Effects of a synthetic PEG-ylated Tie-2 agonist peptide on endotoxemic lung injury and mortality

In Vivo Animal Studies

All experiments were approved by the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee. Eight-week-old male C57BL6 mice weighing 20–25 g purchased from Charles River Laboratories International (Wilmington, MA) were acclimated to the facility for at least a week before beginning an experiment. Eight-week-old male Tie-2 heterozygous mice and their wild-type littermates (CD-1 background, generated in Dumont Laboratory) were also studied (14).

Synthesis of VT

Peptide NH2-CHHHRHSF-COOH (Genescript, Piscataway, NJ) was reacted with four-armed, PEG (polydisperse, average molecular mass 10 kDa) (Sunbright PTE-100MA, NOF, Tokyo, Japan) in a 12:1 molar ratio to generate the illustrated product whose molecular weight is 14 kDa (Fig. 1). Covalent attachment of the peptide to the activated maleimide groups took place in PBS, pH 6.4, at room temperature with agitation for 4 h. Products were purified by way of dialysis (Slide-A-Lyzer, 7,000 Da, MWCO, Pierce Biotechnology, Rockford, IL), at 4°C with stirring. Products were first dialyzed against PBS, pH 7.4 (2 exchanges, 300× volume, 3 h each) and then double distilled water (8 exchanges, 300× volume, 6 h each). Dialyzed products were then frozen, lyophilized, and weighed to calculate product yield. Validation of VT was assessed by MALDI-TOF to determine the potential presence of free peptide impurities and to measure the molecular size distribution of the final product (efficiency of conjugation). A control version of VT was similarly prepared by conjugating 10-kDa PEG with cysteine (PEG-Cys).

Antibodies and Reagents

Antibodies used for immunoblotting and/or immunocyto- or histochemistry were purchased from the following manufacturers: Tie-2 (C-20) and actin (C-11) (Santa Cruz Biotechnology, Santa Cruz, CA), phosphotyrosine (4G10, Upstate/Millipore, Temecula, CA), vascular endothelial (VE)-cadherin (BD Bioscience, San Jose, CA). Akt and phospho-Akt (Ser 473) antibodies were purchased from Cell Signaling Technology (Danvers, MA); fluorophore- or horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Invitrogen (Carlsbad, CA), as well as phalloidin and 4′,6-diamidino-2-phenylindole (DAPI).

Cell Culture

Passage 5–6 dermal HMVECs (Lonza, Basel, Switzerland) were cultured in EBM-2 media (Lonza) supplemented with 5% fetal bovine serum (FBS) and growth factors according to the manufacturer's instructions at 37°C and 5% CO₂. Before experimental treatments, HMVECs were starved for 2 h in EBM-2 containing 1% FBS.

Western Blot Analysis

Cells were washed with ice-cold PBS twice and lysed with ice-cold RIPA buffer [50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA] supplemented with protease inhibitors (Roche Diagnostics, Indianapolis, IN), 1 mM NaVO₄, and 1 mM NaF. Lysates were sonicated and centrifuged at 10,000 rpm for 10 min at 4°C, and supernatants were collected. Protein concentrations were determined by Bradford protein assay with bovine serum albumin as a standard (Bio-Rad). A mixture of lysate, NuPAGE reducing agent, and NuPAGE sample buffer were heated at 70°C for 10 min, electrophoresed in NuPAGE 4–12% Novex Bis-Tris Gels (all from Invitrogen), transferred to nitrocellulose membrane, and immunoblotted with specific primary antibodies. Binding of primary antibodies was detected by using HRP-conjugated secondary antibodies (Amersham Pharmacia Biotech) and SuperSignal WestDura (Pierce) reagents as chemiluminescence substrates.

Immunoprecipitation

For measurement of phospho-Tie-2, murine lungs were homogenized in the above lysis buffer, and 1.5 mg of total protein was incubated with anti-Tie-2 antibody for 12 h at 4°C and then precipitated with magnetic Dynabeads conjugated with protein G (Invitrogen) for 45 min at 4°C. After the beads were washed, proteins were eluted by heating in sample buffer and detected by Western blot analysis with anti-phospho-tyrosine (4G10) as described above.

RNA Extraction and Quantitative PCR

Total RNA was extracted by using Trizol (Invitrogen, Carlsbad, CA) followed by RNeasy Mini Kit (Qiagen, Valencia, CA) with on-column DNAse-I treatment per the manufacturers' instructions. Quantification and purity of total RNA was assessed by A₂₆₀/A₂₈₀ absorption. Total RNA (2 μg) was then reverse transcribed by using the Omniscript Reverse Transcriptase kit (Qiagen). Quantitative PCR was performed by the Multi-Gene Transcriptional Profiling core (MGTP, http://cvbr.hms.harvard.edu) at Beth Israel Deaconess Medical Center by using the ABI 7000 Sequence Detection System (Applied Biosystems). Results were analyzed by the comparative threshold cycle method. Transcript abundances were normalized per 10⁶ 18S rRNA copies.

Immunohistochemistry

Air-dried PLP-fixed cryosections from lungs were blocked for 60 min in 10% donkey serum, incubated with primary antibodies overnight at 4°C, followed by fluorescence-conjugated affinity-purified secondary antibody labeling (60 min, 1:500, Jackson), and mounted with ProLong Gold/DAPI. All images were taken by a Zeiss LSM510 META confocal system at ×63 magnification. Of note, all images were obtained with the same laser power, gain, and offset conditions.

Immunocytochemistry

HMVECs were grown to confluence on glass coverslips coated with collagen type I. After starvation the cells were first treated with VT (100 ng/ml), equimolar PEG-Cys (70 ng/ml), or PBS 90 min prior to challenge with the following permeability mediators: LPS (100 ng/ml) plus LPS-binding protein (100 ng/ml) plus CD14 (10 ng/ml); thrombin (1 U/ml); and human septic serum (5%). A second dose of VT (or PEG-Cys) was given when permeability mediators were added. For the septic serum treatment, EBM-2 media was supplemented with 5% prefiltered human serum from a patient with septic shock collected for a prior published study (26).

Thirty minutes after mediator treatment, cells were fixed for 10 min in 2.5% paraformaldehyde and permeabilized for 5 min in 0.2% Triton X-100 in PBS. Cells were blocked overnight at 4°C with a blocking buffer (1% BSA, Triton, sodium azide), then incubated for 12 h with primary antibody, serial washes in PBS, then 60 min incubation with secondary Alexa-antibody and phalloidin. The coverslips were mounted by using ProLong Gold/DAPI. All images were taken by a Zeiss LSM510 META confocal system at ×63. Of note, all images were obtained with the same laser power, gain, and offset conditions.

TER

HMVECs were grown to confluence in polycarbonate wells containing evaporated gold microelectrodes in series with a large gold counter connected to a phase-sensitive lock-in amplifier as described previously (10, 11, 32). VT (100 ng/ml), equimolar PEG-Cys (70 ng/ml), or PBS were added to wells 90 min prior to LPS and again at the time that LPS was added. Measurements of transendothelial electrical resistance (TER) were performed by using an electrical cell-substrate impedance sensing system (ECIS; Applied BioPhysics, Troy, New York). Briefly, current was applied across the electrodes by a 4,000-Hz AC voltage source with amplitude of 1 V in series with a 1 MΩ resistance to approximate a constant current source (∼1 μA). The in-phase and out-of-phase voltages between the electrodes were monitored in real time with the lock-in amplifier and subsequently converted to scalar measurements of transendothelial impedance, of which resistance was the primary focus.

These measurements provide a highly sensitive biophysical assay that indicates the state of cell shape and focal adhesion. Values from each microelectrode were pooled at discrete time points and either plotted vs. time as means ± SD or reported as bar graphs at the time point of maximal response to a given stimulus as described elsewhere in detail (10).

Transwell Permeability Assay

Confluent HMVEC monolayers were grown on type I collagen-coated Costar Transwell membranes (polyester 0.4-μm filter, Corning, Corning, NY) and permeability was determined by measurement of fluorometric signal in the luminal and abluminal chambers at the indicated time point after luminal addition of 1 mg/ml FITC-labeled human serum albumin (Sigma) as described previously (22). Relative fluorescence units were used in the following equation to determine the permeability coefficient of albumin (P_a):

where [A] is abluminal concentration, [L] is luminal concentration, V is volume of abluminal chamber, t is time in hours, and A is area of membrane in square centimeters.

Statistical Analysis

Results are presented as means ± SE. Statistical significance was evaluated by unpaired two sided t-test unless otherwise stated. Survival data were analyzed by log-rank test and visualized by Kaplan-Meier curves. Analysis was performed in SPSS (SPSS, Chicago, IL) and graphs were made in GraphPad Prism (GraphPad Prism Software, San Diego, CA).

VT Prevents Endothelial Structural Rearrangements Induced by Sepsis Mediators

Given the important roles of cytoskeletal changes and VE-cadherin localization in endothelial barrier defense (5, 6), we studied these parameters on HMVECs treated with LPS in the presence of PBS, PEG-Cys, or VT. Only VT prevented the formation of F-actin stress fibers, enhanced the junctional staining of VE-cadherin, and prevented the formation of interendothelial gaps associated with LPS (Fig. 3). We also found that VT prevented morphological changes in HMVECs induced by thrombin or 5% human septic serum (Supplemental Fig. S1; the online version of this article contains supplemental data).

VT Ameliorates Endotoxin-Induced Endothelial Barrier Dysfunction

Having observed structural evidence of VT-mediated endothelial barrier defense, we next evaluated its performance in functional assays of the same. We first used TER to characterize the dose- and time-dependent reduction of monolayer electrical resistance induced by LPS and to identify an optimal dose of VT (Supplemental Fig. S2). From these data, we determined a time window in which to observe the maximum effect of LPS (Fig. 4A); 100 ng/ml VT prevented LPS-induced endothelial barrier dysfunction whereas an equimolar concentration of PEG-Cys had no effect (Fig. 4B). We then used a Transwell experiment to confirm our TER findings. Whereas TER reports instantaneous resistance at frequent time intervals, the Transwell assay provides a cumulative measure of monolayer leakiness to macromolecules (FITC-labeled albumin in this assay). We again found that VT prevented the barrier dysfunction induced by LPS (LPS + saline vs. LPS + VT, P = 0.001; Fig. 4C).

VT Reduces Lung Vascular Leakage Following Endotoxin Exposure

After initial dose-ranging studies, 8-wk-old male C57BL6 mice were administered 500 ng VT (or equal volume of sterile saline) intraperitoneally 7 h prior to 15 mg/kg LPS. Mice were euthanized 16 h after LPS administration to perform several measurements. Having already observed strong evidence of endothelial barrier defense against LPS in vitro, we first surveyed vascular leakage in the viscera of these animals by the Evans blue assay (Fig. 5A). Although most organs showed a trend toward increased leakage with LPS, the lungs were affected most prominently, showing more than fourfold increase in dye extravasation (n = 4 control mice, n = 6 LPS-treated mice, P = 0.03). Notably, the lungs were also the most protected by VT (n = 6 LPS + VT treated mice, P = 0.04 vs. LPS alone).

On the basis of these findings, we further evaluated the lungs of these mice for structural evidence of barrier dysfunction. As seen in vitro (Fig. 3A), VE-cadherin staining in lung endothelium (confirmed by CD31 costaining) was markedly attenuated by LPS exposure and restored to near-normal by VT pretreatment (Fig. 5B).

Finally, to discern the contribution of local inflammation to the permeability increase induced by LPS, we measured an array of inflammatory markers by quantitative PCR (Fig. 5C). As expected, LPS induced all of the markers in a highly significant fashion. However, VT pretreatment did not diminish the expression of any of these markers. These data suggest that the VT-mediated reversal of LPS-induced pulmonary vascular hyperpermeability proceeds independently of changes in the local inflammatory milieu.

VT Augments Tie-2 Signaling In Vivo

To explore the signaling changes induced by LPS and VT in the lungs, we began by performing coimmunofluorescence for Tie-2 and phosphotyrosine (pY) on lung tissue obtained 16 h after LPS challenge. Illustrative images of n = 5 mice per group are shown in Fig. 6A. Control tissue (left column) demonstrated colocalization of Tie-2 (red) and pY (green) staining, resulting in abundant yellow signal (left column, inset of merged image). LPS exposure (middle column) resulted in considerable attenuation of both Tie-2 and pY. The merged image showed distinct patches of red signal, indicative that the two stains do not colocalize and suggesting that Tie-2 is dephosphorylated. VT pretreatment (left column) enhanced pY staining to near-control intensity and reduced the patches of red signal in the merged image.

We next quantified these observations by immunoprecipitation studies on lung lysates obtained from these mice. The phosphorylated fraction of Tie-2 was suppressed by systemic exposure to LPS (P = 0.03 vs. control) and fully restored by VT (P = 0.007 vs. LPS alone; Fig. 6B). LPS reduced Tie-2 protein abundance by 62.6% (P < 0.0001 vs. control; Fig. 6C). This was partially prevented by VT pretreatment (P = 0.002 vs. LPS alone).

To evaluate the LPS-induced suppression of Tie-2 expression further, we performed quantitative PCR on lung homogenates from animals treated with LPS with or without VT (Fig. 6D). Endotoxemia strongly suppressed Tie-2 transcript abundance (P < 0.0001 vs. control), and VT pretreatment did not prevent this drop (P < 0.0001 vs. control and nonsignificant vs. LPS).

To resolve the impact of the changes in Tie-2 activation and protein abundance on downstream signaling, we measured the activation of Akt in lung lysates (Fig. 6E). The total level of Akt was unchanged across all three conditions. However, Akt was deactivated during endotoxemia and restored to a normal level by VT administration (LPS + saline vs. LPS + VT, P = 0.02). Together, these results suggest that the decreased Tie-2 protein abundance induced by LPS is overcome by increased activation of the remaining Tie-2 molecules when VT is given, thereby preserving net flux through this signaling pathway.

VT May Ameliorate Endotoxemic Cardiac Dysfunction

Two prior publications have suggested a cardiotropic role of exogenously administered Angpt-1 in rodents, one in the endotoxemia model (4, 37). We therefore performed serial echocardiography in mice before LPS and 16 h after LPS. Eight mice were pretreated with VT and nine with an equal volume of saline. In the group that only received LPS, and not VT, virtually all measured cardiac parameters were suppressed (Supplemental Fig. S3). Comparing the two post-LPS groups (without VT = 9, with VT = 8), we found that measures of cardiac contraction, namely ejection fraction (EF) and stroke volume (SV), were improved in the mice receiving VT compared with the LPS-only group in a statistically significant fashion (Fig. 7, P = 0.02 and 0.04, respectively).

As opposed to the catheter-based technique in the prior report that evaluated Angpt-1 in endotoxemia (4, 37), the present method of assessing cardiac performance enabled repeated measurements in a single mouse. We therefore analyzed the data in a different fashion (Table 1). Within each treatment arm, we calculated a “delta” (Δ) for each parameter (e.g., ΔEF = EF_pre-LPS − EF_post-LPS). When we compared ΔEF or ΔSV between VT and non-VT treated groups, we found trends that suggested a benefit of VT, but nothing that reached statistical significance. Overall, these data suggest that VT could improve cardiac contractility during endotoxemia, but analysis of the change in performance attributable to VT itself yielded encouraging trends that did not meet statistical significance.

VT Improves Survival of Endotoxemic Mice

Lastly, we asked whether the specific in vivo benefits of VT could be associated with increased survival. Of mice pretreated with VT followed by 15 mg/kg LPS (n = 15), 71.4% survived indefinitely whereas only 30.0% of PBS pretreated mice lived (n = 30; P = 0.02; Fig. 8A). To confirm the specificity of VT's action in vivo, we repeated this experiment in Tie-2 heterozygous mice and found that VT-mediated protection from LPS lethality was abolished (Fig. 8B). In a second set of experiments, mice were first treated with 15 mg/kg LPS, after which they received two injections of VT (500 ng 2 h post-LPS followed by 250 ng 14 h post-LPS) or equivalent volume of sterile PBS (Fig. 8C). Of the VT-treated mice 85.0% lived whereas only 55.0% of sham-treated mice survived (n = 20 per group, P = 0.051). Finally, we used high-titer recombinant adenoviruses to confirm the results of Witzenbichler et al. (37), namely that excess human Angpt-1 could also prevent LPS-induced lethality (Supplemental Fig. S4).