Regression of Hypertrophied Rat Pulmonary Arteries in Organ Culture Is Associated With Suppression of Proteolytic Activity, Inhibition of Tenascin-C, and Smooth Muscle Cell Apoptosis

Preparation of Explants

PAs were collected and prepared for organ culture as previously described.¹³ Freshly harvested porcine PAs obtained from the local slaughterhouse were transported to the laboratory in sterile PBS and 2% antibiotics/antimycotics (GIBCO). While the endothelial surface was kept moist and protected, the periadventitial fat was removed and PAs were cut along their long axes. Rectangular pieces of full-thickness vessel wall (4×10 mm) were cut and placed in culture.

In the second series of experiments, PAs were harvested from adult male Sprague-Dawley rats (300 g) (Charles River Laboratories, Saint-Laurent, Quebec, Canada) 21 days after a subcutaneous injection of the alkaloid toxin monocrotaline (MCT) (60 mg/kg) or saline (controls). MCT-induced PVD is well established,^{14 15} and at 21 days after injection of the toxin up to a doubling of the wall thickness of the PA is expected.⁵ The rats were sacrificed by a lethal intraperitoneal injection of sodium pentobarbital and central PAs were harvested and cleaned and explants prepared as described above. The protocols were approved in accordance with the guidelines of the Animal Care Committee of The Hospital for Sick Children (Toronto, Ontario, Canada).

Each explant was cultured in a 35-mm diameter tissue culture dish embedded in a type I collagen gel prepared as recommended (Vitrogen 100, Collagen Corp). The collagen preparation (2 mL for porcine PAs and 1 mL for rat PAs) was dispersed in culture dishes, and PAs were placed endothelium side up in the viscous solution and subsequently settled slightly into the gel during collagen fibrillogenesis, which was initiated by warming at 37°C for 2 hours. Once set, the PAs were located at the interface between the atmosphere and the top of the gel. Medium 199 (2.5 mL; GIBCO) was added, replaced after 1 hour, supplemented with 5% FBS and 2% antibiotics/antimycotics, and changed every 3 days. After 1 day, half of the cultures were “floated.” The edges of the gel were released by moving the end of a spatula around the perimeter of the dish followed by gradually loosening the bottom of the gel. PAs and conditioned media from all cultures were collected at 3, 5, and 8 days.

Movat Pentachrome Staining

This stain includes iron hematoxylin to identify elastin. Measurements of intimal and medial thicknesses were calculated. In porcine PAs, 3 equidistant measurements were recorded, and in rat PAs, 25 measurements in consecutive fields of view (290-μm² fields at an original magnification of ×400) were recorded and means calculated. Elastic laminae were assessed by counting the number of continuous elastic laminae per PA. The total amount of elastin was assessed by planimeterization of both elastic laminae and interlamellar fragments of elastin. These assessments were performed in 5 random fields per vessel at the above magnification.

Assessment of Elastolytic Activity

Conditioned media (380 μL) were incubated at 37°C with 20 μL (200 μg) [³H]elastin produced by radiolabeling purified insoluble elastin from bovine nuchal ligament (Elastin Products Co) with [³H]NaBH₄ (New England Nuclear) as previously described.¹⁶ After 24 hours, the radioactivity in 250 μL of the supernatant was determined by liquid scintillation spectrometry as a measure of elastolysis. To control for nonenzymatic degradation, radiolabeled elastin was incubated with medium from tissue-free cultures. Elastase activity was related to a standard curve generated with human leukocyte elastase (0.075 to 5.0 ng) (Elastin Products Co).

MMP Activity

Tissue extracts were prepared by homogenizing PAs at 4°C in homogenization buffer (50 mmol/L Tris-Cl, 0.2% Triton X-100, 10 mmol/L CaCl₂, and 2 mol/L guanidine HCl, pH 7.5). The supernatants were dialyzed and used for gelatin substrate zymography, as previously described.¹⁰ To identify tissue inhibitors of MMPs (TIMPs), similar gels were incubated in 4-aminophenyl mercuric acetate–activated conditioned medium for 3 hours at 37°C. After staining, dark anti-gelatinolytic bands were detected.

Western Immunoblotting

MMP-2, MMP-9, and TIMP-1 Western immunoblotting was performed on 12% polyacrylamide gels using anti-human monoclonal antibodies (1:50) (Oncogene Science) for porcine extracts and an anti-MMP-2 rabbit polyclonal antiserum for rat extracts (a kind gift from Drs M. Silverman and M. Ailenburg [Department of Medicine, University of Toronto]). An anti-human cellular TN polyclonal antibody (1:50) (Sigma) for porcine tissue and an anti-rat polyclonal antibody raised against formaldehyde-fixed TN (1:100) (a generous gift from Dr Harold Erickson [Duke University Medical Center, Durham, NC]) was used to detect TN separated on 6% gels. Visualization of immunoreactive bands was achieved with a horseradish peroxidase–conjugated secondary antibody (GIBCO) followed by enhanced chemiluminescence (Amersham) and normalized by comparison with a Coomassie blue–stained gel.

Northern Blotting

From culture, PAs were immediately placed in 5 mL of TRIzol, total RNA was extracted according to that protocol (GIBCO-BRL), and Northern blotting was performed as previously described.⁹ A 250-bp cDNA probe derived from the seventh fibronectin type III constant domain of rat TN was used on 8-μg samples. The relative quantity of TN mRNA in each sample was analyzed by densitometry and corrected for loading conditions by direct comparison with ethidium bromide–stained 28S rRNA and by hybridization with a 600-bp cDNA probe for rat GAPDH.

Immunohistochemistry

Samples were removed from the collagen gels and fixed in 2% paraformaldehyde. They were then embedded in paraffin and cut into 5-μm-thick sections. Immunohistochemistry for TN was performed with the species-specific TN primary antibodies described above (same dilutions) in an overnight incubation at 4°C. Binding was visualized using the Vectastain ABC System (Vector Laboratories). Control sections were treated with normal rabbit isotypic IgG (DAKO). Nuclei were counterstained using hematoxylin.

The relative abundance of TN was graded quantitatively in 5 random fields (290-μm² fields at an original magnification of ×400) per sample using the Image-Pro Plus program for a Macintosh computer (Media Cybernetics). Planimetry and densitometry were performed on positive staining above the designated “background.” Multiplication of the total positive area and the average density provided a relative densitometric measurement of TN deposition for each field, and means were calculated.

PCNA was detected after nuclease digestion and incubation with an anti-PCNA monoclonal primary antibody (1:100) (DAKO). Control sections were incubated with normal mouse isotypic IgG (DAKO), and a normal human skin section was used as a positive control. Antibody binding was visualized using 3,3′-diaminobenzidine (Sigma) and a substrate intensifier (Amersham). Cytoplasmic counterstaining was performed using eosin.

Apoptosis was detected by an in situ detection system following the manufacturer’s protocol (Apoptag, Oncor). On porcine sections, the secondary antibody was peroxidase-conjugated, whereas we used a fluorescein-conjugated secondary antibody on rat sections. A normal skin sample was used as a positive control, and sections incubated in the absence of either antibody or enzyme were used as negative controls. Nuclear counterstaining was performed on porcine sections with methylene green and on rat sections with propidium iodide.

The presence of proliferating and apoptotic cells was also quantitatively assessed in 10 randomly selected fields (290-μm² fields at an original magnification of ×400). The number of positive cells and the total number of cells (nuclei identified by propidium iodide counterstaining) per field were counted, a percentage of positive cells was calculated, and a mean percentage was generated.

Detection of Necrosis

Cellular necrosis was assessed with a lactate dehydrogenase release assay (Sigma) performed on 120 μL of conditioned medium and according to the manufacturer’s specifications. Background, determined using both serum-free medium and medium containing 5% FBS cultured in the absence of a tissue section, was subtracted. Assay sensitivity was determined using supernatants from PA SMCs after freeze-thaw cycles.

Statistical Analysis

Experiments were performed at least 3 times (exact numbers are indicated in figure legends), and values are expressed as mean±SEM. Statistical significance was determined using 1-way ANOVA followed by the Fisher least significant difference test of multiple comparisons to establish differences between individual groups.

MMP Activity in Porcine PAs

Because MMP-2 regulates TN deposition in cultured PA SMCs,¹⁰ net MMP activity was assessed in PA tissue extracts by gelatin zymography and reverse zymography. Gelatinolytic bands at ≈83 kDa and a triplet at ≈52, 56, and 60 kDa were detected (Figure 1⇓) and confirmed to result from MMP activity through their abrogation after incubation in EDTA (data not shown). Furthermore, some of these bands were characterized by native Western immunoblotting to be MMP-9 (83 kDa) and the active and latent (52- and 56-kDa) forms of MMP-2, respectively (data not shown). The enzyme responsible for the 60-kDa gelatinolytic band was not identified. Qualitative analysis of the zymograms showed a reproducible increase in MMP-2 activity in attached cultures on day 5 compared with day 0. This increase in MMP-2 was evident in both attached and floating cultures by day 8; however, in attached cultures, but not in floating cultures, an increase in MMP-9 was now observed. These differences were confirmed as significant (P<0.05) by densitometric analysis, the interpretation of which is limited by the nonlinear nature of the assay (data not shown).

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Figure 1.

Representative gelatin zymogram of porcine PA organ culture tissue collected from attached and floating cultures at 0, 1, 3, 5, and 8 days is shown. Bands at ≈83, 56, and 52 kDa reflecting MMP-9 and MMP-2 (active and latent form), respectively, are evident in both attached (Att) and floating (Fl) conditions. S indicates molecular weight standards.

Reverse gelatin zymography was performed to detect changes in native MMP inhibitors that would influence net MMP activity. Multiple bands corresponding to bound and free native inhibitors of MMPs were identified (data not shown). Western immunoblots for TIMP-1 were performed, identifying anti-gelatinolytic bands containing TIMP-1. However, no significant difference between attached and floating cultures was detected in any of the bands (data not shown).

TN Protein Deposition in Porcine PAs

Western immunoblotting and immunohistochemistry for TN was performed to determine whether attached versus floating culture conditions affect temporal and spatial deposition of TN. On Western immunoblot, TN was identified as a ≈220-kDa immunoreactive band that progressively increased in tissue cultured on attached collagen (Figure 2⇓). On day 5 there was a significant increase in both attached and floating gels relative to day 0 (P<0.05) but on day 8, values in floating cultures had fallen to control levels. There was, therefore, a >2-fold increase in TN on attached relative to floating PA organ culture (P<0.05).

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Figure 2.

A, Representative Western immunoblot for TN in porcine PA organ culture tissue collected from attached and floating cultures at 0, 1, 3, 5, and 8 days. B, Densitometric analyses from 3 different experiments. TN was identified in these tissues as a ≈220-kDa immunoreactive band that was increased in attached cultures and, in contrast, was reduced in floating cultures. Data are mean+SEM of 3 vessels at each time point. *P<0.05 comparing attached and floating cultures; †P<0.05 compared with day 0.

Immunolocalization of TN revealed focal rather than homogenous deposition (Figure 3A⇓ and 3B⇓). We therefore developed a semiquantitative method to evaluate the sections to take this into account. Planimeterization and densitometry of TN-immunopositive regions revealed an increase in TN on attached collagen gels that was significant by day 5 (P<0.05) (Figure 3E⇓), whereas PAs on floating collagen displayed low levels of immunostaining. The decrease in TN immunostaining preceded the decrease in TN on Western immunoblot, which suggests either a difference in the sensitivity of the methods or an initial conformational change in TN in PAs on floating collagen gels, making it less immunodense on tissue sections.

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Figure 3.

Photomicrographs of porcine PA organ culture tissue sections, although taken from an attached culture, are representative of samples collected from attached and floating cultures at 8 days and immunostained for TN (A and B) and PCNA (C) and after TUNEL assay (D). Magnification: A, ×100; B, C, and D, ×400. In panels B, C, and D, the boundary between a TN-rich and TN-poor region is illustrated on serially stained sections. C and D show that PCNA positivity (C) codistributes with the TN-rich area and apoptotic cells (D) with the TN-poor area. Bars=25 μm. Planimetric and densitometric grading of TN from porcine attached and floating cultures from days 0 to 8 is shown in panel E, and counts of proliferating or apoptotic cells calculated from numerous TN-rich and TN-poor regions (see Materials and Methods) are illustrated in panel F. Data are mean+SEM of 3 vessels at each time point (n=8 for panel F). *P<0.05 comparing attached and floating cultures; †P<0.05 compared with day 0.

Tenascin, Proliferating Cells, and Apoptosis

To assess whether TN deposition correlated with proliferating cells, immunostaining for PCNA was used, whereas apoptosis was detected by in situ terminal deoxynucleotidyl transferase–mediated deoxyuridine nick end labeling (TUNEL) assays. TN, accumulating in foci (Figure 3A⇑ and 3B⇑), colocalized directly with PCNA-positive cells (Figure 3C⇑), and inversely with apoptotic cells (Figure 3D⇑). Planimetry and densitometry of TN-rich and TN-poor foci further confirmed this association, as regions with high TN deposition had a similarly high proliferation index and low levels of apoptosis, whereas areas with reduced TN exhibited almost an absence of proliferation and had a very high percentage of apoptotic cells (Figure 3F⇑). Despite these correlations, changes in wall thickness were not observed when attached and floating vessels were compared (data not shown). If we divide TN staining into negative, minimal, moderate, and intense classifications as judged by relative densitometric units (<0.01, 0.01 to 1, >1 to 10, and >10, respectively [Figure 3F⇑]), it is conceivable that floating cultures were composed of negative and moderate patches of TN and attached cultures were composed of minimal and intense patches. Thus, in both cultures, proliferating and apoptotic cells were balanced (supported by total counts of PCNA-positive and apoptotic cells; data not shown).

Progression and Regression of Hypertrophied Rat PAs

Recent studies suggest that actively remodeling vessels are uniquely dependent on survival signals provided through interaction with the ECM.¹⁷ Consequently, we investigated whether hypertensive vessels, already in a state of active remodeling, may be more responsive to manipulation of TN by attached versus floating conditions. After MCT treatment, a 30% increase in medial hypertrophy was observed when compared with saline controls (P<0.05) (Figure 4A⇓, 4B⇓, and 4E⇓). This hypertrophy was not only perpetuated on attached collagen gels, but there was a further 25% increase by day 8 (P<0.05) (Figure 4C⇓ and 4E⇓), whereas a progressive regression of medial hypertrophy over this time frame was observed on floating collagen (40% decrease over day 0) (P<0.05) (Figure 4D⇓ and 4E⇓). The progression and regression were not recapitulated by rat saline control vessels cultured in a similar fashion (data not shown) and indicated that normotensive rat PAs behave like the porcine PAs described previously in detail.

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Figure 4.

Representative photomicrographs of Movat-stained rat PA organ culture tissue collected from attached and floating cultures at 0 and 8 days. Rat PAs taken at 21 days, the point at which they would be placed into culture, show medial hypertrophy (A), in contrast to PAs from saline-injected rats (B). On attached collagen gels, these vessels continue to thicken over the 8 days (C and E). Vessels on floating gels, however, show a progressive regression of medial thickness over the 8 days (D and E). Bar=25 μm. Measurements of medial hypertrophy (E) showed significant corresponding differences and are associated with modulation of matrix, represented as increases in elastin in attached cultures and decreases in floating cultures. This is assessed by the number of elastic laminae (F) and by relative densitometric units of elastin in the vessel wall (data not shown). Data are mean+SEM of 3 vessels at each time point (n=8 for graph of medial thickness). *P<0.05 comparing attached and floating conditions; †P<0.05 compared with day 0.

Progressive hypertrophy on attached gels was associated with a trend toward an increase in the number of elastic laminae (Figure 4A⇑, 4C⇑, and 4F⇑) and deposition of elastin (data not shown). Regression of medial hypertrophy in floating cultures was associated with resorption of elastin (Figure 4A⇑ and 4D⇑) to values comparable with those in saline control vessels in terms of number of laminae (Figure 4F⇑) and elastin densitometry (data not shown).

Elastase and MMP-2 Activity

Because serine elastases are increased in hypertensive PAs,^{3 4} they may play a direct role in the upregulation of TN through the activation of MMPs.^{11 12} Thus, elastolytic activity was examined by elastase assays comparing attached and floating cultures at 8 days. There was a 60% increase in elastin-degrading activity in attached versus floating cultures (P<0.05) (Figure 5A⇓). To determine whether differences in MMPs exist in hypertensive PAs on attached and floating collagen gels, gelatin zymography was performed on tissues harvested at day 8 (Figure 5B⇓). A predominant gelatinolytic doublet was observed at ≈56 and 52 kDa. Western immunoblotting of a similar native gel identified these bands as the latent and active forms of MMP-2, respectively (not shown); on a reducing and denaturing gel, these forms of MMP-2 migrate as ≈72- and 66-kDa species (data not shown). A decrease in the active form of the enzyme was evident by either detection method in floating relative to attached cultures (83% by Western immunoblot) (P<0.05), whereas both conditions retained similar amounts of latent enzyme. Release of MMPs into the culture medium was also assessed. Only latent MMP-2 was detected by zymography in conditioned medium from attached and floating cultures and in similar amounts. The active form of MMP-2 was not observed (data not shown).

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Figure 5.

Elastase assays were performed on MCT-injected rat PAs after 8 days in organ culture and showed reduced elastolytic activity in floating vs attached conditions (A). A decrease in proteolytic activity in floating cultures was also observed after Western immunoblotting for MMP-2 (not shown) and gelatin zymography (B). Densitometric quantification of differences in the active form of MMP-2 are shown in panel B; the amount of latent enzyme is similar in tissues on attached and floating gels. Data are mean+SEM of 3 vessels. *P<0.05 compared with attached conditions.

Deposition of Tenascin

TN deposition, assessed by immunohistochemistry, was largely negative in PAs from saline-injected rats (Figure 6B⇓). TN accumulated after MCT injection and was deposited throughout the vessel wall in a cell-associated fashion (Figure 6A⇓). TN deposition is further enhanced on attached collagen (Figure 6C⇓), in contrast to floating cultures in which TN is suppressed (Figure 6D⇓). The marked reduction of TN in floating cultures was consistent with Northern and Western immunoblotting for TN (Figure 6E⇓ and 6F⇓). There was a >50% decrease in total TN mRNA, identified as ≈7.3- and 6.4-kb alternatively spliced isoforms, with a loss of TN-immunoreactive bands (≈230, 220, and 180 kDa) in 3 independent experiments. Extrusion of TN from these cultures, examined by immunoblotting conditioned medium, was not evident.

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Figure 6.

Representative photomicrographs of TN immunohistochemistry in rat PA tissue reveal an increase in TN deposition 21 days after MCT injection (A) relative to saline-injected controls (B). PA tissue harvested after 8 days in organ culture indicate an accumulation of TN in cultures on attached gels (C), whereas floating PAs reduce TN deposition such that it is nearly absent at day 8 (D). Bar=25 μm. A representative TN Northern blot shows a decrease of total TN mRNA (≈7.3- and 6.4-kb alternatively spliced transcripts observed) in tissue on floating compared with attached collagen gels, as compared with 28S rRNA loading control (E). A marked reduction in TN protein (≈230-, 220-, and 180-kDa immunoreactive bands) was observed on Western immunoblots (F). Data are mean+SEM of 3 vessels. *P<0.05 compared with attached conditions.

Proliferation and Apoptosis

PCNA immunostaining was performed and showed a significant induction of proliferation in PAs from MCT-injected rats at 21 days compared with saline-injected controls (Figure 7⇓). The number of medial proliferating cells progressively increased on attached collagen (>2-fold by day 8) (P<0.05), whereas PAs cultured on floating collagen showed minimal evidence of proliferation, with PCNA values similar to those of saline controls (P<0.05).

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Figure 7.

Quantification of the percentage of PCNA-positive cells in PA organ culture tissue in either attached or floating conditions as determined by immunohistochemistry. PCNA positivity is observed in PAs after MCT injection, but not from saline-injected rats. Attached cultures show a progressive increase in positivity at days 3, 5, and 8, whereas floating cultures show reduced positivity on day 3, with PCNA staining being absent by days 5 and 8. Data are mean+SEM of 3 vessels at each time point. *P<0.05 comparing attached and floating; †P<0.05 compared with day 0.

We next sought to determine, by performing TUNEL assays, whether apoptosis was related to the regression in medial hypertrophy and suppression of TN in these hypertrophied rat PAs when they were floated on collagen gels (Figure 8A⇓ through 8E). To confirm that the in situ TUNEL assays were not aberrantly identifying necrotic cells as apoptotic, lactate dehydrogenase assays were performed. No significant difference was detected between attached and floating cultures and the tissue-free control (data not shown), indicating that the observed vascular regression was not due to the onset of necrosis. Normalized counts of medial apoptotic cells revealed low levels of apoptosis in MCT- and saline-injected rat PAs (Figure 8A⇓, 8B⇓, and 8E⇓). When MCT-injected rat PAs were cultured on attached collagen, there was minimal or absent apoptosis (Figure 8C⇓ and 8E⇓). Conversely, floating cultures displayed an early and sustained >6-fold induction of medial apoptosis from day 3 (P<0.05) (Figure 8D⇓ and 8E⇓). In addition, normotensive rat PAs (saline injected) did not show an induction of apoptosis on either attached or floating gels (data not shown).

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Figure 8.

Apoptosis was monitored by TUNEL assay in PA organ culture tissue. A and B, Nuclei from the PAs of MCT-injected (A) and saline-injected control (B) rats after 21 days appear normal as stained by propidium iodide. There were few TUNEL-positive cells in PA tissues on attached collagen gels in organ culture as shown on day 3 (C) and as quantified in panel E. With regression of medial thickness, apoptotic cells were identified as bright green fluorescent nuclei beginning on day 3 (D) and are quantified in panel E. Bar=25 μm. Quantification of the percentage apoptotic cells at each time point indicates that these observed differences were statistically significant (E). Data are mean+SEM of 3 vessels at each time point. *P<0.05 comparing attached and floating conditions; †P<0.05 compared with day 0.