Alterations in TGF-{beta}1 expression in lambs with increased pulmonary blood flow and pulmonary hypertension

Eugenia Mata-Greenwood,1 Barbara Meyrick,2 Robin H. Steinhorn,1 Jeffrey R. Fineman,3 and Stephen M. Black1,4

Departments of 1Pediatrics and 4Molecular Pharmacology, Northwestern University, Chicago, Illinois 60611-3008; 2Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2650; and 3Department of Pediatrics, University of California San Francisco, San Francisco, California 94143-0106

Submitted 3 June 2002 ; accepted in final form 25 March 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanisms responsible for pulmonary vascular remodeling in congenital heart disease with increased pulmonary blood flow remain unclear. We developed a lamb model of congenital heart disease and increased pulmonary blood flow utilizing an in utero placed aortopulmonary vascular graft (shunted lambs). Morphometric analysis of barium-injected pulmonary arteries indicated that by 4 wk of age, shunts had twice the pulmonary arterial density of controls (P < 0.05), and their pulmonary vessels showed increased muscularization and medial thickness at both 4 and 8 wk of age (P < 0.05). To determine the potential role of TGF-{beta}1 in this vascular remodeling, we investigated vascular changes in expression and localization of TGF-{beta}1 and its receptors T{beta}RI, ALK-1, and T{beta}RII in lungs of shunted and control lambs at 1 day and 1, 4, and 8 wk of life. Western blots demonstrated that TGF-{beta}1 and ALK-1 expression was elevated in shunts compared with control at 1 and 4 wk of age (P < 0.05). In contrast, the antiangiogenic signaling receptor T{beta}RI was decreased at 4 wk of age (P < 0.05). Immunohistochemistry demonstrated shunts had increased TGF-{beta}1 and T{beta}RI expression in smooth muscle layer and increased TGF-{beta}1 and ALK-1 in endothelium of small pulmonary arteries at 1 and 4 wk of age. Moreover, T{beta}RI expression was significantly reduced in endothelium of pulmonary arteries in the shunt at 1 and 4 wk. Our data suggest that increased pulmonary blood flow dysregulates TGF-{beta}1 signaling, producing imbalance between pro- and antiangiogenic signaling that may be important in vascular remodeling in shunted lambs.

shunted lambs


THE DEVELOPMENT OF PULMONARY hypertension and its associated increased vascular reactivity is a common accompaniment of congenital heart disease with increased pulmonary blood flow (10). After birth, the presence of a systemic to pulmonary communication results in increasing pulmonary blood flow as pulmonary vascular resistance normally decreases. This abnormal postnatal hemodynamic state results in progressive structural and functional abnormalities of the pulmonary vascular bed (22, 29). Although the vascular morphology of pulmonary hypertension is well described, the mechanisms of vascular remodeling remain incompletely understood. A clearer understanding of these mechanisms would provide potential new avenues for the prevention and treatment of this disorder.

Members of the transforming growth factor (TGF)-{beta} superfamily play a critical role in the regulation of cellular growth and differentiation in a wide range of biological systems, including the vasculature (1820, 32, 33). However, the actions of TGF-{beta} in vivo are complex and largely dependent on the environment of individual target cells (1820). TGF-{beta}1 has shown a crucial role in the development of various pulmonary diseases, including fibrotic pulmonary disease due to various injuries (28, 37). In addition, some clinical studies have demonstrated an association between increased or decreased expression of TGF-{beta}1 in adult patients suffering from pulmonary hypertension (41).

Active TGF-{beta}1 signals through a heteromeric complex consisting of two types of transmembrane serine/threonine kinases, known as type I and type II receptors (18). The type II TGF-{beta} receptor (T{beta}RII) is the primary receptor target for TGF-{beta}. On binding to TGF-{beta}1, T{beta}RII recruits type I TGF-{beta} receptor (T{beta}RI) (19, 20). T{beta}RI, also called activin receptor-like kinase 5 (ALK-5), is a widely expressed type I receptor for TGF-{beta}1 (13, 19). In addition, endothelial cells express activin receptor-like kinase 1 (ALK-1), another type I receptor that has recently been shown to bind TGF-{beta}1 (16) and to be present in a receptor complex in association with T{beta}RII (26). T{beta}RI has been attributed antiangiogenic properties, whereas ALK-1 has shown proangiogenic effects in endothelial cells (8).

We have developed an animal model of pulmonary hypertension by inserting an aortopulmonary vascular graft in the late-gestational fetal lamb (3, 4, 28). Postnatally, these lambs have increased pulmonary blood flow and pressure. In addition, they display pulmonary vascular remodeling characterized by increased medial wall thickness of the small muscular pulmonary arteries and abnormal extension of muscle to peripheral pulmonary arteries (18, 27, 28). Last, at 4 wk of age, these lambs have a transient increase in the number of barium-filled intra-acinar pulmonary arteries, which may represent an early adaptive angiogenesis and/or vessel recruitment (28). Thus we hypothesized that the expression of TGF-{beta}1 and its proangiogenic receptors would be increased in lambs with increased pulmonary blood flow. Therefore, in the present study, we investigated the relationship between the development of muscle and medial thickening of pulmonary arteries and changes in arterial number with alterations in the expression of TGF-{beta}1 and its signaling receptors T{beta}RII, T{beta}RI, and ALK-1 from 1 day to 8 wk in lambs with increased pulmonary blood flow.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Surgical preparation and care. Forty mixed-breed, pregnant Western ewes (137–141 days gestation, term = 145 days) were operated on under sterile conditions as previously described (30). After spontaneous delivery of the lambs (2–7 days after surgery), antibiotics [106 units of penicillin G potassium and 25 mg of gentamicin sulfate intramuscularly (im)] were administered for 2 days. The lambs were weighed daily, and respiratory rate and heart rate were measured. Furosemide (1 mg/kg im) was administered daily, and elemental iron (50 mg im) was given weekly. At 1 day and at 1, 4, or 8 wk of age, the lambs were anesthetized with intravenous infusions of ketamine hydrochloride (~1mg·kg-1·min-1) and diazepam (0.002 mg · kg-1 · h-1), intubated with a 5.5-mm outer diameter endotracheal tube, and mechanically ventilated with a pediatric, time-cycled, pressure-limited ventilator at 21% O2 (Healthdyne, Marietta, GA). Ventilation rate was adjusted to maintain an arterial PCO2 (Paco2) between 35 and 45 Torr. Via a midsternotomy incision, the lambs were instrumented to measure pulmonary and systemic arterial pressure, right and left atrial pressure, heart rate, and left pulmonary blood flow, as previously described (31). After 60 min of recovery, baseline hemodynamic variables and O2 saturation values were obtained. The lambs were then euthanized with an intravenous injection of pentobarbital sodium (Euthanasia CII; Central City Medical, Union City, CA) and subjected to bilateral thoracotomy. An autopsy was performed to confirm patency of the vascular graft. The lungs were removed and prepared for RNA preparation, Western blot analysis, and immunohistochemistry. All procedures and protocols were approved by the Committee on Animal Research of the University of California, San Francisco and Northwestern University.

Measurements. Pulmonary and systemic arterial and right and left atrial pressures were measured using Sorenson Neonatal Transducers (Abbott Critical Care Systems, Chicago, IL). Mean pressures were obtained by electrical integration. Heart rate was measured by a cardiotachometer triggered from the phasic systemic arterial pressure pulse wave. Left pulmonary blood flow was measured on an ultrasonic flow meter (Transonic Systems, Ithaca, NY). All hemodynamic variables were recorded continuously on a Gould multichannel electrostatic recorder (Gould, Cleveland, OH). Systemic arterial blood gases and pH were measured on a Radiometer ABL5 pH/blood gas analyzer (Radiometer, Copenhagen, Denmark). Hemoglobin concentration and oxygen saturation were measured by a hemoximeter (270; CIBA-Corning). The ratio of pulmonary to systemic blood flow (Qp/Qs) was calculated using the Fick equation. Pulmonary vascular resistance was calculated using standard formulas. It should be noted that ketamine anesthesia increases systemic vascular resistance, which may falsely increase the Qp/Qs.

Tissue preparation for immunohistochemistry. The heart and lungs were removed en bloc. The lungs were dissected with care to preserve the integrity of the vascular endothelium. Sections (2–3 g) from each lobe of the lung were removed. These tissues were snap-frozen in liquid N2 and stored at -70°C until analysis.

For immunohistochemistry, the pulmonary vascular tree was rinsed with cold (4°C) PBS to remove blood and was fixed by perfusion with cold (4°C) 4% paraformaldehyde. The pulmonary artery was then clamped. The airways were fixed at 20 cm of H2O pressure by filling the trachea with cold (4°C) 4% paraformaldehyde. When the lungs were distended at this pressure, the trachea was clamped. The lungs were fixed for 24 h at 4°C by immersion in 4% paraformaldehyde. Representative slices from each lobe were removed, placed in 30% sucrose until they sank, placed in optimum cutting temperature compound, frozen on dry ice, and stored at -70°C until they were sectioned. Five- to 10-µm sections were cut using a cryostat, transferred to aminoalkylsilane-treated slides (Superfrost Plus; Fisher Scientific, Santa Clara, CA), and stored at -70°C (1).

Structural studies (morphometric analysis). The lungs, heart, and trachea were removed intact from four shunt and four control lambs each at 1, 4, and 8 wk of age, and the pulmonary arterial bed was distended with a barium gelatin suspension (563 ml micropaque powder, Nicholas Picker, Stoughton, MA; 50 g gelatin, Bloom 8-G, Fisher Scientific, Fairlawn, NJ; 387 ml distilled water; and a few crystals of phenol) at 60°C from a pressure of 70 mmHg for 2 min (11). This mixture has been shown to not cross the capillary bed and to fill small arteries down to a lumen of 15- to 20-µm internal diameter. Use of the hypertensive pressure ensures that the arteries are fixed in the fully distended state, thereby allowing application of morphometric techniques (20a). After arterial injection, the lungs were inflated by way of the trachea with 10% formol-saline from a pressure of 35 cmH2O and placed in a bath of formalin for fixation.

After fixation, the lungs were cut into longitudinal 2-cm slices, and approximately six random blocks were taken from each lung for routine light microscopy. Two 5-µm sections were cut from each block, one was stained with hematoxylin and eosin, and the other was stained with Verhoff's elastin stain, followed by van Gieson. The sections were then examined for the characteristic structural changes of chronic pulmonary hypertension using well-established quantitative techniques (11). Briefly, external diameter of at least 100 arterial profiles was measured as well as medial thickness of the muscular and partially muscular arteries. Medial thickness was then related to arterial size using the calculation: percent medial thickness = 2 x medial thickness/external diameter x 100. The structure of each artery was also noted: muscular, partially muscular, and nonmuscular, as was the structure of the accompanying airway: bronchus, bronchiolus, terminal bronchiolus, respiratory bronchiolus, alveolar duct, and alveolar wall. The density of the barium-filled intra-acinar arteries was also assessed. With the use of a x25 objective and an eyepiece reticule, the number of barium-filled arteries of <200-µm external diameter was counted and related to the number of alveolar profiles in these same fields. At least 25 consecutive microscopic fields were counted for each animal.

Western blot analysis. Protein extracts (100 µg) were separated on 4–20% (TGF-{beta}1) and 12% (T{beta}RI, T{beta}RII, and ALK-1) SDS-polyacrylamide gel and electrophoretically transferred to polyvinylidene difluoride membranes (Amersham). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T). After 1 h of blocking, the membranes were incubated at 4°C for 2 consecutive days with 1:500 (~3.4 µg/ml) dilution of rabbit polyclonal anti-human TGF-{beta}1 antibody (2 µg/ml in blocking solution, Santa Cruz). Alternatively, membranes were probed with goat polyclonal antibodies against human T{beta}RI and T{beta}RII (1 µg/ml, Santa Cruz) and ALK-1 (1 µg/ml, R&D Systems) for 2h at room temperature. Membranes were washed 3 x 15 min with TBS-T and then hybridized with anti-rabbit or anti-goat horseradish peroxidase antibody for 45 min. After 3 x 15 min washes, bands were visualized with chemiluminescence using a Kodak Digital Science Image Station (NEN). The following size bands were obtained: 12.5 and 25 kDa for monomeric and dimeric TGF-{beta}1, 50 kDa for T{beta}RI, 62 kDa for ALK-1, and 70 kDa for T{beta}RII.

To compare the various protein levels obtained from controls and shunts (n = 5, total of 10 samples per age) from various ages (1-day-old and 1-, 4-, and 8-wk-olds, total of 40 samples), four different gels were run (1 for each age containing protein samples from both control and shunted lambs). Also, included on each gel was an internal control (a lung extract prepared from a 1-day-old shunted lamb). Each one of the four membranes was probed for the particular protein of interest and then reprobed the next day for {beta}-actin. Each densitometric value was divided by its {beta}-actin value to obtain a relative value for TGF-{beta}1, T{beta}RI, T{beta}RII, and ALK-1 protein levels. Relative values were averaged and then corrected using a factor (relative protein level of internal control obtained from that particular blot divided by relative protein level of internal control from the original day 1 membrane). In this way, differences in time exposure leading to different protein levels were observed and corrected for. Standard deviations were also corrected by using the same factor.

Immunohistochemistry. Immunohistochemistry was performed as previously described (1, 2). Studies were done on serial sections of control and shunted ovine lung using rabbit polyclonal anti-TGF-{beta}1 (Santa Cruz) or goat polyclonal anti-T{beta}RI, ALK-1, and T{beta}RII. Frozen tissue sections (7 µm) were allowed to come to room temperature. Samples were fixed for 10 min in cold acetone and then washed 3x with PBS. To eliminate nonspecific binding of the primary antiserum to tissue proteins, tissue sections were incubated with 1% horse serum in PBS (blocking solution) for 1 h. The tissue sections were then incubated with primary antibodies (5 µg/ml) in the presence of monoclonal smooth muscle cell-actin antibody (1:400, Sigma) in blocking solution at 4°C overnight. After three washes with PBS x 5 min, samples were visualized with a combination of Rhodamine Red-X goat anti-rabbit and Oregon Green 488 goat anti-mouse secondary antibodies (Molecular Probes) at a concentration of 1:400 in blocking solution for 45 min at room temperature. Alternatively, a combination of Rhodamine Red-X rabbit anti-goat and Alexa Green 488 rabbit anti-mouse secondary antibodies was used to localize TGF-{beta} receptors. After three further washes with PBS, an antifading solution was added, and samples were visualized by fluorescence microscopy. For each tissue section, a parallel experiment was carried out in which the primary antibody was omitted. This served as the negative control. A minimum of three different sets of control and shunted lung tissues were prepared and examined. Because it is difficult to utilize immunohistochemistry for qualitative measurements on protein expression, we used this technique only to determine protein localization and to determine whether there were differences between shunt and control lambs in the numbers of vessels expressing for TGF-{beta}1, T{beta}RI, T{beta}RII, and ALK-1. To carry out this procedure, small, muscularized pulmonary arteries were visualized next to airways, and at least 30 vessels (representing at least 10 fields) were counted as positively immunoreactive or nonreactive for a particular protein. The number of vessels immunoreactive for each protein as a proportion of the total vessels counted was determined. Results were then calculated as the average number of immunoreactive vessels ± SE (n = at least 3 different lambs from each age group), and statistical significance was calculated as described below.

Data analysis. For each age studied (1, 4, and 8 wk), the mean value was calculated for each structural variable. Quantitation of protein expression was performed using a Kodak image station 440CF and KDS1D imaging software. This allows a pixel density from 1–103 instead of 256 gray scale of autoradiographic film. The response is linear within this range. In all experiments, means ± SD were calculated, and comparisons among control and shunted lambs were made by ANOVA for repeated measures. When differences were present among study groups, Student-Newman-Keuls post hoc testing was performed. P < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Functional changes. Spontaneous delivery occurred 2–9 days after fetal surgery. All shunted lambs had an audible continuous murmur and an increase in oxygen saturation between the right ventricle and the distal pulmonary artery. In shunted lambs, mean pulmonary arterial pressure and left pulmonary blood flow were greater than age-matched controls at all ages. Left pulmonary vascular resistance was decreased in shunted lambs at 4 and 8 wk of age. Biventricular cardiomegaly was present in shunted lambs aged 1–8 wk and tended to increase with age (Table 1). Compared with 4-wk-old shunted lambs, mean pulmonary arterial pressure and left pulmonary vascular resistance was significantly higher in 8-wk-old shunted lambs (P < 0.05).


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Table 1. General hemodynamics in 1-day-old and 1-, 4-, and 8-wk-old lambs

 

Structural changes. Previous morphometric analysis showed a significant increase in number of barium-filled peripheral arteries per unit area in 4-wk-old shunted lambs compared with age-matched controls (31). However, this increase in vessel number per unit area was not yet present in 1-wk-old lambs and did not persist in 8-wk-old lambs compared with age-matched controls (Fig. 1A). Alveolar number was similar in each group of shunted animals compared with age-matched controls. However, at 4 wk, a burst of alveolar multiplication had occurred compared with the values at 1 wk, and by 8 wk, alveolar number was reduced, likely indicating an enlargement of the alveoli between 4 and 8 wk (Table 2). Previously, in 4-wk-old shunted lambs, we demonstrated that the percent medial thickness of arteries <200-µm external diameter was approximately twice that of age-matched controls (31). In this study, we found that medial thickness was still increased in the 8-wk-old shunted lambs, and although values for percent medial thickness of controls and shunted animals was less than at 4 wk, the increase remained twice that seen in age-matched controls. At 1 wk, we found that medial thickness in control sheep was generally less than at either 4 or 8 wk, and no significant difference was noted between control and shunted animals at that time (Fig. 1B). Previous analysis of the structure of the intra-acinar arteries related to airway level (Table 3) established the appearance of muscle in the walls of smaller and more peripheral arteries than normal in the 4-wk-old shunted lambs. Although arterial muscularity of the 1-wk shunted animals was similar to age-matched controls, at 8-wk, increased muscularity of the intra-acinar arteries was still apparent, although at the alveolar duct and alveolar wall level, this difference was not as pronounced as at 4 wk (Table 3). Together, these data indicate that the 4- and 8-wk shunted animals show the structural changes of pulmonary hypertension. Since the structural changes were not apparent in the shunted sheep at 1 wk, we did not examine 1-day-old shunted animals.



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Fig. 1. Morphometric analysis of vascular development in the shunt model. A: increase in blood vessel number according to age development in control and shunted lambs. Values are means ± SE. *P < 0.05 vs. control lambs. B: increase in percent medial thickness in shunt modeling according to age-stage of development. Four sets of control and shunt twins were evaluated for medial thickness according to diameter size of blood vessel. *P < 0.05 vs. control.

 

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Table 2. Structural changes in shunted and control lambs

 

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Table 3. Percentage of fully muscular, partially muscular, and nonmuscular pulmonary arteries in shunted and control lambs

 

Expression and localization of TGF-{beta}1. TGF-{beta}1 protein levels were significantly increased in peripheral lung in the shunted lambs at 1 wk (125%) and 4 wk (120%, Fig. 2) but were comparable to control samples at 1 day and 8 wk (Fig. 2). In agreement with these data, TGF-{beta}1 mRNA expression was increased by 65% in 1-wk-old shunted lambs and 310% in 4-wk-old shunted lambs compared with age-matched controls (P < 0.05; Fig. 3) but was similar in shunted and control lambs at 1 day and 8 wk (Fig. 3). Immunohistochemical analysis revealed that TGF-{beta}1 was highly expressed in the lung of both control and shunted lambs. No differences were observed between shunted and control lambs at 1 day of age. However, TGF-{beta}1-specific staining appeared to be more intense in both smooth muscle and endothelium of small pulmonary arteries as well as in the smooth muscle of airways in 1- and 4-wk-old shunted lambs compared with age-matched twin controls (Fig. 4, A–F). At 8 wk of age, TGF-{beta}1 stained brightly in the airway epithelium of shunted lambs but not in control lambs. This contrasts with the Western blot analysis of peripheral lung tissue in which TGF-{beta}1 expression at 8 wk of age was similar to controls.



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Fig. 2. Western blot analysis for transforming growth factor (TGF)-{beta}1 in lung tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs (control and, after insertion of an aorta-to-pulmonary artery vascular graft in utero, shunt). A: representative Western blots are shown from protein extracts (100 µg) prepared from lung tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs (1 control and 1 shunt from each age), separated on a 4–20% SDS-polyacrylamide gradient gel, electrophoretically transferred to Hybond membranes, and analyzed using a polyclonal rabbit anti-TGF-{beta}1. TGF-{beta}1 protein expression was increased in shunted lambs only at 1 and 4 wk. Con, control; Sh, shunt. B: densitometric values for relative TGF-{beta}1 protein from 5 control and 5 shunted lambs at each age. In shunted lambs, relative TGF-{beta}1 protein is increased by 125% at 1 wk and by 120% at 4 wk (P < 0.05). Values are means ± SE. *P < 0.05 control vs. shunt.

 


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Fig. 3. RNase protection assay for TGF-{beta}1 in lung tissue RNA from 1-day-old and 1-, 4-, and 8-wk-old lambs (control and, after insertion of an aorta-to-pulmonary artery vascular graft in utero, shunt). A: representative RNase protection assays are shown from a cRNA probe for ovine TGF-{beta}1 that was hybridized overnight to 50 µg of total lung RNA prepared from 1-day-old and 1-, 4-, and 8-wk-old lambs (1 control and 1 shunt from each age). TGF-{beta}1 mRNA expression was increased in shunted lambs only at 1 and 4 wk. There were no protected fragments detected in the lanes where the probe was hybridized without RNA (PA) or in the presence of tRNA. TGF-{beta}1 is undigested probe. A cRNA for ovine 18S was also hybridized to serve as a control for RNA loading. B: densitometric values for relative TGF-{beta}1 mRNA (normalized to 18S mRNA and to control values) from 5 control and 5 shunted lambs at each age. In shunted lambs, relative TGF-{beta}1 mRNA increased by 65% at 1 wk and by 230% at 4 wk (P < 0.05). Values are means ± SE. *P < 0.05 control vs. shunt.

 


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Fig. 4. TGF-{beta}1 protein expression in the lung in vivo from tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs (control and, after insertion of an aorta-to-pulmonary artery vascular graft in utero, shunt). Immunohistochemical localization of TGF-{beta}1 expression in the lung in vivo from 1-day-old (A and B), 1-wk-old (C and D), 4-wk-old (E and F), and 8-wk-old (G and H) lambs (control: A, C, E, and G; shunted: B, D, F, and H). Polyclonal rabbit anti-TGF-{beta}1 and monoclonal mouse anti-smooth muscle cell-actin antibodies were used to localize expression. TGF-{beta}1 expression is shown in red and smooth muscle cell-actin expression is shown in green. Colocalization is shown in yellow. Magnification, x200. Results are representative of 3 different sets of twin matches (control and shunt). AW, airway; V, vessel; Sm, smooth muscle cell layer; End, endothelium; Epi, epithelium.

 

TGF receptor expression. Active TGF-{beta}1 binds to T{beta}RII, inducing its dimerization with either T{beta}RI or ALK-1 (8). Because the formation of either complex leads to different biological functions, we investigated the changes in expression of all three receptors by immunoblotting. T{beta}RII expression was unchanged between shunted and control lambs over all developmental periods (Fig. 5). Immunohistochemical analysis localized T{beta}RII expression mainly in the endothelial layers of medium-sized (500–200 µm), small-sized (<200 µm), and microvessels (capillaries, <10 µm), in both control and shunt samples (Fig. 6). Differences in T{beta}RII localization were observed between shunt and controls. Specifically, compared with age-matched controls, decreased expression of T{beta}RII was observed in the capillaries of 1-wk-old shunts (Fig. 6, C and D), whereas increased staining of T{beta}RII was found in the small pulmonary arterial endothelium of 4-wk-old shunts. Also, we observed an increased localization of T{beta}RII in the smooth muscle layer of vessels and airways in 8-wk-old shunts (Fig. 6, G and H).



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Fig. 5. Western blot analysis for type II TGF-{beta} receptor (T{beta}RII) in lung tissue protein from 1-day-old and 1-, 4-, and 8-wk-old lambs (control and, after insertion of an aorta-to-pulmonary vascular graft in utero, shunt). A: representative Western blots are shown from protein extracts (100 µg) prepared from lung tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs (1 control and 1 shunt from each age), separated on a 12% SDS-polyacrylamide gradient gel, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against T{beta}RII. B: densitometric values for relative T{beta}RII protein from 5 control and 5 shunted lambs at each age. There were no significant differences in relative T{beta}RII protein in the shunts compared with controls. Values are means ± SE.

 


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Fig. 6. T{beta}RII protein expression in the lung in vivo from tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs (control and, after insertion of an aorta-to-pulmonary artery vascular graft in utero, shunt). Immunohistochemical localization of T{beta}RII expression in the lung in vivo from 1-day-old (A and B), 1-wk-old (C and D), 4-wk-old (E and F), and 8-wk-old (G and H) lambs (control: A, C, E, and G; shunted: B, D, F, and H). Polyclonal goat anti-T{beta}RII antiserum and monoclonal mouse anti-smooth muscle cell-actin antibody were used to localize expression of T{beta}RII. T{beta}RII expression is shown in red and smooth muscle cell-actin expression is shown in green. Colocalization is shown in yellow. Magnification, x200. Results are representative of at least 2 different sets of twin matches (control and shunt). Arrows indicate pulmonary arteries.

 

Although T{beta}RI protein expression showed little change in control animals over time, shunted lambs showed a significant decrease in expression of T{beta}RI in 4-wk-old shunted animals relative to age-matched controls (226% decrease in the shunt compared with controls, P < 0.05, Fig. 7). Studies using immunohistochemistry showed that T{beta}RI was highly expressed in the endothelium of pulmonary vessels, including capillaries, of normal lambs (Fig. 8, A, C, E, and G). However, pulmonary vessels of shunted lambs showed a marked absence of expression of T{beta}RI in the endothelium of pulmonary vessels, in particular small pulmonary arteries of less than 200-µm diameter. At 1 wk of age, 69.4 ± 2.6% immunoreactive arteries were found in the controls compared with only 36.3 ± 6.9% in the shunts (P < 0.05). At 4 wk of age, 56.1 ± 5.9% immunoreactive arteries were observed in the controls compared with only 25.7 ± 6.4% in the shunts (P < 0.05, Fig. 8, D and F). In addition, 15.2 ± 5.2% of small pulmonary arteries from 1-wk-old shunts showed increased immunoreactivity for T{beta}RI in their smooth muscle layer compared with only 1.5 ± 0.1% in agematched controls (P < 0.05, Fig. 8, C and D). T{beta}RI expression in the endothelium of microvessels of shunted lambs was somewhat decreased in the shunt at 1 wk of age but otherwise normal to control lambs at other developmental ages (Fig. 8, A–H).



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Fig. 7. Western blot analysis for type I TGF-{beta} receptor (T{beta}RI) in lung tissue protein from 1-day-old and 1-, 4-, and 8-wk-old lambs (control and, after insertion of an aorta-to-pulmonary artery vascular graft in utero, shunt). A: representative Western blots are shown from protein extracts (100 µg) prepared from lung tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs (1 control and 1 shunt from each age), separated on a 12% SDS-polyacrylamide gradient gel, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against T{beta}RI. T{beta}RI protein expression was decreased in shunted lambs only at 4 wk. B: densitometric values for relative T{beta}RI protein from 4 control and 4 shunted lambs at each age. In shunted lambs, relative T{beta}RI protein is decreased by 221% at 4 wk (P < 0.05). Values are means ± SE. *P < 0.05 control vs. shunt.

 


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Fig. 8. T{beta}RI protein expression in the lung in vivo from tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs (control and, after insertion of an aorta-to-pulmonary artery vascular graft in utero, shunt). Immunohistochemical localization of T{beta}RI expression in the lung in vivo from 1-day-old (A and B), 1-wk-old (C and D), 4-wk-old (E and F), and 8-wk-old (G and H) lambs (control: A, C, E, and G; shunted: B, D, F, and H). Polyclonal goat anti-T{beta}RI antiserum and monoclonal mouse anti-smooth muscle cell-actin antibody were used to localize expression of T{beta}RI. T{beta}RI expression is shown in red and smooth muscle cell-actin expression is shown in green. Colocalization is shown in yellow. Magnification, x200. Results are representative of at least 2 different sets of twin matches (control and shunt). Arrows, pulmonary arteries. Note that 1- and 4-wk-old controls, but not shunts, show intense T{beta}RI staining in the endothelium of pulmonary arteries.

 

Finally, we analyzed the expression of ALK-1, which has been shown to mediate TGF-{beta}1 signaling in the endothelium. Immunoblotting experiments showed that ALK-1 protein expression in the control lambs decreases with age (Fig. 9). Compared with control values, an increase in ALK-1 protein expression was evident at 1 day (150% increase) and was significant at 1 and 4 wk of age (400% and 86% increase, respectively, P < 0.05, Fig. 10). Therefore, ALK-1 and T{beta}RI protein expression appear to be regulated in opposing directions in control compared with shunted lambs. Immunohistochemistry localized ALK-1 to the endothelium layer of pulmonary microvessels in both control and shunt samples (Fig. 10, A–H). However, at 1 and 4 wk of age, control lambs exhibited little expression of ALK-1 (43.7 ± 10.2% positively stained arteries at 1 wk and 19.2 ± 7.4% at 4 wk of age) in the endothelium of small-to-large pulmonary vessels (Fig. 10, C and E). In shunted lambs, ALK-1 was highly expressed in small pulmonary arteries with 76.3 ± 15.3% immunoreactive arteries at 1 wk and 58.4 ± 6.5% at 4 wk of age (P < 0.05 compared with controls, Fig. 10, D and F). There were no detectable differences in expression of ALK-1 in endothelium of small pulmonary arteries between control and shunted lambs at 8 wk of age (Fig. 10, D and G).



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Fig. 9. Western blot analysis for activin receptor-like kinase 1 (ALK-1) in lung tissue protein from 1-day-old and 1-, 4-, and 8-wk-old lambs (control and, after insertion of an aorta-to-pulmonary artery vascular graft in utero, shunt). A: representative Western blots are shown from protein extracts (100 µg) prepared from lung tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs (1 control and 1 shunt from each age), separated on a 12% SDS-polyacrylamide gradient gel, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against ALK-1. ALK-1 protein expression was increased in shunted lambs only a 1 and 4 wk. B: densitometric values for relative ALK-1 protein from 5 control and 5 shunted lambs at each age. In shunted lambs, relative ALK-1 protein is increased by 407% at 1 wk and by 86% at 4 wk (P < 0.05). Values are means ± SE. *P < 0.05 control vs. shunt.

 


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Fig. 10. ALK-1 protein expression in the lung in vivo from tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs (control and, after insertion of an aorta-to-pulmonary artery vascular graft in utero, shunt). Immunohistochemical localization of ALK-1 expression in the lung in vivo from 1-day-old (A and B), 1-wk-old (C and D), 4-wk-old (E and F), and 8-wk-old (G and H) lambs (control: A, C, E, and G; shunted: B, D, F, and H). Polyclonal goat anti-ALK-1 antiserum and monoclonal mouse anti-smooth muscle cell-actin antibody were used to localize expression of ALK-1. ALK-1 expression is shown in red and smooth muscle cell-actin expression is shown in green. Colocalization is shown in yellow. Magnification, x200. Results are representative of at least 2 different sets of twin matches (control and shunt). Arrows, pulmonary arteries. Note that 1-day-old and 1- and 4-wk-old shunts, but not controls, have intense ALK-1 (red) staining in the endothelium of pulmonary arteries.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The progressive structural abnormalities of the pulmonary vascular bed associated with congenital heart disease and increased pulmonary blood flow are well characterized. In 1958, Heath and Edwards (9) described a progressive structural classification that ranged from reversible medial hypertrophy to terminal changes such as angiomatoid formation and fibrinoid necrosis. In 1978, Rabinovitch et al. (30) described a modification based on lung biopsy specimens that featured alterations in normal remodeling and growth. The three progressive stages (A–C) that have been correlated with perioperative hemodynamics are characterized by progressive medial hypertrophy, abnormal muscle extension, and reduced artery size and concentration. The vascular remodeling at 4 and 8 wk of age described in the present study would be characterized as grade B alterations, with medial hypertrophy ~2 times normal and abnormal appearance of muscle in the walls of intra-acinar arteries. Between 4 and 8 wk of life, the pulmonary-to-systemic blood flow ratio, mean pulmonary arterial pressure, and left pulmonary vascular resistance all tended to increase in shunted lambs, suggesting a persistent remodeling stimulus. However, since there is a normal thinning of the medial layer with age, the less dramatic medial thickness at 8 wk most likely represents changes relative to normal development. The increase in pulmonary vessel number per unit area observed in 4-wk-old shunted lambs has not been reported in children with pulmonary hypertension and increased pulmonary blood flow (22, 31) and may represent an early adaptive angiogenic and/or recruitment response to incorporate the increase in pulmonary blood flow. During the second month of life, when alveolar growth is significant, arterial vessel growth and/or recruitment was not maintained at the same degree, such that 8-wk-old shunted lambs had similar vessel number per unit area as age-matched control lambs.

Hemodynamic insult, as in increased blood flow and/or pressure, has been known to play a critical role in the increase in smooth muscle hypertrophy and hyperplasia and phenotypic changes of vascular cells (23, 39); however, little is known about the mechanisms by which biomechanical forces transduce intracellular signals leading to gene regulation. TGF-{beta}1 increases have been observed in response to laminar shear stress in endothelial cells and conduit vessels (4, 15) and to cyclic stretch in cardiomyocytes (42). In support of these findings, we have observed in our model of increased pulmonary blood flow an increase in TGF-{beta}1 expression in the pulmonary vessels of shunted lambs compared with age-matched controls. In our studies, a relatively normal arterial morphology was found in the 1-wk-old shunted lambs, a time when TGF-{beta}1 expression starts to increase. Four-week-old shunted lambs have increased vessel number per unit area, a significant increase in muscularity of the intraacinar arteries, and a significant increase in medial thickness of small pulmonary arteries, a time when TGF-{beta}1 expression peaks. At 8 wk, the shunted lambs have normal vessel number per unit area but maintain medial thickening and increased muscularity of the intra-acinar arteries. However, at this age the levels of TGF-{beta}1 expression are similar in shunted and control lambs. These data suggest that dysregulation of TGF-{beta}1 signaling pathways is an early event that precedes the development of vascular remodeling induced by increased pulmonary blood flow secondary to congenital heart disease. Furthermore, studies will be required to determine whether TGF-{beta}1 is a stimulus or a marker of remodeling.

TGF-{beta}1 plays a pivotal role in vascular homeostasis by regulating the synthesis of extracellular matrix proteins that stabilize interactions between endothelial, mesenchymal, and smooth muscle cells of the vessel wall (25). TGF-{beta}1 has been ascribed both antiangiogenic and proangiogenic effects in vivo and in vitro (6, 8, 1921, 33). However, the role of TGF-{beta}1 in vascular remodeling is not well understood. Previous observations have shown a biphasic in vitro effect of TGF-{beta}1 on endothelial cell proliferation, where low doses of TGF-{beta}1 stimulate proliferation and migration and high doses inhibit these processes (8, 26). Recently, it has been shown that the biphasic effect of TGF-{beta}1 on angiogenesis is due to differences in receptor signaling (8). Active TGF-{beta}1 binds to T{beta}RII, which then recruits a type I receptor. In the endothelial cell, two type I receptors have been described: T{beta}RI and ALK-1 (8, 1920). Both type I receptors have been shown to heterodimerize with T{beta}RII and endoglin (a type III receptor) and to bind TGF-{beta}1 and TGF-{beta}3 (8, 1920, 24). However, ALK-1 and T{beta}RI have opposing signaling events and biological functions (8). T{beta}RI phosphorylates Smad 2 and Smad 3, leading to transcriptional activation of extracellular matrix proteins (collagen and fibrin) and plasminogen activator inhibitor-1 (PAI-1) (8). ALK-1 induces the phosphorylation of Smad 1 and Smad 5, leading to enhanced gene expression of Id-1, a cell differentiation inhibitor (8). T{beta}RI activation leads to inhibitory functions in endothelial cell migration and proliferation and, therefore, signals the antiangiogenic effects of TGF-{beta}1 (8, 38). Conversely, ALK-1 activation and Id-1 upregulation leads to the opposite effects of T{beta}RI signaling, thereby showing proangiogenic effects (8). In agreement with these studies, we have observed in our shunt model of increased pulmonary blood flow a profound imbalance between the expression of T{beta}RI and ALK-I in endothelial cells of small pulmonary arteries in shunted lambs in which structural differences were indicative of an active angiogenic process. In addition, we have observed that plasminogen activator inhibitor-1 is decreased in the shunt model as early as 1 wk of age, in conjunction with T{beta}RI downregulation (data not shown). Together, our data suggest that the decrease in T{beta}RI and increase in ALK-1 signaling would lead to endothelial activation and increased production of extracellular matrix protein degradation necessary for new blood vessel formation.

Other studies have suggested that TGF-{beta}1 induces proangiogenic effects indirectly by upregulating VEGF expression (3, 12, 27). Moreover, TGF-{beta}1 has been shown to activate VEGF transcription in vascular smooth muscle cells through activation of Smads 2 and 3, in conjunction with other transcriptional factors, including hypoxia-inducible factor 1 (HIF-1) and activator protein-1 (34). These studies suggest that increased VEGF transcription occurs through T{beta}RI signaling. Our immunohistochemical analysis demonstrated that, while T{beta}RI is downregulated in endothelial cells, the opposite is true in vascular smooth muscle cells of ~15% of small pulmonary arteries in shunted lambs of 1–4 wk of age. In addition, we have observed an increase in VEGF expression in similar vessels. In support of our observations, various reports show parallel increased expression of TGF-{beta}1 and VEGF in models of atrial fibrillation and adult pulmonary hypertension (35). In addition, in vitro studies have shown that cyclic stretch-induced VEGF expression is dependent on previous activation of TGF-{beta}1 since a TGF-{beta}1 neutralizing antibody abolished stretch-induced increases in VEGF mRNA expression and protein secretion (42). This suggests that TGF-{beta}1 might be inducing proangiogenic effects by upregulating VEGF expression in vascular smooth muscle cells of small pulmonary arteries in models of increased blood flow, similar to HIF-1{alpha} factors in hypoxia-induced pulmonary hypertension (36). Increased TGF-{beta}1 signaling in the smooth muscle cell layer, through TBRI, could also account for hyperplasia and increased extracellular matrix protein production (14, 23).

Considering that TGF-{beta}1 signaling is fundamental for the homeostasis of intimal, medial, and adventitial layers of vessels, we propose that the dysregulation of TGF-{beta}1 and its receptors due to increased blood flow is likely to play an important role in the development of the pulmonary vascular remodeling in our model of pulmonary hypertension secondary to increased pulmonary blood flow. Further understanding of these mechanisms could lead to potential new therapies for the management of secondary pulmonary hypertension due to congenital heart disease.


    ACKNOWLEDGMENTS
 
The authors thank Michael J. Johengen for expert technical assistance.

This research was supported in part by National Institutes of Health Grants HL-60190 (S. M. Black), HL-67841 (S. M. Black), HD-398110 (S. M. Black), and HL-61284 (J. R. Fineman), March of Dimes Grant FY00-98 (S. M. Black), and American Heart Association, Midwest Affiliate Grant 0051409Z (S. M. Black).

S. M. Black is a member of the Feinberg Cardiovascular Research Institute.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. M. Black, Division of Neonatology, Northwestern Univ. Medical School, Ward 12-191, 303 E. Chicago Ave., Chicago, IL 60611-3008 (E-mail: steveblack{at}northwestern.edu).

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


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