September 2011
Volume 52, Issue 10
Free
Retina  |   September 2011
Transforming Growth Factor-β Inhibition Decreases Diode Laser–Induced Choroidal Neovascularization Development in Rats: P17 and P144 Peptides
Author Affiliations & Notes
  • Sergio Recalde
    From the Experimental Ophthalmology Laboratory,
  • Javier Zarranz-Ventura
    From the Experimental Ophthalmology Laboratory,
    the Department of Ophthalmology, Clínica Universidad de Navarra, and
  • Patricia Fernández-Robredo
    From the Experimental Ophthalmology Laboratory,
  • Pío J. García-Gómez
    the Department of Ophthalmology, Clínica Universidad de Navarra, and
  • Angel Salinas-Alamán
    the Department of Ophthalmology, Clínica Universidad de Navarra, and
  • Francisco Borrás-Cuesta
    the Division of Hepatology and Gene Therapy, Center for Applied Medical Research (CIMA), Universidad de Navarra, Pamplona, Spain; and
  • Javier Dotor
    Digna Biotech, Madrid, Spain.
  • Alfredo García-Layana
    From the Experimental Ophthalmology Laboratory,
    the Department of Ophthalmology, Clínica Universidad de Navarra, and
  • Corresponding author: Alfredo García-Layana, Department of Ophthalmology, Clínica Universidad de Navarra, Universidad de Navarra, Avda Pío XII, 36, 31008, Pamplona, Spain; aglayana@unav.es
  • Footnotes
    2  These authors contributed equally to this project and should be considered equivalent first authors.
Investigative Ophthalmology & Visual Science September 2011, Vol.52, 7090-7097. doi:10.1167/iovs.11-7300
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      Sergio Recalde, Javier Zarranz-Ventura, Patricia Fernández-Robredo, Pío J. García-Gómez, Angel Salinas-Alamán, Francisco Borrás-Cuesta, Javier Dotor, Alfredo García-Layana; Transforming Growth Factor-β Inhibition Decreases Diode Laser–Induced Choroidal Neovascularization Development in Rats: P17 and P144 Peptides. Invest. Ophthalmol. Vis. Sci. 2011;52(10):7090-7097. doi: 10.1167/iovs.11-7300.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To assess the effect of transforming growth factor (TGF)-β inhibitor peptides (P17 and P144) on the development of laser-induced choroidal neovascularization (LI-CNV) in a rat model.

Methods.: Sixty-one Long-Evans rats underwent diode LI-CNV model. Forty-eight hours later, treatment was administered. The intravenous control group (IV-control) and intravenous P17 group (IV-17) received five doses (0.2 mg every 72 hours) of vehicle and P17, respectively. Four groups received intravitreal injections of P17 low-dose (LD-17; 1 mg/mL) and high-dose (HD-17; 20 mg/mL) and P144 low-dose (LD-144; 1 mg/mL) and high-dose (HD-144; 3 mg/mL), and fellow eyes received vehicle. CNV evolution was assessed weekly by fluorescein angiography (FA). After death, VEGF, TGF-β and PDGF protein levels were measured by ELISA in RPE and retina homogenates. Data were analyzed with commercially available statistical analysis software.

Results.: The mean CNV area, measured in pixels, was significantly lower at the second and fourth weeks in IV-17 (P < 0.05) and from the second week in HD-17 (P < 0.05), whereas LD-144 and HD-144 showed significant differences at every time point (P < 0.05). LD-17 showed significantly lower protein levels of TGF-β in retina and PDGF in RPE (P < 0.05), whereas HD-17 showed lower levels of VEGF (RPE and retina; P < 0.05), TGF-β (RPE and retina; P < 0.05), and PDGF (RPE; P < 0.05). HD-144 showed lower VEGF levels in the retina (P < 0.05).

Conclusions.: TGF-β inhibition with these peptides represents a promising new therapeutic line for CNV targeting a different pathway than current therapies. More studies are needed to assess this effect on early CNV, alone or in combination with anti-VEGF.

Choroidal neovascularization (CNV) is the common end point of several retinal diseases, among which age-related macular degeneration (AMD) and high myopia (HM) are prominent. 1 5 The etiopathogeny of CNV has been widely studied, and currently it is considered a multifactorial process, with advanced age, genetics, ethnicity, and smoking as main risk factors in AMD and patchy atrophy and lacquer cracks in HM. 6 13 Through different triggering mechanisms, in both diseases, CNV development produces fluid leakage into the outer subretinal space, affecting the macula. The growth of new vascular tissue is followed by a fibrous process with progressive macular destruction. As a result, central vision can be compromised, with associated substantial loss of autonomy and quality of life. 14,15 Both angiogenic and fibrotic processes are regulated by complex pathways that involve multiple growth factors. In normal eyes, the resting retina's homeostasis is regulated by the retinal pigment epithelium (RPE) and other cells, which maintain a global antiangiogenic state. This is achieved through a balance of proangiogenic factors, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), angiopoietin, and b-fibroblast growth factor (b-FGF), and antiangiogenic factors, such as pigment epithelium-derived factor (PEDF), thrombospondin-1, and others that have been studied with increasing interest in recent years. 16 18 In CNV, upregulation of proangiogenic molecules and decreased angiogenesis inhibitors produces an imbalance that favors neovascular growth, especially in eyes with defects in Bruch's membrane, increased oxidative stress, and hypoxia, as in aging retinas. Among the proangiogenic factors related with CNV, VEGF is prominent and has become the target of current therapies. High VEGF levels have been reported in cellular cultures, surgically excised neovascular membranes, and CNV animal model studies. Thus, recent studies have suggested that VEGF upregulation can be enhanced by other growth factors, among which is transforming growth factor (TGF)-β. This factor, usually related to fibrosis, also exerts a strong proangiogenic effect through VEGF activation and extracellular matrix remodeling. 19,20 TGF-β is present in surgically excised human neovascular membranes, and several studies in human RPE cultures have reported that it significantly enhances VEGF secretion by promoting mRNA transcription, involving the MAP-kinase pathway. 20 22 Recently, a synergistic association with b-FGF and tumor necrosis factor (TNF)-α over VEGF transcription has been reported, suggesting that TGF-β may work in concert with other angiogenic cytokines in VEGF upregulation. 20,23 These findings suggest that selective blockade of TGF-β signaling may abate CNV development, affecting different cytokines involved in the process. 
To test this hypothesis, we selected two specific TGF-β inhibitor peptides: P17, a soluble hydrophilic peptide derived from a phage display peptide library, and P144, a hydrophobic peptide derived from the sequence of the extracellular region of type III receptor for TGF-β (β-glycan, amino acids 730-743). The different hydrophilic nature of P17 allows its administration in both intravenous and intravitreal injections, whereas P144 can be administered only intravitreally because of its poor solubility. P17 inhibits TGF-β, with a relative binding affinity of 100% for TGF-β1, 80% for TGF-β2, and 30% for TGF-β3 in surface plasmon resonance (SPR) assays, 24 whereas similar binding assays are currently being performed for P144 (Digna Biotech S.L., unpublished data). These peptides have demonstrated a strong TGF-β inhibitory effect in different animal models and an antagonist effect over TGF-β-dependent angiogenesis in cellular cultures and in vivo angiogenesis assays in a synthetic matrix (Matrigel; BD Biosciences, Madrid, Spain). 25 30  
We assessed the effect of the anti-TGF-β P17 and P144 peptides in a diode laser-induced CNV model in rats that was previously established in our laboratory. 31 This is the first study to assess the antiangiogenic effect of these peptides in a CNV animal model. 
Methods
Study Animals
The present study was conducted according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and was approved by the Ethics Committee for Animal Research of the University of Navarra. Sixty-one pigmented-retina, Long-Evans male rats (age, 12 weeks; weight, ∼320 grams) where randomized into two intravenous and four intravitreal study groups. The rats were intraperitoneally anesthetized with a mixture of ketamine (75 mg/kg, Imalgene 1000; Merial, Lyon, France) and xylazine (Rompun 2%, 10 mg/kg; Bayer Animal Health, Leverkusen, Germany) for all procedures. Injections were performed with a 1-mL syringe and a 25-gauge needle. 
Diode Laser-Induced CNV Model
All study eyes were dilated with tropicamide 1% eye drops, and 8 to 10 laser photocoagulation sites were placed concentrically around the optic disc to induce CNVs. A diode laser (810 nm) connected to a slit lamp was used with a relative potency scale of 250 mW, an exposure time of 0.05 second, and a spot size of 75 μm, as established by protocol. 31 Laser spots were focused with crystal covers to avoid laser beam dispersion. Bubble formation confirmed the rupture of Bruch's membrane. Laser rupture sites which had hemorrhages or subretinal bleeding at the time of laser application were excluded from analysis. The number of laser photocoagulation sites that developed CNV was assessed at every time point and differences between treated and control eyes for each study group were analyzed. 
Study Groups
Intravenous Treatment Groups.
Two intravenous groups were included in the study: control and intravenous P17. The intravenous control group (IV-control) received five intravenous injections of 0.2 mL of saline every 72 hours over 2 weeks, representative of natural laser-induced CNV evolution. The intravenous P17 group (IV-17) received five intravenous injections of 0.2 mL of P17 (1 mg/mL), suspended in 0.2 mL of saline, every 72 hours over 2 weeks. Eight rats were included in the IV-control group and six were included in the IV-17 group. 
Intravitreal Treatment Groups.
Four groups were included: two P17 and two P144. Two different doses of each peptide were intravitreally injected, as follows: P17 low dose (LD-17; 1 mg/mL) and P17 high dose (HD-17; 20 mg/mL); and P144 low dose (LD-144; 1 mg/mL) and P144 high dose (HD-144; 3 mg/mL). These rats received a single dose of 7 μL of the peptide solution in the treated eye and 7 μL of its respective vehicle in the fellow eye of the same animal. The hydrophilic P17 peptide was suspended in saline, and the hydrophobic P144 peptide was suspended in a 75% of dimethyl sulfoxide saline solution. Ten rats were included in LD-17, 12 in HD-17, 12 in LD-144, and 13 in HD-144. 
Injection Procedures
Treatments were administered 48 hours after laser application in all study groups. Intravenous injections were performed in the tail vein with a 1-mL syringe and 30-gauge needle. Five consecutive injections of 0.2 mL (each dose every 72 hours) were administered in both intravenous control and intravenous P17 groups (IV-control and IV-P17, respectively). Intravitreal injections were performed with a high-precision syringe (Gastight 1702L; Hamilton Co., Reno, NV) and 30-gauge needle. The injection site was located 1 mm posterior to the corneoscleral limbus. A single 7-μL injection of different peptide concentrations was administered in the treated eye and the same volume of vehicle solution in the fellow eye of the same animal (intravitreal controls) in all intravitreal study groups (LD-17, HD-17, LD-144, and HD-144). The treatment eye was randomly selected for each rat. 
Peptides
P144 (TSLDASIIWAMMQN), a hydrophobic peptide derived from the sequence of the extracellular region of type III receptor for TGF-β (β-glycan, amino acids 730-743), 26 and P17 (KRIWFIPRSSWYERA), a soluble hydrophilic peptide identified using a random phage display peptide library, 27 were synthesized by the solid-phase method, using the Fmoc alternative by PolyPeptide Laboratories (Strasbourg, France). 32,33 Peptides were at least 95% pure, as determined by HPLC. 
Fluorescein Angiographies
Images were obtained using a retinograph (CF-60 ZA; Canon Inc., Kawasaki City, Japan). For FA evaluations, 0.2 mL of intravenous sodium fluorescein 2% (200 mL/kg) was administered into the tail vein 2 minutes before the images were captured. FA images were digitalized and converted to TIFF files (Photoshop 7.0; Adobe Inc., San Jose, CA), and the CNV leakage area was calculated by measuring the pixel area within the best-fitting polygon of each rupture site 34 (shape-definition tool of ImageQuantTL; GE Healthcare, Uppsala, Sweden) software by two independent, trained, masked observers (SR, JZV). Mean CNV area was determined for every study group and time point. FA was performed weekly for four consecutive weeks, to assess the induced-CNV evolution in all study groups. 
VEGF, TGF-β, and PDGF Protein Measurement by ELISA
Fifty micrograms of total protein were used for measurement of VEGF, TGF-β and PDGF protein by ELISA (R&D Systems, Minneapolis, MN) on RPE and retinal homogenates supernatants. All results are shown in nanograms per microgram of total protein. The methods used followed manufacturers' instructions. 
Statistical Analysis
Differences in the intravenous treatment groups were assessed with independent Student's t-test with equal variance. Differences between treated and control eyes in the intravitreal treatment groups were assessed with paired Student's t-test with equal variance or Wilcoxon's test in cases in which nonparametric tests were required. P < 0.05 was considered statistically significant (all statistical analysis performed using SPSS 15.0 software; SPSS Inc., Chicago, IL). 
Results
Fluorescein Angiographies
Mean CNV areas measured in pixels for all study groups are summarized in Table 1. FA images of control and P17-treated eyes are shown in Figure 1 and those of control and P144-treated eyes are shown in Figure 2. In the intravenous treatment groups, significant differences were found at the second and fourth weeks in the IV-17 group, compared with the IV-control group, whereas at the third week, differences did not reach the significance level. In the intravitreal study groups, mean CNV analysis in the LD-17 group did not show significant differences between controls and treated eyes at any time point. At high dose, significant differences were found from the second week in the HD-17-treated eyes compared with their respective controls. Both the LD- and HD-144 groups reveal significant differences between treated and control eyes during the entire study period (Fig. 3). 
Table 1.
 
CNV Areas Assessed by Fluorescein Angiographies
Table 1.
 
CNV Areas Assessed by Fluorescein Angiographies
Study Group 1st Week 2nd Week 3rd Week 4th Week
Control Eyes Treated Eyes P Control Eyes Treated Eyes P Control Eyes Treated Eyes P Control Eyes Treated Eyes P
IV-Control Intravenous control 9447 ± 2068 NS 9955 ± 2878 <0.05* 10254 ± 2443 0.06 10206 ± 2035 <0.05*
IV-17 Intravenous P17 7816 ± 1345 6762 ± 1991 7796 ± 1769 7400 ± 2019
LD-17 Low-dose intravitreal P17 10689 ± 3503 9821 ± 3086 NS 12006 ± 3675 10157 ± 2649 NS 10722 ± 3194 10202 ± 2647 NS 11260 ± 2852 8962 ± 2117 0.06
HD-17 High-dose intravitreal P17 8816 ± 2176 7174 ± 2981 NS 9142 ± 2537 6673 ± 2809 <0.05* 9218 ± 2354 6560 ± 1967 <0.01† 9341 ± 3020 6817 ± 2447 <0.01†
LD-144 Low-dose intravitreal P144 10862 ± 1980 8771 ± 858 <0.05* 10822 ± 2208 7964 ± 1183 <0.01† 10561 ± 2846 8196 ± 1394 <0.05* 10279 ± 2313 8077 ± 1061 <0.05*
HD-144 High-dose intravitreal P144 9853 ± 3444 6599 ± 1945 <0.01† 9628 ± 1637 7534 ± 1650 <0.01† 9536 ± 2859 7677 ± 2446 <0.05* 9494 ± 3102 7600 ± 1896 <0.05*
Figure 1.
 
FA images of control and P17-treated eyes. (A) The IV-control group; (B) the IV-17 intravenous P17 group; (C, D) control and treated eyes in the LD-17 intravitreal low-dose P17 group; (E, F) control and treated eyes in the HD-17 intravitreal high-dose P17 group.
Figure 1.
 
FA images of control and P17-treated eyes. (A) The IV-control group; (B) the IV-17 intravenous P17 group; (C, D) control and treated eyes in the LD-17 intravitreal low-dose P17 group; (E, F) control and treated eyes in the HD-17 intravitreal high-dose P17 group.
Figure 2.
 
FA images of control and P144-treated eyes. (A, B) Control and treated eyes in the LD-144 intravitreal low-dose P144 group; (C, D) control and treated eyes in the HD-144 high-dose intravitreal P144 group.
Figure 2.
 
FA images of control and P144-treated eyes. (A, B) Control and treated eyes in the LD-144 intravitreal low-dose P144 group; (C, D) control and treated eyes in the HD-144 high-dose intravitreal P144 group.
Figure 3.
 
Mean CNV areas assessed by fluorescein angiographies in all study groups (measured in pixels). Top left: intravenous controls versus intravenous P17 (IV-control versus IV-17); middle left: low-dose intravitreal P17 (LD-17); middle right: high-dose intravitreal P17 (HD-17); bottom left: low-dose intravitreal P144 (LD-144); bottom right: high-dose intravitreal P144 (HD-144). Differences between treated and untreated eyes: NS, not significant; *P < 0.05; **P < 0.01.
Figure 3.
 
Mean CNV areas assessed by fluorescein angiographies in all study groups (measured in pixels). Top left: intravenous controls versus intravenous P17 (IV-control versus IV-17); middle left: low-dose intravitreal P17 (LD-17); middle right: high-dose intravitreal P17 (HD-17); bottom left: low-dose intravitreal P144 (LD-144); bottom right: high-dose intravitreal P144 (HD-144). Differences between treated and untreated eyes: NS, not significant; *P < 0.05; **P < 0.01.
Enzyme-Linked Immunosorbent Assay
ELISA results for mean VEGF, TGF-β, and PDGF protein levels (nanograms/microgram total protein) in treated and control eyes for all study groups in both RPE and rat retinas are summarized in Table 2
Table 2.
 
Mean VEGF, TGF-β, and PDGF Levels Measured by ELISA in RPE and Retina
Table 2.
 
Mean VEGF, TGF-β, and PDGF Levels Measured by ELISA in RPE and Retina
Study Group Eyes VEGF TGF-β PDGF
RPE P Retina P RPE P Retina P RPE P Retina P
IV-Control Intravenous control Control 50.84 ± 8.95 NS 47.87 ± 8.98 NS 272.87 ± 3.95 NS 30.23 ± 0.58 NS 5.82 ± 1.09 NS 4.55 ± 0.28 NS
IV-17 Intravenous P17 Treated 41.59 ± 4.85 42.76 ± 2.24 269.08 ± 5.44 27.64 ± 2.84 5.76 ± 0.53 4.40 ± 0.15
LD-17 Low-dose intravitreal P17 Control 41.54 ± 2.28 NS 43.90 ± 4.31 0.08 254.96 ± 14.85 0.080 32.62 ± 1.84 <0.05* 6.67 ± 0.76 <0.05* 4.55 ± 0.24 NS
Treated 41.86 ± 1.87 36.89 ± 1.86 241.71 ± 6.92 25.42 ± 6.81 5.10 ± 0.61 4.51 ± 0.71
HD-17 High-dose intravitreal P17 Control 45.56 ± 5.03 <0.05* 44.90 ± 3.65 <0.05* 261.67 ± 14.63 <0.05* 34.10 ± 1.73 <0.05* 5.45 ± 0.46 <0.05* 4.80 ± 0.32 NS
Treated 34.54 ± 1.35 41.27 ± 2.57 192.30 ± 20.88 26.08 ± 4.45 4.92 ± 0.16 4.60 ± 0.15
LD-144 Low-dose intravitreal P144 Control 45.91 ± 6.84 NS 43.21 ± 2.27 NS 247.54 ± 3.62 NS 38.86 ± 12.08 NS 5.14 ± 0.44 NS 4.95 ± 0.61 NS
Treated 42.22 ± 5.06 42.79 ± 3.84 235.22 ± 34.97 37.45 ± 8.32 4.89 ± 0.37 4.80 ± 0.56
HD-144 High-dose intravitreal P144 Control 51.43 ± 15.59 NS 52.33 ± 10.80 <0.05* 321.09 ± 63.98 NS 33.38 ± 2.57 NS 5.24 ± 0.25 NS 5.20 ± 0.59 NS
Treated 47.08 ± 5.41 40.66 ± 5.07 318.99 ± 31.39 31.94 ± 1.57 5.19 ± 0.28 4.99 ± 0.43
Vascular Endothelial Growth Factor.
The mean VEGF protein level measured in RPE was significantly lower in the HD-17-treated eyes than in the control eyes. No significant differences in RPE were found in any other group. The mean VEGF protein level measured in rat retinas was significantly lower than in their respective controls in HD-17- and -144-treated eyes, whereas in the LD-17 group the differences did not reach the significance level. No differences were observed in other study groups. 
Transforming Growth Factor-β.
The mean TGF-β levels measured in RPE were significantly lower in the HD-17 group. In the LD-17 group, differences did not reach the significance level. None of the other study groups showed significant differences between treated and control eyes. The mean TGF-β levels measured in rat retinas were significantly lower in both the LD-and HD-17 groups. No significant differences were found in any of the other groups. 
Platelet-Derived Growth Factor.
In RPE, the mean PDGF protein level was significantly lower in treated eyes than in the controls in both the LD-and HD-17 groups. No significant differences were observed in any other group in RPE. None of the study groups showed significant differences in PDGF levels measured in rat retinas. 
Statistically significant results for the mean VEGF, TGF-β, and PDGF protein levels between treated and control eyes in all study groups are presented in Figures 4 to 6
Figure 4.
 
Mean TGF-β protein levels in rat retinas and mean PDGF protein levels in RPE measured by ELISA in the low-dose intravitreal P17 group (LD-17; 1 mg/mL). *P < 0.05.
Figure 4.
 
Mean TGF-β protein levels in rat retinas and mean PDGF protein levels in RPE measured by ELISA in the low-dose intravitreal P17 group (LD-17; 1 mg/mL). *P < 0.05.
Figure 5.
 
Mean VEGF, TGF-β, and PDGF protein levels measured by ELISA in RPE and rat retinas in the high-dose intravitreal P17 group (HD-17; 20 mg/mL). NS, not significant; *P < 0.05.
Figure 5.
 
Mean VEGF, TGF-β, and PDGF protein levels measured by ELISA in RPE and rat retinas in the high-dose intravitreal P17 group (HD-17; 20 mg/mL). NS, not significant; *P < 0.05.
Figure 6.
 
Mean VEGF protein levels measured by ELISA in rat retinas in the HD-144 intravitreal P144 high-dose group (3 mg/mL). NS, not significant; *P < 0.05.
Figure 6.
 
Mean VEGF protein levels measured by ELISA in rat retinas in the HD-144 intravitreal P144 high-dose group (3 mg/mL). NS, not significant; *P < 0.05.
Discussion
Our study describes that peptides P17 and P144 can decrease CNV development in a laser-induced rat model, an effect associated with downregulation of VEGF, TGF-β, and PDGF cytokines. The positive effects reported suggest that TGF-β inhibition is a novel therapeutic approach to the treatment of CNV. 
Intravitreal injections of anti-VEGF drugs are currently considered the gold standard treatment for CNV, albeit with limited results in terms of visual recovery. 35 37 Thus, a deeper understanding of CNV etiopathogeny is warranted, to develop more effective therapeutic strategies. Stemming from recent mechanistic studies, new therapeutic avenues such as anti-sphingosine-1-phosphate antibodies, rapamycin, and homoisoflavanone have been proposed, whereas other studies have suggested complement system as a possible target. 38 41 Concomitantly, a better understanding of the biology of neovascular and fibrous processes in subretinal neovascular membranes revealed a role for TGF-β in CNV development. In normal eyes, TGF-β is a multifunctional regulator that mediates cellular proliferation, differentiation, apoptotic death, and angiogenesis. 18 22 In contrast to these physiological functions, in CNV, TGF-β is not only the main growth factor responsible for scar tissue formation; recent studies in human RPE cultured cells have also reported an enhancer effect on VEGF secretion. 20,23 The purpose of the present study was to inhibit this proangiogenic effect in vivo in an established animal CNV model. This is the first animal study specifically directed toward assessing the antiangiogenic effect of these peptides through TGF-β inhibition in CNV. To select the anti-TGF-β study drugs, we reviewed previous work in this area and found promising experimental results reported with P17 and P144 peptides. They have shown good results in distinct processes in the lung, heart, liver, and skin, and are currently under evaluation in clinical trials. 25 30 We postulated that these peptides may also block TGF-β's pathogenic effects in our study area. We tested the efficacy of the peptides, using a diode laser-induced CNV model in pigmented retinas of Long-Evans rats previously developed by our research group. 31  
The results obtained with both peptides are promising. Regarding the P17 peptide, significant differences in the mean CNV areas assessed by FA and the mean VEGF, TGF-β, and PDGF protein levels were found, suggesting that this peptide could inhibit CNV, especially with high-dose intravitreal injections. In the intravenous treatment group differences in FA were significant at week 2, when the peptide dosage is completed after the fifth injection, and at week 4. However, ELISA results did not show any differences in any of the cytokines analyzed in this group. In the low-dose intravitreal P17 group differences were not significant in FA, but a mild effect on TGF-β levels in retina and PDGF levels in RPE could be observed. In the high-dose intravitreal P17 group, differences in FA were obtained from week 2, with a concordant decrease in the mean VEGF, TGF-β, and PDGF protein levels in rat retinas in treated eyes compared with levels in their respective controls. This group shows the most consistent differences between treated and control eyes in the present study. The hydrophilic nature of the P17 peptide that confers high solubility in saline solutions may explain the differences obtained, suggesting that repeated injections in the intravenous group or high-dose intravitreal injections are needed to obtain a proper effect. Solubility may also account for different diffusion characteristics regarding effects on deep structures, such as RPE, where lower levels of all cytokines analyzed were found in the high-dose–injected group. On the other hand, both low- and high-dose intravitreal P144 groups showed significant differences in mean CNV areas assessed by FA along the entire study period compared with their respective controls. Although this effect was evident from week 1 after treatment, and persisted during the 4 study weeks, ELISA results did not show a good correlation with angiographic findings. Mean VEGF protein level in retina was significantly lower in the high-dose group, but none of the other cytokines showed significant differences. Its low solubility in physiologic solutions may explain the early and lasting effect of P144 in CNV lesions in FA, and also its poor diffusion along retinal structures. The hydrophobic characteristics that make P144 unsuitable for intravenous injection in conventional vehicles make it possible to achieve a lasting effect locally with lower dosage in small volumes, as with intravitreal injections. This feature may explain the lack of differences observed by ELISA, limiting the conclusions that can be drawn from biochemical analyses. Nevertheless, cytokine levels were measured for 4 weeks after peptide injection in all study groups. The time between treatment injection and sample collection may also explain the mild differences found in some cases and especially in the intravenous P17 group, which are probably due to the high clearance rate of the peptide. In future studies, it will be interesting to assess the protein levels at earlier time points after peptide injection, when the therapeutic effect becomes evident. 
Previous studies in rats have not found any macroscopic signs of toxicity with the P144 peptide. No alterations were observed in different organs, such as liver, lung, kidney, spleen, intestine, tongue, or testes, suggesting that its administration is safe at the stated doses. 30 Although minor results have been reported about the systemic safety of P17 peptide, further studies should evaluate the safety and toxicity of both peptides over retinal structures. The findings should be confirmed through specific structural and molecular studies, to determine the local and systemic safety of these drugs. 
These data suggest a strong effect of the peptides over some of the cytokines involved in the CNV pathways, and especially VEGF, the target of current treatments. 35 37 Additional inhibition of TGF-β and PDGF, other molecules that have shown an enhancer effect over VEGF secretion in cellular cultures, could provide an added benefit to anti-VEGF drugs. 19 23 The results reported here establish a baseline for development of new therapeutic approaches in CNV as a primary or coadjuvant treatment with anti-VEGF drugs acting through a mechanism different from that of current treatments and blocking an alternative VEGF upregulation pathway. In addition to interspecies differences between rat and human retina, the laser-induced CNV model may produce iatrogenic neovascularization in young healthy rats, in which the RPE is unaffected, physiologically slowing the CNV process via the release of antiangiogenic growth factors. Moreover, these animal have no drusen, characteristic of CNV development by complement system activation. 42 However, laser-induced CNV reproduces the stages of the wound-healing process and involves growth factors similar to those in pathologic CNV, making this model useful as a first approach to assess the peptide effect on VEGF upregulation in vivo. 18,20 22 Nevertheless, if the process of laser-induced CNV follows a parallel course with AMD and high myopia lesions, even if the triggering mechanism differs in both processes, the inhibition of TGF-β by the peptides should be comparable. 
In summary, we report for the first time that TGF-β inhibition decreases CNV development in a laser-induced rat model and that this decrease is associated with a downregulation of VEGF, TGF-β, and PDGF expression. The future of CNV management may require combined therapies, with several drugs acting on different mediators of CNV, such as VEGF, complement system, TGF-β, PDGF, and TNF-α. Thus, in light of the current results, the anti-TGF-β peptides could be considered in this multivariate CNV treatment, but further studies are needed to assess their effect in early and established lesions. 
Footnotes
 Supported by Digna Biotech.
Footnotes
 Disclosure: S. Recalde, None; J. Zarranz-Ventura, None; P. Fernández-Robredo, None; P.J. García-Gómez, None; A. Salinas-Alamán, None; F. Borrás-Cuesta, None; J. Dotor, Digna Biotech (F, E), P; A. García-Layana, None
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Figure 1.
 
FA images of control and P17-treated eyes. (A) The IV-control group; (B) the IV-17 intravenous P17 group; (C, D) control and treated eyes in the LD-17 intravitreal low-dose P17 group; (E, F) control and treated eyes in the HD-17 intravitreal high-dose P17 group.
Figure 1.
 
FA images of control and P17-treated eyes. (A) The IV-control group; (B) the IV-17 intravenous P17 group; (C, D) control and treated eyes in the LD-17 intravitreal low-dose P17 group; (E, F) control and treated eyes in the HD-17 intravitreal high-dose P17 group.
Figure 2.
 
FA images of control and P144-treated eyes. (A, B) Control and treated eyes in the LD-144 intravitreal low-dose P144 group; (C, D) control and treated eyes in the HD-144 high-dose intravitreal P144 group.
Figure 2.
 
FA images of control and P144-treated eyes. (A, B) Control and treated eyes in the LD-144 intravitreal low-dose P144 group; (C, D) control and treated eyes in the HD-144 high-dose intravitreal P144 group.
Figure 3.
 
Mean CNV areas assessed by fluorescein angiographies in all study groups (measured in pixels). Top left: intravenous controls versus intravenous P17 (IV-control versus IV-17); middle left: low-dose intravitreal P17 (LD-17); middle right: high-dose intravitreal P17 (HD-17); bottom left: low-dose intravitreal P144 (LD-144); bottom right: high-dose intravitreal P144 (HD-144). Differences between treated and untreated eyes: NS, not significant; *P < 0.05; **P < 0.01.
Figure 3.
 
Mean CNV areas assessed by fluorescein angiographies in all study groups (measured in pixels). Top left: intravenous controls versus intravenous P17 (IV-control versus IV-17); middle left: low-dose intravitreal P17 (LD-17); middle right: high-dose intravitreal P17 (HD-17); bottom left: low-dose intravitreal P144 (LD-144); bottom right: high-dose intravitreal P144 (HD-144). Differences between treated and untreated eyes: NS, not significant; *P < 0.05; **P < 0.01.
Figure 4.
 
Mean TGF-β protein levels in rat retinas and mean PDGF protein levels in RPE measured by ELISA in the low-dose intravitreal P17 group (LD-17; 1 mg/mL). *P < 0.05.
Figure 4.
 
Mean TGF-β protein levels in rat retinas and mean PDGF protein levels in RPE measured by ELISA in the low-dose intravitreal P17 group (LD-17; 1 mg/mL). *P < 0.05.
Figure 5.
 
Mean VEGF, TGF-β, and PDGF protein levels measured by ELISA in RPE and rat retinas in the high-dose intravitreal P17 group (HD-17; 20 mg/mL). NS, not significant; *P < 0.05.
Figure 5.
 
Mean VEGF, TGF-β, and PDGF protein levels measured by ELISA in RPE and rat retinas in the high-dose intravitreal P17 group (HD-17; 20 mg/mL). NS, not significant; *P < 0.05.
Figure 6.
 
Mean VEGF protein levels measured by ELISA in rat retinas in the HD-144 intravitreal P144 high-dose group (3 mg/mL). NS, not significant; *P < 0.05.
Figure 6.
 
Mean VEGF protein levels measured by ELISA in rat retinas in the HD-144 intravitreal P144 high-dose group (3 mg/mL). NS, not significant; *P < 0.05.
Table 1.
 
CNV Areas Assessed by Fluorescein Angiographies
Table 1.
 
CNV Areas Assessed by Fluorescein Angiographies
Study Group 1st Week 2nd Week 3rd Week 4th Week
Control Eyes Treated Eyes P Control Eyes Treated Eyes P Control Eyes Treated Eyes P Control Eyes Treated Eyes P
IV-Control Intravenous control 9447 ± 2068 NS 9955 ± 2878 <0.05* 10254 ± 2443 0.06 10206 ± 2035 <0.05*
IV-17 Intravenous P17 7816 ± 1345 6762 ± 1991 7796 ± 1769 7400 ± 2019
LD-17 Low-dose intravitreal P17 10689 ± 3503 9821 ± 3086 NS 12006 ± 3675 10157 ± 2649 NS 10722 ± 3194 10202 ± 2647 NS 11260 ± 2852 8962 ± 2117 0.06
HD-17 High-dose intravitreal P17 8816 ± 2176 7174 ± 2981 NS 9142 ± 2537 6673 ± 2809 <0.05* 9218 ± 2354 6560 ± 1967 <0.01† 9341 ± 3020 6817 ± 2447 <0.01†
LD-144 Low-dose intravitreal P144 10862 ± 1980 8771 ± 858 <0.05* 10822 ± 2208 7964 ± 1183 <0.01† 10561 ± 2846 8196 ± 1394 <0.05* 10279 ± 2313 8077 ± 1061 <0.05*
HD-144 High-dose intravitreal P144 9853 ± 3444 6599 ± 1945 <0.01† 9628 ± 1637 7534 ± 1650 <0.01† 9536 ± 2859 7677 ± 2446 <0.05* 9494 ± 3102 7600 ± 1896 <0.05*
Table 2.
 
Mean VEGF, TGF-β, and PDGF Levels Measured by ELISA in RPE and Retina
Table 2.
 
Mean VEGF, TGF-β, and PDGF Levels Measured by ELISA in RPE and Retina
Study Group Eyes VEGF TGF-β PDGF
RPE P Retina P RPE P Retina P RPE P Retina P
IV-Control Intravenous control Control 50.84 ± 8.95 NS 47.87 ± 8.98 NS 272.87 ± 3.95 NS 30.23 ± 0.58 NS 5.82 ± 1.09 NS 4.55 ± 0.28 NS
IV-17 Intravenous P17 Treated 41.59 ± 4.85 42.76 ± 2.24 269.08 ± 5.44 27.64 ± 2.84 5.76 ± 0.53 4.40 ± 0.15
LD-17 Low-dose intravitreal P17 Control 41.54 ± 2.28 NS 43.90 ± 4.31 0.08 254.96 ± 14.85 0.080 32.62 ± 1.84 <0.05* 6.67 ± 0.76 <0.05* 4.55 ± 0.24 NS
Treated 41.86 ± 1.87 36.89 ± 1.86 241.71 ± 6.92 25.42 ± 6.81 5.10 ± 0.61 4.51 ± 0.71
HD-17 High-dose intravitreal P17 Control 45.56 ± 5.03 <0.05* 44.90 ± 3.65 <0.05* 261.67 ± 14.63 <0.05* 34.10 ± 1.73 <0.05* 5.45 ± 0.46 <0.05* 4.80 ± 0.32 NS
Treated 34.54 ± 1.35 41.27 ± 2.57 192.30 ± 20.88 26.08 ± 4.45 4.92 ± 0.16 4.60 ± 0.15
LD-144 Low-dose intravitreal P144 Control 45.91 ± 6.84 NS 43.21 ± 2.27 NS 247.54 ± 3.62 NS 38.86 ± 12.08 NS 5.14 ± 0.44 NS 4.95 ± 0.61 NS
Treated 42.22 ± 5.06 42.79 ± 3.84 235.22 ± 34.97 37.45 ± 8.32 4.89 ± 0.37 4.80 ± 0.56
HD-144 High-dose intravitreal P144 Control 51.43 ± 15.59 NS 52.33 ± 10.80 <0.05* 321.09 ± 63.98 NS 33.38 ± 2.57 NS 5.24 ± 0.25 NS 5.20 ± 0.59 NS
Treated 47.08 ± 5.41 40.66 ± 5.07 318.99 ± 31.39 31.94 ± 1.57 5.19 ± 0.28 4.99 ± 0.43
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