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Cardiovascular Research Advance Access first published online on February 10, 2008
This version [Corrected Proof] published online on March 13, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn034
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Adiponectin inhibits vascular endothelial growth factor-induced migration of human coronary artery endothelial cells

Kalyankar Mahadev1,*, Xiangdong Wu1, Sylvia Donnelly1, Raogo Ouedraogo1, Andrea D. Eckhart2 and Barry J. Goldstein1,*

1 Division of Endocrinology, Diabetes and Metabolic Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Suite 320, Curtis Building, 1015 Walnut Street, Philadelphia, PA 19107, USA
2 Eugene Feiner Laboratory of Vascular Biology and Thrombosis, Center for Translational Medicine, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA

* Corresponding authors. Tel: +1 215 503 1272; fax: +1 215 923 7932. E-mail addresses: barry.goldstein{at}jefferson.edu (B.J.G); mahadev.kalyankar{at}jefferson.edu (K.M.)

Received 19 November 2007; revised 16 January 2008; accepted 6 February 2008

Time for primary review: 27 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: Vascular endothelial growth factor (VEGF)-induced endothelial cell migration and angiogenesis are associated with the vascular complications of diabetes mellitus, and adiponectin is an abundant plasma adipokine that exhibits salutary effects on endothelial function. We investigated whether adiponectin suppresses VEGF-induced migration and related signal transduction responses in human coronary artery endothelial cells (HCAECs).

Methods and results: Using a modified Boyden chamber technique and a monolayer ‘wound-healing’ assay, both the recombinant adiponectin globular domain and full-length adiponectin protein potently suppressed the migration of HCAEC induced by VEGF. Adiponectin did not increase endothelial cell apoptosis, as measured by Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labelling assay. Adiponectin also suppressed VEGF-induced reactive oxygen species generation, activation of Akt, the mitogen-activated protein kinase ERK and the RhoGTPase RhoA, and induction of the formation of actin stress fibres and focal cellular adhesions. VEGF-stimulated cell migration was inhibited by activation of adenylyl cyclase with forskolin, and adiponectin treatment increased cellular cyclic adenosine monophosphate (cAMP) levels and protein kinase A (PKA) enzymatic activity. Pharmacological inhibition of either adenylyl cyclase or PKA significantly abrogated the effect of adiponectin globular domain to suppress VEGF-induced cell migration.

Conclusion: Adiponectin suppresses VEGF-stimulated HCAEC migration via cAMP/PKA-dependent signalling, an important effect with implications for a regulatory role of adiponectin in vascular processes associated with diabetes and atherosclerosis.

KEYWORDS Adiponectin; acrp30; VEGF; Endothelium; Reactive oxygen species; Apoptosis; Focal adhesions; Actin stress fibres; RhoA


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Adiponectin is an abundant circulating plasma protein secreted by adipose tissue that exhibits beneficial effects in the vasculature.1,2 Circulating levels of adiponectin are decreased in individuals with obesity and type 2 diabetes, suggesting a potential role for adiponectin in the vascular diseases that frequently accompany these disorders.3 In endothelial cells, adiponectin suppresses adverse effects of inflammatory cytokines and reduces oxidative stress induced by oxidized low-density lipoprotein (LDL) or high glucose.4–7

Migration of endothelial cells contributes to diverse vascular physiology and pathology such as wound healing, restenosis in grafted and injured vessels and atherosclerosis.8 Vascular endothelial growth factor (VEGF)3 is involved in cellular mobilization via disruption of cell–cell and cell–substrate contacts and cellular proliferation, mediated primarily through its receptor VEGFR2 and activation of key signalling enzymes including mitogen-activated protein kinase (MAPK), focal adhesion kinase (FAK), Akt and RhoA activity.9,10 Cell stimulation by VEGF also elicits the generation of reactive oxygen species (ROS) that serve as secondary messengers in signal transduction.11,12

Although the 5'-AMP-activated protein kinase pathway mediates much of the metabolic effect of adiponectin in skeletal muscle and liver cells,1,13 and is involved in the stimulation of endothelial NO availability,14 a growing body of data has supported a major role for the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway in mediating at least some of the anti-inflammatory and anti-oxidant effects of adiponectin in endothelial cells,2 including suppression of tumour necrosis factor-{alpha} (TNF-{alpha})-induced adhesion molecule expression and inflammatory signalling4,5 and reduction of cellular ROS levels.7 In the current study, we investigated whether the globular domain and the full-length forms of adiponectin (fAd) suppress VEGF-induced migration of human coronary artery endothelial cells (HCAECs) and using a variety of pharmacological inhibitors, we tested the potential role of cAMP/PKA signalling in this process.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
2.1 Materials
The human recombinant globular domain of adiponectin (gAd) from an Escherichia coli expression system was from PeproTech (Rocky Hill, NJ, USA). fAd from a eukaryotic expression system was from BioVendor (Candler, NC, USA). Phospho-ERK1/2 MAPK (Thr202/Tyr204) and phospho-Akt (Ser473) antibodies were from Cell Signaling Technology (Danvers, MA, USA). Reagents for enhanced chemiluminescence (ECL) were from Perkin-Elmer Life Sciences (Boston, MA, USA). [{gamma}-32P]ATP used in the PKA assay was from GE Amersham (Piscataway, NJ, USA). All other reagents, unless otherwise noted, were obtained from Sigma–Aldrich (St Louis, MO, USA).

2.2 Cell culture, treatment, and western blot analysis
HCAECs (Cambrex BioScience, Walkersville, MD, USA) were cultured in endothelial cell growth medium EBM-2 supplemented with growth factors and 5% (v/v) fetal bovine serum (FBS) to 70–80% confluency. FBS was changed to 0.5% (w/v) bovine serum albumin (BSA) for 16 h along with the addition of gAd or fAd where indicated. In some experiments, cells were pretreated with Rp-adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMP; 10 µM), 2',3'-dideoxyadenosine (ddAdo; 100 µM), or H2O2 (100 µM) for 30 min prior to cell lysis and sodium dodecyl sulphate–polyacrylamide gel electrophoresis as reported.7 Proteins were transferred to polyvinylidene difluoride membranes, probed with primary antibody followed by secondary antibody conjugated with horseradish peroxidase and ECL and quantitated using an Image Station 440 (Kodak, Rochester, NY, USA). The molecular mass of the adiponectin isoforms was analysed by non-denaturing electrophoresis in 3–8% Tris-acetate gels essentially as described by Lara-Castro et al.15

2.3 Quantitation of human coronary artery endothelial cells migration
Cell migration assay was performed as described previously using modified Boyden chambers (Costar) coated on the lower side with type I collagen.16

2.4 Wound-healing assay
The wound-healing assay was performed as described previously using six-well dishes coated with collagen type I.17

2.5 Terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling apoptosis assay
The TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labelling) assay was performed according to the manufacturer's instructions using a TUNEL Apoptosis Detection Kit (Upstate, Temecula, CA, USA). Briefly, subconfluent HCAECs were grown on two-well chamber slides before incubation for 16 h in EBM2 basal medium containing 0.5% FBS and 3.0 µg/mL gAd or fAd. For a positive control, cells were incubated with DNAse I for 1 h at 37°C. Nuclear contrast was obtained by 4',6'-diamidino-2-phenylindole (DAPI) staining (10 µg/mL; Sigma–Aldrich).

2.6 Measurement of reactive oxygen species in human coronary artery endothelial cells
5,6-Chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCF-DA) (Molecular Probes/Invitrogen, Carlsbad, CA, USA) fluorescence was performed as reported previously.18

2.7 Protein kinase A activity assay
PKA enzyme activity was measured using a kit and instructions provided by the manufacturer (Upstate, Lake Placid, NY, USA). Cell lysates containing 400 µg of protein were immunoprecipitated with 2.0 µg of anti-PKAc-{alpha} polyclonal rabbit antibody and adsorbed to protein A agarose beads prior to kinase assay with kemptide substrate and 10 µCi [{gamma}-32P] ATP for 15 min at 30°C. PKA activity was expressed as the phosphorylation of kemptide (dpm) per 15 min/µg of protein.

2.8 Measurement of cellular cyclic adenosine monophosphate content
cAMP was measured in the HCAECs using a direct enzyme immunoassay kit and instructions provided by the manufacturer (GE Healthcare/Amersham Biosciences).

2.9 Fluorescent staining of F-actin and paxillin in human coronary artery endothelial cells
HCAECs grown on glass cover-slips were starved for 16 h in serum-free medium containing 0.5% BSA. Some of the cells were pretreated with gAd or fAd for 16 h, followed by treatment with VEGF or other activators and inhibitors for 2 h. After cell treatments, they were fixed with 3.7% paraformaldehyde for 15 min, washed three times with phosphate-buffered saline (PBS) and permeabilized with 0.2% Triton X 100 for 5 min at room temperature, and blocked with 2% BSA in PBS for 30 min. Incubation with paxillin antibody was performed in blocking solution for 1 h at room temperature followed by staining with Alexa Fluor 555 conjugated secondary antibody. Actin filaments were stained with Alexa Fluor 594 phalloidin and nuclei with Hoechst 33342 for 1 h at room temperature.

2.10 Measurement of RhoA activity in human coronary artery endothelial cells
A RhoA activation assay kit (Cytoskeleton, Denver, CO, USA) based on methods described by Ren et al.,19 was used for the measurement of RhoA activity in subconfluent HCAECs cultured in six-well plates.

2.11 Statistical analyses
Data are presented as mean ± SE for at least three experiments. Statistical analyses were based on Student's t-test for comparison of two groups. A P-value less than 0.05 was used to determine statistical significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Globular and full-length adiponectin oligomeric isoforms used as reagents in this study
In order to characterize the recombinant adiponectin used in these experiments, non-denaturing gradient gel electrophoresis was performed (see Supplementary material online, Figure S1). The recombinant gAd migrated as a single band near the expected molecular weight of ~16 kDa. The full-length protein, as purified from a eukaryotic expression system, undergoes post-translational processing by glycosylation, cross-linking, and assembly into multiple high-molecular weight oligomeric isoforms.1,15,20 The fAd preparation used in these studies exhibited a typical pattern of high molecular weight oligomers, with major components at ~120 kDa and >250 kDa in non-denaturing gel analysis.21

3.2 Adiponectin suppression of VEGF-induced endothelial cell migration
Initially, we determined whether VEGF-stimulated HCAEC cell migration was suppressed by incubation with gAd or fAd using the modified Boyden chamber assay. The primary data showing cells stained with Crystal Violet allows visualization of the dose response between 0 and 3.0 µg/mL adiponectin (see Supplementary material online, Figure S2). Stimulation with VEGF alone increased cell migration by 2.2-fold (120%) over basal rate of cell migration (P < 0.001) (Figure 1A). Incubation with gAd or fAd alone had no effect on the basal rate of cell migration (not shown). However, the lowest dose of gAd or fAd tested (0.5 µg/mL) reduced migration by 24% and 20%, respectively (both P < 0.01). The VEGF-stimulated migration of HCAECs in the Boyden chamber assay was then progressively inhibited by increasing doses of gAd or fAd to the level of HCAECs prior to stimulation with VEGF at doses of 3.0 µg/mL for each of the adiponectin isoforms.


Figure 1
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  		Figure 1 Effect of adiponectin on vascular endothelial growth factor (VEGF)-induced cell migration. (A) Dose-response of adiponectin isoforms on VEGF-induced human coronary artery endothelial cell (HCAEC) migration in the Boyden chamber derived from primary data shown in Supplementary material online, Figure S2. Migration analysis was performed as described in Methods in the presence of increasing doses of globular domain of adiponectin (gAd) or full-length forms of adiponectin (fAd) and VEGF (50 ng/mL) as indicated. After 24 h, cells that migrated were quantitated. *P < 0.001; **P < 0.01; vs. control VEGF-stimulated migration. (B) Effect of various pharmacological agents including forskolin (2 µM), Rp-cAMP (10 µM), ddAdo (100 µM), H2O2 (100 µM), DPI (10 µM), and apocynin (500 µM) in the presence or absence of gAd and VEGF on HCAEC migration for 24 h. The data shown are the mean values from replicate experiments. *P < 0.001; **P < 0.01, vs. control VEGF-stimulated migration. (C) ‘Wound-healing’ assay of VEGF-stimulated HCAEC migration in culture. An interruption in the culture monolayer (the ‘wound’) was created by manually scraping the surface of the plate with a sterile p200 pipette tip. Cells were then treated with 3 µg/mL of gAd or fAd and the other indicated reagents as described in Methods and allowed to migrate into the denuded area for 8 h. Representative phase-contrast images of the cell monolayer were then acquired using the Nikon Eclipse TE 200-U microscope fitted with a 4X objective. The bar in the lower right corner indicates 5 µm.

 
3.3 Effect of inhibition of protein kinase A or adenylyl cyclase on the suppressive effect of globular domain of adiponectin on VEGF-induced human coronary artery endothelial cells migration
Further studies were performed to examine whether cAMP/PKA signalling was involved in the suppressive effect of adiponectin on VEGF-induced cell migration (Figure 1B). In these experiments, co-treatment of gAd with VEGF for the 24 h incubation potently suppressed the VEGF-induced increase in cell migration by 82% (P < 0.001). Consistent with the hypothesis that cAMP signalling can reduce VEGF-induced cell migration, activation of adenylyl cyclase with forskolin reduced VEGF-induced cell migration by 50% (P < 0.01). During treatment with gAd and VEGF, HCAECs were also incubated in the presence of the PKA inhibitor Rp-cAMP or the adenylyl cyclase inhibitor ddAdo for 24 h prior to the assessment of cell migration in the modified Boyden chamber. Inhibition of either PKA or adenylyl cyclase significantly abrogated the effect of gAd to suppress VEGF-induced cell migration by 46–47% (both P < 0.001). Thus, suppression of VEGF-induced cell migration by gAd is substantially dependent on signalling via the cAMP and PKA pathways.

3.4 Wound-healing assay of VEGF-stimulated human coronary artery endothelial cells migration
An additional test of endothelial cell migration was performed by disrupting the monolayer with a pipette tip and assessing the directional migration of the cells into the denuded region (Figure 1C). Compared with control medium, during the 8 h incubation period, VEGF stimulated the migration of HCAECs into the plate area from which the attached cells were removed and this process was inhibited by treatment with gAd or fAd. In addition, the effect of VEGF on migration was blocked by diphenylene iodonium (DPI) and apocynin, two agents that inhibit ROS generation by blocking the NADPH oxidase activity implicated in VEGF action.22 Importantly, the suppression of VEGF-induced migration by gAd was inhibited by Rp-cAMP or ddAdo, further implicating a cAMP/PKA pathway in this action of gAd.

3.5 Adiponectin does not increase apoptosis of human coronary artery endothelial cells
A TUNEL assay was used to evaluate whether adiponectin caused apoptotic death of HCAECs which might be responsible for the suppressed cell movement in the Boyden chamber or the wound-healing assays (Figure 2A). After 16 h, there was no evidence of apoptotic DNA fragmentation in the HCAECs treated with 3.0 µg/mL gAd or fAd in EBM2 basal medium containing 0.5% FBS. Increased TUNEL staining was clearly observed in the positive control samples treated with DNAse I.


Figure 2
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  		Figure 2 (A) Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labelling (TUNEL) assay to evaluate apoptosis in human coronary artery endothelial cells (HCAECs), following various treatments as indicated. TUNEL labelling with counterstaining of cell nuclei using 4',6'-diamidino-2-phenylindole (DAPI) was performed as described in Methods. Positive control cells were incubated with DNAse I to create DNA fragmentation. Representative fluorescent photomicrographs are shown. The white arrows indicate positive sites of endonucleolytic cleavage of chromatin. The bar in the lower right corner indicates 10 µm. (B) Suppression of VEGF-induced ROS generation by adiponectin in HCAECs measured by dichlorodihydrofluorescein (DCF) fluorescence. Intracellular generation of H2O2 in HCAECs was detected by fluorescence of 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCF-DA; catalogue number C-6827, Molecular Probes). Cells were loaded with CM-H2DCF-DA as described above prior to treatment with VEGF, and the ROS signal was recorded by fluorescence microscopy. To avoid photo-oxidation, the fluorescence image was collected by a single rapid scan with identical parameters for all samples.

 
3.6 Role of reactive oxygen species in the suppression of VEGF-induced human coronary artery endothelial cells migration by globular domain of adiponectin
As ROS have been shown to be integral to VEGF signalling in vascular cells, we also tested whether inhibition of ROS generation by gAd might also play a role in the suppression of VEGF-induced HCAEC migration (Figure 1B). Incubation of cells with H2O2 (100 µM) for 24 h mimicked the effect of VEGF, increasing cell migration by 74% over basal rate of cell migration (P < 0.001). Treatment with either DPI (10 µM) or apocynin (500 µM) blocked VEGF-induced cell migration to the same degree, by 83% (P < 0.001). These results are consistent with prior studies showing that VEGF-induced cell migration is ROS-dependent.23 Interestingly, gAd was also effective in blocking H2O2-induced cell migration by 61%, suggesting that it may act downstream of NADPH oxidase, or via cross-talk between signalling pathways converging on the cell migration response.

3.7 Adiponectin suppression of VEGF-induced generation of reactive oxygen species in endothelial cells
Intracellular generation of H2O2 in HCAECs was measured by the dichlorodihydrofluorescein (DCF) loading technique (Figure 2B). These data show a rapid stimulation of ROS production with VEGF stimulation that is completely suppressed by treatment with either gAd or fAd. VEGF-stimulated DCF fluorescence was blocked by the NADPH oxidase inhibitors DPI or apocynin. In addition, the effect of gAd to suppress VEGF-stimulated ROS production was inhibited by Rp-cAMP or ddAdo, implicating cAMP/PKA signalling in the mechanism of action of gAd.

3.8 Stimulation of cellular protein kinase A activity by adiponectin in human coronary artery endothelial cells
Several of the experiments described above implicate PKA signalling in the mechanism of action of gAd and fAd suppression of VEGF-induced responses in HCAECs. We directly measured PKA activity using a peptide substrate (kemptide) assay (Figure 3). VEGF did not affect cellular PKA activity. Treatment with either gAd or fAd, with or without VEGF stimulation, consistently elicited a 2.1-fold increase in PKA activity (P < 0.001). As expected, the adenylyl cyclase activator forskolin also increased PKA activity. These data are consistent with the results of the cAMP/PKA signalling inhibitors and further implicate PKA activation in the cellular mechanism of action of adiponectin.


Figure 3
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  		Figure 3 Cellular protein kinase A (PKA) activity in human coronary artery endothelial cells after various treatments. PKA activity in cell lysates was measured as phosphorylation of kemptide with [{gamma}-32P]ATP as described in Methods using a reagent kit and instructions provided by the manufacturer (Upstate). PKA activity was expressed as the phosphorylation of peptide (dpm) per 15 min/µg of protein. **P < 0.01; *P < 0.001 vs. the respective control samples without treatment with VEGF, adiponectin, or pharmacological agents.

 
3.9 Adiponectin induction of increased cyclic adenosine monophosphate levels in human coronary artery endothelial cells
Since adiponectin suppression of VEGF-induced cell migration was blocked by inhibitors of cAMP/PKA signalling, it was important to further demonstrate that adiponectin also increased cAMP levels in HCAECs. Compared with basal levels of cellular cAMP content (3.17 ± 0.05 pmoles/µg protein), treatment of HCAECs for 24 h with 3 µg/mL gAd increased cAMP levels by 56% to 4.93 ± 0.17 (P < 0.001); similarly, treatment with 3 µg/mL fAd increased cAMP levels by 61% to 5.10 ± 0.55 (P < 0.01). This result supports the cAMP/PKA pathway in the mechanism of adiponectin action in endothelial cells and indicates that adiponectin activates PKA via an upstream increase in cellular cAMP concentration.

3.10 Suppression of VEGF-induced ERK phosphorylation by adiponectin in human coronary artery endothelial cells
Activation of the MAPK pathway is also integral to endothelial cell migration and is known to be elicited by VEGF stimulation. We tested whether treatment of HCAECs with gAd or fAd reduced VEGF-induced ERK (Thr202/Tyr204) phosphorylation, and also if that reduction was dependent on cAMP and PKA signalling (Figure 4A). After serum deprivation for 16 h, VEGF stimulation of HCAECs for 15 min increased ERK phosphorylation ~19-fold (P < 0.001) over control. During the serum starvation period, HCAECs were also incubated with gAd or fAd for 16 h prior to stimulation with VEGF for 15 min. VEGF-induced ERK phosphorylation was dramatically suppressed by 92% following pre-treatment with gAd or fAd (P < 0.001). Furthermore, pre-treating the cells with the inhibitors of PKA or adenylyl cyclase, Rp-cAMP and ddAdo, respectively, for 30 min prior to stimulation with VEGF, significantly abrogated the suppressive effect of gAd on VEGF-induced ERK phosphorylation by 56 and 65%, respectively (P < 0.001). H2O2 alone also potently stimulated ERK phosphorylation by 22-fold over basal. Interestingly, gAd was ineffective at suppressing ERK phosphorylation following cellular stimulation with H2O2.


Figure 4
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  		Figure 4 (A) Effect of adiponectin on vascular endothelial growth factor (VEGF)-induced ERK phosphorylation. Human coronary artery endothelial cells (HCAECs) were serum-starved with or without globular domain of adiponectin or full-length forms of adiponectin for 16 h and then stimulated with VEGF in the presence of the indicated reagents as described in Methods. After stimulation, sodium dodecyl sulphate–polyacrylamide gel electrophoresis and western blot analysis were performed using antibodies specific to phospho-ERK1/2 and ERK1/2. Similar results were obtained from replicate experiments and the mean data are shown. *P < 0.001 vs. VEGF-stimulated ERK phosphorylation. (B) Effect of adiponectin on VEGF-induced Akt phosphorylation. HCAECs were cultured and treated as described in the legend to Figure 4A with VEGF without or with adiponectin isoforms as indicated. Western blot analysis was performed using antibodies specific to phospho-Akt (p-Ser473) and Akt total protein. A representative experiment is shown along with mean data from replicate experiments. *P < 0.001 vs. VEGF-stimulated Akt phosphorylation.

 
3.11 Suppression of VEGF-induced Akt phosphorylation by adiponectin in human coronary artery endothelial cells
Akt has also been strongly implicated in the migration response to VEGF in endothelial cells. Accordingly, we tested whether treatment of HCAECs with gAd or fAd affected VEGF-stimulated Akt phosphorylation at the activating Ser473 site (Figure 4B). VEGF stimulation increased phospho-Akt levels 3.2-fold over control. Treatment with either gAd or fAd for 16 h, prior to VEGF stimulation, for the last 15 min of the experiment resulted in 29% and 48% suppression of the Akt phosphorylation induced by VEGF alone (P < 0.001).

3.12 Suppression of VEGF-induced stress fibre formation by adiponectin
In addition to signalling pathways, we next studied whether adiponectin can suppress VEGF-induced formation of actin stress fibres. HCAECs were subjected to serum deprivation with or without gAd or fAd for 16 h before and during stimulation with VEGF for 2 h. Actin stress fibres were visualized by staining cells for F-actin filaments using fluorescently-labelled Alexa Fluor 594 phalloidin (Figure 5A). As expected, stimulation of HCAECs with VEGF clearly increased stress fibre formation, which was suppressed by co-incubation with either gAd or fAd, as shown.


Figure 5
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  		Figure 5 (A) Inhibition of vascular endothelial growth factor (VEGF)-induced actin stress fibre formation by adiponectin. Human coronary artery endothelial cells (HCAECs) grown on cover-slips were starved for 16 h in serum-free medium containing 0.5% BSA. Some of the cells were pre-treated with globular domain of adiponectin and full-length form of adiponectin for 16 h, followed by treatment with VEGF for 15 min in the presence of activators or inhibitors as indicated and described in Methods. Actin filaments were stained with Alexa Fluor 594 phalloidin and nuclei with Hoechst 33342. (B) Inhibition of VEGF-induced focal adhesion formation by adiponectin. Cells were treated as in (A) but paxillin primary antibody was used to visualize focal adhesions, which are indicated by the white arrows. Alexa Fluor 555 conjugated probe was used as secondary antibody (Molecular Probes). The bar in the lower right corner represents 10 µm.

 
Inhibitors of PKA and adenylyl cyclase also reduced the effect of gAd on suppression of VEGF-induced stress fibre formation (Figure 5A). Furthermore, the adenylyl cyclase activator forskolin reduced stress fibre formation by VEGF that is consistent with the hypothesis that the effect of gAd on stress fibre formation is at least partially via the cAMP signalling pathway. Hydrogen peroxide (100 µM) treatment increased the stress fibre formation over basal in a manner that was not suppressed by incubation with gAd.

3.13 Suppression of VEGF-induced focal adhesions by adiponectin
To examine the abundance of focal adhesions, cells were stained for paxillin (Figure 5B). VEGF stimulation of HCAECs for 2 h significantly increased the number and size of focal adhesions compared with untreated cells. Incubation of cells for 16 h with gAd before and during VEGF stimulation completely blocked the effect of VEGF on focal adhesion formation. Consistent with the effects of Rp-cAMP and ddAdo on gAd suppression of stress fibre formation by VEGF, these inhibitors also abolished the gAd suppression of focal adhesion formation by VEGF. Similarly, forskolin prevented the increase in focal adhesion formation induced by VEGF, also consistent with the hypothesis that the effect of gAd is transduced via the cAMP signalling pathway. H2O2 (100 µM) treatment dramatically increased the focal adhesion formation and their size when compared with basal. Incubation with gAd did not influence focal adhesion formation induced by H2O2.

3.14 Suppression of VEGF-induced RhoA activation by adiponectin
We determined whether VEGF-induced RhoA activity can also be suppressed by adiponectin, and if this suppression is also dependent on cAMP and PKA signalling. RhoA activity was assayed using a pull-down assay with the fusion protein glutathione-S-transferase-Ras binding domain (GST-RBD), which recognizes only RhoA-GTP, the active form of RhoA.19 VEGF stimulation of HCAECs for 15 min increased RhoA activity by 2.1-fold (Figure 6). Pre-incubation with gAd for 16 h and during VEGF stimulation suppressed basal RhoA-GTP accumulation by 81% and completely abrogated the stimulation of RhoA-GTP formation by VEGF. Pretreatment of cells with Rp-cAMP or ddAdo reduced the suppressive effect of gAd on VEGF-stimulated accumulation of active RhoA by 55% and 62%, respectively.


Figure 6
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  		Figure 6 Suppression of VEGF-induced RhoA activation by adiponectin in human coronary artery endothelial cells (HCAECs). HCAECs were serum-starved and pre-incubated with or without globular domain of adiponectin for 16 h and then stimulated with VEGF for 15 min in the presence of Rp-cAMP or ddAdo. After treatment, cleared cell lysates were incubated with rhotekin-RBD beads for 1 h at 4°C on a rotator. Rhotekin-RBD beads were collected, washed, and subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis, followed by western blotting with anti-RhoA monoclonal antibody. Parallel samples of the cell lysates were blotted for total RhoA protein. The mean results of duplicate determinations normalized to total RhoA protein are shown in the bar graph.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
Recombinant gAd and fAd both suppressed endothelial cell migration induced by VEGF. Pharmacological inhibition of either adenylyl cyclase or PKA significantly abrogated the effect of gAd to suppress VEGF-induced cell migration. Similarly, adiponectin suppressed VEGF-induced ROS generation, activation of ERK/MAPK and Akt, induction of formation of actin stress fibres and focal cellular adhesions, and activation of RhoA processes that are all integral to VEGF-induced endothelial cell migration, were each significantly abrogated by inhibition of cAMP generation or PKA activity. Direct measurements also showed that treatment of HCAECs with gAd or fAd increased cellular levels of cAMP and the enzymatic activity of PKA. Overall, these data demonstrate that adiponectin suppresses VEGF-stimulated cell migration in a cAMP/PKA-dependent manner and support an important role for adiponectin in endothelial responsiveness to VEGF.

In obesity and diabetes, cytokines such as TNF-{alpha}, high circulating levels of free fatty acids, and increased plasma glucose levels contribute to the early stages of vascular impairment in atherogenesis.24,25 As each of these agonists of endothelial function act via unique mechanisms, we have been interested in understanding the mechanisms by which adiponectin can suppress their collective adverse cellular effects. Early studies by Ouchi et al.4 showed that adiponectin inhibited TNF-{alpha}-induced expression of adhesion molecules VCAM-1, E-selectin, and ICAM-1 on the endothelial cell surface and adhesion of monocytic THP-1 cells to cultured endothelial cells, processes that are characteristic of inflammatory endothelial responses.26 Other studies have highlighted the anti-inflammatory nature of adiponectin signalling to oppose activation of the I{kappa}B-NF{kappa}B cascade.5,27 Recently, we also demonstrated that replenishment of adiponectin exhibits anti-inflammatory signalling in vivo in a knock-out mouse model by reversing a baseline state of endothelial dysfunction characterized by upregulation of endothelial cell adhesion molecules and deficient NO generation.28

Our laboratory initially reported that adiponectin suppresses cellular ROS generation stimulated by oxidized LDL or high glucose, a cellular effect that may enhance the protective effects of adiponectin on endothelial function.6,7 Various agonists known to induce endothelial dysfunction, initiate inflammatory signalling and impair NO generation, including glucose, cytokines, and free fatty acids, also cause excessive systemic and cellular ROS production.29 A major finding in the present study is that adiponectin also suppresses ROS production associated with cellular VEGF treatment, a signalling transduction pathway integral to VEGF-induced cell migration,23 suggesting that adiponectin may play a broad antioxidant role in the vasculature. Adiponectin suppression of ROS generation induced by high glucose in endothelial cells was mediated by increased cAMP-content PKA activity,7 which also plays a role in adiponectin signalling in macrophages.30

The signalling mechanism by which adiponectin suppresses VEGF-induced ROS is unknown. Both forms of adiponectin effectively suppressed VEGF-induced fluorescence of DCF, reflecting inhibition of overall cellular ROS availability. As we previously showed, gAd suppresses endothelial ROS generation in high glucose conditions, which is believed to be derived from overproduction of mitochondrial superoxide.31,32 In contrast, an NADPH-oxidase linked mechanism has been linked to many, but perhaps not all of VEGF signalling in endothelial cells via ROS generation in endothelial cells.23,33 Recent studies have also proposed a link between PKA and suppression of cellular ROS generation, e.g. downstream of adrenomedullin action in vascular smooth muscle cells.34

Controversy exists over whether gAd is a naturally occurring product of cleavage of fAd in vivo, or acts as a pharmacological agent.1,35,36 Nevertheless, many studies have demonstrated that gAd exerts potent anti-inflammatory pharmacological effects in vascular cells, including our recent report in vivo in a mouse model.28 It is also possible that the effect of fAd on vascular endothelial responses are mediated via the globular domain of the protein.

Our findings are generally consistent with those of Bråkenhielm et al.37 who showed that fAd inhibited migration of porcine aortic endothelial (PAE) cells overexpressing VEGF-2 receptors. These authors also found adiponectin suppressed fibroblast growth factor (FGF)-2-stimulated proliferation of bovine capillary endothelial (BCE) cells and FGF-2-induced proliferation of FGF receptor 1-overexpressing PAE cells. We also previously reported that adiponectin suppressed oxidized LDL-induced proliferation of bovine aortic endothelial cells, assessed by thymidine incorporation and determination of cell numbers.6 Bråkenhielm et al.37 also reported that adiponectin induced apoptotic body formation in BCE and human dermal microvascular endothelial cells and caspase activation in BCE cells. However, in the present study, we did not detect apoptosis in adiponectin-treated HCAECs by TUNEL assay, in agreement with the report by Kobayashi et al.38 in serum-starved human umbilical vein endothelial cells that adiponectin suppressed caspase-3 activation and apoptosis. The discrepant results on apoptosis may represent cell type-specific or technical differences; nevertheless, the finding of adiponectin suppression of endothelial cell proliferation and migration is now reproducibly demonstrated in a variety of experimental settings.

The protective role of cAMP-dependent PKA in maintaining vascular integrity and preventing vascular leakage induced by inflammatory mediators is well established.39 PKA phosphorylates myosin light chain kinase, reducing its activity and stimulates phosphorylation of the actin-binding proteins, filamin and adductin; the focal adhesion proteins, paxillin and FAK; and promotes the disappearance of stress fibres and F-actin accumulation in membrane ruffles. PKA-mediated modulation of Rho GTPase activity also plays an essential role in the regulation of actin cytoskeletal organization.40,41 PKA phosphorylates RhoA and decreases Rho association with Rho-kinase and increasing the interaction of RhoA with Rho-GDP dissociation inhibitor (Rho-GDI), which enhances RhoA translocation from the membrane to the cytosol.42,43 Thus, PKA inhibits RhoA activity and stabilizes the actin cytoskeleton, inhibiting endothelial cell mobilization and migration. Our findings in the present work support a key role for adiponectin in this process via a cascade of cAMP/PKA signal transduction.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
National Institutes of Health (DK63018 to B.J.G.; DK71360 to B.J.G.).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

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