Endothelial bioreactor system ameliorates multiple organ dysfunction in septic rats
- Shuai Ma†1, 3,
- Yuli Lin†2,
- Bo Deng1, 3,
- Yin Zheng1, 3,
- Chuanming Hao3,
- Rui He2Email author and
- Feng Ding1Email author
© The Author(s). 2016
Received: 19 April 2016
Accepted: 13 July 2016
Published: 22 July 2016
The endothelium is a potentially valuable target for sepsis therapy. We have previously studied an extracorporeal endothelial cell therapy system, called the endothelial bioreactor (EBR), which prolonged the survival time of endotoxemia sepsis in swine. To further study of the therapeutic effects and possible mechanisms, we established a miniature EBR system for septic rats induced by cecal ligation and puncture (CLP).
In the miniature EBR system, the extracorporeal circulation first passed through a mini-hemofilter, and the ultrafiltrate (UF) was separated, then the UF passed through an EBR (a 1-mL cartridge containing approximately 2 × 106 endothelial cells grown on microcarriers) and interact with endothelial cells. Eighteen hours after CLP, the rats were treated for 4 h with this extracorporeal system containing either endothelial cells (EBR group) or no cells (sham EBR group). Physiologic and biochemical parameters, cytokines, endothelial functions, and 7-day survival time were monitored. In vitro, the pulmonary endothelial cells of the septic rats were treated with the EBR system and the resulting changes in their functions were monitored.
The EBR system ameliorated CLP-induced sepsis compared with the sham EBR system. After CLP, the 7-day survival rate of sham-treated rats was only 25.0 %, while in the EBR-treated group, it increased to 57.1 % (p = 0.04). The EBR system protected the liver and renal function and ameliorated the kidney and lung injury. Meanwhile, this therapy reduced pulmonary vascular leakage and alleviated the infiltration of inflammatory cells in the lungs, especially neutrophils. Furthermore, after the EBR treatment both in vivo and in vitro, the expression of intercellular adhesion molecule-1 and the secretion of CXCL1 and CXCL2 of pulmonary endothelium decreased, which helped to alleviate the adhesion and chemotaxis of neutrophils. In addition, the EBR system decreased CD11b expression and intracellular free calcium level of peripheral blood neutrophils, modulated the activation of these neutrophils.
The EBR system significantly ameliorated CLP-induced sepsis and improved survival and organ functions. Compared with the sham EBR system, this extracorporeal endothelial therapy may be involved in modulating the function of pulmonary endothelial cells, reducing the adhesion and chemotaxis of neutrophil, and modulating the activation of peripheral blood neutrophils.
Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection  and is the most common cause of death in the intensive care unit . Despite the progress in the development of antibiotics and in critical care therapy, sepsis is still associated with a high mortality rate .
Cell-based therapy has developed into a new therapeutic platform for the treatment of a vast array of clinical disorders, which may prove to be a more successful strategy by providing a dynamic, interactive, and individualized therapeutic approach . In animal studies, stem/progenitor technologies, including exogenous infusion and endogenous recruitment, have shown significant promise as treatment strategies in critical care medicine [4–7]. In terms of safety, extracorporeal cell therapies may be a better choice, which not only replace the function of injury cells or modulate the pathophysiological processes but also provide an immunoprotective barrier. In swine models of sepsis, extracorporeal cell therapy with granulocytes  or renal proximal tubule cells  improved survival duration. Furthermore, in a phase II multicenter clinical trial, involving critically ill patients with acute renal failure, this extracorporeal renal-tubule cell therapy was proved to be efficacious and safe . Nevertheless, the difficulties in large animal experiments keep the mechanism study elucidation insufficient.
General dysfunction of the endothelium is a key event in the pathogenesis of sepsis . Once the endothelium becomes activated during the development of sepsis, it transforms into a procoagulant, antifibrinolytic, and proadhesive state . The endothelial activation in sepsis is associated with changes in hemostatic balance, leukocyte trafficking, vascular permeability, inflammatory processes, and microcirculatory flow . Thus, the endothelium plays a key role in mediating the sepsis phenotype  and is a potentially valuable target of sepsis therapy.
We previously studied an endothelial cell therapy system , called the endothelial bioreactor (EBR), involving a nonwoven fabric polytetrafluoroethylene (PTFE) hollow fiber cartridge containing endothelial cells in an extracorporeal circuit. Timely use of EBR therapy may improve cardiovascular performance and prolong the survival time of endotoxemia sepsis in swine . Nevertheless, the difficulties in large animal experiments keep its elucidation insufficient. Therefore, we established a miniature EBR system for septic rats to study the therapeutic effects and possible mechanisms of this endothelial cell-based therapy.
Cecal ligation and puncture
Adult male Sprague-Dawley rats (450–550 g) were used for the studies. The experimental procedures were in accordance with the Animal Care and Use Committee of Shanghai Jiaotong University, Shanghai, China. All surgical procedures were carried out under general anesthesia induced by 4 % chloral hydrate (0.9 mL/100 g intraperitoneally). The cecum was identified and ligated with a 3-0 silk tie at 25 % length of the cecum. A double puncture of the cecal wall was performed with a 20-gauge needle, and the cecum was gently squeezed to ensure that a small amount of feces was extruded onto the surface of the bowel. Lactated Ringer’s solution (2 mL/100 g) was given subcutaneously as fluid resuscitation [16, 17]. As sham cecal ligation and puncture (CLP) rats, the cecum was minimally handled without ligation and puncture.
Culturing of endothelial cells on microcarriers
Endothelial cells (human umbilical vein endothelial cells (HUVECs)) were a gift from Professor Lijun Jia (Department of Immunology, Shanghai Medical College, Fudan University, Shanghai, China).
For endothelial cell culture, protocols were adapted based on the recommendations for microcarrier culture using Cytodex-3 (GE Healthcare, Piscataway, USA). Cytodex-3 is a microporous microcarrier, made up of a dextran matrix with a collagen layer at the surface, for expansion of cells. For stirred microcarrier cultures, 100-mL spinner flasks (Bellco Glass, Vineland, USA) were used, with a final medium volume of 80 mL and a stirring speed of 40 rpm. Required quantities of Cytodex-3 were weighted (final concentrations of 1.0 mg/mL), hydrated, and sterilized by autoclaving as recommended by the manufacturers. Microcarriers were equilibrated in the culture medium for at least 30 min prior to the addition of the cells in order to maximize cell attachment. The cells were cultured in DMEM media containing 10 % heat-inactivated fetal bovine serum (GIBCO, Gaithersburg, USA), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine. Incubation was conducted at 37 °C in a 5 % CO2, humidified atmosphere. The expansion of endothelial cells was performed as previously described .
The experimental protocol of the EBR system
Eighteen hours after CLP, the animals were re-anesthetized with chloral hydrate. The carotid artery and the femoral vein were isolated by dissection and cannulated with 0.97-mm polyethylene-50 tubing (Becton Dickinson, San Diego, USA) for implementation of extracorporeal circulation.
The EBR system consisted of a mini-hemofilter (polyether sulphone high-flux membrane, membrane surface area 0.02 m2, cutoff point 40 kD; PEONY, Shanghai, China), an EBR (a 1-mL cartridge containing approximately 2 × 106 endothelial cells grown on microcarriers), tubing lines, and mini-pumps (VWR, West Chester, USA).
Analysis of bronchoalveolar lavage fluid
Bronchoalveolar lavage fluid (BALF) was obtained by lavaging the left lung once with 2 mL PBS and three times with 4 mL PBS. Total cells were counted under optical microscopes. Total protein in the first BALF was measured by bicinchoninic acid kit. TNF-α, IL-1β, and IL-6 levels in the first BALF were measured by an enzyme-linked immunosorbent assay (ELISA) from eBioscience (San Diego, USA).
Flow cytometric analysis
Fluorochrome-labeled antibodies CD4, CD8, CD25, Foxp3, CD45, CD11b, and Granulocyte Marker (eBioscience, San Diego, USA) were used for surface and intracellular staining, according to the manufacturer’s instructions. After staining, cells were analyzed by flow cytometer (Becton Dickinson, San Diego, USA).
Quantitative real-time RT-PCR analysis
Total RNA was extracted using Micro Scale RNAqueous Isolation kit, and then cDNA was synthesized with the High-Capacity cDNA Reverse Transcription kit (both from Applied Biosystems, Foster City, USA). Quantitative real-time polymerase chain reaction was performed with the SYBR green Gene Expression Assay (Applied Biosystems, Foster City, USA). The relative expressions of target genes were calculated using the 2ΔC(t) method. The sequences of primers for polymerase chain reaction are as follows: intercellular adhesion molecule (ICAM)-1, 5′-CTCATCCTGCGCTGTCTGGT-3′ (forward), 5′-CCGGAGCTGCCTGACCTCGG-3′ (reverse); CXCL1, 5′-GCGTTTCATCGATGGTCGTT-3′ (forward), 5′-CTTCTTCCCGCTCAACACCT-3′ (reverse); and CXCL2, 5′-AACCATCAGGGTACAGGGGT-3′ (forward), 5′-GGGCTTCAGGGTTGAGACAA-3′ (reverse).
Western blot analysis for ICAM-1
Pulmonary endothelial cells or lung tissue were lysed with RIPA buffer. Proteins were separated by electrophoresis in a denaturing polyacrylamide gel and transferred to a PVDF membrane. After blocking with 5 % milk-Tris-buffered saline and Tween 20 (TBST) and washing in TBST, membranes were then incubated in the appropriate primary antibodies (anti-ICAM-1; both from R&D, Emeryville, USA) and anti-GAPDH (Cell Signal Technology, Beverly, USA) at 4 °C overnight. After washing, membranes were incubated with the appropriate HRP-conjugated secondary antibodies and analyzed by ECL development.
Isolation of rat pulmonary endothelial cells
The lungs were harvested at 18 h after CLP, finely minced, and digested in 10 ml collagenase type II (2 mg/ml, Sigma-Aldrich, St. Louis, USA). The isolated cells were incubated with Anti-Rat CD31 Biotin (eBioscience, San Diego, USA), and endothelial cells bound with antibody were magnetically separated with Anti-Biotin Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) as described previously [20, 21]. Pulmonary endothelial cells of healthy rats were also isolated as control.
Simulation of EBR treatment in vitro
Eighteen hours after CLP, the rats were cannulated, and the extracorporeal circulation passed through a mini-hemofilter with the ultrafiltration rate at 0.25 mL/min. The UF of the rats were harvested (sham-UF). The UF of healthy rats were also harvested as a control (control-UF). HUVECs were stimulated with the UF for 0, 0.5, 1, 2, or 4 h. Then, the supernatant (EBR-UF) was collected and used to stimulate the isolated pulmonary endothelial cells. After 24 h of stimulation with EBR-UF, the cells were harvested for Western blot and the supernatant was used to analyze the concentrations of CXCL1 and CXCL2 using ELISA kits (R&D, Emeryville, USA).
Isolation of rat peripheral blood neutrophils and detection of intracellular free calcium level
Rat neutrophils were isolated from the peripheral blood at 18 h after CLP by Percoll density gradient as previously described . The purity of isolated neutrophils was checked by fluorochrome-labeled antibodies CD45 and Granulocyte Marker (eBioscience, San Diego, USA) using flow cytometer (Becton Dickinson, San Diego, USA). Neutrophils of healthy rats were also isolated as a control.
The neutrophils were incubated with Fluo-3 AM (Invitrogen) for 30 min in darkness at room temperature, and the resulting fluorescence as the indicator of Ca2+ was monitored using a flow cytometer (Becton Dickinson, San Diego, USA) at excitation wavelength 488 nm and the emission wavelength of 525 nm [23, 24].
Histopathologic analysis of the lung and kidney
Lung sections were stained with hematoxylin and eosin. All lung fields at ×200 magnification were examined for each sample. Assessment of histological lung injury was performed as follows: 1 = normal; 2 = focal (<50 % lung section) interstitial congestion and inflammatory cell infiltration; 3 = diffuse (>50 % lung section) interstitial congestion and inflammatory cell infiltration; 4 = focal (<50 % lung section) consolidation and inflammatory cell infiltration; 5 = diffuse (>50 % lung section) consolidation and inflammatory cell infiltration .
Kidney sections were stained with periodic acid and Schiff’s reagent. All kidney fields at ×400 magnification were examined for each sample. Damaged renal tubules were identified by diffuse tubular dilatation, intraluminal casts and/or tubular cell blebbing, vacuolization, and detachment, in cortex and outer medulla, as follows: 0 = none; 1 = <10 %; 2 = 11–25 %; 3 = 26–45 %; 4 = 46–75 %; 5 = >76 % [26, 27].
Measurements of the liver and renal functions
Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), and blood urea nitrogen (BUN) were measured with an automated chemical analyzer (Vitros-950, Johnson & Johnson, New Brunswick, USA).
All numerical data are expressed as mean ± SEM. The Student’s t tests were used for comparisons between two groups. Multiple-group comparisons were performed by one-way ANOVA followed by a post hoc Tukey’s test to compare each group. The survival analysis was performed by the Kaplan-Meier method and log-rank test. All statistical analyses were performed with GraphPad Prism software, and a two-sided p < 0.05 was considered significant.
Construction of the EBR system for CLP-induced sepsis in rats
In order to estimate the potential harmful effect of this extracorporeal circulation on survival time, the rats were divided into two groups: the CLP group (CLP, without extracorporeal circulation) and the sham EBR group (CLP, EBR system without any cells); then, survival was analyzed until 7 days after CLP. We found that survival duration was not statistically different between the sham EBR and control animals (25.0 vs. 33.3 %, p = 0.54; Fig. 1c), which meant this circulation system was safe for the septic rats. The EBR was a 1-mL cartridge, whereas we need approximately 2 × 106 endothelial cells in it. Thus, we cultured endothelial cells on microcarriers to increase the surface-area-to-volume ratio. During the culturing of endothelial cells on microcarriers, we analyzed the concentrations of glucose, lactate, and nitric oxide (NO) in different time points for analyzing the metabolic activity. We found that endothelial cells could grow adhering to the surface of microcarriers after seeding; lactate and NO levels in the culture media rose continuously, and the glucose concentration decreased with time, indicating metabolic activity residing in the microcarrier culture (data not shown). For testing whether the EBR system treatment will affect the adhesion of endothelial cells on the microcarrier, we collected microcarriers and stained them with DAPI before and after the extracorporeal circulation. We observed that there were a large number of endothelial cells growing on the microcarriers before the circulation. Similar observations after the extracorporeal circuit revealed no obvious decrease in the density of the cells (Fig. 1d). These data showed that we successfully constructed an EBR system for rats with CLP-induced sepsis.
The EBR system ameliorated CLP-induced sepsis
The EBR system alleviated inflammation in the lungs
The EBR system modulated the function of pulmonary endothelial cells and peripheral blood neutrophils
A previous study by our laboratory has showed that timely EBR therapy improves cardiovascular performance and prolongs the survival time of endotoxemia sepsis in swine . In the present study, we established a new EBR system for septic rats and demonstrated that the EBR system ameliorated CLP-induced sepsis and improved survival and organ functions. In animal models, the cytokine response is fully activated by 16–18 h after CLP , and the ligated cecum begins to heal after 24 h [19, 43]. In clinical practice, patients often do not come to the hospital at the early stage of infection. In order to simulate clinical practice, we started the treatment at 18 h after CLP. The therapeutic use of extracorporeal bioreactors has a long tradition. Different cell-based extracorporeal organ support systems involving either hepatocytes or renal tubular cells have been used against acute liver failure [44–46] and acute renal failure associated with sepsis [9, 47]. An extracorporeal bioartificial kidney consisting of a conventional hemofilter connected to a renal tubule assist device has shown its ability to successfully replace the filtration, transport, metabolic, and endocrine functions of the kidney in critically ill patients with acute renal failure in phase I/II clinical trials [10, 47]. Moreover, an extracorporeal treatment with donor granulocytes appears to be well tolerated and shows promising efficacy results in patients with septic shock, thus encouraging further studies .
The EBR system is an extracorporeal cell therapy with endothelial cells. We found that the EBR system alleviates inflammation in the lungs and reduces the infiltration of inflammatory cells, especially neutrophils. In sepsis, a number of different mechanisms may induce functional modulation of the endothelium, including increased expression of cell adhesion molecules and trafficking of leukocytes . Endothelial cells are critical in maintaining a delicate balance between vasoconstriction and vasodilation, blood cell adherence and non-adherence, anticoagulation and procoagulation, and permeability and tightness . These cells are not inert, rather they may adapt their function upon interaction with inflammatory mediators, and these changes are referred to as activation. They encompass a change from an anti- into a procoagulant surface; the expression of adhesion molecules; the production of inflammatory mediators, including chemoattractant agents; and the production of vasoactive compounds . Two studies in cell-based treatments showed that transplanting endothelial cells to mice with ischemic acute kidney injury could change the function of renal endothelial cells and improve the renal function [51, 52]. The dysfunction or activation of endothelial cells plays a major role in the pathogenesis of sepsis [11, 14, 50]. Thus, endothelial cells represent an attractive therapeutic target in sepsis.
ICAM-1 is a cell-surface protein that is expressed at very low levels on pulmonary endothelium. The expression of ICAM-1 is upregulated after stimulation by inflammatory mediators such as cytokines and bacterial lipopolysaccharides during septic processes [53, 54]. ICAM-1 mediates inflammatory responses via adhesion of leukocytes to activated endothelium and subsequent leukocyte transmigration through the pulmonary endothelial layer . The increased expression of endothelial adhesion molecules either at the membrane level or in the plasma typifies different models of sepsis . Moreover, there is a close relationship between plasma levels of adhesion molecules and outcomes of sepsis. In human sepsis, studies have shown that the higher the plasma levels of ICAM-1, the greater the number of organs damaged and the mortality [13, 56]. The present study showed that the level of ICAM-1 in the lung tissue was increased after CLP, while the EBR treatment reduced its expression compared with the sham treatment. Furthermore, the level of pulmonary endothelial ICAM-1 significantly decreased after the EBR treatment in vitro. In other words, our findings indicate that this extracorporeal endothelial therapy may be involved in reducing the expression of ICAM-1 in the pulmonary endothelium, and this change should help to alleviate the adhesion of neutrophils. Moreover, in vivo and in vitro, we also found that this treatment decreased the secretion of CXCL1 and CXCL2 of septic pulmonary endothelial cells compared with the sham treatment. CXCL1 and CXCL2 are important neutrophil-attracting chemokines. Recent studies showed that CXCR2 (the receptor of CXCL1 and CXCL2) blockade results in impaired neutrophil recruitment in lipopolysaccharide-induced acute lung injury [57, 32]. Our data indicate that this EBR therapy may be involved in reducing the chemotaxis of neutrophils in the lung tissue via decreasing the secretion of CXCL1 and CXCL2 of pulmonary endothelial cells. Neutrophils are known to play an important role in inflammatory responses . However, excessive activation of neutrophils is a direct cause of tissue damage and organ failure in sepsis [39, 58]. Mac-1 (CD11b/CD18), the major subtype of integrins, is responsible for the firm adhesion of neutrophils to endothelium . Once the neutrophils are activated, the shape of these cells changed and the amount of Mac-1 increased, resulting in enhanced adhesion to the endothelium and transmigration and infiltration of these neutrophils . Another parameter commonly used for the assessment of neutrophil activation is intracellular free calcium level. Ca2+-dependent functions include activation of the membrane-associated superoxide-generating electron transporter, NADPH oxidase, and adhesion to the endothelium . The present study showed that the EBR system decreased the CD11b expression and intracellular free calcium level of peripheral blood neutrophils compared with the sham treatment, which might modulate the activation of these neutrophils.
In the early phase of sepsis, the overwhelming inflammatory response is initiated after microbial infection , while the late phase is characterized by T cell hyporesponsiveness and defective antigen presentation [60, 61]. This phase is considered a state of immunosuppression or immunoparalysis of the host . Massive apoptosis of lymphocytes is one of the main drivers leading to immunoparalysis . To assess the adaptive immune response, circulating and splenic levels of CD4+ helper T cells, CD8+ cytotoxic T cells, and CD4+CD25+Foxp3+ T regulatory cells were measured at 72 h after CLP. However, there was no significant difference between EBR-treated and sham-treated rats either in the peripheral circulation or in the spleen (Additional file 1: Figure S1). Thus, the 4-h EBR system treatment had no effect on the levels of T lymphocytes in the current rat model.
In summary, these data suggested that EBR system ameliorates CLP-induced sepsis and improves survival and organ functions compared with the sham EBR system. The EBR system alleviates inflammation in the lungs and reduces the infiltration of inflammatory cells and may be involved in modulating the function of pulmonary endothelial cells and reducing the adhesion and chemotaxis of neutrophils. In the same time, the EBR system might probably modulate the activation of peripheral blood neutrophils directly.
We are grateful to Professor Lijun Jia (Department of Immunology, Shanghai Medical College, Fudan University, Shanghai, China) for providing HUVECs; Professor H. David Humes (Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA) for helping to construct the EBR system; and Doctor Zhiyong Peng and Professor John A. Kellum (The CRISMA Center, Department of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA) for supporting the extracorporeal circulation of rats.
This study was supported, in part, by grants from the National Natural Science Foundation of China (No: 81070609, 81270850, 81470990), the National Key Technology R&D Program (No: 2011BAI10B08), the Shanghai Pudong New Area Program (No: PW2015D-4), and the Shanghai Jiaotong University Key Program (YG2014ZD06).
FD, RH, and CH conceived and designed the research. SM, BD, YL, and YZ performed the research. SM, BD, and FD analyzed the data. SM wrote the paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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