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Circulation Research. 1996;79:1131-1142

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(Circulation Research. 1996;79:1131-1142.)
© 1996 American Heart Association, Inc.

Articles

Tenascin-C Is Induced With Progressive Pulmonary Vascular Disease in Rats and Is Functionally Related to Increased Smooth Muscle Cell Proliferation

Peter Lloyd Jones, Marlene Rabinovitch

the Division of Cardiovascular Research, Research Institute, The Hospital for Sick Children and Departments of Pediatrics, Pathology, and Medicine, University of Toronto (Canada).

Correspondence to Marlene Rabinovitch, MD, Division of Cardiovascular Research, The Hospital for Sick Children, 555 University Ave, Toronto, Ontario, Canada, M5G 1X8. E-mail mr@sickkids.on.ca.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Tenascin-C, an extracellular matrix glycoprotein prominent during tissue remodeling, has been linked to cell migration, proliferation, and apoptosis. To determine its potential role in the pathobiology of pulmonary hypertension, we compared tenascin expression in adult and infant rat pulmonary arteries (PAs) after injection of the toxin monocrotaline. Immunohistochemistry, in situ hybridization, and Northern blot analysis demonstrated induction of tenascin in adult rat central and peripheral PA. Tenascin was not, however, detected in infant vessels, which show spontaneous regression of vascular lesions. To determine a function for tenascin, we correlated its expression with evidence of apoptosis and cell proliferation using the TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay and 5-bromo-2'-deoxyuridine labeling, respectively. Apoptosis was observed only in the adult rat PA endothelial cell layer, preceding the induction of tenascin, which colocalized both temporally and spatially with proliferating smooth muscle cells (SMCs). A cause-and-effect relationship was documented in cultured rat PA SMCs, where tenascin promoted growth in response to basic fibroblast growth factor and was a prerequisite for epidermal growth factor–induced proliferation. These data provide novel functional information suggesting that endothelial cell apoptosis precedes progressive pulmonary hypertension and that induction of tenascin may be critical to growth factor–dependent SMC proliferation.


Key Words: pulmonary hypertension • extracellular matrix • tenascin-C • cell proliferation • apoptosis


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Pulmonary hypertension may occur as a progressively fatal condition of unexplained etiology or, more often, as a major complication linked to a variety of disorders that affect the heart and lungs of children and adults (reviewed in Reference 1). In common with other vascular diseases, including atherosclerosis, restenosis, and post–cardiac transplant coronary arteriopathy, pulmonary hypertension is characterized, in its advanced stages, by intimal thickening related to increased migration and proliferation of vascular SMCs. This switch in SMC phenotype appears to be initiated by structural and functional alterations in endothelial cells2 3 and may be accompanied by influx and accumulation of inflammatory cells in the developing lesion,4 5 induction of elastases,6 which release growth factors,7 and increased expression and deposition of specific ECM components, including elastin, collagen, and fibronectin.8 9 10

In addition, ECM components may regulate endothelial and SMC behavior directly through interactions with a myriad of cell surface receptors, which transduce extracellular information to the intracellular machinery that controls cell growth, differentiation, and survival. For example, during permanent closure of the ductus arteriosus, SMC migration is accompanied by accumulation of endothelial cell hyaluronan and SMC fibronectin and by cell surface expression of the receptor for hyaluronan-mediated motility.11 Similarly, a number of studies have shown that increased deposition of fibronectin in the subendothelium and inner media of coronary arteries plays a pivotal role in the development of intimal lesions after cardiac transplantation.12 13

TN-C, variously known as hexabrachion or cytotactin (reviewed in Reference 14), is another ECM component that is prominent during normal and pathological tissue restructuring, including embryonic development,15 16 17 epithelial-mesenchymal interactions,18 wound healing,19 20 and cancer,21 22 where it is believed to modulate morphogenetic events including cell migration and proliferation. More recently, tissue culture studies demonstrate that TN-C may modulate endothelial cell sprouting,23 matrix metalloproteinase gene expression in fibroblasts,24 and ECM-dependent tissue-specific gene expression in mammary epithelial cells.25 However, in hypertensive blood vessels, efforts to define a functional role for TN-C have been limited to descriptive alterations of TN-C synthesis after wound injury26 and to the study of extrinsic factors that modulate its expression in vascular cell cultures.27 28

Although TN-C expression has thereby been implicated in growth/motility-related patterns of behavior in blood vessels, recent studies in mammary epithelium show that induction of TN-C occurs concomitantly with tissue involution,25 a developmental process characterized by high levels of apoptosis.29 30 Furthermore, TN-C–treated mammary epithelial cells are induced to undergo apoptosis in tissue culture.31 These findings are noteworthy because recent work also suggests that apoptosis may act to counterbalance SMC proliferation during vascular development32 and disease.33 34 35 36

To begin to define a functional role for TN-C in pulmonary hypertension, we compared the pattern of TN-C expression in central PAs isolated from adult and infant rats treated with the toxin monocrotaline. The development of vascular changes in both age groups is likely initiated by endothelial cell injury and fragmentation of the internal elastic lamina by 4 days.3 6 By day 8, there is extension of muscle into peripheral normally nonmuscular arteries and, by day 14, medial hypertrophy of more proximal arteries as well as increased PA pressure, resistance, and reactivity associated with right ventricular hypertrophy. Thereafter, adult and infant rats diverge in their response to monocrotaline. Whereas adult rats continue to develop progressive pulmonary hypertension and vascular changes, infant rats show spontaneous regression of pulmonary vascular changes and a lack of progression of right ventricular hypertrophy.6 Therefore, examining the temporal and spatial relationships among TN-C protein expression, apoptosis, and cell proliferation in adult and infant rats should indicate whether this matrix component is associated with the progression or regression of monocrotaline-induced pulmonary vascular disease.

We now report by immunohistochemistry that increased TN-C expression accompanies progressive vascular changes observed in the adult rats and that TN-C is expressed in regions of active SMC proliferation rather than in regions with apoptosis, which occurred within the endothelial cell layer at an earlier time point. In parallel studies, we used Northern blot analyses to show that induction and increased deposition of TN-C protein correlated with increased steady state levels of TN-C mRNA. In situ hybridization was used to confirm the induction of SMC-derived TN-C mRNA within adult central PAs and intrapulmonary vessels. To test for a cause-and-effect relationship between TN-C and stimulation of PA SMC growth, we isolated and cultured rat PA SMCs and showed that TN-C potentiates the growth of these cells in response to bFGF and appears to be a requirement for EGF-induced proliferation.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Experimental Animal Model
The rat monocrotaline model of pulmonary hypertension has been previously described in detail.3 6 In the present study, 60 adult male Sprague-Dawley rats (Charles River Breeding Laboratories Inc, Montreal, Quebec, Canada) and 30 infant rats (male and female) were assigned at random to receive either monocrotaline or physiological saline (0.9% NaCl). Adult rats were injected at 8 weeks of age; infant rats, at 8 days of age. All injections were administered subcutaneously in the hind flank; the dose of monocrotaline (Transworld Chemicals) was 60 mg/kg body wt, and the saline injection was of an equal volume. At the end of the designated experimental time point, the animals to be analyzed were killed by overdose with sodium pentobarbital (300 mg/kg). To verify the model, assessment of right ventricular hypertrophy was carried out at 14 and 28 days after injection. Immunohistochemistry, Apoptag (Oncor) staining, and BrdU studies were carried out at 7, 14, 21, and 28 days after injection of saline or monocrotaline; in situ hybridization, at 7 and 14 days after injection; and Northern blot analyses, at 7, 14, and 28 days after injection. The number of animals in each group assessed at each time point for each measurement is indicated in the text and/or figure legends.

Assessment of Right Ventricular Hypertrophy
The heart and lungs were removed en bloc, rinsed in PBS (pH 7.6), and fixed overnight in 2% paraformaldehyde at room temperature. The right ventricle and left ventricle with septum were then dissected and weighed separately, and the ventricular weights were expressed as the ratio of the right ventricle to the left ventricle plus septum.

Tissue Isolation, Routine Histology and Immunohistochemistry
Main and branch PAs were removed at the hila and were immediately fixed overnight in 2% paraformaldehyde, whereas infant tissues were perfusion-fixed in 2% paraformaldehyde. All tissues were infused with 18% and 30% sucrose before being embedded and frozen in Tissue-Tek OCT compound (Miles Scientific Division). Three 5-µm sections, one each from the right, left, and central PA, were cut onto glass slides and either stained with hemotoxylin and eosin or stored at -70°C until used.

Primary antisera that recognize TN-C (rabbit polyclonal serum, pK7) and {alpha}-smooth-muscle actin (FITC-conjugated mouse monoclonal IgG2A, clone IA4, Sigma Chemical Co) were diluted in PBSA. The empirically derived optimal concentration for each antibody was determined in pilot studies. The specificity of anti–TN-C antisera has been characterized in previous studies.25 Negative controls included omission of primary antibody and substitution of irrelevant IgG antisera (Dako Corp). Nuclei were counterstained with 0.5 mg/mL DAPI (Sigma). For TN-C immunostaining, frozen tissue sections were air-dried for 10 minutes at room temperature, and nonspecific binding sites were blocked with PBSA and 10% goat serum (Sigma). Sections were incubated overnight at 4°C with primary antibody diluted 1:1000. TN-C protein was visualized by indirect immunofluorescence using a 1:100 dilution of FITC-conjugated goat anti-rabbit antisera (Pierce). For {alpha}-smooth muscle actin staining, frozen tissue sections were fixed in acetone at -20°C for 5 minutes, rinsed in PBS, and incubated overnight at 4°C with FITC-conjugated anti–{alpha}-smooth muscle actin antibody (Sigma) diluted 1:400. Immunofluorescence was evaluated by epifluorescence using standard fluorescein excitation and emission filters. For each tissue section examined, the immunostaining for TN-C was scored as positive or negative, and the extent of localization (ie, medial-adventitial, medial, or intimal) was recorded by a consensus of two independent observers (P.L. Jones and M. Rabinovitch). An average assessment of the three sections from each animal (n=3 per condition per time point) was recorded and used in the evaluation of each group. {alpha}-Smooth muscle actin was used only to confirm whether the cells that were positive for TN-C, Apoptag, or BrdU were smooth muscle or non–smooth muscle in origin.

Northern Blot Analysis
To extract total RNA for Northern blot analysis, six central and branch rat PAs (base of main PA to hila) were taken, as well as a slice of the apex of lung tissue from six separate animals. For PAs, the adventitia was gently removed by scraping with a scalpel. These tissues (arteries and lung) were separately homogenized in 3 mL of RNAzol/100 mg tissue (Cinna/Biotex). The homogenate was mixed with 1/10 vol of chloroform and centrifuged at 12 000g for 15 minutes at 4°C. The aqueous phase was mixed with an equal volume of isopropanol before precipitation at -20°C overnight. Samples were centrifuged at 12 000g for 15 minutes at 4°C, and the resulting RNA pellets were washed twice in 75% ethanol. A 15-µg sample of total RNA per lane was loaded on a 1% agarose/formaldehyde gel and transferred to a nylon membrane (Hybond-N, Amersham Life Science Inc) by capillary transfer for 12 hours and then cross-linked by exposure to UV radiation. Hybridizations were performed with a 32P-labeled random-primed probe prepared from a 250-bp cDNA derived from the seventh fibronectin type III constant domain of rat TN-C. The relative quantity of TN-C mRNA in each sample was analyzed by densitometry and corrected for loading conditions by direct comparison with 28S rRNA measurements detected after ethidium bromide staining of agarose gels.

Riboprobe Preparation and In Situ Hybridization
Rat TN-C cDNA corresponding to the seventh universal fibronectin type III domain subcloned into the EcoRI site of pGMEM-7Zf was linearized with XhoI and BamHI (Promega Corp) to produce antisense and sense DNA templates, respectively. Labeled RNA probes were synthesized by in vitro transcription with SP6 or T7 RNA polymerases using digoxigenin-labeled UTP as substrate (DIG RNA labeling kit [SP6/T7], Boehringer-Mannheim). After labeling, riboprobes were treated with RNase-free DNase for 15 minutes at 37°C, ethanol-precipitated, and resuspended in distilled water at 37°C for 30 minutes. The transcripts were analyzed on agarose gels after ethidium bromide staining, and the yield was estimated densitometrically by comparison with a control RNA of known concentration (Boehringer-Mannheim). Immunohistochemical detection of digoxigenin-labeled RNA on nylon membranes revealed equivalent levels of labeling between sense and anti-sense RNAs.

PAs or lungs isolated from monocrotaline-treated adult rats were fixed overnight in 4% paraformaldehyde at 4°C and sequentially dehydrated in 70% ethanol and xylene before being embedded in paraffin. Tissue sections of 5 µm each were mounted on silanized slides and dried overnight. Next, sections were deparaffinized in xylene and graded alcohols (100% to 50%), rinsed in distilled water, and treated with 1 mg/mL proteinase K in TE (10 mmol/L Tris [pH 7.5] and 1 mmol/L EDTA) at 37°C for 25 minutes. Prehybridization was carried out at 45°C for 1 hour in buffer containing 50% deionized formamide, 3 mmol/L NaCl, 10 mmol/L Tris (pH 7.5), 1 mmol/L EDTA, 10% dextran sulfate, 1% blocking reagent (DIG detection system, Boehringer-Mannheim), 160 µg/mL tRNA, and 1 mg/mL yeast total RNA. Probes were denatured in hybridization buffer (300 ng/mL) at 80°C for 10 minutes, cooled on ice, and incubated with tissue sections overnight at 45°C. After hybridization, sections were washed once with 2x SSPE (300 mmol/L NaCl, 20 mmol/L NaH2PO4·2H2O, and 2.5 mmol/L EDTA) at room temperature for 5 minutes and twice with 0.2x SSPE at 50°C for 1 hour each. Sections were then washed in PBS (pH 7.6) for 5 minutes at room temperature, incubated in blocking buffer (2% blocking reagent, 100 mmol/L Tris [pH 7.5], and 150 mmol/L NaCl) for 45 minutes at room temperature, and rinsed at room temperature in BSA wash buffer containing 1% BSA, 0.3% Triton X-100, 100 mmol/L Tris (pH 7.5), and 150 mmol/L NaCl. Alkaline phosphatase–conjugated anti-digoxigenin antibody (DIG nucleic acid detection kit, Boehringer-Mannheim) diluted 1:500 in blocking buffer was incubated with sections overnight at room temperature. Sections were rinsed three times for 2 minutes each in BSA wash buffer and then incubated in blocking buffer for 30 minutes at room temperature. Before detection of alkaline phosphatase/digoxigenin-labeled RNAs, sections were prewashed in 100 mmol/L Tris (pH 7.5), 100 mmol/L NaCl, and 50 mmol/L MgCl2 and then treated for 30 minutes in color substrate (nitro blue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate toluidine salt in dimethylformamide) diluted in prewash buffer. Once the desired color intensity was attained, sections were washed in TE at pH 8 (10 mmol/L Tris and 1 mmol/L EDTA), rinsed in distilled water, and dehydrated in graded alcohols (50% to 100%) and xylene before mounting in Permount reagent (Fisher Scientific).

Detection of Apoptotic Cells
DNA fragmentation is a characteristic feature of apoptotic cells and was detected using the Apoptag kit (Oncor). Briefly, frozen PA tissue sections derived from saline- and monocrotaline-treated adult or infant rats were postfixed in a 2:1 mixture of ethanol/acetic acid at -20°C for 5 minutes. Tissue sections were next incubated with equilibration buffer for 1 hour at 37°C with terminal deoxynucleotidyltransferase in the presence of digoxigenin-11–dUTP and –dATP and thereafter in stop buffer for 30 minutes at 37°C. Next, sections were incubated with anti-digoxigenin fluorescein antibody for 30 minutes at ambient temperature, washed in PBS, mounted in Elvanol, and viewed by epifluorescence using standard fluorescein excitation and emission filters. We assessed each section at each time point for the presence of fluorescent apoptotic nuclei, and in parallel sections, we determined their origin (smooth muscle, non–smooth muscle, or endothelial) as described above.

Assessment of Cell Proliferation
Identification of proliferating cells in PA tissue isolated from saline- and monocrotaline-treated rats was achieved using in vivo labeling of animals with BrdU, followed by immunohistochemical detection with an anti-BrdU mouse monoclonal antibody (cell proliferation kit, Amersham Life Science Inc). Briefly, rats were given an intraperitoneal injection of labeling reagent (Amersham Life Science Inc) at a dose of 1 mL/100 g body wt and killed 2 hours later. PAs were isolated and processed for sectioning as described above. Frozen tissue sections were immediately treated with 100% acetone at room temperature, and endogenous peroxidase activity was quenched with 2% H2O2 in methanol for 25 minutes at room temperature. Sections were rinsed in PBS (pH 7.6) and incubated with nuclease/anti–5-BrdU antibody for 1 hour at ambient temperature. Samples were then incubated sequentially with a peroxidase-conjugated anti-mouse IgG and 0.5 mg/mL of diaminobenzidine substrate (Sigma). Similar sections were used to colocalize proliferating cells with TN-C, apoptosis, or {alpha}-smooth muscle actin.

Cell Culture
Vascular SMCs were isolated from PAs of 300-g adult male Sprague-Dawley rats. Briefly, arteries were harvested from anesthetized animals and placed in cold sterile PBS (pH 7.6). Endothelium was removed by gently scraping the luminal surface with a scalpel blade, and the adventitia was also removed from the vessel. The medial layer was minced using scalpel blades and incubated at 37°C in M199 (GIBCO-BRL) supplemented with 0.1% collagenase I (Sigma) and 0.1% BSA (Boehringer-Mannheim) for 1 hour with gentle rotation. Tissue was collected by centrifugation at 300g, resuspended in M199/collagenase I solution, and incubated overnight at 37°C with gentle rotation. SMCs were harvested by centrifugation and were routinely maintained in M199 containing 10% heat-inactivated FBS (Intergen), 10 U/mL penicillin G sodium, 10 µg/mL streptomycin sulfate, 0.25 µg/mL amphotericin B, and 0.1 mg/mL gentamicin sulfate (GIBCO-BRL). Cells were passaged by trypsinization using 0.05% trypsin/EDTA (GIBCO-BRL). Vascular SMCs were identified by their characteristic hill-and-valley morphology and immunohistochemical staining for {alpha}-smooth muscle actin. For attachment efficiency and growth studies, cells were cultured in M199 supplemented with 0.1% BSA plus antibiotics and antimycotics. All experiments were performed in triplicate using cells between passages 2 and 4.

Preparation of Collagen Gels
Collagen gels were prepared according to the methods of Wren et al37 and Elsdale and Bard.38 Briefly, 0.8 mL of a 3.1 mg/mL solution of bovine dermal type I collagen (Vitrogen 100, Collagen Corp), 0.1 mL of 0.1 mol/L NaOH, and 0.1 mL of 10x PBS were mixed at 4°C for a final collagen concentration of 2.48 mg/mL. To determine the effect of TN-C on SMC attachment and growth, neutralized collagen was supplemented with 15 µg/mL of human TN-C protein (Chemicon International). Pilot studies showed that plating SMCs on TN-C alone caused a variable response perhaps due to inconsistencies in the concentration of TN-C to which the cells were exposed. The concentration of TN-C chosen to supplement the gels was the first that gave a response in preliminary studies, where a range of concentrates was used. Aliquots (1 mL) of collagen (±TN-C) were added to each 35-mm-diameter tissue culture dish, and fibrillogenesis was initiated overnight in a humid 5% CO2 environment at 37°C. Before use, the gels were rinsed three times with M199 containing 0.1% BSA.

Analysis of Smooth Muscle Attachment and Growth on Collagen Gels
Confluent cultures of rat PA SMCs were serum-starved in M199/0.1% BSA for 48 hours. Cells were collected by trypsinization and centrifugation, and cell number was determined using an improved Neubauer hemocytometer (American Optical). Cell pellets were resuspended in M199/0.1% BSA plus 0.5% serum, and a 1 mL aliquot containing 2x104 cells was seeded onto the surface of each gel. All experiments were performed in triplicate. Three hours after seeding of cells onto the gels, attachment efficiencies were determined by counting the number of cells in the medium using a hemocytometer. The number of attached cells (number of cells seeded minus the number of cells in the medium) was expressed as a percentage of the number of cells seeded. To assess the number of cells retained in the collagen, gels were digested with 1 mg/mL collagenase type II (Sigma) for 1 hour at 37°C. Free cells were pelleted by centrifugation at 4°C for 10 minutes at 300g and suspended in PBS containing 0.05% trypsin, and cell number was determined by counting aliquots in triplicate using a hemocytometer.

To determine the effect of TN-C and growth factors (bFGF and EGF) on SMC proliferation, 2x104 cells were plated in triplicate onto gels as described above. Three hours later, cells were rinsed in M199 and cultured for an additional 21 hours in M199/0.1% BSA. Twenty-four hours after plating, cells were cultured in M199/0.1% BSA either with or without 2 ng/mL bFGF (Upstate Biotechnology Inc) or 50 ng/mL EGF (GIBCO-BRL). The number of cells retained on collagen gels was determined 72 hours after plating. The doses of EGF and bFGF chosen to be stimulatory to SMC growth were determined in pilot studies and previously published by our group.7

Statistical Analyses
Measurements of right ventricular hypertrophy in the animal groups and SMC attachment efficiency and growth in cultured cells were compared by oneway ANOVA and Student-Newman-Keuls post hoc analysis. A value of P<.05 was considered statistically significant. Mean±SEM values are given in the figures.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Measurement of Right Ventricular Hypertrophy
In monocrotaline-treated adult rats, the development of right ventricular hypertrophy was observed between 14 and 28 days after injection (P<.05), as judged by an increase in the ratio of right ventricular weight to left ventricle plus septum weight (n=6 per group) (Fig 1ADown). In contrast, right ventricular hypertrophy did not develop in saline-treated adult rats (n=6 per group) (Fig 1ADown) or in infant rats (n=4 per group) (Fig 1BDown) over this time frame. Therefore, consistent with our previous studies,3 6 these data indicate that adult rats differ from infants in their development of progressive pulmonary vascular disease following injection of monocrotaline.



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  		Figure 1. Right ventricular hypertrophy in monocrotaline-treated adult rats. A, In adult rats, the ratios of right ventricular weight relative to the left ventricle plus septum weight increased significantly between 14 and 28 days after monocrotaline injection. No significant differences were observed in saline-treated adult rats. Values are mean±SEM (n=6 per time point, *P<.05). B, In infant rats, no significant differences in the ratios of right ventricular weight relative to the left ventricle plus septum weight were noted between 14 and 28 days after either saline or monocrotaline injection. Values are mean±SEM (n=4 per time point, P<.05).

Tenascin Protein Deposition in Monocrotaline-Treated Adult Rat PAs
The potential contribution of TN-C to progressive versus nonprogressive pulmonary hypertension was first assessed by comparing the spatial and temporal deposition of TN-C protein in saline- and monocrotaline-treated adult and infant rat central PAs (three animals per group per time point). Immunohistochemical staining for TN-C was negative in PAs from control (Fig 2ADown) and 7-day post–monocrotaline-treated adult rats (data not shown). In addition, TN-C staining was not observed in infant rats in either the control or monocrotaline-treated groups at each time point examined (data not shown). In contrast, high-level focal expression of TN-C was consistently seen in the adventitial and outer medial cell layers of all monocrotaline-treated adult rats from day 14 onward (Fig 2BDown). By day 21, abundant TN-C staining was also detected in the medial cell layer (Fig 2CDown), and by day 28, TN-C was apparent at high levels in the vicinity of the developing neointima (Fig 2DDown). Similar sections stained with an antibody to {alpha}-smooth muscle actin identified tenascin-positive cells as likely to be smooth muscle in origin (data not shown).



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  		Figure 2. Representative photomicrographs of immunofluorescent staining for TN-C in PAs of saline (A) and monocrotaline-treated (B through D) adult rats. Negative staining for TN-C was seen in PA tissue isolated from saline-treated animals (A). The elastic laminae show intense autofluorescence. In contrast, by day 14, focal deposition of TN-C was observed in the outer medial and adventitial layers of monocrotaline-treated rats (B). By day 21, TN-C was seen throughout the media (C), and by day 28, it was also apparent in close proximity to the developing neointima (arrow, D). Bar=35 µm (n=3 per time point).

Tenascin mRNA Expression in Monocrotaline-Treated Adult Rat PAs and Lung Tissue
Northern blot analysis showed that TN-C mRNA expression increased during monocrotaline-induced progressive pulmonary vascular disease both in PA and lung tissue (Fig 3ADown). In PA tissue, TN-C mRNAs were detected from day 14 onward (Fig 3ADown, top). These data indicate that induction and/or increased expression of steady state levels of TN-C mRNA parallel the increase in TN-C protein deposition observed by immunohistochemistry in PAs. In lung tissue, TN-C mRNAs were detected by day 7 after the injection of monocrotaline (Fig 3ADown, bottom). In addition, two TN-C isoforms, with sizes of {approx}7.3 and 6.4 kb, were expressed in both tissues, suggesting that alternative splicing of TN-C mRNA may accompany its upregulation during progression of the disease. Furthermore, densitometric analysis revealed an {approx}1.5-fold increase in TN-C mRNA, both in PA and lung tissue, between days 14 and 28 after monocrotaline injection (Fig 3BDown).



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  		Figure 3. A, Northern blot analyses for TN-C in adult pulmonary artery (top) and lung (bottom) tissue isolated at 7, 14, and 28 days after monocrotaline injection are shown. Two TN-C isoforms, with approximate sizes of 6.4 and 7.3 kb, were observed from 14 days after injection in PA and from 7 days after injection in lung tissue. B, Densitometric analysis of the 7.3-kb TN-C isoform from autoradiograms shown in panel A, normalized to 28S rRNA loading controls, showed that steady state TN-C mRNA levels increase with progressive pulmonary vascular disease.

Next, by in situ hybridization, we determined the spa-tial pattern of TN-C mRNA expression within adult lung tissue. Consistent with our immunohistochemical and Northern blot analyses, at 14 days after monocrotaline injection, TN-C mRNA localized to cells in the outer media and inner adventitia of large muscularized arteries (Fig 4ADown) to SMCs of small arteries (Fig 4BDown) as well as to large airway epithelial cells (Fig 4CDown). The specificity of the TN-C anti-sense riboprobe was demonstrated by hybridization with a TN-C sense riboprobe in an adjacent tissue section in which no signal for TN-C was observed either in PA (data not shown) or lung tissue (Fig 4DDown). Similarly, in tissue isolated from control animals, TN-C mRNA was not detected either in large or peripheral intrapulmonary arteries or in the airways using either antisense (Fig 5A and 5BDownDown) or sense riboprobes (Fig 5C and 5DDownDown). Together, these Northern blot and in situ hybridization data establish that during the development of progressive monocrotaline-induced pulmonary hypertension, TN-C mRNA is induced and upregulated in central and peripheral (intrapulmonary) PAs.



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  		Figure 4. Representative photomicrographs of in situ hybridization with digoxigenin-labeled TN-C antisense (A through C) and sense (D) riboprobes in lung tissue isolated from monocrotaline-treated rats at 14 days after injection. Rat TN-C cDNA corresponding to the seventh universal fibronectin type III domain subcloned into the EcoRI site of pGMEM-7Zf was linearized to produce antisense and sense DNA templates, respectively. Labeled RNA probes were synthesized by in vitro transcription with SP6 or T7 RNA polymerases using digoxigenin-labeled UTP as substrate. TN-C–positive cells contain numerous dark blue granules. TN-C mRNA transcripts are localized to outer medial cells in a large intrapulmonary artery (A) and to smooth muscle cells in a small artery at alveolar wall level (B) as well as to large airway epithelial cells (C). Note the absence of TN-C mRNA transcripts in the artery or airway with the sense probe (D). L indicates lumen. Bar=25 µm.



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  		Figure 5. Representative photomicrographs from saline-treated control rats at 14 days after monocrotaline injection. Rat TN-C cDNA corresponding to the seventh universal fibronectin type III domain subcloned into the EcoRI site of pGMEM-7Zf was linearized to produce antisense and sense DNA templates, respectively. Labeled RNA probes were synthesized by in vitro transcription with SP6 or T7 RNA polymerases using digoxigenin-labeled UTP as substrate. In situ hybridization with antisense (A and B) and sense (C and D) riboprobes revealed negative staining for TN-C mRNA in intrapulmonary vessels (A and C) and large airway epithelial cells (B and D). Bar=25 µm.

Apoptosis Within the Endothelial Cell Layer Precedes the Onset of TN-C Expression
Recent studies indicate that actively remodeling vascular tissue contains apoptotic cells (reviewed in Reference 39). Also, since TN-C is known to induce apoptosis in fully differentiated epithelial cells,31 we next determined whether increased TN-C protein deposition coincided spatially and temporally with apoptosis. To achieve this, we evaluated apoptosis in PAs isolated from saline- and monocrotaline-treated adult and infant rats using in situ end-labeling of fragmented DNA. No apoptotic nuclei were detected in PAs of control adult animals (Fig 6ADown and 6B) or in control and experimental groups of infant rats (data not shown). In contrast, apoptotic cells were consistently observed on the luminal vessel surface of monocrotaline-treated adult rat PAs from 7 days onward (Fig 6C and 6DDownDown). These apoptotic cells were negative for {alpha}-smooth muscle actin (data not shown), suggesting, on the basis of their location, that they are likely endothelial in origin. This observed temporal and spatial dissociation of apoptosis and TN-C deposition indicates that although programmed cell death may be a key event in the early development of progressive monocrotaline-induced hypertensive pulmonary vascular disease, TN-C does not appear to regulate this process directly. However, we cannot exclude the possibility that at 28 days after monocrotaline injection, TN-C, which is now apparent in the subendothelium, may be contributing to the apoptosis seen in the endothelial cell layer.



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  		Figure 6. Representative photomicrographs showing Apoptag (Oncor) staining of PAs isolated from saline-treated (A and B) and monocrotaline-treated (C and D) adult rats. Apoptosis was assessed at 7 days (A and C) and 28 days (B and D) after injection. Apoptotic cells, identified by the presence of fluorescent nuclei (arrow), were observed within the endothelial cell layer in monocrotaline-treated adult rat PAs from 7 days after injection. Apoptotic nuclei are indicated with an arrow. Bar=25 µm.

Proliferating Cells Colocalize With Tenascin-Rich Regions of the Vessel Wall
To determine whether TN-C contributes to progressive hypertensive pulmonary vascular disease related to increased SMC proliferation, we assessed the spatial relationships between TN-C deposition, BrdU incorporation, and expression of {alpha}-smooth muscle actin. Immunostaining of serial tissue sections for TN-C and BrdU at 14 days (Fig 7ADown) and 28 days (Fig 7BDown) after monocrotaline injection showed that pockets of proliferating cells colocalized exclusively with TN-C-rich regions of the vessel wall (Fig 7A and 7BDownDown, insets). Furthermore, immunostaining of adjacent tissue sections with anti–{alpha}-smooth muscle actin antibody demonstrated that proliferating cells in monocrotaline-treated rats, including those that appeared adjacent to the luminal surface of the vessel, were likely to be smooth muscle in origin (Fig 8Down). These data support our initial observation that induction of TN-C accompanies progressive structural alterations in hypertensive blood vessels and indicate that TN-C alone, or in concert with other factors, may provide a microenvironment conducive to SMC growth.



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  		Figure 7. Representative photomicrographs showing colocalization of BrdU-positive cells (A and B) with regions of TN-C protein deposition (insets, A and B) in PAs isolated at 14 days (A) and 28 days (B) after monocrotaline injection. Cells that are actively proliferating incorporate BrdU and were detected using immunoperoxidase staining as indicated with arrows. Note the overlapping distribution of TN-C and proliferating cells in monocrotaline-treated PAs. Bar=20 µm (n=3 per time point).



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  		Figure 8. Representative photomicrographs of immunofluorescent staining for SMCs using a fluorescein-conjugated anti–{alpha}-smooth muscle actin antibody. Cell nuclei stained with DAPI appear blue. PAs isolated from a saline rat (A) and a monocrotaline-treated rat (B) at 28 days after injection are shown. In the saline-treated control vessel (A), {alpha}-smooth muscle actin–positive cells (green and blue) are restricted to the medial SMC layer, whereas in the vessel from the monocrotaline-treated rat (B), {alpha}-smooth muscle actin–positive cells are occasionally seen on the luminal surface of the vessel (thin arrows) and in the inner adventitia (thick arrow). Bar=50 µm (n=3 per time point).

Tenascin Cooperates With bFGF and EGF to Promote Vascular SMC Growth
Having established that TN-C is upregulated in progressive hypertensive pulmonary vascular disease and is intimately associated with proliferating cells, we reasoned that TN-C may contribute to this disease by promoting SMC proliferation. To test this directly, we first compared the attachment efficiencies of adult PA SMCs on type I collagen matrices, either with or without the incorporation of exogenous TN-C. Approximately 70% of SMCs attached to type I collagen substrates within 3 hours after seeding (Fig 9ADown). Incorporation of exogenous TN-C into collagen matrices did not significantly affect the ability of rat PA SMCs to attach to this substrate (Fig 9ADown). Therefore, any differences in cell number that may occur in longer-term culture could not be attributed to differences in cell adhesion to TN-C.



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  		Figure 9. Effect of exogenous TN-C (15 µg/mL) on PA SMC attachment (A) and growth factor–dependent proliferation (B). No significant differences (P<.05) in attachment efficiency were noted between cells plated on type I collagen alone or on TN-C-supplemented gels (A). Similarly, SMC growth in serum-free medium (SFM) was unaffected by addition of exogenous TN-C. In contrast, addition of bFGF or EGF to TN-C-treated cultures resulted in a significant increase in cell number. Values represent mean±SEM. *P<.05 vs corresponding SFM level; {dagger}P<.05 for difference related to TN-C.

Next, we determined the ability of TN-C to support SMC growth under serum-free conditions. No significant differences in cell number were noted on collagen- and TN-C–supplemented gels over the 3-day time course of the proliferation experiments (Fig 9BUp). In addition, no significant differences in cell number were observed in cells cultured on TN-C substrate alone, even in response to a higher concentration than that used in the above experiments (data not shown). This would minimize the possibility of contaminating growth factors in the TN-C substrate. In contrast, by supplementing serum-free medium with either bFGF or EGF, significant increases in cell proliferation were observed on TN-C–enriched matrices (Fig 9BUp). In addition, whereas bFGF promoted cell growth on both substrate types, EGF-dependent SMC proliferation was highly dependent on incorporation of exogenous TN-C into the collagen gels (Fig 9BUp).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Previous studies have documented TN-C expression in vascular pathology, but its functional significance during the development of pulmonary vascular disease has remained obscure. Our present data indicate that induction of TN-C is associated with progressive vascular changes related to SMC proliferation. We have established both a temporal and spatial pattern for the induction of TN-C and have correlated the localization of TN-C protein with increased mRNA levels in the PAs and intrapulmonary vessels of monocrotaline-treated adult rats. Although expression of TN-C protein was apparent over a time in which we have previously documented active cell proliferation,6 we now show a focal pattern of colocalization of BrdU-positive cells with TN-C deposition. These features are absent in monocrotaline-treated infant rats, which fail to develop progressive vascular changes. In addition, a direct relationship between TN-C and SMC proliferation was documented in cell culture studies in which we showed that the response of quiescent adult rat PA SMCs to mitogens is enhanced, in the case of bFGF, and dependent, in the case of EGF, upon contact with TN-C; these features have been reproduced in subsequent studies. Whereas in other cell and tissue systems, TN-C has been related to the onset of apoptosis,31 we now provide evidence that dissociates these features pathophysiologically. Apoptosis was absent in infant rats, which fail to develop pulmonary vascular changes, but it did occur within the endothelial cell layer of adult animals, where it preceded the induction of TN-C both temporally and spatially. Therefore, these results suggest that apoptosis may arise as a consequence of progressive pulmonary vascular changes or that it may contribute to the disease mechanism. We do not know why apoptosis was absent in infant rats; however, the greater regenerative potential of infant tissue compared with adult tissue may account for the differences observed.

The link between TN-C expression and cell proliferation is supported by studies in cultured cells40 41 and in actively remodeling tissues.42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 For example, recent studies by Chung et al40 show that bFGF-dependent endothelial cell growth is facilitated by the alternatively spliced region of TN-C, and TN-C also appears to cooperate with EGF to promote proliferation of aortic SMCs derived from spontaneously hypertensive rats.41 In vivo, TN-C expression is associated with the proliferating uterine epithelium,42 and in denervated skeletal muscle, increased proliferation of fibroblasts coincides with the onset of TN-C synthesis.43 TN-C is also found in the adult gut, close to migrating and constantly renewing cells of the epithelium,44 and in proliferating epidermal cells in hyperproliferative skin disorders.45 46 47 TN-C expression is increased in the stroma that surrounds pulmonary fibroses,48 and in tumors it is often associated with the malignant but not benign state.21 49 50 In cardiovascular tissues, TN-C expression has been described in the developing heart,51 52 in normal blood vessels,53 54 and in carotid arteries after experimental balloon injury26 and has recently been linked to cell proliferation in neointimal lesions in polytetrafluoroethylene grafts55 and in vessels from spontaneously hypertensive rats,56 but by immunohistochemistry only. Apoptosis has also been described in the restenosis model,33 but the relationship between TN-C, proliferation, and apoptosis has not been investigated.

Our data indicate that the initial induction of TN-C mRNA and accumulation of TN-C protein in the adventitia and outer media is focal, and only with disease progression is TN-C protein distributed more widely throughout the vessel wall. A number of factors may have contributed to this initial heterogeneous pattern of TN-C deposition in the arteries of experimental animals in our studies. Interleukin-1ß has been shown to induce TN-C expression in a variety of cell types,57 including SMCs (P.L. Jones and M. Rabinovitch, unpublished data). Therefore, recruitment of inflammatory cells may take place in the adventitia, and the soluble factors produced by these cells, including interleukin-1ß, may then stimulate TN-C expression. Further, the induction of a more homogeneous pattern of expression of TN-C with progressive disease may be related to the previously described paracrine induction of expression, which has been documented in vascular and other pathologies.58 59 This concept is reinforced by the fact that interleukin-1 has been shown to play a role in the progression of pulmonary hypertension in monocrotaline-treated adult rats.60

TN-C expression may also be regulated by endogenous vascular elastase, which plays a critical role in the pathophysiology of monocrotaline-induced pulmonary hypertension in adult rats.61 62 63 We have shown that endogenous elastases produced by SMCs liberate biologically active bFGF from SMC ECM stores.7 bFGF is a potent vascular SMC mitogen and is also known to stimulate TN-C expression.64 65 Therefore, the second increase in elastase induction observed later in the course of monocrotaline-induced adult (but not infant) disease is temporally associated with the induction of TN-C and suggests a cooperative interaction with elastase-liberated growth factors in stimulating TN-C expression and SMC proliferation. It is also possible, as we have shown in coronary arteries and cultured SMCs, that cooperative interaction between elastase activity, elastin degradation products, and cytokine expression is required for induction of matrix glycoproteins.66

Since onset and increased expression of TN-C correlated temporally with the development and progression of pulmonary hypertension, mechanical factors should also be considered.67 68 Recently, Chiquet-Ehrismann et al69 demonstrated that increased mechanical stress positively regulates TN-C expression in fibroblasts. Similarly, we have demonstrated that TN-C is suppressed in cultured rat PA SMCs after stress-relaxation.70 In addition, it has been shown that annexin II, a TN-C receptor,71 is upregulated in rat aortas after mechanical stretch (F.W. Keeley, personal communication). Therefore, increased mechanical stress and hemodynamic forces, which may accompany monocrotaline-induced pulmonary hypertension, may also lead to the induction of TN-C and one of its cognate receptors.

The focal pattern of TN-C expression and cell proliferation may also be a reflection of functional SMC heterogeneity previously documented in vivo and in tissue culture.9 72 73 74 75 76 The idea that distinct SMC subpopulations have different TN-C–expressing capabilities is also supported by recent studies in fibroblasts and other cell types.54 Together, these results indicate that TN-C expression may be governed not only by the availability of multiple extrinsic signals but also by intrinsic factors such as cell lineage and cell type–specific differences in signaling pathways.54

Northern blot analyses not only determined that the induction and upregulation of TN-C in monocrotaline-treated adult rat PAs and lungs is a reflection of increased steady state levels of TN-C mRNA but also suggested that alternative splicing of TN-C mRNAs occurs. Two TN-C mRNA isoforms were expressed in PA and lung tissue isolated from monocrotaline-treated adult rats, with the higher molecular weight isoform predominating. Although the relative functional significance of these isoforms in vascular pathology remains to be proven, previous studies have shown that the lower TN-C mRNA isoform is more prevalent in quiescent cells, whereas in remodeling embryonic tissues, transformed cells, and tumors, the larger isoform predominates.77 78 79 80 Murphy-Ullrich et al81 demonstrated that the alternatively spliced domain of TN-C present in the larger isoform can induce loss of focal contacts in cultured cells. Given that motile and transformed cells have fewer focal contacts than quiescent cells, a possibility exists that generation of higher molecular weight TN-C protein isoforms via alternative RNA splicing would allow TN-C to modulate motility as well as growth-related forms of behavior during tissue remodeling via its effects on cell adhesion.

Previous studies indicate that during the first postnatal week of fetal rat lung development, high levels of TN-C coincide with the peak of alveolization.82 Thereafter, TN-C expression declines82 and is absent in normal adult lung tissue.83 In situ hybridization with TN-C riboprobes in saline-treated adult lung tissue has established that TN-C is absent under control conditions. Therefore, these findings further support our immunohistochemical and Northern hybridization data by showing that TN-C mRNA is induced in cells of the outer medial and adventitial layers within central PAs isolated from monocrotaline-treated rats and in peripheral PAs and the large airway epithelium of the lung.

Apoptosis plays a pivotal role in tissue homeostasis and may occur in developmental and pathological conditions pertaining to the vasculature.32 33 34 35 39 84 Although it is widely believed that SMC apoptosis in blood vessels contributes to tissue homeostasis or regression of intimal lesions, our present studies indicate that apoptosis within the endothelial cell layer is an early event in the development of progressive monocrotaline-induced pulmonary hypertension in adult rats. This was substantiated by examining apoptosis in monocrotaline-treated infant rats, which fail to show progressive vascular changes and do not show any evidence of apoptosis. Previously, we and others were able to observe ultrastructural changes in the arterial endothelium of monocrotaline-treated adult rats3 6 85 that are reminiscent of many of the features already ascribed to apoptotic cells, including swelling and blebbing of nuclei and other organelles.3 6 85 In addition, with progressive pulmonary vascular disease, endothelial injury was more evident, whereas endothelial changes were not observed to the same extent in infant rats.6 The depletion of endothelium by an apoptotic mechanism in adult rats may lead to loss of barrier function. In turn, the subsequent "serum and endothelial cell leakage," which has been attributed to the induction of elastase activity in vivo and in cultured SMCs, may be responsible for the persistent increase in elastase activity observed in monocrotaline-injected adult rats exhibiting progressive disease.86

Having established the temporal and spatial link between TN-C and proliferation in vivo, studies were carried out to determine whether a functional association between these features existed in tissue culture. Cell contact with TN-C potentiated and permitted PA SMC proliferation on type I collagen gels in response to bFGF and EGF, respectively. These growth factors have previously been linked to SMC proliferation in tissue culture.41 87 88 It is of interest that in the study by Quinn et al,88 bovine PA SMC proliferation in response to EGF was minimal relative to the PDGF-induced response despite a similar increase in pHi. This suggested that other factors are important, eg, the ECM microenvironment. In the case of rat PA SMCs, we have shown that the expression of TN-C is a critical modulating factor. Consistent with our findings, increasing evidence suggests that cell behavior may be dictated by a combination of synergistic or antagonistic factors derived from cell-ECM interactions and soluble factors.25 89 90 91 92 Furthermore, binding of ECM ligands to integrin receptors may result in the accumulation of growth factor receptors and intracellular signaling components at the binding site.93 94 For example, in related tissue culture studies, we have shown that SMC interactions with TN-C via the {alpha}vß3 integrin receptor culminate in alterations in the actin-based cytoskeleton and EGF receptor clustering, a prerequisite for EGF receptor activation.94 In addition, the role of TN-C as an SMC survival factor was shown by studies in which withdrawal of endogenous TN-C resulted in apoptosis, whereas its addition prevented this process.94

Finally, our recent studies using graded lung biopsy tissue isolated from patients with congenital heart defects and pulmonary hypertension support these present findings by demonstrating that induction of TN-C accompanies progressive pulmonary vascular changes and also show that TN-C colocalizes with EGF and the proliferating cell nuclear antigen, especially in the neointima.94 These clinical data therefore suggest that our observations made in experimental pulmonary hypertension and in cell culture may be highly relevant to progressive pulmonary hypertension regardless of etiology. On the basis of these studies, we therefore suggest that TN-C and its receptors may be prime targets for therapy in inhibiting the SMC proliferation that is associated with progressive pulmonary vascular disease.


*    Selected Abbreviations and Acronyms
 
bFGF = basic fibroblast growth factor
BrdU = 5-bromo-2'-deoxyuridine
DAPI = 4,6-diamidino-2-phenylindole
ECM = extracellular matrix
EGF = epidermal growth factor
M199 = medium 199
PA = pulmonary artery
PBSA = PBS supplemented with 2% BSA
SFM = serum-free medium
SMC = smooth muscle cell
TN-C = tenascin-C


*    Acknowledgments
 
This study was supported by program grant T2229 from the Heart and Stroke Foundation of Canada. We would like to thank Dr Ike Aukhil of the University of North Carolina for providing rat TN-C cDNAs and Dr Melitta Schachner for the gift of anti-TN-C polyclonal antisera. We also thank Lily Morikawa of the Department of Pathology, The Hospital for Sick Children, for sectioning tissues, and Claire Coulber, Joan Jowlabar, and Susy Taylor for their help in preparing the manuscript.


*    Footnotes
 
Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995, and published in abstract form (Circulation. 1995;92[suppl I]:I-111).

Received June 27, 1996; accepted September 26, 1996.


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 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
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