Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

Pulmonary arteries exposed to high blood pressure or increased flow become thickened by increased cellular and extracellular components. These structural features are reversible if the heightened hemodynamic stimulus is removed, as occurs after surgical repair of mitral stenosis (1) and congenital heart disease (2). The structural changes may recede passively by reduced rates of turnover of tissue components (cellular senescence, reduced matrix synthesis), by active tissue breakdown (apoptosis, degradation of extracellular matrix proteins), or by switching from a hypertrophied cell phenotype to one that modulates vessel tone. If active breakdown of tissue components occurs after blood pressure is reduced, it might suggest that blood vessels are endowed with the capacity to restore vascular architecture in response to lowered blood pressure.

We hypothesized that proteolysis of extracellular matrix proteins occurs in pulmonary arteries after reduction in pulmonary blood pressure. We tested this postulate in rats in which remodeling of the pulmonary artery was induced by hypoxic exposure which regressed after return to normoxia. In this model, leukocytic and monocytic infiltration and cellular necrosis are notably absent in pulmonary arteries and microvessels (3), suggesting that inflammatory cells may not be an important source of proteolytic activity. Rat interstitial collagenase and elastase-like activity were examined in pulmonary artery tissue during the regression phase of structural remodeling. Rat and mouse interstitial collagenase, produced by uterine smooth muscle cells and fibroblasts (4, 5), are identical to each other but are very different from human fibroblast collagenase (matrix metalloproteinase-1 [MMP-1]). We found increased activities of collagenase and elastolytic enzyme within days of reversal of hypoxic pulmonary hypertension, coincident with marked decreases in collagen and elastin contents in pulmonary arteries. The predominant source of the increased collagenase appeared to be increased numbers of connective tissue-type mast cells, which may act as key effector cells in mediating degradation of vascular collagen.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Animals and Reagents

Outbred 6-wk-old male Sprague-Dawley rats of 200-350 g body wt, 15-17-d pregnant Sprague-Dawley rats, and adult male Hartley strain guinea pigs were obtained from Charles River Breeding Laboratories, Wilmington, MA. Metrizamide was supplied by Aldrich Chemical Company, Inc., Milwaukee, WI. Oligo(dT)_12-18 and nucleotide triphosphates (dNTPs) were purchased from Pharmacia LKB Biotechnology Inc., Piscataway, NJ. Epon-araldite was from Polysciences, Inc., Warrington, PA. 4-Aminophenylmercuric acetate (APMA), bovine serum albumin (BSA), ethylenediaminetetraacetic acid (EDTA), L-trans-epoxy-succinyl-L-leucylamido-4-guanidobutane (E-64), elastatinol, N-(2-hydroxyethyl) piperazine-N'-3-propanesulfonic acid (Hepes), -naphthyl butyrate esterase, naphthol AS-D chloroacetate esterase, NP-40, penicillin G, pepstatin A, 1,10-phenantroline, phosphotungstic acid, pepsin, and streptomycin were obtained from Sigma Chemical Co., St. Louis, MO. Porcine antimouse stomach desmin monoclonal antibody, [¹⁴C]acetic anhydride (108 mCi/mmol), and 30 nm goat antirabbit and 20 nm rabbit antirat IgG conjugated beads were from Amersham Corp., Arlington Heights, IL. Moloney murine leukemia virus (MMLV) reverse transcriptase was purchased from GIBCO BRL, Gaithersburg, MD. We obtained sodium citrate and formaldehyde from EM Science, Gibbstown, NJ, and guanidine isothiocyanate from International Biotechnologies, Inc., New Haven, CT. The source of pentobarbital sodium was Abbott Laboratories, North Chicago, IL. Heparin sodium was obtained from Rugby Laboratories, Inc., Rockville Center, NY. We bought nitrocellulose from Schleicher & Schuell, Inc., Keene, NH. Polyvinylidene difluoride (PVDF) membranes were purchased from Millipore Corp., Bedford, MA. Bovine nuchal ligament elastin was obtained from Elastin Products Co., Inc., Owensville, MO. T-Max 400 photographic film was obtained from Kodak, Inc., Rochester, NY. Toluidine blue and Lowicryl K4M were from Electron Microscopic Sciences, Fort Washington, PA. SDS, Tween 20, agarose, and acrylamide were purchased from Bio-Rad Laboratories, Hercules, CA. O.C.T. was purchased from Miles Scientific, Naperville, IL. [¹⁴C]Sodium formaldehyde (0.3 µCi/mmol) and Econofluor scintillation fluid were from I.E. du Pont de Nemours & Co., Wilmington, DE. [¹⁴C]Proline (260 mCi/mmol) was obtained from New England Nuclear, Boston, MA. Ethyl maleimide, -mercaptoethanol, phenylmethysulfanyl fluoride (PMSF), ¹²⁵I-protein A (0.1 µCi/ml), ¹²⁵I-protein G (0.1 µCi/ml), [³²P]d-cytosine triphosphate (3,000 Ci/mmol), and nonimmune rabbit and goat sera were received from ICN Biomedicals Inc., Costa Mesa, CA. Taq DNA polymerase was purchased from Perkin-Elmer Corp., Norwalk, CT. All other chemicals were reagent grade.

Antibodies

Rabbit antirat interstitial collagenase polyclonal antibody and rat uterine smooth muscle cell collagenase (6) were provided by J. Jeffrey, Albany Medical College, Albany, NY. Goat antimouse tissue inhibitor of metalloproteinase (TIMP)-1 polyclonal antibody was provided by D. Denhardt, Rutgers University, Piscataway, NJ (7). Rabbit antirat mast-cell chymase I polyclonal antibody (8) was provided by L. Schwartz, Virginia Commonwealth University, Richmond, VA. Porcine antimouse stomach desmin monoclonal antibody was from Amersham Corp.

Nucleic Acid Probes

The probes were a 900 bp Hind III complementary DNA (cDNA) fragment of rat pancreatic elastase I (9), a 1250 bp EcoR I cDNA fragment of rat uterine smooth muscle collagenase cDNA (UMRCase54) (4), a 1500 bp EcoR I cDNA fragment of human pro1(I) collagen (Hf677) (10), a 600 bp Pst I fragment of the cDNA subcloned in pIBI coding for human pro1(III) collagen (11), a 700 bp BamH 1-Hind III cDNA fragment of human -actin cDNA (pHF-A-3'ut) (12), and a 650 bp EcoR I cDNA fragment of human neutrophil elastase (pPB15) (13).

Preparation of Neutrophil Elastase Antibody

Human neutrophil elastase, prepared according to the method of Baugh and Travis (14), was further purified by separation on a carboxymethyl-cellulose column by NaCl gradient elution in pH 5.0 buffers followed by filtration in a Sephadex G-75 column. The 29-kD protein was used for immunizing rabbits, and those sera showing a single arc against purified human neutrophil elastase were used to prepare IgG fractions by ammonium sulfate precipitation.

Hypoxic Exposure and Hemodynamic Measurements

After being retained in animal quarters for 1 wk, rats were exposed to hypoxia (10% O₂, 90% N₂) at ambient pressure (15). One group was studied prior to exposure (Day 0); others were exposed to hypoxia for 1, 3, or 10 d or were exposed to hypoxia for 10 d and allowed to recover in air for 3, 7, or 14 d (referred to as Days 11, 13, 17, and 24, respectively). The number of animals used for the hemodynamic measurements was n = 5 for Day 0 and n = 6 for each time point at Days 10-24. Hypoxic animals were freely fed standard rat chow and water. Age-matched control animals were exposed to air. Final body weights were matched by feeding the amount of food consumed by the hypoxic group to the control group. Mean right ventricular pressure (RVP) (measured in animals anesthetized with 50 mg/kg pentobarbital sodium, intraperitoneally), the ratio of weights of cardiac ventricles (ratio of right ventricle to left ventricle plus septum, RV/[LV+S]), and hematocrit were measured (15). The procedures followed were in accordance with institutional guidelines.

Thickness of Muscular Pulmonary Arterioles

Thickness of muscular pulmonary arterioles (vessels with external diameters 20-120 µm) was measured as previously described (15). The number of animals used for the wall thickness measurements was n = 4 at each time point on Days 0, 10, and 24. 

Preparation of Tissues and Cells

The pulmonary artery trunk, the entire left extrapulmonary artery, and the proximal 3 mm of the right pulmonary artery were excised en bloc. All studies were done on individual tissues, except that pulmonary arteries, pancreas, and uteri (n = 6 for each organ) were pooled for extraction of total RNA and pulmonary arteries (n = 6) were pooled for immunoblots. Messenger RNAs (mRNAs) from pancreatic and uterine tissues were used as positive controls in hybridization studies using radiolabeled cDNA probes for pancreatic elastase and rat collagenase, respectively. The body of the uterus was removed from rats 30- 34 h after parturition for measurement of collagenolytic activity. Lungs were distended and fixed by perfusing the pulmonary artery with fixative at 100 cm H₂O (15), and sections were taken for immunohistochemical examination of the main pulmonary artery trunk and peripheral lung sections for muscular pulmonary arteries. For quantitation of mast cells, lungs were perfused with 10% formaldehyde (15). For immunocytology at the light level, lungs were perfused with O.C.T. and frozen in liquid nitrogen. For transmission electron microscopy, lungs were perfused with 2% paraformaldehyde and 0.2% glutaraldehyde for 30 min, dehydrated in a graded series of ethanols, and embedded at 35°C in Lowicryl K4M or at 65°C in Epon-araldite. Purified peritoneal mast cells were placed on gelatin-coated slides and stained with 0.1% toluidine blue or prepared for immunohistochemistry in a 3:7 glycine/glycerol mounting medium (0.1 M glycine, 0.1 M CaCl₂, glycerol, pH 8.6).

Hydroxyproline, Protein, and Desmosine Measurements and Collagen Synthesis

Pulmonary artery segments were homogenized; hydrolyzed in 6 normal HCl at 116°C for 48 h; and total protein, hydroxyproline, and desmosine were assayed (16). Relative collagen synthesis in pulmonary artery explants was measured by incorporation of [¹⁴C]proline into newly synthesized collagen (16). The hydroxyproline and protein were measured in the same tissues, and separate tissues were used to measure desmosine and the rate of collagen synthesis. Six animals were used for both hydroxyproline and protein assays and six animals for desmosine for each of Days 0, 10, 13, and 17, and an additional desmosine assay was done on Day 11. Measurements of hydroxyproline in air-breathing animals were performed (n = 6) on Days 10, 13, and 17 to insure there were no changes in collagen content related to growth.

Collagenolytic Activity

Degradation of soluble [¹⁴C]collagen by pulmonary artery homogenates was employed as an assay of collagenolytic activity. Type I collagen, extracted from guinea-pig skin by acetic acid solubilization and NaCl precipitation (17), was labeled with [¹⁴C]acetic anhydride (18). The sample was digested with pepsin (19), and pepsin-resistant [¹⁴C]collagen (specific activity, 6.7 × 10⁴ cpm/mg) was used for the collagenase assay. Pulmonary artery segments were excised and homogenized (Model PT 10/35; Brinkman Instruments, Inc., Westbury, NY) in 0.15 mol NaCl and centrifuged at 6,000 × g for 20 min at 4°C. The pellet was washed in 0.15 M NaCl and resuspended at 10 mg/ml in a 0.04 M Tris buffer with 0.15 M NaCl, 0.01 M CaCl₂, 250 µg/ml streptomycin, and 200 U/ml penicillin G, pH 7.5. The homogenates were frozen in liquid nitrogen and stored at 70°C until assayed. Fifty microliters of tissue homogenate were incubated with 50 µl [¹⁴C]collagen, 2 mmol APMA, and 350 µl of buffer (0.04 mol Tris, 0.15 M NaCl, 10 mmol CaCl₂, 5 mmol MgCl₂, pH 7.5) for 48 h at 37°C. After incubation, 2 M EDTA was added, and the mixture was centrifuged at 10,000 × g for 10 min. Fifty microliters of 0.04 M phosphotungstic acid and 50 µl of 2 normal HCl were added to the supernatant, which was then centrifuged for 10 min at 10,000 × g, and the supernatant was counted by liquid scintillation spectrometry (model LS 6000IC; Beckman Instruments, Inc., Fullerton, CA). The number of animals used for the collagenolytic activity was n = 6 for each of Days 0, 10, 11, 13, and 17.

Elastolytic Assay

Degradation of [¹⁴C]elastin by pulmonary artery homogenates provided a measure of elastolytic activity. Bovine nuchal ligament elastin was labeled with [¹⁴C]sodium formaldehyde by reduction with NaBH₄ (20) to a specific activity of 4.5 × 10⁵ cpm/mg. Pulmonary arteries (10 µl) were added to 500 µl of buffer (0.1 M Tris, 0.15 M NaCl, 5 mmol MgCl₂, 100 U/ml penicillin G, and 100 µg/ml streptomycin, pH 7.8) and incubated with 50 µl [¹⁴C]elastin substrate for 48 h at 37°C, and the supernatant was counted in a liquid scintillation spectrometer. Activity was defined as degradation of 1 mg elastin by 1 U porcine pancreatic elastase per 20 min. Class-specific elastolytic activity was assayed by adding the following inhibitors (21) to homogenates of pulmonary arteries obtained from Day-13 animals: PMSF (5 mmol), EDTA (1 mmol), 1,10-phenantroline (40 mmol), elastatinol (0.2 µmol), E-64 (3 µmol), and pepstatin A (1.5 µmol). Percent inhibition was estimated as ([cpm without inhibitor  cpm with inhibitor]/ cpm without inhibitor) × 100. The number of animals used for elastolytic activity was n = 6 for each of Days 0, 10, 11, 13, and 17.

Immunohistochemistry

Blocks of peripheral lung tissue, cut on a cryostat microtome (HistoSTAT; Reichert Scientific Instruments Co., Inc., Buffalo, NY) to 5-6-µm-thick sections, were fixed in 70% ethanol, washed twice with phosphate-buffered saline (PBS), and incubated for 30 min at 4°C with either rabbit or goat nonimmune serum to block Fc receptors in the tissue. Tissues were washed twice with PBS, reacted with primary antibody overnight at 4°C, washed again, reacted for 2 h with an appropriate secondary antibody conjugated with rhodamine or Texas red, and mounted using glycine/glycerol medium. Immunofluorescence was observed with a microscope fitted for epifluorescence, and images were photographed on Kodak T-Max 400 film. Tissues from 4-6 animals were studied at Days 0, 10, and 13.

Several control experiments were performed to evaluate the specificity of collagenase antibody. First, to determine the immunologic specificity of antirat collagenase antibody, the antibody was absorbed with rat collagenase prior to staining. Second, in separate experiments, rat collagenase antibody (1:50) was incubated with rat collagenase in PBS for 30 min at 37°C, held overnight at 4°C, and centrifuged at 1,500 × g for 10 min. The supernatants were tested by staining sections of pulmonary artery tissues obtained from Day 10 animals. No specific fluorescence was observed on tissues, indicating that the antibody was immunologically specific for rat collagenase. Third, an unrelated antibody, antimouse desmin, was used to stain pulmonary artery tissue, and antidesmin did not stain mast-cell granules. Antihuman neutrophil elastase was also employed as an unrelated antibody and to identify neutrophil elastase in tissue. Fourth, tissues were observed in fluorescence prior to reacting with secondary antibody to assess autofluorescence. Preincubated tissue sections showed an absence of fluorescence. Fifth, fluorescence with the conjugated secondary antibody was examined in each experiment. Sixth, the tissues were reacted with an appropriate nonimmune serum followed by conjugated secondary antibody. For all of these control experiments, weak, nonspecific fluorescence was observed. The presence of mast cells in all the tissue sections was verified by toluidine blue staining in adjacent sections. Since mature rat mast cells average ~ 13 µm in diameter (22) and tissue sections were cut at 5-6 µm thickness, it was possible to identify a mast cell in adjacent sections.

Quantitation of Mast Cells in Pulmonary Artery Trunk

Using light microscopy, we counted the total number of mast cells per cross section of main trunk pulmonary artery, the total number of cells, and computed percent mast cells. Animals were studied at Days 0, 10, 13, and 17. A segment of pulmonary artery from the outlet of the pulmonic valve to the first 3 mm of the left and right main branches was dissected and prepared for light microscopic examination as previously described (16). Six sections that were cut perpendicular to the vascular lumen were stained with toluidine blue (0.1%), and one section was randomly selected for analysis. Each cross section was examined at ×400 magnification, and cells were enumerated using an eyepiece fitted with a square graticule that allowed a field of view measuring 0.1 mm². Cells only in the tunica adventitia, the area from the external elastic lamina to the outer margin of the adventitia, were counted, since mast cells were predominately in the outermost wall of the vessels. Mast cells were identified by metachromatic staining of granules, and total vascular cells by staining of nuclei with toluidine blue (16). Counting, done in a blinded manner, was performed systematically by moving the viewing field over the entire area of the tunica adventitia. For each cross section, the number of mast cells, total cells, and the percent mast cells were determined. Results for 5-6 rats/ group were averaged.

Immunoelectron Microscopy

Sections (0.1 µm) of Lowicryl-embedded peripheral lung were reacted with rabbit antirat collagenase and with rabbit antirat mast cell chymase I antibody. In the double-labeled experiment, the secondary antibody for the collagenase was conjugated with 30-nm gold particles; the secondary antibody for mast cell chymase I was conjugated to 20-nm gold particles. Immunoreactivity was localized with a transmission electron microscope (Model CM12; Philips, Inc., Mahwah, NJ). In these experiments, immunologic specificity of rabbit antirat collagenase antibody was tested by absorption with rat collagenase prior to staining (see previous discussion) and by examining the tissue after incubation with conjugated nonimmune sera. Specificity of the goat antirat mast cell chymase I antibody has previously been demonstrated (23). Tissues from two animals at Day 10 were studied.

Enzyme Histochemical Assays

To identify mast cells, tissue sections were stained with toluidine blue (0.1%) or stained as described by Beckstead and associates (24), using the following reagents: -naphthyl butyrate esterase, which localizes esterases in monocytes and macrophages; naphthol AS-D chloroacetate esterase, which identifies trypsin-like enzymes in mast cells and neutrophils; peroxidase, which detects myeloperoxidase in monocytes and neutrophils; and acid phosphatase, which detects phosphatases in lysosomes of monocytes and macrophages. Cells were counterstained with hematoxylin, and reactivity was observed with light microscopy and photographed on Kodak T-Max 400 film.

RNA Analysis

Six pulmonary arteries were pooled, total RNA was extracted by the guanidine isothiocyanate method, and Northern blot analysis was performed as previously described (16) with 20 µg of total RNA. Nitrocellulose filters were hybridized to nick-translated cDNA probes that were labeled with a commercial kit (Boehringer Mannheim, Indianapolis, IN) to specific activities > 1 × 10⁸ cpm/µg. After hybridization, the nitrocellulose filters were washed under high-stringency conditions of 0.1× standard saline-citrate buffer (SSC 1× is 150 mmol sodium chloride, 15 mmol sodium citrate, pH 7.2) in 0.1% sodium dodecyl sulfate (SDS) at 65°C. Estimates of mRNA levels using slot-blot analysis were quantitated as previously described (16). Southern blot analysis (25) was performed using ³²P-labeled rat uterine smooth-muscle collagenase cDNA. The pooled specimens from six animals at each time point were studied (n = 3) at Days 0, 10, 13, and 17.

Polymerase Chain Reaction

Total RNA (50 µg) from Days 0 (control), 10, 13, 17, and 24 rat pulmonary arteries was employed to synthesize single-stranded cDNA by reverse transcriptase-polymerase chain reaction (RT-PCR) by adding 750 U MMLV reverse transcriptase and 50-100 pmol of oligo(dT)_12-18. An aliquot of the single-stranded cDNA was primed with the two oligonucleotide primers 5'-GACCTCATGTTCATCTTTAG-3' and 5'-CACCACAATAAGGAATTCGT-3' complementary to the rat uterine smooth-muscle collagenase cDNA (4). Amplification was performed in a 100-µl reaction mixture (50 mmol NaCl, 10 mmol KCl, 10 mmol Tris-HCl, 1.5 mmol MgCl₂, 3 mmol dithiothreitol, gelatin, 200 µmol each of dNTPs, pH 8.8) containing 2.5 U of Taq polymerase. The reaction was carried out on a DNA thermal cycler (Model 480; Perkin-Elmer Cetus Instruments, Emeryville, CA) for 35 cycles as follows: denaturation at 94°C, 1.5 min; annealing at 54°C, 1 min; extension at 75°C, 1.5 min; and final extension, 10 min. The RT-PCR products were electrophoresed on an agarose gel and blotted onto nitrocellulose, and Southern blot analysis was performed. The analyses were done on pooled tissues from six animals at Days 0, 3, 10, 13, 17, and 24.

Isolation of Peritoneal Mast Cells

Mast cells were obtained by peritoneal lavage (26) from air-exposed rats anesthetized with 50 mg/kg pentobarbital sodium administered intraperitoneally. Peritoneal cavities were instilled with 10 ml of a mast cell medium containing 10 mmol Hepes-buffered saline, 0.1% (wt/vol) gelatin, and 10 µ/ml heparin, pH 6.8. Aspirates from 3-4 rats were pooled, centrifuged at 150 × g for 15 min at 24°C, washed 3× with mast cell medium, and purified using continuous 3-9% metrizamide gradients (26). Mast cell purity, assessed by toluidine blue staining, was  96%; the yield was ~ 7 × 10⁶ mast cells per rat.

Western Blot Analysis

Western blot analysis was performed on pulmonary artery tissue and peritoneal mast cells. Pulmonary arteries were homogenized in NP-40 buffer (0.1 M Tris, pH 8.0, 1% NP-40, 0.15 M NaCl) containing 5 mmol ethylmaleimide and 2 mmol PMSF. The homogenate and peritoneal mast-cell lysate were incubated for 1 h at 4°C, centrifuged at 27,000 × g for 20 min, and stored at 70°C. Cell lysates were homogenized and incubated in the same way, and some cell lysates were incubated with 2 mmol APMA for 10 min at room temperature. Samples were denatured for 10-15 min in Laemmli's buffer containing -mercaptoethanol and subjected to SDS-polyacrylamide gel electrophoresis on a 12.5% acrylamide gel (27). After electrophoresis, the gels were blotted onto PVDF, which was blocked for 1 h with 4% dry milk powder in 0.2% Tween 20-PBS. The nitrocellulose strips were incubated overnight at 4°C with nonimmune sera, antirat collagenase, anti-mast cell chymase I antibody, or antimouse TIMP-1. The strips were then washed and incubated for 1 h with ¹²⁵I-protein G for anti-mast cell chymase and antimouse TIMP-1, or with ¹²⁵I-protein A for antirat collagenase. The strips were washed extensively, air-dried, and exposed to X-ray film for 24 h. The analyses were done on tissues pooled from six animals at Days 0, 3, 10, 13, 17, and 24. Studies were repeated four times for each antibody.

Statistical Analysis

Values are mean ± SEM. Chi-square analysis (²) was used to assess nonparametric data (28). One-way analysis of variance was done (28), followed by a Duncan's post-hoc test (29). A P value  0.05 was considered significant.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

Animals

Survival was 100% in control animals (n = 114) and was 97% in hypoxic animals (n = 386) (² = 2.45, P = 0.86, NS). No animals died after Day 5. Animals lost ~ 5% body weight in the first 3 d of exposure but regained the lost weight by Day 10. Mean body weights were not significantly different in age-matched hypoxic and control groups.

Hemodynamics and Medial Wall Thickness

Reversal of pulmonary hypertension and vascular remodeling was evaluated by hemodynamic measurements and thickness of arterial walls. Mean RVP was increased 2.5-fold at Day 10, decreased significantly by Day 11, and was at control levels at Day 24 (Figure 1A). The RV/(LV+S) ratio, hematocrit, and medial wall thickness of muscular pulmonary arterioles were increased at Day 10, decreased significantly during recovery, but remained above control at Day 24 (Figures 1B through 1D).

View larger version (27K): [in this window] [in a new window]   Figure 1.   Graphs showing measurements of regression of pulmonary hypertension. Groups of rats were exposed to air (Day 0), 10% O2 (hypoxia) at ambient pressure for 10 d, or hypoxia for 10 d, and allowed to recover in air for 1, 3, 7, or 14 d (designated Days 11, 13, 17, and 24, respectively). (A) Mean RVP. (B) Ratios of weights of the right ventricle to left ventricle plus septum (RV/[LV+S]). (C) Hematocrit. (D) Medial wall thickness of muscular pulmonary arteries. Closed circles or solid bars, animals exposed to hypoxia. Open circles or open bars, animals exposed to air. Abscissa, time in days; ordinate, measured variables. Data points and bars, mean; brackets, ± 1 SEM; (n = 5-6 for each data point in panels A through C and n = 4 for data in panel D). *P  0.05 compared with age-matched control. P  0.05 compared with Day 10 hypoxic group.

Connective Tissue Protein Turnover

Biochemical measurements were used to determine the balance between connective tissue synthesis and degradation during recovery from hypoxia. Hydroxyproline content of main pulmonary artery increased 2.5-fold and protein content increased 1.5-fold at Day 10, were reduced at Day 13 compared with Day 10, and were further decreased at Day 17 (Figures 2A and 2B). The ratio of hydroxyproline to protein (52 ± 5 µg/mg in control, n = 6) was not different in any experimental group compared with control (not shown), suggesting that collagen maintains a normal relationship to protein during recovery from hypoxia. Collagenolytic activity was not different from control at Days 10 and 11, but increased 2-fold at Day 13, decreased below control level at Day 17 (Figure 2C), and was at control level at Day 24 (not shown). Desmosine content was increased 2.5-fold at Day 10, and decreased to control between Days 11 and 13 (Figure 2D). Elastase-like activity was increased 2-fold at Day 11 and 7-fold at Day 13, and was at control levels at Day 17 (Figure 2E). Elastolytic activity was primarily of the serine protease and MMP classes, as shown by 55 ± 6% (n = 6) inhibition by 5 mmol PMSF, a serine protease inhibitor; 43 ± 9% (n = 6) by 1 mmol EDTA, a MMP inhibitor; and 21 ± 6% (n = 6) by 40 mmol 1,10-phenantroline, a MMP inhibitor. There was 89 ± 7% (n = 7) inhibition by 2 µmol elastatinol, an elastase inhibitor, and  3 ± 4% (n = 4 each) inhibition each by E-64 and pepstatin, inhibitors of cysteine and aspartyl proteases, respectively. The rate of collagen synthesis and levels of types I and III procollagen mRNAs were not increased at Days 10, 13, or 17 (Figures 2F and 2G), a finding consistent with previous observations that collagen synthesis is markedly increased early in exposure to hypoxia but returns to normal by Day 10 of hypoxia (16).

View larger version (34K): [in this window] [in a new window]   Figure 2.   Graphs depicting biochemical and mRNA changes in pulmonary arteries. Exposure conditions and designations of animal groups are described in Figure 1. (A) Hydroxyproline content. (B) Protein. (C) Collagenolytic activity. (D) Desmosine content. (E) Elastase-like activity. (F) Rate of collagen synthesis. (G) mRNA levels for type I procollagen (I) (closed triangles), type III procollagen (III) (closed circles), and -actin () (closed squares). Abscissa, time in days; ordinate, measured variables. Data points, mean; bracket, ± 1 SEM; n = 6 for each data point in panels A through E; n = 3 for data in panel F; n = 3 for six pooled samples each in panel G.

Immunoblots for Collagenase and TIMP-1

The presence of active and latent forms of collagenase was examined by immunoblot to assess activation of latent collagenase during the hypertensive episode. Immunoblots showed little detectable immunoreactive collagenase at Day 0, and a ~ 52-kD collagenase band at Days 3 to 24 (Figure 3A). The ~ 52-kD band was more prominent at Day 13 and decreased in intensity by Day 24 (Figure 3A). A second band of ~ 42 kD, consistent with the activated form of collagenase (30), was observed at Day 13 (Figure 3A). Immunoreactive TIMP-1, observed as a ~ 28.5-kD band, was faint at Day 0, increased in density at Days 10 and 13, and decreased by Day 24 (Figure 3B). Four immunoblots prepared from pooled tissues of separate animals showed similar results for collagenase and TIMP-1. Thus, collagenase was expressed during active remodeling, the active form of the enzyme was detected at the time of peak collagenolytic activity, and TIMP-1 was expressed during the time of greatest collagenase expression.

View larger version (53K): [in this window] [in a new window]   Figure 3.   Autoradiograms of immunoblots for collagenase and TIMP-1. (A) Collagenase. (B) TIMP-1. Homogenates of pulmonary artery tissues (15 µg protein) were examined during hypoxia and recovery from hypoxia. Lanes indicate samples obtained from animals exposed to air at Day 0 (C), exposed to hypoxia (10% O2) for 3 or 10 d, or exposed to hypoxia for 10 d, followed by recovery in air for 3, 7, or 14 d (designated Days 13, 17, and 24, respectively). Results of six pooled pulmonary arteries at each time are shown, and similar results were observed in three other assays of six pulmonary arteries each. In panel A, higher Mr form of collagenase (~ 52 kD) representing latent collagenase was present in tissues from animals at Days 3, 10, 13, 17, and 24. Lower Mr form of collagenase (~ 42 kD) representing active enzyme was present at Day 13. In panel B, TIMP-1 protein is faintly visible at Day 0, was increased at Days 10 and 13, and returned toward control on Days 17 and 24.

Immunolocalization of Enzymes

The cellular source of collagenase was assessed by immunolocalization. In control rats, immunoreactive collagenase was localized diffusely in the media and adventitia of muscular pulmonary arteries (Figure 4A). Little immunofluorescence was present using secondary antibody alone (Figure 4B). At Days 10 and 13, immunoreactive antibody was bright and granular (Figures 4C and 4D). Experiments with absorbed antibody confirmed the specificity of the immune reactivity (Figure 5). Findings similar to those in muscular pulmonary arteries were seen in main trunk pulmonary arteries (Figure 6). Diffuse immunofluorescence appeared at all times, and at Days 10 and 13 staining of mast cells with procollagenase antibody was apparent in the adventitia of main pulmonary arteries (Figure 6). The identity of the immunopositive-staining granular cells as mast cells was confirmed by reaction of these cells with toluidine blue and chloroacetate esterase in adjacent tissue sections (not shown). The diffusely localized immunoreactive collagenase appeared not to change during remodeling, suggesting that collagenase in non-mast cells was not increased. Immunoelectron microscopy confirmed this observation, since collagenase localized almost exclusively to mast cell granules at Day 10, with little antibody in other cells (Figure 7). Localization of the collagenase in mast cell granules was confirmed by colocalization with rat mast cell chymase I, a selective marker for rat connective tissue-type mast cells (31) (Figure 8). Specificity of the antirat collagenase antibody was demonstrated by electron microscopy as substantially reduced antibody deposition when the antibody had been absorbed by rat collagenase antigen (Figure 9). To evaluate whether a neutrophil-derived protease was present in pulmonary artery tissue from hypoxic animals, fluorescence to human neutrophil elastase was examined in Day 13 tissues; no difference in neutrophil elastase immunoreactivity was observed compared with control tissues (results not shown).

View larger version (132K): [in this window] [in a new window]   Figure 4.   Localization of rabbit antirat collagenase in pulmonary arteries. (A) Control pulmonary artery. Diffuse fluorescent staining of the tunica media of muscular pulmonary artery (180 µm external diameter). The location of the blood vessel (bv) lumen is indicated. (B) Fluorescent staining with secondary antibody (goat antirabbit IgG) is barely visible. The location of the bv lumen is indicated. (C) Day 10. Open arrows indicate locations of collagenase antibody deposition in granules of cells in tunica adventitia of bv walls. Location of alveolus (al) is indicated. (D) Day 13. Open arrows indicate locations of collagenase antibody deposition in granules and cells in the media and adventitia of the bv wall. Pattern is similar to that observed at Day 10. Bar = 20 µm, applies to all panels.

View larger version (112K): [in this window] [in a new window]   Figure 5.   Specificity of rabbit antirat collagenase in pulmonary arteries. (A) Ten-day hypoxic pulmonary artery. Immunofluorescence of blood vessel wall and granular cell reacted with rabbit antirat collagenase. (B) Adjacent tissue section reacted with the collagenase antibody, which had been absorbed with rat collagenase. The tissues exhibit a weak, nonspecific fluorescence, and the cell is faintly outlined. Bar = 20 µm.

View larger version (119K): [in this window] [in a new window]   Figure 6.   Immunofluorescence microscopy of main trunk pulmonary artery. (A) Control pulmonary artery. Fluorescent staining with secondary antibody (goat antirabbit IgG) is shown. The tunica adventitia is located at the top of each blood vessel in all panels. (B) Control pulmonary artery stained with procollagenase antibody. Diffuse fluorescent staining of the tunica media and adventitia. (C) Day 10. Open arrows indicate locations of procollagenase antibody deposition in granules of cells in adventitia of blood vessel walls. (D) Day 13. Open arrows indicate locations of collagenase antibody deposition in cells in the adventitia of the blood vessel wall. Pattern is similar to that observed at Day 10. Bar = 20 µm, applies to all panels.

View larger version (164K): [in this window] [in a new window]   Figure 7.   Immunofluorescence microscopy of muscular pulmonary artery from rat exposed to hypoxia for 10 d. Mast cell is recognized by its typical granular appearance and the centrally located nucleus. Antirat collagenase antibody (closed arrowheads) localized almost exclusively to mast cell granules. Other cells in the vicinity of the mast cell are unreactive. Inset shows a cluster of granules at higher magnification.

View larger version (154K): [in this window] [in a new window]   Figure 8.   Double-label immunoelectron microscopy of mast cell in muscular pulmonary artery of rat exposed to hypoxia for 10 d. Localization of rabbit antirat collagenase antibody (large arrowheads) and goat antirat mast cell chymase I antibody (small arrowheads) in pulmonary artery tissue from Day 11 rats. Deposition patterns of antibodies to rat mast cell chymase I and collagenase are similar and are located primarily in cytoplasmic granules, indicating the cell is a mast cell. Open arrows indicate granules with decreased staining. Bar = 1 µm.

View larger version (129K): [in this window] [in a new window]   Figure 9.   Immunoelectron microscopy of muscular pulmonary artery. (Top panel ) Muscular pulmonary artery tissue from Day 10 rat treated with rabbit antirat collagenase antibody that had been absorbed with the antigen rat collagenase, followed by an immunogold (10 nm)-labeled secondary antibody. Arrowheads indicate relatively fewer areas of antibody deposition in the cytoplasmic granules when compared with Figure 7. Immunogold particles in the extracellular matrix are also reduced from that in tissues reacted with unabsorbed antibody. (Bottom panel ) Magnified area of cytoplasm showing relative absence of immunoreactivity. Arrowheads correspond to those in top panel.

Quantitation of Mast Cells in Main Trunk Pulmonary Artery

The relative proportions of mast cells to total cells in main trunk pulmonary arteries were increased at Days 10, 13, and 17 compared with Day 0 (Table 1).

Identification of Collagenase and Chymase I in Mast Cells

Lung mast cells were not obtained in sufficient quantities for analysis. Instead, we used peritoneal mast cells ( 96% purity) for analysis of collagenase, since lung and peritoneal mast cells are both of connective tissue type (31). We identified immunoreactive collagenase ~ 52 kD and a less dense ~ 42-kD band which increased in intensity after addition of APMA, consistent with activation of the enzyme (Figure 10A, lanes 1, 2). A ~ 28-kD protein that reacted with rat mast cell chymase I antibody was also present, thus identifying connective tissue-type mast cells (Figure 10A, lane 3). Immunoreactive collagenase was distributed in a granular pattern in peritoneal mast cells, and only faint fluorescence was present in non-mast cells (Figure 10B).

View larger version (68K): [in this window] [in a new window]   Figure 10.   Collagenase in mast cells. (A) Autoradiogram of immunoblot of collagenase in rat peritoneal mast cells. Samples of lysates of peritoneal mast cells were examined for the presence of collagenase and rat mast cell chymase I by immunoblot. Lane 1: Collagenase antigen. Lane 2: Collagenase staining after activation by 2 mmol 4-aminophenylmercuric acetate. Note presence of more intense ~ 42-kb band indicating active form of collagenase. Lane 3: Rat mast cell chymase I as indicated by presence of ~ 30-kD band. (B) Texas red immunofluorescence localization of collagenase in intact rat peritoneal mast cells shows granular deposition. Inset: Note weak fluorescence in the smaller-sized cells, probably contaminating lymphocytes. Bar = 20 µm.

Analysis of mRNA Levels

Messenger RNA was used to assess collagenase and neutrophil elastase expression in pulmonary arteries. Transcripts for collagenase and neutrophil elastase were not detected in pulmonary artery tissues at any time by Northern blot analyses under low-stringency conditions, but transcripts were present in control tissues: a 2.9-kb transcript for collagenase in postpartum uterine tissue and a 1.25-kb transcript for pancreatic elastase in pancreas (not shown). Collagenase mRNA of the predicted 380-bp size (4) was not detected by RT-PCR analysis of control and hypoxic pulmonary artery tissues at 35 cycles (not shown). These results indicate low levels of collagenase mRNA in pulmonary artery tissue.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

In these studies of reversal of pulmonary vascular remodeling, we observed a clear shift in the balance of synthesis and degradation of extracellular matrix proteins in pulmonary arteries toward degradation. At Day 13, a time when collagen synthetic rate and the level of procollagen mRNAs were at control levels, collagenolytic and elastolytic activities were increased 2- and 7-fold, respectively. We also observed increased numbers of mast cells and an increased amount of immunoreactive collagenase in mast cells during development of pulmonary vascular remodeling. The activated form of collagenase was identified in pulmonary arteries just preceding the rapid decrease in collagen content following return to normoxia. These results suggest that collagenase derived from interstitial mast cells may mediate the collagenolysis that occurs during regression of pulmonary vascular remodeling.

The immunoblots showed increasing levels of both collagenase and TIMP-1 during the hypoxic period, possibly explaining the lack of collagenolytic activity during this period. Collagen breakdown was clearly evident at Day 13, a time when collagen synthesis and levels of procollagen mRNAs were not elevated. At Day 13, the active form of collagenase was apparent without a further increase in TIMP-1 level, suggesting a role for activation of collagenase in rapid breakdown of vascular collagen. After Day 13, the active forms of collagenase and collagenolysis were not detectable, and collagenase and TIMP-1 levels remained elevated. We suggest that collagenase is increased in pulmonary arteries during the hypoxic period and at Days 17 and 24, but collagenolysis is suppressed by increased levels of TIMP-1. It is the transient activation of collagenase at Day 13 that may be the key event in initiating collagen breakdown and leading to reduced vascular collagen content.

We observed an increase in percent of mast cells in main trunk pulmonary arteries of rats at Day 10 of hypoxia, which persisted until Day 24. Increased mast-cell density has been noted in pulmonary arterioles following chronic hypoxia in rats (32), in plexogenic pulmonary arteriopathy (33), and in myocardium of pressure-induced right ventricular hypertrophy in a variety of species (34). In our study, mast cells appeared to be the major source of increased immunoreactive collagenase in the main trunk pulmonary artery. Diffuse immunoreactive collagenase was observed, but this immunoreactivity did not change during hypoxia, suggesting that mast cell-derived collagenase was the major source of increased collagenase in remodeled pulmonary arteries. The mast cells were of connective-tissue type, as indicated by colocalization of collagenase and rat mast-cell chymase I, a neutral protease that distinguishes connective tissue-type from mucosal mast cells (31). Since lung mast cells could not be obtained from rats in sufficient purity for analysis, we used peritoneal mast cells, which are phenotypically identical to pulmonary artery mast cells (31). In peritoneal mast cells, collagenase was detected by immunoblot, the active form of collagenase was induced by APMA, and immunoreactive collagenase was localized in mast cell granules. These findings indirectly support our conclusion that pulmonary artery mast cells of rats express collagenase. Whether collagenase is released from mast cells at Day 13 to account for the increased collagenolytic activity cannot be determined from our results. Piecemeal degranulation, a non-IgE-mediated process found in a variety of inflammatory and neoplastic disorders, could be a mechanism for the slow release of mast-cell granule contents (22). Released collagenase may be subsequently activated extracellularly by several proteolytic enzymes such as stromelysin and rodent tryptase, a mast cell neutral protease that lacks the ability to degrade native collagen but activates prostromelysin (35).

Mammalian mast cells originate from precursors in the bone marrow and are distributed primarily in normal connective tissues (36). The connective tissue-rich microenvironment acquired by pulmonary arteries during early remodeling may mediate mast cell recruitment, since fibroblasts are important for the development and maintenance of mature mast cells (36). Our data show that collagenase mRNA was not detected by Northern blot analyses and RT-PCR in normal and remodeled pulmonary artery tissue. The absence of detectable collagenase in pulmonary arteries may indicate that the mast cells store, but do not make, collagenase. Collagenase might be synthesized in precursor cells in bone marrow. Bone is one of few adult tissues with collagenolytic activity (37), but its cellular source is not known. We speculate that the newly synthesized fibrous tissue that accumulates in early remodeling does "recruit" and maintain mast cells containing preformed collagenase. Upon removal of the stimulus that elicited remodeling, mast cells release stored collagenase, which degrades the fibrous tissue "signal" and leads to partial restoration of tissue architecture.

The increase in serine-type elastolytic activity occurred within one day of recovery from hypoxia and peaked at Day 3 recovery, concomitant with a decrease in desmosine levels. The increased elastolytic activity appeared to occur somewhat earlier (Day 11) than collagenolytic activity (Day 13). The nature of the elastolytic activity was not fully explored. Failure to detect neutrophil elastase mRNA probably indicates that elastolytic activity was not due to increased expression of neutrophil elastase. It is possible that other serine proteinasessuch as adipsin, which is expressed in hypertensive pulmonary arteries (38)may contribute to the elastolytic activity.

It is interesting to speculate whether the physiologic processes controlling proteolysis in pulmonary arteries following reduction in blood pressure are analogous to those mediating the rapid resorption of collagen and elastin from other tissues, such as the postpartum uterus. During uterine involution, collagenase is activated within 24 h of parturition (39), and collagen breakdown occurs rapidly for 48 h (40). The sources of uterine collagenase are smooth-muscle cells and fibroblasts (6). Reduced distending force may be a common physiologic stimulus to "involution" of uterus and blood vessels. Uterine involution can be delayed by inserting a foreign object into the uterine cavity, and removal of the object initiates involution (41). Abrupt reduction in wall tension in isolated segments of remodeled pulmonary arteries stimulates proteolysis (42). It is known that alteration in cell shape influences the expression of certain genes, and shape change in fibroblasts is correlated with reversible expression of the collagenase gene (43). After relief of hypoxic vasoconstriction, it is conceivable that abrupt changes in cytoskeletal architecture in responsive pulmonary artery cells could trigger the release of collagenase and related MMPs.

In conclusion, our findings suggest that pulmonary arteries acquire the capacity to break down rapidly the excess collagen and elastin that accumulate during a 10-d period of high blood pressure. The process may involve recruitment and/or maturation of pulmonary artery mast cells that serve as compartmentalized storage sites for matrix-degrading proteases. Release of stored proteases may lead to temporally controlled collagenolysis without widespread tissue damage. Although we observed an association between collagenolysis and collagen breakdown, causality has not been established. Future studies, such as in vivo demonstration that inhibition of collagenolytic activity prevents breakdown of vascular collagen, may provide direct evidence for a role of endogenous proteases in regulating vascular wall remodeling.