Abstract
Purpose
To evaluate the effect of placental growth factor (PlGF) gene knockdown in a murine model of laser-induced choroidal neovascularization.
Methods
Choroidal neovascularization was induced in the left eyes of 11 mice by infrared laser. Small interfering RNA (siRNA, 20 picomoles/10 μl) corresponding to PlGF mRNA was administered intravitreally by Hamilton syringe in all subjects. One month later, fluorescein angiography and histolologic examination were performed.
Results
No leakage was apparent in the 11 eyes treated with siRNA cognate to PlGF. The results of histological evaluation were consistent with angiographic findings showing absence of choroidal neovascularization.
Conclusion
Knockdown of the PlGF gene can inhibit the growth of laser-induced choroidal neovascularization in mice.
Keywords: Choroidal Neovascularization, Placental Growth Factor, Small Interfering RNA
INTRODUCTION
The vascular endothelial growth factor (VEGF) is a well-recognized inciting angiogenic stimulus for the growth of choroidal neovascularization (CNV).1 The VEGF family includes VEGF-A, -B, -C, and -D in addition to the placental growth factor (PlGF). The presence of these proteins has recently been reported within human CNV membranes.2 PlGF is a glycoprotein synthesized in the inner portion of the neural retina and plays a key role in promoting monocyte chemotaxis, collateral vessel growth, and endothelial cell growth and migration.3,4 PlGF and VEGF demonstrate synergistic effects inpathological angiogenesis amplifying VEGF- driven angiogenesis and activation of vascular endothelial cells.5 Furthermore, PlGF mRNA expression is present in the intact choroid and is significantly up-regulated during experimental induction of CNV.6
RNA interference (RNAi) results in destruction of mRNAs that share homologous sequences with the double strand RNA (dsRNA). It has been established that synthetic RNAs, 21 and 22 nucleotides in length, called small interfering RNAs (siRNAs), are able to mediate cleavage of the target RNA.7 An in vitro study revealed that anti PlGF siRNA can hinder new vessel formation by inhibiting PlGF production.8
In the present study we evaluated the efficacy of siRNA directed against the PlGF gene in a mouse model of laser-induced CNV.
METHODS
The study was performed on 11 adult mice. All animal experiments were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Animals were anesthetized by a mixture of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight). Pupillary dilatation was achieved by administrating tropicamide 1% eye drops (SinaDarou, Tehran, Iran) prior to fundus examination, fluorescein angiography and retinal laser photocoagulation.
Choroidal neovascular lesions were induced at 1, 3, 5 o’clock positions at a distance of two disc diameters from the optic disc margin using an infrared diode laser (Keeler Microlase, Windsor, UK). Three laser burns were applied at 0.1 second duration with spot size of 100 μm and power settings ranging from 450 mW to 850 mW in order to induce a break in Bruch’s membrane indicated by the formation of a vapor bubble and a small hemorrhage. Failure to produce breaks in Bruch’s membrane or severe bleeding during laser photocoagulation resulted in exclusion of the animal from the study.
Immediately after laser photocoagulation, 11 mice received 20 picomoles /10 microliter of PlGF-specific siRNA (Qiagen, Hilden, Germany). Each siRNA solution was injected into the mid- vitreous cavity using a 10 microliter Hamilton syringe and a 30-gauge needle. Injections were monitored for evidence of reflux or inadequate injection; eyes with extensive substance reflux were excluded from the analysis.
Fluorescein angiography was performed 30 days following laser induction for each animal using the Heidelberg Retina Angiograph (HRA2, Heidelberg Engineering, Heidelberg, Germany). The angiograms were taken by intravenous injection of 10% fluorescein sodium (0.1 mL/kg).
On the same day, the animals were sacrificed under deep anesthesia and their eyes were carefully enucleated and placed in 10% neutral- buffered formalin overnight. Subsequently, each globe was processed and embedded in paraffin. Serial sections were performed and stained with hematoxylin and eosin. Slides were observed and photographed under bright field microscopy.
RESULTS
No choroidal neovascularization was visualized on fluorescein angiography in any of the 11 eyes that had received siRNA cognate to PlGF (Fig. 1).
Figure 1.
Fluorescein angiogram taken 30 days after laser injury; no leakage is observed.
Histological evaluation of the tissue samples was consistent with angiographic findings. Histologic features of CNV such as telangiectatic vessels in the choriocapillaris, subretinal hemorrhage and presence of focal subretinal exudates adjacent to the disrupted retinal pigment epithelium were not observed in any of the 11 treated eyes that had received siRNA cognate to PlGF (Figure 2).
Figure 2.
Note the absence of telangiectatic vessels in the choriocapillaris, and lack of subretinal hemorrhage and focal subretinal exudate (arrows) in an eye which had received siRNA cognate to placental growth factor [hematoxylin and eosin stain; Í100 (A) and Í400 (B)].
DISCUSSION
The development of choroidal neovascularization in eyes treated with intravitreal siRNA corresponding to PlGF mRNA was not observed in the current study, which indicates that siRNA cognate to PlGF may hinder formation of laser induced CNV.
Placental growth factor is thought to play an important role in the angiogenesis process; it could individually enhance angiogenesis through activation of VEGF receptor type 1(VEGFR-1) by forming PlGF/VEGF-A heterodimers.5 Rakic et al demonstrated that both deficient PlGF expression and PlGF receptor neutralization can significantly reduce the incidence and severity of laser-induced CNV.6 VEGF and PlGF were revealed to acquire different roles during retinal vascular development that could be justified by activation of distinct receptors; VEGF-A binds to both VEGF-R1 and VEGF-R2 whereas PlGF is unable to activate VEGF-R2 which is necessary for mitogenic and proliferative responses in endothelial cells.3 Brave et al clarified that PlGF may play a fundamental role in driving VEGF-A dependent angiogenesis when the local concentration of VEGF is low.9 Choroidal PlGF levels were upregulated three days after laser injury in a study by Van de Veire and colleagues.10 They showed that intravitreal anti-PlGF antibody could inhibit laser induced CNV and choroidal vessel leakage. Besides, they argued in favor of the efficacy and specificity of anti-PlGF monoclonal antibody focusing on the results of their study, and emphasized that delivery of monoclonal antibody and PlGF loss could induce similar mechanisms with contextual differences. They stated that angiogenesis was inhibited comparably by loss or inhibition of PlGF in choroidal neovascularization.10
VEGFR-2 expression has been detected in non-endothelial retinal cells such as ganglion cells, neural photoreceptors and Muller cells which illustrates the non-vascular function of VEGF within the retina; therefore, VEGFR-2 blockade may produce potentially harmful effects. Currently available anti-VEGF drugs for treatment of CNV do not differentiate various signaling systems mediated by VEGFR-1 and VEGFR-2. In addition, long-term exposure to VEGFR-2 blockade could have serious consequences for the retinal nerve system.11 These considerations along with PlGF binding specificity for VEGFR-1, makes the latter receptor a safer target for treating neovascular disorders.
It has been demonstrated that PlGF knockdown within the retinal pigment epithelium (RPE) has no impact on the proliferation and apoptosis of these cells. This could signify minimal side effects from PlGF knockdown on the morphology and functionality of RPE cells. Consequently, suppression of PlGF gene did not affect RPE cell proliferation and survival, nor alter the transcript levels of RPE65, cellular retinaldehyde-binding protein (CRALBP) or tyrosinase in cultures treated by siRNA cognate to PlGF.8
A major restriction of intravitreal injection is the limited volume of drugs which can be administered in the confined vitreous cavity. By applying siRNA, 100 to 1000 molecules of the target protein can be inhibited by each mRNA molecule whereas only one molecule of the target protein can be inhibited by each molecule of the antibody. Efficacy of siRNA targeting VEGF in decreasing the extent of experimental CNV has already been revealed.12 In an in vitro study by Akrami et al, almost 92% of RPE cells were successfully transfected using 20 pmol/ ml siRNA; analysis of PlGF gene expression by real-time reverse-transcription polymerase chain reaction (PCR) indicated that PlGF transcripts reached their lowest level (10% of controls) after 36 hours and that vascular tube formation was efficiently reduced with siRNA cognate to PlGF.8
Immune stimulation, especially involvement of toll-like receptor3 (TLR3), has recently been a concern in the RNAi field. According to a recent study, siRNA-based treatment for wet type AMD does not provoke an immune response. This study delineated that naked siRNA could enter the target cells in the eye and inhibit its target gene but was unable to activate TLR3; intravitreal injection of the siRNA, targeting RTP 801 (either naked or lipofectamine formulated), resulted in detection of siRNA molecules in retinal ganglion cells, RPE and retinal and choroidal endothelial cells. However, no TLR3 receptor activation was observed (Feinstein et al, Proceedings of the Association for Research in Vision & Ophthalmology 2009, Fort Lauderdale).
In summary, we used siRNA cognate to PlGF to investigate the effect of PlGF knockdown in a murine model of laser-induced choroidal neovascularization. This was performed based on our previous in vitro study, in which we
evaluated the specificity of desired PlGF siRNA and outlined no off-target effects on RPE cultures. According to our experimental study, the naked siRNA cognate to PlGF may have a significant value in the treatment of CNV due to different etiologies. However, further studies with larger sample size and a control group are required to investigate this issue.
Acknowledgments
The authors would like to thank Zohreh Salimi for performing fluorescein angiography and Omid Saffarian for technical assistance.
Footnotes
Conflicts of Interest
None.
REFERENCES
- 1.Spilsbury K, Garrett KL, Shen WY, Constable IJ, Rakoczy PE. Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to the development of choroidal neovascularization. Am J Pathol. 2000;157:135–144. doi: 10.1016/S0002-9440(10)64525-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Otani A, Takagi H, Oh H, Koyama S, Ogura Y, Matumura M, et al. Vascular endothelial growth factor family and receptor expression in human choroidal neovascular membranes. Microvasc Res. 2002;64:162–169. doi: 10.1006/mvre.2002.2407. [DOI] [PubMed] [Google Scholar]
- 3.Feeney SA, Simpson DA, Gardiner TA, Boyle C, Jamison P, Stitt AW. Role of vascular endothelial growth factor and placental growth factors during retinal vascular development and hyaloids regression. Invest Ophthalmol Vis Sci. 2003;44:839–847. doi: 10.1167/iovs.02-0040. [DOI] [PubMed] [Google Scholar]
- 4.Ziche M, Maglione D, Ribatti D, Morbidelli L, Lago CT, Battisti M, et al. Placenta growth factor-1 is chemotactic, mitogenic and angiogenic. Lab Invest. 1997;76:517–531. [PubMed] [Google Scholar]
- 5.Carmeliet P, Moons L, Luttun A, Vincenti V, Compernolle V, De Mol, et al. Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat Med. 2001;7:575–583. doi: 10.1038/87904. [DOI] [PubMed] [Google Scholar]
- 6.Rakic JM, Lambert V, Devy L, Luttun A, Carmeliet P, Claes C, et al. Placental growth factor, a member of the VEGF family, contributes to the development of choroidal neovascularization. Invest Ophthalmol Vis Sci. 2003;44:3186–3193. doi: 10.1167/iovs.02-1092. [DOI] [PubMed] [Google Scholar]
- 7.Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22- nucleotide RNAs. Genes Dev. 2001;15:188–200. doi: 10.1101/gad.862301. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Akrami H, Soheili ZS, Sadeghizadeh M, Ahmadieh H, Rezaeikanavi M, Samiei S, et al. PlGF gene knockdown in human retinal pigment epithelial cells. Graefes Arch Clin Exp Ophthalmol. 2011;249:537–546. doi: 10.1007/s00417-010-1567-7. [DOI] [PubMed] [Google Scholar]
- 9.Brave SR, Eberlein C, Shibuya M, Wedge SR, Barry ST. Placental growth factor neutralizing antibodies give limited antiangiogenic effects in an in vitro organotypic angiogenesis model. Angiogenesis. 2010;13:337–347. doi: 10.1007/s10456-010-9190-0. [DOI] [PubMed] [Google Scholar]
- 10.Van de Veire S, Stalmans I, Heindryckx F, Oura H, Tijeras-Raballand A, Schmidt T, et al. Further pharmacological and genetic evidence for the efficacy of PLGF inhibition in cancer and eye disease. Cell. 2010;141:178–190. doi: 10.1016/j.cell.2010.02.039. [DOI] [PubMed] [Google Scholar]
- 11.Cao R, Xue Y, Hedlund EM, Zhong Z, Tritsaris K, Tondelli B, et al. VEGFR1-mediated pericyte ablation links VEGF and PLGF to cancer associated retinopathy. Proc Natl Acad Sci USA. 2010;107:856–861. doi: 10.1073/pnas.0911661107. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Tolentino MJ, Brucker AJ, Fosnot J, Ying GS, Wu IH, Malik G, et al. Intravitreal injection of vascular endothelial growth factor small interfering RNA inhibits growth and leakage in a nonhuman primate laser-induced model of choroidal neovascularization. Retina. 2004;24:132–138. doi: 10.1097/00006982-200402000-00018. [DOI] [PubMed] [Google Scholar]