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Host's Endogenous Caveolin-1 Expression is Downregulated in the Lung During Sepsis to Promote Cytoprotection

Kataki, Agapi*; Karagiannidis, Ioannis*; Memos, Nikolaos*; Koniaris, Efthymios; Antonakis, Pantelis*; Papalois, Apostolos; Zografos, George C.§; Konstadoulakis, Manoussos M.§

doi: 10.1097/SHK.0000000000001005
Basic Science Aspects
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ABSTRACT The present study focuses on the profile of “endogeneous” caveolin-1 protein in septic lung (CLP model).

Caveolin-1, CD25, pP38, pAkt, and 14-3-3b protein expression profiles were studied using flow cytometry and immunohistochemistry 6, 12, 24, 36, and 48 h after sepsis induction. Cell viability was determined by 7-AAD staining and fibrosis by Masson trichrome stain. The effect of protein C zymogen concentrate (PC) on caveolin-1 expression was also investigated given that PC, once dissociated from caveolin-1, elicits a PAR-1-mediated protective signaling by forming a complex with endothelial protein C receptor (EPCR).

CLP treatment increased lung inflammation and cell apoptosis. Fibrosis was apparent in vessels and alveoli. Caveolin-1+ cells presented reduced protein expression, especially 12 h post-CLP (P = 0.002). Immunohistochemistry revealed caveolin-1 positive expression mainly in regions with strong inflammatory reaction. Early induction of pP38+ cell population (P = 0.014) and gradual increase of CD25+ cells were also observed. Alternations in 14-3-3b expression related to apoptosis were apparent and accompanied by increased AKT phosphorylation activity late during sepsis progression.

After PC administration, cell apoptosis was reduced (P = 0.004) and both the percentile and expression intensity of caveolin-1 positive cells were compromised (P = 0.009 and P = 0.027, respectively). 14-3-3b, CD25, and pP38 protein expression were decreased (P = 0.014, P = 0.004, and P = 0.007, respectively), whereas pAkt expression was induced (P = 0.032).

The observed decline of endogenous caveolin-1 protein expression during sepsis implies its involvement in host's cytoprotective reaction either directly, by controlling caveolae population to decrease bacterial burden, or indirectly via regulating 14-3-3b-dependent apoptosis and EPCR-PAR-1-dependent protective signaling.

*Laboratory of Surgical Research, 1st Department of Propaedeutic Surgery, University of Athens, Hippocration Hospital, Athens, Greece

Department of Pathology, Henry Dunant Hospital Center, Athens, Greece

Experimental Research Center, ELPEN Pharmaceuticals, Pikermi, Greece

§1st Department of Propaedeutic Surgery, University of Athens, Hippocration Hospital, Athens, Greece

Address reprint requests to Agapi Kataki, MSc, PhD, Laboratory of Experimental Surgery, 1st Department of Propaedeutic Surgery, University of Athens, Hippocration Hospital, 114 Queen's Sofia Avenue, 11527 Athens, Greece. E-mail: akataki@med.uoa.gr

Received 10 May, 2017

Revised 25 May, 2017

Accepted 20 September, 2017

This work was financially supported by the Special Account for Research Grants 70/3/8316 of the National and Kapodistrian University of Athens.

The authors report no conflicts of interest.

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INTRODUCTION

Sepsis is a clinical entity currently defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection (1). Therefore, better understanding of host–pathogen interaction, host's immunity, coagulation cascade, and cell signaling during sepsis could identify cytoprotective mechanisms contributing toward the reduction of sepsis mortality.

Currently, the role of caveolin-1, the main constituent of caveolae membrane microdomains, is being investigated in sepsis, knowing that caveolar vesicles are implicated in transendothelial immune cell trafficking, vascular barrier homeostasis, and inflammatory signaling (2). Research data showed that various bacterial pathogens, such as those involved in sepsis, can gain access into the eukaryotic cells by binding to caveolae and initiating endocytosis, avoiding lysosomal degradation (3). In addition, the “caveolae signaling hypothesis” underlined the role of caveolae as lipid-based signaling platforms that facilitates intracellular trafficking and regulation of signaling pathways leading to antimicrobial responses (3, 4). In the lung, caveolin-1 was shown to reduce pulmonary fibrosis and to act beneficially in the fibrotic phase of lung injury (5, 6).

Many studies have suggested a cytoprotective role for caveolin-1 during sepsis. Using the cecal ligation and puncture (CLP) model Feng et al. (7) reported that caveolin-1 knock out mice were more susceptible to polymicrobial septic death than their wild littermates presenting exaggerated inflammatory cytokine generation, altering lymphocyte homeostasis and elevating bacterial burdens in liver and spleen. Furthermore, caveolin-1(−/−) mice challenged with Salmonella enterica serovar Typhimurium, P. aeruginosa or Pseudomonas aeruginosa had increased mortality rates (8–10). In contrast, LPS-challenged caveolin-1 knock out animals showed decreased mortality compared with wild ones (11–12).

The present study uses the CLP procedure for inducing sepsis in nongenetically altered rats and is designed to monitor the expression of host's endogeneous caveolin-1 protein expression in the lung during sepsis progression and in relation to the expression profiles of various proteins regulating inflammation, cell signaling, and apoptosis.

Furthermore, based on the aforementioned evidence, supporting the protective role of caveolin-1 in the inflammatory response, the present study was extended to investigate caveolin-1 expression profiles in response to protein C, a known anti-inflammatory and cytoprotective substance. Previous studies have shown that activated protein C (APC) treatment could increase survival by influencing host's defense during experimental pneumococcal pneumonia (13, 14). Research data showed that both PC and its activated form APC exert cytoprotective effects by supporting endothelial barrier function, decreasing plasma inflammatory cytokines levels and reducing apoptosis (15, 16).

In our study, 7AAD cell staining was used to assess cell viability, whereas the apoptotic process was also studied indirectly by monitoring the expression of 14-3-3b protein, a member of the 14-3-3 protein family purposed to bind and sequester phosphorylated Bad or Bax in the cytosol and thereby reducing Bad/Bax-induced apoptosis (17). Lung inflammation was evaluated by changes in the immunopositivity profile of S100 proteins as they are related to proinflammatory mechanisms and are significantly overexpressed in monocytes and macrophages (18). Host's attempts to reduce bacterial burden in cells during sepsis was estimated by monitoring CD25+ cell population in septic lung for it is known to recognize a large variety of cells, including Treg cells, activated T cells, B cells, and macrophages (19). Finally, the importance of caveolae in interacting with and organizing signaling molecules was evaluated by monitoring the activation of important signaling pathways as P38 and Akt kinases (20). The P38MAPK pathway seems to be important for caveolin-1 antiflammatory role as the use of P38 inhibitor suppresses the anti-inflammatory effect of caveolin-1 on LPS-induced cytokine production in an in vitro system (21).

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MATERIALS AND METHODS

Sepsis induction

Sepsis animal group: Sepsis was induced by the procedure of CLP in pathogen-free Wistar rats (average weight 250 g). Anesthesia was induced by ketamine (60 mg/kg body weight) and xylazine (3 mg/kg body weight) intramuscularly. Briefly, the cecum was ligated just distal to the ilieocecal valve, punctured twice with an 18-gauge needle, and compressed gently to extrude cecal contents before returning back to the abdomen. The abdominal incision was then closed in layers and the animals received 2 mL/100 g body weight saline solution subcutaneously to maintain normal circulation and to assure the presence of a hyperdynamic phase circulatory state. Animals were returned to their cages and monitored for signs of distress. Timing of the CLP procedure was in between 10 and 15 min. No antibiotics were administrated. Blood cultures were positive for enteric organisms, including Eschericia Coli, Enterococcus casseliflavus, and Proteous species. Based on previous experience, 60-h survival rate was anticipated at 54% in the CLP-group (15). Five animals were euthanized under deep anesthesia at time points of 6, 12, 24, 36, and 48 h after sepsis induction. Only survivors at the specific time points were included in the study. Three sham-operated animals, without ligation nor puncture receiving fluid resuscitation, served as the control group.

CLP-PC animal group: 100 IU/kg of human protein C concentrate (PC, Ceprotin, Baxter, Hellas) was administered intravenously via the penile vein in each animal and sepsis was induced by CLP after 1 h. Two additional doses of 100 IU/kg PC were likewise administered at 6 and 12 h after sepsis induction. Animals were previously sedated using the volatile anesthetic sevoflurane (Sevorane, Abbott Laboratories, Hellas). Treated animals were euthanized 24, 36, and 48 h after sepsis induction. The 60-h survival rate was anticipated 75% in the CLP-PC group (15).

The experimental protocol was reviewed by Animal Welfare Committee of Athens University Medical School and approved by the Prefecture of Athens (Directorate of Veterinary Services, K3282003) according to Greek legislation and the EU directive on the protection of animals used for scientific purposes and all the procedures were carried out according to the National Institute of Health Guidelines on Laboratory Animals.

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Tissue collection

Immediately postmortem, both lungs were excised: one was snap-frozen and stored at −80°C, whereas the other was fixed in 10% formalin buffer and embedded in a paraffin block. Blood samples were collected by exsanguination via heart puncture after animal anesthetization.

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Flow cytometry

Tissue preparation

Four to five pieces of frozen lung tissue sections (approximately 2.5 mm3 in size) were homogenized using the Medimachine system (BD Biosciences, San Jose, Calif). Resulting cell suspensions were filtered (Filcon, BD Biosciences), washed twice with 5 mL 1× PBS and counted with a hemocytometer. Samples of approximately 10 × 106 cells/mL were processed for flow cytometry analysis.

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Cell staining and analysis

Ten microliters of 7AAD (BD Biosciences, Cat # 559925) were added to aliquots of alveolar cell suspension and steps of incubation (10 min in the dark), wash, and fixation (6% formaldehyde for 10 min at room temperature) followed. 7-AAD dye exclusion was used during flow cytometry analysis to monitor the effects of experimental polymicrobial sepsis on cell viability. Cells were divided to viable, apoptotic, and necrotic cells based on the integrity of their membrane to 7-AAD (−), dim and (+), respectively. Pelleted cells were washed using 1 × PBS, resuspended in 500 μL ice-cold methanol, and incubated for 30 min at −20°C. Fixed/permeabilized cells were washed with 1× PBS, before staining with the following primary antibodies: Caveolin 1-FITC (Santa Cruz Biotechnology Inc sc-70516-FITC, dilution 1:100), Phospho-P38 MAPK Alexa Fluor 488 Conjugate (Cell Signaling Technology #4551, dilution 1:50), Phospho-Akt Alexa Fluor 488 Conjugate (Cell Signaling Technology #2336, dilution 1:50), 14-3-3β-PE (Santa Cruz Biotechnology, Inc., SC-1657PE, dilution 1:100) for 45 min at room temperature. Cells were then washed with 1× PBS and analyzed. For anti-CD25 and pP38 double staining, aliquots were incubated with 10 μL CD25-PE (BD Biosciences 554866) for 20 min at 4°C before performing the fixation-permeabilization steps.

Negative controls were included in all staining procedures. Fluorescence intensity measurements were performed on a COULTER EPICS XL-MCL. Fluorochromes were excited with a single 488-nm Argon laser. FITC fluorescence was collected through a 530 ± 15-nm band pass filter while RPE fluorescence was detected via a 575-nm band pass filter. A broad gate was established for data collection using a Cartesian plot of FSC (forward scatter) versus SSC (side scatter) to exclude cellular fragments and debris from the cell population. For each sample, at least 10,000 events were counted.

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Immunohistochemical detection

Paraffin sections (5 μm thick) were deparaffinized in xylol, rehydrated in a graded ethanol series, and subjected to microwave antigen retrieval (3 × 10 min, 800 W, in 0.1 M citrate acid buffer solution, pH 6). Endogenous peroxidase activity was blocked using 0.1% hydrogen peroxide. Sections were incubated overnight at 4°C with the following primary antibodies: Caveolin 1 (Cell Signalling 3267, 1:200), phospho Akt (Cell Signaling Technology #4060 dilution 1:50), phospho P38 (Cell Signaling Technology #4511, dilution 1:50), and S100 (DAKO Z0311, dilution 1:100). Immunohistochemical staining was performed using the Dako REAL EnVision Detection System Peroxidase/DAB+ (DAKO, Denmark Cat# K5007) as instructed by manufacturer. Finally, sections were counterstained with Mayer hematoxylin, dehydrated, and mounted.

To assess the degree of fibrosis in septic lung, paraffin sections were stained with Masson's trichrome according to the protocol suggested by the manufacturer (Masson trichrome with aniline blue; Bio-Optica, Italy Cat #04-010802). Cytoplasm was stained red, collagen blue, and cell nuclei black.

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Determination of IL6 plasma levels

Plasma levels of interleukin-6 (IL-6) were determined with rat IL-6 ELISA kit (Quantikine M Immunoassay; R&D Systems, Mineapolis, Minn). Data were expressed as picograms of the measured cytokine per milliliter of plasma (pg/mL).

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Statistical Analysis

Data were presented as mean ± SEM values. Nonparametric statistics were used for analysis of quantitative variables. Differences between groups were analyzed by Mann-Whitney U test. Time distribution of a variable within group was analyzed using the Kruskal–Wallis test, whereas curve estimation was performed with polynomial functions of time. Differences were considered to be statistically significant when the two-sided probability value was P < 0.05.

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RESULTS

Impact of CLP treatment on lung

Lung inflammation was increased after CLP treatment

Persisting inflammation was shown in lung tissue by studying the expression of S100 family proteins at 6, 12, 24, 36, and 48 h after exposing rats to CLP treatment. Control animals were negative for S100 protein expression (Fig. 1A). S100alpha and S100beta proteins were not detectable in the smooth muscle cells or in the respiratory epithelium. In contrast, expression pattern of S100 family proteins was amplified and extended in CLP group, indicating a prominent inflammatory response (Fig. 1, B–D). Morphologically, both the shape and architecture of the alveoli were seen distorted. Fibrosis, as demonstrated by Masson trichrome stain, was detected mainly around the vessels and the alveoli in septic lung and was increased during disease progression (Fig. 1, E and F).

Fig. 1

Fig. 1

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Cell viability was reduced in the CLP group

CLP-treated animals presented decreased cell viability in the lung compared with sham-operated animals as early as 6 h after sepsis induction (P = 0.009, Fig. 2A). Observed reduction in cell viability was mainly due to apoptosis. The lowest percentage of viable cells was identified 12 h post-CLP treatment (89.72 ± 0.61 vs. 95.27 ± 0.22, P = 0.009), when both apoptosis and necrosis were significantly induced, as compared with controls (9.73 ± 0.56 vs. 9.7 ± 0.56, P = 0.009 and 0.055 ± 0.09 vs. 0.19 ± 0.04, P = 0.028 respectively; Fig. 2B).

Fig. 2

Fig. 2

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Caveolin-1 positive cells exhibited reduced protein expression in the septic lung

Within observation period, a nonstatistical significant variation in the percentage of cells expressing caveolin-1 was observed in the lungs of septic animals as compared with control group (P > 0.05; Fig. 2C). In contrast, single-cell analysis revealed significantly reduced caveolin-1 protein expression throughout sepsis progression (Mean MnIX: 1.84 ± 0.15 vs. 4.13 ± 0.22, P = 0.002). This decline became apparent as early as 6 h post-CLP compared with the sham-operated animals (1.66 ± 0.108, P = 0.021) and reached its lowest value 12 h after sepsis induction (1.25 ± 0.15, P = 0.021; Fig. 2D).

Within CLP group, caveolin-1 protein expression was intensified 24 and 36 h post-CLP compared with 12 h after sepsis, with single-cell values increasing from 1.25 ± 0.14 to 2.61 ± 0.32 (P = 0.021) and 2.37 ± 0.31 (P = 0.02). Still they remained significantly lower compared with controls (P = 0.021, P = 0.02, and P = 0.02).

Apoptosis of caveolin-1 positive cells increased significantly 12 h after sepsis induction (P = 0.02), whereas cell necrosis increased, though not reaching a statistically significant level (P = 0.083).

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Immunostaining of caveolin-1 was modified in septic lung during disease progression

Caveolin-1 was expressed in alveolar capillary endothelial cells in the control group (Fig. 2E).

Twelve hours post-CLP, caveolin-1 expression remained intense in inflamed areas, whereas it was reduced in alveolar epithelial type I cells. The pseudostratified epithelium of the bronchi and vessel walls were negative (Fig. 2F). Focal staining of caveolin-1 was seen in smooth muscle cells 24 h (Fig. 2G) after sepsis, whereas vessel walls exhibited intense staining 36 and 48 h after sepsis (Fig. 2H). Regions with prominent inflammation retained their caveolin-1 immuno-positivity throughout sepsis progression.

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Early induction of pP38 positive cells after CLP treatment

A 2.6-fold increase of pP38 positive cells was observed 6 h post-CLP as compared with controls (11.23 ± 1.44 vs. 4.28 ± 0.49, P = 0.014) that was then reduced 12 h after sepsis (4.06 ± 1.11, P = 0.014) and remained at basal levels throughout studied period (Fig. 3A). Still single-cell analysis revealed a gradual increase of pP38 expression within cells, indicating an intracellular activation of P38 MAPK kinase (Fig. 3B). This rise was statistically significant 24 and 36 h after CLP treatment, as compared with sham-operated animals (2.02 ± 0.52 and 3.08 ± 0.53 vs. 1.19 ± 0.13, P = 0.016 and P = 0.009, respectively). During sepsis, caveolin-1 expression significantly correlated to P38 activation within cells (Spearman test correlation coefficient 0.548, P = 0.012). Immunohistochemical analysis confirmed the results of flow cytometry analysis. The limited number of activated P38 lung cells seen under physiologic conditions was further induced 6 h after CLP administration. pP38 nuclear immunoreactivity was observed in inflamed areas as indicated by septal thickening (Fig. 3, C and D). Furthermore, between 24 and 36 h after CLP treatment, signal intensity showed a time-dependent enhancement, although the percentage of immunopositive cells was reduced.

Fig. 3

Fig. 3

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Late AKT phosphorylation activity during sepsis progression

In the early phase of sepsis, Akt phosphorylation was not altered considerably, whereas it was significantly induced 12 h after CLP treatment and remained increased thereafter (12, 24, 36, and 48 h after sepsis induction) (P = 0.009, P = 0.016, P = 0.016, and P = 0.02, respectively; Fig. 3E). In addition, pAKT mean fluorescent intensity was significantly induced 24 h post-CLP (P = 0.016; Fig. 3F). No immunoreactivity was detected 6 h post-CLP (Fig. 3G), whereas after 36 h, induction of both the cytoplasmic and nuclear immunopositivity was observed in regions presenting distortion and inflammation (Fig. 3H).

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Differential 14-3-3beta protein expression after CLP treatment

Populations of 14-3-3 beta-positive cells presented a gradual increase during the first 24 h of sepsis evolution, which was significantly compared with sham-operated animals 36 h post-CLP (24.4 ± 0.55 vs. 16.18 ± 1.09, P = 0.025; Fig. 3I). However, protein expression of 14-3-3beta showed important fluctuations within cells. Gene silencing 12 h after sepsis induction was alternated by gene amplification 24 h post-CLP (P = 0.009) (Fig. 3J). Also, low levels of 14-3-3beta protein expression were detected 36 and 48 h after sepsis induction (P = 0.05 and P = 0.009, respectively). By immunohistochemistry, an induction in immunoreactivity was observed in septic animals. 14-3-3beta cytoplasmic immunopositivity increased considerably in the pneumonocytes of the alveoli as compared with controls (Fig. 3, K and L).

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CD25+ cell population was induced during sepsis progression

A gradual increase in the percentage of CD25+ immune cells was observed throughout sepsis evolution, which became significant beyond 12 h post-CLP (Fig. 4A). Within cells however, an induction of CD25 expression was already detectable 6 h after CLP treatment (P = 0.009; Fig. 4B). CD25+ cells increased by 1.8-fold between 6 and 12 h after sepsis induction (P = 0.014). The peak of their expression was seen 36 h after sepsis (30.4 ± 6.6 vs. 6.77 ± 0.33, P = 0.014) but 48 h post-CLP they were drastically reduced (P = 0.014), even though levels remained high compared with controls (15.12 ± 2.64, P = 0.009). CD25+cells presented increased apoptosis 6 h post-CLP (27.16 ± 4.16 vs. 43.42 ± 4.6, P = 0.036). Throughout sepsis progression CD25+cell population significantly correlated with pP38 expression (Spearman Rank Correlation test rho: 0.685, P < 0.001).

Fig. 4

Fig. 4

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Impact of PC administration on septic lung

Cytoprotective effects of PC treatment

Systemic inflammation was reduced after PC treatment. The proinflammatory cytokine IL-6 was measured in plasma during sepsis progression to confirm systemic inflammation. A biphasic distribution was observed as during sepsis IL-6 serum levels were high in the first 12 h post-CLP, whereas a significant reduction was detected later on (1467.34 ± 159.35 pg/mL and 932.8 ± 130.65 pg/mL, P = 0.028 at 24 and 36 h, respectively). The lowest levels were observed 48 h post-CLP (117.58 ± 80.07 pg/mL). IL-6 levels were significantly reduced after PC treatment (24 h after CLP induction: 770 ± 74.51 pg/mL, P = 0.028), but presented no significant differences 36 and 48 h after CLP treatment.

Furthermore, the cytoprotective effect of protein C administration in the septic lung was apparent 24 h post-CLP treatment as cell viability was significantly increased compared with septic group (96.35 ± 0.58 vs. 92.57 ± 0.063, P = 0.004; Fig. 5A). The percentages of both apoptotic and necrotic cells in the CLP-PC group diminished compared with CLP animals (3.47 ± 0.58 vs. 6.92 ± 0.07, P = 0.004 and 0.5 ± 0.05 vs. 0.17 ± 0.08, P = 0.004, respectively; Fig. 5B). Pulmonary fibrosis existed in vessels and alveoli, but it was less prominent; Fig. 5, C and D).

Fig. 5

Fig. 5

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The population of caveolin-1 positive cells was diminished and caveolin-1 protein expression was reduced in the septic lung after PC administration

Septic animals treated with PC showed a significant decrease of caveolin-1 expression in the lung. Flow cytometric analysis showed reduction in the percentage of caveolin-1 positive cells 24 h after sepsis, not only compared with matching CLP group (15.1 ± 1.36 vs. 24.06 ± 0.92, P = 0.009), but also compared with the control group (23.22 ± 1.21, P = 0.014; Fig. 5E). In addition, 24 h post-CLP, a statistically significant decline of caveolin-1 protein expression within caveolin-1 positive cells was detected in comparison to time matching septic and control groups (1.15 ± 0.29 vs. 2.61 ± 0.31, P = 0.027 and 4.13 ± 0.21, P = 0.014, respectively; Fig. 5F). The effect of PC treatment was less dramatic 36 and 48 h post-CLP as only caveolin-1 expression was significantly reduced compared with control (1.79 ± 0.17 and 1.15 ± 0.15 vs. 4.13 ± 0.21, P = 0.014 and P = 0.011, respectively), whereas the population of caveolin-1 positive cells did not vary significantly. Even though the values of caveolin-1 expression in the CLP-PC group were still significantly decreased 36 h after sepsis as compared with the control group, no statistical difference was observed between the CLP and CLP-PC groups. Accordingly, decreased intensity in caveolin-1 immunopositivity was observed 24 h post-CLP that was reestablished 48 h post-CLP in the CLP-PC group (Fig. 5, G and H). Caveolin-1 positive cells presented significantly reduced apoptosis 24 and 36 h after sepsis compared with their septic counterparts (P = 0.014 and P = 0.049, respectively). Forty-eight hours post-CLP though, only the value of caveolin-1 MnIX between the CLP-PC and control group remained significantly reduced (1.15 ± 0.15 vs. 4.13 ± 0.21, P = 0.011).

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pP38 positive cells were induced 36 h after CLP-treatment in the CLP-PC group

In the CLP-PC group, the population of pP38+ cells was found increased compared with sham-operated animals (P = 0. 047; Fig. 6A). A significant impact of PC administration on P38 activation was apparent later at 36 h when the concentration of pP38+ cells presented a significant increase compared with CLP and control animals (P = 0.006 and P = 0.011, respectively). This rise continued to be statistically significant compared with control group 48 h post-CLP (P = 0.018). In contrast, PC administration significantly diminished single cell expression in treated animals as declining values were within control range (P = 0.007 and P = 0.018 at 24 and 36 h post-CLP group, respectively; Fig. 6B).

Fig. 6

Fig. 6

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pAkt expression but not cell population was induced after PC administration.

In the CLP-PC group, the population of pAkt positive cells significantly diminished 48 h after sepsis induction as compared with CLP group (P = 0.032; Fig. 6C), but within pAkt positive cells increased protein activation was observed (P = 0.032; Fig. 6D). The values of pAkt single-cell expression persistently increased compared with control group 36 and 48 h after CLP treatment (P = 0.006 and P = 0.044, respectively).

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Silencing of 14-3-3b after PC administration

The percentage of 14-3-3b+ cells remained practically unaffected, as their decrease was not significant (Fig. 6E). In contrast, 14-3-3b protein expression levels dropped dramatically in the CLP-PC group, compared with their septic partners 24 h post-CLP (P = 0.014), maintaing protein levels low throughout sepsis progression (Fig. 6F).

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Silencing of CD25 protein expression after PC administration

The percentage of CD25+ cells remained high compared with control group (P = 0.004, P = 0.006, and P = 0.006 at 24, 36, and 48 h post-CLP, respectively) and unaltered compared with CLP group (Fig. 5G). Instead, CD25 protein expression decreased radically within positive cells 24 h after sepsis induction (P = 0.008; Fig. 6H). Apoptosis was reduced among the CD25+ subpopulation at 24 h post-CLP (P = 0.004).

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DISCUSSION

Despite the plethora of research evidence, obtained particularly with the caveolin-1 knock out systems (7–12), the role of caveolin-1 in sepsis remains a debatable issue. Results on the cytoprotective effect of caveolin-1 were often contradictory and seemed to depend on the septic model used (2). The present study, using the CLP model for sepsis in nongenetically modified rats, initially examined the behavior of host's “endogeneous” caveolin-1 in the lung during sepsis progression and in relation to important proteins of inflammation, cell signaling, and apoptosis. The CLP model has been successfully developed in several species including rat and differs from other models such as endotoxemia as it produces a biphasic hemodynamic response instead of an immediate hypotention (22) Therefore, it is generally considered to be one of the best representations of human sepsis as it closely assimilates human sepsis progression and characteristics, although it cannot fully reproduce the clinical complexity and intrinsic heterogeneity of septic patients.

In our septic animals, immunohistochemical distribution of S100 family proteins indicated increased inflammation in the lung. Alveolar septae, where gas exchange takes place, were markedly thickened. Similar pathomorphological defects with extended areas of markedly thickened lung septa, due to an uncontrolled proliferation of endothelial cells, have also been described in caveolin-1 knockout animals (23). Animals subjected to CLP procedure showed increased IL-6 plasma levels during the first 24 h, possibly attributable to the amplified bacterial load and to increased necrosis. Survival rates were within accepted ranges based on the strain and size of needle used.

The herein presented data showed that the percentile of caveolin-1 positive cells remained stable in the lung of nongenetically modified rats during the course of the disease, but caveolin-1 positive cells expressed reduced protein levels. Similar results were previously observed in normal pigs infected with Haemophilus parasuis, showing particularly impaired caveolin-1 protein expression in infected porcine lung (24). In our CLP group, single-cell analysis with flow cytometry showed that caveolin-1 reduction was more prominent up to 12 h after CLP treatment, when IL-6 levels were induced. These findings further support an anti-inflammatory role for caveolin-1 protein (18, 21). Immunohistochemical analysis verified this decline indicating faint immunopositivity within the cell populations of smooth muscle cells and pseudostratified epithelium of the bronchi. Masson trichrome stain showed induced fibrosis around the vessels and the alveoli and further supported the observed reduced caveolin-1 protein expression, given that decreased caveolin-1 expression has also been previously reported in fibrotic diseases such as idiopathic pulmonary fibrosis (5).

Based on the knowledge that caveolin-1 is essential for caveolae formation, the reduction in caveolin-1 expression observed in our study most probably reflects a shrinkage in the number of caveolae present in cells. It has been demonstrated that many pathogenic bacteria and viruses choose caveolae as an entry port to host cells in their effort to find intracellular compartments where they can grow and replicate under optimal conditions, without being degraded by lysosomes (25). It stands to reason therefore that the observed decrease of caveolin-1 protein expression represents an immediate act of the host to diminish the intracellular bacterial burden by withdrawing the available for endocytosis caveolae. The entry of the intestinal pathogen Campylobacter jejuni into host cells was shown to be significantly inhibited by caveolin disrupters (26). Up to date, many studies have demonstrated the capacity of different pathogens to co-opt caveolae for entering and trafficking within host cells as specific pathogen receptors were shown to be localized within the caveolae (2, 3).

Furthermore, it has been previously demonstrated that the biophysical properties of caveolae could be co-opted by pathogens to expose host cells to relative high concentrations of toxins without the requirement of a close contact between the bacterial and host cell. In similar cases, necrosis of the target cells was induced by high concentrations of toxin, whereas an inflammatory response and/or apoptosis was more prominent by sublytic concentrations of a toxin (2, 27, 28). Therefore, it can be assumed that regulation of caveolin-1 expression during sepsis helps to maintain the balance between host response and tissue damage at the site of inflammation. The data herein reported presented significantly elevated apoptosis in septic lung. Initially, increased apoptosis of CD25 positive cells attributed to their activation state was observed 6 h after sepsis induction in the CLP-treated animals. The peak of apoptosis was observed 12 h after sepsis induction. Caveolin-1 positive cells presented high apoptosis and increased necrosis at 12 h post-CLP, probably due to the presence of microorganisms and to increased concentrations of toxins in these cells as a result of a caveolae-mediated endocytosis mechanism. Examination of the anti-apoptotic 14-3-3b expression 12 h post-CLP showed a significant decline. Apoptosis of caveolin-1 positive cells was also increased. It is known that caveolae are most numerous in the microvascular endothelia of the lung and that prostacyclin PGI2 defends endothelial survival and protects vascular smooth muscle cells from apoptosis via peroxisome-proliferator-activated receptors and 14-3-3 upregulation (29). Several studies have shown that the cytokine cascade induced by foreign microorganisms and toxins as well as the endogenously produced cytokines and immune mediators cause endothelial cell “stimulation” (early event) and “activation” (later event) leading to apoptosis (2, 30).

Based on the pivotal role of caveolae in signal transduction (2), changes in the intracellular activation of MAP kinase P38 and pAkt, on a time-dependent basis, were also assessed in the present study. Shortly after lung exposure to inflammatory agents, the percentage of pP38+ cells in the lung was significantly elevated compared with control group. Activation of P38MAPK signaling pathway has been linked to the acute phase of inflammation during sepsis progression and was reported to promote neutrophils chemotaxis per se. P38 activation was recognized as a coordinator of CXC chemokine-dependent leukocyte recruitment in septic lung injury and a contributing factor in the formation of lung edema, further justifying its early induction during sepsis (31). The functional significance of caveolae on the activation of P38MAPK signaling was also evident in the present study, as the percentage of cells presenting activation of P38 MAPK was radically reduced, when caveolin-1 intensity was drastically diminished. Late Akt phosphorylation activity was also observed in the present study. Akt activation was demonstrated to mediate the launch and migration of immune/inflammatory cells and to further decrease apoptosis and promote cell survival in an inflamed environment supporting the repair of the airways after lung injury (21). Therefore, induction of apoptotic signals in septic animals could explain the constantly upregulated pAkt beyond 12 h post-CLP. This increase could also contribute to the observed lung fibrosis. It was demonstrated that in idiopathic pulmonary fibrosis, decreased caveolin expression augments levels of phosphorylated Akt by regulating membrane-associated PTEN activity (5).

In addition, our study explored the expression profile of endogeneous caveolin-1 protein after the administration of three doses of protein C zymogen in CLP-treated animals. In the past PC was shown to present anti-apoptotic and anti-inflammatory activities and to improve survival in CLP-treated animals (15, 16). In the present study, PC administration significantly diminished cell apoptosis. Furthermore, the high plasma levels of IL-6 found in CLP-treated animals were significantly reduced in septic lung after PC administration. In a mouse CLP model it was documented that high levels of IL-6 in the early phases of sepsis in experimental animal models could predict early mortality (32). However, the beneficial effect of PC administration in sepsis resulted in decreased plasma inflammatory cytokines levels and reduced apoptosis was escorted by a fall of caveolin-1 protein expression as not only the population of caveolin-1 positive cells was diminished but also intracellular protein expression was reduced in the septic lung. Research data have shown that PC exerts its anti-inflammatory and cytoprotective signaling in endothelial cells through activation of PAR-1. Experiments in wild-type and PAR1−/− mice demonstrated that intravenous injection of APC leads to PAR1-dependent gene induction in the lung (33). Thrombin cleavage of PAR-1 engages endothelial protein C receptor (EPCR) that interacts with caveolin-1. Dissociation of EPCR from caveolin-1 facilitates the occupancy of EPCR by PC. Cellular models suggest that PAR-1 would elicit only protective signaling responses in endothelial cells if they express EPCR and the receptor is occupied by its ligand PC/APC (34, 35). Thus, we suggest that in our PC-CLP-treated animals caveolin-1 expression was reduced by the presence of excess PC to facilitate the interaction between EPCR and PC, driving signaling of PAR-1 toward cell protection, and bringing about an anti-inflammatory response.

Furthermore, our data on PC administration further support the role of caveolae as lipid-based signaling platforms that regulate diverse signaling pathways because the expression profile of the signaling molecules investigated were modified. In agreement with a previous study, P38 kinase activation was considerably attenuated in the lung of those animals pretreated with PC (5).

In conclusion and with the understanding that the complexity of sepsis in humans cannot be fully reproduced by animal models, the herein presented results indicate that part of host's defensive reaction during sepsis is directed toward the control of caveolin-1 protein expression because downregulation of caveolin-1 mirrors withdrawing of available caveolae to control pathogens entry in host cells; control of caveolae in the microvascular endothelia of the lung and adjustment of 14-3-3b expression reveals regulation of vascular smooth muscle cells apoptosis; reduction of caveolin-1 switches the specificity of PAR-1 to initiate protective response in endothelial cells by altering the occupancy of EPCR. Thus, adjustment of caveolin-1 expression via different cytoprotective mechanisms can apparently modify pulmonary function both at cell and at systemic levels and supports caveolin-1 cytoprotective profile, which makes it a promising candidate in the battle against sepsis-induced mortality.

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Keywords:

14-3-3b; caveolin-1; CLP model; pAkt; PC; pP38; S100; sepsis; −; negative stained; +; positive stained; 7AAD; 7-aminoactinomycin D protein; APC; activated protein C; CLP; cecal ligation and puncture; EPCR; endothelial protein C receptor; FITC; fluorescein isothiocyanate; MAPK; mitogen-activated protein kinase; MFI; mean fluorescent intensity; pAkt; phospho-Akt; PAR-1; protease-activated receptor-1; PBS; phosphate-buffered saline; PC; human protein C concentrate; PE; phycoerythrin; pP38; phosphorylated-P38; rhAPC; recombinant human-activated protein C; SEM value; standard error of the mean; Treg; regulatory T cell

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