ORIGINAL RESEARCH Critical Role of Vascular Endothelial Growth Factor Secreted by Mesenchymal Stem Cells in Hyperoxic Lung Injury Yun Sil Chang1,2*, So Yoon Ahn1*, Hong Bae Jeon3, Dong Kyung Sung2, Eun Sun Kim4, Se In Sung1, Hye Soo Yoo1, Soo Jin Choi3, Won Il Oh3, and Won Soon Park1,2 1
Department of Pediatrics, Samsung Medical Center, and 2Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Korea; 3Biomedical Research Institute, MEDIPOST Co., Ltd., Seoul, Korea; and 4Department of Pediatrics, CHA Gangnam Medical Center, CHA University School of Medicine, Seoul, Korea
Abstract Intratracheal transplantation of human umbilical cord blood (UCB)–derived mesenchymal stem cells (MSCs) protects against neonatal hyperoxic lung injury by a paracrine rather than a regenerative mechanism. However, the role of paracrine factors produced by the MSCs, such as vascular endothelial growth factor (VEGF), has not been delineated. This study examined whether VEGF secreted by MSCs plays a pivotal role in protecting against neonatal hyperoxic lung injury. VEGF was knocked down in human UCB–derived MSCs by transfection with small interfering RNA specific for human VEGF. The in vitro effects of MSCs with or without VEGF knockdown or neutralizing antibody were evaluated in a rat lung epithelial (L2) cell line challenged with H2O2. To confirm these results in vivo, newborn Sprague-Dawley rats were exposed to hyperoxia (90% O2) for 14 days. MSCs (1 3 105 cells) with or without VEGF knockdown were administered intratracheally at postnatal Day 5. Lungs were serially harvested for biochemical and histologic analyses. VEGF knockdown and antibody abolished the in vitro benefits of MSCs on H2O2–induced cell death and the upregulation of inflammatory cytokines in L2 cells. VEGF knockdown also abolished the in vivo protective effects of MSCs in hyperoxic lung injury, such as the attenuation of impaired alveolarization and angiogenesis, reduction in the number of terminal deoxynucleotidyl
transferase dUTP nick end labeling–positive and ED-1–positive cells, and down-regulation of proinflammatory cytokine levels. Our data indicate that VEGF secreted by transplanted MSCs is one of the critical paracrine factors that play seminal roles in attenuating hyperoxic lung injuries in neonatal rats. Keywords: stem cells; cell transplantation; newborn; lung injury;
vascular endothelial growth factor
Clinical Relevance Intratracheal transplantation of human umbilical cord blood–derived mesenchymal stem cells (MSCs) protects against hyperoxic lung injury in neonatal rats. These protective effects represent a paracrine rather than a regenerative mechanism. However, the role of paracrine factors secreted by MSCs, such as vascular endothelial growth factor (VEGF), has not been delineated. VEGF secreted by transplanted MSCs is at least one of the critical factors that mediate the protective effects of MSCs against hyperoxic lung injury, such as impaired alveolarization and inflammatory responses in newborn rats.
( Received in original form September 5, 2013; accepted in final form March 24, 2014 ) *These authors contributed equally to this work. This work was supported by grants HI12C1821 (A121968) from the Korean Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea; by grant NRF-2011–0014046 from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology; and by grants 20 by 20 Project (Best #3, GFO1140091) from Samsung Medical Center. Correspondence and requests for reprints should be addressed to Won Soon Park, M.D., Ph.D., Department of Pediatrics, Samsung Medical Center; Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul 135-710, Korea. E-mail: [email protected], [email protected] Author Contributions: Y.S.C. and S.Y.A. contributed equally as co-first authors in data collection and analysis and in manuscript writing and revision. W.S.P. contributed the study idea, design, and hypothesis and critically reviewed and revised the manuscript. D.K.S. contributed biochemical analysis, wrote a portion of the manuscript, and critically reviewed and approved the final manuscript. H.S.Y., S.I.S., and E.S.K. contributed animal experiments and data interpretation and critically reviewed and revised the manuscript. H.B.J., S.J.C, and W.I.O. contributed material support including stem cells, participated in in vitro experiments, and contributed to manuscript revision. All authors listed above have read and approved the manuscript. This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Cell Mol Biol Vol 51, Iss 3, pp 391–399, Sep 2014 Copyright © 2014 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2013-0385OC on March 26, 2014 Internet address: www.atsjournals.org
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ORIGINAL RESEARCH Bronchopulmonary dysplasia (BPD) is a chronic lung disease characterized by impaired alveolar and vascular growth in extremely preterm infants who are treated with prolonged ventilator and oxygen therapy. Despite recent advances in neonatal intensive care medicine, BPD remains a major cause of mortality and morbidity in premature infants, with few clinically effective treatments (1, 2). New therapies are urgently needed to improve the prognosis of this serious disease. Recently, we showed that xenotransplantation of human umbilical cord blood (UCB)–derived mesenchymal
stem cells (MSCs) in immunocompetent neonatal rats attenuates hyperoxia-induced lung injuries, such as impaired alveolarization, increased apoptosis, inflammatory responses, oxidative stress, and fibrosis, all of which simulate BPD in human infants (3–6). Although the benefits of MSC transplantation are primarily mediated by a paracrine rather than a regenerative mechanism (3), the specific factors responsible for the protective action of MSC transplantation have not been elucidated. Disrupted angiogenesis might be the primary cause of impaired alveolarization
in BPD (7). Vascular endothelial growth factor (VEGF) is essential for lung angiogenesis and alveolar development (8, 9). Reduced VEGF levels were observed in premature infants with BPD (8, 10–12), and exogenous VEGF supplementation significantly attenuated hyperoxic lung injury in animals (9, 13, 14). Taken together, these findings suggest that decreased VEGF and the resultant disruption of angiogenesis might be the critical pathogenetic factor for the development of BPD. Our previous research showing that intratracheal transplantation of human UCB–derived
Figure 1. Induction of vascular endothelial growth factor (VEGF) in rat lung epithelial L2 cells by VEGF-secreted by human mesenchymal stem cells (MSCs) rescues oxidative injury in vitro. Rat epithelial L2 cells were treated with H2O2 for 1 hour to induce oxidative stress and cocultured with nontransfected human MSCs with or without VEGF-blocking antibody, scrambled small interfering RNA (siRNA)–transfected MSCs, VEGF siRNA–transfected MSCs with or without recombinant human (rh)VEGF, MRC5 cells, or rhVEGF. (A) Expression of human VEGF from MSCs or MRC5 cells and (B) expression of rat VEGF from L2 cells in the culture supernatant was evaluated in each group. (C) Cell survival rate in each group was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (D–G) Inflammatory cytokines, including IL-1a (D), IL-1b (E), TNF-a (F), and IL-6 (G), were measured in the culture supernatant of each group. Data are presented as mean 6 SEM. *P , 0.05 compared with normoxia control. † P , 0.05 compared with H2O2 control. ‡P , 0.05 compared with H2O2 1 human umbilical cord blood (UCB)–derived MSCs. a, normoxia control; b, H2O2 control; c, H2O2 1 human UCB–derived MSCs; d, H2O2 1 scrambled siRNA–transfected MSCs; e, H2O2 1 VEGF siRNA–transfected MSCs; f, H2O2 1 human fibroblast (MRC5); g, H2O2 1 human UCB–derived MSCs 1 immunoglobulin G; h, H2O2 1 human VEGF blocking antibody; i, H2O2 1 VEGF siRNA MSCs 1 rhVEGF 1 ng; j, H2O2 1 VEGF siRNA MSCs 1 rhVEGF 10 ng; k, H2O2 1 rhVEGF 1 ng; l, H2O2 1 rhVEGF 10 ng.
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ORIGINAL RESEARCH MSCs in rats significantly restores levels of rat VEGF in hyperoxic lungs (5) supports this conclusion. However, because we could not detect human VEGF secreted from the transplanted MSCs at the end of the experiment, it was not clear whether VEGF secreted from transplanted MSCs was a critical paracrine factor mediating the protective effects of MSC transplantation. In this study, we hypothesized that VEGF secreted by transplanted MSCs plays a pivotal role in protection against hyperoxic lung injury in newborn rats. To test this hypothesis, VEGF was specifically knocked down in human UCB–derived MSCs by transfection with small interfering RNA (siRNA) specific for human VEGF. The protective effects of MSCs with or without VEGF knockdown or VEGF blocking antibody were evaluated in vitro in a rat lung epithelial (L2) cell line challenged with H2O2. To confirm these results in vivo, the protective effects of MSCs with or without VEGF knockdown were evaluated in newborn rats exposed to hyperoxia.
Materials and Methods Cell Preparation and Culture Conditions
Human UCB was obtained from healthy, normal, full-term newborns as previously reported (3) after obtaining written informed parental consent. UCB-derived MSCs were isolated and expanded according to a previously reported procedure (15, 16).
antibody (Abcam, Cambridge, MA) or control IgG) (Dako, Carpinteria, CA) was added to the coculture of H2O2–treated L2 cells and nontransfected MSCs at a concentration of 200 ng per well. Low-dose (1 ng/ml) or high-dose (10 ng/ml) recombinant human (rh)VEGF (R&D Systems, Minneapolis, MN) was added to the H2O2–treated L2 cells with or without cocultured VEGF siRNA–transfected MSCs.
intratracheally. An equal volume of PBS was administered to the control groups in the same manner. Morphometry
Alveolarization was assessed by measuring the mean linear index and mean alveolar volume as previously described (3–6). A minimum of three sections per rat and six fields per section were randomly evaluated in a blinded manner.
Animal Model
Animal procedures were approved by the Animal Care and Use Committee of Samsung Biomedical Research Institute, Seoul, Korea. This study was performed in accordance with institutional and National Institutes of Health guidelines for laboratory animal care. Timed pregnant Sprague-Dawley rats (Orient Co., Seoul, Korea) spontaneously delivered rat pups. Newborn rats were allocated to five experimental groups: normoxia control, hyperoxia control, hyperoxia with human UCB–derived MSCs, hyperoxia with scrambled siRNA–transfected MSCs, and hyperoxia with VEGF siRNA–transfected MSCs. Normoxic rat pups were kept in room air, and hyperoxic rat pups were raised in hyperoxic chambers (90% O2) from birth until postnatal day (P)14. At P5, 5 3 105 nontransfected, VEGF siRNA–transfected, or scrambled siRNA–transfected MSCs were administered
Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling Assay
Details are described in the online supplement. Immunohistochemistry
Details are described in the online supplement. ELISA
Frozen lungs were homogenized, and the total protein content in the supernatant was determined as previously reported (3–5). The amount of human- and rat-specific VEGF, IL-1a, IL-1b, IL-6, and TNF-a was measured as described in the online supplement. Statistical Analyses
Data are expressed as the mean 6 SEM. For continuous variables with a normal
VEGF Silencing with siRNA
MSCs (3 3 106) were seeded in 100-mm plates with 1 ml Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA). After cells reached 40 to 80% confluency, MSCs were transfected with siRNA targeting VEGF or scrambled siRNA using Lipofectamine (Invitrogen) as described in the online supplement. In Vitro Cell Culture
Cultured L2 rat lung epithelial cells were treated with 100 mM H2O2 for 60 minutes and then incubated in complete media alone or supplemented with 1 3 103 MRC5 cells (human fibroblast cell line), nontransfected MSCs, scrambled siRNA–transfected MSCs, or VEGF siRNA–transfected MSCs. VEGF blocking
Figure 2. Induction of rat VEGF in rat host lungs by human VEGF from transplanted human MSCs. The figure shows a temporal profile of human VEGF (A) and rat VEGF (B) in rat lungs from each group. Data are presented as mean 6 SEM. *P , 0.05 compared with normal. †P , 0.05 compared with hyperoxia control. ‡P , 0.05 compared with hyperoxia 1 MSCs. xP , 0.05 compared with scrambled siRNA MSCs.
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ORIGINAL RESEARCH distribution, the groups were compared using one-way ANOVA followed by a least significant difference multiple comparison test. A P value , 0.05 was considered significant. SPSS version 17.0 (SPSS Institute, Chicago, IL) was used for all analyses.
Results
significant differences in the number of PKH26-positive donor cells in the rat lung tissue at P14 among the MSC transplantation groups (Figure E4). The amount of rat VEGF in hyperoxic lung tissue gradually decreased over time and was significantly lower than that in the normoxia control group after P7. The decrease in rat VEGF was significantly
attenuated in the nontransfected and scrambled siRNA–transfected MSC groups but not in the VEGF siRNA–transfected MSC group (Figure 2B). Protective Effects of MSC Transplantation In Vitro and In Vivo
In cultured rat L2 cells, H2O2 exposure significantly reduced cell survival compared
VEGF Knockdown and Human and Rat VEGF Level
VEGF levels in the culture medium of nontransfected or scrambled siRNA–transfected MSCs increased over time, whereas transfection of MSCs with VEGF siRNA significantly decreased VEGF production for up to 7 days (see Figure E1 in the online supplement). No significant differences in VEGF production were noted between the nontransfected and scrambled siRNA–transfected groups. No human VEGF level was detected in the supernatant of normoxic or H2O2–treated rat L2 cells cultured alone. Human VEGF was detected in the supernatant of H2O2–treated rat L2 cells that were cocultured with nontransfected or scrambled siRNA–transfected human MSCs and at significantly higher levels than in the supernatant of H2O2–treated cells cocultured with VEGF siRNA–transfected MSCs or human fibroblasts (MRC5) (Figure 1A). The significant decrease in rat VEGF level in the H2O2 control group compared with the normoxic control group was significantly improved by coculture with nontransfected or scrambled siRNA–transfected MSCs but not by coculture with MRC5 or VEGF siRNA–transfected MSCs (Figure 1B). In rat lung tissue, human VEGF secreted from the transplanted MSCs was detected in the nontransfected and scrambled siRNA–transfected groups, and its concentration decreased over time. Human VEGF was not detected in the VEGF siRNA–transfected group (Figures 2A and E2). PKH26-positive donor cells were colocalized with ED-positive rather than terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in P14 rat lungs, indicating that transplanted MSCs are phagocytosed directly or undergo apoptosis/necrosis and that the cell fragments are phagocytosed by the macrophages (Figure E3). There were no 394
Figure 3. Pulmonary angiogenesis in the lungs of postnatal day (P)14 rats. (A) Representative immunofluorescence photomicrographs of von Willebrand factor (vWF) staining in the lungs of P14 rats in each group. vWF was labeled with the fluorescent marker 5(6)-carboxyfluoresceindiacetate N-succinimidyl ester (green), and nuclei were labeled with 49,6-diamidino-2-phenylindole (blue) (scale bars, 25 mm). 1, normal; 2, hyperoxia control; 3, hyperoxia 1 MSC; 4, hyperoxia 1 scramble siRNA MSC; 5, hyperoxia 1 VEGF siRNA MSC. (B) The mean optical density of vWF immunofluorescence per one high power field in each group. Data are presented as mean 6 SEM. *P , 0.05 compared with normal. †P , 0.05 compared with hyperoxia control. ‡P , 0.05 compared with hyperoxia 1 MSCs. xP , 0.05 compared with scrambled siRNA MSCs.
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ORIGINAL RESEARCH with normoxic conditions. This oxidative stress–induced cell death was significantly lower in the L2 cells cocultured with nontransfected or scrambled siRNA–transfected MSCs but not in cells cocultured with MRC5 cells or VEGF siRNA–transfected MSCs. The protective effect of MSCs against H2O2–induced cell death was abolished by VEGF neutralizing antibody but not by control IgG. Supplementation with 1 or 10 ng rhVEGF restored the protective effects of VEGF knockdown MSCs against hyperoxic L2 cell injury in a dose-dependent manner. For rhVEGF supplementation alone, 10 ng (but not 1 ng) significantly attenuated H2O2–induced L2 cell death (Figure 1C). In rat lung tissue, expression of von Willebrand factor was significantly reduced, indicating impaired angiogenesis, and mean linear index and alveolar volume were increased, indicating impaired alveolarization, in the hyperoxic groups compared with the normoxic group. These effects were significantly improved by transplantation with nontransfected MSCs. The protective effects of transplanted MSCs were abolished by transfection with VEGF siRNA but not with scrambled siRNA (Figures 3 and 4). The hyperoxiainduced increase in the number of TUNEL-positive cells in rat lung tissue was significantly attenuated by transplantation with nontransfected or scrambled siRNA–transfected MSCs but not by VEGF siRNA–transfected MSCs (Figure 5A). Inflammatory Responses
In rat L2 cells, the increased expression of inflammatory cytokines, such as IL-1a, IL-1b, TNF-a, and IL-6, in the H2O2–exposed groups were significantly attenuated in cells that were cocultured with nontransfected or scrambled siRNA–transfected MSCs but not in groups that were cocultured with MRC5 cells or VEGF siRNA–transfected MSCs. Similarly, the anti-inflammatory effects of MSCs, as evidenced by reduced IL-1a, IL-1b, IL-6, and TNF-a levels, were abolished by VEGF neutralizing antibody but not by IgG (Figures 1D–1G). Furthermore, supplementation with 1 or 10 ng rhVEGF restored the anti-inflammatory effects of VEGF knockdown MSCs in a dose-dependent manner. For rhVEGF supplementation alone, 10 ng (but
Figure 4. Histologic and morphometric analysis of P14 rat lungs. (A) Representative optical microscopy photomicrographs of lungs from rats in each group stained with hematoxylin and eosin (scale bar, 25 mm). 1, normal; 2, hyperoxia control; 3, hyperoxia 1 MSC; 4, hyperoxia 1 scramble siRNA MSC; 5, hyperoxia 1 VEGF siRNA MSC. Degree of alveolarization assessed by the mean linear intercept (B) and mean alveolar volume (C). Data are presented as mean 6 SEM. *P , 0.05 compared with normal. †P , 0.05 compared with hyperoxia control. ‡P , 0.05 compared with hyperoxia 1 MSCs. xP , 0.05 compared with scrambled siRNA MSCs.
not 1 ng) significantly attenuated the H2O2–induced increase in inflammatory cytokines (Figures 1D–1G). In rat lung tissue, the hyperoxia-induced increase in ED-1–positive cells and inflammatory cytokines such as IL-1a, IL-1b, IL-6, and TNF-a was significantly attenuated by transplantation with nontransfected MSCs. The antiinflammatory effects of transplanted MSCs were abolished by transfection with VEGF
Chang, Ahn, Jeon, et al.: Critical Role of VEGF from MSCs
siRNA but not by transfection with scrambled siRNA (Figures 5C, 5D, and 6).
Discussion In the present study, we show that human UCB–derived MSCs, but not human fibroblasts, significantly improved cell survival in H2O2–treated rat lung epithelial (L2) cells, suggesting that the protective 395
ORIGINAL RESEARCH
Figure 5. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and pulmonary macrophages in the distal lungs of P14 rat pups. (A, C) Representative photomicrographs of TUNEL-positive cells (A) and ED-1–positive cells (C) in the lungs of P14 rats in each group. TUNEL-positive cells were labeled with FITC (green), and ED-1–positive alveolar macrophages were labeled with 5(6)-carboxyfluoresceindiacetate N-succinimidyl ester (green). The cell nuclei were labeled with DAPI (blue) (scale bar, 25 mm). 1, normal; 2, hyperoxia control; 3, hyperoxia 1 MSC; 4, hyperoxia 1 scramble siRNA MSC; 5, hyperoxia 1 VEGF siRNA MSC. (B, D) The average number of TUNEL-positive cells (B) and ED-1–positive cells (D) per high-power field (HPF) in each group. Data are presented as mean 6 SEM. *P , 0.05 compared with normal. †P , 0.05 compared with hyperoxia control. ‡P , 0.05 compared with hyperoxia 1 MSCs. xP , 0.05 compared with scrambled siRNA MSCs.
effects may be specific to MSCs. Moreover, the protective effects of MSCs observed in vitro in L2 cells exposed by H2O2 were abolished by VEGF knockdown or VEGFneutralizing antibody and were restored by rhVEGF supplementation. The beneficial effects of MSCs were also abolished in vivo in a newborn Sprague-Dawley rat model of hyperoxic lung injury by VEGF knockdown with VEGF siRNA but not by scrambled siRNA. Taken together, these results support the hypothesis that VEGF secreted by transplanted MSCs might be a critical paracrine factor in protection against neonatal hyperoxic lung injury, such as that in BPD. A substantial increase in microvascular endothelial cell proliferation is necessary for 396
normal neonatal lung growth, which is characterized by rapid angiogenesis and alveolarization (22). VEGF, a specific mitogen for endothelial cells that is mainly expressed by alveolar epithelial cells adjacent to capillary beds, plays a critical role in postnatal lung angiogenesis (22, 23). These findings suggest a close link between lung angiogenesis and alveolarization in normal conditions (22) and in disease conditions, such as BPD (7). VEGF secreted by transplanted stem cells is also a crucial paracrine mediator in facilitating recovery in various animal models of disease, such as myocardial injury (17), acute kidney injury (18), and stroke (19). These findings suggest that VEGF secreted by transplanted
stem cells might be a universal paracrine mediator of the protective effects of stem cell transplantation in various diseases (20) and could also be used as a biomarker of the potency of stem cell transplants (17). Although the VEGF level was increased in the MSC transplantation group in the present study, it was still much lower than that in the normoxia group. Overexpression of VEGF in MSCs has been reported to significantly enhance stem cell–mediated therapeutic efficacy in neural and cardiac repair (21, 22). Therefore, further studies might be necessary to investigate whether transplantation of VEGF-overexpressing MSCs enhances their beneficial effects in this BPD model.
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ORIGINAL RESEARCH
Figure 6. Temporal profile of inflammatory cytokines in rat lungs at P7, P10, and P14. Expression of IL-1a (A), IL-1b (B), IL-6 (C), and TNF-a (D) in rat lungs at P7, P10, and P14 for each group. Data are presented as mean 6 SEM. *P , 0.05 compared with normal. †P , 0.05 compared with hyperoxia control. ‡P , 0.05 compared with hyperoxia 1 MSCs. xP , 0.05 compared with scrambled siRNA MSCs.
The results of the present study demonstrated a significantly lower level of human VEGF in the VEGF siRNA transfection group, but not in the scrambled siRNA transfection group, up to 7 days after transplantation. Human VEGF expression colocalized with PKH26-positive donor cells in rats transplanted with nontransfected or scrambled siRNA–transfected MSCs but not in rats transplanted with VEGF siRNA–transfected MSCs. Overall, these findings indicate that VEGF knockdown in MSCs by siRNA transfection was effective and specific. In a similar study by Pierro and colleagues (23), there was a dramatic decrease in MSCs during the first day after intratracheal engraftment, and MSCs were almost undetectable 4 days after transplantation. We also previously showed
that the number of MSCs steadily decreased (5) and that transplanted cells were virtually undetectable 10 weeks after intratracheal transplantation (6). Taken together, these findings suggest that the gradual decrease in human VEGF in the rat lung tissue in nontransfected and scrambled siRNA–transfected groups might be attributed to a loss of the transplanted MSCs. Therefore, improving donor cell survival might improve the protective effects of transplanted MSCs (24). Understanding the mechanism by which human VEGF secreted by exogenous MSCs preserves rat VEGF and protects lung tissue against hyperoxic injury is of particular interest. Although the human VEGF ELISA kit does not cross-react with rat VEGF, there is . 85% homology between human and rat VEGF (25), and
Chang, Ahn, Jeon, et al.: Critical Role of VEGF from MSCs
their functional action of mechanism is quite similar (13, 14). Because VEGF is an obligatory survival factor for lung alveolar and vascular endothelium, a reduction in rat VEGF below the threshold level for survival might be primarily responsible for hyperoxia-induced lung injuries (26, 27). Therefore, human VEGF secreted by the transplanted MSCs might initially maintain VEGF levels within the local microenvironment above the threshold level essential for rat lung tissue survival against hyperoxic injury (28) and thus might indirectly restore the rat VEGF level. In our previous study, we showed that, although donor cells rapidly faded away after transplantation, the favorable effects of the MSCs persisted up to P21 (5). These findings suggest that the paracrine effects induced by the stem cells might initially 397
ORIGINAL RESEARCH play a pivotal role in tissue repair, and as the donor cells are lost the intact host tissue that was protected by MSC transplantation is able to sustain up-regulation of rat VEGF expression (29). Inhibition of apoptosis might be another protective mechanism of VEGF. Our results show that transplantation of MSCs significantly attenuated the hyperoxia-induced increase in the number of TUNEL-positive cells, which was abolished by VEGF siRNA but not by scrambled siRNA (21, 30, 31). The antiapoptotic effects of VEGF might be mediated by inhibiting the expression of proapoptotic proteins and promoting antiapoptotic and cell survival factors (32–34). Transplantation of MSCs also significantly attenuated the hyperoxia-induced increase in expression of inflammatory cytokines in vitro and in vivo and attenuated the increase in ED-1–positive alveolar macrophages in lung tissue. These decreases in inflammatory responses were abolished by transfection with VEGF siRNA but not by scrambled siRNA. Taken together, these findings suggest that VEGF secreted by MSCs may play a role in down-regulating hyperoxia-induced lung injury in the newborn rats. The anti-inflammatory effects of VEGF observed in the present study and in previous studies (19, 35) are contradictory
to the conventional proinflammatory effects of VEGF (36). However, Manoonkitiwongsa and colleagues (37) reported dose-dependent pro- or antiinflammatory effects of VEGF, showing that treatment of ischemic brain with low doses of VEGF reduced macrophage infiltration, whereas higher doses increased macrophage density. Therefore, the antiinflammatory effects of VEGF observed in the present study might be explained by the fact that the VEGF level, although improved, was still much lower in the MSC transplantation group compared with the normoxia group. For VEGF treatment alone, repetitive high doses are required for beneficial effects against hyperoxic lung injury in newborn rats (13, 14). Moreover, VEGF also increases lung permeability, leading to pulmonary edema (14). We have previously shown that a single transplantation with human UCB–derived MSCs attenuates Escherichia coli–induced pulmonary edema (38) and that this beneficial effect might be mediated by keratinocyte growth factor and angiopoietin-1 secreted by MSCs (39–41). In particular, human UCB–derived MSCs secrete exceptionally high amounts of angiopoietin-1 compared with bone marrow–derived or adipose tissue–derived MSCs (41). We have also shown that MSC transplantation simultaneously rescues the hyperoxia-
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induced reduction in VEGF and hepatocyte growth factor (5), another critical growth factor for lung alveolarization (42). In the present study, supplementation with a low dose of rhVEGF (1 ng) restored the protective effects of VEGF knockdown MSCs against hyperoxic L2 cell injury, but low-dose VEGF supplementation alone did not significantly attenuate H2O2–induced L2 cell death. Taken together, these findings suggest that MSC transplantation is more effective than rhVEGF treatment alone in protecting against hyperoxic injury. Furthermore, although the critical role of human VEGF secreted from MSCs for the protection of hyperoxic lung injury was demonstrated in our study, there may be other critical secreted factors that act similarly, and our data do not preclude this possibility, which requires further study. In conclusion, VEGF secreted by transplanted human UCB–derived MSCs is the key paracrine factor in protection against hyperoxia induced in vitro in a rat alveolar epithelial cell line challenged with H2O2 and against hyperoxia-induced lung injury in vivo in neonatal rats. The antiapoptotic and anti-inflammatory effects of VEGF might contribute to these protective effects. n Author disclosures are available with the text of this article at www.atsjournals.org.
8. Thebaud ´ B. Angiogenesis in lung development, injury and repair: implications for chronic lung disease of prematurity. Neonatology 2007;91:291–297. 9. Thebaud ´ B, Ladha F, Michelakis ED, Sawicka M, Thurston G, Eaton F, Hashimoto K, Harry G, Haromy A, Korbutt G, et al. Vascular endothelial growth factor gene therapy increases survival, promotes lung angiogenesis, and prevents alveolar damage in hyperoxiainduced lung injury: evidence that angiogenesis participates in alveolarization. Circulation 2005;112:2477–2486. 10. Lassus P, Ristimaki ¨ A, Ylikorkala O, Viinikka L, Andersson S. Vascular endothelial growth factor in human preterm lung. Am J Respir Crit Care Med 1999;159:1429–1433. 11. Lassus P, Turanlahti M, Heikkila¨ P, Andersson LC, Nupponen I, Sarnesto A, Andersson S. Pulmonary vascular endothelial growth factor and Flt-1 in fetuses, in acute and chronic lung disease, and in persistent pulmonary hypertension of the newborn. Am J Respir Crit Care Med 2001;164:1981–1987. 12. Bhatt AJ, Pryhuber GS, Huyck H, Watkins RH, Metlay LA, Maniscalco WM. Disrupted pulmonary vasculature and decreased vascular endothelial growth factor, Flt-1, and TIE-2 in human infants dying with bronchopulmonary dysplasia. Am J Respir Crit Care Med 2001; 164:1971–1980. 13. Kunig AM, Balasubramaniam V, Markham NE, Morgan D, Montgomery G, Grover TR, Abman SH. Recombinant human VEGF treatment enhances alveolarization after hyperoxic lung injury in neonatal rats. Am J Physiol Lung Cell Mol Physiol 2005; 289:L529–L535.
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ORIGINAL RESEARCH 14. Kunig AM, Balasubramaniam V, Markham NE, Seedorf G, Gien J, Abman SH. Recombinant human VEGF treatment transiently increases lung edema but enhances lung structure after neonatal hyperoxia. Am J Physiol Lung Cell Mol Physiol 2006;291: L1068–L1078. 15. Yang SE, Ha CW, Jung M, Jin HJ, Lee M, Song H, Choi S, Oh W, Yang YS. Mesenchymal stem/progenitor cells developed in cultures from UC blood. Cytotherapy 2004;6:476–486. 16. Chang YS, Ahn SY, Yoo HS, Sung SI, Choi SJ, Oh WI, Park WS. Mesenchymal stem cells for bronchopulmonary dysplasia: phase 1 dose-escalation clinical trial. J Pediatr 2014;164:966–972. 17. Markel TA, Wang Y, Herrmann JL, Crisostomo PR, Wang M, Novotny NM, Herring CM, Tan J, Lahm T, Meldrum DR. VEGF is critical for stem cell-mediated cardioprotection and a crucial paracrine factor for defining the age threshold in adult and neonatal stem cell function. Am J Physiol Heart Circ Physiol 2008;295:H2308–H2314. 18. Togel ¨ F, Zhang P, Hu Z, Westenfelder C. VEGF is a mediator of the renoprotective effects of multipotent marrow stromal cells in acute kidney injury. J Cell Mol Med 2009;13:2109–2114. 19. Horie N, Pereira MP, Niizuma K, Sun G, Keren-Gill H, Encarnacion A, Shamloo M, Hamilton SA, Jiang K, Huhn S, et al. Transplanted stem cell-secreted vascular endothelial growth factor effects poststroke recovery, inflammation, and vascular repair. Stem Cells 2011;29: 274–285. 20. Song SY, Chung HM, Sung JH. The pivotal role of VEGF in adiposederived-stem-cell-mediated regeneration. Expert Opin Biol Ther 2010;10:1529–1537. 21. Lee HJ, Kim KS, Park IH, Kim SU. Human neural stem cells overexpressing VEGF provide neuroprotection, angiogenesis and functional recovery in mouse stroke model. PLoS ONE 2007;2:e156. 22. Zisa D, Shabbir A, Suzuki G, Lee T. Vascular endothelial growth factor (VEGF) as a key therapeutic trophic factor in bone marrow mesenchymal stem cell-mediated cardiac repair. Biochem Biophys Res Commun 2009;390:834–838. 23. Pierro M, Ionescu L, Montemurro T, Vadivel A, Weissmann G, Oudit G, Emery D, Bodiga S, Eaton F, Peault ´ B, et al. Short-term, long-term and paracrine effect of human umbilical cord-derived stem cells in lung injury prevention and repair in experimental bronchopulmonary dysplasia. Thorax 2013;68:475–484. 24. Burst VR, Gillis M, Putsch ¨ F, Herzog R, Fischer JH, Heid P, Muller¨ Ehmsen J, Schenk K, Fries JW, Baldamus CA, et al. Poor cell survival limits the beneficial impact of mesenchymal stem cell transplantation on acute kidney injury. Nephron, Exp Nephrol 2010;114:e107–e116. 25. Avivi A, Resnick MB, Nevo E, Joel A, Levy AP. Adaptive hypoxic tolerance in the subterranean mole rat Spalax ehrenbergi: the role of vascular endothelial growth factor. FEBS Lett 1999;452:133–140. 26. Maniscalco WM, Watkins RH, D’Angio CT, Ryan RM. Hyperoxic injury decreases alveolar epithelial cell expression of vascular endothelial growth factor (VEGF) in neonatal rabbit lung. Am J Respir Cell Mol Biol 1997;16:557–567. 27. Voelkel NF, Cool C, Taraceviene-Stewart L, Geraci MW, Yeager M, Bull T, Kasper M, Tuder RM. Janus face of vascular endothelial growth factor: the obligatory survival factor for lung vascular endothelium controls precapillary artery remodeling in severe pulmonary hypertension. Crit Care Med 2002;30(5 Suppl):S251–S256.
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28. von Degenfeld G, Banfi A, Springer ML, Wagner RA, Jacobi J, Ozawa CR, Merchant MJ, Cooke JP, Blau HM. Microenvironmental VEGF distribution is critical for stable and functional vessel growth in ischemia. FASEB J 2006;20:2657–2659. 29. Cho HJ, Lee N, Lee JY, Choi YJ, Ii M, Wecker A, Jeong JO, Curry C, Qin G, Yoon YS. Role of host tissues for sustained humoral effects after endothelial progenitor cell transplantation into the ischemic heart. J Exp Med 2007;204:3257–3269. 30. Esquibies AE, Bazzy-Asaad A, Ghassemi F, Nishio H, Karihaloo A, Cantley LG. VEGF attenuates hyperoxic injury through decreased apoptosis in explanted rat embryonic lung. Pediatr Res 2008;63: 20–25. 31. Farkas L, Farkas D, Ask K, Moller ¨ A, Gauldie J, Margetts P, Inman M, Kolb M. VEGF ameliorates pulmonary hypertension through inhibition of endothelial apoptosis in experimental lung fibrosis in rats. J Clin Invest 2009;119:1298–1311. 32. Marti HH. Vascular endothelial growth factor. Adv Exp Med Biol 2002; 513:375–394. 33. Harrigan MR, Ennis SR, Sullivan SE, Keep RF. Effects of intraventricular infusion of vascular endothelial growth factor on cerebral blood flow, edema, and infarct volume. Acta Neurochir (Wien) 2003;145:49–53. 34. Rosenstein JM, Krum JM. New roles for VEGF in nervous tissue: beyond blood vessels. Exp Neurol 2004;187:246–253. 35. Ahn SY, Chang YS, Sung DK, Sung SI, Yoo HS, Lee JH, Oh WI, Park WS. Mesenchymal stem cells prevent hydrocephalus after severe intraventricular hemorrhage. Stroke 2013;44:497–504. 36. Hao Q, Wang L, Tang H. Vascular endothelial growth factor induces protein kinase D-dependent production of proinflammatory cytokines in endothelial cells. Am J Physiol Cell Physiol 2009;296: C821–C827. 37. Manoonkitiwongsa PS, Schultz RL, Whitter EF, Lyden PD. Contraindications of VEGF-based therapeutic angiogenesis: effects on macrophage density and histology of normal and ischemic brains. Vascul Pharmacol 2006;44:316–325. 38. Kim ES, Chang YS, Choi SJ, Kim JK, Yoo HS, Ahn SY, Sung DK, Kim SY, Park YR, Park WS. Intratracheal transplantation of human umbilical cord blood-derived mesenchymal stem cells attenuates Escherichia coli-induced acute lung injury in mice. Respir Res 2011; 12:108. 39. Mei SH, McCarter SD, Deng Y, Parker CH, Liles WC, Stewart DJ. Prevention of LPS-induced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin 1. PLoS Med 2007;4:e269. 40. Gillis P, Savla U, Volpert OV, Jimenez B, Waters CM, Panos RJ, Bouck NP. Keratinocyte growth factor induces angiogenesis and protects endothelial barrier function. J Cell Sci 1999;112:2049–2057. 41. Jin HJ, Bae YK, Kim M, Kwon SJ, Jeon HB, Choi SJ, Kim SW, Yang YS, Oh W, Chang JW. Comparative analysis of human mesenchymal stem cells from bone marrow, adipose tissue, and umbilical cord blood as sources of cell therapy. Int J Mol Sci 2013; 14:17986–18001. 42. Ohki Y, Mayuzumi H, Tokuyama K, Yoshizawa Y, Arakawa H, Mochizuki H, Morikawa A. Hepatocyte growth factor treatment improves alveolarization in a newborn murine model of bronchopulmonary dysplasia. Neonatology 2009;95:332–338.
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Department of Pediatrics, Samsung Medical Center, and 2Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Korea; 3Biomedical Research Institute, MEDIPOST Co., Ltd., Seoul, Korea; and 4Department of Pediatrics, CHA Gangnam Medical Center, CHA University School of Medicine, Seoul, Korea
Abstract Intratracheal transplantation of human umbilical cord blood (UCB)–derived mesenchymal stem cells (MSCs) protects against neonatal hyperoxic lung injury by a paracrine rather than a regenerative mechanism. However, the role of paracrine factors produced by the MSCs, such as vascular endothelial growth factor (VEGF), has not been delineated. This study examined whether VEGF secreted by MSCs plays a pivotal role in protecting against neonatal hyperoxic lung injury. VEGF was knocked down in human UCB–derived MSCs by transfection with small interfering RNA specific for human VEGF. The in vitro effects of MSCs with or without VEGF knockdown or neutralizing antibody were evaluated in a rat lung epithelial (L2) cell line challenged with H2O2. To confirm these results in vivo, newborn Sprague-Dawley rats were exposed to hyperoxia (90% O2) for 14 days. MSCs (1 3 105 cells) with or without VEGF knockdown were administered intratracheally at postnatal Day 5. Lungs were serially harvested for biochemical and histologic analyses. VEGF knockdown and antibody abolished the in vitro benefits of MSCs on H2O2–induced cell death and the upregulation of inflammatory cytokines in L2 cells. VEGF knockdown also abolished the in vivo protective effects of MSCs in hyperoxic lung injury, such as the attenuation of impaired alveolarization and angiogenesis, reduction in the number of terminal deoxynucleotidyl
transferase dUTP nick end labeling–positive and ED-1–positive cells, and down-regulation of proinflammatory cytokine levels. Our data indicate that VEGF secreted by transplanted MSCs is one of the critical paracrine factors that play seminal roles in attenuating hyperoxic lung injuries in neonatal rats. Keywords: stem cells; cell transplantation; newborn; lung injury;
vascular endothelial growth factor
Clinical Relevance Intratracheal transplantation of human umbilical cord blood–derived mesenchymal stem cells (MSCs) protects against hyperoxic lung injury in neonatal rats. These protective effects represent a paracrine rather than a regenerative mechanism. However, the role of paracrine factors secreted by MSCs, such as vascular endothelial growth factor (VEGF), has not been delineated. VEGF secreted by transplanted MSCs is at least one of the critical factors that mediate the protective effects of MSCs against hyperoxic lung injury, such as impaired alveolarization and inflammatory responses in newborn rats.
( Received in original form September 5, 2013; accepted in final form March 24, 2014 ) *These authors contributed equally to this work. This work was supported by grants HI12C1821 (A121968) from the Korean Healthcare Technology R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea; by grant NRF-2011–0014046 from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology; and by grants 20 by 20 Project (Best #3, GFO1140091) from Samsung Medical Center. Correspondence and requests for reprints should be addressed to Won Soon Park, M.D., Ph.D., Department of Pediatrics, Samsung Medical Center; Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul 135-710, Korea. E-mail: [email protected], [email protected] Author Contributions: Y.S.C. and S.Y.A. contributed equally as co-first authors in data collection and analysis and in manuscript writing and revision. W.S.P. contributed the study idea, design, and hypothesis and critically reviewed and revised the manuscript. D.K.S. contributed biochemical analysis, wrote a portion of the manuscript, and critically reviewed and approved the final manuscript. H.S.Y., S.I.S., and E.S.K. contributed animal experiments and data interpretation and critically reviewed and revised the manuscript. H.B.J., S.J.C, and W.I.O. contributed material support including stem cells, participated in in vitro experiments, and contributed to manuscript revision. All authors listed above have read and approved the manuscript. This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Cell Mol Biol Vol 51, Iss 3, pp 391–399, Sep 2014 Copyright © 2014 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2013-0385OC on March 26, 2014 Internet address: www.atsjournals.org
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ORIGINAL RESEARCH Bronchopulmonary dysplasia (BPD) is a chronic lung disease characterized by impaired alveolar and vascular growth in extremely preterm infants who are treated with prolonged ventilator and oxygen therapy. Despite recent advances in neonatal intensive care medicine, BPD remains a major cause of mortality and morbidity in premature infants, with few clinically effective treatments (1, 2). New therapies are urgently needed to improve the prognosis of this serious disease. Recently, we showed that xenotransplantation of human umbilical cord blood (UCB)–derived mesenchymal
stem cells (MSCs) in immunocompetent neonatal rats attenuates hyperoxia-induced lung injuries, such as impaired alveolarization, increased apoptosis, inflammatory responses, oxidative stress, and fibrosis, all of which simulate BPD in human infants (3–6). Although the benefits of MSC transplantation are primarily mediated by a paracrine rather than a regenerative mechanism (3), the specific factors responsible for the protective action of MSC transplantation have not been elucidated. Disrupted angiogenesis might be the primary cause of impaired alveolarization
in BPD (7). Vascular endothelial growth factor (VEGF) is essential for lung angiogenesis and alveolar development (8, 9). Reduced VEGF levels were observed in premature infants with BPD (8, 10–12), and exogenous VEGF supplementation significantly attenuated hyperoxic lung injury in animals (9, 13, 14). Taken together, these findings suggest that decreased VEGF and the resultant disruption of angiogenesis might be the critical pathogenetic factor for the development of BPD. Our previous research showing that intratracheal transplantation of human UCB–derived
Figure 1. Induction of vascular endothelial growth factor (VEGF) in rat lung epithelial L2 cells by VEGF-secreted by human mesenchymal stem cells (MSCs) rescues oxidative injury in vitro. Rat epithelial L2 cells were treated with H2O2 for 1 hour to induce oxidative stress and cocultured with nontransfected human MSCs with or without VEGF-blocking antibody, scrambled small interfering RNA (siRNA)–transfected MSCs, VEGF siRNA–transfected MSCs with or without recombinant human (rh)VEGF, MRC5 cells, or rhVEGF. (A) Expression of human VEGF from MSCs or MRC5 cells and (B) expression of rat VEGF from L2 cells in the culture supernatant was evaluated in each group. (C) Cell survival rate in each group was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (D–G) Inflammatory cytokines, including IL-1a (D), IL-1b (E), TNF-a (F), and IL-6 (G), were measured in the culture supernatant of each group. Data are presented as mean 6 SEM. *P , 0.05 compared with normoxia control. † P , 0.05 compared with H2O2 control. ‡P , 0.05 compared with H2O2 1 human umbilical cord blood (UCB)–derived MSCs. a, normoxia control; b, H2O2 control; c, H2O2 1 human UCB–derived MSCs; d, H2O2 1 scrambled siRNA–transfected MSCs; e, H2O2 1 VEGF siRNA–transfected MSCs; f, H2O2 1 human fibroblast (MRC5); g, H2O2 1 human UCB–derived MSCs 1 immunoglobulin G; h, H2O2 1 human VEGF blocking antibody; i, H2O2 1 VEGF siRNA MSCs 1 rhVEGF 1 ng; j, H2O2 1 VEGF siRNA MSCs 1 rhVEGF 10 ng; k, H2O2 1 rhVEGF 1 ng; l, H2O2 1 rhVEGF 10 ng.
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ORIGINAL RESEARCH MSCs in rats significantly restores levels of rat VEGF in hyperoxic lungs (5) supports this conclusion. However, because we could not detect human VEGF secreted from the transplanted MSCs at the end of the experiment, it was not clear whether VEGF secreted from transplanted MSCs was a critical paracrine factor mediating the protective effects of MSC transplantation. In this study, we hypothesized that VEGF secreted by transplanted MSCs plays a pivotal role in protection against hyperoxic lung injury in newborn rats. To test this hypothesis, VEGF was specifically knocked down in human UCB–derived MSCs by transfection with small interfering RNA (siRNA) specific for human VEGF. The protective effects of MSCs with or without VEGF knockdown or VEGF blocking antibody were evaluated in vitro in a rat lung epithelial (L2) cell line challenged with H2O2. To confirm these results in vivo, the protective effects of MSCs with or without VEGF knockdown were evaluated in newborn rats exposed to hyperoxia.
Materials and Methods Cell Preparation and Culture Conditions
Human UCB was obtained from healthy, normal, full-term newborns as previously reported (3) after obtaining written informed parental consent. UCB-derived MSCs were isolated and expanded according to a previously reported procedure (15, 16).
antibody (Abcam, Cambridge, MA) or control IgG) (Dako, Carpinteria, CA) was added to the coculture of H2O2–treated L2 cells and nontransfected MSCs at a concentration of 200 ng per well. Low-dose (1 ng/ml) or high-dose (10 ng/ml) recombinant human (rh)VEGF (R&D Systems, Minneapolis, MN) was added to the H2O2–treated L2 cells with or without cocultured VEGF siRNA–transfected MSCs.
intratracheally. An equal volume of PBS was administered to the control groups in the same manner. Morphometry
Alveolarization was assessed by measuring the mean linear index and mean alveolar volume as previously described (3–6). A minimum of three sections per rat and six fields per section were randomly evaluated in a blinded manner.
Animal Model
Animal procedures were approved by the Animal Care and Use Committee of Samsung Biomedical Research Institute, Seoul, Korea. This study was performed in accordance with institutional and National Institutes of Health guidelines for laboratory animal care. Timed pregnant Sprague-Dawley rats (Orient Co., Seoul, Korea) spontaneously delivered rat pups. Newborn rats were allocated to five experimental groups: normoxia control, hyperoxia control, hyperoxia with human UCB–derived MSCs, hyperoxia with scrambled siRNA–transfected MSCs, and hyperoxia with VEGF siRNA–transfected MSCs. Normoxic rat pups were kept in room air, and hyperoxic rat pups were raised in hyperoxic chambers (90% O2) from birth until postnatal day (P)14. At P5, 5 3 105 nontransfected, VEGF siRNA–transfected, or scrambled siRNA–transfected MSCs were administered
Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling Assay
Details are described in the online supplement. Immunohistochemistry
Details are described in the online supplement. ELISA
Frozen lungs were homogenized, and the total protein content in the supernatant was determined as previously reported (3–5). The amount of human- and rat-specific VEGF, IL-1a, IL-1b, IL-6, and TNF-a was measured as described in the online supplement. Statistical Analyses
Data are expressed as the mean 6 SEM. For continuous variables with a normal
VEGF Silencing with siRNA
MSCs (3 3 106) were seeded in 100-mm plates with 1 ml Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA). After cells reached 40 to 80% confluency, MSCs were transfected with siRNA targeting VEGF or scrambled siRNA using Lipofectamine (Invitrogen) as described in the online supplement. In Vitro Cell Culture
Cultured L2 rat lung epithelial cells were treated with 100 mM H2O2 for 60 minutes and then incubated in complete media alone or supplemented with 1 3 103 MRC5 cells (human fibroblast cell line), nontransfected MSCs, scrambled siRNA–transfected MSCs, or VEGF siRNA–transfected MSCs. VEGF blocking
Figure 2. Induction of rat VEGF in rat host lungs by human VEGF from transplanted human MSCs. The figure shows a temporal profile of human VEGF (A) and rat VEGF (B) in rat lungs from each group. Data are presented as mean 6 SEM. *P , 0.05 compared with normal. †P , 0.05 compared with hyperoxia control. ‡P , 0.05 compared with hyperoxia 1 MSCs. xP , 0.05 compared with scrambled siRNA MSCs.
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ORIGINAL RESEARCH distribution, the groups were compared using one-way ANOVA followed by a least significant difference multiple comparison test. A P value , 0.05 was considered significant. SPSS version 17.0 (SPSS Institute, Chicago, IL) was used for all analyses.
Results
significant differences in the number of PKH26-positive donor cells in the rat lung tissue at P14 among the MSC transplantation groups (Figure E4). The amount of rat VEGF in hyperoxic lung tissue gradually decreased over time and was significantly lower than that in the normoxia control group after P7. The decrease in rat VEGF was significantly
attenuated in the nontransfected and scrambled siRNA–transfected MSC groups but not in the VEGF siRNA–transfected MSC group (Figure 2B). Protective Effects of MSC Transplantation In Vitro and In Vivo
In cultured rat L2 cells, H2O2 exposure significantly reduced cell survival compared
VEGF Knockdown and Human and Rat VEGF Level
VEGF levels in the culture medium of nontransfected or scrambled siRNA–transfected MSCs increased over time, whereas transfection of MSCs with VEGF siRNA significantly decreased VEGF production for up to 7 days (see Figure E1 in the online supplement). No significant differences in VEGF production were noted between the nontransfected and scrambled siRNA–transfected groups. No human VEGF level was detected in the supernatant of normoxic or H2O2–treated rat L2 cells cultured alone. Human VEGF was detected in the supernatant of H2O2–treated rat L2 cells that were cocultured with nontransfected or scrambled siRNA–transfected human MSCs and at significantly higher levels than in the supernatant of H2O2–treated cells cocultured with VEGF siRNA–transfected MSCs or human fibroblasts (MRC5) (Figure 1A). The significant decrease in rat VEGF level in the H2O2 control group compared with the normoxic control group was significantly improved by coculture with nontransfected or scrambled siRNA–transfected MSCs but not by coculture with MRC5 or VEGF siRNA–transfected MSCs (Figure 1B). In rat lung tissue, human VEGF secreted from the transplanted MSCs was detected in the nontransfected and scrambled siRNA–transfected groups, and its concentration decreased over time. Human VEGF was not detected in the VEGF siRNA–transfected group (Figures 2A and E2). PKH26-positive donor cells were colocalized with ED-positive rather than terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in P14 rat lungs, indicating that transplanted MSCs are phagocytosed directly or undergo apoptosis/necrosis and that the cell fragments are phagocytosed by the macrophages (Figure E3). There were no 394
Figure 3. Pulmonary angiogenesis in the lungs of postnatal day (P)14 rats. (A) Representative immunofluorescence photomicrographs of von Willebrand factor (vWF) staining in the lungs of P14 rats in each group. vWF was labeled with the fluorescent marker 5(6)-carboxyfluoresceindiacetate N-succinimidyl ester (green), and nuclei were labeled with 49,6-diamidino-2-phenylindole (blue) (scale bars, 25 mm). 1, normal; 2, hyperoxia control; 3, hyperoxia 1 MSC; 4, hyperoxia 1 scramble siRNA MSC; 5, hyperoxia 1 VEGF siRNA MSC. (B) The mean optical density of vWF immunofluorescence per one high power field in each group. Data are presented as mean 6 SEM. *P , 0.05 compared with normal. †P , 0.05 compared with hyperoxia control. ‡P , 0.05 compared with hyperoxia 1 MSCs. xP , 0.05 compared with scrambled siRNA MSCs.
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ORIGINAL RESEARCH with normoxic conditions. This oxidative stress–induced cell death was significantly lower in the L2 cells cocultured with nontransfected or scrambled siRNA–transfected MSCs but not in cells cocultured with MRC5 cells or VEGF siRNA–transfected MSCs. The protective effect of MSCs against H2O2–induced cell death was abolished by VEGF neutralizing antibody but not by control IgG. Supplementation with 1 or 10 ng rhVEGF restored the protective effects of VEGF knockdown MSCs against hyperoxic L2 cell injury in a dose-dependent manner. For rhVEGF supplementation alone, 10 ng (but not 1 ng) significantly attenuated H2O2–induced L2 cell death (Figure 1C). In rat lung tissue, expression of von Willebrand factor was significantly reduced, indicating impaired angiogenesis, and mean linear index and alveolar volume were increased, indicating impaired alveolarization, in the hyperoxic groups compared with the normoxic group. These effects were significantly improved by transplantation with nontransfected MSCs. The protective effects of transplanted MSCs were abolished by transfection with VEGF siRNA but not with scrambled siRNA (Figures 3 and 4). The hyperoxiainduced increase in the number of TUNEL-positive cells in rat lung tissue was significantly attenuated by transplantation with nontransfected or scrambled siRNA–transfected MSCs but not by VEGF siRNA–transfected MSCs (Figure 5A). Inflammatory Responses
In rat L2 cells, the increased expression of inflammatory cytokines, such as IL-1a, IL-1b, TNF-a, and IL-6, in the H2O2–exposed groups were significantly attenuated in cells that were cocultured with nontransfected or scrambled siRNA–transfected MSCs but not in groups that were cocultured with MRC5 cells or VEGF siRNA–transfected MSCs. Similarly, the anti-inflammatory effects of MSCs, as evidenced by reduced IL-1a, IL-1b, IL-6, and TNF-a levels, were abolished by VEGF neutralizing antibody but not by IgG (Figures 1D–1G). Furthermore, supplementation with 1 or 10 ng rhVEGF restored the anti-inflammatory effects of VEGF knockdown MSCs in a dose-dependent manner. For rhVEGF supplementation alone, 10 ng (but
Figure 4. Histologic and morphometric analysis of P14 rat lungs. (A) Representative optical microscopy photomicrographs of lungs from rats in each group stained with hematoxylin and eosin (scale bar, 25 mm). 1, normal; 2, hyperoxia control; 3, hyperoxia 1 MSC; 4, hyperoxia 1 scramble siRNA MSC; 5, hyperoxia 1 VEGF siRNA MSC. Degree of alveolarization assessed by the mean linear intercept (B) and mean alveolar volume (C). Data are presented as mean 6 SEM. *P , 0.05 compared with normal. †P , 0.05 compared with hyperoxia control. ‡P , 0.05 compared with hyperoxia 1 MSCs. xP , 0.05 compared with scrambled siRNA MSCs.
not 1 ng) significantly attenuated the H2O2–induced increase in inflammatory cytokines (Figures 1D–1G). In rat lung tissue, the hyperoxia-induced increase in ED-1–positive cells and inflammatory cytokines such as IL-1a, IL-1b, IL-6, and TNF-a was significantly attenuated by transplantation with nontransfected MSCs. The antiinflammatory effects of transplanted MSCs were abolished by transfection with VEGF
Chang, Ahn, Jeon, et al.: Critical Role of VEGF from MSCs
siRNA but not by transfection with scrambled siRNA (Figures 5C, 5D, and 6).
Discussion In the present study, we show that human UCB–derived MSCs, but not human fibroblasts, significantly improved cell survival in H2O2–treated rat lung epithelial (L2) cells, suggesting that the protective 395
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Figure 5. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and pulmonary macrophages in the distal lungs of P14 rat pups. (A, C) Representative photomicrographs of TUNEL-positive cells (A) and ED-1–positive cells (C) in the lungs of P14 rats in each group. TUNEL-positive cells were labeled with FITC (green), and ED-1–positive alveolar macrophages were labeled with 5(6)-carboxyfluoresceindiacetate N-succinimidyl ester (green). The cell nuclei were labeled with DAPI (blue) (scale bar, 25 mm). 1, normal; 2, hyperoxia control; 3, hyperoxia 1 MSC; 4, hyperoxia 1 scramble siRNA MSC; 5, hyperoxia 1 VEGF siRNA MSC. (B, D) The average number of TUNEL-positive cells (B) and ED-1–positive cells (D) per high-power field (HPF) in each group. Data are presented as mean 6 SEM. *P , 0.05 compared with normal. †P , 0.05 compared with hyperoxia control. ‡P , 0.05 compared with hyperoxia 1 MSCs. xP , 0.05 compared with scrambled siRNA MSCs.
effects may be specific to MSCs. Moreover, the protective effects of MSCs observed in vitro in L2 cells exposed by H2O2 were abolished by VEGF knockdown or VEGFneutralizing antibody and were restored by rhVEGF supplementation. The beneficial effects of MSCs were also abolished in vivo in a newborn Sprague-Dawley rat model of hyperoxic lung injury by VEGF knockdown with VEGF siRNA but not by scrambled siRNA. Taken together, these results support the hypothesis that VEGF secreted by transplanted MSCs might be a critical paracrine factor in protection against neonatal hyperoxic lung injury, such as that in BPD. A substantial increase in microvascular endothelial cell proliferation is necessary for 396
normal neonatal lung growth, which is characterized by rapid angiogenesis and alveolarization (22). VEGF, a specific mitogen for endothelial cells that is mainly expressed by alveolar epithelial cells adjacent to capillary beds, plays a critical role in postnatal lung angiogenesis (22, 23). These findings suggest a close link between lung angiogenesis and alveolarization in normal conditions (22) and in disease conditions, such as BPD (7). VEGF secreted by transplanted stem cells is also a crucial paracrine mediator in facilitating recovery in various animal models of disease, such as myocardial injury (17), acute kidney injury (18), and stroke (19). These findings suggest that VEGF secreted by transplanted
stem cells might be a universal paracrine mediator of the protective effects of stem cell transplantation in various diseases (20) and could also be used as a biomarker of the potency of stem cell transplants (17). Although the VEGF level was increased in the MSC transplantation group in the present study, it was still much lower than that in the normoxia group. Overexpression of VEGF in MSCs has been reported to significantly enhance stem cell–mediated therapeutic efficacy in neural and cardiac repair (21, 22). Therefore, further studies might be necessary to investigate whether transplantation of VEGF-overexpressing MSCs enhances their beneficial effects in this BPD model.
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Figure 6. Temporal profile of inflammatory cytokines in rat lungs at P7, P10, and P14. Expression of IL-1a (A), IL-1b (B), IL-6 (C), and TNF-a (D) in rat lungs at P7, P10, and P14 for each group. Data are presented as mean 6 SEM. *P , 0.05 compared with normal. †P , 0.05 compared with hyperoxia control. ‡P , 0.05 compared with hyperoxia 1 MSCs. xP , 0.05 compared with scrambled siRNA MSCs.
The results of the present study demonstrated a significantly lower level of human VEGF in the VEGF siRNA transfection group, but not in the scrambled siRNA transfection group, up to 7 days after transplantation. Human VEGF expression colocalized with PKH26-positive donor cells in rats transplanted with nontransfected or scrambled siRNA–transfected MSCs but not in rats transplanted with VEGF siRNA–transfected MSCs. Overall, these findings indicate that VEGF knockdown in MSCs by siRNA transfection was effective and specific. In a similar study by Pierro and colleagues (23), there was a dramatic decrease in MSCs during the first day after intratracheal engraftment, and MSCs were almost undetectable 4 days after transplantation. We also previously showed
that the number of MSCs steadily decreased (5) and that transplanted cells were virtually undetectable 10 weeks after intratracheal transplantation (6). Taken together, these findings suggest that the gradual decrease in human VEGF in the rat lung tissue in nontransfected and scrambled siRNA–transfected groups might be attributed to a loss of the transplanted MSCs. Therefore, improving donor cell survival might improve the protective effects of transplanted MSCs (24). Understanding the mechanism by which human VEGF secreted by exogenous MSCs preserves rat VEGF and protects lung tissue against hyperoxic injury is of particular interest. Although the human VEGF ELISA kit does not cross-react with rat VEGF, there is . 85% homology between human and rat VEGF (25), and
Chang, Ahn, Jeon, et al.: Critical Role of VEGF from MSCs
their functional action of mechanism is quite similar (13, 14). Because VEGF is an obligatory survival factor for lung alveolar and vascular endothelium, a reduction in rat VEGF below the threshold level for survival might be primarily responsible for hyperoxia-induced lung injuries (26, 27). Therefore, human VEGF secreted by the transplanted MSCs might initially maintain VEGF levels within the local microenvironment above the threshold level essential for rat lung tissue survival against hyperoxic injury (28) and thus might indirectly restore the rat VEGF level. In our previous study, we showed that, although donor cells rapidly faded away after transplantation, the favorable effects of the MSCs persisted up to P21 (5). These findings suggest that the paracrine effects induced by the stem cells might initially 397
ORIGINAL RESEARCH play a pivotal role in tissue repair, and as the donor cells are lost the intact host tissue that was protected by MSC transplantation is able to sustain up-regulation of rat VEGF expression (29). Inhibition of apoptosis might be another protective mechanism of VEGF. Our results show that transplantation of MSCs significantly attenuated the hyperoxia-induced increase in the number of TUNEL-positive cells, which was abolished by VEGF siRNA but not by scrambled siRNA (21, 30, 31). The antiapoptotic effects of VEGF might be mediated by inhibiting the expression of proapoptotic proteins and promoting antiapoptotic and cell survival factors (32–34). Transplantation of MSCs also significantly attenuated the hyperoxia-induced increase in expression of inflammatory cytokines in vitro and in vivo and attenuated the increase in ED-1–positive alveolar macrophages in lung tissue. These decreases in inflammatory responses were abolished by transfection with VEGF siRNA but not by scrambled siRNA. Taken together, these findings suggest that VEGF secreted by MSCs may play a role in down-regulating hyperoxia-induced lung injury in the newborn rats. The anti-inflammatory effects of VEGF observed in the present study and in previous studies (19, 35) are contradictory
to the conventional proinflammatory effects of VEGF (36). However, Manoonkitiwongsa and colleagues (37) reported dose-dependent pro- or antiinflammatory effects of VEGF, showing that treatment of ischemic brain with low doses of VEGF reduced macrophage infiltration, whereas higher doses increased macrophage density. Therefore, the antiinflammatory effects of VEGF observed in the present study might be explained by the fact that the VEGF level, although improved, was still much lower in the MSC transplantation group compared with the normoxia group. For VEGF treatment alone, repetitive high doses are required for beneficial effects against hyperoxic lung injury in newborn rats (13, 14). Moreover, VEGF also increases lung permeability, leading to pulmonary edema (14). We have previously shown that a single transplantation with human UCB–derived MSCs attenuates Escherichia coli–induced pulmonary edema (38) and that this beneficial effect might be mediated by keratinocyte growth factor and angiopoietin-1 secreted by MSCs (39–41). In particular, human UCB–derived MSCs secrete exceptionally high amounts of angiopoietin-1 compared with bone marrow–derived or adipose tissue–derived MSCs (41). We have also shown that MSC transplantation simultaneously rescues the hyperoxia-
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