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RGD PEPTIDES PROTECTS AGAINST ACUTE LUNG INJURY IN SEPTIC MICE THROUGH WISP1-INTEGRIN "6 PATHWAY INHIBITION Xibing Ding,* Xin Wang,† Xiang Zhao,* Shuqing Jin,† Yao Tong,† Hao Ren,† Zhixia Chen,* and Quan Li* *Department of Anesthesiology, East Hospital; and † Department of Anesthesiology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, China Received 30 Aug 2014; first review completed 16 Sep 2014; accepted in final form 5 Dec 2014 ABSTRACT—Acute lung injury is a common consequence of sepsis, a life-threatening inflammatory response caused by severe infection. In this study, we elucidate the attenuating effects of synthetic Arg-Gly-Asp-Ser peptides (RGDs) on acute lung injury in a sepsis mouse model. We further reveal that the beneficial effects of RGDs stem from their negative regulation of the Wisp1 (WNT1-inducible signaling pathway)Yintegrin "6 pathway. After inducing sepsis using cecal ligation and puncture (CLP), mice were randomized into experimental and control groups, and survival rates were recorded over 7 days, whereas only 20% of mice subjected to CLP survived when compared with untreated controls; the addition of RGDs to this treatment regimen dramatically increased the survival rate to 80%. Histological analysis revealed acute lung injury in CLPtreated mice, whereas those subjected to the combined treatment of CLP and RGDs showed a considerable decrease in lung injury severity. The addition of RGDs also dramatically attenuated other common sepsis-associated effects, such as increased white blood cell number in bronchoalveolar lavage fluid and decreased pulmonary capillary barrier function. Furthermore, treatment with RGDs decreased the serum and bronchoalveolar lavage fluid levels of inflammatory cytokines such as tumor necrosis factor ! and interleukin 6, contrary to the CLP treatment alone that increased the levels of these proteins. Interestingly, however, RGDs had no detectable effect on bacterial invasion following sepsis induction. In addition, mice treated with RGDs showed decreased levels of wisp1 and integrin "6 when compared with CLP-treated mice. In the present study, a linkage between Wisp1 and integrin "6 was evaluated in vivo. Most strikingly, RGDs resulted in a decreased association of Wisp1 with integrin "6 based on coimmunoprecipitation analyses. These data suggest that RGDs ameliorate acute lung injury in a sepsis mouse model by inhibiting the Wisp1Yintegrin "6 pathway. KEYWORDS—Arg-Gly-Asp (RGDs), acute lung injury (ALI), cecal ligation and puncture (CLP), sepsis, Wisp1, integrin "6 ABBREVIATIONS—ALI V acute lung injury; ARDS V acute respiratory distress syndrome; BALF V bronchoalveolar lavage fluid; CLP V cecal ligation and puncture; EBA V Evans blue albumin; ECM V extracellular matrix; ELISA V enzyme-linked immunosorbent assay; IL-6 V interleukin 6; RGD V Arg-Gly-Asp-Ser; TNF-! V tumor necrosis factor !; Wisp1 V WNT1-inducible signaling pathway
INTRODUCTION
The Wisp1 (WNT1-inducible signaling pathway) protein is a cysteine-rich, secreted matricellular protein. Matricellular proteins are a subset of the extracellular matrix (ECM) proteins responsible for modulating cellular responses, such as cell growth, differentiation, and survival, but do not exhibit structural function (7). Wisp1 was initially identified in carcinoma tissues, but subsequent studies found an association between Wisp1 expression and nonmalignant diseases, in particular those involving the heart, bone, and lung. Study of ventilator-induced lung injury revealed Wisp1 upregulation (8), and Wisp1 was found to be primarily expressed in the alveolar epithelium in experimental and human idiopathic pulmonary disease (9). Cells interact with ECM proteins through a variety of adhesion receptors, including integrins. During the process of acute lung injury, the "6 integrin family member is known to play an important role in regulating lung inflammation, macrophage protease expression, and pulmonary edema. The "6 integrins recognize their ligands through the linear tripeptide sequence arginine-glycine-aspartic acid (RGD), contained within the ligands (10). As such, proteins containing the RGD sequence can compete with adhesive proteins for binding to these integrin receptors (11), thereby inhibiting the integrin-related functions in different cell systems. Previous reports have suggested the possibility that RGD-containing proteins may alter systemic
Sepsis and septic shock are major causes of morbidity and mortality among patients with severe trauma, burns, or blood loss (1). Sepsis is a complex pathophysiological condition characterized by profound hypotension, progressive metabolic acidosis, systemic inflammatory response syndrome, tissue damage, multiple organ dysfunction syndrome, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), and even death (2). Due to the lack of effective treatments for severe sepsis, the mortality rates of patients suffering from this condition are extremely high (3, 4), due in part to the hyperactivation of inflammatory cytokines (5). Controlling the release of inflammatory cytokines during acute respiratory failure is an important therapeutic target for treating sepsis. While there have been clinical trials aimed at controlling inflammation by modulating cytokine levels, an effective pharmacologic agent has not been successfully developed (6).
Address reprint requests to Quan Li, MD, PhD, Department of Anesthesiology, East Hospital, Tongji University School of Medicine, 150 Jimo Rd, Pudong, Shanghai, China. E-mail: [email protected]. Funding: National Natural Science Foundation (81270135) and Shanghai Education Committee Key Project (13ZZ024). DOI: 10.1097/SHK.0000000000000313 Copyright Ó 2015 by the Shock Society
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inflammation by attenuating lipopolysaccharides (LPSs) and mechanical ventilationYinduced lung injury (12, 13). However, studies have not yet addressed the effect of RGDs on pulmonary inflammation during cecal ligation and puncture (CLP)Yinduced lung injury. In this study, we used an animal model of polymicrobial sepsis that utilizes induction of ALI by CLP to test whether RGD peptides (RGDs) prevent CLP-induced ALI through inhibition of the Wisp1Yintegrin "6 pathway. MATERIALS AND METHODS Experimental animals All procedures were approved by the Institutional Animal Care and Use Committee of Tongji University. Male pathogen-free C57BL/6 mice (8Y12 weeks old) were obtained from the Laboratory Animal Research Center of Shanghai. The animals were housed in cages in an air-conditioned room (20-C T 1-C) with controlled 12-h light-dark cycles and ad libitum access to water and rat chow (Global Diet, Shanghai, China). All animal handling procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Bethesda, Md).
Experimental design The mice were divided into seven groups: (A) sham-operated control group, (B) sham-operated control group with RGD treatment, (C) CLP treatment group, (D) RGD and CLP combined treatment group, (E) sham-operated control group with immunoglobulin G (IgG) treatment, (F) Wisp1 antibody and CLP combined treatment group, and (G) "6 antibody and CLP combined treatment group. Each distinct treatment group was housed in separate cages.
Sepsis model Polymicrobial sepsis was induced by the established cecal ligation and twohole puncture method described by Wichterman et al. (14), with the following minor modifications (15). The mice were anesthetized by i.p. administration of 100 mg/kg ketamine and 10 mg/kg xylazine. After the abdominal fur was shaved, a 2-cm midline incision was made through the skin and peritoneum. The cecum was then isolated and ligated with a 4-0 silk ligature at half the distance between the distal pole and the base of the cecum. Cecal puncture (Bthrough-and-through[) was initiated at the mesentery and proceeded in the antimesenteric direction after medium ligation by a 21-gauge needle. The cecum was then returned to the peritoneal cavity, and the abdominal incision was closed with a 4-0 sterile synthetic absorbable suture. Laparotomies were performed on the sham-operated control groups, and while the cecum was manipulated, it was neither ligated nor perforated. All animals were injected subcutaneously with prewarmed normal saline (37-C; 5 mL/100 g body weight) for fluid resuscitation, and buprenorphine (0.05 mg/kg body weight) for postoperative analgesia. Mice in the control + RGD and CLP + RGD groups received 5 mg/kg of RGDs via i.p. injection in saline. RGDs were administered once 1 h following CLP treatment (13). An equal volume of saline was administered to the control and CLP groups as a vehicle. Mice in the CLP + Wisp1 antibody, CLP + "6 antibody, and CLP + IgG groups were administered anti-Wisp1 monoclonal antibody, anti-"6 monoclonal antibody, and serum IgG 1 h following CLP treatment (0.5 2g/g in 50-2L intratracheal instillation). Postoperatively, the mice had unlimited access to food and water. Ten mice per group were followed for 7 days to calculate their survival rate. Five mice per group were used to measure the alveolar-capillary permeability. The remaining animals were killed at 24 h after CLP using an anesthetic overdose of ketamine and xylazine. Five mice were killed at every different time (0, 6, 8, 12, 24, and 48 h) after CLP. A 20-gauge sterile catheter was inserted into the trachea to collect bronchoalveolar lavage fluid (BALF). Cardiac blood samples were immediately collected and transferred to the laboratory for analysis of tumor necrosis factor ! (TNF-!) and interleukin 6 (IL-6) levels in both serum and BALF. The lungs were rapidly removed from all mice and washed in ice-cold saline. Half of the lung tissues were stored at j80-C prior to biochemical analyses, whereas the other half of the lung tissue was fixed in 10% formalin solution in preparation for histopathologic analyses.
Measurement of alveolar-capillary permeability Alveolar-capillary permeability was assessed with Evans blue albumin (EBA) as previous described (16). Evans blue (0.5% Evans blue; SigmaAldrich, St Louis, Mo) was dissolved in Ca2+/Mg2+-free phosphate-buffered saline (PBS; Sigma-Aldrich) and conjugated to albumin (4% EBA) that was prepared by adding bovine serum albumin (Sigma-Aldrich). After thoroughly
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dissolving by gently stirring with a magnetic bar, the EBA solution was filtered through a 0.22-2m syringe filter, and aliquots were stored at j80-C until use. Each aliquot was used only once for each experiment. To evaluate alveolarcapillary barrier function, EBA (25 mg/kg body weight) was injected into the internal jugular vein 1 h before the animals were killed and lung harvesting. Blood samples were obtained from the right heart, and the pulmonary vasculature was subsequently infused with 1 mL PBS. The right lung was ligated at the level of the right mainstem bronchus, excised, blotted dry, weighed, and stored in liquid nitrogen until these samples were used for EBA analysis. After freeze/thaw, the lung tissue was homogenized in 2 mL PBS and incubated with an additional 2 mL of formamide (Sigma-Aldrich) (18 h; 60-C). Formamide extracts were centrifuged (15,000g 30 min; 4-C), and the centrifuged supernatants were collected to quantify lung EBA content using a dualwavelength (620 and 740 nm) spectrophotometric method. Pulmonary EBA absorbance at 620 nm was corrected by a correction factor with EBA absorbance at 740 nm. The EBA permeability index was calculated by dividing pulmonary EBA absorbance at 620 nm/g of lung tissue by plasma EBA absorbance at 620 nm.
Analysis of BALF The BALF procedure was performed with instillation of 1 mL Ca2+/Mg2+free PBS using 10 mice. Approximately 80% of the instilled volume was retrieved. All samples were kept on ice until processing. Recovered BAL fluids were centrifuged (1,000g 5 min, 4-C), and the supernatants were frozen immediately on dry ice before storing at j80-C. These samples were used to determine both total protein concentration using a bicinchoninic acid assay (BCA) assay, as well as total cytokine levels by enzyme-linked immunosorbent assay (ELISA). After pellets were resuspended in PBS, the total cell number was quantified using a hemocytometer.
Histopathologic examination of injured lung tissue For histological analysis, lung tissue samples were fixed in 4% paraformaldehyde in PBS overnight at 4-C. The samples were then dehydrated, embedded in paraffin, and cut into 5-2m sections. After deparaffinization, the tissues were stained with hematoxylin-eosin for histological analysis. To summarize briefly, the histological alterations of lung parenchyma were quantitatively graded on a scale from 0 to 4 (0, absent and appears normal; 1, light; 2, moderate; 3, strong; and 4, intense) for four items: alveolar congestion, hemorrhage, infiltration or aggregation of neutrophils in airspaces or vessel walls, and thickness of alveolar wall membrane formation. We calculated the sum of all grades for each parameter as the total score, ranging between 0 (least severe) and 16 (most severe).
Measurement of lung wet-to-dry weight ratio The severity of pulmonary edema was assessed by determining the wet-todry (W/D) weight ratio of the lung. The left lung was weighed before drying in a drying oven at 65-C for 48 h. The lung was then reweighed, and the W/D weight ratio was calculated.
Bacterial counts Bacterial load was determined in blood and peritoneal fluid at 24 h following CLP. Briefly, blood and peritoneal fluid were serially diluted and plated on tryptic soy agar plates before incubating at 37-C for 48 h. The number of colony forming units per milliliter was quantified to determine bacterial load.
Measurement of TNF-! and IL-6 cytokine in serum and BALF Serum and BALF samples collected from each of the groups were separated and stored at j80-C prior to analysis. Interleukin 6 and TNF-! levels were measured using highly sensitive ELISA kits that have been specifically tailored towards analysis of mouse cytokines (R&D Systems, Minneapolis, Minn).
Western blot analysis The Western blotting for Wisp1 and integrin "6 were performed as previously described (8). Frozen lung tissues were thawed and homogenized in radioimmunoprecipitation lysis buffer, protease inhibitors (Roche, Mannheim, Germany), and phenylmethylsulfonyl fluoride. Protein concentrations were subsequently determined by standard BCA assay. After addition of 6 sodium dodecyl sulfate (SDS) loading buffer, equivalent amounts of protein were heated (100-C; 5 min) and separated by gel electrophoresis using a 10% SDSYpolyacrylamide electrophoresis gel. Resolved proteins were then transferred to a nitrocellulose membrane and blocked with Tris-buffered saline containing Tween-20 (TBST) and 5% nonfat milk (1 h; 24-C). Nitrocellulose membranes were incubated overnight at 4-C with either rabbit polyclonal primary antibody against Wisp1 (ab178547; Abcam, Hong Kong, China), rat monoclonal primary antibody against integrin "6 (MAB 2389; R&D Systems), or mouse monoclonal antibody against "-actin(ab8226; Abcam). The membranes
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FIG. 1. Wisp1 expression in lung at different time point post-CLP. Wisp1 expression in lung at 0, 6, 8, 12, 24, and 48 h by RT-PCR and Western blot. It increased significantly, peaking at 24 h after CLP, a finding that is consistent with IL-6 in BALF by ELISA. The expression of Wisp1 in lung and IL-6 secretion in BALF decreased gradually at 48 h after CLP. Graphed values represent the mean T SD. Five mice were analyzed per group. *P G 0.05, **P G 0.01, ***P G 0.001.
were washed in TBST three times, incubated with secondary antibody (926-32219 IRDye 800CW goat antiYrat IgG and 926-32221 IRDye 680 antiYrabbit secondary antibody; Licor Biosciences, Lincoln, Neb) for 1 h at 37-C and then washed in TBST three additional times. The membranes were determined by using an Odyssey image analysis system (Licor Biosciences). Western blots were quantitated using Quantity One software (Bio-Rad, Foster City, Calif) and normalized to "-actin signal.
Coimmunoprecipitation Lung samples were lysed with NP-40 buffer containing protease inhibitors (Roche) and phenylmethylsulfonyl fluoride. Protein concentrations were determined using BCA protein assay. Equal protein amounts of supernatants were then incubated overnight with protein A/G beads in the presence of antiWisp1 (sc-8866; Santa Cruz Biotechnology, Santa Cruz, Calif) or anti-"6 (sc15329; Santa Cruz Biotechnology), respectively. After centrifugation and addition of 6 SDS loading buffer, equivalent amounts of protein were heated (100-C; 5 min) and separated by gel electrophoresis using a 10% SDSYpolyacrylamide electrophoresis gel. Resolved proteins were then transferred to a nitrocellulose membrane and blocked with TBST and 5% nonfat milk (1 h; 24-C). Nitrocellulose membranes were incubated overnight at 4-C with either rat monoclonal primary antibody against integrin "6 (MAB 2389; R&D Systems) or goat polyclonal primary antibody against Wisp1 (sc-8866; Santa Cruz Biotechnology). The membranes were washed in TBST three times, incubated with secondary antibody for 1 h at 37-C and then washed in TBST three additional times. The membranes were determined by using an Odyssey image analysis system (Licor Biosciences).
Reverse transcriptionYpolymerase chain reaction Total RNA was extracted from lung tissues using the TRIzol reagent (Sigma-Aldrich) and following the manufacturer_s instructions. Total RNA was then reverse transcribed using a PrimeScript RT reagent kit (TaKaRa Bio Inc, Shiga, Japan). Primers for wisp1 amplification (137 bp) were as follows: position 280 forward 5¶-CAGCACCACTAGAGGAAACGA-3¶, position 394 reverse 5¶-CTGGGCACATATCTTACAGCATT-3¶. Primers for integrin "6 amplification (186 base pairs) were as follows: position 1266 forward 5¶-CAACTA TCGGCCAACTCATTGA-3¶, position 1324 reverse 5¶-GCAGTTCTTCATAA GCGGAGAT-3¶. Primers for "-actin amplification (154 base pairs) were as follows: position 163 forward 5¶-GGCTGTATTCCCCTCCATCG-3¶, position 295 reverse 5¶-CCAGTTGGTAACAATGCCATGT-3¶. The product of reverse
transcription was amplified by following the Premix Taq version 2.0 instructions (TaKaRa Bio Inc). Polymerase chain reaction (PCR) products were separated using a 2% agarose gel and identified by SYBR green staining. Expression of mRNA was quantitated using Image Lab software (Bio-Rad) and normalized to the "-actin signal.
Statistical analysis Survival rates were evaluated using the Kaplan-Meier method, and significance was determined by the generalized Wilcoxon method. Data are presented as the mean T SD and analyzed using either a one-way or two-way analysis of variance; post hoc testing was performed with Bonferroni correction of the t test. The individual studies performed throughout this work represent at least five independent studies. Power analyses were performed by using a type I error probability of 0.05, with a power of 0.9, to determine the sample size necessary to reject the null hypothesis. All statistical analyses were carried out using the GraphPad Prism 5 program.
RESULTS Wisp1 expression in lung at different time after CLP
Lung tissues were harvested 0, 6, 8, 12, 24, and 48 h after CLP to determine Wisp1 expression using reverse transcription (RT)YPCR and Western Blot. Wisp1 expression in lung increased significantly, peaking at 24 h after CLP (Fig. 1, A and B), a finding that is consistent with IL-6 in BALF by ELISA (Fig. 1C). It means Wisp1 can reflect the lung injury like IL-6 in BALF. But the expression of Wisp1 in lung and IL-6 secretion in BALF decreased gradually at 48 h after CLP. So we chose 24 h as the time point in the next experiment. RGDs improve the survival rates of CLP-treated mice
We assessed and compared the life span of CLP-treated mice with control and CLP-treated mice over a 7-day period. We found an 80% mortality rate for the CLP-treated group
FIG. 2. RGDs prolong the life span of CLP-treated mice. Cecal ligation and punctureYtreated mice showed significantly reduced survival rates when compared with control mice. When RGDs (5 mg/kg), anti-Wisp1, and anti-"6 were added in with CLP treatment; however, survival was dramatically improved. Ten mice were analyzed per treatment group.
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FIG. 3. RGDs decrease the levels of TNF-! and IL-6 in serum and BALF. Serum and BALF samples were collected from mice in the untreated control, control + RGD, CLP, and CLP + RGD groups. A significant increase in both TNF-! and IL-6 protein levels from both sample types was seen in CLP-treated mice. Nevertheless, the addition of RGDs (5 mg/kg) to CLP-treated mice significantly reduced the levels of TNF-! and IL-6. Graphed values represent the mean T SD. Five mice were analyzed per treatment group. For comparing the CLP group with the untreated control group, *P G 0.05, **P G 0.01, ***P G 0.001. For comparing the CLP + RGD group with the CLP group, †P G 0.05, †††P G 0.001.
over this time period, compared with a significantly reduced 20% mortality rate in mice treated with RGDs in addition to CLP (P G 0.01). We also observed a significantly reduced 50% and 40% mortality rate in mice treated with Wisp1 antibody (P G 0.05) and "6 antibody (P G 0.05), respectively. All control, control + RGD, and control + IgG mice survived throughout the 7-day period. The survival rate of the CLP group was significantly different from that of the control group (P G 0.001) (Fig. 2).
treated mice were also treated with RGDs (CLP + RGD group) (serum TNF-!: 13.08 T 4.15 vs 84.52 T 37.62, P G 0.05; BALF TNF-!: 11.97 T 0.25 vs. 45.93 T 9.59, P G 0.05; serum IL-6: 203.10 T 93.21 vs 485.34 T 148.64, P G 0.05; BALF IL-6: 83.76 T 8.82 vs. 213.92 T 39.86, P G 0.001). When RGDs were administered without CLP treatment, the serum and BALF cytokine levels from this treatment group showed no significant deviation from the controls.
RGDs reduce TNF-! and IL-6 levels in both serum and BALF in CLP-treated mice
RGDs improve the histopathology of lung tissue in CLP-treated mice
We evaluated serum and BALF levels of the proinflammatory cytokines TNF-! and IL-6 following CLP treatment. Levels of both cytokines in serum and BALF samples were significantly increased in the CLP-treated group when compared with the control group (serum TNF-!: 84.52 T 37.62 vs. 15.21 T 13.35, P G 0.05; BALF TNF-!: 45.93 T 9.59 vs. 6.37 T 0.79, P G 0.01; serum IL-6: 485.34 T 148.64 vs. 54.08 T 12.65, P G 0.01; BALF IL-6: 213.92 T 39.86 vs. 31.95 T 4.12, P G 0.001) (Fig. 3). In contrast to the CLP-treated group, both TNF-! and IL-6 levels in serum and BALF were significantly decreased when CLP-
To determine the effect of CLP and RGD, Wisp1, and "6 antibody treatment on lung histology, lung tissue specimens were obtained 24 h after CLP treatment. No abnormal histological alterations of the lung samples were observed in the control group (Fig. 4A). In the CLP-treated lungs, however, diffuse interstitial edema and inflammatory cell infiltration were apparent (Fig. 4B). Interestingly, when RGDs were introduced into mice following CLP treatment, lung samples isolated from these mice appeared similar to the controls (Fig. 4C). In addition, Wisp1 and "6 antibody treatment also improved the
FIG. 4. RGDs rescue abnormal lung tissue histopathology in CLP-induced sepsis. AYE, Hematoxylin-eosin staining of lung tissue samples collected 24 h following no treatment (A), CLP treatment (B), CLP + RGD treatment (C), CLP + anti-Wisp1 treatment (D), or CLP + anti-"6 treatment (E). Whereas no histological alterations of the lung were observed in the untreated control group, diffuse interstitial edema and inflammatory cell infiltration were observed in the CLP group. These histological alterations were attenuated in RGD, anti-Wisp1, and anti-"6 group. F, Histological scores of lung injury in the groups. Graphs values represent the means T SDs. Five mice were analyzed per group. **P G 0.001, ***P G 0.001. All images were acquired using 400 magnification.
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FIG. 5. RGD peptides reduces EBA permeability of lung tissue in CLP-induced sepsis. A significant increase in EBA permeability was seen in CLPtreated mice compared with controls. Treatment with RGDs (5 mg/kg), however, significantly reduced EBA permeability back to control levels (Fig. 4A). This increase was also inhibited by anti-Wisp1 and anti-"6 (Fig. 4B). Graphed values represent the means T SDs. Five mice were analyzed per group. For comparing the CLP group with the untreated control group, **P G 0.01. For comparing the CLP + RGD group with the CLP group, ††P G 0.01.
histopathology when compared with the CLP group. Consistent with these histological findings, the histological scoring scheme that we utilized to analyze various categories of the lung tissue_s appearance revealed scores that were significantly higher following CLP treatment (1 T 1 vs. 15 T 1, P G 0.001) when compared with controls (Fig. 4F). Taken together, these data suggest that administration of RGDs reduces the severity of tissue defects following CLP-induced injury (15 T 1 vs. 8.7 T 0.6, P G 0.001) through Wisp1Yintegrin "6 signaling (Fig. 4F).
RGDs decrease EBA permeability in lung tissue from CLP-treated mice
Evans blue albumin permeability, which reflects the damage degree of pulmonary capillary well, was increased in CLPtreated mice when compared with controls (0.63 T 0.04 vs. 0.34 T 0.04, P G 0.01); however, this effect was ameliorated by RGD treatment (0.36 T 0.06 vs. 0.63 T 0.04, P G 0.01) (Fig. 5). We did not find any statistical difference between the control and the control + RGD treatment groups. The EBA permeability was
FIG. 6. RGDs decrease blood cell number, total BALF protein concentration, and lung wet/dry in CLP-induced sepsis of mice. The white blood cell number (A), total BALF protein concentration (B), and lung W/D weight ratio (C) were significantly increased in the CLP-treated group. Treatment with RGDs (5 mg/kg) in addition to CLP, but resulted in a significant reduction of these three categories. These categories were also inhibited by anti-Wisp1 and anti-"6 (DYF). Graphed values represent the means T SDs. Five mice were analyzed per group. For comparing the CLP group with the untreated control group, ***P G 0.001. For comparing the CLP + RGD group with the CLP group, †P G 0.05, ††P G 0.01.
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FIG. 7. RGDs have no effect on bacterial loads in blood and peritoneal fluid. RGD (5 mg/kg) treatment showed no significant effect on bacterial load in blood and peritoneal fluid. Graphed values represent the means T SDs. Five mice were analyzed per group.
decreased by Wisp1 and "6 antibody (P G 0.01) intratracheal instillation directly. RGDs decrease white blood cell number, total protein concentration in BALF, and lung W/D weight ratio in CLP-treated mice
White blood cell number (7.92 T 0.31 vs. 1.17 T 0.24, P G 0.001), total protein concentration in BALF (7.15 T 1.06 vs. 1.8 T 0.3, P G 0.001), and lung W/D weight ratio (8.2 T 0.79 vs. 4.1 T 0.66, P G 0.001) were found to be increased in the CLPtreated group when compared with control group. Notably, the addition of RGDs to CLP-treated mice was sufficient to suppress the increases seen in CLP alone (5.15 T 0.64 vs. 7.92 T 0.31, P G 0.01; 3.5 T 0.42 vs. 7.15 T 1.06, P G 0.01; 6.1 T 0.3 vs. 8.2 T 0.79, P G 0.05; respectively) (Fig. 6). No statistical differences were evident between the control and control + RGD groups. These indicators above were also decreased significantly when given antibodies through intratracheal instillation (Fig. 6, DYF). RGDs do not affect bacterial load in blood or peritoneal fluid
Interestingly, there were no significant differences in bacterial load between the CLP-treated group and CLP + RGD group at the 24-h time point tested (Fig. 7). Taken together, our data suggest that while RGDs modulate inflammation in sepsis, they do not affect bacterial load.
RGDs downregulate Wisp1/integrin"6 signaling in CLP-induced lung injury
Cecal ligation and puncture treatment resulted in a significant increase in both wisp1 and integrin "6 mRNA and protein expression in the lung compared with controls when analyzed 24 h after injury (wisp1 mRNA: 0.98 T 0.13 vs. 0.34 T 0.02, P G 0.01; integrin "6 mRNA: 0.88 T 0.25 vs. 0.23 T 0.06, P G 0.01; Wisp1 protein: 0.44 T 0.04 vs. 0.22 T 0.04, P G 0.05; integrin "6 protein: 0.69 T 0.03 vs. 0.34 T 0.02, P G 0.001). Conversely, treatment with RGDs 1 h following CLP induction resulted in a significant decrease in wisp1, integrin "6 mRNA, and protein expression (wisp1 mRNA: 0.44 T 0.03 vs. 0.98 T 0.13, P G 0.01; integrin "6 mRNA: 0.42 T 0.12 vs. 0.88 T 0.25, P G 0.05; Wisp1 protein: 0.27 T 0.06 vs. 0.44 T 0.04; P G 0.05; integrin "6 protein: 0.5 T 0.01 vs. 0.69 T 0.03, P G 0.01) (Fig. 8). No statistical difference was found between the control and control + RGD group. Most important, Wisp1 expression (0.67 T 0.06 vs. 0.38 T 0.04, P G 0.01) significantly decreases when treated with "6 antibody (Fig. 9). RGDs decrease the interaction between Wisp1 and integrin"6 in CLP-treated mice
Coimmunoprecipitation was performed in order to assess if Wisp1 interacts directly with"6. Lung tissue lysates were immunoprecipitated with anti-"6 or anti-Wisp1 and then analyzed for
FIG. 8. RGDs significantly decrease mRNA and protein levels of Wisp1 and integrin "6 following CLP-induced lung injury. Lung tissue was harvested from mice in untreated control, control + RGD, CLP, and CLP + RGD treatment groups. Twenty-four hours following CLP treatment, wisp1 and "6 mRNA (A, B) and protein (C, D) expressions in the lung were detected by RT-PCR and Western blotting analysis, respectively. Whereas CLP treatment caused an increase in both wisp1 and integrin "6 mRNA and protein expression, the addition of RGDs counteracted CLP’s effect. Graphed values represent the means T SDs. Five mice were analyzed per group. For comparing the CLP group with the untreated control group, *P G 0.05, **P G 0.01, ***P G 0.001. For comparing the CLP + RGD group with the CLP group, †P G 0.05, ††P G 0.01.
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FIG. 9. Anti-"6 significantly decreases mRNA and protein levels of Wisp1 following CLP-induced lung injury. Lung tissue was harvested from mice in untreated control, control + IgG, CLP, and CLP + anti-"6 treatment groups. Twenty-four hours following CLP treatment, wisp1 mRNA (B) and protein (A) expressions in the lung were detected by RT-PCR and Western blotting analysis, respectively. Whereas CLP treatment caused an increase in wisp1 mRNA and protein expression, the addition of anti-"6 counteracted CLP’s effect. Graphed values represent the means T SDs. Five mice were analyzed per group. For comparing the CLP group with the untreated control group, **P G 0.01. For comparing the CLP + anti-"6 group with the CLP group, ††P G 0.01.
Wisp1 or"6 by immunoblotting. As shown in Figure 10, Wisp1 and integrin"6 interact at baseline, and the interaction, which was enhanced by CLP, was decreased by RGD treatment. DISCUSSION In this study, we reveal that RGD peptides helps to ameliorate many of the negative effects associated with CLP-induced lung injury. Our findings also suggest that the inhibition of Wisp1 and integrin "6 expression by RGDs plays an important role in attenuation of CLP-induced lung injury. Acute lung injury/ARDS associated with sepsis is considered to be a major factor affecting the development and prognosis of critically ill patients in intensive care units (17). Approximately 40% of patients with sepsis develop ALI/ ARDS (18Y20), so it is clinically relevant to use a sepsisinduced ALI model to investigate the pathogenesis of this condition with the hope of contributing to future therapeutic advancements. Inducing sepsis in mice using CLP injury is a widely accepted model that closely mimics bowel perforation and bacterial infection prevalent in septic humans (21). The distinction between the mortality rates of the CLP + RGDYtreated mice
and the mice treated with CLP only is significant. The early release of macrophage-derived proinflammatory cytokines, such as TNF-! and IL-6, is important in the pathogenesis of septic shock and multiple organ failure (22). In our study, treatment with RGDs results in a significant decrease in proinflammatory (TNF-! and IL-6) cytokine levels following CLP treatment. These data suggest that the protective effect of RGDs in response to CLPinduced sepsis accounts for a considerable decrease in cytokinerelated lung injury, which likely contributes to the significant increase in survival rate seen for RGD-treated septic mice. We determine that CLP treatment results in a considerable amount of injury in the treated mice_s lungs when compared with the control group, whereas the severity of the injury is dramatically improved in the CLP + RGDYtreated group when compared with the CLP-treated group. We obtain similar results when we assess the expression of Wisp1 and integrin "6, which both increase significantly in CLP-treated group but are downregulated in the CLP + RGD group. Wisp1 is a secreted matricellular protein that has been associated with cell proliferation, differentiation, ECM deposition, and ECM turnover (23). Konigshoff et al. (9) reported that Wisp1 was increased in bleomycin-induced pulmonary
FIG. 10. RGDs decrease the interaction between Wisp1 and integrin"6 in CLP-treated mice. Lung tissues were from C57 mice in control, CLP, and CLP + RGD group. A, Western blot analysis (immunoblotting) for Wisp1 of lung tissue lysates coimmunoprecipitated with anti-"6(B). Reverse direction immunoprecipitation using anti-Wisp1 followed by Western blot analysis for"6. C, Immunoblot of Wisp1 and integrin"6 total protein expression used as input for the experiments in A, B. Wisp1 and integrin"6 interact at baseline, and the interaction, which was enhanced by CLP, was decreased by RGD treatment.
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fibrosis. In addition, this study revealed that Wisp1 antibody was sufficient to attenuate pulmonary fibrosis while also improving lung function and increasing survival in mice. Heise et al. (24) reported that Wisp1 mRNA increased in mechanically stretched mouse alveolar type II cells, whereas furthermore Li et al. (8) demonstrated that Wisp1 acts as an adjuvant adaptor molecule that contributes to ventilation-induced lung injury in mice, most likely by modulating and/or amplifying TLR4-mediated cellular functions. Collectively, these findings suggest that the secreted Wisp1 protein plays a critical role in the pathogenesis of acute lung injury in mice. Our in vitro study reveals that by treating RAW 264.7 cells with LPS, the increase observed in Wisp1 mRNA expression displays a time- and dose-effect relationship (unpublished results). We therefore use a CLP model to simulate in patients with clinical sepsis to study Wisp1 in vivo by using mice as a mammalian model system. Integrins are a family of cell surface receptors that mediate either cell-cell adhesion or cell-ECM interactions (10, 25). Integrins are believed to modulate cell differentiation, gene expression, proliferation, and survival. The "6 integrin subunit was first identified in cultured epithelial cells as part of the !v"6 heterodimer, which was shown to interact with fibronectin (26, 27). Breuss et al. (28) reported that "6 expression is part of an early epithelial response to injury and/or inflammation. It is particularly interesting that "6 is exclusively upregulated in epithelia that express "6 during development (distal tubules, collecting ducts, airways, alveolar epithelium). Hogmalm et al. (29) demonstrated that the absence of the "6 integrin subunit alleviates IL-1"Yinduced inflammation and injury in the lungs of infant mice. In addition, Miller et al. (30) reported that lung epithelial cell expression of "6 integrin is associated with sites of neutrophil recruitment. RGD-based strategies include antagonist drugs (peptidic or peptidomimetic) that specifically target RGD motifs and RGD conjugates, as well as the grafting of the RGD peptides to the surface of nanocarriers (31). Wang et al. (13) found that S247, an RGD-peptidomimetic agent, can significantly attenuate ventilation-induced lung injury in rats by interfering with integrins. Moon et al. (12) reported that RGDs inhibit integrindependent induction of immune cell migration into the lungs, while also inhibiting proinflammatory mediator production. In addition, we find that treating mice with RGDs following CLP induction is highly effective at reducing the inflammatory responses that have been associated with integrin signaling and MAP kinase pathway activation during LPS-induced development of acute lung injury. Our study reveals that administration of RGD peptides ameliorates acute lung injury in a mice model of sepsis induced by CLP, partially through inhibition of Wisp1 and integrin "6 expression. Our data further suggest that RGDs mediate inflammation in sepsis without affecting the bacterial load. In conclusion, we confirm that Wisp1 and integrin "6 expression levels increase in lung tissue as a response to CLP-induced sepsis in mice. Administration of RGDs is sufficient to inhibit the production of inflammatory cytokines, such as TNF-! and IL-6, while also reducing Wisp1 and integrin "6 expression and interaction. This results in the attenuation of ALI (one of the major complications that
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occur as a result of septic shock) following CLP-induced sepsis in mice. Further investigation should involve randomized clinically controlled trials that will help add further insight into the mechanism behind RGDs_ protective function during sepsis-induced ALI.
ACKNOWLEDGMENTS The authors thank the National Natural Science Foundation (81270135) and the Shanghai Education Committee Key Project (13ZZ024) for the financial support. They also thank Weifan Xiao, Yue Zhang, Ying Long, and Feng Li for their valuable discussion and technical assistance.
REFERENCES 1. Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR: Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med 29:1303Y1310, 2001. 2. Ozdulger A, Cinel I, Koksel O, Cinel L, Avlan D, Unlu A, Okcu H, Dikmengil M, Oral U: The protective effect of N-acetylcysteine on apoptotic lung injury in cecal ligation and punctureYinduced sepsis model. Shock 19:366Y372, 2003. 3. Das UN: Recent advances in the pathobiology of septicemia and septic shock. J Assoc Physicians India 48:1181Y1184, 2000. 4. Vincent JL: Is the current management of severe sepsis and septic shock really evidence based. PLoS Med 3:e346, 2006. 5. Dellinger RP, Levy MM, Carlet JM, Bion J, Parker MM, Jaeschke R: Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008. Crit Care Med 36:296Y327, 2008. 6. Riedemann NC, Guo RF, Ward PA: The enigma of sepsis. J Clin Invest 112: 460Y467, 2003. 7. Chen CC, Lau LF: Functions and mechanisms of action of CCN matricellular proteins. Int J Biochem Cell Biol 41:771Y783, 2009. 8. Li HH, Li Q, Liu P, Liu Y, Li J, Wasserloos K, Chao W, You M, Oury TD, Chhinder S, et al.: WNT1-inducible signaling pathway protein 1 contributes to ventilator-induced lung injury. Am J Respir Cell Mol Biol 47:528Y535, 2012. 9. Konigshoff M, Kramer M, Balsara N, Wilhelm J, Amarie OV, Jahn A, Rose F, Fink L, Seeger W, Schaefer L, et al.: WNT1-inducible signaling protein-1 mediates pulmonary fibrosis in mice and is upregulated in humans with idiopathic pulmonary fibrosis. J Clin Invest 119:772Y787, 2009. 10. Hynes RO: Integrins: versatility modulation, and signaling in cell adhesion. Cell 69:11Y25, 1992. 11. Chung A, Gao Q, Kao WJ: Macrophage matrix metalloproteinase-2/-9 gene and protein expression following adhesion to ECM-derived multifunctional matrices via integrin complexation. Biomaterials 28:285Y298, 2007. 12. Moon C, Han JR, Park HJ, Hah JS, Kang JL: Synthetic RGDS peptide attenuates lipopolysaccharide-induced pulmonary inflammation by inhibiting integrin signaled MAP kinase pathways. Respir Res 10:18Y32, 2009. 13. Wang B, Wan JY, Zhang L, Min S: Synthetic RGDS peptide attenuates mechanical ventilationYinduced lung injury in rats. Exp Lung Res 38:204Y210, 2012. 14. Wichterman KA, Baue AE, Chaudry IH: Sepsis and septic shockVa review of laboratory models and a proposal. J Surg Res 29:189Y201, 1980. 15. Rittirsch D, Huber-Lang MS, Flierl MA, Ward PA: Immunodesign of experimental sepsis by cecal ligation and puncture. Nat Protoc 4:31Y36, 2009. 16. Li H, Su X, Yan X, Wasserloos K, Chao W, Kaynar AM, Liu ZQ, Leikauf GD, Pitt BR, Zhang LM: Toll-like receptor 4-myeloid differentiation factor 88 signaling contributes to ventilator-induced lung injury in mice. Anesthesiology 113: 619Y629, 2010. 17. Xu L, Bao HG, Si YN, Han L, Zhang R, Cai MM, Shen Y: Effects of adiponectin on acute lung injury in cecal ligation and punctureYinduced sepsis rats. J Surg Res 183:752Y759, 2013. 18. Hudson LD, Milberg JA, Anardi D, Maunder RJ: Clinical risks for development of the acute respiratory distress syndrome. Am J Respir Crit Care Med 151:293Y301, 1995. 19. Newman JH: Sepsis and pulmonary edema. Clin Chest Med 6:371Y391, 1985. 20. Tsushima K, King LS, Aggarwal NR, De Gorordo A, D_Alessio FR, Kubo K: Acute lung injury review. Intern Med 48:621Y630, 2009. 21. Chen J, Raj N, Kim P, Andrejko KM, Deutschman CS: Intrahepatic nuclear factor-kappa B activity and alpha 1-acid glycoprotein transcription do not predict outcome after cecal ligation and puncture in the rat. Crit Care Med 29:589Y596, 2001.
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22. Damas P, Ledoux D, Nys M, Vrindts Y, De Groote D, Franchimont P, Lamy M: Cytokine serum level during severe sepsis in human IL-6 as a marker of severity. Ann Surg 215:356Y362, 1992. 23. Berschneider B, Konigshoff M: WNT1 inducible signaling pathway protein 1 (Wisp1): a novel mediator linking development and disease. Int J Biochem Cell Biol 43:306Y309, 2011. 24. Heise RL, Stober V, Cheluvaraju C, Hollingsworth JW, Garantziotis S: Mechanical stretch induces epithelialYmesenchymal transition in alveolar epithelia via hyaluronan activation of innate immunity. J Biol Chem 286:17435Y17444, 2011. 25. Giancotti FG, Mainiero F: Integrin-mediated adhesion and signaling in tumorigenesis. Biochim Biophys Acta 1198:47Y64, 1994. 26. Sheppard D, Rozzo C, Starr L, Quaranta V, Erle DJ, Pytela R: Complete amino acid sequence of a novel integrin beta subunit (beta 6) identified in epithelial cells using the polymerase chain reaction. J Biol Chem 265:11502Y11507, 1990.
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27. Busk M, Pytela R, Sheppard D: Characterization of the integrin alpha v beta 6 as a fibronectin-binding protein. J Biol Chem 267:5790Y5796, 1992. 28. Breuss JM, Gallo J, DeLisser HM, Klimanskaya IV, Folkesson HG, Pittet JF, Nishimura SL, Aldape K, Landers DV, Carpenter W: Expression of the beta 6 integrin subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling. J Cell Sci 2241Y2251, 1995. 29. Hogmalm A, Sheppard D, Lappalainen U, Bry K: beta6 Integrin subunit deficiency alleviates lung injury in a mouse model of bronchopulmonary dysplasia. Am J Respir Cell Mol Biol 43:88Y98, 2010. 30. Miller LA, Barnett NL, Sheppard D, Hyde DM: Expression of the beta6 integrin subunit is associated with sites of neutrophil influx in lung epithelium. J Histochem Cytochem 49:41Y48, 2001. 31. Danhier F, Le Breton A, Pre´at V: RGD-based strategies to target alpha(v) beta(3) integrin in cancer therapy and diagnosis. Mol Pharm 9:2961Y2973, 2012.
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RGD PEPTIDES PROTECTS AGAINST ACUTE LUNG INJURY IN SEPTIC MICE THROUGH WISP1-INTEGRIN "6 PATHWAY INHIBITION Xibing Ding,* Xin Wang,† Xiang Zhao,* Shuqing Jin,† Yao Tong,† Hao Ren,† Zhixia Chen,* and Quan Li* *Department of Anesthesiology, East Hospital; and † Department of Anesthesiology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, China Received 30 Aug 2014; first review completed 16 Sep 2014; accepted in final form 5 Dec 2014 ABSTRACT—Acute lung injury is a common consequence of sepsis, a life-threatening inflammatory response caused by severe infection. In this study, we elucidate the attenuating effects of synthetic Arg-Gly-Asp-Ser peptides (RGDs) on acute lung injury in a sepsis mouse model. We further reveal that the beneficial effects of RGDs stem from their negative regulation of the Wisp1 (WNT1-inducible signaling pathway)Yintegrin "6 pathway. After inducing sepsis using cecal ligation and puncture (CLP), mice were randomized into experimental and control groups, and survival rates were recorded over 7 days, whereas only 20% of mice subjected to CLP survived when compared with untreated controls; the addition of RGDs to this treatment regimen dramatically increased the survival rate to 80%. Histological analysis revealed acute lung injury in CLPtreated mice, whereas those subjected to the combined treatment of CLP and RGDs showed a considerable decrease in lung injury severity. The addition of RGDs also dramatically attenuated other common sepsis-associated effects, such as increased white blood cell number in bronchoalveolar lavage fluid and decreased pulmonary capillary barrier function. Furthermore, treatment with RGDs decreased the serum and bronchoalveolar lavage fluid levels of inflammatory cytokines such as tumor necrosis factor ! and interleukin 6, contrary to the CLP treatment alone that increased the levels of these proteins. Interestingly, however, RGDs had no detectable effect on bacterial invasion following sepsis induction. In addition, mice treated with RGDs showed decreased levels of wisp1 and integrin "6 when compared with CLP-treated mice. In the present study, a linkage between Wisp1 and integrin "6 was evaluated in vivo. Most strikingly, RGDs resulted in a decreased association of Wisp1 with integrin "6 based on coimmunoprecipitation analyses. These data suggest that RGDs ameliorate acute lung injury in a sepsis mouse model by inhibiting the Wisp1Yintegrin "6 pathway. KEYWORDS—Arg-Gly-Asp (RGDs), acute lung injury (ALI), cecal ligation and puncture (CLP), sepsis, Wisp1, integrin "6 ABBREVIATIONS—ALI V acute lung injury; ARDS V acute respiratory distress syndrome; BALF V bronchoalveolar lavage fluid; CLP V cecal ligation and puncture; EBA V Evans blue albumin; ECM V extracellular matrix; ELISA V enzyme-linked immunosorbent assay; IL-6 V interleukin 6; RGD V Arg-Gly-Asp-Ser; TNF-! V tumor necrosis factor !; Wisp1 V WNT1-inducible signaling pathway
INTRODUCTION
The Wisp1 (WNT1-inducible signaling pathway) protein is a cysteine-rich, secreted matricellular protein. Matricellular proteins are a subset of the extracellular matrix (ECM) proteins responsible for modulating cellular responses, such as cell growth, differentiation, and survival, but do not exhibit structural function (7). Wisp1 was initially identified in carcinoma tissues, but subsequent studies found an association between Wisp1 expression and nonmalignant diseases, in particular those involving the heart, bone, and lung. Study of ventilator-induced lung injury revealed Wisp1 upregulation (8), and Wisp1 was found to be primarily expressed in the alveolar epithelium in experimental and human idiopathic pulmonary disease (9). Cells interact with ECM proteins through a variety of adhesion receptors, including integrins. During the process of acute lung injury, the "6 integrin family member is known to play an important role in regulating lung inflammation, macrophage protease expression, and pulmonary edema. The "6 integrins recognize their ligands through the linear tripeptide sequence arginine-glycine-aspartic acid (RGD), contained within the ligands (10). As such, proteins containing the RGD sequence can compete with adhesive proteins for binding to these integrin receptors (11), thereby inhibiting the integrin-related functions in different cell systems. Previous reports have suggested the possibility that RGD-containing proteins may alter systemic
Sepsis and septic shock are major causes of morbidity and mortality among patients with severe trauma, burns, or blood loss (1). Sepsis is a complex pathophysiological condition characterized by profound hypotension, progressive metabolic acidosis, systemic inflammatory response syndrome, tissue damage, multiple organ dysfunction syndrome, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), and even death (2). Due to the lack of effective treatments for severe sepsis, the mortality rates of patients suffering from this condition are extremely high (3, 4), due in part to the hyperactivation of inflammatory cytokines (5). Controlling the release of inflammatory cytokines during acute respiratory failure is an important therapeutic target for treating sepsis. While there have been clinical trials aimed at controlling inflammation by modulating cytokine levels, an effective pharmacologic agent has not been successfully developed (6).
Address reprint requests to Quan Li, MD, PhD, Department of Anesthesiology, East Hospital, Tongji University School of Medicine, 150 Jimo Rd, Pudong, Shanghai, China. E-mail: [email protected]. Funding: National Natural Science Foundation (81270135) and Shanghai Education Committee Key Project (13ZZ024). DOI: 10.1097/SHK.0000000000000313 Copyright Ó 2015 by the Shock Society
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inflammation by attenuating lipopolysaccharides (LPSs) and mechanical ventilationYinduced lung injury (12, 13). However, studies have not yet addressed the effect of RGDs on pulmonary inflammation during cecal ligation and puncture (CLP)Yinduced lung injury. In this study, we used an animal model of polymicrobial sepsis that utilizes induction of ALI by CLP to test whether RGD peptides (RGDs) prevent CLP-induced ALI through inhibition of the Wisp1Yintegrin "6 pathway. MATERIALS AND METHODS Experimental animals All procedures were approved by the Institutional Animal Care and Use Committee of Tongji University. Male pathogen-free C57BL/6 mice (8Y12 weeks old) were obtained from the Laboratory Animal Research Center of Shanghai. The animals were housed in cages in an air-conditioned room (20-C T 1-C) with controlled 12-h light-dark cycles and ad libitum access to water and rat chow (Global Diet, Shanghai, China). All animal handling procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Bethesda, Md).
Experimental design The mice were divided into seven groups: (A) sham-operated control group, (B) sham-operated control group with RGD treatment, (C) CLP treatment group, (D) RGD and CLP combined treatment group, (E) sham-operated control group with immunoglobulin G (IgG) treatment, (F) Wisp1 antibody and CLP combined treatment group, and (G) "6 antibody and CLP combined treatment group. Each distinct treatment group was housed in separate cages.
Sepsis model Polymicrobial sepsis was induced by the established cecal ligation and twohole puncture method described by Wichterman et al. (14), with the following minor modifications (15). The mice were anesthetized by i.p. administration of 100 mg/kg ketamine and 10 mg/kg xylazine. After the abdominal fur was shaved, a 2-cm midline incision was made through the skin and peritoneum. The cecum was then isolated and ligated with a 4-0 silk ligature at half the distance between the distal pole and the base of the cecum. Cecal puncture (Bthrough-and-through[) was initiated at the mesentery and proceeded in the antimesenteric direction after medium ligation by a 21-gauge needle. The cecum was then returned to the peritoneal cavity, and the abdominal incision was closed with a 4-0 sterile synthetic absorbable suture. Laparotomies were performed on the sham-operated control groups, and while the cecum was manipulated, it was neither ligated nor perforated. All animals were injected subcutaneously with prewarmed normal saline (37-C; 5 mL/100 g body weight) for fluid resuscitation, and buprenorphine (0.05 mg/kg body weight) for postoperative analgesia. Mice in the control + RGD and CLP + RGD groups received 5 mg/kg of RGDs via i.p. injection in saline. RGDs were administered once 1 h following CLP treatment (13). An equal volume of saline was administered to the control and CLP groups as a vehicle. Mice in the CLP + Wisp1 antibody, CLP + "6 antibody, and CLP + IgG groups were administered anti-Wisp1 monoclonal antibody, anti-"6 monoclonal antibody, and serum IgG 1 h following CLP treatment (0.5 2g/g in 50-2L intratracheal instillation). Postoperatively, the mice had unlimited access to food and water. Ten mice per group were followed for 7 days to calculate their survival rate. Five mice per group were used to measure the alveolar-capillary permeability. The remaining animals were killed at 24 h after CLP using an anesthetic overdose of ketamine and xylazine. Five mice were killed at every different time (0, 6, 8, 12, 24, and 48 h) after CLP. A 20-gauge sterile catheter was inserted into the trachea to collect bronchoalveolar lavage fluid (BALF). Cardiac blood samples were immediately collected and transferred to the laboratory for analysis of tumor necrosis factor ! (TNF-!) and interleukin 6 (IL-6) levels in both serum and BALF. The lungs were rapidly removed from all mice and washed in ice-cold saline. Half of the lung tissues were stored at j80-C prior to biochemical analyses, whereas the other half of the lung tissue was fixed in 10% formalin solution in preparation for histopathologic analyses.
Measurement of alveolar-capillary permeability Alveolar-capillary permeability was assessed with Evans blue albumin (EBA) as previous described (16). Evans blue (0.5% Evans blue; SigmaAldrich, St Louis, Mo) was dissolved in Ca2+/Mg2+-free phosphate-buffered saline (PBS; Sigma-Aldrich) and conjugated to albumin (4% EBA) that was prepared by adding bovine serum albumin (Sigma-Aldrich). After thoroughly
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dissolving by gently stirring with a magnetic bar, the EBA solution was filtered through a 0.22-2m syringe filter, and aliquots were stored at j80-C until use. Each aliquot was used only once for each experiment. To evaluate alveolarcapillary barrier function, EBA (25 mg/kg body weight) was injected into the internal jugular vein 1 h before the animals were killed and lung harvesting. Blood samples were obtained from the right heart, and the pulmonary vasculature was subsequently infused with 1 mL PBS. The right lung was ligated at the level of the right mainstem bronchus, excised, blotted dry, weighed, and stored in liquid nitrogen until these samples were used for EBA analysis. After freeze/thaw, the lung tissue was homogenized in 2 mL PBS and incubated with an additional 2 mL of formamide (Sigma-Aldrich) (18 h; 60-C). Formamide extracts were centrifuged (15,000g 30 min; 4-C), and the centrifuged supernatants were collected to quantify lung EBA content using a dualwavelength (620 and 740 nm) spectrophotometric method. Pulmonary EBA absorbance at 620 nm was corrected by a correction factor with EBA absorbance at 740 nm. The EBA permeability index was calculated by dividing pulmonary EBA absorbance at 620 nm/g of lung tissue by plasma EBA absorbance at 620 nm.
Analysis of BALF The BALF procedure was performed with instillation of 1 mL Ca2+/Mg2+free PBS using 10 mice. Approximately 80% of the instilled volume was retrieved. All samples were kept on ice until processing. Recovered BAL fluids were centrifuged (1,000g 5 min, 4-C), and the supernatants were frozen immediately on dry ice before storing at j80-C. These samples were used to determine both total protein concentration using a bicinchoninic acid assay (BCA) assay, as well as total cytokine levels by enzyme-linked immunosorbent assay (ELISA). After pellets were resuspended in PBS, the total cell number was quantified using a hemocytometer.
Histopathologic examination of injured lung tissue For histological analysis, lung tissue samples were fixed in 4% paraformaldehyde in PBS overnight at 4-C. The samples were then dehydrated, embedded in paraffin, and cut into 5-2m sections. After deparaffinization, the tissues were stained with hematoxylin-eosin for histological analysis. To summarize briefly, the histological alterations of lung parenchyma were quantitatively graded on a scale from 0 to 4 (0, absent and appears normal; 1, light; 2, moderate; 3, strong; and 4, intense) for four items: alveolar congestion, hemorrhage, infiltration or aggregation of neutrophils in airspaces or vessel walls, and thickness of alveolar wall membrane formation. We calculated the sum of all grades for each parameter as the total score, ranging between 0 (least severe) and 16 (most severe).
Measurement of lung wet-to-dry weight ratio The severity of pulmonary edema was assessed by determining the wet-todry (W/D) weight ratio of the lung. The left lung was weighed before drying in a drying oven at 65-C for 48 h. The lung was then reweighed, and the W/D weight ratio was calculated.
Bacterial counts Bacterial load was determined in blood and peritoneal fluid at 24 h following CLP. Briefly, blood and peritoneal fluid were serially diluted and plated on tryptic soy agar plates before incubating at 37-C for 48 h. The number of colony forming units per milliliter was quantified to determine bacterial load.
Measurement of TNF-! and IL-6 cytokine in serum and BALF Serum and BALF samples collected from each of the groups were separated and stored at j80-C prior to analysis. Interleukin 6 and TNF-! levels were measured using highly sensitive ELISA kits that have been specifically tailored towards analysis of mouse cytokines (R&D Systems, Minneapolis, Minn).
Western blot analysis The Western blotting for Wisp1 and integrin "6 were performed as previously described (8). Frozen lung tissues were thawed and homogenized in radioimmunoprecipitation lysis buffer, protease inhibitors (Roche, Mannheim, Germany), and phenylmethylsulfonyl fluoride. Protein concentrations were subsequently determined by standard BCA assay. After addition of 6 sodium dodecyl sulfate (SDS) loading buffer, equivalent amounts of protein were heated (100-C; 5 min) and separated by gel electrophoresis using a 10% SDSYpolyacrylamide electrophoresis gel. Resolved proteins were then transferred to a nitrocellulose membrane and blocked with Tris-buffered saline containing Tween-20 (TBST) and 5% nonfat milk (1 h; 24-C). Nitrocellulose membranes were incubated overnight at 4-C with either rabbit polyclonal primary antibody against Wisp1 (ab178547; Abcam, Hong Kong, China), rat monoclonal primary antibody against integrin "6 (MAB 2389; R&D Systems), or mouse monoclonal antibody against "-actin(ab8226; Abcam). The membranes
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FIG. 1. Wisp1 expression in lung at different time point post-CLP. Wisp1 expression in lung at 0, 6, 8, 12, 24, and 48 h by RT-PCR and Western blot. It increased significantly, peaking at 24 h after CLP, a finding that is consistent with IL-6 in BALF by ELISA. The expression of Wisp1 in lung and IL-6 secretion in BALF decreased gradually at 48 h after CLP. Graphed values represent the mean T SD. Five mice were analyzed per group. *P G 0.05, **P G 0.01, ***P G 0.001.
were washed in TBST three times, incubated with secondary antibody (926-32219 IRDye 800CW goat antiYrat IgG and 926-32221 IRDye 680 antiYrabbit secondary antibody; Licor Biosciences, Lincoln, Neb) for 1 h at 37-C and then washed in TBST three additional times. The membranes were determined by using an Odyssey image analysis system (Licor Biosciences). Western blots were quantitated using Quantity One software (Bio-Rad, Foster City, Calif) and normalized to "-actin signal.
Coimmunoprecipitation Lung samples were lysed with NP-40 buffer containing protease inhibitors (Roche) and phenylmethylsulfonyl fluoride. Protein concentrations were determined using BCA protein assay. Equal protein amounts of supernatants were then incubated overnight with protein A/G beads in the presence of antiWisp1 (sc-8866; Santa Cruz Biotechnology, Santa Cruz, Calif) or anti-"6 (sc15329; Santa Cruz Biotechnology), respectively. After centrifugation and addition of 6 SDS loading buffer, equivalent amounts of protein were heated (100-C; 5 min) and separated by gel electrophoresis using a 10% SDSYpolyacrylamide electrophoresis gel. Resolved proteins were then transferred to a nitrocellulose membrane and blocked with TBST and 5% nonfat milk (1 h; 24-C). Nitrocellulose membranes were incubated overnight at 4-C with either rat monoclonal primary antibody against integrin "6 (MAB 2389; R&D Systems) or goat polyclonal primary antibody against Wisp1 (sc-8866; Santa Cruz Biotechnology). The membranes were washed in TBST three times, incubated with secondary antibody for 1 h at 37-C and then washed in TBST three additional times. The membranes were determined by using an Odyssey image analysis system (Licor Biosciences).
Reverse transcriptionYpolymerase chain reaction Total RNA was extracted from lung tissues using the TRIzol reagent (Sigma-Aldrich) and following the manufacturer_s instructions. Total RNA was then reverse transcribed using a PrimeScript RT reagent kit (TaKaRa Bio Inc, Shiga, Japan). Primers for wisp1 amplification (137 bp) were as follows: position 280 forward 5¶-CAGCACCACTAGAGGAAACGA-3¶, position 394 reverse 5¶-CTGGGCACATATCTTACAGCATT-3¶. Primers for integrin "6 amplification (186 base pairs) were as follows: position 1266 forward 5¶-CAACTA TCGGCCAACTCATTGA-3¶, position 1324 reverse 5¶-GCAGTTCTTCATAA GCGGAGAT-3¶. Primers for "-actin amplification (154 base pairs) were as follows: position 163 forward 5¶-GGCTGTATTCCCCTCCATCG-3¶, position 295 reverse 5¶-CCAGTTGGTAACAATGCCATGT-3¶. The product of reverse
transcription was amplified by following the Premix Taq version 2.0 instructions (TaKaRa Bio Inc). Polymerase chain reaction (PCR) products were separated using a 2% agarose gel and identified by SYBR green staining. Expression of mRNA was quantitated using Image Lab software (Bio-Rad) and normalized to the "-actin signal.
Statistical analysis Survival rates were evaluated using the Kaplan-Meier method, and significance was determined by the generalized Wilcoxon method. Data are presented as the mean T SD and analyzed using either a one-way or two-way analysis of variance; post hoc testing was performed with Bonferroni correction of the t test. The individual studies performed throughout this work represent at least five independent studies. Power analyses were performed by using a type I error probability of 0.05, with a power of 0.9, to determine the sample size necessary to reject the null hypothesis. All statistical analyses were carried out using the GraphPad Prism 5 program.
RESULTS Wisp1 expression in lung at different time after CLP
Lung tissues were harvested 0, 6, 8, 12, 24, and 48 h after CLP to determine Wisp1 expression using reverse transcription (RT)YPCR and Western Blot. Wisp1 expression in lung increased significantly, peaking at 24 h after CLP (Fig. 1, A and B), a finding that is consistent with IL-6 in BALF by ELISA (Fig. 1C). It means Wisp1 can reflect the lung injury like IL-6 in BALF. But the expression of Wisp1 in lung and IL-6 secretion in BALF decreased gradually at 48 h after CLP. So we chose 24 h as the time point in the next experiment. RGDs improve the survival rates of CLP-treated mice
We assessed and compared the life span of CLP-treated mice with control and CLP-treated mice over a 7-day period. We found an 80% mortality rate for the CLP-treated group
FIG. 2. RGDs prolong the life span of CLP-treated mice. Cecal ligation and punctureYtreated mice showed significantly reduced survival rates when compared with control mice. When RGDs (5 mg/kg), anti-Wisp1, and anti-"6 were added in with CLP treatment; however, survival was dramatically improved. Ten mice were analyzed per treatment group.
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FIG. 3. RGDs decrease the levels of TNF-! and IL-6 in serum and BALF. Serum and BALF samples were collected from mice in the untreated control, control + RGD, CLP, and CLP + RGD groups. A significant increase in both TNF-! and IL-6 protein levels from both sample types was seen in CLP-treated mice. Nevertheless, the addition of RGDs (5 mg/kg) to CLP-treated mice significantly reduced the levels of TNF-! and IL-6. Graphed values represent the mean T SD. Five mice were analyzed per treatment group. For comparing the CLP group with the untreated control group, *P G 0.05, **P G 0.01, ***P G 0.001. For comparing the CLP + RGD group with the CLP group, †P G 0.05, †††P G 0.001.
over this time period, compared with a significantly reduced 20% mortality rate in mice treated with RGDs in addition to CLP (P G 0.01). We also observed a significantly reduced 50% and 40% mortality rate in mice treated with Wisp1 antibody (P G 0.05) and "6 antibody (P G 0.05), respectively. All control, control + RGD, and control + IgG mice survived throughout the 7-day period. The survival rate of the CLP group was significantly different from that of the control group (P G 0.001) (Fig. 2).
treated mice were also treated with RGDs (CLP + RGD group) (serum TNF-!: 13.08 T 4.15 vs 84.52 T 37.62, P G 0.05; BALF TNF-!: 11.97 T 0.25 vs. 45.93 T 9.59, P G 0.05; serum IL-6: 203.10 T 93.21 vs 485.34 T 148.64, P G 0.05; BALF IL-6: 83.76 T 8.82 vs. 213.92 T 39.86, P G 0.001). When RGDs were administered without CLP treatment, the serum and BALF cytokine levels from this treatment group showed no significant deviation from the controls.
RGDs reduce TNF-! and IL-6 levels in both serum and BALF in CLP-treated mice
RGDs improve the histopathology of lung tissue in CLP-treated mice
We evaluated serum and BALF levels of the proinflammatory cytokines TNF-! and IL-6 following CLP treatment. Levels of both cytokines in serum and BALF samples were significantly increased in the CLP-treated group when compared with the control group (serum TNF-!: 84.52 T 37.62 vs. 15.21 T 13.35, P G 0.05; BALF TNF-!: 45.93 T 9.59 vs. 6.37 T 0.79, P G 0.01; serum IL-6: 485.34 T 148.64 vs. 54.08 T 12.65, P G 0.01; BALF IL-6: 213.92 T 39.86 vs. 31.95 T 4.12, P G 0.001) (Fig. 3). In contrast to the CLP-treated group, both TNF-! and IL-6 levels in serum and BALF were significantly decreased when CLP-
To determine the effect of CLP and RGD, Wisp1, and "6 antibody treatment on lung histology, lung tissue specimens were obtained 24 h after CLP treatment. No abnormal histological alterations of the lung samples were observed in the control group (Fig. 4A). In the CLP-treated lungs, however, diffuse interstitial edema and inflammatory cell infiltration were apparent (Fig. 4B). Interestingly, when RGDs were introduced into mice following CLP treatment, lung samples isolated from these mice appeared similar to the controls (Fig. 4C). In addition, Wisp1 and "6 antibody treatment also improved the
FIG. 4. RGDs rescue abnormal lung tissue histopathology in CLP-induced sepsis. AYE, Hematoxylin-eosin staining of lung tissue samples collected 24 h following no treatment (A), CLP treatment (B), CLP + RGD treatment (C), CLP + anti-Wisp1 treatment (D), or CLP + anti-"6 treatment (E). Whereas no histological alterations of the lung were observed in the untreated control group, diffuse interstitial edema and inflammatory cell infiltration were observed in the CLP group. These histological alterations were attenuated in RGD, anti-Wisp1, and anti-"6 group. F, Histological scores of lung injury in the groups. Graphs values represent the means T SDs. Five mice were analyzed per group. **P G 0.001, ***P G 0.001. All images were acquired using 400 magnification.
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FIG. 5. RGD peptides reduces EBA permeability of lung tissue in CLP-induced sepsis. A significant increase in EBA permeability was seen in CLPtreated mice compared with controls. Treatment with RGDs (5 mg/kg), however, significantly reduced EBA permeability back to control levels (Fig. 4A). This increase was also inhibited by anti-Wisp1 and anti-"6 (Fig. 4B). Graphed values represent the means T SDs. Five mice were analyzed per group. For comparing the CLP group with the untreated control group, **P G 0.01. For comparing the CLP + RGD group with the CLP group, ††P G 0.01.
histopathology when compared with the CLP group. Consistent with these histological findings, the histological scoring scheme that we utilized to analyze various categories of the lung tissue_s appearance revealed scores that were significantly higher following CLP treatment (1 T 1 vs. 15 T 1, P G 0.001) when compared with controls (Fig. 4F). Taken together, these data suggest that administration of RGDs reduces the severity of tissue defects following CLP-induced injury (15 T 1 vs. 8.7 T 0.6, P G 0.001) through Wisp1Yintegrin "6 signaling (Fig. 4F).
RGDs decrease EBA permeability in lung tissue from CLP-treated mice
Evans blue albumin permeability, which reflects the damage degree of pulmonary capillary well, was increased in CLPtreated mice when compared with controls (0.63 T 0.04 vs. 0.34 T 0.04, P G 0.01); however, this effect was ameliorated by RGD treatment (0.36 T 0.06 vs. 0.63 T 0.04, P G 0.01) (Fig. 5). We did not find any statistical difference between the control and the control + RGD treatment groups. The EBA permeability was
FIG. 6. RGDs decrease blood cell number, total BALF protein concentration, and lung wet/dry in CLP-induced sepsis of mice. The white blood cell number (A), total BALF protein concentration (B), and lung W/D weight ratio (C) were significantly increased in the CLP-treated group. Treatment with RGDs (5 mg/kg) in addition to CLP, but resulted in a significant reduction of these three categories. These categories were also inhibited by anti-Wisp1 and anti-"6 (DYF). Graphed values represent the means T SDs. Five mice were analyzed per group. For comparing the CLP group with the untreated control group, ***P G 0.001. For comparing the CLP + RGD group with the CLP group, †P G 0.05, ††P G 0.01.
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FIG. 7. RGDs have no effect on bacterial loads in blood and peritoneal fluid. RGD (5 mg/kg) treatment showed no significant effect on bacterial load in blood and peritoneal fluid. Graphed values represent the means T SDs. Five mice were analyzed per group.
decreased by Wisp1 and "6 antibody (P G 0.01) intratracheal instillation directly. RGDs decrease white blood cell number, total protein concentration in BALF, and lung W/D weight ratio in CLP-treated mice
White blood cell number (7.92 T 0.31 vs. 1.17 T 0.24, P G 0.001), total protein concentration in BALF (7.15 T 1.06 vs. 1.8 T 0.3, P G 0.001), and lung W/D weight ratio (8.2 T 0.79 vs. 4.1 T 0.66, P G 0.001) were found to be increased in the CLPtreated group when compared with control group. Notably, the addition of RGDs to CLP-treated mice was sufficient to suppress the increases seen in CLP alone (5.15 T 0.64 vs. 7.92 T 0.31, P G 0.01; 3.5 T 0.42 vs. 7.15 T 1.06, P G 0.01; 6.1 T 0.3 vs. 8.2 T 0.79, P G 0.05; respectively) (Fig. 6). No statistical differences were evident between the control and control + RGD groups. These indicators above were also decreased significantly when given antibodies through intratracheal instillation (Fig. 6, DYF). RGDs do not affect bacterial load in blood or peritoneal fluid
Interestingly, there were no significant differences in bacterial load between the CLP-treated group and CLP + RGD group at the 24-h time point tested (Fig. 7). Taken together, our data suggest that while RGDs modulate inflammation in sepsis, they do not affect bacterial load.
RGDs downregulate Wisp1/integrin"6 signaling in CLP-induced lung injury
Cecal ligation and puncture treatment resulted in a significant increase in both wisp1 and integrin "6 mRNA and protein expression in the lung compared with controls when analyzed 24 h after injury (wisp1 mRNA: 0.98 T 0.13 vs. 0.34 T 0.02, P G 0.01; integrin "6 mRNA: 0.88 T 0.25 vs. 0.23 T 0.06, P G 0.01; Wisp1 protein: 0.44 T 0.04 vs. 0.22 T 0.04, P G 0.05; integrin "6 protein: 0.69 T 0.03 vs. 0.34 T 0.02, P G 0.001). Conversely, treatment with RGDs 1 h following CLP induction resulted in a significant decrease in wisp1, integrin "6 mRNA, and protein expression (wisp1 mRNA: 0.44 T 0.03 vs. 0.98 T 0.13, P G 0.01; integrin "6 mRNA: 0.42 T 0.12 vs. 0.88 T 0.25, P G 0.05; Wisp1 protein: 0.27 T 0.06 vs. 0.44 T 0.04; P G 0.05; integrin "6 protein: 0.5 T 0.01 vs. 0.69 T 0.03, P G 0.01) (Fig. 8). No statistical difference was found between the control and control + RGD group. Most important, Wisp1 expression (0.67 T 0.06 vs. 0.38 T 0.04, P G 0.01) significantly decreases when treated with "6 antibody (Fig. 9). RGDs decrease the interaction between Wisp1 and integrin"6 in CLP-treated mice
Coimmunoprecipitation was performed in order to assess if Wisp1 interacts directly with"6. Lung tissue lysates were immunoprecipitated with anti-"6 or anti-Wisp1 and then analyzed for
FIG. 8. RGDs significantly decrease mRNA and protein levels of Wisp1 and integrin "6 following CLP-induced lung injury. Lung tissue was harvested from mice in untreated control, control + RGD, CLP, and CLP + RGD treatment groups. Twenty-four hours following CLP treatment, wisp1 and "6 mRNA (A, B) and protein (C, D) expressions in the lung were detected by RT-PCR and Western blotting analysis, respectively. Whereas CLP treatment caused an increase in both wisp1 and integrin "6 mRNA and protein expression, the addition of RGDs counteracted CLP’s effect. Graphed values represent the means T SDs. Five mice were analyzed per group. For comparing the CLP group with the untreated control group, *P G 0.05, **P G 0.01, ***P G 0.001. For comparing the CLP + RGD group with the CLP group, †P G 0.05, ††P G 0.01.
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FIG. 9. Anti-"6 significantly decreases mRNA and protein levels of Wisp1 following CLP-induced lung injury. Lung tissue was harvested from mice in untreated control, control + IgG, CLP, and CLP + anti-"6 treatment groups. Twenty-four hours following CLP treatment, wisp1 mRNA (B) and protein (A) expressions in the lung were detected by RT-PCR and Western blotting analysis, respectively. Whereas CLP treatment caused an increase in wisp1 mRNA and protein expression, the addition of anti-"6 counteracted CLP’s effect. Graphed values represent the means T SDs. Five mice were analyzed per group. For comparing the CLP group with the untreated control group, **P G 0.01. For comparing the CLP + anti-"6 group with the CLP group, ††P G 0.01.
Wisp1 or"6 by immunoblotting. As shown in Figure 10, Wisp1 and integrin"6 interact at baseline, and the interaction, which was enhanced by CLP, was decreased by RGD treatment. DISCUSSION In this study, we reveal that RGD peptides helps to ameliorate many of the negative effects associated with CLP-induced lung injury. Our findings also suggest that the inhibition of Wisp1 and integrin "6 expression by RGDs plays an important role in attenuation of CLP-induced lung injury. Acute lung injury/ARDS associated with sepsis is considered to be a major factor affecting the development and prognosis of critically ill patients in intensive care units (17). Approximately 40% of patients with sepsis develop ALI/ ARDS (18Y20), so it is clinically relevant to use a sepsisinduced ALI model to investigate the pathogenesis of this condition with the hope of contributing to future therapeutic advancements. Inducing sepsis in mice using CLP injury is a widely accepted model that closely mimics bowel perforation and bacterial infection prevalent in septic humans (21). The distinction between the mortality rates of the CLP + RGDYtreated mice
and the mice treated with CLP only is significant. The early release of macrophage-derived proinflammatory cytokines, such as TNF-! and IL-6, is important in the pathogenesis of septic shock and multiple organ failure (22). In our study, treatment with RGDs results in a significant decrease in proinflammatory (TNF-! and IL-6) cytokine levels following CLP treatment. These data suggest that the protective effect of RGDs in response to CLPinduced sepsis accounts for a considerable decrease in cytokinerelated lung injury, which likely contributes to the significant increase in survival rate seen for RGD-treated septic mice. We determine that CLP treatment results in a considerable amount of injury in the treated mice_s lungs when compared with the control group, whereas the severity of the injury is dramatically improved in the CLP + RGDYtreated group when compared with the CLP-treated group. We obtain similar results when we assess the expression of Wisp1 and integrin "6, which both increase significantly in CLP-treated group but are downregulated in the CLP + RGD group. Wisp1 is a secreted matricellular protein that has been associated with cell proliferation, differentiation, ECM deposition, and ECM turnover (23). Konigshoff et al. (9) reported that Wisp1 was increased in bleomycin-induced pulmonary
FIG. 10. RGDs decrease the interaction between Wisp1 and integrin"6 in CLP-treated mice. Lung tissues were from C57 mice in control, CLP, and CLP + RGD group. A, Western blot analysis (immunoblotting) for Wisp1 of lung tissue lysates coimmunoprecipitated with anti-"6(B). Reverse direction immunoprecipitation using anti-Wisp1 followed by Western blot analysis for"6. C, Immunoblot of Wisp1 and integrin"6 total protein expression used as input for the experiments in A, B. Wisp1 and integrin"6 interact at baseline, and the interaction, which was enhanced by CLP, was decreased by RGD treatment.
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fibrosis. In addition, this study revealed that Wisp1 antibody was sufficient to attenuate pulmonary fibrosis while also improving lung function and increasing survival in mice. Heise et al. (24) reported that Wisp1 mRNA increased in mechanically stretched mouse alveolar type II cells, whereas furthermore Li et al. (8) demonstrated that Wisp1 acts as an adjuvant adaptor molecule that contributes to ventilation-induced lung injury in mice, most likely by modulating and/or amplifying TLR4-mediated cellular functions. Collectively, these findings suggest that the secreted Wisp1 protein plays a critical role in the pathogenesis of acute lung injury in mice. Our in vitro study reveals that by treating RAW 264.7 cells with LPS, the increase observed in Wisp1 mRNA expression displays a time- and dose-effect relationship (unpublished results). We therefore use a CLP model to simulate in patients with clinical sepsis to study Wisp1 in vivo by using mice as a mammalian model system. Integrins are a family of cell surface receptors that mediate either cell-cell adhesion or cell-ECM interactions (10, 25). Integrins are believed to modulate cell differentiation, gene expression, proliferation, and survival. The "6 integrin subunit was first identified in cultured epithelial cells as part of the !v"6 heterodimer, which was shown to interact with fibronectin (26, 27). Breuss et al. (28) reported that "6 expression is part of an early epithelial response to injury and/or inflammation. It is particularly interesting that "6 is exclusively upregulated in epithelia that express "6 during development (distal tubules, collecting ducts, airways, alveolar epithelium). Hogmalm et al. (29) demonstrated that the absence of the "6 integrin subunit alleviates IL-1"Yinduced inflammation and injury in the lungs of infant mice. In addition, Miller et al. (30) reported that lung epithelial cell expression of "6 integrin is associated with sites of neutrophil recruitment. RGD-based strategies include antagonist drugs (peptidic or peptidomimetic) that specifically target RGD motifs and RGD conjugates, as well as the grafting of the RGD peptides to the surface of nanocarriers (31). Wang et al. (13) found that S247, an RGD-peptidomimetic agent, can significantly attenuate ventilation-induced lung injury in rats by interfering with integrins. Moon et al. (12) reported that RGDs inhibit integrindependent induction of immune cell migration into the lungs, while also inhibiting proinflammatory mediator production. In addition, we find that treating mice with RGDs following CLP induction is highly effective at reducing the inflammatory responses that have been associated with integrin signaling and MAP kinase pathway activation during LPS-induced development of acute lung injury. Our study reveals that administration of RGD peptides ameliorates acute lung injury in a mice model of sepsis induced by CLP, partially through inhibition of Wisp1 and integrin "6 expression. Our data further suggest that RGDs mediate inflammation in sepsis without affecting the bacterial load. In conclusion, we confirm that Wisp1 and integrin "6 expression levels increase in lung tissue as a response to CLP-induced sepsis in mice. Administration of RGDs is sufficient to inhibit the production of inflammatory cytokines, such as TNF-! and IL-6, while also reducing Wisp1 and integrin "6 expression and interaction. This results in the attenuation of ALI (one of the major complications that
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occur as a result of septic shock) following CLP-induced sepsis in mice. Further investigation should involve randomized clinically controlled trials that will help add further insight into the mechanism behind RGDs_ protective function during sepsis-induced ALI.
ACKNOWLEDGMENTS The authors thank the National Natural Science Foundation (81270135) and the Shanghai Education Committee Key Project (13ZZ024) for the financial support. They also thank Weifan Xiao, Yue Zhang, Ying Long, and Feng Li for their valuable discussion and technical assistance.
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