The FASEB Journal • Research Communication
Role of sphingolipids in murine radiation-induced lung injury: protection by sphingosine 1-phosphate analogs Biji Mathew,*,1 Jeffrey R. Jacobson,*,1 Evgeny Berdyshev,* Yong Huang,‡ Xiaoguang Sun,* Yutong Zhao,* Lynnette M. Gerhold,§ Jessica Siegler,* Carrie Evenoski,* Ting Wang,* Tong Zhou,* Rafe Zaidi,* Liliana Moreno-Vinasco,* Robert Bittman,¶ Chin Tu Chen,§ Patrick J. LaRiviere,§ Saad Sammani,* Yves A. Lussier,‡ Steven M. Dudek,* Viswanathan Natarajan,*,† Ralph R. Weichselbaum,储,2 and Joe G. N. Garcia*,2,3 *Institute for Personalized Respiratory Medicine, Section of Pulmonary, Critical Care, and Sleep Medicine, and †Department of Pharmacology, University of Illinois at Chicago, Chicago, Illinois, USA; ‡Department of Medicine, §Department of Radiology, and 储Department of Radiation Oncology, University of Chicago, Chicago, Illinois, USA; and ¶Department of Chemistry and Biochemistry, Queens College, City University of New York, Flushing, New York, USA Clinically significant radiation-induced lung injury (RILI) is a common toxicity in patients administered thoracic radiotherapy. Although the molecular etiology is poorly understood, we previously characterized a murine model of RILI in which alterations in lung barrier integrity surfaced as a potentially important pathobiological event and genome-wide lung gene mRNA levels identified dysregulation of sphingolipid metabolic pathway genes. We hypothesized that sphingolipid signaling components serve as modulators and novel therapeutic targets of RILI. Sphingolipid involvement in murine RILI was confirmed by radiation-induced increases in lung expression of sphingosine kinase (SphK) isoforms 1 and 2 and increases in the ratio of ceramide to sphingosine 1-phosphate (S1P) and dihydro-S1P (DHS1P) levels in plasma, bronchoalveolar lavage fluid, and lung tissue. Mice with a targeted deletion of SphK1 (SphK1ⴚ/ⴚ) or with reduced expression of S1P receptors (S1PR1ⴙ/ⴚ, S1PR2ⴚ/ⴚ, and S1PR3ⴚ/ⴚ) exhibited marked RILI susceptibility. Finally, studies of 3 potent vascular barrier-protective S1P analogs, FTY720, (S)-FTY720-phosphonate (fTyS), and SEW-2871, identified significant RILI attenuation and radiation-induced gene dysregulation by the phosphonate analog, fTyS (0.1 and 1 mg/kg i.p., 2ⴛ/wk) and to a lesser degree by SEW-2871 (1 mg/kg i.p., 2ⴛ/wk), compared with those in controls. These results support the targeting of S1P signaling as a novel therapeutic strategy in RILI.—Mathew, B., Jacobson, J. R., Berdyshev, E., Huang, Y., Sun, X., Zhao, Y., Gerhold, L. M., Siegler, J., Evenoski, C., Wang, T., Zhou, T., Zaidi, R., Moreno-Vinasco, L., Bittman, R., Chen, C. T., LaRiviere, P. J., Sammani, S., Lussier, Y. A., Dudek, S. M., Natarajan, V., Weichselbaum, R. R., Garcia, J. G. N. Role of sphingolipids in murine radiation-induced lung injury: protection by sphingosine 1-phosphate analogs. FASEB J. 25, 3388 –3400 (2011). www.fasebj.org ABSTRACT
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Key Words: gene dysregulation 䡠 S1P receptors 䡠 fTysiponate 䡠 FTY720
Radiation-induced lung injury (RILI) is a serious, dose-limiting toxicity of thoracic radiotherapy characterized by a subacute course developing over several weeks and associated with the potential for significant morbidity or death (1). At the molecular level, RILI is associated with increased generation of reactive oxygen and nitrogen species, secretion of inflammatory cytokines and chemokines, increased lung vascular permeability, and inflammatory cell recruitment into the lung parenchyma (2). A precise understanding of the mechanisms underlying these events, however, remains elusive and has served to impede the successful identification of novel therapeutic targets. Unfortunately, the treatment most commonly administered, corticosteroids, has limited efficacy and serious side effects (3). We recently reported that statins, a class of 3-hydroxy3-methylglutaryl (HMG)-CoA reductase inhibitors, may represent a novel therapy for RILI because this class of drugs exerts potent anti-inflammatory and vascularprotective properties (4, 5) and has been proven to be effective in rodent models of acute lung injury (6), including murine RILI (7). In these studies, we identified the attenuation of several radiation-induced dysregulated lung gene pathways by simvastatin, including genes involved in sphingolipid metabolism, a 1
These authors contributed equally to this work. These authors contributed equally to this work. 3 Correspondence: Department of Medicine, Pharmacology, and Bioengineering, University of Illinois at Chicago, 1737 W. Polk St., 305D AOB, Chicago, IL 60612-7227. E-mail:
[email protected] doi: 10.1096/fj.11-183970 2
0892-6638/11/0025-3388 © FASEB
pathway previously implicated in cellular responses to radiation (8, 9). Sphinoglipids are plasma membrane components recognized to mediate a wide range of cell signaling events. We have previously demonstrated the potent vascular barrier-protective effects of sphingosine 1-phosphate (S1P), a bioactive phospholipid, both in vitro and in preclinical models of inflammatory lung injury (10 –12). S1P is formed by phosphorylation of sphingosine via sphingosine kinase (SphK), whereas the N-acylation of sphingosine produces ceramide, a proapoptotic molecule in the lung (13). Because ceramide coupling to phosphocholine results in sphingomyelin, ceramide can be produced from sphingomyelin via enzymatic activities of sphingomyelinases (14). Endothelial cells (ECs) are rich in acid sphingomyelinase (ASMase; ref. 15), and ASMase activities in serum or directly in lung tissues are increased in several animal models of acute lung injury (ALI; refs. 16, 17). In addition, increased lung vascular permeability has been linked to ASMase-dependent production of ceramide in murine ALI (18). Notably, dihydrosphingosine, a precursor of ceramide and converted by SphK1 to dihydrosphingosine 1-phosphate (DHS1P), is a prosurvival molecule like S1P (19). S1P-mediated EC barrier enhancement involves ligation of specific S1P receptor subtypes (S1PR1, S1PR2, and S1PR3; refs. 20, 21); however, the relevance of these effects to RILI is poorly understood. In this study, we characterized the effect of RILI on S1P pathway components and assessed levels of S1P-related molecules in vivo. In addition, we examined the role of S1P receptor ligation and downstream signaling in modulating murine RILI. We have translated these findings into an exploration of the therapeutic effects of S1PR1 agonists such as SEW-2871 (SEW) and two S1P analogs, FTY720 (FTY) and (S)-FTY720-phosphonate [fTysiponate (fTyS)], in a preclinical model of RILI. These results confirm sphingolipid signaling as a critical modulator of RILI and S1P signaling elements as novel therapeutic targets in RILI. MATERIALS AND METHODS Animals and reagents All experiments and animal care procedures were approved by the University of Chicago Animal Care and Use Committee. Female C57BL/6 (20 –25 g) mice, 8 –10 wk old, were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and housed in cages with free access to food and water in a temperature-controlled room with a 12-h dark-light cycle. FTY720 was purchased from Novartis (New York, NY, USA), SEW was procured from Cayman Chemical (Ann Arbor, MI, USA), and fTyS was generated as we have described previously (22). SphK1⫺/⫺, S1PR2⫺/⫺, S1PR3⫺/⫺, and S1PR1⫹/⫺ mice were generously provided by Dr. Richard Proia (U.S. National Institutes of Health, Bethesda, MD, USA). Murine RILI model and S1P analog administration Mice were anesthetized with ketamine (100 mg/kg) and acepromazine (1.5 mg/kg), and radiation (10 –25 Gy) was MODULATION OF MURINE RILI BY SPHINGOLIPID COMPONENTS
administered to the thorax as described previously (7). A 5-mm-thick lead block was used to shield the rest of the animal while the thorax, between the clavicles and below the sternum, was irradiated with a 250-kV X-ray beam at a dose rate of 2 Gy/min using an orthovoltage animal irradiator. Each experimental group consisted of 10 mice irradiated to a single dose of 10, 20, or 25 Gy. The variation of the dose delivered within the lung was estimated to be within ⫾5% of the prescribed dose using thermoluminescence dosimeters. Select mice were treated with 0.01 or 0.1 mg/kg FTY720, SEW, or fTyS via intraperitoneal injection 2⫻/wk beginning 1 wk before irradiation and continuing for a period up to 6 wk afterward. Mice were then sacrificed, and indices of lung vascular leak and inflammation were assessed via bronchoalveolar lavage (BAL) fluid protein levels and cell counts at 4 – 6 wk, as described previously (23). Lungs were harvested and stored at ⫺80°C for histological evaluation. Lung histology To characterize histological alterations, lungs from each experimental group were inflated to 30 cm H2O with 10% formalin for histological evaluation by hematoxylin and eosin staining. Fluorescence molecular tomography (FMT) imaging To assess microvascular changes associated with RILI in real time, mice were imaged with a VisEn FMT 1500 Quantitative Tomography In Vivo Imaging System (Perkin Elmer, Bedford, MA, USA). IntegriSense 750 NIR (Perkin Elmer) was used as a probe, which targets the vasculature as a selective nonpeptide small molecule integrin ␣v3 antagonist and a near-infrared fluorochrome. Mice were injected with probe (2 nM i.v.) at 6 wk postradiation (single dose of 25Gy) and imaged 24 h later. Each experimental group consisted of three mice. Probe intensity was quantified using TrueQuant 3D software (Perkin Elmer). Western blotting of S1P lyase and SphK protein in lung tissue Lung tissues were homogenized in a TissueRuptor (Qiagen, Valencia, CA, USA) with a buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EGTA, 5 mM -glycerophosphate, 1 mM MgCl2, 1% Triton X-100, 1 mM sodium orthovanadate, 10 g/ml protease inhibitors, 1 g/ml aprotinin, 1 g/ml leupeptin, and 1 g/ml pepstatin. Lysates were centrifuged at 500 g for 5 min at 4°C, equal amounts of protein (20 g) were loaded onto 10% SDS-PAGE gels, and Western blotting was performed according to standard protocols. Analysis of sphingoid bases, sphingoid base-1-phosphates, and ceramides Sphingolipid analyses were performed via combined liquid chromatography-tandem mass spectrometry on an API4000 Q-trap hybrid triple quadrupole linear ion-trap mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with a TurboIonSpray ionization source interfaced with an automated Agilent 1100 series liquid chromatograph and autosampler (Agilent Technologies, Wilmington, DE, USA). The sphingolipids were ionized via electrospray ionization (ESI) with detection via multiple reaction monitoring (MRM). Analysis of sphingoid bases used ESI in positive ions with MRM analysis as we described previously (19, 24). BAL and plasma samples were collected, and lungs were harvested at 6 wk postradiation. Each group consisted of 5 animals. 3389
RNA isolation and microarray analysis Total RNA was isolated from whole lungs at 6 wk postradiation (25 Gy) for microarray analyses as described previously (23), using Affymetrix Mouse 430 2.0 arrays and protocols (Affymetrix, Santa Clara, CA, USA). Chips were scanned using a GeneChip Scanner 3000 (Affymetrix). Chip quality and present calls were determined by Affymetrix GCOS software. The chip data were normalized by the rank invariant set method using dChip software (25). The potential batch effect was corrected by Combat software (26). The microarray data have been submitted to the Gene Expression Omnibus repository of the National Center for Biotechnology Information (GSE25295). The differentially expressed genes between two experimental groups were identified using significance analysis of microarrays (SAM; ref. 27). Dysregulated genes were uploaded into the Ingenuity Pathway Analysis (IPA) software (http:// www.ingenuity.com), and canonical pathways were determined as described previously (28). Principal component analysis (PCA) on the experimental conditions was performed using R package Ade4. Statistical analysis Two-way ANOVA was used to compare the means of data from 2 or more experimental groups. If a significant difference was present by ANOVA (P⬍0.05), a least significant differences test was performed post hoc. Subsequently, differences between groups were considered statistically significant at values of P ⬍ 0.05. Results are expressed as means ⫾ se.
RESULTS Identification of S1P pathway-related biomarkers in RILI Initial studies investigated potential S1P pathway-related RILI biomarkers and identified increased expres-
sion of the two SphK isoforms, key sphingolipid pathway components that regulate S1P production via phosphorylation of sphingosine. Protein levels of both SphK1 and SphK2 isoforms were increased in whole lung homogenates of RILI-challenged mice at 6 wk (Fig. 1). Interestingly, SphK1 expression was further augmented in RILI-challenged mice that received simvastatin, whereas expression of SphK2 was significantly reduced by this intervention. Given the primacy for SphK1 in increasing cellular S1P levels compared with SphK2 (recently inferred to participate in apoptosis; ref. 29), these findings suggest that simvastatin reverses the RILI-mediated dysregulation of sphingolipid homeostasis. We next assessed potential radiation-induced effects on ceramide and S1P levels in biological fluids. One week postradiation, S1P levels were significantly increased in lung homogenates and decreased significantly in BAL (Fig. 2A, B). No significant changes were detected in plasma at any time point (Fig. 2C). In contrast, ceramide levels were significantly decreased ⬍1 wk postradiation in lung homogenates but were unchanged at later time points (Fig. 2D), whereas BAL ceramide levels were significantly increased at 3– 4 wk (Fig. 2E). Plasma ceramide levels were significantly increased only at 4 wk postirradiation (Fig. 2F). Nonetheless, the ratio of ceramide to cumulative S1P and DHS1P levels was significantly increased 3– 6 wk postradiation in lung homogenates and BAL fluid and plasma (Fig. 2G–I) and were attenuated by simvastatin, findings consistent with prior reports that radiation increases tissue ceramide levels and alters sphingolipid homeostasis (30). These results provide further evidence that the beneficial effects of simvastatin in RILI may be linked to an attenuation of radiation-mediated changes in sphingolipid metabolism.
Figure 1. SphK1 and SphK2 are biomarkers for murine RILI. A) Western blotting of lung homogenates from RILI-challenged mice (25 Gy) reveals increases in SphK1 and Sphk2 but not S1P lyase (S1PL) expression at 6 wk. B) Significant increases in SphK1 and SphK2 were confirmed by densitometry. Simvastatin treatment (10 mg/kg i.p., 3⫻/wk beginning 1 wk before irradiation) resulted in a significant increase in lung SphK1 but a significant decrease in lung SphK2 at 6 wk compared with RILI-challenged controls; n⫽3/group. *P ⬍0.05 vs. control; **P ⬍ 0.05 vs. RILI alone. 3390
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Figure 2. Alterations in sphingolipid homeostasis in murine RILI. Mice were administered a single dose of thoracic radiation (25 Gy), and samples were collected at various time points as shown. A, B) At 1 wk after radiation, S1P levels were significantly increased in lung homogenates (A) and significantly decreased in BAL (B). C) There were no significant changes detected in plasma at any time point. D, E). In contrast, ceramide levels were significantly decreased ⬍1 wk postradiation in lung homogenates but were unchanged at later time points (D), whereas BAL ceramide levels were significantly increased at 3–4 wk (E). F) Plasma ceramide levels were significantly increased only at 4 wk. (G–I) Ratio of ceramide to cumulative S1P and DHS1P levels was significantly increased 3–6 wk postradiation in lung homogenates (G), BAL fluid (H), and plasma (I). J–L) Graphic representation of ratio of ceramide to cumulative S1P and DHS1P levels in lung homogenates (J), BAL fluid (K), and plasma (L). n⫽5/group. *P ⬍ 0.05, **P ⬍ 0.01 vs. control. MODULATION OF MURINE RILI BY SPHINGOLIPID COMPONENTS
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Role of sphingolipid pathway components in RILI pathogenesis To further characterize the role of specific sphingolipid pathway components in the elaboration of RILI, we exposed genetically engineered mice with complete or partial targeted deletion of alleles for SphK1 (SphK1⫺/⫺), S1PR1 (S1PR⫹/⫺), S1PR2 (S1PR2⫺/⫺), or S1PR3 (S1PR3⫺/⫺) to a single dose of thoracic irradiation (10 –25 Gy) and assessed responses at 4 – 6 wk. Although the differences in BAL protein and cell counts were not significant at baseline, these indices were significantly increased at 4 – 6 wk postradiation in each group compared with those in wild-type controls, consistent with increased susceptibility to RILI in S1P pathway-modified mice (Fig. 3). The degree of increased RILI
susceptibility was relatively comparable across the strains of genetically engineered mice, albeit at variable time points and radiation dosing. Effect of S1P analogs on severity of murine RILI We previously reported the vascular-protective effects of FTY720 in a murine model of lipopolysaccharide (LPS)-induced acute lung injury (⬍24 h; ref. 11). Unfortunately, subsequent studies in chronic models of lung injury (2 wk) have suggested that FTY induces increased lymphopenia and bradycardia, as well as mortality, thereby limiting its potential utility in human disease (31, 32). As a result, we subsequently characterized robust effects of SEW, an S1PR1 agonist, and fTyS, a phosphonate S1P analog, in reducing vascular leak
Figure 3. Aberrant sphingolipid signaling is associated with increased susceptibility to RILI in vivo. A–D) Significantly increased BAL fluid protein content (A, C) and cell counts (B, D) were observed in RILI-challenged (25 Gy) SphK⫺/⫺ mice at 6 wk (A, B) and S1PR1⫹/⫺ (10 Gy) at 4 wk (C, D) compared with the respective WT RILI-challenged control animals. E–H) Likewise, S1PR2⫺/⫺ (E, F) and S1PR3⫺/⫺ (G, H) mice demonstrated significantly increased BAL protein (E, G) and cell counts (F, H) 6 wk after irradiation (20 Gy). n⫽3–5/ group. *P ⬍ 0.05 vs. WT control; **P ⬍ 0.05 vs. RILI-WT.
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and inflammation in both in vitro and in vivo in murine models of LPS-induced ALI as well as in a preclinical brain death model of lung injury (20, 33). Accordingly, we investigated the potential protective effects of FTY, SEW, and fTyS in murine RILI. Analogs were administered in either low or high concentrations (0.01 or 0.1 mg/kg, respectively) to RILI-challenged mice and assessed at 6 wk via BAL fluid protein levels and cell counts (Fig. 4). These studies confirmed significant dose-dependent protective effects of both fTyS and SEW. In contrast, FTY did not confer significant protection at comparable concentrations. Notably, no adverse effects were evident with respect to the administration of any of the analogs. Lung histological examination as well as VisEn FMT lung imaging corroborated biochemical and cellular levels of S1P analog protection at 6 wk (Fig. 5) with abundant areas of inflammatory cell infiltration into the lung interstitium induced by radiation, which were markedly attenuated by fTyS and to a lesser extent by SEW and FTY. Separately, VisEn FMT imaging demonstrated significant probe signal localized to the thorax in RILI-challenged mice consistent with increased lung vascular permeability. Quantification of probe intensity confirmed significant decreases in radiation-induced probe extravasation in animals treated with either SEW or fTyS. In contrast, significant differences were not identified between FTY-treated mice and RILI-challenged control animals.
Modulation of RILI-induced lung gene dysregulation by S1P analogs To link the protective effects of the S1P analogs in murine RILI to genomic influences of these interventions, we conducted genome-wide mRNA profiling from lung tissues after RILI. Genes potentially differentially regulated were identified by 2-group comparison using SAM software, and 92 genes with increased mRNA levels and 158 genes with decreased mRNA levels (ⱖ1.8 fold change, 5% false discovery rate) were identified in response to radiation alone at 6 wk (Table 1). The 250 RILI dysregulated genes were uploaded into Ingenuity software, and deregulated canonical pathways were identified (Tables 2 and 3), including leukocyte extravasation signaling, IL-10 signaling, and hepatic-inducible factor (HIF) 1␣ signaling. The pathways that were most prominently attenuated (decreased mRNA levels) included B-cell development and PKA signaling. Heat map analysis of S1P analog effects on RILI gene mRNA levels (Fig. 6A) revealed significant radiationmediated genomic effects that are strongly attenuated with varying potency by S1PR1 agonism via FTY, SEW, and fTyS. Whereas analog effects were minimal in the absence of radiation, the effects of fTyS on radiationinduced gene mRNA levels were the most robust with 54 genes significantly dysregulated by both radiation alone (compared with controls) and by fTyS in irradi-
Figure 4. Protective effects of S1P analogs on BAL protein and cell counts in murine RILI. C57BL/6 mice were pretreated with fTyS (A, B), SEW (C, D), or FTY720 (E, F) (0.01 or 0.1 mg/kg i.p.) 2⫻/wk beginning 1 wk before irradiation (IR; 20 Gy). BAL fluid was then collected at 6 wk and assessed for total protein (A, C, E) and cell counts (B, D, F). A–D) Compared with RILI-challenged controls, mice treated with fTyS had significant decreases in both BAL protein (A) and cell counts (B) at both high and low dosing, whereas treatment with SEW (C, D) resulted in a dose-dependent protective effect. E, F) In contrast, treatment with FTY720 did not confer significant protection at either low or high dosing. n⫽5/group. *P ⬍ 0.05 vs. uninjured control; **P ⬍ 0.05 vs. RILI control. MODULATION OF MURINE RILI BY SPHINGOLIPID COMPONENTS
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Figure 5. Protective effects of S1P analogs on lung histology and VisEn FMT imaging in murine RILI. A) Hematoxylin and eosin staining of lung sections from mice administered a single dose of thoracic radiation (25 Gy) demonstrate modest interstitial edema but a prominent influx of inflammatory cells (arrow) at 6 wk compared with that in uninjured controls. These changes are visibly attenuated in RILI-challenged mice treated with SEW or fTyS (0.1 mg/kg i.p., administered 2⫻/wk beginning 1 wk before irradiation). In contrast, lungs from RILI-challenged mice treated with FTY720 (0.1 mg/kg) were not significantly different in appearance at 6 wk from lungs of mice subjected to radiation alone. B) In separate experiments, RILI-challenged mice (25 Gy) were injected with an intravascular probe (IntegriSense 750) 6 wk postradiation and then subjected to VisEn FMT imaging 6 h later, which demonstrated evidence of extravasation of dye into the surrounding lung tissue. C) SEW and fTyS treatment (0.1 mg/kg) significantly decreased the radiation-induced dye extravasation. There was no evidence of protection in animals treated with FTY720 (0.1 mg/kg) compared with RILI controls.
ated animals compared with radiation alone. Remarkably, all of these genes demonstrated opposing directional changes in these two analyses as there were 33 genes with increased mRNA levels in response to radiation that were decreased in response to fTyS and 21 genes with decreased mRNA levels in response to radiation that were increased by fTyS. Additional filtering was then applied to identify the most dysregulated genes (fold change⬎3; Fig. 6B). Included in these gene sets were IL-1, previously found to be markedly in-
TABLE 1.
creased in the lungs of mice subjected to a single dose of thoracic irradiation (34), and matrix metalloproteinase (MMP-9), a gene that we previously identified as activated in our murine RILI model (7). IL-1 and MMP-9 mRNA levels were both increased by radiation compared with those in controls (4.4- and 3.8-fold change, respectively) but decreased by fTyS in irradiated mice compared with those in untreated irradiated control animals (0.33-fold change for both). To further characterize the effects of the S1P analogs
Genomic changes associated with RILI and treatment with sphingolipid analogs
Comparison
IR vs. CTR IR-FTY vs. CTR IR-SEW vs. CTR IR-fTyS vs. CTR FTY vs. CTR SEW vs. CTR fTyS vs. CTR IR-fTyS vs. IR
FDR (%)
FC
Probe sets
Up
Down
5 5 5 5 5 5 5 7
1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8
250 420 10 3 1 44 19 103
92 186 7 3 0 8 19 50
158 234 3 0 1 36 0 53
Genes with differential mRNA levels were identified by 2-group comparison using SAM software. IR, irradiation; CTR, control; FDR, false discovery rate; FC, fold change.
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TABLE 2.
Canonical pathways represented by genes with increased lung mRNA levels in response to radiation
Canonical pathway
Leukocyte extravasation signaling Atherosclerosis signaling TREM1 signaling IL-10 signaling Dendritic cell maturation Liver X receptor; RXR/retinoid X receptor activation Natural killer cell signaling Graft-vs.-host disease signaling HIF1␣ ⫾ signaling Systemic lupus erythematosus signaling Airway pathology in chronic obstructive pulmonary disease Communication between innate and adaptive immune cells Eicosanoid signaling Role of pattern recognition receptors in recognition of bacteria and viruses Pathogenesis of multiple sclerosis IL-8 signaling
⫺log (P)
Molecules
3.70 2.88 2.63 2.49 2.38 2.36 2.05 1.97 1.94 1.83 1.45 1.45 1.40
ITGAM, NCF2, NCF4, MMP12, MMP9, SELPLG IL1B, CCR2, MMP9, SELPLG TREM1, TYROBP, IL1B CCR1, FCGR2A, IL1B FCGR2A, TYROBP, FCER1G, IL1B IL1B, APOC2, MMP9 TYROBP, CD244, FCER1G FCER1G, IL1B MMP12, MMP9, SLC2A3 FCGR2A, FCER1G, IL1B MMP9 FCER1G, IL1B FPR2, ALOX5AP
1.36 1.35 1.34
NLRP3, IL1B CCR1 ITGAM, NCF2, MMP9
The 92 probe sets with increased mRNA levels from lungs of mice 6 wk after single dose thoracic radiation (25 Gy) in response to radiation compared with controls were uploaded into Ingenuity software to identify deregulated canonical pathways. The most prominently represented pathways are shown.
on the genomic changes induced by radiation, we performed PCA using the 250 probe sets dysregulated by radiation exposure (Fig. 7). In the 3-dimensional (3D) scatterplot of the PCA analysis, the first component represents the primary variable affecting sample conditions (lung injury induced by radiation). Twogroup comparison by t test between radiation alone and uninjured controls as well as between each drugtreated, irradiated group and radiation alone revealed that the principal component of the radiation-alone group was substantially higher than that of uninjured controls (as expected) but was significantly reduced by both SEW and fTyS interventions but not by FTY (P⫽0.07). Moreover, consistent with our previous findings, these data again suggest a more potent effect of fTyS compared with that of SEW because the fTyStreated samples are grouped more closely to the controls with respect to the first component. There were no TABLE 3.
significant differences between the principal component of radiation with FTY and radiation alone, indicating that, similar to its effects on direct histological and biochemical indices of lung injury, the genomic effects of FTY on the radiation response were marginal.
DISCUSSION RILI represents a common complication of thoracic radiotherapy for which current therapies have demonstrated limited efficacy. In preclinical models of ALI, our laboratory has demonstrated that strategies designed to target lung vascular barrier regulation (6, 11, 12, 35, 36) hold promise in potentially attenuating acute inflammatory lung injury. We recently characterized a murine model of RILI and identified features in common with ALI, including increased lung vascular
Canonical pathways represented by genes with decreased lung mRNA levels in response to radiation
Canonical pathway
Primary immunodeficiency signaling B-cell development Glycerophospholipid metabolism Systemic lupus erythematosus signaling Melatonin signaling Cellular effects of sildenafil PKA signaling Citrate cycle G-protein signaling mediated by tubby Phospholipid degradation d-Glutamine and d-glutamate metabolism Mechanisms of viral exit from host cells Synaptic long-term potentiation Estrogen receptor signaling
⫺log (P)
Molecules
3.79 3.06 2.66 2.20 1.95 1.93 1.83 1.73 1.65 1.65 1.54 1.53 1.43 1.40
LCK, IGKC, IGHM, IGK-V28 IGKC, IGHM, IGK-V28 PLCB4, DGKD, PLA2G2D, CHKA, ETNK1 LCK, IGKC, IGHM, IGK-V28 PRKACB, PLCB4, RORA SLC4A5, PRKACB, PLCB4, MYH2 PRKACB, PLCB4, MYH2, PDE7A, CREBBP, AKAP9 SUCLA2, PCK1 LCK, PLCB4 PLCB4, DGKD (includes EG:8527), PLA2G2D GLS CHMP4C, NEDD4 PRKACB, PLCB4, CREBBP CREBBP, PCK1, NR3C1
The 158 probe sets with decreased mRNA levels in response to radiation compared with the control were uploaded into Ingenuity software to identify deregulated canonical pathways. The most prominently represented pathways are shown.
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Figure 6. Attenuation of RILI-mediated gene dysregulation by S1P analogs. A) Hierarchical clustering of genes dysregulated by radiation (20 Gy) at 6 wk across experimental conditions is shown as identified by SAM (Table 1). Genes were displayed by dChip software and classified into two clusters (genes with increased mRNA levels and genes with decreased mRNA levels). Blue, white, and red represent mRNA levels below, at, and above the average level of the corresponding gene, respectively. Consistent with the corresponding physiological data, treatment with fTyS and SEW (0.1 mg/kg) significantly blunted the effects of radiation on lung gene dysregulation, whereas FTY720 had only a marginal effect. B) Filtering of genes with ⬎3-fold change identified specific genes that were most dysregulated by radiation. n⫽3– 4/group. Cont, control; Veh, vehicle.
leak and inflammation (7). Subsequently, we confirmed a protective effect in RILI of simvastatin, an agonist that directly augments vascular barrier function and is protective in other models of inflammatory lung injury (6, 10). Our results now extend these prior studies and confirm that the targeting of sphingolipid 3396
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components may identify novel RILI biomarkers, novel and effective therapeutic strategies in RILI, and unique insights into RILI pathogenesis. We further provide evidence of RILI protection by specific S1P analogs associated with the attenuation of RILI-associated physiological and genomic derangements.
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Figure 7. PCA of genes dysregulated in murine RILI and effects of S1P analogs. A) 3D scatterplot of PCA; each triangle represents a sample [black, control; red, radiation (IR) alone; green, radiation⫹fTyS; brown, radiation⫹SEW; blue, radiation⫹FTY720]. B) Corresponding principal component changes, expressed as the linear gene-specific weight over the mRNA levels of all analyzed genes. These demonstrate significant gene dysregulation associated with radiation alone (25 Gy) at 6 wk that was not significantly altered in FTY720-treated animals (0.1 mg/kg). In contrast, gene dysregulation associated with radiation was significantly attenuated by both SEW and fTyS. n⫽3/group. *P ⬍ 0.05 vs. radiation alone.
Originally characterized as a potent angiogenic factor (37), S1P ligates specific G-protein-coupled receptors on the surface of ECs to initiate a series of cytoskeletal and adhesive protein rearrangements that result in decreased EC permeability. Activation of the small GTPase Rac and its downstream target, p21activated kinase (PAK), is critical for formation of a prominent cortical actin ring that accompanies S1Pinduced EC barrier enhancement (38). Moreover, Rac activation stimulates translocation of the multidomain actin-binding protein cortactin to the cortical actin ring where its ability to interact with other cytoskeletal proteins appears to be essential for maximal S1Pmediated barrier enhancement (10). However, in addition to inducing actin cytoskeletal rearrangement, S1P alters cell-cell and cell-matrix contacts in association with EC permeability reduction. S1P dramatically increases localization of vascular endothelial-cadherin and of ␣-, -, and ␥-catenin at EC cell-cell junctions (39) while increasing interaction of these adherens junction proteins with the cortical actin-based cytoskeleton. S1P also induces focal adhesion protein rearrangement in association with cortical actin ring formation (40). Thus, consistent with our previously reported potent EC barrier-enhancing effects of S1P (7, 10, 38) with protective effects in animal ALI models (11, 12), we hypothesized that S1P analogs may represent effective therapeutic agonists in RILI. Our hypothesis was further supported by reports linking sphingolipid signaling to the cellular responses to radiation (30, 41). In the current study, a significant increase in levels of SphK isoforms, SphK1 and SphK2, in lung tissues was observed in RILI-challenged mice after 6 wk. Although the mechanisms underlying the differential effects of simvastatin on lung SphK1 and SphK2 expression are unclear, evidence of these MODULATION OF MURINE RILI BY SPHINGOLIPID COMPONENTS
changes, in response to an agent we have previously confirmed attenuates RILI, supports the idea that SphK1 and SphK2 could potentially serve as useful clinical biomarkers. Moreover, radiation-induced increases in the ratio of ceramide to cumulative S1P and DHS1P levels in murine plasma, BAL fluid, and lung homogenates were attenuated by simvastatin. Numerous other investigators have pursued studies designed to identify reliable clinical RILI biomarkers. For example, it has previously been hypothesized that RILI is the result of a sustained cytokine cascade due to an inflammatory response activated by radiation, with a large body of experimental data implicating a number of chemokines and cytokines, including TGF-, IL-1, IL-6, and TNF-␣ (42– 44). However, neither the prediction nor the amelioration of radiation pneumonitis has been consistently correlated with cytokine levels or specific neutralization of individual cytokines (45). Therefore, our findings represent an important advance and suggest that sphingolipids may potentially serve as clinical biomarkers for both RILI and for monitoring responses to therapy. Consistent with the importance of the sphingolipid pathway in RILI pathogenesis, genetically engineered mice with targeted S1P receptor deletions, including S1PR1, S1PR2, and S1PR3 as well as SphK1, all demonstrated increased susceptibility to RILI. Although the association of deficiencies in SphK1 or S1PR1 with increased RILI injury is consistent with our prediction of the role of these S1P signaling pathway components, the observed deleterious effects of S1PR2 or S1PR3 depletion were less intuitive and appear to be in conflict with our recent report that S1PR2⫺/⫺ and S1PR3⫺/⫺ mice exhibit reduced injury in a LPS-induced preclinical ALI model (20). In these studies, S1PR2⫺/⫺ mice administered intratracheal LPS were 3397
found to have elevated BAL fluid total protein levels compared with LPS-treated wild-type animals, although no difference was detected with respect to BAL cell counts. Likewise, in animals administered S1PR3 siRNA via angiotensin 1-converting enzyme antibody-conjugated nanocarriers, LPS-induced elevations in BAL fluid albumin and total protein levels were significantly reduced compared with those of LPS-treated controls (20). These results strongly suggest unique, differential roles for S1PR2 and S1PR3 in specific models of murine inflammatory lung injury. A potential explanation for the conflicting roles of S1P receptors in ALI and RILI responses includes the real possibility that alveolar epithelial cell barrier function and vascular endothelial cell barrier function are distinct in susceptibility to ionizing radiation and LPS treatment. We speculate that S1PR2 and S1PR3 probably exert complex, possibly injury-, cell-, and speciesspecific barrier regulatory properties, potentially due to their ability to activate multiple multimeric G proteins (46). Nonetheless, our data support the idea that S1P-related compounds may represent effective and novel therapeutic agents in radiation pneumonitis and that S1P receptors are critical to RILI pathobiology. Despite its known vascular-protective effects both in vivo and in vitro, endogenous S1P produces a multitude of effects, several of which limit its usefulness in patients. Increased concentrations of S1P disrupt EC monolayer integrity in vitro through S1P3R ligation and resultant Rho activation, limiting the therapeutic window for its barrier-enhancing properties (33, 40, 47). In addition, S1P induces bradycardia through activation of S1PR3 in the heart (48, 49). Finally, S1P worsens airway hyperresponsiveness in mice by stimulating contraction of human airway smooth muscle cells (50, 51), raising concerns that it may exacerbate airway obstruction in patients with underlying lung disease. Given these limitations of S1P, we investigated structurally similar compounds such as FTY, SEW, and fTyS as potential novel therapies for RILI. We confirmed a protective effect of SEW and fTyS in our murine model of RILI as assessed by both indices of lung vascular permeability and inflammation and by effects on radiation-induced lung gene dysregulation. Microarray studies identified specific genes dysregulated by radiation with attenuation of these radiationinduced changes by S1P analogs, particularly fTyS. As noted, these include MMP-9, with increased mRNA levels in the lungs of mice after irradiation, changes that were significantly blunted by fTyS. Because T-cell activation contributes to the elaboration of radiation pneumonitis (52), an important role for MMP-9 in the pathogenesis of RILI is suggested by evidence that MMP-9 regulates lung injury mediated by T cells (53). Separately, similar changes were observed with respect to both S100a8 and S100a9, also known as myeloidrelated proteins 8 and 14 (MRP8 and MRP14), respectively, which are increased in a variety of inflammatory states and contribute to EC barrier disruption and apoptosis (54). In addition, increased expression of 3398
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S100a9 has been reported in the BAL from patients with fibrotic lung diseases (55), which is certainly relevant to the long-term effects of radiation on the lung. Collectively, these findings suggest that the protective effects of S1P analogs in our model are mediated in part by genomic alterations that result in the attenuation of radiation-induced lung inflammatory changes. We recognize that a limitation of our microarray analyses is the reliance on relatively small sample sizes (n⫽3/experimental group). However, validation of our microarray findings is not only provided by the corroborative effects of FTY, SEW, and fTyS on lung vascular leak and inflammation in our murine RILI model but also by the identification of relevant pathways that were dysregulated by radiation. For example, increased HIF1␣ signaling induced by radiation in our model is notable because increased HIF1␣ expression has previously been found in the lungs of rats administered thoracic radiation and was also found to correlate with the degree of lung inflammation in these animals (56). In addition, decreased PKA signaling by radiation is consistent with the important role for this pathway in responses to radiation as evidence of increased PKA expression has been linked to poor clinical responses to radiotherapy in some patient populations (57). In summary, our results provide evidence of significant protection conferred by fTyS and, to a lesser extent, by SEW, with minimal efficacy of FTY. Protection against RILI by specific S1P analogs is consistent with our previous findings in an LPS-induced ALI model (20, 33) and offer strong support for preliminary studies to explore the safety and efficacy of these novel agonists in relevant patient populations exposed to thoracic radiation. The authors are grateful to Eddie T. Chiang for outstanding technical assistance. This work was supported by U.S. National Institutes of Health grants R01 HL079396 (V.N.), P01 HL098050 (V.N., J.G.N., S.D., J.R.J., and R.R.W.), HL083187 (R.B.), HL58094, HL09805, K22 LM008308-04, and 5U54CA121852-05. Microarray data have been submitted to the Gene Expression Omnibus repository of the National Center for Biotechnology Information (GSE25295).
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Received for publication May 4, 2011. Accepted for publication June 9, 2011.
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