Sepsis is a systemic inflammatory response syndrome indicating that the body overreacts to infection of pathogens and is one of the most common causes of mortality of patients in the intensive care unit. In the past decade, even though some progresses have been made in understanding the physiological and pathological mechanisms and treatment of sepsis (1), the hospitalization rate of patients with sepsis is increasing every year, and their mortality rate also remains at a high level (2). In addition, patients with acute sepsis who survived after treatment have high risks of long-term physical and mental dysfunction and decreased life expectancy (3, 4). Therefore, it is urgently needed in clinics to have positive and effective treatment measures.
We have previously conducted proteomic analysis of serum samples of dead septic rats and septic rats that survived and found 28 serum proteins were deferentially expressed (5). By further subnetwork enrichment analysis of the 28 proteins, it was found that endostatin is an important node protein, suggesting it may be a potential intervention target with therapeutic significance for sepsis.
Endostatin was initially purified by O’Reilly et al. (6) as a vascular endothelial inhibitor from the culture supernatant of mouse tumor vascular endothelial cells. It is an endogenous 20-kd fragment of noncollagen feature domain at the carboxy segment of collagen XVIII and composed of 184-amino-acid residues. The current mainstream researches on endostatin are focused on the field of antitumor therapy and have demonstrated that it could attenuate the proliferation and migration and induce apoptosis of endothelial cells, thereby inhibiting angiogenesis and tumor growth (6, 7).
It is worthy of attention that endostatin may have beneficial effects on patients with chronic inflammatory diseases. Endostatin reduces inflammatory responses and angiogenesis induced by injecting human rheumatoid arthritis synovial fluid into immunodeficient mice as well as in cyclophosphamide-induced hemorrhagic cystitis mouse model (8, 9). In addition, subcutaneous application of endostatin reduces lung allergic inflammatory responses in asthma mouse model and attenuates the levels of interleukin 4 (IL-4), IL-10, monocyte chemotactic factors, and other inflammatory mediators in lung tissues (10). Recent studies also showed that endostatin reduces bleomycin- and radiotherapy-induced pulmonary fibrosis and the level of lung inflammatory cells (11, 12). All of these studies strongly suggest that endostatin may participate and have an impact on inflammation-related mechanisms. Moreover, studies on endostatin pathways found that endostatin influences cell behaviors through multiple pathways. It interacts with many protein receptors on the cell membrane such as integrin α5β1, vascular endothelial growth factor (VEGF) receptors, β-catenin, and matrix metalloproteinase precursors (13–15), which are all related to changes of vascular permeability and the pathophysiology of sepsis-related aspects, suggesting that endostatin may become a sepsis-associated therapeutic intervention target.
In this study, we, for the first time, evaluated the effects of endostatin on mouse sepsis model established by cecal ligation and puncture (CLP) and explored its implications for treatment of sepsis. We found that endostatin could increase the survival rate, mitigate multiple organ damages, and inhibit early inflammation and changes in vascular permeability of CLP-induced septic mice. These findings provide new research ideas and experimental clues for treatment of sepsis.
MATERIALS AND METHODS
The mouse strain Kunming, an outbred strain of Swiss mouse and a widely used mouse strain for animal experiments in China, was used to establish the CLP-induced sepsis model. Male Kunming mice, aged 6 to 8 weeks (20–22 g), were purchased from the Experimental Animal Center of Central South University (Changsha, China). Mice were fed with standard rodent diet and water ad libitum and kept in an animal facility and maintained at 21°C ± 2°C on a 12-h light-dark cycle. All experiments were performed in accordance with the Guide for Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Central South University Xiangya Medical School.
CLP-induced sepsis model
Mice were housed in strict accordance with the standard procedures of animal-feeding operations. To establish CLP-induced sepsis model, mice were anesthetized by intraperitoneally injecting 10% chloral hydrate (The Third Xiangya Hospital, Changsha, China) at 30 mg/kg body weight after 8 h of fasting and away from water. Mouse abdomen was shaved and disinfected. Under aseptic conditions, a 1.0-cm abdominal incision was created along the ventral midline to expose the cecum with adjoining intestine. The cecum was tightly ligated with a 4-0 silk suture in the cecal vascular arcades 1.0 cm away from cecum blind end and perforated with a 20-gauge needle on the same side of the cecum. The cecum was squeezed to extrude a small amount of feces from the puncture site. After disinfection with alcohol swab, the cecum was returned to the peritoneal cavity, and the peritoneum was closed layer by layer. Mice in the endostatin intervention group were subcutaneously injected 0.5 mL phosphate-buffered saline (PBS) solution containing recombinant human endostatin (Simcere Pharmaceutical Research Co, Ltd, Shandong, China) at different concentrations to observe its dose and time effects. Mice in the model control group were injected with 0.5 mL PBS. Morphine was injected (0.1 mg/kg s.c.) for postoperative analgesia every 6 h for 48 h. Mice in the sham group underwent similar surgical procedure except CLP. The survival rate of mice in all groups was observed for 72 h. To avoid differences in the surgical procedure, surgical operation was conducted by the same person. In addition, some mice in each group were anesthetized at different septic time points with 10% chloral hydrate, and approximately 1 mL whole blood was collected from their heart through thoracotomy and used to separate serum. Meanwhile, heart, kidney, liver, and lung tissues were retained for further examination.
Serum indicator detection
After incubation at room temperature for 2 h, the whole blood was centrifuged at 2,000g for 15 min. The supernatant was stored at −80°C and used to measure the levels of blood urea nitrogen (BUN), creatinine (Cre), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine kinase isoforms enzyme (CK-MB) using the Olympus AU5400 Automatic Biochemical Analyzer (Olympus, Japan).
Enzyme-linked immunosorbent assay
The expression levels of tumor necrosis factor α (TNF-α), IL-1β, and IL-6 in mouse serum were measured using enzyme-linked immunosorbent assay (ELISA) kits from Boster according to the instructions provided by the manufacturer, and the expression levels of endostatin and VEGF were measured using ELISA kits from USCN Life Science (Wuhan, China) as recommended. Each experiment was repeated at least five times.
The right lower lung specimens were fixed in 4% paraformaldehyde solution at 4°C for 24 h. Paraffin-embedded sections (5 μm) were stained with hematoxylin-eosin, mounted, and observed with Olympus optical microscope (Olympus). Each slide was evaluated by two expert investigators blinded to the experiment groups. Lung injury was evaluated in 10 fields to grade the injury degree based on a modified scoring system (16), which includes four different categories, that is, edema, hemorrhage, leukocyte infiltration, and alveolar septal thickening. Each category was scored from 0 to 4, and the total lung injury score was calculated by adding the individual scores for each category.
Lung wet-to-dry weight ratio
The right upper lung tissues were rapidly collected and weighed on an electronic scale after removing the surface water and blood using absorbent paper. The tissues were then dried to constant weight at 80°C for 72 h and weighed again. The wet-to-dry (W/D) weight ratio was calculated.
Pulmonary microvascular leakage test
Pulmonary microvascular leakage test was performed using Evans blue (EB) (Dingguo Biotechnology Co, Ltd, Beijing, China) dye overflow techniques (17). In brief, mice were injected 50 μg/g EB through the tail vein. Thirty minutes after injection, they were infused with 10 mL PBS to rinse pulmonary circulation. Their lung tissues were then collected, rinsed with PBS, and rapidly frozen in liquid nitrogen. The frozen lung tissues were homogenized on ice in PBS, incubated with formamide at 60°C for 16 h, and centrifuged at 7,000 g for 25 min. The supernatants were collected, and their absorptions at 620 nm were measured to calculate EB content.
Western blot analysis of protein expression
Thirty micrograms of protein of lung supernatants was mixed with two times the sodium dodecyl sulfate sample buffer, incubated at 100°C for 5 min, subjected to 10% to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride membranes. After 2-h blocking at room temperature, the membranes were incubated overnight with appropriate primary antibodies at 4°C. After washing three times with Tris-buffered saline with Tween for 5 min each, the membranes were then incubated with appropriate horseradish peroxidase–conjugated secondary antibodies for 1 h. After washing three times with Tris-buffered saline with Tween for 5 min each, the labeled proteins were visualized using chemiluminescence and exposed on x-ray film, and their expressions were quantitatively analyzed using optical density scanning. All antibodies were from Cell Signaling (Danvers, Mass).
Data were expressed as mean ± SD. Multiple comparisons were analyzed for significant differences by using the one-way analysis of variance with Turkey post hoc test. The Mann-Whitney U test was applied to data with nonnormal distributions. Kaplan-Meier plots were used to illustrate survival of mice between treatment groups, and statistical assessment was performed by the log-rank test. All tests were two-sided, and significance was accepted at P < 0.05. GraphPad Prism version 5.02 (GraphPad Prism Software Inc, San Diego, Calif) was used for data analysis and figure preparation.
Endostatin improves survival and organ function of CLP mice
First, we observed the effects of endostatin on survival of mice with CLP-induced sepsis. Application of different doses of endostatin immediately after operation (Fig. 1A) significantly improved the survival of septic mice compared with control mice in a dose-dependent manner (P < 0.05), as demonstrated that the septic mice treated with 2 and 5 mg/kg endostatin had higher survival rate than did the septic mice treated with 0.2 mg/kg endostatin (P < 0.01). Moreover, the impact of endostatin on the survival of septic mice also showed a time-dependent manner (Fig. 1B). Septic mice treated with endostatin immediately and at 3 h after operation had significantly higher survival rate than did the septic mice without endostatin treatment (P < 0.05). In addition, septic mice treated with endostatin at 6 and 12 h after operation also had slightly higher survival rate (53.3% and 46.6%) than did the untreated septic mice, although not reaching statistical significance.
Because sepsis-induced fatal injury is often associated with multiple organ damage, we examined the serum markers commonly seen in patients with organ dysfunction in clinics. We used serum Cre and BUN to evaluate renal function. As shown in Figure 2, A and B, endostatin treatment of septic mice significantly lowered the levels of serum Cre and BUN at 3, 6, 12, and 24 h after operation compared with untreated septic mice (P < 0.05), suggesting that endostatin has a protective effect on renal function. Similarly, endostatin treatment significantly reduced the levels of serum ALT and AST, the two indicators of liver function, at 3, 6, 12, and 24 h after operation in septic mice (Fig. 2, C and D, P < 0.05), prompting that application of endostatin can reduce liver damage in CLP-induced septic mice. The protective effects of endostatin were also seen in the myocardial injury indicators CK-MB and LDH, showing significant differences in CK-MB at 6 and 12 h after operation and in LDH at 12 and 24 h after operation (Fig. 2, E and F).
Among the multiple organ dysfunctions in sepsis, lung damage is often the first sign and shows the highest incidence (18). Evans blue leakage in lung tissues of septic mice at 12 h after operation was significantly increased compared with that of sham-operated mice, and application of endostatin significantly inhibited this increase (Fig. 3A, P < 0.05). Similar effects and trends were also seen in lung W/D weight ratio (Fig. 3B, P < 0.05). Histological examination revealed no evidence of lung injury in the sham group, but different degrees of lung injury with marked edema, hemorrhage, leukocyte infiltration, and alveolar septal thickening in the septic groups. In addition, the degrees of hemorrhage, leukocyte infiltration, and alveolar septal thickening were much lower in endostatin-treated septic mice than in untreated septic mice, with statistical significance in the lung injury score between the two groups (Fig. 3C, P < 0.05), indicating that endostatin treatment significantly reduced pathological lesions of lung tissues in septic mice. The representative photomicrographs of each group are presented in Figure 3D.
Overall, these results strongly suggest that endostatin can significantly increase the survival rate of mice with CLP-induced sepsis and mitigate sepsis-caused multiple organ damage in mice.
Endostatin affects the levels of serum VEGF and inflammatory mediators in septic mice
To evaluate the actual changes of endostatin during the development of sepsis in mice, we collected serum of septic mice at different postoperation time and measured dynamic changes of endogenous endostatin by ELISA. As shown in Figure 4A, serum endostatin level was significantly enhanced at 3, 6, 12, and 24 h after operation compared with the baseline (P < 0.05) and returned to basal level at 48 h after operation (P > 0.05).
Because endostatin has an antagonistic role of VEGF and is relevant with the body's inflammation and vascular permeability, to understand the effects of endostatin on VEGF and their relationship, we examined serum levels of VEGF-A and VEGF-C in sham-operated mice, septic mice, and endostatin-treated septic mice at different time points of postoperation. The results showed that serum VEGF-A level in septic mice was significantly higher at each time point than that of sham-operated mice, and this enhancement was not affected by endostatin treatment (Fig. 4B). In addition, VEGF-C level in septic mice was significantly increased at multiple postoperation time points, and this enhancement was reversed to basal level by endostatin intervention (P < 0.05, Fig. 4C), suggesting that endostatin treatment could significantly inhibit the increase in serum VEGF-C, but not VEGF-A, in septic mice.
Excessive inflammatory response is an important mechanism leading to organ dysfunction and poor prognosis in sepsis because it could significantly promote the production of inflammatory mediators such as IL-1β, TNF-α, and IL-6 (18). In this study, the serum levels of these inflammatory mediators at different postoperation time points were examined to explore the effect of endostatin on excessive inflammatory responses in sepsis and its possible underlying mechanisms. The results showed that serum levels of IL-1β, TNF-α, and IL-6 in CLP-induced septic mice were significantly increased compared with those of sham-operated mice, and endostatin treatment significantly lowered the levels of these inflammatory cytokines (P < 0.05, Fig. 4D), prompting that endostatin could inhibit the upregulation of these inflammatory mediators in septic mice.
Effects of endostatin on mitogen-activated protein kinase signaling pathway
ERK, JNK, and p38 mitogen-activated protein kinase (MAPK) signaling pathways are important for the body's inflammatory responses. They could be activated in development of sepsis. To further study the mechanisms of endostatin on sepsis, we measured the changes in phosphorylation of JNK, p38, and ERKl/2 proteins in lung tissues of mice with sepsis. Figure 5A shows the immunoblot results of the total and phosphorylated p38, ERK1/2, and JNK in lung tissues of shame-operated mice, septic mice, and endostatin-treated mice at different postoperation times. Semiquantitative gray analysis showed that all the three proteins were activated at 1 h after operation and remained in active state until 24 h after operation. In addition, endostatin treatment could inhibit sepsis-induced phosphorylation of p38, significantly reduce the ratio of phosphorylated and total p38 (Fig. 5B, P < 0.05), and markedly inhibit sepsis-induced ERK phosphorylation, especially at 1, 6, 12, and 24 h after operation (Fig. 5C, P < 0.05). However, endostatin treatment apparently showed no significant effects on sepsis-induced JNK phosphorylation (Fig. 5D).
Endostatin is an endogenous angiogenesis inhibitor discovered nearly 20 years ago. Numerous animal and clinical studies have shown that endostatin had a good ability to inhibit the growth of more than 60 kinds of tumors (19). Endostatin-targeted pathways are associated with the pathological and physiological processes of inflammation; thus, besides its wide application in antitumor therapy in clinics, endostatin is also used in basic researches for chronic inflammatory diseases and has achieved notable results in animal and cell experiments. However, its application for treatment of sepsis has not been reported.
In this study, we intentionally used a sepsis model with a high mortality rate. The reason for using a severe model of sepsis was that we wanted to examine if endostatin would have any salutary effects on the survival of animals with severe sepsis. We also postulated that if endostatin helps even in a model of severe sepsis it will also be effective in a model of mild sepsis. Accordingly, we examined a dose-effect relationship between endostatin and survival rates. The results clearly showed that endostatin could reduce mortality of septic mice in a dose-dependent manner. In this experiment, the dosage of endostatin was up to 5 mg/kg, strikingly below its maximum dosage to have adverse effects in antitumor therapy (100–500 mg/kg per day) (20). This low-dose treatment may be more secure and possible to reduce the adverse effects. For drugs used in clinics to treat sepsis, we expect valid effects upon delayed administration because diagnosis and treatment often occur after onset of the disease. Therefore, we examined its time responses and found that administration of endostatin as late as 3 h after onset of sepsis still could significantly increase the survival rate of septic mice. Although administration of endostatin at 6 and 12 h after operation still has a tendency to increase the survival rate of septic mice, its effect gradually declined with delayed administration. All these may be related to its inhibitory effects on inflammatory responses at early stage of sepsis, vascular permeability, and other pathophysiological processes. We speculate that patients who were identified at earlier course of sepsis might benefit more from endostatin treatment. In addition, in clinical practice, all patients with sepsis received antibiotics as a part of their therapies. In this study, however, we did not add antibiotics because we wished to examine the effects of endostatin per se without the contributory effect of antibiotics.
The functions of main body organs are often compromised during sepsis. Consistent with previous reports, we also observed impaired performances of a number of organ systems including liver, kidney, heart, and lung in mice with CLP-induced sepsis, and application of endostatin treatment protected septic mice from such damages. These protective roles were significant at early time points (0–24 h), but not statistically significant in multiple serum indicators at 48 h after operation. This may be due to the smaller number of mice that survived to 48 h after operation, thus limiting the significance of statistic analysis. Meanwhile, most of these mice had strong resistance to sepsis and mild organ damages. The protective effects of endostatin on lung injury in septic mice were significant. Endostatin treatment improved EB leakage and reduced water content in lung tissues of septic mice. Moreover, lung tissue biopsy also showed that endostatin treatment significantly improved the pathological manifestations of septic mice such as widened interstitium, alveolar collapse, and accumulation of inflammatory cells. These results suggest that endostatin has significant protective effects on multiple organ dysfunction in septic mice.
Excessive inflammation is an important pathophysiological change in sepsis. We for the first time demonstrated that endostatin reduced serum levels of inflammatory cytokines such as IL-1β, TNF-α, and IL-6 in septic mice, suggesting that the protective effects of endostatin on mortality and organ dysfunction of septic mice might be related with its inhibition on the body's excessive inflammatory responses.
In recent years, some researchers applied endostatin for intervention of chronic inflammatory diseases and found that endostatin has the ability to improve vascular permeability and reduce the release of inflammatory mediators and infiltration of local inflammatory cells (8, 10–12, 21, 22). In this study, we also found that endostatin could increase the survival rate and reduce multiple organ damages of mice with sepsis, and these effects may be related to its ability to inhibit systemic inflammation responses and vascular permeability. Perkins et al. (23) found that endostatin content was elevated in serum and/or bronchoalveolar lavage fluid of patients with acute lung injury (ALI), consistent with our observation that endostatin level was increased in mice with CLP-induced sepsis. Different from Perkins et al., we believe that increased endostatin level in patients with ALI and septic mice is a compensatory protective response against injury rather than a harmful factor leading to ALI and sepsis.
Vascular endothelial growth factor is a molecule closely related to the body's inflammation, increased vascular permeability, and angiogenesis. Its family consists of VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF (phosphatidylinositol-glycan biosynthesis class F), and so on. Studies have found that increased serum VEGF-A level is related to the severity of sepsis (24), and application of VEGF antibody can reduce the mortality of animals with severe sepsis and mitigate the body's inflammatory responses (25). Although endostatin is often applied as an antagonist of VEGF-induced changes in vascular permeability and inflammation, we found that endostatin had no significant effects on upregulation of serum VEGF-A in mice with sepsis, suggesting that endostatin may exert its protective effects by competitively binding to VEGF receptor rather than lowering serum VEGF-A level, therefore blocking the biological effectiveness of VEGF-A (14). Vascular endothelial growth factor C could stimulate endothelial cell migration, increase vascular permeability, promote proliferation of lymphatic endothelial cells (26), and participate in lymphangiogenesis. Recently, Zhang et al. (27) found VEGF-C level was enhanced in gram-negative bacteria– or LPS-treated mice and patients with sepsis. However, the significance of VEGF-C in sepsis is not clear. We found that serum VEGF-C level was significantly higher in septic mice, and application of endostatin reversed this enhancement, which probably has the following significance: (i) VEGF-C may be the causative factor of systemic inflammatory responses and increased vascular permeability in sepsis; (ii) besides binding to VEGF receptor to block the biological functions of VEGF-C, endostatin could directly downregulate VEGF-C expression in septic mice, thus playing the downstream effects of anti-inflammation and inhibiting increased vascular permeability. However, which cells are most important for endostatin's effects are still unknown and need further studies.
It is well known that MAPKs are important signal transduction mediators in organisms and have crucial roles in all cellular processes. At present, the most widely studied MAPK signal transduction pathways include JNK, p38, and ERKl/2 pathways. Activation of one or more of these pathways could initiate inflammatory cascades in sepsis (28). A number of studies have found that endostatin could inhibit phosphorylation of MAPKs at the cellular level, resulting in mitigation of vascular permeability and/or angiogenesis (11, 29–31). Our study found that in mice with sepsis endostatin could decrease ERK and p38 phosphorylation in lung tissues. Based on literature and our findings, it is reasonable to speculate that endostatin exerts its protective functions probably through the following mechanisms: on the one hand, it may block VEGF receptor, thereby inhibiting the activation of its downstream inflammatory MAPK signaling pathways and increased microvascular permeability; on the other hand, it may directly or indirectly inhibit VEGF-C expression, thus breaking the vicious cycle in the pathogenesis of sepsis. The study also found that phosphorylation of JNK in lung tissues of septic mice is increased, but this increase is not affected by endostatin treatment. At present, only a few scholars studied the relationship between endostatin and JNK pathway (31). Thus, the effects of endostatin on JNK phosphorylation in sepsis need to be further studied.
To our best knowledge, this is the first report evaluating the effects of endostatin on mouse sepsis model and revealed the potential roles for endostatin in the treatment of sepsis. However, there were some limitations: First, in our sepsis models, the majority of deaths occur within 24 to 48 h. Because this phenomenon is typical in septic mice at hyperinflammatory phase, endostatin might be beneficial in mice at this phase. In clinical sepsis, many deaths are due to suppressing immunity and later hospital-acquired infections. Whether implications of endostatin have effects on immunity in sepsis is unclear and needs to be further investigated. Second, surgical patients are prone to suffering from sepsis, and wound healing is an unignorable issue. Although a few researches have shown that endostatin can delay long-bone development and excisional cutaneous wound healing (32), its influence on these sepsis groups needs to be further explored.
All in all, endostatin can increase the survival rate of septic mice in a dose- and time-dependent manner. Meanwhile, in septic mice, it can (i) reduce serum indicators of multiple organ damages as well as lung tissue EB leakage, W/D weight ratio, and inflammation; (ii) decrease serum TNF-α, IL-1β, IL-6, and VEGF-C levels; and (iii) inhibit phosphorylation of p38 and ERK1/2 in lung tissues. To our knowledge, the study for the first time demonstrated the protective effects of endostatin on sepsis and analyzed its possible mechanisms from the aspects of inhibiting inflammatory responses, blocking VEGF receptor, and reducing VEGF-C expression, as well as their resulted decrease in vascular permeability. Thus, the study provides a new research idea for endostatin treatment of sepsis.
This study was performed at the Translational Medicine Center of Sepsis, Department of Pathophysiology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, People's Republic of China.
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