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Am J Physiol Heart Circ Physiol 282: H739-H748, 2002; doi:10.1152/ajpheart.00656.2001
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Vol. 282, Issue 2, H739-H748, February 2002

Angiotensin II stimulates migration of retinal microvascular pericytes: involvement of TGF-beta and PDGF-BB

Jose A. Nadal, G. M. Scicli, L. A. Carbini, and A. Guillermo Scicli

Eye Care Services Research, Henry Ford Health System, Detroit, Michigan 48202-3450


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We studied the promigratory effect of angiotensin II (ANG II) on cultured bovine retinal microvascular pericytes. ANG II stimulated migration of pericytes by 86% at 10-8 M, but this effect was lost at 10-4 M. Migratory responses were inhibited by the ANG II type 1 (AT1) receptor antagonist losartan but not by PD-123319, an AT2 antagonist. Addition of PD-123319 to the 10-4 M ANG II dose restored migratory responses. The promigratory effect of ANG II (10-7 M) was reduced by 59% in absence of gradient. Although ANG II augmented the latent matrix metalloproteinase-2 (MMP-2) activity of the pericyte by 35%, it also doubled tissue inhibitors of MMPs. ANG II-induced migration was not altered by a broad-spectrum MMP inhibitor (GM6001); it was inhibited by ~50% by antibodies against transforming growth factor (TGF)-beta 1/2/3 and was abolished by antibodies against platelet-derived growth factor (PDGF)-BB. We conclude that ANG II induces chemotactic responses on retinal microvascular pericytes acting through the AT1 receptor. This effect is opposed by the AT2 receptor. ANG II-induced chemotaxis is mediated by PDGF-BB and involves TGF-beta , but it is independent of MMP activity. It is also independent of vascular endothelial growth factor (VEGF) because VEGF did not stimulate pericyte migration. ANG II can contribute to the regulation of retinal neovascularization by stimulating pericyte migration.

vasoactive peptides; angiogenesis; eye; renin-angiotensin system; transforming growth factor-beta ; platelet-derived growth factor-BB


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE RENIN-ANGIOTENSIN SYSTEM is present in the eye, and concentrations of angiotensin II (ANG II) in the retina are reportedly much higher than in plasma (11). ANG II is produced by angiotensin-converting enzyme (ACE) from the inactive circulating precursor ANG I. There are two ANG II receptor subtypes: AT1 and AT2. Most of the traditional vascular effects of ANG II are mediated by the AT1 receptor, but an increasing number of data suggest that some cellular responses are mediated by the AT2 receptor (13, 18).

Treatment with ACE inhibitors has been reported to be beneficial in diabetic retinopathy, a disease characterized by neovascularization, vascular leakage, and pericyte fall off (9, 20, 65). ACE inhibitors also decrease hyperoxic retinopathy in rodents (50, 54). ACE degrades bradykinin, N-acetyl-seryl-aspartyl-lysyl-proline (SDKP), and other peptides, but most of the cardiovascular effects of ACE inhibitors are mimicked by ANG II AT1 receptor antagonists (19). This suggests that most of the chronic effects induced by ACE inhibitors involve lowering ANG II concentrations. Translated to the eye, these data suggest that ANG II plays a role in the regulation of retinal neovascularization, although the mechanism involved remains unclear.

Microvessels are formed by two main cell types: endothelial cells and perimural cells, which wrap the endothelium. Under normal conditions, the endothelial cells forming the capillary wall are quiescent. It is thought that perimural cells contribute to stabilization of the endothelium by inhibiting endothelial cell migration and proliferation (14, 29, 57, 63). Angiogenesis involves a coordinated sequence of events beginning with detachment and migration of perimural cells, which is thought to render the endothelial cells responsive to growth factors. If growth factors such as vascular endothelial growth factor (VEGF) are present, this may lead to an angiogenic response (2, 7). The final steps in angiogenesis also involve differential recruitment of associated supporting perimural cells to the newly formed vasculature and their attachment, thus leading to a stable (mature) neovascular network (21, 27, 29). Transforming growth factor (TGF)-beta and platelet-derived growth factor (PDGF)-BB are involved in these processes. In retinal microvessels, the perimural cells are pericytes. Consequently, substances capable of stimulating migration of pericytes would also be potentially involved in regulating retinal neovascularization.

Locally and systemically generated ANG II may influence vascular function in both health and disease by a number of mechanisms, including its well-documented ability to induce migration of perimural cells such as vascular smooth muscle cells (VSMC) and glomerular mesangial cells (41, 42). Extending these data to retinal microvessels, we asked whether ANG II influences migration of retinal microvascular pericytes. Here, we show that ANG II increases migration of cultured retinal pericytes via its AT1 receptors, acting as a chemoattractant. We further characterized the mechanisms whereby ANG II stimulates migratory behavior of retinal pericytes in vitro. We studied whether its effects on pericyte migration involve alteration of the pattern of expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) and implicate TGF-beta , PDGF-BB, and VEGF.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Human ANG II was purchased from Bachem (Torrance, CA). The AT1 receptor antagonist losartan was obtained from DuPont-Merck (Wilmington, DE). The AT2 receptor antagonist PD-123319 was a generous gift from Warner-Lambert, a division of Pfizer (Ann Arbor, MI). PDGF-BB was obtained from Collaborative Biomedical Products (Bedford, MA), and the neutralizing sheep IgG anti-human PDGF-BB (not cross-reacting with PDGF-A) was obtained from Biomedical Technologies (Stoughton, MA). TGF-beta 2 and the neutralizing antibody against TGF-beta 1/2/3 (Celtrix; Santa Clara, CA) were provided by Dr. Bruce Riser (61). A second antibody against TGF-beta 1/2/3, rabbit IgG, was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against AT1 and AT2 ANG II receptors and rabbit IgG were purchased from Alpha Diagnostic International (San Antonio, TX). The broad-spectrum MMP inhibitor N-[(2R)-2-(hydroxyamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophanmethylamide (GM6001) was purchased from Calbiochem (Temecula, CA). 3G5 antibody was a kind gift from Dr. R. C. Nayak (Joslin Diabetes Center; Boston, MA). Anti-human smooth muscle cell alpha -actin antibody, clone A14 mouse IgG was obtained from Sigma (St. Louis, MO). Fetal bovine serum was purchased from HyClone (Logan, UT), and DMEM was purchased from GIBCO-BRL (Gaithersburg, MD). All other reagents were obtained from Sigma.

Isolation and Culture of Bovine Retina-Derived Pericytes

Bovine retina-derived microvascular pericytes were obtained by a modification of the method described by Kennedy et al. (37). Eyes from recently slaughtered calves were immersed in Betadine, rinsed in sterile 0.9% saline, and stored in sterile saline overnight at 4°C. The following morning, the eyes were cleaned of fat, rinsed with 70% ethanol, and dissected, and the retinas were harvested under sterile conditions. Retinas were homogenized at a ratio of 1 retina to 10 ml of cold homogenization buffer [HB; containing 1× Hank's salt solution, 26 mM HEPES, 4.0 mM NaHCO3, and 0.13 g/l BSA (fraction V, Calbiochem); pH 7.4] using a Wheaton glass-Teflon homogenizer. The homogenate was sequentially sieved through 210-µm (Spectra/Mesh polypropylene) and 70-µm filters (Spectra/Mesh fluorocarbon; both from Spectrum Laboratories; Laguna Hills, CA). The microvessel-enriched fraction retained on the 70-µm mesh was recovered by washings with HB and plated on four T-25 flasks precovered with 2% gelatin and incubated at 37°C in a 95% air-5% CO2 atmosphere in 20% fetal bovine serum and DMEM (4.5 g/l glucose). Confluent cells were split into two T-75 flasks. Up to the second subculture, cells were grown with medium supplemented with 150 U/ml penicillin, 150 µg/ml streptomycin, and 250 µg/ml gentamycin. Cells from three to six passages were used in all experiments.

Immunocytochemistry

Autoclaved 13-mm glass coverslips were placed in a 24-well plate and precoated with 2% gelatin. Fifty thousand pericytes per well were grown in normal growth medium to a density of ~50%. The medium was removed, and cells were fixed with 3.7% p-formaldehyde for 10 min and then washed with glycine-phosphate-buffered saline (PBS) (0.15 M glycine in 0.1 M PBS).

Smooth muscle cell alpha -actin. After fixation, cells were extracted with 0.1% saponin, washed with PBS, incubated with 1/400 anti-smooth muscle cell alpha -actin antibody clone A14 in PBS and 1% BSA. After PBS washings, FITC-conjugated secondary antibody was applied for 30 min and washed with PBS. Coverslips were mounted in anti-fade medium.

3G5 membrane ganglioside. After fixation, cells were washed in PBS, incubated with 1/40 anti-3G5 antibody for 1 h, and then washed again in PBS. After FITC-conjugated secondary antibody was applied for 30 min, cells were washed, and coverslips were mounted in anti-fade medium (39, 56). Images were taken with a Sony 3CCD camera attached to a Nikon microscope.

Cell Migration Assay

Tested substances were added to the bottom wells of a 48-well microchemotaxis chamber (Neuro Probe; Gaithersburg, MD) while 50 µl of a cell suspension (approx 600,000 cells/ml in DMEM, 1 g/l glucose, and 1 g/l BSA) were added to the top wells. A polyester membrane (pore size: 10 µm; Osmonics; Livermore, CA) separated the top and bottom wells. Before use, the membranes were wet with 0.1 mg/ml collagen IV (Trevigen; Gaithersburg, MD) and air dried.

In all experiments, cell migration was allowed to proceed for 6 h at 37°C in a 95% air-5% CO2 atmosphere. After this time, cells were fixed and stained with Diff-Quick (Dade Behring; Deerfield, IL). The cells on the top of the membrane were wiped off, and those that migrated (located on the underside of the membrane) were counted under a microscope. Every experiment included a control, either vehicle alone or normal mouse IgG at the same concentration as the tested antibody when studying the effects of antibodies on migration. Control and treatments were tested by triplicate in every assay. To reduce variability, control and experimental points were always assessed in the same Boyden chamber. The number of cells that migrated after each treatment (average of the triplicates) was expressed as a percentage of the number of migrated cells in the control sample, which was considered 100% in every chamber.

Migration protocol for ANG II. ANG II treatments were placed in the lower compartment, and 30,000 cells in DMEM and 0.1% BSA were placed in the upper compartment. Antagonists were placed in the lower compartment together with ANG II.

Migration protocol for TGF-beta , PDGF-BB, and VEGF. These agonists were placed in the lower compartment at the doses indicated in the corresponding figures, and 30,000 cells in DMEM and 0.1% BSA were placed in the upper compartment.

Migration protocol for chemotaxis and chemokinesis. Thirty thousand cells were placed in the upper compartment. ANG II (10-7 M) was placed in both the upper and lower compartments for "no gradient" treatment and only the lower compartment for "gradient" treatment. A gradient is established when ANG II is present only in the lower chamber, opposite the cells. If the cells migrate as a function of the gradient, this represents chemotaxis; if they migrate no matter where the agonist is located and movement is not affected by the absence of a gradient (same concentrations on both sides of the membrane), this indicates that they move via a chemokinetic mechanism. A pure chemotactic compound will not stimulate cell movement in the absence of a gradient (70).

Migration protocol for the MMP inhibitor GM6001 and PDGF-BB and TGF-beta antibodies. Thirty thousand cells were placed in the upper compartment along with either GM6001 (25 µM), anti-PDGF-BB (5 µg/ml, the concentration determined to neutralize PDGF in pilot experiments), or anti-TGF-beta 1/2/3 (10 µg/ml) (61). Lower compartments contained either vehicle (DMEM and 0.1% BSA) or 10-7 M ANG II.

Zymography

Pericytes were grown to 80% confluence on culture dishes precovered with 2% gelatin. Growth medium was then replaced with DMEM, 4.5 g/l glucose, and 1% fetal bovine serum. Twenty-four hours later, sterile ANG II or PBS was added to the plates, and incubation continued for 18 h. After treatment, cells were lysed in 120 mM Tris · HCl (pH 8.7), 0.1% Triton X-100, 5% glycerol, and 0.01% sodium azide, and protein concentration was determined by bicinchoninic acid (Pierce; Rockford, IL). Samples from the lysates were loaded in precast gelatin-10% polyacrylamide or casein-12% polyacrylamide gels (Bio-Rad; Hercules, CA) and fractionated in SDS-PAGE. Gels were then washed in 2.5% Triton X-100, rinsed with water, and incubated at 37°C for 36 h in 50 mM Tris · HCl, 200 mM NaCl, 5 mM CaCl2, and 0.02% Brij-35. To confirm that zymograms were reflecting metalloproteinase activity, controls were run in the presence of 5 mM EDTA.

After incubation, gels were stained with Coomassie blue R-250 and destained using a 40% methanol-10% acetic acid solution until white bands were clearly visible. Gels were then scanned, and differences were determined by computerized densitometry using imaging analysis software (Sigma ScanPro, Jandel Sciences).

Western Blotting

Cells were grown to 80% confluence in culture dishes. Plates were scraped in the presence of 10 ml of cold PBS, and the resultant suspension was centrifuged for 5 min at 20,000 g and 4°C. Pellets were resuspended in RIPA buffer [20 mM HEPES (pH 7.4), 100 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1% deoxycholic acid, 10% glycerol, 1 mM EDTA, 1 mM NaVO3, 50 mM NaF, and Protease Inhibitors Set 1 (Calbiochem)] for 30 min and then sonicated. Homogenates were centrifuged for 10 min at 20,000 g and 4°C. The resultant pellets were discarded, and protein concentration in the supernatant was determined by BCA. Samples were mixed 5:1 with loading buffer [0.3 M Tris base (pH 6.8), 10% SDS, 50% glycerol, 0.03% bromophenol blue, and 5% beta -mercaptoethanol] heated to 90°C for 3 min and run in 14% SDS-PAGE at 100 V with running buffer (25 mM Tris base, 190 mM glycine, and 0.025% SDS). Protein was then transferred overnight to a nylon polyvinylidene difluoride Immobilon-P membrane (Millipore; Bedford, MA) at 23 V and 4°C in transfer buffer (20% methanol, 20 mM Tris base, and 140 mM glycine). The transferred membrane was incubated in blocking buffer [0.2% I-Block reagent (Tropix; Bedford, MA) and 0.1% Tween 20 in PBS] for 1 h before the addition of the primary antibody (1:2,000-1:5,000 dilution, following the manufacturer's instructions). The membrane was thoroughly washed with PBS-0.1% Tween 20 and then incubated for 1 h with secondary antibody (horseradish peroxidase-conjugated immunoglobulin anti-IgG) in 1:20,000 dilution for TIMP detection or 1:300,000 dilution for AT1/AT2 receptor detection. After several washes with PBS-0.1% Tween 20, the membrane was treated with enhanced chemiluminescence detection substrate (Pierce Pico Signal for TIMP blots, Pierce Femto Signal for AT1/AT2 receptors) and developed on Kodak luminescence-sensitive film.

Statistical Analysis

For the migration experiments, statistical tests are based upon ratio estimates. The unit of analysis is the mean of the triplicates comprising every experimental value. Statistical differences were analyzed using the Hochberg method (30). Cells migrating in the presence of vehicle alone were taken as 100%. Changes in migration were expressed as the percent increase from control. Western blots and zymograms were analyzed by paired t-test. For both zymograms and Western blots, the density of the bands in the control group was taken as 100% in every experiment, and changes were expressed as the percent change from control. Data are means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of Cultured Retinal Pericytes

The retinal cells we used were identified as pericytes because of their morphology, prominent cytoskeleton, slow growth rate, and positive immunostaining for alpha -actin and 3G5 (Fig. 1).


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  		Fig. 1.   Characterization of pericytes. A: isolated retinal pericytes showing the typical morphological appearance of pericytes with light microscopy (×20). Retinal pericytes were stained for smooth muscle alpha -actin (B) and the specific marker ganglioside 3G5 (×20; C) and (×40; D). No immunostaining was observed using nonspecific immunoglobulins. These characteristics verify that the cells are pericytes.

Expression of ANG II AT1 and AT2 Receptors by Retinal Pericytes

Primary cultures of retinal pericytes (3-6 passages) were tested for the expression of ANG II AT1 and AT2 receptors. Western blots showed that both subtypes were present in homogenates of pericytes (Fig. 2).


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  		Fig. 2.   Western blot for angiotensin (ANG) II type 1 (AT1) and type 2 (AT2) receptors. Primary cultures of pericytes express both AT1 (40 kDa) and AT2 receptors (41 kDa).

Effect of ANG II on Migration of Retinal Pericytes

ANG II stimulated migration of retinal pericytes in a dose-dependent manner (10-9 M: 44 ± 13%; 10-8 M: 86 ± 10%; 10-7 M: 53 ± 7%; 10-6 M: 30 ± 7%; and 10-5 M: 24 ± 8%, P < 0.001, n = 5-10). Although a variable migratory response was already observed at 10-10 M, a significant promigratory effect was documented at a dose of 10-9 M, reached a maximum value at 10-8-10-7 M, and then progressively decreased as ANG II concentration increased. This yielded a bell-shaped dose-response curve in which cells did not show a migratory response to ANG II at doses higher than 10-5 M (Fig. 3).


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  		Fig. 3.   Effect of ANG II on migration of retinal pericytes. ANG II stimulated migration of retinal pericytes (n = 5-10). This effect disappeared at 10-4 M. *P < 0.001 vs. control.

Effect of ANG II AT1 and AT2 Receptor Antagonists

To test the effects of ANG II antagonists on ANG II-induced migratory responses, we used a concentration of 10-7 M ANG II. By itself, neither antagonist, losartan or PD-123319, had any effect on pericyte migration. The AT1 receptor antagonist losartan (10-6 M) completely abolished the effect of ANG II on pericyte migration, reducing the response to 1 ± 7% (P < 0.005, n = 4), whereas the AT2 receptor antagonist PD-123319 (10-6 M) was ineffective (52 ± 7%, nonsignificant vs. ANG II alone, n = 7) (Fig. 4A). Because ANG II at 10-4 M failed to stimulate migration of retinal pericytes (4 ± 7%, n = 10), we used this dose to study whether responses were altered in the presence of the ANG II receptor antagonists. With losartan, the absence of migratory responses to this high concentration of ANG II remained unaltered (11 ± 6%, n = 4). In contrast, migratory responses to 10-4 M ANG II were revealed in the presence of the AT2 receptor antagonist PD-123319 (42 ± 9%, P < 0.001, n = 9) (Fig. 4B).


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  		Fig. 4.   AT1 and AT2 receptor antagonists. A: migratory responses of retinal pericytes to ANG II were blocked by the AT1 receptor antagonist losartan (10-6 M, n = 4) but not affected by the AT2 receptor antagonist PD-123319 (10-6 M, n = 7). *P < 0.002 vs. control; #P < 0.005 vs. 10-7 M ANG II. B: in the presence of 10-4 M ANG II, losartan (n = 4) did not modify migratory responses, but addition of PD-123319 (n = 9) revealed a promigratory response to ANG II. *P < 0.001 vs. control; #P < 0.005 vs. 10-4 M ANG II.

Chemotaxis/Chemokinesis

The promigratory effect of ANG II on retinal pericytes was reduced by 59 ± 19% (P < 0.01, n = 5) when concentrations of the peptide were the same on both sides of the membrane (no gradient) instead of the standard setup in which the chemoattractant is only placed opposite the cells (gradient) (Fig. 5).


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  		Fig. 5.   Chemotaxis/chemokinetics. The promigratory effect of ANG II was significantly reduced in the absence of a concentration gradient (n = 5). *P < 0.01 vs. control; #P < 0.001 vs. ANG II gradient.

Effect of ANG II on MMP Activity (Zymography)

Zymography of pericyte lysates showed one band at ~70 kDa, which corresponded to the molecular mass of latent MMP-2. No active MMP isoform was observed. Conditioned medium also showed one band at the same molecular mass. Treatment of cultured pericytes with 10-7 M ANG II for 18 h resulted in a 35 ± 13% increase (P < 0.05, n = 6) in the ~70-kDa band observed in the pericyte lysate. No changes were observed in the conditioned medium (Fig. 6). Incubation with 5 mM EDTA eliminated all protease activity. No proteolysis was observed on zymograms obtained using casein as a substrate (n = 2; data not shown).


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  		Fig. 6.   Matrix metalloproteinases (MMPs) (zymography). Changes in MMP activity were studied in lysates of pericytes and conditioned medium by zymography using gelatin as a substrate. Lysates and conditioned medium showed a ~70-kDa band of metalloenzyme proteolytic activity corresponding to latent MMP-2 [inset: C, control; A, 10-7 M ANG II; MMP-2, standard MMP-2 (72-kDa latent and 64- kDa active form)]. ANG II increased the activity of latent MMP-2 in cell lysates (n = 6). *P < 0.05 vs. control. odot , Individual experiments.

Effect of ANG II on TIMP-1 and TIMP-2

We next studied whether 6 h of incubation with 10-7 M ANG II would alter the concentration of the MMP inhibitors TIMP-1 and TIMP-2 in pericyte homogenates. ANG II increased TIMP-1 levels in cultured retinal pericytes by 102 ± 17% (P < 0.01, n = 4). Changes in TIMP-2 were not significant (27 ± 28%, n = 6) (Fig. 7).


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  		Fig. 7.   Effect of ANG II on tissue inhibitor of MMP (TIMP)-1 and TIMP-2. Western blots of pericyte lysates show that treatment with ANG II increased TIMP-1 (n = 5) but not TIMP-2 (n = 6). For calculation of statistical significance, the highest value in the TIMP-1 experiments () was excluded because it was considered an outlier (n = 4). *P = 0.01 vs. control.

Migratory Responses in the Presence of a Broad-Spectrum MMP Inhibitor

Migratory responses of retinal pericytes to ANG II and PDGF remained unaffected in the presence of 25 µM of the broad-spectrum MMP inhibitor GM6001 (n = 3) (Fig. 8).


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  		Fig. 8.   Effect of GM6001, a broad-spectrum MMP inhibitor. Inhibition of MMPs had no significant effect on the promigratory effects of ANG II or platelet-derived growth factor (PDGF)-BB on retinal microvascular pericytes (n = 3).

Effect of Anti-TGF-beta Antibodies on ANG II-Induced Migration of Retinal Pericytes

IgG anti-TGF-beta 1/2/3 reduced the migratory response of retinal pericytes to 10-7 M ANG II by 49 ± 17% (Fig. 9). The first anti-TGF-beta 1/2/3 antibody used (Celtrix) decreased migratory responses to ANG II by 52 ± 23% (n = 6). A second anti-TGF-beta 1/2/3 antibody (Santa Cruz) decreased responses by 42 ± 6% (n = 2). Because the responses to both antibodies were similar, the data were pooled for statistical analysis (P < 0.02, ANG II vs. ANG II + anti-TGF-beta 1/2/3 antibodies, n = 8). Normal IgG had no effect on the migratory response. Although TGF-beta 2 tended to stimulate migration of pericytes by 35 ± 15% (n = 5), these data did not reach statistical significance.


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  		Fig. 9.   Effect of transforming growth factor (TGF)-beta antibodies. The migratory response of retinal pericytes to ANG II was decreased by two different antibodies against all TGF-beta isoforms. For statistical purposes, the results obtained with both antibodies were pooled (n = 8). *P < 0.02 vs. control; #P < 0.02 vs. ANG II. TGF-beta 2 tended to increase migration, but results were not statistically significant (n = 5).

Effect of anti-PDGF-BB Antibodies on ANG II-Induced Migration of Retinal Pericytes

IgG against PDGF-BB decreased promigratory pericyte responses to 10-7 M ANG II by 95 ± 12% (P < 0.001, n = 7). PDGF-BB (5 ng/ml) stimulated migration by 47 ± 13% (P < 0.01, n = 7), and this effect was also eliminated by anti-PDGF-BB (P < 0.05, n = 3; data not shown) (Fig. 10).


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  		Fig. 10.   Effect of PDGF-BB antibodies. Neutralizing antibodies against PDGF-BB eliminated the promigratory effect of ANG II on retinal pericytes (n = 7). *P < 0.01 vs. control; #P < 0.001 vs. ANG II. PDGF-BB stimulated migration of retinal pericytes, which in turn was blocked by antibodies against PDGF-BB (n = 3; data not shown).

Effect of VEGF on Pericyte Migration

VEGF (100 ng/ml) failed to induce migratory responses in retinal pericytes (n = 4) (Fig. 11).


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  		Fig. 11.   Effect of vascular endothelial growth factor (VEGF) on migration of retinal microvascular pericytes. VEGF (100 ng/ml) had no effect on migration of pericytes. ANG II was included in these experiments for comparison (n = 4). *P < 0.02 vs. control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It is currently accepted that pericytes, the perimural cells of the retinal microvessels, participate in the regulation of angiogenesis by stabilizing the vessel wall (2, 29). The present study demonstrates that ANG II acts as a migratory factor for retinal microvascular pericytes through interactions with PDGF-BB and TGF-beta but independently from VEGF and MMP activity.

Pericytes employed in this study were isolated from the retinas of freshly killed calves using previously described methods (59). Pure pericyte cultures were obtained after three passages. In an effort to minimize the phenotypic changes known to occur in cells passaged repeatedly, we used fewer than six passages. There were no differences in the apparent content of alpha -actin between cells from these passages. The cells were identified as pericytes because of their morphology (39), slow growth, robust cytoskeleton, and positive immunoreactivity for alpha -actin and 3G5 ganglioside, the latter considered a relatively specific pericyte marker (56).

The migration experiments were performed using Boyden chambers in which the cells were placed in one of two chambers separated by a microporous membrane. In an effort to mimic in vivo conditions more closely, the membrane was coated with collagen IV, a major component of the vascular extracellular matrix (6, 32). This method has some advantages over migration based on cell wounding, because intracellular components are eliminated.

Studies designed to determine dose dependency showed a bell-shaped dose-response curve, with maximal effects at 10-8-10-7 M and no stimulation of migration at the highest dose used, 10-4 M.

Two main subtypes of ANG II receptor subtypes have been identified: AT1 and AT2 (31). Western blots indicated that both receptors were present in our cultured retinal pericytes. To clarify how the migratory responses elicited by ANG II in retinal pericytes are mediated, we used the highly selective nonpeptide ANG II antagonists losartan and PD-123319 as discrete inhibitors of AT1 and AT2 receptors, respectively. AT1 receptrors mediate most of the known ANG II-induced biological effects (13). Consistent with this, the promigratory effects of ANG II were obliterated by losartan, indicating that they were mediated by the AT1 receptor. PD-123319 had no effect on the promigratory effects induced by ANG II. Interestingly, when high ANG II concentrations that do not stimulate pericyte migration were tested in the presence of the AT2 receptor antagonist, the promigratory effect was restored, transforming the bell-shaped dose-response curve into a hyperbola. A similar biphasic response to ANG II has been described for both human and rat aortic smooth muscle cells, but in these cells both pro- and antimigratory effects were mediated by the AT1 receptor (49).

The AT1 and AT2 receptors share only ~34% of their amino acid sequence and have distinct signal transduction pathways. Both have similar affinity for the ANG II ligand. The AT2 receptor has been proposed to counterbalance some of the effects of the AT1 receptor, although this is still controversial (3, 13, 18). The present results suggest that stimulation of the AT2 receptor counteracts the promigratory effects induced by AT1 receptor activation and point to an opposing role of AT1 and AT2 receptors in influencing retinal microvascular pericyte function. However, the AT2 receptor-mediated effects were only observed at very high, unphysiological ANG II concentrations. At concentrations closer to those that might be found in vivo, the predominant effect of ANG II was to induce retinal pericyte migration via the AT1 receptor. It should be noted that both AT1 and AT2 receptors are expressed in the choroid and retina (68).

ANG II has been reported to be chemotactic for a number of cells, including VSMC (43), neonatal cardiac fibroblasts (22), and monocytes (62), closely resembling the effects of PDGF (71). We found that ANG II acted mostly as a chemotactic agent for retinal pericytes, because the promigratory effect of ANG II on pericytes was markedly reduced when no gradient was present. However, we cannot exclude the possibility that ANG II also induces random cell movement (chemokinesis), because there was still significant pericyte migration in the absence of a gradient. Systematic examination of the cellular signaling pathways activated by ANG II in retinal pericytes is needed.

A determinant aspect in cell migration may be degradation of extracellular matrix by MMPs. Production and activation of MMPs, together with their tissue inhibitors (TIMPs), establish a fine regulation of extracellular matrix degradation. Thus changes in the MMP/TIMP balance may determine cell movement within the extracellular matrix and therefore migration (55). However, a recent study (34) of VSMC suggests that cellular migration may not necessarily involve MMP activity. Thus to determine whether MMP activation is involved in the promigratory effects of ANG II, we studied whether it alters MMPs and TIMPs in ways that would increase the proteolytic capability of pericytes. We found that ANG II induced a modest increase (~20-30%) in a cell-associated MMP, which, based on its molecular weight, was identified as latent MMP-2. No active form was observed. Furthermore, ANG II doubled TIMP-1 concentrations in cell lysates, as has been reported for heart endothelial cells (10). These results suggest that ANG II does not stimulate pericyte migration by a mechanism involving increases in MMP activity. It may be argued that concomitant changes in TIMP-1 made it difficult to observe changes in active MMPs, but treatment with a broad-spectrum metalloproteinase inhibitor, GM6001 (1, 17), did not alter migratory responses to ANG II. Likewise, migratory responses to PDGF-BB were not affected by MMP inhibition.

There is general agreement that MMPs are important in the implementation of cell invasion (66). The assay we used to study migration does not address invasion but rather movement through a collagen-coated porous barrier. In smooth muscle cells, such movement has been reported to be independent of MMP activity (34). The present data also suggest that the effects of both ANG II and PDGF-BB on pericyte migration are likely independent of MMP activation. One possibility is that pericytes move by haptotaxis, using collagen as an adhesive substrate (8).

Multiple studies support the notion that ANG II can stimulate vascular remodeling via the regulation of growth factors, including TGF-beta and PDGF-BB. Some of the effects of ANG II in both VSMC and mesangial cells are associated with changes in TGF-beta and PDGF (4, 12, 33, 35, 40, 42). Pericytes may have a similar embryological origin and morphological and functional properties to both VSMC and mesangial cells (27, 29, 46, 47). We therefore hypothesize that responses to ANG II would also be related to TGF-beta and PDGF in pericytes. Because of the selective effect of PDGF-BB on pericytes, we focused on this isoform. With the use of two different blocking antibodies against TGF-beta 1/2/3, we found that both partially inhibited ANG II-induced pericyte migration. Consistent with the present results, it has been reported that TGF-beta increases migration of VSMC in culture (44, 49); however, these results are not universal (51, 52).

TGF-beta 2 is the predominant TGF-beta isoform in the eye and is produced by bovine retinal pericytes in culture (36). Retinal pericytes reportedly synthesize latent TGF-beta (63). In VSMC, ANG II induces expression of both TGF-beta and TGF-beta receptors (16, 64). It is reasonable to assume that treatment with ANG II resulted in some activation of the latent TGF-beta complex; however, it is still not know how (or if) ANG II alters TGF-beta levels, activity, and TGF-beta receptor expression in retinal microvascular pericytes.

Unlike the partial blockade observed with antibodies against TGF-beta , blocking antibodies against PDGF-BB abolished ANG II-induced pericyte migration. PDGF-BB induces migration at ~10 to 100 times lower concentrations than ANG II. Thus small changes in PDGF-BB may result in significant changes in pericyte migration.

There is evidence of a link among ANG II, PDGF-B homodimer, and PDGF receptors in both VSMC and mesangial cells (28, 38, 53). The present data suggest that a link between the renin-angiotensin system and PDGF also exists in retinal microvascular pericytes.

PDGF is a potent stimulator of proliferation and migration of several types of cells, including VSMC and pericytes. Moreover, PDGF-BB has been proposed to mediate pericyte recruitment to newly formed microvessels (27, 45-47). The signaling cascades elicited by ANG II resemble those characteristic of growth factor stimulation. It has been demonstrated that ANG II induces activation of the PDGF-beta receptor independently from PDGF-BB autocrine secretion (26, 48). It is highly unlikely that ANG II activated the PDGF-BB receptor, because antibodies against the ligand, PDGF-BB, abolished the migratory responses to ANG II. This suggests that ANG II induces the autocrine release of PDGF-BB in retinal pericytes.

Hahn et al. (25) reported that a single exposure of quiescent VSMC to ANG II resulted in prolonged induction of PDGF and TGF-beta transcripts, which was maximal 5-6 h after stimulation. In the migration experiments reported here, retinal pericytes were exposed to ANG II for 6 h, within the time frame described by Hahn et al. (25) .

VEGF plays an important role in hypoxia-stimulated neovascularization. VEGF exerts its effect through two known high-affinity tyrosine kinase receptors, kinase insert domain-containing receptor (KDR) and fms-like tyrosine kinase (Flt). VEGF receptors are located primarily on endothelial cells. Takagi et al. (67) demonstrated the expression of Flt but not KDR in bovine retinal pericytes. Grosskreutz et al. (24) showed that both KDR and Flt VEGF receptors are present in vascular smooth muscle and that VEGF influences migration of these cells, acting as a chemoattractant. Furthermore, Otani et al. (58) reported that ANG II significantly increased VEGF mRNA in retinal pericytes. To determine whether the effects of ANG II are linked to VEGF, we tested whether VEGF would induce migration of retinal pericytes but found no differences in the rate of migration between basal and VEGF-treated cells. These results differ from those obtained by Grosskreutz et al. (24) in vascular smooth muscle and by Yamagishi et al. (69) in retinal pericytes. Perhaps migration induced by VEGF in vascular smooth muscle is mediated by KDR, not present in pericytes. Differences in experimental conditions may explain the variance with the data of Yamagishi et al. (69), because these authors passaged pericytes 10-15 times, whereas we used cells passaged fewer than six times. Although there are no data on retinal pericytes, marked differences in migratory responses between subcultured cells and primary cultures of VSMC have been reported, suggesting profound phenotype changes after prolonged passages (5). In preliminary experiments (n = 2), costimulation with VEGF and ANG II or PDGF-BB failed to augment and in fact decreased both ANG II- and PDGF-BB-induced migration (data not shown). These data suggest that VEGF and VEGF receptors are not involved in ANG II-induced migration of retinal pericytes.

It has been suggested that ANG II is involved in the regulation of angiogenesis (15, 23, 50, 54, 60). The data presented here show that ANG II is part of a loop together with PDGF-BB and TGF-beta that influences microvascular retinal pericyte migration and therefore could be involved in regulation of neovascularization in the retina. These data may help understand a recent report (9) showing that inhibition of the formation of ANG II may have protective effects in diabetic retinopathy.


    ACKNOWLEDGEMENTS

We thank Dr. R. Frank for help in setting up the bovine retinal pericyte cultures. We are grateful to Dr. B. Riser for help with TGF-beta and its blocking antibody and to Dr. R. C. Nayak for the generous donation of the 3G5 ganglioside antibodies. We also appreciate the support provided by Drs. J. J. Nussbaum and F. Whitehouse.


    FOOTNOTES

This research was supported by a grant from the Gurwin Foundation and Henry Ford Health Systems. J. A. Nadal was supported by the American Heart Association, Midwest Affiliate.

Initial preliminary data leading to this work were presented in Biochem Biophys Res Commun 266: 382-385, 1999.

Address for reprint requests and other correspondence: A. G. Scicli, Eye Care Services Research, Henry Ford Health System, One Ford Place 4D, Detroit, MI 48202-3450 (E-mail: gscicli1{at}hfhs.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpheart.00656.2001

Received 26 July 2001; accepted in final form 15 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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