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Am J Physiol Heart Circ Physiol 291: H2272-H2281, 2006. First published June 23, 2006; doi:10.1152/ajpheart.00404.2006
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Enhanced {alpha}1-adrenergic trophic activity in pulmonary artery of hypoxic pulmonary hypertensive rats

James E. Faber,1 Caroline L. Szymeczek,2 Sundeep S. Salvi,3 and Hua Zhang1

Departments of 1Cell and Molecular Physiology, School of Medicine and 2Biology, University of North Carolina, Chapel Hill, North Carolina; and 3Chest Research Foundation, Pune, India

Submitted 19 April 2006 ; accepted in final form 12 June 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mechanisms that induce the excessive proliferation of vascular wall cells in hypoxic pulmonary hypertension (PH) are not fully understood. Alveolar hypoxia causes sympathoexcitation, and norepinephrine can stimulate {alpha}1-adrenoceptor ({alpha}1-AR)-dependent hypertrophy/hyperplasia of smooth muscle cells and adventitial fibroblasts. Adrenergic trophic activity is augmented in systemic arteries by injury and altered shear stress, which are key pathogenic stimuli in hypoxic PH, and contributes to neointimal formation and flow-mediated hypertrophic remodeling. Here we examined whether norepinephrine stimulates growth of the pulmonary artery (PA) and whether this is augmented in PH. PA from normoxic and hypoxic rats [9 days of 0.1 fraction of inspired O2 (FIO2)] was studied in organ culture, where wall tension, PO2, and PCO2 were maintained at values present in normal and hypoxic PH rats. Norepinephrine treatment for 72 h increased DNA and protein content modestly in normoxic PA (+10%, P < 0.05). In hypoxic PA, these effects were augmented threefold (P < 0.05), and protein synthesis was increased 34-fold (P < 0.05). Inferior thoracic vena cava from normoxic or hypoxic rats was unaffected. Norepinephrine-induced growth in hypoxic PA was dose dependent, had efficacy greater than or equal to endothelin-1, required the presence of wall tension, and was inhibited by {alpha}1A-AR antagonist. In hypoxic pulmonary vasculature, {alpha}1A-AR was downregulated the least among {alpha}1-AR subtypes. These data demonstrate that norepinephrine has trophic activity in the PA that is augmented by PH. If evident in vivo in the pulmonary vasculature, adrenergic-induced growth may contribute to the vascular hyperplasia that participates in hypoxic PH.

vascular smooth muscle; hypertrophy; catecholamines; adrenergic receptors; hypoxia

CHRONIC OBSTRUCTIVE DISEASES, hypoventilatory disorders, and extended exposure to high altitude lead to hypoxic pulmonary hypertension (PH), the most common type of PH not arising from left ventricular dysfunction. Chronic alveolar hypoxia is thought to cause hypoxic-metabolic and/or hemodynamic "injury" to the endothelium and vascular wall, including enhanced production of reactive oxidative species (for a review, see Refs. 6, 14, 19, 33, 36, 37, and 40). Induction of multiple intracellular and autocrine and paracrine signals leads to proliferation of endothelial cells, vascular smooth muscle cells (VSMCs), fibroblasts, intermediate cells, and pericytes. Proliferation, migration, and matrix accumulation elicit wall hypertrophy, fibrosis, and distal muscularization in the pulmonary arterial circulation. These changes cause structural lumen loss, enhanced VSMC tone, and reduced compliance, which, together with polycythemia, increase pulmonary vascular resistance, right heart hypertrophy, and risk of right heart failure. Although a number of signaling pathways have been implicated, the pathogenesis of the hypertrophic vascular changes is complex and unclear. Treatment options have evolved for patients with hypoxic PH wherein the primary hypoxic disturbance cannot be corrected (14, 15, 19). However, current therapies are limited and the prognosis is often grave. Thus research continues toward identifying the pathophysiological mechanisms causing hypoxic PH.

Enhanced activity of the sympathetic nervous system in hypoxic PH is not usually mentioned as a potential contributor, although adrenergic catecholamines have been proposed to participate (19, 33, 44). No studies have tested this possibility despite a number of clues supporting it. Norepinephrine (NE) induces growth of VSMCs and adventitial fibroblasts (5, 7, 8, 10, 11, 12, 42, 43, 46–50; discussed below). NE concentration in pulmonary artery (PA) blood (i.e., mixed venous blood) is ~40% higher than in arterial blood (21), owing to its diffusion into the blood from peripheral nerves and adrenomedullary chromaffin cells, followed by partial metabolism in the liver and lungs. This constitutively higher level of NE in pulmonary arterial blood is further increased by chemoreflex-dependent sympathoexcitation during exposure to reduced FIO2 in experimental studies and in patients with primary and secondary PH (see Refs. 31, 36 and references therein, and 44). Hypoxia also increases expression of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis (36), and increases expression of {alpha}1-adrenoceptors (ARs) in VSMCs in vitro and in aorta in vivo (9). The incidence of PH is increased by certain appetite suppressants (aminorex and fenfluramine), cocaine, and liver disease, which all increase plasma catecholamines (36, 37). Although adrenergic innervation of PAs is less dense than most other vascular beds (25), the contractile sensitivity of medium-sized PAs to NE, which is {alpha}1-AR dependent (28), is the highest among 12 rabbit arteries and veins studied in vitro (up to 200-fold greater) (3). It is not known whether the trophic sensitivity of systemic arteries to NE extends to PAs or whether such sensitivity is increased in hypoxic PH.

Certain G protein-coupled receptors, including angiotensin, endothelin-1, and serotonin, exert growth factor-like actions on VSMCs, and have been implicated in the vascular hypertrophy of hypoxic PH (2, 14, 19, 24, 30, 35, 38, 39). More recently, catecholamines have also been shown to stimulate growth of arterial VSMCs and adventitial fibroblasts. NE induces {alpha}1-AR-dependent proliferation, hypertrophy, and migration of VSMCs and adventitial fibroblasts of rat aorta studied in cell and organ culture (7, 12, 46–48) and in vivo (10, 43). The trophic action of NE is mediated by reactive oxidative species-dependent transactivation of EGF receptors (5, 49). Furthermore, adrenergic growth is strongly augmented in aorta (47) and carotid artery (10, 11, 42, 50) after either balloon injury or altered shear stress, contributing significantly to the subsequent hypertrophic remodeling in these vessels, and in small collateral arteries undergoing shear stress-induced outward remodeling (8). Local or systemic pharmacological blockade or gene deletion of dopamine beta-hydroxylase or specific {alpha}1-AR subtypes ({alpha}1A-AR in rat and {alpha}1B-AR in mouse) sharply reduces neointimal, medial, and adventitial growth and lumen loss after carotid injury (10, 11, 42, 50). Interestingly, nonsubtype-specific {alpha}1-AR antagonists reduce wall hypertrophy, resistance, and pressure in the pulmonary circulation of patients with PH (for review, see Ref. 36). However, the mechanisms underlying these beneficial effects have not been investigated, presumably because hypotensive side effects of these agents have precluded their use in patients with PH. These observations suggest that NE not only mediates VSMC contraction for control of pulmonary vascular compliance but also may exert trophic activity on pulmonary vessels.

Despite the above considerations, no studies have examined whether NE exerts trophic activity on PAs and whether this is augmented in hypoxic PH. Hypertrophy and constriction of the small PAs and arterioles account for the major increase in pulmonary resistance underlying hypoxic PH. The main PA similarly exhibits pronounced hypertrophy of the intima, media, and adventitia and is often studied in vitro as a model for pulmonary arterial vessels (27, 29, 32, 39, 40, 45). Herein, the left PA from normoxic and hypoxic rats was maintained in organ culture under conditions simulating transmural pressure and blood gases present in vivo in normoxia and chronic hypoxia. NE stimulated DNA and protein synthesis in normoxic PA. These effects were augmented in hypoxic PA and inhibited by {alpha}1A-AR antagonist, consistent with mRNA expression of this receptor in extra- and intrapulmonary arteries. These data support the hypothesis that nerve-released and/or blood-borne NE may contribute to excessive wall growth in hypoxic PH.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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 REFERENCES
 
Hypoxic PH. Male Sprague-Dawley rats (450–500 g) were maintained in a normobaric 0.1 FIO2 environment, except for two ~15-min exposures to normoxia to allow changing of bedding and replenishment of food, water, and absorbents for CO2 and water vapor. All experimental procedures were approved by the University of North Carolina Institutional Animal Care and Use Committee. Animals were anesthetized 9 days later with a mixture ketamine (150 mg/kg) and acepromazine (1.50 mg/kg im). After sterile thoracotomy, hematocrit was determined, the ascending aorta was cannulated via the left ventricle, the open chest was flooded with 4°C phosphate-buffered solution (PBS), and the vasculature was perfused with cold PBS at 100 mmHg for exsanguination and to cool the PA and inferior vena cava (VC). The VC, which is exposed to the same blood gas, hormone, and plasma catecholamine concentrations in vivo as PA, was studied as a nonpulmonary vessel to determine whether the augmented trophic effects of NE in hypoxic rats were specific to PAs.

PA and VC organ culture. All procedures were carried out under sterile conditions at 4°C. The thoracic cavity was suffused with 4°C PBS, and the proximal left PA and inferior VC were isolated from surrounding tissues and fat with a stereomicroscope, excised, and placed in 4°C PBS. Hearts were removed to 4% paraformaldehyde for later determination of right ventricle and left ventricle-plus-septum wet weights. The PA was trimmed to 3-mm length and placed into an organ culture system that permits application of circumferential wall tension (preload) (7, 47). Vehicle-treated and drug-treated PA were from separate rats, because the PA is too small to be transected and still yield enough material for biochemical analysis. Preloads were set at 0.72 g for PA from normoxic rats and 1.44 g for hypoxic PAs, based on normal PA mean pressure of 16 mmHg, average intrapleural pressure of –4 mmHg (for a transmural pressure of 20 mmHg), and the reported doubling of average PA pressure in rats after 9–10 days in 0.1 FIO2, respectively (29, 32, 45). These preloads are similar to those used by others for PA ring contractile studies (29, 45). Maintenance of arteries in vitro under preload favors the quiescent VSMC phenotype (4, 7, 47), whereas dedifferentiation, proliferation, and centripetal migration of VSMCs occur when arteries are cultured in the absence of load (23). VCs were placed into organ culture without load because mean transmural thoracic VC pressure is normally near zero (3–4 mmHg) and because the thin-walled VC in culture media does not maintain a longitudinal orientation required for preloading. PAs and VCs were maintained for 72 h in serum-free media consisting of 50% Dulbecco's modified Eagle's medium and 50% F-12 medium, supplemented with 5 mg/l transferrin (Sigma, St. Louis, MO), 17.6 mg/l ascorbic acid, 6 ng/ml selenium, 100 U/ml penicillin, and 100 µg/ml streptomycin, in a 37°C incubator. Oxygen and CO2 were maintained at, respectively, 40 and 46 mmHg (pH 7.35) for normoxia and 30 and 24 mmHg (pH 7.5), according to reported values for mixed venous blood gases in rats and humans acclimatized to 0.1 FIO2 (17, 41).

The following drugs were dissolved in PBS, unless stated otherwise: NE (Sigma; dissolved in 100 µmol/l ascorbated PBS, made fresh daily and maintained in the dark at 4°C), endothelin-1 (Sigma), {alpha}1A-AR antagonist KMD-3213 (kindly provided by Dr. Y. Kurashina, Kissei Pharmaceutical, Matsumoto-City, Japan), and {alpha}1D-AR antagonist BMY-7378 (RBI Biochemical, Natick, MA). We have previously confirmed the potency and selectivity of these antagonists at the concentrations used herein (see RESULTS) (12, 42, 47, 48). Media were changed and drugs were readded at 24 h and 40 h; media were also changed to low methionine (2.24 mg/l) at 40 h. [35S]methionine (1,000 Ci mmol/l, Amersham, Arlington Heights, IL) was added at a final concentration of 1.25 µCi/ml at 48 h. Twenty-four hours later, PAs and VCs were removed from organ culture and immersed in 4°C PBS. Digital images of submerged PAs were acquired in several seconds (Kodak Image Station 440; ImageJ, National Institutes of Health) for determination of length and diameter (this could not be done for VCs because their thin wall did not maintain vessel shape). Vessels were blotted, weighed, and frozen in liquid nitrogen and stored at –80°C.

Protein synthesis, protein content, and DNA content. The following assays were detailed previously (46, 47). Frozen vessels were pulverized in liquid nitrogen and added to 4°C PBS containing 1% nonidet p40 (Sigma), 0.5% sodium deoxycholate (Sigma), 1% sodium dodecyl sulfate (GIBCO-BRL, Grand Island, NY), and proteinase inhibitors [30 ul/ml aprotinin, 100 µg/ml PMSF, and 1 mM sodium orthovanadate (final concentrations; Sigma)], and homogenized (model TH, Omni, Atlanta, GA) on ice at maximal speed for 25 s. Procedures described below were carried out at 4°C. Homogenates were passed through a 21-gauge needle three times to shear DNA and allowed to rest for 20 min. After centrifugation for 20 min at 14,000 rpm, protein content of the supernatant [representing primarily soluble, intracellular protein (47)] was determined in duplicate by bicinchoninic acid assay (Pierce, Rockford, IL). DNA content was determined in triplicate (Hoefer DyNA Quant 200; Amersham Pharmacia, San Francisco, CA). Protein synthesis (measured for the last 24-h interval in culture) was determined in duplicate by precipitation with trichloroacetic acid and collection on GF/C filters (Whatman, Clifton, NJ), followed by scintillometry (Ecoscint H, National Diagnostics, Atlanta, GA).

{alpha}1-AR expression. In separate rats, relative mRNA levels for {alpha}1-ARs were determined with RT-PCR in PA, intralung hilar branches of the PA, and the periphery of whole lung lobe. Rats were transcardially perfused with sterile, diethyl pyrocarbonate-treated 4°C PBS containing RNase inhibitors (PBS+). The above tissues were isolated with a stereomicroscope during superfusion of the heart and lungs with cold PBS+. Tissues were frozen and pulverized in liquid N2, and total RNA was extracted and treated with RQ1 RNase-free DNase (1 U for 10 ug RNA) for 45 min at 37°C as described previously (12, 13). RNA concentration was determined spectrophotometrically at an optical density of 260 nm. Samples were accepted for purity and quality if the A260/A280 ratio was >1.8 and if electrophoresis on 1.2% formaldehyde denatured agarose gels yielded intact ribosomal RNA. RT-PCR of {alpha}1A-, {alpha}1B-, {alpha}1D-, and 18S ribosomal transcripts was performed as detailed previously (12, 13). In preliminary studies, cycle number-product curves were obtained for each target in each tissue type by using primer and competimer concentrations determined previously. The cycle numbers on the midpoint of the curves were then chosen for subsequent relative RT-PCR analysis. Cycle numbers for {alpha}1A-AR, {alpha}1B-AR, {alpha}1D-AR, and 18S transcripts were, respectively, as follows: for PA, 40, 34, 32, and 32; for intralung hilar arteries, 36, 34, 32, and 30; for whole lung, 36 for all transcripts. No-reverse transcriptase controls verified no genomic contamination. The primer pairs employed give similarly sized products with similar efficiencies of amplification (12, 13).

Data are given as means ± SE for n number of vessels or animals (1 vessel per animal) unless stated otherwise. Significance (P < 0.05) was determined by unpaired Student's t-tests or ANOVA followed by Bonferroni protected t-tests.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Baseline data for hypoxic PH rats. As expected after 9 days of hypoxia (32), hematocrit increased by 26% and wet weight of the right ventricle and left PA increased by 34% and 67%, respectively (Fig. 1). By comparison, the thoracic inferior VC, which averaged 4.8 mm in length, had similar weight in normoxic and hypoxic rats (11.0 ± 0.4 mg, n = 9; and 11.2 ± 0.5 mg, n = 13, respectively). The wet weight of left ventricle-plus-septum decreased slightly in hypoxic rats, in accordance with the 12% decrease in body weight (Fig. 1), resulting in no effect on the Fulton ratio. Although PA length was cut to ~3 mm, length variation could alter comparisons of absolute vessel weights and the other measures reported below. Therefore, length and diameter were determined by digital image analysis in a subset of the vessels for the data in Fig. 1 (and in all vessels for data shown in the subsequent figures): In this subset, lengths normalized to either diameter or vessel weight in normoxic PA (n = 9) were 3.1 ± 0.2 mm and 1.8 ± 0.1 mm/mg, respectively, and in hypoxic PA (n = 5) were 3.0 ± 0.6 mm and 3.4 ± 0.3 mm/mg, resulting in hypertrophy (in mg/mm) in hypoxic rats of 82 ± 21% (P < 0.01). Similar changes in hematocrit and in weight of heart, PA, and whole body have been reported previously in rats for this duration of 0.1 FIO2 (e.g., 27, 32, 39). Thus PA, but not VC, undergoes hypertrophy in hypoxic PH. The VC data are consistent with an absence of hypertrophic changes in rat aorta in hypoxic PH (39).


Figure 1
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  		Fig. 1. Baseline data (means ± SE here and in other figures) after 9 days of ambient hypoxia (fraction of inspired O2 = 0.1). The n sizes at base of columns are number of animals, hearts, or vessels (in this and in other figures). RBC, red blood cells. **P < 0.01 and ***P < 0.001 vs. open bars.

 
Hypoxia augments trophic response of PA to NE. Exposure of PA to 1 µmol/l NE for 72 h caused a similar increase (compared with vehicle) in DNA and protein content (10% in both) in normoxic PA, suggesting stimulation of modest proliferation (P < 0.05; Fig. 2). Protein synthesis, which presumably increased during the initial 48h of NE exposure as we have observed in rat aorta (47), was not elevated during the final 24 h of NE exposure. In PAs from hypoxic rats, NE-induced increases in DNA and protein content were augmented approximately threefold over normoxic PAs. Protein synthesis increased 34-fold. To determine whether differences in culture conditions contribute to this augmentation, vessels from normoxic rats were maintained for 72 h under the same increased preload, increased pH, and altered gas conditions as hypoxic vessels (see MATERIALS AND METHODS). Similar trophic responses to NE were obtained for vessels taken from normoxic rats and maintained in hypoxic culture conditions (Fig. 2). Thus alterations in the PA, e.g., hypertrophy or increased adrenergic responsiveness of wall cells, created by in vivo conditions during chronic hypoxia, rather than culture conditions, conferred the augmented trophic sensitivity to NE.


Figure 2
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  		Fig. 2. Norepinephrine (NE; 1 µmol/l, 72-h exposure in organ culture)-induced hyperplasia (proportional increases in DNA and protein content) in pulmonary artery (PA) from normoxic rats is augmented in PA from rats exposed to 9 days of chronic hypoxia. Data normalized to values obtained for an equal number of vehicle-treated vessels. DNA and protein contents were determined from soluble (~cellular) SDS-lysis buffer extracts. Protein synthesis ([35S]methionine incorporation) was determined for final 24-h interval. Media were changed and drugs readded at ~24-h intervals (here and in studies shown in other figures). Amount of tonic load (preload; in g) applied to vessels and the O2 and CO2 (in mmHg) and pH conditions of the culture bath were, respectively, as follows: for normoxia, 0.72, 40, 46, and 7.35; for hypoxia, 1.44, 30, 24, and 7.50. Third bar in each panel is for PA from normoxic rats maintained in the same hypoxic culture conditions as vessels from hypoxic rats. Baseline data for this experiment are shown in Fig. 5. *P < 0.05, **P <0.01, and ***P <0.001 vs. vehicle-treated vessels; #P < 0.05 and ###P <0.001 vs. normoxia.

 
VC is exposed to similar blood gas, hormone, and plasma catecholamine concentrations in vivo as PA. However, NE caused no significant increase in DNA or protein content or protein synthesis in normoxic VC. In hypoxic VC, only a small increase in protein content occurred (+7 ± 3%, P < 0.05); DNA content (+6 ± 5%) and protein synthesis (+10 ± 6%, P = 0.06) were unaffected. Thus VC was not examined further. These data suggest that augmented trophic responsiveness of the PA to NE is not conferred to nonpulmonary vessels by blood conditions present during chronic hypoxia. It is possible that VCs did not respond to NE because they could not be maintained in culture under preload (see MATERIALS AND METHODS), which, at least for PA, is required for trophic responsiveness (GoFig. 4; discussed below). Also, unlike PA, VCs did not experience hypertrophy during hypoxia in vivo.


Figure 3
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  		Fig. 3. Top left: Growth response of PA from hypoxic rats after 72-h exposure to NE or endothelin-1 (ET1) in vessels maintained under hypoxic culture conditions (see Fig. 2). Data normalized to values obtained from an equal number of vehicle-treated vessels. Top right and bottom left and right: trophic effect of NE on PA from hypoxic rats is blocked by {alpha}1A-adrenoceptor ({alpha}1A-AR) antagonist. PA from hypoxic rats were placed in hypoxic culture conditions (see Fig. 2), pretreated for 30 min with 0.1 µM KMD-3213, and then exposed to 1 µM NE for 72 h (solid bars). Data for NE alone (open bars) are from Fig. 2. Data normalized to values obtained from an equal number of vehicle-treated vessels. OD, optical density; cpm, counts/min. **P < 0.01 and ***P < 0.001 vs. vehicle groups.

 

Figure 4
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  		Fig. 4. Effect of maintaining wall tension during in vitro study of trophic responsiveness of PA to NE. PA from normoxic and hypoxic rats were placed in normoxia or hypoxia culture conditions (see Fig. 2) in the absence of wall tension (no preload) and exposed to NE or vehicle for 72 h. Data for preload groups are from Fig. 2. Data normalized to values obtained from an equal number of vehicle-treated vessels. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. vehicle; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. no preload.

 
Trophic effect of NE is inhibited by {alpha}1A-AR antagonist. NE-mediated growth in hypoxic PA extended over a concentration range (Fig. 3) similar to that for contraction (12). At concentrations of NE (1 µmol/l) and endothelin-1 (0.01 µmol/l) that cause near-maximal contraction of rat PA in vitro (39), NE had greater trophic activity than endothelin-1 (Fig. 3). The {alpha}1A-AR antagonist KMD-3213 (0.1 µmol/l), which is 583-fold and 56-fold selective for {alpha}1A-ARs over {alpha}1B- and {alpha}1D-ARs, respectively (see Ref. 47 and references therein), abolished NE-induced growth (Fig. 3). In contrast, NE in the presence of the {alpha}1D-AR antagonist BMY-7378 (0.1 µmol/l), which is 267-fold selective for {alpha}1D-ARs over {alpha}1A- and {alpha}1B-ARs (47), caused similar increases in DNA content (18 ± 8%), protein synthesis (23 ± 3%), and protein content (16 ± 8%) to those in the presence of antagonists alone (n = 4 for each group). Neither antagonist, alone, had an effect on PAs exposed to vehicle. Our previous studies of rat aorta in organ culture (47) and in cultured VSMCs (12, 46, 48) have confirmed the selectivity of these antagonist concentrations and absence of effects of antagonists alone. We did not test {alpha}1B-AR blockade because of lack of availability of an antagonist with high selectivity. These results suggest that the growth effects of NE may be mediated by the {alpha}1A-AR.

Wall tension maintains trophic responsiveness of hypoxic PA in vitro. Preload was maintained twofold higher in hypoxic versus normoxic PA to reflect the higher average transmural pressures seen in vivo in rats with hypoxic PH of 9 days' duration as studied herein (29, 32, 45). To determine whether wall tension influences the trophic responsiveness to NE, PAs from normoxic and hypoxic rats were placed, respectively, in normoxic or hypoxic culture conditions in the absence of preload and exposed to NE or vehicle for 72 h. Figure 4 shows responses to NE compared with Fig. 2 data for PAs maintained under preload. NE had a similar small growth effect on vessels from normoxic rats maintained in normoxic culture conditions, irrespective of whether preload was present or absent. In contrast, the heightened trophic response to NE in the hypoxic PA was absent when vessels were maintained in culture without preload. The tendency for the trophic effects of NE in the normoxic vessels to be greater in the absence of preload may extend from the known dedifferentiation and induction of proliferation of smooth muscle cells when vessels are placed in culture in the absence of load (23). This state may have augmented the growth response to NE. In previous studies, we have demonstrated that conditions that promote the proliferative phenotype in SMCs and fibroblasts in intact systemic arteries heightens their trophic response to NE (5, 10, 11, 47, 50). The loss of the trophic effect of NE in hypoxic PAs maintained in the absence of preload may have occurred because these PAs, which were hypertrophied PA from rats with hypoxic PH (Fig. 1), lost weight during culture: Weight of the hypertrophied PAs from hypoxic rats declined by 15.7 ± 2.5% (n = 15; P < 0.0001) after 3 days in culture in the absence of preload. This likely occurred from cell atrophy and/or reduction of extracellular matrix (discussed below for Fig. 5 data) when cultured in the absence of preload. In contrast, VC from hypoxic rats, which, unlike PA, does not develop hypertrophy in vivo, did not lose weight over 72 h in culture in the absence of preload (–3.1 ± 1.9%, n = 15). Thus the catabolic conditions created by absence of preload in hypoxic PAs may have opposed the trophic effect of NE, resulting in no measured growth to NE.


Figure 5
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  		Fig. 5. Absolute DNA and protein content (normalized to wet wt) for normoxic PA in Fig. 2 that were exposed to NE for 72 h while maintained under circumferential tension (preload) and vessels in Fig. 4 that were not maintained under tension (no preload). **P < 0.01 vs. normoxia; ##P < 0.01 vs. no preload.

 
Absolute DNA content and protein content (normalized for tissue weight) for the data in Fig. 4 are shown in Fig. 5. Minimal differences were evident between normoxic and hypoxic vessels maintained under preload. Thus augmented NE growth in hypoxic PAs (Fig. 4) does not arise from baseline differences. Also, the increase in hypoxic PA weight (Fig. 1) is not consistent with an increase in cell density or cell size, because DNA and protein content (per unit of vessel weight in Fig. 5) are the same for normoxic and hypoxic vessels maintained under preload. Rather, the data suggest that PA hypertrophy after 9 days of alveolar hypoxia is due to a proportionate increase in total cell number and extracellular matrix (39). In contrast, the absence of preload in hypoxic PAs caused DNA content, but not protein content, to increase, presumably reflecting proliferation into initially smaller daughter cells. However, potential effects of PH and subsequent placement in organ culture on DNA polyploidy in PA could modify these interpretations. Irrespective, the data in Figs. 4 and 5 suggest that maintaining wall tension is important when studying trophic effects in PA in organ culture.

{alpha}1-AR expression is reduced in hypoxic PH. Relative mRNA levels for {alpha}1-ARs were examined to determine whether {alpha}1A-AR expression is increased by chronic hypoxic PH as a possible mechanism for the increased trophic sensitivity of hypoxic PAs to NE that was blocked by {alpha}1A-antagonist. Expression of the three {alpha}1-AR subtypes was similar in PA, intrapulmonary hilar arteries, and tissue samples from peripheral lung lobe. Vascular segments in the latter consist predominantly of small arterioles, capillaries, and venules (Fig. 6), whereas RNA from whole peripheral lung lobe of mice primarily reflects that of endothelial, epithelial, and interstitial cells (16). Levels of {alpha}1A-AR mRNA were lowest, and {alpha}1D-AR levels were generally highest. After 9 days of hypoxia, {alpha}1B- and {alpha}1D- but not {alpha}1A-AR expression was reduced in PA and whole lung, whereas all three mRNA levels were reduced, and to a greater degree, in intralung hilar arteries (Fig. 7). Hypoxia had no effect on 18S ribosomal RNA in PA and whole lung but increased it in hilar arteries (Fig. 7), presumably reflecting cell proliferation and/or hypertrophy that can continue beyond 10 days of hypoxia in small arteries while becoming complete by 3–5 days in PA (27, 40).


Figure 6
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  		Fig. 6. Relative mRNA levels, as determined by RT-PCR, for {alpha}1-ARs in PA, intralung hilar branches, and whole lung taken from periphery of a lobe. All tissues were from normoxic rats. Top left: actual agarose gels. A–C: results from band densitometry, with normalization to 18S ribosomal mRNA levels. n, Number of RNA samples, each derived from 3–4 rats.

 

Figure 7
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  		Fig. 7. Relative mRNA levels, as determined by RT-PCR, for {alpha}1-ARs in PA, intralung hilar branches, and whole lung taken from periphery of a lobe. All tissues were from hypoxic rats. Levels were normalized to 18S ribosomal mRNA levels and were expressed relative to (and statistically tested against) levels in normoxic rat from Fig. 6. n, Number of RNA samples, each derived from 3–4 rats; n sizes and labels for columns in top panels are the same as for columns in bottom panels. *P < 0.05 and ***P < 0.001 vs. normoxic tissue levels.

 
{alpha}-AR mRNA and receptor levels are too low, especially in the pulmonary vasculature, to permit localization by in situ hybridization or receptor autoradiography. Also, no antibodies are available that are capable of identifying {alpha}1-AR subtypes in blood vessels by histology or immunoblot. In addition, the small size and wall thickness of rat PAs prevents use of competition binding studies to confirm these mRNA data at the receptor level. However, densities of {alpha}1-AR subtypes, as determined by radioligand binding assay, follow their mRNA levels in normal rat aorta and carotid, as well as in response to balloon injury, which causes {alpha}1-AR downregulation (12, 13). Assuming this is also true for the pulmonary vasculature, the present data indicate that increased trophic sensitivity of hypoxic PA to NE is not due to increased {alpha}1A-AR expression but rather occurs despite a trend toward reduced expression.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The present study examined PAs from normoxic and hypoxic rats that were maintained in organ culture at preload tensions and blood gases/pH similar to those present in vivo in normoxia and hypoxia. Although lacking pulsatile wall tension and flow, this model permits exposure of vessels to vasoactive agents (e.g., NE, endothelin, and adrenergic antagonists) that, if administered in vivo, would cause changes in arterial pressure capable themselves of inducing vascular trophic effects. NE produced a modest increase in DNA and protein content in normoxic PA that was strongly augmented in hypoxic PA, that was dose dependent, and that was more potent that endothelin-1 when compared at concentrations equieffective for constriction. We also found that the extralung PAs and their intralung hilar branches express all three {alpha}1-AR mRNAs and that they are downregulated in the hypoxic PA (albeit {alpha}1A-AR was reduced the least or not at all). The trophic effects of NE in hypoxic PA appear to be mediated by the {alpha}1A-AR subtype, based on analysis with pharmacological antagonists. Lastly, maintenance of preload in arteries studied in organ culture is required to permit detection of adrenergic trophic activity, at least over the 3 days studied herein. These data support our hypothesis that chronic alveolar hypoxia confers increased adrenergic trophic sensitivity that, in turn, may contribute to the excessive vascular wall growth in PH.

The trophic effect of NE in rat PA is similar to the response of rat thoracic aorta studied in organ culture (47). In that study, NE induced a modest SMC hypertrophy (10% over 48-h treatment) that was also strongly augmented (up to 20-fold increases in DNA and protein content and protein synthesis) in vessels that had received balloon injury in vivo 4 or 12 days before removal into organ culture. The effects were dose dependent, had potency comparable to angiotensin, were associated with downregulation of proteins associated with the contractile phenotype of VSMCs, and were unaffected by beta-, {alpha}2-, {alpha}1D-, or {alpha}1B-AR antagonists but were abolished by {alpha}1A-AR blockade (47). Judging from previous in vivo studies (10, 43), the modest trophic effect in PA of normoxic rats observed in the current study may only be evident in vivo when sympathoexcitation is maintained for prolonged duration. Thus chronic local in vivo administration of {alpha}1-antagonists to the normal carotid artery wall had no effect on wall dimensions in adult rats (10). However, chronic local elevation of wall NE levels caused a 25% increase in wall-to-lumen ratio (10), as did chronic systemic catecholamine infusion at levels subthreshold for elevating arterial pressure (43). Such effects may promote normalization of wall stress in the presence of elevated arterial and PA pressures that accompany sympathoexcitation.

NE increased DNA and protein contents that were almost identical in magnitude (NE also increased protein synthesis). Similar increases in DNA and protein content are known to correlate well with cell proliferation in the intact vascular wall, although induction of polyploid hypertrophy in some VSMCs can contribute to such changes. We do not know the relative contributions of proliferation versus polyploidy and hypertrophy of smooth muscle cells. However, adventitial fibroblasts do not undergo hypertrophy (47), or to our knowledge, polyploidy. To be sure, a small fraction of VSMCs can undergo polyploidy rather than proliferation in response to growth stimuli and may be doing so to NE. However, proliferation, polyploidy, and hypertrophy are all part of the overall trophic response of VSMCs to certain growth stimuli. The main finding of our study, i.e., that NE induces growth of the PA and that this effect is augmented in hypoxic PH, could arise from a combination of these effects as well as proliferation of adventitial fibroblasts.

Trophic AR subtype. Pharmacological antagonists were used to examine the adrenergic receptor type(s) mediating trophic activity of NE. KMD-3213 has the highest {alpha}1A-AR selectivity available, i.e., ~600-fold selective for {alpha}1A over {alpha}1B and ~60-fold selective for {alpha}1A over {alpha}1D (see Ref. 47 and references therein). Although KMD-3213 is less selective for the {alpha}1D-AR, BMY-7378, which is 267-fold selective for the {alpha}1D-AR (47), had no effect. Although we did not test an {alpha}1B-AR antagonist because none exists with high selectivity, these results suggest that growth effects of NE may be mediated by the {alpha}1A-AR. This is in agreement with our previous studies in rat aorta and carotid artery (10, 42, 47). Among {alpha}1-ARs, mRNA for the {alpha}1A-AR subtype also showed the least downregulation in the hypoxic PAs. We did not examine antagonists or mRNAs for {alpha}2- or beta-ARs because KMD-3213 abolished trophic actions of NE and because {alpha}2- or beta-AR antagonists had no effect on the growth effects of NE on rat aorta and carotid (10, 42, 47).

Expression of {alpha}1-AR subtypes. mRNA levels for {alpha}1-ARs were examined to determine whether {alpha}1A-AR expression increases in PH to account for enhanced trophic activity of NE. In normal rats, relative expression of the three {alpha}1-AR subtypes was similar in the extrapulmonary arteries, intralung hilar branches, and in peripheral lung tissue where small arterioles, capillaries, and venules make up the vascular compartment. Transcript levels for {alpha}1A-ARs were lowest and {alpha}1D-AR levels highest in all three tissue samples. This expression pattern is similar to the medial layer of rat thoracic aorta and carotid (12, 13). In rats with hypoxic PH, mRNAs for {alpha}1B- and {alpha}1D- were reduced, with less or no effect on {alpha}1A-AR levels. We were unable to determine if these mRNA levels predict receptor subtype densities because the small size of PAs does not yield sufficient membrane protein for competition binding assays and because no antibodies exist that distinguish among the subtypes in blood vessels. However, densities of {alpha}1-AR subtypes follow their mRNA levels in rat aorta and VSMCs cultured from it (12), which may extend to the pulmonary vasculature. In support of this, NE-induced contraction of isolated rat PAs, which is {alpha}1D-AR dependent, showed no change in potency or affinity in rats with monocrotaline-induced PH (see Ref. 28 and references therein). However, maximal response and receptor reserve were reduced, which is consistent with the reduced {alpha}1D-AR expression we observed. Contractile responses to potassium chloride, serotonin, or PGF2{alpha} were not reduced (see Ref 28 and references therein). Others have found that maximal contraction to NE is reduced in isolated PAs from rats with hypoxic PH, although maximal responses to endothelin-1 and thromboxane agonist were also reduced (39).

Our expression data indicate that increased trophic sensitivity of hypoxic PA to NE is not due to increased {alpha}1A-AR expression, because mRNA levels were either unchanged or tended toward reduction. Only a transient several-day decline in {alpha}1A-AR mRNAs occurred in vivo after balloon injury of rat thoracic aorta, whereas {alpha}1B- and {alpha}1D-AR mRNAS underwent sustained reduction (13). Like the present results, this was associated with a strongly increased {alpha}1A-AR-dependent trophic sensitivity to NE that contributes significantly to neointimal formation and lumen narrowing (10, 42, 47, 50). Elevated catecholamine levels in PH (20, 44), together with modulation of VSMCs to the proliferative phenotype during PA hypertrophy (19, 40), likely contribute to the general {alpha}1-AR downregulation we observed. We have observed similar results in the balloon-injured rat aorta (12, 13), a procedure that is unlikely to increase systemic sympathetic activity. On the other hand, decreased {alpha}1-AR density occurs in the right and left ventricles of rats exposed to 15 days of hypoxia (26), which may reflect reduced expression from receptor downregulation due to elevated catecholamines, because only the right ventricle undergoes hypertrophy. Despite no increase in {alpha}1A-AR mRNA, we find that trophic adrenergic sensitivity is strongly increased in hypoxic PH. Our pharmacological analysis suggests that this is signaled by {alpha}1A-ARs, a conclusion supported by similar results in rat aorta and carotid (10, 42, 47) and by the maintained expression of this subtype that we observed. Interestingly, unlike transcription of the other {alpha}1-AR genes, {alpha}1A-AR expression is resistant to downregulation when cardiomyocytes are exposed to NE for 24 to 72 h (1, 34).

Importance of wall tension in vitro. We and others have previously demonstrated that wall tension is required for maintenance of contractile phenotype and quiescence of VSMCs in aorta organ culture (4, 7, 47). Our current results extend this to trophic responsiveness to NE. Regression of hypertrophy was also evident in hypoxic PAs cultured without preload. It is possible that signals directing the reversal of hypertrophy may interfere with or oppose trophic actions of NE, leading to the loss of NE responsiveness. In addition to effects on NE-mediated growth in vitro, maintenance of preload prevented an increase in baseline DNA content in normoxic and hypoxic vessels that otherwise occurred in the absence of load. This may reflect proliferation of VSMCs or fibroblasts into initially smaller daughter cells. These data are consistent with evidence that wall tension maintains quiescence in vitro (4, 23, 47). Our results demonstrate the importance of maintaining wall tension when studying trophic effects in PA in organ culture.

Potential mechanisms augmenting adrenergic trophic activity. Hypoxic PH involves multiple pathophysiological mechanisms (6, 14, 19, 24, 30, 33, 38, 40). An important initiating disturbance may involve repeated episodes of hypoxic pulmonary vasoconstriction followed by dilation, with consequential fluctuations in shear stress and oxidative stress, endothelial cell injury, and resultant deficits in endothelial nitric oxide and prostacyclin activity (6, 14, 15, 19, 22, 40). Endothelial cell activation and decline in tonic activity of nitric oxide and prostacyclin favor increased smooth muscle tone, oxidative stress, platelet/leukocyte activation and adhesion, and proliferation of vascular wall cells, as well as increased levels of cytokines, inflammatory mediators, receptor tyrosine kinase growth factors, and the trophic G protein-coupled receptor agonists angiotensin, endothelin, and serotonin that activate downstream mitogen-activated protein kinases (MAPK) (24, 27, 30, 35, 38, 39). These changes and the intracellular pathways activated by them could augment the trophic activity of catecholamines on pulmonary vessels in several ways. NE-induced growth of VSMCs has thus far been defined as follows: {alpha}1-AR -> NADPH-oxidase -> superoxide and H2O2 -> HB-EGF -> EGF receptor -> Raf1/MEKK/ERK1/2 -> proliferation and protein synthesis (5, 49). In adventitial fibroblasts, which also express abundant {alpha}1-ARs (12), the pathway does not require HB-EGF for activation of EGF receptor; in addition, ERK1/2 and p38 and JNK MAPKs each contribute to the {alpha}1-AR-induced proliferation and protein synthesis of adventitial fibroblasts (5, 49). Increased reactive oxygen species resulting from reduced nitric oxide production, from increased leukocytes and inflammatory cytokines, and from growth factors and G protein-coupled receptors agonists that signal through NADPH-oxidase activation could augment trophic activity of NE. Hypoxic PH has also been shown to be accompanied by elevated HB-EGF levels (31). In addition, endothelin, which contributes to hypoxic pulmonary vascular remodeling (14, 19, 30, 38), interacts synergistically at the postreceptor level to amplify vasoconstriction by NE of VSMCs, and NE is similarly synergistic for constrictor activities of endothelin (18). Whether trophic synergism exists among these agonists, which activate similar but not identical pathways signaling cell growth, or with serotonin and its 5-HT2B receptor, which have also been implicated significantly in hypoxic PH (24), has not been determined.

Besides interactions among pathways activated by {alpha}1-ARs and the above factors induced in hypoxic PH, other conditions could augment trophic activity of NE. Although hypoxia causes sympathoexcitation, chronic alveolar hypoxia does not increase growth of sympathetic nerves, at least in rabbit pulmonary vessels (25). However, reduced oxygen increases expression of tyrosine hydroxylase, the rate-limiting enzyme in NE synthesis (36), which could elevate NE levels in and released from pulmonary adrenergic nerves.

In conclusion, hypoxic PH augmented adrenergic trophic activity in PAs. This may reflect "injury" of pulmonary precapillary vessels by conditions present in developing hypoxic PH, for example, from increased pressure, pressure-induced hypertrophy, oxidative stress, and disturbed shear stress (from increased hematocrit/viscosity and/or fluctuations in regional constriction and dilation). We propose that this injury alters activity of autocrine and paracrine factors and cellular signaling pathways regulating smooth muscle cell growth, resulting in increased sensitivity of precapillary pulmonary vessels to the trophic effects of catecholamines. Thus adrenergic growth effects may participate in the development or progression of PH. Our conclusions are limited, however, because the PA was studied in vitro and because it may not reflect mechanisms active in the smaller precapillary vessels that primarily account for elevated pulmonary resistance in hypoxic PH. An additional limitation is that, unlike carotid artery and aorta (12, 13, 47), the small size and wall thickness of rat PAs prevent separation of media and adventitia for analysis of trophic effects of NE on VSMCs and adventitial fibroblasts. Studies examining the smaller PAs and arterioles, together with the development of methods to reduce or block expression of {alpha}1-AR subtypes selectively in the pulmonary circulation in vivo, are needed to further investigate this hypothesis.


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This research was supported by National Heart, Lung, and Blood Institute Grant HL-62584 (to J. E. Faber).


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. E. Faber, Dept. of Cell and Molecular Physiology, 6309 MBRB, Univ. of North Carolina, Chapel Hill, NC 27599-7545 (e-mail: jefaber{at}med.unc.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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J. E. Faber, C. L. Szymeczek, S. Cotecchia, S. A. Thomas, A. Tanoue, G. Tsujimoto, and H. Zhang
{alpha}1-Adrenoceptor-dependent vascular hypertrophy and remodeling in murine hypoxic pulmonary hypertension
Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2316 - H2323.
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