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Am J Physiol Lung Cell Mol Physiol 294: L955-L963, 2008. First published February 29, 2008; doi:10.1152/ajplung.00449.2007
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Regulation of angiopoietin expression by bacterial lipopolysaccharide

Mahroo Mofarrahi, Thamir Nouh, Salman Qureshi, Loic Guillot, Dominique Mayaki, and Sabah N. A. Hussain

Critical Care and Respiratory Divisions, McGill University Health Centre, Meakins-Christie Laboratories, McGill University, Montréal, Québec, Canada

Submitted 29 October 2007 ; accepted in final form 27 February 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Angiopoietins are ligands for Tie-2 receptors and play important roles in angiogenesis and inflammation. While angiopoietin-1 (Ang-1) inhibits inflammatory responses, angiopoietin-2 (Ang-2) promotes cytokine production and vascular leakage. In this study, we evaluated in vivo and in vitro effects of Escherichia coli lipopolysaccharides (LPS) on angiopoietin expression. Wild-type C57/BL6 mice were injected with saline (control) or E. coli LPS (20 mg/ml ip) and killed 6, 12, and 24 h later. The diaphragm, lung, and liver were excised and assayed for mRNA and protein expression of Ang-1, Ang-2, and Tie-2 protein and tyrosine phosphorylation. LPS injection elicited a severalfold rise in Ang-2 mRNA and protein levels in the three organs. By comparison, both Ang-1 and Tie-2 levels in the diaphragm, liver, and lung were significantly attenuated by LPS administration. In addition, Tie-2 tyrosine phosphorylation in the lung was significantly reduced in response to LPS injection. In vitro exposure to E. coli LPS elicited cell-specific changes in Ang-1 expression, with significant induction in Ang-1 expression being observed in cultured human epithelial cells, whereas significant attenuation of Ang-1 expression was observed in response to E. coli LPS exposure in primary human skeletal myoblasts. In both cell types, E. coli LPS elicited substantial induction of Ang-2 mRNA, a response that was mediated in part through NF-{kappa}B. We conclude that in vivo endotoxemia triggers functional inhibition of the Ang-1/Tie-2 receptor pathway by reducing Ang-1 and Tie-2 expression and inducing Ang-2 levels and that this response may contribute to enhanced vascular leakage in sepsis.

sepsis; inflammation; angiopoietins; nuclear factor-{kappa}B

ANGIOPOIETIN-1, -2, -3, AND -4 are oligomeric-secreted glycoproteins that are ligands for endothelial cell-specific Tie-2 receptors (4). Angiopoietin-1 (Ang-1) is released by fibroblasts, vascular smooth muscles, thyrocytes, tumor cells, and endothelial (EC) cells, whereas angiopoietin-2 (Ang-2) is produced primarily by ECs (4). Ang-3 and its human ortholog Ang-4 are more recently identified members of the angiopoietin family, with Ang-3 having wide tissue distribution in mice and Ang-4 being most strongly expressed in human lungs (33). Ang-1 and Ang-2 bind Tie-2 receptors with equal affinity, although Ang-2 elicits only context-sensitive phosphorylation of these receptors and can competitively inhibit Ang-1-induced phosphorylation of Tie-2 (21).

In addition to its crucial roles in embryonic vascular development (29), the Ang-1/Tie-2 receptor pathway promotes EC migration, proliferation, survival, and differentiation (28). Moreover, Ang-1 protects adult peripheral vasculature from vascular leakage (31) and inhibits the effects of proinflammatory cytokines on ECs. Indeed, when Ang-1 is coincubated with vascular endothelial growth factor (VEGF) or tumor necrosis factor (TNF{alpha}), leukocyte adhesion to ECs, migration of leukocytes across ECs, and the expression of adhesion molecules are all strongly inhibited (16, 17). Unlike Ang-1, the effect of Ang-2 on angiogenesis is more complex and is context dependent; when Ang-2 levels are elevated, enhanced angiogenesis is apparent when VEGF is present, whereas vascular regression has been observed in its absence (11).

There is increasing evidence that Ang-2 may promote EC vascular leakage both in vitro and in vivo (24, 26). Ang-2 also promotes adhesion of leukocytes to ECs by sensitizing the latter towards TNF{alpha} and increases expression of adhesion molecules on the surface of ECs (6). The biological roles of Ang-3 and Ang-4 remain unclear. Although initial reports suggest that Ang-4 stimulates Tie-2 phosphorylation in a fashion similar to that elicited by Ang-1, and that Ang-3 does not activate Tie-2 receptors (33), later studies have confirmed that both Ang-3 and Ang-4 promote EC survival, proliferation, and differentiation (14).

Septic shock is caused by an exaggerated systemic inflammatory response to gram-negative bacteria and the cell wall component, LPS. In the United States, severe sepsis and septic shock are major causes of mortality in intensive care units and account for more than 750,000 cases per year, with an estimated mortality of ~30% (2). Septic shock is characterized by increased capillary permeability, widespread EC dysfunction, alveolar and interstitial pulmonary edema, and increased expression of proinflammatory cytokines and chemokines, including TNF{alpha}, IL-1, IL-6, and interferon-{gamma}. The involvement of angiopoietins in the pathogenesis of septic shock is as yet unclear, primarily because little information is available regarding in vivo regulation of angiopoietin production in various organs during sepsis or septic shock. In vitro studies have revealed opposing effects of proinflammatory cytokines such as TNF{alpha}, IL-1β, and IFN{gamma} on Ang-1 gene expression. While TNF{alpha} and IL-1β stimulate Ang-1 expression in synovial fibroblasts and ECs (5, 9), a combination of TNF{alpha} and IFN{gamma} reduces Ang-1 levels in cultured osteoblasts (13). As for Ang-2 gene expression, it has been shown that proinflammatory stimuli strongly activate Ang-2 transcription in ECs (9, 15, 22), and two recent studies have documented elevated circulating levels of Ang-2 protein in patients with sepsis (23, 24).

Despite the importance of bacterial LPS in the pathogenesis of sepsis and septic shock, little is known about the influence of LPS on the regulation of angiopoietin expression. Brown et al. (5) have reported that Escherichia coli LPS induced Ang-1 mRNA expression and promoter activity in cultured fibroblasts. In contrast, Karmpaliotis et al. (12) have studied the in vivo regulation of angiopoietins in a murine model of LPS-induced acute lung injury and have reported a significant decline in epithelial Ang-1 and Ang-4 mRNA and protein expressions 96 h after administration of nebulized LPS (12). These authors have also found that in response to LPS administration, Ang-2 expression in airway epithelial cells declines significantly, whereas that of alveolar cells increases (12). These studies leave many important questions regarding the influence of LPS on angiopoietin expression unanswered. In this study, we used a murine model of LPS-induced sepsis and cultured cell models to address the influence of in vivo and in vitro effects of LPS on angiopoietin expression and to evaluate the role of NF-{kappa}B in this regulation. Our hypothesis was that LPS administration would elicit downregulation of Ang-1 expression while simultaneously inducing Ang-2 expression, with the overall response being inhibition of the Ang-1/Tie-2 receptor pathway. We also hypothesized, on the basis of previous studies on Ang-1 and Ang-2 promoters (5, 10, 22), that NF-{kappa}B plays an important role in LPS-induced Ang-2 expression, but not in the downregulation of Ang-1 expression brought on by LPS administration.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal Preparation

Adult (8- to 12-wk-old) male wild-type C57/BL6 (n = 36) mice were studied at the McGill University Animal Facility and were divided into two major groups. Animals in group 1 were injected intraperitoneally (ip) with saline and killed 6, 12, and 24 h later (n = 6 at each time point). Animals in group 2 received ip injections of E. coli LPS (20 mg/kg, serotype 055:B5; Sigma-Aldrich, Oakville, ON) and were killed 6, 12, and 24 h later (n = 6 at each time point). All animals were euthanized with pentobarbital sodium followed by quick excision of the liver, diaphragm, and lungs, which were flash-frozen in liquid nitrogen and stored at –80°C until further analysis. All protocols were approved by the Animal Care Committee of McGill University.

Cell Culture

It has been well established that systemic exposure to LPS initiates a rapid, coordinated recruitment of inflammatory cells and overproduction of proinflammatory mediators that might indirectly regulate tissue angiopoietin expression in response to in vivo LPS administration. To study the primary regulation of angiopoietin gene expression by LPS signaling, we evaluated angiopoietin gene expression in cultured epithelial cells (representative of pulmonary cells) and skeletal muscle myoblasts (representative of skeletal muscles) exposed to E. coli LPS for up to 24 h. We have verified in pilot experiments that these two cell types produce mainly Ang-1 and have no detectable Tie-2 receptor expression.

Epithelial cell culture. The adeno12 SV40-transformed human bronchial epithelial cell line, BEAS-2B, was a gift from Dr. C. Harris (National Cancer Institute, Bethesda, MD) and was cultured in DMEM/F-12 (Invitrogen, Carlsbad, CA) supplemented with 5% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). BEAS-2B cells were exposed to either PBS (control) or 0.1 µg/ml of E. coli LPS (serotype 055:B5, Sigma-Aldrich) for 6, 12, and 24 h. To evaluate gene expression of ESE-1 transcription factor, cells were exposed to PBS or LPS for 3, 6, and 12 h. Cells were then harvested and total RNA was extracted (see below). We verified in preliminary experiments that this concentration of LPS elicits a substantial induction of IL-8 protein production in the culture medium as measured by ELISA (R&D Systems, Minneapolis, MN). To evaluate the involvement of the NF-{kappa}B pathway in LPS-induced regulation of angiopoietin expression in epithelial cells, we used adenoviruses expressing GFP (Ad-GFP) or a dominant negative mutant form (K44A) of I{kappa}B kinase β (IKKβ) (Ad-dnIKKβ) (27). BEAS-2B cells (60–70% confluent) were infected overnight at a multiplicity of infection (MOI) of 100 in serum-free medium. The virus-containing medium was then replaced with complete medium containing 5% FBS and incubated for 24 h. Cells were then exposed for 6 h to either PBS (control) or E. coli LPS, as described above.

Human skeletal muscle myoblasts. Primary human skeletal muscle myoblasts, immortalized by expression of the E6E7 early region of human papillomavirus type 16, were a kind gift from Dr. E. Shoubridge (McGill Univ., Montréal, QC). Myoblasts were cultured in SkBM using the SkBM Bulletkit (Cambrex Research Products, Walkersville, MD) supplemented with 15% inactivated FBS. Subconfluent myoblasts were exposed either to PBS (control) or 10 µg/ml of E. coli LPS (serotype 055:B5, Sigma-Aldrich) for 6, 12, and 24 h. To evaluate gene expression of ESE-1 transcription factor, cells were exposed to PBS or LPS for 3, 6, and 12 h. Cells were then lysed, and total RNA was extracted (see below). To evaluate the involvement of NF-{kappa}B transcription factor in LPS-induced changes in angiopoietin expression in myoblasts, cells were infected overnight (1,000 MOI) with adenoviruses expressing GFP and a dominant-negative form of IKKβ, as described above. The virus-containing medium was then replaced with complete medium containing 15% FBS. Cells were incubated for 24 h and then exposed for 6 h to either PBS (control) or E. coli LPS, as described above.

RNA Extraction and Real-Time PCR

Total RNA was extracted from mouse liver, diaphragm, and lung samples, as well as from cultured cells using a GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich). Quantification and purity of total RNA was assessed by A260/A280 absorption. Total RNA (2 µg) was then reverse transcribed using Superscript II Reverse Transcriptase kits and random primers (Invitrogen). Reactions were incubated at 42°C for 50 min and at 90°C for 5 min. Real-time PCR was performed using a Prism 7000 Sequence Detection System from Applied Biosystems (Foster City, CA). To quantify the expressions of 18S (endogenous control), murine Ang-1, Ang-2, Ang-3, and VEGF, as well as human Ang-1, Ang-2, and Ang-4 transcripts, we used Applied Biosystems TaqMan gene expression assays (cat. no. 4352930E, Mm00456503_ m1, Mm00545822_m1, Mm00507766_m1, Mm00437304_m1, Hs00181613_m1, Hs00169867_m1, and Hs00211115_m1, respectively). For ESE-1 (ELF3) transcription factor, we used TaqMan expression assay cat. no. Hs00963882_g1. The thermal profile was as follows: 50°C for 2 min, 95°C for 10 min and 40 cycles (95°C for 15 s and 60°C for 1 min). Each PCR reaction was carried out in triplicate on one plate, and the results presented are combined from each treatment group. Dissociation curve analyses were performed to show the specificity of amplification. Results were analyzed in two ways. First, we used the comparative threshold cycle (CT) method to calculate fold changes in expression in the LPS groups compared with the saline groups (20), where:

Formula

Formula

Fold changes in gene expression in the LPS groups were then calculated as 2Formula. All real-time PCR experiments were performed in triplicate. A similar approach was used to calculate fold changes in expression in LPS-treated cells compared with control cells. Second, to determine the absolute copy numbers of Ang-1, Ang-2, and Ang-3 mRNA transcripts in tissue or cell samples, we established standard curves relating the CT values of these genes to the copy numbers. These curves were generated by performing real-time PCR analysis using angiopoietin TaqMan primers on samples with known copy numbers of plasmids containing full coding sequences of murine and human Ang-1, Ang-2, Ang-3, and Ang-4 cDNA. Plasmids were diluted serially to generate copy numbers ranging from 30 to 300,000. Copy numbers of angiopoietins were then calculated using these curves and were normalized per 1 ng of total RNA obtained from tissue and cell samples.

Immunoblotting

Frozen liver, diaphragm, and lung tissues were homogenized in six volumes (wt/vol) of homogenization buffer (pH 7.4, 10 mM HEPES buffer, 0.1 mM EDTA, 1 mM dithiothreitol, 1 mg/ml phenylmethylsulfonyl fluoride, 0.32 mM sucrose, and 10 µg/ml each of leupeptin, aprotinin, and pepstatin A). Crude homogenates were centrifuged at 4°C for 15 min at 5,000 rpm. Supernatants were then collected and used for immunoblotting. Tissue proteins (100 µg) were heated for 5 min at 90°C and then loaded onto Tris-glycine SDS-polyacrylamide gels, electrophoretically separated, transferred to polyvinylidene difluoride membranes, blocked with 5% nonfat dry milk, and subsequently incubated overnight at 4°C with primary monoclonal antibodies to Ang-1 and Ang-2 (R&D Systems) and a polyclonal anti-Tie-2 receptor antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Preliminary experiments indicated that commercially available anti-Ang-3 antibodies are not selective to this protein. Specific proteins were then detected with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies and enhanced chemiluminescence reagents provided with an ECL kit from Amersham Canada (Oakville, ON). Loading of equal amounts of proteins was confirmed by stripping the membranes and reprobing with anti-{alpha}-tubulin antibody (Sigma-Aldrich). The blots were scanned with an imaging densitometer, and protein band optical densities was quantified with Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Predetermined molecular mass standards were used as markers.

Immunoprecipitation

To investigate whether LPS administration alters Tie-2 receptor tyrosine phosphorylation, we immunoprecipitated Tie-2 receptors and probed them with anti-phosphotyrosine antibodies. Tissue homogenates (500 µg) were incubated with primary polyclonal anti-Tie-2 receptor antibody for 12 h at 4°C. Protein A-agarose conjugates were then added, and samples were incubated for a further 3 h. After centrifugation, pellets were washed three times with a buffer containing 125 mM Tris·HCl, pH 8.1, 500 mM NaCl, 0.5% Triton X-100, 10 mM EDTA, and 0.02% NaN3. The final wash was performed with water. Proteins were eluted with electrophoresis sample buffer, and immunoblotting of supernatant and eluted proteins was undertaken as described above. Membranes were probed with monoclonal anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology, Lake Placid, NY). Proper negative controls included omission of primary antibody and omission of protein A-agarose conjugates.

Data Analysis

Data are presented as means ± SD. Six separate animals were studied in each group for each time point, and three different organs (liver, diaphragm, and lung) were sampled in each animal. At a given time point, differences between control (saline-injected) and LPS-injected animals in terms of angiopoietin and VEGF expressions were compared using two-way analysis of variance, and P values less than 0.05 were considered significant. Similar analysis in terms of angiopoietin expression was used to compare control (PBS-treated) and LPS-treated epithelial cells and skeletal muscle myoblasts.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Influence of In Vivo LPS Injection

Figure 1 illustrates the influence of in vivo LPS injection on angiopoietin and VEGF mRNA expression in the liver, diaphragm, and lung. Expression of Ang-1 mRNA increased significantly after 6 h of LPS injection only in the diaphragm. After 12 and 24 h of LPS administration, Ang-1 mRNA declined significantly in the liver and diaphragm and after 12 h in the lung (Fig. 1). Similarly, Ang-3 mRNA in the diaphragm and lung declined after 6, 12, and 24 h and in the liver after 6 and 12 h of LPS administration. The time course of VEGF mRNA expression in the diaphragm was very similar to that of Ang-1 with an initial increase and a subsequent decline after 12 and 24 h of LPS administration (Fig. 1). Expression of VEGF in the liver and lung remained unchanged in response to LPS administration. In comparison, Ang-2 mRNA increased substantially in the three organs through the time course of LPS injection (Fig. 1). To validate whether these changes in mRNA expression were associated with similar changes in protein levels, we performed immunoblotting for Ang-1 and Ang-2 proteins using selective antibodies. Figure 2 shows that injection of LPS after 12 and 24 h resulted in a significant decline in Ang-1 and an increase in Ang-2 protein levels in the liver, diaphragm, and lung.


Figure 1
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  		Fig. 1. Changes in the expression of angiopoietin (Ang)-1, Ang-2, Ang-3, and VEGF mRNA in the lung, liver, and diaphragm of mice injected with Escherichia coli LPS and killed 6, 12, and 24 h later. Results are expressed as fold changes from those measured in mice injected with saline and killed at the same time. *P < 0.05 compared with those injected with saline. Note the substantial induction of Ang-2 mRNA, whereas Ang-1 and Ang-3 mRNA levels declined significantly.

 

Figure 2
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  		Fig. 2. Changes in the protein levels of Ang-1 and Ang-2 in the lung (A), liver (B), and diaphragm (C) measured after 12 and 24 h of E. coli LPS or saline administration in mice. Tubulin detection was used to evaluate equal protein loading. Note the decline in Ang-1 protein levels, whereas Ang-2 protein expression increased significantly in response to LPS administration.

 
Liver, diaphragm, and lung Tie-2 protein levels declined significantly in response to LPS administration (Fig. 3). We were only able to detect Tie-2 tyrosine phosphorylation in lung samples. This could be due to a relatively higher abundance of Tie-2 expression in the lung compared with other tissues. LPS administration resulted in a significant decline in Tie-2 tyrosine phosphorylation (Fig. 3C). This reduction is not simply due to a reduction in Tie-2 protein levels since the ratio of tyrosine-phosphorylated Tie-2 to total Tie-2 declined significantly in the lungs of LPS-injected animals (P < 0.05 compared with saline-injected animals).


Figure 3
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  		Fig. 3. A and B: representative samples and mean values of Tie-2 protein levels in the diaphragm, liver, and lung measured after 6, 12, and 24 h of E. coli LPS and saline administration. *P < 0.05 compared with animals injected with saline. C: total and tyrosine-phosphorylated Tie-2 protein levels in the lungs of mice injected 12 and 24 h earlier with saline or E. coli LPS. D: means ± SD of tyrosine-phosphorylated/total Tie-2 ratios of lung samples obtained after 12 and 24 h of saline and E. coli LPS administration in mice. *P < 0.05 compared with saline-injected animals.

 
Effects of In Vitro LPS Exposure

Table 1 lists the abundance of Ang-1, -2, and -4 mRNA transcripts in BEAS-2B epithelial cells and primary human skeletal myoblasts. BEAS-2B cells express relatively lower levels of these transcripts compared with primary skeletal myoblasts (Table 1). Exposure of BEAS-2B cells to LPS elicited a significant induction in Ang-1 and Ang-2 mRNA expression, which peaked after 12 h of LPS administration (Fig. 4A). In comparison, Ang-4 mRNA expression declined significantly after 12 and 24 h of LPS exposure (Fig. 4). Inhibition of the NF-{kappa}B transcription factor activation using a dominant-negative form of IKKβ had no influence on the 12-h response of Ang-1 and Ang-4 transcripts to LPS administration; however, it significantly attenuated LPS-induced induction of Ang-2 mRNA (Fig. 4B).


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  		Table 1. mRNA expression (expressed as copies per 1,000 copies of 18S) of angiopoietins measured as baseline values in the livers, diaphragms, and lungs of wild-type C57/BL6 mice and in BEAS-2B epithelial cells and human skeletal myoblasts

 

Figure 4
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  		Fig. 4. A: expression of Ang-1, Ang-2, and Ang-4 mRNAs in BEAS-2B epithelial cells measured after 6, 12, and 24 h of E. coli LPS exposure. Results are expressed as fold changes from values measured after 6-, 12-, and 24-h exposure to PBS (control). *P < 0.05 compared with control. B: BEAS-2B cells were infected with adenoviruses expressing GFP or a dominant-negative (dn) form of IKKβ and were then exposed to PBS (control) or E. coli LPS. Expression of Ang-1, Ang-2, and Ang-4 mRNA levels in these cells was the measured with real-time PCR and expressed as fold changes from PBS-exposed (control) cells. *P < 0.05 compared with control cells. Note that infection with adenoviruses expressing a dominant-negative form of IKKβ had no influence on LPS-triggered changes in Ang-1 and Ang-4 levels but completely blocked LPS-induced Ang-2 expression.

 
Primary human skeletal myoblasts express relatively higher levels of Ang-1 mRNA transcripts compared with those of Ang-2 and Ang-4 (Table 1). LPS exposure of these cells significantly attenuated Ang-1 and Ang-4 mRNA expressions, whereas Ang-2 mRNA was significantly induced by LPS in these cells (Fig. 5). Expression of a dominant-negative form of IKKβ had no influence on the decline in Ang-1 and Ang-4 expression, but it completely eliminated LPS-induced Ang-2 expression (Fig. 5B).


Figure 5
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  		Fig. 5. A: expression of Ang-1, Ang-2, and Ang-4 mRNAs in primary human skeletal myoblasts measured after 6, 12, and 24 h of E. coli LPS exposure. Results are expressed as fold changes from values measured after 6-, 12-, and 24-h exposure to PBS (control). *P < 0.05 compared with control. B: primary human skeletal myoblast cells were infected with adenoviruses expressing GFP or a dominant-negative form of IKKβ and were then exposed to PBS (control) or E. coli LPS. Expression of Ang-1, Ang-2, and Ang-4 mRNAs in these cells were the measured with real-time PCR and expressed as fold changes from PBS-exposed (control) cells. *P < 0.05 compared with control cells. Note that infection with adenoviruses expressing a dominant-negative form of IKKβ had no influence on LPS-triggered changes in Ang-1 and Ang-4 levels but completely reversed LPS-induced Ang-2 expression.

 
Figure 6A illustrates the time course of mRNA expression of ESE-1 (ELF3) transcription factor in both BEAS-2B epithelial cells and skeletal myoblasts in response to PBS (control) and LPS exposure. ESE-1 expression rose substantially after 3 h of LPS exposure with a decline in the expression thereafter. After 12 h of LPS exposure, ESE-1 levels measured in response to LPS exposure in both cells types were not different from those measured in the presence of PBS. Infection of epithelial and skeletal myoblast cells with adenoviruses expressing GFP did not alter the induction of ESE-1 mRNA in response to 3-h exposure to LPS (Fig. 6B). In comparison, expression of a dominant-negative form of IKKβ completely blocked the effect of LPS exposure on ESE-1 mRNA expression (Fig. 6B). These results indicate that LPS-induced ESE-1 mRNA expression in both cell types is dependent on NF-{kappa}B activation.


Figure 6
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  		Fig. 6. A: time course of ESE-1 transcription factor mRNA expression in BEAS-2B epithelial cells and primary skeletal myoblasts after 3, 6, and 12 h of E. coli LPS exposure. Results are expressed as fold changes from values measured in cells exposed for 3, 6, and 12 h to PBS (control). *P < 0.05 compared with control. B: BEAS-2B epithelial cells and primary human skeletal myoblast cells were infected with adenoviruses expressing GFP or a dominant-negative form of IKKβ and were then exposed to PBS (control) or E. coli LPS. Expression of ESE mRNA in these cells was then measured with real-time PCR and expressed as fold changes from PBS-exposed (control) cells. *P < 0.05 compared with control cells. Note that infection with adenoviruses expressing a dominant-negative form of IKKβ completely blocked LPS-induced ESE-1 expression.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main findings of this study include: 1) in vivo LPS administration elicits differential effects on angiopoietin and Tie-2 receptor expressions. While expressions of Ang-1, Ang-3, and Tie-2 receptors decline significantly in the liver, diaphragm, and lung, Ang-2 mRNA expression in these organs is significantly induced; 2) in vitro LPS exposure elicits cell-specific changes in Ang-1 expression. Ang-1 mRNA is induced in BEAS-2B epithelial cells but is significantly attenuated in skeletal myoblasts. In comparison, LPS induces Ang-2 mRNA levels and attenuates Ang-4 expression in both cell types. While the induction of Ang-2 expression in both cell types is mediated in part through NF-{kappa}B, this transcription factor had no direct role in the influence of LPS on Ang-1 and Ang-4 expressions in either cell type; and 3) LPS elicits a significant induction of ESE-1 mRNA expression in epithelial cells and skeletal myoblasts, a response that is dependent on NF-{kappa}B activation.

In addition to their roles in the regulation of angiogenesis, there is increasing evidence that angiopoietins regulate inflammation, with Ang-1 exerting anti-inflammatory effects by inhibiting leukocyte-endothelial cell adhesion and transmigration, reducing inflammatory mediator-induced vascular leakage and cytokine production, and by attenuating EC adhesion molecule expression (7, 16, 17, 25). Moreover, overexpression of Ang-1 improves survival and homodynamic functions, reduces lung injury, and attenuates the expression of adhesion molecules in mice with LPS-induced acute lung injury (32). In contrast, upregulation of Ang-2 production by ECs exposed to proinflammatory mediators such as TNF{alpha}, thrombin, and angiotensin II suggests that Ang-2 may promote inflammation (15, 22). This suggestion is supported by findings of elevated circulating Ang-2 levels in patients with severe sepsis and in children with septic shock, and by the observation that injection of Ang-2 protein in vivo elicits a significant increase in edema formation in the mouse paw (8, 23, 26). Finally, Ang-2-deficient mice have an impaired ability to express cytokine-inducible adhesion molecules on EC surfaces after inflammatory activation, further implicating Ang-2 in promoting inflammation (6). No information is as yet available as to whether Ang-3 and Ang-4 regulate inflammation; however, the observations that both Ang-3 and Ang-4 activate Tie-2 receptors suggest that these angiopoietins may have similar anti-inflammatory effects to those of Ang-1 (19).

Despite increasing evidence of important roles for angiopoietins in regulating inflammation, little is known about how proinflammatory stimuli such as LPS influence endogenous angiopoietin production. Thus far, two studies have addressed the influence of LPS on Ang-1 expression but have resulted in contradictory conclusions. Brown et al. (5) reported a significant induction of Ang-1 mRNA expression by LPS in fibroblasts. In comparison, in vivo LPS administration into murine lungs was reported to attenuate pulmonary epithelial Ang-1 expression, a response that was associated with increased vascular leakage and infiltration of lung interstitia by inflammatory cells (12). The present study is the first to describe both the in vivo and in vitro effects of LPS on the expression of the four angiopoietins in various organs and cells. We report here that in vivo administration of LPS in mice is associated with attenuation of Ang-1 and Ang-3 expressions in at least three organs, liver, diaphragm, and lung, whereas Ang-2 expression in these organs increases significantly (Fig. 1). These changes in angiopoietin expression are associated with downregulation of Tie-2 receptor expression and reduction in Tie-2 tyrosine phosphorylation. We conclude, on the basis of these findings, that in vivo LPS administration causes functional inhibition of the angiopoietin/Tie-2 receptor pathway in at least these three organs and that this inhibition is mediated by three mechanisms, namely, reduced expression of Tie-2 receptor agonists (Ang-1 and Ang-3), induction of Ang-1 antagonist (Ang-2), and reduction in Tie-2 receptor expression. Although we did not directly assess the implications of an elevation of Ang-2 expression and downregulation of Ang-1, Ang-3, and Tie-2, we speculate that these alterations likely contribute to hemodynamic derangements and enhancements of inflammatory responses in sepsis since attenuation of Ang-1 and increased Ang-2 expression are likely to result in enhanced vascular leakage and augmented expression of proinflammatory mediators and adhesion molecules on the surface of ECs.

It should be pointed out that the decline in pulmonary Ang-1 and Ang-3 expression found in our study is qualitatively similar to that observed by Karmpaliotis et al. (12) in mice exposed to inhaled LPS. However, unlike the significant induction of lung Ang-2 expression observed in our study, these authors failed to detect changes in pulmonary Ang-2 expression in their study. We attribute these variances in pulmonary Ang-2 expression to methodological differences in terms of the routes of LPS administration. Ang-2 originates primarily from endothelial cells, and pulmonary endothelial cells are likely to be the largest contributor to total Ang-2 mRNA expression measured in lung samples. Pulmonary endothelial cells and Ang-2 promoter activity are likely to be strongly activated by circulating LPS administered systemically as in our study compared with inhaled LPS administration where LPS are likely to be deposited mainly on airway and pulmonary epithelial cells. Another issue that could explain the differences in Ang-2 regulation is that we studied Ang-2 expression during early phases of endotoxemia (6–24 h), whereas Karmpaliotis et al. studied the late phases of LPS-induced acute lung injury (24–96 h). It is possible that LPS-induced upregulation of Ang-2 expression is a transient phenomenon and might not be detected during the late phases of endotoxemia.

The results shown in Fig. 1 reveal three new aspects of in vivo regulation of angiopoietins in sepsis. First, the expression of both Ang-1 and Ang-3 mRNA in the three organs declines after 12 and 24 h of in vivo LPS administration, suggesting the existence of common mechanisms involved in regulating these two angiopoietins in response to LPS administration. The nature of these mechanisms remains to be elucidated. We should point out that the similarity in the time course of Ang-1 and Ang-3 expression in mice injected with LPS has never before been reported. In fact, opposite regulation of these changes has been observed in the lungs of hypoxic rats, where Ang-3 expression is significantly induced, whereas that of Ang-1 expression is attenuated (1). Second, the fact that LPS injection elicits a significant upregulation of Ang-2 mRNA and protein in at least three organs implies that increased circulating levels of this protein in patients with severe sepsis may originate from more than one organ (8, 23). Third, there is qualitative similarity in the responses of Ang-1 and VEGF expressions in the diaphragm to in vivo LPS administration, with the expression of both genes being induced within 6 h, followed by significant attenuation thereafter. These results suggest that previously reported upregulation of VEGF expression in septic animals is only a transient response (3) and that prolonged effects of LPS include the inhibition of VEGF expression. The similarity in the time course of Ang-1 and VEGF expression in the diaphragm during the course of endotoxemia also suggests that common pathways are involved in regulating these two proangiogenesis factors in the diaphragm.

The exact mechanisms through which in vivo LPS exposure elicits a decline in Ang-1 and Ang-3 expression and the induction of Ang-2 expression remain unclear. To investigate the involvement of the NF-{kappa}B transcription factor in these processes, we used in vitro cultured epithelial cells and skeletal myoblasts and infected these cells with adenoviruses expressing a dominant-negative form of IKKβ. This construct has been shown to inhibit LPS-induced NF-{kappa}B-mediated signaling in macrophages (27). Our results indicate that in both cell types, LPS-induced elevation of Ang-2 mRNA is completely eliminated by expression of dominant-negative IKKβ, whereas LPS-induced changes in Ang-1 and Ang-4 levels are not altered by this intervention (Figs. 4 and 5).

Molecular cloning of human Ang-2 promoter has revealed putative binding sites for the Ets family of transcription factors and also for GATA factors, c-Rel, Smad3, Smad4, AP1–1, AP-2, and Sp1 in a fragment spanning 650 bp around the transcription start site (–109 to +476) (10). This fragment was also identified to be sufficient to control endothelial cell-specific cytokine induction of Ang-2. The involvement of NF-{kappa}B transcription factor in the regulation of Ang-2 expression is not clearly understood because thus far analyses of human Ang-2 promoter have not revealed abundant putative sites for NF-{kappa}B binding. It is possible, however, that NF-{kappa}B transcription factor is involved indirectly in the activation of Ang-2 promoter through cooperative interactions with other transcription factors, particularly Ets proteins. Indeed, both NF-{kappa}B and Ets-1 (a strong inducer of Ang-2 expression) cooperate in inducing inducible nitric oxide synthase in embryonic ventricular myocytes exposed to LPS (30).

The failure of a dominant-negative form of IKKβ to inhibit LPS-induced changes in Ang-1 expression suggests that NF-{kappa}B transcription factor is not a major regulator of Ang-1 expression during in vitro LPS exposure. Brown et al. (5) have recently concluded that NF-{kappa}B may not directly bind to Ang-1 promoter but that it could induce the expression of Ang-1 indirectly through the induction of ESE-1 transcription factor, which binds directly to Ang-1 promoter in fibroblasts exposed to LPS, TNF{alpha}, and IL-1β. To evaluate the involvement of ESE-1 in LPS-induced changes in Ang-1 expression, we measured the time course of ESE-1 mRNA expression in cultured epithelial cells and skeletal myoblasts. In both cell types, LPS exposure elicited a strong, albeit transient, induction of ESE-1 mRNA expression (Fig. 6). In addition, ESE-1 induction by LPS was strongly inhibited in cells expressing a dominant-negative form of IKKβ, thereby confirming the involvement of NF-{kappa}B in the regulation of ESE-1 expression. This observation, along with the fact that expression of a dominant-negative IKKβ had no effect on LPS-induced alterations in Ang-1 expression in epithelial cells and skeletal myoblasts, suggests that, in these cell types, ESE-1 is not responsible for the regulation of Ang-1 and Ang-4 expression in response to in vitro LPS exposure. Clearly, additional studies are required to elucidate the mechanisms through which LPS regulates Ang-1 and Ang-4 (Ang-3) expression in both in vivo and in vitro conditions.

It should be noted that whereas LPS exposure triggered similar changes in Ang-1 expression in skeletal myoblasts and the diaphragms of LPS-injected animals, regulation of Ang-1 expression by LPS in cultured epithelial cells was qualitatively different from that observed in the lungs of LPS-injected mice (Figs. 1 and 4). The reasons behind these differences are not clear. One possibility is that LPS-induced Ang-1 expression by LPS in cultured epithelial cells is the outcome of primary signaling events triggered by direct activation of TLR-4 receptors, whereas secondary responses elicited by mediators such as cytokines, chemokines, growth factors, and thrombotic agents released by parenchymal cells and inflammatory cells infiltrating the lungs might have been responsible for reduction in pulmonary Ang-1 expression in mice (Fig. 1). Recent studies suggest that two cytokines whose expressions are usually induced by LPS administration may inhibit Ang-1 expression. Indeed, a combination of TNF{alpha} and interferon-{gamma} triggered a significant inhibition of Ang-1 expression in osteoblasts, and this effect was mediated by induction of the inducible nitric oxide synthase and enhanced nitric oxide production (13). It is possible that these two cytokines and the resulting increase in pulmonary nitric oxide production, which usually peaks after 12 h of LPS administration in mice (18), could have been involved in the reduction of pulmonary Ang-1 expression in mice. Additional experiments are clearly required to explore the influence of these mediators on the expression of angiopoietins in lung cells.

In summary, our results indicate that LPS administration elicits differential changes in angiopoietin expression in both in vivo and in vitro settings and that the overall response to LPS administration is a functional inhibition of the Ang-1/Tie-2 receptor pathway. We speculate, on the basis of these results, that inhibition of this pathway may have deleterious effects on vascular function in the settings of sepsis and septic shock.


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 ABSTRACT
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This study was supported by a grant from the Canadian Institutes of Health Research.


   ACKNOWLEDGMENTS
 
We are grateful to L. Franchi for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Hussain, Rm. L3.05, Critical Care Division, Royal Victoria Hospital, 687 Pine Ave West, Montréal, Québec, Canada H3A 1A1 (e-mail: sabah.hussain{at}muhc.mcgill.ca)

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.


   REFERENCES
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 ABSTRACT
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 DISCUSSION
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 REFERENCES
 

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