Wnt/Β-Catenin Signaling Is Modulated by Mechanical Ventilation in an Experimental Model

ESM-Wnt/β-catenin-Villar et al-ICM 2010-00828.R2

Wnt/β-catenin signaling is modulated by mechanical ventilation in an experimental model of acute lung injury

- Electronic Supplementary Material -

1,2,3 Jesús Villar*, MD, PhD, FCCM 1,2 Nuria E. Cabrera*, PhD 1,2 Milena Casula, PhD 1,4 Francisco Valladares, PhD 1,5 Carlos Flores, PhD 1,6 Josefina López-Aguilar, PhD 1,6 Lluis Blanch, MD, PhD 3 Haibo Zhang, MD, PhD 7 Robert M. Kacmarek, RRT, PhD 3,8,9 Arthur S. Slutsky, MD

From (1) CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid, Spain; (2) Multidisciplinary Organ Dysfunction Evaluation Research Network, Research Unit, Hospital Universitario Dr. Negrin, Las Palmas de Gran Canaria, Spain; (3) Keenan Research Center at the Li Ka Shing Knowledge Institute of St. Michael´s Hospital, Toronto, Canada; (4) Department of Anatomy, Pathology & Histology, University of La Laguna, Tenerife, Spain; (5) Research Unit, Hospital Universitario N.S. de Candelaria, Tenerife, Spain; (6) Critical Care Center, Corporació Sanitaria Parc Taulí, Sabadell, Barcelona, Spain; (7) Department of Respiratory Care, Massachusetts General Hospital, Boston, USA. (8) Interdepartmental Division of Critical Care Medicine, University of Toronto, Toronto, Canada; (9) Adjunct Professor, King Saud University, Riyadh, Saudi Arabia.

* JV and NEC have equally contributed to this work. ESM-Wnt/β-catenin-Villar et al-ICM 2010-00828.R2

METHODS

Experimental Model

The cecal ligation and puncture (CLP) model is a well established approach for ALI induction.

Sepsis was produced using a modification of the cecal ligation and perforation (CLP) technique described by Chaudry et al (1) and Bohnen et al (2). With the animals breathing spontaneously, we performed a laparotomoy through a midline abdominal incision using an aseptic technique. Then, we ligated the cecum just below the ileocecal valve with 3-0 silk ligature, so that intestinal continuity was maintained. Using an 18-gauge needle, the cecum was perforated in two locations, 1 cm apart, on the antimesenteric surface of the cecum, and the cecum was gently compressed until feces were extruded. The bowel was then returned to the abdomen and the incision was closed with a layer of prolene sutures for the muscles and 2-0 silk for the skin. We performed all surgical manipulations with the animals lying supine on a restraining board inclined 20º from the horizontal. Afterward, we observed the animals in a recovery cage for 2 h. We did not administer antibiotics to any of the animals.

In the three groups of septic animals, the peritoneal cavity was reopened in all surviving animals 18 hrs after CLP with the animal anesthetized as described above. All animals that were re- explored had a considerable amount of ascetic fluid when the peritoneal cavity was reopened 18 h after CLP. The cecum was then excised distal to the ligature and removed. We washed the peritoneal cavity with 20 ml of warm, normal saline, and gently squeezed several times. After closing the abdomen, each animal received 10 ml normal saline subcutaneously for fluid resuscitation. All animals had free access to food and water after the surgical procedures. Control animals were sham operated

(anesthesia, laparotomy, and manipulation of cecum without being ligated and perforated).

The CLP model results in peritonitis with systemic manifestations (3). It is the single best animal model of sepsis and organ injury. In general, animals become progressively leukopenic (4) with relatively preserved blood pressure (5). Blood cultures are usually positive for multiple organism, the most common being enteric gram negative rods. CLP results systemic inflammation and high levels of circulating cytokines, and in mild lung injury similar to ALI/ARDS. The lungs show inflammatory cell influx and loss of the alveolar-capillary barrier, increased permeability, and neutrophil accumulation in the interstitium and the alveolar spaces within 18 to 72 hours. Mortality is high, ranging from 25-30% at

18 hours to 70 to 90% in rats within 30 hours of the operation. The necrotic/ischemic cecum is ESM-Wnt/β-catenin-Villar et al-ICM 2010-00828.R2 removed at 18 h as part of the surgical treatment of the septic process, in order to prevent additional deaths as a result of a severe peritonitis and sepsis-induced multiple organ failure during the 4-h period of MV. In a previous study by our group (6) using the same CLP model and comparing the effects of the same two ventilatory strategies on the inflammatory responses, we found a marked increased of local expression of cytokines in the lungs and increased cytokine systemic levels in animals ventilated with high VT, as reported also by Haitsma et al (7). On the other hand, the use of a protective ventilation (VT=6 ml/kg plus PEEP=10 cmH2O) was accompanied by a marked attenuation of the sepsis-induced pulmonary and systemic inflammatory response.

Histological examination

At the end of the 4-hour observation and ventilation period, a midline thoracotomy/laparotomy was performed in the first 6 surviving rats in each experimental group. In mechanically ventilated animals, blood samples for blood gases were obtained by cardiac puncture from the left ventricle of a beating heart. The abdominal vessels were transected and the hearts and lungs were removed en bloc from the thorax. The lungs were isolated, the trachea was cannulated and the right lung was fixed by intratracheal instillation of 3 ml of 10% neutral buffered formalin. After fixation, the lungs were floated in 10% formalin for a week. Lungs were sampled in multiple areas, serially sliced from apex to base and specimens were embedded in paraffin, then cut (3 mm thickness), stained with hematoxylin- eosin and with the Masson-Goldner trichrome technique. Slides were viewed using a Nikon Optiphot light microscope (Tokyo, Japan) and photographed with a Nikon Digital DS-5M camera (Tokyo, Japan) at x200 magnification. A semiquantitative morphometric analysis of lung injury was performed by a pathologist (FV) blinded to the experimental history of the lungs examined. Three random sections of the right lung from each animal were examined with particular reference to alveolar and interstitial damage defined as cellular inflammatory infiltrates, pulmonary edema, atelectasis, alveolar overdistension, alveolar rupture, hemorrhage, and fibrosis. By scoring from 0 to 4 (none, light, moderate, severe, very severe) for each of these parameters, a total histological injury score was obtained by adding the individual scores in every animal and averaging the total values in each group.

Western blot analysis

Left lungs were excised, washed with saline, frozen in liquid nitrogen, and stored at -80ºC for subsequent protein extraction. Lungs were homogenized in 1 ml per lung of ice-cold Nonidet P-40 lysis buffer containing 1% Nonidet P-40, 25 mM Tris-HCl (pH 7.5), 150 mM sodium chloride, 1 mM ESM-Wnt/β-catenin-Villar et al-ICM 2010-00828.R2

EDTA, 5 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride plus

Protease Inhibitor Cocktail (Roche Molecular Biochemicals). Proteins were extracted by centrifugation

(14.000 rpm) for 5 min at 4ºC and protein concentrations in each experimental condition were determined by the Bio-Rad DC Protein Assay. Samples containing 30 μg of protein were separated by electrophoresis on a 10% SDS-PAGE gel. The separated proteins were transferred to PVDF membranes and blocked with 10% skim milk in Tris-buffered saline plus 0.1% Tween 20 (TBS-T).

After incubation with WNT5A (Abcam, Cambridge, UK), total β-Catenin (Santa Cruz Biotechnology,

Santa Cruz, CA) and MMP7 (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies, respectively, blots were incubated with secondary antibody linked to HRP (Goat Anti-rabbit IgG-HRP;

Santa Cruz Biotechnology). Proteins were then visualized by enhanced chemiluminescence detection

(Amersham ECL Western Blotting Detection Reagents, GE Healthcare). Prior to reprobing, PVDF membranes were stripped using Restore Western Blot Stripping Buffer. As loading controls, membranes were reprobed with β-actin primary antibody (Cell Signaling Technology). Densitometry in each experimental condition was performed using the Scion Image software package. Western blots were repeated three times.

Immunohistochemistry for total β-catenin, WNT5A and MMP7

Immunohistochemical stains for total β-catenin, WNT5A and MMP7 were performed by applying a standard avidin-biotin complex (ABC) technique. Paraffin-embedded rat lung sections (3

μm thick) were deparaffinized and rehydrated; slides were placed in citrate buffer solution (0.01 M, pH

6.0). After blocking endogenous peroxidase activity (10 min in 0.3% hydrogen peroxide), sections were incubated for 1 hour with diluted rabbit polyclonal primary antibodies directed against WNT5A

(dilution 1:150, Abcam, Cambridge, UK), β-catenin (dilution 1:400, Santa Cruz Biotechnology, Santa

Cruz, CA) and MMP7 (dilution 1:100, Santa Cruz Biotechnology, Santa Cruz, CA), respectively, then washed in PBS and incubated for 10 min with a biotinylated secondary antibody (Santa Cruz

Biotechnology, Santa Cruz, CA). Following another washing cycle, slides were incubated for 13 min with streptavidin-horseradish peroxidase (HRP) (Zymed, San Francisco, CA). Staining was visualized using the 3-amino-9-ethylcarbazole AEC+/substrate Chromogen (Dako, Hamburg, Germany). Finally, sections were rinsed in distilled water, counterstained with Mayer's hematoxylin (Dako, Hamburg,

Germany), and mounted with mounting media. Slides were viewed using an Olympus (BX50) microscope and photographed with an Olympus Camedia digital camera at x400 magnification. ESM-Wnt/β-catenin-Villar et al-ICM 2010-00828.R2

RESULTS

Cyclin D1 and VEGF protein levels

The pulmonary cyclin D1 protein levels increased in septic unventilated rats (p<0.01) compared to the non-septic animals. The highest cyclin D1 protein levels were found in septic rats ventilated with high VT for 4 h (p<0.001), when compared to septic animals ventilated with low VT, while the protective ventilation (low VT+PEEP) reduced cyclin D1 protein levels (p<0.01).

Furthermore, total lung VEFG protein levels were also significantly increased in septic rats

(p<0.001) when compared to non-septic control animals. The highest VEGF protein levels were found in septic rats ventilated with high VT (p<0.001) when compared to septic animals ventilated for 4 h with low VT+PEEP (p<0.001).

Our data on cyclin D1 and VEGF are in agreement with previous studies. Using epithelial cells from patients with idiopathic pulmonary fibrosis, Königshoff et al (8) showed that Western blot analysis of Wnt target genes cyclin D1, Mmp7 or fibronectin 1 demonstrated increased functional Wnt/β-catenin signaling in pulmonary fibrosis compared with control patients. Similarly, Stockmann et al (9) reported that detection of VEGF protein by Western blot in whole lung lysates revealed markedly elevated

VEGF levels after intraperitoneal bleomycin treatment in mice. ESM-Wnt/β-catenin-Villar et al-ICM 2010-00828.R2

REFERENCES

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RM (2010) Mechanical ventilation modulates Toll-like receptor signaling pathway in a sepsis-

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Functional Wnt signaling is increased in idiopathic pulmonary fibrosis. PLoS ONE 3:e2142

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Collado AX, Iruela-Arispe L, Nizet V, Johnson RS (2010) Loss of myeloid cell-derived vascular

endothelial growth factor accelerates fibrosis. Proc Natl Acad Sci USA 107:4329-4334 ESM-Wnt/β-catenin-Villar et al-ICM 2010-00828.R2

FIGURE S1. Western blotting showing the effects of mechanical ventilation on pulmonary cyclin D1 and vascular endothelial growth factor (VEGF) protein levels in several groups of rats: healthy unventilated (N) , septic unventilated (S), septic ventilated with low tidal volume (VT) plus PEEP and septic ventilated with high VT. Bars represent densitometry values (mean±standard deviation) of six rats per group calculated as a ratio between protein levels and β-actin and expressed as fold changes in relation to healthy, spontaneous breathing rats. PEEP= positive end-expiratory pressure. Cyclin D1:

** p<0.01 vs. healthy rats; † p<0.01 vs. septic unventilated rats; *** p<0.001 vs. septic animals ventilated with low VT. VEGF: ***p<0.001 vs. healthy rats and vs. septic animals ventilated with low

VT; * p<0.05 vs. septic unventilated animals; # p<0.001 vs. septic animals ventilated with low VT.

ls/ ve

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