that TGF-beta 1 downregulated ET-1 selective binding sites in a vascular smooth muscle-derived cell line. Thus it is likely that the downregulation of ET-1-stimulated intracellular ET-1 by pretreatment with TGF-beta 1 is mediated at the receptor level. Our findings suggest that in the inner media of normal pulmonary artery, regulation of ET-1-induced ET-1 synthesis, and presumably vascular tone is modulated by this peptide. The paucity of TGF-beta 1 transcripts and expression in the intimal layer of normal pulmonary artery (27) is in line with the lack of response of this factor on the L1 cells.

In summary, cells cultured from the subendothelial and inner medial layers of control sheep main pulmonary artery have well-developed but different ET-1-ECE-1 systems. The L2 cells are more responsive to exogenous ET-1 than the L1 cells, whereas the L1 cells produce greater levels of ET-1 than the L2 cells. These distinctions suggest diverse functions for the two cell types. It is likely that the ET-1-responsive L2 cells are contractile in nature and contribute to normal vascular tone. The high level of ET-1 produced by the subendothelial cell suggests that, like the endothelial cell, it is likely to be a local source of ET-1. It is possible that under pathological conditions, where endothelial injury occurs, the subendothelial cells proliferate and the thickened intima becomes the major local source of ET-1.


    ACKNOWLEDGEMENTS

We thank Tamara Lasakow for editorial assistance with the manuscript.


    FOOTNOTES

This work was supported by the National Heart, Lung, and Blood Institute Grant HL-48536. The confocal microscopy was carried out using the Cell Imaging Resource at Vanderbilt University Medical Center and studies were supported in part by the National Institutes of Health Grants CA-68485 and DK-20593.

The antibody to SM myosin was generously provided by Dr. R. S. Adelstein, Laboratory of Molecular Cardiology, National Institutes of Health, Bethesda, MD (20).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: B. Meyrick, Center for Lung Research, Vanderbilt Univ. Medical Center, MCN T-1217, Nashville, TN 37232-2650 (E-mail: barbara.meyrick{at}mcmail.vanderbilt.edu).

Received 7 June 1999; accepted in final form 4 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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