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. Author manuscript; available in PMC: 2014 Jan 1.
Published in final edited form as: Dev Biol. 2012 Oct 24;373(1):163–175. doi: 10.1016/j.ydbio.2012.10.020

VEGF-mediated phosphorylation of eNOS regulates angioblasts and embryonic endothelial cell proliferation

Carmine Gentile 1, Robin C Muise-Helmericks 1, Christopher J Drake 1,2
PMCID: PMC3523730  NIHMSID: NIHMS418618  PMID: 23103584

Abstract

To evaluate potential roles of nitric oxide (NO) in the regulation of the endothelial lineage and neovascular processes (vasculogenesis and angiogenesis) we evaluated endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (p-eNOS) expression in 7.2–8.5 days post-coitum (dpc) mouse embryos. Analysis revealed that p-eNOS(S1177) but not P-eNOS(S617) or P-eNOS(T495) was expressed in a subpopulation of angioblasts (TAL-1+/Flk-1+/CD31−/CD34−/VE-Cadherin−) at 7.2 dpc. A role of the VEGF/Akt1/eNOS signaling pathway in the regulation of the endothelial cell (EC) lineage was suggested by the strong correlation observed between cell division and p-eNOS(S1177) expression in both angioblasts and embryonic endothelial cells (EECs, TAL-1+/Flk-1+/CD31+/CD34+/VE-Cadherin+). Our studies using Akt1 null mouse embryos show a reduction in p-eNOS(S1177) expression in angioblast and EECs that is correlated with a decrease in endothelial cell proliferation and results in changes in VEGF-induced vascular patterning. Further, we show that VEGF-mediated cell proliferation in Flk-1+ cells in allantoic cultures is decreased by pharmacological inhibitors of the VEGF/Akt1/eNOS signaling pathways. Taken together, our findings suggest that VEGF-mediated eNOS phosphorylation on Ser1177 regulates angioblast and EEC division, which underlies the formation of blood vessels and vascular networks.

Keywords: Angioblasts, Endothelial Nitric Oxide Synthase, VEGF, Proliferation, Vasculogenesis, Embryo

INTRODUCTION

The production of NO in endothelial cells (ECs) is maintained during homeostasis through endothelial Nitric Oxide Synthase (eNOS), which is regulated by mechanical stimuli, cytokines and growth factors (Balligand et al., 2009; Dudzinski and Michel, 2007; Kolluru et al., 2010). eNOS acts to regulate a number of biological activities that are relevant to neovascular process, including gene expression (Braam et al., 2004; Illi et al., 2008), stem cell/mesoderm differentiation (Guthrie et al., 2005; Spallotta et al.), cell growth (Garcia-Cardena and Folkman, 1998; Hsieh et al.; Iwakiri; Oliveira et al., 2008; Papapetropoulos et al., 1997) and matrix remodeling (Chen and Wang, 2004). Despite the important roles played by NO in adult ECs, its potential role(s) during embryonic vascular development has not been extensively studied. Nath, et al., (2004) have shown that eNOS is expressed at 8.0 days post-coitum (dpc) in ECs of the forming yolk sac vascular plexus, and that inhibition of NO using NG-monomethyl-L-arginine (L-NMMA), altered yolk sac vascularization.

The differentiation from mesoderm to angioblasts to embryonic endothelial cells (EECs) is associated with the temporal expression of the transcription factor SCL/TAL-1 (Drake et al., 1997; Kallianpur et al., 1994), the receptor tyrosine kinase VEGFR2 (also known as Flk-1/KDR) (Shalaby et al., 1995) and cell–cell adhesion molecules such as CD31/PECAM (Baldwin et al., 1994), CD34 (Wood et al., 1997; Young et al., 1995) and VE-Cadherin (Lampugnani et al., 1992; Vittet et al., 1997). Using the expression of key markers we have previously defined angioblasts as TAL-1+/Flk-1+/CD31−/CD34−/VE-Cadherin− cells and EECs as TAL-1+/Flk-1+/CD31+/CD34+/VE-Cadherin+ cells (Drake and Fleming, 2000). Given the prominent role of vascular endothelial growth factor (VEGF) in the regulation of neovascular processes and the fact that VEGF has been shown to regulate NO production, it is reasonable that NO acts downstream of the VEGF/Flk-1 signaling pathway in the EC lineage. VEGF signaling has been shown to regulate NO levels by both increasing eNOS mRNA expression levels (Papapetropoulos et al., 1997) and by post-transcriptional modifications (Gelinas et al., 2002), in part mediated by VEGF-dependent activation of the PI3 Kinase/Akt pathway (Garcia-Cardena and Folkman, 1998; Lin and Sessa, 2006; Papapetropoulos et al., 1997). As part of a multimolecular complex (the “signalosome”) following VEGF stimulation, activated Akt1 acts to phosphorylate eNOS on Ser617 and Ser1177, whereas phosphorylation of Thr495 by PKC has been shown to have an inhibitory effect on NO production (Lin et al., 2003; Sessa, 2004). Moreover, eNOS can be phosphorylated on Ser1177 by multiple kinases (i.e., Akt1, AMPK, PKA, PKG, and PKC (Lin et al., 2003; Schleicher et al., 2009; Sessa, 2004). The phosphorylated isoform of eNOS on Ser1177, p-eNOS(S1177), acts to convert L-Arginine to L-Citrulline and NO (Eglen, 2006; Forstermann and Sessa, 2011; Wyatt et al., 2004). This activity is enhanced by HSP90 binding to p-eNOS(S1177), which inhibits the dephosphorylation of p-eNOS(S1177) (Fontana et al., 2002). Additionally, HSP90 association with p-eNOS(S1177) has been shown to enhance the S-nitrosylation of proteins by providing a more favorable steric setting for the enzyme and the target protein (Fontana et al., 2002).

A role for eNOS in the regulation of VEGF-mediated neovascular processes is suggested by the study of Murohara, et al., (1998) which demonstrated that in animal models of hindlimb ischemia eNOS modulates angiogenesis in response to tissue ischemia. Mechanistically the study of Taylor, et al., (2010) showing that VEGF/Flk-1 signaling through Akt1, has a mitogenic effect on ECs, and the study of Dai, et al., (2010) showing that mice deficient in eNOS exhibited decreased expression of 40 out of 44 cell cycle related genes following femoral artery ligation suggest that VEGF-mediated NO signaling acts to regulate angiogenesis by controlling EC proliferation.

Given the prominent role of VEGF/Flk-1 signaling in the development of the EC lineage and its role in the regulation of eNOS we hypothesized that VEGF/Akt1-mediated NO signaling may also act to control angioblast and EEC proliferation, which is critical for the proper patterning of blood vessel. Our in vivo studies using wild type and Akt1 null murine embryos show that the expression of p-eNOS(S1177) is critical for cell proliferation in angioblasts and EECs and in dependent on Akt1 signaling pathway, whereas our in vitro studies using wild type, heterozygous and homozygous Akt1 allantoic cultures show that the measured changes in p-eNOS(S1177) expression and NO production following VEGF treatment are responsible for the alteration in vascular patterning via VEGF/Akt1 signaling pathway.

MATERIALS AND METHODS

Drugs and Reagents

Primary antibodies: rabbit polyclonal antimouse TAL-1/SCL was obtained from Stephen J. Brandt (Vanderbilt University and Veterans Affairs Medical Center, Nashville, TN, USA). Rat monoclonal antimouse CD31/PECAM, rat monoclonal antimouse CD102/ICAM2, mouse monoclonal antihuman eNOS, rat monoclonal antimouse Flk-1 and rat monoclonal antimouse FITC-conjugated Flk-1 were purchased from BD Pharmingen (San Diego, CA, USA). Rabbit polyclonal antimouse p-eNOS(S1177), was purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal antimouse phospho-histone H3-Ser10, rabbit polyclonal antibovine p-eNOS(S617) and rabbit polyclonal antibovine p-eNOS(T495) were purchased from Millipore (Billerica, MA, USA). Rabbit polyclonal antimouse iNOS was purchased from Abcam (Cambridge, MA, USA). Secondary antibodies: donkey anti-rabbit, antimouse and anti-rat secondary fluorochrome-conjugated antibodies (Jackson Immunological Research Labs, Inc., West Grove, PA, USA). Growth factors and inhibitors: Vascular Endothelial Growth Factor-165 (VEGF-A, used at 50 ng/ml) and recombinant mouse sFlt-1 (used at 3 μg/ml) were purchased from R&D Systems (Minneapolis, MN, USA). Drugs: L-NIO (N5-(1-iminoethyl)-L-ornithine, dihydrochloride, used at 100 μM) was purchased from EMD Chemicals (Rockland, MA, USA). Resveratrol (3,5,4′-trihydroxy-trans-stilbene, used at 20 μM) and LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one, used at 20 μM) were purchased from Sigma-Aldrich (St. Louis, MO, USA). SU1498 (used at 10 μM) was purchased by Calbiochem (La Jolla, CA, USA).

Whole-mount immunolabeling

Isolation of mouse embryos was performed as previously described (Drake and Fleming, 2000). Briefly, CD1/ICR pregnant mice (Harlan Laboratories, Indianapolis, IN, USA) and Akt1 knockout mice (kindly provided by Dr. Philip N. Tsichlis, Molecular Oncology Research Institute, Tufts-New England Medical Center, Boston, MA, USA (Mao et al., 2007) and backcrossed onto C57Bl6 background) were sacrificed by cervical dislocation and embryos at 7.0 to 8.5 days post-coitum (dpc) (0.5dpc, plug date) were dissected free of the uterine muscle and decidua and placed into embryonic phosphate-buffered saline (EPBS, 4°C). Reichert’s membrane and the ectoplacental cone were removed and the embryos flattened by cutting the yolk sac either lateral (7.0 dpc) or perpendicular to the embryonic axis (8.5 dpc) and removing the amniotic sac. Flattened embryos were fixed in 4% paraformaldehyde for 60 minutes and permeabilized in phosphate buffered saline/0.01% sodium azide (PBSA) containing 0.02% Triton-X-100 (60 minutes). Embryos were then exposed to a blocking solution (3% bovine serum albumin (BSA)/PBSA, 5% donkey serum, 0.1% Triton-X-100) and then to appropriate primary (SCL/TAL-1 10 μg/ml, eNOS 5 μg/ml, Flk-1 15 μg/ml, CD31 15 μg/ml, p-eNOS 15 μg/ml, phospho-histone H3-Ser10 20 μg/ml) and secondary (10 μg/ml) antibodies (overnight at 4°C). For eNOS and p-eNOS immunolabeling, embryos were first exposed to eNOS primary antibodies followed by secondary antibodies (overnight, 4°C), washed three times for 20 minutes and then exposed to p-eNOS primary and secondary antibodies (overnight, 4°C). Embryos were incubated with Hoechst stain (Invitrogen, Carlsbad, CA, USA) for 1 hour at room temperature. Embryos were mounted ventral side up using Fluorogel (Electron Microscopy Sciences, Hatfield, PA, USA). All aspects of animal research were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, NIH Publication No. 86-23, 1985 edition or succeeding revised editions) and with guidelines set by the Institutional Animal Care and Use Committee at the Medical University of South Carolina.

Allantoic isolation, culture and immunolabeling

For experiments conducted as part of this study we utilized two modified allantoic culture systems (Argraves et al., 2002). For morphometric analyses, allantoides were excised from embryos isolated as described above, washed in PBSA (4°C) and then transferred into fibronectin pre-coated Nunc 4-chambered culture slides (Fisher Scientific Co., Suwanee, GA, USA) containing 500 μl complete Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen Ltd, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS, Atlanta Geologics, Atlanta, GA, USA), 1% penicillin/streptomycin (P/S) and 1% L-Glutamine. For measurements of the numbers of Flk-1-positive cells expressing p-eNOS(S1177), phospho-histone H3 or NO, allantoic cultures (N=10 per sample) were pooled together, dissociated into single cells by trypsinization and then plated into fibronectin pre-coated Nunc 4-chambered culture slides containing 500 μl complete DMEM. Explants and single cells cultures were cultured at 37°C in a 5% CO2 incubator overnight. The following day the media was replaced with serum starvation (SS) media (DMEM containing 1% FBS). Combinations of compounds (VEGF alone and VEGF plus eNOS inhibitors) were prepared in SS media and then added to individual wells. After 5 hours, allantoic cultures were fixed in 4% paraformaldehyde (1 hour), permeabilized in 0.02% Triton-X/PBSA (20 minutes) blocked in 3% BSA/PBSA (1 hour), and exposed sequentially to the appropriate primary (CD102/ICAM2 15 μg/ml, eNOS 5 μg/ml, P-eNOS 15 μg/ml, phospho-histone H3-Ser10 20 μg/ml) and secondary (10 μg/ml) antibodies (overnight, 4°C), and then stained with Hoechst stain and mounted as described above.

Intracellular NO detection by fluorescence imaging

NO was detected by using DAF-FM diacetate using an approach similar to the one proposed by Paul, D.M., et al. (Paul et al.). For morphological analysis of allantoic cultures, intact allantoides were plated after their isolation, whereas for measurement of the numbers of Flk-1+ cells producing NO, allantoides were pulled together, tryspinized and then plated. Allantoic cells (either intact allantoic tissues or following trysinization to obtain single cell culture) were plated on Nunc 8-chambered culture slides pre-coated with fibronectin containing 200 μl complete DMEM and allowed to adhere for 18hrs in an incubator at 37°C in a 5% CO2 incubator. Cells were then rinsed twice with DPBS, and 1 μM DAF-FM diacetate solution was prepared freshly in Phenol Red Free DMEM (Invitrogen Ltd, Carlsbad, CA, USA) containing 0.5% FBS together with antibiotics and L-glutamine. After 30 minutes, DAF-FM diacetate solution was removed, cells were rinsed twice with DPBS and fresh PRF-SSM was added for 30 minutes. Chemical inhibitors (i.e., L-NIO, resveratrol, SU1498, LY294002) were added to the PRF-SSM prior to VEGF stimulation. Only rmsFlt-1 was added together with VEGF. VEGF was added to the cells for 1hr in presence or absence of the inhibitors. Cells were then fixed in 2% PFA for 1hr and then immunostained using antibodies to Flk-1 and p-eNOS(S1177). For measurements of NO producing cells, 15 fields were imaged per sample using a Leica DMI 4000 B fluorescence microscope (Leica Microsystems, Inc., Buffalo Grove, IL, USA) and NO-producing Flk-1+ cells were counted.

Microscopy and image processing

Conventional fluorescence images of some allantoic cultures were obtained using a Leica DMR research grade microscope equipped with Leica objectives (×5, ×10, ×20/0.7, ×40/0.85) and a SPOT-RT camera (Vashaw Scientific, Raleigh, NC, USA). Images were acquired using SPOT-RT 4.6.1.40 software. Embryos and some allantoic cultures were analyzed using a Leica TCS SP5 AOBS Laser Scanning Confocal Microscope (Leica Microsystems GmbH, Wetzlar, Germany). Optical sectioning along the Z axis was performed and the images collapsed into a single focal plane using the manufacturer’s software. Images were processed using NIH Image 1.37v software (National Institutes of Health, Bethesda, MD, USA) and Adobe Photoshop CS5 (Adobe Systems, Inc., San Jose, CA, USA). For 3D reconstructions, images generated by using Leica TCS SP5 LSCM were processed by AMIRA 5.3.3 (Visage Imaging, Inc., San Diego, CA, USA).

Mitotic Index calculations in single cell cultures

To determine mitotic index (MI) of Flk-1+ cells using fluorescence imaging in single cell allantoic cultures, pooled allantoic cultures were treated and subjected to immunostaining with antibodies to Flk-1 and phospho-histone H3 as described above. Ten fields for sample (N=3) were imaged and phospho-histone H3/Flk-1-positive cells were counted over total Flk-1 population. MI was calculated as the ratio of proliferative (phospho-histone H3/Flk-1-positive cells) over the total number of Flk-1+ cells and expressed as a percentage.

Flow Cytometric analysis

Ten allantoides were pooled and plated in one well of 6-well plate pre-coated with fibronectin (Greiner Bio-One, Monroe, NC, USA). The allantoides were cultured and treated as described above. After 5 hours, cells were harvested, washed three times with FACS buffer (PBS containing 0.1% BSA) and then incubated in the dark at 0°C for 30 minutes with FITC-conjugated rat antibodies to antimouse-Flk-1 (15 μg/ml) in FACS buffer. Analyses of the Flk-1+ cell population were performed using FACScan cytometer (BD Biosciences, San Jose, CA, USA) and the data analyzed using BD Cell Quest (BD Biosciences, San Jose, CA, USA). Resulting data were normalized by considering the VEGF-mediated response as 100% of maximal response.

To determine mitotic index (MI) in Flk-1+ cells using flow cytometric analyses, pooled allantoic cultures were treated and subjected to immunostaining as described above and then stained with propidium iodide (PI)/RNAse solution (BD Biosciences, San Jose, CA, USA). Cell cycle analyses of the Flk-1+ cells were performed using a MoFlo Astrios High Speed Cell Sorter (Beckman Coulter, Brea, CA, USA). MI was calculated as the ratio of Flk-1+ cells in the G2/M phase over the total number of Flk-1+ cells and expressed as a percentage.

Morphogenic analysis

Allantoides were cultured overnight on fibronectin-precoated Nunc 4-chambered culture slides in 1 ml complete DMEM at 37°C, 5% CO2. The following day the media was changed and the cells were then cultured in either SS media alone, or SS media containing VEGF, or SM media containing VEGF and individual eNOS inhibitors. After 5 hours, the cultures were fixed and permeabilized as described above. The cultures were then blocked in 3% BSA/PBS (1 hour), exposed to primary and secondary antibodies to ICAM2 (15 μg/ml, overnight, 4°C), stained with Hoechst stain and mounted. Cultures were imaged using a Leica TCS SP5 AOBS LSCM and images were then collapsed into a single focal plane. To evaluate potential inhibitory effects on vascular patterning the ratio of the vascular area to avascular area was calculated for each culture. This was achieved by inverting the images so that the ICAM2-positive vascular areas were black and the ICAM2-negative avascular area were white and then using Image J to calculate the black to white ratio in the area delineated by the perimeter of the allantoides. Resulting data were normalized by considering VEGF-mediated response as 100% of maximal response.

Statistical Analysis

Experimental data are expressed as the mean ± s.e.m. from at least three independent experiments.

RESULTS

eNOS and p-eNOS(S1177) are expressed in angioblasts and embryonic endothelial cells

Using antibodies to the angioblast/early endothelial cell marker TAL-1 (Drake et al., 1997) and to eNOS the first expression of eNOS in the endothelial lineage was evident at 7.2 dpc in a subpopulation of TAL-1+ cells (data not shown). By 8.2–8.5 dpc three distinct populations of TAL-1+ cells could be identified based on eNOS expression and by their position in the embryo (Drake and Fleming, 2000) (Fig. 1A and Fig. 1B). These cell types were TAL-1+/eNOS− (Fig. 1B, arrows) and TAL-1+/eNOS+ angioblasts (Fig. 1B, arrowheads) found in the lateral mesoderm, and TAL-1+/eNOS+ embryonic endothelial cells (EECs) of the dorsal aortae (Fig. 1B, asterisks). We also observed a population of eNOS expressing cells that did not express TAL-1 (Fig. 1B, blue arrowheads). To further define eNOS expression in the context of the endothelial lineage, 8.5 dpc embryos were immunolabeled with antibodies to eNOS and Flk-1 (Fig. 1C). As was the case with TAL-1 three distinct populations of Flk-1+ cells could be identified based on eNOS expression and their position in the embryo; they were both Flk-1+/eNOS− (Fig. 1C, arrows) and Flk-1+/eNOS+ (Fig. 1C, arrowheads) angioblasts found in the lateral mesoderm, and Flk-1+/eNOS+ EECs of the dorsal aortae (Fig. 1C, asterisks). Next, we evaluated if eNOS in Flk-1+ cells was phosphorylated. To achieve this, 8.5 dpc embryos were immunolabeled with antibodies to Flk-1 and antibodies specific for eNOS phosphorylated on Ser1177, Ser617 and Thr495, which are the major downstream targets of the VEGF/Flk-1 signaling pathway in ECs (Sessa, 2004). Analysis revealed that phosphorylated Ser1177 was observed in a subpopulation of the Flk-1+ EECs (Fig. 1D, asterisks), and of the Flk-1+ angioblasts (Fig. 1D, white arrowheads). We also observed a population of isolated P-eNOS expressing cell that did not express Flk-1 (Fig. 1D, blue arrowheads). In contrast, analysis of the other phosphorylation sites on eNOS regulated by VEGF signaling pathway showed that neither phosphorylated Ser617 nor Thr495 were detected in Flk-1+ cells at 8.5dpc (Supplementary Fig. S1A–H). Taken together these findings establish that both eNOS and eNOS phosphorylated on S1177, hereafter referred to as p-eNOS(S1177), are expressed in angioblasts and EECs.

Figure 1. eNOS is expressed in a subpopulation of angioblasts and EECs.

Figure 1

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A and B) En face views of a whole mounted 8.5 dpc embryo (N = 4) labeled with antibodies to TAL-1 (green) and eNOS (red). B) Higher magnification image of the boxed area depicted in (A), showing low eNOS (white arrowheads) or no eNOS (arrows) expression in TAL-1+ angioblasts and high eNOS expression in EECs of the dorsal aorta (asterisks). Blue arrowheads designate eNOS+/TAL-1− cells. C) an en face view of a whole mounted 8.5 dpc embryo (N = 4) labeled with antibodies to Flk-1 (green) and eNOS (red), showing both Flk-1+/eNOS+ (arrowheads) and Flk-1+/eNOS− (arrows) angioblasts and Flk-1+/eNOS+ EECs (asterisks). D) an en face view of a whole mounted 8.5 dpc embryo (N = 4) labeled with antibodies to Flk-1 (green) and p-eNOS(S1177) (red), showing both Flk-1+/p-eNOS(S1177)+ angioblasts (white arrowheads) and Flk-1+/P-eNOS− angioblasts (arrows), and Flk-1+/p-eNOS(S1177)+ expression in a subpopulation of EECs (asterisks). Blue arrowheads designate population of p-eNOS(S1177)+ cells not expressing Flk-1. Magnification bars equal 150 μm (A) and 50 μm (B, C, D).

p-eNOS(S1177) expression in the allantoic vasculature recapitulates the pattern observed in the endothelial lineage of the embryo proper

In previous studies we established the utility of cultured murine allantois as a model system for studying vasculogenesis and angiogenesis (Argraves et al., 2004; Argraves et al., 2002; Crosby et al., 2005). To determine its suitability as an experimental model for the studying of eNOS/p-eNOS regulation we evaluated if the expression profile of p-eNOS(S1177) in the allantois recapitulated that observed in the embryo proper. Immunolabeling of 8.5 dpc allantois with antibodies to Flk-1 and to p-eNOS(S1177) (Fig. 2A) showed that as was the case in the embryo proper only a subpopulation (arrows) of Flk-1+ (green) EECs expressed p-eNOS(S1177) (red). 3D AMIRA reconstructions of the allantois depicted in Figure 2A (Fig. 2B, Fig. 2C and Movie1), serve to highlight the Flk-1+/p-eNOS(S1177)+ EECs (arrows) and distinguish them from the Flk-1−/p-eNOS(S1177)+ cells (arrowheads), which were often positioned closely apposed to forming blood vessels.

Figure 2. p-eNOS(S1177) expression in the allantois.

Figure 2

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A–C) Images of an 8.5dpc allantois (N=3) labeled with antibodies to Flk-1 (green) and to p-eNOS(S1177) (red). A) Confocal image depicting Flk-1+/p-eNOS(S1177)+ cells (arrows) and Flk-1−/p-eNOS(S1177)+ cells (arrowheads). B and C) 3D AMIRA reconstructions of the allantois represented in (A) depicting Flk-1+/p-eNOS(S1177)+ cells (arrows), and Flk-1−/p-eNOS(S1177)+ cells (arrowheads). C) Side view of upper left hand area depicted in (B). Magnification bars equal 50 μm (A).

A role for eNOS in regulating cell proliferation in the EC lineage

Given that VEGF is both an upstream regulator of eNOS/NO and mediator of EC proliferation we investigated whether there was a correlation between angioblast and/or EEC proliferation and eNOS expression. To evaluate this 8.5 dpc embryos were immunolabeled with antibodies to Flk-1 and phospho-histone H3, a marker of cell proliferation. As seen in Figure 3A both Flk-1 (green) and phospho-histone H3 (red) expression was detected in a subpopulation of Flk-1+ angioblasts (arrowheads) and aortic EECs (asterisks). Similarly to what observed in the embryo proper, analysis of eNOS and phospho-histone H3 expression in the 8.5 dpc allantois (Fig. 3B) revealed that a subpopulation of eNOS+ EECs were phospho-histone H3+(arrows). In addition to proliferating EECs we also observed a population of isolated eNOS+/phospho-histone H3+ cells (arrowhead) that were adjacent to eNOS+ allantoic endothelium Taken together these findings suggest that there is a correlation between eNOS expression and proliferation in cells of the EC lineage.

Figure 3. Cell proliferation and eNOS expression in the embryo proper and in the allantois.

Figure 3

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A) Image of an 8.5 dpc embryo labeled with antibodies to Flk-1 (green) and phospho-histone H3 (red), showing co-expression of Flk-1 and phospho-histone H3 in a subpopulation of EECs (asterisks) and angioblasts (arrowheads). B) Image of an 8.5 dpc allantois labeled with antibodies to eNOS (green) and phospho-histone H3 (red), showing co-expression of eNOS and phospho-histone H3 in EECs (arrows) and in a subpopulation of cells adjacent to the forming vessels (arrowheads). Magnification bars equal 50 μm.

VEGF regulates P-eNOS-S1177 expression in Flk-1+ cells

To assess the role of VEGF/Flk-1 signaling in the regulation of p-eNOS(S1177) in cells of the EC lineage, allantoides were cultured with and without VEGF followed by immunolabeling with antibodies against p-eNOS(S1177) and the EC marker ICAM2 (Bautch et al., 2000). As depicted in Figure 4, p-eNOS expression in untreated cultures was observed in a subpopulation of EECs of forming vessels (Fig. 4A, arrow). Analysis of the VEGF treated cultures showed an apparent increase in the number of p-eNOS(S1177)+ EECs (Fig. 4B, arrows). Also evident in these cultures was a population of p-eNOS(S1177)+ cells which were close apposed to developing blood vessels that did not express ICAM2. That p-eNOS(S1177)+ expression in EECs is dependent on VEGF/Flk-1 signaling is supported by our finding that p-eNOS(S1177) expression (red) was confined to Flk-1+ cells (green) in VEGF-treated allantoides cultures (Fig. 4C, arrows). It is noteworthy that as was the case in vivo we also observed p-eNOS(S1177) expression in isolated Flk-1− cells (Supplementary Fig. S2, arrowheads).

Figure 4. VEGF regulates p-eNOS(S1177) expression in angioblasts and EECs.

Figure 4

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A–B) Images of serum starved (SS, A) 8.5 dpc allantoides culture and VEGF-treated (50 ng/ml, B) 8.5 dpc allantoides culture labeled with antibodies to ICAM2 (green), p-eNOS(S1177) (red) and stained with Hoechst (blue). Evident in both (A) and (B) are isolated ICAM2+ EECs that co-express p-eNOS(S1177) (arrows). C) VEGF-treated 8.5 dpc allantoic culture labeled with antibodies to Flk-1 (green) and p-eNOS(S1177) (red) and stained with Hoechst (blue). Flk-1+ EECs that co-express p-eNOS(S1177) are indicated by arrows. Magnification bars equal 50 μm.

p-eNOS(S1177) expression is correlated with NO production

Having observed a correlation between cell proliferation and eNOS expression we next sought to establish if a similar correlation existed between p-eNOS(S1177) expression and NO production. To achieve this allantoic cultures were exposed to DAF-FM diacetate, treated with VEGF and then immunostained with antibodies to Flk-1 and p-eNOS(S1177). The immunolabeling depicted in Figure 5 shows an apparent increase in the number of Flk-1+ cells producing NO following VEGF treatment (50ng/ml, 30 minutes, Fig. 5D) versus control cultures (Fig. 5C). In order to quantify this increase of NO producing Flk-1+ cells, allantoides were pooled (N=10 per sample) and trypsinized. Following 18 hours in culture, cells were cultured in presence of DAF-FM diacetate and then treated with VEGF (50ng/ml, 30 minutes). As seen in Supplementary Figure S3A and S3B, VEGF induced an increase in NO producing Flk-1+ cells. Supplementary Figure S3C shows that the observed VEGF-induced NO production was statistically significant (~50%) and it was correlated with an increase in p-eNOS(S1177) expression levels (~55%). To exclude a role for iNOS-mediated NO production allantoic cultures were immunostained with antibodies to Flk-1 and iNOS. Analysis revealed that VEGF responsive Flk-1+ cells do not express iNOS in these culture conditions (Supplementary Fig. S4A–C). However, iNOS expression was detected in a population of Flk-1− cells (Supplementary Fig. S4A–C, arrows). The lack of iNOS expression in Flk-1+ cells of the allantoic culture is consistent with our in vivo analysis of iNOS expression, which also showed that Flk-1+ cells did not express iNOS, whereas iNOS expression was detected in a subpopulation of Flk-1− cells (Supplementary Fig. S5A–F).

Figure 5. VEGF regulates intracellular NO production in angioblasts and EECs.

Figure 5

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A–F) Images of control (A, C, E) and VEGF-treated (50 ng/ml, 30 minutes, B, D, F) 8.5 dpc allantoic cultures cultured in the presence DAF-FM diacetate for the detection of intracellular NO production (green), labeled with antibodies to Flk-1 (red) and stained with Hoechst (blue). (C) and (D) are images of NO producing cells from fields showed in (A) and (B), respectively. (E) and (F) are images of Flk-1+ cells from fields showed in (A) and (B), respectively. Flk-1+ cells that produce NO are indicated by arrowheads. Magnification bars equal 150 μm.

Antagonists of NO production decrease VEGF-mediated cell division in angioblast and embryonic endothelial cells

Our findings that VEGF acts to increase the levels of p-eNOS(S1177) and NO in Flk-1+ cells and that p-eNOS(S1177) expression is associated with EC division, led us to assess the effects of eNOS/NO inhibitors on VEGF-mediated angioblast and/or EEC division. Figure 6A shows representative FACS analyses of the Flk-1+ cell population in serum starved allantoic cultures (Fig. 6A), VEGF-treated cultures, and cultures treated with VEGF plus L-NIO, an eNOS inhibitor, or resveratrol, an NO scavenger. Our analysis showed that VEGF increased the number of Flk-1+ cells as compared to serum starved control cultures and that this increase was attenuated by the eNOS/NO inhibition. Quantitation in Figure 6B showed that VEGF treatment increased the number of Flk-1+ cells by 30%, while inhibition of eNOS/NO under all conditions resulted in reductions in Flk-1+ cells. While co-treatment with resveratrol did not result in a statistically significant reduction, L-NIO decreased the VEGF-mediated increase in Flk-1+ population by 32%, respectively. FACS analyses revealed that the inhibitory effects of L-NIO and resveratrol were not due to an increase in apoptosis as determined by propidium iodide staining (data not shown).

Figure 6. VEGF mediated proliferation in Flk-1-positive cells is attenuated by NO antagonists.

Figure 6

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A) Representative FACS analyses of the Flk-1+ cells from pooled allantois cultures treated for 5 hours with: serum starvation (SS) media only, VEGF (50 ng/ml), VEGF plus L-NIO (100 μM) and VEGF plus resveratrol (20 μM). B) Statistical analysis of the FACS data from three independent experiments conducted following methods described in (A). C) Mitotic indexes derived from FACS analysis of DNA content of fixed Flk-1+ cells (N = 3) from serum starved HUVECs treated with: serum starvation (SS) media only, VEGF (50 ng/ml) and VEGF plus L-NIO (100 μM). Significance was set at *p < 0.05 (N = 3).

To investigate if the effects of eNOS/NO antagonist on Flk-1+ cell numbers were due to reduced cell proliferation, Flk-1+ cells were isolated from VEGF-treated allantoic cultures co-treated with L-NIO. Isolated Flk-1+ cells were labeled with propidium iodide and analyzed for changes in cell cycle by flow cytometry. The mitotic indexes (MIs) were determined by calculating the ratio of Flk-1+ in the G2/M phase and are expressed as a percentage of total number of Flk-1+ cells (for details see Materials and Methods section). As shown in Figure 6C, VEGF increased the number of mitotic cells by 14%. VEGF-mediated effects were reduced by L-NIO (~4%). Taken together our findings suggest that VEGF effects on cell proliferation are in part mediated by NO.

VEGF-mediated cell division in the endothelial lineage is dependent on phosphorylation of eNOS on Ser1177 by Akt1

Given that Akt1 acts to regulate the phosphorylation of eNOS on Ser1177 in adult ECs (Schleicher et al., 2009) and is a downstream target of VEGF and our findings that VEGF acts to increase the levels of p-eNOS(S1177) and NO in Flk-1+ cells and that NO production is associated with EC division, led us to assess whether the VEGF induced responses we had observed in angioblasts and EECs were mediated by Akt1. To evaluate this we utilized Akt1 null mice (Mao et al., 2007). We first evaluated the expression pattern of p-eNOS(S1177) in 8.5 dpc Akt1 null embryos (Supplementary Fig. S6A–C). In contrast to wild type embryos, p-eNOS(S1177) expression was almost completely absent in angioblasts and EECs of the Akt1 null embryos. Consistent with a reduction in p-eNOS(S1177), analysis of phospho-histone H3 expression in vivo in 8.5dpc Akt1 nulls showed reduced cell proliferation in both angioblasts and EECs (Supplementary Fig. S7).

To evaluate the effects of the loss of Akt1 on VEGF-mediated p-eNOS(S1177) expression and NO production, 8.5 dpc allantoic cultures generated from 8.5dpc Akt1 null embryos were treated with 50ng/ml VEGF and immunolabeled with antibodies to Flk-1 and p-eNOS(S1177) (Figs. 7A and 7B). Comparison of the numbers of Flk-1+ cells expressing p-eNOS(S1177) between wild type, heterozygous and homozygous allantoic cultures (Fig. 7C) showed that p-eNOS(S1177) expression was respectively reduced by 45% and 80% in heterozygous and homozygous allantoic cultures compared to controls. Next, to evaluate the relationship between the PI3K/Akt1-mediated p-eNOS(S1177) expression and cell proliferation, cell proliferation in VEGF-treated allantoic cultures was inhibited pharmacologically and genetically, respectively by using the PI3K inhibitor LY294002 in wild type allantoides and allantoides isolated from Akt1 heterozygous and homozygous mouse embryos. Mitotic Indexes generated by calculating the ratio of proliferative (phospho-histone H3+) over the total Flk-1+ cells indicated that Akt1 expression is necessary for the increase of cell proliferation in angioblasts and EECs (Fig.7D). Consistent with these results our analysis of NO producing Flk-1+ cells in the above cultures demonstrate a similar relationship between Akt1 and NO production (Supplementary Fig. S8A–C). A similar inhibitory response on VEGF-mediated increase in p-eNOS(S1177) expression and NO production was observed by using chemical inhibitors of the VEGF, Akt1 and eNOS signaling pathways (Supplementary Fig. S9A and S9B).

Figure 7. VEGF mediated p-eNOS(S1177) expression and cell proliferation in Flk-1-positive cells is dependent on Akt1.

Figure 7

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A and B) Confocal images of an intact Akt1 homozygous allantoic culture (N=4) that was treated with VEGF (50 ng/ml, 30 minutes), labeled with antibodies to Flk-1 (green) and p-eNOS(S1177) (red) and stained with Hoechst (blue). C) Statistical analysis of isolated Flk-1+ allantoic cells that were treated with VEGF (50 ng/ml, 30 minutes), labeled with antibodies to Flk-1 and p-eNOS(S1177) and stained with Hoechst, showing the differences in p-eNOS(S1177)+/Flk-1+ cell ratios following VEGF treatment (30 minutes) between wild type, heterozygous and homozygous. D) Mitotic indexes derived from immunohistochemical analysis of phospho-histone H3+/Flk-1+ wild type cells treated with serum starved media only (SSM), VEGF, VEGF plus LY294002, and phospho-histone H3+/Flk-1+ heterozygous and homozygous cell following VEGF treatment. Significance was set at *p < 0.05 (N = 3).

VEGF-mediated vascular patterning is regulated by Akt1-mediated P-eNOS expression and NO production during development

As the number of Flk-1+ cells (angioblasts and/or EECs) is relevant to the patterning of blood vessels (LaRue et al., 2004; LaRue et al., 2003), we next sought to determine if the decrease in Flk-1+ cell proliferation observed in response to PI3K/Akt1 deletion was relevant to the VEGF-mediated vascular pattern. Analysis of 8.5 dpc wild type and Akt1 null allantoic cultures treated with VEGF and immunolabeded with antibodies to Flk-1 showed that the absence of Akt1 dramatically changed the number and patterning of allantoic vessels (Supplementary Fig. S10).

To investigate the role of NO in VEGF/Akt-1-mediated vascular patterning we used pharmacological inhibitors of Akt1 and NO (Fig. 8A). Comparison of control, serum starved cultures to VEGF-treated cultures showed that VEGF dramatically increased the vascularized area (black areas) of the culture with a concomitant loss of the avascular area (white areas). VEGF-treated cultures co-treated with LY294002, L-NIO or resveratrol showed alterations in the patterning of the blood vessels as compared to the VEGF control. Comparisons between vascularized and avascular areas revealed that exogenous VEGF increased vascularized areas as compared to control by 70% and that LY294002, L-NIO and resveratrol reduced the VEGF-induced ratio by 57%, 79% and 68%, respectively (Fig. 8B). It is important to note that although the resveratrol-mediated inhibition was 68% it was not found to be statistically significant based on three independent experiments.

Figure 8. VEGF mediated vascular patterning is altered by PI3K/Akt1 and eNOS/NO antagonists.

Figure 8

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A) Representative images of allantois cultures treated for 5 hours with: serum starvation (SS) media only, VEGF (50 ng/ml), VEGF plus LY294002 (20 μM), VEGF plus L-NIO (100 μM) and VEGF plus resveratrol (20 μM), and labeled with antibody to ICAM2. B) Statistical analysis of vascular patterns (analyzed using black versus white ratio) from six independent experiments (N = 6) conducted as in (A). Significance was set at *p < 0.05 (N = 3).

DISCUSSION

Our study is the first to show that eNOS and its phosphorylated isoform on Ser1177, p-eNOS(S1177), are expressed in endothelial progenitor cells (angioblasts) and that phosphorylation on Ser1177 in angioblasts and embryonic endothelial cells (EECs) plays a prominent role in regulating NO production. The expression of both eNOS and p-eNOS(S1177) at the earliest stages of vascular development (7.2–8.5 dpc) establishes that the expression of eNOS in angioblasts and EECs is not dependent on the initiation of blood flow which occurs at 9.0–9.5 dpc (Mu and Adamson, 2006), nor signaling by vascular smooth muscle cells (SMCs), which first appears at 10.5 dpc (Esner et al., 2006; Wasteson et al., 2008). This finding suggested that NO had role(s) in vascular development that have not previously been described. The fact that we observed both TAL-1+/eNOS− and Flk-1+/eNOS− angioblasts suggest that NO is not involved in EC lineage determination. Compatible with the studies of others showing that NO can mediate EC division (Dai and Faber, 2010; Oliveira et al., 2008; Papapetropoulos et al., 1997; Polytarchou and Papadimitriou, 2005), we then hypothesized that NO acted to mediate angioblast and EEC division and thus vasculogenic and/or angiogenic blood vessel formation. In support of such a role, we showed that following VEGF treatment p-eNOS(S1177) levels are increased and that this increase is correlated with elevated NO production in both proliferating angioblasts and/or EECs in vivo. It is important to note that our immunostaining highlighted the presences of p-eNOS(S1177) expressing cells that were neither Flk-1 nor TAL-1 positive. This observation suggests that in addition to previously described cell types that express eNOS, such as cardiac myocytes, neurons, and renal epithelial cells (Michel and Feron, 1997), there may be additional cell types and/or progenitor cells that fall outside the endothelial-hematopoietic lineage that also express eNOS.

A role for NO as a downstream target of VEGF/Flk-1-mediated angioblast and/or EEC division is highlighted in our in vitro studies evaluating the effects of PI3K/Akt1 and eNOS/NO antagonists on VEGF/Flk-1 signaling pathway. These studies showed that pharmacological inhibition of the eNOS/NO pathway decreased the number Flk-1+ cells in VEGF treated allantoic cultures (Fig. 6) and that the deletion of Akt1 dramatically reduced VEFG-mediated Flk-1+ cell division (Fig. 7). Based on the pattern of Flk-1 and eNOS expression in angioblasts and EECs, and the effects of eNOS/NO antagonist on VEGF–mediated cell proliferation (Fig. 6C) we speculate that the reduction in Flk-1+ cell numbers observed in response to L-NIO and resveratrol is due to decreased proliferation due to reduced VEGF-mediated intracellular NO. Additionally, based on the effects of both pharmacological (i.e., LY294002) and genetic inhibition of Akt1 on VEGF-mediated cell proliferation and vascular pattern (Fig. 7D), we conclude that the VEGF/eNOS-mediated response is dependent on Akt1. In support of this the study of Papapetropoulos, et al., (1997) showed that NO is a downstream target of VEGF/Flk-1-mediated EC division.

Our studies also suggest a mechanism by which VEGF/Flk-1 signaling acts to mediate EC division by regulating the phosphorylation of eNOS. This is based on our findings that VEGF acts within 30 minutes to increase p-eNOS(S1177) levels and the mitotic index in the Flk-1+ cell population isolated from allantoic cultures treated with VEGF (as determined by both FACS and IHC analyses, Fig. 6C and 7D, respectively). That the mitogenic effects we observed in response to VEGF are specific to VEGF-mediated phosphorylation eNOS on Ser1177 is supported by our findings that other potential phosphorylation sites on eNOS (i.e., Ser617 and Thr495) remain unphosphorylated in these cells at this embryonic stage (Supplementary Fig. S1). Further, our findings demonstrating that Flk-1+ cells don’t express iNOS in vitro and in vivo (Supplementary Fig. S3A–C and S4A–C, respectively) at stages relevant to this study exclude a VEGF mediated response through iNOS.

A role for NO as a mediator of VEGF-induced angioblast and/or EEC proliferation is also consistent with the changes in VEGF mediated patterning induced by eNOS/NO antagonist (fig. 8). In previous studies, we have demonstrated that the proper patterning of embryonic blood vessels is dependent on the correct number of ECs (Giles et al., 2005; LaRue et al., 2004; LaRue et al., 2003). This number can be altered by either increased/decreased EC lineage commitment or EC proliferation. The presence of thinner and more isolated vessel segments, as reflected in the black versus white ratio analysis, induced by the eNOS/NO and Akt1 antagonists is indicative of a reduction in EC numbers and is in accordance with our findings (Fig. 6). That this reduction in cell numbers is due to inhibition of proliferation in angioblasts and/or EECs is consistent with the study of Yue and Tomanek (2001), that showed the ability of VEGF to induce neovascularization in a model of cultured embryonic quail hearts by increasing the numbers of ECs in a VEGF dose-dependent manner and by our studies demonstrating diminished proliferation in these cells following inhibition of Akt1 (Fig. 7D). Based on these findings we conclude that the changes in the patterning of allantoic vessels observed following treatment with VEGF is due in part to VEGF/Akt1-mediated NO production that acts to increase angioblasts and/or EEC proliferation. Conversely, we speculate that the alterations to the VEGF-mediated patterning of allantoic vessels observed in cultures treated with NO and Akt1 inhibitors are due to decreased NO production and decreased proliferation in angioblasts and/or EECs.

This study extends the role played by NO in VEGF-mediated proliferation to the earliest stages of vascular development and suggests that phosphorylation of eNOS on Ser1177 is a key regulator of VEGF-mediated vasculogenesis. In support of this are our findings showing a strong correlation between VEGF-mediated p-eNOS(S1177) expression and eNOS activity (i.e., NO production) and function (i.e., VEGF-mediated Flk-1+ cell proliferation and vascular pattern).

Supplementary Material

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Highlights.

  • Endothelial progenitor cells (angioblasts) express eNOS.

  • VEGF regulates eNOS phosphorylation in angioblasts and embryonic endothelial cells.

  • VEGF-mediated eNOS phosphorylation regulates angioblast and EEC division.

    Inhibition of eNOS/nitric oxide alters VEGF-induced vascular patterning.

Acknowledgments

This work was supported by NIH HL080168 and NSF EPS-0903795 to CJD, NIH HL084565 to RMH and by AHA 11PRE7530048 to CG. The authors wish to thank Dr. Philip N. Tsichlis (Tufts-New England Medical Center) for the Akt1 knockout mice, and the Regenerative Medicine Flow Cytometry Core (Chris Fuchs), the Regenerative Medicine imaging facility (Dr. Thomas Trusk) and Stephen Romeo (MUSC) for their assistance.

Footnotes

Authorship Contributions and Disclosure of Conflicts of Interest

C.G. designed and performed experiments, analyzed data and wrote the manuscript, R. M.H. designed experiments, analyzed data and wrote the manuscript, C.J.D. designed experiments, analyzed data and wrote the manuscript. The authors have no conflict of interest.

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Supplementary Materials

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NIHMS418618-supplement-04.pdf (1.2MB, pdf)
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NIHMS418618-supplement-12.pdf (1.4MB, pdf)

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