Introduction

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Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and permeability factor whose expression is highly regulated by hypoxia (1). Its target cell specificity is conferred by binding to at least two highly homologous receptors, Flk-1 and Flt-1, expressed by endothelial cells. Both VEGF receptors are transmembrane tyrosine kinases and bind VEGF with high affinity, but whereas Flt-1 is more closely associated with cell differentiation, Flk-1 is thought to have a more important role in VEGF-mediated endothelial cell proliferation (4).

The VEGF receptors are expressed at high levels in a variety of pathologic conditions that are characterized by hypoxia (5). The Flk-1 receptor was shown to be induced in vascular endothelial cells by conditioned media obtained from hypoxic smooth muscle cells (8). The role of VEGF in hypoxia-induced angiogenesis has been extensively studied in a variety of disease entities such as diabetic retinopathy (9), wound healing (10), and solid tumor vascularization (6, 7, 11). However, even though VEGF is abundantly expressed in the lung, its physiologic role in pulmonary vascular homeostasis and its aberrations are only beginning to be characterized. Specifically, the role of VEGF in the development of hypoxia-induced pulmonary hypertension and the accompanying vascular remodeling is incompletely understood. Although the most striking vascular morphologic feature in pulmonary hypertension is the increased medial thickness of pulmonary arterioles, ultrastructural studies of remodeled pulmonary vessels revealed intimal hyperplasia and subendothelial edema, which suggest increased endothelial cell proliferation and possibly increased permeability (12). Tuder and co-workers (13) reported increased [³H]thymidine incorporation in endothelial cells of muscularized arteries of hypoxic animals, which suggests increased endothelial cell proliferation in response to hypoxia in this model. Although intimal hyperplasia due to increased endothelial cell proliferation can physically impose significant resistance to pulmonary blood flow, leakage of plasma proteins in the subendothelial space is more likely to trigger a cascade of cellular events that may contribute to structural remodeling (14).

We conducted this in vivo study to (1) test the hypothesis that hypoxia regulates VEGF and its receptor Flk-1 in the lungs of animals with hypoxia-induced pulmonary hypertension; (2) define the time course of this hypoxic regulation; (3) identify the specific cell sources of VEGF mRNA within the lung and localize VEGF protein in the setting of hypoxia; and (4) correlate exposure to hypoxia with circulating VEGF levels and in vivo lung permeability.

	Materials and Methods

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Animals and Hypoxic Exposure

Adult virus-free male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 350-375 g were exposed for the specified time periods (3 h, 9 h, 15 h, 48 h, 1 wk, 2 wk, 3 wk) to normobaric hypoxia (10% oxygen environment) in a ventilated Plexiglas box (dimensions, 23 × 16 × 11 in.one animal per box) while age- and weight-matched control rats were maintained in 21% oxygen. Four animals were studied in each group. To establish the hypoxic conditions the chamber was flushed with a gas mixture of room air and nitrogen from a liquid nitrogen reservoir and an oxygen analyzer (Fyrite gas analyzer; Bacharach, Pittsburgh, PA) was used to monitor the chamber environment. CO₂ was removed with soda lime, excess humidity prevented by Drierite granules, and boric acid was used to keep ammonia levels within the chamber to a minimum. The chamber was opened every 2-3 d for 10-15 min to clean the cages and replenish food and water. The normoxic control rats were not kept in Plexiglas boxes but were subjected to the same light-dark cycle in the same virus-free room. The animals had free access to food and water. At the end of each time point the animals were killed with intraperitoneal pentobarbital (75 mg/kg) and their lungs were harvested for Northern analysis, in situ hybridization, and immunohistochemistry. The left main stem bronchus was ligated and the left lung was excised, flash frozen in liquid nitrogen, and stored at 80°C until RNA extraction. The pulmonary artery was infused with normal saline to clear the blood from the pulmonary vessels. The pulmonary veins were subsequently ligated and the pulmonary artery and trachea were perfused with 4% paraformaldehyde at constant pressure (100 cm H₂O for the pulmonary artery and 25 cm H₂O for the trachea) to fully distend the pulmonary blood vessels and airway, respectively. Peripheral lung specimens were then excised, fixed in 4% paraformaldehyde, and paraffin embedded for in situ hybridization and immunohistochemistry. Lung sections were also stained with hematoxylin and eosin (H&E) for morphologic studies to assess the degree of muscularization of distal arterioles. Blood specimens for serum VEGF measurements were collected directly from the right and left ventricles before lung fixation.

Northern Analysis

Total lung RNA was isolated by guanidinium isothiocyanate extraction as previously reported (15), fractionated by formaldehyde gel electrophoresis, and transferred onto Duralose UV membranes (Stratagene, La Jolla, CA). Fifteen micrograms of total RNA were loaded in each lane. The filters were hybridized with VEGF cDNA probes specific for rat VEGF gene (16) and the rat Flk-1 gene (17). The mouse -actin gene (18) was used for normalization of RNA loading. The cDNA fragments were labeled with [-³²P]dCTP, using a standard random-primed reaction, to a specific activity of 1-2 × 10⁹ cpm/µg. The membranes were hybridized for 2 h at 68°C in Quik Hyb solution (Stratagene) with 2 × 10⁶ cpm/ml of probe, washed twice in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at room temperture for 15 min, followed by 0.1× SSC-0.1% sodium dodecyl sulfate (SDS) at 60°C for 30 min, and were then exposed to film (X-Omat AR; Eastman Kodak, Rochester, NY) with intensifying screens at 80°C. For quantitation, the autoradiographs were scanned with a laser densitometer (Ultrascan XL; LKB Instruments, Bromma, Sweden) running the Gel Scan SL software package (Pharmacia LKB Biotechnology, Piscataway, NJ).

In Situ Hybridization

Six-micrometer lung sections were rehydrated, rendered permeable with proteinase K and acetylated in 0.1 M triethanolamine and acetic anhydride, dehydrated in ethanol, and air dried. They were subsequently hybridized at 52°C overnight with an ³⁵S-labeled single-stranded RNA probe (1 × 10⁶ cpm/section) synthesized in vitro with T7 RNA polymerase using a 300-bp rat VEGF fragment cloned in pGEM3Zf(+) vector (Promega, Madison, WI) (16). Unbound cRNA probe was digested by incubation in RNase A and the slides were subsequently washed, coated with photographic emulsion, exposed for 2 wk at 4°C, and then developed and counterstained with hematoxylin. Lung sections hybridized with a sense probe were used as negative controls.

Serum VEGF Measurements

Blood specimens collected from the right and left ventricles were centrifuged at 2,500 × g for 5 min and serum collected and stored at 80°C until assayed. The samples were processed in a single batch using a commercially available sandwich VEGF ELISA detection kit (R&D Systems, Minneapolis, MN) according to manufacturer instructions. The sensitivity of the assay was 9 pg/ml and the intraassay variability was < 10%. We collected blood specimens from seven normoxic animals and five hypoxic animals (3-wk exposure) for serum VEGF determination.

Immunohistochemistry

Rat lung sections were subjected to deparaffinization, hydration, and incubation with blocking serum (3% horse serum) and biotin-avidin blocking solutions (Vector Laboratories, Burlingame, CA) before incubation with primary antibody or preimmune serum. We used a commercially available goat anti-mouse VEGF antibody (R&D Systems) at a concentration of 0.1 µg/ml. As negative controls, we used preimmune serum from goat or purified goat IgG at the same concentration as the primary antibody. After overnight incubation at 4°C, the slides were washed in phosphate-buffered saline (PBS) and incubated for 30 min with a biotinylated horse anti-goat antibody diluted 1:300 in PBS (Vector Laboratories). We then used the avidin-biotin- glucose oxidase detection method in combination with the glucose oxidase substrate kit (ABC-GO kit; Vector Laboratories) to detect VEGF localization. Sections were subsequently counterstained with hematoxylin, dehydrated, and mounted.

In Vivo Lung Permeability

Permeability assays were performed using Evans Blue (EB) extravasation as an indicator of albumin leak from pulmonary vessels as previously reported (19, 20). The animals were placed in the same hypoxic chamber for 3 wk (n = 11). At the end of the hypoxic exposure and after metofane anesthesia and intraperitoneal pentobarbital injection (25 mg/kg), a midline laparotomy was performed and 20 mg/kg of EB dye in normal saline was injected in the inferior vena cava. After 5 min the animals were sacrificed by exsanguination and the heart and lungs were removed en bloc. A 22-gauge catheter was placed in the pulmonary artery and 10 ml of normal saline was infused at a constant pressure of 90 cm H₂O to remove the dye from the intravascular space. The lungs were then removed, weighed, and incubated in formamide (4 ml/g of wet tissue) for 16-18 h to extract the dye. The optical density at 620 nm (OD₆₂₀) was subsequently determined by spectrophotometry to quantitate EB extravasation as previously reported (21). Serial dilutions of EB in formamide were made and a standard curve of EB concentration versus OD₆₂₀ was generated. Optical density at 620 nm was found to be directly proportional to EB concentration as previously reported (22). The results were then plotted on the standard curve and EB concentration was calculated. The same procedure was performed with normoxic control rats (n = 9).

Statistical Methods

VEGF serum concentrations were compared between the normoxic and hypoxic animals, using the Wilcoxon rank sum test with a level of significance of P < 0.05. The same statistical test was used to compare OD₆₂₀ in the lung effluent in the two groups of animals.

	Results

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Hypoxia Increases VEGF and Flk-1 mRNA Levels in Hypoxic Rat Lungs

Interaction of VEGF with its receptor Flk-1 is the basis of the mitogenic and permeability action of VEGF. To assess the effect of hypoxia on overall VEGF and Flk-1 mRNA levels in the lung, we performed Northern analysis of total lung RNA. Animals were exposed to hypoxia (10% O₂) or to normoxia (21% O₂) for various periods of time and the lungs were removed for RNA isolation. VEGF and Flk-1 mRNA levels remained unchanged throughout a 3 wk period under normoxia (data not shown). However, increased levels of VEGF and Flk-1 mRNA were noted in response to hypoxia. A time course of VEGF induction is shown in Figure 1: VEGF mRNA levels did not change significantly after acute hypoxia (3-48 h) but a steady increase was observed after 1-3 wk. The response of Flk-1 mRNA to hypoxia appeared to be biphasic with an initial increase as early as 9 h after hypoxic exposure followed by a transient decline at 48 h and a subsequent rise at 1-3 wk of continued hypoxic exposure. Thus, there appears to be an induction of the VEGF receptor Flk-1 mRNA in the lung that precedes the induction of VEGF mRNA. Levels of -actin mRNA remained unchanged throughout the entire exposure period and were used to correct for RNA loading. As shown in Figure 1, equal amounts of RNA were loaded in each lane (ethidium bromide staining of RNA [28S] gel).

View larger version (35K): [in this window] [in a new window]   Figure 1.   (A) Time course of hypoxic induction of VEGF and Flk-1 mRNA in rat lungs. Northern blot analysis of VEGF and Flk-1 mRNA in the lungs of rats exposed to 10% oxygen for the following time periods: lane 1, 0 h (normoxic control); lane 2, 3 h; lane 3, 9 h; lane 4, 15 h; lane 5, 48 h; lane 6, 1 wk; lane 7, 2 wk; lane 8, 3 wk. Each lane represents total lung RNA from a different animal. Four animals were studied in each group. Relative mRNA levels as quantified by densitometry and normalized to -actin are shown for the corresponding Northern blots. Levels of -actin mRNA remained unchanged. Ethidium bromide staining of RNA (28S) gel shows even loading of RNA in all lanes. (B) Mean VEGF and Flk-1 relative mRNA levels from the three different experiments are shown. * P = 0.01; ** P = 0.0001.

VEGF Is Mostly Expressed in Alveolar Epithelial Cells in the Setting of Hypoxia

Cellular localization of VEGF transcript in the lung was done by in situ hybridization. Using the VEGF antisense probe, we detected abundant signal throughout the lung parenchyma under normoxia (Figures 2A and 2B). A higher density of silver grains was, however, noted in the lungs of hypoxic animals after a 3-wk exposure period compared with the normoxic controls. As shown in Figures 2C and 2D, in a hypoxic animal, the VEGF mRNA signal was localized mostly in alveolar epithelial cells as well as intraalveolar cells that probably represent macrophages. We used peripheral lung tissue sections, and therefore no large airways were included in the studied areas. To assess nonspecific hybridization, we used a sense probe that resulted in a low density of silver grains as shown in Figures 2E and 2F (negative control). H&E (Figure 3) staining clearly depicts the increase in smooth muscle cell layers surrounding distal pulmonary arterioles of the hypoxic animals compared with those breathing room air (Figures 3A and 3B, respectively). All animals exposed to hypoxia for 1-3 wk manifested similar extensive muscularization of distal arterioles along with excess matrix deposition.

View larger version (62K): [in this window] [in a new window]   View larger version (63K): [in this window] [in a new window]   Figure 2.   VEGF in situ hybridization experiments in rat lungs. Dark-field (A, C, and E) and corresponding bright-field (B, D, and F) photomicrographs are shown. Sections are counterstained with hematoxylin. VEGF mRNA signal is present in alveolar epithelial cells of the normoxic rat lung (A and B). Increased density of silver grains representing increased levels of VEGF mRNA over alveolar epithelial cells (arrowheads) is evident after 3 wk of hypoxic exposure (C and D). Low levels of nonspecific hybridization using a sense probe are shown in E and F. Original magnification: ×400. Three animals in each condition were examined, and representative data are shown here.

View larger version (67K): [in this window] [in a new window]   Figure 3.   Morphology of pulmonary arterioles by H&E staining of peripheral lung sections in hypoxic and normoxic animals. Dramatic medial hyperplasia is shown in a pulmonary arteriole at the alveolar level in the hypoxic animal (A). Thin medial layer is noted in a pulmonary arteriole of the normoxic animal (B). Three animals in each condition were examined and representative data are presented here. Original magnification: ×400.

VEGF Peptide Is Abundantly Present in Hypoxic Rat Lungs

VEGF-like immunoreactivity was detected throughout the lung parenchyma after 3 wk of hypoxic exposure (Figures 4A and 4B). As shown in Figure 4, VEGF immunoreactivity is present in alveolar epithelial cells (Figures 4A and 4C), vascular structures (Figures 4A and 4B), as well as in large airways (Figure 4B) of hypoxic animals. We localized VEGF staining in endothelial cells, smooth muscle cells, type II cells, and bronchiolar epithelial cells. In contrast, a low degree of staining was seen in the lungs of normoxic animals (Figures 4C and 4D). Nonspecific staining was minimal in the sections incubated with preimmune serum (Figures 4E and 4F).

View larger version (113K): [in this window] [in a new window]   Figure 4.   Immunohistochemical staining with VEGF antibody. Abundant staining is evident throughout the lung parenchyma in the hypoxic animals (A and B). Staining is minimal in the normoxic controls (C and D) as well as in sections incubated with preimmune serum (E and F). Structures stained for VEGF in hypoxic rat lung include the following: alveolar epithelial cells (arrowheads, A and C), vascular structures (black arrows, A and B), as well as large airways (open arrows, B). We localized VEGF staining in endothelial, smooth muscle, type II, and bronchiolar epithelial cells. The photomicrographs are representative of three animals examined under each condition. Original magnification: ×400.

VEGF Serum Levels Are Higher in Hypoxic Animals Compared with Normoxic Controls

To determine if the increased production of VEGF in the lung parenchyma could be reflected in the systemic circulation, we measured VEGF concentrations in the serum of hypoxic and control animals. After a 3-wk study period, serum was collected and VEGF assayed using an ELISA. As shown in Figure 5, the median serum VEGF concentration was significantly higher (34 pg/ml; n = 5) in hypoxic animals compared with normoxic controls (median, 19 pg/ ml; n = 7; P = 0.03). The higher circulating VEGF levels in hypoxic animals paralleled the increased VEGF mRNA transcripts in the lung, which were maximal after 3 wk of hypoxic exposure. They also correlated with the abundant presence of VEGF peptide in the lung after 3 wk of hypoxic exposure.

View larger version (15K): [in this window] [in a new window]   Figure 5.   Circulating VEGF concentrations in hypoxic versus normoxic rats after a 3-wk study period. Median VEGF concentration in hypoxic animals was 34 pg/ml, and 19 pg/ml in normoxic controls. The difference between median values is statistically significant (P = 0.03). Individual values are shown for each condition (n = 5 for hypoxia, n = 7 for normoxia).

Vascular Permeability in Hypoxic Rat Lungs Compared with Controls

Because VEGF is also known as a permeability factor, we wanted to examine whether the increased peptide levels detected in the lung and serum resulted in increased vascular permeability. Animals previously exposed to hypoxia or to ambient air were anesthetized and injected with EB dye in the inferior vena cava. Optical density at 620 nm was found to be directly proportional to EB concentration as previously reported (22). We therefore compared OD₆₂₀ between normoxic and hypoxic animals as a measure of vascular permeability. As shown in Figure 6, the values tended to be higher in the hypoxic animals (median OD₆₂₀, 0.3) compared with the normoxic controls (median OD₆₂₀, 0.16), but owing to the high variability between animals these differences were not significant for this sample size (n = 9 for normoxia; n = 11 for hypoxia; P = 0.19). To assess the role of VEGF in lung permeability we treated normoxic animals with recombinant VEGF intravenously (1.5 µg/kg) and measured vascular permeability as described previously. Median OD₆₂₀ following treatment with VEGF was not significantly different from untreated animals (median OD₆₂₀, 0.23; n = 8; P = 0.14).

View larger version (13K): [in this window] [in a new window]   Figure 6.   In vivo permeability assays. Optical density at 620 nm (OD620) is proportional to EB extravasation in the rat lungs. Bars indicate median levels, and individual values are shown for each condition (n = 9 for normoxia, n = 11 for hypoxia). Median OD620 is 0.3 for hypoxic animals and 0.16 for normoxic controls (P = 0.19).

	Discussion

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Regulation of pulmonary vascular tone and homeostasis is complex and multifactorial. It involves a balance of vasoconstrictors, relaxing agents, and growth factors released from different cell types that interact through feedback loop mechanisms in autocrine and paracrine ways. Hypoxia is a key regulator of such interactions in the pulmonary vasculature shifting the balance toward aberrant blood vessel structure and function leading to pulmonary hypertension. In the setting of hypoxia there is impairment of endothelium-dependent relaxation (23), intimal and medial hyperplasia (24, 25), subendothelial edema (12), and a switch of smooth muscle cell phenotype to a more synthetic type resulting in excessive extracellular matrix deposition (26).

The vascular endothelium through elaboration of vasoactive mediators and growth factors plays a crucial role in mediating the effects of hypoxia on the smooth muscle cells and matrix in the vessel wall. For example, hypoxia has been reported to increase the production of vasoconstrictors and smooth muscle cell mitogens such as endothelin 1 (ET-1) and platelet-derived growth factor B (PDGF-B) from endothelial cells (18, 27), whereas it decreases the production of the endothelial-derived relaxing factor nitric oxide (NO) both in vitro and in vivo (23, 28, 29). However, the vascular endothelium is not only a regulator of hypoxic responses in the vasculature but also a target of paracrine effects from the adjacent smooth muscle cell layer. We have reported that vascular smooth muscle cells respond to hypoxia by increasing the expression of heme oxygenase 1 (HO-1), which leads to increased carbon monoxide (CO) production (30). In turn, smooth muscle cell-derived CO was shown to modulate the hypoxic induction of endothelial cell gene expression (ET-1, PDGF-B) in a paracrine way (31).

In this article we have shown that VEGF/vascular permeability factor, an endothelial cell mitogen and permeability factor, is upregulated by hypoxia in the lung and that the main cellular sources of VEGF are the alveolar epithelial cells. We propose that alveolar epithelial cells participate in the hypoxic responses of the lung and affect endothelial cell behavior in a paracrine manner. In addition, we found increased mRNA levels of the VEGF receptor Flk-1 in the lungs of hypoxic animals compared with normoxic controls. The hypoxic induction of Flk-1 mRNA preceded the induction of VEGF mRNA with an early significant response at 9 h of exposure, followed by a transient decrease and subsequent rise with maximal message at 3 wk of hypoxic exposure. This is the first in vivo study characterizing the time course of VEGF and Flk-1 mRNA expression in the lung in response to acute hypoxia. A previous report by Tuder and colleagues (13), using an ex vivo preparation of isolated perfused rat lungs showed increased VEGF and Flk-1 mRNA levels after exposure to 0% oxygen for 2 h. In contrast, by using an in vivo system and exposure to 10% oxygen, we did not detect any increase in VEGF or Flk-1 mRNA levels before 9 h of hypoxic exposure. The difference between the two studies on the observed effects of acute hypoxia may be related to the different models used. It is possible that the ex vivo preparation may not adequately account for circulating factors that may regulate VEGF and Flk-1 mRNA levels in vivo, or that the earlier induction observed by Tuder and coworkers was a result of a more potent stimulus used (anoxia versus hypoxia). However, in agreement with the previous study, exposure of rats to prolonged hypoxia in vivo leads to a sustained induction of VEGF and Flk-1 mRNA expression in the lungs.

We observed a significant induction of VEGF mRNA only after 1 wk of hypoxic exposure, whereas Flk-1 mRNA was regulated in a biphasic manner starting at 9 h of hypoxic exposure. The mechanisms underlying the different kinetics of the hypoxic response are not clear but it is possible that the induction of VEGF mRNA in the lung is modulated by other endothelial or smooth muscle cell- derived factors induced in the setting of hypoxia. Specifically, we have reported that hypoxia increases the production of CO by smooth muscle cells, which in a coculture system of endothelial and smooth muscle cells serves to inhibit the induction of both ET-1 and PDGF-B by endothelial cells (31). It is possible that smooth muscle cell-derived CO inhibits the hypoxic induction of VEGF in the lung as it does in vascular cells in vitro (our unpublished observations). Also, the endogenous endothelial-derived NO can modulate hypoxic gene expression (32), but because it is suppressed in the setting of hypoxia (28), we think it is less likely to play a critical role in VEGF gene regulation in this setting. However, exogenous NO, as shown by Tuder and colleagues, reduced the hypoxic induction of VEGF mRNA in the rat lung whereas further inhibition of NO synthesis under hypoxia led to VEGF mRNA superinduction (13).

In the current study we found significantly higher VEGF serum levels in the hypoxic animals compared with the normoxic controls. Although serum levels do not generally reflect the pulmonary concentrations, our immunohistochemistry results confirmed the abundant presence of VEGF peptide in the lung parenchyma in the setting of hypoxia. Furthermore, the lung is known to be the predominant source of VEGF mRNA in the rat (33). We therefore suggest that increased lung VEGF production may lead to elevated serum VEGF levels under hypoxic conditions. We collected the serum samples after 3 wk of hypoxic exposure, which coincided with the time of maximal VEGF mRNA signal in the lungs. Vasoactive factors produced in the lung can be detected in the systemic circulation. For instance, infants receiving inhaled NO responded with increased circulating levels of cGMP (34), and patients with pulmonary hypertension had elevated ET-1 in their arterial plasma, suggesting that the lungs were the source of ET-1 production (35).

The role of VEGF in the pathogenesis of hypoxia- induced pulmonary hypertension is poorly understood. We hypothesized that VEGF-induced endothelial cell proliferation and alterations in microvascular permeability could contribute to the pathogenesis of pulmonary hypertension. It has been reported that subendothelial accumulation of serum activates endogenous elastolytic activity which in turn "liberates" multiple growth factors such as basic fibroblast growth factor and transforming growth factor , which could clearly contribute to the pathophysiology of the disease process (14, 36).

To assess vascular permeability in the lung in the setting of in vivo hypoxia and increased VEGF production, we performed EB dye extravasation studies as previously reported (21). EB is inexpensive and easily quantitated spectrophotometrically and has been used as a marker of albumin clearance both in cultured endothelium and in isolated rat lungs (19). This method was used in reports of increased bronchial (21) and parenchymal pulmonary vascular permeability (39) in response to administration of platelet-activating factor. Potential limitations of this assay include the possibility of altered EB extravasation due to differences in albumin levels, flow, and vascular pressure. Although the hypoxic animals in our study tended to have increased lung permeability, which could be due to higher vascular pressure compared with normoxic controls, we found wide variability in EB extravasation among normoxic and hypoxic animals with no statistically significant difference. This variability in lung permeability did not correlate with variability in circulating levels of VEGF. To explore further the limitations of this assay, we treated animals with intravenous injections of recombinant VEGF and, to our surprise, found no statistically significant increase in lung permeability compared with untreated controls. We concluded that either VEGF does not increase lung permeability during hypoxia in this model or this method was not sufficiently sensitive to detect potentially physiologically important alterations in microvascular permeability in response to hypoxia. Dallal and Chang (22) reported that EB may not be a reliable marker of vascular permeability in perfused rat lungs owing to rapid binding of EB dye to lung tissue proteins. In that report, high variability among animals was also noted. More importantly, this method did not detect the increase in lung permeability induced by treatment with lipopolysaccharide (22). In addition, EB extravasation may not adequately distinguish between bronchial and pulmonary vascular permeability. We used a different measure of lung permeability (i.e., the wet-to-dry lung weight ratio) to assess for the presence of enhanced lung permeability in the presence of hypoxia. Again, although a tendency toward higher ratios was observed in the hypoxic animals, no statistically significant difference was detected between the two groups of animals (data not shown).

In conclusion, although the physiologic function of VEGF in the lung is unknown, its abundant expression throughout the pulmonary parenchyma suggests a possible role for VEGF in the maintenance of the unique balance of microvascular permeability and endothelial cell function. In the hypoxic setting, increased VEGF mRNA levels were detected in the alveolar epithelial cells and increased protein signal was noted throughout the lung parenchyma including vascular structures. Taken together, these results point to the lung epithelial cells as the source of VEGF, and to the lung endothelium as the cell type targeted by VEGF action. Given that VEGF may increase cGMP levels indirectly by stimulating NO synthesis (40, 41), increased VEGF levels by pulmonary epithelial cells may compensate for the suppressive effect of hypoxia on endothelial-derived NO. Alternatively, increased VEGF production by alveolar epithelial cells may contribute to the pathogenesis of hypoxia-induced pulmonary hypertension by modulating endothelial cell behavior. Early alterations in endothelial cell barrier function and proliferation may initiate a sequence of cellular events leading to pulmonary hypertension. Intervention at this stage of the disease may prevent the development of structural changes and thus the progression to chronic pulmonary hypertension.