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To determine if β-adrenergic blockade inhibits choroidal neovascularization (CNV) in a mouse model of laser-induced CNV, and to investigate the mechanism by which β-adrenoreceptor antagonism blunts CNV.
The impact of β-adrenoreceptor blockade on CNV was determined using the laser-induced CNV model. Briefly, mice were subjected to laser burns, inducing CNV, and treated with daily intraperitoneal injections of propranolol. Neovascularization was measured on choroidal-sclera flat mounts using intercellular adhesion molecule-2 immunofluorescence staining. The impact of β-adrenergic receptor signaling on expression of vascular endothelial growth factor (VEGF) was investigated using primary mouse choroidal endothelial cells (ChEC) and retinal pigment epithelial (RPE) cells. These cells were incubated with β-adrenoreceptor agonists and/or antagonists, and assayed for VEGF mRNA and protein levels.
Propranolol-treated mice demonstrated a 50% reduction in laser-induced CNV. Norepinephrine treatment stimulated VEGF mRNA expression and protein secretion in both ChEC and RPE cells. This effect was blocked by β2-adrenoreceptor antagonism and mimicked by β2-adrenergic receptor agonists.
β-Adrenergic blockade attenuated CNV. β2-Adrenergic receptors regulated VEGF expression in ChEC and RPE cells. Antagonists of β-adrenergic receptors are safe and well tolerated in patients with glaucoma and cardiovascular disease. Thus, blockade of β-adrenoreceptors may provide a new avenue to inhibit VEGF expression in CNV.
Exudative age-related macular degeneration (AMD) is one of the leading causes of blindness worldwide1. Exudative AMD develops when vascular tissue from the choriocapillaris invades through a break in Bruch’s membrane into the sub-retinal pigment epithelium (RPE) and/or sub-retinal space; this process is termed choroidal neovascularization (CNV). Leakage of blood, serous fluid, and lipid from CNV causes central vision loss and ultimately leads to an irreversible fibrovascular scar1.
Histopathological studies of CNV membranes have provided valuable insight into the pathogenesis of exudative AMD. Several studies identified choroidal endothelial cells (EC), retinal pigment epithelium (RPE) cells, fibroblasts, myofibroblasts, photoreceptors, glial cells, and macrophages within surgically excised CNV membranes2–6. Follow up studies focusing upon growth factor expression demonstrated interleukin-1β7, tumor necrosis factor-α7, tissue factor8, monocyte chemotactic protein8, transforming growth factor-β9,10, acidic and basic fibroblast growth factors9,10, platelet-derived growth factor10, and vascular endothelial growth factor (VEGF)6–8,10–13 within these CNV membranes. VEGF expression localized to RPE, choroidal endothelium, fibroblasts, and macrophages 6–8,10–13. Since VEGF is a secreted protein that stimulates vascular permeability and angiogenesis14,15, it was hypothesized to play a central role in CNV.
Experimental models demonstrate a causative role for VEGF in CNV. In the laser-induced model, VEGF is up-regulated in RPE, choroidal endothelium, fibroblasts, and macrophages16. In this same model, inhibition of VEGF signaling prevents CNV17. These studies demonstrate that VEGF is correlated with and necessary for CNV. Overexpression of VEGF in photoreceptors18 and in the RPE19 produces retinal and intrachoroidal neovascularization, respectively, that did not cross Bruch’s membrane. However, adenoviral injection into the sub-retinal space, which both damages Bruch’s membrane and stimulates overexpression of VEGF in the RPE, causes CNV20. These studies demonstrate that a break in Bruch’s membrane and elevated VEGF levels are sufficient for CNV. These studies led to the ANCHOR21 and MARINA22 trials, which established anti-VEGF therapy as the gold standard for treatment of exudative AMD.
Despite the significant breakthrough of anti-VEGF therapy, patients must undergo a large treatment burden, consisting of monthly intravitreal injections. Recently, it was serendipitously discovered that the β-adrenergic receptor blocker propranolol regresses periocular hemangiomas23,24. The putative mechanism for this phenotype is the inhibitory action of β-adrenergic blockers on VEGF production in multiple non-ocular cell types25–27, including EC28–31. In the retina, propranolol or β2-adrenoreceptor blockade inhibit neovascularization in the oxygen-induced retinopathy (OIR) model via reduced VEGF production32,33. These studies suggest that β-adrenergic receptor blockade could be an alternative or adjunctive anti-VEGF treatment.
To test this hypothesis, we determined if propranolol could suppress CNV in the laser-induced model. Since RPE and ChEC have consistently been shown to be a source of VEGF production in CNV, we established a model of norepinephrine-induced VEGF production in primary mouse RPE and ChEC. We finally investigated which β-adrenergic receptor drives norepinephrine-stimulated VEGF expression.
Norepinephrine (10 mM; A7257, Sigma, St. Louis, MO) was dissolved in 0.5 M HCl. β-adrenoreceptor agonists and antagonists propranolol (1 mM; P0884, Sigma), CGP 20712 (1 mM; β1-adrenoreceptor antagonist; #1024, Tocris, R&D Systems, Minneapolis, MN), ICI 118,551 (100 μM; β2-adrenoreceptor antagonist; #0812, Tocris), SR 59230A (100 μM; β3-adrenoreceptor antagonist; #1511, Tocris), xamoterol (100 μM; β1-adrenoreceptor agonist; #0950, Tocris), formoterol (100 μM; β2-adrenoreceptor agonist; #1448, Tocris), and BRL 37344 (1 mM; β3-adrenoreceptor agonist; #0948, Tocris) were dissolved in water.
All research using mouse models of CNV was carried out in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and was approved by the Institutional Animal Use and Care Committee of the University of Wisconsin School of Medicine and Public Health. Wild-type 6 week-old female C57BL/6j mice were housed on a 12-hour light-dark cycle and provided with food and water ad libitum. Laser-induced CNV experiments were performed as previously described34. Briefly, mice were anesthetized and treated with 3 focal laser burns in each eye. Propranolol was dissolved in citrate buffer and delivered daily via intraperitoneal injection at 20 mg/kg. After 14 days, mice were sacrificed and CNV was measured on choroidal-sclera flat mounts using intercellular adhesion molecule-2 (ICAM-2; BD BioSciences) immunofluorescence staining. Images were analyzed using ImageJ software.
Sheep anti-rat Dynabeads (Invitrogen) were washed 3 times with serum-free DMEM (Dulbecco’s Modified Eagle’s Medium, Invitrogen) and then incubated with PECAM-1 antibody (MEC13.3, BD Pharmingen) overnight at 4°C (10 μl beads in 1 mL DMEM). Following incubation, beads were washed 3 times with DMEM containing 10% fetal bovine serum (FBS), re-suspended in the same medium, and stored at 4°C.
Eyes from one litter (6 to 10 pups) of 4 week-old immortomice (C57BL/6j) were enucleated and all connective tissue and muscle was removed from the sclera. In cold DMEM, the anterior eye was removed, followed by the lens, vitreous, retina and optic nerve, leaving only a tissue composed of RPE, choroid, and sclera. At this point, ChEC were isolated from the RPE-choroid-sclera complex identically to retinal EC as previously described, using anti-PECAM-1 antibody-coated magnetic beds. ChECs were analyzed by FACS to document purity and grown identically to retinal EC35.
For RPE cells, two sets of tweezers were used to remove the RPE sheet, which was then digested in 5 ml of type I collagenase (1 mg/ml in serum-free DMEM, Worthington) for 45 minutes at 37°C. Following digestion, DMEM with 10% FBS was added and cells were centrifuged at 400 xg for 10 min. The cellular digest was filtered through a double layer of sterile 40 μm nylon mesh (Sefar America Inc., Fisher Scientific), centrifuged, and washed twice with DMEM containing 10% FBS. RPE cells were plated into a single well of a 24 well plate pre-coated with 2 μg/ml of human fibronectin (BD Biosciences, San Jose, CA). To document purity, RPE cells were analyzed by FACS as previously described35 using anti-bestrophin (MAB5466, Millipore, Billerica, MA) and anti-RPE65 (MAB5428, Millipore) antibodies. RPE cells were grown in low glucose DMEM containing 10% FBS, 2 mM L-glutamine, 100 μg/ml streptomycin, 100 U/ml penicillin, and murine recombinant interferon-γ at 44 units/ml.
ChEC and RPE cells were maintained at 33°C with 5% CO2 in 1% gelatin-coated 60 mm dishes. Cells were not allowed to grow beyond 20 passages. Prior to experiments, cells were serum starved overnight in serum-free medium. Serum-free medium was identical to growth medium described above except it lacked 10% FBS, and the ChEC medium also lacked EC growth supplement.
For mRNA analysis, cells were pre-incubated for 30 minutes and/or incubated for 2 hours in 24 well plates. Cells were then washed with 1X PBS, lysed in RLT plus, and frozen at −20°C. mRNA was extracted using RNeasy Plus Mini Kit (Qiagen, Valencia, CA). The cDNA was synthesized using Sprint RT Complete-Double PrePrimed (Clontech, Mountain View, CA). VEGF mRNA was measured by quantitative PCR (Eppendorf, Hauppauge, NY) and normalized to the housekeeping gene RpL13A by generating a ΔCt value. Fold values were generated by normalizing to the vehicle control. Vehicle control samples were used to assay for baseline levels of VEGF and β-adrenergic receptors. For VEGF secreted protein analysis, cells were incubated for 24 hours in 12 well plates. VEGF levels were measured as previously described35. VEGF levels were normalized to total protein from cell lysates using the BCA Protein Assay (Pierce, Rockford, IL). Fold values were generated by normalizing to the vehicle control.
For CNV and gene expression comparisons between cell lines, Student’s unpaired t-test was performed. For cell culture, each biological N was generated by an experiment on a unique passage day. Thus, Student’s paired t-test (two tailed) was performed to compare two groups. For multiple comparisons, Repeated Measures ANOVA was performed using Dunnett’s multiple comparison post-test against the vehicle control.
In order to determine if β-adrenergic signaling plays a role in AMD, we tested propranolol in the in vivo laser-induced CNV model. Briefly, mice were anesthetized and treated with focal laser burns, which rupture Bruch’s membrane and induce CNV. Mice were treated with propranolol or citrate vehicle control for 14 days and assessed for CNV. We used intraperitoneal drug delivery because prior studies demonstrated positive results with this method32,33. We found that propranolol reduced the average CNV area by 2-fold (Fig. 1).
We next sought to investigate the mechanism by which propranolol inhibits CNV. As discussed earlier, the choroidal endothelium and RPE have been consistently identified as key sources of VEGF production in histopathological studies of AMD. We thus began our in vitro analysis by assessing the levels of VEGF and β-adrenergic receptors in primary mouse ChEC and RPE cells. We found that ChEC and RPE cells express Vegf and the β2-adrenergic receptor at equal mRNA levels (Fig. 2). However, ChEC displayed significantly less β1- and β3-adrenoreceptor mRNA compared with RPE cells (Fig. 2B & 2D).
Since primary ChEC and RPE cells express VEGF and all three β-adrenoreceptors, these cells are a good in vitro model to investigate the role of β-adrenergic signaling in VEGF production. To test this hypothesis, we incubated ChEC and RPE cells with norepinephrine, a naturally produced neurotransmitter and hormone that stimulates α- and β-adrenergic receptors. We found that norepinephrine increased Vegf mRNA production by 4-fold in ChEC and RPE cells (Fig. 3A & 3C). In conditioned medium from these cells, norepinephrine incubation augmented VEGF secretion by 1.6- and 1.2-fold in RPE and ChEC cells, respectively (Fig. 3B & 3D).
We next sought to inhibit the above effect with propranolol to replicate our CNV model in vitro and confirm that this effect is driven by β-adrenoreceptors. Propranolol completely inhibited norepinephrine-stimulated Vegf mRNA production in ChEC and RPE cells (Fig. 4). Furthermore, propranolol only prevented Vegf mRNA production in the presence of norepinephrine and had no effect on baseline Vegf mRNA levels.
Since propranolol is a non-selective β-adrenergic receptor blocker, we next investigated which β-adrenoreceptor drives VEGF production. Prior to norepinephrine stimulation, we pre- incubated ChEC and RPE cells with selective β-adrenoreceptor antagonists: CGP 20712 (β1-adrenoreceptor), ICI 118,551 (β2-adrenoreceptor), and SR 59230A (β3-adrenoreceptor). We found that only the β2-adrenoreceptor antagonist was able to prevent norepinephrine-stimulated Vegf mRNA production (Fig. 5A & 5B). To confirm this result, we stimulated ChEC and RPE cells with selective β-adrenoreceptor agonists: xamoterol (β1-adrenoreceptor), formoterol (β2-adrenoreceptor), and BRL 37344 (β3-adrenoreceptor). Only the β2-selective agonist formoterol significantly increased Vegf mRNA production (Fig. 5C & 5D). Interestingly, treatment with the β3-adrenoreceptor agonist also trended toward a small positive effect on Vegf mRNA production (Fig. 5C & 5D).
Here we demonstrated that β-adrenoreceptor antagonism was effective to diminish CNV in the laser-induced mouse model. Furthermore, we showed that norepinephrine stimulates VEGF mRNA production and secretion via the β2-adrenergic receptor in primary ChEC and RPE cells. These results suggest that β2-adrenoreceptor blockers could be effective anti-VEGF therapeutics in CNV-driven diseases like exudative AMD, traumatic choroidal rupture, myopic CNV, and ocular histoplasmosis.
The use of β-adrenergic antagonism to suppress VEGF production and neovascularization has been previously shown in rodent models of retinopathy of prematurity. In the rat, topical timolol (a β-adrenergic receptor antagonist with highest affinity for the β2-adrenoreceptor36) diminishes retinal neovascularization during OIR37. More recently, the Bagnoli group showed that propranolol32 and a selective β2-adrenoreceptor blocker33 can both attenuate retinal neovascularization during OIR in mice by decreasing VEGF production. However, another group failed to demonstrate that propranolol could prevent retinal neovascularization in OIR mice38. Although Ristori et al hypothesized that the β3-adrenoreceptor stimulated VEGF expression because the β3-adrenergic receptor was up-regulated in OIR mice32, Martini et al conclusively demonstrated that the β2-adrenoreceptor modulates this effect via receptor-specific antagonism33. Although initially counter-intuitive, Dal Monte et al showed that chronic isoproterenol (a β1 and β2 adrenoreceptor agonist) treatment attenuates retinal neovascularization in OIR mice. This occurs by agonist-induced desensitization of the β2-adrenoreceptor39. Our results agree with the findings of Martini and Dal Monte et al, that the β2-adrenoreceptor is key in regulating VEGF expression. However, we do see a trend toward increased VEGF mRNA production with β3-adrenoreceptor agonism (Fig. 5), similar to the Ristori, et al. hypothesis. There may be a minor biological role for the β3-adrenoreceptor in VEGF production or we are simply seeing off-target effects because the pharmacological modulators of β2- and β3-adrenoreceptors have some overlap with each other. In summary, our data agree with prior reports, which demonstrate a role for β2-adrenoreceptor driven VEGF expression in OIR mice, and extend these results to the laser-induced CNV model.
There are several published reports, examining models of reduced ocular adrenergic signaling, but the results are conflicting. Chronic loss of adrenergic signaling in rats via surgical sympathectomy40 or propranolol41 treatment increases choroidal and retinal vascularity. Contrary to our findings, these results suggest that β-adrenergic signaling is anti-angiogenic. On the other hand, there are several reports that agree with our findings, showing that β-adrenergic signaling is pro-angiogenic. First, β3-adrenoreceptor agonism stimulates elongation, migration, and proliferation of human retinal42 and choroidal43 EC. In addition, surgical sympathectomy in rats decreases VEGF levels44, while isoproterenol treatment in human ChEC increases VEGF expression45. These studies demonstrate that β-adrenergic receptor signaling is pro-angiogenic and drives VEGF expression, in agreement with our results.
In addition, surgical sympathectomy in rats46, Dbh null mice (which cannot synthesize norepinephrine)47, and mice null for β1-adrenoreceptors48 display degenerate capillaries, pericyte loss, and increased basement membrane thickness, which are all hallmarks of non-proliferative diabetic retinopathy. In agreement, β1-adrenergic agonism reduces human retinal EC apoptosis49,50. These results suggest that loss of β1-adrenergic signaling can mimic early diabetic retinopathy. Our results identify the β2-adrenoreceptor as a driver of VEGF expression and specific therapeutic antagonism of the β2 adrenoreceptor should avoid the deleterious effects of β1-adrenergic blockade.
Despite the wealth of knowledge about VEGF and its expression, the specific driver of VEGF expression in exudative AMD remains unknown. In proliferative diabetic retinopathy and central retinal vessel occlusions, VEGF expression is driven by hypoxia51. However, the choroid provides more blood flow than is necessary and the role of hypoxia in AMD is unclear. Alternative hypotheses for drivers of VEGF expression in AMD include oxidative stress52, insulin-like growth factor-153, inflammatory cytokines7, transforming growth factor- β54, age55, and contact with choroidal endothelium55. Our results suggest that adrenergic signaling could also contribute to VEGF expression in CNV.
A potential negative consequence of anti-VEGF therapy is the inhibition of RPE-derived VEGF for normal visual function. Complete loss of RPE-derived VEGF expression causes absence of choriocapillaris development and poor vision56. Loss of RPE-derived soluble VEGF leads to choriocapillaris atrophy, RPE and Bruch’s membrane abnormalities, and photoreceptor death57. Importantly, β-adrenergic antagonism does not suppress baseline VEGF expression in RPE cells (Fig. 4), suggesting that therapeutic β2-adrenergic blockade would not have these complications.
Our studies have several important limitations and considerations for clinical translation. First, the laser-induced CNV model more closely approximates post-traumatic CNV after choroidal rupture and post-inflammatory CNV in the context of diseases like histoplasmosis than it does exudative AMD. Second, our studies used systemic, intraperitoneal drug delivery. Prior studies show that systemic propranolol affects retinal phenotypes, including the electroretinogram58 and NV32,33, demonstrating that systemic propranolol penetrates to the posterior eye. Additionally, pharmacokinetic studies find that highly lipophilic β-adrenoreceptor blockers, like propranolol, are most highly concentrated in the RPE and choroid after either intravitreal or subconjunctival injection59,60. Therefore, local propranolol could be highly effective for CNV due to its lipophilic nature; we will assess this hypothesis in future studies. Finally, our studies used primary mouse RPE cells after passaging. We recognize that primary RPE cells lose some characteristic phenotypes with passaging, but our cells maintain bestrophin and RPE65 expression at up to 20 passages. We will assess this limitation in future studies using fresh human fetal RPE cells.
In conclusion, we have identified β2-adrenergic signaling as a potential driver of RPE and ChEC VEGF expression in CNV. Future studies will extend these results to human models and expand the mechanism beyond the β2-adrenoreceptor.
This work was supported by grants EY016995, EY021357 (NS), and P30-EY16665, from the National Institutes of Health and an unrestricted departmental award from Research to Prevent Blindness. JAL was supported by the Medical Scientist Training Program. NS is a recipient of a Research Award from American Diabetes Association, 1-10-BS-160 and Retina Research Foundation. CMS is supported by a grant from American Heart Association, 0950057G. We thank Elizabeth A. Scheef for her expertise and help with cell culture studies. We thank SunYoung Park for her assistance with VEGF ELISA assays. NS had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.