Ocular neovascularization, comprising retinal, choroidal, and corneal neovascularization, is associated with enormous public health problems that severely affect the quality of life of the patients.20
For example, retinal neovascularization is developed in ischemic retinopathies such as diabetic retinopathy and retinopathy of prematurity, which represent the leading causes of severe vision loss and blindness in adults of working age and premature neonates, respectively.21,22
CNV complicates several diseases, such as age-related macular degeneration, the most common cause of severe loss of vision in patients older than 60 in developed countries.23
Corneal neovascularization is a serious complication of many corneal diseases as a result of mechanical- or chemical-induced corneal injury and is associated with a high risk factor for graft rejection after corneal allograft transplantation.24,25
The present study provides the first strong evidence that parstatin, a synthetic peptide that corresponds to the cleaved peptide on PAR1 activation, has therapeutic potential in the treatment of neovascular ocular diseases. Intraocular injection of parstatin strongly suppressed ischemia-induced retinal neovascularization in neonatal mice. Dose dependency with high potency and efficacy were observed in the ischemic retinopathy model in which maximal inhibition of approximately 60% was achieved with 3 μg parstatin. Similarly, intravitreal or subconjunctival administration of parstatin inhibited CNV at Bruch's membrane rupture sites in mice and potently delayed the onset and progression of neovascularization in rat corneas with chemical burn–induced trauma. The antiangiogenic effects of parstatin in these experimental models of ocular neovascularization are comparable to those of the most effective treatments currently known, such as anti-VEGF, anti-VEGF receptor-2, and anti-PIGF.26,27
The extent of the inhibition of ocular neovascularization by parstatin attainable in these experiments may be an underestimation of the inhibition expected in primates and in humans. Parstatin peptide used in these experimental rodent models corresponds to the cleaved fragment of human PAR1, which shares 63% and 67% homology to the mouse and rat parstatin, respectively. We have demonstrated that, despite the cross-species activity of parstatin, there is considerable species specificity in vitro.8
Maximum inhibition of corneal neovascularization was demonstrated using 200 μg parstatin. This dose would be considered high enough compared with doses used in retinal and choroidal neovascularization models. It is possible that periocular (subconjunctival) or intraocular (intravitreal) administration of parstatin may result in differences in parstatin potency; therefore, a higher dose may be needed for the corneal model to achieve regression of neovascularization. Dratviman-Storobinsky et al.28
reported that different routes of bevacizumab administration in the same animal model of corneal neovascularization resulted in differences in the effectiveness of the drug. In this regard, it should also be noted that the retinal/choroidal neovascularization models were developed in mice whereas the corneal neovascularization model was established in rats, which have a much greater eye size and subsequently a larger volume distribution for the administered parstatin. In addition, our results have shown that the application of parstatin in adult animal eyes was well tolerated and without detectable immunoresponses or toxicity in the cornea or retina. However, in an ischemia-induced retinal neovascularization model in neonatal mice, parstatin exhibited toxicity in the retina at concentrations greater than 10 μg. The different toxicologic profile of parstatin between ocular neovascularization animal models cannot be explained, but it is likely that the immature retina is more sensitive to parstatin than the adult tissues. This may be related to the fact that the neonatal retina is still actively developing, and parstatin may interfere with this development. Alternatively, this apparent toxicity may be related to metabolic or permeability differences in the immature retina. No adverse effects, however, have been observed when parstatin is administered to adult animals. Nevertheless, more experimental studies are needed to evaluate the long-term results and safety of parstatin for the treatment of ocular diseases.
We can speculate on the possible mechanisms involved in the inhibitory activity of parstatin on ocular neovascularization. Many factors have been proposed to mediate ocular neovascularization, but VEGF seems to play a critical role since increased VEGF levels have been shown to be a common pathologic factor in neovascular ocular diseases in humans and in animal models, and signaling through VEGF receptors is both necessary and sufficient for the development of aberrant ocular neovascularization.10,29–31
In line with these observations, the development of potent VEGF antagonists has revolutionized the treatment of CNV caused by age-related macular degeneration32
and corneal neovascularization caused by various etiologies.33
However, despite these impressive recent advances, anti-VEGF therapies seem to stabilize the disease process rather than improve vision, indicating that proangiogenic factors other than VEGF may be involved. For example, although the overexpression of FGF2 in the eye does not stimulate neovascularization because it is sequestered,9,34
FGF2 does contribute to CNV when there is tissue disruption from the disease process itself or attempts at treatment.35
Parstatin has been demonstrated to have direct effects on endothelial cells.8
It inhibits survival, proliferation, migration, and tube formation of cultured vascular endothelial cells, and it rapidly localizes to the cell surface, penetrates the cell membrane, and accumulates in the intracellular space. These events may explain the specific interactions between parstatin and signaling mediated by VEGF and FGF2. Pretreatment of endothelial cells with parstatin blocked the activation of Erk1/2 stimulated by either VEGF or FGF2 but had no effect on epidermal growth factor (EGF)- and heparin-binding EGF-driven mitogenic responses. Therefore, combined blockage of VEGF and FGF2 receptor signaling by parstatin may provide greater efficacy for the treatment of ocular neovascularization than does targeting VEGF alone. The precise molecular targets that mediate these cellular effects of parstatin are under investigation. Furthermore, parstatin has been shown to promote cell cycle arrest and apoptosis in endothelial cells through a mechanism involving, at least in part, the activation of caspase-3.8
However, we have no evidence whether parstatin functions in the same way in animal models of neovascularization. We are planning to perform apoptosis experiments in an oxygen-induced retinal neovascularization model to investigate this issue.
In addition, parstatin is likely to suppress ocular neovascularization through the inhibition of ocular inflammation. Because inflammatory cells play an essential role in the formation of choroidal and retinal neovascularization, the prevention of inflammatory cell recruitment and infiltration into ocular tissues may ameliorate the development of ocular neovascularization.12,36–38
During inflammation, leukocytes are recruited to the retina in a cascade-like fashion, starting with rolling, followed by firm adhesion and extravasation.39,40
We have shown in the present study that intraocular injection of parstatin significantly reduced VEGF-induced leukocyte influx into the retina. Furthermore, we found that the application of parstatin markedly reduced the number of inflammatory cells in rat corneas after chemical cauterization. Given that the development of new blood vessels toward the area of the burn is closely associated with increased inflammatory cells within the cornea,17
our findings suggest that the inhibition of corneal neovascularization by parstatin may be due, at least in part, to the suppression of inflammatory cell recruitment.
In conclusion, in this study we evaluated the pharmacologic efficacy of parstatin for inhibiting ocular neovascularization induced in three well-established animal models. We demonstrated that parstatin effectively suppresses retinal, choroidal, and corneal neovascularization and reduces inflammatory cell recruitment to the ocular lesions. These findings suggest that parstatin may represent a novel attractive and potent therapeutic strategy in the treatment of ocular neovascular and inflammatory diseases.