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Published ahead of print on July 8, 2004, doi:10.1165/rcmb.2004-0051OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 501-509, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0051OC

Cigarette Smoke Induces Persisting Increases of Vasoactive Mediators in Pulmonary Arteries

Joanne L. Wright, Hsin Tai and Andrew Churg

Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada

Address correspondence to: J.L. Wright, Department of Pathology, 2211 Wesbrook Mall, Vancouver, BC, Canada V6T 2B5. E-mail: jlwright{at}interchange.ubc.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The pathogenesis of cigarette smoke–induced pulmonary hypertension is not understood. We previously reported that a single smoke exposure acutely but transiently upregulated gene expression of the vasoconstrictor/vasoproliferative agents endothelin (ET) and vascular endothelial growth factor in pulmonary arteries from rat lungs. To determine whether similar changes occurred with chronic smoke exposure, we exposed Hartley guinea pigs, an outbred strain that develops pulmonary hypertension, to smoke for 2, 4, or 12 wk. Small intrapulmonary artery branches were isolated using laser capture microdissection, and gene expression was determined by real-time polymerase chain reaction. In smoke-exposed animals, there were significantly elevated but variable increases in gene expression, with some animals demonstrating 30- to 50-fold increases. Increases in ET and vascular endothelial growth factor expression occurred early and persisted through the exposure period, whereas increases in expression of the vasodilator, endothelial nitric oxide synthase, developed more slowly. Protein levels of these mediators were also elevated by immunohistochemical staining and correlated with increases in gene expression levels. We conclude that, in some animals, cigarette smoke induces persisting and marked vascular production of mediators that control vascular muscularization and contraction/dilation. These changes may be important in the development of smoke-induced pulmonary hypertension.

Abbreviations: chronic obstructive pulmonary disease, COPD • endothelial nitric oxide synthase, eNOS • endothelin, ET • laser capture microdissection, LCM • muscularized vessels, MV • polymerase chain reaction, PCR • partially muscularized vessels, PMV • vascular endothelial growth factor, VEGF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Epidemiologic studies have demonstrated progressive increases in pulmonary arterial pressure in some cigarette smokers who have chronic obstructive lung disease (1, 2). The pathogenesis of this process is unclear. The traditional view is that pulmonary hypertension is secondary to loss of vascular bed from emphysema, but we have shown that there is no loss of capillary vascular bed in a guinea pig model of chronic cigarette smoke exposure, despite the fact that some of these animals develop pulmonary hypertension (3). In addition, a study from the National Emphysema Treatment Trial (2) failed to find any correlation between mean pulmonary artery pressure and emphysema.

Other evidence in animals and in humans suggests that pulmonary hypertension might result from a direct effect of cigarette smoke on the pulmonary arteries, with increased production of the vasoconstrictive/proliferative peptide endothelin (ET) and stimulation of vascular cell proliferation by upregulation of vascular endothelial growth factor (VEGF) (4, 5), along with abnormal regulation of endothelial nitric oxide synthase (eNOS, otherwise known as NOS3), which produces the vasodilator, nitric oxide. Santos and colleagues (5) demonstrated immunohistochemically increased expression of VEGF protein in the pulmonary arteries of smokers and patients with moderate chronic obstructive pulmonary disease (COPD), and also found increased levels of VEGF mRNA and protein in bulk lung samples.

Using an animal model, we have previously examined the effects of cigarette smoke on the pulmonary vasculature, searching for evidence of changes in gene expression of vasoconstrictors and vasodilators. In bulk lung samples, we showed that smoke transiently increased inducible NOS expression but produced sustained increases in eNOS expression up to 1 mo. (6); however, these experiments did not indicate the anatomic origin of the mediators, an important issue, as these substances are widely expressed in the lung. Subsequently, we utilized a hand microdissection technique and demonstrated that a single smoke exposure induced an immediate (within 2 h of exposure) upregulation of ET, VEGF, and VEGF-receptor 2 gene expression in the main pulmonary artery and the larger medium-sized intrapulmonary arteries in rats (4). These data suggested that smoke directly and rapidly affects production of mediators that modify vasoconstriction and proliferation of vascular cells. These changes were, however, transient, with clear evidence of reversion toward control levels by 24 h after smoke exposure.

Whether these changes persist with chronic smoke exposure and how they may relate to the development of pulmonary hypertension is unknown. Guinea pigs, a species that develops pulmonary hypertension, do show progressive muscularization of the small vessels adjacent to the alveolar ducts, but no significant alteration of the muscular arteries adjacent to the membranous bronchioles (7), after long-term smoke exposure, implying that increased production of vasoproliferative agents, such as VEGF and ET, might be occurring on a long-term basis, at least in some particular sizes of vessels.

The present study was designed to determine: first, whether cigarette smoke exposure would result in a long-term alteration of gene expression of the vasoconstrictor/vasoproliferative/vasodilator mediators ET, VEGF, and eNOS in small intrapulmonary arteries; second, whether this expression would alter over a time course of months; and, third, whether expression levels differed depending upon vessel size. We also examined whether increased gene expression levels lead to increased protein visible by immunochemistry, and whether gene expression and protein levels correlated with structural changes in the small arteries.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Smoke Exposure and Tissue Collection
The research protocol was approved by the University of British Columbia. We exposed groups of three Hartley strain guinea pigs to the whole smoke of seven 2R1 research cigarettes (University of Kentucky) 5 d/wk for 2, 4, and 12 wk using our previously published exposure protocol (7). Groups of guinea pigs were exposed to room air as controls for the same periods. After killing with an overdose of urethane anesthesia (10 mg/kg), the lungs were removed and inflated with cold 100% ethanol (use of 100% ethanol is necessary to prevent RNA degradation because of the high levels of endogenous RNAases in guinea pig lungs). After fixation, slices of lung were embedded in paraffin and multiple 5-µm thick sections were cut onto diethyl pyrocarbonate (Sigma-Aldrich, Oakville, ON, Canada)–treated water and collected on cleaned slides. One section was deparaffinized and stained with hematoxylin and eosin to serve as a dissection guide. The remaining sections were deparaffinized and stained using the Arcturus stain and protocol (Arcturus, Mountain View, CA).

Microdissection Procedure
We utilized the Acturus Pixcell II (Arcturus) laser capture microdissection (LCM) apparatus. For the muscularized vessels (MV) adjacent to the membranous or respiratory bronchioles, we collected all vessels present on 15 histologic sections for each animal, whereas for the small, partially muscularized vessels (PMV) adjacent to the alveolar ducts, we collected all vessels present on 10 sections for each animal. Tissue samples were collected onto LCM caps. Each sample collection cap was then placed on an Eppendorf (Mississauga, ON, Canada) 500 µl tube and stored at –80°C until the RNA extraction and isolation procedure.

We used the PicoPure RNA Isolation kit (Arcturus) for RNA extraction and isolation. For RNA extraction, the frozen LCM samples were thawed and brought to room temperature. A 10 µl aliquot of extraction buffer from the PicoPure kit was pipetted onto the cap and the samples were incubated for 30 min at 42°C. After each sample was centrifuged at 800 x g for 2 min, all samples from an individual animal were combined in to one for each of the respective sites, and cell extract was collected into 1.5 ml microcentrifuge tubes.

The RNA purification column in the PicoPure kit was preconditioned by adding 250 µl conditioning buffer followed by incubation for 5 min at room temperature and centrifugation at 16,000 x g for 1 min, after which 10 µl of 70% ethanol was added to the cell extract tube and mixed well. The cell extract and ethanol mixture was then added to the preconditioned purification column and centrifuged at 16,000 x g for 1 min. Two sequential wash of buffers (1 and 2) of 100 µl each were added into the purification column and centrifuged at 8,000 x g for 1 min. A further wash step consisted of 100 µl wash buffer 2, followed by centrifugation at 16,000 x g for 2 min. The column was then transferred to a 0.5 ml microcentrifuge tube and 11 µl of elution buffer was added, followed by a 5 min incubation and final centrifugation at 10,000 x g for 1 min.

Reverse Transcription Reaction
First-strand cDNA was synthesized using superscript II RNase H– reverse transcriptase (Invitrogen, Burlington, ON, Canada). Ten-microliter RNA aliquots were added to a reaction mixture of 1X first strand buffer, 3 µg random primers (Invitrogen), 0.5 mM each of dATP, dTTP, dGTP and dCTP, 0.1M DTT plus water to a 20 µl total volume. A total of 200 U superscript RT was added and the reaction incubated at 42°C for 1 h.

Real-Time Reverse-Transcriptase–Polymerase Chain Reaction
Polymerase chain reactions (PCRs) were performed in a final volume of 20 µl in LightCycler (Roche, Mannheim, Germany) glass capillaries. The reaction mixture consisted of 2 µl LightCycler-FastStart DNA Master SYBR Green I (Roche), 2.4 µl 25 mM MgCl2 stock solution, 11.6 µl sterile PCR-grade H2O, 2 µl of the cDNA template for each gene of interest, and 1 µl of 10 µM of each primer. The cycling program consisted of initial denaturation for 300 ~ 600 s at 95°C followed by 55 cycles of 95°C for 5 s, 55 ~ 60°C for 10 s, and 72°C for 15 s, with 20°C/s slope. The program for analytical melting was 95°C for 0 s, 55 ~ 60°C for 30 s, increased to 98°C for 10 s at a 0.1°C/s ramp rate. The program for cooling was 40°C for 30 s.

Each set of PCR reactions included water as a negative control and five dilutions of standard. Standards were created by cloning part of the transcript of interest into a cloning vector (Invitrogen). Each insert was generated by PCR from cDNA. Known amounts of DNA were then isolated and diluted to provide standards and a regression curve of crossing points versus concentration generated with the LightCycler. ß-actin was used as a housekeeping standard and was similarly cloned.

Primer Sequences:

ß-actin: 116bp

Forward: 5' TTG TTA CCA ACT GGG ACG ACA TG 3'

Reverse: 5' GGG TCA TCT TCT CAC GGT TGG 3'

VEGF: 226bp

Forward: 5' ATG GCA GAA GGA GAG CAG AAG CC 3'

Reverse: 5' TGC ATG GTG ATG TTG AAC TCC TC 3'

ET-1: 251bp

Forward: 5' GTG GTC TCT GGA GCG GAG CTC AGC 3'

Reverse: 5' CTT GGC AGA AAT TCC AGC ACT TCT 3'

eNOS: 222bp

Forward: 5' CTT GAA GAG TGT GGG CCA GGA GCC 3'

Reverse: 5' CCA GTT CTT CAC TCG AGG GAA CTT 3'

Immunohistochemical Analysis of Mediator Production
eNOS and VEGF protein were immunohistochemically evaluated on paraffin sections using goat anti-eNOS and anti-VEGF antibody (R&D Systems, Minneapolis, MN) at a dilution of 10 µg/ml and 15 µg/ml, respectively. Rabbit anti-ET antibody was purchased from Biodesign (Saco, ME) and used at a dilution of 1:100. Staining reactions were visualized using standard immunohistochemical procedures with appropriate controls (8).

To evaluate the staining, we utilized a grading system based upon extent and intensity of staining reaction as compared with background. Each vessel of the appropriate size in the histologic section was examined and assigned a grade. Grade 0 was defined as no staining, whereas Grade 3 was defined as intense and diffuse vessel staining. A final grade was obtained by summation of all grades and expressed as a percentage of the maximum possible grade for that number of vessels.

Immunohistochemistry and Morphometric Analysis of Muscularization of PMV
Muscularization of the small vessels adjacent to the alveolar ducts was determined by immunohistochemical staining with monoclonal mouse anti-human {alpha}–smooth muscle actin (catalog no. M0851; DAKO, Mississauga, ON, Canada: diluted 1:200) using standard immunohistochemical procedures with appropriate positive and negative controls (8). In each animal, we examined 25 vessels adjacent to alveolar ducts identified by random light microscopic fields using a 20x objective and 10x eyepieces. Each vessel was categorized into quartiles for the percentage of the vessel circumference surrounded by stained cells, and the final numbers of vessels in each quartile was calculated. To develop a final case score, we then weighted each quartile by 1–4 respectively, and summed the values.

Cell proliferation was determined by immunochemical staining with polyclonal antibodies against proliferating cell nuclear antigen (DAKO, Mississauga, ON; 1:100 dilution). We examined 15 MV adjacent to the small noncartilagenous bronchioles, and 15 PMV adjacent to the alveolar ducts, chosen randomly, for each animal. For each vessel, using 1,000x magnification, we counted the numbers of stained nuclei in the endothelial cell and vessel wall compartments and expressed the positive nuclei as a percentage of the total number of nuclei. To develop a final case score for each of the MV and PMV sites, we summed the individual vessel data of percentages of positive cells.

Statistical Analysis
Using the crossing point curves values generated by the LightCycler and the standard curves, relative concentrations of each RNA of interest were determined and corrected for loading with the corresponding actin value. For both the gene expression and immunochemical analysis, we compared the data from control animals and smoke-exposed animals at each time period using an analysis of variance. Relationships between the morphometric data and the gene expression levels or immunohistochemistry scores, and between the gene expression levels and immunohistochemistry scores were determined by regression analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Figures 1–3 show the relative levels of gene expression for the three animals in each group at each time point, separated into PMV and MV. For ease of interpretation, the data are presented with the median control animal level for each time/vessel size/gene normalized to a value of 1.0 and other values adjusted accordingly; but all statistical analyses were performed from the raw data sets. At all time periods and treatments, the controls showed relatively little animal-to-animal variation in gene expression levels and, overall, gene expression levels for all three mediators were increased in the smoke-exposed animals, but for some times and sizes of vessel, considerable variation was seen with smoke exposure.



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  		Figure 1. ET gene expression levels at 2, 4, and 12 wk. Note the tight clustering of the control values and the marked spread in the smoke-exposed animals. Values are corrected for actin and normalized to a median control value of 1.0 for each size of vessel at each time point. Error bars indicate mean ± SD. *P <= 0.05 compared with control vessels of the same size.

 


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  		Figure 2. VEGF gene expression levels at 2, 4, and 12 wk. As with ET, there is considerable spread in the smoke-exposed animals. Values are corrected for actin and normalized to a median control value of 1.0 for each size of vessel at each time point. Error bars indicate mean ± SD. *P <= 0.05 compared with control vessels of the same size.

 


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  		Figure 3. eNOS gene expression levels at 2, 4, and 12 wk. eNOS expression levels show a different pattern from ET and VEGF, with minimal upregulation at 2 wk. Values are corrected for actin and normalized to a median control value of 1.0 for each size of vessel at each time point. Error bars indicate mean ± SD. *P <= 0.05 compared with control vessels of the same size.

 
Figure 1 shows data for ET. At the two-week time period, smoke exposure increased ET gene expression levels an average of ~ 5-fold over control levels in the PMV, and ~ 15-fold in the MV. At 4 and 12 wk, the mean levels in the smoke-exposed animals were slightly increased from the two-week time period (6- and 7-fold increases, respectively, compared with control) in the PMV. Expression levels in the MV demonstrated a greater spread of data at the 4-wk time period, with a mean 19-fold increase, compared with control, whereas at 12 wk, the data had little variation, with a mean 8-fold increase in the smoke-exposed animals. The increases in expression levels in animals exposed to smoke were significantly greater than controls at all times and in all sizes of vessel.

For VEGF, levels of gene expression tended to be increased, but with considerable variability at some time periods (Figure 2). The smoke-exposed animals showed a significant increase in mean VEGF gene expression levels at 2 wk in both the PMV and MV (22- and 11-fold respectively). Individual animals showed greater variability at 4 and 12 wk in the PMV, with 1 animal having a value within the range of control and the other 2 increases of over 40-fold at 4 wk, whereas at 12 wk all animals were at least 4-fold increased over the control levels. The MV also demonstrated a wide degree of variability at 4 wk, although all animals were at least 12-fold greater than control, whereas at 12 wk, variability decreased, with all animals at least 4-fold greater than control.

eNOS levels (Figure 3) at 2 wk were tightly clustered, with significant but minor increases for either MV or PMV. At 4 wk exposure, these levels increased markedly (up to 27 times control in the PMV and 20 times control in the MV), with, again, considerable animal-to-animal variation. At 12 wk, there was much less animal-to-animal variation, and expression levels were increased ~ 10-fold.

Figures 4–6 illustrate the immunohistochemical-based grading analysis for these same mediators, and Figure 7 shows representative images. In the MV, there was significantly increased staining for ET in the smoke-exposed animals at all three time periods (Figure 4). In the PMV, there was significantly increased staining for ET at the 2-wk and 12-wk points, whereas the staining grade did not achieve significance at the 4-wk time period. VEGF staining (Figure 5) was significantly increased at all three time periods for both the PMV and MV.



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  		Figure 4. Immunohistochemical staining analysis of ET as assessed by a grading analysis. The vessels from the smoke-exposed animals show an overall significant increase in the degree of staining compared with the control animals. *P < 0.05 or less compared with control vessels of the same size.

 


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  		Figure 5. Immunohistochemical staining analysis of VEGF as assessed by a grading analysis. The vessels from the smoke-exposed animals show a greater degree of staining compared with the control animals. *P <= 0.05 compared with control vessels of the same size.

 


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  		Figure 6. Immunohistochemical staining analysis of eNOS as assessed by a grading analysis. Increases in immunochemically detectable protein develop much more slowly over time than is the case for ET and VEGF. *P <= 0.05 compared with control vessels of the same size.

 


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  		Figure 7. Photographic panel illustrating immunohistochemical staining for ET (A: control; D: smoke-exposed), VEGF (B: control; E: smoke-exposed), eNOS (C: control; F: smoke-exposed), and smooth muscle actin (G: control; H: smoke-exposed).

 
Grading for eNOS (Figure 6) demonstrated prominent and widespread vascular expression in both control and smoke-exposed animals, and did not show significant differences between control and smoke-exposed animals for either vessel size at the 2- and 4-wk time periods, although there was clearly a trend toward increased staining at 4 wk. At twelve weeks, there was a significant increase for both PMV and MV sizes.

Table 1 demonstrates the correlations between the gene expression levels and immunohistochemical staining scores. Statistically significant, and often quite strong, correlations were seen for all three mediators in both the MV and PMV.


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  		TABLE 1. Correlation of gene expression levels and immunohistochemistry scores

 
Table 2 provides data on the degree of muscularization of the PMV adjacent to the alveolar ducts as determined by smooth muscle actin staining. There was a significant difference between the control and smoke-exposed animals in the degree of muscularization at each time period (P < 0.02 each time).


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  		TABLE 2. Muscularization indices in partially muscularized vessels

 
Table 3 documents the cell proliferation data in both vessel sizes at the three time periods. The data represent a summation of the proliferation in both the endothelium and vascular wall compartments. When these compartments were analyzed separately, a similar pattern of change was found. There was a significant difference in cell proliferation between the control and smoke-exposed animals at each time period in both the MV (P < 0.03, 0.01, and 0.04, respectively) and in the PMV (P < 0.01, 0.01, and 0.02, respectively) compared with controls. The increase in cell proliferation is more marked in PMV (8- to 10-fold) compared with the MV (4- to 5-fold) at every time period.


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  		TABLE 3. Cell proliferation indices in muscularized and partially muscularized vessels

 
Table 4 documents the relationships of the structural and proliferation data with gene expression and protein (immunochemistry) levels. Cell proliferation in the PMV correlated strongly with levels of gene expression levels and histochemistry scores for all of the mediators. Muscularization of these vessels correlated with gene expression levels of both eNOS and ET, and approached but did not achieve significance for the levels of VEGF. Cell proliferation in the MV correlated significantly with both the gene expression levels and immunohistochemistry scores for almost all of the proteins. Muscularization correlated significantly with immunohistochemistry scores for eNOS and VEGF, but not for ET.


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  		TABLE 4. Correlations between gene expression/immunochemistry levels and indicators of vascular structural changes

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have shown that chronic exposure to cigarette smoke leads to persisting upregulation of gene expression, along with protein production, for a set of mediators that control vasoconstriction, vasodilatation, and proliferation of vascular cells, and that this process occurs down to the very smallest branches of the pulmonary arterial tree. These data extend our previous study (6) and demonstrate that the reaction to smoke continues for a long time period and is not species-dependent.

One of the notable features of both the gene expression and immunochemical data is the variability of the smoke response from animal to animal. While this might appear to be an analytical error, in fact the control values are tightly clustered, and repeated analysis of the gene expression levels showed high reproducibility in any given animal. These observations indicate that the variability in the response to cigarette smoke is real, a phenomenon that probably reflects the fact that Hartley guinea pigs are an outbred strain. By the same token, only a portion of the Hartley-strain guinea pig population develops pulmonary hypertension after chronic cigarette smoke exposure. Although this problem complicates interpretation of the data, it is probably a close analogy of the situation in humans.

Our data indicate that smoke exposure leads to upregulation of ET expression in both the MV and PMV of guinea pigs. ET is believed to be an important mediator of pulmonary hypertension, both in its role as a vasoconstrictor and its role as a mitogen. Vascular remodeling induced by hypoxia, emboli, or monocrotaline can be decreased by administration of ET receptor inhibitors (reviewed in Ref. 9). ET acts to increase DNA synthesis in the smooth muscle cells of the pulmonary arterial tree, and ET activity appears to be altered in patients with pulmonary hypertension because such individuals have an increased concentration of ET receptors in the lung parenchyma (10).

We have previously shown that administration of the ET antagonist BQ610 will reduce the vascular cell proliferation induced by an acute exposure to cigarette smoke (11), and we have also demonstrated a transient increase in ET mRNA levels in the main pulmonary artery and larger intrapulmonary arteries after acute smoke exposure in the rat (4). Although the role of ET in the genesis of cigarette smoke–induced pulmonary hypertension is not clear, an increased amount of circulating ET in cigarette smokers (12) and in patients with COPD (13) has been reported. In addition, cigarette smoke has been found to induce ET-1 production in endothelial cell cultures (14). Our data serve to confirm the close relationship of smoke exposure and upregulated ET production in the pulmonary vessels.

eNOS is a generator of nitric oxide, a vasodilator. Few data are available on the effects of cigarette smoke on eNOS production. In endothelial cell cultures, cigarette smoke extract has been shown to diminish eNOS activity in addition to decreasing both mRNA and protein levels (15). Impairment of eNOS associated vasodilation appears to be an important factor in pulmonary hypertension induced by long-term smoke exposure; in human smokers who have developed COPD, the pulmonary arteries show decreased immunohistochemical expression and decreased bulk lung protein content of eNOS (16) accompanied by a decreased response to nitric oxide (17), with impaired endothelium dependent relaxation (18, 19). In our previous microdissection study in rats, eNOS gene transcription in the main and larger pulmonary arteries was not significantly elevated 24 h after smoke exposure (4), but the present study demonstrates a fairly slowly developing increase in eNOS gene expression and an even slower (and relatively small) increase in protein production in animals exposed for longer periods of time.

However, the relationship of eNOS gene expression to the possible development of pulmonary hypertension is not simple, because what may be important is the relative increase in the generation of vasodilators compared with vasoconstrictors (20). The regulation and physiologic reaction of smooth muscle cells requires an appropriate balance between ET and nitric oxide (21, 22). ET stimulates production and release of nitric oxide, while nitric oxide dissociates ET from its receptors (23), and interferes with the calcium mobilization pathway and inhibiting end-responses to ET (24). A strong relationship has been found between eNOS upregulation and the onset of vascular remodeling in experimental animal models of hypoxic hypertension (25), and vascular remodeling distal to a ligature is associated with an increase in both ET receptor and eNOS expression (26). In this regard, it is noteworthy that some animals in our study showed quite marked increases in ET and VEGF gene expression levels compared with eNOS gene expression levels. The delay in eNOS upregulation relative to ET and VEGF upregulation may also be important, as it could potentially lead to a period of unopposed vasoconstriction and vascular cell proliferation, as suggested by the observation of early muscularization of the PMV.

VEGF exerts a number of functions, including maintenance of normal endothelial cell structure. Through a complex relationship with metalloproteinases, VEGF can induce the proliferation of vascular smooth muscle cells and enable them to migrate into the muscle media (27). Because of its role as a mediator of vascular remodeling, VEGF potentially is an important factor in pulmonary hypertension (28), and increased VEGF vascular expression has been identified in patients with pulmonary hypertension secondary to a variety of etiologies (29). An increased immunohistochemical expression of VEGF has been demonstrated in the medial smooth muscle of arteries in the lungs of human smokers with and without COPD, and increased expression levels correlate with vessel wall thickness (5). Increased levels of VEGF have also been found in the plasma after smoking in some, but not all, cigarette smokers (30); interestingly, VEGF expression is upregulated by cigarette smoke in endothelial cell cultures (31).

Again the relationship of VEGF to other mediators of vascular function is complex. VEGF expression is affected in a paracrine fashion by other protein mediators in that nitric oxide decreases expression of both VEGF and VEGFR, similar to its effect on ET (32). Conversely, VEGF appears to augment nitric oxide release from the endothelium (33), and VEGF and VEGF-receptor activation produce an angiogenic response via a nitric oxide–mediated mechanism (34). In the present study, there is, as is true of ET, a variability in gene expression increases in response to cigarette smoke; but some animals show very marked upregulation, and there is a consistent increase in VEGF protein measured by immunochemistry.

Our data also confirm our previous findings that cigarette smoke causes cell proliferation, more marked in the PMV compared with the MV, along with rapid muscularization of the PMV (35). Because the smooth muscle actin antibody will stain both smooth muscle cells and myofibroblasts, we were unable to determine which cells were proliferating in the muscle layer. Interestingly, we found correlations between cell proliferation in the PMV and both gene expression levels and protein immunohistochemistry scoring of all three proteins. Similar correlations were found in the MV. This probably reflects the complex interaction among the three proteins as described above, because both ET and VEGF are known proliferative agents, but this is not true of eNOS. It is also possible that the crucial effects are related to the ratio of proliferative/constrictive compared with vasodilator mediators, and this is quite varied. In addition, it is as yet unclear whether morphologically visible anatomic changes in the vasculature correlate in a simple way with the presence of pulmonary hypertension, or, as our previous studies have suggested, there is also a dynamic component related to active vasoconstriction, which may not be reflected by morphometric analysis (35).

These questions need to be addressed in longer-term studies that can examine the relationship between gene expression levels, protein levels, morphologic changes, and the presence or absence of pulmonary hypertension. Nonetheless, the present study shows clearly that smoke directly and markedly affects the expression of factors that control vascular tone and cellularity in the pulmonary arterial tree, and that this effect persists over long periods.


   Acknowledgments
 
Supported by grant 62693 from the Canadian Institutes for Health Research.


   Footnotes
 
Conflict of Interest Statement: J.L.W. is supported on another project by AstraZeneca, but the work performed in the present study does not have any relationship to the supported project; H.T. has no declared conflicts of interest; and A.C. has no declared conflicts of interest.

Received in original form February 10, 2004

Received in final form May 30, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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