Angiogenic growth factors in the pathophysiology of a murine model of acute lung injury

Dimitrios Karmpaliotis, Ioanna Kosmidou, Edward P. Ingenito, Kailin Hong, Atul Malhotra, Mary E. Sunday, and Kathleen J. Haley

Division of Pulmonary and Critical Care Medicine, Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02364


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Capillary leakage and alveolar edema are hallmarks of acute lung injury (ALI). Neutrophils and serum macromolecules enter alveoli, promoting inflammation. Vascular endothelial growth factor (VEGF) causes plasma leakage in extrapulmonary vessels. Angiopoietin (Ang)-1 and -4 stabilize vessels, attenuating capillary leakage. We hypothesized that VEGF and Ang-1 and -4 modulate vessel leakage in the lung, contributing to the pathogenesis of ALI. We examined a murine model of lipopolysaccharide (LPS)-induced ALI. C57BL/6 and 129/J mice were studied at baseline and 24, 48, and 96 h after single or multiple doses of aerosolized LPS. Both strains exhibited time- and dose-dependent increases in inflammation and a deterioration of lung mechanics. Bronchoalveolar lavage (BAL) protein levels increased significantly, suggesting capillary leakage. Increased BAL neutrophil and total protein content correlated with time-dependent increased tissue VEGF and decreased Ang-1 and -4 levels, with peak VEGF and minimum Ang-1 and -4 expression after 96 h of LPS challenge. These data suggest that changes in the balance between VEGF and Ang-1 and -4 after LPS exposure may modulate neutrophil influx, protein leakage, and alveolar flooding during early ALI.

pulmonary edema; vascular growth factors; cytokines; inflammation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ACUTE LUNG INJURY (ALI) can result from either a primary pulmonary process or a systemic insult (9, 12, 25). ALI is characterized by increased capillary permeability, interstitial and alveolar edema, influx of circulating inflammatory cells, and formation of hyaline membranes. In some patients, compensatory mechanisms reestablish the integrity of the epithelial-endothelial barrier and clear the edema without progression to a more chronic phase. In others, injury proceeds through subacute and chronic stages that evolve over weeks to months and are associated with considerable morbidity (15).

Studies during the past decade have suggested that the respiratory epithelium is principally responsible for maintaining an effective barrier that prevents leakage of fluid and solutes into the lung. This critical function has been ascribed to the junctional complex, which consists of subcellular structures, including tight and intermediate junctions and desmosomes. These structures are common to epithelial cells throughout the respiratory tract (17) and function to inhibit passage of water, electrolytes, and peptides into the airways and alveoli under physiological conditions (24).

Less attention has been focused on the role of the endothelial barrier for several reasons. Paracellular pores through the endothelial surface are significantly larger than epithelial pores and thus allow freer passage of hydrophilic solutes such as small sugars, electrolytes, and other charged particles under baseline conditions. Estimates of reflection coefficients for serum proteins across the pulmonary endothelium are also significantly less than those of the epithelium (0.5-0.7 vs. 1.0) (5). Thus it has been concluded that the epithelium is the more important of the two barriers with respect to limiting permeability and preventing capillary leakage.

Despite this, loss of integrity in the endothelial barrier is clearly a prerequisite for development of interstitial edema, is associated with a marked increase in permeability in experimental models, and can trigger ALI-like responses. Recent observations suggest the endothelial barrier may play an important role in the pathogenesis of ALI. Endothelial-specific structures, known as intercellular adherens junction complexes (AJCs), regulate endothelial cell growth patterns and angiogenesis during embryonic development as well as permeability of mature vessels, in response to inflammatory stimuli (7). These complexes, consisting of vascular endothelial cadherin (VE-cadherin) and components of the "armadillo" protein family, are linked to cytoplasmic microfilaments and join endothelial cells together in either a "tight" or "loose" configuration, rendering the vasculature nonpermeable or permeable, respectively.

The angiogenic growth factors, vascular endothelial growth factor (VEGF), angiopoietin (Ang)-1, Ang-2, and Ang-4 have recently been shown to modulate endothelial permeability of extrapulmonary vessels by altering the state of the AJC (4, 26). VEGF is a potent endothelial cell mitogen (16) that, through binding to its receptor, VEGF-R2/fetal liver kinase (flk), induces capillary leakage (19) and fluid accumulation in models of ischemic organ injury (27) or systemic inflammation (22). Recently, adenovirus-mediated expression of VEGF within the lung has been shown to result in profound pulmonary edema (11). Conversely, Ang-1, a ligand of the endothelium-specific Tie-2 receptor (20), promotes vessel maturation through its antiapoptotic action (18) and inhibits vessel leakage in response to VEGF or other proinflammatory agents by stabilizing the AJC (20, 22, 23). The same actions have been proposed for Ang-4, whereas Ang-2 promotes vascular leakage by antagonizing the actions of both Ang-1 and Ang-4 through competitive binding to the Tie-2 receptor.

Based on the observation that the expression of angiogenic growth factors modulates endothelial integrity in several extrapulmonary vascular beds, we hypothesized that there might be a potential association between vascular growth factors, specifically VEGF and the angiopoietins, and the pulmonary edema that occurs during ALI. We tested this hypothesis by examining the expression of VEGF and the angiopoietins in a murine model of intrapulmonary lipopolysaccharide (LPS)-induced ALI. Because responses to inhaled LPS vary among murine strains, we used two strains for these investigations, C57BL/6 and 129/J. Both strains have been demonstrated to develop pulmonary edema and neutrophilic-predominant infiltrates after aerosolized LPS (3), consistent with ALI. We have previously shown that the 129/J strain demonstrates significant lung dysfunction in response to inhaled LPS (10), consistent with a "high responder." The C57BL/6 strain has also been demonstrated to respond to LPS with neutrophilic lung infiltrates and increased pulmonary vascular permeability (3). We reasoned that, if vascular growth factors were important to the development of pulmonary edema in ALI, then that association should be demonstrable in multiple murine strains. Our data indicate that the C57BL/6 strain has a substantially decreased response to inhaled LPS compared with the 129/J strain but that the two strains have similar expression changes among the vascular growth factors after inhaled LPS. Observations summarized here support the notion that changes in the balance between the "proleakage" factor VEGF and "antileakage" factors Ang-1 and Ang-4 might contribute to the pathophysiology of ALI after intrapulmonary endotoxin exposure.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Experimental protocol. The experimental protocol was approved by our institution's animal studies committee. Twenty-four 129/J mice (Jackson Laboratories) weighing 18.8 ± 2.1 g (16-24 g) and 24 C57BL/6 mice weighing 22.3 ± 3.4 g (18-32 g) were divided into four groups: 1) control (n = 6); 2) animals exposed to endotoxin (Sigma Chemical) 24 h before study (24-h LPS, n = 6); 3) animals exposed twice, 24 and 48 h before study (48-h LPS, n = 6); and 4) animals exposed four times, 24, 48, 72, and 96 h before study (96-h LPS, n = 6). Additionally, we also studied the bronchoalveolar lavages (BALs) of six 129/J and six C57BL/6 mice 2 h after LPS exposure for cytokine expression. Endotoxin was administered as previously described (10).

Lung mechanics. Animals were anesthetized and ventilated as previously described (13). Measurements of lung function were performed using the optimal ventilator waveform method of Lutchen et al. (13).

BAL cell count. Cells were removed by centrifugation (400 g, 4°C, 10 min), and the supernatant was stored (-70°C). Recovery (2.82 ± 0.1/3 ml) was similar among groups. Red blood cells were lysed, cytospins were prepared and stained using Diff-Quik (Fisher Scientific, Fairlawn, NJ), and differential and total leukocyte counts were determined by manual counting (10).

Total protein. Determinations were performed using the bicinchoninic acid microassay method (Pierce Chemical, Rockford, IL) according to the manufacturer's instructions (10).

BAL cytokines. Tumor necrosis factor (TNF)-alpha , interleukin (IL)-1beta , IL-6, IL-10, and interferon (IFN)-gamma were determined in BAL supernatants using an ELISA kit (Endogen, Woburn, MA) according to the manufacturer's instructions (10).

Immunohistochemistry. Three lungs from each time point were embedded in paraffin, and 3-µm-thick sections were cut. Primary goat polyclonal antisera included anti-VEGF (1:400), anti-Ang-4 (1: 100), and anti-Ang-2 (1:400; Santa Cruz Biotechnology, Santa Cruz, CA). Primary antisera used for identifying cell types included the panleukocyte marker monoclonal rat anti-CD45R/B220 (1:25; clone 30-F11; PharMingen, San Diego, CA) and the mesenchymal cell marker monoclonal murine anti-vimentin (1:75; clone V9, Dako, Carpinteria, CA). Negative controls consisted of preabsorbing primary antibody with specific peptide before immunostaining for VEGF. VEGF, CD45, and Ang-2 slides underwent microwave antigen retrieval. Slides were blocked in 10% horse serum and incubated overnight with the primary antibody (4°C). They were then incubated in biotinylated donkey anti-goat secondary antibody (1:500) (for VEGF, Ang-2, Ang-4) or mouse anti-rat (1:500 for CD45; 2 h, 4°C; Jackson Immunoresearch). Endogenous peroxidases were blocked by slide incubation in 3% H2O2-methanol (10 min, 25°C).

Slides were incubated for 30 min in streptavidin-horseradish peroxidase (1:1,000), and VEGF samples were amplified using biotinylated tyramide (TSA Biotin System; NEN, Boston, MA). Immunopositivity was visualized using chromagen diaminobenzidine (0.025%) in PBS and 0.1% H2O2. All sections were counterstained with 2% methyl green (Sigma).

mRNA measurements. We examined 12 129/J animals, i.e., 3 at each time point (baseline, 24, 48, and 96 h after LPS exposure) and 8 C57BL/6 animals, i.e., 4 at baseline, 2 at 24 h, and 2 at 48 h after LPS exposure. Total lung RNA was extracted after the RNeasy protocol for animal tissues (Qiagen, Valencia, CA). cDNA templates were synthesized by reverse transcriptase (Clontech, Palo Alto, CA). PCR amplification was performed using the following primers: 1) Ang-1, 3'-gaggcgcattcgctgta-5' (sense) and 3'-tgcgtcaaaccaccagc-5'; 2) Ang-2, 3'-tccagaacacgacggga-5' (sense) and 3'-atctgggcccatctccga-5'; 3) VEGF, 3'-caggctgctgtaacgatgaa-5' (sense) and 3'-tgcgtcaaaccaccagc-5'; and 4) beta -actin, 5'-gtgggccgctctaggcaccaa-3' (sense) and 5'-ctctttgatgtcacgcacgatttc-3'. PCR amplification conditions were denaturation at 94°C (5 min) followed by 31 cycles at 1-min denaturation (94°C), 1-min annealing (55°C), and 1-min primer extension (72°C). Final extension was 7 min (72°C). PCR products were resolved in 1.5% agarose gel along with 100-bp DNA ladder.

Statistics. Comparisons at different time points were performed by two-way ANOVA. Statistical significance was defined as P < 0.05.


    RESULTS
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Pulmonary physiological response to LPS administration. Both murine strains demonstrated physiological responses typical of ALI, summarized in Fig. 1. Airway resistance (Raw), tissue resistance (expressed in terms of the dissipative modulus G), and dynamic elastance (expressed in terms of the elastic modulus H) at each time point are shown. Both strains demonstrated similar baseline physiology. Within 24 h of LPS exposure, G and H increased significantly in C57BL/6 mice (G: 6.1 ± 0.6 to 15.5 ± 4.7 cmH2O/ml, P = 0.002, and H: 44.5 ± 3.4 to 89.4 ± 18.0 cmH2O/ml, P = 0.001) and 129/J mice (G: 5.9 ± 0.5 to 13.1 ± 2.2 cmH2O/ml, P = 0.003, and H: 32.7 ± 3.3 to 73.2 ± 5.2 cmH2O/ml, P = 0.001). Raw did not change significantly after LPS exposure, although total lung resistance, the sum of airway and tissue components, did increase due to the change in the tissue component. In C57BL/6 mice, the physiological response peaked at 24 h and persisted without additional change throughout the 96-h exposure period. In 129/J mice, tissue resistance and lung elastance, that is, the change in pressure per the change in volume, continued to increase with serial LPS exposure such that peak responses were observed at 96 h.


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Fig. 1.   Histogram comparing lung physiology of 129/J mice and C57BL/6 mice after lipopolysaccharide (LPS) exposure. Airway resistance (Raw) is shown in A, tissue resistance (G) is shown in B, and dynamic elastance (H) is shown in C. Values were similar in the 2 strains at baseline. After LPS exposure, G (P = 0.002 for C57BL/6 and P = 0.003 for 129/J mice at 24 h) and H (P = 0.001 for C57BL/6 and P = 0.001 for 129/J mice at 24 h) increased significantly, whereas Raw did not change.

Alteration in BAL cell counts/differentials after nebulized LPS. BAL fluid white blood cell counts and differentials are summarized in Fig. 2. Before LPS exposure, BAL in both 129/J and C57BL/6 mice revealed a predominance of macrophages, with similar total counts and differentials. Within 24 h of LPS exposure, total counts in C57BL/6 [(0.6 ± 0.2 to 8.8 ± 2.9) × 105 cells/ml, P < 0.001] and 129/J [(0.3 ± 0.1 to 14.4 ± 4.3) × 105 cells/ml, P < 0.001] mice increased by a factor of 10-15 due to a marked influx of neutrophils. In C57BL/6 mice, neutrophil counts continued to increase with each LPS exposure up to 96 h, whereas among 129/J mice, neutrophil counts peaked at 48 h and began to decline toward baseline thereafter. Although the percentage of mononuclear leukocytes (macrophages and lymphocytes) decreased in both strains after LPS, absolute numbers increased such that at 96 h, the number of macrophages plus lymphocytes was four- to fivefold greater than baseline (C57BL/6: 0.59 ± 0.06 to 2.36 ± 0.45 × 105 cells/ml, P < 0.001 and 129/J: 0.28 ± 0.06 to 2.61 ± 0.43 × 105 cells/ml, P < 0.001).


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Fig. 2.   Total cell counts and differentials for 129/J (A) and C57BL/6 (B) mice. Within 24 h of LPS exposure, bronchoalveolar lavage (BAL) leukocytes increased >10-fold in both strains (P < 0.001), due primarily to the influx of neutrophils. In both strains, leukocyte numbers remained elevated throughout the 96-h testing period. Although neutrophils remained the predominant cell type at all time points after LPS exposure, mononuclear leukocytes also increased. PMN, polymorphonuclear leukocyte; lymph, lymphocyte; eos, eosinophil; mac, macrophage.

Evidence of capillary leakage after nebulized LPS. Inhaled LPS treatment caused a graded increase in BAL total protein content among both 129/J and C57BL/6 mice (Fig. 3). In both strains, peak protein levels were observed after 96 h of LPS exposure (C57BL/6: 111 ± 129 mg/ml at baseline to 1,236 ± 223 mg/ml at 96 h, P < 0.001; 129/J: 59 ± 36 mg/ml at baseline to 865 ± 160 mg/ml at 96 h).


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Fig. 3.   BAL total protein content after LPS exposure is summarized. Protein levels, used as an index of capillary permeability, steadily increased with successive LPS doses. In both strains (C57BL/6 and 129/J), compared with baseline, peak levels were observed at 96 h after 4 doses of inhaled LPS (P < 0.001).

Lung wet weight normalized to body weight is summarized in Fig. 4. Increases paralleled changes in BAL protein levels and corresponded with the number of LPS exposures. Measurements at 48 (C57BL/6: 19.2 ± 0.8 mg/g; 129/J: 17.8 ± 3.4 mg/g) and 96 (C57BL/6: 23.7 ± 1.9 mg/g; 129/J: 26.8 ± 1.8 mg/g) h were significantly different from baseline (C57BL/6: 12.8 ± 1.6 mg/g; 129/J: 14.3 ± 2.5 mg/g) in both strains. Peak increases, observed at 96 h, were 1.5-to-2-fold times baseline, similar to values reported among patients with ALI (22).


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Fig. 4.   Lung weight normalized to animal body wt. In both strains (C57BL/6 and 129/J), LPS exposure resulted in a marked increase in lung weight. By 96 h, lung weight had approximately doubled.

Changes in BAL cytokines after nebulized LPS. BAL levels of inflammatory cytokines were similar among the two strains of mice (Fig. 5). After LPS exposure, TNF-alpha and IL-6 BAL levels increased rapidly. TNF-alpha levels increased within 2 h after LPS exposure and returned toward baseline by 24 h in a pattern consistent with that previously described (1, 21, 22). IL-6 increased by 2 h and remained persistently elevated at all subsequent time points. INF-gamma , IL-10, and IL-1beta increased more gradually, but levels were slightly greater with each successive LPS exposure, suggesting the potential for continued increase beyond 96 h.


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Fig. 5.   Cytokine levels at different time points after LPS exposure in 129/J (B) and C57BL/6 mice (A). Although the 2 strains demonstrated differences in absolute cytokine levels, patterns of release were similar. Cytokine levels are shown as a function of time after LPS exposure in 129/J mice (B) and C57BL/6 mice (A) with the concentration (conc) expressed as pg/ml. Although the two strains demonstrated differences in absolute cytokine levels, patterns of release were similar. Tumor necrosis factor (TNF)-alpha increased within 2 h after LPS exposure and returned toward normal by 24 h. Interleukin (IL)-6 also increased immediately after LPS but remained persistently elevated at all time points. IL-1beta , IL-10, and interferon (IFN)-gamma increased more gradually, but levels were greater with each successive LPS dose, suggesting the potential for continued increase beyond 96 h.

Alteration in VEGF and Ang expression after nebulized LPS. Protein expression for VEGF, Ang-4, and Ang-2 was analyzed using immunostaining. Results of immunoperoxidase staining for C57BL/6 mice are summarized in Figs. 6 (VEGF), 7, 8 (Ang-4), and 9 (Ang-2). At baseline, VEGF (Fig. 6, A and D), Ang-4 (Fig. 7, A and C), and Ang-2 (Fig. 9, A and D) were readily detected in the airway epithelium and in the alveolar epithelial cells. Immunostaining patterns for VEGF and Ang-4 changed substantially after exposure to inhaled LPS. VEGF increased within the alveolar compartment of both strains after sequential LPS exposures, with the greatest intensity noted in the 96-h samples (Fig. 6, B and E). The increases in immunoreactivity were due to greater numbers of peripheral mononuclear cells and neutrophils staining in the alveolar interstitium and alveolar spaces. These cells were identified by serial staining as both mesenchymal cells (vimentin-positive cells) and leukocytes (large mononuclear cells positive for CD45 and polymorphonuclear leukocytes identified by CD45-positive cells with nuclear appearance typical for polymorphonuclear cells). No obvious increase in VEGF expression was noted in epithelial or endothelial cells. Preabsorbing the primary antibody ablated the immunostaining for VEGF (Fig. 6, C and F). The staining patterns for 129/J mice were similar to those observed in the C57BL/6 mice (data not shown).


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Fig. 6.   Representative immunoperoxidase staining is shown for vascular endothelial growth factor (VEGF) in C57BL/6 mice. At baseline, abundant immunostaining is detected in airway epithelial cells, indicated by arrowheads, with negligible staining in the alveolar compartment (A). After daily exposure to aerosolized LPS for 4 days (96-h time point), the airway epithelial immunostaining persists, indicated by arrowheads, with a substantial increase in immunopositive cells in the alveolar compartment within mononuclear cells and neutrophils, indicated by arrows (B). The immunostaining was ablated after preabsorbing the primary antibody against specific peptide (C). Magnification, ×200. High power views are shown in D-F to illustrate the immunostaining in the alveolar compartment. At baseline, little immunostaining is detected in alveolar cells, designated by arrows (D). After daily exposure to aerosolized LPS for 4 days, the immunostaining is substantially increased in the alveolar compartment, particularly in mononuclear inflammatory cells, designated by arrows (E). Immunostaining is ablated after preabsorbing the primary antibody against specific peptide (F). Magnification, ×400.



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Fig. 7.   Representative immunostaining is shown for angiopoietin (Ang)-4 in C57BL/6 mice treated with LPS. Ang-4 shows abundant immunostaining in the airway epithelial cells and in the interstitial cells of the alveolar compartment at baseline (A). After daily exposure to aerosolized LPS for 4 days (96-h time point), the immunostaining substantially decreases in both the airway epithelial cells and in the alveolar compartment (B). Magnification, ×200. High power views are shown in C and D to illustrate the immunostaining in the alveolar compartment. At baseline, immunostaining, designated by arrows, is readily detected in the alveolar compartment (C). After daily exposure to aerosolized LPS for 4 days, the immunostaining in the alveolar compartment substantially diminishes (D). Magnification, ×400.

By contrast, immunostaining for Ang-4 in C57BL/6 mice (Fig. 7) was readily detectable in the alveolar compartment and airway epithelium at baseline (Fig. 7, A and C) but decreased with successive exposures to LPS. The greatest decrease in immunopositive cells was noted in 96-h samples and involved both the bronchial epithelial cells and cells within the alveolar compartment (Fig. 7, B and D). Preabsorbing the primary antibody with specific peptide ablated the immunostaining for Ang-4 (Fig. 8, A-D ). These staining patterns were observed in both C57BL/6 and 129/J mice.


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Fig. 8.   Representative sections demonstrating decreased immunostaining in murine lung after preabsorbing antisera for Ang-4 with specific peptide. At baseline, abundant Ang-4 immunostaining is seen in the epithelial cells (A). This immunostaining is ablated after preabsorbing with specific peptide (B). Magnification, ×200. High power views are shown in C and D to illustrate the immunostaining in the alveolar compartment. At baseline, immunostaining, designated by arrows, is readily detected in the alveolar compartment (C). Immunostaining is substantially decreased after preabsorbing the primary antibody with specific peptide (D). Magnification, ×400.

Immunostaining for Ang-2 was abundant at baseline (Fig. 9, A and D). Similar to Ang-4 immunostaining, Ang-2 was expressed by both bronchial epithelial cells and cells within the alveolar compartment. In C57BL/6 mice, the pattern for Ang-2 after LPS exposure differed from both VEGF and Ang-4 in that epithelial cell immunoreactivity decreased (Fig. 9B), but immunoreactivity within alveolar cells increased (Fig. 9E) at 96 h. Staining of serial sections with CD45 demonstrated that the increased distal immunostaining was largely due to increased macrophage expression (data not shown). In 129/J mice, there was little change in the Ang-2 immunostaining after LPS (data not shown).


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Fig. 9.   Representative sections demonstrating immunostaining for Ang-2 in C57BL/6 mice. At baseline, abundant immunoreactivity is apparent in the epithelial cells, indicated by arrowheads (A). After daily exposure to LPS for 4 days (96-h time point), the epithelial immunostaining, indicated by arrowheads, is decreased, whereas the peripheral mononuclear cells, indicated by arrows, demonstrate increased immunoreactivity (B). The immunostaining was ablated by preabsorbing the primary antibody with specific peptide (C). Magnification, ×200. High power views are shown in D-F to illustrate the immunostaining in the alveolar compartment. At baseline, constitutive immunoreactivity for Ang-2 is detected in the alveolar compartment (D). After daily exposure to LPS for 4 days, substantially increased immunostaining for Ang-2 is noted in the alveolar compartment, particularly in the mononuclear inflammatory cells, designated by arrows (E). Immunostaining is ablated after preabsorbing the primary antibody with the specific peptide (F). Magnification, ×400.

We then examined whether alterations in VEGF and Ang-4 and Ang-2 protein expression were associated with similar changes in mRNA expression. In addition, levels of mRNA encoding Ang-1 were assessed. Messenger RNA levels assayed by RT-PCR are summarized in Fig. 10 for both 129/J and C57BL/6 mice. Similar responses to LPS were observed between both strains, with VEGF mRNA tending to increase, whereas levels of Ang-1 and Ang-4 tended to decrease.


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Fig. 10.   RT-PCR results for total lung mRNA levels of vascular growth factors for 129/J (A) and C57BL/6 (B) mice. In A, lanes 1-3 represent baseline measurements, lanes 4-6 are from the 24-h time point, lanes 7-9 are from the 48-h time point, and lanes 10-12 are from the 96-h time point. In B, lanes 1-4 represent baseline measurements, lanes 5 and 6 are from the 24-h time point, and lanes 7 and 8 are from the 48-h time point. Both strains demonstrate similar patterns. The housekeeping gene beta -actin was unchanged after LPS exposure. At baseline, mRNA encoding VEGF is expressed at a low level but increased substantially after LPS exposure at 24, 48, and 96 h. Ang-1 mRNA and Ang-4 mRNA tend to show the opposite after LPS exposure, with expression greatest at baseline but declining in response to serial LPS exposures. Total mRNA encoding Ang-2 expression did not vary significantly after LPS exposure in either strain.

At baseline, mRNA encoding VEGF was detected at low levels. After exposure to LPS, this mRNA increased at the 24-h time point and remained elevated at 48 and 96 h. The mRNA encoding Ang-1 was abundantly expressed at baseline and became undetectable in C57BL/6 mice by the 48-h time point. A similar pattern was observed in 129/J mice, with the mRNA encoding Ang-1 becoming markedly decreased, but still detectable, by 96 h. The mRNA encoding Ang-4 was similarly decreased at 48 and 96 h in both strains. Thus the patterns of expression for the mRNAs encoding VEGF, Ang-1, and Ang-4 paralleled those observed with immunostaining for VEGF and Ang-4. This suggests that the increased (or decreased) immunostaining reflects increased protein expression due to increased (or decreased) transcription. In contrast, the total mRNA encoding Ang-2 was slightly decreased at the 48-h time point in the C57BL/6 mice and was largely unchanged after LPS exposure in the 129/J mice. Levels of the housekeeping gene beta -actin were unchanged after LPS exposure.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Vascular growth factors, which include VEGF, Ang-1, Ang-2, and Ang-4, are known to regulate vasculogenesis and angiogenesis during embryonic development as well as new vessel growth and proliferation in certain tumors and during tissue healing (26). More recently, it has been demonstrated that these peptides modulate epithelial integrity in adult vascular beds and influence the course of diseases in which vascular leakage is important. Systemic expression of VEGF has been shown to cause widespread multiorgan capillary leakage, an effect that is attenuated by concomitant expression of Ang-1 (23). Ang-1 has also been shown to abrogate capillary leakage within the dermis after administration of nonspecific irritants (22). Together, these observations suggest that a balance between the proleakage effects of VEGF and antileakage effects of the angiopoietins, in part, modulates vascular integrity and ultimately oxygen delivery. The present study examines the role of VEGF and the angiopoietins in modulating capillary leakage and inflammation during the development of ALI after LPS exposure.

To examine this question, we utilized a previously validated murine model (10) that closely replicates many of the physiological and biological aspects of human ALI (8). In two common strains of mice (C57BL/6 and 129/J), nebulized LPS produced two- to fivefold increases in tissue resistance and dynamic elastance and smaller changes in airway resistance. The magnitude of change in physiological parameters was more pronounced in 129/J mice than in C57BL/6 mice, although patterns were similar in both and paralleled changes in human patients with ALI/acute respiratory distress syndrome (ARDS) in which the predominant abnormality involves the parenchyma. The marked frequency dependence of elastance indicates physiological heterogeneity, also a characteristic of ALI (2).

LPS exposure was associated with cellular inflammation and an increase in BAL cytokines. The large increase in total cells was due primarily to a rise in the number of neutrophils. BAL levels of TNF-alpha , IL-1beta , IL-6, and IFN-gamma increased in both strains after LPS exposure, consistent with acute inflammation. The patterns of cytokine expression in BAL fluid were similar among the two strains. TNF-alpha levels peaked within hours of LPS exposure, returning toward normal within 24 h, whereas IL-1beta , IL-6, and IFN-gamma increased steadily with sequential exposures. Together, these observations confirm that both C57BL/6 and 129/J mice develop an inflammatory response to LPS physiologically and biologically similar to ALI.

We further show that this inflammatory response is accompanied by pronounced capillary leakage. Lavage fluid protein, a nonspecific marker of breakdown in the epithelial-endothelial barrier, was significantly elevated after LPS exposure at all time points compared with baseline. Protein levels increased with serial LPS exposures. Lung weights normalized to body weight also progressively increased with each LPS dosage. In both C57BL/6 and 129/J mice, lung weight doubled by 96 h.

The extent of the physiological and biological responses to LPS were substantially greater in the 129/J mice compared with the C57BL/6 mice. In this context, it is notable that the patterns of expression for VEGF and the angiopoietins following LPS in these two strains are remarkably similar. The pattern is consistent with the observed changes in lung wet-to-dry ratios, which correlates with the degree of pulmonary edema in this model. These findings suggest that the changes in the expression patterns of vascular growth pattern are likely not strain dependent but are probably part of the pulmonary response to inhaled LPS in this model of murine ALI. These data are also consistent with the hypothesis that the vascular growth factors, VEGF and the angiopoietins, modulate capillary permeability in ALI.

Of note, the changes in cytokine expression, BAL cellular profiles and total protein, elastance, and tissue resistance preceded the changes in the vascular growth factor mRNA. There are at least two possible reasons for this observation. RT-PCR is a semiquantitative technique, so small changes in gene expression between groups may not have been detected. Alternatively, alterations in vascular growth factor expression may be one of several concurrently acting processes contributing to the pathophysiology of ALI.

To test the hypothesis that LPS induces differential protein expression of VEGF and the angiopoietins, immunostaining was performed. Our data demonstrate that after LPS and coincident with the development of inflammation, capillary leakage, and lung edema, VEGF expression increased significantly in resident mononuclear cells and neutrophils within the lung parenchyma. VEGF was also detected in the bronchial epithelium but was unchanged from baseline after exposure to LPS. The increased immunostaining was paralleled by increased mRNA expression, consistent with a transcriptional mechanism for the alterations in VEGF expression. This is supported by the increased intensity of immunostaining for VEGF within mononuclear inflammatory cells after exposure to LPS compared with the immunostaining detected in the mononuclear inflammatory cells in untreated lung tissue samples. However, we cannot exclude an increase in the overall expression of mRNA encoding VEGF due solely to the influx of inflammatory cells after exposure to inhaled LPS. By contrast, Ang-4 immunostaining, which was readily evident at baseline, decreased over time after LPS exposure as lung inflammation progressed. In control samples, Ang-4 staining was demonstrated predominantly by epithelial cells, and to a lesser extent by interstitial cells, but not by resident pulmonary macrophages or neutrophils. After LPS exposure, Ang-4 expression by epithelial cells decreased substantially. As observed with VEGF, the decreased immunostaining was paralleled by decreased mRNA expression, consistent with a transcriptional mechanism for the alteration in Ang-4 expression. Immunostaining for Ang-2, which is associated with increased vascular permeability, did not change after LPS exposure in 129/J mice. In C57BL/6 mice, bronchiolar epithelial immunostaining for Ang-2 decreased, but peripheral mononuclear cell staining increased after LPS exposure. Thus in the C57BL/6 mice, Ang-2 immunostaining increased in the alveolar compartment, which is the site of capillary leakage in pulmonary edema. In these animals, the mRNA for Ang-2 did not substantially change in the 129/J mice and showed a slight decrease in the C57BL/6 mice at the 48-h time point baseline, which may reflect the divergent expression patterns observed in airway and parenchymal cells after endotoxin.

The pattern of VEGF upregulation accompanied by Ang-1 and Ang-4 downregulation after LPS exposure within the lung is consistent with patterns previously reported to promote capillary leakage and tissue edema in other organs (11, 27). This suggests that the relative balance between proleakage (VEGF and Ang-2) and antileakage (Ang-1 and Ang-4) vascular growth factors may be important in the determination of capillary integrity. That is, changes in any given factor may be less important than the overall balance between the two types of factors. Overexpression of VEGF induces plasma leakage (11) through binding to the endothelial cell-specific tyrosine kinase receptor flk-1/VEGF-R2 (16). VEGF-R2 activation leads to phosphorylation of AJC proteins (4, 6, 21), decreased association between VE-cadherin and intracellular catexin, and a weakening of intercellular junctions with increased paracellular leakage (4). In contrast, Ang-1, which promotes stabilization of endothelial junctional complexes (4, 7), was downregulated in our model. Other investigators have shown that Ang-1 promotes vascular integrity even in the face of increased VEGF expression (4, 7). Our data suggest that the combination of decreased Ang-1 and increased VEGF might favor capillary leakage due to unopposed action of VEGF on the endothelium. In addition, Ang-4, which is known to be specifically expressed in lung tissue but has not previously been examined in the context of a model of ALI, is also decreased in this model. We speculate that, similar to Ang-1, Ang-4 may modulate pulmonary vascular integrity.

Our findings are in agreement with those of Thickett and colleagues (21) who demonstrated that plasma VEGF was significantly elevated in patients with ARDS compared with patients who had risk factors to develop ARDS, normal control subjects, and ventilated patients without ARDS. In the same study, peripheral blood monocytes from patients with ARDS produced significantly more VEGF in vitro than those of at risk patients. In addition, albumin flux across human pulmonary endothelial cell monolayers was significantly increased after the addition of plasma from patients with ARDS compared with plasma from controls, an effect that was partially inhibited by the addition of a soluble VEGF inhibitor (21).

In contrast, a recent study by Maitre and colleagues (14) showed decreased levels of VEGF expression in a rat model of infection-associated severe lung injury and in the BAL from patients with early ARDS. Although these results would appear to conflict with the current study, there are several important methodological differences between the two studies. First, the investigation by Maitre and colleagues (14) examined rat lung tissue and human BAL, whereas the current study examines murine tissue. Also, the expression of mediators in BAL may not be completely representative of the expression of mediators in the tissue compartment. Also, the kinetics of the two studies differ in that Maitre and colleagues focused on very early time points, whereas the current study focused on a longer time course.

The observations presented here do not prove a causal role for these vascular growth factors in the pathophysiology of ALI. In contrast to other organs in which the endothelium represents the primary barrier to capillary leakage and vascular growth factors appear to play a dominant regulatory function, the lung has a dual epithelial-endothelial barrier. The epithelial barrier has traditionally been recognized as being primarily responsible for regulating fluid balance within the lung. Therefore, it is unclear whether isolated disruption of the endothelial barrier would be sufficient to allow capillary leakage of the type that occurs in ALI. Nevertheless, alterations in the endothelial barrier are ultimately necessary to allow marked changes in permeability. It is, therefore, interesting that the pulmonary epithelial cell appears to be primarily responsible for expression of Ang-1 and Ang-4. This expression pattern suggests that the epithelium is an important regulator of the state of "leakiness" of the endothelial barrier, and thus permeability of the serially aligned epithelial-endothelial barrier systems may change in a synchronous fashion. Decreases in Ang-1 and Ang-4, resulting from changes in epithelial cell expression due to cell death or altered rates of synthesis and/or secretion, might act permissively to increase permeability. Although this regulatory configuration facilitates a rapid immunological response to alveolar pathogens by permitting transcapillary access for neutrophils, complement proteins, serum immunoglobulins, and fibrin, it also tends to promote development of exudative pulmonary edema as seen in ALI and described here in our animal model.

In summary, our study demonstrates that inhaled LPS causes physiological and biological responses in mice similar to those reported in human ALI. The observed neutrophil infiltration, capillary leakage, and lung edema are accompanied by increased production of VEGF and downregulation of Ang-1 and Ang-4 in a time- and dose-dependent manner. Further studies are required to clarify the potential mechanisms regulating expression of these vascular growth factors as well as their direct mode of action on the lung endothelium, both under physiological conditions and during acute inflammation. Our results suggest that the differential expression of VEGF, Ang-1, Ang-2, and Ang-4 by activated mononuclear phagocytes, neutrophils, and epithelial cells within the lung may contribute to the development of ALI.


    ACKNOWLEDGEMENTS

The authors thank Michael Cullivan for technical assistance and Yolanda Marzan for assistance with animal studies.


    FOOTNOTES

This work was supported by funding from the Thoracic Foundation, National Heart, Lung, and Blood Institute Grant KO8 HL-67910 (to K. Hong), and by State Scholarship Foundation of Greece Grant 82106.12.1 (to I. Kosmidou).

Address for reprint requests and other correspondence: K. J. Haley, Division of Pulmonary and Critical Care Medicine, 75 Francis St., Brigham and Women's Hospital, Boston, MA 02115 (E-mail: khaley{at}rics.bwh.harvard.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.

May 3, 2002;10.1152/ajplung.00048.2002

Received 1 February 2002; accepted in final form 25 April 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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