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TRANSLATIONAL AND CLINICAL RESEARCH: MESENCHYMAL STEM CELLS SERIES

Angiogenic Effects of Human Multipotent Stromal Cell Conditioned Medium Activate the PI3K-Akt Pathway in Hypoxic Endothelial Cells to Inhibit Apoptosis, Increase Survival, and Stimulate Angiogenesis

^aCenter for Gene Therapy, Tulane University Health Science Center, New Orleans, Louisiana, USA;
^bStem Cell Laboratory, Veterans General Hospital-Taipei, Taipei, Taiwan;
^cInstitute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan

Key Words. Bone marrow stromal cells • Hypoxia • Endothelial cell • Angiogenesis • Apoptosis • Human aortic endothelial cells Interleukin-6 • Therapeutic potential

Correspondence: Darwin J. Prockop, Ph.D., M.D., Center for Gene Therapy, Tulane University Health Sciences Center, 1430 Tulane Avenue, SL-99, New Orleans, Louisiana 70112, USA. Telephone: (504) 988-7711; Fax: (504) 988-7710; e-mail: dprocko{at}tulane.edu; or Shih-Chieh Hung, Ph.D., M.D., Department of Medical Research and Education, Veteran General Hospital-Taipei, 201, Sec2, Shih-Pai Road, Taipei, Taiwan. Telephone: 886-2-28757396; Fax: 886-2-28757396; e-mail: hungsc{at}vghtpe.gov.tw

Received on October 26, 2006; accepted for publication on May 18, 2007.

First published online in STEM CELLS EXPRESS  May 31, 2007.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Recent reports indicated that vascular remodeling and angiogenesis are promoted by conditioned medium from the cells referred to as multipotent stromal cells (MSCs). However, the molecular events triggered by MSC-conditioned medium (CdM) were not defined. We examined the effects of CdM from human MSCs on cultures of primary human aortic endothelial cells (HAECs). The CdM inhibited hypoxia-induced apoptosis and cell death of HAECs. It also promoted tube formation by HAECs in an assay in vitro. Conditioned medium from multipotent stromal cells incubated under hypoxic conditions in serum-free endothelial basal medium for 2 days (CdM^Hyp) from hypoxic culture of MSCs was more effective than conditioned medium from MSCs incubated under normoxic conditions in serum-free endothelial basal medium for 2 days from normoxic cultures of MSCs, an observation in part explained by its higher content of antiapoptotic and angiogenic factors, such as interleukin (IL)-6, vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein (MCP)-1. The effects of CdM^Hyp on hypoxic HAECs were partially duplicated by the addition of IL-6 in a dose-dependent manner; however, anti-IL-6, anti-MCP-1, and anti-VEGF blocking antibodies added independently did not attenuate the effects. Also, addition of CdM^Hyp activated the PI3K-Akt pathway; the levels of p-Akt and several of its downstream targets were increased by CdM^Hyp, and both the increase in p-Akt and the increase in angiogenesis were blocked by an inhibitor of PI3K-Akt or by expression of a dominant negative gene for PI3K. CdM^Hyp also increased the levels of p-extracellular signal-regulated kinase (ERK), but there was a minimal effect on p-signal transducer and activator of transcription-3, and an inhibitor of the ERK1/2 pathway had no effect on hypoxia-induced apoptosis of the HAECs. The results are consistent with suggestions that administration of MSCs or factors secreted by MSCs may provide a therapeutic method of decreasing apoptosis and enhancing angiogenesis.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

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The plastic adherent cells from bone marrow referred to as mesenchymal stem cells or multipotent stromal cells (MSCs) are capable of self-renewing and have the potential to differentiate into mesenchymal and nonmesenchymal tissues [1]. After systemic administration, they home to injured tissues such as heart [2], brain [3], and skeletal tissues [4] and appear to enhance regeneration of the tissues. MSCs were tested in animal models for a number of diseases and in clinical trials for osteogenesis imperfecta [5], an autosomal recessive mucopolysaccharidosis (Hurler syndrome) [6], and myocardial ischemia [7]. Promising results were reported both in animal models and in clinical trials, but the level of engraftment and differentiation of the cells was generally low. For example, i.v. administration of MSCs stimulated revascularization of ischemic tissues [2, 8], and small numbers of the cells were found to differentiate into both cardiomyocytes [2] and endothelial cells [9]. However, the effects in ischemic limb [10] and myocardial infarction [11, 12] were recently attributed to endocrine or paracrine factors produced by MSCs instead of differentiation of the cells. For these reasons, we have examined the effects of conditioned medium (CdM) from human MSCs on human endothelial cells exposed to hypoxia.

	MATERIALS AND METHODS

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Cell Culture Conditions
Frozen vials of previously standardized passage 1 MSCs [13] from three normal human volunteers were obtained from the Tulane Center for Preparation and Distribution of Adult Stem Cells (http://www.som.tulane.edu/gene_therapy/distribut.shtml). The cells (approximately 1 million) were plated on a 15-cm diameter plate (Nalgene Nunc International, Rochester, NY, http://www.nalgenunc.com) in complete culture medium (-minimal essential medium; Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com), 17% fetal bovine serum, lot selected for rapid growth of MSCs (Atlanta Biologicals Inc., Norcross, GA, http://www.atlantabio.com), 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (Gibco-BRL). After incubation for 24 hours, the viable adherent cells were lifted by incubation with 0.25% trypsin and 1 mM EDTA at 37°C for approximately 5 minutes. The cells were recovered by centrifugation and replated at 100 cells per cm², incubated in complete growth medium (CCM) with a change in medium every 2–3 days, and recovered as passage 2 cells after they reached approximately 80% confluency in 8–9 days. For some experiments, the cells were further expanded under the same conditions and used at passage 3 or 4. Human aortic endothelial cells (HAECs) harvested from the aorta of two normal males were obtained from a commercial source (catalog number 304-05a; HAOEC; Cell Applications Inc, Bath, U.K., http://www.cellapplications.com). They were positive for factor VIII and Dil-Ac-LDL uptake. Frozen vials of the cells were plated at 5,000 cells per cm² and cultured in endothelial growth medium (catalog number 211-500; Cell Applications, Inc.) that contained 2% fetal bovine serum and referred to here as eGM. When the cells reached 80%–90% confluency, they were lifted with trypsin/EDTA, diluted 1:3, and expanded to passages 3–6 under the same conditions.

Culture Under Hypoxic Conditions
Both the MSCs and HAECs were expanded under standard normoxic conditions for cell culture at 37°C in a humidified incubator containing 21% O₂, 5% CO₂, and 74% N₂. To examine hypoxia-induced apoptosis of MSCs, the cells were plated at 2 x 10⁴ cells per well in gelatin-coated 96-well microtiter plates (Nalgene Nunc International) and incubated for 1 day under standard conditions. The incubations were then continued under normoxic conditions or hypoxic conditions for 48 hours in CCM. The hypoxic conditions consisted of culture in an atmosphere-controlled CO₂ incubator (Forma Series II) containing a gas mixture composed of 94% N₂, 5% CO₂, and 1% O₂. The nitrogen was provided with a feed from a tank of liquid N_2. To examine hypoxia-induced apoptosis of HAECs, the cells were incubated under the same conditions except the culture for the first day was in serum containing eGM and then the cultures were continued either in the same medium or in fetal bovine serum-free endothelial basal medium (eBM^sf) (catalog number 210–500; Endothelial Cell Basal Medium; Cell Applications Inc).

Preparation of Conditioned Medium
MSCs were replated at 5,000 cells per cm² and incubated in CCM for 1 day. The attached cells were washed three times with phosphate-buffered saline (PBS), and the medium was replaced with eBM^sf to generate CdM that was serum free and compatible for culture of HAECs. The incubation was then continued under either normoxic conditions, to prepare conditioned medium from MSCs incubated under normoxic conditions in eBM^sf for 2 days (CdM^Nor), or hypoxic conditions, to prepare conditioned medium from MSCs incubated under hypoxic conditions in eBM^sf for 2 days (CdM^Hyp). To prepare conditioned medium from HAECs, the cells were recovered and replated at 5,000 cells per cm² and incubated in eGM for 1 day. The medium was replaced with eBM^sf and incubated under normoxic or hypoxic conditions. After 2 days, the medium was harvested as HAEC-CdM.

Assays for Apoptosis and Cell Survival
Apoptosis was assayed with a cellular dye that detects membrane alterations (phosphatidylserine flip) and stains apoptotic cells (APOPercentage; Accurate Chemical & Scientific Corporation, New York, http://www.accuratechemical.com/accuratechemical). Stained cells were assayed on a colorimetric plate reader (Benchmark Microplate Reader; Bio-Rad, Hercules, CA, http://www.bio-rad.com). Alternatively, cells were harvested by trypsin treatment and pooled with floating cells to analyze the degree of apoptosis in the entire cell population. Cells were then stained with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay (In Situ Cell Death Detection Kit; Roche Applied Science, Indianapolis, IN, http://roche-applied-science.com) or double-stained with propidium iodide (PI) and fluorescein isothiocyanate-conjugated Annexin V (Sigma, St. Louis, http://www.sigmaaldrich.com) according to the manufacturer's instructions and analyzed by flow cytometry. We defined PI–/Annexin V+ as early apoptotic cells, PI +/Annexin V+ as apoptotic cells, and PI +/Annexin V– as dead cells. Total apoptotic cells were the sum of early apoptotic cells and apoptotic cells. We also stained nuclei with 4', 6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI; 1:1,000 in PBS; Sigma) by incubation of the cells for 10 minutes in the dark at 0°C–5°C. Apoptotic cells with condensed and fragmented nuclei were assayed by epifluorescence microscopy. For the detection of the active form of caspase 3 and 9, protein lysates were prepared and detected by Western blotting assays with the primary antibodies purchased from Cell Signaling Technology (Beverly, MA, http://www.cellsignal.com) and Chemicon (Temecula, CA, http://www.chemicon.com), respectively. Cell survival was assayed by trypan blue exclusion of cells counted by phase microscopy. Alternatively, cell survival was assayed with a nuclear dye (CyQuant; Molecular Probes, Eugene, OR, http://probes.invitrogen.com) and a fluorescence plate reader (FLUORstar OPTIMA; BMG Labtech Inc., Durham, NC, http://www.bmglabtech.com).

Assays of Cytokines
Samples of CdM^Nor or CdM^Hyp were centrifuged at 1,500g for 10 minutes to remove cell debris and concentrated 40x by tangential flow dialysis (Bio-Rad). Membranes from a human protein cytokine array kit (Human Cytokine Antibody Array; RayBiotech, Norcross, GA, http://www.raybiotech.com) were blocked with a blocking buffer and incubated with 1 ml of the concentrated CdM at room temperature for 2 hours. The membranes were then washed three times with wash buffer I and twice with wash buffer II, both at room temperature and 5 minutes for each time of wash, and assayed by chemiluminescence. To verify the results, the same samples and concentrated HAEC-CdM were assayed with enzyme-linked immunosorbent assay (ELISA) kits for interleukin (IL)-6 (RayBiotech), monocyte chemoattractant protein (MCP)-1 (RayBiotech), and vascular endothelial growth factor (VEGF) (Endogen, Woburn, MA, http://www.endogen.com).

Real-Time Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated (RNAqueous; Ambion, Austin, TX, http://www.ambion.com) from MSCs and HAECs and reverse transcribed to cDNA by using M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). The assays were performed using an automated instrument (ABI PRISM 7700; Applied Biosystems, Bedford, MA, http://www.appliedbiosystems.com) and a commercial kit (TaqMan 2-Step; Applied Biosystems). A 50-µl volume reaction was used containing 200 ng of the sample cDNA; TaqMan buffer; 200 mmol/l each of deoxy-ATP, deoxycytidine triphosphate, and deoxyguanosine triphosphate; 400 mmol/l deoxyuridine triphosphate; 5.5 mmol/l magnesium chloride; 0.025 U/ml polymerase (AmpliTaq Gold DNA Polymerase; Applied Biosystems); 0.01 U/l N-glycosylase (AmpErase; Applied Biosystems); 200 nmol/l forward primers and reverse primers; and 100 nmol/l fluorescent probe. The polymerase chain reaction (PCR) cycling conditions included an initial phase of 2 minutes at 50°C followed by 10 minutes at 95°C for the N-glycosylase reaction, 40 cycles of 15 minutes at 95°C, and 1 minute at 60°C. A commercial supplier (Biosource International, San Jose, CA, http://www.biosource.com) was used for primers for IL-6 and controls (GHC0064) with IL-6 fluorescence resonance energy transfer (FRET) probe (GHC0065); primers for ß-actin and control (GHO0124) with ß-actin FRET probe (GHO0125); and primers for VEGF and control (GHG0114) with VEGF FRET probe (GHG0115). MCP-1 primers were designed using designated software (TaqMan probes; Primer Express; Applied Biosystems): forward, 5'-c ccagtcacctgctgttata-3'; reverse, 5'-tgctggtgattcttctata-3'; and MCP-1 FRET probe, aggaagatctcagtgcagaggct.

Cell Proliferation Assays
HAECs were plated at 2 x 10⁴ cells per well in gelatin-coated 96-well microtiter plates (Nalgene Nunc International) and incubated in eGM. After 24 hours, eGM was replaced with eBM^sf or CdM^Hyp with or without PI3K inhibitor (LY294002; Cell Signaling Technology), and cells were exposed to hypoxia for another 16 hours. Cell number was assayed with a nuclear dye (CyQuant).

Tube Formation Assay
Ninety-six-well microtiter plates were coated with 100 µl per well of Matrigel or Growth Factor Reduced Matrigel (GFR-Matrigel; BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). HAECs were suspended in basal medium or CdM with and without IL-6-, MCP-1-, or VEGF-neutralizing antibodies and plated at 4 x 10⁴ per cm² in triplicate. Cells were incubated under hypoxic conditions. After 24–48 hours, the formation of tube-like structures was examined microscopically. Photographs were taken of each well, and the lengths of >100 tubes were measured and assayed with a software program (Photoshop; Adobe, San Jose, CA, http://www.adobe.com).

Effects of Neutralizing Antibodies to IL-6, MCP-1, or VEGF
CdM was combined with neutralizing antibodies from a commercial source (R&D Systems, Hornby, Ontario, Canada, http://www.rndsystems.com) against IL-6 (MAB206), MCP-1 (MAB279), and VEGF (MAB293). The final concentrations of the antibodies were 10 µg/ml and therefore were 25- to 1,000-fold greater than the concentrations for half-maximal inhibition of 10 ng/ml of the recombinant proteins (R&D Systems). The mixtures were then added to cultures of HAECs for incubation for 1–2 days.

Western Blotting Assays
HAECs were incubated in hypoxia for 1 day in eBM^sf with or without IL-6 or in CdM^Hyp with or without an inhibitor of PI3K (Cell Signaling Technology) or the inhibitor of mitogen-activated protein kinase kinase (MAPKK), also known as MAPK/extracellular signal-regulated kinase (ERK) kinase or MEK kinase (PD98059; Cell Signaling Technology). The cells were lysed in buffer (M-PER or NE-PER; Pierce Biotechnology, Rockford, IL, http://www.piercenet.com) supplemented with protease inhibitor cocktail (Halt; Pierce Biotechnology), and protein concentration was determined (Micro BCA Kit; Pierce Biotechnology). The cell lysate (20 µg of protein) was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (NuPAGE; 4%–12% Bis-Tris gels; Invitrogen). The sample was transferred to a filter (Immobilon P; Millipore, Bedford, MA, http://www.millipore.com) by electroblotting (XCell II Blot Module; Invitrogen). The filter was blocked for 1 hour with Tris-buffered saline (TBS) containing 5% nonfat dry milk and 0.05% Tween 20 and then incubated overnight at 4°C with the primary antibodies. The primary antibodies purchased from Cell Signaling Technology were against p-Akt (1:1,000; Ser473), Akt (1:1,000), p-signal transducer and activator of transcription (STAT)-3 (1:1,000; Ser727), p-ERK (1:2,000; Thr202/Tyr204), ERK (1:1,000), p-glycogen synthase kinase (GSK)-3ß (1:1,000; Ser9), p-forkhead transcription factors (FKHR) (1:1,000; Ser256), p-acute-lymphocytic-leukemia-1 fused gene from chromosome X (AFX) (1:1,000; Ser193), and p-endothelial nitric oxide synthase (eNOS) (1:1,000; Ser1177). The filter was washed three times for 10 minutes each with TBS containing 0.1% Tween 20. Bound primary antibodies were detected by incubating for 1 hour with horseradish peroxidase-conjugated goat anti-mouse (1:3,000) or anti-rabbit IgG (1:2,000; BD Biosciences). The filter was washed and developed using a chemiluminescence assay (West-Femto Detection Kit; Pierce Biotechnology).

Adenoviral Vectors and Transfection
Adenoviral vectors expressing a dominant negative mutant of the p85 subunit of PI3K (AdGFP-p85) and constitutively activated mutant of PI3K (AdGFP-p110) were kindly provided by Dr. Patrice Delafontaine (Department of Medicine, Tulane University Health Sciences Center, New Orleans). An adenoviral vector encoding eGFP gene (green fluorescent protein) was used as a control vector. The HAECs were transduced at a multiplicity of infection of 100 at 48 hours prior to the experiment. The transduction efficiency was over 90% as assayed by green fluorescent protein-positive cells by epifluorescence microscopy.

Statistics
Data and results are reported as means ± SD. Statistical comparisons between two groups were performed with unpaired Student's t test. Pearson product moment correlation coefficients were obtained to estimate linear correlations among variables.

	RESULTS

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Effect of CdM on Hypoxia-Induced Apoptosis and Cell Death
To test whether hypoxia induces apoptosis, MSCs and HAECs were plated at 2 x 10⁴ cells per well in gelatin-coated 96-well plates and cultured in CCM and eGM, respectively. The next day, the cells were incubated in air or hypoxia for 48 hours in the same medium with a decrease in serum concentration. The decrease of serum concentration increased the level of apoptosis both in MSCs and HAECs. With MSCs, exposure to hypoxia induced a significant decrease in apoptosis at all tested serum concentrations (Fig. 1A). With HAECs, exposure to hypoxia further increased the apoptosis both in serum-containing and serum-free media (Fig. 1A). We also found that MSCs survived and proliferated in hypoxia for long-term culture; however, most of the HAECs and tumor cell lines did not survive under hypoxic conditions. Since the inhibitor of protein synthesis, cycloheximide at 20 µM or PI3K inhibitor LY294002 at 20 µM, did not alter the susceptibility of MSCs to hypoxia-induced apoptosis (supplemental online Fig. 1), the mechanisms by which MSCs protect themselves from hypoxia-induced apoptosis do not need new protein synthesis and are not mediated via the PI3K-Akt pathway.

View larger version (34K): [in this window] [in a new window]   Figure 1. Conditioned medium inhibits hypoxia-induced apoptosis and increases survival of HAECs. (A): MSCs were cultured for 2 days under normoxic or hypoxic conditions in 16%, 5%, or 0% FBS-supplemented -minimal essential medium. HAECs were grown under normoxic or hypoxic conditions in endothelial cell growth medium with or without serum. Apoptosis assayed by APOPercentage assay method. Values are mean ± SD of n = 3. * p < .05, ** p < .01 versus control under normoxic conditions. (B–E): HAECs were incubated under hypoxic conditions for 16 hours and in eBMsf, CdMNor, CdMHyp, or in a 1:1 mixture of CdMNor/Hyp. (B): Apoptosis assayed by APOPercentage assay method. (C): Survival of HAECs assayed by trypan blue exclusion. Values are mean ± SD of n = 3; * p < .05, ** p < .01 versus cells in eBMsf. (D): Apoptosis assayed by TUNEL assay. (E): The detection of activated caspase 3 and 9 and actin by Western blotting assays. Abbreviations: BM, basal medium; CdMHyp, conditioned medium prepared under hypoxic conditions; CdMNor, conditioned medium prepared under normoxic conditions; CdMNor/Hyp, conditioned medium prepared under normoxic or hypoxic conditions; eBMsf, serum-free endothelial basal medium; eGM, endothelial growth medium containing 2% fetal bovine serum; FBS, fetal bovine serum; HAEC, human aortic endothelial cell; Hyp, hypoxic; MSC, multipotent stromal cell; Nor, normoxic; OD, optical density; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

CdM Promotes Angiogenesis by HAECs
In the presence of GFR-Matrigel, hypoxic HAECs did not demonstrate tube formation (Fig. 2A). In contrast, the replacement of eBM^sf with CdM^Hyp or CdM^Nor stimulated tube formation (Fig. 2A). Since the tube formation is incomplete in CdM^Nor and complete in CdM^Hyp, the angiogenic effect was therefore greater with CdM^Hyp than with CdM^Nor. In the presence of Matrigel containing growth factors, the hypoxic HAECs developed tubules when incubated either in eBM^sf or in CdM (Fig. 2B). The tube length was 172 ± 92 µm, 207 ± 82 µm, and 265 ± 109 µm in eBM^sf, in CdM^Nor, and in CdM^Hyp, respectively (n > 40). The tube length was slightly greater in CdM than in eBM^sf and was significantly greater in CdM^Hyp than in CdM^Nor (p < .01, Student's t test).

View larger version (92K): [in this window] [in a new window]   Figure 2. Conditioned medium promotes tube formation by hypoxic human aortic endothelial cells in Growth Factor Reduced Matrigel (GFR-Matrigel) assay. (A): HAECs suspended in eBMsf, CdMNor, or CdMHyp were seeded on GFR-Matrigel in 96-well plates at 4 x 104 cells per well and cultured in hypoxic conditions for 48 hours. The cells were photographed by phase microscopy at x4 magnification. (B): Same as (A) but cells seeded in standard Matrigel. Abbreviations: CdMHyp, conditioned medium prepared under hypoxic conditions; CdMNor, conditioned medium prepared under normoxic conditions; eBMsf, serum-free endothelial basal medium.

View larger version (51K): [in this window] [in a new window]   Figure 3. Release of cytokines from MSCs in normoxic and hypoxic conditions. (A): Human protein cytokine array. CdMNor and CdMHyp were blotted onto two membranes, which are arrayed in duplicate with 120 human cytokines. The rectangles highlight cytokines higher in the CdMHyp than in CdMNor. (B–D): Enzyme-linked immunosorbent assays for IL-6, MCP-1, and VEGF from conditioned medium from MSCs and HAECs. ** p < .01 versus MSC-CdM or HAEC-CdM prepared under normoxic conditions or hypoxic conditions. (E): Real-time RT-PCR for mRNAs for IL-6, MCP-1, and VEGF. Abbreviations: Acrp30, adipocyte complement-related protein of 30 kDa; CdM, conditioned medium; CdMHyp, conditioned medium prepared under hypoxic conditions; CdMNor, conditioned medium prepared under normoxic conditions; eBMsf, serum-free endothelial basal medium; HAEC, human aortic endothelial cell; Hyp, hypoxic; IGFBP, insulin-like growth factor binding protein; IL, interleukin; MCP, monocyte chemoattractant protein; MSC, multipotent stromal cell; NAP, neutrophil activating protein; Nor, normoxic; RT-PCR, reverse transcription-polymerase chain reaction; TIMP, tissue inhibitor of metalloproteinase; uPAR, urokinase plasminogen activator receptor; VEGF, vascular endothelial growth factor.

CdM Induces Akt Phosphorylation and Increased Activation of Downstream Targets of Akt
We next tested the hypothesis that one or more of the active factors in CdM may activate the PI3K-Akt signaling pathway that plays a crucial role in survival, proliferation, microvascular permeability, and angiogenesis in HAECs and other endothelial cells [15, 16]. Incubation of HAECs in CdM^Hyp induced phosphorylation of Akt (Ser473), ERK, and STAT-3 (Fig. 4A). The effects occurred as early as 5 minutes after the addition of CdM^Hyp, and they were not reproduced by the addition of IL-6 to HAECs cultured in eBM^sf. The results further supported the conclusion that the effects of CdM^Hyp on HAECs were not entirely explained by the presence of IL-6. To further support the observation that CdM^Hyp activates the PI3K-Akt pathway, we next examined whether CdM^Hyp also induced phosphorylation of downstream targets in the signaling cascade. HAECs incubated in hypoxia and eBM^sf had either undetectable or low constitutive levels of phosphorylated GSK-3ß, FKHR, and AFX (Fig. 4B). Incubation in CdM^Hyp induced phosphorylation of GSK-3ß, FKHR, and AFX in a time-dependent manner. Importantly, the induced phosphorylation of Akt and its downstream targets was inhibited by pretreatment for 1 hour with the PI3K inhibitor LY294002 (10 µM). In contrast, the addition of the PI3K inhibitor LY294002 did not inhibit the phosphorylation of ERK, suggesting that the PI3K-Akt pathway was not targeting to the MEK/ERK pathway. Hypoxic HAECs cultured in eBM^sf had a high level of phosphorylated eNOS, another downstream target of Akt (Fig. 4B). CdM^Hyp slightly increased the levels of p-eNOS, and the effect was partially blocked by LY294002.

View larger version (66K): [in this window] [in a new window]   Figure 4. Conditioned medium activates Akt and its downstream targets via PI3K-Akt signaling in human aortic endothelial cells (HAECs). (A): HAECs were incubated under hypoxia in serum-free endothelial basal medium (eBMsf) for 18 hours followed by incubation in CdMHyp for 5 and 15 minutes or in eBMsf with IL-6 (10 or 100 ng/ml) for 15 minutes. Cells were lysed, electrophoresed, and immunoblotted. (B): HAECs were incubated under hypoxic conditions first in eBMsf for 18 hours, then in eBMsf with either vehicle (0.01% DMSO) or LY294002 (10 µM) in vehicle for 1 hour, and finally in CdMHyp for 0–30 minutes. Cells were lysed and immunoblotted. Abbreviations: AFX, acute-lymphocytic-leukemia-1 fused gene from chromosome X; CdMHyp, conditioned medium prepared under hypoxic conditions; DMSO, dimethyl sulfoxide; eNOS, endothelial nitric oxide synthase; ERK, extracellular signal-regulated kinase; FKHR, forkhead transcription factors; GSK, glycogen synthase kinase; IL, interleukin; min, minutes; STAT, signal transducer and activator of transcription.

View larger version (31K): [in this window] [in a new window]   Figure 5. LY294002 inhibits CdMHyp induced survival and angiogenesis. Human aortic endothelial cells (HAECs) were incubated for 16 hours under hypoxia in eBMsf or in CdMHyp without or with the addition of the PI3K inhibitor LY294002 (2, 10, 50 µM). (A): Apoptosis and cell death were evaluated quantitatively by flow cytometry after PI/Annexin V staining (medium/LY294002 at µM). Data are gated for apoptotic cells (PI± and Annexin+, red) and dead cells (PI+ and Annexin V–, blue). (B): Percentage of total apoptotic cells and dead cells in each condition. (C): Survival of HAECs by counting the total cell number with CyQuant cell assay. Data represent mean ± SD of three independent experiments. ** p < .01 versus cells in eBMsf; # p < .05, ## p < .01 versus cells in CdMHyp. (D): Tube formation by HAECs under hypoxia for 24 hours in Growth Factor Reduced-Matrigel and CdMHyp in the presence or absence of LY294002 (2, 10, or 50 µM). The addition of LY294002 at all tested concentrations blocked tube formation. Abbreviations: CdMHyp, conditioned medium prepared under hypoxic conditions; eBMsf, serum-free endothelial basal medium; Hyp, hypoxic; LY, LY294002; Nor, normoxic; PI, propidium iodide.

View larger version (39K): [in this window] [in a new window]   Figure 6. Overexpression and downregulation of PI3K-Akt signaling. (A): Western blot assays of human aortic endothelial cells (HAECs) at day 3 after adenoviral transduction. HAECs transfected with the activated PI3K gene (AdGFP-p110) and cultured in hypoxia and eBMsf showed enhanced phosphorylation of Akt and its downstream targets FKHR and GSK-3ß but not ERK. HAECs transfected with the dominant negative gene (AdGFP-p85) and cultured in CdMHyp had decreased levels of Akt and its downstream targets FKHR and GSK-3ß but not ERK. (B): HAECs were cultured in eBMsf under hypoxic conditions for 16 hours and apoptosis was assayed by DAPI staining. Images of cells expressing GFP fluorescence (green) and nuclei stained with DAPI (blue); x600. HAECs transfected with AdGFP-p110 and cultured in eBMsf showed no apoptosis. HAECs transduced with AdGFP-p85 and cultured in CdMHyp showed apoptotic cells with nuclear condensation. (C): HAECs transduced with AdGFP-p110 and cultured in eBMsf and hypoxia had an increase in tube formation in Growth Factor Reduced-Matrigel as compared with cells transduced with AdGFP. HAECs transduced with AdGFP-p85 had a decrease in tube formation as compared with cells infected with AdGFP. Photomicrographs of identical fields by phase and GFP fluorescence are shown. Abbreviations: AFX, acute-lymphocytic-leukemia-1 fused gene from chromosome X; CdMHyp, conditioned medium prepared under hypoxic conditions; DAPI, 4,6-diamidino-2-phenylindole; eBMsf, serum-free endothelial basal medium; ERK, extracellular signal-regulated kinase; FKHR, forkhead transcription factors; GFP, green fluorescent protein; GSK, glycogen synthase kinase; O/E, overexpression.

	DISCUSSION

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Incubation of hypoxic endothelial cells with CdM^Hyp also increased the phosphorylation of ERK and briefly produced a small increase in the phosphorylation of STAT-3. However, inhibition of PI3K with LY294002 did not decrease phosphorylation of ERK, and inhibition of ERK activity with the MAPKK inhibitor PD98059 had no effect on the suppression of apoptosis in hypoxic endothelial cells. Therefore, it is unlikely that activation of the ERK pathway made a major contribution to the observed effects.

An incidental observation was that the effects of CdM^Hyp on endothelial cells were not explained by activation of eNOS, another downstream target of Akt that was previously shown to mediate stromal cell-derived factor-1 enhanced ischemic vasculogenesis and angiogenesis [24]. The results support previous suggestions [10, 11] that MSCs or the components of CdM can potentially provide therapeutic benefits in pathological conditions that involve endothelial cells, such as wound repair, angiogenesis in ischemic tissues, microvascular permeability, vascular protection, and hemostasis.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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This work was supported in part by Grants from the NIH (HL73755 and HL075161), the NSC of Taiwan (NSC95-2314-B-075-047-MY3), HCA the Healthcare Company, and the Louisiana Gene Therapy Research Consortium. We thank Dr. Patrice Delafontaine (Division of Cardiology, School of Medicine, Tulane University Health Sciences Center, New Orleans) for providing the adenoviral vectors.

	REFERENCES

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1. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276:71–74.[Abstract/Free Full Text]

2. Nagaya N, Fujii T, Iwase T et al. Intravenous administration of mesenchymal stem cells improves cardiac function in rats with acute myocardial infarction through angiogenesis and myogenesis. Am J Physiol Heart Circ Physiol 2004;287:H2670–2676.[Abstract/Free Full Text]

3. Iihoshi S, Honmou O, Houkin K et al. A therapeutic window for intravenous administration of autologous bone marrow after cerebral ischemia in adult rats. Brain Res 2004;1007:1–9.[CrossRef][Medline]

4. Murphy JM, Fink DJ, Hunziker EB et al. Stem cell therapy in a caprine model of osteoarthritis. Arthritis Rheum 2003;48:3464–3474.[CrossRef][Medline]

5. Horwitz EM, Prockop DJ, Fitzpatrick LA et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999;5:309–313.[CrossRef][Medline]

6. Koc ON, Day J, Nieder M et al. Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH). Bone Marrow Transplant 2002;30:215–222.[CrossRef][Medline]

7. Wollert KC, Meyer GP, Lotz J et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: The BOOST randomised controlled clinical trial. Lancet 2004;364:141–148.[CrossRef][Medline]

8. Chen J, Zhang ZG, Li Y et al. Intravenous administration of human bone marrow stromal cells induces angiogenesis in the ischemic boundary zone after stroke in rats. Circ Res 2003;92:692–699.[Abstract/Free Full Text]

9. Silva GV, Litovsky S, Assad JAR et al. Mesenchymal stem cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model. Circulation 2005;111:150–156.[Abstract/Free Full Text]

10. Kinnaird T, Stabile E, Burnett MS et al. Local delivery of marrow-derived stromal cells augments collateral perfusion through paracrine mechanisms. Circulation 2004;109:1543–1549.[Abstract/Free Full Text]

11. Gnecchi M, He H, Liang OD et al. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nat Med 2005;11:367–368.[CrossRef][Medline]

12. Tang YL, Zhao Q, Qin X et al. Paracrine action enhances the effects of autologous mesenchymal stem cell transplantation on vascular regeneration in rat model of myocardial infarction. Ann Thorac Surg 2005;80:229–236.[Abstract/Free Full Text]

13. Sekiya I, Larson BL, Smith JR et al. Expansion of human adult stem cells from bone marrow stroma: Conditions that maximize the yields of early progenitors and evaluate their quality. STEM CELLS 2002;20:530–541.[Abstract/Free Full Text]

14. Schwab G, Siegall CB, Aarden LA et al. Characterization of an interleukin-6-mediated autocrine growth loop in the human multiple myeloma cell line, U266. Blood 1991;77:587–593.[Abstract/Free Full Text]

15. Franke TF, Kaplan DR, Cantley LC et al. Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science 1997;275:665–668.[Abstract/Free Full Text]

16. Jiang BH, Zheng JZ, Aoki M et al. Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells. Proc Natl Acad Sci U S A 2000;97:1749–1753.[Abstract/Free Full Text]

17. Gruber R, Kandler B, Holzmann P et al. Bone marrow stromal cells can provide a local environment that favors migration and formation of tubular structures of endothelial cells. Tissue Eng 2005;11:896–903.[CrossRef][Medline]

18. Majumdar MK, Thiede MA, Haynesworth SE et al. Human marrow-derived mesenchymal stem cells (MSCs) express hematopoietic cytokines and support long-term hematopoiesis when differentiated toward stromal and osteogenic lineages. J Hematother Stem Cell Res 2000;9:841–848.[CrossRef][Medline]

19. Takai K, Hara J, Matsumoto K et al. Hepatocyte growth factor is constitutively produced by human bone marrow stromal cells and indirectly promotes hematopoiesis. Blood 1997;89:1560–1565.[Abstract/Free Full Text]

20. Labouyrie E, Dubus P, Groppi A et al. Expression of neurotrophins and their receptors in human bone marrow. Am J Pathol 1999;154:405–415.[Abstract/Free Full Text]

21. Kim HS, Skurk C, Thomas SR et al. Regulation of angiogenesis by glycogen synthase kinase-3beta. J Biol Chem 2002;277:41888–41896.[Abstract/Free Full Text]

22. Kops GJ, Dansen TB, Polderman PE et al. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 2002;419:316–321.[CrossRef][Medline]

23. Potente M, Urbich C, Sasaki K et al. Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularization. J Clin Invest 2005;115:2382–2392.[CrossRef][Medline]

24. Hiasa K, Ishibashi M, Ohtani K et al. Gene transfer of stromal cell-derived factor-1alpha enhances ischemic vasculogenesis and angiogenesis via vascular endothelial growth factor/endothelial nitric oxide synthase-related pathway: Next-generation chemokine therapy for therapeutic neovascularization. Circulation 2004;109:2454–2461.[Abstract/Free Full Text]

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