From the
Departments of Medical Physiology and ¶Internal Medicine in the Cardiovascular Research Institute, College of Medicine, The Texas A&M University System Health Science Center, Temple, Texas 76504
Received for publication, January 13, 2003 , and in revised form, March 21, 2003.
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ABSTRACT |
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INTRODUCTION |
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Ocular angiogenesis in the posterior region likely comes from either choroidal or retinal vascular beds. The current opinion is that age-related macular degeneration results from choroidal neovascularization, which destroys retinal pigmented epithelial cells, and blood vessels that invade the photoreceptor layer (3). Diabetic retinopathy, on the other hand, is thought to result from the abnormal growth of retinal blood vessels (1). Both, however, produce a significant loss of vision or blindness. Although many hypotheses have been put forward to explain the abnormal vascular growth in both of these diseases, a definitive answer is not yet available. Most will agree that both of these diseases result from an imbalance of angiogenic versus angiostatic factors.
While investigating sympathetic nerve regulation of choroidal blood flow, we found that blood flow to the treated eye increased over 4-fold following superior cervical ganglionectomy and was not altered in other cranial structures (9). Sympathectomy also produced significant increases in the number of choroidal venules and arterioles and the retinal capillaries in the outer nuclear layer (9). Additionally, we determined that the -adrenergic receptor antagonist propranolol (which blocks
1- and
2-adrenergic receptors) produced changes in the choroid similar to those after a sympathectomy (10). Because the
3-adrenergic receptor was thought to be found primarily in white and brown adipose tissues (11), we did not characterize the role of this receptor after sympathectomy.
-adrenergic receptor subtypes have been located on some types of endothelial cells (12, 13). In the present study, we sought to determine which subtypes of
-adrenergic receptors were present on retinal endothelial cells. We also characterized the signaling pathways associated with the activation of these receptors. Finally, we determined whether activation of
-adrenergic receptors in cultured retinal endothelial cells could mediate proliferation and migration, two markers of an angiogenic phenotype.
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EXPERIMENTAL PROCEDURES |
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Human microvascular endothelial cells were isolated by enzymatic digestion of blood vessels taken from the mesentery of the small bowel and cloned by limiting dilution (designated MM1 cells). Endothelial cell identity was verified by positive staining for a factor VIII-related antigen.
Pharmacological Activators and InhibitorsStimulation of the 3-adrenergic receptor was achieved by application of the specific receptor agonist BRL37344 (10 µM). Xamoterol (10 µM) was used as a specific
1-adrenergic receptor agonist. Both activators were obtained from Tocris (Ellisville, MO).
Protein kinases A and G were blocked with KT5720 (1 µM) and KT5823 (1 µM). Src was blocked by PP2 (1 µM), and the inactive isoform PP3 (1 µM) served as a negative control. Wortmannin (100 nM) or LY294002 (2 µM) were used to inhibit phosphatidylinositol-3-kinase activity; the inactive isoform LY303511 (100 µM) served as a negative control. The phosphatidylinositol ether analog 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (10 µM), referred to as Akt-I1 in the remainder of the text, was used as a potent and selective inhibitor of Akt (protein kinase B). The MAP kinase pathway was blocked using the specific MEK inhibitor PD98059 (10 µM). Matrix metalloproteinases (MMPs) 2 and 9 were blocked with MMP-2/MMP-9 inhibitor III (1 µM), abbreviated as MMP-I in the remainder of the text. All inhibitors were obtained from Calbiochem, except wortmannin (Tocris, Ellisville, MO).
-Adrenergic Receptor Protein ExpressionWestern blotting was conducted to determine which
-adrenergic receptor subtypes were present on retinal endothelial cells (REC; passage 36), choroidal endothelial cells (ChEC; passage 26), or human microvascular mesenteric endothelial cells (MM1; passage 1015). Cells in 60-mm dishes were lysed (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml each of aprotinin, leupeptin, and pepstatin and 1 mM Na3VO4,1mM NaF, and 0.1% SDS), and 50 µg of protein was separated on a 412% pre-cast polyacrylamide gel (Invitrogen), blotted onto a nitrocellulose membrane, and blocked with Super BlockTM (Pierce) for 1 h at room temperature. Primary antibodies to the
1-(A-20; 5 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA),
2-(H-73; 5 µg/ml; Santa Cruz Biotechnology), or
3-adrenergic receptor (C-20; 5 µg/ml; Santa Cruz Biotechnology) were applied overnight at 4 °C. Membranes were probed with horseradish peroxidase-conjugated anti-rabbit secondary antibodies applied at a 1:10,000 dilution for 2 h at room temperature. Immunoreactive bands were detected by enhanced chemiluminescence (LumniGlo, Cell Signaling, Beverly, MA) using Kodak BioMax ML film and scanned into the computer using reflectance scanning.
Western blots to evaluate the phosphorylation states of MAPK (phospho p42/44,1:1000, ERK1/2, Cell Signaling), Src (phospho Src-416, 1:1000, Cell Signaling), or Akt (phospho-Akt, Ser473, 1:1000, BIO-SOURCE) were done as described above following stimulation of the 3-adrenergic receptor.
Radioligand Binding Assays-adrenergic subtype levels were quantified on intact human retinal endothelial cells by modification of previously described methods (14, 15) using the hydrophilic
-adrenoreceptor ligand ()[5,7-3H]CGP 12177 (33 Ci/mmol; PerkinElmer Life Sciences). To prevent receptor internalization, binding studies were performed at 4 °C. Cells were grown in 12-well plates and serum-starved for 24 h. For determination of saturation binding, cells were incubated in 0.3 ml of serum-free medium (1% protease-free bovine serum albumin, 25 mM HEPES (pH 7.4 at 4 °C), 100 IU/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B) and increasing amounts of [3H]CGP 12177 per well. To assess displacement of [3H]CGP 12177 binding by the
3-adrenergic receptor agonist BRL37344 and the
1/2-receptor antagonist propranolol, cells were incubated with 25 nM [3H]CGP 12177. Three wells of cells were used per condition, and all binding assays were performed three times. Binding was carried out for 6 h, as preliminary studies established that equilibrium binding was reached at that time. Afterward, the binding medium was aspirated, and the cells were washed twice with 0.5 ml of ice-cold Hanks' balanced salt solution with 1% bovine serum albumin. Cells were digested by incubation at 22 °C for 10 min in 0.5 ml of 1% Triton X-100 with 1% bovine serum albumin. Radioactivity in cell digests was measured in a liquid scintillation counter (Beckman Coulter LS6500), using Ecolite(+) mixture (ICN Pharmaceuticals). Specific [3H]CGP 12177 binding was defined as the difference between total and nonspecific binding, with the latter determined by incubating cells with various concentrations of [3H]CGP 12177 and 100 µM nonlabeled ligand. Data obtained from the saturation binding experiments were analyzed by Prism 3.0 (GraphPad Software). Binding results were best fitted to a two-site model (r2 = 0.89 ± 0.05), which was verified by Scatchard analysis.
Retinal Endothelial Cell MigrationAll migration assays were done using BD BioCoat angiogenesis system endothelial cell invasion plates (BD Biosciences) according to supplied protocols with little variation. Briefly, 100,000 retinal endothelial cells were added to top chambers in 250 µl of medium. Starvation medium, agonists, and inhibitors were added to each of the lower chambers. All inhibitors were added for 30 min before agonist application to allow for complete pathway blockade. No cells were added to the bottom chamber so that background fluorescence could be subtracted from post-migration values. Plates were allowed to incubate for 2629 h at 37 °C to allow for migration through the Matrigel-coated membrane. Chambers were transferred to wells containing calcein AM, a fluorophore taken up by cells that have invaded the membrane (16, 17) (Molecular Probes, Eugene, OR), in Hank's balanced salt solution for 1.5 h and then read on a fluorescent plate reader (Bio-Tek, Winooski, VT, Model FL600, gain of 100) at 485/530 nm. Migration of cells was expressed as a percentage of control (those receiving only starvation medium) after background fluorescence was subtracted. Data were analyzed using Prism software (GraphPad, San Diego, CA) and presented with significance at p < 0.05.
Proliferation AssayEndothelial cell proliferation was assessed using an assay based on the cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases. Expansion in the number of viable cells results in an increase in the overall activity of the mitochondrial dehydrogenases in the sample. The augmentation in enzyme activity leads to an increase in the formazan dye formed. The formazan dye produced by viable cells can be quantified by a multiwell spectrophotometer by measuring the absorbance of the dye solution at 440 nm.
To perform the experiments, an aliquot of 50,000 retinal endothelial cells was added to each well of a 96-well tray in medium with 10% fetal bovine serum. After cells attached to the 96-well tray, the cells were washed, and high serum medium was replaced with starvation medium overnight. All wells were rinsed with phosphate-buffered saline. Negative control wells received starvation medium, and positive control wells received agonist only. Inhibitors were added 30 min prior to application of an agonist to allow for complete blockade. Controls treated with inhibitor alone were also included to determine their effect on proliferation. Cells were allowed to incubate for 48 h. At this time, the WST-1 reagent, dissolved in Electro Coupling Solution (Chemicon), was applied for 4 h to measure cell proliferation. The plates were read on a spectrophotometer, and data were presented as a percentage of negative control proliferation with p < 0.05 being significant.
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RESULTS |
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Binding of [3H]CGP 12177 to Human Retinal Endothelial CellsSaturation radioligand binding studies were performed to quantify the levels of -adrenergic receptor subtypes on human retinal endothelial cells. Fig. 2A shows the specific binding of the hydrophilic
-adrenoreceptor ligand [3H]CGP 12177 to human retinal endothelial cells. Analysis of the saturation binding results revealed the presence of two populations of binding sites with high and low affinity for [3H]CGP 12177. The Kd of the high affinity site was determined to be 0.06 ± 0.02 nM (n = 3), and the Kd of the low affinity site was 25 ± 4 nM (n = 3). The presence of two populations of [3H]CGP 12177 binding sites of high and low affinity was confirmed by Scatchard analysis (Fig. 2B). The total number (Bmax) of high and low affinity binding sites was determined to be 0.037 ± 0.003 and 0.071 ± 0.023 fmol/µg protein, respectively. Based on comparable Kd values reported by others (15), the high and low affinity sites would represent binding of [3H]CGP 12177 to
1- and
3-adrenergic receptors, respectively. Using a concentration of [3H]CGP 12177 equal to the Kd of the low affinity binding site (25 nM), the
3-adrenergic receptor agonist BRL37344 at 10 µM displaced 37.0 ± 5.3% of specific radioligand binding. The remainder of the specific binding was displaceable with the
1/2-receptor antagonist, propranolol.
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Stimulation of 3-Adrenergic Receptors with BRL37344 Phosphorylates Several Known Signaling MoleculesAdministration of BRL37344 produced significant phosphorylation of Akt versus non-treated retinal endothelial cells (Fig. 3, A and B). Similarly, activation of
3-adrenergic receptors also increased phosphorylation of both ERK1/2 (Fig. 3, C and D) and Src (Fig. 3, E and F; *, p < 0.05 versus non-treated controls).
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3-Adrenergic Receptor But Not
1-Adrenergic Receptor Stimulation Increases Retinal Endothelial Cell MigrationActivation of the
3-receptor produced a 43 ± 7% increase in retinal endothelial cell migration (p < 0.001, Fig. 4A). This could be attenuated by inhibition of phosphatidylinositol 3-kinase (PI3K) with either LY294002 (p < 0.001) or wortmannin (p < 0.001), whereas the non-active isoform LY303511 had no effect. Retinal endothelial cell migration elicited by
3-adrenergic receptor activation was also diminished by inhibition of Akt (p < 0.05), MMP2/MMP9 (p < 0.001), or MEK (p < 0.01). Migration induced by BRL3744 was not diminished in the presence of blockers of protein kinase A, protein kinase G, or Src. With the exception of MMP-I, the inhibitors alone had no significant effect on migration (Fig. 4B). Treatment of retinal endothelial cells with the
1-adrenergic agonist xamoterol did not produce a significant increase in migration over control values.
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3-Adrenergic Receptor But Not
1-Receptor Activation Promotes Retinal Endothelial Cell ProliferationStimulation of
3-adrenergic receptors on retinal endothelial cells with BRL37344 produced a 28 ± 4% increase in proliferation (p < 0.001, Fig. 5A). This proliferation could be completely blocked by inhibitors of Src (p < 0.01), PI3K (LY294002, p < 0.01; wortmannin, p < 0.001), or MEK (p < 0.001). The administration of Akt-I, KT5720, or KT5823 did not abolish the BRL37344-stimulated retinal endothelial cell proliferation. No pharmacological agents altered proliferation when given in the absence of BRL37344 (Fig. 5B). Stimulation of
1-adrenergic receptors with xamoterol did not significantly affect retinal endothelial cell proliferation (Fig. 5C).
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DISCUSSION |
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3-Adrenergic Receptors in Retinal Endothelial Cell ProliferationOthers have noted that
3-adrenergic receptors linked to G-coupled proteins promote phosphorylation of Src and the subsequent activation of ERK1/2 of the MAPK pathway (18) in adipose tissue. It was assumed that this activation was responsible for lipolysis and thermoregulation. Activation of these receptors in retinal endothelial cells by BRL37344 promoted a similar response (Fig. 5A), which was comparable with that noted in response to Eph B4 stimulation.2 Since LY294002 and wortmannin prevented retinal endothelial cell proliferation, it is clear that PI3K is involved. LY294002 and wortmannin prevented cell proliferation, it is clear that PI3K is involved. It has been reported that Src can phosphorylate PI3K in addition to activating ERK1/2 (19) in receptors linked to G-proteins. Therefore, this may be the pathway for ERK1/2 regulation of retinal endothelial cell proliferation after the administration of BRL37344. Although ERK1/2 can be stimulated via nitric oxide production and protein kinase G activation after Akt phosphorylation (20), this is not likely in these retinal endothelial cells, because neither the Akt-inhibitor or KT5823 blocked proliferation. Furthermore, the protein kinase A inhibitor KT5720 did not affect proliferation, suggesting that cAMP is not involved in retinal endothelial cell proliferation following
3-adrenergic receptor stimulation. Stimulation of cultured retinal endothelial cells with xamoterol, a
1-adrenergic receptor agonist, did not stimulate proliferation. Therefore,
3-adrenergic receptors appear to mediate the changes in retinal endothelial cell proliferation via Src, PI3K, and MAPK, whereas Akt, protein kinase G, protein kinase A, and
1-adrenergic receptors have little effect.
3-Adrenergic Receptor-mediated Migration of Retinal Endothelial CellsRelative to proliferation, much less has been demonstrated for the role of G-protein-coupled receptors in cell migration. Using BRL37344 to stimulate the
3-adrenergic receptors on retinal endothelial cells, we showed that these receptors could regulate migration. Stimulation increased migration through a Matrigel-coated membrane by >37%. The increased migration in response to BRL37344 is similar to that noted after Eph B4 treatment of this same cell type.3 The BRL37344-mediated migration could be antagonized by prior administration of inhibitors of PI3K, Akt, MEK, and MMP-2/MMP-9. PI3K appears to be directly linked to the
3-adrenergic receptor. Similar signaling designs have been reported previously in models of heart disease and
-adrenergic receptors (21).
-adrenergic receptors may exhibit kinase activity and are designated as
ARK. In this situation,
ARK recruits PI3K to the plasma membrane of the myocardium and leads to its phosphorylation (21). PI3K is then able to phosphorylate Akt and lead to the induction of matrix metalloproteinase 2 and 9 (20). Additionally, Akt can activate ERK1/2 of the MAPK pathway, as occurs in retinal endothelial cells stimulated with Eph B4.2 The Src inhibitor PP2 did not alter retinal endothelial cell migration. Unlike what was observed with proliferation, G-protein-coupled phosphorylation of Src does not initiate the signaling for migration. As was noted in retinal endothelial cell proliferation, activation of the
1-adrenergic receptors does not appear to modulate cell migration. Thus, it appears that
3-adrenergic receptors, not
1-adrenergic receptors, are involved in retinal endothelial cell migration via PI3K, Akt, MMP 2 or 9, and ERK1/2.
The results from these studies demonstrate for the first time that 3-adrenergic receptors exist on human retinal endothelial cells. Alterations in sympathetic nerve activity may contribute to vascular complications of diabetes. It may be that diabetic retinopathy results from changes in
3-adrenergic receptor signaling. If these in vitro studies on cultured cells can be extrapolated to the intact retinal circulation, then the signaling of
3-adrenergic receptors may be capable of initiating retinal endothelial cell proliferation and migration, both of which are critical stages of angiogenesis.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Medical Physiology, Cardiovascular Research Inst., The Texas A&M University System Health Science Center, 702 S.W. H. K. Dodgen Loop, Rm. 202A, Temple, TX 76504. Tel.: 254-742-7144; Fax: 254-742-7145; E-mail: jsteinle{at}tamu.edu.
1 The abbreviations used are: Akt-I, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; MMP, matrix metalloproteinase; PI3K, phosphatidylinositol 3-kinase.
2 Steinle, J. J., Meininger, C. J., Chowdhury, U., Wu, G., and Granger, H. J. (2003) Cell. Signal., in press.
3 J. J. Steinle, C. J. Meininger, U. Chowdhury, G. Wu, and H. J. Granger, submitted for publication.
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REFERENCES |
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