mscs modified with ace2 restore endothelial function ... · (mscs)-based therapy is a promising...

MSCs Modified With ACE2Restore Endothelial FunctionFollowing LPS Challenge byInhibiting the Activation of RASHONG-LI HE, LING LIU, QI-HONG CHEN, SHI-XIA CAI, JI-BIN HAN, SHU-LING HU,PAN CHUN, YI YANG, FENG-MEI GUO, YING-ZI HUANG, AND HAI-BO QIU*Department of Critical Care Medicine, Zhong-Da Hospital, School of Medicine, Southeast University, Nanjing, Jiangsu, China

Angiotensin (Ang) II plays an important role in the process of endothelial dysfunction in acute lung injury (ALI) and is degraded byangiotensin-converting enzyme2 (ACE2). However, treatments that target ACE2 to injured endothelium and promote endothelial repairof ALI are lacking. Mesenchymal stem cells (MSCs) are capable of homing to the injured site and delivering a protective gene. Our studyaimed to evaluate the effects of genetically modified MSCs, which overexpress the ACE2 protein in a sustained manner via a lentiviralvector, on Ang II production in endothelium and in vitro repair of lipopolysaccharide (LPS)-induced endothelial injury. We found that theefficiency of lentiviral vector transduction of MSCs was as high as 97.8% and was well maintained over 30 passages. MSCs modified withACE2 showed a sustained high expression of ACE2 mRNA and protein. The modified MSCs secreted soluble ACE2 protein into theculture medium, which reduced the concentration of Ang II and increased the production of Ang 1–7. MSCs modified with ACE2 weremore effective at restoring endothelial function than were unmodified MSCs, as shown by the enhanced survival of endothelial cells; thedownregulated production of inflammatory mediators, including ICAM-1, VCAM-1, TNF-a, and IL-6; reduced paracellular permeability;and increased expression of VE-cadherin. These data demonstrate that MSCs modified to overexpress the ACE2 gene can producebiologically active ACE2 protein over a sustained period of time and have an enhanced ability to promote endothelial repair after LPSchallenge. These results encourage further testing of the beneficial effects of ACE2-modified MSCs in an ALI animal model.J. Cell. Physiol. 230: 691–701, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company

Acute lung injury (ALI) and its severe form, acute respiratorydistress syndrome (ARDS), are lethal diseases in critically illpatients and are characterized by increased lung permeability,pulmonary edema, and diffuse inflammation (Ware andMatthay, 2000; Matthay et al., 2012). Numerous noxiousagents, such as bacterial lipopolysaccharide (LPS), may alterearly pulmonary endothelial functions inducing a shift from anormal anti-inflammatory state to an “activated” phenotypecharacterized by pro-adhesive properties, the production ofinflammatory mediators and a high permeability (Feletou andVanhoutte, 2006). This endothelial dysfunction subsequentlycontributes to the development of ALI/ARDS (Orfanos et al.,2004; Maniatis et al., 2008). Currently, few specific targets forpharmacological therapy that inhibit endothelium activationand promote endothelial repair have been identified.

The activation of the local renin–angiotensin system (RAS) inthe endothelium plays an important role in endothelialdysfunction in ARDS (Morrison and Ulevitch, 1978). It has beenreported that Ang II levels greatly increase in the circulationand lung tissue during ALI (Schaller et al., 1985; Zhang and Sun,2005). Ang II, generated mainly by the angiotensin-convertingenzyme (ACE) of the endothelium, is a strong pro-inflammatory mediator that induces the expression of anumber of cytokines, chemokines, and adhesion molecules thatregulate endothelial inflammation and apoptosis through theAng II type 1 receptor (AT1R). Furthermore, Ang II can alsoincrease vascular permeability, exacerbate tissue edema,and induce leukocyte infiltration. The inflammatory cascadeinduced by endothelial dysfunction finally results in ARDS(Imai et al., 2008; Liu et al., 2009). Thus, decreasing Ang II levelscan inhibit endothelial activation and promote endothelialrepair.

Angiotensin converting enzyme 2 (ACE2), a first homologueof ACE, degrades Ang II into Ang 1–7 peptides and abates thedetrimental effects of Ang II in ALI animal models (Imai et al.,2005; Imai et al., 2008). However, the systematic infusion of

recombinant ACE2 may affect vasoconstriction (Rentzschet al., 2008; Imai et al., 2010). Therefore, sustained and localhigh expression of ACE2 protein in lung tissue may reduce AngII levels in the pulmonary endothelium without changing Ang IIlevels in the serum, thus achieving the optimal therapeuticeffect by suppressing endothelial activation and enhancingpulmonary endothelial function.

Currently, bone marrow-derived mesenchymal stem cells(MSCs)-based therapy is a promising therapeutic strategy inthe treatment of ARDS, not only because MSCs suppressinflammation in lung tissues and attenuate lung injury, mainly

Hong-li He and Ling Liu contributed equally to this work.

Contract grant sponsor: National Natural Science Foundation ofChina;Contract grant numbers: 81000828, 81070049, 81170057,81372093.Contract grant sponsor: Natural Science Foundation of JiangsuProvince of China;Contract grant number: BK20131302.Contract grant sponsor: Graduate Innovation Project in JiangsuProvince of China;Contract grant numbers: CXLX_0151, CXLX_0153.

*Correspondence to: Hai-bo Qiu, Department of Critical CareMedicine, Zhong-Da Hospital, School of Medicine, SoutheastUniversity, #87 Ding Jiaqiao, Nanjing 210009, Jiangsu, China.E-mail: [email protected]

Manuscript Received: 16 January 2014Manuscript Accepted: 5 September 2014

Accepted manuscript online in Wiley Online Library(wileyonlinelibrary.com): 9 September 2014.DOI: 10.1002/jcp.24794

ORIGINAL RESEARCH ARTICLE 691J o u r n a l o fJ o u r n a l o f

CellularPhysiologyCellularPhysiology

© 2 0 1 4 W I L E Y P E R I O D I C A L S , I N C . , A W I L E Y C O M P A N Y

through paracrine functions (Lee et al., 2011), but alsobecause MSCs can target protective genes to injured lungtissues, which enhances the therapeutic effects (Xu et al.,2008). Therefore, treatment with sustained highly expressedACE2 protein, delivered by MSCs, may target the injuredpulmonary endothelium and reduce Ang II production, thuspreventing endothelium-mediated inflammation and facilitatingoptimal endothelial repair.

The first aim of the present study was to establish sustainedACE2-overexpressing MSC lines using a human translationelongation factor 1a(EF1a) promoter-dependent lentiviralvector. The second study aim was to evaluate the effects ofACE2 gene modified MSCs, using in vitro experimentalapproaches, on the regulation of the RAS in endothelium, aswell as the inhibition of LPS-induced pulmonary endothelium-mediated inflammation and the repair of injured endothelialcells in vitro. These approaches provide a basis for furthertesting the beneficial effects of ACE2-transduced MSCs in anALI animal model.

Materials and MethodsTransduction of MSCs with lentiviral vectors

Mouse MSCs and 293FT cells used in this study were purchasedfrom Cyagen Biosciences Inc. (Guangzhou, China) as previouslyreported (Liu et al., 2013). These MSCs were isolated from thebonemarrow of C57BL/6mice, were uniformly positive for CD29,CD34, CD44, and Sca-1 antigens and negative for CD117 antigen,and possessed the potential to differentiate into osteocytes,adipocytes, and chondrocytes, as demonstrated in assaysperformed by the supplier.

The lentiviral expression vector was created based on thehuman translation elongation factor 1a(EF1a), a promoter-dependent third-generation self-inactivating humanimmunodeficiency virus (HIV) vector pLV.EX3d.P/neo (CyagenBiosciences Inc.) and cloned with the full-length coding sequenceof ACE2 (NM_001130513.1, 2418 bp). The Gateway cloningsystem including BP reaction (Gateway1 BP ClonaseTM IIEnzyme Mix, Invitrogen Life Technologies, Carlsbad, CA) and LRreaction (Gateway1 LR ClonaseTM II Plus Enzyme Mix,Invitrogen Life Technologies) were used to transfect theACE2 gene into the expression vector between the EF1apromoter and the internal ribosomal entry site (IRES)-dependenteGFP as previously described (Hartley et al., 2000). Then,293FT cells were transduced with the lentiviral expressionvector and the other three packaging plasmids, pLV/helper-SL3,pLV/helper-SL4, and pLV/helper-SL5 (www.addgene.org),using Lipofectamine 2000 (Invitrogen Life Technologies) toobtain high titer recombinant lentiviral vectors and transducedMSCs. G418 (0.5 mg/ml) was used for 7–14 days to purifythe MSCs carrying eGFP (MSC-GFP) or both the ACE2 geneand eGFP (MSC-ACE2), and cells were harvested afterselection.

The expression of eGFP was detected using a fluorescentmicroscope to evaluate the long-term transduction efficiency oftransduced MSCs cultured in complete culture medium for over30 passages. The expression of the ACE2 mRNA wasdetermined by reverse transcription-polymerase chain reaction(RT-PCR) using the following primer sequences: ACE2 (254 bp):forward, 50-TGGTAGTGGTTGGCATCATCATCC-30 andreverse, 50-ACGCACACCGGCCTTATTCC-30; b-actin(243 bp): forward, 50-ATCGTGGGCCGCCCTAGGCA-30 andreverse, 50-TGGCCTTAGGGTTCAGGGGGG-30. Theexpression of ACE2 protein was measured by Western blotting.ACE2 protein levels secreted into the conditioned medium werealso quantified using an ELISA kit (Westang Bio-tech. Co. Ltd.,Shanghai, China) by following the instructions of themanufacturer. MSC-GFP and MSC-ACE2 from passages 7–10were used in this study.

Primary human pulmonary microvascular endothelialcell culture and treatment with LPS

First-passage human pulmonary microvascular endothelial cells(ECs) were purchased from ScienCellTM Research Laboratories(Carlsbad, CA). ECs were routinely characterized by expressionof CD31 and vWF, as assessed by immunofluorescence assaysperformed by the supplier. The ECs (2� 105 cells per well)were seeded in 6-well cell culture plates and cultured at 37 °Cin a 5% CO2 incubator overnight with Dulbecco’s modifiedEagle media/nutrient mixture F-12 (DMEM/F12) (Wisent, Inc.,St-Bruno, Quebec, Canada) containing 10% FBS (Wisent, Inc.)and 1% antibiotics (streptomycin and penicillin, Wisent, Inc.).Then, the medium was changed with DMEM/F12 supplementedwith 10% FBS containing 100 ng/ml lipopolysaccharide (LPS,Escherichia coli 0111:B4; Sigma–Aldrich, St. Louis, MO) andcultured for 0, 2, 4, 6, 12, and 24 h. Viability and paracellularpermeability of ECs was measured at different time points tochoose the best duration of LPS stimulation that resulted indamage of ECs.We found that stimulation with 100 ng/ml LPS for6 h significantly reduces the viability of ECs and increases theparacellular permeability of the ECmonolayer. Thus, we used 6 has the intervention time point in our subsequent experiments(Figs. 2A–D).

Transwell and co-culture of ECs and MSCs

ECs were co-cultured indirectly with the stem cells (MSCs, MSC-GFP, or MSC-ACE2) in collagen-coated 0.4mm pore sizetranswell systems (Corning Inc., NY) (Fig. 1F). Briefly, 5� 104

ECs per well were seeded in 24-well culture plates in 400mlDMEM/F12 supplemented with 10% FBS. Then, 1� 104 stem cellswere planted in the upper chamber of the transwell inserts with200ml culture medium and were co-cultured with the ECs for24 h at 37 °C in a 5% CO2 incubator. Afterwards, the co-cultureswere treated with 100 ng/ml of LPS for 6 h. The ECs cultured inthe culture medium were negative controls. The ECs treatedwith LPS but without co-culture with stem cells were the positivecontrols. After exposure to LPS, the supernatants were collectedfor use in enzyme linked immunosorbent assays (ELISA) forAng II, Ang 1–7, tumor-necrosis factor-a(TNF-a), interleukin-6(IL-6), and vascular endothelial growth factor (VEGF). Viabilityand apoptosis evaluations of ECs were performed. The ECswere also harvested for quantitative real-time polymerasechain reaction (qPCR) assays of inflammatory mediators andAng II receptors, and used for Western blotting of VE-cadherin.Experiments were run in triplicate and repeated at least threetimes.

Measurements of Ang II, Ang 1–7, TNF-a, IL-6, VEGF byELISA

Human EC-derived Ang II levels in the conditioned medium of theco-culture system were assessed using an Ang II ELISA kit(Westang Bio-tech. Co. Ltd.), the concentrations of Ang 1–7 in theco-culture system were detected using an Ang 1–7 ELISA kit(JanCheng Bioengineering Co., Nanjing, China), and the levels ofTNF-a, IL-6, VEGF in the co-culture system were measured usingthe appropriate ELISA kits (ExCellBio, Shanghai, China). All ELISAswere performed strictly according to the instructions of themanufacturer.

Viability and apoptosis assays of ECs

The viability of ECs was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT; Sigma–Aldrich) assay, andsenescence-associated b-galactosidase (SAb-gal) assay. TheMTT assay can detect cell survival and growth.The ECs wereco-cultured with MSCs, MSC-GFP, or MSC-ACE2 for 24 h in

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24-well culture plates. The ECs, at approximately 75–85%confluence, were treated with LPS for 6 h, after which 60ml of5mg/ml MTT was added to each well, and the plates wereincubated at 37 °C for 4 h. Then, 200ml dimethyl sulfoxide(DMSO; Sigma–Aldrich) was added to the wells and incubated for15min. Cell viability was assessed by measuring the sampleabsorbance at 570 nm, using the absorbance at 630 nm as areference. The SAb-gal assay is used to evaluate cellularsenescence, whereby cells completely lose the ability to divide andbecome nonfunctional, although the cells may survive for a longtime. ECs senescence was detected using a senescence cellshistochemical staining kit (Sigma–Aldrich) according to themanufacturers’ protocols. The SAb-gal positive cells wereconsidered senescent and visualized using a light microscope. Anindependent investigator counted the stained cells in five randomlychosen fields (200�) and the cell counts are represented as apercentage of the total number of cells.

We used the annexin V-FITC assay kit (Sigma–Aldrich) to assessthe percentage of apoptotic cells in the ECs cultures, according to

the manufacturer’s instructions. The LPS-treated ECs in the co-culture system were harvested and washed in PBS. The cells weresuspended in 1� binding buffer at a concentration of�1� 106 cells/ml, and 5ml annexin V FITC conjugate (annexin V)and 10ml propidiumiodide solution (PI) were added to each cellsuspension. After mixing, the cells were incubated for 10min atroom temperature in the dark and analyzed using a flow cytometer(BD Biosciences, Mountain View, CA) within 1 h after staining. Thelive cells stain with neither PI nor annexin V. The cells that are earlyin the apoptotic process stain with annexin V. Late apoptotic ornecrotic cells stain with both PI and annexin V.

qPCRanalysis of adhesionmolecules, cytokines andAng IIreceptors of ECs

The expression level of intercellular cell adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), TNF-a,IL-6, Ang II type 1 receptor (AT1R), and AT2R in ECs wereanalyzed via qPCR. First, total RNA was extracted from the cells

Fig. 1. Stable genetic modification of MSCs with a lentiviral vector mediates ACE2 gene transduction. (A, B) Examples of transduced MSC-ACE2 morphology at passage 30 under phase control (A) and fluorescence microscopy (B). The transduction efficiency as assessed bydetecting eGFP expression was as high as 97.8%, indicating that the transduction efficiency was well maintained over 30 passages and thelentiviral vector-mediated ACE2 gene transduction of MSCs was stable. (C) High levels of ACE2 mRNA expression were detected by reversetranscription-polymerase chain reaction. (D) The expression of ACE2 protein in MSCs was measured by Western blotting. (E) The secretedsoluble ACE2 protein was measured using an ELISA kit. Approximately 3.2-fold higher levels were present in the MSC-ACE2 group than theMSC and MSC-GFP groups (n¼ 6; *P< 0.01 vs. MSC; #P< 0.01 vs. MSC-GFP). (F) An indirect co-culture model system was used to investigatethe therapeutic effects of MSCs, MSC-GFP, and MSC-ACE2 on the restoration of endothelial function induced by LPS.


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using Trizol-reagent (Takara Bio, Inc., Kyoto, Japan). TheRevertAid First Strand cDNASynthesis Kit (Thermo FisherScientific Co., Ltd, China) was used for cDNA synthesis, andcDNA amplification was performed using the IQ SYBR GreenSupermix (Bio-Rad Laboratories, Inc. Hercules, CA). Threeindependent experiments were performed, and the geneexpression was normalized by comparison with GAPDH. Allresults are displayed as ratios to the control group. The primersused for qPCR had the following sequences:

Measurement of EC monolayer permeability

The permeability of ECmonolayer was determined by measuringthe diffusion of 40-kDa fluorescein isothiocyanate (FITC)-Dextran (Sigma–Aldrich) through a confluent EC monolayer

(Pati et al., 2011b). 5� 104 ECs per well were plated in insertswith 200ml DMEM/F12 supplemented with 10% FBS in 24-wellculture plates, and 1� 104 stem cells were plated in the lowerchamber with 400ml culture medium and co-cultured at 37 °C ina 5% CO2 incubator. When the ECs reached 100% confluence,100 ng/ml of LPS was added to the co-cultures for 6 h. The ECmonolayer permeability was tested by adding 5ml of 5 mg/ml40-kDa FITC-Dextran to the upper chamber of each well for40min. After the incubation, 100ml of the medium waswithdrawn from the upper and lower wells and added to a black-bottomed 96-well plate. The fluorescence intensity wasdetermined using a Multiscan Spectrum (InfiniteR 200 PRO,Tecan Group Ltd., Mannedorf, Zurich, Switzerland) at excitationand emission wavelengths of 485 and 530 nm, respectively. Thedata represent the mean standard deviation (SD) from threeindependent experiments.

Western blotting analysis of VE-cadherin

The expression of VE-cadherin on ECs was detected by Westernblotting as previously described (Liu et al., 2013). Total protein wasextracted and quantified using the BCA method. The proteinswere separated using 10% sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) and were transferred onto PVDFmembranes (Millipore, Bedford, MA). The membranes wereblocked in Tris-1 buffer at pH 7.4 containing 0.1% Tween20 (TBST)and 5% bovine serum albumin for 1 h at room temperature andwere then incubated with a primary antibody against VE-cadherin(1:50 dilution; Abcam Ltd., Cambridge, UK) at 4 °C overnight. The

Fig. 2. LPS stimulation results in endothelial injury. (A, B) Senescent ECs (SAb-galþ) present at different time points after 100 ng/ml LPSintervention. (C) The viability of ECs as detected by MTT assay at different time points after treatment with 100ng/ml LPS. (D) Paracellularpermeability of ECs at different time points after treatment with 100ng/ml LPS. We found that LPS treatment induced EC senescence,reduced the viability of ECs and increased the paracellular permeability of ECs in a time-dependent manner. Significant differences wereobserved at 6, 12, and 24h. LPS significantly induced EC senescence even as early as 4 h after LPS stimulation. At 6h, the shortest time of LPSexposure that significantly decreased the viability and increased the permeability of ECs was chosen for subsequent experiments.(n¼ 3;*P< 0.05 vs. 0 h).

ICAM-1 Forward, 50-TGGTAGCAGCCGCAGTCATA-30

(377 bp: NM_000201.2) Reverse, 50-CTCCTTCCTGGCTTAGT-30

VCAM-1 Forward, 50-GCTGAGAGGCAGACTTCC-30

(127 bp: NM_001078.3) Reverse, 50-GGCAGTTACTGTTCTTCAGG-30

TNF-a Forward, 50-TACTGAACTTCGGGGTGATTG GTCC-30

(248 bp: NM_000594.3) Reverse, 50-CAGCCTTGTCCCTTGAAGAG AACC-30

IL-6 Forward, 50-CCTTCGGTCCAGTTGCCTTCT-30

(234 bp: NM_000600.3) Reverse, 50-CCAGTGCCTCTTTGCTGCTTTC-30

AT1R Forward, 50-ATAATGTAAGCTCATCCACC-30

(205 bp: NM_000685.4) Reverse, 50-GAGATTGCATTTCTGTCAGT-30

AT2R Forward, 50-AACTGGCACCAATGAGTCCG-30

(210 bp: NM_000686.4) Reverse, 50-CCAAAAGGAGTAAGTCAG CCAAG-30

GAPDH Forward, 50-CCATGTTCGTCATGG GTGTGAACCA-30

(251 bp: NM_001256799.1) Reverse, 50-GCCAGTAGAGGCAGGGA TGATGTTC-30


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blot waswashedwith TBST and incubatedwith goat anti-rabbit IgGconjugated with horseradish peroxidase (Zhongshan GoldenBridge Biotechnology Co., Ltd., Beijing, China). Immunoreactivecomplexes were visualized using chemiluminescence reagents(Thermo Scientific). Experiments were repeated at least threetimes.

Statistical analysis

Statistical analyses were performed using SPSS 16.0. The datawere presented as the mean� standard deviation (SD). Forcomparison among groups, statistical analyses were performedby one-way ANOVA followed by Tukey’s multiple comparisontests. P values< 0.05 were considered statistically significant.

ResultsACE2 gene modification of MSCs

An example of the morphology of transduced MSC-ACE2 atpassage 6 under phase control and fluorescence microscopy isshown in Figures 1A and B. The transduction efficiency of MSC-ACE2 at passage 30, as assessed by detecting the expression ofeGFP, was as high as 97.8%. This result indicates that thetransduction efficiency was well maintained over 30 passagesand the lentiviral vector-mediated ACE2 gene transduction ofMSCs was very stable. The results of the RT-PCR analysis(Fig. 1C) and Western blotting (Fig. 1D) showed highexpression levels of the ACE2 mRNA and protein after ACE2gene transduction.

TransducedMSCs secreted soluble ACE2 into the culturemedium

We measured the concentration of ACE2 protein in theculture medium using an ELISA kit. The results demonstratethat MSCs modified by a lentiviral vector to express the ACE2gene could secrete soluble ACE2 protein into the culturemedium at levels approximately 3.2-fold higher than the MSCgroup (*P< 0.01 vs. MSC) and 2.6-fold higher than the MSC-GFP group (#P< 0.01 vs. MSC-GFP) (Fig. 1E). Next, we used anindirect co-culture model system to investigate the therapeuticeffects of MSCs, MSC-GFP, and MSC-ACE2 on endothelialrepair of LPS-induced injury (Fig. 1F).

LPS stimulation resulted in endothelial injury

LPS was used to induce endothelial injury in the present study.To choose the best duration of LPS stimulation, the viability ofECs, as assessed by ECs proliferation and ECs senescence, andthe paracellular permeability of ECs at different time pointsafter LPS treatment were measured. We found that LPS(100 ng/ml) reduced the proliferation of ECs, induced ECsenescence, and increased the paracellular permeability of ECsin a time-dependent manner. We observed significantdifferences between 6, 12, and 24 h versus 0 h in both theviability and paracellular permeability, and LPS significantlyincreased EC senescence even as early as 4 h after LPSstimulation(*P< 0.05 vs. 0 h) (Figs. 2A–D). Thus, 6 h, theshortest time of LPS exposure that significantly inhibited theviability and increased the permeability of ECs, was chosen forsubsequent experiments.

MSC-ACE2 inhibited the activation of RAS in theendothelium by degrading Ang II into Ang 1–7

It was found that the concentration of Ang II in the culturemedium increased significantly (~5-fold) after stimulation withLPS (*P< 0.05 vs. EC) (Fig. 3A), which accompanied thedecrease of Ang 1–7 levels in the culture medium (*P< 0.05 vs.

EC) (Fig. 3B). Significant differences in the expression of AT1Rand AT2R were not observed (Figs. 3C and D). Theaccumulation of Ang II in LPS-stimulated ECs cultured withMSC-ACE2 was significantly reduced (~1.9-fold) compared toLPS-stimulated ECs cultured alone (~5-fold) (#P< 0.05 vs. LPS)or LPS-stimulated ECs co-cultured with either MSCs (~3.6-fold) ($P< 0.05 vs. MSC) orMSC-GFP (~3.9-fold) (&P< 0.05 vs.MSC-GFP) (Fig. 3A). In contrast, the production of Ang 1–7 inthe EC culture medium was significantly increased after co-culture with MSC-ACE2 when compare with LPS group(#P< 0.05), MSC group ($P< 0.05) or MSC-GFP group(&P< 0.05) (Fig. 3B). Differences in the concentrations of Ang IIand Ang 1–7 were not observed between the LPS group andthe MSC or MSC-GFP groups (Figs. 3A and B). These resultssuggest that the increased Ang II was degraded into Ang 1–7. Itwas also found that the co-culture of ECs with the three typesof MSCs did not affect the expression of AT1R or AT2R(Figs. 3C and D).

MSC-ACE2 enhanced the survival of ECs by decreasingEC apoptosis and improving cell viability

The survival of ECs was evaluated using cell apoptosis and cellviability assays. The protective effect of MSCs, MSC-GFP, andMSC-ACE2 against apoptosis of ECs was assessed by using anAnnexin V-FITC assay kit. The results show that LPS inducedearly apoptosis in approximately 21% and necrosis inapproximately 11% of the cells, and there were significantdifferences between the LPS group and the EC group(*P< 0.05 vs. EC). Treatment with MSCs or MSC-GFPreduced the number of early apoptotic cells as shown by thereduced staining with annexin V alone (#P< 0.05 vs. LPS).However, the percentages of late apoptotic or necrotic cellswere not affected as shown by staining with both PI andannexin V. In comparison to the MSCs or MSC-GFP co-cultures, the MSC-ACE2 further reduced the number of earlyapoptotic cells and tended to decrease the number ofnecrotic cells ($P< 0.05 vs. MSC; &P< 0.05 vs. MSC-GFP)(Figs. 4A–C). The cell viability results by MTT assay and SAb-gal assay also confirmed that MSC-ACE2 restored cell viabilityto a greater extent than the MSCs and MSC-GFP treatmentsafter LPS stimulation (Figs. 4D–F).

MSC-ACE2 downregulated the production ofinflammatory mediators

The gene transcription levels of inflammatory mediatorsincluding the adhesion molecules ICAM-1 and VCAM-1 andthe cytokines IL-6 and TNF-a were measured by qPCR. Wefound that the expression of ICAM-1, VCAM-1, IL-6, andTNF-a mRNA in ECs was significantly elevated after LPSstimulation and, when co-cultured with MSC-ACE2,decreased almost to baseline (*P< 0.05 vs. EC; #P< 0.05 vs.LPS; $P< 0.05 vs. MSC; &P< 0.05 vs. MSC-GFP). Although, theproduction of inflammatory mediators decreased in the MSCand MSC-GFP groups, significant differences were notobserved except in the expression of ICAM-1 (Figs. 5A–D).The protein expressions of IL-6 and TNF-a in the culturemedium were measured with ELISA kits, and the resultsshowed changes similar to the changes in gene expression(Figs. 5E and F).

MSC-ACE2 restored the integrity of EC monolayers

The integrity of the EC monolayers was assessed by measuringthe paracellular permeability and the expression of VE-cadherin, the main component of adhesive junctions(Vandenbroucke et al., 2008). The paracellular permeability ofthe EC monolayers was measured in unstimulated conditions



and following LPS stimulation by the diffusion of 40-kDadextran-FITC.We found that LPS greatly increased monolayerpermeability to 40-kDa dextran-FITC (*P< 0.05 vs. EC). TheECs co-cultured with MSC-ACE2 were significantly lesspermeable to the 40-kDa dextran-FITC than the LPS group(#P< 0.05), while differences were not observed in the MSCand MSC-GFP groups, although the permeability decreased(Fig. 6A). The expression of VE-cadherin detected by Westernblotting was dramatically decreased in the LPS group (*P< 0.05vs. EC), while VE-cadherin was restored almost to normallevels in ECs that were co-cultured with MSC-ACE2 (#P< 0.05vs. LPS) (Fig. 6B). The level of VEGF, the main regulator ofendothelial permeability, was also determined using an ELISAkit. The result shows that LPS stimulation increased theexpression of VEGF significantly and was decreased bytreatment with MSC-ACE2. Difference in VEGF expressionwas not observed after treating withMSC andMSC-GFP, which

suggests that the benefits of MSC-ACE2 on the restoration ofEC monolayers were associated with the downregulation ofVEGF (Fig. 6C).

Discussion

The major findings of our study can be summarized as follows:(1) the local renin–angiotensin system (RAS) of the pulmonaryendothelium was activated after treatment with LPS; (2) MSCstransduced with the ACE2 gene by an EF1a promoterdependent lentiviral vector had a high transduction efficiencyand stably overexpressed ACE2 protein long-term; and (3) theprotective effect of MSC-ACE2 against endothelial damageinduced by LPS was more significant than that of MSC or MSC-GFP treatment. These findings indicate thatMSCsmodifiedwiththe ACE2 gene can sustainably overexpress the ACE2 proteinand inhibit the activationof the local RAS in endothelium, further

Fig. 3. MSC-ACE2 inhibits the activation of RAS in the endothelium. (A) The concentration of Ang II in the culture medium was significantlyincreased (~5-fold) after stimulation of ECs with LPS. Medium from LPS-exposed ECs cultured with MSC-ACE2 had a significantly reducedaccumulation of Ang II (~1.9-fold) compared to ECs cultured alone (~5-fold), or co-cultured with MSCs (~3.6-fold) or MSC-GFP (~3.9-fold). Nodifferences were observed between the LPS group and the MSC or MSC-GFP groups. (B) The production of Ang 1–7 in the culture mediumwas markedly decreased after LPS challenge and significantly increased after co-culture with MSC-ACE2 compare with the LPS group, MSCgroup or MSC-GFP group. No significant improvement was observed after MSC or MSC-GFP treatment. (C) The expression of AT1R in ECs.No significant differences were observed between groups. (D) The expression of AT2R in ECs. No significant differences were observedbetween groups. (n¼ 3;*P< 0.05 vs. EC; #P< 0.05 vs. ECþLPS; $P< 0.05 vs. ECþLPSþMSC; &P< 0.05 vs. ECþLPSþMSC-GFP).


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Fig. 4. MSC-ACE2 improves the survival of ECs after LPS stimulation. (A) Flow cytometric analysis of ECs was performed to detectapoptotic and necrotic cells. The lower right quadrant containing cells staining with annexin V alone shows early apoptotic cells. The upperright quadrant containing cells binding both PI and annexin V shows late apoptotic or necrotic cells (B and C). The results indicate that LPSinduced early apoptosis in approximately 21% and necrosis in approximately 11% of the cells and that there was a significant differencebetween the LPS group and the control group. Treatment with MSCs or MSC-GFP reduced the percentage of early apoptotic cells but had noeffect on the percentage of late apoptotic or necrotic cells. However, when compared with ECs co-cultured with either MSCs or MSC-GFP,co-culture with MSC-ACE2 further reduced the number of early apoptotic cells and tended to decrease the number of necrotic cells. (D, E)The assessment of EC senescence, detected using a SAb-gal assay, demonstrates that MSC-ACE2 reduced the percentage of senescent ECs toa greater extent than either MSCs or MSC-GFP. (F) Measurement of cell viability using the MTT test showed that MSC-ACE2 protected cellviability to a greater extent than either the MSCs or MSC-GFP after LPS stimulation. (n¼ 3; *P< 0.05 vs. EC; #P< 0.05 vs. ECþLPS; $P< 0.05vs. ECþLPSþMSC; &P< 0.05 vs. ECþLPSþMSC-GFP).



Fig. 5. MSC-ACE2 downregulates the production of inflammatory mediators. (A–D) The gene transcription levels of inflammatorymediators including the adhesion molecules ICAM-1 and VCAM-1 and the cytokines IL-6 and TNF-a were measured by qPCR. We found thatthe expression of ICAM-1 (A), VCAM-1 (B), IL-6 (C), and TNF-a (D) mRNA in ECs was significantly elevated after LPS stimulation anddecreased almost to baseline in ECs co-cultured with MSC-ACE2. The production of inflammatory mediators also decreased in MSC andMSC-GFP groups, but no significant differences were observed, with the exception of ICAM-1. (E, F) The changes and trends in proteinexpression of IL-6 and TNF-a correlated with the gene expression results. (n¼ 3; *P< 0.05 vs. EC; #P< 0.05 vs. ECþLPS; $P< 0.05 vs.ECþLPSþMSC; &P< 0.05 vs. ECþLPSþMSC-GFP).


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promoting endothelial repair and attenuating endothelialinflammation.

The pulmonaryendothelium is a dynamic barrier and a majormetabolic organ that is critical for the regulation of pulmonaryand systemic vascular homeostasis. Numerous agents,including mechanical, chemical, or cellular factors, may causeendothelial injury, especially LPS (Pittet et al., 1997; Wort andEvans, 1999). This detrimental effect of LPS on endothelial cellsis related to the activation of the local RAS. Our and others’previous in vivo studies have found that Ang II may contributeto vascular dysfunction after LPS exposure. Additionally,treatment with an AT1R blocker or ACE inhibitors significantlyimproved endothelial dysfunction and lung injury (Wiel et al.,2004; Zhang and Sun, 2005; Lund et al., 2007; Liu et al., 2009). Inthis study, we also find that stimulation with LPS results in alarge increase in Ang II concentration and causes significant ECapoptosis and necrosis, decreases EC viability and productionof inflammatory mediators, and increases EC monolayerpermeability. When treated with MSC-ACE2, which cansecrete ACE2 protein into the culture medium, theaccumulation of Ang II was reduced and its degradationproducts Ang 1–7 increased. The endothelial cell damage wasreversed to some extent, indicating that the attenuation of LPS-induced endothelial injury was mainly due to the degradation ofAng II into Ang 1–7 by overexpressed ACE2. The results of thisin vitro study provide further confirmation that the activationof RAS is an important contributor to endothelial dysfunctionupon LPS exposure.

The underlying mechanism of LPS-induced, Ang II-mediatedEC dysfunction has not been completely elucidated. The Ang II-induced apoptosis was shown to be receptor-subtype- and cell-type-dependent. For pulmonary endothelial cells, Lee and hiscolleagues demonstrated that Ang II activates the intrinsicapoptotic pathway through its AT2R, which downregulates theantiapoptotic Bcl-2family protein Bcl-xL (Lee et al., 2010).Ang II also activates the Cdk4/Rb/E2F1 signaling pathway bydirectly enhancing the transcription of the proapoptoticprotein Bim (Kim and Day, 2012). Nonetheless, LPS-inducedEC apoptosis was not completely eliminated after treatment

with MSC-ACE2. The number of MSC-ACE2, the co-culturedtime and other Ang II-independent signaling pathway such asLPS or TNF-a may initiate the extrinsic apoptotic pathwaydirectly (Bannerman and Goldblum, 2003; Hotchkiss et al.,2009), or the LPS-activated intrinsic apoptotic pathway (Wanget al., 2007) may be involved in this effect. However, LPSinduced the expression of adhesion molecules and the releaseof proinflammatory cytokines may mainly through the Ang IIpathway because the levels of inflammatory mediators werealmost normalized to baseline after treatment with MSC-ACE2. Our previously unpublished observation and otherreport (Dandona et al., 2007) showed that Ang II exerts thiseffect by activating NF-kB via AT1R, which can be blocked withthe AT1R antagonist losartan.

Hyperpermeability is another important characteristic of ECdamage, which can promote pulmonary edema and leukocyteextravasation. In this study, we found that with the increase inAng II levels after LPS stimulation, the endothelial permeabilityincreased greatly. The ECs apoptosis/necrosis and thedisruption of the adherens junction protein VE-cadherin maycontribute to this effect. However, LPS treatment inducedapoptosis in a low percentage of ECs and had a minor effect onthe ECs viability. Conversely, LPS treatment had a significanteffect on VE-cadherin expression. After treatment with MSC-ACE2, the concentration of Ang II decreased and the level ofVE-cadherin was increased to normal levels and thehyperpermeability of ECmonolayers was completely reversed,even though a few apoptotic ECs were present. These resultsindicate that LPS increased the permeability of the ECmonolayers mainly through the activation of the Ang IIpathway, resulting the disruption of VE-cadherin junctions. Themechanism of cell junction impairment induced by Ang IIremains unclear. The increased VEGF expression maybeinvolved in this effect because VEGF is the main regulator ofendothelial permeability, and may induce the tyrosinephosphorylation, internalization, and cleavage of VE-cadherin(Dejana et al., 2008). However, further study is still needed toclarify the role of VEGF in Ang II-induced hyperpermeabilityafter LPS stimulation.

Fig. 6. MSC-ACE2 restores the integrity of EC monolayers. (A) The paracellular permeability of EC monolayers in unstimulated conditionsand following LPS stimulation was measured by the diffusion of 40-kDa dextran-FITC. It was observed that LPS greatly increased thepermeability of EC monolayers to 40-kDa dextran-FITC. ECs co-cultured with MSC-ACE2 were significantly less permeable to 40-kDadextran-FITC than ECs cultured alone. Although permeability decreased, there were no differences observed in the MSCs and MSC-GFP co-culture groups. (B) The expression of VE-cadherin as detected by western blotting was dramatically decreased in the LPS group, while VE-cadherin was restored almost to normal levels in ECs co-cultured with MSC-ACE2. (C) The expression of VEGF detected using an ELISA kitshows that LPS stimulation increased the expression of VEGF significantly. Treatment with MSC-ACE2 decreased VEGF expression. Nodifferences in VEGF expression were observed after treatment with MSC and MSC-GFP (n¼ 3; *P< 0.05 vs. EC; #P< 0.05 vs. ECþLPS;$P< 0.05 vs. ECþLPSþMSC; &P< 0.05 vs. ECþLPSþMSC-GFP).



Because Ang II plays a key role in the activation anddysfunction of the pulmonary endothelium, the regulation ofendothelial Ang II levels may inhibit this activation and promoteendothelial repair. Currently, ACE inhibitors (Molthen et al.,2012), AT1R antagonists (Zhang and Sun, 2005; Liu et al., 2009),and ACE2 (Imai et al., 2005) have been used in preclinical ALIanimal models. ACE inhibitors reduce the generation of Ang IIbut cannot counteract the harmful effects produced by Ang II.AT1R antagonists prevent the binding of Ang II to AT1R, thusinterrupting signal transduction, but cannot increase theexpression of ACE2 in ECs, which is severely decreased in acidaspiration or sepsis induced ALI (Imai et al., 2005). ACE2, thefirst homolog of ACE, inactivates Ang II directly and efficientlyeliminates the detrimental effects of Ang II. Furthermore, theACE2 degradation products Ang 1–7 also have a protectiverole in endothelial damage and lung injury (Imai et al., 2007; Imaiet al., 2010; Clarke and Turner, 2012). Thus, ACE2 is superiorto ACE inhibitors and AT1R antagonists in the regulation ofAng II. However, ACE2-mediated downregulation of Ang II inserum may influence systemic vascular homeostasis (Rentzschet al., 2008; Imai et al., 2010). The targeted localization of ACE2to injured endothelium to locally reduce Ang II concentration isextremely important.

MSC-based gene therapy is a novel therapeutic approach forseveral diseases including ALI (Mei et al., 2007) and pulmonaryarterial hypertension (Zhao et al., 2005). Delivery of aprotective gene by MSCs could overcome the limitations oftransient gene expression, host immuneresponses,inflammation, and nonspecific cell targeting by classical viral ornonviral vectors because MSCs are suggested to be minimallyimmunogenic (Le Blanc et al., 2003) and to home to injuredsites (Xu et al., 2008). Furthermore, previous studies haveshown that MSCs delivered intravenously can improveendothelial dysfunction by suppressing systemic inflammationand reducing peritoneal, renal, lung, and liver vascularpermeability through their paracrine functions (Xu et al., 2007;Nemeth et al., 2009; Zhao et al., 2009). Thus, the combinationof MSCs and the ACE2 gene may not only enhance thetherapeutic effects of MSCs but also achieve durable ACE2expression targeted to the injured endothelium, which maypromote optimal endothelial repair.

Because mesenchymal stem cells have been shown torestore the function of damaged endothelium in variouspreclinical disease models (Zhao et al., 2005; Kanki-Horimotoet al., 2006), the observed protective effects of MSCs in LPS-induced endothelial injury were expected. We found thatMSCs improve EC survival and reduce the expression ofendothelial adhesion molecules, consistent with a previousstudy (Pati et al., 2011a). However, there were no significantdifferences in the levels of TNF-a and IL-6, the recovery ofendothelium permeability or the restoration of VE-cadherinlevels. Many factors may have affected these results such as thenumber of co-cultured MSCs, the co-culture time, and theseverity of endothelial damage, among others. However,interestingly, we also found that the concentration of Ang II inthe co-culture medium does not decrease significantly whenendothelial cells are co-cultured with MSCs or MSC-GFP,despite low levels of ACE2 protein expression and secretion byMSCs and MSC-GFP. These findings indicate for the first timethat the beneficial effect of MSCs in endothelial repair may notbe mainly due to the inhibition of the Ang II pathway and thatthe benefit of non-transduced MSCs in endothelial repair isinadequate, providing an argument for MSC-based genetherapy.

To date, a number of viral (Xu et al., 2008) and nonviral(Campbell et al., 2001) vector systems have been used todeliver transgenes into MSCs. Among these methods, lentiviralvectors have the ability to integrate the exogenous gene intothe host cell genome, enabling prolonged transgene expression

(Lin and Dean, 2011), making it the most suitable gene transfervector for delivery of the ACE2 gene into MSCs. The EF1apromoter has been proven to drive strong gene expression inMSCs and is superior to other promoters including CMV andPGK (McGinley et al., 2011). Thus in this study, we used theEF1a promoter in third generation lentiviral vectors toconstruct stable and long-term ACE2 gene-overexpressingMSC lines. We found that the transduction efficiency, asassessed by detecting eGFP and ACE2 gene co-expression, wasas high as 97.8% and was well maintained over 30 passages,comparable with a previous report (McGinley et al., 2011). Theexpression of ACE2 mRNA and ACE2 protein increasedsignificantly after ACE2 gene transduction and theoverexpressed ACE2 protein possessed biological function(the degradation of Ang II into Ang 1–7), and further promotedendothelial repair and functional recovery in vitro. Based onthese findings, we propose that further studies are needed todetermine if MSCs modified with the ACE2 gene havetherapeutic potential in targeting injured endothelium andrestoring endothelial function in ALI animal models.

Conclusion

In conclusion, we have shown that LPS activates the local RASin the endothelium and induces endothelial dysfunction. Bonemarrow-derived MSCs modified with the ACE2 gene, using anEF1a promoter dependent lentiviral vector, stablyoverexpressed the ACE2 protein over an extended period oftime, maintained the biological activity to reduce Ang II levels,enhanced endothelial repair and restored endothelial functionafter LPS injury. These results encourage the evaluation of thetherapeutic potential of ACE2-overexpressing MSCs in moreclinically relevant ALI animal models. This approach mayprovide a new therapeutic strategy against LPS-induced ALI inthe future.

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (81000828, 81070049, 81170057, and81372093), the Natural Science Foundation of Jiangsu Provinceof China (BK20131302), and the Graduate Innovation Projectin Jiangsu Province of China (CXLX_0151, CXLX_0153).

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mscs modified with ace2 restore endothelial function ... · (mscs)-based therapy is a promising...

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