Cell Transplantation, Vol. 24, pp. 1699–1715, 2015 Printed in the USA. All rights reserved. Copyright Ó 2015 Cognizant Comm. Corp.
0963-6897/15 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368914X685087 E-ISSN 1555-3892 www.cognizantcommunication.com
Mesenchymal Stem Cells Overexpressing Angiotensin-Converting Enzyme 2 Rescue Lipopolysaccharide-Induced Lung Injury Hongli He,1 Ling Liu,1 Qihong Chen, Airan Liu, Shixia Cai, Yi Yang, Xiaomin Lu, and Haibo Qiu Department of Critical Care Medicine, Zhongda Hospital, Southeast University School of Medicine, Nanjing, Jiangsu, P. R. China
Bone marrow-derived mesenchymal stem cells (MSCs), which have beneficial effects in acute lung injury (ALI), can serve as a vehicle for gene therapy. Angiotensin-converting enzyme 2 (ACE2), a counterregulatory enzyme of ACE that degrades angiotensin (Ang) II into Ang 1–7, has a protective role against ALI. Because ACE2 expression is severely reduced in the injured lung, a therapy targeted to improve ACE2 expression in lung might attenuate ALI. We hypothesized that MSCs overexpressing ACE2 would have further benefits in lipopolysaccharide (LPS)-induced ALI mice, when compared with MSCs alone. MSCs were transduced with ACE2 gene (MSC-ACE2) by a lentiviral vector and then infused into wild-type (WT) and ACE2 knockout (ACE2−/y) mice following an LPS-induced intratracheal lung injury. The results demonstrated that the lung injury of ALI mice was alleviated at 24 and 72 h after MSC-ACE2 transplantation. MSC-ACE2 improved the lung histopathology and had additional anti-inflammatory effects when compared with MSCs alone in both WT and ACE2−/y ALI mice. MSC-ACE2 administration also reduced pulmonary vascular permeability, improved endothelial barrier integrity, and normalized lung eNOS expression relative to the MSC group. The beneficial effects of MSC-ACE2 could be attributed to its recruitment into the injured lung and enhanced local expression of ACE2 protein without changing the serum ACE2 levels after MSC-ACE2 transplantation. The biological activity of the increased ACE2 protein decreased the Ang II amount and increased the Ang 1–7 level in the lung when compared with the ALI and MSC-only groups, thereby inhibiting the detrimental effects of accumulating Ang II. Therefore, compared to MSCs alone, the administration of MSCs overexpressing ACE2 resulted in a further improvement in the inflammatory response and pulmonary endothelial function of LPS-induced ALI mice. These additional benefits could be due to the degradation of Ang II that accompanies the targeted overexpression of ACE2 in the lung. Key words: Mesenchymal stem cells (MSCs); Angiotensin-converting enzyme 2 (ACE2); Acute lung injury (ALI); Gene therapy
INTRODUCTION Acute lung injury (ALI) is a devastating clinical syndrome that is characterized by the diffuse damage of lung vascular endothelial cells and alveolar epithelial cells and an excessive inflammatory response in the lung (2,5,40). There are currently few specific pharmacological therapies that attenuate ALI and promote lung repair (6,10,18,35). Experimental and clinical evidence indicates that the renin–angiotensin system (RAS) plays an essential role in the pathogenesis of ALI through an increased level of angiotensin (Ang) II generated by the angiotensinconverting enzyme (ACE). The main active molecule, Ang II, can initiate lung inflammatory responses and impair the function of the pulmonary endothelial barrier via the Ang II type 1 receptor (AT1R) (16,17,23,27,45). ACE2, the first homolog of ACE (a counterregulatory
enzyme of ACE), degrades Ang II into Ang 1–7 and mitigates the detrimental effects of Ang II in ALI animal models (16,17). Moreover, previous studies have found that ACE2 mRNA, protein, and enzymatic activity were severely downregulated in human and experimental lung tissue injuries (17,24). In ACE2 knockout mice, the loss of ACE2 results in higher Ang II levels and a more severe lung injury, indicating that the decrease of ACE2 is a major factor contributing to the pathogenesis of ALI by permitting Ang II accumulation. Accordingly, therapies aimed at increasing ACE2 expression in lung tissue might attenuate ALI. However, the systemic infusion of recombinant ACE2 decreases serum Ang II and may affect blood pressure (37,44). Therefore, we hypothesized that a treatment with ACE2 targeted specifically to the injured lung would achieve the optimal therapeutic effect.
Received January 16, 2014; final acceptance September 20, 2014. Online prepub date: October 6, 2014. 1 These authors provided equal contribution to this work. Address correspondence to Haibo Qiu, Department of Critical Care Medicine, Zhongda Hospital, Southeast University School of Medicine, No. 87 Ding Jiaqiao, Nanjing (210009), Jiangsu, P. R. China. Tel: +86-25-83262551; Fax: +86-25-83272123; E-mail:
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In animal models of lung injury, bone marrow-derived mesenchymal stem cells (MSCs) have been shown to attenuate the severity of lung injury and to reduce mortality. These effects occurred through several mechanisms, including differentiation into mature lung epithelial and endothelial cells and a paracrine function to modulate localized inflammation (11,21,33,42). Importantly, MSCs can also act as a vehicle for delivering a protective gene by overexpressing a transgene at the injured site, which could not only enhance its therapeutic effects but also promote local lung repair (29). Therefore, the combination of MSCs and the protective gene ACE2 may be a potential strategy for the treatment of ALI. In the present study, we tested the hypothesis that MSCs with the stable long-term expression of ACE2 via lentiviral-mediated gene transfer could be targeted to the injured lung, locally overexpress ACE2, and attenuate lung injury by reducing the inflammatory response and improving the function of the lung vascular endothelium in lipopolysaccharide (LPS)-induced ALI mice. MATERIALS AND METHODS Ethics Statement Male wild-type (WT) C57BL/6 mice (Laboratory Animal Center, Academy of Military Medical Sciences, Beijing, China) and male ACE2 knockout C57BL/6 (ACE2−/y) mice (Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences, Beijing, China) were maintained under specific pathogen-free conditions. All experiments involving the use of animals were approved by the Committee of Animal Care and Use of Southeast University. Production of Lentiviral Vectors and Transduction of MSCs MSCs from the bone marrow of male WT C57BL/6 mice and 293FT cells were purchased from Cyagen Biosciences, Inc. (Guangzhou, China) as previously described (25). MSCs from passages 4–7 were used for transduction. The Gateway cloning system, as previously reported (12), was used to transfer the full-length coding sequence of ACE2 (NM_001130513.1, 2418 bp) into the human translation elongation factor 1a (EF1a) promoter-dependent lentiviral expression vector pLV.EX3d.P/neo(Cyagen Biosciences, Inc., Guangzhou, China). The ACE2gene was cloned into the lentiviral expression vector between the EF1a promoter and the internal ribosomal entry site (IRES)-dependent eGFP via the BP reaction (Gateway® BP Clonase™ II Enzyme Mix; Invitrogen Life Technologies, Carlsbad, CA, USA) and the LR reaction (Gateway® LR Clonase™ II Plus Enzyme Mix; Invitrogen Life Technologies). The lentivirus was packaged in 293FT cells with the aid of three packaging plasmids: pLV/helper-SL3, pLV/helperSL4, and pLV/helper-SL5 (http://www.addgene.org).
HE ET AL.
Subsequently, a high titer of the recombinant lentivirus was obtained and used to transfect the MSCs. MSCs carrying either eGFP (MSC-GFP) alone or both ACE2 and eGFP (MSC-ACE2) were harvested after selection using G418 (0.5 mg/ml; Amresco, Solon, OH, USA) for 7–14 days. The transduction efficiency was evaluated by detecting the expression of eGFP with an Olympus IX51 fluorescence microscope (Olympus Co., Tokyo, Japan) and a Becton Dickinson FACS Calibur flow cytometer (FACS Calibur; Becton-Dickinson, Mountain View, CA, USA). The transcription of the ACE2 transgene was evaluated using reverse transcription-polymerase chain reaction (RT-PCR). The expression of ACE2 protein was determined using Western blot analysis, and the secreted ACE2 protein in the culture media was evaluated using an enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Westang Bio-tech. Co., Ltd., Shanghai, China). The primers used for designing the RT-PCR were based on the sequences of the genomic clones: ACE2 (254 bp): forward, 5¢-TGGTAGTGGTTGGCATCATCATCC-3¢ and reverse, 5¢-ACGCACACCGGCCTTATTCC-3¢; b-actin (243 bp): forward, 5¢-ATCGTGGGCCGCCCTAGGCA-3¢ and reverse, 5¢- TGGCCTTAGGGTTCAGGGGGG-3¢. The primary antibodies against ACE2 (1:100 dilution; Abcam Ltd., Cambridge, UK) and b-actin (1:3,000 dilution; Bioworld Technology, Co. Ltd., Nanjing, China) were used in the Western blot analysis. The cells in passages 7–10 were used for the in vivo experiments. Murine Model of LPS-Induced ALI Eight- to 10-week-old WT and ACE2−/y mice received intratracheal instillations of 100 µg of LPS (Escherichia coli 0111:B4; Sigma-Aldrich, St. Louis, MO, USA) dissolved in 50 µl of phosphate-buffered saline (PBS; Wisent Inc., St-Bruno, Quebec, Canada) as previously described to produce the ALI model (9). PBS, MSC-GFP, and MSC-ACE2 (5 × 105 cells resuspended in 100 µl PBS) (42) were infused via tail vein injection 4 h (11) after the LPS challenge. Mice without the LPS instillation were injected with PBS as a control. Either 24 or 72 h after the MSC treatment, the mice were sacrificed, and tissues were collected for further analysis. Tracking of MSCs in the Lung MSC-ACE2 cells (5 × 105) labeled with CellVue NIR815 dye (eBioscience Inc., San Diego, CA, USA) were transplanted into WT ALI mice. Three mice at each time point (30 min, 24 h, and 72 h postinjection) were imaged for whole-body and ex vivo organs, including the lung, heart, kidney, spleen, pancreas, small intestine, and liver using a Maestro In-Vivo Optical Imaging System (excitation = 786 nm; emission = 814 nm; exposition time = 4,000 ms; Caliper Life Sciences, Woburn, MA, USA) (28,38). The autofluorescence spectra were then
Figure 1. Transduction efficiency and measurement of angiotensin-converting enzyme 2 (ACE2) mRNA and ACE2 protein after ACE2 gene transduction. (A) Phase control and fluorescence microscopy of mesenchymal stem cells (MSCs), MSC-GFP, and MSC-ACE2 at passages 4, 6, and 6, respectively (100×, scale bar: 50 µm). (B) Transduction efficiency was evaluated by detecting the expression of eGFP using flow cytometry and was as high as 94.3%. (C) Detection of ACE2 mRNA in MSCs using RT-PCR. The amount of ACE2 mRNA in the MSC-ACE2 group is approximately four times as high as that of the MSC group (n = 3; *p
