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Intravenous delivery of adipose-derived mesenchymal stromal cells attenuates acute radiation-induced lung injury in rats
Cytotherapy, 2015; 17: 560e570
Intravenous delivery of adipose-derived mesenchymal stromal cells attenuates acute radiation-induced lung injury in rats
XINPING JIANG1, XIN JIANG1, CHAO QU1, PENGYU CHANG1, CHU ZHANG3, YAQIN QU1 & YONGJUN LIU2 1
Department of Oncological Radiotherapy, The First Bethune Hospital of Jilin University, Changchun, China, Alliancells Bioscience Co, Ltd, Tianjin, China, and 3Department of Oncological Radiotherapy, The Second Bethune Hospital of Jilin University, Changchun, China
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Abstract Background aims. Radiation-induced lung injury (RILI) commonly occurs in patients with thoracic cancer. However, an effective treatment option has not yet been established. Adipose-derived mesenchymal stromal cells (Ad-MSCs) have significant potential for clinical use, but their role in RILI is currently unknown. We aimed to evaluate the therapeutic capacity of Ad-MSCs to heal acute RILI in rats. Methods. Sprague-Dawley rats were used in this study. Rat Ad-MSCs were delivered through the tail veins of rats 2 h after thorax irradiation. Lung histopathologic findings, pulmonary levels of inflammatory cytokines (interleukin [IL]-1, IL-6, IL-10 and tumor necrosis factor-a), pro-fibrotic factors (transforming growth factor [TGF]-b1, connective tissue growth factor, a-smooth muscle actin and type 1 collagen), pro- or anti-apoptotic mediators (Bcl-2, Bax and caspase-3) and the multifunctional factor hepatocyte growth factor were evaluated after Ad-MSC transplant. Results. Intravenous delivery of Ad-MSCs attenuated acute RILI. Further studies showed that Ad-MSCs had antiinflammation and anti-fibrotic effects and maintained lung epithelium integrity, as indicated by reduced serum levels of the pro-inflammatory cytokines IL-1, IL-6 and tumor necrosis factor-a, increased levels of the anti-inflammatory cytokine IL-10, and downregulated transforming growth factor -b1, a-smooth muscle actin and type 1 collagen levels in irradiated lung tissues. Ad-MSCs also regulated the expression of pro- and anti-apoptotic mediators (Bcl-2, Bax and caspase-3) to protect lung cells from apoptosis. Conclusions. Intravenous Ad-MSC delivery attenuated acute RILI through antiinflammation, anti-fibrosis and anti-apoptosis mechanisms. Key Words: adipose tissue, cell-based transplantation, mesenchymal stromal cells, radiation-induced lung injury
Introduction Radiation-induced lung injury (RILI) is a common major obstacle in thoracic cancer radiotherapy [1,2]. The clinical incidence of radiation-induced pneumonitis ranges from 5% to 10% [3,4]. RILI develops through a complex pathological process, resulting in excessive inflammation or extracellular matrix deposition in the lung interstitium, ultimately leading to impaired lung function and respiratory failure [5]. Thus, it is important to alleviate RILI to improve tumor control and the patient’s quality of life. The current primary approach to manage RILI is hormone treatments to temporarily suppress inflammation [2]. However, there is no known effective therapeutic strategy for RILI. Mesenchymal stromal cells (MSCs) have significant clinical potential [6e8]. Recent studies have
demonstrated that engrafted stem cells have beneficial effects in injured lung tissues [9,10]. Radiation exposure of the lung leads to fibrosis, and fibrosis is a complex pathology driven by numerous biological factors, such as chronic inflammation and hypoxia. MSC anti-fibrotic functions have been described in various organs [11]. MSC cellular therapy would be an ideal approach to treat RILI. However, the clinical use of bone marrowederived MSCs has presented problems, including pain, morbidity and low cell number on harvest [12]. Adipose-derived mesenchymal stromal cells (Ad-MSCs) have also been widely studied because of their ease of isolation, abundant distribution and low immunogenicity [13,14]. In addition, Ad-MSCs have been shown to have expansion capacity superior to that of bone marrowederived MSCs in vitro [15,16]. Therefore,
Correspondence: Yaqin Qu, MD, Department of Oncological Radiotherapy, The First Bethune Hospital of Jilin University, Changchun 130021, China. E-mail: [email protected]; Yongjun Liu, MD, Alliancells Bioscience Co, Ltd, Tianjin, China. E-mail: [email protected] (Received 20 October 2014; accepted 13 February 2015) ISSN 1465-3249 Copyright Ó 2015, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2015.02.011
Adipose-derived MSCs attenuate radiation-induced lung injury Ad-MSCs are a potential candidate for autologous transplant in lung disease. Moreover, autologous AdMSC application is free from ethical or security issues. The therapeutic potential and usefulness of AdMSC delivery in RILI treatment has not been studied. We studied the effects of intravenous (IV) delivery of Ad-MSCs on acute RILI in a Sprague-Dawley rat model. Methods
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Rad-Source). The remainder of the body was shielded by lead strips. Ninety rats were used in the study. Animals were randomly divided into three groups (n ¼ 30 per group). After thoracic irradiation, animals received a 0.5-mL saline injection (radiationþphosphate-buffered saline [PBS] group), 5 106 Ad-MSCs in a 0.5-mL saline injection (radiationþMSC group), or were used as normal controls. Animals were injected through the tail vein within 2 h after irradiation. Peripheral blood and lung tissues were collected from rats on days 1, 3, 7, 14 and 28 for analysis.
Ethical approval Sprague-Dawley rats (female; weight, 180e220 g) were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China). All experiments involving animal subjects were performed in accordance with guidelines approved by our local animal care and use committee. Ad-MSC isolation, culture and characterization
Tracing experiments The cell tracker CM-Dil (Invitrogen Inc) was dissolved in dimethyl sulfoxide (Invitrogen) and added to passage 3 Ad-MSCs according to the manufacturer’s protocol. A total of 5 106 cells were intravenously injected into irradiated and normal rats. The distribution of labeled Ad-MSCs was determined by use of the Xenogen (IVIS, Lumina, Caliper Life Sciences) in vivo optical imaging technique, and frozen lung sections were examined my means of fluorescence microscopy (Olympus).
Fat tissue was obtained from subcutaneous adipose tissue in the inguinal groove of female SpragueDawley rats under sterile conditions. Ad-MSCs were separated by a 200-mesh strainer and suspended in complete medium (90% Dulbecco’s modified Eagle’s medium-low glucose/F-12þ10% fetal bovine serum) (Gibco) and grown with 5% CO2 in a humidified atmosphere at 37 C, as described previously [17]. Cells were passaged every 2 to 3 days, and were then harvested at passage 3 for characterization, identification and IV delivery. The phenotype of Ad-MSCs was evaluated by means of flow cytometry analysis with the use of antirat cluster of differentiation (CD)11b-phycoerythrin (PE), CD29-PE, CD44efluorescein isothiocyanate (FITC) and CD45eantigen-presenting cells. Mouse immunoglobulin G1eFITC and PE were used as isotype controls. All antibodies were purchased from eBioscience. Cells were harvested at passage 3 and plated into six-well plates. Adipogenic, osteogenic and chrondrogenic medium (Trevigen) was added to each well under standard culture conditions according to the manufacturer’s protocol. Cells in the remaining wells served as controls. Adipogenesis, osteogenesis and chondrogenesis were confirmed by oil red O, alizarin red and alcian blue staining, respectively.
Serum was collected from the peripheral blood by centrifugation. IL-1, IL-6, IL-10, tumor necrosis factor (TNF)-a, transforming growth factor (TGF)b1 and hepatocyte growth factor (HGF) (R＆D Systems) levels were measured by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions. For hydroxyproline analysis, the lung tissues were homogenized and assayed by use of hydroxyproline ELISA kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instructions.
Experimental design
Immunohistochemistry
Rats received a single local dose of radiation with 15 Gy of x-rays to the right thorax (160 kV, 25 mA, 1.25 Gy/min, RS-2000 Pro Biological Irradiator,
Paraffin-embedded lung sections were de-waxed and rehydrated before antigen retrieval. The sections were incubated in 0.3% H2O2 to block endogenous
Histopathology For histological examination, irradiated lung tissue was fixed in 10% neutral-buffered formalin for 48 h, paraffin-embedded and sectioned at an average thickness of 5 mm. The sections were stained with hematoxylin and eosin and Masson’s trichrome. The sections were semi-quantitatively analyzed by two blinded observers using five random images per group. Enzyme-linked immunosorbent assay
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Figure 1. Chemotactic homing of Ad-MSCs. (A) The distribution of Ad-MSCs in a RILI rat model were determined by means of the Xenogen in vivo optical imaging technique. (B) Histological analysis by frozen section. Magnification 100. Scale bar, 200 mm. (C) Quantification of CM-Dilelabeled Ad-MSCs in lung section fields on day 3. (D) SDF-1 mRNA expression in lungs 28 days after irradiation. Fold expression values were normalized to the normal control rat. Data represent mean standard deviation (*P < 0.05, **P < 0.01, n ¼ 5 per group). R, radiation; N, normal.
peroxidase and incubated with serum from the host to block nonspecific antigen binding. The primary antibody was diluted according to the manufacturer’s recommendations and was added to the sections and incubated overnight at 4 C. The primary antibodies used for immunohistochemistry (IHC) included TGF-b1, connective tissue growth factor (CTGF), a-smooth muscle actin (SMA), type 1 collagen (Col1al) and hydroxyproline (Abcam). Secondary antibody was added and incubated at 37 C for 2 h. Diaminobenzidine was added to detect positive cells. An IHC staining kit (Abcam) was used to detect TGF-b1, CTGF, a-SMA, Col1al and hydroxyproline.
Apoptosis assay Apoptosis was assessed in paraffin-embedded lung sections with the use of a terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL, Roche) in situ cell death detection kit according to
the manufacturer’s instructions. TUNEL-positive cells were quantified in five random 40 images per group under light microscopy. Real-time polymerase chain reaction A total of 100 mg of irradiated lung tissue was freshly isolated from each sample. Total RNA was isolated with TRIzol reagent (Invitrogen), and 1 mg of total RNA from each sample was used for first-strand complementary DNA synthesis with the use of a real-time polymerase chain reaction (PCR) Kit (Takara Bio Inc). Total complementary DNA was then incubated with primers against stromal cellederived factor-1 (SDF-1), TGF-b1, HGF, aSMA, Col1al or CTGF. The primer sequences were as follows: SDF-1 Forward 50 -ACGGTCTTGAACTACTGGC G-30 Reverse 50 -GGAGGCTTACAGCACGAAAC-30
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Figure 2. Effect of Ad-MSCs on histological changes after thorax irradiation. (A) Histological analysis by hematoxylin and eosin (HE) staining. Magnification 200. Scale bar, 100 mm. (B) Histological analysis by means of Masson staining. Magnification 400. Scale bar, 50 mm. (C) Lung fibrosis was assessed by means of hydroxyproline IHC detection. (D,E) Alveolar thickness and Masson-stained areas were semi-quantified from five random images per group (*P < 0.05, **P < 0.01, n ¼ 5 per group). (F) Hydroxyproline serum levels were detected by means of ELISA assay at day 28. Data are shown as mean standard deviation (*P < 0.05, **P < 0.01, n ¼ 5 per group).
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Figure 3. TNF- a, IL-6, IL-1 and IL-10 serum levels (AeD) were detected by means of ELISA assay on days 1, 3, 7, 14 and 28 after thorax irradiation. Data are shown as mean standard deviation (*P < 0.05, **P < 0.01, n ¼ 5 per group). R, radiation.
TGF-b1 Forward 50 -GGACTACTACGCCAAAG AAG-30 Reverse 50 -TCAAAAGACAGCCACTCAGG-30 HGF
Forward GCT-30
50 -GTGCATCAGAAACAAGG
Reverse 50 -TGGCACTTGATGCCACTCTT-30 a-SMA Forward 50 -GGCATCCACGAAACCAC CTA-30 Reverse 50 -TGAAGGCGCTGATCCACAAA-30 Col1a1 Forward 50 -GCCTCTGCAACAAATCCC CA-30 Reverse 50 -CATGTGTGGCCGATGTTTCC-30 CTGF Forward 50 - GCGCCAAGCAGCTGGG AGAA-30 Reverse 50 - CGGCCCCATCCAGGCAAGTG-30 b-Actin Forward 50 -CACCCGCGAGTACAACC TTC-30 Reverse 50 -CCATACCCACCATCACACCC-30 Expression levels were normalized to b-actin levels. Quantitative PCR was performed with the use of SYBR Green I TaqMan probes (Roche) in 40 amplification cycles in an ABI 7500 Fast (Life Technologies). All reactions were carried out in duplicate, and the results were analyzed by the 2DDCT method.
Western blot For Western blot analysis, lung tissues were homogenized in an appropriate amount of ice-cold lysis buffer. Equivalent amounts of protein (25e40 mg) were separated by use of 15% sodium dodecyl sulfateepolyacrylamide gel electrophoresis and transferred onto a polyvinylidene fuoride membrane (Millipore). Membranes were probed with the following antibodies: caspase3 (1:1000, Santa Cruz), Bcl-2 (1:750, Abcam) and Bax (1:1000, Santa Cruz). b-Actin (Kangchen) was used as an internal control. Immunoreactivity was detected by use of the Odyssey Infrared Imaging System (Gene Company Limited). All blots were exposed for optimal lengths of time for visualization. Statistical analysis All data were analyzed with the use of SPSS 19.0 software (IBM Corp) and expressed as mean standard deviation (SD). One-way analysis of variance was performed to compare data among groups. Statistical significance was defined as P 0.05. Results Features of rat Ad-MSCs Isolated rat Ad-MSCs showed a spindle-like morphology and were able to differentiate into
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Figure 4. Profibrotic expression in serum and lung tissue. (AeD) TGF-b1, a-SMA, Col1a1 and CTGF expression in lung tissue was measured by means of IHC. Magnification 400. Scale bar, 50 mm. (E) Serum TGF-b1 expression was detected by means of ELISA on days 7, 14 and 28. (FeI) TGF-b1, a-SMA, Col1a1 and CTGF mRNA was detected by means of real-time PCR after thorax irradiation with b-actin as the internal control. Fold expression values were normalized to the control. Data represent mean standard deviation (*P < 0.05, **P < 0.01, n ¼ 5 per group).
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Adipose-derived MSCs attenuate radiation-induced lung injury adipocytes, osteoblasts and chondrocytes after 14 days in defined culture media. Flow cytometry revealed that rat Ad-MSCs were CD29-and CD44positive and CD11b- and CD45-negative. All of these results are consistent with the minimal criteria for MSC identification [18]. Ad-MSCs home to injured lung tissue We determined the systemic in vivo Ad-MSC distribution through the use of the Xenogen imaging system (Figure 1A). Within the first 2 h after irradiation, there was no significant difference in Ad-MSC lung distribution between the radiation group and the normal group. The RþMSC group showed that engrafted cells localized primarily to the bone and lymph gland from 2 to 24 h, and the number of engrafted cells in the lung peaked at 72 h. Interestingly, we observed little to no Ad-MSCs in the lung after day 14 in either the radiation group or normal group. However, our fluorescence images confirmed that a large number of transplanted cells had already homed to the injured lung by 12 h (Figure 1B, C). The fluorescence imaging data were inconsistent with the Xenogen data. Previous studies examined large numbers of engrafted cells at the lung in the first 2 h by the Xenogen system in the C57/BL6 mouse [19]. However, the inconsistencies in the engrafted cells at 2 h and 12 h in our study may be due to the thick fur of Sprague-Dawley rats. Moreover, our data showed that engraftment peaked at 72 h in the lung rather than at 12 h; this probably was caused by the number of engrafted cells or the dose of radiation xray, which would affect chemokine release. MSCs express the specific chemokine receptor CXCR4, and they migrate to radiation-injured sites through chemotaxis [20]. The SDF-1/CXCR4 axis is a signaling pathway associated with active stem cell recruitment in MSCs homing to irradiated lungs. Therefore, we evaluated SDF-1 expression in irradiated lung tissues by real-time PCR at various time points after Ad-MSC delivery. SDF-1 expression was significantly higher on day 7 in the radiation group compared with that in the normal group (1.72-fold increase; P < 0.01) (Figure 2D). Ad-MSC therapy attenuates RILI histopathology We evaluated RILI-associated histological changes on day 28 by means of hematoxylin and eosin
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staining, Masson staining and hydroxyproline detection. The alveolar architecture was completely disrupted in the radiationþPBS group compared with the normal control group. In contrast, Ad-MSC treatment allowed for alveolar wall thickening and minimal interstitial edema (Figure 2A). Masson staining and hydroxyproline detection showed marked radiation-induced collagen deposition in lung tissue on day 28. However, MSC treatment attenuated collagen deposition compared with the radiationþPBS group (Figure 2B, C, F). The histology and semi-quantitative analysis are shown in Figure 2D, E. Ad-MSC treatment reduces pro-inflammatory cytokine levels and improves anti-inflammatory cytokine expression We measured pro-inflammatory cytokine serum levels, including IL-1, IL-6 and TNF-a, by means of ELISA on days 1, 3, 7, 14 and 28 after irradiation. Thorax irradiation resulted in the production of the inflammatory cytokines IL-1, IL-6 and TNF-a, whose serum levels significantly increased in RILI rats on day 14 (Figure 3AeC). In contrast, Ad-MSC treatment significantly decreased the irradiationinduced protein release of IL-1, IL-6 and TNF-a. We also measured the serum levels of the antiinflammatory cytokine IL-10 on days 1, 3, 7, 14 and 28. As shown in Figure 3D, baseline IL-10 was similar among groups 1 day after thorax irradiation; however, compared with baseline, serum IL-10 gradually decreased within 7 days after irradiation in the radiationþPBS group. In contrast, serum IL10 levels significantly increased to 180.9 9.67 pg/ mL on day 7 in the radiationþMSC group compared with 28.076.43 pg/mL in the radiationþPBS group (P < 0.01). Ad-MSC therapy reduces profibrotic factor expression We examined TGF-b1 expression in the serum and lung tissue for 28 days after irradiation. As shown in Figure 4A, we measured TGF-b1 expression in the lungs by means of IHC. Irradiation treatment notably increased serum TGF-b1 levels in the radiationþPBS group within 7 days (5723.8 304.7 pg/mL), and levels gradually increased until peaking on day 14 (6570.16 317.8 pg/mL).
Figure 5. Ad-MSCs protected lung tissue cells from apoptosis and mediated HGF expression in injured rats. (A) Apoptosis in lung sections was detected by means of TUNEL staining after irradiation. Magnification 400. Scale bar, 50 mm. (B) The apoptosis index was quantified of five random 40 images per group. Data are mean standard deviation of five random images. (C) Bax, Bcl-2 and caspase-3 protein levels in lungs were analyzed by means of Western blot. (DeF) Relative Bax, Bcl-2 and caspase-3 expression was evaluated by means of densitometric analysis normalized to b-actin for each sample. The Western blots are representative of at least three independent experiments. (G) HGF mRNA in lung tissue was detected on day 3 and normalized to rat b-actin. (H) Serum HGF was detected by means of ELISA on days 1, 3 and 7. Data are shown as mean standard deviation (*P < 0.05, **P < 0.01, n ¼ 5 per group). R, radiation.
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However, peak irradiation-induced TGF-b1 expression significantly decreased in the radiationþMSC group (3164.51 256.3 pg/mL) (Figure 4E). We also compared TGF-b1 expression in injured lungs through the use of real-time PCR. TGF-b1 messenger RNA (mRNA) expression was significantly higher on PBS treatment compared with the control group on day 7 (23.85-fold). In contrast, we only observed a 6.06-fold increase in TGF-b1 expression in the radiationþMSC group compared with the control group (Figure 4F; P < 0.01). We examined CTGF, a-SMA and Col1a1 expression in lung tissue on day 28 by means of IHC (Figure 4BeD). Irradiation induced increased CTGF, a-SMA and Col1a1 protein expression in the injured lung sections. CTGF, a-SMA and Col1a1 mRNA also increased on day 28 in radiationþPBS lung tissue by 8.43-fold, 10.73-fold and 13.09-fold, respectively. However, CTGF, a-SMA and Col1a1 mRNA only increased by 2.94-fold, 3.48-fold and 3.12-fold, respectively, in the radiationþMSC group compared with the radiationþPBS group (Figure 4GeI; P < 0.01). Ad-MSCs protect lung tissue cells from apoptosis and mediate HGF expression TUNEL staining showed that irradiation induced a significant increase in apoptosis in the lung within 28 days compared with the control group and radiationþMSC group (Figure 5A). The results showed that the apoptosis rate was 16.93% 2.61% in the radiationþPBS group. However, only 8.02% 2.47% of cells in the radiationþMSC group and 2.94% 0.93% in the normal control group were apoptotic on day 7 (Figure 5B). We analyzed Bcl-2, Bax and caspase-3 expression by means of Western blot for 28 days after irradiation. The results showed that Ad-MSC treatment significantly improved Bcl-2 expression in lung tissues on day 7 after irradiation, whereas the mean optical densities of Bax and caspase-3 were higher compared with the radiationþPBS group (P < 0.01) (Figure 5CeF). HGF mRNA expression significantly increased in the radiationþMSC group (6.44-fold increase) compared with the radiationþPBS group (0.89-fold increase) on day 3 (Figure 5G). HGF serum expression was significantly higher after MSC treatment (99706.2 1935.2 pg/mL) compared with the radiationþPBS group (56552.33 12094 pg/mL) group on day 7 (Figure 5H; P ¼ 0.004). Discussion This study provides evidence that IV delivery of AdMSCs is a beneficial treatment of acute RILI through
anti-inflammation, anti-fibrotic and anti-apoptotic effects. Sprague-Dawley rats were given a single 15 Gy dose of X-irradiation to the right thorax [21,22], successfully modeling RILI in rats. Previous evidence has shown that the IV route was as effective in treating lung disease as was intratracheal injection [9,10]. IV administration provides better therapy systemically and to the injured lungs, given the ease of IV administration and IV dosing. Therefore, we administered Ad-MSCs through IV delivery with a good safety record. Long-term in vitro MSC culture could lead to malignant transformation and could generate sarcoma in recipient lungs [23]. In the present study, we used Ad-MSCs from passage 3 to minimize unexpected transformation. We did not observe any sarcoma or other tumor type throughout the study. We injected Ad-MSCs within 2 h after irradiation to prevent MSC differentiation into myofibroblasts, which would accelerate lung fibrosis [24]. MSCs can selectively migrate to injured lungs because of several cytokines and their receptors (SDF-1/CXCR4). Previous studies found large numbers of engrafted cells at the lung in the first 1 h through the use of the Xenogen system in the C57BL/6J mouse [19]. However, our data showed that engraftment peaked at 72 h in the lung rather than within 24 h; this probably is due to the number of engrafted cells or the dose of radiation x-ray, which would affect chemokine release. Moreover, the Xenogen and fluorescence imaging data inconsistencies at 2 h and 12 h in our study may have been caused by the thick fur of Sprague-Dawley rats. Future studies will focus on the involvement of other potential cytokines/receptors axes at various radiation doses. Inflammatory cytokines play an important role in mediating, amplifying and maintaining the RILI process. Previous studies have shown that MSCs reduce the inflammatory response in both lipopolysaccharide- and bleomycin-induced acute lung injury models [9,25]. In our study, we found that Ad-MSCs decreased serum IL-1,IL-6 and TNF-a expression within the first 28 days after irradiation. IL-10 is a highly anti-inflammatory cytokine that inhibits macrophage production, and IL-10 treatment can attenuate acute lung injury [26]. However, serum IL-10 levels significantly increased in the radiationþMSC group. Increased serum IL-10 levels on Ad-MSC delivery may involve several mechanisms. Ad-MSCs can inhibit inflammation through autocrine IL-10 signaling [27,28], and they also release signals to several types of allogenic pro-inflammatory cells to alter their cytokine secretion profiles. High IL-10 levels inhibit the
Adipose-derived MSCs attenuate radiation-induced lung injury inflammatory process, such as neutrophil rolling, adhesion and transepithelial migration in the inflammatory host [26]. Lung irradiationeinduced fibrosis is a complex process that involves the activation of various proinflammatory and profibrotic cytokines, which are produced by damaged alveolar epithelial cells, endothelial cells and activated interstitial cells [2,3]. MSCs have been reported to alleviate fibrosis in experimental models [29]. In the present study, we demonstrated that MSCs significantly decreased fibrotic markers (TGF-b1, CTGF, a-SMA and Col1a1) and hydroxyproline content, a direct index reflecting lung fibrosis, suggesting that stem cell therapy has notable effects on fibrosis. Studies have demonstrated that TGF-b1 plays a critical role in radiation-induced lung fibrosis pathogenesis [30,31]. In this study, we observed a peak of radiation-induced TGF-b1 levels in the lung tissue and serum after thoracic irradiation during our experimental time course. We found that TGF-b1 mRNA production peaked on day 7 in the lung tissue. Therefore, the suppression of TGF-b1 expression may be sufficient to reduce irradiation-induced lung damage. Expectedly, we found that Ad-MSC IV administration dramatically inhibited irradiationinduced TGF-b1 expression. Therefore, the optimal time of Ad-MSCs treatment is 1 week after irradiation. Ad-MSC treatment can inhibit lung fibrosis primarily by decreasing TGF-b1 expression but also by avoiding immune rejection from irradiation-induced immune cell activation if injected earlier within 2 h after irradiation. MSCs secrete growth factors with cytoprotective and repair properties, including HGF [32]. HGF is a multifunctional factor that stimulates angiogenesis, inhibits fibrosis and reduces apoptosis [33,34]. HGF treatment improved Bcl-2 expression in lung tissue after treatment, which is an anti-apoptotic Bcl-2 family member that plays a key role in apoptosis regulation [17]. The pro-apoptotic protein Bax exerts the opposite effect of Bcl-2. It has been reported that high Bax expression promotes cell death. The expression of caspase-3, a key apoptosis executioner, can induce apoptosis [35]. In this study, we demonstrated that HGF expression increased within the irradiated lung on day 7 when rats were treated with Ad-MSCs. Moreover, our results show that MSC treatment decreased Bax and caspase-3 expression and increased Bcl-2 expression in the lung. Both cell proliferation and cell death can modulate alveolar repair, and we observed a decrease in apoptosis in radiation-injured lungs after MSC treatment. Previous studies have shown that HGF regulates TGFb1einduced expression of myofibroblast markers,
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such as a-SMA, collagen type 1 and fibronectin, in rat alveolar epithelial cells [33]. TGF-b1 is known to regulate a-SMA expression, but HGF has also been shown to affect expression of other TGFb1esignaling components, including the TGF-b1 signaling inhibitor Smad7. HGF-induced Smad7 expression limits the acquisition of the myofibroblast phenotype in alveolar epithelial cells, and HGF may mediate anti-inflammatory and anti-fibrotic responses [36]. RILI is a complex process that involves different cell types at different stages and diverse parts of RILI [1]. The limitation of our study is the lack of an in vitro investigation into the interaction between AdMSCs and alveolar or endothelial cells, or even other inflammatory cells. In addition, one issue of AdMSC therapy for RILI patients in clinical application is whether cancer patientederived Ad-MSCs would be as useful as those from healthy persons. Our future study will focus on whether Ad-MSCs from patients with cancer have similar capacity, safety and efficacy for autologous clinical application. In conclusion, Ad-MSCs have therapeutic potential in RILI management. We showed the beneficial role of IV delivery in treating RILI, including anti-inflammation, anti-fibrosis and anti-apoptosis roles. Acknowledgments This work was financially supported by the Natural Science Foundation of China (grant no. 81201218/ H1101) and the Science and Technology Department of Jilin Province (grant No. 20130413032GH). Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article.
References [1] Tsoutsou P, Koukourakis M. Radiation pneumonitis and fibrosis: mechanisms underlying its pathogenesis and implications for future research. Int J Radiat Oncol Biol Phys 2006;66:1281e93. [2] Graves PR, Siddiqui F, Anscher MS, Movsas B. Radiation pulmonary toxicity: from mechanisms to management. Semin Radiat Oncol 2010;20:201e7. [3] Abratt RP, Morga GW. Lung toxicity following chest irradiation in patients with lung cancer. Lung Cancer 2002;35: 103e9. [4] Roach M, Gandara DR, Yuo HS, Swift PS, Kroll S, Shrieve DC, et al. Radiation pneumonitis following combined modality therapy for lung cancer: analysis of prognostic factors. J Clin Oncol 1995;13:2606e12. [5] Cappuccini F, Eldh T, Bruder D, Gereke M, Jastrow H, Schulze-Osthoff K, et al. New insights into the molecular
570
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
X. Jiang et al. pathology of radiation-induced pneumopathy. Radiother Oncol 2011;101:86e92. Yufang S, Gangzheng H, Juanjuan S, Wenzhao L, Qing C, Peishun S, et al. Mesenchymal stem cells: a new strategy for immunosuppression and tissue repair. Cell Research 2010; 20:510e8. Prockop DJ, Oh JY. Mesenchymal stem/stromal cells (MSCs): role as guardians of inflammation. Mol Therapy 2012;20:14e20. Blanc KL, Mougiakakos D. Multipotent mesenchymal stromal cells and the innate immune system. Nat Rev Immunol 2012;12:383e96. Ortiz LA, Gambelli F, McBride C, Gaupp D, Baddoo M, Kaminski N, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci U S A 2003;100:8407e11. Rojas M, Xu J, Woods CR, Mora AL, Spears W, Roman J, et al. Bone marrow-derived mesenchymal stem cells in repair of the injured lung. Am J Respir Cell Mol Biol 2005;33: 145e52. Usunier B, Benderitter M, Tamarat R, Chape A. Management of fibrosis: the mesenchymal stromal cells breakthrough. Stem Cells Int 2014;2014:340257. Yanxia Z, Tianqing Z, Kedong S, Xiubo F, Xuehu M, Zhanfeng C. Adipose-derived stem cell: A better stem cell than BMSC. Cell Research 2008;18:S165. Lee RH, Kim B, Choi I, Kim H, Choi HS, Suh K, et al. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem 2004;14:311e24. Schäffler A, Büchler C. Concise review: adipose tissuederived stromal cellsebasic and clinical implications for novel cell-based therapies. Stem Cells 2007;25:818e27. Morizono K, De Ugarte DA, Zhu M, Zuk P, Elbarbary A, Ashjian P, et al. Multilineage cells from adipose tissue as gene delivery vehicles. Hum Gene Therapy 2003;14:59e66. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001;7: 211e28. Rodriguez AM, Elabd C, Delteil F, Astier J, Vernochet C, Saint-Marc P, et al. Adipocyte differentiation of multipotent cells established from human adipose tissue. Biochem Biophys Res Commun 2004;315:255e63. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:315e7. Hua W, Yue-Feng Y, Li Z, Feng-Jun X, Qun-Wei Z, MingLing W, et al. Hepatocyte Growth factor gene-modified mesenchymal stem cells reduce radiation-induced lung injury. Human Gene Ther 2013;24:343e53. Ji JF, He BP, Dheen ST, Tay SS. Interactions of chemokines and chemokine receptors mediate the migration of mesenchymal stem cells to the impaired site in the brain after hypoglossal nerve injury. Stem Cells 2004;2:415e27. Karvonen RL, Fernandez-Madrid F, Maughan RL, Palmer KC, Fernandez-Madrid I. An animal model of pulmonary radiation fibrosis with biochemical, physiologic,
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31] [32]
[33]
[34]
[35]
[36]
immunologic, and morphologic observations. Radiat Res 1987;111:68e80. Haston CK, Travis EL. Murine susceptibility to radiation -induced pulmonary fibrosis is influenced by a genetic factor implicated in susceptibility to bleomycin-induced pulmonary fibrosis. Cancer Research 1997;57:5286e91. Lee RH, Pulin AA, Seo MJ, Kota DJ, Ylostalo J, Larson BL, et al. Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell 2009; 5:54e63. Johnson LB, Riaz AA, Adawi D, Wittgren L, Back S, Thornberg C, et al. Radiation enteropathy and leucocyteendothelial cell reactions in a refined small bowel model. BMC Surg 2004;4:10. Ortiz LA, Dutrei M, Fattman C, Pandey AC, Torres G, Go K, et al. Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury. Proc Natl Acad Sci U S A 2007;104:11002e7. Li HD, Zhang QX, Mao Z, Xu XJ, Li NY, Zhang H. Exogenous interleukin-10 attenuates hyperoxia-induced acute lung injury in mice. Exp Physiol 2015;100:331e40. Yagi H, Soto-Gutierrez A, Parekkadan B, Kitagawa Y, Tompkins RG, Kobayashi N, et al. Mesenchymal stem cells: mechanisms of immunomodulation and homing. Cell Transplant 2010;19:667e79. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 2005; 105:1815e22. Moodley Y, Atienza D, Manuelpillai U, Samuel CS, Tchongue J, Ilancheran S, et al. Human umbilical cord mesenchymal stem cells reduce fibrosis of bleomycin induced lung injury. Am J Pathol 2009;175:303e13. Rube CE, Uthe D, Schmid KW, Richter KD, Wessel J, Schuck A, et al. Dose-dependent induction of transforming growth factor(TGF-b1) in the lung tissue of fibrosis-prone mice after thoracic irradiation. Int J Radiation Oncology Biol Phys 2000;47:1033e42. Border WA, Noble NA. Transforming growth factor beta in tissue fibrosis. N Eng J Med 1994;331:1286e92. Gazdhar A, Grad I, Tamò L, Gugger M, Feki A, Geiser T. The secretome of induced pluripotent stem cells reduces lung fibrosis in part by hepatocyte growth factor. Stem Cell Res Ther 2014;5:123. Marchand Adam S, Fabre A, Mailleux AA, Marchal J, Quesnel C, Kataoka H, et al. Defect of pro-hepatocyte growth factor activation by fibroblasts in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2006;174: 58e66. Matsumoto K, Nakamura T. Hepatocyte growth factor: Molecular structure, roles in liver regeneration, and other biological functions. Crit Rev Oncog 1992;3:27e54. Wang X, Zhou Y, Kim HP, Song R, Zarnegar R, Ryter SW, et al. Hepatocyte growth factor protects against hypoxia/ reoxygenation-induced apoptosis in endothelial cells. J Biol Chem 2004;279:5237e43. Shukla MN, Rose JL, Ray R, Lathrop KL, Ray A, Ray P. Hepatocyte Growth Factor Inhibits Epithelial to Myofibroblast Transition in Lung Cells via Smad7. Am J Respir Cell Mol Biol 2009;40:643e53.
Intravenous delivery of adipose-derived mesenchymal stromal cells attenuates acute radiation-induced lung injury in rats
XINPING JIANG1, XIN JIANG1, CHAO QU1, PENGYU CHANG1, CHU ZHANG3, YAQIN QU1 & YONGJUN LIU2 1
Department of Oncological Radiotherapy, The First Bethune Hospital of Jilin University, Changchun, China, Alliancells Bioscience Co, Ltd, Tianjin, China, and 3Department of Oncological Radiotherapy, The Second Bethune Hospital of Jilin University, Changchun, China
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Abstract Background aims. Radiation-induced lung injury (RILI) commonly occurs in patients with thoracic cancer. However, an effective treatment option has not yet been established. Adipose-derived mesenchymal stromal cells (Ad-MSCs) have significant potential for clinical use, but their role in RILI is currently unknown. We aimed to evaluate the therapeutic capacity of Ad-MSCs to heal acute RILI in rats. Methods. Sprague-Dawley rats were used in this study. Rat Ad-MSCs were delivered through the tail veins of rats 2 h after thorax irradiation. Lung histopathologic findings, pulmonary levels of inflammatory cytokines (interleukin [IL]-1, IL-6, IL-10 and tumor necrosis factor-a), pro-fibrotic factors (transforming growth factor [TGF]-b1, connective tissue growth factor, a-smooth muscle actin and type 1 collagen), pro- or anti-apoptotic mediators (Bcl-2, Bax and caspase-3) and the multifunctional factor hepatocyte growth factor were evaluated after Ad-MSC transplant. Results. Intravenous delivery of Ad-MSCs attenuated acute RILI. Further studies showed that Ad-MSCs had antiinflammation and anti-fibrotic effects and maintained lung epithelium integrity, as indicated by reduced serum levels of the pro-inflammatory cytokines IL-1, IL-6 and tumor necrosis factor-a, increased levels of the anti-inflammatory cytokine IL-10, and downregulated transforming growth factor -b1, a-smooth muscle actin and type 1 collagen levels in irradiated lung tissues. Ad-MSCs also regulated the expression of pro- and anti-apoptotic mediators (Bcl-2, Bax and caspase-3) to protect lung cells from apoptosis. Conclusions. Intravenous Ad-MSC delivery attenuated acute RILI through antiinflammation, anti-fibrosis and anti-apoptosis mechanisms. Key Words: adipose tissue, cell-based transplantation, mesenchymal stromal cells, radiation-induced lung injury
Introduction Radiation-induced lung injury (RILI) is a common major obstacle in thoracic cancer radiotherapy [1,2]. The clinical incidence of radiation-induced pneumonitis ranges from 5% to 10% [3,4]. RILI develops through a complex pathological process, resulting in excessive inflammation or extracellular matrix deposition in the lung interstitium, ultimately leading to impaired lung function and respiratory failure [5]. Thus, it is important to alleviate RILI to improve tumor control and the patient’s quality of life. The current primary approach to manage RILI is hormone treatments to temporarily suppress inflammation [2]. However, there is no known effective therapeutic strategy for RILI. Mesenchymal stromal cells (MSCs) have significant clinical potential [6e8]. Recent studies have
demonstrated that engrafted stem cells have beneficial effects in injured lung tissues [9,10]. Radiation exposure of the lung leads to fibrosis, and fibrosis is a complex pathology driven by numerous biological factors, such as chronic inflammation and hypoxia. MSC anti-fibrotic functions have been described in various organs [11]. MSC cellular therapy would be an ideal approach to treat RILI. However, the clinical use of bone marrowederived MSCs has presented problems, including pain, morbidity and low cell number on harvest [12]. Adipose-derived mesenchymal stromal cells (Ad-MSCs) have also been widely studied because of their ease of isolation, abundant distribution and low immunogenicity [13,14]. In addition, Ad-MSCs have been shown to have expansion capacity superior to that of bone marrowederived MSCs in vitro [15,16]. Therefore,
Correspondence: Yaqin Qu, MD, Department of Oncological Radiotherapy, The First Bethune Hospital of Jilin University, Changchun 130021, China. E-mail: [email protected]; Yongjun Liu, MD, Alliancells Bioscience Co, Ltd, Tianjin, China. E-mail: [email protected] (Received 20 October 2014; accepted 13 February 2015) ISSN 1465-3249 Copyright Ó 2015, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2015.02.011
Adipose-derived MSCs attenuate radiation-induced lung injury Ad-MSCs are a potential candidate for autologous transplant in lung disease. Moreover, autologous AdMSC application is free from ethical or security issues. The therapeutic potential and usefulness of AdMSC delivery in RILI treatment has not been studied. We studied the effects of intravenous (IV) delivery of Ad-MSCs on acute RILI in a Sprague-Dawley rat model. Methods
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Rad-Source). The remainder of the body was shielded by lead strips. Ninety rats were used in the study. Animals were randomly divided into three groups (n ¼ 30 per group). After thoracic irradiation, animals received a 0.5-mL saline injection (radiationþphosphate-buffered saline [PBS] group), 5 106 Ad-MSCs in a 0.5-mL saline injection (radiationþMSC group), or were used as normal controls. Animals were injected through the tail vein within 2 h after irradiation. Peripheral blood and lung tissues were collected from rats on days 1, 3, 7, 14 and 28 for analysis.
Ethical approval Sprague-Dawley rats (female; weight, 180e220 g) were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China). All experiments involving animal subjects were performed in accordance with guidelines approved by our local animal care and use committee. Ad-MSC isolation, culture and characterization
Tracing experiments The cell tracker CM-Dil (Invitrogen Inc) was dissolved in dimethyl sulfoxide (Invitrogen) and added to passage 3 Ad-MSCs according to the manufacturer’s protocol. A total of 5 106 cells were intravenously injected into irradiated and normal rats. The distribution of labeled Ad-MSCs was determined by use of the Xenogen (IVIS, Lumina, Caliper Life Sciences) in vivo optical imaging technique, and frozen lung sections were examined my means of fluorescence microscopy (Olympus).
Fat tissue was obtained from subcutaneous adipose tissue in the inguinal groove of female SpragueDawley rats under sterile conditions. Ad-MSCs were separated by a 200-mesh strainer and suspended in complete medium (90% Dulbecco’s modified Eagle’s medium-low glucose/F-12þ10% fetal bovine serum) (Gibco) and grown with 5% CO2 in a humidified atmosphere at 37 C, as described previously [17]. Cells were passaged every 2 to 3 days, and were then harvested at passage 3 for characterization, identification and IV delivery. The phenotype of Ad-MSCs was evaluated by means of flow cytometry analysis with the use of antirat cluster of differentiation (CD)11b-phycoerythrin (PE), CD29-PE, CD44efluorescein isothiocyanate (FITC) and CD45eantigen-presenting cells. Mouse immunoglobulin G1eFITC and PE were used as isotype controls. All antibodies were purchased from eBioscience. Cells were harvested at passage 3 and plated into six-well plates. Adipogenic, osteogenic and chrondrogenic medium (Trevigen) was added to each well under standard culture conditions according to the manufacturer’s protocol. Cells in the remaining wells served as controls. Adipogenesis, osteogenesis and chondrogenesis were confirmed by oil red O, alizarin red and alcian blue staining, respectively.
Serum was collected from the peripheral blood by centrifugation. IL-1, IL-6, IL-10, tumor necrosis factor (TNF)-a, transforming growth factor (TGF)b1 and hepatocyte growth factor (HGF) (R＆D Systems) levels were measured by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions. For hydroxyproline analysis, the lung tissues were homogenized and assayed by use of hydroxyproline ELISA kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instructions.
Experimental design
Immunohistochemistry
Rats received a single local dose of radiation with 15 Gy of x-rays to the right thorax (160 kV, 25 mA, 1.25 Gy/min, RS-2000 Pro Biological Irradiator,
Paraffin-embedded lung sections were de-waxed and rehydrated before antigen retrieval. The sections were incubated in 0.3% H2O2 to block endogenous
Histopathology For histological examination, irradiated lung tissue was fixed in 10% neutral-buffered formalin for 48 h, paraffin-embedded and sectioned at an average thickness of 5 mm. The sections were stained with hematoxylin and eosin and Masson’s trichrome. The sections were semi-quantitatively analyzed by two blinded observers using five random images per group. Enzyme-linked immunosorbent assay
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Figure 1. Chemotactic homing of Ad-MSCs. (A) The distribution of Ad-MSCs in a RILI rat model were determined by means of the Xenogen in vivo optical imaging technique. (B) Histological analysis by frozen section. Magnification 100. Scale bar, 200 mm. (C) Quantification of CM-Dilelabeled Ad-MSCs in lung section fields on day 3. (D) SDF-1 mRNA expression in lungs 28 days after irradiation. Fold expression values were normalized to the normal control rat. Data represent mean standard deviation (*P < 0.05, **P < 0.01, n ¼ 5 per group). R, radiation; N, normal.
peroxidase and incubated with serum from the host to block nonspecific antigen binding. The primary antibody was diluted according to the manufacturer’s recommendations and was added to the sections and incubated overnight at 4 C. The primary antibodies used for immunohistochemistry (IHC) included TGF-b1, connective tissue growth factor (CTGF), a-smooth muscle actin (SMA), type 1 collagen (Col1al) and hydroxyproline (Abcam). Secondary antibody was added and incubated at 37 C for 2 h. Diaminobenzidine was added to detect positive cells. An IHC staining kit (Abcam) was used to detect TGF-b1, CTGF, a-SMA, Col1al and hydroxyproline.
Apoptosis assay Apoptosis was assessed in paraffin-embedded lung sections with the use of a terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL, Roche) in situ cell death detection kit according to
the manufacturer’s instructions. TUNEL-positive cells were quantified in five random 40 images per group under light microscopy. Real-time polymerase chain reaction A total of 100 mg of irradiated lung tissue was freshly isolated from each sample. Total RNA was isolated with TRIzol reagent (Invitrogen), and 1 mg of total RNA from each sample was used for first-strand complementary DNA synthesis with the use of a real-time polymerase chain reaction (PCR) Kit (Takara Bio Inc). Total complementary DNA was then incubated with primers against stromal cellederived factor-1 (SDF-1), TGF-b1, HGF, aSMA, Col1al or CTGF. The primer sequences were as follows: SDF-1 Forward 50 -ACGGTCTTGAACTACTGGC G-30 Reverse 50 -GGAGGCTTACAGCACGAAAC-30
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Figure 2. Effect of Ad-MSCs on histological changes after thorax irradiation. (A) Histological analysis by hematoxylin and eosin (HE) staining. Magnification 200. Scale bar, 100 mm. (B) Histological analysis by means of Masson staining. Magnification 400. Scale bar, 50 mm. (C) Lung fibrosis was assessed by means of hydroxyproline IHC detection. (D,E) Alveolar thickness and Masson-stained areas were semi-quantified from five random images per group (*P < 0.05, **P < 0.01, n ¼ 5 per group). (F) Hydroxyproline serum levels were detected by means of ELISA assay at day 28. Data are shown as mean standard deviation (*P < 0.05, **P < 0.01, n ¼ 5 per group).
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Figure 3. TNF- a, IL-6, IL-1 and IL-10 serum levels (AeD) were detected by means of ELISA assay on days 1, 3, 7, 14 and 28 after thorax irradiation. Data are shown as mean standard deviation (*P < 0.05, **P < 0.01, n ¼ 5 per group). R, radiation.
TGF-b1 Forward 50 -GGACTACTACGCCAAAG AAG-30 Reverse 50 -TCAAAAGACAGCCACTCAGG-30 HGF
Forward GCT-30
50 -GTGCATCAGAAACAAGG
Reverse 50 -TGGCACTTGATGCCACTCTT-30 a-SMA Forward 50 -GGCATCCACGAAACCAC CTA-30 Reverse 50 -TGAAGGCGCTGATCCACAAA-30 Col1a1 Forward 50 -GCCTCTGCAACAAATCCC CA-30 Reverse 50 -CATGTGTGGCCGATGTTTCC-30 CTGF Forward 50 - GCGCCAAGCAGCTGGG AGAA-30 Reverse 50 - CGGCCCCATCCAGGCAAGTG-30 b-Actin Forward 50 -CACCCGCGAGTACAACC TTC-30 Reverse 50 -CCATACCCACCATCACACCC-30 Expression levels were normalized to b-actin levels. Quantitative PCR was performed with the use of SYBR Green I TaqMan probes (Roche) in 40 amplification cycles in an ABI 7500 Fast (Life Technologies). All reactions were carried out in duplicate, and the results were analyzed by the 2DDCT method.
Western blot For Western blot analysis, lung tissues were homogenized in an appropriate amount of ice-cold lysis buffer. Equivalent amounts of protein (25e40 mg) were separated by use of 15% sodium dodecyl sulfateepolyacrylamide gel electrophoresis and transferred onto a polyvinylidene fuoride membrane (Millipore). Membranes were probed with the following antibodies: caspase3 (1:1000, Santa Cruz), Bcl-2 (1:750, Abcam) and Bax (1:1000, Santa Cruz). b-Actin (Kangchen) was used as an internal control. Immunoreactivity was detected by use of the Odyssey Infrared Imaging System (Gene Company Limited). All blots were exposed for optimal lengths of time for visualization. Statistical analysis All data were analyzed with the use of SPSS 19.0 software (IBM Corp) and expressed as mean standard deviation (SD). One-way analysis of variance was performed to compare data among groups. Statistical significance was defined as P 0.05. Results Features of rat Ad-MSCs Isolated rat Ad-MSCs showed a spindle-like morphology and were able to differentiate into
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Figure 4. Profibrotic expression in serum and lung tissue. (AeD) TGF-b1, a-SMA, Col1a1 and CTGF expression in lung tissue was measured by means of IHC. Magnification 400. Scale bar, 50 mm. (E) Serum TGF-b1 expression was detected by means of ELISA on days 7, 14 and 28. (FeI) TGF-b1, a-SMA, Col1a1 and CTGF mRNA was detected by means of real-time PCR after thorax irradiation with b-actin as the internal control. Fold expression values were normalized to the control. Data represent mean standard deviation (*P < 0.05, **P < 0.01, n ¼ 5 per group).
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Adipose-derived MSCs attenuate radiation-induced lung injury adipocytes, osteoblasts and chondrocytes after 14 days in defined culture media. Flow cytometry revealed that rat Ad-MSCs were CD29-and CD44positive and CD11b- and CD45-negative. All of these results are consistent with the minimal criteria for MSC identification [18]. Ad-MSCs home to injured lung tissue We determined the systemic in vivo Ad-MSC distribution through the use of the Xenogen imaging system (Figure 1A). Within the first 2 h after irradiation, there was no significant difference in Ad-MSC lung distribution between the radiation group and the normal group. The RþMSC group showed that engrafted cells localized primarily to the bone and lymph gland from 2 to 24 h, and the number of engrafted cells in the lung peaked at 72 h. Interestingly, we observed little to no Ad-MSCs in the lung after day 14 in either the radiation group or normal group. However, our fluorescence images confirmed that a large number of transplanted cells had already homed to the injured lung by 12 h (Figure 1B, C). The fluorescence imaging data were inconsistent with the Xenogen data. Previous studies examined large numbers of engrafted cells at the lung in the first 2 h by the Xenogen system in the C57/BL6 mouse [19]. However, the inconsistencies in the engrafted cells at 2 h and 12 h in our study may be due to the thick fur of Sprague-Dawley rats. Moreover, our data showed that engraftment peaked at 72 h in the lung rather than at 12 h; this probably was caused by the number of engrafted cells or the dose of radiation xray, which would affect chemokine release. MSCs express the specific chemokine receptor CXCR4, and they migrate to radiation-injured sites through chemotaxis [20]. The SDF-1/CXCR4 axis is a signaling pathway associated with active stem cell recruitment in MSCs homing to irradiated lungs. Therefore, we evaluated SDF-1 expression in irradiated lung tissues by real-time PCR at various time points after Ad-MSC delivery. SDF-1 expression was significantly higher on day 7 in the radiation group compared with that in the normal group (1.72-fold increase; P < 0.01) (Figure 2D). Ad-MSC therapy attenuates RILI histopathology We evaluated RILI-associated histological changes on day 28 by means of hematoxylin and eosin
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staining, Masson staining and hydroxyproline detection. The alveolar architecture was completely disrupted in the radiationþPBS group compared with the normal control group. In contrast, Ad-MSC treatment allowed for alveolar wall thickening and minimal interstitial edema (Figure 2A). Masson staining and hydroxyproline detection showed marked radiation-induced collagen deposition in lung tissue on day 28. However, MSC treatment attenuated collagen deposition compared with the radiationþPBS group (Figure 2B, C, F). The histology and semi-quantitative analysis are shown in Figure 2D, E. Ad-MSC treatment reduces pro-inflammatory cytokine levels and improves anti-inflammatory cytokine expression We measured pro-inflammatory cytokine serum levels, including IL-1, IL-6 and TNF-a, by means of ELISA on days 1, 3, 7, 14 and 28 after irradiation. Thorax irradiation resulted in the production of the inflammatory cytokines IL-1, IL-6 and TNF-a, whose serum levels significantly increased in RILI rats on day 14 (Figure 3AeC). In contrast, Ad-MSC treatment significantly decreased the irradiationinduced protein release of IL-1, IL-6 and TNF-a. We also measured the serum levels of the antiinflammatory cytokine IL-10 on days 1, 3, 7, 14 and 28. As shown in Figure 3D, baseline IL-10 was similar among groups 1 day after thorax irradiation; however, compared with baseline, serum IL-10 gradually decreased within 7 days after irradiation in the radiationþPBS group. In contrast, serum IL10 levels significantly increased to 180.9 9.67 pg/ mL on day 7 in the radiationþMSC group compared with 28.076.43 pg/mL in the radiationþPBS group (P < 0.01). Ad-MSC therapy reduces profibrotic factor expression We examined TGF-b1 expression in the serum and lung tissue for 28 days after irradiation. As shown in Figure 4A, we measured TGF-b1 expression in the lungs by means of IHC. Irradiation treatment notably increased serum TGF-b1 levels in the radiationþPBS group within 7 days (5723.8 304.7 pg/mL), and levels gradually increased until peaking on day 14 (6570.16 317.8 pg/mL).
Figure 5. Ad-MSCs protected lung tissue cells from apoptosis and mediated HGF expression in injured rats. (A) Apoptosis in lung sections was detected by means of TUNEL staining after irradiation. Magnification 400. Scale bar, 50 mm. (B) The apoptosis index was quantified of five random 40 images per group. Data are mean standard deviation of five random images. (C) Bax, Bcl-2 and caspase-3 protein levels in lungs were analyzed by means of Western blot. (DeF) Relative Bax, Bcl-2 and caspase-3 expression was evaluated by means of densitometric analysis normalized to b-actin for each sample. The Western blots are representative of at least three independent experiments. (G) HGF mRNA in lung tissue was detected on day 3 and normalized to rat b-actin. (H) Serum HGF was detected by means of ELISA on days 1, 3 and 7. Data are shown as mean standard deviation (*P < 0.05, **P < 0.01, n ¼ 5 per group). R, radiation.
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However, peak irradiation-induced TGF-b1 expression significantly decreased in the radiationþMSC group (3164.51 256.3 pg/mL) (Figure 4E). We also compared TGF-b1 expression in injured lungs through the use of real-time PCR. TGF-b1 messenger RNA (mRNA) expression was significantly higher on PBS treatment compared with the control group on day 7 (23.85-fold). In contrast, we only observed a 6.06-fold increase in TGF-b1 expression in the radiationþMSC group compared with the control group (Figure 4F; P < 0.01). We examined CTGF, a-SMA and Col1a1 expression in lung tissue on day 28 by means of IHC (Figure 4BeD). Irradiation induced increased CTGF, a-SMA and Col1a1 protein expression in the injured lung sections. CTGF, a-SMA and Col1a1 mRNA also increased on day 28 in radiationþPBS lung tissue by 8.43-fold, 10.73-fold and 13.09-fold, respectively. However, CTGF, a-SMA and Col1a1 mRNA only increased by 2.94-fold, 3.48-fold and 3.12-fold, respectively, in the radiationþMSC group compared with the radiationþPBS group (Figure 4GeI; P < 0.01). Ad-MSCs protect lung tissue cells from apoptosis and mediate HGF expression TUNEL staining showed that irradiation induced a significant increase in apoptosis in the lung within 28 days compared with the control group and radiationþMSC group (Figure 5A). The results showed that the apoptosis rate was 16.93% 2.61% in the radiationþPBS group. However, only 8.02% 2.47% of cells in the radiationþMSC group and 2.94% 0.93% in the normal control group were apoptotic on day 7 (Figure 5B). We analyzed Bcl-2, Bax and caspase-3 expression by means of Western blot for 28 days after irradiation. The results showed that Ad-MSC treatment significantly improved Bcl-2 expression in lung tissues on day 7 after irradiation, whereas the mean optical densities of Bax and caspase-3 were higher compared with the radiationþPBS group (P < 0.01) (Figure 5CeF). HGF mRNA expression significantly increased in the radiationþMSC group (6.44-fold increase) compared with the radiationþPBS group (0.89-fold increase) on day 3 (Figure 5G). HGF serum expression was significantly higher after MSC treatment (99706.2 1935.2 pg/mL) compared with the radiationþPBS group (56552.33 12094 pg/mL) group on day 7 (Figure 5H; P ¼ 0.004). Discussion This study provides evidence that IV delivery of AdMSCs is a beneficial treatment of acute RILI through
anti-inflammation, anti-fibrotic and anti-apoptotic effects. Sprague-Dawley rats were given a single 15 Gy dose of X-irradiation to the right thorax [21,22], successfully modeling RILI in rats. Previous evidence has shown that the IV route was as effective in treating lung disease as was intratracheal injection [9,10]. IV administration provides better therapy systemically and to the injured lungs, given the ease of IV administration and IV dosing. Therefore, we administered Ad-MSCs through IV delivery with a good safety record. Long-term in vitro MSC culture could lead to malignant transformation and could generate sarcoma in recipient lungs [23]. In the present study, we used Ad-MSCs from passage 3 to minimize unexpected transformation. We did not observe any sarcoma or other tumor type throughout the study. We injected Ad-MSCs within 2 h after irradiation to prevent MSC differentiation into myofibroblasts, which would accelerate lung fibrosis [24]. MSCs can selectively migrate to injured lungs because of several cytokines and their receptors (SDF-1/CXCR4). Previous studies found large numbers of engrafted cells at the lung in the first 1 h through the use of the Xenogen system in the C57BL/6J mouse [19]. However, our data showed that engraftment peaked at 72 h in the lung rather than within 24 h; this probably is due to the number of engrafted cells or the dose of radiation x-ray, which would affect chemokine release. Moreover, the Xenogen and fluorescence imaging data inconsistencies at 2 h and 12 h in our study may have been caused by the thick fur of Sprague-Dawley rats. Future studies will focus on the involvement of other potential cytokines/receptors axes at various radiation doses. Inflammatory cytokines play an important role in mediating, amplifying and maintaining the RILI process. Previous studies have shown that MSCs reduce the inflammatory response in both lipopolysaccharide- and bleomycin-induced acute lung injury models [9,25]. In our study, we found that Ad-MSCs decreased serum IL-1,IL-6 and TNF-a expression within the first 28 days after irradiation. IL-10 is a highly anti-inflammatory cytokine that inhibits macrophage production, and IL-10 treatment can attenuate acute lung injury [26]. However, serum IL-10 levels significantly increased in the radiationþMSC group. Increased serum IL-10 levels on Ad-MSC delivery may involve several mechanisms. Ad-MSCs can inhibit inflammation through autocrine IL-10 signaling [27,28], and they also release signals to several types of allogenic pro-inflammatory cells to alter their cytokine secretion profiles. High IL-10 levels inhibit the
Adipose-derived MSCs attenuate radiation-induced lung injury inflammatory process, such as neutrophil rolling, adhesion and transepithelial migration in the inflammatory host [26]. Lung irradiationeinduced fibrosis is a complex process that involves the activation of various proinflammatory and profibrotic cytokines, which are produced by damaged alveolar epithelial cells, endothelial cells and activated interstitial cells [2,3]. MSCs have been reported to alleviate fibrosis in experimental models [29]. In the present study, we demonstrated that MSCs significantly decreased fibrotic markers (TGF-b1, CTGF, a-SMA and Col1a1) and hydroxyproline content, a direct index reflecting lung fibrosis, suggesting that stem cell therapy has notable effects on fibrosis. Studies have demonstrated that TGF-b1 plays a critical role in radiation-induced lung fibrosis pathogenesis [30,31]. In this study, we observed a peak of radiation-induced TGF-b1 levels in the lung tissue and serum after thoracic irradiation during our experimental time course. We found that TGF-b1 mRNA production peaked on day 7 in the lung tissue. Therefore, the suppression of TGF-b1 expression may be sufficient to reduce irradiation-induced lung damage. Expectedly, we found that Ad-MSC IV administration dramatically inhibited irradiationinduced TGF-b1 expression. Therefore, the optimal time of Ad-MSCs treatment is 1 week after irradiation. Ad-MSC treatment can inhibit lung fibrosis primarily by decreasing TGF-b1 expression but also by avoiding immune rejection from irradiation-induced immune cell activation if injected earlier within 2 h after irradiation. MSCs secrete growth factors with cytoprotective and repair properties, including HGF [32]. HGF is a multifunctional factor that stimulates angiogenesis, inhibits fibrosis and reduces apoptosis [33,34]. HGF treatment improved Bcl-2 expression in lung tissue after treatment, which is an anti-apoptotic Bcl-2 family member that plays a key role in apoptosis regulation [17]. The pro-apoptotic protein Bax exerts the opposite effect of Bcl-2. It has been reported that high Bax expression promotes cell death. The expression of caspase-3, a key apoptosis executioner, can induce apoptosis [35]. In this study, we demonstrated that HGF expression increased within the irradiated lung on day 7 when rats were treated with Ad-MSCs. Moreover, our results show that MSC treatment decreased Bax and caspase-3 expression and increased Bcl-2 expression in the lung. Both cell proliferation and cell death can modulate alveolar repair, and we observed a decrease in apoptosis in radiation-injured lungs after MSC treatment. Previous studies have shown that HGF regulates TGFb1einduced expression of myofibroblast markers,
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such as a-SMA, collagen type 1 and fibronectin, in rat alveolar epithelial cells [33]. TGF-b1 is known to regulate a-SMA expression, but HGF has also been shown to affect expression of other TGFb1esignaling components, including the TGF-b1 signaling inhibitor Smad7. HGF-induced Smad7 expression limits the acquisition of the myofibroblast phenotype in alveolar epithelial cells, and HGF may mediate anti-inflammatory and anti-fibrotic responses [36]. RILI is a complex process that involves different cell types at different stages and diverse parts of RILI [1]. The limitation of our study is the lack of an in vitro investigation into the interaction between AdMSCs and alveolar or endothelial cells, or even other inflammatory cells. In addition, one issue of AdMSC therapy for RILI patients in clinical application is whether cancer patientederived Ad-MSCs would be as useful as those from healthy persons. Our future study will focus on whether Ad-MSCs from patients with cancer have similar capacity, safety and efficacy for autologous clinical application. In conclusion, Ad-MSCs have therapeutic potential in RILI management. We showed the beneficial role of IV delivery in treating RILI, including anti-inflammation, anti-fibrosis and anti-apoptosis roles. Acknowledgments This work was financially supported by the Natural Science Foundation of China (grant no. 81201218/ H1101) and the Science and Technology Department of Jilin Province (grant No. 20130413032GH). Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article.
References [1] Tsoutsou P, Koukourakis M. Radiation pneumonitis and fibrosis: mechanisms underlying its pathogenesis and implications for future research. Int J Radiat Oncol Biol Phys 2006;66:1281e93. [2] Graves PR, Siddiqui F, Anscher MS, Movsas B. Radiation pulmonary toxicity: from mechanisms to management. Semin Radiat Oncol 2010;20:201e7. [3] Abratt RP, Morga GW. Lung toxicity following chest irradiation in patients with lung cancer. Lung Cancer 2002;35: 103e9. [4] Roach M, Gandara DR, Yuo HS, Swift PS, Kroll S, Shrieve DC, et al. Radiation pneumonitis following combined modality therapy for lung cancer: analysis of prognostic factors. J Clin Oncol 1995;13:2606e12. [5] Cappuccini F, Eldh T, Bruder D, Gereke M, Jastrow H, Schulze-Osthoff K, et al. New insights into the molecular
570
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
X. Jiang et al. pathology of radiation-induced pneumopathy. Radiother Oncol 2011;101:86e92. Yufang S, Gangzheng H, Juanjuan S, Wenzhao L, Qing C, Peishun S, et al. Mesenchymal stem cells: a new strategy for immunosuppression and tissue repair. Cell Research 2010; 20:510e8. Prockop DJ, Oh JY. Mesenchymal stem/stromal cells (MSCs): role as guardians of inflammation. Mol Therapy 2012;20:14e20. Blanc KL, Mougiakakos D. Multipotent mesenchymal stromal cells and the innate immune system. Nat Rev Immunol 2012;12:383e96. Ortiz LA, Gambelli F, McBride C, Gaupp D, Baddoo M, Kaminski N, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci U S A 2003;100:8407e11. Rojas M, Xu J, Woods CR, Mora AL, Spears W, Roman J, et al. Bone marrow-derived mesenchymal stem cells in repair of the injured lung. Am J Respir Cell Mol Biol 2005;33: 145e52. Usunier B, Benderitter M, Tamarat R, Chape A. Management of fibrosis: the mesenchymal stromal cells breakthrough. Stem Cells Int 2014;2014:340257. Yanxia Z, Tianqing Z, Kedong S, Xiubo F, Xuehu M, Zhanfeng C. Adipose-derived stem cell: A better stem cell than BMSC. Cell Research 2008;18:S165. Lee RH, Kim B, Choi I, Kim H, Choi HS, Suh K, et al. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem 2004;14:311e24. Schäffler A, Büchler C. Concise review: adipose tissuederived stromal cellsebasic and clinical implications for novel cell-based therapies. Stem Cells 2007;25:818e27. Morizono K, De Ugarte DA, Zhu M, Zuk P, Elbarbary A, Ashjian P, et al. Multilineage cells from adipose tissue as gene delivery vehicles. Hum Gene Therapy 2003;14:59e66. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001;7: 211e28. Rodriguez AM, Elabd C, Delteil F, Astier J, Vernochet C, Saint-Marc P, et al. Adipocyte differentiation of multipotent cells established from human adipose tissue. Biochem Biophys Res Commun 2004;315:255e63. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:315e7. Hua W, Yue-Feng Y, Li Z, Feng-Jun X, Qun-Wei Z, MingLing W, et al. Hepatocyte Growth factor gene-modified mesenchymal stem cells reduce radiation-induced lung injury. Human Gene Ther 2013;24:343e53. Ji JF, He BP, Dheen ST, Tay SS. Interactions of chemokines and chemokine receptors mediate the migration of mesenchymal stem cells to the impaired site in the brain after hypoglossal nerve injury. Stem Cells 2004;2:415e27. Karvonen RL, Fernandez-Madrid F, Maughan RL, Palmer KC, Fernandez-Madrid I. An animal model of pulmonary radiation fibrosis with biochemical, physiologic,
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31] [32]
[33]
[34]
[35]
[36]
immunologic, and morphologic observations. Radiat Res 1987;111:68e80. Haston CK, Travis EL. Murine susceptibility to radiation -induced pulmonary fibrosis is influenced by a genetic factor implicated in susceptibility to bleomycin-induced pulmonary fibrosis. Cancer Research 1997;57:5286e91. Lee RH, Pulin AA, Seo MJ, Kota DJ, Ylostalo J, Larson BL, et al. Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell 2009; 5:54e63. Johnson LB, Riaz AA, Adawi D, Wittgren L, Back S, Thornberg C, et al. Radiation enteropathy and leucocyteendothelial cell reactions in a refined small bowel model. BMC Surg 2004;4:10. Ortiz LA, Dutrei M, Fattman C, Pandey AC, Torres G, Go K, et al. Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury. Proc Natl Acad Sci U S A 2007;104:11002e7. Li HD, Zhang QX, Mao Z, Xu XJ, Li NY, Zhang H. Exogenous interleukin-10 attenuates hyperoxia-induced acute lung injury in mice. Exp Physiol 2015;100:331e40. Yagi H, Soto-Gutierrez A, Parekkadan B, Kitagawa Y, Tompkins RG, Kobayashi N, et al. Mesenchymal stem cells: mechanisms of immunomodulation and homing. Cell Transplant 2010;19:667e79. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 2005; 105:1815e22. Moodley Y, Atienza D, Manuelpillai U, Samuel CS, Tchongue J, Ilancheran S, et al. Human umbilical cord mesenchymal stem cells reduce fibrosis of bleomycin induced lung injury. Am J Pathol 2009;175:303e13. Rube CE, Uthe D, Schmid KW, Richter KD, Wessel J, Schuck A, et al. Dose-dependent induction of transforming growth factor(TGF-b1) in the lung tissue of fibrosis-prone mice after thoracic irradiation. Int J Radiation Oncology Biol Phys 2000;47:1033e42. Border WA, Noble NA. Transforming growth factor beta in tissue fibrosis. N Eng J Med 1994;331:1286e92. Gazdhar A, Grad I, Tamò L, Gugger M, Feki A, Geiser T. The secretome of induced pluripotent stem cells reduces lung fibrosis in part by hepatocyte growth factor. Stem Cell Res Ther 2014;5:123. Marchand Adam S, Fabre A, Mailleux AA, Marchal J, Quesnel C, Kataoka H, et al. Defect of pro-hepatocyte growth factor activation by fibroblasts in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2006;174: 58e66. Matsumoto K, Nakamura T. Hepatocyte growth factor: Molecular structure, roles in liver regeneration, and other biological functions. Crit Rev Oncog 1992;3:27e54. Wang X, Zhou Y, Kim HP, Song R, Zarnegar R, Ryter SW, et al. Hepatocyte growth factor protects against hypoxia/ reoxygenation-induced apoptosis in endothelial cells. J Biol Chem 2004;279:5237e43. Shukla MN, Rose JL, Ray R, Lathrop KL, Ray A, Ray P. Hepatocyte Growth Factor Inhibits Epithelial to Myofibroblast Transition in Lung Cells via Smad7. Am J Respir Cell Mol Biol 2009;40:643e53.